STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[001] This invention was made with government support under project number ZIA

BC005562-28 awarded by the National Institutes of Health, National Cancer Institute. The government has certain rights in the invention.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

[002] This application claims priority to U.S. Provisional Application No. 62/465,009, filed February 28, 2017. All publications, patent applications, patents, figures and other references cited or referenced herein and all documents cited or referenced in the herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated by reference, and may be employed in the practice of the invention.

BACKGROUND

1. Field of the Discovery.

[003] The present invention relates to methods of treating and preventing obesity, insulin resistance and fatty liver disease. Present disclosure shows that intestine HIF-2a and neu3 would be viable target for NAFLD therapy, NASH therapy, obesity and insulin resistance. The disclosure presents methods and compositions of matter relating to the targeted inhibition and/or otherwise inactivation of HIF-2a for treating and/or preventing obesity, insulin resistance, NAFLD and NASH.

2. Background Information.

[004] Dramatic changes in the lifestyle and diet of the global population are fueling a worldwide epidemic of obesity and the increasing prevalence of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). It has been predicted that the next epidemic in chronic liver disease will be a direct result of this increased incidence of obesity, NAFLD and NASH. Fatty deposits in the liver result in a disease spectrum ranging from simple steatosis with no symptoms, through to fibrosis and cirrhosis, which can result in liver cancer, liver failure and death. Importantly, NAFLD and NASH are set to replace viral hepatitis as the primary causes of end-stage liver disease and liver transplantation over the next decade or so, with the disease affecting both adults and children. The challenge now facing hepatologists worldwide is what can be done to help reverse this trend.

[005] Treatment of NAFLD with diet and exercise is not an effective at all stages and in all cases. Alternative broad spectrum medications and treatments are needed.

[006] The intestine is supported by a complex vascular system that undergoes dynamic and transient daily shifts in blood perfusion, depending on the metabolic state. The hypoxic transcription factors hypoxia- inducible factor (HIF)-la and HIF-2a are essential in maintaining intestinal homeostasis. HIF-Ια and HIF-2a are also highly expressed in a wide variety of solid tumors, including those of the colon, breast, lung, and pancreas. In renal cell carcinoma (RCC) which is deficient in VHL, which stabilizes HIF-Ια and HIF-2a in normoxic conditions, HIF-la has an antitumor role and decreases tumor growth by increasing expression of proapoptotic genes. HIF-2a is essential for RCC tumor growth and promotes tumor cell proliferation through augmented c-Myc activity. However, in lung cancer, HIF-2a exerts a tumor suppressive effect. These studies demonstrate the careful need to evaluate the tumor specific roles for HIF-Ια and HIF-2a for therapeutic targeting.

[007] HIF-2a is essential in maintaining proper micronutrient balance, inflammatory response, and the regenerative and proliferative capacity of the intestine following an acute injury.

However, chronic activation of HIF-2a leads to enhanced proinflammatory response, intestinal injury, and colorectal cancer. The role of hypoxia- inducible factor (HIF)-la in intestinal homeostasis is known. HIF-la-in white adipose tissue activation has already been disclosed as a potential target for treating diabetes, obesity, insulin resistance and reducing body weight. HIF- 2a activation on the other hand has shown negative and opposing effects (i.e., increased obesity and insulin resistance). There are a number of HIF-2a inhibitors, including PT2385, and its analogs, PT2567, PT2399 and PT2977. HIF-2 a inhibitors have been studied for the treatment of cancer but not for treating obesity, NAFLD, NASH or insulin resistance.

[008] Accordingly, the disclosure presents methods and compositions of matter relating to the targeted inhibition and/or otherwise degradation of HIF-2a for treating and/or preventing obesity, insulin resistance, NAFLD and NASH. SUMMARY

[009] Disclosed herein are methods of treating and preventing obesity, insulin resistance and hepatic lipid metabolism and inflammation. Non-alcoholic fatty liver disease (NAFLD) is becoming the most common chronic liver disease in western countries with limited therapeutic options. NAFLD can lead to NASH, fibrosis, liver cancer and even liver failure and death. Here a new role for intestinal hypoxia-inducible factor (HIF) in NAFLD is uncovered. Human intestine biopsies from patients with or without obesity revealed a relationship between activated HIF-2a but not HIF- la in increased body mass index and hepatic lipid toxicity. The causality of this correlation was verified in mice with an intestine- specific HIF-2a-disruption, in which high- fat diet-induced hepatic steatosis and obesity were substantially decreased. PT2385, an HIF-2a- specific inhibitor, had preventive and therapeutic effects on metabolic disorders dependent on intestine HIF-2a. Intestine HIF-2a inhibition markedly reduced intestine and serum ceramide levels. Mechanistically, intestine HIF-2a regulates ceramide metabolism mainly from the salvage pathway, which was revealed by the identification of the novel HIF-2a target gene encoding neuraminidase 3 (Neu3). These results suggest for the first time that intestine HIF-2a- neu3 axis would be a viable target for NAFLD therapy, obesity and insulin resistance.

[0010] One aspect discloses a method for treating or preventing obesity, insulin resistance, and non-alcoholic fatty liver disease in a subject, comprising selectively decreasing the expression or inhibiting the activity of intestine specific HIF-2a, thereby decreasing obesity, insulin resistance, non-alcoholic fatty acid disease and non-alcoholic steatohepatitis in the subject.

[0011] Another aspect discloses a method of decreasing obesity, insulin resistance, non-alcoholic fatty liver disease and non-alcoholic steatohepatitis in a subject, comprising administering a therapeutically effective amount of PT2385, thereby decreasing obesity, insulin resistance, nonalcoholic fatty acid disease and non-alcoholic steatohepatitis in the subject.

[0012] Certain embodiments disclose a method of decreasing obesity, insulin resistance, and non-alcoholic fatty acid disease comprises decreasing ceramide synthesis, decreased fatty acid transport and lipogenesis, and inhibition of salvage pathway or a combination of two or more thereof in the subject as compared to a control.

[0013] In various embodiments, the method discloses that intestine specific HIF-2a and neu3 inhibition attenuates hepatic steatosis. [0014] In some embodiments, the subject has non-alcoholic fatty liver disease (NAFLD).

[0015] In various embodiments, the method discloses that use of at least one of PT2385, PT2567, PT2399, PT2977 or a combination thereof to decrease HIF-2a expression or activity relative to a control.

[0016] In certain additional embodiments, the method depicts that decreasing obesity, insulin resistance, and non-alcoholic fatty acid disease comprises inhibition of NEU3 activity.

[0017] In some specific embodiments, NEU3 activity is inhibited by 2,3-didehydro-N-acetyl- neuraminic acid (DANA) or naringin.

[0018] Some additional embodiments disclose that NEU3 expression is decreased by intestine- specific HIF-2a inhibitor for treating or preventing hepatic steatosis.

[0019] In some embodiments, the method of decreasing obesity, insulin resistance, non-alcoholic fatty acid disease and non-alcoholic steatohepatitis comprises decreased or inhibited ceramide synthesis.

[0020] In some additional embodiments, the method discloses that decreasing obesity, insulin resistance, and non-alcoholic fatty acid and non-alcoholic steatohepatitis disease comprises decreased fatty acid transport and lipogenesis.

[0021] The method of decreasing obesity, insulin resistance, non-alcoholic fatty acid disease and non-alcoholic steatohepatitis in some embodiments comprises inhibition of salvage pathway.

[0022] In specific embodiments, the method discloses that intestine specific HIF-2a inhibitor is administered orally.

[0023] In certain additional embodiments, the intestine specific HIF-2a inhibitor is PT2385.

[0024] In any of the embodiments, the subject is a mammal.

[0025] In certain additional embodiments, the subject is a human.

[0026] In some embodiments, the method uses a composition comprising 1 mg/kg- 100 mg/kg dose of at least one of PT2385, PT2567, PT2399, PT2977 or a combination thereof. In some embodiments, the method uses a composition comprising 20 mg/kg. In still other embodiments, the method uses composition comprising about 5 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, about 60 mg/kg, about 70 mg/kg, about 80 mg/kg, about 90 mg/kg, or about 100 mg/kg or more.

[0027] In any of the embodiments, the composition further comprises a pharmaceutically acceptable carrier. [0028] One aspect discloses a method of decreasing obesity, insulin resistance, and nonalcoholic fatty acid disease in a subject, comprising administering a therapeutically effective amount of at least one of PT2385, PT2567, PT2399, PT2977 or a combination thereof, thereby decreasing obesity, insulin resistance, and non-alcoholic fatty acid disease in the subject.

[0029] In another aspect, the disclosure provides a method of decreasing obesity in a subject, comprising administering a therapeutically effective amount of at least one of PT2385, PT2567, PT2399, PT2977 or a combination thereof, DANA or naringin, thereby decreasing obesity in the subject.

[0030] In still another aspect, the disclosure provides a method of decreasing insulin resistance in a subject, comprising administering a therapeutically effective amount of at least one of PT2385, PT2567, PT2399, PT2977 or a combination thereof, DANA or naringin, thereby decreasing insulin resistance in the subject.

[0031] In yet another aspect, the disclosure provides a method of decreasing non-alcoholic fatty acid disease and non-alcoholic steatohepatitis in a subject, comprising administering a therapeutically effective amount of at least one of PT2385, PT2567, PT2399, PT2977 or a combination thereof, thereby decreasing non-alcoholic fatty acid disease including non-alcoholic steatohepatitis in the subject.

[0032] Another aspect discloses a method of decreasing obesity, insulin resistance, non-alcoholic fatty acid disease and non-alcoholic steatohepatitis in a subject, comprising selectively decreasing the expression or inhibiting the activity of HIF-2a in the intestine, thereby decreasing obesity, insulin resistance, non-alcoholic fatty acid disease including non-alcoholic

steatohepatitis in the subject.

[0033] In still another aspect, the disclosure provides a method of decreasing obesity in a subject, comprising selectively decreasing the expression or inhibiting the activity of HIF-2a in the intestine, thereby decreasing obesity in the subject.

[0034] In another aspect, the disclosure provides a method of decreasing insulin resistance in a subject, comprising selectively decreasing the expression or inhibiting the activity of HIF-2a in the intestine, thereby decreasing insulin resistance in the subject.

[0035] In yet another aspect, the disclosure provides a method of decreasing non-alcoholic fatty acid disease inclduing non-alcoholic steatohepatitis in a subject, comprising selectively decreasing the expression or inhibiting the activity of HIF-2a and Neu3 in the intestine, thereby decreasing non-alcoholic fatty acid disease and non-alcoholic steatohepatitis in the subject.

[0036] In various embodiments, the method of decreasing obesity, insulin resistance, and nonalcoholic fatty acid disease results in decreased ceramide levels, decreased fatty acid transport and lipogenesis, and inhibition of salvage pathway or a combination of two or more thereof in the subject as compared to a control.

[0037] In other embodiments, HIF-2a and neu3 inhibition attenuates hepatic steatosis.

[0038] In various embodiments, the subject treatable by the methods provided herein have nonalcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) or are at risk for NAFLD and NASH.

[0039] In various other embodiments, the subject treatable by the methods provided herein suffers from obesity or is at risk of becoming obese.

[0040] In still other embodiments, the subject treatable by the methods provided herein is resistant to insulin.

[0041] In other embodiments, the subject treatable by the methods provided herein has type II diabetes or is at risk for developing type II diabetes.

[0042] In various embodiments, the step of selectively decreasing the expression or inhibiting the activity of HIF-2a in the intestine comprises administering a therapeutically effective amount of an HIF-2a inhibitor. In various embodiments, the HIF-2a inhibitor may be administered in a selective manner that results in selective delivery to intestine epithelia cells but miminizes delivery to other cell types. In various embodiments, the HIF-2a inhibitor may be delivered by way of a pharmaceutical composition or vehicle which comprises one or more intestinal- specific targeting moieties. Moieties can include, for example, intestinal-specific antibodies or ligands.

[0043] In certain embodiments, the HIF-2a inhibitor is PT2385 or a functional derivative thereof having the same or equivalent effect as PT2385.

[0044] In certain embodiments, the step of selectively decreasing the expression or inhibiting the activity of HIF-2a in the intestine comprises administering a therapeutically effective amount of a pharmaceutical composition comprising a HIF-2a inhibitor and a pharmaceutically acceptable carrier. [0045] The pharmaceutical compositions described herein may further comprise a hepatic- specific targeting moiety which can include a hepatic-specific antibody that recognizes and binds to a hepatic target, or a hepatic- specific ligand.

[0046] Other aspects of the invention are described in, or are obvious from, the following disclosure, and are within the ambit of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0047] The above and other features of the present invention will now be described in detail with reference to certain exemplary embodiments thereof illustrated the accompanying drawings which are given herein below by way of illustration only, and thus are not limitative of the present invention. Those skilled in the art to which the subject disclosure appertains will readily understand how to make and use the devices and methods of the subject disclosure without undue experimentation, embodiments thereof will be described in detail herein below with reference to certain figures, wherein:

[0048] FIG. 1 Increased HIF-2a signaling in human ileum biopsies is correlated with obesity, (a) Representative immunohistochemical staining for the expression of HIF-2a and HIF-Ια in human ileum biopsies from cohort 1 (n = 6 subjects/group, 3 images/subject), (b) Representative western blot analysis of HIF-2a and HIF-Ια protein expression in three individual ileum biopsies from cohort 1. n = 6/group for blot quantification, (c) mRNA expression levels of HIF-2a target gene DMTl, DCYTB, and HIF-Ια target gene PDKl in human ileum biopsies from individuals without obesity (n = 18) and with obesity (n = 17) (cohort 2). **P < 0.01, relative nonobese individuals, by two-tailed Student's i-test. (d) Heat map of the correlative analysis of DMTl, DCYTB, and PDKl mRNAs in human ileum biopsies (cohort 2) with BMI and clinical biochemistry parameters (n = 35). Correlations were assessed by nonparametric Spearman's test, (e) The relative luciferase activities in small intestine (ileum) from ODD-luciferase transgenic mice fed a chow diet or HFD (n = 4/group). (f) Western blot analysis of HIF-2a and HIF-la protein expression (n = 3/group) and mRNA expression analysis (n = 5/group) of their target genes in small intestine from chow-diet or HFD-fed mice (8 weeks). Data are presented as mean + s.e.m. For box plots, the midline represents the median; box represents the interquartile range (IQR) between the first and third quartiles, and whiskers represent the lowest or highest values within 1.5 times interquartile range (IQR) from the first or third quartiles. *P < 0.05, **P < 0.01 relative to a chow diet, by two-tailed Student' s i-test.

[0049] FIG. 2 Intestine-specific HIF-2a disruption ameliorates the development of hepatic steatosis, (a) Representative H&E staining (left two panels) and Oil Red O staining (right two panels) of liver sections (n = 5 mice/group, 3 images/mouse). Scale bars, 100 μιη. (b) Liver weights, (c) Liver weight-to-body weight ratios. (d,e) Liver (d) and serum (e) triglyceride content. (f,g) Liver (f) and serum (g) cholesterol content, (h) Serum ALT levels, (i) Hepatic expression of mRNAs encoded by hepatic fatty acid transport and lipogenesis-related genes, j) Hepatic expression of mRNAs encoded by hepatic fatty acid β -oxidation-related genes, (k) Hepatic expression of mRNAs encoded by inflammatory cytokine and chemokine genes. HFD fed Hif2afUfl and Hif2aAJE mice, n = 5/group. For box plots, the midline represents the median; box represents the IQR between the first and third quartiles, and whiskers represent the lowest or highest values within 1.5 times IQR from the first or third quartiles. *P < 0.05, **P < 0.01 relative to Hif2afUfl mice, by two tailed Student' s i-test.

[0050] FIG. 3 Intestinal HIF-2a deficiency reduces ceramide synthesis in the small intestine, (a) Score scatter plot of a PCA model of the intestinal metabolites between Hif2 fUfl (circle) and Hif2 AIE (square) mice. Each point represents an individual mouse sample, (b) S-plot of an orthogonal partial least- squares discriminant analysis (OPLS-DA) model of the intestinal metabolites. Each point represents a metabolite ion. Insert shows a scaled region with metabolites M2-M6. (c,d) Quantitation of ceramide concentrations in the small intestine (c) and serum (d). (e) Scheme for ceramide- synthesis pathways. (f,g) The relative levels of

sphingomyelin (f) and glucosylceramide (g) in the small intestine, (h) Intestinal expression of intestinal mRNAs encoding ceramide salvage-related enzymes. Hif2afUfl and Hif2a ΙΈ mice fed a HFD for 12 weeks, n = 6/group. Data are presented as mean + s.e.m. For box plots, the midline represents the median; box represents the IQR between the first and third quartiles, and whiskers represent the lowest or highest values within 1.5 times IQR from the first or third quartiles. *P < 0.05, **P < 0.01 relative to ΗΐβαΆΙΆ mice, by two-tailed Student's t-test.

[0051] FIG. 4 The ceramide-synthesis-related gene Neu3 is a novel HIF-2a target gene in the small intestine, (a) Expression of NEU3 mRNA in human ileum biopsies from nonobese individuals (n = 18) and individuals with obesity (n = 17) (cohort 2). **P < 0.01, versus healthy subjects, by two-tailed Student' s i-test. (b-f) Correlative analysis of ileum NEU3 mRNA levels with BMI (b), ALT (c), AST (d), DMT1 mRNA (e), and DCYTB mRNA (f). n = 35. Correlations were assessed by nonparametric Spearman's test, (g) Western blot analysis of NEU3 protein expression in small intestine of chow-diet or HFD-fed mice (8 weeks, n = 3/group). (h) Western blot analysis of NEU3 protein expression in the small intestine of HFD fed-Hzy2afl/fl and Hif2aAIE mice (12 weeks, n = 3/group). Data are presented as means + s.e.m. **P < 0.01 relative to Hif2afUfl mice, by two-tailed Student's i-test. (i) Schematic diagram of the mouse Neu3 promoter illustrating the HREs in the regulatory region; the upstream regions are numbered in relation to the transcription initiation site, which is designated +1. (j) Luciferase reporter gene assay of Neu3 promoter activity (n = 5/group). **P < 0.01, by two-tailed Student's i-test. (k) In vivo ChIP assays on small intestinal extracts from Vhl ^wa and Vhl ^m mice (n = 3/group). **P < 0.01, by two-tailed Student's i-test. (1) Ceramide levels in HCT116 cells treated with vehicle, PT2385, or DANA and exposed to either vehicle or CoC12 (n = 5/group). **P < 0.01 relative to normoxia + vehicle, ##P < 0.01 relative to hypoxia (CoC12) + vehicle, by one way ANOVA with Tukey' s correction, (m) Ceramide levels in HCT116 cells transfected with control or siNEU3 and exposed to either vehicle and CoC12 (n = 5/group). **P < 0.01 versus normoxia + control, ##P < 0.01 versus hypoxia (CoC12) + control, by one-way ANOVA with Tukey' s correction. Data are presented as mean + s.e.m. For box plots, the midline represents the median; box represents the IQR between the first and third quartiles, and whiskers represent the lowest or highest values within 1.5 times IQR from the first or third quartiles.

[0052] FIG. 5 Administration of ceramide reverses the protective effects of intestinal HIF-2a inhibition on the development of HFD -induced hepatic steatosis, (a) Representative H&E staining (upper) and Oil Red O staining (lower) of liver sections (n = 5 mice/group, 3

images/mouse). Scale bars, 100 μιη. (b) Liver weights, (c) Liver weight-to-body weight ratios. (d,e) Liver (d) and serum (e) triglyceride content. (f,g) Liver (f) and serum (g) cholesterol content, (h) Serum ALT levels, (i) Hepatic expression of mRNAs encoded by hepatic fatty acid transport and lipogenesis-related genes, (j) Hepatic expression of mRNAs encoded by inflammatory cytokine and chemokine genes. Ceramide-treated HFD-fed Hif2afUfl and

Hif2aAIE mice, n = 5/group. For box plots, the midline represents the median; box represents the IQR between the first and third quartiles, and whiskers represent the lowest or highest values within 1.5 times IQR from the first or third quartiles. *P < 0.05, **P < 0.01 relative to vehicle treated Hif2af\/f mice, #P < 0.05, ##P < 0.01 relative to vehicle-treated Hif2aAJE mice, by one way ANOVA with Tukey's correction.

[0053] FIG. 6 PT2385 reverses HFD-induced hepatic steatosis, (a) Representative H&E staining (left two panels) and Oil Red O staining (right two panels) of liver sections (n = 4 mice for vehicle group, n = 5 mice for PT2385 group, 3 images/mouse). Lipids stain positive (red color) with Oil Red O. Scale bars, 100 μιη. P = 0.06 for steatosis score, (b) Liver weights, (c) Liver weight-to-body weight ratios. (d,e) Liver (d) and serum (e) triglyceride content. (f,g) Liver (f) and serum (g) cholesterol content, (h) Serum ALT levels, (i) Quantitation of ceramide concentrations in the small intestine, (]) Intestinal expression of mRNAs encoding ceramide salvage-related enzymes. PT2385-treated HFD-fed mice with obesity, n = 4 for vehicle group, n= 5 for PT2385 group. For box plots, the midline represents the median; box represents the IQR between the first and third quartiles, and whiskers represent the lowest or highest values within 1.5 times IQR from the first or third quartiles. (k) Western blot analysis of NEU3 protein expression in the small intestine (n = 3/group). (1) A schematic diagram summarizing the findings that obesity promotes a HIF-2a-NEU3-ceramide pathway that contributes to NAFLD progression. Data are presented as mean + s.e.m. *P < 0.05, **P < 0.01 relative to vehicle treatment, by two-tailed Student's i-test.

[0054] FIG. 7 Increased HIF2a signaling in human ileum biopsies is correlated with obesity, (a) Correlative analysis of ileum DMT1, DCYTB, and PDK1 mRNA levels with BMI, ALT, AST, triglycerides, cholesterol, HDL, LDL, and glucose, n = 35. Correlations were assessed by nonparametric Spearman's test, (b) Western blot analysis of HIF2a and HIF1 a protein expression (n = 3/group) and mRNA expression analysis of their target genes in small intestine from chow or HFD-fed mice (1 week), n = 4 for chow and n = 5 for HFD. For box plots, the midline represents the median; box represents the interquartile range (IQR) between the first and third quartiles, and whiskers represent the lowest or highest values within 1.5 times IQR from the first or third quartiles. *P < 0.05, **P < 0.01 versus chow, by two-tailed Student's i-test.

[0055] FIG. 8 Lack of intestinal HIF2a prevents HFD-induced obesity and improves metabolic homeostasis, (a) Growth curves of HFD-fed Hif2a ^Wii and Hif2a ^Am mice, (b) Glucose tolerance testf HFD-fed and Hif2a ^Am mice, (c) Insulin tolerance testf HFD-fed Hif2a ^Wii and Hif2a mice, (d) Hif2a mRNA expression in different tissues from HFD-fed Hif2a ^Wii and Hif2a ^Am mice, (e) Western blot analysis of liver HIF2a and HIFla from f HFD-fed Ηίβα^ and Hif2a mice (n = 3/group). (f) Growth curves of chow-fed Hif2a and Hif2a mice, (g) Representative H&E staining of liver sections of chow-fed Ηίβα^ and Hif2a ^Am mice (n = 15, 3 images/mouse). Scale bars, 100 μιη. (h) Liver weights of chow-fed Hif2a ^wa and Hif2a ^Am mice, (i) Liver weight to body weight ratios of chow-fed Ηίβα^ and Hif2a ^Am mice, (j, k) Liver (j) and serum (k) triglyceride content of chow-fed Ηίβα^ and Hif2a ^Am mice. (l,m) Liver (1) and serum (m) cholesterol content of chow-fed Hif2a ^wa and Hif2a ^Am mice, (n) Serum ALT levels of chow- fed Ηίβα^ and Hif2a ^Am mice, n = 5/group. Data are presented as the mean + sem. For box plots, the midline represents the median; box represents the IQR between the first and third quartiles, and whiskers represent the lowest or highest values within 1.5 times IQR from the first or third quartiles. *P < 0.05, **P < 0.01 versus Ηίβα^ mice, by two-tailed Student's i-test. Figure 9 depicts that intestine-specific HIF-2a disruption does not affect metabolic homeostasis without HFD feeding. Ηΐβα ^Ά and Hif2a ^Am mice were fed a chow diet for 12 weeks (n = 5/group). (a) Growth curves, (b) Representative H&E staining of liver sections. Scale bars: 100 μιη. (c) Liver weights, (d) Liver weight to body weight ratios, (e, f) Liver (e) and serum (f) triglyceride content, (g, h) Liver (g) and serum (h) cholesterol content, (i) Serum ALT levels, (j) Hepatic expression of mRNAs encoded by hepatic fatty acid transport and lipogenesis-related genes, (k) Hepatic expression of mRNAs encoded by hepatic fatty acid β -oxidation-related genes. (1) Hepatic expression of mRNAs encoded by inflammatory cytokine and chemokine genes. The data are presented as the mean + sem. Two-tailed Student's t-test.

[0056] FIG. 9 Loss of HIF2a in the intestine affects ceramide metabolism in HFD-fed Ηίβα^ and Hif2a ΔΙΕ mice, (a) Score scatter plot of a PCA model of the serum metabolites between Ηίβα^ (circle) and Hif2a ^Am (square) mice, (b) S-plot of an OPLS-DA model of the serum metabolites. (c,d) The relative levels of sphingomyelin (c) and glucosylceramide (d) in serum, (e) Expression of intestinal Hif2a mRNA and HIF2a target gene mRNAs. (f,g) Intestinal expression of mRNAs encoded by ceramide synthesis-related genes, including the de-novo pathway (f) and the sphingomyelinase pathway (g). (h) Intestinal expression of mRNAs encoded by ceramide catabolism-related genes, n = 6/group. For box plots, the midline represents the median; box represents the IQR between the first and third quartiles, and whiskers represent the lowest or highest values within 1.5 times IQR from the first or third quartiles. *P < 0.05, **P < 0.01 versus Ηίβα^ mice, by two-tailed Student's i-test. [0057] FIG. 10 Inhibition of the intestinal HIF2a substantially increases the metabolic rate and decreases hepatic steatosis independent of body weight changes in HFD-fed Ηίβα^ and

Hif2a ΔΙΕ mice, (a) body weight, (b) Glucose tolerance test, (c) Insulin tolerance test, (d)

Cumulative food intake, (e) Activity, (f) Energy expenditure, (g) Oxygen consumption rate, (h) Carbon dioxide production rate, (i) Liver weights, (j) Liver weight to body weight ratios. (k,l) Liver (k) and serum (1) triglyceride content. (m,n) Liver (m) and serum (n) cholesterol content, (o) Serum ALT levels (P = 0.08). (p) Hepatic expression of mRNAs encoding fatty acid transport and lipogenesis. (q) Hepatic expression of mRNAs encoding fatty acid oxidation- related enzymes, (r) Hepatic expression of mRNAs encoding inflammatory cytokines and chemokines. n = 6 for Hif2a ^wa group and n = 5 for Hif2a ^Am group. For box plots, the midline represents the median; box represents the IQR between the first and third quartiles, and whiskers represent the lowest or highest values within 1.5 times IQR from the first or third quartiles. *P < 0.05, **P < 0.01 versus Hifta^ mice, by two-tailed Student's t-test.

[0058] FIG. 11 Intestinal HIF2a deficiency reduces ceramide synthesis in the small intestine independent of body weight changes in HFD-fed Ηίβα^ and Hif2a ^Am mice, (a) Expression of intestinal Hif2a mRNA and HIF2a target gene mRNAs. (b-d) Intestinal expression of mRNAs encoded by ceramide synthesis-related genes, including the de-novo pathway (b), the

sphingomyelinase pathway (c), and the salvage pathway (d). (e-g) Ceramide levels in the small intestine (e), systematic serum (f), and portal serum (g). (h-j) Thermogenic gene expression in scWAT (h), BAT (i), and eWAT (j). (k) Western blot analysis of UCP1 protein expression in scWAT (n = 3/group). Data are presented as the mean + sem. n = 6 for Hif2a ^wa group and n = 5 for Hif2a ΔΙΕ group. For box plots, the midline represents the median; box represents the IQR between the first and third quartiles, and whiskers represent the lowest or highest values within 1.5 times IQR from the first or third quartiles. *P < 0.05, **P < 0.01 versus Ηίβα^ mice, by two-tailed Student's i-test. (1) Representative UCP1 immunohistochemistry staining of scWAT sections (n = 3 images/mice). Scale, 100 μιη.

[0059] FIG. 12 HIF2a regulates the ceramide synthesis in the small intestine, (a) Intestinal expression of Vhl, Hifla, Hif2a, Dmtl and Dcytb mRNAs. (b-d) Intestinal expression of mRNAs encoded by ceramide synthesis-related genes, including the de-novo pathway (b), the

sphingomyelinase pathway (c), and the salvage pathway (d). Male Vhl/Hifl a ^wa and Vhl/ Hifla ^Am mice fed a chow diet were treated with or without PT2385 (20 mg/kg) for three consecutive days (n = 4 to 6/group). *P < 0.05, **P < 0.01 versus vehicle-treated Vhl/Hifla ^u mice, #P < 0.05,

##P < 0.01 versus vehicle-treated Vhl/Hifla ΔΙΕ mice, by one-way ANOVA with Tukey's correction, (e-j) Intestinal mRNA expression levels of Vhl (e), Hif2a (f), Hifla (g), Dmtl (h), Pdkl (i), and Neu3 (j) in Vhl™, Vhl ^Am , Vhl/Hifla™, Vhl/Hifla ^Am , Vhl/Hifla™, and

Vhl/Hif2a ΔΙΕ mice fed a chow diet (n = 4 to 6/group). (k) The relative levels of lactosylceramide C16:0 in the small intestine from Hifla™ and Hifla ^Am mice fed a HFD for 12 weeks (n = 6/group). **P < 0.01 versus Hifla™ mice, by two-tailed Student's i-test. (1) mRNA expression of DMT1 , DCYTB, and NEU3 in HCT116 cells treated with vehicle or PT2385 and exposed to either vehicle or CoCl ₂ (n = 5/group). **P < 0.01 versus Normoxia + Vehicle treatment, #P < 0.05, ##P < 0.01 versus Hypoxia (CoCl ₂ ) + Vehicle treatment, by one-way ANOVA with Tukey's correction, (m) The knockdown efficiency of siNEU3 in HCT116 cells. **P < 0.01, by two-tailed Student's t-test. For box plots, the midline represents the median; box represents the IQR between the first and third quartiles, and whiskers represent the lowest or highest values within 1.5 times IQR from the first or third quartiles.

[0060] FIG. 13 NEU3 inhibitor DANA and naringin treatments protect mice from HFD-induced obesity and hepatic steatosis, (a-c) Neuraminidase activities in intestine (a), liver (b), and white adipose tissue (c). (d,e) Ceramide levels in the small intestine (d) and serum (e). (f) Growth curves, (g) Representative H&E staining of liver sections (n = 3 images/mouse). Scale bars, 100 μιη. (h) Liver weights, (i) Liver weight to body weight ratios. j,k) Liver (j) and serum (k) triglyceride content. (l,m) Liver (1) and serum (m) cholesterol content, (n) Serum ALT levels, (o) Hepatic expression of mRNAs encoding fatty acid transport and lipogenesis. (p) Hepatic expression of mRNAs encoding inflammatory cytokines and chemokines. n = 6/group. Data are presented as the mean + sem. For box plots, the midline represents the median; box represents the IQR between the first and third quartiles, and whiskers represent the lowest or highest values within 1.5 times IQR from the first or third quartiles. *P < 0.05, **P < 0.01 versus vehicle treatment, by two-tailed Student's i-test.

[0061] FIG. 14 Administration of ceramide reverses the protective effects of intestinal HIF2a inhibition on the HFD-induced obesity and insulin resistance in Hifla™ and Hifla ^Am . (aDb) Ceramide levels in the small intestine (a) and serum (b). (c) Growth curves. (d,e) Glucose tolerance test (d) and glucose AUC (e). (f) Insulin tolerance test, n = 5/group. Data are presented as the mean + sem. For box plots, the midline represents the median; box represents the IQR between the first and third quartiles, and whiskers represent the lowest or highest values within 1.5 times IQR from the first or third quartiles. *P < 0.05, **P < 0.01 versus vehicle-treated Hif2a ^em mice, #P < 0.05, ## < 0.01 versus vehicle-treated Hif2a ^Am mice, by one-way ANOVA with Tukey's correction.

[0062] FIG. 15 PT2385 prevents mice from HFD-induced obesity and hepatic steatosis through inhibition of the intestinal HIF2a-ceramide axis in Ηΐβα^ and Hif2a ^Am mice, (a) Growth curves, (b) Glucose tolerance test, (c) Insulin tolerance test, (d) Representative H&E staining of liver sections (n = 3 images/mouse). Scale bars: 100 μιη. (e) Liver weights, (f) Liver weight to body weight ratios. (g,h) Liver (g) and serum (h) triglyceride content. (i,j) Liver (i) and serum (j) cholesterol content, (k) Serum ALT levels. (1) Quantitation of ceramide concentrations in the intestine, (m) Quantitation of ceramide concentrations in serum, n = 5/group. Data are presented as the mean + sd. For box plots, the midline represents the median; box represents the IQR between the first and third quartiles, and whiskers represent the lowest or highest values within 1.5 times IQR from the first or third quartiles. *P < 0.05, **P < 0.01 versus vehicle-treated Ηίβα^ mice, by one-way ANOVA with Tukey's correction.

[0063] FIG. 16 PT2385 inhibits ceramide synthesis in the small intestine and alters fatty acid synthesis, metabolism, and inflammation in the liver dependent on intestinal HIF2a in Ηίβα^ and Hif2a ΔΙΕ mice, (a) Expression of Hif2a mRNA and its target gene mRNAs in the intestine, (b-d) Intestinal expression of mRNAs encoded by ceramide synthesis-related genes, including the de-novo pathway (b), the sphingomyelinase pathway (c) and the salvage pathway (d). (e) Hepatic expression of mRNAs encoding fatty acid transport and lipogenesis. (f) Hepatic expression of mRNAs encoding inflammatory cytokines and chemokines. n = 5/group. For box plots, the midline represents the median; box represents the IQR between the first and third quartiles, and whiskers represent the lowest or highest values within 1.5 times IQR from the first or third quartiles. *P < 0.05, **P < 0.01 versus vehicle-treated Hif2a ^wa mice, by one-way ANOVA with Tukey's correction.

[0064] FIG. 17 PT2385 reverses metabolic dysfunctions in HFD-induced obese mice, (a) Growth curves, (b) glucose tolerance test, (c) Insulin tolerance test, (d) Expression levels of HIF2a target gene mRNAs in the intestine, (e) Quantitation of ceramide concentrations in serum. (f,g) Intestinal expression of mRNAs encoded by ceramide synthesis-related genes, including the de novo pathway (f) and the sphingomyelinase pathway (g). (h) Hepatic expression of mRNA encoding fatty acid transport and lipogenesis-related enzymes, (i) Hepatic expression of mRNAs encoding inflammatory cytokines and chemokines. n = 4 for vehicle group, n = 5 for PT2385 group. Data are presented as the mean + sem. For box plots, the midline represents the median; box represents the IQR between the first and third quartiles, and whiskers represent the lowest or highest values within 1.5 times IQR from the first or third quartiles. *P < 0.05, **P < 0.01 versus vehicle treatment, by two-tailed Student's i-test.

[0065] FIG. 18 Full western blot gel panels, (a-d) HIF2a, HIFla, NEU3 and β-ACTIN from which the data in FIG lb (a), FIG. If (b), FIG. 10b (c), FIG. 4g (b) and FIG. 8e (d) were derived, (e, f) NUE3 and β-ACTIN from which the data in FIG. 4h (e) and FIG. 6k (f) were derived, (g) UCP1 and EIF5 from which the data in FIG. lOv were derived.

[0066] FIG. 19 Demographic characteristics of the herein subjects.

[0067] FIG. 20 Clinical biochemistry of the herein subjects.

[0068] FIG. 21 provides the sequences of oligonucleotide primers used herein.

[0069] FIG. 22 Intestine- specific HIF-2oc disruption ameliorates high-fat-diet induced obesity, insulin resistance and activate the beiging process of adipose tissues, (a) body mass (b) body fat and (c) body fat rate of intestine- specific HIF-2oc knockout mice after 8 -week high-fat-diet feeding, (d) Glucose Tolerance Test (GTT) of intestine- specific HIF-2oc knockout mice and (e) glucose area under the curve (AUC). (f) insulin tolerance test (ΓΤΤ ) of intestine-specific HIF-2oc knockout mice, (g) subcutaneous adipose tissues expression of mRNAs relative to beiging. (h) Western blot analysis of UCP1 protein expression in subcutaneous adipose tissues, (i) subcutaneous adipose tissues and (j) visceral adipose tissues expression of mRNAs involved in the futile creatine cycle, a UCP1 independent thermogenesis process, (k) Western blot analysis of CKMT2 protein expression in visceral adipose tissues.

[0070] FIG. 23 (a) Anal temperature of intestine- specific HIF-2oc knockout mice during cold stimulation (12°C). (b) representative UCP1 immunohistochemical staining of scWAT sections, (c) expression of mRNAs relative to beiging in subcutaneous adipose tissues and (d) visceral adipose tissues, (e) expression of mRNAs in liver involved in bile acid synthesis and transport, from intestine-specific HIF-2oc knockout mice after a short term 2- week high-fat-diet treatment, (f) expression of HIF-2oc target gene mRNAs in intestine. [0071] FIG. 24 Intestine- specific HIF-2oc disruption alters bile acid compositions and its relative expression bile acid transport and metabolism genes in mice, (a) expression of mRNAs in intestine involved in bile acid transport, (b) liver expression of mRNAs in liver involved in bile acid synthesis and transport, bile acid profiles in serum (c) ileum (d) and feces (e) after an 8- week high-fat-diet treatment.

[0072] FIG. 25 Transfer of intestine-specific HIF-2 D disrupted mice fecal bacterium

ameliorates high-fat-diet induced obesity, insulin resistance and activate the beiging process of adipose tissues, (a) body mass (b) body fat and (c) body fat rate of fecal transfer mice, (d) Glucose Tolerance Test (GTT) of fecal transfer mice and (e) glucose area under the curve (AUC). (f) insulin tolerance test (ITT) of fecal transfer mice, (g) subcutaneous adipose tissues expression of mRNAs relative to beiging. (h) Western blot analysis of UCP1 protein expression in subcutaneous adipose tissues, (i) subcutaneous adipose tissues and (j) visceral adipose tissues expression of mRNAs relative to futile creatine cycle, a UCP1 independent thermogenesis process, (k) Western blot analysis of CKMT2 protein expression in visceral adipose tissues.

[0073] FIG. 26 Fecal bacterium transplanted mice have altered bile acid and its relative gene expression, (a) expression of mRNAs in liver involved in bile acid synthesis and transport, bile acid profiles in serum (b) ileum (c) and feces (d) after fecal microbiota transfer.

[0074] FIG. 27 Depletion of the microbiota by antibiotic treatment reverses the therapeutic effects of intestine-specific HIF-2oc disruption, (a) expression of mRNAs in liver involved in bile acid synthesis and transport, (b) expression of mRNAs in intestine involved in bile acid transport, (c) expression of mRNAs subcutaneous adipose involved in beiging. (d) expression of mRNAs visceral adipose tissues involved in beiging. (e) body mass (f) Glucose Tolerance Test (GTT) (g) glucose area under the curve (AUC) and (h) insulin tolerance test (ITT) of antibiotic treatment mice.

[0075] FIG. 28 Intestine-specific HIF-2oc disruption and fecal microbiota transplantation upregulates TGR5 signaling in both subcutaneous adipose tissues and visceral adipose tissues, (a) subcutaneous adipose tissues and (b) visceral adipose tissues expression of Tgr5 and Dio2 mRNAs in intestine- specific HIF-2oc knockout mice, (c) subcutaneous adipose tissues and (d) visceral adipose tissues expression of Tgr5 and Dio2 mRNAs in microbiota-transplanted mice. DETAILED DESCRIPTION

[0076] The following is a detailed description of the invention provided to aid those skilled in the art in practicing the present invention. Those of ordinary skill in the art may make modifications and variations in the embodiments described herein without departing from the spirit or scope of the present invention. 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. The terminology used in the description of the invention herein is for describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents, figures and other references mentioned herein are expressly incorporated by reference in their entirety.

[0077] Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and described the methods and/or materials in connection with which the publications are cited.

[0078] The liver plays a central role in maintaining overall organism energy balance by controlling carbohydrate and lipid metabolism. Liver disease is a growing global health problem, as deaths from end-stage liver cirrhosis and cancer are rising across the world. At present, pharmacologic approaches to effectively treat or prevent liver disease are extremely limited. Given the high prevalence and rising incidence of NAFLD, the absence of approved therapies is striking. NAFLD is a complex disease, with considerable variation in severity amongst individuals. NAFLD can lead to NASH. To manage NAFLD and reverse its destructive effects on the liver, the underlying disease mechanisms need to be properly understood.

[0079] Based upon current understanding, NAFLD therapies typically target four main pathways. The dominant approach is targeting hepatic fat accumulation and the resultant metabolic stress. Medications in this group include peroxisome proliferator-activator receptor agonists (eg, pioglitazone, elafibranor, saroglitazar), medications targeting the bile acid-farnesoid X receptor axis (obeticholic acid), inhibitors of de novo lipogenesis (aramchol, NDI-010976), incretins (liraglutide) and fibroblast growth factor (FGF)-21 or FGF-19 analogues.

[0080] A second approach is targeting the oxidative stress, inflammation and injury that follow the metabolic stress. Medications from this group include antioxidants (vitamin E), medications with a target in the tumour necrosis factor a pathway (emricasan, pentoxifylline) and immune modulators (amlexanox, cenicriviroc).

[0081] A third group has a target in the gut, including antiobesity agents such as orlistat or gut microbiome modulators (IMM-124e, faecal microbial transplant, solithromycin). Finally, as the ongoing injury leads to fibrosis approaching to liver-related morbidity and mortality, antifibrotics (simtuzumab and GR-MD-02) make fourth group of therapy.

[0082] Despite these state of the art therapeutic approaches, their overall effectiveness is poor. Thus, there remains a need for better therapeutic strategies which can target NAFLD across the entire spectrum of disease, as well as associated and related metabolic disorders including obesity and insulin resistance.

[0083] Hypoxia-inducible factor (HIF) is a transcription factor that regulates diverse signaling pathways enabling adaptive cellular responses to perturbations of the tissue microenvironment. HIF is a heterodimeric complex consisting of a constitutively expressed β-subunit and an oxygen-sensitive a-subunit. To date, three isoforms of HIF-a subunit have been described, in which HIF- la and HIF-2a are the best characterized.

[0084] Hypoxia and expression of HIF- la and HIF-2a are characteristic features of all solid tumors. HIF signaling serves as a major adaptive mechanism in tumor growth in a hypoxic microenvironment. HIFs represent a critical signaling node in the switch to protumorigenic inflammatory responses through recruitment of protumor immune cells and altered immune cell effector functions to suppress antitumor immune responses and promote tumor growth through direct growth-promoting cytokine production, angiogenesis, and ROS production. Role of HIF- 2a in Renal Cell Carcinoma(RCC) using HIF-2a inhibitors, including PT2385, is known in the art.

[0085] HIF activation through hypoxia-dependent and hypoxia-independent signals have been reported in liver disease of diverse etiologies. Increased expression of HIF- la and HIF-2a has been reported in many liver diseases, including nonalcoholic fatty liver disease (NAFLD), alcoholic liver disease (ALD), IR-induced liver injury, and hepatocellular carcinoma. A common feature of these liver diseases is tissue hypoxia due to an imbalance of metabolic demand and supply.

[0086] The liver expresses all three HIF-a family members, HIF- la, -2a, and -3a, under physiologic and pathophysiologic conditions, suggesting that HIFs are important mediators of normal liver function and disease. With regard to metabolism in the liver, HIF-1 regulates the expression of glucose transporters as well as glycolytic enzymes and is thought to contribute to the glycolytic phenotype of hepatocellular carcinomas. In addition, recent studies have suggested a role for HIF-1 a, and HIF-2a, in the regulation of hepatic lipid metabolism. HIF-1 a is shown to inhibit adipose tissue resulting in reduction of obesity and insulin resistance whereas inhibiting adipose HIF-2a has been observed to lead to increased body weight, glucose intolerance, and insulin resistance. Indeed, the state of the art suggests that HIF-1 a and HIF-2a have opposing effects in certain contexts such as macrophage polarization. HIF-1 a inhibition leads to steatohepatitis associated with impaired fatty acid β-oxidation, decreased lipogenic gene expression, and increased lipid storage capacity. The present invention discloses that inactivation of HIF-2a significantly suppresses the development of hepatic steatosis, by decreasing ceramide synthesis, decreasing fatty acid transport and lipogenesis, and inhibition of salvage pathway or a combination of two or more, resulting in regulation of hepatic lipid metabolism in vivo, reduced obsesity, and decreased insulin resistance (or increase insulin sensitivity).

[0087] Definitions

[0088] 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. The following references, the entire disclosures of which are incorporated herein by reference, provide one of skill with a general definition of many of the terms (unless defined otherwise herein) used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, the Harper Collins Dictionary of Biology (1991). Generally, the procedures of molecular biology methods described or inherent herein and the like are common methods used in the art. Such standard techniques can be found in reference manuals such as for example Sambrook et al., (2000, Molecular Cloning— A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratories); and Ausubel et al., (1994, Current Protocols in Molecular Biology, John Wiley & Sons, New- York).

[0089] The terminology used in the description is for describing particular embodiments only and is not intended to be limiting of the invention. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise (such as in the case of a group containing a number of carbon atoms in which case each carbon atom number falling within the range is provided), between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.

[0090] The following terms are used to describe the present invention. In instances where a term is not specifically defined herein, that term is given an art-recognized meaning by those of ordinary skill applying that term in context to its use in describing the present invention.

[0091] The articles "a" and "an" as used herein and in the appended claims are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article unless the context clearly indicates otherwise. By way of example, "an element" means one element or more than one element.

[0092] The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and/or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and/or B", when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0093] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of."

[0094] In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding,"

"composed of," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of and "consisting essentially of shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0095] As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from anyone or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a nonlimiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0096] It should also be understood that, in certain methods described herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited unless the context indicates otherwise.

[0097] The terms "co-administration", "co-administered" and "co-administering" or

"combination therapy" refer to both concurrent administration (administration of two or more agents at the same time) and time varied administration (administration of one or more agents at a time different from that of the administration of an additional agent or agents), as long as the agents are present in the area to be treated to some extent, preferably at effective amounts, at the same time. In certain preferred aspects, one or more of the present agents described herein, are co-administered in combination with at least one additional bioactive agent, especially including an antifungal, antibacterial, and/or biocide. In particularly preferred aspects, the coadministration of agents

[0098] The term "therapeutically effective amount" means the amount required to achieve a therapeutic effect. The therapeutic effect could be any therapeutic effect ranging from

prevention, symptom amelioration, symptom treatment, to disease termination or cure, e.g., the reduction, amelioration, or otherwise decrease the level of obesity, insulin resistance, and NAFLD.

[0099] As used herein, the term "administering" is meant to refer to a means of providing the composition to the subject in a manner that results in the composition being inside the subject's body. Such an administration can be by any route including, without limitation, subcutaneous, intradermal, intravenous, intra-arterial, intraperitoneal, and intramuscular.

[00100] The term "patient" or "subject" is used throughout the specification to describe an animal, preferably a human or a domesticated animal, to whom treatment, including prophylactic treatment, with the compositions according to the present disclosure is provided. For treatment of conditions or disease states which are specific for a specific animal such as a human patient, the term patient refers to that specific animal, including a domesticated animal such as a dog or cat or a farm animal such as a horse, cow, sheep, etc. In general, in the present disclosure, the term patient refers to a human patient unless otherwise stated or implied from the context of the use of the term.

[00101] The term "effective" is used to describe an amount of a compound, composition or component which, when used within the context of its intended use, effects an intended result. The term effective subsumes all other effective amount or effective concentration terms, which are otherwise described or used in the present application.

[00102] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements. "Consisting essentially of", when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. "Consisting of" shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the

compositions of this invention. Embodiments defined by each of these transition terms are within the scope of this invention.

[00103] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

[00104] As used herein, the term "functionalities" or "moieties" or "functional moieties" refer to substances or agents which are capable of being contained in, or attached, to another compound or composition of the disclosure, e.g., a HIF-2a and neu3 inhibitor or to a

composition or vehicle comprising said HIF-2a and neu3 inhibitor. In an example, the moiety can be a "targeting moiety," which includes any ligand, antibody, or otherwise agent which can be attached to a HIF-2a and neu3 inhibitor or to a composition or vehicle comprising said HIF- 2a inhibitor and neu3 that allows the selective binding and attachment of the HIF-2a and neu3 inhibitor or otherwise other "cargo" to a target cell or tissue, but not to other non-desired cells or tissues.

[00105] As used herein, "kits" are understood to contain at least the non-standard laboratory reagents of the invention and one or more non-standard laboratory reagents for use in the methods of the invention.

[00106] The term "obtaining" is understood herein as manufacturing, purchasing, or otherwise coming into possession of.

[00107] As used herein, the term "treated," "treating" or "treatment" includes the diminishment or alleviation of at least one symptom associated or caused by the state, disorder or disease being treated. A subject that has been treated can exhibit a partial or total alleviation of symptoms (for example, NAFLD or obesity), or symptoms can remain static following treatment according to the invention. The term "treatment" is intended to encompass prophylaxis, therapy and cure. [00108] As used herein, the term "control" refers to a sample or standard used for comparison with an experimental sample. In some embodiments, the control is a sample obtained from a healthy patient. In other embodiments, the control is a historical control or standard reference value or range of values (such as a previously tested sample, subject, or group of samples or subjects).

[00109] As used herein, a disease caused by a relative or absolute lack of insulin leading to uncontrolled carbohydrate metabolism, commonly simplified to "diabetes," though diabetes mellitus should not be confused with diabetes insipidus. As used herein, "diabetes" refers to diabetes mellitus, unless otherwise indicated. A "diabetic condition" includes pre-diabetes and diabetes. Type 1 diabetes (sometimes referred to as "insulin-dependent diabetes" or "juvenile- onset diabetes") is an auto-immune disease characterized by destruction of the pancreatic β cells that leads to a total or near total lack of insulin. In type 2 diabetes (T2DM; sometimes referred to as "non-insulin-dependent diabetes" or "adult-onset diabetes"), the body does not respond to insulin, though it is present. Symptoms of diabetes include: excessive thirst (polydipsia);

frequent urination (polyuria); extreme hunger or constant eating (polyphagia); unexplained weight loss; presence of glucose in the urine (glycosuria); tiredness or fatigue; changes in vision; numbness or tingling in the extremities (hands, feet); slow-healing wounds or sores; and abnormally high frequency of infection. Diabetes may be clinically diagnosed by a fasting plasma glucose (FPG) concentration of greater than or equal to 7.0 mmol/L (126 mg/dL), or a plasma glucose concentration of greater than or equal to 11.1 mmol/L (200 mg/dL) at about two hours after an oral glucose tolerance test (OGTT) with a 75 g load. A more detailed description of diabetes may be found in Cecil Textbook of Medicine, J.B. Wyngaarden, et al., eds. (W.B. Saunders Co., Philadelphia, 1992, 19 ^th ed.).

[00110] As used herein, a hypoxia-inducible factor 1 (HIF-1): A transcription factor found in mammalian cells cultured under reduced oxygen tension (hypoxia) that plays a role in cellular and systemic response to hypoxia. HIF- 1 is a heterodimer composed of an alpha subunit and a beta subunit. The beta subunit is the aryl hydrocarbon receptor nuclear translocator (Arnt; also known as HIF-Ιβ). Arnt is constitutively present in the cell nucleus. There are three HIF alpha subunits, HIF- la, HIF-2a (also known an EPAS 1), and HIF-3a, which accumulate in hypoxic conditions. Under normoxic conditions, HIF alpha subunits are hydroxylated by a prolyl hydroxylase and targeted for proteasome dependent degradation. The prolyl hydroxylase is inhibited in hypoxia, leading to accumulation of HIF alpha subunits. HIF alpha subunits dimerize with Arnt to form a functional transcription factor capable of binding DNA at hypoxia response elements (HRE) and transcriptional activation.

[00111] HIF-1 nucleic acid and protein sequences are publicly available. For example,

GenBank Accession Nos. NM_001530 and NM_181054 disclose exemplary human HIF-la nucleic acid sequences and GenBank Accession Nos. NP_001521 and NP_851397 disclose exemplary human HIF-Ια protein sequences. GenBank Accession No. NM_001430 discloses an exemplary human HIF-2a nucleic acid sequence and GenBank Accession No. NP_001421 discloses an exemplary human HIF-2a protein sequence. GenBank Accession Nos. NM_152795, NM_022462, and NM_152794 disclose exemplary human HIF3a nucleic acid sequences and GenBank Accession Nos. NP_690008, NP_071907, and NP_690007 disclose exemplary human HIF3a. protein sequences. GenBank Accession Nos. NM_001668 and NM_178427 disclose exemplary human Arnt nucleic acid sequences and GenBank Accession Nos. NP_001659 and NP_848514 disclose exemplary human Arnt protein sequences. Each of these sequences is incorporated by reference herein, as present in GenBank on December 16, 2010.

[00112] As used herein, the term "inhibitor" as in an HIF-2a inhibitor refers to any chemical compound, nucleic acid molecule, or peptide (such as an antibody), specific for a gene product that can reduce activity of a gene product or directly interfere with expression of a gene, such as genes that encode HIF, such as HIF-2a. An inhibitor of the disclosure, for example, can inhibit the activity of a HIF-2a protein either directly or indirectly. Direct inhibition can be accomplished, for example, by binding to a HIF-2a protein and thereby preventing the protein from binding an intended target, such as a dimerization partner (e.g., Arnt) or a DNA sequence (such as a HRE). Indirect inhibition can be accomplished, for example, by binding to a HIF-2a protein intended target, such as a receptor or binding partner, thereby blocking or reducing activity of HIF-2a. Furthermore, an inhibitor of the disclosure can inhibit a HIF-2a gene by reducing or inhibiting expression of the gene, inter alia by interfering with gene expression (transcription, processing, translation, post-translational modification), for example, by interfering with the mRNA and blocking translation of the HIF-2a gene product or by altering post-translational modification of a HIF-2a gene product (such as prolyl hydroxylation), or by causing changes in intracellular localization. [00113] As used herein, reference to "insulin resistance" refers to a state in which the cells of a subject do not respond appropriately to insulin, and increased amounts of insulin are required for glucose to be taken up by the cells. In some examples, insulin resistance is defined as a state where 200 units of insulin per day or more are required to attain glycemic control and prevent ketosis. Subjects with insulin resistance often have increased plasma glucose levels, increased plasma insulin levels, or both, as compared with a subject without insulin resistance or standard normal ranges. In some examples, insulin resistance is determined by measuring blood glucose (such as fasting plasma glucose) and/or blood insulin (such as fasting plasma insulin) levels. In other examples, insulin resistance is determined by oral glucose tolerance test, glucose clamp (such as hyperinsulinemic euglycemic clamp), modified insulin suppression test, homeostatic model assessment, or quantitative insulin sensitivity check index (QUICKI).

[00114] As used herein, reference to the condition of "obesity" refers to a condition in which excess body fat may put a person at health risk (see Barlow and Dietz, Pediatrics 102: E29, 1998; National Institutes of Health, Obes. Res. 6 (suppl. 2):51S-209S, 1998). Excess body fat is a result of an imbalance of energy intake and energy expenditure. In one embodiment in humans, the Body Mass Index (BMI) is used to assess obesity. In one embodiment, a BMI of 25.0 kg/m 2 to 29.9 kg/m 2 is overweight (also called grade I obesity), while a BMI of 30 kg/m or more is truly obese (also called grade II obesity). In another embodiment in humans, waist circumference is used to assess obesity. In this embodiment, in men a waist circumference of 102 cm or more is considered obese, while in women a waist circumference of 89 cm or more is considered obese. Strong evidence shows that obesity affects both the morbidity and mortality of individuals. For example, an obese individual is at increased risk for heart disease, non-insulin dependent (type 2) diabetes, hypertension, stroke, cancer (e.g. endometrial, breast, prostate, and colon cancer), dyslipidemia, gall bladder disease, sleep apnea, reduced fertility, and

osteoarthritis, amongst others (see Lyznicki et al., Am. Fam. Phys. 63:2185, 2001).

[00115] As used herein, the term "non-alcoholic fatty liver disease" or NAFLD is the term for a range of conditions caused by a build-up of fat in the liver. NAFLD includes simple fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), fibrosis and cirrhosis.

[00116] As used herein, the term "non-alcoholic steatohepatitis" or NASH is a condition that causes inflammation and accumulation of fat and fibrous (scar) tissue in the liver. Liver enzyme levels in the blood may be more elevated than the mild elevations seen with nonalcoholic fatty liver disease (NAFLD). NAFLD can lead to NASH.

[00117] As used herein, "neuraminidase 3" or "NEU3" (the protein) or (the gene) or aliase terms SIAL3, neuraminidase 3 (membrane sialidase), neuraminidase 3, membrane sialidase, and the like, refer to a glycohydrolytic enzyme which remove sialic acid residues from glycoproteins and glycolipids. It is localized in the plasma membrane, and its activity is specific for gangliosides. It may play a role in modulating the ganglioside content of the lipid bilayer. Mechanistically, intestine HIF-2a regulates ceramide metabolism mainly from the salvage pathway, which was revealed by the identification of the novel HIF-2a target gene encoding neuraminidase 3 (Neu3).

[00118] As used herein, the term "pharmaceutically acceptable carrier" refers to conventional convention such carriers for any type of active agent, as described for example in Remington: The Science and Practice of Pharmacy, The University of the Sciences in

Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21 ^st Edition (2005), describes compositions and formulations suitable for pharmaceutical delivery of compounds, such as an inhibitor of HIF-2a (for example, PT2385). In general, the nature of the carrier will depend on the particular mode of administration employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, pH buffering agents, or the like, for example sodium acetate or sorbitan monolaurate.

[00119] As used herein, the term "purified" does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified preparation of a HIF-2a inhibitor is one in which the HIF-2a inhibitor is more enriched than in its environment within a cell or other preparation, such as the environment in which it is synthesized. Preferably, a preparation is purified such that the HIF-2a inhibitor represents at least 50% of the total content of the preparation, for example, at least 50% by weight. In one embodiment, the HIF-2a inhibitor is at least 50%, for example at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more free of proteins, lipids, carbohydrates or other materials with which it is originally associated.

[00120] As used herein, "RNA interference (RNAi)" refers to a cellular process that inhibits expression of genes, including cellular and viral genes. RNAi is a form of antisense- mediated gene silencing involving the introduction of double stranded RNA-like

oligonucleotides leading to the sequence- specific reduction of RNA transcripts. Double- stranded RNA molecules that inhibit gene expression through the RNAi pathway include siRNAs, miRNAs, and shRNAs. Typically, the sequence of a siRNA is substantially identical to a portion of a transcript of a target gene (mRNA) for which interference or inhibition of expression is desired. For example, small, double stranded RNAs of about 15 to about 40 nucleotides in length (the length of each of the individual strands of the dsRNA), such as about 15 to about 25 nucleotides in length (for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides), that interfere with, or inhibit, expression of a target sequence. The RNA backbone and/or component nucleotides can be unmodified or modified. For instance, the dsRNA can contain one or more deoxynucleic acids. Synthetic small dsRNAs may be used to induce gene-specific inhibition of expression. The dsRNAs can be formed from complementary single stranded RNAs ("ssRNAs") or from a ssRNA that forms a hairpin or from expression from a DNA vector. In certain examples, these small interfering nucleotide sequences have 3' and/or 5' overhangs on each strand of the duplex. These overhangs can be 0 nucleotides (that is, blunt ends) to 5 nucleotides in length. Such siRNA molecules can be used as reverse genetic and therapeutic tools in mammalian cells, including human cells, both in vitro and in vivo. These small interfering nucleotide sequences are suitable for interference or inhibition of expression of a target gene wherein the sequence of the small interfering nucleotide sequence is substantially identical to a portion of an mRNA or transcript of the target gene for which interference or inhibition of expression is desired.

[00121] In addition to native nucleotide molecules, nucleotides suitable for inhibiting or interfering with the expression of a target sequence include nucleotide derivatives and analogs. For example, a non-natural linkage between nucleotide residues can be used, such as a phosphorothioate linkage. The nucleotide strand can be derivatized with a reactive functional group or a reporter group, such as a fluorophore. For example, the 2'-hydroxyl at the 3' terminus can be readily and selectively derivatized with a variety of groups. Other useful nucleotide derivatives incorporate nucleotides having modified carbohydrate moieties, such as 2'-0- alkylated residues or 2'-deoxy-2'-halogenated derivatives. Particular examples of such

carbohydrate moieties include 2'-0-methyl ribosyl derivatives and 2'-0-fluoro ribosyl derivatives.

[00122] The nucleotide bases can be modified. Any modified base useful for inhibiting or interfering with the expression of a target sequence can be used. For example, halogenated bases, such as 5-bromouracil and 5-iodouracil can be incorporated. The bases may also be alkylated, for example, 7-methylguanosine may be incorporated in place of a guanosine residue. Non-natural bases that yield successful inhibition can also be incorporated.

[00123] Reference will now be made in detail to exemplary embodiments of the invention.

While the invention will be described in conjunction with the exemplary embodiments, it will be understood that it is not intended to limit the invention to those embodiments. To the contrary, it is intended to cover alternatives, modifications, and equivalents as may be included within the spirit and scope of the invention as defined by the appended claims.

[00124] Exemplary compositions and methods of the present invention are described in more detail below.

[00125] Current invention discloses a method of treating

[00126] HIF-2a Inhibitors and Compositions Thereof

[00127] The present disclosure contemplates any suitable known HIF-2a inhibitor for use in the methods of the present disclosure. The present disclosure also contemplates any functional derivatives or variants of known HIF-2a inhibitors for use in the methods of the present disclosure.

[00128] As used herein, the term "HIF-2a" refers to a monomelic protein that contains several conserved structured domains: basic helix-loop-helix (bHLH), and two Per-ARNT-Sim (PAS) domains designated PAS -A and PAS-B, in addition to C-terminal regulatory regions. "HIF-2a" is also alternatively known by several other names in the scientific literature, including Endothelial PAS Domain Protein 1 (EPAS 1), HIF2A, PASD2, HIF-2-Alpha, HIF2- Alpha, HLF, Hypoxia- Inducible Factor 2- Alpha, HIF-1 alpha- Like Factor, and MOP2. As a member of the bHLH/PAS family of transcription factors, "HIF-2a " forms an active heterodimeric transcription factor complex by binding to the ARNT (also known as HIF-Ιβ) protein through non-covalent interactions.

[00129] As used herein, "HIF-2a activity" has its ordinary meaning in the art. HIF-2a activity, for example, includes activation of gene transcription mediated by HIF-2a. The term "inhibiting HIF-2a activity", as used herein, refers to slowing, reducing, altering, as well as completely eliminating and/or preventing HIF-2a activity.

[00130] The HIF-2a inhibitor is not limited to any type of molecule, including small molecular weight compounds, peptides, polypeptides, antibodies or functional fragments thereof which directly interact and/or bind to HIF-2a thereby blocking, inhibiting, or otherwise interfering with its activity. Inhibitors of HIF-2a may also include nucleic acid based inhibitors, e.g., siRNA based inhibitors which block, inhibit, or otherwise modulate the expression of HIF- 2a inhibitor. Blocking the expression of HIF-2a may include strategies for blocking the transcription of the HIF-2a gene, RNA-based inhibitors that block translation or otherwise lead to transcript degradation (e.g., siRNA processes), or protein-degradation-based strategies (e.g., ubiquitination-based proteosome-mediated degradation of the encoded HIF-2a. Such strategies are well known in the art and as such, their further detailed description is not necessary to enable or support disclosure.

[00131] In one embodiment, the present disclosure contemplates the use of any HIF-2a described in WO2015/035223 (Aryl Ethers and Uses Thereof), which is incorporated herein by reference in its entirety. This includes the disclosure of PT2385.

[00132] In certain embodiments, the HIF-2a inhibitor can have the following structure of formula I:

[00133] or a pharmaceutically acceptable salt thereof, wherein:

[00134] Ri is aryl or heteroaryl;

[00135] R ₂ is nitro, carboxaldehyde, carboxylic acid, ester, amido, cyano, halo, sulfonyl, alkyl or heteroalkyl; [00136] R ₃ is hydrogen, halo, cyano, alkyl, heteroalkyl, alkenyl, alkynyl, alkylamino, carboxaldehyde, carboxylic acid, oxime, ester, amido or acyl, or R ₂ /R ₃ and atoms they are attached to form a 5- or 6- membered carbocycle with at least one sp hybridized carbon;

[00137] R4 is nitro, halo, cyano, alkyl, sulfinyl, sulfonamide, sulfonyl or sulfoximinyl; and

R5 is hydrogen, halo or alkyl. Said formula I embodies PT2385.

[00138] In a specific embodiment, the HIF-2a inhibitor is at least one of PT2385, PT2567,

PT2399, PT2977 or a combination thereof, and any functional derivative, salt, or polymorph thereof, which i shown as having the following structure:

[00139] (PT2385)

[00140] All chemical terms have their commonly understood meaning known in the art.

[00141] The methods and formulations comprising HIF-2a inhibitors described herein include the use of N-oxides, crystalline forms (also known as polymorphs), or pharmaceutically acceptable salts of compounds having the structure of formulae described herein, as well as active metabolites of these compounds having the same type of activity. In addition, the compounds described herein can exist in unsolvated as well as solvated forms with

pharmaceutically acceptable solvents such as water, ethanol, and the like. The solvated forms of the compounds presented herein are also considered to be disclosed herein.

[00142] The compounds described herein may exhibit their natural isotopic abundance, or one or more of the atoms may be artificially enriched in a particular isotope having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number predominantly found in nature. The present invention is meant to include all suitable isotopic variations of the compounds described herein. For example, hydrogen has three naturally occurring isotopes, denoted Ή (protium), 2 H (deuterium), and 3 H (tritium). Protium is the most abundant isotope in nature. Enriching for deuterium may afford certain therapeutic advantages, such as increased in vivo half-life and/or exposure, or may provide a compound useful for investigating in vivo routes of drug elimination and metabolism.

[00143] Isotopically-enriched compounds may be prepared by conventional techniques well known to those skilled in the art or by processes analogous to those described in the

Schemes and Examples herein using appropriate isotopically-enriched reagents and/or intermediates. See Pleiss and Voger, Synthesis and Applications of lsotopically Labeled

Compounds, Vol. 7, Wiley, ISBN-10: 0471495018, published on March 14, 2001.

[00144] Unless otherwise specified, chemical entities described herein may include, but are not limited to, when possible, their optical isomers, such as enantiomers and diastereomers, mixtures of enantiomers, including racemates, mixtures of diastereomers, and other mixtures thereof, to the extent they can be made by one of ordinary skill in the art by routine

experimentation. In those situations, the single enantiomers or diastereomers, i.e., optically active forms, can be obtained by asymmetric synthesis or by resolution of the racemates or mixtures of diastereomers. Resolution of the racemates or mixtures of diastereomers, if needed, can be accomplished, for example, by conventional methods such as crystallization in the presence of a resolving agent, or chromatography, using, for example, a chiral high-pressure liquid chromatography (HPLC) column. In addition, chemical entities having carbon-carbon double bonds or carbon-nitrogen double bonds may exist in Z- or inform (or cis- or trans- form). Furthermore, some chemical entities may exist in various tautomeric forms. Unless otherwise specified, chemical entities described herein are intended to include all Z-, E- and tautomeric forms as well.

[00145] HIF-2a inhibitors can include small organic molecules. Some small molecule inhibitors may inhibit multiple HIF alpha subunits (such as HIF-Ια, HIF-2a, and/or HIF-3a.), while others may be specific for HIF-2a. In some examples, a small molecule inhibitor inhibits more than one HIF alpha subunit, including HIF-2a. In particular examples, a small molecule inhibitor specifically inhibits HIF-2a expression or activity (such as dimerization, DNA binding, and/or transcriptional activity).

[00146] In some examples, the small molecule inhibitor of HIF-2a is a previously identified HIF-2a inhibitor. In one non-limiting examples, the inhibitor is at least one of PT2385, PT2567, PT2399, PT2977 or a combination thereof. In other examples, a HIF-2a small molecule inhibitor is a cardiac glycoside, such as digoxin, ouabain, proscillaridin A, digitoxin, acetydigitoxin, convallatoxin, peruvoside, strophanthin K, nerifolin, cymarin, or periplocymarin (see e.g., Zhang et al., Proc. Natl. Acad. Sci. USA 105: 19579-19586, 2008), rapamycin or an analog thereof (such as rapamycin, everolimus, temsirolimus, or tacrolimus), an anthracycline or analog thereof (such as doxorubicin or daunorubicin), a proteasome inhibitor (such as

bortezomib (PS-341)), or camptothecin or an analog thereof (such as CRLX-101, SN-38, EZN- 2208, irinotecan, or topotecan). In additional examples, a HIF-2a small molecule inhibitor includes echinomycin (see, e.g., Kong et al., Cancer Res. 65:9047-9055, 2005), 17-allylamino- 17-demethoxygeldanamycin (see, e.g., Liu et al., Mol. Cell 25:207-217, 2007), 17- dimethylaminoethylamino-17-demethoxygeldanamycin (e.g., WO 02/079167), NSC 644221 (see, e.g., Creighton-Gutteridge et al., Clin. Cancer Res. 13: 1010-1018, 2007), YC-1 (e.g., Yeo et al., J. Natl. Cancer Inst. 95:498-499, 2003), PX-478 (see, e.g., U.S. Pat. 7,399,785), 2- methoxyestradiol or derivatives thereof (e.g., ENMD-1198 or ENMD-2076), wondonin (e.g., Jun et al., FEBS Lett. 581:4977-4982, 2007), Palomid-529 (Paloma Pharmaceuticals), CLT-003 (Charlesson), cyclopentabenzofuranes (e.g., IMD-026260; WO 2010/063471), furoquinoline- based molecules (e.g., Lohar et al., Bioorg. Med. Chem. Lett. 18:3603-3606, 2008), BAY 87- 2243, BTG-6228 (BTG), or KC7F2 (e.g., Naria et al., Clin. Cancer Res. 15:6128-6136, 2009); alpha-ketoglutarates (e.g., WO 06/016143); P3155 or P2630 (e.g., Kumar et al., Bioorg. Med. Chem. Lett. 20:6426-6429, 2010); EL- 102 (McLaughlin et al., American Association for Cancer Research, Abstract LB-385, 2011); CX-4715 or CX-3800 series compounds (Cylene

Pharmaceuticals). In another example, a small molecule inhibitor of HIFla is aminoflavone (e.g., Terzuoli et al., Cancer Res. 20:6837-6848, 2010). It is to be understood that HIF-2a inhibitors for use in the present disclosure also include novel HIF-2a small molecule inhibitors developed in the future.

[00147] HIF-2a inhibitors also include nucleic acid molecules, including, but not limited to antisense molecules. Some HIF-2a nucleic acid inhibitors may inhibit multiple HIF alpha subunits (such as HIF- la, HIF-2a, and/or HIF-3a.), while others may be specific for HIF-2a. In some examples, a nucleic acid inhibitor inhibits more than one HIF alpha subunit, including HIF- 2a. In particular examples, a nucleic acid inhibitor specifically inhibits HIF-2a expression or activity (such as dimerization, DNA binding, and/or transcriptional activity). [00148] In some examples, the nucleic acid inhibitor of HIF-2a decreases expression of

HIF-2a, such as an antisense molecule or an RNAi molecule (such as siRNA, miRNA, or shRNA). In a particular non-limiting example, a HIF-2a siRNA includes SEQ ID NOs: 70 or 71. In other examples, the inhibitor is RX-0047 or RX-0149 (see, e.g., U.S. Pat. No. 7,205,283), double-stranded HIF decoy oligonucleotides (e.g., WO 2005/056795), ACU-HHY-011 (Opko Health), or EZN-2968 (see, e.g., U.S. Pat. Nos. 7,589,190 and 7,737,264). In further examples, the nucleic acid inhibitor of HIF-2a decreases activity of HIF-2a, such as a G-rich

oligodeoxynucleotide (for example, a G-quartet structure). In particular, non-limiting examples, the inhibitor is JG243 or JG244 (see, e.g., Guan et al., Mol. Ther. 18: 188-197, 2010). It is to be understood that HIF-2a inhibitors for use in the present disclosure also include novel HIF-2a nucleic acid molecule inhibitors developed in the future.

[00149] In additional examples, inhibitors of HIF-2a can include HIF-2a-specific binding agents, such as polyclonal or monoclonal antibodies. Specific examples of HIF-2a-specific binding agents include HIF-2a-specific antibodies or functional fragments thereof, for instance monoclonal antibodies or fragments of monoclonal antibodies, including humanized monoclonal antibodies. Methods for producing antibodies (including monoclonal antibodies) using standard procedures are described in a number of texts, including Harlow and Lane (Antibodies, A Laboratory Manual, CSHL, New York, 1988). Techniques for producing humanized monoclonal antibodies are described, for example, by Jones et al., Nature 321:522, 1986; Riechmann et al., Nature 332:323, 1988; Verhoeyen et al., Science 239: 1534, 1988; Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285, 1992; Sandhu, Crit. Rev. Biotech.12:437, 1992; and Singer et al., J.

Immunol.150:2844, 1993. Monoclonal or polyclonal antibodies may be produced to either the normal HIF-2a protein or mutant forms of this protein, for instance particular portions that contain a mutation and therefore may provide a distinguishing epitope. Optimally, antibodies raised against these proteins or peptides specifically detect the protein or peptide with which the antibodies are generated. That is, an antibody generated to the HIF-2a protein or a fragment thereof would recognize and bind the HIF-2a protein and would not substantially recognize or bind to other proteins found in mammalian cells (for example, human cells).

[00150] Methods of making antibodies that can be used clinically are known in the art.

Antibodies for HIF-2a can also be obtained from commercially available sources, including from Santa Cruz Biotechnology (Santa Cruz, CA), Abeam (Cambridge, MA), and Millipore (Billerica, MA). It is to be understood that HIF-2a antibodies for use in the present disclosure also include novel anti-HIF-2a antibodies developed in the future.

[00151] In some embodiments, the HIF-2a inhibitor is targeted to adipose tissue (such as white adipose tissue). WAT targeting motifs include peptides that preferentially bind to WAT cells or WAT vasculature. In some examples, the targeting peptide includes CKGGRAKDC, CMLAGWIPC, and CWLGEWLGC, See, e.g., Kolonin et al., Nat. Med. 10:625-632, 2004 and Nie et al., Stem Cells 26:2735-2745, 2008; U.S. Pat. No. 7,951,362; each of which is

incorporated herein by reference. In other examples, an adipose tissue targeting molecule includes an antibody that preferentially binds to adipose tissue (such as WAT), for example an antibody that preferentially binds to resistin. In some examples, a HIF-2a inhibitor is coupled to an adipose tissue targeting molecule (such as a peptide or antibody). Methods of coupling molecules are well known to one of skill in the art. This includes, but is not limited to, covalently bonding one molecule to another molecule (for example, directly or via a linker molecule), noncovalently bonding one molecule to another (e.g. electrostatically bonding) (see, for example, U.S. Patent No. 6,921,496, which discloses methods for electrostatic conjugation), noncovalently bonding one molecule to another molecule by hydrogen bonding, non-covalently bonding one molecule to another molecule by van der Waals forces, and any and all

combinations of such couplings.

[00152] In other embodiments, the HIF-2a inhibitor is targeted to hepatic tissue. Hepatic- specific ligands or antibodies that bind to one or more specific hepatic cell target molecules (e.g., cell surface protein unique to hepatic cells) can be used. Such "targeting moieties" can be coupled directly to the HIF-2a inhibitor or to the pharmaceutical composition or vehicle used to deliver the HIF-2a inhibitor to the body. Such targeting strategies specifically for hepatic cells can be found described in the state of the art, for example, in Mishra et al., "Efficient hepatic delivery of drugs: novel strategies and their significance," BioMed Research International, Vol. 2013 (2013), Article ID 382184, the entire contents of which are incorporated by reference.

[00153] Pharmaceutical Compositions and Administration

[00154] Pharmaceutical compositions that include an inhibitor of HIF-2a can be formulated with an appropriate pharmaceutically acceptable carrier, depending upon the particular mode of administration chosen. In one example, the pharmaceutical composition includes a HIF-2a inhibitor and a pharmaceutically acceptable carrier. In some examples, the pharmaceutical composition consists essentially of an inhibitor of HIF-2a and a pharmaceutically acceptable carrier.

[00155] The pharmaceutically acceptable carriers and excipients useful in this disclosure are conventional. See, e.g., Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21 ^st Edition (2005). For instance, parenteral formulations usually comprise injectable fluids that are pharmaceutically and physiologically acceptable fluid vehicles such as water, physiological saline, other balanced salt solutions, aqueous dextrose, glycerol or the like. For solid

compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non- toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, pH buffering agents, or the like, for example sodium acetate or sorbitan monolaurate. Excipients that can be included are, for instance, other proteins, such as human serum albumin or plasma preparations.

[00156] In some embodiments, the HIF-2a inhibitor is included in a controlled release formulation, for example, a microencapsulated formulation. Various types of biodegradable and biocompatible polymers, methods can be used, and methods of encapsulating a variety of synthetic compounds, proteins and nucleic acids, have been well described in the art (see, for example, U.S. Pat. Publication Nos. 2007/0148074; 2007/0092575; and 2006/0246139; U.S. Patent Nos. 4,522, 811 ; 5,753,234; and 7,081,489; PCT Publication No. WO/2006/052285; Benita, Microencapsulation: Methods and Industrial Applications, 2 ^nd ed., CRC Press, 2006).

[00157] In other examples, the HIF-2a inhibitor is included in a nanodispersion system.

Nanodispersion systems and methods for producing such nanodispersions are well known to one of skill in the art. See, e.g., U.S. Pat. No. 6,780,324; U.S. Pat. Publication No. 2009/0175953. For example, a nanodispersion system includes a biologically active agent and a dispersing agent (such as a polymer, copolymer, or low molecular weight surfactant). Exemplary polymers or copolymers include polyvinylpyrrolidone (PVP), poly(D,L-lactic acid) (PLA), poly(D,L-lactic- co-glycolic acid (PLGA), poly(ethylene glycol). Exemplary low molecular weight surfactants include sodium dodecyl sulfate, hexadecyl pyridinium chloride, polysorbates, sorbitans, poly(oxyethylene) alkyl ethers, poly(oxyethylene) alkyl esters, and combinations thereof. In one example, the nanodispersion system includes PVP and a HIF- Ια inhibitor (such as 80/20 w/w). In some examples, the nanodispersion is prepared using the solvent evaporation method. See, e.g., Kanaze et al., Drug Dev. Indus. Pharm. 36:292-301, 2010; Kanaze et al., J. Appl. Polymer Sci. 102:460-471, 2006.

[00158] In some examples, the HIF-2a inhibitor includes pharmaceutically acceptable salts of such compounds. "Pharmaceutically acceptable salts" of the presently disclosed compounds include those formed from cations such as sodium, potassium, aluminum, calcium, lithium, magnesium, zinc, and from bases such as ammonia, ethylenediamine, N-methyl- glutamine, lysine, arginine, ornithine, choline, Ν,Ν'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine,

tris(hydroxymethyl)aminomethane, and tetramethylammonium hydroxide. These salts may be prepared by standard procedures, for example by reacting the free acid with a suitable organic or inorganic base. Any chemical compound recited in this specification may alternatively be administered as a pharmaceutically acceptable salt thereof. "Pharmaceutically acceptable salts" are also inclusive of the free acid, base, and zwitterionic forms. Description of suitable pharmaceutically acceptable salts can be found in Handbook of Pharmaceutical Salts, Properties, Selection and Use, Wiley VCH (2002).

[00159] In some examples, the pharmaceutical compositions disclosed herein comprise a

HIF-2a inhibitor and at least one pharmaceutically acceptable carrier. In other examples, the composition consists essentially of a HIF-2a inhibitor and at least one pharmaceutically acceptable carrier. In the present disclosure, "consists essentially of indicates that additional active compounds (for example additional inhibitors of HIF-Ια) are not included in the composition, but that other inert agents (such as fillers, wetting agents, or the like) can be included, and "consists of indicates that additional agents are not included in the composition.

[00160] The dosage form of the pharmaceutical composition will be determined by the mode of administration chosen. For instance, in addition to injectable fluids, topical, inhalation, oral and suppository formulations can be employed. Topical preparations can include eye drops, ointments, sprays, patches and the like.

[00161] Inhalation preparations can be liquid (e.g., solutions or suspensions) and include mists, sprays and the like. Oral formulations can be liquid (e.g., syrups, solutions or

suspensions), or solid (e.g., powders, pills, tablets, or capsules). Suppository preparations can also be solid, gel, or in a suspension form. For solid compositions, conventional non-toxic solid carriers can include pharmaceutical grades of mannitol, lactose, cellulose, starch, or magnesium stearate. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art.

[00162] The pharmaceutical compositions that include an inhibitor of HIF-2a can be formulated in unit dosage form, suitable for individual administration of precise dosages. In one specific, non-limiting example, a unit dosage contains from about 1 mg to about 1 g of an HIF- 2a inhibitor (such as about 10 mg to about 100 mg, about 50 mg to about 500 mg, about 100 mg to about 900 mg, about 250 mg to about 750 mg, or about 400 mg to about 600 mg HIF-2a inhibitor). The amount of active compound(s) administered will be dependent on the subject being treated, the severity of the affliction, and the manner of administration, and is best left to the judgment of the prescribing clinician. Within these bounds, the formulation to be

administered will contain a quantity of the active component(s) in amounts effective to achieve the desired effect in the subject being treated.

[00163] The compositions of this disclosure including an inhibitor of HIF-2a can be administered to humans or other animals on whose tissues they are effective in various manners such as orally, intravenously, intramuscularly, intraperitoneally, intranasally, intradermally, intrathecally, subcutaneously, via inhalation or via suppository. In one non-limiting example, the composition is administered orally. In further examples, site-specific administration of the composition can be used, for example by administering a HIF-2a inhibitor to adipose tissue (for example by injection in adipose tissue, such as visceral adipose tissue). The particular mode of administration and the dosage regimen will be selected by the attending clinician, taking into account the particulars of the case (e.g. the subject, the disease, the disease state involved, the particular treatment, and whether the treatment is prophylactic). Treatment can involve daily or multi-daily doses of compound(s) over a period of a few days to months, or even years. In a particular non-limiting example, treatment involves once daily dose of a HIF-2a inhibitor (such as acriflavine).

[00164] In some examples, a therapeutically effective amount of a HIF-2a inhibitor is about 0.01 mg/kg to about 50 mg/kg (for example, about 0.5 mg/kg to about 25 mg/kg or about 1 mg/kg to about 10 mg/kg). In a specific example, a therapeutically effective amount of a HIF-2a inhibitor is about 1 mg/kg to about 5 mg/kg, for example about 2 mg/kg. In a particular example, a therapeutically effective amount of a HIF-2a inhibitor includes about 1 mg/kg to about 10 mg/kg acriflavine, such as about 2 mg/kg acriflavine.

[00165] A therapeutically effective amount of a HIF-2a inhibitor can be the amount of a

HIF-2a inhibitor necessary to treat diabetes or reduce body weight in a subject or treat NAFLD. A therapeutically effective amount of an inhibitor of HIF-2a can be administered in a single dose, or in several doses, for example daily, during a course of treatment. However, the therapeutically effective amount will be dependent on the subject being treated, the severity and type of the affliction, and the manner of administration of the therapeutic(s).

[00166] The present disclosure also includes combinations of a HIF-2a inhibitor with one or more other agents useful in the treatment of diabetes or insulin resistance or in the reduction of body weight or NAFLD. For example, the compounds of this disclosure can be administered in combination with effective doses of anti-diabetic agents (such as biguanides,

thiazolidinediones, or incretins) and/or lipid lowering compounds (such as statins or fibrates). The term "administration in combination" or "co-administration" refers to both concurrent and sequential administration of the active agents. Administration of a HIF-2a inhibitor may also be in combination with lifestyle modifications, such as increased physical activity, low fat diet, and smoking cessation.

[00167] A subject that has diabetes or a subject with insulin resistance (for example, a fasting plasma glucose of > 100 mg/dL) is a candidate for treatment using the therapeutic methods disclosed herein. A subject in need of a reduction in body weight, for example, a subject with overweight or obesity (for example, a subject with a body mass index of 25 kg/m or more) is a candidate for treatment using the therapeutic methods herein.

[00168] Methods of Treating Obesity, NAFLD, NASH, and Insulin Resistance

[00169] In another aspect, the present disclosure provides a method of inhibiting the activities of HIF-2a in a cell, comprising contacting the cell with an effective amount of a compound described herein, e.g., at least one of PT2385, PT2567, PT2399, PT2977 or a combination thereof. In various embodiments, the inhibition of HIF-2a in the intestine results in reduced obesity, insulin resistance, and NAFLD.

[00170] The disclosed methods include administering a therapeutically effective amount of an inhibitor of HIF-2a to a subject. HIF-2a inhibitors include compounds that decrease the expression, longevity (e.g., half-life) or activity of HIF-2a (directly or indirectly), for example, relative to a control. Direct inhibition can be accomplished, for example, by binding to a HIF-2a protein and thereby preventing the protein from binding an intended target, such as a

dimerization partner or a DNA sequence. Indirect inhibition can be accomplished, for example, by binding to a HIF-2a protein intended target, such as a receptor or binding partner, thereby blocking or reducing activity of the protein. Furthermore, an inhibitor of the disclosure can inhibit a HIF— 2a gene by reducing or inhibiting expression of the gene, inter alia by interfering with gene expression (transcription, processing, translation, post-translational modification, or stability), for example, by interfering with the mRNA and blocking translation of the HIF-2a gene product or by altering post-translational modification of a HIF-2a gene product (such as prolyl hydroxylation), or by causing changes in intracellular localization.

[00171] Disclosed herein are methods for reducing obesity or decreasing body weight of a subject (for example an overweight or obese subject) utilizing an inhibitor of HIF-2a. Also disclosed are methods for treating insulin resistance and/or diabetes in a subject (for example, type 2 diabetes) utilizing an inhibitor of HIF-2a. In some examples, the methods include administering a therapeutically effective amount of a composition including a HIF-2a inhibitor to a subject, thereby decreasing body weight of the subject. In other examples, the methods include administering a therapeutically effective amount of a composition including a HIF-2a inhibitor to a subject having diabetes or insulin resistance, thereby treating diabetes or insulin resistance in the subject. In certain embodiments, the administration of the HIF-2a inhibitor is targeted to the intestinal tissue, e.g., vis-a-vis a target moiety specific for a intestinal cell marker and wherein the targeting moiety is complexed or coupled directly to the HIF-2a inhibitor or to the composition or vehicle that is employed to deliver the HIF-2a inhibitor.

[00172] In some embodiments, the disclosure includes decreasing body weight of a subject or reducing obesity by administering a therapeutically effective amount of a composition including an inhibitor of HIF-2a to a subject. In some examples, the method includes selecting a subject in need of decreasing body weight (such as an overweight or obese subject). A subject may be considered overweight or obese if their BMI is greater than 25 kg/m2, their waist circumference is greater than 35 inches (female) or 40 inches (male) or body fat percentage is greater than 25% (male) or 32% (female). In some examples, decreasing body weight includes one or more of decreasing total body weight, decreasing BMI, decreasing waist circumference, and decreasing body fat (such as total body fat, subcutaneous body fat, or visceral body fat). In some embodiments, the disclosed methods include measuring total body weight, BMI, waist circumference, and/or body fat amount in a subject.

[00173] In some examples, decreasing body weight of a subject includes reducing total body weight of the subject by at least about 1% (such as at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, or more). In particular examples, reduction in total body weight is determined relative to the starting total body weight of the subject (for example, prior to treatment with a HIF-2a inhibitor).

[00174] In other examples, decreasing body weight of a subject includes decreasing BMI of the subject by at least about 1% (such as at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, or more). BMI is calculated by dividing weight (in kg) by height 2 (in meters 2). The current standards for both men and women accepted as "normal" are a BMI of 20- 24.9 kg/m". In one embodiment, a BMI of greater than 25 kg/m can be used to identify an obese subject. Grade I obesity (also called "overweight") corresponds to a BMI of 25-29.9 kg/m .

Grade II obesity corresponds to a BMI of 30-40 kg/m ; and Grade III obesity corresponds to a BMI greater than 40 kg/m . In particular examples, reduction in BMI is determined relative to the starting BMI of the subject (for example, prior to treatment with a HIF-2a inhibitor). In other examples, decreasing BMI of a subject includes reduction of BMI from a starting point (for example BMI greater than 30 kg/m ) to a target level (for example, BMI less than 30 kg/m , 29 kg/m ² , 28 kg/m ² , 27 kg/m ² , 26 kg/m ² , or 25 kg/m ² ).

[00175] In further examples, decreasing body weight of a subject includes decreasing waist circumference by at least 1% (such as at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, or more). In particular examples, reduction in waist circumference is determined relative to the starting waist circumference of the subject (for example, prior to treatment with a HIF-2a inhibitor). In other examples, decreasing waist circumference of a subject includes reduction of waist circumference from a starting point (for example greater than 40 inches for men or greater than 35 inches for women) to a target level (for example, waist circumference less than 40 inches for men or less than 35 inches for women).

[00176] In additional examples, decreasing body weight of a subject includes decreasing body fat (such as total body fat, subcutaneous body fat, or visceral body fat) of the subject by at least 1% (such as at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, or more). Methods of determining body fat (such as body fat percentage) are known to one of skill in the art. Such methods include near-infrared interactance, dual energy X-ray absorptiometry, body average density measurement, bioelectrical impedance analysis, skinfold tests (for example, Durnin-Womersley skinfold method or Jackson-Pollock skinfold method), and U.S. Navy circumference method. In particular examples, reduction in body fat is determined relative to the starting body fat of the subject (for example, prior to treatment with a HIF-2a inhibitor). In other examples, decreasing body fat of a subject includes reduction of body fat from a starting point (for example greater than about 25% body fat for men or greater than about 32% body fat for women) to a target level (for example, body fat of less than about 25% for men or less than about 32% for women). In some examples, a target body fat level may be about 14-24% body fat for men or about 21-31% body fat for women.

[00177] In some embodiments, the disclosure includes treating diabetes or reducing insulin resistance in a subject by administering a therapeutically effective amount of a composition including an inhibitor of HIF-2a to the subject. In some examples, the method includes selecting a subject with diabetes or at risk for diabetes (such as a subject with prediabetes or shows insulin resistance). In some examples, a subject with diabetes may be clinically diagnosed by a fasting plasma glucose (FPG) concentration of greater than or equal to 7.0 mmol/L (126 mg/dL), or a plasma glucose concentration of greater than or equal to 11.1 mmol/L (200 mg/dL) at about two hours after an oral glucose tolerance test (OGTT) with a 75 g load, or in a patient with classic symptoms of hyperglycemia or hyperglycemic crisis, a random plasma glucose concentration of greater than or equal to 11.1 mmol/L (200 mg/dL), or HbAlc levels of greater than or equal to 6.5%. In other examples, a subject with pre-diabetes may be diagnosed by impaired glucose tolerance (IGT). An OGTT two-hour plasma glucose of greater than or equal to 140 mg/dL and less than 200 mg/dL (7.8-11.0 mM), or a fasting plasma glucose (FPG) concentration of greater than or equal to 100 mg/dL and less than 125 mg/dL (5.6-6.9 mmol/L), or HbAlc levels of greater than or equal to 5.7% and less than 6.4% (5.7-6.4%) is considered to be IGT, and indicates that a subject has pre-diabetes. A more detailed description of diabetes may be found in Standards of Medical Care in Diabetes— 2010 (American Diabetes Association, Diabetes Care 33:S 11-61, 2010). In some examples, treating diabetes includes one or more of increasing glucose tolerance, decreasing insulin resistance (for example, decreasing plasma glucose levels, decreasing plasma insulin levels, or a combination thereof), decreasing serum triglycerides, decreasing free fatty acid levels, and decreasing HbAlc levels in the subject. In some embodiments, the disclosed methods include measuring glucose tolerance, insulin resistance, plasma glucose levels, plasma insulin levels, serum triglycerides, free fatty acids, and/or HbAlc levels in a subject.

[00178] In some examples, administration of a HIF-2a inhibitor treats diabetes by increasing glucose tolerance, for example, by decreasing blood glucose levels (such as two-hour plasma glucose in an OGTT or FPG) in a subject. In some examples, the method includes decreasing blood glucose by at least 5% (such as at least 10%, 15%, 20%, 25%, 30%, 35%, or more) as compared with a control. In particular examples, a decrease in blood glucose level is determined relative to the starting blood glucose level of the subject (for example, prior to treatment with a HIF-2a inhibitor). In other examples, decreasing blood glucose levels of a subject includes reduction of blood glucose from a starting point (for example greater than about 126 mg/dL FPG or greater than about 200 mg/dL OGTT two-hour plasma glucose) to a target level (for example, FPG of less than 126 mg/dL or OGTT two-hour plasma glucose of less than 200 mg/dL). In some examples, a target FPG may be less than 100 mg/dL. In other examples, a target OGTT two-hour plasma glucose may be less than 140 mg/dL. Methods to measure blood glucose levels in a subject (for example, in a blood sample from a subject) are routine.

[00179] In some embodiments, the disclosed methods include treating a subject with diabetes by decreasing insulin resistance in the subject. In some examples, a subject with insulin resistance is a subject with diabetes, while in other examples, a subject with insulin resistance does not have diabetes, but may, for example, be pre-diabetic. Insulin resistance is a decreased sensitivity or responsiveness to the metabolic actions of insulin. In some examples, insulin resistance results in increased blood glucose and/or increased blood insulin levels (such as fasting blood glucose or fasting blood insulin levels).

[00180] In some examples, insulin resistance is determined by hyperinsulinemic euglycemic clamp (glucose clamp), which measures the amount of glucose necessary to compensate for increased insulin levels without causing hypoglycemia (see, e.g., DeFronzo et al., Am. J. Physiol. 237:E214-E223, 1979). In one example, the glucose clamp method includes infusing insulin in a subject at 10-120 mU/m /min and infusing 20% glucose to maintain blood glucose levels between about 90-100 mg/dL. If low levels of glucose (such as <4 mg/min) are required to maintain blood glucose levels, then the subject is considered insulin resistant. High levels of glucose (such as>7.5 mg/min) indicate that the subject is insulin sensitive, while between 4-7.5 mg/min of glucose is considered to indicate impaired glucose tolerance (IGT), which is an early sign of insulin resistance.

[00181] In some examples of the disclosed method, administration of an inhibitor of HIFla decreases insulin resistance by increasing the amount of glucose required to maintain blood glucose levels in a glucose clamp in a subject, for example, by at least 5% (such as at least 10%, 15%, 20%, 25%, 30%, 35%, or more) as compared with a control. In some examples, the method includes increasing the amount of glucose required to maintain blood glucose levels in a glucose clamp to >4 mg/min glucose. In other examples, the method includes increasing the amount of glucose required to maintain blood glucose levels in a glucose clamp to>7.5 mg/min glucose.

[00182] In another example, insulin resistance is determined by the frequently sampled intravenous glucose tolerance test (FSIVGTT; Bergman, Diabetes 38: 1512-1527, 1989).

FSIVGTT is performed by administering intravenous glucose with frequent blood sampling to determine glucose and insulin levels. Insulin is injected 20 minutes after the start of glucose administration. The insulin sensitivity index (SI), reflecting increase in fractional glucose disappearance per unit of insulin increase, is calculated. In some examples, an SI value of <2 μυ/min/mL indicates insulin resistance. In some examples of the disclosed method,

administration of a HIF-2a inhibitor decreases insulin resistance by increasing the insulin sensitivity index of a subject, for example, by at least 5% (such as at least 10%, 15%, 20%, 25%, 30%, 35%, or more) as compared with a control (such as the subject prior to administration of the HIF-2a inhibitor). In some examples, the method includes increasing the insulin sensitivity index to >2 μυ/min/mL.

[00183] In other examples, insulin resistance is determined by QUICKI (Katz et al., J.

Clin. Endocrinol. Metab. 85:2402-2410, 2000). QUICKI is calculated from fasting glucose and fasting insulin levels: QUICKI = l/[(log(Io) + (log(Go)] wherein Io is the fasting plasma insulin level (μυ/mL) and Go is the fasting blood glucose level (mg/dL). In some examples of the disclosed method, administration of an inhibitor of HIF-Ια decreases insulin resistance by increasing the QUICKI value in a subject by at least 5% (such as at least 10%, 15%, 20%, 25%, 30%, 35%, or more) as compared with a control (such as the subject prior to administration of the HIFla inhibitor). In some examples, the method includes increasing the subject's QUICKI to >0.350. [00184] In other examples, insulin resistance is determined by the homeostasis model assessment (HOMA-IR; Matthews et al., Diabetologia 28:412-429, 1985). HOMA-IR is calculated from fasting glucose and fasting insulin levels: HOMA-IR = [fasting plasma insulin x fasting plasma glucose]/22.5 wherein fasting plasma insulin is expressed as μ Ι/mL and fasting plasma glucose is expressed as mM. In some examples of the disclosed method, administration of an inhibitor of HIF-2a decreases insulin resistance by decreasing the HOMA-IR value in a subject by at least 5% (such as at least 10%, 15%, 20%, 25%, 30%, 35%, or more) as compared with a control (such as the subject prior to administration of the HIF-2a inhibitor). In some examples, the method includes decreasing HOMA-IR to <4.

[00185] In additional examples, administration of an inhibitor of HIF-2a decreases insulin resistance by decreasing plasma insulin levels (such as fasting plasma insulin or 2-hour insulin levels following OGTT) in a subject, for example, decreasing plasma insulin levels by at least 5% (such as at least 10%, 15%, 20%, 25%, 30%, 35%, or more) as compared with a control (such as the subject prior to administration of the HIF-2a inhibitor). In some examples, the method includes decreasing fasting plasma insulin levels to <15 μυ/mL. Methods to measure plasma insulin in a subject (for example, in a blood sample from a subject), such as

immunoassays, are routine.

[00186] One of skill in the art will recognize that because of a lack of standardized assays, interassay variability in insulin measurements can confound defining universal ranges for insulin resistance and insulin sensitivity. Therefore, in some examples, insulin sensitive subjects include the top 25 ^th percentile of insulin sensitive subjects in a given cohort where insulin levels are measured in the same central reference laboratory. Similarly, in some examples, insulin resistant subjects include the bottom 25 ^th percentile of insulin sensitive subjects in a given cohort where insulin levels are measured in the same central reference laboratory. In additional examples, impaired glucose tolerance can be defined according the results of an oral glucose tolerance test using guidelines that are published by the American Diabetes Association. See, e.g., Diabetes Care 33:S62-S69, 2010.

[00187] In some embodiments, the disclosed methods include treating a subject with diabetes by decreasing triglyceride or free fatty acid levels in the subject. In some examples, administration of an inhibitor of HIF-2a treats diabetes by decreasing blood triglyceride levels in a subject, for example decreasing triglyceride levels by at least 5% (such as at least 10%, 15%, 20%, 25%, 30%, 35%, or more) as compared with a control (such as the subject prior to administration of the HIF-2a inhibitor). In some examples, the method includes decreasing triglyceride levels in a subject to < 150 mg/dL. Methods of determining triglyceride levels in a subject (for example in a blood sample from a subject) are routine.

[00188] In further examples, administration of a HIF-2a inhibitor treats diabetes by decreasing blood free fatty acid levels in a subject, for example, decreasing free fatty acid levels by at least 5% (such as at least 10%, 15%, 20%, 25%, 30%, 35%, or more) as compared with a control (such as the subject prior to administration of the a inhibitor). In some examples, the method includes decreasing free fatty acid levels below 0.6 mmol/L (such as about 0.1-0.6 mmol/L). Methods of determining free fatty acid levels in a subject (for example, in a blood sample from a subject) are routine.

[00189] In some embodiments, the disclosed methods include comparing one or more indicators of body weight or obesity (such as body weight, body mass index, waist

circumference, or body fat) to a control, wherein a decrease in the particular indicator relative to the control indicates effective reduction of body weight or treatment of obesity.

[00190] The control can be any suitable control against which to compare the indicator of body weight or obesity in a subject. In some examples, the control is a historical control or standard reference value or range of values (such as a previously tested control sample, such as a group of subjects which are overweight or obese, or group of samples from subjects which are not overweight or obese). In further examples, the control is a reference value, such as a standard value obtained from a population of individuals that is used by those of skill in the art. Similar to a control population, the value of the sample from the subject can be compared to the mean reference value or to a range of reference values (such as the high and low values in the reference group or the 95% confidence interval). In other examples, the control is the subject (or group of subjects) treated with placebo compared to the same subject (or group of subjects) treated with the therapeutic compound in a cross-over study. In further examples, the control is the subject (or group of subjects) prior to treatment with the HIF-2a inhibitor.

[00191] In other embodiments, the disclosed methods include comparing one or more indicator of diabetes to a control, wherein an increase or decrease in the particular indicator relative to the control (as discussed above) indicates effective treatment of diabetes. In particular examples, the disclosed methods include comparing one or more indicator of insulin resistance (such as blood glucose levels, blood insulin levels, insulin sensitivity index, HOMA-IR, or QUICKI) to a control, wherein a decrease in the particular indicator relative to the control (as discussed above) indicates effective treatment of insulin resistance. In further embodiments, the disclosed methods include comparing adiponectin levels to a control, wherein an increase in adiponectin levels (such as total adiponectin, HMW adiponectin, and/or the ratio of HMW to total adiponectin) indicates an effective treatment of diabetes or obesity.

[00192] The control can be any suitable control against which to compare the indicator of diabetes in a subject. In some embodiments, the control is a sample obtained from a healthy subject (such as a subject without diabetes). In some embodiments, the control is a historical control or standard reference value or range of values (such as a previously tested control sample, such as a group of subjects with diabetes, or group of samples from subjects that do not have diabetes). In further examples, the control is a reference value, such as a standard value obtained from a population of normal individuals that is used by those of skill in the art. Similar to a control population, the value of the sample from the subject can be compared to the mean reference value or to a range of reference values (such as the high and low values in the reference group or the 95% confidence interval). In other examples, the control is the subject (or group of subjects) treated with placebo compared to the same subject (or group of subjects) treated with the therapeutic compound in a cross-over study. In further examples, the control is the subject (or group of subjects) prior to treatment with the HIF-2a inhibitor.

[00193] In another aspect, the present disclosure provides a kit comprising a

pharmaceutical composition comprising a compound described herein and a pharmaceutically acceptable carrier or excipient and an instruction for using the composition to treat a subject to reduce obesity, insulin resistance, NAFLD or NASH.

[00194] The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the invention to the particular features or embodiments described.

[00195] EXAMPLES

[00196] Example 1; Treatment of non-alcoholic fatty liver disease

[00197] Non-alcoholic fatty liver disease (NAFLD) is becoming the most common chronic liver disease in western countries with limited therapeutic options. In addition, related metabolic conditions that include obesity, insulin resistance, type 2 diabetes, and non-alcoholic steatohepatitis are also major health concerns throughout the world. This Example demonstrates a new role for intestinal hypoxia-inducible factor (HIF) not only in NAFLD, but also in obesity, insulin resistance, type 2 diabetes, and non-alcoholic steatohepatitis.

[00198] The Example shows in particular that human intestine biopsies from patients with or without obesity revealed a relationship between activated HIF2a but not HIFla in increased body mass index and hepatic toxicity. The causality of this correlation was verified in mice with an intestine-specific H 2<2-disruption, in which high-fat diet-induced hepatic steatosis and obesity were substantially decreased. PT2385, a HIF2a-specific inhibitor, had preventive and therapeutic effects on metabolic disorders dependent on intestine HIF2a. Intestine HIF2a inhibition markedly reduced intestine and serum ceramide levels. Mechanistically, intestine HIF2a regulates ceramide metabolism mainly from the salvage pathway, which was revealed by the identification of the novel HIF2a target gene encoding neuraminidase 3. These results suggest that intestine HIF2a would be viable target for NAFLD therapy. These results also suggest intestine HIF2a inhibition may be a viable treatment target for related conditions including obesity, insulin resistance, type 2 diabetes, and non-alcoholic steatohepatitis.

[00199] Non-alcoholic fatty liver disease (NAFLD), characterized by the accumulation of ectopic triglycerides in the liver without excess alcohol consumption, is becoming the most common chronic liver disease in industrialized countries ¹ . Persistent NAFLD triggers the increased risk of non-alcoholic steatohepatitis (NASH) and end stage liver diseases such as cirrhosis and hepatocellular carcinoma . Obesity is a well-recognized risk factor for NAFLD. Pharmacologic therapy that targets NAFLD remains extremely limited .

[00200] Accumulating reports indicate that hypoxia- inducible factors (HIFs), members of the basic helix -hoop-helix Per-Arnt-Sim (bHLH-PAS) transcription factor family, exert a pivotal role during the pathogenesis of NAFLD ⁴ . HIF is a heterodimer of an oxygen-sensitive a subunit and a constitutively expressed β subunit (HIF-1□ or ARNT) ⁵ ' ⁶ . Under normoxic conditions, HIF- α (HIF- la and HIF-2a) is rapidly hydroxylated and degraded by several prolyl hydroxylase domain enzymes (PHD) followed by conjugation with the von Hippel-Lindau (VHL) E3 ubiquitin ligase complex. Conversely, the HIF proteins are stabilized during hypoxia due to inhibition of PHD activity induced by low 0 ₂ 7-9. Hepatocyte-specific disruption of PHD2 and PHD3 or VHL, which lead to overexpression of both HIF- la and HIF-2a, was demonstrated to promote hepatic steatosis ^10"12 . Hepatic HIF-2a but not HIF- la was further identified as a major regulator of hepatic lipid metabolism through the up-regulation of genes involved in fatty acid synthesis (Srebplc and Fasn) and fatty acid uptake (Cd36) and the down-regulation of genes involved in regulating fatty acid β-oxidation (Ppara and Acoxl) 13.

[00201] Most studies on the relationship between HIF and NAFLD focused on evaluating the effects of liver HIF. However, liver HIF-2a activation was recently observed to ameliorate hyperglycemia through the insulin-dependent pathway with increased insulin receptor substrate- 2 (Irs2), or the insulin-independent pathway with the repression of glucagon action ^14"16 . These studies implied that pharmacological inhibition of liver HIF-2a would not be suitable to exploit for NAFLD therapy, due to the increased risk of increased hepatic glucose production and type 2 diabetes. Several novel targets in the intestine were recently implicated in the development of NAFLD ^17"19 . While both HIF- la and HIF-2a are expressed in the intestinal epithelial cells, the role of the intestine HIFa on the pathogenesis of NAFLD and other metabolic diseases is poorly understood.

[00202] Herein, the intestine- specific knockout or activation of HIF-2a and metabolomics profiling analysis were adopted to clarify the role and dissect the precise mechanism of intestine HIF-2a in NAFLD development. The study revealed that intestine HIF-2a but not HIF- la signaling was activated during obesity. Intestine- specific HIF-2a ablation substantially ameliorated HFD-induced obesity and hepatic steatosis. The amelioration of adverse metabolic phenotypes was correlated with alterations in ceramide metabolism. NEU3, encoding a key enzyme in the ceramide salvage pathway, was identified as a novel target gene of HIF-2a. It was elucidated that the HIF-2a-NEU3 -ceramide axis has a role in NAFLD development. Importantly, a specific HIF-2a inhibitor PT2385, which is in clinical trials for the treatment of renal cancer, was found to prevent and reverse metabolic disorders through the inhibition of intestinal HIF-2a. This work suggests that intestine HIF-2a is a novel target for the treatment of NAFLD, obesity and insulin resistance, as well as related conditions such as possibly type 2 diabetes and nonalcoholic steatohepatitis.

Results

Intestinal HIF-2a signaling is activated in humans with obesity.

[00203] To investigate the potential association between HIF-2a signaling and obesity, we assessed HIF-2a expression in distal ileum biopsies from nonobese individuals and individuals with obesity by immunohistochemical staining (FIG. la) and western blot analysis (FIG. lb), revealing notably higher HIF-2a expression in humans with obesity relative to nonobese controls. The mRNA levels of DMT 1 and DCYTB (officially known as CYBRD1), two genes whose transcription is targeted by HIF-2a, were also markedly upregulated in humans with obesity (FIG. lc).

[00204] By contrast, no change was noted in HIF-2a protein expression or in the mRNA levels of its target gene PDK1 (FIG. la-c). Ileum DMT1 and DCYTB mRNA levels were positively correlated with body-mass index (BMI), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) activities (FIG. Id and FIG. 7a). However, no correlation was observed for PDK1 mRNA level with any of the parameters. The human data indicated the presence of hypoxia and the activation of HIF-2a signaling in the intestine of individuals with obesity.

[00205] To test the hypothesis that the small intestines of mice fed a HFD were hypoxic, we employed the HIF-a oxygen-dependent degradation domain (ODD)-luc mice, expressing the C-terminal portion of the ODD fused to a firefly luciferase (luc) gene ¹⁶ . Under normoxic conditions, the ODD is hydroxylated, which results in ubiquitination and proteasomal degradation, whereas hypoxic stress inhibits hydroxylation, leading to the accumulation of luciferase in the hypoxic tissues.

[00206] As compared to the chow-diet-treated mice, there was robust activation of the luciferase signal in the small intestines from the HFD-treated group (Fig. le), which suggests that a HFD might trigger a hypoxia response in the small intestine. Consistent with the activation of HIF-2a signaling observed in humans with obesity, a rapid and selective induction in HIF-2a expression was demonstrated as early as 1 week following HFD treatment (FIG. 7b). A longer, 8-week treatment resulted in an augmentation in the small intestine of HIF-2a protein levels and mRNA levels encoded by the HIF-2a target genes Dmtl, Dcytb, Epo, and Fpn as compared to chowfed control mice (FIG. If).

[00207] Intestine-specific HIF-2a disruption attenuated steatosis

[00208] To understand the importance of intestinal HIF-2a in the development and progression of metabolic disorders and NAFLD, we treated control mice {Ηίβα^) and intestine- specific Ηίβα-mxM (Hif2a ^Am ) mice with a HFD for 12 weeks. Compared to the Ηίβα^ mice,

Hif2a ΔΙΕ mice exhibited less body-weight gain with HFD feeding (FIG. 8a). A glucose-tolerance test (GTT) and insulin-tolerance test (ITT) revealed that abrogation of intestinal HIF-2a substantially improved insulin sensitivity (FIG. 8b,c). Furthermore, H&E and Oil Red O staining showed a reduction in hepatic lipid droplets in Hif2a ^Am mice (FIG. 2a). Hif2a ^Am mice displayed significantly lower liver weights and liver weight-to-body weight ratios relative to controls (FIG. 2b,c). Hepatic triglyceride levels, hepatic and serum cholesterol levels, and serum ALT levels reflecting hepatic lipotoxicity were markedly lower in Hif2a ΔΙΕ mice, with no significant difference in serum triglyceride levels as compared to the Ηίβα^ mice (FIG. 2d-h).

[00209] Furthermore, the mRNA expression of genes involved in fatty acid transport and anabolism (Srebplc, Cidea, Cd36, Fabpl, Fabp4, Fasn, Scdl, and Elovl6), triglyceride synthesis

(Dgatl and Dgat2), and the lipid-droplet coat (Plin2) were all substantially reduced in Hif2a ΔΙΕ mice as compared to controls (FIG. 2i). By contrast, the mRNA expression of genes involved in fatty acid β-oxidation, such as Acox2, Acsll, and Acaala, were moderately elevated in Hif2a ΔΙΕ mice as compared to the control mice (FIG. 2j). Although a 12- week-period of HFD feeding did not result in obvious inflammatory cell infiltration by microscopic examination, the expression levels of inflammatory cytokines and chemokines, such as Tnfa, Pail, Ccl2, and Ccl3, were significantly lower in Hif2a ΔΙΕ mice than in the controls (FIG. 2k). Disruption of Hi 2 a was restricted to the intestines of Hif2a ΔΙΕ mice, as revealed by lower Hif2a mRNA expression in the intestine, with no changes in expression in other tissues, relative to Ηίβα^ mice (FIG. 8d). Western blot analysis further showed that the disruption of intestinal HIF-2a did not affect HIF- la or HIF-2a protein levels in the livers of the mice fed a HFD (FIG. 8e). In addition, intestinal HIF-2a disruption did not affect body- weight gain nor hepatic steatosis of mice fed a chow diet, even though there was no evident steatosis in chow-fed mice (FIG. 8f-n).

[00210] Intestinal HIF-2a deficiency lowers ceramide levels

[00211] To further decipher the underlying mechanism by which intestinal HIF-2a affects hepatic lipid homeostasis, we employed lipidomics to analyze the metabolites in the small intestines of the Ηίβα^ and Hif2a ^Am mice fed a HFD. Multivariate analysis distinguished different metabolic profiles between the Hif2a ΔΙΕ and control mice (FIG. 3a). The ions leading to the separation of the Ηίβα^ and Hif2a ^Am mice were identified as ceramides, such as C16:0 (Ml, m/z 582.5103); C18: l (M2, m/z 608.5250); C18:0 (M3, m/z 610.5403); C20:0 (M4, mJz 638.5717); C22:0 (M5, m/z 666.6019); and C24: l (M6, m/z 692.6186) (FIG. 3b). The levels of ceramides, especially the most abundant, C16:0 ceramide, were substantially lower in the small intestines of Hif2a ΔΙΕ mice than in those of the controls (FIG. 3c). Similar to what was observed in the small intestine, serum ceramides were also lower in Hif2a mice than in Hif2a mice (FIG. 3d and FIG. 9a,b).

[00212] Ceramides are synthesized through three different pathways (FIG. 3e): a de novo pathway from palmitoyl-CoA and serine, a sphingomyelinase (SMase) pathway generated by the hydrolysis of sphingomyelin and the salvage pathway generated from the catabolism of complex sphingolipids, such as from ganglioside monosialo 3 (GM3) hydrolysis 17, 18. Consequently, sphingomyelins and glucosylceramides, the two sources for ceramide synthesis from SMase and salvage pathways, were also evaluated. There was no notable change in the relative levels of sphingomyelins in the small intestine, and only a modest reduction in serum (FIG. 3f and FIG. 9c), whereas a marked change was detected in the relative levels of glucosylceramides in Hif2a ^m mice (FIG. 3g and FIG. 9d).

[00213] It is well established that activation of the hepatic diacylglycerol- PKC-ε pathway triggers hepatic steatosis and insulin resistancel9. Hepatic acylcarnitine and diacylglyceride contents and PKC-ε translocation remained unaltered in the livers of Hif2a ΔΙΕ mice as compared to in those of Ηίβα^ mice (C.X. and Y.L., unpublished data). The changes in ceramide metabolism in response to the inhibition of intestinal HIF-2a were due neither to altered lipid absorption, as revealed by measurements of intestine and fecal lipids by both lipid-assay kits and 1H-NMR, nor to morphological changes of the intestine, as examined histologically (C.X., Y.T. and A.D.P. unpublished data). Along with diminished HIF-2a signaling in the small intestine (FIG. 9e), many mRNAs encoded by ceramide-synthesis-related genes, including Degs2 in the de novo pathway, Smpdl, Smpd3, Smpd4, and Enpp7 in the SMase pathway and Neu3, Glbl, and

Gba2 in the salvage pathway, were significantly downregulated in Hif2a ΔΙΕ mice as compared to in Hif2a ^wa mice (FIG. 9f,g and FIG. 3h). The mRNAs encoded by genes involved in ceramide catabolism were at similar levels in Hif2a ΔΙΕ mice and in controls (FIG. 9h).

[00214] Decreased steatosis is independent of adiposity to further exclude a decrease in adiposity as a causal factor for the observed beneficial metabolic effects after intestinal- specific

HIF-2a disruption, Ηϊρ,αΆΙΆ and Hif2a ΔΙΕ mice were treated with a HFD for a short duration of 1 week that did not lead to a notable alteration of body weight (FIG. 10a). A GTT and an ΓΤΤ revealed an improvement in glucose intolerance and insulin resistance in HFD-fed Hif2a ΔΙΕ mice as compared to those of Hif2a ^wa mice (FIG. 10b,c). The energy expenditure was substantially enhanced in Hif2a ΔΙΕ mice, without significant changes in cumulative food intake and ambulatory activity (FIG. lOd-h). Hif2a mice exhibited lower hepatic triglyceride levels and trended toward a reduction in serum ALT levels without significant changes in liver weights and liver weight-to-body weight ratios, hepatic and serum cholesterol levels, and serum triglyceride levels, as compared to Ηίβα^ mice (FIG. lOi-o). The expression of hepatic Srebplc, Cidea,

Cd36, Acly, Acaca, Fasn, Scdl, Elovl6, Dgatl, Dgat2, and Tnfa were lower in the Hif2a ΔΙΕ mice relative to the controls (FIG. ΙΟρ-r). Accompanied by the restrained HIF-2a signaling in the small intestine, the Neu3, Glbl Smpdl, Smpd3, Enpp7, and Degs2 mRNAs were substantially lower in the small intestine of the Hif2a ΔΙΕ mice as compared to floxed control mice (FIG. 11a- d), but were unchanged in the liver white adipose tissue (WAT) of Hif2a ΔΙΕ mice (C.X., unpublished data).

[00215] Lipidomics analysis confirmed that small intestinal, portal and systematic ceramides were markedly diminished in Hif2a ΔΙΕ mice (FIG. 1 le-g). It should be noted that the portal ceramides (which are mainly derived from the intestine) were lower to a greater degree than that of systematic ceramides (31% lower relative to 19% lower) in Hif2a ΔΙΕ mice as compared to the floxed controls, which suggested that serum ceramide changes resulted mainly from altered ceramide synthesis in the intestine.

[00216] To explore the mechanism by which the intestinal HIF-2a -ceramide pathway affected energy expenditure, the browning-related genes of different adipose tissues were examined. As compared to the Ηίβα^ mice, an induction of mRNAs encoded by uncoupling protein 1 (Ucpl) and other key thermogenic genes was found in subcutaneous WAT (scWAT) from Hif2a ΔΙΕ mice without activation of thermogenic genes in brown adipose tissue (BAT) and visceral epididymal WAT (eWAT) (FIG. 1 lh-j). Western blot analysis further confirmed the upregulation of UCP1 in scWAT of Hif2a ΔΙΕ mice (FIG. 1 lk), and immunohistochemical analysis showed an increased number of UCP1 -positive beige adipocytes in the scWAT of Hif2a ^Am mice (FIG. 111).

[00217] HIF-2a modulates ceramide synthesis through targeting Neu3

[00218] Genetic models were used to investigate whether intestinal HIF-2a activation regulated ceramide metabolism. Mice with an intestinespecific disruption of Vhl (V7z/DIE) had robust activation of both HIF-Ια and HIF-2a signaling, whereas mice lacking both VHL and HIF-la (Vhl/Hifl a ^Am ) in the intestine induced only functional HIF-2a activation ²⁰ . Consistently, the HIF-2a signaling was markedly activated in the small intestine of Vhl/ Hifla ΔΙΕ mice relative to Vhl/HiflafUf , as revealed by measurements of the HIF-2a target genes Dmtl and Dcytb mRNA expression (FIG. 12a). A similar induction of the mRNA levels of Degs2, Smpdl,

Smpd3, Smpd4, Enpp7, Neu3, Glbl, and Gba2 was observed in Vhl/ Hifla ΔΙΕ mice (FIG. 12b-d). Neu3 mRNA expression in the small intestine was shown to be the most robust (ten-fold) induced among the genes involved in ceramide synthesis as a result of intestine HIF-2a activation (FIG. 12d). These alterations were completely blocked by the HIF-2a antagonist PT2385 (ref. 21).

[00219] Induction of Neu3 was observed in both single-mutant Vhl ΔΙΕ and double-mutant

Vhl/ Hifla ΔΙΕ mice, but not in Vhl/ Hif2a ΔΙΕ mice (FIG. 12e-j). Furthermore, the expression of NEU3 mRNA was notably greater in the ileum biopsies of humans with obesity relative to individuals without obesity and was positively correlated with BMI, ALT, AST, DMT1 mRNA, and DCYTB mRNA expression (FIG. 4a-f). An upregulation of NEU3 expression was also observed in the small intestines of HFD-treated mice as compared to chowfed mice (FIG. 4g). Lactosylceramide is the product of NEU3 and a substrate for GLB 1 in the salvage pathway. Consistent with the geneexpression data, the relative abundance of lactosylceramide C16:0, the predominant lactosylceramide in intestine, was also markedly lower in the small intestine of Hif2a ^Am mice as compared to in those of Hif2a ^wa mice (FIG. 12k), indicating that NEU3 activity was suppressed in Hif2a ΔΙΕ mice. Western blot analysis also confirmed a reduction of NEU3 expression in the small intestine by selective HIF-2a ablation (FIG. 4h).

[00220] There are two putative HIF-response elements (HRE) in the promoter of Neu3

(FIG. 4i), which was analyzed by transient transfection using a Neu3 promoter luciferase reporter construct. In the intestine derived HCT116 cells, the hypoxia mimic CoC12 or co-transfection with a constitutively active HIF-2a triple mutant (TM) expression plasmid markedly induced the luciferase activity (FIG. 4j). The HIF-2a TM induction of luciferase expression was further potentiated in cells incubated with CoC12. HRE1 (AHREl) or HRE2 (AHRE2) single-deletion constructs did not change luciferase activity, whereas the activity in both HRE (AHRE) deletion constructs was markedly suppressed. These results demonstrated that HIF-2a directly regulated Neu3 expression, and the expression was activated with either one or both HREs. Chromatin immunoprecipitation (ChIP) assays were then performed on cross-linked soluble chromatin isolated from the small intestines of Vhf^ or Vhl ^Am mice. Primers flanking both HREs specifically amplified the DNA sequence immunoprecipitated by the HIF-2a antibody in Vhl ΔΙΕ mice whereas no amplification was noted in controls (FIG. 4k), demonstrating that HIF-2a is able to bind the Neu3 HREs in vivo. Increased HIF-2a binding to the HREs in the Neu3 promoter from the small intestine was also found in HFD-fed mice relative to controls (T.Y., unpublished data).

[00221] Intestine-derived ceramides control hepatic steatosis

[00222] To more definitively establish the connection between intestine ceramide metabolism with hepatic steatosis, Neu3 expression and ceramide production were investigated in HCT116 cells treated with the HIF-2a inhibitor PT2385, the NEU3 inhibitor 2,3-didehydro- N-acetyl-neuraminic acid (DANA)22 and siNEU3. PT2385 treatment completely abolished the induction of Neu3 under hypoxia, accompanied by decreased expression of the HIF-2a target gene DMT1 and DCYTB mRNAs (FIG. 121). Treatment with PT2385, siNEU3 and DANA significantly blunted hypoxia-mediated induction of ceramide levels in the intestinal cell line (FIG. 12m and FIG. 41,m). These results suggested that the activation of HIF-2a signaling resulted in elevated ceramide production primarily through increased Neu3 expression in vitro. Furthermore, oral administration of the NEU3 inhibitors DANA and naringin23 specifically inhibited the NEU3 activity in the small intestine, but not in the liver and WAT (FIG. 13a-c). As a result, the small intestine and serum ceramides were substantially reduced after NEU3 -inhibitor treatment as compared to vehicle (FIG. 13d,e). DANA and naringin treatment markedly attenuated hepatic steatosis and obesity in HFD-fed mice (FIG. 13f-p).

[00223] Moreover, ceramide administered by the injection of C16:0 ceramide to Hif2a ΔΙΕ mice fed a HFD for 6 weeks resulted in increased ceramide levels in small intestine and serum that were comparable to those in vehicle-treated HFD-fed Hif2a ^wa mice (FIG. 14a,b). The administration of ceramide substantially reversed the improvement in body weight and insulin resistance in HFD-fed Hif2a ^Am mice as compared to that of Hif2a ^wa mice (FIG. 14c-f). The intestinal- specific HIF-2a -ablation mediated reduction in hepatic lipid droplets, liver weights, ratios of liver weight to body weight, hepatic triglyceride levels, hepatic cholesterol levels, and serum ALT levels was abrogated by ceramide administration (FIG. 5a-h). Ceramide eliminated the downregulation of hepatic expression of Srebplc, Cidea, Cd36, Fabpl, Fabp4, Fasn, Scdl, Elovl6, Plin2, Tnfa, Pail, Cell, and Ccl3 mRNAs (FIG. 5i,j).

[00224] PT2385 improves steatosis by inhibiting intestinal HIF-2a [00225] To assess the role of intestinal HIF-2a in the PT2385-improved NAFLD, we treated HFD-fed Hif2a ^njii and Hif2a ^Am mice withvehicle or PT2385. PT2385 substantially prevented HFD-induced body- weight increase and insulin resistance in Hif2a ^wa mice, but not in

Hif2a ΔΙΕ mice (FIG. 15a-c). Histology analysis showed that PT2385 eliminated hepatic lipid accumulation in HFD-fed H Ηίβα^ mice, but had no further inhibition on Hif2a ^Am mice (FIG.

15d). Hif2a ΔΙΕ mice exhibited lower liver weights, ratios of liver weight to body weight, hepatic triglycerides, hepatic and serum cholesterol content, and ALT versus Ηίβα^ mice, and were unresponsive to the inhibition of PT2385 treatment (FIG. 15e-k). Further, small intestine and serum ceramide levels were markedly reduced by PT2385 in the Hif2a ^wa mice, but not in the

Hif2a ΔΙΕ mice (FIG. 151,m). Accordingly, the mRNA levels of Degs2, Smpdl, Smpd3, Smpd4, Enpp7, Neu3, Glbl, and Gba2 were substantially inhibited in the HFD-fed Ηίβα^ mice by

PT2385 treatment, but remained similar in the HFD-fed Hif2a ΔΙΕ mice (FIG. 16a-d). In the liver, PT2385 downregulated the mRNA expression of Srebplc, Cidea, Cd36, Fabp4, Fasn, Scdl, Elovl6, Plin2, Tnfa, Pail, Ccl2, and Ccl3 in the Ηίβα^ mice, whereas no change was found in Hif2a ^m mice treated with PT2385 (FIG. 16e,f).

[00226] To further address whether selective inhibition of intestinal HIF-2a could be a therapeutic target for HFD-induced NAFLD, and to confirm whether it is a suitable drug target, we treated HFD-fed mice with obesity and hepatic steatosis with PT2385. Oral administration of PT2385 resulted in reduced body weight and improved insulin sensitivity (FIG. 17a-c). Liver histological analysis by H&E and Oil Red O staining indicated a reduction in hepatic lipid droplets after PT2385 treatment (FIG. 6a), which was reflected by lower liver weights, ratios of liver weight to body weight, and hepatic triglycerides relative to controls (FIG. 6b-e). Hepatic and serum cholesterol levels, and serum ALT were markedly reduced after PT2385 treatment (FIG. 6f-h). PT2385 treatment substantially inhibited HIF-2a signaling as indicated by the decreased target gene Dmtl and Dcytb mRNAs in the small intestine (FIG. 17d). As a result, PT2385-treated mice exhibited lower ceramide levels in both the small intestine and serum relative to vehicle-treated mice, owing to suppressed HIF-2a signaling (FIG. 6i and FIG. 17e). Consistently, the expression of mRNAs encoded by Degs2, Smpdl, Smpd3, Smpd4, Enpp7, Neu3, and Glbl was substantially suppressed in PT2385-treated mice (FIG. 17f,g and FIG. 6j). Western blot confirmed the reduction of NEU3 expression by PT2385 (FIG. 6k). Hepatic mRNA expression of the genes Srebplc, Cidea, Cd36, Fabp3, Fabp4, Fasn, Scdl, Elovl6, Plin2, Tnfa, Pail, Ccl2, and Ccl3 was lower in the PT2385-treatment group than in the vehicle-treated control group (FIG. 17h,i).

[00227] Studies with hypoxic probes indicated that there is a low p0 ₂ at the villi tips, and inflammation and tumors further elevate epithelial hypoxia 24. The current study demonstrated that HFD treatment promotes HIF-2a activation; however, it does not affect HIF-Ια signaling in the intestine. The precise mechanism by which a HFD activates intestinal HIF-2a signaling remains unclear. The gut-bacterial-derived shortchain fatty acids (SCFAs), notably butyrate, were reported to deplete 02 levels and activate HIF signaling in the intestinal epithelium ²⁵ ' ²⁶ . SCFAs produced by the gut microbiota might contribute to induce intestinal HIF-2a expression and activation under the condition of HFD. Other gut-microbiota-derived metabolites from tryptophan and indole metabolic pathways activate the aryl-hydrocarbon receptor (AhR) after

HFD treatment 27. HIF-2a and AhR as heterodimeric transcription factors share the same heterodimer, partner HIF-Ια. Thus, there is the potential for cross-talk between the HIF-2a and AhR signaling pathways that might influence HIF-2a signaling.

[00228] Intestinal HIF-2a depletion results in less susceptibility to HFD induced hepatic fatty liver and obesity, accompanied by a downregulation of intestine and serum ceramide levels. The underlying mechanism revealed that intestine HIF-2a but not HIF-la inhibition markedly suppressed intestinal-derived ceramides by directly targeting ceramide biosynthesis by the key enzyme in the ceramide salvage pathway, NEU3. Furthermore, inhibition of intestinal HIF-2a signaling by PT2385 had both preventive and therapeutic effects on NAFLD and obesity (FIG. 61). There is a positive correlation between HIF-2a signaling in human intestine biopsies and obesity. Considering the close link between obesity and NAFLD, these findings indicate that intestinal HIF-2a signaling is activated in obesity and NAFLD and so could be a promising therapy target in humans. Several studies highlight the potential role for compromised epithelial barrier function and immune response in NAFLD pathogenesis 14 ' 28 ' 29. HIF-la and HIF-2a were shown to influence intestinal epithelial permeability and inflammation 30 ' 31. HIF-la exerts a potent protective function at the epithelial barrier by regulating adherensj unction and tight- junction genes, including claudin 1 (Cldnl), mucin 3 (Muc3), trefoil factor 1 (Tjfl), and 5- ectonucleotidase (Cd73) 32—35. It was reported that HIF-2a has a dual role in barrier function. Acute activation of HIF-2a results in the maintenance of tight-junctionassemblies and barrier integrity through the upregulation of creatine kinase ³⁶ , whereas chronic activation of HIF-2a disrupts the tight junctions through an increase of caveolin 1 (ref. 37). Several reports showed that mice with intestinal epithelial HIF-Ια ablation are more susceptible to intestinal injury and inflammation, whereas HIF-Ια activation leads to an anti-inflammatory response in

inflammatory bowel disease 38. Prolonged HIF-2a activation in intestinal epithelial cells triggers a spontaneous inflammatory response, whereas intestinal HIF-2a deficiency substantially reduces inflammation through the direct regulation of inflammatory mediators, including tumor necrosis factor-a, microsomal prostaglandin synthase 1, and cyclooxygenase 2 in colitis models 20 ' 39. It cannot be excluded that improvement of the intestinal epithelial permeability and inflammation might contribute to the metabolic benefits of intestinal HIF-2a inhibition in the mouse model of obesity.

[00229] It is well established that there is a positive relationship between ceramide levels and metabolic diseases in humans and mice ⁴⁰ ' ⁴¹ . In patients with NAFLD, the serum ceramide levels are markedly increased ⁴² ' ⁴³ . A causal role of ceramides in NAFLD development was further demonstrated by using pharmacological or genetic inhibition of enzymes involved in ceramide metabolism ⁴⁴ . Overexpression of acid ceramidase in either the liver or adipose tissue protects against HFD-induced hepatic lipid accumulation and insulin resistance by reducing ceramide synthesis in adipose tissue ⁴⁵ . The elevated C16:0-ceramide levels induced by overexpressing ceramide synthase enzymes 6 (Cers6) leads to hepatic steatohepatitis and insulin resistance, whereas liver- or adipose- specific Cers6 disruption improves fatty liver and obesity by boosting fatty acid β-oxidation ⁴⁶ ' ⁴⁷ . Mice lacking dihydroceramide desaturase 1 (DEGS 1) are also resistant to HFD-induced obesity and insulin resistance, owing to lower levels of ceramides 48. Ceramides substantially upregulate fatty acid uptake and synthesis through direct or indirect modulation of CD36 and SREBP1C signaling, respectively ¹⁵ ' ⁴⁶ . The lower levels of intestinal ceramide production led to less hepatic lipid accumulation in a gutmicrobiota- remodeling mouse model ¹⁵ . Beside the effects of ceramide on NAFLD, ceramide was also shown to impair beige-fat function, thereby lowering energy expenditure ⁴⁹ ' ⁵⁰ . It is also well established that enhanced energy expenditure can improve hepatic steatosis and insulin resistance in rodent models of NAFLD 51 ' 52. Mice with adipocyte- specific disruption of Sptlc2 involved in the de novo ceramide- synthesis pathway displayed improved beige-fat thermogenesis and hepatic steatosis following the inhibition of adipocyte ceramide synthesis 52. Supporting this view, the current study showed that mice lacking intestine HIF-2a are resistant to HFD-induced hepatic steatosis and obesity, which is correlated with lower intestine and serum ceramides, with suppressed fatty acid synthesis and uptake, and with higher 'beiging' and thermogenic capacity. However, expression of the fatty acid β-oxidation-related genes are not changed in the livers of Hif2a ^wa and Hif2a ΔΙΕ mice fed a HFD for 1 week, whereas most genes encoding fatty acid-synthesis- related enzymes are substantially downregulated in the livers of Hif2a ΔΙΕ mice, which suggests that the HIF-2a -ceramide axis mainly regulates hepatic fatty acid synthesis.

[00230] Furthermore, intestine HIF-2a but not HIF-la was defined as a novel regulator of the ceramide salvage pathway, as revealed by measuring Neu3, Glbl, and Gba2 mRNA expression. Notably, NEU3 catalyzes the hydrolysis of GM3 into lactosylceramides, which can be converted into glucosylceramides by GLB 1, whereas GBA2 catalyzes the generation of ceramides from glucosylceramides 53. This study revealed that Neu3 is a direct target gene of HIF-2a. Interestingly, NEU3 overexpression in liver was observed to increase hepatic lipid accumulation and liver weight ⁵⁴ . In the current study, direct inhibition of intestinal NEU3 substantially ameliorated hepatic steatosis. It was demonstrated that the ceramides themselves represent a more central modulator of obesity and hepatic steatosis than glucosylceramides, lactosylceramides, or sphingomyelins ⁴⁹ . Therefore, the decrease of intestine-derived ceramide levels might contribute to the improvement of NAFLD after the inhibition of intestinal HIF-2a. Besides, NEU3 was found to be an upstream activator of HIF-Ια in muscle cells ⁵⁵ . Although HFD treatment elevated NEU3 expression, increased expression of HIF-Ια protein was not found. The cell types and differential expression of the Hifla genes may be crucial for the regulation of HIF-Ια. HIF-2a, a bHLH-PAS domain protein, was considered undruggable until the discovery of a class of compounds, including PT2385 (ref. 21). PT2385 is an orally bioavailable HIF-2a antagonist that specially inhibits HIF-2a transcriptional activity by allosterically blocking the heterodimerization between HIF-2a and HIF-Ια, while having no effect on HIF-Ια. Recent reports revealed that PT2385 and the closely related analog PT2399 inhibits tumor growth and displays better efficacy than sunitinib, a currently approved first-line antiangiogenesis drug ²¹ ' ⁵⁶ ' ⁵⁷ . PT2385 is well tolerated without toxicities in a phase 1 clinical trial to treat renal cell carcinoma. In the present study, the inhibition of intestinal HIF-2a signaling by PT2385 substantially prevents and reverses obesity and hepatic steatosis, followed by a reduction of intestine and serum ceramide levels. Thus, this study revealed an essential role for intestinal HIF-2a in regulating obesity, insulin resistance and hepatic lipid metabolism, and provided a potential therapeutic approach for treating metabolic disorders.

[00231] Methods and Materials

[00232] Subjects

[00233] Distal ileum mucosa biopsies were taken from 39 individuals who underwent colonoscopy. The subjects were between 18 to 65 years, and all had a BMI between 18.0 and

37.6 kg/m 2. Lean subjects (n=21) BMI are defined as under 25 kg/m 2 , while the BMI of obese subjects (n=18), are over 25 kg/m . The genders and ages were at similar levels at baseline between the groups as disclosed in FIG. 19. The clinical biochemistry viariables are listed in FIG. 20. All subjects fulfilled the following inclusion criteria: (1) no significant acute or chronic inflammatory disease; (2) no significant alcohol consumption (the definition of significant alcohol consumption has been inconsistent and ranged from > 1 alcoholic beverage at 10 grams of alcohol per one drink unit) per day to > 40 grams per day); (3) no medical history of hypertension (diastolic blood pressure <85 mm Hg and systolic blood pressure <140 mm Hg ); (4) no clinical evidence of either cardiovascular or peripheral artery disease; (5) no

gastrointestinal disease; (6) no thyroid dysfunction; and (7) no pregnancy. The study was approved by Conjoint Health Research Ethics Board of Peking University People's Hospital, and written informed consents were given all subjects before participation in this study.

[00234] Mouse studies

[00235] Hifta ^m , Vhf^, VhUHifla , and Vhl/Hifta^ mice were previously described.

For intestine-specific disruption, Ηΐβα^, Vhl^, Vhl/Hifla ^Wii , and Vhl Hif2a ^em were crossed with mice harboring the Cre recombinase under control of the villin promoter to obtain the Hif2a ^Am , Vhl ^Am , Vhl/Hifla ^A , and Vhl/Hif2a ^Am mice. The Vhf" ^& , Vhl ^Am , Vhl/Hifla^,

Vhl/Hifla ^]E , Vhl Hifta ^m , and Vhl/Hif2a ^m were in a mixed Svl29 and C57BL/6 background. The Ηΐβα^ and Hif2a ^Am were on a C57BL/6N background, after backcrossing with C57BL/6N mice for over five generations. HFD (60% kcal from fat) was purchased from Bioserv. Inc (Flemington, NJ). Eight- to 10- weeks-old male littermate Ηίβα^ and Hif2a ^Am mice were fed a chow or HFD for 1 or 12 weeks to induce hepatic steatosis. For the ceramide turnover study, C16:0 ceramide, purchased from Avanti Polar Lipids (Alabaster, AL), was suspended in saline with 0.5% sodium carboxymethyl cellulose. 8- to 10- weeks-old male littermate Ηίβα^ and

Hif2a ΔΙΕ mice fed a HFD were intraperitoneally injected every other day with vehicle or C 16:0 ceramide (10 mg/kg) for 6 weeks. For the NEU3 inhibitor study, DANA and naringin were purchased from Sigma- Aldrich (St. Louis, MO). DANA was dissolved in saline and naringin was suspended in saline with 0.5% sodium carboxymethyl cellulose and 5% dimethyl sulfoxide. C57B6/N mice fed a HFD were gavaged with vehicle, DANA (20 mg/kg o.p.d.), or naringin (200 mg/kg o.p.d.) for 4 weeks. For the HIF-2oc inhibitor studies, PT2385, purchased from MedChem Express (Monmouth Junction, NJ), was suspended in saline with 0.5% sodium carboxymethyl cellulose, 2.5% Tween 80, and 2.5% dimethyl sulfoxide. For treatment of hepatic steatosis, obese C57BL/6N mice fed a HFD for 8 weeks were administered vehicle or PT2385 (20 mg/kg o.p.d.) by gavage for another 4 weeks. To determine whether the action of PT2385 was HIF-2a dependent, 8- to 10- weeks-old male littermate Ηίβα^ and Hif2a ^Am mice, were fed a HFD and administered vehicle or PT2385 (20 mg/kg o.p.d.) by gavage for 12 weeks. For the short-term treatment, 8- to 10-weeks-old male littermate Vhl/Hifla^, Vhl/Hifla ^Am mice, were fed a chow diet and administered vehicle or PT2385 (20 mg/kg o.p.d.) by gavage for 3 days. All mice were randomly assigned to experimental groups (at least 4 mice per group) and the groups did not present differences in body weights before the treatments. All mouse studies were approved by the NCI Animal Care and Use Committee and performed in accordance with the Institute of Laboratory Animal Resources guidelines. All mice were fed ad-libitum and kept in a 12-h light- dark cycle.

[00236] ODD-luciferase transgenic mice study

[00237] ODD-luciferase transgenic mice were obtained from Jackson Laboratories (Bar

Harbor, ME). Ten- week-old male littermate mice were fed a chow or HFD for 1 week. Small intestines were collected, extracted with lysis buffer and the luciferase activities were measured by use of the luciferase assay system (Promega).

[00238] Metabolic assays

[00239] For the glucose tolerance test (GTT), mice were fasted overnight for 16 hours. For the insulin tolerance test (ITT), mice were fasted overnight for 4 hours. Glucose at 2 g/kg, or insulin (Eli Lilly, Washington, DC) at 0.8 U/kg, in saline were injected intraperitoneally to conscious animals and from tail vein blood glucose at was measured at 15, 30, 60, and 90 min post injection using a Glucometer (Bayer, Pittsburgh, PA).

[00240] Indirect calorimetry [00241] Indirect calorimetry was performed on Hif2a and Hif2a mice fed a HFD for 1 week using a 12-chamber Environment Controlled CLAMS (Columbus Instruments, Columbus, OH). After 48-h acclimatization, mice were monitored for 24 h at 22 °C. During testing, food and water were provided ad libitum.

[00242] Histological analysis

[00243] Formalin fixed paraffin embedded liver sections was stained by Hematoxylin and eosin (H&E) and OCT embedded frozen liver sections was stained by Oil O Red according to standard protocols followed by microscopic examination. At least three discontinuous liver sections were evaluated for each mouse.

[00244] Clinical chemistry measurements

[00245] Liver injury was evaluated by measuring alanine aminotransferase (ALT) in serum (Catachem Inc., Oxford, CT). Hepatic and serum triglycerides were determined with a triglyceride colorimetric assay kit (Bioassay Systems, Hayward, CA). Hepatic and serum cholesterol were measured using assay kit from Wako Diagnostics (Wako Chemicals USA, Inc., Richmond, VA).

[00246] Neuraminidase activity assays

[00247] Intestine neuraminidase activity was determined in the intestine homogenates using a Neuraminidase Activity Assay kit (Sigma- Aldrich). Inhibition of NEU3 in the intestines may be a treatment strategy for metabolic disorders.

[00248] Luciferase reporter gene assays

[00249] The NEU3 promoter region was predicted by FANTOM5 mouse promoterome.

Hypoxia response elements (HREs) in the promoter region were further identified by HIF-2a CHIP-seq (see below). The NEU3 promoter and the HRE-lacking promoter fragments were amplified by PCR from mouse genomic DNA. The primer sequences are listed in FIG. 21. The amplified fragments were digested by Kpn I and Xho I restriction enzymes (New England Biolabs), and then cloned into the pGL4. l l luciferase vector (Promega). NEU3 reporter vectors and phRL-TK Renilla luciferase control vector (Promega) were co-transfected into HCTl 16 cells (ATCC CCL-247) by use of Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific). In addition, either constitutively-active HIF-2a triple mutants (HIF2aTM) expression vector or the empty backbone vector (pcDNA3) were co-transfected into the cells and cobalt (II) chloride hexahydrate (Sigma-Aldrich) was added to culture medium at a 100 μΜ final concentration to mimic hypoxia. Empty vector (pGL4.11) was used as a negative control and the standard. After 24 hours from the transfection, luciferase assays were performed by use of the Dual-luciferase assay system (Promega). Firefly and Renilla luciferase activities were measured by Veritas microplate luminometer (Turner Biosystems).

[00250] Cell treatment

[00251] HCT116 cells were seeded in 12-well plates (for gene expression analysis) or 6- well plates (for lipidomics analysis). Cells were treated with vehicle, PT2385 (10 μΜ), or DANA (100 μΜ), or transfected with siNEU3 (20 nM, Thermo Fisher Scientific, Waltham, MA) and exposed to either vehicle or CoCl ₂ (100 μΜ) for 24 hours.

[00252] Real-time PCR analysis

[00253] The intestine mucosa was gently scraped and liver flash frozen in liquid nitrogen and both stored at -80°C. Total RNA from frozen intestine and liver was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA). cDNA was synthesized from 1 μg total RNA using qScript™ cDNA SuperMix (Gaithersburg, MD). Real-time PCR primer sequences are included in FIG. 21. The relative amounts of each mRNA was calculated after normalizing to their corresponding β- actin mRNA and the results expressed as fold change relative to the control group.

[00254] Western blot analysis

[00255] Intestine samples were lysed in RIPA buffer with protease and phosphatase inhibitors, and then the protein extracts were separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane. The membrane was incubated overnight at 4°C with antibodies against HIF2a, HIFla (Novus Biologicals, LLC, Littleton, CO), NEU3 (Origene Technologies, Rockville, MD), and ytf-ACTIN (Cell Signaling, Danvers, MA).

[00256] ChIP Assay

[00257] ChIP was performed as described previously on duodenal epithelium scrapings using 1% formaldehyde in IX PBS as a cross -linker ⁴⁰ . The primary antibody for HIF2a (Novus Biologicals) was used for immunoprecipitation. The precipitated DNA samples were incubated with RNase A and proteinase K, purified using PCR clean-up column (Qiagen), and 2 μL· of sample was used for real-time PCR using primers listed in FIG. 21.

[00258] Metabolomics analysis

[00259] The lipidomics analysis was undertaken as previously described ¹⁹ . For global lipidomics, the multivariate data matrix was analyzed by SIMCA-P+14 software (Umetrics, Kinnelon, NJ). For ceramide quantitation, the data was analyzed by TargetLynx software, a subroutine of the MassLynx software (Waters Corp.). The ceramide standards including C16:0, C18:0, C18: l, C20:0, C22:0, C24:0 and C24: l were obtained from Avanti Polar Lipids

(Alabaster, AL).

[00260] Data analysis

[00261] Statistical analysis was performed using Prism version 7.0 (GraphPad Software,

San Diego, CA) and power analysis was performed using StatMate version 2.0 (GraphPad Software, San Diego, CA). Experimental values are presented as mean + sem. The investigators involved in this study were not completely blinded during sample collection or data analysis. All data shown were representative results from at least three independent experiments. No animal or sample was excluded from the analysis. The sample distribution was determined by

Kolmogorov-Smirnov normality test. Correlations were assessed by nonparametric Spearman's test. Statistical significance between two groups was determined using two-tailed Student's t-test. One-way ANOVA followed by Tukey's post-hoc correction was applied for multi-group comparisons. P- values of less than 0.05 were considered significant.

[00262] EQUIVALENTS

[00263] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. It is understood that the detailed examples and embodiments described herein are given by way of example for illustrative purposes only, and are in no way considered to be limiting to the invention. Various modifications or changes in light thereof will be suggested to persons skilled in the art and are included within the spirit and purview of this application and are considered within the scope of the appended claims. For example, the relative quantities of the ingredients may be varied to optimize the desired effects, additional ingredients may be added, and/or similar ingredients may be substituted for one or more of the ingredients described. Additional advantageous features and functionalities associated with the systems, methods, and processes of the present invention will be apparent from the appended claims. Moreover, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. [00264] References

[00265] Each of the following references (as cited to in the Examples) are incorporated herein by reference in their entireties:

1. Ray, K. NAFLD-the next global epidemic. Nat. Rev. Gastroenterol. Hepatol. 10, 621

(2013).

2. Chalasani, N. et al. The diagnosis and management of non-alcoholic fatty liver disease: practice guideline by the American Gastroenterological Association, American

Association for the Study of Liver Diseases, and American College of Gastroenterology.

Gastroenterology 142, 1592-1609 (2012).

3. Rotman, Y. & Sanyal, A.J. Current and upcoming pharmacotherapy for non-alcoholic fatty liver disease. Gut 66, 180-190 (2017).

4. Ju, C, Colgan, S.P. & Eltzschig, H.K. Hypoxia-inducible factors as molecular targets for liver diseases. J. Mol. Med. (Bed.) 94, 613-627 (2016). 5. Semenza, G.L. & Wang, G.L. A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human

erythropoietin gene enhancer at a site required for transcriptional activation. Mol. Cell. Biol. 12, 5447-5454 (1992).

6. Ivan, M. et al. HIFa targeted for VHL-mediated destruction by proline hydroxylation: implications for 02 sensing. Science 292, 464-468 (2001).

7. Jaakkola, P. et al. Targeting of HIF-a to the von Hippel-Lindau ubiquitylation complex by 02-regulated prolyl hydroxylation. Science 292, 468-472 (2001).

8. Minamishima, Y.A. et al. A feedback loop involving the Phd3 prolyl hydroxylase tunes the mammalian hypoxic response in vivo. Mol. Cell. Biol. 29, 5729-5741

(2009).

9. Qu, A. et al. Hypoxia-inducible transcription factor 2a promotes steatohepatitis through augmenting lipid accumulation, inflammation, and fibrosis. Hepatology 54,

472-483 (2011).

10. Ramakrishnan, S.K. et al. HIF2a is an essential molecular brake for postprandial hepatic glucagon response independent of insulin signaling. Cell Metab. 23,

505-516 (2016).

11. Taniguchi, CM. et al. Cross-talk between hypoxia and insulin signaling through Phd3 regulates hepatic glucose and lipid metabolism and ameliorates diabetes. Nat. Med. 19, 1325-1330 (2013).

12. Wei, K. et al. A liver Hif-2a-Irs2 pathway sensitizes hepatic insulin signaling and is modulated by Vegf inhibition. Nat. Med. 19, 1331-1337 (2013).

13. Gonzalez, F.J., Jiang, C. & Patterson, A.D. An intestinal microbiota-farnesoid X receptor axis modulates metabolic disease. Gastroenterology 151, 845-859 (2016).

14. Singh, V. et al. Microbiota-dependent hepatic lipogenesis mediated by stearoyl CoA desaturase 1 (SCD1) promotes metabolic syndrome in TLR5-deficient mice. Cell

Metab. 22, 983-996 (2015).

15. Jiang, C. et al. Intestinal farnesoid X receptor signaling promotes nonalcoholic fatty liver disease. J. Clin. Invest. 125, 386-402 (2015).

16. Safran, M. et al. Mouse model for noninvasive imaging of HIF prolyl hydroxylase activity: assessment of an oral agent that stimulates erythropoietin production. Proc.

Natl. Acad. Sci. USA 103, 105-110 (2006).

17. Pagadala, M., Kasumov, T., McCullough, A.J., Zein, N.N. & Kirwan, J.P. Role of ceramides in nonalcoholic fatty liver disease. Trends Endocrinol. Metab. 23, 365-371 (2012).

18. Kitatani, K., Idkowiak-Baldys, J. & Hannun, Y.A. The sphingolipid salvage pathway in ceramide metabolism and signaling. Cell. Signal. 20, 1010-1018 (2008).

19. Jornayvaz, F.R. & Shulman, G.I. Diacylglycerol activation of protein kinase Ce and hepatic insulin resistance. Cell Metab. 15, 574-584 (2012).

20. Xue, X. et al. Endothelial PAS domain protein 1 activates the inflammatory response in the intestinal epithelium to promote colitis in mice. Gastroenterology 145, 831-841 (2013).

21. Wallace, E.M. et al. A small-molecule antagonist of HIF2a is efficacious in preclinical models of renal cell carcinoma. Cancer Res. 76, 5491-5500 (2016).

22. Zou, Y., Albohy, A., Sandbhor, M. & Cairo, C.W. Inhibition of human neuraminidase 3 (NEU3) by C9-triazole derivatives of 2,3-didehydro-N-acetyl-neuraminic acid.

Bioorg. Med. Chem. Lett. 20, 7529-7533 (2010).

23. Yoshinaga, A. et al. NEU3 inhibitory effect of naringin suppresses cancer cell growth by attenuation of EGFR signaling through GM3 ganglioside accumulation. Eur. J. Pharmacol. 782, 21-29 (2016).

24. Triner, D. & Shah, Y.M. Hypoxia-inducible factors: a central link between inflammation and cancer. . Clin. Invest. 126, 3689-3698 (2016). 25. Kelly, C.J. et al. Crosstalk between microbiota-derived short-chain fatty acids and intestinal epithelial HIF augments tissue barrier function. Cell Host Microbe 17, 662-671 (2015).

26. Rivera-Chavez, F. et al. Depletion of butyrate-producing Clostridia from the gut microbiota drives an aerobic luminal expansion of salmonella. Cell Host Microbe 19, 443-454 (2016).

27. Hubbard, T.D., Murray, I.A. & Perdew, G.H. Indole and tryptophan metabolism: endogenous and dietary routes to Ah receptor activation. Drug Metab. Dispos. 43, 1522-1535 (2015).

28. Kawano, Y. et al. Colonic pro-inflammatory macrophages cause insulin resistance in an intestinal Ccl2/Ccr2-dependent manner. Cell Metab. 24, 295-310 (2016).

29. Rahman, K. et al. Loss of junctional adhesion molecule A promotes severe steatohepatitis in mice on a diet high in saturated fat, fructose, and cholesterol.

Gastroenterology 151, 733-746 (2016).

30. Ramakrishnan, S.K. & Shah, Y.M. Role of intestinal HIF-2a in health and disease. Annu. Rev. Physiol. 78, 301-325 (2016).

31. Glover, L.E., Lee, J.S. & Colgan, S.P. Oxygen metabolism and barrier regulation in the intestinal mucosa. J. Clin. Invest. 126, 3680-3688 (2016).

32. Furuta, G.T. et al. Hypoxia-inducible factor 1-dependent induction of intestinal trefoil factor protects barrier function during hypoxia. . Exp. Med. 193, 1027-1034

(2001).

33. Synnestvedt, K. et al. Ecto-5 '-nucleotidase (CD73) regulation by hypoxia-inducible factor- 1 mediates permeability changes in intestinal epithelia. . Clin. Invest. 110,

993-1002 (2002).

34. Robinson, A. et al. Mucosal protection by hypoxia-inducible factor prolyl

hydroxylase inhibition. Gastroenterology 134, 145-155 (2008).

35. Karhausen, J. et al. Epithelial hypoxia-inducible factor- 1 is protective in murine experimental colitis. J. Clin. Invest. 114, 1098-1106 (2004).

36. Glover, L.E. et al. Control of creatine metabolism by HIF is an endogenous mechanism of barrier regulation in colitis. Proc. Natl. Acad. Sci. USA 110, 19820-19825 (2013). 37. Xie, L. et al. Hypoxia-inducible factor/MAZ-dependent induction of caveolin-1 regulates colon permeability through suppression of occludin, leading to hypoxiainduced inflammation. Mol. Cell. Biol. 34, 3013-3023 (2014).

38. Cummins, E.P. et al. The hydroxylase inhibitor dimethyloxalylglycine is protective in a murine model of colitis. Gastroenterology 134, 156-165 (2008).

39. Shah, Y.M. et al. Hypoxia-inducible factor augments experimental colitis through an MIF-dependent inflammatory signaling cascade. Gastroenterology 134, 2036-2048. e3 (2008).

40. Summers, S.A. & Goodpaster, B.H. CrossTalk proposal: Intramyocellular ceramide accumulation does modulate insulin resistance. J. Physiol. (Lond.) 594, 3167-3170 (2016).

41. Chavez, J.A. & Summers, S.A. A ceramide-centric view of insulin resistance. Cell Metab. 15, 585-594 (2012).

42. Gorden, D.L. et al. Biomarkers of NAFLD progression: a lipidomics approach to an epidemic. J. Lipid Res. 56, 722-736 (2015).

43. Haus, J.M. et al. Plasma ceramides are elevated in obese subjects with type 2 diabetes and correlate with the severity of insulin resistance. Diabetes 58, 337-343 (2009).

44. Chaurasia, B. & Summers, S.A. Ceramides— lipotoxic inducers of metabolic disorders. Trends Endocrinol. Metab. 26, 538-550 (2015).

45. Xia, J.Y. et al. Targeted induction of ceramide degradation leads to improved systemic metabolism and reduced hepatic steatosis. Cell Metab. 22, 266-278 (2015).

46. Turpin, S.M. et al. Obesity-induced CerS6-dependent C16:0 ceramide production promotes weight gain and glucose intolerance. Cell Metab. 20, 678-686 (2014).

47. Raichur, S. et al. CerS2 haploinsufficiency inhibits β-oxidation and confers susceptibility to diet-induced steatohepatitis and insulin resistance. Cell Metab. 20, 687-695 (2014).

48. Holland, W.L. et al. Inhibition of ceramide synthesis ameliorates glucocorticoid-, saturated-fat-, and obesity-induced insulin resistance. Cell Metab. 5, 167-179

(2007).

49. Chaurasia, B. et al. Adipocyte ceramides regulate subcutaneous adipose browning, inflammation, and metabolism. Cell Metab. 24, 820-834 (2016).

50. Jiang, C. et al. Intestine- selective farnesoid X receptor inhibition improves obesityrelated metabolic dysfunction. Nat. Commun. 6, 10166 (2015).

51. Perry, R.J., Samuel, V.T., Petersen, K.F. & Shulman, G.I. The role of hepatic lipids in hepatic insulin resistance and type 2 diabetes. Nature 510, 84-91(2014).

52. Samuel, V.T. & Shulman, G.I. Mechanisms for insulin resistance: common threads and missing links. Cell 148, 852-871 (2012).

53. Giussani, P., Tringali, C, Riboni, L., Viani, P. & Venerando, B. Sphingolipids: key regulators of apoptosis and pivotal players in cancer drug resistance. Int. J. Mol.

Sci. 15, 4356-4392 (2014).

54. Yoshizumi, S. et al. Increased hepatic expression of ganglioside-specific sialidase, NEU3, improves insulin sensitivity and glucose tolerance in mice. Metabolism 56,

420-429 (2007).

55. Scaringi, R. et al. NEU3 sialidase is activated under hypoxia and protects skeletal muscle cells from apoptosis through the activation of the epidermal growth factor

receptor signaling pathway and the hypoxia- inducible factor (HIF)-la. J. Biol. Chem.

288, 3153-3162 (2013).

56. Cho, H. et al. On-target efficacy of a HIF2a antagonist in preclinical kidney cancer models. Nature 539, 107-111 (2016).

57. Chen, W. et al. Targeting renal cell carcinoma with a HIF-2 antagonist. Nature 539, 112-117 (2016).