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Effect of ethanol on lipid metabolism

  • Min You
    Correspondence
    Corresponding authors. Addresses: Department of Pharmaceutical Sciences, College of Pharmacy, Northeast Ohio Medical University, Rootstown, OH, USA (M. You), or Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh PA, USA (G. Arteel).
    Affiliations
    Department of Pharmaceutical Sciences, College of Pharmacy, Northeast Ohio Medical University, Rootstown, OH, USA
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  • Gavin E. Arteel
    Correspondence
    Corresponding authors. Addresses: Department of Pharmaceutical Sciences, College of Pharmacy, Northeast Ohio Medical University, Rootstown, OH, USA (M. You), or Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh PA, USA (G. Arteel).
    Affiliations
    Department of Medicine, Division of Gastroenterology, Hepatology and Nutrition, University of Pittsburgh, Pittsburgh, PA, USA

    Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, PA, USA
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      Summary

      Hepatic lipid metabolism is a series of complex processes that control influx and efflux of not only hepatic lipid pools, but also organismal pools. Lipid homeostasis is usually tightly controlled by expression, substrate supply, oxidation and secretion that keep hepatic lipid pools relatively constant. However, perturbations of any of these processes can lead to lipid accumulation in the liver. Although it is thought that these responses are hepatic arms of the ‘thrifty genome’, they are maladaptive in the context of chronic fatty liver diseases. Ethanol is likely unique among toxins, in that it perturbs almost all aspects of hepatic lipid metabolism. This complex response is due in part to the large metabolic demand placed on the organ by alcohol metabolism, but also appears to involve more nuanced changes in expression and substrate supply. The net effect is that steatosis is a rapid response to alcohol abuse. Although transient steatosis is largely an inert pathology, the chronicity of alcohol-related liver disease seems to require steatosis. Better and more specific understanding of the mechanisms by which alcohol causes steatosis may therefore translate into targeted therapies to treat alcohol-related liver disease and/or prevent its progression.

      Keywords

      Introduction

      Alcohol is highly prevalent in most societies and more than 50% of Americans consume alcohol at least once a month.

      Services USDoHaH. Results from the 2010 National Survey on Drug Use and Health; 2010, 2010.

      Heavy alcohol consumption associated with alcohol dependence and/or abuse (i.e., binge drinking) is well known to damage the liver. Alcohol-related liver disease (ALD) affects more than 10 million Americans each year, while treating the medical consequences of the disease costs more than $166 billion annually.
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      The first and most common hepatic change caused by alcohol consumption is steatosis, or fatty liver (Fig. 1). The prevalence of steatosis is essentially 100% in those who consume alcohol at levels that increase their risk of liver disease.
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      These facts challenge the assumption that steatosis is an inert pathology. Hepatic fat accumulation can invoke metabolic changes that sensitise the liver to further injury (see below). Therefore, a full understanding of how alcohol induces steatosis could be key in preventing progression to later stages of ALD.
      The initial hepatic change caused by excessive alcohol consumption is steatosis, which occurs in almost all patients who consume harmful levels of alcohol.
      Figure thumbnail gr1
      Fig. 1Steatosis in alcohol-related liver disease. Representative pictures of liver biopsies from patients with ALD and different degrees of steatosis. In all cases macro- and microsteatosis are present. Photomicrographs courtesy of Dr. John Woosley, University of North Carolina at Chapel Hill.
      The liver plays a central role in lipid metabolism for the entire organism. Hepatic free fatty acids (FAs) are not only directly synthesised from glycolytic end products and hepatic catabolism (e.g., autophagy), but are also actively taken up by the liver from dietary, and extrahepatic (e.g., adipose tissue lipolysis) sources. This pool of FAs can either be used for energy via β-oxidation, membrane synthesis or esterification into triglycerides by hepatocytes. Triglycerides are subsequently packaged as very low-density lipoproteins (VLDLs) that can be secreted into the bloodstream or serve as precursors for primary bile acids, which facilitate the emulsification of dietary lipids for delivery to the liver and extrahepatic sites. There is intricate cross-talk between these systems. Hepatic lipid metabolism is controlled by a complex interplay of hormones, nuclear receptors, intracellular signalling pathways and transcription factors. Under homeostatic conditions, hepatic lipid flux maintains relatively low concentrations of hepatic lipid pools. However, dysregulation of this flux can cause lipids to accumulate in hepatocytes, leading to steatosis (Fig. 2).
      Figure thumbnail gr2
      Fig. 2Intricate regulation of lipid metabolism, and the impact of ethanol exposure. The liver plays a central role in lipid metabolism for the entire organism. Hepatic free FAs are not only directly synthesised (lipogenesis), but are also actively taken up by the liver. This pool of FAs can either be used for energy (FA oxidation), membrane synthesis or for esterification into triglycerides by hepatocytes. Triglycerides are subsequently packaged as VLDLs to be secreted. There is intricate cross-talk between these systems and hepatic lipid metabolism is controlled by a complex interplay of hormones, nuclear receptors, intracellular signalling pathways and transcription factors. Alcohol directly and indirectly impacts numerous aspects of hepatic lipid flux that ultimately leads to lipid accumulation. FA, fatty acid; VLDL, very low-density lipoprotein.
      Alcohol directly and indirectly impacts numerous aspects of hepatic lipid flux that ultimately lead to lipid accumulation. The simplest example is that alcohol metabolism itself directly causes steatosis. Concentrations of alcohol can easily reach the mM range in the portal/hepatic circulation during alcohol consumption. In the process of metabolising ethanol to acetate, 2 equivalents of reduced NADH are generated per equivalent of ethanol oxidised. This metabolism robustly increases the ratio of NADH:NAD+ within the cell, which then favours inhibition of FA β-oxidation in the liver. Furthermore, ethanol metabolism also increases the rate of esterification of Fas.
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      The net effect is to favour triglyceride accumulation in the hepatocytes. However, the impact of alcohol exposure on lipid metabolism is far more complex than simple redox inhibition of β-oxidation. The purpose of this review is to summarise the known impacts of ethanol on this process.

      Effects of ethanol on fatty acid transporters

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      Effects of ethanol on FA and triglyceride synthesis: potential key players

      As mentioned, the liver can generate FAs from non-lipid precursors via de novo lipogenesis. This process is predominantly regulated by insulin and glucose flux in the liver and serves to provide a storage source of energy during times of fasting. Pyruvate from glycolysis enters the citric acid cycle and is converted to citrate, which is subsequently converted to acetyl- and malonyl-CoA and used to synthesise FAs. Rate-limiting enzymes in this process include acetyl-CoA carboxylases 1 and 2 (ACC-1 and -2 which convert acetyl-CoA to malonyl-CoA), FA synthase (FASN which synthesise saturated FAs from malonyl-CoA), and steryl-CoA-desaturase-1 (SCD-1 which converts saturated FAs to monounsaturated FAs). The synthesis of glycerolipid (i.e., triglycerides) from FAs is mediated by key acetyltransferases (e.g., GPAT, AGPAT and DGAT) and phosphatidate phosphatases (e.g., lipin-1).

      SREBP-1c and ChREBP and transcriptional control of lipogenesis

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      Lipin-1

      Lipin-1 protein plays a pivotal role in lipid synthesis as a mammalian Mg2+-dependent phosphatidic acid phosphohydrolase (PAP), which catalyses the penultimate step in triglyceride synthesis.
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      • et al.
      DEP domain-containing mTOR-interacting protein suppresses lipogenesis and ameliorates hepatic steatosis and acute-on-chronic liver injury in alcoholic liver disease.
      Ethanol also blocks lipin-1 nuclear entry, inhibits nuclear lipin-1-mediated FA oxidation and perturbs VLDL secretion in mouse liver.
      • Hu M.
      • Yin H.
      • Mitra M.S.
      • Liang X.
      • Ajmo J.M.
      • Nadra K.
      • et al.
      Hepatic-specific lipin-1 deficiency exacerbates experimental alcohol-induced steatohepatitis in mice.
      Furthermore, ethanol suppresses lipin-1 alternative pre-mRNA splicing and subsequently increases the ratio of lipin-1β/α by disrupting the SIRT1-SFRS10 axis.
      • Everitt H.
      • Hu M.
      • Ajmo J.M.
      • Rogers C.Q.
      • Liang X.
      • Zhang R.
      • et al.
      Ethanol administration exacerbates the abnormalities in hepatic lipid oxidation in genetically obese mice.
      • Yin H.
      • Hu M.
      • Liang X.
      • Ajmo J.M.
      • Li X.
      • Bataller R.
      • et al.
      Deletion of SIRT1 from hepatocytes in mice disrupts lipin-1 signaling and aggravates alcoholic fatty liver.
      Abnormalities in lipin-1 are also involved in the ethanol-induced production of a panel of pro-inflammatory cytokines.
      • Yin H.
      • Liang X.
      • Jogasuria A.
      • Davidson N.O.
      • You M.
      miR-217 regulates ethanol-induced hepatic inflammation by disrupting sirtuin 1-lipin-1 signaling.
      These ethanol-mediated alterations in lipin-1 promote steatosis, exacerbate inflammation and cause liver injury.

      ER stress and the UPR

      The endoplasmic reticulum (ER) is critically involved in the proper folding and assembly of secreted and membrane proteins. Homeostasis between the protein load and the capacity of the ER to process this load must be maintained to ensure proper protein folding. Physiological and pathological stimuli can disrupt this homeostasis causing misfolded or unfolded proteins to accumulate, leading to ER stress. In attempts to re-establish homeostasis, the ER activates a signalling network known as the unfolded protein response (UPR). One downstream effect of activation of the UPR by ER stress is the insulin-independent proteolytic activation of SREBP-1c. This effect of the UPR makes teleological sense, in that increasing lipogenesis would increase lipid substrate supply to the ER for protein processing.
      • Ferre P.
      • Foufelle F.
      Hepatic steatosis: a role for de novo lipogenesis and the transcription factor SREBP-1c.
      It has been shown that alcohol induces ER stress in the liver, at least in part by causing hyperhomocysteinaemia.
      • Ji C.
      • Kaplowitz N.
      Betaine decreases hyperhomocysteinemia, endoplasmic reticulum stress, and liver injury in alcohol-fed mice.
      • Kaplowitz N.
      • Ji C.
      Unfolding new mechanisms of alcoholic liver disease in the endoplasmic reticulum.

      TNFα

      It is well known that both the basal and lipopolysaccharide-stimulated production of TNFα (or TNF) are increased in humans consuming alcohol and in experimental ALD.
      • McClain C.J.
      • Cohen D.A.
      Increased tumor necrosis factor production by monocytes in alcoholic hepatitis.
      • Nanji A.
      • Zhoa S.
      • Sadrzadeh S.
      • Waxman D.
      Use of reverse transcription-polymerase chain reaction to evaluate in vivo cytokin gene expression in rats fed ethanol for long periods.
      The role of TNFα and other pro-inflammatory cytokines in hepatic inflammation is well known. However, studies in experimental ALD indicate that they may also contribute to lipogenesis. Specifically, genetic or pharmacologic inhibition of TNFα signalling blunted steatosis caused by alcohol.
      • Iimuro Y.
      • Gallucci R.M.
      • Luster M.I.
      • Kono H.
      • Thurman R.G.
      Antibodies to tumor necrosis factor-a attenuate hepatic necrosis and inflammation due to chronic exposure to ethanol in the rat.
      • Ji C.
      • Deng Q.
      • Kaplowitz N.
      Role of TNF-alpha in ethanol-induced hyperhomocysteinemia and murine alcoholic liver injury.
      • Yin M.
      • Wheeler M.D.
      • Kono H.
      • Bradford B.U.
      • Gallucci R.M.
      • Luster M.I.
      • et al.
      Essential role of tumor necrosis factor alpha in alcohol-induced liver injury in mice.
      This effect of TNFα may be mediated at several levels of lipid metabolism. For example, TNFα increases free FA release from adipocytes in the periphery,
      • Hardardottir I.
      • Doerrler W.
      • Feingold K.R.
      • Grunfeld C.
      Cytokines stimulate lipolysis and decrease lipoprotein lipase activity in cultured fat cells by a prostaglandin independent mechanism.
      increases lipogenesis in hepatocytes,
      • Feingold K.R.
      • Serio M.K.
      • Adi S.
      • Moser A.H.
      • Grunfeld C.
      Tumor necrosis factor stimulates hepatic lipid synthesis and secretion.
      and inhibits β-oxidation of Fas.
      • Nachiappan V.
      • Curtiss D.
      • Corkey B.E.
      • Kilpatrick L.
      Cytokines inhibit fatty acid oxidation in isolated rat hepatocytes: synergy among TNF, IL-6, and IL-1.
      Moreover, prooxidant production stimulated by TNFα in hepatocytes could impair mitochondrial electron flow and cause lipid peroxidation, processes that could also slow the metabolism of fat by mitochondria. Other studies demonstrated transcription and activation of SREBP-1c is enhanced by TNFα in hepatocytes,
      • Lawler Jr., J.F.
      • Yin M.
      • Diehl A.M.
      • Roberts E.
      • Chatterjee S.
      Tumor necrosis factor-alpha stimulates the maturation of sterol regulatory element binding protein-1 in human hepatocytes through the action of neutral sphingomyelinase.
      • Endo M.
      • Masaki T.
      • Seike M.
      • Yoshimatsu H.
      TNF-alpha induces hepatic steatosis in mice by enhancing gene expression of sterol regulatory element binding protein-1c (SREBP-1c).
      which yields another mechanistic link between TNFα and lipogenesis. Other cytokines induced by alcohol (e.g., IL-1 and IL-6) may also impair transport and secretion of triglycerides.
      • Navasa M.
      • Gordon D.A.
      • Hariharan N.
      • Jamil H.
      • Shigenaga J.K.
      • Moser A.
      • et al.
      Regulation of microsomal triglyceride transfer protein mRNA expression by endotoxin and cytokines.

      PPARγ

      Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear hormone receptor that is known to impact on lipid metabolism and glucose homeostasis. The PPARG gene encodes 2 splice isoforms of the protein product, PPARγ1 and PPARγ2; the former is constitutively expressed at low levels in most tissues, whereas the latter is expressed predominantly in adipose tissue under basal conditions.
      • Vidal-Puig A.J.
      • Considine R.V.
      • Jimenez-Linan M.
      • Werman A.
      • Pories W.J.
      • Caro J.F.
      • et al.
      Peroxisome proliferator-activated receptor gene expression in human tissues. Effects of obesity, weight loss, and regulation by insulin and glucocorticoids.
      Although the liver normally expresses low levels of PPARγ2, expression is elevated in steatotic livers, both alcoholic and non-alcoholic.
      • Vidal-Puig A.J.
      • Considine R.V.
      • Jimenez-Linan M.
      • Werman A.
      • Pories W.J.
      • Caro J.F.
      • et al.
      Peroxisome proliferator-activated receptor gene expression in human tissues. Effects of obesity, weight loss, and regulation by insulin and glucocorticoids.
      • Pettinelli P.
      • Videla L.A.
      Up-regulation of PPAR-gamma mRNA expression in the liver of obese patients: an additional reinforcing lipogenic mechanism to SREBP-1c induction.
      • Zhang W.
      • Sun Q.
      • Zhong W.
      • Sun X.
      • Zhou Z.
      Hepatic peroxisome proliferator-activated receptor gamma signaling contributes to alcohol-induced hepatic steatosis and inflammation in mice.
      The activation of PPARγ may be pleiotropic in fatty liver disease. Specifically, PPARγ agonists exert beneficial effects in both diet-induced and alcohol-induced fatty liver injury;
      • Bouskila M.
      • Pajvani U.B.
      • Scherer P.E.
      Adiponectin: a relevant player in PPARgamma-agonist-mediated improvements in hepatic insulin sensitivity?.
      • Wang Y.
      • Lam K.S.
      • Yau M.H.
      • Xu A.
      Post-translational modifications of adiponectin: mechanisms and functional implications.
      • Enomoto N.
      • Takei Y.
      • Hirose M.
      • Konno A.
      • Shibuya T.
      • Matsuyama S.
      • et al.
      Prevention of ethanol-induced liver injury in rats by an agonist of peroxisome proliferator-activated receptor-gamma, pioglitazone.
      these protective effects are largely attributed to increasing adiponectin production in adipocytes (
      • Rogers C.Q.
      • Ajmo J.M.
      • You M.
      Adiponectin and alcoholic fatty liver disease.
      ; see later). In contrast, studies in hepatocyte-specific knockout mice indicate that PPARγ2 activation is detrimental to the liver in experimental alcoholic and non-alcoholic liver disease.
      • Zhou S.L.
      • Gordon R.E.
      • Bradbury M.
      • Stump D.
      • Kiang C.L.
      • Berk P.D.
      Ethanol up-regulates fatty acid uptake and plasma membrane expression and export of mitochondrial aspartate aminotransferase in HepG2 cells.
      This hepatic effect of PPARγ appears to be mediated via induction of SREBP-1c and other genes key to lipogenesis.

      AMPK and SIRT1

      The protein kinase complex, AMPK, provides another level of control over lipid metabolism. . AMPK acts as a “sensor” of cellular energy status and helps to maintain homeostasis.
      • Hardie D.G.
      AMP-activated/SNF1 protein kinases: conserved guardians of cellular energy.
      In general, the downstream effects of AMPK activation are considered catabolic and favour ATP generation during energy depletion. For example, glycolysis is enhanced by AMPK. Signalling downstream of AMPK also inhibits ATP-consuming processes, such as de novo lipogenesis.
      • Krause U.
      • Bertrand L.
      • Hue L.
      Control of p70 ribosomal protein S6 kinase and acetyl-CoA carboxylase by AMP-activated protein kinase and protein phosphatases in isolated hepatocytes.
      More specifically, AMPK phosphorylates a number of serine residues on both isoforms of ACC (ACC-1 and ACC-2), which inhibits their activity, even in the presence of citrate.
      • Park S.H.
      • Gammon S.R.
      • Knippers J.D.
      • Paulsen S.R.
      • Rubink D.S.
      • Winder W.W.
      Phosphorylation-activity relationships of AMPK and acetyl-CoA carboxylase in muscle.
      In addition to blocking the activity of key lipogenic enzymes, AMPK indirectly decreases lipogenesis by phosphorylating ChREBP, thereby hindering its nuclear translocation and transcriptional activity.
      • Kawaguchi T.
      • Osatomi K.
      • Yamashita H.
      • Kabashima T.
      • Uyeda K.
      Mechanism for fatty acid “sparing” effect on glucose-induced transcription: regulation of carbohydrate-responsive element-binding protein by AMP-activated protein kinase.
      Likewise, AMPK directly phosphorylates SREBP-1c, which also causes an inhibition of this factor’s transcriptional activity.
      • Li Y.
      • Xu S.
      • Mihaylova M.M.
      • Zheng B.
      • Hou X.
      • Jiang B.
      • et al.
      AMPK phosphorylates and inhibits SREBP activity to attenuate hepatic steatosis and atherosclerosis in diet-induced insulin-resistant mice.
      Ethanol has been demonstrated to inhibit AMPK phosphorylation, thereby inhibiting ACC, SREBP-1c and ChREBP.
      • Hu M.
      • Wang F.
      • Li X.
      • Rogers C.Q.
      • Liang X.
      • Finck B.N.
      • et al.
      Regulation of hepatic lipin-1 by ethanol: role of AMP-activated protein kinase/sterol regulatory element-binding protein 1 signaling in mice.
      • You M.
      • Crabb D.W.
      Recent advances in alcoholic liver disease II. Minireview: molecular mechanisms of alcoholic fatty liver.
      • You M.
      • Matsumoto M.
      • Pacold C.M.
      • Cho W.K.
      • Crabb D.W.
      The role of AMP-activated protein kinase in the action of ethanol in the liver.
      • Liangpunsakul S.
      • Ross R.A.
      • Crabb D.W.
      Activation of carbohydrate response element-binding protein by ethanol.
      The mechanisms appear to involve activation of the dephosphorylase PP2A via aSMase-mediated ceramide signalling
      • Supakul R.
      • Liangpunsakul S.
      Alcoholic-induced hepatic steatosis-role of ceramide and protein phosphatase 2A.
      • Yang L.
      • Jin G.H.
      • Zhou J.Y.
      The role of ceramide in the pathogenesis of alcoholic liver disease.
      and and/or via inhibition of upstream activation pathways (e.g., LKB1
      • Liangpunsakul S.
      • Wou S.E.
      • Zeng Y.
      • Ross R.A.
      • Jayaram H.N.
      • Crabb D.W.
      Effect of ethanol on hydrogen peroxide-induced AMPK phosphorylation.
      ).
      SIRT-1 is an NAD+-dependent protein deactylase. Targets of its deactylase activity include several key players in SREBP-1 and ChREBP-1 signalling.
      • Ponugoti B.
      • Kim D.H.
      • Xiao Z.
      • Smith Z.
      • Miao J.
      • Zang M.
      • et al.
      SIRT1 deacetylates and inhibits SREBP-1C activity in regulation of hepatic lipid metabolism.
      • You M.
      • Jogasuria A.
      • Taylor C.
      • Wu J.
      Sirtuin 1 signaling and alcoholic fatty liver disease.
      • Bouras T.
      • Fu M.F.
      • Sauve A.A.
      • Wang F.
      • Quong A.A.
      • Perkins N.D.
      • et al.
      SIRT1 deacetylation and repression of p300 involves lysine residues 1020/1024 within the cell cycle regulatory domain 1.
      • Wang R.H.
      • Li C.
      • Deng C.X.
      Liver steatosis and increased ChREBP expression in mice carrying a liver specific SIRT1 null mutation under a normal feeding condition.
      SIRT-1 also deacetylates histones, namely H3 and H4, which could epigenetically increase expression of lipogenic genes (e.g., SREBF1
      • You M.
      • Jogasuria A.
      • Taylor C.
      • Wu J.
      Sirtuin 1 signaling and alcoholic fatty liver disease.
      ). Ethanol exposure downregulates expression of SIRT-1,
      • You M.
      • Jogasuria A.
      • Taylor C.
      • Wu J.
      Sirtuin 1 signaling and alcoholic fatty liver disease.
      • Lieber C.S.
      • Leo M.A.
      • Wang X.
      • Decarli L.M.
      Effect of chronic alcohol consumption on Hepatic SIRT1 and PGC-1alpha in rats.
      likely at multiple levels of control.
      • You M.
      • Jogasuria A.
      • Taylor C.
      • Wu J.
      Sirtuin 1 signaling and alcoholic fatty liver disease.
      Additionally, the deactylase activity of SIRT-1 is sensitive to the NADH redox state of the cell.
      • Revollo J.R.
      • Li X.
      The ways and means that fine tune Sirt1 activity.
      Thus, the increased ratio of NADH:NAD+ in the more reduced state caused by ethanol metabolism may not only blunt FA oxidation, but also directly contribute to increased de novo lipogenesis by blunting SIRT-1 activity. AMPK and SIRT-1 share many overlapping targets of regulation, the former via phosphorylation and the latter via deacetylation. Indeed, it is thought that these overlapping functions are at least permissive to each other and that maximal inhibition of lipogenesis is only affected when both AMPK and SIRT-1 are activated.
      • Ruderman N.B.
      • Xu X.J.
      • Nelson L.
      • Cacicedo J.M.
      • Saha A.K.
      • Lan F.
      • et al.
      AMPK and SIRT1: a long-standing partnership?.
      Thus, the fact that both are inhibited by ethanol implies that lipogenesis will be effectively disinhibited.

      Molecular chaperones

      Stress induced heat shock proteins (Hsps) such as Hsp90, Hsp70, and Hsp72 are ubiquitous and highly conserved, and can be induced by a wide variety of physiological and environmental insults.
      • Krijgsheld K.R.
      • Scholtens E.
      • Mulder G.J.
      An evaluation of methods to decrease the availability of inorganic sulphate for sulphate conjugation in the rat in vivo.
      Heat shock factors (HSFs) upregulate a family of Hsp genes by binding to the heat shock-binding element (HSE).
      • Henstridge D.C.
      • Whitham M.
      • Febbraio M.A.
      Chaperoning to the metabolic party: the emerging therapeutic role of heat-shock proteins in obesity and type 2 diabetes.
      • Kuan Y.C.
      • Hashidume T.
      • Shibata T.
      • Uchida K.
      • Shimizu M.
      • Inoue J.
      • et al.
      Heat Shock protein 90 modulates lipid homeostasis by regulating the stability and function of sterol regulatory element-binding protein (SREBP) and SREBP cleavage-activating protein.
      • Mandrekar P.
      Signaling mechanisms in alcoholic liver injury: role of transcription factors, kinases and heat shock proteins.
      Hsps serve as chaperones that maintain the function of signalling molecules in lipid metabolism. For instance, Hsp90 alters lipid homeostasis by regulating SREBP-1.
      • Kuan Y.C.
      • Hashidume T.
      • Shibata T.
      • Uchida K.
      • Shimizu M.
      • Inoue J.
      • et al.
      Heat Shock protein 90 modulates lipid homeostasis by regulating the stability and function of sterol regulatory element-binding protein (SREBP) and SREBP cleavage-activating protein.
      Hsps play pivotal pathophysiological roles in AFLD.
      • Mandrekar P.
      Signaling mechanisms in alcoholic liver injury: role of transcription factors, kinases and heat shock proteins.
      Like other stress signals, ethanol consumption results in accumulation of stress proteins such as hepatic Hsp70, Hsp72, Hsp90 and HSF-1 in human and experimental murine AFLD.
      • Mandrekar P.
      Signaling mechanisms in alcoholic liver injury: role of transcription factors, kinases and heat shock proteins.
      • Porras N.
      • Strauss M.
      • Rodriguez M.
      • Anselmi G.
      Hsp70 accumulation and ultrastructural features of lung and liver induced by ethanol treatment with and without L-carnitine protection in rats.
      • Mikami T.
      • Sumida S.
      • Ishibashi Y.
      • Ohta S.
      Endurance exercise training inhibits activity of plasma GOT and liver caspase-3 of mice [correction of rats] exposed to stress by induction of heat shock protein 70.
      • Ikeyama S.
      • Kusumoto K.
      • Miyake H.
      • Rokutan K.
      • Tashiro S.
      A non-toxic heat shock protein 70 inducer, geranylgeranylacetone, suppresses apoptosis of cultured rat hepatocytes caused by hydrogen peroxide and ethanol.
      • Yao X.
      • Bai Q.
      • Yan D.
      • Li G.
      • Lu C.
      • Xu H.
      Solanesol protects human hepatic L02 cells from ethanol-induced oxidative injury via upregulation of HO-1 and Hsp70.
      • Kitam V.O.
      • Maksymchuk O.V.
      • Chashchyn M.O.
      The possible mechanisms of CYP2E1 interactions with HSP90 and the influence of ethanol on them.
      • Islam A.
      • Abraham P.
      • Hapner C.D.
      • Deuster P.A.
      • Chen Y.
      Tissue-specific upregulation of HSP72 in mice following short-term administration of alcohol.
      • Bukong T.N.
      • Hou W.
      • Kodys K.
      • Szabo G.
      Ethanol facilitates hepatitis C virus replication via up-regulation of GW182 and heat shock protein 90 in human hepatoma cells.
      • Carbone D.L.
      • Doorn J.A.
      • Kiebler Z.
      • Ickes B.R.
      • Petersen D.R.
      Modification of heat shock protein 90 by 4-hydroxynonenal in a rat model of chronic alcoholic liver disease.
      • Mandrekar P.
      • Catalano D.
      • Jeliazkova V.
      • Kodys K.
      Alcohol exposure regulates heat shock transcription factor binding and heat shock proteins 70 and 90 in monocytes and macrophages: implication for TNF-alpha regulation.
      • Ambade A.
      • Catalano D.
      • Lim A.
      • Kopoyan A.
      • Shaffer S.A.
      • Mandrekar P.
      Inhibition of heat shock protein 90 alleviates steatosis and macrophage activation in murine alcoholic liver injury.
      For example, ethanol exposure induces hepatic Hsp90 in mice and contributes to the development of steatosis and liver injury via dysregulation of molecules important in lipid metabolism, including SREBP-1, SCD-1, FASN and ACC-1.
      • Ambade A.
      • Catalano D.
      • Lim A.
      • Kopoyan A.
      • Shaffer S.A.
      • Mandrekar P.
      Inhibition of heat shock protein 90 alleviates steatosis and macrophage activation in murine alcoholic liver injury.
      Pharmacologic inhibition of Hsp90 ameliorates fatty liver injury during chronic or acute ethanol exposure in rodents. These studies have demonstrated the clear and direct regulation of hepatic lipid metabolism by Hsps in rodents in response to ethanol challenge. In addition to Hsps, sestrins are a family of stress-sensitive genes regulating lipid metabolism.
      • Ambade A.
      • Catalano D.
      • Lim A.
      • Kopoyan A.
      • Shaffer S.A.
      • Mandrekar P.
      Inhibition of heat shock protein 90 alleviates steatosis and macrophage activation in murine alcoholic liver injury.
      The inhibitory effect of ethanol on sestrin 3 contributes to the development of steatosis by disrupting AMPK signalling, which leads to alterations in the genes involved in FA synthesis and oxidation.
      • Kang X.
      • Petyaykina K.
      • Tao R.
      • Xiong X.
      • Dong X.C.
      • Liangpunsakul S.
      The inhibitory effect of ethanol on Sestrin3 in the pathogenesis of ethanol-induced liver injury.
      Future studies are needed to delineate the precise role of Hsps and sestrins in lipid metabolism and its contribution to alcoholic steatosis.

      Adiponectin and FGF-15 axis

      Adiponectin is an adipose-derived hormone that circulates in the plasma as low, middle, and high molecular weight multimers.
      • Tao C.
      • Sifuentes A.
      • Holland W.L.
      Regulation of glucose and lipid homeostasis by adiponectin: effects on hepatocytes, pancreatic beta cells and adipocytes.
      • You M.
      • Rogers C.Q.
      Adiponectin: a key adipokine in alcoholic fatty liver.
      Adiponectin is a pivotal player in the regulation of lipid metabolism (Fig. 1). After reaching the liver, adiponectin transduces signals via 2 major adiponectin receptors AdipoR1 and AdipoR2. Adiponectin inhibits lipid synthesis and stimulates FA oxidation, in part by activating SIRT1, AMPK, PGC-1α and PPARα, and suppressing SREBP-1.
      • Tao C.
      • Sifuentes A.
      • Holland W.L.
      Regulation of glucose and lipid homeostasis by adiponectin: effects on hepatocytes, pancreatic beta cells and adipocytes.
      • You M.
      • Rogers C.Q.
      Adiponectin: a key adipokine in alcoholic fatty liver.
      Fibroblast growth factor (FGF) 15 (human homolog FGF19), is a terminal small intestine (ileum)-derived hormone.
      • Markan K.R.
      • Potthoff M.J.
      Metabolic fibroblast growth factors (FGFs): Mediators of energy homeostasis.
      Circulating FGF15/19 signalling regulates bile acid and lipid metabolism in the liver through activation of a receptor complex comprised of fibroblast growth factor receptor 4 (FGFR4)/β-Klotho.
      • Markan K.R.
      • Potthoff M.J.
      Metabolic fibroblast growth factors (FGFs): Mediators of energy homeostasis.
      Ethanol impairs adiponectin synthesis and production in adipocytes and downregulates hepatic adiponectin receptors.
      • Shen Z.
      • Liang X.
      • Rogers C.Q.
      • Rideout D.
      • You M.
      Involvement of adiponectin-SIRT1-AMPK signaling in the protective action of rosiglitazone against alcoholic fatty liver in mice.
      • Jiang Z.
      • Zhou J.
      • Zhou D.
      • Zhu Z.
      • Sun L.
      • Nanji A.A.
      The adiponectin-SIRT1-AMPK pathway in alcoholic fatty liver disease in the rat.
      • Ambade A.
      • Catalano D.
      • Lim A.
      • Kopoyan A.
      • Shaffer S.A.
      • Mandrekar P.
      Inhibition of heat shock protein 90 alleviates steatosis and macrophage activation in murine alcoholic liver injury.
      • You M.
      • Rogers C.Q.
      Adiponectin: a key adipokine in alcoholic fatty liver.
      • Wang M.
      • Zhang X.J.
      • Feng K.
      • He C.W.
      • Li P.
      • Hu Y.J.
      • et al.
      Dietary alpha-linolenic acid-rich flaxseed oil prevents against alcoholic hepatic steatosis via ameliorating lipid homeostasis at adipose tissue-liver axis in mice.
      • Correnti J.M.
      • Juskeviciute E.
      • Swarup A.
      • Hoek J.B.
      Pharmacological ceramide reduction alleviates alcohol-induced steatosis and hepatomegaly in adiponectin knockout mice.
      • Shearn C.T.
      • Smathers R.L.
      • Jiang H.
      • Orlicky D.J.
      • Maclean K.N.
      • Petersen D.R.
      Increased dietary fat contributes to dysregulation of the LKB1/AMPK pathway and increased damage in a mouse model of early-stage ethanol-mediated steatosis.
      • Esfandiari F.
      • You M.
      • Villanueva J.A.
      • Wong D.H.
      • French S.W.
      • Halsted C.H.
      S-adenosylmethionine attenuates hepatic lipid synthesis in micropigs fed ethanol with a folate-deficient diet.
      • Xu J.
      • Lai K.K.Y.
      • Verlinsky A.
      • Lugea A.
      • French S.W.
      • Cooper M.P.
      • et al.
      Synergistic steatohepatitis by moderate obesity and alcohol in mice despite increased adiponectin and p-AMPK.
      Adiponectin elicits profound lipid lowing effects in rodents administered ethanol and in patients with AFLD.
      • Shen Z.
      • Liang X.
      • Rogers C.Q.
      • Rideout D.
      • You M.
      Involvement of adiponectin-SIRT1-AMPK signaling in the protective action of rosiglitazone against alcoholic fatty liver in mice.
      • Jiang Z.
      • Zhou J.
      • Zhou D.
      • Zhu Z.
      • Sun L.
      • Nanji A.A.
      The adiponectin-SIRT1-AMPK pathway in alcoholic fatty liver disease in the rat.
      • Ambade A.
      • Catalano D.
      • Lim A.
      • Kopoyan A.
      • Shaffer S.A.
      • Mandrekar P.
      Inhibition of heat shock protein 90 alleviates steatosis and macrophage activation in murine alcoholic liver injury.
      • You M.
      • Rogers C.Q.
      Adiponectin: a key adipokine in alcoholic fatty liver.
      • Wang M.
      • Zhang X.J.
      • Feng K.
      • He C.W.
      • Li P.
      • Hu Y.J.
      • et al.
      Dietary alpha-linolenic acid-rich flaxseed oil prevents against alcoholic hepatic steatosis via ameliorating lipid homeostasis at adipose tissue-liver axis in mice.
      • Correnti J.M.
      • Juskeviciute E.
      • Swarup A.
      • Hoek J.B.
      Pharmacological ceramide reduction alleviates alcohol-induced steatosis and hepatomegaly in adiponectin knockout mice.
      • Shearn C.T.
      • Smathers R.L.
      • Jiang H.
      • Orlicky D.J.
      • Maclean K.N.
      • Petersen D.R.
      Increased dietary fat contributes to dysregulation of the LKB1/AMPK pathway and increased damage in a mouse model of early-stage ethanol-mediated steatosis.
      • Esfandiari F.
      • You M.
      • Villanueva J.A.
      • Wong D.H.
      • French S.W.
      • Halsted C.H.
      S-adenosylmethionine attenuates hepatic lipid synthesis in micropigs fed ethanol with a folate-deficient diet.
      • Xu J.
      • Lai K.K.Y.
      • Verlinsky A.
      • Lugea A.
      • French S.W.
      • Cooper M.P.
      • et al.
      Synergistic steatohepatitis by moderate obesity and alcohol in mice despite increased adiponectin and p-AMPK.
      Aberrant hepatic adiponectin signalling is associated with lower activities of AMPK and SIRT1 and elevated levels of downstream molecules such as SREBP-1, ACC and lipin-1β in the livers of ethanol-fed rodents and patients with AFLD.
      • Shen Z.
      • Liang X.
      • Rogers C.Q.
      • Rideout D.
      • You M.
      Involvement of adiponectin-SIRT1-AMPK signaling in the protective action of rosiglitazone against alcoholic fatty liver in mice.
      • Jiang Z.
      • Zhou J.
      • Zhou D.
      • Zhu Z.
      • Sun L.
      • Nanji A.A.
      The adiponectin-SIRT1-AMPK pathway in alcoholic fatty liver disease in the rat.
      • Ambade A.
      • Catalano D.
      • Lim A.
      • Kopoyan A.
      • Shaffer S.A.
      • Mandrekar P.
      Inhibition of heat shock protein 90 alleviates steatosis and macrophage activation in murine alcoholic liver injury.
      • You M.
      • Rogers C.Q.
      Adiponectin: a key adipokine in alcoholic fatty liver.
      • Wang M.
      • Zhang X.J.
      • Feng K.
      • He C.W.
      • Li P.
      • Hu Y.J.
      • et al.
      Dietary alpha-linolenic acid-rich flaxseed oil prevents against alcoholic hepatic steatosis via ameliorating lipid homeostasis at adipose tissue-liver axis in mice.
      • Correnti J.M.
      • Juskeviciute E.
      • Swarup A.
      • Hoek J.B.
      Pharmacological ceramide reduction alleviates alcohol-induced steatosis and hepatomegaly in adiponectin knockout mice.
      • Shearn C.T.
      • Smathers R.L.
      • Jiang H.
      • Orlicky D.J.
      • Maclean K.N.
      • Petersen D.R.
      Increased dietary fat contributes to dysregulation of the LKB1/AMPK pathway and increased damage in a mouse model of early-stage ethanol-mediated steatosis.
      • Esfandiari F.
      • You M.
      • Villanueva J.A.
      • Wong D.H.
      • French S.W.
      • Halsted C.H.
      S-adenosylmethionine attenuates hepatic lipid synthesis in micropigs fed ethanol with a folate-deficient diet.
      • Xu J.
      • Lai K.K.Y.
      • Verlinsky A.
      • Lugea A.
      • French S.W.
      • Cooper M.P.
      • et al.
      Synergistic steatohepatitis by moderate obesity and alcohol in mice despite increased adiponectin and p-AMPK.
      These findings all point to a critical link between altered hepatic adiponectin signalling and AFLD.
      Adipose-derived adiponectin and gut-derived FGF15/19 associate with each other, with the endocrine adiponectin-FGF15/19 axis a pivotal regulator of lipid metabolism.
      • Ge H.
      • Zhang J.
      • Gong Y.
      • Gupte J.
      • Ye J.
      • Weiszmann J.
      • et al.
      Fibroblast growth factor receptor 4 (FGFR4) deficiency improves insulin resistance and glucose metabolism under diet-induced obesity conditions.
      • Luo Y.
      • Yang C.
      • Ye M.
      • Jin C.
      • Abbruzzese J.L.
      • Lee M.H.
      • et al.
      Deficiency of metabolic regulator FGFR4 delays breast cancer progression through systemic and microenvironmental metabolic alterations.
      Chronic or chronic-binge ethanol feeding concomitantly reduces adiponectin and FGF15/19 levels in mice.
      • Hu X.
      • Jogasuria A.
      • Wang J.
      • Kim C.
      • Han Y.
      • Shen H.
      • et al.
      MitoNEET deficiency alleviates experimental alcoholic steatohepatitis in mice by stimulating endocrine adiponectin-Fgf15 axis.
      • Wang J.
      • Kim C.
      • Jogasuria A.
      • Han Y.
      • Hu X.
      • Wu J.
      • et al.
      Myeloid cell-specific Lipin-1 deficiency stimulates endocrine adiponectin-FGF15 axis and ameliorates ethanol-induced liver injury in mice.
      • Hartmann P.
      • Hochrath K.
      • Horvath A.
      • Chen P.
      • Seebauer C.T.
      • Llorente C.
      • et al.
      Modulation of the intestinal bile acid/farnesoid X receptor/fibroblast growth factor 15 axis improves alcoholic liver disease in mice.
      Remarkably, the concurrent elevation of adiponectin and FGF15 is associated with inhibition of the genes involved in lipid uptake (e.g. CD36/FAT) and activation of the genes (e.g. PPARα and medium chain acyl-CoA dehydrogenase) implicated in lipid oxidation and the presence of ethanol-induced steatohepatitis in Cisd1 knockout mice.
      • Hu X.
      • Jogasuria A.
      • Wang J.
      • Kim C.
      • Han Y.
      • Shen H.
      • et al.
      MitoNEET deficiency alleviates experimental alcoholic steatohepatitis in mice by stimulating endocrine adiponectin-Fgf15 axis.
      These findings suggest that endocrine adiponectin-FGF15/19 signalling protects against AFLD, at least in part by ameliorating the ethanol-induced abnormality in lipid metabolism.

      Overall effect of ethanol exposure on lipogenesis

      In summary, the net effect of ethanol is to activate (e.g., via ER stress, TNFα and/or hepatic PPARγ) de novo lipogenesis, while concomitantly inhibiting processes that block this response (e.g., AMPK and SIRT1). Although some of this net effect results from the direct action of ethanol on lipogenic enzymes (e.g., disinhibition of ACC by AMPK inhibition), it is primarily the result of ethanol activating the transcriptional activity of SREBP-1c and ChREBP. This explains why these transcription factors are activated even when ethanol decreases the canonical inducers of these pathways (see earlier). In NAFLD, a similar loss of negative regulation of SREBP-1c and ChREBP is hypothesised to contribute to de novo lipogenesis, even in the fasting state.
      • Lambert J.E.
      • Ramos-Roman M.A.
      • Browning J.D.
      • Parks E.J.
      Increased de novo lipogenesis is a distinct characteristic of individuals with nonalcoholic fatty liver disease.
      Although the effect of ethanol on fasting de novo lipogenesis is less clear, a similar mechanism which contributes to the loss of diurnal regulation of lipid metabolism could be in play (see later).
      Ethanol activates de novo lipogenesis via a number of processes, leading to lipid accumulation in the liver.

      Effects of ethanol on mitochondrial β-oxidation: potential key players

      Mitochondrial β-oxidation shortens FAs into acetyl-CoA subunits, which can either enter the citric acid cycle, or be used to synthesise ketone bodies.
      • Fromenty B.
      • Pessayre D.
      Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.
      Although short-chain FAs can readily cross the outer and inner mitochondrial membranes, medium- and long-chain FAs are actively transported into the inner mitochondrial space via the carnitine shuttle. The rate-limiting enzyme in this process is carnitine palmytoyl transferase I (CPTI), which is regulated both transcriptionally and post-transcriptionally. Ethanol causes several changes that can directly or indirectly impair β-oxidation.

      Transcriptional inhibition of mitochondrial β-oxidation by ethanol

      Despite a net increase in the supply of FAs for β-oxidation, there is no apparent induction of β-oxidation genes during alcohol exposure. The major mechanism of action underlying this effect is hypothesised to be the inhibition of peroxisome proliferator-activated receptor alpha (PPARα) signalling.
      • Crabb D.W.
      • Galli A.
      • Fischer M.
      • You M.
      Molecular mechanisms of alcoholic fatty liver: role of peroxisome proliferator-activated receptor alpha.
      PPARα is a nuclear hormone receptor that regulates expression of numerous genes involved in mitochondrial β-oxidation.
      • Yu S.
      • Rao S.
      • Reddy J.K.
      Peroxisome proliferator-activated receptors, fatty acid oxidation, steatohepatitis and hepatocarcinogenesis.
      • Aoyama T.
      • Peters J.M.
      • Iritani N.
      • Nakajima T.
      • Furihata K.
      • Hashimoto T.
      • et al.
      Altered constitutive expression of fatty acid-metabolizing enzymes in mice lacking the peroxisome proliferator-activated receptor alpha (PPARalpha).
      Ethanol exposure decreases PPARα DNA binding activity, without decreasing PPARα expression;
      • Fischer M.
      • You M.
      • Matsumoto M.
      • Crabb D.W.
      Peroxisome proliferator-activated receptor alpha (PPARalpha) agonist treatment reverses PPARalpha dysfunction and abnormalities in hepatic lipid metabolism in ethanol-fed mice.
      this effect is potentially mediated via decreasing protein levels of the retinoid X receptor (RXR), which heterodimerises with PPARα to bind to target DNA.
      • Fischer M.
      • You M.
      • Matsumoto M.
      • Crabb D.W.
      Peroxisome proliferator-activated receptor alpha (PPARalpha) agonist treatment reverses PPARalpha dysfunction and abnormalities in hepatic lipid metabolism in ethanol-fed mice.

      Nutritional deficiencies

      Alcoholics replace in excess of 50% of their total daily calories with ethanol.
      • Patek Jr., A.J.
      Alcohol, malnutrition, and alcoholic cirrhosis.
      Furthermore, alcohol consumption often causes malabsorption,
      • Bujanda L.
      The effects of alcohol consumption upon the gastrointestinal tract.
      which may further exacerbate nutrient deficiencies. As the name implies, the carnitine shuttle requires carnitine as a cofactor. Roughly 25% of carnitine is synthesised endogenously from lysine and methionine, with the remainder derived from dietary sources.
      • Flanagan J.L.
      • Simmons P.A.
      • Vehige J.
      • Willcox M.D.
      • Garrett Q.
      Role of carnitine in disease.
      Several experimental lines of evidence support the hypothesis that nutritional deficiencies may lead to functional carnitine deficiency, via restricting precursor supply and/or carnitine proper.
      • Sachan D.S.
      • Rhew T.H.
      • Ruark R.A.
      Ameliorating effects of carnitine and its precursors on alcohol-induced fatty liver.
      • Bykov I.
      • Jarvelainen H.
      • Lindros K.
      L-carnitine alleviates alcohol-induced liver damage in rats: role of tumour necrosis factor-alpha.
      In contrast, the impact of alcohol on circulating levels of carnitine metabolites is equivocal at this time.
      • Alonso dlP.
      • Rozas I.
      • Alvarez-Prechous A.
      • Pardinas M.C.
      • Paz J.M.
      • Rodriguez-Segade S.
      Free carnitine and acylcarnitine levels in sera of alcoholics.
      • Fuller R.K.
      • Hoppel C.L.
      Plasma carnitine in alcoholism.
      • Kepka A.
      • Waszkiewicz N.
      • Zalewska-Szajda B.
      • Chojnowska S.
      • Pludowski P.
      • Konarzewska E.
      • et al.
      Plasma carnitine concentrations after chronic alcohol intoxication.
      Nevertheless, alcohol consumption may cause nutritional deficiencies that potentially impair mitochondrial β-oxidation.

      Inhibition of β-oxidation activity

      As mentioned, the increase in the NADH:NAD+ratio caused by alcohol metabolism directly inhibits mitochondrial β-oxidation. This effect is thought to be predominantly mediated by the NAD+ reducing enzyme, 3-hydroxy-CoA dehydrogenase, the final step in generating acetyl-CoA during β-oxidation.
      • Latipaa P.M.
      • Karki T.T.
      • Hiltunen J.K.
      • Hassinen I.E.
      Regulation of palmitoylcarnitine oxidation in isolated rat liver mitochondria. Role of the redox state of NAD(H).
      Furthermore, the disinhibition of ACC caused by impairing AMPK activity (see earlier) increases the carboxylation of acetyl-CoA to malonyl-CoA, which inhibits CPTI activity.
      • You M.
      • Matsumoto M.
      • Pacold C.M.
      • Cho W.K.
      • Crabb D.W.
      The role of AMP-activated protein kinase in the action of ethanol in the liver.
      • Foster D.W.
      Malonyl-CoA: the regulator of fatty acid synthesis and oxidation.
      Coupled to the activity of CPTI, voltage-dependent anion channels (VDACs) are required to transport acyl-CoA esters through the outer membrane to the intermembrane space. Ethanol and acetaldehyde cause VDACs on hepatocyte mitochondria to close, which also impairs mitochondrial β-oxidation.
      • Holmuhamedov E.L.
      • Czerny C.
      • Beeson C.C.
      • Lemasters J.J.
      Ethanol suppresses ureagenesis in rat hepatocytes: role of acetaldehyde.
      • Lemasters J.J.
      • Holmuhamedov E.
      Voltage-dependent anion channel (VDAC) as mitochondrial governator–thinking outside the box.
      Lastly, ethanol exposure damages the mitochondria and leads to mitochondria dysfunction;
      • Garcia-Ruiz C.
      • Kaplowitz N.
      • Fernandez-Checa J.C.
      Role of mitochondria in alcoholic liver disease.
      this impact on mitochondrial function can indirectly impair the ability of the organelles to oxidise free FAs. This latter point is likely exacerbated by the impaired autophagy of damaged mitochondria that is associated with alcohol exposure.
      • Williams J.A.
      • Ding W.X.
      A mechanistic review of mitophagy and its role in protection against alcoholic liver disease.

      Effect of ethanol exposure on mitochondrial β-oxidation

      In summary, the net effect of ethanol is to inhibit mitochondrial β-oxidation by blunting the induction of β-oxidation genes, even in the context of increased FA supply (e.g., via inhibition of PPARα signalling), through potential functional deficiencies in critical cofactors for β-oxidation (e.g., carnitine), directly (e.g., increased NADH malonyl-CoA), and indirectly (via VDAC closure and mitochondrial dysfunction). Ethanol’s myriad of inhibitory effects on mitochondrial β-oxidation likely explain the continued inhibition of this process during chronic ethanol consumption, even after the ratio of NADH:NAD+ appears to normalise.
      • Salaspuro M.P.
      • Shaw S.
      • Jayatilleke E.
      • Ross W.A.
      • Lieber C.S.
      Attenuation of the ethanol-induced hepatic redox change after chronic alcohol consumption in baboons: metabolic consequences in vivo and in vitro.
      The net effect of ethanol is to inhibit mitochondrial β-oxidation, even in the context of increased fatty acid supply, reducing the utilisation of lipid.

      Effects of ethanol on cholesterol synthesis and secretion

      Another mechanism by which lipids can accumulate in the liver is via alterations in the packaging of triglycerides into lipoproteins to form cholesterol. Some studies have indicated that chronic experimental ethanol impairs hepatic cholesterol synthesis,
      • Li Q.
      • Zhong W.
      • Qiu Y.
      • Kang X.
      • Sun X.
      • Tan X.
      • et al.
      Preservation of hepatocyte nuclear factor-4alpha contributes to the beneficial effect of dietary medium chain triglyceride on alcohol-induced hepatic lipid dyshomeostasis in rats.
      • Tomita K.
      • Azuma T.
      • Kitamura N.
      • Nishida J.
      • Tamiya G.
      • Oka A.
      • et al.
      Pioglitazone prevents alcohol-induced fatty liver in rats through up-regulation of c-Met.
      whereas others have shown no effect.
      • Bergheim I.
      • Guo L.
      • Davis M.A.
      • Lambert J.C.
      • Beier J.I.
      • Duveau I.
      • et al.
      Metformin prevents alcohol-induced liver injury in the mouse: critical role of plasminogen activator inhibitor-1.
      • Lieber C.S.
      Interference of ethanol in hepatic cellular metabolism.
      However, few studies suggest that hepatic cholesterol synthesis is increased by alcohol. In this context, the lack of response of this system to the increase in lipid flux through the hepatocyte may contribute indirectly to the steatosis caused by ethanol consumption. The rate of cholesterol synthesis and release is controlled predominantly by the supply of apoliprotein B and the activity of microsomal triglyceride transfer protein (MTTP). A key regulator of both processes is hepatocyte growth factor (HGF) signalling via its receptor c-Met.
      • Bottaro D.P.
      • Rubin J.S.
      • Faletto D.L.
      • Chan A.M.
      • Kmiecik T.E.
      • Vande Woude G.F.
      • et al.
      Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product.
      The activation of hepatic nuclear receptor 4α (HNF-4α) is also hypothesised to play a key role in this process.
      • Sheena V.
      • Hertz R.
      • Nousbeck J.
      • Berman I.
      • Magenheim J.
      • Bar-Tana J.
      Transcriptional regulation of human microsomal triglyceride transfer protein by hepatocyte nuclear factor-4alpha.
      • Yu D.
      • Chen G.
      • Pan M.
      • Zhang J.
      • He W.
      • Liu Y.
      • et al.
      High fat diet-induced oxidative stress blocks hepatocyte nuclear factor 4alpha and leads to hepatic steatosis in mice.
      Activation of c-Met by HGF stimulates VLDL synthesis in hepatocytes through upregulation of apoliprotein B synthesis.
      • Kaibori M.
      • Kwon A.H.
      • Oda M.
      • Kamiyama Y.
      • Kitamura N.
      • Okumura T.
      Hepatocyte growth factor stimulates synthesis of lipids and secretion of lipoproteins in rat hepatocytes.
      HGF administration has also been shown to enhance the rate of recovery from experimental alcohol-induced fatty liver and is associated with increased synthesis and secretion of apolipoprotein B and subsequent formation of VLDL.
      • Tahara M.
      • Matsumoto K.
      • Nukiwa T.
      • Nakamura T.
      Hepatocyte growth factor leads to recovery from alcohol-induced fatty liver in rats.
      • Sugimoto T.
      • Yamashita S.
      • Ishigami M.
      • Sakai N.
      • Hirano K.
      • Tahara M.
      • et al.
      Decreased microsomal triglyceride transfer protein activity contributes to initiation of alcoholic liver steatosis in rats.
      The protective effect of medium chain triglycerides
      • Li Q.
      • Zhong W.
      • Qiu Y.
      • Kang X.
      • Sun X.
      • Tan X.
      • et al.
      Preservation of hepatocyte nuclear factor-4alpha contributes to the beneficial effect of dietary medium chain triglyceride on alcohol-induced hepatic lipid dyshomeostasis in rats.
      and the PPARγ agonist pioglitazone
      • Tomita K.
      • Azuma T.
      • Kitamura N.
      • Nishida J.
      • Tamiya G.
      • Oka A.
      • et al.
      Pioglitazone prevents alcohol-induced fatty liver in rats through up-regulation of c-Met.
      are hypothesised to be mediated, at least in part, by enhancing the capacity of hepatocytes to synthesise cholesterol. Enhancing the post-translational formation of HGF has also been shown to be protective against ethanol-induced steatosis. For example, although the canonical role of plasminogen activator inhibitor-1 (PAI-1) is to inhibit fibrinolysis by plasminogen activators, such as urokinase plasminogen activator (uPA),
      • Kruithof E.K.
      Plasminogen activator inhibitors–a review.
      uPA also activates pro-HGF to mature HGF.
      • Naldini L.
      • Vigna E.
      • Bardelli A.
      • Follenzi A.
      • Galimi F.
      • Comoglio P.M.
      Biological activation of pro-HGF (hepatocyte growth factor) by urokinase is controlled by a stoichiometric reaction.
      • Taniyama Y.
      • Morishita R.
      • Nakagami H.
      • Moriguchi A.
      • Sakonjo H.
      • Shokei K.
      • et al.
      Potential contribution of a novel antifibrotic factor, hepatocyte growth factor, to prevention of myocardial fibrosis by angiotensin II blockade in cardiomyopathic hamsters.
      Indeed, genetic or pharmacologic inhibition of PAI-1 prevents ethanol-induced steatosis, in part, by enhancing HGF-mediated VLDL synthesis.
      • Bergheim I.
      • Guo L.
      • Davis M.A.
      • Lambert J.C.
      • Beier J.I.
      • Duveau I.
      • et al.
      Metformin prevents alcohol-induced liver injury in the mouse: critical role of plasminogen activator inhibitor-1.
      It is highly likely that other processes impacted by alcohol exposure (e.g., ER stress
      • Kaplowitz N.
      • Ji C.
      Unfolding new mechanisms of alcoholic liver disease in the endoplasmic reticulum.
      ) contribute to altered/impaired VLDL synthesis during ALD. This area of research has been somewhat underappreciated partly because of the difficultly in studying cholesterol metabolism in intact organisms. The advent of more advanced stable isotope labelling approaches and lipidomic analyses may now make this possible.
      • Wang H.Y.
      • Quan C.
      • Hu C.
      • Xie B.
      • Du Y.
      • Chen L.
      • et al.
      A lipidomics study reveals hepatic lipid signatures associating with deficiency of the LDL receptor in a rat model.

      Other mechanisms by which ethanol impacts lipid metabolism

      Lipocalin-2

      Lipocalin-2 is an important innate immune protein belonging to the lipocalin family.
      • Asimakopoulou A.
      • Weiskirchen S.
      • Weiskirchen R.
      Lipocalin 2 (LCN2) expression in hepatic malfunction and therapy.
      Emerging evidences demonstrate a pivotal and multifunctional role of lipocalin-2 in the early stages of ALD and in alcoholic steatosis.
      • Hu X.
      • Jogasuria A.
      • Wang J.
      • Kim C.
      • Han Y.
      • Shen H.
      • et al.
      MitoNEET deficiency alleviates experimental alcoholic steatohepatitis in mice by stimulating endocrine adiponectin-Fgf15 axis.
      • Wang J.
      • Kim C.
      • Jogasuria A.
      • Han Y.
      • Hu X.
      • Wu J.
      • et al.
      Myeloid cell-specific Lipin-1 deficiency stimulates endocrine adiponectin-FGF15 axis and ameliorates ethanol-induced liver injury in mice.
      • Glavind E.
      • Vilstrup H.
      • Gronbaek H.
      • Hamilton-Dutoit S.
      • Magnusson N.E.
      • Thomsen K.L.
      Long-term ethanol exposure decreases the endotoxin-induced hepatic acute phase response in rats.
      • Yin H.
      • Hu M.
      • Liang X.
      • Ajmo J.M.
      • Li X.
      • Bataller R.
      • et al.
      Deletion of SIRT1 from hepatocytes in mice disrupts lipin-1 signaling and aggravates alcoholic fatty liver.
      • Jiang Z.
      • Zhou J.
      • Zhou D.
      • Zhu Z.
      • Sun L.
      • Nanji A.A.
      The adiponectin-SIRT1-AMPK pathway in alcoholic fatty liver disease in the rat.
      Ethanol administration in mice or rats markedly increases liver and adipose lipocalin-2 expression and elevates circulating lipocalin-2 levels.
      • Hu X.
      • Jogasuria A.
      • Wang J.
      • Kim C.
      • Han Y.
      • Shen H.
      • et al.
      MitoNEET deficiency alleviates experimental alcoholic steatohepatitis in mice by stimulating endocrine adiponectin-Fgf15 axis.
      • Wang J.
      • Kim C.
      • Jogasuria A.
      • Han Y.
      • Hu X.
      • Wu J.
      • et al.
      Myeloid cell-specific Lipin-1 deficiency stimulates endocrine adiponectin-FGF15 axis and ameliorates ethanol-induced liver injury in mice.
      • Glavind E.
      • Vilstrup H.
      • Gronbaek H.
      • Hamilton-Dutoit S.
      • Magnusson N.E.
      • Thomsen K.L.
      Long-term ethanol exposure decreases the endotoxin-induced hepatic acute phase response in rats.
      • Yin H.
      • Hu M.
      • Liang X.
      • Ajmo J.M.
      • Li X.
      • Bataller R.
      • et al.
      Deletion of SIRT1 from hepatocytes in mice disrupts lipin-1 signaling and aggravates alcoholic fatty liver.
      • Jiang Z.
      • Zhou J.
      • Zhou D.
      • Zhu Z.
      • Sun L.
      • Nanji A.A.
      The adiponectin-SIRT1-AMPK pathway in alcoholic fatty liver disease in the rat.
      In a cellular model of alcoholic steatosis, recombinant lipocalin-2 or over-expression of lipocalin-2 exacerbates the ethanol-induced fat accumulation, whereas knocking down lipocalin-2 prevents steatosis in hepatocytes exposed to ethanol.
      • Cai Y.
      • Jogasuria A.
      • Yin H.
      • Xu M.J.
      • Hu X.
      • Wang J.
      • et al.
      The detrimental role played by lipocalin-2 in alcoholic fatty liver in mice.
      Consistently, global ablation of lipocalin-2 partially but significantly prevents experimental alcoholic fatty liver injury in mice.
      • Cai Y.
      • Jogasuria A.
      • Yin H.
      • Xu M.J.
      • Hu X.
      • Wang J.
      • et al.
      The detrimental role played by lipocalin-2 in alcoholic fatty liver in mice.
      Lipocalin-2 also promotes liver inflammation after alcohol intake by mediating neutrophil infiltration into liver and prolonging neutrophil lifespan in rodents and humans.
      • Wieser V.
      • Tymoszuk P.
      • Adolph T.E.
      • Grander C.
      • Grabherr F.
      • Enrich B.
      • et al.
      Lipocalin 2 drives neutrophilic inflammation in alcoholic liver disease.
      Mechanistically, abnormally elevated lipocalin-2 plays a causative role in the experimental cellular and animal models of alcoholic steatosis by disrupting signalling cascades involved in lipid metabolism, including the phosphoribosyltransferase-SIRT1 axis, chaperone-mediated autophagy, FA oxidation and endocrine metabolic regulatory hepatic FGF15/19 signalling.
      • Cai Y.
      • Jogasuria A.
      • Yin H.
      • Xu M.J.
      • Hu X.
      • Wang J.
      • et al.
      The detrimental role played by lipocalin-2 in alcoholic fatty liver in mice.

      Autophagy

      Macroautophagy (herein, referred to as autophagy) is a genetically programmed and highly conserved intracellular lysosomal degradation mechanism.
      • Wang L.
      • Khambu B.
      • Zhang H.
      • Yin X.M.
      Autophagy in alcoholic liver disease, self-eating triggered by drinking.
      • Manley S.
      • Ding W.
      Role of farnesoid X receptor and bile acids in alcoholic liver disease.
      Autophagy maintains normal cellular functions and regulates lipid homeostasis, including lipid droplet turnover and formation. Aberrant autophagic machinery is associated with the development and progression of AFLD.
      • Cai Y.
      • Jogasuria A.
      • Yin H.
      • Xu M.J.
      • Hu X.
      • Wang J.
      • et al.
      The detrimental role played by lipocalin-2 in alcoholic fatty liver in mice.
      • Wang L.
      • Khambu B.
      • Zhang H.
      • Yin X.M.
      Autophagy in alcoholic liver disease, self-eating triggered by drinking.
      • Manley S.
      • Ding W.
      Role of farnesoid X receptor and bile acids in alcoholic liver disease.
      • Ding W.X.
      • Li M.
      • Yin X.M.
      Selective taste of ethanol-induced autophagy for mitochondria and lipid droplets.
      • Ding W.X.
      • Li M.
      • Chen X.
      • Ni H.M.
      • Lin C.W.
      • Gao W.
      • et al.
      Autophagy reduces acute ethanol-induced hepatotoxicity and steatosis in mice.
      • Ni H.M.
      • Du K.
      • You M.
      • Ding W.X.
      Critical role of FoxO3a in alcohol-induced autophagy and hepatotoxicity.
      • Lin C.W.
      • Zhang H.
      • Li M.
      • Xiong X.
      • Chen X.
      • Chen X.
      • et al.
      Pharmacological promotion of autophagy alleviates steatosis and injury in alcoholic and non-alcoholic fatty liver conditions in mice.
      • Eid N.
      • Ito Y.
      • Maemura K.
      • Otsuki Y.
      Elevated autophagic sequestration of mitochondria and lipid droplets in steatotic hepatocytes of chronic ethanol-treated rats: an immunohistochemical and electron microscopic study.
      • Thomes P.G.
      • Trambly C.S.
      • Fox H.S.
      • Tuma D.J.
      • Donohue Jr., T.M.
      Acute and chronic ethanol administration differentially modulate hepatic autophagy and transcription factor EB.
      • Li Y.
      • Ding W.X.
      A gene transcription program decides the differential regulation of autophagy by acute versus chronic ethanol?.
      • Lu Y.
      • Cederbaum A.I.
      Autophagy protects against CYP2E1/chronic ethanol-induced hepatotoxicity.
      • Tang L.
      • Yang F.
      • Fang Z.
      • Hu C.
      Resveratrol ameliorates alcoholic fatty liver by inducing autophagy.
      • Kong X.
      • Yang Y.
      • Ren L.
      • Shao T.
      • Li F.
      • Zhao C.
      • et al.
      Activation of autophagy attenuates EtOH-LPS-induced hepatic steatosis and injury through MD2 associated TLR4 signaling.
      • Cho H.I.
      • Seo M.J.
      • Lee S.M.
      2-Methoxyestradiol protects against ischemia/reperfusion injury in alcoholic fatty liver by enhancing sirtuin 1-mediated autophagy.
      However, because of the complexity of autophagic machinery and differences in animal AFLD models, experimental findings are controversial. The induction of autophagy by acute ethanol treatment eliminates hepatic intracellular lipid droplets and reduces lipid accumulation in rodents.
      • Ding W.X.
      • Li M.
      • Yin X.M.
      Selective taste of ethanol-induced autophagy for mitochondria and lipid droplets.
      • Ding W.X.
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      Autophagy reduces acute ethanol-induced hepatotoxicity and steatosis in mice.
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      Critical role of FoxO3a in alcohol-induced autophagy and hepatotoxicity.
      However, chronic ethanol administration at higher dosages inhibits autophagy, coupled with accumulation of hepatic triglycerides in mice.
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      The detrimental role played by lipocalin-2 in alcoholic fatty liver in mice.
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      Autophagy reduces acute ethanol-induced hepatotoxicity and steatosis in mice.
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      Critical role of FoxO3a in alcohol-induced autophagy and hepatotoxicity.
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      Pharmacological promotion of autophagy alleviates steatosis and injury in alcoholic and non-alcoholic fatty liver conditions in mice.
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      Elevated autophagic sequestration of mitochondria and lipid droplets in steatotic hepatocytes of chronic ethanol-treated rats: an immunohistochemical and electron microscopic study.
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      Acute and chronic ethanol administration differentially modulate hepatic autophagy and transcription factor EB.
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      A gene transcription program decides the differential regulation of autophagy by acute versus chronic ethanol?.
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      Autophagy protects against CYP2E1/chronic ethanol-induced hepatotoxicity.
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      Resveratrol ameliorates alcoholic fatty liver by inducing autophagy.
      • Kong X.
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      Activation of autophagy attenuates EtOH-LPS-induced hepatic steatosis and injury through MD2 associated TLR4 signaling.
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      2-Methoxyestradiol protects against ischemia/reperfusion injury in alcoholic fatty liver by enhancing sirtuin 1-mediated autophagy.
      Although the mechanisms by which ethanol regulates autophagic machinery are not fully understood, ethanol metabolism-induced oxidative stress is likely to participate in the activation of autophagy.
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      Autophagy protects against CYP2E1/chronic ethanol-induced hepatotoxicity.
      In addition, regulation of autophagy by acute vs. chronic ethanol exposure may be determined by a gene transcription programme in liver.
      • Thomes P.G.
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      • Fox H.S.
      • Tuma D.J.
      • Donohue Jr., T.M.
      Acute and chronic ethanol administration differentially modulate hepatic autophagy and transcription factor EB.
      • Li Y.
      • Ding W.X.
      A gene transcription program decides the differential regulation of autophagy by acute versus chronic ethanol?.

      Circadian clock

      The circadian clock regulates circadian rhythms and is maintained by a complex circuitry of transcriptional/translational regulatory loops at molecular levels.
      • Mayeuf-Louchart A.
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      Circadian control of metabolism and pathological consequences of clock perturbations.
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      Crosstalk between components of circadian and metabolic cycles in mammals.
      The circadian clock plays an essential role in orchestrating many physiological processes, including lipid metabolism. Derangements in the finely tuned circadian clock can contribute to dyslipidaemia and liver diseases.
      • Mayeuf-Louchart A.
      • Zecchin M.
      • Staels B.
      • Duez H.
      Circadian control of metabolism and pathological consequences of clock perturbations.
      • Asher G.
      • Schibler U.
      Crosstalk between components of circadian and metabolic cycles in mammals.
      Circadian clock disruption is an important contributor to aberrant lipid metabolism and ethanol-induced steatosis.
      • Udoh U.S.
      • Valcin J.A.
      • Gamble K.L.
      • Bailey S.M.
      The molecular circadian clock and alcohol-induced liver injury.
      Chronic ethanol exposure results in the disturbance of the hepatic circadian clock and time-of-day specific regulation of lipid homeostasis in rodents.
      • Filiano A.N.
      • Millender-Swain T.
      • Johnson Jr., R.
      • Young M.E.
      • Gamble K.L.
      • Bailey S.M.
      Chronic ethanol consumption disrupts the core molecular clock and diurnal rhythms of metabolic genes in the liver without affecting the suprachiasmatic nucleus.
      • Wang T.
      • Yang P.
      • Zhan Y.
      • Xia L.
      • Hua Z.
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      Deletion of circadian gene Per1 alleviates acute ethanol-induced hepatotoxicity in mice.
      • Zhou P.
      • Ross R.A.
      • Pywell C.M.
      • Liangpunsakul S.
      • Duffield G.E.
      Disturbances in the murine hepatic circadian clock in alcohol-induced hepatic steatosis.
      • Udoh U.S.
      • Swain T.M.
      • Filiano A.N.
      • Gamble K.L.
      • Young M.E.
      • Bailey S.M.
      Chronic ethanol consumption disrupts diurnal rhythms of hepatic glycogen metabolism in mice.
      • Tran M.
      • Yang Z.
      • Liangpunsakul S.
      • Wang L.
      Metabolomics analysis revealed distinct cyclic changes of metabolites altered by chronic ethanol-plus-binge and Shp deficiency.
      Large time-of-day-dependent increases in triglyceride and cholesterol levels have been demonstrated in the livers of mice receiving chronic ethanol-administration.
      • Filiano A.N.
      • Millender-Swain T.
      • Johnson Jr., R.
      • Young M.E.
      • Gamble K.L.
      • Bailey S.M.
      Chronic ethanol consumption disrupts the core molecular clock and diurnal rhythms of metabolic genes in the liver without affecting the suprachiasmatic nucleus.
      • Wang T.
      • Yang P.
      • Zhan Y.
      • Xia L.
      • Hua Z.
      • Zhang J.
      Deletion of circadian gene Per1 alleviates acute ethanol-induced hepatotoxicity in mice.
      • Zhou P.
      • Ross R.A.
      • Pywell C.M.
      • Liangpunsakul S.
      • Duffield G.E.
      Disturbances in the murine hepatic circadian clock in alcohol-induced hepatic steatosis.
      • Udoh U.S.
      • Swain T.M.
      • Filiano A.N.
      • Gamble K.L.
      • Young M.E.
      • Bailey S.M.
      Chronic ethanol consumption disrupts diurnal rhythms of hepatic glycogen metabolism in mice.
      • Tran M.
      • Yang Z.
      • Liangpunsakul S.
      • Wang L.
      Metabolomics analysis revealed distinct cyclic changes of metabolites altered by chronic ethanol-plus-binge and Shp deficiency.
      Changes in the diurnal oscillations of core clock genes (Arntl, Clock, Cry1, Cry2, Per1, Per2) and clock-controlled genes (e.g. Dbp, Hlf, Noct, Npas2, Nr1d1, Tef) were observed in the steatotic livers of ethanol-fed rodents.
      • Filiano A.N.
      • Millender-Swain T.
      • Johnson Jr., R.
      • Young M.E.
      • Gamble K.L.
      • Bailey S.M.
      Chronic ethanol consumption disrupts the core molecular clock and diurnal rhythms of metabolic genes in the liver without affecting the suprachiasmatic nucleus.
      Per1 knockout mice have lower levels of triglyceride synthesis genes following acute alcohol administration.
      • Wang T.
      • Yang P.
      • Zhan Y.
      • Xia L.
      • Hua Z.
      • Zhang J.
      Deletion of circadian gene Per1 alleviates acute ethanol-induced hepatotoxicity in mice.
      Chronic ethanol administration to mice disrupts diurnal rhythms in hepatic lipid metabolism at gene and protein levels.
      • Zhou P.
      • Ross R.A.
      • Pywell C.M.
      • Liangpunsakul S.
      • Duffield G.E.
      Disturbances in the murine hepatic circadian clock in alcohol-induced hepatic steatosis.
      • Udoh U.S.
      • Swain T.M.
      • Filiano A.N.
      • Gamble K.L.
      • Young M.E.
      • Bailey S.M.
      Chronic ethanol consumption disrupts diurnal rhythms of hepatic glycogen metabolism in mice.
      • Summa K.C.
      • Voigt R.M.
      • Forsyth C.B.
      • Shaikh M.
      • Cavanaugh K.
      • Tang Y.
      • et al.
      Disruption of the circadian clock in mice increases intestinal permeability and promotes alcohol-induced hepatic pathology and inflammation.
      Ethanol-mediated alterations in the hepatic NAD+/NADH ratio are also under clock control.
      • Zhou P.
      • Ross R.A.
      • Pywell C.M.
      • Liangpunsakul S.
      • Duffield G.E.
      Disturbances in the murine hepatic circadian clock in alcohol-induced hepatic steatosis.
      The exact underlying mechanisms through which ethanol negatively impacts circadian clock-mediated lipid metabolism and contributes to steatosis, remain to be elucidated. Ethanol-mediated alterations in 2 key energy sensing metabolites, NAD + and ATP, may disturb the liver circadian clock by disrupting post-transcriptional modification events (e.g. acetylation, and ADP-ribosylation, and phosphorylation) mediated by the molecules involved in lipid metabolism (e.g. SIRT1, AMPK and poly ADP-ribose polymerase 1).
      • Udoh U.S.
      • Valcin J.A.
      • Gamble K.L.
      • Bailey S.M.
      The molecular circadian clock and alcohol-induced liver injury.
      • Filiano A.N.
      • Millender-Swain T.
      • Johnson Jr., R.
      • Young M.E.
      • Gamble K.L.
      • Bailey S.M.
      Chronic ethanol consumption disrupts the core molecular clock and diurnal rhythms of metabolic genes in the liver without affecting the suprachiasmatic nucleus.
      • Zhou P.
      • Ross R.A.
      • Pywell C.M.
      • Liangpunsakul S.
      • Duffield G.E.
      Disturbances in the murine hepatic circadian clock in alcohol-induced hepatic steatosis.
      Further, deciphering the mechanisms that link ethanol, lipid metabolism and circadian responses will provide valuable insights for the development of innovative therapeutic strategies.
      A number of emerging research areas deserve further investigation in the context of alcohol and steatosis, including long noncoding RNAs, pre-mRNA splicing and gut microbiota.

      Emerging areas

      There are several new areas, including long noncoding RNAs, pre-mRNA splicing, and gut microbiota that deserve further investigation in the context of alcohol and steatosis.

      Alternate mRNA processing

      A regulatory role for microRNAs in AFLD has been suggested.
      • Natarajan S.K.
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      • Mott J.L.
      Role of microRNAs in alcohol-induced multi-organ injury.
      For example, microRNA-217 promotes ethanol-induced fat accumulation in hepatocytes by disrupting the SIRT1-lipin-1 axis.
      • Yin H.
      • Hu M.
      • Zhang R.
      • Shen Z.
      • Flatow L.
      • You M.
      MicroRNA-217 promotes ethanol-induced fat accumulation in hepatocytes by down-regulating SIRT1.
      Whether and how ethanol-mediated alterations in specific microRNA expression are linked to dysregulated lipid metabolism in alcoholic steatosis will need further investigation. Long noncoding RNAs (lncRNAs) influence lipid homeostasis by controlling the lipid metabolism-related gene expression, either by base-pairing with RNA and DNA or by binding to proteins.
      • Chen Z.
      Progress and prospects of long noncoding RNAs in lipid homeostasis.
      • Smekalova E.M.
      • Kotelevtsev Y.V.
      • Leboeuf D.
      • Shcherbinina E.Y.
      • Fefilova A.S.
      • Zatsepin T.S.
      • et al.
      lncRNA in the liver: prospects for fundamental research and therapy by RNA interference.
      Alterations in lncRNA expression have been linked to a number of liver diseases including ALD.
      • Smekalova E.M.
      • Kotelevtsev Y.V.
      • Leboeuf D.
      • Shcherbinina E.Y.
      • Fefilova A.S.
      • Zatsepin T.S.
      • et al.
      lncRNA in the liver: prospects for fundamental research and therapy by RNA interference.
      • Mayfield R.D.
      Emerging roles for ncRNAs in alcohol use disorders.
      It is worthwhile exploring whether, and how, ethanol disrupts hepatic IncRNAs and subsequently causes fatty liver injury. Alternative splicing of precursor messenger RNA (pre-mRNA) is a pivotal step in gene expression, eliminating the introns and ligating the exons to form mature mRNAs that can be translated into proteins.
      • Calandra S.
      • Tarugi P.
      • Bertolini S.
      Altered mRNA splicing in lipoprotein disorders.
      • Elizalde M.
      • Urtasun R.
      • Azkona M.
      • Latasa M.U.
      • Goni S.
      • Garcia-Irigoyen O.
      • et al.
      Splicing regulator SLU7 is essential for maintaining liver homeostasis.
      Defects in the pre-mRNA splicing machinery can impact on lipid homeostasis and contribute to steatosis.
      • Elizalde M.
      • Urtasun R.
      • Azkona M.
      • Latasa M.U.
      • Goni S.
      • Garcia-Irigoyen O.
      • et al.
      Splicing regulator SLU7 is essential for maintaining liver homeostasis.
      • Sen S.
      • Jumaa H.
      • Webster N.J.G.
      Splicing factor SRSF3 is crucial for hepatocyte differentiation and metabolic function.
      • Sen S.
      • Langiewicz M.
      • Jumaa H.
      • Webster N.J.
      Deletion of serine/arginine-rich splicing factor 3 in hepatocytes predisposes to hepatocellular carcinoma in mice.
      Ethanol exposure causes changes in pre-mRNA splicing.
      • Pleiss J.A.
      • Whitworth G.B.
      • Bergkessel M.
      • Guthrie C.
      Rapid, transcript-specific changes in splicing in response to environmental stress.
      However, alternative pre-mRNA splicing is an underappreciated mechanism in the pathogenesis of AFLD.
      • Starkel P.
      • Schnabl B.
      Bidirectional communication between liver and gut during alcoholic liver disease.
      It will be of importance to investigate whether aberrant splicing machinery contributes to ethanol-mediated dysregulation of lipid metabolism and alcoholic steatosis.

      Microbiome

      Growing evidence demonstrates the involvement of gut microbiota in the development and progression of ALD.
      • Starkel P.
      • Schnabl B.
      Bidirectional communication between liver and gut during alcoholic liver disease.
      The influences of gut microbiota on ethanol-mediated dysregulation of lipid metabolism and the relationship between gut microbiota and AFLD warrant future investigation. Undoubtedly, illuminating the mechanistic connections between these newly understood machineries and ethanol will provide a more cohesive picture of how ethanol deranges hepatic lipid metabolism and results in steatosis and liver injury.

      Concluding remarks

      Hepatic lipid metabolism is a series of complex processes that control influx and efflux of not only hepatic lipid pools, but also organismal pools. As mentioned, lipid homeostasis is usually tightly controlled by expression, substrate supply, oxidation and secretion that keeps hepatic lipid poolsrelatively constant. However, perturbations of any of these processes can lead to lipid accumulation in the liver. Although it is thought that these responses are hepatic arms of the ‘thrifty genome’, they are maladaptive in the context of chronic fatty liver diseases.
      • Chakravarthy M.V.
      • Booth F.W.
      Eating exercise, and, “thrifty” genotypes: connecting the dots toward an evolutionary understanding of modern chronic diseases.
      • van Ginneken V.J.
      Liver fattening during feast and famine: an evolutionary paradox.
      Ethanol is likely unique among toxins, in that it perturbs almost all aspects of hepatic lipid metabolism. This complex response is due in part to the large metabolic demand placed on the organ by alcohol metabolism, but also appears to involve more nuanced changes in expression and substrate supply. The net effect is that steatosis is a rapid response to alcohol abuse. Although transient steatosis is largely an inert pathology, the chronicity of ALD seems to require steatosis. Better and more specific understanding of the mechanisms by which alcohol causes steatosis may therefore translate into targeted therapies to treat ALD and/or prevent its progression.

      Financial support

      Supported, in part, by grants ( AA021978 , AA013623 and AA015951 ) and centers (P50 AA024333 P50 AA024337) funded by National Institute of Alcohol Abuse and Alcoholism (NIAAA, USA).

      Conflict of interest

      Dr Arteel and Dr You report grants from National Institutes of Health, during the conduct of the study.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Supplementary data

      The following are the Supplementary data to this article:

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