Elsevier

Journal of Hepatology

Volume 39, Issue 6, December 2003, Pages 978-983
Journal of Hepatology

Diet associated hepatic steatosis sensitizes to Fas mediated liver injury in mice

https://doi.org/10.1016/S0168-8278(03)00460-4Get rights and content

Abstract

Background/Aims: Hepatic steatosis sensitizes the liver to injury and inflammation by unclear mechanisms. Because Fas has been linked to liver injury and inflammation, Fas expression and sensitization to Fas signaling was examined in models of hepatic steatosis.

Methods: Mice were fed a carbohydrate diet while control animals received standard chow. Sensitization to Fas was examined following administration of Jo2 antibody. For the in vitro experiments, HepG2 cells were incubated with or without a mixture of long chain fatty acids (2:1 oleate:palmitate). Sensitization of the cells to Fas was examined using the CH11 antibody.

Results: Mice fed a high caloric diet developed hepatic steatosis, hyperlipidemia, insulin resistance, and hyperleptinemia, all features of the human syndrome. Fas expression in hepatocytes was increased as compared to lean animals and was coupled to cytotoxic signaling. Indeed, hepatocyte apoptosis, liver injury and chemokine generation were all accentuated in obese animals following administration of Jo-2, a Fas agonist. Hep G2 cells cultured in the presence of free fatty acids also developed ‘cellular steatosis’, upregulated Fas expression and were more sensitive to apoptosis by a Fas agonist.

Conclusions: Collectively, these data implicate Fas as a link between obesity associated fatty liver and increased susceptibility to liver damage.

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.

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