Ion V. Deaciuc

Monocyte activation in alcoholic liver disease

Activated monocytes and macrophages have been postulated to play an important role in the pathogenesis of alcoholic liver disease (ALD). Monocyte activation can be documented by measurement of neopterin, adhesion cell molecules, and certain proinflammatory cytokines and chemokines. We first became interested in the role of monocytes and monocyte-derived cytokines in ALD in relation to altered zinc metabolism that occurs regularly in ALD. Patients with ALD have hypozincemia, which responds poorly to oral zinc supplementation. We have shown that in ALD monocytes make a low-molecular-weight substance that, when injected into rabbits, causes prominent hypozincemia. Subsequently, multiple cytokines [especially tumor necrosis factor (TNF) and interleukin (IL)-8] have been shown to be overproduced by monocytes in ALD. We initially showed that monocytes in ALD spontaneously produce TNF and overproduce TNF in response to a lipopolysaccharide (LPS) stimulus, and this could be attenuated by antioxidants in vitro and in vivo. Alterations in the endotoxin-binding protein LPS-binding protein, in CD14, and in the endotoxin receptor Toll-like receptor 4 all may play roles in enhanced proinflammatory cytokine signaling in ALD. Moreover, several groups have documented increased TNF receptor density in monocytes in ALD. Inadequate negative regulation of TNF occurs at multiple levels in ALD. This includes decreased monocyte production of the important antiinflammatory cytokine IL-10 and blunted response to the antiinflammatory properties of adenosine. Finally, generation of reactive oxygen species (which occurs during alcohol metabolism) and products of lipid peroxidation induce production of cytokines, such as TNF and IL-8. In conclusion, there are multiple overlapping potential mechanisms for enhanced proinflammatory cytokine production by monocytes in ALD. We postulate that activation of monocytes and macrophages with subsequent proinflammatory cytokine production plays an important role in certain metabolic complications of ALD and is a component of the liver injury of ALD.

Integrated hepatic transcriptome and proteome analysis of mice with high-fat diet-induced nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is the most common form of liver disease in the US and refers to a wide spectrum of liver damage, including simple steatosis, steatohepatitis, fibrosis and cirrhosis. The goal of the present study was to achieve a more detailed understanding of the molecular changes in response to high fat-induced liver steatosis through the identification of a differentially expressed liver transcriptome and proteome. Male C57/BL6 mice fed a high-fat lard diet for 8 weeks developed visceral obesity and hepatic steatosis characterized by significantly increased liver and plasma free fatty acid and triglyceride levels and plasma alanine aminotransferase activities. Transcriptome analysis demonstrated that, compared to the control diet (CD), high-fat diet changed the expression of 309 genes (132 up- and 177 down-regulated; by a twofold change and more, P<.05). Multiple genes encoding proteins involved in lipogenesis were down-regulated, whereas genes involved in fatty acid oxidation were up-regulated. Proteomic analysis revealed 12 proteins which were differentially expressed. Of these, glutathione S-transferases mu1 and pi1 and selenium-binding protein 2 were decreased at both the gene and protein levels. This is the first study to perform a parallel transcriptomic and proteomic analysis of diet-induced hepatic steatosis. Several key pathways involving xenobiotic and lipid metabolism, the inflammatory response and cell-cycle control were identified. These pathways provide targets for future mechanistic and therapeutic studies as related to the development and prevention of NAFLD.

Inhibition of Adiponectin Production by Homocysteine : A Potential Mechanism for Alcoholic Liver Disease

Although recent evidence suggests that down‐regulation of production of the adipocyte hormone adiponectin has pathophysiological consequences for the development of alcoholic liver disease (ALD), the underlying mechanisms are elusive. Abnormal hepatic methionine‐homocysteine metabolism induced by prolonged alcohol exposure has been reported both in clinical and experimental studies of ALD. Here, we conducted both in vivo and in vitro experiments to examine the effects of prolonged alcohol exposure on homocysteine levels in adipose tissue, its potential involvement in regulating adiponectin production, and the consequences for ALD. Chronic alcohol exposure decreased the circulating adiponectin concentration and adiponectin messenger RNA (mRNA) and protein levels in epididymal fat pads. Alcohol feeding induced modest hyperhomocysteinemia and increased homocysteine levels in the epididymal fat pad, which was associated with decreased mRNA levels of cystationine β‐synthase. Betaine supplementation (1.5%, wt/vol) in the alcohol‐fed mice reduced homocysteine accumulation in adipose tissue and improved adiponectin levels. Moreover, exogenous homocysteine administration reduced gene expression, protein levels, and secretion of adiponectin in primary adipocytes. Furthermore, rats fed a high‐methionine diet (2%, wt/wt) were hyperhomocysteinemic and had decreased adiponectin levels in both plasma and adipose tissue, which was associated with suppressed AMP‐activated protein kinase activation in the liver. Mechanistic studies revealed that both inactivation of the extracellular signal regulated kinase 1/2 pathway and induction of endoplasmic reticulum stress response, specifically C/EBP homologous protein expression, may contribute to the inhibitory effect exerted by homocysteine. Conclusion: Chronic alcohol feeding caused abnormal accumulation of homocysteine in adipocytes, which contributes to decreased adiponectin production in ALD. (HEPATOLOGY 2008.)

Good fat/bad fat

The hallmark of nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH) is hepatic steatosis, usually macrovesicular fat. The clinical factors associated with NAFLD/NASH are multiple and ever increasing, including not only obesity/ metabolic syndrome, but also drugs (e.g., Amiodarone, Tamoxifen), chemotherapy-associated steatohepatitis, and environmental toxins to name only a few.1-4 Why some patients progress from simple steatosis to more aggressive liver disease is unknown. The prevailing concept is the 2-hit theory in which there is a baseline of steatosis plus one or more second hits (e.g., cytokines, oxidative stress, mitochondrial dysfunction1,5). A rapidly-evolving concept, which has been much more intensively investigated in other forms of organ injury, is lipotoxicity.6-10 Herein, lipids such as fatty acids are the noxious agents, and triglycerides actually serve as a “sink” or protective pathway in lipid metabolism.6,7 There are multiple definitions for lipotoxicity; a frequently used one is adverse effects of fatty acid accumulation in nonadipose tissues (Fig. 1). Many novel strategies are being investigated to prevent/treat NAFLD/NASH, including small molecules that alter lipid metabolism. Previous studies by Yu et al. published in HEPATOLOGY used antisense oligonucleotides to decrease the expression and activity of diacylglycerol acyltransferase (DGAT) 2 (Enzyme Commission number EC 2.3.1.20), which markedly reduced hepatic steatosis in obese (ob/ob) mice.10 Thus, there was some optimism that blocking DGAT2, which catalyzes the final step in hepatic triglyceride production, may be beneficial in NAFLD/NASH. In the study by Yamaguchi et al. in this issue of HEPATOLOGY, a more complex model of steatosis was used, employing db/db mice fed a methionine-restricted, choline-deficient (MCD) diet.11 Animal models for NAFLD/NASH are multiple, with genetic and dietary manipulations being most frequently used. The db/db mouse is an animal model of the metabolic syndrome, having obesity, hyperglycemia, insulin resistance, and hyperleptinemia, and modest increases of hepatic triglycerides. The db/db mouse also has increased intestinal permeability, portal vein endotoxemia, and increased levels of circulating inflammatory cytokines.12 Feeding the MCD diet has been shown to markedly augment steatosis, inflammation, and fibrosis in the db/db mouse.13 Feeding the MCD diet also sensitizes rodents to lipopolysaccharide, resulting in increased tumor necrosis factor production and hepatotoxicity.14 Animals fed MCD diets develop low levels of the critical methylating agent S-adenosylmethionine (SAM) and an altered SAM: S-adenosylhomocysteine ratio, which is associated with decreased activity of most methyltransferases.14 Rats remaining on the MCD diet over the long term can develop severe fibrosis/cirrhosis and even hepatocellular carcinoma. Similarly, mice deficient in the enzyme that converts methionine to SAM in the liver (MAT1A) also develop steatosis, subsequent steatohepatitis, and in the long term, may develop hepatocellular carcinoma.15 Moreover, gut microbiota (through reducing bioavailability of choline) have recently been shown to play a critical role in fatty liver development in a frequently used insulin-resistant mouse strain (129S6).16 Patients with alcoholic and nonalcoholic steatohepatitis often have alterations in hepatic methionine metabolism.1 Thus, the NASH model (db/db mouse fed the MCD diet) used by Yamaguchi and coworkers in this issue is a unique combination of a genetic and dietary model of NASH that may have important mechanistic and clinical relevance. Feeding db/db mice the MCD diet in the Yamaguchi study caused hepatic steatosis, hepatocyte injury and cell death, oxidative stress, and fibrosis.11 The elevated hepatic triglyceride content decreased with duration of study and with increasing severity of liver injury (similar to human NASH and cryptogenic cirrhosis). Although treatment with DGAT2 antisense oligonucleotides produced an expected reduction in hepatic triglyceride content, antisense-treated animals also developed significantly greater hepatic inflammation, fibrosis, elevated alanine aminotransferase levels, terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling staining, and very prominent increases in cytochrome P450 2E1 and 4-hydroxynonenal. The MCD-fed animals (with antisense therapy) had lower body weights, lower glucose and lower insulin levels, and an improved overall insulin sensitivity profile (disassociating liver injury and insulin sensitivity). Animals receiving antisense therapy Abbreviations: CLA, conjugated linoleic acid; DGAT, diacylglycerol acyltransferase; LCFA, long-chain fatty acids; MCD, methionine-restricted, choline-deficient; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; SAM, S-adenosylmethionine. Address reprint requests to: Craig McClain, M.D., Department of Internal Medicine, University of Louisville Medical Center, Louisville, KY 40292. E-mail: craig.mcclain@louisville.edu; fax: 502-852-8927. Copyright

Alcohol and cytokine networks

Experimental and clinical data pertaining to alcohol (ethanol, EtOH) interference with cytokine networks are analyzed from the viewpoint of their importance for understanding the deleterious effects of EtOH on various functions. The data are grouped and analysed according to several major directions that have emerged in the last decade since the research on EtOH interference with cytokine networks started. These are: cytokine secretion, including the effects of the drug on in vivo cytokine levels and on in vitro cytokine secretion by various cells; cytokine-receptor interaction; intracellular fate of cytokines; and cellular responses to cytokines. Correspondingly, the data are presented in four tables. Attention is paid to the fact that the effects of EtOH on various aspects of cytokine biology (e.g., secretion by various cells) are diversified. Contradictory data have been reported but the cause for discrepancy is poorly understood. EtOH effects vary with the cytokine studied, the method of EtOH administration (i.e., acute or chronic), the species used, or with other experimental conditions. It is important to note, however, that the vast majority of experimental and clinical data show that EtOH interferes with cytokine networks and that research on such interference may lead to important steps in understanding the mechanisms of action of EtOH. An attempt is made herein to select aspects of cytokine biology, as affected by EtOH, that have been studied to a lesser extent, thus calling for more research efforts in those areas.

Chronic alcohol exposure of rats exacerbates apoptosis in hepatocytes and sinusoidal endothelial cells

Background/aims: The liver apoptotic response to chronic alcohol consumption remains poorly characterized. The purpose of this study was to determine in rats the effects of chronic alcohol consumption on the relative magnitude of apoptosis in two major targets of alcohol-induced liver injury: the hepatocyte (Hep) and sinusoidal endothelial cell (SEC). Methods: Rats were fed a liquid diet containing either alcohol or isocaloric amounts of maltose-dextrin for 14 weeks. Hep and SEC were isolated by liver perfusion with collagenase followed by centrifugal elutriation. The state of the liver was assessed on the basis of light microscopic appearance, plasma liver enzymes (alanine and aspartate:2-oxoglutarate amino transferases), and the content of malondialdehyde in Hep. Apoptosis was assessed on the basis of DNA fragmentation in the whole organ (TUNEL), and caspase-3 and -8 activity in isolated cells. A mechanistic approach was also undertaken by measuring mRNA expression and the amount of protein for Fas/CD95, Fas ligand, caspase-3, Bax, Bcl-X(L), and Bcl-2 in the isolated Hep and SEC. Results: The livers of alcohol-fed rats displayed prominent steatosis. Oxidative stress was also present as reflected by an increase in the malondialdehyde content of Hep. Alcohol consumption increased apoptosis in the whole liver assessed on the basis of TUNEL procedure and in Hep and SEC as reflected by significant increase in caspase-3 activity. Of the multiple pro- and anti-apoptotic factors determined in this study, significant changes as assessed by both mRNA expression and the amount of proteins, were observed only in the SEC compartment. Conclusions: The data presented in this study indicate that: (1) chronic alcohol consumption in rats leads to a moderate augmentation of apoptosis in the whole liver and in two liver cell types which are targets for injury in alcoholic liver disease: Hep and SEC; (2) the mechanisms recruited/activated by these two types of liver cells to initiate and execute apoptosis in response to alcohol vary with the cell type.

Effects of acute alcohol intoxication on gluconeogenesis and its hormonal responsiveness in isolated, perfused rat liver

Rats were acutely administered ethanol as a primed constant infusion in order to produce sustained blood ethanol levels of 8-12 or 55-65 mM. At the end of ethanol infusion the livers were either freeze-clamped in vivo or isolated and perfused for metabolic studies. The rate of gluconeogenesis and its responsiveness to phenylephrine (10 microM), prostaglandin F2 alpha (5 microM) and glucagon (10 nM), as well as the redox state of the cytosolic NAD(+)-NADH system were assessed in livers isolated from acutely ethanol-treated rats, and subsequently perfused without ethanol. For liver clamped in vivo, high- but not low-ethanol treatment decreased the ATP content by 31% and slightly increased ADP and AMP content, resulting in a decreased energy charge (11%). Glutamate and aspartate content was also increased in high-dose ethanol-infused rats with no changes in malate and 2-oxoglutarate content. Gluconeogenesis with saturating concentrations of lactate (4 mM)+pyruvate (0.4 mM) was delayed in reaching a plateau in the livers of high-dose ethanol-treated rats and its response to all three stimulators was impaired. Low-dose ethanol treatment only decreased the liver response to phenylephrine. While the perfused livers of low-dose ethanol-treated rats displayed no changes in adenine nucleotide content, the livers of high-dose ethanol-treated rats had a decreased ATP (35%) and an increased AMP (77%) content, paralleled by a fall in the total adenine nucleotides (14%) and energy charge (14%). No differences were observed between the saline- and ethanol-treated rats with respect to malate-aspartate shuttle intermediate concentration in perfused livers. Also, the livers of high-, but not low-dose ethanol-treated rats had a more negative value of NAD(+)-NADH redox state as compared to the livers of control rats. The data suggest that acute ethanol intoxication produces changes in liver metabolism and its responsiveness to hormones/agonists that are demonstrable for at least 2 hr after isolation and perfusion of the liver.

Effects of exogenous superoxide anion and nitric oxide on the scavenging function and electron microscopic appearance of the sinusoidal endothelium in the isolated, perfused rat liver

BACKGROUND/AIMS Functional and morphological alterations of the hepatic sinusoidal endothelial cell occur in several models of experimental liver injury and in clinical settings. The causes of these alterations are multiple. The aim of this study was to test the hypothesis that the early functional impairment and morphological alterations of the sinusoidal endothelial cell and hepatic sinusoid associated with liver injury are mediated by free radical species, such as superoxide anion and nitric oxide. METHODS Isolated rat livers were perfused by recirculation with hemoglobin-free, Krebs-Henseleit bicarbonate buffer and presented with a source of superoxide anion (xanthine oxidase+hypoxanthine) or nitric oxide (S-nitroso-N-acetyl penicillamine). Hyaluronan uptake (an index of sinusoidal endothelial cell scavenging function), thiobarbituric acid-reactive substances content of the tissue (a marker of lipid peroxidation), reduced and oxidized glutathione (a marker of the thiol system oxidation/reduction state), lactate dehydrogenase and alanine aminotransferase activities (markers of cytolysis), as well as scanning and transmission electron microscopic appearance of the sinusoid were evaluated. RESULTS At the high concentrations used, both free radical generating systems suppressed hyaluronan uptake, increased malondialdehyde content of the tissue, enhanced the release of both liver enzymes, decreased the total glutathione content of the liver, and altered the ratio of reduced/oxidized glutathione. Both free radical species induced dose-dependent morphological alterations of the sinusoid, consisting of the appearance of large gaps replacing the sieve-plated fenestration. CONCLUSIONS The free radical species-induced functional impairment and morphological alterations of the liver sinusoid, presented in this study, closely resemble the early in vivo changes associated with liver injury under a variety of conditions, such as preservation and reperfusion, or administration of hepatotoxicants such as D-galactosamine, Gram-negative bacterial lipopolysaccharides, acetaminophen, alcohol and others. Therefore, we suggest that early liver sinusoid injury, observed under these conditions, can be attributed to the action of free radicals, such as superoxide anion and nitric oxide.

Effect of chronic alcohol consumption by rats on tumor necrosis factor-α and interleukin-6 clearance in vivo and by the isolated, perfused liver☆

The effects of chronic (16-week) alcohol consumption by rats on [125I]tumor necrosis factor (TNF)-α and [125I]interleukin (IL)-6 plasma clearance and organ distribution in vivo and uptake and metabolism by the isolated, perfused liver were studied. Alcohol was administered to rats in a liquid diet for 16 weeks, and caused a decreased (48%) plasma clearance rate of IL-6 and converted the plasma clearance kinetics of the cytokine from a biphasic exponential in normal rats to a monophasic exponential decay. Alcohol feeding significantly increased (101%) plasma clearance of TNF-α, which followed a biphasic exponential decay and decreased the T12 for both the α (67%) and β (76%) elimination components. The distribution of both cytokines in trichloroacetic acid precipitable and non-precipitable fractions of liver, spleen, stomach, small intestine (ileum), lung, kidney, and blood was also studied. The only effect of alcohol treatment was a significant decrease in IL-6 uptake and metabolism by the small intestine. Perfused livers, isolated from alcohol-fed rats, took up and metabolized larger amounts of IL-6 than did livers isolated from pair-fed rats. TNF-α uptake and metabolism by the isolated, perfused liver were not affected by chronic alcohol consumption. Regardless of the animal treatment, the isolated perfused liver took up and metabolized significantly larger (17-fold) amounts of TNF-α than IL-6, in spite of identical concentrations of cytokines (6 nM) in the perfusion medium. The data presented in this study along with our previous results demonstrating the effects of alcohol consumption on TNF-α and IL-6 receptors on various liver cells suggest that the effects of chronic alcohol treatment on cytokine clearance cannot be ascribed to changes in the receptors for the two cytokines. Also, no correlation was found between the effects of alcohol treatment on plasma cytokine clearance and uptake and metabolism of cytokines by the isolated, perfused liver. Experimental data and theoretical considerations suggest that cytokine receptor recycling may play an important role in mediating alcohol effects on cytokine clearance.

Ethanol exposure modulates hepatic S-adenosylmethionine and S-adenosylhomocysteine levels in the isolated perfused rat liver through changes in the redox state of the NADH/NAD+ system

Methionine metabolism is disrupted in patients with alcoholic liver disease, resulting in altered hepatic concentrations of S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), and other metabolites. The present study tested the hypothesis that reductive stress mediates the effects of ethanol on liver methionine metabolism. Isolated rat livers were perfused with ethanol or propanol to induce a reductive stress by increasing the NADH/NAD(+) ratio, and the concentrations of SAM and SAH in the liver tissue were determined by high-performance liquid chromatography. The increase in the NADH/NAD(+) ratio induced by ethanol or propanol was associated with a marked decrease in SAM and an increase in SAH liver content. 4-Methylpyrazole, an inhibitor the NAD(+)-dependent enzyme alcohol dehydrogenase, blocked the increase in the NADH/NAD(+) ratio and prevented the alterations in SAM and SAH. Similarly, co-infusion of pyruvate, which is metabolized by the NADH-dependent enzyme lactate dehydrogenase, restored the NADH/NAD(+) ratio and normalized SAM and SAH levels. The data establish an initial link between the effects of ethanol on the NADH/NAD(+) redox couple and the effects of ethanol on methionine metabolism in the liver.