Structurally-engineered fatty acid 1024 (SEFA-1024) improves diet-induced obesity, insulin resistance, and fatty liver disease

Abstract

Obesity is a global epidemic that drives morbidity and mortality through cardiovascular disease, diabetes, and non-alcoholic fatty liver disease (NAFLD). No definitive therapy has been approved to improve glycemic control and treat NAFLD in obese patients. Here, we investigated a semi-synthetic, long chain, structurally-engineered fatty acid-1024 (SEFA-1024), as a treatment for obesity-induced hyperglycemia, insulin-resistance, and fatty liver disease in rodent models. A single dose of SEFA-1024 was administered to evaluate glucose tolerance and active glucagon-like peptide 1 (GLP-1) in lean rats in the presence and absence of a DPP-4 inhibitor. The effects of SEFA-1024 on weight loss and glycemic control were assessed in genetic (ob/ob) and environmental (high-fat diet) murine models of obesity. Liver histology, serum liver enzymes, liver lipidomics, and hepatic gene expression were also assessed in the high-fat diet murine model. SEFA-1024 reversed obesity-associated insulin resistance and improved glycemic control. SEFA-1024 increased active GLP-1. In a long-term model of diet-induced obesity, SEFA-1024 reversed excessive weight gain, hepatic steatosis, elevated liver enzymes, hepatic lipotoxicity, and promoted fatty acid metabolism. SEFA-1024 is an enterohepatic-targeted, eicosapentaenoic acid derivative that reverses obesity-induced dysregulated glucose metabolism and hepatic lipotoxicity in genetic and dietary rodent models of obesity. The mechanism by which SEFA-1024 works may include increasing aGLP-1, promoting fatty acid oxidation, and inhibiting hepatic triglyceride formation. SEFA-1024 may serve as a potential treatment for obesity-related diabetes and NAFLD.

Keywords: fatty acid metabolism; free fatty acid receptors (FFARs); high-fat diet (HFD); hyperglycemia; hyperinsulinemia; lipidomics; lipotoxicity; obesity; structurally-engineered fatty acid (SEFA).

FIGURE 1

Comparison of the chemical structures of eicosapentaenoic acid (EPA) and structurally-engineered fatty acid 1024 (SEFA-1024). Note the modifications at the alpha and beta carbons in SEFA-1024. These modifications are intended to prevent esterification and β-oxidation

FIGURE 2

Summarizes the timeline of events

FIGURE 3

SEFA-1024 improves glycemic control in ob/ob mice. After 29 days of treatment, high-dose SEFA-1024 (251.2 mg/kg) reduced fasting blood glucose in ob/ob mice (a). Fasting serum insulin was reduced at 29 days in pioglitazone-treated but not SEFA-1024-treated mice (b). Change in serum insulin from day 1 to day 29 was greatest in pioglitazone-treated mice but also reduced in SEFA-1024-treated mice relative to control (c). Glucose tolerance testing (d) showed significantly improved glucose tolerance (reduced glucose AUC, e) in mice treated with SEFA-1024. Treatment with SEFA-1024 resulted in a dose-dependent decrease in post-prandial glucose (f). * = p < 0.05, ** = p < 0.01 relative to vehicle. (a, b) are two-way ANOVA, (c, e) are one-way ANOVA

FIGURE 4

A single dose of SEFA-1024 increases active glucagon-like peptide 1 (aGLP-1) in lean Sprague–Dawley rats. Prior to glucose tolerance testing (GTT), rats were pre-treated with a dose of the DPPIV inhibitor linagliptin (3 mg/kg) or vehicle and then a single dose of SEFA-1024 (251.2 mg/kg) or vehicle. On GTT, no differences were seen in blood glucose (a, d) or insulin (b, e). aGLP-1 (c, f) was increased in mice treated with DPPIV inhibitor and SEFA-1024 but not SEFA-1024 alone or DPPIV inhibitor alone. DPPIV inhibitors prevent inactivation of aGLP-1 by dipeptidyl peptidase which normally occurs within 1–2 min of activation. About 24 h after treatment, no differences were seen in glucose (g) or insulin (h). aGLP-1 (i) was significantly increased in mice treated with SEFA-1024 alone and DPPIV inhibitor with SEFA-1024. * = p < 0.05, ** = p< 0.01 by one-way ANOVA relative to vehicle 1 + vehicle 2

FIGURE 5

SEFA-1024 reverses obesity caused by a high fat diet (HFD) in wild-type mice. After 15 weeks, HFD mice exhibited significantly greater body mass than mice on chow (a). Treatment with SEFA-1024 was initiated at week 15 (dotted line) and resulted in weight loss to body mass similar to chow. No significant differences were seen in food consumption across groups. Accordingly, weight loss induced by SEFA-1024 was not the result of an anorexiant effect. ** = p < 0.01 by one-way ANOVA relative to chow

FIGURE 6

SEFA-1024 improves glucose metabolism and reduces insulin resistance in wild-type mice on a high fat diet (HFD). After 5 weeks of treatment with SEFA-1024 or control, mice underwent glucose tolerance testing (GTT) with serial measurement of blood glucose, insulin, and total glucagon-like peptide 1 (GLP-1). HFD mice exhibited marked hyperglycemia (a, d) and hyperinsulinemia (b, e) in response to orogastric glucose which was attenuated by treatment with SEFA-1024. HFD mice demonstrated fasting hyperinsulinemia (b, time = 0 min) which was reduced by SEFA-1024. Total GLP-1 was reduced in mice treated with SEFA-1024 (f). ** = p < 0.01, **** = p < 0.0001 by one-way ANOVA relative to chow

FIGURE 7

SEFA-1024 reverses high fat diet-induced steatohepatitis. Serum aspartate aminotransferase (AST, a) and alanine aminotransferase (ALT, b) were elevated by HFD and reduced by SEFA-1024. Mice on chow (c) demonstrated normal liver histology with little to no steatosis. HFD mice (d) had marked macrovesicular (arrow) and microvesicular hepatic steatosis. SEFA-1024 treated mice (e) had little to steatosis similar to chow mice. (c)–(d) Hematoxylin and eosin-stained liver tissue imaged at 400× magnification. * = p < 0.05 by one-way ANOVA relative to chow

FIGURE 8

SEFA-1024 prevents high-fat diet (HFD)-induced hepatic lipotoxicity. Mass spectroscopy was used to analyze hepatic lipids. Mice treated with SEFA-1024 had reduced hepatic cholesterol esters (a), triglycerides (b), and bile acids (c). Hepatic acylcarnitines (e), omega-3 fatty acids (f), and the omega-3 to omega-6 fatty acid ratio (g) were all increased by SEFA-1024. Acylcarnitines are compounds that shuttle fatty acids to the mitochondrial membrane for oxidation. Increased acylcarnitines indicate increased fatty acid metabolism. * = p < 0.05, ** = p < 0.01, **** = p < 0.0001 by one-way ANOVA relative to chow. ## = p < 0.05 by one-way ANOVA relative to HFD

FIGURE 9

SEFA-1024 prevents HFD-induced changes in hepatic transcription. The COLA1 gene encodes type I alpha collagen and increased COLA1 transcription is seen in progressive NAFLD. Hepatic COLA1 transcription (a) was increased by HFD and reduced with SEFA-1024. The ACACB gene encodes acetyl-CoA carboxylase (ACCα) which catalyzes the rate-limiting step in fatty acid synthesis. ACCα transcription (b) was decreased by HFD but not when treated with SEFA-1024. Sterol-regulatory element binding protein 1 (SREBP1) is a key regulator of lipid homeostasis. SEFA-1024 reduced transcription of SREBP1 (c), perhaps through feedback inhibition, as SEFA-1024 increased SREBP1 expression (Figure 10b). CD36 (also known as fatty acid translocase) transcription (d) was increased by SEFA-1024. CD36 metabolizes very long chain fatty acids (VLCFA) in the cytoplasm and increased transcription indicates increased VLCFA metabolism. Peroxisome proliferator-activated receptor γ (PPARγ) is the target of the thiazolidinedione class of medications. PPARγ transcription (e) was similarly elevated in HFD mice with and without SEFA-1024 treatment. CYP7A1 is the cytochrome that catalyzes the rate-limiting step in the conversion of cholesterol to bile within the liver. CYP7A1 transcription was increased by SEFA-1024 but this did not reach statistical significance. ABCC, PPARα, MAP2K, SCD1, ARRB2, CASP1, and NRLH4 are regulators hepatic lipid and bile homeostasis and were not significantly altered by HFD and/or SEFA-1024 treatment. * = p < 0.05, ** = p < 0.01, *** = p < 0.001 by one-way ANOVA relative to chow. # = p < 0.05 by one-way ANOVA relative to HFD

FIGURE 10

SEFA-1024 alters hepatic expression of ACCα, SREBP1, and NFκB. ACCα expression was decreased by HFD but not when treated with SEFA-1024 (a). SREBP1 expression was not affect by HFD but was increased by SEFA-1024 (b). Nuclear factor kappa B (NFκB) was not affected by HFD but increased by SEFA-1024. No significant differences were observed in phosphorylated AMPK, AMPK, phosphorylated TAK, Gαq11/14, and phosphorylated AKT in HFD or SEFA-1024-treated mice. * = p < 0.05, ** = p < 0.01 by one-way ANOVA relative to chow