WO2008137075A2 - Compositions and methods for the treatment of metabolic disorders and inflammation - Google Patents

Abstract

The invention relates to the discovery of the role of STAMP2 in coordination of responses to both inflammatory and nutritional stimuli. The invention provides for the use of STAMP2 deficient cells and STAMP2 deficient mice for screening for agents to modulate biomarkers associated with inflammation, oxidative stress, and metabolism. The invention also provides for the use of STAMP containing cells, particularly adipose, hepatic, or muscle cells, to identify agents that modulate STAMP2. The invention provide agents identified by the methods of the invention and their use as therapeutic agents for the treatment of metabolic disorders, which frequently include an inflammatory component. The invention also provide kits including STAMP 2 deficient cells.

Description

COMPOSITIONS AND METHODS FOR THE TREATMENT OF METABOLIC DISORDERS AND INFLAMMATION

Cross Reference to Related Applications

[0001] This application claims the benefit of U.S. Provisional Application Serial No.

60/927,398, filed May 2, 2007, the entire contents of which are incorporated herein by reference.

Government Support

[0002] This invention was made with government support under grant 5 ROl

DK052539-08 awarded by the National Institutes of Health. The government has certain rights in the invention.

Technical Field

[0003] The invention generally relates to the discovery that a coordinated response to nutrient and inflammatory stimuli is at least partially mediated by a six-transmembrane protein of prostate 2 (STAMP2). More specifically, the invention relates to methods for screening for compounds that modulate metabolic, oxidative stress, or inflammatory biomarkers using STAMP2 deficient cells.

Background

[0004] Cells and organisms must strike an appropriate balance between nutrient sufficiency and surplus. Adequate amounts of nutrients must be obtained in order to survive and function normally. On the other hand, chronic nutrient overload can lead to obesity, as well as to an array of associated metabolic disorders, including insulin resistance, hepatosteatosis, type 2 diabetes, and cardiovascular disease. This cluster of chronic diseases now constitutes the largest global health threat.

[0005] In the past decade, it has been clearly recognized that metabolic disease, particularly obesity and diabetes, has strong inflammatory underpinnings (Wellen and Hotamisligil 2005; Hotamisligil 2006). For example, obesity is associated with a state of chronic low-grade inflammation, and insulin action can be inhibited by several inflammatory signaling molecules, including the JNK, IKK, and SOCS family proteins (Emanuelli et al. 2000; Mooney et al. 2001; Yuan et al. 2001; Hotamisligil 2006). Experiments with loss of function mouse models have demonstrated the central role of these inflammatory pathways in metabolic diseases (Yuan et al. 2001; Hirosumi et al. 2002; Howard et al. 2004; Arkan et al. 2005; Cai et al. 2005). Inflammatory signaling pathways can be activated in obesity both by cytokines such as TNFα, as well as by nutrients such as lipids and glucose, particularly when blood or cellular levels are chronically elevated (Brownlee 2001; Hotamisligil 2006).

[0006] Presently available therapeutic interventions do not address the inflammatory component of metabolic disorders. Chronic inflammation is known to damage tissues resulting in many of the conditions associated with metabolic disorders. There is a need to address this aspect of metabolic disease to reduce the deleterious systemic effects of metabolic disorders.

Summary of the Invention

[0007] The invention generally provides methods for identifying an agent that increases the expression of a STAMP2 nucleic acid molecule in a cell. The method includes contacting a cell containing a STAMP2 promoter operably linked to a nucleic acid sequence for transcription with an agent and detecting an increase in transcription from the STAMP2 promoter relative to a control cell. In an embodiment, the STAMP2 promoter is operably linked to a reporter gene. In an embodiment, the STAMP2 promoter is operably linked to a nucleic acid sequence operably linked to a nucleic acid sequence encoding a STAMP2 polypeptide, and measuring an increase in expression of the STAMP2 nucleic acid molecule in the cell relative to a corresponding control cell. The agent may, for example, increase STAMP2 transcription or translation.

[0008] The invention further provides methods for identifying an agent that increases the expression or biological activity of a STAMP2 polypeptide. The method includes contacting a cell expressing a STAMP2 polypeptide with a candidate agent and measuring an increase in the expression or the biological activity of the STAMP2 polypeptide in the cell relative to a corresponding control cell. The increase can be measured, for example, in an immunological assay to detect expression of a STAMP2 polypeptide. Alternatively, STAMP2 biological activity can be detected by detecting a modulation of at least one biomarker of inflammation, oxidative stress, or metabolism.

[0009] The invention also provides methods for identifying an agent that binds a

STAMP2 polypeptide. The method comprises the steps of contacting a candidate compound with the isolated STAMP2 polypeptide under conditions that allow binding and detecting binding of the candidate compound to the polypeptide.

[0010] The invention provides methods for identification of an agent related to modulating the expression or activity of STAMP2, or that binds to STAMP2. The method comprises administering a compound to a cell in vivo and detecting a desirable metabolic change. For example, the metabolic change can be detected by measuring insulin responsiveness, serum insulin, blood glucose, serum triglycerides, cholesterol, glucose tolerance, insulin tolerance, distribution of lipoprotein particles in cells, tissues, or organs, liver lipid accumulation, liver triglyceride, body weight, or body fat accumulation or localization (e.g., visceral vs. subcutaneous fat accumulation). The desirable metabolic change for example can include a decrease in at least one sign or symptom of a metabolic or inflammatory disorder such as insulin resistance, glucose intolerance, fatty liver, type 2 diabetes, hyperlipidemia, inflammation, or visceral obesity.

[0011] The invention further provides a method of identifying an agent regulated by

STAMP2, the method comprising contacting a cell with STAMP2; and measuring an increase in the expression or activity of the agent. For example, a cell can be contacted with STAMP2 by expressing STAMP2 from an expression construct having a constitutive promoter. In an embodiment, the soluble factor is expressed in a cell, for example in an adipocyte.

[0012] The invention further provides a method of treating a metabolic or inflammatory disorder in an animal. The method comprises the steps of administering to the animal a therapeutically effective amount of an agent that increases the expression or activity of a STAMP2 polypeptide. The invention includes the use of such an agent for the preparation of a medicament for the treatment of a disorder, particularly a metabolic or inflammatory disorder. In an embodiment, the agent is a small molecule, polypeptide, or nucleic acid molecule or fragment thereof. For example, the agent can be a polypeptide that binds STAMP2.

[0013] The invention further provides a method of identifying an agent that treats a metabolic disorder, the method comprising contacting an animal having a metabolic disorder with an agent; and detecting a reduction in at least one sign or symptom of the metabolic disorder of the animal. The invention includes the use of an agent identified by the screening methods of the invention for the preparation of a medicament for the treatment of a disorder, particularly a metabolic or inflammatory disorder. A reduction in the metabolic disorder is detected, for example, by measuring insulin responsiveness, serum insulin, blood glucose, serum triglycerides, cholesterol, glucose tolerance, insulin tolerance, distribution of lipoprotein particles in cells, tissues, or organs, liver lipid accumulation, liver triglyceride, body weight, or body fat accumulation or body fat localization. For example, the agent may reduce insulin resistance, fatty liver, type 2 diabetes, hyperlipidemia, inflammation, or visceral obesity.

[0014] The invention generally provides methods of screening for agents to treat or prevent a metabolic disorder comprising: providing an agent to be screened; contacting a STAMP2 deficient cell with the agent; and determining whether at least one inflammatory, oxidative stress, and/or metabolic biomarker is modulated. The invention includes the use of an agent identified by the screening method for the preparation of a medicament for the treatment of a disorder, particularly a metabolic or inflammatory disorder. Cells include STAMP2 deficient cell is in culture or in a transgenic mouse. The cells may also include STAMP2 deficient cells in leptin deficient, JNK deficient, XBP-I deficient, or db/db mouse. STAMP2 deficient cells can include a mutation or deletion in the STAMP2 gene. Alternatively, STAMP2 deficient cells can be generated by treating cells with an agent, such as an antisense nucleic acid, or an siRNA or shRNA compound to reduce or eliminate the expression of STAMP2. Cells can include, for example, liver cells and adipose cells. Metabolic disorders include, for example, diabetes, obesity, insulin resistance, glucose intolerance, dyslipidemia, hyperglycemia, fatty liver disease, and hypercholesterolemia. The invention further includes obtaining agents or cells for screening.

[0015] The invention also provides methods of screening for agents that modulate

(e.g., increase or decrease) expression of one or more biomarkers related to one or more of inflammation, oxidative stress, or metabolic disease or dysfunction in STAMP 2 deficient cells. The invention includes the use of an agent identified by the screening method for the preparation of a medicament for the treatment of a disorder, particularly a metabolic or inflammatory disorder. Biomarkers related to inflammation include, for example, interleukin (IL)-6, IL- lβ, tumor necrosis factor (TNF)-α, TNF receptor 1, TNF receptor 2, MCP-I, haptoglobin, SOCS-3, Mac-1, CD68, and adipokines. Biomarkers related to oxidative stress include, for example, glutathione-S-transferase (GST), superoxide dismutase-1 (SOD-I), nicotinamide (NADPH), thiobarbituric acid reactive substances (TBARS), and lipid peroxidation. Biomarkers related to metabolism include, for example, Akt, GLUT4, adiponectin, fatty acid synthase, fatty acid transporter 1 , PPARγ, FATP4, leptin, fatty acid synthase (FAS), stearoyl CoA desaturase (SCD-I), resistin.

[0016] The invention further provides an agent for treating or preventing a metabolic disorder, wherein the agent modulates at least one inflammatory, oxidative stress, and/or metabolic biomarker, said agent having been identified by a screening method comprising: contacting the agent with a STAMP2 deficient cell; and determining whether the agent modulates at least one inflammatory, oxidative stress, and/or metabolic biomarker. The invention includes the use of an agent identified by the screening method for the preparation of a medicament for the treatment of a disorder, particularly a metabolic or inflammatory disorder. Metabolic disorders include, for example, diabetes, obesity, insulin resistance, glucose intolerance, dyslipidemia, hyperglycemia, fatty liver disease, and hypercholesterolemia. The invention still further provides prevention and/or treatment of a metabolic disorder comprising administering to a subject in need thereof a therapeutically effective amount of an agent identified by the screening methods of the invention.

[0017] The invention provides methods of screening for agents to treat or prevent a metabolic disorder comprising: providing an agent to be screened; contacting a STAMP2 containing cell with the agent; and determining whether STAMP2 expression or activity is modulated. The invention includes the use of an agent identified by the screening method for the preparation of a medicament for the prevention of a disorder, particularly a metabolic or inflammatory disorder. Preferably, the agent increases STAMP2 transcription, translation, or biological activity. In an aspect, the method includes further determining if at least one inflammatory, oxidative stress, and/or metabolic biomarker is modulated. In an aspect, the STAMP2 containing cell is an adipose, hepatic, or muscle cell wherein the cell may have a metabolic defect or be derived from or be part of an animal with a metabolic defect, either genetic (e.g., leptin-, JNK, or XBP-I -deficient mouse or db/db mouse) or induced (e.g., high fat fed animal). In an aspect, the agent is tested in a cell being subjected to a nutritional, metabolic, and/or inflammatory stimulus.

[0018] The invention further provides the use of any agent identified in any of the screening methods above or in other screening methods set forth herein that modulate the activity and/of expression of STAMP2 for the preparation of a medicament for the prevention and/or treatment of a metabolic or inflammatory disorder. In an embodiment, the medicament can include other agents for the treatment of a metabolic or inflammatory disease.

[0019] The invention also provides a pharmaceutical composition for the prevention and/or treatment of a metabolic or inflammatory disorder including any agent identified in any of the screening methods above or in other screening methods set forth herein to identify agents that modulate the activity and/of expression of STAMP2. The composition can further comprise a pharmaceutically acceptable carrier. In an embodiment, the pharmaceutical composition can include other agents for the treatment of a metabolic or inflammatory disease.

[0020] The invention also provides a kit comprising a STAMP2 deficient cell and instructions for use. Instructions can include instructions for growth of the cells and/or for methods of screening related to the instant invention.

[0021] In various embodiments, the invention provides methods that are a combination of two or more of the screening methods of the invention.

[0022] The invention further provides a transgenic mouse having a metabolic disorder, such as a leptin-, JNK, or XBP-I -deficient mouse or db/db mouse, further comprising a STAMP2 deficiency. STAMP2 deficient mice can include a mutation or deletion in the STAMP2 gene. Alternatively, STAMP2 deficient mice can be mice treated with an agent, such as an antisense or an siRNA compound to reduce or eliminate the expression of STAMP2. In an embodiment, the mouse can be heterozygous for the mutation resulting in the metabolic disorder and/or heterozygous for STAMP2 deficiency. The invention still further provides methods of making such mice and their use as models for metabolic and/or inflammatory disease.

[0023] The invention provides a transgenic mouse having an inflammatory disorder and/or a deficiency in an inflammatory mediator such as TNFα and a STAMP2 deficiency. STAMP2 deficient mice can include a mutation or deletion in the STAMP2 gene.

Alternatively, STAMP2 deficient mice can be mice treated with an agent, such as an antisense or an siRNA compound to reduce or eliminate the expression of STAMP2. In an embodiment, the mouse can be heterozygous for the mutation resulting in the metabolic disorder and/or heterozygous for STAMP2 deficiency. The invention still further provides methods of making such mice and their use as models for inflammatory and/or metabolic disease.

[0024] The invention also provides methods for identification of a subject prone to a metabolic disorder comprising testing a subject for an alteration in STAMP2 gene expression.

Brief Description of the Drawings [0025] Figure 1: Regulation of STAMP2 expression. (A) Regulation of adipose tissue STAMP2 mRNA expression in ob/ob and ob/ob-TNFά'^' treated with vehicle or thiazolidinediones, assessed by Northern blot. (B) Northern blot showing tissue distribution of STAMP2 mRNA in wt mice. (C) STAMP2 expression by Northern blot during differentiation of 3T3-L1 preadipocytes into adipocytes. (D) STAMP2 expression in adipocyte and stromal- vascular fractions of adipose tissue. (E) Activation of the STAMP2 promoter.

Responsiveness of the -2kb STAMP2 promoter to transcription factors CEBPα, LXRα, and PPARγ was analyzed by luciferase reporter assays in HeLa cells. T0901317 is a synthetic LXR agonist and pioglitzaone (Pio) is an agonist of PPARγ. (F) Regulation of STAMP2 expression in response to 4mM or 25 mM glucose or 6-hour treatments of 100 nM insulin, 300 μM oleic acid, 20% serum, or 10 ng/ml TNFα, assessed by quantitative RT-PCR (qPCR). Data indicates mean ± s.e.m. ** p <0.005. (G) Regulation of STAMP2 expression by in WAT in fed and fasted conditions (Northern blot). (F=Fed; S=Starved). (H) Immunostaining for adipose tissue STAMP2 protein in fed and fasted mice. [0026] Figure 2: STAMP2 deficiency results in elevated inflammatory gene expression and reduced insulin action in cultured adipocytes. (A) Efficient knockdown of STAMP2 mRNA confirmed by qPCR following electroporation of control and STAMP2- specific siRNAs into 3T3-L1 adipocytes. (B) Knockdown of STAMP2 protein confirmed by immunofluorescence. (C) Knockdown of Flag-STAMP2 expressed in 3T3-F442A adipocytes confirmed by Western blot analysis. (D) IL-6 expression measured by qPCR after overnight incubation in high glucose serum-free medium, and stimulation with TNFα (10 ng/ml, 6h). (E) IL-6 expression in adipocytes in high and low glucose conditions (F) IL-6 secretion measured by ELISA in cell supernatants collected under the same conditions as panel E. (G) Insulin-stimulated of ³H-2-deoxyglucose uptake in cultured adipocytes upon suppression of STAMP2 (H) Insulin stimulated myc-Glut4-GFP translocation to the plasma membrane assessed by immunohistochemistry and confocal microscopy (I) Quantitation of experiments shown in panel H in 3 independent experiments. Data represent mean ± s.e.m. Open bars- control siRNA; closed bars- STAMP2 siRNA. * indicates p<0.05; ** indicates p<0.005. [0027] Figure 3: Adipose tissue of STAMP2-I- mice exhibits elevated expression of inflammatory genes and accumulation of mononuclear cells. Tissues were harvested from 5-6 month old mice. (A) STAMP2 mRNA expression in the adipose tissue of STAMPl'^' (KO) and wild type (WT) mice examined by Northern blot analysis (B) Expression of STAMP family members in VWAT. (C) Inflammatory gene expression in VWAT and SWAT, examined by qPCR. (Open bars- WT; closed bars-STAMP2-/-). (D) Tissue sections from WT and STAMP2-/- mice were stained with hemotoxylin and eosin (E) F4/80 antigen positivity in WT and STAMP2^'1' VWAT, detected by immunohistochemistry (F) Gene expression related to oxidative stress was evaluated in VWAT (open bars- WT; closed bars- STAMPl'^') (G) Levels of TBARS in VWAT (n=5 animals/ genotype). All data presented as mean ± s.e.m. For all qPCR experiments, 5-6 animals in each genotype were examined, and mRNA expression of each gene was normalized to 18S rRNA levels. * indicates p< 0.05; ** p<0.005; *** p<0.0005.

[0028] Figure 4: Impaired insulin action in the visceral WAT of STAMP2^V~ mice. (A) PBS or intralipid and glucose (LG) were injected intraperitoneally into wt and STAMPl'^' mice followed by measurement of IL-6 and SOCS-3 mRNA levels in VWAT by qPCR. Data pooled from 2 independent experiments, shown as mean ± s.e.m. (B) Glucose transport in primary adipocytes. (C) Insulin stimulated Akt phosphorylation in VWAT and SWAT. Representative animals are shown in the Western blot, with each lane representing one animal. Phosphorylation of Akt normalized to total Akt protein was quantified and both absolute (Fig. 10) and fold insulin-stimulated Akt phosphorylation graphed, with data pooled from 2 independent experiments, represented as mean ± s.e.m. Expression of (D) metabolic and (E) macrophage-specific genes in VWAT was determined by qPCR. For each gene 6-9 animals/ genotype were examined, shown as mean ± s.e.m. For B-E, open bars- WT, closed bars- STAMP2-/-. * indicates p< 0.05; ** p< 0.005; *** p< 0.0005.

[0029] Figure 5: Development of metabolic disease in STAMP2^V~ mice. (A) Wild type (n=l 1) and STAMP2^"Λ (n=9) mice were weighed weekly until 20 weeks of age. (B)

Percent body fat was measured in a separate group of mice aged 2 and 5 months using DEXA analysis. (C) Every 4 weeks, blood was collected after a 6 hour fast. At 4, 12, and 20 weeks of age, serum insulin measured by ELISA. (D) Blood glucose was measured every 4 weeks after a 6 hour fast. (E) Serum triglycerides and cholesterol were measured and (F) distribution of lipoprotein particles profiled at 12 weeks of age. (G) Glucose and (H) insulin tolerance tests were performed on mice aged 12 and 17 weeks respectively. All data are presented as mean ± s.e.m. In A, D, G & H- squares- wt, triangles- STAMPI^1'; in B, C, & E, open bars- wt, closed bars- STAMPI^1'. * p<0.05; ** p<0.005; *** p<0.0005.

[0030] Figure 6: Liver insulin action and hyperinsulinemic-euglycemic clamp. (A) Insulin action in the liver following insulin injection into the portal vein of anesthetized mice. Each lane represents one animal. (B) Hyperinsulinemic-euglycemic clamp experiments. Rate of glucose infusion was higher in wild type than in STAMPI^1' mice over the 2 h course of the clamp. (C) Average GIR and Rd were calculated. (D) Hepatic glucose production at basal and clamp conditions, as well as (E) percent suppression of hepatic glucose production by hyperinsulinemic clamp were calculated. (F) Muscle and (G) adipose tissue glucose uptake during the clamp were assessed by measuring uptake of a ¹⁴C-2- deoxyglucose tracer. * p<0.05; ** p<0.005. Data are graphed as mean ± s.e.m. For parts C- G, open bars- wt; closed bars- STAMPI^1'. For part B, open circles- wt; closed squares- STAMPI^1'. [0031] Figure 7: Liver lipid accumulation in STAMP2^"'^" mice, and absence of

STAMP2 exacerbates metabolic phenotype of ob/ob mice. (A) Liver sections were stained with hemotoxylin and eosin. (B) Quantitation of liver triglyceride, with ob/ob liver used as a control. (C) FAS and SCD-I expression determined by qPCR in liver at 6 months of age. (D) Generation of ob/ob (n=4) and ST AMPl'^' ob/ob (n=5) mice. In 12-week-old mice, (D) body weight was similar between genotypes, though (E) body fat was significantly higher in mice lacking STAMP2. (F) Blood glucose after a 6 hour fast was significantly higher in the ST AMP2^~'^~ ob/ob mice. (G) H & E staining of liver sections showed higher lipid accumulation with larger lipid droplets in the mice lacking STAMP2. All bar graph data represent mean ± s.e.m. [0032] Figure 8: STAMP2 expression during feeding and fasting in mice fed regular or high fat diet. Expression of STAMP2 in mice fed regular or high fat diet for 16 weeks was examined by quantitative PCR and normalized to 18S in adipose tissue.

[0033] Figure 9: Impaired insulin action in the absence of STAMP2. (A) Signaling in response to insulin was examined after transfection of control and STAMP2 siRNA. Quantitation of pAkt/Akt represents combined data from 2 independent experiments. Error bars represent s.e.m. * indicates p < 0.05. Open bars - basal conditions, closed bars- insulin stimulated. (B) Conditioned media collected from STAMP2 knockdown adipocytes was applied to Fao liver cells. Fao cells were treated with insulin an phosphorylation of Akt was examined.

[0034] Figure 10: Expression of oxidative stress-related genes in subcutaneous

(SWAT) of STAMP2^V~ mice and in vivo insulin action. (A) Oxidative stress-related gene expression levels in SWAT were measured by qPCR in 6-month old male mice. Open bars represent STAMPI^1' (KO) * indicates p < 0.05. (B) Absolute quantification of in vivo insulin signaling shown in Figure 4.

[0035] Figure 11: Metabolic analysis of wild type and STAMP2^V~ mice. Mice were evaluated in metabolic cages for 48 hours. (A) Food intake, (B) VO₂ and VCO₂, and (C) movement along X, Y, and Z axes were measured throughout the duration of the experiment. (D) Differences in movement between genotypes were particularly noticeable during dark periods. * indicates p < 0.05. For parts A, B, and C, open bars represent wt and closed bars represent KO. For part D, closed squares represent wt and open triangles represent KO.

[0036] Figure 12: Hyperinsulinemic-eugenic clamp studies. (A) Body weight was equal in both genotypes at time of catheterization surgery and at time of clamp. (B) Blood glucose before and during the clamp studies.

Detailed Description

Definitions

[0037] As used herein, "adipose" is understood to refer to fat, such as adipose tissue or an adipose cell. Adipose tissue can be further divided by location in the body and/or biochemical markers into white adipose tissue (WAT) and brown adipose tissue (BAT).

WAT can be further subdivided into subcutaneous white adipose tissue (SWAT) which is present below the skin, and visceral white adipose tissue (VWAT) which is present around the internal organs. [0038] As used herein, "agents to treat metabolic disorders" or "agents to modulate

STAMP2 activity" include small molecules, proteins, nucleic acids, or fragments thereof, and any other chemical compounds. Agents can include known drugs or compounds. An agent can be rationally designed or selected. An agent can be a compound or a series of compounds comprising a library of small molecules, peptides, nucleic acids, or other chemical compounds. An agent can be a compound already identified as having a therapeutic use for the treatment of a disease or condition not necessarily related to the disfunction or altered expression of STAMP2.

[0039] As used herein, "alteration" is meant a change (increase or decrease) in the expression levels of a gene or polypeptide as detected by standard art known methods such as those described above. The change can also be in the post-synthetic modification state of a protein or other biological molecule. Modifications can include for example, phosphorylation, ubiquitination, peroxidation, and cleavage. As used herein, an alteration includes about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or more change in the level of expression or post-synthetic modification.

[0040] As used herein, "animal" is understood as preferably a mammal. A mammal can be human or non-human including, but not limited to laboratory and/or commercially important mammals, such as mouse, rat, rabbit, monkey, dog, cat, pig, cow, sheep, and goat.

[0041] As used herein, a "biomarker" is a distinctive biological or biologically derived indicator (e.g., protein or metabolite) of a process, event (e.g., feeding or starvation), or condition (e.g., obesity, oxidative stress or inflammation). A biomarker can include a protein for which expression is modulated (i.e., increased or decreased) in response to a stimulus, either acute (e.g., feeding or starvation) or persistent (e.g., mutation of a gene). Biomarkers can also be modulated by a post synthetic modification in a reversible (e.g., phosphorylation of a protein) or irreversible (e.g., peroxidation of lipids) manner. Biomarkers can also be modulated by disruption of localization or translocation in a cell (e.g., retained in the cytoplasm rather than translocation to the plasma membrane, internalization of receptors). Biomarkers may be present in a cell or secreted, for example into growth media. Inflammatory biomarkers include, but are not limited to, interleukin (IL)-6, IL-I β, tumor necrosis factor (TNF)-α, TNF receptor 1, TNF receptor 2, MCP (monocyte chemoattractant protein)-l , haptoglobin, suppressors of cytokine signaling (SOCS)-3, Mac-1 , CD68, and adipokines. Metabolic biomarkers include, but are not limited to, Akt, GLUT4 (glucose transporter 4), adiponectin, fatty acid synthase, fatty acid transporter protein (FATP) 1 , FATP4, peroxisome proliferator activated receptor (PPAR)-γ, leptin, fatty acid synthase (FAS), stearoyl CoA desarurase (SCD-I), resistin. Oxidative stress biomarkers include glutathione-S-transferase (GST), superoxide dismutase-1 (SOD-I), nicotinamide (NADPH), thiobarbituric acid reactive substances (TBARS), and lipid peroxidation.

[0042] As used herein, "cell culture" is understood to mean grown outside of the body in a dish, flask, or other container in the presence of growth media. Cell culture can be performed with transformed or immortalized cell lines. Cell culture can also be performed with "primary cells" removed from an animal, such as a mammal, and are not transformed or immortalized. Primary cells can be dividing or non-dividing cells. For example, the cells can be hepatic or adipose cells.

[0043] As used herein, "contacting a cell" refers to placing the agent in proximity to the cell, either directly or indirectly. In culture, contacting a cell can include adding or incorporating the agent into growth media. In an animal, such as a mammal, contacting can include administration of the agent to the animal by an enteral (e.g., oral) or parenteral (e.g., injection, topical) route so that the agent contacts a cell.

[0044] By "STAMP2 polypeptide" is meant a protein or fragment thereof having at least 85% identity to the amino acid sequence of STAMP2 and having STAMP2 biological activity. An exemplary STAMP2 amino acid sequence is provided at NCBI Accession No. AAQ04063. Preferably, the STAMP2 polypeptide has at least 90%, 95%, or even 100 % identity to a STAMP2 polypeptide over the entire length of the polypeptide or fragment thereof. STAMP2 biological activity includes the regulation of metabolism or inflammation (e.g., as evidenced by an effect on insulin resistance, glucose intolerance, mild hyperglycemia, dyslipidemia, or fatty liver disease), oxidoreductase/ metalloreductase activity, or the regulation of cell proliferation. Methods of detecting an alteration in STAMP2 biological activity include, but are not limited to, measuring glucose transport, translocation of glut4, production of reactive oxygen species, thiobarbituric acid reactive substances (TBARs), measuring the expression of genes or polypeptides involved in oxidative stress, measuring insulin signaling, and measuring oxidoreductase/ metalloreductase activity. Alternatively, an increase in STAMP2 biological activity can be detected by measuring insulin resistance, fatty liver, type 2 diabetes, hyperlipidemia, inflammation, and visceral obesity. [0045] As used herein, a "cell deficient in STAMP2" or a "STAMP2 deficient cell" is understood as a cell that has lower expression of STAMP2 relative to a wild-type cell of the same type (e.g., adipose, liver, muscle) under comparable conditions, in culture or in vivo. It is preferred that a "STAMP2 deficient cell" expresses less than about 50%, about 40%, about 30%, about 20%, or about 10% of the amount of STAMP2 expressed in a wild type cell under comparable conditions. In one embodiment, a STAMP2 deficient cell fails to express STAMP2 or expresses a reduced level of a biologically active STAMP2 polypeptide. A deficiency in STAMP2 can be induced transiently using an siRNA or antisense oligonucleotide. A deficiency in STAMP2 can alternatively be induced by disruption of the gene encoding STAMP2. This disruption can be in regulatory, coding, and/or intronic sequences of the gene. A STAMP2 deficient cell can be homozygous or heterozygous for the disruption. Methods of detection and quantitation of expression levels of genes or proteins are well known to those skilled in the art. A cell deficient in TNFα, leptin, JNK, or XBP-I is similarly understood.

[0046] As used herein, a "STAMP2 containing cell" is a cell in which STAMP2 expression is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% of the level of expression of STAMP2 in a wild-type cell. STAMP2 containing cells can include other mutations or defects. For example, STAMP2 containing cells can be derived from or contained in an animal with a metabolic and/or inflammatory disorder. Such animals include animals with genetic defects (heterozygous or homozygous), such as leptin (ob) deficient, JNK-deficient, XPB-I -deficient and db/db mice.. Animals with inflammatory disorders include, for example, heterozygous or homozygous TNFα deficient mice. Induced models of metabolic and inflammatory disease are also known. For example, high fat fed animals develop metabolic and eventually inflammatory disorders. [0047] As used herein, a "STAMP2 expressing cell" is a cell that expresses a detectable level of STAMP2. The expression of STAMP2 is preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more relative to a normal cell.

[0048] As used herein, a "STAMP2 promoter region" or "STAMP2 promoter" and the like are understood as at least the minimal nucleic acid sequence that controls transcription of the STAMP2 gene in vivo. A STAMP2 promoter region can optionally include additional enhancer or repressor sequences not absolutely required for the transcription of STAMP2. It is understood that a STAMP2 promoter region can include mutations, deletions, insertions, truncations, and/ or other alterations while retaining the function of a native, wild-type STAMP2 promoter and would be understood to fall within the definition of a STAMP2 promoter. A STAMP2 promoter region can be identified in any animal expressing STAMP2, preferably a mammal expressing STAMP2. Such sequences can be readily identified by one of skill in the art using routine methods and publicly available databases. An example of a STAMP2 promoter sequence is provided in SEQ ID NO: 1 which includes the human STAMP2 promoter region. SEQ ID NO: 1 GenBank Accession No. EF121762. (incorporated herein by reference). Identification of promoter sequences from other animals that express STAMP2, e.g., humans, is well within the skill of the art. Agents that modulate transcription through the mouse STAMP2 promoter are likely to modulate transcription through the human STAMP2 promoter.

[0049] As used herein, "metabolic disorder" is understood as a disease or condition related to an imbalance in energy intake and output in an animal. Metabolic diseases include, for example, diabetes, especially type 2 diabetes, obesity, insulin resistance, glucose intolerance, dyslipedemia, hyperglycemia, fatty liver disease, and hypercholesterolemia. Insulin resistance, glucose intolerance, and hyperglycemia can be caused by factors other than type 1 diabetes, including, for example, excess weight including obesity and type 2 diabetes. It is also noted that insulin resistance and glucose intolerance may persist after weight loss in some individuals. Metabolic disorders can be the result of genetic factors or predisposition, lifestyle, or a combination thereof. Genetic mouse models of metabolic disease include the naturally occurring db/db mouse, and both heterozygous and homozygous transgeneic leptin knockout mouse, JNK knockout mouse (Hirosumi et al., Nature, 420:333-6, 2002), and XBP- 1 knockout mouse (Ozcan et al., Science,306:457-61, 2004) [0050] As used herein, an "inflammatory disorder" is understood as a disease or condition related to swelling, redness, pain, heat, and loss of function and associated with the presence of inflammatory cells including mononuclear immune cells (monocytes, macrophages, lymphocytes, and plasma cells), and inflammatory mediators including interleukins and cytokines. Most inflammatory disorders are associated with chronic inflammation which is also associated with attempted wound healing and formation of scar tissue. Inflammation can be due to injury, autoimmune disorder, or other systemic stress (e.g., metabolic disorder).

[0051] As used herein, "modulate," "modulated," or "modulation" of a biomarker and the like are understood to mean increase or decrease expression or activity of a biomarker; modify a biomarker reversibly or irreversibly after synthesis possibly altering activity and interaction with downstream targets; and/or disrupt targeting or translocation of a biomarker.

[0052] As used herein, "modulation of STAMP2" and the like is understood to mean alteration of expression or biological activity of STAMP2. In a preferred embodiment, increased expression or activity is an increase of about at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or more activity as compared to cells or animals not treated with an agent to modulate STAMP2. Modulation of STAMP2 activity is preferably an increase in STAMP2 activity. An agent may act directly on STAMP2 or the STAMP2 promoter to increase activity or expression of STAMP2. Alternatively an agent may interact with a STAMP2 interacting protein, for example to increase STAMP2 activity or half life. [0053] As used herein, an "increase biological activity" mimics the phenotype of an increase in expression of STAMP2 causing a STAMP2 deficient cell to have a phenotype more closely mimicking or identical to a cell expressing a normal level of STAMP2. Phenotypic markers of STAMP2 deficient and normal cells include biomarkers of inflammation, oxidative stress, and metabolism. An increase in biological activity in an animal having at least one sign or symptom of a metabolic or inflammatory disease will decrease at least one sign or symptom of the metabolic or inflammatory disease.

[0054] As used herein, "obesity" is understood as a condition characterized by the excessive accumulation and storage of fat in the body. It is commonly defined as a body mass index (weight divided by height squared) of 30 kg/m² or higher. Overweight is typically defined as a BMI of 25-29.9 kg/m².

[0055] As used herein, "obtaining" as in "obtaining an agent" or "obtaining a cell" refers to purchasing, synthesizing, or otherwise procuring an agent or cell.

[0056] As used herein, "operably linked" is understood as juxtaposition a nucleic acid sequence including at least one transcriptional regulatory sequence (e.g., promoter sequence, repressor sequence, enhancer sequence) to a nucleic acid sequence for transcription including a transcriptional start site, and optionally a translational start site, such that transcription of the nucleic acid sequence is regulated by sequences in the regulatory region. For example, in a cell containing no mutations in the STAMP2 transcriptional regulatory region, the STAMP2 promoter is operably linked to the STAMP2 gene. The STAMP2 promoter sequence can be moved to an expression vector and can be operably linked to a sequence for expression of a reporter gene (e.g., beta-galactosidase, luciferase, alkaline phosphatase) to result in expression of the reporter gene upon activation of the promoter sequence. Alternatively, promoter activation can be detected using quantitative RT-PCR to detect a transcript expressed under the control of the STAMP2 promoter.

[0057] As used herein, "detecting" is understood as performing an assay to determine the presence or absence of a compound in a sample, such as a cell lysate, a tissue sample, or an animal. Detecting can include the determination that the amount of compound present is none or below the detection level of the assay method. Detecting can include determining the presence or absence of post-translational modifications.

[0058] According to the present invention, a "peptide" or "protein" comprises a string of at least three amino acids linked together by peptide bonds. The terms "protein" and "peptide" may be used interchangeably. [0059] As used herein, "polynucleotide" or "oligonucleotide" refers to a polymer of nucleotides, either synthetic or naturally occuring. A "short interfering RNA (siRNA)" is a double stranded RNA polynucleotide compound. An "antisense oligonucleotide" is typically a single stranded nucleotide polymer. An "shRNA" is a short hairpin RNA polynucleotide polymer that can adopt a folded, self-complementary structure. SiRNA, shRNA, and antisense oligonucleotides can be referred to collectively as "nucleic acid therapeutics." Nucleic acid therapeutics can also include ribozymes, aptamers, and longer double stranded RNA compounds (e.g., 25-35 nucleotides in length, see e.g., Kim et al., Nat Biotechnol. 23:222-6, 2005). The polynucleotide may include natural nucleosides, nucleoside analogs, chemically modified bases, biologically modified bases, intercalated bases, modified sugars, and/or modified phosphate groups. The polynucleotide can act to reduce expression of a target gene and/or protein, resulting in deficiency in a target gene and/or protein.

[0060] As used herein, the terms "prevent," "preventing," "prevention,"

"prophylactic treatment" and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition. Prevention or prophylactic treatment can require administration of more than one dose of the compositions of the invention.

[0061] As used herein a subject "prone to or suffering from" a metabolic disorder is an individual susceptible to developing a metabolic disorder due to any of a number of factors such as family history, ethnicity, or lifestyle (e.g., poor diet and/or exercise habits). A subject prone to a metabolic disorder can also show early signs or indicators of metabolic disorder such as excessive weight or body mass index, elevated cholesterol or triglyceride level, glucose intolerance, insulin resistance, dyslipedemia, or hyperglycemia, excess visceral fat, or a combination thereof, wherein the signs or symptoms do not yet constitute metabolic disease. A subject suffering from a metabolic disorder has at least one sign or indicator of the disorder such as excessive weight or body mass index, elevated cholesterol or triglyceride level, glucose intolerance, insulin resistance, diabetes, especially type 2 diabetes, and glucose intolerance and/or insulin resistance related to excess body weight, obesity, or type 2 diabetes, dyslipedemia, hyperglycemia, fatty liver disease, excess visceral fat, and hypercholesterolemia. Subjects prone to or suffering from a metabolic disorder exist have signs and/or symptoms that exist along a continuum such that no discrete distinction is required.

[0062] A "reporter gene" is a sequence that is operably linked to transcriptional control sequences that encodes a protein that is easily, and typically quantitatively, detectable using a colorimetric, luminescent, alkaline phosphatase, or fluorescent substrate. Commonly used reporter genes include, but are not limited to, beta-galactosidase, luciferase, and green fluorescent protein. A "reporter construct" is a nucleic acid including a reporter gene operably linked to a transcriptional control sequence. Typically reporter constructs are DNA plasmids. [0063] As used herein, the term "small molecule" refers to organic compounds, whether naturally-occurring or artificially created (e.g., via chemical synthesis) that have relatively low molecular weight and that are not proteins, polypeptides, or nucleic acids. Typically, small molecules have a molecular weight of less than about 1500 g/mol, 1000 g/mol, or 500 g/mol. Also, small molecules typically have multiple carbon-carbon bonds. [0064] As used herein, a "subject" is a mammal, preferably a human.

[0065] As used herein, "treat," "treating," "treatment" and the like are meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease, or to reduce at least one sign or symptom of a disease. Treatment can include administration of more than one dose at regular or irregular intervals. Treatment can include prophylaxis.

[0066] As used herein, "a", "an", and "the" are understood to be either singular or plural unless otherwise obvious from context.

[0067] As used herein, "or" is meant to be inclusive unless otherwise obvious from context. [0068] As used herein, ranges are understood to include all values within the range.

For example, 1 to 50 is understood to mean 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, and 50. A series of values are understood to represent a range, and thereby all of the values within the range unless otherwise obvious from context. [0069] Any two or more methods or embodiments of the invention set forth herein can be combined within the scope of the invention.

Screening Assays

[0070] Applicants have discovered that mice lacking STAMP2 exhibit metabolic disorders and related inflammatory changes, as well as disruptions in the expression of genes and polypeptides whose expression is altered in a STAMP2-defϊcient mouse (i.e., "STAMP2 regulated gene or polypeptide"). Accordingly, the invention provides compositions and methods for identifying agents that treat or prevent metabolic or inflammatory disorders by increasing STAMP2 expression or activity. In one preferred embodiment, the agent is a polypeptide that specifically binds STAMP2 and induces STAMP2 biological activity. In addition, the invention further provides methods for identifying agents that modulate the expression of genes and polypeptides whose expression is altered in a STAMP2-deficient mouse. Preferably, such agents "normalize" the expression of these genes and polypeptides, such that their expression is substantially similar to (i.e., is at least about 75%, 80%, 85%, 90%, or 95%) the level present in a "reference cell or animal," i.e., a normal control cell or animal not having a STAMP2 deficiency, a metabolic disorder, or an inflammatory disorder. A "normal" animal is typically referred to as a "wild-type" animal. [0071] As reported in more detail below, STAMP2 regulates the production of soluble molecules expressed by adipocytes. These soluble molecules regulate insulin action in liver. Accordingly, the invention provides methods for identifying and using such agents to promote insulin action, to increase metabolism, or to reduce inflammation, or a combination thereof. [0072] Agents identified by the methods described herein produce desirable changes in the metabolism of a subject. Metabolic changes are detected using methods known in the art and described herein. Such methods include measuring insulin responsiveness, serum insulin (e.g., by ELISA), blood glucose, serum triglycerides and cholesterol, glucose and insulin tolerance tests, distribution of lipoprotein particles in cells, tissues, or organs (e.g., liver lipid accumulation), quantitation of liver triglyceride, body weight, or body fat accumulation or localization (e.g., visceral or subcutaneous).

[0073] Methods of the invention are useful for the high-throughput low-cost screening of candidate agents that produce desirable metabolic changes (e.g., by enhancing insulin responsiveness, reducing hyperglycemia, or reducing body fat or obesity), that reduce inflammation, or that regulate the expression of a STAMP2 regulated gene or polypeptide. In one embodiment, the invention provides methods of identifying an agent that specifically binds to STAMP2 and stimulates STAMP2 activity. The agent is then isolated and tested for activity in an in vitro assay or in vivo assay for its ability to induce desirable metabolic changes, reduce inflammation, or normalize the expression of STAMP2 regulated genes or polypeptides. One skilled in the art appreciates that the effects of a candidate agent on a cell is typically compared to a corresponding control cell not contacted with the candidate agent. Thus, the screening methods include comparing a cell or animal contacted by a candidate agent to an untreated control cell or animal. In certain embodiments, the control cell or animal is contacted with a vehicle control, i.e., the solvent in which the agent is dissolved. [0074] In other embodiments, the expression or activity of STAMP2 in a cell treated with a candidate agent is compared to untreated control samples to identify a candidate compound that increases the expression or activity of STAMP2 in the contacted cell. Polypeptide expression or activity can be compared by procedures well known in the art, such as Western blotting, flow cytometry, immunocytochemistry, binding to magnetic and/or STAMP2 -specific antibody-coated beads, in situ hybridization, fluorescence in situ hybridization (FISH), ELISA, microarray analysis, RT-PCR, Northern blotting, or colorimetric assays, such as the Bradford Assay and Lowry Assay. Reporter constructs can also be used to identify agents that increase expression from a STAMP2 promoter. [0075] In one working example, one or more candidate agents are added at varying concentrations to the culture medium containing a STAMP2 expressing cell. An agent that promotes the expression of a STAMP2 polypeptide expressed in the cell is considered useful in the invention; such an agent may be used, for example, as a therapeutic to prevent, delay, ameliorate, stabilize, or treat a metabolic or inflammatory disease or disorder characterized by a deficiency in insulin responsiveness, an increase in insulin resistance, an increase in inflammatory changes, or a decrease in the expression of a STAMP2 polypeptide. Once identified, agents of the invention (e.g., agents that specifically bind to and/or stimulate STAMP2 activity or expression) may be used to increase metabolism, insulin responsiveness, or reduce inflammation in a patient in need thereof. An agent identified according to a method of the invention is locally or systemically delivered to increase insulin responsiveness or reduce inflammation in a subject.

[0076] In one embodiment, the effect of a candidate agent may, in the alternative, be measured at the level of STAMP2 polypeptide production using the same general approach and standard immunological techniques, such as Western blotting or immunoprecipitation with an antibody specific for STAMP2. For example, immunoassays may be used to detect or monitor the expression of STAMP2 in an adipose cell or other STAMP2-expressing cell. In one embodiment, the invention identifies a polyclonal or monoclonal antibody (produced using methods in the art, see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratories, 1988; O'Brien and Aitken, Antibody Phage Display: Methods and Protocols, Humana Press, 2002) that is capable of binding to and activating a STAMP2 polypeptide. An agent that promotes an increase in the expression or activity of a STAMP2 polypeptide is considered particularly useful. Again, such an agent may be used, for example, as a therapeutic to combat a metabolic disorder or inflammation.

[0077] Alternatively, or in addition, candidate agents may be identified by first assaying those that specifically bind to and activate a STAMP2 polypeptide of the invention in vitro and subsequently testing their effect on metabolism in vivo as described in the Examples (e.g., measuring insulin responsiveness, serum insulin, for example, by ELISA, blood glucose, serum triglycerides and cholesterol, glucose and insulin tolerance tests, distribution of lipoprotein particles in cells, tissues, or organs (e.g., liver lipid accumulation), quantitation of liver triglyceride, body weight, or body fat accumulation or localization (e.g., visceral or subcutaneous). In one embodiment, the efficacy of a candidate agent is dependent upon its ability to interact with the STAMP2 polypeptide. Such an interaction can be readily assayed using any number of standard binding techniques and functional assays (e.g., those described in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 2000). For example, a candidate compound may be tested in vitro for interaction and binding with a polypeptide of the invention and its ability to modulate metabolism may be assayed by any standard assays (e.g., those described herein).

[0078] Potential insulin responsiveness agonists include organic molecules, peptides, peptide mimetics, polypeptides, nucleic acid ligands, aptamers, and antibodies that bind to a STAMP2 polypeptide and stimulate its activity. In one particular example, a candidate compound that binds to a STAMP2 polypeptidemay be identified using a chromatography-based technique. For example, a recombinant STAMP2 polypeptide of the invention can be purified by standard techniques from cells engineered to express the polypeptide, or may be chemically synthesized. Once purified, the peptide, optionally fused to a peptide sequence to facilitate binding to a solid support, is immobilized on a column. A solution of candidate agents is then passed through the column, and an agent that specifically binds the STAMP2 polypeptide or a fragment thereof is identified on the basis of its ability to bind to STAMP2 polypeptide and to be immobilized on the column. To isolate the agent, the column is washed to remove non-specifically bound molecules, and the agent of interest is then released from the column and collected. Agents isolated by this method (or any other appropriate method) may, if desired, be further purified (e.g., by high performance liquid chromatography). In addition, these agents may be tested for their ability to modulate metabolism or inflammation(e.g., as described herein). Agents isolated by this approach may also be used, for example, as therapeutics to treat or prevent the onset of a disease or disorder characterized by a reduction in insulin responsiveness or to treat or prevent inflammation.

Compounds that are identified as binding to a STAMP2 polypeptide with an affinity constant less than or equal to 1 nM, 5 nM, 10 nM, 100 nM, 1 μM or 10 μM are considered particularly useful in the invention.

[0079] A polynucleotide sequence encoding STAMP2 may also be used in the discovery and development of therapeutics or prophylactics. The encoded STAMP2 protein, upon expression, can be used as a target for the screening of drugs to enhance metabolism or reduce inflammation. The STAMP2 agonists of the invention may be employed, for instance, to inhibit and treat a variety of metabolic disorders, including diabetes and obesity.

Test Compounds and Extracts

[0080] In general, agents of the invention, such as agents that increase the expression or activity of a STAMP2 polypeptide, STAMP2 agonists (e.g., agents that specifically bind and stimulate a STAMP2 polypeptide) or agents that normalize the expression of a STAMP2 regulated gene or polypeptide are identified from large libraries of natural product or synthetic (or semi-synthetic) extracts or chemical libraries or from polypeptide or nucleic acid libraries, according to methods known in the art. Clinical compound based libraries including known pharmaceutical agents, preferably approved for use in humans have been found to be useful for the identification of new uses for approved compounds. Those skilled in the field of drug discovery and development will understand that the precise source of test extracts or compounds is not critical to the screening procedure(s) of the invention. Agents used in screens may include known those known as therapeutics for the treatment of metabolic disorders or inflammation. Alternatively, virtually any number of unknown chemical extracts or compounds can be screened using the methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal- based extracts, fermentation broths, and synthetic compounds, as well as the modification of existing polypeptides. [0081] Libraries of natural polypeptides in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, FIa.), and PharmaMar, U.S.A. (Cambridge, Mass.). Such polypeptides can be modified to include a protein transduction domain using methods known in the art and described herein. In addition, natural and synthetically produced libraries are produced, if desired, according to methods known in the art, e.g., by standard extraction and fractionation methods. Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al., Proc. Natl. Acad. Sci. U.S. A 90:6909, 1993; Erb et al., Proc. Natl. Acad. Sci. USA 91 : 11422, 1994; Zuckermann et al., J. Med. Chem 37:2678, 1994; Cho et al., Science 261 : 1303, 1993; Carrell et al., Angew. Chem. Int. Ed. Engl. 33:2059, 1994; Carell et al.,

Angew. Chem. Int. Ed. Engl. 33:2061, 1994; and Gallop et al., J. Med. Chem. 37:1233, 1994. Furthermore, if desired, any library or compound is readily modified using standard chemical, physical, or biochemical methods.

[0082] Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of polypeptides, chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds. Synthetic compound libraries are commercially available from Brandon Associates (Merrimack, N.H.) and Aldrich Chemical (Milwaukee, Wis.). Alternatively, chemical compounds to be used as candidate compounds can be synthesized from readily available starting materials using standard synthetic techniques and methodologies known to those of ordinary skill in the art. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the compounds identified by the methods described herein are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2nd ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof.

[0083] Libraries of compounds may be presented in solution (e.g., Houghten, Biotechniques 13:412-421, 1992), or on beads (Lam, Nature 354:82-84, 1991), chips (Fodor, Nature 364:555-556, 1993), bacteria (Ladner, U.S. Patent No. 5,223,409), spores (Ladner U.S. Patent No. 5,223,409), plasmids (Cull et al., Proc Natl Acad Sci USA 89:1865-1869, 1992) or on phage (Scott and Smith, Science 249:386-390, 1990; Devlin, Science 249:404- 406, 1990; Cwirla et al. Proc. Natl. Acad. Sci. 87:6378-6382, 1990; Felici, J. MoI. Biol. 222:301-310, 1991; Ladner supra.).

[0084] In addition, those skilled in the art of drug discovery and development readily understand that methods for dereplication (e.g., taxonomic dereplication, biological dereplication, and chemical dereplication, or any combination thereof) or the elimination of replicates or repeats of materials already known for their activity should be employed whenever possible.

[0085] When a crude extract is found to have STAMP2 binding and/or stimulating activity further fractionation of the positive lead extract is necessary to isolate molecular constituents responsible for the observed effect. Thus, the goal of the extraction, fractionation, and purification process is the careful characterization and identification of a chemical entity within the crude extract that enhances insulin responsiveness, normalizes or increases metabolism, or reduces inflammation. Methods of fractionation and purification of such heterogeneous extracts are known in the art. If desired, compounds shown to be useful as therapeutics are chemically modified according to methods known in the art. In vitro and In vivo Models of Metabolic Disease

[0086] The invention is based, at least in part, on the observation that STAMP2 deficient cells and mice are useful for the identification of therapeutics for the treatment or prevention of metabolic or inflammatory disorders. Cells having reduced expression of a gene of interest are generated using any method known in the art. In one embodiment, a targeting vector is used that creates a knockout mutation in a STAMP2 gene. The targeting vector is introduced into a suitable cell (e.g., ES cell) or cell line to generate one or more cell lines that carry a knockout mutation. By a "knockout mutation" is meant an artificially- induced alteration in a nucleic acid molecule (created by recombinant DNA technology or deliberate exposure to a mutagen) that reduces the biological activity of the STAMP2 polypeptide normally encoded therefrom by at least about 50%, 75%, 80%, 90%, 95%, or more relative to the unmutated gene. The mutation can be, without limitation, an insertion, deletion, frameshift mutation, or a missense mutation. The targeting construct may result in the disruption of the gene of interest, e.g., by insertion of a heterologous sequence containing stop codons, or the construct may be used to replace the wild-type gene with a mutant form of the same gene, e.g. a "knock-in." In some embodiments, dominant negative versions of the protein can be generated that interfere with the function of the wild-type protein, preventing it from having its usual effect.

[0087] In another example, FRT sequences may be introduced into the cell such that they flank the gene of interest. Transient or continuous expression of the FLP protein is then used to induce site-directed recombination, resulting in the excision of the gene of interest. The use of the FLP/FRT system is well established in the art and is described in, for example, U.S. Pat. No. 5,527,695, and in Lyznik et al. (Nucleic Acid Research 24:3784-3789, 1996).

[0088] Furthermore, the targeting construct may contain a sequence that allows for conditional expression of the gene of interest. For example, a sequence may be inserted into the gene of interest that results in the protein not being expressed in the presence of tetracycline. Such conditional expression of a gene is described in, for example, Yamamoto et al. (Cell 101 :57-66, 2000)).

[0089] Conditional knockout cells are also produced using the Cre-lox recombination system. Cre is an enzyme that excises DNA between two recognition sites termed loxP. The cre transgene may be under the control of an inducible, developmentally regulated, tissue specific, or cell-type specific promoter. In the presence of Cre, the gene, for example a nucleic acid sequence described herein, flanked by loxP sites is excised, generating a knockout. This system is described, for example, in Kilby et al. (Trends in Genetics 9:413- 421, 1993). [0090] Construction of transgenes can be accomplished using any suitable genetic engineering technique, such as those described in Ausubel et al. (Current Protocols in Molecular Biology, John Wiley & Sons, New York, 2000). Many techniques of transgene construction and of expression constructs for transfection or transformation in general are known and may be used for the disclosed constructs.

[0091] One skilled in the art will appreciate that a promoter is chosen that directs expression of the chosen gene in a cell of interest. Any promoter that regulates expression of a nucleic acid sequence described herein can be used in the expression constructs of the present invention. One skilled in the art would be aware that the modular nature of transcriptional regulatory elements and the absence of position-dependence of the function of some regulatory elements, such as enhancers, make modifications such as, for example, rearrangements, deletions of some elements or extraneous sequences, and insertion of heterologous elements possible. Numerous techniques are available for dissecting the regulatory elements of genes to determine their location and function. Such information can be used to direct modification of the elements, if desired. It is advantageous, however, that an intact region of the transcriptional regulatory elements of a gene is used. Once a suitable transgene construct has been made, any suitable technique for introducing this construct into cells can be used.

Inhibitory Nucleic Acids

[0092] Cells or animals having reduced levels of STAMP2 may be generated using inhibitory polynucleotides that reduce the expression or activity of a STAMP2 polypeptide. Inhibitory nucleic acid molecules are those oligonucleotides that inhibit the expression or activity of a STAMP2 polypeptide. Such oligonucleotides include single and double stranded nucleic acid molecules (e.g., DNA, RNA, PNA, and analogs thereof) that bind a nucleic acid molecule that encodes a STAMP2 polypeptide (e.g., antisense molecules, siRNA, shRNA) as well as nucleic acid molecules that bind directly to a STAMP2 polypeptide to modulate its biological activity (e.g., aptamers).

[0093] Short twenty-one to twenty-five nucleotide double-stranded RNAs are effective at down-regulating gene expression (Zamore et al., Cell 101 : 25-33; Elbashir et al., Nature 411 : 494-498, 2001 , hereby incorporated by reference). The therapeutic effectiveness of an siRNA approach in mammals was demonstrated in vivo by McCaffrey et al. (Nature 418: 38-39.2002), Reich et al. (MoI. Vis. 9:210-216, 2003), and Zimmerman et al. (Nature, 441 : 111 -114, 2006). Twenty nucleotide antisense oligonucleotides have been demonstrated to have therapeutic effectiveness in vivo by Kastelein et al. (Circulation, 114: 1729-35, 2006). [0094] Given the sequence of a target gene, nucleic acid therapeutics may be designed to inactivate that gene. Such nucleic acid therapeutics, for example, can be administered directly to an affected tissue, or administered systemically. The nucleic acid sequence of an STAMP2 gene can be used to design double stranded RNAs of the desired length (about 21 to about 35 nucleotides in length) or single stranded oligonucleotides. The nucleic acid therapeutics may be used, for example, as therapeutics to treat a metabolic disease and/or inflammatory disease.

[0095] The inhibitory nucleic acid molecules of the present invention may be employed as double-stranded RNAs for RNA interference (RNAi)-mediated knock-down of STAMP2 expression. In one embodiment, STAMP2 expression is reduced in an adiopocyte cell or other cell type. RNAi is a method for decreasing the cellular expression of specific proteins of interest (reviewed in Tuschl, Chembiochem 2:239-245, 2001 ; Sharp, Genes & Devel. 15:485-490, 2000; Hutvagner and Zamore, Curr. Opin. Genet. Devel. 12:225-232, 2002; and Harmon, Nature 418:244-251, 2002). The introduction of siRNAs into cells either by transfection of dsRNAs or through expression of siRNAs using a plasmid-based expression system is increasingly being used to create loss-of-function phenotypes in mammalian cells.

[0096] In one embodiment of the invention, double-stranded RNA (dsRNA) molecule is made that includes between eight and nineteen consecutive nucleobases of a nucleobase oligomer of the invention. The dsRNA can be two distinct strands of RNA that have duplexed, or a single RNA strand that has self-duplexed (small hairpin (sh)RNA). Typically, dsRNAs are about 21 or 22 base pairs, but may be shorter or longer (up to about 29 nucleobases) if desired. In some embodiments, the dsRNA can be about 25 to about 35 nucleotides in length, or longer (US Patent Publication 20050244858 and 20050277610). dsRNA can be made using standard techniques (e.g., chemical synthesis or in vitro transcription). Kits are available, for example, from Ambion (Austin, Tex.) and Epicentre (Madison, Wis.). Methods for expressing dsRNA in mammalian cells are described in Brummelkamp et al. Science 296:550-553, 2002; Paddison et al. Genes & Devel. 16:948-958, 2002. Paul et al. Nature Biotechnol. 20:505-508, 2002; Sui et al. Proc. Natl. Acad. Sci. USA 99:5515-5520, 2002; Yu et al. Proc. Natl. Acad. Sci. USA 99:6047-6052, 2002; Miyagishi et al. Nature Biotechnol. 20:497-500, 2002; and Lee et al. Nature Biotechnol. 20:500-505 2002, each of which is hereby incorporated by reference.

[0097] Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a STAMP2 polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having "substantial identity" to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By "hybridize" is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).

[0098] For example, stringent salt concentration will ordinarily be less than about

750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 37° C, and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred: embodiment, hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate, 1 % SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

[0099] For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, more preferably of at least about 42° C, and even more preferably of at least about 68° C. In a preferred embodiment, wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[00100] By "substantially identical" is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison. [00101] Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e^"3 and e^"100 indicating a closely related sequence.

Delivery of Inhibitory Nucleic Acid Molecules

[00102] Naked nucleic acid therapeutics or inhibitory nucleic acids (e.g., siRNA, shRNA, other double stranded RNA therapeutics, or antisense polynucleotides), or analogs thereof, are capable of entering mammalian cells and inhibiting expression of a gene of interest. Nonetheless, it may be desirable to utilize a formulation that aids in the delivery of oligonucleotides or other nucleobase oligomers to cells (see, e.g., U.S. Pat. Nos. 5,656,611, 5,753,613, 5,785,992, 6,120,798, 6,221,959, 6,346,613, and 6,353,055, each of which is hereby incorporated by reference) Methods and compositions for formulation and delivery of phamraceutical agents is provided, for example, in Remington's Pharmaceutical Sciences by Mack Publishing (21^st Edition, 2005). Therapeutic Methods

[00103] Agents identified as binding and/or increasing the expression or activity of a

STAMP2 polypeptide are useful for preventing or ameliorating a disease associated with insulin resistance or increased inflammation. In one therapeutic approach, an agent identified as described herein is administered to the site of a potential or actual disease-affected tissue or is administered systemically. The dosage of the administered agent depends on a number of factors, including the size and health of the individual patient. For any particular subject, the specific dosage regimes should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions.

[00104] Accordingly, the present invention provides methods of treating a metabolic or inflammatory disease and/or disorders or symptoms thereof which comprise administering a therapeutically effective amount of a pharmaceutical composition comprising an agent identified according to a method of the invention to a subject (e.g., a mammal, such as a human). Thus, one embodiment is a method of treating a subject suffering from or susceptible to a metabolic or inflammatory disease or disorder or symptom thereof. The method includes the step of administering to the mammal a therapeutic amount of an amount of a compound herein sufficient to treat the disease or disorder, or sign or symptom thereof, under conditions such that the disease or disorder is treated.

[00105] The methods herein include administering to the subject (including a subject identified as in need of such treatment) an effective amount of an agent described herein, or a composition described herein to produce such effect. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method).

[00106] The therapeutic methods of the invention (which include prophylactic treatment) in general comprise administration of a therapeutically effective amount of the agents herein, such as an agent identified using the methods of the invention to a subject (e.g., animal, human) in need thereof, including a mammal, particularly a human. Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a disease, or disorder, or at least one sign or symptom thereof. Determination of those subjects "at risk" can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, biomarker (as defined herein), family history, and the like). The compounds herein may be also used in the treatment of any other disorders in which an increase in insulin resistance, an increase in obesity, or an increase in inflammation may be implicated.

[00107] In one embodiment, the invention provides a method of monitoring treatment progress. The method includes the step of determining a level of diagnostic marker (Marker) (e.g., any target delineated herein modulated by a compound herein, a protein or indicator thereof, a biomarker, etc.) or diagnostic measurement (e.g., screen, assay) in a subject suffering from or susceptible to a disorder or symptoms thereof associated with an increase in insulin resistance, an increase in obesity, or an increase in inflammation, in which the subject has been administered a therapeutic amount of a compound herein sufficient to treat the disease or symptoms thereof. The level of Marker determined in the method can be compared to known levels of Marker in either healthy normal controls or in other afflicted patients to establish the subject's disease status. In preferred embodiments, a second level of Marker in the subject is determined at a time point later than the determination of the first level, and the two levels are compared to monitor the course of disease or the efficacy of the therapy. In certain preferred embodiments, a pre-treatment level of Marker in the subject is determined prior to beginning treatment according to this invention; this pre-treatment level of Marker can then be compared to the level of Marker in the subject after the treatment commences, to determine the efficacy of the treatment.

Pharmaceutical Therapeutics

[00108] The invention provides a simple means for identifying agents (including nucleic acids, peptides, and small molecule agonists) capable of binding to an activating STAMP2, enhancing insulin responsiveness, increasing metabolism, or acting as therapeutics or medicaments for the treatment or prevention of a metabolic or inflammatory disorder. Accordingly, a chemical entity or agent discovered to have medicinal value using the methods described herein is useful as a drug or as information for structural modification of existing compounds, e.g., by rational drug design. Such methods are useful for screening agents having an effect on a variety of conditions characterized by a reduction in metabolism or an increase in insulin resistance or inflammation. [00109] For therapeutic uses, the compositions or agents identified using the methods disclosed herein may be administered systemically, for example, formulated in a pharmaceutically-acceptable buffer such as physiological saline. Preferable routes of administration include, for example, subcutaneous, intravenous, interperitoneally, intramuscular, or intradermal injections that can in some embodiments provide continuous, sustained levels of the drug in the patient. Treatment of human patients or other animals will be carried out using a therapeutically effective amount of a therapeutic identified herein in a physiologically-acceptable carrier. Suitable carriers and their formulation are described, for example, in Remington's Pharmaceutical Sciences by E. W. Martin. The amount of the therapeutic agent to be administered varies depending upon the manner of administration, the age and body weight of the patient, and with the clinical symptoms of the metabolic disorder or inflammation. Generally, amounts will be in the range of those used for other agents used in the treatment of other diseases associated with a metabolic disorder or inflammation, although in certain instances lower amounts will be needed because of the increased specificity of the compound. A compound is administered at a dosage that activates STAMP2 or that increases insulin action as determined by a method known to one skilled in the art, or using any assay that measures the expression or the biological activity of a STAMP2 polypeptide.

Formulation of Pharmaceutical Compositions [00110] The administration of an agent for the treatment of a metabolic disorder or inflammation may be by any suitable means that results in a concentration of the therapeutic that, combined with other components, is effective in ameliorating, reducing, or stabilizing a metabolic disorder or inflammation. The compound may be contained in any appropriate amount in any suitable carrier substance, and is generally present in an amount of 1-95% by weight of the total weight of the composition. The composition may be provided in a dosage form that is suitable for parenteral (e.g., subcutaneously, intravenously, intramuscularly, or intraperitoneally) administration route. The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000; Goodman and Gilman: The Pharmacological Basis of Therpeutics by Laurence Brunton; and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).

[00111] Pharmaceutical compositions according to the invention may be formulated to release the active compound substantially immediately upon administration or at any predetermined time or time period after administration. The latter types of compositions are generally known as controlled release formulations, which include (i) formulations that create a substantially constant concentration of the drug within the body over an extended period of time; (ii) formulations that after a predetermined lag time create a substantially constant concentration of the drug within the body over an extended period of time; (iii) formulations that sustain action during a predetermined time period by maintaining a relatively, constant, effective level in the body with concomitant minimization of undesirable side effects associated with fluctuations in the plasma level of the active substance (sawtooth kinetic pattern); (iv) formulations that localize action by, e.g., spatial placement of a controlled release composition adjacent to or in contact with an adipose tissue; (v) formulations that allow for convenient dosing, such that doses are administered, for example, once every one or two weeks; and (vi) formulations that target a metabolic disorder or inflammation by using carriers or chemical derivatives to deliver the therapeutic agent to a particular cell type (e.g., adipose cell). For some applications, controlled release formulations obviate the need for frequent dosing during the day to sustain the plasma level at a therapeutic level. [00112] Any of a number of strategies can be pursued in order to obtain controlled release in which the rate of release outweighs the rate of metabolism of the compound in question. In one example, controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, e.g., various types of controlled release compositions and coatings. Thus, the therapeutic is formulated with appropriate excipients into a pharmaceutical composition that, upon administration, releases the therapeutic in a controlled manner. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, patches, and liposomes.

Parenteral Compositions

[00113] The pharmaceutical composition may be administered parenterally by injection, infusion or implantation (subcutaneous, intravenous, intramuscular, intraperitoneal, or the like) in dosage forms, formulations, or via suitable delivery devices or implants containing conventional, non-toxic pharmaceutically acceptable carriers and adjuvants. The formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulation. Formulations can be found in Remington: The Science and Practice of Pharmacy, supra.

[00114] Compositions for parenteral use may be provided in unit dosage forms (e.g., in single-dose ampoules), or in vials containing several doses and in which a suitable preservative may be added as is well known in the art. The composition may be in the form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation, or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use. Apart from the active agent that reduces or ameliorates a metabolic disorder or inflammation, the composition may include suitable parenterally acceptable carriers and/or excipients. The active therapeutic agent(s) may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for controlled release. Furthermore, the composition may include suspending, solubilizing, stabilizing, pH-adjusting agents, tonicity adjusting agents, and/or dispersing, agents.

[00115] As indicated above, the pharmaceutical compositions according to the invention may be in the form suitable for sterile injection. To prepare such a composition, the suitable active therapeutic(s) are dissolved or suspended in a parenterally acceptable liquid vehicle. Among acceptable vehicles and solvents that may be employed are water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution, and isotonic sodium chloride solution and dextrose solution. The aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate). In cases where one of the compounds is only sparingly or slightly soluble in water, a dissolution enhancing or solubilizing agent can be added, or the solvent may include 10-60% w/w of propylene glycol or the like.

Combination Therapies

[00116] Optionally, a therapeutic of the invention is administered in combination with any other standard metabolic or inflammatory therapy; such methods are known to the skilled artisan and described in Remington's Pharmaceutical Sciences (supra).

Kits

[00117] The invention provides kits for the treatment or prevention of a metabolic or inflammatory disease or disorder. In one embodiment, the kit includes a therapeutic or prophylactic composition or medicament containing an effective amount of an agent that increases STAMP2 expression or activity in unit dosage form. In some embodiments, the kit comprises a sterile container which contains a therapeutic or prophylactic cellular composition; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

[00118] If desired an agent of the invention is provided together with instructions for administering the agent or medicament to a subject having or at risk of developing metabolic or inflammatory disease or disorder. The instructions will generally include information about the use of the composition for the treatment or prevention of the disease. In other embodiments, the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for treatment or prevention of a metabolic or inflammatory disease or symptoms thereof; precautions; warnings; indications; counter-indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

[00119] In some embodiments kits can include kits for screening compounds for modulation of STAMP2 activity or expression. Kits can include, for example, an expression plasmid in which a reporter gene is expressed under the control of a STAMP2 promoter, in appropriate packaging. The kit can further include, for example at least one of, instructions for use, a control plasmid, a STAMP2 deficient cell, or reagents for detecting expression of the reporter gene.

General Considerations

[00120] Given the potential for nutrients to stimulate inflammatory pathways and the importance of keeping these pathways in check, it was hypothesized that previously unrecognized counter-regulatory mechanisms might exist to protect cells from activation of inflammatory pathways during physiological fluctuations in nutrient exposure or in nutrient- rich conditions. It was reasoned that a factor participating in such a coordinating mechanism between nutrient and inflammatory responses would be expected to meet several criteria: 1) The gene product should be present in tissue types responsible for nutrient clearance from blood, such as adipose tissue. 2) Expression or activity should be regulated by both nutritional and inflammatory stimuli. 3) The factor should regulate inflammatory signaling components and/or gene expression. Cells or tissues lacking such a factor would then exhibit excess or prolonged inflammation in response to challenges by both nutrients and inflammatory mediators. 4) The factor should regulate cellular metabolism, and its absence should result in impaired cellular metabolic function. 5) Through regulation of metabolic function in particular cell-types and organs, the factor should also impact systemic metabolism.

[00121] A six-transmembrane protein of prostate 2 (STAMP2) was identified as a novel factor meeting these criteria. STAMP2, also known as TNF-induced adipose related protein (TIARP) or six transmembrane epithelial antigen of prostate 4 (STEAP4), belongs to a family of six transmembrane proteins, termed either the STAMP or STEAP family (Moldes et al. 2001 ; Korkmaz et al. 2005; Ohgami et al. 2006). Three of the four family members, including STAMP2, have recently been characterized as metalloreductases playing a role in cellular import of iron and copper (Ohgami et al. 2006). Here, it is reported that STAMP2 coordinates inflammatory responses with metabolic function in adipocytes and is essential for maintenance of systemic metabolic homeostasis.

[00122] The results of this study position STAMP2 as modulating inflammatory responses and protecting metabolic function in adipocytes. Recent studies have indicated the relevance of nutrients in activating inflammatory pathways (Hotamisligil 2006). Much evidence has shown the role of hyperglycemia in mediating diabetic complications, and recently the ability of glucose to activate oxidative stress and inflammatory pathways in adipocytes has also been shown (Brownlee 2001; Lin et al. 2004). Additionally, free fatty acids can activate inflammatory responses and impair insulin action in adipocytes (Nguyen et al. 2005). Thus, excess nutrients can be damaging. It is demonstrated herein that STAMP2 likely plays an important role in the adipocyte defense arsenal against nutrient surplus. The results disclosed herein demonstrate not only that acute nutritional challenges can cause excessive inflammation in the adipose tissue of STAMP2^"/" mice, but that even in conditions of ad lib feeding on a standard diet, STAMP2 prevents excessive inflammation and protects adipocyte insulin sensitivity and systemic glucose homeostasis.

[00123] The findings that STAMP2 both protects against excessive inflammation and is also upregulated under inflammatory and nutrient-rich conditions may appear paradoxical at first. The results herein demonstrate that STAMP2 acts in a regulatory role, not to block activation of inflammatory pathways, but to restrict the degree of their activity. This is exemplified in the regulation of STAMP2 by TNFα. TNFα induces both STAMP2 and inflammatory cytokine expression; yet, in the absence of STAMP2, the ability of TNFα to promote IL-6 expression is more potent. STAMP2 acts in a similar regulatory capacity in response to acute nutritional challenges or chronic hyperglycemia, ob/ob mice lacking

STAMP2 exhibit an exacerbated metabolic phenotype as compared to ob/ob mice with intact STAMP2 function. It is important to note that ob/ob mice experience much more rapid and severe onset of obesity than would be typical for either mice fed a high fat diet or for most human obesity.

[00124] STAMP2 belongs to a family of four mammalian proteins, which have been described within recent years under several different names, including STAMPl (STEAP2), STAMP2 (TIARP; STEAP4), STEAP, and STEAP3 (pHyde; TSAP6) (Hubert et al. 1999; Steiner et al. 2000; Moldes et al. 2001; Korkmaz et al. 2002; Passer et al. 2003; Korkmaz et al. 2005; Ohgami et al. 2005). While all members have been characterized in the prostate, STAMP2 is the only family member that has been previously reported to be expressed in adipocytes and adipose tissue (Moldes et al. 2001). No role for STAMP2 had been previously described prior to this report.

[00125] STAMP2 deficiency in mice results in impaired insulin action at tissues critical for glucose homeostasis: fat, liver, and muscle. Although STAMP2 expression is highest in WAT in mice, it is also present in liver and muscle.

[00126] Finally, this study also highlights the differences in biology between visceral and subcutaneous WAT and the unique involvement of STAMP2 in this distinction. Clearly, the visceral depot has a much stronger phenotype than the subcutaneous depot in STAMP2 deficiency. Without wishing to be bound by mechanism, this suggests that the visceral depot is either more susceptible to environmental insults such as hyperglycemia, due to fundamental metabolic differences between the depots, or more exposed to such stressors, due perhaps to ^' differences in vascularization or circulation. In addition to the clinical association between visceral obesity and metabolic syndrome, there are differences in gene expression patterns between the visceral and subcutaeous depots (Lefebvre et al. 1998; Despres and Lemieux 2006).

[00127] The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, "Molecular Cloning: A Laboratory Manual", second edition (Sambrook, 1989);

"Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction", (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991), and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratories, 1988. These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

[00128] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Example 1: STAMP2^"7" and ob/ob mice

[00129] STAMPI^1' mice were purchased from Deltagen, Inc. (San Carlos, CA)

(Deltagen tracking number ZBB519) where they were generated by gene targeting into embryonic stem cells derived from 129P2/OlaHsd mice and injected into C57B1/6 blastocysts. Two heterozygous breeding pairs from Deltagen were obtained, which were used to generate WT and STAMP2^'1' mice. Mice were given free access to the standard rodent diet (PicoLab Mouse Diet 20, containing 9% fat by weight [21.6% kcal from fat]). Age-matched lean and obese ob/ob (leptin deficient) male mice used in feeding/ fasting experiment were obtained from Jackson Labs. To generate STAMP2^~/~ob/ob mice, STAMPI^1' mice were intercrossed with ob^+/' mice and Fl double heterozygotes were then used to generate the STAMPI^{1 '}ob/ob and ob/ob genotypes. The Institutional Animal Care and Use Committee (Harvard School of Public Health) approved all studies.

Example 2: STAMP2 promoter-linked reporter assays. [00130] A 2kb fragment of mouse 5'-STAMP2 promoter region was cloned into a reporter plasmid [Accession No: EF121762, SEQ ID NO: I]. Promoter activity was evaluated in HeIa cells (ATCC) cultured in DMEM supplemented with 10% fetal calf serum. Cells were seeded in 6-well plates, grown to -50% confluency and transfected with plasmid DNA with the indicated transcription factors and ligands for LXR (T0901317, lOμM), PPARγ (pioglitazone, 1.5μM), or DMSO followed by luciferase assays, as described previously (Slagsvold et al. 2001).

Example 3: Histology and immunostaining.

[00131] Five to six month old wild type and STAMP2^"'^" mice were sacrificed by ketamine/xylazine injection. Tissue samples were fixed in formalin immediately upon dissection from animals. Paraffin embedding, mounting, and hematoxylin and eosin staining were carried out by Harvard Medical School Rodent Histopathology core facility. For F4/80 staining, samples were first paraffin embedded and 5- micron slices mounted on slides. Samples were deparafinized and immunoperoxidase staining was conducted using anti-F4/80 primary antibody (Serotec) and VECTASTAIN Elite ABC kit (Vector Laboratories, Burlingame, CA).

[00132] For detection of STAMP2 in cells, cells were first fixed in 2% paraformaldehyde for 20 minutes at room temperature and permeabilized in 0.5% Triton-X for 5 minutes on ice. Slides were blocked in 4% donkey serum plus 2% BSA and STAMP2 was detected using the anti-TIARP antibody followed by anti-rabbit Alexa-488.

[00133] For STAMP2 staining in tissue sections, first, nonspecific antigenic sites were blocked by 5% goat serum diluted in 2% bovine serum albumin in phosphate buffered saline at room temperature for 1 hour. The slides were then incubated in primary antibody (anti-TIARP, 1 :200) at 4°C overnight. The sections were then incubated at room temperature with a biotinylated secondary antibody (Vector) and with the ABC-complex for 30 minutes respectively. Finally, staining was done by using the Vector Red Alkaline Phosphatase Substrate kit.

Example 4: Quantitative PCR.

[00134] RNA was isolated from tissues and cells using Trizol (Invitrogen). cDNA was synthesized using either Thermoscript (Invitrogen) or iScript (BioRad). Q-PCR was performed using a BioRad iCycler. Primers were synthesized by Integrated DNA Technologies. Primer sequences were:

Adiponectin (F- GAT GGC AGA GAT GGC ACT CC (SEQ ID NO: 2); R- CTT GCC AGT GCT GCC GTC AT); (SEQ ID NO: 3)

Catalase (F- AGC GAC CAG ATG AAG CAG TG (SEQ ID NO: 4); R- TCC GCT CTC TGT CAA AGT GTG); (SEQ ID NO: 5) FAS (F- GGA GGT GGT GAT AGC CGG TAT (SEQ ID NO: 6); R- TGG GTA ATC CAT AGA GCC CAG); (SEQ ID NO: 7)

GST (F- AAG AAT GGA GCC TAT CCG GTG (SEQ ID NO: 8); R- CCA TCA CTT CGT AAC CTT GCC); (SEQ ID NO: 9)

IL-6 (F-ACA ACC ACG GCC TTC CCT ACT T (SEQ ID NO: 10); R- CAC GAT TTC CCA GAG AAC ATG TG); (SEQ ID NO: 11 )

MCP-I (F- CCA CTC ACC TGC TGC TAC TCA T (SEQ ID NO: 12); R- TGG TGA TCC TCT TGT AGC TCT CC); (SEQ ID NO: 13)

NADPH oxidase 1 (F- GGT TGG GGC TGA ACA TTT TTC (SEQ ID NO: 14) ; R- TCG ACA CAC AGG AAT CAG GAT); (SEQ ID NO: 15) SCD-I (TTC TTG CGA TAC ACT CTG GTG C (SEQ ID NO: 16); R- CGG GAT TGA ATG TTC TTG TCG T); (SEQ ID NO: 17) SOCS-3 (F- CAC AGC AAG TTT CCC GCC GCC (SEQ ID NO: 18); R- GTG CAC CAG CTT GAG TAC ACA); (SEQ ID NO: 19)

SOD-I (F- AAC CAG TTG TGT TGT CAG GAC (SEQ ID NO: 20); R- CCA CCATGT TTC TTA GAG TGA GG); (SEQ ID NO: 21) STAMP2 (F- TCA AAT GCG GAA TAC CTT GCT (SEQ ID NO: 22); R- GCA TCT AGT GTT CCT GAC TGG A); (SEQ ID NO: 23)

STEAP (F- GGT CGC CAT TAC CCT CTT GG (SEQ ID NO: 24); R- GGT ATG AGA GAC TGT AAA CAG CG); (SEQ ID NO: 25)

STEAP3 (F- CCC GTC CAT TGC TAA TTC CCT (SEQ ID NO: 26); R- CAG AAA AGA GAC CCG AAC CCA); (SEQ ID NO: 27)

TNFα (F- CCC TCA CAC TCA GAT CAT CTT CT (SEQ ID NO: 28); R- GCT ACG ACG TGG GCT ACA G) (SEQ ID NO: 29).

Example 5: Cell culture and siRNA STAMP2 knockdown. [00135] 3T3-L1 preadipocytes were maintained in DMEM supplemented with 10% calf serum. To differentiate, cells were grown to confluence and placed into induction medium (DMEM, 10% cosmic calf serum, 5 μg/ml insulin, 0.5 mM IBMX, 1 μM Dexamethazone, and 10 μM TZD. After 2 days, cells were switched into differentiation medium (DMEM, 10% cosmic calf serum, 5 μg/ml insulin) until used for experimentation. [00136] For insulin signaling experiments, cells were serum-starved overnight, treated with insulin for 100 seconds, and immediately frozen in liquid nitrogen prior to preparation of protein lysate.

[00137] For RNAi experiments, siRNA specific to STAMP2 (target sequence: AAG

CAG CAT CCA AGT CTG ACA; SEQ ID NO: 30) and non-specific control were synthesized by Qiagen. An Amaxa Nucleofector was used to electroporate siRNA into 3T3- Ll adipocytes 5 days after inducing differentiation. Experiments were performed 36-48 hours after electroporation.

Example 6: Glucose uptake and Glut4 trafficking experiments. [00138] For experiments in cultured cells, adipocytes were electroporated with

STAMP2 or control siRNA 5 days after induction of differentiation. The next day, cells were washed and switched into serum-free medium for overnight incubation. [00139] Cells were washed 3x in warm Krebs-Ringer Hepes (KRH) buffer, followed by treatment with 100 nM insulin in warm KRH buffer. In some wells, 50μM cytochalasin B was added as a control to inhibit glucose transporter-mediated glucose uptake. Cells were incubated for 20 min at 37⁰C. ³H-2-deoxyglucose was then added to each well for an additional 10 minutes. Experiment was terminated by placing cells on ice and washing with ice-cold KRH buffer. After washing cells 3x in cold KRH buffer and removing final wash, cells were lysed in 0.1% SDS and uptake determined by scintillation counting.

[00140] For experiments in primary adipocytes, adipocytes were isolated from either subcutaneous or epididymal fat pads by the following method. Mice were killed and fat pads removed. Fat pads were placed into KRP buffer containing 1 mg/ml collagenase (Liberase RI, Roche), 2.5% BSA and 200 M adenosine (isolation buffer, IB), chopped thoroughly with scissors, and incubated for 45 min with gentle shaking at 37⁰C. Fat cells were passed through nylon mesh and then washed 3x in IB (without collagenase), each time allowing fat cells to rise to the surface and then removing infranatant. 3 volumes of IB (without collagenase) were added to fat cells and they were placed at 37⁰C. Cells were then treated with or without 100 nM insulin for 30 min. After insulin stimulation, ³H-2-deoxyglucose was added for 45 min. Glucose uptake was ended with the addition of 500 μl Fluka reagent (Sigma). Cells were spun for 2 min at 1000 rpm. Floating fat cakes were collected, transferred into scintillation vials, and counted. [00141] For Glut4 trafficking experiments 3T3-L1 adipocytes were co-electroporated with myc-Glut4-GFP construct (gift of Dr. M. Czech) and either control or STAMP2-specifϊc siRNA, and cells were plated on coverslips. One day later, cells were placed into serum-free DMEM for overnight incubation. Cells were then treated with 160 nM insulin for 20 minutes and cells fixed in 2% paraformaldehyde. [00142] For immunofluorescence, non permeabilized cells were incubated with mouse monoclonal anti-myc antibody (Santa Cruz) followed by anti-mouse Cy3 (Jackson ImmunoResearch). Confocal images were obtained using a Zeiss LSM-410 at 40Ox magnification. For quantitation, in 3 independent experiments, a total of 500 GFP positive cells were scored in each condition (plus or minus) for myc rim staining.

Example 7: Injections of insulin and lipid/ glucose administration.

[00143] Three month-old male mice were anesthetized with tribromoethanol, and 2

IU/kg insulin or PBS infused into the portal vein. After 3 minutes, liver, VWAT, and SWAT were collected, in that order, and immediately frozen in liquid nitrogen. Lysates were prepared and used for Western blotting. Lipid and glucose administrations were done in 2 month old WT and STAMP2^'1' mice. After overnight fasting, mice were injected intraperitoneally (i.p.) with either 2 ml PBS or 2 ml 20% intralipid (gift of Baxter Healthcare, Deerfield, IL). After 4 hours, either PBS or 2 mg/kg glucose was injected i.p., and 90 minutes later animals were sacrificed. Tissues were removed and immediately frozen in liquid nitrogen. Data were pooled from 2 independent experiments, with total 4 mice/ genotype receiving PBS and 6 mice/ genotype receiving lipid and glucose.

Example 8: Hyperinsulinemic-euglycemic clamp studies. [00144] Surgery was performed to catheterize the jugular vein of 4-month-old wild type (n=8) and STAMP!^1' (n=7) mice. After four days recovery period, clamp experiments were performed as previously described (Ozcan et al. 2006). To determine [³H] -glucose and 2-[¹⁴C]-DG concentrations, plasma was deproteinized with ZnSC>₄ and Ba(OH)₂, dried to remove ³H₂O, re-suspended in water, and counted in scintillation fluid for detection of ³H and 1⁴C. The plasma concentration of ³H₂O was calculated by the difference between ³H counts with and without drying. Tissue 2-[¹⁴C]-DG-6-phosphate (2-DG-6-P) content was determined in homogenized samples that were subjected to an ion-exchange column to separate 2-DG-6-P from 2-[¹⁴C]-DG. Calculations and experimental details are described in the supplement.

Example 9: Glucose tolerance tests

[00145] For glucose tolerance tests, male 12-week-old wild type (n=9) and STAMP2^'

'^' (n=10) mice were fasted overnight (14 hours) and injected intraperitoneally (i.p.) with 2 g/kg glucose. For insulin tolerance tests, male 17-week-old wild type (n=4) and STAMP!^1' (n=5) mice were fasted for 6 hours and injected i.p. with 0.75 IU/kg insulin. For both, blood was collected from the tail vein at the indicated times and glucose measured using an

Ascensia glucometer. Body fat was measured in mice anethesthetized with ketamine/xylazine using duel energy X-ray absorbitometry (DEXA) (age 2 months: n=4 wt, n=6 KO; age 5 mos: n=6 wt, n=7 KO).

Example 10: Determination of liver triglyceride levels.

[00146] Lipid extraction was performed using the Bligh-Dyer method (Bligh and

Dyer 1959). Briefly, livers were homogenized in chloroform: MeOH: H₂O (1 :2:0.8) at room temperature. Samples were spun and supernatants saved. Equal amounts of chloroform and water were added to supernatant. Samples were vortexed and centrifuged. Chloroform layer was collected and samples dried completely in vacuum oven. Samples were resuspended in 90% isopropanol: 10% Triton-X. Triglyceride concentration was measured using Sigma Kit TROlOO, per manufacturers' instructions.

Example 11: Regulation ofSTAMP2 expression in cultured adipocytes and adipose tissue in response to inflammatory and nutritional stimuli

[00147] In attempts to identify factors that might participate in coordinated regulation of metabolism and inflammation, a screen for genes differentially regulated in obese mice 1) lacking a key inflammatory molecule, TNFα, or receptors responsible for TNF action or 2) treated with insulin-sensitizing thiazolidinedione (TZD) drugs was performed (Wellen et al. 2004). The focus was narrowed to genes that are exclusive to or highly enriched in adipose tissue. STAMP2 was identified as a candidate molecule. Adipose tissue STAMP2 expression was reduced in obese animals either deficient in TNFα function or treated with TZD, demonstrating that STAMP2 is regulated by TNFα in vivo (Fig. Ia). Consistent with these findings, previous reports have demonstrated that STAMP2 is expressed in adipocytes in vitro and regulated by cytokines (Moldes et al. 2001; Fasshauer et al. 2004).

[00148] To further explore the regulation of STAMP2, its expression was examined in mouse tissues and in cultured adipocytes during differentiation. STAMP2 is expressed in white adipose tissue (WAT) at more abundant levels than any other tissue examined (Fig. Ib). In addition, STAMP2 expression was absent in preadipocytes but strongly induced during adipocyte differentiation in 3T3-L1 cells, in agreement with earlier studies (Moldes et al. 2001) (Fig. Ic). In adipose tissue, the principal source of STAMP2 expression was also found in mature adipocytes although a low level expression was detected in the stromal- vascular fraction (Fig. Id). [00149] Adipocyte differentiation is predominantly coordinated by PPARγ and

C/EBP family member transcription factors, with PPARγ and C/EBPα engaging in a positive feedback loop to drive differentiation forward (Rosen et al. 2000). Since STAMP2 expression is induced during differentiation, it was asked whether one or both of these adipogenic transcription factors might control STAMP2 expression. While no portion of the 2kb 5'- promoter sequences (SEQ ID NO: 1) that were examined responded to PPARγ even in the presence of synthetic agonists, STAMP2 promoter activity was strongly induced by CEBPα (Fig. Ie). Interestingly, it was found that the promoter was also responsive to LXRα, another transcription factor implicated in adipocyte gene expression, although this was smaller in magnitude compared to CEBPα (Fig. Ie). Since PPARγ does not appear to directly regulate the STAMP2 promoter, it is likely that reduced expression of STAMP2 seen in the TZD- treated ob/ob WAT is indirect (Fig. Ia). Together, these results establish STAMP2 as a differentiation-dependent molecule present in adipocytes in vitro and in vivo.

[00150] To investigate whether STAMP2 expression is regulated by nutritional conditions in vitro, 3T3-L1 adipocytes were treated with various stimuli and examined for STAMP2 expression. While glucose and insulin treatments alone produced minimal regulation of STAMP2 expression in adipocytes, high serum and fatty acids markedly induced STAMP2 levels (Fig. If). Similarly, treatment with TNFα increased STAMP2 levels in adipocytes to levels comparable to or higher than those obtained by these nutritional stimuli (Fig. If). [00151] STAMP2 expression was examined during feeding and fasting in lean as well as genetically obese, leptin-defϊcient (ob/ob) mice. In lean mice, STAMP2 expression, was elevated in the fed as compared to fasted state, particularly in visceral adipose depot (VWAT), a site often considered as the most relevant depot for metabolic pathologies (Fig. Ig) (Despres and Lemieux 2006). STAMP2 protein levels, as assessed by immunohistochemistry, correlated with the RNA levels (Fig. Ih). In ob/ob mice, however, nutritional regulation of STAMP2 expression was completely lost (Fig. Ig). A similar loss of nutritional regulation was also observed in high-fat diet-induced obesity (Fig 8). Thus, STAMP2 expression is responsive to both nutritional and inflammatory signals both in cultured adipocytes and adipose tissue.

Example 12: Aberrant inflammatory and metabolic responses in the absence of STAMP2 in vitro

[00152] To investigate whether STAMP2 has a direct role in modulating inflammation in adipocytes, an siRNA targeted to the sequence of SEQ ID NO: 30 was used to reduce STAMP2 expression in 3T3-L1 adipocytes. After administration of siRNA, we confirmed efficient (>80%) knockdown of STAMP2 mRNA expression (Fig. 2a). Protein levels, as assessed by immunofluorescence analysis, were reduced (Figure 2b). Finally, Western blotting demonstrated that Flag-tagged STAMP2 exogenously expressed in adipocytes can be almost entirely suppressed using this siRNA-based experimental system (Fig. 2c).

[00153] Expression of inflammatory cytokine production in adipocytes, in the presence of either inflammatory (TNFα stimulation) or nutritional (hyperglycemic) challenges was examined. Upon treatment with TNFα, significantly greater induction of IL-6 expression in STAMP2-knockdown versus control cells was observed (Fig. 2d). When cultured in a physiological low glucose medium, STAMP2 knockdown and control cells expressed similar amounts of interleukin-6 (IL-6) mRNA..However, in high glucose conditions, STAMP2- defϊcient cells expressed approximately twice as much IL-6 mRNA as control cells (Fig. 2e). Secretion of IL-6 protein was measured. It was found that in high glucose conditions, IL-6 secretion was indeed augmented in the absence of STAMP2 (Fig. 2f). Interestingly, this effect appears to be somewhat selective, as other TNFα-regulated genes, such as MCP-I and IL-I β, as well as the TNFRl and TNFR2, were not differentially regulated in these conditions.

[00154] Insulin action was examined in 3T3-L1 adipocytes transfected with

STAMP2-specific or control siRNA. STAMP2 deficiency resulted in moderate but consistent impairment of insulin-stimulated glucose transport in adipocytes (Fig. 2g). Insulin-induced translocation of the glucose transporter Glut4 to the plasma membrane was assessed in the absence of STAMP2 to determine is role, if any, in the observed glucose transport impairment.

[00155] An ectopically expressed Glut4 (tagged with a myc epitope in the first exo facial loop as well as GFP, for tracking) exhibited reduced insulin-stimulated translocation to the plasma membrane in the absence of STAMP2 (Fig. 2h). This reduction was significant when STAMP-deficient cells with detectable Glut4 translocation were quantitated compared to controls (Fig. 2i). The insulin receptor signaling pathway was also examined. In control cells, insulin stimulation results in dose dependent increases in the tyrosine phosphorylation of insulin receptor and serine phosphorylation of Akt. In STAMP2-deficient cells, the effects of insulin were modestly but significantly reduced (Figure 9a). These results indicate that STAMP2 deficiency impairs insulin action and disrupts glucose transport in adipocytes.

Example 13: Increased inflammation and reduced insulin action in the genetic absence of STAMP2 in vivo

[00156] To confirm the function of STAMP2 in regulating adipose tissue inflammation and metabolism in vivo, mice with homozygous targeted null mutations in the STAMP2 locus were examined. Northern blot analysis confirmed that in adipose tissue these mice did lack STAMP2 expression (Fig. 3a). STAMP2^"A mice were fully viable and fertile and reproduced at the expected Mendelian ratios with no visible abnormalities. At 5-6 months of age, wild type and STAMP2^"7" mice were sacrificed and tissues harvested for analysis of gene expression, biochemical studies, and histological analysis. Having confirmed the absence of STAMP2, the expression of the three other STAMP family members was analyzed for possible compensatory regulation. Interestingly, all of the STAMP family members are expressed in adipose tissue, but only STEAP3 was consistently and significantly upregulated in the absence of STAMP2 (Fig. 3b). On the other hand, STEAP and STAMPl tended to be suppressed in STAMP2^"'^" VWAT (Fig. 3b).

[00157] Next, inflammatory gene expression in both subcutaneous and visceral WAT depots was examined. It was found that inflammation due to STAMP2 deficiency was much more pronounced in this in vivo setting compared to that observed in 3T3-L1 adipocytes. In STAMP2^"'^" animals, expression of multiple inflammatory factors, including IL-6, TNFα, MCP-I, haptoglobin and SOCS-3 were significantly elevated in VWAT (Fig. 3c). In contrast to VWAT, alterations in inflammatory gene expression in SWAT in STAMP2^"'^" animals were minimal; while there was a trend for higher levels of some genes, these differences were subtle and did not reach statistical significance (Fig. 3c). Thus, the primary site of STAMP2 expression and regulation in vivo coincides with that of marked inflammatory abnormalities in its absence.

[00158] The VWAT depot also manifested striking histological differences between

STAMP2^"'^" and WT animals. Though no clear differences in adipocyte cell size were observed, in STAMP2^"A mice, VWAT, but not SWAT, contained markedly increased numbers of mononuclear cells among adipocytes (Fig. 3d).

[00159] Recent evidence has indicated that adipose tissue, particularly in obesity, can accumulate macrophages (Weisberg et al. 2003; Xu et al. 2003). To test whether the mononuclear cells present in the VWAT of STAMP2^"7" mice might be infiltrating macrophages, samples were tested for the presence of the macrophage-specific antigen F4/80 by immunohistochemistry (Fig. 3e). There was strong staining for F4/80 in the interstitial space between adipocytes in VWAT of most STAMP2^"'^" animals, suggesting that STAMP2^"'^" VWAT indeed becomes infiltrated by macrophages, defined as F4/80-positive cells. In contrast, no noticeable macrophage accumulation was observed in any WT mice. Interestingly, similar to inflammatory gene expression, macrophage infiltration was also seen only in STAMP2^"A VWAT and absent from SWAT (Fig. 3d, e).

[00160] Metabolic dysfunction in adipocytes and adipose tissue is frequently associated with oxidative stress (Furukawa et al. 2004; Houstis et al. 2006). Therefore, the expression of antioxidant genes was measured in adipose tissues. These experiments demonstrated that expression of catalase, glutathione-S-transferase (GST), and superoxide dismutase 1 (SODl), were significantly reduced in the VWAT of mice lacking STAMP2, whereas NADPH oxidase 1 was significantly upregulated (Fig. 3f). These expression patterns are similar to observations previously reported in obesity (Furukawa et al. 2004). Interestingly, no significant changes were observed in these genes in the subcutaneous adipose tissue (Figure 10 a). To test whether STAMP2 ^A mice might have increased oxidative stress in VWAT as compared to WT mice, we measured levels of thiobarbituric acid reactive substances (TBARS) as an indicator of lipid peroxidation. Indeed, STAMP2^''^" mice displayed significantly higher levels of lipid peroxidation in VWAT than WT mice (Fig. 3g). These results suggest that in the absence of STAMP2, adipose tissue is susceptible to oxidative stress.

Example 14: Increased inflammation in STAMP2/^' mice in response to nutritional challenge

[00161] Intralipid and glucose were administered into WT and STAMP2^"'^" mice and inflammatory gene expression was examined in adipose tissue compared to saline-treated control mice in both genotypes. Combined administration of glucose and lipid stimulated expression of IL-6 and SOCS-3 in both genotypes, although to a significantly greater extent in the adipose tissue of STAMP2^'A mice (Fig. 4a). These data support the model that even a short-term nutritional challenge can stimulate an inflammatory response in WAT in vivo, and that STAMP2 plays a role in modulating this response. To determine whether STAMP2 is also necessary for metabolic regulation in vivo, primary adipocytes were isolated from WT and STAMP2^"'^" mice and examined glucose transport. Adipocytes isolated from STAMP2 ^~'^~ mice exhibited severely defective insulin-stimulated glucose transport. Interestingly, defective glucose uptake was far more profound in the STAMP2^"/" adipocytes obtained from the visceral depot, although it was also reduced in subcutaneous adipocytes (Fig. 4b).

Example 15: Insulin receptor signaling in vivo is disrupted in STAMP2^V~ mice.

[00162] In WT mice, insulin action as measured by increased phosphorylation of Akt was clear and uniform in both adipose depots. In STAMP2^"'^" mice, however, basal and insulin-stimulated levels of Akt phosphorylation were rather heterogeneous between animals, and basal Akt phosphorylation levels were overall elevated (Fig. 4c and 10b). Hence, insulin had a significantly impaired stimulatory impact on Akt phosphorylation in the VWAT of STAMP2^"A mice (Fig. 4c). In contrast, insulin signaling in SWAT in the same STAMP2^"A mice was normal (Fig. 4c). The alterations in insulin signaling may contribute to the severe suppression of insulin-stimulated glucose transport in visceral adipocytes in the absence of STAMP2.

[00163] Significantly decreased expression of several genes critical for metabolic function were also found, including Glut4, adiponectin, fatty acid synthase, and fatty acid transporter protein 1 , and PPARγ, though others, such as FATP4 and leptin were not significantly regulated (Fig. 4d). It is likely that reduced expression of Glut4 also contributes to the pronounced glucose transport defect in STAMP2^''^" visceral adipocytes. Gene expression experiments were performed in the entire fat pad without separating out adipocytes specifically, it is possible that the increased presence of macrophages in adipose tissue contributed to adipocyte-specific gene expression patterns. As would be anticipated, macrophage-specific gene expression, such as Mac-1 and CD68, is also elevated in the VWAT of STAMP2 ^A mice (Fig. 4e).

Example 16: STAMP2 deficiency causes spontaneous metabolic disease in mice [00164] Body weight, systemic glucose metabolism and lipid levels were examined in whole animals. Upon weaning, mice were placed on a standard rodent diet, and monitored to 20 weeks of age. Throughout the experimental period, there was minimal difference in total body weight between genotypes which did not reach statistical significance at any age (Fig. 5a). Body composition in mice was analyzed by performing dual energy x-ray absorbiton (DEXA) analysis. No differences in adiposity were observed at 2 months of age; however, by 5 months, STAMP2^*7" mice tended to accumulate more body fat than WT mice (Fig. 5b). Subcutaneous (WT: 0.021±0.002; KO: 0.032±0.002 g/g body weight; p=0.004) but not visceral (WT: 0.039±0.003; KO: 0.041±0.004 g/g body weight; p= 0.6) adipose tissue weight was significantly higher in STAMP2^''^" mice. It is possible that the extra body fat later on in life accumulates subcutaneously, rather than viscerally, due to the severe defects in insulin action in visceral fat in STAMP2^"7" mice. In addition, liver weight was significantly higher in STAMP2^"7" mice (WT: 0.043±0.004; KO: 0.057±0.004 g/g body weight; p=0.038).

[00165] To gain additional insights into the metabolism of these mice and possible reasons for the tendency to accumulate body fat, 6-week old STAMP2^"'^" and WT mice were monitored for 48 hours in metabolic cages which enable determination of both energy intake (eating) and output (metabolic rate). Although there were no differences in food intake, VO₂ and VCO₂ tended to be lower in STAMP2^"'^" mice, suggesting that they may have a lower metabolic rate (Figure 11a and b). There was also a reduced rate of physical activity in these animals, particularly during the dark cycle. These results suggest that the principal cause of higher adiposity in STAMP2^"A mice may be reduced energy expenditure.

[00166] Insulin resistance is a central feature of metabolic syndrome. To evaluate the status of insulin action, plasma insulin and glucose levels were examined throughout the experimental period. At 12 weeks of age, plasma insulin levels were significantly elevated in STAMP2^"'^" mice compared with WT mice and this pattern was progressively accentuated until the end of the experiment at 20 weeks (Fig. 5c). Plasma glucose concentrations were also slightly but significantly higher in the STAMP2^7" mice compared to WT animals by 16 weeks of age and continued to increase at 20 weeks (Fig. 5d). Higher blood glucose concentrations in the presence of hyperinsulinemia in the STAMP2^7" mice is indicative of systemic insulin resistance. [00167] Steady state levels of plasma lipids were also determined, to assess whether

STAMP2-deficiency causes dyslipidemia. Plasma triglycerides and cholesterol concentrations were moderately but significantly elevated at 12 weeks of age in STAMP2^7" mice compared to control animals (Fig. 5e). While total levels of cholesterol were elevated, FPLC analysis revealed no clear difference in lipoprotein particle distribution (Fig. 5f). Plasma free fatty acid levels did not differ between genotypes (WT: 0.82±0.1; KO: 0.84±0.4 mM).

Example 17: Insulin and glucose tolerance tests demonstrate abnormal metabolism in STAMP2+ mice.

[00168] Insulin tolerance tests (ITT) and glucose (GTT) tolerance tests were performed in each genotype. Glucose disposal curves upon administration of insulin were significantly reduced in STAMP2 ^Λ mice compared to WT controls, demonstrating the presence of insulin resistance (Fig. 5g). Similarly, in GTT, the glucose disposal patterns observed in STAMP2^7" mice indicates significant glucose intolerance (Fig. 5h). These results clearly show that STAMP2-deficiency disturbs systemic insulin action in the absence of additional stress.

[00169] Given the clear impairment in systemic glucose metabolism in STAMP2^"A mice, it was suspected that insulin action at metabolic tissues other than adipose tissue may also be impaired. To address whether liver function may be altered in STAMP2^"A mice, insulin action was investigated at this site in vivo. In these experiments, a severe impairment of insulin receptor signaling was observed in the livers of STAMP2^"A mice (Fig. 6a).

[00170] These results indicate that insulin action is impaired in STAMP2-deficient mice in at least two metabolically relevant tissues, visceral adipose tissue and liver. An important question for understanding the role of STAMP2 in regulation of systemic metabolism is which tissues are critical in mediating the development of systemic insulin resistance. To elucidate the organs responsible for the systemic metabolic phenotype hyperinsulinemic-euglycemic clamp experiments were performed in WT and STAMP2^"'^" mice. As expected, no differences in body weight were observed between genotypes in the animals used for the clamp experiments, either at the time of catheterization surgery or experiment (Fig. 12a). Experiments were performed with 4 month old STAMP2^"7" mice in the fasted state, and blood glucose values were normalized during the clamp (Fig. 12b). Glucose infusion rate was significantly lower in STAMP2^"'^" mice than WT controls throughout the duration of the clamp, confirming systemic insulin resistance (Fig. 6b). The rate of glucose disposal was also lower in STAMP2^"'^" animals, but did not achieve statistical significance (p=0.06, Fig. 6c). Hepatic glucose production was significantly higher in STAMP2^"'^" mice in both basal and clamp conditions, and ability to suppress hepatic glucose production in hyperinsulinemic conditions was significantly compromised, demonstrating the presence of liver insulin resistance (Fig. 6d,e). Insulin-stimulated glucose uptake into muscle and fat were found to be altered in these mice by determining tissue content of ¹⁴C-2-deoxyglucose tracer. As expected and in agreement with the rest of the data herein, glucose uptake into adipose tissue was impaired in the absence of STAMP2 (Fig. 6g). Muscle glucose uptake was also significantly reduced in STAMP2 '^" animals (Fig. 6f). These results indicate that the moderate reduction observed in the rate of glucose disposal in STAMP2^"'^" mice is biologically significant, since both muscle and fat are significantly impaired in glucose uptake capacity. Overall, the results of these clamp experiments indicate that both liver glucose production and peripheral glucose disposal contribute to the systemic insulin resistance in STAMP2^"7" animals.

Example 18: STAMP2-deficiency causes fatty accumulation in liver

[00171] In STAMP2^"Λ mice, liver sections exhibited notable fatty accumulation, particularly as the animals aged (Fig. 7a). At 6 months of age total liver triglyceride content in STAMP2 ^A mice was significantly elevated (Fig. 7b). Expression of fatty acid synthase (FAS) and stearoyl CoA desaturase (SCD-I), enzymes important in fatty acid and triglyceride synthesis respectively, were significantly elevated in the livers of STAMP2^''^" mice (Fig. 7c). As the signs of insulin resistance precede the time of marked steatosis, we next asked whether the defective insulin signaling in liver might be the result an effect mediated by altered endocrine activity of adipose tissue in STAMP2-deficiency. To explore this possibility, rat Fao liver cells were treated with conditioned medium that was collected from 3T3-L1 adipocytes transfected with either control or STAMP2 siRNA. Liver cells cultured with conditioned medium from STAMP2 -deficient adipocytes exhibited reduced insulin signaling compared to controls, suggesting that the absence of STAMP2 in adipocytes may regulate insulin sensitivity in liver cells (Fig 9b). Plasma levels of various adipokines were examined, reasoning that increased inflammatory gene expression in adipose tissue may be reflected in increased plasma levels thus contributing to systemic effects. Although elevated adipose expression of some inflammatory cytokines did not translate into detectably higher levels in circulation in these experiments, both leptin and resistin were found to be significantly elevated in STAMP2^"Λ mice (Table 1 below). ,-/-

Table 1. Adipokine levels (ng/ml) in 12 week-old wild type and STAMP2 ^" mice

*p < 0.05; nd = not detectable

[00173] Resistin has been implicated in impairing glucose homeostasis, particularly through regulation of hepatic glucose production (Steppan and Lazar 2004); hence, it is possible that elevated resistin levels may contribute to some of the liver phenotypes of the STAMP2^"A mouse.

Example 19: Metabolic stress exacerbates STAMP2 ιV-^' phenotype

[00174] If STAMP2 indeed serves as a regulatory factor to preserve metabolic function, we would predict that under conditions of metabolic stress, such as obesity, that the absence of STAMP2 would exacerbate the phenotype. Alternatively, and particularly considering the loss of regulation of STAMP2 expression in obesity (Fig. Ig), it is possible that STAMP2 action is only relevant during relatively short-term or acute challenges. The function of STAMP2 in ob/ob mice, a model of severe obesity with moderate hyperglycemia, was evaluated. A small group of STAMP2^"/"o6/oZ) mice was compared to age-matched ob/ob controls. Although the two groups were similar in total body weight at 12 weeks of age (Fig. 7d), the ob/ob mice lacking STAMP2 had significantly higher body fat percentage (Fig. 7e). On the C57/B16 genetic background, ob/ob mice develop only mild hyperglycemia. Remarkably however, STAMPT' ob/ob mice had extremely high blood glucose levels, averaging nearly 500 mg/dl after a 6 hour fast (Fig. 7f). Comparison of liver sections also indicated even more pronounced lipid accumulation in the STAMP2^~'^~ob/ob mice (Fig. 7g).

Example 20: Expression ofSTAMP2 is inhibited by shRNAs targeted to STAMP2

[00175] Cos7 cells were cotransfected with an expression vector encoding human STAMP2 tagged with the HA epitope and an shRNA vector which simultaneously encodes GFP using FuGeneό (Roche Diagnostics) per manufacturer's instructions.

5'- TGC TGT TGA CAG TGA GCG CGC ACT TCC TCT TAC TAT GAA TTA GTG AAG CCA CAG ATG TAA TTC ATA GTA AGA GGA AGT GCA TGC CTA CTG CCT CGG A-3' (SEQ ID NO: 33, hybridizes to nt 127-147 of SEQ ID NO: 37) 5'-TGC TGT TGA CAG TGA GCG CGC CAA GAA GTC TGA CAT CAT ATA GTG AAG CCA CAG ATG TAT ATG ATG TCA GAC TTC TTG GCT TGC CTA CTG CCT CGG A-3' (SEQ ID NO: 34 , hybridizes to nucleotides 316-336 of SEQ ID NO: 37)

[00176] Underlined sequences are the portions that hybridize to the indicated portion of human ST AMP2.

[00177] Media was changed and cells were cultured to allow for expression of the

GFP-shRNA and STAMP2-HA fusion constructs. Cells were fixed and stained using an anti- HA antibody. GFP positive cells were scored by immunoflourescence microscopy for HA- tagged STAMP2 expression. Both of the shRNA constructs were demonstrated to decrease expression of ST AMP2.

Example 21: Identification of agents that increase expression ofSTAMP2 using a reporter construct

[00178] Cells containing a reporter construct including a STAMP2 promoter region functionally linked to a reporter gene are cultured in a multi-well plate, typically a 96- or 384- well plate. Cells can be STAMP2 deficient cells or normal cells, primary cells or immortalized cells. Cells can be derived from an animal having a disease or an animal model of a disease (e.g., a high fat fed mouse, an ob/ob mouse). The effects of compounds on normal, STAMP2 deficient, and diseased cells can be compared. [00179] The reporter construct is transfected into the cells and a stable cell line may be generated. Transfection methods are selected based on the cell type. If the cell type is difficult to transfect (e.g., most primary cell lines), a plasmid expressing a marker construct such as green fluorescent protein under the control of a constitutive promoter can be co- transfected with the reporter construct to allow for monitoring of transfection efficiecy. [00180] The cells are contaced with a library of agents and proper controls (e.g., vehicle control as a negative control). Preferably, the agents are tested in duplicate or triplicate. Cells can be contacted with the agents for a single time point or for multiple time points. After exposure, the cells are washed and tested for the presence of the expression product from the reporter construct. For example, beta-galactosidase, luciferase, or alkaline phosphatase can be quantitatively detected using commercially available reagents and kits per manufacture's instructions. Agents identified to induce expression from the STAMP2 promoter can be further tested in vitro or in vivo using methods herein. Example 22: Identification of agents that increase expression ofSTAMP2 by RT-qPCR

[00181] Cells expressing at least some STAMP2 from an endogenous promoter are grown in culturein a multi-well plate, typically a 24- or 96-well plate. Cells can be STAMP2 deficient cells that express some STAMP2 (e.g., a heterozygous STAMP2^+/" mouse) or normal cells, primary cells or immortalized cells. Cells can be derived from an animal having a disease or an animal model of a disease (e.g., a high fat fed mouse, a leptin-, JNK-, or XPB-I deficient mouse, db/db mouse). The effects of compounds on normal, STAMP2 deficient, and diseased cells can be compared.

[00182] The cells are contaced with a library of agents and proper controls (e.g., vehicle control as a negative control). Preferably, the agents are tested in duplicate or triplicate. Cells can be contacted with the agents for a single time point or for multiple time points. After exposure, the cells are washed and total RNA is isolated, cDNA is transcribed, and Q-PCR is performed using any of a number of commercially available kits and devices using STAMP2 specific probes such as those provided in Example 4. Compounds that significantly increase the expression of STAMP2 are further tested using the in vitro or in vivo methods taught herein. Alternatively, RNA expression can be tested by northern blot. Agents identified to induce expression of STAMP2 can be further tested in vitro or in vivo using methods herein.

Example 23: Identification of agents that increase activity of STAMP2

[00183] Cells expressing at least some STAMP2 from an endogenous promoter are grown in culture in a multi-well plate, typically a 24-, 96-, or 384-well plate. Cells can be STAMP2 deficient cells that express some STAMP2 (e.g., a heterozygous STAMP2^+/' mouse) or normal cells, primary cells or immortalized cells. Cells can be derived from an animal having a disease or an animal model of a disease (e.g., a high fat fed mouse, a leptin-, JNK-, or XPB-I deficient mouse, db/db mouse). The effects of compounds on normal, STAMP2 deficient, and diseased cells can be compared.

[00184] The cells are contacted with a library of agents and proper controls (e.g., vehicle control as a negative control). Preferably, the agents are tested in duplicate or triplicate. Cells can be contacted with the agents for a single time point or for multiple time points.

[00185] Cells are analyzed by any of a number of methods to detect an increase in

STAMP2 activity. For example, cells can be tested for changes in glucose trafficking and export as in Example 4. Calls can be tested for signalling through the insulin receptor as in Example 5. Cells can be tested for a decrease in the release of IL-6 in response to glucose as in Example 6. Cells can be tested for an increase in phosporylation of Akt using western blots or other methods. Cells can be tested for changes in response to inflammatory and nutritional stimuli as in Example 11. Cells can be tested for insensitivity to the effects of conditioned media from STAMP2 deficient cells as in Example 18. Agents identified to increase activity of STAMP2 promoter can be further tested in vitro or in vivo using methods herein.

Example 24: In vivo testing of agents identified using in vitro screens to alter STAMP2 expression or activity [00186] A number of animal models of metabolic disease, both genetic and induced are known, for example high fat fed mouse model, db/db mouse, leptin deficient mouse, JNK deficient mouse (Hirosumi et al., Nature, 420:333-6, 2002), XBP-I deficient mouse (Ozcan et al., Science,306:457-61, 2004). Methods for crossing animals with various genotypes to create desired animal models is known in the art. Animal models of various diseases can be further placed on diets to induce or exacerbate disease conditions.

[00187] Agents identified using in vitro screening methods such as those set forth in the examples above can be tested for activity in models of metabolic disease using the assay methods such as those set forth in the examples above. Identified agents and control agents can be administered to mice prior to and/or during various challenges to determine if at least one sign or symptom of a metabolic and/or an inflammatory disease is reduced.

[00188] For example, mice can be tested using hyperinsulinemic-eugenic clamp studies as in Example 8. Mice can be tested for glucose tolerance and insulin tolerance as in Examples 9 and 17. Liver and serum triglyceride and cholesterol levels, and hepatic fat accumulation can be determined as in Examples 10 and 18. Mice can be tested for insulin resistance as in Example 13. Mice can bet tested for release of inflammatory mediators in response to nutritional challenge in Example 14. Mice can be tested for insulin receptor signaling as in Example 15. Mice can be tested for expression of adipokine levels as in Example 18. Mice can be tested for expression of a number of RNAs or proteins including fatty acid synthase and stearoyl CoA desaturase 1. Mice can be tested to determine a shift in fat deposition from VWAT to SWAT.

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(2001). "Reversal of obesity- and diet-induced insulin resistance with salicylates or targeted disruption of Dckbeta." Science 293(5535): 1673-7. The following GenBank numbers and sequences presented therein as of April 30, 2007 are incorporated herein by reference.

NM_054098 (SEQ ID NO: 35 and 36) and BC006651 (Mus musculus STAMP2)

NM 024636 (SEQ ID NO: 37 and 38) and BC020600 (SEQ ID NO: 39 and 40) (Homo sapien STAMP2)

DQ400413 (SEQ ID NO: 41 and 42) and ABD64619.1 (Oncorhynchus mykiss STAMP2).

[00189] Taken together, these results demonstrate that STAMP2-defϊciency is sufficient to spontaneously recapitulate many cardinal features of the metabolic syndrome, including insulin resistance, glucose intolerance, mild hyperglycemia, dyslipidemia, and fatty infiltration of liver and markedly exacerbate the metabolic abnormalities of the ob/ob model of severe obesity.

[00190] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

[00191] All references, patents, patent applications, and GenBank numbers cited are incorporated herein by reference in their entirety.

Claims

ClaimsWe claim:

1. A method for identifying an agent that increases the expression of a STAMP2 in a cell, the method comprising: a) contacting a cell with a candidate agent, wherein the cell comprises a STAMP2 promoter sequence operably linked to a nucleic acid sequence for transcription, ; and b) detecting an increase in transcription of the nucleic acid sequence for transcription relative to a corresponding control cell.

2. The method of claim 1, wherein the nucleic acid sequence for transcription comprises a nucleic acid sequence encoding a STAMP2 polypeptide.

3. The method of claim 1 or 2, wherein the method identifies a compound that increases STAMP2 transcription or translation.

4. The method of any of claims 1 to 3, wherein the cell expressing STAMP2 is a cell of an animal comprising at least one sign or symptom of a metabolic disorder.

5. The method of claim 4, wherein the metabolic disorder is selected from the group consisting of type 1 diabetes, type 2 diabetes, obesity, insulin resistance, glucose intolerance, dyslipedemia, hyperglycemia, fatty liver disease, and hypercholesterolemia.

6. The method of claim 4 or 5, wherein the cell is in culture.

7. The method of claim 4 or 5, wherein the cell is in an animal.

8. The method of claim 1 , wherein the nucleic acid sequence for transcription comprises a reporter gene.

9. The method of claim 8, wherein the reporter gene is selected from the group consisting of beta-galactosidase, luciferase, alkaline phosphatase, and green fluorescent protein.

10. A method for identifying a compound that increases the expression or biological activity of a STAMP2 polypeptide, the method comprising: a) contacting a cell expressing a STAMP2 polypeptide with a candidate compound; and b) measuring an increase in the expression or the biological activity of the STAMP2 polypeptide in the cell relative to a control cell.

11. The method of claim 10, wherein the increase is measured in an immunological assay.

12. The method of claim 10, wherein STAMP2 biological activity is detected by measuring a modulation of at least one biomarker selected from the group consisting of inflammatory biomarker, oxidative stress biomarker and metabolic biomarker.

13. The method of claim 10, wherein the cell expressing STAMP2 is a cell of an animal comprising a metabolic disorder.

14. The method of claim 13, wherein the metabolic disorder is selected from the group consisting of type 1 diabetes, type 2 diabetes, obesity, insulin resistance, glucose intolerance, dyslipedemia, hyperglycemia, fatty liver disease, visceral obesity, and hypercholesterolemia.

15. The method of claim 13 or 14, wherein the cell is in culture.

16. The method of claim 13 or 14, wherein the cell is in an animal.

17. A method for identifying an agent that binds a STAMP2 polypeptide, said method comprising the steps of:

(a) contacting a candidate agent with the isolated STAMP2 polypeptide under conditions that allow binding; and

(b) detecting binding of the candidate agent to the polypeptide.

18. The method of any one of claims 1 to 17, further comprising the step of administering the agent to a cell in an animal and detecting a modulation of at least one sign or symptom of a metabolic or inflammatory disease.

19. The method of claim 18, wherein the sign or symptom of a metabolic or inflammatory disease is selected from the group consisting of insulin responsiveness, serum insulin, blood glucose, serum triglycerides, cholesterol, glucose tolerance, insulin tolerance, distribution of lipoprotein particles in cells, tissues, or organs, liver lipid accumulation, liver triglyceride, body weight, or body fat accumulation or localization.

20. The method of any one of claims 1 to 19, wherein the modulation of a sign or symptom of a metabolic or inflammatory disease is selected from the group consisting of a decrease in insulin resistance, fatty liver, type 2 diabetes, hyperlipidemia, inflammation, visceral obesity, and any combination thereof.

21. A method of treating a metabolic or inflammatory disorder in a subject, said method comprising the steps of administering to said mammal a therapeutically effective amount of an agent identified by the method of any one of claims 1 to 20 that increases the expression or biological activity of a STAMP2 polypeptide.

22. The method of claim 21, wherein the agent is a small molecule, polypeptide, or nucleic acid molecule, or fragment thereof.

23. The method of claim 21 , wherein the agent is a polypeptide that binds STAMP2.

24. A method of identifying a downstream target regulated by STAMP2, the method comprising:

(a) contacting a cell with an agent that increases expression or biological activity of STAMP2; and

(b) detecting an increase in the expression or activity of the downstream target.

25. The method of claim 24, wherein the cell is selected from the group consisting of adipocyte, hepatocyte, and myocyte.

26. The method of claim 24 or 25, wherein the downstream target is a soluble factor expressed in the cell.

27. A method of identifying an agent that treats a metabolic disorder, the method comprising: (a) contacting an animal having a metabolic disorder with an agent; and

(b) detecting a reduction in at least one sign or a symptom of the metabolic disorder.

28. The method of claim 27, wherein a reduction in at least one sign or symptom of the metabolic disorder is detected by measuring at least one of insulin responsiveness, serum insulin, blood glucose, serum triglycerides, cholesterol, glucose tolerance, insulin tolerance, distribution of lipoprotein particles in cells, tissues, or organs, liver lipid accumulation, liver triglyceride, body weight, body fat accumulation, and body fat localization.

29. The method of claim 27 or 28, wherein the agent reduces insulin resistance, fatty liver, type 2 diabetes, hyperlipidemia, inflammation, or visceral obesity.

30. The method of any of claims 27 to 29 wherein the agent was identified using any of the methods of claim 1 to 17.

31. A method of identifying an agent to treat or prevent a metabolic disorder comprising: contacting a STAMP2 deficient cell with the agent; determining whether at least one inflammatory, oxidative stress, and/or metabolic biomarker is modulated.

32. The method of claim 31 , wherein the STAMP2 deficient cell is in culture.

33. The method of claim 31 or 32, wherein the culture is a primary cell culture.

34. The method of claim 31, wherein the cell is in a STAMP2 deficient mouse.

35. The method of claim 34, wherein the STAMP2 deficient mouse further comprises a genetic mouse model for metabolic disease

36. The method of claim 34 or 35, wherein the STAMP2 deficient mouse comprises a STAMP2 deficient mouse further comprising a deficiency selected from the group consisting of leptin-deficiency, JNK-deficiency, XPB-I -deficiency, db/db genotype.

37. The method of any of claims 31 to 36, wherein the STAMP2 deficient cell includes a disruption of the STAMP2 gene.

38. The method of any of claims 31 to 36, wherein the STAMP2 deficient cell is generated by contacting a STAMP2 expressing cell with an agent that reduces expression of STAMP2.

39. The method of claim 38, wherein the agent is an inhibitory nucleic acid molecule.

40. The method of claim 38 or 39, wherein the nucleic acid molecule is an siRNA, shRNA, or antisense compound.

41. The method of any of claims 12 to 16, 20 to 23, and 31 to 40, wherein the inflammatory biomarker is selected from the group consisting of interleukin (IL)-6, IL-I β, tumor necrosis factor (TNF)-α, TNF receptor 1, TNF receptor 2, MCP-I, haptoglobin, SOCS- 3, Mac-1, CD68, and adipokines.

42. The method of any of claims 12 to 16, 20 to 23, and 31 to 40, wherein the oxidative stress biomarker is selected from the group consisting of glutathione-S-transferase (GST), superoxide dismutase-1 (SOD-I), nicotinamide (NADPH), thiobarbituric acid reactive substances (TBARS), and lipid peroxidation.

43. The method of any of claims 12 to 16, 20 to 23, and 31 to 40, wherein the metabolic biomarker is selected from the group consisting of Akt, GLUT4, adiponectin, fatty acid synthase, fatty acid transporter 1, PPARγ, FATP4, leptin, fatty acid synthase (FAS), stearoyl CoA desaturase (SCD-I), and resistin.

44. The method of any of claims 12 to 16, 20 to 23, and 31 to 43, wherein modulation of the biomarker comprises an increase in the expression, stability, or biological activity of of the biomarker.

45. The method of any of claims 12 to 16, 20 to 23, and 31 to 43, wherein modulation of the biomarker comprises a decrease in the expression, stability, or biological activity of the biomarker.

46. The method of any of claims 12 to 16, 20 to 23, and 31 to 43, wherein modulation of the biomarker comprises a post-synthesis modification of the biomarker.

47 The method of any of claims 31 to 46, wherein the metabolic disorder is selected from the group consisting of diabetes, obesity, insulin resistance, glucose intolerance, dyslipidemia, hyperglycemia, fatty liver disease, visceral obesity, and hypercholesterolemia.

48. The method of any of claims 31 to 44, wherein the cell is selected from the group consisting of adipocyte, hepatocyte, and myocyte.

49. The method of any of claims 31 to 44, further comprising obtaining said STAMP 2 deficient cell.

50. An agent for treating or preventing a metabolic disorder, wherein the agent modulates at least one inflammatory, oxidative stress, and/or metabolic biomarker, said agent having been identified by a screening method comprising: contacting the agent with a STAMP2 deficient cell; and determining whether the agent modulates at least one inflammatory, oxidative stress, and/or metabolic biomarker.

51. The agent of claim 50, wherein the agent is selected from the group consisting of small molecules, proteins, nucleic acids, and peptides.

52. The agent of claim 50, wherein the metabolic disorder is selected from the group consisting of diabetes, obesity, insulin resistance, glucose intolerance, dyslipidemia, hyperglycemia, fatty liver disease, visceral fat deposition, and hypercholesterolemia.

53. A method for treating or preventing a metabolic disorder comprising administering to a subject in need thereof a therapeutically effective amount of the agent of claim 50.

54. The method of claim 53, wherein the metabolic disorder is sleeted from the group consisting of diabetes, obesity, insulin resistance, glucose intolerance, dyslipidemia, hyperglycemia, fatty liver disease, visceral fat deposition, and hypercholesterolemia.

55. A pharmaceutical composition comprising a therapeutically effective amount of the agent of claim 50 and a pharmaceutically acceptable carrier or excipient.

56. A pharmaceutical composition comprising a therapeutically effective amount of the agent identified by any of the methods of claims 1 to 49 and a pharmaceutically acceptable carrier or excipient.

57. A method of identifying an agent that treats or prevents a metabolic disorder comprising: contacting a STAMP2 expressing cell with the agent; and detecting an increase in STAMP2 expression or biological activity, thereby identifying the agent as treating or preventing a metaolic disorder.

58. The method of claim 57, wherein the increase in STAMP2 expression is detected by measuring an increase in the level of a STAMP2 encoding nucleic acid molecule.

59. The method of claim 57, wherein the increase in STAMP2 expression is detected by measuring an increase in the level of a STAMP2 polypeptide.

60. The method of claim 57 or 59, wherein the increase in the level of STAMP2 polypeptide is measured in an immunoassay.

61. The method of claim 57 to 60, wherein the cell is in culture.

62. The method of claim 61 , wherein the culture is a primary cell culture.

63. The method of claim 57 to 60, wherein the cell is in an animal.

64. The method of any of claim 63, wherein the mammal is prone to or suffering from a metabolic disorder.

65. The method of claim 63 or 64, wherein the mammal is a db/db mouse, a leptin- deficient mouse, a JNK-deficient mouse, an XBP-I -deficient mouse, and a high fat fed mouse.

66. The method of any of claims 57 to 65, wherein the cell is selectect from the group consisting of adipocyte, hepatocyte, and myocyte.

67. The method of any of claims 57 to 66, further comprising determining whether at least one inflammatory, oxidative stress, or metabolic biomarker is modulated.

68. The method of claim 67, wherein the inflammatory biomarker is selected from the group consisting of interleukin (IL)-6, IL-I β, tumor necrosis factor (TNF)-α, TNF receptor 1, TNF receptor 2, MCP-I, haptoglobin, SOCS-3, Mac-1, CD68, and adipokines.

69. The method of claim 67, wherein the oxidative stress biomarker is selected from the group consisting of glutathione-S-transferase (GST), superoxide dismutase-1 (SOD-I), nicotinamide (NADPH), thiobarbituric acid reactive substances (TBARS), and lipid peroxidation.

70. The method of claim 67, wherein the metabolic biomarker is selected from the group consisting of Akt, GLUT4, adiponectin, fatty acid synthase, fatty acid transporter 1, PPARγ, FATP4, leptin, fatty acid synthase (FAS), stearoyl CoA desaturase (SCD-I), resistin.

71. The method of any of claims 57 to 70, wherein modulation of the biomarker comprises an increase of expression or presence of the biomarker.

72. The method of any of claims 57 to 70, wherein modulation of the biomarker comprises a decrease in the expression or presence of the biomarker.

73. The method of any of claims 57 to 70, wherein modulation of the biomarker comprises a post-synthesis modification of the biomarker.

74. The method of any of claims 57 to 73, wherein metabolic disorder is selected from the group consisting of diabetes, obesity, insulin resistance, glucose intolerance, dyslipidemia, hyperglycemia, fatty liver disease, visceral fat, and hypercholesterolemia.

75. The method of any of claims 57 to 74, further comprising obtaining said cell.

76. The method of any of claims 57 to 75, wherein the cell is a STAMP2 containing cell.

77. An agent for treating or preventing a metabolic disorder, wherein the agent modulates STAMP2, said agent having been identified by a screening method comprising: contacting the agent with a STAMP2 deficient cell; and determining whether the agent modulates at least one inflammatory, oxidative stress, and/or metabolic biomarker.

78. The agent of claim 77, wherein the agent is selected from the group consisting of small molecules, proteins, nucleic acids, and peptides.

79. The agent of claim 77 or 78, wherein the agent further modulates at least one inflammatory, oxidative stress, and/or metabolic biomarker.

80. The agent of any of claims 77 to 79, wherein the metabolic disorder is selected from the group consisting of diabetes, obesity, insulin resistance, glucose intolerance, dyslipidemia, hyperglycemia, fatty liver disease, and hypercholesterolemia.

81. A method for treating or preventing a metabolic disorder comprising administering to a subject in need thereof a therapeutically effective amount of the agent of any of claims 77 to 80.

82. The method of claim 81 , wherein the metabolic disorder is sleeted from the group consisting of diabetes, obesity, insulin resistance, glucose intolerance, dyslipidemia, hyperglycemia, fatty liver disease, visceral fat, and hypercholesterolemia.

83. A pharmaceutical composition comprising a therapeutically effective amount of the agent of any of claims 77 to 80 and a pharmaceutically acceptable carrier or excipient.

84. A use of a STAMP2 deficient mouse as a model of metabolic disorder.

85. A kit comprising a STAMP2 deficient cell and instructions for use in accordance with the method of any one of claims 31-49.