Effects of Monensin on Ethanol-Induced Alterations in Function of Hepatocellular Asialoglycoprotein Receptor Subpopulations

Chronic ethanol consumption is associated with multiple impairments in receptor-mediated endocytosis (RME) in hepatocytes. RME mediated by the asialoglycoprotein receptor seems to be especially impaired by ethanol. In the present study, we determined susceptibility of RME to alterations in ethanol-fed and pair-fed control animals by the addition of a carboxylic ionophore, monensin. Monensin inhibits acidification of prelysosomal vesicular compartments, which results in a decrease in the rate of receptor-ligand dissociation within the cell. Low levels (25 μM) of monensin have been shown to preferentially affect receptor-ligand dissociation of one subset (state 2) of asialoglycoprotein receptor, whereas dissociation in a second subset (state 1 receptors) seems to be relatively unaffected. We examined whether ethanol treatment preferentially affected one or another of these receptor subpopulations. Male Wistar rats were fed a standard ethanol-containing (36% of calories) or control diet for 10 to 14 weeks, and hepatocytes were prepared from the animals. Similar to previous results from our laboratory, surface and total ligand and antibody binding were decreased by 30 to 45% (p < 0.01) in ethanol animals, compared with controls. An ethanol-induced impairment of receptor-ligand dissociation was also apparent in these cells, as shown by increased ratios of bound-to-free ligand during continuous endocytosis. After monensin treatment, surface receptors on both ethanol and control cells showed a similar pattern of redistribution to the cell interior and intracellular inactivation. When kinetics of intracellular receptor-ligand dissociation were examined upon addition of monensin, the bound-to-free ligand ratios in both control and ethanol cells increased dramatically and to an equal extent. These results indicate that, in the ethanol animals, the pattern of sensitivity to monensin is unchanged relative to controls. Thus, the relative proportion of state 1 and state 2 receptor populations do not seem to be affected after long-term feeding, and ethanol may be a perturbant that affects both state 1 and state 2 receptor function.     

Equilibrative Adenosine Transport in Rat Hepatocytes after Chronic Ethanol Feeding

Acute treatment of cells with ethanol in vitro inhibits adenosine uptake via equilibrative nucleoside transporters. After longer periods of exposure to ethanol in culture, rechallenge with ethanol no longer inhibits adenosine uptake. Herein, we have investigated the long-term effects of ethanol consumption in vivo on equilibrative nucleoside transport. Rats were fed a liquid diet containing 35% of calories as ethanol (ethanol-fed). Control rats were pair-fed a liquid diet that isocalorically substituted maltose dextrins for ethanol. After 4 weeks of ethanol consumption, nucleoside transport was measured in isolated hepatocytes. Uptake of [³H]adenosine was lower in ethanol-fed rats compared with control. Influx of the nonmetabolizable nucleoside analog, [³H]formycin B, was also decreased after ethanol feeding. However, neither the number of nitrobenzylthioinosine (NBMPR) binding sites or inhibition of adenosine uptake by NBMPR were affected by ethanol feeding. In controls, acute treatment of isolated hepatocytes with 100 itim ethanol inhibited [³H]adenosine uptake by 30–40%. However, in ethanol-fed rats, acute challenge with ethanol did not inhibit [³H]adenosine uptake. These data demonstrate that long-term ethanol feeding decreases equilibrative nucleoside transport in hepatocytes independent of a change in the number of nucleoside transporters and renders adenosine uptake insensitive to inhibition by ethanol.     

Ethanol Feeding Selectively Impairs the Spreading of Rat Perivenous Hepatocytes on Extracellular Matrix Substrates

BACKGROUND: Hepatocytes require attachment and subsequent spreading on an extracellular matrix for their proper growth, function and survival. Our previous studies have shown that ethanol feeding selectively impairs perivenule hepatocyte attachment to various extracellular matrices. This study was undertaken to determine whether zonal differences in hepatocyte spreading in response to ethanol feeding occurs and to ascertain the influence of ethanol consumption on the zonal expression of the beta1 subunit of integrins, which are the major surface receptors responsible for matrix binding and subsequent interactions. METHODS: Hepatocytes from the perivenous and periportal regions of the liver were isolated by digitonin/collagenase perfusion from rats that were pair-fed for 2 to 3 weeks with a liquid diet containing either ethanol or isocaloric carbohydrate. The ability of perivenous and periportal hepatocytes to spread on plates coated with either type IV collagen, laminin, fibronectin or polylysine was determined. In addition, the isolated cells were used for the analysis of total cellular and surface beta1 integrin expression. RESULTS: With all of the matrix substrates tested, the spreading of perivenous hepatocytes isolated from the ethanol-fed animals was markedly impaired, while the spreading of periportal hepatocytes was essentially unaffected by ethanol feeding. Both the total cellular as well as the surface expression of the beta1 integrin subunit in perivenous cells from the ethanol-fed rats were significantly higher than from the perivenous control cells, whereas the total and surface expression of the beta1 integrin in periportal cells isolated from ethanol-fed and control rats were not significantly different. CONCLUSIONS: The results indicated that in addition to impairing hepatocyte attachment, ethanol feeding also impairs another critical step of the adhesion process, that of hepatocyte spreading on extracellular matrix substrates. This defect occurred preferentially in perivenous cells and not periportal cells and was associated with an increase in beta1 integrin expression, suggesting that a compensatory mechanism occurs as an attempt by the perivenous cells to overcome impaired cell-matrix interactions caused by ethanol. Overall, these alterations in extracellular matrix-hepatocyte interactions could lead to alterations of hepatocyte structure and function and potentially play a role in alcoholic liver injury.     

Differential inhibition of epidermal growth factor signaling pathways in rat hepatocytes by long-term ethanol treatment

BACKGROUND & AIMS: Long-term ethanol intake suppresses liver regeneration in vivo and ethanol interferes with epidermal growth factor (EGF)-induced DNA synthesis in vitro. Therefore, the effects of long-term ethanol treatment on EGF-activated signaling reactions in rat hepatocytes were investigated. METHODS: Hepatocytes from long-term ethanol-fed rats and pair-fed controls were stimulated with EGF (0.5-20 nmol/L) for 15-120 seconds. Tyrosine phosphorylation of EGF receptor (EGFR), Shc, and phospholipase-C gamma1 (PLC gamma), and growth factor receptor binding protein 2 (Grb2) coprecipitation with EGFR and Shc were analyzed by Western blotting. RESULTS: EGFR autophosphorylation was suppressed at all EGF concentrations in ethanol-fed cells compared with pair-fed cells, without significant differences in total EGFR protein or EGFR tyrosine kinase activity detected in cell lysates, suggesting that intracellular factors suppressed EGFR function. EGF- induced PLC gamma tyrosine phosphorylation and inositol 1,4,5- trisphosphate (InsP3) formation were suppressed, but cytosolic [Ca2+]c elevation was little affected, indicating enhanced InsP3-mediated intracellular Ca2+ release in ethanol-fed cells. Grb2 binding to EGFR was suppressed, but EGF-induced Shc tyrosine phosphorylation and Grb2 association with Shc were not significantly decreased. CONCLUSIONS: Long-term ethanol feeding suppressed EGF-induced receptor autophosphorylation in rat hepatocytes with differential inhibition of downstream signaling processes mediated by PLC gamma, Shc, and Grb2. Altered patterns of downstream signals emanating from EGFR may contribute to deficient liver regeneration in chronic alcoholism. (Gastroenterology 1997 Jun;112(6):2073-88)     

Zonal Differences in Ethanol-Induced Impairments in Fluid-Phase Endocytosis in Rat Hepatocytes

We have previously shown that ethanol administration impairs the processes of fluid-phase endocytosis (FPE) and receptor-mediated endocytosis (RME) in isolated rat hepatocytes after as early as 1 week of ethanol administration. The defects in RME were most prominent in the perivenule (PV) region of the liver lobule, the area wherein alcoholic liver disease has been shown to start and predominate. We undertook the present study to see if changes in FPE were likewise more apparent in the PV versus the periportal (PP) region of the liver. For these studies, we fed male Sprague-Dawley rats with an ethanol-supplemented liquid diet or an isocaloric control diet for 1 or 5 weeks. PV and PP hepatocytes were isolated using a digitonin-collagenase perfusion method. Internalization and efflux of the marker dye, Lucifer Yellow, was then examined in the cell populations. After as early as 1 week of feeding, cells from the PV region in ethanol-fed animals showed dramatic impairments in the net internalization of dye, compared with PV cells from the pair-fed controls, and these changes persisted throughout the 5-week feeding period. In contrast, internalization of Lucifer Yellow in cells from the PP region of the liver were not different between control and ethanol animals. Because net internalization represents the balance between uptake into the cells versus efflux from the cells, we examined these components individually. Early uptake of the dye into the cell was not altered by ethanol treatment. The decreased net internalization seemed to be caused by enhanced efflux of the dye, which was significantly increased in PV cells, compared with the same cell type in control animals. Cells from the PP region of the ethanol-fed animals did not exhibit altered efflux after either 1 or 5 weeks of feeding. These results indicate that ethanol-induced impairments in FPE are more dramatic in the PV region of the liver, and these impairments seem to result from an ethanol-induced enhancement of efflux.     

Relationship of Alcoholic Hyperlipidemia to the Feed-Back Regulation of Hepatic Cholesterol Synthesis by Chylomicron Remnants

Pairfeeding of ethanol liquid diet for 6 weeks to male Wistar rats resulted in defective extrahepatic as well as hepatic catabolism of rat lymph chylomicrons. Based upon the exponential decay curves of the chylomicrons in the blood compartment it was concluded that chronic ethanol feeding caused 30 and 67% decreases in the rate of degradation of the triacylglycerol and cholesterol moieties, respectively. As a consequence, abnormal chylomicron remnants accumulated in chronic ethanol-fed but not in control animals. These abnormal remnants were not as efficient as the normal remnants in causing the feedback inhibition of cholesterol synthesis in hepatocytes from normal meal-fed rats. Furthermore, the hepatocytes from chronic ethanol-fed animals exhibited defective feedback regulation of cholesterol synthesis by normal chylomicron remnants. The net result of all these abnormalities caused by chronic ethanol feeding would be the delayed clearance of triacylglycerol-rich lipoproteins from the blood compartment and enhanced synthesis and secretion of the triacylglycerol-rich lipoproteins by the liver.     

Effects of acute and chronic ethanol administration on the gastrointestinal hormones gastrin, enteroglucagon, pancreatic glucagon and peptide YY in the rat

The effect of acute and chronic ethanol administration on the gastrointestinal hormones gastrin, enteroglucagon (EG), pancreatic glucagon (PG) and peptide YY (PYY) was studied in the rat alcohol model. Plasma levels of gastrin and PYY were not significantly changed under chronic and/or acute alcohol, while PG was stimulated by acute intraperitoneal ethanol injections in control animals as well as in chronically ethanol-fed rats (8 +/- 1 vs. 28 +/- 6 pmol/l, p less than or equal to 0.05, and 7 +/- 1 vs. 21 +/- 4 pmol/l, p less than or equal to 0.05). EG levels were significantly raised after chronic ethanol feeding (45 +/- 5 vs. 73 +/- 8 pmol/l, p less than or equal to 0.01) and even further elevated if an acute dose of alcohol was given to chronically ethanol-fed rats (73 +/- 8 vs. 168 +/- 29 pmol/l, p less than or equal to 0.05). The immunohistologically evaluated numbers of the respective hormone-producing cells were not significantly changed by alcohol feeding. The ethanol-dependent elevations of EG and PG may contribute, at least in part, to the intestinal hyper-regeneration, motility disturbances and altered glucose metabolism observed after alcohol consumption.     

Separate analysis of asialoglycoprotein receptors in the right and left hepatic lobes using Tc-GSA SPECT

The asialoglycoprotein receptor (ASGPR) is abundantly expressed on the sinusoidal surfaces of hepatocytes. We aimed to clarify the clinical significance of the regional distribution of ASGPRs in the human liver, especially in chronic viral hepatitis. Eighteen volunteers, 34 patients with chronic hepatitis, and 33 patients with cirrhosis (11/Child-Pugh A, 11/Child-Pugh B, 11/Child-Pugh C) were studied using a newly developed, conventional technetium-99m-diethylenetriaminepentaacetic acid-galactosyl human serum albumin ((99m)Tc-GSA), single photon emission computed tomography (SPECT) method. Using Cantlie's line as a guide, ASGPR dynamics were analyzed separately in the right and left lobes, as well as in the whole liver, using novel indices (the liver uptake ratio [LUR] and the liver uptake density [LUD], which reflect the amount and density of ASGPRs in the liver, respectively). Mean LUR and LUD values for the whole liver and the right and left lobes decreased with increasing progression of chronic viral hepatitis. The LUR for the whole liver correlated well with parameters measuring the hepatic functional reserve and the platelet count. The right LUR correlated particularly well with conventional liver function tests, and comparison of the right LUD with histologic findings showed that it was a good indicator of periportal and/or bridging necrosis and fibrosis. In conclusion, our (99m)Tc-GSA SPECT method was clinically useful in evaluating regional hepatic function and the progression of chronic viral hepatitis using dynamic changes in ASGPRs.     

Separate analysis of asialoglycoprotein receptors in the right and left hepatic lobes using (99m)Tc-GSA SPECT in patients with acute hepatic damage.

The asialoglycoprotein receptor (ASGPR) is abundantly expressed on the sinusoidal surfaces of hepatocytes. However, regional expression and clinical significance of the ASGPR in acute hepatic damage is presently unknown. Our aim was to clarify the clinical significance of the regional expression of ASGPR in human livers with acute hepatitis (AH) and fulminant hepatic failure (FHF). Eighteen volunteers, 42 patients with AH and 10 with FHF were studied using a newly developed, conventional (99m)Tc-GSA SPECT analysis. Using Cantlie's line as a guide, ASGPR expression was analyzed separately in the right and left hepatic lobes, as well as in the whole liver, using novel indices (the liver uptake ratio [LUR] and liver uptake density [LUD], which reflect the amount and density of ASGPRs in the liver, respectively). Mean LUR and LUD values for the whole liver and the right and left lobes decreased in accordance with the severity of acute hepatic damage. In the FHF group, the reduction in LUR and LUD values in the right lobes was more significant than in the left lobes. The LUR and LUD values for the whole liver correlated well with hepatic functional reserve and total bilirubin levels. The right LUR and LUD values in particular correlated well with these parameters. A time-course observation of 13 patients with either AH or FHF revealed that the expression of ASGPRs in the right lobe recovered faster than in the left. We first evaluated the regional expression of AGSPRs by (99m)Tc-GSA SPECT analysis in both AH and FHF patients, which is a clinically useful and reliable indicator for assessing the severity of regional hepatic damage and evaluating regional liver regeneration.     

Chronic Ethanol-Induced Impairments in Receptor-Mediated Endocytosis of Insulin in Rat Hepatocytes

The effects of chronic ethanol administration on the receptor-mediated endocytosis of insulin were investigated in isolated hepatocytes. When hepatocytes were isolated from rats that were fed an ethanol liquid diet for 5-6 weeks, these cells bound 25% less insulin to their surface membrane than did cells from the chow-fed or pair-fed controls. This decreased binding was likely a result of reduced surface receptor number rather than changes in receptor affinity. Rates of insulin degradation were also reduced by 25-30% in hepatocytes from the ethanol-fed animals. In addition, chronic ethanol feeding induced a defect in the internalization of the receptor-insulin complex and altered the hepatocellular processing of the internalized insulin. These results indicate that chronic ethanol administration impairs both the surface binding and the endocytosis of insulin by the liver.     

Zonal heterogeneity of the effects of chronic ethanol feeding on hepatic fatty acid metabolism

Periportal and perivenous hepatocytes were isolated from rats fed a high-fat, ethanol-containing diet to investigate the acinar heterogeneity of the effects of prolonged ethanol administration on lipid metabolism. Chronic feeding of ethanol caused a rather selective accumulation of triacylglycerols in the perivenous zone of the liver. In control animals the rate of lipogenesis and the activity of acetyl-CoA carboxylase were higher in perivenous than in periportal hepatocytes, whereas the rate of fatty acid oxidation and the activity of carnitine palmitoyltransferase I were higher in periportal than in perivenous cells; however, no zonation was evident for very-low-density-lipoprotein-lipid secretion. Prolonged ethanol administration abolished the zonal asymmetry of the lipogenic process and inverted the acinar distribution of the fatty acid-oxidative process (i.e., in ethanol-fed animals the rate of fatty acid oxidation and the activity of carnitine palmitoyltransferase I were higher in perivenous than in periportal hepatocytes). Moreover, chronic feeding of ethanol led to a marked and selective inhibition of very-low-density-lipoprotein-triacylglycerol secretion by the perivenous zone of the liver. Nevertheless, no zonal differences were observed between control and ethanol-fed animals with respect to the effects of acute doses of ethanol and acetaldehyde on lipid metabolism. In conclusion, our results show that chronic ethanol intake produces important alterations in the acinar distribution of the different fatty acid-metabolizing pathways.     

Hepatic Adenosine in Rats Fed Ethanol: Effect of Acute Hyperoxia or Hypoxia

The level of adenosine was measured in monthly biopsied livers from rats fed ethanol and a high fat/low protein diet in order to test a hypothesis that hepatic adenosine is increased due to enhanced breakdown of adenine nucleotides in which ATP and total adenylate pool were decreased by chronic ethanol feeding. The ethanol-fed rats showed a significantly higher average level of adenosine compared to the pair-fed controls. When investigated monthly, however, adenosine in ethanol-fed rats increased only after the decrease in ATP had stabilized and AMP remained unchanged, indicating that these changes were not temporarily related. The average percentage of change in adenosine after acute hyperoxia or hypoxia were variable both in ethanol-fed and pair-fed rats. There was a tendency for a positive correlation between the percentage of change of adenosine and AMP after hyperoxia regardless of ethanol feeding. A negative correlation between the percentage of change of adenosine and energy charge, and a positive correlation between the percentage of change of adenosine and AMP were seen after hypoxia regardless of ethanol feeding. Adenosine levels changed rapidly in response to changes in systemic of pO2 in both the ethanol-fed and control rats, indicating that the liver maintained its normal response to the changes in energy state. The results indicate that chronic ethanol feeding does increase the level of adenosine in the liver and that this level remains responsive to acute changes in pO2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ethanol potentiates tumor necrosis factor-alpha cytotoxicity in hepatoma cells and primary rat hepatocytes by promoting induction of the mitochondrial permeability transition

In the present study, tumor necrosis factor-alpha (TNF-alpha) cytotoxicity is shown to be potentiated by ethanol exposure in vitro in the human hepatoma cell line, HepG2, and in rat primary hepatocytes. Exposure of HepG2 cells and primary hepatocytes for 48 hours to concentrations of ethanol ranging between 50 and 100 mmol/L significantly increased TNF-alpha cytotoxicity compared with cells treated with TNF-alpha alone. The cell killing was associated with, and dependent on, the development of the mitochondrial permeability transition (MPT). Two inhibitors of MPT pore opening, cyclosporin A and bongkrekic acid, prevented TNF-alpha cytotoxicity in the presence of ethanol. In addition to inhibiting cell death caused by TNF-alpha, blockade of MPT pore opening prevented mitochondrial depolarization, cytochrome c redistribution from the mitochondria to the cytosol, caspase 3 activation, and oligonucleosomal DNA fragmentation. Unlike the potentiation of TNF-alpha cytotoxicity by the translational inhibitor cycloheximide, ethanol promoted TNF-alpha-induced cell killing by a mechanism that was independent of caspase-8 activity. HepG2 cells overexpressing cytochrome-P4502E1 were even more sensitized by ethanol to induction of the MPT by TNF-alpha and the resultant cytotoxicity than wild-type HepG2 cells. In addition, primary hepatocytes isolated from chronically ethanol-fed rats showed enhanced susceptibility to TNF-alpha cytotoxicity compared with their isocalorically matched controls. Again as with the HepG2 cells, inhibiting MPT pore opening prevented the cytotoxicity of TNF-alpha in the primary hepatocytes isolated from ethanol-fed animals.     

Centrilobular liver necrosis induced by hypoxia in chronic ethanol-fed rats

Rats fed ethanol from 21 to 130 days were subjected to one or more episodes of hypoxia (6% O2) in order to determine if ethanol predisposed to centrilobular liver necrosis induced by hypoxia. Pair-fed control rats were fed the diet regimen in parallel with the ethanol-fed rats through an indwelling gastric cannula. The diet and ethanol were fed continuously 24 hr per day so as to maintain high blood alcohol levels in the ethanol-fed rats. Serum enzyme levels, SGOT and SGPT were measured before and after the hypoxic episodes as an indicator of centrilobular necrosis. Animal livers were studied for centrilobular necrosis by light and electron microscopy. Necrosis was documented to be present when flocculent densities were found in hepatocytic mitochondria or the plasma membrane permitted lanthanum entrance into the cell. The results showed that ethanol feeding to maintain high blood alcohol levels did increase the propensity of the liver to undergo centrilobular necrosis when the rats were subjected to hypoxia (1 hr 45 min to 5 hr 30 min). Centrilobular necrosis was observed in the ethanol-fed rats only. Serum enzyme levels (SGPT and SGOT) rose to very high levels in these rats when they were permitted to die of hypoxia. Serum sediment from the ethanol-fed rats contained numerous cell fragments and free organelles. Since the plasma membranes were missing along the sinusoidal face of centrilobular hepatocytes and microbodies were present, it was concluded that the cell fragments in the blood had originated from necrotic hepatocytes.     

Effect of Dietary Fat on Chronic Ethanol-Induced Oxidative Stress in Hepatocytes

BACKGROUND: Although oxidative stress and deficits in hepatic energy metabolism have been implicated as important factors in the initiation of alcoholic liver disease, their relative contribution to ethanol-induced cell death is not known. The purpose of this study was to examine the effects of chronic ethanol administration on hepatocyte reactive oxygen species (ROS) generation, energy state, and viability, as well as the effect of dietary fat on these parameters. METHODS: Male Sprague-Dawley rats were fed liquid diets that provided 36% total calories as ethanol, with fat as either 12% (low fat) or 35% (high fat) of total calories. Pair-fed controls received liquid diets in which maltose-dextrin was substituted for ethanol calories. The fluorescent probe 2',7'-dichlorofluorescin diacetate was used to detect ROS, lactate dehydrogenase leakage was used to assess viability, and ATP levels were used as a measure of the energy state. The effect of chronic ethanol feeding on these parameters was determined by incubating hepatocytes under a 5% oxygen-containing atmosphere or an atmosphere < or = 1% oxygen for 60 min. RESULTS: In general, chronic ethanol feeding stimulated ROS production and decreased ATP concentrations, which were associated with decreased viability in hepatocytes isolated from rats fed either high- or low-fat, ethanol-containing diets, compared to the corresponding controls. Incubation under an atmosphere < or = 1% oxygen and/or ethanol (10 mM) augmented these effects in both high- and low-fat control and ethanol-fed hepatocytes. The addition of antimycin to the incubations increased ROS production, decreased ATP concentrations, and accelerated loss of hepatocyte viability. Viability loss under all conditions used in this study was correlated with decreases in cellular ATP. CONCLUSIONS: Comparisons of incubations performed under the two oxygenation conditions revealed that viability loss was inversely associated with ROS production, which indicates that ATP loss and not ROS production was a better predictor of loss in cell integrity. This study also demonstrates that the level of dietary fat has only minor effects on generation of ROS and the cellular energy state. In contrast, ethanol consumption had significant effects on generation of ROS, energy state, and hepatocyte viability.     

Feeding S-adenosyl-l-methionine attenuates both ethanol-induced depletion of mitochondrial glutathione and mitochondrial dysfunction in periportal and perivenous rat hepatocytes

Mitochondrial glutathione plays an important role in maintaining a functionally competent organelle. Previous studies have shown that ethanol feeding selectively depletes the mitochondrial glutathione pool, more predominantly in mitochondria from perivenous hepatocytes. Because S-adenosyl-l-methionine (SAM) is a glutathione precursor and maintains the structure and function of biological membranes, the purpose of the present study was to determine the effects of SAM on glutathione and function of perivenous (PV) and peri-portal (PP) mitochondria from chronic ethanol-fed rats. SAM administration resulted in a significant increase in the basal cytosol and mitochondrial glutathione in both PP and PV cells from both pair-fed or ethanol-fed groups. When hepatocytes from ethanol-fed rats supplemented with SAM were incubated with methionine plus serine or N-acetylcysteine, mitochondrial glutathione increased in parallel with cytosol, an effect not observed in cells from ethanol-fed rats without SAM. Feeding equimolar N-acetylcysteine raised cytosol glutathione but did not prevent the mitochondrial glutathione defect. In addition, SAM feeding resulted in significant preservation of cellular adenosine triphosphate (ATP) levels (23% to 43%), mitochondrial membrane potential (17% to 25%), and the uncoupler control ratio (UCR) of respiration (from 5.1 ± 0.7 to 7.3 ± 0.6 and 2.1 ± 0.3 to 6.1 ± 0.7) for PP and PV mitochondria, respectively. Thus, these effects of SAM suggest that it may be a useful agent to preserve the disturbed mitochondrial integrity in liver disease caused by alcoholism through maintenance of mitochondrial glutathione transport.     

Serum high-density lipoprotein particles of alcohol-fed rats are deficient in apolipoprotein E

Effects of chronic ethanol consumption on serum lipoproteins have been studied in the rat. The serum levels of triglycerides, cholesterol, phospholipids and apolipoproteins AI and AIV increased significantly after 1 week of ethanol feeding, and they remained elevated up to 7 weeks of alcohol drinking. By contrast, serum total apolipoprotein E decreased or, sometimes, did not change. Very-low-density lipoprotein cholesterol, triglycerides and very-low-density lipoprotein apolipoprotein E of the alcohol-fed rats increased in parallel and were about 2- to 2.5-fold over the controls. Whereas high-density lipoprotein cholesterol, phospholipids, apolipoprotein AI and AIV increased 1.2-fold by chronic alcohol feeding, the level of high-density lipoprotein apolipoprotein E decreased to 70% of that of the control rats. The rates of secretion of apolipoprotein AI, E and AIV into the culture medium by hepatocytes isolated from ethanol-fed rats were 1.8-, 1.3- and 1.1-fold higher than those from control rats. These data indicate that (i) chronic ethanol feeding increases very-low-density lipoprotein and high-density lipoprotein in the rat; (ii) serum high-density lipoprotein particles of the ethanol-fed rats are deficient in apolipoprotein E, and (iii) chronic ethanol feeding increases hepatic secretion of apolipoprotein AI, E and AIV. Since the steady-state serum level of apolipoprotein E decreases or remains unchanged in the presence of increased hepatic apolipoprotein E secretion, this imbalance suggests that alcohol feeding either accelerates the rate of degradation of serum apolipoprotein E or suppresses apolipoprotein E synthesis by nonhepatic tissues.     

Zonal differences in ethanol-induced impairments in receptor-mediated endocytosis of asialoglycoproteins in isolated rat hepatocytes

We have shown previously that ethanol-induced defects in receptor-mediated endocytosis of asialoorosomucoid occurred as early as 1 wk after ethanol feeding. This study was undertaken as an initial attempt to establish a possible role of defective receptor-mediated endocytosis in liver injury by investigating whether differences exist in the effects of ethanol on receptor-mediated endocytosis in hepatocytes isolated from different regions of the liver. Perivenule cells, present in the distal half of the liver, are thought to be more susceptible to ethanol-induced liver injury than are the periportal cells located in the proximal half of the liver acini. For these studies, we fed male Sprague-Dawley rats for 7 days with liquid diets containing either ethanol (36% of calories) or isocaloric carbohydrate. Perivenule and periportal hepatocytes were then isolated using a digitonin-collagenase perfusion method. In control animals, cells isolated from the perivenule region bound significantly more ligand than did cells from the periportal region. Amounts of ligand internalized and degraded were also greater in perivenule than in periportal cells in these animals. After ethanol feeding, cells isolated from both the perivenule and periportal regions bound significantly less ligand than their respective controls. This impairment in surface and total binding was more pronounced in perivenule than in periportal cells. Internalization and degradation of the ligand were also more adversely affected in the centrilobular region as shown by decreases of greater than 60% in perivenule cells and by only 20% to 30% in periportal cells of ethanol-fed animals compared with controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Chronic Ethanol Feeding on Kupffer Cell-Mediated Antitumor Cell Activity

We have previously reported that the Kupffer cell has antitumor activity through mitochondrial damage to tumor cells by nitric oxide production. In this study, the effect of chronic ethanol feeding on antihepatoma cell activity of the Kupffer cell was examined in rats. Male rats of the Wistar strain were fed ethanol chronically for 8 weeks by liquid diets. Kupffer cells were isolated from the control rat or the ethanol-fed rat, and cocultured with AH 70 cells, a rat hepatoma cell line. Fluorescence of rhodamine 123 or propidium iodide was observed as indicators of the mitochondrial damage or cell membrane injury, respectively, by a laser scanning confocal microscopy. Mitochondrial damage of AH 70 cells as indicated by reduction of rhodamine 123 fluorescence was smaller by the coculture with Kupffer cell from the ethanol rat than that from the control. Cell membrane barrier dysfunction of AH 70 cell was less frequently observed with the Kupffer cell from ethanol-fed rats. A metabolite of nitric oxide (nitrite and nitrate) was less in the cultured medium with the ethanol Kupffer cell than with the control Kupffer cell. Ca²⁺ mobilization, which induces inducible nitric oxide synthase and observed by the fluorescence of fluo-3, in Kupffer cells cocultured with AH 70 cells was suppressed in ethanol-fed rats. These result suggests that chronic ethanol feeding suppresses antitumor cell activity of Kupffer cell through the impairment of Ca²⁺ mobilization and nitric oxide production.