MicroRNA 122, Regulated by GRLH2, Protects Livers of Mice and Patients From Ethanol-Induced Liver Disease.
ABSTRACT: BACKGROUND & AIMS:Chronic, excessive alcohol consumption leads to alcoholic liver disease (ALD) characterized by steatosis, inflammation, and eventually cirrhosis. The hepatocyte specific microRNA 122 (MIR122) regulates hepatocyte differentiation and metabolism. We investigated whether an alcohol-induced decrease in level of MIR122 contributes to development of ALD. METHODS:We obtained liver samples from 12 patients with ALD and cirrhosis and 9 healthy individuals (controls) and analyzed them by histology and immunohistochemistry. C57Bl/6 mice were placed on a Lieber-DeCarli liquid diet, in which they were fed ethanol for 8 weeks, as a model of ALD, or a control diet. These mice were also given injections of CCl4, to increase liver fibrosis, for 8 weeks. On day 28, mice with ethanol-induced liver disease and advanced fibrosis, and controls, were given injections of recombinant adeno-associated virus 8 vector that expressed the primary miR-122 transcript (pri-MIR122, to overexpress MIR122 in hepatocytes) or vector (control). Two weeks before ethanol feeding, some mice were given injections of a vector that expressed an anti-MIR122, to knock down its expression. Serum and liver tissues were collected; hepatocytes and liver mononuclear cells were analyzed by histology, immunoblots, and confocal microscopy. We performed in silico analyses to identify targets of MIR122 and chromatin immunoprecipitation quantitative polymerase chain reaction analyses in Huh-7 cells. RESULTS:Levels of MIR122 were decreased in liver samples from patients with ALD and mice on the Lieber-DeCarli diet, compared with controls. Transgenic expression of MIR122 in hepatocytes of mice with ethanol-induced liver disease and advanced fibrosis significantly reduced serum levels of alanine aminotransferase (ALT) and liver steatosis and fibrosis, compared with mice given injections of the control vector. Ethanol feeding reduced expression of pri-MIR122 by increasing expression of the spliced form of the transcription factor grainyhead like transcription factor 2 (GRHL2) in liver tissues from mice. Levels of GRHL2 also were increased in liver tissues from patients with ALD, compared with controls; increases correlated with decreases in levels of MIR122 in human liver. Mice given injections of the anti-MIR122 before ethanol feeding had increased steatosis, inflammation, and serum levels of alanine aminotransferase compared with mice given a control vector. Levels of hypoxia-inducible factor 1 alpha (HIF1?) mRNA, a target of MIR122, were increased in liver tissues from patients and mice with ALD, compared with controls. Mice with hepatocyte-specific disruption of Hif1? developed less-severe liver injury following administration of ethanol, injection of anti-MIR122, or both. CONCLUSIONS:Levels of MIR122 decrease in livers from patients with ALD and mice with ethanol-induced liver disease, compared with controls. Transcription of MIR122 is inhibited by GRHL2, which is increased in livers of mice and patients with ALD. Expression of an anti-MIR122 worsened the severity of liver damage following ethanol feeding in mice. MIR122 appears to protect the liver from ethanol-induced damage by reducing levels of HIF1?. These processes might be manipulated to reduce the severity of ALD in patients.
Project description:BACKGROUND: We have previously developed an oncolytic serotype 5 adenovirus (Ad5) with chromogranin-A (CgA) promoter-controlled E1A expression, Ad[CgA-E1A], with the intention to treat neuroendocrine tumors, including carcinoids. Since carcinoids tend to metastasize to the liver it is important to fully repress viral replication in hepatocytes to avoid adenovirus-related liver toxicity. Herein, we explore miRNA-based regulation of E1A expression as a complementary mechanism to promoter-based transcriptional control. METHODOLOGY/PRINCIPAL FINDINGS: Ad[CgA-E1A-miR122], where E1A expression is further controlled by six tandem repeats of the target sequence for the liver-specific miR122, was constructed and compared to Ad[CgA-E1A]. We observed E1A suppression and replication arrest of the miR122-detargeted adenovirus in normal hepatocytes, while the two viruses killed carcinoid cells to the same degree. Repeated intravenous injections of Ad[CgA-E1A] induced liver toxicity in mice while Ad[CgA-E1A-miR122] injections did not. Furthermore, a miR122-detargeted adenovirus with the wild-type E1A promoter showed reduced replication in hepatic cells compared to wild-type Ad5 but not to the same extent as the miR122-detargeted adenovirus with the neuroendocrine-selective CgA promoter. CONCLUSIONS/SIGNIFICANCE: A combination of transcriptional (promoter) and post-transcriptional (miRNA target) regulation to control virus replication may allow for the use of higher doses of adenovirus for efficient tumors treatment without liver toxicity.
Project description:BACKGROUND & AIMS:Aging is known to exacerbate the progression of alcoholic liver disease (ALD), but the underlying mechanisms remain obscure. The aim of this study was to use a chronic plus binge ethanol feeding model in mice to evaluate the effects of aging on alcohol-induced liver injury. METHODS:C57BL/6 mice were subjected to short-term (10days) ethanol plus one binge or long-term (8weeks) ethanol plus multiple binges of ethanol. Liver injury and fibrosis were determined. Hepatic stellate cells (HSCs) were isolated and used in in vitro studies. RESULTS:Middle-aged (12-14months) and old-aged (>16months) mice were more susceptible to liver injury, inflammation, and oxidative stress induced by short-term plus one binge or long-term plus multiple binges of ethanol feeding when compared to young (8-12weeks) mice. Long-term plus multiple binges of ethanol feeding induced greater liver fibrosis in middle-aged mice than that in young mice. Hepatic expression of sirtuin 1 (SIRT1) protein was downregulated in the middle-aged mice compared to young mice. Restoration of SIRT1 expression via the administration of adenovirus-SIRT1 vector ameliorated short-term plus binge ethanol-induced liver injury and fibrosis in middle-aged mice. HSCs isolated from middle-aged mice expressed lower levels of SIRT1 protein and were more susceptible to spontaneous activation in in vitro culture than those from young mice. Overexpression of SIRT1 reduced activation of HSCs from middle-aged mice in vitro with downregulation of PDGFR-? and c-Myc, while deletion of SIRT1 activated HSCs isolated from young mice in vitro. Finally, HSC-specific SIRT1 knockout mice were more susceptible to long-term chronic-plus-multiple binges of ethanol-induced liver fibrosis with upregulation of PDGFR-? expression. CONCLUSIONS:Aging exacerbates ALD in mice through the downregulation of SIRT1 in hepatocytes and HSCs. Activation of SIRT1 may serve as a novel target for the treatment of ALD. LAY SUMMARY:Aged mice are more susceptible to alcohol-induced liver injury and fibrosis, which is, at least in part, due to lower levels of sirtuin 1 protein in hepatocytes and hepatic stellate cells. Our findings suggest that sirtuin 1 activators may have beneficial effects for the treatment of alcoholic liver disease in aged patients.
Project description:Alcohol abuse is a major cause of liver disease and mortality worldwide and is a significant public health issue. Patients with alcoholic liver disease (ALD) have severe hepatic lipid accumulation, inflammation, and fibrosis. Therapies for ALD are very limited and even abstinence from alcohol consumption does not necessarily protect patients from progression of the disease. We sought to evaluate the efficacy of a liver X receptor (LXR) inverse agonist, SR9238, in an animal model of ALD. SR9238 suppresses hepatic lipogenesis, a pathological hallmark of ALD, and we hypothesized that targeting suppression of hepatic metabolic pathways that are activated in ALD may be an effective treatment for the disease. A chronic ethanol diet with or without a final ethanol binge treatment was used to induce ALD in mice. Mice were administered the liver specific LXR inverse agonist SR9238 for 4 weeks after the mice had been maintained on the ethanol diet for 14 days. Mice developed all the hallmarks of advanced ALD demonstrating significant pathophysiology and hepatotoxicity. SR9238 significantly attenuated liver injury and hepatic steatosis and fibrosis was nearly eliminated in SR9238 treated mice. SR9238 treatment reversed the damage associated with chronic ethanol use returning the liver to near normal morphology. These results indicate that inhibiting LXR activity using the inverse agonist has a hepatoprotective effect in rodent models of ALD; thus, this pharmacological approach may be efficacious for treatment of ALD in humans.
Project description:The hypoxia-sensing transcriptional factor HIF1? is implicated in a variety of hepato-pathological conditions; however, the contribution of hepatocyte-derived HIF1? during progression of alcoholic liver injury is still controversial. HIF1? induces a variety of genes including those involved in apoptosis via p53 activation. Increased hepatocyte apoptosis is critical for progression of liver inflammation, stellate cell activation and fibrosis. Using hepatocyte-specific HIF1?-deficient mice (?HepHIF1?-/-), here we investigated the contribution of HIF1? to ethanol-induced hepatocyte apoptosis and its role in amplification of fibrosis after carbon tetrachloride (CCl4) exposure. Moderate ethanol feeding (11% of Kcal) induced accumulation of hypoxia-sensitive pimonidazole adducts and HIF1? expression in the liver within 4 days of ethanol feeding. Chronic CCl4 treatment increased M30-positive cells, a marker of hepatocyte apoptosis in pair-fed control mice. Concomitant ethanol feeding (11% of Kcal) amplified CCl4-induced hepatocyte apoptosis in livers of wild-type mice, associated with elevated p53(K386)acetylation, PUMA expression and Ly6c+ cell infiltration. Subsequent to increased apoptosis, ethanol enhanced induction of pro-fibrotic markers including stellate cell activation, collagen 1 expression and extracellular matrix deposition, following CCl4 exposure. Ethanol-induced exacerbation of hepatocyte apoptosis, p53(K386) acetylation and PUMA expression following CCl4 exposure was attenuated in livers of ?HepHIF1?-/- mice. This protection was also associated with a reduction in Ly6c(+) cell infiltration and decreased fibrosis in livers of ?HepHIF1?-/- mice. In summary, these results indicate that moderate ethanol exposure leads to hypoxia/HIF1?-mediated signaling in hepatocytes and induction of p53-dependent apoptosis of hepatocytes, resulting in increased hepatic fibrosis during chronic CCl4 exposure.
Project description:Alcohol liver disease (ALD) is one of the major chronic liver diseases worldwide, ranging from fatty liver, alcoholic hepatitis, cirrhosis, and potentially, hepatocellular carcinoma. Epidemiological studies suggest a potential link between ALD and impaired circadian rhythms, but the role of hepatic circadian proteins in the pathogenesis of ALD remains unknown. Here we show that the circadian clock protein BMAL1 in hepatocytes is both necessary and sufficient to protect mice from ALD. Ethanol diet-fed mice with liver-specific knockout (Bmal1-LKO) or depletion of Bmal1 develop more severe liver steatosis and injury as well as a simultaneous suppression of both de novo lipogenesis and fatty acid oxidation, which can be rescued by the supplementation of synthetic PPAR? ligands. Restoring de novo lipogenesis in the liver of Bmal1-LKO mice by constitutively active AKT not only elevates hepatic fatty acid oxidation but also alleviates ethanol-induced fatty liver and liver injury. Furthermore, hepatic over-expression of lipogenic transcription factor ChREBP, but not SREBP-1c, in the liver of Bmal1-LKO mice also increases fatty acid oxidation and partially reduces ethanol-induced fatty liver and liver injury. Conclusion: we identified a protective role of BMAL1 in hepatocytes against ALD. The protective action of BMAL1 during alcohol consumption depends on its ability to couple ChREBP-induced de novo lipogenesis with PPAR?-mediated fatty oxidation. (Hepatology 2018).
Project description:Argininosuccinate synthase (ASS) is the rate-limiting enzyme in both the urea and the L-citrulline/nitric oxide (NO·) cycles regulating protein catabolism, ammonia levels, and NO· generation. Because a proteomics analysis identified ASS and nitric oxide synthase-2 (NOS2) as coinduced in rat hepatocytes by chronic ethanol consumption, which also occurred in alcoholic liver disease (ALD) and in cirrhosis patients, we hypothesized that ASS could play a role in ethanol binge and chronic ethanol-induced liver damage. To investigate the contribution of ASS to the pathophysiology of ALD, wildtype (WT) and Ass(+/-) mice (Ass(-/-) are lethal due to hyperammonemia) were exposed to an ethanol binge or to chronic ethanol drinking. Compared with WT, Ass(+/-) mice given an ethanol binge exhibited decreased steatosis, lower NOS2 induction, and less 3-nitrotyrosine (3-NT) protein residues, indicating that reducing nitrosative stress by way of the L-citrulline/NO· pathway plays a significant role in preventing liver damage. However, chronic ethanol-treated Ass(+/-) mice displayed enhanced liver injury compared with WT mice. This was due to hyperammonemia, lower phosphorylated AMP-activated protein kinase alpha (pAMPK?) to total AMPK? ratio, decreased sirtuin-1 (Sirt-1) and peroxisomal proliferator-activated receptor coactivator-1? (Pgc1?) messenger RNAs (mRNAs), lower fatty acid ?-oxidation due to down-regulation of carnitine palmitoyl transferase-II (CPT-II), decreased antioxidant defense, and elevated lipid peroxidation end-products in spite of comparable nitrosative stress but likely reduced NOS3.Partial Ass ablation protects only in acute ethanol-induced liver injury by decreasing nitrosative stress but not in a more chronic scenario where oxidative stress and impaired fatty acid ?-oxidation are key events.
Project description:BACKGROUND & AIMS:Defects in lysosome function and autophagy contribute to the pathogenesis of alcoholic liver disease. We investigated the mechanisms by which alcohol consumption affects these processes by evaluating the functions of transcription factor EB (TFEB), which regulates lysosomal biogenesis. METHODS:We performed studies with GFP-LC3 mice, mice with liver-specific deletion of TFEB, mice with disruption of the transcription factor E3 gene (TFE3-knockout mice), mice with disruption of the Tefb and Tfe3 genes (TFEB and TFE3 double-knockout mice), and Tfebflox/flox albumin cre-negative mice (controls). TFEB was overexpressed from adenoviral vectors or knocked down with small interfering RNAs in mouse livers. Mice were placed on diets of regular ethanol feeding plus an acute binge to induce liver damage (ethanol diet); some mice also were given injections of torin-1, an inhibitor of the kinase activity of the mechanistic target of rapamycin (mTOR). Liver tissues were collected and analyzed by immunohistochemistry, immunoblots, and quantitative real-time polymerase chain reaction to monitor lysosome biogenesis. We analyzed levels of TFEB in liver tissues from patients with alcoholic hepatitis and from healthy donors (controls) by immunohistochemistry. RESULTS:Liver tissues from mice on the ethanol diet had lower levels of total and nuclear TFEB compared with control mice, and hepatocytes had decreased lysosome biogenesis and autophagy. Hepatocytes from mice on the ethanol diet had increased translocation of mTOR into lysosomes, resulting in increased mTOR activation. Administration of torin-1 increased liver levels of TFEB and decreased steatosis and liver injury induced by ethanol. Mice that overexpressed TFEB in the liver developed less severe ethanol-induced liver injury and had increased lysosomal biogenesis and mitochondrial bioenergetics compared with mice carrying a control vector. Mice with knockdown of TFEB and TFEB-TFE3 double-knockout mice developed more severe liver injury in response to the ethanol diet than control mice. Liver tissues from patients with alcohol-induced hepatitis had lower nuclear levels of TFEB than control tissues. CONCLUSIONS:We found that ethanol feeding plus an acute binge decreased hepatic expression of TFEB, which is required for lysosomal biogenesis and autophagy. Strategies to block mTOR activity or increase levels of TFEB might be developed to protect the liver from ethanol-induced damage.
Project description:Nogo-B (Reticulon 4B) is an endoplasmic reticulum (ER) resident protein that regulates ER structure and function. Because ER stress is known to induce M2 macrophage polarization, we examined whether Nogo-B regulates M1/M2 polarization of Kupffer cells and alters the pathogenesis of alcoholic liver disease (ALD). M1 and M2 phenotypes were assessed in relation to Nogo-B expression and disease severity in liver specimens from ALD patients (NCT01875211). Liver specimens from wild-type (WT) and Nogo-B knockout (KO) mice fed a control or Lieber-DeCarli ethanol liquid diet (5% ethanol) for 6 weeks were analyzed for liver injury and steatosis. Kupffer cells isolated from WT and Nogo-B KO mice were assessed for M1 and M2 activation. A significant positive correlation was observed between Nogo-B positive Kupffer cells and disease severity in ALD patients (n = 30, r = 0.66, P = 0.048). Furthermore, Nogo-B-positive Kupffer cells were correlated with M1 activation (inducible nitric oxide synthase) (r = 0.50, P = 0.05) and negatively with markers of M2 status (CD163) (r = -0.48, P = 0.07) in these patients. WT mice exhibited significantly increased liver injury (P < 0.05) and higher hepatic triglyceride levels (P < 0.01) compared with Nogo-B KO mice in response to chronic ethanol feeding. Nogo-B in Kupffer cells promoted M1 polarization, whereas absence of Nogo-B increased ER stress and M2 polarization in Kupffer cells.Nogo-B is permissive of M1 polarization of Kupffer cells, thereby accentuating liver injury in ALD in humans and mice. Nogo-B in Kupffer cells may represent a new therapeutic target for ALD. (Hepatology 2017;65:1720-1734).
Project description:Chronic ethanol consumption is a prominent cause of liver disease worldwide. Dysregulation of an important lipid uptake and trafficking gene, liver-fatty acid binding protein (L-FABP), may contribute to alterations in lipid homeostasis during early-stage alcoholic liver. We have reported the detrimental effects of ethanol on the expression of L-FABP and hypothesize this may deleteriously impact metabolic networks regulating fatty acids. Male wild-type (WT) and L-FABP(-/-) mice were fed a modified Lieber-DeCarli liquid diet for six weeks. To assess the response to chronic ethanol ingestion, standard biochemical indicators for alcoholic liver disease (ALD) and oxidative stress were measured. Ethanol ingestion resulted in attenuation of hepatic triglyceride accumulation and elevation of cholesterol in L-FABP(-/-) mice. Lipidomics analysis validated multiple alterations in hepatic lipids resulting from ethanol treatment. Increased immunohistochemical staining for the reactive aldehydes 4-hydroxynonenal and malondialdehyde were observed in WT mice ingesting ethanol; however, L-FABP(-/-) mice displayed prominent protein adducts in liver sections evaluated from pair-fed and ethanol-fed mice. Likewise, alterations in glutathione, thiobarbituric acid reactive substances (TBARS), 8-isoprostanes, and protein carbonyl content all indicated L-FABP(-/-) mice exhibit high sustained oxidative stress in the liver. These data establish that L-FABP is an indirect antioxidant protein essential for sequestering FFA and that its impairment could contribute to in the pathogenesis of ALD.
Project description:BACKGROUND & AIMS:As a nicotinamide adenine dinucleotide-dependent deacetylase and a key epigenetic regulator, sirtuin 6 (SIRT6) has been implicated in the regulation of metabolism, DNA repair, and inflammation. However, the role of SIRT6 in alcohol-related liver disease (ALD) remains unclear. The aim of this study was to investigate the function and mechanism of SIRT6 in ALD pathogenesis. METHODS:We developed and characterized Sirt6 knockout (KO) and transgenic mouse models that were treated with either control or ethanol diet. Hepatic steatosis, inflammation, and oxidative stress were analyzed using biochemical and histological methods. Gene regulation was analyzed by luciferase reporter and chromatin immunoprecipitation assays. RESULTS:The Sirt6 KO mice developed severe liver injury characterized by a remarkable increase of oxidative stress and inflammation, whereas the Sirt6 transgenic mice were protected from ALD via normalization of hepatic lipids, inflammatory response, and oxidative stress. Our molecular analysis has identified a number of novel Sirt6-regulated genes that are involved in antioxidative stress, including metallothionein 1 and 2 (Mt1 and Mt2). Mt1/2 genes were downregulated in the livers of Sirt6 KO mice and patients with alcoholic hepatitis. Overexpression of Mt1 in the liver of Sirt6 KO mice improved ALD by reducing hepatic oxidative stress and inflammation. We also identified a critical link between SIRT6 and metal regulatory transcription factor 1 (Mtf1) via a physical interaction and functional coactivation. Mt1/2 promoter reporter assays showed a strong synergistic effect of SIRT6 on the transcriptional activity of Mtf1. CONCLUSIONS:Our data suggest that SIRT6 plays a critical protective role against ALD and it may serve as a potential therapeutic target for ALD. LAY SUMMARY:The liver, the primary organ for ethanol metabolism, can be damaged by the byproducts of ethanol metabolism, including reactive oxygen species. In this study, we have identified a key epigenetic regulator SIRT6 that plays a critical role in protecting the liver from oxidative stress-induced liver injury. Thus, our data suggest that SIRT6 may be a potential therapeutic target for alcohol-related liver disease.