HEPATIC STEATOSIS

RELATED APPLICATIONS

[0001 ] This application claims the benefit of priority from U.S. provisional application no. 62/133,630 filed on March 16 ^th , 2015, which is incorporated herein by reference in its entirety.

FIELD

[0002] The present invention relates to field of metabolic disorders and more specifically to the use of 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF) for the treatment or prevention of disorders related to lipid metabolism.

INTRODUCTION

[0003] Excessive caloric intake combined with increasingly sedentary lifestyles is producing an epidemic of overweight and obesity, affecting more than 35% of the world's adults (Ng et al., 2014). The cluster of metabolic disturbances associated with increased adiposity, called the metabolic syndrome (MetS), is characterized by high abdominal adiposity, hypertension, high fasting plasma glucose, hypertriglyceridemia, and low high-density cholesterol (HDL) (Despres and Lemieux, 2006). These factors substantially increase the risk of diabetes, fatty liver disease, stroke, and atherosclerosis, which are directly attributed to 1 in 5 deaths in the United States annually (Eckel et al., 2005; Masters et al., 2013). The underlying pathophysiology of MetS is based in dysregulated lipid metabolism, resulting in aberrant lipid storage in the liver and muscle, increased insulin resistance, and altered circulating lipoprotein levels (Avramoglu et al., 2006; Bergman and Ader, 2000; Cao et al., 2008; Ginsberg, 2006). While this dyslipidemia is largely attributed to a ubiquitous increase in release of free fatty acids (FFAs) from the adipose tissue, systemic lipid profiling has revealed strong correlations between MetS and a specific fatty acid signature of serum lipids, rather than elevated lipids in general (Warensjo et al., 2005). Thus, individual lipids from the diet, micobiome, and de novo synthesis influenced by genetics and environment that are characteristic of the MetS plasma lipomic signature may play a more significant role in the regulation of systemic metabolic homeostasis than previously thought.

[0004] Metabolomic screening has now become a widely employed method for examining the lipid signature of MetS, insulin resistance, and diabetes (Bain et al., 2009; Fiehn et al., 2010; Friedrich, 2012; Prentice et al., 2014; Wang et al., 201 1 ). Several specific fatty acids, as well as some of their metabolic by-products, have been strongly implicated in directly altering metabolic homeostasis. For example, C16:0 (palmitic acid) and C18:1 n9 (oleic acid) are ubiquitously used to model dyslipidemia and to induce insulin resistance in rodent models (Joseph et al., 2004; Prentki and Nolan, 2006; Tang et al., 2007). Interestingly, metabolic by-products of palmitic acid, also shown to be dysregulated in MetS, insulin resistance and diabetes, also have distinctive effects on metabolic homeostasis. The elongation of palmitic acid through an SREBP-1 -mediated pathway in the liver results in the production of C18:0 (stearic acid), which is associated with increased hepatic insulin resistance (Chu et al., 2013). Reduction of stearic acid synthesis resulted in amelioration of insulin resistance in otherwise hyperlipidemic mice (Chu et al., 2013). Conversely, an unsaturated fatty acid by-product of palmitic acid, C16: 1 n7 (palmitoleate), has beneficial effect on hepatic metabolism (Cao et al., 2008; Yang et al., 201 1 ). Administration of high concentrations of palmitoleate to genetically obese, hyperlipidemic KK-Ay mice for 4 weeks reduced hepatic lipid accumulation and serum inflammatory markers, resulting in significantly improved insulin sensitivity (Yang et al., 201 1 ). While these studies point to important roles for endogenous lipids in the regulation of metabolism, manipulations are often genetically mediated, or rely on the chronic administration of supra-physiological levels of the metabolite, limiting their therapeutic potential.

[0005] Hepatic steatosis, also known as fatty liver disease, can be broadly subdivided into 3 categories: simple fatty liver disease (also referred to as non-alcoholic fatty liver disease (NAFLD)), non-alcoholic steatohepatitis (NASH), and alcoholic fatty liver disease. NAFLD is closely associated with being overweight or obese, as it is caused by excessive fat deposition in the liver. This occurs when fat intake is greater than can be processed by the adipose tissue alone, or when the liver is unable to processes fat for excretion, leading to inappropriate fat storage (Angulo, 2002). Increased oxidative stress and cytokines are also thought to be contributing factors. The World Gastroenterology Organization (WGO) estimates that 30% of the global population has NAFLD, with up to 90% of those with obesity or diabetes having this condition. While NAFLD is benign from the standpoint of hepatic function, in up to 25% of cases it progresses into NASH. NASH is characterized by fatty liver with associated inflammation, and is associated with elevated rates of morbidity and mortality due to cirrhosis, liver failure, and hepatocellular carcinoma (HCC) (WGO Guidelines). Alcoholic fatty liver disease, on the other hand, is caused by excessive long-term alcohol consumption. It is associated with increased inflammation leading to liver scarring and cirrhosis, and is not associated with BMI.

[0006] NAFLD alone is strongly associated with the insulin resistance characteristic of obesity and diabetes (Angulo, 2002). In fact, insulin-sensitive and insulin-resistant obese subjects can be stratified based on muscle and liver lipid accumulation alone. The mechanism underling NAFLD-induced insulin resistance is associated with increased diacylglycerols (DAG) accumulation, which induces activation of ΡΚΟε and results in inhibition of insulin receptor signaling (Birkenfeld et al., 2014). This pathway has been elucidated in numerous rodent models of high-fat diet induced NAFLD (Birkenfeld et al., 2014; Alves et al., 201 1 ; Erion, et al., 2009, Jornayvaz et al., 2010; Samuel et al., 2007). The fact that NAFLD induces insulin resistance makes it a critical target for the prevention and treatment of diabetes.

[0007] In humans, diagnosis of NAFLD can be difficult. As obesity is the most common risk factor (with 75-90% of obese individuals having NAFLD), this line of screening is the most prevalent diagnostic tool (Torres et al, 2008). Blood tests demonstrating mild to moderate elevations in serum aminotransferase levels is most commonly associated with NAFLD in addition to mild changes in serum alkaline phosphatase and g-glutamyl transpeptidase. As NAFLD is commonly associated with insulin resistance and metabolic syndrome, other parameters including fasting cholesterol and triglycerides, glucose and insulin are likely to also be abnormal. Further tests including liver ultrasound and more rarely liver biopsy can also be used for clinical diagnosis. However, these advances tests are commonly reserved for the investigation of NASH, due to the elevated risk of morbidity and mortality.

[0008] There are limited treatment options and/or preventative strategies for NAFLD. The primary preventative option is modification of diet to limit fat intake, and sustained weight loss. While it is widely believed that some medications prescribed for diabetes, including Metformin, may be beneficial for the treatment of NAFLD, there is no evidence that this is the case (Lavine et al., 201 1 ). Clinical trials investigating the effect of other diabetic medications Rosiglitazone (Avandia) and Pioglitazone (Actos), which treat insulin resistance, and the lipid-lowering drug Atorvastatin (Lipitor), have shown promising results associated with these treatments and improvements in liver enzyme levels, however none have shown a direct targeting of these drugs for lowering hepatic lipid accumulation (Bayard et al., 2006).

[0009] There remains a need for new and improved therapies for the treatment and/or prevention of hepatic steatosis, including NAFLD and NASH. There also remains a need for new and improved therapies for the treatment or prevention of insulin resistance.

SUMMARY

[0010] The fu ran fatty acid metabolite 3-carboxy-4-methyl-5-propyl-2- furanpropanoic acid (CMPF) has been shown to be elevated in the plasma of patients with gestational (GDM), and type 2 diabetes (T2D), as well as impaired glucose tolerant (IGT) individuals (Prentice et al., 2014). Elevated concentrations of CMPF induce beta cell dysfunction through altering insulin biosynthesis, thus reducing insulin secretion and resulting in a failure to maintain glucose homeostasis.

[001 1 ] It has now been determined that CMPF alters whole body glucose metabolism, resulting in preferential fatty acid utilization and significantly improved insulin sensitivity in rodent models of obesity and insulin resistance. Administration of CMPF also correlates with a significant reduction in fat accumulation in the liver, in terms of both eliminating existing fat deposits, as well as preventing the accumulation of new fat deposits, in mice that are fed a high fat diet. As shown in Example 1 , one week of CMPF treatment followed by a high fat diet increases energy expenditure and lipid metabolism in mice. This corresponds to improved insulin sensitivity, and protection against the development of hepatic steatosis. CMPF also appears to act as an allosteric inhibitor of Acetyl-CoA Carboxylase (ACC) to drive lipolysis and prevent triglyceride synthesis. CMPF induces FGF21 expression and potentiates lipid uptake into hepatocytes. Increased lipolysis has also been shown to be associated with induction of FGF21 expression and secretion, which acts locally on the liver to drive lipid metabolism and protect against the development of steatosis.

[0012] As set out in Example 2, mice placed in a high fat diet prior to and concurrently with CMPF administration exhibited dramatically improved insulin sensitivity (comparable to mice fed a normal control diet), relative to control mice on the same high fat diet. Insulin sensitivity in high fat diet fed mice treated with CMPF was similar to mice fed a normal control diet, and significantly improved compared to control mice fed a high fat diet. Example 2 also demonstrates the effects of CMPF on reducing hepatic steatosis and improving insulin sensitivity in leptin knockout Ob/Ob mice, which is a genetic model of obesity and insulin resistance.

[0013] Accordingly, in one aspect of the disclosure, there is provided a method for the treatment or prevention of hepatic steatosis. In one embodiment, the method comprises administering to a subject in need thereof 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF). Also provided is the use of CMPF for the treatment or prevention of hepatic steatosis in a subject in need thereof. Optionally, the subject has, or is at risk of developing non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH). In one embodiment, the subject has a liver with a fat content of 5% or greater by mass. In one embodiment, the subject has a liver with a fat content of 7% or greater by mass, or a fat content between 5% and 10%. In one embodiment, the subject has metabolic syndrome, insulin resistance, is obese and/or has a high fat diet.

[0014] In another aspect of the disclosure there is provided a method for the treatment or prevention of insulin resistance. Also provided is a method for improving insulin sensitivity. In one embodiment, the method comprises administering CMPF to a subject in need thereof. Also provided is the use of CMPF for the treatment or prevention of insulin resistance in a subject in need thereof. Also provided is the use of CMPF for improving insulin sensitivity in a subject in need thereof. In one embodiment, the subject has insulin resistance and/or impaired insulin sensitivity. For example, in some embodiments the subject has a fasting serum insulin level greater than 25mlU/L or 174 pmol/L. In one embodiment, the subject has impaired glucose tolerance. In some embodiments the subject has metabolic syndrome, is obese and/or has a high fat diet.

[0015] In another aspect, there is provided a method of increasing lipid metabolism. In one embodiment, the method comprises administering CMPF to a subject. Also provided is the use of CMPF for increasing lipid metabolism in a subject in need thereof. In one embodiment, lipid metabolism is increased relative to carbohydrate metabolism in the subject. In one embodiment, CMPF increases lipolysis and/or decreases lipogenesis in the subject. In one embodiment, CMPF increases lipid metabolism in the liver. In one embodiment, CMPF increases fatty acid oxidation in the islets cells of the pancreas. Also provided are methods and uses for increasing lipid metabolism of cell in vivo, in vitro or ex vivo by administrating or contacting the cells with CMPF. In one embodiment, the cells are liver cells such as hepatocytes. In one embodiment, the methods and uses described herein include contacting a liver, or part thereof, in vivo, in vitro or ex vivo with CMPF to increase lipid metabolism and/or reduce the fat content of the liver or part thereof.

[0016] In one embodiment, CMPF inhibits Acetyl-CoA Carboxylase 1 or 2 (ACC1 and ACC2). In one embodiment CMPF induces expression of FGF21 . In one embodiment, CMPF inhibits ACC in liver cells in vivo, in vitro or ex vivo. In one embodiment, CMPF induces expression of FGF21 in liver cells in vivo, in vitro or ex vivo. In one embodiment, the liver cells are hepatocytes.

[0017] Optionally, the methods and uses described herein include the use and/or administration of a composition comprising CMPF and a pharmaceutically acceptable carrier.

[0018] Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 shows acute treatment with CMPF induces persistent changes in whole body metabolism. A) Illustration of the treatment protocol with one week of CMPF treatment prior to 4 weeks of high fat diet feeding. B) Plasma CMPF concentration following intraperitoneal injection (n=4-6). C) Weekly weight gain through injection period and following placement on 60% high fat diet (n=20/group). D) Food and (E) water intake over 24 hours four weeks following final injection (n=4/group). F) MRI scan images and (G) quantification of fat distribution (n=4/group). H) Activity over 24 hour period in x, y, and z planes (n=4/group). I) Traces and (J) quantification of respiratory exchange ratio (RER) calculated as VC02/V02 over 24 hour period (n=4/group). K) Fasting plasma leptin, (L) adiponectin, (M) free fatty acid (FFA), and (N) triglyceride (TG) levels 4 weeks following final injection (n=8/group). *P<0.05, **P<0.01 , ***P<0.001 . All error bars SEM.

[0020] Figure 2 shows that CMPF treatment protects insulin sensitivity on a high fat diet. A) 16hr fasting blood glucose and (B) fasting plasma insulin levels (N=8/group). C) Blood glucose levels during intraperitoneal insulin tolerance test (IpITT) (n=8/group). Western blot and quantification of (D) skeletal muscle and (E) liver Akt activation following in vivo insulin stimulation (n=4/group). F) IpITT showing that treatment with 2,5-Furandicarboxylic acid, a control furan acid, does not have the same effect as CMPF on improving insulin sensitivity during IpITT (n=4/group) *P<0.05, **P<0.01 , ***P<0.001 . All error bars SEM.

[0021 ] Figure 3 shows CMPF treatment prevents the development of high fat diet-induced steatosis. A) Representative liver morphology of freshly isolated livers from mice 4 weeks following final injection. B) H&E and oil red O staining of liver sections (n=8/group). C) Triglyercide content from isolated livers (n=8/group) D) Serum ALT from mice following 4 weeks of HFD feeding. TUNEL positive pixel staining in (e) liver and (f) white adipose tissue. *P<0.05, **P<0.01 , ***P<0.001 . All error bars SEM.

[0022] Figure 4 shows the acute effect of CMPF on hepatocyte metabolism. A) Fatty acid uptake and (B) oxidation per hour in isolated hepatocytes following 24 hour treatment (n=4/group). C) Glucose oxidation per hour (n=4/group). D) Demonstration of specificity of fatty acid oxidation measurements by inhibition with the CPT1 inhibitor etomoxir (N=4). E) Chemical structures of CMPF and the ACC inhibitor TOFA. Western blot and quantification of (F) ACC and (G) AMPK phosphorylation in isolated hepatocytes treated for 24 hours with CMPF or TOFA (n=4/group). H) Fatty acid oxidation rate and (I) fold change over control in isolated hepatocytes treated for 24 hours with modulators of AMPK activation Compound C and AICAR. *P<0.05, **P<0.01 , ***P<0.001 . All error bars SEM.

[0023] Figure 5 shows the persistent effect of CMPF is associated with increased FGF21 and decreased ACC abundance. A) Concentration of CMPF in the liver over time as determined by SRM-MS (n=4- 6). Western blot and quantification of B) AMPK activity and abundance and C) ACC and in livers isolated from mice 4 weeks following final injection (n=3-4/group). D) Heat map showing significantly altered genes associated with lipogenesis and lipolysis in livers from mice isolated 4 weeks following treatment as determined by microarray (n=3/group). E) Quantitative PCR (qPCR) validation of differential gene expression in liver 4 weeks following final injection (n=8/group). F) FGF21 levels in fasting serum at the time of sacrifice and (G) gene expression in 24 hour treated hepatocytes. H) Serum FGF21 levels in the days following first injection of CMPF, vehicle, or 2,5- Furandicarboxylic acid. I) qPCR showing expression of FGF21 , ACC, and GCK in livers isolated from mice immediately following 7 day injection period (n=8/group). *P<0.05, **P<0.01 , ***P<0.001 . All error bars SEM.

[0024] Figure 6 shows that CMPF prevents steatosis through FGF21 A)

Schematic of the treatment protocol used for the treatment of FGF21 KO and c57 control (WT) mice. B) Weight gain through the injection and follow up periods (N=4-7/group). C) Blood glucose during ipITT and (D) plasma insulin following 14 hour fast (n=4-7/group). E) Photographs and oil red O staining of liver sections (n=10-13/group) and (F) quantification of oil red O and G) liver triglyceride content (n=10-13/group). H) Western blot of total ACC from isolated liver tissue (n=3/group). *P<0.05, **P<0.01 , ***P<0.001 . All error bars SEM.

[0025] Figure 7 shows that CMPF must enter the liver through OAT transporters. A) Weight gain through the injection and 6-week follow-up periods (N=2-3/group). B) Blood glucose during ipITT (N=2-3/group). C) Photographs and Oil red O staining of liver sections (N=2-3/group). D) Liver weight as a percentage of body weight and (E) quantification of liver triglyceride (N=2-3/group). *P<0.05. All error bars SEM.

[0026] Figure 8 shows that CMPF alters whole-body metabolism in diet-induced obese mice. A) Mice on a high fat diet (HFD) gain significantly more weight than chow fed mice. Injection with CMPF (HFD- CMPF) has no effect on body weight compared to HFD-Control injected mice (N=20-23/group). B) Activity over a 24hour period summed in the x, y and z planes (N=3-4/group). C) 24 hour food consumption per mouse house in metabolic cages (N=3-4/group). D) Representative MRI images showing adipose tissue distribution and (E) quantification of adipose area (N=4/group). F) Adipocyte size calculated from subcutaneous fat pads (N=8/group). Plasma (G) leptin and (H) adiponectin levels at the end of the treatment period (N=12/group). I) Summed RER over 24 hour period with corresponding average (J) V02 and (K) VC02 as determine in metabolic cages (N=3- 4/group). * indicates significance compared to Chow-Control, # indicates significance compared to HFD-Control. *,#P<0.05, **P<0.01 , ***,### P<0.001 .

[0027] Figure 9 shows that CMPF reverses insulin resistance in diet- induced obese mice. 16hour fasting (A) blood glucose and (B) plasma insulin levels (N=8/group). C) Random plasma insulin levels, following a 4hr fast. D) Blood glucose during ipITT (N=12/group).

[0028] Figure 10 shows that CMPF treatment ameliorates steatosis and improves hepatic insulin sensitivity in high fat diet fed mice. A) Photographs and Oil red O staining of liver sections (N=8/group). B) Liver weight expressed as percentage of body weight (N=4/group). C) In vivo insulin signaling shown as pAKT in (C) liver and (D) muscle samples (N=4/group). *P<0.05.

[0029] Figure 1 1 shows metabolomic and microarray analysis of diet- induced obese livers. A) Heat map showing fold change in select metabolites from the pentose phosphate pathway, pentose metabolism, and glutathione metabolism (N=4/group). Dark grey indicates significant change at P<0.05, light grey indicates change at P<0.10. B) Schematic showing the proposed mechanism underlying alterations in the PPP and glutathione pathways. C) Increase in Lipinl expression as determined by microarray (N=3/group) and (d) confirmation of increased Lipinl translocation to the nucleus with CMPF treatment (N=2/group).

[0030] Figure 12 shows that CMPF improves insulin sensitivity in ob/ob mice. A) Body weight before and after the two week injection period (N=4- 7/group). B) Representative MRI images showing adipose distribution and (C) quantification of adipose area in MRI images (N=4-7). D) Plasma adiponectin levels at the end of the treatment period. E) Random blood glucose and 16hour fasting (F) blood glucose and (F) plasma insulin levels (N=4-7). G) Blood glucose levels during ipITT (N=4-7). *P<0.05, **P<0.01 .

[0031 ] Figure 13 shows that CMPF reduces steatosis in ob/ob mice. A) Photographs and (b) Oil red O staining of liver sections (N=8-14). C) Liver weight expressed as percentage of body weight (N=4-7). *P<0.05.

[0032] Figure 14 shows that treatment with CMPF prior to a HFD impairs glucose tolerance. A) Schematic showing the treatment protocol with 7 days of CMPF or vehicle treatment prior to 4 weeks of 60%kcal from fat HFD feeding. B) Blood glucose and (C) corresponding plasma insulin values during intraperitoneal glucose tolerance test (N=8/group). *,#P<0.05

[0033] Figure 15 shows that islets from CMPF-injected mice placed on a high fat diet exhibit impaired glucose-stimulated insulin secretion (GSIS) and enhanced palmitate-stimulated insulin secretion (PSIS). Static secretion assays with A) low glucose (LG; 2mM Glucose) and high glucose (HG; 20mM) stimulation, or with B) low glucose (LG; 2mM glucose) and low glucose with palmitate (PAL; 2mM glucose plus 400uM Palmitate) showing significantly reduced GSIS and enhanced PSIS in CMPF-HFD islets (N=4/group). C) CMPF-HFD islets have significantly reduced intracellular insulin content (N=4/group). D) Islets isolated from treated mice have no difference in ROS accumulation (N=4/group), however E) CMPF- HFD islets are significantly smaller than both Control-Chow and Control- HFD islets (N=4/group). F) Mitochondrial membrane potential measurements in intact islets loaded with Rhodamine123 show CMPF-HFD islets have impaired response to the addition of high glucose (N=3-4 islets/mouse with 4 mice/group). *P<0.05, ***P<0.001 .

[0034] Figure 16 shows that HFD mice treated with CMPF have further impairment in glucose tolerance. A) Schematic showing the treatment protocol. Mice were placed on a 60%kcal from fat HFD for 6 weeks, followed by 2 weeks of vehicle or CMPF injection concurrent with HFD feeding. B) Blood glucose and (C) corresponding plasma insulin values during an oral glucose tolerance test (OGTT) (N=8/group). D) Blood glucose and (E) corresponding plasma insulin values during intraperitoneal glucose tolerance test (IpGTT) (N=8/group). *P<0.05 vs. chow, #P<0.05 vs. HFD-Control.

[0035] Figure 1 7 shows that CMPF treatment in high fat diet-fed mice causes a significant decrease in islet mass and pancreatic insulin area. A) Total intracellular insulin content in islets isolated from mice treated following diet intervention (N=13-14/group). B) Insulin positive area calculated using positive pixel analysis of immunohistochemically (IHC) stained whole pancreatic sections show HFD-CMPF mice have significantly reduced insulin area relative to total pancreatic area (N=5- 6/group). C) Insulin staining of whole pancreatic sections show reduced islet size in HFD-CMPF mice (N=5-6/group), as quantified in D). E) Stratification of islet size as determined based on insulin positive staining of pancreatic sections shows HFD-CMPF mice have a significant increase in small islets (N=5-6/group). * indicates significance compared to Chow- Control, # indicates significance compared to HFD-Control. *,#P<0.05

[0036] Figure 18 shows that differences in islet size are not due to proliferation. A) BrdU positive cells in pancreatic sections (N=4 mice/group). B) Quantification of average cell size determined by islet area divided by number of nuclei (N=8/mice group). C) Quantification of number of cells per islet (N=8/group). *P<0.05, **P<0.01 , ***P<0.001 .

[0037] Figure 19 shows that CMPF treatment impairs glucose metabolism and promotes fatty acid metabolism in the beta cell of high fat diet-fed mice treated with CMPF. A) Isolated islets show no signs of apoptosis as measured with cleaved Caspase 3/7 activity (N=8/group). B) Static secretion assays with low glucose (LG; 2m M Glucose), high glucose (HG; 20mM) and high glucose with KCI treatment (KCI; 20mM glucose plus 30mM KCI), with C) fold change in insulin secretion (high glucose/low glucose) (N=13-14/group). D) Static secretion assays with low glucose (LG; 2m M glucose) and low glucose with palmitate (PAL; 2m M glucose plus 400uM Palmitate), with E) fold change in insulin secretion (PAL/LG) (N=4/group). Mitochondrial membrane potential measurements in intact islets loaded with Rhodamine123 show F) HFD-CMPF islets have impaired response to the addition of high glucose, and G) enhanced response to the addition of palmitate compared to both Chow- and HFD-Control islets (N=4-5 islets/mouse with 4 mice/group). H) Reactive oxygen species accumulation in isolated islets as measured based on DCF fluorescence, with representative images (N=4-5 islets/mouse with 4 mice/group). * indicates significance compared to Chow-Control, # indicates significance compared to HFD-Control. *,#P<0.05, ***,### P<0.001 .

[0038] Figure 20 shows that CMPF treatment worsens glucose intolerance in leptin knockout Ob/Ob mice. A) Schematic diagram showing the treatment protocol. 7 week old ob/ob mice were injected with CMPF or vehicle for 2 weeks before analysis. B) Glucose tolerance was tested by OGTT and demonstrated significant impairment of glucose tolerance in Ob/Ob-CMPF mice despite (C) no difference in plasma insulin during the OGTT (N=4-7/group). D) Glucose tolerance was also evaluated by IpGTT and was also worsened. E) This corresponds to further impairment in glucose stimulated insulin secretion (N=4-7/group). *P<0.05, **P<0.01 .

[0039] Figure 21 shows that islets from CMPF-treated Ob/Ob Mice have enhanced palmitate-stimulated and reduced glucose-stimulated insulin secretion. A) Islets isolated from Ob/Ob-CMPF mice are significantly smaller than islets from Ob/Ob-Control mice (N=4-7/group). B) Fold change in glucose-stimulated insulin secretion (HG/LG) during static secretion assays show Ob/Ob-CMPF islets have reduced GSIS (N=4- 7/group). C) Total intracellular insulin content in isolated islets (N=4- 7/group). D) Fold change in palmitate-stimulated insulin secretion (PAL/LG) during static secretion assays show Ob/Ob-CMPF islets have enhanced PSIS (N=4-7/group). E) Mitochondrial membrane potential measurements in intact islets loaded with Rhodamine123 show Ob/Ob-CMPF islets have impaired response to the addition of high glucose (N=3-4 islets/mouse with 4-7 mice/group). F) Reactive oxygen species accumulation in isolated islets as measured based on DCF fluorescence, with representative images (N=4-5 islets/mouse with 4-7 mice/group). *P<0.05, **P<0.01 , ***P<0.001 .

DESCRIPTION OF VARIOUS EMBODIMENTS

Definitions

[0040] As used herein, "hepatic steatosis", also known as "fatty liver disease", refers to a condition characterized by excessive amounts of triglycerides and other fats inside liver cells. In some embodiments, subjects with hepatic steatosis exhibit elevated levels of SGOT (serum glutamic- oxaloacetic transaminase), SGPT (serum glutamic pyruvic transaminase) and/or alkaline phosphatase. In some embodiments, subjects with hepatic steatosis exhibit elevated serum levels of aspartate aminotransferase (AST) and/or serum alanine aminotransferase (ALT). In some embodiments, subjects will have an altered AST/ALT ratio.

[0041 ] As used herein, "non-alcoholic fatty liver disease" (NAFLD) refers to hepatic steatosis wherein at least 5% of the mass of the liver is fat. In some embodiments, NAFLD refers to hepatic steatosis wherein between 5- 10% of the mass of the liver is fat. In some embodiments, NAFLD refers to hepatic steatosis wherein at least about 5%, 6%, 7%, 8%, 9% or 10% of the mass of the liver is fat. In one embodiment, subjects with NAFLD have an aspartate aminotransferase-to-alanine aminotransferase (AST/ALT) ratio >1 . [0042] As used herein "non-alcoholic steatohepatitis" (NASH) refers to hepatic steatosis characterized by fatty liver with associated inflammation and fibrosis. In subjects with NASH, typically at least 5% of the mass of the liver is fat, or between 5-10% of the liver is fat.

[0043] As used herein, "insulin resistance" refers to a physiological condition wherein insulin becomes less effective at lowering blood sugar levels. The term "insulin sensitivity" is related to insulin resistance and refers to the effectiveness of insulin to lower blood sugar levels. In some embodiments, a subject with insulin resistance has a fasting serum insulin level greater than 25 lU/ml or 174 pmol/L. In one embodiment, subjects with insulin resistance may be identified using the homeostasis model assessment-estimated insulin resistance (HOMA-IR) index. In one embodiment, HOMA-IR is calculated by multiplying fasting plasma insulin (FPI) by fasting plasma glucose (FPG), then dividing by the constant 22.5, i.e. HOMA-IR = (FPIxFPG)/22.5. In one embodiment, subjects with a HOMA-I R index greater than 2.60 are identified as having insulin resistance.

[0044] As used herein "impaired glucose tolerance" refers to pre- diabetic state associated with insulin resistance. In one embodiment, "impaired glucose tolerance" refers to a subject with a fasting blood glucose level >1 10 mg/dL.

[0045] As used herein "obese" refers to a condition wherein a subject has excess body fat to the extent that it has a negative effect on health. In one embodiment a subject is considered obese if they have a body mass index equal or greater to 30 kg/m ²

[0046] As used herein, the term "subject" refers to any member of the animal kingdom that has a liver capable of converting acetyl-CoA to fatty acids. In one embodiment, the subject is any member of the animal kingdom that has β-cells which store and release insulin in order to control levels of glucose in the blood. In one embodiment, the subject is any member of the animal kingdom wherein CMPF induces the expression of FGF21 . In one embodiment, the subject is a human. In one embodiment, the subject is an animal such as a rat or mouse. In one embodiment, the subject has, or is suspected of having hepatic steatosis. In one embodiment, the subject has or is suspected of having a metabolic disorder. In one embodiment, the subject has insulin resistance and/or impaired glucose tolerance.

[0047] "Treating" or "Treatment" as used herein and as is well understood in the art, means an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease (e.g. maintaining physiologically normal levels of fat in the liver and/or physiologically normal levels of blood insulin), preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission (whether partial or total), whether detectable or undetectable. "Treating" and "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. "Treating" and "treatment" as used herein also include prophylactic treatment. In one embodiment, treatment methods comprise administering to a subject a therapeutically effective amount of CMPF as described herein and optionally consists of a single administration, or alternatively comprises a series of administrations.

[0048] As used herein, the phrase "effective amount" or "therapeutically effective amount" means an amount effective, at dosages and for periods of time necessary to achieve the desired result. For example in the context of treating hepatic steatosis, an effective amount is an amount that helps reduce the level of fat in in the liver compared to the response obtained without administration of CMPF. In one embodiment, an effective amount is an amount that helps reduce the level of fat in the liver to less than about 5% by mass. In one embodiment, an effective amount is an amount that changes the presence of biomarkers for hepatic steatosis in the subject. For example, in one embodiment an effective amount of CMPF is an amount that measurably reduces levels of serum aminotransferase and/or g-glutamyl transpeptidase in the subject. In one embodiment, an effective amount of CMPF is an amount that measurably reduces serum triglyceride levels in the subject. In the context of treating insulin resistance, an effective amount is an amount that helps improve glucose homeostasis, reduces the fasting amount of insulin in the blood and/or improves HOMA-I R in the subject. Effective amounts may vary according to factors such as the disease state, age, sex and weight of the subject. The amount of a given compound that will correspond to such an amount will vary depending upon various factors, such as the given drug or compound, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject or host being treated, and the like, but can nevertheless be routinely determined by one skilled in the art.

CMPF for the Treatment of Hepatic Steatosis and/or Insulin Resistance

[0049] 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF) treatment followed by a high fat diet has been shown to increase energy expenditure and lipid metabolism, which corresponds to dramatically improved insulin sensitivity, and protection against the development of hepatic steatosis. As set out in Example 1 , CMPF has been shown to alter fat deposition and utilization. CMPF has also been shown to prevent high-fat diet-induced steatosis. CMPF-treated mice fed a high fat diet did not show any signs of steatosis and had no fat deposits or inflammation in their livers unlike Control-HFD mice (Figure 3, parts A, B). Unexpectedly, despite exhibiting significantly elevated fasting glucose and plasma insulin levels, CMPF-HFD mice exhibited dramatically improved insulin sensitivity compared to Control-HFD mice (Figure 2, part C). [0050] Furthermore, as shown in Example 2 CMPF alters whole body glucose metabolism in rodent models of obesity and insulin resistance, resulting in preferential fatty acid utilization and significantly improved insulin sensitivity. In one embodiment, this correlates with a significant reduction in fat accumulation in the liver, in terms of both eliminating existing fat deposits, as well as preventing the accumulation of new fat deposits, while mice are fed a high fat diet. CMPF has also been demonstrated to alter lipid metabolism and improve insulin sensitivity in ob/ob mice, which represents a genetic model of obesity and insulin resistance. CMPF therefore appears to act on cells in the liver and beta cells in the pancreas but may also have actions beyond these cells.

[0051 ] The present description therefore provides uses and methods for the treatment or prevention of hepatic steatosis with CMPF. In one embodiment, CMPF is for use or administered to a subject with hepatic steatosis or a subject at risk of developing hepatic steatosis. For example, in one embodiment, the subject has, or is at risk of developing, non-alcoholic fatty liver disease (NAFLD). The methods and uses described herein can also be for the treatment or prevention of more severe forms of hepatic steatosis such as non-alcoholic steatohepatitis (NASH). In one embodiment, subjects at risk of developing hepatic steatosis or who may benefit from the use or administration of CMPF include subjects with metabolic syndrome, insulin resistance, and/or a high fat diet.

[0052] The present description also provides uses and methods for improving insulin sensitivity with CMPF in a subject in need thereof. Also provided are uses and methods for treating insulin resistance with CMPF in a subject in need thereof. For example, in one embodiment, CMPF is for use or administered to a subject with insulin resistance with a fasting serum insulin level greater than 25 IU/L or 174 pmol/L. In one embodiment, the subject has impaired glucose tolerance. In one embodiment, subjects who may benefit from improving insulin sensitivity or from the use or administration of CMPF include subjects with metabolic syndrome, insulin resistance, obesity and/or a high fat diet.

[0053] CMPF has also been demonstrated to alter lipid metabolism resulting in preferential fatty acid utilization. Accordingly, in one embodiment the present description provides uses and methods for increasing lipid metabolism with CMPF. In one embodiment, the method comprises administering CMPF to a subject. In one embodiment, the use or administration of CMPF increases the metabolism of lipids relative to that of carbohydrates. For example, in one embodiment CMPF increases beta oxidation and inhibits glucose oxidation. In one embodiment, CMPF increases lipolysis and/or decreases lipogenesis. In one embodiment, CMPF increases lipolysis and/or decreases lipogenesis in the liver or pancreas. Also provided are uses and methods for increasing lipid metabolism with CMPF in vivo, in vitro or ex vivo. In one embodiment, CMPF increases lipid metabolism in liver cells (hepatocytes). In one embodiment, CMPF increases lipid metabolism in the pancreas. For example, in one embodiment, CMPF increases fatty acid oxidation in the islets cells of the pancreas. In one embodiment, subjects who may benefit from the use or administration of CMPF for increasing lipid metabolism include subjects with metabolic syndrome, insulin resistance, obesity and/or a high fat diet. In one embodiment, CMPF may be used to alter lipid metabolism and/or reduce fat content in a liver, or part thereof, in vitro or ex vivo.

[0054] CMPF has also been demonstrated to have an effect on the expression or activity of proteins associated with lipid metabolism. For example, in one embodiment CMPF inhibits Acetyl-CoA Carboxylase (ACC). In one embodiment, ACC expression and/or activity is reduced in a subject even after CMPF has been removed from the circulation. In one embodiment, CMPF induces the expression of Fibroblast Growth Factor-21 (FGF21 ). In one embodiment, CMPF increases the expression of one or more genes selected from leptin receptor (Lepr), PCK1 and CPT1 a. In one embodiment, CMPF decreases the expression or protein levels of one or more genes selected from ACC1 , ACC2, Gck and Scd1 .

[0055] CMPF may be formulated for use and/or prepared for administration to a subject in need thereof as known in the art. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington's Pharmaceutical Sciences (2003 - 20th edition) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999.

[0056] Administration can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell(s) being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician, veterinarian, or clinician.

[0057] Routes of administration include, but are not limited to, oral (e.g., by ingestion); buccal; sublingual; transdermal (including, e.g., by a patch, plaster, etc.); transmucosal (including, e.g., by a patch, plaster, etc.); intranasal (e.g., by nasal spray, drops or from an atomiser or dry powder delivery device); ocular (e.g., by eyedrops); pulmonary (e.g., by inhalation or insufflation therapy using, e.g., an aerosol, e.g., through the mouth or nose); rectal (e.g., by suppository or enema); vaginal (e.g., by pessary); parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot or reservoir, for example, subcutaneously or intramuscularly. In one embodiment, the route of administration is oral (e.g., by ingestion). In another embodiment, the route of administration is parenteral (e.g., by injection). [0058] Also provided are compositions comprising CMPF and a pharmaceutically acceptable carrier. In one embodiment, the composition comprises CMPF and a pharmaceutically acceptable carrier for the treatment or prevention of hepatic steatosis. In one embodiment, the composition comprises CMPF and a pharmaceutically acceptable carrier for improving insulin sensitivity. In one embodiment, the composition comprises CMPF and a pharmaceutically acceptable carrier for increasing lipid metabolism. Also provided is the use of CMPF for the manufacture of a medicament for the treatment or prevention of one or more conditions as described herein.

[0059] The pharmaceutical compositions include, albeit not exclusively, solutions of CMPF in association with one or more pharmaceutically acceptable vehicles or diluents, and optionally contained in buffered solutions with a suitable pH. Optionally, the compositions include described herein one or more pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents, colouring agents, flavouring agents, and sweetening agents. The formulation may further comprise other active agents, for example, other therapeutic or prophylactic agents.

[0060] The present disclosure also includes a kit comprising CMPF, or a composition comprising CMPF, e.g., preferably provided in a suitable container and/or with suitable packaging; and (b) instructions for use, e.g., written instructions on the use or administration of CMPF for e.g. the treatment or prevention of hepatic steatosis, the treatment or prevention of insulin resistance and/or for increasing lipid metabolism as described herein.

[0061 ] The above disclosure generally describes the present application. A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely for the purpose of illustration and are not intended to limit the scope of the application. Changes in form and substitution of equivalents are contemplated as circumstances might suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.

[0062] The following non-limiting examples are illustrative of the present application:

EXAMPLE 1 : CMPF enhances lipid metabolism, protects insulin sensitivity and induces FGF21 to prevent steatosis

Materials and Methods

[0063] For the intraperitoneal injection of CMPF and tolerance tests, mice were injected intraperitoneally (IP) with 6mg/kg CMPF or vehicle control at 24h intervals for seven days and assessed for body weight, blood glucose and plasma insulin. All experiments were approved by the Animal Care Committee (University of Toronto) and animals handled according to the Canadian Council of Animal Care guidelines.

CMPF Preparation

[0064] CMPF was purchased from Cayman Chemical (product number 10007133) and dissolved in 70% ethanol to a stock concentration of 100mM. CMPF was stored at 4C. For injections, CMPF was dissolved in 10Oul sterile saline within an insulin syringe for injection.

Intraperitoneal injection of CMPF and Tolerance Tests

[0065] Seven-week-old male CD1 mice were purchased from Charles River and allowed to acclimate for one week prior to the beginning of experiments. Mice were maintained on standard chow diet while being injected intraperitoneally (i.p.) with 6mg/kg CMPF or vehicle control at 24h intervals for seven days, as previously described (Prentice et al., 2014). 24 hours following the final injection, mice were switched to either a 60%kcal from fat HFD (HFD; OpenSource diets D12492, Research Diets Inc, USA) or a sucrose-matched control (OpenSource diets D12450J, Research Diets Inc, USA) for 4 weeks. Mice were also monitored weekly for individual body weights and whole-cage food consumption. [0066] At the end of the follow-up period, mice were fasted overnight for 14h before an intraperitoneal glucose tolerance test (ipGTT). Mice were injected ip with 1 g/kg sucrose. IpGTTs, and measurement of plasma insulin were performed as previously described (Allister et al., 2013). Blood (<25ul) was collected from the tail vein at 0 (fasting), 10, 20 and 30 minutes from quantification of plasma insulin and blood glucose. Blood glucose was also measured at 60 and 120 minutes post injection. IP insulin tolerance tests (ipITT) were performed following a 4h fast. 1 .5IU/kg insulin was injected and blood glucose was measured at 0, 10, 20, 30, 45, 60 and 120 minutes. Plasma insulin was quantified by ELISA (Alpco Ultrasensitive Insulin ELISA). All experiments were approved by the Animal Care Committee at the University of Toronto and animals handled according to the Canadian Council of Animal Care guidelines.

Fat Distribution Analysis by MRI

[0067] Mice were anesthetized using isofluorance and imaged by a 7 tesla pre-clinical MRI at the STTARR facility at the University Health Network, Toronto, ON. A heating pad through the imaging process maintained body temperature. 26 images were captured from each mouse, and fat distribution was assessed using TissueStudio where area of visceral and subcutaneous fat depots was quantified in each section for each animal. Total areas were summed and averaged within groups.

CLAMs

[0068] CLAM experiments were performed as previous described. Briefly, mice were housed individually in CLAMs with monitoring of food and water consumption, activity in x,y,and z planes, oxygen consumption and C02 production were measured every 10 minutes over 48 hours. The cages were housed in a room with a standard 12 hour light-dark cycle. Mice were provided with the same diet they were maintained on before entering the cages. The first 24 hours of data was removed from analysis while the mice were acclimated to the cages. Analysis was performed on the final 24 hours within the cages. Quantification of Circulating Factors

[0069] Circulating leptin, adiponectin, FGF21 , and triglyceride were measured in 14hr fasting samples obtained at the time of sacrifice. Total free fatty acids were quantified from 14hr fasting samples and following a 1 hr reefed with ad libitum access to food. Assays were performed according to manufacturer protocols (BioVision, USA).

Western Blotting

[0070] To evaluate in vivo insulin signaling, mice were given a 1 1 U/kg insulin bolus by tail vein injection. Mice were sacrificed 10 minutes following injection, tissues isolated and flash frozen in liquid nitrogen. Liver and muscle tissues for analysis were ground in liquid nitrogen and lysed in RIPA buffer (Cell Signaling, USA) containing protease inhibitor cocktail (Roche, Canada) and stored at -20C prior to use. Lysates were spun at 12,000rpm and supernatant was evaluated for protein content by Bradford assay (BioRad, Canada). Equal amount of protein were then combined with sample buffer containing DTT and loaded onto a 4-15% SDS-PAGE gradient gel (BioRad, Canada) and run at increasing voltages of 50V for 30minutes, 75V for 30 minutes, and then 100V for 30 minutes. Proteins were then transferred onto PVDF membrane using a Turbo Blotter (BioRad, Canada). The membrane was probed with antibodies and imaged using Kodak Imager 4000pro (Carestream, USA).

Quantification of Liver Triglyceride

[0071 ] Livers were isolated, embedded into TissueTek and frozen. Staining of sections was performed as previously described. For H&E staining, livers were weighed and fixed in 10% neutral buffered formalin at the time of sacrifice. Tissue processing and immunostaining for insulin have been described previously (Luu et al., 2013). Briefly, pancreata were dehydrated, cleared and embedded in paraffin. Paraffin sections were cut at 4um from the middle of the pancreas. Sections were then dried and deparaffinized for staining. Images of each section were acquired using Aperio Imagescope at 40x magnification (Aperio Image scope).

Fatty Acid Uptake and Oxidation

[0072] Fatty acid and glucose uptake and oxidation experiments were performed as previously described. Briefly, primary hepatocytes were isolated from 7-9 week old wild type mice and plated at a density of 400,000 cells per well in 12-well plates. Cells were allowed to attach in DMEM media supplemented with 10% FBS for 4 hours. Cells were then washed and cultured in Williams Solution without FBS. Cell were treated with vehicle or 200uM for CMPF starting at this time point for 18 hours. Compound C and AICAR were incubated for 4 hours prior to oxidation and uptake. CMPF was conjugated to fatty acid free BSA for 4 hours prior to starting treatment. At the end of the treatment period, cells were incubated with transport solution containing 20uM oleic acid and 0.5uCi/ml of 14C oleic acid, conjugated to BSA for 4 hours. Uptake experiments were performed at room temperature for 3 minutes, oxidation experiments were performed at 37C for 90 minutes. Data was normalized to protein content determined by Bradford assay.

Gene Expression

[0073] Total RNA was extracted from isolated islets using the Qiagen RNeasy Plus mini kit (Hilden, Germany). Reverse transcription from total RNA and quantitative real time PCR (qPCR) analysis was performed as previously described (Basford et al., 2012). Reverse transcription was performed using a M-MLV kit according to manufacturer's instructions (Sigma Aldrich, Canada). Primers were designed using Primer3 software (NCBI) and are listed in Table. Data were normalized to β actin mRNA. Microarray analysis was performed as previously described (Basford et al., 2012) using the Affymethx Mouse 430 2.0 Gene Chip at the University Health Network microarray center (Toronto, Canada). Significant changes were defined as P<0.05.

FGF21KO Mice [0074] Six to nine week old male FGF21 KO mice were obtained from Taconic along with age-matched c57 controls. Mice were allowed to acclimate for one week prior to the beginning of the treatment period and randomized to chow and HFD control and CMPF treatment groups. Mice were injected with 6mg/kg/day CMPF for seven days and changed to HFD and matched sucrose control as described above. Mice were maintained on the diet for six weeks prior to evaluation. FGF21 deletion was confirmed by PCR.

Statistics

[0075] Statistical significance was assessed using either the Student's t-test, or a two-way ANOVA for repeated measures followed by a Bonferroni post-test comparison where required. P<0.05 was considered significant. All data is mean ± SEM unless otherwise specified.

Results

CMPF is Rapidly Cleared from Circulation

[0076] Acute treatment with the furan fatty acid metabolite CMPF has been shown to alter whole-body glucose utilization, including a reduction in both glucose appearance and disappearance rates in a hyperinsulinemic, euglycemic clamp condition (Prentice et al., 2014). Reduced glucose utilization may be consistent with increased reliance on alternate energy sources, including fatty acids. This is consistent with an elevation of CMPF in gestational diabetes, as during pregnancy maternal metabolism switches to preferential fat oxidation to preserve glucose for the developing fetus. To determine the role of CMPF in altered substrate utilization, mice were treated with CMPF acutely, as described previously (Prentice et al., 2014), and then placed on a high fat diet (HFD; 60% kcal from fat) for four-six weeks to induce insulin resistance (Figure 1 , part A). Following intraperitoneal injection of CMPF, plasma concentrations peaked within one hour of injection to concentrations observed in diabetic populations (Prentice et al. , 2014), and declined rapidly thereafter, returning to baseline within 24 hours of injection (Figure 1 , part B). CMPF was not elevated in the plasma for the duration of dietary intervention.

CMPF Alters Fat Deposition and Utilization

[0077] Both CMPF and vehicle control mice placed on a HFD (CMPF-

HFD and Control-HFD, respectively) gained significantly more weight than vehicle injected mice maintained on a chow diet (Control-Chow) (Figure 1 , part C). Interestingly, the CMPF-HFD group gained slightly, but significantly less weight than the Control-HFD group. This did not correlate to any difference in food or water consumption at any time (Figure 1 parts D, E). To determine if the difference in body weight was due to altered body composition, mice were placed in MRI scanners to examine body fat distribution (Figure 1 , part F). CMPF-HFD mice had significantly lower total fat area than Control-HFD mice, which was specifically due to a significant reduction in subcutaneous fat area (Figure 1 , part G). There was no difference in visceral fat area. Mice were placed in metabolic CLAMs for 48hrs at the end of the 4 week diet period to assess changes in whole body metabolism. CMPF-HFD mice were significantly more active than Control- HFD mice, particularly during the light phase (Figure 1 , part H). Consistent with a decrease in whole-body glucose utilization, CMPF-HFD had a significantly reduced respiratory exchange ratio (RER) compared to Control- HFD and Control-Chow mice, suggesting preferential fatty acid utilization (Figure 1 , parts I and J). In spite of reduced subcutaneous fat area, there were no differences in circulating leptin, adiponectin, or free fatty acid levels (Figure 1 , parts K-M). CMPF-HFD mice had significantly reduced circulating TG than Control-HFD, with no difference compared to chow-fed animals (Fig. 1 N).

Insulin Sensitivity is Protected with Previous CMPF Treatment

[0078] CMPF-HFD mice exhibited significantly elevated fasting blood glucose and no change in plasma insulin levels (Figure 2, parts A, B). Interestingly, in spite impaired glucose tolerance, CMPF-HFD mice exhibited dramatically improved insulin sensitivity compared to Control-HFD mice during intraperitoneal insulin tolerance test (IpITT) (Figure 2, part C). The rate of glucose clearance in response to insulin was equal in the Control-Chow and CMPF-HFD groups.

[0079] To further quantify the improvement in insulin sensitivity with CMPF treatment, tissues were isolated from mice following an intravenous bolus of insulin, and insulin signaling was evaluated. Enhanced insulin sensitivity was observed in both skeletal muscle and liver in the CMPF-HFD mice compared to Control-HFD mice, as evaluated based on enhanced serine phosphorylation of Akt, observed in both skeletal muscle and liver of the CMPF-HFD mice compared to Control-HFD mice, suggesting enhanced insulin signaling through insulin receptor (Figure 2, parts D,E)

[0080] To determine if this effect of CMPF is specific or a general effect of furan dicarboxylic acids, we utilized 2,5-Furandicarboxylic acid (FDCA) as a control. Mice were treated according to the same protocol as CMPF, with 7 days of 6mg/kg treatment followed by 4 weeks of HFD. FDCA-HFD mice had no difference in insulin sensitivity by IpITT compared to Control-HFD animals, implying that the effect of CMPF is not common among this class of molecules (Fig. 2F).

High-Fat Diet-Induced Steatosis is Prevented by CMPF

[0081 ] To determine the source of the increased insulin sensitivity, tissues from the CMPF-HFD and control mice following 4 weeks of diet intervention were compared. Consistent with the previous observation that CMPF accumulates in the liver more than most other tissue types (Prentice et al., 2014), the most striking difference between CMPF-HFD and Control-HFD mice was in the appearance of the liver (Figure 3, part A). Remarkably, livers from the CMPF-HFD mice appear nearly identical to Control-Chow livers, with no visual evidence of fat deposits or inflammation. This was confirmed by histological analysis with both H&E and Oil red O staining for triglyceride revealing that the HFD induced massive steatosis in the Control-HFD livers, with little intracellular lipid accumulation in the CMPF-HFD (Figure 3, part B). In fact, livers from CMPF-HFD mice had reduced triglyceride accumulation compared to Control-Chow mice. There was no difference in any of the other tissues examined with the exception of significantly reduced adipocyte size within the subcutaneous fat pads, consistent with the reduction in area (data not shown).

[0082] Furthermore, quantification of hepatic TG content showed comparable levels between Control-Chow and CMPF-HFD animals (Fig. 3C). This difference in TG accumulation did not correspond to increases in hepatic stress markers, as there was no difference in serum ALT (Fig. 3D), and in fact, a significant reduction in TUNEL positive staining (Figure 3E). Interestingly, the reduction in subcutaneous fat mass was also not associated with any differences in TUNEL staining, suggesting that reduced adipocyte mass is not due to increased cell death (Figure 3F).

CMPF Acutely Inhibits Acetyl-CoA Carboxylase

[0083] Primary hepatocytes isolated from chow-fed mice were used to examine the mechanism underlying the increase in fatty acid utilization and resulting reduction in hepatic lipid accumulation with CMPF treatment. Treatment for 24 hours with CMPF stimulated fatty acid uptake compared to vehicle treatment (Figure 4, part A). This corresponded to a significant increase in beta oxidation (Figure 4, part B), and a significant impairment in glucose oxidation (Figure 4, part C). TOFA, an allosteric inhibitor of acetyl- CoA carboxylase (ACC), the master regulatory proteins of lipolysis and lipogenesis, produced a similar increase in beta-oxidation with no effect on glucose oxidation compared to control (Figure 4, parts B and C). Specificity of the assay for measuring beta-oxidation was confirmed through co-treatment with the CPT1 a inhibitor etoxomir, which reduced control and blocked any stimulation in beta-oxidation (Figure 4, part D). Due to the structural similarity (Figure 4, part E), and similar effect on potentiating fat metabolism, whether CMPF may also act as a direct inhibitor of ACC activity was investigated. Acute 24 hour treatment with either CMPF or TOFA stimulated beta oxidation in the absence of an increase in ACC phosphorylation (Figure 4, part F), suggesting that inhibition of activity may be direct. Intriguingly, despite no change in phosphorylated ACC, a significant increase in phosphorylated AMPK in hepatocytes treated with CMPF or TOFA was observed compared to control (Figure 4, part G). Modulation of AMPK activity with the inhibitor Compound C, or the activator AICAR revealed that the acute effect of CMPF is independent of AMPK activity, as the fold increase in beta oxidation remained constant with AMPK manipulation (Figure 4, parts H and I). The effect of TOFA to increase beta-oxidation, however, is dependent on AMPK activation, as co-treatment with Compound C eliminated the ability of TOFA to stimulate beta-oxidation (Figure 4, parts H and I). Together, this suggests that CMPF may act through ACC inhibition to acutely increase beta-oxidation in hepatocytes.

ACC is Diminished Weeks After CMPF Treatment

[0084] Direct inhibition of ACC by CMPF may be responsible for the increase in fat utilization during the 7 day injection period, however, CMPF is eliminated both from circulation (Figure 1 , part B) and the liver while the mice are maintained on a high fat diet (Figure 5, part A). To determine how CMPF protects against the development of steatosis in vivo even when it is no longer present, the AMPK- ACC pathway was investigated in liver isolated from CMPF-HFD mice and controls at the end of the 4 week diet period. Activation of AMPK leads to downstream inhibition of ACC transcription, which may potentiate a feedback loop to promote beta-oxidation. Indeed CMPF-HFD mice had both increased levels of phosphorylated AMPK (Figure 5, part B), as well as lower total ACC protein, with a higher ratio of phosphorylated ACC to total ACC (Figure 5, part C). The AMPK-ACC pathway is therefore likely critical for the prevention of steatosis development by CMPF. CMPF may act to directly modulate the pyruvate dehydrogenase complex, regulating the switch between glucose and lipid oxidation.

CMPF Induces FGF21 Expression to Maintain Insulin Sensitivity

[0085] To further elucidate the mechanism underlying the loss of ACC abundance, microarray analysis of livers isolated from Control-Chow, Control-HFD, and CMPF-HFD groups was performed. Overall, significant differences in gene expression were observed between the Control-HFD and CMPF-HFD groups, particularly in terms of genes involved in lipid processing as anticipated based on the loss of ACC. Remarkably, expression of FGF21 was found to be dramatically induced in the CMPF- HFD condition compared to both control groups (Figure 5, parts D, E). This was further validated in the plasma of the CMPF-HFD group, where circulating levels of FGF21 were also found to be significantly elevated (~7- fold) (Figure 5, part F). FGF21 is known to attenuate hepatic steatosis through regulation of lipolysis in addition to an increase in beta-oxidation (Fisher et al., 201 1 ). Closer examination of FGF21 target genes indicated that FGF21 acts locally on the liver in CMPF-HFD mice to regulate lipolysis (Fig. 5D and E). This includes potent changes in key FGF21 target genes including leptin receptor (Lepr) and Sterol-CoA Desaturase 1 (Scd1 ), which were significantly increased and decreased, respectively. Furthermore, pronounced alterations in expression of a number of genes were observed including increased expression of PCK1 , and CPT1 a, as well as reduced expression of both ACC1 and ACC2, Gck, and Cyp7a1 (Fig. 5D, E). Therefore, increased lipolysis and diminished lipogenesis observed in CMPF- HFD mice is likely mediated through FGF21 .

[0086] It was first confirmed that enhanced gene expression of FGF21 corresponded to increased circulating FGF21 in the serum from mice at the end of the 4 week diet period (Figure 5, part F). The induction of FGF21 expression with CMPF treatment may be acute or the result of chronically enhanced lipolysis rates. To determine when FGF21 expression is increased with CMPF treatment, lysates from the livers of mice following 7 days of CMPF treatment while maintained on a chow diet were observed, as well as isolated hepatocytes from chow-fed mice treated with CMPF for 24 hours. FGF21 was significantly increased with CMPF treatment under both conditions (Figure 5, parts G, I). This prompted a time-course experiment to determine when FGF21 becomes elevated in circulation. Three days following the first CMPF injection, plasma levels of FGF21 are significantly increased (2.57-fold) in the CMPF treated group compared to vehicle and FDCA controls, and remain elevated at least one week into the HFD feeding period (Fig 5H).ln addition, expression of key genes regulating lipolysis and glucose metabolism were significantly altered in 24 hour treated hepatocytes, including ACC1 and ACC2, and Gck (Figure 5, part I). Together this suggests that CMPF induces the expression of FGF21 acutely following the start of treatment, and therefore, FGF21 is likely essential for the long-term prevention of development of steatosis when CMPF is cleared from circulation.

[0087] FGF21 expression is normally elevated under fasting or nutrient restrictive conditions. Treatment with CMPF may mimic this status in the liver of chow-fed mice, as ACC inhibition drives beta-oxidation, the primary fuel source during fasting, as well as inhibits glucose utilization. This "fasting-like state" may drive FGF21 expression, activating a feedback loop that continues after CMPF is eliminated.

FGF21KO Mice are Resistant to CMPF Treatment

[0088] To determine if FGF21 is explicitly required for the long-term prevention of steatosis development, CMPF (6mg/kg) was administered to mice with global deletion of FGF21 (FGF21 KO) and age-matched c57 controls (WT) for 7 days, followed by 6 weeks of 60% kcal from fat HFD feeding (Fig. 6a). As observed in the CD1 animals, both WT and FGF21 KO mice treated with CMPF gained slightly less weight than HFD-controls (Fig. 6b). At the end of the protocol, insulin sensitivity was evaluated by IplTT. WT controls treated with CMPF had significantly improved insulin sensitivity compared to both HFD controls, as observed previously (Figure 6C), however, CMPF treatment had the opposite effect of worsening insulin sensitivity in FGF21 KO mice when compared to FGF21 KO-HFD controls (Fig. 6C). The lack of improvement in insulin sensitivity corresponded to both worsening of fasting hyperinsulinemia (Fig. 6D) and no difference in hepatic triglyceride content in FGF21 KO-CMPF mice (Fig. 6E-G). CMPF treatment significantly reduced triglyceride content in HFD-fed WT control mice, as observed in CD1 mice (Fig. 6E-G). Consistent with induction of FGF21 regulating ACC expression and activity, livers from CMPF-treated FGF21 KO mice had no significant difference in total ACC content compared to HFD- controls (Fig. 6H). Therefore, CMPF prevents the development of steatosis through induction of FGF21 .

CMPF Enters the Liver through OAT Transporters

[0089] To establish how CMPF mediates its effects on hepatic lipid accumulation and insulin sensitivity, experiments were performed to inhibit CMPF transport into the liver. CMPF is known to be a substrate for the Organic Anion Transporter (OAT) family of transporters, particularly through OAT1 (SLC22A6) and OAT3 (SLC22A8) (outlined in Prentice et al., 2014). Probenecid, an FDA-approved drug for the treatment of gout, is a nonspecific OAT inhibitor. To determine if OAT-mediated CMPF entry into the liver is mediated by OATs is required for its activity, 8-week-old male c57bl6 mice were treated twice daily with 30mg/kg probenecid for 3 days, prior to initiating treatment for 7 days with once daily 6mg/kg CMPF simultaneously with continued Probenecid treatment. 24 hours following the final injection, mice were placed on a 60% kcal from fat HFD for 6 weeks to induce insulin resistance. Probenecid-treated control (PBN-CON) and CMPF-treated (PBN- CMPF) mice had no significant difference in weight gain over the HFD period (Fig. 7A). At the end of 6 weeks of HFD-feeding, while CMPF-controls had improved insulin sensitivity compared to HFD-Control mice, mice treated with PBN had no significant difference between control and CMPF-treated groups (Fig. 7B). Evaluation of hepatic lipid accumulation by Oil red O staining revealed no difference in triglyceride accumulation between PBN-groups, while there was a significant improvement in CMPF-treated controls compared to HFD-controls (Fig. 7C). Consistent with this observation, there was no difference in liver weight or liver triglyceride between PBN-CON and PBN-CMPF mice (Fig. 7 D, E). Overall, it appears that CMPF must enter the liver through OAT transporters in order to mediate its effect on reducing lipid accumulation and improving insulin sensitivity. Discussion

[0090] These findings suggest that CMPF may play dual roles in the context of metabolic disorders, with a beneficial effect on hepatic insulin sensitivity and lipid metabolism, and a conversely detrimental role for beta cell function (Prentice et al., 2014). Interestingly, the underlying mechanism of CMPF in inducing fatty acid metabolism while reducing glucose metabolism appears to be constant between these two tissues. In the liver, the driving force allowing for long-term protection against the development of steatosis is dependent on induction of FGF21 expression and activity. FGF21 expression is normally elevated under fasting or nutrient restrictive conditions (Galman et al., 2008). Treatment with CMPF may mimic this status in the liver of chow- fed mice through increasing beta-oxidation, the primary fuel source during fasting, as well as inhibition of glucose utilization. Importantly, this "fasting-like state" is induced directly, rapidly, and independently of FGF21 , as increases in fatty acid uptake and oxidation are observed during in vitro treatment of isolated hepatocytes from FGF21 KO animals (data not shown). This potent stimulation of beta-oxidation is likely to stimulate FGF21 expression and secretion. Increased FGF21 , combined with a persistent drive toward beta- oxidation induced by CMPF over the chronic 7-day treatment period likely activates a feedback loop that continues to perpetuate after CMPF is eliminated. FGF21 activates AMPK, resulting in inhibition of SREBPI c, and reduced expression of ACC 1 and 2 (Potthoff et al., 2009). These enzymes are rate-limiting for the production of malonyl-CoA, which is required for both triglyceride synthesis, as well as inhibition of beta-oxidation (Abu-Elheiga et al., 2001 ). Livers isolated from mice treated with CMPF have a significant reduction in total ACC levels, while those from FGF21 KO livers have no significant difference, supporting this hypothesis. Loss of ACC likely explains the inability of CMPF-treated mice to accumulate hepatic triglyceride, even when maintained on a 60% kcal from fat HFD. EXAMPLE 2: CMPF for the Treatment of Steatosis, Insulin Resistance and Increasing Fatty Acid Metabolism

[0091 ] As CMPF is found to be elevated in conditions of Gestational and Type 2 Diabetes, two conditions characterized by insulin insufficiency in the face of insulin resistance, the relationship between CMPF and insulin resistance was investigated.

[0092] In non-obese, chow-fed mice, 7 days of CMPF injection had no effect on insulin tolerance, as determined using intraperitoneal insulin tolerance tests (IplTTs) and hyperinsulinemic euglycemic clamp studies. Interestingly, however, the clamp studies demonstrated that the CMPF- injected mice developed an increased reliance on non-glucose energy source, suggesting a switch in metabolism from primarily carbohydrates to fats or amino acids. This correlates with the switch in metabolism observed in gestational diabetes, which is established in order to preserve glucose for the developing fetus. To examine the effect of CMPF under conditions of insulin resistance, mice were injected after the induction of insulin resistance using either high-fat diet (HFD) or genetic induction of obesity.

CMPF Treatment in Obese Animals

[0093] Two models of obesity and insulin resistance were used to evaluate the effect of CMPF. In the first model of diet-induced obesity (DIO), eight-week old male CD1 mice were placed on a high fat diet (60% of calories from fat) for six weeks, prior to two weeks of daily injections of 6mg/kg CMPF. At the time of injection, mice fed HFD were significantly heavier than chow- fed controls (Figure 8, part A). Following CMPF injection, there was no significant difference in body weight between CMPF and vehicle control injected HFD-fed mice (HFD-CMPF and HFD-Control respectively). Both groups remained significantly heavier than chow-fed, vehicle-injected controls (Chow-Control) (Figure 8, part A).

[0094] To evaluate the effect of CMPF on whole-body metabolism in mice were placed in metabolic cages. Mice were observed to have significantly reduced activity (in the x,y,z planes) in the light phase as compared to HFD-Control mice (Fig. 8b). This reduction in activity did not correspond to any changes in food consumption (Fig. 8c). Consistent with reduced activity, HFD-CMPF mice had greater total adipose tissue area by MRI imaging, primarily attributed to the subcutaneous depot (Fig. 8d,e). This increase in adipose area did not correspond to changes in adipocyte size (Fig. 8f), or circulating levels of leptin or adiponectin (Fig. 8g,h). Overall calculation of the respiratory exchange ratio (RER) revealed a significant reduction in the HFD-Controls compared to Chow-controls, consistent with increased fatty acid metabolism. The RER was even significantly reduced to an even greater extent in the HFD-CMPF group compared to HFD-Controls (Fig. 8i). This significant reduction in RER, V02 and VC02 (Fig 8 j,k), suggest that CMPF treatment enhances whole-body fat metabolism, consistent with previous observations in CMPF-HFD mice (examined in Example 1 ) and chow-fed mice (Prentice et al., 2014)

[0095] Following two weeks of injections, 16hr fasting blood glucose and fasting plasma insulin trended, but were not significantly higher compared to HFD-controls (Fig 9a, b). Random plasma insulin was also trending higher (Fig. 9c). This prompted us to evaluate insulin sensitivity by IplTT. Unexpectedly, the HFD-CMPF mice exhibited dramatically improved insulin sensitivity as compared to the HFD-control mice, with insulin sensitivity comparable to Chow-Control mice (Figure 9, part d).

CMPF Treatment Reverses Diet-Induced NAFLD and Improves Insulin Sensitivity

[0096] NAFLD is the prominent source of insulin resistance in the DIO murine models of diabetes, thus we examined whether the liver was the source of the observed improvement in insulin sensitivity. HFD-Control mice had significantly greater lipid accumulation in the hepatocytes compared to Chow-Control livers, as determined by gross anatomy, as well as histological sections and Oil Red-0 staining of whole liver sections (Figure 10a). Quite unexpectedly, the livers of the HFD-CMPF treated mice were remarkably similar to Chow-Controls, both in visual appearance, as well as with Oil Red- O Staining of lipid droplets. The reduced hepatic lipid accumulation corresponded to a significant reduction in liver weight in HFD-CMPF mice compared to HFD-Controls, with weight following CMPF treatment reduced even compared to Chow-controls (Fig. 10b). To confirm that the reduced lipid accumulation corresponded to improved insulin sensitivity, mice were injected intravenously with a bolus of insulin and sacrificed 10 minutes later. Harvested tissues were evaluated for pAkt as a marker of insulin signalling. Levels of pAkt were significantly increased in the livers of the HFD-CMPF mice compared to HFD-Controls, consistent with improved insulin sensitivity (Fig. 10c). Interestingly, there was no difference in insulin signalling in the muscle, suggesting that the improved phenotype is due to the liver and not other peripheral insulin-sensitive tissues (Fig 10d). Therefore, CMPF rescues diet-induced NAFLD to improve insulin sensitivity.

CMPF Treatment Alters the Liver Metabolomic Profile Indicating Alternate Glucose Utilization

[0097] To determine the mechanism underlying the reversal of steatosis and insulin resistance in DIO with CMPF treatment, we employed two discovery-based approaches: global metabolomics profiling and microarray analysis.

[0098] Global metabolomics profiling revealed significant changes in a number of pathways associated with CMPF treatment. The two primary categories of interest were in glucose metabolism and glutathione metabolism. Consistent with previous hyperinsulinemic euglycemic clamp data in lean animals, HFD-CMPF mice appear to shunt glucose through alternate, non-oxidation pathways. Several intermediate metabolites of the pentose phosphate pathway (PPP) were significantly increased with in HFD- CMPF mice compared to both Chow- and HFD-Controls (Fig. 1 1 a). Flux through the PPP is initiated when glucose-6-phosphate (G6P) is oxidized to 6-phosphgluconate, both of which are increased with CMPF. Increased flux through this pathway alters NADPH availability and can affect nucleotide biosynthesis. Thus, CMPF treatment in obese animals induces alternate glucose utilization, which may potentiate lipid clearance and improved insulin sensitivity.

[0099] Alterations in the glutathione pathway, including lower levels of several gamma glutamyl amino acids and the glutathione catabolite 5- oxoproline suggests that CMPF induces alterations in glutathione synthesis. Glutathione is essential for maintaining the redox status of cells, thus alterations in this pathway are consistent with CMPF inducing a state of oxidative stress. Increases in flux through the PPP may be occurring in response to the decreased level of glutathione synthesis to increase NADPH levels (pathway illustrated in Figure 1 1 , part B). Overall, CMPF induces an altered metabolic state in the liver of obese animals.

[00100] Microarray analysis was performed on the livers of Chow- and HFD-controls, as well as HFD-CMPF mice at the end of the 6 week diet period. Consistent with alterations in glucose and amino acid metabolism, several genes involved in metabolic processes were significantly altered. Of primary interest, the mTORcl target gene Lipinl was significantly increased in HFD-CMPF livers (Fig.1 1 c). Furthermore, increased activity of this gene, as determined by increased nuclear localization, was confirmed by western blot in livers HFD-CMPF mice compared to controls (Fig. 1 1 d). Lipinl is a regulator of numerous downstream transcription factors that regulate hepatic lipid accumulation. Thus, CMPF may alter mTOR signaling to reduce hepatic lipid accumulation and increase insulin sensitivity in obese mice.

CMPF Treatment Improves Insulin Sensitivity in a Genetic Model of Insulin Resistance

[00101 ] Leptin is a hormone produced by the adipose tissue that acts to regulate fat storage through influencing satiety signals. Deletion of the leptin gene (ob/ob mouse strain) induces a loss of satiety signals and results in massive obesity due to overeating on a standard chow diet. The ob/ob mice are a common model of obesity, diabetes, and NAFLD ¹¹ . Therefore, the effect of CMPF was examined in the ob/ob mouse strain to determine if the 'switch' from carbohydrate to fat metabolism induced by CMPF is sufficient to overcome genetic-induced obesity and NAFLD. Seven week old ob/ob mice were injected intraperitoneally with 6mg/kg CMPF for two weeks. At the end of the injection period, the CMPF-injected ob/ob mice (ob/ob-CMPF) weighed significantly more than vehicle- injected controls (ob/ob-Control) (Figure 12, part A). This increased weight gain corresponded to a modest increase in adipose area by MRI imaging, though no difference in circulating adiponectin (Fig. 12b-d). Interestingly, ob/ob-CMPF mice had a significantly elevated random blood glucose level (Figure 12, Part E), however, unlike previous models of CMPF- treatment, ob/ob-CMPF mice had no difference in either fasting blood glucose or fasting plasma insulin levels (Figure 12, parts F,G). Consistent with CMPF-treatment in the DIO model, the ob/ob-CMPF treated mice exhibited dramatically improved insulin sensitivity during IpITT (Figure

12, part H).

CMPF Treatment Reduces Hepatic Liver Content and Alters Whole-Body Fat Distribution in a Genetic Model of Insulin Resistance

[00102] In spite of the increased body weight, CMPF is able to improve insulin sensitivity and reduce hepatic lipid accumulation in ob/ob mice. Livers from ob/ob-control and -CMPF mice were isolated and a reduction in visible lipid accumulation in the livers of ob/ob- CMPF mice was observed (Figure

13, part A). This reduced steatosis was further confirmed by reduced Oil red O staining (Fig 13b), and consistent with reduced liver weight (Fig 13c).

[00103] CMPF appears to cause a shift in whole-body metabolism from being glucose to fat-driven. In the beta cell this corresponds to impaired glucose sensing, and enhanced insulin secretion with palmitate stimulation. In the peripheral tissues, this shifted metabolism enhances fat metabolism in the liver, resulting in either elimination of fat deposits, or prevention from fat accumulation. Importantly, this reduction in hepatic lipid accumulation can occur in the presence of excess fat intake associated with a HFD, as well as under conditions of genetic-induced obesity. EXAMPLE 3: CMPF alters Islet Metabolism to Induce Preferential Fatty Acid Oxidation and Reduce Glucose Metabolism

[00104] Previously, CMPF was demonstrated to impair pancreatic beta cell function in chow-fed mice, leading to impaired insulin biosynthesis and secretion, ultimately resulting in the development of diabetes. Because CMPF was originally identified as being elevated in gestational (GDM) and type 2 diabetes (T2D), conditions characterized by insulin resistance, we wanted to explore the effect of CMPF on islet function under these conditions. Furthermore, due to the observed effect of CMPF on increasing whole-body fatty acid metabolism, as outlined in examples 1 and 2, we investigated whether CMPF may induce beta cell function through enhancing lipid metabolism at the expense of glucose oxidation, and thus glucose-stimulated insulin secretion.

CMPF Treatment Induces a Long-Term Impairment in Beta Cell Function

[00105] Eight week old male CD1 mice were treated once daily for 7 days with either 6mg/kg CMPF (Prentice et al., 2014) or vehicle while maintained on a chow diet. At the end of the treatment period, mice were placed on a 60%kcal from fat HFD for 4-5 weeks to induce insulin resistance (outlined in Figure 14, part A). As anticipated, control-HFD mice exhibited significant glucose intolerance during IpGTT (Figure 14, part B). Interestingly, despite improved insulin sensitivity (outlined in Example 2), CMPF-HFD mice had worsened glucose tolerance compared to Control-HFD mice (Figure 14, part B). Consistent with previous observations in chow-fed mice, CMPF-HFD mice had an absence of glucose-stimulated insulin secretion (GSIS) during IpGTT (Figure 14, part C). Thus, despite the fact CMPF is no longer in circulation, and mice have significantly improved insulin sensitivity, CMPF still worsens glucose tolerance, likely through impairment of beta cell function.

CMPF Treatment Enhances Palmitate-Stimulated Insulin Secretion and Decreases Islet Mass [00106] Consistent with in vivo observations, islets isolated from CMPF- HFD mice exhibited significantly reduced GSIS compared to islets from Control-HFD mice (Figure 15, part A). To determine whether CMPF induced a 'switch' in metabolism from carbohydrate to preferential fat utilization, a palmitate-stimulated insulin secretion (PSIS) was performed on isolated islets. CMPF-HFD islets had significantly greater insulin secretion with palmitate stimulation compared to both Control-Chow and Control-HFD islets (Figure 15, part B). This corresponded to a decrease in total insulin content in isolated islets (Figure 15, part C). However, CMPF-HFD islets did not exhibit a significant difference in ROS (Figure 15, part D). This may be due to beta cell compensation for increased ROS in the form of increase antioxidant expression, as observed in the 7-day treated chow-fed mice (Prentice et al., 2014). As anticipated, islets from the Control-HFD mice were significantly larger than those from the Control-Chow mice. However, intriguingly, islets from the CMPF-HFD mice were significantly smaller than both those from the Control-HFD and Control-Chow mice, (Figure 15, part E). This observation may be due to the improvement in insulin sensitivity. Consistent with the static secretion assays, CMPF-HFD islets had an impaired hyperpolarization of the mitochondrial membrane potential (MMP) in response to the addition of high glucose as compared to both Control-Chow and Control-HFD groups (Figure 15, part F). Thus, previous treatment with CMPF induces a shift in islet metabolism, decreasing glucose oxidation and thus GSIS, while enhancing fatty acid metabolism and PSIS.

CMPF Treatment Exacerbates Glucose Intolerance in HFD-fed Mice

[00107] Under conditions of insulin resistance, as observed in diet- induced obesity (DIO), the pancreatic islets exhibit compensatory increases in mass and function to maintain euglycemia. To determine the effect of CMPF on islets that are in a compensatory state, we treated mice fed a HFD for 6 weeks with either vehicle or 6mg/kg CMPF once daily for 2 weeks (outlined in Figure 16, part A). Mice were maintained on the HFD throughout the injection period. As anticipated, HFD-control mice exhibited worsened glucose tolerance compared to Chow-control mice during both OGTT and IpGTT (Figure 16, parts B,D). Intriguingly, though consistent with the CMPF-HFD model, treatment with CMPF worsened glucose tolerance compared to HFD- controls by both tests (Figure 16, parts B,D). Interestingly, while GSIS was abolished in the HFD-CMPF mice during IpGTT compared to both control groups (Figure 16, part E), GSIS was enhanced from HFD-CMPF mice during OGTT (Figure 16, part C). This difference may be explained by alterations in incretin action, or the release of lipids from the intestine following oral gavage of glucose. Chylomicron release from the intestines may act to potentiate insulin secretion through delivery of fatty acids to the beta cells.

CMPF Treatment Following High Fat Diet Decreases Islet Mass

[00108] In order to compensate for increased insulin resistance, pancreatic islets are known to increase in size and secretory capacity in DIO (HFD) models. Previously, it was observed that islets from 7-day CMPF- treated chow-fed mice exhibited impaired glucose-stimulated insulin secretion, which was attributed to decreased insulin biosynthesis, as well as impaired glucose sensing. To characterize the effect of CMPF under conditions of diet-induced obesity (DIO), islets were isolated from the mice and examined ex vivo. Interestingly, no correspondence to any significant difference in total insulin content in the isolated islets (Figure 17, part A) was observed. However, immunohistochemical staining of whole pancreatic sections revealed a significant decrease in insulin positive area (Figure 1 7 , part B), which corresponded to an overall decrease in islet size in HFD- CMPF islets compared to both Chow- and HFD-Controls (Figure 1 7, parts C, D). When islet size was stratified from the pancreatic sections, a significant increase was observed in the number of large islets in the HFD-Control compared to Chow-Control, as expected based on previous observations. Unexpectedly, however, not only was a loss of these large islets observed in the HFD-CMPF mice, but also a significant increase in very small islets (<1000 pixels in area) compared to both Chow- and HFD-Controls (Figure 1 7, part E). This suggests that CMPF may either be inducing the large islets to decrease in size, or may be inducing apoptosis of the large islets as well as promoting islet neogenesis to stimulate the formation of new, small islets.

[00109] To evaluate whether the smaller islets observed in HFD-CMPF mice were new islets produced through replication, mice were dosed with BrdU throughout the treatment period with CMPF. Interestingly, there was no difference in BrdU accumulation in the islets, suggesting that these smaller islets are not being produced via replication (Fig. 18A). Quantification of individual beta cell size, determined by number of nuclei per islet area, revealed that the individual beta cells were smaller in HFD-CMPF mice compared to HFD-Controls, while the number of cells was significantly increased (Fig. 18B, C). Overall, smaller beta cell number and reduced islet size is consistent with enhanced insulin sensitivity with CMPF treatment. Importantly, there were no signs of apoptosis in the isolated islets, as indicated by cleaved Caspase 3/7 activity at the end of the treatment period (Figure 19, part A).

CMPF Treatment Promotes Fatty Acid Metabolism

[001 10] Islets isolated from the HFD-CMPF mice exhibited significantly impaired glucose-stimulated insulin secretion (GSIS), as well as impaired KCI-induced insulin secretion compared to Chow-Control and HFD-Control islets (Figure 19, parts B, C). Based on hyperinsulinemic euglycemic clamp studies CMPF may be inducing a 'switch' in substrate utilization from carbohydrate-driven metabolism to preferential utilization of other substrates such as fats or amino acids. To see if preferential fatty acid oxidation is occurring in the islets of the HFD-CMPF mice, static palmitate- stimulated insulin secretion assays (PSIS) was performed. Consistent with a 'switch' in metabolism, islets HFD-CMPF mice had greater PSIS as compared to both Chow- and HFD-Controls (Figure 19, parts D, E). Further to this observation, mitochondrial membrane potential measurements (MMP), which reflect mitochondrial substrate utilization, demonstrated that islets from HFD-CMPF mice had a greater depolarization of the MMP in response to the addition of palmitate than in response to high glucose. In contrast, both the Chow- and HFD-controls had a greater response to the addition of high glucose (Figure 19, parts F, G). Further, islets isolated from HFD-CMPF mice had greater levels of reactive oxygen species (ROS) than either Chow- or HFD-Controls. Excess substrate for the TCA cycle, as observed with enhanced beta oxidation of free fatty acids, is known to elevate ROS production and induce a state of oxidative stress, thus these data are consistent with increased fat metabolism in islets (Figure 19, part H). Together, these results support the conclusion that CMPF is inducing a 'switch' in metabolism to preferential fatty acid oxidation, as evidenced by impaired glucose- and enhanced lipid metabolism.

CMPF Treatment Worsens Glucose Tolerance in a Genetic Model of Insulin Resistance

[001 1 1 ] Seven week old leptin-deficient ob/ob mice were injected intraperitoneally with 6mg/kg CMPF for two weeks to evaluate the effect on glucose tolerance and beta cell function (illustrated in Figure 20, part A). At the end of the injection period, an OGTT was performed to determine if CMPF treatment had an effect on glucose tolerance. As observed previously, CMPF treatment caused a significant impairment in glucose tolerance compared to ob/ob-Controls (Figure 20, part B). Paradoxically, this worsening in glucose tolerance corresponded to increased insulin secretion during the OGTT (Figure 20, part C). This increase in insulin secretion in the first 30 minutes post-glucose bolus is likely responsible for the improved glucose tolerance observed in the latter time points of the OGTT (60-120 minutes post-gavage) (Figure 20, part C). To further explore this unexpected finding of increased insulin secretion during OGTT, intraperitoneal glucose tolerance tests (IpGTTs) were performed on mice at the end of the 2 week treatment period. Consistent with the results of the OGTT, CMPF-treated mice were again more glucose intolerant during IpGTT (Figure 20, part D). However, consistent with previous observations, glucose-stimulated insulin secretion was blunted in ob/ob-CMPF mice compared to ob/ob-controls (Figure 20, Part E). Therefore, CMPF worsens glucose tolerance in ob/ob mice and alters glucose-stimulated insulin secretion. The mechanisms underlying the differences in insulin secretion between OGTT and IpGTT remain to be explored.

CMPF Treatment Enhances Palmitate-Stimulated Insulin Secretion and Decreases Islet Mass in a Genetic Model of Insulin Resistance

[001 12] The ob/ob mouse model is a genetic induction of insulin resistance and obesity. The effect of CMPF on the islets of ob/ob mice closely mirrors the phenotype observed in islets isolated from the HFD- CMPF mice. As seen in the HFD-CMPF mice, islets isolated from the ob/ob-CMPF mice were significantly smaller compared to controls (Figure 21 , part A). Static GSIS assays demonstrated a significantly lower fold change in insulin secretion under high glucose conditions in the ob/ob-CMPF islets compared to the ob/ob-Controls (Figure 21 , part B), due to significantly greater insulin secretion in the basal, low glucose condition. This corresponded to significantly greater total intracellular insulin content (Figure 21 , part C). Consistent with previous observations of increased response to fatty acid stimulation, ob/ob-CMPF islets had significantly greater insulin secretion when stimulated with palmitate compared to ob/ob-controls (Figure 21 , part D). As observed in the HFD-CMPF treated mouse islets, and consistent with the secretion results, islets from the ob/ob-CMPF mice have a significant impairment in high-glucose-induced mitochondrial membrane depolarization (Figure 21 , part E). Together, these results suggest that CMPF is able to impair glucose sensing in ob/ob mouse beta cells, and induce a 'switch' to preferential fat oxidation. In line with this, we also observed a significant increase in ROS in the islets of ob/ob-CMPF mice compared to ob/ob- Controls, again consistent with increased fat oxidation (Figure 21 , part F).

[001 13] CMPF appears to cause a shift in whole-body metabolism from being glucose to fat-driven. In the beta cell this corresponds to impaired glucose sensing, and enhanced insulin secretion with palmitate stimulation. In the peripheral tissues, this shifted metabolism enhances fat metabolism in the liver, resulting in either elimination of fat deposits, or prevention from fat accumulation. Importantly, this reduction in hepatic lipid accumulation can occur in the presence of excess fat intake associated with a HFD, as well as under conditions of genetic-induced obesity.

[001 14] Increased fasting insulin secretion is a common feature in CMPF-injected mice. In light of the apparent increase in hepatic lipolysis, it is possible that there is an elevation in circulating lipids which are sensed by the beta cell to stimulate insulin secretion. During the OGTT, both HFD-CMPF and ob/ob- CMPF mice exhibit either unchanged or elevated insulin secretion. However, this is not observed during IpGTT, where insulin secretion is significantly lower. This raises the possibility that oral gavage of glucose stimulates fat release from the intestine to stimulate insulin secretion. Chylomicron release may be responsible for this effect which remains to be determined. Remarkably, despite impairing beta cell function and insulin secretion, CMPF prevents and improves insulin resistance and prevents and improves fatty liver. CMPF likely has a direct effect on the liver but may act by reducing insulin secretion to improve liver function and IR. While the present disclosure has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the disclosure is not limited to the disclosed examples. To the contrary, the disclosure is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[001 15] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. References Abu-Elheiga, L, Matzuk, M.M., Abo-Hashema, K.A., and Wakil, S.J. (2001 ). Continuous fatty acid oxidation and reduced fat storage in mice lacking acetyl- CoA carboxylase 2. Science 291, 2613-2616.

Alves, T. C. et al. Regulation of hepatic fat and glucose oxidation in rats with lipid-induced hepatic insulin resistance. Hepatology 53, 1 175-1 181 , doi: 10.1002/hep.24170 (201 1 ).

Angulo, P. Nonalcoholic fatty liver disease. The New England journal of medicine 346, 1221 - 1231 , doi: 10.1056/NEJMra01 1775 (2002).

Avramoglu, R.K., Basciano, H., and Adeli, K. (2006). Lipid and lipoprotein dysregulation in insulin resistant states. Clinica chimica acta; international journal of clinical chemistry 368, 1 -19. Bain, J.R., Stevens, R.D., Wenner, B.R., llkayeva, O., Muoio, D.M., and Newgard, C.B. (2009). Metabolomics applied to diabetes research: moving from information to knowledge. Diabetes 58, 2429-2443.

Bayard, M., Holt, J. & Boroughs, E. Nonalcoholic fatty liver disease.

American family physician, 73, 1961 -1968 (2006).

Bergman, R.N., and Ader, M. (2000). Free fatty acids and pathogenesis of type 2 diabetes mellitus. Trends in endocrinology and metabolism: TEM 11, 351 -356. Birkenfeld, A. L. & Shulman, G. I. Nonalcoholic fatty liver disease, hepatic insulin resistance, and type 2 diabetes. Hepatology 59, 713-723, doi: 10.1002/hep.26672 (2014).

Cao, H., Gerhold, K., Mayers, J.R., Wiest, M.M., Watkins, S.M., and Hotamisligil, G.S. (2008). Identification of a lipokine, a lipid hormone linking adipose tissue to systemic metabolism. Cell 134, 933-944. Chu, X., Liu, L , Na, L, Lu, H., Li, S., Li, Y., and Sun, C. (2013). Sterol regulatory element- binding protein-1 c mediates increase of postprandial stearic acid, a potential target for improving insulin resistance, in hyperlipidemia. Diabetes 62, 561 -571 . Deguchi, T., Kouno, Y., Terasaki, T., Takadate, A., and Otagiri, M. (2005). Differential contributions of rOatl (Slc22a6) and rOat3 (Slc22a8) to the in vivo renal uptake of uremic toxins in rats. Pharmaceutical research 22, 619-627. Despres, J. P. , and Lemieux, I . (2006). Abdominal obesity and metabolic syndrome. Nature 444, 881 -887.

Eckel, R.H., Grundy, S.M., and Zimmet, P.Z. (2005). The metabolic syndrome. Lancet 365, 1415- 1428.

Erion, D. M. et al. Prevention of hepatic steatosis and hepatic insulin resistance by knockdown of cAMP response element-binding protein. Cell metabolism 10, 499-506, doi: 10.1016/j.cmet.2009.10.007 (2009).

Fiehn, O., Garvey, W.T., Newman, J.W., Lok, K.H., Hoppel, C.L., and Adams, S.H. (2010). Plasma metabolomic profiles reflective of glucose homeostasis in non-diabetic and type 2 diabetic obese African-American women. PloS one 5, e15234. Friedrich, N. (2012). Metabolomics in diabetes research. The Journal of endocrinology 215, 29- 42.

Fisher, F.M., Estall, J.L., Adams, A.C., Antonellis, P.J., Bina, H.A., Flier, J.S., Kharitonenkov, A., Spiegelman, B.M., and Maratos-Flier, E. (201 1 ). Integrated regulation of hepatic metabolism by fibroblast growth factor 21 (FGF21 ) in vivo. Endocrinology 152, 2996-3004.

Galman, C, Lundasen, T., Kharitonenkov, A., Bina, H.A., Eriksson, M. , Hafstrom, I., Dahlin, M., Amark, P., Angelin, B., and Rudling, M.

(2008). The circulating metabolic regulator FGF21 is induced by prolonged fasting and PPARalpha activation in man. Cell metabolism 8, 169-174

Ginsberg, H.N. (2006). Is the slippery slope from steatosis to steatohepatitis paved with triglyceride or cholesterol? Cell metabolism 4, 179-181 .

Jornayvaz, F. R. et al. A high-fat, ketogenic diet causes hepatic insulin resistance in mice, despite increasing energy expenditure and preventing weight gain. American journal of physiology. Endocrinology and metabolism 299, E808-815, doi: 10.1 152/ajpendo.00361 .2010 (2010).

Joseph, J.W., Koshkin, V., Saleh, M.C., Sivitz, W.I., Zhang, C.Y., Lowell, B.B., Chan, C.B., and Wheeler, M. B. (2004). Free fatty acid-induced beta- cell defects are dependent on uncoupling protein 2 expression. The Journal of biological chemistry 279, 51049-51056.

Lavine, J. E. et al. Effect of vitamin E or metformin for treatment of nonalcoholic fatty liver disease in children and adolescents: the TONIC randomized controlled trial. JAMA : the journal of the American Medical Association 305, 1659-1668, doi: 10.1001 /jama.201 1 .520 (201 1 ).

Masters, R.K. , Powers, D.A. , and Link, B.G. (2013). Obesity and US mortality risk over the adult life course. American journal of epidemiology 177, 431 -442.

Ng, M., Fleming, T., Robinson, M., Thomson, B., Graetz, N., Margono, C, Mullany, E.C., Biryukov, S., Abbafati, C, Abera, S.F., et al. (2014). Global, regional, and national prevalence of overweight and obesity in children and adults during 1980-2013: a systematic analysis for the Global Burden of Disease Study 2013. Lancet 384, 766-781 .

Potthoff, M.J., Inagaki, T., Satapati, S., Ding, X., He, T., Goetz, R., Mohammadi, M., Finck, B.N., Mangelsdorf, D.J., Kliewer, S.A., et al. (2009). FGF21 induces PGC-1 alpha and regulates carbohydrate and fatty acid metabolism during the adaptive starvation response. Proceedings of the National Academy of Sciences of the United States of America 106, 10853- 10858 Prentice, K. J. et al. The Furan Fatty Acid Metabolite CMPF Is Elevated in Diabetes and Induces beta Cell Dysfunction. Cell metabolism 19, 653- 666, doi: 10.1016/j.cmet.2014.03.008 (2014).

Prentki, M., and Nolan, C.J. (2006). Islet beta cell failure in type 2 diabetes. The Journal of clinical investigation 116, 1802-1812. Samuel, V. T. et al. Inhibition of protein kinase Cepsilon prevents hepatic insulin resistance in nonalcoholic fatty liver disease. The Journal of clinical investigation 1 17, 739-745, doi: 10.1 172/JCI30400 (2007).

Tang, C, Han, P., Oprescu, A.I., Lee, S.C., Gyulkhandanyan, A.V. , Chan, G.N., Wheeler, M.B., and Giacca, A. (2007). Evidence for a role of superoxide generation in glucose-induced beta-cell dysfunction in vivo. Diabetes 56, 2722-2731 .

Torres, D. M. & Harrison, S. A. Diagnosis and therapy of nonalcoholic steatohepatitis. Gastroenterology 134, 1682-1698, doi: 10.1053/j.gastro.2008.02.077 (2008). Wang, T.J., Larson, M.G., Vasan, R.S., Cheng, S., Rhee, E.P., McCabe, E., Lewis, G. D., Fox, C.S., Jacques, P.F., Fernandez, C, et al. (201 1 ). Metabolite profiles and the risk of developing diabetes. Nature medicine 17, 448-453. Warensjo, E., Riserus, U., and Vessby, B. (2005). Fatty acid composition of serum lipids predicts the development of the metabolic syndrome in men. Diabetologia 48, 1999-2005.

Yang, Z.H., Miyahara, H., Takemura, S., and Hatanaka, A. (201 1 ). Dietary saury oil reduces hyperglycemia and hyperlipidemia in diabetic KKAy mice and in diet-induced obese C57BL/6J mice by altering gene expression. Lipids 46, 425-434.