Alcohol-induced microtubule acetylation leads to the accumulation of large, immobile lipid droplets.

Although steatosis (fatty liver) is a clinically well-described early stage of alcoholic liver disease, surprisingly little is known about how it promotes hepatotoxicity. We have shown that ethanol consumption leads to microtubule hyperacetylation that can explain ethanol-induced defects in protein trafficking. Because almost all steps of the lipid droplet life cycle are microtubule dependent and because microtubule acetylation promotes adipogenesis, we examined droplet dynamics in ethanol-treated cells. In WIF-B cells treated with ethanol and/or oleic acid (a fatty acid associated with the "Western" diet), we found that ethanol dramatically increased lipid droplet numbers and led to the formation of large, peripherally located droplets. Enhanced droplet formation required alcohol dehydrogenase-mediated ethanol metabolism, and peripheral droplet distributions required intact microtubules. We also determined that ethanol-induced microtubule acetylation led to impaired droplet degradation. Live-cell imaging revealed that droplet motility was microtubule dependent and that droplets were virtually stationary in ethanol-treated cells. To determine more directly whether microtubule hyperacetylation could explain impaired droplet motility, we overexpressed the tubulin-specific acetyltransferase αTAT1 to promote microtubule acetylation in the absence of alcohol. Droplet motility was impaired in αTAT1-expressing cells but to a lesser extent than in ethanol-treated cells. However, in both cases, the large immotile droplets (but not small motile ones) colocalized with dynein and dynactin (but not kinesin), implying that altered droplet-motor microtubule interactions may explain altered dynamics. These studies further suggest that modulating cellular acetylation is a potential strategy for treating alcoholic liver disease.NEW & NOTEWORTHY Chronic alcohol consumption with the "Western diet" enhances the development of fatty liver and leads to impaired droplet motility, which may have serious deletrious effects on hepatocyte function.

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.

Ethanol metabolism by alcohol dehydrogenase or cytochrome P450 2E1 differentially impairs hepatic protein trafficking and growth hormone signaling.

The liver metabolizes alcohol using alcohol dehydrogenase (ADH) and cytochrome P450 2E1 (CYP2E1). Both enzymes metabolize ethanol into acetaldehyde, but CYP2E1 activity also results in the production of reactive oxygen species (ROS) that promote oxidative stress. We have previously shown that microtubules are hyperacetylated in ethanol-treated polarized, hepatic WIF-B cells and livers from ethanol-fed rats. We have also shown that enhanced protein acetylation correlates with impaired clathrin-mediated endocytosis, constitutive secretion, and nuclear translocation and that the defects are likely mediated by acetaldehyde. However, the roles of CYP2E1-generated metabolites and ROS in microtubule acetylation and these alcohol-induced impairments have not been examined. To determine if CYP2E1-mediated alcohol metabolism is required for enhanced acetylation and the trafficking defects, we coincubated cells with ethanol and diallyl sulfide (DAS; a CYP2E1 inhibitor) or N-acetyl cysteine (NAC; an antioxidant). Both agents failed to prevent microtubule hyperacetylation in ethanol-treated cells and also failed to prevent impaired secretion or clathrin-mediated endocytosis. Somewhat surprisingly, both DAS and NAC prevented impaired STAT5B nuclear translocation. Further examination of microtubule-independent steps of the pathway revealed that Jak2/STAT5B activation by growth hormone was prevented by DAS and NAC. These results were confirmed in ethanol-exposed HepG2 cells expressing only ADH or CYP2E1. Using quantitative RT-PCR, we further determined that ethanol exposure led to blunted growth hormone-mediated gene expression. In conclusion, we determined that alcohol-induced microtubule acetylation and associated defects in microtubule-dependent trafficking are mediated by ADH metabolism whereas impaired microtubule-independent Jak2/STAT5B activation is mediated by CYP2E1 activity.NEW & NOTEWORTHY Impaired growth hormone-mediated signaling is observed in ethanol-exposed hepatocytes and is explained by differential effects of alcohol dehydrogenase (ADH)- and cytochrome P450 2E1 (CYP2E1)-mediated ethanol metabolism on the Jak2/STAT5B pathway.

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.

Review: Precision Cut Liver Slices for the Evaluation of Fatty Liver and Fibrosis.

INTRODUCTION: Ethanol metabolism in the liver results in oxidative stress, altered cytokine production and fat accumulation in the liver. Thus, it is thought that the accumulation of benign fat into the liver in conjunction with a second hit leads to liver failure. However, we have recently developed the use of precision-cut liver slices (PCLSs) as an in vitro culture model in which to investigate the pathophysiology of alcohol-induced liver injury. In this review, these studies will be discussed and newer data presented. METHODS: Original investigations into the use of PCLS were obtained from chow fed rats (200-300g). PCLSs were cultured 24-96h in media, 25 mM ethanol, or 25 mM ethanol and 0.5 mM 4- methylpyrazole (4-MP). PCLSs were examined for at different times and evaluated for glutathione (GSH) levels, extent of lipid peroxidation (TBARS assay), cytokine production (ELISA and RT-PCR) and myofibroblast activation. Age-matched rats were fed high fat diets for 13 months, PCLSs were prepared, and evaluated as outlined above. In recently, human and mouse PCLSs were cut, equilibrated, and evaluated using the methods outlined as above. RESULTS: In these studies, it was shown that the PCLSs from rats, mice and human livers retained excellent viability over a 96 hour period of incubation. During this time period, alcohol dehydrogenase, aldehyde dehydrogenase, and cytochrome P4502E1 levels were viable. After 24 hours of ethanol exposure, fatty livers and fibrogenic responses developed and could be prevented/reversed with the 4-MP. In a separate study using overly obese rats, ethanol metabolism was decreased in PCLSs as compared to age-matched controls (AMC). However, higher levels of triglycerides and lipid peroxidation were found in PCLSs from obese rats compared to AMC. Also, increased concentrations of the proinflammatory cytokines (TNF-α and IL-6) were found in the culture supernatants. In contrast, decreased levels of reduced glutathione (GSH) and heme oxygenase I (HO-1) levels were detected. CONCLUSION: Within 24h of incubation, ethanol metabolism by PCLSs initiates fat accumulation in the liver at which point there is an activation of myofibroblasts. Thus, fatty liver is the first response to ethanol and sensitizes the liver to other products of oxidative stress that result in inflammation and the start of liver failure ending in cirrhosis. Thus, from these studies it appears that PCLSs can be utilized to determine the mechanisms(s) by which ethanol exposure leads to the development and/or progression of alcoholic liver disease (ALD).

Prolonged feeding with guanidinoacetate, a methyl group consumer, exacerbates ethanol-induced liver injury.

AIM: To investigate the hypothesis that exposure to guanidinoacetate (GAA, a potent methyl-group consumer) either alone or combined with ethanol intake for a prolonged period of time would cause more advanced liver pathology thus identifying methylation defects as the initiator and stimulator for progressive liver damage. METHODS: Adult male Wistar rats were fed the control or ethanol Lieber DeCarli diet in the absence or presence of GAA supplementation. At the end of 6 wk of the feeding regimen, various biochemical and histological analyses were conducted. RESULTS: Contrary to our expectations, we observed that GAA treatment alone resulted in a histologically normal liver without evidence of hepatosteatosis despite persistence of some abnormal biochemical parameters. This protection could result from the generation of creatine from the ingested GAA. Ethanol treatment for 6 wk exhibited changes in liver methionine metabolism and persistence of histological and biochemical defects as reported before. Further, when the rats were fed the GAA-supplemented ethanol diet, similar histological and biochemical changes as observed after 2 wk of combined treatment, including inflammation, macro- and micro-vesicular steatosis and a marked decrease in the methylation index were noted. In addition, rats on the combined treatment exhibited increased liver toxicity and even early fibrotic changes in a subset of animals in this group. The worsening liver pathology could be related to the profound reduction in the hepatic methylation index, an increased accumulation of GAA and the inability of creatine generated to exert its hepato-protective effects in the setting of ethanol. CONCLUSION: To conclude, prolonged exposure to a methyl consumer superimposed on chronic ethanol consumption causes persistent and pronounced liver damage.

Acetaldehyde Disrupts Interferon Alpha Signaling in Hepatitis C Virus-Infected Liver Cells by Up-Regulating USP18.

BACKGROUND: Alcohol consumption exacerbates the pathogenesis of hepatitis C virus (HCV) infection and worsens disease outcomes. The exact reasons are not clear yet, but they might be partially attributed to the ability of alcohol to further suppress the innate immunity. Innate immunity is known to be already decreased by HCV in liver cells. METHODS: In this study, we aimed to explore the mechanisms of how alcohol metabolism dysregulates IFNα signaling (STAT1 phosphorylation) in HCV+ hepatoma cells. To this end, CYP2E1+ Huh7.5 cells were infected with HCV and exposed to the acetaldehyde (Ach) generating system (AGS). RESULTS: Continuously produced Ach suppressed IFNα-induced STAT1 phosphorylation, but increased the level of a protease, USP18 (both measured by Western blot), which interferes with IFNα signaling. Induction of USP18 by Ach was confirmed in primary human hepatocyte cultures and in livers of ethanol-fed HCV transgenic mice. Silencing of USP18 by specific siRNA attenuated the pSTAT1 suppression by Ach. The mechanism by which Ach down-regulates pSTAT1 is related to an enhanced interaction between IFNαR2 and USP18 that finally dysregulates the cross talk between the IFN receptor on the cell surface and STAT1. Furthermore, Ach decreases ISGylation of STAT1 (protein conjugation of a small ubiquitin-like modifier, ISG15, Western blot), which preserves STAT1 activation. Suppressed ISGylation leads to an increase in STAT1 K48 polyubiquitination which allows pSTAT1 degrading by proteasome. CONCLUSIONS: We conclude that Ach disrupts IFNα-induced STAT1 phosphorylation by the up-regulation of USP18 to block the innate immunity protection in HCV-infected liver cells, thereby contributing to HCV-alcohol pathogenesis. This, in part, may explain the mechanism of HCV-infection exacerbation/progression in alcohol-abusing patients.

Creatine Supplementation Does Not Prevent the Development of Alcoholic Steatosis.

BACKGROUND: Alcohol-induced reduction in the hepatocellular S-adenosylmethionine (SAM):S-adenosylhomocysteine (SAH) ratio impairs the activities of many SAM-dependent methyltransferases. These impairments ultimately lead to the generation of several hallmark features of alcoholic liver injury including steatosis. Guanidinoacetate methyltransferase (GAMT) is an important enzyme that catalyzes the final reaction in the creatine biosynthetic process. The liver is a major site for creatine synthesis which places a substantial methylation burden on this organ as GAMT-mediated reactions consume as much as 40% of all the SAM-derived methyl groups. We hypothesized that dietary creatine supplementation could potentially spare SAM, preserve the hepatocellular SAM:SAH ratio, and thereby prevent the development of alcoholic steatosis and other consequences of impaired methylation reactions. METHODS: For these studies, male Wistar rats were pair-fed the Lieber-DeCarli control or ethanol (EtOH) diet with or without 1% creatine supplementation. At the end of 4 to 5 weeks of feeding, relevant biochemical and histological analyses were performed. RESULTS: We observed that creatine supplementation neither prevented alcoholic steatosis nor attenuated the alcohol-induced impairments in proteasome activity. The lower hepatocellular SAM:SAH ratio seen in the EtOH-fed rats was also not normalized or SAM levels spared when these rats were fed the creatine-supplemented EtOH diet. However, a >10-fold increased level of creatine was observed in the liver, serum, and hearts of rats fed the creatine-supplemented diets. CONCLUSIONS: Overall, dietary creatine supplementation did not prevent alcoholic liver injury despite its known efficacy in preventing high-fat-diet-induced steatosis. Betaine, a promethylating agent that maintains the hepatocellular SAM:SAH, still remains our best option for treating alcoholic steatosis.

Role of apoptotic hepatocytes in HCV dissemination: regulation by acetaldehyde.

Alcohol consumption exacerbates hepatitis C virus (HCV) pathogenesis and promotes disease progression, although the mechanisms are not quite clear. We have previously observed that acetaldehyde (Ach) continuously produced by the acetaldehyde-generating system (AGS), temporarily enhanced HCV RNA levels, followed by a decrease to normal or lower levels, which corresponded to apoptosis induction. Here, we studied whether Ach-induced apoptosis caused depletion of HCV-infected cells and what role apoptotic bodies (AB) play in HCV-alcohol crosstalk. In liver cells exposed to AGS, we observed the induction of miR-122 and miR-34a. As miR-34a has been associated with apoptotic signaling and miR-122 with HCV replication, these findings may suggest that cells with intensive viral replication undergo apoptosis. Furthermore, when AGS-induced apoptosis was blocked by a pan-caspase inhibitor, the expression of HCV RNA was not changed. AB from HCV-infected cells contained HCV core protein and the assembled HCV particle that infect intact hepatocytes, thereby promoting the spread of infection. In addition, AB are captured by macrophages to switch their cytokine profile to the proinflammatory one. Macrophages exposed to HCV(+) AB expressed more IL-1ß, IL-18, IL-6, and IL-10 mRNAs compared with those exposed to HCV(-) AB. The generation of AB from AGS-treated HCV-infected cells even enhanced the induction of aforementioned cytokines. We conclude that HCV and alcohol metabolites trigger the formation of AB containing HCV particles. The consequent spread of HCV to neighboring hepatocytes via infected AB, as well as the induction of liver inflammation by AB-mediated macrophage activation potentially exacerbate the HCV infection course by alcohol and worsen disease progression.

Acute and Chronic Ethanol Administration Differentially Modulate Hepatic Autophagy and Transcription Factor EB.

BACKGROUND: Chronic ethanol (EtOH) consumption decelerates the catabolism of long-lived proteins, indicating that it slows hepatic macroautophagy (hereafter called autophagy) a crucial lysosomal catabolic pathway in most eukaryotic cells. Autophagy and lysosome biogenesis are linked. Both are regulated by the transcription factor EB (TFEB). Here, we tested whether TFEB can be used as a singular indicator of autophagic activity, by quantifying its nuclear content in livers of mice subjected to acute and chronic EtOH administration. We correlated nuclear TFEB to specific indices of autophagy. METHODS: In acute experiments, we gavaged GFP-LC3(tg) mice with a single dose of EtOH or with phosphate buffered saline (PBS). We fed mice chronically by feeding them control or EtOH liquid diets. RESULTS: Compared with PBS-gavaged controls, livers of EtOH-gavaged mice exhibited greater autophagosome (AV) numbers, a higher incidence of AV-lysosome co-localization, and elevated levels of free GFP, all indicating enhanced autophagy, which correlated with a higher nuclear content of TFEB. Compared with pair-fed controls, livers of EtOH-fed mice exhibited higher AV numbers, but had lower lysosome numbers, lower AV-lysosome co-localization, higher P62/SQSTM1 levels, and lower free GFP levels. The latter findings correlated with lower nuclear TFEB levels in EtOH-fed mice. Thus, enhanced autophagy after acute EtOH gavage correlated with a higher nuclear TFEB content. Conversely, chronic EtOH feeding inhibited hepatic autophagy, associated with a lower nuclear TFEB content. CONCLUSIONS: Our findings suggest that the effect of acute EtOH gavage on hepatic autophagy differs significantly from that after chronic EtOH feeding. Each regimen distinctly affects TFEB localization, which in turn, regulates hepatic autophagy and lysosome biogenesis.

Acetaldehyde accelerates HCV-induced impairment of innate immunity by suppressing methylation reactions in liver cells.

Alcohol exposure worsens the course and outcomes of hepatitis C virus (HCV) infection. Activation of protective antiviral genes is induced by IFN-α signaling, which is altered in liver cells by either HCV or ethanol exposure. However, the mechanisms of the combined effects of HCV and ethanol metabolism in IFN-α signaling modulation are not well elucidated. Here, we explored a possibility that ethanol metabolism potentiates HCV-mediated dysregulation of IFN-α signaling in liver cells via impairment of methylation reactions. HCV-infected Huh7.5 CYP2E1(+) cells and human hepatocytes were exposed to acetaldehyde (Ach)-generating system (AGS) and stimulated with IFN-α to activate IFN-sensitive genes (ISG) via the Jak-STAT-1 pathway. We observed significant suppression of signaling events by Ach. Ach exposure decreased STAT-1 methylation via activation of protein phosphatase 2A and increased the protein inhibitor of activated STAT-1 (PIAS-1)-STAT-1 complex formation in both HCV(+) and HCV(-) cells, preventing ISG activation. Treatment with a promethylating agent, betaine, attenuated all examined Ach-induced defects. Ethanol metabolism-induced changes in ISGs are methylation related and confirmed by in vivo studies on HCV(+) transgenic mice. HCV- and Ach-induced impairment of IFN signaling temporarily increased HCV RNA levels followed by apoptosis of heavily infected cells. We concluded that Ach potentiates the suppressive effects of HCV on activation of ISGs attributable to methylation-dependent dysregulation of IFN-α signaling. A temporary increase in HCV RNA sensitizes the liver cells to Ach-induced apoptosis. Betaine reverses the inhibitory effects of Ach on IFN signaling and thus can be used for treatment of HCV(+) alcohol-abusing patients.

Role of defective methylation reactions in ethanol-induced dysregulation of intestinal barrier integrity.

Alcoholic liver disease (ALD) is a major healthcare challenge worldwide. Emerging evidence reveals that ethanol administration disrupts the intestinal epithelial tight junction (TJ) complex; this defect allows for the paracellular translocation of gut-derived pathogenic molecules to reach the liver to cause inflammation and progressive liver injury. We have previously demonstrated a causative role of impairments in liver transmethylation reactions in the pathogenesis of ALD. We have further shown that treatment with betaine, a methylation agent that normalizes liver methylation potential, can attenuate ethanol-induced liver injury. Herein, we explored whether alterations in methylation reactions play a causative role in disrupting intestinal mucosal barrier function by employing an intestinal epithelial cell line. Monolayers of Caco-2 cells were exposed to ethanol or a-pan methylation reaction inhibitor, tubercidin, in the presence and absence of betaine. The structural and functional integrity of intestinal epithelial barrier was then examined. We observed that exposure to either ethanol or tubercidin disrupted TJ integrity and function by decreasing the localization of TJ protein occludin-1 to the intracellular junctions, reducing transepithelial electrical resistance and increasing dextran influx. All these detrimental effects of ethanol and tubercidin were attenuated by co-treatment with betaine. We further show that the mechanism of betaine protection was through BHMT-mediated catalysis. Collectively, our data suggest a novel mechanism for alcohol-induced gut leakiness and identifies the importance of normal methylation reactions in maintaining TJ integrity. We also propose betaine as a potential therapeutic option for leaky gut in alcohol-consuming patients who are at the risk of developing ALD.

Isoaspartate, carbamoyl phosphate synthase-1, and carbonic anhydrase-III as biomarkers of liver injury.

We had previously shown that alcohol consumption can induce cellular isoaspartate protein damage via an impairment of the activity of protein isoaspartyl methyltransferase (PIMT), an enzyme that triggers repair of isoaspartate protein damage. To further investigate the mechanism of isoaspartate accumulation, hepatocytes cultured from control or 4-week ethanol-fed rats were incubated in vitro with tubercidin or adenosine. Both these agents, known to elevate intracellular S-adenosylhomocysteine levels, increased cellular isoaspartate damage over that recorded following ethanol consumption in vivo. Increased isoaspartate damage was attenuated by treatment with betaine. To characterize isoaspartate-damaged proteins that accumulate after ethanol administration, rat liver cytosolic proteins were methylated using exogenous PIMT and (3)H-S-adenosylmethionine and proteins resolved by gel electrophoresis. Three major protein bands of ∼ 75-80 kDa, ∼ 95-100 kDa, and ∼ 155-160 kDa were identified by autoradiography. Column chromatography used to enrich isoaspartate-damaged proteins indicated that damaged proteins from ethanol-fed rats were similar to those that accrued in the livers of PIMT knockout (KO) mice. Carbamoyl phosphate synthase-1 (CPS-1) was partially purified and identified as the ∼ 160 kDa protein target of PIMT in ethanol-fed rats and in PIMT KO mice. Analysis of the liver proteome of 4-week ethanol-fed rats and PIMT KO mice demonstrated elevated cytosolic CPS-1 and betaine homocysteine S-methyltransferase-1 when compared to their respective controls, and a significant reduction of carbonic anhydrase-III (CA-III) evident only in ethanol-fed rats. Ethanol feeding of rats for 8 weeks resulted in a larger (∼ 2.3-fold) increase in CPS-1 levels compared to 4-week ethanol feeding indicating that CPS-1 accumulation correlated with the duration of ethanol consumption. Collectively, our results suggest that elevated isoaspartate and CPS-1, and reduced CA-III levels could serve as biomarkers of hepatocellular injury.

Alcohol, carcinoembryonic antigen processing and colorectal liver metastases.

It is well established that alcohol consumption is related to the development of alcoholic liver disease. Additionally, it is appreciated that other major health issues are associated with alcohol abuse, including colorectal cancer (CRC) and its metastatic growth to the liver. Although a correlation exists between alcohol use and the development of diseases, the search continues for a better understanding of specific mechanisms. Concerning the role of alcohol in CRC liver metastases, recent research is aimed at characterizing the processing of carcinoembryonic antigen (CEA), a glycoprotein that is associated with and secreted by CRC cells. A positive correlation exists between serum CEA levels, liver metastasis, and alcohol consumption in CRC patients, although the mechanism is not understood. It is known that circulating CEA is processed primarily by the liver, first by nonparenchymal Kupffer cells (KCs) and secondarily, by hepatocytes via the asialoglycoprotein receptor (ASGPR). Since both KCs and hepatocytes are known to be significantly impacted by alcohol, it is hypothesized that alcohol-related effects to these liver cells will lead to altered CEA processing, including impaired asialo-CEA degradation, resulting in changes to the liver microenvironment and the metastatic potential of CRC cells. Also, it is predicted that CEA processing will affect cytokine production in the alcohol-injured liver, resulting in pro-metastatic changes such as enhanced adhesion molecule expression on the hepatic sinusoidal endothelium. This chapter examines the potential role that alcohol-induced liver cell impairments can have in the processing of CEA and associated mechanisms involved in CEA-related colorectal cancer liver metastasis.

Malondialdehyde-acetaldehyde adducts and anti-malondialdehyde-acetaldehyde antibodies in rheumatoid arthritis.

OBJECTIVE: Malondialdehyde-acetaldehyde (MAA) adducts are a product of oxidative stress associated with tolerance loss in several disease states. This study was undertaken to investigate the presence of MAA adducts and circulating anti-MAA antibodies in patients with rheumatoid arthritis (RA). METHODS: Synovial tissue from patients with RA and patients with osteoarthritis (OA) were examined for the presence of MAA-modified and citrullinated proteins. Anti-MAA antibody isotypes were measured in RA patients (n = 1,720) and healthy controls (n = 80) by enzyme-linked immunosorbent assay. Antigen-specific anti-citrullinated protein antibodies (ACPAs) were measured in RA patients using a multiplex antigen array. Anti-MAA isotype concentrations were compared in a subset of RA patients (n = 80) and matched healthy controls (n = 80). Associations of anti-MAA antibody isotypes with disease characteristics, including ACPA positivity, were examined in all RA patients. RESULTS: Expression of MAA adducts was increased in RA synovial tissue compared to OA synovial tissue, and colocalization with citrullinated proteins was found. Increased levels of anti-MAA antibody isotypes were observed in RA patients compared to controls (P < 0.001). Among RA patients, anti-MAA antibody isotypes were associated with seropositivity for ACPAs and rheumatoid factor (P < 0.001) in addition to select measures of disease activity. Higher anti-MAA antibody concentrations were associated with a greater number of positive antigen-specific ACPA analytes (expressed at high titer) (P < 0.001) and a higher ACPA score (P < 0.001), independent of other covariates. CONCLUSION: MAA adduct formation is increased in RA and appears to result in robust antibody responses that are strongly associated with ACPAs. These results support speculation that MAA formation may be a cofactor that drives tolerance loss, resulting in the autoimmune responses characteristic of RA.

Alcohol-induced defects in hepatic transcytosis may be explained by impaired dynein function.

Alcoholic liver disease has been clinically well described, but the molecular mechanisms leading to hepatotoxicity have not been fully elucidated. Previously, we determined that microtubules are hyperacetylated and more stable in ethanol-treated WIF-B cells, VL-17A cells, liver slices, and in livers from ethanol-fed rats. From our recent studies, we believe that these modifications can explain alcohol-induced defects in microtubule motor-dependent protein trafficking including nuclear translocation of a subset of transcription factors. Since cytoplasmic dynein/dynactin is known to mediate both microtubule-dependent translocation and basolateral to apical/canalicular transcytosis, we predicted that transcytosis is impaired in ethanol-treated hepatic cells. We monitored transcytosis of three classes of newly synthesized canalicular proteins in polarized, hepatic WIF-B cells, an emerging model system for the study of liver disease. As predicted, canalicular delivery of all proteins tested was impaired in ethanol-treated cells. Unlike in control cells, transcytosing proteins were observed in discrete sub-canalicular puncta en route to the canalicular surface that aligned along acetylated microtubules. We further determined that the stalled transcytosing proteins colocalized with dynein/dynactin in treated cells. No changes in vesicle association were observed for either dynein or dynactin in ethanol-treated cells, but significantly enhanced dynein binding to microtubules was observed. From these results, we propose that enhanced dynein binding to microtubules in ethanol-treated cells leads to decreased motor processivity resulting in vesicle stalling and in impaired canalicular delivery. Our studies also importantly indicate that modulating cellular acetylation levels with clinically tolerated deacetylase agonists may be a novel therapeutic strategy for treating alcoholic liver disease.

Increased methylation demand exacerbates ethanol-induced liver injury.

We previously reported that chronic ethanol intake lowers hepatocellular S-adenosylmethionine to S-adenosylhomocysteine ratio and significantly impairs many liver methylation reactions. One such reaction, catalyzed by guanidinoacetate methyltransferase (GAMT), is a major consumer of methyl groups and utilizes as much as 40% of the SAM-derived groups to convert guanidinoacetate (GAA) to creatine. The exposure to methyl-group consuming compounds has substantially increased over the past decade that puts additional stresses on the cellular methylation potential. The purpose of our study was to investigate whether increased ingestion of a methyl-group consumer (GAA) either alone or combined with ethanol intake, plays a role in the pathogenesis of liver injury. Adult male Wistar rats were pair-fed the Lieber DeCarli control or ethanol diet in the presence or absence of GAA for 2weeks. At the end of the feeding regimen, biochemical and histological analyses were conducted. We observed that 2 weeks of GAA- or ethanol-alone treatment increases hepatic triglyceride accumulation by 4.5 and 7-fold, respectively as compared with the pair-fed controls. However, supplementing GAA in the ethanol diet produced panlobular macro- and micro-vesicular steatosis, a marked decrease in the methylation potential and a 28-fold increased triglyceride accumulation. These GAA-supplemented ethanol diet-fed rats displayed inflammatory changes and significantly increased liver toxicity compared to the other groups. In conclusion, increased methylation demand superimposed on chronic ethanol consumption causes more pronounced liver injury. Thus, alcoholic patients should be cautioned for increased dietary intake of methyl-group consuming compounds even for a short period of time.

Alcohol consumption decreases rat hepatic creatine biosynthesis via altered guanidinoacetate methyltransferase activity.

BACKGROUND: We have previously shown that decreased S-adenosylmethionine (SAM):S-adenosylhomocysteine (SAH) ratio generated in livers of alcohol-fed rats can impair the activities of many SAM-dependent methyltransferases. One such methyltransferase is guanidinoacetate methyltransferase (GAMT) that catalyzes the last step of creatine synthesis. As GAMT is the major utilizer of SAM, the purpose of the study was to examine the effects of ethanol (EtOH) on liver creatine levels and GAMT activity. METHODS: Male Wistar rats were pair-fed the Lieber-DeCarli control and EtOH diet for 4 to 5 weeks. At the end of the feeding regimen, the liver, kidney, and blood were removed from these rats for subsequent biochemical analyses. RESULTS: We observed ~60% decrease in creatine levels in the livers from EtOH-fed rats as compared to controls. The reduction in creatine levels correlated with lower SAM:SAH ratio observed in the livers of the EtOH-fed rats. Further, in vitro experiments with cell-free system and hepatic cells revealed it is indeed elevated SAH and lower SAM:SAH ratio that directly impairs GAMT activity and significantly reduces creatine synthesis. EtOH intake also slightly decreases the hepatocellular uptake of the creatine precursor, guanidinoacetate (GAA), and the GAMT enzyme expression that could additionally contribute to reduced liver creatine synthesis. The consequences of impaired hepatic creatine synthesis by chronic EtOH consumption include (i) increased toxicity due to GAA accumulation in the liver; (ii) reduced protection due to lower creatine levels in the liver, and (iii) reduced circulating and cardiac creatine levels. CONCLUSIONS: Chronic EtOH consumption affects the hepatic creatine biosynthetic pathway leading to detrimental consequences not only in the liver but could also affect distal organs such as the heart that depend on a steady supply of creatine from the liver.

Rab GTPases associate with isolated lipid droplets (LDs) and show altered content after ethanol administration: potential role in alcohol-impaired LD metabolism.

BACKGROUND: Alcoholic liver disease is manifested by the presence of fatty liver, primarily due to accumulation of hepatocellular lipid droplets (LDs). The presence of membrane-trafficking proteins (e.g., Rab GTPases) with LDs indicates that LDs may be involved in trafficking pathways known to be altered in ethanol (EtOH) damaged hepatocytes. As these Rab GTPases are crucial regulators of protein trafficking, we examined the effect EtOH administration has on hepatic Rab protein content and association with LDs. METHODS: Male Wistar rats were pair-fed Lieber-DeCarli diets for 5 to 8 weeks. Whole liver and isolated LD fractions were analyzed. Identification of LDs and associated Rab proteins was performed in frozen liver or paraffin-embedded sections followed by immunohistochemical analysis. RESULTS: Lipid accumulation was characterized by larger LD vacuoles and increased total triglyceride content in EtOH-fed rats. Rabs 1, 2, 3d, 5, 7, and 18 were analyzed in postnuclear supernatant (PNS) as well as LDs. All of the Rabs were found in the PNS, and Rabs 1, 2, 5, and 7 did not show alcohol-altered content, while Rab 3d content was reduced by over 80%, and Rab 18 also showed EtOH-induced reduction in content. Rab 3d was not found to associate with LDs, while all other Rabs were found in the LD fractions, and several showed an EtOH-related decrease (Rabs 2, 5, 7, 18). Immunohistochemical analysis revealed the enhanced content of a LD-associated protein, perilipin 2 (PLIN2) that was paralleled with an associated decrease of Rab 18 in EtOH-fed rat sections. CONCLUSIONS: Chronic EtOH feeding was associated with increased PLIN2 and altered Rab GTPase content in enriched LD fractions. Although mechanisms driving these changes are not established, further studies on intracellular protein trafficking and LD biology after alcohol administration will likely contribute to our understanding of fatty liver disease.

Relaxin decreases the severity of established hepatic fibrosis in mice.

BACKGROUND & AIMS: Hepatic fibrosis is characterized by excess collagen deposition, decreased extracellular matrix degradation and activation of the hepatic stellate cells. The hormone relaxin has shown promise in the treatment of fibrosis in a number of tissues, but the effect of relaxin on established hepatic fibrosis is unknown. The aim of this study was to determine the effect of relaxin on an in vivo model after establishing hepatic fibrosis METHODS: Male mice were made fibrotic by carbon tetrachloride treatment for 4 weeks, followed by treatment with two doses of relaxin (25 or 75 µg/kg/day) or vehicle for 4 weeks, with continued administration of carbon tetrachloride. RESULTS: Relaxin significantly decreased total hepatic collagen and smooth muscle actin content at both doses, and suppressed collagen I expression at the higher dose. Relaxin increased the expression of the matrix metalloproteinases MMP13 and MMP3, decreased the expression of MMP2 and tissue inhibitor of metalloproteinase 2 (TIMP2) and increased the overall level of collagen-degrading activity. Relaxin decreased TGFß-induced Smad2 nuclear localization in mouse hepatic stellate cells. CONCLUSIONS: The results suggest that relaxin reduced collagen deposition and HSC activation in established hepatic fibrosis despite the presence of continued hepatic insult. This reduced fibrosis was associated with increased expression of the fibrillar collagen-degrading enzyme MMP13, decreased expression of TIMP2, and enhanced collagen-degrading activity, and impaired TGFß signalling, consistent with relaxin's effects on activated fibroblastic cells. The results suggest that relaxin may be an effective treatment for the treatment of established hepatic fibrosis.