Binge Alcohol Is More Injurious to Liver in Female than in Male Rats: Histopathological, Pharmacologic, and Epigenetic Profiles.

Binge alcohol consumption is a health problem, but differences between the sexes remain poorly defined. We have examined the in vivo effects of three acute, repeat binge alcohol administration on the liver in male and female rats. Sprague-Dawley rats were gavaged with alcohol (5 g/kg body weight) three times at 12-hour intervals. Blood and liver tissues were collected 4 hours after the last binge ethanol. Subsequently, several variables were analyzed. Compared with male rats, females had higher levels of blood alcohol, alanine aminotransferase, and triglycerides. Liver histology showed increased lipid vesicles that were larger in females. Protein levels of liver cytochrome P4502E1 were higher in the liver of females than in the liver of males after binge. Hepatic phospho-extracellular signal-regulated kinase 1/2 and phosph-p38 mitogen-activated protein kinase levels were lower in females compared with males after binge alcohol, but no differences were found in the phospho-C-jun N-terminal kinase levels. Peroxisome proliferator-activated receptor γ-coactivator 1α and cyclic AMP response element binding (CREB) protein levels increased more in female than in male livers; however, increases in phospho-CREB levels were lower in females. Remarkably, c-fos was reduced substantially in the livers of females, but no differences in c-myc protein were found. Binge ethanol caused elevation in acetylated (H3AcK9) and phosphoacetylated (H3AcK9PS10) histone H3 in both sexes but without any difference. Binge alcohol caused differential alterations in the levels of various species of phosphatidylethanol and a larger increase in the diacylglycerol kinase-α protein levels in the liver of female rats compared with male rats. These data demonstrate, for the first time, similarities and differences in the sex-specific responses to repeat binge alcohol leading to an increased susceptibility of female rats to have liver injury in vivo. SIGNIFICANCE STATEMENT: This study examines the molecular responses of male and female rat livers to acute binge alcohol in vivo and demonstrates significant differences in the susceptibility between sexes.

Different mechanisms for histone acetylation by ethanol and its metabolite acetate in rat primary hepatocytes.

Ethanol and its major metabolite acetate both induced histone H3 acetylation in primary culture of rat hepatocytes. The acetylation by ethanol was dependent on the reactive oxygen species and mitogen-activated protein kinase pathway, whereas that by acetate was independent of both pathways. Ethanol increased CYP2E1 protein expression but acetate had negligible effect. The level of phospho-H2AX, an indicator of DNA breaks, was elevated by ethanol but not by acetate. Ethanol and acetate differentially activated mRNA expression for different genes, e.g., IL-6, PPARγ, c-Fos, Egr-1, and PNPLA3 in hepatocytes. The most striking increase (3-fold) was in PNPLA3 mRNA by ethanol with little change by acetate. It was further shown that acetate inhibited histone deacetylase activity. Taken together, these data establish for the first time that ethanol and acetate exhibit differences in their effects on hepatocytes in gene expression, P-H2AX levels, and the mechanism of histone H3 acetylation. The implications of these differences in the actions of ethanol in liver are discussed.

Binge ethanol and liver: new molecular developments.

Binge consumption of alcohol is an alarming global health problem. Binge (acute) ethanol (EtOH) is implicated in the pathophysiology of alcoholic liver disease (ALD). New studies from experimental animals and from humans indicate that binge EtOH has profound effects on immunological, signaling, and epigenetic parameters of the liver. This is in addition to the known metabolic effects of acute EtOH. Binge EtOH alters the levels of several cellular components and dramatically amplifies liver injury in chronically EtOH exposed liver. These studies highlight the importance of molecular investigations into binge effects of EtOH for a better understanding of ALD and also to develop therapeutic strategies to control it. This review summarizes these recent developments.

Histone H3 phosphorylation (Ser10, Ser28) and phosphoacetylation (K9S10) are differentially associated with gene expression in liver of rats treated in vivo with acute ethanol.

The epigenetic histone modification by ethanol is emerging as one of the mechanisms for its deleterious effects in the liver. In this context, we have investigated the role of histone H3 phosphorylation at Ser10 (P-H3-Ser10), and Ser28 (P-H3-Ser28) in liver after acute ethanol treatment in vivo. Ethanol was administered intraperitoneally in male Sprague-Dawley rats. Ethanol dose-response (1-5 g/kg body weight) and time-course (1-4 h) experiments were conducted, and various parameters were monitored. Steatosis and necrosis (serum alanine aminotransferase) of the liver increased in 4 h, suggesting liver injury. There were differences between P-H3-Ser10 and P-H3-Ser28 at 1 h, with the latter being more sensitive to lower ethanol doses. It was noteworthy that phosphorylation of both serines disappeared at the highest dose used (5 g/kg). We also examined phosphoacetylation of histone H3 at K9S10 and observed a dramatic increase. The changes in histone H3 phosphorylation and phosphoacetylation were also accompanied with expression of early response genes (c-fos, c-jun, mitogen-activated protein kinase phosphatase-1). Chromatin immunoprecipitation assays in samples from 1.5 and 4 h of ethanol administration indicated that increased histone H3 phosphorylation at Ser28 was associated with the promoters of c-jun and plasminogen activator inhibitor-1. In conclusion, this study demonstrates for the first time that in vivo exposure of liver to acute ethanol induced phosphorylation and phosphoacetylation of histone H3, and these modifications are differentially involved in the mRNA expression of genes.

A proteomic analysis of liver after ethanol binge in chronically ethanol treated rats.

BACKGROUND: Binge ethanol in rats after chronic ethanol exposure augments necrosis and steatosis in the liver. In this study, two-dimensional gel electrophoresis proteomic profiles of liver of control, chronic ethanol, control-binge, and chronic ethanol- binge were compared. RESULTS: The proteomic analysis identified changes in protein abundance among the groups. The levels of carbonic anhydrase 3 (CA3) were decreased after chronic ethanol and decreased further after chronic ethanol-binge. Ethanol binge alone in control rats had no effect on this protein suggesting its possible role in increased susceptibility to injury by binge after chonic ethanol treatment. A protein spot, in which both cytosolic isocitrate dehydrogenase (IDH1) and glutamine synthetase (GS) were identified, showed a small decrease after chronic ethanol binge but western blot demonstrated significant decrease only for glutamine synthetase in chronic ethanol treated rats. The level of gluathione S-transferase mu isoform (GSTM1) increased after chronic ethanol but was lower after chronic ethanol-binge compared to chronic ethanol treatment. The protein levels of the basic form of protein disulfide isomerase associated protein 3 (PDIA3) were significantly decreased and the acidic forms were increased after chronic ethanol- binge but not in chronic ethanol treated rats or ethanol binge in control rats. The significant changes in proteome profile in chronic ethanol binge were accompanied by a marked increase in liver injury as evidenced by enhanced steatosis, necrosis, increased 4-hydroxynonenal labeled proteins, CYP2E1 expression, and decreased histone H2AX phosphorylation. CONCLUSIONS: Given the role of CA3, IDH1 and GST in oxidative stress; PDIA3 in protein quality control, apoptosis and DNA repair and decreased glutamine synthetase as a sensitive marker of pericentral liver injury this proteome study of chronic ethanol-binge rat model identifies these proteins for the first time as molecular targets with potential role in progression of liver injury by binge ethanol drinking.

Gene-selective histone H3 acetylation in the absence of increase in global histone acetylation in liver of rats chronically fed alcohol.

AIMS: The aim of this study was to determine the effect of chronic ethanol feeding on acetylation of histone H3 at lysine 9 (H3-Lys9) at promoter and coding regions of genes for class I alcohol dehydrogenase (ADH I), inducible nitric oxide synthase (iNOS), Bax, p21, c-met and hepatocyte growth factor in the rat liver. METHODS: Rats were fed ethanol-containing liquid diet (5%, w/v) for 1-4 weeks. The global level of acetylation of H3-Lys9 in the liver was examined by western blot analysis. The levels of mRNA for various genes were measured by real-time reverse transcriptase-polymerase chain reaction. The association of acetylated histone H3-Lys9 with the different regions of genes was monitored by chromatin immunoprecipitation assay. RESULTS: Chronic ethanol treatment increased mRNA expression of genes for iNOS, c-jun and ADH 1. Chronic ethanol treatment did not cause increase in global acetylation of H3-Lys9, but significantly increased the association of acetylated histone H3-Lys9 in the ADH I gene, both in promoter and in coding regions. In contrast, chronic ethanol treatment did not significantly increase the association of acetylated histone H3-Lys9 with iNOS and c-jun genes. CONCLUSION: Chronic ethanol exposure increased the gene-selective association of acetylated H3-Lys9 in the absence of global histone acetylation. Thus, not all genes expressed by ethanol are linked to transcription via histone H3 acetylation at Lys9.

Elevated activation of ERK1 and ERK2 accompany enhanced liver injury following alcohol binge in chronically ethanol-fed rats.

BACKGROUND: Binge drinking after chronic ethanol consumption is one of the important factors contributing to the progression of steatosis to steatohepatitis. The molecular mechanisms of this effect remain poorly understood. We have therefore examined in rats the effect of single and repeat ethanol binge superimposed on chronic ethanol intake on liver injury, activation of mitogen-activated protein kinases (MAPKs), and gene expression. METHODS: Rats were chronically treated with ethanol in liquid diet for 4 weeks followed by single ethanol binge (5 gm/kg body weight) or 3 similar repeated doses of ethanol. Serum alcohol and alanine amino transferase (ALT) levels were determined by enzymatic methods. Steatosis was assessed by histology and hepatic triglycerides. Activation of MAPK, 90S ribosomal kinase (RSK), and caspase 3 were evaluated by Western blot. Levels of mRNA for tumor necrosis factor alpha (TNFα), early growth response-1 (egr-1), and plasminogen activator inhibitor-1 (PAI-1) were measured by real-time qRT-PCR. RESULTS: Chronic ethanol treatment resulted in mild steatosis and necrosis, whereas chronic ethanol followed by binge group exhibited marked steatosis and significant increase in necrosis. Chronic binge group also showed significant increase (compared with chronic ethanol alone) in the phosphorylation of extracellular regulated kinase 1 (ERK1), ERK2, and RSK. Phosphorylation of c-Jun N-terminal kinase (JNK) and p38 MAPK did not increase by the binge. Ethanol binge, after chronic ethanol intake, caused increase in mRNA for egr-1 and PAI-1, but not TNFα. CONCLUSIONS: Chronic ethanol exposure increases the susceptibility of rat liver to increased injury by 1 or 3 repeat binge. Among other alterations, the activated levels of ERK1, and more so ERK2, were remarkably amplified by binge suggesting a role of these isotypes in the binge amplification of the injury. In contrast, p38 MAPK and JNK1/2 activities were not amplified. These binge-induced changes were also reflected in the increases in the RNA levels for egr-1 and PAI-1. This study offers chronic followed by repeat binge as a model for the study of progression of liver injury by ethanol and highlights the involvement of ERK1 and ERK2 isotypes in the amplification of liver injury by binge ethanol.

Citrus flavonoids repress the mRNA for stearoyl-CoA desaturase, a key enzyme in lipid synthesis and obesity control, in rat primary hepatocytes.

Citrus flavonoids have been shown to decrease plasma lipid levels, improve glucose tolerance, and attenuate obesity. One possible mechanism underlying these physiological effects is reduction of hepatic levels of the mRNA for stearoyl-CoA desaturase-1 (SCD1), since repression of this enzyme reduces hyperlipidemia and adiposity. Here, we show that citrus flavonoids of two structural classes reduce SCD1 mRNA concentrations in a dose-dependent manner in rat primary hepatocytes. This is the first demonstration of repression of SCD1 by citrus flavonoids, either in vivo or in cultured cells. Furthermore, it is the first use of freshly-isolated hepatocytes from any animal to examine citrus flavonoid action at the mRNA level. This study demonstrates that regulation of SCD1 gene expression may play a role in control of obesity by citrus flavonoids and that rat primary hepatocytes are a physiologically-relevant model system for analyzing the molecular mechanisms of flavonoid action in the liver.

Differential changes in MAP kinases, histone modifications, and liver injury in rats acutely treated with ethanol.

BACKGROUND: Acute ethanol is known to affect cells and organs but the underlying molecular mechanisms are poorly explored. Recent developments highlight the potential importance of mitogen-activated protein kinases, MAPKs (i.e., ERK1/2, p38, and JNK1/2) signaling, and histone modifications (i.e., acetylation, methylation, and phosphorylation) in the actions of ethanol in hepatocytes. We have therefore investigated significance of these molecular steps in vivo using a model in which rats were acutely administered ethanol intraperitoneally (IP). METHODS: Ethanol was administered IP (3.5 gm/kg body weight) to 12-week-old male Sprague-Dawley rats. Liver was subsequently removed at 1 and 4 hours. Serum was used for alcohol and ALT assays. At the time of the removal of liver, small portions of each liver were formalin-fixed and stained with hematoxylin and eosin (H&E) and used for light microscopy. Western blot analysis was carried out with specific primary antibodies for various parameters. RESULTS: There were clear differences at 1 and 4 hours in blood ethanol, ALT, steatosis, and cleaved caspase 3. Apoptosis at 1 hour was followed by necrosis at 4 hours. Acute alcohol elicited a marked increase in the phosphorylation of ERK1/2 and moderate increases in the phosphorylation of p38 MAPK and JNK. Temporally different phosphorylation of histone H3 at ser-10 and ser-28 occurred and acetylation of histone H3 at lys 9 increased progressively. CONCLUSIONS: There were distinct differences in the behavior of the activation of the 3 MAP kinases and histone modifications after acute short exposure of liver to ethanol in vivo. Although all 3 MAPKs were rapidly activated at 1 hour, the necrosis, occurring at 4 hours, correlated to sustained activation of ERK1/2. Transient activation of p38 is associated with rapid phosphorylation of histone H3, whereas prolonged activation of ERK1/2 is correlated to persistent histone H3 acetylation.

Surrogate alcohols and their metabolites modify histone H3 acetylation: involvement of histone acetyl transferase and histone deacetylase.

BACKGROUND: Ethanol increases histone H3 acetylation in the rat liver. However, the effect of other carbon chain length alcohols, consumed as surrogate alcohols and used in industry, on H3 acetylation is unknown. Hence, we investigated the effect of these alcohols on histone H3 acetylation, cell toxicity and HAT and HDAC activity. METHODS: Primary cultures of rat hepatocytes were incubated with selected concentration (40 mM) of different chain length alcohols with or without inhibitors of alcohol metabolizing enzymes. Cells were also treated with low concentration (2.5 mM) of 1-propanol or 1-butanol or isopentanol, with or without 40 mM ethanol for 24 hours. Effects of the metabolites of these alcohols were also studied. Cytotoxicity was determined by lactate dehydrogenase (LDH) release and mitochondrial activity (MTT assay). The degree of histone H3 acetylation at specific lysine residues were monitored by western bloting using site specific antibodies. Histone acetyltransferase (HAT) and histone deacetylase (HDAC) activities were measured by enzyme-linked immunosorbent assay (ELISA) and colorimetric assay respectively. RESULTS: Alcohols with increasing carbon chain length exhibited a variable effect on the ratio of acetylated lys9 histone H3 to beta-actin. A graded increase (methanol < ethanol < 1-propanol < 1-butanol) followed by a gradual decrease (1-butanol > 1-pentanol > 1-hexanol > 1-octanol) in the ratio was observed. Other lysine sites were not affected. HAT activation also corresponded to the acetylation profile. These alcohols or their metabolites did not significantly alter HDAC activity in the hepatocytes. Low concentration (2.5 mM) of 1-propanol alone did not affect acetylation, but sensitized the ethanol induced H3 acetylation at lysine 9 (H3AcK9). 1-Butanol and isopentanol also increased the response of ethanol induced H3AcK9. Alcohol metabolizing inhibitors attenuated ethanol and propanol induced increase in H3AcK9. Carboxylic acid metabolites of these alcohols also increased HAT activity and histone H3 acetylation at lysine 9. Propionate and butyrate modestly inhibited HDAC activity in an in vitro assay. CONCLUSIONS: Surrogate alcohols modulate H3AcK9 via increasing HAT activity and this is dependent on their metabolism. Furthermore, alcohol metabolites also increased H3AcK9, but in contrast, exhibit both HAT activation and HDAC inhibition.

Emerging role of epigenetics in the actions of alcohol.

This review deals with the recent developments on the epigenetic effects of ethanol. A large body of data have come from studies in liver and in neuronal systems and involve post-translational modifications in histones and methylations in DNA. Ethanol causes site selective acetylation, methylation, and phosphorylation in histone. With respect to methylations the methyl group donating system involving S-adenosyl methionine appears to play a central role. There is contrasting effect of acetylation versus methylation on the same site of histone, as it relates to the transcriptional activation. Epigenetic memory also appears to correlate with liver pathology and Mallory body formation. Experimental evidence supports transcriptional regulation of genes in the CNS by DNA methylations. These studies are contributing towards a better understanding of a novel epigenetic regulation of gene expression in the context of alcohol. The critical steps and the enzymes (e.g., histone acetyltransferase, histone deacetylase, DNA methyltransferase) responsible for the epigenetic modifications are prime targets for intense investigation. The emerging data are also beginning to offer novel insight towards defining the molecular actions of ethanol and may contribute to potential therapeutic targets at the nucleosomal level. These epigenetic studies have opened up a new avenue of investigation in the alcohol field.

Ethanol inhibition of angiotensin II-stimulated Tyr705 and Ser727 STAT3 phosphorylation in cultured rat hepatocytes: relevance to activation of p42/44 mitogen-activated protein kinase.

Angiotensin (Ang) II-stimulated phosphorylation of signal transducer and activator transcription (STAT) 3 in rat hepatocytes and the effects of ethanol on this activation were investigated. Angiotensin II (100 nM) stimulated Tyr705 and Ser727 phosphorylation of STAT3 and formation of sis-inducing factor complexes. In the presence of U-0126 (10microM), a p42/44 mitogen-activated protein kinase (MAPK) kinase inhibitor, Ang II further increased Tyr705 phosphorylation of STAT3 but completely abrogated Ser727 phosphorylation of STAT3. Inhibition of p42/44MAPK also increased STAT3 DNA-binding activity. Pretreatment with ethanol (100mM) for 24h resulted in decrease in Tyr705 phosphorylation of STAT3 by ethanol alone and inhibition of Tyr705 phosphorylation of STAT3 stimulated by Ang II. Although ethanol potentiates Ang II stimulated p42/44 MAPK activation in hepatocytes, ethanol inhibited Ser727 phosphorylation of STAT3 stimulated by Ang II. Angiotensin II-stimulated STAT3-binding activity was not significantly affected by ethanol treatment. These results suggest a negative regulation of Ang II-stimulated STAT3 tyrosine phosphorylation and STAT3-binding activity through p42/44 MAPK activation in hepatocytes. However, ethanol modulation of Ang II-stimulated STAT3 phosphorylation occurs by MAPK independent mechanisms. Ethanol potentiation of MAPK signaling without suppression of STAT3 function may modulate the course of alcoholic liver injury.

Platelets from diabetic pigs exhibit hypersensitivity to thrombin.

Responses of platelets from diabetic and diabetic-hyperlipidemic pigs were studied. Pigs were made diabetic with single dose of alloxan, which acts by selectively destroying insulin-producing pancreatic beta cells thus inducing type 1 diabetes. Pigs were kept for 1 or 12 wk, during which thrombin-induced aggregation was monitored in washed platelets. The platelets showed increased sensitivity to aggregation within 1 wk of induction of diabetes. Hyperlipidemia alone for 12 wk did not increase platelet hypersensitivity, but hyperlipidemia together with diabetes significantly increased thrombin-induced platelet aggregation. Because this hypersensitivity occurred in washed platelets, this characteristic appears to be independent of any contribution by plasma factors or other blood cells. The hypersensitivity of platelets from diabetic pigs correlated with decreased activity of mitogen-activated protein kinase. These studies offer the first evidence that platelet hyperactivity occurs during the early stages (within a week) of induction of diabetes in pigs and before manifestation of other cardiovascular problems.

Acetaldehyde alters MAP kinase signalling and epigenetic histone modifications in hepatocytes.

Although both oxidative and non-oxidative metabolites of ethanol are involved in generating ethanol matabolic stress (Emess), the oxidative metabolite acetaldehyde plays a critical role in the cellular actions of ethanol. We have investigated the effects of acetaldehyde on p42/44 MAP kinase, p46/p54 c-jun N-terminal kinase (JNK1/JNK2) and p38 MAP kinase in hepatocytes. Acetaldehyde caused temporal activation of p42/44 MAPK followed by JNK, but the activation of the p42/44 MAPK was not a prerequisite for the JNK activation. Activation ofJNK1 by acetaldehyde was greater than JNK2. Ethanol and acetaldehyde activatedJNK have opposing roles; ethanol-induced JNK activation increased apoptosis whereas that by acetaldehyde decreased apoptosis. Acetaldehyde also caused histone H3 acetylation at Lys9 and phosphorylation of histone H3 at Serl0 and 28, the latter being dependent on p38 MAP kinase. Phosphorylation at Ser28 was higher than at Serl0. Thus acetaldehyde distinctively alters MAP kinase signalling and histone modifications, processes involved in transcriptional activation.

Histone H3 phosphorylation at serine 10 and serine 28 is mediated by p38 MAPK in rat hepatocytes exposed to ethanol and acetaldehyde.

Ethanol modulates mitogen-activated protein kinases (MAPKs). We have now investigated the influence of ethanol and its metabolite, acetaldehyde on histone H3 phosphorylation to ascertain downstream targets of MAPKs. In primary culture of rat hepatocytes, ethanol and acetaldehyde increased phosphorylation of nuclear histone H3 at serine 10 and serine 28. Specific inhibitors of p38 MAPK, SB203580, PD169316 and SB202190 blocked this phosphorylation. The inactive analogue, SB202474 had no effect. In contrast, c-Jun N-terminal kinase (JNK) inhibitor, SP600125 or MAP/ERK kinase (MEK) 1/2 inhibitor, PD98059 had no effect on the histone H3 phosphorylation. The p38 MAPK activation correlated with upstream activation of MAPK kinase (MKK) 3/6 but was independent of protein synthesis. In the nuclear fraction, the phosphorylation of p38 MAPK and its protein level increased with peak activation at 24 h by ethanol and at 30 min by acetaldehyde. These responses were ethanol and acetaldehyde dose dependent. Surprisingly, the phosphorylation of p38 MAPK was undetectable in the cytosolic fraction suggesting a subcellular selectivity of p38 MAPK signaling. The phosphorylation of JNK and p42/44 MAPK and their protein levels also increased in the nuclear fraction. Although ethanol caused translocation of all three major MAPKs (p42/44 MAPK, JNK, p38 MAPK) into the nucleus, histone H3 phosphorylation at serine 10 and serine 28 was mediated by p38 MAPK. This histone H3 phosphorylation had no influence on ethanol and acetaldehyde induced apoptosis. These studies demonstrate for the first time that ethanol and acetaldehyde stimulated phosphorylation of histone H3 at serine 10 and serine 28 are downstream nuclear response mediated by p38 MAPK in hepatocytes.

Distinct methylation patterns in histone H3 at Lys-4 and Lys-9 correlate with up- & down-regulation of genes by ethanol in hepatocytes.

Ethanol induced liver injury is associated with a global change in gene expression but its mechanisms are not known. We studied whether alcohol-induced gene expression is associated with post-translational methylations of histone H3. Primary culture of rat hepatocytes was treated with ethanol (50 or 100 mM) for 24 h and the status of methylation of H3 at lys 4 (H3dimeK4) or lys 9 (H3dimeK9) was monitored by Western blotting using antibodies to dimethylated histone H3 at lys 4 or lys 9. The cells exposed to ethanol showed strikingly opposing behaviors in methylation patterns; H3dimeK9 methylation was decreased whereas H3dimeK4 increased. Similar results were obtained in the interphase nuclei. Their binding on the metaphase chromosomes exhibits distinct site specific pattern of accumulation. Next, chromatin immunoprecipitation of the ethanol treated samples with antibodies for methylated lys 4 or lys 9 histone H3 followed by amplification of the immunoprecipitated DNA, was used to determine their association with the promoters of genes up- or downregulated by ethanol. Lys4 methylation was associated with ethanol upregulated genes (Adh, GST-yc2) whereas lys 9 methylation with downregulated genes (Lsdh, cytP4502c11) demonstrating a difference between these two methylations. These results suggest that exposure of hepatocytes to ethanol changes the expression of several susceptible genes which are associated with site specific modification of dimethylated forms of histone H3 amino termini at their regulatory regions.

Binge alcohol alters PNPLA3 levels in liver through epigenetic mechanism involving histone H3 acetylation.

The human PNPLA3 (patatin-like phospholipase domain-containing 3) gene codes for a protein which is highly expressed in adipose tissue and liver, and is implicated in lipid homeostasis. While PNPLA3 protein contains regions homologous to functional lipolytic proteins, the regulation of its tissue expression is reflective of lipogenic genes. A naturally occurring genetic variant of PNPLA3 in humans has been linked to increased susceptibility to alcoholic liver disease. We have examined the modulatory effect of alcohol on PNPLA3 protein and mRNA expression as well as the association of its gene promoter with acetylated histone H3K9 by chromatin immunoprecipitation (ChIP) assay in rat hepatocytes in vitro, and in vivo in mouse and rat models of acute binge, chronic, and chronic followed by acute binge ethanol administration. Protein expression of PNPLA3 was significantly increased by alcohol in all three models used. PNPLA3 mRNA also increased, albeit to a varying degree. ChIP assay using H3AcK9 antibody showed increased association with the promoter of PNPLA3 in hepatocytes and in mouse liver. This was less evident in rat livers in vivo except under chronic treatment. It is concluded for the first time that histone acetylation plays a role in the modulation of PNPLA3 levels in the liver exposed to binge ethanol both in vitro and in vivo.

Role of Ras in ethanol modulation of angiotensin II activated p42/p44 MAP kinase in rat hepatocytes.

Angiotensin II plays a role in both liver cell proliferation and liver injury but the effects of ethanol on angiotensin II signaling in liver are not clearly understood. We have investigated the role of Ras in ethanol modulation of p42/p44 mitogen-activated protein kinase (MAPK) stimulated by angiotensin II (Ang II) in primary cultures of rat hepatocytes. Hepatocytes were incubated with ethanol (100 mM) for 24 h, then stimulated with Ang II (100 nM). The level of p42/p44 MAPK phosphorylation was measured by Western blot analysis and Ras activation was assessed by specific binding of Ras-GTP (activated form) to a GST-RBD fusion protein containing Ras-binding domain (RBD) of Raf-1. Ethanol potentiated p42/p44 MAPK activation by Ang II, whereas ethanol alone did not significantly affect phosphorylation of p42/p44 MAPK. Ang II increased Ras activity by about 2 fold. Ethanol exposure increased Ang II stimulated Ras activity by an additional about 2 fold. Ethanol alone elicited a small increase in basal Ras activity. Pretreatment with manumycin A (10 microM), a Ras farnesylation inhibitor, partially blocked both Ang II-activated and ethanol-potentiated MAPK activities. These data provided the first evidence that ethanol potentiation of Ang II stimulated p42/p44 MAPK is mediated, in part, by Ras in hepatocytes.

Epigenetic effects of ethanol on liver and gastrointestinal injury.

Alcohol consumption causes cellular injury. Recent developments indicate that ethanol induces epigenetic alterations, particularly acetylation, methylation of histones, and hypo- and hypermethylation of DNA. This has opened up a new area of interest in ethanol research and is providing novel insight into actions of ethanol at the nucleosomal level in relation to gene expression and patho-physiological consequences. The epigenetic effects are mainly attributable to ethanol metabolic stress (Emess), generated by the oxidative and non-oxidative metabolism of ethanol, and dysregulation of methionine metabolism. Epigenetic changes are important in ethanol-induced hepatic steatosis, fibrosis, carcinoma and gastrointestinal injury. This editorial highlights these new advances and its future potential.

Angiotensin II activation of focal adhesion kinase and pp60c-Src in relation to mitogen-activated protein kinases in hepatocytes.

We have investigated signaling pathways leading to angiotensin II (Ang II) activation of mitogen-activated protein kinase (MAPK) in hepatocytes. MAPK activation by Ang II was abolished by the Ang II type 1 (AT1) receptor antagonist losartan, but not by the Ang II type 2 (AT2) receptor antagonist PD123319. Ang II (100 nM) induced a rapid phosphorylation of Src (peak approximately 2 min) and focal adhesion kinase (FAK, peak approximately 5 min) followed by a decrease to basal levels in 30 min. An increased association between FAK and Src in response to Ang II was detected after 1 min, which declined to basal levels after 30 min. Treatment with the Src kinase inhibitor PP-1 inhibited FAK phosphorylation. Downregulation of PKC, intracellular Ca2+ chelator BAPTA or inhibitors of PKC, Src kinase, MAPK kinase (MEK), Ca2+/calmodulin dependent protein kinase, phosphatidylinositol 3-kinase all blocked Ang II-induced MAPK phosphorylation. In contrast to other cells, there was no evidence for the role of EGF receptor transactivation in the activation of MAPK by Ang II. However, PDGF receptor phosphorylation is involved in the Ang II stimulated MAPK activation. Furthermore, Src/FAK and Ca/CaM kinase activation serve as potential links between the Ang II receptor and MAPK activation. These studies offer insight into the signaling network upstream of MAPK activation by AT1 receptor in hepatocytes.