WO2011038110A2 - Methods of treating metabolic disease - Google Patents

Abstract

It has been discovered that SIRT6 functions as a histone H3K9 deacetylase to control the expression of multiple glycolytic genes. Specifically, SIRT6 appears to function as a co-repressor of the transcription factor Hif1?, a critical regulator of nutrient stress responses. This invention relates generally to methods of reducing expression or activity of SIRT6 to reduce or inhibit hyperglycemia or obesity in a subject.

Description

METHODS OF TREATING METABOLIC DISEASE

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Application Nos. 61/245,098, filed on September 23, 2009, and 61/252,044, filed on October 15, 2009, which are incorporated by reference in their entireties.

TECHNICAL FIELD

This invention relates to methods of treating metabolic disease, e.g., reducing or inhibiting hyperglycemia or obesity, by inhibiting Sirtuin (silent mating type information regulation 2 homolog) 6, also known as SIRT6.

BACKGROUND

Hyperglycemia, or high blood sugar, is a condition in which an excessive amount of glucose circulates in the blood. This is generally a blood glucose level of 10+ mmol/L, but symptoms may not start to become noticeable until levels reach 15-20+ mmol/L. Nevertheless, chronic levels exceeding 7 mmol/L can produce organ damage. Temporary hyperglycemia is often benign and asymptomatic. Blood glucose levels can rise well above normal for significant periods without producing any permanent effects or symptoms. However, chronic hyperglycemia at levels more than slightly above normal can produce a very wide variety of serious complications over a period of years, including kidney damage, neurological damage, cardiovascular damage, and loss of vision.

Obesity, defined as excess adipose tissue, is a leading cause of mortality, morbidity, disability, healthcare utilization, and healthcare costs in the United States. This disorder is associated with numerous adverse health effects, including Type 2 diabetes, high cholesterol, hypertension, gallstones, fatty liver disease, sleep apnea, stress incontinence, heart failure, degenerative joint disease, birth defects, miscarriages, asthma, cancers in men (esophageal, colorectal, liver, pancreatic, lung, prostate, kidney, non- Hodgkin's lymphoma, multiple myeloma, and leukemia), and cancers in women (breast, colorectal, gallbladder, pancreatic, lung, uterine, cervical, ovarian, kidney, non-Hodgkin's lymphoma, and multiple myeloma). Current guidelines recommend drug treatment for individuals, especially those with other obesity-related health conditions, who have failed to respond adequately to dietary and behavioral modifications. A limited number of medications are available for the treatment of obesity. Concerns about side effects have diminished enthusiasm for appetite-suppressant drugs, particularly fenfluramine, which carry serious risks and have been withdrawn from the market.

In order to avoid these serious health conditions, more effective methods to reduce or inhibit hyperglycemia and obesity are desirable.

SUMMARY

This invention is based, at least in part, on the discovery that SIRT6 is a master regulator of glucose homeostasis functioning to control the expression of multiple glycolytic genes and glucose uptake.

In one aspect, the invention features use of a SIRT6 inhibitor for reducing or inhibiting hyperglycemia or obesity in a subject. The use is effective for a variety of subjects including mammals, e.g., humans and other animals, such as laboratory animals, e.g., mice, rats, rabbits, or monkeys, or domesticated and farm animals, e.g., cats, dogs, goats, sheep, pigs, cows, or horses.

In one aspect, the invention features methods of reducing or inhibiting

hyperglycemia or obesity in a subject by administering a therapeutically effective amount of a SIRT6 inhibitor to the subject. The methods are effective for a variety of subjects including mammals, e.g., humans and other animals, such as laboratory animals, e.g., mice, rats, rabbits, or monkeys, or domesticated and farm animals, e.g., cats, dogs, goats, sheep, pigs, cows, or horses.

In some embodiments, the subject has a blood glucose level of 10 mmol/L or greater, e.g., 11 mmol/L or greater, 12 mmol/L or greater, 13 mmol/L or greater,

14 mmol/L or greater, 15 mmol/L or greater, 16 mmol/L or greater, 17 mmol/L or greater, 18 mmol/L or greater, 19 mmol/L or greater, 20 mmol/L or greater, 22 mmol/L or greater, 25 mmol/L or greater, or 30 mmol/L or greater. In some embodiments, the subject has a body mass index of 25 kg/m² or greater, e.g., 26 kg/m² or greater, 27 kg/m² or greater, 28 kg/m² or greater, 29 kg/m² or greater, 30 kg/m² or greater, 31 kg/m² or greater, 32 kg/m² or greater, 33 kg/m² or greater, 34 kg/m² or greater, 35 kg/m² or greater, 36 kg/m² or greater, 37 kg/m² or greater, 38 kg/m² or greater, 39 kg/m² or greater, 40 kg/m² or greater, 45 kg/m² or greater, or 50 kg/m² or greater.

In some embodiments, the methods include an anti-SIRT6 antibody or antigen- binding fragment thereof.

In some embodiments, the methods include an inhibitory nucleic acid (e.g., a small interfering RNA molecule or antisense nucleic acid) effective to specifically reduce expression of SIRT6.

In one aspect, the invention features methods of identifying candidate compounds that inhibit hyperglycemia or obesity. The methods include contacting a sample (e.g., a living cell) comprising a SIRT6 polypeptide and an acetylated histone substrate

(e.g., H3K9) with a test compound under conditions that allow the SIRT6 polypeptide to bind or deacetylate the histone substrate and determining a level of histone deacetylation in the sample in the presence of the test compound. If the test compound decreases the level of histone deacetylation, relative to a level of histone deacetylation in the absence of the test compound, then the test compound is a candidate compound for the inhibition of hyperglycemia or obesity.

In some embodiments, the method further comprises administering the candidate compound to a mammal and evaluating an effect of the candidate compound on glycemia or obesity, wherein a candidate compound that inhibits hyperglycemia or obesity is a candidate therapeutic agent for the treatment of hyperglycemia or obesity.

The invention provides several advantages. The prophylactic and therapeutic methods described herein using a SIRT6 inhibitor are effective in reducing or inhibiting hyperglycemia or obesity and have minimal, if any, side effects. Further, methods described herein are effective to identify candidate compounds that inhibit hyperglycemia or obesity.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other features, objects, and advantages of the invention will be apparent from the detailed description, and from the claims.

DESCRIPTION OF DRAWINGS FIG. 1. Increased glucose uptake in SIRT6 deficient cells and mice. FIG. 1A.

[1,2 ¹³C] labeled glucose trace assay was carried out on 16 day-old SIRT6 wild-type (WT) and knock-out (KO) mice. Mice were i.p. injected with 2 mg/g weight of the labeled glucose, and 30 min. later blood was collected and analyzed for the presence of the labeled glucose by mass spectrometry (see Experimental Methods). For each genotype, n= 4 mice, mean +/- standard deviation shown. FIG. IB. Standard Uptake Value (SUV) ratio of labeled ¹⁸F-Glucose incorporation in WT and KO SIRT6 mice. The different tissues analyzed are indicated. Samples were normalized against brain, which exhibit stable glucose uptake across genotypes. The experiment is an average of three mice per genotype. FIG. 1C is a PET image of 16-day old SIRT6 WT and KO mice 60 minutes following i.v. injection of ¹⁸F-glucose. Dotted lines indicate position of the brown adipose tissue (BAT). *: labeled glucose at site of injection (retro-orbital). FIG. ID. SIRT6 WT and SIRT6 KO mouse embryonic fibroblasts (MEFs) together with SIRT1 WT and KO MEFs were grown in the presence of the fluorescent glucose analog NBDG (Invitrogen) for 1 hr., and glucose uptake was then quantified using flow cytometry (FACS). SIRT1 WT and KO cells were used as controls. Dotted lines are controls without the fluorescent NBDG glucose analog. FIG. IE. One WT and two independently generated SIRT6 KO ES lines (KOI and K02) were treated as in (FIG. ID), and analyzed by FACS.

FIG. IF. 293T cells were stable transfected with a SIRT6 cDNA carrying a H133Y mutation (SIRT6HY) that acts as a dominant negative, under the control of the

Tetracycline promoter. Lower panel: western blot showing that SIRT6 was induced specifically after tetracycline treatment (SIRT6). Empty vector was used as a control (Ctrl). Upper panel: glucose uptake was measured as in (FIG. ID). Note the specific increase in glucose uptake following acute inactivation of SIRT6. Error bars in all graphs indicate the standard error of the mean. FIG. 1G. SIRT6 KO cells were infected with a SIRT6 expressing-lenti virus. Infected cells were sorted for GFP expression, and following expansion, cells were assayed for glucose uptake following 1 hour, incubation with NBDG. Lower panel: quantification of the FACS data. GEO mean values were the average of three independent clones. Error bars indicate the standard error of the mean.

FIG. 2. Increased lactate production and decreased oxygen consumption in SIRT6 deficient cells. FIG. 2A are photomicrographs of SIRT6 WT and KO ES cells immunostained with anti-GLUTl antibody. Images were taken using a confocal microscope with constant laser beam for all images (KR: 39.8; IRIS: 2.0). FIG. 2B is a bar graph showing the amount of GLUTl membrane staining in SIRT6 WT and KO cells. FIG. 2C is a bar graph showing the levels of lactate measured in SIRT6 WT and KO ES cells (KOI and K02) using the colorimetric Lactate Assay Kit (Bio Vision). FIG. 2D is a line graph showing oxygen consumption in live SIRT6 WT and KO ES cells under basal conditions, following the addition of the mitochondrial Fl-FO-ATPase inhibitor oligomycin (μΜ), the uncoupler FCCP (1 μΜ), and the Complex I inhibitor rotenone (rot) (5 μΜ) in combination with the Complex I inhibitor myxothiazol (5μΜ). Oxygen consumption rate (OCR) was measured using the XF24 SeaHorse Analyzer (Seahorse Bioscience). Each data point is the average of five independent measurements. Error bars indicate standard deviation. FIG. 2E. Protein lysates were purified from three

independent samples of WT and KO ES cells and glucose metabolites were analyzed by liquid chromatography-mass spectrometry (LC-MS). Red asterisks: TCA intermediate metabolites. FIG. 2F is a bar graph showing ATP levels in SIRT6 WT and KO ES cells (KOI and K02) that were either in regular media or in low glucose (0.5 g/L) media for 36 hours as measured using the ATP Assay Kit (Sigma).

FIG. 3. SIRT6 directly inhibits expression of glycolytic genes functioning as an H3K9 deacetylase. FIG. 3A is a bar graph showing the expression levels of nine genes. RNA was purified from SIRT6 WT and KO ES cells and real-time PCR (RT-PCR) was performed with primers specific for the indicated genes. Three independent samples were averaged, keeping a threshold of 0.4 as confidence value in the threshold cycle (Ct).

Values were normalized against actin. FIG. 3B. Chromatin immunoprecipitation (ChIP) assays using an antibody against SIRT6 were performed on samples from SIRT6 WT and KO ES cells. Real-time PCR were carried out using primers specific for the promoter regions of the indicated glycolytic genes, except for LDHA-lkb, where primers lying lkb downstream of the 3' UTR of the LDHA gene were used, and served as a negative control. FIG. 3C. ChIP assays were performed as described in FIG. 3B, using an antibody that specifically recognizes acetylated H3 lysine 9 (Abeam). The LDHA- 1Kb primers were used as a negative control. FIG. 3D. High resolution ChIP analysis was performed in the LDHB locus using antibodies against total RNA Polymerase II (RNAP II), phosphorylated Serine 5 form of RNAP II (S5P-CTD), phosphorylated Serine 2 form of RNAP II (S2P- CTD), and acetylated H3 lysine 9. Error bars in all graphs indicate the standard error of the mean.

FIG. 4. SIRT6 is a co-repressor of Hifla. FIG. 4 A. A luciferase reporter gene under the regulation of 3 tandem copies of Hypoxia-Responsive Elements (HRE) was co-transfected with empty vector (CMV), SIRT6 (S6) or SIRT6-HY (catalytic dead) plasmids, and subjected to low-glucose (5 mM) conditions for 24 hours. Extracts were analyzed for Luciferase activity with the Luciferase Assay Kit (Promega). Transfection efficiency was normalized against Renilla activity. FIG. 4B. Upper panel: A Flag control, a SIRT6-Flag or a SIRTl-Flag proteins were either expressed alone or co-expressed with Hifla-Myc in 293T cells, and following immunoprecipitation (IP) with either a Flag or a Myc antibody, extracts were run in a Western blot, and probed with the indicated antibodies. Lower Panel: lysates were prepared from SIRT6 WT and KO mice, and following IP with anti-Hifl a antibody, extracts were run in a western blot probed with anti-SIRT6 antibody. Lysates from SIRT6 deficient muscle (KO) were used as negative controls. FIG. 4C is a photograph of a Western blot with an Hifla antibody of SIRT6 WT or KO ES cells treated with or without the Hifla chemical stabilizer CoCl₂. FIG. 4D are line graphs showing glucose uptake of ES cells (left panel) or 293T cells stably expressing tetracycline inducible SIRT6 catalytic dead allele (S6HY) (right panel) treated with or without the Hifl a inhibitor #77 (Zimmer et al, Mol Cell 32:838-848, 2008), as measured by FACS following 1 hour exposure to NBDG.

FIG. 5. Knocking down Hifl a completely rescues the metabolic phenotype in SIRT6 deficient cells. FIG. 5A. SIRT6 WT and KO ES cells were infected with either a Hifl a-knockdown lentivirus (shHifla ) or vector alone (scr). Independent clones were expanded, and glucose uptake was measured using NBDG, as described before. Lower right panel: Western blot analysis of the different clones with an anti-Hifla antibody. Note that clone #3 failed to down-regulate Hifl a, and thus it served as an internal control. FIG. 5B. RNA was purified from Hifl a-KD clones and glycolytic gene expression levels were examined by RT-PCR. The different analyzed genes are indicated. Fold induction was normalized against actin. Three independent samples were averaged, keeping a threshold of 0.4 as confidence value in the threshold cycle (Ct). Error bars in all graphs indicate the standard error of the mean. FIG. 5C is a bar graph showing that Hifl a recruits SIRT6 to glycolytic promoters. ChIP was performed on wild-type control (WT- ctrl) and Hifl a knockdown cells (WT-shHif) with an antibody against SIRT6. RT- PCR was carried out using primers specific for the promoter region of the Ldhb gene. SIR6 KO cells were used as negative controls in the ChIP assay.

FIG. 6. Increased Hifl a stability and protein synthesis in SIRT6-deficient cells. FIG. 6A is a bar graph showing RNA was purified from SIRT6 WT and KO ES cells and Hifl a expression was analyzed by RT-PCR using primers specific for the mRNA of Hifl a. Results are shown as the mean ± SEM (n = 6). FIG. 6B. Upper panel: Lysates were prepared from SIRT6 WT or KO ES cells, followed by IP and Western blot with an Hifl a antibody. Samples were either left untreated or treated with the Hifl a stabilizer CoC12 (150 mM) for 24 hours prior to lysate preparation. Lower panel: Quantitative densitometric analysis of Hifl a levels from the upper panel blot. FIG. 6C. Wild-type (WT) and SIRT6-deficient (KO) cells were cotransfected with an empty 5 ' UTR-Luc vector or Hifl a 5' UTR-Luc reporters and shifted 6 hour post-transfection to no glucose- hypoxia conditions for 24 hours for measurement of luciferase activity. FIG. 6D.

Polysome profile analysis of WT and Sirt6-deficient (KO) ES cells. Lower panel: WT and KO cells were treated with cycloheximide (CHX) for 10 minutes before collection. The lysates were processed for polysome analysis by velocity sedimentation on sucrose gradients. Gradients were fractionated by scanning at 254 nm, and the resulting absorbance profiles are shown with sedimentation from left to right. Upper panel:

Quantitative RT-PCR was performed to assess distribution of Hifl mRNA. Error bars in all graphs indicate SEM.

FIG. 7. Increased expression of glycolytic genes and lactate production in SIRT6 deficient mice. FIG. 7A. Lysates were prepared from muscles of 4 littermate -pairs of SIRT6 WT and KO mice. Western analysis was carried out with antibodies against the indicated proteins. Tubulin was used as a loading control. FIG. 7B. Immunostaining with a GLUT1 antibody (green) was carried out on muscles from SIRT6 WT and KO mice. Nuclei were stained with DAPI (blue). Images were taken using a confocal microscope with constant laser beam for all images (KR: 39.8; IRIS: 2.0). FIG. 7C. Serum was purified from SIRT6 WT and KO mice, and lactate was measured using the Lactate Assay Kit (Bio Vision). Error bars indicate the standard error of the mean, n = 4 for each genotype. FIG. 7D. A Hifl small-molecule inhibitor rescues the glucose phenotype in SIRT6-deficient mice. Hifl inhibitor #77 (20 mg/g weight) was injected

intraperitoneally (i.p.) in 19-day-old WT and SIRT6 KO mice, and 30 minutes later, blood was withdrawn for glucose measurement. Five percent DMSO (dilution solution) was injected as control. FIG. 7E. Model. Under normal nutrient conditions, SIRT6 inhibits expression of glycolytic genes acting as a histone deacetylase to co-repress Hifla. This maintains proper flux of glucose to the TCA cycle. Under conditions of nutrient stress, SIRT6 is inactivated, allowing activation of Hifla, recruitment of p300, acetylation of H3K9 at the promoters and increased expression of multiple metabolic genes, causing increased glycolysis and decreased mitochondrial respiration.

FIG. 8. SIRT6 functions as a histone H3K9 deacetylase in vitro and in vivo.

FIG. 8A. In vitro deacetylation assay. Flag-tagged purified SIRT6 was incubated with 10μg HeLa-purified histones (Millipore) for 1 hour at 37°C, in the presence or absence of NAD⁺. Western analysis showing SIRT6 deacetylation of H3K9 on full-length histone H3 in vitro. Reactions with NAD⁺, SIRT6, or the catalytic H133Y SIRT6 mutant protein (SIRT6-HY) are indicated. FIGs. 8B and 8C. Whole cell extracts from SIRT6 wild type (WT) and KO ES cells (B) and MEFs (C) were blotted with the indicated antibodies. FIG. 9. SIRT6 deficient mice fed with a high fat diet (HFD) weigh less and have lower blood glucose levels than wild type littermates. FIG. 9 A is a line graph showing that SIRT6 KO-HFD animals exhibit lower weight than wild type littermates. FIG. 9B is a bar graph showing that SIRT6 KO-HFD animals have lower blood glucose levels than wild type littermates.

FIG. 10 is a photograph of a Coomassie Blue-stained Western blot, with immunoprecipitated FLAG-SIRT6 purified protein. Total Protein: SIRT6-HY, 13 μg; and SIRT6-WT, 280 μg.

DETAILED DESCRIPTION

SIRT6 is a member of a highly conserved family of NAD⁺-dependent

deacetylases with various roles in metabolism, stress resistance, and life span. SIRT6- deficient mice develop normally but succumb to lethal hypoglycemia early in life. This invention relates to the role of SIRT6 as a histone H3K9 deacetylase to control the expression of multiple glycolytic genes. Specifically, SIRT6 functions as a corepressor of the transcription factor Hifl , a critical regulator of nutrient stress responses. SIRT6- deficient cells exhibit increased Hifl activity and show increased glucose uptake with upregulation of glycolysis and diminished mitochondrial respiration. A role for the chromatin factor SIRT6 as a master regulator of glucose homeostasis provides, inter alia, a basis for therapeutic approaches against metabolic diseases, such as hyperglycemia and obesity.

HYPERGLYCEMIA

Glucose levels vary before and after meals, and at various times of day; in general, the normal range for most fasting adults is about 4 to 6 mmol/L. A subject with a consistent range above 7 mmol/L is generally considered to have hyperglycemia, whereas a consistent range below 4 mmol/L is considered hypoglycemic. The definition of acute hyperglycemia varies by study, with mmol/L levels from 8 to 15. In fasting adults, blood plasma glucose should not exceed 7 mmol/L. Sustained higher levels of blood sugar cause damage to the blood vessels and to the organs they supply. Causes of Hyperglycemia

Hyperglycemia can be associated with a number of conditions, including diabetes mellitus, drugs, critical illness, and physiological stress.

A. Diabetes Mellitus

Chronic hyperglycemia that persists even in fasting states is most commonly caused by diabetes mellitus, and in fact chronic hyperglycemia is the defining characteristic of the disease. Intermittent hyperglycemia may be present in prediabetic states. Acute episodes of hyperglycemia without an obvious cause may indicate developing diabetes or a predisposition to the disorder.

In diabetes mellitus, hyperglycemia is usually caused by low insulin levels and/or by resistance to insulin at the cellular level, depending on the type and state of the disease. Insulin causes cells in the liver, muscle, and fat tissue to take up glucose from the blood, storing it as glycogen in the liver and muscle, and stopping use of fat as an energy source. Low insulin levels (Type 1 diabetes) and/or insulin resistance (Type 2 diabetes) prevent the body from converting glucose into glycogen, which in turn inhibits the removal of excess glucose from the blood. With normal glucose levels, the total amount of glucose in the blood at any given moment is only enough to provide energy to the body for 20-30 minutes. Therefore, glucose levels must be precisely maintained by the body's internal control mechanisms. When the mechanisms fail in a way that allows glucose to rise to abnormal levels, hyperglycemia is the result.

B. Drugs

Certain medications increase the risk of hyperglycemia, including beta blockers, thiazide diuretics, corticosteroids, niacin, pentamidine, protease inhibitors, L- asparaginase, and some antipsychotic agents.

C. Critical Illness

A high proportion of patients suffering an acute stress such as stroke or myocardial infarction may develop hyperglycemia, even in the absence of a diagnosis of diabetes. Human and animal studies suggest that stress-induced hyperglycemia is associated with a high risk of mortality after both stroke and myocardial infarction. D. Physiological Stress

Hyperglycemia occurs naturally during times of infection and inflammation. Endogenous catecholamines are released upon stress, which amongst other things, serve to raise the blood glucose levels. The amount of increase varies from one individual to another and also, from one inflammatory response to the next. As such, no patient with first-time hyperglycemia should be diagnosed immediately with diabetes if that patient is concomitantly ill with another ailment. Further testing, such as a fasting plasma glucose, random plasma glucose, or two-hour postprandial plasma glucose level, must be performed.

Measurement of Blood Glucose Levels

Blood glucose levels can be measured using methods known in the art, e.g., via a HbAlc test (hemoglobin Ale test, glycosylated hemoglobin Ale test, glycohemoglobin Ale test, or Ale test). The HbAlc test is a lab test that reveals average blood glucose over a period of two to three months. Specifically, it measures the number of glucose molecules attached to hemoglobin. The test takes advantage of the lifecycle of red blood cells. Although constantly replaced, individual cells live for about four months. By measuring attached glucose in a current blood sample, average blood sugar levels over the previous two to three months can be determined.

OBESITY

Obesity is a medical condition in which excess body fat has accumulated to the extent that it may have an adverse effect on health, leading to reduced life expectancy and/or increased health problems. Body mass index (BMI), a measurement that compares weight and height, defines people as overweight (pre-obese) when their BMI is between 25 kg/m² and 30 kg/m², and obese when it is greater than 30 kg/m².

Obesity increases the likelihood of various diseases, particularly heart disease, Type 2 diabetes, breathing difficulties during sleep, certain types of cancer, and osteoarthritis. Obesity is most commonly caused by a combination of excessive dietary calories, lack of physical activity, and genetic susceptibility, although a few cases are caused primarily by genes, endocrine disorders, medications, or psychiatric illness. The primary treatment for obesity is dieting and physical exercise. To supplement this, or in case of failure, anti-obesity drugs may be taken to reduce appetite or inhibit fat absorption. In severe cases, surgery is performed or an intragastric balloon is placed to reduce stomach volume and/or bowel length, leading to earlier satiation and reduced ability to absorb nutrients from food.

Causes of Obesity

A combination of excessive caloric intake and a lack of physical activity is thought to explain most cases of obesity. A limited number of cases are due primarily to genetics, medical reasons, or psychiatric illness. In contrast, increasing rates of obesity at a societal level are felt to be due to an easily accessible and palatable diet, increased reliance on cars, and mechanized manufacturing.

Some other possible contributors to the recent increase of obesity are:

(1) insufficient sleep, (2) endocrine disruptors (environmental pollutants that interfere with lipid metabolism), (3) decreased variability in ambient temperature, (4) decreased rates of smoking, because smoking suppresses appetite, (5) increased use of medications that can cause weight gain (e.g., atypical antipsychotics), (6) proportional increases in ethnic and age groups that tend to be heavier, (7) pregnancy at a later age (which may cause susceptibility to obesity in children), (8) epigenetic risk factors passed on generationally, (9) natural selection for higher BMI, and (10) assortative mating leading to increased concentration of obesity risk factors (this would not necessarily increase the number of obese people, but would increase the average population weight). There is substantial evidence supporting the influence of these mechanisms on the increased prevalence of obesity, however, the evidence is still inconclusive.

SIRT6

Little is known about the function of the mammalian Sirtuin 6 (SIRT6) protein. SIRT6 is a nuclear, chromatin-bound protein (Mostoslavsky et al., Cell 124:315-329, 2006). Among the sirtuins, SIRT6 deficiency causes the most striking phenotype. SIRT6 deficient mice are born normally, but at around 3 weeks of age they develop several acute degenerative processes, dying before one month of age. The defects include a severe metabolic imbalance, with low levels of serum IGF-1, complete loss of subcutaneous fat, lymphopenia, osteopenia, and acute onset of hypoglycemia, leading to death

(Mostoslavsky et al, Cell 124:315-329, 2006). Furthermore, SIRT6 promotes resistance to DNA damage and oxidative stress, and suppresses genomic instability in mouse cells, in association with a role in base excision DNA repair (BER) (Mostoslavsky et al., Cell 124:315-329, 2006). Recent studies have demonstrated that SIRT6 is located at the telomeres in human cells, and knock-down of SIRT6 in these cells altered the telomere structure, causing accelerated senescence and telomere-dependent genomic instability. Furthermore, SIRT6 functions as a histone deacetylase, deacetylating histone H3 lysine 9 (H3K9) specifically at telomeres (Michishita et al, Nature 452:492-496, 2008). New studies indicate that SIRT6 can function as a co-repressor of NF-κΒ, silencing NF-KB target genes through deacetylation of H3K9, and decreasing NF-KB-dependent apoptosis and senescence (Kawahara et al., Cell 136:62-74, 2009). Therefore, it appears that SIRT6 can function as a histone H3K9 deacetylase in a cell- and context-dependent manner. At this point, however, the molecular defect underlying the main phenotype in SIRT6 deficient mice, namely the lethal hypoglycemia, remains unclear. Critically, it is not known whether SIRT6 is directly or indirectly involved in the modulation of glucose metabolism.

In the presence of oxygen and glucose, cells convert glucose to pyruvate, which enters the mitochondria, is converted to acetyl coenzyme A, and metabolized via the tricarboxylic acid cycle (TCA), yielding reducing equivalents that are used for oxidative phosphorylation to generate ATR However, under hypoxic or low nutrient conditions, cells shift their metabolism from aerobic to anaerobic metabolism, converting pyruvate instead to lactate (the "Pasteur effect") (Aragones et al., Cell Metabolism 9: 11-22, 2009; Vander et al, Science 324: 1029-1033, 2009). With this energy compensation, cells continue to generate ATP (albeit less efficiently), in an attempt to meet their metabolic demands during this period of stress. The hypoxia-inducible transcription factor Hifla is a key mediator of this cellular adaptation to nutrient and oxygen stress (Lum et al., Genes & Development 21 : 1037-1049, 2007; Seagroves et al, Mol Cell Biol 21 :3436-3444, 2001), functioning as a direct transcriptional activator of multiple genes. On one hand, it enhances glycolytic flux by up-regulating expression of key glycolytic genes, including the glucose transporters GLUT-1 and GLUT-3, lactate dehydrogenase (LDH),

phosphoglycerate kinase (PGK-1), glucose-6-phosphate Isomerase (GPI) and

phosphofructose kinase 1 (PFK-1) (Hu et al, Mol Cell Biol 26:3514-3526, 2006). On the other hand, Hifla directly inhibits mitochondrial respiration by up-regulating expression of the pyruvate dehydrogenase kinase (PDK) gene (Kim et al., Cell Metabolism 3: 177- 185, 2006; Papandreou et al, Cell Metabolism 3: 187-197, 2006). PDK in turn

phosphorylates and inactivates pyruvate dehydrogenase (PDH), a rate-limiting enzyme that converts pyruvate to Acetyl-CoA to fuel the TCA cycle. Recent studies indicate that Hifl a also diminishes mitochondrial activity through inhibition of the Cytochrome Oxidase Subunit Cox4-l and the coactivator PGC-Ιβ (Fukuda et al, Cell 129: 111-122, 2007; Zhang et al., Cancer Cell 11 :407-420, 2007). Overall, Hifla appears to modulate multiple genes in order to activate glycolysis and at the same time repress mitochondrial respiration in a coordinated fashion.

The activity of Hifla is tightly regulated. Under normoxia, Hifla is hydroxylated at multiple prolyl residues by the prolyl-hydroxylase-domain (PHD) proteins. Following hydroxylation, Hifla is recognized by the von-Hippel-Lindau (VHL) ubiquitin ligase, marking Hifla for subsequent proteosome degradation. When oxygen or glucose are low, PHD proteins are inactivated, thereby stabilizing Hifla protein levels (Aragones et al, Cell Metabolism 9: 11-22, 2009). However, even under normoxic and normoglycemic conditions, Hifla regulates basal expression of its target genes (Carmeliet et al, Nature 394:485-490, 1998), suggesting that further mechanisms should be in place to ensure that this stress response is tightly regulated under normal nutrient conditions.

SIRT6 deficiency causes a cell-autonomous up-regulation of glucose uptake, both in vitro and in vivo, triggering a nutrient-stress response and a switch in glucose metabolism towards glycolysis and away of mitochondrial respiration. SIRT6 functions as a co-repressor of Hifla transcriptional activity, deacetylating H3K9 at Hifla target gene promoters. In this way, SIRT6 maintains efficient glucose flux into the TCA cycle under normal nutrient conditions. Regulation of glucose flux by SIRT6 appears critical since SIRT6 deficiency causes a lethal hypoglycemia. In this context, it is striking that deficiency in a single chromatin factor exerts such a severe and specific metabolic phenotype, highlighting the crucial role for SIRT6 in this pathway (FIG. 6D). Seven examples of SIRT6 are highlighted below in Table 1 , and inhibitors to substantially identical nucleotide sequences can also be used. As used herein, "substantially identical" refers to a nucleotide sequence that contains a sufficient or minimum number of identical or equivalent nucleotides to the sequence of SIRT6, such that homologous recombination can occur. For example, nucleotide sequences that are at least about 75% identical to the sequence of SIRT6 are defined herein as substantially identical. In some embodiments, the nucleotide sequences are about 80%, 85%, 90%, 95%, 99%, or 100% identical.

To determine the percent identity of two sequences, the sequences are aligned for optimal comparison purposes (gaps are introduced in one or both of a first and a second amino acid or nucleic acid sequence as required for optimal alignment, and nonhomologous sequences can be disregarded for comparison purposes). The length of a reference sequence aligned for comparison purposes is at least 80%> (in some

embodiments, about 85%, 90%>, 95%, or 100% of the length of the reference sequence) is aligned. The nucleotides or residues at corresponding positions are then compared. When a position in the first sequence is occupied by the same nucleotide or residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two amino acid sequences can be determined using the

Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package, using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. Species Nucleic Acid Amino Acid GenelD

Homo sapiens NM_016539.1 NP_057623.1 51548

Mus musculus NMJ81586.3 NP_853617.1 50721

Rattus norvegicus NM OO 1031649.1 NP OO 1026819.1 299638

Macaca mulatta NC_007876.1 NW OO 1106369.1 714545

Pan troglodytes NC_006486.2 NW_001228145.1 737026

Canis lupus familiaris NC 006602.2 NW_876272.1 485045

Bos Taurus NM OO 1098084.1 NP OO 1091553.1 535416

Table 1. SIRT6 orthologs from seven different species along with their GenBank Ref Seq

Accession Numbers.

Provided herein are methods for reducing or inhibiting hyperglycemia or obesity in a subject by administering to the subject a therapeutically effective amount of a SIRT6 inhibitor.

Subjects to be Treated

In one aspect of the methods described herein, a subject is selected on the basis that they have, or are at risk of developing, hyperglycemia or obesity. A subject that has, or is at risk of developing, hyperglycemia or obesity is one having one or more symptoms of the condition.

Symptoms of hyperglycemia are known to those of skill in the art and include, without limitation, polyphagia (frequent hunger, especially pronounced hunger), polydipsia (frequent thirst, especially excessive thirst), polyuria (frequent urination, especially excessive urination), blurred vision, fatigue/sleepiness, weight loss, poor wound healing (e.g., cuts and scrapes), dry mouth, dry or itchy skin, impotence (male), recurrent infections such as vaginal yeast infections, groin rash, external ear infections (swimmer's ear), Kussmaul hyperventilation (deep, rapid breathing), cardiac arrhythmia, stupor, and coma. Polydipsia and polyuria occur when blood glucose levels rise high enough to result in excretion of excess glucose via the kidneys (glycosuria), producing osmotic diuresis. Symptoms of obesity are known to those of skill in the art and include, without limitation, increased weight, increased BMI, increased abdominal girth, and secondary medical complications (e.g., high cholesterol (including high triglyceride levels), diabetes, high blood pressure, heart disease, stroke, sleep apnea, osteoarthritis, and gallstones).

The methods are effective for a variety of subjects including mammals, e.g., humans and other animals, such as laboratory animals, e.g., mice, rats, rabbits, or monkeys, or domesticated and farm animals, e.g., cats, dogs, goats, sheep, pigs, cows, or horses.

INHIBITORS OF SIRT6

Examples of inhibitors of SIRT6 include antibodies that bind to and/or inhibit a SIRT6, as well as nucleic acids that inhibit SIRT6 gene expression. Such modulators can be provided as a pharmaceutical composition. A. Antibodies to SIRT6

Antibodies can be produced that bind to SIRT6. For example, an antibody can bind to SIRT6 and prevent SIRT6 enzymatic activity or an interaction between SIRT6 and a SIRT6 binding partner (e.g., Hifl ). The term "antibody" as used herein refers to an immunoglobulin molecule or immunologically active portion thereof, i.e., an antigen- binding fragment. Examples of immunologically active portions of immunoglobulin molecules include F(ab') and F(ab')₂ fragments, which retain the ability to bind antigen. Such fragments can be obtained commercially, or using methods known in the art. For example, F(ab')₂ fragments can be generated by treating the antibody with an enzyme such as pepsin, a non-specific endopeptidase that normally produces one F(ab')₂ fragment and numerous small peptides of the Fc portion. The resulting F(ab')₂ fragment is composed of two disulfide-connected Fab units. The Fc fragment is extensively degraded and can be separated from the F(ab')₂ by dialysis, gel filtration or ion exchange chromatography. F(ab') fragments can be generated using papain, a non-specific thiol- endopeptidase that digests IgG molecules, in the presence of a reducing agent, into three fragments of similar size: two Fab fragments and one Fc fragment. When Fc fragments are of interest, papain is the enzyme of choice because it yields a 50,00 Dalton Fc fragment; to isolate the F(ab') fragments, the Fc fragments can be removed, e.g., by affinity purification using protein A/G. A number of kits are available commercially for generating F(ab') fragments, including the ImmunoPure IgGl Fab and F(ab')₂ Preparation Kit (Pierce Biotechnology, Rockford, IL). In addition, commercially available services for generating antigen-binding fragments can be used, e.g., Bio Express, West Lebanon, NH.

The antibody can be a polyclonal, monoclonal, recombinant, e.g., a chimeric, de-immunized or humanized, fully human, non-human, e.g., murine, or single chain antibody. In some embodiments the antibody has effector function and can fix complement. In some embodiments, the antibody has reduced or no ability to bind an Fc receptor. For example, the antibody can be an isotype or subtype, fragment or other mutant, which does not support binding to a Fc receptor, e.g., it has a mutagenized or deleted Fc receptor binding region. The antibody can be coupled to a toxin or imaging agent.

Methods for making suitable antibodies are known in the art. A full-length SIRT6 protein or antigenic peptide fragment thereof can be used as an immunogen, or can be used to identify antibodies made with other immunogens, e.g., cells, membrane preparations, and the like, e.g., E rosette positive purified normal human peripheral T cells, as described in U.S. Patent Nos. 4,361,549 and 4,654,210.

Methods for making monoclonal antibodies are known in the art. Basically, the process involves obtaining antibody-secreting immune cells (lymphocytes) from the spleen of a mammal (e.g., mouse) that has been previously immunized with the antigen of interest (e.g., a cancer-related antigen) either in vivo or in vitro. The antibody- secreting lymphocytes are then fused with myeloma cells or transformed cells that are capable of replicating indefinitely in cell culture, thereby producing an immortal, immunoglobulin-secreting cell line. The resulting fused cells, or hybridomas, are cultured, and the resulting colonies screened for the production of the desired monoclonal antibodies. Colonies producing such antibodies are cloned, and grown either in vivo or in vitro to produce large quantities of antibody. A description of the theoretical basis and practical methodology of fusing such cells is set forth in Kohler and Milstein

(Nature 256:495, 1975), which is hereby incorporated by reference.

Mammalian lymphocytes are immunized by in vivo immunization of the animal (e.g., a mouse) with a cancer-related antigen. Such immunizations are repeated as necessary at intervals of up to several weeks to obtain a sufficient titer of antibodies. Following the last antigen boost, the animals are sacrificed and spleen cells removed.

Fusion with mammalian myeloma cells or other fusion partners capable of replicating indefinitely in cell culture is effected by known techniques, for example, using polyethylene glycol ("PEG") or other fusing agents (See Milstein and Kohler, Eur. J. Immunol. 6:511, 1976, which is hereby incorporated by reference). This immortal cell line, which is preferably murine, but can also be derived from cells of other mammalian species, including but not limited to rats and humans, is selected to be deficient in enzymes necessary for the utilization of certain nutrients, to be capable of rapid growth, and to have good fusion capability. Many such cell lines are known to those skilled in the art, and others are regularly described.

Procedures for raising polyclonal antibodies are also known. Typically, such antibodies can be raised by administering the protein or polypeptide of the present invention subcutaneously to New Zealand white rabbits that have first been bled to obtain pre-immune serum. The antigens can be injected at a total volume of 100 μΐ per site at six different sites. Each injected material will contain synthetic surfactant adjuvant pluronic polyols, or pulverized acrylamide gel containing the protein or polypeptide after SDS-polyacrylamide gel electrophoresis. The rabbits are then bled two weeks after the first injection and periodically boosted with the same antigen three times every six weeks. A sample of serum is then collected 10 days after each boost. Polyclonal antibodies are then recovered from the serum by affinity chromatography using the corresponding antigen to capture the antibody. Ultimately, the rabbits are euthanized, e.g., with pentobarbital 150 mg/kg IV. This and other procedures for raising polyclonal antibodies are disclosed in E. Harlow, et. al., editors, Antibodies: A Laboratory Manual (1988).

In addition to utilizing whole antibodies, the invention encompasses the use of binding portions of such antibodies. Such binding portions include Fab fragments, F(ab')₂ fragments, and Fv fragments. These antibody fragments can be made by conventional procedures, such as proteolytic fragmentation procedures, as described in J. Goding, Monoclonal Antibodies: Principles and Practice, pp. 98-118 (N.Y. Academic Press 1983).

Chimeric, humanized, de -immunized, or completely human antibodies are desirable for applications which include repeated administration, e.g., therapeutic treatment of human subjects.

Chimeric antibodies generally contain portions of two different antibodies, typically of two different species. Generally, such antibodies contain human constant regions and variable regions from another species, e.g., murine variable regions. For example, mouse/human chimeric antibodies have been reported which exhibit binding characteristics of the parental mouse antibody, and effector functions associated with the human constant region. See, e.g., Cabilly et al, U.S. Pat. No. 4,816,567; Shoemaker et al, U.S. Pat. No. 4,978,745; Beavers et al, U.S. Pat. No. 4,975,369; and Boss et al, U.S. Pat. No. 4,816,397, all of which are incorporated by reference herein. Generally, these chimeric antibodies are constructed by preparing a genomic gene library from DNA extracted from pre-existing murine hybridomas (Nishimura et al., Cancer Research, 47:999, 1987). The library is then screened for variable region genes from both heavy and light chains exhibiting the correct antibody fragment rearrangement patterns.

Alternatively, cDNA libraries are prepared from RNA extracted from the hybridomas and screened, or the variable regions are obtained by polymerase chain reaction. The cloned variable region genes are then ligated into an expression vector containing cloned cassettes of the appropriate heavy or light chain human constant region gene. The chimeric genes can then be expressed in a cell line of choice, e.g., a murine myeloma line. Such chimeric antibodies have been used in human therapy.

Humanized antibodies are known in the art. Typically, "humanization" results in an antibody that is less immunogenic, with complete retention of the antigen-binding properties of the original molecule. In order to retain all the antigen-binding properties of the original antibody, the structure of its combining-site has to be faithfully reproduced in the "humanized" version. This can potentially be achieved by transplanting the combining site of the nonhuman antibody onto a human framework, either (a) by grafting the entire nonhuman variable domains onto human constant regions to generate a chimeric antibody (Morrison et al, Proc Natl Acad Sci USA 81 :6801, 1984; Morrison and Oi, Adv Immunol 44:65, 1988) (which preserves the ligand-binding properties, but which also retains the immunogenicity of the nonhuman variable domains); (b) by grafting only the nonhuman CDRs onto human framework and constant regions with or without retention of critical framework residues (Jones et al., Nature 321 :522, 1986;

Verhoeyen et al., Science 239: 1539, 1988); or (c) by transplanting the entire nonhuman variable domains (to preserve ligand-binding properties) but also "cloaking" them with a human- like surface through judicious replacement of exposed residues (to reduce antigenicity) (Padlan, Molec Immunol 28:489, 1991).

Humanization by CDR grafting typically involves transplanting only the CDRs onto human fragment onto human framework and constant regions. Theoretically, this should substantially eliminate immunogenicity (except if allotypic or idiotypic differences exist). However, it has been reported that some framework residues of the original antibody also need to be preserved (Riechmann et al, Nature 332:323, 1988; Queen et al, Proc Natl Acad Sci USA 86: 10,029, 1989). The framework residues which need to be preserved can be identified by computer modeling. Alternatively, critical framework residues may potentially be identified by comparing known antibody combining site structures (Padlan, Mol Immun 31(3): 169-217, 1994). The invention also includes partially humanized antibodies, in which the 6 CDRs of the heavy and light chains and a limited number of structural amino acids of the murine monoclonal antibody are grafted by recombinant technology to the CDR-depleted human IgG scaffold (Jones et al, Nature 321 :522-525, 1986).

Deimmunized antibodies are made by replacing immunogenic epitopes in the murine variable domains with benign amino acid sequences, resulting in a deimmunized variable domain. The deimmunized variable domains are linked genetically to human

IgG constant domains to yield a deimmunized antibody (Biovation, Aberdeen, Scotland).

The antibody can also be a single chain antibody. A single-chain antibody (scFV) can be engineered (see, for example, Colcher et al, Ann NY Acad Sci 880:263-80, 1999; and Reiter, Clin Cancer Res 2:245-52, 1996). The single chain antibody can be dimerized or multimerized to generate multivalent antibodies having specificities for different epitopes of the same target protein. In some embodiments, the antibody is monovalent, e.g., as described in Abbs et al, Ther Immunol 1(6):325-31, 1994, incorporated herein by reference.

SIRT6 antibodies are also commercially available, e.g., from Abeam, Novus Biologicals, Thermo Scientific Pierce Antibodies, and Sigma- Aldrich. These antibodies can be modified as known in the art and disclosed herein, e.g., humanized or

deimmunized.

B. SIRT6 Inhibitory Nucleic Acids

Nucleic acid molecules (e.g., RNA molecules) can be used to inhibit (i.e., reduce) SIRT6 expression or activity. A SIRT6 inhibitor can be a siRNA, antisense RNA, a ribozyme, or aptamer, which can specifically reduce the expression of SIRT6. In some aspects, a cell or subject can be treated with a compound that reduces the expression of SIRT6. Such approaches include oligonucleotide -based therapies such as RNA interference, antisense, ribozymes, and aptamers. i. siRNA Molecules

RNA interference (RNAi) is a process whereby double-stranded RNA (dsRNA, also referred to herein as siRNAs or ds siRNAs, for double-stranded small interfering RNAs) induces the sequence-specific degradation of homologous mRNA in animals and plant cells (Hutvagner and Zamore, Curr Opin Genet Dev 12:225-232, 2002; Sharp, Genes Dev 15:485-490, 2001). In mammalian cells, RNAi can be triggered by 21- nucleotide (nt) duplexes of small interfering RNA (siRNA) (Chiu et al, Mol Cell 10:549- 561, 2002; Elbashir et al, Nature 411 :494-498, 2001), or by micro-RNAs (miRNA), functional small-hairpin RNA (shRNA), or other dsRNAs which are expressed in vivo using DNA templates with RNA polymerase III promoters (Zeng et al., Mol Cell 9: 1327- 1333, 2002; Paddison et al, Genes Dev 16:948-958, 2002; Lee et al, Nature Biotechnol 20:500-505, 2002; Paul et al, Nature Biotechnol 20:505-508, 2002; Tuschl, Nature Biotechnol 20:440-448, 2002; Yu et al, Proc Natl Acad Sci USA 99(9):6047-6052, 2002; McManus et al, RNA 8:842-850, 2002; Sui et al., Proc Natl Acad Sci USA 99(6):5515- 5520, 2002). The nucleic acid molecules or constructs can include dsRNA molecules comprising 16-30, e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in each strand, wherein one of the strands is substantially identical, e.g., at least 75% (or more, e.g., 80%, 85%, 90%, 95%, or 100%) identical, e.g., having 3, 2, 1, or 0 mismatched nucleotide(s), to a target region in the mRNA, and the other strand is complementary to the first strand. The dsRNA molecules can be chemically synthesized, or can transcribed in vitro from a DNA template, or in vivo from, e.g., shRNA. The dsRNA molecules can be designed using any method known in the art; a number of algorithms are known, and are commercially available. Gene walk methods can be used to optimize the inhibitory activity of the siRNA.

The nucleic acid compositions can include both siRNA and modified siRNA derivatives, e.g., siRNAs modified to alter a property such as the pharmacokinetics of the composition, for example, to increase half-life in the body, as well as engineered RNAi precursors.

siRNAs can be delivered into cells by methods known in the art, e.g., cationic liposome transfection and electroporation. siRNA duplexes can be expressed within cells from engineered RNAi precursors, e.g., recombinant DNA constructs using mammalian Pol III promoter systems (e.g., HI or U6/snRNA promoter systems (Tuschl 2002, supra) capable of expressing functional double-stranded siRNAs; (Bagella et al, J Cell Physiol 177:206-213, 1998; Lee et al, 2002, supra; Miyagishi et al, 2002, supra; Paul et al,

2002, supra; Yu et al, 2002, supra; Sui et al, 2002, supra). Transcriptional termination by RNA Pol III occurs at runs of four consecutive T residues in the DNA template, providing a mechanism to end the siRNA transcript at a specific sequence. The siRNA is complementary to the sequence of the target gene in 5 '-3' and 3 '-5' orientations, and the two strands of the siRNA can be expressed in the same construct or in separate constructs. Hairpin siRNAs, driven by HI or U6 snRNA promoter and expressed in cells, can inhibit target gene expression (Bagella et al, 1998, supra; Lee et al, 2002, supra; Miyagishi et al, 2002, supra; Paul et al., 2002, supra; Yu et al., 2002, supra; Sui et al, 2002, supra). Constructs containing siRNA sequence under the control of T7 promoter also make functional siRNAs when cotransfected into the cells with a vector expression T7 RNA polymerase (Jacque, 2002, supra). ii. Antisense Nucleic Acids

An "antisense" nucleic acid can include a nucleotide sequence that is

complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to a SIRT6 mRNA sequence. The antisense nucleic acid can be complementary to an entire coding strand of a target sequence, or to only a portion thereof. In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence (e.g., the 5' and 3' untranslated regions).

An antisense nucleic acid can be designed such that it is complementary to the entire coding region of a target mRNA, but can also be an oligonucleotide that is antisense to only a portion of the coding or noncoding region of the target mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of the target mRNA, e.g., between the -10 and +10 regions of the target gene nucleotide sequence of interest. An antisense oligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length.

An antisense nucleic acid can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. The antisense nucleic acid also can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).

Based upon the sequences disclosed herein, one of skill in the art can easily choose and synthesize any of a number of appropriate antisense molecules for use in accordance with the present invention. For example, a "gene walk" comprising a series of oligonucleotides of 15-30 nucleotides spanning the length of a target nucleic acid can be prepared, followed by testing for inhibition of target gene expression. Optionally, gaps of 5-10 nucleotides can be left between the oligonucleotides to reduce the number of oligonucleotides synthesized and tested.

In some embodiments, the antisense nucleic acid molecule is an a-anomeric nucleic acid molecule. An a-anomeric nucleic acid molecule forms specific double- stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gaultier et al, Nucleic Acids Res 15:6625-6641, 1987). The antisense nucleic acid molecule can also comprise a 2'-o- methylribonucleotide (Inoue et al, Nucleic Acids Res 15:6131-6148, 1987) or a chimeric RNA-DNA analogue (Inoue et al, FEBS Lett 215:327-330, 1987).

In some embodiments, the antisense nucleic acid is a morpholino oligonucleotide (see, e.g., Heasman, Dev Biol 243:209-14, 2002; Iversen, Curr Opin Mol Ther 3:235-8, 2001; Summerton, Biochim Biophys Acta 1489: 141-58, 1999).

Target gene expression can be inhibited by targeting nucleotide sequences complementary to a regulatory region (e.g., promoters and/or enhancers) to form triple helical structures that prevent transcription of the Spt5 gene in target cells. See generally, Helene, Anticancer Drug Des 6:569-84, 1991; Helene, Ann NY Acad Sci 660:27-36, 1992; and Maher, Bioassays 14:807-15, 1992. The potential sequences that can be targeted for triple helix formation can be increased by creating a so called "switchback" nucleic acid molecule. Switchback molecules are synthesized in an alternating 5'-3', 3'-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex. iii. Ribozymes

Ribozymes are a type of RNA that can be engineered to enzymatically cleave and inactivate other RNA targets in a specific, sequence-dependent fashion. By cleaving the target RNA, ribozymes inhibit translation, thus preventing the expression of the target gene. Ribozymes can be chemically synthesized in the laboratory and structurally modified to increase their stability and catalytic activity using methods known in the art. Alternatively, ribozyme genes can be introduced into cells through gene-delivery mechanisms known in the art. A ribozyme having specificity for a target nucleic acid can include one or more sequences complementary to the nucleotide sequence of a cDNA described herein, and a sequence having known catalytic sequence responsible for mRNA cleavage (see U.S. Pat. No. 5,093,246 or Haselhoff and Gerlach, Nature 334:585-591, 1988). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a target mRNA. See, e.g., Cech et al, U.S. Patent

No. 4,987,071; and Cech et al, U.S. Patent No. 5,116,742. Alternatively, a target mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Barrel and Szostak, Science 261 : 1411-1418, 1993. iv. Aptamers

Aptamers are short oligonucleotide sequences that can specifically bind specific proteins. It has been demonstrated that different aptameric sequences can bind specifically to different proteins, for example, the sequence GGNNGG where

N=guanosine (G), cytosine (C), adenosine (A), or thymidine (T) binds specifically to thrombin (Bock et al, Nature 355:564-566, 1992; and U.S. Pat. No. 5,582,981, Toole et al., 1996). Methods for selection and preparation of such RNA aptamers are known in the art (see, e.g., Famulok, Curr Opin Struct Biol 9:324, 1999; Herman and Patel, J Sci 287:820-825, 2000; Kelly et al, J Mol Biol 256:417, 1996; and Feigon et al, Chem Biol 3:611, 1996).

Making and Using Inhibitory Nucleic Acids

The nucleic acid sequences used to practice the methods described herein, whether RNA, cDNA, genomic DNA, vectors, viruses, or hybrids thereof, can be isolated from a variety of sources, genetically engineered, amplified, and/or expressed/generated recombinantly. Recombinant nucleic acid sequences can be individually isolated or cloned and tested for a desired activity. Any recombinant expression system can be used, including e.g. in vitro, bacterial, fungal, mammalian, yeast, insect, or plant cell expression systems. Nucleic acid sequences of the invention can be inserted into delivery vectors and expressed from transcription units within the vectors. The recombinant vectors can be DNA plasmids or viral vectors. Generation of the vector construct can be accomplished using any suitable genetic engineering techniques well known in the art, including, without limitation, the standard techniques of PCR, oligonucleotide synthesis, restriction endonuclease digestion, ligation, transformation, plasmid purification, and DNA sequencing, for example as described in Sambrook et al. Molecular Cloning: A

Laboratory Manual. (1989)), Coffin et al. (Retroviruses. (1997)) and "RNA Viruses: A Practical Approach" (Alan J. Cann, Ed., Oxford University Press, (2000)). As will be apparent to one of ordinary skill in the art, a variety of suitable vectors are available for transferring nucleic acids of the invention into cells. The selection of an appropriate vector to deliver nucleic acids and optimization of the conditions for insertion of the selected expression vector into the cell, are within the scope of one of ordinary skill in the art without the need for undue experimentation. Viral vectors comprise a nucleotide sequence having sequences for the production of recombinant virus in a packaging cell. Viral vectors expressing nucleic acids of the invention can be constructed based on viral backbones including, but not limited to, a retrovirus, lentivirus, adenovirus, adeno- associated virus, pox virus or alpha virus. The recombinant vectors capable of expressing the nucleic acids of the invention can be delivered as described herein, and persist in target cells (e.g., stable trans formants).

Nucleic acid sequences used to practice this invention can be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896;

Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68: 109; Beaucage (1981) Terra. Lett. 22: 1859; U.S. Patent No. 4,458,066.

Nucleic acid sequences of the invention can be stabilized against nucleolytic degradation such as by the incorporation of a modification, e.g., a nucleotide

modification. For example, nucleic acid sequences of the invention includes a phosphorothioate at least the first, second, or third internucleotide linkage at the 5 ' or 3' end of the nucleotide sequence. As another example, the nucleic acid sequence can include a 2'-modified nucleotide, e.g., a 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-0-methyl, 2'-0- methoxyethyl (2'-0-MOE), 2'-0-aminopropyl (2'-0-AP), 2'-0-dimethylaminoethyl (2'-0- DMAOE), 2'-0-dimethylaminopropyl (2'-0-DMAP), 2'-0-dimethylaminoethyloxyethyl (2'-0-DMAEOE), or 2'-0~N-methylacetamido (2'-0~NMA). As another example, the nucleic acid sequence can include at least one 2'-0-methyl-modified nucleotide, and in some embodiments, all of the nucleotides include a 2'-0-methyl modification. In some embodiments, the nucleic acids are "locked," i.e., comprise nucleic acid analogues in which the ribose ring is "locked" by a methylene bridge connecting the 2'-0 atom and the 4'-C atom (see, e.g., Kaupinnen et al, Drug Disc. Today 2(3):287-290 (2005);

Koshkin et al, J. Am. Chem. Soc, 120(50): 13252-13253 (1998)). For additional modifications see US 20100004320, US 20090298916, and US 20090143326.

Techniques for the manipulation of nucleic acids used to practice this invention, such as, e.g., subcloning, labeling probes (e.g., random-primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization and the like are well described in the scientific and patent literature, see, e.g., Sambrook et al, Molecular Cloning; A Laboratory Manual 3d ed. (2001); Current Protocols in Molecular Biology, Ausubel et al, eds. (John Wiley & Sons, Inc., New York 2010); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); Laboratory Techniques In Biochemistry And Molecular Biology: Hybridization With Nucleic Acid Probes, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).

Administration of Inhibitory Nucleic Acid Molecules

The inhibitory nucleic acid molecules described herein can be administered to a subject (e.g., by direct injection at a tissue site), or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a target protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. Alternatively, inhibitory nucleic acid molecules can be modified to target selected cells and then administered systemically. For systemic administration, inhibitory nucleic acid molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the inhibitory nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens. The inhibitory nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the inhibitory nucleic acid molecules, vector constructs in which the inhibitory nucleic acid molecule is placed under the control of a strong promoter can be used.

An "effective amount" is an amount sufficient to effect beneficial or desired results. For example, a therapeutic amount is one that achieves the desired therapeutic effect. This amount can be the same or different from a prophylactically effective amount, which is an amount necessary to prevent onset of disease or disease symptoms. An effective amount can be administered in one or more administrations, applications or dosages. A therapeutically effective amount of a composition depends on the

composition selected. The compositions can be administered from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the compositions described herein can include a single treatment or a series of treatments.

Methods of Screening (Test Compounds)

Included herein are methods for screening test compounds, e.g., polypeptides, polynucleotides (including inhibitory nucleic acids), inorganic or organic large or small molecule test compounds, to identify agents useful in the treatment of metabolic disease, e.g., hyperglycemia and obesity.

As used herein, "small molecules" refers to small organic or inorganic molecules of molecular weight below about 3,000 Daltons. In general, small molecules useful for the invention have a molecular weight of less than 3,000 Daltons (Da). The small molecules can be, e.g., from at least about 100 Da to about 3,000 Da (e.g., between about 100 to about 3,000 Da, about 100 to about 2500 Da, about 100 to about 2,000 Da, about 100 to about 1,750 Da, about 100 to about 1,500 Da, about 100 to about 1,250 Da, about 100 to about 1,000 Da, about 100 to about 750 Da, about 100 to about 500 Da, about 200 to about 1500, about 500 to about 1000, about 300 to about 1000 Da, or about 100 to about 250 Da).

The test compounds can be, e.g., natural products or members of a combinatorial chemistry library. A set of diverse molecules should be used to cover a variety of functions such as charge, aromaticity, hydrogen bonding, flexibility, size, length of side chain, hydrophobicity, and rigidity. Combinatorial techniques suitable for synthesizing small molecules are known in the art, e.g., as exemplified by Obrecht and Villalgordo, Solid-Supported Combinatorial and Parallel Synthesis of Small-Molecular-Weight Compound Libraries, Pergamon-Elsevier Science Limited (1998), and include those such as the "split and pool" or "parallel" synthesis techniques, solid-phase and solution-phase techniques, and encoding techniques (see, for example, Czarnik, Curr. Opin. Chem. Bio. 1 :60-6 (1997)). In addition, a number of small molecule libraries are commercially available. A number of suitable small molecule test compounds are listed in U.S. Patent No. 6,503,713, incorporated herein by reference in its entirety.

Libraries screened using the methods of the present invention can comprise a variety of types of test compounds. A given library can comprise a set of structurally related or unrelated test compounds. In some embodiments, the test compounds are peptide or peptidomimetic molecules. In some embodiments, the test compounds are nucleic acids.

In some embodiments, the test compounds and libraries thereof can be obtained by systematically altering the structure of a first test compound, e.g., a first test compound that is structurally similar to a known natural binding partner of the target polypeptide, or a first small molecule identified as capable of binding the target polypeptide, e.g., using methods known in the art or the methods described herein, and correlating that structure to a resulting biological activity, e.g., a structure-activity relationship study. As one of skill in the art will appreciate, there are a variety of standard methods for creating such a structure-activity relationship. Thus, in some instances, the work may be largely empirical, and in others, the three-dimensional structure of an endogenous polypeptide or portion thereof can be used as a starting point for the rational design of a small molecule compound or compounds. For example, in one embodiment, a general library of small molecules is screened, e.g., using the methods described herein.

In some embodiments, a test compound is applied to a test sample, e.g., a cell, and one or more effects of the test compound is evaluated. In some embodiments, a cultured or primary cell for example, the ability of the test compound to inhibit deacetylation, e.g., of H3K9, can be evaluated. In other embodiments, a cultured or primary cell for example, the ability of the test compound to inhibit SIRT6 expression can be evaluated, e.g., assay SIRT6 mRNA or protein levels. In some embodiments, the test sample comprises an isolated or recombinant SIRT6 polypeptide, e.g., in a cell-free system.

In some embodiments, the test sample is, or is derived from (e.g., a sample taken from) an in vivo model of a disorder as described herein. For example, an animal model, e.g., a rodent such as a rat, can be used.

Methods for evaluating these effects are known in the art. For example, ability to modulate expression of a protein can be evaluated at the gene or protein level, e.g., using quantitative PCR or immunoassay methods. In some embodiments, high throughput methods, e.g., protein or gene chips as are known in the art (see, e.g., Ch. 12, Genomics, in Griffiths et al., Eds. Modern Genetic Analysis, 1999,W. H. Freeman and Company; Ekins and Chu, Trends in Biotechnology, 1999, 17:217-218; MacBeath and Schreiber, Science 2000, 289(5485): 1760-1763; Simpson, Proteins and Proteomics: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 2002; Hardiman, Microarrays Methods and Applications: Nuts & Bolts, DNA Press, 2003), can be used to detect an effect on SIRT6.

A test compound that has been screened by a method described herein and determined to inhibit deacetylation, e.g., of H3K9, can be considered a candidate compound. A candidate compound that has been screened, e.g., in an in vivo model of a disorder, e.g., hyperglycemia or obesity, and determined to have a desirable effect on the disorder, e.g., on one or more symptoms of the disorder, can be considered a candidate therapeutic agent. Candidate therapeutic agents, once screened in a clinical setting, are therapeutic agents. Candidate compounds, candidate therapeutic agents, and therapeutic agents can be optionally optimized and/or derivatized, and formulated with

physiologically acceptable excipients to form pharmaceutical compositions. Thus, test compounds identified as "hits" (e.g., test compounds that inhibit deacetylation, e.g., of H3K9) in a first screen can be selected and systematically altered, e.g., using rational design, to optimize binding affinity, avidity, specificity, or other parameter. Such optimization can also be screened for using the methods described herein. Thus, in one embodiment, the invention includes screening a first library of compounds using a method known in the art and/or described herein, identifying one or more hits in that library, subjecting those hits to systematic structural alteration to create a second library of compounds structurally related to the hit, and screening the second library using the methods described herein.

Test compounds identified as hits can be considered candidate therapeutic compounds, useful in treating metabolic disease as described herein, e.g., hyperglycemia or obesity. A variety of techniques useful for determining the structures of "hits" can be used in the methods described herein, e.g., NMR, mass spectrometry, gas

chromatography equipped with electron capture detectors, fluorescence and absorption spectroscopy. Thus, the invention also includes compounds identified as "hits" by the methods described herein, and methods for their administration and use in the treatment, prevention, or delay of development or progression of a disorder described herein.

Test compounds identified as candidate therapeutic compounds can be further screened by administration to an animal model of a disorder associated with

hyperglycemia or obesity, as described herein. The animal can be monitored for a change in the disorder, e.g., for an improvement in a parameter of the disorder, e.g., a parameter related to clinical outcome. In some embodiments, the parameter is blood glucose level, and an improvement would be euglycemia. In some embodiments, the subject is a human, e.g., a human with diabetes, and the parameter is blood glucose level.

The following are examples of the practice of the invention.

EXAMPLES

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. EXPERIMENTAL PROCEDURES

The following methods were used in the Examples set forth below, unless otherwise noted.

Western and Immunostaining Analysis

Western analysis was carried out as previously described (Cheng et al, Proc Natl Acad Sci 100: 10794-10799, 2003). The antibodies used are: anti-SIRT6 (Abeam), anti- Hifla (Novus), anti-Flag (Sigma), anti-PFKl (Abeam), anti-PDKl (Abeam), anti-TPIl (Abeam), anti-TIGAR (Abeam), and anti-Tubulin (Sigma). For immunostaining of ES cells, cells were grown on coverslips and fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS as described (Bassing et al., Proc Natl Acad Sci USA 99:8173-8178, 2002). Images were taken using a confocal microscope with constant laser beam for all images (KR:39.8; IRIS:2.0).

Lactate Assay

Supernatant of either ES cells or MEFs was collected 24 hours after seeding lxl 0⁶ cells of each genotype, and lactate concentration was determined with the Lactate Assay Kit (Bio Vision). Optical Density was measured at 570nm, 30 min. after addition of substrate.

Oxygen Consumption Assay

4xl0⁵ SIRT6 WT and KO ES cells were seeded, and 24 hours Oxygen

Consumption rate was measured with the Seahorse XF24 instrument (Seahorse

Bioscience), as published (Liu et al, Nature 459:387-392, 2009).

Metabolite Analysis

SIRT6 wild type and KO cells were grown under normal nutrient conditions, and methanol-fixed proteins were analyzed by liquid chromatography-mass spectrometry (LC-MS), as described (Lewis et al, J Clin Inv 118:3503-3512, 2008).

Glucose Uptake Assay in Mice and in Cells

For the assessment of in vivo glucose disposal using [1,2-¹³C] glucose, the mice are given an IP injection (2 mg/gm body weight) containing 50% [l,2-¹³C]-glucose. The mice (n = 4) are retro-orbitally bled at 30 min. The samples are then processed for GC/MS analysis as previously described (Xu et al, Endocrinology 145: 1087-1095, 2004). For the glucose uptake assay in mice, 16-days old SIRT6 WT and KO mice were imaged using a Siemens Inveon PET-CT 45 minutes post injection of approximately 500 of FDG. The PET was set to finish after 600-million events were recorded. List mode files were rebinned in 3D, and the subsequent sinograms were reconstructed using filtered back projection for quantification. Certain images were also reconstructed using 2DOSEM for better visualization. Prior to the PET scan, a CT scan was acquired. The x-ray source was set to a voltage and current of 80 kVp and 500 uA respectively, and it was positioned relative to the CCD detector camera and mouse such that the effective pixel size was 59.73 μιη isotropically. All CT projection data were reconstructed using Filtered Backprojection. The images were interpolated bilinearly, and filtered with a Shepp-Logan filter for higher resolution. PET and CT image reconstructions were converted into a DICOM format and fused using Inveon Research Workplace (Siemens Molecular Imaging, Knoxville, TN). Utilizing the acquired CT for anatomical reference, 3D regions of interested (ROIs) were drawn on the fused images to encompass the following regions on the PET data set: parietal brain, frontal brain, leg muscle, left ventricle (blood pool), brown adipose tissue, bladder, left kidney, myocardium, and liver. The average, PET-derived, activity value for each ROI was recorded, properly decay corrected, and used to calculate an FDG standard uptake value (SUV). Normalizing for variable FDG injection activity, weight, and the ROI volume of each mouse, SUVs were calculated in units of grams/cc^A3 by the following formula: SUV = (mean ROI activity at start of scan/ROI volume)/(total mouse activity at start of scan/mouse weight). All animals were housed at the MGH Animal Facility, and the experiments were performed in accordance with the MGH Subcommittee on Research Animal Care (SRAC) and

Institutional Animal Care an Use Committee (IACUC) guidelines. For glucose uptake assays in cells, cells were grown under normal conditions for 24 hours and 2-NBDG (Invitrogen) was added to the media at 100 μΜ for 2 hours. Fluorescence was measured in a FACSCalibur^™ Analyzer (BD). ATP Concentration Assay

SIRT6 WT or KO ES cells were grown in low glucose (0.5 g/L) media for 24 hours and ATP concentration was measured by Adenosine 5 '-triphosphate (ATP) bioluminescent somatic cell assay kit (Sigma) per manufacturer instructions.

Luciferase Reporter Assay

lxlO⁵ 293T cells were transfected using Trans-IT 293 (Minis Bio LLC) with 1 μg of the following plasmids as described in the text: pGL3::HRE4, pCMV-3xF-SIRT6 and pCMV-3xF-SIRT6HY. 24 hours after transfection, cells were harvested and luciferase activity was determined using the Dual-Luciferase Reporter Assay System (Promega).

ChlPs and Q-RT-PCR

For Q-RT-PCR, total RNA was isolated using RNeasy Mini Kit (Qiagene) and cDNA was generated using QuantiTect^® Reverse Transcription Kit (Qiagene). Q-PCR was carried out using Brilliant SYBR Green QPCR Master Mix Kit (Stratagene). For ChIP assays, cells were fixed with 1% formaldehyde and harvested for whole-cell lysate preparation. Protein lysate was used for ChIP with the following antibodies: anti-SIRT6 antibody (Novus), anti-Hifla antibody (Novus) and anti-H3K9Ac antibody (Millipore). ChlP-enriched DNA was analyzed by Q-PCR as described above. High resolution ChIP analysis was carried out as described in (Donner et al, Mol Cell 27: 121-133, 2007), using the following antibodies: RNA polymerase II (Santa Cruz Biotechnology), S5P-CTD (Covance) and S2P-CTD (Covance).

Retroviral Infection

SIRT6-WT cDNA was amplified by PCR and cloned into the pHAGE2-EFla- dsRed-IRES-tomato vector. Hifla shRNA lentivirus vectors were obtained from The RNAi Consortium Library (MGH). SIRT6-WT and SIRT6-KO ES cells were infected by incubating with virus and 10 μg/ml polybrene. 48 hours later, cells were selected in 2.5 g/ml puromycin and single colonies were picked and plated for various experiments. Immunoprecipitation of Active FLAG-SIRT6 Assays

Cells were transfected with FLAG-SIRT6 construct, using Minis Trans-IT^® (Minis Bio LLC), per manufacturer's instructions. After 48 hours, cells were washed in PBS and lysed with 1 ml of lysis buffer (0.5 M KC1, 50 mM TRIS HC1 (pH 7.5), TSA (2000X), 0.5 mM DTT, 1% NP-40, and inhibitors (EDTA-free)). Cells were rotated for 10 minutes at 4°C and centrifuged at maximum speed for 15 minutes. 50 μΐ of

M2-FLAG beads were added to the supernatant and rotated for two hours at 4°C. The suspension was centrifuged at 4000 rpm for 5 minutes at 4°C, and the beads were washed three times with lysis buffer and once with SDAC Buffer (50 mM TRIS HC1 (pH 9), 4 mM MgCl₂, 50 mM NaCl, 0.5 mM DTT, and inhibitors (EDTA-free)). SIRT6 was eluted by rotating the beads with 100 μΐ SDAC Buffer and 4 μΐ FLAG-peptide for one hour at 4°C before centrifugation at 4000 rpm for 3 minutes at 4°C. The enzyme was stored in 20 μΐ aliquots at -80°C.

Histone Deacetylation Assay

Histone deacetylation reactions were routinely performed as previously described

(Michishita et al., Nature 452:492-496, 2008) with several modifications. 5X deacetylase buffer (50 mM TRIS (pH 8), 50 mM NaCl, 4 mM MgCl₂, 0.5 mM DTT) was used for the assay. Each reaction containing 10 μΐ of the enzyme, 10 μΐ of the buffer, 1 μΐ of 100 μΜ NAD, 1 μΐ of 50X protease inhibitors, 5 μΐ of histones (50 ng/μΐ), TSA (2000X), and distilled H₂0 (18 μΐ to a final volume of 50 μΐ) are incubated for 2.5 hours at 32°C. The reactions are stopped by freezing. Aliquots of 10 μΐ were used in Western blots to analyze deacetylation using anti-H3K9Ac antibody. FIGs. 7A-C are Western blots showing that SIRT6 functions as a histone H3K9 deacetylase in vitro and in vivo. Example 1 : SIRT6 deficiency causes a cell-autonomous increase in glucose uptake

Preliminary studies demonstrated that the most severe defect in SIRT6 deficient animals is lethal hypoglycemia. Although such a phenotype is typically associated with hyperinsulinemia, SIRT6 deficient mice exhibit normal pancreatic islets, and, remarkably, lower blood insulin levels, indicating that low glucose may have triggered a reduction in insulin secretion as an adaptive response. In addition, SIRT6 deficient mice had no defects in glucose absorption in the intestine and did not exhibit increased glucose secretion by the kidney. These observations prompted an analysis to determine whether the mice were experiencing an intrinsic increase in glucose uptake, independent of insulin levels in blood. As seen in FIG. 1A, SIRT6 deficient animals clear 1,2-¹³C Glucose from blood significantly faster than wild-type littermates. Subsequently, ¹⁸F- fiuorodeoxyglucose-positron emission tomography (FDG-PET) was used to determine which tissues had increased glucose uptake. SIRT6 deficient mice exhibit a pronounced increase in glucose uptake both in muscle and brown adipose tissue (FIGs. 1B-C), whereas no changes were observed in the liver, brain or heart (FIG. 1C). This tissue specific increase in glucose uptake could explain the hypoglycemic phenotype of SIRT6 deficient mice.

In order to determine whether SIRT6 influences glucose uptake in a cell- autonomous fashion, glucose uptake in SIRT6 wild-type (WT) and KO cells was measured using a fluorescent glucose analog (2-NBDG) that is incorporated into cells and allows quantification of glucose uptake using fiow-cytometry. Notably, both embryonic stem (ES) cells and mouse embryonic fibroblasts (MEFs) display a striking increase in glucose uptake as early as one hour following addition of the glucose analog (FIGs. 1D- E). Furthermore, this effect appears specific for SIRT6, since SIRT1 deficient MEFs do not show this phenotype. These results indicate that SIRT6 deficiency causes increased glucose uptake in a cell-autonomous fashion. In order to rule out the possibility that this effect on glucose uptake was secondary to adaptation to chronic depletion of SIRT6, cells were generated where SIRT6 can be inactivated in an acute manner. In this system, a previously characterized dominant negative form of SIRT6 (Mostoslavky et al., 2006; Kawahara et al., Cell 136:62-74, 2009) is specifically induced following treatment with tetracycline. Remarkably, 48 hours after expression of this dominant negative mutant, cells exhibit a marked increase in glucose uptake (FIG. IF).

To definitively demonstrate that the glucose phenotype observed was specific to the lack of SIRT6, SIRT6 was re-expressed in SIRT6 KO ES cells, and glucose uptake was tested. Re-expression of SIRT6 rescued the metabolic phenotype, reducing glucose uptake significantly (FIG. 1G). These data show that a lack of SIRT6 in multiple cell types in vivo and in vitro causes a specific and cell-autonomous increase in glucose uptake.

Example 2: Increased membrane expression of the glucose transporter GLUT1 in the absence of SIRT6

SIRT6 deficient cells were assessed to determine whether an increased glucose uptake was associated with elevated expression of glucose transporters. The main glucose transporter in ES cells and MEFs is GLUT1, a receptor that modulates basal uptake of glucose, independent of growth factors or insulin (Pessin and Bell, Ann Rev Phys 54:911-930, 1992). Therefore, cells were stained with an antibody against GLUT1 and confocal microscopy was used to determine quantitative differences in membrane expression of this receptor. SIRT6 KO cells express substantially higher levels of membrane GLUT1 (FIGs. 2A-B), consistent with an increased glucose uptake in these cells.

Example 3: Enhanced glycolysis and reduced mitochondrial respiration in SIRT6 deficient cells

The above results prompted an investigation into how glucose is utilized in SIRT6 deficient cells. Lactate production was measured in order to determine whether glycolysis was enhanced. Indeed, both SIRT6 deficient ES cells (FIG. 2C) and MEFs display significantly higher levels of lactate when compared to WT cells. Concomitantly, lack of SIRT6 causes a reduction in oxygen consumption (FIG. 2D), indicating that in SIRT6 deficient cells, glucose is utilized primarily for glycolysis, whereas mitochondrial respiration is inhibited. To further validate these results, mass-spectrometry was performed based metabolic profiling. Out of 106 metabolites analyzed, 22 showed altered levels in the SIRT6 KO cells (p< 0.05). Among those, multiple TCA metabolites were found that were reduced in SIRT6 KO cells (FIG. 2E), clearly confirming that mitochondrial respiration is inhibited in these cells. Cells switch to glycolysis in order to sustain ATP production under conditions of nutrient stress. Therefore, SIRT6 deficient cells, which exhibit increased glycolysis, were tested to determine whether they are more fit than wild-type cells when exposed to nutrient starvation. Although ATP levels were similar in both cell types when the cells were maintained in normal media, SIRT6 deficient cells produce significantly higher levels of ATP after a few hours in low-glucose (FIG. 2F). Overall, these results indicate that absence of SIRT6 causes a switch towards enhanced glycolysis and reduced mitochondrial respiration, a response usually observed under conditions of nutrient/oxygen stress.

Example 4: Glycolytic genes as putative SIRT6 targets in glucose metabolism

Based on the strong binding of SIRT6 to chromatin (Mostoslavsky et al., Cell 124:315-329, 2006), and the fact that SIRT6 is known to function as a histone H3K9 deacetylase (Kawahara et al, Cell 136:62-74, 2009; Michishita et al, Nature 452:492- 496, 2008), SIRT6 could influence glucose metabolism by controlling expression of key metabolic genes. Therefore, comparative microarray gene expression analysis of WT and SIRT6 KO muscle and ES cells was performed. As previously reported (Kawahara et al, Cell 136:62-74, 2009), multiple pathways appear to be affected in the absence of SIRT6. Notably, there is a statistically significant alteration in regulators of glucose metabolism and clustering analysis of metabolic genes separated the samples based on genotype. When a glucose metabolic filter was applied, the highest difference was observed among key glycolytic genes, such as lactate dehydrogenase (LDH), Triose Phosphate Isomerase (TPI), Aldolase, and the rate limiting glycolytic enzyme phospho-fructo kinase (PFK1). Using real-time PCR, increased expression of all these genes in independent RNA samples was validated (FIG. 3 A). GLUT1 was also increased at the level of RNA, thereby explaining the increased protein levels described in Example 2. Notably, higher levels of the pyruvate dehydrogenase kinase genes PDK1 and PDK4 were observed. As mentioned before, these enzymes phosphorylate and inhibit pyruvate dehydrogenase (PDH), the rate-limiting enzyme that regulates entrance of pyruvate into the TCA cycle. In brief, these results indicate that in the absence of SIRT6, expression of multiple glucose-related genes are up-regulated, causing enhanced glycolysis and in parallel, inhibition of mitochondrial respiration. Example 5: SIRT6 functions as a H3K9 deacetylase to regulate glucose homeostasis

In order to test whether SIRT6 directly controls expression of glycolytic genes, chromatin immunoprecipitation (ChIP) was performed using an antibody to SIRT6. As shown in FIGs. 3B and 8, SIRT6 specifically binds to the promoters of all five of the most upregulated glycolytic genes identified in expression analysis (see Example 4), strongly indicating that SIRT6 functions as a direct transcriptional repressor for these genes. Since previous work has identified SIRT6 as a histone H3K9 deacetylase

(Michishita et al, Nature 452:492-496, 2008), SIRT6 deficient cells were assayed for increased H3K9 acetylation in the promoters of these glycolytic genes. Indeed, ChIP analysis with an anti-H3K9Ac antibody clearly showed increased acetylation at all these putative targets (FIGs. 3C and 8). Together, these results strongly suggest that SIRT6 directly suppresses expression of multiple glucose-metabolic genes by deacetylating H3K9 at their promoters.

To gain insight into the mechanism by which SIRT6 modulates expression of these genes, high-resolution, quantitative ChIP analysis was performed on one of these targets, LDHB. Using qPCR amplicons against eight different locations within this genomic region, the behavior of RNA polymerase II (RNAPII) in WT cells versus SIRT6 KO cells was analyzed. Antibodies against total RNAPII were employed, as well as phospho-specific antibodies recognizing phosphorylation of Ser5 and Ser2 within the C- terminal domain (CTD) repeats of Rbpl, the largest subunit of RNAPII (Donner et al., Mol Cell 27: 121-133, 2007). Interestingly, this analysis revealed that in WT cells the LDHB promoter carries pre-loaded hypophosphorylated RNAPII and that SIRT6 depletion leads to increased RNAPII CTD phosphorylation concomitant with enhanced transcription elongation (FIG. 3D). While RNAPII was readily detectable at the LDHB transcription start site (TSS) in WT and SIRT6-KO cells, transit throughout the intragenic region was observed only in the latter. Furthermore, total RNAPII signals at the TSS were several-fold higher than at any amplicon in the intragenic region, a hallmark of RNAPII pausing at the promoter. Typically, Ser5 phosphorylation occurs at 5' end of genes and is associated with promoter escape by RNAPII. Accordingly, SIRT6 KO cells show significantly higher levels of this mark. Of note, the fold increase in Ser5 phosphorylation surpasses that of total RNAPII, indicating that in WT cells pre-loaded RNAPII exists in a hypophosphorylated state. The fact that LDHB transcription is stimulated at post-RNAPII recruitment steps is reinforced by analysis of Ser2- phosphorylation, a mark of actively elongating RNAPII that is increased several fold in SIRT6 KO cells. Consistent with conventional ChIP results (FIGs. 3C and 8), higher H3- K9 acetylation was also observed in this assay. It is, however, of interest that this increase occurs focally, close to the TSS, without spreading to nearby regions. Overall, these results indicate that SIRT6 action represses transcription of LDHB (and arguably the other target genes) at regulatory steps downstream of RNAPII recruitment.

Example 6: SIRT6 functions as a co-repressor of Hifla

Examples 1-5 indicate that SIRT6 may play a role in re-directing carbohydrate flux from glycolysis to mitochondrial respiration, and in the absence of SIRT6, glycolysis is enhanced and the TCA cycle inhibited, a phenotype usually observed as an adaptation against nutrient or oxygen deprivation. One of the main positive regulators of this switch is the transcription factor Hifla. In this context, all the genes that were up-regulated in the SIRT6 deficient cells are direct targets of Hifla. Therefore, the ability of SIRT6 to modulate a Hifla nutrient stress response was tested. First, SIRT6 was tested to determine whether it could influence expression of a luciferase reporter carrying multiple Hypoxia-Responsive -Elements (HREs), the consensus binding sequence for Hifla. This construct is specifically activated following low glucose/hypoxia, and thus it represents a direct measurement of Hifla activation in these cells (Zimmer et al., Mol Cell 32:838- 848, 2008). As shown in FIG. 4A, exposing the cells to 24 hours of hypoxia elicited robust luciferase activity and notably, SIRT6 co-expression caused significant repression. Such an effect was not observed when a catalytically inactive mutant of SIRT6 was over- expressed, suggesting that the enzymatic activity of SIRT6 was required for this specific effect on the promoter.

Since Hifla appears to maintain basal activity even under normoglycemia (Carmeliet et al, Nature 394:485-490, 1998), it is conceivable that SIRT6 might bind Hifla at the chromatin to regulate its activity. In order to test whether SIRT6 and Hifla interact, FLAG-tagged SIRT6 was co-expressed with Myc-tagged Hifla in 293T cells. Western analysis of the IPs revealed that Myc-Hifla co-precipitated with SIRT6 and, likewise, FLAG-SIRT6 co-precipitated with Hifla (FIG. 4B). This interaction appears specific, since other FLAG-sirtuins did not interact with Hifla under these conditions (FIG. 4B). In order to confirm that these proteins interact under physiological conditions, Hifl a was immunoprecipitated from muscle and tested whether SIRT6 co-precipitated. As shown in FIG. 4B, SIRT6 was readily detected in the Hifla IP, clearly indicating that endogenous Hifl a and SIRT6 can interact.

Conditions of nutrient and oxygen stress cause activation of Hifla, with increased protein levels due to stabilization of the protein (Aragones et al., Cell Metabolism 9: 11- 22, 2009). Since lack of SIRT6 mimics a nutrient stress response, levels of Hifla were measured in SIRT6 deficient cells. Extracts were purified from SIRT6 wild type and KO cells grown under normoglycemic conditions, and Western blot analysis was performed with an antibody specific for Hifla. Cells treated with a low concentration of CoCl₂, a Hifl a stabilizer that helps in visualizing the protein, were also included. As expected for a normoglycemic condition, Hifla was barely detected in wild-type cells, even after treatment with CoCl₂ (FIG. 4C). Remarkably, SIRT6 KO cells express significantly higher levels of Hifla, an effect that was further exacerbated in the presence of the stabilizer (FIG. 4C). These results strongly indicate that under normal nutrient conditions, SIRT6 plays an important inhibitory role upon Hifla-dependent glucose- related gene transcription, and lack of SIRT6 is sufficient to upregulate glycolytic gene- transcription.

Example 7: Down-regulation of Hifla rescues the metabolic phenotypes in SIRT6 deficient cells

The above results suggest that lack of SIRT6 triggers a Hifla-dependent metabolic switch. In order to test whether Hifla plays a critical role in this phenotype, Hifl a was inhibited in SIRT6 KO cells to determine whether the metabolic abnormalities observed in these cells could be reversed. For this purpose, SIRT6 KO ES cells were first treated with a recently described small molecule inhibitor of Hifla/Hif2a (Zimmer et al., Mol Cell 32:838-848, 2008). Treatment with this inhibitor for 24 hours was sufficient to completely revert the glucose uptake increase in SIRT6 KO cells (FIG. 4D, left panel). This effect appears specific, since the compound did not affect wild-type cells.

Furthermore, treatment with AKT or mTOR inhibitors, both modulators of insulin signaling and stress responses, was not able to rescue the metabolic phenotype, strongly indicating that SIRT6 modulates glucose homeostasis specifically through a Hif la- dependent pathway. To further validate these results, a similar experiment was performed in the inducible SIRT6 dominant-negative cells, where acute inactivation of SIRT6 leads to increased glucose uptake (FIG. IF). Similar to what was observed in the KO ES cells, treatment with the Hifla inhibitor readily decreased glucose uptake in these cells as well (FIG. 4D, right panel).

In order to confirm the role of Hifla in this phenotype, Hifla was specifically knocked-down in SIRT6 deficient cells. Multiple independent ES clones obtained following infection with a shRNA-Hifla virus were grown. Notably, in those clones where Hifla was down-regulated, the increased glucose uptake was completely rescued (FIG. 5A, clones #1 and #2). This effect is specific, since wild-type cells show no effect upon Hifla knock-down. Furthermore, in those few clones where the Hifla knockdown failed (as an example, see clone #3, FIG. 5 A), no changes in glucose uptake were observed. To test the transcriptional level the critical modulators of this rescue, RNA from the SIRT6KO/Hif la-knockdown cells was purified and expression of the glycolytic genes previously identified was analyzed. Notably, expression of most of these glycolytic genes was rescued to the levels observed in WT cells (FIG. 5B). One exception is Pdkl, which exhibits no statistical differences between the parental SIRT6 KO and the Hifla knock-down cells, suggesting that in this case, the Pdk4 isoform plays a more dominant role.

Taking advantage of these Hifl -knockdown cells, Hifl was tested to determine whether Hifla is required to recruit SIRT6 to these glycolytic gene promoters. For this purpose, ChIP with anti-SIRT6 antibodies was performed in these cells, and SIRT6 occupancy on those promoters evaluated. As seen in FIG. 5C, lack of Hifla significantly reduced SIRT6 binding to these promoters, indicating that SIRT6 is specifically recruited to these promoters via its physical interaction with Hifla. Example 8: Lack of SIRT6 increases both protein synthesis and stability of Hifla

In order to gain further insight into the increase levels of Hifl that was observed in SIRT6-deficient cells, we first tested whether SIRT6 directly regulates Hifl . For this purpose, RNA levels were analyzed in SIRT6-deficient cells. As shown in FIG. 6A,

Hifla RNA levels were comparable between WT and KO cells, indicating that Hifla is not a direct transcriptional target of SIRT6. Previous studies have indicated that Hifla could itself be acetylated (Jeong et al, Cell 111, 709-720, 2002). However, such findings were later disputed (Arnesen et al, FEBS Letters 579, 6428-6432, 2005; Murray-Rust et al, FEBS Letters 580, 1911-1918, 2006). In this context, Hifla acetylation was not detected in vivo, even in SIRT6 KO cells, where total levels of Hifla were significantly higher; therefore, a direct effect for SIRT6 on Hifl a appears unlikely. The protein stability of Hifla was also tested to determine if Hifla was more stable in SIRT6 KO cells. For this purpose, cells were treated with an iron chelator CoC12, which inhibits the activity of the prolyl-hydroxylases, therefore inhibiting degradation of Hifla. Whereas treatment with the drug robustly increased Hifla in WT cells, this effect was significantly diminished in the SIRT6 KO cells (FIG. 6B), indicating that Hifla is already stabilized in these cells. However, some increase was observed in the KO cells, suggesting that increased stability could only partially account for the higher levels observed in these cells. Therefore, Hifla protein synthesis was examined to determine whether it was also enhanced in the absence of SIRT6. Using a previously described luciferase reporter carrying the 5' untranslated region (UTR) of the Hifla gene (Bert et al, 2006), SIRT6 KO cells were shown to exhibit a significant increase in luciferase activity under basal conditions, similar to the levels observed in WT cells following nutrient/oxygen stress (FIG. 6C). As an independent assay, the polysomes fraction of ribosomes were purified and the rate of Hifla translation in both WT and KO cells was quantified (Serikawa et al., 2003). Consistent with the previous assay, SIRT6 KO cells exhibit a clear increase in Hifl a translation in this assay as well (FIG. 6D). Altogether, these results indicate that both Hifla protein synthesis and stability are increased in SIRT6-deficient cells.

To test whether the hypoglycemia observed in the SIRT6-deficient animals was dependent on Hifla, as found in vitro, SIRT6-deficient animals were treated with the Hifl a inhibitor described above. Strikingly, treatment with the drug caused a fast and specific increase in blood glucose levels specifically in the KO animals (FIG. 6D). These results indicate that, similar to what was observed in SIRT6 KO ES cells, regulation of glucose metabolism by SIRT6 depends on Hifl a in vivo as well.

Example 9: Increased expression of glycolytic genes and increased lactate production in SIRT6 deficient mice

Results from the in vitro experiments indicate that a lack of SIRT6 causes a switch towards glycolysis, with increased expression of multiple glycolytic genes, including the glucose transporter GLUTl . In order to test whether such a switch was also responsible for the metabolic phenotype observed in mice, in vivo expression of these genes was tested. Protein extracts were purified from muscle of multiple SIRT6 WT and KO mice, and Western blots were performed with antibodies against the different enzymes. Strikingly, the two rate limiting factors PFK1 and PDK1, which were barely detectable in wild-type muscle, were expressed at very high levels in SIRT6 KO muscle (FIG. 7A). As well, higher levels of other glycolytic genes, such as TPI1, strongly indicate that, similar to the observations in vitro, the increased glucose uptake found in SIRT6 deficient animals (FIGs. 1B-C) was likely due to enhanced expression of glycolytic genes. Consistently, expression of the p53 target TIGAR, which was recently shown to inhibit glycolysis (Bensaad et al, Cell 126: 107-120, 2006) was reduced in the KO samples. Using immunostaining, the expression of GLUTl was tested. Similar to what was found in ES cells, muscle from SIRT6 deficient animals expressed significantly higher levels of GLUTl (FIG. 7B), further validating the in vitro results. Finally, lactate production was also increased in the KO animals. As shown in FIG. 7C, SIRT6 KO animals exhibit a modest but statistically significant increased in serum lactate, when compared to wild-type animals, thus supporting the argument that lack of SIRT6 in vivo promotes uncontrolled glucose uptake and a glycolytic switch, consistent with the findings in vitro.

Example 10: Rescue of neonatal lethality of SIRT6 deficient mice by a high fat diet

Maternal mice were fed with a high fat diet (60% fat) beginning at 2 months of age and impregnated at around 3 months of age. Nursing mothers and their litters were kept on the high fat diet throughout the experimental time period, during and after weaning (which occurred around 21 days of age).

While SIRT6 KO offspring of mothers fed a normal diet (ND KO) die around 26 days of age, neonatal lethality in SIRT6 knockout mice was rescued by feeding the nursing mothers, and then the weaned pups, high fat diets. SIRT6 knockout mice raised on the high fat diet (HFD KO) generally lived over 40 days, and up to 144 days.

Strikingly, while the WT mice developed obesity and diabetes (as expected under high fat diet), the body weight and blood glucose levels in HFD KO mice remained low (Table 2 and FIGs. 9A-B).

Table 2. The lifespan, body weight and blood glucose levels of SIRT6 WT and KO mice fed on a high fat diet. Note: Mouse numbers 1 and 2, 3 and 4, and 5 and 6 are littermates, from three different litters.

Example 11: Purification of SIRT6 protein

Enzymatically active SIRT6 was purified as described above with high yield for screening assays for inhibitors (FIG. 10). Purified SIRT6 can be used in a screening assay to develop small molecule inhibitors. Once an inhibitor is identified, the purified protein can be used to validate it. The purified protein could also be used as a positive control when testing other type of inhibitors in vivo (e.g., RNA interference to inhibit SIRT6 in cells). The purified protein could be used as a positive control when testing whether extracts from the knock-down cells do not exhibit deacetylase activity.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. Use of a Sirtuin (silent mating type information regulation 2 homolog) 6 (SIRT6) inhibitor for reducing or inhibiting hyperglycemia or obesity in a subject.

2. The use of claim 1, wherein the SIRT6 inhibitor is an anti-SIRT6 antibody or

antigen-binding fragment thereof.

3. The use of claim 1, wherein the SIRT6 inhibitor is an inhibitory nucleic acid

effective to specifically reduce expression of SIRT6.

4. The use of claim 3, wherein the inhibitory nucleic acid is a small interfering RNA molecule or antisense nucleic acid effective to specifically reduce expression of SIRT6.

5. The use of claim 1, wherein the subject is a mammal.

6. The use of claim 1, wherein the subject is a human.

7. The use of claim 1, wherein the subject has a blood glucose level of 10 mmol/L or greater.

8. The use of claim 1 , wherein the subject has a body mass index of 25 kg/m² or greater.

9. A method of reducing or inhibiting hyperglycemia or obesity in a subject, the

method comprising administering to the subject a therapeutically effective amount of a SIRT6 inhibitor, thereby reducing or inhibiting hyperglycemia or obesity in the subject.

10. The method of claim 9, wherein the SIRT6 inhibitor is an anti-SIRT6 antibody or antigen-binding fragment thereof.

11. The method of claim 9, wherein the SIRT6 inhibitor is an inhibitory nucleic acid effective to specifically reduce expression of SIRT6.

12. The method of claim 11 , wherein the inhibitory nucleic acid is a small interfering RNA molecule or antisense nucleic acid effective to specifically reduce expression of SIRT6.

13. The method of claim 9, wherein the subject is a mammal.

14. The method of claim 9, wherein the subject is a human.

15. The method of claim 9, wherein the subject has a blood glucose level of 10 mmol/L or greater.

16. The method of claim 9, wherein the subject has a body mass index of 25 kg/m² or greater.

17. A method of identifying a candidate compound that inhibits hyperglycemia or

obesity, the method comprising:

providing a sample comprising a SIRT6 polypeptide and an acetylated histone substrate;

contacting the sample with a test compound under conditions that allow the SIRT6 polypeptide to bind or deacetylate the histone substrate;

determining a level of histone deacetylation in the sample in the presence of the test compound; and

if the test compound decreases the level of histone deacetylation, relative to a level of histone deacetylation in the absence of the test compound, then the test compound is a candidate compound for the inhibition of hyperglycemia or obesity.

18. The method of claim 17, wherein the sample is a living cell.

19. The method of claim 17, wherein the acetylated histone substrate is a histone 3 protein acetylated at position lysine 9 (H3K9).

20. The method of claim 17, further comprising:

selecting a candidate compound;

administering the candidate compound to a mammal; and

evaluating an effect of the candidate compound on glycemia or obesity,

wherein a candidate compound that inhibits hyperglycemia or obesity is a candidate therapeutic agent for the treatment of hyperglycemia or obesity.