Exenatide ameliorates hepatic steatosis and attenuates fat mass and FTO gene expression through PI3K signaling pathway in nonalcoholic fatty liver disease

Abstract

Non-alcoholic fatty liver disease (NAFLD) is a common disease associated with metabolic syndrome and can lead to life-threatening complications like hepatic carcinoma and cirrhosis. Exenatide, a glucagon-like peptide-1 (GLP-1) receptor agonist antidiabetic drug, has the capacity to overcome insulin resistance and attenuate hepatic steatosis but the specific underlying mechanism is unclear. This study was designed to investigate the underlying molecular mechanisms of exenatide therapy on NAFLD. We used in vivo and in vitro techniques to investigate the protective effects of exenatide on fatty liver via fat mass and obesity associated gene (FTO) in a high-fat (HF) diet-induced NAFLD animal model and related cell culture model. Exenatide significantly decreased body weight, serum glucose, insulin, insulin resistance, serum free fatty acid, triglyceride, total cholesterol, low-density lipoprotein, aspartate aminotransferase, and alanine aminotransferase levels in HF-induced obese rabbits. Histological analysis showed that exenatide significantly reversed HF-induced lipid accumulation and inflammatory changes accompanied by decreased FTO mRNA and protein expression, which were abrogated by PI3K inhibitor LY294002. This study indicated that pharmacological interventions with GLP-1 may represent a promising therapeutic strategy for NAFLD.

Figure 1. Changes in body weight, food intake, and liver index in exenatide (Ex)-treated high fat (HF)-induced obese rabbits (n=8). A, Body weight during HF diet feeding detected every 2 weeks. B, Average food intake during HF diet feeding. C, Average energy intake during the experimental period. D, Upon sacrifice at the end of 20 weeks, the liver index, which is equal to the liver weight/body weight x 100, was calculated. Data are reported as means±SD. *P<0.05, **P<0.01 compared with the control group; ^#P<0.05 compared with the HF group (ANOVA).

Figure 2. Effects of exenatide (Ex) on biochemical parameters of non-alcoholic fatty liver disease rabbits (n=8). At the end of the 8-week treatment, blood samples were collected for analysis of glucose (A), insulin (B), homeostasis model of assessment of insulin resistance (HOMA-IR) (C), free fatty acid (FFA) (D), total cholesterol (TC) (E), triglyceride (TG) (F), alanine aminotransferase (ALT) (G), aspartate aminotransferase (AST) (H), and low-density lipoprotein (LDL) (I) levels after overnight starvation. Data are reported as means±SD. *P<0.05, **P<0.01 compared with the control group; ^#P<0.05, ^{# #}P<0.01 compared with the high fat (HF) group (ANOVA).

Figure 3. Histopathology of each group. A, Liver specimens from rabbits in each group are shown. B, Liver sections with hematoxylin and eosin (H&E) staining. Arrows indicate lipid droplets. The original magnification was ×200. Scale bars=500 μm. C, Non-alcoholic fatty liver disease activity scores were evaluated semi-quantitatively: steatosis (0–3), lobular inflammation (0–2), and hepatocellular ballooning (0–2). N.D., not detected. D, Hepatic triglycerides (TG) were extracted from frozen tissue and measured by enzymatic assays. Values of TG were normalized relative to protein concentration. Data are reported as means±SD (n=8). **P<0.01 compared with the control group; ^{# #}P<0.01 compared with the high fat (HF) group (ANOVA).

Figure 4. Effects of exenatide (Ex) on high fat (HF)-induced liver oxidative stress (n=8). After the 20-week study period, the contents of A, superoxide dismutase (SOD) and B, malondialdehyde (MDA) in livers from rabbits in each group were estimated. Data are reported as means±SD. *P<0.05, **P<0.01 compared with the control group; ^{# #}P<0.01 compared with the HF group (ANOVA).

Figure 5. Effect of exenatide (Ex) on high fat (HF)-induced mass and obesity associated gene (FTO) expression in rabbit liver tissue (n=8). A and B, FTO protein expression; C and D, pAKT and AKT protein expression. Data were normalized relative to the GAPDH level for each sample and are reported as means±SD. **P<0.01 compared with the control group; ^{# #}P<0.01 compared with the HF group (one-way ANOVA and paired t-test).

Figure 6. LY294002 attenuated effects of exenatide (Ex) on palmitic acid-induced lipid accumulation, liver enzyme, and triglyceride (TG) levels in L02 cells. A, Lipid accumulation in L02 cells was observed by Oil Red O staining. L02 cells stained with Oil Red O were examined by light microscopy (magnification ×400). Scale bars=500 μm. B–F, Serum contents of TG, alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and alkaline phosphatase (ALP) were detected. Data are reported as means±SD (n=6). **P<0.01 compared with the control group; ^{# #}P<0.01 compared with the high fat (HF) group; ⁺P<0.05, ⁺⁺P<0.01 compared with the HF+Ex group (paired t-test).

Figure 7. PI3K inhibitor LY294002 partially reversed exenatide (Ex)-dependent inhibition of palmitic acid-induced mass and obesity associated (FTO) gene expression in L02 cells. Total RNA and protein were extracted from L02 cells treated in each group and used to assess A, FTO mRNA expression and B, FTO protein expression by qRT-PCR and western blot, respectively. Data were normalized relative to the GAPDH level for each sample and are reported as means±SD (n=6). **P<0.01 compared with the control group; ^{# #}P<0.01 compared with the high fat (HF) group; ⁺⁺P<0.01 compared with the HF+Ex group (paired t-test).