WO2008109689A2 - Methods for identifying agents of use in treating obesity and diabetes - Google Patents

Methods for identifying agents of use in treating obesity and diabetes Download PDF

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Publication number
WO2008109689A2
WO2008109689A2 PCT/US2008/055941 US2008055941W WO2008109689A2 WO 2008109689 A2 WO2008109689 A2 WO 2008109689A2 US 2008055941 W US2008055941 W US 2008055941W WO 2008109689 A2 WO2008109689 A2 WO 2008109689A2
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msh
agrp
npy
brain tissue
mice
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PCT/US2008/055941
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French (fr)
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WO2008109689A3 (en
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Michael Cowley
Pablo Enriori
Kevin Grove
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Oregon Health & Science University
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    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G01N2333/5755—Neuropeptide Y
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/665—Assays involving proteins derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
    • G01N2333/68—Melanocyte-stimulating hormone [MSH]
    • G01N2333/685—Melanocyte-stimulating hormone [MSH] alpha-Melanotropin
    • 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

  • FIELD This application relates to the field of obesity and diabetes, specifically methods of screening for agents that can be used to treat obesity, diabetes, and related disorders are disclosed.
  • obesity is complex and multi-factorial. Increasing evidence suggests that obesity is not a simple problem of self-control but is a complex disorder involving appetite regulation and energy metabolism. In addition, obesity is associated with a variety of conditions associated with increased morbidity and mortality in a population. Although the etiology of obesity is not definitively established, genetic, metabolic, biochemical, cultural and psychosocial factors are believed to contribute. In general, obesity has been described as a condition in which excess body fat puts an individual at a health risk.
  • the disclosed methods include contacting a test agent to brain tissue in vitro in media that includes artificial cerebrospinal fluid (aCSF).
  • the brain tissue includes both proopiomelanocortin (POMC) neurons and neuropeptide Y/Agouti related peptide (NP Y/ AgRP) neurons.
  • POMC proopiomelanocortin
  • NP Y/ AgRP neuropeptide Y/Agouti related peptide
  • the amount and/or concentration of the neuropeptides AgRP, NPY and alpha-melanotropin ( ⁇ -MSH) secreted by the brain tissue is measured.
  • the ratio of ⁇ -MSH secreted from the brain tissue to AgRP secreted from the brain tissue is determined and compared to a control, for example, a standard value, or the ratio of ⁇ -MSH secreted from the brain tissue to AgRP secreted from the brain tissue not contacted with a test agent.
  • the amount of NPY secreted by the brain tissue is compared to a control, for example, a standard value, or the amount of NPY secreted by brain tissue not contacted with a test agent.
  • Figure 1A-1D is a set of graphs showing body weights and feeding characteristics for mice fed regular chow (Control) versus (vs.) mice fed a high fat diet (HFD) (diet-induced obese-restored (DIO-R) and diet-induced obese (DIO)).
  • Figure IA is graph showing body weight distribution of Control (12% fat diet) and DIO-R/DIO (45% fat diet) mice after 20 weeks. Normality test (DIO-R/DIO) was p ⁇ 0.0001.
  • Figure IB is graph showing the body weight change of Control, DIO-R, and DIO mice over 20 weeks. Results are expressed as mean ⁇ standard error in the mean (SEM) (*p ⁇ 0.001).
  • Figure 1C is graph showing total calorie, total fat, total protein, and total carbohydrate calorie intake for Control, DIO-R, and DIO groups after 20 weeks. Results are expressed as mean ⁇ SEM (*p ⁇ 0.001).
  • Figure ID is graph showing the feed efficiency (weight gained/kcal consumed) of Control, DIO- R, and DIO groups.
  • Figure 2A-2D is a set of graphs showing the effect of peripheral and central leptin on body weight and calorie intake in Control, DIO-R, and DIO mice
  • Figure 2A is a graph showing body weight change (%) 24 hours after intraperitoneal (i.p.) saline or leptin (2 ⁇ g/g/d).
  • Figure 2B is a graph showing calorie intake two days after i.p. leptin (2 ⁇ g/g/d). Data from each group were normalized to their own saline control.
  • Figure 2C is a graph showing body weight change (%) 24 hours after intracerebroventricular (i.c.v.) artificial cerebrospinal fluid (aCSF) or leptin (0.1 ⁇ g).
  • Figure 2D is a graph showing calorie intake one day after i.c.v. leptin (0.1 ⁇ g). Data from each group were normalized to their own aCSF control.
  • Figure 3A-3J is a set of graphs showing leptin modulation of hypothalamic neuropeptide secretion in Control, DIO-R, and DIO groups. Data from each group were normalized to baseline levels.
  • Figures 3A-3 J represent four independent experiments.
  • Figure 3A-C is a set of graphs showing leptin modulation of AgRP secretion.
  • Figure 3D-3F is a set of graphs showing leptin modulation of NPY secretion. The horizontal bar in Figure 3D show the approximate basal level of NPY secretion.
  • Figure 3G-3I is a set of graphs showing leptin modulation of ⁇ - MSH secretion.
  • Figure 3 J is a graph showing the ⁇ -MSH/AgRP ratio of secretion. The horizontal bar in Figure 3 J show the approximate basal ratio of ⁇ -MSH/AgRP secretion.
  • Figure 4A-4E is a set of graphs showing mRNA neuropeptide expression after i.p. leptin (2 ⁇ g/g), expression of leptin obese receptor (ObRb) mRNA, and concentration of desacetyl- ⁇ -MSH in the hypothalamic arcuate nucleus (ARH) of Control, DIO-R and DIO groups. Results are expressed as mean ⁇ SEM (*p ⁇ 0.05, ***p ⁇ 0.001).
  • Figure 4A is a graph showing caudal AgRP mRNA expression after i.p. lep tin/saline.
  • Figure 4B is a graph showing total NPY mRNA expression after i.p. lep tin/saline.
  • Figure 4C is a graph showing total proopiomelanocortin (POMC) mRNA expression after i.p. leptin/saline.
  • Figure 4D is a graph showing baseline leptin obese receptor (ObRb) expression.
  • Figure 4E is a graph showing desacetyl- ⁇ -MSH concentration.
  • Figure 5A-5D is a set of images and graphs showing the effect of i.p. leptin (2 ⁇ g/g) on signal transduction in the ARH. Results expressed as mean ⁇ SEM. Representative microphotographs of hypothalamic sections are shown from Control, DIO-R, and DIO groups (*p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ## p ⁇ O-(M saline Control vs. saline DIO).
  • Figure 5A shows expression of c-Fos 30 minutes after i.p. leptin/saline.
  • Figure 5B shows expression of pStat3 30 minutes after i.p. leptin/saline.
  • Figure 5C shows expression of phospho MAP kinase (pMAPK) 30 minutes after i.p. leptin/saline.
  • Figure 5D shows expression of suppressor of cytokine signaling-3 (SOCS-3) mRNA 45 minutes after i.p. saline/leptin.
  • Figure 6A-6B is a set of graphs and images showing melanocortin system integrity (***p ⁇ 0.001).
  • Figure 6A is a graph showing the effect of i.p. melanotan-II (MTII) (1 ⁇ g/g) over 24 hours of food intake in Control, DIO-R, and DIO groups. Data from each group were normalized to their own saline control.
  • Figure 6B is a representative microphotograph and a graph of results of baseline Melanocortin 4 receptor (MC4R) mRNA expression in paraventricular nucleus of the hypothalamus (PVH) of Control, DIO-R, and DIO groups. Results expressed as mean ⁇ SEM.
  • M4R Melanocortin 4 receptor
  • Figure 7A- 7F is a set of graphs showing the restoration of leptin sensitivity after changing from HFD to regular chow (*p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001).
  • Figure 7A is a graph showing body weight change of Control, DIO, and Restored mice at 37 weeks. Results are expressed as mean ⁇ SEM.
  • Figure 7B is a graph showing glucose tolerance in DIO and Restored mice after 14 hours of fasting. Results are expressed as mean ⁇ SEM.
  • Figure 7C is a graph showing body weight change (%) in Control, Restored, and DIO mice 24 hours after i.p. saline or leptin (2 ⁇ g/g/d).
  • Figure 7D is a graph showing leptin (100 nM) modulation of AgRP secretion in Control, Restored, and DIO groups. Data from each group is normalized to baseline levels.
  • Figure 7E is a graph showing leptin (100 nM) modulation of NPY secretion in Control, Restored, and DIO groups. Data from each group is normalized to baseline levels.
  • Figure 7F is a graph showing leptin (100 nM) modulation of ⁇ -MSH secretion in Control, Restored, and DIO groups. Data from each group normalized to baseline levels.
  • Figure 8 is a graph showing the relationship of initial body weight and final weight of identically aged mice on HFD.
  • Figure 9A-9C is a set of graphs showing baseline neuropeptide secretion from hypothalamic explants in Control, DIO-R, and DIO groups. Data were pooled from four independent experiments. Data are represented as mean ⁇ SEM.
  • Figure 9A is a graph showing AgRP baseline secretion.
  • Figure 9B is a graph showing NPY baseline secretion.
  • Figure 9C is a graph showing ⁇ -MSH baseline secretion.
  • Figure 10A-10B is a set of graphs showing the concentration of POMC peptide-derivatives in the ARH were measured by radio immunoassay (RIA) after high performance liquid chromatography (HPLC) separation in hypothalamic explants. Synthetic peptides were injected on the HPLC to determine retention times. Predicted retention times allowed for analysis of specific regions along the gradient for RIA analysis.
  • Figure 1OA is a graph showing the production of desacetyl- ⁇ -MSH, and acetyl- ⁇ -MSH in ARH of Control, DIO-R, and DIO mice.
  • Figure 1OB is a graph showing the production of adrenocorticotropic hormone (ACTH) and CLIP in ARH of Control, DIO-R, and DIO mice.
  • ACTH adrenocorticotropic hormone
  • Figure 1 IA-I IB is a set of bar graphs showing glucose sensing is lost in POMC-mut-Kir6.2 neurons.
  • Figure 12A-12B is a set of bar graphs showing that glucose-sensing is lost in POMC neurons of mice on HFD.
  • Figure 12A is two bar graphs showing that glucose-induced ⁇ -MSH release from hypothalamic slices of wild-type C57BL/6 mice fed chow or a high-fat diet for 20 weeks.
  • Figure 12B is a bar graph showing the percentage of POMC neurons activated by 5 mM glucose in loose-patch recordings from POMC-GFP mice fed either chow or HFD for eight weeks.
  • Figure 13A-13B is a set of bar graphs showing acute inhibition or genetic deletion of UCP2 restores or prevents loss of glucose sensing in POMC neurons as a result of obesity induced by HFD.
  • ⁇ -MSH secretion from hypothalamic slices from wild-type ( Figure 13A WT) and Ucp2 ⁇ ' ⁇ ( Figure 13B) mice in response to glucose, with or without genipin (20 ⁇ M). Data are presented as mean ⁇ SEM, n 6 mice for each experimental condition. Asterisk, P ⁇ 0.05.
  • Figure 14A-14C are a set of plots showing the average baseline secretion of
  • aCSF Artificial cerebrospinal fluid
  • ACTH Adrenocorticotropic hormone
  • AgRP Agouti-related peptide
  • ⁇ -MSH alpha-melanotropin
  • ARH Hypothalamic arcuate nucleus
  • ISH In situ Hybridization
  • MC4R Melanocortin 4 receptor
  • NPY Neuropeptide Y
  • ObRb Leptin obese receptor
  • PAM Peptidyl- ⁇ -monooxygenase
  • POMC Proopiomelanocortin
  • PVH Paraventricular nucleus of the hypothalamus
  • Antagonist A substance that tends to nullify the action of another, such as an agent that binds to a cell receptor without eliciting a biological response, for example to block the specific binding of substances that could elicit such responses.
  • AgRP is an antagonist of a melanocortin receptor, such as the melanocortin 4 receptor (MC4R).
  • M4R melanocortin 4 receptor
  • an antagonist decreases signal transduction from the receptor it binds.
  • Agent or Test Agent Any polypeptide, compound, small molecule, organic compound, salt, polynucleotide, or other molecule of interest.
  • Agonist A substance that specifically binds to a specific receptor and triggers a response in the cell, for example the binding of ⁇ -MSH to a melanocortin receptor on the surface of a cell.
  • ⁇ -MSH is an agonist of a melanocortin receptor, such as the melanocortin 4 receptor (MC4R).
  • Agouti-related peptide A neuropeptide made in the arcuate nucleus of the brain that increases appetite and decreases metabolism.
  • Agouti- related protein is an antagonist of the melanocortin-3 and melanocortin-4 receptor.
  • An exemplary amino acid sequence of agouti-related peptide can be found at
  • Animal Living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds.
  • the term mammal includes both human and non-human mammals.
  • subject includes both human and veterinary subjects, for example mice.
  • Antibody A polypeptide ligand comprising at least a light chain or heavy chain immunoglobulin variable region which specifically binds an epitope of an antigen, such as one of a AgRP, NPY, or ⁇ -MSH neuropeptide or a fragment thereof.
  • an AgRP specific binding agent binds substantially only to a defined target, such as an AgRP, NPY, or ⁇ -MSH neuropeptide.
  • an AgRP specific binding agent is an agent that binds substantially to an AgRP polypeptide. If an agent, such as an antibody, specifically binds AgRP it does not specifically bind other peptides including NPY and ⁇ -MSH neuropeptides.
  • an NPY specific binding agent is an agent that binds substantially to an NPY neuropeptide, but not AgRP and ⁇ -MSH neuropeptides.
  • an ⁇ -MSH specific binding agent is an agent that binds substantially to an ⁇ -MSH neuropeptide, but not AgRP and NPY neuropeptides.
  • a minor degree of non-specific interaction may occur between a molecule, such as a specific binding agent, and a non-target polypeptide.
  • Specific binding can be distinguished as mediated through specific recognition of the antigen. Although selectively reactive antibodies bind antigen, they can do so with low affinity.
  • Specific binding typically results in greater than 2- fold, such as greater than 5-fold, greater than 10-fold, or greater than 100-fold increase in amount of bound antibody or other ligand (per unit time) to an AgRP, NPY, or ⁇ -MSH neuropeptide.
  • a variety of immunoassay formats are appropriate for selecting antibodies specifically immunoreactive with a particular protein.
  • solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York
  • Antibodies can include a heavy chain and a light chain, each of which has a variable region, termed the variable heavy (VH) region and the variable light (VL) region. Together, the VH region and the VL region are responsible for binding the antigen recognized by the antibody.
  • VH variable heavy
  • VL variable light
  • a scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains.
  • the term also includes recombinant forms such as chimeric antibodies (for example, humanized murine antibodies), heteroconjugate antibodies (such as, bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, Immunology, 3rd Ed., W. H. Freeman & Co., New York, 1997.
  • a "monoclonal antibody” is an antibody produced by a single clone of
  • Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody- forming cells from a fusion of myeloma cells with immune spleen cells. These fused cells and their progeny are termed "hybridomas.” Monoclonal antibodies include humanized monoclonal antibodies.
  • Alpha-melanotropin (Alpha-melanocyte stimulating hormone, ⁇ -MSH, or ⁇ -MSH): An agonist peptide ligand for melanocortin receptors, such as MC4R. ⁇ -MSH along with adrenocorticotropic hormone (ACTH), ⁇ -MSH and ⁇ -MSH are processed from the pre-prohormone proopiomelanocortin (POMC).
  • ⁇ -MSH melanocortin receptors
  • ACTH adrenocorticotropic hormone
  • POMC pre-prohormone proopiomelanocortin
  • Artificial cerebrospinal fluid Cell culture medium that approximates the physiological conditions of cerebrospinal fluid.
  • artificial cerebrospinal fluid can include NaCl, Na 2 HPO 4 , KCl, CaCl 2 , MgSO 4 , NaHCO 3 , glucose, ascorbic acid, and aprotinin.
  • Binding A specific interaction between two or more molecules, such as the binding of an antibody and an antigen (for example an antibody to NPY, ⁇ -MSH or AgRP) or the binding of a ligand to a receptor, such as the binding of AgRP or ⁇ - MSH to a melanocortin receptor, such as MC4R.
  • binding affinity is calculated by a modification of the Scatchard method described by Frankel et al, MoI. Immunol, 16:101-106, 1979.
  • binding affinity is measured by an antigen/antibody dissociation rate.
  • a high binding affinity is measured by a competition radioimmunoassay (RIA).
  • a high binding affinity is at least about 1 x 10 ⁇ 8 M.
  • a high binding affinity is at least about 1.5 x 10 ⁇ 8 , at least about 2.0 x 10 ⁇ 8 , at least about 2.5 x 10 ⁇ 8 , at least about 3.O x 10 "8 , at least about 3.5 x 10 "8 , at least about 4.0 x 10 "8 , at least about 4.5 x 10 "8 , or at least about 5.0 x 10 "8 M.
  • Body Mass Index A mathematical formula for measuring body mass in humans, also sometimes called Quetelet's Index. BMI is calculated by dividing weight (in kg) by height (in meters ). The current standards for both men and women accepted as "normal" are a BMI of 20-24.9 kg/m 2 . In one embodiment, a BMI of greater than 25 kg/m can be used to identify an obese subject. Grade I obesity corresponds to a BMI of 25-29.9 kg/m 2 . Grade II obesity corresponds to a BMI of 30-40 kg/m ; and Grade III obesity corresponds to a BMI greater than 40 kg/m 2 (Jequier, Am. J CHn. Nutr., 45 : 1035-47, 1987). Ideal body weight will vary among species and individuals based on height, body build, bone structure, and sex.
  • Caloric intake or calorie intake The number of calories (energy) consumed by an individual.
  • Calorie A unit of measurement in food.
  • Chromatography The process of separating a mixture, for example a mixture containing AgRP, NPY and ⁇ -MSH. It involves passing a mixture through a stationary phase, which separates molecules of interest from other molecules in the mixture and allows one or more molecules of interest to be isolated.
  • Examples of methods of chromatographic separation include capillary-action chromatography, such as paper chromatography, thin layer chromatography (TLC), column chromatography, fast protein liquid chromatography (FPLC), nano-reversed phase liquid chromatography, ion exchange chromatography, gel chromatography, such as gel filtration chromatography, size exclusion chromatography, affinity chromatography, high performance liquid chromatography (HPLC), and reverse phase high performance liquid chromatography (RP-HPLC) amongst others.
  • capillary-action chromatography such as paper chromatography, thin layer chromatography (TLC), column chromatography, fast protein liquid chromatography (FPLC), nano-reversed phase liquid chromatography, ion exchange chromatography, gel chromatography, such as gel filtration chromatography, size exclusion chromatography, affinity chromatography, high performance liquid chromatography (HPLC), and reverse phase high performance liquid chromatography (RP-HPLC) amongst others.
  • Contacting includes in solution and solid phase, for example contacting brain tissue with a test agent.
  • the test agent may also be a combinatorial library for screening a plurality of compounds.
  • contacting includes contacting a sample with an antibody, for example contacting a sample that contains or is suspected of containing AgRP, NPY and/or ⁇ -MSH, with an antibody that specifically binds AgRP, NPY or ⁇ -MSH.
  • a control can be a known value indicative of basal secretion of a neuropeptide from brain tissue, such as the amount of NPY, AgRP or ⁇ -MSH secreted from brain tissue not treated with an agent.
  • a control can also be a known value indicative of the ratio of the basal secretion of one neuropeptide to another neuropeptide, such as the ratio the amount of ⁇ -MSH secreted to the amount of AgRP secreted from brain tissue not treated with an agent.
  • a difference between a test sample (such as brain tissue contacted with an agent) and a control can be an increase or conversely a decrease. The difference can be a qualitative difference or a quantitative difference, for example a statistically significant difference.
  • a difference is an increase or decrease in amount, relative to a control, of at least about 1 %, such as at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, or greater then 500%.
  • a difference is an increase or decrease in ratio, relative to a control, of at least about 1 %, such as at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, or greater then 500%.
  • Diabetes A failure of cells to transport endogenous glucose across their membranes either because of an endogenous deficiency of insulin and/or a defect in insulin sensitivity. Diabetes is a chronic syndrome of impaired carbohydrate, protein, and fat metabolism owing to insufficient secretion of insulin or to target tissue insulin resistance. It occurs in two major forms: insulin-dependent diabetes mellitus (IDDM, type I) and non-insulin dependent diabetes mellitus (NIDDM, type II) which differ in etiology, pathology, genetics, age of onset, and treatment. The two major forms of diabetes are both characterized by an inability to deliver insulin in an amount and with the precise timing that is needed for control of glucose homeostasis.
  • IDDM insulin-dependent diabetes mellitus
  • NIDDM non-insulin dependent diabetes mellitus
  • Diabetes type I or insulin dependent diabetes mellitus (IDDM) is caused by the destruction of ⁇ cells, which results in insufficient levels of endogenous insulin.
  • Diabetes type II or non-insulin dependent diabetes, results from a defect in both the body's sensitivity to insulin, and a relative deficiency in insulin production.
  • Food intake The amount of food consumed by an individual. Food intake can be measured by volume or by weight. In one embodiment, food intake is the total amount of food consumed by an individual. In another embodiment, food intake is the amount of proteins, fat, carbohydrates, cholesterol, vitamins, minerals, or any other food component, of the individual. “Protein intake” refers to the amount of protein consumed by an individual. Similarly, “fat intake,” “carbohydrate intake,” “cholesterol intake,” “vitamin intake,” and “mineral intake” refer to the amount of proteins, fat, carbohydrates, cholesterol, vitamins, or minerals consumed by an individual.
  • Glucose resistance The loss of the ability of cells and/or tissues to take up glucose, for example in a subject suffering from diabetes.
  • High throughput technique Through a combination of modern robotics, data processing and control software, liquid handling devices, and sensitive detectors, high throughput techniques allows the rapid screening of potential pharmaceutical agents in a short period of time. Through this process one can rapidly identify active compounds, which affect the secretion of neuropeptides, for example the secretion of NPY, AgRP and/or ⁇ -MSH from brain tissue.
  • Isolated An isolated biological component (such as a nucleic acid, peptide or protein) has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, for example the separation of a peptide, such as NPY, AgRP or ⁇ -MSH from brain tissue.
  • Peptides and proteins that have been isolated include nucleic acids and proteins purified by standard purification methods, such as chromatography, for example high performance liquid chromatography (HPLC) and the like.
  • HPLC high performance liquid chromatography
  • the term also embraces peptides, and proteins prepared by recombinant expression in a host cell as well as chemically synthesized peptide and nucleic acids.
  • isolated does not imply that the biological component is free of trace contamination, and can include molecules that are at least 50% isolated, such as at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or even 100% isolated.
  • Immunoassay A biochemical test that measures the presence or concentration of a substance in a sample, such as a biological sample, using the reaction of an antibody to its cognate antigen, for example the specific binding of an antibody to a neuropeptide, such as NPY, ⁇ -MSH, or AgRP. Both the presence of antigen or the amount of antigen present can be measured.
  • a neuropeptide such as NPY, ⁇ -MSH, or AgRP.
  • the neuropeptide is the antigen and the presence and amount of the neuropeptide is determined or measured.
  • Measuring the quantity of antigen can be achieved by a variety of methods.
  • One of the most common is to label either the antigen or antibody with a detectable label.
  • Specific, non-limiting examples of labels include fluorescent tags, enzymatic linkages, and radioactive isotopes (for example 14 C, 32 P, 125 I, and 3 H isotopes and the like).
  • NPY, AgRP and/or ⁇ -MSH is labeled with a radioactive isotope, such as 14 C, 32 P, 125 1, 3 H isotope.
  • an antibody that specifically binds NPY, ⁇ -MSH, or AgRP is labeled.
  • Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed for example in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989) Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998), and Harlow & Lane, (Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, 1988)),
  • a “competitive radioimmunoassay (RIA)” is a type of immunoassay used to test for antigens (for example, neuropeptides present in a sample, such as a biological sample).
  • antigens for example, neuropeptides present in a sample, such as a biological sample.
  • it involves mixing known quantities of radioactive antigen (for example a radioactively labeled neuropeptide, such as a 125 I labeled neuropeptide, for example 125 I labeled , NPY, AgRP or ⁇ -MSH), with antibody to that antigen, then adding unlabeled or "cold” antigen (for example unlabeled antigen present in a sample, such as biological sample obtained from a subject, for example a sample of brain tissue obtained from a subject) and measuring the amount of labeled antigen displaced by the unlabeled antigen.
  • radioactive antigen for example a radioactively labeled neuropeptide, such as a 125 I labeled neuro
  • the radioactive antigen is bound to the antibodies.
  • "cold” i.e. unlabeled
  • the two compete for antibody binding sites - at higher concentrations of "cold” antigen, more of it binds to the antibody, displacing the radioactive variant.
  • the bound antigens are isolated from the unbound ones and the amount of radioactivity measured.
  • a radioimmunoassay can be used to calculate the amount of an antigen in a sample.
  • a radioimmunoassay is used to measure the amount of NPY present in a sample, such as a biological sample, for example brain tissue.
  • a radioimmunoassay is used to measure the amount of ⁇ -MSH present in a sample, such as a biological sample, for example brain tissue.
  • a radioimmunoassay is used to measure the amount of AgRP present in a sample, such as a biological sample, for example brain tissue.
  • Label A detectable compound or composition that is conjugated directly or indirectly to another molecule, such as an antibody or a protein, to facilitate detection of that molecule.
  • Specific, non-limiting examples of labels include fluorescent tags, enzymatic linkages, and radioactive isotopes (for example 14 C, 32 P, 125 1, 3 H isotopes and the like).
  • NPY, AgRP and/or ⁇ -MSH is labeled with a radioactive isotope, such as 14 C, 32 P, 125 1, 3 H isotope.
  • an antibody that specifically binds NPY, AgRP and/or ⁇ -MSH is labeled.
  • Leptin A 16-kD protein that plays a role in the regulation of body weight by inhibiting food intake and stimulating energy expenditure. Defects in leptin production cause severe hereditary obesity in rodents and humans. In addition to its effects on body weight, leptin has a variety of other functions, including the regulation of hematopoiesis, angiogenesis, wound healing, and the immune and inflammatory response. Leptin acts through the leptin receptor, a single- transmembrane-domain receptor of the cytokine receptor family, which is found in many tissues in several alternatively spliced forms. Exemplary amino acid sequences of leptin can be found in GENBANK® at accession nos.
  • Leptin resistance The inability of cells and/or tissue to propagate leptin signaling in response contact of the cells and/or tissue with leptin.
  • leptin resistance in a subject can occur when circulating leptin fails to reach its targets in the brain or that there is a failure of components of the intracellular leptin receptors (ObRb) signaling cascade, for example due to loss of expression or presentation of leptin receptors or other mechanism.
  • ObRb intracellular leptin receptors
  • Neuropeptide Y A 36-amino acid peptide that is a neuropeptide identified in the mammalian brain. NPY is believed to be an important regulator in both the central and peripheral nervous systems and influences a diverse range of physiological parameters, including effects on psychomotor activity, food intake, central endocrine secretion, and vasoactivity in the cardiovascular system. High concentrations of NPY are found in the sympathetic nerves supplying the coronary, cerebral, and renal vasculature and have contributed to vasoconstriction. NPY binding sites have been identified in a variety of tissues, including spleen, intestinal membranes, brain, aortic smooth muscle, kidney, testis, and placenta.
  • NPY neuropeptide Y receptor
  • PYY peptide YY
  • PP pancreatic polypeptide
  • NPY binds to several receptors, including the Yl, Y2, Y3, Y4 (PP), Y5, Y6, and Y7 receptors. These receptors are recognized based on binding affinities, pharmacology, and sequence. Most, if not all of these receptors are G protein coupled receptors.
  • the Yl receptor is generally considered to be postsynaptic and mediates many of the known actions of neuropeptide Y in the periphery. Originally, this receptor was described as having poor affinity for C-terminal fragments of neuropeptide Y, such as the 13-36 fragment, but interacts with the full length neuropeptide Y and peptide YY with equal affinity (e.g. see Patent Cooperation Treaty publication WO 93/09227).
  • NP Y/ AgRP neurons Neurons of the hypothalamic arcuate nucleus named for their ability to produce, neuropeptide Y (NPY) and agouti-related protein (AgRP). These neurons make peptides that potently stimulate food intake not only by increasing neuropeptide Y (NPY) signaling, but by reducing melanocortin signaling via the release of agouti-related peptide (AgRP), an endogenous melanocortin 3/4 receptor antagonist. Since NPY/ AgRP neurons express receptors for leptin and insulin and are inhibited by these hormones, they are activated by a decrease of leptin or insulin signaling.
  • NPY/ AgRP neurons respond to afferent signals reflecting energy deficits (for example low blood glucose) by NPY and AgRP, which exert orexigenic effects (i.e. increased food intake and decreased energy expenditure).
  • Normal Daily Diet The average food intake for an individual of a given species.
  • a normal daily diet can be expressed in terms of caloric intake, protein intake, carbohydrate intake, and/or fat intake.
  • a normal daily diet in humans generally comprises the following: about 2,000, about 2,400, or about 2,800 to significantly more calories.
  • a normal daily diet in humans generally includes about 12 g to about 45 g of protein, about 120 g to about 610 g of carbohydrate, and about 11 g to about 90 g of fat.
  • a low calorie diet would be no more than about 85%, and preferably no more than about 70%, of the normal caloric intake of a human individual.
  • the caloric and nutrient requirements vary depending on the species and size of the animal.
  • the total caloric intake per pound, as well as the percent distribution of protein, carbohydrate and fat varies with the age of the cat and the reproductive state.
  • a general guideline for cats is 40 cal/lb/day (18.2 cal/kg/day).
  • About 30% to about 40% should be protein, about 7% to about 10% should be from carbohydrate, and about 50% to about 62.5% should be derived from fat intake.
  • One of skill in the art can readily identify the normal daily diet of an individual of any species.
  • Obesity A condition in which excess body fat may put a person at health risk (see Barlow and Dietz, Pediatrics 102: E29, 1998; National Institutes of Health, National Heart, Lung, and Blood Institute (NHLBI), Obes. Res. 6 (suppl. 2):51S- 209S, 1998). Excess body fat is a result of an imbalance of energy intake and energy expenditure.
  • the Body Mass Index (BMI) is used to assess obesity.
  • BMI Body Mass Index
  • a BMI of 25.0 kg/m 2 to 29.9 kg/m 2 is overweight, while a BMI of 30 kg/m 2 is obese.
  • waist circumference is used to assess obesity.
  • a waist circumference of 102 cm or more is considered obese, while in women a waist circumference of 89 cm or more is considered obese.
  • Strong evidence shows that obesity affects both the morbidity and mortality of individuals.
  • an obese individual is at increased risk for heart disease, non-insulin dependent (type 2) diabetes, hypertension, stroke, cancer (e.g. endometrial, breast, prostate, and colon cancer), dyslipidemia, gall bladder disease, sleep apnea, reduced fertility, and osteoarthritis, amongst others (see Lyznicki et al., Am. Fam. Phys. 63:2185, 2001).
  • Overweight An individual who weighs more than their ideal body weight.
  • An overweight individual can be obese, but is not necessarily obese.
  • an overweight human individual is any individual who desires to decrease their weight.
  • an overweight human individual is an individual with a BMI of 25.0 kg/m 2 to 29.9 kg/m 2 .
  • Therapeutically effective amount The quantity of a chemical composition or sufficient to achieve a desired effect in a subject being treated. For instance, this can be the amount necessary to treat obesity and/or diabetes or to measurably alter outward symptoms of obesity and/or diabetes.
  • a dosage When administered to a subject, a dosage will generally be used that will achieve target tissue concentrations that has been shown to achieve in vitro inhibition of obesity and/or diabetes or to measurably alter outward symptoms of obesity and/or diabetes.
  • parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like
  • solid compositions such as powder, pill, tablet, or capsule forms
  • conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • Polypeptide A polymer in which the monomers are amino acid residues which are joined together through amide bonds. When the amino acids are alpha- amino acids, either the L-optical isomer or the D-optical isomer can be used, the L- isomers being preferred.
  • the terms "polypeptide” or "protein” or “peptide” as used herein are intended to encompass any amino acid sequence and include modified sequences such as glycoproteins.
  • polypeptide or "protein” or “peptide” is specifically intended to cover naturally occurring proteins, as well as those which are recombinantly or synthetically produced.
  • a peptide is NPY.
  • a peptide is AgRP.
  • a peptide is ⁇ -MSH.
  • POMC Proopiomelanocortin
  • Beta-lipotropin is processed to produce the proteins alpha-lipotropin, beta-endorphins, beta-melanocyte stimulating hormone (MSH), and met-enkephalin.
  • the amino-terminal fragment of POMC is processed to a family of gamma-MSH peptides and to a peptide with putative mitogenic stimulatory activity of the adrenal cortical cells.
  • the biological activity of POMC-derived peptides is further regulated in a tissue-specific manner by acetylation of the amino-terminal amino acid residue and/or amidation of the carboxyterminal amino acid residue by the enzyme peptidyl- ⁇ -monooxygenase (PAM).
  • PAM peptidyl- ⁇ -monooxygenase
  • the POMC gene (human chromosome 2p23) contains three exons and two large introns: one, of about 3.5 kb, interrupts the N- terminal fragment of the common precursor mostly encoded in exon 3.
  • Exon 2 contains the sequence for a portion of the 5' untranslated portion of the mRNA, all of the signal sequence which directs insertion of the precursor protein into the endoplasmic reticulum, and 8 amino acids of the N-terminal fragment.
  • the overall arrangement of introns and exons in the POMC gene is almost identical in all mammalian species.
  • POMC neurons are neurons that secrete POMC or peptides derived from POMC. Exemplary amino acid sequences of POMC and the peptides derived from POMC can be found on GENBANK® at accession nos. NP 001030333, NP 000930, POl 189, POl 193, and NP_032921 as available March 5, 2007, which are incorporated herein by reference.
  • purified does not require absolute purity; rather, it is intended as a relative term.
  • a purified peptide preparation such as a preparation of ⁇ -MSH, NPY or AgRP
  • a purified peptide preparation is one in which the peptide is more pure than the peptide in its natural environment within a cell or tissue, such as brain tissue.
  • Such peptides may be produced, for example, by standard purification techniques (such as chromatography, for example HPLC), and/or by recombinant expression.
  • a preparation of a protein is purified such that the protein represents at least 50%, for example at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 85%, 90%, 95%, 98% or 99%, of the total protein content of the preparation.
  • Tissue A plurality of functionally related cells.
  • a tissue can be a suspension, a semi-solid, or solid.
  • tissue is brain tissue.
  • Therapeutically effective amount A quantity of a specific substance sufficient to achieve a desired effect in a subject being treated. When administered to a subject, a dosage will generally be used that will achieve target tissue concentrations that has been shown to achieve a desired in vitro effect. Treating: Inhibiting the full development of a disease or condition, for example, in a subject who is at risk for a disease such as obesity and/or diabetes. "Treatment” includes a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. The term “ameliorating,” with reference to a disease or pathological condition, refers to any observable beneficial effect of the treatment.
  • the beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, an improvement in the overall health or well-being of the subject, or by other parameters well known in the art that are specific to obesity and/or diabetes.
  • a "prophylactic" treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology.
  • Obesity and diabetes are interrelated metabolic disorders that affect millions of people worldwide, decreasing overall health of the population and resulting incalculable economic costs from lost productivity. In most adults, body weight is relatively constant despite large variations in daily food intake and energy expenditure. Energy balance is regulated by neural and hormonal signals that are integrated in the brain (Seeley and Woods, Nat. Rev. Neurosci. 4:901-909, 2003). Leptin, a hormone secreted primarily by adipocytes, is present in serum concentrations directly proportional to the amount of adipose tissue (Considine et ah, N. Engl. J. Med.
  • leptin-deficient mice In obese leptin-deficient mice, administration of leptin reduces hyperphagia and obesity. In contrast, obese mice that are deficient in the leptin obese receptor (ObRb) do not respond to leptin (Chen et al, Cell 84:491-495, 1996; Halaas et al, Science 269:543-546, 1995). Leptin also affects energy expenditure in rodents and humans (Halaas et al, Science 269, 543-546, 1995; Rosenbaum et al, J. Clin.
  • Activation of central ObRb increases sympathetic nervous system activity, which stimulates energy expenditure in adipose tissue (Commins et al, Endocrinology ⁇ AO:A112-A11%, 1999).
  • the arcuate nucleus in the hypothalamus (ARH) is a major site of leptin sensing (Balthasar et al, Neuron 42:983-991, 2004; Coppari et al, Cell Metab. 1 :63-72, 2005; Cowley et al, Nature 411 :480-484, 2001; van den Top et al, Nat Neurosci 7, 493-494, 2004).
  • ObRb is highly expressed in the ARH (Elmquist et al, J. Comp. Neurol. 395:535-547, 1998; Schwartz et al, J. Clin. Invest. 98:1101- 1106, 1996).
  • the ARH contains at least two key populations of leptin-responsive neurons that have opposite actions on food intake.
  • One population expresses the anorexigenic peptide ⁇ -melanocyte- stimulating hormone ( ⁇ -MSH), derived from the proopiomelanocortin (POMC) precursor.
  • POMC proopiomelanocortin
  • the other population expresses the orexigenic peptides: neuropeptide Y (NPY) and agouti-related peptide (AgRP) (Cone, Nat. Neurosci.
  • Leptin can modulate both POMC and AgRP neurons and can promote the release of ⁇ -MSH, a potent anorexigen at central
  • MC4Rs (Cowley et al, Nature 411 :480-484, 2001; Elias et al, Neuron 23:775-786, 1999; Schwartz et al, Diabetes 46:2119-2123, 1997).
  • leptin resistance Although most obese humans and rodents have very high circulating leptin, this hyperleptinemia neither reduces appetite nor increases energy expenditure. This state has been termed "leptin resistance.” Because leptin resistance appears in obese individuals, leptin treatment alone is not sufficient to treat obesity and related diseases, such as diabetes. Therefore obtaining therapeutic drugs useful treating diabetes and/or obesity, especially in leptin resistant individuals, could have profound beneficial effects on public health and economic efficiency.
  • disorders that can be treated by the administration of such agents include, but are not limited to, cardiovascular disease, (including, but not limited to, hypertension, atherosclerosis, congestive heart failure, and dyslipidemia), stroke, gallbladder disease, osteoarthritis, sleep apnea, reproductive disorders such as, but not limited to, polycystic ovarian syndrome, cancers (for example, breast, prostate, colon, endometrial, kidney, and esophagus cancer), varicose veins, acnthosis nigricans, eczema, exercise intolerance, insulin resistance, hypertension hypercholesterolemia, cholithiasis, osteoarthritis, orthopedic injury, insulin resistance (such as, but not limited to, type 2 diabetes and syndrome X) and thromboembolic disease (see Kopelman, Nature 404:635-43; Rissanen et al, British Med.
  • cardiovascular disease including, but not limited to, hypertension, atherosclerosis, congestive heart
  • the method includes contacting a test agent to brain tissue in vitro in media that that includes cerebrospinal fluid, such as artificial cerebrospinal fluid (aCSF), and measuring the amount and/or concentration of NPY, AgRP, and ⁇ -MSH secreted from the brain tissue.
  • cerebrospinal fluid such as artificial cerebrospinal fluid (aCSF)
  • NPY neurotrophic factor
  • AgRP ⁇ -MSH secreted from the brain tissue.
  • ⁇ -MSH secreted from the brain tissue either natural or artificial cerebrospinal fluid can be used, but artificial cerebrospinal fluid is preferred because it can be more readily obtained in the quantities used in the disclosed assay and methods.
  • the artificial cerebrospinal fluid can include NaCl, Na 2 HPO 4 , KCl, CaCl 2 , MgSO 4 , NaHCO 3 , glucose, and ascorbic acid.
  • the aCSF has a concentration of about 120 to about 130 mM NaCl, a concentration of about 15 to about 25 mM NaHCO 3 , a concentration of about 2mM to about 3 mM KCl, a concentration of about 1 mM to about 2 mM NaH 2 PO 4 , a concentration of about 1 mM to about 2 mM MgCl 2 , a concentration of about 2 mM to about 3 mM CaCl 2 , and a concentration of about 5 mM to about 15 mM D-glucose and a pH from about 7 to about 8.
  • the aCSF can include a protease inhibitor, for example aprotinin, for example a concentration of about 0.1 to about 1.0 trypsin inhibitor units (TIU) aprotinin/ml.
  • a protease inhibitor cocktail is used, such as those commercially available from Roche Bioscience, Pierce Biotechnology and the like.
  • POMC proopiomelanocortin
  • NPY/ AgRP neuropeptide peptide Y/ Agouti-related peptide
  • the amount and/or concentration of NPY, AgRP, and ⁇ -MSH secreted from brain tissue is normalized to the amount of brain tissue contacted with the agent, for example the amount and/or concentration of NPY, AgRP, and ⁇ -MSH per gram of brain tissue.
  • the amount and/or concentration of alpha-melanotropin ( ⁇ -MSH) secreted from the brain tissue contacted with the test agent is measured, for example using an immunoassay specific for ⁇ -MSH, such as a radio immunoassay (RIA) specific for ⁇ -MSH.
  • the amount and/or concentration of AgRP secreted from the brain tissue contacted with the test agent is also measured, for example using an immunoassay specific for AgRP, such as a RIA specific for AgRP.
  • the ratio of the amount and/or concentration of ⁇ -MSH secreted from the brain tissue to the amount and/or concentration of AgRP secreted from the brain tissue contacted with the test agent is determined.
  • This ratio is compared to a control, for example, a standard value for the ratio of the amount and/or concentration of ⁇ -MSH secreted from brain tissue to the amount and/or concentration of AgRP secreted from brain tissue, or the ratio of the amount and/or concentration of ⁇ -MSH secreted to the amount and/or concentration of AgRP secreted from brain tissue not contacted with a test agent.
  • the amount and/or concentration of NPY secreted from brain tissue contacted with the test agent is also measured, for example using an immunoassay specific for NPY, such as a RIA specific for NPY.
  • This amount and/or concentration is compared to a control, for example, a standard value for the amount and/or concentration of NPY secretion from brain tissue, or the amount and/or concentration of NPY secreted from brain tissue not contacted with a test agent.
  • An increase in the ratio of the amount and/or concentration of ⁇ -MSH secreted from brain tissue to the amount and/or concentration of AgRP secreted from brain tissue contacted with a test agent relative to a control and a corresponding decrease in the amount and/or concentration NPY secreted from brain tissue contacted with a test agent relative to a control identifies the test agent as being of use for the treatment of diabetes, obesity, or a combination thereof.
  • a test agent that increases the ratio of the amount and/or concentration ⁇ -MSH secreted from the brain tissue to the amount and/or concentration of AgRP secreted from the brain tissue test and decreases the amount and/or concentration of NPY secreted from the brain tissue relative a control is identified as being of use for treating obesity.
  • a test agent that increases the ratio of the amount and/or concentration ⁇ -MSH secreted from the brain tissue to the amount and/or concentration of AgRP secreted from the brain tissue relative to a control and decreases the amount and/or concentration of NPY secreted from the brain tissue relative a control is identified as being of use for treating diabetes.
  • control is a standard value indicative of the basal level of NPY secreted by brain tissue. In some embodiments, the control is a standard value indicative of the basal ratio of ⁇ -MSH to AgRP secreted by brain tissue. In some embodiments, a control is the ratio of ⁇ -MSH to AgRP secreted by brain tissue in vitro not contacted with the test agent. In some embodiments, a control is the amount of NPY secreted by brain tissue in vitro not contacted with the test agent.
  • control is expressed in amount and/or concentration of NPY, AgRP, and ⁇ -MSH secreted from brain tissue is normalized to the amount of brain tissue, for example the amount and/or concentration of NPY, AgRP, and ⁇ -MSH per gram of brain tissue.
  • a test agent can induce a statistically significant difference in the ratio of the amount of ⁇ -MSH secreted from the brain tissue to the amount of AgRP secreted from the brain tissue contacted with the test agent, as compared to the control, such brain tissue not contacted with the test agent (such as an brain tissue contacted with carrier alone).
  • the difference between the ratio of the amount of ⁇ -MSH secreted from the brain tissue to the amount of AgRP secreted from the brain tissue contacted with the test agent relative to a control is at least about 10%, such as at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, or at least about 500% or even greater then 500%.
  • a test agent can induce a statistically significant decrease in the amount of NPY secreted from the brain tissue contacted with the test agent, as compared to the control, such brain tissue not contacted with the test agent (such as an brain tissue contacted with carrier alone).
  • the decrease in the amount of NPY secreted from the brain tissue contacted with the test agent relative to a control is at least about 10%, such as at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500% or even greater then 500%.
  • a 2 fold or greater increase in the ratio of the amount and/or concentration of ⁇ -MSH secreted from brain tissue to the amount and/or concentration of AgRP secreted from brain tissue contacted with a test agent relative to a control and a corresponding 50% decrease in the amount and/or concentration of NPY secreted from brain tissue contacted with a test agent relative to a control identifies the test agent as being of use for the treatment of diabetes, obesity, or a combination thereof.
  • the brain tissue is contacted with the test agent at a concentration of about 1 picomolar to about 100 mmolar, such as a concentration of test agent of about 1 picomolar, about 10 picomolar, about 100 picomolar, about 1 nanomolar, about 10 nanomolar, about 100 nanomolar, about 1 micromolar, about 10 micromolar, about 100 micromolar, 1 millimolar, about 10 millimolar, or even about 100 millimolar.
  • brain tissue such as different portions of brain tissue, is contacted with different concentrations of the test agent, for example to determine the secretion of NPY, AgRP, and ⁇ -MSH as a function of test agent concentration.
  • the amount and/or concentration of AgRP, NPY, and ⁇ -MSH can be measured at any concentration of test agent or any number of concentrations of test agent and that the concentrations given above are exemplary.
  • the brain tissue is contacted with the test agent for at least about 1 second, such as at least about 5 seconds, at least about 10 seconds, at least about 30 seconds, at least about 1 minute, at least about 5 minutes, at least about 10 minutes, at least about 30 minutes, at least about 60 minutes, at least about 120 minutes, at least about 3 hours, at least about 6 hours, at least about 12 hours, or even about 24 hours or more.
  • brain tissue can be contacted with the test agent for any amount of time and that periods given above are exemplary.
  • multiple time points are determined.
  • the amount and/or concentration of AgRP, NPY and ⁇ -MSH secreted from brain tissue is measured at various time points (for example to determine a time course for the secretion of AgRP, NPY, and ⁇ -MSH in response to contact with a test agent), such at about 10 seconds, at about 30 seconds, at about 1 minute, at about 5 minutes, at about 10 minutes, at about 30 minutes, at about 60 minutes, at about 2 hours, or even at least 24 hours after contact with the test agent.
  • the amount and/or concentration of AgRP, NPY, and ⁇ -MSH can be measured at any time point or number of time points and that the time points given above are exemplary.
  • the brain tissue can be obtained from any subject, including both human and veterinary mammalian subjects.
  • the subject can be a human, or can be a non- human primate, a farm animal such as swine, cattle, and poultry, a sport animal or pet such as dogs, cats, horses, hamsters, or laboratory animal such as a rodent, for example a mouse, rat, guinea pig and the like.
  • the brain tissue includes both POMC (neurons that secrete ⁇ -MSH) neurons and AgRP/NPY neurons (i.e. neurons that secrete AgRP and NPY).
  • the brain tissue includes the paraventricular nucleus of the hypothalamus (PVH) (or a portion thereof) and the hypothalamic arcuate nucleus (ARH) (or a portion thereof).
  • a portion of brain tissue is a histological section obtained from the brain of a subject, for example a section that is between about lmm and about 20mm thick, such as about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 10 mm, about 15 mm, or about 20 mm thick.
  • the brain tissue is mouse brain tissue (such as brain tissue from an obese mouse, for example, a diet induced obese (DIO) mouse, or a brain tissue from a diabetic mouse).
  • the brain tissue is human brain tissue (such as brain tissue from an obese human or a brain tissue from a diabetic human).
  • the brain tissue is leptin resistant, such that contacting the brain tissue with leptin does not stimulate the secretion of neuropeptides, such as AgRP, NPY and/or ⁇ -MSH.
  • the brain tissue is glucose resistant, for example obtained from a subject with diabetes.
  • the brain tissue is obtained from a diabetic subject, such as a diabetic human subject or a diabetic laboratory animal, for example a diabetic mouse.
  • the amount and/or concentration of AgRP, NPY, and ⁇ -MSH can be measured using any method known to one of skill in the art.
  • the amount of AgRP, NPY, and ⁇ -MSH secreted from brain tissue is measured using an antibody that specifically binds AgRP, NPY, or ⁇ -MSH, such as a monoclonal or polyclonal antibody.
  • the antibody that specifically binds AgRP does not specifically bind NPY, or ⁇ -MSH.
  • the antibody that specifically binds ⁇ -MSH does not specifically bind NPY, or AgRP.
  • the antibody that specifically binds NPY does not specifically bind AgRP, or ⁇ -MSH.
  • the presence of antibody: antigen complexes can be determined using methods known in the art.
  • the antibody can include a detectable label, such as a fluorophore, radiolabel, or enzyme, which permits detection of the antibody, for example using ELISA.
  • a detectable label such as a fluorophore, radiolabel, or enzyme
  • multiple antibodies are incubated with the supernatant, and the presence of multiple complexes detected simultaneously, such that the amount of AgRP, NPY, and ⁇ -MSH (or any combination thereof) can be measured in a single sample, such as in a single tube or well of a microtiter plate.
  • the amount of AgRP, NPY, and ⁇ -MSH is measured using an immunoassay (such as a radioimmunoassay, for example competitive radioimmunoassay), for example using antibodies that specifically bind the neuropeptides AgRP, NPY, and ⁇ -MSH.
  • an immunoassay such as a radioimmunoassay, for example competitive radioimmunoassay
  • the presence of ⁇ -MSH, NPY, and/or AgRP is measured using a competition immunoassay, such as a competition radioimmunoassay (RIA).
  • a competition immunoassay a sample of pure or nearly pure antigen (such as ⁇ -MSH, NPY, or AgRP) that has been labeled with a detectable label is used.
  • the antigen is labeled with a radioisotope (for example ⁇ -MSH, NPY, and/or AgRP is been labeled) for example labeled with two or more 125 I atoms.
  • a constant amount (known amount) of the labeled antigen is mixed with a test solution that contains an unknown amount of unlabeled antigen, such as a solution containing neuropeptide secreted from brain tissue, for example ⁇ -MSH, NPY, and/or AgRP secreted from brain tissue.
  • the solution is then allowed to bind to a subsaturating amount of an antibody specific for the antigen, such as an antibody specific for ⁇ - MSH, NPY, or AgRP. High levels of unlabeled antigen present in the solution will reduce the amount of labeled antigen bound to the antibody specific for the antigen.
  • the bound antigen (both labeled and unlabeled) is separated from the unbound antigen and the amount of labeled antigen can then be determined by measurement, for example if the antigen is labeled with a radioisotope the radioactivity emitted from the bound labeled antigen is proportional to the amount of labeled antigen bound.
  • the sensitivity of a competition assay typically varies depending on three factors, including (1) the number of antibodies used to detect antigen, (2) the avidity of the antigen for the antibody, and (3) the specific activity of the labeled antigen. Thus, one of skill in the art can adjust these parameters depending on the sensitivity and dynamic range required.
  • a sample of antigen such as a sample containing secreted neuropeptides, such as ⁇ -MSH, NPY, and/or AgRP, is contacted with a first antibody that has been labeled with a detectable label.
  • the sample is contacted with a second unlabeled antibody that competes with the binding of the first antibody. High levels of unlabeled antibody present in the solution will reduce the amount of labeled antibody bound to the antigen.
  • the bound antibody (both labeled and unlabeled) is separated from the unbound antibody and the amount of labeled antibody can then be determined by measurement, for example if the antibody is labeled with a radioisotope the radioactivity emitted from the bound labeled antibody is proportional to the amount of labeled antibody bound to the antigen.
  • the amount of ⁇ -MSH is measured with a competitive radioimmunoassay.
  • the radioimmunoassay uses ⁇ -MSH peptide exogenous to the brain tissue that is labeled with two or more 125 I atoms.
  • the amount of NPY is measured with a competitive radioimmunoassay.
  • the radioimmunoassay uses NPY peptide exogenous to the brain tissue that is labeled with two or more 125 I atoms. In some embodiments, the amount of AgRP is measured with a competitive radioimmunoassay. In some embodiments, the radioimmunoassay uses AgRP peptide exogenous to the brain tissue that is labeled with two or more 125 I atoms. Iodine labeling of antigens, such as AgRP, NPY and ⁇ -MSH, and antibodies is well know in the art, and exemplary procedures can be found in Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York (1988).
  • the disclosed immunoassays are particularly sensitive and can detect the neuropeptides AgRP, NPY, and ⁇ -MSH at sub-nanomolar levels, for example at picomolar levels or even sub-picomolar levels. In other words the disclosed immunoassays can detect AgRP, NPY, and ⁇ -MSH at a concentration of about 0.1 picomolar or greater.
  • the disclosed immunoassays are capable of detecting an amount of AgRP at a concentration of about 0.1 picomolar to about 10 picomolar or greater, such as about 0.1 picomolar, about 0.2 picomolar, about 0.3 picomolar, about 0.4 picomolar, about 0.5 picomolar, about 0.6 picomolar, about 0.7 picomolar, about 0.8 picomolar, about 0.9 picomolar, about 1.0 picomolar, about 2.0 picomolar, about 3.0 picomolar, about 4.0 picomolar, about 5.0 picomolar, about 6.0 picomolar, about 7.0 picomolar, about 8.0 picomolar, about 9.0 picomolar, or about 10.0 picomolar or greater.
  • the disclosed immunoassays are capable of detecting an amount of AgRP from about 0.1 picomolar to about 10 picomolar, from about 0.1 picomolar to about 100 picomolar, 0.1 picomolar to about 1000 picomolar, or even greater.
  • the disclosed immunoassays are capable of detecting an amount of NPY at a concentration of about 0.1 picomolar to about 10 picomolar or greater, such as about 0.1 picomolar, about 0.2 picomolar, about 0.3 picomolar, about 0.4 picomolar, about 0.5 picomolar, about 0.6 picomolar, about 0.7 picomolar, about 0.8 picomolar, about 0.9 picomolar, about 1.0 picomolar, about 2.0 picomolar, about 3.0 picomolar, about 4.0 picomolar, about 5.0 picomolar, about 6.0 picomolar, about 7.0 picomolar, about 8.0 picomolar, about 9.0 picomolar, or about 10.0 picomolar or greater.
  • the disclosed immunoassays are capable of detecting an amount of AgRP from about 0.1 picomolar to about 10 picomolar, from about 0.1 picomolar to about 100 picomolar, 0.1 picomolar to about 1000 picomolar, or even greater.
  • the disclosed immunoassays are capable of detecting an amount of NPY at a concentration of about 0.1 picomolar to about 10 picomolar or greater, such as about 0.1 picomolar, about 0.2 picomolar, about 0.3 picomolar, about 0.4 picomolar, about 0.5 picomolar, about 0.6 picomolar, about 0.7 picomolar, about 0.8 picomolar, about 0.9 picomolar, about 1.0 picomolar, about 2.0 picomolar, about 3.0 picomolar, about 4.0 picomolar, about 5.0 picomolar, about 6.0 picomolar, about 7.0 picomolar, about 8.0 picomolar, about 9.0 picomolar, or about 10.0 picomolar or greater.
  • the disclosed immunoassays are capable of detecting an amount of AgRP from about 0.1 picomolar to about 10 picomolar, from about 0.1 picomolar to about 100 picomolar, 0.1 picomolar to about 1000 picomolar, or even greater.
  • Western blotting is used to quantify the amount of AgRP, NPY and ⁇ -MSH, such as AgRP, NPY and ⁇ -MSH present brain tissue. Briefly, the brain tissue or an extract thereof is resolved by SDS-PAGE, and the peptides transferred to an appropriate medium, such as nitrocellulose. The nitrocellulose is incubated with the appropriate antibody (which itself can have a label, or which can be detected by using the appropriate labeled secondary antibody), which permits detection of the antibody-protein complex.
  • the appropriate antibody which itself can have a label, or which can be detected by using the appropriate labeled secondary antibody
  • a colorimetric assay is used to quantify the amount of AgRP, NPY and ⁇ -MSH, such as AgRP, NPY and ⁇ -MSH present brain tissue. Briefly, the brain tissue or an extract thereof is exposed to a material that will produce a colorimetric reaction if AgRP, NPY or ⁇ -MSH is present, for example at a particular concentration.
  • the antibody specific for AgRP, NPY, or ⁇ -MSH is attached to a solid support, such as a multiwell plate (such as, a microtiter plate, for example, 96 well, 384 well, etc bead, membrane or the like).
  • a multiwell plate such as, a microtiter plate, for example, 96 well, 384 well, etc bead, membrane or the like.
  • microtiter plates may conveniently be utilized as the solid phase. The surfaces may be prepared in advance, stored, and shipped to another location(s).
  • the sample Prior to determining the amount of ⁇ -MSH, NPY, and/or AgRP in the sample, such as a sample of brain tissue, the sample can be subjected to one or more dimensions of chromatographic separation, for example, one or more dimensions of liquid or size exclusion chromatography, for example to isolate ⁇ -MSH, NPY, and/or AgRP from constituents of the sample, such as other peptides, proteins, nucleic acids and cellular components.
  • chromatographic separation for example, one or more dimensions of liquid or size exclusion chromatography
  • chromatographic separation examples include paper chromatography, thin layer chromatography (TLC), liquid chromatography, column chromatography, fast protein liquid chromatography (FPLC), ion exchange chromatography, size exclusion chromatography, affinity chromatography, high performance liquid chromatography (HPLC), nano-reverse phase liquid chromatography (nano-RPLC), poly acrylamide gel electrophoresis (PAGE), capillary electrophoresis (CE), reverse phase high performance liquid chromatography (RP-HPLC) or other suitable chromatographic techniques.
  • TLC thin layer chromatography
  • FPLC fast protein liquid chromatography
  • ion exchange chromatography size exclusion chromatography
  • affinity chromatography affinity chromatography
  • HPLC high performance liquid chromatography
  • nano-RPLC nano-reverse phase liquid chromatography
  • PAGE poly acrylamide gel electrophoresis
  • CE capillary electrophoresis
  • RP-HPLC reverse phase high performance liquid chromatography
  • ⁇ -MSH, AgRP and NPY is isolated from the brain tissue prior to measuring the amount of ⁇ -MSH, AgRP and NPY secreted from the brain tissue, for example using a chromatography technique such as HPLC.
  • test agents involves testing a combinatorial library containing a large number of potential modulator compounds.
  • a combinatorial chemical library may be 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 (for example the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.
  • Appropriate test agents can be contained in libraries, for example, synthetic or natural compounds in a combinatorial library.
  • libraries are commercially available or can be readily produced; means for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides, such as antisense oligonucleotides and oligopeptides, also are known.
  • libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or can be readily produced.
  • natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Such libraries are useful for the screening of a large number of different compounds.
  • Libraries useful in the disclosed methods include, but are not limited to, peptide libraries (see, e.g., U.S. Patent No. 5,010,175; Furka, Int. J. Pept. Prot. Res., 37:487-493, 1991; Houghton et al, Nature, 354:84-88, 1991; PCT Publication No.
  • WO 91/19735 (see, e.g., Lam et al, Nature, 354:82-84, 1991; Houghten et al, Nature, 354:84-86, 1991), and combinatorial chemistry-derived molecular library made of D-and/or L- conf ⁇ guration amino acids, phosphopeptides (including, but not limited to, members of random or partially degenerate, directed phosphopeptide libraries; see, e.g., Songyang et al., Cell, 12:161-11%, 1993), antibodies (including, but not limited to, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and Fab, F(ab') 2 and Fab expression library fragments, and epitope-binding fragments thereof), small organic or inorganic molecules (such as, so-called natural products or members of chemical combinatorial libraries), molecular complexes (such as protein complexes), or nucleic acids, encoded peptid
  • Libraries useful for the disclosed screening methods can be produced in a variety of manners including, but not limited to, spatially arrayed multipin peptide synthesis (Geysen, et al, Proc. Natl. Acad. Sci., 81(13):3998-4002, 1984), "tea bag” peptide synthesis (Houghten, Proc. Natl. Acad. Sci., 82(15):5131-5135, 1985), phage display (Scott and Smith, Science, 249:386-390, 1990), spot or disc synthesis (Dittrich et al, Bioorg. Med. Chem.
  • Libraries can include a varying number of compositions (members), such as up to about 100 members, such as up to about 1000 members, such as up to about 5000 members, such as up to about 10,000 members, such as up to about 100,000 members, such as up to about 500,000 members, or even more than 500,000 members.
  • members such as up to about 100 members, such as up to about 1000 members, such as up to about 5000 members, such as up to about 10,000 members, such as up to about 100,000 members, such as up to about 500,000 members, or even more than 500,000 members.
  • the methods can involve providing a combinatorial chemical or peptide library containing a large number of potential therapeutic compounds. Such combinatorial libraries are then screened by the methods disclosed herein to identify those library members (particularly chemical species or subclasses) that display a desired characteristic activity.
  • the compounds identified using the methods disclosed herein can serve as conventional "lead compounds" or can themselves be used as potential or actual therapeutics.
  • pools of candidate test agents can be identified and further screened to determine which individual or subpools of test agents in the collective have a desired activity.
  • Control reactions can be performed in combination with the libraries. Such optional control reactions are appropriate and can increase the reliability of the screening. Accordingly, disclosed methods can include such a control reaction.
  • the control reaction may be a negative control reaction that measures the secretion of
  • the control reaction may also be a positive control reaction that measures the secretion of AgRP, NPY and ⁇ -MSH in response to a known agent that affects AgRP, NPY and ⁇ -MSH secretion, such as leptin.
  • kits for identifying test agents that are useful in treating obesity and/or diabetes include antibodies that specifically bind AgRP, NPY and ⁇ -MSH.
  • the kits may further include additional components such as instructional materials and additional reagents, for example specific binding agents, such as antibodies, labeled peptides (such as radiolabeled peptide, for example 125 I labeled peptide, such as 125 I AgRP, NPY and ⁇ -MSH) or a means for labeling peptides.
  • the kits may also include additional components to facilitate the particular application for which the kit is designed (for example microtiter plates). Such kits and appropriate contents are well known to those of skill in the art.
  • the instructional materials may be written, in an electronic form (such as a computer diskette or compact disk) or may be visual (such as video files).
  • This disclosure also provides integrated systems for high-throughput screening of test agents for an effect on AgRP, NPY and ⁇ -MSH secretion.
  • the systems typically include a robotic armature that transfers fluid from a source to a destination, a controller that controls the robotic armature, a tag detector, a data storage unit that records tag detection, and an assay component such as a microtiter dish comprising a well having a reaction mixture for example brain tissue in aCSF.
  • a number of robotic fluid transfer systems are available, or can easily be made from existing components.
  • a Zymate XP Zymark Corporation; Hopkinton, Mass.
  • a Microlab 2200 Hamilton; Reno, Nev.
  • pipetting station can be used to transfer parallel samples to 96 well microtiter plates to set up several parallel simultaneous assays, for example to assay for the effect of one or more test agents on the secretion of AgRP, NPY and ⁇ -MSH.
  • a variety of commercially available peripheral equipment and software is available for digitizing, storing and analyzing a digitized video or digitized optical image, e.g., using PC (Intelx86 or Pentium chip-compatible DOSTM, OS2 TM WINDOWS TM, WINDOWS NT TM or WINDOWS95TM based computers), MACINTOSHTM, or UNIX based (for example, a SUNTM, a SGITM, or other work station) computers.
  • PC Intelx86 or Pentium chip-compatible DOSTM, OS2 TM WINDOWS TM, WINDOWS NT TM or WINDOWS95TM based computers
  • MACINTOSHTM or UNIX based (for example, a SUNTM, a SGITM, or other work station) computers.
  • mice At six weeks of age, C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME) were fed a regular diet (PURINA® Lab Chow #5001, Ralston Purina Corp, St. Louis, MO) or a high fat diet (HFD) (Rodent Chow #D 12451, RESEARCH DIETSTM INC., New Brunswick, NJ) for 20 or 37 weeks.
  • Regular diet provided 3.3 kcal/grams of energy (59.8% carbohydrate, 28.0% protein, and 12.1% fat).
  • the HFD provided 4.75 kcal/grams of energy (35.0% carbohydrate, 20.0% protein, and 45.0% fat).
  • mice were housed (5/cage) in a controlled environment.
  • ⁇ -MSH immunoreactivity was measured with a rabbit anti- ⁇ -MSH (Phoenix Pharmaceuticals, Inc., Belmont, CA).
  • the antibody cross-reacts fully with the acetylated ⁇ -MSH and partially (46%) with desacetylated ⁇ -MSH, but not with NPY nor AgRP.
  • NPY immunoreactivity was measured with a rabbit anti-NP Y.
  • the antibody does not cross-react with AgRP or ⁇ -MSH.
  • AgRP immunoreactivity was measured with a rabbit anti-AgRP (82-131)-NH2 (Phoenix Pharmaceuticals, Inc., Belmont, CA). The antibody does not cross-react with ⁇ -MSH or NPY.
  • 125 I-labeled ⁇ -MSH, AgRP, and NPY were prepared by the iodogen method and purified by high-pressure liquid chromatography ( ⁇ -MSH and NPY) or by AG 1X8 ion exchange resin (AgRP).
  • ⁇ -MSH RIA the lowest detectable level (LDL) that could be distinguished from the zero standard was 0.30 fmol/tube.
  • LDL low detectable level
  • the inter assay-variation (CV%) was 10.7% and 12.1% for the range of value measured.
  • the LDL was 0.32 fmol/tube.
  • the LDL was 0.70 fmol/tube.
  • the CV% was 13.4% and 13.5% for the range of values measured.
  • mice 28 Control, 10 DIO-R, and 10 DIO were individually housed and sham injected for five days prior to drug treatment. Mice were divided into two groups which received i.p. injections of recombinant murine leptin (PEPROTECH®, 2.0 ⁇ g/g body weight) or saline for 2 days. Body weights and calorie intake were measured daily.
  • PEPROTECH® recombinant murine leptin
  • mice (8 Control, 8 DIO-R and 8 DIO) were individually housed and fasted overnight. Following five days of daily sham injection, half of the mice received i.p.
  • MTII (NeoMPS Inc., San Diego, CA, 1.0 ⁇ g/g) and the other half received i.p. saline. Fifteen minutes after the injection, food was reoffered. Food intake was measured during the 24 hours after the injection.
  • a sterile guide cannula (15 millimeters long, 22 gauge, PLASTICS ONE®, Inc. Roanoke, VA) was stereotaxically implanted into the lateral ventricle (-0.5 millimeters posterior, 1 millimeters lateral to bregma, and 1.5 millimeters below the surface of the skull, in accordance with Franklin and Paxinos (Paxinos and Franklin, The Mouse Brain in Stereotaxic Coordinates, San Diego: Academic Press, 2001) of Control, DIO-R, and DIO mice. A 28-gauge obturator was inserted into each cannula. Cannula position was verified at the end of the experiment by dye administration and histological analysis.
  • mice received i.c.v. leptin (0.1 ⁇ g) or aCSF in 1 ⁇ l.
  • food intake and body weight were measured after 24 hours. Two days later, treatments were crossed over in all mice.
  • mice were perfused 45 minutes after i.c.v. leptin administration for IHC.
  • mice Eight na ⁇ ve mice were given increasing doses of MTII i.c.v. (0.01-500 ng). The minimum dose that produced significant modification of food intake in Control mice (0.1 ng) was selected for the follow-up analysis. Mice (Control, DIO-R and DIO) were divided into two groups, each receiving i.c.v. MTII or aCSF after an overnight fast. Food intake was recorded during 24 hours after the injection.
  • mice were sacrificed by decapitation and whole brains immediately removed. The hypothalamus was cut from the removed brains. Care was taken to ensure that there was no contamination of the hypothalamic portion with residual pituitary.
  • a 2 millimeter thick slice of mediobasal forebrain was prepared using a vibrating microtome (Leica VS 1000, Leica Microsystems, Inc., Bannockburn, IL). Each slice was taken from the base of the brain to include the PVH and ARH.
  • hypothalami were treated separately with aCSF (w/ 0.6 TIU aprotinin/ml), equilibrated with 95% O 2 and 5% CO 2 and incubated at 37°C. After a 1 hour equilibration period, hypothalami were incubated for 45 minutes in aCSF (basal period) before being challenged with a single concentration of leptin (0.1 to 100 nM) for 45 minutes. Tissue viability was verified by exposure to 56 mM KCl for 45 minutes. Each experiment was repeated four times. Treatments were performed in quadruplicate. At the end of each period, supernatants were removed and frozen until assayed. Hypothalamic explants that failed to show peptide release 3X above that of basal in response to KCl were excluded from analysis.
  • mice (8 Control, 8 DIO-R, and 9 DIO) were injected i.p. with 2 ⁇ g/g leptin or saline. After 30 min, mice were killed under pentobarbital anesthesia by cardiac perfusion with PBS then paraformaldehyde in NaPO 4 buffer, pH 7.4. Brains were removed, dehydrated in 25% sucrose, frozen and stored at -80 0 C until sectioned coronally on a microtome (30 ⁇ m).
  • tissue was pre-treated in 1% NaOH+1% H 2 O 2 in H 2 O for 10 min, 0.3% glycine for 10 min, 0.03% SDS for 10 min, and blocked in 3% NDS in PBS/0.25% Triton X- 100/0.02% NaN 3 .
  • tissue was incubated in blocking buffer for 20 minutes (KPBS+0.4% Triton X-100 + 2% donkey serum) to reduce background.
  • Antibodies (Phospho-Stat3 rabbit #9135, 1 : 1000, Phospho-p44/42 Map Kinase rabbit #9101,1 :1000, CELL SIGNALING TECHNOLOGY®, Beverly, MA; c-Fos rabbit #SC-52, 1 :10000, SANTA CRUZ BIOTECHNOLOGY®, Inc., Santa Cruz, CA) were added in blocking solution and incubated overnight at room temp. Sections were washed, incubated with Biotin-SP-conjugated Aff ⁇ niPure F(ab !
  • mice 23 Control, 21 DIO-R, and 22 DIO were injected i.p. with 2 ⁇ g/g leptin or saline. After 2 hours, mice were killed and brains were frozen on dry ice. Immunohistochemistry (ISH) was performed as described in Grove, et al. ⁇ Endocrinology 142:4771-4776, 2001).
  • rat NPY cRNA 400 bp
  • rat POMC cRNA 925 bp
  • mouse AgRP cRNA 807 bp
  • rat leptin receptor ObRb
  • mouse MC4R cRNA -520 bp
  • SOCS-3 cRNA 450 bp
  • Tissue was then washed in SSC, RNAse A at 37 0 C, then O.lx SSC at 55-6O 0 C.
  • Tissue was dehydrated and exposed to autoradiographic film for 1-8 days then dipped in Kodak NBT2 emulsion and stored in light-tight boxes for 7-60 days. Slides were developed and counter-stained with cresyl violet.
  • a COOLSNAPTM HQ camera POTOMETRICS®, Westchester, PA
  • Metamorph Software Universal Imaging Corp
  • mice 14 Control and 14 DIO were injected i.p. with 2 ⁇ g/g leptin or saline. After 45 minutes, mice were anesthetized and brains were rapidly removed. The hypothalamus was cut from the rest of the brain. 0.4 millimeter slices were prepared in aCSF using a vibrating microtome and then placed in RNALATER® (AMBION®, Austin, TX). Using a dissecting microscope, the ARH and PVH were cut from each slice using the fornix, optic tracts, and third ventricle as landmarks. ARH and PVH pieces were then put into separate microcentrifuge tubes and stored at -80 0 C.
  • ABI 7900HT Applied Biosystems Foster City, CA
  • GTT GlucoseTolerance Test
  • ITT Insulin Tolerance Test
  • the hypothalami in acetic acid supplemented with a protease inhibitor, were heated at 95°C for 15 minutes and sonicated. Cell disruption was performed using a Dounce homogenizer. Samples were then centrifuged at 15000rpm at 4°C for 30 minutes. Supernatants were collected and protein concentrations were determined by Bradford assay (Coomassie Protein Assay Reagent, Pierce, Rockford, IL, USA). Supernatants were then evaporated using a speed vacuum and reconstituted in 1 milliliter of 0.1% trifluoracetic acid (TFA) solution. 200 ⁇ g of total protein was injected into a Varian ProStar Gradient HPLC System equipped with a C 18 reverse phase column (MICROSORB® MV 300-5; Varian Inc.
  • TFA trifluoracetic acid
  • the assays used for ⁇ -MSH and ACTH-derived peptides were developed using peptides and primary antibodies. Each purified peptide was iodinated with 125 I using the Chloramine T oxidation-reduction method, purified by HPLC, and used as tracer.
  • the ⁇ -MSH RIA was performed in 0.5 milliliters of phosphate buffer (pH 7.4) at 500 mg/L sodium azide at 2.5 g/L bovine serum albumin (BSA), with primary anti- ⁇ -MSH antiserum (1 :20,000), and 5000 counts per minute (cpm) of 125 I des- ⁇ MSH tracer.
  • the sensitivity of the assays was approximately 11.5 pg/tube, and the intra- and inter-assay variability were approximately 5-7% and 10-11%, respectively.
  • the ⁇ -MSH assay used in this condition can detect both acetyl- and des- ⁇ -MSH forms.
  • the ACTH RIA was also performed in 0.5 milliliters of the same RIA buffer by using the anti-ACTH antiserum (1 :30,000) and 5000 cpm of 125I-ACTH tracer.
  • the sensitivity of the assays was approximately 10.0 pg/tube, and the intra- and inter-assay variability were approximately 5-7% and 10-11%, respectively.
  • mice fed HFD had a wide distribution in body weight gain showing bimodal characteristics.
  • the heavier group on the HFD was named “diet- induced obese” (DIO) mice.
  • Others on HFD remained almost as lean as Controls, those fed regular chow ( Figure IA).
  • DIO-R diet-induced obese resistant mice
  • the small number of mice ( ⁇ 5%) that overlapped in the bimodal distribution of body weight was excluded from experiments.
  • the frequency of DIO-R and DIO per cage was normally distributed, and the same ratio occurred when the mice were individually housed.
  • mice fed HFD The largest fat gain was obtained by the DIO group (10.5 g), around time times that of the Control fat gain (p ⁇ 0.001).
  • the extra weight gain in mice fed HFD was mostly due to accumulation of body fat.
  • Example 5 Hypothalamic secretion experiments in mice after 20 or 37 weeks on HFD This example describes the detection of neuropeptide secretion using the disclosed methods.
  • DIO-R mice had a 3-fold higher ratio of ⁇ -MSH to AgRP secretion than DIO mice (Figure 3D).
  • Figure 14A-14C shows average baseline secretion of ArRP, NPY, and ⁇ -MSH expressed as fmoles per gram of tissue.
  • Example 6 Leptin effect (i.p.) on ARH niRNA expression This example describes the effect of i.p. leptin on ARH mRNA expression in mice after 20 wk on a high fat diet.
  • This example describes the effect of i.p. leptin on ARH signal transduction in mice after 20 weeks on a high fat diet.
  • ISH data were inconclusive, higher levels of SOCS-3 mRNA in baseline conditions of DIO mice using Real-Time PCR were found.
  • Control mice had elevated SOCS-3 levels while levels remained unchanged in DIO mice.
  • the expression of pStat3 (signal transducer and activator of transcription) in arcuate neurons was selectively reduced in leptin-treated diet-induced obese (DIO) mice, but not in neurons within the VMH or DMH, suggesting that the ARH is a major site of leptin resistance.
  • the heterozygous SOCS-3 deficient mouse was more sensitive to the weight-reducing effects of leptin and was resistant to the development of DIO.
  • This example describes the determination of diet induced obesity on the melanocortin pathway in mice unresponsive to leptin.
  • the unresponsiveness to leptin in DIO mice may be due to a primary insensitivity or a lack of secondary neuronal targets.
  • the melanocortin pathway is a well-established pathway that mediates leptin' s actions.
  • MTII melanocortin agonist
  • calorie intake was reduced 45% in Control, -65% in DIO-R, and 90% in DIO.
  • the orexigenic effect of MTII was maintained for four hours in Control and DIO-R groups, and continued over eight hours in the DIO group ( Figure 6A).
  • mice When mice were given central MTII, they responded similarly to the peripheral injection: DIO mice showed a greater and prolonged response to MTII than Control or DIO-R mice. To determine if this exaggerated response in DIO mice was due to an up-regulation of MC4R mRNA expression, the expression in the PVH by ISH was measured. Levels of MC4R mRNA were very low in Control and DIO-R mice, but were strikingly high in DIO mice ( Figure 6B). An additional analysis of this gene expression was performing using Real-Time PCR on microdissected PVH samples. The MC4R expression was significantly higher in DIO mice than in Control mice (p ⁇ 0.05). Example 10 Restoration of leptin sensitivity
  • This example describes the restoration of leptin sensitivity in response to a dietary change from a high fat diet to a regular diet.
  • mice After 20 weeks on HFD, DIO mice were intolerant to glucose and insulin resistant. This intolerance and resistance worsened by week 37, causing more than 70% of the mice to be diabetic. Mice were classified as diabetic when glucose AUC was 3 SD higher than the mean of Control mice. At the end of 37 weeks, the Restored group responded normally to glucose and insulin challenges (Figure 7B). Thus, the diabetes/obesity syndrome appears reversible at this stage.
  • Leptin sensitivity of the Restored group was determined by injecting mice with leptin. After the second day of i.p. leptin, body weight of the Restored group was significantly reduced. This was identical to the sensitivity of the Control group ( Figure 7C). In addition, 100 nM leptin inhibited secretion of AgRP and NPY and stimulated secretion of ⁇ -MSH from hypothalamic explants in Restored mice. In contrast, leptin did not modulate the secretion of AgRP, NPY, nor ⁇ -MSH from hypothalami of DIO mice (at 37 weeks) ( Figure 7D, E, F). These results show that sensitivity to leptin in the ARH was re-established in the Restored group.
  • mice fed a high fat diet exhibit increased body adiposity along with other characteristics of human obesity, such as diabetes mellitus (>70% of mice on HFD).
  • mice on HFD lose weight when the fat content of the diet decreases, independent of total calorie intake.
  • the development of obesity and leptin resistance in C57B1/6J mice on HFD can be divided into three stages. In the early stage, mice gain weight (adiposity), but maintain an adequate response to anorectic effects of peripheral leptin injection. In the middle stage, mice lose peripheral leptin sensitivity while retaining the capacity to respond to central leptin. Finally, in the late stage, mice demonstrate central leptin resistance.
  • Leptin modulates NPY/ AgRP and ⁇ -MSH secretion from the ARH in mice fed regular chow (Control) and mice that remain lean when fed HFD (DIO-R). Leptin stimulated ⁇ -MSH secretion and inhibited NP Y/ AgRP secretion in a dose- dependent manner. Leptin failed to modulate the secretion of melanocortin peptides in obese mice (DIO). Secretion can occur from ARH neurons though it is recognize that NPY secretion might be due to other neuron populations within the hypothalamus. NPY/AgRP and ⁇ -MSH secretion in DIO mice was not modulated by leptin (in all doses studied).
  • DIO mice have increased adiposity and high plasma leptin concentrations, but the endogenous hyperleptinemia fails to curtail the progression of obesity. Moreover, DIO mice are unresponsive to peripheral or central leptin injection since they exhibit neither a decrease in food intake nor a decrease in body weight. While c-Fos expression is induced in arcuate neurons of Control and DIO-R mice after leptin treatment (presumably due to activation of POMC neurons), leptin treatment does not alter c-Fos expression in arcuate neurons of DIO mice.
  • the best-defined signal transduction pathway for leptin is the Janus kinase-STAT3 pathway.
  • Hyperleptinemia produces leptin resistance by a down-regulation of ObRb, because ObRb mRNA expression was not different between Control and DIO mice.
  • the magnitude of decreased ObRb expression -30% when it has been shown, is likely not enough to explain the leptin resistance phenotype. It was determined if leptin resistance might be due to a disruption downstream of leptin receptor binding.
  • SOCS-3 a negative regulator of leptin signal transduction
  • Higher baseline SOCS-3 mRNA levels were found in the ARH of DIO mice. After i.p. leptin, only the Control mice showed an increase of SOCS-3.
  • the melanocortin pathway mediates some of leptin' s actions. Since ⁇ -MSH is a MC4R agonist and AgRP is a MC4R inverse agonist, the secretion ratio ⁇ - MSH/ AgRP is an index of MC4R activation. At most leptin concentrations, DIO mice have 3 times less activation of this receptor compared with Control or DIO-R mice. The DIO 's leptin resistance prevents a leptin-induced increase of ⁇ -MSH secretion while preventing a decrease in AgRP secretion. This leads to weakened central melanocortin activation. It was also confirmed that the distal components of the melanocortin system were intact.
  • DIO-R mice Despite genetically identical backgrounds, some C57BL/6J mice on HFD are able to maintain a regular body weight - only 7% heavier than Control mice. DIO-R mice also retain the same "normal" response to exogenous leptin and glycemic control as Control mice. The ability of DIO-R mice to maintain their regular body weight is not due to over-expression of melanocortin peptides, because there was a decrease in the biosynthesis of prohormones (acetyl- and desacetyl- ⁇ -MSH, ACTH and CLIP). DIO mice become leptin resistant and most of them become diabetic. But, they can lose weight by decreasing the fat content of their diet.
  • the Restored group lost weight by eating the same amount of calories as the DIO group, but less fat. This indicates that it is macronutrient composition that causes weight gain in DIO mice and not the total calories consumed. After losing weight, the response to a glucose challenge was normalized, as well as leptin sensitivity, shown by a decrease in body weight after i.p. leptin. Restored mice showed a re-established response to leptin-regulated neuropeptide secretion. This recovery of leptin sensitivity was in parallel with the recovery of NP Y/ AgRP and POMC neurons' response to leptin actions. In summary, leptin sensitivity of POMC and NP Y/ AgRP neurons in the
  • This example describes exemplary procedures and reagents used in the Example 13.
  • POMC-mut-Kir6.2 mice For generation of POMC-mut-Kir6.2 mice, the mut-Kir6.2 cassette (Kir[ ⁇ 2- 30,Kl 85Q]-GFP) was inserted into a POMC BAC genomic clone so that the ATG codon replaced that of POMC, as described in Balthasar et al. ⁇ Neuron 42:983-991 (2004).
  • POMC-mut-Kir6.2 BAC DNA was prepared using a commercially available kit (QIAGEN®) and microinjected into pronuclei of fertilized one-cell- stage embryos of FVB mice (Jackson Laboratories), resulting in the generation of two POMC-mut-Kir6.2 lines that were maintained on an FVB inbred background.
  • POMC-GFP and NPY-GFP mice were generated by insertion of hrGFP into a POMC or NPY BAC, respectively, as described above.
  • Ucp2 ⁇ ' ⁇ mice heterozygous POMC-GFP transgenic mice were crossed with heterozygous Ucp2 ⁇ ' ⁇ mice.
  • mice were placed on a high-fat rodent diet (45% kcal from fat; Research Diets Inc; D12451) at 4 weeks of age for a total of 20 weeks (or 8 weeks for electrophysiological studies).
  • Brain slices were prepared from young adult mice (4-7 weeks old or 12 weeks old for high- fat diet studies). Briefly, mice were anesthetized with Isoflurane before decapitation and removal of the entire brain. Brains were immediately submerged in ice-cold, carbogen-saturated (95% O 2 , 5% CO 2 ) high-sucrose solution (238 mM sucrose, 26 mM NaHCO 3 , 2.5 mM KCl, 1.0 mM NaH 2 PO 4 , 5.0 mM MgCl 2 , 1.0 mM CaCl 2 , 11 mM D-glucose).
  • Coronal sections (200 ⁇ m thick were cut with a Leica VTlOOOS Vibratome and incubated in oxygenated recording aCSF (126 mM NaCl, 21.4 mM NaHCO 3 , 2.5 mM KCl, 1.2 mM NaH 2 PO 4 , 1.2 mM MgCl 2 , 2.4 mM CaCl 2 , 10 mM D-glucose) at room temperature (21 0 C) for at least 1 hour before recording.
  • aCSF oxygenated recording
  • the pipette solution for loose-patch recording contained 150 mM NaCl, 3.5 mM KCl, 10 mM HEPES, 10 mM glucose, 2.5 mM CaCl 2 , 1.3 mM MgCl 2 (pH 7.3).
  • Seal resistance was in the range of 8-30 M ⁇ and was checked during the recordings; cells that showed deviations in seal resistance were not included in the data analysis. Recordings that showed no recovery in spike activity after return to 5 mM glucose were also excluded from data analysis.
  • Firing rate averaged for every 20 seconds was taken as one data point; 9-18 data points taken from the last 3-6 minutes of each experimental condition (5 mM or 3 mM glucose, with or without addition of drugs) were compared using unpaired t-tests, with P ⁇ 0.05 considered a statistically significant change.
  • a 2-mm slice was prepared using a vibrating microtome (Leica VS 100) taken from the base of the brain to include the PVH and arcuate nucleus. Each hypothalamic slice was treated separately and incubated in artificial cerebrospinal fluid (aCSF: 126 mM NaCl, 0.09 mM Na 2 HPO, 6 mM KCl, 4 mM CaCl 2 , 0.09 mM MgSO 4 , 20 mM NaHCOs, 8 mM glucose, 0.18 mg ml "1 ascorbic acid, 0.6 trypsin inhibitor units (TIU) aprotinin ml "1 ), pre-equilibrated with 95% O 2 and 5% CO 2 at 37 0 C for 1 hour.
  • aCSF artificial cerebrospinal fluid
  • TIU trypsin inhibitor units
  • mice were fasted overnight (16 hours) and injected intraperitoneally with a 20% (w/v) glucose solution at 1 g kg "1 body weight. Blood glucose levels were measured before and 15, 30, 45, 90, 120 and 180 minutes after glucose injection.
  • Coronal mouse brain sections (25 ⁇ m) were washed in PBS six times before blocking in 0.25% Triton X-100 in PBS containing 3% (w/v) normal donkey serum (PBT-azide) for 2 hours. Sections were incubated overnight in rabbit anti- ⁇ - endorphin IgG (Peninsula Laboratories Inc; 1 :5,000) in PBT-azide containing 3% (w/v) normal donkey serum at 21 0 C, followed by a 2 hour incubation in Cy3- conjugated donkey anti-rabbit IgG (ImmunoResearch Laboratories; 1 :500.
  • a Ucp2 riboprobe was generated by PCR amplification of a 527-base pair (bp) DNA fragment complementary to exons 3 and 4 of Ucp2 from mouse brain cDNA. This amplicon was subcloned into pcR4-TOPO vector (INVITROGEN®), and a 35 S-labelled cRNA probe generated.
  • pcR4-TOPO vector INVITROGEN®
  • 35 S-labelled cRNA probe generated.
  • ⁇ -endorphin immunohistochemistry sections processed for in situ hybridization were washed twice in PBS and pre- treated with 0.3% (v/v) hydrogen peroxide in PBS for 30 minutes, then incubated in 3% normal donkey serum in PBT-azide for 2 hours.
  • Sections were then incubated overnight with rabbit anti- ⁇ -endorphin primary antiserum (Peninsula Laboratories; 1 :4,000) in PBT-azide. Sections were washed in PBS six times before a 2 hours incubation in biotinylated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories; 1 : 1 ,000). Sections were then washed three times in PBS and incubated with avidin-biotin complex (VECTASTAIN® Elite ABC Kit, Vector Laboratories; 1 :500) in PBS for 1 hour.
  • rabbit anti- ⁇ -endorphin primary antiserum Peninsula Laboratories; 1 :4,000
  • biotinylated donkey anti-rabbit IgG Jackson ImmunoResearch Laboratories; 1 : 1 ,000
  • Sections were then washed three times in PBS and incubated with avidin-biotin complex (VECTASTAIN® Elite ABC Kit, Vector Laboratories
  • RNA STAT-60TM Tel-Test Inc
  • AMBION® DNA-Free
  • Ucp2 was amplified from 0.5 ng of reverse-transcribed total RNA using TAQMANTM Universal PCR Mastermix (Applied Biosystems) with Ucp2 sense and antisense primers, dual- labelled probe (5'-FAM, 3'-TAMRA) (Applied Biosystems; assay on demand Mn00495907_gl). Standard curves were constructed by amplifying serial dilutions of cDNA (5 ng to 0.32 pg) and plotting cycle threshold (CT) values as a function of starting reverse-transcribed RNA. mRNA expression of Ucp2 was normalized to levels of the 18S ribosomal RNA housekeeping gene.
  • This example describes the measurement of ⁇ -MSH secretion in brain tissue induced by glucose.
  • Glucose stimulated release of ⁇ -MSH from hypothalamic slices of chow- fed, wild-type mice ( Figure 12A). However, glucose failed to stimulate release of ⁇ -MSH from wild-type mice fed a high-fat diet ( Figure 12A). Electrophysiological techniques were also used to independently assess glucose sensing in POMC neurons of mice on a high- fat diet. In this case, four- week-old mice were fed chow or a high-fat diet for eight weeks.
  • Brian tissue is obtained from mice. Two-mm slices are prepared using a vibrating microtome taken from the base of the brain to include the PVH and arcuate nucleus. Each hypothalamic slice is treated separately with a test agent and incubated in artificial cerebrospinal fluid (aCSF: 126 mM NaCl, 0.09 mM Na 2 HPO, 6 mM KCl, 4 mM CaCl 2 , 0.09 mM MgSO 4 , 20 mM NaHCO 3 , 8 mM glucose, 0.18 mg ml "1 ascorbic acid, 0.6 trypsin inhibitor units (TIU) aprotinin ml "1 ), pre- equilibrated with 95% O 2 and 5% CO 2 at 37 0 C for 1 hour.
  • aCSF artificial cerebrospinal fluid
  • TIU trypsin inhibitor units
  • aCSF containing 1 pM to 1 mM test agent are then incubated for 45 minutes in 700 ⁇ l aCSF containing 1 pM to 1 mM test agent.
  • the viability of the tissue is verified by a 45 -minute incubation in aCSF containing 56 mM KCl.
  • supernatants are removed and tested for AgRP, NPY and ⁇ -MSH secretion by radioimmunoassay as described in the Examples above.
  • Potential therapeutic agents identified with these or other approaches are used as lead compounds to identify other agents having even greater effects on AgRP, NPY and ⁇ -MSH.
  • chemical analogs of identified chemical entities, or variant, fragments of fusions of peptide agents are tested for their activity in the assays described herein.
  • Candidate agents also can be tested in the animal models of obesity and diabetes described herein.

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Abstract

Methods are disclosed for identifying agents of use for the treatment of diabetes, obesity, or a combination thereof. The method includes contacting a test agent to brain tissue in vitro in artificial cerebrospinal fluid. The brain tissue includes both POMC neurons and NPY/ AgRP neurons, such that the neuropeptides secreted from these neurons can be measured. The amount of the neuropeptide NPY secreted is measured, and the ratio of α-MSH secreted to AgRP secreted is also measured. An increase in ratio of α-MSH to AgRP relative to a control and a corresponding decrease in NPY relative to a control identifies the test agent of use for the treatment of diabetes, obesity, or a combination thereof.

Description

METHODS FOR IDENTIFYING AGENTS OF USE IN TREATING OBESITY AND DIABETES
PRIORITY CLAIM The application claims the benefit of U.S. Provisional Application No.
60/983,084, filed March 5, 2007, which is incorporated by reference herein in its entirety.
STATEMENT OF GOVERNMENT SUPPORT This invention was made with United States Government support under grants DK 62202 and TW/HD-00668, awarded by the National Institutes of Health. The Government has certain rights in the invention.
FIELD This application relates to the field of obesity and diabetes, specifically methods of screening for agents that can be used to treat obesity, diabetes, and related disorders are disclosed.
BACKGROUND In the United States, sixty percent of men and fifty-one percent of women of the age of 20 or older are either overweight or obese. In addition, a large percentage of children in the United States are overweight or obese.
The cause of obesity is complex and multi-factorial. Increasing evidence suggests that obesity is not a simple problem of self-control but is a complex disorder involving appetite regulation and energy metabolism. In addition, obesity is associated with a variety of conditions associated with increased morbidity and mortality in a population. Although the etiology of obesity is not definitively established, genetic, metabolic, biochemical, cultural and psychosocial factors are believed to contribute. In general, obesity has been described as a condition in which excess body fat puts an individual at a health risk.
There is strong evidence that obesity is associated with increased morbidity and mortality. Disease risk, such as cardiovascular disease risk and type 2 diabetes disease risk, increases independently with increased body mass index. Indeed, this risk has been quantified as a five percent increase in the risk of cardiac disease for males, and a seven percent increase in the risk of cardiac disease for females, for each point of a body mass index (BMI) greater than 24.9 (see Kenchaiah et at., N. Engl. J. Med. 347:305, 2002; Massie, N. Engl. J. Med. 347:358, 2002). In addition, there is substantial evidence that weight loss in obese persons reduces important disease risk factors. Even a small weight loss, such as 10% of the initial body weight, in both overweight and obese adults has been associated with a decrease in risk factors such as hypertension, hyperlipidemia, and hyperglycemia. Although diet and exercise provide a simple process to decrease weight gain, overweight and obese individuals often cannot sufficiently control these factors to effectively lose weight. Weight loss surgery is an option in carefully selected patients with clinically severe obesity. However, these treatments are high-risk, and suitable for use in only a limited number of patients. Limited pharmacotherapy is available; several weight loss drugs have been approved by the Food and Drug Administration that can be used as part of a comprehensive weight loss program. However, there remains a need for agents that can be used to effect weight loss in overweight and obese subjects and related conditions such as diabetes.
SUMMARY
Methods are disclosed for identifying agents that affect obesity and/or diabetes. These methods are used to identify agents of use in treating obesity, or that can be used to decrease the weight of a subject. These methods are also of use to identify agents of use in treating diabetes. In several embodiments, the disclosed methods include contacting a test agent to brain tissue in vitro in media that includes artificial cerebrospinal fluid (aCSF). The brain tissue includes both proopiomelanocortin (POMC) neurons and neuropeptide Y/Agouti related peptide (NP Y/ AgRP) neurons. The amount and/or concentration of the neuropeptides AgRP, NPY and alpha-melanotropin (α-MSH) secreted by the brain tissue is measured. The ratio of α-MSH secreted from the brain tissue to AgRP secreted from the brain tissue is determined and compared to a control, for example, a standard value, or the ratio of α-MSH secreted from the brain tissue to AgRP secreted from the brain tissue not contacted with a test agent. The amount of NPY secreted by the brain tissue is compared to a control, for example, a standard value, or the amount of NPY secreted by brain tissue not contacted with a test agent. An increase in the ratio of α-MSH to AgRP relative to a control and a corresponding decrease in NPY relative to a control identifies the test agent as of use for the treatment of diabetes, obesity, or a combination thereof.
The foregoing and other objects, features, and advantages of the invention will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1A-1D is a set of graphs showing body weights and feeding characteristics for mice fed regular chow (Control) versus (vs.) mice fed a high fat diet (HFD) (diet-induced obese-restored (DIO-R) and diet-induced obese (DIO)). Figure IA is graph showing body weight distribution of Control (12% fat diet) and DIO-R/DIO (45% fat diet) mice after 20 weeks. Normality test (DIO-R/DIO) was p<0.0001. Figure IB is graph showing the body weight change of Control, DIO-R, and DIO mice over 20 weeks. Results are expressed as mean ± standard error in the mean (SEM) (*p<0.001). Figure 1C is graph showing total calorie, total fat, total protein, and total carbohydrate calorie intake for Control, DIO-R, and DIO groups after 20 weeks. Results are expressed as mean ± SEM (*p<0.001). Figure ID is graph showing the feed efficiency (weight gained/kcal consumed) of Control, DIO- R, and DIO groups.
Figure 2A-2D is a set of graphs showing the effect of peripheral and central leptin on body weight and calorie intake in Control, DIO-R, and DIO mice
(**p<0.01, ***p<0.001). Figure 2A is a graph showing body weight change (%) 24 hours after intraperitoneal (i.p.) saline or leptin (2 μg/g/d). Figure 2B is a graph showing calorie intake two days after i.p. leptin (2 μg/g/d). Data from each group were normalized to their own saline control. Figure 2C is a graph showing body weight change (%) 24 hours after intracerebroventricular (i.c.v.) artificial cerebrospinal fluid (aCSF) or leptin (0.1 μg). Figure 2D is a graph showing calorie intake one day after i.c.v. leptin (0.1 μg). Data from each group were normalized to their own aCSF control.
Figure 3A-3J is a set of graphs showing leptin modulation of hypothalamic neuropeptide secretion in Control, DIO-R, and DIO groups. Data from each group were normalized to baseline levels. Figures 3A-3 J represent four independent experiments. Figure 3A-C is a set of graphs showing leptin modulation of AgRP secretion. Figure 3D-3F is a set of graphs showing leptin modulation of NPY secretion. The horizontal bar in Figure 3D show the approximate basal level of NPY secretion. Figure 3G-3I is a set of graphs showing leptin modulation of α- MSH secretion. Figure 3 J is a graph showing the α-MSH/AgRP ratio of secretion. The horizontal bar in Figure 3 J show the approximate basal ratio of α-MSH/AgRP secretion.
Figure 4A-4E is a set of graphs showing mRNA neuropeptide expression after i.p. leptin (2 μg/g), expression of leptin obese receptor (ObRb) mRNA, and concentration of desacetyl-α-MSH in the hypothalamic arcuate nucleus (ARH) of Control, DIO-R and DIO groups. Results are expressed as mean ± SEM (*p<0.05, ***p<0.001). Figure 4A is a graph showing caudal AgRP mRNA expression after i.p. lep tin/saline. Figure 4B is a graph showing total NPY mRNA expression after i.p. lep tin/saline. Figure 4C is a graph showing total proopiomelanocortin (POMC) mRNA expression after i.p. leptin/saline. Figure 4D is a graph showing baseline leptin obese receptor (ObRb) expression. Figure 4E is a graph showing desacetyl- α-MSH concentration.
Figure 5A-5D is a set of images and graphs showing the effect of i.p. leptin (2 μg/g) on signal transduction in the ARH. Results expressed as mean ± SEM. Representative microphotographs of hypothalamic sections are shown from Control, DIO-R, and DIO groups (*p<0.05, **p<0.01, ***p<0.001, ##p<O-(M saline Control vs. saline DIO). Figure 5A shows expression of c-Fos 30 minutes after i.p. leptin/saline. Figure 5B shows expression of pStat3 30 minutes after i.p. leptin/saline. Figure 5C shows expression of phospho MAP kinase (pMAPK) 30 minutes after i.p. leptin/saline. Figure 5D shows expression of suppressor of cytokine signaling-3 (SOCS-3) mRNA 45 minutes after i.p. saline/leptin. Figure 6A-6B is a set of graphs and images showing melanocortin system integrity (***p<0.001). Figure 6A is a graph showing the effect of i.p. melanotan-II (MTII) (1 μg/g) over 24 hours of food intake in Control, DIO-R, and DIO groups. Data from each group were normalized to their own saline control. Figure 6B is a representative microphotograph and a graph of results of baseline Melanocortin 4 receptor (MC4R) mRNA expression in paraventricular nucleus of the hypothalamus (PVH) of Control, DIO-R, and DIO groups. Results expressed as mean ± SEM.
Figure 7A- 7F is a set of graphs showing the restoration of leptin sensitivity after changing from HFD to regular chow (*p<0.05, **p<0.01, ***p<0.001). Figure 7A is a graph showing body weight change of Control, DIO, and Restored mice at 37 weeks. Results are expressed as mean ± SEM. Figure 7B is a graph showing glucose tolerance in DIO and Restored mice after 14 hours of fasting. Results are expressed as mean ± SEM. Figure 7C is a graph showing body weight change (%) in Control, Restored, and DIO mice 24 hours after i.p. saline or leptin (2 μg/g/d). Figure 7D is a graph showing leptin (100 nM) modulation of AgRP secretion in Control, Restored, and DIO groups. Data from each group is normalized to baseline levels. Figure 7E is a graph showing leptin (100 nM) modulation of NPY secretion in Control, Restored, and DIO groups. Data from each group is normalized to baseline levels. Figure 7F is a graph showing leptin (100 nM) modulation of α-MSH secretion in Control, Restored, and DIO groups. Data from each group normalized to baseline levels.
Figure 8 is a graph showing the relationship of initial body weight and final weight of identically aged mice on HFD.
Figure 9A-9C is a set of graphs showing baseline neuropeptide secretion from hypothalamic explants in Control, DIO-R, and DIO groups. Data were pooled from four independent experiments. Data are represented as mean ± SEM. Figure 9A is a graph showing AgRP baseline secretion. Figure 9B is a graph showing NPY baseline secretion. Figure 9C is a graph showing α-MSH baseline secretion.
Figure 10A-10B is a set of graphs showing the concentration of POMC peptide-derivatives in the ARH were measured by radio immunoassay (RIA) after high performance liquid chromatography (HPLC) separation in hypothalamic explants. Synthetic peptides were injected on the HPLC to determine retention times. Predicted retention times allowed for analysis of specific regions along the gradient for RIA analysis. Figure 1OA is a graph showing the production of desacetyl-α-MSH, and acetyl-α-MSH in ARH of Control, DIO-R, and DIO mice. Figure 1OB is a graph showing the production of adrenocorticotropic hormone (ACTH) and CLIP in ARH of Control, DIO-R, and DIO mice.
Figure 1 IA-I IB is a set of bar graphs showing glucose sensing is lost in POMC-mut-Kir6.2 neurons. Figure HA is a bar graph showing α-MSH release from hypothalamic slices of wild-type and POMC-mut-Kir6.2 mice (n = 3 hypothalamic slices per data point, ± SEM). Figure HB is a bar graph showing representative glucose tolerance curves from eight-week-old male wild-type and POMC-mut-Kir6.2 littermates (n = 8-10 mice per genotype, ± SEM). Asterisk, P < 0.05; two asterisks, P < 0.01 compared with wild-type at a given time point.
Figure 12A-12B is a set of bar graphs showing that glucose-sensing is lost in POMC neurons of mice on HFD. Figure 12A is two bar graphs showing that glucose-induced α-MSH release from hypothalamic slices of wild-type C57BL/6 mice fed chow or a high-fat diet for 20 weeks. Figure 12B is a bar graph showing the percentage of POMC neurons activated by 5 mM glucose in loose-patch recordings from POMC-GFP mice fed either chow or HFD for eight weeks.
Figure 13A-13B is a set of bar graphs showing acute inhibition or genetic deletion of UCP2 restores or prevents loss of glucose sensing in POMC neurons as a result of obesity induced by HFD. α-MSH secretion from hypothalamic slices from wild-type (Figure 13A WT) and Ucp2~'~ (Figure 13B) mice in response to glucose, with or without genipin (20 μM). Data are presented as mean ± SEM, n = 6 mice for each experimental condition. Asterisk, P < 0.05. Figure 14A-14C are a set of plots showing the average baseline secretion of
ArRP, NPY, and α-MSH expressed as fmoles per gram of tissue.
DETAILED DESCRIPTION
/. Abbreviations aCSF: Artificial cerebrospinal fluid ACTH: Adrenocorticotropic hormone AgRP: Agouti-related peptide α-MSH: alpha-melanotropin
ARH: Hypothalamic arcuate nucleus
DIO: Diet-Induced Obese DIO-R: Diet-Induced Obese Restored
HFD: High fat diet
HPLC: High performance liquid chromatography
IHC: Immunohistochemical
ISH: In Situ Hybridization MC4R: Melanocortin 4 receptor
MTII: Melanotan-II
NPY: Neuropeptide Y
ObRb: Leptin obese receptor
PAM: Peptidyl-α-monooxygenase POMC: Proopiomelanocortin
PVH: Paraventricular nucleus of the hypothalamus
SEM: Standard error in the mean i.p.: Intraperitoneal i.c.v.: Intracerebro ventricular RIA: Radioimmunoassay
SEM: Standard Error in the Mean
WT: Wild-type
//. Terms Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes VII, published by Oxford University Press, 1999; Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994; and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995; and other similar references. Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular forms "a," "an," and "the," refer to both the singular as well as plural, unless the context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. As used herein, the term "comprises" means "includes." Hence comprising A or B" means A, B, or A and B. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described below. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
To facilitate review of the various embodiments of the invention, the following explanations of terms are provided: Antagonist: A substance that tends to nullify the action of another, such as an agent that binds to a cell receptor without eliciting a biological response, for example to block the specific binding of substances that could elicit such responses. In one example AgRP is an antagonist of a melanocortin receptor, such as the melanocortin 4 receptor (MC4R). In some examples, an antagonist decreases signal transduction from the receptor it binds.
Agent or Test Agent: Any polypeptide, compound, small molecule, organic compound, salt, polynucleotide, or other molecule of interest.
Agonist: A substance that specifically binds to a specific receptor and triggers a response in the cell, for example the binding of α-MSH to a melanocortin receptor on the surface of a cell. In one example α-MSH is an agonist of a melanocortin receptor, such as the melanocortin 4 receptor (MC4R).
Agouti-related peptide (AgRP): A neuropeptide made in the arcuate nucleus of the brain that increases appetite and decreases metabolism. Agouti- related protein is an antagonist of the melanocortin-3 and melanocortin-4 receptor. An exemplary amino acid sequence of agouti-related peptide can be found at
GENBANK® accession No. NP_031453 as available March 5, 2007, incorporated herein by reference. Animal: Living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term mammal includes both human and non-human mammals. Similarly, the term "subject" includes both human and veterinary subjects, for example mice. Antibody: A polypeptide ligand comprising at least a light chain or heavy chain immunoglobulin variable region which specifically binds an epitope of an antigen, such as one of a AgRP, NPY, or α-MSH neuropeptide or a fragment thereof. The term "specifically binds" refers to, with respect to an antigen such as an AgRP, NPY, or α-MSH neuropeptide, the preferential association of an antibody or other ligand, in whole or part, with the AgRP, NPY, or α-MSH neuropeptide. A specific binding agent binds substantially only to a defined target, such as an AgRP, NPY, or α-MSH neuropeptide. Thus, in one example an AgRP specific binding agent is an agent that binds substantially to an AgRP polypeptide. If an agent, such as an antibody, specifically binds AgRP it does not specifically bind other peptides including NPY and α-MSH neuropeptides. In another example, an NPY specific binding agent is an agent that binds substantially to an NPY neuropeptide, but not AgRP and α-MSH neuropeptides. In yet another example, an α-MSH specific binding agent is an agent that binds substantially to an α-MSH neuropeptide, but not AgRP and NPY neuropeptides. A minor degree of non-specific interaction may occur between a molecule, such as a specific binding agent, and a non-target polypeptide. Specific binding can be distinguished as mediated through specific recognition of the antigen. Although selectively reactive antibodies bind antigen, they can do so with low affinity. Specific binding typically results in greater than 2- fold, such as greater than 5-fold, greater than 10-fold, or greater than 100-fold increase in amount of bound antibody or other ligand (per unit time) to an AgRP, NPY, or α-MSH neuropeptide. A variety of immunoassay formats are appropriate for selecting antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York
(1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity. Antibodies can include a heavy chain and a light chain, each of which has a variable region, termed the variable heavy (VH) region and the variable light (VL) region. Together, the VH region and the VL region are responsible for binding the antigen recognized by the antibody. This includes intact immunoglobulins and the variants and portions of them well known in the art, such as Fab' fragments, F(ab)'2 fragments, single chain Fv proteins ("scFv"), and disulfide stabilized Fv proteins ("dsFv"). A scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains. The term also includes recombinant forms such as chimeric antibodies (for example, humanized murine antibodies), heteroconjugate antibodies (such as, bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, Immunology, 3rd Ed., W. H. Freeman & Co., New York, 1997. A "monoclonal antibody" is an antibody produced by a single clone of
B-lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody- forming cells from a fusion of myeloma cells with immune spleen cells. These fused cells and their progeny are termed "hybridomas." Monoclonal antibodies include humanized monoclonal antibodies.
Alpha-melanotropin (Alpha-melanocyte stimulating hormone, α-MSH, or α-MSH): An agonist peptide ligand for melanocortin receptors, such as MC4R. α-MSH along with adrenocorticotropic hormone (ACTH), β-MSH and γ-MSH are processed from the pre-prohormone proopiomelanocortin (POMC).
Artificial cerebrospinal fluid: Cell culture medium that approximates the physiological conditions of cerebrospinal fluid. In one embodiment, artificial cerebrospinal fluid can include NaCl, Na2HPO4, KCl, CaCl2, MgSO4, NaHCO3, glucose, ascorbic acid, and aprotinin. Binding: A specific interaction between two or more molecules, such as the binding of an antibody and an antigen (for example an antibody to NPY, α-MSH or AgRP) or the binding of a ligand to a receptor, such as the binding of AgRP or α- MSH to a melanocortin receptor, such as MC4R. In one embodiment, specific binding is identified by a dissociation constant (Kd). In one embodiment, binding affinity is calculated by a modification of the Scatchard method described by Frankel et al, MoI. Immunol, 16:101-106, 1979. In another embodiment, binding affinity is measured by an antigen/antibody dissociation rate. In yet another embodiment, a high binding affinity is measured by a competition radioimmunoassay (RIA). In several examples, a high binding affinity is at least about 1 x 10~8 M. In other embodiments, a high binding affinity is at least about 1.5 x 10~8, at least about 2.0 x 10~8, at least about 2.5 x 10~8, at least about 3.O x 10"8, at least about 3.5 x 10"8, at least about 4.0 x 10"8, at least about 4.5 x 10"8, or at least about 5.0 x 10"8 M.
Body Mass Index (BMI): A mathematical formula for measuring body mass in humans, also sometimes called Quetelet's Index. BMI is calculated by dividing weight (in kg) by height (in meters ). The current standards for both men and women accepted as "normal" are a BMI of 20-24.9 kg/m2. In one embodiment, a BMI of greater than 25 kg/m can be used to identify an obese subject. Grade I obesity corresponds to a BMI of 25-29.9 kg/m2. Grade II obesity corresponds to a BMI of 30-40 kg/m ; and Grade III obesity corresponds to a BMI greater than 40 kg/m2 (Jequier, Am. J CHn. Nutr., 45 : 1035-47, 1987). Ideal body weight will vary among species and individuals based on height, body build, bone structure, and sex.
Caloric intake or calorie intake: The number of calories (energy) consumed by an individual.
Calorie: A unit of measurement in food. A standard calorie is defined as 4.184 absolute joules, or the amount of energy it takes to raise the temperature of one gram of water from 15 to 16° C (orl/100th the amount of energy needed to raise the temperature of one gram of water at one atmosphere pressure from 0° C to 100° C), food calories are actually equal to 1,000 standard calories (1 food calorie = 1 kilocalorie).
Chromatography: The process of separating a mixture, for example a mixture containing AgRP, NPY and α-MSH. It involves passing a mixture through a stationary phase, which separates molecules of interest from other molecules in the mixture and allows one or more molecules of interest to be isolated. Examples of methods of chromatographic separation include capillary-action chromatography, such as paper chromatography, thin layer chromatography (TLC), column chromatography, fast protein liquid chromatography (FPLC), nano-reversed phase liquid chromatography, ion exchange chromatography, gel chromatography, such as gel filtration chromatography, size exclusion chromatography, affinity chromatography, high performance liquid chromatography (HPLC), and reverse phase high performance liquid chromatography (RP-HPLC) amongst others.
Contacting: "Contacting" includes in solution and solid phase, for example contacting brain tissue with a test agent. The test agent may also be a combinatorial library for screening a plurality of compounds. In another example, contacting includes contacting a sample with an antibody, for example contacting a sample that contains or is suspected of containing AgRP, NPY and/or α-MSH, with an antibody that specifically binds AgRP, NPY or α-MSH.
Control: A reference standard. A control can be a known value indicative of basal secretion of a neuropeptide from brain tissue, such as the amount of NPY, AgRP or α-MSH secreted from brain tissue not treated with an agent. A control can also be a known value indicative of the ratio of the basal secretion of one neuropeptide to another neuropeptide, such as the ratio the amount of α-MSH secreted to the amount of AgRP secreted from brain tissue not treated with an agent. A difference between a test sample (such as brain tissue contacted with an agent) and a control can be an increase or conversely a decrease. The difference can be a qualitative difference or a quantitative difference, for example a statistically significant difference. In some examples, a difference is an increase or decrease in amount, relative to a control, of at least about 1 %, such as at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, or greater then 500%. In some examples, a difference is an increase or decrease in ratio, relative to a control, of at least about 1 %, such as at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, or greater then 500%.
Diabetes: A failure of cells to transport endogenous glucose across their membranes either because of an endogenous deficiency of insulin and/or a defect in insulin sensitivity. Diabetes is a chronic syndrome of impaired carbohydrate, protein, and fat metabolism owing to insufficient secretion of insulin or to target tissue insulin resistance. It occurs in two major forms: insulin-dependent diabetes mellitus (IDDM, type I) and non-insulin dependent diabetes mellitus (NIDDM, type II) which differ in etiology, pathology, genetics, age of onset, and treatment. The two major forms of diabetes are both characterized by an inability to deliver insulin in an amount and with the precise timing that is needed for control of glucose homeostasis. Diabetes type I, or insulin dependent diabetes mellitus (IDDM) is caused by the destruction of β cells, which results in insufficient levels of endogenous insulin. Diabetes type II, or non-insulin dependent diabetes, results from a defect in both the body's sensitivity to insulin, and a relative deficiency in insulin production.
Food intake: The amount of food consumed by an individual. Food intake can be measured by volume or by weight. In one embodiment, food intake is the total amount of food consumed by an individual. In another embodiment, food intake is the amount of proteins, fat, carbohydrates, cholesterol, vitamins, minerals, or any other food component, of the individual. "Protein intake" refers to the amount of protein consumed by an individual. Similarly, "fat intake," "carbohydrate intake," "cholesterol intake," "vitamin intake," and "mineral intake" refer to the amount of proteins, fat, carbohydrates, cholesterol, vitamins, or minerals consumed by an individual.
Glucose resistance: The loss of the ability of cells and/or tissues to take up glucose, for example in a subject suffering from diabetes.
High throughput technique: Through a combination of modern robotics, data processing and control software, liquid handling devices, and sensitive detectors, high throughput techniques allows the rapid screening of potential pharmaceutical agents in a short period of time. Through this process one can rapidly identify active compounds, which affect the secretion of neuropeptides, for example the secretion of NPY, AgRP and/or α-MSH from brain tissue.
Isolated: An isolated biological component (such as a nucleic acid, peptide or protein) has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, for example the separation of a peptide, such as NPY, AgRP or α-MSH from brain tissue. Peptides and proteins that have been isolated include nucleic acids and proteins purified by standard purification methods, such as chromatography, for example high performance liquid chromatography (HPLC) and the like. The term also embraces peptides, and proteins prepared by recombinant expression in a host cell as well as chemically synthesized peptide and nucleic acids. It is understood that the term "isolated" does not imply that the biological component is free of trace contamination, and can include molecules that are at least 50% isolated, such as at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or even 100% isolated.
Immunoassay: A biochemical test that measures the presence or concentration of a substance in a sample, such as a biological sample, using the reaction of an antibody to its cognate antigen, for example the specific binding of an antibody to a neuropeptide, such as NPY, α-MSH, or AgRP. Both the presence of antigen or the amount of antigen present can be measured. For measuring neuropeptides, such as NPY, α-MSH, or AgRP, the neuropeptide is the antigen and the presence and amount of the neuropeptide is determined or measured.
Measuring the quantity of antigen (such as a neuropeptide, for example NPY, α-MSH, or AgRP) can be achieved by a variety of methods. One of the most common is to label either the antigen or antibody with a detectable label. Specific, non-limiting examples of labels include fluorescent tags, enzymatic linkages, and radioactive isotopes (for example 14C, 32P, 125I, and 3H isotopes and the like). In some examples NPY, AgRP and/or α-MSH is labeled with a radioactive isotope, such as 14C, 32P, 1251, 3H isotope. In other examples an antibody that specifically binds NPY, α-MSH, or AgRP is labeled. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed for example in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989) Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998), and Harlow & Lane, (Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, 1988)),
A "competitive radioimmunoassay (RIA)" is a type of immunoassay used to test for antigens (for example, neuropeptides present in a sample, such as a biological sample). In some examples it involves mixing known quantities of radioactive antigen (for example a radioactively labeled neuropeptide, such as a 125I labeled neuropeptide, for example 125I labeled , NPY, AgRP or α-MSH), with antibody to that antigen, then adding unlabeled or "cold" antigen (for example unlabeled antigen present in a sample, such as biological sample obtained from a subject, for example a sample of brain tissue obtained from a subject) and measuring the amount of labeled antigen displaced by the unlabeled antigen.
Initially, the radioactive antigen is bound to the antibodies. When "cold" (i.e. unlabeled) antigen is added, the two compete for antibody binding sites - at higher concentrations of "cold" antigen, more of it binds to the antibody, displacing the radioactive variant. The bound antigens are isolated from the unbound ones and the amount of radioactivity measured. A radioimmunoassay can be used to calculate the amount of an antigen in a sample. In some disclosed examples, a radioimmunoassay is used to measure the amount of NPY present in a sample, such as a biological sample, for example brain tissue. In some disclosed examples, a radioimmunoassay is used to measure the amount of α-MSH present in a sample, such as a biological sample, for example brain tissue. In some disclosed examples, a radioimmunoassay is used to measure the amount of AgRP present in a sample, such as a biological sample, for example brain tissue. Label: A detectable compound or composition that is conjugated directly or indirectly to another molecule, such as an antibody or a protein, to facilitate detection of that molecule. Specific, non-limiting examples of labels include fluorescent tags, enzymatic linkages, and radioactive isotopes (for example 14C, 32P, 1251, 3H isotopes and the like). In some examples NPY, AgRP and/or α-MSH is labeled with a radioactive isotope, such as 14C, 32P, 1251, 3H isotope. In some examples an antibody that specifically binds NPY, AgRP and/or α-MSH is labeled. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed for example in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998), Harlow & Lane (Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, 1988),.
Leptin: A 16-kD protein that plays a role in the regulation of body weight by inhibiting food intake and stimulating energy expenditure. Defects in leptin production cause severe hereditary obesity in rodents and humans. In addition to its effects on body weight, leptin has a variety of other functions, including the regulation of hematopoiesis, angiogenesis, wound healing, and the immune and inflammatory response. Leptin acts through the leptin receptor, a single- transmembrane-domain receptor of the cytokine receptor family, which is found in many tissues in several alternatively spliced forms. Exemplary amino acid sequences of leptin can be found in GENBANK® at accession nos. NP OO 1003680, NP_001003679, NP_002294, AAY46797, BAA09787, NP_032519, NP_666258, and NP_034834, as available March 5, 2007, which are incorporated herein by reference.
Leptin resistance: The inability of cells and/or tissue to propagate leptin signaling in response contact of the cells and/or tissue with leptin. For example, leptin resistance in a subject can occur when circulating leptin fails to reach its targets in the brain or that there is a failure of components of the intracellular leptin receptors (ObRb) signaling cascade, for example due to loss of expression or presentation of leptin receptors or other mechanism.
Neuropeptide Y (NPY): A 36-amino acid peptide that is a neuropeptide identified in the mammalian brain. NPY is believed to be an important regulator in both the central and peripheral nervous systems and influences a diverse range of physiological parameters, including effects on psychomotor activity, food intake, central endocrine secretion, and vasoactivity in the cardiovascular system. High concentrations of NPY are found in the sympathetic nerves supplying the coronary, cerebral, and renal vasculature and have contributed to vasoconstriction. NPY binding sites have been identified in a variety of tissues, including spleen, intestinal membranes, brain, aortic smooth muscle, kidney, testis, and placenta. In addition, binding sites have been reported in a number of rat and human cell lines. Exemplary amino acids sequences for NPY can be found at GENBANK® accession Nos. NP 075945, AAH43012 and NP_000896 as available March 5, 2007, which are incorporated herein by reference. Neuropeptide Y (NPY) receptor has structure/activity relationships within the pancreatic polypeptide family. This family includes NPY, which is synthesized primarily in neurons; peptide YY (PYY), which is synthesized primarily by endocrine cells in the gut; and pancreatic polypeptide (PP), which is synthesized primarily by endocrine cells in the pancreas. These 36 amino acid peptides have a compact helical structure involving an amino acid structure, termed a "PP-fold" in the middle of the peptide.
NPY binds to several receptors, including the Yl, Y2, Y3, Y4 (PP), Y5, Y6, and Y7 receptors. These receptors are recognized based on binding affinities, pharmacology, and sequence. Most, if not all of these receptors are G protein coupled receptors. The Yl receptor is generally considered to be postsynaptic and mediates many of the known actions of neuropeptide Y in the periphery. Originally, this receptor was described as having poor affinity for C-terminal fragments of neuropeptide Y, such as the 13-36 fragment, but interacts with the full length neuropeptide Y and peptide YY with equal affinity (e.g. see Patent Cooperation Treaty publication WO 93/09227).
NP Y/ AgRP neurons: Neurons of the hypothalamic arcuate nucleus named for their ability to produce, neuropeptide Y (NPY) and agouti-related protein (AgRP). These neurons make peptides that potently stimulate food intake not only by increasing neuropeptide Y (NPY) signaling, but by reducing melanocortin signaling via the release of agouti-related peptide (AgRP), an endogenous melanocortin 3/4 receptor antagonist. Since NPY/ AgRP neurons express receptors for leptin and insulin and are inhibited by these hormones, they are activated by a decrease of leptin or insulin signaling. Fasting, uncontrolled diabetes, and genetic leptin deficiency are examples of conditions in which food intake increases via a mechanism involving NPY/ AgRP neurons. The NPY/ AgRP neurons respond to afferent signals reflecting energy deficits (for example low blood glucose) by NPY and AgRP, which exert orexigenic effects (i.e. increased food intake and decreased energy expenditure).
Normal Daily Diet: The average food intake for an individual of a given species. A normal daily diet can be expressed in terms of caloric intake, protein intake, carbohydrate intake, and/or fat intake. A normal daily diet in humans generally comprises the following: about 2,000, about 2,400, or about 2,800 to significantly more calories. In addition, a normal daily diet in humans generally includes about 12 g to about 45 g of protein, about 120 g to about 610 g of carbohydrate, and about 11 g to about 90 g of fat. A low calorie diet would be no more than about 85%, and preferably no more than about 70%, of the normal caloric intake of a human individual.
In animals, the caloric and nutrient requirements vary depending on the species and size of the animal. For example, in cats, the total caloric intake per pound, as well as the percent distribution of protein, carbohydrate and fat varies with the age of the cat and the reproductive state. A general guideline for cats, however, is 40 cal/lb/day (18.2 cal/kg/day). About 30% to about 40% should be protein, about 7% to about 10% should be from carbohydrate, and about 50% to about 62.5% should be derived from fat intake. One of skill in the art can readily identify the normal daily diet of an individual of any species. Obesity: A condition in which excess body fat may put a person at health risk (see Barlow and Dietz, Pediatrics 102: E29, 1998; National Institutes of Health, National Heart, Lung, and Blood Institute (NHLBI), Obes. Res. 6 (suppl. 2):51S- 209S, 1998). Excess body fat is a result of an imbalance of energy intake and energy expenditure. In one embodiment in humans, the Body Mass Index (BMI) is used to assess obesity. In one embodiment, a BMI of 25.0 kg/m2 to 29.9 kg/m2 is overweight, while a BMI of 30 kg/m2 is obese.
In another embodiment in humans, waist circumference is used to assess obesity. In this embodiment, in men a waist circumference of 102 cm or more is considered obese, while in women a waist circumference of 89 cm or more is considered obese. Strong evidence shows that obesity affects both the morbidity and mortality of individuals. For example, an obese individual is at increased risk for heart disease, non-insulin dependent (type 2) diabetes, hypertension, stroke, cancer (e.g. endometrial, breast, prostate, and colon cancer), dyslipidemia, gall bladder disease, sleep apnea, reduced fertility, and osteoarthritis, amongst others (see Lyznicki et al., Am. Fam. Phys. 63:2185, 2001).
Overweight: An individual who weighs more than their ideal body weight. An overweight individual can be obese, but is not necessarily obese. In one embodiment, an overweight human individual is any individual who desires to decrease their weight. In another embodiment, an overweight human individual is an individual with a BMI of 25.0 kg/m2 to 29.9 kg/m2.
Therapeutically effective amount: The quantity of a chemical composition or sufficient to achieve a desired effect in a subject being treated. For instance, this can be the amount necessary to treat obesity and/or diabetes or to measurably alter outward symptoms of obesity and/or diabetes. When administered to a subject, a dosage will generally be used that will achieve target tissue concentrations that has been shown to achieve in vitro inhibition of obesity and/or diabetes or to measurably alter outward symptoms of obesity and/or diabetes.
Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers of use are conventional. Remington 's Pharmaceutical Sciences, by E.W. Martin, Mack Publishing Co., Easton, PA, 15th Edition, 1975, describes compositions and formulations suitable for pharmaceutical delivery of the compositions disclosed herein.
In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (such as powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. Polypeptide: A polymer in which the monomers are amino acid residues which are joined together through amide bonds. When the amino acids are alpha- amino acids, either the L-optical isomer or the D-optical isomer can be used, the L- isomers being preferred. The terms "polypeptide" or "protein" or "peptide" as used herein are intended to encompass any amino acid sequence and include modified sequences such as glycoproteins. The term "polypeptide" or "protein" or "peptide" is specifically intended to cover naturally occurring proteins, as well as those which are recombinantly or synthetically produced. In some disclosed examples, a peptide is NPY. In some disclosed examples, a peptide is AgRP. In some disclosed examples, a peptide is α-MSH.
Proopiomelanocortin (POMC): A glycosylated protein of a molecular weight of about 31 kDa protein. POMC is synthesized mainly in the anterior pituitary but also found in the hypothalamus and brainstem. This protein is a precursor protein, post-translational processing of POMC yields several neuroactive peptides upon specific cleavage. The POMC coding sequence includes the amino acid sequences of adrenocoroticotropic (ACTH) hormone and beta-lipotropin. ACTH is processed to produce the proteins alpha-melanotropin (α-MSH), corticotrophin-like intermediate lobe peptide. Beta-lipotropin is processed to produce the proteins alpha-lipotropin, beta-endorphins, beta-melanocyte stimulating hormone (MSH), and met-enkephalin. The amino-terminal fragment of POMC is processed to a family of gamma-MSH peptides and to a peptide with putative mitogenic stimulatory activity of the adrenal cortical cells. The biological activity of POMC-derived peptides is further regulated in a tissue-specific manner by acetylation of the amino-terminal amino acid residue and/or amidation of the carboxyterminal amino acid residue by the enzyme peptidyl-α-monooxygenase (PAM).
The POMC gene (human chromosome 2p23) contains three exons and two large introns: one, of about 3.5 kb, interrupts the N- terminal fragment of the common precursor mostly encoded in exon 3. Exon 2 contains the sequence for a portion of the 5' untranslated portion of the mRNA, all of the signal sequence which directs insertion of the precursor protein into the endoplasmic reticulum, and 8 amino acids of the N-terminal fragment. The overall arrangement of introns and exons in the POMC gene is almost identical in all mammalian species. Hormonal control of POMC gene transcription and release of peptide products derived from the POMC precursor is tissue-specific; for example, glucocorticoids specifically inhibit anterior but not intermediate pituitary POMC transcription. POMC neurons are neurons that secrete POMC or peptides derived from POMC. Exemplary amino acid sequences of POMC and the peptides derived from POMC can be found on GENBANK® at accession nos. NP 001030333, NP 000930, POl 189, POl 193, and NP_032921 as available March 5, 2007, which are incorporated herein by reference.
Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide preparation (such as a preparation of α-MSH, NPY or AgRP) is one in which the peptide is more pure than the peptide in its natural environment within a cell or tissue, such as brain tissue. Such peptides may be produced, for example, by standard purification techniques (such as chromatography, for example HPLC), and/or by recombinant expression. In some embodiments, a preparation of a protein is purified such that the protein represents at least 50%, for example at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 85%, 90%, 95%, 98% or 99%, of the total protein content of the preparation.
Tissue: A plurality of functionally related cells. A tissue can be a suspension, a semi-solid, or solid. In one embodiment, tissue is brain tissue.
Therapeutically effective amount: A quantity of a specific substance sufficient to achieve a desired effect in a subject being treated. When administered to a subject, a dosage will generally be used that will achieve target tissue concentrations that has been shown to achieve a desired in vitro effect. Treating: Inhibiting the full development of a disease or condition, for example, in a subject who is at risk for a disease such as obesity and/or diabetes. "Treatment" includes a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. The term "ameliorating," with reference to a disease or pathological condition, refers to any observable beneficial effect of the treatment. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, an improvement in the overall health or well-being of the subject, or by other parameters well known in the art that are specific to obesity and/or diabetes. A "prophylactic" treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology.
///. Description of several embodiments
Obesity and diabetes are interrelated metabolic disorders that affect millions of people worldwide, decreasing overall health of the population and resulting incalculable economic costs from lost productivity. In most adults, body weight is relatively constant despite large variations in daily food intake and energy expenditure. Energy balance is regulated by neural and hormonal signals that are integrated in the brain (Seeley and Woods, Nat. Rev. Neurosci. 4:901-909, 2003). Leptin, a hormone secreted primarily by adipocytes, is present in serum concentrations directly proportional to the amount of adipose tissue (Considine et ah, N. Engl. J. Med. 334:292-295, 1996), signaling to the central nervous system (CNS) the relative extent of energy (adipose) stores in the body (Spiegelman and Flier, Cell 104:531-543, 2001). Leptin regulates food intake by binding to CNS receptors and modulating the activity of neurons in appetite control centers in the brain (de Luca et al, J Clin Invest 115:3484-3493, 2005).
In obese leptin-deficient mice, administration of leptin reduces hyperphagia and obesity. In contrast, obese mice that are deficient in the leptin obese receptor (ObRb) do not respond to leptin (Chen et al, Cell 84:491-495, 1996; Halaas et al, Science 269:543-546, 1995). Leptin also affects energy expenditure in rodents and humans (Halaas et al, Science 269, 543-546, 1995; Rosenbaum et al, J. Clin.
Invest. 115:3579-3586, 2005). Activation of central ObRb increases sympathetic nervous system activity, which stimulates energy expenditure in adipose tissue (Commins et al, Endocrinology \AO:A112-A11%, 1999). The arcuate nucleus in the hypothalamus (ARH) is a major site of leptin sensing (Balthasar et al, Neuron 42:983-991, 2004; Coppari et al, Cell Metab. 1 :63-72, 2005; Cowley et al, Nature 411 :480-484, 2001; van den Top et al, Nat Neurosci 7, 493-494, 2004). ObRb is highly expressed in the ARH (Elmquist et al, J. Comp. Neurol. 395:535-547, 1998; Schwartz et al, J. Clin. Invest. 98:1101- 1106, 1996). The ARH contains at least two key populations of leptin-responsive neurons that have opposite actions on food intake. One population expresses the anorexigenic peptide α-melanocyte- stimulating hormone (α-MSH), derived from the proopiomelanocortin (POMC) precursor. The other population expresses the orexigenic peptides: neuropeptide Y (NPY) and agouti-related peptide (AgRP) (Cone, Nat. Neurosci. 8:571-578, 2005). Arcuate neurons subsequently innervate various second order hypothalamic targets that express melanocortin-4 receptors (MC4R) and NPY receptors (Liu et al, J. Neurosci. 23:7143-7154, 2003). Leptin can modulate both POMC and AgRP neurons and can promote the release of α-MSH, a potent anorexigen at central
MC4Rs (Cowley et al, Nature 411 :480-484, 2001; Elias et al, Neuron 23:775-786, 1999; Schwartz et al, Diabetes 46:2119-2123, 1997).
Although most obese humans and rodents have very high circulating leptin, this hyperleptinemia neither reduces appetite nor increases energy expenditure. This state has been termed "leptin resistance." Because leptin resistance appears in obese individuals, leptin treatment alone is not sufficient to treat obesity and related diseases, such as diabetes. Therefore obtaining therapeutic drugs useful treating diabetes and/or obesity, especially in leptin resistant individuals, could have profound beneficial effects on public health and economic efficiency. Disorders that can be treated by the administration of such agents include, but are not limited to, cardiovascular disease, (including, but not limited to, hypertension, atherosclerosis, congestive heart failure, and dyslipidemia), stroke, gallbladder disease, osteoarthritis, sleep apnea, reproductive disorders such as, but not limited to, polycystic ovarian syndrome, cancers (for example, breast, prostate, colon, endometrial, kidney, and esophagus cancer), varicose veins, acnthosis nigricans, eczema, exercise intolerance, insulin resistance, hypertension hypercholesterolemia, cholithiasis, osteoarthritis, orthopedic injury, insulin resistance (such as, but not limited to, type 2 diabetes and syndrome X) and thromboembolic disease (see Kopelman, Nature 404:635-43; Rissanen et al, British Med. J. 301 :835, 1990). It is disclosed herein that because the NPY and AgRP work in opposition to α-MSH to decrease metabolism and increase appetite, agents that effect the secretion of NPY, AgRP, α-MSH, for example by decreasing the secretion of NPY and AgRP while increasing the secretion of α-MSH are of use in treating obesity and/or diabetes. Disclosed herein is an assay developed to screen for such agents. This assay is highly specific because at measures the amount of all three of these neuropeptides simultaneously. In addition, by taking the ratio of the agonist/antagonist pair α-MSH/ AgRP this assay is extremely sensitive in measuring differences in the activation of the melanocortin pathway through activation of MC4R.
Methods of Identifying Agents of use Disclosed herein is a method for identifying an agent for the treatment of diabetes, obesity, or a combination thereof. The method includes contacting a test agent to brain tissue in vitro in media that that includes cerebrospinal fluid, such as artificial cerebrospinal fluid (aCSF), and measuring the amount and/or concentration of NPY, AgRP, and α-MSH secreted from the brain tissue. Either natural or artificial cerebrospinal fluid can be used, but artificial cerebrospinal fluid is preferred because it can be more readily obtained in the quantities used in the disclosed assay and methods. In some embodiments, the artificial cerebrospinal fluid (aCSF) can include NaCl, Na2HPO4, KCl, CaCl2, MgSO4, NaHCO3, glucose, and ascorbic acid. In some embodiments, the aCSF has a concentration of about 120 to about 130 mM NaCl, a concentration of about 15 to about 25 mM NaHCO3, a concentration of about 2mM to about 3 mM KCl, a concentration of about 1 mM to about 2 mM NaH2PO4, a concentration of about 1 mM to about 2 mM MgCl2, a concentration of about 2 mM to about 3 mM CaCl2, and a concentration of about 5 mM to about 15 mM D-glucose and a pH from about 7 to about 8. In some embodiments, the aCSF can include a protease inhibitor, for example aprotinin, for example a concentration of about 0.1 to about 1.0 trypsin inhibitor units (TIU) aprotinin/ml. In some examples, a protease inhibitor cocktail is used, such as those commercially available from Roche Bioscience, Pierce Biotechnology and the like. The brain tissue includes both proopiomelanocortin (POMC) neurons and neuropeptide peptide Y/ Agouti-related peptide (NPY/ AgRP) neurons, such that the amount and/or concentration of NPY, AgRP, and α-MSH secreted from these neurons can be measured. In some embodiments, the amount and/or concentration of NPY, AgRP, and α-MSH secreted from brain tissue is normalized to the amount of brain tissue contacted with the agent, for example the amount and/or concentration of NPY, AgRP, and α-MSH per gram of brain tissue.
The amount and/or concentration of alpha-melanotropin (α-MSH) secreted from the brain tissue contacted with the test agent is measured, for example using an immunoassay specific for α-MSH, such as a radio immunoassay (RIA) specific for α-MSH. The amount and/or concentration of AgRP secreted from the brain tissue contacted with the test agent is also measured, for example using an immunoassay specific for AgRP, such as a RIA specific for AgRP. The ratio of the amount and/or concentration of α-MSH secreted from the brain tissue to the amount and/or concentration of AgRP secreted from the brain tissue contacted with the test agent is determined. This ratio is compared to a control, for example, a standard value for the ratio of the amount and/or concentration of α-MSH secreted from brain tissue to the amount and/or concentration of AgRP secreted from brain tissue, or the ratio of the amount and/or concentration of α-MSH secreted to the amount and/or concentration of AgRP secreted from brain tissue not contacted with a test agent. The amount and/or concentration of NPY secreted from brain tissue contacted with the test agent is also measured, for example using an immunoassay specific for NPY, such as a RIA specific for NPY. This amount and/or concentration is compared to a control, for example, a standard value for the amount and/or concentration of NPY secretion from brain tissue, or the amount and/or concentration of NPY secreted from brain tissue not contacted with a test agent.
An increase in the ratio of the amount and/or concentration of α-MSH secreted from brain tissue to the amount and/or concentration of AgRP secreted from brain tissue contacted with a test agent relative to a control and a corresponding decrease in the amount and/or concentration NPY secreted from brain tissue contacted with a test agent relative to a control identifies the test agent as being of use for the treatment of diabetes, obesity, or a combination thereof. In one embodiment, a test agent that increases the ratio of the amount and/or concentration α-MSH secreted from the brain tissue to the amount and/or concentration of AgRP secreted from the brain tissue test and decreases the amount and/or concentration of NPY secreted from the brain tissue relative a control is identified as being of use for treating obesity. In another embodiment, a test agent that increases the ratio of the amount and/or concentration α-MSH secreted from the brain tissue to the amount and/or concentration of AgRP secreted from the brain tissue relative to a control and decreases the amount and/or concentration of NPY secreted from the brain tissue relative a control is identified as being of use for treating diabetes.
In some embodiments, the control is a standard value indicative of the basal level of NPY secreted by brain tissue. In some embodiments, the control is a standard value indicative of the basal ratio of α-MSH to AgRP secreted by brain tissue. In some embodiments, a control is the ratio of α-MSH to AgRP secreted by brain tissue in vitro not contacted with the test agent. In some embodiments, a control is the amount of NPY secreted by brain tissue in vitro not contacted with the test agent. In some embodiments, the control is expressed in amount and/or concentration of NPY, AgRP, and α-MSH secreted from brain tissue is normalized to the amount of brain tissue, for example the amount and/or concentration of NPY, AgRP, and α-MSH per gram of brain tissue.
In some embodiments, a test agent can induce a statistically significant difference in the ratio of the amount of α-MSH secreted from the brain tissue to the amount of AgRP secreted from the brain tissue contacted with the test agent, as compared to the control, such brain tissue not contacted with the test agent (such as an brain tissue contacted with carrier alone). In some embodiments, the difference between the ratio of the amount of α-MSH secreted from the brain tissue to the amount of AgRP secreted from the brain tissue contacted with the test agent relative to a control is at least about 10%, such as at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, or at least about 500% or even greater then 500%.
In some embodiments, a test agent can induce a statistically significant decrease in the amount of NPY secreted from the brain tissue contacted with the test agent, as compared to the control, such brain tissue not contacted with the test agent (such as an brain tissue contacted with carrier alone). In some embodiments, the decrease in the amount of NPY secreted from the brain tissue contacted with the test agent relative to a control is at least about 10%, such as at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500% or even greater then 500%.
In some examples, a 2 fold or greater increase in the ratio of the amount and/or concentration of α-MSH secreted from brain tissue to the amount and/or concentration of AgRP secreted from brain tissue contacted with a test agent relative to a control and a corresponding 50% decrease in the amount and/or concentration of NPY secreted from brain tissue contacted with a test agent relative to a control identifies the test agent as being of use for the treatment of diabetes, obesity, or a combination thereof.
In some examples, the brain tissue is contacted with the test agent at a concentration of about 1 picomolar to about 100 mmolar, such as a concentration of test agent of about 1 picomolar, about 10 picomolar, about 100 picomolar, about 1 nanomolar, about 10 nanomolar, about 100 nanomolar, about 1 micromolar, about 10 micromolar, about 100 micromolar, 1 millimolar, about 10 millimolar, or even about 100 millimolar. In some examples, brain tissue, such as different portions of brain tissue, is contacted with different concentrations of the test agent, for example to determine the secretion of NPY, AgRP, and α-MSH as a function of test agent concentration. One of skill in the art will understand that the amount and/or concentration of AgRP, NPY, and α-MSH can be measured at any concentration of test agent or any number of concentrations of test agent and that the concentrations given above are exemplary. In some examples, the brain tissue is contacted with the test agent for at least about 1 second, such as at least about 5 seconds, at least about 10 seconds, at least about 30 seconds, at least about 1 minute, at least about 5 minutes, at least about 10 minutes, at least about 30 minutes, at least about 60 minutes, at least about 120 minutes, at least about 3 hours, at least about 6 hours, at least about 12 hours, or even about 24 hours or more. One of skill in the art will understand that brain tissue can be contacted with the test agent for any amount of time and that periods given above are exemplary. In some examples, multiple time points are determined. For example, following incubation with the one or more test agents, the amount and/or concentration of AgRP, NPY and α-MSH secreted from brain tissue is measured at various time points (for example to determine a time course for the secretion of AgRP, NPY, and α-MSH in response to contact with a test agent), such at about 10 seconds, at about 30 seconds, at about 1 minute, at about 5 minutes, at about 10 minutes, at about 30 minutes, at about 60 minutes, at about 2 hours, or even at least 24 hours after contact with the test agent. One of skill in the art will understand that the amount and/or concentration of AgRP, NPY, and α-MSH can be measured at any time point or number of time points and that the time points given above are exemplary.
The brain tissue can be obtained from any subject, including both human and veterinary mammalian subjects. Thus, the subject can be a human, or can be a non- human primate, a farm animal such as swine, cattle, and poultry, a sport animal or pet such as dogs, cats, horses, hamsters, or laboratory animal such as a rodent, for example a mouse, rat, guinea pig and the like.
The brain tissue includes both POMC (neurons that secrete α-MSH) neurons and AgRP/NPY neurons (i.e. neurons that secrete AgRP and NPY). In some examples, the brain tissue includes the paraventricular nucleus of the hypothalamus (PVH) (or a portion thereof) and the hypothalamic arcuate nucleus (ARH) (or a portion thereof). In some examples a portion of brain tissue is a histological section obtained from the brain of a subject, for example a section that is between about lmm and about 20mm thick, such as about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 10 mm, about 15 mm, or about 20 mm thick. In some examples, the brain tissue is mouse brain tissue (such as brain tissue from an obese mouse, for example, a diet induced obese (DIO) mouse, or a brain tissue from a diabetic mouse). In some examples, the brain tissue is human brain tissue (such as brain tissue from an obese human or a brain tissue from a diabetic human). In some examples, the brain tissue is leptin resistant, such that contacting the brain tissue with leptin does not stimulate the secretion of neuropeptides, such as AgRP, NPY and/or α-MSH. In some examples, the brain tissue is glucose resistant, for example obtained from a subject with diabetes. Thus in some examples, the brain tissue is obtained from a diabetic subject, such as a diabetic human subject or a diabetic laboratory animal, for example a diabetic mouse.
The amount and/or concentration of AgRP, NPY, and α-MSH can be measured using any method known to one of skill in the art. In several examples, the amount of AgRP, NPY, and α-MSH secreted from brain tissue is measured using an antibody that specifically binds AgRP, NPY, or α-MSH, such as a monoclonal or polyclonal antibody. In some embodiments, the antibody that specifically binds AgRP does not specifically bind NPY, or α-MSH. In some embodiments, the antibody that specifically binds α-MSH, does not specifically bind NPY, or AgRP. In some embodiments, the antibody that specifically binds NPY does not specifically bind AgRP, or α-MSH. The presence of antibody: antigen complexes can be determined using methods known in the art. For example, the antibody can include a detectable label, such as a fluorophore, radiolabel, or enzyme, which permits detection of the antibody, for example using ELISA. In particular examples, multiple antibodies (each with a unique detectable label) are incubated with the supernatant, and the presence of multiple complexes detected simultaneously, such that the amount of AgRP, NPY, and α-MSH (or any combination thereof) can be measured in a single sample, such as in a single tube or well of a microtiter plate. In some embodiments, the amount of AgRP, NPY, and α-MSH (and therefore the ratio of α-MSH to AgRP) is measured using an immunoassay (such as a radioimmunoassay, for example competitive radioimmunoassay), for example using antibodies that specifically bind the neuropeptides AgRP, NPY, and α-MSH.
In some examples, the presence of α-MSH, NPY, and/or AgRP is measured using a competition immunoassay, such as a competition radioimmunoassay (RIA). In one example of a competition immunoassay, a sample of pure or nearly pure antigen (such as α-MSH, NPY, or AgRP) that has been labeled with a detectable label is used. In one example of a competition radioimmunoassay, the antigen is labeled with a radioisotope (for example α-MSH, NPY, and/or AgRP is been labeled) for example labeled with two or more 125I atoms. Typically, a constant amount (known amount) of the labeled antigen is mixed with a test solution that contains an unknown amount of unlabeled antigen, such as a solution containing neuropeptide secreted from brain tissue, for example α-MSH, NPY, and/or AgRP secreted from brain tissue. The solution is then allowed to bind to a subsaturating amount of an antibody specific for the antigen, such as an antibody specific for α- MSH, NPY, or AgRP. High levels of unlabeled antigen present in the solution will reduce the amount of labeled antigen bound to the antibody specific for the antigen. The bound antigen (both labeled and unlabeled) is separated from the unbound antigen and the amount of labeled antigen can then be determined by measurement, for example if the antigen is labeled with a radioisotope the radioactivity emitted from the bound labeled antigen is proportional to the amount of labeled antigen bound. The sensitivity of a competition assay typically varies depending on three factors, including (1) the number of antibodies used to detect antigen, (2) the avidity of the antigen for the antibody, and (3) the specific activity of the labeled antigen. Thus, one of skill in the art can adjust these parameters depending on the sensitivity and dynamic range required.
In another example of a competition immunoassay, a sample of antigen, such as a sample containing secreted neuropeptides, such as α-MSH, NPY, and/or AgRP, is contacted with a first antibody that has been labeled with a detectable label. The sample is contacted with a second unlabeled antibody that competes with the binding of the first antibody. High levels of unlabeled antibody present in the solution will reduce the amount of labeled antibody bound to the antigen. The bound antibody (both labeled and unlabeled) is separated from the unbound antibody and the amount of labeled antibody can then be determined by measurement, for example if the antibody is labeled with a radioisotope the radioactivity emitted from the bound labeled antibody is proportional to the amount of labeled antibody bound to the antigen. In some embodiments, the amount of α-MSH is measured with a competitive radioimmunoassay. In some embodiments, the radioimmunoassay uses α-MSH peptide exogenous to the brain tissue that is labeled with two or more 125I atoms. In some embodiments, the amount of NPY is measured with a competitive radioimmunoassay. In some embodiments, the radioimmunoassay uses NPY peptide exogenous to the brain tissue that is labeled with two or more 125I atoms. In some embodiments, the amount of AgRP is measured with a competitive radioimmunoassay. In some embodiments, the radioimmunoassay uses AgRP peptide exogenous to the brain tissue that is labeled with two or more 125I atoms. Iodine labeling of antigens, such as AgRP, NPY and α-MSH, and antibodies is well know in the art, and exemplary procedures can be found in Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York (1988).
The disclosed immunoassays are particularly sensitive and can detect the neuropeptides AgRP, NPY, and α-MSH at sub-nanomolar levels, for example at picomolar levels or even sub-picomolar levels. In other words the disclosed immunoassays can detect AgRP, NPY, and α-MSH at a concentration of about 0.1 picomolar or greater. In some embodiments, the disclosed immunoassays are capable of detecting an amount of AgRP at a concentration of about 0.1 picomolar to about 10 picomolar or greater, such as about 0.1 picomolar, about 0.2 picomolar, about 0.3 picomolar, about 0.4 picomolar, about 0.5 picomolar, about 0.6 picomolar, about 0.7 picomolar, about 0.8 picomolar, about 0.9 picomolar, about 1.0 picomolar, about 2.0 picomolar, about 3.0 picomolar, about 4.0 picomolar, about 5.0 picomolar, about 6.0 picomolar, about 7.0 picomolar, about 8.0 picomolar, about 9.0 picomolar, or about 10.0 picomolar or greater. For example, the disclosed immunoassays are capable of detecting an amount of AgRP from about 0.1 picomolar to about 10 picomolar, from about 0.1 picomolar to about 100 picomolar, 0.1 picomolar to about 1000 picomolar, or even greater. In some embodiments, the disclosed immunoassays are capable of detecting an amount of NPY at a concentration of about 0.1 picomolar to about 10 picomolar or greater, such as about 0.1 picomolar, about 0.2 picomolar, about 0.3 picomolar, about 0.4 picomolar, about 0.5 picomolar, about 0.6 picomolar, about 0.7 picomolar, about 0.8 picomolar, about 0.9 picomolar, about 1.0 picomolar, about 2.0 picomolar, about 3.0 picomolar, about 4.0 picomolar, about 5.0 picomolar, about 6.0 picomolar, about 7.0 picomolar, about 8.0 picomolar, about 9.0 picomolar, or about 10.0 picomolar or greater. For example, the disclosed immunoassays are capable of detecting an amount of AgRP from about 0.1 picomolar to about 10 picomolar, from about 0.1 picomolar to about 100 picomolar, 0.1 picomolar to about 1000 picomolar, or even greater. In some embodiments, the disclosed immunoassays are capable of detecting an amount of NPY at a concentration of about 0.1 picomolar to about 10 picomolar or greater, such as about 0.1 picomolar, about 0.2 picomolar, about 0.3 picomolar, about 0.4 picomolar, about 0.5 picomolar, about 0.6 picomolar, about 0.7 picomolar, about 0.8 picomolar, about 0.9 picomolar, about 1.0 picomolar, about 2.0 picomolar, about 3.0 picomolar, about 4.0 picomolar, about 5.0 picomolar, about 6.0 picomolar, about 7.0 picomolar, about 8.0 picomolar, about 9.0 picomolar, or about 10.0 picomolar or greater. For example, the disclosed immunoassays are capable of detecting an amount of AgRP from about 0.1 picomolar to about 10 picomolar, from about 0.1 picomolar to about 100 picomolar, 0.1 picomolar to about 1000 picomolar, or even greater.
In some examples, Western blotting is used to quantify the amount of AgRP, NPY and α-MSH, such as AgRP, NPY and α-MSH present brain tissue. Briefly, the brain tissue or an extract thereof is resolved by SDS-PAGE, and the peptides transferred to an appropriate medium, such as nitrocellulose. The nitrocellulose is incubated with the appropriate antibody (which itself can have a label, or which can be detected by using the appropriate labeled secondary antibody), which permits detection of the antibody-protein complex.
In another example, a colorimetric assay is used to quantify the amount of AgRP, NPY and α-MSH, such as AgRP, NPY and α-MSH present brain tissue. Briefly, the brain tissue or an extract thereof is exposed to a material that will produce a colorimetric reaction if AgRP, NPY or α-MSH is present, for example at a particular concentration.
In some embodiments, the antibody specific for AgRP, NPY, or α-MSH is attached to a solid support, such as a multiwell plate (such as, a microtiter plate, for example, 96 well, 384 well, etc bead, membrane or the like). In some embodiments, the AgRP, NPY, and/or α-MSH that was secreted from the brain tissue is attached to a solid support, such as a multiwell plate (such as, a microtiter plate, for example, 96 well, 384 well, etc bead, membrane or the like). In practice, microtiter plates may conveniently be utilized as the solid phase. The surfaces may be prepared in advance, stored, and shipped to another location(s). Methods of attaching antibodies and antigens to solid surfaces are well know in the art, and exemplary procedures can be found in Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York (1988). Prior to determining the amount of α-MSH, NPY, and/or AgRP in the sample, such as a sample of brain tissue, the sample can be subjected to one or more dimensions of chromatographic separation, for example, one or more dimensions of liquid or size exclusion chromatography, for example to isolate α-MSH, NPY, and/or AgRP from constituents of the sample, such as other peptides, proteins, nucleic acids and cellular components. Representative examples of chromatographic separation include paper chromatography, thin layer chromatography (TLC), liquid chromatography, column chromatography, fast protein liquid chromatography (FPLC), ion exchange chromatography, size exclusion chromatography, affinity chromatography, high performance liquid chromatography (HPLC), nano-reverse phase liquid chromatography (nano-RPLC), poly acrylamide gel electrophoresis (PAGE), capillary electrophoresis (CE), reverse phase high performance liquid chromatography (RP-HPLC) or other suitable chromatographic techniques. Thus, in some embodiments α-MSH, AgRP and NPY is isolated from the brain tissue prior to measuring the amount of α-MSH, AgRP and NPY secreted from the brain tissue, for example using a chromatography technique such as HPLC.
Test Agents In some embodiments, screening of test agents involves testing a combinatorial library containing a large number of potential modulator compounds. A combinatorial chemical library may be 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. For example, 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 (for example the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks. Appropriate test agents can be contained in libraries, for example, synthetic or natural compounds in a combinatorial library. Numerous libraries are commercially available or can be readily produced; means for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides, such as antisense oligonucleotides and oligopeptides, also are known. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or can be readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Such libraries are useful for the screening of a large number of different compounds.
Preparation and screening of combinatorial libraries is well known to those of skill in the art. Libraries (such as combinatorial chemical libraries) useful in the disclosed methods include, but are not limited to, peptide libraries (see, e.g., U.S. Patent No. 5,010,175; Furka, Int. J. Pept. Prot. Res., 37:487-493, 1991; Houghton et al, Nature, 354:84-88, 1991; PCT Publication No. WO 91/19735), (see, e.g., Lam et al, Nature, 354:82-84, 1991; Houghten et al, Nature, 354:84-86, 1991), and combinatorial chemistry-derived molecular library made of D-and/or L- confϊguration amino acids, phosphopeptides (including, but not limited to, members of random or partially degenerate, directed phosphopeptide libraries; see, e.g., Songyang et al., Cell, 12:161-11%, 1993), antibodies (including, but not limited to, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and Fab, F(ab')2 and Fab expression library fragments, and epitope-binding fragments thereof), small organic or inorganic molecules (such as, so-called natural products or members of chemical combinatorial libraries), molecular complexes (such as protein complexes), or nucleic acids, 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. Patent No.
No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al, Proc. Natl. Acad. ScL USA, 90:6909-6913, 1993), vinylogous polypeptides (Hagihara et al., J. Am. Chem. Soc, 114:6568, 1992), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al., J. Am. Chem. Soc, 114:9217-9218, 1992), analogous organic syntheses of small compound libraries (Chen et al, J. Am. Chem. Soc, 116:2661, 1994), oligocarbamates (Cho et al, Science, 261 :1303, 1003), and/or peptidyl phosphonates (Campbell et al, J. Org. Chem., 59:658, 1994), nucleic acid libraries (see Sambrook et al Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press, N. Y., 1989; Ausubel et al. , Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y., 1989), peptide nucleic acid libraries (see, e.g., U.S. Patent No. 5,539,083), antibody libraries (see, e.g., Vaughn et al, Nat. Biotechnol, 14:309- 314, 1996; PCT App. No. PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al, Science, 274:1520-1522, 1996; U.S. Patent No. 5,593,853), small organic molecule libraries (see, e.g., benzodiazepines, Baum, C&EN, Jan 18, page 33, 1993; isoprenoids, U.S. Patent No. 5,569,588; thiazolidionones and methathiazones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Patent Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. U.S. Patent No. 5,506,337; benzodiazepines, 5,288,514) and the like.
Libraries useful for the disclosed screening methods can be produced in a variety of manners including, but not limited to, spatially arrayed multipin peptide synthesis (Geysen, et al, Proc. Natl. Acad. Sci., 81(13):3998-4002, 1984), "tea bag" peptide synthesis (Houghten, Proc. Natl. Acad. Sci., 82(15):5131-5135, 1985), phage display (Scott and Smith, Science, 249:386-390, 1990), spot or disc synthesis (Dittrich et al, Bioorg. Med. Chem. Lett., 8(17):2351-2356, 1998), or split and mix solid phase synthesis on beads (Furka et al, Int. J. Pept. Protein Res., 37(6):487-493, 1991; Lam et al, Chem. Rev., 97(2):411-448, 1997).
Devices for the preparation of combinatorial libraries are also commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition, numerous combinatorial libraries are themselves commercially available (see, for example, ComGenex,
Princeton, N. J., Asinex, Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.).
Libraries can include a varying number of compositions (members), such as up to about 100 members, such as up to about 1000 members, such as up to about 5000 members, such as up to about 10,000 members, such as up to about 100,000 members, such as up to about 500,000 members, or even more than 500,000 members.
In one example, the methods can involve providing a combinatorial chemical or peptide library containing a large number of potential therapeutic compounds. Such combinatorial libraries are then screened by the methods disclosed herein to identify those library members (particularly chemical species or subclasses) that display a desired characteristic activity.
The compounds identified using the methods disclosed herein can serve as conventional "lead compounds" or can themselves be used as potential or actual therapeutics. In some instances, pools of candidate test agents can be identified and further screened to determine which individual or subpools of test agents in the collective have a desired activity.
Control reactions can be performed in combination with the libraries. Such optional control reactions are appropriate and can increase the reliability of the screening. Accordingly, disclosed methods can include such a control reaction. The control reaction may be a negative control reaction that measures the secretion of
AgRP, NPY and α-MSH independent of a test agent. The control reaction may also be a positive control reaction that measures the secretion of AgRP, NPY and α-MSH in response to a known agent that affects AgRP, NPY and α-MSH secretion, such as leptin.
Kits and High Throughput Systems
This disclosure also provides kits for identifying test agents that are useful in treating obesity and/or diabetes. The kits include antibodies that specifically bind AgRP, NPY and α-MSH. The kits may further include additional components such as instructional materials and additional reagents, for example specific binding agents, such as antibodies, labeled peptides (such as radiolabeled peptide, for example 125I labeled peptide, such as 125I AgRP, NPY and α-MSH) or a means for labeling peptides. The kits may also include additional components to facilitate the particular application for which the kit is designed (for example microtiter plates). Such kits and appropriate contents are well known to those of skill in the art. The instructional materials may be written, in an electronic form (such as a computer diskette or compact disk) or may be visual (such as video files).
This disclosure also provides integrated systems for high-throughput screening of test agents for an effect on AgRP, NPY and α-MSH secretion. The systems typically include a robotic armature that transfers fluid from a source to a destination, a controller that controls the robotic armature, a tag detector, a data storage unit that records tag detection, and an assay component such as a microtiter dish comprising a well having a reaction mixture for example brain tissue in aCSF. A number of robotic fluid transfer systems are available, or can easily be made from existing components. For example, a Zymate XP (Zymark Corporation; Hopkinton, Mass.) automated robot using a Microlab 2200 (Hamilton; Reno, Nev.) pipetting station can be used to transfer parallel samples to 96 well microtiter plates to set up several parallel simultaneous assays, for example to assay for the effect of one or more test agents on the secretion of AgRP, NPY and α-MSH. A variety of commercially available peripheral equipment and software is available for digitizing, storing and analyzing a digitized video or digitized optical image, e.g., using PC (Intelx86 or Pentium chip-compatible DOS™, OS2 ™ WINDOWS ™, WINDOWS NT ™ or WINDOWS95™ based computers), MACINTOSH™, or UNIX based (for example, a SUN™, a SGI™, or other work station) computers.
The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the invention to the particular features or embodiments described.
EXAMPLES
Example 1 Materials and Methods
This example describes exemplary procedures and reagents used in the Examples 2-11. Animals: Diet and Experimental Procedures
At six weeks of age, C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME) were fed a regular diet (PURINA® Lab Chow #5001, Ralston Purina Corp, St. Louis, MO) or a high fat diet (HFD) (Rodent Chow #D 12451, RESEARCH DIETS™ INC., New Brunswick, NJ) for 20 or 37 weeks. Regular diet provided 3.3 kcal/grams of energy (59.8% carbohydrate, 28.0% protein, and 12.1% fat). The HFD provided 4.75 kcal/grams of energy (35.0% carbohydrate, 20.0% protein, and 45.0% fat). In seven independent experiments, mice were housed (5/cage) in a controlled environment. Food and water were available ad libitum unless otherwise indicated. Body weights were measured weekly. To measure food intake, some mice were individually housed. In two experiments, groups of diet induced obese (DIO) mice that had been on HFD for 20 weeks were then switched to regular diet for 17 weeks (until week 37). Mouse procedures were performed in accordance with the guidelines and approval of the Oregon National Primate Research Center's Institutional Animal Care and Use Committee. Body composition was measured using dual X-ray absortiometry (DXA) (Lunar Piximus, GE Medical Systems, Madison, WI).
RIAs of samples from secretion experiment α-MSH immunoreactivity was measured with a rabbit anti-α-MSH (Phoenix Pharmaceuticals, Inc., Belmont, CA). The antibody cross-reacts fully with the acetylated α-MSH and partially (46%) with desacetylated α-MSH, but not with NPY nor AgRP. NPY immunoreactivity was measured with a rabbit anti-NP Y. The antibody does not cross-react with AgRP or α-MSH. AgRP immunoreactivity was measured with a rabbit anti-AgRP (82-131)-NH2 (Phoenix Pharmaceuticals, Inc., Belmont, CA). The antibody does not cross-react with α-MSH or NPY. 125I-labeled α-MSH, AgRP, and NPY were prepared by the iodogen method and purified by high-pressure liquid chromatography (α-MSH and NPY) or by AG 1X8 ion exchange resin (AgRP). For the α-MSH RIA, the lowest detectable level (LDL) that could be distinguished from the zero standard was 0.30 fmol/tube. The intra-assay variation (CV%) was determined by replicate analysis (n=10) of two samples at α- MSH concentrations of 2 and 10 fmol/tube, and the results were 7.8% and 7.5%, respectively. The inter assay-variation (CV%) was 10.7% and 12.1% for the range of value measured. For NPY, the LDL was 0.32 fmol/tube. The CV% was determined by replicate analysis (n=10) of one sample at a NPY concentration of 5 fmol/tube and the result was 9.1 %. The CV% was 11.1%. For AgRP, the LDL was 0.70 fmol/tube. The CV% was determined by replicate analysis (n=10) of two samples at AgRP concentrations of 2 and 10 fmol/tube, and the results were 9.1% and 9.8%, respectively. The CV% was 13.4% and 13.5% for the range of values measured.
Intraperitoneal (i.p.) leptin in mice before and after 20 or 37 weeks on HFD
Mice (28 Control, 10 DIO-R, and 10 DIO) were individually housed and sham injected for five days prior to drug treatment. Mice were divided into two groups which received i.p. injections of recombinant murine leptin (PEPROTECH®, 2.0 μg/g body weight) or saline for 2 days. Body weights and calorie intake were measured daily.
Intraperitoneal Melanotan-II (MTII) in mice after 20 or 37 wk on HFD
Mice (8 Control, 8 DIO-R and 8 DIO) were individually housed and fasted overnight. Following five days of daily sham injection, half of the mice received i.p.
MTII (NeoMPS Inc., San Diego, CA, 1.0 μg/g) and the other half received i.p. saline. Fifteen minutes after the injection, food was reoffered. Food intake was measured during the 24 hours after the injection.
Intracerebroventricular (i.c.v.) leptin/MTII in mice after 20 wk on HFD
Under isoflurane anesthesia, a sterile guide cannula (15 millimeters long, 22 gauge, PLASTICS ONE®, Inc. Roanoke, VA) was stereotaxically implanted into the lateral ventricle (-0.5 millimeters posterior, 1 millimeters lateral to bregma, and 1.5 millimeters below the surface of the skull, in accordance with Franklin and Paxinos (Paxinos and Franklin, The Mouse Brain in Stereotaxic Coordinates, San Diego: Academic Press, 2001) of Control, DIO-R, and DIO mice. A 28-gauge obturator was inserted into each cannula. Cannula position was verified at the end of the experiment by dye administration and histological analysis. After about 2 weeks, mice received i.c.v. leptin (0.1 μg) or aCSF in 1 μl. In one trial, food intake and body weight were measured after 24 hours. Two days later, treatments were crossed over in all mice. In another trial, mice were perfused 45 minutes after i.c.v. leptin administration for IHC.
Eight naϊve mice were given increasing doses of MTII i.c.v. (0.01-500 ng). The minimum dose that produced significant modification of food intake in Control mice (0.1 ng) was selected for the follow-up analysis. Mice (Control, DIO-R and DIO) were divided into two groups, each receiving i.c.v. MTII or aCSF after an overnight fast. Food intake was recorded during 24 hours after the injection.
Static incubation of hypothalamic explants after 20 or 37 weeks on HFD After 20 weeks on regular or HFD diet, in five duplicate trials, 620 mice were sacrificed by decapitation and whole brains immediately removed. The hypothalamus was cut from the removed brains. Care was taken to ensure that there was no contamination of the hypothalamic portion with residual pituitary. A 2 millimeter thick slice of mediobasal forebrain was prepared using a vibrating microtome (Leica VS 1000, Leica Microsystems, Inc., Bannockburn, IL). Each slice was taken from the base of the brain to include the PVH and ARH. Hypothalami were treated separately with aCSF (w/ 0.6 TIU aprotinin/ml), equilibrated with 95% O2 and 5% CO2 and incubated at 37°C. After a 1 hour equilibration period, hypothalami were incubated for 45 minutes in aCSF (basal period) before being challenged with a single concentration of leptin (0.1 to 100 nM) for 45 minutes. Tissue viability was verified by exposure to 56 mM KCl for 45 minutes. Each experiment was repeated four times. Treatments were performed in quadruplicate. At the end of each period, supernatants were removed and frozen until assayed. Hypothalamic explants that failed to show peptide release 3X above that of basal in response to KCl were excluded from analysis.
Immunohistochemical (IHC) studies after 20 on HFD
Mice (8 Control, 8 DIO-R, and 9 DIO) were injected i.p. with 2 μg/g leptin or saline. After 30 min, mice were killed under pentobarbital anesthesia by cardiac perfusion with PBS then paraformaldehyde in NaPO4 buffer, pH 7.4. Brains were removed, dehydrated in 25% sucrose, frozen and stored at -800C until sectioned coronally on a microtome (30 μm). For pStat3 and pMAPK IHC, free-floating tissue was pre-treated in 1% NaOH+1% H2O2 in H2O for 10 min, 0.3% glycine for 10 min, 0.03% SDS for 10 min, and blocked in 3% NDS in PBS/0.25% Triton X- 100/0.02% NaN3. For c-Fos IHC, tissue was incubated in blocking buffer for 20 minutes (KPBS+0.4% Triton X-100 + 2% donkey serum) to reduce background. Antibodies (Phospho-Stat3 rabbit #9135, 1 : 1000, Phospho-p44/42 Map Kinase rabbit #9101,1 :1000, CELL SIGNALING TECHNOLOGY®, Beverly, MA; c-Fos rabbit #SC-52, 1 :10000, SANTA CRUZ BIOTECHNOLOGY®, Inc., Santa Cruz, CA) were added in blocking solution and incubated overnight at room temp. Sections were washed, incubated with Biotin-SP-conjugated AffϊniPure F(ab!)2 Fragment Donkey Anti-Rabbit IgG secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) 1 : 1000 in blocking solution, without NaN3, for 1 hour. Tissue was treated with VECTASTAIN® Elite ABC Kit (Vector Labs, Burlingame, CA) for 1 hour, and the signal developed by MSO4/DAB solution. Tissue was mounted on subbed slides, cover slipped, and photographed.
In Situ Hybridization (ISH) after 20 days on HFD
Mice (23 Control, 21 DIO-R, and 22 DIO) were injected i.p. with 2 μg/g leptin or saline. After 2 hours, mice were killed and brains were frozen on dry ice. Immunohistochemistry (ISH) was performed as described in Grove, et al. {Endocrinology 142:4771-4776, 2001). Six riboprobes were transcribed from cDNA as follows: rat NPY cRNA (400 bp) and rat POMC cRNA (925 bp), in which 25% of the UTP was 35S-labeled, and mouse AgRP cRNA (807 bp), rat leptin receptor (ObRb) cRNA (-400 bp), mouse MC4R cRNA (-520 bp) and SOCS-3 cRNA (450 bp) in which 100% of the UTP was 33P-labeled. Tissue was incubated in anti-sense probe (20 million counts per ml labeled probe) overnight at 550C. Tissue was then washed in SSC, RNAse A at 370C, then O.lx SSC at 55-6O0C. Tissue was dehydrated and exposed to autoradiographic film for 1-8 days then dipped in Kodak NBT2 emulsion and stored in light-tight boxes for 7-60 days. Slides were developed and counter-stained with cresyl violet. A COOLSNAP™ HQ camera (PHOTOMETRICS®, Westchester, PA) coupled with Metamorph Software (Universal Imaging Corp) was used to quantify autoradiograms. The area of exposed film and the average gray level density of labeling (integrated optical density (OD)) in each region studied were measured using a constant sampling box encompassing the entire labeled area. Background labeling was subtracted from OD values. For the PVH and ARH measurements, the sampling box encompassed the entire nucleus, except for the SOCS-3 sampling box which contained one hemisphere of the ARH. Mean integrated OD for each brain region was obtained from sections that were anatomically matched between mice.
Micro-dissection and Real-Time PCR after 20 wk on HFD
Mice (14 Control and 14 DIO) were injected i.p. with 2 μg/g leptin or saline. After 45 minutes, mice were anesthetized and brains were rapidly removed. The hypothalamus was cut from the rest of the brain. 0.4 millimeter slices were prepared in aCSF using a vibrating microtome and then placed in RNALATER® (AMBION®, Austin, TX). Using a dissecting microscope, the ARH and PVH were cut from each slice using the fornix, optic tracts, and third ventricle as landmarks. ARH and PVH pieces were then put into separate microcentrifuge tubes and stored at -800C. Total RNA was isolated from each sample using an RNEAS Y® Mini Kit (QIAGEN® Valencia, CA). Standard RT-PCR was performed using 0.1 μg of total RNA. Samples were bioanalyzed on a RNA 6000 Nano LABCHIP® kit (Agilent 2100 Bioanalyzer, Agilent Technologies, Inc., Palo Alto, CA) to check for integrity and concentration. Real-time PCR was performed on the ABI 7900HT (Applied Biosystems Foster City, CA) using the SOCS-3 and MC4R primer/probe set (cat. #mm00545913 si, #mm 00457483 si, ABI) for 45 cycles and using 18S as an internal control.
GlucoseTolerance Test (GTT) and Insulin Tolerance Test (ITT)
After a 14 hour fast, samples were obtained in the morning from saphenous vein bleeds. Blood glucose was measured using a glucometer (ACCU-CHEK®, Roche Diagnostic Corporation, Indianapolis, IN) at 0, 15, 30, 60 and 120 minutes after an i.p. injection of glucose (1 mg/g). Samples (30 μl of blood) were obtained at each timepoint to check for insulin levels by RIA (Linco Research Inc). For the ITT, blood glucose measurements were taken at 0, 15 and 30 minutes after injection of human insulin (1.0 U/kg; Lilly, Indianapolis, IN). HPLC fractionation and RIA
The hypothalami, in acetic acid supplemented with a protease inhibitor, were heated at 95°C for 15 minutes and sonicated. Cell disruption was performed using a Dounce homogenizer. Samples were then centrifuged at 15000rpm at 4°C for 30 minutes. Supernatants were collected and protein concentrations were determined by Bradford assay (Coomassie Protein Assay Reagent, Pierce, Rockford, IL, USA). Supernatants were then evaporated using a speed vacuum and reconstituted in 1 milliliter of 0.1% trifluoracetic acid (TFA) solution. 200 μg of total protein was injected into a Varian ProStar Gradient HPLC System equipped with a C 18 reverse phase column (MICROSORB® MV 300-5; Varian Inc. Palo Alto, CA, USA) used to fractionate the tissue samples. For POMC-derivate peptide elution, a linear gradient was used from 20-40% B in 20 minute using the following mobile phases: (A) 0% acetonitrile/0.1% TFA and (B) 100% acetonitrile/0.1% TFA. The flow rate was 1.5 milliliters/minute and there were equilibration times used on either side of the gradient. Fractions (0.75 milliliters) were collected over the entire 20 minute gradient. These fractions were then evaporated using a speed vacuum and reconstituted in buffer used for RIA. Synthetic peptides were injected on the HPLC to determine retention times. Predicted retention times allowed for analysis of specific regions along the gradient for RIA analysis.
The assays used for α-MSH and ACTH-derived peptides were developed using peptides and primary antibodies. Each purified peptide was iodinated with 125I using the Chloramine T oxidation-reduction method, purified by HPLC, and used as tracer. The α-MSH RIA was performed in 0.5 milliliters of phosphate buffer (pH 7.4) at 500 mg/L sodium azide at 2.5 g/L bovine serum albumin (BSA), with primary anti-α-MSH antiserum (1 :20,000), and 5000 counts per minute (cpm) of 125I des-αMSH tracer. The sensitivity of the assays was approximately 11.5 pg/tube, and the intra- and inter-assay variability were approximately 5-7% and 10-11%, respectively. The α-MSH assay used in this condition can detect both acetyl- and des-α-MSH forms. The ACTH RIA was also performed in 0.5 milliliters of the same RIA buffer by using the anti-ACTH antiserum (1 :30,000) and 5000 cpm of 125I-ACTH tracer. The sensitivity of the assays was approximately 10.0 pg/tube, and the intra- and inter-assay variability were approximately 5-7% and 10-11%, respectively.
Statistical analysis
All values are expressed as mean ± SEM. Data were analyzed by two-way analysis of variance (ANOVA) for body weight change over time, calorie intake over time, i.p./i.c.v leptin effect on food intake, body weight, mRNA expression, IHC cell counts, and i.p./i.c.v. MTII effect on food intake. One-way ANOVA was used for final body weights, total calorie intake, feeding efficiency, blood leptin levels, and leptin sensitivity, followed by Bonferroni's Multiple Comparison Test. Body weight change correlation was analyzed using linear regression test. Secretion experiments were analyzed using Nonlinear Regression (sigmoidal dose-response best-fit curve). Data for the bimodal distribution was assessed by the DAgostino & Pearson omnibus normality test. For glucose and insulin tolerance tests, areas under the curve (AUC) were calculated by trapezoid analysis and were compared by oneway ANOVA. Probability values <0.05 were considered statistically significant. Analyses were performed with statistical software (GraphPad Prism 4.0, GraphPad Software, Inc., San Diego, CA).
Example 2
Effects of HFD on body composition
This example describes the effect of a high fat diet on body weight, fat tissue and leptin levels. At 20 weeks, mice fed HFD had a wide distribution in body weight gain showing bimodal characteristics. The heavier group on the HFD was named "diet- induced obese" (DIO) mice. Others on HFD remained almost as lean as Controls, those fed regular chow (Figure IA). These mice were defined these as "diet-induced obese resistant" (DIO-R) mice, those with body weights ranging between the average ±3 standard deviations of the Control group. The small number of mice (~5%) that overlapped in the bimodal distribution of body weight was excluded from experiments. The frequency of DIO-R and DIO per cage was normally distributed, and the same ratio occurred when the mice were individually housed. Interestingly, initial body weight of identically aged mice significantly correlated to final weight and was the only factor that predicted DIO-R vs. DIO (Figure 8). No pre-existing differences to leptin sensitivity were seen between highest and lowest weight mice preceding HFD exposure, weight loss and decrease in food intake was similar with i.p. leptin.
Over a period of 20 weeks, the DIO-R group became only 6.7% heavier than the Control group, while the DIO group became 32.9% heavier (Figure IB). The number of mice that became obese on the HFD steadily increased over time. By 13- 15 weeks on HFD, -65% of mice had become obese and this percentage remained stable past 20 wk. At this point, the lean mass gain was similar for every group (Control 6.7 ± 2.1 g, DIO-R 6.1 ± 1.1 g, DIO 8.0 ± 1.5 gs). In contrast, fat gain was significantly different among the three groups. Control mice gained 1.7 g of fat and DIO-R mice gained 4.5 g of fat, (p < 0.001). The largest fat gain was obtained by the DIO group (10.5 g), around time times that of the Control fat gain (p < 0.001). Thus, the extra weight gain in mice fed HFD was mostly due to accumulation of body fat. In parallel, leptin levels were elevated by 20 weeks on HFD and were significantly different between every group (Control 3.7+ 2.0 ng/ml, n=64; DIO-R 14.3 ± 9.1 ng/ml, n=56 and DIO 36.7 ± 10.0 ng/ml, n= 58, p<0.001).
Example 3
Effects of HFD on food intake
This example describes the effect a high fat diet has on the food intake of mice. In DIO-R mice, calorie intake increased and paralleled that of Control for the first four weeks; then, calorie intake decreased significantly from the fifth weeks until the end of the experiment. Although calorie intake in DIO mice was no different from Control, because of the different macronutrient composition of the diets, they were consuming significantly more fat (Figure 1C). Thus, obesity in C57BL/6J mice fed a HFD is not a result of hyperphagia but increased feeding efficiency (weight gained/kcal consumed) (Figure ID). Example 4 Leptin effect
This example describes the effect of i.p. and i.c.v.leptin on food intake and body weight in mice after 20 weeks on high fat diet. The minimum dose that could modify body weight or food intake in control mice (2 μg/g body weight for 2 days) was determined. After the second day of leptin injection, the Control and DIO-R mice significantly lost weight. In contrast, the DIO group did not show any change in body weight (Figure 2A). Calorie intake after leptin treatment was reduced significantly in the Control and DIO-R mice. However, no significant difference was found in the DIO group (Figure 2B). Two hours after i.p. leptin, leptin plasma concentrations in all groups were between 160- 190 ng/mg. This represented a 40-fold increase from endogenous levels in Control mice, a 12-fold increase in DIO-R mice, and a 5 -fold increase in DIO mice (p< 0.001). After i.c.v. leptin, Control and DIO-R mice had a significant reduction of body weight at 24 hours. However, the DIO group showed no change (Figure 3C). Calorie intake after 24 hours was reduced by -20% in Control and DIO-R mice. No significant difference was found in the DIO group (Figure 2D). To confirm this lack of i.c.v. leptin responsiveness, the immediate-early gene c-Fos (a marker of neuronal activation) in the ARH was measured. After i.c.v. leptin, c-Fos expression significantly increased in Control mice, but remained unchanged in DIO mice.
Example 5 Hypothalamic secretion experiments in mice after 20 or 37 weeks on HFD This example describes the detection of neuropeptide secretion using the disclosed methods.
The baseline secretion of AgRP, NPY and α-MSH from hypothalamic blocks was also determined. There was no difference in the baseline, non-stimulated secretion of NPY, AgRP, or α-MSH in DIO-R mice or DIO mice when compared with Controls (Figure 9). Incubation with leptin caused a dose-dependent inhibition of AgRP and NPY secretion in the Control group (EC50 = 23 nM, EC50=17 nM, respectively). The DIO-R group showed similar curves and similar EC50s (24 nM, 20 nM respectively). On the contrary, AgRP and NPY secretion in the DIO group did not change with an increase in leptin concentration (Figure 3A, 3B). The sensitivity of POMC neurons to leptin was studied by measuring α-MSH secretion. Incubation with leptin caused a dose-dependent increase of α-MSH levels in the Control and DIO-R groups (EC50=46 nM, EC50= 37 nM, respectively). The DIO group, however, was again resistant to the effects of leptin (Figure 3C). The α- MSH/ AgRP secretion ratio as an index of activation of the MC4R was defined. DIO-R mice had a 3-fold higher ratio of α-MSH to AgRP secretion than DIO mice (Figure 3D). Figure 14A-14C shows average baseline secretion of ArRP, NPY, and α-MSH expressed as fmoles per gram of tissue.
Example 6 Leptin effect (i.p.) on ARH niRNA expression This example describes the effect of i.p. leptin on ARH mRNA expression in mice after 20 wk on a high fat diet.
Since AgRP mRNA expression increases from rostral to caudal within the ARH, neuropeptide expression was divided into caudal and rostral regions for analysis. After i.p. leptin, caudal levels of AgRP expression were decreased by -30% in both Control and DIO-R mice. In contrast, DIO mice showed no expression changes (Figure 4A). Rostral areas of the ARH showed no differences in AgRP levels after leptin. Baseline levels of NPY expression were not significantly different between Control and DIO groups. After leptin injection, NPY mRNA expression decreased 50% in Control. There was no change in NPY mRNA expression in the DIO group (Figure 4B).
Neither baseline nor leptin-induced POMC mRNA expression was significantly different among groups when the entire extent of the ARH or just rostral sections (Figure 4C) was considered. ObRb expression was not significantly different among groups in baseline levels (Figure 4D) nor after leptin injection. Example 7 Synthesis of POMC-derived peptides in ARH
In order to test if the obesity-resistant phenotype (DIO-R) is due to a sustained level of anorexigenic POMC-derived peptides, ACTH, CLIP, acetyl and desacetyl-α-MSH. Desacetyl-α-MSH levels in DIO and DIO-R mice were significantly lower than Control mice (Figure 4E) were measured. The same decrease of ACTH, CLIP, and acetyl α-MSH were also seen in DIO-R and DIO mice (Figure 9).
Example 8
Leptin effect (i.p.) on ARH signal transduction
This example describes the effect of i.p. leptin on ARH signal transduction in mice after 20 weeks on a high fat diet.
In order to assess leptin-induced activation of ARH cells, c-Fos, phosphorylation of Stat3 (a well-known mediator of leptin activation) and pMAPK (a measure of the MAP Kinase pathway activation) in leptin-treated mice was measured. Baseline conditions showed no significant differences among groups in the number of cells expressing c-Fos, pStat3, or pMAPK. After i.p. leptin, c-Fos expression in Control and DIO-R groups increased significantly whereas there was no change in the DIO group (Figure 5A). pStat3 expression was dramatically increased in Control and moderately elevated in the DIO-R leptin-treated group. No change was observed in the DIO group (Figure 5B). The Control group showed a 2- fold increase in the positive pMAPK cells after leptin, whereas the DIO-R and DIO groups showed no difference (Figure 5C). SOCS-3 levels were determined by ISH and Real-Time PCR in the ARH.
Although the ISH data were inconclusive, higher levels of SOCS-3 mRNA in baseline conditions of DIO mice using Real-Time PCR were found. After i.p. leptin, Control mice had elevated SOCS-3 levels while levels remained unchanged in DIO mice. The expression of pStat3 (signal transducer and activator of transcription) in arcuate neurons was selectively reduced in leptin-treated diet-induced obese (DIO) mice, but not in neurons within the VMH or DMH, suggesting that the ARH is a major site of leptin resistance. The heterozygous SOCS-3 deficient mouse was more sensitive to the weight-reducing effects of leptin and was resistant to the development of DIO. Moreover, using a SOCS-3 deficient (within the brain) mouse, Mori et al. showed that the level of POMC induced by leptin was greater in SOCS-3 deficient mice than in wild-type mice. These data indicate that SOCS-3 is a negative regulator of leptin-induced pStat3 signaling in the hypothalamus and that excessive activity of SOCS-3 may be a potential mechanism for leptin resistance.
Example 9 Integrity of the melanocortin pathway
This example describes the determination of diet induced obesity on the melanocortin pathway in mice unresponsive to leptin.
The unresponsiveness to leptin in DIO mice may be due to a primary insensitivity or a lack of secondary neuronal targets. The melanocortin pathway is a well-established pathway that mediates leptin' s actions. Using a very low dose of MTII, a potent melanocortin agonist, it was determined if the melanocortin system downstream of POMC neurons is intact. One hour after i.p. MTII, calorie intake was reduced 45% in Control, -65% in DIO-R, and 90% in DIO. The orexigenic effect of MTII was maintained for four hours in Control and DIO-R groups, and continued over eight hours in the DIO group (Figure 6A).
When mice were given central MTII, they responded similarly to the peripheral injection: DIO mice showed a greater and prolonged response to MTII than Control or DIO-R mice. To determine if this exaggerated response in DIO mice was due to an up-regulation of MC4R mRNA expression, the expression in the PVH by ISH was measured. Levels of MC4R mRNA were very low in Control and DIO-R mice, but were strikingly high in DIO mice (Figure 6B). An additional analysis of this gene expression was performing using Real-Time PCR on microdissected PVH samples. The MC4R expression was significantly higher in DIO mice than in Control mice (p<0.05). Example 10 Restoration of leptin sensitivity
This example describes the restoration of leptin sensitivity in response to a dietary change from a high fat diet to a regular diet. To determine whether the effects of a HFD on energy balance were reversible, after 20 weeks on HFD one cohort of mice was continued on HFD (DIO) and the other cohort was returned to regular chow diet (Restored). Restored mice continuously lost weight and after -seven weeks showed similar body weights to Control (those maintained on regular chow during the whole experiment) (Figure 7A). Although total calorie intake of Restored mice (weeks 20 to 37) was similar to that of DIO mice, total fat calorie intake was five times lower in the Restored group (p<0.001).
After 20 weeks on HFD, DIO mice were intolerant to glucose and insulin resistant. This intolerance and resistance worsened by week 37, causing more than 70% of the mice to be diabetic. Mice were classified as diabetic when glucose AUC was 3 SD higher than the mean of Control mice. At the end of 37 weeks, the Restored group responded normally to glucose and insulin challenges (Figure 7B). Thus, the diabetes/obesity syndrome appears reversible at this stage.
Leptin sensitivity of the Restored group was determined by injecting mice with leptin. After the second day of i.p. leptin, body weight of the Restored group was significantly reduced. This was identical to the sensitivity of the Control group (Figure 7C). In addition, 100 nM leptin inhibited secretion of AgRP and NPY and stimulated secretion of α-MSH from hypothalamic explants in Restored mice. In contrast, leptin did not modulate the secretion of AgRP, NPY, nor α-MSH from hypothalami of DIO mice (at 37 weeks) (Figure 7D, E, F). These results show that sensitivity to leptin in the ARH was re-established in the Restored group.
Mouse models of dietary obesity provide an excellent system to identify how leptin signaling becomes compromised when leptin resistance occurs. C57B1/6J mice fed a high fat diet (HFD) exhibit increased body adiposity along with other characteristics of human obesity, such as diabetes mellitus (>70% of mice on HFD). Notably, mice on HFD lose weight when the fat content of the diet decreases, independent of total calorie intake. The development of obesity and leptin resistance in C57B1/6J mice on HFD can be divided into three stages. In the early stage, mice gain weight (adiposity), but maintain an adequate response to anorectic effects of peripheral leptin injection. In the middle stage, mice lose peripheral leptin sensitivity while retaining the capacity to respond to central leptin. Finally, in the late stage, mice demonstrate central leptin resistance.
Example 11
Leptin modulates NPY/ AgRP and α-MSH secretion from the ARH in mice fed regular chow (Control) and mice that remain lean when fed HFD (DIO-R). Leptin stimulated α-MSH secretion and inhibited NP Y/ AgRP secretion in a dose- dependent manner. Leptin failed to modulate the secretion of melanocortin peptides in obese mice (DIO). Secretion can occur from ARH neurons though it is recognize that NPY secretion might be due to other neuron populations within the hypothalamus. NPY/AgRP and α-MSH secretion in DIO mice was not modulated by leptin (in all doses studied).
DIO mice have increased adiposity and high plasma leptin concentrations, but the endogenous hyperleptinemia fails to curtail the progression of obesity. Moreover, DIO mice are unresponsive to peripheral or central leptin injection since they exhibit neither a decrease in food intake nor a decrease in body weight. While c-Fos expression is induced in arcuate neurons of Control and DIO-R mice after leptin treatment (presumably due to activation of POMC neurons), leptin treatment does not alter c-Fos expression in arcuate neurons of DIO mice. The best-defined signal transduction pathway for leptin is the Janus kinase-STAT3 pathway. It is believed to be essential for mediating leptin's effects on homeostatic energy regulation. It is demonstrated herein, that once DIO is established, the ability of leptin to activate hypothalamic pStat3 signaling is also diminished. Notably, the dose of leptin used to produce a response caused supraphysio logical serum leptin levels (around 40-fold above endogenous Control levels and 5 -fold above DIO levels), indicating that endogenous leptin signaling in the ARH of DIO mice must be severely impaired since they are unresponsive to such high leptin levels.
Hyperleptinemia produces leptin resistance by a down-regulation of ObRb, because ObRb mRNA expression was not different between Control and DIO mice. The magnitude of decreased ObRb expression, -30% when it has been shown, is likely not enough to explain the leptin resistance phenotype. It was determined if leptin resistance might be due to a disruption downstream of leptin receptor binding. In normal rats, an induction of SOCS-3 (a negative regulator of leptin signal transduction) occurs following i.p. leptin in areas involved in body weight regulation. Higher baseline SOCS-3 mRNA levels were found in the ARH of DIO mice. After i.p. leptin, only the Control mice showed an increase of SOCS-3. Interestingly, deletion of SOCS-3 in POMC neurons caused a reduction in the rate of weight gain in mice fed HFD, and the mutation had beneficial effects on glucose homeostasis. The complete lack of leptin responsiveness of the ARH in DIO mice seems to possibly be due to increased SOCS-3 expression, yet cannot be proven by our current data.
To examine changes in neuropeptide expression with leptin treatment, it was determined if major ARH neurons were involved in energy homeostasis. In Control and DIO mice, NPY, AgRP and POMC mRNA expression were not different in baseline conditions. After leptin, NPY and AgRP mRNA expression decreased in Control mice, but had no effect on DIO mice. This result is in agreement with the secretion studies, which demonstrated decreased release only in the Control mice.
The melanocortin pathway mediates some of leptin' s actions. Since α-MSH is a MC4R agonist and AgRP is a MC4R inverse agonist, the secretion ratio α- MSH/ AgRP is an index of MC4R activation. At most leptin concentrations, DIO mice have 3 times less activation of this receptor compared with Control or DIO-R mice. The DIO 's leptin resistance prevents a leptin-induced increase of α-MSH secretion while preventing a decrease in AgRP secretion. This leads to weakened central melanocortin activation. It was also confirmed that the distal components of the melanocortin system were intact. This increased responsiveness is likely due to the observed over-expression of MC4R mRNA seen in the PVH of DIO mice. Leptin resistance leads to some type of hypersensitivity in the melanocortin pathway in response to pharmacological stimulation of the MC4R, at least partially caused by an over-expression of MC4R. This series of studies strongly suggested a primary failure of leptin to modulate the melanocortin system in DIO mice, and further suggests that the distal melanocortin system remains intact and capable of regulating energy balance.
Despite genetically identical backgrounds, some C57BL/6J mice on HFD are able to maintain a regular body weight - only 7% heavier than Control mice. DIO-R mice also retain the same "normal" response to exogenous leptin and glycemic control as Control mice. The ability of DIO-R mice to maintain their regular body weight is not due to over-expression of melanocortin peptides, because there was a decrease in the biosynthesis of prohormones (acetyl- and desacetyl-α-MSH, ACTH and CLIP). DIO mice become leptin resistant and most of them become diabetic. But, they can lose weight by decreasing the fat content of their diet. The Restored group lost weight by eating the same amount of calories as the DIO group, but less fat. This indicates that it is macronutrient composition that causes weight gain in DIO mice and not the total calories consumed. After losing weight, the response to a glucose challenge was normalized, as well as leptin sensitivity, shown by a decrease in body weight after i.p. leptin. Restored mice showed a re-established response to leptin-regulated neuropeptide secretion. This recovery of leptin sensitivity was in parallel with the recovery of NP Y/ AgRP and POMC neurons' response to leptin actions. In summary, leptin sensitivity of POMC and NP Y/ AgRP neurons in the
ARH is drastically decreased in DIO, but not in DIO-R mice. The melanocortin system downstream of the ARH in DIO mice is over-responsive possibly due to up- regulation of MC4R in the PVH. Lastly, the recovery of leptin resistance is due to a recovery of NPY/ AgRP and POMC neuron sensitivity to leptin's actions. These studies indicate a primary failure of leptin to modulate the melanocortin system in DIO mice, and further suggest that the distal melanocortin system remains intact and capable of regulating energy balance. These results demonstrate that the methods disclosed herein are particularly suited for identifying agents that are useful in the treatment of obesity and or diabetes. This disclosure provides methods for identifying agents of use in treating obesity and related disorders, such as diabetes. Example 12 Materials and Methods
This example describes exemplary procedures and reagents used in the Example 13.
Generation of POMC-mut-Kir6.2 mice
For generation of POMC-mut-Kir6.2 mice, the mut-Kir6.2 cassette (Kir[Δ2- 30,Kl 85Q]-GFP) was inserted into a POMC BAC genomic clone so that the ATG codon replaced that of POMC, as described in Balthasar et al. {Neuron 42:983-991 (2004). POMC-mut-Kir6.2 BAC DNA was prepared using a commercially available kit (QIAGEN®) and microinjected into pronuclei of fertilized one-cell- stage embryos of FVB mice (Jackson Laboratories), resulting in the generation of two POMC-mut-Kir6.2 lines that were maintained on an FVB inbred background. POMC-GFP and NPY-GFP mice were generated by insertion of hrGFP into a POMC or NPY BAC, respectively, as described above. To generate POMC-GFP; Ucp2~'~ mice, heterozygous POMC-GFP transgenic mice were crossed with heterozygous Ucp2~'~ mice. For high-fat diet feeding experiments, mice were placed on a high-fat rodent diet (45% kcal from fat; Research Diets Inc; D12451) at 4 weeks of age for a total of 20 weeks (or 8 weeks for electrophysiological studies).
Electrophysiology
Brain slices were prepared from young adult mice (4-7 weeks old or 12 weeks old for high- fat diet studies). Briefly, mice were anesthetized with Isoflurane before decapitation and removal of the entire brain. Brains were immediately submerged in ice-cold, carbogen-saturated (95% O2, 5% CO2) high-sucrose solution (238 mM sucrose, 26 mM NaHCO3, 2.5 mM KCl, 1.0 mM NaH2PO4, 5.0 mM MgCl2, 1.0 mM CaCl2, 11 mM D-glucose). Coronal sections (200 μm thick were cut with a Leica VTlOOOS Vibratome and incubated in oxygenated recording aCSF (126 mM NaCl, 21.4 mM NaHCO3, 2.5 mM KCl, 1.2 mM NaH2PO4, 1.2 mM MgCl2, 2.4 mM CaCl2, 10 mM D-glucose) at room temperature (21 0C) for at least 1 hour before recording. Slices were transferred to the recording chamber and bathed in oxygenated recording aCSF heated to approximately 34 0C, at a flow rate of approximately 2 ml min \ GFP-positive neurons were visualized using epifluorescence imaging and patched under infrared differential interference contrast (IR-DIC) optics. Recordings were made using a MultiClamp 700B amplifier using pClamp 9.0 software (both Axon Instruments). Bath perfusion was used for the addition of glucose, genipin and leptin during recording. Equal molar NaCl was replaced with 56 mM KCl to prepare "high-K+ aCSF". The pipette solution for loose-patch recording contained 150 mM NaCl, 3.5 mM KCl, 10 mM HEPES, 10 mM glucose, 2.5 mM CaCl2, 1.3 mM MgCl2 (pH 7.3). Loose-patch recordings were performed in /= 0 current-clamp mode, which maintains an average 0 pA holding current. Seal resistance was in the range of 8-30 MΩ and was checked during the recordings; cells that showed deviations in seal resistance were not included in the data analysis. Recordings that showed no recovery in spike activity after return to 5 mM glucose were also excluded from data analysis. Firing rate averaged for every 20 seconds was taken as one data point; 9-18 data points taken from the last 3-6 minutes of each experimental condition (5 mM or 3 mM glucose, with or without addition of drugs) were compared using unpaired t-tests, with P < 0.05 considered a statistically significant change.
Static incubation of hypothalamic explants
Mice were killed by decapitation and whole brains removed immediately. A 2-mm slice was prepared using a vibrating microtome (Leica VS 100) taken from the base of the brain to include the PVH and arcuate nucleus. Each hypothalamic slice was treated separately and incubated in artificial cerebrospinal fluid (aCSF: 126 mM NaCl, 0.09 mM Na2HPO, 6 mM KCl, 4 mM CaCl2, 0.09 mM MgSO4, 20 mM NaHCOs, 8 mM glucose, 0.18 mg ml"1 ascorbic acid, 0.6 trypsin inhibitor units (TIU) aprotinin ml"1), pre-equilibrated with 95% O2 and 5% CO2 at 37 0C for 1 hour. Slices were then incubated for 45 minutes in 700 μl aCSF containing 8 mM glucose (baseline) followed by a 45-minute incubation in aCSF containing 0.1, 0.5, 1.0, 5.0 or 10 mM glucose for dose-response experiments, or glucose (3, 8 or
15 mM) or genipin (20 μM) for feeding experiments (high-fat diet versus regular chow). Finally, the viability of the tissue was verified by a 45-minute incubation in aCSF containing 56 rnM KCl. At the end of each incubation period, supernatants were removed and tested for α-MSH release by radioimmunoassay as described in the Examples above. α-MSH secretion from each individual hypothalamus was normalized to the amount of α-MSH secreted during the baseline period (8 mM glucose). Only hypothalami that showed a 300% secretion over baseline in response to 56 mM KCl were used in the analysis. Individual experiments were repeated a total of four times.
Glucose tolerance testing
Mice were fasted overnight (16 hours) and injected intraperitoneally with a 20% (w/v) glucose solution at 1 g kg"1 body weight. Blood glucose levels were measured before and 15, 30, 45, 90, 120 and 180 minutes after glucose injection.
Immunohistochemistry
Coronal mouse brain sections (25 μm) were washed in PBS six times before blocking in 0.25% Triton X-100 in PBS containing 3% (w/v) normal donkey serum (PBT-azide) for 2 hours. Sections were incubated overnight in rabbit anti-β- endorphin IgG (Peninsula Laboratories Inc; 1 :5,000) in PBT-azide containing 3% (w/v) normal donkey serum at 21 0C, followed by a 2 hour incubation in Cy3- conjugated donkey anti-rabbit IgG (ImmunoResearch Laboratories; 1 :500. After three washes in PBS, sections were blocked in PBT-azide containing 3% (w/v) normal donkey serum and incubated overnight in chicken anti-GFP IgG (Upstate Laboratories; 1 :5,000) at 21 0C and Cy2-conjugated streptavidin (Jackson ImmunoResearch Laboratories; 1 :500) for 1 hour. Sections were mounted onto SUPERFROST® slides and visualized on an inverted microscope with a digital camera (Axioscope, Zeiss). A total of four brains for each POMC-mut-Kir6.2 transgenic line were processed and imaged. The total number of both GFP- and β- endorphin-positive neurons was counted for a total of four series per brain. For each of the two transgenic lines that were used for further study, the percentage of β- endorphin-positive cells that co-expressed GFP was >95% and the percentage of GFP -positive cells that were negative for β-endorphin expression was <1%. Ucp2 in situ hybridization
A Ucp2 riboprobe was generated by PCR amplification of a 527-base pair (bp) DNA fragment complementary to exons 3 and 4 of Ucp2 from mouse brain cDNA. This amplicon was subcloned into pcR4-TOPO vector (INVITROGEN®), and a 35S-labelled cRNA probe generated. For β-endorphin immunohistochemistry, sections processed for in situ hybridization were washed twice in PBS and pre- treated with 0.3% (v/v) hydrogen peroxide in PBS for 30 minutes, then incubated in 3% normal donkey serum in PBT-azide for 2 hours. Sections were then incubated overnight with rabbit anti-β -endorphin primary antiserum (Peninsula Laboratories; 1 :4,000) in PBT-azide. Sections were washed in PBS six times before a 2 hours incubation in biotinylated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories; 1 : 1 ,000). Sections were then washed three times in PBS and incubated with avidin-biotin complex (VECTASTAIN® Elite ABC Kit, Vector Laboratories; 1 :500) in PBS for 1 hour. Finally, sections were washed three times in PBS and incubated in 0.04% diaminobenzidine tetrahydrochloride (DAB; Sigma) and 0.01% hydrogen peroxide in PBS. The DAB reaction was quenched by two washes with PBS.
Quantitative real-time PCR
Hypothalami were removed and snap-frozen in liquid nitrogen before isolation of total RNA using RNA STAT-60™ (Tel-Test Inc) according to the manufacturer's instructions. Samples were then treated with DNA-Free (AMBION®) to remove any contaminating genomic DNA, and complementary DNA was synthesized from 1 μg of total RNA using SUPERSCRIPT™ III
FirstStrand cDNA Synthesis Kit (INVITROGEN®). Ucp2 was amplified from 0.5 ng of reverse-transcribed total RNA using TAQMAN™ Universal PCR Mastermix (Applied Biosystems) with Ucp2 sense and antisense primers, dual- labelled probe (5'-FAM, 3'-TAMRA) (Applied Biosystems; assay on demand Mn00495907_gl). Standard curves were constructed by amplifying serial dilutions of cDNA (5 ng to 0.32 pg) and plotting cycle threshold (CT) values as a function of starting reverse-transcribed RNA. mRNA expression of Ucp2 was normalized to levels of the 18S ribosomal RNA housekeeping gene.
Example 13 Glucose induced secretion of α-MSH
This example describes the measurement of α-MSH secretion in brain tissue induced by glucose.
POMC neurons in the arcuate nucleus of the hypothalamus have been shown to be excited by glucose. To test glucose-sensing by POMC neurons in POMC-mut- Kir6.2 mice the release of α-melanocyte stimulating hormone α-MSH) from hypothalamic slices was measured in response to increasing glucose concentrations using the disclosed explant assay. In slices from wild-type mice, glucose stimulated α-MSH secretion in a dose-dependent manner (Figure 1 IA). In contrast, in hypothalamic slices from POMC-mut-Kir6.2 mice, glucose did not result in stimulation of α-MSH release (Figure 1 IA). Together, these findings confirm that glucose sensing in POMC-mut-Kir6.2 neurons is disrupted, and provide direct evidence that ATP-induced closure of KATP channels is required for glucose- excitation of POMC neurons.
The effects of defective glucose sensing in POMC neurons on whole-body glucose homeostasis was also investigated. Of note, body weight is normal in POMC-mut-Kir6.2 mice (wild-type, 22.3 ± 0.3 g; mut-Kir6.2 mice, 22.1 ± 0.3 g; mean ± SEM). Intraperitoneal glucose tolerance tests were performed on male POMC-mut-Kir6.2 mice and their wild-type littermates at eight weeks of age. After an exogenous load of glucose (1 g, glucose per kg body weight), mice lacking glucose sensing in POMC neurons (POMC-mut-Kir6.2 mice) showed impaired glucose tolerance (Figure 1 IB). This phenotype was also observed in a second line of POMC-mut-Kir6.2 mice. These findings demonstrate that glucose sensing in glucose-excited POMC neurons is required for the normal handling of a systemic glucose load. Abnormal glucose homeostasis in the face of normal body weight regulation raises the possibility that POMC neurons are heterogeneous with respect to function. Glucose sensing in pancreatic β-cells is lost during the development of obesity-induced type 2 diabetes. Given this, and given the role of POMC neurons in handing a systemic glucose load, it was tested whether obesity induces a similar impairment in glucose sensing in POMC neurons, wild-type mice were placed on a high-fat diet for 20 weeks and the release of α-MSH in hypothalamic slices in response to elevated glucose was measured. Glucose stimulated release of α-MSH from hypothalamic slices of chow- fed, wild-type mice (Figure 12A). However, glucose failed to stimulate release of α-MSH from wild-type mice fed a high-fat diet (Figure 12A). Electrophysiological techniques were also used to independently assess glucose sensing in POMC neurons of mice on a high- fat diet. In this case, four- week-old mice were fed chow or a high-fat diet for eight weeks. Approximately 46% (5 out of 11) of POMC neurons from chow-fed, control mice significantly increased their firing rate with an increase in glucose concentration from 3 to 5 mM (fold increase, 2.17 ± 0.06), compared to only 10% of POMC neurons from mice on a high- fat diet (1 out of 10) (Figure 12B). Notably, the fold increase in firing rate of the single glucose-responsive POMC neuron from the high-fat diet group was much lower that that of the chow-fed group (1.47-fold increase). These results indicate that agents that can stimulate release of α-MSH are of use in treating both obesity and diabetes.
As shown in Figure 13A and observed previously (Figure 12A), glucose- stimulated release of α-MSH was lost in hypothalamic slices from wild-type mice on a high-fat diet. This defective response was fully restored by the acute addition of an effective amount of genipin (Figure 13 A, right panel). Ucp2~'~ mice were completely protected from diet-induced loss of glucose sensing in POMC neurons (Figure 13B, right panel). These findings, which are analogous to previous observations in pancreatic β-cells, indicate that increased UCP2 activity is causally linked to loss of glucose sensing in POMC neurons induced by a high- fat diet.
We have shown that glucose sensing in POMC neurons has an important role in controlling systemic glucose homeostasis. Second, glucose sensing in these neurons is lost with obesity linked to a high- fat diet. Finally, UCP2 is involved in this loss of glucose sensing, perhaps by decreasing ATP production in POMC neurons. As POMC neurons represent only a fraction of all glucose-excited neurons in the brain (which include melanin-concentrating hormone (MCH) neurons in the lateral hypothalamus, neurons in the ventromedial hypothalamus, and neurons in the hindbrain), the UCP2-mediated loss of glucose sensing in glucose-excited neurons is believed to be an important pathogenic component of type 2 diabetes.
Example 14 Identification of agents that affect AgRP, NPY and α-MSH secretion
Brian tissue is obtained from mice. Two-mm slices are prepared using a vibrating microtome taken from the base of the brain to include the PVH and arcuate nucleus. Each hypothalamic slice is treated separately with a test agent and incubated in artificial cerebrospinal fluid (aCSF: 126 mM NaCl, 0.09 mM Na2HPO, 6 mM KCl, 4 mM CaCl2, 0.09 mM MgSO4, 20 mM NaHCO3, 8 mM glucose, 0.18 mg ml"1 ascorbic acid, 0.6 trypsin inhibitor units (TIU) aprotinin ml"1), pre- equilibrated with 95% O2 and 5% CO2 at 37 0C for 1 hour. Slices are then incubated for 45 minutes in 700 μl aCSF containing 1 pM to 1 mM test agent. Optionally, the viability of the tissue is verified by a 45 -minute incubation in aCSF containing 56 mM KCl. At the end of each incubation period, supernatants are removed and tested for AgRP, NPY and α-MSH secretion by radioimmunoassay as described in the Examples above.
Potential therapeutic agents identified with these or other approaches, including the specific assays and screening systems described herein, are used as lead compounds to identify other agents having even greater effects on AgRP, NPY and α-MSH. For example, chemical analogs of identified chemical entities, or variant, fragments of fusions of peptide agents, are tested for their activity in the assays described herein. Candidate agents also can be tested in the animal models of obesity and diabetes described herein.
In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

We claim:
1. A method for identifying an agent for the treatment of diabetes, obesity, or a combination thereof, comprising: contacting a test agent to brain tissue in vitro in artificial cerebrospinal fluid
(aCSF), wherein the brain tissue comprises proopiomelanocortin (POMC) neurons and neuropeptide peptide Y/ Agouti-related peptide (NPY/ AgRP) neurons; measuring the amount of alpha-melanotropin (α-MSH) secreted from the brain tissue using an immunoassay specific for α-MSH; measuring the amount of AgRP secreted from the brain tissue using an immunoassay specific for AgRP; determining the ratio of the amount of α-MSH to the amount of AgRP; comparing the ratio of the amount of α-MSH secreted to the amount of AgRP with a first control, measuring the amount of NPY secreted from the brain tissue using an immunoassay specific for NPY; comparing the amount of NPY with a second control; and wherein an increase in the ratio of the amount of α-MSH to the amount of AgRP relative to the first control and a decrease in the relative amount NPY relative to the second control identifies the test agent as being of use for the treatment of diabetes, obesity, or a combination thereof.
2. The method of claim 1, further comprising isolating α-MSH, AgRP and NPY from the brain tissue prior to measuring the amount of α-MSH, AgRP and NPY secreted from the brain tissue.
3. The method of claim 2, wherein isolating α-MSH, AgRP and NPY from the brain tissue comprises high performance liquid chromatography (HPLC).
4. The method of claim 1 , wherein the immunoassay for α-MSH is a competitive radioimmunoassay (RIA).
5. The method of claim 4, wherein the competitive RIA for α-MSH comprises the use of a radiolabeled α-MSH peptide exogenous to the brain tissue that is labeled with two or more 125I atoms.
6. The method of claim 1 , wherein the immunoassay for AgRP is a competitive RIA.
7. The method of claim 6, wherein the competitive RIA for AgRP comprises the use of a radiolabeled AgRP peptide exogenous to the brain tissue that is labeled with two or more I atoms.
8. The method of claim 1 , wherein the immunoassay for NPY comprises a competitive RIA.
9. The method of claim 8, wherein the competitive RIA for NPY comprises the use of a radiolabeled NPY peptide exogenous to the brain tissue that is labeled with two or more 125I atoms.
10. The method of claim 1 , wherein the first control is the ratio of α- MSH to AgRP secreted by brain tissue in vitro not contacted with the test agent, and the second control is the amount of NPY secreted by brain tissue in vitro not contacted with the test agent.
11. The method of claim 1 , wherein method identifies an agent of use for treating diabetes.
12. The method of claim 1, wherein method identifies an agent of use for treating obesity.
13. The method of claim 1 , wherein the brain tissue comprises the paraventricular nucleus of the hypothalamus (PVH) and the hypothalamic arcuate nucleus (ARH).
14. The method of claim 1 , wherein the brain tissue is mouse brain tissue.
15. The method of claim 1 , wherein the brain tissue is human brain tissue.
16. The method of claim 1 , wherein the brain tissue is leptin resistant.
17. The method of claim 1 , wherein the brain tissue is obtained from an obese subject.
18. The method of claim 17, wherein the obese subject is an obese mouse.
19. The method of claim 1 , wherein the brain tissue is glucose resistant.
20. The method of claim 1 , wherein the brain tissue is obtained from a diabetic subject.
21. The method of claim 20, wherein the diabetic subject is a diabetic mouse.
22. The method of claim 1 , wherein the immunoassay is capable of detecting AgRP at a concentration of about 0.2 picomolar to about 2 picomolar or greater.
23. The method of claim 1, wherein the immunoassay is capable of detecting NPY at a concentration of about 0.2 picomolar to about 2 picomolar or greater.
24. The method of claim 1 , wherein the immunoassay is capable of detecting α-MSH at a concentration of about 0.2 picomolar to about 2 picomolar or greater.
25. The method of claim 1, wherein the artificial cerebrospinal fluid comprises NaCl, Na2HPO4, KCl, CaCl2, MgSO4, NaHCO3, glucose, and ascorbic acid.
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Title
ENRIORI ET AL.: 'Diet-induced obesity causes severe bit reversible leptin resitsance in arcuate melanocortin neurons' CELL METABOLISM vol. 5, 2007, pages 181 - 194 *
ENRIORI ET AL.: 'Leptin resistance and obesity' OBESITY vol. 14, 2006, pages 254S - 258S *

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