WO2006017171A2 - Methodes de diagnostic et de traitement de l'obesite, du diabete et de l'insulinoresistance - Google Patents

Methodes de diagnostic et de traitement de l'obesite, du diabete et de l'insulinoresistance Download PDF

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WO2006017171A2
WO2006017171A2 PCT/US2005/024256 US2005024256W WO2006017171A2 WO 2006017171 A2 WO2006017171 A2 WO 2006017171A2 US 2005024256 W US2005024256 W US 2005024256W WO 2006017171 A2 WO2006017171 A2 WO 2006017171A2
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expression
polypeptide
indicates
mean
protein
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PCT/US2005/024256
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WO2006017171A3 (fr
Inventor
Shonna A. Moodie
Fang Zhang
Paul G. Rack
Jin Shang
Daniel M. Joo
Chi-Wai Wong
Francine Gregoire
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Metabolex, Inc.
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Priority to JP2007521515A priority Critical patent/JP2008506949A/ja
Publication of WO2006017171A2 publication Critical patent/WO2006017171A2/fr
Publication of WO2006017171A3 publication Critical patent/WO2006017171A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/426—1,3-Thiazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/044—Hyperlipemia or hypolipemia, e.g. dyslipidaemia, obesity

Definitions

  • Obesity has reached epidemic proportions globally with more than 1 billion adults overweight- at least 300 million of them clinical obese- and is a major contributor to the global burden of chronic disease and disability.
  • Overweight and obesity leads to adverse metabolic effect on blood pressure, cholesterol, triglycerides and insulin resistance.
  • the non-fatal but debilitating health problems associated with obesity include respiratory difficulties, chronic musculoskeletal problems, skin problems and infertility.
  • the more life-threatening problems fall into four main areas: cardiovascular disease problems, conditions associated with insulin resistance such as Type 2 diabetes, certain types of cancers especially the hormonally related and large-bowel cancers, and gall bladder disease.
  • Type 2 diabetes and hypertension rises steeply with increasing body fatness.
  • Weight reduction leads to correction of a number of obesity- associated endocrine and metabolic disorders.
  • Effective weight management for individuals and groups at risk of developing obesity involves a range of long term strategies. These include prevention, weight maintenance, management of co-morbidities and weight loss.
  • Existing treatment strategies include calorific restriction programs, surgery (gastric stapling) and drug intervention.
  • the currently available anti-obesity drugs can be divided into two classes: central acting and peripheral acting. Three marketed drugs are Xenical (Orlistat), Merida (Sibutramine) and Adipex-P (Phentermine).
  • Xenical is a non-systemic acting GI lipase inhibitor which is indicated for short and long term obesity management. Merida reduces food intake by re ⁇ uptake inhibition of primarily norepinephrine and serotonin.
  • Adipex-P is a phenteramine with sympathomimetic activities and suppresses appetite. It is indicated only for short term use. A more drastic solution to permanent weight loss is surgery and a gastric by-pass which limits absorption of calories through massive reduction in stomach size. [04] Carrying extra body weight and body fat go hand and hand with the development of diabetes. People who are overweight (BMI greater than 25) are at a much greater risk of developing type 2 diabetes than normal weight individuals. Almost 90% of people with type 2 diabetes are overweight. [05] Diabetes mellitus can be divided into two clinical syndromes, Type 1 and Type 2 diabetes mellitus.
  • Type 1 diabetes mellitus is a chronic autoimmune disease characterized by the extensive loss of beta cells in the pancreatic Islets of Langerhans, which produce insulin. As these cells are progressively destroyed, the amount of secreted insulin decreases, eventually leading to hyperglycemia (abnormally high level of glucose in the blood) when the amount of secreted insulin drops below the level required for euglycemia (normal blood glucose level).
  • hyperglycemia abnormally high level of glucose in the blood
  • euglycemia normal blood glucose level
  • Type 2 diabetes also referred to as non-insulin dependent diabetes mellitus (NIDDM)
  • NIDDM non-insulin dependent diabetes mellitus
  • This failure to respond may be due to reduced numbers of insulin receptors on these cells, or a dysfunction of signaling pathways within the cells, or both.
  • the beta cells initially compensate for this insulin resistance by increasing insulin output. Over time, these cells become unable to produce enough insulin to maintain normal glucose levels, indicating progression to Type 2 diabetes.
  • Type 2 diabetes is brought on by a combination of genetic and acquired risk factors - including a high-fat diet, lack of exercise, and aging. Worldwide, Type 2 diabetes has become an epidemic, driven by increases in obesity and a sedentary lifestyle, widespread adoption of western dietary habits, and the general aging of the population in many countries. In 1985, an estimated 30 million people worldwide had diabetes ⁇ by 2000, this figure had increased 5-fold, to an estimated 154 million people. The number of people with diabetes is expected to double between now and 2025, to about 300 million. [08] Type 2 diabetes is a complex disease characterized by defects in glucose and lipid metabolism.
  • TZD thiazolidinedione
  • the principal effect of these drugs is to improve glucose homeostasis.
  • PPAR gamma The molecular target of TZDs is a member of the PPAR family of ligand-activated transcription factors called PPAR gamma.
  • This transcription factor is highly expressed in adipose tissue with much lower levels being observed in muscle. Binding of TZDs to PPAR gamma in target cells and tissues such as fat and muscle brings about a change in gene expression. The link between TZD-altered gene expression in fat and muscle and increased insulin sensitivity is unknown.
  • the present invention addresses this and other problems.
  • the present invention provides methods for identifying an agent for treating an obese, diabetic or pre-diabetic individual.
  • the method comprises the steps of: (i) contacting an agent to a polypeptide encoded by a polynucleotide that is substantially identical to or hybridizes to a nucleic acid encoding a polypeptide listed in Table 1 under hybridization conditions of 50% formamide, 5X SSC, and 1% SDS at 42 0 C followed by a wash in 0.2X SSC, and 0.1% SDS at 55 0 C, wherein the polypeptide optionally has the activity listed in Table 1; and (ii) selecting an agent that modulates the expression or activity of the polypeptide or that binds to the polypeptide, thereby identifying an agent for treating an obese, diabetic or pre-diabetic individual.
  • Table 1 List of Polypeptides, SEQ ID numbers and Proposed Activity
  • the polypeptide comprises an amino acid sequence at least 95% identical to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129 or a protein domain thereof.
  • the method further comprises detecting whether the selected agent modulates weight and/or obesity. In some embodiments, the method further comprises detecting whether the selected agent modulates insulin sensitivity. [13] In some embodiments, step (ii) comprises selecting an agent that modulates expression of the polypeptide. In some embodiments, step (ii) comprises selecting an agent that modulates the activity of the polypeptide. In some embodiments, step (ii) comprises selecting an agent that specifically binds to the polypeptide.
  • the polypeptide is expressed in a cell and the cell is contacted with the agent.
  • the polypeptide comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127 or 129.
  • the present invention also provides methods of reducing body weight in an animal.
  • the methods comprise administering to the animal an effective amount of an agent that modulates the activity or expression of cell and the cell is contacted with the agent,
  • the polypeptide comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127 or 129.
  • the agent is selected by a method comprising (i) contacting an agent to a mixture comprising a polypeptide encoded by a polynucleotide substantially identical to, or that hybridizes to, a nucleic acid encoding a polypeptide listed in Table 1 under hybridization conditions of 50% formamide, 5X SSC, and 1% SDS at 42 0 C followed by a wash in 0.2X SSC, and 0.1% SDS at 55 0 C, wherein the polypeptide optionally has the activity listed in Table 1 ; and (ii) selecting an agent that modulates the expression or activity of the polypeptide or that binds to the polypeptide.
  • the agent is an antibody.
  • the antibody is a monoclonal antibody, hi some embodiments, the animal is a human.
  • the present invention also provides methods of treating a diabetic or pre-diabetic animal.
  • the method comprising administering to the animal a therapeutically effective amount of an agent that modulates the activity or expression of cell and the cell is contacted with the agent.
  • the polypeptide comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127 or 129.
  • the polypeptide comprises an amino acid sequence at least 95% identical to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129 or a protein domain thereof.
  • the agent is selected by a method comprising (i) contacting an agent to a mixture comprising a polypeptide encoded by a polynucleotide that hybridizes to a nucleic acid encoding cell and the cell is contacted with the agent.
  • the polypeptide comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127 or 129 in 50% formamide, 5X SSC, and 1% SDS at 42 0 C followed by a wash in 0.2X SSC, and 0.1% SDS at 55 0 C; and (ii) selecting an agent that modulates the expression or activity of the polypeptide or that binds to the polypeptide.
  • the agent is an antibody.
  • the antibody is a monoclonal antibody
  • the animal is a human.
  • the present invention also provides methods of introducing an expression cassette into a cell.
  • the methods comprise introducing into the cell an expression cassette comprising a promoter operably linked to a polynucleotide encoding a polypeptide, wherein the polynucleotide is substantially identical to or hybridizes to a nucleic acid encoding a polypeptide listed in Table 1 under hybridization conditions of 50% formamide, 5X SSC, and 1% SDS at 42 0 C followed by a wash in 0.2X SSC, and 0.1% SDS at 55 0 C, and the polypeptide optionally has the activity listed in Table 1.
  • the polypeptide comprises an amino acid sequence at least 95% identical to
  • polypeptide is expressed in a cell and the cell is contacted with the agent.
  • the polypeptide comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127 or 129.
  • the cell is selected from the group consisting of an adipocyte and a skeletal muscle cell.
  • the method further comprises introducing the cell into a human.
  • the human is obese, hi some embodiments, the human is diabetic. In some embodiments, the human is prediabetic. In some embodiments, the cell is from the human.
  • the present invention also provides methods of diagnosing an individual who has obesity, Type 2 diabetes or has a predisposition for diabetes or obesity, hi some embodiments, the method comprises detecting in a sample from the individual the level of a polypeptide or the level of a polynucleotide encoding the polypeptide, wherein the polynucleotide is substantially identical to or hybridizes to a nucleic acid encoding a polypeptide listed in Table 1 under hybridization conditions of 50% formamide, 5X SSC, and 1% SDS at 42 0 C followed by a wash in 0.2X SSC, and 0.1% SDS at 55 0 C, wherein a modulated level of the polypeptide or polynucleotide in the sample compared to a level of the polypeptide or polynucleotide in either a lean individual or a previous sample from the individual indicates that the individual is obese or diabetic or has a predisposition for diabetes or obesity.
  • the detecting step comprises contacting the sample with an antibody that specifically binds to the polypeptide.
  • the amino acid sequence is expressed in a cell and the cell is contacted with the agent.
  • the polypeptide comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127 or 129.
  • the detecting step comprises quantifying mRNA encoding the polypeptide.
  • the mRNA is reverse transcribed and amplified in a polymerase chain reaction.
  • the sample is a blood, urine or tissue sample.
  • the present invention provides for an isolated nucleic acid that is substantially identical to or hybridizes to a nucleic acid encoding a polypeptide listed in Table 1 under hybridization conditions of 50% formamide, 5X SSC, and 1% SDS at 42 0 C followed by a wash in 0.2X SSC, and 0.1% SDS at 55 0 C.
  • the polynucleotide comprises SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126 or 128.
  • the polynucleotide is encoded by a cell and the cell is contacted with the agent.
  • the polypeptide comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127 or 129.
  • the present invention also provides expression cassettes comprising a heterologous promoter operably linked to a nucleic acid that is substantially identical to or hybridizes to a nucleic acid encoding a polypeptide listed in Table 1 under hybridization conditions of 50% formamide, 5X SSC, and 1% SDS at 42 0 C followed by a wash in 0.2X SSC, and 0.1% SDS at 55 0 C.
  • the present invention also provides host cells transfected with nucleic acids that is substantially identical to or hybridizes to a nucleic acid encoding a polypeptide listed in Table 1 under hybridization conditions of 50% formamide, 5X SSC, and 1% SDS at 42 0 C followed by a wash in 0.2X SSC, and 0.1% SDS at 55 0 C.
  • the host cell is a human cell.
  • the host cell is a bacterium.
  • the present invention also provides isolated polypeptides comprising an amino acid sequence at least 70% identical to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129 or fragments thereof.
  • the polypeptide is encoded by a cell and the cell is contacted with the agent.
  • the polypeptide comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127 or 129.
  • the present invention also provides antibodies that specifically bind to a polypeptide selected from the groups consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127 or 129.
  • a polypeptide selected from the groups consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 58, 60, 62, 64, 66, 68, 70,
  • the present invention also provides pharmaceutical compositions comprising polypeptides comprising an amino acid sequence at least 70% identical to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129 or fragments thereof, and a pharmaceutically-acceptable excipient.
  • Insulin sensitivity refers to the ability of a cell or tissue to respond to insulin. Responses include, e.g., glucose uptake of a cell or tissue in response to insulin stimulation. Sensitivity can be determined at an organismal, tissue or cellular level. For example, blood or urine glucose levels following a glucose tolerance test are indicative of insulin sensitivity. Other methods of measuring insulin sensitivity include, e.g., measuring glucose uptake (see, e.g., Garcia de Herreros, A., and Birnbaum, M. J. J. Biol. Chem. 264, 19994-19999 (1989); Klip, A., Li, G., and Logan, WJ. Am. J. Physiol.
  • BMI body mass index
  • WHR wear-to-hip ratio
  • a waist-to-hip ratio is the ratio of a person's waist circumference to hip circumference, . For most people, carrying extra weight around their middle increases health risks more than carrying extra weight around their hips or thighs. For both men and women, a waist-to-hip ratio of 1.0 or higher is considered “at risk” or in the danger zone for undesirable health consequences, such as heart disease and other ailments connected with being overweight.
  • adipogenic when used in reference to cells refers to a cell which can become an adipocyte.
  • An “adipogenic factor” refers to a factor (including, e.g., a protein (or glycoprotein)) that can induce or stimulate the differentiation of cells into an adipocyte.
  • lipid metabolism refers to the in vivo process of catabolism (decomposition) and anabolism (accumulation) of lipids (e.g., triglycerides derived from food) and is intended to include, in the broad sense, reactions for transforming lipids into energy, biosynthesis of fatty acids, acylglycerol, phospholipid metabolism and cholesterol metabolism.
  • Activity of a polypeptide of the invention refers to structural, regulatory, or biochemical functions of a polypeptide in its native cell or tissue.
  • Examples of activity of a polypeptide include both direct activities and indirect activities.
  • Exemplary direct activities are the result of direct interaction with the polypeptide, , e.g., enzymatic activity, ligand binding, production or depletion of second messengers (e.g., cAMP, cGMP, IP 3 , DAG, or Ca 2+ ), ion flux, phosphorylation levels, transcription levels, and the like.
  • second messengers e.g., cAMP, cGMP, IP 3 , DAG, or Ca 2+
  • Exemplary indirect activities are observed as a change in phenotype or response in a cell or tissue to a polypeptide's directed activity, e.g., loss of body weight or molecular events associated with loss of body weight or obesity or modulating insulin sensitivity of a cell as a result of the interaction of the polypeptide with other cellular or tissue components.
  • Predisposition for diabetes occurs in a person when the person is at high risk for developing diabetes.
  • a number of risk factors are known to those of skill in the art and include: genetic factors (e.g., carrying alleles that result in a higher occurrence of diabetes than in the average population or having parents or siblings with diabetes); overweight (e.g., body mass index (BMI) greater or equal to 25 kg/m 2 ); habitual physical inactivity, race/ethnicity (e.g., African- American, Hispanic- American, Native Americans, Asian- Americans, Pacific Islanders); previously identified impaired fasting glucose or impaired glucose tolerance, hypertension (e.g., greater or equal to 140/90 mmHg in adults); HDL cholesterol less than or equal to 35 mg/dl; triglyceride levels greater or equal to 250 mg/dl; a history of gestational diabetes or delivery of a baby over nine pounds; and/or polycystic ovary syndrome. See, e.g., "Report of the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus" and “Screening for Diabetes” Diabetes Care 25(1)
  • a “2 hour PG” refers to the level of blood glucose after challenging a patient to a glucose load containing the equivalent of 75g anhydrous glucose dissolved in water. The overall test is generally referred to as an oral glucose tolerance test (OGTT). See, e.g., Diabetes Care, 2003, 26(11 ) : 3160-3167 (2003).
  • the level of a polypeptide in a lean individual can be a reading from a single individual, but is typically a statistically relevant average from a group of lean individuals.
  • the level of a polypeptide in a lean individual can be represented by a value, for example in a computer program.
  • An "agonist” refers to an agent that binds to, stimulates, increases, activates, facilitates, enhances activation, sensitizes or up regulates the activity or expression of a polypeptide of the invention.
  • An “antagonist” refers to an agent that binds to, partially or totally blocks stimulation, decreases, prevents, delays activation, inactivates, desensitizes, or down regulates the activity or expression of a polypeptide of the invention.
  • Antibody refers to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof which specifically bind and recognize an analyte (antigen).
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • An exemplary immunoglobulin (antibody) structural unit comprises a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy” chain (about 50-70 kD).
  • the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the terms variable light chain (V L ) and variable heavy chain (VH) refer to these light and heavy chains respectively.
  • Antibodies exist, e.g., as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases.
  • pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)' 2; a dimer of Fab which itself is a light chain joined to V H -C H I by a disulfide bond.
  • the F(ab)' 2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)' 2 dimer into an Fab' monomer.
  • the Fab' monomer is essentially an Fab with part of the hinge region (see, Paul (Ed.) Fundamental Immunology, Third Edition, Raven Press, NY (1993)). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv). [47] The terms "peptidomimetic” and “mimetic” refer to a synthetic chemical compound that has substantially the same structural and functional characteristics of the antagonists or agonists of the invention.
  • Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compound are termed “peptide mimetics” or “peptidomimetics” (Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber and Freidinger 2BVS p. 392 (1985); and Evans et al. J. Med. Chan. 30:1229 (1987), which are incorporated herein by reference). Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce an equivalent or enhanced therapeutic or prophylactic effect.
  • a paradigm polypeptide i.e., a polypeptide that has a biological or pharmacological activity
  • the mimetic can be either entirely composed of synthetic, non-natural analogues of amino acids, or, is a chimeric molecule of partly natural peptide amino acids and partly non-natural analogs of amino acids.
  • the mimetic can also incorporate any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantially alter the mimetic' s structure and/or activity.
  • a mimetic composition is within the scope of the invention if it is capable of carrying out the binding or other activities of an agonist or antagonist of a polypeptide of the invention.
  • gene means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).
  • nucleic acid or protein when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It is preferably in a homogeneous state although it can be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified. In particular, an isolated gene is separated from open reading frames that flank the gene and encode a protein other than the gene of interest. The term “purified” denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.
  • nucleic acid or “polynucleotide” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double- stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzcr et at., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et ah, 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.
  • polypeptide refers to a polymer of amino acid residues.
  • the terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.
  • the terms encompass amino acid chains of any length, including full-length proteins (i.e., antigens), wherein the amino acid residues are linked by covalent peptide bonds.
  • 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.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, ⁇ -carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an ⁇ carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain 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 which functions in a manner similar to a naturally occurring amino acid.
  • Amino acids may be referred to herein by either the commonly known three letter symbols or by the one-letter symbols recommended by the IUP AC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
  • Constantly modified variants applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, “conservatively modified variants” refers to those nucleic acids that encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
  • nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
  • TGG which is ordinarily the only codon for tryptophan
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
  • Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions ⁇ i.e., gaps) as compared to the reference sequence (e.g., a polypeptide of the invention), which does not comprise additions or deletions, for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same sequences. Sequences are "substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same ⁇ i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection, or across the entire sequence where not indicated.
  • the invention provides polypeptides or polynucleotides that are substantially identical to the polypeptides or polynucleotides, respectively, exemplified herein (e.g., SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 61, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well known in the art.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. MoI. Biol.
  • BLAST and BLAST 2.0 algorithms Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. MoI. Biol. 215:403-410, respectively.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence.
  • HSPs high scoring sequence pairs
  • T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score.
  • Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative- scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g. , Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to 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.
  • nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below.
  • a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by 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 to” refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (e.g., total cellular or library DNA or RNA).
  • stringent hybridization conditions refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found 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 thermal melting point (T m ) for the specific sequence at a defined ionic strength pH.
  • T m thermal melting point
  • the T m is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T m , 50% of the probes are occupied at equilibrium).
  • Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 3O 0 C for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C for long probes (e.g., greater than 50 nucleotides).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • a positive signal is at least two times background, optionally 10 times background hybridization.
  • Exemplary stringent hybridization conditions can be as following: 50% formamide, 5X SSC, and 1% SDS, incubating at 42 0 C, or 5X SSC, 1% SDS, incubating at 65 0 C, with wash in 0.2X SSC, and 0.1% SDS at 55 0 C, 6O 0 C, or 65 0 C. Such washes can be performed for 5, 15, 30, 60, 120, or more minutes.
  • Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides that they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions.
  • Exemplary "moderately stringent hybridization conditions" include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37 0 C, and a wash in IX SSC at 45 0 C. Such washes can be performed for 5, 15, 30, 60, 120, or more minutes. A positive hybridization is at least twice background.
  • a nucleic acid sequence encoding refers to a nucleic acid which contains sequence information for a structural RNA such as rRNA, a tRNA, or the primary amino acid sequence of a specific protein or peptide, or a binding site for a trans ⁇ acting regulatory agent. This phrase specifically encompasses degenerate codons (i.e., different codons which encode a single amino acid) of the native sequence or sequences that may be introduced to conform with codon preference in a specific host cell.
  • recombinant when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified.
  • recombinant cells express genes that are not found within the native (nonrecombinant) form of the cell or express native genes that are otherwise abnormally expressed, under-expressed or not expressed at all.
  • heterologous when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature.
  • the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source.
  • a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).
  • An "expression vector” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host cell.
  • the expression vector can be part of a plasmid, virus, or nucleic acid fragment.
  • the expression vector includes a nucleic acid to be transcribed operably linked to a promoter.
  • the specified antibodies bind to a particular protein and do not bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein.
  • antibodies raised against a protein having an amino acid sequence encoded by any of the polynucleotides of the invention can be selected to obtain antibodies specifically immunoreactive with that protein and not with other proteins, except for polymorphic variants.
  • a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
  • solid-phase ELISA immunoassays, Western blots, or immunohistochemistry are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See, Harlow and Lane Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, NY (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
  • a specific or selective reaction will be at least twice the background signal or noise and more typically more than 10 to 100 times background.
  • “Inhibitors,” “activators,” and “modulators” of expression or of activity are used to refer to inhibitory, activating, or modulating molecules, respectively, of expression of the polypeptides of the invention as determined using in vitro or in vivo assays to monitor expression or activity. Modulators encompass e.g., ligands, agonists, antagonists, their homologs and mimetics, as well as the polypeptides of the invention, or fragments thereof with antagonist activity or that act to increase overall polypeptide activity (i.e., fragments that have at least some of the activity of the full-length protein).
  • fragments of the polypeptides of the invention are at least 20, 50, 75 or 100 amino acids in length.
  • modulator includes inhibitors and activators. Inhibitors are agents that, e.g., inhibit expression of a polypeptide of the invention or bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity of a polypeptide of the invention, e.g., antagonists.
  • Activators are agents that, e.g., induce or activate the expression of a polypeptide of the invention or bind to, stimulate, increase, open, activate, facilitate, or enhance activation, sensitize or up regulate the activity of a polypeptide of the invention, e.g., agonists.
  • Modulators include naturally occurring and synthetic ligands, antagonists, agonists, small chemical molecules and the like.
  • assays for inhibitors and activators include, e.g., applying putative modulator compounds to cells expressing a polypeptide of the invention and then determining the functional effects on a polypeptide of the invention activity, as described above.
  • Samples or assays comprising a polypeptide of the invention that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of effect.
  • Control samples (untreated with modulators) are assigned a relative activity value of 100%.
  • Inhibition of a polypeptide of the invention is achieved when the polypeptide activity value relative to the control is about 80%, optionally 50% or 25, 10%, 5% or 1%.
  • Activation of the polypeptide is achieved when the polypeptide activity value relative to the control is 110%, optionally 150%, optionally 200, 300%, 400%, 500%, or 1000-3000% or more higher.
  • the present application demonstrates that, surprisingly, modulated levels of mRNA comprising sequences of the invention occur in human adipose tissue collected from either insulin resistant obese non-diabetics or from type 2 diabetic individuals compared to levels of the mRNA in the lean, non-diabetic individuals. Insulin resistant obese individuals are generally predisposed to become type II diabetics. Therefore, the modulation of the sequences in the study described herein indicates the sequences' involvement in obesity, diabetes and/or pre-diabetes.
  • modulation of the expression or activity of the polypeptides or polynucleotides of the invention is beneficial in treating obesity, diabetic, pre-diabetic or insulin resistant, non-diabetic patients.
  • modulated levels of the polypeptides of the invention are indicative of insulin resistance, obesity, diabetes or a predisposition for obesity and/or diabetes.
  • the detection of a polypeptide of the invention is useful for diagnosis of obesity, predisposition for obesity and/or diabetes, diabetes and/or insulin resistance.
  • This invention also provides methods of using polypeptides of the invention and modulators of the polypeptides of the invention to diagnose and treat obesity, diabetes, pre-diabetes (including insulin resistant individuals) and related metabolic diseases.
  • the present method also provides methods of identifying modulators of expression or activity of the polypeptides of the invention.
  • modulators are useful for treating obesity and/or Type 2 diabetes as well as the pathological aspects of obesity (e.g., increased risk for cardiovascular disease, hypertension or cancer) and/or diabetes (e.g., insulin resistance).
  • nucleic acids encoding a polypeptide of the present invention will be isolated and cloned using recombinant methods. Such embodiments are used, e.g., to isolate polynucleotides identical or substantially identical to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126 or 128 for protein expression or during the generation of variants, derivatives, expression cassettes, or other sequences derived from an polypeptide or polynucleotide of the invention, to
  • sequences encoding the polypeptides of the invention are operably linked to a heterologous promoter.
  • fragments of the polypeptides of the invention are at least 20, 50, 75 or 100 amino acids in length.
  • the polypeptides of the invention can be linked to heterologous amino acid sequences using recombinant DNA technology.
  • the nucleic acids of the invention are from any mammal, including, in particular, e.g., a human, a mouse, a rat, etc.
  • Polynucleotides, including expression cassettes, encoding polypeptides of the invention can be introduced into cells and optionally expressed in the cells.
  • Polynucleotides of the invention can be introduced into eukaryotic or prokaryotic cells, including adipocyte or muscle cells.
  • the cells can be primary cells or cell lines.
  • nucleic acids sizes are given in either kilobases (kb) or base pairs (bp). These are estimates derived from agarose or acrylamide gel electrophoresis, from sequenced nucleic acids, or from published DNA sequences.
  • kb kilobases
  • bp base pairs
  • proteins sizes are given in kilodaltons (kDa) or amino acid residue numbers. Proteins sizes are estimated from gel electrophoresis, from sequenced proteins, from derived amino acid sequences, or from published protein sequences.
  • Oligonucleotides that are not commercially available can be chemically synthesized according to the solid phase phosphoramidite triester method first described by Beaucage & Caruthers, Tetrahedron Letts. 22:1859-1862 (1981), using an automated synthesizer, as described in Van Devanter et. al, Nucleic Acids Res. 12:6159-6168 (1984). Purification of oligonucleotides is by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson & Reanier, J. Chrom. 255:137-149 (1983).
  • the nucleic acids encoding the subject proteins are cloned from DNA sequence libraries that are made to encode cDNA or genomic DNA.
  • the particular sequences can be located by hybridizing with an oligonucleotide probe, the sequence of which can be derived from the sequences disclosed herein, which provide a reference for PCR primers and defines suitable regions for isolating probes specific for the polypeptides or polynucleotides of the invention.
  • the sequence is cloned into an expression library
  • the expressed recombinant protein can be detected immunologically with antisera or purified antibodies made against a polypeptide of interest, including those disclosed herein.
  • cDNA library a source that is rich in mRNA.
  • the mRNA can then be made into cDNA, ligated into a recombinant vector, and transfected into a recombinant host for propagation, screening and cloning.
  • genomic library the DNA is extracted from a suitable tissue and either mechanically sheared or enzymatically digested to yield fragments of preferably about 5-100 kb.
  • the fragments are then separated by gradient centrifugation from undesired sizes and are constructed in bacteriophage lambda vectors.
  • These vectors and phage are packaged in vitro, and the recombinant phages are analyzed by plaque hybridization. Colony hybridization is carried out as generally described in Grunstein et ah, Proc. Natl. Acad. Sd. USA., 72:3961-3965
  • An alternative method combines the use of synthetic oligonucleotide primers with polymerase extension on an mRNA or DNA template.
  • Suitable primers can be designed from specific sequences disclosed herein.
  • This polymerase chain reaction (PCR) method amplifies the nucleic acids encoding the protein of interest directly from mRNA, cDNA, genomic libraries or cDNA libraries. Restriction endonuclease sites can be incorporated into the primers.
  • Polymerase chain reaction or other in vitro amplification methods may also be useful, for example, to clone nucleic acids encoding specific proteins and express said proteins, to synthesize nucleic acids that will be used as probes for detecting the presence of mRNA encoding a polypeptide of the invention in physiological samples, for nucleic acid sequencing, or for other purposes (see, U.S. Patent Nos. 4,683,195 and
  • Genes amplified by a PCR reaction can be purified from agarose gels and cloned into an appropriate vector.
  • Appropriate primers and probes for identifying the genes encoding a polypeptide of the invention from mammalian tissues can be derived from the sequences provided herein. For a general overview of PCR, see, Innis et al. PCR Protocols: A Guide to
  • Synthetic oligonucleotides can be used to construct genes. This is done using a series of overlapping oligonucleotides, usually 40-120 bp in length, representing both the sense and anti-sense strands of the gene. These DNA fragments are then annealed, ligated and cloned.
  • a polynucleotide encoding a polypeptide of the invention can be cloned using intermediate vectors before transformation into mammalian cells for expression. These intermediate vectors are typically prokaryote vectors or shuttle vectors. The proteins can be expressed in either prokaryotes or eukaryotes, using standard methods well known to those of skill in the art.
  • Naturally occurring polypeptides of the invention can be purified from any source (e.g., tissues of an organism expressing an ortholog).
  • Recombinant polypeptides can be purified from any suitable expression system. etal
  • polypeptides of the invention may be purified to substantial purity by standard techniques, including selective precipitation with such substances as ammonium sulfate; column chromatography, immunopurification methods, and others (see, e.g., Scopes, Protein Purification: Principles and Practice (1982); U.S. Patent No. 4,673,641; Ausubel et ah, supra; and Sambrook et ah, supra).
  • proteins having established molecular adhesion properties can be reversibly fused to a polypeptide of the invention.
  • either protein can be selectively adsorbed to a purification column and then freed from the column in a relatively pure form. The fused protein may be then removed by enzymatic activity.
  • polypeptides can be purified using immunoaffinity columns.
  • inclusion bodies typically involves the extraction, separation and/or purification of inclusion bodies by disruption of bacterial cells typically, but not limited to, by incubation in a buffer of about 100-150 ⁇ g/ml lysozyme and 0.1% Nonidet P40, a non-ionic detergent.
  • the cell suspension can be ground using a Polytron grinder (Brinkman Instruments, Westbury, NY). Alternatively, the cells can be sonicated on ice. Alternate methods of lysing bacteria are described in Ausubel and Sambrook et ah, both supra, and will be apparent to those of skill in the art.
  • the cell suspension is generally centrifuged and the pellet containing the inclusion bodies resuspended in buffer which does not dissolve but washes the inclusion bodies, e.g., 20 mM Tris-HCl (pH 7.2), 1 mM EDTA, 150 mM NaCl and 2% Triton-X 100, a non-ionic detergent. It may be necessary to repeat the wash step to remove as much cellular debris as possible.
  • the remaining pellet of inclusion bodies may be resuspended in an appropriate buffer (e.g., 20 mM sodium phosphate, pH 6.8, 150 mM NaCl).
  • an appropriate buffer e.g., 20 mM sodium phosphate, pH 6.8, 150 mM NaCl.
  • Other appropriate buffers will be apparent to those of skill in the art.
  • the inclusion bodies are solubilized by the addition of a solvent that is both a strong hydrogen acceptor and a strong hydrogen donor (or a combination of solvents each having one of these properties).
  • a solvent that is both a strong hydrogen acceptor and a strong hydrogen donor or a combination of solvents each having one of these properties.
  • the proteins that formed the inclusion bodies may then be renatured by dilution or dialysis with a compatible buffer.
  • Suitable solvents include, but are not limited to, urea (from about 4 M to about 8 M), formamide (at least about 80%, volume/volume basis), and guanidine hydrochloride (from about 4 M to about 8 M).
  • Some solvents that are capable of solubilizing aggregate-forming proteins are inappropriate for use in this procedure due to the possibility of irreversible denaturation of the proteins, accompanied by a lack of immunogenicity and/or activity.
  • SDS sodium dodecyl sulfate
  • 70% formic acid Some solvents that are capable of solubilizing aggregate-forming proteins, such as SDS (sodium dodecyl sulfate) and 70% formic acid, are inappropriate for use in this procedure due to the possibility of irreversible denaturation of the proteins, accompanied by a lack of immunogenicity and/or activity.
  • guanidine hydrochloride and similar agents are denaturants, this denaturation is not irreversible and renaturation may occur upon removal (by dialysis, for example) or dilution of the denaturant, allowing re-formation of the immunologically and/or biologically active protein of interest.
  • the protein can be separated from other bacterial proteins by standard separation techniques.
  • the periplasmic fraction of the bacteria can be isolated by cold osmotic shock in addition to other methods known to those of skill in the art (see, Ausubel et al, supra).
  • the bacterial cells are centrifuged to form a pellet. The pellet is resuspended in a buffer containing 20% sucrose.
  • the bacteria are centrifuged and the pellet is resuspended in ice-cold 5 mM MgSO 4 and kept in an ice bath for approximately 10 minutes.
  • the cell suspension is centrifuged and the supernatant decanted and saved.
  • the recombinant proteins present in the supernatant can be separated from the host proteins by standard separation techniques well known to those of skill in the art.
  • Proteins can also be purified from eukaryotic gene expression systems as described in, e.g., Fernandez and Hoeffler, Gene Expression Systems (1999).
  • baculo virus expression systems are used to isolate proteins of the invention.
  • Recombinant baculoviruses are generally generated by replacing the polyhedrin coding sequence of a baculovirus with a gene to be expressed (e.g., encoding a polypeptide of the invention).
  • Viruses lacking the polyhedrin gene have a unique plaque morphology making them easy to recognize.
  • a recombinant baculovirus is generated by first cloning a polynucleotide of interest into a transfer vector (e.g., a pUC based vector) such that the polynucleotide is operably linked to a polyhedrin promoter.
  • the transfer vector is transfected with wildtype DNA into an insect cell (e.g., Sf9, Sf21 or BT1-TN-5B1-4 cells), resulting in homologous recombination and replacement of the polyhedrin gene in the wildtype viral DNA with the polynucleotide of interest.
  • Virus can then be generated and plaque purified. Protein expression results upon viral infection of insect cells. Expressed proteins can be harvested from cell supernatant if secreted, or from cell lysates if intracellular. See, e.g., Ausubel et al. and Fernandez and Hoeffler, supra.
  • Polypeptides of the invention and in particular, secreted proteins of the invention can be readily purified from mammalian cells expressing the polypeptides.
  • Expression of the polypeptides can be the result of either transient or stable expression of the protein from a recombinant expression cassette introduced into the cells.
  • Secreted proteins can generally be isolated using standard procedures to purify the proteins from the cell culture medium.
  • an initial salt fractionation can separate many of the unwanted host cell proteins (or proteins derived from the cell culture media) from the recombinant protein of interest.
  • the preferred salt is ammonium sulfate.
  • Ammonium sulfate precipitates proteins by effectively reducing the amount of water in the protein mixture. Proteins then precipitate on the basis of their solubility. The more hydrophobic a protein is, the more likely it is to precipitate at lower ammonium sulfate concentrations.
  • a typical protocol is to add saturated ammonium sulfate to a protein solution so that the resultant ammonium sulfate concentration is between 20-30%. This will precipitate the most hydrophobic proteins.
  • the precipitate is discarded (unless the protein of interest is hydrophobic) and ammonium sulfate is added to the supernatant to a concentration known to precipitate the protein of interest.
  • the precipitate is then solubilized in buffer and the excess salt removed if necessary, through either dialysis or diaf ⁇ ltration.
  • Other methods that rely on solubility of proteins, such as cold ethanol precipitation, are well known to those of skill in the art and can be used to fractionate complex protein mixtures.
  • a protein of greater and lesser size can be isolated using ultrafiltration through membranes of different pore sizes (for example, Amicon or Millipore membranes).
  • the protein mixture is ultrafiltered through a membrane with a pore size that has a lower molecular weight cut-off than the molecular weight of the protein of interest.
  • the retentate of the ultrafiltration is then ultrafiltered against a membrane with a molecular cut off greater than the molecular weight of the protein of interest.
  • the recombinant protein will pass through the membrane into the filtrate.
  • the filtrate can then be chromatographed as described below. 3.
  • the proteins of interest can also be separated from other proteins on the basis of their size, net surface charge, hydrophobicity and affinity for ligands.
  • antibodies raised against proteins can be conjugated to column matrices and the proteins immunopurified. AU of these methods are well known in the art.
  • Immunoaffinity chromatography using antibodies raised to a variety of affinity tags such as hemagglutinin (HA), FLAG, Xpress, Myc, hexahistidine (His), glutathione S transferase (GST) and the like can be used to purify polypeptides.
  • the His tag will also act as a chelating agent for certain metals (e.g., Ni) and thus the metals can also be used to purify His-containing polypeptides. After purification, the tag is optionally removed by specific proteolytic cleavage.
  • metals e.g., Ni
  • DNA and RNA measurement A variety of methods of specific DNA and RNA measurement that use nucleic acid hybridization techniques are known to those of skill in the art (see, Sambrook, supra). Some methods involve an electrophoretic separation (e.g., Southern blot for detecting DNA, and Northern blot for detecting RNA), but measurement of DNA and RNA can also be carried out in the absence of electrophoretic separation (e.g., by dot blot). Southern blot of genomic DNA (e.g., from a human) can be used for screening for restriction fragment length polymorphism (RFLP) to detect the presence of a genetic disorder affecting a polypeptide of the invention.
  • RFLP restriction fragment length polymorphism
  • nucleic acid hybridization format The selection of a nucleic acid hybridization format is not critical. A variety of nucleic acid hybridization formats are known to those skilled in the art. For example, common formats include sandwich assays and competition or displacement assays. Hybridization techniques are generally described in Hames and Higgins Nucleic Acid Hybridization, A Practical Approach, IRL Press (1985); Gall and Pardue, Proc. Natl. Acad. ScL U.S.A., 63:378-383 (1969); and John et al. Nature, 223:582-587 (1969).
  • Detection of a hybridization complex may require the binding of a signal-generating complex to a duplex of target and probe polynucleotides or nucleic acids. Typically, such binding occurs through ligand and anti-ligand interactions as between a ligand-conjugated probe and an anti-ligand conjugated with a signal.
  • the binding of the signal generation complex is also readily amenable to accelerations by exposure to ultrasonic energy.
  • the label may also allow indirect detection of the hybridization complex.
  • the label is a hapten or antigen
  • the sample can be detected by using antibodies.
  • a signal is generated by attaching fluorescent or enzyme molecules to the antibodies or in some cases, by attachment to a radioactive label (see, e.g., Tijssen, "Practice and Theory of Enzyme Immunoassays " Laboratory Techniques in Biochemistry and Molecular Biology, Burdon and van Knippenberg Eds., Elsevier (1985), pp. 9-20).
  • the probes are typically labeled either directly, as with isotopes, chromophores, lumiphores, chromogens, or indirectly, such as with biotin, to which a streptavidin complex may later bind.
  • the detectable labels used in the assays of the present invention can be primary labels (where the label comprises an element that is detected directly or that produces a directly detectable element) or secondary labels (where the detected label binds to a primary label, e.g., as is common in immunological labeling).
  • labeled signal nucleic acids are used to detect hybridization.
  • Complementary nucleic acids or signal nucleic acids may be labeled by any one of several methods typically used to detect the presence of hybridized polynucleotides. The most common method of detection is the use of autoradiography with H, I, S, C, or P-labeled probes or the like.
  • Other labels include, e.g., ligands that bind to labeled antibodies, fluorophores, chemiluminescent agents, enzymes, and antibodies that can serve as specific binding pair members for a labeled ligand.
  • a detector that monitors a particular probe or probe combination is used to detect the detection reagent label.
  • Typical detectors include spectrophotometers, phototubes and photodiodes, microscopes, scintillation counters, cameras, film and the like, as well as combinations thereof. Examples of suitable detectors are widely available from a variety of commercial sources known to persons of skill in the art.
  • an optical image of a substrate comprising bound labeling moieties is digitized for subsequent computer analysis.
  • the amount of, for example, an RNA is measured by quantifying the amount of label fixed to the solid support by binding of the detection reagent.
  • the presence of a modulator during incubation will increase or decrease the amount of label fixed to the solid support relative to a control incubation that does not comprise the modulator, or as compared to a baseline established for a particular reaction type.
  • Means of detecting and quantifying labels are well known to those of skill in the art.
  • the target nucleic acid or the probe is immobilized on a solid support.
  • Solid supports suitable for use in the assays of the invention are known to those of skill in the art. As used herein, a solid support is a matrix of material in a substantially fixed arrangement.
  • VLSIP STM very large scale immobilized polymer arrays
  • Affymetrix, Inc. in Santa Clara, CA can be used to detect changes in expression levels of a plurality of genes involved in the same regulatory pathways simultaneously. See, 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
  • spotted cDNA arrays can also be used to monitor expression of a plurality of genes.
  • the array elements are organized in an ordered fashion so that each element is present at a specified location on the substrate. Because the array elements are at specified locations on the substrate, the hybridization patterns and intensities (which together create a unique expression profile) can be interpreted in terms of expression levels of particular genes and can be correlated with a particular disease or condition or treatment. See, e.g., Schena et al, Science 270: 467-470 (1995)) and (Lockhart et al, Nature Biotech. 14: 1675-1680 (1996)).
  • Hybridization specificity can be evaluated by comparing the hybridization of specificity-control polynucleotide sequences to specificity-control polynucleotide probes that are added to a sample in a known amount.
  • the specificity-control target polynucleotides may have one or more sequence mismatches compared with the corresponding polynucleotide sequences. In this manner, whether only complementary target polynucleotides are hybridizing to the polynucleotide sequences or whether mismatched hybrid duplexes are forming is determined.
  • Hybridization reactions can be performed in absolute or differential hybridization formats.
  • polynucleotide probes from one sample are hybridized to the sequences in a microarray format and signals detected after hybridization complex formation correlate to polynucleotide probe levels in a sample.
  • differential hybridization format the differential expression of a set of genes in two biological samples is analyzed.
  • polynucleotide probes from both biological samples are prepared and labeled with different labeling moieties.
  • a mixture of the two labeled polynucleotide probes is added to a microarray. The microarray is then examined under conditions in which the emissions from the two different labels are individually detectable.
  • the labels are fluorescent labels with distinguishable emission spectra, such as Cy3 and Cy5 fluorophores.
  • the microarray is washed to remove nonhybridized nucleic acids and complex formation between the hybridizable array elements and the polynucleotide probes is detected.
  • Methods for detecting complex formation are well known to those skilled in the art.
  • the polynucleotide probes are labeled with a fluorescent label and measurement of levels and patterns of fluorescence indicative of complex formation is accomplished by fluorescence microscopy, such as confocal fluorescence microscopy.
  • polynucleotide probes from two or more different biological samples are labeled with two or more different fluorescent labels with different emission wavelengths. Fluorescent signals are detected separately with different photomultipliers set to detect specific wavelengths. The relative abundances/expression levels of the polynucleotide probes in two or more samples are obtained.
  • microarray fluorescence intensities can be normalized to take into account variations in hybridization intensities when more than one microarray is used under similar test conditions.
  • individual polynucleotide probe/target complex hybridization intensities are normalized using the intensities derived from internal normalization controls contained on each microarray.
  • Detection of nucleic acids can also be accomplished, for example, by using a labeled detection moiety that binds specifically to duplex nucleic acids (e.g., an antibody that is specific for RNA-DNA duplexes).
  • a labeled detection moiety that binds specifically to duplex nucleic acids
  • an antibody that is specific for RNA-DNA duplexes e.g., an antibody that is specific for RNA-DNA duplexes.
  • the nucleic acids used in this invention can be either positive or negative probes. Positive probes bind to their targets and the presence of duplex formation is evidence of the presence of the target. Negative probes fail to bind to the suspect target and the absence of duplex formation is evidence of the presence of the target.
  • the use of a wild type specific nucleic acid probe or PCR primers may serve as a negative probe in an assay sample where only the nucleotide sequence of interest is present.
  • the sensitivity of the hybridization assays may be enhanced through use of a nucleic acid amplification system that multiplies the target nucleic acid being detected. Examples of such systems include the polymerase chain reaction (PCR) system and the ligase chain reaction (LCR) system.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • nucleic acid sequence based amplification NASBA, Cangene, Mississauga, Ontario
  • Q Beta Replicase systems can be used to directly identify mutants where the PCR or LCR primers are designed to be extended or ligated only when a selected sequence is present.
  • the selected sequences can be generally amplified using, for example, nonspecific PCR primers and the amplified target region later probed for a specific sequence indicative of a mutation.
  • detection probes including Taqman and molecular beacon probes can be used to monitor amplification reaction products, e.g., in real time.
  • An alternative means for determining the level of expression of the nucleic acids of the present invention is in situ hybridization.
  • In situ hybridization assays are well known and are generally described in Angerer et al, Methods Enzymol. 152:649-660 (1987).
  • cells preferentially human cells from the cerebellum or the hippocampus, are fixed to a solid support, typically a glass slide. IfDNA is to be probed, the cells are denatured with heat or alkali. The cells are then contacted with a hybridization solution at a moderate temperature to permit annealing of specific probes that are labeled.
  • the probes are preferably labeled with radioisotopes or fluorescent reporters.
  • Single nucleotide polymorphism (SNP) analysis is also useful for detecting differences between alleles of the polynucleotides (e.g., genes) of the invention.
  • SNPs linked to genes encoding polypeptides of the invention are useful, for instance, for diagnosis of diseases (e.g., diabetes) whose occurrence is linked to the gene sequences of the invention.
  • diseases e.g., diabetes
  • the individual is likely predisposed for one or more of those diseases.
  • the individual is homozygous for a disease-linked SNP, the individual is particularly predisposed for occurrence of that disease (e.g., diabetes).
  • the SNP associated with the gene sequences of the invention is located within 300,000; 200,000; 100,000; 75,000; 50,000; or 10,000 base pairs from the gene sequence.
  • Various real-time PCR methods including, e.g., Taqman or molecular beacon-based assays (e.g., U.S. Patent Nos. 5,210,015; 5,487,972; Tyagi et al, Nature Biotechnology 14:303 (1996); and PCT WO 95/13399 are useful to monitor for the presence of absence of a SNP.
  • Additional SNP detection methods include, e.g., DNA sequencing, sequencing by hybridization, dot blotting, oligonucleotide array (DNA Chip) hybridization analysis, or are described in, e.g., U.S. Patent No.
  • Immunoassays can be used to qualitatively or quantitatively analyze polypeptides of the invention. A general overview of the applicable technology can be found in Harlow & Lane, Antibodies: A Laboratory Manual (1988).
  • a recombinant protein is produced in a transformed cell line.
  • An inbred strain of mice or rabbits is immunized with the protein using a standard adjuvant, such as Freund's adjuvant, and a standard immunization protocol.
  • a synthetic peptide derived from the sequences disclosed herein is conjugated to a carrier protein and used as an immunogen.
  • Polyclonal sera are collected and titered against the immunogen in an immunoassay, for example, a solid phase immunoassay with the immunogen immobilized on a solid support.
  • Polyclonal antisera with a titer of 10 4 or greater are selected and tested for their crossreactivity against proteins other than the polypeptides of the invention or even other homologous proteins from other organisms, using a competitive binding immunoassay.
  • Specific monoclonal and polyclonal antibodies and antisera will usually bind with a K D of at least about 0.1 mM, more usually at least about 1 ⁇ M, preferably at least about 0.1 ⁇ M or better, and most preferably, 0.01 ⁇ M or better.
  • a number of proteins of the invention comprising immunogens may be used to produce antibodies specifically or selectively reactive with the proteins of interest.
  • Recombinant protein is the preferred immunogen for the production of monoclonal or polyclonal antibodies.
  • Naturally occurring protein may also be used either in pure or impure form.
  • Synthetic peptides made using the protein sequences described herein may also be used as an immunogen for the production of antibodies to the protein.
  • Recombinant protein can be expressed in eukaryotic or prokaryotic cells and purified as generally described supra. The product is then injected into an animal capable of producing antibodies. Either monoclonal or polyclonal antibodies may be generated for subsequent use in immunoassays to measure the protein.
  • an immunogen preferably a purified protein
  • an adjuvant preferably an adjuvant
  • animals are immunized.
  • the animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to polypeptides of the invention.
  • blood is collected from the animal and antisera are prepared. Further fractionation of the antisera to enrich for antibodies reactive to the protein can be done if desired ⁇ see, Harlow and Lane, supra).
  • Monoclonal antibodies may be obtained using various techniques familiar to those of skill in the art.
  • spleen cells from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell (see, Kohler and Milstein, Eur. J. Immunol. 6:511-519 (1976)).
  • Alternative methods of immortalization include, e.g., transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other methods well known in the art.
  • Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells may be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host.
  • the immunogen can be measured by a variety of immunoassay methods with qualitative and quantitative results available to the clinician. For a review of immunological and immunoassay procedures in general see, Stites, supra. Moreover, the immunoassays of the present invention can be performed in any of several configurations, which are reviewed extensively in Maggio Enzyme Immunoassay, CRC Press, Boca Raton, Florida (1980); Tijssen, supra; and Harlow and Lane, supra. [135] Immunoassays to measure target proteins in a human sample may use a polyclonal antiserum that was raised to full-length polypeptides of the invention or a fragment thereof. This antiserum is selected to have low cross-reactivity against other proteins and any such cross-reactivity is removed by immunoabsorption prior to use in the immunoassay.
  • a protein of interest is detected and/or quantified using any of a number of well-known immunological binding assays (see, e.g., U.S. Patents 4,366,241; 4,376,110; 4,517,288; and 4,837,168).
  • Immunological binding assays typically utilize a "capture agent" to specifically bind to and often immobilize the analyte (e.g., full-length polypeptides of the present invention, or antigenic subsequences thereof).
  • the capture agent is a moiety that specifically binds to the analyte.
  • the antibody may be produced by any of a number of means well known to those of skill in the art and as described above.
  • Immunoassays also often utilize a labeling agent to bind specifically to and label the binding complex formed by the capture agent and the analyte.
  • the labeling agent may itself be one of the moieties comprising the antibody/analyte complex.
  • the labeling agent may be a third moiety, such as another antibody, that specifically binds to the antibody/protein complex.
  • the labeling agent is a second antibody bearing a label.
  • the second antibody may lack a label, but it may, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived.
  • the second antibody can be modified with a detectable moiety, such as biotin, to which a third labeled molecule can specifically bind, such as enzyme-labeled streptavidin.
  • Other proteins capable of specifically binding immunoglobulin constant regions, such as protein A or protein G, can also be used as the label agents. These proteins are normal constituents of the cell walls of streptococcal bacteria.
  • incubation and/or washing steps may be required after each combination of reagents. Incubation steps can vary from about 5 seconds to several hours, preferably from about 5 minutes to about 24 hours. The incubation time will depend upon the assay format, analyte, volume of solution, concentrations, and the like. Usually, the assays will be carried out at ambient temperature, although they can be conducted over a range of temperatures, such as 10°C to 40°C. 1.
  • Immunoassays for detecting proteins or analytes of interest from tissue samples may be either competitive or noncompetitive.
  • Noncompetitive immunoassays are assays in which the amount of captured protein or analyte is directly measured.
  • the capture agent e.g., antibodies specific for the polypeptides of the invention
  • the capture agent can be bound directly to a solid substrate where it is immobilized. These immobilized antibodies then capture the polypeptide present in the test sample.
  • the polypeptide of the invention thus immobilized is then bound by a labeling agent, such as a second labeled antibody specific for the polypeptide.
  • the second antibody may lack a label, but it may, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived.
  • the second can be modified with a detectable moiety, such as biotin, to which a third labeled molecule can specifically bind, such as enzyme-labeled streptavidin.
  • the amount of protein or analyte present in the sample is measured indirectly by measuring the amount of an added (exogenous) protein or analyte displaced (or competed away) from a specific capture agent (e.g., antibodies specific for a polypeptide of the invention) by the protein or analyte present in the sample.
  • a specific capture agent e.g., antibodies specific for a polypeptide of the invention
  • the amount of immunogen bound to the antibody is inversely proportional to the concentration of immunogen present in the sample.
  • the antibody is immobilized on a solid substrate.
  • the amount of analyte may be detected by providing a labeled analyte molecule.
  • labels can include, e.g., radioactive labels as well as peptide or other tags that can be recognized by detection reagents such as antibodies.
  • Immunoassays in the competitive binding format can be used for cross- reactivity determinations.
  • the protein encoded by the sequences described herein can be immobilized on a solid support. Proteins are added to the assay and compete with the binding of the antisera to the immobilized antigen. The ability of the above proteins to compete with the binding of the antisera to the immobilized protein is compared to that of the protein encoded by any of the sequences described herein. The percent cross-reactivity for the above proteins is calculated, using standard calculations. Those antisera with less than 10% cross-reactivity with each of the proteins listed above are selected and pooled. The cross-reacting antibodies are optionally removed from the pooled antisera by imrnunoabsorption with the considered proteins, e.g., distantly related homologs.
  • the immunoabsorbed and pooled antisera are then used in a competitive binding immunoassay as described above to compare a second protein, thought to be perhaps a protein of the present invention, to the immunogen protein, hi order to make this comparison, the two proteins are each assayed at a wide range of concentrations and the amount of each protein required to inhibit 50% of the binding of the antisera to the immobilized protein is determined. If the amount of the second protein required is less than 10 times the amount of the protein partially encoded by a sequence herein that is required, then the second protein is said to specifically bind to an antibody generated to an immunogen consisting of the target protein. 3. Other Assay Formats
  • western blot (immunoblot) analysis is used to detect and quantify the presence of a polypeptide of the invention in the sample.
  • the technique generally comprises separating sample proteins by gel electrophoresis on the basis of molecular weight, transferring the separated proteins to a suitable solid support (such as, e.g., a nitrocellulose filter, a nylon filter, or a derivatized nylon filter) and incubating the sample with the antibodies that specifically bind the protein of interest.
  • a suitable solid support such as, e.g., a nitrocellulose filter, a nylon filter, or a derivatized nylon filter
  • antibodies are selected that specifically bind to the polypeptides of the invention on the solid support.
  • These antibodies may be directly labeled or alternatively may be subsequently detected using labeled antibodies (e.g., labeled sheep anti-mouse antibodies) that specifically bind to the antibodies against the protein of interest.
  • LOA liposome immunoassays
  • the particular label or detectable group used in the assay is not a critical 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 material having a detectable physical or chemical property.
  • Such detectable labels have been well-developed in the field of immunoassays and, in general, most labels useful in such methods can be applied to the present invention.
  • a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • Useful labels in the present invention include magnetic beads (e.g., DynabeadsTM), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g., 3 H, 125 1, 35 S, 14 C, or 32 P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.
  • fluorescent dyes e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like
  • radiolabels e.g., 3 H, 125 1, 35 S, 14 C, or 32 P
  • enzymes e.g., horse radish peroxidase, alkaline phosphatase and others commonly used
  • 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 may be used, with the choice of label depending on the sensitivity required, the ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions. [149] Non-radioactive labels are often attached by indirect means.
  • the molecules can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorescent compound. A variety of enzymes and fluorescent compounds can be used with the methods of the present invention and are well-known to those of skill in the art (for a review of various labeling or signal producing systems which may be used, see, e.g., U.S. Patent No. 4,391,904).
  • Means of detecting labels are well known to those of skill in the art.
  • means for detection include a scintillation counter or photographic film as in autoradiography.
  • the label is a fluorescent label, it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence may be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like.
  • CCDs charge coupled devices
  • enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product.
  • simple colorimetric labels may be detected directly by observing the color associated with the label. Thus, in various dipstick assays, conjugated gold often appears pink, while various conjugated beads appear the color of the bead.
  • agglutination assays can be used to detect the presence of the target antibodies.
  • antigen-coated particles are agglutinated by samples comprising the target antibodies.
  • none of the components need to be labeled and the presence of the target antibody is detected by simple visual inspection.
  • Modulators of a polypeptide of the invention i.e. agonists or antagonists of a polypeptide's activity, or polypeptide's or polynucleotide's expression or full-length polypeptides of the invention or fragments thereof, are useful for treating a number of human diseases, including diabetes or obesity.
  • administration of modulators can be used to treat diabetic patients or prediabetic individuals to prevent progression, and therefore symptoms, associated with diabetes (including insulin resistance).
  • Modulators of the invention can also be used to reduce obesity as well as the various diseases associated with obesity (e.g., gallbladder disease, cancer, sleep apnea, atherosclerosis, diabetes, and hypertension).
  • the modulators of the invention are used to regulate body physiology to reduce the chance of obesity-related diseases.
  • the modulators can be used to regulate serum lipids (total cholesterol, low-density lipoprotein (LDL), cholesterol, LDL/high density lipoprotein ratio and triglycerides).
  • agents tested as modulators of polypeptides of the invention can be any small chemical compound, or a biological entity, such as a protein, sugar, nucleic acid or lipid.
  • any chemical compound can be used as a potential modulator or ligand in the assays of the invention, although most often compounds that can be dissolved in aqueous or organic (especially DMSO-based) solutions are used.
  • Modulators include agents designed to reduce the level of mRNA encoding a polypeptide of the invention (e.g.
  • Modulators of the invention also include antibodies that specific bind to and/or inhibit or activate the polypeptides of the invention.
  • Other modulators include the polypeptides of the invention themselves, fragments thereof, or fusion proteins comprising the polypeptides or fragments thereof (e.g., in some embodiments, comprising at least 25, 50, or 100 amino acids of the polypeptide).
  • polypeptides of the invention that are receptors
  • soluble fragments of the polypeptides i.e., lacking a transmembrane domain
  • polypeptides of the invention that are secreted both full length and fragments with biological activity can act as modulators.
  • Sigma Sigma (St. Louis, MO), Aldrich (St. Louis, MO), Sigma- Aldrich (St. Louis, MO), Fluka Chemika-Biochemica Analytika (Buchs, Switzerland) and the like.
  • high throughput screening methods involve providing a combinatorial chemical or peptide library containing a large number of potential therapeutic compounds (potential modulator compounds). Such "combinatorial chemical libraries” or “ligand libraries” are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional "lead compounds” or can themselves be used as potential or actual therapeutics.
  • a combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks" such as reagents.
  • a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.
  • Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S.
  • Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (e.g., PCT Publication No. WO 91/19735), encoded peptides (e.g., PCT Publication WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091), benzodiazepines (e.g., U.S. Pat. No.
  • Patent 5,539,083) antibody libraries (see, e.g., Vaughn et al, Nature Biotechnology, 14(3):309-314 (1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al, Science, 274:1520-1522 (1996) and U.S. Patent 5,593,853), small organic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, Jan 18, page 33 (1993); isoprenoids, U.S. Patent 5,569,588; thiazolidinones and metathiazanones, U.S. Patent 5,549,974; pyrrolidines, U.S. Patents 5,525,735 and 5,519,134; morpholino compounds, U.S. Patent 5,506,337; benzodiazepines, 5,288,514, and the like).
  • antibody libraries see, e.g., Vaughn et al, Nature Biotechnology, 14(3):
  • a number of different screening protocols can be utilized to identify agents that modulate the level of expression or activity of a polynucleotide of a polypeptide of the invention in cells, particularly mammalian cells, and especially human cells.
  • the screening methods involve screening a plurality of agents to identify an agent that modulates the activity of a polypeptide of the invention by, e.g., binding to the polypeptide, preventing an inhibitor or activator from binding to the polypeptide, increasing association of an inhibitor or activator with the polypeptide, or activating or inhibiting expression of the polypeptide.
  • the assays can be designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source to assays, which are typically run in parallel (e.g., in microtiter formats on microtiter plates in robotic assays).
  • any cell expressing a full-length polypeptide of the invention or a fragment thereof can be used to identify modulators.
  • the cells are eukaryotic cells lines (e.g., CHO or HEK293) transformed to express a heterologous polypeptide of the invention.
  • a cell expressing an endogenous polypeptide of the invention is used in screens.
  • modulators are screened for their ability to affect insulin responses.
  • modulators are screened for their ability to effect body weight (as measured by BMI or waist-to-hip ratio) and secretion of a variety of obesity markers (e.g., leptin, IL-6 or TNF alpha), hi other embodiments, modulators are screened for their ability to effect lipid metabolism. In other embodiments, modulators are screened for their ability to effect the secretion and activity of adipogenic factors.
  • body weight as measured by BMI or waist-to-hip ratio
  • secretion of a variety of obesity markers e.g., leptin, IL-6 or TNF alpha
  • modulators are screened for their ability to effect lipid metabolism.
  • modulators are screened for their ability to effect the secretion and activity of adipogenic factors.
  • modulators of ADPN may be identified using, e.g., modulator binding assays, expression assays or promoter-reporter assays.
  • modulators of ALOX5 may be identified using, e.g., modulator binding assays, expression assays or promoter-reporter assays.
  • Enzyme assays can be carried out after contacting either purified recombinant ALOX5 protein, or an intact cell with a modulator using, e.g., arachidonic acid as a substrate or measuring the production of either LTB4 or cysteinyl leukotrienes.
  • modulators of CMAl may be identified using, e.g., modulator binding assays, expression assays or promoter-reporter assays. Enzyme assays can be carried out after contacting either purified recombinant CMAl protein, or an intact cell with a modulator using e.g. angiotensin I as a substrate or measuring the production of angiotensin II.
  • modulators of DUSP4 may be identified using, e.g., modulator binding assays, expression assays or promoter-reporter assays. Enzyme assays can be carried out after contacting either purified recombinant DUSP4 protein, or an intact cell with a modulator and using a screening assay based on a receptor protein tyrosine phosphatase activity or phosphorylation and activity of MAPK.
  • modulators of ECHDCl may be identified using, e.g., modulator binding assays, expression assays or promoter-reporter assays.
  • modulators of ECHDC3 may be identified using, e.g., modulator binding assays, expression assays or promoter-reporter assays.
  • modulators of HADHSC may be identified using, e.g., modulator binding assays, expression assays or promoter-reporter assays.
  • Enzyme assays can be carried out after contacting either purified recombinant HADHSC protein, or an intact cell with a modulator and using a screening assay based on dehydrogenation of 3-hydroxyacyl- CoAs to their corresponding 3-ketoacyl-CoAs activity and/or measuring NADH levels.
  • modulators of LGLL338 may be identified using, e.g., modulator binding assays, expression assays or promoter-reporter assays.
  • modulators of MGCl 0946 may be identified using, e.g., modulator binding assays, expression assays or promoter-reporter assays.
  • modulators of NPRl may be identified using, e.g., modulator binding assays, expression assays or promoter-reporter assays. Modulators can be screened either by binding assays or methods that monitor modulator-induced fluctuation of intracellular cyclic GMP concentration or activity of protein kinase G. Modulators which bind to the NPRl can be screened by a ligand binding assay method using e.g. ANP or BNP.
  • modulators of PLD3 may be identified using, e.g., modulator binding assays, expression assays or promoter-reporter assays. Modulators can be screened by methods that monitor modulator-induced fluctuation of intracellular phosphatidic acid concentrations.
  • modulators of PTGER2 may be identified using, e.g., modulator binding assays, expression assays or promoter-reporter assays. Modulators can be screened by methods that monitor modulator-induced fluctuation of intracellular cyclic AMP concentrations or phosphorylation and activity of MAPK. Modulators which bind to the PTGER2 can be screened by a ligand binding assay method using e.g. prostaglandin E2.
  • modulators of PTGER3 may be identified using, e.g., modulator binding assays, expression assays or promoter-reporter assays. Modulators can be screened by methods that monitor modulator-induced fluctuation of intracellular cyclic AMP and/or calcium concentrations. Modulators which bind to the PTGER3 can be screened by a ligand binding assay method using, e.g., prostaglandin E2.
  • modulators of PTGER4 may be identified using, e.g., modulator binding assays, expression assays or promoter-reporter assays. Modulators can be screened by methods that monitor modulator-induced fluctuation of intracellular cyclic AMP concentrations or phosphorylation and activity of MAPK. Modulators which bind to the PTGER4 can be screened by a ligand binding assay method using e.g. prostaglandin E2.
  • modulators of RARRES2 may be identified using, e.g., modulator binding assays, expression assays or promoter-reporter assays. Modulators can be screened by methods that monitor modulator-induced fluctuation of intracellular calcium and /or cyclic AMP concentrations or phosphorylation and activation of MAPK. Modulators which bind to the RARRES2 can be screened by a ligand binding assay method using, e.g., ChemR23, the G-protein coupled receptor known to bind to RARRES2.
  • modulators of SCRN2 may be identified using, e.g., modulator binding assays, expression assays or promoter-reporter assays.
  • modulators of TLR8 e.g., comprising the amino acid sequence of SEQ ID NO: 109, 111 or 113
  • modulator binding assays may be identified using, e.g., modulator binding assays, expression assays or promoter-reporter assays.
  • modulators of TM7SF2 may be identified using, e.g., modulator binding assays, expression assays or promoter-reporter assays.
  • modulators of TMND e.g., comprising the amino acid sequence of SEQ ID NO: 125, 127 or 129
  • modulator binding assays e.g., comprising the amino acid sequence of SEQ ID NO: 125, 127 or 129
  • expression assays e.g., expression assays or promoter-reporter assays.
  • Preliminary screens can be conducted by screening for agents capable of binding to polypeptides of the invention, as at least some of the agents so identified are likely modulators of a polypeptide of the invention.
  • Binding assays are also useful, e.g., for identifying endogenous proteins that interact with polypeptides of the invention. For example, antibodies, receptors or other molecules that bind polypeptides of the invention can be identified in binding assays.
  • Binding assays usually involve contacting a polypeptide of the invention with one or more test agents and allowing sufficient time for the protein and test agents to form a binding complex. Any binding complexes formed can be detected using any of a number of established analytical techniques. Protein binding assays include, but are not limited to, methods that measure co-precipitation or co-migration on non-denaturing SDS- polyacrylamide gels, and co-migration on Western blots ⁇ see, e.g., Bennet, J.P. and Yamamura, H.I. (1985) "Neurotransmitter, Hormone or Drug Receptor Binding Methods," in Neurotransmitter Receptor Binding (Yamamura, H.
  • binding assays involve the use of mass spectrometry or NMR techniques to identify molecules bound to a polypeptide of the invention or displacement of labeled substrates.
  • the polypeptides of the invention utilized in such assays can be naturally expressed, cloned or synthesized.
  • mammalian or yeast two-hybrid approaches can be used to identify polypeptides or other molecules that interact or bind when expressed together in a host cell.
  • polypeptides of the invention can be assessed using a variety of in vitro and in vivo assays to determine functional, chemical, and physical effects, e.g., measuring ligand binding ⁇ e.g., radioactive or otherwise labeled ligand binding), second messengers ⁇ e.g., cAMP, cGMP, P 3 , DAG, or Ca 2+ ), ion flux, phosphorylation levels, transcription levels, and the like.
  • ligand binding e.g., radioactive or otherwise labeled ligand binding
  • second messengers e.g., cAMP, cGMP, P 3 , DAG, or Ca 2+
  • ion flux phosphorylation levels
  • transcription levels e.g., transcription levels, and the like.
  • assays can be used to test for inhibitors and activators of the polypeptides of the invention.
  • Modulators can also be genetically altered versions of polypeptides of the invention.
  • the polypeptide of the assay will be selected from a polypeptide with substantial identity to a sequence of cell and the cell is contacted with the agent.
  • the polypeptide comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129 or other conservatively modified variants thereof.
  • the amino acid sequence identity will be at least 70%, optionally at least 85%, optionally at least 90, or optionally at least 95% to the polypeptides exemplified herein.
  • the polypeptide of the assays will comprise a fragment of a polypeptide of the invention, such as an extracellular domain, transmembrane domain, cytoplasmic domain, ligand binding domain, subunit association domain, active site, and the like. Either a polypeptide of the invention or a domain thereof can be covalently linked to a heterologous protein to create a chimeric protein used in the assays described herein.
  • Modulators of polypeptide activity are tested using either recombinant or naturally occurring polypeptides of the invention.
  • the protein can be isolated, expressed in a cell, expressed in a membrane derived from a cell, expressed in tissue or in an animal, either recombinant or naturally occurring.
  • tissue slices, dissociated cells, e.g., from tissues expressing polypeptides of the invention, transformed cells, or membranes can be used. Modulation is tested using one of the in vitro or in vivo assays described herein.
  • Modulator binding to polypeptides of the invention, a domain, or chimeric protein can be tested in solution, in a bilayer membrane, attached to a solid phase, in a lipid monolayer, or in vesicles. Binding of a modulator can be tested using, e.g. , changes in spectroscopic characteristics ⁇ e.g., fluorescence, absorbance, refractive index), hydrodynamic ⁇ e.g., shape), chromatographic, or solubility properties.
  • Samples or assays that are treated with a potential modulator e.g., a potential modulator
  • test compound are compared to control samples without the test compound, to examine the extent of modulation.
  • Control samples untreated with activators or inhibitors
  • Inhibition of the polypeptides of the invention is achieved when the activity value relative to the control is about 90%, optionally 50%, optionally 25- 0%.
  • Activation of the polypeptides of the invention is achieved when the activity value relative to the control is 110%, optionally 150%, 200%, 300%, 400%, 500%, or 1000-2000%.
  • Screening for a compound that modulates the expression of a polynucleotide or a polypeptide of the invention is also provided. Screening methods generally involve conducting cell-based assays in which test compounds are contacted with one or more cells expressing a polynucleotide or a polypeptide of the invention, and then detecting an increase or decrease in expression (either transcript or translation product). Assays can be performed with any cells that express a polynucleotide or a polypeptide of the invention.
  • Expression can be detected in a number of different ways.
  • the expression level of a polynucleotide of the invention in a cell can be determined by probing the mRNA expressed in a cell with a probe that specifically hybridizes with a transcript (or complementary nucleic acid derived there from) of a polynucleotide of the invention. Probing can be conducted by lysing the cells and conducting Northern blots or without lysing the cells using in szYw-hybridization techniques.
  • a polypeptide of the invention can be detected using immunological methods in which a cell lysate is probed with antibodies that specifically bind to the polypeptide.
  • Promoter-reporter assays can be carried out using mammalian cells transfected with a reporter gene operably linked to sequences derived from the promoter regions of genes encoding the polypeptides of the invention.
  • the increased or decreased expression of the reporter gene can be detected in the presence and absence of the modulator.
  • Expression of reporter genes may be detected by hybridization to a complementary nucleic acid, by using an immunological reagent, by assaying for an activity of the reporter gene product, or other methods known to those in the art
  • the level of expression or activity of a polynucleotide or a polypeptide of the invention can be compared to a baseline value.
  • the baseline value can be a value for a control sample or a statistical value that is representative of expression levels of a polynucleotide or a polypeptide of the invention for a control population (e.g., lean individuals as described herein) or cells (e.g., tissue culture cells not exposed to a modulator). Expression levels can also be determined for cells that do not express the polynucleotide or a polypeptide of the invention as a negative control. Such cells generally are otherwise substantially genetically the same as the test cells. [191] A variety of different types of cells can be utilized in the reporter assays.
  • Cells that do not endogenously express a polypeptide of the invention can be prokaryotic, but are preferably eukaryotic.
  • the eukaryotic cells can be any of the cells typically utilized in generating cells that harbor recombinant nucleic acid constructs.
  • Exemplary eukaryotic cells include, but are not limited to, yeast, and various higher eukaryotic cells such as the HEK293, HepG2, COS, CHO and HeLa cell lines.
  • Agents that are initially identified by any of the foregoing screening methods can be further tested to validate the apparent activity.
  • potential modulators can be tested initially using the forgoing validation assays without preliminary screening.
  • Modulators that are selected for further study can be tested for anti ⁇ diabetic effects using the "classic" insulin responsive cell line, mouse 3T3-L1 adipocytes, muscle cells such as L6 cells and the like.
  • Cells e.g., adipocytes or muscle cells
  • adipocytes or muscle cells are pre- incubated with the modulators and tested for acute (up to 4 hours) and chronic (overnight) effects on basal and insulin-stimulated GLUT4 translocation and glucose uptake.
  • Modulators that are selected for further study can be tested for anti- obesity effects using any adipocyte or adipogenic cell, e.g., mouse cell line 3T3-L1 adipocytes, freshly isolated rodent or human adipocytes, undifferentiated adipogenic cells and the like.
  • Cells e.g., adipocytes cells
  • adipocytes cells are pre-incubated with the modulators and tested for acute (up to 4 hours) and chronic (overnight or longer) effects on basal and insulin-stimulated release of adipogenic factors, adipocyte cell size, leptin and TNF alpha release, and/or lipid metabolism.
  • Undifferentiated adipogenic cells can be pre-incubated with the modulators and tested for effects on differentiation into adipocytes (including changes in differentiation markers) and/or triglyceride accumulation.
  • the response of this increase in body weight can be determined at an organismal, tissue or cellular level.
  • increased fasting blood leptin levels are indicative of obesity.
  • Other methods of measuring obesity include, e.g., calculation of BMI, waist-to-hip ratio, total body fat, measuring the blood levels of a variety of secreted proteins which have been shown to correlate to obesity (IL-6, TNF alpha) and measuring the fasted blood levels of free fatty acids.
  • Monogenic models of diabetes e.g., ob/ob and db/db mice, Zucker rats and Zucker Diabetic Fatty rats, etc.
  • polygenic models of diabetes e.g., OLETF rats, GK rats, NSY mice, and KK mice
  • transgenic animals expressing human polypeptides of the invention can be used to further validate drug candidates.
  • each well of a microtiter plate can be used to run a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator.
  • a single standard microtiter plate can assay about 100 (e.g., 96) modulators. If 1536 well plates are used, then a single plate can easily assay from about 100 to about 1500 different compounds. It is possible to assay several different plates per day; assay screens for up to about 6,000-20,000 or more different compounds are possible using the integrated systems of the invention.
  • microfluidic approaches to reagent manipulation can be used.
  • a molecule of interest e.g., a polypeptide or polynucleotide of the invention, or a modulator thereof
  • a tag can be any of a variety of components.
  • a molecule that binds the tag (a tag binder) is fixed to a solid support, and the tagged molecule of interest is attached to the solid support by interaction of the tag and the tag binder.
  • a number of tags and tag binders can be used, based upon known molecular interactions well described in the literature.
  • a tag has a natural binder, for example, biotin, protein A, or protein G
  • tag binders avidin, streptavidin, neutravidin, the Fc region of an immunoglobulin, poly-His, etc.
  • Antibodies to molecules with natural binders such as biotin are also widely available and appropriate tag binders (see, SIGMA Immunochemicals 1998 catalogue SIGMA, St. Louis MO).
  • 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 additional antibodies are described in the literature.
  • the tag is a first antibody and the tag binder is a second antibody that recognizes the first antibody.
  • receptor-ligand interactions are also appropriate as tag and tag-binder pairs, such as agonists and antagonists of cell membrane receptors (e.g., cell receptor-ligand interactions such as transferrin, c-kit, viral receptor ligands, cytokine receptors, chemokine receptors, interleukin receptors, immunoglobulin receptors and antibodies, the cadherin family, the integrin family, the selectin family, and the like; see, e.g., Pigott & Power, The Adhesion Molecule Facts Book I (1993)).
  • cell membrane receptors e.g., cell receptor-ligand interactions such as transferrin, c-kit, viral receptor ligands, cytokine receptors, chemokine receptors, interleukin receptors, immunoglobulin receptors and antibodies, the cadherin family, the integrin family, the selectin family, and the like; see, e.g., Pigott & Power, The Adhesion Molecule
  • toxins and venoms can all interact with various cell receptors.
  • hormones e.g., opiates, steroids, etc.
  • intracellular receptors e.g., which mediate the effects of various small ligands, including steroids, thyroid hormone, retinoids and vitamin D; peptides
  • lectins e.g., which mediate the effects of various small ligands, including steroids, thyroid hormone, retinoids and vitamin D; peptides
  • drugs lectins
  • sugars e.g., nucleic acids (both linear and cyclic polymer configurations), oligosaccharides, proteins, phospholipids and antibodies
  • nucleic acids both linear and cyclic polymer configurations
  • oligosaccharides oligosaccharides
  • proteins e.g.
  • Synthetic polymers such as polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, and polyacetates can also form an appropriate tag or tag binder. Many other tag/tag binder pairs are also useful in assay systems described herein, as would be apparent to one of skill upon review of this disclosure.
  • Common linkers such as peptides, polyethers, and the like can also serve as tags, and include polypeptide sequences, such as poly-gly sequences of between about 5 and 200 amino acids.
  • Such flexible linkers are known to those of skill in the art. For example, ⁇ oly(ethelyne glycol) linkers are available from Shearwater Polymers, Inc., Huntsville, Alabama. These linkers optionally have amide linkages, sulfhydryl linkages, or heterofunctional linkages.
  • Tag binders are fixed to solid substrates using any of a variety of methods currently available.
  • Solid substrates are commonly derivatized or functionalized by exposing all or a portion of the substrate to a chemical reagent that fixes a chemical group to the surface that is reactive with a portion of the tag binder.
  • groups that are suitable for attachment to a longer chain portion would include amines, hydroxyl, thiol, and carboxyl groups.
  • Amino alkylsilanes and hydroxyalkylsilanes can be used to functionalize a variety of surfaces, such as glass surfaces. The construction of such solid phase biopolymer arrays is well described in the literature ⁇ see, e.g., Merrifield, J. Am. Chem. Soc.
  • Non-chemical approaches for fixing tag binders to substrates include other common methods, such as heat, cross-linking by UV radiation, and the like.
  • the invention provides in vitro assays for identifying, in a high throughput format, compounds that can modulate the expression or activity of a polypeptide of the invention.
  • Control reactions that measure activity of a polypeptide of the invention in a cell in a reaction that does not include a potential modulator are optional, as the assays are highly uniform. Such optional control reactions are appropriate and increase the reliability of the assay. Accordingly, in some embodiments, the methods of the invention include such a control reaction.
  • "no modulator" control reactions that do not include a modulator provide a background level of binding activity.
  • a known activator of a polypeptide or a polynucleotide of the invention can be incubated with one sample of the assay, and the resulting increase in signal resulting from an increased expression level or activity of a polypeptide or a polynucleotide of the invention are determined according to the methods herein.
  • a known inhibitor of a polypeptide or a polynucleotide of the invention can be added, and the resulting decrease in signal for the expression or activity of a polypeptide or a polynucleotide of the invention can be similarly detected.
  • modulators can also be combined with activators or inhibitors to find modulators that inhibit the increase or decrease that is otherwise caused by the presence of the known modulator of a polypeptide or a polynucleotide of the invention.
  • the invention provides compositions, kits and integrated systems for practicing the assays described herein using nucleic acids or polypeptides of the invention, antibodies, etc.
  • the invention provides assay compositions for use in solid phase assays; such compositions can include, for example, one or more nucleic acids encoding a polypeptide of the invention immobilized on a solid support, and a labeling reagent.
  • the assay compositions can also include additional reagents that are desirable for hybridization. Modulators of expression or activity of a polypeptide of the invention can also be included in the assay compositions.
  • kits for carrying out the assays of the invention typically include a probe that comprises (1) an antibody that specifically binds to a polypeptide of the invention or (2) a polynucleotide sequence encoding at least a fragment of such polypeptides, and a label for detecting the presence of the probe.
  • the kits may include at least one polynucleotide sequence encoding a polypeptide of the invention.
  • Kits can include any of the compositions noted above, and optionally further include additional components such as instructions to practice a high-throughput method of assaying for an effect on expression of the genes encoding a polypeptide of the invention, or on activity of a polypeptide of the invention, one or more containers or compartments (e.g., to hold the probe, labels, or the like), a control modulator of the expression or activity of a polypeptide of the invention, a robotic armature for mixing kit components or the like.
  • the invention also provides integrated systems for high-throughput screening of potential modulators for an effect on the expression or activity of a polypeptide of the invention.
  • the systems can include a robotic armature which transfers fluid from a source to a destination, a controller which controls the robotic armature, a label detector, a data storage unit which records label detection, and an assay component such as a microtiter dish comprising a well having a reaction mixture or a substrate comprising a fixed nucleic acid or immobilization moiety.
  • a number of robotic fluid transfer systems are available, or can easily be made from existing components.
  • a Zymate XP Zymark Corporation; Hopkinton, MA
  • a Microlab 2200 Hamilton; Reno, NV
  • pipetting station can be used to transfer parallel samples to 96 well microtiter plates to set up several parallel simultaneous binding assays.
  • Optical images viewed (and, optionally, recorded) by a camera or other recording device are optionally further processed in any of the embodiments herein, e.g., by digitizing the image and storing and analyzing the image on a computer.
  • a camera or other recording device e.g., a photodiode and data storage device
  • a variety of commercially available peripheral equipment and software is available for digitizing, storing and analyzing a digitized video or digitized optical image.
  • One conventional system carries light from the specimen field to a cooled charge-coupled device (CCD) camera, in common use in the art.
  • a CCD camera includes an array of picture elements (pixels). The light from the specimen is imaged on the CCD. Particular pixels corresponding to regions of the specimen (e.g., individual hybridization sites on an array of biological polymers) are sampled to obtain light intensity readings for each position. Multiple pixels are processed in parallel to increase speed.
  • the apparatus and methods of the invention are easily used for viewing any sample, e.g., by fluorescent or dark field microscopic techniques.
  • Modulators of the polypeptides of the invention can be administered directly to the mammalian subject (typically in need thereof due to a pre-diabetic, diabetic or obese condition) for modulation of activity of a polypeptide of the invention in vivo.
  • Administration is by any of the routes normally used for introducing a modulator compound into ultimate contact with the tissue to be treated and is well known to those of skill in the art.
  • compositions of the 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 are a wide variety of suitable formulations of pharmaceutical compositions of the present invention ⁇ see, e.g., Remington 's Pharmaceutical Sciences, 17 th ed. 1985)).
  • the modulators e.g., agonists or antagonists of the expression or activity of a polypeptide of the invention, alone or in combination with other suitable components, can be prepared for injection or for use in a pump device.
  • Pump devices also known as "insulin pumps" are commonly used to administer insulin to patients and therefore can be easily adapted to include compositions of the present invention.
  • Manufacturers of insulin pumps include Animas, Disetronic and MiniMed.
  • the modulators e.g., agonists or antagonists of the expression or activity of a polypeptide of the invention, alone or in combination with other suitable components, can be made into aerosol formulations (i.e., they can be "nebulized") to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
  • Formulations suitable for administration include aqueous and non ⁇ aqueous solutions, isotonic sterile solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • compositions can be administered, for example, orally, nasally, topically, intravenously, intraperitoneally, or intrathecally.
  • the formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials. Solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • the modulators can also be administered as part of a prepared food or drug.
  • the dose administered to a patient should be sufficient to induce a beneficial response in the subject over time.
  • the optimal dose level for any patient will depend on a variety of factors including the efficacy of the specific modulator employed, the age, body weight, physical activity, and diet of the patient, on a possible combination with other drugs, and on the severity of the case of diabetes. It is recommended that the daily dosage of the modulator be determined for each individual patient by those skilled in the art in a similar way as for known insulin compositions.
  • the size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular compound or vector in a particular subject.
  • a physician may evaluate circulating plasma levels of the modulator, modulator toxicity, and the production of anti-modulator antibodies.
  • the dose equivalent of a modulator is from about 1 ng/kg to 10 mg/kg for a typical subject.
  • modulators of the present invention can be administered at a rate determined by the LD-50 of the modulator, and the side-effects of the modulator at various concentrations, as applied to the mass and overall health of the subject. Administration can be accomplished via single or divided doses.
  • the compounds of the present invention can also be used effectively in combination with one or more additional active agents depending on the desired target therapy (see, e.g., Turner, N. et al. Prog. Drug Res. (1998) 51: 33-94; Hafmer, S. Diabetes Care (1998) 21: 160-178; and DeFronzo, R. et al. (eds.), Diabetes Reviews (1997) Vol. 5 No. 4).
  • a number of studies have investigated the benefits of combination therapies with oral agents (see, e.g., Mahler, R., J. Clin. Endocrinol. Metab. (1999) 84: 1165-71; United Kingdom Prospective Diabetes Study Group: UKPDS 28, Diabetes Care (1998) 21: 87-92; Bardin, C.
  • Combination therapy includes administration of a single pharmaceutical dosage formulation that contains a modulator of the invention and one or more additional active agents, as well as administration of a modulator and each active agent in its own separate pharmaceutical dosage formulation.
  • a modulator and a thiazolidinedione can be administered to the human subject together in a single oral dosage composition, such as a tablet or capsule, or each agent can be administered in separate oral dosage formulations.
  • a modulator and one or more additional active agents can be administered at essentially the same time (i.e., concurrently), or at separately staggered times (i.e., sequentially).
  • Combination therapy is understood to include all these regimens.
  • One example of combination therapy can be seen in treating pre- diabetic individuals (e.g., to prevent progression into type 2 diabetes) or diabetic individuals (or treating diabetes and its related symptoms, complications, and disorders), wherein the modulators can be effectively used in combination with, for example, sulfonylureas (such as chlorpropamide, tolbutamide, acetohexamide, tolazamide, glyburide, gliclazide, glynase, glimepiride, and glipizide); biguanides (such as metformin); a PPAR beta delta agonist; a ligand or agonist of PPAR gamma such as thiazolidinediones (such as ciglitazone, pioglitazone (see, e.g., U.S.
  • sulfonylureas such as chlorpropamide, tolbutamide, acetohexamide, tola
  • Patent No. 6,218,409 troglitazone, and rosiglitazone (see, e.g., U.S. Patent No. 5,859,037)); PPAR alpha agonists such as clofibrate, gemfibrozil, fenofibrate, ciprofibrate, and bezafibrate; dehydroepiandrosterone (also referred to as DHEA or its conjugated sulphate ester, DHEA-SO4); antiglucocorticoids; TNF ⁇ inhibitors; ⁇ -glucosidase inhibitors (such as acarbose, miglitol, and voglibose); amylin and amylin derivatives (such as pramlintide, (see, also, U.S.
  • the modulators of the invention can also be combined with anti- obesity drugs (e.g., Xenical (Orlistat), Merida (Sibutramine) or Adipex-P (Phentermine)) or appetite-suppressing drugs.
  • nucleic acids encoding engineered amino acid sequences comprising the polypeptides of the invention can be used to introduce nucleic acids encoding engineered amino acid sequences comprising the polypeptides of the invention in mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding amino acid sequences comprising polypeptides of the invention to cells in vitro. In some embodiments, the nucleic acids encoding amino acid sequences comprising polypeptides of the invention are administered for in vivo or ex vivo gene therapy uses.
  • Non- viral vector delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome.
  • Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell.
  • DNA and RNA viruses which have either episomal or integrated genomes after delivery to the cell.
  • RNA viruses which have either episomal or integrated genomes after delivery to the cell.
  • Methods of non- viral delivery of nucleic acids encoding engineered polypeptides of the invention include lipofection, microinjection, biolistics, virosomes, liposomes, irnmunoliposom.es, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA.
  • Lipofection is described in e.g., US 5,049,386, US 4,946,787; and US 4,897,355) and lipofection reagents are sold commercially (e.g., TransfectamTM and LipofectinTM).
  • Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91/17424, WO 91/16024. Delivery can be to cells (ex vivo administration) or target tissues (in vivo administration).
  • RNA or DNA viral based systems for the delivery of nucleic acids encoding engineered polypeptides of the invention take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus.
  • Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro and the modified cells are administered to patients (ex vivo).
  • Conventional viral based systems for the delivery of polypeptides of the invention could include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer.
  • Viral vectors are currently the most efficient and versatile method of gene transfer in target cells and tissues. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.
  • Lenti viral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system would therefore depend on the target tissue. Retroviral vectors are comprised of czs-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum ex ⁇ acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression.
  • Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), human immuno deficiency virus (HIV), and combinations thereof ⁇ see, e.g., 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); PCT/US94/05700).
  • MiLV murine leukemia virus
  • GaLV gibbon ape leukemia virus
  • SIV Simian Immuno deficiency virus
  • HAV human immuno deficiency virus
  • Adenoviral based systems are typically used.
  • Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and levels of expression have been obtained.
  • This vector can be produced in large quantities in a relatively simple system.
  • Adeno-associated virus (“AAV”) vectors are also used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Patent No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest.
  • pLASN and MFG-S are examples are retroviral vectors that have been used in clinical trials (Dunbar et al, Blood 85:3048-305 (1995); Kohn et al, Nat. Med. 1:1017-102 (1995); Malech et ⁇ /., PNAS 94:22 12133-12138 (1997)).
  • PA317/pLASN was the first therapeutic vector used in a gene therapy trial. (Blaese et al, Science 270:475-480 (1995)). Transduction efficiencies of 50% or greater have been observed for MFG-S packaged vectors. (Ellem et al, Immunol Immunother. 44(l):10-20 (1997); Dranoff et al, Hum. Gene Ther. 1:111-2 (1997).
  • rAAV Recombinant adeno-associated virus vectors
  • AU vectors are derived from a plasmid that retains only the AAV 145 bp inverted terminal repeats flanking the transgene expression cassette. Efficient gene transfer and stable transgene delivery due to integration into the genomes of the transduced cell are key features for this vector system.
  • Replication-deficient recombinant adenoviral vectors can be engineered such that a transgene replaces the Ad EIa, EIb, and E3 genes; subsequently the replication defector vector is propagated in human 293 cells that supply deleted gene function in trans.
  • Ad vectors can transduce multiply types of tissues in vivo, including nondividing, differentiated cells such as those found in the liver, kidney and muscle system tissues. Conventional Ad vectors have a large carrying capacity.
  • An example of the use of an Ad vector in a clinical trial involved polynucleotide therapy for antitumor immunization with intramuscular injection (Sterman et al, Hum. Gene Ther. 7:1083-9 (1998)).
  • adenovirus 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 virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and ⁇ 2 cells or P A317 cells, which package retrovirus.
  • Viral vectors used in gene therapy are usually generated by producer cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the protein to be expressed. The missing viral functions are supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome.
  • Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences.
  • the cell line is also infected with adenovirus as a helper.
  • the helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid.
  • the helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV. [237]
  • a viral vector is typically modified to have specificity for a given cell type by expressing a ligand as a fusion protein with a viral coat protein on the viruses outer surface.
  • the ligand is chosen to have affinity for a receptor known to be present on the cell type of interest.
  • Han et al, PNAS 92:9747-9751 (1995) reported that Moloney murine leukemia virus can be modified to express human heregulin fused to gp70, and the recombinant virus infects certain human breast cancer cells expressing human epidermal growth factor receptor. This principle can be extended to other pairs of virus expressing a ligand fusion protein and target cell expressing a receptor.
  • filamentous phage can be engineered to display antibody fragments (e.g., FAB or Fv) having specific binding affinity for virtually any chosen cellular receptor.
  • antibody fragments e.g., FAB or Fv
  • nonviral vectors Such vectors can be engineered to contain specific uptake sequences thought to favor uptake by specific target cells.
  • Gene therapy vectors can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application, as described below.
  • vectors can be delivered to cells ex vivo, such as cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, tissue biopsy) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient, usually after selection for cells which have incorporated the vector.
  • Ex vivo cell transfection for diagnostics, research, or for gene therapy is well known to those of skill in the art.
  • cells are isolated from the subject organism, transfected with a nucleic acid (gene or cDNA) encoding a polypeptides of the invention, and re-infused back into the subject organism (e.g., patient).
  • a nucleic acid gene or cDNA
  • Various cell types suitable for ex vivo transfection are well known to those of skill in the art ⁇ see, e.g., Freshney et al., Culture of Animal Cells, A Manual of Basic Technique (3rd ed.
  • stem cells are used in ex vivo procedures for cell transfection and gene therapy.
  • the advantage to using stem cells is that they can be differentiated into other cell types in vitro, or can be introduced into a mammal (such as the donor of the cells) where they will engraft in the bone marrow.
  • Methods for differentiating CD34+ cells in vitro into clinically important immune cell types using cytokines such as GM- CSF, IFN- ⁇ and TNF- ⁇ are known (see Inaba et al, J. Exp. Med. 176:1693-1702 (1992)).
  • Stem cells are isolated for transduction and differentiation using known methods. For example, stem cells are isolated from bone marrow cells by panning the bone marrow cells with antibodies which bind unwanted cells, such as CD4+ and CD8+ (T cells), CD45+ (panB cells), GR-I (granulocytes), and lad (differentiated antigen presenting cells) (see Inaba et al, J. Exp. Med. 176:1693-1702 (1992)).
  • T cells CD4+ and CD8+
  • CD45+ panB cells
  • GR-I granulocytes
  • lad differentiated antigen presenting cells
  • Vectors e.g., retroviruses, adenoviruses, liposomes, etc.
  • therapeutic nucleic acids can be also administered directly to the organism for transduction of cells in vivo.
  • 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. Suitable methods of administering such nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.
  • 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 variety of suitable formulations of pharmaceutical compositions of the present invention, as described below (see, e.g., Remington 's Pharmaceutical Sciences, 17th ed., 1989).
  • the present invention also provides methods of diagnosing diabetes or obesity, or a predisposition of at least some of the pathologies of diabetes and/or obesity. Diagnosis can involve determination of a genotype of an individual (e.g., with SNPs) and comparison of the genotype with alleles known to have an association with the occurrence of obesity and/or diabetes. Alternatively, diagnosis also involves determining the level of a polypeptide or polynucleotide of the invention in a patient and then comparing the level to a baseline or range. Typically, the baseline value is representative of a polypeptide or polynucleotide of the invention in a healthy (e.g., lean) person.
  • a healthy e.g., lean
  • level of a polypeptide or polynucleotide of the invention indicates that the patient is either obese, at risk for becoming obese, diabetic or at risk of developing at least some of the pathologies of diabetes (e.g., pre-diabetic).
  • the level of a polypeptide in a lean individual can be a reading from a single individual, but is typically a statistically relevant average from a group of lean individuals.
  • the level of a polypeptide in a lean individual can be represented by a value, for example in a computer program.
  • the level of polypeptide or polynucleotide of the invention is measured by taking a blood, urine or tissue sample from a patient and measuring the amount of a polypeptide or polynucleotide of the invention in the sample using any number of detection methods, such as those discussed herein. For instance, fasting and fed blood or urine levels can be tested.
  • the baseline level and the level in a lean sample from an individual, or at least two samples from the same individual differ by at least about 5%, 10%, 20%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500%, 1000% or more.
  • the sample from the individual is greater by at least one of the above- listed percentages relative to the baseline level. In some embodiments, the sample from the individual is lower by at least one of the above-listed percentages relative to the baseline level.
  • the level of a polypeptide or polynucleotide of the invention is used to monitor the effectiveness of either anti-obese therapies such as orlistat or sibutramine, or, antidiabetic therapies such as thiazolidinediones, metformin, sulfonylureas and other standard therapies.
  • anti-obese therapies such as orlistat or sibutramine
  • antidiabetic therapies such as thiazolidinediones, metformin, sulfonylureas and other standard therapies.
  • the activity or expression of a polypeptide or polynucleotide of the invention will be measured prior to and after treatment of an obese patient with antiobese therapies, or, diabetic or pre-diabetic patients with antidiabetic therapies as a surrogate marker of clinical effectiveness.
  • the greater the reduction in expression or activity of a polypeptide of the invention indicates greater effectiveness.
  • Glucose/insulin tolerance tests can also be used to detect the effect of glucose levels on levels of a polypeptide or polynucleotide of the invention, m glucose tolerance tests, the patient's ability to tolerate a standard oral glucose load is evaluated by assessing serum and urine specimens for glucose levels. Blood samples are taken before the glucose is ingested, glucose is given by mouth, and blood or urine glucose levels are tested at set intervals after glucose ingestion. Similarly, meal tolerance tests can also be used to detect the effect of insulin or food, respectively, on levels of a polypeptide or polynucleotide of the invention.
  • Body weight or other indicators of obesity can also be used to detect the effect of modulating the levels of a polypeptide or polynucleotide of the invention. Measurement of a subject's response can be evaluated by assessing serum for altered levels of obesity-associated gene products, e.g., leptin, TNF alpha or IL-6.
  • obesity-associated gene products e.g., leptin, TNF alpha or IL-6.
  • peripheral tissues especially muscle and fat
  • peripheral tissues are known to have an impaired ability to respond to insulin and hence to take up glucose.
  • This defect in glucose metabolism is usually compensated for by increased secretion of insulin from the pancreas, thereby maintaining normal glucose levels.
  • the majority of glucose disposal occurs in the muscle.
  • a number of obese insulin resistant patients will progress to overt diabetics with time.
  • the molecular defects underlying this peripheral insulin resistance in both the obese and type II diabetics are not well defined.
  • Genes in muscle or fat whose expression is altered in either or both the obese or type II diabetics when compared to lean individuals can be causative genes for either obesity, insulin resistance and/or diabetes and are able to predict the transition to diabetes.
  • Modulators of such genes have the ability to reverse obesity, insulin resistance and restore normal insulin sensitivity, thereby improving whole body glucose homeostasis including for example insulin secretion. Modulators of such genes also have the ability to be used to pre-empt the transition from obesity-induced insulin resistance to diabetes. Modulators of such genes also have the ability to be used to reverse metabolic obesity-related diseases such as cardiovascular disease, hypertension or obesity-related cancer. [255] The molecular mechanism by which thiazolidinediones (TZDs) cause an increase in peripheral insulin sensitivity was studied. Genes in muscle or fat whose expression is altered by TZDs may lie on a pathway leading from TZD treatment to increased insulin sensitivity. Modulators of such genes can elicit the same effect as TZD treatment.
  • TZDs thiazolidinediones
  • Such modulators can lack some of the side effects of TZD.
  • Gene expression profiling in cultures of primary human adipocytes treated with either pioglitazone or rosiglitazone were used to identify genes important for TZD action and therefore treatment of obesity, diabetes and/or insulin resistance.
  • tissue samples subcutaneous adipose samples obtained from lean, obese and diabetic individuals. Two studies were performed. In the first study, samples were isolated from all individuals after a 5 hour hyperinsulinemic euglycemic clamp.
  • adipose samples were obtained from lean (BMK 25) and obese (BMI>30) individuals after an overnight fast.
  • samples were obtained from human subcutaneous and omental adipose tissues. Genes expressed only, or enriched, in fat can lie on pathways involved in insulin sensitivity, appetite suppression or lipid metabolism in the adipose itself or other peripheral tissues (e.g., muscle, liver, brain). For all tissue samples mRNA was isolated from these adipose samples and converted to cRNA by standard procedures. The gene expression profile for each individual was determined by hybridization of cRNA to commercial and custom synthesized Affymetrix chips.
  • Gene expression profile differences were calculated as follows. The expression level of a particular gene is indicated by its 'signal intensity'. The raw data was analyzed by a statistical test to remove Outliers'. The mean 'signal intensity' was then calculated from the signal intensities for all individuals in a particular treatment group.
  • Genes were determined to be changed in the first two studies by calculating the Students t test statistic between the two conditions and selecting those with t less than or equal to 0.05.
  • the fold change was determined as the ratio of mean signal intensity in condition 2 to the mean signal intensity in condition 1.
  • three comparisons was undertaken: diabetics (condition 1) versus leans (condition 2), obese (condition 1) versus lean (condition 2) and diabetics (condition 1) versus obese (condition 2).
  • the second study comparison is lean (condition 1) versus obese (condition 2).
  • the third comparison is identification of fat specific or fat enriched genes when comparing the expression profile of human subcutaneous and omental adipose tissues to at last 12 other human adult tissues.
  • Genes were determined to be meeting the criteria cut-off when the mean signal intensity of the human adipose samples was 3 fold greater than the mean signal intensity of all the other human adult tissues profiled or called present only in the adipose samples and absent in all others by the Affymetrix software program.
  • Probe set 233030 detects ADPN nucleic acid sequences. Expression of ADPN transcripts was decreased in diabetic compared to lean patients in the gene profiling experiment.
  • B/C indicates sample is from Basal or Clamp; "Mean Expr” indicates mean expression; “SEM” indicates standard error of mean; “n” indicates number of patient samples; “Fold Change” indicates fold change of diabetics in comparison to lean patients.
  • ADPN was also evaluated using real-time PCR. The results further show that ADPN is significantly under-expressed in subcutaneous adipose from diabetic individuals when compared to subcutaneous adipose from lean individuals.
  • “Fold Change” indicates the fold expression calculated as the ratio of the mean diabetic expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.
  • Probe set 233030 detects ADPN nucleic acid sequences. Expression of ADPN transcripts was decreased in obese compared to lean patients in the gene profiling experiment.
  • ADPN was also evaluated using real-time PCR. The results further show that ADPN is significantly under-expressed in subcutaneous adipose from obese individuals when compared to subcutaneous adipose from lean individuals.
  • “Fold Change” indicates the fold expression calculated as the ratio of the mean obese expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.
  • ADPN contains the following protein domains (designated with reference to SEQ ID NO:2): Patatin-like phospholipase (PF01734) at amino acids 10 to 179.
  • Adiponutrin is a newly identified nonsecreted adipocyte protein regulated by changes in energy balance in rodents (Bauieri, S. et ah, J Biol Chem., 276:33336-44 (2001)).
  • Probe set 204446 detects ALOX5 nucleic acid sequences. Expression of ALOX5 transcripts was increased in diabetic compared to lean patients in the gene profiling experiment.
  • B/C indicates sample is from Basal or Clamp; "Mean Expr” indicates mean expression; “SEM” indicates standard error of mean; “n” indicates number of patient samples; “Fold Change” indicates fold change of diabetics in comparison to lean patients.
  • ALOX5 was also evaluated using real-time PCR. The results further show that ALOX5 is significantly over-expressed in subcutaneous adipose from diabetic individuals when compared to subcutaneous adipose from lean individuals.
  • “Fold Change” indicates the fold expression calculated as the ratio of the mean diabetic expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.
  • ALOX5 contains the following protein domains (designated with reference to SEQ ID NO:8): PLAT/LH2 domain (PF01477) at amino acids 2 to 115; and Lipoxygenase (PF00305) at amino acids 125 to 666.
  • the leukotrienes arise from oxidative metabolism of arachidonic acid through the action of ALOX5, leading to the unstable allylic epoxide leukotriene A4.
  • This intermediate represents the substrate for two different specific enzymes, namely leukotriene A4-hydrolase and leukotriene C4-synthase, generating LTB4 and cysteinyl leukotrienes, respectively.
  • LTB(4) is a potent chemotactic and chemokinetic agent for a variety of leukocytes whereas the cysteinyl-leukotrienes C, D(4) and E(4) are known mediators of vascular permeability and smooth muscle contraction (Werz, O., Curr Drug Targets Inflamm Allergy 1 :23-44 (2002)).
  • Probe set 214533 detects CMAl nucleic acid sequences. Expression of
  • CMAl transcripts was increased in obese compared to lean patients in the gene profiling experiment.
  • B/C indicates sample is from Basal or Clamp; "Mean Expr” indicates mean expression; “SEM” indicates standard error of mean; “n” indicates number of patient samples; “Fold Change” indicates fold change of obese in comparison to lean patients.
  • CMAl was also evaluated using real-time PCR. The results further show that CMAl is significantly over-expressed in subcutaneous adipose from obese individuals when compared to subcutaneous adipose from lean individuals.
  • “Fold Change” indicates the fold expression calculated as the ratio of the mean obese expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.
  • CMAl contains the following protein domains (designated with reference to SEQ ID NO: 14): Signal peptide at amino acids 1 to 19; and Trypsin (PF00089) at amino acids 22 to 240. A soluble active secreted form of CMAl has been detected (Caughey, GM. Et al, J Biol Chem. 1991 JuI 15;266(20):12956-63) and this is displayed in SEQ ID NO: 15.
  • CMAl is a proteinase and is found highly expressed in mast cells and thought to function in the degradation of the extracellular matrix, the regulation of submucosal gland secretion, and the generation of vasoactive peptides.
  • CMAl is largely responsible for converting angiotensin I to the vasoactive peptide angiotensin II.
  • Angiotensin II has been implicated in blood pressure control and in the pathogenesis of hypertension, cardiac hypertrophy, and heart failure.
  • Probe set 204014 detects DUSP4 nucleic acid sequences. Expression of DUSP4 transcripts was decreased in diabetic compared to lean patients in the gene profiling experiment.
  • B/C indicates sample is from Basal or Clamp; "Mean Expr” indicates mean expression; “SEM” indicates standard error of mean; “n” indicates number of patient samples; “Fold Change” indicates fold change of diabetics in comparison to lean patients.
  • DUSP4 was also evaluated using real-time PCR. The results further show that DUSP4 is significantly under-expressed in subcutaneous adipose from diabetic individuals when compared to subcutaneous adipose from lean individuals.
  • “Fold Change” indicates the fold expression calculated as the ratio of the mean diabetic expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.
  • Probe set 204014 detects DUSP4 nucleic acid sequences. Expression of DUSP4 transcripts was decreased in obese compared to lean patients in the gene profiling experiment.
  • B/C indicates sample is from Basal or Clamp; "Mean Expr” indicates mean expression; “SEM” indicates standard error of mean; “n” indicates number of patient samples; “Fold Change” indicates fold change of obese in comparison to lean patients.
  • DUSP4 was also evaluated using real-time PCR. The results further show that DUSP4 is significantly under-expressed in subcutaneous adipose from obese individuals when compared to subcutaneous adipose from lean individuals.
  • “Fold Change” indicates the fold expression calculated as the ratio of the mean obese expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.
  • Probe set 204014 detects DUSP4 nucleic acid sequences. Expression of DUSP4 transcripts was increased in adipose tissues compared to all other human adult tissues in the gene profiling experiment.
  • Mean Expr indicates mean expression; “SEM” indicates standard error of mean; “n” indicates number of tissue samples; “Fold Change” indicates fold change of adipose tissues in comparison to all other human adult tissues profiled.
  • DUSP4 was also evaluated using real-time PCR. The results further show that DUSP4 is significantly over-expressed in adipose tissues when compared to all other human adult tissues.
  • “Fold Change” indicates the fold expression calculated as the ratio of the mean adipose tissues expression/ mean other tissues expression. Numbers in parentheses indicates the number of human adult tissue samples analyzed by real-time PCR.
  • DUSP4 contains the following protein domains (designated with reference to SEQ ID NO:21): Dual specificity phosphatase, catalytic domain (PF00782) at amino acids 195 to 333; Rhodanese-like domain (PF00581) at amino acids 33 to 153; and Protein-tyrosine phosphatase (PFOOl 02) at amino acids 159 to 337.
  • DUSP4 is a member of the dual specificity protein phosphatase subfamily. DUSP4 has been reported to negatively regulate members of the mitogen-activated protein (MAP) kinase superfamily (MAPK/ERK, SAPK/JNK, p38), which are associated with cellular proliferation and differentiation. Two alternatively spliced transcript variants, encoding distinct isoforms, have been observed for this gene.
  • MAP mitogen-activated protein
  • Probe set 223087 detects ECHDCl nucleic acid sequences. Expression of ECHDCl transcripts was decreased in patients with insulin resistance compared to normal patients in the gene profiling experiment.
  • B/C indicates sample is from Basal or Clamp; "Corr Co-efficient” indicates the relationship between glucose disposal rate (Rd) and signal intensities. A positive co-efficient indicates down regulation whereas a negative co-efficient indicates up regulation of the gene with increasing insulin resistance; "n" indicates number of patient samples.
  • Probe set 223087 detects ECHDCl nucleic acid sequences. Expression of ECHDCl transcripts was decreased in diabetic compared to lean patients in the gene profiling experiment.
  • B/C indicates sample is from Basal or Clamp; "Mean Expr” indicates mean expression; “SEM” indicates standard error of mean; “n” indicates number of patient samples; “Fold Change” indicates fold change of diabetics in comparison to lean patients.
  • Probe set 223087 detects ECHDCl nucleic acid sequences. Expression of ECHDCl transcripts was increased in adipose tissues compared to all other human adult tissues in the gene profiling experiment.
  • Mean Expr indicates mean expression; “SEM” indicates standard error of mean; “n” indicates number of tissue samples; “Fold Change” indicates fold change of adipose tissues in comparison to all other human adult tissues profiled.
  • ECHDCl was also evaluated using real-time PCR. The results further show that ECHDCl is significantly over-expressed in adipose tissues when compared to all other human adult tissues.
  • “Fold Change” indicates the fold expression calculated as the ratio of the mean adipose tissues expression/ mean other tissues expression. Numbers in parentheses indicates the number of human adult tissue samples analyzed by real-time PCR.
  • ECHDCl contains the following protein domains (designated with reference to SEQ ID NO:29): Enoyl-CoA hydratase/isomerase family (PF00378) at amino acids 59 to 213. It is possible that ECHDCl has similar activity as to enoyl-CoA hydratase which catalyzes the second step in beta-oxidation of fatty acids.
  • Probe set 219298 detects ECHDC3 nucleic acid sequences. Expression of ECHDC3 transcripts was decreased in obese compared to lean patients in the gene profiling experiment.
  • B/C indicates sample is from Basal or Clamp; "Mean Expr” indicates mean expression; “SEM” indicates standard error of mean; “n” indicates number of patient samples; “Fold Change” indicates fold change of obese in comparison to lean patients.
  • ECHDC3 was also evaluated using real-time PCR. The results further show that ECHDC3 is significantly under-expressed in subcutaneous adipose from obese individuals when compared to subcutaneous adipose from lean individuals.
  • ECHDC3 contains the following protein domains (designated with reference to SEQ ID NO:39): Enoyl-CoA hydratase/isomerase family (PF00378) at amino acids 57 to 225. It is possible that ECHDC3 has similar activity as to enoyl-CoA hydratase which catalyzes the second step in beta-oxidation of fatty acids.
  • Probe set 211569 detects HADHSC nucleic acid sequences. Expression of HADHSC transcripts was decreased in diabetic compared to lean patients in the gene profiling experiment.
  • B/C indicates sample is from Basal or Clamp; "Mean Expr” indicates mean expression; “SEM” indicates standard error of mean; “n” indicates number of patient samples; “Fold Change” indicates fold change of diabetics in comparison to lean patients.
  • HADHSC was also evaluated using real-time PCR. The results further show that HADHSC is significantly under-expressed in subcutaneous adipose from diabetic individuals when compared to subcutaneous adipose from lean individuals.
  • “Fold Change” indicates the fold expression calculated as the ratio of the mean diabetic expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.
  • Probe set 211569 detects HADHSC nucleic acid sequences.
  • HADHSC transcripts was decreased in obese compared to lean patients in the gene profiling experiment.
  • B/C indicates sample is from Basal or Clamp; "Mean Expr” indicates mean expression; “SEM” indicates standard error of mean; “n” indicates number of patient samples; “Fold Change” indicates fold change of obese in comparison to lean patients.
  • HADHSC contains the following protein domains (designated with reference to SEQ ID NO:43): 3-hydroxyacyl-CoA dehydrogenase, NAD binding domain (PF02737) at amino acids 25 to 214; and 3-hydroxyacyl-CoA dehydrogenase, C-terminal domain (PF00725) at amino acids 216 to 313.
  • HADHSC plays an essential role in the mitochondrial beta-oxidation of short chain fatty acids. It catalyzes the reversible dehydrogenation of 3-hydroxyacyl-CoAs to their corresponding 3-ketoacyl-CoAs with concomitant reduction of NAD to NADH and exerts it highest activity toward 3- hydroxybutyryl-CoA.
  • Probe set 235496 detects LGLL338 nucleic acid sequences. Expression of LGLL338 transcripts was increased in diabetic compared to lean patients in the gene profiling experiment.
  • B/C indicates sample is from Basal or Clamp; "Mean Expr” indicates mean expression; “SEM” indicates standard error of mean; “n” indicates number of patient samples; “Fold Change” indicates fold change of diabetics in comparison to lean patients.
  • LGLL338 was also evaluated using real-time PCR. The results further show that LGLL338 is significantly over-expressed in subcutaneous adipose from diabetic individuals when compared to subcutaneous adipose from lean individuals.
  • “Fold Change” indicates the fold expression calculated as the ratio of the mean diabetic expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.
  • Probe set 235496 detects LGLL338 nucleic acid sequences. Expression of LGLL338 transcripts was increased in adipose tissues compared to all other human adult tissues in the gene profiling experiment.
  • Mean Expr indicates mean expression; “SEM” indicates standard error of mean; “n” indicates number of tissue samples; “Fold Change” indicates fold change of adipose tissues in comparison to all other human adult tissues profiled.
  • LGLL338 contains the following protein domains (designated with reference to SEQ ID NO:51): 1 transmembrane domain (TMHMM2.0) at amino acids 10 to 32.
  • Probe set MBXHUMFAT06172 detects MGC10946 nucleic acid sequences. Expression of MGC 10946 transcripts was decreased in diabetic compared to lean patients in the gene profiling experiment.
  • MGC 10946 was also evaluated using real-time PCR. The results further show that MGC 10946 is significantly under-expressed in subcutaneous adipose from diabetic individuals when compared to subcutaneous adipose from lean individuals.
  • “Fold Change” indicates the fold expression calculated as the ratio of the mean diabetic expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.
  • MGCl 0946 contains the following protein domains (designated with reference to SEQ ID NO:57): Signal peptide at amino acids 1 to 26; and 1 transmembrane domain (TMHMM2.0) at amino acids 4 to 26.
  • TSHMM2.0 transmembrane domain
  • Probe set 204648 detects NPRl nucleic acid sequences. Expression of NPRl transcripts was decreased in diabetic compared to lean patients in the gene profiling experiment.
  • B/C indicates sample is from Basal or Clamp; "Mean Expr” indicates mean expression; “SEM” indicates standard error of mean; “n” indicates number of patient samples; “Fold Change” indicates fold change of diabetics in comparison to lean patients.
  • NPRl was also evaluated using real-time PCR. The results further show that NPRl is significantly under-expressed in subcutaneous adipose from diabetic individuals when compared to subcutaneous adipose from lean individuals.
  • “Fold Change” indicates the fold expression calculated as the ratio of the mean diabetic expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.
  • Probe set 204648 detects NPRl nucleic acid sequences. Expression of NPRl transcripts was increased in adipose tissues compared to all other human adult tissues in the gene profiling experiment.
  • Mean Expr indicates mean expression; “SEM” indicates standard error of mean; “n” indicates number of tissue samples; “Fold Change” indicates fold change of adipose tissues in comparison to all other human adult tissues profiled.
  • NPRl was also evaluated using real-time PCR. The results further show that NPRl is significantly over-expressed in adipose tissues when compared to all other human adult tissues.
  • “Fold Change” indicates the fold expression calculated as the ratio of the mean adipose tissues expression/ mean other tissues expression. Numbers in parentheses indicates the number of human adult tissue samples analyzed by real-time PCR.
  • NPRl contains the following protein domains (designated with reference to SEQ ID NO: 64): Receptor family ligand binding region (PFOl 094) at amino acids 54 to 417; Adenylate and Guanylate cyclase catalytic domain (PF00211) at amino acids 867 to 1053; and Protein kinase domain (PF00069) at amino acids 538 to 801.
  • NPRl is a membrane-bound guanylate cyclase that serves as the receptor for both atrial and brain natriuretic peptides.
  • Atrial natriuretic peptide initiates natriuresis, diuresis, and vasodilation, all of which contribute to lowering blood pressure whereas the structurally related peptide, brain natriuretic peptide has similar effects but mainly functions in the cardiac ventricles.
  • Probe set 201050 detects PLD3 nucleic acid sequences. Expression of PLD3 transcripts was increased in diabetic compared to lean patients in the gene profiling experiment.
  • B/C indicates sample is from Basal or Clamp; "Mean Expr” indicates mean expression; “SEM” indicates standard error of mean; “n” indicates number of patient samples; “Fold Change” indicates fold change of diabetics in comparison to lean patients.
  • PLD3 was also evaluated using real-time PCR. The results further show that PLD3 is significantly over-expressed in subcutaneous adipose from diabetic individuals when compared to subcutaneous adipose from lean individuals.
  • “Fold Change” indicates the fold expression calculated as the ratio of the mean diabetic expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.
  • PLD3 contains the following protein domains (designated with reference to SEQ ID NO:70): Phospholipase D. Active site motif (PF00614) at amino acids 143 to 170, 358 to 384. This domain is found in other enzymes which are members of the phospholipase superfamily of enzymes which are known to hydrolyze the terminal phosphodiester bond of phospholipids to phosphatidic acid. Phosphatidic acid is a lipid mediator involved in signal transduction. PTGER2
  • Probe set 206631 detects PTGER2 nucleic acid sequences. Expression of PTGER2 transcripts was increased in rosi compared to vehicle treated cultures of primary human adipocytes in the gene profiling experiment.
  • B/C indicates sample is from Basal or Clamp; "Pre-Rosi” and “Post-Rosi” indicates sample was taken before or after 24 hours of rosiglitazone treatment; “Mean Expr” indicates mean expression; “SEM” indicates standard error of mean; “n” indicates number of patient samples; “Fold Change” indicates fold change of primary human adipocytes post-rosi in comparison to pre-rosi samples.
  • PTGER2 was also evaluated using real-time PCR. The results further show that PTGER2 is significantly over-expressed in primary cultured human adipocytes treated with rosi when compared to vehicle.
  • “Fold Change” indicates the fold expression calculated as the ratio of the mean rosi expression/ mean vehicle expression. Numbers in parentheses indicates the number of primary human adipocyte samples analyzed by real-time PCR.
  • PTGER2 was over-expressed in 3T3-L1 adipocytes and the effect on basal and insulin stimulated glucose transport and Glut 4 translocation was determined.
  • Con indicates control 3T3-L1 adipocytes that do not express hPTGER2.
  • FC indicates the fold change defined as the following ratio; glucose transport in hPTGER2-expressing cells incubated for 3 hours with 1 uM butaprost free acid/glucose transport in non-PTGER2-expressing cells incubated for 3 hours with 1 uM butaprost free acid, h" is human, “n” is the number of experiments. SEM is the standard error of the mean.
  • PTGER2 contains the following protein domains (designated with reference to SEQ ID NO:78): 7 transmembrane receptor (rhodopsin family) (PFOOOOl) at amino acids 38 to 315. Mice deficient in the PTGER2 displayed resting systolic blood pressure that was significantly lower than that in wildtype controls.
  • PTGER2 is a G protein coupled receptor, activation of which increases intracellular cyclic AMP ⁇ see, e.g., Bastien, et al., J. Biol.Chem 269:11873-11877 (1994), Katsuyama, et al. FEBS Lett. 372:151-156 (1995)). Agonists of PTGER2 can therefore be identified, e.g., by screening cells with high levels of PTGER2. to identify compounds that increase intracellular cyclic AMP.
  • prostaglandin E2 include, for example, prostaglandin El; butaprost free acid (GR32191B, 9-oxo -1 l ⁇ , l ⁇ R-dihdroxy- ⁇ -cyclobutyl-prost-lSE-en-l-oic acid), Regan, et al. MoI Pharmacol. 46:213-220 (1994); 16,16, dimethyl prostaglandin E2, Wilson, et al. Eur. J.
  • Probe set 210374 detects PTGER3 nucleic acid sequences. Expression of PTGER3 transcripts was increased in adipose tissues compared to all other human adult tissues in the gene profiling experiment.
  • Mean Expr indicates mean expression; “SEM” indicates standard error of mean; “n” indicates number of tissue samples; “Fold Change” indicates fold change of adipose tissues in comparison to all other human adult tissues profiled.
  • PTGER3 was also evaluated using real-time PCR. The results further show that PTGER3 is significantly over-expressed in adipose tissues when compared to all other human adult tissues.
  • “Fold Change” indicates the fold expression calculated as the ratio of the mean adipose tissues expression/ mean other tissues expression. Numbers in parentheses indicates the number of human adult tissue samples analyzed by real-time PCR.
  • PTGER3 was over-expressed in 3T3-L1 adipocytes and the effect on basal and insulin stimulated glucose transport and Glut 4 translocation was determined.
  • Con indicates control 3T3-L1 adipocytes that do not express hPTGER3.
  • FC indicates the fold change defined as the following ratio; glucose transport in hPTGER3-expressing cells incubated for 1 hour with 1 uM sulprostone /glucose transport in non-PTGER3 -expressing cells incubated for 1 hour with 1 uM sulprostone.
  • h is human
  • n is the number of experiments. SEM is the standard error of the mean.
  • PTGER3 contains the following protein domains (designated with reference to SEQ ID NO:84): 7 transmembrane receptor (rhodopsin family) (PFOOOOl) at amino acids 65 to 346. This receptor may have many biological functions, which involve digestion, nervous system, kidney reabsorption, and uterine contraction activities.
  • the PTGER3 receptor is a G protein coupled receptor linked to the inhibition of adenylate cyclase (see, e.g., Kunapuli, et al. Biochem. J. 298:263-267 (1994)). Agonists of the PTGER3 receptor can therefore be identified, e.g., by screening cells with high levels of the PTGER3 receptor and treated with forskolin to identify compounds that decrease the levels of cyclic AMP.
  • PTRGE3 selective agonists have been described.
  • Probe set 204897 detects PTGER4 nucleic acid sequences. Expression of PTGER4 transcripts was decreased in pio compared to vehicle treated cultures of primary human adipocytes in the gene profiling experiment.
  • B/C indicates sample is from Basal or Clamp; "Pre-Pio” and “Post-Pio” indicates sample was taken before or after 24 hours of pioglitazone treatment; “Mean Expr” indicates mean expression; “SEM” indicates standard error of mean; “n” indicates number of patient samples; “Fold Change” indicates fold change of primary human adipocytes post-pio in comparison to pre-pio samples.
  • PTGER4 was also evaluated using real-time PCR. The results further show that PTGER4 is significantly under-expressed in primary cultured human adipocytes treated with pio when compared to vehicle.
  • “Fold Change” indicates the fold expression calculated as the ratio of the mean pio expression/ mean vehicle expression. Numbers in parentheses indicates the number of primary human adipocyte samples analyzed by real-time PCR.
  • Probe set 204897 detects PTGER4 nucleic acid sequences. Expression of PTGER4 transcripts was decreased in rosi compared to vehicle treated cultures of primary human adipocytes in the gene profiling experiment.
  • B/C indicates sample is from Basal or Clamp; "Pre-Rosi” and “Post-Rosi” indicates sample was taken before or after 24 hours of rosiglitazone treatment; “Mean Expr” indicates mean expression; “SEM” indicates standard error of mean; “n” indicates number of patient samples; “Fold Change” indicates fold change of primary human adipocytes post-rosi in comparison to pre-rosi samples.
  • PTGER4 was also evaluated using real-time PCR. The results further show that PTGER4 is significantly under-expressed in primary cultured human adipocytes treated with rosi when compared to vehicle.
  • “Fold Change” indicates the fold expression calculated as the ratio of the mean rosi expression/ mean vehicle expression. Numbers in parentheses indicates the number of primary human adipocyte samples analyzed by real-time PCR.
  • PTGER4 contains the following protein domains (designated with reference to SEQ ID NO:90): C.elegans Srg family integral membrane protein (PF02118) at amino acids 1 to 293; 7TM chemoreceptor (PFOl 604) at amino acids 19 to 299; and 7 transmembrane receptor (rhodopsin family) (PFOOOOl) at amino acids 34 to 329.
  • This receptor can activate T-cell factor signaling. It has been shown to mediate PGE2 induced expression of early growth response 1 (EGRl), regulate the level and stability of cyclooxygenase-2 mRNA, and lead to the phosphorylation of glycogen synthase kinase-3. Knockout studies in mice suggest that this receptor may be involved in the neonatal adaptation of circulatory system, osteoporosis, as well as initiation of skin immune responses.
  • EGRl early growth response 1
  • Probe set 209496 detects RARRES2 nucleic acid sequences. Expression of RARRES2 transcripts was decreased in diabetic compared to lean patients in the gene profiling experiment.
  • B/C indicates sample is from Basal or Clamp; "Mean Expr” indicates mean expression; “SEM” indicates standard error of mean; “n” indicates number of patient samples; “Fold Change” indicates fold change of diabetics in comparison to lean patients.
  • RARRES2 was also evaluated using real-time PCR. The results further show that RARRES2 is significantly under-expressed in subcutaneous adipose from diabetic individuals when compared to subcutaneous adipose from lean individuals.
  • “Fold Change” indicates the fold expression calculated as the ratio of the mean diabetic expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.
  • Probe set 209496 detects RARRES2 nucleic acid sequences.
  • B/C indicates sample is from Basal or Clamp; "Mean Expr” indicates mean expression; “SEM” indicates standard error of mean; “n” indicates number of patient samples; “Fold Change” indicates fold change of obese in comparison to lean patients.
  • RARRES2 was also evaluated using real-time PCR. The results further show that RARRES2 is significantly under-expressed in subcutaneous adipose from obese individuals when compared to subcutaneous adipose from lean individuals.
  • “Fold Change” indicates the fold expression calculated as the ratio of the mean obese expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.
  • Probe set 209496 detects RARRES2 nucleic acid sequences. Expression of RARRES2 transcripts was increased in adipose tissues compared to all other human adult tissues in the gene profiling experiment.
  • Mean Expr indicates mean expression; “SEM” indicates standard error of mean; “n” indicates number of tissue samples; “Fold Change” indicates fold change of adipose tissues in comparison to all other human adult tissues profiled.
  • RARRES2 was also evaluated using real-time PCR. The results further show that RARRES2 is significantly over-expressed in adipose tissues when compared to all other human adult tissues.
  • “Fold Change” indicates the fold expression calculated as the ratio of the mean adipose tissues expression/ mean other tissues expression. Numbers in parentheses indicates the number of human adult tissue samples analyzed by real-time PCR.
  • RARRES2 contains the following protein domains (designated with reference to SEQ ID NO:96): Signal peptide at amino acids 1 to 20. A soluble active secreted form of RARRES2 has been detected (Meder, W. et al, FEBS Lett. 2003 Dec 18;555(3):495- 9) and this is displayed in SEQ ID NO:97.
  • RARRES2 is the ligand for ChemR23. ChemR23 is a putative chemoattractant receptor relatively specific for antigen-presenting cells and it could play an important role in the recruitment or trafficking of these cell populations (Samson, M. et al, Eur J Immunol. 28:1689-7000 (1998); Wittamer, V., et al, J Exp Med. 198(7):977-85 (2003)).
  • Probe set 228730 detects SCRN2 nucleic acid sequences. Expression of SCRN2 transcripts was decreased in diabetic compared to lean patients in the gene profiling experiment.
  • B/C indicates sample is from Basal or Clamp; "Mean Expr” indicates mean expression; “SEM” indicates standard error of mean; “n” indicates number of patient samples; “Fold Change” indicates fold change of diabetics in comparison to lean patients.
  • SCRN2 was also evaluated using real-time PCR. The results further show that SCRN2 is significantly under-expressed in subcutaneous adipose from diabetic individuals when compared to subcutaneous adipose from lean individuals.
  • “Fold Change” indicates the fold expression calculated as the ratio of the mean diabetic expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.
  • Probe set 228730 detects SCRN2 nucleic acid sequences. Expression of SCRN2 transcripts was increased in adipose tissues compared to all other human adult tissues in the gene profiling experiment.
  • Mean Expr indicates mean expression; “SEM” indicates standard error of mean; “n” indicates number of tissue samples; “Fold Change” indicates fold change of adipose tissues in comparison to all other human adult tissues profiled.
  • SCRN2 was also evaluated using real-time PCR. The results further show that SCRN2 is significantly over-expressed in adipose tissues when compared to all other human adult tissues.
  • “Fold Change” indicates the fold expression calculated as the ratio of the mean adipose tissues expression/ mean other tissues expression. Numbers in parentheses indicates the number of human adult tissue samples analyzed by real-time PCR.
  • SCRN2 contains the following protein domains (designated with reference to SEQ ID NO:103): Peptidase family U34 (PF03577) at amino acids 11 to 370.
  • Probe set 229560 detects TLR8 nucleic acid sequences. Expression of TLR8 transcripts was increased in diabetic compared to lean patients in the gene profiling experiment.
  • TLR8 was also evaluated using real-time PCR. The results further show that TLR8 is significantly over-expressed in subcutaneous adipose from diabetic individuals when compared to subcutaneous adipose from lean individuals.
  • “Fold Change” indicates the fold expression calculated as the ratio of the mean diabetic expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.
  • TLR8 contains the following protein domains (designated with reference to SEQ ID NO:109): TIR domain (PF01582) at amino acids 900 to 1039; Leucine Rich Repeat (PF00560) at amino acids 82 to 105, 106 to 143, 241 to 264, 265 to 305, 306 to 329, 330 to 354, 658 to 681, 731 to 754; and 1 transmembrane domain (TMHMM2.0) at amino acids 844 to 866.
  • TLR8 is thought to recognize pathogen-associated molecular patterns (PAMPs) that are expressed on infectious agents and mediate the production of cytokines necessary for the development of an effective immune response.
  • PAMPs pathogen-associated molecular patterns
  • Probe set 210130 detects TM7SF2 nucleic acid sequences. Expression of TM7SF2 transcripts was decreased in diabetic compared to lean patients in the gene profiling experiment.
  • B/C indicates sample is from Basal or Clamp; "Mean Expr” indicates mean expression; “SEM” indicates standard error of mean; “n” indicates number of patient samples; “Fold Change” indicates fold change of diabetics in comparison to lean patients.
  • TM7SF2 was also evaluated using real-time PCR. The results further show that TM7SF2 is significantly under-expressed in subcutaneous adipose from diabetic individuals when compared to subcutaneous adipose from lean individuals.
  • “Fold Change” indicates the fold expression calculated as the ratio of the mean diabetic expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.
  • Probe set 210130 detects TM7SF2 nucleic acid sequences. Expression of TM7SF2 transcripts was decreased in obese compared to lean patients in the gene profiling experiment.
  • B/C indicates sample is from Basal or Clamp; "Mean Expr” indicates mean expression; “SEM” indicates standard error of mean; “n” indicates number of patient samples; “Fold Change” indicates fold change of obese in comparison to lean patients.
  • Probe set 210130 detects TM7SF2 nucleic acid sequences. Expression of TM7SF2 transcripts was increased in adipose tissues compared to all other human adult tissues in the gene profiling experiment.
  • Mean Expr indicates mean expression; “SEM” indicates standard error of mean; “n” indicates number of tissue samples; “Fold Change” indicates fold change of adipose tissues in comparison to all other human adult tissues profiled.
  • TM7SF2 contains the following protein domains (designated with reference to SEQ ID NO:115): Protein of unknown function (DUF1295) (PF06966) at amino acids 200 to 409; Ergosterol biosynthesis ERG4/ERG24 family (PFO 1222) at amino acids 7 to 418; and 7 transmembrane domains (TMHMM2.0) at amino acids 13 to 35, 62 to 81, 102 to 124, 129 to 148, 255 to 277, 287 to 304, 355 to 377.
  • the transmembrane region shares 59% identity with the transmembrane region of the lamin B receptor and 38 to 46% identity with the transmembrane regions of the C14 sterol reductases from different species. This suggests TM7SF2 may play a role in sterol metabolism.
  • Probe set 220065 detects TNMD nucleic acid sequences. Expression of
  • TNMD transcripts was increased in diabetic compared to lean patients in the gene profiling experiment.
  • B/C indicates sample is from Basal or Clamp; "Mean Expr” indicates mean expression; “SEM” indicates standard error of mean; “n” indicates number of patient samples; “Fold Change” indicates fold change of diabetics in comparison to lean patients.
  • TNMD was also evaluated using real-time PCR. The results further show that TNMD is significantly over-expressed in subcutaneous adipose from diabetic individuals when compared to subcutaneous adipose from lean individuals.
  • “Fold Change” indicates the fold expression calculated as the ratio of the mean diabetic expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.
  • Probe set 220065 detects TNMD nucleic acid sequences. Expression of TNMD transcripts was increased in obese compared to lean patients in the gene profiling experiment.
  • B/C indicates sample is from Basal or Clamp; "Mean Expr” indicates mean expression; “SEM” indicates standard error of mean; “n” indicates number of patient samples; “Fold Change” indicates fold change of obese in comparison to lean patients.
  • Probe set 220065 detects TNMD nucleic acid sequences. Expression of TNMD transcripts was increased in adipose tissues compared to all other human adult tissues in the gene profiling experiment.
  • Mean Expr indicates mean expression; “SEM” indicates standard error of mean; “n” indicates number of tissue samples; “Fold Change” indicates fold change of adipose tissues in comparison to all other human adult tissues profiled.
  • TNMD contains the following protein domains (designated with reference to SEQ ID NO: 125): BRICHOS domain (PF04089) at amino acids 93 to 186; and 1 transmembrane domain (TMHMM2.0) at amino acids 31 to 50.
  • TNMD may function as a type II transmembrane protein on cell surface (Shukunami, C. et ah, Biochem Biophys Res Commun. 280:1323-7 (2001)).
  • Amino acid sequence of rat ADPN encoded by the DNA sequence shown in SEQ ID NO: 5.
  • SEQ ID NO: 15 Amino acid sequence of human CMAl, a soluble active secreted form derived from SEQ ID NO: 14.
  • SEQIDNO: 19 AminoacidsequenceofratCMAl encodedbytheDNAsequenceshowninSEQIDNO: 18.
  • SEQIDNO:21 Amino acid sequence of human DUSP4 encoded by the DNA sequence shown in SEQ ID NO: 20.
  • Amino acid sequence of mouse DUSP4 encoded by the DNA sequence shown in SEQ ID NO: 24.
  • SEQIDNO:27 Amino acid sequence of rat DUSP4 encoded by the DNA sequence shown in SEQ ID NO: 26.

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Abstract

La présente invention concerne des compositions et des méthodes destinées au diagnostic et au traitement de l'obésité, du diabète et de l'insulinorésistance. L'invention concerne plus particulièrement des méthodes destinées à l'identification de modulateurs des polynucléotides ou polypeptides de l'invention et à l'utilisation de ces modulateurs pour le traitement de l'obésité et/ou du diabète, ainsi que des méthodes de diagnostic de l'obésité et/ou du diabète, consistant à mesurer les niveaux de polynucléotides ou de polypeptides de l'invention chez un patient.
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EP1687330A1 (fr) * 2003-10-28 2006-08-09 Protemix Discovery Limited Peptides avec action contre l'obesite et autre utilisations apparentees
WO2007027630A2 (fr) * 2005-08-30 2007-03-08 Smithkline Beecham Corporation Genes associes au diabete de type ii
WO2007106488A2 (fr) * 2006-03-13 2007-09-20 Wyeth Modulateurs de la gluconeogenese
WO2008061902A2 (fr) * 2006-11-24 2008-05-29 Vrije Universiteit Brussel Ciblage de l-3-hydroxyacyl-coenzyme a déshydrogénase, à chaîne courte (hadhsc) dans les troubles de l'homéostasie glucosique
WO2009057461A1 (fr) * 2007-10-31 2009-05-07 Kobe University Agent thérapeutique pour le diabète
WO2012051567A3 (fr) * 2010-10-15 2012-12-06 The Trustees Of Columbia University In The City Of New York Gènes de l'obésité, leurs protéines et leurs utilisations
EP2710029B1 (fr) * 2011-05-20 2017-11-08 Covagen AG Nouveaux composés de liaison à une chymase et leurs utilisations médicales
US11154591B2 (en) 2016-10-14 2021-10-26 The Trustees Of Columbia University In The City Of New York Methods of treating alcohol abuse disorder

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JP2018068235A (ja) * 2016-10-31 2018-05-10 国立大学法人神戸大学 抗インスリン抵抗性物質スクリーニング方法
SG11202010729XA (en) * 2018-05-07 2020-11-27 Jennewein Biotechnologie Gmbh A SIMPLE METHOD FOR THE PURIFICATION OF LACTO-N-NEOTETRAOSE (LNnT) FROM CARBOHYDRATES OBTAINED BY MICROBIAL FERMENTATION

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US6854933B2 (en) * 2002-08-07 2005-02-15 Deepwater Technologies, Inc. Vertically restrained centerwell SPAR
WO2004053124A1 (fr) * 2002-12-06 2004-06-24 Shionogi & Co., Ltd. Procede de criblage d'un remede ou d'un agent preventif contre le diabete

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WO2004028479A2 (fr) * 2002-09-25 2004-04-08 Genentech, Inc. Nouvelles compositions et methodes de traitement du psoriasis

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EP1687330A4 (fr) * 2003-10-28 2007-03-14 Protemix Discovery Ltd Peptides avec action contre l'obesite et autre utilisations apparentees
EP1687330A1 (fr) * 2003-10-28 2006-08-09 Protemix Discovery Limited Peptides avec action contre l'obesite et autre utilisations apparentees
WO2007027630A2 (fr) * 2005-08-30 2007-03-08 Smithkline Beecham Corporation Genes associes au diabete de type ii
WO2007027630A3 (fr) * 2005-08-30 2007-07-12 Smithkline Beecham Corp Genes associes au diabete de type ii
US7763441B2 (en) 2006-03-13 2010-07-27 Wyeth Modulators of gluconeogenesis
WO2007106488A2 (fr) * 2006-03-13 2007-09-20 Wyeth Modulateurs de la gluconeogenese
WO2007106488A3 (fr) * 2006-03-13 2007-11-01 Wyeth Corp Modulateurs de la gluconeogenese
WO2008061902A2 (fr) * 2006-11-24 2008-05-29 Vrije Universiteit Brussel Ciblage de l-3-hydroxyacyl-coenzyme a déshydrogénase, à chaîne courte (hadhsc) dans les troubles de l'homéostasie glucosique
WO2008061902A3 (fr) * 2006-11-24 2009-03-05 Univ Bruxelles Ciblage de l-3-hydroxyacyl-coenzyme a déshydrogénase, à chaîne courte (hadhsc) dans les troubles de l'homéostasie glucosique
WO2009057461A1 (fr) * 2007-10-31 2009-05-07 Kobe University Agent thérapeutique pour le diabète
JP5209636B2 (ja) * 2007-10-31 2013-06-12 国立大学法人神戸大学 糖尿病治療剤
US8569232B2 (en) 2007-10-31 2013-10-29 Kobe University Therapeutic agent for diabetes
WO2012051567A3 (fr) * 2010-10-15 2012-12-06 The Trustees Of Columbia University In The City Of New York Gènes de l'obésité, leurs protéines et leurs utilisations
US20130274181A1 (en) * 2010-10-15 2013-10-17 The Trustees Of Columbia University In The City Of New York Obesity-related genes and their proteins and uses thereof
CN103391784A (zh) * 2010-10-15 2013-11-13 纽约市哥伦比亚大学理事会 肥胖症-相关的基因和它们的蛋白和其用途
CN108404115A (zh) * 2010-10-15 2018-08-17 纽约市哥伦比亚大学理事会 肥胖症-相关的基因和它们的蛋白和其用途
US10912816B2 (en) 2010-10-15 2021-02-09 The Trustees Of Columbia University In The City Of New York Obesity-related genes and their proteins and uses thereof
EP2710029B1 (fr) * 2011-05-20 2017-11-08 Covagen AG Nouveaux composés de liaison à une chymase et leurs utilisations médicales
US11154591B2 (en) 2016-10-14 2021-10-26 The Trustees Of Columbia University In The City Of New York Methods of treating alcohol abuse disorder

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