Gehua Jiejiu Dizhi decoction ameliorates alcoholic fatty liver in mice by regulating lipid and bile acid metabolism and with exertion of antioxidant stress based on 4DLabel-free quantitative proteomic study.

OBJECTIVE: To analyze the effect and molecular mechanism of Gehua Jiejiu Dizhi decoction (, GJDD) on alcoholic fatty live disease (AFLD) by using proteomic methods. METHODS: The male C57BL/6J mouse were randomly divided into four groups: control group, model group, GJDD group and resveratrol group. After the AFLD model was successfully prepared by intragastric administration of alcohol once on the basis of the Lieber-DeCarli classical method, the GJDD group and resveratrol group were intragastrically administered with GJDD (4900 mg/kg) and resveratrol (400 mg/kg) respectively, once a day for 9 d. The fat deposition of liver tissue was observed and evaluated by oil red O (ORO) staining. 4DLabel-free quantitative proteome method was used to determine and quantify the protein expression in liver tissue of each experimental group. The differentially expressed proteins were screened according to protein expression differential multiples, and then analyzed by Gene ontology classification and Kyoto Encyclopedia of Genes and Genomes pathway enrichment. Finally, expression validation of the differentially co-expressed proteins from control group, model group and GJDD group were verified by targeted proteomics quantification techniques. RESULTS: In semiquantitative analyses of ORO, all kinds of steatosis (ToS, MaS, and MiS) were evaluated higher in AFLD mice compared to those in GJDD or resveratrol-treated mice. 4DLabel-free proteomics analysis results showed that a total of 4513 proteins were identified, of which 3763 proteins were quantified and 946 differentially expressed proteins were screened. Compared with the control group, 145 proteins were up-regulated and 148 proteins were down-regulated in the liver tissue of model group. In addition, compared with the model group, 92 proteins were up-regulated and 135 proteins were down-regulated in the liver tissue of the GJDD group. 15 differentially co-expressed proteins were found between every two groups (model group vs control group, GJDD group vs model group and GJDD group vs control group), which were involved in many biological processes. Among them, 11 differentially co-expressed key proteins (Aox3, H1-5, Fabp5, Ces3a, Nudt7, Serpinb1a, Fkbp11, Rpl22l1, Keg1, Acss2 and Slco1a1) were further identified by targeted proteomic quantitative technology and their expression patterns were consistent with the results of 4D label-free proteomic analysis. CONCLUSIONS: Our study provided proteomics-based evidence that GJDD alleviated AFLD by modulating liver protein expression, likely through the modulation of lipid metabolism, bile acid metabolism and with exertion of antioxidant stress.

Hepatocyte-specific mitogen-activated protein kinase phosphatase 1 in sexual dimorphism and susceptibility to alcohol induced liver injury.

Background: It is well established that females are more susceptible to the toxic effects of alcohol, although the exact mechanisms are still poorly understood. Previous studies noted that alcohol reduces the expression of mitogen-activated protein kinase phosphatase 1 (MKP1), a negative regulator of mitogen-activated protein kinases (MAPK) in the liver. However, the role of hepatocyte- specific MKP1 in the pathogenesis of alcohol-associated liver disease (ALD) remains uncharacterized. This study aimed to evaluate the role of hepatocyte-specific MKP1 in the susceptibility and sexual dimorphism in alcohol-induced liver injury. Methods: C57Bl/6 mice were used in an intragastric ethanol feeding model of alcohol-associated steatohepatitis (ASH). Hepatocyte-specific Mkp1-/- knockout and (Mkp1+/+ "f/f" male and female mice were subjected to the NIAAA chronic plus binge model. Primary mouse hepatocytes were used for in vitro studies. Liver RNA sequencing was performed on an Illumina NextSeq 500. Liver injury was evaluated by plasma alanine transaminase (ALT), hepatic ER stress and inflammation markers. Statistical analysis was carried out using ANOVA and the unpaired Student's t-test. Results: ASH was associated with the severe injury accompanied by increased endoplasmic reticulum (ER) stress and significant downregulation of Dusp1 mRNA expression. In vitro, ethanol treatment resulted in a time-dependent decrease in Dusp1 mRNA and protein expression in primary hepatocytes in both males and females; however, this effect was significantly more pronounced in hepatocytes from females. In vivo, female mice developed more liver injury in a chronic plus binge model which was accompanied by a significant decrease in liver Dusp1 mRNA expression. In comparison, liver Dusp1 was not changed in male mice, while they developed milder injury to alcohol. Mkp1 deletion in hepatocytes led to increased alcohol induced liver injury, ER stress and inflammation in both sexes. Conclusion: Hepatocyte Mkp1 plays a significant role in alcohol induced liver injury. Alcohol downregulates Mkp1 expression in hepatocytes in a sex dependent manner and could play a role in sexual dimorphism in increased female susceptibility to alcohol.

Diagnostic significance of alanine aminotransferase isoenzymes in alcoholic and non-alcoholic fatty liver cancers.

OBJECTIVES: Alanine aminotransferase (ALT) expression is highly elevated in the serum of patients with hepatocellular carcinoma. However, the role of ALT isoenzymes in the total ALT activity remains unclear. In the present study, we systematically investigated the role of ALT isoenzymes in alcoholic and non-alcoholic fatty liver cancer. MATERIALS AND METHODS: The expression of ALT1 and ALT2 at the mRNA and protein levels in 25 paired primary liver cancer tissues was detected by reverse transcription quantitative PCR (RT-qPCR), Western blotting, and immunohistochemistry. Serum ALT activity was determined using an automated biochemical analyzer. RESULTS: The mRNA and protein expression levels of ALT1 and ALT2 were lower in the tissues of alcoholic and non-alcoholic fatty liver cancers than in the paracancerous tissues. Notably, ALT2 was highly expressed in non-alcoholic fatty liver cancer tissues compared with alcoholic fatty liver cancer tissues. Total serum ALT activity was mainly contributed by ALT1 in alcoholic fatty liver cancer, whereas ALT1 contributed only marginally more to the total ALT activity than ALT2 in non-alcoholic fatty liver cancer. ALT2/ALT1 ratio can well discriminate normal control group, alcoholic liver cancer and non-alcoholic liver cancer. CONCLUSION: ALT1 contributed more to the total ALT activity than ALT2 in both alcoholic and non-alcoholic fatty liver cancer. Serum ALT2 to ALT activity was higher in non-alcoholic fatty liver cancer than that in alcoholic fatty liver cancer. ALT2/ALT1 ratio has some diagnostic significance for alcoholic and non-alcoholic liver cancer.

Inhibition of ACSS2 attenuates alcoholic liver steatosis via epigenetically regulating de novo lipogenesis.

BACKGROUND AND AIMS: Steatosis is the early pathological change in alcohol-associated liver disease. However, its precise mechanism is still unclear. The present study is aimed to explore the role and mechanism of acetyl-CoA synthetase 2 (ACSS2) in acute alcohol-induced lipogenesis. METHODS: The increase in ACSS2 nuclear import and histone H3 acetylation were observed in mice after intraperitoneally injected with 2 g/kg ethanol or oral administration of 5 g/kg ethanol and also validated in hepatocytes stimulated with ethanol or acetate. The role of ACSS2 was further explored in liver-specific ACSS2 knockdown mice fed with ethanol-containing diet. RESULTS: Alcohol challenge induced hepatic lipid deposition and upregulated lipogenic genes in mice. It also promoted ACSS2 nuclear import and increased histone H3 acetylation. In hepatocytes, ethanol induced similar phenomena whereas ACSS2 knockdown blocked histone acetylation and lipogenic gene induction. P300/CBP associated factor (PCAF), but not general control nonderepressible 5, CREB-binding protein (CBP) and p300, facilitated H3K9 acetylation responding to ethanol challenge. CUT&RUN assay showed the enrichment of acetylated histone H3K9 surrounding Fasn and Acaca promoters. These results indicated that ethanol metabolism promoted ACSS2 nuclear import to support lipogenesis via H3K9 acetylation. In alcohol-feeding mice, liver-specific ACSS2 knockdown blocked the interaction between PCAF and H3K9 and suppressed lipogenic gene induction in the liver, demonstrating the critical role of ACSS2 in lipogenesis. CONCLUSIONS: Our study demonstrated that alcohol metabolism generated acetyl-CoA in the nucleus dependently on nuclear ACSS2, contributing to epigenetic regulation of lipogenesis in hepatic steatosis. Targeting ACSS2 may be a potential therapeutical strategy for acute alcoholic liver steatosis.

p53 protects against alcoholic fatty liver disease via ALDH2 inhibition.

The tumor suppressor p53 is critical for tumor suppression, but the regulatory role of p53 in alcohol-induced fatty liver remains unclear. Here, we show a role for p53 in regulating ethanol metabolism via acetaldehyde dehydrogenase 2 (ALDH2), a key enzyme responsible for the oxidization of alcohol. By repressing ethanol oxidization, p53 suppresses intracellular levels of acetyl-CoA and histone acetylation, leading to the inhibition of the stearoyl-CoA desaturase-1 (SCD1) gene expression. Mechanistically, p53 directly binds to ALDH2 and prevents the formation of its active tetramer and indirectly limits the production of pyruvate that promotes the activity of ALDH2. Notably, p53-deficient mice exhibit increased lipid accumulation, which can be reversed by ALDH2 depletion. Moreover, liver-specific knockdown of SCD1 alleviates ethanol-induced hepatic steatosis caused by p53 loss. By contrast, overexpression of SCD1 in liver promotes ethanol-induced fatty liver development in wild-type mice, while it has a mild effect on p53-/- or ALDH2-/- mice. Overall, our findings reveal a previously unrecognized function of p53 in alcohol-induced fatty liver and uncover pyruvate as a natural regulator of ALDH2.

1-Methylnicotinamide promotes hepatic steatosis in mice: A potential mechanism in chronic alcohol-induced fatty liver disease.

Alcohol abuse and its related diseases are the major risk factors for human health. Alcohol-related liver disease (ALD) is a leading cause of morbidity and mortality worldwide. Although the mechanism of ALD has been widely investigated, liver metabolites associated with long-term alcohol intake-induced hepatic steatosis have not been well explored. In this study, we aimed to investigate the role and mechanisms of 1-methylnicotinamide (1-MNA), a metabolite during nicotinamide adenine dinucleotide (NAD+) metabolism, in the pathogenesis of ALD. C57BL/6 wild-type mice were subjected to chronic alcohol feeding with or without 1-MNA (50 mg/kg/day). Our data showed that 1-MNA administration significantly enhanced chronic alcohol consumption-induced hepatic steatosis. Mechanistic studies revealed that alcohol-increased hepatic protein levels of sterol regulatory element-binding transcription factor (SREBP-1c), a key enzyme that regulates lipid lipogenesis, were enhanced in mice administered with 1-MNA, regardless of alcohol feeding. Consistently, alcohol-increased mRNA and protein levels of hepatic diacylglycerol o-acyltransferase 2 (DGAT2) and very low-density lipoprotein receptor (VLDLR) were also exacerbated by 1-MNA administration. Alcohol-induced hepatic endoplasmic reticulum (ER) stress was enhanced by 1-MNA administration, which was evidenced by increased protein levels of binding immunoglobulin protein (BIP), phosphorylated- protein kinase r-like ER kinase (PERK), activating transcription factor 4 (ATF4), and C/EBP-homologous protein (CHOP) in the mouse liver. Overall, this study demonstrated that 1-MNA serves as a pathogenic factor in the development of ALD. Targeting liver 1-MNA levels may serve as a promising therapeutic approach for improving hepatic steatosis in ALD.

FNDC3B protects steatosis and ferroptosis via the AMPK pathway in alcoholic fatty liver disease.

BACKGROUND: Alcoholic liver disease (ALD) is a leading cause of chronic liver disease worldwide with limited therapeutic options. The role of fibronectin type III domain-containing protein 3B (FNDC3B), an important regulator of metabolism, in ALD, and the underlying mechanism as well as its potential implication in ALD therapeutic strategies remain unknown. METHODS: Hepatocyte-specific FNDC3B knockdown or control C57BL/6 N mice received a Lieber-DeCarli diet for four weeks, followed by oral gavage (chronic-binge). Primary mouse hepatocytes and cell lines were used for in vitro studies. Liver injury, hepatic steatosis, and lipid peroxidation were assessed. RESULTS: In cultured cells and mouse livers, alcohol exposure increased FNDC3B expression. Hepatocyte-specific FNDC3B deletion aggravated alcohol-induced liver steatosis via AMP-activated protein kinase (AMPK) inhibition. In vitro, FNDC3B expression was negatively regulated by miR-192-5p. Furthermore, FNDC3B deletion significantly exacerbated ethanol-mediated lipid peroxidation. The RNA sequence assay revealed a connection between FNDC3B and ferroptosis, which was verified by the administration of the ferroptosis inhibitor ferrostatin-1 (Fer-1). Additionally, FNDC3B inhibition-mediated AMPK inactivation downregulated transferrin expression, which was associated with marked iron overload and ferroptosis. CONCLUSIONS: This study elucidated the critical role of FNDC3B in preventing hepatic steatosis and ferroptosis in response to chronic alcohol consumption. Our findings indicate that FNDC3B is a potential therapeutic target for ALD.

Tangeretin Protects Mice from Alcohol-Induced Fatty Liver by Activating Mitophagy through the AMPK-ULK1 Pathway.

Alcoholic beverages are widely consumed all over the world, but continuous ethanol exposure leads to hepatic steatosis that, without proper treatment, will later develop into severe liver disorders. In this study, we investigated the potential protective effect of tangeretin, a flavonoid derived from citrus peel, against alcoholic fatty liver. The in vivo effects of tangeretin were analyzed by oral intake in a chronic-binge alcohol feeding C57BL/6j mouse model, while the underlying mechanism was explored by in vitro studies performed on ethanol-treated hepatic AML-12 cells. Ethanol feeding increased the serum alanine aminotransferase and aspartate aminotransferase levels, the liver weight, and the serum and liver triacylglycerol contents, whereas 20 and 40 mg/kg tangeretin treatment promoted a dose-dependent suppression of these effects. Interestingly, tangeretin prevented increases in the liver oxidative stress level and protected the hepatocyte mitochondria from ethanol-induced morphologic abnormalities. A mechanistic study showed that 20 µM tangeretin treatment activated mitophagy through an AMP-activated protein kinase (AMPK)-uncoordinated 51-like kinase 1 (Ulk1) pathway, thereby restoring mitochondria respiratory function and suppressing steatosis. By contrast, blocking the AMPK-Ulk1 pathway with compound C reversed the hepatoprotective effect of tangeretin. Overall, tangeretin activated mitophagy and protected against ethanol-induced hepatic steatosis through an AMPK-Ulk1-dependent mechanism.

Identification of differentially expressed miRNAs and key genes involved in the progression of alcoholic fatty liver disease using rat models.

BACKGROUND: Alcoholic fatty liver disease (AFLD) is a liver disease caused by prolonged heavy drinking and has a poor prognosis in the clinic. This study aimed to explore the differential miRNAs expression profiles in the AFLD rat model. METHODS: The rat model of AFLD was established by ethanol intragastric administration and was used to explore the differential miRNAs expression profiles. We further analyzed the potential target mRNAs using the bioinformatics technique. GO and KEGG pathway enrichment analyses were carried out to better understand the biological function of differential expression genes (DEGs). We used the human Gene Expression Omnibus (GEO) dataset GSE28619 to further screen the key differentially expressed genes. The integration between the differentially expressed genes from the AFLD model and GEO was conducted and the key genes were identified. RESULTS: The serum ALT, AST, TG, and TC levels in the AFLD model group were significantly higher than those in the normal control group. There are 45 miRNAs with significant changes including 26 upregulated and 19 down-regulated miRNAs. GO and KEGG enrichment showed various metabolic processes and signaling pathways were enriched in the progression of AFLD. After integrating the results of GSE28619 and DEGs, we observed that there are 12 genes with significant changes in two data sets, including PSAT1, TKFC, PTTG1, LCN2, CXCL1, NR4A1, RGS1, VCAN, FOS, CXCL10, ATF3, and CYP1A1. CONCLUSION: AFLD showed differentially expressed miRNAs, which may be involved in the occurrence and progression of AFLD. Meanwhile, some signal metabolic pathways may be related to the pathogenesis of AFLD.

miRNA-432 and SLC38A1 as Predictors of Hepatocellular Carcinoma Complicated with Alcoholic Steatohepatitis.

Alcoholic steatohepatitis (ASH) is asymptomatic in the early stages and is typically advanced at the time of diagnosis. With the global rise in alcohol abuse, ASH is currently among the most detrimental diseases around the world. Hepatocellular carcinoma (HCC) is one of the final outcomes of numerous liver diseases. However, at present, HCC screening is mostly focused on liver cancer development. Moreover, there is no effective biomarker to predict the prognosis and recurrence of liver cancer. Meanwhile, there are limited studies on the prognosis and recurrence of HCC patients complicated with ASH. In this study, using bioinformatic analysis as well as cellular and animal models, we screened the differentially expressed (DE) miRNA-432 and SLC38A1 gene in ASH. Based on our analysis, miRNA-432 targeted SLC38A1, and the levels of miRNA-432 and SLC38A1 could accurately predict the overall survival (OS) and relapse free survival (RFS) in patients with liver cancer. Hence, these two genetic elements have the potential to synergistically predict the prognosis and recurrence of HCC complicated with ASH.

MyD88 in hepatic stellate cells promotes the development of alcoholic fatty liver via the AKT pathway.

Myeloid differentiation primary response gene 88 (MyD88), an adaptor protein in the Toll-like receptors (TLRs) signalling pathway, is expressed in various liver cells including hepatocytes, Kupffer cells and hepatic stellate cells (HSCs). And yet, the functional role of MyD88 in HSCs is poorly elucidated in alcoholic fatty liver (AFL). Here, to study the functional role of MyD88 in HSCs and the molecular mechanism related to the development of AFL, chronic-binge ethanol mouse models were established in mice with specific MyD88 knockout in quiescent (MyD88GFAP-KO) and activated HSCs (MyD88SMA-KO), respectively. Our results clearly showed an elevated expression of MyD88 in liver tissues of ethanol treated mouse model which harbours the wild type. Intriguingly, ethanol treatment profoundly inhibited inflammation in both MyD88GFAP-KO and MyD88SMA-KO mice, but the suppression of lipogenesis was only observed in MyD88GFAP-KO mice. Molecularly, our study indicated that MyD88 induced osteopontin (OPN) secretion in HSCs, which consequently resulted in activation of AKT signalling pathway and accumulation of fat in hepatocytes. Additionally, our data also suggested that OPN promoted inflammation by activating p-STAT1. Thus, targeting MyD88 may be a potentially represent a promising strategy for the prevention and treatment of AFL. KEY MESSAGES: The expression of MyD88 in HSCs was significantly increased in ethanol-induced liver tissues of wild-type mice. MyD88 deficiency in quiescent HSCs inhibited inflammation and lipogenesis under the ethanol feeding condition. MyD88 deficiency in activated HSCs only inhibited inflammation under the ethanol feeding condition. MyD88 promoted the OPN secretion of HSCs, which further activated the AKT signalling pathway of hepatocytes and upregulated lipogenic gene expression to promote fat accumulation. OPN also promotes inflammation by activating p-STAT1.

Knockdown of UCA1 attenuated the progression of alcoholic fatty disease by sponging miR-214.

Alcoholic fatty liver (AFL) is the initial manifestation of Alcoholic liver disease which can develop into alcoholic cirrhosis even extensive necrosis of liver cells, which induces liver failure finally. This study aims to focus on the role of long noncoding RNA UCA1 in AFL and further explored possible mechanism of this disease. We first downloaded GSE28619 to identify the expression of UCA1 in patients with AFL and use lncRNAs microarray to confirm UCA1 expression in serum of patients with AFL. Then we established ethanol-induced L02 cell model to mimic hepatocyte injury condition. By conducting qRT-PCR, we measured the expression of LncRNA UCA1 and miR-214 in serum of patients and ethanol-induced L02 cell. MTT assay, transwell migration, ELISA, qRT-PCR, and western blotting analysis were applied to evaluating the effect of UCA1 on ethanol-induced L02 cell. The bioinformatics analysis and the rescue experiment were devoted to the underlying mechanism. In this study, we first detected the expression of UCA1 was up-regulated in serum of patients with AFL and ethanol-induced L02 cells. And knockdown of UCA1 reversed the inhibiting effect of ethanol on the biological behavior of L02 cells including cell proliferation, migration, and apoptosis. Besides, lncRNA UCA1 regulated the expression of KLF5 by sponging miR-214. LncRNA UCA1 regulated the biological behavior of ethanol-induced L02 cells by sponging miR-214, which may provide novel therapeutic strategies for alcoholic fatty liver.

ZFP36L1 Regulates Fgf21 mRNA Turnover and Modulates Alcoholic Hepatic Steatosis and Inflammation in Mice.

Zinc finger protein 36 like 1 (ZFP36L1) enhances the turnover of mRNAs containing AU-rich elements (AREs) in their 3'-untranslated regions (3'UTR). The physiological and pathological functions of ZFP36L1 in liver, however, remain largely unknown. Liver-specific ZFP36L1-deficient (Zfp36l1flox/flox/Cre+; L1LKO) mice were generated to investigate the role of ZFP36L1 in liver physiology and pathology. Under normal conditions, the L1LKO mice and their littermate controls (Zfp36l1flox/flox/Cre-; L1FLX) appeared normal. When fed a Lieber-DeCarli liquid diet containing alcohol, L1LKO mice were significantly protected from developing alcohol-induced hepatic steatosis, injury, and inflammation compared with L1FLX mice. Most importantly, fibroblast growth factor 21 (Fgf21) mRNA was significantly increased in the livers of alcohol diet-fed L1LKO mice compared with the alcohol diet-fed L1FLX group. The Fgf21 mRNA contains three AREs in its 3'UTR, and Fgf21 3'UTR was directly regulated by ZFP36L1 in luciferase reporter assays. Steady-state levels of Fgf21 mRNA were significantly decreased by wild-type ZFP36L1, but not by a non-binding zinc finger ZFP36L1 mutant. Finally, wild-type ZFP36L1, but not the ZFP36L1 mutant, bound to the Fgf21 3'UTR ARE RNA probe. These results demonstrate that ZFP36L1 inactivation protects against alcohol-induced hepatic steatosis and liver injury and inflammation, possibly by stabilizing Fgf21 mRNA. These findings suggest that the modulation of ZFP36L1 may be beneficial in the prevention or treatment of human alcoholic liver disease.

Metabolic and Genetic Risk Factors Are the Strongest Predictors of Severity of Alcohol-Related Liver Fibrosis.

BACKGROUND & AIMS: Individual risk for developing alcohol-related liver disease (ALD) varies greatly. We hypothesized that metabolic risk factors and genetic polymorphisms predict severity of ALD. METHODS: Biopsy-controlled, cross-sectional study in patients with a history of excessive drinking. We measured the homeostatic model assessment of insulin resistance (HOMA-IR), plasma triglycerides, high- and low-density lipoproteins (HDL, LDL), and total cholesterol. Moreover, we genotyped four single nucleotide polymorphisms in PNPLA3 (rs738409C>G), TM6SF2 (rs58542926C>T), MBOAT7 (rs641738C>T), and HSD17B13 (rs72613567T>TA). We assessed predictors of higher fibrosis stage using multivariable ordered logistic regression. RESULTS: Of 325 included patients, 25% had severe fibrosis or cirrhosis and 59% had HOMA-IR ≥2.5. HOMA-IR increased for each fibrosis stage, while there was a similar decrease in LDL and total cholesterol. Individuals with risk variant PNPLA3 rs738409-G or TM6SF2 rs58542926-T had higher fibrosis stage. In multivariable regression, HOMA-IR ≥2.5 (OR = 3.04, 95% CI 1.90-4.87), LDL <2.60 mmol/L (OR = 2.05, 95% CI 1.33-3.16), TM6SF2 rs58542926-T (OR = 1.99, 95% CI 1.17-3.37), age above 50 years (OR = 1.66, 95% CI 1.03-2.70), and PNPLA3 rs738409-G (OR = 1.54, 95% CI 1.11-2.12) independently predicted higher fibrosis stage. Independent predictors of hepatic inflammatory activity were HOMA-IR, active drinking, age, and PNPLA3 risk variant. Active drinking, elevated triglycerides, and PNPLA3 risk variant predicted steatosis. CONCLUSIONS: Insulin resistance is the strongest predictor of liver fibrosis stage and hepatic inflammation in patients with alcohol-related liver disease. Genetic susceptibility further aggravates this risk. These data highlight the clinical value of detailed metabolic and genetic profiling of patients with excessive alcohol use.

Depdc5 deficiency exacerbates alcohol-induced hepatic steatosis via suppression of PPARα pathway.

Alcohol-related liver disease (ALD), a condition caused by alcohol overconsumption, occurs in three stages of liver injury including steatosis, hepatitis, and cirrhosis. DEP domain-containing protein 5 (DEPDC5), a component of GAP activities towards Rags 1 (GATOR1) complex, is a repressor of amino acid-sensing branch of the mammalian target of rapamycin complex 1 (mTORC1) pathway. In the current study, we found that aberrant activation of mTORC1 was likely attributed to the reduction of DEPDC5 in the livers of ethanol-fed mice or ALD patients. To further define the in vivo role of DEPDC5 in ALD development, we generated Depdc5 hepatocyte-specific knockout mouse model (Depdc5-LKO) in which mTORC1 pathway was constitutively activated through loss of the inhibitory effect of GATOR1. Hepatic Depdc5 ablation leads to mild hepatomegaly and liver injury and protects against diet-induced liver steatosis. In contrast, ethanol-fed Depdc5-LKO mice developed severe hepatic steatosis and inflammation. Pharmacological intervention with Torin 1 suppressed mTORC1 activity and remarkably ameliorated ethanol-induced hepatic steatosis and inflammation in both control and Depdc5-LKO mice. The pathological effect of sustained mTORC1 activity in ALD may be attributed to the suppression of peroxisome proliferator activated receptor α (PPARα), the master regulator of fatty acid oxidation in hepatocytes, because fenofibrate (PPARα agonist) treatment reverses ethanol-induced liver steatosis and inflammation in Depdc5-LKO mice. These findings provide novel insights into the in vivo role of hepatic DEPDC5 in the development of ALD.

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.

Alcoholic fatty liver disease inhibited the co-expression of Fmo5 and PPARα to activate the NF-κB signaling pathway, thereby reducing liver injury via inducing gut microbiota disturbance.

BACKGROUND: Alcohol-induced intestinal dysbiosis disrupts and inflammatory responses are essential in the development of alcoholic fatty liver disease (AFLD). Here, we investigated the effects of Fmo5 on changes in enteric microbiome composition in a model of AFLD and dissected the pathogenic role of Fmo5 in AFLD-induced liver pathology. METHODS: The expression profile data of GSE8006 and GSE40334 datasets were downloaded from the GEO database. The WGCNA approach allowed us to investigate the AFLD-correlated module. DEGs were used to perform KEGG pathway enrichment analyses. Four PPI networks were constructed using the STRING database and visualized using Cytoscape software. The Cytohubba plug-in was used to identify the hub genes. Western blot and immunohistochemistry assays were used to detect protein expression. ELISA assay was used to detect the levels of serum inflammatory cytokines. Lipid droplets in the cytoplasm were observed using Oil Red O staining. Apoptosis was detected using a TUNEL assay and flow cytometry analysis. ROS levels were detected using flow cytometry analysis. Nuclear translocation of NF-κB p65 was observed using immunofluorescence staining. Co-immunoprecipitation was used to detect the co-expression of PPARα and Fmo5 in L02 cells. 16S rDNA sequencing defined the bacterial communities in mice with AFLD. RESULTS: Fmo5 is a key DEG and is closely associated with the gut microbiota and PPAR signaling pathway. Gut microbiome function in AFLD was significantly related to the PPAR signaling pathway. AFLD induced shifts in various bacterial phyla in the cecum, including a reduction in Bacteroidetes and increased Firmicutes. Fmo5 and PPARα co-expression in cell and animal models with AFLD, which decreased significantly. Silencing of Fmo5 and PPARα aggravated the functions of AFLD inducing apoptosis and inflammatory response, promoting liver injury, and activating the NF-κB signaling pathway in vivo and in vitro. The NF-κB inhibitor abolished the functions of silencing of Fmo5 and PPARα promoting AFLD-induced apoptosis, inflammatory response, and liver injury. CONCLUSION: Our data indicated that the co-expression of Fmo5 and PPARα was involved in AFLD-related gut microbiota composition and alleviated AFLD-induced liver injury, apoptosis, and inflammatory response by inhibiting the nuclear translocation of NF-κB p65 to inhibit the NF-κB signaling pathway.

The critical role of redox regulation of PTEN and peroxiredoxin III in alcoholic fatty liver.

Hepatic steatosis and subsequent fatty liver disease are developed in response to alcohol consumption. Reactive oxygen species (ROS) are thought to play an important role in the alcoholic fatty liver disease (AFLD). However, the molecular targets of ROS and the underlying cellular mechanisms are unknown. Here, we investigate roles of peroxiredoxin III and redox regulation of phosphatase and tension homolog deleted on chromosome 10 (PTEN) in the alcoholic fatty liver. Alcohol-induced mitochondrial oxidative stress was found to contribute to reversible oxidation of PTEN, which results in Akt and MAPK hyperactivation with elevated levels of the lipogenesis regulators SREBP1c and PPARγ. Moreover, mitochondrial peroxiredoxin III was found to have antagonistic effects on lipogenesis via the redox regulation of PTEN by removing ROS, upon alcohol exposure. This study demonstrated that redox regulation of PTEN and peroxiredoxin III play crucial roles in the development of AFLD.

Live Confocal Imaging as a Novel Tool to Assess Liver Quality: Insights From a Murine Model.

BACKGROUND: In an experimental murine liver clamping model, we aimed to investigate the efficacy of real-time confocal microscopy (RCM) in assessing viability of steatotic livers in comparison to standard assessment tools, including histopathological evaluation. METHODS: C57Bl/6 mice were subjected to a methionine-choline-deficient diet causing nonalcoholic fatty liver disease or to Lieber DeCarli diet causing ethanol-induced liver injury. Untreated animals served as controls. Liver biopsies were analyzed following challenge with 45 min of warm ischemia time and either 4 h of reperfusion or 24 h of cold storage. Organ quality assessment was performed at defined time points by RCM, histological staining, measurement of serum alanine aminotransferase activity, and expression analyses of proinflammatory cytokines. Additionally, survival analysis was performed. RESULTS: Cold as well as warm ischemia time resulted in a significant decrease in cell viability when compared with naive livers as well as nonischemic-challenged steatotic livers (P < 0.05) as assessed by RCM. Furthermore, RCM revealed the actual cellular damage at early time points, while established methods including H&E-staining and serum transaminase profile failed. CONCLUSIONS: In a translational attempt, we demonstrate that RCM is a suitable diagnostic tool to obtain information about functional damage of the liver apart from standard approaches.

Chronic-plus-binge alcohol intake induces production of proinflammatory mtDNA-enriched extracellular vesicles and steatohepatitis via ASK1/p38MAPKα-dependent mechanisms.

Alcohol-associated liver disease is a spectrum of liver disorders with histopathological changes ranging from simple steatosis to steatohepatitis, cirrhosis, and hepatocellular carcinoma. Recent data suggest that chronic-plus-binge ethanol intake induces steatohepatitis by promoting release by hepatocytes of proinflammatory mitochondrial DNA-enriched (mtDNA-enriched) extracellular vesicles (EVs). The aim of the present study was to investigate the role of the stress kinase apoptosis signal-regulating kinase 1 (ASK1) and p38 mitogen-activated protein kinase (p38) in chronic-plus-binge ethanol-induced steatohepatitis and mtDNA-enriched EV release. Microarray analysis revealed the greatest hepatic upregulation of metallothionein 1 and 2 (Mt1/2), which encode 2 of the most potent antioxidant proteins. Genetic deletion of the Mt1 and Mt2 genes aggravated ethanol-induced liver injury, as evidenced by elevation of serum ALT, neutrophil infiltration, oxidative stress, and ASK1/p38 activation in the liver. Inhibition or genetic deletion of Ask1 or p38 ameliorated ethanol-induced liver injury, inflammation, ROS levels, and expression of phagocytic oxidase and ER stress markers in the liver. In addition, inhibition of ASK1 or p38 also attenuated ethanol-induced mtDNA-enriched EV secretion from hepatocytes. Taken together, these findings indicate that induction of hepatic mtDNA-enriched EVs by ethanol is dependent on ASK1 and p38, thereby promoting alcoholic steatohepatitis.