Methods for Reducing Brain Inflammation, Increasing Insulin Sensitivity, and Reducing Ceramide Levels

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of United States Provisional Application Serial No. 61/623,896, filed April 13, 2012, herein incorporated by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

The field of the invention is related to methods for reducing brain inflammation, increasing insulin sensitivity, and reducing ceramide levels in a subject including by administering one or more compounds of Formula I, Formula II, or both.

Current research indicates that obesity in large part results from a mismatch of food intake versus energy utilization, resulting in energy or caloric excess and ultimately obesity. This affects many organ systems that become disregulated as regards the ability to process nutrients and in turn signal the appropriate physiological responses to food intake to the liver, muscle, heart, adipose tissue, and the brain. Sustained exposure to excess dietary fatty acids (FA) causes lipid accumulation in non-adipose tissues with limited storage capacity. This lipotoxicity causes cellular stresses and inflammation that lead to cell damage (Schaffer (2003) Current Opin. Lipidol, 14:281-287), and in peripheral tissues contributes to insulin resistance and metabolic syndrome (de Luca et al. (2008) , FEBS Lett. 582:97-105 ; Shoelson et al. (2007) Gastroenterology 132:2169-2180).

The brain plays a central role in regulating food intake and metabolism by integrating multiple signals from peripheral organs that metabolize or store nutrients. Recent evidence demonstrates that nutrient excess, especially excess fatty acids, has a deleterious effect on the ability of neurons in the brain to perceive and respond to nutrient signaling. The

hypothalamus is similarly vulnerable to lipotoxicity. The hypothalamus senses availability of nutrients, including fat, to control food intake and regulate energy balance (Lam et al. (2005) Nat. Neurosci. 8:579-584; Lopez et al. (2007) Bioessays 29:248-261). However, elevated saturated FA is sufficient to induce lipotoxic stress and attenuate hypothalamic responses to insulin and leptin negative feedback, contributing to dietary-induced obesity (DIO) and attendant metabolic dysfunction (Milanski et al. (2009) J. Neurosci. 29:359-370; Posey et al. (2009) Am. J. Physiol. Endocrinol. Metab. 296:E1003-E1012; Zhang et al. (2008) Cell 135:61-73). Long-chain FA signal nutrient surplus in hypothalamus and adjusting FA catabolic and anabolic processing alters feeding behavior (Lopez et al (2007) Bioessays 29:248-261; Ronnett et al. (2005) Physiol. Behav. 8 :25-35). Fatty acid synthase (FASN, EC 2.1.38).) catalyzes ATP- and NADH-dependent palmitate synthesis (Wakil (1989) Biochemistry 28:4523-4530). Carnitine palmitoyltransferase- 1 (CPT-1) catalyzes long-chain FA translocation into mitochondria for β-oxidation (McGarry et al. (1997) Eur. J. Biochem. 244: 1-14) . Without being limited to any particular theory or mechanism, C75 is a FAS inhibitor and CPT-1 stimulator (Landree et al, (2004) J. Biol. Chem. 279:3817-3827) that decreases expression of orexigenic neuropeptides agouti-related protein (AgRP) and neuropeptide Y (NPY) (Aja et al. (2006) Am. J. Physiol. Regul. Integr. Comp. Physiol.

29LR148-R154; Kim et al. (2004) J. Biol. Chem. 279:19970-19976) to decrease food intake and increase energy expenditure (Thupari et al. (2004) Am J. Physiol. Endocrinol Metab. 287: E97-E104). C75's effects rely less on FAS inhibition than on CPT-1 stimulation and FAOx (Landree et al. (2004) J. Biol. Chem. 279:3817-3827; Thupari et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99:9498-9502); intracerebroventricular (i.c.v) injection of C89b, a CPT-1 stimulator that does not affect FA synthesis, elicits persistent hypophagia and weight loss (Aja et al (2008) Am. J. Physiol Regul Integr. Comp. Physiol. 294:R352-R361). Glycerol- 3 -phosphate acyltransferases (GPATs) have emerged as another target for appetite suppression and weight loss. GPATs catalyze the first esterification step for acylglycerol and phospholipid syntheses (Bell et al (1980) Annu. Rev. Biochem. 49:459-487). GPAT inhibitor FSG67 (Wydysh et al. (2009) J. Med. Chem. 52:3317-3327) given by

intraperitoneal (i.p.) injection or intracerebroventricular (i.c.v.) injection elicits hypophagia and weight loss (Kuhajda et al (2011) Am. J. Physiol Regul. Integr. Comp. Physiol.

301 :R116-R130). There are a number of GPATs known including GPAT1 (GPAM), GPAT2, GPAT3 and GPAT4 in humans. As used herein, unless provided otherwise, GPAT includes GPATs 1 to 4 and alternative spliced forms thereof.

Fluctuating ATP level may be a common signal in hypothalamic nutrient sensing and appetite control (MacLean et al (2004) Brain Res. 1020:1-11), by altering activity of AMP- activated protein kinase (AMPK), an energy-sensor that ensures intracellular and body energy balance (Hardie (2008) Int. J. Obes. 32:S7-S 12). With high AMP: ATP ratio, AMPK is phosphorylated and activated (pAMPK) to preserve and produce ATP by multiple means, including fat catabolism. Food restriction decreases hypothalamic ATP (MacLean et al. (2004) Brain Res. 1020:1-11), and whereas food restriction and orexigenic signals increase hypothalamic pAMP to increase ingestion, nutrients and other negative feedback signals decrease hypothalamic pAMPK and food intake (Minokoshi et al. (2004) Nature 428:569- 574).

While oxidative metabolism produces ATP, it also generates reactive oxygen species (ROS). Sustained high levels of ROS lead to oxidative stress and impaired mitochondrial function and ATP production (Lin et al. (2006) Nature 443:787-795). Increased ROS can also cause unfolded or misfolded proteins to accumulate in the endoplasmic reticulum (ER). This ER stress initiates the unfolded protein response (UPR) (Rutkowski et al. (2007) Trends Biochem. Sci. 32:469-476) that reduces protein translation generally, but upregulates expression of transcription factors X-box binding protein- 1 (XBP1) and activating transcriptional factor (ATF) 4 and ATF6, to increase ER chaperone and degradation machinery that optimize protein folding. Overnutrition induces hypothalamic ER stress, leading to insulin and leptin resistance and obesity (Zhang et al. (2008) Cell 135:61-73). Excess palmitate induces ER stress in the mHypoE-44 hypothalamic cell line (Mayer et al. (2010) Endocrinology 151 :576-585), and CNS administration of an ER stress inducer inhibits hypophagic effects of leptin and insulin (Won et al. (2009) Obesity 17:1861-1865).

Overnutrition also leads to inflammation, characterized by elevated interleukin (IL) 6, IL1 β, and tumor-necrosis factor-a (TNFa). Inflammation, potentially mediated by ER stress (de Luca et al. (2008) FEBSLett. 582:97-105), is involved in development and pathogenesis of insulin resistance and metabolic syndrome (Shoelson et al. (2007) Gastroenterology 132:2169-2180). The hypothalamus is susceptible to inflammation from saturated FA (Milanski et al. (2009) J. Neurosci. 29:359-370; Posey et al. (2009) Am. J. Physiol.

Endocrinol. Metab. 296:E1003-E1012). Mice with hypothalamic FAS deletion are protected from DIO and inflammation (Chakravarthy et al. (2009) J. Lipid Res. 50:530-640); therefore, controlling hypothalamic FA metabolism might prevent neuronal inflammation and its contribution to DIO.

SUMMARY OF THE INVENTION

The present disclosure provides for, and includes the use of a compound of Formula I, Formula II, or both for the manufacture of a medicine for reducing inflammation. In certain aspects, the compound of Formula I is FSG67 and the compound of Formula II is C75

The present disclosure also provides for, and includes, the use of a compound of Formula I, Formula II, or both for the manufacture of a medicine for the activation of palmitate oxidation in a subject in need. The present disclosure also provides for, and includes the use of a compound of Formula I, Formula II, or both for the manufacture of a medicine for improving glucose or insulin tolerance in a subject in need of improved glucose or insulin tolerance.

The present disclosure also provides for, and includes the use of a compound of Formula I, Formula II, or both for the manufacture of a medicine for increasing insulin sensitivity in a subject in need of increased insulin sensitivity.

The present disclosure also provides for, and includes methods for activating palmitate oxidation in a subject in need thereof comprising administering to a subject an inhibitor fatty acid synthesis or an inhibitor of triglyceride biosynthesis wherein the inhibitor activates palmitate oxidation.

The present disclosure also provides for, and includes methods for reducing ceramide levels in a subject in need, comprising administering to a subject an inhibitor fatty acid synthesis or an inhibitor of triglyceride biosynthesis.

The present disclosure further provides for, and includes methods for reducing inflammation comprising administering an inhibitor of fatty acid synthesis or triglyceride biosynthesis.

In addition, the present disclosure provides for, and includes methods for improving hypothalamic function in a subject comprising administering an inhibitor of fatty acid synthesis or triglyceride biosynthesis.

The present disclosure also provides for, and includes methods for improving glutathione turnover in a subject comprising administering an inhibitor of fatty acid synthesis or triglyceride biosynthesis.

The present disclosure also provides for, and includes methods for improving glucose tolerance to a subject in need comprising administering to a subject in need an inhibitor of fatty acid synthesis or triglyceride biosynthesis.

The present disclosure also provides for, and includes methods for improving increasing insulin sensitivity to a subject in need comprising administering to a subject in need an inhibitor of fatty acid synthesis or triglyceride biosynthesis.

The present disclosure also provides for, and includes methods decreasing leptin levels sensitivity to a subject in need comprising administering to a subject in need an inhibitor of fatty acid synthesis or triglyceride biosynthesis.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A and IB are micrographs of immunostained primary hypothalamus neurons (PHNs) showing (A) 85% MAP2-positive neurons, 0.41% OX42-positive microglia, and (B) 4.8% GFAP -positive astrocytes. Hoechst 33342 stained nuclear DNA.

Figure 1C and ID are graphs showing the effect of a treatment of PHNs with (C) 70 μΜ C75 or (D) 160 μΜ FSG67 on cell viability after 24 hours according to an aspect of the present disclosure.

Figure IE and IF are graphs showing the effect of an acute treatment of PHNs with C75 (IE) and FAG67 (IF) on cFOS expression.

Figures 1G and 1H show the effects of C75 (1G) and FSG67 (1H) on fatty acid (FA) synthesis after exposure for two hours.

Figures II and 1 J are graphs showing the effect of C75 (II) or FSG67 (1 J) on palmitate oxidation in PHN after 4 h.

Figures I to IN show the effects of C75 and FSG67 on ATP levels relative to an untreated PHN control and AMPK activation over time.

Figures 2A and 2B show the relative levels of CPT 1 a, CPT lb, and CPT 1 c in the (A) brain tissues from rat and in (B) PHN cultures.

Figures 2C and 2D show the effects of (C) C75 or (D) FSG67 on CPT-1 expression in

PHN.

Figures 2E and 2F show the relative expression of GPAT homologues 1 to 4 in (E) Brain tissues and (F) PHN.

Figures 2G and 2H show the effects of (G) C75 and (H) FSG67 on the expression of GPAT isoforms.

Figures 21 and 2J show the effects of (I) C75 and (J) FSG67 on the expression of SREBPlc and FAS in PHN.

Figures 3A and 3B show the effects of exposure to C16:0 in combination with (A)

C75 or (B) FSG67 on OS production.

Figure 3C is a graph a showing the effects of acute C75 on SOD activity with or without C16:0 according to an aspect of the present disclosure.

Figures 3D, and 3E show the effects of (D) C75 or (E) FSG67 on mitochondrial membrane potential.

Figure 4A is a heat map of a targeted lipidomic analysis of PHN treated with vehicle (control), C16:0, or C16:0 + C75 (70 μΜ). Figures 4B to 4E, show representative (B) free FA, (C) acylglycerol, (D) ceramide, and (E) cholesterol ester data from Figure 4A.

Figure 4F is a heat map of untargeted metabolomics analysis of PFiN treated with vehicle (control) or C75.

Figure 5 A is a graph showing the effects of TG on the UPR.

Figure 5B shows the effects of CI 6:0 in the absence or presence of C75 on ATF4 and ATF6 levels.

Figures 5C and 5F shows the effects of C16:0 in the absence or presence of FSG67 on (C) ATF4 and ATF6 levels and (F) XPB1 splicing.

Figure 5D shows the effects of C89b on ATF4 and ATF 6.

Figure 5E shows the effects of C16:0 in the absence or presence of C75 on XPB1 splicing.

Figure 5F shows the effect of C16:0 in the absence or presence of FSG67 on XPB1 splicing.

Figures 6A and 6B shows the effects of (A) C75 and (B) FSG67 on C16:0-induced mRNA expression of TNFa and IL1 β according to an aspect of the present disclosure.

Figures 6C and 6D shows the effects of (C) C75 and (D) FSG67 on IL6 and ILi protein levels.

Figure 6E shows the effect of the selective CPT-1 stimulator C89b on cytokine expression.

Figures 7A to 71 shows the effects of C75 on (A) food intake, (B) body weight, (C) activated muscle AMP a, (D) inactivated ACCa, (E) MCD mRNA expression, (F) GLUT4, (G) blood glucose, (H) serum insulin, and (I) serum leptin levels.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure provides for and includes methods for activating palmitate oxidation in a subject in need by administering an inhibitor of fatty acid synthesis or triglyceride biosynthesis. In an aspect, an inhibitor of fatty acid synthesis or triglyceride biosynthesis is an inhibitor of glycerol-3 -phosphate acyltransferase (GPAT). In an aspect, the inhibitor of fatty acid synthesis or triglyceride biosynthesis may be a compound of Formula I, Formula II, or both. In certain aspects, the fatty acid synthesis or triglyceride biosynthesis inhibitor may be the compound C75. In another aspect, the fatty acid synthesis or triglyceride biosynthesis inhibitor may be the compound FSG67. In yet another aspect, the inhibitor may be a combination of compounds C75 and FSG67. In an aspect according to the present disclosure, a subject in need of increased oxidation of palmitate may be a person having excess fatty acids (FA) in the blood, tissues, or organs. In some aspects the subject may have excess FA due to an excess of FA in the diet. In other aspects, the subject may have an excess FA due to metabolic disregulation. In an aspect, the subject in need may have an increase in the levels of palmitic acid (C 16:0) in the blood, tissues, or organs. In further aspects, the subject in need may have increased palmitoleic acid (C16: 1). In an aspect, the subject in need may have increased stearic (C18:0), oleic acid (C18: l), or linoleic acids (C18:2). In other aspects, the subject in need may have increased arachidonic (C20:4) or eicosapentaenoic acids (C20:5). In yet another aspect, the subject in need may have increased docosahexaenoic acid (C22:6). In a further aspect according the present disclosure, the subject in need may have one or more increased fatty acids selected from palmitic acid (C16:0), palmitoleic acid (C16:l), increased stearic (C18:0), oleic acid (C18: l), linoleic acids (C18:2), arachidonic (C20:4), eicosapentaenoic acids (C20:5), or docosahexaenoic acid (C22:6). Increased fatty acids may be measured by methods generally known in the art.

In aspects according to the present disclosure, a subject in need may be identified by performing an oral glucose tolerance test (OGTT). Oral glucose tolerance tests reflect the uptake of glucose from blood by tissues, along with suppression of release of endogenously produced glucose into blood from tissues, as provided in the clearance rate of an exogenous glucose load from the bloodstream. The OGTT is widely used in clinical practice. This approach does not provide information regarding the specific metabolic fate or consequences of the glucose administered nor provide information about the mechanisms underlying impaired glucose tolerance but may be used to identify subjects in need of increased palmitate oxidation.

In other aspects according to the present disclosure, a subject in need of increased palmitate oxidation may be identified by performing fat tolerance testing that measures the uptake of fatty acids from blood by tissues. As with the OGTT, the test does not provide information about the specific metabolic fate or consequences of the fat administered.

Similarly, no information about the mechanisms underlying impaired fat tolerance. Fat tolerance testing has mostly been used to assess the clearance of dietary fat from blood in the context of evaluating hyperlipidemia. Fat tolerance testing provides for the identification of subjects having a need for increased palmitate oxidation. In one particular aspect, the subject in need of increased palmitate oxidation is an individual diagnosed with type 1 diabetes mellitus. In another particular aspect, the subject is an individual diagnosed with type 2 diabetes mellitus, Maturity Onset Diabetes of the Young (MODY), Latent Autoimmune Diabetes of Adults (LAD A), or pre-diabetes. In one aspect, the subject may be 1) an individual diagnosed with one or more of the conditions selected from the group consisting of overweight, obesity, visceral obesity and abdominal obesity; 2) an individual who shows one, two or more of the following conditions: a) a fasting blood glucose or serum glucose concentration greater than 100 mg/dL, in particular greater than 125 mg/dL, b) a postprandial plasma glucose equal to or greater than 140 mg dL, or c) an HbAlc value equal to or greater than 6.0%, in particular equal to or greater than 6.5%, in particular equal to or greater than 8.0%; 3) an individual wherein one, two, three or more of the following conditions are present: a) obesity, visceral obesity and/or abdominal obesity, b) a triglyceride blood level greater than or equal to 150 mg/dL, c) high density lipoprotein (HDL)-cholesterol blood level <40 mg/dL in female patients and <50 mg/dL in male patients, d) a systolic blood pressure greater than or equal to 130 mm Hg and a diastolic blood pressure greater than or equal to 85 mm Hg, or e) a fasting blood glucose level greater than or equal to 100 mg/dL; or 4) an individual with morbid obesity.

In yet other aspects according to the present disclosure, a person in need of increased palmitate oxidation may be a subject having a metabolic disease, obesity or an obesity related condition. In one aspect, such a person could have a body mass index (BMI, body mass divided by height-squared, kg/m ² ) above the upper limit of "normal" range, i.e. over 25 (25- 30 = "overweight", over 30 = "obese") (World Health Organization). In another aspect, a person in need may have a measured or estimated body fat percentage of 24% and above for a women or 25% and above for a man.

In yet other aspects according to the present disclosure, a person in need of increased palmitate oxidation may be a subject having increased levels of brain inflammation. One of ordinary skill in the art would understand how to measure brain inflammation including by magnetic resonance imaging (MRI), positron emission tomography (PET scan), X-ray computed tomography (CT scan), or angiography. In some aspects, a person in need may have brain atrophy, hyperintensities in the white matter and lacunae, or amyloid deposits visible through standard neuroimaging techniques. In yet other aspects according to the present disclosure, a person in need of increased palmitate oxidation may be a subject having decreased cognitive functions including waxing/waning delirium and dementia. In a further aspect, a subject in need of activated palmitate oxidation may need to modify one or more measures of metabolic homeostasis selected from the group consisting of reducing inflammation, increasing SOD activity, increasing fatty acid oxidation, increasing glutathione turnover, reducing ceramide levels, decreasing C16:0 and C18:0 cholesterol esters levels, increasing CPT-1 activity, increasing insulin sensitivity, decreasing leptin levels, improving glucose tolerance, improving insulin signaling, reducing inflammation, increasing SOD activity, and improving leptin signaling. In a further aspect, a subject in need of improved glucose tolerance may need to modify at least two or more of said physiological parameters selected from the group above. In other aspects, a subject in need may need to modify at least three or more of said physiological parameters selected from the group above. In yet other aspects, the subject in need may need to modify at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten of said physiological parameters selected from the group consisting of reducing inflammation, increasing SOD activity, increasing fatty acid oxidation, increasing palmitate oxidation, increasing glutathione turnover, reducing ceramide levels, decreasing CI 6:0 and CI 8:0 cholesterol esters levels, increasing CPT-1 activity, increasing insulin sensitivity, decreasing leptin levels, improving glucose tolerance, improving insulin signaling, reducing inflammation, increasing SOD activity, and improving leptin signaling.

In aspects according to the present disclosure, inhibition of fatty acid synthesis or triglyceride biosynthesis and activation of palmitate oxidation decreases fatty acid levels in a sample obtained from a subject. In certain aspects, the level of palmitate in a sample obtained from a subject decreases relative to an initial level of palmitate in a sample obtained from a subject. In other aspects, the level of palmitate measured in a sample is decreased relative to samples obtained from a normalized population or may be decreased relative to a initial palmitate level determined in a subject based on one or more pre-treatment measurements. In some aspects, an initial pre-treatment level may be determined for one or more fatty acids selected from the group consisting of palmitic acid (CI 6:0), palmitoleic acid (C16:l), increased stearic (C18:0), oleic acid (C18: l), linoleic acids (C18:2), arachidonic (C20:4), eicosapentaenoic acids (C20:5), and docosahexaenoic acid (C22:6) is determined and used as a baseline. In some aspects, two or more fatty acid levels may be determined. In other aspects, three or more fatty acid levels may be measured. In some aspects, this includes the standard lipids measured in panel screens, such as triglycerides, cholesterol, LDL, HDL, and also metabolomics screening which evaluates all lipids in serum samples. In one aspect of the present disclosure, increased palmitate oxidation is measured by screening serum samples with a metabolomics panel known to those of ordinary skill in the art. According to aspects of the present disclosure, increased levels of palmitate oxidation following administration of a compound of Formula I, Formula II, or both relative to an initial level may be presented as percentages. In an aspect, palmitate oxidation may increase by 5% or more relative to the pre-treatment level. In other aspects, palmitate oxidation may increase by 10% or more relative to the pre-treatment level. In some aspects, palmitate oxidation may increase 15%, 20% or more. In some aspects, palmitate oxidation will increase by 2.5, 5%, 7.5% or 10% of the pre-treatment level. In further aspects according to the present disclosure, palmitate oxidation may increase from 1 to 5% or 1 to 10%. In other aspects, palmitate oxidation may increase from 5 to 10%, 5 to 20%, or 5 to 30%. In other aspects, palmitate oxidation may increase from 10 to 15%, 10 to 20%, 15 to 20%, or 15 to 30%. See, e.g., Lightle et al., (2003) Archives ofBiochem and Biophysics 419, 120 to 128, the content of which is herein incorporated by reference in its entirety.

The present disclosure further includes and provides for methods for reducing ceramide levels in a subject in need by administering an inhibitor fatty acid synthesis or triglyceride biosynthesis. In an aspect, the methods further provide for reducing ceramide derivative levels. In other aspects, the methods provide for reducing ceramide and ceramide derivative levels. In an aspect, an inhibitor of fatty acid synthesis or triglyceride biosynthesis is an inhibitor of glycerol-3 -phosphate acyltransferase (GPAT), where the inhibitor results in a reduction in ceramide levels in the subject. In an aspect, the inhibitor of fatty acid synthesis or triglyceride biosynthesis may be a compound of Formula I, Formula II, or both. In certain aspects, the fatty acid synthesis or triglyceride biosynthesis inhibitor may be the compound C75. In another aspect, the fatty acid synthesis or triglyceride biosynthesis inhibitor may be the compound FSG67. In yet another aspect, the inhibitor may be a combination of compounds C75 and FSG67.

In an aspect, the reduction of ceramide may be direct or indirect. In an aspect, a person in need has increased levels of ceramide in a sample. In certain aspects, the sample may be a blood sample. In aspects according to the present disclosure, increased ceramide or ceramide derivative levels in a biological sample may be determined. In some aspects, the increased plasma ceramide level may be associated with obesity and Type 2 diabetes. In some aspects, the increased plasma ceramide level may be associated with serum markers of inflammation, as demonstrated with T2DM (Haus et al., 2009, Diabetes 58; 337 to 343, the content of which is herein incorporated by reference in its entirety), as well as in adolescents at risk for metabolic syndrome (Majumdar and Mastrandrea, 2012, Endocrine 41; 441 to 449, the content of which is herein incorporated by reference in its entirety).

In aspects according to the present disclosure, a subject in need of reduced ceramide levels may have an increased level of ceramide in a sample relative to the levels of ceramide measured in samples from a normal population. A subject in need of reduced ceramide levels may have levels of ceramide that are 5% or greater than the normal level of ceramide. In other aspects, a subject may have levels of ceramide that are 10% or greater than the normal level of ceramide. In an aspect, the level of ceramide may be 20% higher than the normal level. In other aspects, the ceramide levels may be 30 or 40% higher than normal. In aspects according to the present disclosure, a subject may have from 1 to 5%, 1 to 10%, 2.5 to 10%>, 5 to 10% or greater than 10% higher levels than a normal population. In aspects according to the present disclosure, a subject may have from 2.5 to 7.5%, 2.5 to 10%, 5 to 10%, 7.5 to 10%) or greater than 15% higher levels than a normal population. In further aspects, the level of ceramide in a subject in need may be from 50 to 100% higher. In yet another aspect the level of ceramide may be greater than 50%, greater than 75% or greater than 90% higher than normal.

In an aspect, a subject having increased levels of ceramide may have increased levels of dihydroceramide. In another aspect, a subject may have an increased level of glycosyl ceramide (GL). In certain aspects, ceramide may be a plasma ceramide level and may be glucosyl ceramide (GL-1) where the normal mean level is 0.98±0.09, lactosyl ceramide (GL- 2) where the normal mean level is 0.55±.09, ceramide trihexoside (GL-3) where the normal mean level is 0.76±.21, or tetrahexosyl ceramide (GL-4) where the normal mean level is 0.31±.09. In other aspects, the ceramide level may be a red blood cell level of ceramide.

Methods to determine the levels of ceramide species in biological samples are known in the art, for example, as provided in Kasumov et ah, "Quantification of Ceramide Species in Biological Samples by Liquid Chromatography-Electrospray Tandem Mass

Spectrometry," Anal. Biochem. 401(1): 154-161 (2010) or Hu, W., et al, (2009) J. Lipid. Res. 50, 1852-1862, herein incorporated by reference in their entireties. Quantitative analyses of ceramides, sphingolipids, DAG, TAG, cholesterol or cholesterol esters may be performed by high pressure liquid chromatography (HPLC) coupled to a turbo ion electro spray source of a triple stage quadrupole tandem mass spectrometer operated in positive ionization mode, and a mass spectrometer operated in negative ionization mode for the analysis of free fatty acids. Lipid analytes may be monitored in multiple reaction monitoring (MRM).

In one particular aspect, the subject in need of a reduction in ceramide levels is an individual diagnosed with type 1 diabetes mellitus. In another particular aspect, the subject is an individual diagnosed with type 2 diabetes mellitus, Maturity Onset Diabetes of the Young (MODY), Latent Autoimmune Diabetes of Adults (LAD A) or pre-diabetes. In one aspect, the subject may be 1) an individual diagnosed with one or more of the conditions selected from the group consisting of overweight, obesity, visceral obesity and abdominal obesity; 2) an individual who shows one, two or more of the following conditions: a) a fasting blood glucose or serum glucose concentration greater than 100 mg/dL, in particular greater than 125 mg/dL, b) a postprandial plasma glucose equal to or greater than 140 mg dL, c) an HbAlc value equal to or greater than 6.0%, in particular equal to or greater than 6.5%, in particular equal to or greater than 8.0%; 3) an individual wherein one, two, three or more of the following conditions are present: a) obesity, visceral obesity and/or abdominal obesity, b) a triglyceride blood level greater than or equal to 150 mg/dL, c) HDL-cholesterol blood level <40 mg/dL in female patients and <50 mg/dL in male patients, d) a systolic blood pressure greater than or equal to 130 mm Hg and a diastolic blood pressure greater than or equal to 85 mm Hg, or e) a fasting blood glucose level greater than or equal to 100 mg/dL; or 4) an individual with morbid obesity. Subjects in need of reduced ceramide levels may also be identified by OGTT and fat tolerance tests.

In a further aspect, a subject in need of reduced ceramide levels may need to modify one or more physiological parameters selected from the group consisting of reducing inflammation, increasing SOD activity, increasing fatty acid oxidation, increasing glutathione turnover, decreasing C16:0 and C18:0 cholesterol esters levels, increasing CPT-1 activity, increasing insulin sensitivity, decreasing leptin levels, improving glucose tolerance, improving insulin signaling, reducing inflammation, increasing SOD activity, and improving leptin signaling. In a further aspect, a subject in need of improved glucose tolerance may need to modify at least two or more of said physiological parameters selected from the group above. In other aspects, a subject in need may need to modify at least three or more of said physiological parameters selected from the group above. In yet other aspects, the subject in need may need to modify at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten of said physiological parameters selected from the group consisting of reducing inflammation, increasing SOD activity, increasing fatty acid oxidation, increasing palmatate oxidation, increasing glutathione turnover, decreasing CI 6:0 and CI 8:0 cholesterol esters levels, increasing CPT-1 activity, increasing insulin sensitivity, decreasing leptin levels, improving glucose tolerance, improving insulin signaling, reducing inflammation, increasing SOD activity, and improving leptin signaling.

Also provided herein are methods for decreasing cholesterol or cholesterol esters concentrations in a subject in need comprising the administration of an inhibitor of fatty acid synthesis or triglyceride biosynthesis. In an aspect, an inhibitor of fatty acid synthesis or triglyceride biosynthesis for decreasing cholesterol or cholesterol ester concentrations is an inhibitor of glycerol-3 -phosphate acyltransferase (GPAT). In some aspects the cholesterol or cholesterol ester being reduced are selected from the group consisting of C14:0, C16:0, C18:0, C18: l, C20:4, and C22:4. In some aspects, the methods comprise decreasing the circulating cholesterol and cholesterol ester concentrations wherein the circulating cholesterol and cholesterol ester concentrations in the serum are decreased by at least about 2.5% to about 50%) compared to placebo or baseline circulating concentrations. In certain aspects, the methods comprise decreasing the circulating cholesterol and cholesterol ester concentrations wherein the circulating cholesterol and cholesterol ester concentrations are decreased by at least 10% compared to placebo or baseline circulating concentrations. In certain aspects, the methods comprise decreasing the circulating cholesterol and cholesterol ester concentrations wherein the circulating cholesterol and cholesterol ester concentrations are decreased by at least 15% compared to placebo or baseline circulating concentrations. In certain aspects, the methods comprise decreasing the circulating cholesterol and cholesterol ester concentrations by at least 25% compared to placebo or baseline circulating concentrations.

The present disclosure also includes and provides for methods for reducing chronic inflammation in a subject in need by administering an inhibitor of fatty acid synthesis or triglyceride biosynthesis. In an aspect, an inhibitor of fatty acid synthesis or triglyceride biosynthesis is an inhibitor of glycerol-3 -phosphate acyltransferase (GPAT), where the inhibitor results in a reduction in inflammation in the subject. In an aspect, the inhibitor of fatty acid synthesis or triglyceride biosynthesis may be a compound of Formula I, Formula II, or both. In certain aspects, the fatty acid synthesis or triglyceride biosynthesis inhibitor may be the compound C75. In another aspect, the fatty acid synthesis or triglyceride biosynthesis inhibitor may be the compound FSG67. In yet another aspect, the inhibitor may be a combination of compounds C75 and FSG67. In an aspect, the inflammation may be inflammation of the hypothalamus, the central nervous system (CNS), or peripheral nervous system (PNS). In some aspects, the inflammation may be chronic. In an aspect, the chronic inflammation may be systemic. In certain aspects, the inflammation may be characterizable by elevated interleukin 6 (IL6 ) levels in the blood. In another aspect, the inflammation may be characterizable by elevated interleukin 1-beta (Ι β). In yet another aspect, inflammation may be characterizable by an elevated level of tumor-necrosis factor-a (TNF ). Not to be limited by mechanism, inflammation may be the result of overnutrition. In other aspects, inflammation may be the result of diabetes or obesity. In an aspect, a person in need of reducing chronic inflammation may have elevated white blood cell levels as described in Johannsen et al., 2010, Br J Sports Med. 44(8): 588 to 593, the content of which is herein incorporated in by reference in its entirety.

In one aspect, the level of inflammation is determined through routine methods in the art including by quantifying peripheral markers such as C-reactive protein (CRP), IL6, intracellular adhesion molecule- 1 (ICAM1) or using the erythrocyte sedimentation. See, e.g., Salas-Salvado et al., (2008) European Journal of Clinical Nutrition 62, 651-659, the content of which is herein incorporated in by reference in its entirety.

In another aspect, the subject in need of reduced inflammation is an individual having an elevated serum level of CRP. In one particular aspect, the subject in need has a serum CRP concentration of 1.5 mg/1 or more. In another aspect, the individual in need has a serum CRP concentration of 3 mg/1, 5 mg/1, 7.5 mg/1, 10 mg/1, 15 mg/1, or 20 mg/1 or more.

In one particular aspect, the subject in need of reduced inflammation is an individual diagnosed with type 1 diabetes mellitus. In another particular aspect, the subject is an individual diagnosed with type 2 diabetes mellitus, Maturity Onset Diabetes of the Young (MODY), Latent Autoimmune Diabetes of Adults (LAD A), or pre-diabetes. In one aspect, the subject may be 1) an individual diagnosed with one or more of the conditions selected from the group consisting of overweight, obesity, visceral obesity and abdominal obesity; or 2) an individual who shows one, two or more of the following conditions: a) a fasting blood glucose or serum glucose concentration greater than 100 mg/dL, in particular greater than 125 mg/dL, b) a postprandial plasma glucose equal to or greater than 140 mg/dL, or c) an HbAlc value equal to or greater than 6.0%, in particular equal to or greater than 6.5%, in particular equal to or greater than 8.0%; 3) an individual wherein one, two, three or more of the following conditions are present: a) obesity, visceral obesity and/or abdominal obesity, b) a triglyceride blood level greater than or equal to 150 mg/dL, c) HDL-cholesterol blood level <40 mg/dL in female patients and <50 mg/dL in male patients, d) a systolic blood pressure greater than or equal to 130 mm Hg and a diastolic blood pressure greater than or equal to 85 mm Hg, or e) a fasting blood glucose level greater than or equal to 100 mg/dL; or 4) an individual with morbid obesity.

In a further aspect, a subject in need of reducing inflammation may need to modify one or more physiological parameters selected from the group consisting of increasing SOD activity, increasing fatty acid oxidation, increasing glutathione turnover, reducing ceramide levels, decreasing CI 6:0 and CI 8:0 cholesterol esters levels, increasing CPT-1 activity, increasing insulin sensitivity, decreasing leptin levels, improving glucose tolerance, improving insulin signaling, reducing inflammation, increasing SOD activity, and improving leptin signaling. In a further aspect, a subject in need of improved glucose tolerance may need to modify at least two or more of said physiological parameters selected from the group above. In other aspects, a subject in need may need to modify at least three or more of said physiological parameters selected from the group above. In yet other aspects, the subject in need may need to modify at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten of said physiological parameters selected from the group consisting of increasing SOD activity, increasing fatty acid oxidation, increasing palmatate oxidation, increasing glutathione turnover, reducing ceramide levels, decreasing C16:0 and CI 8:0 cholesterol esters levels, increasing CPT-1 activity, increasing insulin sensitivity, decreasing leptin levels, improving glucose tolerance, improving insulin signaling, reducing

inflammation, increasing SOD activity, and improving leptin signaling.

The present disclosure further includes and provides for methods for improving hypothalamic function comprising administering an inhibitor of fatty acid synthesis or triglyceride biosynthesis. In an aspect, an inhibitor of fatty acid synthesis or triglyceride biosynthesis is an inhibitor of glycerol-3 -phosphate acyltransferase (GPAT). In an aspect, the inhibitor of fatty acid synthesis or triglyceride biosynthesis may be a compound of Formula I, Formula II, or both. In certain aspects, the fatty acid synthesis or triglyceride biosynthesis inhibitor may be the compound C75. In another aspect, the fatty acid synthesis or triglyceride biosynthesis inhibitor may be the compound FSG67. In yet another aspect, the inhibitor may be a combination of compounds C75 and FSG67.

In one particular aspect, the subject in need of improved hypothalamic function is an individual diagnosed with type 1 diabetes mellitus. In another particular aspect, the subject is an individual diagnosed with type 2 diabetes mellitus, Maturity Onset Diabetes of the Young (MODY), Latent Autoimmune Diabetes of Adults (LAD A), or pre-diabetes. In one aspect, the subject may be 1) an individual diagnosed with one or more of the conditions selected from the group consisting of overweight, obesity, visceral obesity and abdominal obesity; 2) an individual who shows one, two or more of the following conditions: a) a fasting blood glucose or serum glucose concentration greater than 100 mg/dL, in particular greater than 125 mg/dL; b) a postprandial plasma glucose equal to or greater than 140 mg/dL; or c) an HbAlc value equal to or greater than 6.0%, in particular equal to or greater than 6.5%, in particular equal to or greater than 8.0%; 3) an individual wherein one, two, three or more of the following conditions are present: a) obesity, visceral obesity and/or abdominal obesity, b) triglyceride blood level greater than or equal to 150 mg/dL, c) HDL-cholesterol blood level <40 mg/dL in female patients and <50 mg/dL in male patients, d) a systolic blood pressure greater than or equal to 130 mm Hg and a diastolic blood pressure greater than or equal to 85 mm Hg, or e) a fasting blood glucose level greater than or equal to 100 mg/dL; or 4) is an individual with morbid obesity.

A number of tissues have been implicated in the pathophysiology of obesity and type 2 diabetes, and of particular interest is the hypothalamus. The hypothalamus is a key brain area in the regulation of energy intake (Stellar, Psychol Rev 61: 5-22, 1954) and the hypothalamus plays a central role in energy homeostasis, integrating and coordinating a large number of factors produced by and/or acting on the hypothalamus. A number of these factors have been investigated for their role in energy balance and body weight regulation, including neuropeptide Y, corticotropin-releasing hormone, melanin-concentrating hormone, leptin and insulin. It has been proposed that genetic alterations perturbing the metabolic pathways regulating energy balance in the hypothalamus could contribute to the development of obesity, and subsequently diabetes. Thus, an important step in understanding the function of the hypothalamus in regulating the metabolism of an animal requires the identification of the targets of these hormones. Such targets may be whole organs, and genes whose expression is regulated by the presence of these hormones.

In aspects according to the present disclosure, improved hypothalamic function may be evaluated by measuring neuropeptide levels, hormone levels, or both. In some aspects, improved hypothalamic function may be evaluated by measuring melanocortin or neuropeptide Y (NPY) levels, or both. In some aspects, improved hypothalamic function may be evaluated by determining the levels of melanocortin, neuropeptide Y (NPY), leptin, adiponectin, menstrual hormones, corticotropins, growth hormone, vasopressin and oxytocin. In certain aspects, the menstrual hormones may include one or more of the hormones luteinizing hormone (LH), follicle-stimulating hormone (FSH) or thyroid- stimulating hormone (TSH). In an aspect, improved hypothalamic function may be evaluated by determining the levels of one or more of the levels of melanocortin, NPY, leptin, adiponectin, menstrual hormones, corticotrophins, growth hormone, vasopressin and oxytocin. In an aspect, improved hypothalamic function may be evaluated by determining the levels of two or more, three or more, or four or more of the levels of melanocortin, NPY, leptin, adiponectin, menstrual hormones, corticotrophins, growth hormone, vasopressin and oxytocin. Comparisons to placebo, initial, baseline levels, or pre-treatment levels may be used to evaluate improvement according to methods known in the art.

In aspects according to the present disclosure, hypothalamic function improves in a subject in need following administration of an inhibitor of fatty acid synthesis or triglyceride biosynthesis. In an aspect, an inhibitor of fatty acid synthesis or triglyceride biosynthesis is an inhibitor of glycerol-3 -phosphate acyltransferase (GPAT). In some aspects, hypothalamic function is improved by at least about 2.5% to about 50 % compared to initial or baseline hypothalamic function test results. In certain aspects, administration of a compound of Formula I, Formula II, or both improve hypothalamic function by at least 2.5% compared to initial, baseline hypothalamic function test results, or pre-treatment levels. In certain aspects, the methods comprise improving hypothalamic function wherein hypothalamic function is improved by at least 5% compared to initial or baseline hypothalamic function test results. In certain aspects, hypothalamic function is improved by at least 10% compared to initial or baseline hypothalamic function test results. In certain aspects, the methods provide for improved hypothalamic function by at least 20% compared to initial or baseline hypothalamic function test results. In certain aspects, the methods comprise improving hypothalamic function by at least 30% compared to initial or baseline hypothalamic function test results. In aspects according to the present disclosure, hypothalamic function may improve from 1 to 5%, 1 to 10%, 2.5 to 10%, 5 to 10% or greater than 10% from initial or baseline

hypothalamic function test results or pre-treatment levels.

In an aspect, hypothalamic function may increase by 5% or more relative to the pre- treatment level. In other aspects, hypothalamic function may increase by 10% or more relative to the pre-treatment level. In some aspects, hypothalamic function may increase 15%), 20% or more. In some aspects, hypothalamic function will increase by 2.5, 5%, 7.5%) or 10% of the pre-treatment level. In further aspects according to the present disclosure, hypothalamic function may increase from 1 to 5% or 1 to 10%. In other aspects,

hypothalamic function may increase from 5 to 10%>, 5 to 20%, or 5 to 30%>. In other aspects, hypothalamic function may increase from 10 to 15%, 10 to 20%, 15 to 20%, or 15 to 30%.

In a further aspect, a subject in need of improving hypothalamic function may need to modify one or more physiological parameters selected from the group consisting of reducing inflammation, increasing SOD activity, increasing fatty acid oxidation, increasing glutathione turnover, reducing ceramide levels, decreasing CI 6:0 and CI 8:0 cholesterol esters levels, increasing CPT-1 activity, increasing insulin sensitivity, decreasing leptin levels, improving glucose tolerance, improving insulin signaling, reducing inflammation, increasing SOD activity, and improving leptin signaling. In a further aspect, a subject in need of improved glucose tolerance may need to modify at least two or more of said physiological parameters selected from the group above. In other aspects, a subject in need may need to modify at least three or more of said physiological parameters selected from the group above. In yet other aspects, the subject in need may need to modify at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten of said physiological parameters selected from the group consisting of reducing inflammation, increasing SOD activity, increasing fatty acid oxidation, increasing palmatate oxidation, increasing glutathione turnover, reducing ceramide levels, decreasing CI 6:0 and CI 8:0 cholesterol esters levels, increasing CPT-1 activity, increasing insulin sensitivity, decreasing leptin levels, improving glucose tolerance, improving insulin signaling, reducing inflammation, increasing SOD activity, and improving leptin signaling.

The present disclosure further includes and provides for methods for improving glucose tolerance in a subject in need, comprising administering to a subject in need, a compound that is an inhibitor of fatty acid synthesis or triglyceride biosynthesis. In an aspect, an inhibitor of fatty acid synthesis or triglyceride biosynthesis is an inhibitor of glycerol-3 -phosphate acyltransferase (GPAT). In an aspect, the inhibitor of fatty acid synthesis or triglyceride biosynthesis may be a compound of Formula I, Formula II, or both. In certain aspects, the fatty acid synthesis or triglyceride biosynthesis inhibitor may be the compound C75. In another aspect, the fatty acid synthesis or triglyceride biosynthesis inhibitor may be the compound FSG67. In yet another aspect, the inhibitor may be a combination of compounds C75 and FSG67. In one particular aspect, the subject in need of improved glucose tolerance is an individual diagnosed with type 1 diabetes mellitus. In another particular aspect, the subject is an individual diagnosed with type 2 diabetes mellitus, Maturity Onset Diabetes of the Young (MODY), Latent Autoimmune Diabetes of Adults (LAD A), or pre-diabetes. In one aspect, the subject may be 1) an individual diagnosed of one or more of the conditions selected from the group consisting of overweight, obesity, visceral obesity and abdominal obesity; or 2) an individual who shows one, two or more of the following conditions: a) a fasting blood glucose or serum glucose concentration greater than 100 mg/dL, in particular greater than 125 mg/dL; b) a postprandial plasma glucose equal to or greater than 140 mg/dL; c) an HbAlc value equal to or greater than 6.0%, in particular equal to or greater than 6.5%, in particular equal to or greater than 8.0%; 3) an individual wherein one, two, three or more of the following conditions are present: a) obesity, visceral obesity and/or abdominal obesity, b) triglyceride blood level greater than or equal to 150 mg/dL, c) HDL-cholesterol blood level <40 mg/dL in female patients and <50 mg/dL in male patients, d) a systolic blood pressure greater than or equal to 130 mm Hg and a diastolic blood pressure greater than or equal to 85 mm Hg, e) a fasting blood glucose level greater than or equal to 100 mg/dL; or 4) is an individual with morbid obesity. Subjects in need of improved glucose tolerance may be identified by OGTT and fat tolerence tests.

In aspects according to the present disclosure, glucose tolerance improves in a subject in need following administration of an inhibitor of fatty acid synthesis or triglyceride biosynthesis. In an aspect, glucose tolerance improves in a subject following administratoin of an inhibitor of glycerol-3-phosphate acyltransferase (GPAT). In some aspects, glucose tolerance is improved by at least about 2.5% to about 50 % compared to initial or baseline glucose tolerance test results. In certain aspects, the methods comprise improving glucose tolerance by at least 2.5 % compared to initial, baseline glucose tolerance test results, or pre- treatment levels. In certain aspects, the methods comprise improving glucose tolerance wherein glucose tolerance is improved by at least 5 % compared to initial or baseline glucose tolerance test results. In certain aspects, glucose tolerance is improved by at least 10 % compared to initial or baseline glucose tolerance test results. In other aspects, the methods provide for improved glucose tolerance by at least 20 % compared to initial or baseline glucose tolerance test results. In yet other aspects, the methods comprise improving glucose tolerance by at least 30 % compared to initial or baseline glucose tolerance test results. In aspects according to the present disclosure, glucose tolerance may improve from 1 to 5%, 1 to 10%, 2.5 to 10%, 5 to 10%) or greater than 10% from initial or baseline glucose tolerance test results or pre-treatment levels.

In an aspect, glucose tolerance may increase by a 5% or more relative to the pre- treatment level. In other aspects, glucose tolerance may increase by a 10%> or more relative to the pre-treatment level. In some aspects, glucose tolerance may increase 15%, 20% or more. In some aspects, glucose tolerance will increase by 2.5, 5%, 7.5% or 10%> of the pre- treatment level. In further aspects according to the present disclosure, glucose tolerance may increase from 1 to 5% or 1 to 10%. In other aspects, glucose tolerance may increase from 5 to 10%, 5 to 20%), or 5 to 30%. In other aspects, glucose tolerance may increase from 10 to 15%, 10 to 20%, 15 to 20%, or 15 to 30%.

In some aspects, the methods comprise decreasing the circulating glucose

concentrations to an individual in need by administering an inhibitor of fatty acid synthesis or triglyceride biosynthesis. In an aspect, administration of an inhibitor of glycerol-3 -phosphate acyltransferase (GPAT) decreases circulating glucose concentrations. In an aspect, the inhibitor of fatty acid synthesis or triglyceride biosynthesis may be a compound of Formula I, Formula II, or both. In certain aspects, the fatty acid synthesis or triglyceride biosynthesis inhibitor may be the compound C75. In another aspect, the fatty acid synthesis or triglyceride biosynthesis inhibitor may be the compound FSG67. In yet another aspect, the inhibitor may be a combination of compounds C75 and FSG67. In some aspects, the circulating glucose concentrations are decreased by at least about 2.5% to about 50 % compared to placebo or baseline circulating concentrations. In certain aspects, the methods comprise decreasing the circulating glucose concentrations wherein the circulating glucose concentrations are decreased by at least 2.5 % compared to placebo or baseline circulating concentrations. In certain aspects, the methods comprise decreasing the circulating glucose concentrations wherein the circulating glucose concentrations are decreased by at least 5 % compared to placebo or baseline circulating concentrations. In certain aspects, the methods comprise decreasing the circulating glucose concentrations wherein the circulating glucose concentrations are decreased by at least 10 % compared to placebo or baseline circulating concentrations. In certain aspects, the methods comprise decreasing the circulating glucose concentrations wherein the circulating glucose concentrations are decreased by at least 20 % compared to placebo or baseline circulating concentrations. In certain aspects, the methods comprise decreasing the circulating glucose concentrations wherein the circulating glucose concentrations are decreased by at least 30 % compared to placebo or baseline circulating concentrations.

In a further aspect, a subject in need of improving glucose tolerance may need to modify one or more physiological parameters selected from the group consisting of reducing inflammation, increasing SOD activity, increasing fatty acid oxidation, increasing glutathione turnover, reducing ceramide levels, decreasing CI 6:0 and CI 8:0 cholesterol esters levels, increasing CPT-1 activity, increasing insulin sensitivity, decreasing leptin levels, improving glucose tolerance, improving insulin signaling, reducing inflammation, increasing SOD activity, and improving leptin signaling. In a further aspect, a subject in need of improved glucose tolerance may need to modify at least two or more of said physiological parameters selected from the group above. In other aspects, a subject in need may need to modify at least three or more of said physiological parameters selected from the group above. In yet other aspects, the subject in need may need to modify at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten of said physiological parameters selected from the group consisting of reducing inflammation, increasing SOD activity, increasing fatty acid oxidation, increasing palmatate oxidation, increasing glutathione turnover, reducing ceramide levels, decreasing CI 6:0 and CI 8:0 cholesterol esters levels, increasing CPT-1 activity, increasing insulin sensitivity, decreasing leptin levels, improving insulin signaling, reducing inflammation, increasing SOD activity, and improving leptin signaling.

The present disclosure further includes and provides for methods for improving glutathione turnover in a subject comprising administering to a subject in need thereof an inhibitor of fatty acid synthesis or triglyceride biosynthesis. In an aspect, an inhibitor of fatty acid synthesis or triglyceride biosynthesis is an inhibitor of glycerol-3-phosphate

acyltransferase (GPAT). In an aspect, the inhibitor of fatty acid synthesis or triglyceride biosynthesis may be a compound of Formula I, Formula II, or both. In certain aspects, the fatty acid synthesis or triglyceride biosynthesis inhibitor may be the compound C75. In another aspect, the fatty acid synthesis or triglyceride biosynthesis inhibitor may be the compound FSG67. In yet another aspect, the inhibitor may be a combination of compounds C75 and FSG67. In aspects according to the present disclosure, improved glutathione turnover may be characterized by an increase in the concentration γ-glutamyl amino acids. In some aspects, improved glutathione turnover may be characterized by an increase in levels of one or more substrates selected from the group consisting of 5-oxoproline, cysteine glutathione disulfide, γ-glutamylisoleucine, γ-glutamylleucine, γ-glutamylthreonine, γ- glutamylvaline, oxidized glutathione, and reduced glutathione. In some aspects, at least two of said substrates are increased. In other aspects, at least three of said substrates are increased. In yet other aspects, at least four, at least five, or at least six substrates selected from the group consisting of 5-oxoproline, cysteine glutathione disulfide, γ- glutamylisoleucine, γ-glutamylleucine, γ-glutamylthreonine, γ-glutamylvaline, oxidized glutathione, and reduced glutathione are increased.

In aspects according to the present disclosure, the need for increased turnover may be determined by increased levels of inflammatory markers or insulin insensitivity. In one aspect, the levels of inflammatory markers may be determined through routine methods in the art including by quantifying peripheral markers such as C-reactive protein (CRP), IL6, intracellular adhesion molecule- 1 (ICAM1), or using the erythrocyte sedimentation technique. See, e.g., Salas-Salvado et al., (2008) European Journal of Clinical Nutrition 62, 651-659, the content of which is herein incorporated in by reference in its entirety.

In another aspect, a subject in need of increased glutathione turnover may be an individual having an elevated serum level of CRP. In one particular aspect, the subject in need may have a serum CRP concentration of 1.5 mg/1 or more. In another aspect, the individual in need may have a serum CRP concentration of 3 mg/1, 5 mg/1, 7.5 mg/1, 10 mg/1, 1 mg/1, or 20 mg/1 or more.

In one particular aspect, a subject in need of increased glutathione turnover may be an individual diagnosed with type 1 diabetes mellitus. In another particular aspect, the subject may be an individual diagnosed with type 2 diabetes mellitus, Maturity Onset Diabetes of the Young (MODY), Latent Autoimmune Diabetes of Adults (LAD A), or pre-diabetes. In one aspect, the subject may be 1) an individual diagnosed with one or more of the conditions selected from the group consisting of overweight, obesity, visceral obesity and abdominal obesity; 2) an individual who shows one, two or more of the following conditions: a) a fasting blood glucose or serum glucose concentration greater than 100 mg/dL, in particular greater than 125 mg/dL, b) a postprandial plasma glucose equal to or greater than 140 mg/dL, or c) an HbAlc value equal to or greater than 6.0%, in particular equal to or greater than 6.5%, in particular equal to or greater than 8.0%; 3) an individual wherein one, two, three or more of the following conditions are present: a) obesity, visceral obesity and/or abdominal obesity, b) a triglyceride blood level greater than or equal to 150 mg/dL, c) HDL-cholesterol blood level <40 mg/dL in female patients and <50 mg dL in male patients, d) a systolic blood pressure greater than or equal to 130 mm Hg and a diastolic blood pressure greater than or equal to 85 mm Hg, or e) a fasting blood glucose level greater than or equal to 100 mg/dL; or 4) an individual with morbid obesity.

Reactive oxygen species (ROS) generated by physiological processes in a subject may indicate a need for increased glutathione turnover. For example, oxidative metabolism produces ATP and may also generate reactive oxygen species (ROS). In aspects according to the present disclosure, a subject may be protected from increased reactive oxygen species by increasing the levels of one or more substrates selected from the group consisting of 5- oxoproline, cysteine glutathione disulfide, γ-glutamylisoleucine, γ-glutamylleucine, γ- glutamylthreonine, γ-glutamylvaline, oxidized glutathione, and reduced glutathione. In aspects according to the present disclosure, both the oxidized and reduced forms of glutathione, and increased levels of γ-glutamyl amino acids regenerate glutathione and may counteract increased ROS, generated by, for example, increased fatty acid oxidation. In an aspect, increased 5-oxoproline, a marker of glutathione degradation may indicate a need for an increased glutathione turnover in a subject. In other aspects, increased levels of cysteine- glutathione disulfide, an indicator of oxidative stress, may indicate a need for increased glutathione turnover. Other indications of a need for increased glutathione turnover include urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG), and activities of superoxide dismutase and glutathione peroxidase and the concentrations of total reduced glutathione and protein-bound thiols in serum. See, e.g., Chen et al., Clinical Chemistry 51(4): 759-767 (2005), Rafii et al., J Chromatogr B Analyt Technol Biomed Life Sci. 2009 Oct 15;877(28):3282-91, Vallejo M, et al., J Chromatogr A. 2008 Apr 11;1187(l-2):267-74, herein incorporated by reference in their entireties.

In other aspects, a need for increased glutathione turnover may be indicated by enteral or parenteral cystine, methionine, N-acetyl-cysteine, and l-2-oxothiazolidine-4-carboxylate. In other aspects, a need for increased glutathione turnover may be indicated by oxidative stress associated with aging and the pathogenesis of kwashiorkor, seizure, Alzheimer's disease, Parkinson's disease, liver disease, cystic fibrosis, sickle cell anemia, HI, AIDS, cancer, heart attack, stroke, and diabetes. See, e.g., Gu et al., J. Nutr. March 1, 2004 vol. 134 no. 3 489-492, herein incorporated by reference in its entirety.

Also provided herein are methods for increasing glutathione turnover in a subject in need comprising the administration of a fatty acid synthesis or triglyceride biosynthesis inhibitor. In an aspect, an inhibitor of fatty acid synthesis or triglyceride biosynthesis is an inhibitor of glycerol-3 -phosphate acyltransferase (GPAT). In some aspects, the methods comprise increasing glutathione turnover by at least about 2.5% to about 100% compared to placebo or baseline glutathione turnover rates. In other aspects, the methods comprise increasing glutathione turnover by at least about 5% to about 75% compared to placebo or baseline glutathione turnover rates. In further aspects, the methods comprise increasing glutathione turnover by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, or by at least about 50% compared to placebo or baseline glutathione turnover rates.

Improved glutathione turnover may be assessed by determining the amounts of one or more components of the gamma glutamyl cycle in a sample. In aspects according to the present disclosure, an increase in one or more substrates selected from the group consisting of 5-oxoproline, cysteine glutathione disulfide, γ-glutamylisoleucine, γ-glutamylleucine, γ- glutamylthreonine, γ-glutamylvaline, oxidized glutathione, and reduced glutathione indicates improved glutathione turnover. In some aspects, the one or more substrates may be compared to an initial, pre-treatment baseline level, or to a value or range determined in a normal population. See, e.g., Lauterburg et al. The Journal of Pharmacology and

Experimental Therapeutics (1980) 213(1): 54 to 58, herein incorporated by reference in its entirety.

In some aspects, glutathione turnover may be improved by at least about 2.5% to about 50%) compared to initial or baseline glutathione turnover test result or compared to one or more substrates selected from the group consisting of 5-oxoproline, cysteine glutathione disulfide, γ-glutamylisoleucine, γ-glutamylleucine, γ-glutamylthreonine, γ-glutamylvaline, oxidized glutathione, and reduced glutathione. In certain aspects, the methods provide for improved glutathione turnover of at least 2.5% compared to initial or baseline glutathione turnover test results or substrate levels. In certain aspects, the methods comprise improving glutathione turnover wherein glutathione turnover is improved by at least 5% compared to initial or baseline glutathione turnover test results or substrate levels. In certain aspects, glutathione turnover is improved by at least 10% compared to initial or baseline glutathione turnover test results or substrate levels. In certain aspects, the methods provide for improved glutathione turnover by at least 20% compared to initial or baseline glutathione turnover test results or substrate levels. In certain aspects, the methods comprise improving glutathione turnover by at least 30%> compared to initial or baseline glutathione turnover test results or substrate levels. In aspects according to the present disclosure, glutathione turnover may improve from 1 to 5%, 1 to 10%, 2.5 to 10%>, 5 to 10% or greater than 10% from initial or baseline glutathione turnover test results or substrate levels.

In an aspect, glutathione turnover may increase by 5% or more relative to the pre- treatment level. In other aspects, glutathione turnover may increase by 10% or more relative to the pre-treatment level. In some aspects, glutathione turnover may increase 1 %, 20% or more. In some aspects, glutathione turnover may increase by 2.5%, 5%, 7.5% or 10% of the pre-treatment level. In further aspects according to the present disclosure, glutathione turnover may increase from 1 to 5% or 1 to 10%. In other aspects, glutathione turnover may increase from 5 to 10%, 5 to 20%, or 5 to 30%. In other aspects, glutathione turnover may increase from 10 to 15%, 10 to 20%, 15 to 20%, or 15 to 30%.

The present disclosure further includes and provides for methods for altering phospholipid synthesis in a subject comprising administering to a subject in need thereof an inhibitor of fatty acid synthesis or triglyceride biosynthesis. In an aspect, an inhibitor of fatty acid synthesis or triglyceride biosynthesis is an inhibitor of glycerol-3-phosphate

acyltransferase (GPAT). In an aspect, the inhibitor of fatty acid synthesis or triglyceride biosynthesis may be a compound of Formula I, Formula II, or both. In certain aspects, the fatty acid synthesis or triglyceride biosynthesis inhibitor may be the compound C75. In another aspect, the fatty acid synthesis or triglyceride biosynthesis inhibitor may be the compound FSG67. In yet another aspect, the inhibitor may be a combination of compounds C75 and FSG67.

In a further aspect, a subject in need of altering phospholipid synthesis may have an elevated crude plasma concentration of one or more fatty acids selected from the group consisting of palmatate (16:0), linoleic acid (18:2n-6), stearic acid (18:0), arachadonic acid (20:4n-6), and oleic acid ( cis 18: ln-9). See, e.g., Saadation-Elahi et al., (2009) American Journal of Clinical Nutrition 89: 331-346, the contents of which are herein incorporated in by reference in their entirety. In one aspect, said subject has an elevated level of at least two of said fatty acids. In certain aspects, said subject has an elevated level of at least three, at least four, or all five said fatty acids.

In one particular aspect, the subject in need of altered phospholipid synthesis is an individual diagnosed with type 1 diabetes mellitus. In another particular aspect, the subject is an individual diagnosed with type 2 diabetes mellitus, Maturity Onset Diabetes of the Young (MODY), Latent Autoimmune Diabetes of Adults (LAD A), or pre-diabetes. In one aspect, the subject may be 1) an individual diagnosed of one or more of the conditions selected from the group consisting of overweight, obesity, visceral obesity and abdominal obesity; or 2) an individual who shows one, two or more of the following conditions: a) a fasting blood glucose or serum glucose concentration greater than 100 mg/dL, in particular greater than 125 mg/dL; b) a postprandial plasma glucose equal to or greater than 140 mg/dL; c) an HbAlc value equal to or greater than 6.0%, in particular equal to or greater than 6.5%, in particular equal to or greater than 8.0%; 3) an individual wherein one, two, three or more of the following conditions are present: a) obesity, visceral obesity and/or abdominal obesity, b) triglyceride blood level greater than or equal to 150 mg/dL, c) HDL-cholesterol blood level <40 mg/dL in female patients and <50 mg/dL in male patients, d) a systolic blood pressure greater than or equal to 130 mm Hg and a diastolic blood pressure greater than or equal to 85 mm Hg, e) a fasting blood glucose level greater than or equal to 100 mg/dL; or 4) is an individual with morbid obesity.

In a further aspect, a subject in need of altering phospholipid synthesis may need to modify one or more physiological parameters selected from the group consisting of reducing inflammation, increasing SOD activity, increasing fatty acid oxidation, increasing glutathione turnover, reducing ceramide levels, decreasing CI 6:0 and CI 8:0 cholesterol esters levels, increasing CPT-1 activity, increasing insulin sensitivity, decreasing leptin levels, improving glucose tolerance, improving insulin signaling, reducing inflammation, increasing SOD activity, and improving leptin signaling. In a further aspect, a subject in need of improved glucose tolerance may need to modify at least two or more of said physiological parameters selected from the group above. In other aspects, a subject in need may need to modify at least three or more of said physiological parameters selected from the group above. In yet other aspects, the subject in need may need to modify at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten of said physiological parameters selected from the group consisting of reducing inflammation, increasing SOD activity, increasing fatty acid oxidation, increasing palmitate oxidation, increasing glutathione turnover, reducing ceramide levels, decreasing CI 6:0 and CI 8:0 cholesterol esters levels, increasing CPT-1 activity, increasing insulin sensitivity, decreasing leptin levels, improving glucose tolerance, improving insulin signaling, reducing inflammation, increasing SOD activity, and improving leptin signaling.

The present disclosure provides for and includes methods for increasing insulin sensitivity in a subject in need, comprising administering to a subject in need a compound that is an inhibitor of fatty acid synthesis or triglyceride biosynthesis. In an aspect, an inhibitor of fatty acid synthesis or triglyceride biosynthesis is an inhibitor of glycerol-3- phosphate acyltransferase (GPAT) In an aspect, the inhibitor of fatty acid synthesis or triglyceride biosynthesis may be a compound of Formula I, Formula II, or both. In certain aspects, the fatty acid synthesis or triglyceride biosynthesis inhibitor may be the compound C75. In another aspect, the fatty acid synthesis or triglyceride biosynthesis inhibitor may be the compound FSG67. In yet another aspect, the inhibitor may be a combination of compounds C75 and FSG67.

In an aspect according to the present disclosure, a subject in need of increased insulin sensitivity may be a subject with insulin resistance, a subject with mild Type 2 diabetes, a subject with severe Type 2 diabetes, or a subject with mild, moderate or severe obesity.

In aspects according to the present disclosure, a subject in having insulin resistance or mild Type 2 diabetes, the responses of both plasma glucose and plasma insulin are markedly exaggerated compared to the normal patient during the three hour period following ingestion of 100 grams of glucose. Much more insulin is required to dispose of the same amount of glucose. When subjects in need

In an example of the method, a subject having a metabolic disease, such as type 1 diabetes, may be administered a therapeutically effective amount of a compound of Formula I or Formula II or other compounds capable of modulating or inhibiting fatty acid synthesis or triglyceride biosynthesis, such as glycerol-3-phosphate acyltransferase (GPAT). In other aspects, a compound may further stimulate carnitine palmitoyltransferase- 1 (CPT-1) activity, either directly or indirectly.

In aspects according to the present disclosure, a subject in need may be identified by performing an oral glucose tolerance test (OGTT). Oral glucose tolerance tests reflect the uptake of glucose from blood by tissues, along with suppression of release of endogenously produced glucose into blood from tissues, as provided in the clearance rate of an exogenous glucose load from the bloodstream. This approach does not provide information regarding the specific metabolic fate or consequences of the glucose administered nor provide information about the mechanisms underlying impaired glucose tolerance. The OGTT is widely used in clinical practice.

In aspects according to the present disclosure, a subject in need may be identified by performing fat tolerance testing that measures the uptake of fatty acids from blood by tissues. As with the OGTT, the test does not provide information about the specific metabolic fate or consequences of the fat administered. Similarly, no information about the mechanisms underlying impaired fat tolerance. Fat tolerance testing has mostly been used to assess the clearance of dietary fat from blood in context of evaluating hyperlipidemia. Fat tolerance testing provides for the identification of subjects having a need for increased insulin sensitivity.

In one particular aspect, the subject in need of increase insulin sensitivity is an individual diagnosed with type 1 diabetes mellitus. In another particular aspect, the subject is an individual diagnosed with type 2 diabetes mellitus, Maturity Onset Diabetes of the Young (MODY), Latent Autoimmune Diabetes of Adults (LAD A), or pre-diabetes. In one aspect, the subject may be 1) an individual diagnosed of one or more of the conditions selected from the group consisting of overweight, obesity, visceral obesity and abdominal obesity; or 2) an individual who shows one, two or more of the following conditions: a) a fasting blood glucose or serum glucose concentration greater than 100 mg/dL, in particular greater than 125 mg/dL; b) a postprandial plasma glucose equal to or greater than 140 mg/dL; c) an HbAlc value equal to or greater than 6.0%, in particular equal to or greater than 6.5%, in particular equal to or greater than 8.0%; 3) an individual wherein one, two, three or more of the following conditions are present: a) obesity, visceral obesity and/or abdominal obesity, b) triglyceride blood level greater than or equal to 150 mg/dL, c) HDL-cholesterol blood level <40 mg/dL in female patients and <50 mg/dL in male patients, d) a systolic blood pressure greater than or equal to 130 mm Hg and a diastolic blood pressure greater than or equal to 85 mm Hg, e) a fasting blood glucose level greater than or equal to 100 mg/dL; or 4) is an individual with morbid obesity.

In some aspects, insulin sensitivity is improved by at least about 2.5% to about 50 % compared to initial or baseline insulin sensitivity test result. In certain aspects, the methods provide for improved insulin sensitivity of at least 2.5 % compared to initial or baseline insulin sensitivity test results. In certain aspects, the methods comprise improving insulin sensitivity wherein insulin sensitivity is improved by at least 5 % compared to initial or baseline insulin sensitivity test results. In certain aspects, insulin sensitivity is improved by at least 10 % compared to initial or baseline insulin sensitivity test results. In certain aspects, the methods provide for improved insulin sensitivity by at least 20 % compared to initial or baseline g insulin sensitivity test results. In certain aspects, the methods comprise improving insulin sensitivity by at least 30 % compared to initial or baseline insulin sensitivity test results. In aspects according to the present disclosure, insulin sensitivity may improve from 1 to 5%, 1 to 10%, 2.5 to 10%, 5 to 10% or greater than 10% from initial or baseline insulin sensitivity test results.

In an aspect, insulin sensitivity may increase by a 5% or more relative to the pre- treatment level. In other aspects, insulin sensitivity may increase by a 10% or more relative to the pre-treatment level. In some aspects, insulin sensitivity may increase 15%, 20% or more. In some aspects, insulin sensitivity may increase by 2.5, 5%, 7.5% or 10%> of the pre- treatment level. In further aspects according to the present disclosure, insulin sensitivity may increase from 1 to 5% or 1 to 10%. In other aspects, insulin sensitivity may increase from 5 to 10%, 5 to 20%), or 5 to 30%>. In other aspects, insulin sensitivity may increase from 10 to 15%, 10 to 20%, 15 to 20%, or 15 to 30%.

In a further aspect, a subject in need of increasing insulin sensitivity may need to modify one or more physiological parameters selected from the group consisting of reducing inflammation, increasing SOD activity, increasing fatty acid oxidation, increasing glutathione turnover, reducing ceramide levels, decreasing CI 6:0 and CI 8:0 cholesterol esters levels, increasing CPT-1 activity, decreasing leptin levels, improving glucose tolerance, improving insulin signaling, reducing inflammation, increasing SOD activity, and improving leptin signaling. In a further aspect, a subject in need of improved glucose tolerance may need to modify at least two or more of said physiological parameters selected from the group above. In other aspects, a subject in need may need to modify at least three or more of said physiological parameters selected from the group above. In yet other aspects, the subject in need may need to modify at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten of said physiological parameters selected from the group consisting of reducing inflammation, increasing SOD activity, increasing fatty acid oxidation, increasing palmitate oxidation, increasing glutathione turnover, reducing ceramide levels, decreasing C16:0 and C18:0 cholesterol esters levels, increasing CPT-1 activity, decreasing leptin levels, improving glucose tolerance, improving insulin signaling, reducing

inflammation, increasing SOD activity, and improving leptin signaling.

The present disclosure further includes and provides for methods for decreasing leptin levels in a subject in need comprising administering to said subject a compound that is an inhibitor of fatty acid synthesis or triglyceride biosynthesis. In an aspect, an inhibitor of fatty acid synthesis or triglyceride biosynthesis is an inhibitor of glycerol-3-phosphate

acyltransferase (GPAT). In an aspect, the inhibitor of fatty acid synthesis or triglyceride biosynthesis may be a compound of Formula I, Formula II, or both. In certain aspects, the fatty acid synthesis or triglyceride biosynthesis inhibitor may be the compound C75. In another aspect, the fatty acid synthesis or triglyceride biosynthesis inhibitor may be the compound FSG67. In yet another aspect, the inhibitor may be a combination of compounds C75 and FSG67.

In one aspect, the subject in need of decreased leptin concentration is an individual with an elevated body mass index (BMI). In one aspect, the individual in need of decreased circulating leptin concentration has a BMI of 40 or more. In another aspect, the subject in need of decreased leptin concentration is an individual having a BMI of 35 or more. In another aspect, the subject in need of decreased leptin concentration is an individual having a BMI of 25 to 35. In another aspect, the subject in need of decreased leptin concentration is an individual having a BMI of 30 or more.

In another aspect, the subject in need of decreased leptin concentration is an individual having a serum leptin concentration of 10 ng/ml or more. In another aspect, the individual in need has a serum leptin concentration of 15 ng/ml or more. In another aspect, the individual in need has a serum leptin concentration of 20 ng/ml or more. In another aspect, the individual in need has a serum leptin concentration of 30 ng/ml or more. In another aspect, the individual in need has a serum leptin concentration of 40 ng/ml, 45 ng/ml, 50 ng/ml, 60 ng/ml, 75 ng/ml, or 100 ng/ml or more. In another aspect, the subject in need of decreased leptin concentration is an individual having a elevated serum level of C-reactive protein (CRP). In one particular aspect, the subject in need has a serum CRP concentration of 1.5 mg 1 or more. In another aspect, the individual in need has a serum CRP concentration of 3 mg/1, 5 mg/1, 7.5 mg/1, 10 mg/1, 15 mg/1, or 20 mg/1 or more. In aspects according to the present disclosure, a subject in need has leptin concentrations that are above the normal range in healthy individuals. In healthy individuals, baseline leptin levels are generally between 1 and 5 ng/dl in men and 7 and 13 ng/dl in women. In a study of "normal weight" (BMI = 23.0±2.5) vs "obese" (BMI = 35.1±7.2) subjects (cohort had men, and women...), normals averaged srum leptin of 7.5 ng/ml, and obese subjects averaged 31+ ng/ml. See, e.g. , Considine et al., 1996. N Engl J Med 334: 292-295, 1996, hereby incorporated by reference in its entirety.

In some aspects, leptin concentration is improved by at least about 2.5% to about 50

% compared to initial or baseline leptin concentration test result. In certain aspects, the methods provide for improved leptin concentration of at least 2.5 % compared to initial or baseline leptin concentration test results. In certain aspects, the methods comprise improving leptin concentration wherein leptin concentration is improved by at least 5 % compared to initial or baseline leptin concentration test results. In certain aspects, leptin concentration is improved by at least 10 % compared to initial or baseline leptin concentration test results. In certain aspects, the methods provide for improved leptin concentration by at least 20 % compared to initial or baseline leptin concentration test results. In certain aspects, the methods comprise improving leptin concentration by at least 30 % compared to initial or baseline leptin concentration test results. In aspects according to the present disclosure, leptin concentration may improve from 1 to 5%, 1 to 10%, 2.5 to 10%, 5 to 10% or greater than 10%) from initial or baseline leptin concentration test results.

In an aspect, leptin concentration may decrease by a 5% or more relative to the pre- treatment level. In other aspects, leptin concentration may decrease by a 10% or more relative to the pre -treatment level. In some aspects, leptin concentration may decrease 15%>, 20%) or more. In some aspects, leptin concentration may decrease by 2.5, 5%, 7.5% or 10% of the pre-treatment level. In further aspects according to the present disclosure, leptin concentration may decrease from 1 to 5% or 1 to 10%. In other aspects, leptin concentration may decrease from 5 to 10%>, 5 to 20%, or 5 to 30%. In other aspects, leptin concentration may decrease from 10 to 15%, 10 to 20%, 15 to 20%, or 15 to 30%. In an aspect, the leptin concentration may decrease from 5 to 20%. In other aspects, the leptin concentration my decrease by 10% or more, 20% or more, or 30% or more.

In one particular aspect, the subject in need of decreased leptin concentration is an individual diagnosed with type 1 diabetes mellitus. In another particular aspect, the subject is an individual diagnosed with type 2 diabetes mellitus, Maturity Onset Diabetes of the Young (MODY), Latent Autoimmune Diabetes of Adults (LAD A), or pre-diabetes. In one aspect, the subject may be 1) an individual diagnosed of one or more of the conditions selected from the group consisting of overweight, obesity, visceral obesity and abdominal obesity; or 2) an individual who shows one, two or more of the following conditions: a) a fasting blood glucose or serum glucose concentration greater than 100 mg/dL, in particular greater than 125 mg/dL; b) a postprandial plasma glucose equal to or greater than 140 mg/dL; c) an HbAlc value equal to or greater than 6.0%, in particular equal to or greater than 6.5%, in particular equal to or greater than 8.0%; 3) an individual wherein one, two, three or more of the following conditions are present: a) obesity, visceral obesity and/or abdominal obesity, b) triglyceride blood level greater than or equal to 150 mg/dL, c) HDL-cholesterol blood level <40 mg/dL in female patients and <50 mg/dL in male patients, d) a systolic blood pressure greater than or equal to 130 mm Hg and a diastolic blood pressure greater than or equal to 85 mm Hg, e) a fasting blood glucose level greater than or equal to 100 mg/dL; or 4) is an individual with morbid obesity. Subjects in need of reduced decreased leptin concentration may be identified by OGTT and fat tolerance tests.

In some aspects, leptin is decreased by at least about 2.5 % to 50 % compared to baseline circulating concentration. In certain aspects, the circulating concentration of leptin is decreased by at least about 5 % to 40% compared to baseline circulating concentration. In certain aspects, the circulating concentration of leptin is decreased by at least about 10 % to 35 % compared to baseline circulating concentration. In certain aspects, the circulating concentration of leptin is decreased by at least about 15 % to 30 % compared to baseline circulating concentration. In certain aspects, the circulating concentration of leptin is decreased by at least about 20% to 25 % compared to baseline circulating concentration. In some aspects, leptin is decreased by at least about 2 ng/ml or more. In another aspect, leptin is decreased by at least about 5 ng/ml or more. In another aspect, leptin is decreased by at least about 7.5 ng/ml, 10 ng/ml, 15 ng ml, 20 ng/ml, 25 ng/ml, or 50 ng/ml or more.

In a further aspect, a subject in need of decreased leptin concentration may need to modify one or more physiological parameters selected from the group consisting of reducing inflammation, increasing SOD activity, increasing fatty acid oxidation, increasing glutathione turnover, reducing ceramide levels, decreasing CI 6:0 and CI 8:0 cholesterol esters levels, increasing CPT-1 activity, increasing insulin sensitivity, improving glucose tolerance, improving insulin signaling, reducing inflammation, increasing SOD activity, and improving leptin signaling. In a further aspect, a subject in need of improved glucose tolerance may need to modify at least two or more of said physiological parameters selected from the group above. In other aspects, a subject in need may need to modify at least three or more of said physiological parameters selected from the group above. In yet other aspects, the subject in need may need to modify at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten of said physiological parameters selected from the group consisting of reducing inflammation, increasing SOD activity, increasing fatty acid oxidation, increasing palmitate oxidation, increasing glutathione turnover, reducing ceramide levels, decreasing C16:0 and C18:0 cholesterol esters levels, increasing CPT-1 activity, increasing insulin sensitivity, decreasing, improving glucose tolerance, improving insulin signaling, reducing inflammation, increasing SOD activity, and improving leptin signaling. The present disclosure further includes and provides for methods for decreasing levels of cholesterol or cholesterol esters in a subject in need comprising administering to said subject a compound that is an inhibitor of fatty acid synthesis or triglyceride biosynthesis. In an aspect, an inhibitor of fatty acid synthesis or triglyceride biosynthesis is an inhibitor of glycerol-3 -phosphate acyltransferase (GPAT). In an aspect, the inhibitor of fatty acid synthesis or triglyceride biosynthesis may be a compound of Formula I, Formula II, or both. In certain aspects, the fatty acid synthesis or triglyceride biosynthesis inhibitor may be the compound C75. In another aspect, the fatty acid synthesis or triglyceride biosynthesis inhibitor may be the compound FSG67. In yet another aspect, the inhibitor may be a combination of compounds C75 and FSG67.

In aspects according to the present disclosure, administering a compound that is an inhibitor of fatty acid synthesis or triglyceride biosynthesis decreases the levels of cholesterol esters that are esters having a chain length of 1 to 24 carbons (e.g., CI to C24). In other aspects, a subject may be in need of decreased cholesterol, cholesterol esters or both. In some aspects, the cholesterol esters that are in need of being decreased are medium-chained unsaturated cholesterol esters of chain lengths 14 (CI 4) to 24 (C:24) carbons. In other aspects, the cholesterol esters are C16:0 or C18:0 cholesterol esters. In further aspects, the cholesterol esters reduced are C14, C 16, C18, C18: l , C20:4 or C22:4. In an aspect, the cholesterol ester in need of reduction is an ester having a chain length of CI 8. In an aspect, the cholesterol ester in need of reduction is an ester having a chain length of CI 6.

In aspects according to the present disclosure, a subject in need may have a mean serum low density lipoproteins (LDL) concentration greater than 130 mg/dL. In one aspect, a subject in need may have a mean serum LDL concentration greater than 140 mg/dL. In yet another aspect, a subject in need may have a mean serum LDL concentration greater than about 150 mg dL, 160 mg/dL, 170 mg/dL, 180 mg/dL, 190 mg/dL or more. In one aspect, a subject in need may have a mean serum high density lipoproteins (HDL) concentration less than 60 mg/dL. In one aspect, a subject in need may have a mean serum HDL concentration less than 55 mg/dL. In one aspect, a subject in need may have a mean serum HDL concentration less than 50 mg/dL. In yet another aspect, a subject in need may have a mean serum HDL concentration less than 45 mg/dL, or even less than 40 mg/dL.

In certain aspects, the methods comprise decreasing the circulating cholesterol and cholesterol ester concentrations by at least 2.5 mg/dL compared to placebo, initial, baseline levels, or pre-treatment levels. In other aspects, the methods comprise decreasing the circulating cholesterol and cholesterol ester concentrations by at least 5 mg/dL. In other aspects, the methods comprise decreasing the circulating cholesterol and cholesterol ester concentrations by at least 10 mg/dL. In certain other aspects, the methods comprise decreasing the circulating cholesterol and cholesterol ester concentrations by at least 15 mg/dL, at least 20 mg/dL, at least 25 mg dL, at least 30 mg/dL, or at least 35 mg/dL or more.

In aspects according to the present disclosure, a subject in need of decreased levels of cholesterol or cholesterol esters may have decreased LDL and increased HDL levels in response to administration of an inhibitor of fatty acid synthesis or triglyceride biosynthesis that is a compound of Formula I, Formula II, or both. In an aspect, LDL concentration may decrease by 5% or more relative to a placebo, initial, baseline, or pre-treatment level. In other aspects, LDL concentration may decrease by 10% or more relative to a placebo, initial, baseline, or pre-treatment level. In some aspects, LDL concentration may decrease 15%, 20%), 25%o or more. In some aspects, LDL concentration may decrease by 2.5%, 5%>, 7.5% or 10%) of a placebo, initial, baseline, or pre-treatment level. In further aspects according to the present disclosure, LDL concentration may decrease from 5 to 50% or 10 to 25%. In other aspects, LDL concentration may decrease from 10 to 30%, 15 to 25%>, or 5 to 50%. In other aspects, LDL concentration may decrease by about 5 mg/dL, 10 mg/dL, 15 mg/dL, 20 mg/dL, 25 mg/dL, 30 mg/dL, 50 mg/dL or more relative to a placebo, initial, baseline, or pre- treatment level. In another aspect, the mean serum HDL concentration may increase by 10% or more relative to a placebo, initial, baseline, or pre-treatment level. In another aspect, the mean serum HDL concentration may increase by a 20% or more relative to a placebo, initial, baseline, or pre-treatment level. In another aspect, the mean serum HDL concentration may increase by 25%, 30%, 35%, 40%, 45%, 50%), or more relative to a placebo, initial, baseline, or pre-treatment level. In other aspects, HDL concentration may increase by about 5 mg/dL or more relative to a placebo, initial, baseline, or pre-treatment level. In other aspects, HDL concentration may increase by about 10 mg/dL, 15 mg/dL, 20 mg/dL, 25 mg/dL, 30 mg/dL, 50 mg/dL or more relative to the pre-treatment level.

In aspects according to the present disclosure, a subject in need of decreased levels of cholesterol or cholesterol esters may have decreased LDL and decreased HDL levels in response to administration of an inhibitor of fatty acid synthesis or triglyceride biosynthesis that is a compound of Formula I, Formula II, or both. In an aspect, LDL concentration may decrease by 5%> or more relative to a placebo, initial, baseline, or pre-treatment level. In other aspects, LDL concentration may decrease by 10% or more relative to a placebo, initial, baseline, or pre-treatment level. In some aspects, LDL concentration may decrease 15%, 20%), 25%o or more. In some aspects, LDL concentration may decrease by 2.5%, 5%, 7.5% or 10%) of a placebo, initial, baseline, or pre-treatment level. In further aspects according to the present disclosure, LDL concentration may decrease from 5 to 50% or 10 to 25%. In other aspects, LDL concentration may decrease from 10 to 30%, 15 to 25%, or 5 to 50%). In other aspects, LDL concentration may decrease by about 5 mg/dL, 10 mg/dL, 15 mg/dL, 20 mg/dL, 25 mg/dL, 30 mg/dL, 50 mg/dL or more relative to a placebo, initial, baseline, or pre- treatment level. In another aspect, the mean serum HDL concentration may decrease by 10%> or more relative to a placebo, initial, baseline, or pre-treatment level. In another aspect, the mean serum HDL concentration may decrease by a 20% or more relative to a placebo, initial, baseline, or pre-treatment level. In another aspect, the mean serum HDL concentration may decrease by 25%, 30%>, 35%, 40%>, 45%, 50%, or more relative to a placebo, initial, baseline, or pre-treatment level. In other aspects, HDL concentration may decrease by about 5 mg/dL or more relative to a placebo, initial, baseline, or pre-treatment level. In other aspects, HDL concentration may decrease by about 10 mg/dL, 15 mg/dL, 20 mg/dL, 25 mg/dL, 30 mg/dL, 50 mg/dL or more relative to the pre-treatment level.

In aspects according to the present disclosure, a subject in need of decreased levels of cholesterol or cholesterol esters may have an increased ratio of HDL to LDL (HDL:LDL ratio) in response to administration of an inhibitor of fatty acid synthesis or triglyceride biosynthesis that is a compound of Formula I, Formula II, or both. In some aspects, the

HDL:LDL ratio may decrease by 5% or more relative to a placebo, initial, baseline, or pre- treatment level. In other aspects, the HDL:LDL ratio may decrease by 10% or more relative to a placebo, initial, baseline, or pre-treatment level. In some aspects, the HDL:LDL ratio may decrease 15%), 20%), 25% or more. In some aspects, the HDL:LDL ratio may decrease by 2.5%, 5%, 7.5% or 10% of a placebo, initial, baseline, or pre-treatment level. In further aspects according to the present disclosure, the HDL:LDL ratio may decrease from 5 to 50%) or 10 to 25%. In other aspects, the HDL:LDL ratio may decrease from 10 to 30%, 15 to 25%, or 5 to 50%.

According to the present disclosure, a subject having an excess or increased level can be identifiable by comparison with a normalized population. In an aspect, a normalized population may be an age and sex normalized population. In other aspects, a normalized population may be selected based on weight, glucose levels, glucose tolerance, insulin sensitivity, levels of one or more fatty acids, cholesterol levels and types, ceramide levels and types, leptin levels, phospholipid levels and composition, measures of hypothalamic function, or inflammation levels. Normalized populations may be selected based on the indicator of a subject in need. For example a normalized population may be selected based on average fatty acid levels or one or more fatty acids. In an aspect according to the present disclosure, a normalized population may be selected from a group having glucose tolerance test in the normal range, an insulin tolerance test result in the normal range, a normal range of serum insulin, a normal range of serum glucose and a BMI of less than 20%.

In other aspects of the present disclosure, the level of glucose, glucose tolerance, insulin sensitivity, fat, ceramide, leptin, phospholipid, hypothalamic function, inflammation or other indication of need as provided above, can be compared to a predetermined value to determine whether a subject is in need of administration of a compound of Formula I, Formula II, or both. A predetermined value can be based upon analyte levels in comparable samples obtained from the general population or from a select population of humans. For example, a select population may be comprised of apparently healthy individuals.

"Apparently healthy," as used herein, means individuals who have not previously had any signs or symptoms indicating the presence of a disease such as metabolic disease, cardiovascular disease, obesity, diabetes, etc.

In certain aspects according to the present disclosure, a predetermined value can be related to a value used to characterize a level of an analyte in a sample obtained from a subject with a need of administration of a compound of Formula I, Formula II, or both. Thus, if the level of an analyte is a representative value, such as an arbitrary unit obtained from, for example, an OGTT, fat tolerance test, or blood test, the predetermined value may also be based on the representative value. Subjects in need of administration of a compound of Formula I, Formula II, or both, may be identified based on genetic tests, for example, for susceptibility to metabolic disease, obesity, cardiovascular disease, liver disease, or pancreatic disease.

A predetermined value can take a variety of forms. A predetermined value can be a single cut-off value, such a as a median or mean. A predetermined value can be established based upon comparative groups such as where the level of the analyte (e.g., fatty acid level in the blood, glucose levels, glucose tolerance, leptin levels, etc.) in one defined group is half the level of the corresponding analyte in another defined group. A predetermined value can be a range, for example, where the general population is divided equally (or unequally) into groups, or into quadrants, the lowest quadrant being individuals with the lowest levels of the analyte, the highest quadrant being subjects with the highest levels of the analyte.

In aspects according to the present disclosure, a predetermined value can be derived by determining the respective analyte level in the general population. Alternatively, a predetermined value can be derived by determining the respective analyte level in a select population. For example, a predetermined value may be determined from analyte levels obtained from subject populations in need of administration of a compound of Formula I, Formula II, or both. Accordingly, predetermined values selected may take into account the category in which a subject falls. Appropriate ranges and categories can be selected with no more than routine experimentation by those of ordinary skill in the art.

Predetermined values, such as mean levels, median levels, or "cut-off levels, may be established by assaying samples of subjects in a general population or a select population. Analyte levels determined in a sample test may be compared to a single predetermined value or to a range of predetermined values. In some aspects, the comparison may be to an initial analyte level determined in a subject based on one or more pre -treatment measurements. If the level of the marker in the sample is lower than a predetermined value or range of predetermined values, then a subject may be identified as being in need of administration of an inhibitor of fatty acid synthesis or triglyceride biosynthesis, for example, glycerol-3 - phosphate acyltransferase (GPAT), for example a subject with a particular metabolic disease or, alternatively, diagnosed as being at risk for developing a metabolic disease.

The extent of the difference between the analyte level and a predetermined value may also be useful for characterizing the extent of need for administration of an inhibitor of fatty acid synthesis or triglyceride biosynthesis, for example, glycerol-3 -phosphate acyltransferase (GPAT). For example, an analyte level may be below a certain cut-off value or in the higher tertile or quartile of a "normal range." In this instance, such an analyte level may indicate an elevated risk for developing a need for administration of an inhibitor of fatty acid synthesis or triglyceride biosynthesis, for example, glycerol-3 -phosphate acyltransferase (GPAT).

The term "modulate" as used herein refers to affecting a change in the level, activity, amount or other characteristic of a desired target, such as a molecule or cell. For example, "modulate" may refer to increasing or decreasing the level of fatty acid synthesis or triglyceride biosynthesis activity, glucose tolerance, insulin sensitivity, fatty acid level, ceramide level, leptin level, phospholipid composition, hypothalamic function, inflammation or other indication of need. A "modulator" of a pathway refers to a substance or agent which modulates the activity of one or more cellular proteins mapped to the same specific signal transduction pathway. A modulator may augment or suppress the activity and/or expression level or pattern of a signaling molecule. A modulator can activate a component in a pathway by directly binding to the component. A modulator can also indirectly activate a component in a pathway by interacting with one or more associated components. The output of the pathway can be measured in terms of the expression or activity level of proteins. The expression level of a protein in a pathway can be reflected by levels of corresponding mRNA or related transcription factors as well as the level of the protein in a subcellular location. For instance, certain proteins are activated by translocating in or out of a specific subcellular component, including but not limited to nucleus, mitochondria, endosome, lysosome or other

membranous structure of a cell. The output of the pathway can also be measured in terms of physiological effects, such as mitochondrial biogenesis, fatty acid oxidation, or glucose uptake.

An "activator" refers to a modulator that influences a pathway in a manner that increases the pathway output. Activation of a particular target may be direct (e.g. , by interaction with the target) or indirect (e.g. by interaction with a protein upstream of the target in a signaling pathway including the target).

The term "about" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ± 20% or ± 10%, more preferably ± 5%, even more preferably ± 1%, and still more preferably ± 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

The term "therapeutically effective amount" refers to an amount of a molecule (e.g., a compound of Formula I, Formula II, or both) that is sufficient to modulate the level and/or activity of an enzyme in the fatty acid synthesis or triglyceride biosynthesis pathways. In certain aspects, a therapeutically effective amount modulates that activity GPAT and/or CPT- 1 in at least one cell of a subject. In other aspects according to the present disclosure, the term "effective amount" or "therapeutically effective amount" refers to that amount of a compound of Formula I, Formula II, or both described herein that is sufficient to effect the intended application including but not limited to disease treatment, as defined herein. The therapeutically effective amount may vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated, e.g. , the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The term also applies to a dose that will induce a particular response in target cells, e.g., reduction of inflammation, or increased oxidation of palmitate. A specific dose will vary depending on the particular compounds chosen, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, the tissue to which it is administered, and the physical delivery system in which it is carried.

The term "sample" or "biological sample" generally indicates a specimen of tissue, cells, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, blood cells, hair, tumors, organs, other material of biological origin that contains polynucleotides, or in vitro cell culture constituents of any of these. A sample can further be in a semi-purified or purified form. A "biological sample" encompasses a variety of sample types obtained from an individual. The definition encompasses blood and other liquid samples of biological origin, that are accessible from an individual through sampling by minimally invasive or non-invasive approaches (e.g., urine collection, blood drawing, needle aspiration, and other procedures involving minimal risk, discomfort or effort). The definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components, such as proteins or polynucleotides. The term "biological sample" also encompasses a clinical sample such as serum, plasma, other biological fluid, or tissue samples, and also includes cells in culture, cell supernatants and cell lysates. A sample can be isolated from a mammal, such as a human or an animal. In a preferred aspect, a sample is obtained from a human.

The term "metabolic disease" refers to a group of identified disorders in which errors of metabolism, imbalances in metabolism, or sub-optimal metabolism occur. A metabolic disease as described herein also include diseases that can be treated through the modulation of metabolism, although the disease itself may or may not be caused by a specific metabolic defect. Such metabolic diseases may involve, for example, glucose and fatty acid oxidation pathways.

The term "obesity" as used herein is defined in the WHO classifications of weight. Underweight is less than 18.5 BMI (thin); healthy is 18.5-24.9 BMI (normal); grade 1 overweight is 25.0-29.9 BMI (overweight); grade 2 overweight is 30.0-39.0 BMI (obesity); grade 3 overweight is greater than or equal to 40.0 BMI. BMI is body mass index (morbid obesity) and is kg/m ² . Waist circumference can also be used to indicate a risk of metabolic complications. Waist circumference can be measured (in cm) at midpoint between the lower border of ribs and the upper border of the pelvis. Other measures of obesity include, but are not limited to, skinfold thickness and bioimpedance, which is based on the principle that lean mass conducts current better than fat mass because it is primarily an electrolyte solution.

The term "obesity-related condition" refers to any disease or condition that is caused by or associated with (e.g., by biochemical or molecular association) obesity or that is caused by or associated with weight gain and/or related biological processes that precede clinical obesity. Examples of obesity-related conditions include, but are not limited to, diabetes (e.g., type 1 diabetes, type 2 diabetes, and gestational diabetes), Syndrome X, hyperglycemia, hyperinsulinemia, impaired glucose tolerance, impaired fasting glucose, dyslipidemia, hypertriglyceridemia, insulin resistance, hypercholesterolemia, atherosclerosis, coronary artery disease, peripheral vascular disease, and hypertension.

The term "subject" refers to a mammal, such as a human being. As also used herein, the term "subject" may refer to a patient.

The terms "administer", "administered", "administers" and "administering" are defined as the providing a composition to a subject via a route known in the art, including but not limited to intravenous, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, or intraperitoneal routes of administration. In certain aspects of the subject application, oral routes of administering a composition may be preferred.

In accordance with the methods of the present invention, a compound of Formula I, Formula II, or both may be administered in any manner known in the art that renders a compound biologically available to the subject or sample in an effective amount. For example, a compound of the present disclosure may be administered to a subject via any central or peripheral route known in the art including, but not limited to: oral, parenteral, transdermal, transmucosal, or pulmonary routes. In an aspect the administration is parenteral administration. Exemplary routes of administration include oral, ocular, rectal, buccal, topical, nasal, ophthalmic, subcutaneous, intramuscular, intraveneous, intracerebral, transdermal, and pulmonary. In certain aspects the route of administration is subcutaneous. Further, a compound of the present disclosure can be administered to a sample via pouring, pipetting, immersing, injecting, infusing, perfusing, or any other means known in the art. Determination of the appropriate administration method is usually made upon consideration of the condition (e.g., disease or disorder) to be treated, the stage of the condition (e.g., disease or disorder), the comfort of the subject, and other factors known to those of skill in the art.

Administration by the methods of the present invention can be intermittent or continuous, both on an acute and/or basis. One mode of administration of a compound of the present disclosure is continuous. Continuous intravenous or subcutaneous infusion, and continuous transcutaneous infusion are exemplary aspects of administration for use in the methods of the present invention. In certain aspects, subcutaneous infusions, both acute and chronic, provide for continuous administration. Another exemplary mode of administration is intermittent subcutaneous injection. In another exemplary mode of administration, a compound of the present disclosure is formulated for extended or sustained release.

Exemplary formulations are reported for example in WO2005000222, US20040228833, US20040208929, US 20050031549, and US20050002927, the entireties of which are incorporated herein by reference.

The term "effective amount" refers to an amount of a compound of the present disclosure used to treat, ameliorate, prevent, or eliminate the identified condition (e.g., disease or disorder), or to exhibit a detectable therapeutic or preventative effect. The effect can be detected by, for example, chemical markers, antigen levels, cardiac function, physical measurements of the heart, or time to a measurable event, such as morbidity or mortality. Therapeutic effects include, for example, increased palmitate oxidation, improved glucose tolerance, increased insulin sensitivity, reduced ceramide levels, decreased leptin levels, improved hypothalamic function, and decreased inflammation. The precise effective amount for a subject will depend upon the subject's body weight, size, and health; the nature and extent of the condition; and the therapeutic or combination of therapeutics selected for administration. Effective amounts for a given situation can be determined by routine experimentation that is within the skill and judgment of the clinician.

For any compound of the present disclosure, the effective amount can be estimated initially either in cell culture assays, e.g., in animal models, such as rat or mouse models. An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

Efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED ₅₀ (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, ED50/LD50. Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies may be used in formulating a range of doses for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include an ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.

More specifically, the concentration-biological effect relationships observed with regard to compounds of the present disclosure employed in the methods of the present invention indicate an initial target plasma concentration ranging from about 5 pM to about 400 pM, preferably from about 20 pM to about 200 pM, more preferably from about 80 pM to about 100 pM. To achieve such plasma concentrations in the methods of the present invention, a compound of the present disclosure may be administered at doses that vary from about 0.25 pmol/kg/min to about 10 nmol/kg min, more preferably about 0.45 pmol/kg/min to about 4.5 nmol/kg/min, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is generally available to practitioners in the art and is provided herein.

In general, for continuous subcutaneous infusion, the dose will be in the range of about 0.2 pmol/kg/min to about 3 pmol/kg/min, or from about 0.3 pmol/kg/min to about 30 pmol/kg/min, or preferably from about 0.45 pmol/kg/min to about 25 pmol/kg/min. For acute subcutaneous infusion, the dose will generally be in the range of about 2.5 pmol/kg/min to about 7 nmol/kg/min, or from about 3.5 pmol/kg/min to about 6 pmol/kg/min, or preferably from about 5 pmol/kg/min to about 4.5 nmol/kg/min. Exemplary treatment regimens include, but are not limited to, administration via injection to achieve a dose of from about 0.1 μg/kg to about 0.5 μΒ/kg or from about 0.005 μg/kg to about 0.2 μΒ/kg of the compound of the present disclosure. Other exemplary treatment regimens include, but are not limited to, administration via injection to achieve a dose of from about 1 μg/day to about 1 mg/day or from about 500 μg day to about 12,000 μg/day of thecompound of the present disclosure in a single or divided dose.

Still other exemplary treatment regimens include, but are not limited to, pulmonary administration to achieve a dose from about 100 μg/day to about 12,000 μg day of the compound of the present disclosure in a single or divided dose; nasal administration to achieve a dose from about 10 μg/day to about 12,000 μg/day of the compound of the present disclosure in a single or divided dose; and buccal administration to achieve a dose from about 100 μg day to about 12,000 μg day of the compound of the present disclosure in a single or divided dose.

The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment.

As mentioned above, the compound of the present disclosure may be administered as a result of an acute event or a chronic condition. Whether it is an acute event or a chronic condition, methods of the invention include chronic treatment with a compound of the present disclosure. Thus, length of chronic treatment may include the time when the event has passed and the subject is considered to have recovered from the acute event or recovered from the chronic condition.

Chronic administration of or treatment with the compounds of the present disclosure for the prevention, attenuation, delay, or amelioration of metabolic dysfunction may be warranted where no particular transient event or transient condition associated with metabolic dysfunction is identified. Chronic administration includes administration of the compounds of the present disclosure over an indefinite period of time on the basis of a general predisposition to metabolic dysfunction or on the basis of a predisposing condition that is non-transient (e.g. , a condition that is non-transient may be unidentified or unamenable to elimination, such as diabetes), regardless of etiology. Chronic administration of a compound of the present disclosure for the prevention or amelioration of metabolic or hypothalamic dysfunction may also be implicated in diabetics at risk for palmitate oxidation, glucose tolerance, insulin sensitivity, ceramide reduction, leptin reduction, phospholipid, improved hypothalamic function, decreased inflammation or other indication of need When a compound of the present disclosure is administered chronically, administration may continue for any length of time. However, chronic administration often occurs for an extended period of time. For example, in a preferred aspect, chronic administration continues for 6 months, 1 year, 2 years or longer.

In a further aspect of the present invention, prophylactic and therapeutic methods are provided. Treatment on an acute or chronic basis is contemplated. In addition, treatment on an acute basis may be extended to chronic treatment, if so indicated. Chronic treatment is contemplated as being longer than 2 weeks. In certain aspects, chronic treatment may be longer than 1 month, 3 months, 6 months, 1 year, 2 years, 5 years, or over a life. The method generally comprises administering to the subject an amount of a compound of the present disclosure effective to prevent or ameliorate metabolic or hypothalamic dysfunction, wherein the condition associated with metabolic or hypothalamic dysfunction is thereby improved, prevented or delayed.

In yet another aspect of the invention, the methods of the present invention further comprise the identification of a subject in need of treatment. Any effective criteria may be used to determine that a subject may benefit from administration of a compound of the present disclosure. Methods for the diagnosis metabolic dysfunction, for example diabetes, as well as procedures for the identification of individuals at risk for development of these conditions, are well known to those in the art. Such procedures may include clinical tests, physical examination, personal interviews and assessment of family history.

A compound of the present disclosures may be formulated as pharmaceutical compositions for use in conjunction with the methods of the present invention. The pharmaceutical compositions may be formulated with pharmaceutically acceptable excipients such as carriers, solvents, stabilizers, adjuvants, diluents, etc., depending upon the particular mode of administration and dosage form. The pharmaceutical compositions should generally be formulated to achieve a physiologically compatible pH, and may range from a pH of about 3 to a pH of about 11, or from about pH 3 to about pH 7, depending on the formulation and route of administration. In alternative aspects, the pH may be adjusted to a range from about pH 5.0 to about pH 8.0.

In an aspect, a pharmaceutical composition of the invention comprises an effective amount of at least one compound of the present disclosure, together with one or more pharmaceutically acceptable excipients. Optionally, a pharmaceutical composition may include a second active ingredient useful in the prevention or treatment of diabetes, obesity, or another metabolic syndrome.

The pharmaceutical compositions may be formulated for administration in any manner known in the art. By way of example, when formulated for oral administration or parenteral administration, the pharmaceutical composition is most typically a solid, liquid solution, emulsion or suspension, while inhalable formulations for pulmonary or nasal administration are generally liquids or powders. A pharmaceutical composition may also be formulated as a lyophilized solid that is reconstituted with a physiologically compatible solvent prior to administration. Alternative pharmaceutical compositions of the invention may be formulated as syrups, creams, ointments, tablets, and the like. As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agent of the Federal or state government, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, such as humans. The term "carrier" refers to a diluent, adjuvant, excipient, stabilizer, or vehicle with which the agent is formulated for administration. Pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a typical carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rich, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. Pharmaceutical compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. The composition can also be formulated as a suppository, with traditional binders and carriers such as triglycerides.

The term "pharmaceutically acceptable excipient" refers to an excipient for administration of a pharmaceutical agent, such as a compound of the present disclosure. The term refers to any pharmaceutical excipient that may be administered without undue toxicity. Pharmaceutically acceptable excipients are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there exist a wide variety of suitable formulations of pharmaceutical compositions for use in the methods of the present invention (see, e.g., Remington's Pharmaceutical Sciences).

Suitable excipients may be carrier molecules that include large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Other exemplary excipients include antioxidants such as ascorbic acid; chelating agents such as EDTA; carbohydrates such as dextrin, hydroxyalkylcellulose, hydroxyalkylmethylcellulose, and stearic acid; liquids such as oils, water, saline, glycerol and ethanol; wetting or emulsifying agents; pH buffering substances; and the like. Liposomes are also included within the definition of pharmaceutically acceptable excipients. More particularly, when intended for oral use, e.g., tablets, troches, lozenges, aqueous or oil suspensions, non-aqueous solutions, dispersible powders or granules (including micronized particles or nanoparticles), emulsions, hard or soft capsules, syrups or elixirs may be prepared. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions, and such compositions may contain one or more agents including sweetening agents, flavoring agents, coloring agents and preserving agents, in order to provide a palatable preparation.

Pharmaceutically acceptable excipients particularly suitable for use in conjunction with tablets include, for example, inert diluents, such as celluloses, calcium or sodium carbonate, lactose, calcium or sodium phosphate; disintegrating agents, such as

croscarmellose sodium, cross-linked povidone, maize starch, and alginic acid; binding agents, such as povidone, starch, gelatin and acacia; and lubricating agents, such as magnesium stearate, stearic acid and talc. Tablets may be uncoated or may be coated by known techniques including microencapsulation to delay disintegration and adsorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax may be employed.

Formulations for oral use may be also presented as hard gelatin capsules where the active ingredient is mixed with an inert solid diluent, for example celluloses, lactose, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with non-aqueous or oil medium, such as glycerin, propylene glycol, polyethylene glycol, peanut oil, liquid paraffin and olive oil.

In another aspect, the pharmaceutical composition of the invention may be formulated as a suspension comprising a compound of the present disclosure in admixture with at least one pharmaceutically acceptable excipient suitable for the manufacture of a suspension. In yet another aspect, a compound of the present disclosure may be formulated as dispersible powder and granules suitable for preparation of a suspension by the addition of suitable excipients.

The pharmaceutical composition may also be formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, pharmaceutical compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the pharmaceutical composition can also include a solubilizing agent. Generally, the ingredients are supplied either separately or mixed together in unit dosage form as, for example, dry lyophilized powder or water-free concentrate in a hermetically sealed container (e.g., an ampoule or sachette) indicating the quantity of the active agent. Where the pharmaceutical composition is to be administered by infusion, the pharmaceutical composition can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the pharmaceutical composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

Excipients suitable for use in connection with suspensions include suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcelluose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g. , polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g. , heptadecaethyleneoxycethanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan monooleate); and thickening agents, such as carbomer, beeswax, hard paraffin or cetyl alcohol. The suspensions may also contain one or more preservatives such as acetic acid, methyl and/or n-propyl p-hydroxy-benzoate; one or more coloring agents; one or more flavoring agents; and one or more sweetening agents such as sucrose and saccharin.

The pharmaceutical composition of the present invention may also be in the form of an oil-in-water emulsion. The oily phase may be a vegetable oil, such as olive oil and arachis oil, a mineral oil, such as liquid paraffin, or a mixture of these. Suitable emulsifying agents include naturally-occurring gums, such as gum acacia and gum tragacanth; naturally occurring phosphatides, such as soybean lecithin, esters and partial esters derived from fatty acids; hexitol anhydrides, such as sorbitan monooleate; and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. The emulsion may also contain sweetening and flavoring agents. Syrups and elixirs may be formulated with sweetening agents, such as glycerol, sorbitol and sucrose. Such formulations may also contain a demulcent, a preservative, a flavoring or a coloring agent.

In another aspect, the pharmaceutical composition of the invention may be formulated as a sterile injectable preparation, such as a sterile injectable aqueous emulsion or oleaginous suspension. This emulsion or suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents such as those that have been mentioned above. In another preferred aspect, the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,2-propane-diol. The sterile injectable preparation may also be prepared as a lyophilized powder. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils may be employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may likewise be used in the preparation of injectables.

Certain compounds of the present disclosure may be substantially insoluble in water and sparingly soluble in most pharmaceutically acceptable protic solvents and in vegetable oils. However, the compounds may be soluble in medium chain fatty acids (e.g., caprylic and capric acids) or triglycerides and have high solubility in propylene glycol esters of medium chain fatty acids. Also contemplated for use in the methods of the invention are

compositions, which have been modified by substitutions or additions of chemical or biochemical moieties which make them more suitable for delivery (e.g., increase solubility, bioactivity, palatability, decrease adverse reactions, etc.), for example by esterification, glycation, PEGylation, etc.

A compound of the present disclosure may also be formulated for oral administration in a self-emulsifying drug delivery system (SEDDS). Lipid-based formulations such as SEDDS are particularly suitable for low solubility compounds, and can generally enhance the oral bioavailability of such compounds.

In an alternative aspect, cyclodextrins may be added as aqueous solubility enhancers. Cyclodextrins include hydroxypropyl, hydroxyethyl, glucosyl, maltosyl and maltotriosyl derivatives of α-, β-, and γ-cyclodextrin. A preferred cyclodextrin solubility enhancer is hydroxypropyl-P-cyclodextrin (HPBC), which may be added to any of the above-described compositions to further improve the aqueous solubility characteristics of a GLP-1 molecule or agonist thereof. In one aspect, the composition comprises 0.1% to 20% hydroxypropyl- β- cyclodextrin, more preferably 1% to 15% hydroxypropyl-P-cyclodextrin, and even more preferably from 2.5% to 10% hydroxypropyl- -cyclodextrin. The amount of solubility enhancer employed will depend on the amount of compound of the present disclosure in the composition.

Dosage and administration are adjusted to provide sufficient levels of the active agent(s) in a pharmaceutical composition or to maintain the desired effect. Factors that may be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Whether an administration is acute or chronic may also be considered in determining dosage. Long- acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation. In a preferred aspect, compounds of the present disclosures used in the methods of the present invention are formulated for sustained release.

Exemplary treatment regimens include, but are not limited to, administration via injection to achieve a dose of from about 0.1 μg/kg to about 0.5 μg/kg or from about 0.005 μg/kg to about 0.2 μg/kg of the compound of the present disclosure. Other exemplary treatment regimens include, but are not limited to, administration via injection to achieve a dose of from about 1 μg/day to about 1 mg/day or from about 500 μg/day to about 12,000 μg/day of the compound of the present disclosure in a single or divided dose.

Still other exemplary treatment regimens include, but are not limited to, pulmonary administration to achieve a dose from about 100 μg/day to about 12,000 μg/day of the compound of the present disclosure in a single or divided dose; nasal administration to achieve a dose from about 10 μg/day to about 12,000 μg/day of the compound of the present disclosure in a single or divided dose; and buccal administration to achieve a dose from about 100 μg/day to about 12,000 μg day of the compound of the present disclosure in a single or divided dose.

The compositions of the aspects described herein may be co-administered with known therapies for the treatment of any of the conditions described herein. Co-administration can also provide for additive or synergistic effects, resulting in the need for lower dosages of a known therapy, the compositions described herein, or both. Additional benefits of co administration include the reduction in toxicities associated with any of the known therapies.

Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which both agents are present. Thus, in some aspects, compositions described herein and a known therapy are administered in a single treatment. In some aspects, the compositions described herein and a known therapy are admixed in a resulting composition.

In some aspects, compositions described herein and the known therapy are administered in separate compositions or administrations. Administration of compositions described herein and known therapies described herein may be by any suitable means. Administration of a composition described herein and a second compound (e.g., diabetes drug or obesity drug) may be by any suitable means. If the compositions described herein and a second compound are administered as separate compositions, they may be administered by the same route or by different routes. If the compositions described herein and a second compound are administered in a single composition, they may be administered by any suitable route such as, for example, oral administration. Therapies, drugs and compounds useful for the treatment of diabetes, metabolic syndrome (including glucose intolerance, insulin resistance, and dyslipidemia), and/or diseases or conditions associated therewith may be administered with a fatty acid synthesis, triglyceride biosynthesis, or GPAT inhibitor. Diabetic therapies drugs and compounds include, but are not limited to, those that decrease triglyceride concentrations, decrease glucose concentrations, and/or modulate insulin (e.g., stimulate insulin production, mimic insulin, enhance glucose-dependent insulin secretion, suppress glucagon secretion or action, improve insulin action or insulin sensitizers, or are exogenous forms of insulin).

Drugs that decrease triglyceride level include but are not limited to ascorbic acid, asparaginase, clofibrate, colestipol, fenofibrate mevastatin, pravastatin, simvastatin, fluvastatin, or omega-3 fatty acid. Drugs that decrease LDL cholesterol level include but are not limited to clofibrate, gemfibrozil, and fenofibrate, nicotinic acid, mevinolin, mevastatin, pravastatin, simvastatin, fluvastatin, lovastatin, cholestyrine, colestipol or probucol.

In another aspect, compositions of the aspects described herein may be administered in combination with glucose-lowering compounds.

The medication classes of thiazolidinediones (also called glitazones), sulfonylureas, meglitinides, biguanides, alpha-glucosidase inhibitors, DPP-IV inhibitors, and incretin mimetics have been used as adjunctive therapies for hyperglycemia and diabetes mellitus (type 2) and related diseases.

Drugs that decrease glucose level include but are not limited to glipizides, glyburides, exenatide (Byetta®), incretins, sitagliptin (Januvia®), pioglitizone, glimepiride, rosiglitazone, metformin, vildagliptin, saxagliptin (OnglyzaTM), sulfonylureas, meglitinide (e.g.,

Prandin®) glucosidase inhibitor, biguanides (e.g., Glucophage®), repaglinide, acarbose, troglitazone, nateglinide, natural, synthetic or recombinant insulin and derivatives thereof, and amylin and amylin derivatives. In certain instances, fatty acid synthesis, triglyceride biosynthesis, or GPAT inhibitor compositions provided herein are used in combination with biguanides. Biguanides include metformin, phenformin, buformin and related compounds. In certain instances, fatty acid synthesis, triglyceride biosynthesis, or GPAT inhibitor compositions provided herein are used in combination with metformin.

When administered sequentially, the combination may be administered in two or more administrations. In an alternative aspect, it is possible to administer one or more fatty acid synthesis, triglyceride biosynthesis, or GPAT inhibitors and one or more additional active ingredients by different routes. The skilled artisan will also recognize that a variety of active ingredients may be administered in combination with one or more fatty acid synthesis, triglyceride biosynthesis, or GPAT inhibitors that may act to augment or synergistically enhance the control, prevention, amelioration, attenuation, or treatment of obesity or eating disorders or conditions.

According to the methods provided herein, when co-administered with at least one other obesity reducing (or anti-obesity) or weight reducing drug, a fatty acid synthesis, triglyceride biosynthesis, or GPAT inhibitor may be: (1) co-formulated and administered or delivered simultaneously in a combined formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by any other combination therapy regimen known in the art. When delivered in alternation therapy, the methods provided may comprise administering or delivering the active ingredients sequentially, e.g., in separate solution, emulsion, suspension, tablets, pills or capsules, or by different injections in separate syringes. In general, during alternation therapy, an effective dosage of each active ingredient is administered sequentially, i.e., serially, whereas in simultaneous therapy, effective dosages of two or more active ingredients are administered together. Various sequences of intermittent combination therapy may also be used.

In certain aspects, compositions provided herein may be used with other

commercially available diet aids or other anti-obesity agents, such as, by way of example, PYY and PYY agonists, GLP-1 and GLP-1 agonists, a DPPIV inhibitor, CCK and CCK agonists, exendin and exendin agonists, GIP and GIP agonists, amylin and amylin agonists, ghrelin modulators {e.g., inhibitors) and leptin and leptin agonists. In certain instances, fatty acid synthesis, triglyceride biosynthesis, or GPAT inhibitor composition as provided herein may be used in combination with amylin, amylin agonists or mimetics. Exemplary amylin agonists or mimetics include pramlintide and related compounds. In certain instances, fatty acid synthesis, triglyceride biosynthesis, or GPAT inhibitor compositions provided herein are used in combination with leptin, leptin agonists or mimetics. Additional leptin agonists or mimetics can be identified using the methods described by U.S. Pat. No. 7,247,427 which is incorporated by reference herein.

In further instances, fatty acid synthesis, triglyceride biosynthesis, or inhibitor compositions provided herein increase leptin sensitivity and increase effectiveness of leptin, leptin agonists or mimetics.

Additional anti-obesity agents for use in the methods provided that are in current development are also of interest in the methods of the present disclosure. Other anti-obesity agents include, alone or any combination of, phentermine, fenfluramine, sibutramine, rimonabant, topiramate, zonisamide bupropion, naltrexone, lorcaserin, and orlistat.

Therapies, drugs and compounds useful for the treatment of weight loss, binge eating, food addictions and cravings may be administered with the compositions described herein. For example, the subject may further be administered at least one other drug which is known to suppress hunger or control appetite. Such therapies, drugs and compounds include but are not limited to phenteramines such as Meridia® and Xenical®. Additional therapies, drugs and compounds are known in the art and contemplated herein.

In some aspects, a composition comprises an amount of a sirtuin pathway activator, such as a polyphenol (e.g., resveratrol). The amount of sirtuin pathway activator may be a subtherapeutic amount, and/or an amount that is synergistic with one or more other compounds in the composition or one or more other compounds administered simultaneously or in close temporal proximity with the composition. In some aspects, the sirtuin pathway activator is administered in a low dose, a medium dose, or a high dose, which describes the relationship between two doses, and generally do not define any particular dose range. For example, a daily low dose of resveratrol may comprise about, less than about, or more than about 0.5 mg/kg, 1 mg kg, 2.5 mg/kg, 5 mg/kg, 7.5 mg/kg, 10 mg/kg, 12.5 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 50 mg/kg, 75 mg/kg, 100 mg/kg, or more; a daily medium dose of resveratrol may comprise about, less than about, or more than about 20 mg/kg, 25 mg/kg, 50 mg/kg, 75 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 250 mg/kg, or more; and a daily high dose of resveratrol may comprise about, less than about, or more than about 1 0 mg kg, 175 mg/kg, 200 mg/kg, 225 mg/kg, 250 mg/kg, 300 mg/kg, 350 mg/kg, 400 mg/kg, or more. As used herein, "an alkyl group" denotes both straight and branched carbon chains with one or more carbon atoms, but reference to an individual radical such as "propyl" embraces only the straight chain radical, a branched chain isomer such as "isopropyl" specifically referring to only the branched chain radical. A "substituted alkyl" is an alkyl group wherein one or more hydrogens of the alkyl group are substituted with one or more substituent groups as otherwise defined herein.

As used herein, "an alkoxy group" refers to a group of the Formula alkyl-O-, where alkyl is as defined herein. A "substituted alkoxy" is an alkoxy group wherein one or more hydrogens are substituted with one or more of the substituent groups otherwise defined herein.

As used herein, "alkenyl" refers to a partially unsaturated alkyl radical derived by the removal of one or more hydrogen atoms from a alkyl chain such that it contains at least one carbon-carbon double bond.

As used herein, "an aryl group" denotes a structure derived from an aromatic ring containing six carbon atoms. Examples include, but are not limited to a phenyl or benzyl radical and derivatives thereof.

As used herein, "arylalkyl" denotes an aryl group having one or more alkyl groups not at the point of attachment of the aryl group.

As used herein, "alkylaryl" denotes an aryl group having an alkyl group at the point of attachment.

A used herein, "carboxylate" denotes salt or ester of an organic acid, containing the radical— COOR, wherein R may be, but is not limited to, a H, an alkyl group, an alkenyl group, or any other residue otherwise known in the art.

As used herein, "carboxylic acid" denotes an organic functional group comprising the following structure:— COOH or— CO ₂ H. As used herein, "cyano" denotes an organic functional group comprising the following structure: C=N

As used herein, "cycloalkyl" refers to a monovalent or polycyclic saturated or partially unsaturated cyclic non-aromatic group containing all carbon atoms in the ring structure, which may be substituted with one or more substituent groups defined herein. In certain non-limiting aspects the number of carbons comprising the cycloalkyl group may be between 3 and 7. As used herein, "cycloalkenyl" refers to a partially unsaturated cycloalkyl radical derived by the removal of one or more hydrogen atoms from a cycloalkyl ring system such that it contains at least one carbon-carbon double bond.

As used herein, "halogen" or "halide" denotes any one or more of a fluorine, chlorine, bromine, or iodine atoms.

As used herein, "heterocyclic" refers to a monovalent saturated or partially unsaturated cyclic aromatic or non-aromatic carbon ring group which contains at least one heteroatom, in certain aspects between 1 to 4 heteroatoms, which may be but is not limited to one or more of the following: nitrogen, oxygen, sulfur, phosphorus, boron, chlorine, bromine, or iodine. In further non-limiting aspects, the hetercyclic ring may be comprised of between 1 and 10 carbon atoms.

As used herein, "hydroxyl" denotes an organic functional group comprising the following structure:— OH.

As used herein, "phosphonate" denotes an organic functional group comprising the following structure:— P0 ₃ H ₂ or— PO(OH) ₂ .

As used herein, "phosphate" denotes an organic functional group comprising the following structure:— OP0 ₃ H ₂ or— OPO(OH) ₂ .

The present disclosure relates to novel compounds, pharmaceutical compositions containing the same, and methods of use by inhibiting the enzymatic activity of Glycerol 3- phosphate acyltransferase (GPAT). Such compounds, compositions, and methods have a variety of therapeutically valuable uses including, but not limited to, treating inflammation, increasing insulin sensitivity, treating obesity, increasing palmitate oxidation, improving glucose tolerance. The class of compounds of the present disclosure are comprised of Formula I:

Formula I

wherein n is either 0 or 1. A is selected from the group consisting of NRl, O, and S, wherein Rl is either a H, hydroxyl, Ci-Cio alkyl, Ci-Cio alkoxy, alkenyl, aryl, alkylaryl or arylalkyl. X is selected from the group consisting of a carboxylate residue, a phosphonate residue, a phosphate residue, or a Ci-Cio alkyl residue which is optionally substituted with one or more carboxylate, phosphonate or phosphate residues. Y is selected from the group consisting of C1-C20 alkyl, alkenyl, halide, hydroxyl, C1-C20 alkoxy, aryl, alkylaryl, arylalkyl, cycloalkyl, cycloalkenyl, or a heterocyclic ring. In aspects where Y is a C1-C20 alkyl, alkenyl, C1-C20 alkoxy, aryl, alkylaryl, arylalkyl, cycloalkyl, cycloalkenyl, or a heterocyclic ring, it is optionally substituted at one or more positions with a halide. Z is selected from the group consisting of a H, a hydroxyl group, a halide, an aryl group, an alkylaryl group, an arylalkyl group, a cycloalkyl group, a cycloalkenyl group or a heterocyclic ring. In aspects, where Z is an aryl group, an alkylaryl group, an arylalkyl group, a cycloalkyl group, a cycloalkenyl group or a heterocyclic ring, the ring moiety may be substituted with one or more substituent groups selected from a C1-C1 ₀ alkyl group, C1-C1 ₀ alkoxy group, a hydroxyl group, a cyano group, a carboxylate group, a halide, an aryl group, an alkylaryl group, an arylalkyl group, a cycloalkyl group, a cycloalkenyl group or a heterocyclic ring.

In certain aspects, X is comprised of either a carboxylic acid residue or a phosphonate residue. In alternative aspects, X may include a C1-C10 alkyl group, which is substituted at one or more positions with either a phosphonate residue or carboxylate. In further aspects, the alkyl group may comprise between 1 and 3 carbons. In any of the foregoing, X may be positioned on the phenyl ring in either the ortho, meta, or para position with respect to the sulfonyl linker. As shown below, in certain non-limiting aspects X occupies either the ortho or meta position.

In further non-limiting aspects, Y is comprised of a C1-C20 alkyl group, which may be either a CH3, C5H11, C%-Hn, C14H29, and C16H33. Alternatively, Y may be comprised of an aryl ring system, which is optionally substituted with one or more halogen atoms. In even further alternative aspects, Y is comprised of an alkylaryl residue, wherein the alkyl moiety connects the aryl ring to the Y position. The alkyl chain may have between 1 to 3 carbon atoms, with certain aspects having 1 or 2 carbon atoms. The aryl residue in this latter aspect may be substituted with one or more halogen atoms.

In even further non-limiting aspects, Z is either a hydrogen atom, a hydroxyl group, a halogen atom, an optionally substituted aryl group or an optionally substituted heterocyclic ring. In any of the foregoing, Z may be position on the phenyl ring in either the ortho, meta, or para position with respect to the sulfonyl linker. As shown below, in certain nonlimiting aspects Z occupies either the meta or para position with respect to the sulfonyl linker of the phenyl ring. In even further aspects, Z occupies either the meta or para position with respect to both the sulfonyl linker and X positions.

Based on the foregoing, one compound of the present disclosure is C-67 or FSG67 and is comprised of the following structure:

In another aspect, the compounds of the present disclosure may be comprised of the following structures:

In an even further aspect, the compounds of the present disclosure may be comprised of one or more of the following:

, and

Based on the foregoing, in certain non- limiting aspects of Formula I, A is comprised of NRl w herein Rl is any of the aspects defined above. In further aspects Rl is a hydrogen atom. To this end, certain aspects of the compounds of the present disclosure may be represented by Formula lb

wherein each of n, X, Y, and Z are any of the aspects defined above.

In alternative aspects of Formula I, n is comprised of 0. To this end, certain compounds of the present disclosure may be represented by Formula Ic:

wherein each of A, X, Y, and Z are any of the aspects defined above.

In even further aspects of Formula I, X is comprised of a carboxylic acid residue at either the ortho, meta or para positions with respect to the sulfonyl linker of the phenyl ring. Accordingly, certain compounds of the present disclosure may be represented by Formula Id:

wherein each of n, A, Y, and Z are any of the aspects defined above.

While the carboxylic acid residue may occupy either the ortho, meta, or para positions, in certain aspects it occupies the ortho position with respect to the sulfonyl linker. To this end, certain compounds of the present disclosure may be represented by Formula Ie:

wherein each of n, A, Y, and Z are any of the aspects defined above.

Similarly, although it may occupy either the ortho, meta, or para positions, in certain compounds of the present disclosure Z occupies either the meta or the para postions with respect to both the sulfonyl linker and X, as set forth below in Formulas If and Ig:

wherein each of n, A, Y, and Z are any of the aspects defined above.

Based on the foregoing structures of Formulas If and Ig compounds of the present disclosure may be comprised of one or more of the following:

, . and

In further aspects of Formula I, X is comprised of either a phosphate group or an alkyl residue having 1 to 3 carbon atoms, which is substituted with a phosphonate group. Such compounds of the present disclosure may be represented by Formula Ih:

wherein m is comprised of either 0, 1, 2, or 3 and each of n, A, Y and Z are any of the aspects defined above.

Accordingly, compounds of the present disclosure may be comprised of one or more of the following:

A further aspect of the present disclosure is the class of compounds comprised of Formula II:

Formula II

wherein:

W is selected from the group consisting of a saturated linear C3-C18 alkyl; a saturated branch -C18 alkyl; an unsaturated linear or unsaturated branched C3-C18 alkyl;

wherein ¾ and R2 each are H, CH ₃ , C ₂ H ₅ , C ₃ H ₇ , C ₄ H ₉ , CF ₃ , OCH ₃ , F, CI, or Br. R is H, CH ₃ , C ₂ H ₅ , C ₃ H ₇ , C4H9, COOH, COOCH ₃ , COOC2H5, COOC H ₇ , or COOC4H9. R4 is H, CH ₃ , C ₂ H ₅ , C ₃ H ₇ , or C4H9. X is N, S or O. Z is CH ₂ , O, NH or S. i is 1 to 5, j is 0 to 10, k 1 to 10, m is 1 to 13; and n is 1 to 15, and Ri and R ₂ may be the same or different.

Based on the foregoing, one compound of the present disclosure is tetrahydro-3- methylene-2-oxo-5-n-octyl-4-furancarboxylic acid (C-75) comprised of the following structure:

EXAMPLES

Example 1: Neuron cultures

Hypothalami are dissected from embryonic (El 7) Sprague-Dawley rats (Harlan) and digested in papain (20.1 units/ml) in Earle's balanced salt solution with 10 μg/ml DNAse for 18 min in a 37°C water bath. Tissues from the hypothalamus, cerebral cortex, and cerebellum from 7 adult rats are saved for RNA analysis. Digested hypothalamic cells are plated onto poly-D-lysine-coated Nunclon™ plates, dishes, or flasks at 2.0 x 10 ⁵ cells/cm ² to

3.0 5 2

x 10 cells/cm , depending on application, and cultured in Neurobasal-A medium with no glucose (Invitrogen™), supplemented with 3 mM glucose, 2% B27 (Invitrogen™, Gran Island, NY), 2 mM glutamax-I (Invitrogen™, Gran Island, NY), 100 units/ml penicillin, and 100 μ§/πύ streptomycin (Invitrogen™, Gran Island, NY). Hypothalamic cells are maintained at 37°C in a humidified incubator under 5% C0 ₂ , 5% 0 ₂ , and 90% N ₂ . Media glucose levels are measured immediately prior to feeding of cells(Accu-Chek™ glucometer, Roche, Indianapolis, IN). Hypothalamic cells are fed every 3 days by restorative feeding to maintain final glucose at 3 mM. On the third day in vitro (DIV), hypothalamic cells are treated with 1 μΜ cytosine arabinoside to inhibit astrocyte proliferation. Assays are performed at DIV 9 or 10. For immortalized hopothalamic neuronal cell lines N38HN and R7HN, neurons are plated on plastic Nunclon™ culture plates or dishes at 1.6 ^x 10 ⁴ cells/cm ² . Hypothalamic cells are cultured in Dulbeccos ME medium (DMEM) with 10% fetal bovine serum (FBS), 5 mM glucose, 1% penicillin/streptomycin and maintained at 37°C in a humidified incubator with 5%> CO ₂ , 5% O ₂ , and 90% N ₂ gas mixture. When hypothalamic cells are 90% confluent, FBS is removed, treatments are applied, and assays are performed.

Example 2: Immunocytochemistry

At DIV 10, media is removed and primary hypothalamic neurons (PHN) are cultured in 24-well plates and rinsed with phosphate buffered saline (PBS) and then fixed with 4% paraformladahyde (PFA) for 20 min. After a dextrose phosphate buffered saline (DPBS) rinse, PHN are placed in 10% normal goat serum and 0.1% Triton X-100™ for 60 min, followed by overnight incubation with primary antibodies in the same solution at 4°C [co- incubations are rabbit anti-MAP2 (1 :1,000; Millipore, Billerica, MA) plus mouse anti-OX-42 (1 :250; Millipore, Billerica, MA), or mouse anti-MAP2 (1 :200; Millipore, Billerica, MA) plus rabbit anti-GFAP (1 : 10; ImmunoStar, Hudson, WI)]. The following day, PHN are rinsed with DPBS and incubated with Alexa 488 conjugated goat anti-rabbit, or Alexa 488 anti-mouse secondary antibody, coincubated with Cy3 goat anti-mouse or Cy3 goat anti- rabbit IgG, respectively, for 60 min. Cellular nuclei of the PHN are counterstained with Hoechst 33342 (1 :2000) for 15 min. Immunostained PHN are observed with fluorescent microscope (Axiovert 200; Zeiss, Thornwood, NY). Five fields from each well are randomly selected for quantitative analysis. Image-J software is used to count Hoechst stained nuclei, immunolabeled neurons, and glia.

Example 3: Radiolabeled substrate assays

For fatty acid (FA) oxidation (FAOx), adherent neurons in T25 flasks are treated with compounds for 2 hours in Ham's-FlO media. During the last 30 minutes of treatment with the compounds of interest or controls, 0.5 μ^ηιΐ (20 nmol) of [l- ¹⁴ C]-palmitate (Moravek Biochemicals, Brea, CA) complexed to 1% bovine serum albumen (BSA) and 2 μΜ carnitine is added. Flasks are fitted with serum stoppers and plastic center wells containing benzethonium hydroxide. Following incubation, 7% HCIO4 is injected into flasks and ¹⁴ C0 ₂ is trapped for 2 h at 37°C. Trapped ¹⁴ CC>2 is removed and quantified by liquid scintillation counting. Flask contents are then hydrolyzed with 4 N KOH and neutralized using H2SO4. Acid soluble products are extracted using chloroform:methanol (2:1, v:v) and dH ₂ 0 and quantified by liquid scintillation counting. Total FA oxidation is obtained by addition of C0 ₂ and acid soluble products. For measurement of FA synthesis, neurons cultured in 6- well plates are labeled with 100 μΜ [ ³ H]-acetic acid (PerkinElmer Life Sciences, Waltham, MA) for an additional 2 hours. Lipids are extracted with chloroform/methanol, dried under N ₂ , and counted using a liquid scintillation counter. Example 4: Immunoblotting

PFiN are grown on 6-well plates and washed on ice with DPBS with 50 mM NaF then scraped in lysis buffer (20 mM Tris-HCl, pH 7.4, 50 mM NaCl, 50 mM NaF, 5 mM Na ₄ 0 ₇ P ₂ , 250 mM sucrose, 1% Triton X-100™ (v/v), 500 mM dithiothreitol, and protease inhibitor (1 pill/ 10 mL of buffer; Roche, Indianapolis, IN). PUN cellular lysate is centrifuged at 15,000 x g for 15 min. Supernatant from the centrifuged PHN cellular lysate is stored at -80°C for protein quantification and immunoblotting. To ensure equal loading of PHN cellular protein, the BCA kit (Bio-Rad, Hercules, CA) is used for protein quantification. For protein immunoblotting, PHN cellular lysates are thawed on ice and mixed with 5X sample buffer (312.5 mM Tris-HCl, pH 6.8, 50% glycerol, 10% sodium dodecyl sulfate, 5% β- mercaptoethanol, and trace amounts of bromophenol blue), boiled, and run on Tris-HCl 4- 15%) linear gradient polyacrylamide gels (Bio-Rad, Hercules, CA). Following protein transfer from gels to PVDF membranes (Bio-Rad, Hercules, CA), blots are successively probed with the following antibodies: 1 : 1 ,000 antiphosphorylated-ΑΜΡΚ (Cell Signaling, Boston, MA), 1 : 1500 AMPKa (residues 2-20 of l and a2; Covance, Chantilly, VA), 1 :200 IL6, 1 :200 IL 1 B (Santa Cruz Biotechnology, Santa Cruz, CA), or 1 : 1000 CHOP (Thermo Scientific, Rockford, IL) in TBST containing 0.1% Tween-20, 5% protease free BSA and 50 mM NaF. Blots are visualized using SuperSignal chemiluminescence kits (Pierce, Rockford, IL). A L IO dilution of the Femto kit is used to detect the pAMPK signal and the undiluted Pico kit is used to detect all other proteins. Example 5: Metabolomics

For targeted lipidomics, PHN treated with compounds of interest or controls are grown on 60 mm dishes and washed with ice-cold DPBS, scraped in HPLC water, then centrifuged at 16,000 x g for 5 min. Supernatant is aspirated, and the pellet is frozen in liquid nitrogen and stored at -80°C until extraction. Total lipids are extracted according to a modified Bligh and Dyer procedure(Haughey et al., 2004). Briefly, each sample is homogenized (Sonic Dismembrator, Fisher Scientific, Pittsburg, PA) with one or two 5- second pulses at room temperature in 200 of deionized water, followed by addition of 600 μΐ _^ of methanol containing 53mM ammonium formate with appropriate internal standards, then vortexed. Chloroform (800 μί) is then added, and the mixture further vortexed then centrifuged at 1,000 x g for 5 min. The bottom chloroform layer is separated and dried under nitrogen evaporation stream drier. The aqueous supernatant with protein is dried by vacuum and the protein pellet is re-suspended in deionized water and used for protein quantification by BCA assay. The dried bottom chloroform layer is re-suspended in pure methanol and stored at -80°C until analysis by LC/ESI/MS/MS. Briefly, chromatographic separations are performed by reverse-phase C 18 liquid chromatography columns as a stationary phase and the following mobile elution phases (A) water: methanol: formic acid (59:40: 1, v/v/v) with 5mM ammonium formate); (B) methanohformic acid (99: 1, v/v) with 5mM ammonium formate is used for the separation of sphingo lipids; (C) methanol and 0.1% formic acid, v/v with 5mM ammonium formate, (D) methanol: chloroform (75:25, v/v) and 0.1% formic acid with 5mM ammonium formate is used for the separation of acyglcyerols, cholesterol and esters and (E) water and 0.01% formic acid, v/v (F) methanol and 0.01% formic acid, v/v used for the separation of free fatty acids. All solutes are separated by using the gradient elution conditions. Quantitative analyses of ceramides, sphingolipids, DAG, TAG, cholesterol or cholesterol esters are performed by high pressure liquid chromatography (HPLC) coupled to a turbo ion electro spray source of a triple stage quadrupole tandem mass spectrometer API3000 PE Sciex (Applied Biosystems, Grand Island, NY) operated in positive ionization mode, and a 4000Qtrap mass spectrometer (Applied Biosystems, Grand Island, NY) operated in negative ionization mode for the analysis of free fatty acids. Lipid analytes are monitored in multiple reaction monitoring (MRM). Instrument control and data acquisition are performed using Analyst 1.5.1 software.

For untargeted metabolomics, PHN treated with compounds of interest are grown on 100 mm dishes and washed with ice-cold DPBS then soaked in extraction solvent (80% methanol and 20% ultrapure water with internal standards (D,L-2-fluorophenylglycine, D,L- 4-chlorophenylalanine, tridecanoic acid, D6 cholesterol) for exactly 5 min at room temperature. Solvent is collected and stored at -80°C until analysis. The remaining monolayer is scraped in water for protein quantification by Bradford assay. Global metabolomic profiling is carried out by Metabolon, Inc. (Durham, NC) on three independent instrument platforms: gas chromatography/mass spectrometry (GC/MS), and ultrahigh performance liquid chromatography/tandem mass spectrometry (UHLC/MS/MS2) optimized for basic or acidic species. Example 6: Effects of C75 or FSG67 on Fatty Acid Catabolism

Primary hypothalamic neurons (PHN) as described in Example 1 are cultured with glucose and oxygen levels physiological for brain are cultured. PHN are used to study neuronal metabolism in cultures having 85% neurons, 0.4% microglia and 4.8% astrocytes (Figure 1 A, IB). Validated hypothalamic neuronal lines are used. Treatment of PHN for 24 h with C75 up to 70 μΜ or FSG67 up to 160 μΜ does not alter cell viability (Figure 1C, ID). cFos mRNA in PHN is measured to assess neuronal activation. C75 for 6 h increases cFos expression (Figure IE), but FSG67 does not (Figure IF).

C75 decreases acetate incorporation into lipids, and thus FA synthesis, in PHN (Figure 1G). FSG67 does not affect acetate incorporation (Figure 1H). C75 increases palmitate oxidation in PHN (Figure II) and N38HN (Figure S1A). FSG67 also increases palmitate oxidation in PFfN (Fig. 1 J). FSG67 may enhance FA availability to CPT-1 by decreasing esterification.

FSG67, like C75, can increase ATP in hypothalamic neurons. C75 increases ATP in PHN (Figure IK), and decreases active pAMPK (Figure 1L). FSG67 likewise increases ATP and decreases pAMPK (Figure 1M, IN). In N38HN, both compounds produce biphasic responses in ATP levels and pAMPK phosphorylation. ATP decreases, and then increases, with reciprocal changes in pAMPK (Figure SIB, SIC). For all data: p < 0.001; **, p <

0.01; *, p < 0.05. Data are represented as means ± SEM.

Example 7: C75 and FSG67 alter transcription of CPT-1 and GPAT isoforms in PHN CPT-1 is required for β-oxidation of long-chain FA. CPT-1 a and CPT-lb catalyze acyl transfer from CoA to carnitine for transport across the outer mitochondrial membrane.

CPT-la is predominant in rat hypothalamus, cerebral cortex, and cerebellum (Figure 2A).

CPT-1 c is most prevalent in PHN (Figure 2B), consistent with neuronal enrichment. C75 and FSG67 increase CPT-la expression in PHN (Figure 2C, 2D), an effect that would support increased palmitate oxidation. C75 decreases CPT-1 c expression (Figure 2C), which can denote FA flux shift toward mitochondrial uptake, away from ER.

GPAT inhibition alters FA metabolism in PHN. In rat hypothalamus, cortex, cerebellum, and PHN, the GPAT homologues GPATl and GPAT4 are predominant (Figure 2E, 2F). Chronic C75 increases PHN expression of GPAT3 (Figure 2G); this may, without being limited to any particular theory or mechanism, ensure membrane integrity during increased β-oxidation and inhibited FA synthesis. FSG67 does not alter GPAT expression

(Figure 2H). Sterol regulatory element-binding protein- lc (SREBPlc) controls gene transcription for lipogenic enzymes such as acetyl-CoA carboxylase (ACC), FAS, and GPAT. C75 decreases sterol regulatory element binding transcription factor lc (SREBPlc) and FAS transcription (Figure 21). Decreased FAS expression would decrease flux through the FA synthetic pathway and preserve ATP. FSG67 does not alter expression of SREBPlc or FAS (Figure 2J). For tissue mRNA data, n = 6/tissue. CPT-la and GPAT1 mRNA levels were baselines for comparisons within tissue and within isoform. Other data were from two independent experiments, six replicates each. For isoform distributions, treatment differences are signified by differing superscripts within tissue, p < 0.01. For all data: p < 0.001; **, p < 0.01; *, p < 0.05. Data are represented as means ± SEM.

Example 8: Enhanced FAOx increases ROS, but not oxidative stress

C75 and FSG67 increase FAOx. Exposing PHN to palmitate (C16:0; FA excess) increases ROS, an effect greatly potentiated with C75 or FSG67 (Figure 3A, 3B). Responses are similar in PHN cultured with B27 (supplement with antioxidants, linoleate and linolenate) during analysis (data not shown), and in R7HN (data not shown). ROS can result in oxidative stress. Presence of CI 6:0 or C75 alone does not increase SOD activity; however, C75 for 18 h in the presence of CI 6:0 increases SOD activity (Figure 3C). Although increased ATP with C75 or FSG67 does not suggest compromised oxidative phosphorylation, increased ROS can impair mitochondria. C75 and FSG67 (in media with FA from B27) do not effect mitochondrial membrane potential (Figure 3D, 3E). Results suggest without being limited to any particular theory or mechanism that although these agents can potentiate ROS production, they do not compromise mitochondrial function. ROS data were from two independent experiments, five replicates each. SOD activity data were from two

experiments, three replicates each. For all data: ***, p < 0.001; **, p < 0.01; *, p < 0.05. Data are represented as means ± SEM.

Example 9: Increasing FAOx shifts metabolic flux away from anabolic processing

Addition of CI 6:0 increases intracellular free CI 6:0 and CI 6: 1 (Figure 4B), and C75 blocks this effect (Figure 4B). C75 also attenuates C16:0-induced formation of

monoacylglycerol (MAG, i.e., glyceryl- 1-stearate, Figure 4C). Excess C16:0 increases glyceryl tripalmitate 35-fold, indicating significant fat storage in PHN. PHNs in excess

C16:0 have increased ceramides, and C75 reverses this outcome (Figure 4D). Lastly, overall cholesterol level is not affected by exposure to C16:0, but levels of C16:0 and C18:0 cholesterol esters do increase (Figure 4E), and C75 tends to mitigate this, while not being limited by any particular mechanism. Lipidomic data indicates without being limited to any particular theory or mechanismthat FA flux shift away from anabolism overall with a FAOx stimulator.

Modifying FA flux can alter phospholipid synthesis and thus affect cell membranes

(Figure 4F). Levels of 1 -palmitoylglycerophosphoethanolamine and 1- oleoylglycerophosphoethanolamine decrease with C75, consistent with lower free C16:0 and C18:l, but acylglycerophosphocholines increase.

C75 remodels the PHN metabolome in multiple ways to support oxidative metabolism yet prevents oxidative stress (Figure 4F). C75 increases oxidized NAD ⁺ , as well as decreases citric acid cycle intermediates acetyl-CoA and acetyl-carnitine, with a concomitant increase in citrate. Without being limited to any particular theory or mechanism, this suggests increased acetyl-CoA utilization via upregulated CAC flux to support the increased ATP. C75 increases 3-hydroxy-3-methyl-glutarate, indicating increased β-oxidation and ketone production, and consistent with rapid acetyl-CoA utilization. FAOx -induced increases in ROS can promote oxidative stress. C75 increases both the oxidized and reduced forms of glutathione, and increases levels of γ-glutamyl amino acids to regenerate glutathione. C75 also tends to increase 5-oxoproline, a marker of glutathione degradation. Finally, cysteine- glutathione disulfide, an indicator of oxidative stress, does not increase in PFIN in response to FAOx with C75. For all data: ***, p < 0.001; **, p < 0.01; *, p < 0.05. Data are represented as means ± SEM.

Example 10: Enhanced FAOx does not increase ER stress in PHN

TG upregulates expression of UPR markers ATF4, ATF6, C/EBP homologous protein (CHOP, pro-apoptotic marker), and spliced XBP1 (Figure 5A). Excess dietary fat leads to lipid accumulation and abnormal intracellular metabolic fluxes that contribute to ER stress. In PHN, excess C16:0 increases expression of ATF4 and ATF6 (Figure 5B). C75 and FSG67, which increase ROS in PHN, do not induce ATF6 transcription (Figure 5B, 5C). Treatment with a selective CPT- 1 stimulator has a minimal effect on ATF transcription (Figure 5D). CI 6:0 increases XBP1 splicing in PHN. Treatment with C75 or FSG67 does not stimulate XBP1 processing (Figure 5E, 5F). Furthermore, C75 partially reverses C16:0- induced XBP1 splicing, a protective response. The data show that the compounds do not induce UPR, suggesting without being limited to any particular theory or mechanism, no elevation of ER stress. Within the heat maps, rows represent metabolite level and columns correspond to the mean of three pooled replicates (i.e. n = 6, 3 per column). Heat maps are calibrated on a twenty-five point color gradient with highest and lowest metabolite levels as bright red and bright green, respectively. Comparisons are made solely within metabolite. For all data, statistics were performed on the log of normalized, median-scaled data. For all data: ***, p < 0.001; **, p < 0.01; *, p < 0.05. Data are represented as means ± SEM.

Example 11: Increased FAOx prevents C16:0-induced inflammation

PHN exposed to C16:0 for 18 h display elevated TNFa, ΠΛβ, and IL6 mRNAs (Figure 6A). These cytokines elicit pro-inflammatory responses. Without being limited to any particular theory or mechanism, hypothalamic IL6 reduces neuronal inflammation and ER stress to improve insulin and leptin signaling and improve energy balance. C16:0 also increases cytokine expression in R7HN (data not shown). C75, in presence or absence of C16:0, suppresses TNFa and ILi mRNA expression completely in PHN (Figure 6A). As with treatment with C16:0, IL6 mRNA increases with C75 treatment alone; the IL6 mRNA increase is greatly potentiated when PHN in CI 6:0 are treated with C75 (Figure 6A). This pattern of potentiated IL6 expression in response to C 16:0 plus C75 is similar to changes in ATP and ROS, without being limited to any particular theory or mechanism, suggesting a relationship between IL6 and mitochondrial function. Treatment with FSG67 also reverses C16:0-induced TNFa and ILip mRNA but, unlike C75, does not influence IL6 mRNA expression (Figure 6B). R7HN has similar responses to FAOx stimulators, except IL6 expression is not potentiated with C75 and C16:0 (data not shown). C75 increases IL6 and decreases ILi in PHN (Figure 6C, 6D). CPT-1 stimulator C89b produces a cytokine expression profile similar to that with C75 (Figure 6E). These compounds decrease food intake, without being limited to any particular theory or mechanism, FAOx and downstream anti-inflammatory response as a way to reverse the impaired negative feedback signaling on food intake that occurs in response to high-fat diet. Data were collected from two independent experiments, three replicates each. For all data: p < 0.001; **, p < 0.01; *, p < 0.05. Data are represented as means ± SEM.

Example 12: Daily C75 increases muscle AMPK activity and FAOx, improves glucose tolerance, and reverses hyperinsulinemia in DIO

Chronic high-fat diet provides an in vivo model to evaluate how surplus FA alter glucose and FA metabolism, which influence insulin sensitivity in insulin-target tissues. In mice treated with daily intraperitoneal administration of C75, C75 decreased high-fat diet intake by 50% on day 1; eating remains low until day 4, then normalizes despite continued C75 (Figure 7A). Despite renormalized food intake, C75 produces weight loss throughout treatment (Figure 7B). At day 4, when C75 hypophagia is still maximal, active pAMPK is elevated in muscle (Figure 7C) versus vehicle ad libitum and PF controls, thus the increased pAMPK in muscle is not due to decreased food intake.

Consistent with the increased muscle pAMPK, the AMPK target ACC is more phosphorylated in muscle from C75-treated mice than in control groups (Figure 7D). A resulting decrease in C16:0 synthesis without being limited to any particular theory or mechanism would support FAOx in muscle due to decreased malonyl-CoA and subsequent CPT-1 disinhibition. ACC catalyzes malonyl-CoA production, but degradation back to acetyl-CoA is catalyzed by malonyl-CoA decarboxylase (MCD). C75 increases muscle MCD mRNA after 4 days of treatment (Figure 7E), an effect that would decrease malonyl- CoA and further without being limited to any particular theory or mechanism support FAOx.

Concurrent with increased pAMPK in muscle at day 4, C75 also increases GLUT4 mRNA (Figure 7F). C7 -treated DIO mice are less hyperglycemic in response to a glucose tolerance test (Figure 7G), and had lower levels of insulin and leptin in circulation (Figure 7H, 71); PF controls have elevated insulin and leptin like ad libitum vehicle controls, so these C75 effects are not due to hypophagia. Protein and mRNA data are shown as fold-change versus control within time point; data were collected from independent experiments (N = 9) performed in triplicate. Glucose tolerance tests were performed 4 days post i.p. injection of vehicle or C75 (n = 5-6). For all data: ***, p < 0.001; **, p < 0.01; *, p < 0.05. Data are represented as means ± SEM.

Having now generally described the invention, the same will be more readily understood through reference to the following examples that are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified.

Each periodical, patent, and other document or reference cited herein is herein incorporated by reference in its entirety.