Chronic ethanol consumption enhances interleukin-1-mediated signal transduction in rat liver and in cultured hepatocytes

BACKGROUND: Interleukin-1 (IL-1) is a central mediator of the inflammatory process. Increased serum levels of IL-1 have been reported in alcoholics with liver damage, but it remains unknown whether chronic ethanol intake, in the presence or absence of lipopolysaccharide (LPS), activates IL-1 release and signaling in the hepatocyte. METHODS: IL-1beta and IL-10 release, expression of their receptors (IL-1RI and IL-10R), and the IL-1RI signal transduction response were evaluated in livers and cultured hepatocytes from ethanol-fed or pair-fed rats exposed in vivo or in vitro to LPS, ethanol, or both. RESULTS: Chronic ethanol intake increased both the serum levels of IL-1beta and IL-10 and the expression of IL-1RI, but not of IL-10R, in the liver microsomal fraction. In vivo LPS administration potentiated the ethanol-induced release of plasma cytokines. It is interesting to note that ethanol, either given in a single dose or chronically fed, stimulated IL-1beta and IL-10 release from cultured hepatocytes. Stimulation of hepatocytes with IL-1beta caused a higher activation of IL-1-associated kinase, extracellular receptor-activated kinases 1 and 2, and nuclear factor-kappaB (NF-kappaB) in hepatocytes from alcohol-fed animals than from controls. Furthermore, in the absence of any stimulation, hepatocytes from alcohol-fed animals showed an activation of both kinases, as well as an increase in NF-kappaB binding. Our results suggest the participation of the extracellular signal-regulated kinase (ERK)1/2 pathway in ethanol-induced NF-kappaB activation, because treatment with PD-98059, an ERK1/2 inhibitor, partially suppressed IL-1beta-induced NF-kappaB expression. CONCLUSIONS: Chronic ethanol intake potentiates the action of the proinflammatory cytokine IL-1beta, enhancing the release and signaling response of IL-1beta in the hepatocyte, which in conjunction with other cytokines or LPS may exacerbate the inflammatory damage associated with alcoholic liver disease.     

Alcohol consumption induces hepatocyte apoptosis in patients with chronic hepatitis C infection

BACKGROUND: Epidemiological studies have established that heavy alcohol consumption in persons with chronic hepatitis C infection is associated with advanced liver disease, including cirrhosis. The cellular mechanisms underlying this process, which appear to occur over decades, are unknown. Increased hepatocyte apoptosis has been observed in association with hepatitis C infection. The aim of this study was to evaluate the relationship between alcohol consumption and hepatocyte apoptosis in hepatitis C-infected patients. METHODS: Liver tissue from 20 hepatitis C-infected patients with variable alcohol consumption, and 10 normal control subjects was examined for hepatocyte apoptosis, proliferation and bcl-2 expression. RESULTS: Hepatocyte apoptosis was significantly greater in hepatitis C-infected patients than in controls. In hepatitis C-infected patients, significantly more hepatocyte apoptosis was seen in those consuming at least 30 g per day of alcohol compared with those drinking less than 10 g daily. Bcl-2, an inhibitor of apoptosis, was not detected in liver tissue from patients with the highest ethanol intake and rate of hepatocyte apoptosis. In contrast, patients drinking lesser amounts of ethanol had lower rates of hepatocyte apoptosis and more frequent bcl-2 expression. CONCLUSIONS: This study confirms that both hepatitis C infection and ethanol consumption induce hepatocyte apoptosis in humans. Ethanol-induced hepatocyte apoptosis has previously been shown only in animal models of alcohol-related liver injury. The precise role of apoptosis in the pathogenesis of hepatitis C-related liver injury remains unclear, but its induction may be related to downregulation of bcl-2 expression associated with ethanol consumption.     

Correlation Between Adenosine Triphosphate Content and Apoptosis in Liver of Rats Treated With Alcohol

Background: Chronic alcohol consumption depresses adenosine triphosphate (ATP) synthesis and induces mitochondrial DNA (Mt-DNA) deletion. ATP content in cells may play a critical role in inducing cell death, apoptosis, or necrosis. However, it is unknown which type of cell death occurs in alcoholic liver disease. In this study, the deletions of hepatic Mt-DNA, hepatic ATP content, and the number of single-stranded DNA (ss-DNA) of hepatocytes in rats treated with ethanol were determined to elucidate the relationship among Mt-DNA deletion, ATP synthesis, and/or hepatic apoptosis.
Methods: Sixteen male Wistar rats were fed with a liquid diet containing 36% ethanol (E group) or liquid diet without ethanol (C group) for 5 weeks. Hepatic ATP content was measured and the deletions of Mt-DNA encoding complexes I, IV, and V were determined in fresh liver tissue, and ss-DNA was stained in paraffin sections.
Results: Fatty change was observed in the E group, but not in the C group. Hepatic ATP content in the E group was 0.44 μmol/g of liver, which was significantly lower than that in the C group (0.84 μmole/g of liver). However, no deletion of Mt-DNA encoding complexes I, IV, and V was detected in either the E or the C group. ss-DNA staining was clearly observed in the nuclei of hepatocytes in both groups. The number of ss-DNA-positive hepatocytes in the E group was 5.6 ± 1.8/10,000 hepatocytes, which was significantly less than that in the C group: 20.6 ± 4.8/10,000 hepatocytes. There was a positive correlation between hepatic ATP contents and the number of ss-DNA-positive cells.
Conclusions: The results suggest that mitochondrial function, at least in part ATP synthesis, was depressed before the damage of Mt-DNA by chronic ethanol consumption. Chronic ethanol consumption may not be responsible for the apoptosis of hepatocytes.     

Loss of Dicer1 impairs hepatocyte survival and leads to chronic inflammation and progenitor cell activation

AIM: To investigate the continuous hepatic histopathological processes which occur in response to the loss of Dicer1.METHODS: We generated a hepatocyte-selective Dicer1 knockout mouse and observed the gradual hepatic histopathological changes in the mutant liver. Immunohistochemistry and Western blotting were performed to detect Dicer1 expression. We performed hematoxylin and eosin staining, Periodic acid-Schiff staining, Oil Red O staining, and Masson’s trichrome staining to detect histological changes in Dicer1-deficient livers. Ki67 immunohistochemistry, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, and Western blotting were used to determine hepatocyte proliferation and apoptosis. Serum biochemistry, cytokine assays, and flow cytometric analysis were performed to quantity liver necrosis and inflammation. Fibrogenic markers were determined by Western blotting and qPCR. CK19, CD133, and OV6 immunofluorescence were used to observe liver progenitor cells. Immunofluorescence and qPCR were performed to reveal embryonic gene expression. We also performed histological staining and Western blotting to analyze hepatocellular carcinoma (HCC) development.RESULTS: Dicer1 inactivation resulted in significant architecture disorganization and metabolism disruption in the liver. Dicer1 disruption impaired hepatocyte survival and resulted in profound cell apoptosis and continuous necrosis. In contrast to previous reports, the mutant liver exhibited chronic inflammation and progressive fibrosis, and could not be repopulated by Dicer1-positive cells. In addition, extensive activation of hepatic progenitor cells was observed. Primary HCC was observed as early as 4 mo after birth.CONCLUSION: Hepatic loss of Dicer1 results in complex chronic pathological processes, including hepatocyte death, inflammatory infiltration, chronic fibrosis, compensatory proliferation, progenitor activation, and spontaneous hepatocarcinogenesis.     

Hepatic estrogen receptors and alcohol intake

Human liver contains estrogen receptors (ER) which render it sensitive to estrogen. Chronic ethanol ingestion in humans and rats results in alterations of circulating sex steroid levels and expression of sex hormone-dependent phenotype. The analysis and quantitation of hepatic estrogen receptor (ER) activity and sex hormone-responsive proteins have been performed over the past two decades. Alcohol abuse appears to induce an increase in ER content of human liver, especially in patients with alcoholic hepatitis actively drinking. This observation is reproduced in an experimental model of chronic alcohol feeding of rats. In male rat liver, the increased ER expression induced by alcohol is associated with an elevated proliferation rate of the hepatocytes. In female liver, the ER content is not affected by alcohol intake and apoptosis prevails over proliferation. The feminization of the liver in males may protect the liver from the severe alcohol-induced liver injury seen in females.     

Insulin prevents liver damage and preserves liver function in lipopolysaccharide-induced endotoxemic rats

BACKGROUND/AIMS: Liver integrity and function are crucial for survival of patients suffering from trauma, operations or infections. Insulin decreased mortality and prevented the incidence of multi organ failure and infection in critically ill patients. The aim of the present study was to determine whether insulin exerts positive effects on hepatic homeostasis and function during endotoxemia. METHODS: Endotoxemic rats received either saline or insulin. Hepatic morphology and function was determined by measuring the effect of insulin on liver proteins, enzymes, hepatocyte apoptosis and proliferation including caspases-3 and -9 and Bcl-2. Intrahepatic ATP, glucose and lactate concentration were determined by bioluminescence. To determine possible molecular changes the effect of insulin on hepatic cytokine mRNA and gene profile analysis were assessed. RESULTS: Insulin significantly improved hepatic protein synthesis by increasing albumin and decreasing c-reactive protein, P<0.05. Insulin attenuated hepatic damage by decreasing AST and ALT, P<0.05. Improved liver morphology was due to decreased hepatocyte apoptosis along with decreased caspase-3 concentration and increased hepatocyte proliferation along with Bcl-2 concentration, P<0.05. Insulin decreased hepatic IL-1beta, IL-6 and MIF mRNA and improved hepatic glucose metabolism and glycolysis, P<0.05. GeneChip analysis revealed an anti-inflammatory effect of insulin. CONCLUSIONS: Insulin improves hepatic integrity, hepatic glucose metabolism and hepatic function by increasing cell survival and attenuating the hepatic inflammatory response in endotoxemic rats.