Differential contributions of C3, C5, and decay-accelerating factor to ethanol-induced fatty liver in mice

BACKGROUND AND AIMS: The complement pathway is an important component of the innate and adaptive immune response. Here we tested the hypothesis that activation of complement is required for development of ethanol-induced fatty liver. METHODS: Wild-type mice and mice lacking the third (C3) or fifth (C5) components of the complement activation pathway, as well as mice lacking decay-accelerating factor (CD55/DAF), a complement regulatory protein, were fed Lieber-DeCarli ethanol-containing diets for 6 weeks or pair-fed control diets. RESULTS: Ethanol feeding to wild-type mice increased C3a in plasma. Wild-type and C5-/- mice fed the ethanol diet developed hepatic steatosis characterized by microvesicular and macrovesicular lipid accumulation and increased triglyceride content. C3-/- mice did not develop steatosis, while CD55/DAF-/- mice accumulated even more hepatic triglyceride after ethanol feeding than wild-type mice. Levels of serum alanine aminotransferase and hepatic tumor necrosis factor alpha, indicators of hepatocyte injury and inflammation, respectively, were increased in wild-type and CD55/DAF-/- mice but not in C5-/- mice after ethanol feeding. In contrast to the protective effect of C3-/- against ethanol-induced steatosis, levels of both alanine aminotransferase and tumor necrosis factor alpha were increased in C3-/- mice after ethanol feeding. CONCLUSIONS: Here we have identified several elements of the complement system as important contributors to ethanol-induced fatty liver. C3 contributed primarily to the accumulation of triglyceride in the liver, whereas C5 was involved in inflammation and injury to hepatocytes. Further, the absence of CD55/DAF exacerbated these responses, suggesting that CD55/DAF serves as a barrier to ethanol-induced fatty liver.     

Hepatocyte-specific hypoxia-inducible factor-1α is a determinant of lipid accumulation and liver injury in alcohol-induced steatosis in mice

Chronic alcohol causes hepatic steatosis and liver hypoxia. Hypoxia-regulated hypoxia-inducible factor 1-α, (HIF-1α) may regulate liporegulatory genes, but the relationship of HIF-1 to steatosis remains unknown. We investigated HIF-1α in alcohol-induced hepatic lipid accumulation. Alcohol administration resulted in steatosis, increased liver triglyceride levels, and increased serum alanine aminotransferase (ALT) levels, suggesting liver injury in wild-type (WT) mice. There was increased hepatic HIF-1α messenger RNA (mRNA), protein, and DNA-binding activity in alcohol-fed mice compared with controls. Mice engineered with hepatocyte-specific HIF-1 activation (HIF1dPA) had increased HIF-1α mRNA, protein, and DNA-binding activity, and alcohol feeding in HIF1dPA mice increased hepatomegaly and hepatic triglyceride compared with WT mice. In contrast, hepatocyte-specific deletion of HIF-1α [HIF-1α(Hep(-/-) )], protected mice from alcohol- and lipopolysaccharide (LPS)-induced liver damage, serum ALT elevation, hepatomegaly, and lipid accumulation. HIF-1α(Hep(-/-) ), WT, and HIF1dPA mice had equally suppressed levels of peroxisome proliferator-activated receptor α mRNA after chronic ethanol, whereas the HIF target, adipocyte differentiation-related protein, was up-regulated in WT mice but not HIF-1α(Hep(-/-) ) ethanol-fed/LPS-challenged mice. The chemokine monocyte chemoattractant protein-1 (MCP-1) was cooperatively induced by alcohol feeding and LPS in WT but not HIF-1α(Hep(-/-) ) mice. Using Huh7 hepatoma cells in vitro, we found that MCP-1 treatment induced lipid accumulation and increased HIF-1α protein expression as well as DNA-binding activity. Small interfering RNA inhibition of HIF-1α prevented MCP-1-induced lipid accumulation, suggesting a mechanistic role for HIF-1α in hepatocyte lipid accumulation. CONCLUSION: Alcohol feeding results in lipid accumulation in hepatocytes involving HIF-1α activation. The alcohol-induced chemokine MCP-1 triggers lipid accumulation in hepatocytes via HIF-1α activation, suggesting a mechanistic link between inflammation and hepatic steatosis in alcoholic liver disease.     

Increased lipopolysaccharide sensitivity in alcoholic fatty livers is independent of leptin deficiency and toll-like receptor 4 (TLR4) or TLR2 mRNA expression

BACKGROUND: Both alcoholic (AFL) and nonalcoholic (NAFL) fatty livers show increased sensitivity to endotoxin-induced injury. Lipopolysaccharide (LPS) is recognized by toll-like receptor 4 (TLR4), whereas lipopeptide triggers TLR2 to induce common downstream activation of nuclear factor (NF)-kappaB and pro-inflammatory pathways that are activated in AFL and NAFL. METHODS: Serum alanine aminotransferase (ALT), tumor necrosis factor (TNF)-alpha, and interleukin (IL)-6 levels; hepatic NF-kappaB activity; and expression of TLR2, TLR4, inducible nitric oxide synthase (iNOS), and heme oxygenase (HO)-1 mRNAs were investigated in lean and leptin-deficient ob/ob mice after LPS challenge in combination with acute or chronic alcohol feeding. RESULTS: Increased LPS sensitivity in AFL and NAFL was characterized by elevated serum TNF-alpha and IL-6 induction. However, there was no difference in TLR2 and TLR4 mRNA levels between lean and ob/ob livers at baseline and after acute or chronic alcohol treatment. LPS increased TLR2, but not TLR4, mRNA levels in all groups. Chronic alcohol feeding and LPS increased serum ALT and TNF-alpha levels in lean but not in ob/ob mice compared with pair-fed controls. Hepatic NF-kappaB activation was increased in both ob/ob and lean mice after chronic alcohol feeding compared with pair-fed controls. Expression of iNOS, an inducer of oxidative stress, and HO-1, a cytoprotective protein, were higher in ob/ob compared with lean mice after chronic alcohol feeding. However, LPS-induced HO-1, but not iNOS, expression was attenuated in ob/ob compared with lean mice. CONCLUSION: These results imply that the increased sensitivity of AFL to LPS occurs without up-regulation of TLR2 or TLR4 genes and may be related to an imbalance of pro-inflammatory/oxidative and cytoprotective mechanisms.     

Kupffer cell inactivation alleviates ethanol-induced steatosis and CYP2E1 induction but not inflammatory responses in rat liver

BACKGROUND/AIMS: Gadolinium chloride inactivates Kupffer cells and alleviates alcohol-induced liver lesions. We investigated the mechanism of gadolinium chloride protection after oral ethanol feeding. METHODS: Rats were maintained ethanol-intoxicated for 6 weeks by feeding ethanol in a low-carbohydrate/high-fat liquid diet. Macrophages were inactivated by intravenous administrations of gadolinium chloride. At termination, liver samples and cell lysates obtained from the periportal and perivenous region were analyzed for histopathology, mRNA expression of endotoxin-associated parameters and cytokines and for enzymes involved in oxidative stress. RESULTS: Ethanol treatment alone caused marked microvesicular/macrovacuolar steatosis and focal inflammation. Gadolinium significantly alleviated pathology, by reducing steatosis but not inflammation. Gadolinium treatment eliminated ED2 immunopositive Kupffer cells, which were larger and more frequent periportally. Ethanol significantly increased the mRNA expression of the endotoxin (LPS) receptor CD14 and the LPS binding protein LBP, but not that of the pro-inflammatory cytokines TNF-alpha and IL-1beta. The mRNA of CD14 was found to be expressed preferentially in the perivenous region, but gadolinium treatment had no significant effect on the expression or the distribution. However, gadolinium significantly moderated the ethanol induction of CYP2E1 and this effect correlated to the degree of steatosis. Ethanol increased glutathione transferase and reduced glutathione peroxidase activity, but these changes persisted after gadolinium treatment. CONCLUSIONS: Our results suggest that gadolinium chloride reduces symptoms of ALD mainly by counteracting steatosis, and that CD14-positive Kupffer cell populations are not involved in gadolinium protection. The strong correlation between pathology and CYP2E1 induction might suggest a steatopathogenic role for this enzyme.     

Ethanol suppression of the hypothalamic proopiomelanocortin level and the splenic NK cell cytolytic activity is associated with a reduction in the expression of proinflammatory cytokines but not anti-inflammatory cytokines in neuroendocrine and immune cells

BACKGROUND: Immune signals activate a network of cytokines in the central nervous system (CNS) that in turn causes release of neurotransmitters and hormones to modulate immune cell functions. We have recently shown that hypothalamic beta-endorphin neurons, via inhibition of the sympathetic neuronal activity, activate natural killer (NK) cell function in the spleen, and this communication is disrupted following chronic ethanol administration. Beta-endorphin neuronal function is known to be regulated by various proinflammatory and anti-inflammatory cytokines. The effects of ethanol on the proinflammatory and anti-inflammatory cytokines known to control beta-endorphin neuronal and NK cell functions during immune challenges have not been determined. METHODS: In the present study, we evaluated the effects of chronic ethanol consumption on the basal and lipopolysaccharide (LPS)-activated NK cells' functions in the spleen, the beta-endorphin peptide precursor proopiomelanocortin (POMC) gene expression in the arcuate nucleus (ARC) of the hypothalamus, and mRNA levels of proinflammatory cytokines interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), and anti-inflammatory cytokines IL-6 and IL-10 in the spleen and in the ARC. Male rats were ad libitum fed rat chow (ad lib-fed), pair-fed an isocaloric liquid diet, or fed an ethanol-containing liquid diet, and each was treated with LPS (100 microg/kg body weight). After 2 hours, splenocytes and ARC tissues were isolated and used for this study. Splenocytes were used to determine mRNA levels of IL-1beta, TNF-alpha, IL-6, IL-10, granzyme B, and perforin using the real-time RT-PCR assays. Splenocytes were also used to determine the cytolytic activity using a standard 4-hour (51)Cr release assay against YAC-1 lymphoma target cells. Arcuate nuclei were used to determine IL-1beta, TNF-alpha, IL-6, IL-10, and POMC mRNA levels using real-time RT-PCR assays. RESULTS: The results demonstrate that ethanol feeding via a liquid diet for 2 weeks suppressed both basal and LPS-stimulated NK cell cytolytic functions and the levels of cytotoxicity-regulatory perforin and granzyme B mRNAs in the spleen. Ethanol feeding reduced the basal and LPS-stimulated levels of POMC mRNA in the ARC. Ethanol also impaired LPS-induced levels of IL-1beta and TNF-alpha mRNAs both in the spleen and in the ARC. In contrast, ethanol feeding did not cause any significant changes in basal and the LPS-stimulated expression of IL-6 and IL-10 mRNAs in the spleen and of IL-6 mRNA levels in the ARC. These results indicate that ethanol suppression of hypothalamic POMC levels and splenic NK cell functions is associated with a reduced expression of proinflammatory cytokines in neuroendocrine and immune cells.     

Toll-like receptor 4 is involved in the mechanism of early alcohol-induced liver injury in mice.

Chronic alcohol administration increases gut-derived endotoxin in the portal blood, which activates Kupffer cells and causes liver injury. Mice (C3H/HeJ) with mutations in toll-like receptor 4 (TLR4) are hyporesponsive to endotoxin. To test the hypothesis that TLR4 is involved in early alcohol-induced liver injury, the long-term intragastric ethanol feeding protocol developed by Tsukamoto and French for rats was adapted to mice. Animals with nonfunctional TLR4 and wild-type mice (C3H/HeOuJ) were compared. Two-month-old female mice were fed a high-fat liquid diet with either ethanol or isocaloric maltose-dextrin as control continuously for 4 weeks. There was no difference in mean urine alcohol concentrations between the groups. Dietary alcohol significantly increased liver-to-body weight ratios and serum alanine transaminase (ALT) levels in wild-type mice (109 +/- 18 U/L) over high-fat controls (40 +/- 3 U/L), effects that were blunted significantly in mice with a mutation of TLR4 (55 +/- 9 U/L). While no significant pathologic changes were observed in high-fat controls, dietary ethanol caused steatosis, mild inflammation, and focal necrosis in wild-type animals (pathology score = 5.2 +/- 1.2). These pathologic changes were significantly lower in TLR4-deficient mice fed ethanol (score = 2.0 +/- 1.3). Endotoxin levels in the portal vein were increased significantly after 4 weeks in both groups fed ethanol. Moreover, ethanol increased tumor necrosis factor alpha (TNF-alpha) mRNA expression in wild-type, but not in TLR4-deficient, mice. These data are consistent with the hypothesis that Kupffer cell activation by endotoxin via TLR4 is involved in early alcohol-induced liver injury.     

The role of iNOS in alcohol-dependent hepatotoxicity and mitochondrial dysfunction in mice

Nitric oxide (NO) is now known to control both mitochondrial respiration and organelle biogenesis. Under conditions of ethanol-dependent hepatic dysfunction, steatosis is increased, and this is associated with increased expression of inducible nitric oxide synthase (iNOS). We have previously shown that after chronic exposure to ethanol, the sensitivity of mitochondrial respiration to inhibition by NO is enhanced, and we have proposed that this contributes to ethanol-dependent hypoxia. This study examines the role of iNOS in controlling the NO-dependent modification of mitochondrial function. Mitochondria were isolated from the livers of both wild-type (WT) and iNOS knockout (iNOS-/-) mice that were fed an isocaloric ethanol-containing diet for a period of 5 weeks. All animals that consumed ethanol showed some evidence of fatty liver; however, this was to a lesser extent in the iNOS-/- mice compared to controls. At this early stage in ethanol-dependent hepatic dysfunction, infiltration of inflammatory cells and the formation of nitrated proteins was also decreased in response to ethanol feeding in the iNOS-/- animals. Mitochondria isolated from wild-type ethanol-fed mice showed a significant decrease in respiratory control ratio and an increased sensitivity to NO-dependent inhibition of respiration relative to their pair-fed controls. In contrast, liver mitochondria isolated from iNOS-/- mice fed ethanol showed no change in the sensitivity to NO-dependent inhibition of respiration. In conclusion, the hepatic response to chronic alcohol-dependent cytotoxicity involves a change in mitochondrial function dependent on the induction of iNOS.     

Metformin prevents alcohol-induced liver injury in the mouse: Critical role of plasminogen activator inhibitor-1

BACKGROUND & AIMS: The biguanide drug metformin has recently been found to improve steatosis and liver damage in animal models and in humans with nonalcoholic steatohepatitis. METHODS: The aim of the present study was to determine whether metformin also prevents steatosis and liver damage in mouse models of acute and chronic alcohol exposure. RESULTS: Acute ethanol exposure caused a >20-fold increase in hepatic lipids, peaking 12 hours after administration. Metformin treatment significantly blunted the ethanol effect by >60%. Although metformin is a known inducer of AMP kinase (AMPK) activity, the hepatoprotective property of metformin did not correlate with activation of AMPK or of AMPK-dependent pathways. Instead, the protective effects of metformin correlated with complete prevention of the upregulation of plasminogen activator inhibitor (PAI)-1 caused by ethanol. Indeed, a similar protective effect against acute alcohol-induced lipid accumulation was observed in PAI-1-/- mice. Hepatic fat accumulation caused by chronic enteral ethanol feeding was also prevented by metformin or by knocking out PAI-1. Under these conditions, necroinflammatory changes caused by ethanol were also significantly attenuated. CONCLUSIONS: Taken together, these findings suggest a novel mechanism of action for metformin and identify a new role of PAI-1 in hepatic injury caused by ethanol.