Ethanol disrupts hepatocellular lipophagy by altering Rab5-centric LD-lysosome trafficking.

BACKGROUND: Previous reports suggest that lipid droplets (LDs) in the hepatocyte can be catabolized by a direct engulfment from nearby endolysosomes (microlipophagy). Further, it is likely that this process is compromised by chronic ethanol (EtOH) exposure leading to hepatic steatosis. This study investigates the hepatocellular machinery supporting microlipophagy and EtOH-induced alterations in this process with a focus on the small, endosome-associated, GTPase Rab5. METHODS AND RESULTS: Here we report that this small Ras-related GTPase is a resident component of LDs, and its activity is important for hepatocellular LD-lysosome proximity and physical interactions. We find that Rab5 siRNA knockdown causes an accumulation of LDs in hepatocytes by inhibiting lysosome dependent LD catabolism. Importantly, Rab5 appears to support this process by mediating the recruitment of early endosomal and or multivesicular body compartments to the LD surface before lysosome fusion. Interestingly, while wild-type or a constituently active GTPase form (Q79L) of Rab5 supports LD-lysosome transport, this process is markedly reduced in cells expressing a GTPase dead (S34N) Rab5 protein or in hepatocytes exposed to chronic EtOH. CONCLUSIONS: These findings support the novel premise of an early endosomal/multivesicular body intermediate compartment on the LD surface that provides a "docking" site for lysosomal trafficking, not unlike the process that occurs during the hepatocellular degradation of endocytosed ligands that is also known to be compromised by EtOH exposure.

Role of ghrelin hormone in the development of alcohol-associated liver disease.

Fatty liver is the earliest response of the liver to excessive alcohol consumption. Previously we identified that chronic alcohol administration increases levels of stomach-derived hormone, ghrelin, which by reducing circulating insulin levels, ultimately contributes to the development of alcohol-associated liver disease (ALD). In addition, ghrelin directly promotes fat accumulation in hepatocytes by enhancing de novo lipogenesis. Other than promoting ALD, ghrelin is known to increase alcohol craving and intake. In this study, we used a ghrelin receptor (GHSR) knockout (KO) rat model to characterize the specific contribution of ghrelin in the development of ALD with emphasis on energy homeostasis. Male Wistar wild type (WT) and GHSR-KO rats were pair-fed the Lieber-DeCarli control or ethanol diet for 6 weeks. At the end of the feeding period, glucose tolerance test was conducted, and tissue samples were collected. We observed reduced alcohol intake by GHSR-KOs compared to a previous study where WT rats were fed ethanol diet ad libitum. Further, when the WTs were pair-fed to GHSR-KOs, the KO rats exhibited resistance to develop ALD through improving insulin secretion/sensitivity to reduce adipose lipolysis and hepatic fatty acid uptake/synthesis and increase fatty acid oxidation. Furthermore, proteomic data revealed that ethanol-fed KO exhibit less alcohol-induced mitochondrial dysfunction and oxidative stress than WT rats. Proteomic data also confirmed that the ethanol-fed KOs are insulin sensitive and are resistant to hepatic steatosis development compared to WT rats. Together, these data confirm that inhibiting ghrelin action prevent alcohol-induced liver and adipose dysfunction independent of reducing alcohol intake.

Alcohol-induced Golgiphagy is triggered by the downregulation of Golgi GTPase RAB3D.

The development of alcohol-associated liver disease (ALD) is associated with disorganized Golgi apparatus and accelerated phagophore formation. While Golgi membranes may contribute to phagophores, association between Golgi alterations and macroautophagy/autophagy remains unclear. GOLGA4/p230 (golgin A4), a dimeric Golgi matrix protein, participates in phagophore formation, but the underlying mechanism is elusive. Our prior research identified ethanol (EtOH)-induced Golgi scattering, disrupting intra-Golgi trafficking and depleting RAB3D GTPase from the trans-Golgi. Employing various techniques, we analyzed diverse cellular and animal models representing chronic and chronic/binge alcohol consumption. In trans-Golgi of non-treated hepatocytes, we found a triple complex formed between RAB3D, GOLGA4, and MYH10/NMIIB (myosin, heavy polypeptide 10, non-muscle). However, EtOH-induced RAB3D downregulation led to MYH10 segregation from the Golgi, accompanied by Golgi fragmentation and tethering of the MYH10 isoform, MYH9/NMIIA, to dispersed Golgi membranes. EtOH-activated autophagic flux is evident through increased WIPI2 recruitment to the Golgi, phagophore formation, enhanced LC3B lipidation, and reduced SQSTM1/p62. Although GOLGA4 dimerization and intra-Golgi localization are unaffected, loss of RAB3D leads to an extension of the cytoplasmic N terminal domain of GOLGA4, forming GOLGA4-positive phagophores. Autophagy inhibition by hydroxychloroquine (HCQ) prevents alcohol-mediated Golgi disorganization, restores distribution of ASGR (asialoglycoprotein receptor), and mitigates COL (collagen) deposition and steatosis. In contrast to short-term exposure to HCQ, extended co-treatment with both EtOH and HCQ results in the depletion of LC3B protein via proteasomal degradation. Thus, (a) RAB3D deficiency and GOLGA4 conformational changes are pivotal in MYH9-driven, EtOH-mediated Golgiphagy, and (b) HCQ treatment holds promise as a therapeutic approach for alcohol-induced liver injury.Abbreviation: ACTB: actin, beta; ALD: alcohol-associated liver disease; ASGR: asialoglycoprotein receptor; AV: autophagic vacuoles; EM: electron microscopy; ER: endoplasmic reticulum; EtOH: ethanol; HCQ: hydroxychloroquine; IP: immunoprecipitation; KD: knockdown; KO: knockout; MYH10/NMIIB: myosin, heavy polypeptide 10, non-muscle; MYH9/NMIIA: myosin, heavy polypeptide 9, non-muscle; PLA: proximity ligation assay; ORO: Oil Red O staining; PM: plasma membrane; TGN: trans-Golgi network; SIM: structured illumination super-resolution microscopy.

Glucagon-like peptide 1 receptor agonist, exendin-4, reduces alcohol-associated fatty liver disease.

Fatty liver is the earliest response to excessive ethanol consumption, which increases the susceptibility of the liver to develop advanced stage of liver disease. Our previous studies have revealed that chronic alcohol administration alters metabolic hormone levels and their functions. Of current interest to our laboratory is glucagon-like peptide 1 (GLP-1), a widely studied hormone known to reduce insulin resistance and hepatic fat accumulation in patients with metabolic-associated fatty liver disease. In this study, we examined the beneficial effects of exendin-4 (a GLP-1 receptor agonist) in an experimental rat model of ALD. Male Wistar rats were pair-fed the Lieber-DeCarli control or ethanol diet. After 4 weeks of this feeding regimen, a subset of rats in each group were intraperitoneally injected every other day with either saline or exendin-4 at a dose of 3 nmol/kg/day (total 13 doses) while still being fed their respective diet. At the end of the treatment, rats were fasted for 6 h and glucose tolerance test was conducted. The following day, the rats were euthanized, and the blood and tissue samples collected for subsequent analysis. We found that exendin-4 treatment had no significant effect on body weight gain among the experimental groups. Exendin-4-treated ethanol rats exhibited improved alcohol-induced alterations in liver/body weight and adipose/body weight ratio, serum ALT, NEFA, insulin, adiponectin and hepatic triglyceride levels. Reduction in indices of hepatic steatosis in exendin-4 treated ethanol-fed rats was attributed to improved insulin signaling and fat metabolism. These results strongly suggest that exendin-4 mitigates alcohol-associated hepatic steatosis by regulating fat metabolism.

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.

Role of the ghrelin system in alcohol use disorder and alcohol-associated liver disease: A narrative review.

Unhealthy alcohol consumption is a global health problem. Adverse individual, public health, and socioeconomic consequences are attributable to harmful alcohol use. Epidemiological studies have shown that alcohol use disorder (AUD) and alcohol-associated liver disease (ALD) are the top two pathologies among alcohol-related diseases. Consistent with the major role that the liver plays in alcohol metabolism, uncontrolled drinking may cause significant damage to the liver. This damage is initiated by excessive fat accumulation in the liver, which can further progress to advanced liver disease. The only effective therapeutic strategies currently available for ALD are alcohol abstinence or liver transplantation. Any molecule with dual-pronged effects at the central and peripheral organs controlling addictive behaviors and associated metabolic pathways are a potentially important therapeutic target for treating AUD and ALD. Ghrelin, a hormone primarily derived from the stomach, has such properties, and regulates both behavioral and metabolic functions. In this review, we highlight recent advances in understanding the peripheral and central functions of the ghrelin system and its role in AUD and ALD pathogenesis. We first discuss the correlation between blood ghrelin concentrations and alcohol use or abstinence. Next, we discuss the role of ghrelin in alcohol-seeking behaviors and finally its role in the development of fatty liver by metabolic regulations and organ crosstalk. We propose that a better understanding of the ghrelin system could open an innovative avenue for improved treatments for AUD and associated medical consequences, including ALD.

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.

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.

Ethanol attenuates presentation of cytotoxic T-lymphocyte epitopes on hepatocytes of HBV-infected humanized mice.

BACKGROUND AND AIMS: Approximately 3.5% of the global population is chronically infected with Hepatitis B Virus (HBV), which puts them at high risk of end-stage liver disease, with the risk of persistent infection potentiated by alcohol consumption. However, the mechanisms underlying the effects of alcohol on HBV persistence remain unclear. Here, we aimed to establish in vivo/ex vivo evidence that alcohol suppresses HBV peptides-major histocompatibility complex (MHC) class I antigen display on primary human hepatocytes (PHH), which diminishes the recognition and clearance of HBV-infected hepatocytes by cytotoxic T-lymphocytes (CTLs). METHODS: We used fumarylacetoacetate hydrolase (Fah)-/-, Rag2-/-, common cytokine receptor gamma chain knock-out (FRG-KO) humanized mice transplanted with human leukocyte antigen-A2 (HLA-A2)-positive hepatocytes. The mice were HBV-infected and fed control and alcohol diets. Isolated hepatocytes were exposed ex vivo to HBV 18-27-HLA-A2-restricted CTLs to quantify cytotoxicity. For mechanistic studies, we measured proteasome activities, unfolded protein response (UPR), and endoplasmic reticulum (ER) stress in hepatocytes from HBV-infected humanized mouse livers. RESULTS AND CONCLUSIONS: We found that alcohol feeding attenuated HBV core 18-27-HLA-A2 complex presentation on infected hepatocytes due to the suppression of proteasome function and ER stress induction, which diminished both the processing of HBV peptides and trafficking of HBV-MHC class I complexes to the hepatocyte surface. This alcohol-mediated decrease in MHC class I-restricted antigen presentation of the CTL epitope on target hepatocytes reduced the CTL-specific elimination of infected cells, potentially leading to HBV-infection persistence, which promotes end-stage liver disease outcomes.

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.

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.

The non-canonical mechanism of ER stress-mediated progression of prostate cancer.

BACKGROUND: The development of persistent endoplasmic reticulum (ER) stress is one of the cornerstones of prostate carcinogenesis; however, the mechanism is missing. Also, alcohol is a physiological ER stress inducer, and the link between alcoholism and progression of prostate cancer (PCa) is well documented but not well characterized. According to the canonical model, the mediator of ER stress, ATF6, is cleaved sequentially in the Golgi by S1P and S2P proteases; thereafter, the genes responsible for unfolded protein response (UPR) undergo transactivation. METHODS: Cell lines used were non-malignant prostate epithelial RWPE-1 cells, androgen-responsive LNCaP, and 22RV1 cells, as well as androgen-refractory PC-3 cells. We also utilized PCa tissue sections from patients with different Gleason scores and alcohol consumption backgrounds. Several sophisticated approaches were employed, including Structured illumination superresolution microscopy, Proximity ligation assay, Atomic force microscopy, and Nuclear magnetic resonance spectroscopy. RESULTS: Herein, we identified the trans-Golgi matrix dimeric protein GCC185 as a Golgi retention partner for both S1P and S2P, and in cells lacking GCC185, these enzymes lose intra-Golgi situation. Progression of prostate cancer (PCa) is associated with overproduction of S1P and S2P but monomerization of GCC185 and its downregulation. Utilizing different ER stress models, including ethanol administration, we found that PCa cells employ an elegant mechanism that auto-activates ER stress by fragmentation of Golgi, translocation of S1P and S2P from Golgi to ER, followed by intra-ER cleavage of ATF6, accelerated UPR, and cell proliferation. The segregation of S1P and S2P from Golgi and activation of ATF6 are positively correlated with androgen receptor signaling, different disease stages, and alcohol consumption. Finally, depletion of ATF6 significantly retarded the growth of xenograft prostate tumors and blocks production of pro-metastatic metabolites. CONCLUSIONS: We found that progression of PCa associates with translocation of S1P and S2P proteases to the ER and subsequent ATF6 cleavage. This obviates the need for ATF6 transport to the Golgi and enhances UPR and cell proliferation. Thus, we provide the novel mechanistic model of ATF6 activation and ER stress implication in the progression of PCa, suggesting ATF6 is a novel promising target for prostate cancer therapy.

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.

Direct lysosome-based autophagy of lipid droplets in hepatocytes.

Hepatocytes metabolize energy-rich cytoplasmic lipid droplets (LDs) in the lysosome-directed process of autophagy. An organelle-selective form of this process (macrolipophagy) results in the engulfment of LDs within double-membrane delimited structures (autophagosomes) before lysosomal fusion. Whether this is an exclusive autophagic mechanism used by hepatocytes to catabolize LDs is unclear. It is also unknown whether lysosomes alone might be sufficient to mediate LD turnover in the absence of an autophagosomal intermediate. We performed live-cell microscopy of hepatocytes to monitor the dynamic interactions between lysosomes and LDs in real-time. We additionally used a fluorescent variant of the LD-specific protein (PLIN2) that exhibits altered fluorescence in response to LD interactions with the lysosome. We find that mammalian lysosomes and LDs undergo interactions during which proteins and lipids can be transferred from LDs directly into lysosomes. Electron microscopy (EM) of primary hepatocytes or hepatocyte-derived cell lines supports the existence of these interactions. It reveals a dramatic process whereby the lipid contents of the LD can be "extruded" directly into the lysosomal lumen under nutrient-limited conditions. Significantly, these interactions are not affected by perturbations to crucial components of the canonical macroautophagy machinery and can occur in the absence of double-membrane lipoautophagosomes. These findings implicate the existence of an autophagic mechanism used by mammalian cells for the direct transfer of LD components into the lysosome for breakdown. This process further emphasizes the critical role of lysosomes in hepatic LD catabolism and provides insights into the mechanisms underlying lipid homeostasis in the liver.

Acetaldehyde suppresses HBV-MHC class I complex presentation on hepatocytes via induction of ER stress and Golgi fragmentation.

Alcohol consumption worsens hepatitis B virus (HBV) infection pathogenesis. We have recently reported that acetaldehyde suppressed HBV peptide-major histocompatibility complex I (MHC class I) complex display on hepatocytes, limiting recognition and subsequent removal of the infected hepatocytes by HBV-specific cytotoxic T lymphocytes (CTLs). This suppression was attributed to impaired processing of antigenic peptides by the proteasome. However, in addition to proteasome dysfunction, alcohol may induce endoplasmic reticulum (ER) stress and Golgi fragmentation in HBV-infected liver cells to reduce uploading of viral peptides to MHC class I and/or trafficking of this complex to the hepatocyte surface. Hence, the aim of this study was to elucidate whether alcohol-induced ER stress and Golgi fragmentation affect HBV peptide-MHC class I complex presentation on HBV+ hepatocytes. Here, we demonstrate that, while both acetaldehyde and HBV independently cause ER stress and Golgi fragmentation, the combined exposure provided an additive effect. Thus we observed an activation of the inositol-requiring enzyme 1α-X-box binding protein 1 and activation transcription factor (ATF)6α, but not the phospho PKR-like ER kinase-phospho eukaryotic initiation factor 2α-ATF4-C/EBP homologous protein arms of ER stress in HBV-transfected cells treated with acetaldehyde-generating system (AGS). In addition, Golgi proteins trans-Golgi network 46, GM130, and Giantin revealed punctate distribution, indicating Golgi fragmentation upon AGS exposure. Furthermore, the effects of acetaldehyde were reproduced by treatment with ER stress inducers, thapsigargin and tunicamycin, which also decreased the display of this complex and MHC class I turnover in HepG2.2.15 cells and HBV-infected primary human hepatocytes. Taken together, alcohol-induced ER stress and Golgi fragmentation contribute to the suppression of HBV peptide-MHC class I complex presentation on HBV+ hepatocytes, which may diminish their recognition by CTLs and promote persistence of HBV infection in hepatocytes.NEW & NOTEWORTHY Our current findings show that acetaldehyde accelerates endoplasmic reticulum (ER) stress by activating the unfolded protein response arms inositol-requiring enzyme 1α-X-box binding protein 1 and activation transcription factor (ATF)6α but not phospho PKR-like ER kinase-p eukaryotic initiation factor 2α-ATF4-C/EBP homologous protein in hepatitis B virus (HBV)-transfected HepG2.2.15 cells. It also potentiates Golgi fragmentation, as evident by punctate distribution of Golgi proteins, GM130, trans-Golgi network 46, and Giantin. While concomitantly increasing HBV DNA and HBV surface antigen titers, acetaldehyde-induced ER stress suppresses the presentation of HBV peptide-major histocompatibility complex I complexes on hepatocyte surfaces, thereby promoting the persistence of HBV infection in the liver.

Recent Advances in Understanding the Complexity of Alcohol-Induced Pancreatic Dysfunction and Pancreatitis Development.

Chronic excessive alcohol use is a well-recognized risk factor for pancreatic dysfunction and pancreatitis development. Evidence from in vivo and in vitro studies indicates that the detrimental effects of alcohol on the pancreas are from the direct toxic effects of metabolites and byproducts of ethanol metabolism such as reactive oxygen species. Pancreatic dysfunction and pancreatitis development are now increasingly thought to be multifactorial conditions, where alcohol, genetics, lifestyle, and infectious agents may determine the initiation and course of the disease. In this review, we first highlight the role of nonoxidative ethanol metabolism in the generation and accumulation of fatty acid ethyl esters (FAEEs) that cause multi-organellar dysfunction in the pancreas which ultimately leads to pancreatitis development. Further, we discuss how alcohol-mediated altered autophagy leads to the development of pancreatitis. We also provide insights into how alcohol interactions with other co-morbidities such as smoking or viral infections may negatively affect exocrine and endocrine pancreatic function. Finally, we present potential strategies to ameliorate organellar dysfunction which could attenuate pancreatic dysfunction and pancreatitis severity.

Ghrelin regulates adipose tissue metabolism: Role in hepatic steatosis.

Fatty liver is the earliest and most common response of the liver to consumption of excessive alcohol. Steatosis can predispose the fatty liver to develop progressive liver damage. Chief among the many mechanisms involved in development of hepatic steatosis is dysregulation of insulin-mediated adipose tissue metabolism. Particularly, it is the enhanced adipose lipolysis-derived free fatty acids and their delivery to the liver that ultimately results in hepatic steatosis. The adipose-liver axis is modulated by hormones, particularly insulin and adiponectin. In recent studies, we demonstrated that an alcohol-induced increase in serum ghrelin levels impairs insulin secretion from pancreatic ß-cells. The consequent reduction in circulating insulin levels promotes adipose lipolysis and mobilization of fatty acids to the liver to ultimately contribute to hepatic steatosis. Because many tissues, including adipose tissue, express ghrelin receptor we hypothesized that ghrelin may directly affect energy metabolism in adipocytes. We have exciting new preliminary data which shows that treatment of premature 3T3-L1 adipocytes with ghrelin impairs adipocyte differentiation and inhibits lipid accumulation in the tissue designed to store energy in the form of fat. We further observed that ghrelin treatment of differentiated adipocytes significantly inhibited secretion of adiponectin, a hepatoprotective hormone that reduces lipid synthesis and promotes lipid oxidation. These results were corroborated by our observations of a significant increase in serum adiponectin levels in ethanol-fed rats treated with a ghrelin receptor antagonist verses the un-treated ethanol-fed rats. Interestingly, in adipocytes, ghrelin also increases secretion of interleukin-6 (IL-6) and CCL2 (chemokine [C-C motif] ligand 2), cytokines which promote hepatic inflammation and progression of liver disease. To summarize, the alcohol-induced increase in serum ghrelin levels dysregulates adipose-liver interaction and promotes hepatic steatosis by increasing the free fatty acid released from adipose for hepatic uptake, and by altering adiponectin and cytokine secretion. Taken together, our data indicates that targeting the activity of ghrelin may be a powerful treatment strategy.