WO2006007400A2 - Methods of diagnosing and treating obesity, diabetes and insulin resistance - Google Patents

Abstract

The present invention provides compositions and methods for the diagnosis and treatment of obesity, diabetes, and insulin resistance. In particular, the invention relates to methods for identifying modulators of polynucleotides or polypeptides of the invention, as well as the use of such modulators for treating obesity and / or diabetes. The invention further relates to methods of diagnosing obesity and / or diabetes by measuring the levels of polynucleotides or polypeptides of the invention in a patient.

Description

Methods of Diagnosing & Treating Obesity, Diabetes and Insulin

Resistance

CROSS-REFERENCE TO RELATED PATENT APPLCIATIONS [01] The present application claims priority to U.S. Provisional Patent

Application No. 60/ 580,448, filed June 16, 2004, which is incorporated by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION [02] Obesity has reached epidemic proportions globally with more than 1 billion adults overweight- at least 300 million of them clinical obese- and is a major contributor to the global burden of chronic disease and disability. Overweight and obesity leads to adverse metabolic effect on blood pressure, cholesterol, triglycerides and insulin resistance. The non- fatal but debilitating health problems associated with obesity include respiratory difficulties, chronic musculoskeletal problems, skin problems and infertility. The more life-threatening problems fall into four main areas: cardiovascular disease problems, conditions associated with insulin resistance such as Type 2 diabetes, certain types of cancers especially the hormonally related and large-bowel cancers, and gall bladder disease. The likelihood of developing Type 2 diabetes and hypertension rises steeply with increasing body fatness. Weight reduction leads to correction of a number of obesity- associated endocrine and metabolic disorders.

[03] Effective weight management for individuals and groups at risk of developing obesity involves a range of long term strategies. These include prevention, weight maintenance, management of co-morbidities and weight loss. Existing treatment strategies include calorific restriction programs, surgery (gastric stapling) and drug intervention. The currently available anti-obesity drugs can be divided into two classes: central acting and peripheral acting. Three marketed drugs are Xenical (Orlistat), Merida (Sibutramine) and Adipex-P (Phentermine). Xenical is a non-systemic acting GI lipase inhibitor which is indicated for short and long term obesity management. Merida reduces food intake by re- uptake inhibition of primarily norepinephrine and serotonin. Adipex-P is a phenteramine with sympathomimetic activities and suppresses appetite. It is indicated only for short term use. A more drastic solution to permanent weight loss is surgery and a gastric by-pass which limits absorption of calories through massive reduction in stomach size.

[04] Carrying extra body weight and body fat go hand and hand with the development of diabetes. People who are overweight (BMI greater than 25) are at a much greater risk of developing type 2 diabetes than normal weight individuals. Almost 90% of people with type 2 diabetes are overweight.

[05] Diabetes mellitus can be divided into two clinical syndromes, Type 1 and Type 2 diabetes mellitus. Type 1, or insulin-dependent diabetes mellitus (DDDM), is a chronic autoimmune disease characterized by the extensive loss of beta cells in the pancreatic Islets of Langerhans, which produce insulin. As these cells are progressively destroyed, the amount of secreted insulin decreases, eventually leading to hyperglycemia (abnormally high level of glucose in the blood) when the amount of secreted insulin drops below the level required for euglycemia (normal blood glucose level). Although the exact trigger for this immune response is not known, patients with IDDM have high levels of antibodies against proteins expressed in pancreatic beta cells. However, not all patients with high levels of these antibodies develop IDDM.

[06] Type 2 diabetes (also referred to as non-insulin dependent diabetes mellitus (NDDDM)) develops when muscle, fat and liver cells fail to respond normally to insulin. This failure to respond (called insulin resistance) may be due to reduced numbers of insulin receptors on these cells, or a dysfunction of signaling pathways within the cells, or both. The beta cells initially compensate for this insulin resistance by increasing insulin output. Over time, these cells become unable to produce enough insulin to maintain normal glucose levels, indicating progression to Type 2 diabetes.

[07] Type 2 diabetes is brought on by a combination of genetic and acquired risk factors - including a high-fat diet, lack of exercise, and aging. Worldwide, Type 2 diabetes has become an epidemic, driven by increases in obesity and a sedentary lifestyle, widespread adoption of western dietary habits, and the general aging of the population in many countries. In 1985, an estimated 30 million people worldwide had diabetes — by 2000, this figure had increased 5-fold, to an estimated 154 million people. The number of people with diabetes is expected to double between now and 2025, to about 300 million.

[08] Type 2 diabetes is a complex disease characterized by defects in glucose and lipid metabolism. Typically there are perturbations in many metabolic parameters including increases in fasting plasma glucose levels, free fatty acid levels and triglyceride levels, as well as a decrease in the ratio of HDL/LDL. As discussed above, one of the principal underlying causes of diabetes is thought to be an increase in insulin resistance in peripheral tissues, principally muscle and fat.

[09] Therapies aimed at reducing peripheral insulin resistance are available. The most relevant to this invention are drugs of the thiazolidinedione (TZD) class namely troglitazone, pioglitazone, and rosiglitazone. In the US these have been marketed under the names Rezulin™, Avandia™ and Actos™, respectively. The principal effect of these drugs is to improve glucose homeostasis. Notably in diabetics treated with TZDs there are increases in peripheral glucose disposal rates indicative of increased insulin sensitivity in both muscle and fat.

[10] The molecular target of TZDs is a member of the PPAR family of ligand-activated transcription factors called PPAR gamma. This transcription factor is highly expressed in adipose tissue with much lower levels being observed in muscle. Binding of TZDs to PPAR gamma in target cells and tissues such as fat and muscle brings about a change in gene expression. The link between TZD-altered gene expression in fat and muscle and increased insulin sensitivity is unknown. The present invention addresses this and other problems.

BRIEF SUMMARY OF THE INVENTION The present invention provides methods for identifying an agent for treating an obese, diabetic or pre-diabetic individual. In some embodiments, the method comprising the steps of: (i) contacting an agent to a polypeptide encoded by a polynucleotide that is substantially identical to or hybridizes to a nucleic acid encoding a polypeptide listed in Table 1 under hybridization conditions of 50% formamide, 5X SSC, and 1% SDS at 42⁰C followed by a wash in 0.2X SSC, and 0.1 % SDS at 55⁰C, wherein the polypeptide optionally has the activity listed in Table 1 ; and (ii) selecting an agent that modulates the expression or activity of the polypeptide or that binds to the polypeptide, thereby identifying an agent for treating an obese, diabetic or pre-diabetic individual. Table 1: List of Polypeptides, SEQ ID numbers and Proposed Activity

In some embodiments, the polypeptide comprises an amino acid sequence at least 95% identical to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 35, 37, 39, 40, 42, 44, 46, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 182, 184, 186, 188, 190, 192, 194, 196, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 230, 232, 234, 236, 238, 240, 242, 243, 245, 247, 249, 251, 252, 254, 256, 258, 260, 262, 263, 265, 267, 269, 270, 272, 274 or a protein domain thereof. In some embodiments, the method further comprises detecting whether the selected agent modulates weight and/or obesity. In some embodiments, the method further comprising detecting whether the selected agent modulates insulin sensitivity.

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

[12] In some embodiments, the polypeptide is expressed in a cell and the cell is contacted with the agent. In some embodiments, the polypeptide comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 35, 37, 39, 40, 42, 44, 46, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 182, 184, 186, 188, 190, 192, 194, 196, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 230, 232, 234, 236, 238, 240, 242, 243, 245, 247, 249, 251, 252, 254, 256, 258, 260, 262, 263, 265, 267, 269, 270, 272 or 274.

[13] The present invention also provides methods of reducing body weight in an animal. In some embodiments, the methods comprise administering to the animal an effective amount of an agent that modulates the activity or expression of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 35, 37, 39, 40, 42, 44, 46, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 182, 184, 186, 188, 190, 192, 194, 196, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 230, 232, 234, 236, 238, 240, 242, 243, 245, 247, 249, 251, 252, 254, 256, 258, 260, 262, 263, 265, 267, 269, 270, 272 or 274.

[14] In some embodiments, the agent is selected by a method comprising (i) contacting an agent to a mixture comprising a polypeptide encoded by a polynucleotide is substantially identical to or hybridizes to a nucleic acid encoding a polypeptide listed in Table 1 under hybridization conditions of 50% formamide, 5X SSC, and 1% SDS at 42⁰C followed by a wash in 0.2X SSC, and 0.1% SDS at 55⁰C, wherein the polypeptide optionally has the activity listed in Table 1 ; and (ii) selecting an agent that modulates the expression or activity of the polypeptide or that binds to the polypeptide.

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

[16] The present invention also provides methods of treating a diabetic or pre-diabetic animal. In some embodiments, the method comprising administering to the animal a therapeutically effective amount of an agent that modulates the activity or expression of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 35, 37, 39, 40, 42, 44, 46, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 182, 184, 186, 188, 190, 192, 194, 196, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 230, 232, 234, 236, 238, 240, 242, 243, 245, 247, 249, 251, 252, 254, 256, 258, 260, 262, 263, 265, 267, 269, 270, 272 or 274. In some embodiments, the polypeptide comprises an amino acid sequence at least 95% identical to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 35, 37, 39, 40, 42, 44, 46, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 182, 184, 186, 188, 190, 192, 194, 196, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 230, 232, 234, 236, 238, 240, 242, 243, 245, 247, 249, 251, 252, 254, 256, 258, 260, 262, 263, 265, 267, 269, 270, 272, 274 or a protein domain thereof.

[17] In some embodiments, the agent is selected by a method comprising (i) contacting an agent to a mixture comprising a polypeptide encoded by a polynucleotide that hybridizes to a nucleic acid encoding SEQ E) NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 35, 37, 39, 40, 42, 44, 46, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 182, 184, 186, 188, 190, 192, 194, 196, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 230, 232, 234, 236, 238, 240, 242, 243, 245, 247, 249, 251, 252, 254, 256, 258, 260, 262, 263, 265, 267, 269, 270, 272 or 274 in 50% formamide, 5X SSC, and 1% SDS at 42°C followed by a wash in 0.2X SSC, and 0.1% SDS at 55⁰C; and (ii) selecting an agent that modulates the expression or activity of the polypeptide or that binds to the polypeptide. [18] In some embodiments, the agent is an antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the animal is a human. [19] The present invention also provides methods of introducing an expression cassette into a cell. In some embodiments, the methods comprise introducing into the cell an expression cassette comprising a promoter operably linked to a polynucleotide encoding a polypeptide, wherein the polynucleotide is substantially identical to or hybridizes to a nucleic acid encoding a polypeptide listed in Table 1 under hybridization conditions of 50% formamide, 5X SSC, and 1% SDS at 42⁰C followed by a wash in 0.2X SSC, and 0.1% SDS at 55⁰C, and the polypeptide optionally has the activity listed in Table 1. In some embodiments, the polypeptide comprises an amino acid sequence at least 95% identical to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 35, 37, 39, 40, 42, 44, 46, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 182, 184, 186, 188, 190, 192, 194, 196, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 230, 232, 234, 236, 238, 240, 242, 243, 245, 247, 249, 251, 252, 254, 256, 258, 260, 262, 263, 265, 267, 269, 270, 272, 274 or a protein domain thereof.

[20] In some embodiments, the polypeptide comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 35, 37, 39, 40, 42, 44, 46, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 182, 184, 186, 188, 190, 192, 194, 196, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 230, 232, 234, 236, 238, 240, 242, 243, 245, 247, 249, 251, 252, 254, 256, 258, 260, 262, 263, 265, 267, 269, 270, 272 or 274.

[21] In some embodiments, the cell is selected from the group consisting of an adipocyte and a skeletal muscle cell.

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

[23] The present invention also provides methods of diagnosing an individual who has obesity, Type 2 diabetes or has a predisposition for diabetes or obesity. In some embodiments, the method comprises detecting in a sample from the individual the level of a polypeptide or the level of a polynucleotide encoding the polypeptide, wherein the polynucleotide is substantially identical to or hybridizes to a nucleic acid encoding a polypeptide listed in Table 1 under hybridization conditions of 50% formamide, 5X SSC, and 1% SDS at 42⁰C followed by a wash in 0.2X SSC, and 0.1% SDS at 55⁰C, wherein a modulated level of the polypeptide or polynucleotide in the sample compared to a level of the polypeptide or polynucleotide in either a lean individual or a previous sample from the individual indicates that the individual is obese or diabetic or has a predisposition for diabetes or obesity.

[24] hi some embodiments, the detecting step comprises contacting the sample with an antibody that specifically binds to the polypeptide. In some embodiments, the amino acid sequence comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 35, 37, 39, 40, 42, 44, 46, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 182, 184, 186, 188, 190, 192, 194, 196, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 230, 232, 234, 236, 238, 240, 242, 243, 245, 247, 249, 251, 252, 254, 256, 258, 260, 262, 263, 265, 267, 269, 270, 272 or 274. In some embodiments, the detecting step comprises quantifying mRNA encoding the polypeptide. In some embodiments, the mRNA is reverse transcribed and amplified in a polymerase chain reaction. In some embodiments, the sample is a blood, urine or tissue sample.

[25] The present invention provides for an isolated nucleic acid that is substantially identical to or hybridizes to a nucleic acid encoding a polypeptide listed in Table 1 under hybridization conditions of 50% formamide, 5X SSC, and 1 % SDS at 42°C followed by a wash in 0.2X SSC, and 0.1% SDS at 55⁰C. In some embodiments, the polynucleotide comprises SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 34, 36, 38, 41, 43, 45, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 183, 185, 187, 189, 191, 193, 195, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 231, 233, 235, 237, 239, 241, 244, 246, 248, 250, 253, 255, 257, 259, 261, 264, 266, 268, 271 or 273. In some embodiments, the polynucleotide encodes SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 35, 37, 39, 40, 42, 44, 46, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 182, 184, 186, 188, 190, 192, 194, 196, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 230, 232, 234, 236, 238, 240, 242, 243, 245, 247, 249, 251, 252, 254, 256, 258, 260, 262, 263, 265, 267, 269, 270, 272 or 274.

[26] The present invention also provides expression cassettes comprising a heterologous promoter operably linked to a nucleic acid that is substantially identical to or hybridizes to a nucleic acid encoding a polypeptide listed in Table 1 under hybridization conditions of 50% formamide, 5X SSC, and 1 % SDS at 42⁰C followed by a wash in 0.2X SSC, and 0.1% SDS at 55⁰C.

[27] The present invention also provides host cells transfected with nucleic acids that is substantially identical to or hybridizes to a nucleic acid encoding a polypeptide listed in Table 1 under hybridization conditions of 50% formamide, 5X SSC, and 1% SDS at 42⁰C followed by a wash in 0.2X SSC, and 0.1% SDS at 55⁰C. In some embodiments, the host cell is a human cell, hi some embodiments, the host cell is a bacterium.

[28] The present invention also provides isolated polypeptides comprising an amino acid sequence at least 70% identical to SEQ ED NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 35, 37, 39, 40, 42, 44, 46, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 182, 184, 186, 188, 190, 192, 194, 196, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 230, 232, 234, 236, 238, 240, 242, 243, 245, 247, 249, 251, 252, 254, 256, 258, 260, 262, 263, 265, 267, 269, 270, 272, 274 or fragments thereof, hi some embodiments, the polypeptide comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 35, 37, 39, 40, 42, 44, 46, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 182, 184, 186, 188, 190, 192, 194, 196, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 230, 232, 234, 236, 238, 240, 242, 243, 245, 247, 249, 251, 252, 254, 256, 258, 260, 262, 263, 265, 267, 269, 270, 272 or 274. [29] The present invention also provides antibodies that specifically bind to a polypeptide selected from the groups consisting of SEQ ED NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 35, 37, 39, 40, 42, 44, 46, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 182, 184, 186, 188, 190, 192, 194, 196, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 230, 232, 234, 236, 238, 240, 242, 243, 245, 247, 249, 251, 252, 254, 256, 258, 260, 262, 263, 265, 267, 269, 270, 272 or 274.

[30] The present invention also provides pharmaceutical compositions comprising polypeptides comprising an amino acid sequence at least 70% identical to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 35, 37, 39, 40, 42, 44, 46, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 182, 184, 186, 188, 190, 192, 194, 196, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 230, 232, 234, 236, 238, 240, 242, 243, 245, 247, 249, 251, 252, 254, 256, 258, 260, 262, 263, 265, 267, 269, 270, 272, 274 or fragments thereof, and a pharmaceutically-acceptable excipient.

DEFINITIONS

[31] "Insulin sensitivity" refers to the ability of a cell or tissue to respond to insulin. Responses include, e.g., glucose uptake of a cell or tissue in response to insulin stimulation. Sensitivity can be determined at an organismal, tissue or cellular level. For example, blood or urine glucose levels following a glucose tolerance test are indicative of insulin sensitivity. Other methods of measuring insulin sensitivity include, e.g., measuring glucose uptake (see, e.g., Garcia de Herreros, A., and Birnbaum, M. J. J. Biol. Chem. 264, 19994-19999 (1989); Klip, A., Li, G., and Logan, WJ. Am. J. Physiol. 247, E291-296 (1984)), measuring the glucose infusion rate (GINF) into tissue such as the skeletal muscle (see, e.g., Ludvik et ah, J. Clin. Invest. 100:2354 (1997); Frias et al, Diabetes Care 23:64, (2000)) and measuring sensitivity of GLUT4 translocation (e.g., as described herein) in response to insulin.

[32] As used herein, an overweight person has a body mass index (BMI) > 25 and an "obese" person has a BMI >30. BMI is calculated as the weight in kilograms divided by the square of the height in meters.

[33] The term "waist-to-hip ratio or WHR" is the ratio of a person's waist circumference to hip circumference. For most people, carrying extra weight around their middle increases health risks more than carrying extra weight around their hips or thighs. For both men and women, a waist-to-hip ratio of 1.0 or higher is considered "at risk" or in the danger zone for undesirable health consequences, such as heart disease and other ailments connected with being overweight.

[34] The term "adipogenic," when used in reference to cells refers to a cell which can become an adipocyte. An "adipogenic factor" refers to a factor (including, e.g., a protein (or glycoprotein)) that can induce or stimulate the differentiation of cells into an adipocyte. Exemplary adipogenic factors include, e.g., _Wntl0b, Pref-l,ADF and TNF-alpha. [35] The term "lipid metabolism" refers to the in vivo process of catab<->lism (decomposition) and anabolism (accumulation) of lipids (e.g., triglycerides derived from food) and is intended to include, in the broad sense, reactions for transforming lipids into energy, biosynthesis of fatty acids, acylglycerol, phospholipid metabolism and cholesterol metabolism.

[36] "Activity" of a polypeptide of the invention refers to structural, regulatory, or biochemical functions of a polypeptide in its native cell or tissue. Examples of activity of a polypeptide include both direct activities and indirect activities. Exemplary direct activities are the result of direct interaction with the polypeptide, , e.g., enzymatic activity, ligand binding, production or depletion of second messengers (e.g., cAMP, cGMP, IP₃, DAG, or Ca²⁺), ion flux, phosphorylation levels, transcription levels, and the like. Exemplary indirect activities are observed as a change in phenotype or response in a cell or tissue to a polypeptide's directed activity, e.g., loss of body weight or molecular events associated with loss of body weight or obesity or modulating insulin sensitivity of a cell as a result of the interaction of the polypeptide with other cellular or tissue components.

[37] "Predisposition for diabetes" occurs in a person when the person is at high risk for developing diabetes. A number of risk factors are known to those of skill in the art and include: genetic factors (e.g., carrying alleles that result in a higher occurrence of diabetes than in the average population or having parents or siblings with diabetes); overweight (e.g., body mass index (BMI) greater or equal to 25 kg/m²); habitual physical inactivity, race/ethnicity (e.g., African- American, Hispanic-American, Native Americans, Asian- Americans, Pacific Islanders); previously identified impaired fasting glucose or impaired glucose tolerance, hypertension (e.g., greater or equal to 140/90 mmHg in adults); HDL cholesterol less than or equal to 35 mg/dl; triglyceride levels greater or equal to 250 mg/dl; a history of gestational diabetes or delivery of a baby over nine pounds; and/or polycystic ovary syndrome. See, e.g., "Report of the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus" and "Screening for Diabetes" Diabetes Care 25(1): S5- S24 (2002).

[38] A "lean individual," when used to compare with a sample from a patient, refers to an adult with a fasting blood glucose level less than 100 mg/dl or a 2 hour PG reading of 140 mg/dl. "Fasting" refers to no caloric intake for at least 8 hours. A "2 hour PG" refers to the level of blood glucose after challenging a patient to a glucose load containing the equivalent of 75g anhydrous glucose dissolved in water. The overall test is generally referred to as an oral glucose tolerance test (OGTT;. See, e.g., Diabetes Care, 2003, 26(11): 3160-3167 (2003). The level of a polypeptide in a lean individual can be a reading from a single individual, but is typically a statistically relevant average from a group of lean individuals. The level of a polypeptide in a lean individual can be represented by a value, for example in a computer program.

[39] A "pre-diabetic individual," when used to compare with a sample from a patient, refers to an adult with a fasting blood glucose level greater than 100 mg/dl but less than 126 mg/dl or a 2 hour PG reading of greater than 140 mg/dl but less than 200mg/dl. A "diabetic individual," when used to compare with a sample from a patient, refers to an adult with a fasting blood glucose level greater than 126 mg/dl or a 2 hour PG reading of greater than 200 mg/dl.

[40] An "agonist" refers to an agent that binds to, stimulates, increases, activates, facilitates, enhances activation, sensitizes or up regulates the activity or expression of a polypeptide of the invention.

[41] An "antagonist" refers to an agent that binds to, partially or totally blocks stimulation, decreases, prevents, delays activation, inactivates, desensitizes, or down regulates the activity or expression of a polypeptide of the invention. [42] "Antibody" refers to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof which specifically bind and recognize an analyte (antigen). The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

[43] An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (V_L) and variable heavy chain (V_H) refer to these light and heavy chains respectively.

[44] Antibodies exist, e.g., as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'_2; a dimer of Fab which itself is a light chain joined to Vκ-€:^'jl by a disulfide bond. The F(ab)'₂ may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)'₂ dimer into an Fab' monomer. The Fab' monomer is essentially an Fab with part of the hinge region (see, Paul (Ed.) Fundamental Immunology, Third Edition, Raven Press, NY (1993)). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv).

[45] The terms "peptidomimetic" and "mimetic" refer to a synthetic chemical compound that has substantially the same structural and functional characteristics of the antagonists or agonists of the invention. Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compound are termed "peptide mimetics" or "peptidomimetics" (Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber and Freidinger TINS p. 392 (1985); and Evans et al. J. Med. Chem. 30:1229 (1987), which are incorporated herein by reference). Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce an equivalent or enhanced therapeutic or prophylactic effect. Generally, peptidomimetics are structurally similar to a paradigm polypeptide (i.e., a polypeptide that has a biological or pharmacological activity), such as a polypeptide exemplified in this application, but have one or more peptide linkages optionally replaced by a linkage selected from the group consisting of, e.g., -CH2NH-, -CH2S-, -CH2-CH2-, - CH=CH- (cis and trans), -COCH2-, -CH(OH)CH2-, and -CH2SO-. The mimetic can be either entirely composed of synthetic, non-natural analogues of amino acids, or, is a chimeric molecule of partly natural peptide amino acids and partly non-natural analogs of amino acids. The mimetic can also incorporate any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantially alter the mimetic 's structure and/or activity. For example, a mimetic composition is within the scope of the invention if it is capable of carrying out the binding or other activities of an agonist or antagonist of a polypeptide of the invention.

[46] The term "gene" means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (?xons).

[47] The term "isolated," when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It may be in a homogeneous state although it can be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified. In particular, an isolated gene is separated from open reading frames that flank the gene and encode a protein other than the gene of interest. The term "purified" denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.

[48] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double- stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260.2605-2608 (1985); and Cassol et al. (1992); Rossolini et al., MoI. Cell. Probes 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.

[49] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, the terms encompass amino acid chains of any length, including full-length proteins {i.e., antigens), wherein the amino acid residues are linked by covalent peptide bonds. [50] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g. , hydroxyproline, γ -carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. "Amino acid mimetics" refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but which functions in a manner similar to a naturally occurring amino acid.

[51] Amino acids may be referred to herein by either the commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB

Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[52] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, "conservatively modified variants" refers to those nucleic acids that encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.

[53] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which altcs, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.

[54] The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and

8) Cysteine (C), Methionine (M) {see, e.g., Creighton, Proteins (1984)).

[55] "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (e.g., a polypeptide of the invention), which does not comprise additions or deletions, for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[56] The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same sequences. Sequences are "substantially identical" if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection, or across the entire sequence where not indicated. The invention provides polypeptides or polynucleotides that are substantially identical to the polypeptides or polynucleotides, respectively, exemplified herein (e.g., SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273 or 274). This definition also refers to the complement of a test sequence. Optionally, the identity exists over a region that is at least about 50 nucleotides in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides in length.

[57] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[58] A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. MoI. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Nat l. Acad. ScL USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection {see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).

[59] Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. MoI. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative- scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11 , an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix {see Henikoff and Henikoff (1989) Proc. Natl. Acad. ScL USA 89:10915) alignments (B) of 50, expectation (E) of 10, M=S, N=-4, and a comparison of both strands. [60] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences {see, e.g.,

Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[61] An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

[62] The phrase "selectively (or specifically) hybridizes to" refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (e.g., total cellular or library DNA or RNA).

[63] The phrase "stringent hybridization conditions" refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology— Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). Generally, stringent conditions are selected to be about 5-10° C lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength pH. The T_m is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T_m, 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodiurr- ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 3O⁰C for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, optionally 10 times background hybridization. Exemplary stringent hybridization conditions can be as following: 50% formamide, 5X SSC, and 1% SDS, incubating at 42⁰C, or 5X SSC, 1% SDS, incubating at 65°C, with wash in 0.2X SSC, and 0.1% SDS at 55°C, 60⁰C, or 65⁰C. Such washes can be performed for 5, 15, 30, 60, 120, or more minutes.

[64] Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides that they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code, hi such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions. Exemplary "moderately stringent hybridization conditions" include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37⁰C, and a wash in IX SSC at 45⁰C. Such washes can be performed for 5, 15, 30, 60, 120, or more minutes. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency. [65] The phrase "a nucleic acid sequence encoding" refers to a nucleic acid which contains sequence information for a structural RNA such as rRNA, a tRNA, or the primary amino acid sequence of a specific protein or peptide, or a binding site for a trans- acting regulatory agent. This phrase specifically encompasses degenerate codons (i.e., different codons which encode a single amino acid) of the native sequence or sequences that may be introduced to conform with codon preference in a specific host cell.

[66] The term "recombinant" when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (nonrecombinant) form of the cell or express native genes that are otherwise abnormally expressed, under-expressed or not expressed at all. [67] The term "heterologous" when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[68] An "expression vector" is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, the expression vector includes a nucleic acid to be transcribed operably linked to a promoter.

[69] The phrase "specifically (or selectively) binds to an antibody" or "specifically (or selectively) immunoreactive with", when referring to a protein or peptide, refers to a binding reaction which is determinative of the presence of the protein in the presence of a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein and do not bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. For example, antibodies raised against a protein having an amino acid sequence encoded by any of the polynucleotides of the invention can be selected to obtain antibodies specifically immunoreactive with that protein and not with other proteins, except for polymorphic variants. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays, Western blots, or immunohistochemistry are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See, Harlow and Lane Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, NY (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity. Typically, a specific or selective reaction will be at least twice the background signal or noise and more typically more than 10 to 100 times background. [70] "Inhibitors," "activators," and "modulators" of expression or of activity are used to refer to inhibitory, activating, or modulating molecules, respectively, of expression of the polypeptides of the invention as determined using in vitro or in vivo assays ¹O monitor expression or activity. Modulators encompass e.g., ligands, agonists, antagonists, rhoir homologs and mimetics, as well as the polypeptides of the invention, or fragments thereof with antagonist activity or that act to increase overall polypeptide activity (i.e., fragments that have at least some of the activity of the full-length protein). In some cases, fragments of the polypeptides of the invention are at least 20, 50, 75 or 100 amino acids in length. The term "modulator" includes inhibitors and activators. Inhibitors are agents that, e.g., inhibit expression of a polypeptide of the invention or bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity of a polypeptide of the invention, e.g., antagonists. Activators are agents that, e.g., induce or activate the expression of a polypeptide of the invention or bind to, stimulate, increase, open, activate, facilitate, or enhance activation, sensitize or up regulate the activity of a polypeptide of the invention, e.g., agonists. Modulators include naturally occurring and synthetic ligands, antagonists, agonists, small chemical molecules and the like. Such assays for inhibitors and activators include, e.g., applying putative modulator compounds to cells expressing a polypeptide of the invention and then determining the functional effects on a polypeptide of the invention activity, as described above. Samples or assays comprising a polypeptide of the invention that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of effect. Control samples (untreated with modulators) are assigned a relative activity value of 100%. Inhibition of a polypeptide of the invention is achieved when the polypeptide activity value relative to the control is about 80%, optionally 50% or 25, 10%, 5% or 1%. Activation of the polypeptide is achieved when the polypeptide activity value relative to the control is 110%, optionally 150%, optionally 200, 300%, 400%, 500%, or 1000-3000% or more higher.

DETAILED DESCRIPTION OF THE INVENTION I. INTRODUCTION

[71] The present application demonstrates that, surprisingly, modulated levels of mRNA comprising sequences of the invention occur in human adipose tissue collected from either insulin resistant obese non-diabetics or from type 2 diabetic individuals compared to levels of the mRNA in the lean, non-diabetic individuals. Insulin resistant obese individuals are generally predisposed to become type II diabetics. Therefore, the modulation of the sequences in the study described herein indicates the sequences' involvement in obesity, diabetes and/or pre-diabetes. [72] Without intending to limit the invention to a particular mechanism of action, it :<-. believed that modulation of the expression or activity of the polypeptides or polynucleotides of the invention is beneficial in treating obesity, diabetic, pre-diabetic or insulin resistant, non-diabetic patients. Furthermore, modulated levels of the polypeptides of the invention are indicative of insulin resistance, obesity, diabetes or a predisposition for obesity and/or diabetes. Thus, the detection of a polypeptide of the invention is useful for diagnosis of obesity, predisposition for obesity and/or diabetes, diabetes and/or insulin resistance.

[73] This invention also provides methods of using polypeptides of the invention and modulators of the polypeptides of the invention to diagnose and treat obesity, diabetes, pre-diabetes (including insulin resistant individuals) and related metabolic diseases. The present method also provides methods of identifying modulators of expression or activity of the polypeptides of the invention. Such modulators are useful for treating obesity and/or Type 2 diabetes as well as the pathological aspects of obesity (e.g., increased risk for cardiovascular disease, hypertension or cancer) and/or diabetes (e.g., insulin resistance).

II. GENERAL RECOMBINANT NUCLEIC ACID METHODS FOR USE WITH THE INVENTION

[74] In numerous embodiments of the present invention, nucleic acids encoding a polypeptide of the present invention will be isolated and cloned using recombinant methods. Such embodiments are used, e.g., to isolate polynucleotides identical or substantially identical to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 34, 36, 38, 41, 43, 45, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 183, 185, 187, 189, 191, 193, 195, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 231, 233, 235, 237, 239, 241, 244, 246, 248, 250, 253, 255, 257, 259, 261, 264, 266, 268, 271 or 273 for protein expression or during the generation of variants, derivatives, expression cassettes, or other sequences derived from an polypeptide or polynucleotide of the invention, to monitor gene expression, for the isolation or detection of sequences in different species, for diagnostic purposes in a patient, e.g., to detect mutations in a polypeptide or polynucleotide of the invention or to detect expression levels of nucleic acids or polypeptides. In some embodiments, the sequences encoding the polypeptides of the invention (or polypeptides comprising fragments of the polypeptides of the invention) are operably linked to a heterologous promoter. In some cases, fragments of the polypeptides of the invention are at least 20, 50, 75 or 100 amino acids in length. The polypeptides of the invention can be linked to heterologous amino acid sequences using recombinant DNA technology. In one embodiment, the nucleic acids of the invention are from any mammal, including, in particular, e.g., a human, a mouse, a rat, etc. [75] Polynucleotides, including expression cassettes, encoding polypeptides of the invention can be introduced into cells and optionally expressed in the cells. Polynucleotides of the invention can be introduced into eukaryotic or prokaryotic cells, including adipocyte or muscle cells. The cells can be primary cells or cell lines. A. General Recombinant Nucleic Acid Methods

[76] This invention relies on routine techniques in the field of recombinant genetics. Basic texts disclosing the general methods of use in this invention include Sambrook et al., Molecular Cloning, A Laboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al, eds., 1994)).

[77] For nucleic acids, sizes are given in either kilobases (kb) or base pairs (bp). These are estimates derived from agarose or acrylamide gel electrophoresis, from sequenced nucleic acids, or from published DNA sequences. For proteins, sizes are given in kilodaltons (kDa) or amino acid residue numbers. Proteins sizes are estimated from gel electrophoresis, from sequenced proteins, from derived amino acid sequences, or from published protein sequences.

[78] Oligonucleotides that are not commercially available can be chemically synthesized according to the solid phase phosphoramidite triester method first described by Beaucage & Caruthers, Tetrahedron Letts. 22:1859-1862 (1981), using an automated synthesizer, as described in Van Devanter et. al., Nucleic Acids Res. 12:6159-6168 (1984). Purification of oligonucleotides is by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson & Reanier, J. Chrom. 255:137-149 (1983).

[79] The sequence of the cloned genes and synthetic oligonucleotides can be verified after cloning using, e.g., the chain termination method for sequencing double- stranded templates of Wallace et al, Gene 16:21-26 (1981).

B. Cloning Methods for the Isolation of Nucleotide Sequences Encoding Desired Proteins

[80] In general, the nucleic acids encoding the subject proteins are cloned from DNA sequence libraries that are made to encode cDNA or genomic DNA. The particular sequences can be located by hybridizing with an oligonucleotide probe, the sequence of which can be derived from the sequences disclosed herein, which provide a reference for PCR primers and defines suitable regions for isolating probes specific for the polypeptides or polynucleotides of the invention. Alternatively, where the sequence is cloned into an expression library, the expressed recombinant protein can be detected immunologically with antisera or purified antibodies made against a polypeptide of interest, including those disclosed herein.

[81] Methods for making and screening genomic and cDNA libraries are well known to those of skill in the art (see, e.g., Gubler and Hoffman Gene 25:263-269 (1983); Benton and Davis Science, 196:180-182 (1977); and Sambrook, supra).

[82] Briefly, to make the cDNA library, one should choose a source that is rich in mRNA. The mRNA can then be made into cDNA, ligated into a recombinant vector, and transfected into a recombinant host for propagation, screening and cloning. For a genomic library, the DNA is extracted from a suitable tissue and either mechanically sheared or enzymatically digested to yield fragments of preferably about 5-100 kb. The fragments are then separated by gradient centrifugation from undesired sizes and are constructed in bacteriophage lambda vectors. These vectors and phage are packaged in vitro, and the recombinant phages are analyzed by plaque hybridization. Colony hybridization is carried out as generally described in Grunstein et al., Proc. Natl. Acad. ScL USA., 72:3961-3965 (1975).

[83] An alternative method combines the use of synthetic oligonucleotide primers with polymerase extension on an mRNA or DNA template. Suitable primers can be designed from specific sequences disclosed herein. This polymerase chain reaction (PCR) method amplifies the nucleic acids encoding the protein of interest directly from mRNA, cDNA, genomic libraries or cDNA libraries. Restriction endonuclease sites can be incorporated into the primers. Polymerase chain reaction or other in vitro amplification methods may also be useful, for example, to clone nucleic acids encoding specific proteins and express said proteins, to synthesize nucleic acids that will be used as probes for detecting the presence of mRNA encoding a polypeptide of the invention in physiological samples, for nucleic acid sequencing, or for other purposes (see, U.S. Patent Nos. 4,683,195 and 4,683,202). Genes amplified by a PCR reaction can be purified from agarose gels and cloned into an appropriate vector.

[84] Appropriate primers and probes for identifying the genes encoding a polypeptide of the invention from mammalian tissues can be derived from the sequences provided herein. For a general overview of PCR, see, Innis et al. PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego (1990).

[85] Synthetic oligonucleotides can be used to construct genes. This is done using a series of overlapping oligonucleotides, usually 40-120 bp in length, representing both the sense and anti-sense strands of the gene. These DNA fragments are then annealed, ligated and cloned.

[86] A polynucleotide encoding a polypeptide of the invention can be cloned using intermediate vectors before transformation into mammalian cells for expression. These intermediate vectors are typically prokaryote vectors or shuttle vectors. The proteins can be expressed in either prokaryotes or eukaryotes, using standard methods well known to those of skill in the art.

III. PURIFICATION OF PROTEINS OF THE INVENTION

[87] Either naturally occurring or recombinant polypeptides of the invention can be purified for use in functional assays. Naturally occurring polypeptides of the invention can be purified from any source (e.g., tissues of an organism expressing an ortholog). Recombinant polypeptides can be purified from any suitable expression system.

[88] The polypeptides of the invention may be purified to substantial purity by standard techniques, including selective precipitation with such substances as ammonium sulfate; column chromatography, immunopurification methods, and others (see, e.g., Scopes, Protein Purification: Principles and Practice (1982); U.S. Patent No. 4,673,641; Ausubel et ah, supra; and Sambrook et al, supra).

[89] A number of procedures can be employed when recombinant polypeptides are being purified. For example, proteins having established molecular adhesion properties can be reversibly fused to a polypeptide of the invention. With the appropriate ligand, either protein can be selectively adsorbed to a purification column and then freed from the column in a relatively pure form. The fused protein may be then removed by enzymatic activity. Finally polypeptides can be purified using immunoaffϊnity columns.

A. Purification of Proteins from Recombinant Bacteria [90] When recombinant proteins are expressed by the transformed bacteria in large amounts, typically after promoter induction, although expression can be constitutive, the proteins may form insoluble aggregates. There are several protocols that are suitable for purification of protein inclusion bodies. For example, purification of aggregate proteins (hereinafter referred to as inclusion bodies) typically involves the extraction, separation and/or purification of inclusion bodies by disruption oi^'bacterial cells typically, but not limited to, by incubation in a buffer of about 100-150 μg/ml lysozyme and 0.1% Nonidet P40, a non-ionic detergent. The cell suspension can be ground using a Polytron grinder (Brinkman Instruments, Westbury, NY). Alternatively, the cells can be sonicated on ice. Alternate methods of lysing bacteria are described in Ausubel et al. and Sambrook et al, both supra, and will be apparent to those of skill in the art.

[91] The cell suspension is generally centrifuged and the pellet containing the inclusion bodies resuspended in buffer which does not dissolve but washes the inclusion bodies, e.g., 20 niM Tris-HCl (pH 7.2), 1 mM EDTA, 150 mM NaCl and 2% Triton-X 100, a non-ionic detergent. It may be necessary to repeat the wash step to remove as much cellular debris as possible. The remaining pellet of inclusion bodies may be resuspended in an appropriate buffer (e.g., 20 mM sodium phosphate, pH 6.8, 150 mM NaCl). Other appropriate buffers will be apparent to those of skill in the art.

[92] Following the washing step, the inclusion bodies are solubilized by the addition of a solvent that is both a strong hydrogen acceptor and a strong hydrogen donor (or a combination of solvents each having one of these properties). The proteins that formed the inclusion bodies may then be renatured by dilution or dialysis with a compatible buffer. Suitable solvents include, but are not limited to, urea (from about 4 M to about 8 M), formamide (at least about 80%, volume/volume basis), and guanidine hydrochloride (from about 4 M to about 8 M). Some solvents that are capable of solubilizing aggregate-forming proteins, such as SDS (sodium dodecyl sulfate) and 70% formic acid, are inappropriate for use in this procedure due to the possibility of irreversible denaturation of the proteins, accompanied by a lack of immunogenicity and/or activity. Although guanidine hydrochloride and similar agents are denaturants, this denaturation is not irreversible and renaturation may occur upon removal (by dialysis, for example) or dilution of the denaturant, allowing re-formation of the immunologically and/or biologically active protein of interest. After solubilization, the protein can be separated from other bacterial proteins by standard separation techniques.

[93] Alternatively, it is possible to purify proteins from bacteria periplasm. Where the protein is exported into the periplasm of the bacteria, the periplasmic fraction of the bacteria can be isolated by cold osmotic shock in addition to other methods known to those of skill in the art (see, Ausubel et al., supra). To isolate recombinant proteins from the periplasm, the bacterial cells are centrifuged to form a pellet. The pellet is resuspended in a buffer containing 20% sucrose. To lyse the cells, the bacteria are centrifuged and the pellet is resuspended in ice-cold 5 mM MgSO₄ and kept in an ice bath for approximately 10 minutes. The cell suspension is centrifuged and the supernatant decanted and saved. The recombinant proteins present in the supernatant can be separated from the host proteins by standard separation techniques well known to those of skill in the art.

B. Purification of Proteins from Insect Cells [94] Proteins can also be purified from eukaryotic gene expression systems as described in, e.g., Fernandez and Hoeffler, Gene Expression Systems (1999). In some embodiments, baculovirus expression systems are used to isolate proteins of the invention. Recombinant baculoviruses are generally generated by replacing the polyhedrin coding sequence of a baculovirus with a gene to be expressed (e.g., encoding a polypeptide of the invention). Viruses lacking the polyhedrin gene have a unique plaque morphology making them easy to recognize. In some embodiments, a recombinant baculovirus is generated by first cloning a polynucleotide of interest into a transfer vector (e.g., a pUC based vector) such that the polynucleotide is operably linked to a polyhedrin promoter. The transfer vector is transfected with wildtype DNA into an insect cell (e.g., Sf9, Sf21 or BT1-TN-5B1-4 cells), resulting in homologous recombination and replacement of the polyhedrin gene in the wildtype viral DNA with the polynucleotide of interest. Virus can then be generated and plaque purified. Protein expression results upon viral infection of insect cells. Expressed proteins can be harvested from cell supernatant if secreted, or from cell lysates if intracellular. See, e.g., Ausubel et al. and Fernandez and Hoeffler, supra.

C. Purification of secreted proteins from mammalian cells

[95] Polypeptides of the invention, and in particular, secreted proteins of the invention can be readily purified from mammalian cells expressing the polypeptides. Expression of the polypeptides can be the result of either transient or stable expression of the protein from a recombinant expression cassette introduced into the cells. Secreted proteins can generally be isolated using standard procedures to purify the proteins from the cell culture medium.

D. Standard Protein Separation Techniques For Purifying Proteins 1. Solubility Fractionation

[96] Often as an initial step, and if the protein mixture is complex, an initial salt fractionation can separate many of the unwanted host cell proteins (or proteins derived from the cell culture media) from the recombinant protein of interest. The preferred salt is ammonium sulfate. Ammonium sulfate precipitates proteins by effectively reducing the amount of water in the protein mixture. Proteins then precipitate on the basis of their solubility. The more hydrophobic a protein is, the more likely it is to precipitate at lower ammonium sulfate concentrations. A typical protocol is to add saturated ammonium sulfate to a protein solution so that the resultant ammonium sulfate concentration is between 20-30%. This will precipitate the most hydrophobic proteins. The precipitate is discarded (unless the protein of interest is hydrophobic) and ammonium sulfate is added to the supernatant to a concentration known to precipitate the protein of interest. The precipitate is then solubilized in buffer and the excess salt removed if necessary, through either dialysis or diafiltration. Other methods that rely on solubility of proteins, such as cold ethanol precipitation, are well known to those of skill in the art and can be used to fractionate complex protein mixtures.

2. Size Differential Filtration

[97] Based on a calculated molecular weight, a protein of greater and lesser size can be isolated using ultrafiltration through membranes of different pore sizes (for example, Amicon or Millipore membranes). As a first step, the protein mixture is ultrafiltered through a membrane with a pore size that has a lower molecular weight cut-off than the molecular weight of the protein of interest. The retentate of the ultrafiltration is then ultrafiltered against a membrane with a molecular cut off greater than the molecular weight of the protein of interest. The recombinant protein will pass through the membrane into the filtrate. The filtrate can then be chromatographed as described below.

3. Column Chromatography

[98] The proteins of interest can also be separated from other proteins on the basis of their size, net surface charge, hydrophobicity and affinity for ligands. In addition, antibodies raised against proteins can be conjugated to column matrices and the proteins immunopurified. All of these methods are well known in the art.

[99] Immunoaffinity chromatography using antibodies raised to a variety of affinity tags such as hemagglutinin (HA), FLAG, Xpress, Myc, hexahistidine (His), glutathione S transferase (GST) and the like can be used to purify polypeptides. The His tag will also act as a chelating agent for certain metals (e.g., Ni) and thus the metals can also be used to purify His-containing polypeptides. After purification, the tag is optionally removed ^•oy specific proteolytic cleavage.

[100] It will be apparent to one of skill that chromatographic techniques can be performed at any scale and using equipment from many different manufacturers (e.g., Pharmacia Biotech). IV. DETECTION OF POLYNUCLEOTIDES OF THE INVENTION

[101] Those of skill in the art will recognize that detection of expression of polynucleotides and polypeptides of the invention has many uses. For example, as discussed herein, detection of levels of polynucleotides and polypeptides of the invention in a patient is useful for diagnosing diabetes or a predisposition for at least some of the pathological effects of diabetes. Moreover, detection of gene expression is useful to identify modulators of expression of polynucleotides and polypeptides of the invention.

[102] A variety of methods of specific DNA and RNA measurement that use ^' nucleic acid hybridization techniques are known to those of skill in the art (see, Sambrook, supra). Some methods involve an electrophoretic separation (e.g., Southern blot for detecting DNA, and Northern blot for detecting RNA), but measurement of DNA and RNA can also be carried out in the absence of electrophoretic separation (e.g., by dot blot). Southern blot of genomic DNA (e.g., from a human) can be used for screening for restriction fragment length polymorphism (RFLP) to detect the presence of a genetic disorder affecting a polypeptide of the invention.

[103] The selection of a nucleic acid hybridization format is not critical. A variety of nucleic acid hybridization formats are known to those skilled in the art. For example, common formats include sandwich assays and competition or displacement assays. Hybridization techniques are generally described in Hames and Higgins Nucleic Acid Hybridization, A Practical Approach, TRL Press (1985); Gall and Pardue, Proc. Natl. Acad. ScL U.S.A., 63:378-383 (1969); and John et al. Nature, 223:582-587 (1969).

[104] Detection of a hybridization complex may require the binding of a signal-generating complex to a duplex of target and probe polynucleotides or nucleic acids. Typically, such binding occurs through ligand and anti-ligand interactions as between a ligand-conjugated probe and an anti-ligand conjugated with a signal. The binding of the signal generation complex is also readily amenable to accelerations by exposure to ultrasonic energy.

[105] The label may also allow indirect detection of the hybridization complex. For example, where the label is a hapten or antigen, the sample can be detected by using antibodies. In these systems, a signal is generated by attaching fluorescent or enzyme molecules to the antibodies or in some cases, by attachment to a radioactive label (see, e.g., Tijssen, "Practice and Theory of Enzyme Immunoassays," Laboratory Techniques in Biochemistry and Molecular Biology, Burdon and van Knippenberg Eds., Elsevier (1985), pp. 9-20).

[106] The probes are typically labeled either directly, as with isotopes, chromophores, lumiphores, chromogens, or indirectly, such as with biotin, to which a streptavidin complex may later bind. Thus, the detectable labels used in the assays of the present invention can be primary labels (where the label comprises an element that is detected directly or that produces a directly detectable element) or secondary labels (where the detected label binds to a primary label, e.g., as is common in immunological labeling). Typically, labeled signal nucleic acids are used to detect hybridization. Complementary nucleic acids or signal nucleic acids may be labeled by any one of several methods typically used to detect the presence of hybridized polynucleotides. The most common method of detection is the use of autoradiography with ³H, ¹²⁵1, ³⁵S, ¹⁴C, or ³²P-labeled probes or the like.

[107] Other labels include, e.g., ligands that bind to labeled antibodies, fluorophores, chemiluminescent agents, enzymes, and antibodies that can serve as specific binding pair members for a labeled ligand. An introduction to labels, labeling procedures and detection of labels is found in Polak and Van Noorden Introduction to Immunocytochemistry, 2nd ed., Springer Verlag, NY (1997); and in Haugland Handbook of Fluorescent Probes and Research Chemicals, a combined handbook and catalogue Published by Molecular Probes, Inc. (1996). [108] In general, a detector that monitors a particular probe or probe combination is used to detect the detection reagent label. Typical detectors include spectrophotometers, phototubes and photodiodes, microscopes, scintillation counters, cameras, film and the like, as well as combinations thereof. Examples of suitable detectors are widely available from a variety of commercial sources known to persons of skill in the art. Commonly, an optical image of a substrate comprising bound labeling moieties is digitized for subsequent computer analysis.

[109] The amount of, for example, an RNA is measured by quantifying the amount of label fixed to the solid support by binding of the detection reagent. Typically, the presence of a modulator during incubation will increase or decrease the amount of label fixed to the solid support relative to a control incubation that does not comprise the modulator, or as compared to a baseline established for a particular reaction type. Means of detecting and quantifying labels are well known to those of skill in the art.

[110] In some embodiments, the target nucleic acid or the probe is immobilized on a solid support. Solid supports suitable for use in the assays of the invention are known to those of skill in the art. As used herein, a solid support is a matrix of material in a substantially fixed arrangement.

[Ill] A variety of automated solid-phase assay techniques are also appropriate. For instance, very large scale immobilized polymer arrays (VLSIPS™), i.e. Gene Chips or microarrays, available from Affymetrix, Inc. in Santa Clara, CA can be used to detect changes in expression levels of a plurality of genes involved in the same regulatory pathways simultaneously. See, Tijssen, supra., Fodor et al. (1991) Science, 251: 767- 777; Sheldon et al. (1993) Clinical Chemistry 39(4): 718-719, and Kozal et al. (1996) Nature Medicine 2(7): 753-759. Similarly, spotted cDNA arrays (arrays of cDNA sequences bound to nylon, glass or another solid support) can also be used to monitor expression of a plurality of genes.

[112] Typically, the array elements are organized in an ordered fashion so that each element is present at a specified location on the substrate. Because the array elements are at specified locations on the substrate, the hybridization patterns and intensities (which together create a unique expression profile) can be interpreted in terms of expression levels of particular genes and can be correlated with a particular disease or condition or treatment. See, e.g., Schena et al, Science 270: 467-470 (1995)) and (Lockhart et al., Nature Biotech. 14: 1675-1680 (1996)).

[113] Hybridization specificity can be evaluated by comparing the hybridization of specificity-control polynucleotide sequences to specificity-control polynucleotide probes that are added to a sample in a known amount. The specificity-control target polynucleotides may have one or more sequence mismatches compared with the corresponding polynucleotide sequences. In this manner, whether only complementary target polynucleotides are hybridizing to the polynucleotide sequences or whether mismatched hybrid duplexes are forming is determined.

[114] Hybridization reactions can be performed in absolute or differential hybridization formats. In the absolute hybridization format, polynucleotide probes from one sample are hybridized to the sequences in a microarray format and signals detected after hybridization complex formation correlate to polynucleotide probe levels in a sample. In the differential hybridization formal, the differential expression of a set of genes in two biological samples is analyzed. L⁷or differential hybridization, polynucleotide probes from both biological samples are prepared and labeled with different labeling moieties. A mixture of the two labeled polynucleotide probes is added to a microarray. The microarray is then examined under conditions in which the emissions from the two different labels are individually detectable. Sequences in the microarray that are hybridized to substantially equal numbers of polynucleotide probes derived from both biological samples give a distinct combined fluorescence (Shalon et al. PCT publication WO95/35505). In some embodiments, the labels are fluorescent labels with distinguishable emission spectra, such as Cy3 and Cy5 fluorophores.

[115] After hybridization, the microarray is washed to remove nonhybridized nucleic acids and complex formation between the hybridizable array elements and the polynucleotide probes is detected. Methods for detecting complex formation are well known to those skilled in the art. In some embodiments, the polynucleotide probes are labeled with a fluorescent label and measurement of levels and patterns of fluorescence indicative of complex formation is accomplished by fluorescence microscopy, such as confocal fluorescence microscopy.

[116] In a differential hybridization experiment, polynucleotide probes from two or more different biological samples are labeled with two or more different fluorescent labels with different emission wavelengths. Fluorescent signals are detected separately with different photomultipliers set to detect specific wavelengths. The relative abundances/expression levels of the polynucleotide probes in two or more samples are obtained.

[117] Typically, microarray fluorescence intensities can be normalized to take into account variations in hybridization intensities when more than one microarray is used under similar test conditions. In some embodiments, individual polynucleotide probe/target complex hybridization intensities are normalized using the intensities derived from internal normalization controls contained on each microarray.

[118] Detection of nucleic acids can also be accomplished, for example, by using a labeled detection moiety that binds specifically to duplex nucleic acids {e.g., an antibody that is specific for RNA-DNA duplexes). One example uses an antibody that recognizes DNA-RNA heteroduplexes in which the antibody is linked to an enzyme (typically by recombinant or covalent chemical bonding). The antibody is detected when the enzyme reacts with its substrate, producing a detectable product. Coutlee et al. (1989) Analytical Biochemistry 181 :153-162; Bogulavski (1986) et al. J. Immunol. Methods 89:123- 130; Prooijen-Knegt (1982) Exp. Cell Res. 141 :397-407; Rudkin (1976) Nature 265:472-473, Stollar (1970) PNAS 65:993-1000; Ballard (1982) MoI. Immunol. 19:793-799; Pisetsky and Caster (1982) MoI. Immunol. 19:645-650; Viscidi et al. (1988) J. Clin. Microbial. 41 :199- 209; and Kiney et al. (1989) J. Clin. Microbiol. 27:6-12 describe antibodies to RNA duplexes, including homo and heteroduplexes. Kits comprising antibodies specific for DNA:RNA hybrids are available, e.g., from Digene Diagnostics, Inc. (Beltsville, MD).

[119] In addition to available antibodies, one of skill in the art can easily make antibodies specific for nucleic acid duplexes using existing techniques, or modify those antibodies that are commercially or publicly available. In addition to the art referenced above, general methods for producing polyclonal and monoclonal antibodies are known to those of skill in the art (see, e.g., Paul (ed) Fundamental Immunology, Third Edition Raven Press, Ltd., NY (1993); Coligan Current Protocols in Immunology Wiley/Greene, NY (1991); Harlow and Lane Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY (1989); Stites et al. (eds.) Basic and Clinical Immunology (4th ed.) Lange Medical Publications, Los Altos, CA, and references cited therein; Goding Monoclonal Antibodies: Principles and Practice (2d ed.) Academic Press, New York, NY, (1986); and Kohler and Milstein Nature 256: 495- 497 (1975)). Other suitable techniques for antibody preparation include selection of libraries of recombinant antibodies in phage or similar vectors (see, Huse et al. Science 246:1275- 1281 (1989); and Ward et al. Nature 341 :544-546 (1989)). Specific monoclonal and polyclonal antibodies and antisera will usually bind with a K_D of at least about 0.1 μM, preferably at least about 0.01 μM or better, and most typically and preferably, 0.001 μM or better.

[120] The nucleic acids used in this invention can be either positive or negative probes. Positive probes bind to their targets and the presence of duplex formation is evidence of the presence of the target. Negative probes fail to bind to the suspect target and the absence of duplex formation is evidence of the presence of the target. For example, the use of a wild type specific nucleic acid probe or PCR primers may serve as a negative probe in an assay sample where only the nucleotide sequence of interest is present. [121] The sensitivity of the hybridization assays may be enhanced through use of a nucleic acid amplification system that multiplies the target nucleic acid being detected. Examples of such systems include the polymerase chain reaction (PCR) system and the ligase chain reaction (LCR) system. Other methods recently described in the art are the nucleic acid sequence based amplification (NASBA, Cangene, Mississauga, Ontario) and Q Beta Replicase systems. These systems can be used to directly identify mutants where the PCR or LCR primers are designed to be extended c* Iigated only when a selected sequence is present. Alternatively, the selected sequences can be generally amplified using, for example, nonspecific PCR primers and the amplified target region later probed for a specific sequence indicative of a mutation. It is understood that various detection probes, including Taqman and molecular beacon probes can be used to monitor amplification reaction products, e.g., in real time.

[122] An alternative means for determining the level of expression of the nucleic acids of the present invention is in situ hybridization. In situ hybridization assays are well known and are generally described in Angerer et al, Methods Enzymol. 152:649-660 (1987). In an in situ hybridization assay, cells, preferentially human cells from the cerebellum or the hippocampus, are fixed to a solid support, typically a glass slide. If DNA is to be probed, the cells are denatured with heat or alkali. The cells are then contacted with a hybridization solution at a moderate temperature to permit annealing of specific probes that are labeled. The probes are preferably labeled with radioisotopes or fluorescent reporters.

[123] Single nucleotide polymorphism (SNP) analysis is also useful for detecting differences between alleles of the polynucleotides (e.g., genes) of the invention. SNPs linked to genes encoding polypeptides of the invention are useful, for instance, for diagnosis of diseases (e.g., diabetes) whose occurrence is linked to the gene sequences of the invention. For example, if an individual carries at least one SNP linked to a disease- associated allele of the gene sequences of the invention, the individual is likely predisposed for one or more of those diseases. If the individual is homozygous for a disease-linked SNP, the individual is particularly predisposed for occurrence of that disease (e.g., diabetes). In some embodiments, the SNP associated with the gene sequences of the invention is located within 300,000; 200,000; 100,000; 75,000; 50,000; or 10,000 base pairs from the gene sequence.

[124] Various real-time PCR methods including, e.g., Taqman or molecular beacon-based assays (e.g., U.S. Patent Nos. 5,210,015; 5,487,972; Tyagi et al, Nature Biotechnology 14:303 (1996); and PCT WO 95/13399 are useful to monitor for the presence of absence of a SNP. Additional SNP detection methods include, e.g., DNA sequencing, sequencing by hybridization, dot blotting, oligonucleotide array (DNA Chip) hybridization analysis, or are described in, e.g., U.S. Patent No. 6,177,249; Landegren et al, Genome Research, 8:769-776 (1998); Botstein et al, Am J Human Genetics 32:314-331 (1980); Meyers et al, Methods in Enzymology 155:501-527 (1987); Keen et al, Trends in Genetics 7:5 (1991); Myers et al, Science 230:1242-1246 (1985); and Kwok et al, Genomics 23:138- 144 (1994). V. DETECTION OF POLYPEPTIDES OF THE INVENTION

[125] In addition to the detection of polynucleotides of the invention and gene expression using nucleic acid hybridization technology, one can also use immunoassays to detect polypeptides of the invention. Immunoassays can be used to qualitatively or quantitatively analyze polypeptides of the invention. A general overview of the applicable technology can be found in Harlow & Lane, Antibodies: A Laboratory Manual (1988).

A. Antibodies to Target Proteins or other immunogens

[126] Methods for producing polyclonal and monoclonal antibodies that react specifically with a protein of interest or other immunogen are known to those of skill in the art {see, e.g., Coligan, supra; and Harlow and Lane, supra; Stites et ah, supra and references cited therein; Goding, supra; and Kohler and Milstein Nature, 256:495-497 (1975)). Such techniques include antibody preparation by selection of antibodies from libraries of recombinant antibodies in phage or similar vectors {see, Huse et ah, supra; and Ward et al, supra). For example, in order to produce antisera for use in an immunoassay, the protein of interest or an antigenic fragment thereof, is isolated as described herein. For example, a recombinant protein is produced in a transformed cell line. An inbred strain of mice or rabbits is immunized with the protein using a standard adjuvant, such as Freund's adjuvant, and a standard immunization protocol. Alternatively, a synthetic peptide derived from the sequences disclosed herein is conjugated to a carrier protein and used as an immunogen.

[127] Polyclonal sera are collected and titered against the immunogen in an immunoassay, for example, a solid phase immunoassay with the immunogen immobilized on a solid support. Polyclonal antisera with a titer of 10⁴ or greater are selected and tested for their crossreactivity against proteins other than the polypeptides of the invention or even other homologous proteins from other organisms, using a competitive binding immunoassay. Specific monoclonal and polyclonal antibodies and antisera will usually bind with a K_D of at least about 0.1 mM, more usually at least about 1 μM, preferably at least about 0.1 μM or better, and most preferably, 0.01 μM or better.

[128] A number of proteins of the invention comprising immunogens may be used to produce antibodies specifically or selectively reactive with the proteins of interest. Recombinant protein is the preferred immunogen for the production of monoclonal or polyclonal antibodies. Naturally occurring protein may also be used either in pure or impure form. Synthetic peptides made using the protein sequences described herein may also be used as an immunogen for the production of antibodies to the protein. Recombinant protein can be expressed in eukaryotic or prokaryotic cells and purified as generally described supra. The product is then injected into an animal capable of producing antibodies. Either monoclonal or polyclonal antibodies may be generated for subsequent use in immunoassays to measure the protein.

[129] Methods of production of polyclonal antibodies are known to those of skill in the art. In brief, an immunogen, preferably a purified protein, is mixed with an adjuvant and animals are immunized. The animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to polypeptides of the invention. When appropriately high titers of antibody to the immunogen are obtained, blood is collected from the animal and antisera are prepared. Further fractionation of the antisera to enrich for antibodies reactive to the protein can be done if desired {see, Harlow and Lane, supra).

[130] Monoclonal antibodies may be obtained using various techniques familiar to those of skill in the art. Typically, spleen cells from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell (see, Kohler and Milstein, Eur. J. Immunol. 6:511-519 (1976)). Alternative methods of immortalization include, e.g. , transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other methods well known in the art. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells may be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host. Alternatively, one may isolate DNA sequences that encode a monoclonal antibody or a binding fragment thereof by screening a DNA library from human B cells according to the general protocol outlined by Huse et al., supra.

[131] Once target immunogen-specific antibodies are available, the immunogen can be measured by a variety of immunoassay methods with qualitative and quantitative results available to the clinician. For a review of immunological and immunoassay procedures in general see, Stites, supra. Moreover, the immunoassays' of the present invention can be performed in any of several configurations, which are reviewed "xtensively in Maggio Enzyme Immunoassay, CRC Press, Boca Raton, Florida (19SO)," Tijssen, supra; and Harlow and Lane, supra. [132] Immunoassays to measure target proteins in a human sample may use a polyclonal antiserum that was raised to full-length polypeptides of the invention or a fragment thereof. This antiserum is selected to have low cross-reactivity against other proteins and any such cross-reactivity is removed by immunoabsorption prior to use in the immunoassay.

B. Immunological Binding Assays

[133] In some embodiments, a protein of interest is detected and/or quantified using any of a number of well-known immunological binding assays (see, e.g., U.S. Patents 4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review of the general immunoassays, see also Asai Methods in Cell Biology Volume 37: Antibodies in Cell Biology, Academic Press, Inc. NY (1993); Stites, supra. Immunological binding assays (or immunoassays) typically utilize a "capture agent" to specifically bind to and often immobilize the analyte (e.g., full-length polypeptides of the present invention, or antigenic subsequences thereof). The capture agent is a moiety that specifically binds to the analyte. The antibody may be produced by any of a number of means well known to those of skill in the art and as described above.

[134] Immunoassays also often utilize a labeling agent to bind specifically to and label the binding complex formed by the capture agent and the analyte. The labeling agent may itself be one of the moieties comprising the antibody/analyte complex.

Alternatively, the labeling agent may be a third moiety, such as another antibody, that specifically binds to the antibody/protein complex.

[135] In a preferred embodiment, the labeling agent is a second antibody bearing a label. Alternatively, the second antibody may lack a label, but it may, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived. The second antibody can be modified with a detectable moiety, such as biotin, to which a third labeled molecule can specifically bind, such as enzyme-labeled streptavidin.

[136] Other proteins capable of specifically binding immunoglobulin constant regions, such as protein A or protein G, can also be used as the label agents. These proteins are normal constituents of the cell walls of streptococcal bacteria. They exhibit a strong non-immunogenic reactivity with immunoglobulin constant regions from a variety of species {see, generally, Kronval, et al. J. Immunol, 111 :1401-1406 (1973); and Akerstrom, et al. J. Immunol, 135:2589-2542 (1985)).

[137] Throughout the assays, incubation and/or washing steps may be required after each combination of reagents. Incubation steps can vary from about 5 seconds to several hours, preferably from about 5 minutes to about 24 hours. The incubation time will depend upon the assay format, analyte, volume of solution, concentrations, and the like. Usually, the assays will be carried out at ambient temperature, although they can be conducted over a range of temperatures, such as 10°C to 4O⁰C.

1. Non-Competitive Assay Formats [138] Immunoassays for detecting proteins or analytes of interest from tissue samples may be either competitive or noncompetitive. Noncompetitive immunoassays are assays in which the amount of captured protein or analyte is directly measured. In one preferred "sandwich" assay, for example, the capture agent {e.g., antibodies specific for the polypeptides of the invention) can be bound directly to a solid substrate where it is immobilized. These immobilized antibodies then capture the polypeptide present in the test sample. The polypeptide of the invention thus immobilized is then bound by a labeling agent, such as a second labeled antibody specific for the polypeptide. Alternatively, the second antibody may lack a label, but it may, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived. The second can be modified with a detectable moiety, such as biotin, to which a third labeled molecule can specifically bind, such as enzyme-labeled streptavidin.

2. Competitive Assay Formats

[139] In competitive assays, the amount of protein or analyte present in the sample is measured indirectly by measuring the amount of an added (exogenous) protein or analyte displaced (or competed away) from a specific capture agent (e.g., antibodies specific for a polypeptide of the invention) by the protein or analyte present in the sample. The amount of immunogen bound to the antibody is inversely proportional to the concentration of immunogen present in the sample. In a particularly preferred embodiment, the antibody is immobilized on a solid substrate. The amount of analyte may be detected by providing a labeled analyte molecule. It is understood that labels can include, e.g., radioactive labels as well as peptide or otb^.r tags that can be recognized by detection reagents such as antibodies.

[140] Immunoassays in the competitive binding format can be used for cross- reactivity determinations. For example, the protein encoded by the sequences described herein can be immobilized on a solid support. Proteins are added to the assay and compete with the binding of the antisera to the immobilized antigen. The ability of the above proteins to compete with the binding of the antisera to the immobilized protein is compared to that of the protein encoded by any of the sequences described herein. The percent cross-reactivity for the above proteins is calculated, using standard calculations. Those antisera with less than 10% cross-reactivity with each of the proteins listed above are selected and pooled. The cross-reacting antibodies are optionally removed from the pooled antisera by immunoabsorption with the considered proteins, e.g., distantly related homologs.

[141] The immunoabsorbed and pooled antisera are then used in a competitive binding immunoassay as described above to compare a second protein, thought to be perhaps a protein of the present invention, to the immunogen protein. In order to make this comparison, the two proteins are each assayed at a wide range of concentrations and the amount of each protein required to inhibit 50% of the binding of the antisera to the immobilized protein is determined. If the amount of the second protein required is less than 10 times the amount of the protein partially encoded by a sequence herein that is required, then the second protein is said to specifically bind to an antibody generated to an immunogen consisting of the target protein.

3. Other Assay Formats

[142] In some embodiments, western blot (immunoblot) analysis is used to detect and quantify the presence of a polypeptide of the invention in the sample. The technique generally comprises separating sample proteins by gel electrophoresis on the basis of molecular weight, transferring the separated proteins to a suitable solid support (such as, e.g., a nitrocellulose filter, a nylon filter, or a derivatized nylon filter) and incubating the sample with the antibodies that specifically bind the protein of interest. For example, antibodies are selected that specifically bind to the polypeptides of the invention on the solid support. These antibodies may be directly labeled or alternatively may be subsequently detected using labeled antibodies (e.g., labeled sheep anti-mouse antibodies) that specifically bind to the antibodies against the protein of interest.

[143] Other assay formats include liposome immunoassays (LIA), which use liposomes designed to bind specific molecules (e.g., antibodies) and release encapsulated reagents or markers. The released chemicals are then detected according to standard techniques (see, Monroe et al. ( ^ 9S6) Amer. Clin. Prod. Rev. 5:34-41).

4. Labels

[144] The particular label or detectable group used in the assay is not a critical aspect of the invention, as long as it does not significantly interfere with the specific binding of the antibody used in the assay. The detectable group can be any material having a detectable physical or chemical property. Such detectable labels have been well-developed in the field of immunoassays and, in general, most labels useful in such methods can be applied to the present invention. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the present invention include magnetic beads (e.g., Dynabeads™), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵1, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.

[145] The label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. As indicated above, a wide variety of labels may be used, with the choice of label depending on the sensitivity required, the ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions.

[146] Non-radioactive labels are often attached by indirect means. The molecules can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorescent compound. A variety of enzymes and fluorescent compounds can be used with the methods of the present invention and are well-known to those of skill in the art (for a review of various labeling or signal producing systems which may be used, see, e.g., U.S. Patent No. 4,391,904).

[147] Means of detecting labels are well known to those of skill in the art. Thus, for example, where the label is a radioactive label, means for detection include a scintillation counter or photographic film as in autoradiography. Where the label is a fluorescent label, it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence may be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like. Similarly, enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product. Finally simple colorimetric labels may be detected directly by observing the color associated with the label. Thus, in various dipstick assays, conjugated gold often appears pink, while various conjugated beads appear the color of the bead.

[148] Some assay formats do not require the use of labeled components. For instance, agglutination assays can be used to detect the presence of the target antibodies. In this case, antigen-coated particles are agglutinated by samples comprising the target antibodies. In this format, none of the components need to be labeled and the presence of the target antibody is detected by simple visual inspection.

VI. IDENTIFICATION OF MODULATORS OF POLYPEPTIDES OF THE INVENTION

[149] Modulators of a polypeptide of the invention, i.e. agonists or antagonists of a polypeptide's activity, or polypeptide's or polynucleotide's expression or full-length polypeptides of the invention or fragments thereof, are useful for treating a number of human diseases, including diabetes or obesity. For example, administration of modulators can be used to treat diabetic patients or prediabetic individuals to prevent progression, and therefore symptoms, associated with diabetes (including insulin resistance). Modulators of the invention can also be used to reduce obesity as well as the various diseases associated with obesity (e.g., gallbladder disease, cancer, sleep apnea, atherosclerosis, diabetes, and hypertension). In some cases, the modulators of the invention are used to regulate body physiology to reduce the chance of obesity-related diseases. For example, the modulators can be used to regulate serum lipids (total cholesterol, low-density lipoprotein (LDL), cholesterol, LDL/high density lipoprotein ratio and triglycerides).

A. Agents that Modulate Polypeptides of the Invention

[150] The agents tested as modulators of polypeptides of the invention can be any small chemical compound, or a biological entity, such as a protein, sugar, nucleic acid or lipid. Essentially any chemical compound can be used as a potential modulator or ligand in the assays of the invention, although most often compounds that can be dissolved in aqueous or organic (especially DMSO-based) solutions are used. Modulators include agents designed to reduce the level of mRNA encoding a polypeptide of the invention (e.g. antisense molecules, ribozymes, DNAzymes, small inhibitory RNAs and the like) or the level of translation from an mRNA (e.g., translation blockers such as an antisense molecules that are complementary to translation start or other sequences on an mRNA molecule). Modulators of the invention also include antibodies that specific bind to and/or inhibit or activate the polypeptides of the invention. Other modulators include the polypeptides of the invention themselves, fragments thereof, or fusion proteins comprising the polypeptides or fragments thereof (e.g., in some embodiments, comprising at least 25, 50, or 100 amino acids of the polypeptide). For polypeptides of the invention that are receptors, soluble fragments of the polypeptides (i.e., lacking a transmembrane domain) can act as modulators of polypeptide signaling activity. For polypeptides of the invention that are secreted, both full length and fragments with biological activity can act as modulators. It will be appreciated that there are many suppliers of chemical compounds, including Sigma (St. Louis, MO), Aldrich (St. Louis, MO), Sigma- Aldrich (St. Louis, MO), Fluka Chemika-Biochemica Analytika (Buchs, Switzerland) and the like.

[151] In some embodiments, high throughput screening methods involve providing a combinatorial chemical or peptide library containing a large number of potential therapeutic compounds (potential modulator compounds). Such "combinatorial chemical libraries" or "ligand libraries" are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional "lead compounds" or can themselves be used as potential or actual therapeutics.

[152] A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks" such as reagents. 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 (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks. [153] Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Patent 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493 (1991) and Houghton et al, Nature 354:84-88 (1991)). Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (e.g., PCT Publication No. WO 91/19735), encoded peptides (e.g., PCT Publication WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al, J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al, J. Amer. Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of small compound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates (Cho et al, Science 261 :1303 (1993)), and/or peptidyl phosphonates (Campbell et al, J. Org. Chem. 59:658 (1994)), nucleic acid libraries (see Ausubel, Berger and Sambrook, all supra), peptide nucleic acid libraries (see, e.g., U.S. Patent 5,539,083), antibody libraries (see, e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314 (1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al, Science, 274:1520-1522 (1996) and U.S. Patent 5,593,853), small organic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, Jan 18, page 33 (1993); isoprenoids, U.S. Patent 5,569,588; thiazolidinones and metathiazanones, U.S. Patent

5,549,974; pyrrolidines, U.S. Patents 5,525,735 and 5,519,134; morpholino compounds, U.S. Patent 5,506,337; benzodiazepines, 5,288,514, and the like).

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

B. Methods of Screening for Modulators of the Polypeptides of the Invention

[155] A number of different screening protocols can be utilized to identify agents that modulate the level of expression or activity of a polynucleotide of a polypeptide of the invention in cells, particularly mammalian cells, and especially human cells. In general terms, the screening methods involve screening a plurality of agents to identify an agent that modulates the activity of a polypeptide of the invention by, e.g., binding to the polypeptide, preventing an inhibitor or activator from binding to the polypeptide, increasing association of an inhibitor or activator with the polypeptide, or activating or inhibiting expression of the polypeptide. The assays can be designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source to assays, which are typically run in parallel (e.g., in microtiter formats on microtiter plates in robotic assays).

[156] Any cell expressing a full-length polypeptide of the invention or a fragment thereof can be used to identify modulators. In some embodiments, the cells are eukaryotic cells lines (e.g., CHO or HEK293) transformed to express a heterologous polypeptide of the invention, hi some embodiments, a cell expressing an endogenous polypeptide of the invention is used in screens. In other embodiments, modulators are screened for their ability to affect insulin responses, hi other embodiments, modulators are screened for their ability to effect body weight (as measured by BMI or waist-to-hip ratio) and secretion of a variety of obesity markers (e.g., leptin, IL-6 or TNF alpha). In other embodiments, modulators are screened for their ability to effect lipid metabolism. In other embodiments, modulators are screened for their ability to effect the secretion and activity of adipogenic factors. [157] In some embodiments, modulators of ADLICAN comprising the amino acid sequence of SEQ DD NO: 2, 4, or 6, may be identified using, e.g., modulator binding assays, expression assays or promoter-reporter assays.

[158] In some embodiments, modulators of ALDHl A3 comprising the amino acid sequence of SEQ ID NO: 8, 10, or 12, may be identified using, e.g., modulator binding assays, expression assays, promoter-reporter assays, or assays based on the retinoic acid production.

[159] In some embodiments, modulators of ALK7 comprising the amino acid sequence of SEQ ID NO: 14, 16, or 18, may be identified using, e.g., expression assays, promoter-reporter assays, modulator binding assays or kinase assays based on Smad2 phosphorylation. Kinase assays can be carried out after contacting either purified recombinant ALK7 protein, or an intact cell with the modulator. Modulators which bind to the ALK7 can be screened by a ligand binding assay method using e.g. nodal as the ligand. [160] In some embodiments, modulators of C3AR1 comprising the amino acid sequence of SEQ ID NO: 20, 22, or 24, may be identified using, e.g., the expression assays, promoter-reporter assays, binding assays, or screening methods that monitor modulator-induced fluctuation of intracellular Ca⁺⁺ concentration. Modulators which bind to the C3AR1 can be screened by a ligand binding assay method using e.g. complement anaphylatoxin C3a as the ligand.

[161] In some embodiments, modulators of CALCRL comprising the amino acid sequence of SEQ ED NO: 26, 28, or 30, may be identified using, e.g., expression assays, promoter-reporter assays, modulator binding assays, or screening methods that monitor modulator-induced fluctuation of intracellular cAMP concentration. Modulators which bind to the CALCRL can be screened by a ligand binding assay method using, e.g., adrenomedullin or calcitonin gene related peptide as ligands. [162] In some embodiments, modulators of CCL13 comprising the amino acid sequence of SEQ ID NO: 32, 33, 35, or 37, may be identified using, e.g., expression assays promoter-reporter assays, modulator binding assays, or screening methods that monitor modulator-induced fluctuation of intracellular Ca⁺* concentration. Modulators which bind to the CCLl 3 can be screened by a ligand binding assay method using, e.g., CCRl or other C-C G-protein coupled receptors known to bind to CCLl 3.

[163] In some embodiments, modulators of CCL8 comprising the amino acid sequence of SEQ ID NO: 39, 40, 42, or 44, may be identified using, e.g., expression assays, promoter-reporter assays, modulator binding assays, or screening methods that monitor modulator-induced fluctuation of intracellular Ca⁺⁺ concentration. Modulators which bind to the CCL8 can be screened by a ligand binding assay method using, e.g., CCRl or other C-C G-protein coupled receptors known to bind to CCL8.

[164] In some embodiments, modulators of CHI3L1 comprising the amino acid sequence of SEQ ID NO: 46, 47, 49, or 51, may be identified using, e.g., expression assays, promoter-reporter assays, modulator binding assays, or screening methods that monitor modulator-induced fluctuation of phosphorylation and activity of MAPK and/or AKT (see, e.g., Recklies, A.D. et al, Biochem J. 365:119-26 (2002)).

[165] Li some embodiments, modulators of CRl comprising the amino acid sequence of SEQ ID NO: 53 or 55, may be identified using, e.g., expression assays, promoter-reporter assays, or modulator binding assays. Modulators which bind to the CRl can be screened by a ligand binding assay method using, e.g., complement component C3b as ligand.

[166] In some embodiments, modulators of CSFRl comprising the amino acid sequence of SEQ ID NO: 57, 59 or 61, may be identified using, e.g., expression assays, promoter-reporter assays, modulator binding assays or activity assays. Kinase assays can be carried out after contacting either purified recombinant CSFRl protein or an intact cell with modulators. Modulators which bind to the CSFRl can be screened by a ligand binding assay method using e.g clony stimulating factor as ligand. [167] In some embodiments, modulators of CTSK comprising the amino acid sequence of SEQ ID NO: 63, 64, 66 or 68, may be identified using, e.g., expression assays, promoter-reporter assays, modulator binding assays, or enzyme assays. Enzyme assays can be carried out after contacting either purified recombinant CTSK protein, or an intact cell with a modulator using e.g. fibrinogen as a substrate. [168] hi some embodiments, modulators of CXCR4 comprising the amino acid sequence of SEQ ID NO: 70, 72 or 74, may be identified using, e.g., expression assays, promoter-reporter assays, modulator binding assays, or screening methods that monitor modulator-induced fluctuation of intracellular Ca⁺⁺ concentration or phosphorylation and activity of MAPK and/or AKT. Modulators which bind to the CXCR4 can be screened by a ligand binding assay method using e.g. CXCLl 2 as a ligand.

[169] In some embodiments, modulators of DDAH2 comprising the amino acid sequence of SEQ ID NO: 76, 78 or 80, may be identified using, e.g., expression assays, promoter-reporter assays, modulator binding assays, or activity assays. Modulators which effect DDAH2 activity can be screened by measuring the conversation of ADMA to citrulline and methylamines.

[170] In some embodiments, modulators of DERP7 comprising the amino acid sequence of SEQ ID NO: 82, 84 or 86, may be identified using, e.g., expression assays, modulator binding assays, or promoter-reporter assays.

[171] hi some embodiments, modulators of ENDOGLYXl comprising the amino acid sequence of SEQ ID NO: 88, 90 or 92, may be identified using, e.g., expression assays, modulator binding assays, promoter-reporter or activity assays based on angiogenesis (see, e.g., Christian, S. et al, J. Biol. Chem. 276: 48588-48595 (2001)). [172] In some embodiments, modulators of ETL comprising the amino acid sequence of SEQ ID NO: 94, 96 or 98, may be identified using, e.g., expression assays, promoter-reporter assays, modulator binding assays, or assays based on a G-protein coupled receptor activity.

[173] hi some embodiments, modulators of FLJ12389 comprising the amino acid sequence of SEQ ID NO: 100, 102, 104, 106 or 108, may be identified using, e.g., expression assays, promoter-reporter assays, modulator binding assays, or assays based on an AMP binding or an acetoacetate-CoA ligase activity.

[174] hi some embodiments, modulators of FZD4 comprising the amino acid sequence of SEQ ID NO: 110, 112, 114 or 116, may be identified using, e.g., expression assays, promoter-reporter assays or modulator binding assays. Modulators which bind to the FZD4 can be screened by a ligand binding assay method using e.g. norrin as a ligand.

[175] In some embodiments, modulators of GLIPRl comprising the amino acid sequence of SEQ ID NO: 118, 120 or 122, may be identified using, e.g., expression assays, promoter-reporter assays, modulator binding assays, or activity assays based on an induction of apoptosis.

[176] In some embodiments, modulators of GPRl 05 -.omprising the amino acid sequence of SEQ ID NO: 124, 126 or 128, may be identified using, e.g., expression assays, promoter-reporetr assays, modulator binding assays, or screening methods that monitor modulator-induced fluctuation of intracellular Ca⁺⁺ concentration. Modulators which bind to the GPR105 can be screened by a ligand binding assay method using, e.g., UDP-glucose as ligand.

[177] In some embodiments, modulators of GPRl 46 comprising the amino acid sequence of SEQ ID NO: 130, 132 or 134, may be identified using, e.g., expression assays, promoter-reporter assays, modulator binding assays, or assays based on a G-protein coupled receptor activity.

[178] In some embodiments, modulators of GPR30 comprising the amino acid sequence of SEQ ID NO: 136, 138 or 140, may be identified using, e.g., expression assays, promoter-reporter assays, modulator binding assays, or screening methods that monitor modulator-induced fluctuation of intracellular cAMP concentration or phosphorylation and activity of MAPK.

[179] In some embodiments, modulators of GPR65 comprising the amino acid sequence of SEQ ID NO: 142, 144 or 146, may be identified using, e.g., expression assays, promoter-reporter assays or modulator binding assays. Modulators which bind to the GPR65 can be screened by a ligand binding assay method using, e.g., psychosine as ligand.

[180] In some embodiments, modulators of HTR2B comprising the amino acid sequence of SEQ ID NO: 148, 150 or 152, may be identified using, e.g., expression assays, promoter-reporter assays or modulator binding assays. Modulators which bind to the HTR2B can be screened by a ligand binding assay method using, e.g., serotonin as ligand. Assays detecting phosphoinositide phospholipase C activity can be used.

[181] In some embodiments, modulators of ITGB2 comprising the amino acid sequence of SEQ ID NO: 154, 156 or 158, may be identified using, e.g., expression assays, promoter-reporter assays or modulator binding assays. Modulators which bind to the ITGB2 can be screened by a ligand binding assay method using, e.g., ITG alpha chain protein. [182] In some embodiments, modulators of ITIH5 comprising the amino acid sequence of SEQ ID NO: 160, 161 or 163, may be identified using, e.g., expression assays, promoter-reporter assays, or modulator binding assays.

[183] In some embodiments, modulators of LGALS 12 comprising the amino acid sequence of SEQ ID NO: 165, 167, 169, 171, 173, 175, 177 or 179, may be identified using, e.g., expression assays, promoter-reporter assays, modulator binding assays, or screening methods that monitor modulator-induced fluctuation in apoptosis.

[184] In some embodiments, modulators of NMB comprising the amino acid sequence of SEQ BD NO: 181, 182, 184 or 186, may be identified using, e.g., expression assays, promoter-reporter assays, modulator binding assays, screening methods that monitor modulator-induced fluctuation of intracellular Ca⁺⁺ concentration or binding assays. Modulators which bind to the NMB can be screened by a ligand binding assay method using e.g. NMBR.

[185] In some embodiments, modulators of NNAT comprising the amino acid sequence of SEQ ED NO: 188, 190, 192 or 194, may be identified using, e.g., expression assays, promoter-reporter assays, modulator binding assays.

[186] In some embodiments, modulators of OLFM2 comprising the amino acid sequence of SEQ ID NO: 196, 197, 199 or 201, may be identified using, e.g., expression assays, promoter-reporter assays, or modulator binding assays. [187] In some embodiments, modulators of OPN3 comprising the amino acid sequence of SEQ ID NO: 203, 205, 207, 209 or 211, may be identified using, e.g., expression assays, promoter-reporter assays, or modulator binding assays.

[188] In some embodiments, modulators of PTPRE comprising the amino acid sequence of SEQ ID NO: 213, 215, 217, 219 or 221, may be identified using, e.g., expression assays, promoter-reporter assays, modulator binding assays, or screening assays based on a receptor protein tyrosine phosphatase activity or phosphorylation and activity of MAPK.

[189] In some embodiments, modulators of RDCl comprising the amino acid sequence of SEQ ID NO: 223, 225 or 227, may be identified using, e.g., expression assays, promoter-reporter assays, or modulator binding assays.

[190] In some embodiments, modulators of SLIT2 comprising the amino acid sequence of SEQ ID NO: 229, 230, 232 or 234, may be identified using, e.g., expression assays, promoter-reporter assays or modulator binding assays. Modulators which bind to the SLIT2 can be screened by a ligand binding assay method using, e.g., roundabout receptor ROBOl.

[191] In some embodiments, modulators of TNFRSF21 comprising the amino acid sequence of SEQ DD NO: 236, 238 or 240, may be identified using, e.g., expression assays, promoter-reporter assays, modulator binding assays, or screening methods that monitor modulator-induced fluctuation in apoptosis and activation of both NF-kappaB and JNK.

[192] In some embodiments, modulators of TNFSF 13B comprising the amino acid sequence of SEQ ID NO: 242, 243, 245, 247 or 249, may be identified using, e.g., expression assays, promoter-reporter assays, modulator binding assays based on a receptor activity using e.g. TNFRSF13b, TNFRSF13c or TNFRSF17 or screening methods that monitor modulator-induced fluctuation in activation of NF-kappaB.

[193] In some embodiments, modulators of TNFSF14 comprising the amino acid sequence of SEQ ID NO: 251, 252, 254, 256, 258 or 260, may be identified using, e.g., expression assays, promoter-reporter assays, or modulator binding assays based on a receptor activity using e.g. TNFRSF 14.

[194] In some embodiments, modulators of TPSB2 comprising the amino acid sequence of SEQ ID NO: 262, 263, 265 or 267, may be identified using, e.g., expression assays, promoter-reporter assays, modulator binding assays, or screening methods that monitor modulator-induced fluctuation in serine-type peptidase activity.

[195] In some embodiments, modulators of WISP2 comprising the amino acid sequence of SEQ ID NO: 269, 270, 272 or 274, may be identified using, e.g., expression assays, promoter-reporter assays, modulator binding assays, or screening methods that monitor modulator-induced fluctuation in proliferative rate of vascular smooth muscle cells.

1. Polypeptide Binding Assays

[196] Preliminary screens can be conducted by screening for agents capable of binding to polypeptides of the invention, as at least some of the agents so identified are likely modulators of a polypeptide of the invention. Binding assays are also useful, e.g., for identifying endogenous proteins that interact with polypeptides of the invention. For example, antibodies, receptors or other molecules that bind polypeptides of the invention can be identified in binding assays.

[197] Binding assays usually involve contacting a polypeptide of the invention with one or more test agents and allowing sufficient time for the protein and test agents to form a binding complex. Any binding complexes formed can be detected using any of a number of established analytical techniques. Protein binding assays include, but are not limited to, methods that measure co-precipitation or co-migration on non-denaturing SDS- polyacrylamide gels, and co-migration on Western blots {see, e.g., Bennet, J.P. and Yamamura, H.I. (1985) "Neurotransmitter, Hormone or Drug Receptor Binding Methods," in Neurotransmitter Receptor Binding (Yamamura, H. I., et al., eds.), pp. 61-89. Other binding assays involve the use of mass spectrometry or NMR techniques to identify molecules bound to a polypeptide of the invention or displacement of labeled substrates. The polypeptides of the invention utilized in such assays can be naturally expressed, cloned or synthesized. [198] In addition, mammalian or yeast two-hybrid approaches (see, e.g., Bartel, P. L. et. al. Methods Enzymol, 254:241 (1995)) can be used to identify polypeptides or other molecules that interact or bind when expressed together in a host cell.

2. Polypeptide Activity

[199] The activity of polypeptides of the invention can be assessed using a variety of in vitro and in vivo assays to determine functional, chemical, and physical effects, e.g., measuring ligand binding {e.g., radioactive or otherwise labeled ligand binding), second messengers (e.g., cAMP, cGMP, IP₃, DAG, or Ca ), ion flux, phosphorylation levels, transcription levels, and the like. Measurement of such functional, chemical and/or physical effects may be direct (e.g., directly detecting calcium flux) or indirect (e.g., detecting changes in expression or activity of gene products that are known to be modulated by the effects such as calcium flux or others listed above). Furthermore, such assays can be used to test for inhibitors and activators of the polypeptides of the invention. Modulators can also be genetically altered versions of polypeptides of the invention.

[200] The polypeptide of the assay will be selected from a polypeptide with substantial identity to a sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 35, 37, 39, 40, 42, 44, 46, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 182, 184, 186, 188, 190, 192, 194, 196, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 230, 232, 234, 236, 238, 240, 242, 243, 245, 247, 249, 251, 252, 254, 256, 258, 260, 262, 263, 265, 267, 269, 270, 272, 274 or other conservatively modified variants thereof. Generally, the amino acid sequence identity will be at least 70%, optionally at least 85%, optionally at least 90, or optionally at least 95% to the polypeptides exemplified herein. Optionally, the polypeptide of the assays will comprise a fragment of a polypeptide of the invention, such as an extracellular domain, transmembrane domain, cytoplasmic domain, ligand binding domain,, subunit association domain, active site, and the like. Either a polypeptide of the invention or a domain thereof can be covalently linked to a heterologous protein to create a chimeric protein used in the assays described herein.

[201] Modulators of polypeptide activity are tested using either recombinant or naturally occurring polypeptides of the invention. The protein can be isolated, expressed in a cell, expressed in a membrane derived from a cell, expressed in tissue or in an animal, either recombinant or naturally occurring. For example, tissue slices, dissociated cells, e.g., from tissues expressing polypeptides of the invention, transformed cells, or membranes can be used. Modulation is tested using one of the in vitro or in vivo assays described herein.

[202] Modulator binding to polypeptides of the invention, a domain, or chimeric protein can be tested in solution, in a bilayer membrane, attached to a solid phase, in a lipid monolayer, or in vesicles. Binding of a modulator can be tested using, e.g., changes in spectroscopic characteristics {e.g., fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape), chromatographic, or solubility properties.

[203] Samples or assays that are treated with a potential modulator (e.g., a "test compound") are compared to control samples without the test compound, to examine the extent of modulation. Control samples (untreated with activators or inhibitors) are assigned a relative activity value of 100. Inhibition of the polypeptides of the invention is achieved when the activity value relative to the control is about 90%, optionally 50%, optionally 25- 0%. Activation of the polypeptides of the invention is achieved when the activity value relative to the control is 110%, optionally 150%, 200%, 300%, 400%, 500%, or 1000-2000%.

3. Expression Assays

[204] Screening for a compound that modulates the expression of a polynucleotide or a polypeptide of the invention is also provided. Screening methods generally involve conducting cell-based assays in which test compounds are contacted with one or more cells expressing a polynucleotide or a polypeptide of the invention, and then detecting an increase or decrease in expression (either transcript or translation product). Assays can be performed with any cells that express a polynucleotide or a polypeptide of the invention. [205] Expression can be detected in a number of different ways. As described infra, the expression level of a polynucleotide of the invention in a cell can be determined by probing the mRNA expressed in a cell with a probe that specifically hybridizes with a transcript (or complementary nucleic acid derived there from) of a polynucleotide of the invention. Probing can be conducted by lysing the cells and conducting Northern blots or without lysing the cells using in s/tM-hybridization techniques. Alternatively, a polypeptide of the invention can be detected using : immunological methods in which a cell lysate is probed with antibodies that specifically bind to the polypeptide.

[206] Promoter-reporter assays can be carried out using mammalian cells transfected with a reporter gene operably linked to sequences derived from the promoter regions of genes encoding the polypeptides of the invention. The increased or decreased expression of the reporter gene can be detected in the presence and absence of the modulator. Expression of reporter genes may be detected by hybridization to a complementary nucleic acid, by using an immunological reagent, by assaying for an activity of the reporter gene product, or other methods known to those in the art .

[207] The level of expression or activity of a polynucleotide or a polypeptide of the invention can be compared to a baseline value. The baseline value can be a value for a control sample or a statistical value that is representative of expression levels of a polynucleotide or a polypeptide of the invention for a control population (e.g., lean individuals as described herein) or cells (e.g., tissue culture cells not exposed to a modulator). Expression levels can also be determined for cells that do not express the polynucleotide or a polypeptide of the invention as a negative control. Such cells generally are otherwise substantially genetically the same as the test cells.

[208] A variety of different types of cells can be utilized in the reporter assays. Cells that do not endogenously express a polypeptide of the invention can be prokaryotic, but are preferably eukaryotic. The eukaryotic cells can be any of the cells typically utilized in generating cells that harbor recombinant nucleic acid constructs. Exemplary eukaryotic cells include, but are not limited to, yeast, and various higher eukaryotic cells such as the HEK293, HepG2, COS, CHO and HeLa cell lines. [209] Various controls can be conducted to ensure that an observed activity is authentic including running parallel reactions with cells that lack the reporter construct or by not contacting a cell harboring the reporter construct with test compound. Compounds can also be further validated as described below.

4. Validation

[210] Agents that are initially identified by any of the foregoing screening methods can be further tested to validate the apparent activity. Alternatively, potential modulators can be tested initially using the forgoing validation assays without preliminary screening. [211] Modulators that are selected for further study can be tested for anti¬ diabetic effects using the "classic" insulin responr^ve cell line, mouse 3T3-L1 adipocytes, muscle cells such as L6 cells and the like. Cells (e.g., adipocytes or muscle cells) are pre- incubated with the modulators and tested for acute (up to 4 hours) and chronic (overnight) effects on basal and insulin-stimulated GLUT4 translocation and glucose uptake. [212] Modulators that are selected for further study can be tested for anti- obesity effects using any adipocyte or adipogenic cell, e.g., mouse cell line 3T3-L1 adipocytes, freshly isolated rodent or human adipocytes, undifferentiated adipogenic cells and the like. Cells (e.g., adipocytes cells) are pre-incubated with the modulators and tested for acute (up to 4 hours) and chronic (overnight or longer) effects on basal and insulin-stimulated release of adipogenic factors, adipocyte cell size, leptin and TNF alpha release, and/or lipid metabolism. Undifferentiated adipogenic cells can be pre-incubated with the modulators and tested for effects on differentiation into adipocytes (including changes in differentiation markers) and/or triglyceride accumulation. [213] The response of this increase in body weight can be determined at an organismal, tissue or cellular level. For example, increased fasting blood leptin levels are indicative of obesity. Other methods of measuring obesity include, e.g., calculation of BMI, waist-to-hip ratio, total body fat, measuring the blood levels of a variety of secreted proteins which have been shown to correlate to obesity (IL-6, TNF alpha) and measuring the fasted blood levels of free fatty acids.

[214] Following such studies, validity of the modulators is tested in suitable animal models. The basic format of such methods involves administering a lead compound identified during an initial screen to an animal that serves as a model for humans and then determining if expression of activity of a polypeptide of the invention is in fact modulated. [215] The effect of the compound will be assessed in either obese animals, diabetic animals or in diet induced insulin resistant animals. The body weight loss, blood glucose and insulin levels will be determined. The animal models utilized in validation studies generally are mammals of any kind. Specific examples of suitable animals include, but are not limited to, primates, mice and rats. Monogenic models of diabetes (e.g., ob/ob and db/db mice, Zucker rats and Zucker Diabetic Fatty rats, etc.) or polygenic models of diabetes (e.g., OLETF rats, GK rats, NSY mice, and KK mice) can be useful for validating modulation of a polypeptide of the invention in a diabetic or insulin resistant animal. In addition, transgenic animals expressing human polypeptides of the invention can be used to further validate drug candidates. [216] Monogenic models of obesity (e.g., OLETF, tubby, mahogany, agouti, ob/ob and db/db mice etc) or polygenic models of obesity (e.g., high fat diet-induced obese animals, NZO mice, KK mice, Wellesley mice, GK rats, etc.)can be useful for validating modulation of a polypeptide of the invention in an obese animal. The most widely used criteria for assessing the efficacy of anti-obesity treatments are those from the FDA. The FDA defines a body weight loss of >5% as statistically significant compared to placebo. However, it will be appreciated that any detectable change in body weight following administration of a modulator of the invention can be considered a relevant result.

C. Solid Phase and Soluble High Throughput Assays

[217] In the high throughput assays of the invention, it is possible to screen up to several thousand different modulators or ligands in a single day. In particular, each well of a microtiter plate can be used to run a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator. Thus, a single standard microtiter plate can assay about 100 (e.g., 96) modulators. If 1536 well plates are used, then a single plate can easily assay from about 100 to about 1500 different compounds. It is possible to assay several different plates per day; assay screens for up to about 6,000-20,000 or more different compounds are possible using the integrated systems of the invention. In addition, micro fluidic approaches to reagent manipulation can be used.

~ [218] A molecule of interest (e.g., a polypeptide or polynucleotide of the invention, or a modulator thereof) can be bound to the solid-state component, directly or indirectly, via covalent or non-covalent linkage, e.g., via a tag. The tag can be any of a variety of components. In general, a molecule that binds the tag (a tag binder) is fixed to a solid support, and the tagged molecule of interest is attached to the solid support by interaction of the tag and the tag binder.

[219] A number of tags and tag binders can be used, based upon known molecular interactions well described in the literature. For example, where a tag has a natural binder, for example, biotin, protein A, or protein G, it can be used in conjunction with appropriate tag binders (avidin, streptavidin, neutravidin, the Fc region of an immunoglobulin, poly-His, etc.) Antibodies to molecules with natural binders such as biotin are also widely available and appropriate tag binders (see, SIGMA Immunochemicals 1998 catalogue SIGMA, St. Louis MO).

[220] Similarly, any haptenic or antigenic compound can be used in combination with an appropriate antibody to form a tag/tag binder pair. Thousands of specific antibodies are commercially available and many additional antibodies are described in the literature. For example, in one common configuration, the tag is a first antibody and the tag binder is a second antibody that recognizes the first antibody. In addition to antibody- antigen interactions, receptor-ligand interactions are also appropriate as tag and tag-binder pairs, such as agonists and antagonists of cell membrane receptors (e.g., cell receptor-ligand interactions such as transferrin, c-kit, viral receptor ligands, cytokine receptors, chemokine receptors, interleukin receptors, immunoglobulin receptors and antibodies, the cadherin family, the integrin family, the selectin family, and the like; see, e.g., Pigott & Power, The Adhesion Molecule Facts Book I (1993)). Similarly, toxins and venoms, viral epitopes, hormones (e.g., opiates, steroids, etc.), intracellular receptors (e.g., which mediate the effects of various small ligands, including steroids, thyroid hormone, retinoids and vitamin D; peptides), drugs, lectins, sugars, nucleic acids (both linear and cyclic polymer configurations), oligosaccharides, proteins, phospholipids and antibodies can all interact with various cell receptors.

[221] Synthetic polymers, such as polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, and polyacetates can also form an appropriate tag or tag binder. Many other tag/tag binder pairs are also useful in assay systems described herein, as would be apparent to one of skill upon review of this disclosure.

[222] Common linkers such as peptides, polyethers, and the like can also serve as tags, and include polypeptide sequences, such as poly-gly sequences of between about 5 and 200 amino acids. Such flexible linkers are known to those of skill in the art. For example, poly(ethelyne glycol) linkers are available from Shearwater Polymers, Inc.,

Huntsville, Alabama. These linkers optionally have amide linkages, sulfhydryl linkages, or heterofunctional linkages.

[223] Tag binders are fixed to solid substrates using any of a variety of methods currently available. Solid substrates are commonly derivatized or functionalized by exposing all or a portion of the substrate to a chemical reagent that fixes a chemical group to the surface that is reactive with a portion of the tag binder. For example, groups that are suitable for attachment to a longer chain portion would include amines, hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanes and hydroxyalkylsilanes can be used to functionalize a variety of surfaces, such as glass surfaces. The construction of such solid phase biopolymer arrays is well described in the literature (see, e.g., Merrifield, J. Am. Chem. Soc. 85:2149- 2154 (1963) (describing solid phase synthesis of, e.g., peptides); Geysen et al, J. Immun. Meth. 102:259-274 (1987) (describing synthesis of solid phase components on pins); Frank and Doring, Tetrahedron 44:60316040 (1988) (describing synthesis of various peptide sequences on cellulose disks); Fodor et al, Science, 251:767-777 (1991); Sheldon et al., Clinical Chemistry 39(4):718-719 (1993); and Kozal et al, Nature Medicine 2(7):753759 (1996) (all describing arrays of biopolymers fixed to solid substrates). Non-chemical approaches for fixing tag binders to substrates include other common methods, such as heat, cross-linking by UV radiation, and the like. [224] The invention provides in vitro assays for identifying, in a high throughput format, compounds that can modulate the expression or activity of a polypeptide of the invention. Control reactions that measure activity of a polypeptide of the invention in a cell in a reaction that does not include a potential modulator are optional, as the assays are highly uniform. Such optional control reactions are appropriate and increase the reliability of the assay. Accordingly, in some embodiments, the methods of the invention include such a control reaction. For each of the assay formats described, "no modulator" control reactions that do not include a modulator provide a background level of binding activity.

[225] In some assays it will be desirable to have positive controls. At least two types of positive controls are appropriate. First, a known activator of a polypeptide or a polynucleotide of the invention can be incubated with one sample of the assay, and the resulting increase in signal resulting from an increased expression level or activity of a polypeptide or a polynucleotide of the invention are determined according to the methods herein. Second, a known inhibitor of a polypeptide or a polynucleotide of the invention can be added, and the resulting decrease in signal for the expression or activity of a polypeptide or a polynucleotide of the invention can be similarly detected. It will be appreciated that modulators can also be combined with activators or inhibitors to find modulators that inhibit the increase or decrease that is otherwise caused by the presence of the known modulator of a polypeptide or a polynucleotide of the invention.

VII. COMPOSITIONS, KITS AND INTEGRATED SYSTEMS [226] The invention provides compositions, kits and integrated systems for practicing the assays described herein using nucleic acids or polypeptides of the invention, antibodies, etc.

[227] The invention provides assay compositions for use in solid phase assays; such compositions can include, for example, one or more nucleic acids encoding a polypeptide of the invention immobilized on a solid support, and a labeling reagent. In each case, the assay compositions can also include additional reagents that are desirable for hybridization. Modulators of expression or activity of a polypeptide of the invention can also be included in the assay compositions. [228] The invention also provides kits for carrying out the assays of the invention. The kits typically include a probe that comprises (1) an antibody that specifically binds to a polypeptide of the invention or (2) a polynucleotide sequence encoding at least a fragment of such polypeptides, and a label for detecting the presence of the probe. The kits may include at least one polynucleotide sequence encoding a polypeptide of the invention. Kits can include any of the compositions noted above, and optionally further include additional components such as instructions to practice a high-throughput method of assaying for an effect on expression of the genes encoding a polypeptide of the invention, or on activity of a polypeptide of the invention, one or more containers or compartments (e.g., to hold the probe, labels, or the like), a control modulator of the expression or activity of a polypeptide of the invention, a robotic armature for mixing kit components or the like.

[229] The invention also provides integrated systems for high-throughput screening of potential modulators for an effect on the expression or activity of a polypeptide of the invention. The systems can include a robotic armature which transfers fluid from a source to a destination, a controller which controls the robotic armature, a label detector, a data storage unit which records label detection, and an assay component such as a microtiter dish comprising a well having a reaction mixture or a substrate comprising a fixed nucleic acid or immobilization moiety.

[230] 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, MA) automated robot using a Microlab 2200 (Hamilton; Reno, NV) pipetting station can be used to transfer parallel samples to 96 well microtiter plates to set up several parallel simultaneous binding assays.

[231] Optical images viewed (and, optionally, recorded) by a camera or other recording device (e.g., a photodiode and data storage device) are optionally further processed in any of the embodiments herein, e.g., by digitizing the image and storing and analyzing the image on a computer. A variety of commercially available peripheral equipment and software is available for digitizing, storing and analyzing a digitized video or digitized optical image. [232] One conventional system carries light from the specimen field to a cooled charge-coupled- device (CCD) camera, in common use in the art. A CCD camera includes an array of picture elements (pixels). The light from the specimen is imaged on the CCD. Particular pixels corresponding to regions of the specimen (e.g., individual hybridization sites on an array of biological polymers) are sampled to obtain light intensity readings for each position. Multiple pixels are processed in parallel to increase speed. The apparatus and methods of the invention are easily used for viewing any sample, e.g., by fluorescent or dark field microscopic techniques.

VIII. ADMINISTRATION AND PHARMACEUTICAL COMPOSITIONS [233] Modulators of the polypeptides of the invention (e.g., antagonists or agonists including polypeptides of the invention, fragments thereof, or fusions comprising the polypeptides or fragments which have antagonist activity or an additive effect on overall polypeptide activity) can be administered directly to the mammalian subject (typically in need thereof due to a pre-diabetic, diabetic or obese condition) for modulation of activity of a polypeptide of the invention in vivo. Administration is by any of the routes normally used for introducing a modulator compound into ultimate contact with the tissue to be treated and is well known to those of skill in the art. Although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route. [234] The pharmaceutical compositions of the invention may comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there are a wide variety of suitable formulations of pharmaceutical compositions of the present invention {see, e.g., Remington 's Pharmaceutical Sciences, 17^th ed. 1985)).

[235] The modulators (e.g., agonists or antagonists) of the expression or activity of a polypeptide of the invention, alone or in combination with other suitable components, can be prepared for injection or for use in a pump device. Pump devices (also known as "insulin pumps") are commonly used to administer insulin to patients and therefore can be easily adapted to include compositions of the present invention. Manufacturers of insulin pumps include Animas, Disetronic and MiniMed.

[236] The modulators (e.g., agonists or antagonists) of the expression or activity of a polypeptide of the invention, alone or in combination with other suitable components, can be made into aerosol formulations {i.e., they can be "nebulized") to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.

[237] Formulations suitable for administration include aqueous and non¬ aqueous solutions, isotonic sterile solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. In the practice of this invention, compositions can be administered, for example, orally, nasally, topically, intravenously, intraperitoneally, or intrathecally. The formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials. Solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. The modulators can also be administered as part of a prepared food or drug.

[238] The dose administered to a patient, in the context of the present invention should be sufficient to induce a beneficial response in the subject over time. The optimal dose level for any patient will depend on a variety of factors including the efficacy of the specific modulator employed, the age, body weight, physical activity, and diet of the patient, on a possible combination with other drugs, and on the severity of the case of diabetes. It is recommended that the daily dosage of the modulator be determined for each individual patient by those skilled in the art in a similar way as for known insulin compositions. The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular compound or vector in a particular subject.

[239] In determining the effective amount of the modulator to be administered a physician may evaluate circulating plasma levels of the modulator, modulator toxicity, and the production of anti-modulator antibodies, hi general, the dose equivalent of a modulator is from about 1 ng/kg to 10 mg/kg for a typical subject.

[240] For administration, modulators of the present invention can be administered at a rate determined by the LD-50 of the modulator, and the side-effects of the modulator at various concentrations, as applied to the mass and overall health of the subject. Administration can be accomplished via single or divided doses.

[241] The compounds of the present invention can also be used effectively in combination with one or more additional active agents depending on the desired target therapy (see, e.g., Turner, N. et al. Prog. Drug Res. (1998) 51: 33-94; Haffher, S. Diabetes Care (1998) 21 : 160-178; and DeFronzo, R. et al. (eds.), Diabetes Reviews (1997) Vol. 5 No. 4). A number of studies have investigated the benefits of combination therapies with oral agents (see, e.g., Mahler, R., J. Clin. Endocrinol. Metab. (1999) 84: 1165-71; United Kingdom Prospective Diabetes Study Group: UKPDS 28, Diabetes Care (1998) 21 : 87-92; Bardin, C. W.,(ed.), Current Therapy In Endocrinology And Metabolism, 6th Edition (Mosby - Year Book, Inc., St. Louis, MO 1997); Chiasson, J. et al., Ann. Intern. Med. (1994) 121 : 928-935; Coniff, R. et al., Clin. Ther. (1997) 19: 16-26; Coniff, R. et al., Am. J. Med. (1995) 98: 443-451; and Iwamoto, Y. et al., Diabet. Med. (1996) 13 365-370; Kwiterovich, P. Am. J. Cardiol (1998) 82(12A): 3U-17U). These studies indicate that modulation of diabetes, among other diseases, can be further improved by the addition of a second agent to the therapeutic regimen. Combination therapy includes administration of a single pharmaceutical dosage formulation that contains a modulator of the invention and one or more additional active agents, as well as administration of a modulator and each active agent in its own separate pharmaceutical dosage formulation. For example, a modulator and a thiazolidinedione can be administered to the human subject together in a single oral dosage composition, such as a tablet or capsule, or each agent can be administered in separate oral dosage formulations. Where separate dosage formulations are used, a modulator and one or more additional active agents can be administered at essentially the same time (i.e., concurrently), or at separately staggered times (i.e., sequentially). Combination therapy is understood to include all these regimens.

[242] One example of combination therapy can be seen in treating pre- diabetic individuals (e.g., to prevent progression into type 2 diabetes) or diabetic individuals (or treating diabetes and its related symptoms, complications, and disorders), wherein the modulators can be effectively used in combination with, for example, sulfonylureas (such as chlorpropamide, tolbutamide, acetohexamide, tolazamide, glyburide, gliclazide, glynase, glimepiride, and glipizide); biguanides (such as metformin); a PPAR beta delta agonist; a ligand or agonist of PPAR gamma such as thiazolidinediones (such as ciglitazone, pioglitazone {see, e.g., U.S. Patent No. 6,218,409), troglitazone, and rosiglitazone (see, e.g., U.S. Patent No. 5,859,037)); PPAR alpha agonists such as clofϊbrate, gemfibrozil, fenofibrate, ciprofibrate, and bezafibrate; dehydroepiandrosterone (also referred to as DHEA or its conjugated sulphate ester, DHEA-SO4); antiglucocorticoids; TNFα inhibitors; α-glucosidase inhibitors (such as acarbose, miglitol, and voglibose); amylin and amylin derivatives (such as pramlintide, (see, also, U.S. Patent Nos. 5,902,726; 5,124,314; 5,175,145 and 6,143,718.)); insulin secretogogues (such as repaglinide, gliquidone, and nateglinide (see, also, U.S. Patent Nos. 6,251,856; 6,251,865; 6,221,633; 6,174,856)), and insulin.

[243] The modulators of the invention can also be combined with anti- obesity drugs (e.g., Xenical (Orlistat), Merida (Sibutramine) or Adipex-P (Phentermine)) or appetite-suppressing drugs. IX. GENE THERAPY

[244] Conventional viral and non- viral based gene transfer methods can be used to introduce nucleic acids encoding engineered amino acid sequences comprising the polypeptides of the invention in mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding amino acid sequences comprising polypeptides of the invention to cells in vitro. In some embodiments, the nucleic acids encoding amino acid sequences comprising polypeptides of the invention are administered for in vivo or ex vivo gene therapy uses. Non- viral vector delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. For a review of gene therapy procedures, see Anderson, Science 256:808-813 (1992); Nabel & Feigner, TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10):l 149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin 51(l):31-44 (1995); Haddada et al., in Current Topics in Microbiology and Immunology Doerfler and Bόhm (eds) (1995); and Yu et al, Gene Therapy 1:13-26 (1994).

[245] Methods of non- viral delivery of nucleic acids encoding engineered polypeptides of the invention include lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., US 5,049,386, US 4,946,787; and US 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91/17424, WO 91/16024. Delivery can be to cells {ex vivo administration) or target tissues (in vivo administration).

[246] The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art {see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al, Cancer Gene Ther. 2:291-297 (1995); Behr et al, Bioconjugate Chem. 5:382-389 (1994); Remy et al, Bioconjugate Chem. 5:647- 654 (1994); Gao et al, Gene Therapy 2:710-722 (1995); Ahmad et al, Cancer Res. 52:4817- 4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787). [247] The use of RNA or DNA viral based systems for the delivery of nucleic acids encoding engineered polypeptides of the invention take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro and the modified cells are administered to patients (ex vivo). Conventional viral based systems for the delivery of polypeptides of the invention could include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer. Viral vectors are currently the most efficient and versatile method of gene transfer in target cells and tissues. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.

[248] The tropism of a retrovirus can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system would therefore depend on the target tissue. Retroviral vectors are comprised of czs-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum ex¬ acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), human immuno deficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al, J. Virol. 66:2731-2739 (1992); Johann et al, J. Virol. 66:1635-1640 (1992); Sommerfelt et al, Virol 176:58-59 (1990); Wilson et al, J. Virol. 63:2374-2378 (1989); Miller et al, J. Virol. 65:2220-2224 (1991); PCT/US94/05700).

[249] In applications where transient expression of the polypeptides of the invention is preferred, adenoviral based systems are typically used. Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system Adeno-associated virus ("AAV") vectors are also used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al, Virology 160:38-47 (1987); U.S. Patent No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994)). Construction of recombinant AAV vectors are described in a number of publications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al, MoI. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al, J. Virol. 63:03822-3828 (1989).

[250] pLASN and MFG-S are examples are retroviral vectors that have been used in clinical trials (Dunbar et al, Blood 85:3048-305 (1995); Kohn et al, Nat. Med. 1:1017-102 (1995); Malech et a/., /WΛS 94:22 12133-12138 (1997)). P A317/pLASN was the first therapeutic vector used in a gene therapy trial. (Blaese et al, Science 270:475-480 (1995)). Transduction efficiencies of 50% or greater have been observed for MFG-S packaged vectors. (Ellem et al, Immunol Immunother. 44(1): 10-20 (1997); Dranoff et al, Hum. Gene Ther. 1:111-2 (1997).

[251] Recombinant adeno-associated virus vectors (rAAV) are a promising alternative gene delivery systems based on the defective and nonpathogenic parvovirus adeno-associated type 2 virus. All vectors are derived from a plasmid that retains only the AAV 145 bp inverted terminal repeats flanking the transgene expression cassette. Efficient gene transfer and stable transgene delivery due to integration into the genomes of the transduced cell are key features for this vector system. (Wagner et al, Lancet 351 :9117 1702- 3 (1998), Kearns et al, Gene Ther. 9:748-55 (1996)). [252] Replication-deficient recombinant adenoviral vectors (Ad) can be engineered such that a transgene replaces the Ad EIa, EIb, and E3 genes; subsequently the replication defector vector is propagated in human 293 cells that supply deleted gene function in trans. Ad vectors can transduce multiply types of tissues in vivo, including nondividing, differentiated cells such as those found in the liver, kidney and muscle system tissues. Conventional Ad vectors have a large carrying capacity. An example of the use of an Ad vector in a clinical trial involved polynucleotide therapy for antitumor immunization with intramuscular injection (Sterman et al, Hum. Gene Ther. 7:1083-9 (1998)). Additional examples of the use of adenovirus vectors for gene transfer in clinical trials include Rosenecker et al, Infection 24:1 5-10 (1996); Sterman et al, Hum. Gene Ther. 9:7 1083- 1089 (1998); Welsh et al, Hum. Gene Ther. 2:205-18 (1995); Alvarez et al, Hum. Gene Ther. 5:597-613 (1997); Topf et al, Gene Ther. 5:507-513 (1998); Sterman et al, Hw:: Gene Ther. 7:1083-1089 (1998).

[253] Packaging cells are used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and ψ2 cells or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by producer cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the protein to be expressed. The missing viral functions are supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome. Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line is also infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV. [254] In many gene therapy applications, it is desirable that the gene therapy vector be delivered with a high degree of specificity to a particular tissue type. A viral vector is typically modified to have specificity for a given cell type by expressing a ligand as a fusion protein with a viral coat protein on the viruses outer surface. The ligand is chosen to have affinity for a receptor known to be present on the cell type of interest. For example, Han et al, PNAS 92:9747-9751 (1995), reported that Moloney murine leukemia virus can be modified to express human heregulin fused to gp70, and the recombinant virus infects certain human breast cancer cells expressing human epidermal growth factor receptor. This principle can be extended to other pairs of virus expressing a ligand fusion protein and target cell expressing a receptor. For example, filamentous phage can be engineered to display antibody fragments (e.g., FAB or Fv) having specific binding affinity for virtually any chosen cellular receptor. Although the above description applies primarily to viral vectors, the same principles can be applied to nonviral vectors. Such vectors can be engineered to contain specific uptake sequences thought to favor uptake by specific target cells.

[255] Gene therapy vectors can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramur-oular, subdermal, or intracranial infusion) or topical application, as described bekv". ^" Alternatively, vectors can be delivered to cells ex vivo, such as cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, tissue biopsy) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient, usually after selection for cells which have incorporated the vector.

[256] Ex vivo cell transfection for diagnostics, research, or for gene therapy (e.g., via re-infusion of the transfected cells into the host organism) is well known to those of skill in the art. In some embodiments, cells are isolated from the subject organism, transfected with a nucleic acid (gene or cDNA) encoding a polypeptides of the invention, and re-infused back into the subject organism (e.g., patient). Various cell types suitable for ex vivo transfection are well known to those of skill in the art {see, e.g., Freshney et ah, Culture of Animal Cells, A Manual of Basic Technique (3rd ed. 1994)) and the references cited therein for a discussion of how to isolate and culture cells from patients).

[257] In one embodiment, stem cells are used in ex vivo procedures for cell transfection and gene therapy. The advantage to using stem cells is that they can be differentiated into other cell types in vitro, or can be introduced into a mammal (such as the donor of the cells) where they will engraft in the bone marrow. Methods for differentiating CD34+ cells in vitro into clinically important immune cell types using cytokines such a GM- CSF, IFN-γand TNF-αare known {see Inaba et ah, J. Exp. Med. 176:1693-1702 (1992)).

[258] Stem cells are isolated for transduction and differentiation using known methods. For example, stem cells are isolated from bone marrow cells by panning the bone marrow cells with antibodies which bind unwanted cells, such as CD4+ and CD8+ (T cells), CD45+ (panB cells), GR-I (granulocytes), and lad (differentiated antigen presenting cells) {see Inaba et ah, J. Exp. Med. 176:1693-1702 (1992)).

[259] Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containing therapeutic nucleic acids can be also administered directly to the organism for transduction of cells in vivo. Alternatively, naked DNA can be administered. Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells. Suitable methods of administering such nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route. [260] Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention, as described below {see, e.g., Remington 's Pharmaceutical Sciences, 17th ed., 1989). X. DIAGNOSIS OF OBESITY AND/OR DIABETES

[261] The present invention also provides methods of diagnosing diabetes or obesity, or a predisposition of at least some of the pathologies of diabetes and/or obesity. Diagnosis can involve determination of a genotype of an individual (e.g., with SNPs) and comparison of the genotype with alleles known to have an association with the occurrence of obesity and/or diabetes. Alternatively, diagnosis also involves determining the level of a polypeptide or polynucleotide of the invention in a patient and then comparing the level to a baseline or range. Typically, the baseline value is representative of a polypeptide or polynucleotide of the invention in a healthy (e.g., lean) person. [262] As discussed above, variation of levels (e.g., low or high levels) of a polypeptide or polynucleotide of the invention compared to the baseline range indicates that the patient is either obese, at risk for becoming obese, diabetic or at risk of developing at least some of the pathologies of diabetes (e.g., pre-diabetic). The level of a polypeptide in a lean individual can be a reading from a single individual, but is typically a statistically relevant average from a group of lean individuals. The level of a polypeptide in a lean individual can be represented by a value, for example in a computer program.

[263] In some embodiments, the level of polypeptide or polynucleotide of the invention is measured by taking a blood, urine or tissue sample from a patient and measuring the amount of a polypeptide or polynucleotide of the invention in the sample using any number of detection methods, such as those discussed herein. For instance, fasting and fed blood or urine levels can be tested.

[264] In some embodiments, the baseline level and the level in a lean sample from an individual, or at least two samples from the same individual differ by at least about 5%, 10%, 20%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500%, 1000% or more. In some embodiments, the sample from the individual is greater by at least one of the above- listed percentages relative to the baseline level. In some embodiments, the sample from the individual is lower by at least one of the above- listed percentages relative to the baseline level.

[265] In some embodiments, the level of a polypeptide or polynucleotide of the invention is used to monitor the effectiveness of either anti-obese therapies such as orlistat or sibutramine, or, antidiabetic therapies such as thiazolidinediones, metformin, sulfonylureas and other standard therapies. In some embodiments the activity or expression of a polypeptide or polynucleotide of the invention will be measured prior to and after treatment of an obese patient with antiobese therapies, or, diabetic or pre-diabetic patients with antidiabetic therapies as a surrogate marker of clinical effectiveness. For example, the greater the reduction in expression or activity of a polypeptide of the invention indicates greater effectiveness.

[266] Glucose/insulin tolerance tests can also be used to detect the effect of glucose levels on levels of a polypeptide or polynucleotide of the invention. In glucose tolerance tests, the patient's ability to tolerate a standard oral glucose load is evaluated by assessing serum and urine specimens for glucose levels. Blood samples are taken before the glucose is ingested, glucose is given by mouth, and blood or urine glucose levels are tested at set intervals after glucose ingestion. Similarly, meal tolerance tests can also be used to detect the effect of insulin or food, respectively, on levels of a polypeptide or polynucleotide of the invention.

[267] Body weight or other indicators of obesity can also be used to detect the effect of modulating the levels of a polypeptide or polynucleotide of the invention. Measurement of a subject's response can be evaluated by assessing serum for altered levels of obesity-associated gene products, e.g., leptin, TNF alpha or IL-6.

[268] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[269] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

EXAMPLES

[270] The following examples are offered to illustrate, but not to limit the claimed invention.

[271] In either obese insulin-resistant or type II diabetics, peripheral tissues, especially muscle and fat, are known to have an impaired ability to respond to insulin and hence to take up glucose. This defect in glucose metabolism is usually compensated for by increased secretion of insulin from the pancreas, thereby maintaining normal glucose levels. The majority of glucose disposal occurs in the muscle. A number of obese insulin resistant patients will progress to overt diabetics with time. The molecular defects underlying this peripheral insulin resistance in both the obese and type II diabetics are not well defined. Genes in muscle or fat whose expression is altered in either or both the obese or type II diabetics when compared to lean individuals can be causative genes for either obesity, insulin resistance and/or diabetes and are able to predict the transition to diabetes. Modulators of such genes have the ability to reverse obesity, insulin resistance and restore normal insulin sensitivity, thereby improving whole body glucose homeostasis including for example insulin secretion. Modulators of such genes also have the ability to be used to pre-empt the transition from obesity-induced insulin resistance to diabetes. Modulators of such genes also have the ability to be used to reverse metabolic obesity-related diseases such as cardiovascular disease, hypertension or obesity-related cancer. [272] The molecular mechanism by which thiazolidinediones (TZDs) cause an increase in peripheral insulin sensitivity was studied. Genes in muscle or fat whose expression is altered by TZDs may lie on a pathway leading from TZD treatment to increased insulin sensitivity. Modulators of such genes can elicit the same effect as TZD treatment. Moreover, such modulators can lack some of the side effects of TZD. Gene expression profiling in cultures of primary human adipocytes treated with either pioglitazone or rosiglitazone were used to identify genes important for TZD action and therefore treatment of obesity, diabetes and/or insulin resistance.

[273] Gene expression profiling was performed on tissue samples (subcutaneous adipose samples) obtained from lean, obese and diabetic individuals. Two studies were performed, hi the first study, samples were isolated from all individuals after a 5 hour hyperinsulinemic euglycemic clamp.

[274] In the second study, subcutaneous adipose samples were obtained from lean (BMK 25) and obese (BMI>30) individuals after an overnight fast.

[275] In a third study samples were obtained from human subcutaneous and omental adipose tissues. Genes expressed only, or enriched, in fat can lie on pathways involved in insulin sensitivity, appetite suppression or lipid metabolism in the adipose itself or other peripheral tissues (e.g., muscle, liver, brain). For all tissue samples mRNA was isolated from these adipose samples and converted to cRNA by standard procedures. The gene expression profile for each individual was determined by hybridization of cRNA to commercial and custom synthesized Affymetrix chips.

[276] Gene expression profile di^fferences were calculated as follows. The expression level of a particular gene is indicated by its 'signal intensity'. The raw data was analyzed by a statistical test to remove Outliers'. The mean 'signal intensity' was then calculated from the signal intensities for all individuals in a particular treatment group. Genes were determined to be changed in the first two studies by calculating the Students t test statistic between the two conditions and selecting those with t less than or equal to 0.05. The fold change was determined as the ratio of mean signal intensity in condition 2 to the mean signal intensity in condition 1. In the first study three comparisons was undertaken: diabetics (condition 1) versus leans (condition 2), obese (condition 1) versus lean (condition 2) and diabetics (condition 1) versus obese (condition 2). The second study comparison is lean (condition 1) versus obese (condition 2). The third comparison is identification of fat specific or fat enriched genes when comparing the expression profile of human subcutaneous and omental adipose tissues to at last 12 other human adult tissues. Genes were determined to be meeting the criteria cut-off when the mean signal intensity of the human adipose samples was 3 fold greater than the mean signal intensity of all the other human adult tissues profiled or called present only in the adipose samples and absent in all others by the Affymetrix software program.

ADLICAN [277] Probe set 209596 detects ADLICAN nucleic acid sequences.

Expression of ADLICAN transcripts was increased in diabetic compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of diabetics in comparison to lean patients.

[278] ADLICAN was also evaluated using real-time PCR. The results further show that ADLICAN is significantly over-expressed in subcutaneous adipose from diabetic individuals when compared to subcutaneous adipose from lean individuals.

"Fold Change" indicates the fold expression calculated as the ratio of the mean diabetic expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR. [279] Probe set 209596 detects ADLICAN nucleic acid sequences.

Expression of ADLICAN transcripts was increased in obese compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of obese in comparison to lean patients.

[280] ADLICAN was also evaluated using real-time PCR. The results further show that ADLICAN is significantly over-expressed in subcutaneous adipose from obese individuals when compared to subcutaneous adipose from lean individuals.

"Fold Change" indicates the fold expression calculated as the ratio of the mean obese expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.

[281] Probe set 209596 detects ADLICAN nucleic acid sequences. Expression of ADLICAN transcripts was decreased in pio compared to vehicle treated cultures of primary human adipocytes in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Pre-Pio" and "Post-Pio" indicates sample was taken before or after 24 hours of pioglitazone treatment; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of primary human adipocytes post-pio in comparison to pre-pio samples.

[282] Probe set 209596 detects ADLICAN nucleic acid sequences. Expression of ADLICAN transcripts was increased in adipose tissues compared to all other human adult tissues in the gene profiling experiment.

"Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of tissue samples; "Fold Change" indicates fold change of adipose tissues in comparison to all other human adult tissues profiled.

[283] ADLICAN contains the following protein domains (designated with reference to SEQ ID NO:2): Atrophin-1 family (PF03154) at amino acids 1405 to 2232; Leucine rich repeat N-terminal domain (PF01462) at amino acids 26 to 54; Geminivirus AL2 protein (PF01440) at amino acids 1317 to 1428; Leucine Rich Repeat (PF00560) at amino acids 80 to 103, 128 to 151; and Immunoglobulin domain (PF00047) at amino acids 494 to 557, 592 to 653, 1868 to 1930, 1965 to 2027, 2062 to 2124, 2161 to 2223, 2258 to 2326, 2361 to 2420, 2459 to 2520, 2557 to 2618, 2652 to 2713, 2748 to 2812. ADLICAN is a protein which contains many domains which mediate protein-protein binding.

ALDH1A3

[284] Probe set 203180 detects ALDHl A3 nucleic acid sequences. Expression of ALDHl A3 transcripts was increased in diabetic compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of diabetics in comparison to lean patients.

[285] ALDHl A3 was also evaluated using real-time PCR. The results further show that ALDH1A3 is significantly over-expressed in subcutaneous adipose from diabetic individuals when compared to subcutaneous adipose from lean individuals.

"Fold Change" indicates the fold expression calculated as the ratio of the mean diabetic expression/ mean lean expression. Numbers in parentheses indicates the number υf patient samples analyzed by real-time PCR. [286] Probe set 203180 detects ALDHl A3 nucleic acid sequences.

Expression of ALDHl A3 transcripts was increased in obese compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of obese in comparison to lean patients.

[287] ALDHl A3 was also evaluated using real-time PCR. The results further show that ALDHl A3 is significantly over-expressed in subcutaneous adipose from obese individuals when compared to subcutaneous adipose from lean individuals.

"Fold Change" indicates the fold expression calculated as the ratio of the mean obese expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.

[288] Probe set 203180 detects ALDHl A3 nucleic acid sequences.

Expression of ALDHl A3 transcripts was increased in obese compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of obese in comparison to lean patients.

[289] ALDHl A3 was over-expressed in L6 myotubes and the effect on basal and insulin stimulated glucose transport was determined

Glucose Transport Analysis in L6 Mvotubes

100 I 1.29 I 0.001 I 1.14 0.05.7 1.03 0.634 1.15±0.08

Legend "Con" indicates control L6 myotubes that do not express hALDHlA3. "FC" indicates the fold change defined as the following ratio; glucose transport in hALDHl A3- expressing cells/glucose transport in non-hALDHl A3 -expressing cells, h" is human, "n" is the number of experiments. SEM is the standard error of the mean.

[290] The results show that increased levels of ALDHl A3 in a cell such as a muscle cell leads to a corresponding increase in glucose uptake. This indicates that increasing the levels or activity of ALDHl A3 in tissues of insulin resistant patients or diabetic patients will increase the ability of such tissues to take up glucose and hence, will provide an effective treatment for insulin resistance and diabetes.

[291] ALDHl A3 contains the following protein domains (designated with reference to SEQ ID NO:8): Aldehyde dehydrogenase family (PF00171) at amino acids 40 to 507. ALDHl A3 is a retinaldehyde dehydrogenase that catalyzes the oxidation of all-trans- retinaldehyde to retinoic acid and may have a role in cell differentiation and proliferation (Grun, F., et alJ Biol Chem. 275: 41210-8 (2000); Rexer, B. N., et al Cancer Res. 61 : 7065- 7070 (2001).

[292] It has been established that the mRNA for ALDHl A3 can be induced in hepatocytes by agents such as omeprazole (Nishimura et al Yakugaku Zasshi. 122 :339-61 (2002)). Thus, an exemplary method in which ALDHl A3 activators can be identified comprises treating hepatocytes with candidate compounds and measuring increases in ALDH1A3 mRNA.

ALK7

[293] Probe set MBXHUMF AT04495 detects ALK7 nucleic acid sequences. Expression of ALK7 transcripts was decreased in diabetic compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of diabetics in comparison to lean patients. [294] ALK7 was also evaluated using real-time PCR. The results further show that ALK7 is significantly under-expressed in subcutaneous adipose from diabetic individuals when compared to subcutaneous adipose from lean individuals.

"Fold Change" indicates the fold expression calculated as the ratio of the mean diabetic expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.

[295] Probe set MBXHUMF AT04495 detects ALK7 nucleic acid sequences. Expression of ALK7 transcripts was decreased in patients with insulin resistance compared to normal patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Corr Co-efficient" indicates the relationship between glucose disposal rate (Rd) and signal intensities. A positive co-efficient indicates down regulation whereas a negative co-efficient indicates up regulation of the gene with increasing insulin resistance; "n" indicates number of patient samples.

[296] ALK7 was also evaluated using real-time PCR. The results further show that ALK7 is significantly decreased in patients with insulin resistance compared to normal patients.

"Corr Co-efficient" indicates the relationship between Rd and signal intensities. A positive co-efficient indicates down regulation whereas a negative co-efficient indicates up regulation of the gene with increasing insulin resistance. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.

[297] Probe set MBXHUMF AT04495 detects ALK7 nucleic acid sequences. Expression of ALK7 transcripts was decreased in obese compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of obese in comparison to lean patients. [298] Probe set MBXHUMF AT04495 detects ALK7 nucleic acid sequences. Expression of ALK7 transcripts was increased in adipose tissues compared to all other human adult tissues in the gene profiling experiment.

"Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of tissue samples; "Fold Change" indicates fold change of adipose tissues in comparison to all other human adult tissues profiled.

[299] ALK7 was over-expressed in L6 myotubes and the effect on basal and insulin stimulated glucose transport was determined

Glucose Transport Analysis in L6 Mvotubes

Legend "Con" indicates control L6 myotubes that do not express hALK7. "FC" indicates the fold change defined as the following ratio; glucose transport in hALK7-expressing L6 myotubes/glucose transport in non-ALK7-expressing L6 myotubes. h" is human, "n" is the number of experiments. SD is the standard deviation.

[300] The results show that increased levels of ALK7 in a cell such as a muscle cell leads to a corresponding decrease in glucose uptake. This indicates that decreasing the levels or activity of ALK7 in tissues of insulin resistant patients or diabetic patients will increase the ability of such tissues to take up glucose and hence, will provide an effective treatment for insulin resistance and diabetes.

[301] ALK7 contains the following protein domains (designated with reference to SEQ ID NO: 14): Signal peptide at amino acids 1 to 25; Activin types I and II receptor domain (PFO 1064) at amino acids 15 to 100; Protein kinase domain (PF00069) at amino acids 195 to 482; u-PAR/Ly-6 domain (PF00021) at amino acids 30 to 94; and 1 transmembrane domain (TMHMM2.0) at amino acids 114 to 136. ALK7 is a transmembrane receptor protein serine-threonine kinase for the transforming growth factor-beta (TGF-beta) superfamily related growth factors and signals through SMAD2 (Bondestam J. et al,

11 Cytogenet. Cell Genet., 95: 157-162 (2001). Nodal was identified as the ligand for ALK7. ALK7 may play a role in proliferation and apoptosis (Munir, S., et al, J Biol Chem. May 18 Epub (2004); Jornvall, H., et al., J Biol Chem. 276: 5140-6. (2001)).

[302] ALK7 is a type I serine/threonine kinase receptor of the transforming growth factor (TGF)-beta family. Signalling from the ALK7 receptor involves phosphorylation of SMAD2 and SMAD3 (see, e.g., Kim J, et al., J. Biol. Chem 279: 28458- 28465 (2004)) Inhibitors of ALK7 kinase activity can thus be identified, for example, by using an in vitro phosphorylation assay containing recombinant ALK7 incubated with recombinant SMAD2 or SMAD3 and radio-labelled ATP

[303] Inhibitors of ALK7 kinase activity such as SB 505124 (2-(5-benzo[l, 3] dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride (e.g., Byfield et al., MoL Pharmacol 65 744-752 (2004) and SB 431542 (4-(5-benzol[l,3] dioxol-5-yl-4- pyridin-2-yl-lH-imidazol-2-yl)-benzamide (Inman et al., MoI Pharmacol 62 65-74 (2002)) are known. Such inhibitors as well as other ALK7 kinase inhibitors, e.g., identified using screening assays as described herein can be used to treat insulin resistance and diabetes.

C3AR1

[304] Probe set 209906 detects C3AR1 nucleic acid sequences. Expression of C3AR1 transcripts was increased in diabetic compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of diabetics in comparison to lean patients.

[305] C3 ARl was also evaluated using real-time PCR. The results further show that C3AR1 is significantly over-expressed in subcutaneous adipose from diabetic individuals when compared to subcutaneous adipose from lean individuals.

"Fold Change" indicates the fold expression calculated as the ratio of the mean diabetic expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR. [306] Probe set 209906 detects C3AR1 nucleic acid sequences. Expression of C3AR1 transcripts was increased in obese compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of obese in comparison to lean patients.

[307] The cellular level of C3AR1 was reduced in 3T3-L1 adipocytes using siRNA directed against C3 ARl and the effect on basal and insulin stimulated glucose transport was determined

C3AR1 mRNA Level in 3T3-L1 Adipocytes Transfected with siRNA Oli onucleotides

Legend: "siRNA" indicates Dharmacon Smartpool siRNA oligonucleotides directed against murine C3AR1. "Scr" indicates the Dharmacon Scramble siRNA Control. "FC" indicates the fold change defined as the following ratio; Level of C3AR1 mRNA in C3AR1 siRNA transfected 3T3-L1 adipocytes/Level of C3AR1 mRNA in Scramble siRNA transfected 3T3- Ll adipocytes, "n" is the number of experiments. SEM is the standard error of the mean.

Glucose Transport in 3T3-L1 Adipocytes Transfected with siRNA Oligonucleotides

Legend: "siRNA" indicates Dharmacon Smartpool siRNA oligonucleotides directed against murine C3AR1. "Scr" indicates Dharmacon Scramble siRNA Control oligonucleotides. "FC" indicates the fold change defined as the following ratio; glucose transport in C3AR1 siRNA transfected 3T3-L1 adipocytes/glucose transport in Scramble siRNA transfected 3T3-L1 adipocytes, "n" is the number of experiments. SEM is the standard error of the mean.

[308] These results show that decreasing the levels of C3AR1 in a cell such as an adipocyte leads to a corresponding decrease in glucose uptake. This indicates that increasing the levels or activity of C3AR1 in tissues of insulin resistant patients or diabetic patients will increase the ability of such tissues to take up glucose and hence, will provide an effective treatment for insulin resistance and diabetes.

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

[310] C3AR1 is a G protein coupled receptor, activation of which results in the release of intracellular Ca2+ in HMC-I cells {see, e.g., Legler, D.F. et al., Eur.J.Immunol 26: 753-758 (1996)). Agonists of the C3AR1 can therefore be identified, for example, using assays that measure changes in intracellular calcium. An exemplary assay is a cell based assay in which cells over-expressing C3AR1, such as HMC-I cells, are treated with compounds and an increase in intracellular Ca2+ is measured using Ca2+ sensitive dyes such as Calcium 3.

CALCIlL

[311] Probe set 210815 detects CALCRL nucleic acid sequences. Expression of CALCRL transcripts was decreased in diabetic compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of diabetics in comparison to lean patients. [312] CALCRL was also evaluated using real-time PCR. The results further show that CALCRL is significantly under-expressed in subcutaneous adipose from diabetic individuals when compared to subcutaneous adipose from lean individuals.

"Fold Change" indicates the fold expression calculated as the ratio of the mean diabetic expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.

[313] CALCRL was over-expressed in 3T3-L1 adipocytes and the effect on basal and insulin stimulated glucose transport was determined. Glucose Transport Analysis in 3T3-L1 Adipocytes:

Legend: "Con" indicates control 3T3-L1 adipocytes that do not express hCALCRL. "FC" indicates the fold change defined as the following ratio; glucose transport in hC ALCRL- expressing 3T3-L1 adipocytes /glucose transport in non-hCALCRL expressing 3T3-L1 adipocytes, h" is human, "n" is the number of experiments. SEM is the standard error of the mean.

[314] The results show that increasing the levels of CALCRL in a cell such as an adipocyte leads to a corresponding increase in glucose uptake. This indicates that increasing the levels or activity of CALCRL in tissues of insulin resistant patients or diabetic patients will increase the ability of such tissues to take up glucose and hence, will provide an effective treatment for insulin resistance and diabetes.

[315] CALCRL contains the following protein domains (designated with reference to SEQ ID NO:26): Signal peptide at amino acids 1 to 22; Hormone receptor domain (PF02793) at amino acids 62 to 132; 7 transmembrane receptor (Secretiu family) (PF00002) at amino acids 138 to 391; and 7 transmembrane domains (TMHMM2.0) at amino acids 144 to 166, 179 to 198, 225 to 247, 254 to 276, 291 to 313, 334 to 352, 367 to 389. CALCRL is the G protein-coupled receptor which binds calcitonin-gene-related peptide (CGRP) or adrenomedullin (ADM) depending upon interaction with either of the accessory proteins, RAMPl and RAMP2 and stimulates adenylyl cyclase (Kamitani, S., et al, FEBS Lett. 448: 111-114 (1999); Kuwasako, K., et al, MoI Pharmacol. 65: 207-13 (2004); Flahaut, M., et al, Biochemistry. 42: 10333-41 (2003)).

CCL13

[316] Probe set 206407 detects CCL13 nucleic acid sequences. Expression of CCLl 3 transcripts was increased in diabetic compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of diabetics in comparison to lean patients.

[317] Probe set 206407 detects CCL13 nucleic acid sequences. Expression of CCLl 3 transcripts was increased in obese compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of obese in comparison to lean patients.

[318] CCLl 3 was also evaluated using real-time PCR. The results further show that CCLl 3 is significantly over-expressed in subcutaneous adipose from obese individuals when compared to subcutaneous adipose from lean individuals.

"Fold Change" indicates the fold expression calculated as the ratio of the mean obese expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.

[319] CCLl 3 contains the following protein domains (designated with reference to SEQ ED NO:32): Signal peptide at amino acids 1 to 23; and Small cytokines (intecrine/chemokine), interleukin-8 like (PF00048) at amino acids 24 to 89. A soluble active secreted form of CCLl 3 has been detected (Berkhout, T. A., et al, J Biol Chem. 272:16404- 13 (1997)) and this is displayed in SEQ ID NO:33. CCL13 displays chemotactic activity for monocytes, lymphocytes, basophils and eosinophils, but not neutrophils. This chemokine plays a role in accumulation of leukocytes during inflammation. It may also be involved in the recruitment of monocytes into the arterial wall during artherosclerosis (Garcia-Zepeda, E. A. et al, J Immunol 157: 5613-5626 (1996); White, J. R. et al, J Biol Chem 275: 36626- 36631 (2000); Wain, J.H., et al, Clin Exp Immunol. Ml: 436-44 (2002)).

CCL8

[320] Probe set 214038 detects CCL8 nucleic acid sequences. Expression of CCL8 transcripts was decreased in pio compared to vehicle treated cultures of primary human adipocytes in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Pre-Pio" and "Post-Pio" indicates sample was taken before or after 24 hours of pioglitazone treatment; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of primary human adipocytes post-pio in comparison to pre-pio samples.

[321] CCL8 was also evaluated using real-time PCR. The results further show that CCL8 is significantly under-expressed in primary cultured human adipocytes treated with pio when compared to vehicle.

"Fold Change" indicates the fold expression calculated as the ratio of the mean pio expression/ mean vehicle expression. Numbers in parentheses indicates the number of primary human adipocyte samples analyzed by real-time PCR.

[322] Probe set 214038 detects CCL8 nucleic acid sequences. Expression of CCL8 transcripts was decreased in rosi compared to vehicle treated cultures of primary human adipocytes in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Pre-Rosi" and "Post-Rosi" indicates sample was taken before or after 24 hours of rosiglitazone treatment; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of primary human adipocytes post-rosi in comparison to pre-rosi samples.

[323] CCL8 was also evaluated using real-time PCR. The results further show that CCL8 is significantly under-expressed in primary cultured human adipocytes treated with rosi when compared to vehicle.

"Fold Change" indicates the fold expression calculated as the ratio of the mean rosi expression/ mean vehicle expression. Numbers in parentheses indicates the number of primary human adipocyte samples analyzed by real-time PCR.

[324] Probe set 214038 detects CCL8 nucleic acid sequences. Expression of CCL8 transcripts was increased in adipose tissues compared to all other human adult tissues in the gene profiling experiment.

"Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of tissue samples; "Fold Change" indicates fold change of adipose tissues in comparison to all other human adult tissues profiled.

[325] CCL8 contains the following protein domains (designated with reference to SEQ ID NO:39): Signal peptide at amino acids 1 to 23; and Small cytokines (intecrine/chemokine), interleukin-8 like (PF00048) at amino acids 24 to 90. A soluble active secreted form of CCL8 has been detected (Van Damme, J. et al, J Exp Med. 176: :59-65 (1992)) and this is displayed in SEQ ID NO:40. CCL8 displays chemotactic activity for monocytes, lymphocytes, basophils and eosinophils. By recruiting leukocytes to sites of inflammation this cytokine rnaycontribute to tumor-associated leukocyte infiltration and to the antiviral state against HIV infection ( Noso, N. et al, Biochem. Biophys. Res. Commun. 200: 1470-1476 (1994); Yang, O. et al., J. Infect. Dis. 185: 1174-1178 (2002)). CHI3L1

[326] Probe set 209395 detects CHI3L1 nucleic acid sequences. Expression of CHI3L1 transcripts was increased in diabetic compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of diabetics in comparison to lean patients.

[327] CHI3L1 was also evaluated using real-time PCR. The results further show that CHI3L1 is significantly over-expressed in subcutaneous adipose from diabetic individuals when compared to subcutaneous adipose from lean individuals.

"Fold Change" indicates the fold expression calculated as the ratio of the mean diabetic expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.

[328] Probe set 209395 detects CHI3L1 nucleic acid sequences. Expression of CHI3L1 transcripts was increased in obese compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of obese in comparison to lean patients.

[329] Probe set 209395 detects CHI3L1 nucleic acid sequences. Expression of CHI3L1 transcripts was decreased in pio compared to vehicle treated cultures of primary human adipocytes in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Pre-Pio" and "Post-Pio" indicates sample was taken before or after 24 hours of pioglitazone treatment; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of primary human adipocytes post-pio in comparison to pre-pio samples.

[330] CHDLl was also evaluated using real-time PCR. The results further show that CHD Ll is significantly under-expressed in primary cultured human adipocytes treated with pio when compared to vehicle.

"Fold Change" indicates the fold expression calculated as the ratio of the mean pio expression/ mean vehicle expression. Numbers in parentheses indicates the number of primary human adipocyte samples analyzed by real-time PCR.

[331] Probe set 209395 detects CHDLl nucleic acid sequences. Expression of CHDLl transcripts was decreased in rosi compared to vehicle treated cultures of primary human adipocytes in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Pre-Rosi" and "Post-Rosi" indicates sample was taken before or after 24 hours of rosiglitazone treatment; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of primary human adipocytes post-rosi in comparison to pre-rosi samples.

[332] CHDLl was also evaluated using real-time PCR. The results further show that CHDLl is significantly under-expressed in primary cultured human adipocytes treated with rosi when compared to vehicle.

"Fold Change" indicates the fold expression calculated as the ratio of the mean rosi expression/ mean vehicle expression. Numbers in parentheses indicates the number of primary human adipocyte samples analyzed by real-time PCR.

[333] CHDLl was over-expressed in L6 myotubes and the effect on basal and insulin stimulated glucose transport was determined. Glucose Transport Analysis in L6 Myotubes

Legend "Con" indicates control L6 myotubes that do not express CHI3L1. "FC" indicates the fold change defined as the following ratio; glucose transport in CHI3L1 -expressing cells/glucose transport in non-CHI3 Ll -expressing cells, h" is human, "n" is the number of experiments. SEM is the standard error of the mean.

[334] The results show that increasing the levels of CHI3 Ll in a cell such as a muscle cells leads to a corresponding increase in glucose uptake. This indicates that increasing the levels or activity of CHI3L1 in tissues of insulin resistant patients or diabetic patients will increase the ability of such tissues to take up glucose and hence, will provide an effective treatment for insulin resistance and diabetes.

[335] CHI3L1 contains the following protein domains (designated with reference to SEQ ID NO:46): Signal peptide at amino acids 1 to 21; and Glycosyl hydrolases family 18 (PF00704) at amino acids 22 to 357. A soluble active secreted form of CHI3L1 has been detected (Hakala, B.E., et al, J Biol Chem. 268:25803-10 (1993)) and this is displayed in SEQ ID NO:47. CHI3L1 is a glycoprotein secreted by a variety of cells including articular chondrocytes, synoviocytes and macrophages (Recklies, A.D., et al, Biochem J. 365: 119-26 (2002)) and is associated with conditions of increased matrix turnover and tissue remodeling for example, arthritis (Punzi, L., et al, Ann Rheum Dis. 62: 1224-6 (2003); Ling, H. and Recklies, A.D., Biochem J. Mar 12;Pt. Epub (2004)).

[336] The CHI3L1 gene has been cloned and the proximal promoter has been identified and shown to contain binding sites for transcription factors such as PU.1, SpI, Sp3, USF, AML-I and C/EBP proteins {see, e.g., Rehli M., et al. Genomics. 43: 221-225 (1997); Rehli, M, et al J. Biol. Chem 278: 44058-44067 (2003)). An exemplary method of screening for CHI3L1 regulators comprises an assay as follows: A CHI3L1 promoter is inserted upstream of a reporter gene such as β-galactosidase and expressed in cells. Compounds that up-regulate the activity of the promoter can thus be identified by measuing increased β- galactosidase activity. CRl

[337] Probe set 244313 detects CRl nucleic acid sequences. Expression of CRl transcripts was increased in diabetic compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of diabetics in comparison to lean patients.

[338] CRl was also evaluated using real-time PCR. The results further show that CRl is significantly over-expressed in subcutaneous adipose from diabetic individuals when compared to subcutaneous adipose from lean individuals.

"Fold Change" indicates the fold expression calculated as the ratio of the mean diabetic expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.

[339] Probe set 244313 detects CRl nucleic acid sequences. Expression of CRl transcripts was increased in obese compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of obese in comparison to lean patients.

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

CSFRl

[341] Probe set 203104 detects CSFRl nucleic acid sequences. Expression of CSFRl transcripts was increased in diabetic compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of diabetics in comparison to lean patients.

[342] CSFRl was also evaluated using real-time PCR. The results further show that CSFRl is significantly over-expressed in subcutaneous adipose from diabetic individuals when compared to subcutaneous adipose from lean individuals.

"Fold Change" indicates the fold expression calculated as the ratio of the mean diabetic expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.

[343] Probe set 203104 detects CSFRl nucleic acid sequences. Expression of CSFRl transcripts was increased in obese compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of obese in comparison to lean patients.

[344] CSFlR was over-expressed in 3T3-L1 adipocytes and cells were then treated with CSFl . The effect on basal and insulin stimulated glucose transport and Glut 4 translocation was then determined Glucose Transport Analysis in 3T3-L1 Adipocytes:

Legend: "Con" indicates control 3T3-L1 adipocytes that do not express CSFlR. "FC" indicates the fold change defined as the following ratio; glucose transport in hCSFlR- expressing cells stimulated with 100 ng/ml hCSFl for 24 hours/glucose transport in non- hCSFlR-expressing cells stimulated with 100 ng/ml hCSFl for 24 hours, h" is human, "n" is the number of experiments. SEM is the standard error of the mean.

Glut4 Translocation Anal sis:

Legend "Fold Change" indicates the following ratio; (Mean % of hCSFlR-expressing cells incubated with 100 ng/mL murine CSFl for 24 hours that were scored positive for cell surface Glut4)/(Mean % of LacZ-expressing cells cells incubated with 100 ng/mL murine CSFl for 24 hours that were scored positive for cell surface Glut4). "h" is human, "n" is the number of experiments

[345] The results show that increasing the levels of CSFlR in a cell such as a adipocyie leads to a corresponding increase in glucose uptake. This indicates that increasing the levels or activity of CSFlR in tissues of insulin resistant patients or diabetic patients will increase the ability of such tissues to take up glucose and hence, will provide an effective treatment for insulin resistance and diabetes.

[346] CSFRl contains the following protein domains (designated with reference to SEQ ID NO:57): Signal peptide at amino acids 1 to 19; Protein kinase domain (PF00069) at amino acids 582 to 910; two immunoglobulin domain (PF00047) at amino acids 217 to 280, 412 to 487 and 1 transmembrane domain (TMHMM2.0) at amino acids 515 to 537. CSFRl is the tyrosine kinase receptor for colony stimulating factor 1, a cytokine which controls the production, differentiation, and function of macrophages and may be associated with advanced-stage breast carcinoma and myeloid leukemia (Sapi, E., Exp Biol Med. 229:1- 11 (2004); Boultwood, J. et al, Proc Natl Acad Sci U S A 88: 6176-6180 (1991); Sapi, E. et al, Cancer Res. 59: 5578-85 (1999); Fixe, P. αm/ Praloran, V. Cytokine 10:32-7 (1998)).

[347] Cells over-expressing CSFlR can be generated (see, e.g., Murray LJ. , et al. Clin Exp Metastasis. 20: 757-66 (2003). Agonists of the CSFlR can be identified, e.g., by screening such cells for compounds that have the ability to induce autophosphorylation of the CSFlR.

CTSK

[348] Probe set 202450 detects CTSK nucleic acid sequences. Expression of CTSK transcripts was increased in diabetic compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of diabetics in comparison to lean patients.

[349] CTSK was also evaluated using real-time PCR. The results further show that CTSK is significantly over-expressed in subcutaneous adipose from diabetic individuals when compared to subcutaneous adipose from lean individuals.

"Fold Change" indicates the fold expression calculated as the ratio of the mean diabetic expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR. [350] Probe set 202450 detects CTSK nucleic acid sequences. Expression of CTSK transcripts was increased in patients with insulin resistance compared to normal patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Corr Co-efficient" indicates the relationship between glucose disposal rate (Rd) and signal intensities. A positive co-efficient indicates down regulation whereas a negative co-efficient indicates up regulation of the gene with increasing insulin resistance; "n" indicates number of patient samples.

[351] CTSK was also evaluated using real-time PCR. The results further show that CTSK is significantly increased in patients with insulin resistance compared to normal patients.

"Corr Co-efficient" indicates the relationship between Rd and signal intensities. A positive co-efficient indicates down regulation whereas a negative co-efficient indicates up regulation of the gene with increasing insulin resistance. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.

[352] Probe set 202450 detects CTSK nucleic acid sequences. Expression of CTSK transcripts was increased in adipose tissues compared to all other human adult tissues in the gene profiling experiment.

"Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of tissue samples; "Fold Change" indicates fold change of adipose tissues in comparison to all other human adult tissues profiled.

[353] CTSK contains the following protein domains (designated with reference to SEQ ID NO:63): Signal peptide at amino acids 1 to 23; Outer membrane lipoprotein LoIB (PF03550) at amino acids 4 to 158; and Papain family cysteine protease (PFOOl 12) at amino acids 115 to 328. A soluble active secreted form of CTSK has been detected and this is displayed in SEQ ID NO:64. CTSK is a cysteine (thiol) protease involved in bone remodeling and reabsorption, acts as a collagenase towards cartilage proteoglycans and may play a role in extracellular matrix degradation. Mutations in this gene are the cause of pycnodysostosis, an autosomal recessive disease characterized by osteosclerosis and short stature. (Motyckova, G. and Fisher, D.E., CurrMol Med. 2: 407-21(2002); Soderstrom, M. et al, Biochim Biophys Acta 1446: 35-46 (1999); Hou, W.S., et al., Biol Chem. 384: 891-7 (2003)).

CXCR4

[354] Probe set 211919 detects CXCR4 nucleic acid sequences. Expression of CXCR4 transcripts was increased in obese compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of obese in comparison to lean patients.

[355] CXCR4 was also evaluated using real-time PCR. The results further show that CXCR4 is significantly over-expressed in subcutaneous adipose from obese individuals when compared to subcutaneous adipose from lean individuals.

"Fold Change" indicates the fold expression calculated as the ratio of the mean obese expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR

[356] CXCR4 was over-expressed in 3T3-L1 adipocytes and the cells were treated with SDFl, a ligand for CXCR4. The effects on basal and insulin stimulated glucose transport and Glut 4 translocation were determined. Glucose Transport Analysis in 3T3-L1 Adipocytes:

Legend: "Con" indicates control 3T3-L1 adipocytes that do not express hCXCR.4. "FC" indicates the fold change defined as the following ratio; glucose transport in hCXCR4- expressing cells incubated for 24 hours with 20 nM SDFl /glucose transport in non-CXCR4- expressing cells incubated for 24 hours with 20 nM SDFl . h" is human, "n" is the number of experiments. SEM is the standard error of the mean.

Glut4 Translocation Analysis:

Legend "Fold Change" indicates the following ratio; (Mean % of hCXCR4-expressing cells incubated with 20 nM SDFl for 24 hours that were scored positive for cell surface

Glut4)/(Mean % of LacZ-expressing cells cells incubated with 20 nM SDFl for 24 hours that were scored positive for cell surface Glut4). "h" is human, "n" is the number of experiments.

[357] The results show that in a cell such as an adipocyte, increasing the levels of CXCR4 in the presence of the CXCR4 ligand SDFl leads to a corresponding increase in glucose uptake. This indicates that increasing the levels or activity of CXCR4 in tissues of insulin resistant patients or diabetic patients will increase the ability of such tissues to take up glucose and hence, will provide an effective treatment for insulin resistance and diabetes. [358] CXCR4 contains the following protein domains (designated with reference to SEQ ID NO:70): 7 transmembrane receptor (Secretin family) (PF00002) at amino acids 45 to 280; and 7 transmembrane domains (TMHMM2.0) at amino acids 43 to 65, 78 to 96, 111 to 132, 155 to 174, 199 to 221, 242 to 264, 284 to 306. CXCR4 is a G protein- coupled receptor that binds the CXC cytokine, CXCLl 2. CXCR4 may be required for hematopoiesis and organ vascularization (Tachibana, K. et al., Nature 393: 591-4(1998). It is known to act as a coreceptor for HIV (Moriuchi, M.et al., J. Immunol. 159: 4322-4329 (1997)) and inhibition of this receptor may be therapeutic for invasive breast cancer (Tamamura, H., et al, FEBS Lett. 550: 79-83 (2003)).

[359] Stimulation of the CXCR4 receptor with its ligand SDFl -alpha leads to an increase in intracellular Ca2+ (see, e.g., Princsn K. et al. J Exp Med. 20;186: 1383-1388 (1997)). Agonists of the CXCR4 receptor can therefore be identified, e.g., by screening cells with high levels of the CXCR4 receptor to identify compounds that increases intracellular Ca2+ using a calcium sensitive dye such as Calcium 3 or Fluo 3. DDAH2

[360] Probe set 214909 detects DDAH2 nucleic acid sequences. Expression of DDAH2 transcripts was increased in diabetic compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of diabetics in comparison to lean patients.

[361] Probe set 214909 detects DDAH2 nucleic acid sequences. Expression of DDAH2 transcripts was increased in patients with insulin resistance compared to normal patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Corr Co-efficient" indicates the relationship between glucose disposal rate (Rd) and signal intensities. A positive co-efficient indicates down regulation whereas a negative co-efficient indicates up regulation of the gene with increasing insulin resistance; "n" indicates number of patient samples.

[362] DDAH2 was also evaluated using real-time PCR. The results further show that DDAH2 is significantly increased in patients with insulin resistance compared to normal patients.

"Corr Co-efficient" indicates the relationship between Rd and signal intensities. A positive co-efficient indicates down regulation whereas a negative co-efficient indicates up regulation of the gene with increasing insulin resistance. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.

[363] Probe set 214909 detects DDAH2 nucleic acid sequences. Expression of DDAH2 transcripts was increased in adipose tissues compared to all other human adult tissues in the gene profiling experiment.

"Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of tissue samples; "Fold Change" indicates fold change of adipose tissues in comparison to all other human adult tissues profiled.

[364] DDAH2 contains the following protein domains (designated with reference to SEQ ID NO:76): Amidinotransferase (PF02274) at amino acids 6 to 281.

DDAH2 regulates cellular methylarginine concentrations, which in turn inhibit nitric oxide synthase. DDAH2 expression predominates in more highly vascularized tissues and in immune tissues (Leiper, J.M., et al, Biochem J. 343: 209-14 (1999).

DERP7 [365] Probe set 219410 detects DERP7 nucleic acid sequences. Expression of DERP7 transcripts was increased in obese compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of obese in comparison to lean patients.

[366] DERP7 was also evaluated using real-time PCR. The results further show that DERP7 is significantly over-expressed in subcutaneous adipose from obese individuals when compared to subcutaneous adipose from lean individuals.

"Fold Change" indicates the fold expression calculated as the ratio of the mean obese expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.

[367] Probe set 219410 detects DERP7 nucleic acid sequences. Expression of DERP7 transcripts was decreased in pio compared to vehicle treated cultures of primary human adipocytes in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Pre-Pio" and "Post-Pio" indicates sample was taken before or after 24 hours of pioglitazone treatment; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of primary human adipocytes post-pio in comparison to pre-pio samples.

[368] DERP7 contains the following protein domains (designated with reference to SEQ ED NO:82): Family of unknown function (DUF716) (PF04819) at amino acids 113 to 251; and 7 transmembrane domains (TMHMM2.0) at amino acids 4 to 23, 44 to 66, 91 to 113, 118 to 140, 150 to 172, 181 to 203, 218 to 240. DERP7 has high similarity to an uncharacterized mouse protein, p.19.5. This is a putative membrane protein which was shown to be differentially expressed in two closely related T lymphoma cell clones (MacLeod CL. et al, Cell Growth Differ., 1(6): 271-279 (1990)).

ENDOGLYXl

[369] Probe set 219091 detects ENDOGLYXl nucleic acid sequences. Expression of ENDOGLYXl transcripts was increased in obese compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of obese in comparison to lean patients.

[370] ENDOGLYXl was also evaluated using real-time PCR. The results further show that ENDOGLYXl is significantly over-expressed in subcutaneous adipose from obese individuals when compared to subcutaneous adipose from lean individuals.

"Fold Change" indicates the fold expression calculated as the ratio of the mean obese- expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR. [371] Probe set 219091 detects ENDOGLYXl nucleic acid sequences. Expression of ENDOGLYXl transcripts was increased in adipose tissues compared to all other human adult tissues in the gene profiling experiment.

"Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of tissue samples; "Fold Change" indicates fold change of adipose tissues in comparison to all other human adult tissues profiled.

[372] ENDOGLYXl contains the following protein domains (designated with reference to SEQ ID NO:88): Seryl-tRNA synthetase N-terminal domain (PF02403) at amino acids 499 to 600; Myosin tail (PFO 1576) at amino acids 143 to 814; Apolipoprotein A1/A4/E family (PF01442) at amino acids 200 to 469; CIq domain (PF00386) at amino acids 827 to 946; TNF(Tumour Necrosis Factor) family (PF00229) at amino acids 835 to 946; and Intermediate filament protein (PF00038) at amino acids 360 to 645. ENDOGLYXl is a cell surface glycoprotein which is attached to the extracellular matrix and capable of forming homo- and heteromers via disulfide bonding. It may play a role in angiogenesis, vasculogenesis, cell-matrix adhesion, and hemostasis (Christian, S. et ai, J Biol Chem 276: 48588-95 (2001); Leimeister, C, et al, Dev Biol. 249: 204-18 (2002)).

ETL

[373] Probe set MBXHUMFAT01286 detects ETL nucleic acid sequences. Expression of ETL transcripts was increased in obese compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of obese in comparison to lean patients.

[374] Probe set MBXHUMF ATOl 286 detects ETL nucleic acid sequences. Expression of ETL transcripts was increased in adipose tissues compared to all other human adult tissues in the gene profiling experiment.

"Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of tissue samples; "Fold Change" indicates fold change of adipose tissues in comparison to all other human adult tissues profiled.

[375] ETL was also evaluated using real-time PCR. The results further show that ETL is significantly over-expressed in adipose tissues when compared to all other human adult tissues.

"Fold Change" indicates the fold expression calculated as the ratio of the mean adipose tissues expression/ mean other tissues expression. Numbers in parentheses indicates the number of human adult tissue samples analyzed by real-time PCR. [376] ETL contains the following protein domains (designated with reference to SEQ ID NO:94): BphX-like (PF06139) at amino acids 629 to 728; Latrophilin/CL-1-like GPS domain (PFOl 825) at amino acids 487 to 539; EGF-like domain (PF00008) at amino acids 183 to 220; 7 transmembrane receptor (Secretin family) (PF00002) at amino acids 545 to 792; and 7 transmembrane domains (TMHMM2.0) at amino acids 552 to 574, 587 to 606, 621 to 643, 655 to 677, 692 to 714, 740 to 762, 766 to 788. ETL belongs to the secretin family of G-protein-coupled peptide hormone receptors and the EGF-TM7 subfamily of receptors. The latter are characterized by a variable number of extracellular EGF and cell surface domains and conserved seven transmembrane-spanning regions. (Nechiporuk, T. et al, J Biol Chem 276: 4150-7 (2001)).

FLJl 2389

[377] Probe set 218434 detects FLJ12389 nucleic acid sequences. Expression of FLJl 2389 transcripts was decreased in diabetic compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of diabetics in comparison to lean patients. [378] FLJ12389 was also evaluated using real-time PCR. The results further show that FLJ 12389 is significantly under-expressed in subcutaneous adipose from diabetic individuals when compared to subcutaneous adipose from lean individuals.

"Fold Change" indicates the fold expression calculated as the ratio of the mean diabetic expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.

[379] Probe set 218434 detects FLJ 12389 nucleic acid sequences. Expression of FLJ12389 transcripts was decreased in obese compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of obese in comparison to lean patients.

[380] FLJ12389 was also evaluated using real-time PCR. The results further show that FLJ12389 is significantly under-expressed in subcutaneous adipose from obese individuals when compared to subcutaneous adipose from lean individuals.

"Fold Change" indicates the fold expression calculated as the ratio of the mean obese expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.

[381] Probe set 218434 detects FLJ 12389 nucleic acid sequences. Expression of FLJ 12389 transcripts was increased in adipose tissues compared to all other human adult tissues in the gene profiling experiment.

"Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of tissue samples; "Fold Change" indicates fold change of adipose tissues in comparison to all other human adult tissues profiled. [382] FLJ12389 contains the following protein domains (designated with reference to SEQ H) NO: 100): AMP-binding enzyme (PF00501) at amino acids 130 to 571. FLJl 2389 has some sequence similarity to acetyl coenzyme A synthetases and is predicted to contain ATP/GTP and AMP binding sites. FLJ 12389 may be a ketone body-utilizing enzyme of which the physiological role of remains unclear (Ohgami, M. et al, Biochem Pharmacol 65: 989-994 (2003)).

FZD4

[383] Probe set 218665 detects FZD4 nucleic acid sequences. Expression of FZD4 transcripts was decreased in diabetic compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of diabetics in comparison to lean patients.

[384] FZD4 was also evaluated using real-time PCR. The results further show that FZD4 is significantly under-expressed in subcutaneous adipose from diabetic individuals when compared to subcutaneous adipose from lean individuals.

"Fold Change" indicates the fold expression calculated as the ratio of the mean diabetic expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.

[385] Probe set 218665 detects FZD4 nucleic acid sequences. Expression of FZD4 transcripts was decreased in patients with insulin resistance compared to normal patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Corr Co-efficient" indicates the relationship between glucose disposal rate (Rd) and signal intensities. A positive co-efficient indicates down regulation whereas a negative co-efficient indicates up regulation of the gene with increasing insulin resistance; "n" indicates number of patient samples. [386] FZD4 was also evaluated using real-time PCR. The results further show that FZD4 is significantly decreased in patients with insulin resistance compared to normal patients.

"Corr Co-efficient" indicates the relationship between Rd and signal intensities. A positive co-efficient indicates down regulation whereas a negative co-efficient indicates up regulation of the gene with increasing insulin resistance. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.

[387] Probe set 218665 detects FZD4 nucleic acid sequences. Expression of FZD4 transcripts was increased in adipose tissues compared to all other human adult tissues in the gene profiling experiment.

"Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of tissue samples; "Fold Change" indicates fold change of adipose tissues in comparison to all other human adult tissues profiled.

[388] FZD4 contains the following protein domains (designated with reference to SEQ ID NO:110): Signal peptide at amino acids 1 to 36; Frizzled/Smoothened family membrane region (PF01534) at amino acids 209 to 514; Fz domain (PF01392) at amino acids 35 to 159; and 7 transmembrane domains (TMHMM2.0) at amino acids 10 to 32, 221 to 243, 253 to 275, 301 to 323, 394 to 416, 437 to 459, 474 to 496. FZD4 encodes a 7- transmembrane domain protein and is a receptor for Wnt signaling proteins. The auditory and cerebellar phenotypes of FZD4 null mice implicate Frizzled signaling in maintaining the viability and integrity of the nervous system in later life and retinal angiogenesis (Wang, Y., et al, JNeurosci. 21: 4761-71 (2001); Singaraja, R.R., et al, Nat Genet. 32: 326-30 (2002)).

GLIPRl

[389] Probe set 226142 detects GLIPRl nucleic acid sequences. Expression of GLIPRl transcripts was increased in diabetic compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of diabetics in comparison to lean patients.

[390] GLIPRl was also evaluated using real-time PCR. The results further show that GLIPRl is significantly over-expressed in subcutaneous adipose from diabetic individuals when compared to subcutaneous adipose from lean individuals.

"Fold Change" indicates the fold expression calculated as the ratio of the mean diabetic expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.

[391] Probe set 226142 detects GLIPRl nucleic acid sequences. Expression of GLIPRl transcripts was increased in obese compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of obese in comparison to lean patients.

[392] GLIPRl was also evaluated using real-time PCR. The results further show that GLIPRl is significantly over-expressed in subcutaneous adipose from obese individuals when compared to subcutaneous adipose from lean individuals.

"Fold Change" indicates the fold expression calculated as the ratio of the mean obese expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.

[393] Probe set 226142 detects GLIPRl nucleic acid sequences. Expression of GLIPRl transcripts was decreased in pio+insulin compared to insulin treated cultures of primary human adipocytes in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Pre-Pio" and "Post-Pio" indicates sample was taken before or after 24 hours of pioglitazone treatment; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of primary human adipocytes post-pio in comparison to pre-pio samples.

[394] Probe set 226142 detects GLIPRl nucleic acid sequences. Expression of GLIPRl transcripts was decreased in rosi+insulin compared to insulin treated cultures of primary human adipocytes in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Pre-Rosi" and "Post-Rosi" indicates sample was taken before or after 24 hours of rosiglitazone treatment; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of primary human adipocytes post-rosi in comparison to pre-rosi samples.

[395] GLEPRl contains the following protein domains (designated with reference to SEQ ID NO:118): Signal peptide at amino acids 1 to 21; SCP-like extracellular protein (PF00188) at amino acids 38 to 174; and 1 transmembrane domain (TMHMM2.0) at amino acids 235 to 257. GLIPRl is a putative secreted protein that may play a role in inhibition of malignant growth and progression through its proapoptotic activities (Ren, C. et al, MoI Cell Biol 22: 3345-57 (2002)).

GPRl 05

[396] Probe set 206637 detects GPRl 05 nucleic acid sequences. Expression of GPRl 05 transcripts was increased in diabetic compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of diabetics in comparison to lean patients.

[397] GPRl 05 was also evaluated using real-time PCR. The results further show that GPRl 05 is significantly over-expressed in subcutaneous adipose from diabetic individuals when compared to subcutaneous adipose from lean individuals.

"Fold Change" indicates the fold expression calculated as the ratio of the mean diabetic expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.

[398] GPRl 05 contains the following protein domains (designated with reference to SEQ ID NO:124): 7 transmembrane receptor (rhodopsin family) (PFOOOOl) at amino acids 39 to 295; and 7 transmembrane domains (TMHMM2.0) at amino acids 27 to 49, 56 to 78, 98 to 117, 137 to 159, 187 to 209, 235 to 257, 279 to 298. GPRl 05 is a G(i/o)- G protein-coupled receptor that is activated by extracellular UDP-sugars. Activation of the receptor stimulates intracellular calcium and may mediate primitive hematopoietic cell responses to microenvironments (Chambers, J. K. et al, J Biol Chem 275: 10767-71 (2000); Lee, B. C. et al, Genes D ev 17: 1592-604 (2003); Skelton, L. et aL, J Immunol 171: 1941-9 (2003); Moore, D. J. et al, Brain Res MoI Brain Res 118: 10-23 (2003)).

GPR146

[399] Probe set 228770 detects GPRl 46 nucleic acid sequences. Expression of GPRl 46 transcripts was decreased in diabetic compared to lean patients in the gene profiling experiment.

8/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of diabetics in comparison to lean patients. [400] GPRl 46 was also evaluated using real-time PCR. The results further show that GPR146 is significantly under- expressed in subcutaneous adipose from diabetic individuals when compared to subcutaneous adipose from lean individuals.

"Fold Change" indicates the fold expression calculated as the ratio of the mean diabetic expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.

[401] Probe set 228770 detects GPR146 nucleic acid sequences. Expression of GPRl 46 transcripts was decreased in patients with insulin resistance compared to normal patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Corr Co-efficient" indicates the relationship between glucose disposal rate (Rd) and signal intensities. A positive co-efficient indicates down regulation whereas a negative co-efficient indicates up regulation of the gene with increasing insulin resistance; "n" indicates number of patient samples.

[402] GPRl 46 was also evaluated using real-time PCR. The results further show that GPR 146 is significantly decreased in patients with insulin resistance compared to normal patients.

"Corr Co-efficient" indicates the relationship between Rd and signal intensities. A positive co-efficient indicates down regulation whereas a negative co-efficient indicates up regulation of the gene with increasing insulin resistance. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.

[403] The cellular level of GPRl 46 was reduced in 3T3-L1 adipocytes using siRNA directed against GPRl 46 and the effect on basal and insulin stimulated glucose transport was determined GPRl 46 mRNA Level in 3T3-L1 Adipocytes Transfected with siRNA Oligonucleotides

Legend: "siRNA" indicates Dharmacon Smartpool siRNA oligonucleotides directed against murine GPR146. "Scr" indicates the Dharmacon Scramble siRNA Control. "FC" indicates the fold change defined as the following ratio; Level of GPRl 46 mRNA in GPRl 46 siRNA transfected 3T3-L1 adipocytes/Level of GPR146 mRNA in Scramble siRNA transfected 3T3- Ll adipocytes, "n" is the number of experiments. SEM is the standard error of the mean.

Glucose Transport in 3T3-L1 Adipocytes Transfected with siRNA Oligonucleotides

Legend: "siRNA" indicates Dharmacon Smartpool siRNA oligonucleotides directed against murine GPRl 46. "Scr" indicates Dharmacon Scramble siRNA Control oligonucleotides. "FC" indicates the fold change defined as the following ratio; glucose transport in GPRl 46 siRNA transfected cells/glucose transport in Scramble siRNA transfected cells, "n" is the number of experiments. SEM is the standard error of the mean.

[404] The results show that decreasing the levels of GPRl 46 in a cell such as an adipocyte leads to a corresponding decrease in glucose uptake. This indicates that increasing the levels of GPRl 46 in tissues of insulin resistant patients or diabetic patients will increase the ability of such tissues to take up glucose and hence, will provide an effective treatment for insulin resistance and diabetes.

[405] GPRl 46 contains the following protein domains (designated with reference to SEQ ID NO: 130): Signal peptide at amino acids 1 to 37; and a 7 transmembrane receptor (rhodopsin family) (PFOOOOl) at amino acids 44 to 296. GPR146 is a member of the rhodopsin family of G protein-coupled receptors (GPCR) with very low sequence similiarity to GPR30. [406] GPRl 46 is an orphan GPCR with unknown coupling to G proteins (Goriam DE et al. Biochim Biophys Acta.1722:235-46 (2005)). Agonists of GPR146 can be detected, e.g., using cells over-expressing GPR146 along with either a promiscuous G protein (such as Gαl6, Kostenis E. Trends Pharmacol Sd. 22:560-4 (2001) or a chimeric G protein (such as Gαl6z or Gαl6s) (see, e.g., Liu AM et al. JBiomol Screen. 8:39-49 (2003), Hazari A et al. Cell Signal. 16:51-62 (2004)). Screening of such cells will detect GPR146 agonists, for example either by measuring intracellular Ca2+ with a Ca2+ sensitive dye such as Calcium 3 or by measuring intracellular cyclic AMP.

GPR30

[407] Probe set 210640 detects GPR30 nucleic acid sequences. Expression of GPR30 transcripts was decreased in diabetic compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of diabetics in comparison to lean patients.

[408] GPR30 was also evaluated using real-time PCR. The results further show that GPR30 is significantly under-expressed in subcutaneous adipose from diabetic individuals when compared to subcutaneous adipose from lean individuals.

"Fold Change" indicates the fold expression calculated as the ratio of the mean diabetic expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.

[409] Probe set 210640 detects GPR30 nucleic acid sequences. Expression of GPR30 transcripts was decreased in patients with insulin resistance compared to normal patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Corr Co-efficient" indicates the relationship between glucose disposal rate (Rd) and signal intensities. A positive co-efficient indicates down regulation whereas a negative co-efficient indicates up regulation of the gene with increasing insulin resistance; "n" indicates number of patient samples.

[410] GPR30 was also evaluated using real-time PCR. The results further show that GPR30 is significantly decreased in patients with insulin resistance compared to normal patients.

"Corr Co-efficient" indicates the relationship between Rd and signal intensities. A positive co-efficient indicates down regulation whereas a negative co-efficient indicates up regulation of the gene with increasing insulin resistance. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.

[411] Probe set 210640 detects GPR30 nucleic acid sequences. Expression of GPR30 transcripts was increased in adipose tissues compared to all other human adult tissues in the gene profiling experiment.

"Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of tissue samples; "Fold Change" indicates fold change of adipose tissues in comparison to all other human adult tissues profiled.

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

GPR65

[413] Probe set 214467 detects GPR65 nucleic acid sequences. Expression of GPR65 transcripts was increased in obese compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of obese in comparison to lean patients.

[414] GPR65 was also evaluated using real-time PCR. The results further show that GPR65 is significantly over-expressed in subcutaneous adipose from obese individuals when compared to subcutaneous adipose from lean individuals.

"Fold Change" indicates the fold expression calculated as the ratio of the mean obese expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.

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

HTR2B

[416] Probe set 206638 detects HTR2B nucleic acid sequences. Expression of HTR2B transcripts was decreased in pio compared to vehicle treated cultures of primary human adipocytes in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Pre-Pio" and "Post-Pio" indicates sample was taken before or after 24 hours of pioglitazone treatment; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of primary human adipocytes post-pio in comparison to pre-pio samples.

[417] HTR2B was also evaluated using real-time PCR. The results further show that HTR2B is significantly under-expressed in primary cultured human adipocytes treated with pio when compared to vehicle.

"Fold Change" indicates the fold expression calculated as the ratio of the mean pio expression/ mean vehicle expression. Numbers in parentheses indicates the number of primary human adipocyte samples analyzed by real-time PCR.

[418] HTR2B was over-expressed in 3T3-L1 adipocytes and the cells were then treated with 5-HT and the effect on basal and insulin stimulated glucose transport was determined. Glucose Transport Analysis in 3T3-L1 Adipocytes:

Legend: "Con" indicates control 3T3-L1 adipocytes that do not express hHTR2B. "FC" indicates the fold change defined as the following ratio; glucose transport in HTR2B- expressing cells stimulated with 1 uM 5HT for 3 hours/glucose transport in non-HTR2B- expressing cells stimulated with IuM 5HT for 3 hours, h" is human, "n" is the number of experiments. SEM is the standard error of the mean.

[419] These results show that in a cell such as a adipocyte, increasing the levels of HTR2B in the presence of its ligand 5-HT leads to a corresponding increase in glucose uptake. This indicates that increasing the levels or activity of HTR2B in tissues of insulin resistant patients or diabetic patients will increase the ability of such tissues to take up glucose and hence, will provide an effective treatment for insulin resistance and diabetes.

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

[421] The HTR2B receptor is a G protein coupled receptor linked to the mobilization of intracellular Ca2+ {see, e.g., Schmuck K. et al FEBS Lett.342 :85-90 (1994)). Agonists of the HTR2B receptor can therefore be identified, e.g., by screening cells with high levels of the HTR2B receptor to identify compounds that increase intracellular Ca2+ using a calcium sensitive dye such as Calcium 3 or Fluo 3.

ITGB2

[422] Probe set 202803 detects ITGB2 nucleic acid sequences. Expression of ITGB2 transcripts was increased in diabetic compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of diabetics in comparison to lean patients.

[423] ITGB2 was also evaluated using real-time PCR. The results further show that ITGB2 is significantly over-expressed in subcutaneous adipose from diabetic individuals when compared to subcutaneous adipose from lean individuals.

"Fold Change" indicates the fold expression calculated as the ratio of the mean diabetic expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.

[424]" Probe set 202803 detects ITGB2 nucleic acid sequences. Expression of ITGB2 transcripts was increased in obese compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of obese in comparison to lean patients.

[425] ITGB2 was also evaluated using real-time PCR. The results further show that ITGB2 is significantly over-expressed in subcutaneous adipose from obese individuals when compared to subcutaneous adipose from lean individuals.

"Fold Change" indicates the fold expression calculated as the ratio of the mean obese expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.

[426] ICAM-I, the ligand for ITGB2-containing integrin complexes such as LFA-I, was added to cultures of 3T3-L1 adipocytes and the effect on glucose transport and Glut 4 translocation was determined. Glut4 Translocation Anal sis:

Legend "Fold Change" indicates the following ratio; (Mean % of 3T3-L1 adipocytes incubated 3 hours with 10 ug/ml ICAM + 200 uM Mn2+ scored positive for cell surface Glut4)/(Mean % 3T3-L1 adipocytes incubated 3 hours with 200 uM Mn2+ scored positive for cell surface Glut4). "h" is human, "n" is the number of experiments.

[427] Incubating 3T3-L1 adipocytes with 200 uM Mn2+ for 3 hours enhanced cell surface Glut4 in the absence of insulin. In contrast, including 10 ug/ml ICAM inhibited the increase observed with Mn alone. Glucose Transport Analysis in 3T3-L1 Adipocytes:

Legend: "FC" indicates the fold change defined as the following ratio; glucose transport in 3T3-L1 adipocytes incubated with 10 ug/ml ICAMl + 200 uM Mn for 3 hours/glucose transport in 3T3-L1 adipocytes incubated with 200 uM Mn for 3 hours, h" is human, "n" is the number of experiments. SD is the standard deviation.

[428] The results show that increasing the activity of ITGB2 containing integrins such as LFA-I in a cell such as an adipocyte leads to a corresponding decrease in glucose uptake. This indicates that decreasing the levels or activty of ITGB2-containing integrins such as LFA-I in tissues of insulin resistant patients or diabetic patients will increase the ability of such tissues to take up glucose and hence, will provide an effective treatment for insulin resistance and diabetes.

[429] ITGB2 contains the following protein domains (designated with reference to SEQ ID NO: 154): Signal peptide at amino acids 1 to 22; Plexin repeat (PF01437) at amino acids 24 to 63; Integrin, beta chain (PF00362) at amino acids 32 to

447; EGF- like domain (PF00008) at amino acids 582 to 612; and 1 transmembrane domain (TMHMM2.0) at amino acids 701 to 723. The ITGB2 protein product is the integrin beta chain beta 2. Integrins are integral cell-surface proteins composed of an alpha chain and a beta chain. A given chain may combine with multiple partners resulting in different integrins. For example, beta 2 combines with the alpha L chain to form the integrin LFA-I, and combines with the alpha M chain to form the integrin Mac-1. Integrins are known to participate in cell adhesion as well as cell-surface mediated signalling. The regulation of interaction mediated by adhesion molecules may provide new targets for controlling inflammatory and immune responses (Bunting, M. et ah, Curr Opin Hematol. 9: 30-5 (2002); Tsuji T. et al, Blood 91(4): 1263-71 (1998); Zhang L. and Plow E.F. Biochemistry 38(25): 8064-71 (1999)).

[430] Inhibitors of LFA-I can be detected using a variety of assays, such as those that detect the ability of candidate compounds to disrupt the association of LFA-I with the ligand ICAM-I. For example, purified LFA-I can be immobilized and compounds that cause the inhibition of biotinylated ICAM-I binding can be detected.

[431] Inhibitors of the LFA-I integrin complex (alphaL/beta2) have been developed. These include inhibitors such as BIRT0377, LFA703 and A-286982 (Shimakoa et al Nat. Rev. Drug Discovery 2: 703-716 (2003). Such inhibitors, as well as other inhibitors of LFA-I integrin complex, are useful as agents for the treatment for insulin resistance and diabetes.

ITIH5

[432] Probe set MBXHUMFAT04252 detects ITIH5 nucleic acid sequences. Expression of ITIH5 transcripts was increased in diabetic compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of diabetics in comparison to lean patients.

[433] ITIH5 was also evaluated using real-time PCR. The results further show that ITIH5 is significantly over-expressed in subcutaneous adipose from diabetic individuals when compared to subcutaneous adipose from lean individuals.

"Fold Change" indicates the fold expression calculated as the ratio of the mean diabetic expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.

[434] Probe set MBXHUMFAT04252 detects ITIH5 nucleic acid sequences. Expression of ITIH5 transcripts was increased in obese compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of obese in comparison to lean patients. [435] ITIH5 was also evaluated using real-time PCR. The results further show that ITIH5 is significantly over-expressed in subcutaneous adipose from obese individuals when compared to subcutaneous adipose from lean individuals.

"Fold Change" indicates the fold expression calculated as the ratio of the mean obese expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.

[436] Probe set MBXHUMFAT04252 detects ITffl5 nucleic acid sequences. Expression of ITIH5 transcripts was increased in adipose tissues compared to all other human adult tissues in the gene profiling experiment.

"Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of tissue samples; "Fold Change" indicates fold change of adipose tissues in comparison to all other human adult tissues profiled

[437] ITIH5 was over-expressed in 3T3-L1 adipocytes and the effect on basal and insulin stimulated glucose transport and Glut 4 translocation was determinred

Glucose Transport Analysis in 3T3-L1 Adipocytes:

Legend: "Con" indicates control 3T3-L1 adipocytes that do not express ITIH5. "FC" indicates the fold change defined as the following ratio; glucose transport in ITIH5- expressing 3T3-L1 adipocytes /glucose transport in non-PTPRE-expressing 3T3-L1 adipocytes, h" is human, "n" is the number of experiments. SEM is the standard error of the mean. Glut4 Translocation Analysis:

Legend "Fold Change" indicates the following ratio; (Mean % of ITIH5-expressing cells scored positive for cell surface Glut4)/(Mean % of LacZ-expressing cells scored positive for cell surface Glut4). "h" is human, "n" is the number of experiments.

[438] The results show that increasing the levels of ITIH5 in a cell such as an adipocyte leads to a corresponding increase in glucose uptake. This indicates that increasing the levels or activty of ITIH5 in tissues of insulin resistant patients or diabetic patients will increase the ability of such tissues to take up glucose and hence, will provide an effective treatment for insulin resistance and diabetes.

[439] ITIH5 contains the following protein domains (designated with reference to SEQ ID NO: 160): Signal peptide at amino acids 1 to 21; Inter-alpha-trypsin inhibitor heavy chain C-terminus (PF06668) at amino acids 715 to 909; T-box (PF00907) at amino acids 310 to 426; and von Willebrand factor type A domain (PF00092) at amino acids 295 to 478. A soluble active secreted form of ITIH5 has been detected and this is displayed in SEQ ID NO: 161. ITIH5 belongs to the inter-alpha-trypsin inhibitor (ITI) family constitutes a group of proteins built up from one light chain and a variable set of heavy chains. Originally identified as plasma protease inhibitors, recent data indicate that ITI proteins play a role in extracellular matrix (ECM) stabilization and in prevention of tumor metastasis. ITIH5 expression was found to be consistently downregulated in invasive mammary ductal carcinomas (Himmelfarb M. et al, Cancer Lett. 204(1): 69-77 (2004)).

LGALS12

[440] Probe set 223828 detects LGALS 12 nucleic acid sequences. Expression of LGALS 12 transcripts was decreased in diabetic compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of diabetics in comparison to lean patients. [441] LGALS 12 was also evaluated using real-time PCR. The results further show that LGALS 12 is significantly under-expressed in subcutaneous adipose from diabetic individuals when compared to subcutaneous adipose from lean individuals.

"Fold Change" indicates the fold expression calculated as the ratio of the mean diabetic expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.

[442] Probe set 223828 detects LGALS 12 nucleic acid sequences.

Expression of LGALS 12 transcripts was decreased in obese compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of obese in comparison to lean patients.

[443] LGALS 12 was also evaluated using real-time PCR. The results further show that LGALS 12 is significantly under-expressed in subcutaneous adipose from obese individuals when compared to subcutaneous adipose from lean individuals.

"Fold Change" indicates the fold expression calculated as the ratio of the mean obese expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.

[444] Probe set 223828 detects LGALS 12 nucleic acid sequences.

Expression of LGALS 12 transcripts was decreased in obese compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of obese in comparison to lean patients. [445] Probe set 223828 detects LGALS 12 nucleic acid sequences. Expression of LGALS 12 transcripts was increased in adipose tissues compared to all other human adult tissues in the gene profiling experiment.

"Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of tissue samples; "Fold Change" indicates fold change of adipose tissues in comparison to all other human adult tissues profiled.

[446] LGALS 12 contains the following protein domains (designated with reference to SEQ ID NO: 165): Galactoside-binding lectin (PF00337) at amino acids 48 to 182. LGALS 12 is a a member of the beta- galactoside-binding lectin family and may be an apoptosis activator that negatively regulates the cell cycle and cell proliferation (Yang R. Y. et al, J Biol Chem., 276(23): 20252-60 (2001); Hotta K. et al., J Biol Chem., 276(36): 34089-97 (2001); Liu, F.T., et al., Biochim Biophys Acta .1572: 263-73 (2002)).

NMB

[447] Probe set 205204 detects NMB nucleic acid sequences. Expression of NMB transcripts was increased in diabetic compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of diabetics in comparison to lean patients.

[448] Probe set 205204 detects NMB nucleic acid sequences. Expression of NMB transcripts was increased in adipose tissues compared to all other human adult tissues in the gene profiling experiment.

"Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of tissue samples; "Fold Change" indicates fold change of adipose tissues in comparison to all other human adult tissues profiled.

[449] NMB was also evaluated using real-time PCR. The results further show that NMB is significantly over-expressed in adipose tissues when compared to all other human adult tissues.

"Fold Change" indicates the fold expression calculated as the ratio of the mean adipose tissues expression/ mean other tissues expression. Numbers in parentheses indicates the number of human adult tissue samples analyzed by real-time PCR. [450] NMB contains the following protein domains (designated with reference to SEQ ED NO:181): Signal peptide at amino acids 1 to 26; and Bombesin-like peptide (PF02044) at amino acids 47 to 60. A soluble active secreted form of NMB has been detected and this is displayed in SEQ ID NO: 182. NMB is a bombesin-related neuropeptide which induces calcium flux and phosphatidylinositol turnover leading to stimulatation of cell proliferation through the G-protein coupled neuromedin B receptor (Ohki-Hamazaki H. Prog. Neurobiol. 62(3): 297-312 (2000); Mason S. et ah, Eur J Pharmacol, 438(1-2): 25-34 (2002)).

NNAT

[451] Probe set 204239 detects NNAT nucleic acid sequences. Expression of NNAT transcripts was decreased in diabetic compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of diabetics in comparison to lean patients. [452] NNAT was also evaluated using real-time PCR. The results further show that NNAT is significantly under-expressed in subcutaneous adipose from diabetic individuals when compared to subcutaneous adipose from lean individuals.

"Fold Change" indicates the fold expression calculated as the ratio of the mean diabetic expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.

[453] Probe set 204239 detects NNAT nucleic acid sequences. Expression of NNAT transcripts was increased in rosi compared to vehicle treated cultures of primary human adipocytes in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Pre-Rosi" and "Post-Rosi" indicates sample was taken before or after 24 hours of rosiglitazone treatment; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of primary human adipocytes post-rosi in comparison to pre-rosi samples. [454] NNAT was also evaluated using real-time PCR. The results further show that NNAT is significantly over-expressed in primary cultured human adipocytes treated with rosi when compared to vehicle.

"Fold Change" indicates the fold expression calculated as the ratio of the mean rosi expression/ mean vehicle expression. Numbers in parentheses indicates the number of primary human adipocyte samples analyzed by real-time PCR.

[455] Probe set 204239 detects NNAT nucleic acid sequences. Expression of NNAT transcripts was increased in adipose tissues compared to all other human adult tissues in the gene profiling experiment.

"Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of tissue samples; "Fold Change" indicates fold change of adipose tissues in comparison to all other human adult tissues profiled.

[456] NNAT contains the following protein domains (designated with reference to SEQ ID NO: 188): Signal peptide at amino acids 1 to 23; and 1 transmembrane domain (TMHMM2.0) at amino acids 13 to 35. NNAT is a putative proteolipid that may regulate ion channels during brain development. It is found to be highly expressed in pituitary adenomas and is frequently hypermethylated in childhood myeloid and lymphoid acute leukemias (Dou D. and Joseph R. Genomics 33(2): 292-7 (1996); Usui H. et al, JMoI Neurosci. 9(\): 55-60 (1997); Evans H.K. et al, Genomics 77(1-2): 99-104 (2001)).

OLFM2

[457] Probe set 223601 detects OLFM2 nucleic acid sequences. Expression of OLFM2 transcripts was decreased in diabetic compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of diabetics in comparison to lean patients.

[458] OLFM2 was also evaluated using real-time PCR. The results further show that OLFM2 is significantly under-expressed in subcutaneous adipose from diabetic individuals when compared to subcutaneous adipose from lean individuals.

"Fold Change" indicates the fold expression calculated as the ratio of the mean diabetic expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.

[459] Probe set 223601 detects OLFM2 nucleic acid sequences. Expression of OLFM2 transcripts was increased in adipose tissues compared to all other human adult tissues in the gene profiling experiment.

"Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of tissue samples; "Fold Change" indicates fold change of adipose tissues in comparison to all other human adult tissues profiled.

[460] OLFM2 was over-expressed in 3T3-L1 adipocytes and the effect on basal and insulin stimulated glucose transport was determined

Glucose Transport Analysis in 3T3-L1 Adipocytes:

Legend: "Con" indicates control 3T3-L1 adipocytes that do not express OLFM2. "FC" indicates the fold change defined as the following ratio; glucose transport in OLFM2- expressing 3T3-L1 adipocytes /glucose transport in non-OLFM2-expressing 3T3-L1 adipocytes, h" is human, "n" is the number of experiments. SEM is the standard error of the mean.

[461] The results show that increasing the levels of OLFM2 in a cell such as an adipocyte leads to a corresponding decrease in glucose uptake. This indicates that decreasing the levels or activity of OLFM2 in tissues of insulin resistant patients or diabetic patients will increase the ability of such tissues to take up glucose and hence, will provide an effective treatment for insulin resistance and diabetes.

[462] 0LFM2 contains the following protein domains (designated with reference to SEQ ID NO: 196): Signal peptide at amino acids 1 to 24; and Olfactomedin-like domain (PF02191) at amino acids 196 to 446. A soluble active secreted form of OLFM2 has been predicted and this is displayed in SEQ ED NO: 197. OLFM2 is a protein which possess high similarity to olfactomedin 3 (rat 01fm3), which is known to interact with myocilin and is associated with glaucoma and disorders involving the anterior segment of the eye and the retina (Ortego J. et al, FEBS Lett. 413(2): 349-53 (1997). OPN3

[463] Probe set 219032 detects OPN3 nucleic acid sequences. Expression of OPN3 transcripts was increased in obese compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of obese in comparison to lean patients.

[464] OPN3 was also evaluated using real-time PCR. The results further show that OPN3 is significantly over-expressed in subcutaneous adipose from obese individuals when compared to subcutaneous adipose from lean individuals.

"Fold Change" indicates the fold expression calculated as the ratio of the mean obese expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.

[465] OPN3 contains the following protein domains (designated with reference to SEQ ID NO:203): 7 transmembrane receptor (rhodopsin family) (PFOOOOl) at -amino acids 58 to 309; and 7 transmembrane domains (TMHMM2.0) at amino acids 44 to 66, 78 to 100, 115 to 137, 158 to 180, 200 to 222, 258 to 280, 290 to 312. OPN3 is a member of the opsins receptor cluster of G protein-coupled receptors which may play a role in non- visual photic processes such as the entrainment of circadian rhythm or the regulation of pineal melatonin production (Blackshaw S., and Snyder S.H., JNeurosci. 19(10): 3681-90 (1999); Halford S. et al, Genomics 72(2): 203-8 (2001)).

PTPRE

[466] Probe set 221840 detects PTPRE nucleic acid sequences. Expression of PTPRE transcripts was increased in diabetic compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of diabetics in comparison to lean patients.

[467] PTPRE was also evaluated using real-time PCR. The results further show that PTPRE is significantly over-expressed in subcutaneous adipose from diabetic individuals when compared to subcutaneous adipose from lean individuals.

[468] "Fold Change" indicates the fold expression calculated as the ratio of the mean diabetic expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR

[469] PTPRE was over-expressed in 3T3-L1 adipocytes and the effect on basal and insulin stimulated glucose transport and Glut 4 translocation were determined. Glut4 Translocation Analysis:

Legend "Fold Change" indicates the following ratio; (Mean % of hPTPRE-expressing 3T3- Ll adipocytes that were scored positive for cell surface Glut4)/(Mean % of LacZ-expressing 3T3-L1 adipocytes that were scored positive for cell surface Glut4). "h" is human, "n" is the number of experiments.

[470] Increasing the levels of hPTPRE in 3T3-L1 adipocytes significantly inhibits basal and insulin-stimulated Glut4 translocation to the cell surface.

Glucose Transport Analysis in 3T3-L1 Adipocytes:

Legend: "Con" indicates control 3T3-L1 adipocytes that do not express hPTPRE. "FC" indicates the fold change defined as the following ratio; glucose transport in hPTPRE- expressing 3T3-L1 adipocytes /glucose transport in non-PTPRE-expressing 3T3-L1 adipocytes, h" is human, "n" is the number of experiments. SEM is the standard error of the mean.

[471] The results show that increasing the levels of PTPRE in a cell such as an adipocyte leads to a corresponding decrease in glucose uptake. This indicates that decreasing the levels or activity of PTPRE in tissues of insulin resistant patients or diabetic patients will increase the ability of such tissues to take up glucose and hence, will provide an effective treatment for insulin resistance and diabetes.

[472] PTPRE contains the following protein domains (designated with reference to SEQ ID NO:213): Signal peptide at amino acids 1 to 19; Protein-tyrosine phosphatase (PFOO 102) at amino acids 159 to 393, 451 to 688; and 1 transmembrane domain (TMHMM2.0) at amino acids 47 to 69. PTPRE is found to exist in both a soluble, cytoplasmic and a transmembrane form. PTPRE is induced by ILl and TNFA treatment in astrocytoma cells suggesting a role in the inflammatory response of the brain (Schumann,G. et al, Brain Res MoI Brain Res. 62: 56-64 (1988)). Other studies suggest a role for PTPRE in RAS related signal transduction pathways, cytokines induced signaling, activation of voltage-gated K+ channels and vascular development and angiogenesis (Tiran, ZJ. et al, J. Biol Chem. 278: 17509-14(2003); Toledano-Katchalski,H., et al, MoI Cancer Res. 1 : 541-50 (2003); Thompson, L.J., et al, Am J Physiol Heart Circ Physiol. 281 : H396-403 (2001)). [473] PTPRE can dephosphorylate the tyrosine phosphorylated insulin receptor (see, e.g., Nakagawa Y. et al. Zoolog Sd. 22:169-75 (2005). Thus, an exemplary assay for inhibitors of PTPRE comprises determining the ability of candidate compounds to inhibit the ability of purified PTPRE to dephosphorylate a tyrosine phosphorylated peptide, e.g., a tyrosine phosphorylated peptide derived from the sequence of the insulin receptor and containing autophosphorylation sites such as Tyrosines 11158, 1162 and 1163.

RDCl

[474] Probe set 212977 detects RDCl nucleic acid sequences. Expression of RDCl transcripts was decreased in diabetic compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of diabetics in comparison to lean patients.

[475] RDCl was also evaluated using real-time PCR. The results further show that RDCl is significantly under-expressed in subcutaneous adipose from diabetic individuals when compared to subcutaneous adipose from lean individuals.

"Fold Change" indicates the fold expression calculated as the ratio of the mean diabetic expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.

[476] Probe set 212977 detects RDCl nucleic acid sequences. Expression of RDCl transcripts was decreased in patients with insulin resistance compared to normal patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Corr Co-efficient" indicates the relationship between glucose disposal rate (Rd) and signal intensities. A positive co-efficient indicates down regulation whereas a negative co-efficient indicates up regulation of the gene with increasing insulin resistance; "n" indicates number of patient samples.

[477] RDCl was also evaluated using real-time PCR. The results further show that RDCl is significantly decreased in patients with insulin resistance compared to normal patients.

"Corr Co-efficient" indicates the relationship between Rd and signal intensities. A positive co-efficient indicates down regulation whereas a negative co-efficient indicates up regulation of the gene with increasing insulin resistance. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.

[478] Probe set 212977 detects RDCl nucleic acid sequences. Expression of RDCl transcripts was increased in obese compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of obese in comparison to lean patients.

[479] Probe set 212977 detects RDCl nucleic acid sequences. Expression of RDCl transcripts was increased in adipose tissues compared to all other human adult tissues in the gene profiling experiment.

"Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of tissue samples; "Fold Change" indicates fold change of adipose tissues in comparison to all other human adult tissues profiled.

[480] RDCl contains the following protein domains (designated with reference to SEQ ID NO:223): 7 transmembrane receptor (rhodopsin family) (PFOOOOl) at amino acids 61 to 315; and 7 transmembrane domains (TMHMM2.0) at amino acids 47 to 69, 82 to 104, 119 to 140, 160 to 182, 214 to 236, 255 to 277, 297 to 319. RDCl is considered to be a new member of the rhodopsin family of G-protein coupled receptors. The protein is a co- receptor for human immunodeficiency viruses (HFV). Translocations involving this gene and HMGA2 on chromosome 12 have been observed in lipomas (Broberg, K., et al, Int J Oncol. 21 : 321-6 (2002); Shimizu, N., et al, J Virol. IA: 619-26 (2000)). SLIT2

[481] Probe set 209897 detects SLIT2 nucleic acid sequences. Expression of SLIT2 transcripts was increased in obese compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of obese in comparison to lean patients.

[482] SLIT2 was also evaluated using real-time PCR. The results further show that SLIT2 is significantly over-expressed in subcutaneous adipose from obese . individuals when compared to subcutaneous adipose from lean individuals.

"Fold Change" indicates the fold expression calculated as the ratio of the mean obese expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.

[483] Cellular levels of SLIT2 were reduced in 3T3-L1 adipocytes using siRNA directed against SLIT2 and the effect on basal and insulin stimulated glucose transport was determined

SLIT2 mRNA Level in 3T3-L1 Adipocytes Transfected with siRNA Oli onucleotides

Legend: "siRNA" indicates Dharmacon Smartpool siRNA oligonucleotides directed against murine SLIT2. "Scr" indicates the Dharmacon Scramble siRNA Control. "FC" indicates the fold change defined as the following ratio; Level of SLIT2 mRNA in SLIT2 siRNA transfected 3T3-L1 adipocytes/Level of SLIT2 mRNA in Scramble siRNA transfected 3T3- Ll adipocytes, "n" is the number of experiments. SEM is the standard error of the mean. Glucose Transport in 3T3-L1 Adipocytes Transfected with siRNA Oligonucleotides

Legend: "siRNA" indicates Dharmacon Smartpool siRNA oligonucleotides directed against murine SLIT2. "Scr" indicates Dharmacon Scramble siRNA Control oligonucleotides. "FC" indicates the fold change defined as the following ratio; glucose transport in SLIT2 siRNA transfected 3T3-L1 adipocytes/glucose transport in Scramble siRNA transfected 3T3- Ll adipocytes, "n" is the number of experiments. SEM is the standard error of the mean.

[484] The results show that decreasing the levels of SLIT2 in a cell such as a adipocyte leads to a corresponding decrease in glucose uptake. This indicates that increasing the levels or activity of SLIT2 in tissues of insulin resistant patients or diabetic patients will increase the ability of such tissues to take up glucose and hence, will provide an effective treatment for insulin resistance and diabetes.

[485] SLIT2 contains the following protein domains (designated with reference to SEQ K) NO:229): Signal peptide at amino acids 1 to 30; Leucine rich repeat C- terminal domain (PFOl 463) at amino acids 233 to 258, 454 to 479, 688 to 713, 883 to 908; Leucine rich repeat N-terminal domain (PF01462) at amino acids 27 to 54, 272 to 299, 505 to 532, 726 to 753; Leucine Rich Repeat (PF00560) at amino acids 56 to 79, 128 to 151, 176 to 199, 325 to 348, 349 to 372, 559 to 582, 607 to 630, 778 to 801, 802 to 825, 826 to 849; Laminin G domain (PF00054) at amino acids 1188 to 1319; and EGF-like domain (PF00008) at amino acids 922 to 954, 961 to 995, 1002 to 1033, 1040 to 1073, 1080 to 1111, 1125 to 1156, 1336 to 1367, 1375 to 1406, 1416 to 1447. A soluble active secreted form of SLIT2 has been detected (Nguyen Ba-Charvet, K.T. et al, J. Neurosc, 21: 4281-4289 (2001)) and this is displayed in SEQ ID NO:230. SLIT2 is the ligand for roundabout receptor ROBOl. Mammalian SLIT proteins may participate in the formation and maintenance of the nervous and endocrine systems by protein-protein interactions. SLIT2 has been reported to be a chemorepellant for neuronal migration in vivo induces branching of dorsal root ganglia axons (Nguyen Ba-Charvet K.T. et al, JNeurosci., 21(12): 4281-9 (2001); Nguyen Ba- Charvet K.T. et al, J Physiol Paris. 96: 91-8 (2002)), SLIT2 also found to inhibit leukocyte chemotaxis induced by chemotactic factors (Wu, J.Y., et al, Nature 410: 948-52 (2001).

[486] [The human SLIT2 gene has been cloned and the proximal promoter has been identified {see, e.g, Dallol A. et al Cancer Res. 62:5874-80 (2002). Therefore, one example of a method of screening for SLIT2 regulators is as follows. A SLIT2 promoter can be inserted upstream of a reporter gene such as β-galactosidase and expressed in cells. Compounds that up-regulate the activity of the promoter may therefore be identified by measuing increased β-galactosidase activity.

TNFRSF21

[487] Probe set 214581 detects TNFRSF21 nucleic acid sequences.

Expression of TNFRSF21 transcripts was increased in obese compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of obese in comparison to lean patients.

[488] Probe set 214581 detects TNFRSF21 nucleic acid sequences. Expression of TNFRSF21 transcripts was increased in rosi compared to vehicle treated cultures of primary human adipocytes in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Pre-Rosi" and "Post-Rosi" indicates sample was taken before or after 24 hours of rosiglitazone treatment; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of primary human adipocytes post-rosi in comparison to pre-rosi samples.

[489] TNFRSF21 was also evaluated using real-time PCR. The results further show that TNFRSF21 is significantly over- expressed in primary cultured human adipocytes treated with rosi when compared to vehicle.

"Fold Change" indicates the fold expression calculated as the ratio of the mean rosi expression/ mean vehicle expression. Numbers in parentheses indicates the number of primary human adipocyte samples analyzed by real-time PCR.

[490] Probe set 214581 detects TNFRSF21 nucleic acid sequences. Expression of TNFRSF21 transcripts was increased in adipose tissues compared to all other human adult tissues in the gene profiling experiment.

"Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of tissue samples; "Fold Change" indicates fold change of adipose tissues in comparison to all other human adult tissues profiled.

[491] TNFRSF21 was also evaluated using real-time PCR. The results further show that TNFRSF21 is significantly over-expressed in adipose tissues when compared to all other human adult tissues.

"Fold Change" indicates the fold expression calculated as the ratio of the mean adipose tissues expression/ mean other tissues expression. Numbers in parentheses indicates the number of human adult tissue samples analyzed by real-time PCR.

[492] TNFRSF21 was over-expressed in 3T3-L1 adipocytes and the effect on basal and insulin stimulated glucose transport was determined.

Glucose Transport

Legend: "Con" indicates control 3T3-L1 adipocytes that do not express hTNFRSF21. "FC" indicates the fold change defined as the following ratio; glucose transport in hTNFRSF21- expressing cells/glucose transport in non-TNFRSF21 -expressing cells, h" is human, "n" is the number of experiments. SEM is the standard error of the mean.

[493] The results show that increasing the levels of TNFRSF21 in a cell such as an adipocyte leads to a corresponding decrease in glucose uptake. This indicates that decreasing the levels or activity of TNFRSF21 in tissues of insulin resistant patients or diabetic patients will increase the ability of such tissues to take up glucose and hence, will provide an effective treatment for insulin resistance and diabetes. [494] TNFRSF21 contains the following protein domains (designated with reference to SEQ ID NO:236): Signal peptide at amino acids 1 to 41; Death domain (PFOO531) at amino acids 416 to 498; TNFR/NGFR cysteine-rich region (PF00020) at amino acids 50 to 88, 91 to 131, 133 to 168, 171 to 211; and 1 transmembrane domain (TMHMM2.0) at amino acids 350 to 369. TNFRSF21 has been shown to activate NF-kappaB and MAPK8/JNK, and induce cell apoptosis. Through its death domain, this receptor interacts with TRADD protein, which is known to serve as an adaptor that mediates signal transduction of TNF-receptors. Knockout studies in mice suggested that this gene plays a role in T-helper cell activation, and may be involved in inflammation and immune regulation (Pan G. et al, FEBS Lett. 431(3): 351-356 (1998); Kasof G.M. et al, Oncogene 20(55): 7965- 7975 (2001)).

[495] TNFRSF21 is up-regulated by TNFalpha. which itself has been associated with states of insulin resistance {see, e.g., Hotamisligil GS. J Intern Med. 245:621- 625 (1999);' .Kasof GM et al. Oncogene. 20 :7965-7975 (2001)). An exemplary assay to identify compounds that suppress TNFRSF21 can thus employ cells such as LnCAP to screen candidate compounds for the ability to inhibit TNFalpha-induced up-regulation of TNFRSF21 mRNA or protein.

TNFSF13B

[496] Probe set 223501 detects TNFSF13B nucleic acid sequences. Expression of TNFSF 13B transcripts was increased in diabetic compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of diabetics in comparison to lean patients.

[497] TNFSFl 3B was also evaluated using real-time PCR. The results further show that TNFSF 13B is significantly over-expressed in subcutaneous adipose from diabetic individuals when compared to subcutaneous adipose from lean individuals.

"Fold Change" indicates the fold expression calculated as the ratio of the mean diabetic expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.

[498] TNFSF 13B contains the following protein domains (designated with reference to SEQ ID NO:242): Signal anchor at amino acids 0 to 0; TNF(Tumour Necrosis Factor) family (PF00229) at amino acids 166 to 284; and 1 transmembrane domain (TMHMM2.0) at amino acids 48 to 70. A soluble active secreted form of TNFSF 13B has been detected (Schneider, P. et ah, J Exp. Med., 189: 1747-1756 (1999)) and this is displayed in SEQ ID NO:243. TNFSF13B is a ligand for multiple receptors including TNFRSF13B/TACI, TNFRSF 17/BCMA, and TNFRSFl 3 C/B AFFR. This cytokine is expressed in B cell lineage cells, and acts as a potent B cell activator. It has been also shown to play an important role in the proliferation and differentiation of B cells (Patke, A., et al, Curr Opin Immunol. 16: 251-5 (2004); Schneider, P. αm/ Tschopp, J. Immunol Lett. 88: 57- 62 (2003)).

TNFSF14

[499] Probe set 207907 detects TNFSF 14 nucleic acid sequences. Expression of TNFSF 14 transcripts was increased in omental tissue compared to all other human adult tissues in the gene profiling experiment.

"Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of tissue samples; "Fold Change" indicates fold change of omental tissues in comparison to all other human adult tissues profiled.

[500] TNFSF 14 was also evaluated using real-time PCR. The results further show that TNFSF 14 is significantly over-expressed in omental tissue when compared to all other human adult tissues.

"Fold Change" indicates the fold expression calculated as the ratio of the mean omental tissue expression/ mean other tissues expression. Numbers in parentheses indicates the number of human adult tissue samples analyzed by real-time PCR. [501] TNFSF 14 contains the following protein domains (designated with reference to SEQ ED NO:251): Signal anchor at amino acids 0 to 0; TNF(Tumour Necrosis Factor) family (PF00229) at amino acids 93 to 240; and 1 transmembrane domain (TMHMM2.0) at amino acids 36 to 58. A soluble active secreted form of TNFSF14 has been detected (Harrop, J.A., et al, J Biol Chem.273:27548-56 (1998)) and this is displayed in SEQ ID NO:252. TNFSF14 is the ligand for HVEM (TNFRSF14) and is reported to induce lymphocyte proliferationinduces apoptosis and suppresses in vivo tumor formation and facilitate herpes virus entry (Mauri D.N. et al, Immunity 8(1): 21-30 (1998); Zhai Y. et al, J Clin Invest. 102(6): 1142-51 (1998);Castellano, R. et al, J Biol Chem. 277(45): 42841-51 (2001)).

TPSB2

[502] Probe set 205683 detects TPSB2 nucleic acid sequences. Expression of TPSB2 transcripts was increased in obese compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of obese in comparison to lean patients.

[503] TPSB2 was also evaluated using real-time PCR. The results further show that TPSB2 is significantly over-expressed in subcutaneous adipose from obese individuals when compared to subcutaneous adipose from lean individuals.

"Fold Change" indicates the fold expression calculated as the ratio of the mean obese expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.

[504] Probe set 205683 detects TPSB2 nucleic acid sequences. Expression of TPSB2 transcripts was increased in adipose tissues compared to all other human adult tissues in the gene profiling experiment.

"Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of tissue samples; "Fold Change" indicates fold change of adipose tissues in comparison to all other human adult tissues profiled.

[505] TPSB2 contains the following protein domains (designated with reference to SEQ ID NO:262): Signal peptide at amino acids 1 to 18; and Trypsin (PF00089) at amino acids 31 to 267. A soluble active secreted form of TPSB2 has been detected and this is displayed in SEQ ED NO:263. TPSB2 is a tryptase beta 2 and belongs to the family of mast cell serine proteases which have been implicated as mediators in the pathogenesis of asthma and other allergic and inflammatory disorders. Beta tryptases appear to be the main isoenzymes expressed in mast cells (Pallaoro M. et al, J Biol Chem., 274(6): 3355-62 (1999).

WISP2

[506] Probe set 205792 detects WISP2 nucleic acid sequences. Expression of WISP2 transcripts was increased in obese compared to lean patients in the gene profiling experiment.

B/C indicates sample is from Basal or Clamp; "Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of patient samples; "Fold Change" indicates fold change of obese in comparison to lean patients.

[507] WISP2 was also evaluated using real-time PCR. The results further show that WISP2 is significantly over-expressed in subcutaneous adipose from obese individuals when compared to subcutaneous adipose from lean individuals.

"Fold Change" indicates the fold expression calculated as the ratio of the mean obese expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.

[508] Probe set 205792 detects WISP2 nucleic acid sequences. Expression of WISP2 transcripts was increased in adipose tissues compared to all other human adult tissues in the gene profiling experiment.

"Mean Expr" indicates mean expression; "SEM" indicates standard error of mean; "n" indicates number of tissue samples; "Fold Change" indicates fold change of adipose tissues in comparison to all other human adult tissues profiled.

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

Claims

WHAT IS CLAIMED IS:

L A method for identifying an agent for treating an obese, diabetic or pre-diabetic individual, the method comprising the steps of: (i) contacting an agent to a polypeptide encoded by a polynucleotide that hybridizes to a nucleic acid encoding SEQ ID NO: 213, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 35, 37, 39, 40, 42, 44, 46, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 182, 184, 186, 188, 190, 192, 194, 196, 197, 199, 201, 203, 205, 207, 209, 211, 215, 217, 219, 221, 223, 225, 227, 229, 230, 232, 234, 236, 238, 240, 242, 243, 245, 247, 249, 251, 252, 254, 256, 258, 260, 262, 263, 265, 267, 269, 270, 272 or 274 in 50% formamide, 5X SSC, and 1% SDS at 42⁰C followed by a wash in 0.2X SSC, and 0.1% SDS at 55⁰C; and (ii) selecting an agent that modulates the expression or activity of the polypeptide or that binds to the polypeptide, thereby identifying an agent for treating an obese, diabetic or pre-diabetic individual.

2. The method of claim 1, wherein the polypeptide comprises an amino acid sequence at least 95% identical to SEQ ID NO: 213, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 35, 37, 39, 40, 42, 44, 46, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 182, 184, 186, 188, 190, 192, 194, 196, 197, 199, 201, 203, 205, 207, 209, 211, 215, 217, 219, 221, 223, 225, 227, 229, 230, 232, 234, 236, 238, 240, 242, 243, 245, 247, 249, 251, 252, 254, 256, 258, 260, 262, 263, 265, 267, 269, 270, 272, 274 or a protein domain thereof.

3. The method of claim 1 , the method further comprising detecting whether the selected agent modulates weight and/or obesity.

4. The method of claim 1, the method further comprising detecting whether the selected agent modulates insulin sensitivity.

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5. The method of claim 1, wherein step (ii) comprises selecting an agent that modulates expression of the polypeptide.

6. The method of claim 1, wherein step (ii) comprises selecting an agent that modulates the activity of the polypeptide.

7. The method of claim 1, wherein step (ii) comprises selecting an agent that specifically binds to the polypeptide.

8. The method of claim 1, wherein the polypeptide is expressed in a cell and the cell is contacted with the agent.

9. The method of claim 1 , wherein the polypeptide comprises SEQ ED NO: 213, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 35, 37, 39, 40, 42, 44, 46, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 182, 184, 186, 188, 190, 192, 194, 196, 197, 199, 201, 203, 205, 207, 209, 211, 215, 217, 219, 221, 223, 225, 227, 229, 230, 232, 234, 236, 238, 240, 242, 243, 245, 247, 249, 251 , 252, 254, 256, 258, 260, 262, 263, 265, 267, 269, 270, 272 or 274.

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