Effect of chronic alcohol consumption on Hepatic SIRT1 and PGC-1alpha in rats.

The nuclear genes, NAD-dependent deacetylase Sirtuis 1 (SIRT1) and the peroxisome proliferator-activated receptor-gamma coactivator1alpha (PGC-1alpha) are regulators of energy metabolism. Here, we studied the role of alcohol consumption in expression of these sensing molecules. Alcohol significantly reduced hepatic SIRT1 mRNA by 50% and PGC-1alpha mRNA by 46% and it significantly inhibited the protein expression of SIRT1 and PGC-1alpha, while the transcription factor PPAR-gamma remained unchanged. However, when the lipid composition of the alcohol diet was changed by replacing long-chain triglycerides (LCT) with medium chain triglycerides (MCT), SIRT1 and PGC-1alpha mRNA were restored to near control levels. This study demonstrates that alcohol reduces key energy sensing proteins and that replacement of LCT by MCT affects the transcription of these genes. Since there is a pathophysiological link between SIRT1 and PGC-1alpha and mitochondrial energy, the implication of the study is that mitochondrial dysfunction due to alcohol abuse can be treated by dietary modifications.

Model of nonalcoholic steatohepatitis.

BACKGROUND: Obesity and diabetes are frequently associated with nonalcoholic steatohepatitis (NASH), but studies have been hampered by the absence of a suitable experimental model. OBJECTIVE: Our objective was to create a rat model of NASH. DESIGN: Sprague-Dawley rats were fed a high-fat, liquid diet (71% of energy from fat, 11% from carbohydrates, 18% from protein) or the standard Lieber-DeCarli diet (35% of energy from fat, 47% from carbohydrates, 18% from protein). The diets were given ad libitum or as two-thirds of the amount consumed ad libitum. RESULTS: Rats fed the high-fat diet ad libitum for 3 wk developed panlobular steatosis, whereas those fed the standard diet had few fat droplets. Accordingly, total lipid concentrations with the high-fat and standard diets were 129.9 +/- 9.1 ( +/- SEM) and 66.7 +/- 4.6 mg/g liver, respectively (P < 0.001). The high-fat diet caused abnormal mitochondria and mononuclear inflammation, which were accompanied by increased hepatic tumor necrosis factor alpha (TNF-alpha; P < 0.001), TNF-alpha messenger RNA (mRNA) (P < 0.001), collagen type 1, and alpha1(I) procollagen mRNA (P < 0.001). In addition, these rats had increased cytochrome P4502E1 (CYP2E1) mRNA (P < 0.001), which was accompanied by CYP2E1 induction (P < 0.001) and oxidative stress with increased 4-hydroxynonenal (P < 0.001). Plasma insulin was elevated, which reflected insulin resistance, a NASH pathogenic factor. Rats fed a restricted high-fat diet developed only mild steatosis with attenuated biochemical changes, whereas those given a restricted standard diet had normal livers. CONCLUSION: This rat model reproduces the key features of human NASH and provides a realistic experimental model for elucidating its treatment.

SMART amplification maintains representation of relative gene expression: quantitative validation by real time PCR and application to studies of alcoholic liver disease in primates.

The small quantities of tissue available for most studies of human disease are a significant limitation for meaningful gene expression profiling. The Atlas Switch Mechanism At the 5' end of Reverse Transcript (SMART) probe amplification kit uses as little as 50 ng of total RNA to generate complex cDNA probes for DNA array and other analyses. However, the extent to which this attractive methodology maintains representation of relative gene expression has not been quantified. In this study, we demonstrate using real-time quantitative PCR analysis that the relative expression levels of a range of low- to high-abundance mRNAs are retained after SMART amplification independent of transcript abundance and full-length transcript, coding region and PCR product size. Using this technology, a mean amplification of 3800-fold was achieved in human liver samples, greatly enhancing the ability to perform replicate DNA array experiments. Probes generated with the SMART amplification method were used to detect increased expression of genes involved with inflammation, fibrosis, xenobiotic metabolism, immune function, oxidant stress and endothelium in liver from the baboon model of alcoholic liver disease.

Beneficial effects versus toxicity of medium-chain triacylglycerols in rats with NASH.

BACKGROUND/AIMS: Replacing long-chain triacylglycerols (LCT) with medium-chain triacylglycerols (MCT) reduces alcohol-induced liver injury. Because of the similarity of the pathogenesis of alcohol-induced liver damage and non-alcoholic steatohepatitis (NASH), our aim was to assess whether MCT is also beneficial in NASH. METHODS: We used a rat NASH model in which corn oil (35% of total calories) was isocalorically replaced with MCT. RESULTS: Partial replacement of LCT did not ameliorate hepatic fat accumulation, 4-hydroxynonenal, collagen type I and its mRNA but it increased TNF-alpha and its mRNA (p<0.001). However, in rats given the high-fat diet restricted to 2/3 of the amount they were consuming, these adverse effects decreased, with and without MCT including less liver steatosis and lower triacylglycerols, but without beneficial effects of MCT. When 70% of the fat calories were replaced with MCT with no LCT remaining in the diet, no steatosis developed and hepatic TNF-alpha was low. When all MCT were given with carbohydrates (instead of LCT) hepatic TNF-alpha also decreased (p<0.001). CONCLUSIONS: MCT are not hepatotoxic, provided the diet contains no significant amount of LCT. Total replacement of dietary LCT with MCT fed ad libitum is beneficial whereas partial replacement becomes hepatotoxic, unless the dietary intake is restricted.

Inflammation and repair in viral hepatitis C.

Hepatitis C viral infection (HCV) results in liver damage leading to inflammation and fibrosis of the liver and increasing rates of hepatic decompensation and hepatocellular carcinoma (HCC). However, the host's immune response and viral determinants of liver disease progression are poorly understood. This review will address the determinants of liver injury in chronic HCV infection and the risk factors leading to rapid disease progression. We aim to better understand the factors that distinguish a relatively benign course of HCV from one with progression to cirrhosis. We will accomplish this task by discussion of three topics: (1) the role of cytokines in the adaptive immune response against the HCV infection; (2) the progression of fibrosis; and (3) the risk factors of co-morbidity with alcohol and human immunodeficiency virus (HIV) in HCV-infected individuals. Despite recent improvements in treating HCV infection using pegylated interferon alpha (PEGIFN-alpha) and ribavirin, about half of individuals infected with some genotypes, for example genotypes 1 and 4, will not respond to treatment or cannot be treated because of contraindications. This review will also aim to describe the importance of IFN-alpha-based therapies in HCV infection, ways of monitoring them, and associated complications.

The Combination of S-adenosylmethionine and Dilinoleoylphosphatidylcholine Attenuates Non-alcoholic Steatohepatitis Produced in Rats by a High-Fat Diet.

In the pathogenesis of non-alcoholic steatohepatitis (NASH), oxidative stress resulting from free radicals generated by cytochrome P4502E1 (CYP2E1) plays a major role suggesting the importance of antioxidants. The objective of this study was to assess in a high-fat diet (HF) rat model the effects of the combination of s-adenosylmethionine (SAMe) plus dilinoleoylphosphatidylcholine (DLPC) in the treatment of NASH. To test the hypothesis that these two antioxidants are beneficial in NASH, male Sprague-Dawley rats were fed five different diets for six weeks: control, HF diet and HF plus SAMe and DLPC or their combination. As expected, the HF diet significantly increased hepatic triacylglycerols and CYP2E1 levels. However, only the combination diet opposed this effect, consistent with different actions of the two antioxidants. Next, 24 additional rats divided in two groups were fed the HF or the HF+SAMe+DLPC diets for 3 weeks. Dietary intake was similar, but liver triacylglycerols dropped from 76.1+/-6.8 to 49.4+/-3.5 mg/g (p=0.002) and hepatic CYP2E1 mRNA decreased after treatment (p=0.01) with a trend for less CYP2E1 protein. This was accompanied by a 41% reduction of hepatic 4-hydroxynonenal (4-HNE) (p=0.008), reflecting control of oxidative stress. Furthermore, the hepatic inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) mRNA and TNF-alpha protein decreased (p=0.05 and p=0.01 respectively) with attenuation of alpha1(I) procollagen mRNA and type I collagen levels (p=0.01 and p=0.02, respectively). We concluded that the combination SAMe+DLPC might be beneficial in NASH by reducing oxidative stress and associated liver injury.

Role of medium-chain triglycerides in the alcohol-mediated cytochrome P450 2E1 induction of mitochondria.

BACKGROUND: Chronic alcohol consumption is known to induce cytochrome P450 2E1 (CYP2E1) leading to lipid peroxidation, mitochondrial dysfunction and hepatotoxicity. We showed that replacement of dietary long-chain triglycerides (LCT) by medium-chain triglycerides (MCT) could be protective. We now wondered whether the induction of mitochondrial CYP2E1 plays a role and whether liver injury could be avoided through mitochondrial intervention. METHODS: Rats were fed 4 different isocaloric liquid diets. The control group received our standard dextrin-maltose diet with intake limited to the average consumption of the 3 alcohol groups fed ad libitum the alcohol containing Lieber-DeCarli liquid diet. The fat was either 32% of calories as LCT (alcohol), or 16% as LCT + 16% as MCT (alcohol-MCT 16%), or 32% as MCT only (alcohol-MCT 32%). RESULTS: After 21 days, compared to the controls, the alcohol and both alcohol-MCT groups had a significant increase in mitochondrial CYP2E1 (p < 0.05 for both). As shown before, the same was found for the microsomal CYP2E1. When MCT replaced all the fat, like in the alcohol-MCT 32% group, CYP2E1 was significantly reduced by 40% in mitochondria (p < 0.05) and 30% in microsomes (p < 0.01). In mitochondria, 4-hydroxynonenal (4-HNE), a parameter of oxidative stress, paralleled CYP2E1. Compared to controls, alcohol and alcohol-MCT 16% significantly raised mitochondrial 4-HNE (p < 0.001), whereas the alcohol-MCT 32% diet brought it down to control levels (p < 0.001). Mitochondrial reduced glutathione (GSH) was also significantly lowered by alcohol consumption (p < 0.05), and it increased to almost normal levels with alcohol-MCT 32% (p = 0.006). These changes in the mitochondria reflected the reduction observed in total liver in which alcohol-MCT 32% decreased the alcohol-induced steatosis with a diminution of triglycerides (p < 0.001) and of the pro-inflammatory cytokine tumor necrosis factor-alpha (p < 0.001). CONCLUSION: Mitochondria participate in the induction of CYP2E1 by alcohol and contribute to lipid peroxidation and GSH depletion. Thus, lipid composition of the diet is an important determinant for the beneficial effect of MCT, with a diet containing a mixture of LCT/MCT being ineffective.

DLPC attenuates alcohol-induced cytotoxicity in HepG2 cells expressing CYP2E1.

AIMS: Alcoholic liver injury was shown to result largely from oxidative stress generated by ethanol metabolism via cytochrome P4502E1 (CYP2E1). Our aim was to determine whether this could be overcome by using dilinoleoylphosphatidylcholine (DLPC), an innocuous antioxidant extracted from soybeans. METHODS: To address this question, we determined whether DLPC protects against alcohol-induced cytotoxicity in HepG2 cells expressing CYP2E1. A HepG2 subclone (2E1) expressing CYP2E1 and a control subclone (Neo) were exposed for 2 h to DLPC (10 microM), and then 100 mM ethanol was added for 5 days. RESULTS: Ethanol significantly decreased cell viability in the 2E1 cells and increased apoptosis. These alterations were attenuated by DLPC with the most significant effects in the 2E1 cells. This was accompanied by a reduction of the ethanol-induced oxidative stress, including diminished hydrogen peroxide production in the 2E1, but not in the Neo cells. The mitochondrial membrane potential was significantly diminished by ethanol in both cells. It was also improved after adding DLPC, but only in the 2E1 cells. In these cells, mitochondrial glutathione (GSH) was also partially restored by DLPC, which significantly inhibited the CYP2E1 induction by ethanol. CONCLUSION: DLPC opposes the cytotoxicity induced by alcohol in HepG2 cells expressing CYP2E1, a protective action due, at least in part, to an attenuation of the alcohol-induced oxidative stress and the alteration in the mitochondrial membrane potential. On account of these beneficial effects of DLPC and its innocuity, it is now germane to assess its therapeutic action in alcoholics.

Lycopene attenuates arachidonic acid toxicity in HepG2 cells overexpressing CYP2E1.

Arachidonic acid (AA) was shown to be toxic to HepG2 cells expressing cytochrome P4502E1 (CYP2E1) because of oxidative stress. The aim of this study was to investigate whether lycopene, a carotenoid with high anti-oxidant capacity, protects HepG2 cells expressing CYP2E1 against AA toxicity. In preliminary experiments, lycopene as well as placebo (vehicle) were not toxic in the three types of cells tested: HepG2 cells, HepG2 cells transfected with pCI-neo (Neo) or pCI-neo/2E1 (2E1). AA produced toxic effects, especially in the 2E1 cells, and caused a remarkable increase in hydrogen peroxide production and lipid peroxidation compared to the Neo and HepG2 cells. Lycopene had a protective effect whereas the placebo did not. This was due, at least in part, to inhibition of hydrogen peroxide production and of the resulting lipid peroxidation, confirming the potent anti-oxidant properties of lycopene and its suitability for clinical studies.

Lycopene attenuates alcoholic apoptosis in HepG2 cells expressing CYP2E1.

To test the hypothesis that ethanol-induced hepatic apoptosis is secondary to the oxidative stress generated by cytochrome P4502E1 (CYP2E1), we assessed the effects of the carotenoid lycopene, a potent antioxidant extracted from tomatoes, on oxidative stress and apoptosis in HepG2 cells overexpressing CYP2E1 (2E1 cells). These were exposed for 5 days to 100mM ethanol and 10 microM lycopene or an equal volume of placebo (vehicle). Ethanol significantly increased apoptosis measured by flow cytometry and by TUNEL assay. This was accompanied by an ethanol-induced oxidative stress: hydrogen peroxide production was significantly increased and mitochondrial GSH was strikingly decreased. Both were restored by lycopene, with a significant decrease in apoptosis. The placebo had no protective effect. In conclusion, Lycopene opposes the ethanol-induced oxidative stress and apoptosis in 2E1 cells. The parallelism between these effects suggests a causal link. Furthermore, these beneficial effects and the innocuity of lycopene now justify an in vivo trial.

Cytokeratin 7 staining of hepatocytes predicts progression to more severe fibrosis in alcohol-fed baboons.

BACKGROUND/AIMS: Not all alcoholic patients develop severe liver disease with fibrosis progressing to cirrhosis. It is of practical importance to determine whether some markers can predict progression of liver fibrosis. METHODS: We used a baboon model that mimics human alcoholic liver disease. Cytokeratin 7 and 19 expression and fat deposition were investigated in serial liver biopsies of 18 animals undergoing prolonged alcohol administration (range 2-17 years) and in four controls. Fibrosis was graded histologically and was also assessed quantitatively by image analysis. RESULTS: Ten animals did not show a progression of liver disease even after 17 years of alcohol administration, but eight animals fed alcohol exhibited a progression of liver disease from no fibrosis or perivenular fibrosis to septal fibrosis or cirrhosis within 7 years. In normal liver, cytokeratin 7 and cytokeratin 19 immunostaining is restricted to bile duct cells. Hepatocellular cytokeratin 7 was observed only in those animals which progressed to more severe stages of fibrosis and it anticipated this progression by 4.2 years on average. CONCLUSIONS: In alcohol-fed baboons, cytokeratin 7 staining of hepatocytes (but not cytokeratin 19, nor fat deposition) predicts with a high degree of sensitivity and specificity progression to more severe liver disease.

Silymarin retards the progression of alcohol-induced hepatic fibrosis in baboons.

UNLABELLED: GOAL/BACKGROUND: Hepatoprotective effects of silymarin in patients with alcoholic liver disease are controversial. For strict control, this was assessed in non-human primates. STUDY Twelve baboons were fed alcohol with or without silymarin for 3 years with a nutritionally adequate diet. RESULTS: Silymarin opposed the alcohol-induced oxidative stress (assessed by plasma 4-hydroxynonenal) and the rise in liver lipids and circulating ALT. Alcohol also increased hepatic collagen type I by 50% over the 3 years with a significant rise in mRNA for alpha1 (I) procollagen, both prevented by silymarin. There were corresponding morphologic changes: at 36 months, 2 of 6 animals fed alcohol had cirrhosis and 2 septal fibrosis, with perivenular fibrosis in 2, whereas with alcohol + silymarin, there was only 1 cirrhosis and 1 septal fibrosis, with perivenular fibrosis in 2, and virtually no lesions in the remaining 2. CONCLUSIONS: Silymarin retards the development of alcohol-induced hepatic fibrosis in baboons, consistent with several positive clinical trials. The negative outcome observed in other trials possibly reflects poor compliance resulting in irregular or low silymarin intake. Thus, in view of the innocuity of silymarin, it might be advisable in future clinical studies to insure the controlled administration of sufficient amounts of silymarin.

Acarbose attenuates experimental non-alcoholic steatohepatitis.

The alpha-glucosidase inhibitor acarbose is beneficial in the prevention of type 2 diabetes. To determine whether it attenuates the commonly associated non-alcoholic steatohepatitis (NASH), we used an experimental NASH model. Rats were fed ad libitum a nutritionally adequate high fat diet (71% of calories as fat) with or without acarbose (200 mg/1000 calories) for 3 weeks. All rats given the high fat diet only developed typical NASH whereas acarbose attenuated several of the characteristic hepatic alterations of NASH: there was less steatosis and inflammation, with a significant reduction in the mRNA of the hepatic inflammatory cytokine TNF-alpha and of its protein. There was also a decrease in the CYP2E1 mRNA and in collagen, with similar trends for CYP2E1 protein and procollagen mRNA. Because acarbose attenuates many of the hepatic alterations associated with experimental NASH, it is now indicated to determine whether it exerts similar beneficial effects in patients afflicted by this disease.

Gene expression profiling of alcoholic liver disease in the baboon (Papio hamadryas) and human liver.

The molecular pathogenesis of alcoholic liver disease (ALD) is not well understood. Gene expression profiling has the potential to identify new pathways and altered molecules in ALD. Gene expression profiles of ALD in a baboon model and humans were compared using DNA arrays. Reverse transcriptase-polymerase chain reaction and immunohistochemistry were used for downstream analysis of array results. cDNA array analysis revealed differential expression of several novel genes and pathways in addition to genes known to be involved in ALD pathogenesis. Overall gene expression profiles were similar in both species, with a majority of genes involved with fibrogenesis and xenobiotic metabolism, as well as inflammation, oxidant stress, and cell signaling. Genes associated with stellate cell activation (collagens, matrix metalloproteinases, tissue inhibitors of matrix metalloproteinase) were up-regulated in humans. Decreased expression of several metallothioneins was unexpected. Fourteen molecules related to the annexin family were up-regulated, including annexin A1 and A2. Immunofluorescence revealed a marked overexpression of annexin A2 in proliferating bile duct cells, hepatocyte cell surface, and selective co-localization with CD14-positive cells in human ALD. The gene expression profile of ALD is dominated by alcohol metabolism and inflammation and differs from other liver diseases. Annexins may play a role in the progression of fibrosis in ALD.