mTOR signaling regulates aberrant epithelial cell proliferative and migratory behaviors characteristic of airway mucous metaplasia in asthma.

In asthma, the airway epithelium is hyperplastic, hypertrophied, and lined with numerous large MUC5AC-containing goblet cells (GC). Furthermore, the normal epithelial architecture is disorganized with numerous, what we here describe as, ectopic goblet cells (eGC) deep within the thickened epithelial layer disconnected from the lumenal surface. mTOR is a highly conserved pathway that regulates cell size and proliferation. We hypothesized that the balance between mTOR and autophagy signaling regulates key features of the asthma epithelial layer. Airway histological sections from subjects with asthma had increased frequency of eGC and increased levels of mTOR phosphorylation target-Ribosomal S6. Using human airway epithelial cells (hAECs) with IL-13 stimulation and timed withdrawal to stimulate resolution, we found that multiple key downstream phosphorylation targets downstream from the mTOR complex were increased during early IL-13-mediated mucous metaplasia, and then significantly declined during resolution. The IL-13-mediated changes in mTOR signaling were paralleled by morphologic changes with airway epithelial hypertrophy, hyperplasia, and frequency of eGC. We then examined the relationship between mTOR and autophagy using mice deficient in autophagy protein Atg16L1. Despite having increased cytoplasmic mucins, mouse AECs from Atg16L1 deficient mice had no significant difference in mTOR downstream signaling. mTOR inhibition with rapamycin led to a loss of IL-13-mediated epithelial hypertrophy, hyperplasia, ectopic GC distribution, and reduction in cytoplasmic MUC5AC levels. mTOR inhibition was also associated with a reduction in aberrant IL-13-mediated hAEC proliferation and migration. Our findings demonstrate that mTOR signaling is associated with mucous metaplasia and is crucial to the disorganized airway epithelial structure and function characteristic of muco-obstructive airway diseases such as asthma. Graphical Abstract Key Concepts: The airway epithelium in asthma is disorganized and characterized by cellular proliferation, aberrant migration, and goblet cell mucous metaplasia.mTOR signaling is a dynamic process during IL-13-mediated mucous metaplasia, increasing with IL-13 stimulation and declining during resolution.mTOR signaling is strongly increased in the asthmatic airway epithelium.mTOR signaling is associated with the development of key features of the metaplastic airway epithelium including cell proliferation and ectopic distribution of goblet cells and aberrant cellular migration.Inhibition of mTOR leads to decreased epithelial hypertrophy, reduced ectopic goblet cells, and cellular migration.

Emerging mechanistic insights of selective autophagy in hepatic diseases.

Macroautophagy (hereafter referred to as autophagy), a highly conserved metabolic process, regulates cellular homeostasis by degrading dysfunctional cytosolic constituents and invading pathogens via the lysosomal system. In addition, autophagy selectively recycles specific organelles such as damaged mitochondria (via mitophagy), and lipid droplets (LDs; via lipophagy) or eliminates specialized intracellular pathogenic microorganisms such as hepatitis B virus (HBV) and coronaviruses (via virophagy). Selective autophagy, particularly mitophagy, plays a key role in the preservation of healthy liver physiology, and its dysfunction is connected to the pathogenesis of a wide variety of liver diseases. For example, lipophagy has emerged as a defensive mechanism against chronic liver diseases. There is a prominent role for mitophagy and lipophagy in hepatic pathologies including non-alcoholic fatty liver disease (NAFLD), hepatocellular carcinoma (HCC), and drug-induced liver injury. Moreover, these selective autophagy pathways including virophagy are being investigated in the context of viral hepatitis and, more recently, the coronavirus disease 2019 (COVID-19)-associated hepatic pathologies. The interplay between diverse types of selective autophagy and its impact on liver diseases is briefly addressed. Thus, modulating selective autophagy (e.g., mitophagy) would seem to be effective in improving liver diseases. Considering the prominence of selective autophagy in liver physiology, this review summarizes the current understanding of the molecular mechanisms and functions of selective autophagy (mainly mitophagy and lipophagy) in liver physiology and pathophysiology. This may help in finding therapeutic interventions targeting hepatic diseases via manipulation of selective autophagy.

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.

Hydroxysteroid 17ß-dehydrogenase 11 accumulation on lipid droplets promotes ethanol-induced cellular steatosis.

Lipid droplets (LDs) are fat-storing organelles enclosed by a phospholipid monolayer, which harbors membrane-associated proteins that regulate distinct LD functions. LD proteins are degraded by the ubiquitin-proteasome system (UPS) and/or by lysosomes. Because chronic ethanol (EtOH) consumption diminishes the hepatic functions of the UPS and lysosomes, we hypothesized that continuous EtOH consumption slows the breakdown of lipogenic LD proteins targeted for degradation, thereby causing LD accumulation. Here, we report that LDs from livers of EtOH-fed rats exhibited higher levels of polyubiquitylated-proteins, linked at either lysine 48 (directed to proteasome) or lysine 63 (directed to lysosomes) than LDs from pair-fed control rats. MS proteomics of LD proteins, immunoprecipitated with UB remnant motif antibody (K-ε-GG), identified 75 potential UB proteins, of which 20 were altered by chronic EtOH administration. Among these, hydroxysteroid 17ß-dehydrogenase 11 (HSD17ß11) was prominent. Immunoblot analyses of LD fractions revealed that EtOH administration enriched HSD17ß11 localization to LDs. When we overexpressed HSD17ß11 in EtOH-metabolizing VA-13 cells, the steroid dehydrogenase 11 became principally localized to LDs, resulting in elevated cellular triglycerides (TGs). Ethanol exposure augmented cellular TG, while HSD17ß11 siRNA decreased both control and EtOH-induced TG accumulation. Remarkably, HSD17ß11 overexpression lowered the LD localization of adipose triglyceride lipase. EtOH exposure further reduced this localization. Reactivation of proteasome activity in VA-13 cells blocked the EtOH-induced rises in both HSD17ß11 and TGs. Our findings indicate that EtOH exposure blocks HSD17ß11 degradation by inhibiting the UPS, thereby stabilizing HSD17ß11 on LD membranes, to prevent lipolysis by adipose triglyceride lipase and promote cellular LD accumulation.

Crohn's Disease Patients Uniquely Contain Inflammatory Responses to Flagellin in a CD4 Effector Memory Subset.

BACKGROUND: Specific microbial antigens stimulate production of antibodies indicative of the aberrant immune response in Crohn's disease (CD). We tested for T cell reactivity linkage to B cell responses and now report on the prevalence, functionality, and phenotypic differences of flagellin-specific T cells among CD patients, ulcerative colitis (UC) patients, and control subjects and association with clinical features and flagellin seropositivity within CD patients. METHODS: Sera from non-inflammatory bowel disease control subjects, CD patients, and UC patients were probed for antibody reactivity to gut bacterial recombinant flagellin antigens. Peripheral blood mononuclear cells were measured for flagellin antigen (CBir1, A4 Fla2, FlaX) or control (Candida albicans, and CytoStim) reactivity analyzed by flow cytometry for CD154 and cytokine expression on CD4+ T cells. Supernatants from post-flagellin-stimulated and unstimulated cells were used to measure effects on epithelial barrier function. RESULTS: CD patients had a significantly higher percentage of flagellin-specific CD154+ CD4+ cells that have an effector memory T helper 1 and T helper 17 phenotype compared with UC patients and healthy control subjects. There was a positive correlation between the frequency of flagellin-specific CD154+ CD4+ effector memory T cells and serum levels of anti-flagellin immunoglobulin G in the CD patients. In addition, A4 Fla2-reactive T cells from active CD patients produced cytokines that can decrease barrier function in a gut epithelium. CONCLUSIONS: These findings demonstrate a Crohn's-associated flagellin-reactive CD4 cell subset distinct from UC patients and control subjects. There is a link between these cells and flagellin seropositivity. This CD4 cell subset could reflect a particular endophenotype of CD, leading to novel insight into its pathology and treatment.

Crohn's disease patients display inflammatory cytokine responses to flagellin antigens in an expanded effector memory CD4 subset that is not seen in ulcerative colitis or non­inflammatory bowel disease control subjects. These cells correlate with levels of the specific cognate anti-flagellin antibodies.

Alcohol: basic and translational research; 15th annual Charles Lieber &1st Samuel French satellite symposium.

The present review is based on the research presented at the symposium dedicated to the legacy of the two scientists that made important discoveries in the field of alcohol-induced liver damage: Professors C.S. Lieber and S.W. French. The invited speakers described pharmacological, toxicological and patho-physiological effects of alcohol misuse. Moreover, genetic biomarkers determining adverse drug reactions due to interactions between therapeutics used for chronic or infectious diseases and alcohol exposure were discussed. The researchers presented their work in areas of alcohol-induced impairment in lipid protein trafficking and endocytosis, as well as the role of lipids in the development of fatty liver. The researchers showed that alcohol leads to covalent modifications that promote hepatic dysfunction and injury. We concluded that using new advanced techniques and research ideas leads to important discoveries in science.

A review of alcohol-pathogen interactions: New insights into combined disease pathomechanisms.

Progression of chronic infections to end-stage diseases and poor treatment results are frequently associated with alcohol abuse. Alcohol metabolism suppresses innate and adaptive immunity leading to increased viral load and its spread. In case of hepatotropic infections, viruses accelerate alcohol-induced hepatitis and liver fibrosis, thereby promoting end-stage outcomes, including cirrhosis and hepatocellular carcinoma (HCC). In this review, we concentrate on several unexplored aspects of these phenomena, which illustrate the combined effects of viral/bacterial infections and alcohol in disease development. We review alcohol-induced alterations implicated in immunometabolism as a central mechanism impacting metabolic homeostasis and viral pathogenesis in Simian immunodeficiency virus/human immunodeficiency virus infection. Furthermore, in hepatocytes, both HIV infection and alcohol activate oxidative stress to cause lysosomal dysfunction and leakage and apoptotic cell death, thereby increasing hepatotoxicity. In addition, we discuss the mechanisms of hepatocellular carcinoma and tumor signaling in hepatitis C virus infection. Finally, we analyze studies that review and describe the immune derangements in hepatotropic viral infections focusing on the development of novel targets and strategies to restore effective immunocompetency in alcohol-associated liver disease. In conclusion, alcohol exacerbates the pathogenesis of viral infections, contributing to a chronic course and poor outcomes, but the mechanisms behind these events are virus specific and depend on virus-alcohol interactions, which differ among the various infections.

Alcohol-Induced Liver Injury: Down-regulation and Redistribution of Rab3D Results in Atypical Protein Trafficking.

Previous work from our laboratories has identified multiple defects in endocytosis, protein trafficking, and secretion, along with altered Golgi function after alcohol administration. Manifestation of alcohol-associated liver disease (ALD) is associated with an aberrant function of several hepatic proteins, including asialoglycoprotein receptor (ASGP-R), their atypical distribution at the plasma membrane (PM), and secretion of their abnormally glycosylated forms into the bloodstream, but trafficking mechanism is unknown. Here we report that a small GTPase, Rab3D, known to be involved in exocytosis, secretion, and vesicle trafficking, shows ethanol (EtOH)-impaired function, which plays an important role in Golgi disorganization. We used multiple approaches and cellular/animal models of ALD, along with Rab3D knockout (KO) mice and human tissue from patients with ALD. We found that Rab3D resides primarily in trans- and cis-faces of Golgi; however, EtOH treatment results in Rab3D redistribution from trans-Golgi to cis-medial-Golgi. Cells lacking Rab3D demonstrate enlargement of Golgi, especially its distal compartments. We identified that Rab3D is required for coat protein I (COPI) vesiculation in Golgi, and conversely, COPI is critical for intra-Golgi distribution of Rab3D. Rab3D/COPI association was altered not only in the liver of patients with ALD but also in the donors consuming alcohol without steatosis. In Rab3D KO mice, hepatocytes experience endoplasmic reticulum (ER) stress, and EtOH administration activates apoptosis. Notably, in these cells, ASGP-R, despite incomplete glycosylation, can still reach cell surface through ER-PM junctions. This mimics the effects seen with EtOH-induced liver injury. Conclusion: We revealed that down-regulation of Rab3D contributes significantly to EtOH-induced Golgi disorganization, and abnormally glycosylated ASGP-R is excreted through ER-PM connections, bypassing canonical (ER→Golgi→PM) anterograde transportation. This suggests that ER-PM sites may be a therapeutic target for ALD.

Multiomics Approach Captures Hepatic Metabolic Network Altered by Chronic Ethanol Administration.

Using a multiplatform and multiomics approach, we identified metabolites, lipids, proteins, and metabolic pathways that were altered in the liver after chronic ethanol administration. A functional enrichment analysis of the multiomics dataset revealed that rats treated with ethanol experienced an increase in hepatic fatty acyl content, which is consistent with an initial development of steatosis. The nuclear magnetic resonance spectroscopy (NMR) and liquid chromatography-mass spectrometry (LC-MS) metabolomics data revealed that the chronic ethanol exposure selectively modified toxic substances such as an increase in glucuronidation tyramine and benzoyl; and a depletion in cholesterol-conjugated glucuronides. Similarly, the lipidomics results revealed that ethanol decreased diacylglycerol, and increased triacylglycerol, sterol, and cholesterol biosynthesis. An integrated metabolomics and lipidomics pathway analysis showed that the accumulation of hepatic lipids occurred by ethanol modulation of the upstream lipid regulatory pathways, specifically glycolysis and glucuronides pathways. A proteomics analysis of lipid droplets isolated from control EtOH-fed rats and a subsequent functional enrichment analysis revealed that the proteomics data corroborated the metabolomic and lipidomic findings that chronic ethanol administration altered the glucuronidation pathway.

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.

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.

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.

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.

Chronic alcohol exposure alters circulating insulin and ghrelin levels: role of ghrelin in hepatic steatosis.

Fatty liver is the earliest response of the liver to excessive ethanol consumption. Central in the development of alcoholic steatosis is increased mobilization of nonesterified free fatty acids (NEFAs) to the liver from the adipose tissue. In this study, we hypothesized that ethanol-induced increase in ghrelin by impairing insulin secretion, could be responsible for the altered lipid metabolism observed in adipose and liver tissue. Male Wistar rats were fed for 5-8 wk with control or ethanol Lieber-DeCarli diet, followed by biochemical analyses in serum and liver tissues. In addition, in vitro studies were conducted on pancreatic islets isolated from experimental rats. We found that ethanol increased serum ghrelin and decreased serum insulin levels in both fed and fasting conditions. These results were corroborated by our observations of a significant accumulation of insulin in pancreatic islets of ethanol-fed rats, indicating that its secretion was impaired. Furthermore, ethanol-induced reduction in circulating insulin was associated with lower adipose weight and increased NEFA levels observed in these rats. Additionally, we found that increased concentration of serum ghrelin was due to increased synthesis and maturation in the stomach of the ethanol-fed rats. We also report that in addition to its effect on the pancreas, ghrelin can also directly act on hepatocytes via the ghrelin receptors and promote fat accumulation. In conclusion, alcohol-induced elevation of circulating ghrelin levels impairs insulin secretion. Consequently, reduced circulating insulin levels likely contribute to increased free fatty acid mobilization from adipose tissue to liver, thereby contributing to hepatic steatosis. NEW & NOTEWORTHY Our studies are the first to report that ethanol-induced increases in ghrelin contribute to impaired insulin secretion, which results in the altered lipid metabolism observed in adipose and liver tissue in the setting of alcoholic fatty liver disease.

Giantin Is Required for Post-Alcohol Recovery of Golgi in Liver Cells.

In hepatocytes and alcohol-metabolizing cultured cells, Golgi undergoes ethanol (EtOH)-induced disorganization. Perinuclear and organized Golgi is important in liver homeostasis, but how the Golgi remains intact is unknown. Work from our laboratories showed that EtOH-altered cellular function could be reversed after alcohol removal; we wanted to determine whether this recovery would apply to Golgi. We used alcohol-metabolizing HepG2 (VA-13) cells (cultured with or without EtOH for 72 h) and rat hepatocytes (control and EtOH-fed (Lieber⁻DeCarli diet)). For recovery, EtOH was removed and replenished with control medium (48 h for VA-13 cells) or control diet (10 days for rats). Results: EtOH-induced Golgi disassembly was associated with de-dimerization of the largest Golgi matrix protein giantin, along with impaired transport of selected hepatic proteins. After recovery from EtOH, Golgi regained their compact structure, and alterations in giantin and protein transport were restored. In VA-13 cells, when we knocked down giantin, Rab6a GTPase or non-muscle myosin IIB, minimal changes were observed in control conditions, but post-EtOH recovery was impaired. Conclusions: These data provide a link between Golgi organization and plasma membrane protein expression and identify several proteins whose expression is important to maintain Golgi structure during the recovery phase after EtOH administration.

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.

Dietary fructose augments ethanol-induced liver pathology.

Certain dietary components when combined with alcohol exacerbate alcohol-induced liver injury (ALI). Here, we tested whether fructose, a major ingredient of the western diet, enhances the severity of ALI. We fed mice ethanol for 8 weeks in the following Lieber-DeCarli diets: (a) Regular (contains olive oil); (b) corn oil (contains corn oil); (c) fructose (contains fructose and olive oil) and (d) corn+fructose (contains fructose and corn oil). We compared indices of metabolic function and liver pathology among the different groups. Mice fed fructose-free and fructose-containing ethanol diets exhibited similar levels of blood alcohol, blood glucose and signs of disrupted hepatic insulin signaling. However, only mice given fructose-ethanol diets showed lower insulin levels than their respective controls. Compared with their respective pair-fed controls, all ethanol-fed mice exhibited elevated levels of serum ALT; the inflammatory cytokines TNF-α, MCP-1 and MIP-2; hepatic lipid peroxides and triglycerides. All the latter parameters were significantly higher in mice given fructose-ethanol diets than those fed fructose-free ethanol diets. Mice given fructose-free or fructose-containing ethanol diets each had higher levels of hepatic lipogenic enzymes than controls. However, the level of the lipogenic enzyme fatty acid synthase (FAS) was significantly higher in livers of mice given fructose control and fructose-ethanol diets than in all other groups. Our findings indicate that dietary fructose exacerbates ethanol-induced steatosis, oxidant stress, inflammation and liver injury, irrespective of the dietary fat source, to suggest that inclusion of fructose in or along with alcoholic beverages increases the risk of more severe ALI in heavy drinkers.