Downregulation of long non-coding RNA AIRN promotes mitophagy in alcoholic fatty hepatocytes by promoting ubiquitination of mTOR.

Long non-coding RNAs (lncRNAs) are crucial in chronic liver diseases, but the specific molecular mechanism of lncRNAs in alcoholic fatty liver (AFL) remains unclear. In this study, we investigated the in-depth regulatory mechanism of mTOR affected by AIRN non-protein coding RNA (lncRNA-AIRN) in the development of AFL. LncRNA-AIRN was highly expressed in the liver tissues of AFL C57BL/6mice and oleic acid+alcohol (O+A)treated AML-12cells by using quantitative real-timePCR. RNA pull-down and RNA immunoprecipitation experiments demonstrated that there was an interaction between lncRNA-AIRN and mTOR, and that interference with lncRNA-AIRN could promote the mTOR protein level. Results ofcycloheximide-chase assay showed that the proteinlevel of mTOR was decreased with the treatment time after the knockdown of lncRNA-AIRN. Furthermore, the knockdown of lncRNA-AIRN reducedmTOR protein level by promoting the E3 ubiquitin ligase FBXW7-mediated ubiquitination.The lncRNA-AIRN/mTORaxis was involved in the regulation of the mitophagy of O+A treated hepatocytes, which was confirmed by the cell transfection and the MTT assay.SPSS 16.0 was used for analyzing data. The difference between the two groups was analyzed by performing Student's t-test, and ANOVA was used to analyze the difference when more than two groups. P values < 0.05 were considered to be significantly different.Our findings demonstrated that the knockdown of lncRNA-AIRN influencedmitophagy in AFL by promoting mTOR ubiquitination.

Activity of Total Alcohol Dehydrogenase, Alcohol Dehydrogenase Isoenzymes and Aldehyde Dehydrogenase in the Serum of Patients with Alcoholic Fatty Liver Disease.

Background and objectives: The aim of the current study was to assess the use of determinations of total alcohol dehydrogenase and the activity of its isoenzymes as well as aldehyde dehydrogenase in the serum of patients with alcohol liver disease. Materials and Methods: The testing was performed on the serum of 38 patients with alcoholic fatty liver (26 males and 12 females aged 31-75). The total activity of ADH was determined by the colorimetric method. The activity of ADH I and ADH II, as well as ALDH, was determined by the spectrofluorometric method using fluorogenic specific substrates. The activity of isoenzymes of other classes was determined by spectrophotometric methods using substrates. Results: A statistically significantly higher ADH I activity was noted in the serum of patients with alcoholic fatty liver (4.45 mIU/L) compared to the control group (2.04 mIU/L). A statistically significant increase in the activity was also noted for the class II alcohol dehydrogenase isoenzyme (29.21 mIU/L, control group: 15.56 mIU/L) and the total ADH (1.41 IU/L, control group: 0.63 IU/L). Conclusions: The obtained results imply the diagnostic usefulness of the determination of AHD total, ADH I, and ADH II activity in the serum of patients with alcoholic fatty liver.

Downregulation of mitogen-activated protein kinases (MAPKs) in chronic ethanol-induced fatty liver.

Mitogen-activated protein kinases (MAPKs) are versatile proteins that have been suggested to be involved in the regulation of lipid metabolism. This study was designed to investigate the responses of MAPK signaling to chronic ethanol exposure in vivo and in vitro, and try to explore its role in the pathogenesis of alcoholic fatty liver (AFL). Mice were fed with Lieber-Decarli liquid diet (5% ethanol, w/v) for 4 weeks to induce fatty liver, and the chronological changes of MAPK phosphorylation were measured using western blotting. We found that chronic ethanol feeding led to accumulation of triglyceride (TG), decreased phosphorylation of MAPKs, decreased protein level of peroxisomal proliferator activation receptor α (PPARα), and increased protein expression of cytochrome P4502E1 (CYP2E1) in mice liver. In vitro study showed that overexpression of CYP2E1 blunted the response of MAPKs to ethanol, and MAPK phosphatase 1 (MKP-1) knockdown by siRNA led to upregulation of PPARα protein level. Lastly, epidermal growth factor (EGF), a well-known MAPK activator, significantly suppressed chronic ethanol-induced hepatic fat accumulation and decline of PPARα expression in mice liver. Collectively, MAPK suppression, possibly due to the activation of hepatic CYP2E1, may be involved in chronic ethanol-induced hepatic steatosis.

Nanoformulated SOD1 ameliorates the combined NASH and alcohol-associated liver disease partly via regulating CYP2E1 expression in adipose tissue and liver.

Enhanced free fatty acid (FFA) flux from adipose tissue (AT) to liver plays an important role in the development of nonalcoholic steatohepatitis (NASH) and alcohol-associated liver disease (AALD). We determined the effectiveness of nanoformulated superoxide dismutase 1 (Nano) in attenuating liver injury in a mouse model exhibiting a combination of NASH and AALD. Male C57BL6/J mice were fed a chow diet (CD) or a high-fat diet (HF) for 10 wk followed by pair feeding of the Lieber-DeCarli control (control) or ethanol (ET) diet for 4 wk. Nano was administered once every other day for the last 2 wk of ET feeding. Mice were divided into 1) CD + control diet (CD + Cont), 2) high-fat diet (HF) + control diet (HF + Cont), 3) HF + Cont + Nano, 4) HF + ET diet (HF + ET), and 5) HF + ET + Nano. The total fat mass, visceral AT mass (VAT), and VAT perilipin 1 content were significantly lower only in HF + ET-fed mice but not in HF + ET + Nano-treated mice compared with controls. The HF + ET-fed mice showed an upregulation of VAT CYP2E1 protein, and Nano abrogated this effect. We noted a significant rise in plasma FFAs, ALT, and monocyte chemoattractant protein-1 in HF + ET-fed mice, which was blunted in HF + ET + Nano-treated mice. HF + ET-induced increases in hepatic steatosis and inflammatory markers were attenuated upon Nano treatment. Nano reduced hepatic CYP2E1 and enhanced catalase levels in HF + ET-fed mice with a concomitant increase in SOD1 protein and activity in liver. Nano was effective in attenuating AT and liver injury in mice exhibiting a combination of NASH and AALD, partly via reduced CYP2E1-mediated ET metabolism in these organs.NEW & NOTEWORTHY Increased free fatty acid flux from adipose tissue (AT) to liver accompanied by oxidative stress promotes nonalcoholic steatohepatitis (NASH) and alcohol-associated liver injury (AALD). Obesity increases the severity of AALD. Using a two-hit model involving a high-fat diet and chronic ethanol feeding to mice, and treating them with nanoformulated superoxide dismutase (nanoSOD), we have shown that nanoSOD improves AT lipid storage, reduces CYP2E1 in AT and liver, and attenuates the combined NASH/AALD in mice.

Alcoholic and non-alcoholic fatty liver disease: Focus on ceramide.

Sphingolipids are class of metabolically distinct lipids that play structural and signaling functions in all organisms. Sphingolipid metabolism is deregulated during various diseases such as cancer, neurological and immune disorders, and metabolic syndrome. With the advancement of sphingo-lipidomics and sphingo-genomics, an understanding of the specific roles of ceramide, the quintessential bioactive sphingolipid, in fatty liver disease has taken shape. Two major pathways for ceramide generation, the de novo pathway and the sphingomyelinase pathway are activated in the course of both, the non-alcoholic and the alcoholic, forms of fatty liver disease. The mechanisms of activation of these two pathways are distinct and reflect the different disease etiology in each case; at the same time, common processes impacted by the resulting ceramide overproduction involve lipotoxocity, ER/mitochondrial stress, inflammation, and de-regulation of hepatic lipid metabolism. Studies in human patients and animal models have delineated specific enzymes and ceramide species that are involved at the different stages of the disease, and represent novel pharmaceutical targets for successful management of fatty liver disease.

A novel role for ceramide synthase 6 in mouse and human alcoholic steatosis.

Perilipin 2 (PLIN2) is a lipid-droplet protein that is up-regulated in alcoholic steatosis and associated with hepatic accumulation of ceramides, bioactive lipids implicated in alcoholic liver disease pathogenesis. The specific role of ceramide synthetic enzymes in the regulation of PLIN2 and promotion of hepatocellular lipid accumulation is not well understood. We examined the effects of pharmacologic ceramide synthesis inhibition on hepatic PLIN2 expression, steatosis, and glucose and lipid homeostasis in mice with alcoholic steatosis and in ethanol-incubated human hepatoma VL17A cells. In cells, pharmacologic inhibition of ceramide synthase reduced lipid accumulation by reducing PLIN2 RNA stability. The subtype ceramide synthase (CerS)6 was specifically up-regulated in experimental alcoholic steatosis in vivo and in vitro and was up-regulated in zone 3 hepatocytes in human alcoholic steatosis. In vivo ceramide reduction by inhibition of de novo ceramide synthesis reduced PLIN2 and hepatic steatosis in alcohol-fed mice, but only de novo synthesis inhibition, not sphingomyelin hydrolysis, improved glucose tolerance and dyslipidemia. These findings implicate CerS6 as a novel regulator of PLIN2 and suggest that ceramide synthetic enzymes may promote the earliest stage of alcoholic liver disease, alcoholic steatosis.-Williams, B., Correnti, J., Oranu, A., Lin, A., Scott, V., Annoh, M., Beck, J., Furth, E., Mitchell, V., Senkal, C. E., Obeid, L., Carr, R. M. A novel role for ceramide synthase 6 in mouse and human alcoholic steatosis.

Supplementation of all trans retinoic acid ameliorates ethanol-induced endoplasmic reticulum stress.

CONTEXT: Molecular pathogenesis of chronic alcoholism is linked to increased endoplasmic reticulum stress. Ethanol is a competitive inhibitor of vitamin A metabolism and vitamin A supplementation aggravates existing liver problems. Hence, we probed into the impact of supplementation of all trans retinoic acid (ATRA), the active metabolite of vitamin A on ethanol-induced endoplasmic reticulcum stress. METHODS: Male Sprague-Dawley rats were divided into four groups - I: Control; II: Ethanol; III: ATRA; IV: ATRA + Ethanol. After 90 days the animals were sacrificed to study markers of lipid peroxidation in hepatic microsomal fraction and expression of ER stress proteins and apoptosis in liver. RESULTS AND CONCLUSION: Ethanol caused hepatic hyperlipidemia, enhanced microsomal lipid peroxidation, upregulated expression of unfolded protein response associated proteins and that of apoptosis. Ethanol also led to downregulation of retinoid receptors. ATRA supplementation reversed all these alterations indicating the decrease in ethanol-induced endoplasmic reticulum stress.

Hepatoprotective Effect of Albumin Peptides from Corn Germ Meal on Chronic Alcohol-Induced Liver Injury in Mice.

Despite the fact that chronic and excessive alcohol consumption is a risk factor for many chronic diseases, such as a fatty liver disease, the addictive power of alcohol is strong worldwide. Corn germ meal albumin peptides (CGMAPs), by-products in corn germ oil industry have often been considered as wastes disposal in food processing. The aim of this study was to investigate the hepatoprotective effect of CGMAPs on chronic alcohol-induced liver injury in a mouse model. The corn germ meal-derived albumin was enzymatically hydrolysed, and the albumin peptides fractions (APFs) with Mw < 1 kDa (APF4) was collected. APF4 was an oligopeptide with a high Fischer's ratio (F > 3), rich in glutamic, alanine, leucine and proline. The hydrophobic Q value was 5.1, indicating the property of high enrichment in hydrophobic amino acids. Alcohol administration significantly increased the activities and levels of hepatic aminotransferase (AST), alanine aminotransferase (ALT), malondialdehyde (MDA), and triglycerides (TG) (P < 0.01), and significantly reduced the activities of superoxide dismutase (SOD) and catalase (CAT) and levels of glutathione (GSH) (P < 0.01) compared to the control group. Those changes were significantly reversed by the application of APF4 at 800 mg/kg bw. Thus, APF4 of CGMAPs had a significant protective effect against chronic alcohol-induced liver injury through enhancement of in vivo antioxidant ability as a possible mechanism of action, which therefore suggested that APF4 might be useful as natural sources to protect liver from alcoholic damage. PRACTICAL APPLICATION: Corn germ meal albumin peptides (CGMAPs) of Mw < 1 kDa, a kind of bioactive peptides which could effectively improve alcohol metabolism and protect against the hepatic damage induced by alcohol, might be useful as natural sources to protect liver from alcoholic damage.

Inhibition of aldose reductase ameliorates ethanol­induced steatosis in HepG2 cells.

Aldose reductase (AR) expression is increased in liver tissue of patients with ethanol­induced liver disease. However, the exact role of AR in the development of ethanol­induced liver disease has yet to be elucidated. The present study aimed to determine the effect of an AR inhibitor on ethanol­induced steatosis in HepG2 cells and to identify possible underlying molecular mechanisms. Steatosis was induced in HepG2 cells by stimulating cells with 100 mM absolute ethanol for 48 h. Oil Red O staining was used to detect the lipid droplet accumulation in cells. Western blot analyses were used to determine protein expression levels and reverse transcription­quantitative polymerase chain reaction was used to analyze mRNA expression levels. The results showed that AR protein expression was elevated in HepG2 cells stimulated with ethanol. HepG2 cells exhibited marked improvement of ethanol­induced lipid accumulation following treatment with the AR inhibitor zopolrestat. Phosphorylation levels of 5' adenosine monophosphate­activated protein kinase (AMPK) were markedly higher, whereas the mRNA expression levels of sterol­regulatory element­binding protein (SREBP)­1c and fatty acid synthase (FAS) were significantly lower in zopolrestat­treated and ethanol­stimulated HepG2 cells compared with in untreated ethanol­stimulated HepG2 cells. In addition, zopolrestat inhibited the ethanol­induced expression of tumor necrosis factor (TNF)­α. These results suggested that zopolrestat attenuated ethanol­induced steatosis by activating AMPK and subsequently inhibiting the expression of SREBP­1c and FAS, and by suppressing the expression of TNF­α in HepG2 cells.

Intestinal Alkaline Phosphatase Attenuates Alcohol-Induced Hepatosteatosis in Mice.

BACKGROUND AND AIMS: Bacterially derived factors from the gut play a major role in the activation of inflammatory pathways in the liver and in the pathogenesis of alcoholic liver disease. The intestinal brush-border enzyme intestinal alkaline phosphatase (IAP) detoxifies a variety of bacterial pro-inflammatory factors and also functions to preserve gut barrier function. The aim of this study was to investigate whether oral IAP supplementation could protect against alcohol-induced liver disease. METHODS: Mice underwent acute binge or chronic ethanol exposure to induce alcoholic liver injury and steatosis ± IAP supplementation. Liver tissue was assessed for biochemical, inflammatory, and histopathological changes. An ex vivo co-culture system was used to examine the effects of alcohol and IAP treatment in regard to the activation of hepatic stellate cells and their role in the development of alcoholic liver disease. RESULTS: Pretreatment with IAP resulted in significantly lower serum alanine aminotransferase compared to the ethanol alone group in the acute binge model. IAP treatment attenuated the development of alcohol-induced fatty liver, lowered hepatic pro-inflammatory cytokine and serum LPS levels, and prevented alcohol-induced gut barrier dysfunction. Finally, IAP ameliorated the activation of hepatic stellate cells and prevented their lipogenic effect on hepatocytes. CONCLUSIONS: IAP treatment protected mice from alcohol-induced hepatotoxicity and steatosis. Oral IAP supplementation could represent a novel therapy to prevent alcoholic-related liver disease in humans.

Phosphatase and tensin homolog is a differential diagnostic marker between nonalcoholic and alcoholic fatty liver disease.

AIM: To investigate the protein expression of phosphatase and tensin homolog (PTEN) in human liver biopsies of patients with alcoholic and non-alcoholic liver disease. METHODS: PTEN protein expression was assessed by immunohistochemistry in formalin-fixed, paraffin-embedded liver sections of patients with non-alcoholic fatty liver disease (NAFLD) (n = 44) or alcoholic liver disease (ALD) (n = 25). Liver resections obtained from 3 healthy subjects candidate for partial liver donation served as controls. Histological evaluations were performed by two experienced pathologists, and diagnoses established based on international criteria. The intensity of the PTEN staining in nuclei was compared between steatotic and non-steatotic areas of each liver fragment analyzed. For each liver specimen, the antibody-stained sections were examined and scored blindly by three independent observers, who were unaware of the patients' clinical history. RESULTS: In healthy individuals, PTEN immunostaining was intense in both the cytoplasm and nuclei of all hepatocytes. However, PTEN was strongly downregulated in both the nucleus and the cytoplasm of hepatocytes from steatotic areas in patients with NAFLD, independently of the disease stage. In contrast, no changes in PTEN protein expression were observed in patients with ALD, regardless of the presence of steatosis or the stage of the disease. The degree of PTEN downregulation in hepatocytes of patients with NAFLD correlated with the percentage of steatosis (r = 0.3061, P = 0.0459) and the BMI (r = 0.4268, P = 0.0043). Hovewer, in patients with ALD, PTEN expression was not correlated with the percentage of steatosis with or without obesity as a confounding factor (P = 0.5574). Finally, PTEN expression level in steatotic areas of ALD patients was significantly different from that seen in steatotic areas of NAFLD patients (P < 0.0001). CONCLUSION: PTEN protein expression is downregulated early in NAFLD, but not in ALD. PTEN immunohistochemical detection could help in the differential diagnosis of NAFLD and ALD.

Hepatic Peroxisome Proliferator-Activated Receptor Gamma Signaling Contributes to Alcohol-Induced Hepatic Steatosis and Inflammation in Mice.

BACKGROUND: Peroxisome proliferator-activated receptor gamma (PPARγ) signaling has been shown to regulate lipogenesis and lipid accumulation. Previous studies have shown that hepatic PPARγ is up-regulated in steatotic liver of both animal and human. However, the effects of hepatic PPARγ signaling on alcoholic liver disease (ALD) remain elusive. METHODS: To determine the role of hepatic PPARγ signaling on ALD, wild-type (WT) and hepatocyte-specific PPARγ knockdown (PPARγ∆Hep) mice were fed a modified Lieber-DeCarli alcohol or isocaloric maltose dextrin control liquid diet for 8 weeks to induce ALD. Blood parameters, hepatic steatosis, and inflammation were measured after 8-week alcohol feeding. RESULTS: Alcohol feeding to WT mice resulted in liver damage (alanine aminotransferase [ALT], 94.68 ± 17.05 U/L; aspartate aminotransferase [AST], 55.87 ± 11.29 U/L), which was significantly alleviated by hepatic PPARγ knockdown (ALT, 57.36 ± 14.98 U/L; AST, 38.06 ± 3.35 U/L). Alcohol feeding led to marked lipid accumulation and up-regulation of lipogenic genes including fatty acid transport protein 1 (FATP1), acetyl-CoA carboxylase (ACC), fatty acid synthase (FASN), lipin1 (LIPIN1), diacylglycerol acyltransferase 1 (DGAT1), and diacylglycerol acyltransferase 2 (DGAT2) in the livers of WT mice. Knockdown of hepatic PPARγ significantly alleviated alcohol-induced lipid accumulation and abolished the up-regulation of FASN, DGAT1, and DGAT2. Silencing of PPARγ in FL83B cells significantly decreased ethanol (EtOH)-, linoleic acid-, and EtOH plus linoleic acid-induced lipid accumulation. Knockdown of hepatic PPARγ also significantly reduced alcohol-induced inflammatory chemokine (monocyte chemotactic protein 1 [MCP1], keratinocyte-derived chemokine [KC], interferon gamma-induced protein 10 [IP-10]) and inflammatory infiltration (lymphocyte antigen 6 complex, locus G [Ly6G], and F4/80). CONCLUSIONS: The results suggest that hepatic PPARγ signaling contributes to alcohol-induced liver injury by promoting hepatic steatosis and inflammation.

The role of heme oxygenase-1 in drug metabolizing dysfunction in the alcoholic fatty liver exposed to ischemic injury.

This study was designed to investigate the role of heme oxygenase-1 (HO-1) in hepatic drug metabolizing dysfunction after ischemia/reperfusion (IR) in alcoholic fatty liver (AFL). Rats were fed a Lieber-DeCarli diet for five weeks to allow for development of AFL and were then subjected to 90min of hepatic ischemia and 5h of reperfusion. Rats were pretreated with hemin (HO-1 inducer) or ZnPP (HO-1 inhibitor) for 16h and 3h before hepatic ischemia. After hepatic IR, ethanol diet (ED)-fed rats had higher serum aminotransferase activities and more severe hepatic necrosis compared to the control diet (CD)-fed rats. These changes were attenuated by hemin and exacerbated by ZnPP. The activity and gene expression of HO-1 and its transcription factor (Nrf2) level increased significantly after 5h of reperfusion in CD-fed rats but not in ED-fed rats. After reperfusion, cytochrome P450 (CYP) 1A1, 1A2, and 2B1 activities were reduced to levels lower than those observed in sham group, whereas CYP2E1 activity increased. The decrease in CYP2B1 activity and the increase in CYP2E1 activity were augmented after hepatic IR in ED-fed animals. These changes were significantly attenuated by hemin but aggravated by ZnPP. Finally, CHOP expression and PERK phosphorylation, microsomal lipid peroxidation, and levels of proinflammatory mediators increased in ED-fed rats compared to CD-fed rats after reperfusion. These increases were attenuated by hemin. Our results suggest that AFL exacerbates hepatic drug metabolizing dysfunction during hepatic IR via endoplasmic reticulum stress and lipid peroxidation and this is associated with impaired HO-1 induction.

Identification of cytochrome CYP2E1 as critical mediator of synergistic effects of alcohol and cellular lipid accumulation in hepatocytes in vitro.

Clinical studies propose a causative link between the consumption of alcohol and the development and progression of liver disease in obese individuals. However, it is incompletely understood how alcohol and obesity interact and whether the combined effects are additive or synergistic. In this study, we developed an in vitro model to address this question. Lipid accumulation in primary human hepatocytes was induced by incubation with oleic acid. Subsequently, steatotic and control hepatocytes were incubated with up to 50 mM alcohol. This alcohol concentration on its own revealed only minimal effects but significantly enhanced oleate-induced lipogenesis and cellular triglyceride content compared to control cells. Similarly, lipid peroxidation, oxidative stress and pro-inflammatory gene expression as well as CYP2E1 levels and activity were synergistically induced by alcohol and steatosis. CYP2E1 inhibition blunted these synergistic pathological effects. Notably, alcohol and cellular steatosis also induced autophagy in a synergistic manner, and also this was mediated via CYP2E1. Further induction of autophagy ameliorated the joint effects of alcohol and oleic acid on hepatocellular lipid accumulation and inflammatory gene expression while inhibition of autophagy further enhanced the dual pathological effects. Further analyses revealed that the joint synergistic effect of alcohol and steatosis on autophagy was mediated via activation of the JNK-pathway. In summary, our data indicate that alcohol induces not only pathological but also protective mechanisms in steatotic hepatocytes via CYP2E1. These findings may have important implications on the prognosis and treatment of alcoholic liver disease particularly in obese individuals.

Pharmacological activation of aldehyde dehydrogenase 2 by Alda-1 reverses alcohol-induced hepatic steatosis and cell death in mice.

BACKGROUND & AIMS: Effective therapies for alcoholic liver disease are currently unavailable. The present study tested the efficacy of Alda-1, a specific aldehyde dehydrogenase 2 (ALDH2) activator, in treating alcoholic liver disease. METHODS: Male C57BL/6J mice were exposed to alcohol for a time-course study on aldehyde metabolism. The specificity and efficacy of Alda-1 on activating hepatic ALDH2 and aldehyde clearance were determined by acute treatments. Then, mice were fed alcohol for 8 weeks with Alda-1 administration for the last 10 days to test the therapeutic potential of Alda-1. Lastly, H4IIEC3 cells were treated with ethanol, acetaldehyde, or 4-hydroxynonenal to define the link between aldehydes and hepatotoxicity. RESULTS: Alcohol feeding for 8 weeks induced hepatic ALDH2 dysfunction and aldehyde accumulation. One dose of Alda-1 administration elevated hepatic ALDH activity, which was blocked by the specific ALDH2 inhibitor, daidzin. Alda-1 accelerated acetaldehyde clearance after acute alcohol intoxication. Alda-1 treatment in the 8-week alcohol feeding model reversed liver damage along with reduction of hepatic aldehydes. Alda-1 re-activated transcription factors, upregulated fatty acid oxidation enzymes, and reversed steatosis. Alcohol-induced endoplasmic reticulum stress and apoptotic cell death were also attenuated by Alda-1. Acetaldehyde or 4-hydroxynonenal treatment to H4IIEC3 cells inactivated transcription factors and induced endoplasmic reticulum stress and apoptosis, while ethanol per se showed limited effects. CONCLUSIONS: Pharmacological activation of ALDH2 by Alda-1 reversed alcoholic steatosis and apoptosis through accelerating aldehyde clearance. This study indicates that ALDH2 is a promising molecular target and Alda-1 has therapeutic potential for treating alcoholic liver disease.

FGF21 treatment ameliorates alcoholic fatty liver through activation of AMPK-SIRT1 pathway.

Fibroblast growth factor 21 (FGF21), a recently identified member of the FGF superfamily, is mainly secreted from the liver and adipose tissues and plays an important role in improving metabolic syndrome and homeostasis. The aim of this study is to evaluate the role of FGF21 in alcoholic fatty liver disease (AFLD) and to determine if it has a therapeutic effect on AFLD. In this paper, we tested the effect of FGF21 on alcohol-induced liver injury in a murine model of chronic ethanol gavage and alcohol-treated HepG2 cells. Male KM mice received single dose of 5 g/kg ethanol gavage every day for 6 weeks, which induced significant fatty liver and liver injury. The alcohol-induced fatty liver cell model was achieved by adding ethanol into the medium of HepG2 cell cultures at a final concentration of 75 mM for 9 days. Results showed that treatment with recombinant FGF21 ameliorated alcoholic fatty liver and liver injury both in a murine model of chronic ethanol gavage and alcohol-treated HepG2 cells. In addition, FGF21 treatment down-regulated the hepatic expression of fatty acid synthetic key enzyme, activated hepatic AMPK-SIRT1 pathway and significantly down-regulated hepatic oxidative stress protein. Taken together, FGF21 corrects multiple metabolic parameters of AFLD in vitro and in vivo by activation of the AMPK-SIRT1 pathway.

PKCε contributes to chronic ethanol-induced steatosis in mice but not inflammation and necrosis.

BACKGROUND: Protein kinase C epsilon (PKCε) has been shown to play a role in experimental steatosis by acute alcohol. The "two-hit" hypothesis implies that preventing steatosis should blunt more advanced liver damage (e.g., inflammation and necrosis). However, the role of PKCε in these pathologies is not yet known. The goal of this current work was to address this question in a model of chronic alcohol exposure using antisense oligonucleotides (ASO) against PKCε. METHODS: Accordingly, PKCε ASO- and saline-treated mice were fed high-fat control or ethanol (EtOH)-containing enteral diets for 4 weeks. RESULTS: Chronic EtOH exposure significantly elevated hepatic lipid pools as well as activated PKCε. The PKCε ASO partially blunted the increases in hepatic lipids caused by EtOH. Administration of PKCε ASO also completely prevented the increase in the expression of fatty acid synthase, and tumor necrosis factor α caused by EtOH. Despite these protective effects, the PKCε ASO was unable to prevent the increases in inflammation and necrosis caused by chronic EtOH. These latter results correlated with an inability of the PKCε ASO to blunt the up-regulation of plasminogen activator inhibitor-1 (PAI-1) and the accumulation of fibrin. Importantly, PAI-1 has been previously shown to more robustly mediate inflammation and necrosis (vs. steatosis) after chronic EtOH exposure. CONCLUSIONS: This study identifies a novel potential mechanism where EtOH, independent of steatosis, can contribute to liver damage. These results also suggest that PAI-1 and fibrin accumulation may be at the center of this PKCε-independent pathway.

Aldehyde dehydrogenase 2 deficiency ameliorates alcoholic fatty liver but worsens liver inflammation and fibrosis in mice.

UNLABELLED: Aldehyde dehydrogenase 2 (ALDH2) is the major enzyme that metabolizes acetaldehyde produced from alcohol metabolism. Approximately 40-50% of East Asians carry an inactive ALDH2 gene and exhibit acetaldehyde accumulation after alcohol consumption. However, the role of ALDH2 deficiency in the pathogenesis of alcoholic liver injury remains obscure. In the present study, wild-type and ALDH2(-/-) mice were subjected to ethanol feeding and/or carbon tetrachloride (CCl4 ) treatment, and liver injury was assessed. Compared with wild-type mice, ethanol-fed ALDH2(-/-) mice had higher levels of malondialdehyde-acetaldehyde (MAA) adduct and greater hepatic inflammation, with higher hepatic interleukin (IL)-6 expression but surprisingly lower levels of steatosis and serum alanine aminotransferase (ALT). Higher IL-6 levels were also detected in ethanol-treated precision-cut liver slices from ALDH2(-/-) mice and in Kupffer cells isolated from ethanol-fed ALDH2(-/-) mice than those levels in wild-type mice. In vitro incubation with MAA enhanced the lipopolysaccharide (LPS)-mediated stimulation of IL-6 production in Kupffer cells. In agreement with these findings, hepatic activation of the major IL-6 downstream signaling molecule signal transducer and activator of transcription 3 (STAT3) was higher in ethanol-fed ALDH2(-/-) mice than in wild-type mice. An additional deletion of hepatic STAT3 increased steatosis and hepatocellular damage in ALDH2(-/-) mice. Finally, ethanol-fed ALDH2(-/-) mice were more prone to CCl4 -induced liver inflammation and fibrosis than ethanol-fed wild-type mice. CONCLUSION: ALDH2(-/-) mice are resistant to ethanol-induced steatosis but prone to inflammation and fibrosis by way of MAA-mediated paracrine activation of IL-6 in Kupffer cells. These findings suggest that alcohol, by way of acetaldehyde and its associated adducts, stimulates hepatic inflammation and fibrosis independent from causing hepatocyte death, and that ALDH2-deficient individuals may be resistant to steatosis and blood ALT elevation, but are prone to liver inflammation and fibrosis following alcohol consumption.