A model of insulin resistance and nonalcoholic steatohepatitis in rats: role of peroxisome proliferator-activated receptor-alpha and n-3 polyunsaturated fatty acid treatment on liver injury

Abstract

Insulin resistance induces nonalcoholic fatty liver disease and nonalcoholic steatohepatitis (NASH). We used a high-fat, high-calorie solid diet (HFD) to create a model of insulin resistance and NASH in nongenetically modified rats and to study the relationship between visceral adipose tissue and liver. Obesity and insulin resistance occurred in HFD rats, accompanied by a progressive increase in visceral adipose tissue tumor necrosis factor (TNF)-alpha mRNA and in circulating free fatty acids. HFD also decreased adiponectin mRNA and peroxisome proliferator-activated receptor (PPAR)-alpha expression in the visceral adipose tissue and the liver, respectively, and induced hepatic insulin resistance through TNF-alpha-mediated c-Jun N-terminal kinase (JNK)-dependent insulin receptor substrate-1Ser307 phosphorylation. These modifications lead to hepatic steatosis accompanied by oxidative stress phenomena, necroinflammation, and hepatocyte apoptosis at 4 weeks and by pericentral fibrosis at 6 months. Supplementation of n-3 polyunsaturated fatty acid, a PPARalpha ligand, to HFD-treated animals restored hepatic adiponectin and PPARalpha expression, reduced TNF-alpha hepatic levels, and ameliorated fatty liver and the degree of liver injury. Thus, our model mimics the most common features of NASH in humans and provides an ideal tool to study the role of individual pathogenetic events (as for PPARalpha down-regulation) and to define any future experimental therapy, such as n-3 polyunsaturated fatty acid, which ameliorated the degree of liver injury.

Figures

Figure 1

Effect of HFD on the daily calories consumed (A) and on rat body weight (B) and epididymal fat weight (C). White bars, control animals; black bars, HFD-treated animals. Open circles, control animals; black circles, HFD-treated animals. Data are presented as mean ± SD. *P < 0.05 versus controls.

Figure 2

Effect of HFD on TNF-α and adiponectin mRNA levels (A and B), levels of TNF-α and adiponectin in serum blood (C), and levels of FFA in serum blood (D). No differences were observed between controls at the different time points, which were thus considered as a single control group. A representative agarose gel of RT-PCR for TNF-α and adiponectin mRNA is shown in A. Gray lines, TNF-α; black lines, adiponectin; white bars, FFA. Data are presented as mean ± SD. *P < 0.05 versus controls.

Figure 3

Effect of HFD on PPARα expression in whole-liver homogenates. No differences were observed between controls at the different time points, which were thus considered as a single control group. A representative Western blot is shown. White bar, controls; black bars, HFD-treated animals. Data are presented as mean ± SD. *P < 0.05 versus controls.

Figure 4

H&E staining in control (A) and HFD-treated (B) rat for 3 months. The effect of HFD on triglyceride and cholesterol content in the liver (C and D) and on TBARS and hydroperoxide production (E and F). No alterations were observed in the liver of the rats fed the control diet (A), whereas HFD induced pronounced hepatic steatosis and inflammatory cells infiltrate surrounding steatotic hepatocytes (lipogranuloma). White bars, controls; black bars, HFD-treated animals. Data are presented as mean ± SD. *P < 0.05 versus controls. Final magnification, ×100.

Figure 5

Effect of HFD on phosphorylation of ERK (A), Akt (B), JNK (C), and IRS-1Ser307 (D). On the left, representative Western blots are shown. On the right, the densitometric analysis of phosphorylation level is shown. No differences were observed between controls at the different time points, which were thus considered as a single control group. Data are presented as mean ± SD. *P < 0.05 versus controls.

Figure 6

Effect of TNF-α (30 ng/ml) and insulin (100 nmol/L) on JNK phosphorylation. Hepatocytes were isolated and cultured as described in Materials and Methods and then incubated with TNF-α (30 ng/ml) or insulin (100 nmol/L) for the indicated period of time. Cell lysates (50 μg/lane) were separated by electrophoresis, transferred to nitrocellulose, and then incubated with the specific antibody. β-Actin was used to show equal loading.

Figure 7

Effect of HFD on hepatocyte apoptosis (A), TNF-α protein content in the liver (B), TNF-α mRNA levels in the liver (C and D), and collagen deposition (E and F). A representative agarose gel of RT-PCR for TNF-α mRNA is shown in C. Representative Sirius Red staining is shown in E. No differences were observed between controls at the different time points (A, B, and D), which were thus considered as a single control group. Data are presented as mean ± SD. *P < 0.05 versus controls.

Figure 8

Effect of n-3 PUFA administration on TNF-α and adiponectin mRNA in visceral adipose tissue (A and B), TNF-α and adiponectin levels in portal blood (C), and PPARα expression in nuclear liver extracts (D). Rats were treated for 1 month with HFD and then divided into HFD or HFD/n-3 PUFA group for an additional 2 months. Gray lines, TNF-α; black lines, adiponectin. Data are presented as mean ± SD. *P < 0.05 versus controls.

Figure 9

Effect of n-3 PUFA administration on hepatic deposition of triglyceride (A) and cholesterol (B) and on the histological appearance of liver injury induced by HFD (C and D). Rats were treated for 1 month with HFD and then divided into HFD or HFD/n-3 PUFA group for an additional 2 months. Compared with the histological injury observed with HFD (C), the HFD/n-3 PUFA diet (D) elicited a striking decrease in fat accumulation and inflammatory infiltrate. Data are presented as mean ± SD. *P < 0.05 versus controls. §P < 0.05 versus HFD.

Figure 10

Effect of n-3 PUFA administration on hepatic TNF-α mRNA levels (A and B), necroinflammatory and steatosis score (C), and number of apoptotic bodies (D). Rats were treated for 1 month with HFD and then divided into HFD or HFD/n-3 PUFA group for an additional 2 months. In C, black bars represent steatosis and white bars represent necroinflammation. Data are presented as mean ± SD. *P < 0.05 versus controls. §P < 0.05 versus HFD.