Mitochondrial acetylome analysis in a mouse model of alcohol-induced liver injury utilizing SIRT3 knockout mice

Mitochondrial protein hyperacetylation is a known consequence of sustained ethanol consumption and has been proposed to play a role in the pathogenesis of alcoholic liver disease (ALD). The mechanisms underlying this altered acetylome, however, remain unknown. The mitochondrial deacetylase sirtuin 3 (SIRT3) is reported to be the major regulator of mitochondrial protein deacetylation and remains a central focus for studies on protein acetylation. To investigate the mechanisms underlying ethanol-induced mitochondrial acetylation, we employed a model for ALD in both wild-type (WT) and SIRT3 knockout (KO) mice using a proteomics and bioinformatics approach. Here, WT and SIRT3 KO groups were compared in a mouse model of chronic ethanol consumption, revealing pathways relevant to ALD, including lipid and fatty acid metabolism, antioxidant response, amino acid biosynthesis and the electron-transport chain, each displaying proteins with altered acetylation. Interestingly, protein hyperacetylation resulting from ethanol consumption and SIRT3 ablation suggests ethanol-induced hyperacetylation targets numerous biological processes within the mitochondria, the majority of which are known to be acetylated through SIRT3-dependent mechanisms. These findings reveal overall increases in 91 mitochondrial targets for protein acetylation, identifying numerous critical metabolic and antioxidant pathways associated with ALD, suggesting an important role for mitochondrial protein acetylation in the pathogenesis of ALD.     

白细胞介素-6在酒精性肝病中的作用

酒精性肝病(alcoholic liver disease,ALD)是由于长期过量饮酒导致肝的内部组织发生炎症损伤的慢性肝病.乙醇及其衍生物在代谢过程中直接或间接诱导引起的肝炎症反应可能是ALD发病的重要机制.然而,该过程内在的细胞分子机制尚不明确.最新研究发现,白细胞介素-6(interleukin-6,IL-6)对...     

Role of cAMP and phosphodiesterase signaling in liver health and disease

Liver disease is a significant health problem worldwide with mortality reaching around 2 million deaths a year. Non-alcoholic fatty liver disease (NAFLD) and alcoholic liver disease (ALD) are the major causes of chronic liver disease. Pathologically, NAFLD and ALD share similar patterns of hepatic disorders ranging from simple steatosis to steatohepatitis, fibrosis and cirrhosis. It is becoming increasingly important to identify new pharmacological targets, given that there is no FDA-approved therapy yet for either NAFLD or ALD. Since the evolution of liver diseases is a multifactorial process, several mechanisms involving parenchymal and non-parenchymal hepatic cells contribute to the initiation and progression of liver pathologies. Moreover, certain protective molecular pathways become repressed during liver injury including signaling pathways such as the cyclic adenosine monophosphate (cAMP) pathway. cAMP, a key second messenger molecule, regulates various cellular functions including lipid metabolism, inflammation, cell differentiation and injury by affecting gene/protein expression and function. This review addresses the current understanding of the role of cAMP metabolism and consequent cAMP signaling pathway(s) in the context of liver health and disease. The cAMP pathway is extremely sophisticated and complex with specific cellular functions dictated by numerous factors such abundance, localization and degradation by phosphodiesterases (PDEs). Furthermore, because of the distinct yet divergent roles of both of its effector molecules, the cAMP pathway is extensively targeted in liver injury to modify its role from physiological to therapeutic, depending on the hepatic condition. This review also examines the behavior of the cAMP-dependent pathway in NAFLD, ALD and in other liver diseases and focuses on PDE inhibition as an excellent therapeutic target in these conditions.     

Genetic variants of TNFα, IL10, IL1β, CTLA4 and TGFβ1 modulate the indices of alcohol-induced liver injury in East Indian population

Alcohol induced liver disease or alcoholic liver disease (ALD), a complex trait, encompasses a gamut of pathophysiological alterations in the liver due to continuous exposure to a toxic amount of alcohol (more than 80g per day). Of all chronic heavy drinkers, only 15-20% develops hepatitis or cirrhosis concomitantly or in succession. Several studies revealed that inter-individual as well as inter-ethnic genetic variation is one of the major factors that predispose to ALD. The role of genetic factors in ALD has long been sought for in ethnically distinct population groups. ALD is fast emerging as an important cause of chronic liver disease in India; even in populations such as "Bengalis" who were "culturally immune" earlier. While the genetic involvement in the pathogenesis of ALD is being sought for in different races, the complex pathophysiology of ALD as well as the knowledge of population level diversity of the relevant alcohol metabolizing and inflammatory pathways mandates the need for well designed studies of genetic factors in ethnically distinct population groups. An array of cytokines plays a critical role as mediators of injury, inflammation, fibrosis and cirrhosis in ALD. We, therefore, studied the association of polymorphisms in five relevant cytokine genes with "clinically significant" ALD in an ethnic "Bengali" population in Eastern India. Compared with "alcoholic" controls without liver disease (n=110), TNFα -238AA genotype, IL1β -511CC genotype, TGFβ1 -509CC genotype and IL10 -592AA genotype were significantly overrepresented in ALD patients (n=181; OR=2.4 and 95% CI 1.2-5.5, P(genotype)=0.042, P(allelic)=0.008; OR=2.7 and 95% CI 1.2-5.9, P(genotype)=0.018, P(allelic)=0.023; OR=4.7 and 95% CI 1.7-13.1, P(genotype)=0.003, P(allelic)=0.014; and OR=2.2 and 95% CI 1.1-4.8, P(genotype)=0.04, P(allelic)=0.039 respectively). Moreover a cumulative genetic risk analysis revealed a significant trend for developing ALD with an increase in the number of risk alleles on IL10 and TGFβ1 loci among alcoholics. The risk genotype of IL1β and TGFβ1 also influences the total bilirubin, albumin and alanine aminotransferase levels among alcoholic "Bengalis". The present study is the first case-control study from Eastern India that comprehensively identified polymorphic markers in TNFα, IL10, IL1β and TGFβ1 genes to be associated with ALD in the Bengali population, accentuating the significance of genetic factors in clinical expressions of ALD.     

Pro-inflammatory signalling and gut-liver axis in non-alcoholic and alcoholic steatohepatitis: Differences and similarities along the path

Non-alcoholic fatty liver disease (NAFLD) and alcohol-associated liver disease (ALD) represent a spectrum of injury, ranging from simple steatosis to steatohepatitis and cirrhosis. In humans, in fact, fatty changes in the liver, possibly leading to end-stage disease, were observed after chronic alcohol intake or in conditions of metabolic impairment. In this article, we examined the features and the pro-inflammatory pathways leading to non-alcoholic and alcoholic steatohepatitis. The involvement of several events (hits) and multiple inter-related pathways in the pathogenesis of these diseases suggest that a single therapeutic agent is unlikely to be an effective treatment strategy. Hence, a combination treatment towards multiple pro-inflammatory targets would eventually be required. Gut-liver crosstalk is involved not only in the impairment of lipid and glucose homoeostasis leading to steatogenesis, but also in the initiation of inflammation and fibrogenesis in both NAFLD and ALD. Modulation of the gut-liver axis has been suggested as a possible therapeutic approach since gut-derived components are likely to be involved in both the onset and the progression of liver damage. This review summarizes the translational mechanisms underlying pro-inflammatory signalling and gut-liver axis in non-alcoholic and alcoholic steatohepatitis. With a multitude of people being affected by liver diseases, identification of possible treatments and the elucidation of pathogenic mechanisms are elements of paramount importance.     

Carotenoids and fatty liver disease: Current knowledge and research gaps

Carotenoids form an important part of the human diet, consumption of which has been associated with many health benefits. With the growing global burden of liver disease, increasing attention has been paid on the possible beneficial role that carotenoids may play in the liver. This review focuses on carotenoid actions in non-alcoholic fatty liver disease (NAFLD), and alcoholic liver disease (ALD). Indeed, many human studies have suggested an association between decreased circulating levels of carotenoids and increased incidence of NAFLD and ALD. The literature describing supplementation of individual carotenoids in rodent models of NAFLD and ALD is reviewed, with particular attention paid to β-carotene and lycopene, but also including β-cryptoxanthin, lutein, zeaxanthin, and astaxanthin. The effect of beta-carotene oxygenase 1 and 2 knock-out mice on hepatic lipid metabolism is also discussed. In general, there is evidence to suggest that carotenoids have beneficial effects in animal models of both NAFLD and ALD. Mechanistically, these benefits may occur via three possible modes of action: 1) improved hepatic antioxidative status broadly attributed to carotenoids in general, 2) the generation of vitamin A from β-carotene and β-cryptoxanthin, leading to improved hepatic retinoid signaling, and 3) the generation of apocarotenoid metabolites from β-carotene and lycopene, that may regulate hepatic signaling pathways. Gaps in our knowledge regarding carotenoid mechanisms of action in the liver are highlighted throughout, and the review ends by emphasizing the importance of dose effects, mode of delivery, and mechanism of action as important areas for further study. This article is part of a Special Issue entitled Carotenoids recent advances in cell and molecular biology edited by Johannes von Lintig and Loredana Quadro.     

C26:0-Carnitine Is a New Biomarker for X-Linked Adrenoleukodystrophy in Mice and Man

X-linked adrenoleukodystrophy (ALD), a progressive neurodegenerative disease, is caused by mutations in ABCD1 and characterized by very-long-chain fatty acids (VLCFA) accumulation. Virtually all males develop progressive myelopathy (AMN). A subset of patients, however, develops a fatal cerebral demyelinating disease (cerebral ALD). Hematopoietic stem cell transplantation is curative for cerebral ALD provided the procedure is performed in an early stage of the disease. Unfortunately, this narrow therapeutic window is often missed. Therefore, an increasing number of newborn screening programs are including ALD. To identify new biomarkers for ALD, we developed an Abcd1 knockout mouse with enhanced VLCFA synthesis either ubiquitous or restricted to oligodendrocytes. Biochemical analysis revealed VLCFA accumulation in different lipid classes and acylcarnitines. Both C26:0-lysoPC and C26:0-carnitine were highly elevated in brain, spinal cord, but also in bloodspots. We extended the analysis to patients and confirmed that C26:0-carnitine is also elevated in bloodspots from ALD patients. We anticipate that validation of C26:0-carnitine for the diagnosis of ALD in newborn bloodspots may lead to a faster inclusion of ALD in newborn screening programs in countries that already screen for other inborn errors of metabolism.     

Loss of AMP-activated protein kinase in X-linked adrenoleukodystrophy patient-derived fibroblasts and lymphocytes

X-Adrenoleukodystrophy (X-ALD) is a peroxisomal disorder characterized by accumulation of very-long-chain (VLC) fatty acids, which induces inflammatory disease and alterations in cellular redox, both of which are reported to play a role in the pathogenesis of the severe form of the disease (childhood cerebral ALD). While the mutation defect in ABCD1 gene is common to all forms of X-ALD it fails to account for the spectrum of phenotypic variability seen in X-ALD patients, strongly suggesting a role for as yet unidentified modifier gene(s). Here we report, for the first time, loss of AMP-activated protein kinase alpha1 (AMPKα1) in patient-derived fibroblasts and lymphocytes of the severe cerebral form of X-ALD (ALD), and not in the milder adrenomyeloneuropathy (AMN) form. Decrease in AMPK was observed at both protein and mRNA levels. AMPK loss in ALD patient-derived fibroblasts was associated with increased ubiquitination. Using the Seahorse Bioscience XF^e96 Flux Analyzer for measuring the mitochondrial oxygen consumption and extracellular acidification rate we show that ALD patient-derived fibroblasts have a significantly lower “metabolic state” than AMN fibroblasts. Unstimulated ALD patient-derived lymphocytes had significantly higher proinflammatory gene expression. Selective AMPK loss represents a novel physiopathogenic factor in X-ALD disease mechanism. Strategies aimed at upregulating/recovering AMPK levels might have beneficial therapeutic effects in X-ALD.     

Recent advances in alcoholic liver disease. IV. Dysregulated cytokine metabolism in alcoholic liver disease

Alcoholic liver disease (ALD) remains a leading cause of death from liver disease in the United States for which there is no FDA-approved therapy. Abnormal cytokine metabolism is a major feature of ALD. Elevated serum concentration levels of TNF-alpha and TNF-alpha-inducible cytokines/chemokines, such as IL-6, -8, and -18, have been reported in patients with alcoholic hepatitis and/or cirrhosis, and levels correlated with markers of the acute phase response, liver function, and clinical outcome. Studies in animal models support an etiologic role for cytokines in the liver injury of ALD. Cytokines, such as transforming growth factor-beta, play a critical role in the fibrosis of ALD. Multiple new strategies are under investigation to modulate cytokine metabolism as a form of therapy for ALD.     

Plasma levels of soluble ST2, but not IL‐33, correlate with the severity of alcoholic liver disease

Alcoholic liver disease (ALD) is a complication that is a burden on global health and economy. Interleukin‐33 (IL‐33) is a newly identified member of the IL‐1 cytokine family and is released as an “alarmin” during inflammation. Soluble suppression of tumourigenicity 2 (sST2), an IL‐33 decoy receptor, has been reported as a new biomarker for the severity of systemic and highly inflammatory diseases. Here, we found the levels of plasma sST2, increased with the disease severity from mild to severe ALD. Importantly, the plasma sST2 levels in ALD patients not only correlated with scores for prognostic models (Maddrey's discriminant function, model for end‐stage liver disease and Child‐Pugh scores) and indexes for liver function (total bilirubin, international normalized ratio, albumin, and cholinesterase) but also correlated with neutrophil‐associated factors as well as some proinflammatory cytokines. In vitro, lipopolysaccharide‐activated monocytes down‐regulated transmembrane ST2 receptor but up‐regulated sST2 mRNA and protein expression and produced higher levels of tumour necrosis factor‐α (TNF‐α). By contrast, monocytes pretreated with recombinant sST2 showed decreased TNF‐α production. In addition, although plasma IL‐33 levels were comparable between healthy controls and ALD patients, we found the IL‐33 expression in liver tissues from ALD patients was down‐regulated at both RNA and protein levels. Immunohistochemical staining further showed that the decreased of IL‐33‐positive cells were mainly located in liver lobule area. These results suggested that sST2, but not IL‐33, is closely related to the severity of ALD. Consequently, sST2 could be used as a potential biomarker for predicting the prognosis of ALD.     

Mutational analysis of X-linked adrenoleukodystrophy gene

X-linked adrenoleukodystrophy (ALD) is an inherited peroxisomal disorder characterized by progressive neurological dysfunction, occasionally associated with adrenal insufficiency. The clinical thenotypes of ALD are quite variable, and include childhood ALD, adult-onset ALD, adrenomyeloneuropathy, and Addison's disease only. Although the causative gene for ALD has been identified, the physiological role of the gene product remains to be clarified. Despite many mutations having been identified in patients with these clinical phenotypes, the genotype-phenotype correlations have not been clarified. The authors investigated genotype-phenotype correlatons in ALD by analyses on 29 unrelated Japanese patients with ALD and by a review of the literature. All the phenotypes were associated with mutations leading to protein truncation, as well as those resulting in subtle amino acid changes. Furthermore, there were no differences in phenotypic expression among the natures of the subtle amino acid changes. All these data indicate that no obvious correlations exist between the phenotypes of ALD patients and their geneotypes, suggesting that other genetic or environmental factors may also be involved in determining phenotypic expression in ALD.     

Genetic determinants of ethanol-induced liver damage

BACKGROUND: Although a clear correlation exists between cumulative alcohol intake and liver disease, only some of the alcohol abusers develop signs of ethanol-induced liver damage. To identify some of the genetic variations predisposing persons to alcoholic liver disease (ALD), a genetic study was performed in heavy drinkers from the cohort of the Dionysis study, a survey aimed at evaluating liver disease in the open population of two towns in Northern Italy (6917 individuals). MATERIALS AND METHODS: 158 heavy drinkers (approximately 85% of all heavy drinkers in the population; daily alcohol intake > 120 g in males and >60 g in females) were investigated by the analysis of nine polymorphic regions, mapping in exons III and IX of the alcohol-dehydrogenase (ADH)-2 gene, in exon VIII of the ADH3 gene, in intron VI, in the promoter region of the cytochrome P4502E1 (CYP2E1) gene, and in the promoter region of the tumor necrosis factor-alpha gene. RESULTS: Heavy drinkers with or without ALD significantly differed for the distribution of alleles of the cytochrome P4502E1 (CYP2E1) and alcohol-dehydrogenase-3 (ADH-3) genes. In one town, allele C2 in the promoter region of the CYP2E1 gene had a frequency of 0.06 in healthy heavy drinkers, of 0.19 in heavy drinkers with ALD (p = 0.012), and of 0.33 in heavy drinkers with cirrhosis (p = 0.033). In the other town, whose inhabitants have different genetic derivation, a prominent association between ALD and homozygosity for allele ADH3*2 of ADH3 was found, with a prevalence of 0.31 in heavy drinkers with ALD and of 0.07 in healthy heavy drinkers controls (p = 0.004). CONCLUSIONS. Both heterozygosity for allele C2 of CYP2E1 and homozygosity for allele ADH3*2 of ADH3 are independent risk factors for ALD in alcohol abusers. The relative contribution of these genotypes to ALD is dependent on their frequency in the population. Overall, heavy drinkers lacking either of these two genotypes are 3.2 and 4.3 times more protected from developing ALD and cirrhosis respectively.     

De novo missense mutation Y174S in exon 1 of the adrenoleukodystrophy (ALD) gene

Adrenoleukodystrophy (ALD) is an X-linked disease, characterised by an alteration of the peroxisomal -oxidation of the very long chain fatty acids. The ALD gene has been identified and mutations have been detected in ALD patients. We report here a new missense mutation in the ALD gene of a male patient, predicting a tyrosine to serine substitution at codon 174 (mutation Y174S). The mother of the ALD patient does not have the Y174S mutation in her leukocyte DNA, indicating that Y174S arose de novo in the patient. Y174S is the first reported de novo mutation in the ALD gene.     

Activation of farnesoid X receptor attenuates hepatic injury in a murine model of alcoholic liver disease

Alcoholic liver disease (ALD) is a common cause of advanced liver disease, and considered as a major risk factor of morbidity and mortality worldwide. Hepatic cholestasis is a pathophysiological feature observed in all stages of ALD. The farnesoid X receptor (FXR) is a member of the nuclear hormone receptor superfamily, and plays an essential role in the regulation of bile acid, lipid and glucose homeostasis. However, the role of FXR in the pathogenesis and progression of ALD remains largely unknown. Mice were fed Lieber-DeCarli ethanol diet or an isocaloric control diet. We used a specific agonist of FXR WAY-362450 to study the effect of pharmacological activation of FXR in alcoholic liver disease. In this study, we demonstrated that FXR activity was impaired by chronic ethanol ingestion in a murine model of ALD. Activation of FXR by specific agonist WAY-362450 protected mice from the development of ALD. We also found that WAY-362450 treatment rescued FXR activity, suppressed ethanol-induced Cyp2e1 up-regulation and attenuated oxidative stress in liver. Our results highlight a key role of FXR in the modulation of ALD development, and propose specific FXR agonists for the treatment of ALD patients.     

Noncoding RNAs in alcoholic liver disease

Alcoholic liver disease (ALD) is a complex process with high morbitity and can cause liver dysfunction, which contains a wide spectrum of hepatic lesions, including steatohepatitis, fibrosis, cirrhosis, and eventually hepatocellular carcinoma. To date, the molecular mechanisms for ALD have not been fully explored and an effective therapy is still missing. Overwhelming evidence shows dysregulation of noncoding RNAs (ncRNAs), particularly microRNAs (miRNAs), is correlated with etiopathogenesis and progress of ALD including hepatocyte damage, disrupted lipid metabolism, aggressive inflammatory responses, oxidative stress, programmed cell death, fibrosis, and epigenetic changes induced by alcohol. For example, circulating miRNA-122 is a marker of hepatocyte damage, and miRNA-155 is a potential marker of inflammation, indicating their diagnosis therapeutic potential in ALD. In addition, roles for long noncoding RNAs (lncRNAs) and circular RNAs in ALD are being uncovered. Further, circulating ncRNAs and exosome-derived ncRNAs have attracted more attention lately, suggesting a role in the prevention and treatment of ALD. This review covers the roles of ncRNAs in ALD, and the potential uses as markers for diagnosis and therapeutic options.     

MicroRNAs in alcoholic liver disease: Recent advances and future applications

Alcoholic liver disease (ALD) is characterized by hepatocyte damage, inflammatory cell activation, and increased intestinal permeability leading to the clinical manifestations of alcoholic hepatitis. Selected members of the family of microRNAs (miRNAs) are affected by alcohol, resulting in an abnormal miRNA profile in the liver and circulation in ALD. Increasing evidence suggests that miRNAs that regulate inflammation, lipid metabolism and promote cancer are affected by excessive alcohol administration in mouse models of ALD. This communication highlights recent findings in miRNA expression and functions as they relate to the pathogenesis of ALD. The cell-specific distribution of miRNAs, as well as the significance of circulating extracellular miRNAs, is discussed as potential biomarkers. Finally, the prospects of miRNA-based therapies are evaluated in ALD.     

Characterization of E. coli tetrameric aldehyde dehydrogenases with atypical properties compared to other aldehyde dehydrogenases

Aldehyde dehydrogenases are general detoxifying enzymes, but there are also isoenzymes that are involved in specific metabolic pathways in different organisms. Two of these enzymes are Escherichia coli lactaldehyde (ALD) and phenylacetaldehyde dehydrogenases (PAD), which participate in the metabolism of fucose and phenylalanine, respectively. These isozymes share some properties with the better characterized mammalian enzymes but have kinetic properties that are unique. It was possible to thread the sequences into the known ones for the mammalian isozymes to better understand some structural differences. Both isozymes were homotetramers, but PAD used both NAD+ and NADP+ but with a clear preference for NAD, while ALD used only NAD+. The rate-limiting step for PAD was hydride transfer as indicated by the primary isotopic effect and the absence of a pre-steady-state burst, something not previously found for tetrameric enzymes from other organisms where the rate-limiting step is related to both deacylation and coenzyme dissociation. In contrast, ALD had a pre-steady-state burst indicating that the rate-limiting step was located after the NADH formation, but the rate-limiting step was a combination of deacylation and coenzyme dissociation. Both enzymes possessed esterase activity that was stimulated by NADH; NAD+ stimulated the esterase activity of PAD but not of ALD. Finding enzymes that structurally are similar to the well-characterized mammalian enzymes but have a different rate-limiting step might serve as models to allow us to determine what regulates the rate-limiting step.