JP2008503212A - Methods for diagnosis and treatment of obesity, diabetes and insulin resistance - Google Patents

Abstract

The present invention provides compositions and methods for the diagnosis and treatment of obesity, diabetes and insulin resistance. In particular, the invention relates to a method for identifying modulators of the polynucleotides or polypeptides of the invention and methods for using these modifiers for the treatment of obesity and / or diabetes, as well as the polynucleotides or polypeptides of the invention in patients A method of diagnosing obesity and / or diabetes by measuring levels of is provided.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application is a priority to US Provisional Patent Application No. 60 / 580,448, filed June 16, 2004, which is hereby incorporated by reference in its entirety for all purposes. Insist.

BACKGROUND OF THE INVENTION Obesity has reached a high rate worldwide, with over 1 billion adults being overweight—of which at least 300 million are clinically obese—and chronic diseases and disabilities It is a major cause of global hardship. Overweight and obesity lead to deleterious metabolic effects on blood pressure, cholesterol, triglycerides and insulin resistance. Nonfatal but debilitating health problems associated with obesity include dyspnea, chronic musculoskeletal disorders, skin and infertility. More life-threatening problems can be divided into four main areas: cardiovascular disorders, conditions with insulin resistance (such as type 2 diabetes), certain cancers (especially hormone-related), and Colorectal cancer) as well as gallbladder disease. The likelihood of developing type 2 diabetes and hypertension increases rapidly as physical obesity increases. Weight loss results in correction of a number of endocrine and metabolic diseases associated with obesity.

  Effective weight management for individuals and populations at risk of developing obesity includes a variety of long-term strategies. These include prevention, weight maintenance, management of comorbidities, and weight loss. Existing treatment strategies include calorie restriction programs, surgery (gastric stapling) and drug intervention. Currently available anti-obesity drugs can be divided into two classes: centrally acting and peripherally acting. There are three drugs on the market: Xenical (orlistat), Merida (sibutramine) and Adipex-P (phentermine). Xenical is a non-systemic GI lipase inhibitor, indicated for short-term and long-term obesity management. Merida reduces food intake, primarily through inhibition of norepinephrine and serotonin reuptake. Adipex-P is phenteramine having sympathomimetic activity and suppresses appetite. This is only for short-term use. More radical solutions for permanent weight loss are surgery, and gastric bypass that limits caloric absorption by a significant reduction in stomach size.

  Having excess weight and body fat is closely related to the development of diabetes. People who are overweight (BMI> 25) are at a much higher risk of developing type 2 diabetes than normal-weight individuals. Nearly 90% of people with type 2 diabetes are overweight.

  Diabetes can be divided into two types of clinical syndromes, type 1 diabetes and type 2 diabetes. Type 1 diabetes or insulin-dependent diabetes mellitus (IDDM) is a chronic autoimmune disease characterized by a high loss of insulin-producing beta cells in the pancreatic islets of Langerhans. Since these cells are progressively destroyed, the amount of insulin secreted decreases, and eventually the hyperglycemia when the amount of secreted insulin decreases below that required for normoglycemia (normal blood glucose levels) (Abnormally high blood glucose concentration). The exact trigger of this immune response is unclear, but IDDM patients have high levels of antibodies to proteins expressed in pancreatic β cells. However, not all patients with high levels of these antibodies develop IDDM.

  Type 2 diabetes (also called non-insulin dependent diabetes mellitus (NIDDM)) develops when muscle, fat, and hepatocytes are unable to make a normal response to insulin. This responsive disorder (referred to as insulin resistance) may be due to a decrease in the number of insulin receptors on the surface of these cells, dysfunction of signaling pathways within the cells, or both. Beta cells initially compensate for this insulin resistance by increasing insulin production. Over time, these cells become unable to produce enough insulin to maintain normal blood glucose levels, which means progression to type 2 diabetes.

  Type 2 diabetes is caused by a combination of genetic risk factors and acquired risk factors, including a high-fat diet, lack of exercise, and aging. Type 2 diabetes is widespread worldwide due to an increased lifestyle of obesity and sedentary and underexercise, widespread Western dietary habits, and the overall aging of the population in many countries. As of 1985, it is estimated that there are 30 million diabetics worldwide, and by 2000 this number had increased fivefold to an estimated 154 million. The number of diabetic patients is expected to double by 2025 from now to reach about 300 million.

  Type 2 diabetes is a complex disease characterized by defects in glucose and lipid metabolism. It is common for many metabolic parameters to be disturbed, including fasting plasma glucose levels, free fatty acid and triglyceride levels, and decreased HDL / LDL ratios. As discussed above, one of the main causes underlying diabetes is thought to be an increase in insulin resistance in surrounding tissues, mainly muscle and fat.

  Treatments aimed at reducing peripheral insulin resistance can be used. Of most relevance to the present invention are thiazolidinedione (TZD) class drugs, namely troglitazone, pioglitazone and rosiglitazone. In the United States, they are sold under the names Rezulin (TM), Avandia (TM) and Actos (TM) respectively. The main effect of these drugs is to improve glucose homeostasis. In particular, diabetic patients treated with TZD have an increased rate of peripheral glucose processing that shows increased insulin sensitivity in both muscle and fat.

  The molecular target of TZD is a member of the PPAR family of ligand-activated transcription factors called PPARγ. This transcription factor is highly expressed in adipose tissue, but the levels observed in muscle are much lower. Binding of TZD and PPARγ in target cells and tissues such as fat and muscle results in altered gene expression. The relationship between TZD-modified gene expression in fat and muscle and increased insulin sensitivity is unclear. The present invention addresses this and other issues.

SUMMARY OF THE INVENTION The present invention provides a method for identifying agents for treating obese, diabetic or prediabetic individuals. In some embodiments, the method is (i) substantially the same as a nucleic acid encoding a polypeptide listed in Table 1, or in 50% formamide, 5 × SSC and 1% SDS at 42 ° C. Contacting the agent with a polypeptide encoded by a polynucleotide that hybridizes to it under conditions of washing at 55 ° C. in 0.2 × SSC and 0.1% SDS following hybridization at The polypeptide optionally has the activities listed in Table 1; and (ii) selects an agent that modulates the expression or activity of the polypeptide or binds to the polypeptide, thereby obesity, Identifying an agent for treating a diabetic or prediabetic individual.

Table 1 Polypeptide, SEQ ID number and list of proposed activities

またはそのタンパク質ドメインに対して少なくとも95％同一なアミノ酸配列を含む。いくつかの態様において、本方法はさらに、選択された作用物質が体重および／または肥満を変化させるか否かを検出することを含む。いくつかの態様において、本方法はさらに、選択された作用物質がインスリン感受性を変化させるか否かを検出することを含む。 In some embodiments, the polypeptide is

Or an amino acid sequence that is at least 95% identical to the protein domain. In some embodiments, the method further comprises detecting whether the selected agent alters body weight and / or obesity. In some embodiments, the method further comprises detecting whether the selected agent alters insulin sensitivity.

  In some embodiments, step (ii) comprises selecting an agent that alters the expression of the polypeptide. In some embodiments, step (ii) comprises selecting an agent that alters the activity of the polypeptide. In some embodiments, step (ii) comprises selecting an agent that specifically binds to the polypeptide.

を含む。 In some embodiments, the polypeptide is expressed intracellularly and the cell is contacted with an agent. In some embodiments, the polypeptide is

including.

の活性または発現を変化させる作用物質の有効量を動物に投与することを含む。 The present invention also provides a method for reducing body weight in an animal. In some embodiments, the method comprises:

Administering to the animal an effective amount of an agent that alters the activity or expression of.

  In some embodiments, the agent is (i) substantially identical to a nucleic acid encoding a polypeptide listed in Table 1, or at 42 ° C. in 50% formamide, 5 × SSC and 1% SDS. Contacting the agent with a mixture comprising a polypeptide encoded by a polynucleotide that hybridizes to it under conditions of subsequent washing at 55 ° C. in 0.2 × SSC and 0.1% SDS. Wherein the polypeptide optionally has the activity listed in Table 1; and (ii) selecting an agent that alters the expression or activity of the polypeptide or binds to the polypeptide. Selected by the method of inclusion.

  In some embodiments, the agent is an antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the animal is a human.

の活性または発現を変化させる作用物質の治療的有効量を動物に投与することを含む。いくつかの態様において、ポリペプチドは、

またはそのタンパク質ドメインに対して少なくとも95％同一なアミノ酸配列を含む。 The invention also provides a method of treating a diabetic or prediabetic animal. In some embodiments, the method comprises:

Administering to the animal a therapeutically effective amount of an agent that alters its activity or expression. In some embodiments, the polypeptide is

Or an amino acid sequence that is at least 95% identical to the protein domain.

をコードする核酸とハイブリダイズするポリヌクレオチドによってコードされるポリペプチドを含む混合物と、作用物質を接触させること；および（ii）ポリペプチドの発現もしくは活性を変化させるか、またはポリペプチドと結合する作用物質を選択すること、を含む方法によって選択される。 In some embodiments, the agent is (i) hybridized in 50% formamide, 5 × SSC and 1% SDS at 42 ° C. followed by washing in 0.2 × SSC and 0.1% SDS at 55 ° C. In case

Contacting the agent with a mixture comprising a polypeptide encoded by a polynucleotide that hybridizes with a nucleic acid encoding; and (ii) altering the expression or activity of the polypeptide or binding to the polypeptide. Selecting a substance is selected by a method comprising.

  In some embodiments, the agent is an antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the animal is a human.

またはそのタンパク質ドメインに対して少なくとも95％同一なアミノ酸配列を含む。 The present invention also provides a method for introducing an expression cassette into a cell. In some embodiments, the method comprises introducing into the cell an expression cassette comprising a promoter operably linked to a polynucleotide encoding the polypeptide, wherein the polynucleotide is listed in Table 1. Is substantially identical to the nucleic acid encoding the polypeptide, or hybridization in 50% formamide, 5 × SSC and 1% SDS at 42 ° C. followed by hybridization at 42 ° C. in 0.2 × SSC and 0.1% SDS at 55 ° C. And the polypeptide optionally has the activities listed in Table 1. In some embodiments, the polypeptide is

Or an amino acid sequence that is at least 95% identical to the protein domain.

を含む。 In some embodiments, the polypeptide is

including.

  In some embodiments, the cell is selected from the group consisting of adipocytes and skeletal muscle cells.

  In some embodiments, the method further comprises introducing the cell into a human. In some embodiments, the human is obese. In some embodiments, the human is diabetic. In some embodiments, the human is prediabetic. In some embodiments, the cell is from a human.

  The invention also provides a method of diagnosing an individual who has obesity, type 2 diabetes, or is predisposed to diabetes or obesity. In some embodiments, the method comprises detecting the level of a polypeptide or the level of a polynucleotide encoding the polypeptide in a sample from an individual, wherein the polynucleotide is a polypeptide listed in Table 1. Is substantially identical to the nucleic acid encoding or washed in 50% formamide, 5 × SSC and 1% SDS in 42 ° C. followed by hybridization in 0.2 × SSC and 0.1% SDS at 55 ° C. And the level of the polypeptide or polynucleotide in the sample varies relative to the level of the polypeptide or polynucleotide in a normal body weight lean individual or a previous sample of the same individual. The individual is obese or diabetic or predisposed to obesity or diabetes Is shown.

を含む。いくつかの態様において、検出の段階は、ポリペプチドをコードするmRNAを定量することを含む。いくつかの態様においては、mRNAを逆転写させ、ポリメラーゼ連鎖反応で増幅する。いくつかの態様において、試料は血液試料、尿試料または組織試料である。 In some embodiments, the detecting step comprises contacting the sample with an antibody that specifically binds to the polypeptide. In some embodiments, the amino acid sequence is

including. In some embodiments, the detecting step comprises quantifying mRNA encoding the polypeptide. In some embodiments, the mRNA is reverse transcribed and amplified by polymerase chain reaction. In some embodiments, the sample is a blood sample, a urine sample or a tissue sample.

を含む。いくつかの態様において、ポリヌクレオチドは、

をコードする。 The invention is substantially identical to nucleic acids encoding the polypeptides listed in Table 1, or 0.2 × following hybridization at 42 ° C. in 50% formamide, 5 × SSC and 1% SDS. An isolated nucleic acid is provided that hybridizes to it under conditions of washing at 55 ° C. in SSC and 0.1% SDS. In some embodiments, the polynucleotide is

including. In some embodiments, the polynucleotide is

Code.

  The present invention is also substantially identical to nucleic acids encoding the polypeptides listed in Table 1, or 0.2 following hybridization at 42 ° C. in 50% formamide, 5 × SSC and 1% SDS. Also provided is an expression cassette comprising a nucleic acid that hybridizes under conditions of washing at 55 ° C. in xSSC and 0.1% SDS and a heterologous promoter operably linked.

  The present invention is also substantially identical to nucleic acids encoding the polypeptides listed in Table 1, or 0.2 following hybridization at 42 ° C. in 50% formamide, 5 × SSC and 1% SDS. Also provided are host cells transfected with nucleic acids that hybridize under conditions of washing at 55 ° C. in xSSC and 0.1% SDS. In some embodiments, the host cell is a human cell. In some embodiments, the host cell is a bacterium.

またはその断片に対して少なくとも70％同一なアミノ酸配列を含む、単離されたポリペプチドも提供する。いくつかの態様において、ポリペプチドは、

を含む。 The present invention also provides

Also provided is an isolated polypeptide comprising an amino acid sequence that is at least 70% identical to a fragment thereof. In some embodiments, the polypeptide is

including.

からなる群より選択されるポリペプチドと特異的に結合する抗体も提供する。 The present invention also provides

An antibody that specifically binds to a polypeptide selected from the group consisting of:

またはその断片に対して少なくとも70％同一なアミノ酸配列を含むポリペプチド、および薬学的に許容される添加剤を含む薬学的組成物も提供する。 The present invention also provides

Or a pharmaceutical composition comprising a polypeptide comprising an amino acid sequence at least 70% identical to a fragment thereof and a pharmaceutically acceptable additive.

Definitions “Insulin sensitivity” refers to the ability of a cell or tissue to respond to insulin. The response includes, for example, cellular or tissue glucose uptake in response to insulin stimulation. Sensitivity can be determined at the level of an individual organism, tissue or cell. For example, blood or urine glucose level after a glucose tolerance test is an indicator of insulin sensitivity. Other methods of measuring insulin sensitivity include, for example, measurement of glucose uptake (eg, Garcia de Herreros, A. and Birnbaum, MJJ Biol. Chem. 264, 19994-19999 (1989); Klip, A., Li, G And Logan, WJ Am. J. Physiol. 247, E291-296 (1984)), measurement of glucose leaching rate (GINF) into tissues such as skeletal muscle (see, eg, Ludvik et al., J. Clin. Invest. 100: 2354 (1997); Frias et al., Diabetes Care 23: 64 (2000)) and measurement of the sensitivity of GLUT4 translocation in response to insulin (eg, as described herein).

  As used herein, an overweight person has a body mass index (BMI) ≧ 25 and an “obese” person has a BMI ≧ 30. BMI is calculated as weight (in kg) divided by height (in m) squared.

  The term “waist / hip ratio or WHR” refers to the ratio of a person's waist circumference to the hip circumference. For most people, having extra weight around the torso raises a health risk than having extra weight around the hips or thighs. For both men and women, a waist / hip ratio of 1.0 or higher is a “risk” of undesirable health consequences such as heart disease and other illnesses associated with being overweight. Or considered to be in the danger zone.

  The term “adipogenic”, when used in reference to a cell, refers to a cell that can become an adipocyte. “Adipocyteizing factor” refers to a factor (eg, including a protein (or glycoprotein)) that can induce or stimulate differentiation of cells into adipocytes. Exemplary adipogenic factors include, for example, _Wnt10b, Pref-1, ADF, and TNF-α.

  The term “lipid metabolism” refers to the in vivo process of catabolism (degradation) and assimilation (accumulation) of lipids (eg, food-derived triglycerides), which in a broad sense is a reaction that converts lipids into energy. , Fatty acid, acylglycerol biosynthesis, phospholipid metabolism and cholesterol metabolism.

“Activity” of a polypeptide of the invention refers to the structural, regulatory or biochemical function of the polypeptide in its native cell or tissue. Examples of polypeptide activity include both direct and indirect activity. Examples of direct activity include the result of direct interaction with the polypeptide, eg, production or removal of enzyme activity, ligand binding, second messengers (eg, cAMP, cGMP, IP ₃ , DAG or Ca ²⁺ ) , Ion flow, phosphorylation level, transcription level, and the like. An example of indirect activity is a change in phenotype or response in a cell or tissue to the activity to which the polypeptide is directed, eg, weight loss as a result of the interaction of the polypeptide with other cellular or tissue elements. Or as a molecular event associated with weight loss or obesity, or as the modulation of cellular insulin sensitivity.

“Predisposition to diabetes” is present in a person when the person is at high risk of developing diabetes. Numerous risk factors are known to those of skill in the art and include the following: genetic factors (eg, having an allele that has a higher incidence of diabetes than the average population, or a parent of diabetes Or have siblings); overweight (eg, body mass index (BMI) of 25 kg / m ² or higher); lack of regular exercise, race / ethnicity (eg, African American, Spanish American) People, Native Americans, Asian Americans, Pacific Islanders); fasting glycemic or glucose intolerance previously confirmed, high blood pressure (eg 140 / 90mmHg or higher in adults); HDL cholesterol level 35mg / dl or Lower; Triglyceride levels of 250 mg / dl or higher; Pregnancy diabetes or a history of neonatal delivery greater than 9 pounds; and / or polycystic ovary syndrome. See, for example, “Report of the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus” and “Screening for Diabetes” Diabetes Care 25 (1): S5-S24 (2002).

  A “normal body weight individual (lean individual)” refers to a fasting blood glucose level of less than 100 mg / dl when used in contrast to a sample from a patient, or a 2 hour PG blood glucose reading (2 hour PG). It refers to an adult who is less than 140 mg / dl. “Fast” refers to no caloric intake for at least 8 hours. The “blood glucose level after 2 hours load” refers to the blood glucose level after giving the patient a glucose load including a 75 g anhydrous glucose equivalent dissolved in water. This entire test is commonly referred to as the oral glucose tolerance test (OGTT). See, for example, Diabetes Care, 2003, 26 (11): 3160-3167 (2003). The level of polypeptide in a normal weight glycemic individual may be a reading from a single individual, but is usually a statistically meaningful mean value by a group of normal weight glycemic individuals. The level of polypeptide in a normal weight glycemic individual can be represented by a value, for example in a computer program.

  A “pre-diabetic individual”, when used to compare with a sample from a patient, has a fasting blood glucose level greater than 100 mg / dl but less than 126 mg / dl, or a blood glucose reading after 2 hours of loading. Refers to an adult who is above 140 mg / dl but below 200 mg / dl. A “diabetic individual” is an adult whose fasting blood glucose level is above 126 mg / dl or blood glucose readings above 200 mg / dl after 2 hours of loading when used to compare with samples from patients. Point to.

  An “agonist” is a polypeptide that binds to, activates, increases, activates, promotes, enhances activation, enhances sensitivity, or upregulates its activity or expression. , Refers to the active substance.

  An “antagonist” refers to a polypeptide of the invention that binds, partially or completely prevents its activation, reduces, prevents, delays activation, inactivates, reduces sensitivity, or activates it. Alternatively, it refers to an agent that down-regulates expression.

  “Antibody” refers to a polypeptide or fragment thereof substantially encoded by one or more immunoglobulin genes that specifically binds and recognizes an analyte (antigen). The generally recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as a large number of immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as γ, μ, α, δ, or ε, which define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively.

  A typical immunoglobulin (antibody) structural unit is composed of tetramers. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light chain” (about 25 kDa) and one “heavy chain” (about 50-70 kDa). At the N-terminus of each chain, a variable region consisting of about 100 to 110 amino acids or more, mainly responsible for antigen recognition, is defined. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains, respectively.

Antibodies exist as various well-characterized fragments, eg, produced by digestion with intact immunoglobulins or various peptidases. That is, for example, pepsin cleaves an antibody under a disulfide bond in the hinge region, and produces a Fab dimer, F (ab) ′ ₂ , which is a light chain linked to VH-CH1 by a disulfide bond. F (ab) _'2 was reduced under mild conditions, thereby converting the F (ab)' to break the disulfide linkage in the hinge region ₂ dimer may be converted to Fab 'monomer. The Fab ′ monomer is essentially a Fab with part of the hinge region (see Paul (ed.) “Fundamental Immunology” 3rd edition, Raven Press, NY (1993)). While various antibody fragments have been defined in terms of complete antibody digestion, one skilled in the art will appreciate that such fragments can be synthesized de novo using chemical or recombinant DNA methods. For this reason, the term antibody as used herein includes antibody fragments produced by modification of the entire antibody, or those de novo synthesized using recombinant DNA methods (eg, single chain Fv).

  The terms “peptidomimetic” and “mimetic” refer to a synthetic compound having substantially the same structural and functional characteristics as an antagonist or agonist of the present invention. Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties similar to template peptides. This type of non-peptide compound is called a “peptide mimetic” or “peptide mimetic” (Fauchere, J. Adv. Drug Res. 15: 29 (1986); Veber and Freidinger, TINS p 392 (1985); and Evans et al., J. Med. Chem. 30: 1229 (1987), which are incorporated herein by reference). By using a peptidomimetic that is structurally similar to a therapeutically useful peptide, an equivalent or improved therapeutic or prophylactic effect may be obtained. In general, a peptidomimetic is structurally similar to an exemplary polypeptide (ie, a polypeptide having biological or pharmacological activity), such as the polypeptides exemplified herein, but with CH 2 NH—, —CH 2 S, One or more optionally substituted by a chain selected from the group consisting of -CH2-CH2-, -CH = CH- (cis and trans), -COCH2-, -CH (OH) CH2- and CH2SO- It has the following peptide chain. The mimetics may be composed entirely of synthetic, non-natural amino acid analogs, or may be chimeric molecules, some of which are natural peptide amino acids and some are non-natural amino acid analogs. A mimetic can also include any amount of conservative substitutions of natural amino acids, as long as such substitutions do not substantially alter the mimetic's structure and / or activity. For example, a mimetic composition is within the scope of the present invention if it can achieve the binding or other activity of an agonist or antagonist of a polypeptide of the present invention.

  The term “gene” refers to a segment of DNA involved in the production of a polypeptide chain; this includes regions before and after the coding region (leader and trailer), as well as individual coding segments (exons) ) Intervening sequences (introns) are also included.

  The term “isolated” when applied to a nucleic acid or protein indicates that the nucleic acid or protein is essentially free of other cellular components that naturally accompany it. This may be in a uniform state, but may be dry or in an aqueous solution. Purity and homogeneity are usually determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The protein, the most prevalent species present in the preparation, is substantially purified. In particular, an isolated gene is separated from an open reading frame encoding a protein other than the gene of interest adjacent to the gene. The term “purified” refers to the nucleic acid or protein producing essentially one band in the electrophoresis gel. This means in particular that the purity of the nucleic acid or protein is at least 85%, more preferably at least 95%, most preferably at least 99%.

  The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. Unless specifically limited, the term includes known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to natural nucleotides. Unless otherwise indicated, each nucleic acid sequence implicitly includes conservatively modified variants thereof (eg, degenerate codon substitutions) and complementary sequences in addition to the explicitly specified sequence. . Specifically, degenerate codon substitution is performed by generating a sequence in which the third position of one or more selected (or all) codons is replaced by a mixed base and / or deoxyinosine residue. (Batzer et al., Nucleic Acids Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); and Cassol et al. (1992); Rossolini et al., Mol. Cell. Probes 8 : 91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.

  The terms “polypeptide”, “peptide”, and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. These terms apply to natural amino acid polymers and non-natural amino acid polymers as well as amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding natural amino acid. The As used herein, these terms include amino acid chains of any length, including full-length proteins (ie, antigens), in which amino acid residues are linked by covalent peptide bonds.

  The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Natural amino acids include those encoded by the genetic code, as well as subsequently modified amino acids such as hydroxyproline, γ-carboxyglutamic acid and O-phosphoserine. Amino acid analogs are compounds having the same basic chemical structure as natural amino acids, i.e., compounds having an α carbon bonded to hydrogen, carboxyl group, amino group and R group, such as homoserine, norleucine, methionine sulfoxide, methionine Refers to methylsulfonium. This type of analog has a modified R group (eg, norleucine) or a modified peptide backbone, but retains the same basic chemical structure as a naturally occurring amino acid. “Amino acid mimetics” refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

  Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides are also referenced by their generally accepted single letter symbols.

  “Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With regard to an individual nucleic acid sequence, a “conservatively modified variant” refers to a nucleic acid that encodes the same or essentially the same amino acid sequence, or is essential if the nucleic acid does not encode an amino acid sequence. Refers to the same sequence. Because of the degeneracy of the genetic code, any protein can be encoded by a number of functionally identical nucleic acids. For example, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at any position where alanine is specified by a codon, the codon can be changed to any of the corresponding codons without changing the encoded polypeptide. Such nucleic acid variants are “silent variants” and are a type of conservatively modified variant. Every nucleic acid sequence herein that encodes any polypeptide also describes every possible silent variation of the nucleic acid. One skilled in the art can modify each codon in the nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) to create a functionally identical molecule I understand that. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

  With respect to amino acid sequences, one skilled in the art will recognize individual substitutions, deletions or deletions to a nucleic acid, peptide, polypeptide or protein sequence in which a single amino acid or a small number of amino acids in the encoded sequence are altered, added or removed. It is understood that an adduct is a “conservatively modified variant” in which an amino acid is replaced by a chemically similar amino acid upon modification. Conservative substitution tables resulting in functionally similar amino acids are also well known in the art. Such conservatively modified variants are in addition to the polymorphic variants, interspecies homologs and alleles of the present invention and are not excluded.

The following eight groups each contain amino acids that are conserved from each other:
1) Alanine (A), Glycine (G);
2) Aspartic acid (D), glutamic acid (E);
3) Asparagine (N), glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), leucine (L), methionine (M), valine (V);
6) phenylalanine (F), tyrosine (Y), tryptophan (W);
7) serine (S), threonine (T); and
8) Cysteine (C), methionine (M)
(See, for example, Creighton, “Proteins” (1984)).

  “Percentage of sequence identity” is determined by comparing two optimally aligned sequences over a comparison window, where a portion of the polynucleotide sequence in the comparison region is , For optimal alignment of the two sequences, may include additions or deletions (ie, gaps) relative to a reference sequence (eg, a polypeptide of the invention) that does not contain additions or deletions. This rate is determined by determining the number of positions where the same nucleobase or residue is present in both sequences to determine the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison zone, and Calculated by multiplying the result by 100 to obtain the sequence match rate.

）と実質的に同一であるポリペプチドまたはポリヌクレオチドを提供する。この定義は、被験配列の相補物のことも指す。選択的には、同一性は、少なくとも約50ヌクレオチド長の領域にわたって、またはより好ましくは100〜500ヌクレオチド長もしくは1000ヌクレオチド長またはそれ以上の領域にわたって存在する。 In the context of two or more nucleic acid or polypeptide sequences, the term “identical” or “match” rate refers to two or more sequences or subsequences that are the same sequence. Over a certain comparison area, using one of the following sequence comparison algorithms or over the area specified by manual alignment and visual inspection, or over the entire sequence if not indicated, When compared and aligned as described, the two sequences are in the specified ratio of the same amino acid residues or nucleotides (i.e. 60% over the specified region or over the entire sequence if not specified). Sequences are “substantially identical” if they are identical (optionally 65%, 70%, 75%, 80%, 85%, 90% or 95% identity). The present invention includes polypeptides or polynucleotides each exemplified herein (eg,

A polypeptide or polynucleotide substantially the same as This definition also refers to the complement of the test sequence. Optionally, identity exists over a region that is at least about 50 nucleotides in length, or more preferably over a region that is 100-500 nucleotides in length or 1000 nucleotides in length or longer.

  For sequence comparison, it is common to use one sequence as a reference sequence for comparison with a test sequence. When using a sequence comparison algorithm, a test sequence and a reference sequence are input to a computer, and coordinates of a partial sequence are designated as necessary, and parameters of a sequence algorithm program are designated. Default program parameters can be used, or alternative parameters can be designated. Subsequently, based on the program parameters, the sequence match rate or similarity rate of the test sequence with respect to the reference sequence is calculated by a sequence comparison algorithm.

  As used herein, a “comparison window” is a 20-600 such that after optimal alignment of two sequences, a sequence can be compared to a reference sequence having the same number of consecutive positions. Includes a reference to any one of a number of consecutive locations selected from the group consisting of about 50 to about 200, more usually about 100 to about 150. Methods for alignment of sequences for comparison are well known in the art. The optimal alignment of the sequences for comparison was determined by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2: 482c, according to Needleman and Wunsch (1970) J. Mol. Biol. A homology alignment algorithm was used to calculate the computer implementation of these algorithms (Wisconsin Genetics Software Package (Genetics Computer Package) using the similarity search method of Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci. Group, 575 Science Dr., Madison, Wis.) GAP, BESTFIT, FASTA and TFASTA) or by manual alignment and visual inspection (eg, Ausubel et al., “Current Protocols in Molecular Biology” (1995). See Year Addendum).

  Two examples of algorithms that are suitable for determining sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are Altschul et al., Nuc. Acids Res. 25: 3389-3402 (1977) and Altschul et al., Respectively. J. Mol. Biol. 215: 403-410 (1990). Software for performing BLAST analysis is published in the National Center for Biotechnology Information (http://www.ncbi.n1m.nih.gov/). The algorithm identifies short words of length W in the search sequence that match or meet some positive threshold score T when aligned with words of the same length in the database sequence. First, a high-scoring sequence pair (HSP) is identified. T is referred to as the neighborhood word score threshold (Altschul et al., Supra). These initial neighborhood word hits are the source of initiating searches to find long HSPs containing them. The word search is extended in both directions of each sequence as long as the cumulative alignment score increases. The cumulative score is calculated using the parameters M (reward score for matching residue pairs; always> 0) and N (penalty score for mismatched residues; always <0) for nucleotide sequences. For amino acid sequences, a score matrix is used to calculate the cumulative score. The word search extension in each direction stops when: The cumulative alignment score is below the amount X compared to the maximum achieved value: to accumulate one or more negative score residue alignments When the cumulative score is zero or less; or when either end of the sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses word length (W) 11, expectation (E) 10, M = 5, N = -4 and comparison of both strands as default. For amino acid sequences, the BLASTP program defaults to alignment of word length 3 and expected value (E) 10, and BLOSUM62 score matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)) (B) 50, expected value (E) 10, M = 5, N = -4 and comparison of both strands.

  The BLAST algorithm also performs statistical analysis on the similarity between two sequences (see, eg, Kahn and Altschul (1993), Proc. Natl. Acad. Sci. USA 90: 5873-5787). One measure of similarity obtained by the BLAST algorithm is the smallest sum probability (P (N)), which is a measure of the probability that a match between two nucleotide or amino acid sequences will happen by chance. It becomes. For example, a nucleic acid is considered similar to a reference sequence if the minimum total probability by comparison of the test nucleic acid and the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001. .

  One indication that two nucleic acid sequences or polypeptides are substantially identical is that, as described below, the polypeptide encoded by the first nucleic acid differs from the polypeptide encoded by the second nucleic acid. It is immunologically cross-reactive with the antibodies produced against it. Thus, for example, a polypeptide is generally substantially identical to a second polypeptide if the two peptides differ by only conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

  The phrase “selectively (or specifically) hybridizes” means that the binding, duplex formation or hybridization of a molecule is a complex mixture of sequences (eg, whole cell or library DNA or RNA). When present in, it refers to what occurs only for a specific nucleotide sequence under stringent hybridization conditions.

The phrase “stringent hybridization conditions” generally refers to conditions in a complex mixture of nucleic acids that a probe will hybridize to its target subsequence, but not to other sequences. . Stringent conditions are sequence-dependent and will be different for different environments. Longer sequences are considered to hybridize specifically at higher temperatures. Detailed guidance on nucleic acid hybridization is described in Tijssen, “Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes”, “Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-10 ° C. lower than the melting point (T _m ) for the specific sequence at a defined ionic strength and pH. T _m is the temperature (under a defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize in equilibrium (in excess of the target sequence). Therefore, at _Tm , 50% of the probe is occupied in equilibrium). Stringent conditions include salt concentrations of less than about 1.0M sodium ion concentration, typically about 0.01 to 1.0M sodium ion (or other salt) concentration, pH 7.0 to 8.3, and short temperatures It will be at least about 30 ° C. for probes (eg, 10-50 nucleotides) and at least about 60 ° C. for (eg, greater than 50 nucleotides). Stringent conditions can also be obtained with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least twice background value, optionally 10 times background hybridization. Examples of stringent hybridization conditions include: 50% formamide, 5x SSC and 1% SDS, incubated at 42 ° C, or 5x SSC, 1% SDS, 65 ° C. And then wash at 0.2 × SSC and 0.1% SDS, 55 ° C., 60 ° C., or 65 ° C. Such washing may be performed for 5, 15, 30, 60, 120 minutes or even more fractions.

  Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides that encode them are substantially identical. This occurs, for example, in the case of nucleic acid copies produced using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acid generally hybridizes under moderately stringent hybridization conditions. Examples of “moderately stringent hybridization conditions” include hybridization in 40% formamide, 1M NaCl, 1% SDS, 37 ° C. buffer, and 1 × SSC, wash at 45 ° C. It is. Such washing may be performed for 5, 15, 30, 60, 120 minutes or even more fractions. Positive hybridization is at least twice background. One of ordinary skill in the art will readily recognize that similarly stringent conditions can be obtained using alternative hybridization and wash conditions.

  The phrase “nucleic acid sequence encoding” refers to a nucleic acid that contains sequence information regarding the binding site of a structural RNA, such as rRNA, tRNA, or the primary amino acid sequence of a particular protein or peptide, or a trans-acting regulatory factor. . This phrase specifically includes one or more native sequence degenerate codons (ie, multiple codons that encode a single amino acid) that can be introduced to suit the codon preference in a particular host cell. included.

  The term “recombinant” when used with reference to a cell, nucleic acid, protein or vector, etc. means that the cell, nucleic acid, protein or vector has been modified by introduction of a heterologous nucleic acid or protein or modification of a natural nucleic acid or protein. Or that the cell is derived from a cell so modified. Thus, for example, recombinant cells may express genes that are not found in natural (non-recombinant) cells, or are normally abnormally expressed, underexpressed, or not expressed at all. Expresses natural genes.

  The term “heterologous” when used with reference to a portion of a nucleic acid means that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, a nucleic acid generally has two or more sequences from unrelated genes, such as a promoter from one source and a coding region from another source, resulting in a new functional nucleic acid. Produced by recombinant methods in the deployed form. Similarly, a heterologous protein means that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (eg, a fusion protein).

  An “expression vector” is a nucleic acid construct, produced recombinantly or synthetically, having a series of designated nucleic acid sequences that permit transcription of a particular nucleic acid in a host cell. The expression vector may be part of a plasmid, virus or nucleic acid fragment. Expression vectors generally contain a nucleic acid for transcription that is operably linked to a promoter.

  The phrase “specifically (or selectively) binds to an antibody” or “is specific (or selective) immunoreactive with” when referring to a protein or peptide refers to proteins and other A binding reaction that determines the presence of a protein in the presence of a heterogeneous population of biological material. That is, under the indicated immunoassay conditions, the designated antibody binds to a particular protein present in the sample and does not bind to other proteins in an effective amount. Specific binding to an antibody under such conditions would require an antibody selected for its specificity for a particular protein. For example, by selecting an antibody produced against a protein having an amino acid sequence encoded by any of the polynucleotides of the present invention, other proteins that specifically react with the protein and exclude polymorphic variants Antibodies that do not react with can be obtained. A variety of immunoassay formats may be used to select antigens that are specifically immunoreactive for a particular antibody. For example, flow cytometry and FACS analysis or immunohistochemistry are routinely used to select monoclonal antibodies that specifically immunoreact with a protein. For a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity, see, for example, Harlow and Lane, “Antibodies, Laboratory Manual,” Cold Spring Harbor Publications, NY (1988). Usually, a specific or selective reaction will be at least twice background signal or noise, and more typically more than 10 to 100 times background.

  “Inhibitors”, “activators” and “modifiers” of expression or activity are each determined by using an in vitro or in vivo assay to monitor expression or activity, respectively. Used to refer to an inhibitory, activating or modifying molecule. Modifiers include, for example, ligands, agonists, antagonists, homologues and mimetics thereof, as well as polypeptides of the invention, or fragments thereof that have antagonist activity or act to increase the overall activity of the polypeptide ( That is, a fragment having at least a part of the activity of the full-length protein) is included. In some cases, fragments of polypeptides of the invention are at least 20, 50, 75, or 100 amino acids long. The term “modifier” includes inhibitors and activators. An inhibitory substance is, for example, inhibiting the expression of the polypeptide of the present invention, or binding to the polypeptide of the present invention, partially or completely preventing its activation, reducing or preventing the polypeptide of the present invention, An agent that delays, inactivates, reduces sensitivity, or down-regulates its activity, such as an antagonist. The activator is, for example, inducing or activating the expression of the polypeptide of the present invention, or binding to, activating, increasing, opening, activating, promoting, or activating the polypeptide of the present invention, An agent that enhances activation, increases sensitivity, or upregulates its activity, such as an agonist. Modifiers include natural and synthetic ligands, antagonists, agonists, low molecular weight chemical molecules, and the like. This type of assay for inhibitors and activators includes, for example, applying a putative modifying compound to a cell expressing a polypeptide of the invention, followed by applying a polypeptide of the invention as described above. Determining a functional effect on the activity of the peptide is included. In order to determine the extent of the effect, a sample or assay containing the polypeptide of the present invention treated with a candidate for an activator, inhibitor or modifier is used as a control sample containing no inhibitor, activator or modifier. Compare with A control sample (not treated with an inhibitor) is designated as a relative activity value of 100%. Inhibition of a polypeptide of the invention is achieved when the activity value of the polypeptide is about 80%, optionally 50% or 25%, 10%, 5% or 1% relative to a control. Polypeptide activation occurs when the activity value of the polypeptide is 110%, optionally 150%, optionally 200%, 300%, 400%, 500% or 1000-3000% higher than the control. Achieved in some cases.

Detailed Description of the Invention
I. INTRODUCTION The present application surprisingly found that in human adipose tissue taken from insulin-resistant obese non-diabetic individuals or type 2 diabetic individuals, the level of mRNA comprising the sequences of the present invention has normal body weight lean. It shows that it is changing compared with the level of mRNA in non-diabetic individuals. Insulin resistant obese individuals are generally predisposed to developing type II diabetes. Thus, sequence changes in the tests described herein indicate the involvement of this sequence in obesity, diabetes and / or pre-diabetes.

  While not intending to limit the present invention to a specific mechanism of action, altering the expression or activity of a polypeptide or polynucleotide of the present invention may be obese, diabetic, prediabetic or insulin resistant non-diabetic patients It is thought that it is useful in treating. Furthermore, altered levels of the polypeptide of the invention are also indicative of insulin resistance, obesity, diabetes, or a predisposition to obesity and / or diabetes. Thus, detection of the polypeptides of the present invention is useful for the diagnosis of obesity, predisposition to obesity and / or diabetes, diabetes and / or insulin resistance.

  The present invention also provides a polypeptide of the present invention and a modifier of the polypeptide of the present invention for the diagnosis and treatment of obesity, diabetes, pre-diabetes (including insulin resistant individuals) and related metabolic diseases. A method of use is also provided. The method also provides a method of identifying a modulator for the expression or activity of a polypeptide of the invention. Such modifiers may be used to treat obesity and / or type 2 diabetes, and even obesity (eg, cardiovascular disease, hypertension or cancer) and / or pathological aspects of diabetes (eg, insulin resistance). Useful.

と同一な、または実質的に同一なポリヌクレオチドを、タンパク質の発現または本発明のポリペプチドもしくはポリヌクレオチドに由来する変異体、誘導体、発現カセットもしくは他の配列の作製に際して単離することを目的として遺伝子発現をモニターするために、さまざまな種における配列の単離または検出のために、患者における診断目的のために（例えば、本発明のポリペプチドもしくはポリヌクレオチドにおける変異を検出するために、または核酸もしくはポリペプチドの発現レベルを検出するために）、用いられる。いくつかの態様において、本発明のポリペプチド（または本発明のポリペプチドの断片を含むポリペプチド）をコードする配列は、異種プロモーターと機能的に結合される。場合によっては、本発明のポリペプチドの断片は、少なくとも20、50、75または100アミノ酸長である。本発明のポリペプチドは、組換えDNA技術を用いて異種アミノ酸配列と結び付けることができる。1つの態様において、本発明の核酸は任意の哺乳動物からものであり、これには特に、例えばヒト、マウス、ラットなどが含まれる。 II. General Recombinant Nucleic Acid Methods for Use with the Invention In various aspects of the invention, nucleic acids encoding the polypeptides of the invention are isolated and cloned using recombinant methods. Such an embodiment is, for example,

For the purpose of isolating the same or substantially the same polynucleotide upon expression of the protein or production of a variant, derivative, expression cassette or other sequence derived from the polypeptide or polynucleotide of the invention For monitoring gene expression, for the isolation or detection of sequences in various species, for diagnostic purposes in patients (eg to detect mutations in the polypeptides or polynucleotides of the invention, or in nucleic acids Or to detect the expression level of a polypeptide). In some embodiments, the sequence encoding a polypeptide of the invention (or a polypeptide comprising a fragment of the polypeptide of the invention) is operably linked to a heterologous promoter. In some cases, fragments of polypeptides of the invention are at least 20, 50, 75, or 100 amino acids long. The polypeptides of the present invention can be linked to heterologous amino acid sequences using recombinant DNA techniques. In one embodiment, the nucleic acid of the invention is from any mammal, including in particular humans, mice, rats, and the like.

  A polynucleotide comprising an expression cassette encoding a polypeptide of the present invention can be introduced into a cell and optionally expressed within the cell. The polynucleotides of the present invention can be introduced into eukaryotic or prokaryotic cells, including adipocytes or muscle cells. The cells can be primary cells or cell lines.

A. General recombinant nucleic acid methods The present invention relies on routine techniques in the field of recombinant genetics. Basic texts that disclose general usage in the present invention include Sambrook et al., “Molecular Cloning, Laboratory Manual” (3rd edition, 2001); Kriegler, “Gene Transfer and Expression: A Laboratory Manual”. (1990); and “Current Protocols in Molecular Biology” (Ausubel et al., 1994).

  For nucleic acids, size is expressed in either kilobases (kb) or base pairs (bp). These are estimates obtained from electrophoresis on agarose or acrylamide gels, sequenced nucleic acids, or published DNA sequences. For proteins, size is expressed in kilodaltons (kDa) or amino acid residue numbers. Protein size is deduced from gel electrophoresis, sequenced protein, derived amino acid sequence, or published protein sequence.

  Non-commercial oligonucleotides can be prepared according to the solid phase phosphoramidite triester method first described by Beaucage and Caruthers, Tetrahedron Letts. Chemical synthesis can be performed using an automatic synthesizer described in 6159-6168 (1984). Oligonucleotides are purified by native acrylamide gel electrophoresis or by anion exchange HPLC as described by Pearson and Reanier, J. Chrom. 255: 137-149 (1983).

  The sequence of cloned genes and synthetic oligonucleotides can be confirmed after cloning using, for example, a chain termination method for sequencing of double-stranded templates by Wallace et al., Gene 16: 21-26 (1981). it can.

B. Cloning Methods for Isolation of Nucleotide Sequences Encoding a Desired Protein In general, nucleic acids encoding a subject protein are cloned from a DNA sequence library created to encode cDNA or genomic DNA. The position of a particular sequence can be determined by hybridizing with an oligonucleotide probe, the latter sequence can be derived from the sequences disclosed herein, which serves as a reference for PCR primers, A region suitable for isolating a probe specific for a polypeptide or polynucleotide of the invention is defined. Alternatively, when cloning a sequence into an expression library, the expressed recombinant protein can be used with antisera or purified antibodies made against the polypeptide of interest, including those disclosed herein. It can also be detected immunologically.

  Methods for the generation and screening of genomic and cDNA libraries are well known to those skilled in the art (eg, Gubler and Hoffman, Gene 25: 263-269 (1983); Benton and Davis, Science, 196: 180-182). (1977); and Sambrook, supra).

  Briefly, in order to create a cDNA library, it is necessary to select a source rich in mRNA. Subsequently, the mRNA is converted into cDNA, ligated into a recombinant vector, and transfected into a recombinant host for propagation, screening and cloning. In the case of a genomic library, DNA is extracted from tissue or cells, and fragments of preferably about 5 to 100 kb are obtained by either mechanical shearing or enzymatic digestion. Subsequently, the fragments are separated from those of undesirable size by gradient centrifugation and assembled into bacteriophage lambda vectors. The packaging of these vectors and phage is performed in vitro and the recombinant phage is analyzed by plaque hybridization. Colony hybridization is performed as generally described in Grunstein et al., Proc. Natl. Acad. Sci. USA 72: 3961-3965 (1975).

  One alternative method combines the use of synthetic oligonucleotide primers with the amplification of mRNA or DNA templates. Suitable primers can be designed from the sequences shown above for the specific sequences disclosed herein. In this polymerase chain reaction (PCR) method, a nucleic acid encoding a protein of interest is directly amplified from mRNA, cDNA, genomic library, or cDNA library. Restriction enzyme sites can be incorporated into the primers. Polymerase chain reaction or other in vitro amplification methods may be used, for example, as a probe to detect the presence of mRNA encoding a polypeptide of the invention in a physiological sample, such as by cloning a specific protein to express a precursor protein. It may be useful for synthesizing nucleic acids for use, for nucleic acid sequencing, or for other purposes (see US Pat. Nos. 4,683,195 and 4,683,202). The gene amplified by the PCR reaction can be purified from an agarose gel and cloned into a suitable vector.

  Suitable primers and probes for identifying genes encoding polypeptides of the present invention from mammalian tissue can be derived from the sequences provided herein. For a general review of PCR, see Innis et al., “PCR Protocols: A Guide to Methods and Applications”, Academic Press, San Diego (1990).

  Synthetic oligonucleotides can be used for gene construction. This is done with a series of overlapping oligonucleotides, usually 40-120 bp in length, corresponding to both the sense and non-sense strands of the gene. Subsequently, these DNA fragments are annealed, ligated and cloned.

  A polynucleotide encoding a polypeptide of the invention can be cloned into an intermediate vector prior to transformation into a mammalian cell for expression. These intermediate vectors are generally prokaryotic vectors or shuttle vectors. Proteins can be expressed in prokaryotes or eukaryotes using standard methods well known to those skilled in the art.

III. Purification of Proteins of the Invention Both native and recombinant polypeptides of the invention can be purified for use in functional assays. A naturally occurring polypeptide of the present invention can be purified from any source (eg, tissue of an organism expressing an ortholog). The recombinant polypeptide can be purified from any suitable expression system.

  The polypeptides of the present invention can also be purified to substantial purity by standard techniques including selective precipitation with substances such as ammonium sulfate, column chromatography, immunopurification methods, etc. (eg, Scopes, “Protein Purification: Principles and Practice "(1982); U.S. Pat. No. 4,673,641; Ausubel et al., Supra; and Sambrook et al., Supra).

  Various methods can be used to purify the recombinant polypeptide. For example, a protein with proven molecular adhesion properties can be reversibly fused with a polypeptide of the invention. With the appropriate ligand, either protein can be selectively adsorbed to the purification column, which is then released from the column in a relatively pure form. Subsequently, the fusion protein can be separated by enzymatic activity. Finally, it is possible to purify the polypeptide using an immunoaffinity column.

A. Purification of proteins from recombinant bacteria When recombinant proteins are expressed in large quantities by transformed bacteria (usually by promoter induction, but expression may be constitutive), the protein may form insoluble aggregates. There are several protocols suitable for the purification of protein inclusion bodies. For example, for purification of aggregated proteins (hereinafter referred to as inclusion bodies), a buffer generally containing about 100-150 μg / ml lysosome and 0.1% nonidet P-40 (nonionic surfactant) Inclusion bodies are extracted, separated and / or purified by disruption of bacterial cells by incubation in, but not limited to. Cell suspensions can be disrupted using a Polytron grinder (Brinkman Instruments, Westbury, NY). Alternatively, the cells can be sonicated on ice. Alternative methods of solubilizing bacteria are described in Sambrook et al., Supra and Ausubel et al., Supra, and will be apparent to those skilled in the art.

  The cell suspension is typically centrifuged, and the pellet containing the inclusion bodies is buffered, such as 20 mM Tris-HCl (pH 7.2), 1 mM EDTA, 150 mM NaCl, which does not dissolve the inclusion bodies but can be washed. Resuspend in 2% Triton-X 100 (nonionic surfactant). It may be necessary to repeat the washing step to remove as much necrotic cell debris as possible. The remaining pellet of inclusion bodies may be resuspended in a suitable buffer (eg, 20 mM sodium phosphate, pH 6.8, 150 mM NaCl). Other suitable buffers will be apparent to those skilled in the art.

  After the washing step, the inclusion bodies are solubilized by the addition of a solvent (or combination of solvents each having one of these properties) that is a strong hydrogen acceptor and also a strong hydrogen donor. Subsequently, the protein forming the inclusion bodies can be regenerated by dilution with a compatible buffer or dialysis. Suitable solvents include urea (about 4M to about 8M), formamide (at least about 80% by volume / volume ratio) and guanidine hydrochloride (about 4M to about 8M). Some solvents that can solubilize aggregate-forming proteins, such as SDS (sodium dodecyl sulfate) and 70% formic acid, can irreversibly denature proteins, making them immunogenic and / or non-active Not suitable for use in this procedure. Guanidine hydrochloride and similar drugs are denaturing agents, but this denaturation is not irreversible, and regeneration occurs when the denaturing agent is removed (eg, by dialysis) or diluted to produce the desired immunological and / or biological Active proteins are reconstituted. Following solubilization, the protein can be separated from other bacterial proteins by standard separation methods.

  Alternatively, the polypeptide can be purified from bacterial periplasm. Once the protein has been exported to the bacterial periplasm, the bacterial periplasmic fraction can be isolated by cryo-osmotic shock, as well as other methods known in the art (Ausubel et al., Supra). reference). To isolate the recombinant protein from the periplasm, the bacterial cells are centrifuged and pelleted. The pellet is resuspended in a buffer containing 20% sucrose. To solubilize the cells, the bacteria are centrifuged and the pellet is resuspended in ice-cold 5 mM MgSO4 and placed in an ice bath for about 10 minutes. The cell suspension is centrifuged and the supernatant is decanted and collected. The recombinant protein present in the supernatant can be separated from the host protein by standard separation methods known to those skilled in the art.

B. Purification of proteins from insect cells Proteins can also be purified from eukaryotic gene expression systems such as those described in, for example, Fernandez and Hoeffler, “Gene Expression Systems” (1999). In some embodiments, baculovirus expression systems are used to isolate the proteins of the invention. Recombinant baculoviruses are generally made by replacing the baculovirus polyhedron coding sequence with a gene to be expressed (eg, encoding a polypeptide of the invention). Viruses lacking the polyhedron gene have a unique plaque morphology and are easy to recognize. In some embodiments, the recombinant baculovirus first clones the polynucleotide of interest into a transfer vector (eg, a pUC-based vector) such that the polynucleotide is operably linked to a polyhedron promoter. It is produced by. The transfer vector is transfected into insect cells (for example, Sf9 cells, Sf21 cells, or BT1-TN-5B1-4 cells) together with wild type DNA, and the polyhedron gene in the wild type viral DNA and the polynucleotide of interest are Generate homologous recombination and substitution. The virus can then be generated and the plaque can be purified. Protein expression occurs upon viral infection of insect cells. The expressed protein can be collected from the cell supernatant if it is secreted, or from the cell lysate if it is intracellular. See, for example, Ausubel et al., And Fernandez and Hoeffler, supra.

C. Purification of Secreted Protein from Mammalian Cells The polypeptides of the invention, and in particular the secreted proteins of the invention, can be easily purified from mammalian cells that express the polypeptide. Polypeptide expression may be the result of transient or stable expression of the protein from a recombinant expression cassette introduced into the cell. Secreted proteins can generally be isolated using standard procedures for purifying proteins from cell culture media.

D. Standard protein separation methods for protein purification
1． Separation by solubility Often, if the protein mixture is complex, as the first step, a salt separation is performed first to achieve much of the unwanted host cell protein (or protein from the cell culture medium). From the recombinant protein. A preferred salt can be, for example, ammonium sulfate. Ammonium sulfate precipitates proteins by effectively reducing the amount of water in the protein mixture. The protein then precipitates from the point of solubility. The higher the hydrophobicity of the protein, the more likely it is to precipitate at lower ammonium sulfate concentrations. In a typical protocol, a saturated ammonium sulfate solution is added to the protein solution, resulting in an ammonium sulfate concentration of 20-30%. This is thought to precipitate the most hydrophobic protein. The precipitate is then discarded (if the protein of interest is not hydrophobic) and ammonium sulfate is added to the supernatant to a concentration known to precipitate the protein of interest. Subsequently, the precipitate is dissolved in the buffer and, if necessary, excess salt is removed by dialysis or diafiltration. Other methods that rely on protein solubility, such as low temperature ethanol precipitation, are also known to those skilled in the art and can be used to fractionate complex protein mixtures.

2． Filtration based on size differences Based on the calculated molecular weight, ultrafiltration is passed through membranes of various pore sizes (for example, Amicon or Millipore membranes), and larger or smaller proteins are simply separated. Can be separated. As a first step, ultrafiltration of the protein mixture is performed through a membrane having a pore size lower than the molecular weight of the target protein. Subsequently, ultrafiltration is performed on the retained substance after ultrafiltration on a membrane having a molecular weight cut-off value higher than the molecular weight of the target protein. It is believed that the recombinant protein passes through this membrane and enters the filtrate. Subsequently, the filtrate can be chromatographed as described below.

3． Column Chromatography Proteins of interest can also be separated from other proteins based on their size, net surface charge, hydrophobicity and affinity for foreign molecules. Furthermore, an antibody produced against the protein can be bound to a column substrate to immunopurify the protein. All of these methods are well known in the art.

  Immunoaffinity chromatography using antibodies raised against various affinity tags such as hemagglutinin (HA), FLAG, Xpress, Myc, hexahistidine (His), and glutathione S-transferase (GST) It can be used to purify a polypeptide. The His tag also acts as a chelator for certain metals (eg, Ni), so that metal can also be used to purify His-containing polypeptides. After purification, the tag is optionally removed with a specific proteolytic enzyme.

  It will be apparent to those skilled in the art that chromatographic methods can be performed at any scale and using equipment from various manufacturers (eg, Pharmacia Biotech).

IV. Detection of Polynucleotides of the Invention One skilled in the art will recognize that there are many uses for detecting expression of polynucleotides and polypeptides of the invention. For example, as discussed herein, detection of the levels of polynucleotides and polypeptides of the invention in a patient is useful for diagnosing diabetes or a predisposition to at least some of the pathological effects of diabetes. is there. Furthermore, detection of gene expression is useful for identifying modulators of the expression of polynucleotides and polypeptides of the invention.

  Various methods of measuring specific DNA and RNA using nucleic acid hybridization methods are known to those skilled in the art (see Sambrook, supra). Some methods use electrophoretic separation (eg, Southern blot for detection of DNA and Northern blot for detection of RNA), but measurement of DNA and RNA is done without electrophoretic separation (Eg, by dot blot). Southern blotting of genomic DNA (eg, from humans) can be used to screen for restriction fragment length polymorphisms (RFLPs) to detect the presence of genetic disorders affecting the polypeptides of the invention .

  The choice of nucleic acid hybridization format is not particularly important. Various nucleic acid hybridization formats are known to those skilled in the art. For example, common formats include sandwich assays and competitive or displacement assays. An overview of hybridization methods is provided by Hames and Higgins, “Nucleic Acid Hybridization, A Practical Approach”, IRL Press (1985); Gall and Pardue, Proc. Natl. Acad. Sci. USA, 63: 378-383 (1969); As well as John et al., Nature, 223: 582-587 (1969).

  For detection of the hybridization complex, it appears that the complex that produces the signal must bind to the duplex of the target and the probe polynucleotide or nucleic acid. In general, this type of binding occurs through the interaction of a ligand and an antiligand, such as between a ligand-binding probe and a signal-bound anti-ligand. The binding of the signal producing complex can also be readily applied to facilitating exposure to ultrasonic energy.

  The label also allows indirect detection of the hybridization complex. For example, if the label is a hapten or an antigen, the sample can be detected by using an antibody. In these systems, the signal is generated by the binding of a fluorescent or enzyme molecule to the antibody or, in some cases, by binding to a radioactive label (eg, Tijssen, “Practice and Theory of Enzyme Immunoassays”, “Laboratory Techniques in Biochemistry and Molecular Biology ", edited by Burdon and van Knippenberg, Elsevier (1985), pp. 9-20).

Probes are generally labeled directly, as in the case of isotopes, chromophores, lumiphores, chromophores, or indirectly as in the case of biotin to which the streptavidin complex is bound later. For this reason, the detectable label used in the assay method of the present invention may be a primary label (when the label contains a directly detectable agent or produces a directly detectable agent) or a secondary label (immunological label). As is the case with the label to be detected bound to the primary label). In general, a labeled signal nucleic acid is used for detection of hybridization. Complementary or signal nucleic acids can be labeled by any one of several methods commonly used to detect the presence of hybridized polynucleotides. The most common detection method is the use of autoradiography using probes labeled with ³ H, ¹²⁵ I, ³⁵ S, ¹⁴ C or ³² P.

  Other labels include, for example, a ligand that binds to the labeled antibody, a fluorophore, a chemiluminescent material, an enzyme, and an antibody that acts as a member of a specific binding pair for the labeled ligand. Guidance on labeling, labeling procedures and label detection can be found in Polak and Van Noorden, “Introduction to Immunocytochemistry”, 2nd edition, Springer Verlag, NY (1997); and comprehensive handbooks and catalogs published by Molecular Probes, Inc. Haugland “Handbook of Fluoroscent Probes and Research Chemicals” (1996).

  In general, a detector for observing a specific probe or a combination of probes is used to detect the label of the detection reagent. Typical detectors include spectrophotometers, phototubes and photodiodes, microscopes, scintillation counters, cameras, films, etc., and combinations thereof. Examples of suitable detectors are widely available from various vendors known to those skilled in the art. In general, an optical image of a substrate containing a bound labeling moiety is digitized for subsequent computer analysis.

  For example, the amount of RNA is measured by quantifying the amount of label immobilized on the solid support by binding of the detection reagent. In general, due to the presence of the modifier during the incubation, the amount of label bound to the solid support will be compared to a control incubation without the modifier, or an established baseline for the individual reaction type. Compared to the increase or decrease. Means for label detection and quantification are well known to those of skill in the art.

  In some embodiments, the target nucleic acid or probe is immobilized to a solid support. Suitable solid supports for use in the assay methods of the invention are well known to those skilled in the art. As used herein, a solid support is a matrix of material in a substantially fixed arrangement.

  A variety of automated solid phase assays are also suitable. For example, a very large scale immobilized polymer array (VLSIPS ™), ie a gene chip or microarray available from Affymetrix, Inc. (Santa Clara, Calif.), Can be used to determine the expression levels of multiple genes involved in the same regulatory pathway. It can be used to detect changes simultaneously. Tijssen, supra, Fodor et al. (1991) Science, 251: 767-777; Sheldon et al. (1993) Clinical Chemistry 39 (4): 718-719 and Kozal et al. (1996) Nature Medicine 2 (7): 753-759. Please refer. Similarly, spotted cDNA arrays (cDNA sequence arrays that bind to nylon, glass, or other solid supports) can also be used to monitor the expression of multiple genes.

  Typically, the components of the array are organized in an ordered manner such that each component is in a specified location on the substrate. Because the array components are at specified locations on the substrate, the hybridization pattern and intensity (both of which produce a unique expression profile) can be interpreted with respect to the expression level of a particular gene. Can be associated with other diseases or conditions or treatments. See, for example, Schena et al., Science 270: 467-470 (1995) and (Lockhart et al., Nature Biotech. 14: 1675-1680 (1996)).

  The specificity of hybridization can be assessed by comparing the hybridization of the specificity-control polynucleotide sequence to the specificity-control polynucleotide probe with a known amount of sample added. The specificity-control target polynucleotide may have one or more sequence mismatches relative to the corresponding polynucleotide sequence. In this manner, it is determined whether only complementary target polynucleotides will hybridize to the polynucleotide sequence or a mismatched hybrid duplex will be formed.

  Hybridization reactions can be performed in absolute or differential hybridization formats. In an absolute hybridization format, a polynucleotide probe from one sample is hybridized with a sequence in a microarray format, and the signal detected after formation of the hybridization complex is correlated with the level of the polynucleotide probe in the sample. . In the differential hybridization format, the differential expression of the gene set in the two biological samples is analyzed. For differential hybridization, polynucleotide probes are prepared from both biological samples and labeled with different labeling moieties. A mixture of two labeled polynucleotide probes is added to the microarray. The microarray is then examined under conditions that can detect luminescence from two different labels individually. Sequences in the microarray hybridized with substantially the same number of polynucleotide probes from both biological samples will emit different composite fluorescence (Shalon et al., WO 95/35505). In some embodiments, the label is a fluorescent label having a distinguishable emission spectrum, such as Cy3 and Cy5 fluorophores.

  After hybridization, the microarray is washed to remove unhybridized nucleic acids, and complex formation between the hybridizable array components and the polynucleotide probe is detected. Methods for detection of complex formation are well known to those skilled in the art. In some embodiments, the polynucleotide probe is labeled with a fluorescent label, and the measurement of the level and pattern of fluorescence indicative of complex formation is performed by a fluorescence microscope, such as a confocal fluorescence microscope.

  In differential hybridization experiments, polynucleotide probes from two or more different biological samples are labeled with two or more different fluorescent labels with different emission wavelengths. The fluorescent signal is detected using a different photomultiplier tube set to detect a specific wavelength. The relative amount / expression level of the polynucleotide probe in two or more samples is determined.

  In general, when multiple microarrays are used under similar test conditions, the fluorescence intensity of the microarray can be normalized to take into account variations in hybridization intensity. In some embodiments, the hybridization intensity of individual polynucleotide probes / target complexes is normalized using the intensity obtained from the internal normalization control included on each microarray.

  Nucleic acid can be detected using, for example, a labeled detection moiety that specifically binds to a double-stranded nucleic acid (eg, an antibody specific for an RNA-DNA duplex). In one example, an antibody that recognizes a DNA-RNA heteroduplex in which the antibody is linked to an enzyme is used (usually by recombinant or covalent chemical bonds). An antibody is detected when the enzyme reacts with its substrate to produce a detectable product. Coutlee et al. (1989) Analytical Biochemistry 181: 153-162; Bogulavski (1986) et al., J. Immunol. Methods 89: 123-130; Prooijen-Knegt (1982) Exp. Cell Res. 141: 397-407; Rudkin (1976 ) Nature 265: 472-473, Stollar (1970) PNAS 65: 993-1000; Ballard (1982) Mol. Immunol. 19: 793-799; Pisetsky and Caster (1982) Mol. Immunol. 19: 645-650; Viscidi (1988) J Clin. Microbial. 41: 199-209; and Kiney et al. (1989) J. Clin. Microbiol. 27: 6-12 are directed against RNA duplexes, including homoduplexes and heteroduplexes. Antibodies are described. Kits containing antibodies specific for DNA: RNA hybrids are available, for example, from Digene Diagnostics, Inc. (Beltsville, MD).

In addition to available antibodies, one of ordinary skill in the art can readily generate antibodies specific for nucleic acid duplexes using existing techniques, or commercially or publicly available antibodies. It can also be modified. In addition to the techniques mentioned above, general methods for producing polyclonal and monoclonal antibodies are known to those skilled in the art (eg, Paul (ed.), “Fundamental Immunology, Third Edition” Raven Press, Ltd. ., NY (1993); Coligan, "Current Protocols in Immunology", Wiley / Greene, NY (1991); Harlow and Lane, "Antibodies: A Laboratory Manual" Cold Spring Harbor Press, NY (1989); ) "Basic and Clinical Immunology" (4th edition) Lange Medical Publications, Los Altos, CA and references cited therein; Goding, "Monoclonal Antibodies: Principles and Practice" (2nd edition) Academic Press, New York, NY, (1986); and Kohler and Milstein, Nature 256: 495-497 (1975)). Other techniques suitable for antibody preparation include selection of recombinant antibody libraries in phage vectors or similar vectors (eg, Huse et al., Science 246: 1275-1281 (1989); and Ward et al., Nature 341: 544-546 (1989)). Specific monoclonal and polyclonal antibodies and antisera will usually, K _D of at least about 0.1 [mu] _M, preferably at least about 0.01μM or less, the most common and preferably attached at 0.001μM or K _D of less than Conceivable.

  The nucleic acid used in the present invention may be a positive probe or a negative probe. A positive probe binds to its target, and the presence of duplex formation is evidence of the presence of the target. Negative probes do not bind to suspected targets, and the absence of duplex formation is evidence of the presence of the target. For example, the use of wild type specific nucleic acid probes or PCR primers can serve as a negative probe in an assay sample if only the nucleotide sequence of interest is present.

  The sensitivity of a hybridization assay can also be increased by using a nucleic acid amplification system that increases the target nucleic acid to be detected. Examples of such systems include the polymerase chain reaction (PCR) system and the ligase chain reaction (LCR) system. Other methods recently described in the art include nucleic acid sequence-based amplification (NASBA, Cangene, Mississauga, Ontario) and Q Beta replicase systems. These systems can be used to directly identify variants when PCR or LCR primers are designed to extend or ligate only in the presence of a selected sequence. Alternatively, the selected sequence can be entirely amplified using, for example, non-specific PCR primers, and the amplified target region can then be searched for a specific sequence that is indicative of mutation. It will be appreciated that a variety of detection probes, including Taqman and molecular beacon probes, can be used to monitor amplification reaction products, for example immediately.

  One alternative means for determining the expression level of the nucleic acids of the invention is in situ hybridization. In situ hybridization assays are well known and are outlined in Angerer et al., Methods Enzymol. 152: 649-660 (1987). In an in situ hybridization assay, cells, preferably human cells from the cerebellum or hippocampus, are fixed to a solid support, typically a glass slide. When trying to search for DNA, the cells are denatured by heat or alkali. The cells are then contacted with a moderate temperature hybridization solution that allows annealing of the labeled specific probe. The probe is preferably labeled with a radioisotope or a fluorescent reporter.

  Single nucleotide polymorphism (SNP) analysis is also useful for detecting differences between alleles of the polynucleotides (eg, genes) of the present invention. The SNP associated with the gene encoding the polynucleotide of the present invention is useful, for example, in the diagnosis of a disease whose onset is associated with the gene sequence of the present invention. For example, if an individual has at least one SNP associated with a disease-related allele of the gene sequence of the present invention, the individual is likely to have a predisposition to one or more of these diseases. If an individual is homozygous for a disease-related SNP, the predisposition to the development of the individual's disease (eg, diabetes) is particularly great. In some embodiments, the SNP associated with the gene sequence of the present invention is located within 300,000 bp, 200,000 bp, 100,000 bp, 75,000 bp, 50,000 bp, or 10,000 bp of the gene sequence.

  Various real-time assays including Taqman or molecular beacon assays (eg, US Pat. No. 5,210,015; US Pat. No. 5,487,972; Tyagi et al., Nature Biotechnology 14: 303 (1996); and WO 95/13399) The PCR method is useful for observing the presence or absence of SNP. Other SNP detection methods include, for example, DNA sequencing, sequencing by hybridization, dot blotting, oligonucleotide array (DNA chip) hybridization analysis, or, for example, US Pat. No. 6,177,249; Landegren et al., Genome Research, 8: 769-776 (1998); Botstein et al., Am J Human Genetics 32: 314-331 (1980); Meyers et al., Methods in Enzymology 155: 501-527 (1987); Keen et al., Trends in Genetics 7 : 5 (1991); Myers et al., Science 230: 1242-1246 (1985); and Kwok et al., Genomics 23: 138144 (1994).

V. Detection of the polypeptide of the present invention In addition to the detection of the polynucleotide gene and gene expression of the present invention using a nucleic acid hybridization technique, the polypeptide of the present invention can also be detected using an immunoassay method. Immunoassays can be used to qualitatively or quantitatively analyze the polypeptides of the present invention. A general overview of applicable techniques can be found in Harlow and Lane, “Antibodies: A Laboratory Manual” (1988).

A. Antibodies to target proteins or other immunogens Methods for the production of polyclonal and monoclonal antibodies that react specifically with a protein of interest or other immunogen are known to those skilled in the art (eg, Coligan, supra; Harlow And Lane, supra; see Stites et al., Supra and references cited therein; Goding, supra; and Kohler and Milstein, Nature, 256: 495-497 (1975)). Such techniques include the preparation of antibodies by selection of antibodies from a library of recombinant antibodies in a phage vector or similar vector (see, eg, Huse et al., Supra; and Ward et al., Supra). . For example, to produce an antiserum for use in an immunoassay, the protein of interest or antigenic fragment thereof is isolated as described herein. For example, recombinant proteins are produced in transformed cell lines. Mice or rabbit inbreds are immunized with proteins using standard adjuvants such as Freund's adjuvant and standard immunization protocols. Alternatively, synthetic peptides derived from the sequences disclosed herein are conjugated to carrier proteins and used as immunogens.

Polyclonal serum is collected and titered against the immunogen in an immunoassay, for example, a solid phase immunoassay using an immunogen immobilized on a solid support. Titer selects polyclonal antisera is 10 ⁴ or more, using a competitive binding immunoassay, proteins other than the polypeptide of the present invention or optionally, determine the cross-reactivity with other homologous proteins. Specific polyclonal and monoclonal antibodies are usually, K _D of at least about 0.1 _mM, more usually at least about 1 [mu] M, preferably at least about 0.1μM or less, and most preferably at 0.01μM or K _D of less than It is thought to combine.

  Many proteins of the present invention, including immunogens, can be used to generate antibodies that react specifically or selectively with the protein of interest. Recombinant protein is a preferred immunogen for the production of monoclonal or polyclonal antibodies. Natural proteins may be used in pure or impure state. Synthetic peptides made using the protein sequences described herein can also be used as immunogens for the production of antibodies against that protein. Recombinant proteins can be expressed in eukaryotic or prokaryotic cells and purified as generally described above. Subsequently, the product is injected into the body of an animal capable of producing antibodies. Either monoclonal or polyclonal antibodies may be made for later use in immunoassay methods for measuring proteins.

  Methods for producing polyclonal antibodies are well known to those skilled in the art. Briefly, animals are immunized with an immunogen, preferably purified protein, mixed with an adjuvant. The animal's immune response to the immunogen preparation is monitored by taking a test bleed and measuring the titer of reactivity to the polypeptide of the invention. When antibodies with high titers appropriate for the immunogen are obtained, blood is collected from the animals and antiserum is prepared. If necessary, the antiserum can be further fractionated to concentrate antibodies that react with the protein (see Harlow and Lane, supra).

  Monoclonal antibodies can be obtained by various techniques well known to those skilled in the art. Usually, splenocytes from animals immunized with the desired antigen are immortalized, generally by fusion with myeloma cells (Kohler and Milstein, Eur. J. Immunol. 6: 511-519 ( 1976)). Alternative methods of immortalization include transformation with Epstein-Barr virus, oncogenes or retroviruses, or other methods well known in the art. Clones generated from a single immortalized cell are screened for the desired specificity and affinity for the antigen and the yield of monoclonal antibodies produced by such cells can be determined by a variety of methods including intraperitoneal injection of vertebrate hosts. It can be enhanced by technology. Alternatively, DNA encoding a monoclonal antibody or binding fragment thereof can be screened by screening a human B cell-derived DNA library according to the general protocol outlined in Huse et al., Science 246: 1275-1281 (1989) Sequences can also be isolated.

  Once an antibody specific for the target immunogen is obtained, the immunogen can be measured by various immunoassay methods that yield qualitative and quantitative results available to clinicians. For a review of immunological and immunoassay procedures, see Stites, supra. Further, the immunoassay method of the present invention can be used in any of a number of formats outlined in detail in Maggio, “Enzyme Immunoassay”, CRC Press, Boca Raton, Florida (1980); Tijssen, supra; and Harlow and Lane, supra. It can also be done in the form of

  Polyclonal antisera raised against the full-length polypeptide of the present invention or a fragment thereof may be used in an immunoassay method for measuring a target protein in a human sample. This antiserum is selected to have low cross-reactivity to other proteins, and this type of cross-reactivity is removed by immunoadsorption prior to use in immunoassay methods.

B. Immunological Binding Assays In some embodiments, the protein of interest is detected and / or quantified using any of a variety of well-known immunological binding assays (eg, US patents). 4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review of general immunoassay methods, see Asai, “Methods in Cell Biology Volume 37: Antibodies in Cell Biology”, Academic Press, Inc. NY (1993); Stites, supra. Immunological binding assays (or immunoassays) are generally “capture agents” that specifically bind to and often immobilize analytes (eg, full-length polypeptides of the invention, or antigenic subsequences thereof). (Capture agent) "is used. A capture agent is a moiety that specifically binds to the analyte. Antibodies may be generated using a number of means well known to those skilled in the art and any of the means described above.

  Immunoassay methods often also use a labeling agent that specifically binds to and labels the complex formed by the capture agent and analyte. The labeling agent may itself be one of the moieties comprising the antibody / antigen complex. Alternatively, the labeling agent may be a third moiety such as another antibody that specifically binds to the antibody / protein complex.

  In a preferred embodiment, the labeling agent is a second antibody having a label. Alternatively, the second antibody does not have a label, and instead, a labeled third antibody that is specific to the antibody of the species from which the second antibody is derived may bind. Good. The second antibody can be modified with a labelable moiety, such as biotin, to which a third labeled molecule, such as enzyme-labeled streptavidin, can specifically bind.

  Other proteins such as protein A or protein G that can specifically bind to the immunoglobulin constant region may also be used as the labeling agent. These proteins are normal components of the streptococcal cell wall. They exhibit strong non-immunogenic reactivity against immunoglobulin constant regions from various species (see, for example, Kronval et al., J. Immunol. 111: 1401-1406 (1973); and Akerstrom Et al., J. Immunol. 135: 2589-2542 (1985)).

  Throughout the assay, incubation and / or washing steps may be required after using each combination of reagents. Incubation steps can vary from about 5 seconds to several hours, preferably from about 5 minutes to about 24 hours. Incubation times will depend on assay format, analyte, solution volume, concentration, and the like. Usually, the assay is carried out at room temperature, but can also be carried out at a certain range of temperature, such as 10 ° C to 40 ° C.

1． Non-competitive assay format Immunoassay methods for detecting a protein or analyte of interest from a tissue sample may be competitive or non-competitive. A noncompetitive immunoassay is an assay that directly measures the amount of captured protein or analyte. For example, in one preferred “sandwich” assay, a capture agent (eg, an antibody specific for a polypeptide of the invention) can be bound directly to a solid substrate for immobilizing it. Subsequently, these immobilized antibodies capture the polypeptide present in the test sample. Next, a labeling agent such as a secondary labeled antibody specific for the polypeptide is bound to the polypeptide of the present invention thus immobilized. Alternatively, the second antibody does not have a label, and instead, a third antibody specific for the species of antibody from which the second antibody is derived may be conjugated thereto. The second antibody can be modified with a labelable moiety such as biotin to which a third labeled molecule such as enzyme-labeled streptavidin can specifically bind.

2． Competitive assay format In a competitive assay, a protein or analyte present in a sample dissociates (defeats competition) from a specific capture agent (eg, an antibody specific for a polypeptide of the invention) and is added (exogenous). The amount of protein or analyte present in the sample is indirectly measured by measuring the amount of protein or analyte. The amount of immunogen bound to the antibody is inversely proportional to the concentration of immunogen present in the sample. In one particularly preferred embodiment, the antibody is immobilized on a solid substrate. The amount of analyte can be detected by providing a labeled analyte molecule. It is recognized that the label can include, for example, in addition to a radioactive label, a peptide or other tag label that can be recognized by a detection reagent such as an antibody.

  Competitive binding immunoassay methods can also be used to determine cross-reactivity. For example, the protein encoded by the sequences provided herein can be immobilized on a solid support. Protein is added to the assay system and competes with the binding of antisera to the immobilized antigen. The ability of the above proteins to compete with the binding of antisera to the immobilized protein is compared to that of the protein encoded by any of the sequences provided herein. The ratio of cross-reactivity for the above proteins is calculated using standard calculations. Antisera that have less than 10% cross-reactivity with each of the added proteins listed above are selected and pooled. Cross-reactive antibodies are optionally removed from the pooled antisera by immunoadsorption with the protein of interest, for example, a less closely related homolog.

  The pooled antiserum with immunoadsorption is then used in the competitive binding immunoassay method described above to compare a second protein, presumably a protein of the invention, with the immunogenic protein. To make this comparison, the two proteins are assayed together over a wide range of concentrations to determine the amount of each protein required to inhibit 50% of the binding between the antiserum and the immobilized protein. If the amount of second protein required is less than one-tenth of the amount of protein partially encoded by the protein herein required, then the second protein is against the immunogen consisting of the target protein. It is said to specifically bind to the antibody produced in this way.

3． Other Assay Formats In some embodiments, Western blot (immunoblot) analysis is used to detect and quantify the presence of a polypeptide of the invention in a sample. This technique generally separates the sample protein by gel electrophoresis based on molecular weight, and transfers the separated protein to a suitable solid support (such as a nitrocellulose filter, nylon filter, or derivatized nylon filter). Incubating the sample with an antibody that specifically binds to the protein of interest. For example, an antibody that specifically binds to a polypeptide of the invention on a solid support is selected. These antibodies may be directly labeled or later detected using a labeled antibody that specifically binds to the protein of interest (eg, a labeled sheep anti-mouse antibody).

  Other assay formats include Liposome Immunoassay (LIA) using liposomes designed to bind to specific molecules (eg, antibodies) and release the encapsulated reagent or marker. Subsequently, the released chemical is detected according to standard techniques (see Monroe et al., Amer. Clin. Prod. Rev. 5: 34-41 (1986)).

Four. The particular label or detectable group used in the assay is not a particularly important aspect of the invention, as long as it does not significantly interfere with the specific binding of the antibody used in the assay. The detectable group can be any substance having a detectable physical or chemical property. Such detectable labels are well developed in the field of immunoassays, and usually almost any label useful for such methods can be applied to the present invention. That is, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electronic, engineering or chemical means. Labels useful in the present invention include magnetic beads (eg, Dynabeads ™), fluorescent dyes (eg, fluorescein, isothiocyanate, Texas red, rhodamine, etc.), radiolabels (eg, ³ H, ¹²⁵ I, ³⁵ S ¹⁴ C or ³² P), enzymes (eg, horseradish peroxidase, alkaline phosphatase, and others commonly used in ELISA), and colloidal gold or colored glass or plastic beads (eg, polystyrene, polypropylene, latex, etc.), etc. Of colorimetric labels.

  The label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. As indicated above, a wide variety of labels can be used, and the choice of label depends on the sensitivity required, ease of binding to the compound, stability requirements, available equipment, and disposal considerations. To do.

  Non-radioactive labels are often attached by indirect means. The molecule can also be coupled directly to the signal generating compound, such as by conjugation with an enzyme or fluorophore. A variety of enzymes and fluorescent compounds can be used in the methods of the invention and are well known to those skilled in the art (for a review of various labeling or signal generation systems that can be used, see, eg, US Pat. No. 4,391,904). I want to be)

  Means for detecting labels are well known to those skilled in the art. Thus, for example, if the label is a radioactive label, means for detection include scintillation counters or photographic film in the case of autoradiography. If the label is a fluorescent label, it can be detected by exciting the fluorochrome with light of the appropriate wavelength and detecting the resulting fluorescence. Fluorescence can be detected macroscopically by photographic film by use of an electronic detection device such as a charge coupled device (CCD) or a photomultiplier tube. Similarly, an enzyme label can be detected by providing an appropriate substrate for the enzyme and detecting the resulting reaction product. Furthermore, simple colorimetric labels can be detected by simply observing the color associated with the label. That is, in various test strip assays, the bound gold often appears light red, while the various bound beads appear as bead colors.

  Some assay formats do not require the use of a labeling component. For example, agglutination assays can be used to detect the presence of labeled antibodies. In this case, the antigen-coated particles are aggregated by the sample containing the target antibody. In this format, no component needs to be labeled, and the presence of the target antibody is detected by simple visual inspection.

VI. Identification of the modifiers of the polypeptides of the invention Modifiers of the polypeptides of the invention, ie, the activity of the polypeptides, or the expression of polypeptides or polynucleotides, or agonists or antagonists to the full-length polypeptides of the invention or fragments thereof Are useful for treating a variety of human diseases including diabetes or obesity. For example, the administration of the modifier can be used to treat a diabetic patient or pre-diabetic individual for the purpose of preventing progression and hence for the purpose of preventing symptoms associated with diabetes (including insulin resistance). The modifying substance of the present invention can also be used to reduce obesity and various diseases associated with obesity (eg, gallbladder disease, cancer, sleep apnea, atherosclerosis, diabetes and hypertension). it can. In some cases, the modulators of the present invention can also be used to regulate body physiology in order to reduce the likelihood of obesity-related diseases. For example, the modifiers can be used to regulate serum lipids (total cholesterol, low density lipoprotein (LDL), cholesterol, LDL / high density lipoprotein ratio and triglycerides).

A. Agents that alter the polypeptides of the invention Agents that are tested as modifiers of the polypeptides of the invention may be any low molecular weight compound or biological entity such as a protein, sugar, nucleic acid or lipid. Essentially any compound can be used as a candidate for a modifier or ligand in the assay of the present invention, but using a compound that can be dissolved in an aqueous or organic solution (especially those based on DMSO). Most often. Modifiers include agents designed to reduce the level of mRNA encoding the polypeptides of the invention (eg, antisense molecules, ribozymes, DNAzymes, short inhibitory RNAs, etc.), or Also included are agents designed to reduce the level of translation from the mRNA (eg, translation blockers such as antisense molecules complementary to the translation initiation sequence or other sequences on the mRNA molecule). Other modifiers include a polypeptide of the invention itself, a fragment thereof, or a fusion protein comprising the polypeptide or fragment thereof (eg, in some embodiments, comprises at least 25, 50 or 100 amino acids of the polypeptide). ) Is included. For polypeptides of the invention that are receptors, soluble fragments of the polypeptide (ie, those lacking the transmembrane domain) may act as modulators of the signaling activity of the polypeptide. With respect to secreted polypeptides of the invention, both full-length and biologically active fragments may act as modifiers. Sources of compounds are Sigma (St. Louis, MO), Aldrich (St. Louis, MO), Sigma-Aldrich (St. Louis, MO), Fluka Chemika-Biochemica Analytika (Buchs, Switzerland), etc. Many will be known, including

  In some embodiments, the high-throughput screening method comprises providing a combinatorial chemical library or peptide ligand library comprising a number of therapeutic compound candidates (modifying compound candidates). Subsequently, such “combinatorial chemical libraries” or “ligand libraries” are described herein to identify library members (specific species or subclasses) that exhibit the desired characteristic activity. Screen in one or more assays. The compounds thus identified serve as normal “lead compounds” or can themselves be used as therapeutic candidates or as actual therapeutics.

  A combinatorial chemical library is a collection of diverse compounds generated by chemical or biological synthesis by combining many chemical “building blocks” such as reagents. For example, a linear combinatorial chemical library, such as a polypeptide library, combines a group of constituent chemical units (amino acids) in all possible ways for a given compound length (ie, the number of amino acids in a polypeptide compound). Is generated by Millions of compounds can be synthesized by such combinatorial mixing of constituent chemical units.

  The generation and screening of combinatorial chemical libraries is well known to those skilled in the art. Such combinatorial chemical libraries include peptide libraries (eg, US Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37: 487-493 (1991) and Houghton et al., Nature 354: 84. -88 (1991)) is included without limitation. Other chemicals can also be used to create a diverse chemical library. Such chemicals include peptoids (eg, WO 91/19735), encoded peptides (eg, WO 93/20242), random biooligomers (eg, WO 92/1992). / 00091), benzodiazepines (eg, US Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat. Acad. Sci. USA 90: 6909-6913 (1993)). , Vinylic polypeptides (Hagihara et al., J. Amer. Chem. Soc. 114: 6568 (1992)), non-peptidic peptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer. Chem. Soc. 114: 9217-9218 (1992)), similar organic synthesis of small molecule libraries (Chen et al., J. Amer. Chem. Soc. 116: 2661 (1994)), oligocarbamates (Cho et al., Science 261: 1303 (1993) )), And / or peptidyl Phosphonates (Campbell et al., J. Org. Chem. 59: 658 (1994)), nucleic acid libraries (see Ausubel, Berger and Sambrook, all), peptide nucleic acid libraries (see, eg, US Pat. No. 5,539,083) Reference), antibody libraries (see, eg, Vaughn et al., Nature Biotechnology, 14 (3): 309-314 (1996) and International Patent Application No. PCT / US96 / 10287), carbohydrate libraries (eg, Liang et al., Science 274: 1520-1522 (1996) and US Pat. No. 5,593,853), small organic libraries (eg, benzodiazepines, Baum C & EN, Jan 18, p.33 (1993); isoprenoids, US Pat. No. 5,569,588; Thiazolidinone and metathiazanone, US Pat. No. 5,549,974; pyrrolidine, US Pat. Nos. 5,525,735 and 5,519,134; morpholino compounds, US Pat. No. 5,506,337; benzodiazepines, US Pat. No. 5,288,514, etc. Irradiation) are included in the non-limiting.

  Equipment for the production of combinatorial libraries is commercially available (e.g. 357 MPS, 390 MPS, Advanced Chem Tech, Louisville KY, Symphony, Rainin, Woburn, MA, 433A Applied Biosystems, Foster City, CA, 9050 Plus, Millipore , Bedford, MA). In addition, numerous combinatorial libraries are commercially available per se (eg, ComGenex, Princeton, NJ, Tripos, Inc., St. Louis, MO, 3D Pharmaceuticals, Exton, PA, Martek Biosciences, Columbia, MD, etc. ).

B. Methods of screening for modulators of the polypeptides of the invention To identify agents that alter the level of expression or activity of a polynucleotide of the polypeptides of the invention in cells, particularly mammalian cells, particularly human cells. Numerous types of screening protocols are available. In general, these screening methods include binding to the polypeptide of the invention, preventing the inhibitor or activator from binding to the polypeptide, and association of the inhibitor or activator with the polypeptide. Screening a plurality of agents to identify agents that alter the activity of the polypeptide, such as by increasing or by activating or inhibiting the expression of the polypeptide. The assay automates the steps of the assay and allows compounds from any convenient source to proceed, typically concurrently (eg, in a microtiter format on a microtiter plate in an automated instrument assay). By subjecting it to an assay, it can be designed to screen large chemical libraries.

  Any cell that expresses the full-length polypeptide of the present invention or a fragment thereof can be used to identify the modifier. In some embodiments, the cell is a eukaryotic cell line (eg, CHO or HEK293) transformed to express a heterologous polypeptide of the invention. In some embodiments, cells that express an endogenous polypeptide of the invention are used for screening. In yet another embodiment, the modulator is screened for the ability to affect insulin response. In another embodiment, modulators are screened for the ability to affect body weight (measured by BMI or waist / hip ratio) and secretion of various obesity markers (eg, leptin, IL-6 or TNFα). In another embodiment, the modulator is screened for the ability to affect lipid metabolism. In yet another embodiment, the modulator is screened for the ability to affect the secretion and activity of the adipogenic factor.

  In some embodiments, a modulator of ADLICAN comprising an amino acid sequence of SEQ ID NO: 2, 4, or 6 can be identified using, for example, a modulator binding assay, an expression assay or a promoter-reporter assay.

  In some embodiments, a modulator of ALDH1A3 comprising the amino acid sequence of SEQ ID NO: 8, 10, or 12 uses, for example, a modulator binding assay, an expression assay, a promoter-reporter assay, or an assay based on retinoic acid production. Can be identified.

  In some embodiments, the ALK7 modifier comprising an amino acid sequence of SEQ ID NO: 14, 16, or 18 uses, for example, an expression assay, a promoter-reporter assay, a modifier binding assay or a kinase assay based on Smad2 phosphorylation. Can be identified. The kinase assay can be performed after contacting the purified recombinant ALK7 protein or intact cells with the modifier. A modifying substance that binds to ALK7 can be screened, for example, by a ligand binding assay using nodal as a ligand.

  In some embodiments, the C3AR1 modulator comprising an amino acid sequence of SEQ ID NO: 20, 22, or 24 is an intracellular Ca ++ concentration induced by, for example, an expression assay, a promoter-reporter assay, a binding assay, or a modifier Can be identified using screening methods that monitor fluctuations. Modifiers that bind to C3AR1 can be screened, for example, by ligand binding assays using complement anaphylatoxin C3a as the ligand.

  In some embodiments, the modulator of CALCRL comprising the amino acid sequence of SEQ ID NO: 26, 28, or 30 is, for example, an expression assay, a promoter-reporter assay, a modifier binding assay, or an intracellular induced by the modifier It can be identified using screening methods that monitor fluctuations in cAMP concentration. Modifiers that bind to CALCRL can be screened, for example, by ligand binding assays using peptides related to adrenomedullin or calcitonin genes as ligands.

  In some embodiments, the CCL13 modifier comprising an amino acid sequence of SEQ ID NO: 32, 33, 35, or 37 is, for example, an expression assay promoter-reporter assay, a modifier binding assay, or a cell induced by the modifier. It can be identified using screening methods that monitor fluctuations in internal Ca ++ concentration. Modifiers that bind CCL13 can be screened, for example, by ligand binding assays using CCR1 or other C-CG protein coupled receptors known to bind CCL13.

  In some embodiments, a CCL8 modifier comprising an amino acid sequence of SEQ ID NO: 39, 40, 42, or 44 is derived, for example, by an expression assay, promoter-reporter assay, modifier binding assay, or modifier It can be identified using screening methods that monitor changes in intracellular Ca ++ concentration. Modifiers that bind to CCL8 can be screened, for example, by ligand binding assays using CCR1 or other C-CG protein coupled receptors known to bind CCL8.

  In some embodiments, the modulator of CHI3L1 comprising the amino acid sequence of SEQ ID NO: 46, 47, 49, or 51 is derived from, for example, an expression assay, promoter-reporter assay, modifier binding assay, or modifier It can be identified using screening methods that monitor fluctuations in MAPK and / or AKT phosphorylation and activity (see, eg, Recklies, AD et al., Biochem J. 365: 119-26 (2002)).

  In some embodiments, a CR1 modulator comprising an amino acid sequence of SEQ ID NO: 53 or 55 can be identified using, for example, an expression assay, a promoter-reporter assay, or a modifier binding assay. Modifiers that bind to CR1 can be screened, for example, by ligand binding assays using complement component C3b as the ligand.

  In some embodiments, a modulator of CSFR1 comprising the amino acid sequence of SEQ ID NO: 57, 59, or 61 can be identified using, for example, an expression assay, promoter-reporter assay, modifier binding assay, or activity assay. it can. Kinase assays can be performed after contacting purified recombinant CSFR1 protein or intact cells with a modifier. A modifying substance that binds to CSFR1 can be screened, for example, by a ligand binding assay using a colony stimulating factor as a ligand.

  In some embodiments, a modulator of CTSK comprising an amino acid sequence of SEQ ID NO: 63, 64, 66 or 68 is identified using, for example, an expression assay, promoter-reporter assay, modifier binding assay or enzyme assay. be able to. Enzymatic assays can be performed, for example, using fibrinogen as a substrate after contacting purified recombinant CTSK protein or intact cells with a modifier.

  In some embodiments, the modifier of CXCR4 comprising the amino acid sequence of SEQ ID NO: 70, 72, or 74 is, for example, an expression assay, a promoter-reporter assay, a modifier binding assay, or an intracellular induced by the modifier It can be identified using screening methods that monitor variations in Ca ++ concentration or MAPK and / or AKT phosphorylation and activity. A modifying substance that binds to CXCR4 can be screened, for example, by a ligand binding assay using CXCL12 as a ligand.

  In some embodiments, a modulator of DDAH2 comprising an amino acid sequence of SEQ ID NO: 76, 78, or 80 can be identified using, for example, an expression assay, a promoter-reporter assay, a modifier binding assay, or an activity assay. it can. Modifiers that affect DDAH2 activity can be screened by measuring the conversion of ADMA to citrulline and methylamine.

  In some embodiments, a modulator of DERP7 comprising the amino acid sequence of SEQ ID NO: 82, 84, or 86 can be identified using, for example, an expression assay, a modifier binding assay, or a promoter-reporter assay.

  In some embodiments, a modulator of ENDOGLYX1 comprising the amino acid sequence of SEQ ID NO: 88, 90, or 92 is used, for example, using an expression assay, a modulator binding assay, a promoter-reporter assay, or an angiogenesis-based activity assay. (See, for example, Christian, S. et al., J. Biol. Chem. 276: 48588-48595 (2001)).

  In some embodiments, the ETL modifier comprising the amino acid sequence of SEQ ID NO: 94, 96, or 98 is, for example, an expression assay, a promoter-reporter assay, a modifier binding assay, or an assay based on G protein coupled receptor activity. Can be used to identify.

  In some embodiments, a modifier of FLJ12389 comprising the amino acid sequence of SEQ ID NO: 100, 102, 104, 106 or 108 is, for example, an expression assay, a promoter-reporter assay, a modifier binding assay, or AMP binding or aceto It can be identified using an assay based on acetate-CoA ligase activity.

  In some embodiments, a modulator of FZD4 comprising the amino acid sequence of SEQ ID NO: 110, 112, 114 or 116 can be identified using, for example, an expression assay, a promoter-reporter assay, or a modifier binding assay. . Modifiers that bind to FZD4 can be screened, for example, by a ligand binding assay using norrin as the ligand.

  In some embodiments, the modulator of GLIPR1 comprising the amino acid sequence of SEQ ID NO: 118, 120, or 122 is an activity assay based on, for example, an expression assay, a promoter-reporter assay, a modifier binding assay, or induction of apoptosis. Can be used to identify.

  In some embodiments, the modulator of GPR105 comprising the amino acid sequence of SEQ ID NO: 124, 126, or 128 is, for example, an expression assay, a promoter-reporter assay, a modifier binding assay, or an intracellular induced by the modifier It can be identified using screening methods that monitor variations in Ca ++ concentration. A modifying substance that binds to GPR105 can be screened, for example, by a ligand binding assay using UDP glucose as a ligand.

  In some embodiments, a modulator of GPR146 comprising an amino acid sequence of SEQ ID NO: 130, 132, or 134 is based on, for example, an expression assay, a promoter-reporter assay, a modifier binding assay, or a G protein coupled receptor activity. The assay can be used to identify.

  In some embodiments, a modulator of GPR30 comprising the amino acid sequence of SEQ ID NO: 136, 138, or 140 is, for example, an expression assay, a promoter-reporter assay, a modifier binding assay, or a cell induced by a modifier It can be identified using screening methods that monitor fluctuations in cAMP concentration or MAPK phosphorylation and activity.

  In some embodiments, a modulator of GPR65 comprising the amino acid sequence of SEQ ID NO: 142, 144 or 146 can be identified using, for example, an expression assay, a promoter-reporter assay, or a modifier binding assay. A modifying substance that binds to GPR65 can be screened, for example, by a ligand binding assay using psicosin as a ligand.

  In some embodiments, a modulator of HTR2B comprising the amino acid sequence of SEQ ID NO: 148, 150, or 152 can be identified using, for example, an expression assay, a promoter-reporter assay, or a modifier binding assay. A modifying substance that binds to HTR2B can be screened, for example, by a ligand binding assay using serotonin as a ligand. Assays that detect phosphoinositide phospholipase C activity can be used.

  In some embodiments, a modifier of ITGB2 comprising the amino acid sequence of SEQ ID NO: 154, 156, or 158 can be identified using, for example, an expression assay, a promoter-reporter assay, or a modifier binding assay. Modifiers that bind to ITGB2 can be screened, for example, by ligand binding assays using ITGα chain proteins.

  In some embodiments, a modulator of ITIH5 comprising the amino acid sequence of SEQ ID NO: 160, 161 or 163 can be identified using, for example, an expression assay, a promoter-reporter assay, or a modifier binding assay.

  In some embodiments, a modulator of LGALS12 comprising an amino acid sequence of SEQ ID NO: 165, 167, 169, 171, 173, 175, 177 or 179 includes, for example, an expression assay, a promoter-reporter assay, a modifier binding assay Or screening methods that monitor changes in apoptosis induced by modifiers.

  In some embodiments, the NMB modifier comprising the amino acid sequence of SEQ ID NO: 181, 182, 184, or 186 is, for example, an expression assay, a promoter-reporter assay, a modifier binding assay, a cell induced by the modifier It can be identified using screening methods that monitor fluctuations in internal Ca ++ concentration, or binding assays. Modifiers that bind to NMB can be screened, for example, by a ligand binding assay using NMBR.

  In some embodiments, modulators of NNAT comprising the amino acid sequence of SEQ ID NO: 188, 190, 192 or 194 can be identified using, for example, expression assays, promoter-reporter assays, modifier binding assays. .

  In some embodiments, a modulator of OLFM2 comprising the amino acid sequence of SEQ ID NO: 196, 197, 199 or 201 can be identified using, for example, an expression assay, promoter-reporter assay, or modifier binding assay. .

  In some embodiments, a modulator of OPN3 comprising the amino acid sequence of SEQ ID NO: 203, 205, 207, 209 or 211 is identified using, for example, an expression assay, a promoter-reporter assay, or a modifier binding assay. Can do.

  In some embodiments, the PTPRE modifier comprising the amino acid sequence of SEQ ID NO: 213, 215, 217, 219 or 221 is, for example, an expression assay, a promoter-reporter assay, a modifier binding assay, or a receptor protein tyrosine It can be identified using screening assays based on phosphatase activity or MAPK phosphorylation and activity.

  In some embodiments, a modifier of RDC1 comprising the amino acid sequence of SEQ ID NO: 223, 225, or 227 can be identified using, for example, an expression assay, a promoter-reporter assay, or a modifier binding assay.

  In some embodiments, a modulator of SLIT2 comprising the amino acid sequence of SEQ ID NO: 229, 230, 232, or 234 can be identified using, for example, an expression assay, a promoter-reporter assay, or a modifier binding assay. . Modifiers that bind to SLIT2 can be screened, for example, by ligand binding assays using the roundabout receptor ROBO1.

  In some embodiments, a modulator of TNFRSF21 comprising the amino acid sequence of SEQ ID NO: 236, 238, or 240 is, for example, an expression assay, a promoter-reporter assay, a modifier binding assay, or apoptosis induced by a modifier and It can be identified using screening methods that monitor variations in activation of both NF-κB and JNK.

  In some embodiments, a modulator of TNFSF13B comprising an amino acid sequence of SEQ ID NO: 242, 243, 245, 247, or 249 is received, for example, using an expression assay, a promoter-reporter assay, such as TNFRSF13b, TNFRSF13c, or TNFRSF17 Modifier binding assays based on body activity or screening methods that monitor NF-κB activation variations induced by the modifiers can be identified.

  In some embodiments, the modulator of TNFSF14 comprising the amino acid sequence of SEQ ID NO: 251, 252, 254, 256, 258 or 260 is, for example, an expression assay, a promoter-reporter assay, or a receptor using, for example, TNFRSF14 Activity-based modifier binding assays can be used to identify.

  In some embodiments, the modulator of TPSB2 comprising the amino acid sequence of SEQ ID NO: 262, 263, 265, or 267 is derived from, for example, an expression assay, promoter-reporter assay, modifier binding assay, or modifier It can be identified using screening methods that monitor variations in serine-type peptidase activity.

  In some embodiments, a WISP2 modifier comprising an amino acid sequence of SEQ ID NO: 269, 270, 272, or 274 is derived, for example, by an expression assay, promoter-reporter assay, modifier binding assay, or modifier It can be identified using screening methods that monitor changes in the proliferation rate of vascular smooth muscle cells.

1． Polypeptide Binding Assays Preliminary screening can be performed by screening for agents that can bind to the polypeptides of the present invention, including at least some of the agents so identified. This is because there is a high possibility that the substance is a modified substance of the polypeptide of the invention. Binding assays are also useful, for example, for purposes of identifying endogenous proteins that interact with the polypeptides of the invention. For example, antibodies, receptors or other molecules that bind to the polypeptides of the invention can be identified in binding assays.

  Binding assays typically involve contacting the polypeptide of the invention with one or more test agents and allowing sufficient time for the protein and test agent to form a binding complex. The formed binding complex can be detected using any of a variety of established analytical techniques. Protein binding assays include methods of measuring coprecipitation, co-migration on non-denaturing SDS-polyacrylamide gels, and co-migration on Western blots (eg Bennet, JP and Yamamura, HI (1985) “Neurotransmitter, Hormone or Drug Receptor Binding Methods "," Neurotransmitter Receptor Binding "(Yamamura, HI et al., Pp. 61-89) are included without limitation. Other binding assays include the use of mass spectrometry or NMR methods to identify displacement of molecules or labeled substrates bound to the polypeptides of the invention. The polypeptide of the present invention used in this type of assay may be a naturally expressed polypeptide of the present invention, cloned or synthesized.

  In addition, polypeptides that interact or bind when expressed together in a host cell using the two-hybrid method of mammals or yeast (see, eg, Bartel, PL et al., Methods Enzymol, 254: 241 (1995)). Or it can be used to identify other molecules.

2． Polypeptide Activity The activity of a polypeptide of the invention can be determined, for example, by ligand binding (eg, binding of a radiolabeled or another labeled ligand), second messenger (eg, cAMP, cGMP, IP ₃ , DAG or Ca ²⁺ ), Ion flow, phosphorylation level, measurement of transcription level, and various in vitro and in vivo assays to determine functional, chemical and physical effects can be evaluated. Measurement of such functional, chemical and / or physical effects can be direct (eg, directly detecting calcium flux) or indirectly (eg, calcium flux, or others listed above) Detection of a change in the expression or activity of a gene product known to be altered by the effects of Furthermore, such assays can be used to test for inhibitors and activators of the polypeptides of the invention. The modifying substance may be a genetically altered form of the polypeptide of the present invention.

または保存的に改変されたそれらの他の変異体の配列と実質的な同一性を有するポリペプチドから選択されると考えられる。一般に、アミノ酸配列の同一性は、本明細書に例示したポリペプチドに対して少なくとも70％、選択的には少なくとも85％、選択的には少なくとも90％、または選択的には少なくとも95％であると考えられる。選択的には、アッセイ物のポリペプチドには、細胞外ドメイン、膜貫通ドメイン、細胞質ドメイン、リガンド結合ドメイン、サブユニット会合ドメイン、活性部位といった、本発明のポリペプチドの断片が含まれると考えられる。本明細書に記載のアッセイ法に用いるキメラタンパク質を作出するために、本発明のポリペプチドまたはそのドメインを異種タンパク質と共有結合させることもできる。 The polypeptide of the assay is

Alternatively, it will be selected from polypeptides having substantial identity with the sequence of those other conservatively modified variants. In general, the amino acid sequence identity is at least 70%, optionally at least 85%, optionally at least 90%, or optionally at least 95% relative to the polypeptides exemplified herein. it is conceivable that. Optionally, the polypeptide of the assay will include fragments of the polypeptide of the invention, such as the extracellular domain, transmembrane domain, cytoplasmic domain, ligand binding domain, subunit association domain, active site. . In order to create chimeric proteins for use in the assays described herein, the polypeptides of the invention or domains thereof can be covalently linked to heterologous proteins.

  Testing for a modulator of polypeptide activity is performed using a recombinant polypeptide of the invention or a natural polypeptide. Proteins may be isolated, expressed in cells, expressed in cell-derived membranes, expressed in tissues or animals, and may be recombinant or naturally occurring. For example, a tissue slice, a dissociated cell obtained from a tissue expressing the polypeptide of the present invention, a transformed cell, or a membrane can be used. Testing of the modifying effect is performed using either the in vitro or in vivo assay methods described herein.

  The binding of the modifying substance to the polypeptide, domain or chimeric protein of the present invention can be examined in solution, in a bilayer membrane, in a state bound to a solid phase, in a lipid monolayer, or in a vesicle. Modifier binding can be examined using, for example, changes in spectroscopic characteristics (eg, fluorescence, absorbance, refractive index), hydrodynamic (eg, shape), chromatographic or dissolution properties.

  To determine the extent of the modifying effect, a sample or assay treated with a candidate for a modifier (eg, a “test compound”) is compared to a control sample that does not contain the test compound. Control samples (those not treated with either activator or inhibitor) are designated with a relative activity value of 100. Inhibition of the polypeptides of the invention is achieved when the activity value is about 90%, optionally 50%, optionally 25-0% relative to the control. Activation of the polypeptides of the invention is achieved when the activity value is 110%, optionally 150%, 200%, 300%, 400%, 500% or 1000-2000% compared to the control. The

3． Expression Assays Screening for compounds that modulate the expression of a polynucleotide or polypeptide of the invention is also provided. A screening method is a cell that detects an increase or decrease in expression (either transcript or translation product) after a test compound is contacted with one or more cells that express a polynucleotide or polypeptide of the invention. Carrying out an assay using The assay can be performed using any cell that expresses a polynucleotide or polypeptide of the invention.

  Expression can be detected in a variety of ways. As described below, the level of expression of the polynucleotide of the present invention in a cell can be determined using a probe that specifically hybridizes with a transcript of the polynucleotide of the present invention (or a complementary nucleic acid derived therefrom). It can be determined by searching for mRNA expressed within. Probe searches can be performed by solubilizing cells and performing Northern blotting, or by using in situ hybridization without solubilizing cells. Alternatively, this polypeptide can be detected by using an immunological method for searching for cell lysate using an antibody that specifically binds to the polypeptide of the present invention.

  The promoter-reporter assay can be performed using mammalian cells transfected with a reporter gene operably linked to a sequence derived from the promoter region of the gene encoding the polypeptide of the present invention. An increase or decrease in reporter gene expression can be detected in the presence and absence of the modifier. Reporter gene expression can be detected by hybridization to complementary nucleic acids, by using immunological reagents, by assaying for the activity of the reporter gene product, or by other methods known in the art. Can do.

  The level of expression or activity of a polynucleotide or polypeptide of the invention can be compared to a baseline value. A baseline value is a value for a control sample, or a polynucleotide or polypeptide of the invention for a control population (eg, a normal weight glycemic individual as described herein) or cells (eg, a tissue culture cell not exposed to a modifier). It may be a statistical value representative of the expression level. As a negative control, the expression level for cells that do not express the polynucleotide or polypeptide of the invention can also be determined. This type of cell is generally substantially genetically identical to the test cell in other respects.

  A variety of different cell types may be used for the reporter assay. The cell that does not endogenously express the polypeptide of the present invention may be a prokaryotic cell, but is preferably a eukaryotic cell. The eukaryotic cell may be any of the cells normally used for the production of cells having a recombinant nucleic acid construct. Exemplary eukaryotic cells include yeast and various higher eukaryotic cells such as HEK293, HepG2, COS, CHO and HeLa cell lines.

  To confirm that the observed activity is reliable, a variety of methods can be used, including performing parallel reactions using cells that do not contain the reporter construct, or not contacting cells with the reporter construct with the test compound. A good contrast. The compound can be further validated as follows.

Four. Validation Agents initially identified by any of the screening methods described above can be further tested to perform a clear activity validation. Alternatively, candidates for modifiers can be tested from the beginning using the above assay without performing a preliminary screen.

  Modifiers selected for further testing can be tested for antidiabetic effects using mouse 3T3-L1 adipocytes, muscle cells (such as L6 cells), which are “classical” insulin responsive cell lines. it can. Cells (eg, adipocytes or muscle cells) are pre-incubated with the modifier to examine the acute (up to 4 hours) and chronic (overnight) effects on basal and insulin-responsive GLUT4 translocation and glucose uptake.

  Modifiers selected for further testing include any adipocyte or adipogenic cell, e.g. mouse cell line 3T3-L1 adipocyte, freshly isolated rodent or human adipocyte, undifferentiated fat The produced cells can be used to test for anti-obesity effects. Cells (eg, adipocytes) are pre-incubated with modifiers and acute (longest) to basal and insulin-induced adipogenic factor release, adipocyte size, leptin and TNFα release, and / or lipid metabolism Investigate effects (4 hours) and chronic (overnight or longer). Undifferentiated adipogenic cells can also be pre-incubated with the modifying agent and examined for effects on adipocyte differentiation (including changes in differentiation markers) and / or triglyceride accumulation.

  This response to weight gain can be determined at the organism, tissue or cell level. For example, an increase in fasting blood leptin levels indicates obesity. Other methods of measuring obesity include, for example, BMI, waist / hip ratio, total fat calculation, blood levels of various secreted proteins (IL-6, TNFα) that have been shown to correlate with obesity Measurement and measurement of fasting blood levels of free fatty acids are included.

  After such a test, the validity of the modifier is examined in a suitable animal model. The basic form of this type of method is to administer a lead compound identified during initial screening to an animal that serves as a human model and then determine whether the polypeptide of the invention is actually subject to a modifying effect. Including determining.

  The effect of the compound will be evaluated in either obese animals, diabetic animals or diet-induced insulin resistant animals. Quantify weight loss, blood glucose and insulin levels. The animal model used for the validation test is generally any kind of mammal. Specific examples of suitable animals include, but are not limited to, primates, mice and rats. For example, single gene models of diabetes (such as ob / ob and db / db mice, Zucker rats, and Zucker diabetic obese rats) or multigene models of diabetes (eg, OLETF rats, GK rats, NSY mice and KK) Mice) may be useful for demonstrating the modifying action of the polypeptides of the invention in diabetic or insulin resistant animals. Furthermore, transgenic animals expressing the human polypeptide of the present invention can also be used for further validation of drug candidates.

  Single gene models of obesity (eg, OLETF, tubby, mahogany, agouti, ob / ob and db / db mice) or obese multigene models (eg, obese animals induced by high fat diet, NZO mice, KK Mouse, Wellesley mouse, GK rat, etc.) may be useful for demonstrating the modifying action of the polypeptides of the invention in obese animals. The most widely used standard for assessing the effectiveness of anti-obesity treatment is by the FDA. The FDA defines weight loss greater than 5% as statistically significant compared to placebo. However, it will be understood that any detectable change in body weight after administration of the modulator of the present invention can be considered a relevant result.

C. Solid-phase and solute high-throughput assays The high-throughput assays of the present invention allow up to thousands of different modifiers or ligands to be screened in one day. Specifically, each well of a microtiter plate is used to perform a separate assay on a selected candidate for the modifier, or only if one wishes to observe the effect of concentration or incubation time. Five to ten wells can be used to test one modifier. Thus, it is possible to assay about 100 (eg, 96) modifiers on a single standard microtiter plate. With a 1536 well plate, about 100 to about 1500 different compounds can be assayed on a single plate. If several different plates can be assayed per day, it is possible to screen up to about 6,000 to 20,000 or more different compounds in the assay using the integrated system of the present invention. In addition, a microfluidic approach for reagent manipulation can be used.

  A molecule of interest (eg, a polypeptide or polynucleotide of the present invention, or a modification thereof) can be directly or indirectly bound to a solid component by non-covalent bonding, such as covalent bonding or tag-linked bonding. it can. The tag can be any of a variety of components. In general, a molecule that binds to a tag (tag binding agent) is immobilized on a solid support, and the target molecule to which the tag is attached is bound to the solid support by the interaction between the tag and the tag binding agent.

  Based on the known molecular interactions described in detail in the literature, any of a variety of tags and tag binders can be used. For example, if the tag has a natural binding agent, such as biotin, protein A or protein G, then use an appropriate tag binding agent (avidin, streptavidin, neutravidin, immunoglobulin Fc region, poly-His, etc. ). Antibodies to molecules with natural binding agents such as biotin are also widely available, as are suitable tag binding agents; see, eg, SIGMA Immunochemicals 1998 catalog, SIGMA, St. Louis MO) .

  Similarly, any haptenic or antigenic compound can be used in combination with an appropriate antibody to form a tag / tag binder pair. Thousands of specific antibodies are commercially available, and many other antibodies are described in the literature. For example, in one common configuration, the tag is a first antibody and the tag binding agent is a second antibody that recognizes the first antibody. In addition to antibody-antigen interactions, receptor-ligand interactions such as cell membrane receptor agonists and antagonists are also suitable as tag and tag binder pairs (eg, transferrin, c-kit, viral receptor ligands, cytokines) Cell receptor-ligand interactions such as receptors, chemokine receptors, interleukin receptors, immunoglobulin receptors and antibodies, cadherin family, integrin family, selectin family; eg Pigott and power, “Adhesion Molecule Facts Book 1” (See 1993)). Similarly, mediates the action of various small molecule ligands, including toxins and venoms, viral epitopes, hormones (eg, opiates, steroids, etc.), intracellular receptors (eg, steroids, thyroid hormones, retinoids and vitamin D; peptides) ), Drugs, lectins, sugars, nucleic acids (both linear and cyclic polymers), oligosaccharides, proteins, phospholipids and antibodies can all interact with various cell receptors .

  Synthetic polymers such as polyurethane, polyester, polycarbonate, polyurea, polyamide, polyethyleneimine, polyarylsulfide, polysiloxane, polyimide and polyacetate may also form suitable tags or tag binders. Many other tag / tag binder pairs are also useful in the assay methods described herein, and will be apparent to those of skill in the art upon reviewing the present disclosure.

  Common linkers such as peptides, polyethers, etc. are also useful as tags, including polypeptide sequences such as poly gly sequences of about 5 to 200 amino acids. Such flexible linkers are well known to those skilled in the art. For example, poly (etherin glycol) linkers are commercially available from Sheanvater Polymers, Inc (Huntsville, Alabama). Optionally, these linkers have amide bonds, sulfhydryl bonds or heterofunctional bonds.

  The tag binder is immobilized on the solid substrate using any of a variety of methods currently available. Solid substrates are generally derivatized or functionalized by exposing all or part of the substrate to a chemical reagent that immobilizes on the surface a chemical group that reacts with a portion of the tag binder. For example, groups suitable for attaching relatively long chain moieties may include amine groups, hydroxyl groups, thiol groups, and carboxyl groups. Aminoalkylsilanes and hydroxyalkylsilanes can be used to impart functionality to various surfaces such as glass surfaces. The construction of such solid phase biopolymer arrays has been described in detail in the literature (eg, Merrifield, J. Am. Chem. Soc. 85: 2149-2154 (1963) (which describes solid phase synthesis of peptides etc. Geysen et al., J. Immun. Meth. 102: 259-274 (1987) (describing the synthesis of solid phase components on the pins); Frank and Doring, Tetrahedron 44: 60316040 (1988) ( Describes the synthesis of various peptide sequences on cellulose disks); Fodor et al., Science, 251: 767-777 (1991); Sheldon et al., Clinical Chemistiy 39 (4): 718-719 (1993); See Kozal et al., Nature Medicine 2 (7): 753759 (1996), all describing arrays of biopolymers immobilized on solid substrates, for non-chemical approaches to immobilize tag binders on substrates. Include other common methods such as heating, cross-linking by UV irradiation.

  The present invention provides in vitro assays for identifying, in a high throughput format, compounds that can alter the expression or activity of the polypeptides of the present invention. Because the assay system is highly homogeneous, a control reaction that measures the activity of the polypeptide of the present invention in cells in a reaction that does not include a candidate for a modulator may optionally be selected. Such optional control reactions are appropriate and increase the reliability of the assay. Thus, in some embodiments, the methods of the invention include such a control reaction. For each of the assay formats described, a “no modifier” control reaction without modifier gives a background level of binding activity.

  In some assays, it may be desirable to use a positive control. At least two types of positive controls are suitable. First, a known activator of a polypeptide or polynucleotide of the present invention can be incubated with one assay sample, as a result of increased expression levels or activity of the polypeptide or polynucleotide of the present invention. Signal increase is determined according to the methods described herein. Second, known inhibitors of the polypeptides or polynucleotides of the present invention can be added and the resulting decrease in signal related to expression or activity of the polypeptides or polynucleotides of the present invention can be detected as well. The modifier may be combined with an activator or inhibitor to find a modifier that would normally inhibit the elevation or presence caused by the presence of a known modifier of the polypeptide or polynucleotide of the invention. Is considered to be understood.

VII. Compositions, Kits, and Integrated Systems The present invention provides compositions, kits, and integrated systems for performing the assays described herein using the nucleic acids or polypeptides, antibodies, etc. of the present invention.

  The present invention provides assay compositions for use in solid phase assays; such compositions include, for example, one or more nucleic acids encoding a polypeptide of the invention immobilized on a solid support. And a labeling reagent. In each case, the assay composition can also include other reagents desirable for hybridization. Modifiers for the expression or activity of the polypeptides of the invention can also be included in the assay composition.

  The present invention also provides kits for performing the assay methods of the present invention. The kit typically contains a probe comprising (1) an antibody that specifically binds to a polypeptide of the invention, or (2) a polynucleotide sequence that encodes at least a fragment of such a polypeptide, and the presence of an agent. Contains a label for detection. The kit can include at least one polynucleotide sequence encoding a polypeptide of the invention. The kit can include any of the compositions described above, optionally further comprising a high-throughput assay method for the effect on the expression of a gene encoding the polypeptide of the invention or on the activity of the polypeptide of the invention. Another component, such as instructions for performing, one or more containers or compartments (eg, for holding a probe, label, etc.), a control modifier of the expression or activity of a polypeptide of the invention, a component of a kit Also includes robotic armatures for mixing.

  The invention also provides an integrated system for high-throughput screening of candidate modulators for effects on the expression or activity of the polypeptides of the invention. The system includes a robotic armature for moving liquid from a source to a destination, a controller for controlling the robotic armature, a label detector, a data storage device for recording the label detection, and a well having a reaction mixture Assay components such as microtiter dishes containing or immobilized nucleic acids or substrates containing immobilized moieties.

  Various robotic liquid transport systems are available or can be easily manufactured using existing components. For example, using a Zymate XP (Zymark Corporation; Hopkinton, Mass.) Automated robot using a Microlab 2200 (Hamilton; Reno, NV) pipetting station, parallel samples were transferred to a 96-well microtiter plate. Multiple parallel binding assays can be set up.

  An optical image observed (and optionally recorded) by a camera or other recording device (eg, a photodiode and a data storage device) is optionally further in accordance with any of the aspects herein. For example, by digitizing the image and recording and analyzing the image on a computer. Various commercially available peripherals and software software are available for digitizing, storing and analyzing digitized video images or digitized optical images.

  One conventional system transmits light from a specimen field to a cooled charge coupled device (CCD) camera, commonly used in the art. A CCD camera includes an array of image elements (pixels). The light from the specimen is imaged on the CCD. Individual pixels corresponding to the area of the specimen (eg, individual hybridization sites on the array of biopolymers) are sampled and a light intensity reading is obtained for each location. Many pixels are processed in parallel to increase speed. The apparatus and method of the present invention can be easily used to observe any sample, such as by fluorescence microscopy or dark field microscopy.

VIII. Administration and Pharmaceutical Compositions Modulators of the polypeptides of the invention (eg, polypeptides or fragments of the invention, fragments thereof, or polypeptides or fragments that have an antagonistic or additive effect on the activity of the overall polypeptide) Antagonists or agonists, including fusions comprising a mammalian subject (typically for pre-diabetic, diabetic, or obese conditions) for modification of the activity of a polypeptide of the invention in vivo. To a subject in need thereof). Administration is by any of the routes normally used to introduce a modifying compound and ultimately contact the tissue to be treated and is well known in the art. Multiple routes can be used to administer an individual compound, but one particular route often results in an immediate and more effective response than another route.

  The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide range of suitable formulations for the pharmaceutical compositions of the present invention (see, eg, “Remington's Pharmaceutical Sciences” 17th Edition, 1985).

  Modulators (eg, antagonists or agonists) for the expression or activity of the polypeptides of the invention may be prepared for use in injections or in pump devices, alone or in combination with other suitable components. it can. Pump devices (also known as “insulin pumps”) are commonly used to administer insulin to a patient and are therefore easy to adapt to include the compositions of the present invention. Insulin pump manufacturers include Animas, Disetronic and MiniMed.

  Modulators (eg, antagonists or agonists) for expression or activity of the polypeptides of the invention are aerosol formulations (ie, they are “nebulized”) for administration by inhalation alone or in combination with other suitable ingredients. Can be made). Aerosol formulations can be formulated in pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.

  Formulations suitable for administration include isotonic sterile solutions that may include aqueous and non-aqueous solutions, antioxidants, buffers, bacteriostatic agents, and solutes that make the formulation isotonic, and suspending agents, Aqueous and non-aqueous sterile suspensions may be included, which may include solubilizers, thickeners, stabilizers, and preservatives. In practicing the invention, the composition can be administered, for example, orally, intranasally, topically, intravenously, intraperitoneally, intravesically or intrathecally. Formulations of the compounds can be provided in a unit dose or multiple doses sealed in a container such as an ampoule or vial. Solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. The modifier can also be administered as part of a prepared food or drug.

  The dose administered to the patient needs to be sufficient in the context of the present invention to induce a beneficial response in the subject over a period of time. The dose depends on the effectiveness of the individual modifiers used, various factors including the subject's age, weight, physical activity and dietary content, the possibility of combination with other drugs, and the severity of the diabetic case it is conceivable that. It is recommended that the daily dosage of the modifier be determined for each individual patient by those skilled in the art in a manner similar to that of known insulin compositions. The degree of dosage will also depend on the presence, nature and extent of any adverse side effects associated with the administration of a particular compound or vector in an individual subject.

  In determining the effective amount of modifier to administer, the physician will assess the circulating plasma levels of the modifier, the toxicity of the modifier, and the production of anti-modifier antibodies. In general, doses as modifier equivalents range from about 1 ng / kg to 10 mg / kg for a typical subject.

  With regard to administration, the modulator of the invention may be administered at a rate determined by applying the LD-50 of the modulator, and side effects at various concentrations of the modulator to the subject's weight and general health. it can. Administration can be by single or divided doses.

  The compounds of the invention can also be used effectively in combination with one or more other drugs depending on the desired targeted therapy (eg Turner, N. et al., Prog. Drug Res. (1998) 51 33-94; Haffher, S., Diabetes Care (1998) 21: 160-178; and DeFronzo, R. et al. (Ed.), “Diabetes Reviews” (1997) Vol. 5, No. 4). Numerous trials have examined the benefits of combination therapy with oral drugs (eg Mahier, R., J. Clin. Endocrinol. Metab. (1999) 84: 1165-71; United Kingdom Prospective Diabetes Study Group : UKPDS 28, Diabetes Care (1998) 21: 87-92; Bardin, CW (ed.), “Current Therapy In Endocrinology and Metabolism”, 6th edition (Mosby-Year Book, Inc., St. Louis, MO. 1997) Chiasson, J. et al., Ann. Intern. Med. (1994) 121: 928-935; Coniff, R. et al., Am. Ther. (1997) 19: 16-26; Coniff, R. et al., Am. J; Med. (1995) 98: 443-451; and Iwamoto, Y. et al., Diabet. Med. (1996) 13365-370; Kwiterovich, P., Am. J. Cardiol (1998) 82 (12A): 3U- See 17U). These studies show that diabetes, among other diseases, can be further improved by adding a second drug to the treatment regimen. Combination therapy includes the administration of each drug as a modulator and a separate pharmaceutical formulation, as well as administration of a single pharmaceutical formulation comprising the modulator of the present invention and one or more other drugs. For example, the modifier and thiazolidinedione can be administered to a human subject as a single oral dosage composition, such as a tablet or capsule, or each drug can be administered as a separate oral dosage formulation. When separate dosage formulations are used, the modifier and one or more other agents can be administered at essentially the same time (ie, simultaneously), or separately at different times (ie, It can also be administered sequentially. Combination therapy is interpreted to include all of these regimens.

  One example of combination therapy is when treating a pre-diabetic individual (eg, to prevent progression to type 2 diabetes) or a diabetic individual (or treating diabetes and related symptoms, complications and disorders) In this case, the modifying substance can be effectively combined with, for example, the following: sulfonylureas (chlorpropamide, tolbutamide, acetohexamide, tolazamide, glyburide, gliclazide, glycinase, glimepiride and glipizide ), Biguanides (such as metformin), PPARβδ agonists, ligands or agonists of PPARγ, such as thiazolidinones (ciglitazone, pioglitazone (see, eg, US Pat. No. 6,218,409), troglitazone and rosiglitazone (eg, US Pat. No. 5,859,037) Etc.); PPARα agonists such as lofibrate, gemfibrozil, fenofibrate, sibrofibrate, and bezafibrate, dehydroepiandrosterone (also called DHEA or its conjugated sulfate ester, DHEA-504); antiglucocorticoid; TNFα inhibitor; α-glucosidase inhibitors (acarbose, miglitol and voglibose), amylin and amylin derivatives (pramlintide, etc. (see also US Pat. Nos. 5,902,726; 5,124,314; 5,175,145 and 6,143,718, 6,136,784) ), Insulin secretagogues (repaglinide, gliquidone and nateglinide (US Pat. Nos. 6,251,856; 6,251,865; 6,221,633; see also 6,174,856)) and insulin.

  The modifiers of the invention can also be combined with anti-obesity drugs (eg, Xenical (orlistat), Merida (sibutramine), or Adipex-P (phentermine)) or appetite suppressants.

IX. Gene therapy In order to introduce a nucleic acid encoding a recombinant amino acid sequence containing the polypeptide of the present invention into a mammalian cell or target tissue, conventional gene transfer methods using viruses and non-viral gene transfer methods can be used. . Such methods can be used to administer nucleic acids encoding amino acid sequences comprising the polypeptides of the invention to cells in vitro. In some embodiments, a nucleic acid encoding an amino acid sequence comprising a polypeptide of the invention is administered for in vivo or ex vivo gene therapy uses. Non-viral vector delivery systems include DNA plasmids, naked nucleic acids, and nucleic acids complexed with delivery vehicles such as liposomes. Viral vector delivery systems include DNA and RNA viruses that have episomal or integrative genomes after delivery to the cell. For a review of gene therapy procedures, see Anderson, Science 256: 808-813 (1992); Nabel and Felgner, TIBTECH 11: 211-217 (1993); Mitani and Caskey, TIBTECH 11: 162-166 (1993); Dillon TIBTECH 11: 167-175 (1993); Miller, Nature 357: 455-460 (1992); Van Brunt, Biotechnology 6 (10): 1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8: 35-36 (1995); Kremer and Perricaudet, British Medical Bulletin 51 (1): 31-44 (1995); Haddada et al., Current Topics in Microbiology and Immunology, Doerfier and Bohm (ed.) (1995); and Yu et al., Gene Therapy 1 : See 13-26 (1994).

  Methods for non-viral delivery of nucleic acids encoding recombinant polypeptides of the invention include lipofection, microinjection, biolistic methods, virosomes, liposomes, immunoliposomes, polycations or lipid: nucleic acid conjugates, naked DNA, artificial virions, and drug-enhanced DNA uptake. Lipofection is described, for example, in US Pat. No. 5,049,386, US Pat. No. 4,946,787; and US Pat. No. 4,897,355, and reagents for lipofection are commercially available (eg, Transfectam ™ and Lipofectin ™) . Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides include those of Felgner, WO 91/17424, WO 91/16024 It is. Delivery can be to cells (ex vivo administration) or to target tissues (in vivo administration).

  The preparation of lipid: nucleic acid complexes comprising targeting liposomes such as immunolipid complexes is well known to those skilled in the art (eg, Crystal, Science 270: 404-410 (1995); Blaese et al., Cancer Gene Ther. 2: 291-297 (1995); Behr et al., Bioconjugate Chem. 5: 382-389 (1994); Remy et al., Bioconjugate Chem. 5: 647-654 (1994); Gao et al., Gene Therapy 2: 710-722 (1995). Alhmad et al., Cancer Res. 52: 4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085; No. 4,837,028 and 4,946,787).

  A system for delivery of a nucleic acid encoding a recombinant polypeptide of the present invention using an RNA virus or a DNA virus directs the virus to specific cells in the body and transports the load on the virus to the nucleus In order to take advantage of highly evolved processes. Viral vectors can be administered directly to the patient (in vivo) or the cells can be treated in vitro to administer the modified cells to the patient (ex vivo). Conventional viral systems for delivery of the polypeptides of the invention will include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors and herpes simplex virus vectors for gene transfer. . Viral vectors are currently the most efficient and versatile method for gene transfer into target cells and tissues. Retroviral, lentiviral and adeno-associated virus gene transfer methods allow integration into the host genome, often resulting in long-term expression of the inserted transgene. In addition, high transduction efficiencies have been observed in many different cell types and target tissues.

  Retroviral tropism can be altered by incorporating foreign envelope proteins and expanding the range of potential target populations. Lentiviral vectors are retroviral vectors that can transduce or infect non-dividing cells, and generally provide high viral titers. For this reason, it is considered that the selection of the retroviral gene transfer system depends on the target tissue. Retroviral vectors are composed of cis-acting terminal repeats and are capable of packaging foreign sequences up to 6-10 kb. Minimal cis-acting LTRs are sufficient for vector replication and packaging, which are then used to integrate the therapeutic gene into the target cell and to permanently express the transgene. Commonly used retroviral vectors include those based on murine leukemia virus (MuLV), gibbon leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV) and combinations thereof. Included (eg, Buchscher et al., J. Virol. 66: 2731-2739 (1992); Johann et al., J. Virol. 66: 1635-1640 (1992); Sommerfelt et al., Virol. 176: 58-59 (1990)). Wilson et al., J. Virol. 63: 2374-2378 (1989); Miller et al., J. Virol. 65: 2220-2224 (1991); see International Patent Application No. PCT / US94 / 05700).

  Where transient expression of the polypeptide of the invention is preferred, adenovirus-based systems are generally used. Adenovirus-based vectors provide very high transduction efficiency in many cell types and do not require cell division. High titers and expression levels have been obtained with this type of vector. This vector can be produced in large quantities in a relatively simple system. Adeno-associated virus (“AAV”) vectors have also been used to transduce cells with target nucleic acids, for example, in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures ( For example, West et al., Virology 160: 38-47 (1987); U.S. Patent No. 4,797,368; International Publication No. 93/24641; Kotin, Human Gene Therapy 5: 793-801 (1994); Muzyczka, J. Clin. Invest. 94: 1351 (1994)). Construction of recombinant AAV vectors has been described in various publications, including US Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5: 3251-3260 (1985); Tratschin et al., Mol. Cell Biol. 4: 2072-2081 (1984); Hermonat and Muzyczka, PNAS 81: 6466-6470 (1984); and Samulski et al., J. Virol. 63: 03822-3828 (1989).

  pLASN and MFG-S are examples of retroviral vectors used in clinical trials (Dunbar et al., Blood 85: 3048-305 (1995); Kohn et al., Nat. Med. 1: 1017-102 (1995) Malech et al., PNAS 94: 22 12133-12138 (1997)). PA317 / pLASN is a therapeutic vector first used in gene therapy trials (Blaese et al., Science 270: 475-480 (1995)). For MFG-S package vectors, transduction efficiencies of 50% or higher have been observed (Ellem et al., Immunol Immunother. 44 (1): 10-20 (1997); Dranoff et al., Hum. Gene Ther. 1 : 111-2 (1997).

  Recombinant adeno-associated virus vector (rAAV) is another promising gene delivery system based on type 2 adeno-associated virus, a defective and non-pathogenic parvovirus. All vectors are derived from plasmids that leave only the 145 bp inverted terminal repeat of AAV, sandwiching the transgene expression cassette. Efficient gene transfer and stable transgene delivery resulting from integration into the genome of the transduced cell are key features of this vector system (Wagner et al., Lancet 351: 9117 1702-3 (1998 ), Kearns et al., Gene Ther. 9: 748-55 (1996)).

  A recombinant adenovirus vector (Ad) that is deficient in replication can be prepared by replacing the E1a, E1b, and E3 genes of the transgene with Ad; and then the replication deficient vector is removed. It grows in human 293 cells that complement the trans function. Ad vectors can transduce many types of tissues in vivo, including non-dividing differentiated cells, such as found in liver, kidney and muscular tissue. Ordinary Ad vectors have great loading capabilities. One example of the use of Ad vectors in clinical trials has been associated with polynucleotide therapy for anti-tumor immunization by intramuscular injection (Sterman et al., Hum. Gene Ther. 7: 1083-9 (1998)). Other examples of the use of adenoviral vectors for gene transfer in clinical trials include Rosenecker et al., Infection 24: 1 5-10 (1996); Sterman et al., Hum. Gene Ther. 9: 7 1083-1089 (1998). Welsh et al., Hum. Gene Ther. 2: 205-18 (1995); Alvarez et al., Hum. Gene Ther. 5: 597-613 (1997); Topf et al., Gene Ther. 5: 507-513 (1998); Sterman et al., Hum. Gene Ther. 7: 1083-1089 (1998).

  Packaging cells are used to form viral particles that can infect host cells. Such cells include 293 cells for adenovirus packaging and ψ2 cells or PA317 cells for retrovirus packaging. Viral vectors used in gene therapy are usually made by producer cell lines that package nucleic acid vectors into viral particles. Vectors generally contain the minimal viral sequences necessary for packaging and subsequent integration into the host, other viral sequences that are replaced by the expression cassette of the protein to be expressed. Lost viral function is complemented in trans by the packaging cell line. For example, AAV vectors used for gene therapy generally have only ITR sequences from the AAV genome that are necessary for packaging and integration into the host genome. Viral DNA is packaged into cell lines that contain helper plasmids encoding other AAV genes, ie rep and cap, but no ITR sequences. This cell line is also infected with adenovirus as a helper. Helper virus promotes replication of AAV vectors and expression of AAV genes from helper plasmids. Helper plasmids do not contain ITR sequences and are therefore not packaged as meaningful quantities. Adenovirus contamination can be reduced, for example, by heat treatment (adenovirus is more sensitive than AAV).

  In many gene therapy applications, it is desirable to deliver a gene therapy vector highly specifically to a specific type of tissue. Viral vectors are usually modified to have specificity for a given cell type by expressing a ligand as a fusion protein with a viral coat protein present on the outer surface of the virus. The ligand is selected to have an affinity for a receptor known to be present on the surface of the cell type of interest. For example, Han et al., PNAS 92: 9747-9751 (1995) can modify Moloney murine leukemia virus to express a fusion of human heregulin and gp70, which recombinant virus can accept human epidermal growth factor receptor. It has been reported to infect only certain human breast cancer cells that express the body. This principle may be extended to another pair of virus expressing a ligand fusion protein and target cell expressing a receptor. For example, filamentous phage can be engineered to display antibody fragments (eg, FAB or Fv) having specific binding affinity for virtually any selected cellular receptor. While the above description applies primarily to viral vectors, the same principles can be applied to non-viral vectors. Such vectors can be engineered to contain specific uptake sequences that are thought to preferentially take up by specific target cells.

  Gene therapy vectors are delivered in vivo by administration to an individual patient, as described above, usually by systemic administration (eg, intravenous, intraperitoneal, intramuscular, subcutaneous or intracranial injection) or topical application. Can do. Alternatively, the vector is delivered to ex vivo cells, eg, cells transplanted ex vivo from individual patients (eg, lymphocytes, bone marrow aspirates, biopsy tissue) or universal donor hematopoietic stem cells, which are then usually incorporated into the vector It is also irrelevant to transplant the cells again into the patient's body after selecting the cells obtained.

  Ex vivo cell transfection for diagnostics, research or gene therapy (eg, by reinjecting the transfected cells into the host organism) is well known to those skilled in the art. In some embodiments, cells are isolated from a subject organism, transfected with a nucleic acid (gene or cDNA) encoding a polypeptide of the invention, and then injected again into the subject organism (eg, a patient). Various cell types suitable for ex vivo transfection are well known to those of skill in the art (for a discussion of cell isolation and culture techniques from patients, see, eg, Freshney et al., “Culture of Animal Cells, A Manual of Basic Technique "(3rd edition, 1994)) and references cited therein).

  In one embodiment, stem cells are used for ex vivo procedures for cell transfection and gene therapy. An advantage of using stem cells is that they can differentiate into other cell types in vitro or they can be introduced into a mammal (such as a cell donor) and engrafted in the bone marrow. Methods for differentiating CD34 + cells into clinically important immune cell types in vitro using cytokines such as GM-CSF, IFN-γ and TNF-α are known (Inaba et al., J. Exp. Med. 176: 1693-1702 (1992)).

  Stem cells for transduction and differentiation are isolated using known methods. For example, panning of bone marrow cells with antibodies that bind to unwanted cells such as CD4 + and CD8 + (T cells), CD45 + (whole B cells), GR-1 (granulocytes) and Iad (differentiated antigen presenting cells) By doing so, stem cells are isolated (see Inaba et al., J. Exp. Med. 176: 1693-1702 (1992)).

  Vectors containing therapeutic nucleic acids (eg, retroviruses, adenoviruses, liposomes, etc.) can also be administered directly to an organism for transduction of cells in vivo. Alternatively, naked DNA can be administered. Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells. There are suitable methods of administration for administering such nucleic acids, which are well known to those of skill in the art, and multiple routes can be used to administer a particular composition, although different routes depend on the particular route. Often, an immediate and more effective response than the route is obtained.

  Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, as described below, there are a wide range of suitable formulations for the pharmaceutical compositions of the present invention (see, eg, “Remington's Pharmaceutical Sciences” 17th Edition, 1989).

X. Diagnosis of Obesity and / or Diabetes The present invention also provides a method of diagnosing diabetes or obesity, or at least some predisposition to diabetes and / or obesity pathology. Diagnosis can include determining an individual's genotype (eg, by SNP) and comparing the genotype to an allele known to be associated with the development of obesity and / or diabetes. Alternatively, diagnosis includes measuring the level of a polypeptide or polynucleotide of the invention in a patient and comparing that level to a baseline or range. In general, the baseline value represents the value of the polypeptide or polynucleotide of the present invention in a healthy person (ie, normal body weight blood glucose).

  As discussed above, a difference (eg, high or low) from the baseline range of levels of a polypeptide or polynucleotide of the present invention is whether the patient is obese or at risk of becoming obese, Indicates that the patient is diabetic or at risk of developing at least some of the diabetic conditions (eg, pre-diabetes). The level of polypeptide in a normal weight glycemic individual may be a reading from a single individual, but is usually a statistically meaningful mean value by a group of normal weight glycemic individuals. The level of polypeptide in a normal weight glycemic individual can be represented by a value, for example in a computer program.

  In some embodiments, the level of a polypeptide or polynucleotide of the invention is obtained by taking a blood, urine or tissue sample from a patient and using any of a variety of detection methods as discussed herein. It is measured by measuring the amount of the polypeptide or polynucleotide of the present invention in a sample. For example, fasting and post-feeding blood or urine levels can be examined.

  In some embodiments, the baseline level and the level in a normal weight blood glucose sample from an individual, or at least two samples from the same individual are at least about 5%, 10%, 20%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500%, 1000% or more different. In some embodiments, the sample from the individual exceeds the baseline level by at least one of the percentages listed above. In some embodiments, the sample from the individual is below the baseline level by at least one of the ratios listed above.

  In some embodiments, the level of a polypeptide or polynucleotide of the invention is effective against anti-obesity therapies such as orlistat or sibutramine, or the effectiveness of antidiabetic agents such as thiazolidinediones, metformin, sulfonylureas and other standard therapies. Used to monitor sex. In some embodiments, the activity or expression of a polypeptide or polynucleotide of the invention is used as a surrogate marker of clinical efficacy, by anti-obesity therapy in obese patients, or in the treatment of diabetes in diabetic or prediabetic patients. Measured before and after treatment with drugs. For example, the greater the decrease in expression or activity of the polypeptide of the present invention, the greater the effectiveness.

  A glucose / insulin tolerance test can also be used to detect the effect of glucose levels on the levels of polypeptides or polynucleotides of the invention. In the glucose tolerance test, a patient's tolerance to a standard oral glucose load is assessed by evaluating serum and urine specimens for glucose levels. A blood sample is collected before glucose ingestion, glucose is orally ingested, and the blood or urine glucose level is examined at a designated time after glucose ingestion. Similarly, a meal tolerance test can be used to detect the respective effects of insulin or food on the level of a polypeptide or polynucleotide of the invention.

  Other indicators of weight or obesity can also be used to detect the effect of altering the level of a polypeptide or polynucleotide of the invention. Measurement of a subject's response can be assessed by assessing serum for changes in the level of gene products associated with obesity, such as leptin, TNFα or IL-6.

  All publications and patent applications cited herein are hereby incorporated by reference as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Incorporated in the description.

  For purposes of facilitating understanding, the present invention has been described in some detail by way of illustration and example, but in light of the teachings of the present invention, there are certain variations that do not depart from the spirit or scope of the appended claims. It will be readily apparent to those skilled in the art that changes and modifications in species may be made.

EXAMPLES The following examples are provided to illustrate the claimed invention and are not intended to be limiting.

  In either obese insulin resistant or type II diabetes cases, it is known that the ability of peripheral tissues, particularly muscle and fat, to respond to insulin (and hence the ability to take up glucose) is also impaired. Yes. This deficiency in glucose metabolism is usually compensated by increasing insulin secretion from the pancreas, thereby maintaining normal glucose levels. The majority of glucose processing occurs in muscle. Many obese insulin resistant patients are expected to progress to overt diabetes over time. The molecular defects underlying this peripheral insulin resistance in both obese and type II diabetic cases have not been fully elucidated. Intramuscular or adipose genes whose expression is altered in one or both of obese and type II diabetics compared to normal weight glycemic individuals can be causative genes for obesity, insulin resistance and / or diabetes It is possible to predict the transition to diabetes. Such genetic modifiers have the ability to eliminate obesity, insulin resistance and restore normal insulin sensitivity, thereby improving systemic glucose homeostasis, including, for example, insulin secretion. In addition, such a gene modifying substance can be used to advance the transition from obesity-induced insulin resistance to diabetes. Such gene modifiers can also be used to eliminate metabolic obesity-related diseases such as cardiovascular disease, hypertension or obesity-related cancer.

  The molecular mechanism by which thiazolidinedione (TZD) increases peripheral insulin sensitivity was investigated. Genes in muscle or fat whose expression is altered by TZD may be in a pathway leading from TZD therapy to increased insulin sensitivity. Such genetic modifiers can induce the same effects as TZD therapy. In addition, such modifiers may not have some of the side effects of TZD. Gene expression profiling in cultures of primary human adipocytes treated with pioglitazone or rosiglitazone to identify genes important for the action of TZD and hence for the treatment of obesity, diabetes and / or insulin resistance Used for

  Gene expression profiling was performed on tissue samples (subcutaneous fat samples) obtained from normoweight blood glucose, obesity and diabetic individuals. Two types of tests were conducted. In the first study, samples were isolated from all individuals after a 5 hour hyperinsulinemic euglycemic clamp.

  In the second study, subcutaneous fat samples were obtained after fasting overnight from normoglycemic (BMI <25) and obese (BMI> 30) individuals.

  In the third study, samples were obtained from human subcutaneous and omental adipose tissue. Genes that are expressed exclusively in fat or heavily expressed in fat are present in pathways involved in insulin sensitivity, appetite suppression, or lipid metabolism in fat itself or in other peripheral tissues (eg muscle, liver, brain) there is a possibility. For all tissue samples, mRNA was isolated from these fat samples and converted to cRNA by standard procedures. The gene expression profile for each individual was determined by hybridization of cRNA with commercial and custom-made Affymetrix chips.

  The difference in gene expression profile was calculated as follows. The expression level of a particular gene is indicated by its “signal intensity”. Raw data was analyzed by statistical tests to exclude “outliers”. Subsequently, the average “signal intensity” was calculated from the signal intensity for all individuals in the specific administration group. Student t-test statistic between the two conditions was calculated and the one with t statistic of 0.05 or less was selected to determine that the gene had changed in the first two tests. The fold change was determined as the ratio between the average signal intensity in condition 2 and the average signal intensity in condition 1. In the first study, three comparisons were made: Diabetes (Condition 1) compared with normal weight blood glucose (Condition 2), Obesity (Condition 1) compared with normal weight blood glucose (Condition 2), and diabetes Comparison between (Condition 1) and obesity (Condition 2). The comparison in the second study is a comparison between normal body weight blood glucose (condition 1) and obesity (condition 2). A third comparison is the identification of fat-specific genes or genes that are high in fat when the expression profile of human subcutaneous and omental adipose tissue is compared to at least 12 other adult tissues. A gene is present when the average signal intensity of a human adipose sample is greater than three times the average signal intensity of all other adult tissues that were profiled, or only in adipose tissue and not in all others Was determined by the Affymetrix software program to meet the cutoff criteria.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した糖尿病患者での変化倍数を示す。 ADLICAN
Probe set 209596 detects ADLICAN nucleic acid sequences. In gene profiling experiments, ADLICAN transcript expression was higher in diabetic patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in diabetic patients compared to normal weight blood glucose patients.

「変化倍数」は、糖尿病例での発現の平均値／正常体重血糖例での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 ADLICAN was also evaluated using real-time PCR. The results further show that ADLICAN is significantly over-expressed in subcutaneous fat from diabetic individuals compared to subcutaneous fat from normoglycemic individuals.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in diabetic cases / average value of expression in normal body weight blood glucose cases. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した肥満患者での変化倍数を示す。 Probe set 209596 detects ADLICAN nucleic acid sequences. In gene profiling experiments, ADLICAN transcript expression was higher in obese patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in obese patients compared to normal body weight blood glucose patients.

「変化倍数」は、肥満例での発現の平均値／正常体重血糖例での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 ADLICAN was also evaluated using real-time PCR. The results further show that ADLICAN is significantly over-expressed in subcutaneous fat from obese individuals compared to subcutaneous fat from normal weight glycemic individuals.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in obese cases / average value of expression in normal body weight blood glucose cases. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「Pio前（Pre-Pio）」および「Pio後（Post-Pio）」は、試料をピオグリタゾン処理の前または24時間後に採取したことを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、pio前の試料と比較したpio後の初代ヒト脂肪細胞での変化倍数を示す。 Probe set 209596 detects ADLICAN nucleic acid sequences. In gene profiling experiments, ADLICAN transcript expression was lower in pio than in cultures of primary human adipocytes treated with medium.

B / C indicates whether the sample is a basal sample or a clamp sample; “Pre-Pio” and “Post-Pio” indicate that the sample is 24 hours prior to pioglitazone treatment “Average expression” indicates mean expression; “SEM” indicates standard error of the mean; “n” indicates number of patient samples; “fold change” indicates before pio 2 shows the fold change in primary human adipocytes after pio compared to the other samples.

「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、プロファイリングを行った他のすべての成人組織と比較した脂肪組織での変化倍数を示す。 Probe set 209596 detects ADLICAN nucleic acid sequences. In gene profiling experiments, ADLICAN transcript expression was higher in adipose tissue than in all other adult tissues.

“Average expression” indicates the mean value of expression; “SEM” indicates the standard error of the average value; “n” indicates the number of patient samples; The fold change in adipose tissue compared to tissue is shown.

  ADLICAN contains the following protein domains (designated with reference to SEQ ID NO: 2): the atrophin-1 family (PF03154) at amino acids 1405-2232; the leucine rich repeat N-terminal domain (PF01462) at amino acids 26-54 Geminivirus AL2 protein (PF01440) at amino acids 1317-1428; Leucine rich repeat (PF00560) at amino acids 80103, 128-151; and immunoglobulin domain (PF00047) at amino acids 494-557, 592-653, 1868-1930, 1965-2027, 2062-2124, 2161-2223, 2258-2326, 2361-2420, 2459-2520, 2557-2618, 2652-2713, 2748-2812. ADLICAN is a protein that contains many domains that mediate protein-protein binding.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した糖尿病の変化倍数を示す。 ALDH1A3
Probe set 203180 detects the ALDH1A3 nucleic acid sequence. In gene profiling experiments, ALDH1A3 transcript expression was higher in diabetic patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in diabetes compared to normal weight blood glucose patients.

「変化倍数」は、糖尿病例での発現の平均値／正常体重血糖例での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 ALDH1A3 was also evaluated using real-time PCR. The results further show that ALDH1A3 is significantly over-expressed in subcutaneous fat from diabetic individuals compared to subcutaneous fat from normoglycemic individuals.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in diabetic cases / average value of expression in normal body weight blood glucose cases. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した肥満患者での変化倍数を示す。 Probe set 203180 detects the ALDH1A3 nucleic acid sequence. In gene profiling experiments, ALDH1A3 transcript expression was higher in obese patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in obese patients compared to normal body weight blood glucose patients.

「変化倍数」は、肥満例での発現の平均値／正常体重血糖例での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 ALDH1A3 was also evaluated using real-time PCR. The results further indicate that ALDH1A3 is significantly over-expressed in subcutaneous fat from obese individuals compared to subcutaneous fat from normal weight glycemic individuals.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in obese cases / average value of expression in normal body weight blood glucose cases. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した肥満患者での変化倍数を示す。 Probe set 203180 detects the ALDH1A3 nucleic acid sequence. In gene profiling experiments, ALDH1A3 transcript expression was higher in obese patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in obese patients compared to normal body weight blood glucose patients.

  ALDH1A3 is overexpressed in L6 myotubes and was evaluated for its effects on basal and insulin-induced glucose transport.

説明：「Con」は、hALDH1A3を発現しない対照L6筋管細胞を示す。「FC」は以下の比として定義される変化倍数を示す；hALDH1A3発現細胞におけるグルコース輸送／hALDH1A3を発現しない細胞におけるグルコース輸送。「h」はヒトである。「n」は実験の数である。SEMは平均値の標準誤差である。 Glucose transport analysis in L6 myotube cells

Description: “Con” indicates control L6 myotube cells that do not express hALDH1A3. “FC” indicates the fold change defined as the ratio below: glucose transport in hALDH1A3-expressing cells / glucose transport in cells not expressing hALDH1A3. “H” is human. “N” is the number of experiments. SEM is the standard error of the mean value.

  These results indicate that increasing the level of ALDH1A3 in cells such as muscle cells results in a corresponding increase in glucose uptake. This suggests that increasing the level or activity of ALDH1A3 in tissues of insulin resistant or diabetic patients will increase the ability of such tissues to take up glucose and hence effective treatment for insulin resistance and diabetes It shows that it is thought to bring the law.

  ALDH1A3 contains the following protein domains (designated with reference to SEQ ID NO: 8): the aldehyde dehydrogenase family (PF00171) at amino acids 40-507. ALDH1A3 is a retinaldehyde dehydrogenase that catalyzes the oxidation of all-trans retinaldehyde to retinoic acid and may play a role in cell differentiation and proliferation (Grun, F., et al J Biol Chem. 275: 41210 -8 (2000); Rexer, BN, et al Cancer Res. 61: 7065-7070 (2001).

  It has been demonstrated that ALDH1A3 mRNA can be induced in hepatocytes by drugs such as omeprazole (Nishimura et al Yakugaku Zasshi. 122: 339-61 (2002)). Thus, an exemplary method that can identify an ALDH1A3 activator involves treating hepatocytes with a candidate compound and measuring an increase in ALDH1A3 mRNA.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した糖尿病患者での変化倍数を示す。 ALK7
Probe set MBXHUMFAT04495 detects the ALK7 nucleic acid sequence. In gene profiling experiments, ALK7 transcript expression was lower in diabetic patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in diabetic patients compared to normal weight blood glucose patients.

「変化倍数」は、糖尿病例での発現の平均値／正常体重血糖例での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 ALK7 was also evaluated using real-time PCR. The results further show that ALK7 is significantly under-expressed in subcutaneous fat from diabetic individuals compared to subcutaneous fat from normal weight glycemic individuals.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in diabetic cases / average value of expression in normal body weight blood glucose cases. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「相関係数」は、グルコース処理速度（Rd）とシグナル強度との関係を示す。正の相関係数はインスリン抵抗性の増大に伴う遺伝子のダウンレギュレーションを示し、負の相関係数はアップレギュレーションを示す。「n」は患者試料の数を示す。 Probe set MBXHUMFAT04495 detects the ALK7 nucleic acid sequence. In gene profiling experiments, ALK7 transcript expression was lower in patients with insulin resistance than in normal patients.

B / C indicates whether the sample is a basal sample or a clamped sample; “correlation coefficient” indicates the relationship between glucose processing rate (Rd) and signal intensity. A positive correlation coefficient indicates gene down-regulation with increasing insulin resistance, and a negative correlation coefficient indicates up-regulation. “N” indicates the number of patient samples.

「相関係数」は、Rdとシグナル強度との関係を示す。正の相関係数はインスリン抵抗性の増大に伴う遺伝子のダウンレギュレーションを示し、負の相関係数はアップレギュレーションを示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 ALK7 was also evaluated using real-time PCR. The results further indicate that ALK7 is significantly decreased in insulin resistant patients compared to normal patients.

“Correlation coefficient” indicates the relationship between Rd and signal intensity. A positive correlation coefficient indicates gene down-regulation with increasing insulin resistance, and a negative correlation coefficient indicates up-regulation. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した肥満患者での変化倍数を示す。 Probe set MBXHUMFAT04495 detects the ALK7 nucleic acid sequence. In gene profiling experiments, ALK7 transcript expression was lower in obese patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in obese patients compared to normal body weight blood glucose patients.

「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、プロファイリングを行った他のすべての成人組織と比較した脂肪組織での変化倍数を示す。 Probe set MBXHUMFAT04495 detects the ALK7 nucleic acid sequence. In gene profiling experiments, ALK7 transcript expression was higher in adipose tissue than in all other adult tissues.

“Average expression” indicates the mean value of expression; “SEM” indicates the standard error of the average value; “n” indicates the number of patient samples; The fold change in adipose tissue compared to tissue is shown.

  ALK7 is overexpressed in L6 myotubes and was evaluated for effects on basal and insulin-induced glucose transport.

説明：「Con」は、hALK7を発現しない対照L6筋管細胞を示す。「FC」は以下の比として定義される変化倍数を示す；hALK7発現L6筋管細胞におけるグルコース輸送／ALK7を発現しないL6筋管細胞におけるグルコース輸送。「h」はヒトである。「n」は実験の数である。SDは標準偏差である。 Glucose transport analysis in L6 myotube cells

Description: “Con” indicates control L6 myotube cells that do not express hALK7. “FC” indicates the fold change defined as the ratio: glucose transport in hALK7 expressing L6 myotubes / glucose transport in L6 myotubes not expressing ALK7. “H” is human. “N” is the number of experiments. SD is the standard deviation.

  These results indicate that increasing ALK7 levels in cells such as muscle cells results in a corresponding decrease in glucose uptake. It is believed that reducing the level or activity of ALK7 in tissues of insulin resistant or diabetic patients increases the ability of such tissues to take up glucose and therefore effective against insulin resistance and diabetes. Indicates that it is thought to bring a cure.

  ALK7 contains the following protein domains (designated with reference to SEQ ID NO: 14): signal peptide at amino acids 1-25; activin type I and type II receptor domains (PF01064) at amino acids 15-100; protein Kinase domain (PF00069) at amino acids 195-482. u-PAR / Ly-6 domain (PF00021) at amino acids 30-94; and one transmembrane domain (TMHMM2.0) at amino acids 114-136. ALK7 is a transmembrane receptor protein serine-threonine kinase for signals mediated by transforming growth factor-β (TGF-β) superfamily-related growth factors and SMAD2 (Bondestam J. et al., Cytogenet. Cell Genet , 95: 157-162 (2001) Nodal has been identified as a ligand for ALK7, which may play a role in proliferation and apoptosis (Munir, S., et al., J Biol Chem. May 18 Epub (2004); Jornvall, H., et al., J Biol Chem. 276: 5140-6 (2001)).

  ALK7 is a transforming growth factor (TGF) -β family type I serine / threonine kinase receptor. Signal transduction from the ALK7 receptor involves phosphorylation of SMAD2 and SMAD3 (see, eg, Kim J, et al., J. Biol. Chem 279: 28458-28465 (2004)). Thus, inhibitors of ALK7 kinase activity can be identified, for example, by using an in vitro phosphorylation assay involving recombinant ALK7 incubated with recombinant SMAD2 or SMAD3 and radiolabeled ATP.

  As an inhibitor of ALK7 kinase activity, SB 505124 (2- (5-benzo [1,3] dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl) -6-methylpyridine hydrochloride ( For example, Byfield et al., Mol. Pharmacol 65 744-752 (2004) and SB 431542 (4- (5-Benzol [1,3] dioxol-5-yl-4-pyridin-2-yl-1H-imidazole- 2-yl) -benzamide (Inman et al., Mol Pharmacol 62 65-74 (2002)), etc. Such inhibitors, as well as other ALK7 kinase inhibitors, such as those described herein. Those identified using these screening assays can be used to treat insulin resistance and diabetes.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した糖尿病患者での変化倍数を示す。 C3AR1
Probe set 209906 detects the C3AR1 nucleic acid sequence. In gene profiling experiments, C3AR1 transcript expression was higher in diabetic patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in diabetic patients compared to normal weight blood glucose patients.

「変化倍数」は、糖尿病例での発現の平均値／正常体重血糖例での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 C3AR1 was also evaluated using real-time PCR. The results further indicate that C3AR1 is significantly over-expressed in subcutaneous fat from diabetic individuals compared to subcutaneous fat from normal-weight glycemic individuals.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in diabetic cases / average value of expression in normal body weight blood glucose cases. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した肥満患者での変化倍数を示す。 Probe set 209906 detects the C3AR1 nucleic acid sequence. In gene profiling experiments, C3AR1 transcript expression was higher in obese patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in obese patients compared to normal body weight blood glucose patients.

  The cellular level of C3AR1 was reduced in 3T3-L1 adipocytes using siRNA targeting C3AR1, and the effect on basal and insulin-induced glucose transport was assessed.

説明：「siRNA」は、マウスC3AR1を標的とするDharmacon社のSmartpool siRNAオリゴヌクレオチドを示す。「Scr」は、Dharmacon社のScramble siRNA対照を示す。「FC」は以下の比として定義される変化倍数を示す；C3AR1 siRNAがトランスフェクトされた3T3-L1脂肪細胞におけるC3AR1 mRNAのレベル／Scramble siRNAがトランスフェクトされた3T3-L1脂肪細胞におけるC3AR1 mRNAのレベル。「n」は実験の数である。SEMは平均値の標準誤差である。 C3AR1 mRNA levels in 3T3-L1 adipocytes transfected with siRNA oligonucleotides

Description: “siRNA” refers to a Dharmacon Smartpool siRNA oligonucleotide targeting mouse C3AR1. “Scr” represents the Dharmacon Scramble siRNA control. “FC” indicates the fold change defined as the ratio: C3AR1 mRNA levels in 3T3-L1 adipocytes transfected with C3AR1 siRNA / C3AR1 mRNA in 3T3-L1 adipocytes transfected with Scramble siRNA level. “N” is the number of experiments. SEM is the standard error of the mean value.

説明：「siRNA」は、マウスC3AR1を標的とするDharmacon社のSmartpool siRNAオリゴヌクレオチドを示す。「Scr」は、Dharmacon社のScramble siRNA対照オリゴヌクレオチドを示す。「FC」は以下の比として定義される変化倍数を示す；C3AR1 siRNAがトランスフェクトされた3T3-L1脂肪細胞におけるグルコース輸送／Scramble siRNAがトランスフェクトされた3T3-L1脂肪細胞におけるグルコース輸送。「n」は実験の数である。SEMは平均値の標準誤差である。 Glucose transport in 3T3-L1 adipocytes transfected with siRNA oligonucleotides

Description: “siRNA” refers to a Dharmacon Smartpool siRNA oligonucleotide targeting mouse C3AR1. “Scr” indicates a Dharmacon Scramble siRNA control oligonucleotide. “FC” indicates the fold change defined as the ratio: glucose transport in 3T3-L1 adipocytes transfected with C3AR1 siRNA / glucose transport in 3T3-L1 adipocytes transfected with Scramble siRNA. “N” is the number of experiments. SEM is the standard error of the mean value.

  These results indicate that reducing the level of C3AR1 in cells such as adipocytes results in a corresponding decrease in glucose uptake. This suggests that increasing the level or activity of C3AR1 in tissues of insulin resistant or diabetic patients increases the ability of such tissues to take up glucose and hence effective treatment for insulin resistance and diabetes It shows that it is thought to bring the law.

  C3AR1 contains the following protein domains (designated with reference to SEQ ID NO: 20): 7 transmembrane receptors (rhodopsin family) (PF00001) at amino acids 40-435; and 7 transmembrane domains ( TMHMM2.0) to amino acids 24-46, 59-81, 96-118, 138-160, 338-360, 380-402, 417-439. C3AR1 is a G protein-coupled receptor for complement component 3a and mediates various aspects of the inflammatory response, including complement activation and chemotaxis (Fischer, WH and Hugli TE, J. Immunol. 159: 4279-4286 (1997); Zwirner, J., et al., Eur J Immunol 28: 1570-7 (1998); Crass, T., et al., Eur J Immunol 26: 1944-1950 (1996)).

  C3AR1 is a G protein-coupled receptor and its activation results in the release of intracellular Ca 2+ in HMC-1 cells (eg, Legler, D.F. et al., Eur. J Immunol 26: 753-758 (1996)). Thus, C3AR1 agonists can be identified, for example, using assays that measure changes in intracellular calcium. An exemplary assay is a cell-based assay in which cells such as HMC-1 cells overexpressing C3AR1 are treated with a compound and the increase in intracellular Ca 2+ is measured using a Ca 2+ sensitive dye such as Calcium 3.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した糖尿病患者での変化倍数を示す。 CALCRL
Probe set 210815 detects the CALCRL nucleic acid sequence. In gene profiling experiments, CALCRL transcript expression was lower in diabetic patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in diabetic patients compared to normal weight blood glucose patients.

「変化倍数」は、糖尿病例での発現の平均値／正常体重血糖例での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 CALCRL was also evaluated using real-time PCR. The results further show that CALCRL is significantly under-expressed in subcutaneous fat from diabetic individuals compared to subcutaneous fat from normal weight glycemic individuals.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in diabetic cases / average value of expression in normal body weight blood glucose cases. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

  CALCRL was overexpressed in 3T3-L1 adipocytes and evaluated its effect on basal and insulin-induced glucose transport.

説明：「Con」は、hCALCRLを発現しない対照3T3-L1脂肪細胞を示す。「FC」は以下の比として定義される変化倍数を示す；hCALCRL発現3T3-L1脂肪細胞におけるグルコース輸送／hCALCRLを発現しない3T3-L1脂肪細胞におけるグルコース輸送。「h」はヒトである。「n」は実験の数である。SEMは平均値の標準誤差である。 Glucose transport analysis in 3T3-L1 adipocytes

Description: “Con” indicates control 3T3-L1 adipocytes that do not express hCALCRL. “FC” indicates fold change defined as the ratio: glucose transport in hCALCRL expressing 3T3-L1 adipocytes / glucose transport in 3T3-L1 adipocytes not expressing hCALCRL. “H” is human. “N” is the number of experiments. SEM is the standard error of the mean value.

  These results indicate that increasing the level of CALCRL in cells such as adipocytes results in a corresponding increase in glucose uptake. It is believed that increasing the level or activity of CALCRL in tissues of insulin resistant or diabetic patients will increase the ability of such tissues to take up glucose and hence effective treatment for insulin resistance and diabetes It shows that it is thought to bring the law.

  CALCRL contains the following protein domains (designated with reference to SEQ ID NO: 26): signal peptide at amino acids 1-22; hormone receptor domain (PF02793) at amino acids 62-132; seven transmembrane receptors Body (secretin family) (PF00002) at amino acids 138-391; and seven transmembrane domains (TMHMM2.0) at amino acids 144-166, 179-198, 225-247, 254-276, 291-313, 334 To ~ 352, 367 ~ 389. CALCRL is a G protein-coupled receptor that binds to calcitonin gene-related peptide (CGRP) or adrenomedullin (ADM) and activates adenylyl cyclase depending on the interaction with auxiliary proteins RAMP1 and RAMP2 (Kamitani, S , et al., FEBS Lett. 448: 111-114 (1999); Kuwasako, K., et al., Mol Pharmacol. 65: 207-13 (2004); Flahaut, M., et al., Biochemistry. 42: 10333-41 (2003)).

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した糖尿病患者での変化倍数を示す。 CCL13
Probe set 206407 detects the CCL13 nucleic acid sequence. In gene profiling experiments, CCL13 transcript expression was higher in diabetic patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in diabetic patients compared to normal weight blood glucose patients.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した肥満患者での変化倍数を示す。 Probe set 206407 detects the CCL13 nucleic acid sequence. In gene profiling experiments, CCL13 transcript expression was higher in obese patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in obese patients compared to normal body weight blood glucose patients.

「変化倍数」は、肥満例での発現の平均値／正常体重血糖例での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 CCL13 was also evaluated using real-time PCR. The results further indicate that CCL13 is significantly over-expressed in subcutaneous fat from obese individuals compared to subcutaneous fat from normal weight glycemic individuals.

“Change fold” indicates the fold change calculated as a ratio of the mean value of the expression in obese cases to the mean value of the expression in normal body weight blood glucose cases. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

  CCL13 contains the following protein domains (designated with reference to SEQ ID NO: 32): signal peptide at amino acids 1-23; and low molecular weight cytokines (intechlines / chemokines), interleukin-8 like (PF00048) To amino acids 24-89. A soluble and active secreted form of CCL13 has been detected (Berkhout, T.A., et al., J Biol Chem. 272: 16404-13 (1997)) and is presented in SEQ ID NO: 33. CCL13 exhibits chemotactic activity against monocytes, lymphocytes, basophils and eosinophils but not neutrophils. This chemokine plays a role in the accumulation of white blood cells during inflammation. It also appears to be involved in the recruitment of monocytes to the arterial wall in atherosclerosis (Garcia-Zepeda, EA et al., J Immunol 157: 5613-5626 (1996); White, JR et al., J Biol Chem 275: 36626-36631 (2000); Wain, JH, et al., Clin Exp Immunol. 127: 436-44 (2002)).

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「Pio前」および「Pio後」は、試料をピオグリタゾン処理の前または24時間後に採取したことを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、pio前の試料と比較したpio後の初代ヒト脂肪細胞での変化倍数を示す。 CCL8
Probe set 214038 detects the CCL8 nucleic acid sequence. In gene profiling experiments, CCL8 transcript expression was lower in pio than in cultures of primary human adipocytes treated with medium.

B / C indicates whether the sample is a basal sample or a clamped sample; “before Pio” and “after Pio” indicate that the sample was taken before or 24 hours after pioglitazone treatment; "Means mean expression;" SEM "means standard error of mean;" n "means number of patient samples;" fold change "is the primary human after pio compared to the sample before pio The fold change in adipocytes is shown.

「変化倍数」は、pioでの発現の平均値／媒質での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した初代ヒト脂肪細胞試料の数を示す。 CCL8 was also evaluated using real-time PCR. The results further show that CCL8 is significantly underexpressed in primary cultured human adipocytes treated with pio compared to the medium.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in pio / the average value of expression in medium. Numbers in parentheses indicate the number of primary human adipocyte samples analyzed by real-time PCR.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「Rosi前（Pre-Rosi）」および「Rosi後（Post-Rosi）」は、試料をロシグリタゾン処理の前または24時間後に採取したことを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、rosi前の試料と比較したrosi後の初代ヒト脂肪細胞での変化倍数を示す。 Probe set 214038 detects the CCL8 nucleic acid sequence. In gene profiling experiments, CCL8 transcript expression was lower in rosi than in primary human adipocytes treated with medium.

B / C indicates whether the sample is a basal sample or a clamped sample; “Pre-Rosi” and “Post-Rosi” indicate that the sample is either rosiglitazone treated or 24 “Average expression” indicates the mean value of expression; “SEM” indicates the standard error of the mean value; “n” indicates the number of patient samples; “fold change” indicates rosi The fold change in primary human adipocytes after rosi compared to the previous sample is shown.

「変化倍数」は、rosiでの発現の平均値／媒質での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した初代ヒト脂肪細胞試料の数を示す。 CCL8 was also evaluated using real-time PCR. The results further indicate that CCL8 is significantly under-expressed in primary cultured human adipocytes treated with rosi compared to the medium.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in rosi / the average value of expression in medium. Numbers in parentheses indicate the number of primary human adipocyte samples analyzed by real-time PCR.

「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、プロファイリングを行った他のすべての成人組織と比較した脂肪組織での変化倍数を示す。 Probe set 214038 detects the CCL8 nucleic acid sequence. In gene profiling experiments, CCL8 transcript expression was higher in adipose tissue than in all other adult tissues.

“Average expression” indicates the mean value of expression; “SEM” indicates the standard error of the average value; “n” indicates the number of patient samples; The fold change in adipose tissue compared to tissue is shown.

  CCL8 contains the following protein domains (designated with reference to SEQ ID NO: 39): signal peptide at amino acids 1-23; and low molecular weight cytokines (intechlines / chemokines), interleukin-8 like (PF00048) To amino acids 24-90. A soluble and active secreted form of CCL8 has been detected (Van Damme, J. et al., .J Exp Med. 176: 59-65 (1992)), which is presented in SEQ ID NO: 40 Yes. CCL8 shows chemotactic activity against monocytes, lymphocytes, basophils and eosinophils. By recruiting leukocytes to the site of inflammation, this cytokine appears to contribute to the antiviral state against leukocyte infiltration and HIV infection associated with tumors (Noso, N. et al., Biochem. Biophys. Res. Commun. 200: 1470-1476 (1994); Yang, O. et al., J. Infect. Dis. 185: 1174-1178 (2002)).

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した糖尿病患者での変化倍数を示す。 CHI3L1
Probe set 209395 detects the CHI3L1 nucleic acid sequence. In gene profiling experiments, CHI3L1 transcript expression was higher in diabetic patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in diabetic patients compared to normal weight blood glucose patients.

「変化倍数」は、糖尿病例での発現の平均値／正常体重血糖例での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 CHI3L1 was also evaluated using real-time PCR. The results further indicate that CHI3L1 is significantly over-expressed in subcutaneous fat from diabetic individuals compared to subcutaneous fat from normoglycemic individuals.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in diabetic cases / average value of expression in normal body weight blood glucose cases. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した肥満患者での変化倍数を示す。 Probe set 209395 detects the CHI3L1 nucleic acid sequence. In gene profiling experiments, CHI3L1 transcript expression was higher in obese patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in obese patients compared to normal body weight blood glucose patients.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「Pio前」および「Pio後」は、試料をピオグリタゾン処理の前または24時間後に採取したことを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、pio前の試料と比較したpio後の初代ヒト脂肪細胞での変化倍数を示す。 Probe set 209395 detects the CHI3L1 nucleic acid sequence. In gene profiling experiments, CHI3L1 transcript expression was lower in pio than in cultures of primary human adipocytes treated with medium.

B / C indicates whether the sample is a basal sample or a clamped sample; “before Pio” and “after Pio” indicate that the sample was taken before or 24 hours after pioglitazone treatment; "Means mean expression;" SEM "means standard error of mean;" n "means number of patient samples;" fold change "is the primary human after pio compared to the sample before pio The fold change in adipocytes is shown.

「変化倍数」は、pioでの発現の平均値／媒質での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した初代ヒト脂肪細胞試料の数を示す。 CHI3L1 was also evaluated using real-time PCR. The results further show that CHI3L1 is significantly underexpressed in primary cultured human adipocytes treated with pio compared to the medium.

“Change fold” indicates a fold change calculated as a ratio of the average value of expression in pio / the average value of expression in medium. Numbers in parentheses indicate the number of primary human adipocyte samples analyzed by real-time PCR.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「Rosi前」および「Rosi後」は、試料をロシグリタゾン処理の前または24時間後に採取したことを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、rosi前の試料と比較したrosi後の初代ヒト脂肪細胞での変化倍数を示す。 Probe set 209395 detects the CHI3L1 nucleic acid sequence. In gene profiling experiments, CHI3L1 transcript expression was lower in rosi compared to primary human adipocytes treated with medium.

B / C indicates whether the sample is a basal sample or a clamp sample; “Pre-Rosi” and “Post-Rosi” indicate that the sample was taken before or 24 hours after rosiglitazone treatment; “Mean” indicates the mean expression; “SEM” indicates the standard error of the mean; “n” indicates the number of patient samples; “fold change” indicates the first post-rosi compared to the pre-rosi sample The fold change in human adipocytes is shown.

「変化倍数」は、rosiでの発現の平均値／媒質での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した初代ヒト脂肪細胞試料の数を示す。 CHI3L1 was also evaluated using real-time PCR. The results further indicate that CHI3L1 is significantly underexpressed in primary cultured human adipocytes treated with rosi compared to the medium.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in rosi / the average value of expression in medium. Numbers in parentheses indicate the number of primary human adipocyte samples analyzed by real-time PCR.

  CHI3L1 is overexpressed in L6 myotubes and was evaluated for its effect on basal and insulin-induced glucose transport.

説明：「Con」は、CHI3L1を発現しない対照L6筋管細胞を示す。「FC」は以下の比として定義される変化倍数を示す；CHI3L1発現細胞におけるグルコース輸送／CHI3L1を発現しない細胞におけるグルコース輸送。「h」はヒトである。「n」は実験の数である。SEMは平均値の標準誤差である。 Glucose transport analysis in L6 myotube cells

Description: “Con” indicates control L6 myotube cells that do not express CHI3L1. “FC” indicates fold change defined as the ratio: glucose transport in CHI3L1-expressing cells / glucose transport in cells that do not express CHI3L1. “H” is human. “N” is the number of experiments. SEM is the standard error of the mean value.

  These results indicate that increasing the level of CHI3L1 in cells such as muscle cells results in a corresponding increase in glucose uptake. This suggests that increasing the level or activity of CHI3L1 in tissues of insulin resistant or diabetic patients increases the ability of such tissues to take up glucose, and therefore effective treatment for insulin resistance and diabetes It shows that it is thought to bring the law.

  CHI3L1 contains the following protein domains (designated with reference to SEQ ID NO: 46): signal peptide at amino acids 1-21; and glycosyl hydrolase family 18 (PF00704) at amino acids 22-357. A soluble and active secreted form of CHI3L1 has been detected (Hakala, B.E., et al., J Biol Chem. 268: 25803-10 (1993)) and is presented in SEQ ID NO: 47. CHI3L1 is a glycoprotein secreted by various cells including articular chondrocytes, synoviocytes and macrophages (Recklies, AD, et al., Biochem J. 365: 119-26 (2002)), matrix Associated with conditions that increase turnover and tissue remodeling, such as arthritis (Punzi, L., et al., Ann Rheum Dis. 62: 1224-6 (2003); Ling, H. and Recklies, AD, Biochem J. Mar 12; Pt. Epub (2004)).

  The CHI3L1 gene has been cloned and the proximal promoter has been identified and shown to contain binding sites for transcription factors such as PU.1, Sp1, Sp3, USF, AML-1 and C / EBP proteins (See, for example, Rehli M., et al. Genomics. 43: 221-225 (1997); Rehli, M, et al. J Biol. Chem 278: 44058-44067 (2003)). An exemplary method of screening for CHI3L1 modulators includes the following assay: A CHI3L1 promoter is inserted upstream of a reporter gene such as β-galactosidase and expressed in cells. In this way, compounds that up-regulate promoter activity can be identified by measuring an increase in β-galactosidase activity.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した糖尿病患者での変化倍数を示す。 CR1
Probe set 244313 detects the CR1 nucleic acid sequence. In gene profiling experiments, CR1 transcript expression was higher in diabetic patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in diabetic patients compared to normal weight blood glucose patients.

「変化倍数」は、糖尿病例での発現の平均値／正常体重血糖例での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 CR1 was also evaluated using real-time PCR. The results further show that CR1 is significantly over-expressed in subcutaneous fat from diabetic individuals compared to subcutaneous fat from normal weight glycemic individuals.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in diabetic cases / average value of expression in normal body weight blood glucose cases. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した肥満患者での変化倍数を示す。 Probe set 244313 detects the CR1 nucleic acid sequence. In gene profiling experiments, CR1 transcript expression was higher in obese patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in obese patients compared to normal body weight blood glucose patients.

  CR1 contains the following protein domains (designated with reference to SEQ ID NO: 53): Sushi domain (SCR repeat sequence) (PF00084) amino acids 43-99, 104-161, 166-232, 238-293, 297 ~ 353, 358 ~ 416, 421 ~ 487, 493 ~ 549, 554 ~ 611, 616 ~ 682, 688 ~ 743, 747 ~ 803, 808 ~ 866, 871 ~ 937, 943 ~ 999, 1004 ~ 1061, 1066 ~ 1132 , 1138 to 1193, 1197 to 1253, 1258 to 1316, 1321 to 1387, 1393 to 1449, 1454 to 1511, 1516 to 1582, 1588 to 1643, 1647 to 1703, 1708 to 1766, 1771 to 1837, 1846 to 1902, 1907 To 1964, 1969 to 2035, 2041 to 2096, 2100 to 2156, 2161 to 2219, 2224 to 2290, 2298 to 2354, 2359 to 2415; and one transmembrane domain (TMHMM2.0) to amino acids 2447 to 2489 . Complement receptor 1 (CR1) is a cell surface glycoprotein on erythrocytes, leukocytes and other cells that inhibits both the classical and alternative pathways of complement activation. CR1 also transports C3b-covered immune complexes in erythrocytes, activates phagocytosis of C3b-bearing particles by neutrophils and monocytes, induces interleukin 1 secretion by monocytes, and enhances B cell differentiation Other important immune functions such as (Hamer, I., et al, Biochem. J. 329, 183-190 (1998); Makrides, SC et al. J. Biol. Chem. 267: 24754-24761 ( 1992)).

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した糖尿病患者での変化倍数を示す。 CSFR1
Probe set 203104 detects the CSFR1 nucleic acid sequence. In gene profiling experiments, the expression of the CSFR1 transcript was higher in diabetic patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in diabetic patients compared to normal weight blood glucose patients.

「変化倍数」は、糖尿病例での発現の平均値／正常体重血糖例での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 CSFR1 was also evaluated using real-time PCR. The results further show that CSFR1 is significantly over-expressed in subcutaneous fat from diabetic individuals compared to subcutaneous fat from normal-weight glycemic individuals.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in diabetic cases / average value of expression in normal body weight blood glucose cases. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した肥満患者での変化倍数を示す。 Probe set 203104 detects the CSFR1 nucleic acid sequence. In gene profiling experiments, expression of the CSFR1 transcript was higher in obese patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in obese patients compared to normal body weight blood glucose patients.

  CSF1R was overexpressed in 3T3-L1 adipocytes, and the cells were subsequently treated with CSF1. Subsequently, the effects on basal and insulin-induced glucose transport and Glut4 translocation were evaluated.

説明：「Con」は、CSF1Rを発現しない対照3T3-L1脂肪細胞を示す。「FC」は以下の比として定義される変化倍数を示す；100ng/ml hCSF1で24時間刺激したhCSF1R発現細胞におけるグルコース輸送／100ng/ml hCSF1で24時間刺激した、hCSF1Rを発現しない細胞におけるグルコース輸送。「h」はヒトである。「n」は実験の数である。SEMは平均値の標準誤差である。 Glucose transport analysis in 3T3-L1 adipocytes

Description: “Con” indicates control 3T3-L1 adipocytes that do not express CSF1R. “FC” indicates the fold change defined as the ratio: glucose transport in hCSF1R expressing cells stimulated with 100 ng / ml hCSF1 for 24 hours / glucose transport in cells not expressing hCSF1R stimulated with 100 ng / ml hCSF1 for 24 hours . “H” is human. “N” is the number of experiments. SEM is the standard error of the mean value.

説明：「変化倍数」は、以下の比を示す；（100ng／mLのマウスCSF1とともに24時間インキュベートしたhCSF1R発現細胞のうち細胞表面Glut4に関して陽性と判定された百分率の平均値）／（100ng／mLのマウスCSF1とともに24時間インキュベートしたLacZ発現細胞のうち細胞表面Glut4に関して陽性と判定された百分率の平均値）。「h」はヒトである。「n」は実験の数である。 Glut4 migration analysis

Description: “fold change” indicates the following ratio: (mean value of the percentage of hCSF1R expressing cells that were incubated with 100 ng / mL mouse CSF1 for 24 hours determined positive for cell surface Glut4) / (100 ng / mL Of the LacZ-expressing cells incubated with mouse CSF1 for 24 hours, the average value determined as positive for cell surface Glut4). “H” is human. “N” is the number of experiments.

  These results indicate that increasing the level of CSF1R in cells such as adipocytes results in a corresponding increase in glucose uptake. This suggests that increasing the level or activity of CSF1R in tissues of insulin resistant or diabetic patients increases the ability of such tissues to take up glucose and hence effective treatment for insulin resistance and diabetes It shows that it is thought to bring the law.

  CSFR1 contains the following protein domains (designated with reference to SEQ ID NO: 57): signal peptide at amino acids 1-19; protein kinase domain (PF00069) at amino acids 582-910; two immunoglobulin domains (PF00047) ) To amino acids 217-280, 412-487; and one transmembrane domain (TMHMM2.0) to amino acids 515-537. CSFR1 is a tyrosine kinase receptor for colony-stimulating factor 1, a cytokine that regulates macrophage production, differentiation and function, and may be associated with advanced breast cancer and myeloid leukemia (Sapi, E., Exp Biol Med. 229: 1-11 (2004); Boultwood, J. et al., Proc Natl Acad Sci USA 88: 6176-6180 (1991); Sapi, E. et al., Cancer Res. 59: 5578- 85 (1999); Fixe, P. and Praloran, V. Cytokine 10: 32-7 (1998)).

  Cells that overexpress CSF1R can be generated (see, eg, Murray L.J., et al. Clin Exp Metastasis. 20: 757-66 (2003)). An agonist of CSF1R can be identified, for example, by screening such cells for compounds that have the ability to induce autophosphorylation of CSF1R.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した糖尿病患者での変化倍数を示す。 CTSK
Probe set 202450 detects CTSK nucleic acid sequences. In gene profiling experiments, CTSK transcript expression was higher in diabetic patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in diabetic patients compared to normal weight blood glucose patients.

「変化倍数」は、糖尿病例での発現の平均値／正常体重血糖例での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 CTSK was also evaluated using real-time PCR. The results further show that CTSK is significantly over-expressed in subcutaneous fat from diabetic individuals compared to subcutaneous fat from normal-weight glycemic individuals.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in diabetic cases / average value of expression in normal body weight blood glucose cases. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「相関係数」は、グルコース処理速度（Rd）とシグナル強度との関係を示す。正の相関係数はインスリン抵抗性の増大に伴う遺伝子のダウンレギュレーションを示し、負の相関係数はアップレギュレーションを示す。「n」は患者試料の数を示す。 Probe set 202450 detects CTSK nucleic acid sequences. In gene profiling experiments, CTSK transcript expression was higher in patients with insulin resistance than in normal patients.

B / C indicates whether the sample is a basal sample or a clamped sample; “correlation coefficient” indicates the relationship between glucose processing rate (Rd) and signal intensity. A positive correlation coefficient indicates gene down-regulation with increasing insulin resistance, and a negative correlation coefficient indicates up-regulation. “N” indicates the number of patient samples.

「相関係数」は、Rdとシグナル強度との関係を示す。正の相関係数はインスリン抵抗性の増大に伴う遺伝子のダウンレギュレーションを示し、負の相関係数はアップレギュレーションを示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 CTSK was also evaluated using real-time PCR. The results further indicate that CTSK is significantly increased in insulin resistant patients compared to normal patients.

“Correlation coefficient” indicates the relationship between Rd and signal intensity. A positive correlation coefficient indicates gene down-regulation with increasing insulin resistance, and a negative correlation coefficient indicates up-regulation. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、プロファイリングを行った他のすべての成人組織と比較した脂肪組織での変化倍数を示す。 Probe set 202450 detects CTSK nucleic acid sequences. In gene profiling experiments, CTSK transcript expression was higher in adipose tissue than in all other adult tissues.

“Average expression” indicates the mean value of expression; “SEM” indicates the standard error of the average value; “n” indicates the number of patient samples; The fold change in adipose tissue compared to tissue is shown.

  CTSK contains the following protein domains (designated with reference to SEQ ID NO: 63): signal peptide at amino acids 1-23; outer membrane lipoprotein LolB (PF03550) at amino acids 4-158; and the papain family Cysteine protease (PF00112) to amino acids 115-328. Soluble and active secreted CTSK has been detected and is presented in SEQ ID NO: 64. CTSK is a cysteine (thiol) protease involved in bone remodeling and resorption and acts as a collagenase for cartilage proteoglycans and appears to play a role in extracellular matrix degradation. Mutations in this gene cause Pycnodys Ostosis, an autosomal recessive disease characterized by bone sclerosis and short stature (Motyckova, G. and Fisher, DE, Curr Mol Med. 2: 407-21 (2002 Soderstrom, M. et al., Biochim Biophys Acta 1446: 35-46 (1999); Hou, WS, et al., Biol Chem. 384: 891-7 (2003)).

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した肥満患者での変化倍数を示す。 CXCR4
Probe set 211919 detects the CXCR4 nucleic acid sequence. In gene profiling experiments, CXCR4 transcript expression was higher in obese patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in obese patients compared to normal body weight blood glucose patients.

「変化倍数」は、肥満例での発現の平均値／正常体重血糖例での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 CXCR4 was also evaluated using real-time PCR. The results further show that CXCR4 is significantly over-expressed in subcutaneous fat from obese individuals compared to subcutaneous fat from normal-weight glycemic individuals.

“Change fold” indicates the fold change calculated as a ratio of the mean value of the expression in obese cases to the mean value of the expression in normal body weight blood glucose cases. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

  CXCR4 was overexpressed in 3T3-L1 adipocytes, and the cells were subsequently treated with SDF1, a ligand for CXCR4. Subsequently, the effects on basal and insulin-induced glucose transport and Glut4 translocation were evaluated.

説明：「Con」は、hCXCR4を発現しない対照3T3-L1脂肪細胞を示す。「FC」は以下の比として定義される変化倍数を示す；20nMのSDF1とともに24時間インキュベートしたhCXCR4発現細胞におけるグルコース輸送／20nMのSDF1とともに24時間インキュベートしたCXCR4非発現細胞におけるグルコース輸送。「h」はヒトである。「n」は実験の数である。SEMは平均値の標準誤差である。 Glucose transport analysis in 3T3-L1 adipocytes

Description: “Con” indicates control 3T3-L1 adipocytes that do not express hCXCR4. “FC” indicates the fold change defined as the ratio: glucose transport in hCXCR4-expressing cells incubated with 20 nM SDF1 for 24 hours / glucose transport in CXCR4 non-expressing cells incubated with 20 nM SDF1 for 24 hours. “H” is human. “N” is the number of experiments. SEM is the standard error of the mean value.

説明：「変化倍数」は、以下の比を示す；（20nMのSDF1とともに24時間インキュベートしたhCXCR4発現細胞のうち細胞表面Glut4に関して陽性と判定された百分率の平均値）／（20nMのSDF1とともに24時間インキュベートしたLacZ発現細胞のうち細胞表面Glut4に関して陽性と判定された百分率の平均値）。「h」はヒトである。「n」は実験の数である。 Glut4 migration analysis

Description: “fold change” indicates the ratio: (average of percentages of hCXCR4 expressing cells that were incubated with 20 nM SDF1 for 24 hours that were positive for cell surface Glut4) / (24 hours with 20 nM SDF1) The average value of the percentage of the LacZ-expressing cells that were determined to be positive for cell surface Glut4). “H” is human. “N” is the number of experiments.

  These results indicate that in cells such as adipocytes, increasing the level of CXCR4 in the presence of CXCR4 ligand SDF1 results in a corresponding increase in glucose uptake. This suggests that increasing the level or activity of CXCR4 in tissues of insulin resistant or diabetic patients enhances the ability of such tissues to take up glucose and hence effective treatment for insulin resistance and diabetes It shows that it is thought to bring the law.

  CXCR4 contains the following protein domains (designated with reference to SEQ ID NO: 70): 7 transmembrane receptors (secretin family) (PF00002) at amino acids 45-280; and 7 transmembrane domains ( TMHMM2.0) to amino acids 43-65, 78-96, 111-132, 155-174, 199-221, 242-264, 284-306. CXCR4 is a G protein-coupled receptor that binds to CXCL12, a CXC type cytokine. CXCR4 appears to be necessary for hematopoiesis and organ angiogenesis (Tachibana, K. et al., Nature 393: 591-4 (1998). It is known to act as a co-receptor for HIV ( Moriuchi, M. et al., J Immunol. 159: 4322-4329 (1997)), inhibition of this receptor may have therapeutic effects on invasive breast cancer (Tamamura, H., et al. , FEBS Lett. 550: 79-83 (2003)).

  Stimulation of the CXCR4 receptor with its ligand SDF1-α results in an increase in intracellular Ca 2+ (eg, Princen K. et al. J Exp Med. 20; 186: 1383-1388 (1997)). For this reason, CXCR4 receptor agonists screen cells with high levels of CXCR4 receptor using calcium-sensitive dyes such as Calcium 3 or Fluo 3 to identify compounds that increase intracellular Ca2 +, for example. Can be identified.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した糖尿病患者での変化倍数を示す。 DDAH2
Probe set 214909 detects the DDAH2 nucleic acid sequence. In gene profiling experiments, DDAH2 transcript expression was higher in diabetic patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in diabetic patients compared to normal weight blood glucose patients.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「相関係数」は、グルコース処理速度（Rd）とシグナル強度との関係を示す。正の相関係数はインスリン抵抗性の増大に伴う遺伝子のダウンレギュレーションを示し、負の相関係数はアップレギュレーションを示す。「n」は患者試料の数を示す。 Probe set 214909 detects the DDAH2 nucleic acid sequence. In gene profiling experiments, DDAH2 transcript expression was higher in patients with insulin resistance than in normal patients.

B / C indicates whether the sample is a basal sample or a clamped sample; “correlation coefficient” indicates the relationship between glucose processing rate (Rd) and signal intensity. A positive correlation coefficient indicates gene down-regulation with increasing insulin resistance, and a negative correlation coefficient indicates up-regulation. “N” indicates the number of patient samples.

「相関係数」は、Rdとシグナル強度との関係を示す。正の相関係数はインスリン抵抗性の増大に伴う遺伝子のダウンレギュレーションを示し、負の相関係数はアップレギュレーションを示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 DDAH2 was also evaluated using real-time PCR. The results further indicate that DDAH2 is significantly increased in insulin resistant patients compared to normal patients.

“Correlation coefficient” indicates the relationship between Rd and signal intensity. A positive correlation coefficient indicates gene down-regulation with increasing insulin resistance, and a negative correlation coefficient indicates up-regulation. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、プロファイリングを行った他のすべての成人組織と比較した脂肪組織での変化倍数を示す。 Probe set 214909 detects the DDAH2 nucleic acid sequence. In gene profiling experiments, DDAH2 transcript expression was higher in adipose tissue than in all other adult tissues.

“Average expression” indicates the mean value of expression; “SEM” indicates the standard error of the average value; “n” indicates the number of patient samples; The fold change in adipose tissue compared to tissue is shown.

  DDAH2 contains the following protein domains (designated with reference to SEQ ID NO: 76): amidinotransferase (PF02274) at amino acids 6-281. DDAH2 regulates cellular methylarginine levels, the latter inhibiting nitric oxide synthesis. Expression of DDAH2 is mainly in tissues with relatively developed blood vessels and immune tissues (Leiper, J.M., et al., Biochem J. 343: 209-14 (1999)).

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した肥満患者での変化倍数を示す。 DERP7
Probe set 219410 detects the DERP7 nucleic acid sequence. In gene profiling experiments, the expression of DERP7 transcripts was higher in obese patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in obese patients compared to normal body weight blood glucose patients.

「変化倍数」は、肥満例での発現の平均値／正常体重血糖例での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 DERP7 was also evaluated using real-time PCR. The results further show that DERP7 is significantly over-expressed in subcutaneous fat from obese individuals compared to subcutaneous fat from normal weight glycemic individuals.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in obese cases / average value of expression in normal body weight blood glucose cases. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「Pio前」および「Pio後」は、試料をピオグリタゾン処理の前または24時間後に採取したことを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、pio前の試料と比較したpio後の初代ヒト脂肪細胞での変化倍数を示す。 Probe set 219410 detects the DERP7 nucleic acid sequence. In gene profiling experiments, DERP7 transcript expression was lower in pio than in primary human adipocyte cultures treated with medium.

B / C indicates whether the sample is a basal sample or a clamped sample; “before Pio” and “after Pio” indicate that the sample was taken before or 24 hours after pioglitazone treatment; "Means mean expression;" SEM "means standard error of mean;" n "means number of patient samples;" fold change "is the primary human after pio compared to the sample before pio The fold change in adipocytes is shown.

  DERP7 contains the following protein domains (designated with reference to SEQ ID NO: 82): a family of unknown function (DUF716) (PF04819) at amino acids 113-251; and seven transmembrane domains (TMHMM2.0) To amino acids 4-23, 44-66, 91-113, 118-140, 150-172, 181-203, 218-240. DERP7 is highly similar to the uncharacterized mouse protein p.19.5. It is presumed to be a membrane protein and has been shown to be differentially expressed in two closely related T lymphoma cell clones (MacLeod CL et al., Cell Growth Differ., 1 (6): 271-279 (1990)).

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した肥満患者での変化倍数を示す。 ENDOGLYX1
Probe set 219091 detects the ENDOGLYX1 nucleic acid sequence. In gene profiling experiments, ENDOGLYX1 transcript expression was higher in obese patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in obese patients compared to normal body weight blood glucose patients.

「変化倍数」は、肥満例での発現の平均値／正常体重血糖例での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 ENDOGLYX1 was also evaluated using real-time PCR. The results further indicate that ENDOGLYX1 is significantly overexpressed in subcutaneous fat from obese individuals compared to subcutaneous fat from normal body weight glycemic individuals.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in obese cases / average value of expression in normal body weight blood glucose cases. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、プロファイリングを行った他のすべての成人組織と比較した脂肪組織での変化倍数を示す。 Probe set 219091 detects the ENDOGLYX1 nucleic acid sequence. In gene profiling experiments, ENDOGLYX1 transcript expression was higher in adipose tissue than in all other adult tissues.

“Average expression” indicates the mean value of expression; “SEM” indicates the standard error of the average value; “n” indicates the number of patient samples; The fold change in adipose tissue compared to tissue is shown.

  ENDOGLYX1 contains the following protein domains (designated with reference to SEQ ID NO: 88): seryl-tRNA synthetase N-terminal domain (PF02403) at amino acids 499-600; myosin tail (PF01576) at amino acids 143-814 Apolipoprotein A1 / A4 / E family (PF01442) at amino acids 200-469; C1q domain (PF00386) at amino acids 827-946; TNF (tumor necrosis factor) family (PF00229) at amino acids 835-946; Intermediate filament protein (PF00038) to amino acids 360-645. ENDOGLYX1 is a cell surface glycoprotein that binds to the extracellular matrix and can form homomers and heteromers via disulfide bonds. This may play a role in angiogenesis, angiogenesis, cell matrix adhesion and hemostasis (Christian, S. et al., J Biol Chem 276: 48588-95 (2001); Leimeister, C., et al., Dev Biol. 249: 204-18 (2002)).

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した肥満患者での変化倍数を示す。 ETL
The probe set MBXHUMFAT01286 detects an ETL nucleic acid sequence. In gene profiling experiments, ETL transcript expression was higher in obese patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in obese patients compared to normal body weight blood glucose patients.

「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、プロファイリングを行った他のすべての成人組織と比較した脂肪組織での変化倍数を示す。 The probe set MBXHUMFAT01286 detects an ETL nucleic acid sequence. In gene profiling experiments, ETL transcript expression was higher in adipose tissue than in all other adult tissues.

“Average expression” indicates the mean value of expression; “SEM” indicates the standard error of the average value; “n” indicates the number of patient samples; The fold change in adipose tissue compared to tissue is shown.

「変化倍数」は、脂肪組織での発現の平均値／他の組織での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した成人組織試料の数を示す。 ETL was also evaluated using real-time PCR. The results further indicate that ETL is significantly overexpressed in adipose tissue compared to all other adult tissues.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in adipose tissue / average value of expression in other tissues. Numbers in parentheses indicate the number of adult tissue samples analyzed by real-time PCR.

  The ETL contains the following protein domains (designated with reference to SEQ ID NO: 94): BphX-like (PF06139) to amino acids 629-728; Latrophilin / CL-1-like GPS domain (PF01825) to amino acids 487-539 EGF-like domain (PF00008) to amino acids 183 to 220; 7-transmembrane receptor (secretin family) (PF00002) to amino acids 545 to 792; and 7 transmembrane domains (TMHMM2.0) to amino acids To 552-574, 587-606, 621-643, 655-677, 692-714, 740-762, 766-788. ETL belongs to the secretin family of G protein-coupled peptide hormone receptors and the EGF-TM7 subfamily of receptors. The latter is characterized by a variable number of extracellular EGF and cell surface domains and a conserved seven-transmembrane region (Nechiporuk, T. et al., J Biol Chem 276: 4150-7 (2001)).

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した糖尿病患者での変化倍数を示す。 FLJ12389
Probe set 218434 detects the FLJ12389 nucleic acid sequence. In gene profiling experiments, FLJ12389 transcript expression was lower in diabetic patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in diabetic patients compared to normal weight blood glucose patients.

「変化倍数」は、糖尿病例での発現の平均値／正常体重血糖例での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 FLJ12389 was also evaluated using real-time PCR. The results further indicate that FLJ12389 is significantly under-expressed in subcutaneous fat from diabetic individuals compared to subcutaneous fat from normal weight glycemic individuals.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in diabetic cases / average value of expression in normal body weight blood glucose cases. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した肥満患者での変化倍数を示す。 Probe set 218434 detects the FLJ12389 nucleic acid sequence. In gene profiling experiments, FLJ12389 transcript expression was lower in obese patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in obese patients compared to normal body weight blood glucose patients.

「変化倍数」は、肥満例での発現の平均値／正常体重血糖例での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 FLJ12389 was also evaluated using real-time PCR. The results further indicate that FLJ12389 is significantly under-expressed in subcutaneous fat from obese individuals compared to subcutaneous fat from normal weight glycemic individuals.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in obese cases / average value of expression in normal body weight blood glucose cases. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、プロファイリングを行った他のすべての成人組織と比較した脂肪組織での変化倍数を示す。 Probe set 218434 detects the FLJ12389 nucleic acid sequence. In gene profiling experiments, FLJ12389 transcript expression was higher in adipose tissue than in all other adult tissues.

“Average expression” indicates the mean value of expression; “SEM” indicates the standard error of the average value; “n” indicates the number of patient samples; The fold change in adipose tissue compared to tissue is shown.

  FLJ12389 includes the following protein domains (designated with reference to SEQ ID NO: 100): AMP-conjugated enzyme (PF00501) at amino acids 130-571. FLJ12389 has some sequence similarity to acetyl coenzyme A synthetase and is expected to contain an ATP / GTP binding site and an AMP binding site. Although FLJ12389 may be an enzyme that utilizes ketone bodies, its physiological role is still unclear (Ohgami, M. et al., Biochem Pharmacol 65: 989-994 (2003)).

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した糖尿病患者での変化倍数を示す。 FZD4
Probe set 218665 detects the FZD4 nucleic acid sequence. In gene profiling experiments, FZD4 transcript expression was lower in diabetic patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in diabetic patients compared to normal weight blood glucose patients.

「変化倍数」は、糖尿病例での発現の平均値／正常体重血糖例での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 FZD4 was also evaluated using real-time PCR. The results further indicate that FZD4 is significantly under-expressed in subcutaneous fat from diabetic individuals compared to subcutaneous fat from normal-weight glycemic individuals.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in diabetic cases / average value of expression in normal body weight blood glucose cases. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「相関係数」は、グルコース処理速度（Rd）とシグナル強度との関係を示す。正の相関係数はインスリン抵抗性の増大に伴う遺伝子のダウンレギュレーションを示し、負の相関係数はアップレギュレーションを示す。「n」は患者試料の数を示す。 Probe set 218665 detects the FZD4 nucleic acid sequence. In gene profiling experiments, FZD4 transcript expression was lower in patients with insulin resistance than in normal patients.

B / C indicates whether the sample is a basal sample or a clamped sample; “correlation coefficient” indicates the relationship between glucose processing rate (Rd) and signal intensity. A positive correlation coefficient indicates gene down-regulation with increasing insulin resistance, and a negative correlation coefficient indicates up-regulation. “N” indicates the number of patient samples.

「相関係数」は、Rdとシグナル強度との関係を示す。正の相関係数はインスリン抵抗性の増大に伴う遺伝子のダウンレギュレーションを示し、負の相関係数はアップレギュレーションを示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 FZD4 was also evaluated using real-time PCR. The results further indicate that FZD4 is significantly decreased in insulin resistant patients compared to normal patients.

“Correlation coefficient” indicates the relationship between Rd and signal intensity. A positive correlation coefficient indicates gene down-regulation with increasing insulin resistance, and a negative correlation coefficient indicates up-regulation. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、プロファイリングを行った他のすべての成人組織と比較した脂肪組織での変化倍数を示す。 Probe set 218665 detects the FZD4 nucleic acid sequence. In gene profiling experiments, FZD4 transcript expression was higher in adipose tissue than in all other adult tissues.

“Average expression” indicates the mean value of expression; “SEM” indicates the standard error of the average value; “n” indicates the number of patient samples; The fold change in adipose tissue compared to tissue is shown.

  FZD4 contains the following protein domains (designated with reference to SEQ ID NO: 110): signal peptide at amino acids 1-36; Frizzled / Smoothened family membrane region (PF01534) at amino acids 209-514; Fz domain ( PF01392) at amino acids 35-159; and seven transmembrane domains (TMHMM2.0) at amino acids 10-32, 221-243, 253-275, 301-323, 394-416, 437-459, 474-496 To. FZD4 encodes a 7-transmembrane domain protein, which is a receptor for the Wnt signaling protein. From the auditory and cerebellar phenotypes of FZD4 null mice, Frizzled signaling is thought to be involved in the maintenance of survival and integrity of the nervous system and retinal neovascularization during the rest of the life (Wang, Y., et al , J Neurosci. 21: 4761-71 (2001); Singaraja, RR, et al., Nat Genet. 32: 326-30 (2002)).

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した糖尿病患者での変化倍数を示す。 GLIPR1
Probe set 226142 detects the GLIPR1 nucleic acid sequence. In gene profiling experiments, GLIPR1 transcript expression was higher in diabetic patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in diabetic patients compared to normal weight blood glucose patients.

「変化倍数」は、糖尿病例での発現の平均値／正常体重血糖例での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 GLIPR1 was also evaluated using real-time PCR. The results further show that GLIPR1 is significantly over-expressed in subcutaneous fat from diabetic individuals compared to subcutaneous fat from normal weight glycemic individuals.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in diabetic cases / average value of expression in normal body weight blood glucose cases. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した肥満患者での変化倍数を示す。 Probe set 226142 detects the GLIPR1 nucleic acid sequence. In gene profiling experiments, the expression of GLIPR1 transcript was higher in obese patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in obese patients compared to normal body weight blood glucose patients.

「変化倍数」は、肥満例での発現の平均値／正常体重血糖例での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 GLIPR1 was also evaluated using real-time PCR. The results further indicate that GLIPR1 is significantly over-expressed in subcutaneous fat from obese individuals compared to subcutaneous fat from normal weight glycemic individuals.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in obese cases / average value of expression in normal body weight blood glucose cases. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「Pio前」および「Pio後」は、試料をピオグリタゾン処理の前または24時間後に採取したことを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、pio前の試料と比較したpio後の初代ヒト脂肪細胞での変化倍数を示す。 Probe set 226142 detects the GLIPR1 nucleic acid sequence. In gene profiling experiments, GLIPR1 transcript expression was lower in pio + insulin than in cultures of primary human adipocytes treated with insulin.

B / C indicates whether the sample is a basal sample or a clamped sample; “before Pio” and “after Pio” indicate that the sample was taken before or 24 hours after pioglitazone treatment; "Means mean expression;" SEM "means standard error of mean;" n "means number of patient samples;" fold change "is the primary human after pio compared to the sample before pio The fold change in adipocytes is shown.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「Rosi前」および「Rosi後」は、試料をロシグリタゾン処理の前または24時間後に採取したことを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、rosi前の試料と比較したrosi後の初代ヒト脂肪細胞での変化倍数を示す。 Probe set 226142 detects the GLIPR1 nucleic acid sequence. In gene profiling experiments, expression of the GLIPR1 transcript was lower for rosi + insulin compared to cultures of primary human adipocytes treated with insulin.

B / C indicates whether the sample is a basal sample or a clamp sample; “Pre-Rosi” and “Post-Rosi” indicate that the sample was taken before or 24 hours after rosiglitazone treatment; “Mean” indicates the mean expression; “SEM” indicates the standard error of the mean; “n” indicates the number of patient samples; “fold change” indicates the first post-rosi compared to the pre-rosi sample The fold change in human adipocytes is shown.

  GLIPR1 contains the following protein domains (designated with reference to SEQ ID NO: 118): signal peptide at amino acids 1-21; SCP-like extracellular protein (PF00188) at amino acids 38-174; and one membrane Transmembrane domain (TMHMM2.0) at amino acids 235-257. GLIPR1 is presumed to be a secreted protein and appears to play a role in malignant tumor growth and progression through its apoptosis-inducing activity (Ren, C. et al., Mol Cell Biol 22: 3345-57 (2002)).

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した糖尿病患者での変化倍数を示す。 GPR105
Probe set 206637 detects the GPR105 nucleic acid sequence. In gene profiling experiments, GPR105 transcript expression was higher in diabetic patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in diabetic patients compared to normal weight blood glucose patients.

「変化倍数」は、糖尿病例での発現の平均値／正常体重血糖例での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 GPR105 was also evaluated using real-time PCR. The results further indicate that GPR105 is significantly over-expressed in subcutaneous fat from diabetic individuals compared to subcutaneous fat from normal weight glycemic individuals.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in diabetic cases / average value of expression in normal body weight blood glucose cases. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

  GPR105 contains the following protein domains (designated with reference to SEQ ID NO: 124): 7 transmembrane receptors (rhodopsin family) (PF00001) at amino acids 39-295; and 7 transmembrane domains ( TMHMM2.0) to amino acids 27-49, 56-78, 98-117, 137-159, 187-209, 235-257, 279-298. GPR105 is a G (i / o) -G protein coupled receptor that is activated by extracellular UDP sugars. In addition to stimulating intracellular calcium, activation of this receptor appears to mediate the response of primitive hematopoietic cells to the microenvironment (Chambers, JK et al., J Biol Chem 275: 10767-71 (2000); Lee, BC et al., Genes Dev 17: 1592-604 (2003); Skelton, L., et al, J Immunol 171: 1941-9 (2003); Moore, DJ et al., Brain Res Mol Brain Res 118: 10 -23 (2003)).

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した糖尿病患者での変化倍数を示す。 GPR146
Probe set 228770 detects the GPR146 nucleic acid sequence. In gene profiling experiments, GPR146 transcript expression was lower in diabetic patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in diabetic patients compared to normal weight blood glucose patients.

「変化倍数」は、糖尿病例での発現の平均値／正常体重血糖例での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 GPR146 was also evaluated using real-time PCR. The results further indicate that GPR146 is significantly less expressed in subcutaneous fat from diabetic individuals as compared to subcutaneous fat from normal weight glycemic individuals.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in diabetic cases / average value of expression in normal body weight blood glucose cases. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「相関係数」は、グルコース処理速度（Rd）とシグナル強度との関係を示す。正の相関係数はインスリン抵抗性の増大に伴う遺伝子のダウンレギュレーションを示し、負の相関係数はアップレギュレーションを示す。「n」は患者試料の数を示す。 Probe set 228770 detects the GPR146 nucleic acid sequence. In gene profiling experiments, GPR146 transcript expression was lower in patients with insulin resistance than in normal patients.

B / C indicates whether the sample is a basal sample or a clamped sample; “correlation coefficient” indicates the relationship between glucose processing rate (Rd) and signal intensity. A positive correlation coefficient indicates gene down-regulation with increasing insulin resistance, and a negative correlation coefficient indicates up-regulation. “N” indicates the number of patient samples.

「相関係数」は、Rdとシグナル強度との関係を示す。正の相関係数はインスリン抵抗性の増大に伴う遺伝子のダウンレギュレーションを示し、負の相関係数はアップレギュレーションを示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 GPR146 was also evaluated using real-time PCR. The results further indicate that GPR146 is significantly decreased in insulin resistant patients compared to normal patients.

“Correlation coefficient” indicates the relationship between Rd and signal intensity. A positive correlation coefficient indicates gene down-regulation with increasing insulin resistance, and a negative correlation coefficient indicates up-regulation. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

  The cellular level of GPR146 was reduced in 3T3-L1 adipocytes using siRNA targeting GPR146, and the effect on basal and insulin-induced glucose transport was assessed.

説明：「siRNA」は、マウスGPR146を標的とするDharmacon社のSmartpool siRNAオリゴヌクレオチドを示す。「Scr」は、Dharmacon社のScramble siRNA対照を示す。「FC」は以下の比として定義される変化倍数を示す；GPR146 siRNAがトランスフェクトされた3T3-L1脂肪細胞におけるGPR146 mRNAのレベル／Scramble siRNAがトランスフェクトされた3T3-L1脂肪細胞におけるGPR146 mRNAのレベル。「n」は実験の数である。SEMは平均値の標準誤差である。 GPR146 mRNA levels in 3T3-L1 adipocytes transfected with siRNA oligonucleotides

Description: “siRNA” refers to Dharmacon Smartpool siRNA oligonucleotide targeting mouse GPR146. “Scr” represents the Dharmacon Scramble siRNA control. “FC” indicates the fold change defined as the ratio: GPR146 mRNA level in 3T3-L1 adipocytes transfected with GPR146 siRNA / GPR146 mRNA in 3T3-L1 adipocytes transfected with Scramble siRNA level. “N” is the number of experiments. SEM is the standard error of the mean value.

説明：「siRNA」は、マウスGPR146を標的とするDharmacon社のSmartpool siRNAオリゴヌクレオチドを示す。「Scr」は、Dharmacon社のScramble siRNA対照オリゴヌクレオチドを示す。「FC」は以下の比として定義される変化倍数を示す；GPR146 siRNAがトランスフェクトされた細胞におけるグルコース輸送／Scramble siRNAがトランスフェクトされた細胞におけるグルコース輸送。「n」は実験の数である。SEMは平均値の標準誤差である。 Glucose transport in 3T3-L1 adipocytes transfected with siRNA oligonucleotides

Description: “siRNA” refers to Dharmacon Smartpool siRNA oligonucleotide targeting mouse GPR146. “Scr” indicates a Dharmacon Scramble siRNA control oligonucleotide. “FC” indicates the fold change defined as the following ratio; glucose transport in cells transfected with GPR146 siRNA / glucose transport in cells transfected with Scramble siRNA. “N” is the number of experiments. SEM is the standard error of the mean value.

  These results indicate that reducing the level of GPR146 in cells such as adipocytes results in a corresponding decrease in glucose uptake. It is believed that increasing the level or activity of GPR146 in tissues of insulin resistant or diabetic patients will increase the ability of such tissues to take up glucose and hence effective treatment for insulin resistance and diabetes It shows that it is thought to bring the law.

  GPR146 contains the following protein domains (designated with reference to SEQ ID NO: 130): signal peptide at amino acids 1-37; and 7-transmembrane receptor (rhodopsin family) (PF00001) at amino acids 44- To 296. GPR146 is a member of the rhodopsin family of G protein-coupled receptors (GPCRs) and has very low sequence similarity to GPR30.

  GPR146 is an orphan GPCR that is unknown for conjugation with G protein (Goriam DE et al. Biochim Biophys Acta. 1722: 235-46 (2005)). GPR146 agonists include, for example, promiscuous G proteins (such as Gα16, Kostenis E. Trends Pharmacol Sci. 22: 560-4 (2001)) or chimeric G proteins (such as Gα16z or Gα16s) (eg, Liu AM et al J Biomol Screen. 8: 39-49 (2003), Hazari A et al. Cell Signal. 16: 51-62 (2004)) and can be detected using cells that overexpress GPR146. it can. In such a cell screening, for example, GPR146 agonist is considered to be detected by measuring intracellular Ca2 + using a Ca2 + sensitive dye such as Calcium 3 or measuring intracellular cyclic AMP.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した糖尿病患者での変化倍数を示す。 GPR30
Probe set 210640 detects the GPR30 nucleic acid sequence. In gene profiling experiments, GPR30 transcript expression was lower in diabetic patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in diabetic patients compared to normal weight blood glucose patients.

「変化倍数」は、糖尿病例での発現の平均値／正常体重血糖例での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 GPR30 was also evaluated using real-time PCR. The results further indicate that GPR30 is significantly under-expressed in subcutaneous fat from diabetic individuals compared to subcutaneous fat from normal weight glycemic individuals.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in diabetic cases / average value of expression in normal body weight blood glucose cases. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「相関係数」は、グルコース処理速度（Rd）とシグナル強度との関係を示す。正の相関係数はインスリン抵抗性の増大に伴う遺伝子のダウンレギュレーションを示し、負の相関係数はアップレギュレーションを示す。「n」は患者試料の数を示す。 Probe set 210640 detects the GPR30 nucleic acid sequence. In gene profiling experiments, GPR30 transcript expression was lower in patients with insulin resistance than in normal patients.

B / C indicates whether the sample is a basal sample or a clamped sample; “correlation coefficient” indicates the relationship between glucose processing rate (Rd) and signal intensity. A positive correlation coefficient indicates gene down-regulation with increasing insulin resistance, and a negative correlation coefficient indicates up-regulation. “N” indicates the number of patient samples.

「相関係数」は、Rdとシグナル強度との関係を示す。正の相関係数はインスリン抵抗性の増大に伴う遺伝子のダウンレギュレーションを示し、負の相関係数はアップレギュレーションを示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 GPR30 was also evaluated using real-time PCR. The results further indicate that GPR30 is significantly reduced in insulin resistant patients compared to normal patients.

“Correlation coefficient” indicates the relationship between Rd and signal intensity. A positive correlation coefficient indicates gene down-regulation with increasing insulin resistance, and a negative correlation coefficient indicates up-regulation. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、プロファイリングを行った他のすべての成人組織と比較した脂肪組織での変化倍数を示す。 Probe set 210640 detects the GPR30 nucleic acid sequence. In gene profiling experiments, GPR30 transcript expression was higher in adipose tissue than in all other adult tissues.

“Average expression” indicates the mean value of expression; “SEM” indicates the standard error of the average value; “n” indicates the number of patient samples; The fold change in adipose tissue compared to tissue is shown.

  GPR30 includes the following protein domains (designated with reference to SEQ ID NO: 136): 7 transmembrane receptors (rhodopsin family) (PF00001) at amino acids 76-324; and 7 transmembrane domains ( TMHMM2.0) to amino acids 63-85, 97-119, 134-153, 174-196, 219-241, 262-284, 304-326. GPR30 is a G protein-coupled receptor that activates adenylyl cyclase and mediates the attenuation of Erk-1 / -2 activity by estrogen via Raf-1 inactivation. GPR30 is a progestin target gene whose expression correlates with progestin-induced growth inhibition in breast cancer cells (Filardo, EJ et al., Mol Endocrinol 14: 1649-60 (2000); Ahola, TM et al., Endocrinology 143: 4620-6 (2002)).

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した肥満患者での変化倍数を示す。 GPR65
Probe set 214467 detects the GPR65 nucleic acid sequence. In gene profiling experiments, expression of GPR65 transcripts was higher in obese patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in obese patients compared to normal body weight blood glucose patients.

「変化倍数」は、肥満例での発現の平均値／正常体重血糖例での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 GPR65 was also evaluated using real-time PCR. The results further show that GPR65 is significantly overexpressed in subcutaneous fat from obese individuals compared to subcutaneous fat from normal body weight glycemic individuals.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in obese cases / average value of expression in normal body weight blood glucose cases. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

  GPR65 contains the following protein domains (designated with reference to SEQ ID NO: 142): 7 transmembrane receptors (rhodopsin family) (PF00001) at amino acids 31-290; and 7 transmembrane domains ( TMHMM2.0) to amino acids 15-37, 49-71, 91-110, 130-152, 181-203, 224-246, 271-293. GPR65 is a G protein-coupled receptor activated by the glycosphingolipid pseudocosin. This may act on apoptosis and immunological self-tolerance and play a role in T cell-related diseases and globuloid cell leukodystrophy (Kyaw, H. et al., DNA Cell Biol 17: 493-500 (1998); Im, DS et al., J Cell Biol 153: 429-34 (2001)). GPR65 has been described to be expressed on human primary monocytes and macrophages (Duong, C.Q., et al., Biochim Biophys Acta. 1682: 112-9 (2004)).

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「Pio前」および「Pio後」は、試料をピオグリタゾン処理の前または24時間後に採取したことを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、pio前の試料と比較したpio後の初代ヒト脂肪細胞での変化倍数を示す。 HTR2B
Probe set 206638 detects the HTR2B nucleic acid sequence. In gene profiling experiments, HTR2B transcript expression was lower in pio than in cultures of primary human adipocytes treated with medium.

B / C indicates whether the sample is a basal sample or a clamped sample; “before Pio” and “after Pio” indicate that the sample was taken before or 24 hours after pioglitazone treatment; "Means mean expression;" SEM "means standard error of mean;" n "means number of patient samples;" fold change "is the primary human after pio compared to the sample before pio The fold change in adipocytes is shown.

「変化倍数」は、pioでの発現の平均値／媒質での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した初代ヒト脂肪細胞試料の数を示す。 HTR2B was also evaluated using real-time PCR. The results further show that HTR2B is significantly underexpressed in primary cultured human adipocytes treated with pio compared to the medium.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in pio / the average value of expression in medium. Numbers in parentheses indicate the number of primary human adipocyte samples analyzed by real-time PCR.

  HTR2B was overexpressed in 3T3-L1 adipocytes, and the cells were subsequently treated with 5-HT to assess their effects on basal and insulin-induced glucose transport.

説明：「Con」は、hHTR2Bを発現しない対照3T3-L1脂肪細胞を示す。「FC」は以下の比として定義される変化倍数を示す；1uM 5HTで3時間刺激したHTR2B発現細胞におけるグルコース輸送／1uM 5HTで3時間刺激したHTR2B非発現細胞におけるグルコース輸送。「h」はヒトである。「n」は実験の数である。SEMは平均値の標準誤差である。 Glucose transport analysis in 3T3-L1 adipocytes

Description: “Con” indicates control 3T3-L1 adipocytes that do not express hHTR2B. “FC” indicates fold change defined as the ratio: glucose transport in HTR2B expressing cells stimulated with 1 uM 5HT for 3 hours / glucose transport in HTR2B non-expressing cells stimulated with 1 uM 5HT for 3 hours. “H” is human. “N” is the number of experiments. SEM is the standard error of the mean value.

  These results indicate that in cells such as adipocytes, increasing the level of HTR2B in the presence of its ligand 5-HT results in a corresponding increase in glucose uptake. This suggests that increasing the level or activity of HTR2B in tissues of insulin resistant or diabetic patients enhances the ability of such tissues to take up glucose and hence effective treatment for insulin resistance and diabetes It shows that it is thought to bring the law.

  HTR2B contains the following protein domains (designated with reference to SEQ ID NO: 148): 7 transmembrane receptors (rhodopsin family) (PF00001) at amino acids 71-380; and 7 transmembrane domains ( TMHMM2.0) to amino acids 59-81, 93-115, 130-149, 170-192, 217-239, 327-349, 364-383. HTR2B is a 5-hydroxytryptamine 2B (serotonin) receptor that signals through phospholipase C and is known to induce mitosis and mediate the contractile action of serotonin on gastrointestinal smooth muscle May also play a role in digestion and migraine (Duxon MS et al., Neuroscience 76 (2): 323-9 (1997); Jerman JC et al., Eur J Pharmacol., 414 ( 1): 23-30 (2001); Launay, JM, et al., J Biol Chem. 271: 3141-7 (1996); Nebigil, CG et al., Proc Natl Acad Sci USA. 97: 2591-6 ( 2000); Schaerlinger, B., et al., Br J Pharmacol. 140: 277-84. Epub (2003)).

  The HTR2B receptor is a G protein-coupled receptor that is linked to the mobilization of intracellular Ca2 + (eg, Schmuck K. et al FEBS Lett. 342: 85-90 (1994)). For this reason, agonists of HTR2B receptors screen cells with high levels of HTR2B receptors using calcium sensitive dyes such as Calcium 3 or Fluo 3 to identify compounds that increase intracellular Ca2 +, for example Can be identified.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した糖尿病患者での変化倍数を示す。 ITGB2
Probe set 202803 detects the ITGB2 nucleic acid sequence. In gene profiling experiments, the expression of ITGB2 transcripts was higher in diabetic patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in diabetic patients compared to normal weight blood glucose patients.

「変化倍数」は、糖尿病例での発現の平均値／正常体重血糖例での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 ITGB2 was also evaluated using real-time PCR. The results further show that ITGB2 is significantly over-expressed in subcutaneous fat from diabetic individuals compared to subcutaneous fat from normal weight glycemic individuals.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in diabetic cases / average value of expression in normal body weight blood glucose cases. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した肥満患者での変化倍数を示す。 Probe set 202803 detects the ITGB2 nucleic acid sequence. In gene profiling experiments, ITGB2 transcript expression was higher in obese patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in obese patients compared to normal body weight blood glucose patients.

「変化倍数」は、肥満例での発現の平均値／正常体重血糖例での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 ITGB2 was also evaluated using real-time PCR. The results further show that ITGB2 is significantly over-expressed in subcutaneous fat from obese individuals compared to subcutaneous fat from normal weight glycemic individuals.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in obese cases / average value of expression in normal body weight blood glucose cases. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

  ICAM-1, a ligand for integrin complexes containing ITGB2, such as LFA-1, was added to 3T3-L1 adipocyte cultures and evaluated for effects on glucose transport and Glut4 translocation.

説明：「変化倍数」は、以下の比を示す；（10ug／mlのICAM＋200uMのMn2+とともに3時間インキュベートした3T3-L1脂肪細胞のうち細胞表面Glut4に関して陽性と判定された百分率の平均値）／（200uMのMn2+とともに3時間インキュベートした3T3-L1脂肪細胞のうち細胞表面Glut4に関して陽性と判定された百分率の平均値）。「h」はヒトである。「n」は実験の数である。 Glut4 migration analysis

Description: “fold change” indicates the ratio: (average of the percentages of 3T3-L1 adipocytes that were incubated with 10 ug / ml ICAM + 200 uM Mn2 + for 3 hours determined positive for cell surface Glut4) / ( (Average value of the percentage of 3T3-L1 adipocytes incubated with 200 uM Mn2 + for 3 hours determined positive for cell surface Glut4). “H” is human. “N” is the number of experiments.

  Incubation of 3T3-L1 adipocytes with 200 uM Mn2 + for 3 hours increased cell surface Glut4 in the absence of insulin. In contrast, inclusion of 10 ug / ml ICAM inhibited the increase observed with Mn alone.

説明：「FC」は以下の比として定義される変化倍数を示す；10ug／mlのICAM1＋200uMのMnとともに3時間インキュベートした3T3-L1脂肪細胞におけるグルコース輸送／200uMのMnとともに3時間インキュベートした3T3-L1脂肪細胞におけるグルコース輸送。「h」はヒトである。「n」は実験の数である。SDは標準偏差である。 Glucose transport analysis in 3T3-L1 adipocytes

Description: “FC” indicates the fold change defined as the ratio: glucose transport in 3T3-L1 adipocytes incubated for 3 hours with 10 ug / ml ICAM1 + 200 uM Mn / 3T3-L1 incubated for 3 hours with 200 uM Mn Glucose transport in adipocytes. “H” is human. “N” is the number of experiments. SD is the standard deviation.

  These results indicate that increasing the activity of ITGB2-containing integrin complexes such as LFA-1 in cells such as adipocytes results in a corresponding decrease in glucose uptake. It is believed that reducing the level or activity of ITGB2-containing integrin complexes such as LFA-1 in tissues of insulin resistant or diabetic patients increases the ability of such tissues to take up glucose, which Thus, it has been shown to provide an effective treatment for insulin resistance and diabetes.

  ITGB2 contains the following protein domains (designated with reference to SEQ ID NO: 154): signal peptide at amino acids 1-22; Plexin repeat (PF01437) at amino acids 24-63; integrin beta chain (PF00362) At amino acids 32-447; EGF-like domain (PF00008) at amino acids 582-612; and one transmembrane domain (TMHMM2.0) at amino acids 701-723. The protein product of ITGB2 is the integrin β chain β2. Integrins are endogenous cell surface proteins composed of α and β chains. Any chain can be combined with a number of partners to give a variety of integrins. For example, β2 forms integrin LFA-1 when combined with the α L chain, and integrin Mac-1 when combined with the αM chain. Integrins are known to be involved in cell adhesion and signal transduction through the cell surface. Modulation of interactions mediated by adhesion molecules may be a new target for controlling inflammatory and immune responses (Bunting, M. et al., Curr Opin Hematol. 9: 30-5 ( Tsuji T. et al., Blood 91 (4): 1263-71 (1998); Zhang L. and Plow EF Biochemistry 38 (25): 8064-71 (1999)).

  Inhibitors of LFA-1 can be detected using a variety of assays, including those that detect the ability of candidate compounds to prevent association of LFA-1 with the ligand ICAM-1. For example, purified LFA-1 can be immobilized and compounds that cause inhibition of biotinylated ICAM-1 binding can be detected.

  Inhibitors of the LFA-1 integrin complex (αL / β2) have been developed. These include inhibitors such as BIRT0377, LFA703 and A-286982 (Shimakoa et al Nat. Rev. Drug Discovery 2: 703-716 (2003). Such inhibitors and LFA-1 integrin complexes Other inhibitors of are useful as agents for the treatment of insulin resistance and diabetes.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した糖尿病患者での変化倍数を示す。 ITIH5
The probe set MBXHUMFAT04252 detects the ITIH5 nucleic acid sequence. In gene profiling experiments, ITIH5 transcript expression was higher in diabetic patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in diabetic patients compared to normal weight blood glucose patients.

「変化倍数」は、糖尿病例での発現の平均値／正常体重血糖例での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 ITIH5 was also evaluated using real-time PCR. The results further show that ITIH5 is significantly over-expressed in subcutaneous fat from diabetic individuals compared to subcutaneous fat from normal-weight glycemic individuals.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in diabetic cases / average value of expression in normal body weight blood glucose cases. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した肥満患者での変化倍数を示す。 The probe set MBXHUMFAT04252 detects the ITIH5 nucleic acid sequence. In gene profiling experiments, ITIH5 transcript expression was higher in obese patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in obese patients compared to normal body weight blood glucose patients.

「変化倍数」は、肥満例での発現の平均値／正常体重血糖例での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 ITIH5 was also evaluated using real-time PCR. The results further indicate that ITIH5 is significantly over-expressed in subcutaneous fat from obese individuals compared to subcutaneous fat from normal weight glycemic individuals.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in obese cases / average value of expression in normal body weight blood glucose cases. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、プロファイリングを行った他のすべての成人組織と比較した脂肪組織での変化倍数を示す。 The probe set MBXHUMFAT04252 detects the ITIH5 nucleic acid sequence. In gene profiling experiments, ITIH5 transcript expression was higher in adipose tissue than in all other adult tissues.

“Average expression” indicates the mean value of expression; “SEM” indicates the standard error of the average value; “n” indicates the number of patient samples; The fold change in adipose tissue compared to tissue is shown.

  ITIH5 was overexpressed in 3T3-L1 adipocytes and the effect on basal and insulin-induced glucose transport and Glut4 translocation was assessed.

説明：「Con」は、ITIH5を発現しない対照3T3-L1脂肪細胞を示す。「FC」は以下の比として定義される変化倍数を示す；ITIH5を発現する3T3-L1細胞におけるグルコース輸送／PTPREを発現しない3T3-L1細胞におけるグルコース輸送。「h」はヒトである。「n」は実験の数である。SEMは平均値の標準誤差である。 Glucose transport analysis in 3T3-L1 adipocytes

Description: “Con” indicates control 3T3-L1 adipocytes that do not express ITIH5. “FC” indicates the fold change defined as the ratio: glucose transport in 3T3-L1 cells expressing ITIH5 / glucose transport in 3T3-L1 cells not expressing PTPRE. “H” is human. “N” is the number of experiments. SEM is the standard error of the mean value.

説明：「変化倍数」は、以下の比を示す；（ITIH5発現細胞のうち細胞表面Glut4に関して陽性と判定された百分率の平均値）／LacZ発現細胞のうち細胞表面Glut4に関して陽性と判定された百分率の平均値）。「h」はヒトである。「n」は実験の数である。 Glut4 migration analysis

Explanation: “fold change” indicates the following ratio: (average value of percentage of ITIH5-expressing cells judged positive for cell surface Glut4) / percentage of LacZ expressing cells judged positive for cell surface Glut4 Average value). “H” is human. “N” is the number of experiments.

  These results indicate that increasing ITIH5 levels in cells such as adipocytes results in a corresponding increase in glucose uptake. This suggests that increasing the level or activity of ITIH5 in tissues of insulin resistant or diabetic patients enhances the ability of such tissues to take up glucose and hence effective treatment for insulin resistance and diabetes It shows that it is thought to bring the law.

  ITIH5 contains the following protein domains (designated with reference to SEQ ID NO: 160): signal peptide at amino acids 1-21; inter-α-trypsin inhibitor heavy chain C-terminus (PF06668) at amino acids 715-909; T-box (PF00907) at amino acids 310-426; and von Willebrand factor type A domain (PF00092) at amino acids 295-478. Soluble and active secreted ITIH5 has been detected and is presented in SEQ ID NO: 161. ITIH5 belongs to the inter-α-trypsin inhibitor (ITI) family, a group of proteins composed of one light chain and various sets of heavy chains. Although initially identified as a plasma protease inhibitor, recent data indicate that ITI proteins play a role in extracellular matrix (ECM) stabilization and tumor metastasis prevention. ITIH5 expression is always down-regulated in invasive ductal carcinoma (Himmelfarb M. et al, Cancer Lett. 204 (1): 69-77 (2004)).

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した糖尿病患者での変化倍数を示す。 LGALS12
Probe set 223828 detects the LGALS12 nucleic acid sequence. In gene profiling experiments, LGALS12 transcript expression was lower in diabetic patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in diabetic patients compared to normal weight blood glucose patients.

「変化倍数」は、糖尿病例での発現の平均値／正常体重血糖例での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 LGALS12 was also evaluated using real-time PCR. The results further indicate that LGALS12 is significantly under-expressed in subcutaneous fat from diabetic individuals compared to subcutaneous fat from normal weight glycemic individuals.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in diabetic cases / average value of expression in normal body weight blood glucose cases. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した肥満患者での変化倍数を示す。 Probe set 223828 detects the LGALS12 nucleic acid sequence. In gene profiling experiments, LGALS12 transcript expression was lower in obese patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in obese patients compared to normal body weight blood glucose patients.

「変化倍数」は、肥満例での発現の平均値／正常体重血糖例での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 LGALS12 was also evaluated using real-time PCR. The results further indicate that LGALS12 is significantly under-expressed in subcutaneous fat from obese individuals compared to subcutaneous fat from normal weight glycemic individuals.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in obese cases / average value of expression in normal body weight blood glucose cases. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した肥満患者での変化倍数を示す。 Probe set 223828 detects the LGALS12 nucleic acid sequence. In gene profiling experiments, LGALS12 transcript expression was lower in obese patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in obese patients compared to normal body weight blood glucose patients.

「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、プロファイリングを行った他のすべての成人組織と比較した脂肪組織での変化倍数を示す。 Probe set 223828 detects the LGALS12 nucleic acid sequence. In gene profiling experiments, LGALS12 transcript expression was higher in adipose tissue than in all other adult tissues.

“Average expression” indicates the mean value of expression; “SEM” indicates the standard error of the average value; “n” indicates the number of patient samples; The fold change in adipose tissue compared to tissue is shown.

  LGALS12 contains the following protein domains (designated with reference to SEQ ID NO: 165): galactoside binding lectin (PF00337) at amino acids 48-182. LGALS12 is a member of the β-galactoside-binding lectin family and may be an apoptosis activator that negatively regulates cell cycle and cell proliferation (Yang RY et al., J Biol Chem., 276 (23): 20252-60 (2001); Hotta K. et al., J Biol Chem., 276 (36): 34089-97 (2001); Liu, FT, et al., Biochim Biophys Acta. 1572: 263 -73 (2002)).

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した糖尿病患者での変化倍数を示す。 NMB
Probe set 205204 detects the NMB nucleic acid sequence. In gene profiling experiments, NMB transcript expression was higher in diabetic patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in diabetic patients compared to normal weight blood glucose patients.

「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、プロファイリングを行った他のすべての成人組織と比較した脂肪組織での変化倍数を示す。 Probe set 205204 detects the NMB nucleic acid sequence. In gene profiling experiments, NMB transcript expression was higher in adipose tissue than in all other adult tissues.

“Average expression” indicates the mean value of expression; “SEM” indicates the standard error of the average value; “n” indicates the number of patient samples; The fold change in adipose tissue compared to tissue is shown.

「変化倍数」は、脂肪組織での発現の平均値／他の組織での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した成人組織試料の数を示す。 NMB was also evaluated using real-time PCR. The results further show that NMB is significantly over-expressed in subcutaneous fat from diabetic individuals compared to subcutaneous fat from normal weight glycemic individuals.

“Change fold” indicates the fold change calculated as the ratio of the average value of expression in adipose tissue / the average value of expression in other tissues. Numbers in parentheses indicate the number of adult tissue samples analyzed by real-time PCR.

  NMB contains the following protein domains (designated with reference to SEQ ID NO: 181): signal peptide at amino acids 1-26; and bombesin-like peptide (PF02044) at amino acids 47-60. Soluble and active secreted NMB has been detected and is presented in SEQ ID NO: 182. NMB is a bombesin-related neuropeptide that induces calcium flux and phosphatidylinositol turnover and activates cell proliferation through G protein-coupled neuromedin B receptors (Ohki-Hamazaki H. Prog. Neurobiol. 62 (3): 297 -312 (2000); Mason S. et al., Eur J Pharmacol., 438 (1-2): 25-34 (2002)).

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した糖尿病患者での変化倍数を示す。 NNAT
Probe set 204239 detects the NNAT nucleic acid sequence. In gene profiling experiments, NNAT transcript expression was lower in diabetic patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in diabetic patients compared to normal weight blood glucose patients.

「変化倍数」は、糖尿病例での発現の平均値／正常体重血糖例での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 NNAT was also evaluated using real-time PCR. The results further show that NNAT is significantly under-expressed in subcutaneous fat from diabetic individuals compared to subcutaneous fat from normal-weight glycemic individuals.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in diabetic cases / average value of expression in normal body weight blood glucose cases. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「Rosi前」および「Rosi後」は、試料をロシグリタゾン処理の前または24時間後に採取したことを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、rosi前の試料と比較したrosi後の初代ヒト脂肪細胞での変化倍数を示す。 Probe set 204239 detects the NNAT nucleic acid sequence. In gene profiling experiments, the expression of NNAT transcripts was higher in rosi than in primary human adipocytes treated with medium.

B / C indicates whether the sample is a basal sample or a clamp sample; “Pre-Rosi” and “Post-Rosi” indicate that the sample was taken before or 24 hours after rosiglitazone treatment; “Mean” indicates the mean expression; “SEM” indicates the standard error of the mean; “n” indicates the number of patient samples; “fold change” indicates the first post-rosi compared to the pre-rosi sample The fold change in human adipocytes is shown.

「変化倍数」は、rosiでの発現の平均値／媒質での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した初代ヒト脂肪細胞試料の数を示す。 NNAT was also evaluated using real-time PCR. The results further indicate that NNAT is significantly overexpressed in primary cultured human adipocytes treated with rosi compared to the medium.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in rosi / the average value of expression in medium. Numbers in parentheses indicate the number of primary human adipocyte samples analyzed by real-time PCR.

「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、プロファイリングを行った他のすべての成人組織と比較した脂肪組織での変化倍数を示す。 Probe set 204239 detects the NNAT nucleic acid sequence. In gene profiling experiments, NNAT transcript expression was higher in adipose tissue than in all other adult tissues.

“Average expression” indicates the mean value of expression; “SEM” indicates the standard error of the average value; “n” indicates the number of patient samples; The fold change in adipose tissue compared to tissue is shown.

  NNAT contains the following protein domains (designated with reference to SEQ ID NO: 188): signal peptide at amino acids 1-23; and one transmembrane domain (TMHMM2.0) at amino acids 13-35. NNAT is presumed to be proteolipid and may regulate ion channels during brain development. It has been found to be highly expressed in pituitary adenomas and is frequently hypermethylated in childhood myeloma and lymphocytic acute leukemia (Dou D. and Joseph R. Genomics 33 (2) : 292-7 (1996); Usui H. et al., J Mol Neurosci. 9 (1): 55-60 (1997); Evans HK et al., Genomics 77 (1-2): 99-104 (2001) )).

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した糖尿病の変化倍数を示す。 OLFM2
Probe set 223601 detects the OLFM2 nucleic acid sequence. In gene profiling experiments, the expression of OLFM2 transcripts was lower in diabetic patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in diabetes compared to normal weight blood glucose patients.

「変化倍数」は、糖尿病例での発現の平均値／正常体重血糖例での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 OLFM2 was also evaluated using real-time PCR. The results further show that OLFM2 is significantly under-expressed in subcutaneous fat from diabetic individuals compared to subcutaneous fat from normal weight blood glucose individuals.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in diabetic cases / average value of expression in normal body weight blood glucose cases. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、プロファイリングを行った他のすべての成人組織と比較した脂肪組織での変化倍数を示す。 Probe set 223601 detects the OLFM2 nucleic acid sequence. In gene profiling experiments, OLFM2 transcript expression was higher in adipose tissue than in all other adult tissues.

“Average expression” indicates the mean value of expression; “SEM” indicates the standard error of the average value; “n” indicates the number of patient samples; The fold change in adipose tissue compared to tissue is shown.

  OLFM2 is overexpressed in 3T3-L1 adipocytes and its effect on basal and insulin-induced glucose transport was evaluated.

説明：「Con」は、OLFM2を発現しない対照3T3-L1脂肪細胞を示す。「FC」は以下の比として定義される変化倍数を示す；OLFM2を発現する3T3-L1細胞におけるグルコース輸送／OLFM2を発現しない3T3-L1細胞におけるグルコース輸送。「h」はヒトである。「n」は実験の数である。SEMは平均値の標準誤差を示す。 Glucose transport analysis in 3T3-L1 adipocytes

Description: “Con” indicates control 3T3-L1 adipocytes that do not express OLFM2. “FC” indicates the fold change defined as the ratio: glucose transport in 3T3-L1 cells expressing OLFM2 / glucose transport in 3T3-L1 cells not expressing OLFM2. “H” is human. “N” is the number of experiments. SEM indicates the standard error of the average value.

  These results indicate that increasing the level of OLFM2 in cells such as adipocytes results in a corresponding decrease in glucose uptake. It is believed that reducing the level or activity of OLFM2 in tissues of insulin resistant or diabetic patients increases the ability of such tissues to take up glucose and therefore effective against insulin resistance and diabetes. Indicates that it is thought to bring a cure.

  OLFM2 contains the following protein domains (designated with reference to SEQ ID NO: 196): the signal peptide at amino acids 1-24; and the olfactedin-like domain (PF02191) at amino acids 196-446. Soluble and active secreted OLFM2 has been detected and is presented in SEQ ID NO: 197. OLFM2 is a protein that binds to myosin and has a high degree of similarity to olfactomedin 3 (rat Olfm3), which is known to be associated with glaucoma and diseases that affect the anterior segment and the retina (rat Olfm3). Ortego J. et al., FEBS Lett. 413 (2): 349-53 (1997).

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した肥満患者での変化倍数を示す。 OPN3
Probe set 219032 detects the OPN3 nucleic acid sequence. In gene profiling experiments, OPN3 transcript expression was higher in obese patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in obese patients compared to normal body weight blood glucose patients.

「変化倍数」は、肥満例での発現の平均値／正常体重血糖例での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 OPN3 was also evaluated using real-time PCR. The results further show that OPN3 is significantly over-expressed in subcutaneous fat from obese individuals compared to subcutaneous fat from normal-weight glycemic individuals.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in obese cases / average value of expression in normal body weight blood glucose cases. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

  OPN3 contains the following protein domains (designated with reference to SEQ ID NO: 203): 7 transmembrane receptors (rhodopsin family) (PF00001) at amino acids 58-309; and 7 transmembrane domains (TMHMM2 0.0) to amino acids 44-66, 78-100, 115-137, 158-180, 200-222, 258-280, 290-312. OPN3 is a member of the opsins receptor cluster of G protein-coupled receptors that appears to play a role in non-visual light processes such as circadian rhythm synchronization or pineal melatonin production (Blackshaw S. and Snyder SH, J Neurosci. 19 (10): 3681-90 (1999); Halford S. et al., Genomics 72 (2): 203-8 (2001)).

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した糖尿病の変化倍数を示す。 PTPRE
Probe set 221840 detects PTPRE nucleic acid sequences. In gene profiling experiments, PTPRE transcript expression was higher in diabetic patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in diabetes compared to normal weight blood glucose patients.

「変化倍数」は、糖尿病例での発現の平均値／正常体重血糖例での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 PTPRE was also evaluated using real-time PCR. The results further indicate that PTPRE is significantly over-expressed in subcutaneous fat from diabetic individuals compared to subcutaneous fat from normal-weight glycemic individuals.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in diabetic cases / average value of expression in normal body weight blood glucose cases. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

  PTPRE was overexpressed in 3T3-L1 adipocytes and the effects on basal and insulin-induced glucose transport and Glu4 translocation were evaluated.

説明：「変化倍数」は、以下の比を示す（hPTPRE発現3T3-L1脂肪細胞のうち細胞表面Glut4に関して陽性と判定された百分率の平均値）／（LacZ発現3T3-L1脂肪細胞のうち細胞表面Glut4に関して陽性と判定された百分率の平均値）。「h」はヒトである。「n」は実験の数である。 Glut4 migration analysis

Explanation: “fold change” indicates the following ratio (average percentage of hPTPRE-expressing 3T3-L1 adipocytes determined as positive for cell surface Glut4) / (cell surface of LacZ-expressing 3T3-L1 adipocytes) (Average of percentages tested positive for Glut4). “H” is human. “N” is the number of experiments.

  Increased levels of hPTPRE in 3T3-L1 adipocytes significantly inhibit insulin-induced Glu4 translocation to the cell surface.

説明：「Con」は、hPTPREを発現しない対照3T3-L1脂肪細胞を示す。「FC」は以下の比として定義される変化倍数を示す；hPTPREを発現する3T3-L1脂肪細胞におけるグルコース輸送／PTPREを発現しない3T3-L1脂肪細胞におけるグルコース輸送。「h」はヒトである。「n」は実験の数である。SEMは平均値の標準誤差である。 Glucose transport analysis in 3T3-L1 adipocytes

Description: “Con” indicates control 3T3-L1 adipocytes that do not express hPTPRE. “FC” indicates the fold change defined as the ratio: glucose transport in 3T3-L1 adipocytes expressing hPTPRE / glucose transport in 3T3-L1 adipocytes not expressing PTPRE. “H” is human. “N” is the number of experiments. SEM is the standard error of the mean value.

  These results indicate that increasing the level of PTPRE in cells such as adipocytes results in a corresponding decrease in glucose uptake. It is believed that reducing the level or activity of PTPRE in tissues of insulin resistant or diabetic patients increases the ability of such tissues to take up glucose and therefore effective against insulin resistance and diabetes. Indicates that it is thought to bring a cure.

  PTPRE contains the following protein domains (designated with reference to SEQ ID NO: 213): signal peptide at amino acids 1-19; protein tyrosine phosphatase (PF00102) at amino acids 159-393, 451-688; and 1 One transmembrane domain (TMHMM2.0) at amino acids 47-69. PTPRE has been found to exist in both soluble, cytoplasmic and transmembrane forms. PTPRE is induced in astrocytoma cells by IL1 and TNFA treatment, suggesting a role in the inflammatory response of the brain (Schumann, G. et al., Brain Res Mol Brain Res. 62: 56-64 (1988 )). Other studies suggest a role for PTPRE in RAS-related signaling pathways, cytokine-induced signaling, voltage-gated K + channel activation, and angiogenesis and angiogenesis (Tiran, ZJ et al., J Biol Chem. 278: 17509-14 (2003); Toledano-Katchalski, H., et al., Mol Cancer Res. 1: 541-50 (2003); Thompson, LJ, et al., Am J Physiol Heart Circ Physiol. 281: H396-403 (2001)).

  PTPRE can dephosphorylate tyrosine-phosphorylated insulin receptors (see, eg, Nakagawa Y. et al. Zoolog Sci. 22: 169-75 (2005)). Thus, exemplary assays for inhibitors of PTPRE determine the ability of a candidate compound to inhibit the ability of purified PTPRE to dephosphorylate tyrosine phosphorylated peptides, eg, tyrosine phosphorylated peptides derived from the insulin receptor sequence. And including autophosphorylation sites such as tyrosine 11158, 1162, and 1163.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した糖尿病患者での変化倍数を示す。 RDC1
Probe set 212977 detects the RDC1 nucleic acid sequence. In gene profiling experiments, RDC1 transcript expression was lower in diabetic patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in diabetic patients compared to normal weight blood glucose patients.

「変化倍数」は、糖尿病例での発現の平均値／正常体重血糖例での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 RDC1 was also evaluated using real-time PCR. The results further indicate that RDC1 is significantly under-expressed in subcutaneous fat from diabetic individuals compared to subcutaneous fat from normal-weight glycemic individuals.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in diabetic cases / average value of expression in normal body weight blood glucose cases. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「相関係数」は、グルコース処理速度（Rd）とシグナル強度との関係を示す。正の相関係数はインスリン抵抗性の増大に伴う遺伝子のダウンレギュレーションを示し、負の相関係数はアップレギュレーションを示す。「n」は患者試料の数を示す。 Probe set 212977 detects the RDC1 nucleic acid sequence. In gene profiling experiments, RDC1 transcript expression was lower in patients with insulin resistance than in normal patients.

B / C indicates whether the sample is a basal sample or a clamped sample; “correlation coefficient” indicates the relationship between glucose processing rate (Rd) and signal intensity. A positive correlation coefficient indicates gene down-regulation with increasing insulin resistance, and a negative correlation coefficient indicates up-regulation. “N” indicates the number of patient samples.

「相関係数」は、Rdとシグナル強度との関係を示す。正の相関係数はインスリン抵抗性の増大に伴う遺伝子のダウンレギュレーションを示し、負の相関係数はアップレギュレーションを示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 RDC1 was also evaluated using real-time PCR. The results further indicate that RDC1 is significantly reduced in insulin resistant patients compared to normal patients.

“Correlation coefficient” indicates the relationship between Rd and signal intensity. A positive correlation coefficient indicates gene down-regulation with increasing insulin resistance, and a negative correlation coefficient indicates up-regulation. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した肥満患者での変化倍数を示す。 Probe set 212977 detects the RDC1 nucleic acid sequence. In gene profiling experiments, RDC1 transcript expression was higher in obese patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in obese patients compared to normal body weight blood glucose patients.

「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、プロファイリングを行った他のすべての成人組織と比較した脂肪組織での変化倍数を示す。 Probe set 212977 detects the RDC1 nucleic acid sequence. In gene profiling experiments, RDC1 transcript expression was higher in adipose tissue than in all other adult tissues.

“Average expression” indicates the mean value of expression; “SEM” indicates the standard error of the average value; “n” indicates the number of patient samples; The fold change in adipose tissue compared to tissue is shown.

  RDC1 contains the following protein domains (designated with reference to SEQ ID NO: 223): 7 transmembrane receptors (rhodopsin family) (PF00001) at amino acids 61-315; and 7 transmembrane domains (TMHMM2 0.0) to amino acids 47-69, 82-104, 119-140, 160-182, 214-236, 255-277, 297-319. RDC1 is considered to be a new member of the rhodopsin family of G protein coupled receptors. This protein is a human immunodeficiency virus (HIV) co-receptor. In lipomas, translocations involving this gene and HMGA2 on chromosome 12 have been observed (Broberg, K., et al., Int J Oncol. 21: 321-6 (2002); Shimizu, N., et al. al., J Virol. 74: 619-26 (2000)).

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した肥満患者での変化倍数を示す。 SLIT2
Probe set 209897 detects the SLIT2 nucleic acid sequence. In gene profiling experiments, SLIT2 transcript expression was higher in obese patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in obese patients compared to normal body weight blood glucose patients.

「変化倍数」は、肥満例での発現の平均値／正常体重血糖例での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 SLIT2 was also evaluated using real-time PCR. The results further indicate that SLIT2 is significantly over-expressed in subcutaneous fat from obese individuals compared to subcutaneous fat from normal weight glycemic individuals.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in obese cases / average value of expression in normal body weight blood glucose cases. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

  SLIT2 cellular levels were reduced in 3T3-L1 adipocytes using siRNA targeting SLIT2, and the effect on basal and insulin-induced glucose transport was assessed.

説明：「siRNA」は、マウスSLIT2を標的とするDharmacon社のSmartpool siRNAオリゴヌクレオチドを示す。「Scr」は、Dharmacon社のScramble siRNA対照を示す。「FC」は以下の比として定義される変化倍数を示す；SLIT2 siRNAがトランスフェクトされた3T3-L1脂肪細胞におけるSLIT2 mRNAのレベル／Scramble siRNAがトランスフェクトされた3T3-L1脂肪細胞におけるSLIT2 mRNAのレベル。「n」は実験の数である。SEMは平均値の標準誤差である。 SLIT2 mRNA levels in 3T3-L1 adipocytes transfected with siRNA oligonucleotides

Description: “siRNA” refers to the Dharmacon Smartpool siRNA oligonucleotide targeting mouse SLIT2. “Scr” represents the Dharmacon Scramble siRNA control. “FC” indicates the fold change defined as the ratio: SLIT2 mRNA levels in 3T3-L1 adipocytes transfected with SLIT2 siRNA / SLIT2 mRNA in 3T3-L1 adipocytes transfected with Scramble siRNA level. “N” is the number of experiments. SEM is the standard error of the mean value.

説明：「siRNA」は、マウスSLIT2を標的とするDharmacon社のSmartpool siRNAオリゴヌクレオチドを示す。「Scr」は、Dharmacon社のScramble siRNA対照オリゴヌクレオチドを示す。「FC」は以下の比として定義される変化倍数を示す；SLIT2 siRNAがトランスフェクトされた3T3-L1脂肪細胞におけるグルコース輸送／Scramble siRNAがトランスフェクトされた3T3-L1脂肪細胞におけるグルコース輸送。「n」は実験の数である。SEMは平均値の標準誤差である。 Glucose transport in 3T3-L1 adipocytes transfected with siRNA oligonucleotides

Description: “siRNA” refers to the Dharmacon Smartpool siRNA oligonucleotide targeting mouse SLIT2. “Scr” indicates a Dharmacon Scramble siRNA control oligonucleotide. “FC” indicates the fold change defined as the ratio: glucose transport in 3T3-L1 adipocytes transfected with SLIT2 siRNA / glucose transport in 3T3-L1 adipocytes transfected with Scramble siRNA. “N” is the number of experiments. SEM is the standard error of the mean value.

  These results indicate that reducing the level of SLIT2 in cells such as adipocytes results in a corresponding decrease in glucose uptake. It is believed that increasing the level or activity of SLIT2 in tissues of insulin resistant or diabetic patients enhances the ability of such tissues to take up glucose and hence effective treatment for insulin resistance and diabetes It shows that it is thought to bring the law.

  SLIT2 contains the following protein domains (designated with reference to SEQ ID NO: 229): signal peptide at amino acids 1-30; leucine rich repeat C-terminal domain (PF01463) at amino acids 233-258, 454-479, 688 To 713, 883 to 908; leucine rich repeat N-terminal domain (PF01462) to amino acids 27 to 54, 272 to 299, 505 to 532, 726 to 753; leucine rich repeat (PF00560) to amino acids 56 to 79, 128 to 151, 176-199, 325-348, 349-372, 559-582, 607-630, 778-801, 802-825, 826-849; laminin G domain (PF00054) at amino acids 1188-1319; and EGF-like domain (PF00008) to amino acids 922-954, 961-995, 1002-1033, 1040-173, 1080-1111, 1125-1156, 1336-1367, 1375-1406, 1416-1447. A soluble and active secreted SLIT2 has been detected (Nguyen Ba-Charvet, KT et al., J. Neurosc., 21: 4281-4289 (2001)) and is presented in SEQ ID NO: 230 ing. SLIT2 is a ligand for the roundabout receptor ROBO1. Mammalian SLIT proteins appear to be involved in the formation and maintenance of the nervous and endocrine systems through protein-protein interactions. SLIT2 has been reported to be a repellent chemical for in vivo nerve cell migration that induces dorsal root ganglion axonal branching (Nguyen Ba-Charvet KT et al., J Neurosci., 21 (12): 4281-9 (2001); Nguyen Ba-Charvet KT et al., J Physiol Paris. 96: 91-8 (2002)), SLIT2 is also leukocyte chemotaxis induced by chemotactic factors. (Wu, JY, et al., Nature 410: 948-52 (2001)).

  The human SLIT2 gene has been cloned and a proximal promoter has been identified (eg, Dallol A. et al. Cancer Res. 62: 5874-80 (2002)). For this reason, an example of the screening method regarding a SLIT2 regulator is as follows. The SLIT2 promoter is inserted upstream of a reporter gene such as β-galactosidase and expressed in cells. Thus, compounds that upregulate promoter activity can be identified by measuring an increase in β-galactosidase activity.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した肥満患者での変化倍数を示す。 TNFRSF21
Probe set 214581 detects the TNFRSF21 nucleic acid sequence. In gene profiling experiments, TNFRSF21 transcript expression was higher in obese patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in obese patients compared to normal body weight blood glucose patients.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「Rosi前」および「Rosi後」は、試料をロシグリタゾン処理の前または24時間後に採取したことを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、rosi前の試料と比較したrosi後の初代ヒト脂肪細胞での変化倍数を示す。 Probe set 214581 detects the TNFRSF21 nucleic acid sequence. In gene profiling experiments, TNFRSF21 transcript expression was higher in rosi than in primary human adipocytes treated with medium.

B / C indicates whether the sample is a basal sample or a clamp sample; “Pre-Rosi” and “Post-Rosi” indicate that the sample was taken before or 24 hours after rosiglitazone treatment; “Mean” indicates the mean expression; “SEM” indicates the standard error of the mean; “n” indicates the number of patient samples; “fold change” indicates the first post-rosi compared to the pre-rosi sample The fold change in human adipocytes is shown.

「変化倍数」は、rosiでの発現の平均値／媒質での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した初代ヒト脂肪細胞試料の数を示す。 TNFRSF21 was also evaluated using real-time PCR. The results further show that TNFRSF21 is significantly overexpressed in primary cultured human adipocytes treated with rosi compared to the medium.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in rosi / the average value of expression in medium. Numbers in parentheses indicate the number of primary human adipocyte samples analyzed by real-time PCR.

「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、プロファイリングを行った他のすべての成人組織と比較した脂肪組織での変化倍数を示す。 Probe set 214581 detects the TNFRSF21 nucleic acid sequence. In gene profiling experiments, TNFRSF21 transcript expression was higher in adipose tissue than in all other adult tissues.

“Average expression” indicates the mean value of expression; “SEM” indicates the standard error of the average value; “n” indicates the number of patient samples; The fold change in adipose tissue compared to tissue is shown.

「変化倍数」は、脂肪組織での発現の平均値／他の組織での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した成人組織試料の数を示す。 TNFRSF21 was also evaluated using real-time PCR. The results further indicate that TNFRSF21 is significantly overexpressed in adipose tissue compared to all other adult tissues.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in adipose tissue / average value of expression in other tissues. Numbers in parentheses indicate the number of adult tissue samples analyzed by real-time PCR.

  TNFRSF21 was overexpressed in 3T3-L1 adipocytes and the effect on basal and insulin-induced glucose transport was evaluated.

説明：「Con」は、hTNFRSF21を発現しない対照3T3-L1脂肪細胞を示す。「FC」は以下の比として定義される変化倍数を示す；hTNFRSF21発現細胞におけるグルコース輸送／TNFRSF21を発現しない細胞におけるグルコース輸送。「h」はヒトである。「n」は実験の数である。SEMは平均値の標準誤差である。 Glucose transport

Description: “Con” indicates control 3T3-L1 adipocytes that do not express hTNFRSF21. “FC” indicates the fold change defined as the ratio: glucose transport in hTNFRSF21 expressing cells / glucose transport in cells not expressing TNFRSF21. “H” is human. “N” is the number of experiments. SEM is the standard error of the mean value.

  These results indicate that increasing TNFRSF21 levels in cells such as adipocytes results in a corresponding decrease in glucose uptake. This suggests that reducing the level or activity of TNFRSF21 in tissues of insulin resistant or diabetic patients will increase the ability of such tissues to take up glucose and hence effective against insulin resistance and diabetes. Indicates that it is thought to bring a cure.

  TNFRSF21 contains the following protein domains (designated with reference to SEQ ID NO: 236): signal peptide at amino acids 1-41; Death domain (PF00531) at amino acids 416-498; TNFR / NGFR cysteine rich region (PF00020 ) To amino acids 50-88, 91-131, 133-168, 171-211; and one transmembrane domain (TMHMM2.0) to amino acids 350-369. TNFRSF21 has been shown to activate NF-κB and MAPK8 / JNK and induce cell apoptosis. This receptor interacts with its TRADD protein via its death domain, which is known to act as an adapter that mediates TNF-receptor signaling. Knockout studies in mice suggest that this gene plays a role in helper T cell activation, which may be involved in inflammation and immune regulation (Pan G. et al., FEBS Lett. 431 (3): 351-356 (1998); Kasof GM et al., Oncogene 20 (55): 7965-7975 (2001)).

  TNFRSF21 is upregulated by TNFα and is itself associated with an insulin resistance state (eg, Hotamisligil GS. J Intern Med. 245: 621-625 (1999); Kasof GM et al. Oncogene. 20: 7965-7975 (2001)). Thus, exemplary assays for identifying compounds that inhibit TNFRSF21 include cells such as LnCAP to screen candidate compounds for their ability to inhibit TNFRSF21 mRNA or protein upregulation induced by TNFα. Can be used.

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した糖尿病患者での変化倍数を示す。 TNFSF13B
Probe set 223501 detects the TNFSF13B nucleic acid sequence. In gene profiling experiments, TNFSF13B transcript expression was higher in diabetic patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in diabetic patients compared to normal weight blood glucose patients.

「変化倍数」は、糖尿病例での発現の平均値／正常体重血糖例での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 TNFSF13B was also evaluated using real-time PCR. The results further indicate that TNFSF13B is significantly over-expressed in subcutaneous fat from diabetic individuals compared to subcutaneous fat from normoglycemic individuals.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in diabetic cases / average value of expression in normal body weight blood glucose cases. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

  TNFSF13B includes the following protein domains (designated with reference to SEQ ID NO: 242): signal anchor at amino acids 0-0; TNF (tumor necrosis factor) family (PF00229) at amino acids 166-284; and 1 One transmembrane domain (TMHMM2.0) to amino acids 48-70. A soluble and active secreted TNFSF13B has been detected (Schneider, P. et al., J Exp. Med., 189: 1747-1756 (1999)), which is presented in SEQ ID NO: 243. Yes. TNFSF13B is a ligand for a number of receptors including TNFRSF13B / TACI, TNFRSF17 / BCMA and TNFRSF13C / BAFFR. This cytokine is expressed in B cell lineage cells and acts as a potent B cell activator. It is also known to play an important role in B cell proliferation and differentiation (Patke, A., et al., Curr Opin Immunol. 16: 251-5 (2004); Schneider, P. and Tschopp, J. Immunol Lett. 88: 57-62 (2003)).

「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、プロファイリングを行った他のすべての成人組織と比較した大網組織での変化倍数を示す。 TNFSF14
Probe set 207907 detects the TNFSF14 nucleic acid sequence. In gene profiling experiments, TNFSF14 transcript expression was higher in omentum tissue than in all other adult tissues.

“Average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the number of patient samples; The fold change in the greater omentum compared to the organization is shown.

「変化倍数」は、大網組織での発現の平均値／他の組織での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した成人組織試料の数を示す。 TNFSF14 was also evaluated using real-time PCR. The results further indicate that TNFSF14 is significantly overexpressed in omental tissue compared to all other adult tissues.

“Change multiple” indicates the multiple of change calculated as a ratio of the average value of the expression in the omentum tissue / the average value of the expression in other tissues. Numbers in parentheses indicate the number of adult tissue samples analyzed by real-time PCR.

  TNFSF14 comprises the following protein domains (designated with reference to SEQ ID NO: 251): signal anchor at amino acids 0-0; TNF (tumor necrosis factor) family (PF00229) at amino acids 93-240; and 1 One transmembrane domain (TMHMM2.0) at amino acids 36-58. Soluble and active secreted TNFSF14 has been detected (Harrop, J.A., et al., J Biol Chem. 273: 27548-56 (1998)) and is presented in SEQ ID NO: 252. TNFSF14 is a ligand for HVEM (TNFRSF14) and has been reported to induce lymphocyte proliferation, induce apoptosis, suppress in vivo tumorigenesis, and promote herpesvirus entry (Mauri DN et al. , Immunity 8 (1): 21-30 (1998); Zhai Y. et al., J Clin Invest. 102 (6): 1142-51 (1998); Castellano, R. et al., J Biol Chem. 277 (45): 42841-51 (2001)).

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した肥満患者での変化倍数を示す。 TPSB2
Probe set 205683 detects the TPSB2 nucleic acid sequence. In gene profiling experiments, TPSB2 transcript expression was higher in obese patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in obese patients compared to normal body weight blood glucose patients.

「変化倍数」は、肥満例での発現の平均値／正常体重血糖例での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 TPSB2 was also evaluated using real-time PCR. The results further show that TPSB2 is significantly over-expressed in subcutaneous fat from obese individuals compared to subcutaneous fat from normal weight glycemic individuals.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in obese cases / average value of expression in normal body weight blood glucose cases. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、プロファイリングを行った他のすべての成人組織と比較した脂肪組織での変化倍数を示す。 Probe set 205683 detects the TPSB2 nucleic acid sequence. In gene profiling experiments, TPSB2 transcript expression was higher in adipose tissue than in all other adult tissues.

“Average expression” indicates the mean value of expression; “SEM” indicates the standard error of the average value; “n” indicates the number of patient samples; The fold change in adipose tissue compared to tissue is shown.

  TPSB2 contains the following protein domains (designated with reference to SEQ ID NO: 262): signal peptide at amino acids 1-18; and trypsin (PF00089) at amino acids 31-267. A soluble and active secreted TPSB2 has been detected and is presented in SEQ ID NO: 263. TPSB2 is tryptase β2 and belongs to the family of mast cell serine proteases that are considered mediators in the pathogenesis of asthma and other allergic and inflammatory diseases. β-tryptase appears to be the major isozyme expressed in mast cells (Pallaoro M. et al, J Biol Chem., 274 (6): 3355-62 (1999).

B／Cは試料が基礎試料またはクランプ試料のいずれかであるかを示す；「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、正常体重血糖患者と比較した肥満患者での変化倍数を示す。 WISP2
Probe set 205792 detects the WISP2 nucleic acid sequence. In gene profiling experiments, WISP2 transcript expression was higher in obese patients than in normoglycemic patients.

B / C indicates whether the sample is a basal sample or a clamp sample; “average expression” indicates the average value of expression; “SEM” indicates the standard error of the average value; “n” indicates the patient sample The “fold change” indicates the fold change in obese patients compared to normal body weight blood glucose patients.

「変化倍数」は、肥満例での発現の平均値／正常体重血糖例での発現の平均値という比として算出した変化倍数を示す。括弧内の数字は、リアルタイムPCRによって分析した患者試料の数を示す。 WISP2 was also evaluated using real-time PCR. The results further show that WISP2 is significantly over-expressed in subcutaneous fat from obese individuals compared to subcutaneous fat from normal-weight glycemic individuals.

“Change fold” indicates the fold change calculated as a ratio of the average value of expression in obese cases / average value of expression in normal body weight blood glucose cases. Numbers in parentheses indicate the number of patient samples analyzed by real-time PCR.

「発現平均」は発現の平均値を示す；「SEM」は平均値の標準誤差を示す；「n」は患者試料の数を示す；「変化倍数」は、プロファイリングを行った他のすべての成人組織と比較した脂肪組織での変化倍数を示す。 Probe set 205792 detects the WISP2 nucleic acid sequence. In gene profiling experiments, WISP2 transcript expression was higher in adipose tissue than in all other adult tissues.

“Average expression” indicates the mean value of expression; “SEM” indicates the standard error of the average value; “n” indicates the number of patient samples; The fold change in adipose tissue compared to tissue is shown.

WISP2 contains the following protein domains (designated with reference to SEQ ID NO: 269): signal peptide at amino acids 1-23; insulin-like growth factor binding protein (PF00219) at amino acids 26-96; von Willebrand factor The C-type domain (PF00093) at amino acids 100-163; the thrombospondin type 1 domain (PF00090) at amino acids 196-237; and the TNFR / NGFR cysteine-rich region (PF00020) at amino acids 39-70. A soluble and active secreted form of WISP2 has been detected (Pennica, D., et al., Proc Natl Acad Sci USA. 95: 14717-22 (1998)), which is presented in SEQ ID NO: 270. ing. WISP2 is a member of the CCN family of growth factors and has been found to promote osteoblast adhesion. Decreased expression of WISP2 may have therapeutic effects in the treatment of breast cancer (Kumar S. et al., J Biol Chem., 274 (24): 17123-31 (1999); Pennica D. et al., Proc Natl Acad Sci. USA. 95 (25): 14717-22 (1998)). WISP2 has also been found to suppress vascular smooth muscle cell proliferation, motility and invasiveness (Lake, AC et al., Am J Pathol. 162: 219-31 (2003)).

Claims (9)

A method for identifying an agent for treating an obese, diabetic or prediabetic individual comprising:
(I) SEQ ID NO: 213, 2 when hybridization in 50% formamide, 5 × SSC and 1% SDS at 42 ° C. followed by washing in 0.2 × SSC and 0.1% SDS at 55 ° C. , 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 35, 37, 39, 40, 42, 44, 46, 47, 49 , 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98 , 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148 , 150, 152, 154, 156, 158, 160, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 182, 184, 186, 188, 190, 192, 194, 196 , 197, 199, 201, 203, 205, 207, 209, 211, 215, 217, 219, 221, 223, 225, 227, 229, 230, 232, 234, 236, 238, 240, 242, 243, 245 247, 249, 251, 252, 254, 256, 258, 260, 262, 263, 265, 267, 269, 270, 272 Contacting the agent with a polypeptide encoded by a polynucleotide that hybridizes to a nucleic acid encoding 274; and (ii) modulating or binding to the polypeptide expression or activity Selecting an agent, thereby identifying the agent for treating an obese, diabetic or pre-diabetic individual;
Including methods.   The polypeptide has SEQ ID NO: 213, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 35, 37, 39, 40, 42, 44, 46, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 182, 184, 186, 188, 190, 192, 194, 196, 197, 199, 201, 203, 205, 207, 209, 211, 215, 217, 219, 221, 223, 225, 227, 229, 230, 232, 234, 236, 238, 240, 242, 243, 245, 247, 249, 251, 252, 254, 256, 258, 260, 262, 263, 265, 267, 269, 270, 272, 274 or its protein domain 2. The method of claim 1, comprising an amino acid sequence that is at least 95% identical.   2. The method of claim 1, further comprising detecting whether the selected agent alters body weight and / or obesity.   2. The method of claim 1, further comprising detecting whether the selected agent alters insulin sensitivity.   2. The method of claim 1, wherein step (ii) comprises selecting an agent that alters the expression of the polypeptide.   2. The method of claim 1, wherein step (ii) comprises selecting an agent that alters the activity of the polypeptide.   2. The method of claim 1, wherein step (ii) comprises selecting an agent that specifically binds to the polypeptide.   2. The method of claim 1, wherein the polypeptide is expressed intracellularly and the cell is contacted with an agent.   The polypeptide has SEQ ID NO: 213, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 35, 37, 39, 40, 42, 44, 46, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 182, 184, 186, 188, 190, 192, 194, 196, 197, 199, 201, 203, 205, 207, 209, 211, 215, 217, 219, 221, 223, 225, 227, 229, 230, 232, 234, 236, 238, 240, 242, 243, 245, 247, 249, 251, 252, 254, 256, 258, 260, 262, 263, 265, 267, 269, 270, 272 or 274 the method of.