Diet associated hepatic steatosis sensitizes to Fas mediated liver injury in mice☆
Introduction
Obesity, an increasingly public health problem, is the main risk factor for development of non-alcoholic liver disease (NAFLD) which has recently became the most common liver disease in developed countries [1]. NAFLD represents a spectrum of disorders ranging from simple uncomplicated accumulation of fat in the liver (steatosis), to steatosis with inflammation referred as non-alcoholic steatohepatitis (NASH) which may progress to cirrhosis [2]. Though the pathogenesis of NAFLD remains poorly understood, the current most accepted theory is the ‘two-hits’ hypothesis. The first hit is the development of hepatic steatosis which renders the liver more susceptible to a second not yet defined factor which then triggers an inflammatory response and progressive liver damage [3]. The nature of the ‘second hit’ remains obscure but is of considerable biomedical interest as identification of this process may help identify potential therapeutic targets.
The cardinal pathologic features of NASH include macrosteatosis, mallory bodies, inflammatory cells including neutrophils, and hepatocyte ballooning degeneration [4]. These findings are also present in patients with alcoholic liver disease, and, therefore, the distinction between the two entities based solely on pathologic observations can be difficult. Somewhat unique in chronic liver diseases is the present of neutrophils in both diseases. Thus, the ‘second hit’ in this disease must not only induce damage but also promote inflammation with neutrophilic infiltration.
Hepatocyte apoptosis, a key mechanism contributing to the progression of human liver diseases, was recently identified as a prominent histopathologic feature of NASH and correlated with disease severity [5]. Moreover, increased Fas (CD95) expression, a surface glycoprotein belonging to the tumor necrosis factor receptor family and a specific mediator of apoptosis [6], was also observed in liver specimens from patients with NASH [5]. These data are, however, observational and correlative and cannot relate Fas expression to liver injury in this disease syndrome. Nonetheless, enhanced sensitivity to Fas-mediated apoptosis is an attractive mechanism explaining the progression of steatosis to NASH. Not only would sensitivity to Fas explain the increased apoptotic rate in NASH, but Fas signaling has also been shown to be proinflammatory and promote fibrogenesis [7], [8].
The overall objective of this study was to examine Fas expression and sensitivity to Fas-mediated apoptosis in both an in vivo murine model and an in vitro cell model of hepatocyte steatosis. The data indicate that Fas expression is increased in steatotic hepatocytes. Furthermore, Fas engagement both in vivo and in vitro results in enhanced hepatocyte cytotoxicity. Our data identify the Fas pathway as a potential key link between obesity associated fatty liver and increase susceptibility to liver damage.
Section snippets
Animal studies
These experimental protocols were approved by the Institutional Animal Care and Use Committee at the Mayo Clinic. Male C57/BL mice (Jackson laboratories, Bar Harbor, ME), 20–25 g of body weight, were fed for 8 weeks a high carbohydrate diet ad libidum (consisting of 65% sucrose, 20% casein, 5% corn oil, 4% mineral mixture and 1% vitamin mixture; diet TD 02366, Teklad Mills, Madison, WI) starting at 6–8 weeks of age (n=15). Identical groups of animals (n=15) received standard rodent chow to act
Mice fed a high-carbohydrate diet develop NAFLD
We first generated an animal model, which closely resembles human NAFLD. Mice fed a high carbohydrate diet for 8 weeks developed marked obesity compared to mice kept on a standard rodent diet (32.3±0.6 vs. 25.1±1.6 g body weight, P<0.01; Fig. 1A). Histologic examination of liver tissue from mice on the high-carbohydrate diet (obese mice) showed significant diffuse macro-vesicular steatosis compared to the normal histologic appearance of the liver from mice fed a standard rodent diet (lean mice)
Discussion
Many studies on liver steatosis have employed genetic models deficient in leptin or leptin signaling (e.g. ob/ob mice and fa/fa rats) [19]. However, a large body of evidence implicates leptin in the pathophysiology of NAFLD [20]. In nearly all cases, NAFLD in humans is associated with both hyperinsulinemia and hyperleptinemia [1], [3]. Though the mechanisms leading to accumulation of fat in the liver are not well understood, resistance to the antisteatotic action of leptin and insulin appears
Acknowledgements
The secretarial services of Erin Bungum are gratefully acknowledged.
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This work was supported by NIH grant (DK41876) to GJG, and the Mayo Foundation.