Lipopolysaccharide tolerance and ethanol modulate hepatic nitric oxide production in a gender-dependent manner.

This study was directed at the role of tolerance to endotoxin (lipopolysaccharide, LPS) and ethanol (EtOH) intoxication in modulating hepatic nitric oxide (NO) production, and the demonstration of gender differences. Previous studies demonstrated that tolerance to either LPS or EtOH was associated with reduced hepatic production of superoxide anions. We now tested the hypothesis that the reduced hepatic production of superoxide anions during tolerance to LPS and the altered response to EtOH are accompanied by increased sensitivity of hepatic NO release to stimulation. Age-matched male and female Sprague-Dawley rats were made tolerant to LPS by an i.v. injection of LPS (0.5 or 0.45 mg/kg) 2 days prior to an in vivo EtOH infusion for 3 h (LPS-EtOH group). Control groups were saline-pretreated, saline-infused; saline-pretreated, EtOH-infused; and LPS-pretreated, saline-infused. At the end of the infusion, isolated hepatocytes, Kupffer, and sinusoidal endothelial cells were cultured for 20 h for subsequent measurement of basal (spontaneous) and in vitro-stimulated nitrite release. LPS-tolerance resulted in significantly enhanced stimulated NO production by hepatocytes and Kupffer cells in both male and female rats. EtOH abolished this priming effect in hepatocytes from male, but not from female rats. The priming effect was markedly diminished by EtOH in Kupffer cells of female rats only. LPS-tolerance increased NO production by stimulated endothelial cells of males, and decreased NO production by cells of females. EtOH infusion did not influence NO production by endothelial cells from male rats and it reversed the LPS-tolerance-induced inhibition in females. These data demonstrate that modulation by LPS-tolerance of hepatic NO release in EtOH-treated rats leads to enhanced stimulated NO production, while hepatic superoxide anion release was previously shown to be reduced within the same time frame. Since NO is able to scavenge superoxide, the LPS-tolerance-induced alterations in the EtOH effects on NO production may have a potential significance in modulating - in a time-dependent manner - oxidative injury associated with LPS and acute EtOH intake.

Differential regulation of inducible nitric oxide synthase gene expression by ethanol in the human intestinal epithelial cell line DLD-1.

We have examined the regulation of inducible nitric oxide synthase (iNOS) gene expression by ethanol in monolayers of DLD-1 cells, an epithelial cell line derived from human intestinal adenocarcinoma. Optimum induction of iNOS mRNA in these cells was obtained with IFN-gamma and IL-1beta treatment, while further addition of TNF-alpha did not have significant effect. In a set of experiments to study ethanol effects, DLD-1 monolayers were pretreated with ethanol for 24 h and were then treated with IFN-gamma + IL-1beta for an additional 24 h. Cells pretreated with ethanol showed decreased iNOS mRNA levels, indicating that ethanol may inhibit cytokine-induced iNOS transcription or affect mRNA destabilization. The suppression was ethanol-dose dependent with an IC50 of 50 mM. In another set of experiments to study ethanol effects, DLD-1 monolayers were pretreated with 66 mM ethanol for 24 h. These cells showed significant upregulation of IL-1beta mRNA and protein as detected in the supernatants. Aliquoted supernatants from these cells (i.e., conditioned media) were added to naive DLD-1 monolayers together with IFN-gamma. Conditioned medium from ethanol-treated cells increased the IFN-gamma-induced iNOS mRNA of naive cells by threefold. Two different effects of ethanol are now reported: (a) ethanol inhibits IFN-gamma + IL-1beta-induced iNOS mRNA of the same DLD-1 cells and (b) ethanol induces cellular paracrine signals by releasing IL-1beta into the medium, which in combination with IFN-gamma increases iNOS mRNA levels of the recipient naive DLD-1 cells. Because IFN-gamma and IL-1beta are produced by intestinal immune cells, these findings may have implications for differential in vivo regulation of epithelial iNOS genes by ethanol, depending on the inflammatory and immune status of the host.

Role of Kupffer cells in the ethanol-induced oxidative stress in the liver.

These studies test the hypothesis that acute and chronic alcohol intoxication stimulate the release of oxygen-derived radicals in the liver. Male Sprague-Dawley rats received an intravenous bolus followed by continuous infusion of ethanol to maintain blood alcohol level at about 175 mg/dl for 0-18 hr. They were then allowed to recover from this "alcohol binge" and the release of free radicals during the recovery phase was monitored. In the chronic alcohol intoxication model, rats were fed with 40% ethanol in agar blocks for 16 weeks. Acute ethanol intoxication induced two phases of hepatic superoxide release. The first phase peaked during the first 3 hr of alcohol intoxication, while the second phase reached its maximum at 6 hr of recovery following a 12 hr binge. The recovery period was also associated with elevated serum transaminase activity. Kupffer cells were largely responsible for hepatic superoxide release during the first phase, while both Kupffer and hepatic sinusoidal endothelial cells contributed to the second phase of free radical formation. Acute ethanol intoxication did not induce endotoxemia. During chronic alcohol intoxication, increased levels of serum endotoxin, TNF, IL-1, and transaminase were observed and hepatic superoxide anion release was present. Superoxide release by isolated Kupffer cells, blood and hepatic PMNs of alcoholic rats was also significantly enhanced in the chronic alcoholic rats. These data indicate that acute alcohol intoxication may directly stimulate the release of reactive oxygen intermediates, whereas chronic alcohol may elicit free radical generation through enhanced endotoxin influx and cytokine release. These studies further demonstrate that free radicals produced by hepatic non-parenchymal cells are likely to play an important role in the pathogenesis of hepatic injury in susceptible individuals with alcohol-related liver disorders.

In vivo endotoxin enhances biliary ethanol-dependent free radical generation.

Endotoxemia is associated with alcoholic liver diseases; however, the effect of endotoxin on the oxidation of ethanol is not known. We tested the hypothesis that endotoxin treatment enhances hepatic ethanol radical production. The generation of free radicals by the liver was studied with spin-trapping technique utilizing the primary trap ethanol (0.8 g/kg) and the secondary trap alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone (4-POBN; 500 mg/kg). Electron paramagnetic resonance (EPR) spectra of bile showed six-line signals, which were dependent on ethanol, indicating the trapping of ethanol-dependent radicals. Intravenous injections of Escherichia coli lipopolysaccharide (0.5 mg/kg) 0.5 h before 4-POBN plus ethanol treatment caused threefold increases of biliary radical adducts. EPR analyses of bile from [1-13C]ethanol-treated endotoxic rats showed the presence of species attributable to alpha-hydroxyethyl adduct, carbon-centered adducts, and ascorbate radical. The generation of endotoxin-induced increases of ethanol-dependent radicals was suppressed by 50% on GdCl3 (20 mg/kg i.v.) or desferrioxamine mesylate (1 g/kg i.p.) treatment. Our data show that in vivo endotoxin increases biliary ethanol-dependent free radical formation and that these processes are modulated by Kupffer cell activation and catalytic metals.

Generation of nitro and superoxide radical anions from 2,4,6-trinitrobenzenesulfonic acid by rat gastrointestinal cells.

Reactive oxygen and nitrogen species have been implicated in the inflammation of the gastrointestinal tract. The objective of this study was to investigate mechanisms of free radical formation from the colitis inducer 2,4,6-trinitrobenzene sulfonic acid (TNBS). We showed that TNBS was rapidly metabolized to TNBS nitro radical anion via metabolic reduction by flavinmononucleotide/NADPH, xanthine/xanthine oxidase as well as the rat small intestine and colon. TNBS nitro radical anion was directly detected with electron paramagnetic resonance (EPR) spectroscopy. EPR spectra of TNBS nitro radical anion showed hyperfine coupling constants from the proximal nitrogen, two hydrogens and the two distal nitrogens with respective magnitudes of a(N)(4) = 9.7 G; a(H)(3,5) = 3.2 G (2); and a(N)(2,6) = 0.25 G. EPR spin trapping using 5.5-dimethyl-1-pyrroline N-oxide in aerobic incubations of isolated enterocytes (or colonocytes, or red blood cells) and TNBS, in the presence or absence of NADPH, produced radical adducts characteristic of superoxide and hydroxyl radicals. Our EPR data showing generation of TNBS nitro and superoxide radical anions demonstrate that one-electron reduction of TNBS may be an initial step in the cascade of the in vivo inflammatory events in TNBS-induced colitis.

Alcohol-induced thymocyte apoptosis is accompanied by impaired mitochondrial function.

This study examines the effects of chronic alcohol consumption on thymic apoptosis with or without pretreatment with E. coli lipopolysaccharide (LPS). Apoptotic cell death of thymocytes was monitored by DNA fragments in gel electrophoresis and the appearance of apoptotic cells by flow cytometry. Changes in mitochondrial membrane potential (MMP), as indicated by 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)] uptake, and hydrogen peroxide (H2O2) production as indicated by oxidation of 2',7'-dichlorofluresin diacetate (DCFH-DA), were used to assess altered mitochondrial function. Glutathione levels were also determined to obtain information concerning alterations in the antioxidant potential in the cells. Male Sprague-Dawley rats, fed a nutritionally adequate liquid diet for 8-9 weeks, were divided in four groups: 1) saline-injected, diet controls; 2) LPS-injected, diet controls; 3) saline-injected, alcohol-consuming; and 4) LPS-injected, alcohol-consuming animals. LPS (0.5 mg/kg in 4 ml saline) or saline (4 ml) was continuously infused i.v. for 12 h before the experiments. The results showed that the weight and cell numbers of thymus from the chronic alcoholic rats were significantly less than values found in diet controls. Administration of LPS aggravated thymic apoptosis, as indicated by the presence of significant DNA fragments in gel electrophoresis and increased rate of apoptotic cells in flow cytometry. The alcohol-induced apoptotic changes were also accompanied by decreased MMP, indicating impaired mitochondrial function. Although H2O2 production by the total thymocyte population did not show marked changes among the experimental groups, the subpopulation of thymocytes exhibiting low H2O2 production was increased markedly in the LPS-treated groups. Ethanol consumption or LPS treatment decreased total glutathione concentration in the thymocytes. In summary, 1) chronic administration of alcohol induces atrophy of the thymus gland; 2) apoptosis is a major factor in thymic atrophy under these conditions; 3) chronic alcohol consumption is accompanied by alterations in mitochondrial function of the thymocytes, as indicated by decreased MMP and an increase in the low H2O2-producing cell subpopulation; 4) chronic alcohol abuse may impair intracellular defense mechanisms as reflected by the depletion of the intracellular antioxidant, glutathione; and 5) administration of LPS further enhances thymic apoptosis in chronic alcohol-consuming rats, suggesting that the dual insults of infection and chronic alcoholism exaggerate in vivo immunosuppression.

Cross-tolerance between acute alcohol intoxication and endotoxemia.

This study tests two hypotheses: (1) prior exposure to LPS induces cross-tolerance for the hepatic effects of subsequent short-term alcohol intoxication; and (2) short-term alcohol intoxication renders the liver resistant to the effects of acute endotoxemia, resulting in reduced production of superoxide and tumor necrosis factor. In the first group of experiments, male Sprague-Dawley rats were treated intravenously with E. coli lipopolysaccharide (LPS) (0.5 mg/kg) 48 hr before they were given an intravenous bolus of ethanol (1.75 g/kg), followed by 250-300 mg/kg/hr) for 3-5 hr. Superoxide release in the perfused liver was measured after 3-hr ethanol infusion. Pretreatment with LPS attenuated ethanol-mediated superoxide anion release by the perfused liver. The stimulatory effect of phorbol myristate acetate on hepatic release of superoxide was also decreased. In the second group of experiments, rats previously treated with ethanol for 5 hr, received an intravenous injection of LPS (1 mg/kg). At 90 min after LPS, sera were collected for tumor necrosis factor alpha assay. Hepatic release of superoxide anion was determined 3 hr after LPS. Acute ethanol intoxication for 5 hr significantly reduced LPS-induced serum tumor necrosis factor activity and free radical release by the perfused liver. LPS-induced mortality was also decreased. In both groups of experiments serum corticosteroid levels were reduced during cross-tolerance. These results demonstrate that cross-tolerance develops between acute alcohol intoxication and endotoxemia manifesting in reduced hepatic production of cytotoxic cytokines and superoxide anions.

Chronic alcohol feeding in liquid diet or in drinking water has similar effects on electron microscopic appearance of the hepatic sinusoid in the rat.

Electron microscopic appearance of the liver sinusoid was examined in rats fed alcohol chronically in a complete liquid diet or in sucrose-containing drinking water. The animals were kept on liquid diet (+/- alcohol) for 14 weeks or on sucrose-containing drinking water (+/- alcohol) for 12.5 weeks and sacrificed thereafter. To rule out possible artifact induced by fixation procedure, livers were fixed by immersion (no perfusion), immersion preceded by perfusion, and by perfusion with glutaraldehyde and examined with both scanning and transmission electron microscopy. Regardless of the mode of its administration, and of the fixation procedure used, alcohol induced similar changes in liver sinusoid ultrastructure. Such changes included disruption of the sieve-plate pattern of the sinusoidal endothelial cell fenestrations with the appearance of large gaps and resulting in a meshwork lining, wherein large areas of the sinusoid communicated freely with the underlying hepatocytes. Transmission electron microscopy complemented these findings. The results reported in this study demonstrate that alcohol-induced structural changes of the liver sinusoid in the rat are similar whether alcohol is fed via a liquid diet or in drinking water. Therefore, alcohol administration in drinking water may provide a simple, inexpensive, and convenient method of inducing structural changes in the rat liver sinusoid.

Nitric oxide and liver injury in alcohol-fed rats after lipopolysaccharide administration.

Earlier studies showed that alcohol-fed animals were more susceptible than controls to injurious effects of endotoxin. Increased superoxide radical production by hepatocyte organelles, Kupffer cells, and neutrophils from alcohol-fed animals has been well documented. In this study, electron paramagnetic resonance spectroscopy was used to detect nitrosyl protein complexes indicating nitric oxide (.NO) production. We showed that the concentrations of nitrosyl complexes in whole blood and in liver tissues of alcohol-fed rats treated with lipopolysaccharide (alc + LPS), increased 3-fold, compared with those from rats on control diet treated with LPS (con+LPS). Electron paramagnetic resonance spectra of whole blood and liver tissues from the alc + LPS-treated group exhibited features characteristic of hemoglobin nitrosyl complexes. Plasma levels of the hepatic ASTs and ALTs from the alc + LPS-treated group were increased 2- to 3-fold, compared with those from the con+LPS-treated group. Inhibition of .NO production of aminoguanidine treatment attenuated plasma hepatic enzyme levels in the alc + LPS-treated group. Thus, under the conditions of elevated inflammatory oxidative states caused by chronic alcohol feeding, endotoxin treatment enhanced liver injury as a result of the actions of .NO, and/or the cytotoxic species derived from .NO.

Hepatic sinusoidal endothelial cell in alcoholemia and endotoxemia.

The experimental data reviewed in this study tend to indicate that the hepatic sinusoidal endothelial cell (SEC) is, chronologically, the first hepatic cell that undergoes pathologic changes in alcoholemia. Due to its strategic position in the liver sinusoid, SEC dysfunction and structural alterations have far-reaching repercussions for the whole liver. The authors gather experimental evidence suggesting that alcohol-induced SEC alterations are mostly due to Kupffer cell activation induced by alcohol rather than to a direct action of alcohol on SEC. Once activated, the Kupffer cell secretes a spectrum of mediators that affect both function and structure of SEC. Kupffer cell activation is regarded as a result of both direct and indirect actions of alcohol on the cell. The indirect action of alcohol is ascribed to alcohol-induced elevated plasma levels of Gram-negative bacterial lipopolysaccharide (LPS), a strong activator of Kupffer cell. However, a comparison of alcohol and LPS effects on SEC functions and structure reveals that these two agents may have, under many circumstances, different actions on the SEC, at least in laboratory animals. However, this issue continues to be a matter of debate. Also the review presents justification for the necessity to extend research on mechanisms underlying alcoholic liver disease to the effects of alcohol on the SEC. Finally, several future research directions are suggested in this review to better understand the mechanisms underlying alcohol-induced liver dysfunction.

Postbinge effects of acute alcohol intoxication on hepatic free radical formation.

The present studies were performed to test the hypothesis that Kupffer and endothelial cells are activated after recovery from an acute alcohol binge, which is accompanied by formation of oxygen-derived radicals. These radicals have been implicated in the pathogenesis of alcohol-mediated tissue injury in a number of organs. Male Sprague-Dawley rats received an intravenous injection of 20% ethanol in saline (1.75 g/kg), followed by an intravenous infusion (250 to 300 mg/kg/hr) for 12 hr. At the end of 12-hr infusion, ethanol was replaced by saline, and the infusion was continued for a further 6 hr. This was referred to as the recovery period. The 6-hr recovery period was selected because superoxide anion generation by the perfused liver peaked at this time point. Superoxide anion formation by the perfused liver was measured by the superoxide dismutase-inhibitable reduction of ferricytochrome c. Kupffer and endothelial cells were isolated for the determination of in vivo glucose uptake and in vitro superoxide anion release. Results show that a significant (p < 0.05) amount of superoxide (1.54 nmol/min/g) was generated by the perfused liver at 6 hr recovery after 12 hr of ethanol infusion. Serum ALT activity was also elevated in this treatment group. Time-matched control-saline infused animals or ethanol-treated animals without a recovery period released < 0.2 nmol/min/g of superoxide. The postrecovery superoxide production and an accompanying increase in the in vivo glucose uptake were also observed in isolated Kupffer and endothelial cells. Depletion of Kupffer cells by gadolinium chloride before ethanol treatment and recovery was associated with significant attenuation of free radical formation by the perfused liver and reduction of serum ALT. These studies demonstrate that recovery from an acute alcohol binge has a stimulating effect on hepatic sinusoidal superoxide production, and it may also affect liver function.

Compartmentalization of glucose utilization after intravenous vs. intratracheal challenge with LPS.

The rate of glucose utilization (Rg) of various tissues including lung and bronchoalveolar lavage (BAL) fluids cells was measured, using the 2-deoxyglucose technique in Sprague-Dawley rats 4 h after challenge with either 1 mg/kg intravenous or 0.3 mg/kg intratracheal lipopolysaccharide (LPS). After intravenous LPS, Rg increased in whole lung and nonrespiratory tissues, but was unaltered in BAL cells. After intratracheal LPS, the Rg of nonrespiratory tissues was unchanged, but the Rg of BAL cells increased from 3.7 +/- 0.3 to 71.5 +/- 16.0 nmol/min. This increase in the Rg of BAL cells was explained by a doubling of the macrophage specific Rg, by a 100-fold increase in polymorphonuclear leukocytes (PMN) number, and by a higher Rg in PMN than in macrophages. These results demonstrate that increased glucose utilization after intratracheal LPS is confined to the respiratory system and that intra-alveolar phagocytes participate in this increase.

Endotoxin-induced oxidative stress in the rat small intestine: role of nitric oxide.

Reactive oxygen species have been implicated in the gastrointestinal pathogenesis of septic and endotoxic shock. The objective of this study was to investigate the role of inducible nitric oxide synthase during endotoxin-induced formation of oxidants by cells of the small intestine. After intravenous Escherichia coli lipopolysaccharide (LPS) (1 mg/kg) injection, nitric oxide production was measured as nitrosyl complex formation in the ileum using electron paramagnetic resonance spectroscopy. Oxidative stress biomarkers were determined as duodenal mucosal-reduced thiols, the ileal lipid peroxidation and luminal free radical production using spin trapping methodology. Demonstration of nitrosyl complex formation commenced at 3 h and diminished 24 h post-LPS. Mucosal thiol levels were decreased at 3, 6, 12, and 18 h post-LPS treatment. At these time point, the ileal lipid peroxidation also increased as did luminal formation of hydroxyl radical adduct. Nitric oxide synthase inhibitors reversed the elevation of hydroxyl radical formation and reversed the decrease in mucosal-reduced thiol levels in the LPS-treated rats. Our data indicate that nitric oxide or its oxidant product(s), such as peroxynitrite, contribute to oxidative injury in the small intestine of rats treated with endotoxin.

Nitrosyl complex formation during endotoxin-induced injury in the rat small intestine.

The objective of this study was to demonstrate nitric oxide (NO) production and determine its role in the rat small intestine following endotoxin treatment. By using electron paramagnetic resonance (EPR) spectroscopy, we were able to detect high concentrations of nitrosylated proteins in the small intestines of rats administered 1 mg/kg lipopolysaccharide (LPS) and sacrificed 6 h later. EPR spectra of non-heme and heme nitrosyl complexes were detected in the epithelium layer and intestinal wall. Only EPR spectra characteristic of nitrosyl hemoprotein complexes were detected in the luminal contents of these rats. LPS administration elevated the concentrations of intestinal lipid peroxidation biomarkers, thiobarbituric acid-reactive substances, and conjugated dienes. These changes were attenuated by NO synthase inhibitor treatment. We conclude that oxidants associated with NO formation were at least in part involved in the oxidation of tissue lipids. This process may be one of the mechanisms of intestinal injury induced by LPS.

Decreased production of reactive oxygen intermediates is an early event during in vitro apoptosis of rat thymocytes.

Thymocyte apoptosis is one of the best characterized experimental models of apoptosis that can be induced by a variety of stimuli such as glucocorticoids, ionizing radiation, antibodies, and toxins. Recently, it has been suggested that oxidative stress is a common mediator of apoptosis. However, little is known about the production and possible function of reactive oxygen intermediates (ROI) in thymocytes. We used a highly sensitive flow cytometric assay with the hydrogen peroxide-sensitive dye, 2',7'-dichlorofluorescin diacetate (DCFH-DA), to measure intracellular ROI production in rat thymocytes, to study its primary sources, and to compare ROI levels in normal and apoptotic thymocytes. Apoptosis was induced by incubating the cells in the presence or absence of dexamethasone (Dex) at 37 degrees C in vitro. Normal thymocytes spontaneously produced significant amounts of ROI. Catalase or superoxide dismutase did not affect this intracellular fluorescence, presumably due to their failure to penetrate into the cells. However, N-acetyl-L-cysteine significantly attenuated the fluorescence in a dose-dependent manner. Significant inhibition of the intracellular fluorescence was also observed by addition of N-nitro-L-arginine methyl ester (L-NAME), that could not be reversed by L-arginine. The addition of N-nitro-D-arginine methyl ester (D-NAME) also caused considerable inhibition. This indicates that the inhibition by L-NAME or D-NAME is due to a direct scavenging effect, and nitric oxide production is not likely to be involved. In contrast to neutrophils and macrophages whose superoxide anions are released from membrane-bound NADPH oxidase, the production of ROI in thymocytes is likely to originate mainly from mitochondria, as indicated by the inhibitory effect of the addition of rotenone or antimycin A. The addition of lymphocyte simulators phytohemagglutinin (PHA), concanavalin A (Con A), or phorbol 12-myristate 13-acetate (PMA) enhanced intracellular fluorescence of thymocytes. This increase was abrogated by addition of rotenone or antimycin A. The ROI production was decreased with time after incubation of the thymocytes for 1, 3, and 6 h in vitro. The appearance of apoptosis of thymocytes in vitro, as indicated by DNA content of cells by flow cytometry and DNA ladder formation in agarose gel electrophoresis, was delayed, as compared to the time course of the decreased ROI production. The addition of Dex to the culture medium accelerated both of these processes. The results suggest that a decreased spontaneous production of ROI in thymocytes precedes the spontaneous in vitro apoptosis and Dex exaggerates these changes.

Acute endotoxin tolerance is accompanied by stimulated glucose use in macrophage rich tissues.

A single injection of E. coli LPS at a dose of 10 mg/kg b.w. ("high dose") is lethal in Sprague Dawley rats. However, animals given a sublethal dose of LPS (0.5 mg/kg bw; "low dose") at time zero, followed by a second high dose injection at 48 h, display endotoxin tolerance with 100% survival. The aim of the present study was to assess the relationship between this observed endotoxin tolerance and the endotoxin-induced glucose metabolic response in selected tissues and nonparenchymal hepatic cells. In each experimental group two injections, the first at time zero, the second at 48 h were given in vivo. Four experimental groups constituted these studies: A) saline followed by saline, B) low dose LPS followed by saline, C) saline followed by high dose LPS, and D) low dose LPS followed by high dose LPS. In vivo glucose use in tissues and cells was measured 3h after the last treatments employing the 2-deoxy-glucose tracer technique. Glucose use by liver, lung, spleen and intestine was not different between saline/saline (group A) and low dose LPS/saline injected (group B) animals. Saline/high dose LPS injection (group C) doubled glucose uptake, while the sequential LPS injections (group D) caused an additional, 2-3 fold increase in the glucose use by these tissues. Hepatic endothelial cells showed a similarly elevated glucose use in vivo in both group C and D. Kupffer cells from group D animals, however, displayed markedly elevated glucose use in vivo as compared to cells from group C. Our data indicate that high dose LPS in endotoxin tolerant animals is accompanied by a more markedly stimulated tissue glucose use than found following lethal LPS treatment alone. This increased peripheral glucose use may support cellular functions responsible for the protection of the host.

Liver sinusoid during chronic alcohol consumption in the rat: an electron microscopic study.

Transmission and scanning electron microscopic studies were performed on the liver sinusoid, with emphasis on sinusoidal endothelial cells, in rats fed a liquid diet containing either alcohol or dextrin (control) for 14 weeks. Animals were also treated with either Gram-negative bacterial lipopolysaccharide (LPS; 100 micrograms/100 g body weight, intravenously) or sterile saline (control). All specimens were prepared after perfusion fixation of the liver. Livers of rats fed dextrin-containing liquid diet displayed the ultrastructural features typical of the sinusoid and its endothelial cells. Livers from alcohol-fed animals, however, were characterized by massive loss of sieve-plate architecture of the sinusoidal endothelium, which was virtually replaced with a meshwork of enlarged openings with diameters frequently exceeding 1 micron. Morphological evidence of Kupffer cell activation could also be seen along with significant fatty infiltration of the hepatocyte. Conversely, LPS administration to dextrin-fed animals induced an apparent decrease in fenestration of the sinusoidal endothelial cell, accompanied by morphological evidence of enhanced endocytotic activity and cytoplasmic swelling. The changes seen 3 hr after LPS administration were markedly advanced at 24 hr. LPS administration to alcohol-fed rats accentuated the alterations observed after alcohol treatment alone. Additionally, the presence of platelets in the sinusoid as well as adhering to the hepatocyte microvilli in the space of Disse, along with the presence of Ito and Kupffer cell activation, greater than that observed in the alcohol-treated rats, is morphological evidence consistent with the disruption of vascular integrity in the liver.(ABSTRACT TRUNCATED AT 250 WORDS)