Obesity and the metabolic syndrome are closely correlated with hepatic steatosis. Simple hepatic steatosis in nonalcoholic fatty liver disease can progress to nonalcoholic steatohepatitis (NASH), which can be a precursor to more serious liver diseases, such as cirrhosis and hepatocellular carcinoma. The pathogenic mechanisms underlying progression of steatosis to NASH remain unclear; however, inflammation, proinflammatory cytokines, and oxidative stress have been postulated to play key roles. We previously reported that patients with NASH have elevated serum levels of proinflammatory cytokines, such as interleukin-8 (IL-8), which are likely to contribute to hepatic injury. This study specifically examines the effect of hepatic steatosis on IL-8 production. We induced lipid accumulation in hepatocytes (HepG2, rat primary hepatocytes, and human primary hepatocytes) by exposing them to pathophysiologically relevant concentrations of palmitic acid to simulate the excessive influx of fatty acids into hepatocytes. Significant fat accumulation was documented morphologically by Oil Red O staining in cells exposed to palmitic acid, and it was accompanied by an increase in intracellular triglyceride levels. Importantly S everal studies suggest that elevated levels of circulating free fatty acids (FFAs) contribute to the complications of obesity and the metabolic syndrome by promoting excess fat deposition in nonadipose tissues not suited for fat storage, such as the liver. 1 A strong correlation exists between obesity and fatty liver (hepatic steatosis); indeed, nonalcoholic fatty liver disease is considered to be the hepatic manifestation of the metabolic syndrome. 2 Liver steatosis is often considered a benign condition, but it can progress to nonalcoholic steatohepatitis (NASH), which may be a precursor to more severe liver diseases such as cirrhosis and hepatocellular carcinoma. 3 NASH is a common liver disease in the United States, and its prevalence is on the rise worldwide. NASH is characterized by microvesicular and macrovesicular steatosis, inflammation with mixed cellularity including neutrophils, hepatocyte degeneration and injury, and sometimes fibrosis. 3 The pathogenesis of NASH and the mechanisms of progression of hepatic steatosis to NASH remain unclear. 4 Current understanding supports the "multiple-hit model" for the development of NASH, wherein hepatic steatosis represents the "first hit," whereas the "second" or "subsequent" hits must induce liver damage and promote inflammation with neutrophil infiltration. The nature of the subsequent hits is hypothesized to include direct lipid toxicity, mitochondrial dys-

Nuclear receptors are transcription factors which sense changing environmental or hormonal signals and effect transcriptional changes to regulate core life functions including growth, development, and reproduction. To support this function, following ligand-activation by xenobiotics, members of subfamily 1 nuclear receptors (NR1s) may heterodimerize with the retinoid X receptor (RXR) to regulate transcription of genes involved in energy and xenobiotic metabolism and inflammation. Several of these receptors including the peroxisome proliferator-activated receptors (PPARs), the pregnane and xenobiotic receptor (PXR), the constitutive androstane receptor (CAR), the liver X receptor (LXR) and the farnesoid X receptor (FXR) are key regulators of the gut:liver:adipose axis and serve to coordinate metabolic responses across organ systems between the fed and fasting states. Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease and may progress to cirrhosis and even hepatocellular carcinoma. NAFLD is associated with inappropriate nuclear receptor function and perturbations along the gut:liver:adipose axis including obesity, increased intestinal permeability with systemic inflammation, abnormal hepatic lipid metabolism, and insulin resistance. Environmental chemicals may compound the problem by directly interacting with nuclear receptors leading to metabolic confusion and the inability to differentiate fed from fasting conditions. This review focuses on the impact of nuclear receptors in the pathogenesis and treatment of NAFLD. Clinical trials including PIVENS and FLINT demonstrate that nuclear receptor targeted therapies may lead to the paradoxical dissociation of steatosis, inflammation, fibrosis, insulin resistance, dyslipidemia and obesity. Novel strategies currently under development (including tissue-specific ligands and dual receptor agonists) may be required to separate the beneficial effects of nuclear receptor activation from unwanted metabolic side effects. The impact of nuclear receptor crosstalk in NAFLD is likely to be profound, but requires further elucidation. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie.

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Disruption of the intestinal barrier is a causal factor in the development of alcoholic endotoxemia and hepatitis. This study was undertaken to determine whether zinc deficiency is related to the deleterious effects of alcohol on the intestinal barrier. Mice were pair fed an alcohol or isocaloric liquid diet for 4 wk, and hepatitis was detected in association with elevated blood endotoxin level. Alcohol exposure significantly increased the permeability of the ileum but did not affect the barrier function of the duodenum or jejunum. Reduction of tight-junction proteins at the ileal epithelium was detected in alcohol-fed mice although alcohol exposure did not cause apparent histopathological changes. Alcohol exposure significantly reduced the ileal zinc concentration in association with accumulation of reactive oxygen species. Caco-2 cell culture demonstrated that alcohol exposure increases the intracellular free zinc because of oxidative stress. Zinc deprivation caused epithelial barrier disruption in association with disassembling of tight junction proteins in the Caco-2 monolayer cells. Furthermore, minor zinc deprivation exaggerated the deleterious effect of alcohol on the epithelial barrier. In conclusion, epithelial barrier dysfunction in the distal small intestine plays an important role in alcohol-induced gut leakiness, and zinc deficiency attributable to oxidative stress may interfere with the intestinal barrier function by a direct action on tight junction proteins or by sensitizing to the effects of alcohol.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants associated with non-alcoholic fatty liver disease (NAFLD) in epidemiologic studies. The purpose of this study was to evaluate the hepatic effects of a PCB mixture, Aroclor 1260, whose composition mimics human bioaccumulation patterns, in a mouse model of diet-induced obesity (DIO). Male C57Bl/6J mice were fed control diet or 42% high fat diet (HFD) and exposed to Aroclor 1260 (20 mg/kg or 200 mg/kg in corn oil) for 12 weeks. A glucose tolerance test was performed; plasma/tissues were obtained at necropsy for measurements of adipocytokine levels, histology, and gene expression. Aroclor 1260 exposure was associated with decreased body fat in HFD-fed mice but had no effect on blood glucose/lipid levels. Paradoxically, Aroclor 1260 + HFD co-exposed mice demonstrated increased hepatic inflammatory foci at both doses while the degree of steatosis did not change. Serum cytokines, ALT levels and hepatic expression of IL-6 and TNFα were increased only at 20 mg/kg, suggesting an inhibition of pro-inflammatory cytokine production at the 200 mg/kg exposure. Aroclor 1260 induced hepatic expression of cytochrome P450s including Cyp3a11 (Pregnane-Xenobiotic Receptor target) and Cyp2b10 (constitutive androstane receptor target) but Cyp2b10 inducibility was diminished with HFD-feeding. Cyp1a2 (aryl hydrocarbon Receptor target) was induced only at 200 mg/kg. In summary, Aroclor 1260 worsened hepatic and systemic inflammation in DIO. The results indicated a bimodal response of PCB-diet interactions in the context of inflammation which could potentially be explained by xenobiotic receptor activation. Thus, PCB exposure may be a relevant “second hit” in the transformation of steatosis to steatohepatitis.

Zinc is an essential trace element required for normal cell growth, development, and differentiation. It is involved in DNA synthesis, RNA transcription, and cell division and activation. It is a critical component in many zinc protein/ enzymes, including critical zinc transcription factors. Zinc deficiency/altered metabolism is observed in many types of liver disease, including alcoholic liver disease (ALD) and viral liver disease. Some of the mechanisms for zinc deficiency/ altered metabolism include decreased dietary intake, increased urinary excretion, activation of certain zinc transporters, and induction of hepatic metallothionein. Zinc deficiency may manifest itself in many ways in liver disease, including skin lesions, poor wound healing/liver regeneration, altered mental status, or altered immune function. Zinc supplementation has been documented to block/attenuate experimental ALD through multiple processes, including stabilization of gut-barrier function, decreasing endotoxemia, decreasing proinflammatory cytokine production, decreasing oxidative stress, and attenuating apoptotic hepatocyte death. Clinical trials in human liver disease are limited in size and quality, but it is clear that zinc supplementation reverses clinical signs of zinc deficiency in patients with liver disease. Some studies suggest improvement in liver function in both ALD and hepatitis C following zinc supplementation, and 1 study suggested improved fibrosis markers in hepatitis C patients. The dose of zinc used for treatment of liver disease is usually 50 mg of elemental zinc taken with a meal to decrease the potential side effect of nausea.

Polychlorinated biphenyls (PCBs) are persistent environmental toxicants, present in 100% of U.S. adults and dose-dependently associated with obesity and non-alcoholic fatty liver disease (NAFLD). PCBs are predicted to interact with receptors previously implicated in xenobiotic/energy metabolism and NAFLD. These receptors include the aryl hydrocarbon receptor (AhR), pregnane xenobiotic receptor (PXR), constitutive androstane receptor (CAR), peroxisome proliferator-activated receptors (PPARs), liver-X-receptor (LXRα), and farnesoid-X-receptor (FXR). This study evaluates Aroclor 1260, a PCB mixture with congener composition mimicking that of human adipose tissue, and selected congeners, as potential ligands for these receptors utilizing human hepatoma-derived (HepG2) and primate-derived (COS-1) cell lines, and primary human hepatocytes. Aroclor 1260 (20 μg/ml) activated AhR, and PCB 126, a minor component, was a potent inducer. Aroclor 1260 activated PXR in a simple concentration-dependent manner at concentrations ≥10 μg/ml. Among the congeners tested, PCBs 138, 149, 151, 174, 183, 187, and 196 activated PXR. Aroclor 1260 activated CAR2 and CAR3 variants at lower concentrations and antagonize CAR2 activation by the CAR agonist, CITCO, at higher concentrations (≥20 μg/ml). Additionally, Aroclor 1260 induced CYP2B6 in primary hepatocytes. At subtoxic doses, Aroclor 1260 did not activate LXR or FXR and had no effect on LXR- or FXR-dependent induction by the agonists T0901317 or GW4064, respectively. Aroclor 1260 (20 μg/ml) suppressed PPARα activation by the agonist nafenopin, although none of the congeners tested demonstrated significant inhibition. The results suggest that Aroclor 1260 is a human AhR, PXR and CAR3 agonist, a mixed agonist/antagonist for CAR2, and an antagonist for human PPARα.

Polychlorinated biphenyls (PCBs) are environmental pollutants associated with non-alcoholic-steatohepatitis (NASH), diabetes, and obesity. We previously demonstrated that the PCB mixture, Aroclor 1260, induced steatohepatitis and activated nuclear receptors in a diet-induced obesity mouse model. This study aims to evaluate PCB interactions with the pregnane-xenobiotic receptor (Pxr: Nr1i2) and constitutive androstane receptor (Car: Nr1i3) in NASH. Wild type C57Bl/6 (WT), Pxr(-/-) and Car(-/-) mice were fed the high fat diet (42% milk fat) and exposed to a single dose of Aroclor 1260 (20 mg/kg) in this 12-week study. Metabolic phenotyping and analysis of serum, liver, and adipose was performed. Steatohepatitis was pathologically similar in all Aroclor-exposed groups, while Pxr(-/-) mice displayed higher basal pro-inflammatory cytokine levels. Pxr repressed Car expression as evident by increased basal Car/Cyp2b10 expression in Pxr(-/-) mice. Both Pxr(-/-) and Car(-/-) mice showed decreased basal respiratory exchange rate (RER) consistent with preferential lipid metabolism. Aroclor increased RER and carbohydrate metabolism, associated with increased light cycle activity in both knockouts, and decreased food consumption in the Car(-/-) mice. Aroclor exposure improved insulin sensitivity in WT mice but not glucose tolerance. The Aroclor-exposed, Pxr(-/-) mice displayed increased gluconeogenic gene expression. Lipid-oxidative gene expression was higher in WT and Pxr(-/-) mice although RER was not changed, suggesting PCB-mediated mitochondrial dysfunction. Therefore, Pxr and Car regulated inflammation, behavior, and energy metabolism in PCB-mediated NASH. Future studies should address the 'off-target' effects of PCBs in steatohepatitis.