OBESITY-INDUCED INSULIN RESISTANCE AND PATTY LIVER

This application claims priority of U.S. Provisional Application No. 61/800,180, filed March 15, 2013, the contents of which is hereby incorporated by reference in its entirety.

Throughout this application, various publications are referenced. Full citations for these publications may be found at the end of the specification or at the end of each experimental section. The disclosures of these publications are hereby incorporated by reference into this application to describe more fully the art to which this invention pertains.

Background of the Invention

Metabolic diseases in their protean incarnations are likely to define health, public policy, and economics of the 21st century.1 Aside from surgical remediation, progress in their treatment with lifestyle or pharmacologic therapies has been disappointing.

Altered insulin signaling is often associated with excessive hepatic triglyceride content (hepatosteatosis ) , a correlate of hepatic failure, hepatocellular cancer and need for liver transplantation .2 Activation of the nutrient-sensing mTor pathway stimulates hepatic de novo lipogenesis, providing not only an explanation for how nutrient excess translates into elevated hepatic fat, but also for the apparent paradox whereby increased Akt -an mTor substrate- can simultaneously promote insulin sensitivity and hepatosteatosis .3 Thus, treatment of hepatocytes with rapamycin, an allosteric inhibitor of the mTorcl- dependent functions of mTor, prevents insulin activation of the lipogenic transcription factor 5rep£>2c.3,4 Although interpretation of in vivo studies in rodents chronically treated with rapamycin, and clinical experience in rapamycin-treated patients, is clouded by their effects to disrupt insulin signaling in other tissues, mice with disruptions in hepatic mTor signaling have offered insight into the convergence of mTor and insulin pathways in the combined regulation of glucose and lipid metabolism.5-8 Liver-specific knockout of either the critical mTorcl component Raptor, or the mTorc2-defining subunit Rictor, protect from diet-induced hepatic steatosis, likely due to reduced lipogenesis .9, 10 Interestingly, hepatocyte-specific knockout of the mTor inhibitor, Tscl, activates mTorcl signaling and protects from diet-induced fatty liver due to effects on Insig2a, a regulator of

Srebplc function, suggesting that tight regulation of this pathway is physiologically relevant.11

The bifurcation of the insulin signaling pathways after Akt - to FoxOl for glucose production, and to mTor/Srebplc for lipogenesis - raises the question of whether these pathways have additional inputs. Notch signaling is critical for cell type specification and lineage restriction .12 Cell surface-tethered ligands (Jagged and Delta-like) bind Notch receptors on neighboring cells, resulting in a series of cleavage events that culminate in γ-secretase-dependent liberation of the Notch intracellular domain (NICDJ.13 NICD translocates to the nucleus, where it binds to and co-activates the transcriptional effector Rbp-Jk, promoting expression of the Hairy enhancer of split (Hes) and Hes-related (Hey) family of genes.14 Homozygous null alleles of components of this signaling pathway result in embryonic lethality, demonstrating their importance to normal development .15-17 Importantly, Notch signaling is therapeutically accessible, and inhibitors are in advanced clinical development for cancer.18

Fatty liver disease is a condition where large vacuoles of triglyceride fat accumulate in liver cells via the process of abnormal retention of lipids. Despite having multiple causes, fatty liver can be considered a single disease that occurs worldwide in those with excessive alcohol intake and those who are obese and is diagnosed as either alcoholic fatty liver disease or non-alcoholic fatty liver disease.57

Alcoholic liver diease is the major cause of liver disease in western countries. Non-alcoholic fatty liver disease is the leading cause of elevated liver enzyme levels in U.S. adults and is the most common cause of cirrhosis which cannot be explained by hepatitis, alcohol abuse, toxin exposure, autoimmune disease, congenital liver disease, vascular outflow obstruction, or biliary tract disease.57

Despite recent advancements in treatment, there exists a need for safe and effective treatments for fatty liver disease.

Summary of the Invention

■ ■ The present invention provides a method of treating a subject suffering from a fatty liver disease which comprises administering to the subject an amount of a Notch decoy protein in an amount effective to treat the subject's fatty liver disease.

The present invention provides a composition comprising a pharmaceutically acceptable carrier and an amount of a Notch decoy protein effective to treat a fatty liver disease.

The present invention provides a package comprising:

(a) the pharmaceutical composition of the invention; and

(b) instructions for using the pharmaceutical composition of step (a) to treat the fatty liver disease.

Brief Description of the Figure:

Figures la - 1j : Regulation of hepatic Notch activity, (a) Western blot of cleaved Notchl receptor (NICD) in livers from fasted and refed 9- week-old, chow-fed C57/B16 mice (n=5/group) . (b) Quantification of the data in (a) . Expression of insulin (c) , Srebplc (d) and Notch (e) targets in livers from fasted and refed 9-week-old, chow-fed C57/B16 mice (n=5/group) . (f) Regulation of Notch targets in 16-week-old L-Rbpj and control (Cre-) mice, fasted for 16-h or fasted for 16-h followed by 4-h refeeding (n=6/group) . Fasted values are set arbitrarily at 1 for both groups. *p < 0.05 vs. fasted mice, (g) Western blot of cleaved

Notchl and (h) Notch target gene expression in livers from fasted, 16- week old chow-fed or high-fat diet (HFD)-fed mice (n=12/group) . *p < 0.05 vs. chow-fed mice, (i) Notch target expression in livers from db/db or control (cib/+) mice (n=5/group) , or (j) in hepatocytes from ob/ob or control (wt) mice, all sacrificed in the ad libitum state

(triplicate wells, representative of 2 individual experiments) . *p < 0.05 vs. dbt+ or wt mice. Data show means ± SEM.

Figures 2a - 2b: Nutritional regulation of hepatic Notchl. (a) Western analysis of NICD in 8-week-old, chow-fed C57/BL6 male mice fasted for 24-h, and refed for the indicated times. (b) Protein expression normalized to tubulin. Data show means ± SEM.

Figures 3a - 3o: Decreased hepatic steatosis in HFD-fed L-Rbpj mice, (a) Body weight of Cre- and L-Rbpj male mice (n=6-8/group) on standard chow, or HFD started at weaning, (b) H&E staining from age- and weight-matched L-Rbpj and Cre- mice on HFD. (c) Hepatic lipids (inset shows expanded graph for cholesterol) , and <d) liver and epidydimal white adipose tissue (WAT) weight in mice fed HFD from weaning and sacrificed at 20 weeks after a 16-h fast (n=8/group) . (e) Triglyceride secretion in 12 week-old HFD-fed Cre- and L-Rbpj mice, fasted for 5-h, then injected with Poloxamer 407 (n=6/group) . (f) Fatty acid oxidation determined through 14C02 release after incubation of primary hepatocytes from 16-week old Cre- and L-Rbpj mice with 14C-oleic acid (triplicate wells, representative of 2 individual experiments), (g) Expression of genes regulating fatty acid oxidation in livers of 20- week old, HFD-fed mice sacrificed after 16-h fast, followed in some animals by 4-h refeeding (n=8/group) . (h) β-ΟΗ butyrate levels in 20- week-old, HFD-fed mice sacrificed after a 16-h fast, (i) Hepatic lipogenic protein expression and (j) de novo lipogenesis in livers from 20-week-old, HFD-fed mice sacrificed after a 16-h fast, followed by 6-h refeeding, measuring 3H20 incorporation into fatty acids (n=7/group) . (k) De novo lipogenesis in insulin-treated hepatocytes from chow-fed 16-week-old mice, measuring 14C-acetate incorporation into fatty acids (triplicate wells, representative of 2 individual experiments) . (1) Basal and insulin-stimulated Srebplc expression and

(m) Fasn-luciferase activity in primary hepatocytes from chow-fed, 16- week-old Cre- and L-Rbpj littermates (triplicate wells, representative of 2 individual experiments, (n) Western blot analysis of Akt and mTor signaling in livers from HFD-fed mice sacrificed after a 5-hr fast. (o) Western blots from hepatocytes isolated from 16-week-old mice, transduced with control or Notchl-IC adenovirus, treated with or without ΙΟηΜ insulin or 25nM rapamycin for 4-h. Protein expression normalized to either actin or tubulin. *p < 0.05, **p < 0.01, *** < 0.001 vs. Cre- mice or hepatocytes. Data show means + SEM.

Figures 4a - 4e: Metabolic analyses in L-Rbpj and L-Foxol mice, (a) Serum TG and cholesterol and (b) non-esterified fatty acids (NEFA) in HFD-fed mice after 16-h fast, or 2-h refeeding (n=6-8 /group) . (c) Relative hepatic triglyceride content in chow-fed, 12-week old L-Rbpj, L-Foxol and L-Rbpj/Foxol mice, normalized to respective Cre- littermates. (d) Serum TG before (fasted) and following olive oil gavage ( 300 1/πιου36 ) in HFD-fed mice, (e) Ppary and PPARy target (Ap2, Cd36) expression in primary hepatocytes (triplicate wells, representative of 2 individual experiments) . *p < 0.05, **p < 0.01, ***p < 0.001 vs. Cre- mice. Data show means ± SEM.

Figures 5a - 5f: Metabolic effects of high-fat feeding in L-Rbpj mice, (a) Glucose, (b) insulin, (c) non-esterified fatty acids (NEFA) , (d) hepatic TG, and (e) serum lipid levels in 5-h-fasted Cre- or L-Rbpj mice fed HFD for 3 weeks, (f) β-ΟΗ butyrate levels in Cre- and L-Rbpj mice fed HFD for 3 weeks, fasted (16-h), or refed (4-h). *p < 0.05, **p < 0.01, ***p < 0.001 vs. Cre- mice. Data show means ± SEM.

Figures 6a - 6h: Notch decoy increases insulin sensitivity and decreases hepatic lipid content, (a) Glucose, (b) insulin, (c) liver weight, and (d) hepatic triglyceride 14 d after delivery of Notchl decoy or Fc control adenovirus in HFD-fed mice, fasted for 16-h

(n=6/group). (e) Western blots of liver proteins from HFD-fed mice transduced with Notch decoy or Fc control adenovirus and fasted for 16- h. (f) Expression of fatty acid oxidation genes in livers from fasted mice transduced with Notch decoy (n=6/group) . (g) Srebplc levels in primary mouse hepatocytes transduced with Notch decoy or Fc adenovirus

(triplicate wells, representative of 2 individual experiments) . (h) Western blots of liver protein from HFD-fed mice, sacrificed after a 16-h fast, transduced with Notch decoy or Fc control adenovirus. Protein expression normalized to either Actin or Tubulin. Mice were 12- week-old male C57B1/6, unless otherwise indicated. *p < 0.05 vs. Fc.

Data show means ± SEM.

Figures 7a - 7e: Effect of Notch decoy on weight, adiposity, and lipids, (a) Body and (b) relative epidydimal white adipose tissue (eWAT) weight from fasted mice transduced with Notch decoy or Fc control. (c) Serum TG, and (d) cholesterol measured under the conditions indicated 7-d after transduction of mice with Notchl decoy or Fc adenovirus, (e) Ppary and ΡΡΑΡ,γ target (Ap2, Cd36) expression in primary hepatocytes from 12-week-old mice, transduced with Notch decoy as compared to Fc control adenovirus (triplicate wells, representative of 2 individual experiments). *p < 0.05 vs. Fc. Data show means + SEM.

Figures 8a - 8p: Activation of hepatic Notch increases mTor, lipogenic genes, and steatosis in chow-fed mice, (a) Oil-Red-0 staining, (b) weight, and (c) lipid content in livers of 16-h fasted mice (inset shows expanded graph for cholesterol) 7-d after adenoviral delivery of GFP (control) or constitutively active Notchl (Nl-IC) (n=6/group) . (d)

Liver Western blots and (e) gene expression analysis in mice transduced with GFP or Nl-IC adenovirus, sacrificed after a 16-h fast followed by 2-h refeeding. (n=6/group) . (f) De novo lipogenesis in hepatocytes after transduction with GFP or Nl-IC adenovirus and incubation with ΙΟηΜ insulin (triplicate wells, representative of 2 individual experiments) . (g) Hepatic triglyceride content 7-d after GFP or Nl-IC transduction in 16-h fasted, 24-week-old, chow-fed Cre- and L-Rbpj mice, (h) Gene expression after GFP or Nl-IC transduction of primary hepatocytes from Cre- and L-Rbpj littermates, followed by incubation with lOnM insulin (triplicate wells, representative of 2 individual experiments). *p<0.05 vs. untreated cells, sp<0.05 vs. insulin-treated cells, #p<0.05 vs. Nl-IC transduced, insulin-treated cells, (i) De novo lipogenesis in isolated hepatocytes after transduction with GFP (arbitrarily set to a value of 1) or Nl-IC and incubation with lOnM insulin (triplicate wells, representative of 2 individual experiments) . (j) Western blot from livers of mice transduced with GFP or Nl-IC, fasted for 16-h, or refed for 2-h. (k-m) Quantitation of the phospho/total mTor, 4E-BP1 and S6k levels from the experiments in (j). (n) Western blot from FAO hepatoma cells transduced with Fc (-) or Nl-

IC (Notchl) , incubated in serum-free and amino acid-free medium for 4- h, followed by treatment with lOnM insulin or 4x amino acid mixture for 4-h. (o) Western blot in primary hepatocytes transduced with Fc or Nl- IC, after treatment with lOnM insulin and/or 25nM rapamycin. (p) Fasn- luciferase assays in FAO hepatoma cells transduced with Nl-IC, Nl-decoy or Fc (control) adenovirus and treated with lOnM insulin. *p < 0.05, **p < 0.01, ***p < 0.001 vs. Fc or GFP. Protein expression normalized to either Actin or Tubulin. Mice were 8-week-old C57B1/6 males, unless otherwise indicated. Data show means ± SEM.

Figures 9a - 9£ : mTor inhibition prevents Notch-induced fatty liver.

(a) asn-luciferase in FAO hepatoma cells transfected with either Raptor or scrambled (scr) shRNA, transduced with either Fc (-) or Nl-IC (Notch) adenovirus and treated for 16-h with ΙΟΟηΜ insulin. ***p<0.001 vs. Fc control, &p<0.001 vs. scrambled shRNA, #p<0.001 vs. no insulin. (b) Gene expression in primary hepatocytes after transduction with GFP

(-) or Nl-IC (Notch) adenovirus, followed by incubation with lOnM insulin and/or 25nM rapamycin (triplicate wells, representative of 2 individual experiments). *p<0.05 vs. untreated cells, Sp<0.05 vs. insulin-treated cells, #p<0.05 vs. Nl-IC transduced, insulin-treated cells, (o) Hepatic triglyceride content and (d) gene expression in rapamycin-treated Fc or Nl-IC-transduced mice, sacrificed after a 16-h fast followed by 6-h refeeding. (e) Glucose tolerance test and (f) AUC from GTT in mice transduced with Fc or Nl-IC, injected daily with rapamycin or vehicle. AUC was normalized to Fc-transduced mice for each treatment. Mice were 10-week-old, short-term (3 weeks) HFD-fed C57B1/6 males. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. Cre- mice. Data show means ± SEM.

Figures 10a - 10b: mTor inhibition prevents Notch-induced lipogenic gene expression, (a) Fasn-luciferase activity in FAO hepatoma cells transfected with scrambled (scr) or a second raptor shRNA (raptor2) , transduced with Fc or Nl-IC and treated with ΙΟΟηΜ insulin, or (b) infected with Fc or Nl-IC and treated with ΙΟΟηΜ insulin and/or 25nM rapamycin. ***p<0.001 vs. Fc control, &p<0.001 vs. scrambled shRNA, #p<0.001 vs. no insulin, ^Λ ρ<0.001 vs. insulin, ~p<0.001 vs. Nl-IC tinsulin. Data show means ± SEM.

Figures 11a - lh: Notch induces mTorcl complex stability, (a) Western blots of liver proteins from 5h-fasted L-Rbpj and control mice (mTor and Actin blots reproduced from Figure 2n) . (b) Western blot of liver protein from chow-fed, 12-week-old mice transduced with Fc or Nl-IC, sacrificed at day 7, after overnight fasting, (c) Western blots from FAO hepatoma cells transduced with either Fc or Nl-IC, with or without treatment with MG132 for 4-h. (d) Western blots of primary hepatocytes, transfected with Raptor cDNA, then transduced with Fc or Notchl and treated for 2-h with cycloheximide . (e) Fasn-luciferase activity in FAO hepatoma cells transduced with Fc or Nl-IC, and co-transduced with GFP or Raptor. ***p<0.001 vs. Fc, Sp<0.001 vs. Nl-IC plus GFP. (f) Western blots of HEK 293 cells or (g) primary hepatocytes transfected with Raptor-FLAG, followed by transduction with GFP or Nl-IC and immunoprecipitation with anti-FLAG antibody. Protein expression normalized to either actin or tubulin. Data show means ± SEM. (h) Model of Notch effects on hepatic glucose and lipid metabolism.

Figures 12a - 12d: Notch induces mTorcl complex stability. (a) Endogenous Raptor gene expression in primary hepatocytes transduced with Fc or Nl-IC, then treated with either vehicle (no tx) or insulin (ins) for 2-h. (b) Western blots of primary hepatocytes, transfected with Raptor cDNA, then transduced with control (Fc) or Notchl and treated for 2-h with insulin, (c) Western blots from HEK 293 cells transfected with mTor- yc, followed by transduction with GFP or Nl-IC and iramunoprecipitation with anti- yc antibody in the presence of increasing concentrations of CHAPS (d) . Data show means ± SEM.

Figures 13a - 13c: Hepatocyte specific ablation of Notch signaling protects from obesity-induced glucose tolerance and fatty liver. Despite unchanged body weight (a), HFD-fed L-Rbpj mice show (b) improved glucose tolerance and (c) decreased hepatic steatosis.

Figures 14a — 14d: Reduced Srebplc-induced lipogenesis in L-Rbpj mice, (a) HFD-fed Cre- and L-Rbpj mice were sacrificed after an overnight fast followed by 6 hours of refeeding prior to liver protein extraction and Western blot for lipogenic proteins, (b) De novo lipogenesis in hepatocytes from chow-fed 16-week-old mice (triplicate wells, representative of 2 individual experiments) . (c) Basal and insulin- stimulated Srebplc expression and (d) Fasn-luciferase activity in primary hepatocytes from chow-fed, 16-week-old Cre- and L-Rbpj littermates, transferred to serum-free medium for 16-h, followed by addition of ΙΟηΜ insulin for 6-h prior to lysis. *p<0.05, **p<0.01 and ***p<0.001 as compared to relevant control mice.

Figures 15a - 15b: Inhibition of Notch signaling increases Akt signaling, but reduces mTorcl activity, (a) L-Rbpj mice show higher Akt phosphorylation at the PDK1 site (T308) but lower mTorcl activity, as assessed by p70 S6Kinase T389 or 4E-BP1 T37/46 phosphorylation, (b) Hepatocytes derived from L-Rbpj mice show lower, whereas Cre- hepatocytes infected with Nl-IC adenovirus higher mTorcl activity.

Figures 16a - 16£: Notch Decoy protects from obesity-induced glucose intolerance and fatty liver, (a) Hepatocyte Notch antagonism with Decoy adenoviral transduction improves glucose tolerance and (b) reduces hepatic triglyceride content in HFD-fed mice, independent of change in (c) body weight or (d) adiposity. Notch Decoy (e) Actin increases insulin sensitivity (increased pAkt) while reducing mTorcl signaling (reduced p-S6K) , resulting in (f) less Srebplc cleavage and resultant lower Fasn/Accl gene product expression. *p<0.05 as compared to Fc control . Figures 17a - 17d: Hepatocyte Jagl expression increases with obesity, (a) Livers or (b) primary hepatocytes derived from leptin-deficient obese (ob/ob) mice and wildtype (WT) littermates sacrificed in the ad libitum state, prior to gene expression analysis of canonical Notch target genes, (c) Notch ligand expression in livers from overnight fasted WT mice or (d) obese, non-diabetic patients undergoing liver biopsy at time of bariatric surgery. *p<0.05, **p<0.01 and ***p<0.001 as compared to WT; ND = not detected.

Figures 18a - 18e: Notch decoy variants block ligand-specific Notch signaling. (a) Notch decoy variants schematic. (b) HEK 293 cells transiently transfected with Notchl and a Notch reporter construct (CSLx3-luciferase) were co-cultured with Hela cells transfected with Notch ligand Jaggedl or (c) Delta-like 1; co-cultured cells were treated with gamma-secretase inhibitor (GSI) , negative control (Fc) or parent (Nldl-24) and Notch decoy variants (Nldl-13 or NldlO-24) . (d) Hepalclc7 hepatoma cells transfected with CSLx3-luciferase, then exposed to Fc, parent Notch decoy (1-24) or the two experimental decoy variants (1-13 or 10-24) produced and secreted by HEK 293 cells, (e) Fc control, Nldl-13or NldlO-24 was expressed in liver of highfat diet fed mice by adenoviral transduction, liver RNA isolated and subjected to cD A synthesis and quantitative PCR for Glucose-6-phosphatase (G6pc) and Sterol response element binding protein (Srebplc) expression. *p<0.05, **p<0.01 and ***p<0.001 as compared to no treatment or Fc control .

Detailed Description of the Invention

Terms

As used in this application, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below .

"Administering" may be effected or performed using any of the methods known to one skilled in the art. The methods comprise, for example, intralesional, intramuscular, subcutaneous, intravenous, intraperitoneal, liposome-mediated, transmucosal , intestinal, topical, nasal, oral, anal, ocular or otic means of delivery.

As used herein, the term "composition", as in pharmaceutical composition, is intended to encompass a product comprising the active ingredient ( s ) and the inert ingredient ( s ) that make up the carrier, as well as any product which results, directly or indirectly from combination, complexation, or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients .

As used herein, "effective amount" refers to an amount which is capable of treating a subject having a tumor, a disease or a disorder. Accordingly, the effective amount will vary with the subject being treated, as well as the condition to be treated. A person of ordinary skill in the art can perform routine titration experiments to determine such sufficient amount. The effective amount of a compound will vary depending on the subject and upon the particular route of administration used. Based upon the compound, the amount can be delivered continuously, such as by continuous pump, or at periodic intervals (for example, on one or more separate occasions). Desired time intervals of multiple amounts of a particular compound can be determined without undue experimentation by one skilled in the art. In one embodiment, the effective amount is between about l]iq/ g - 10 mg/kg. In another embodiment, the effective amount is between about 10^g/kg - 1 mg/kg. In a further embodiment, the effective amount is 100ug/kg . "Extracellular domain" as used in connection with Notch receptor protein means all or a portion of Notch which (i) exists extracellularly (i.e. exists neither as a transmembrane portion or an intracellular portion) and (ii) binds to extracellular ligands to which intact Notch receptor protein binds. The extracellular domain of Notch may optionally include a signal peptide ("sp") . "Extracellular domain", "ECD" and "Ectodomain" are synonymous.

"Notch", "Notch protein", and "Notch receptor protein" are synonymous. In addition, the terms "Notch-based fusion protein" and "Notch decoy" are synonymous. The following Notch amino acid sequences are known and hereby incorporated by reference: Notchl (Genbank accession no. S18188 (rat)); Notch2 (Genbank accession no. NP_077334 (rat)); Notch3 (Genbank accession no. Q61982 (mouse)); and Notch4 (Genbank accession no. T09059 (mouse)). The following Notch nucleic acid sequences are known and hereby incorporated by reference: Notchl (Genbank accession no. XM_342392 (rat) and NM_017617 (human)); Notch2 (Genbank accession no. NM_024358 (rat), M99437 (human and AF308601 (human)); Notch3 (Genbank accession no. NM_008716 (mouse) and X _009303 (human)); and Notch4 (Genbank accession no. NM_010929 (mouse) and NM_004557 (human) ) .

"Notch decoy protein", as used herein, means a fusion protein comprising a portion of a Notch receptor protein which lacks intracellular signaling components and acts as a Notch signaling antagonist. Notch decoy proteins comprise all or a portion of a Notch extracellular domain including all or a portion of the EGF-like repeats present in the Notch extracellular domain. Examples of Notch decoy proteins include fusion proteins which comprise (a) amino acids, the sequence of which is identical to the sequence of a portion of the extracellular domain of a human Notch receptor protein and (b) amino acids, the sequence of which is identical to the sequence of an Fc portion of an antibody. In some Notch decoy proteins (b) is located to the carboxy terminal side of (a) . Some Notch decoy proteins further comprise a linker sequence between (a) and (b) . Notch decoy proteins can be selected from the group consisting of human Notchl receptor protein, human Notch2 receptor protein, human Notch3 receptor protein and human Notch4 receptor protein. In some Notch decoy proteins the extracellular domain of the human Notch receptor protein is selected from the group consisting of Notchl EGF-like repeats 1-36, Notchl EGF- like repeats 1-13, Notchl EGF-like repeats 1-24, Notchl EGF-like repeats 9-23, Notchl EGF-like repeats 10-24, Notchl EGF-like repeats 9- 36, Notchl EGF-like repeats 10-36, Notchl EGF-like repeats 14-36,

Notchl EGF-like repeats 13-24, Notchl EGF-like repeats 14-24, Notchl EGF-like repeats 25-36, Notch4 EGF-like repeats 1-29, Notch4 EGF-like repeats 1-13, Notch4 EGF-like repeats 1-23, Notch4 EGF-like repeats 9- 23, Notch4 EGF-like repeats 9-29, Notch4 EGF-like repeats 13-23, and Notch4 EGF-like repeats 21-29.

Examples of Notch decoy proteins can be found in U.S. Patent No. 7,662,919 B2, issued February 16, 2010, U.S. Patent Application Publication No. US 2010-0273990 Al, U.S. Patent Application Publication No. US 2011-0008342 Al, U.S. Patent Application Publication No. US 2011-0223183 Al A D PCT International Application No.

PCT/US2012/058662; the entire contents of each of which are hereby incorporated by reference into this application.

The terms "polypeptide," "peptide" and "protein" are used interchangeably herein, and each means a polymer of amino acid residues. The amino acid residues can be naturally occurring or chemical analogues thereof. Polypeptides, peptides and proteins can also include modifications such as glycosylation, lipid attachment, sulfation, hydroxylation, and ADP-ribosylation .

As used herein, "pharmaceutically acceptable carrier" means that the carrier is compatible with the other ingredients of the formulation and is not deleterious to the recipient thereof, and encompasses any of the standard pharmaceutically accepted carriers. Such carriers include, for example, 0.01-0.1 M and preferably 0.05 M phosphate buffer or 0.8% saline. Additionally, such pharmaceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions and suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases, and the like.

"Subject" shall mean any organism including, without limitation, a mammal such as a mouse, a rat, a dog, a guinea pig, a ferret, a rabbit and a primate. In one embodiment, the subject is a human.

"Treating" means either slowing, stopping or reversing the progression of a disease or disorder. As used herein, "treating" also means the amelioration of symptoms associated with the disease or disorder. Diseases include, but are not limited to. Tumor Angiogenesis , Atherosclerosis, Wound Healing, Retinopathy of Prematurity, Preeclampsia, Diabetic retinopathy, Ischemia, Stroke, Cardiovascular Disease, Psoriasis, lymphedema, tumorigenesis and tumor lymphangiogenesis, age-related macular degeneration (AMD) , wet AMD, pancreatic cancer and breast cancer.

As used herein, an "agents for the treatment of fatty liver disease" are any agent known to or thought to treat a fatty liver disease. Agents for the treatment of obesity include, but are not limited to vitamin E, selenium, betadine, metformin, rosiglitazone, pioglitazone, insulin sensitizers, antioxidants, probiotics, Omega-3 DHA, pentoxif lline, anti-TNF-alpha, FXR agonists and GLP-1 agonists.

Units, prefixes and symbols may be denoted in their SI accepted form. Unless otherwise indicated, nucleic acid sequences are written left to right in 5' to 3' orientation and amino acid sequences are written left to right in amino- to carboxy-terminal orientation. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. Embodiments of the Invention

The present invention provides a method of treating a subject suffering from a fatty liver disease which comprises administering to the subject an amount of a Notch decoy protein in an amount effective to treat the subject's fatty liver disease.

In one or more embodiments the fatty liver disease is alcoholic fatty liver disease.

In one or more embodiments the fatty liver disease is non-alcoholic fatty liver disease.

In one or more embodiments the subject is also suffering from metabolic syndrome .

In one or more embodiments the subject is also suffering from diabetes.

In one or more embodiments the subject is also suffering from hypertension .

In one or more embodiments the subject is also suffering from obesity.

In one or more embodiments the subject is also suffering from dyslipidemia .

In one or more embodiments the Notch decoy protein comprises (a) amino acids, the sequence of which is identical to the sequence of a portion of the extracellular domain of a human Notch receptor protein and (b) amino acids, the sequence of which is identical to the sequence of an Fc portion of an antibody.

In one or more embodiments the human Notch receptor protein is selected from the group consisting of human Notchl receptor protein, human Notch2 receptor protein, human Notch3 receptor protein and human Notch4 receptor protein.

In one or more embodiments the human Notch receptor protein is human Notchl receptor protein.

In one or more embodiments the human Notch receptor protein is human Notch2 receptor protein.

In one or more embodiments the human Notch receptor protein is human

Notch3 receptor protein.

In one or more embodiments the human Notch receptor protein is human Notch4 receptor protein.

In one or more embodiments the Fc portion of the antibody is the Fc portion of a human antibody.

In one or more embodiments (b) is located to the carboxy terminal side of (a) .

In one or more embodiments the Notch decoy protein further comprises a linker sequence between (a) and (b) .

In one or more embodiments the portion of the extracellular domain of the human Notch receptor protein is selected from the group consisting of Notchl EGF-like repeats 1-36, Notchl EGF-like repeats 1-13, Notchl EGF-like repeats 1-24, Notchl EGF-like repeats 9-23, Notchl EGF-like repeats 10-24, Notchl EGF-like repeats 9-36, Notchl EGF-like repeats 10-36, Notchl EGF-like repeats 14-36, Notchl EGF-like repeats 13-24, Notchl EGF-like repeats 14-24, Notchl EGF-like repeats 25-36, Notch4 EGF-like repeats 1-29, Notch4 EGF-like repeats 1-13, Notch4 EGF-like repeats 1-23, Notch4 EGF-like repeats 9-23, Notch4 EGF-like repeats 9-

29, Notch4 EGF-like repeats 13-23, and Notch4 EGF-like repeats 21-29.

The method of claim 18, wherein the portion of the extracellular domain of the human Notch receptor protein is Notchl EGF-like repeats 1-24.

In one or more embodiments the portion of the extracellular domain of the human Notch receptor protein is Notchl EGF-like repeats 1-36.

In one or more embodiments treating comprises reducing hepatic triglycerides .

In one or more embodiments the Notch decoy protein is administered in connection with a diet regimen.

In one or more embodiments the Notch decoy protein is administered in connection with an exercise regimen.

In one or more embodiments the Notch decoy protein is administered as a monotherapy .

In one or more embodiments the Notch decoy protein is administered in combination with one or more additional agents for the treatment of the fatty liver disease.

In one or more embodiments the one or more additional agents for the treatment of the fatty liver disease are selected from the group consisting of vitamin E, selenium, betadine, metformin, rosiglitazone, pioglitazone, insulin sensitizers, antioxidants, probiotics, Omega-3 DHA, pentoxifylline, anti-TNF-alpha, FXR agonists and GLP-1 agonists.

The present invention provides a composition comprising a pharmaceutically acceptable carrier and an amount of a Notch decoy protein effective to treat a fatty liver disease.

The present invention provides a package comprising:

(a) the pharmaceutical composition of the invention; and

(b) instructions for using the pharmaceutical composition of step (a) to treat the fatty liver disease.

For the foregoing embodiments, each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiments. Thus, all combinations of the various elements described herein are within the scope of the invention.

EXPERIMENTAL DETAILS

First Series of Experiments

Materials and Methods For First Series of Experiments

Antibodies

We purchased antibodies to phospho-Aktl (#2965) , phospho-p70 S6K

(#9205), total p70 S6k (#9202), phospho-mTor (#5536), total mTor (#2983), phospho-4E-BPl (#2855), total 4E-BP1 (#9644), raptor (#2280), rictor (#2114), Θβΐ (#3274), fatty acid synthase (#3189), acetyl-CoA carboxylase (#3676), tubulin (#2148), and actin (#8456) from Cell Signaling, FLAG M2 (F1804) and c-Myc (C3956) from Sigma, anti-Srebplc

(NB600-582) from Novus, and anti-Vall744-cleaved Notchl (ab52301) from Abeam.

In vivo inhibitor studies

Dibenzazepine (Syncom, 2μκιο1/!<: body weight), a γ-secretase inhibitor (GSI) , and rapamycin (Enzo, 2mg/kg body weight), were suspended in vehicle - 0.5% Methocel E4M (w/v, Colorcon) and 0.1% Tween-80 (Sigma) solution - and sonicated for two minutes to achieve a homogeneous suspension prior to daily (x5 days) intraperitoneal injection.48

Experimental animals

We crossed ALbumin-cre 23, Rbpjflox 49 and Foxolflox 50 mice on C57BL/6 background to generate albumin (ere) :Rbp-JKflox/flox (L-Rbpj) , albumin (ere) : Foxolflox/ ^' flox {L-Foxol) and albumin (ere) :Rbp-Jxflox/flox Foxolflox/flox [L-Rbpj/Foxol) mice; genotyping primers were previously described 20 and only male mice were studied. Mice were weaned to either standard chow (Purina Mills #5053) or high-fat diet (Harlan

Laboratories TD.06414). Wildtype C57B1/6 (strain #662) and leptin- deficient ob/ob (strain #632) male mice were purchased from Jackson Labs. The Columbia University IACUC has approved all animal procedures.

Metabolic analyses

Blood glucose was measured by glucose meter (OneTouch) and plasma insulin by ELISA (Millipore) . We performed glucose tolerance tests after a 16-hr (6ΡΜ-10Ά ) fast using intraperitoneal injection of 2g/kg body weight glucose. Hepatic lipids were extracted, 51 normalized by either liver weight or protein, and confirmed by Oil Red O staining of snap-frozen liver sections. We used colorimetric assays to measure triglyceride (Thermo) , cholesterol E (Wako) and non-esterified fatty acid (Wako) . Hepatic de novo lipogenesis was determined by measuring the amount of newly synthesized FA, as resolved by TLC, present in the liver 1 h after intraperitoneal injection of lmCi of 3H20.29 Triglyceride secretion rate was measured after injection of Poloxamer

407, with serial measurement of plasma triglycerides .52

Hepatocyte studies

We isolated and cultured primary mouse hepatocytes as described.20 For gene and protein expression studies, we pre-treated hepatocytes with 50nM rapamycin (Cell Signaling) or vehicle for 30 min, followed by 6 h with ΙΟηΜ insulin (Sigma) . We measured fatty acid oxidation as described, 53 with the following modifications: primary hepatocytes were incubated serum-free medium with 1.5% fatty-acid free BSA (Sigma) containing 0.1 mM cold oleic acid and l]iCi 14C-oleic acid (PerkinElmer Life Sciences) for 4 h. Labeled medium was transferred to flasks; 200 μΐ of 70% perchloric acid was injected into the bottom of the flask, lOOuL of 1M OH was injected onto filter paper held by a center well, and the flasks were incubated for an additional 1 hour. Trapped 14C02 on the alkalinized filter paper was measured as described.53 We measured lipogenesis as described, 11 with the following modifications: hepatocytes were stimulated with ΙΟηΜ insulin in serum-free DMEM for 2 h, then labeled with 14C-acetate (PerkinElmer Life Sciences) for 2 h. After incubation with 3:2 hexane : isopropanol for 3 h, extracted lipids were dried under N2 gas, then resuspended in 2:1 chloroform:methanol prior to separation of lipid species by TLC and counting of labeled triglycerides. Counts were normalized to total cellular protein. All primary hepatocyte experiments were finished within 36 h after plating.

Quantitative RT-PCR

We isolated RNA with Trizol (Invitrogen) or RNeasy mini-kit (Qiagen) , synthesized cDNA with Superscript III RT (Invitrogen), and performed qPCR with a DNA Engine Opticon 2 System (Bio-Rad) and DyNAmo HS SYBR green (New England Biolabs) . mRNA levels were normalized to 18s using the ΔΔ0 ( t) method and are presented as relative transcript levels.24 Primer sequences are available upon request.

Adenovirus studies

Notchl-IC, Notch decoy (1-24), Fc and GFP adenoviruses have been described.24, 34, 54 We transduced primary hepatocytes or HEK 293 cells at MOI 5 and FAO hepatoma cells at MOI 200, to achieve 90-100% infection efficiency as assessed by GFP expression. For in vivo studies, we injected 1x109 purified viral particles (Viraquest) /g body weight via orbital sinus; we performed metabolic analysis on days 3-5 and sacrificed the animals at day 7 or 14 post-injection. We limited analysis to mice showing 2-5-fold hepatic Notchl overexpression or detectable hepatic Notch decoy or Fc expression by Western blot. Luciferase assays

We transfected (Lipofectamine 2000, Invitrogen) FAO hepatoma cells or primary hepatocytes with a luciferase construct (Addgene #8890) containing the proximal (-220 to +25) Fasn promoter sequence.55 In some experiments, we co-transfected plasmids containing shRNA to Raptor (Addgene #21339 or #21340) or Rictor (Addgene #21341), with scrambled shRNA (Addgene #1864) as a control, 35 and/or transduced cells with Notchl-IC or control (Fc) adenovirus. 24 h after transfection, FAO cells were transferred to serum-free medium with or without ΙΟΟηΜ insulin (Sigma) for 16 h prior to lysis and luciferase measurements as described.55

Immunoprecipitations

HEK 293, FAO, and primary hepatocytes were lysed in 0.3% or 0.6% CHAPS- containing buffer, 38 followed by immunoprecipitation for 2-h at 4oC, and overnight elution prior to Western blot analysis.56

Statistical Analysis

We used two-way ANOVA to analyze the data. All Westerns were quantitated using NIH ImageJ software. Data represent means ± SEM.

Results

Hepatic Notch action peaks twice, after prolonged fasting and at late refeeding

Notchl activation in liver, as reflected by cleavage at Vall744 and increased expression of Notch targets, increased with fasting.20 In early refeeding (0-2 h) , Notchl cleavage and target gene expression declined, followed by a second peak of Notch activation at later time points (4-12 h) (Fig. la, b and Fig. 2). Notably, Notch activation during fasting coincides with increased gluconeogenic gene expression, while the second peak coincides with expression of Srebplc and its targets (Fatty acid synthase, Fasn; and Acetyl-CoA-carboxylase, Accl) (Fig. lc-e) , as well as activation of mTor (not shown) . This induction was expectedly absent in livers from mice lacking hepatocyte Rbp-Jk (L- Rbpj) (Fig. If), 20 confirming that classical Notch activation is affected by the nutritional state. Livers from mice fed a high-fat diet (HFD) also showed greater Notch activation than chow-fed littermates (Fig. lg, h) , as did hepatocytes and livers from leptin-signaling deficient mice (Fig. li, j), suggesting a cell-autonomous dysregulation of Notch signaling in obesity and fatty liver.

Liver-specific deletion of Rbp-Jk protects from diet-induced steatosis

As whole-body disruption of Rbp-Jk results in embryonic lethality, 16 we generated liver-specific Rbp-Jk knockout [L-Rbpj) mice, in which hepatocyte Rbp-Jk was deleted post-natally, 20 with full recombination by 6-12 weeks of age.23 We have previously shown that chow- or HFD-fed L-Rbpj mice are protected from insulin resistance .20 Given the interaction between Rbp-Jk and Fox01,24 we hypothesized that L-Rbpj mice would have similarly increased hepatic triglyceride as mice lacking liver FoxOs.25,26 L-Rbpj mice showed normal body weight under different diets (Fig. 3a) , but markedly lower HFD-induced hepatic steatosis, due to a 30-50% reduction in hepatic triglycerides without effects on hepatic cholesterol levels (Fig. 3c, d) . L-Rbpj mice showed reduced liver weight without changes in adiposity (Fig. 3d) or serum lipids (Fig. 4a, b) . Reduced hepatic triglyceride content was also seen in chow-fed or short-term (3 week) HFD-fed L-Rbpj mice. Moreover, Rbp- Jk knockout prevented steatosis in mice lacking hepatic FoxOl (Fig. 4c), 25 suggesting that the Notch pathway regulates hepatic lipid deposition independent of FoxOl .

L-Rbpj mice show reduced de novo lipogenesis

We evaluated cell-autonomous and non-autonomous pathways that regulate hepatic triglyceride accumulation .2 , 27 VLDL secretion was unaltered in L-Rbpj mice (Fig. 3e) , as were liver expression of fatty acid oxidation enzymes Acox and Cptla, serum ketones, β-oxidation of exogenous fatty acids in primary hepatocytes (Fig. 3f-h) , and plasma triglyceride levels after olive oil gavage (Fig. 4d) . Next, we studied lipogenesis - L-Rbpj livers showed reduced Fasn and Accl expression (Fig. 3i) , and a trend towards reduced fatty acid production after injection of tritiated water (Fig. 3j). In Rbp-Jk-deficient primary hepatocytes, we found significantly repressed 14C-acetate incorporation into triglyceride (Fig. 3k), reduced insulin-dependent Srebplc expression (Fig. 31) , and reduced expression of a luciferase reporter construct driven by the proximal Fasn promoter containing a consensus Srebplc binding site28 (Fig. 3m) . Alternative lipogenic pathways, including

PPARy signaling, were unaltered in L-Rbpj mice (Fig. 4e).29 These data indicate that blocking hepatic Notch results in reduced hepatic triglyceride, likely due to impaired lipogenesis. We observed a similar protection from insulin resistance associated with reduced hepatic lipid content following short-term HFD (Fig. 5) .

Reduced mTorcl signaling in L-Rbpj mice

We studied the main signaling pathways implicated in lipogenesis, insulin/Akt and nutrient/mTor .3 As we reported, insulin signaling was increased in L-Rbpj liver, with increased Akt phosphorylation at the Pdkl site, T308.20 Conversely, we noted a marked reduction of hepatic mTorcl signaling, as indicated by decreased phosphorylation of mTor and mTorcl targets, p70 S6 kinase and 4E-BP1 (Fig. 3n).30-32 To determine if this effect was cell-autonomous, we isolated primary hepatocytes from Cre- and L-Rbpj mice, and found that Akt phosphorylation was higher (data not shown) , while basal and insulin-stimulated p70 S6k phosphorylation were lower (Fig. 3o) . These data suggest that Notch- dependent transcriptional activity is required for hepatocyte rtiTorcl activit .

Acute Notch inhibition protects from diet-induced insulin resistance and fatty liver

Given these surprising findings, and to exclude the possibility of a developmental phenotype in L-Rbpj mice, we tested if acute inhibition of Notch signaling can similarly protect from diet-induced fatty liver and reduce mTorcl function. We transduced adult mice with a "decoy" Notchl receptor that encodes only the extracellular domain33,34 and acts in a dominant-negative manner by sequestering endogenous ligand. Adenovirus-driven Notchl decoy is preferentially expressed in the liver, and is poorly secreted into the circulation (data not shown) . Consistent with results from L-Rbpj mice, Notch decoy administration to HFD-fed mice lowered glucose and insulin levels (Fig. 6a, b) , and reduced liver weight and triglyceride content (Fig. 6c, d) , without affecting body or adipose weight (Fig. 7a, b) . Notch decoy reduced Srebplc cleavage, and Fasn and Accl expression (Fig. 6e) . We observed no difference in fatty acid oxidation genes (Fig. 6f) or serum lipids (Fig. 7c, d) . We transduced primary hepatocytes with Notch decoy and observed reduced Srebplc expression (Fig. 6g) , but no change in Ppary or its targets (Fig. 7e) , suggesting that acute inhibition of hepatocyte Notch reduces Srebplc-directed lipogenesis in a cell- autonomous manner. Similar to L-Rbpj mice, livers from Notch decoy- transduced mice demonstrated increased pAkt-T308, but lower pS6k-S389 (Fig. 6h) . These data indicate that acute reduction in Notch signaling increases insulin sensitivity, while lowering mTorcl and hepatic triglyceride content.

Hepatic overexpression of Notchl induces mTorcl signaling and fatty liver

Our loss-of-function studies suggest that Notch signaling is permissive for mTorcl activation and diet-induced steatosis. We thus tested whether Notch gain-of-function would be sufficient to increase mTorcl function and induce fatty liver in vivo. Chow-fed mice transduced with adenovirus encoding constitutively active Notchl (Nl-IC) showed a ~40% increase in hepatic triglyceride and increased liver weight (Fig. 8a- c) , without concomitant changes in body weight or composition (data not shown) . Nl-IC-transduced livers demonstrated higher Srebplc cleavage, and increased expression of Srebplc and Fasn (Pig. 8d, e) . Consequently, primary hepatocytes transduced with Nl-IC showed greater lipogenesis (Fig. 8f) . Importantly, Nl-IC expression failed to increase hepatic lipid, gene expression and fatty acid synthesis in L-Rbpj mice and hepatocytes (Fig. 8g-i) , suggesting that Notch-induced lipogenesis requires Rbp-Jk, similar its activation of hepatic glucose production .20

The increase of lipogenic genes induced by Nl-IC was paralleled by increased hepatic mTorcl activity in fasted and (more markedly) refed animals (Fig. 8j-m), consistent with enhanced physiologic regulation of mTor activity. Similarly, activation of mTorcl signaling by insulin and amino acids was potentiated by Nl-IC (Fig. 8n) , suggesting that Notch modulates but does not override endogenous mTor regulation. To confirm that induction in mTorcl signaling and lipogenic gene expression is cell-autonomous, we transduced primary hepatocytes with Nl-IC, and detected increased mTor signaling, greater Srebplc cleavage and higher levels of Fasn protein and mRNA (Fig. 8o) . In hepatoma cells, Notch activity correlated with Fasn-luciferase reporter activation, again consistent with a cell-autonomous effect (Fig. 8p) .

Inhibition of mTor prevents Notch-induced lipogenic gene expression and fatty liver

To test the hypothesis that Notch induction of lipogenic gene expression and fatty liver requires mTorcl signaling, we co-transfected hepatoma cells with Fasn-luciferase and shRNA to Raptor, 35 the defining component of the mTorcl complex, then transduced cells with Nl-IC adenovirus. Nl-IC promoted basal as well as insulin-stimulated Fasn- luciferase activity; Raptor shRNA reversed both effects, which was potentiated by insulin, but reversed by Raptor knockdown (Fig. 9a) . We saw similar results with a second shRNA to Raptor, as well as with rapamycin treatment (Fig. 10a, fa), suggesting that Nl-IC-induced Fasn expression is mTorcl-dependent . Similarly, Notch-induction of endogenous Fasn in primary hepatocytes was augmented by insulin, and suppressed by rapamycin (Fig. 9b) , confirming that Notch activates lipogenesis through mTorcl, and not through secondary effects on insulin signaling.

Based on these data, we hypothesized that the increase in lipogenic gene expression and fatty liver seen in mice transduced with Nl-IC adenovirus would be ameliorated by rapamycin treatment. Indeed, Nl-IC increased hepatic triglyceride and lipogenic gene expression in vehicle-treated mice, while these effects were completely reversed by rapamycin treatment (Fig. 9c, d, as compared to Fig. 8d, e) . The effect of rapamycin was specific to Notch induction of lipogenic genes, as Heyl and Heyl were unaffected (Fig. 9d) . Similarly, although rapamycin induced mild glucose intolerance (data not shown), 5 Nl-IC-transduced mice showed further exacerbation (Fig. 9e, f) . These data show that

Notch induction of hepatic steatosis, but not its induction of glucose intolerance, can be reversed by rapamycin treatment.

Notch increases mTorcl complex stability

To study the mechanism of altered Notch-induced mTorcl activation, we examined mTor complex levels in L-Rbpj mouse liver. We found unchanged levels of the shared mTorcl/mTorc2 components, mTor and Gpl, and of the mTorc2-specific component Rictor, but a surprising reduction in the levels of Raptor protein (Fig. 11a) , independent of changes in Raptor mRNA (not shown) , suggesting that the effects of Rbp-Jk deficiency are post-transcriptional . Conversely, mice transduced with Nl-IC adenovirus demonstrated increased Raptor in liver (Fig. lib) . We found a similar increase of endogenous Raptor protein in hepatoma cells (Fig. lie) or primary hepatocytes (not shown) transduced with Nl-IC, without changes in Raptor mRNA (Fig. 12a) . Transient transfection of Raptor cDNA in primary hepatocytes showed a similar effect, demonstrating that the action of Notch is independent of locus effects (Fig. 12b) . Interestingly, the effect of Nl-IC was independent of proteosomal inhibition by MG132 (Fig. 11c) , but was fully reversed by treatment of hepatocytes with the protein synthesis inhibitor, cycloheximide (Fig. lid) . Raptor overexpression did not suffice to induce Fasn-luciferase, consistent with previous work that Raptor overexpression per se does not increase mTorcl function, 34, 35 whereas co-expression of Nl-IC and Raptor produced a synergistic effect (Fig. lie) . Likewise, overexpression of Raptor was insufficient to activate mTorcl in either primary hepatocytes or HEK 293 cells (data not shown) . We conclude that Notch induction of Raptor levels parallels, but does not cause increased mTorcl activation, and hypothesized that increased Raptor levels are secondary to higher mTorcl complex stability. Indeed, we found that Notch overexpression increased association among mTorcl components in HEK 293 cells (Fig. llf and Fig. 12c) , and primary hepatocytes (Fig. llg) . Notch-stabilized mTorcl complexes were resistant to increasing concentrations of CHAPS detergent known to disrupt the mTor-Raptor interaction (Fig. 12d) .36-38 These data indicate that the Notch stabilizes and activates mTorcl, resulting in increased de novo lipogenesis and fatty liver.

Discussion For First Series of Experiments

The homeostatic functions of Notch in the adult animal have received less attention, except in neoplastic processes .19 We have shown that liver Notch signaling is regulated in response to metabolic stimuli, and that Notchl increases hepatic glucose production by co-activating FoxOl at the Glucose-6-phosphatase promoter.20 Conversely, liver- specific deletion of Rbp-Jk (L-Rbpj mice) , or γ-secretase inhibitor (GSI) treatment improves glucose tolerance, and reduces hepatocyte glucose production.20 Interestingly, previous studies demonstrated that Notchl can activate mTorcl in leukemic cells, whereas GSIs decrease mTorcl activity in breast cancer.21, 22 Thus, we hypothesized that hepatic Notch could modulate the coordinate actions of insulin on gluconeogenesis (via FoxOl) and lipogenesis (via mTorcl) . We describe here that inhibition of hepatic Notch protects from obesity-induced fatty liver, likely through decreased de novo lipogenesis. Conversely, constitutive hepatic Notch signaling increases lipogenesis, fatty liver and activation of hepatic mTorcl signaling, by stabilizing the mTorcl complex. We show that Notch-mediated hepatic steatosis is rapamycin- sensitive, whereas Notch-induced glucose tolerance is mTor-independent . These results establish Notch as a unique pharmacological target in liver, whose inhibition can prevent the twin abnormalities of hepatic insulin resistance - excessive glucose production as well as fatty liver - by virtue of its ability to uncouple Akt from itiTor.

The role of developmental pathways in metabolic homeostasis of adult tissues is only beginning to be appreciated .39 We have shown that genetic or pharmacologic inhibition of Notch protects from diet-induced glucose intolerance, without effects on body weight or adiposity, in a FoxOl-dependent manner.20 In this work, we demonstrate a similar protection from fatty liver with inhibition of hepatic Notch signaling. This is unexpected, as inhibition of hepatic FoxOl is associated with increased hepatic lipid deposition, 25,25,40,41 an increasingly recognized effect of shifting hepatic carbon flux from glucose to lipid production . 2 In this regard, it appears that chronic (L-Rbpj mice) or acute Notch inhibition (Notch decoy) , achieves the long-sought goal of decreasing hepatic glucose production without compensatory increases in hepatic lipid content. Interestingly, GSIs also induce fatty liver, but do so in a Notch-independent fashion (U.B.P., manuscript in preparation) , consistent with the idea that substrates of γ-secretase include Notch-unrelated pathways, and restricting the repertoire of therapeutically viable Notch inhibitors that can be pursued for treatment of metabolic disease. Nonetheless, the many potential benefits of Notch inhibition, which include amelioration of atherosclerosis, 43 provide in our opinion a strong rationale to pursue Notch inhibition as a treatment of the metabolic syndrome.44

The identification of Notch as a regulator of carbon flux towards hepatic glucose or lipid production (Fig. Hi) is a conceptual advance, as is the surprising finding that a molecular pathway thought to be specialized toward differentiation is regulated by physiologic (fasting/re-feeding), as well as pathologic (insulin resistance) metabolic cues in hepatocytes. We hypothesize that in the overfed and insulin-resistant state, Notch signaling is inappropriately activated, and reprises its developmental interactions with FoxOl and mTorcl . The mechanisms underlying nutritional activation of hepatic Notch require further clarification. For example, it should be determined whether Notch activation in the hepatocyte requires input from neighboring hepatocytes or other resident liver cells (endothelial, stellate, Kupffer, etc.) - Similarly, which of the five Notch ligands drives signaling in response to nutrients is unknown, and the possibility that different ligands signal in different metabolic states to direct carbon flux or drive differentiation is teleologically attractive.

Besides the further validation of hepatic Notch as a therapeutic target, our data demonstrate a physiologic, and potentially pharmacologic, means of regulating mTorcl activity and lipogenesis. Previous studies have indicated that tight control of hepatic mTorcl signaling is critical for hepatic lipid metabolism.9, 11 The tandem findings of mTorcl stabilization and activation by Notch deserve further study. Since the identification of Raptor as the mTorcl- regulatory subunit, it has been known that the mTor-Raptor association is sensitive to detergent concentrations 38 subsequent reports have confirmed this finding and identified potential post-translational modifications on Raptor, 36, 37 , 45 but none have been shown to mediate mTor-Raptor interaction. How Notch induces mTorcl stability, and how precisely that translates to greater mTorcl activation remain unclear. The demonstration that Raptor levels are decreased in L-Rbpj mice and that cycloheximide prevents Notch-induced stabilization indicates that a transcriptional target (s) of Notch regulates complex stability.

In summary, Notch antagonism uncouples Akt from mTor activation, suggesting that Notch antagonists from oncology and neuroscience46, 47 may be repurposed to treat fatty liver and diabetes. Furthermore, as Notch-mediated mTorcl activation does not appear to be cell type- specific, modulators of mTorcl processing and degradation may represent a therapeutic avenue to blockmTorcl activity without the metabolic liabilities of current mTor inhibitors.5

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Second Series of Experiments

T2D and NAFLD are inadequately treated with currently available therapy.

Obesity leads to insulin resistance, which begets the fasting hyperglycemia of Type 2 diabetes (T2D) . ¹ In a parallel process, compensatory hyperinsulinemia drives hepatic de novo lipogenesis, ² mediated in part by the nutrient-sensitive mechanistic target of rapamycin (mTOR) pathway. ³ Increased lipogenesis, coupled with excess fatty acid flux to liver and impaired ability to catabolize and export these fatty acids, ⁴ produces non-alcoholic fatty liver disease (NAFLD).

NAFLD may be associated with hepatocellular damage and inflammation which predisposes to cirrhosis and hepatocellular carcinoma, but also further exacerbates hepatic insulin resistance through activation of FoxOl, ⁵ the key transcriptional activator of hepatic glucose production. ¹ This vicious cycle results in coincident NAFLD and T2D, which show independent associations with cardiovascular disease and all-cause mortality. ⁶

There is no approved pharmacologic therapy for NAFLD, and although there are multiple T2D therapies available, none show durability and long-term efficacy. ⁷ Novel pathways are sought to both further our understanding of the pathophysiology of insulin resistance as well as provide potential new pharmaceutical targets to assist in our management of obesity-related morbidity and mortality.

Notch bridges two nutrient-sensitive pathways in hepatocytes insulin/FoxOl and nutrient/mTORCl .

In metabolically healthy liver, insulin represses glucose production, primarily by Akt-mediated phosphorylation and nuclear exclusion of FoxOl, ⁸ and promotes fatty acid synthesis from acetate (lipogenesis) by both transcriptional and non-transcriptional increase in Srebplc activity. ^9"12 In the insulin-resistant state, however, FoxOl-mediated hepatic glucose production is unrestrained, resulting in hyperglycemia, ¹³ but insulin stimulation of Srebplc function is "paradoxically" increased, contributing to fatty liver. ¹ ' ⁵ Notch is an evolutionarily conserved regulatory pathway of normal development, and is inappropriately re-activated in leukemia and other tumors. ¹⁵ It is in these contexts that Notch has been shown to intersect with insulin and nutrient signaling pathways. FoxOl physically interacts with the Notch transcriptional effector, Rbp-Jk, to co-regulate Notch-mediated differentiation processes. ¹⁶ In addition. Notch activates mTOR complex 1 (mTORCl) signaling in leukemic cells, and pharmacologic Notch inhibitors reduce mTORCl-mediated oncogenic potential. ¹⁷ - ¹⁸ These observations provoked our hypothesis that Notch may similarly interact with the FoxOl and mTORCl signaling pathways in normal tissue, and may modulate the coordinate actions of insulin on hepatic glucose production (via FoxOl) and lipid synthesis (via mTORCl) .

The feasibility of this hypothesis depended on whether Notch signaling is present in developed liver - this was a legitimate question, as Notch is so critical to normal differentiation, ^19,20 that its potential role in fully differentiated tissue has not been adequately explored. Our initial characterization in murine liver demonstrated that the Notch pathway is active in healthy, adult mice and physiologically modulated by nutrient availability, but markedly increased in mouse models of obesity and insulin resistance. ^21,22 These results prompted a similar survey in liver biopsy specimens from patients -while Notch activity was evident in all patient liver samples, we observed maximal Notch activation in patients with both T2D and NAFLD . ²³ These data suggested that the Notch pathway is functional in developed rodent and human liver and inappropriately stimulated in metabolic disease.

Inhibition of hepatic Notch signaling leads to weight-independent improvements in hepatic glucose and lipid metabolism.

We next hypothesized that increased Notch activity is causative of, and not just correlated to, obesity-induced T2D and NAFLD. To investigate the repercussion of decreased Notch signaling, the most efficient means is to disrupt the common transcriptional effector of all 4 Notch receptors, Rbp-Jk. ² ' As Rbp-Jk knockout animals show embryonic lethality, ¹⁹ we generated liver-specific Rbp-Jk knockout (Albumin- cre.-Rbpj ^{fl fl} mice, henceforth L-Rbpj) mice, which show gradual, post- natal recombination in hepatocytes . ^2l ' ²⁵ L-Rbpj mice showed no developmental defects, and normal liver histology, and gain weight at a comparable rate as control animals (Figure 13a) . As Rbp-Jk synergizes with FoxOl, the key transcriptional mediator of hepatocyte glucose production, to modulate differentiation, ¹⁶ we hypothesized that L~Rbpj mice would show reduced hepatic glucose production similar to liver- specific FoxOl knockout (L-Foxol) mice. As predicted, L-Rbpj mice are protected from high-fat diet (HFD) -induced glucose intolerance (Figure 13b) , which we established as cell-autonomous, by direct FoxOl- independent binding of Rbp-Jk to the Glucose-6-phosphatase (G6pc) promoter . ²¹

L-Rbpj mice show reduced lipogenesis, leading to protection from fatty liver .

We predicted that L-Rbpj mice would have similarly increased hepatic triglyceride (TG) as mice lacking liver FoxOs. ^26,27 Unexpectedly, L- Rbpj mice were protected from HFD-induced hepatic steatosis (Figure 13c) . We hypothesized a cell-autonomous mechanism for the observed decrease in liver TG, but broadly evaluated potential causes, in L-Rbpj mice. ^2,28 In summary, we found:

Cell non-autonomous:

• Intestinal absorption of dietary, or exogenous (gavaged) , lipids was unaltered.

• Adipose lipolysis was unchanged, with unchanged Atgl and Hsl expression and normal free fatty acids.

Cell-autonomous :

• VLDL secretion was unchanged, leading to normal plasma TG and similar response in Cre ^~ and L-Rbpj mice to lipoprotein lipase inhibition with Poloxamer 407. ²⁹

• Liver expression of fatty acid oxidation enzymes Acox and Cptla, serum ketones and β-oxidation of exogenous fatty acids in primary hepatocytes were unchanged. Next, we studied lipogenesis - first, we measured expression of lipogenic proteins, with focus on Fatty acid synthetase (Fasn) and Acetyl-CoA-Carboxylase (Accl) , rate-limiting enzymes in the manufacture of long-chain fatty acids from two-carbon precursors. ²⁸ L-Rbpj mice express less liver Fasn and Accl (Figure 14a} ; correspondingly, hepatocytes derived from L-Rbpj mice showed lower fatty acid synthesis (Figure 14b) . As Fasn and Accl are transcriptional targets of the insulin and nutrient-activated, lipogenic transcription factor Srebplc, ^9,14,30 we hypothesized that L-Rbpj mice have decreased Srebplc activity. Indeed, we found impaired insulin-dependent Srebplc expression, and activity, as assessed by lower expression of Fasn promoter-driven luciferase containing a consensus Srebplc binding site ³¹ (Figure 14c, d) .

These data suggest that lower hepatic TG in L-Rbpj mice is due to impaired Srebplc-mediated lipogenesis. We next studied pathways that converge on Srebplc - insulin/Akt and nutrient/mTOR. ³ The mTOR protein kinase functions in two multi-protein complexes which have multiple common (mTOR, Οβΐ, deptor) and several unique components, most notably Raptor for mTORCl, and Rictor for mTORC2. ³² Activation of the nutrient- sensing mTORCl pathway stimulates hepatic de novo lipogenesis, ³ regulating insulin and Srebplc-dependent transcription of key lipogenic genes to cause hepatic steatosis. ^3,33 L-Rbpj livers show higher insulin sensitivity, with higher Akt phosphorylation (Figure 15a). ²¹ Conversely, we noted repressed phosphorylation of canonical mTORCl targets, p70 S6 kinase and 4E-BP1, ^34-36 in liver and in primary hepatocytes (Figure 15a, b) . These data suggest that Notch is required for maximal hepatocyte mTORCl activity.

Notch is a novel "druggable target" for both T2D and NAFLD/NASH .

Despite its clear importance in regulation of hepatic insulin sensitivity, FoxOl is a poor drug target due to its nuclear location and the lack of a ligand-binding domain. Similarly, available mTOR inhibitors have multiple liabilities, including non-specificity to the two mTOR-containing complexes (mTORCl and mTORC2) ³⁷ that have distinct functions, ³⁸ resulting in metabolic side-effects including glucose intolerance and dyslipidemia . ^{39, 40} In contrast, Notch signaling is therapeutically accessible, given its plasma membrane location, well- defined ligand-binding domain and equally well-characterized downstream signaling cascade. ¹⁵ Simultaneous improvement in whole-body glucose and lipid metabolism in the absence of altered weight or adiposity, resulting in reduced atherosclerosis, is quite rare, and suggests Notch as a novel means of reducing metabolic disease burden as obesity rates continue to rise.

Notch Decoy prevents diet-induced glucose intolerance and fatty liver.

To confirm our findings in the L-Rbpj mouse model, we tested whether acute inhibition of Notch signaling with Notchl decoy receptor

(henceforth. Decoy) that encodes only the extracellular domain"- ⁴² and acts in a dominant-negative manner by sequestering endogenous ligand, can similarly protect from diet-induced glucose intolerance and fatty liver. Consistent with results from L-Rbpj mice, Notch decoy administration to HFD-fed mice improved glucose tolerance (Figure 16a) and lowered liver TG (Figure 16b) . Body weight and adiposity were unaffected (not shown) , suggesting the metabolic improvements in Decoy- treated mice were mediated through weight-independent effects in the liver. In fact, similar to L-Rbpj liver, Decoy-treated mice showed higher liver insulin sensitivity with a parallel reduction in mTorcl activity (Figure 16c) , leading to reduced lipogenic protein expression (Figure 16d) . Beyond a proof of principle of therapeutic potential of Notch inhibition, these data suggest that Notch ligand-specific inhibitors will reap similar metabolic benefits without known side- effects of non-specific Notch inhibitors. ⁴³ -"

Jaggedl is the primary Notch ligand in liver.

We next hypothesized that specific inhibition of only the relevant Notch ligand in liver may show the same benefit while minimizing potential side-effects. By differential centrifugation after collagenase perfusion, we can achieve near-100% purity of hepatocytes and non-hepatocyte fractions from whole liver. Hepatocytes isolated from obese, insulin-resistant DIO or ob/ob mice show increased Notch activity (not shown and Figure 17a) , and a proportionate increase in expression of Jaggedl {Jagl) ; we observe no meaningful change in other ligands or any ligand in the non-hepatocyte fraction (Figure 17b, and not shown) . This is consistent with recent data demonstrating that Jagl expression in endothelial cells is increased in hyperglycemia, resulting in altered normal Notch-mediated angiogenesis , an effect recapitulated with Jaggedl overexpression or rescued with shRNA to

Jaggedl. ⁴⁵ Of note, Jagl is expressed at 2-3x higher levels in mouse and human liver than the next most abundant ligand, Jag2 (Figure 17c, d) .

Jagged-specific inhibition with the Notch . Decoy variant, Nld 1-13, reduces gluconeogenic and lipogenic gene expression.

Our group has developed Decoy variants Nldi and Nldio based on number of EGF repeats, which block Notch signaling in a ligand-specific manner (Figure 18a) . Whereas parent Notch decoy blocks both Jaggedl/2 and Dll-l/4-mediated Notch signaling, Nldi only inhibits Dll-1/4- induced and Nldio only Jaggedl/2 -induced signaling (Figure 18b, c) .

We transfected hepatoma cells with a Notch-responsive luciferase reporter, then added Notch-decoy transfected and secreted ("conditioned media") from HEK 293 cells. We found that either parent Decoy or Nldio- conditioned media, but not Nldi blocked endogenous Notch activity (Figure 18d) . This critical experiment suggests that hepatocyte- hepatocyte interactions are sufficient to activate Notch signaling in vitro, and that this signal likely arises from Jagged ligands.

We next tested whether Nldio would block gluconeogenic and lipogenic gene expression. We transduced obese mice with Fc (control), Nldi or Nldio adenoviruses . After sacrifice, we isolated livers, and prepared liver cDNA for quantitative PGR of these critical Notch targets. Mice transduced with Nldio but not control or Delta-like-specific adenovirus, showed decreased obesity-induced activation of G6pc and Srebplc expression (Figure 18e) . Of note, the level of inhibition of these key gluconeogenic and lipogenic genes was similar to what was previously seen with parent Decoy (not shown) . This result suggests that Jagged-specific inhibition of hepatic Notch signaling is sufficient to protect from obesity-related metabolic complications, and opens up the possibility of a safe and effective Notch antagonist for treatment of T2D and NAFLD. References For Second Series of Experiments

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