Genetic Differences in Oxidative Stress and Inflammatory Responses to Diet-Induced Obesity Do Not Alter Liver Fibrosis in Mice

Nonalcoholic fatty liver disease (NAFLD) is now the leading cause of chronic liver disease in the developed world and a major indication for liver transplantation (1, 2). However, the outcome of NAFLD is highly variable; only a quarter of individuals will develop progressive disease that is characterized by hepatic necroinflammation, fibrosis and cirrhosis. Elucidating the mechanisms involved in disease progression is, therefore, essential to our understanding of NAFLD and ability to identify those at risk of developing end-stage liver disease. Nonalcoholic fatty liver disease is strongly associated with obesity. The vastmajority of obese individuals with NAFLD will have ‘simple’ hepatic steatosis, which is a stable and benign condition for decades (2). In others, however, hepatic necroinflammation evolves, cumulative liver damage ensues and cirrhosis ultimately results (3, 4). Inter-individual differences in susceptibility to hepatic steatosis, as well as subsequent progression to steatohepatitis and cirrhosis, are thought to reflect variability in exposures and responses to sequential insults (dubbed ‘hits’) (5). Diverse factors that challenge lipid homeostatic mechanisms are the initial ‘hits’ that provoke the onset of hepatic triglyceride accumulation (steatosis) and enhance hepatocyte vulnerability to injury. Progression to steatohepatitis is believed to result from superimposition of secondary ‘hits’, such as metabolic, oxidant and/or inflammatory stresses, that overw-helm cell survival mechanisms and provoke hepatocyte injury and death (6). More recently, our group has proposed that the death of mature hepatocytes provides a ‘third’ hit that enhances the risk of cirrhosis and hepatocellular carcinoma by inducing liver repair mechanisms (7). Genetic factors are likely to modulate fatty liver disease pathogenesis by influencing both exposure and responses to the insults that drive liver disease initiation and progression. This concept is supported by studies of leptin-resistant fa/fa rats and leptin-deficient ob/ob mice. Fa/fa rats are much more susceptible to hepatic steatosis than wild-type rats when fed calorically dense diets enriched with fructose or sucrose (8). Leptindeficient, ob/ob mice are relatively protected from liver fibrosis, despite developing severe steatosis (9). The ability to understand mechanisms that control NAFLD progression in humans would be greatly facilitated by the acquisition of small animal models that develop hepatic steatosis, and subsequent steatohepatitis and cirrhosis when exposed to typical risk factors for human NAFLD (e.g. high-calorie diets). However, reproducible models of progressive NAFLD have been difficult to establish in rodents with diet-induced obesity (10, 11). The reasons for failure remain uncertain. One possibility is that the duration of risk factor exposure was simply insufficient. Typically, rodents have been overfed for 8 weeks or less, a period corresponding to <10% of their life span. Most humans with NAFLD-related cirrhosis, on the other hand, are middle aged or older (4, 12) and have been overeating since at least adolescence, exposing themselves to NAFLD risk factors for a quarter of their lifespan or more. Another possibility is that commonly studied strains of rodents, such as C57BL6 mice, lack genetic factors that convey susceptibility to progressive liver damage. This reasoning suggests that it may be feasible to develop a mouse model of progressive NAFLD by selecting a strain of mice that is naturally predisposed to one or more of the secondary ‘hits’ that trigger progression from steatosis to steatohepatitis (e.g. oxidant/inflammatory stress) and overfeeding them for a quarter of their life span to approximate the duration of risk factor in humans who develop progressive NAFLD. To evaluate this hypothesis, we fed adolescent C57BL6 mice and 129/SVJ mice high-fat (HF) diets for 6 months and compared NAFLD stage at the end of the treatment period in the two groups. C57BL6 mice generally develop steatosis and insulin resistance but little, if any, liver fibrosis when subjected to short periods of diet-induced obesity (13), and hence, were predicted to be relatively resistant to advanced NAFLD. 129/SVJ mice are frequently used as a source of embryonic stem cells (14). The 129/SVJ, specifically, has a mixed genetic background and thus contributes genetic factors that modulate the phenotypes of many transgenes (15). The 129/SVJ strain has been shown to be significantly more sensitive to heavy-metal-induced testicular toxicity and immune-mediated nephritis than the C57BL6 strain (16). These observations suggest that 129/SVJ mice may be predisposed to tissue damage elicited by oxidant and/or inflammatory stress, factors that are believed to promote progressive NAFLD (17, 18). Our results demonstrate that although steatosis, enhanced hepatic oxidant stress, steatohepatitis, T-helper type 2 (Th2) polarization of hepatic cytokine production and overactivation of fibrogenic pathways occurred in 129/SVJ mice during prolonged exposure to HF diets, these events were not sufficient to induce bridging fibrosis/cirrhosis. Similarities in the ultimate outcomes of HF-diet-induced liver damage in C57BL6 mice and 129/SVJ mice despite striking strain-related differences in oxidant stress and relative production of pro-(Th1) and anti-(Th2) inflammatory cytokines suggest the importance of other injury/repair mechanisms for progression of this disease.