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.

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.

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.

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.

Inhibition of Ghrelin Activity by Receptor Antagonist [d-Lys-3] GHRP-6 Attenuates Alcohol-Induced Hepatic Steatosis by Regulating Hepatic Lipid Metabolism.

Alcoholic steatosis, characterized by an accumulation of triglycerides in hepatocytes, is one of the earliest pathological changes in the progression of alcoholic liver disease. In our previous study, we showed that alcohol-induced increase in serum ghrelin levels impair insulin secretion from pancreatic ß-cells. The consequent reduction in the circulating insulin levels promote adipose-derived fatty acid mobilization to ultimately contribute to hepatic steatosis. In this study, we determined whether inhibition of ghrelin activity in chronic alcohol-fed rats could improve hepatic lipid homeostasis at the pancreas-adipose-liver axis. Adult Wistar rats were fed Lieber-DeCarli control or an ethanol liquid diet for 7 weeks. At 6 weeks, a subset of rats in each group were injected with either saline or ghrelin receptor antagonist, [d-Lys-3] GHRP-6 (DLys; 9 mg/kg body weight) for 5 days and all rats were sacrificed 2 days later. DLys treatment of ethanol rats improved pancreatic insulin secretion, normalized serum insulin levels, and the adipose lipid metabolism, as evidenced by the decreased serum free fatty acids (FFA). DLys treatment of ethanol rats also significantly decreased the circulating FFA uptake, de novo hepatic fatty acid synthesis ultimately attenuating alcoholic steatosis. To summarize, inhibition of ghrelin activity reduced alcoholic steatosis by improving insulin secretion, normalizing serum insulin levels, inhibiting adipose lipolysis, and preventing fatty acid uptake and synthesis in the liver. Our studies provided new insights on the important role of ghrelin in modulating the pancreas-adipose-liver, and promoting adipocyte lipolysis and hepatic steatosis. The findings offer a therapeutic approach of not only preventing alcoholic liver injury but also treating it.

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.

Enhanced colorectal cancer metastases in the alcohol-injured liver.

Metastatic liver disease is a major cause of mortality in colorectal cancer (CRC) patients. Alcohol consumption is a noted risk factor for secondary cancers yet the role of alcoholic liver disease (ALD) in colorectal liver metastases (CRLM) is not defined. This work evaluated tumor cell colonization in the alcoholic host liver using a novel preclinical model of human CRC liver metastases. Immunocompromised Rag1-deficient mice were fed either ethanol (E) or isocaloric control (C) diets for 4 weeks prior to intrasplenic injection of LS174T human CRC cells. ALD and CRLM were evaluated 3 or 5 weeks post-LS174T cell injection with continued C/E diet administration. ALD was confirmed by increased serum transaminases, hepatic steatosis and expression of cytochrome P4502E1, a major ethanol-metabolizing enzyme. Alcohol-mediated liver dysfunction was validated by impaired endocytosis of asialoorosomucoid and carcinoembryonic antigen (CEA), indicators of hepatocellular injury and progressive CRC disease, respectively. Strikingly, the rate and burden of CRLM was distinctly enhanced in alcoholic livers with metastases observed earlier and more severely in E-fed mice. Further, alcohol-related increases (1.5-3.0 fold) were observed in the expression of hepatic cytokines (TNF-α, IL-1 beta, IL-6, IL-10) and other factors noted to be involved in the colonization of CRC cells including ICAM-1, CCL-2, CCL-7, MMP-2, and MMP-9. Also, alcoholic liver injury was associated with altered hepatic localization as well as increased circulating levels of CEA released from CRC cells. Altogether, these findings indicate that the alcoholic liver provides a permissive environment for the establishment of CRLM, possibly through CEA-related inflammatory mechanisms.

Effect of chronic ethanol administration on the in vitro production of proinflammatory cytokines by rat Kupffer cells in the presence of apoptotic cells.

BACKGROUND: Chronic ethanol consumption can lead to a variety of pathological consequences by as yet undefined mechanisms. Recently, it has been noted that alcohol-associated liver disease is often accompanied by morphological liver changes that include the increased production of apoptotic cells. Additionally, it has been demonstrated that hepatocellular uptake and removal of potentially damaging apoptotic cells is impaired after ethanol treatment. The aim of the present study was to determine whether the presence of apoptotic cells leads to Kupffer cell (KC) production and release of proinflammatory cytokines that have been linked to hepatocyte damage, such as interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha). METHODS: Kupffer cells were isolated from female rats after an 8-week oral administration of a dextrose control or ethanol-containing fish-oil diet. The isolated KCs were cultured for up to 24 hours in the absence or presence of apoptotic or nonapoptotic hepatoma cells, or lipopolysaccharide. After incubation, media from the cultures were assayed for the presence of TNF-alpha and IL-6 by immunoassay detection. Also, the expression of these cytokines was measured in KC lysates by a quantitative real-time polymerase chain reaction. RESULTS: Kupffer cells cultured for up to 24 hours in the presence of apoptotic cells produced significantly more TNF-alpha and IL-6 (80 and 60%, respectively, p<0.05) when the cells were isolated from ethanol-fed animals compared with controls. Additionally, after as early as 4 hours in culture with apoptotic cells, mRNA levels of both cytokines were increased (2-5-fold) in KCs isolated from ethanol-fed animals compared with controls. CONCLUSIONS: The presence of apoptotic cells results in the in vitro activation of KCs. Additionally, chronic ethanol administration results in an enhanced responsiveness of KCs to produce proinflammatory cytokines indicated by the increased production of inflammatory mediators from KCs obtained from ethanol-fed animals.

Ethanol-induced apoptosis in polarized hepatic cells possibly through regulation of the Fas pathway.

BACKGROUND: It has been noted that alcohol-related liver diseases can be associated with an increase in apoptotic hepatocellular death. Moreover, the promotion of hepatocyte apoptosis may be linked to signals emanating from death receptors, particularly Fas [CD95/apoptosis-inducing protein 1 (APO-1)]. In the present study, we utilized an in vitro hepatic culture model [hybrid of human fibroblast (WI 38) and rat hepatoma (Fao) cells, WIF-B cells] to study potential contributing mechanisms involved in hepatocellular apoptosis following ethanol administration. METHODS: WIF-B cultures (differentiated hepatic cells that efficiently metabolize alcohol) were treated with or without ethanol and specific inhibitors of alcohol metabolism and cysteine protease activity, followed by morphological and biochemical examination of proapoptotic parameters. RESULTS: The results of this work demonstrated that ethanol administration leads to an increase (45%-60%) in caspase-3 activity and that the induction of apoptosis was found to be linked to the metabolism of alcohol. Additionally, increases were observed in the activity of upstream initiator caspases (caspase-2 and caspase-8) that are directly related to membrane signaling events of death receptors such as Fas. Moreover, it was determined that the activation of caspase-3 could be blocked by the presence of a specific caspase-8 inhibitor, again linking death receptor-associated proteases to downstream effector caspase activity in alcohol-related death. Finally, ethanol administration was found to result in an increase in the amount of Fas protein present in the membrane fraction of the cell. The increase in membrane Fas protein indicates ligand-independent membrane targeting of Fas in the alcohol-treated cells that could potentially be a key signaling event in the induction of the proapoptotic caspase cascade. CONCLUSIONS: The data presented here indicate that alcohol metabolism induces apoptosis in WIF-B cells that occurs, in part, by mechanisms involving signals emanating from death receptors.