Gastroenterology

Gastroenterology

Volume 132, Issue 1, January 2007, Pages 282-293
Gastroenterology

Basic–liver, pancreas, and biliary tract
Insulin Resistance Accelerates a Dietary Rat Model of Nonalcoholic Steatohepatitis

https://doi.org/10.1053/j.gastro.2006.10.014Get rights and content

Background & Aims: The increasing prevalence of nonalcoholic steatohepatitis (NASH) is due to the epidemic of obesity and type 2 diabetes, both of which are associated with insulin resistance. Methods: To clarify the causal relationship between insulin resistance and the development of NASH, steatohepatitis was induced in obese diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) and nondiabetic control Long-Evans Tokushima Otsuka (LETO) rats by feeding them a methionine and choline-deficient (MCD) diet. Insulin sensitivity of the rats was altered by adding a high-fat (HF) diet or the peroxisomal-proliferator activated receptor-γ agonist pioglitazone to the MCD diet. Results: The MCD diet-induced steatohepatitis was accelerated in OLETF rats after 8 weeks. Steatosis preceded inflammation, which led to fibrosis and the development of steatohepatitis. The hepatic gene expression for transforming growth factor-β, α1 procollagen and plasminogen activator inhibitor-1 was up-regulated in OLETF rats compared with LETO rats. The MCD + HF diet further enhanced insulin resistance and led to rapid development of pre-cirrhosis in OLETF rats by increasing the triglyceride pool, activating stellate cells, and up-regulating gene expression for sterol regulatory element-binding protein-1c and fatty acid synthase in the liver. In contrast, pioglitazone attenuated the MCD diet-induced steatohepatitis in OLETF rats but not in LETO rats by reversing the underlying pathogenesis involved in this model through improvement of insulin resistance. These results confirm a link between insulin resistance and the development/progression of steatohepatitis, at least partly via up-regulation of genes for lipogenesis, inflammation, and fibrogenesis, in animal models. Conclusions: Insulin resistance and/or diabetes may accelerate the entire pathologic spectrum of NASH.

Section snippets

Animal Model and Experimental Design

Male Otsuka Long-Evans Tokushima Fatty (OLETF) rats, which have been established as an animal model of obese type 2 diabetes20, 21 were used as the experimental subjects (Otsuka Pharmaceutical, Tokushima, Japan). Male Long-Evans Tokushima Otsuka (LETO) rats, which originated from the same colony as OLETF rats by selective mating but did not develop diabetes, were used as control animals (Otsuka, Tokushima, Japan). Four-week-old OLETF and LETO rats were obtained and housed in a room under

Metabolic Parameters

Spontaneous-onset type 2 diabetes OLETF rats manifesting obesity and hyperinsulinemia from 8 weeks of age were compared with nondiabetic control LETO rats. NAFLD has been studied in OLETF rats because these animals show age-dependent increases of fat accumulation in the liver.27, 28 All rats in the OLETF group developed diabetes after 24 weeks of age (data not shown) as described previously.20, 21 Compared with LETO rats, OLETF rats fed a control diet manifested obesity (623 ± 11.4 g vs 504 ±

Progressive Insulin Resistance in Rats Fed MCD + HF Diet and Improved Insulin Sensitivity in Rats Fed MCD + PGZ Diet

Plasma insulin levels were similar between rats fed the MCD, MCD + HF, and MCD + PGZ diets (Table 1). Glucose tolerance tests (GTT) and ITT were conducted after 8 weeks in each group to evaluate insulin sensitivity (Figure 1). In the MCD diet group, blood glucose levels at 30 and 60 minutes increased significantly in OLETF rats compared with the LETO rats (P < .05) indicating that the obese diabetes background of OLETF rats induced postglucose hyperglycemia and insulin resistance (Figure 1A).

Insulin Resistance and Obese Diabetes Accelerate Pathology of Dietary Model of NASH

The MCD diet caused marked macrovesicular steatosis with focal lymphocytic infiltration and hepatocellular dropouts in LETO rats, whereas it caused intense lobular inflammation and perivenular and pericellular fibrosis prominently in zone 3 of the livers of OLETF rats at 8 weeks (Figure 2AC). The MCD diet-induced steatosis and inflammation were more severe in OLETF rats than LETO rats at 4 weeks (P < .05, Figure 2C). The MCD+HF diet accelerated the severities of steatosis, inflammation, and

Insulin Resistance and Obese Diabetes Accelerate Liver Injury Along With Fat Accumulation in the Liver

Rats fed the MCD or MCD + HF diet showed macrovesicular steatosis in the liver (Figure 2A). Therefore, the hepatic triglyceride content was measured to evaluate the progression of steatosis quantitatively after 8 weeks (Figure 4A). The MCD and MCD + HF diet significantly increased hepatic triglyceride content in both LETO and OLETF rats (P < .05 vs the control diet). Hepatic triglyceride levels were more pronounced in OLETF than LETO rats (Figure 4A), although the degree of steatosis (%

Insulin Resistance and Obese Diabetes Activate Stellate Cells More Extensively in the Dietary Model of NASH

To investigate the activation of hepatic stellate cells, which play a central role in liver fibrosis, we performed an immunohistochemical analysis of α-SMA after 8 weeks. Representative photomicrographs of liver sections stained with anti-α-SMA antibody are shown in Figure 5. Activated stellate cells, which express α-SMA and therefore are also called myofibroblast-like cells, showed a prominent proliferation in the liver of rats fed the MCD diet. The activated stellate cells in MCD-OLETF rats

Insulin Resistance and Obese Diabetes Up-Regulate mRNA Expression of Fibrogenic and Lipogenic Genes

To clarify the mechanism by which the MCD diet caused hepatic fibrosis in rats, the expression of TGF-β, a master regulator of fibrogenesis, and its target genes collagen I and PAI-1 in the liver were estimated by quantitative real-time PCR (Figure 6AC). The MCD diet up-regulated mRNA expression for TGF-β, collagen I, and PAI-1 in the livers of LETO rats (12.0-, 6.5-, and 3.9-fold, respectively, compared with the control diet, all P < .01) and further up-regulated expression in OLETF rats

Discussion

In this study, we focused on the pathophysiologic links between insulin resistance and the development of NASH. As reported previously, mice fed the MCD diet develop a malnutrition model of steatohepatitis without whole-body insulin resistance,30 probably due to decreased weight and adiposity in this model. Nevertheless, these mice demonstrate hepatic insulin resistance as evidenced by reduced tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and IRS-2 and reduced phosphorylation

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