Effect of chronic alcohol consumption on Hepatic SIRT1 and PGC-1alpha in rats.

The nuclear genes, NAD-dependent deacetylase Sirtuis 1 (SIRT1) and the peroxisome proliferator-activated receptor-gamma coactivator1alpha (PGC-1alpha) are regulators of energy metabolism. Here, we studied the role of alcohol consumption in expression of these sensing molecules. Alcohol significantly reduced hepatic SIRT1 mRNA by 50% and PGC-1alpha mRNA by 46% and it significantly inhibited the protein expression of SIRT1 and PGC-1alpha, while the transcription factor PPAR-gamma remained unchanged. However, when the lipid composition of the alcohol diet was changed by replacing long-chain triglycerides (LCT) with medium chain triglycerides (MCT), SIRT1 and PGC-1alpha mRNA were restored to near control levels. This study demonstrates that alcohol reduces key energy sensing proteins and that replacement of LCT by MCT affects the transcription of these genes. Since there is a pathophysiological link between SIRT1 and PGC-1alpha and mitochondrial energy, the implication of the study is that mitochondrial dysfunction due to alcohol abuse can be treated by dietary modifications.

Alcohol alters hepatic FoxO1, p53, and mitochondrial SIRT5 deacetylation function.

Chronic alcohol consumption affects the gene expression of a NAD-dependent deacetylase Sirtuis 1 (SIRT1) and the peroxisome proliferator-activated receptor-gamma coactivator1alpha (PGC-1alpha). Our aim was to verify that it also alters the forkhead (FoxO1) and p53 transcription factor proteins, critical in the hepatic response to oxidative stress and regulated by SIRT1 through its deacetylating capacity. Accordingly, rats were pair-fed the Lieber-DeCarli alcohol-containing liquid diets for 28 days. Alcohol increased hepatic mRNA expression of FoxO1 (p=0.003) and p53 (p=0.001) while corresponding protein levels remained unchanged. However phospho-FoxO1 and phospho-Akt (protein kinase) were both decreased by alcohol consumption (p=0.04 and p=0.02, respectively) while hepatic p53 was found hyperacetylated (p=0.017). Furthermore, mitochondrial SIRT5 was reduced (p=0.0025), and PGC-1alpha hyperacetylated (p=0.027), establishing their role in protein modification. Thus, alcohol consumption disrupts nuclear-mitochondrial interactions by post-translation protein modifications, which contribute to alteration of mitochondrial biogenesis through the newly discovered reduction of SIRT5.

Value of fibrosis markers for staging liver fibrosis in patients with precirrhotic alcoholic liver disease.

BACKGROUND: Our aim was to identify markers predictive of fibrosis in alcoholic liver disease (ALD). Percutaneous liver biopsy is the recommended standard for histologic assessment of liver fibrosis. Seven serum markers (tissue inhibitor of matrix metalloproteinase 1 [TIMP1], tenascin, collagen VI, amino-terminal propeptide of type III collagen [PIIINP], matrix metalloproteinases [MMP2], laminin, and hyaluronic acid [HA]) representing various aspects of collagen and extracellular matrix deposition and degradation, have been proposed as noninvasive surrogates for liver biopsy. Moreover, a diagnostic algorithm including 3 serum markers (TIMP1, PIIINP, HA) and age has been proposed to accurately detect fibrosis with acceptable levels of sensitivity/specificity in a chronic hepatitis C subgroup. METHODS: To determine variability of these markers in liver fibrosis with different etiologies, we conducted an evaluation of their correlative properties in a subgroup of patients (n = 247) with biopsy confirmed liver fibrosis resulting from long-term heavy alcohol consumption. Patients were participants in a recently completed VA multicenter clinical trial followed over 2 years with liver biopsy at baseline and 24 months, and with markers assessed every 3 months. RESULTS: Among the markers measured in this alcoholic subgroup all except collagen VI displayed significant correlation with degrees of fibrosis. Three markers, TIMP1, PIIINP and HA adjusted for age, emerged as the most promising predictors of the degree of fibrosis in a population of alcoholics. However, there was little change over time as related to change in fibrosis. The lower than expected accuracy of these markers based on receiver operating curves (ROC) also showed their limited use in this etiologic subgroup. CONCLUSION: In alcoholic patients, various markers have limited value in predicting and diagnosing the stages of fibrosis compared to liver biopsy. Thus, further prospective studies are required to better define the usefulness of each marker or their combination which are possibly affected by alcohol metabolism.

Adipose differentiation-related protein is a reliable lipid droplet marker in alcoholic fatty liver of rats.

BACKGROUND: Adipose differentiation-related protein (ADRP) is a lipid droplet-associated protein that coats cytoplasmic lipid droplets. The present study evaluated whether alcohol feeding enhances ADRP expression and whether ADRP is a lipid droplet marker in alcoholic fatty liver of rats. Because medium-chain triglycerides (MCT) reduce alcoholic hepatosteatosis, their effects on ADRP were also evaluated. METHODS: Fatty liver was induced in rats by the consumption of the Lieber-DeCarli alcohol liquid diet with or without replacement of long-chain triglycerides (LCT) by MCT (32% of calories). Immunohistochemical staining for ADRP was performed in formalin-fixed, paraffin-embedded liver sections. ADRP immunostaining was quantified by image analysis. Triacylglycerol was measured chemically. ADRP mRNA and protein were analyzed by real-time polymerase chain reaction and western blot, respectively. Double staining technique was performed to distinguish ADRP from glycogen in hepatocytes. RESULTS: Alcohol feeding for 21 days increased ADRP staining in the centrilobular and mid zonal regions of the liver lobules coincident with fat deposition in the liver. Replacing LCT in the alcohol diet with MCT diminished ADRP immunostaining in parallel with reduced steatosis. MCT also attenuated the up-regulation of ADRP mRNA and protein after alcohol. In steatotic hepatocytes ADRP selectively stained the surface of macrovesicular and microvesicular lipid droplets. ADRP immunostaining quantitatively correlated with hepatic triacylglycerol levels, validating ADRP as a reliable lipid droplet marker. Compared with hematoxylin and eosin stains, ADRP was more sensitive in detecting microvesicular lipid droplets. ADRP immunostaining also distinguished lipid droplets from glycogen, as demonstrated by double staining for ADRP and glycogen. CONCLUSIONS: Alcohol induction of fatty liver enhances ADRP expression and MCT oppose the alcohol effects. ADRP is a reliable and sensitive marker for lipid droplets in alcoholic fatty liver. ADRP immunostaining permits quantification of fatty change in hepatocytes and can be used as an ancillary technique in assessing the efficacy of diets or drugs against hepatosteatosis.

Metabolism of alcohol.

Most tissues of the body contain enzymes capable of ethanol oxidation or nonoxidative metabolism, but significant activity occurs only in the liver and, to a lesser extent, in the stomach. Hence, medical consequences are predominant in these organs. In the liver, ethanol oxidation generates an excess of reducing equivalents, primarily as NADH, causing hepatotoxicity. An additional system, containing cytochromes P-450 inducible by chronic alcohol feeding, was demonstrated in liver microsomes and found to be a major cause of hepatotoxicity.

S-adenosyl-L-methionine: its role in the treatment of liver disorders.

S-Adenosyl-L-methionine (SAMe) exerts many key functions in the liver, including serving as a precursor for cysteine, 1 of 3 amino acids of glutathione--the major physiologic defense mechanism against oxidative stress. SAMe is particularly important in opposing the toxicity of free oxygen radicals generated by various pathogens, including alcohol, which cause oxidative stress largely by the induction of cytochrome P4502E1 (CYP2E1) and by its metabolite acetaldehyde. SAMe also acts as the main methylating agent in the liver. The precursor of SAMe is methionine, one of the essential amino acids, which is activated by SAMe-synthetase (EC 2.5.1.6). Unfortunately, the activity of this enzyme is significantly decreased as a consequence of liver disease. Because of decreased utilization, methionine accumulates and, simultaneously, there is a decrease in SAMe that acquires the status of an essential nutrient and therefore must be provided exogenously as a supernutrient to compensate for its deficiency. Administration of this innocuous supernutrient results in many beneficial effects in various tissues, mainly in the liver, and especially in the mitochondria. This was shown in alcohol-fed baboons and in other experimental models of liver injury and in clinical trials, some of which are reviewed in other articles in this issue.

S-Adenosylmethionine: molecular, biological, and clinical aspects--an introduction.

In clinical research, a novel approach has emerged: some of the essential nutrients are being used to treat pathologic conditions. Many of these nutrients, including methionine, must first be activated in the liver or in other tissues before they can exert their key functions. However, this activating process is impaired in disease states and, as a consequence, nutritional requirements change. For instance, for methionine to act as the main cellular methyl donor, it must first be activated to S-adenosylmethionine (SAMe; also known as ademethionine). SAMe is required and is of fundamental importance for the metabolism of nucleic acids and polyamines, the structure and function of membranes, and as a precursor of glutathione. These processes are often seriously altered in various pathologic states addressed in this symposium, but they cannot be restored by simply administering methionine. For instance, in liver disease associated with impairment of the enzyme that activates methionine to SAMe, supplementation with methionine is useless and may even become toxic as it accumulates because it is not used. Accordingly, one must correct the lack of SAMe by bypassing the deficiency in enzyme activation; this is done by providing the product of the defective reaction, namely SAMe. Under these pathologic conditions, SAMe becomes crucial for the functioning of the cell. Thus SAMe, which is found in all living organisms, becomes the essential nutrient instead of methionine. This symposium reviewed the biological and corresponding molecular aspects of SAMe metabolism and the clinical consequences of its deficiency or supplementation in various tissues.

Model of nonalcoholic steatohepatitis.

BACKGROUND: Obesity and diabetes are frequently associated with nonalcoholic steatohepatitis (NASH), but studies have been hampered by the absence of a suitable experimental model. OBJECTIVE: Our objective was to create a rat model of NASH. DESIGN: Sprague-Dawley rats were fed a high-fat, liquid diet (71% of energy from fat, 11% from carbohydrates, 18% from protein) or the standard Lieber-DeCarli diet (35% of energy from fat, 47% from carbohydrates, 18% from protein). The diets were given ad libitum or as two-thirds of the amount consumed ad libitum. RESULTS: Rats fed the high-fat diet ad libitum for 3 wk developed panlobular steatosis, whereas those fed the standard diet had few fat droplets. Accordingly, total lipid concentrations with the high-fat and standard diets were 129.9 +/- 9.1 ( +/- SEM) and 66.7 +/- 4.6 mg/g liver, respectively (P < 0.001). The high-fat diet caused abnormal mitochondria and mononuclear inflammation, which were accompanied by increased hepatic tumor necrosis factor alpha (TNF-alpha; P < 0.001), TNF-alpha messenger RNA (mRNA) (P < 0.001), collagen type 1, and alpha1(I) procollagen mRNA (P < 0.001). In addition, these rats had increased cytochrome P4502E1 (CYP2E1) mRNA (P < 0.001), which was accompanied by CYP2E1 induction (P < 0.001) and oxidative stress with increased 4-hydroxynonenal (P < 0.001). Plasma insulin was elevated, which reflected insulin resistance, a NASH pathogenic factor. Rats fed a restricted high-fat diet developed only mild steatosis with attenuated biochemical changes, whereas those given a restricted standard diet had normal livers. CONCLUSION: This rat model reproduces the key features of human NASH and provides a realistic experimental model for elucidating its treatment.

The Research Society on Alcoholism.

Until the 1960s, the small amount of federal funding available in the alcohol field in the USA was provided by a division of the Alcohol, Drug and Mental Health Administration. In the late 1960s, the National Council on Alcoholism (NCA) joined the American Medical Society on Alcoholism (AMSA, which accepted only physicians). A group of physicians at NCA-AMSA felt that progress in our field needed the concerted effort of both physicians and nonphysician scientists, creating the Research Society on Alcoholism (RSA) in 1977. The National Institute on Alcohol Abuse and Alcoholism (NIAAA) had been created a few years earlier. From 250 members in 1978, the RSA has grown to 1500 in 2001, with a ratio of about three PhDs (with specialities that range from gene therapy to social policy) to each MD. The society holds yearly meetings-visitors can find more information on the web page (http://www.RSoA.org). The society publishes the journal Alcoholism: Clinical and Experimental Research. RSA committees are active in presenting the most recent findings to the public and to elected representatives, and in making recommendations on areas of research that need funding.

Mediation by nitric oxide of the stimulatory effects of ethanol on blood flow.

The contribution of nitric oxide (NO) to the hemodynamic effects associated with alcohol oxidation was assessed in rats given either ethanol or water by gastric tube, with and without pre-treatment with either the NO synthase inhibitor N(omega)-monomethyl-L-arginine (L-NMMA; 15 mg/Kg i.p.) or the alcohol dehydrogenase inhibitor 4-methylpyrazole (4-MP; 82 mg/Kg i.p.). Alcohol increased plasma NO (measured with chemiluminescence) by 63%. This was prevented by either L-NMMA or 4-MP. Cardiac output and regional blood flows were determined with 57Cobalt-labeled microspheres. Alcohol markedly enhanced portal blood flow (130 +/- 6 ml/min/Kg vs. 62 +/- 4, in controls; p < 0.01) with no changes in the hepatic, splenic or pancreatic arterial blood flows, indicating that the vasodilatation is mainly mesenteric. In addition, it quadrupled the coronary blood flow, doubled the renal flow and increased cardiac output by 38%, with no significant changes in pulmonary, cerebral or testicular flows. All the stimulatory effects of ethanol on flow, as well as the rise in NO levels, were prevented by L-NMMA, incriminating NO as the mediator of the hemodynamic effects of ethanol oxidation, acting probably via acetate and adenosine.

CYP2E1: from ASH to NASH.

The pathology of the liver in alcoholic steatosis and alcoholic steatohepatitis (ASH) is remarkably similar to that of nonalcoholic fatty liver disease (NAFLD), including nonalcoholic steatohepatitis (NASH), suggesting some common pathogenic mechanism. Studies carried out over the last three decades of possible mechanisms involved revealed one common link, namely the induction of cytochrome P4502E1. Its substrates include fatty acids, ketones and ethanol. These substances, when present chronically in large amounts, induce the activity of the enzyme which thereby contributes to the disposition of these substrates. This reaction, however, is associated with the release of free radicals which can cause lipid peroxidation and liver injury, including mitochondrial damage. Mitochondrial damage in turn exacerbates the oxidative stress. CYP2E1 can also convert various xenobiotics to toxic metabolites. When unchecked, this toxicity eventually results in inflammation and fibrosis, culminating in cirrhosis. Prevention of this disorder is based on limiting the substrates that induce the system, such as excessive fatty acid associated with obesity and excessive alcohol consumption. No effective pharmacologic treatment is presently available but there is ongoing research on possible inhibitors of CYP2E1, innocuous enough to be suitable for chronic human consumption and sufficiently effective to attenuate the CYP2E1 induction to avoid the consequences of its excessive activity while maintaining its physiologic role.

SMART amplification maintains representation of relative gene expression: quantitative validation by real time PCR and application to studies of alcoholic liver disease in primates.

The small quantities of tissue available for most studies of human disease are a significant limitation for meaningful gene expression profiling. The Atlas Switch Mechanism At the 5' end of Reverse Transcript (SMART) probe amplification kit uses as little as 50 ng of total RNA to generate complex cDNA probes for DNA array and other analyses. However, the extent to which this attractive methodology maintains representation of relative gene expression has not been quantified. In this study, we demonstrate using real-time quantitative PCR analysis that the relative expression levels of a range of low- to high-abundance mRNAs are retained after SMART amplification independent of transcript abundance and full-length transcript, coding region and PCR product size. Using this technology, a mean amplification of 3800-fold was achieved in human liver samples, greatly enhancing the ability to perform replicate DNA array experiments. Probes generated with the SMART amplification method were used to detect increased expression of genes involved with inflammation, fibrosis, xenobiotic metabolism, immune function, oxidant stress and endothelium in liver from the baboon model of alcoholic liver disease.

A trial of "standard" outpatient alcoholism treatment vs. a minimal treatment control.

This study sought to examine the effectiveness of a "standard" outpatient alcoholism treatment (ST) program. An outpatient alcoholism treatment as it is commonly practiced in the US (with group and individual therapy, and an emphasis on Alcoholics Anonymous [AA]), was compared with a minimal treatment (MT) approach (weekly alcohol education movies). At 6 months, ST patients surpassed those in MT in terms of complete abstinence, reduction in amount of alcohol consumed, length of sobriety at follow-up, improvement in employment status, number of AA meetings attended, and lower initial drop-out. It is concluded that a ST approach is more helpful than MT in treating severely alcohol-dependent individuals who have not been able to cut down drinking on their own. Those already drinking less appeared to be helped by MT.

Alcoholic fatty liver: its pathogenesis and mechanism of progression to inflammation and fibrosis.

Liver disease in the alcoholic is due not only to malnutrition but also to ethanol's hepatotoxicity linked to its metabolism by means of the alcohol dehydrogenase and cytochrome P450 2E1 (CYP2E1) pathways and the resulting production of toxic acetaldehyde. In addition, alcohol dehydrogenase-mediated ethanol metabolism generates the reduced form of nicotinamide adenine dinucleotide (NADH), which promotes steatosis by stimulating the synthesis of fatty acids and opposing their oxidation. Steatosis is also promoted by excess dietary lipids and can be attenuated by their replacement with medium-chain triglycerides. Through reduction of pyruvate, elevated NADH also increases lactate, which stimulates collagen synthesis in myofibroblasts. Furthermore, CYP2E1 activity is inducible by its substrates, not only ethanol but also fatty acids. Their excess and metabolism by means of this pathway generate release of free radicals, which cause oxidative stress, with peroxidation of lipids and membrane damage, including altered enzyme activities. Products of lipid peroxidation such as 4-hydroxynonenal stimulate collagen generation and fibrosis, which are further increased through diminished feedback inhibition of collagen synthesis because acetaldehyde forms adducts with the carboxyl-terminal propeptide of procollagen in hepatic stellate cells. Acetaldehyde is also toxic to the mitochondria, and it aggravates their oxidative stress by binding to reduced glutathione and promoting its leakage. Oxidative stress and associated cellular injury promote inflammation, which is aggravated by increased production of the proinflammatory cytokine tumor necrosis factor-alpha in the Kupffer cells. These are activated by induction of their CYP2E1 as well as by endotoxin. The endotoxin-stimulated tumor necrosis factor-alpha release is decreased by dilinoleoylphosphatidylcholine, the active phosphatidylcholine (PC) species of polyenylphosphatidylcholine (PPC). Moreover, defense mechanisms provided by peroxisome proliferator-activated receptor alpha and omega fatty acid oxidation are readily overwhelmed, particularly in female rats and also in women who have low hepatic induction of fatty acid-binding protein (L-FABPc). Accordingly, the intracellular concentration of free fatty acids may become high enough to injure membranes, thereby contributing to necrosis, inflammation, and progression to fibrosis and cirrhosis. Eventually, hepatic S-adenosylmethionine and PCs become depleted in the alcoholic, with impairment of their multiple cellular functions, which can be restored by PC replenishment. Thus, prevention and therapy opposing the development of steatosis and its progression to more severe injury can be achieved by a multifactorial approach: control of alcohol consumption, avoidance of obesity and of excess dietary long-chain fatty acids, or their replacement with medium-chain fatty acids, and replenishment of S-adenosylmethionine and PCs by using PPC. Progress in the understanding of the pathogenesis of alcoholic fatty liver and its progression to inflammation and fibrosis has resulted in prospects for their better prevention and treatment.

S-Adenosyl-L-methionine and alcoholic liver disease in animal models: implications for early intervention in human beings.

In patients with severe alcoholic liver disease (i.e., cirrhosis), a deficiency of S-adenosylmethionine (SAMe) develops as a result of decreased SAMe synthetase activity. Whether a sizeable SAMe depletion occurs already at earlier stages of alcoholic liver disease has been the subject of debate. To address this issue, rats were fed alcohol (or isocaloric carbohydrate) in Lieber-DeCarli liquid diets containing adequate amounts of protein, vitamins, and lipotropic factors, including methionine. Alcohol feeding resulted in hepatic steatosis (without fibrosis) and unchanged SAMe synthetase activity, yet SAMe concentration was already greatly decreased. This most likely resulted from oxidative stress associated with the metabolism of alcohol and the induction of cytochrome P4502E1 (CYP2E1), which generates free radicals. Indeed, the decrease in hepatic SAMe correlated with parameters of oxidative stress, such as increased 4-hydroxynonenal (measured by gas chromatography-mass spectrometry) and diminished glutathione (GSH). Decreased GSH, occurring as a result of excessive GSH consumption caused by the oxidative stress, probably generated by enhanced utilization of SAMe, a precursor of GSH, thereby explaining the depletion of SAMe. In view of the known differences between rodents and primates in the metabolism of lipotropes, my colleagues and I have also studied the interaction between alcohol and SAMe in baboons and found again that, at early stages preceding the development of cirrhosis, there was already a significant lowering of hepatic SAMe concentration, associated with a striking oxidative stress documented by decreased levels and accelerated turnover of GSH. This was associated with increased lipid peroxidation and damage to cellular membranes, including those of the mitochondria, assessed by electron microscopy. Oral administration of SAMe resulted in its hepatic repletion with a corresponding attenuation of the ethanol-induced oxidative stress and liver injury, with significantly less GSH depletion, less increases in plasma aspartate aminotransferase (AST) levels, less leakage of mitochondrial glutamic dehydrogenase into the plasma, and fewer megamitochondria. In conclusion, (1) both in rodents and in non-human primates, significant SAMe depletion occurs already at early stages of alcoholic liver disease, despite the consumption of adequate diets; (2) the decreased hepatic SAMe concentration and the associated liver lesions, including mitochondrial injury, can be corrected with SAMe supplementation; and (3) accordingly, therapeutic administration of SAMe should be the subject of a comprehensive clinical trial to assess its capacity to attenuate early stages of alcoholic liver injury in human beings.

Alcohol and health: a drink a day won't keep the doctor away.

We should not advise patients to start drinking alcohol for its alleged cardiovascular benefits. The negative effects of alcohol are well established, and the evidence of alcohol's benefits comes mainly from epidemiologic studies that were not well controlled for other influences, such as lifestyle factors. Moreover, we have other means of lowering cardiovascular risk that are safe and proven. Those who are healthy and whose drinking history shows little risk of developing alcohol dependency may continue to drink moderate amounts. Heavy drinkers should be advised to quit.

Beneficial effects versus toxicity of medium-chain triacylglycerols in rats with NASH.

BACKGROUND/AIMS: Replacing long-chain triacylglycerols (LCT) with medium-chain triacylglycerols (MCT) reduces alcohol-induced liver injury. Because of the similarity of the pathogenesis of alcohol-induced liver damage and non-alcoholic steatohepatitis (NASH), our aim was to assess whether MCT is also beneficial in NASH. METHODS: We used a rat NASH model in which corn oil (35% of total calories) was isocalorically replaced with MCT. RESULTS: Partial replacement of LCT did not ameliorate hepatic fat accumulation, 4-hydroxynonenal, collagen type I and its mRNA but it increased TNF-alpha and its mRNA (p<0.001). However, in rats given the high-fat diet restricted to 2/3 of the amount they were consuming, these adverse effects decreased, with and without MCT including less liver steatosis and lower triacylglycerols, but without beneficial effects of MCT. When 70% of the fat calories were replaced with MCT with no LCT remaining in the diet, no steatosis developed and hepatic TNF-alpha was low. When all MCT were given with carbohydrates (instead of LCT) hepatic TNF-alpha also decreased (p<0.001). CONCLUSIONS: MCT are not hepatotoxic, provided the diet contains no significant amount of LCT. Total replacement of dietary LCT with MCT fed ad libitum is beneficial whereas partial replacement becomes hepatotoxic, unless the dietary intake is restricted.

Inflammation and repair in viral hepatitis C.

Hepatitis C viral infection (HCV) results in liver damage leading to inflammation and fibrosis of the liver and increasing rates of hepatic decompensation and hepatocellular carcinoma (HCC). However, the host's immune response and viral determinants of liver disease progression are poorly understood. This review will address the determinants of liver injury in chronic HCV infection and the risk factors leading to rapid disease progression. We aim to better understand the factors that distinguish a relatively benign course of HCV from one with progression to cirrhosis. We will accomplish this task by discussion of three topics: (1) the role of cytokines in the adaptive immune response against the HCV infection; (2) the progression of fibrosis; and (3) the risk factors of co-morbidity with alcohol and human immunodeficiency virus (HIV) in HCV-infected individuals. Despite recent improvements in treating HCV infection using pegylated interferon alpha (PEGIFN-alpha) and ribavirin, about half of individuals infected with some genotypes, for example genotypes 1 and 4, will not respond to treatment or cannot be treated because of contraindications. This review will also aim to describe the importance of IFN-alpha-based therapies in HCV infection, ways of monitoring them, and associated complications.

Leptin represses matrix metalloproteinase-1 gene expression in LX2 human hepatic stellate cells.

BACKGROUND/AIMS: Collagen accumulation in liver fibrosis is due in part to decreased expression of matrix metalloproteinase (MMP)-1 relative to TIMP-1. LX-2 hepatic stellate cells produce increased amounts of collagen and tissue inhibitor of metalloproteinase (TIMP)-1 in response to leptin. The effect of leptin on MMP-1 production has not been reported. METHODS: LX-2 cells were treated with leptin with or without inhibitors. We determined: phosphorylation of Janus kinase (JAK) 1 and -2, signal transducer and activator of transcription (STAT)3 and -5, extracellular signal-regulated kinase (ERK)1/2 and p38 by Western blot; H2O2 concentration by a colorimetric method; MMP-1 mRNA levels and stability by Northern blot; MMP-1 promoter activity as well as pro-MMP-1 by ELISA; and active MMP-1 by fluorescence. RESULTS: LX-2 cells constitutively expressed the MMP-1 gene and leptin repressed the basal level of MMP-1 mRNA and its promoter activity. The repression was mediated by JAK/STAT pathway in synergism with JAK-mediated H2O2-dependent ERK1/2 and p38 pathways. ERK1/2 inhibited MMP-1 promoter activity, whereas p38 decreased the message stability, contributing to mRNA down-regulation. Inhibition of MMP-1 gene diminished secreted pro-MMP-1 and active MMP-1. CONCLUSIONS: Leptin represses MMP-1 gene expression via the synergistic actions of the JAK/STAT pathway and JAK-mediated H2O2-dependent ERK1/2 and p38 pathways.