ton. Obese and diabetic db/db mice develop marked liver fibrosis in a model of nonalcoholic steatohepatitis: role of short-form leptin receptors and osteopontin. Am J Physiol Gastrointest Liver Physiol 287: G1035-G1043, 2004. First published July 15, 2004 doi:10.1152/ ajpgi.00199.2004.-Obesity and type 2 diabetes are associated with nonalcoholic steatohepatitis (NASH), but an obese/diabetic animal model that mimics human NASH remains undefined. We examined the induction of steatohepatitis and liver fibrosis in obese and type 2 diabetic db/db mice in a nutritional model of NASH and determined the relationship of the expressions of osteopontin (OPN) and leptin receptors to the pathogenesis of NASH. db/db mice and the corresponding lean and nondiabetic db/m mice were fed a diet deficient in methionine and choline (MCD diet) or control diet for 4 wk. Leptindeficient obese and diabetic ob/ob mice fed similar diets were used for comparison. MCD diet-fed db/db mice exhibited significantly greater histological inflammation and higher serum alanine aminotransferase levels than db/m and ob/ob mice. Trichrome staining showed marked pericellular fibrosis in MCD diet-fed db/db mice but no significant fibrosis in db/m or ob/ob mice. Collagen I mRNA expression was increased 10-fold in db/db mice, 4-fold in db/m mice, and was unchanged in ob/ob mice. mRNA expressions of OPN, TNF-␣, TGF-␤, and short-form leptin receptors (Ob-Ra) were significantly increased in db/db mice compared with db/m or ob/ob mice. Parallel increases in OPN and Ob-Ra protein levels were observed in db/db mice. Cultured hepatocytes expressed only Ob-Ra, and leptin stimulated OPN mRNA and protein expression in these cells. In conclusion, our results demonstrate the development of an obese/diabetic experimental model for NASH in db/db mice and suggest an important role for Ob-Ra and OPN in the pathogenesis of NASH. obesity; diabetes; insulin resistance; osteopontin; fibrosis NONALCOHOLIC STEATOHEPATITIS (NASH) is commonly associated with obesity, type 2 diabetes, and the metabolic syndrome (1,41,45,48). However, several studies examining the pathogenesis of NASH have employed lean and nondiabetic strains of mice fed a diet deficient in methionine and choline (MCD diet). We and others have demonstrated that this model produces steatohepatitis and liver fibrosis that is histologically similar to human NASH (11,13,23,25,26,46). In addition, mice lacking methionine adenosyltransferase have been shown to develop steatohepatitis similar to human NASH (31,34,47). The molecular signaling mechanisms that lead to the activation of inflammation and fibrosis in these models of NASH remain poorly defined. Several studies suggest that peroxidative injury may play a role in the development of steatohepatitis in human and in experimental NASH (11,26,47,48). However, we (46) recently assessed oxidative stress during the progression of steatosis to steatohepatitis in MCD diet-fed A/J mice and found that peroxidative injury occurs late in developing steatohepatitis. We have id...

Evidence suggests that most neonatal hemochromatosis (NH) is the phenotypic expression of gestational alloimmune fetal liver injury. Gestational alloimmune diseases are induced by the placental passage of specific reactive immunoglobulin G and often involve the activation of fetal complement by the classical pathway leading to the formation of membrane attack complex (MAC) as the effector of cell injury. We examined liver specimens from cases of NH, from cases of non-NH liver disease, and from infants without liver disease to determine if they would provide evidence that MAC is involved in hepatocyte injury. Sections were immunostained with anti-human C5b-9 complex, the terminal complement cascade (TCC) neoantigen formed in the assembly of MAC. Fetal liver injury in NH cases is associated with a severe loss of hepatocytes. In all NH cases examined, most remaining hepatocytes showed intense staining for TCC neoantigen, whereas hepatocytes in non-NH liver disease cases showed variable light staining. The percentage of hepatocytes containing TCC neoantigen in NH was much greater than that in non-NH liver disease, and there was no overlap between the groups. Findings in both groups suggest that hepatocytes have mechanisms to protect against MAC, including a biliary pathway for its excretion. Conclusion: The finding that all cases of proven NH contained TCC neoantigen far in excess of cases of other neonatal liver diseases suggests that a single process, namely congenital alloimmune hepatitis, is the principal cause of NH. MAC-mediated alloimmune injury in congenital alloimmune hepatitis is a novel mechanism of liver injury that results from an interplay of maternal adaptive immunity and fetal innate immunity.

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Abnormal dietary intake of macronutrients is implicated in the development of obesity and fatty liver disease. Steatosis develops in cultured hepatocytes exposed to medium containing either a high concentration of long chain free fatty acids (HFFA) or medium deficient in methionine and choline (MCD). This study examined the mitochondrial reactive oxygen species (ROS)-dependent regulation of the phosphoinositol (PI) 3-kinase pathway in steatosis induced by exposure of AML-12 mouse hepatocytes to MCD or HFFA medium. Exposure to either MCD or HFFA medium resulted in increased production of superoxide anions and H 2 O 2 , transduction of the PI 3-kinase pathway and steatosis. Inhibition of PI 3-kinase with LY294002 prevented steatosis. Pharmacologically inhibiting electron transport chain complex III production of ROS prevented activation of PI 3-kinase during macronutrient perturbation, whereas pharmacologically promoting electron transport chain complex III ROS production activated PI 3-kinase independent of nutrient input. The data suggest that H 2 O 2 is the ROS species involved in signal transduction; promoting the rapid conversion of superoxide to H 2 O 2 does not inhibit PI 3-kinase pathway activation during nutrient perturbation, and exogenous H 2 O 2 activates it independent of nutrient input. In addition to transducing PI 3-kinase, the ROS-dependent signal cascade amplifies the PI 3-kinase signal by maintaining phosphatase and tensin homolog in its inactive phosphorylated state. Knockdown of phosphatase and tensin homolog by small interfering RNA independently activated the PI 3-kinase pathway. Our findings suggest a common path for response to altered nutrition involving mitochondrial ROS-dependent PI 3-kinase pathway regulation, leading to steatosis.Innumerable epidemiologic studies demonstrate that inappropriate intake of macronutrients and a sedentary lifestyle conspire to produce rampant obesity in the United States. Hepatic steatosis (the accumulation of excess neutral fat within hepatocytes) in obese persons is thought to result from metabolic disturbances attendant to obesity. An estimated 30 million adults and 1.6 million children in the United States have nonalcoholic fatty liver disease (NAFLD), 2 of which an estimated 30% have nonalcoholic steatosis (NASH), which can lead to chronic debilitating liver injury, including cirrhosis (1). Hepatic steatosis is the defining feature of NAFLD (2). It is commonly held that steatosis is the precursor of events that lead to the transition of NAFLD to NASH, including the induction of oxidative stress and activation of inflammatory pathways (3, 4). The development of hepatic steatosis is therefore considered to be the necessary first effect of disturbed nutrition that induces the cascade of events leading to NASH. How disturbed nutrition causes hepatic steatosis is not well understood.Experimental NASH can be produced in laboratory rodents by dietary manipulation, including both restricted intake (i.e. methionine-choline deficient (MCD) diet) (5) and excess ...

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Gestational alloimmune liver disease has emerged as the major cause of antenatal liver injury and failure. It usually manifests as neonatal liver failure with hepatic and extrahepatic iron overload, a clinical presentation called neonatal hemochromatosis. We report on a newborn in whom fetal hepatomegaly was detected during pregnancy and who presented at birth with liver cirrhosis and mild liver dysfunction. Liver biopsy showed the absence of iron overload but strong immunostaining of hepatocytes for the C5b-9 complex, the terminal complement cascade neoantigen occurring specifically during complement activation by the immunoglobulin G-mediated classic pathway, which established the alloimmune nature of the hepatocyte injury. The infant survived with no specific therapy, and follow-up until 36 months showed progressive normalization of all liver parameters. This case report expands the recognized clinical spectrum of congenital alloimmune liver disease to include neonatal liver disease and cirrhosis, even in the absence of siderosis. Such a diagnosis is of utmost importance regarding the necessity for immunotherapy in further pregnancies to avoid recurrence of alloimmune injury.

Sahai, Atul, Xiaomin Pan, Rachelle Paul, Padmini Malladi, Rohit Kohli, and Peter F. Whitington. Roles of phosphatidylinositol 3-kinase and osteopontin in steatosis and aminotransferase release by hepatocytes treated with methionine-choline-deficient medium.