Mitochondrial Dysfunction-Associated Mechanisms in the Development of Chronic Liver Diseases.

The liver is a major metabolic organ that performs many essential biological functions such as detoxification and the synthesis of proteins and biochemicals necessary for digestion and growth. Any disruption in normal liver function can lead to the development of more severe liver disorders. Overall, about 3 million Americans have some type of liver disease and 5.5 million people have progressive liver disease or cirrhosis, in which scar tissue replaces the healthy liver tissue. An estimated 20% to 30% of adults have excess fat in their livers, a condition called steatosis. The most common etiologies for steatosis development are (1) high caloric intake that causes non-alcoholic fatty liver disease (NAFLD) and (2) excessive alcohol consumption, which results in alcohol-associated liver disease (ALD). NAFLD is now termed "metabolic-dysfunction-associated steatotic liver disease" (MASLD), which reflects its association with the metabolic syndrome and conditions including diabetes, high blood pressure, high cholesterol and obesity. ALD represents a spectrum of liver injury that ranges from hepatic steatosis to more advanced liver pathologies, including alcoholic hepatitis (AH), alcohol-associated cirrhosis (AC) and acute AH, presenting as acute-on-chronic liver failure. The predominant liver cells, hepatocytes, comprise more than 70% of the total liver mass in human adults and are the basic metabolic cells. Mitochondria are intracellular organelles that are the principal sources of energy in hepatocytes and play a major role in oxidative metabolism and sustaining liver cell energy needs. In addition to regulating cellular energy homeostasis, mitochondria perform other key physiologic and metabolic activities, including ion homeostasis, reactive oxygen species (ROS) generation, redox signaling and participation in cell injury/death. Here, we discuss the main mechanism of mitochondrial dysfunction in chronic liver disease and some treatment strategies available for targeting mitochondria.

Ethanol Exposure to Ethanol-Oxidizing HEPG2 Cells Induces Intracellular Protein Aggregation.

BACKGROUND: Aggresomes are collections of intracellular protein aggregates. In liver cells of patients with alcoholic hepatitis, aggresomes appear histologically as cellular inclusions known as Mallory-Denk (M-D) bodies. The proteasome is a multicatalytic intracellular protease that catalyzes the degradation of both normal (native) and abnormal (misfolded and/or damaged) proteins. The enzyme minimizes intracellular protein aggregate formation by rapidly degrading abnormal proteins before they form aggregates. When proteasome activity is blocked, either by specific inhibitors or by intracellular oxidants (e.g., peroxynitrite, acetaldehyde), aggresome formation is enhanced. Here, we sought to verify whether inhibition of proteasome activity by ethanol exposure enhances protein aggregate formation in VL-17A cells, which are recombinant, ethanol-oxidizing HepG2 cells that express both alcohol dehydrogenase (ADH) and cytochrome P450 2E1 (CYP2E1). METHODS: We exposed ethanol-non-oxidizing HepG2 cells (ADH-/CYP2E1-) or ethanol-oxidizing VL-17A (ADH+/CYP2E1+) to varying levels of ethanol for 24 h or 72 h. After these treatments, we stained cells for aggresomes (detected microscopically) and quantified their numbers and sizes. We also conducted flow cytometric analyses to confirm our microscopic findings. Additionally, aggresome content in liver cells of patients with alcohol-induced hepatitis was quantified. RESULTS: After we exposed VL-17A cells to increasing doses of ethanol for 24 h or 72 h, 20S proteasome activity declined in response to rising ethanol concentrations. After 24 h of ethanol exposure, aggresome numbers in VL-17A cells were 1.8-fold higher than their untreated controls at all ethanol concentrations employed. After 72 h of ethanol exposure, mean aggresome numbers were 2.5-fold higher than unexposed control cells. The mean aggregate size in all ethanol-exposed VL-17A cells was significantly higher than in unexposed control cells but was unaffected by the duration of ethanol exposure. Co-exposure of cells to EtOH and rapamycin, the latter an autophagy activator, completely prevented EtOH-induced aggresome formation. In the livers of patients with alcohol-induced hepatitis (AH), the staining intensity of aggresomes was 2.2-fold higher than in the livers of patients without alcohol use disorder (AUD). CONCLUSIONS: We conclude that ethanol-induced proteasome inhibition in ethanol-metabolizing VL-17A hepatoma cells causes accumulation of protein aggregates. Notably, autophagy activation removes such aggregates. The significance of these findings is discussed.

Lipidomic Analysis of Liver Lipid Droplets after Chronic Alcohol Consumption with and without Betaine Supplementation.

The earliest manifestation of alcohol-associated liver disease is hepatic steatosis, which is characterized by fat accumulation in specialized organelles called lipid droplets (LDs). Our previous studies reported that alcohol consumption elevates the numbers and sizes of LDs in hepatocytes, which is attenuated by simultaneous treatment with the methyl group donor, betaine. Here, we examined changes in the hepatic lipidome with respect to LD size and dynamics in male Wistar rats fed for 6 weeks with control or ethanol-containing liquid diets that were supplemented with or without 10 mg betaine/mL. At the time of sacrifice, three hepatic LD fractions, LD1 (large droplets), LD2 (medium-sized droplets), and LD3 (small droplets) were isolated from each rat. Untargeted lipidomic analyses revealed that each LD fraction of ethanol-fed rats had higher phospholipids, cholesteryl esters, diacylglycerols, ceramides, and hexosylceramides compared with the corresponding fractions of pair-fed controls. Interestingly, the ratio of phosphatidylcholine to phosphatidylethanolamine (the two most abundant phospholipids on the LD surface) was lower in LD1 fraction compared with LD3 fraction, irrespective of treatment; however, this ratio was significantly lower in ethanol LD fractions compared with their respective control fractions. Betaine supplementation significantly attenuated the ethanol-induced lipidomic changes. These were mainly associated with the regulation of LD surface phospholipids, ceramides, and glycerolipid metabolism in different-sized LD fractions. In conclusion, our results show that ethanol-induced changes in the hepatic LD lipidome likely stabilizes larger-sized LDs during steatosis development. Furthermore, betaine supplementation could effectively reduce the size and dynamics of LDs to attenuate alcohol-associated hepatic steatosis.

Pathogenesis of Alcohol-Associated Liver Disease.

Excessive alcohol consumption is a global healthcare problem with enormous social, economic, and clinical consequences. While chronic, heavy alcohol consumption causes structural damage and/or disrupts normal organ function in virtually every tissue of the body, the liver sustains the greatest damage. This is primarily because the liver is the first to see alcohol absorbed from the gastrointestinal tract via the portal circulation and second, because the liver is the principal site of ethanol metabolism. Alcohol-induced damage remains one of the most prevalent disorders of the liver and a leading cause of death or transplantation from liver disease. Despite extensive research on the pathophysiology of this disease, there are still no targeted therapies available. Given the multifactorial mechanisms for alcohol-associated liver disease pathogenesis, it is conceivable that a multitherapeutic regimen is needed to treat different stages in the spectrum of this disease.

Acute ethanol-induced liver injury is prevented by betaine administration.

Binge drinking is the most common form of excessive alcohol use. Repeated episodes of binge drinking cause multiple organ injuries, including liver damage. We previously demonstrated that chronic ethanol administration causes a decline in the intrahepatic ratio of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH). This decline causes impairments in essential methylation reactions that result in alcohol-induced fatty liver (steatosis) and other features of alcohol-associated liver disease (ALD). Co-treatment with betaine during chronic ethanol feeding, normalizes hepatocellular SAM:SAH ratio and alleviates many features of liver damage including steatosis. Here, we sought to examine whether betaine treatment similarly protects against liver injury in an alcohol binge-drinking model. We hypothesized that ethanol binge with prior or simultaneous betaine administration would prevent or attenuate acute alcohol-induced liver damage. Male C57Bl/6 mice were gavaged twice, 12 h apart, with either 6 g ethanol/kg BW or with an equal volume/kg BW of 0.9% NaCl. Two separate groups of mice (n = 5/group) were gavaged with 4 g betaine/kg BW, either 2 h before or simultaneously with the ethanol or saline gavages. All mice were sacrificed 8 h after the last gavage and serum and liver parameters were quantified. Ethanol binges caused a 50% decrease in hepatic SAM:SAH ratio and a >3-fold rise in liver triglycerides (p ≤ 0.05). These latter changes were accompanied by elevated serum AST and ALT activities and blood alcohol concentrations (BAC) that were ∼three-times higher than the legal limit of intoxication in humans. Mice that were treated with betaine 2 h before or simultaneously with the ethanol binges exhibited similar BAC as in mice given ethanol-alone. Both betaine treatments significantly elevated hepatic SAM levels thereby normalizing the SAM:SAH ratio and attenuating hepatic steatosis and other injury parameters, compared with mice given ethanol alone. Simultaneous betaine co-administration with ethanol was more effective in preventing or attenuating liver injury than betaine given before ethanol gavage. Our findings confirm the potential therapeutic value of betaine administration in preventing liver injury after binge drinking in an animal model.

Alcohol-Induced Lysosomal Damage and Suppression of Lysosome Biogenesis Contribute to Hepatotoxicity in HIV-Exposed Liver Cells.

Although the causes of hepatotoxicity among alcohol-abusing HIV patients are multifactorial, alcohol remains the least explored "second hit" for HIV-related hepatotoxicity. Here, we investigated whether metabolically derived acetaldehyde impairs lysosomes to enhance HIV-induced hepatotoxicity. We exposed Cytochrome P450 2E1 (CYP2E1)-expressing Huh 7.5 (also known as RLW) cells to an acetaldehyde-generating system (AGS) for 24 h. We then infected (or not) the cells with HIV-1ADA then exposed them again to AGS for another 48 h. Lysosome damage was assessed by galectin 3/LAMP1 co-localization and cathepsin leakage. Expression of lysosome biogenesis-transcription factor, TFEB, was measured by its protein levels and by in situ immunofluorescence. Exposure of cells to both AGS + HIV caused the greatest amount of lysosome leakage and its impaired lysosomal biogenesis, leading to intrinsic apoptosis. Furthermore, the movement of TFEB from cytosol to the nucleus via microtubules was impaired by AGS exposure. The latter impairment appeared to occur by acetylation of α-tubulin. Moreover, ZKSCAN3, a repressor of lysosome gene activation by TFEB, was amplified by AGS. Both these changes contributed to AGS-elicited disruption of lysosome biogenesis. Our findings indicate that metabolically generated acetaldehyde damages lysosomes and likely prevents their repair and restoration, thereby exacerbating HIV-induced hepatotoxicity.

A review of the role of ethanol-induced adipose tissue dysfunction in alcohol-associated liver disease.

Alcohol-associated liver disease (AALD) encompasses a spectrum of liver diseases that includes simple steatosis, steatohepatitis, fibrosis, and cirrhosis. The adverse effects of alcohol in liver and the mechanisms by which ethanol (EtOH) promotes liver injury are well studied. Although liver is known to be the primary organ affected by EtOH exposure, alcohol's effects on other organs are also known to contribute significantly to the development of liver injury. It is becoming increasingly evident that adipose tissue (AT) is an important site of EtOH action. Both AT storage and secretory functions are altered by EtOH. For example, AT lipolysis, stimulated by EtOH, contributes to chronic alcohol-induced hepatic steatosis. Adipocytes secrete a wide variety of biologically active molecules known as adipokines. EtOH alters the secretion of these adipokines from AT, which include cytokines and chemokines that exert paracrine effects in liver. In addition, the level of EtOH-metabolizing enzymes, in particular, CYP2E1, rises in the AT of EtOH-fed mice, which promotes oxidative stress and/or inflammation in AT. Thus, AT dysfunction characterized by increased AT lipolysis and free fatty acid mobilization and altered secretion of adipokines can contribute to the severity of AALD. Of note, moderate EtOH exposure results in AT browning and activation of brown adipose tissue which, in turn, can promote thermogenesis. In this review article, we discuss the direct effects of EtOH consumption in AT and the mechanisms by which EtOH impacts the functions of AT, which, in turn, increases the severity of AALD in animal models and humans.

Elevated S-adenosylhomocysteine induces adipocyte dysfunction to promote alcohol-associated liver steatosis.

It has been previously shown that chronic ethanol administration-induced increase in adipose tissue lipolysis and reduction in the secretion of protective adipokines collectively contribute to alcohol-associated liver disease (ALD) pathogenesis. Further studies have revealed that increased adipose S-adenosylhomocysteine (SAH) levels generate methylation defects that promote lipolysis. Here, we hypothesized that increased intracellular SAH alone causes additional related pathological changes in adipose tissue as seen with alcohol administration. To test this, we used 3-deazaadenosine (DZA), which selectively elevates intracellular SAH levels by blocking its hydrolysis. Fully differentiated 3T3-L1 adipocytes were treated in vitro for 48 h with DZA and analysed for lipolysis, adipokine release and differentiation status. DZA treatment enhanced adipocyte lipolysis, as judged by lower levels of intracellular triglycerides, reduced lipid droplet sizes and higher levels of glycerol and free fatty acids released into the culture medium. These findings coincided with activation of both adipose triglyceride lipase and hormone sensitive lipase. DZA treatment also significantly reduced adipocyte differentiation factors, impaired adiponectin and leptin secretion but increased release of pro-inflammatory cytokines, IL-6, TNF and MCP-1. Together, our results demonstrate that elevation of intracellular SAH alone by DZA treatment of 3T3-L1 adipocytes induces lipolysis and dysregulates adipokine secretion. Selective elevation of intracellular SAH by DZA treatment mimics ethanol's effects and induces adipose dysfunction. We conclude that alcohol-induced elevations in adipose SAH levels contribute to the pathogenesis and progression of ALD.

Beneficial Effects of Betaine: A Comprehensive Review.

Medicinal herbs and many food ingredients possess favorable biological properties that contribute to their therapeutic activities. One such natural product is betaine, a stable, nontoxic natural substance that is present in animals, plants, and microorganisms. Betaine is also endogenously synthesized through the metabolism of choline or exogenously consumed through dietary intake. Betaine mainly functions as (i) an osmolyte and (ii) a methyl-group donor. This review describes the major physiological effects of betaine in whole-body health and its ability to protect against both liver- as well as non-liver-related diseases and conditions. Betaine's role in preventing/attenuating both alcohol-induced and metabolic-associated liver diseases has been well studied and is extensively reviewed here. Several studies show that betaine protects against the development of alcohol-induced hepatic steatosis, apoptosis, and accumulation of damaged proteins. Additionally, it can significantly prevent/attenuate progressive liver injury by preserving gut integrity and adipose function. The protective effects are primarily associated with the regulation of methionine metabolism through removing homocysteine and maintaining cellular SAM:SAH ratios. Similarly, betaine prevents metabolic-associated fatty liver disease and its progression. In addition, betaine has a neuroprotective role, preserves myocardial function, and prevents pancreatic steatosis. Betaine also attenuates oxidant stress, endoplasmic reticulum stress, inflammation, and cancer development. To conclude, betaine exerts significant therapeutic and biological effects that are potentially beneficial for alleviating a diverse number of human diseases and conditions.

Natural Recovery by the Liver and Other Organs after Chronic Alcohol Use.

Chronic, heavy alcohol consumption disrupts normal organ function and causes structural damage in virtually every tissue of the body. Current diagnostic terminology states that a person who drinks alcohol excessively has alcohol use disorder. The liver is especially susceptible to alcohol-induced damage. This review summarizes and describes the effects of chronic alcohol use not only on the liver, but also on other selected organs and systems affected by continual heavy drinking-including the gastrointestinal tract, pancreas, heart, and bone. Most significantly, the recovery process after cessation of alcohol consumption (abstinence) is explored. Depending on the organ and whether there is relapse, functional recovery is possible. Even after years of heavy alcohol use, the liver has a remarkable regenerative capacity and, following alcohol removal, can recover a significant portion of its original mass and function. Other organs show recovery after abstinence as well. Data on studies of both heavy alcohol use among humans and animal models of chronic ethanol feeding are discussed. This review describes how (or whether) each organ/tissue metabolizes ethanol, as metabolism influences the organ's degree of injury. Damage sustained by the organ/tissue is reviewed, and evidence for recovery during abstinence is presented.

Contrasting Effects of Fasting on Liver-Adipose Axis in Alcohol-Associated and Non-alcoholic Fatty Liver.

Background: Fatty liver, a major health problem worldwide, is the earliest pathological change in the progression of alcohol-associated (AFL) and non-alcoholic fatty liver disease (NAFL). Though the causes of AFL and NAFL differ, both share similar histological and some common pathophysiological characteristics. In this study, we sought to examine mechanisms responsible for lipid dynamics in liver and adipose tissue in the setting of AFL and NAFL in response to 48 h of fasting. Methods: Male rats were fed Lieber-DeCarli liquid control or alcohol-containing diet (AFL model), chow or high-fat pellet diet (NAFL model). After 6-8 weeks of feeding, half of the rats from each group were fasted for 48 h while the other half remained on their respective diets. Following sacrifice, blood, adipose, and the liver were collected for analysis. Results: Though rats fed AFL and NAFL diets both showed fatty liver, the physiological mechanisms involved in the development of each was different. Here, we show that increased hepatic de novo fatty acid synthesis, increased uptake of adipose-derived free fatty acids, and impaired triglyceride breakdown contribute to the development of AFL. In the case of NAFL, however, increased dietary fatty acid uptake is the major contributor to hepatic steatosis. Likewise, the response to starvation in the two fatty liver disease models also varied. While there was a decrease in hepatic steatosis after fasting in ethanol-fed rats, the control, chow and high-fat diet-fed rats showed higher levels of hepatic steatosis than pair-fed counterparts. This diverse response was a result of increased adipose lipolysis in all experimental groups except fasted ethanol-fed rats. Conclusion: Even though AFL and NAFL are nearly histologically indistinguishable, the physiological mechanisms that cause hepatic fat accumulation are different as are their responses to starvation.

Lipid droplet membrane proteome remodeling parallels ethanol-induced hepatic steatosis and its resolution.

Lipid droplets (LDs) are composed of neutral lipids enclosed in a phospholipid monolayer, which harbors membrane-associated proteins that regulate LD functions. Despite the crucial role of LDs in lipid metabolism, remodeling of LD protein composition in disease contexts, such as steatosis, remains poorly understood. We hypothesized that chronic ethanol consumption, subsequent abstinence from ethanol, or fasting differentially affects the LD membrane proteome content and that these changes influence how LDs interact with other intracellular organelles. Here, male Wistar rats were pair-fed liquid control or ethanol diets for 6 weeks, and then, randomly chosen animals from both groups were either refed a control diet for 7 days or fasted for 48 h before euthanizing. From all groups, LD membrane proteins from purified liver LDs were analyzed immunochemically and by MS proteomics. Liver LD numbers and sizes were greater in ethanol-fed rats than in pair-fed control, 7-day refed, or fasted rats. Compared with control rats, ethanol feeding markedly altered the LD membrane proteome, enriching LD structural perilipins and proteins involved in lipid biosynthesis, while lowering LD lipase levels. Ethanol feeding also lowered LD-associated mitochondrial and lysosomal proteins. In 7-day refed (i.e., ethanol-abstained) or fasted-ethanol-fed rats, we detected distinct remodeling of the LD proteome, as judged by lower levels of lipid biosynthetic proteins, and enhanced LD interaction with mitochondria and lysosomes. Our study reveals evidence of significant remodeling of the LD membrane proteome that regulates ethanol-induced steatosis, its resolution after withdrawal and abstinence, and changes in LD interactions with other intracellular organelles.

Role of Elevated Intracellular S-Adenosylhomocysteine in the Pathogenesis of Alcohol-Related Liver Disease.

BACKGROUND: The earliest manifestation of alcohol-related liver disease (ALD) is steatosis, characterized by the accumulation of lipid droplets (LDs) in hepatocytes. Findings from our laboratory have indicated that many pathological changes, including steatosis, correlate with the alcohol-induced hepatocellular increases in S-adenosylhomocysteine (SAH). Based on these considerations, we hypothesized that an experimental increase in intracellular SAH alone will result in similar steatotic changes to those seen after alcohol exposure. METHODS: Freshly isolated rat hepatocytes grown on collagen-coated plates were exposed to serum-free medium containing 50 µmol/L oleic acid and varying concentrations of 3-deazaadenosine (DZA) to experimentally elevate intracellular SAH levels. RESULTS: Overnight exposure to DZA treatment dose-dependently increased hepatocellular triglyceride accumulation, which was also evident by morphological visualization of larger-sized LDs. The rise in triglycerides and LDs accompanied increases in mRNA and protein levels of several LD-associated proteins known to regulate LD number and size. Furthermore, DZA treatment caused a decline in the levels of lipases that prevent fat accumulation as well as increased the expression of factors involved in lipogenesis and fatty acid mobilization. Collectively, our results indicate that the elevation of intracellular SAH is sufficient to promote fat accumulation in hepatocytes, which is similar to that seen after alcohol exposure.

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

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

Lipophagy and Alcohol-Induced Fatty Liver.

This review describes the influence of ethanol consumption on hepatic lipophagy, a selective form of autophagy during which fat-storing organelles known as lipid droplets (LDs) are degraded in lysosomes. During classical autophagy, also known as macroautophagy, all forms of macromolecules and organelles are sequestered in autophagosomes, which, with their cargo, fuse with lysosomes, forming autolysosomes in which the cargo is degraded. It is well established that excessive drinking accelerates intrahepatic lipid biosynthesis, enhances uptake of fatty acids by the liver from the plasma and impairs hepatic secretion of lipoproteins. All the latter contribute to alcohol-induced fatty liver (steatosis). Here, our principal focus is on lipid catabolism, specifically the impact of excessive ethanol consumption on lipophagy, which significantly influences the pathogenesis alcohol-induced steatosis. We review findings, which demonstrate that chronic ethanol consumption retards lipophagy, thereby exacerbating steatosis. This is important for two reasons: (1) Unlike adipose tissue, the liver is considered a fat-burning, not a fat-storing organ. Thus, under normal conditions, lipophagy in hepatocytes actively prevents lipid droplet accumulation, thereby maintaining lipostasis; (2) Chronic alcohol consumption subverts this fat-burning function by slowing lipophagy while accelerating lipogenesis, both contributing to fatty liver. Steatosis was formerly regarded as a benign consequence of heavy drinking. It is now recognized as the "first hit" in the spectrum of alcohol-induced pathologies that, with continued drinking, progresses to more advanced liver disease, liver failure, and/or liver cancer. Complete lipid droplet breakdown requires that LDs be digested to release their high-energy cargo, consisting principally of cholesteryl esters and triacylglycerols (triglycerides). These subsequently undergo lipolysis, yielding free fatty acids that are oxidized in mitochondria to generate energy. Our review will describe recent findings on the role of lipophagy in LD catabolism, how continuous heavy alcohol consumption affects this process, and the putative mechanism(s) by which this occurs.

Hepatic Inactivation of the Type 2 Deiodinase Confers Resistance to Alcoholic Liver Steatosis.

BACKGROUND: A mouse with hepatocyte-specific deiodinase type II inactivation (Alb-D2KO) is resistant to diet-induced obesity, hepatic steatosis, and hypertriglyceridemia due to perinatal epigenetic modifications in the liver. This phenotype is linked to low levels of Zfp125, a hepatic transcriptional repressor that promotes liver steatosis by inhibiting genes involved in packaging and secretion of very-low-density lipoprotein. METHODS: Here, we used chronic and binge ethanol (EtOH) in mice to cause liver steatosis. RESULTS: The EtOH treatment causes a 2.3-fold increase in hepatic triglyceride content; Zfp125 levels were approximately 50% higher in these animals. In contrast, Alb-D2KO mice did not develop EtOH-induced liver steatosis. They also failed to elevate Zfp125 to the same levels, despite being on the EtOH-containing diet for the same period of time. Their phenotype was associated with 1.3- to 2.9-fold up-regulation of hepatic genes involved in lipid transport and export that are normally repressed by Zfp125, that is, Mttp, Abca1, Ldlr, Apoc1, Apoc3, Apoe, Apoh, and Azgp1. Furthermore, genes involved in the EtOH metabolic pathway, that is, Aldh2 and Acss2, were also 1.6- to 3.1-fold up-regulated in Alb-D2KO EtOH mice compared with control animals kept on EtOH. CONCLUSIONS: EtOH consumption elevates expression of Zfp125. Alb-D2KO animals, which have lower levels of Zfp125, are much less susceptible to EtOH-induced liver steatosis.

Ethanol withdrawal mitigates fatty liver by normalizing lipid catabolism.

We are investigating the changes in hepatic lipid catabolism that contribute to alcohol-induced fatty liver. Following chronic ethanol (EtOH) exposure, abstinence from alcohol resolves steatosis. Here, we investigated the hepatocellular events that lead to this resolution by quantifying specific catabolic parameters that returned to control levels after EtOH was withdrawn. We hypothesized that, after its chronic consumption, EtOH withdrawal reactivates lipid catabolic processes that restore lipostasis. Male Wistar rats were fed control and EtOH liquid diets for 6 wk. Randomly chosen EtOH-fed rats were then fed control diet for 7 days. Liver triglycerides (TG), lipid peroxides, key markers of fatty acid (FA) metabolism, lipophagy, and autophagy were quantified. Compared with controls, EtOH-fed rats had higher hepatic triglycerides, lipid peroxides, and serum free fatty acids (FFA). The latter findings were associated with higher levels of FA transporters (FATP 2, 4, and 5) but lower quantities of peroxisome proliferator-activated receptor-α (PPAR-α), which governs FA oxidation. EtOH-fed animals also had lower nuclear levels of the autophagy-regulating transcription factor EB (TFEB), associated with lower hepatic lipophagy and autophagy. After EtOH-fed rats were refed control diet for 7 days, their serum FFA levels and those of FATPs fell to control (normal) levels, whereas PPAR-α levels rose to normal. Hepatic TG and malondialdehyde levels in EtOH-withdrawn rats declined to near control levels. EtOH withdrawal restored nuclear TFEB content, hepatic lipophagy, and autophagy activity to control levels. EtOH withdrawal reversed aberrant FA metabolism and restored lysosomal function to promote resolution of alcohol-induced fatty liver. NEW & NOTEWORTHY Here, using an animal model, we show mechanisms of reversal of fatty liver and injury following EtOH withdrawal. Our data indicate that reactivation of autophagy and lysosome function through the restoration of transcription factor EB contribute to reversal of fatty liver and injury following EtOH withdrawal.

Effect of nanoformulated copper/zinc superoxide dismutase on chronic ethanol-induced alterations in liver and adipose tissue.

BACKGROUND: We previously reported that nanoformulated copper/zinc superoxide dismutase (Nano) attenuates non-alcoholic fatty liver disease and adipose tissue (AT) inflammation in obese animals. Here, we sought to determine whether Nano treatment attenuates alcohol-associated liver disease (AALD) and AT inflammation in alcohol-fed mice. METHODS: We pre-treated E-47 cells (HepG2 cells that over-express CYP2E1) with native- or nano-superoxide dismutase (SOD) for 6 h, followed by treatment with ethanol and/or linoleic acid (LA), a free fatty acid. For in vivo studies, male C57BL/6 mice were fed the Lieber-DeCarli control or ethanol liquid diet for 4 weeks. The mice received Nano once every 2 days during the last 2 weeks of ethanol feeding. RESULTS: Our in vitro studies revealed that Nano pretreatment reduced LA + ethanol-induced oxidative stress in E-47 cells. Our in vivo experiments showed that ethanol-fed Nano-treated mice had 22% lower hepatic triglyceride levels than mice fed ethanol alone. Nano-treated ethanol-fed mice also had 2-fold lower levels of Cd68 and similarly reduced levels of Ccl2 and Mmp12 mRNAs, than in untreated ethanol-fed mice. We also noted that ethanol feeding caused a remarkable increase in hepatic and/or plasma MCP-1 and CCR2 protein, which was blunted in ethanol + Nano-treated animals. The hepatic content of SREBP-1c, a transcription factor that promotes lipogenesis, was higher in ethanol-fed mice than controls but was attenuated in ethanol + Nano-treated animals. Further, livers of ethanol + Nano-treated mice had significantly higher levels of phosphorylated adenosine monophosphate-activated protein kinase (AMPK) than both control and ethanol-fed mice. In AT, the levels of Il6 mRNA, a hepatoprotective cytokine, and that of Arg1, a marker of anti-inflammatory macrophages, were significantly increased in ethanol + Nano-treated mice compared with control mice. CONCLUSION: Our data indicate that Nano treatment attenuates ethanol-induced steatohepatitis and that this effect is associated with an apparent activation of AMPK signaling. Our data also suggest that Nano induces Arg1 and Il6 expression in AT, suggesting anti-inflammatory effects in this tissue.

Diurnal variations in intestinal barrier integrity and liver pathology in mice: implications for alcohol binge.

Recent studies suggest that circadian rhythms regulate intestinal barrier integrity, but it is not clear whether there are daily variations in barrier integrity. This study investigated daily variations in intestinal barrier integrity, including whether there are differences in alcohol-induced intestinal barrier dysfunction after an alcohol binge at different times of day and whether this is associated with concurrent liver injury. C57BL6/J male mice were fed a standard chow diet, an alcohol-containing liquid diet, or an alcohol control diet for 4 wk. During week 5 (i.e., on days 43-45), mice received three once-daily gavages of alcohol (6 g/kg) or the control (phosphate-buffered saline) at the same time each day. Immediately after the binge on the second day, intestinal permeability was assessed. Four hours after the third and final binge, mice were euthanized and tissue samples collected. The results demonstrated diet-specific and outcome-specific effects of time, alcohol, and/or time by alcohol interaction. Specifically, the alcohol binge robustly influenced markers of intestinal barrier integrity, and liver markers were robustly influenced by time of day. Only intestinal permeability (i.e., sucralose) demonstrated a significant effect of time and also showed a binge by time interaction, suggesting that the time of the alcohol binge influences colonic permeability. NEW & NOTEWORTHY This study investigated daily variations in intestinal barrier integrity, including whether there are differences in alcohol-induced intestinal barrier dysfunction after an alcohol binge at different times of day and whether this is associated with concurrent liver injury. We conclude that 1) alcohol binge significantly impacted markers of intestinal permeability, 2) time of day significantly affected liver outcomes, and 3) the time of day influenced colonic permeability.

Ethanol-induced steatosis involves impairment of lipophagy, associated with reduced Dynamin2 activity.

BACKGROUND: Lipid droplets (LDs), the organelles central to alcoholic steatosis, are broken down by lipophagy, a specialized form of autophagy. Here, we hypothesize that ethanol administration retards lipophagy by down-regulating Dynamin 2 (Dyn2), a protein that facilitates lysosome re-formation, contributing to hepatocellular steatosis. METHODS: Primary hepatocytes were isolated from male Wistar rats fed Lieber-DeCarli control or EtOH liquid diets for 6-8 wk. Hepatocytes were incubated in complete medium (fed) or nutrient-free medium (fasting) with or without the Dyn2 inhibitor Dynasore or the Src inhibitor SU6656. Phosphorylated (active) forms of Src and Dyn2, and markers of autophagy were quantified by Western Blot. Co-localization of LDs-with autophagic machinery was determined by confocal microscopy. RESULTS: In hepatocytes from pair-fed rats, LD breakdown was accelerated during fasting, as judged by smaller LDs and lower TG content when compared to hepatocytes in complete media. Fasting-induced TG loss in control hepatocytes was significantly blocked by either SU6656 or Dynasore. Compared to controls, hepatocytes from EtOH-fed rats had 66% and 40% lower content of pSrc and pDyn2, respectively, coupled with lower rate of fasting-induced TG loss. This slower rate of fasting-induced TG loss was blocked in cells co-incubated with Dynasore. Microscopic examination of EtOH-fed rat hepatocytes revealed increased co-localization of the autophagosome marker LC3 on LDs with a concomitant decrease in lysosome marker LAMP1. Whole livers and LD fractions of EtOH-fed rats exhibited simultaneous increase in LC3II and p62 over that of controls, indicating a block in lipophagy. CONCLUSION: Chronic ethanol administration slowed the rate of hepatocyte lipophagy, owing in part to lower levels of phosphorylated Src kinase available to activate its substrate, Dyn2, thereby causing depletion of lysosomes for LD breakdown.