Lipoic Acid Prevents High-Fat Diet-Induced Hepatic Steatosis in Goto Kakizaki Rats by Reducing Oxidative Stress Through Nrf2 Activation

Abstract

Prevention of hepatic fat accumulation may be an important approach for liver diseases due to the increased relevance of hepatic steatosis in this field. This study was conducted to investigate the effects of the antioxidant α-lipoic acid (α-LA) on hepatic steatosis, hepatocellular function, and oxidative stress in a model of type 2 diabetes fed with a high fat diet (HFD). Goto-Kakizaki rats were randomly divided into four groups. The first group received only a standard rat diet (control GK) including groups 2 (HFD), 3 (vehicle group), and 4 (α-LA group), which were given HFD, ad libitum during three months. Wistar rats are the non-diabetic control group. Carbohydrate and lipid metabolism, liver function, plasma and liver tissue malondialdehyde (MDA), liver GSH, tumor necrosis factor-α (TNF-α) and nuclear factor E2 (erythroid-derived 2)-related factor-2 (Nrf2) levels were assessed in the different groups. Liver function was assessed using quantitative hepatobiliary scintigraphy, serum aspartate, and alanine aminotransferases (AST, ALT), alkaline phosphatase, gamma-glutamyltranspeptidase, and bilirubin levels. Histopathologically steatosis and fibrosis were evaluated. Type 2 diabetic animals fed with HFD showed a marked hepatic steatosis and a diminished hepatic extraction fraction and both were fully prevented with α-LA. Plasma and liver tissue MDA and hepatic TNF-α levels were significantly higher in the HFD group when compared with the control group and significantly lower in the α-LA group. Systemic and hepatic cholesterol, triglycerides, and serum uric acid levels were higher in hyperlipidemic GK rats and fully prevented with α-LA. In addition, nuclear Nrf2 activity was significantly diminished in GK rats and significantly augmented after α-LA treatment. In conclusion, α-LA strikingly ameliorates steatosis in this animal model of diabetes fed with HFD by decrementing the inflammatory marker TNF-α and reducing oxidative stress. α-LA might be considered a useful therapeutic tool to prevent hepatic steatosis by incrementing antioxidant defense systems through Nrf2 and consequently decreasing oxidative stress and inflammation in type 2 diabetes.

Keywords: Nrf2 levels; TNF-α; diabetes; oxidative stress; steatosis; α-lipoic acid.

Figure 1

Effects of α-lipoic acid (α-LA) on liver steatosis. (A–D) H&E stained liver sections. Long-term histological outcome of Goto-kakizaki (GK) rats fed a high fat diet (HFD) with or without vehicle (soybean oil, SO) or α-LA (D) treatment compared with GK control (A, ×100). Normal liver architecture in the GK control group (A, ×100). After three months of HFD (B, ×200) a diffuse micro-vesicular steatosis was observed in the liver (hematoxylin-eosin, ×200) that persists in the vehicle treated group (SO, C, ×200). Note the prevention in steatosis in the α-LA treated group (D, ×200). (E–I) Oil Red O staining of Wistar, GK rats fed with normal or HFD with or without SO or α-LA treatment. Wistar group (E, ×200); GK control group (F, GK, ×200). After 3 months of HFD (G, ×200), an increased lipid accumulation was observed in the liver that persists in the vehicle treated group (H, SO, ×200). Note the decrement in lipid deposition (similar do the control GK group) in the α-LA treated group (I, LA, ×200).

Figure 2

Effects of α-lipoic acid (α-LA) on hepatic cholesterol (A) and triglycerides (B) levels in Wistar, Goto-kakizaki (GK) rats control and GK fed a high fat diet (HFD) with or without vehicle (soybean oil, SO) or α-LA. Data are expressed as mean ± SEM (n = 7 animals per group). *** p < 0.001 vs. Wistar group, §§ p < 0.01, §§§ p < 0.001 vs. GK group, φφφ p < 0.001 vs. GKHFD group, # p < 0.05 vs. GK SO group.

Figure 3

Effects of α-lipoic acid (α-LA) on lipid peroxidation (MDA), glutathione (GSH), and Nrf2 levels in the liver of Goto-kakizaki (GK) rats fed a high fat diet (HFD) compared with nondiabetic Wistar rats. Diabetes and HFD-induced impairment in the nuclear accumulation of Nrf2 and an increment in lipid peroxidation with concomitant reduction in GSH levels and α-LA prevented these effects. (A) Liver malondialdehyde (MDA) levels, (B) liver GSH levels, (C) the nuclear extracts depicted on panel D were also used for the measurement of Nrf2 DNA-binding activity using a TransAm Binding Assay (Active Motif), (D) representative Western blot analysis and average densitometry data (normalized with lamin values) of nuclear Nrf2 protein expression in livers of Wistar, Goto-kakizaki (GK) rats control and GK fed with high fat diet (HFD) with or without vehicle (soybean oil, SO) or α-LA. Data are expressed as mean ± SEM (n = 7 animals per group). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Wistar group, § p < 0.05, §§ p < 0.01, §§§ p < 0.001 vs. GK group; φ p < 0.05, φφ p < 0.01, φφφ p < 0.001 vs. GKHFD group, # p < 0.05, ### p < 0.001 vs. GK SO group.

Figure 4

Effects of α-lipoic acid (α-LA) on tumor necrosis factor-α (TNF-α) levels in the liver of Goto-kakizaki (GK) rats fed a high fat diet (HFD) compared with nondiabetic Wistar rats. (A) representative Western blot analysis of TNF-α levels. (B) Average densitometry data (normalized with β-actin values) expressed as a percentage. (C) Protein TNF-α levels in livers of Wistar, Goto-kakizaki (GK) rats control and GK fed a high fat diet (HFD) with or without vehicle (soybean oil, SO) or α-LA. Data are expressed as mean ± SEM (n = 7 animals per group). * p < 0.05, *** p < 0.001 vs. Wistar group, § p < 0.05, §§§ p < 0.001 vs. GK group, φφ p < 0.01, φφφ p < 0.001 vs. GKHFD group.