3-nitrotyrosine and Fatty-Liver

The aim of this study was to investigate the effect of high cholesterol (CHOL) and CHOL + methionine (MET) diets on atherogenic and oxidative index parameters and on the factors that influence nitric oxide (NO) bioavailability. Also, attempts were made to determine whether dietary betaine (BET) resulted in any improvement in the changes that occurred after CHOL + MET administration.. Guinea pigs were fed chow containing 1.5% CHOL with or without 2% MET for 10 wk. A third group received the CHOL + MET + BET diet. Control groups were given standard chow or standard chow + BET. Arginine, NO, nitrotyrosine (NT), and asymmetric dimethylarginine (ADMA) levels; lipid profile; and dimethylarginine dimethylaminohydrolase (DDAH) activity were measured. The liver and aorta were subjected to histopathologic analysis.. The CHOL + MET diet caused higher serum CHOL and homocysteine levels, but no further increases were seen in aortic CHOL and diene conjugate (DC) levels and histopathologic lesions as compared with the CHOL group. Hepatic lipids and DC levels were also higher, and histopathologic lesions were more severe. CHOL + MET feeding increased ADMA and NT levels as compared with those of the CHOL-fed group. When BET (1 g/kg body weight/d) was added to the CHOL + MET diet, homocysteine and lipid levels decreased and histopathologic changes were reversed. BET diet decreased serum ADMA and hepatic and aortic DC levels and partly restored DDAH activity.. BET supplementation may be effective in preventing hyperlipidemia, disturbed NO availability, oxidative stress, and the development of fatty liver and atherosclerotic lesions that might result from excess amounts of cholesterol and methionine in the diet.

Topics: Animals; Arginine; Atherosclerosis; Betaine; Cholesterol; Cholesterol, Dietary; Diet, High-Fat; Dietary Supplements; Fatty Liver; Guinea Pigs; Hyperlipidemias; Lipid Peroxidation; Liver; Male; Malondialdehyde; Methionine; Nitric Oxide; Tyrosine

Lipid accumulation in the liver and pancreas is primarily caused by combined hyperlipidemia. However, the effect of isolated hypercholesterolemia without hypertriglyceridemia is not fully described. Therefore, our aim was to investigate whether hypercholesterolemia alone leads to alterations both in hepatic and pancreatic lipid panel and histology in rats.. Male Wistar rats were fed with 2% cholesterol +0.25% cholate-supplemented diet or standard chow for 12 weeks. Blood was collected at weeks 0, 4, 8 and 12 to measure serum cholesterol and triglyceride levels. At week 12, both the pancreas and the liver were isolated for further histological and biochemical analysis. Hepatic and plasma fatty acid composition was assessed by gas chromatography. Expression of mRNA of major enzymes involved in saturated/unsaturated fatty acid synthesis was analyzed by qPCR. In separate experiments serum enzyme activities and insulin levels were measured at week 9.. At week 12, rats fed with 2% cholesterol +0.25% cholate-supplemented diet were characterized by elevated serum cholesterol (4.09 ± 0.20 vs. 2.89 ± 0.22 mmol/L, *p < 0.05) while triglyceride (2.27 ± 0.05 vs. 2.03 ± 0.03 mmol/L) and glucose levels (5.32 ± 0.14 vs. 5.23 ± 0.10 mmol/L) remained unchanged. Isolated hypercholesterolemia increased hepatic lipid accumulation, hepatic cholesterol (5.86 ± 0.22 vs. 1.60 ± 0.15 ng/g tissue, *p < 0.05) and triglyceride contents (19.28 ± 1.42 vs. 6.78 ± 0.71 ng/g tissue, *p < 0.05), and hepatic nitrotyrosine level (4.07 ± 0.52 vs. 2.59 ± 0.31 ng/mg protein, *p < 0.05). The histology and tissue lipid content of the pancreas was not affected. Serum total protein level, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities remained unchanged in response to isolated hypercholesterolemia while serum alkaline phosphatase activity (ALP) significantly increased. Plasma insulin levels did not change in response to isolated hypercholesterolemia suggesting an intact endocrine function of the pancreas. Isolated hypercholesterolemia caused a significantly increased hepatic and serum fatty acid level associated with a marked alteration of fatty acid composition. Hepatic expression of Δ9-desaturase (SCD1) was increased 4.92×, while expression of Δ5-desaturase and Δ6-desaturase were decreased (0.447× and 0.577×, respectively) due to isolated hypercholesterolemia.. Isolated hypercholesterolemia leads to hepatic steatosis and marked alterations in the hepatic lipid profile without affecting the pancreas. Altered fatty acid profile might mediate harmful effects of cholesterol in the liver.

Topics: Animals; Blood Glucose; Body Weight; Cholesterol; Enzymes; Fatty Acids; Fatty Liver; Hypercholesterolemia; Insulin; Liver; Male; Nitrosative Stress; Organ Size; Oxidative Stress; Pancreas; Rats, Wistar; RNA, Messenger; Triglycerides; Tyrosine

Intestinal microbiota and barrier functions seem to play an important role in the development of non-alcoholic fatty liver disease (NAFLD). However, whether these changes are an early event in the development of NAFLD or are primarily associated with later stages of the disease, has not yet been clarified. Using a pair-feeding model, we determined the effects of a short-term intake of a fat-, fructose- and cholesterol-rich diet (FFC) on the development of early hepatic steatosis and markers of intestinal barrier function in mice treated with and without non-resorbable antibiotics (AB). For four days, C57BL/6J mice were either pair-fed a control diet or a FFC diet ± AB (92 mg/kg body weight (BW) polymyxin B and 216 mg/kg BW neomycin). Hepatic steatosis and markers of inflammation, lipidperoxidation and intestinal barrier function were assessed. Lipid accumulation and early signs of inflammation found in the livers of FFC-fed mice were markedly attenuated in FFC + AB-fed animals. In FFC-fed mice the development of NAFLD was associated with a significant loss of tight junction proteins and an induction of matrix metalloproteinase-13 in the upper parts of the small intestine as well as significantly higher portal endotoxin levels and an induction of dependent signaling cascades in the liver. As expected, portal endotoxin levels and the expression of dependent signaling cascades in liver tissue were almost at the level of controls in FFC + AB-fed mice. However, FFC + AB-fed mice were also protected from the loss of zonula occludens-1 and partially of occludin protein in small intestine. Our data suggest that the development of early diet-induced hepatic steatosis in mice at least in part results from alterations of intestinal barrier function.

Topics: Animals; Anti-Bacterial Agents; Biomarkers; Cholesterol, Dietary; Diet; Dietary Fats; Disease Models, Animal; Endotoxins; Fatty Liver; Female; Fructose; Gastrointestinal Microbiome; Intestine, Small; Lipid Peroxidation; Lipogenesis; Matrix Metalloproteinase 13; Mice; Mice, Inbred C57BL; Nitric Oxide Synthase Type II; Occludin; Toll-Like Receptor 4; Triglycerides; Tyrosine; Zonula Occludens-1 Protein

Early nicotine exposure causes future obesity and insulin resistance. We evaluated the long-term effect of the maternal nicotine exposure during lactation in liver oxidative status, insulin sensitivity and morphology in adult offspring. Two days after birth, osmotic minipumps were implanted in the dams: nicotine (N), 6 mg/kg/day for 14 days or saline (C). Offspring were killed at 180 days. Protein content of superoxide dismutase, glutathione peroxidase, catalase, nitrotyrosine, 4HNE, IRS1, Akt1 and PPARs were measured. MDA, bound protein carbonyl content, SOD, GPx and catalase activities were determined in liver and plasma. Hepatic morphology and triglycerides content were evaluated. Albumin and bilirubin were determined. In plasma, N offspring had higher catalase activity, and SOD/GPx ratio, albumin and bilirubin levels but lower MDA content. In liver, they presented higher MDA and 4HNE levels, bound protein carbonyl content, SOD activity but lower GPx activity. N offspring presented an increase of lipid droplet, higher triglyceride content and a trend to lower PPARα in liver despite unchanged insulin signaling pathway. Early nicotine exposure causes oxidative stress in liver at adulthood, while protect against oxidative stress at plasma level. In addition, N offspring develop liver microsteatosis, which is related to oxidative stress but not to insulin resistance.

Topics: Animals; Animals, Newborn; Antioxidants; Bilirubin; Catalase; Dose-Response Relationship, Drug; Fatty Liver; Female; Glutathione Peroxidase; Insulin; Insulin Receptor Substrate Proteins; Insulin Resistance; Lactation; Liver; Male; Maternal Exposure; Nicotine; Oxidative Stress; Protein Carbonylation; Proto-Oncogene Proteins c-akt; Rats; Rats, Wistar; Serum Albumin; Signal Transduction; Superoxide Dismutase; Triglycerides; Tyrosine

The mechanism(s) by which alcohol causes cell injury are still not clear but a major mechanism appears to be the role of lipid peroxidation and oxidative stress in alcohol toxicity. CYP2E1-generated ROS contributes to the ethanol-induced oxidant stress and inhibition of CYP2E1 activity decreases ethanol-induced fatty liver. The transcription factor Nrf2 regulates the expression of many cytoprotective enzymes which results in cellular protection against a variety of toxins.. The current study was designed to evaluate the ability of sulforaphane, an activator of Nrf2, to blunt CYP2E1-dependent, ethanol-induced steatosis in vivo and in vitro.. The sulforaphane treatment activated Nrf2, increased levels of the Nrf2 target heme oxygenase-1 and subsequently lowered oxidant stress as shown by the decline in lipid peroxidation and 3-nitrotyrosine protein adducts and an increase in GSH levels after the acute ethanol treatment. It decreased ethanol-elevated liver levels of triglycerides and cholesterol and Oil Red O staining. Similar results were found in vitro as addition of sulforaphane to HepG2 E47 cells, which express CYP2E1, elevated Nrf2 levels and decreased the accumulation of lipid in cells cultured with ethanol. Sulforaphane treatment had no effect on levels of or activity of CYP2E1.. Sulforaphane proved to be an effective in vivo inhibitor of acute ethanol-induced fatty liver in mice.. The possible amelioration of liver injury which occurs under these conditions by chemical activators of Nrf2 is of clinical relevance and worthy of further study.

Topics: Animals; Anti-Infective Agents, Local; Anticarcinogenic Agents; Autophagy; Blotting, Western; Cytochrome P-450 CYP2E1; Ethanol; Fatty Liver; Glutathione; Heme Oxygenase-1; Hep G2 Cells; Humans; Isothiocyanates; Lipid Peroxidation; Liver; Male; Mice; NF-E2-Related Factor 2; Oxidative Stress; Sulfoxides; Tyrosine

Recently a new rat model for type 2 diabetes the Zucker diabetic Sprague-Dawley (ZDSD/Pco) was created. In this study we sought to characterize the development of diabetic neuropathy in ZDSD rats using age-matched Sprague-Dawley rats as a control. Rats were examined at 34 weeks of age 12 weeks after the onset of hyperglycemia in ZDSD rats. At this time ZDSD rats were severely insulin resistant with slowing of both motor and sensory nerve conduction velocities. ZDSD rats also had fatty livers, elevated serum free fatty acids, triglycerides, and cholesterol, and elevated sciatic nerve nitrotyrosine levels. The corneas of ZDSD rats exhibited a decrease in subbasal epithelial corneal nerves and sensitivity. ZDSD rats were hypoalgesic but intraepidermal nerve fibers in the skin of the hindpaw were normal compared to Sprague-Dawley rats. However, the number of Langerhans cells was decreased. Vascular reactivity of epineurial arterioles, blood vessels that provide circulation to the sciatic nerve, to acetylcholine and calcitonin gene-related peptide was impaired in ZDSD rats. These data indicate that ZDSD rats develop many of the neural complications associated with type 2 diabetes and are a good animal model for preclinical investigations of drug development for diabetic neuropathy.

Topics: Age Factors; Animals; Arterioles; Blood Glucose; Cholesterol; Diabetes Mellitus, Type 2; Diabetic Angiopathies; Diabetic Neuropathies; Disease Models, Animal; Fatty Acids, Nonesterified; Fatty Liver; Langerhans Cells; Male; Neural Conduction; Nociception; Rats, Sprague-Dawley; Rats, Zucker; Sciatic Nerve; Time Factors; Triglycerides; Tyrosine; Vasodilation

To investigate the effectiveness of antioxidant compounds in modulating mitochondrial oxidative alterations and lipids accumulation in fatty hepatocytes.. Silybin-phospholipid complex containing vitamin E (Realsil(®)) was daily administered by gavage (one pouch diluted in 3 mL of water and containing 15 mg vitamin E and 47 mg silybin complexed with phospholipids) to rats fed a choline-deprived (CD) or a high fat diet [20% fat, containing 71% total calories as fat, 11% as carbohydrate, and 18% as protein, high fat diet (HFD)] for 30 d and 60 d, respectively. The control group was fed a normal semi-purified diet containing adequate levels of choline (35% total calories as fat, 47% as carbohydrate, and 18% as protein). Circulating and hepatic redox active and nitrogen regulating molecules (thioredoxin, glutathione, glutathione peroxidase), NO metabolites (nitrosothiols, nitrotyrosine), lipid peroxides [malondialdehyde-thiobarbituric (MDA-TBA)], and pro-inflammatory keratins (K-18) were measured on days 0, 7, 14, 30, and 60. Mitochondrial respiratory chain proteins and the extent of hepatic fatty infiltration were evaluated.. Both diet regimens produced liver steatosis (50% and 25% of liver slices with CD and HFD, respectively) with no signs of necro-inflammation: fat infiltration ranged from large droplets at day 14 to disseminated and confluent vacuoles resulting in microvesicular steatosis at day 30 (CD) and day 60 (HFD). In plasma, thioredoxin and nitrosothiols were not significantly changed, while MDA-TBA, nitrotyrosine (from 6 ± 1 nmol/L to 14 ± 3 nmol/L day 30 CD, P < 0.001, and 12 ± 2 nmol/L day 60 HFD, P < 0.001), and K-18 (from 198 ± 20 to 289 ± 21 U/L day 30 CD, P < 0.001, and 242 ± 23 U/L day 60 HFD, P < 0.001) levels increased significantly with ongoing steatosis. In the liver, glutathione was decreased (from 34.0 ± 1.3 to 25.3 ± 1.2 nmol/mg prot day 30 CD, P < 0.001, and 22.4 ± 2.4 nmol/mg prot day 60 HFD, P < 0.001), while thioredoxin and glutathione peroxidase were initially increased and then decreased. Nitrosothiols were constantly increased. MDA-TBA levels were five-fold increased from 9.1 ± 1.2 nmol/g to 75.6 ± 5.4 nmol/g on day 30, P < 0.001 (CD) and doubled with HFD on day 60. Realsil administration significantly lowered the extent of fat infiltration, maintained liver glutathione levels during the first half period, and halved its decrease during the second half. Also, Realsil modulated thioredoxin changes and the production of NO derivatives and significantly lowered MDA-TBA levels both in liver (from 73.6 ± 5.4 to 57.2 ± 6.3 nmol/g day 30 CD, P < 0.01 and from 27.3 ± 2.1 nmol/g to 20.5 ± 2.2 nmol/g day 60 HFD, P < 0.01) and in plasma. Changes in mitochondrial respiratory complexes were also attenuated by Realsil in HFD rats with a major protective effect on Complex II subunit CII-30.. Realsil administration effectively contrasts hepatocyte fat deposition, NO derivatives formation, and mitochondrial alterations, allowing the liver to maintain a better glutathione and thioredoxin antioxidant activity.

Topics: Animals; Biomarkers; Choline Deficiency; Diet, High-Fat; Disease Models, Animal; Fatty Liver; Glutathione; Glutathione Peroxidase; Keratin-18; Liver; Male; Malondialdehyde; Mitochondria, Liver; Mitochondrial Proteins; Nitroso Compounds; Oxidative Phosphorylation; Oxidative Stress; Phospholipids; Rats; Rats, Wistar; Silybin; Silymarin; Sulfhydryl Compounds; Thiobarbituric Acid Reactive Substances; Thioredoxins; Time Factors; Tyrosine; Vitamin E

Genetically diabetic Akita mice, kept on a high-fat and high-cholesterol diet, and treated with the peroxisome proliferator-activated receptor-γ agonist rosiglitazone (10 mg/kg per day during 4 months), displayed rosiglitazone-induced side effects, similar to those observed in patients, including weight and fat gain and early signs of hypertrophic cardiomyopathy. As several cases of hepatotoxicity were reported in patients receiving rosiglitazone treatment, this study evaluated whether rosiglitazone also induced hepatotoxicity in these diabetic animals. Liver structure and function was analysed in wild-type and rosiglitazone-treated and untreated Akita mice, kept for 4 months on the high-fat and high-cholesterol diet. Decreased circulating levels of the liver enzymes aspartate and alanine aminotransferase and increased levels of alkaline phosphatases were observed upon rosiglitazone treatment, whereas liver weight was markedly increased. Rosiglitazone administration was associated with liver steatosis, as demonstrated by triglyceride accumulation. However, gene expression of steatosis markers in liver tissue was not markedly affected by rosiglitazone treatment, while expression of fatty acid transport protein was reduced by rosiglitazone treatment, suggesting an impairment of the fatty acid β-oxidation pathway. mRNA expression of pro- and anti-oxidant enzymes and liver 3-nitrotyrosine content was not affected. Furthermore, gene and protein expression of macrophage markers and of cell adhesion molecules did not indicate progression to steatohepatitis, whereas an unaltered collagen deposition did not suggest steatofibrosis. In conclusion, rosiglitazone treatment of diabetic Akita mice induced liver steatosis without, however, progression to more advanced stages of liver disease.

Topics: Alanine Transaminase; Alkaline Phosphatase; Animals; Aspartate Aminotransferases; Cholesterol, Dietary; Diabetes Mellitus; Diet, High-Fat; Fatty Liver; Gene Expression; Genotype; Liver; Male; Mice; Mice, Inbred C57BL; Organ Size; PPAR gamma; RNA, Messenger; Rosiglitazone; Thiazolidinediones; Triglycerides; Tyrosine

Oxidative and nitrative stress responses resulting from inflammation exacerbate liver injury associated with nonalcoholic steatohepatitis (NASH) by inducing lipid peroxidation and protein nitration. The objective of this study was to investigate whether the anti-inflammatory properties of green tea extract (GTE) would protect against NASH by suppressing oxidative and nitrative damage mediated by proinflammatory enzymes. Obese mice (ob/ob) and their 5-week-old C57BL6 lean littermates were fed 0%, 0.5% or 1% GTE for 6 weeks (n=12-13 mice/group). In obese mice, hepatic lipid accumulation, inflammatory infiltrates and serum alanine aminotransferase activity were markedly increased, whereas these markers of hepatic steatosis, inflammation and injury were significantly reduced among obese mice fed GTE. GTE also normalized hepatic 4-hydroxynonenal and 3-nitro-tyrosine (N-Tyr) concentrations to those observed in lean controls. These oxidative and nitrative damage markers were correlated with alanine aminotransferase (P<.05; r=0.410-0.471). Improvements in oxidative and nitrative damage by GTE were also associated with lower hepatic nicotinamide adenine dinucleotide phosphate oxidase activity. Likewise, GTE reduced protein expression levels of hepatic myeloperoxidase and inducible nitric oxide synthase and decreased the concentrations of nitric oxide metabolites. Correlative relationships between nicotinamide adenine dinucleotide phosphate oxidase and hepatic 4-hydroxynonenal (r=0.364) as well as nitric oxide metabolites and N-Tyr (r=0.598) suggest that GTE mitigates lipid peroxidation and protein nitration by suppressing the generation of reactive oxygen and nitrogen species. Further study is warranted to determine whether GTE can be recommended as an effective dietary strategy to reduce the risk of obesity-triggered NASH.

Topics: Alanine Transaminase; Aldehydes; Animals; Anti-Inflammatory Agents; Fatty Liver; Inflammation; Lipid Peroxidation; Liver; Male; Mice; Mice, Inbred C57BL; Mice, Obese; NADPH Oxidases; Nitric Oxide Synthase Type II; Non-alcoholic Fatty Liver Disease; Obesity; Oxidative Stress; Peroxidase; Plant Extracts; Reactive Oxygen Species; Stress, Physiological; Tea; Tyrosine

This study investigated the role of inducible nitric oxide synthase (iNOS) in failure of ethanol-induced fatty liver grafts. Rat livers were explanted 20 h after gavaging with ethanol (5 g/kg) and storing in UW solution for 24h before implantation. Hepatic oil red O staining-positive areas increased from ∼2 to ∼33% after ethanol treatment, indicating steatosis. iNOS expression increased ∼8-fold after transplantation of lean grafts (LG) and 25-fold in fatty grafts (FG). Alanine aminotransferase release, total bilirubin, hepatic necrosis, TUNEL-positive cells, and cleaved caspase-3 were higher in FG than LG. A specific iNOS inhibitor 1400W (5 μM in the cold-storage solution) blunted these alterations by >42% and increased survival of fatty grafts from 25 to 88%. Serum nitrite/nitrate and hepatic nitrotyrosine adducts increased to a greater extent after transplantation of FG than LG, indicating reactive nitrogen species (RNS) overproduction. Phospho-c-Jun and phospho-c-Jun N-terminal kinase-1/2 (JNK1/2) were higher in FG than in LG, indicating more JNK activation in fatty grafts. RNS formation and JNK activation were blunted by 1400W. Mitochondrial polarization and cell death were visualized by intravital multiphoton microscopy of rhodamine 123 and propidium iodide, respectively. After implantation, viable cells with depolarized mitochondria were 3-fold higher in FG than in LG and 1400W decreased mitochondrial depolarization in FG to the levels of LG. Taken together, iNOS is upregulated after transplantation of FG, leading to excessive RNS formation, JNK activation, mitochondrial dysfunction, and severe graft injury. The iNOS inhibitor 1400W could be an effective therapy for primary nonfunction of fatty liver grafts.

Topics: Adenosine; Alanine Transaminase; Allopurinol; Amidines; Animals; Benzylamines; Bilirubin; Caspase 3; Ethanol; Fatty Liver; Female; Gene Expression; Glutathione; Graft Survival; Insulin; JNK Mitogen-Activated Protein Kinases; Liver Transplantation; Membrane Potential, Mitochondrial; Mitochondria; Nitrates; Nitric Oxide Synthase Type II; Nitrites; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Lew; Reactive Nitrogen Species; Tyrosine

To test the hypothesis that the inducible nitric oxide synthase (iNOS) is involved in mediating the toll-like receptor 4-dependent effects on the liver in the onset of fructose-induced steatosis, wild-type and iNOS knockout (iNOS(-/-)) mice were either fed tap water or 30% fructose solution for 8 weeks. Chronic consumption of 30% fructose solution led to a significant increase in hepatic steatosis and inflammation as well as plasma alanine-aminotransferase levels in wild-type mice. This effect of fructose feeding was markedly attenuated in iNOS(-/-) mice. Hepatic lipidperoxidation, concentration of phospho-IκB, nuclear factor κB activity, and tumor necrosis factor-α mRNA level were significantly increased in fructose-fed wild-type mice, whereas in livers of fructose-fed iNOS(-/-) mice, lipidperoxidation, phospho-IκB, nuclear factor κB activity, and tumor necrosis factor-α expression were almost at the level of controls. However, portal endotoxin levels and hepatic myeloid differentiation factor 88 expression were significantly higher in both fructose-fed groups compared to controls. Taken together, these data suggest that (i) the formation of reactive oxygen species in liver is a key factor in the onset of fatty liver and (ii) iNOS is involved in mediating the endotoxin/toll-like receptor 4-dependent effects in the development of fructose-induced fatty liver.

Topics: Aldehydes; Animals; Cells, Cultured; Coculture Techniques; Endotoxins; Fatty Liver; Fructokinases; Fructose; Glutathione; I-kappa B Proteins; Insulin Resistance; Kupffer Cells; Lipid Peroxidation; Liver; Mice; Mice, Inbred C57BL; Mice, Knockout; Myeloid Differentiation Factor 88; NF-kappa B; Nitric Oxide Synthase Type II; Organ Size; Toll-Like Receptor 4; Transcription, Genetic; Triglycerides; Tumor Necrosis Factor-alpha; Tyrosine; Weight Gain

Numerous steatotic livers are discarded as unsuitable for transplantation (TR) because of their poor tolerance of ischemia/reperfusion (I/R). Cyclic adenosine 3',5'-monophosphate (cAMP)-elevating agents protect against I/R injury both in nonsteatotic livers that have been removed from non-heart-beating donors and subjected to warm ischemia or cold ischemia (CIS) and in perfused, isolated livers. Ischemic preconditioning (PC), which is based on brief periods of I/R, protects steatotic liver grafts, but the mechanism that is responsible is poorly understood. This study examines the role of cAMP in the vulnerability shown by steatotic livers to TR-associated I/R injury and the benefits of PC in this situation. Steatotic livers with or without PC were transplanted into Zucker rats. The hepatic levels of cAMP were measured and altered pharmacologically. Our results indicate that the cAMP levels in the nonsteatotic liver grafts were similar to those found in a sham group. However, high cAMP levels were observed in steatotic liver grafts. The blockage of cAMP generation by adenylate cyclase inhibitor pre-treatment or PC had the following results: reduced hepatic injury and increased survival of steatotic graft recipients; greater preservation of adenosine triphosphate (ATP) and reduced lactate accumulation throughout CI. This blockade of cAMP by a nitric oxide-dependent mechanism protected steatotic liver grafts against oxidative stress and microvascular disorders after reperfusion. In conclusion, cAMP blocking-based strategies could protect patients against the inherent risk of steatotic liver failure after TR.

Topics: Animals; Cyclic AMP; Fatty Liver; Homozygote; Hyaluronic Acid; Ischemia; Ischemic Preconditioning; Liver; Liver Transplantation; Oxidative Stress; Rats; Rats, Zucker; Time Factors; Transaminases; Tyrosine

The pathogenesis of nonalcoholic steatohepatitis (NASH) is still unclear. Recently, the 2-hit hypothesis was proposed, in which nitric oxide production, representing oxidative stress, was proposed as a very important candidate for the second hit.. The total study period was 10 weeks. A total of 20 rats were randomly divided into 2 groups. Group 1 was administered the Choline-Deficient, l-Amino Acid-Defined diet to produce a NASH model, and Group 2 as control received the Choline-Sufficient, l-Amino Acid-defined diet. The blood and tissue concentrations of nitrate + nitrite were measured using the Griess reagent and the expression levels of inducible nitric oxide synthase (iNOS) proteins and mRNA was determined by Western blotting.. In regard to nitric oxide (NO) and NO metabolites, there were significant differences in the blood (especially portal venous blood) as well as tissue (liver and visceral fat) concentrations between the 2 animal groups; the amounts of NO metabolites in the tissues were much higher in the NASH models. The level of nitrotyrosine was much markedly higher in the NASH models than in the controls. In regard to the tissue expression of iNOS a significant difference between the 2 groups was found in the visceral fat, especially in the mesenterium.. Based on these results, we hypothesize that the iNOS expression and NO levels in the visceral fat increase, with increased diffusion of NO and its metabolites into the liver, resulting in increased nitrotyrosine formation in the liver; this, in turn, induces inflammation, apoptosis, and fibrosis in the liver, which are one of the characteristic features of NASH.

Topics: Amino Acids; Animals; Blotting, Western; Choline; Choline Deficiency; Diet; Disease Models, Animal; Fatty Liver; Intra-Abdominal Fat; Liver; Male; Nitrates; Nitric Oxide; Nitric Oxide Synthase Type II; Nitrites; Oxidative Stress; Rats; Rats, Inbred F344; RNA, Messenger; Tyrosine

Steatotic liver grafts are excluded for partial liver transplantation because of increased risk of primary nonfunction. Mechanisms underlying the failure of fatty partial liver grafts (FPG) remain unknown. This study investigated whether inducible nitric oxide synthase (iNOS) plays a role in failure of FPG.. Fatty livers were induced by feeding rats a high-fat high-fructose diet for 2 weeks. Hepatic triglyceride was approximately 9-fold higher in rats fed the high-fat high-fructose diet than those fed a low-fat low-fructose diet. Lean and fatty liver explants were reduced in size ex vivo to approximately one third, stored in the University of Wisconsin cold storage solution for 2 hr, and implanted.. Posttransplantational hepatic iNOS expression and reactive nitrogen species (RNS) formation (nitrite and nitrate levels and 3-nitrotyrosine adducts) increased more profoundly in FPG than in lean partial grafts (LPG). Serum alanine aminotransferase and bilirubin were 2- and 5.5-fold higher after transplantation of FPG than LPG. 5-Bromo-2'-deoxyuridine incorporation was 25% in LPG but only 5% in FPG, and graft weight increased by 64% in LPG while remaining unchanged in FPG. All rats that received FPG died, whereas all those receiving LPG survived. N-(1-naphtyl)ethylendiamine dihydrochloride (5 microM), a specific iNOS inhibitor, largely blunted the production of RNS, prevented the increase of alanine aminotransferase and bilirubin, restored liver regeneration, and improved survival of FPG. Mitochondrial cytochrome c oxidase-IV, ATP synthase-beta, and NADH dehydrogenase-3 decreased markedly in FPG, and these effects were blocked by N-(1-naphtyl)ethylendiamine dihydrochloride.. Thus, hepatic steatosis causes failure of partial liver grafts, most likely by increasing RNS that leads to mitochondrial damage and dysfunction.

Topics: Alanine Transaminase; Animals; Bilirubin; Enzyme Inhibitors; Fatty Liver; Graft Survival; Immunohistochemistry; Liver; Liver Transplantation; Male; Mitochondria; Nitric Oxide Synthase Type II; Rats; Rats, Inbred Lew; Sutures; Triglycerides; Tyrosine

NAFLD (non-alcoholic fatty liver disease), associated with obesity and the cardiometabolic syndrome, is an important medical problem affecting up to 20% of western populations. Evidence indicates that mitochondrial dysfunction plays a critical role in NAFLD initiation and progression to the more serious condition of NASH (non-alcoholic steatohepatitis). Herein we hypothesize that mitochondrial defects induced by exposure to a HFD (high fat diet) contribute to a hypoxic state in liver and this is associated with increased protein modification by RNS (reactive nitrogen species). To test this concept, C57BL/6 mice were pair-fed a control diet and HFD containing 35% and 71% total calories (1 cal approximately 4.184 J) from fat respectively, for 8 or 16 weeks and liver hypoxia, mitochondrial bioenergetics, NO (nitric oxide)-dependent control of respiration, and 3-NT (3-nitrotyrosine), a marker of protein modification by RNS, were examined. Feeding a HFD for 16 weeks induced NASH-like pathology accompanied by elevated triacylglycerols, increased CYP2E1 (cytochrome P450 2E1) and iNOS (inducible nitric oxide synthase) protein, and significantly enhanced hypoxia in the pericentral region of the liver. Mitochondria from the HFD group showed increased sensitivity to NO-dependent inhibition of respiration compared with controls. In addition, accumulation of 3-NT paralleled the hypoxia gradient in vivo and 3-NT levels were increased in mitochondrial proteins. Liver mitochondria from mice fed the HFD for 16 weeks exhibited depressed state 3 respiration, uncoupled respiration, cytochrome c oxidase activity, and mitochondrial membrane potential. These findings indicate that chronic exposure to a HFD negatively affects the bioenergetics of liver mitochondria and this probably contributes to hypoxic stress and deleterious NO-dependent modification of mitochondrial proteins.

Topics: Animals; Cell Respiration; Cytochrome P-450 CYP2E1; Dietary Fats; Fatty Liver; Hypoxia; Liver; Male; Membrane Potential, Mitochondrial; Mice; Mice, Inbred C57BL; Mitochondria, Liver; Mitochondrial Proteins; Nitric Oxide; Nitric Oxide Synthase Type II; Oxidative Stress; Tyrosine

Oxidative stress plays a major role in the pathogenesis of nonalcoholic steatohepatitis (NASH). Mitochondrial abnormality may be associated with the onset and progression of NASH via excessive formation of mitochondrial reactive oxygen species. This study aimed to investigate the role of mitochondrial abnormality in NASH in relation to oxidative stress.. Twenty-six patients with NASH, 11 with simple steatosis, and 10 healthy volunteers underwent clinico-pathological analysis. The liver/spleen ratio, an index of the hepatic fat content, was evaluated with computed tomography. Plasma glutathione levels were measured as an antioxidative marker, and the urinary 8-isoprostane levels and 3-nitrotyrosine staining in the liver as an oxidative stress marker. Mitochondrial abnormality was estimated by serum levels of mitochondria aspartate transaminase (mAST) and the mitochondrial staining in the liver.. Urinary 8-isoprostane levels were higher in NASH than in the healthy volunteers, whereas plasma glutathione levels were similar in the 2 groups. In NASH, urinary 8-isoprostane levels positively correlated with alanine transaminase levels and negatively with the liver/spleen ratio. The 3-nitrotyrosine staining was more advanced in simple steatosis and NASH than in the normal liver, but was similar in simple steatosis and NASH. In contrast to the normal mAST levels in the healthy volunteers and simple steatosis, serum mAST levels were elevated in one-fourth of the NASH patients and positively correlated with urinary 8-isoprostane levels in NASH. Most cases of NASH showed diffuse or focal but intense mitochondrial staining in the liver in contrast to scattered staining in simple steatosis.. Our present study demonstrated that in NASH, the enhanced oxidative stress may be associated with hepatic inflammation and the degree of fat infiltration in the liver. However, simple steatosis and NASH were both exposed to oxidative stress, while NASH alone was associated with mitochondrial abnormality. These findings indicate that mitochondrial abnormality may play a role in the onset and progression of NASH in correlation with oxidative stress.

Topics: Adult; Aspartate Aminotransferases; Biopsy; Dinoprost; Fatty Liver; Female; Glutathione; Humans; Liver; Male; Middle Aged; Mitochondria, Liver; Oxidative Stress; Reference Values; Spleen; Statistics as Topic; Tomography, X-Ray Computed; Tyrosine

Insulin resistance is present in almost all patients with nonalcoholic steatohepatitis (NAFLD), and mitochondrial dysfunction likely plays a critical role in the progression of fatty liver into nonalcoholic steatohepatitis. Rosiglitazone, a selective ligand of peroxisome proliferator-activated receptor gamma (PPARgamma), is an insulin sensitizer drug that has been used in a number of insulin-resistant conditions, including NAFLD. The aim of this study was to analyze the effects of rosiglitazone on the liver histology and mitochondrial function in a model of NAFLD. All studies were carried out in wild-type and leptin-deficient (ob/ob) C57BL/6J mice. Ob/ob mice were treated with 1 mg/kg/day, and activity of mitochondrial respiratory chain (MRC), beta-oxidation, lipid peroxidation, glutathione content in mitochondria, and 3-tyrosine-nitrated proteins in mitochondria were measured. In addition, histological and ultrastructural changes induced by rosiglitazone were also noted. Rosiglitazone treatment increased liver steatosis, particularly microvesicular steatosis. In these animals, mitochondria were markedly swollen with cristae peripherally placed. In ob/ob mice, this drug increased PPARgamma protein expression and lipid peroxide content in liver tissue and decreased glutathione concentration in mitochondria. Rosiglitazone suppressed the activity of complex I of the MRC in ob/ob mice, but did not affect beta-oxidation. 3-Tyrosine nitrated mitochondrial proteins, significantly increased in ob/ob mice, were not modified by rosiglitazone treatment.. Treatment of ob/ob mice with rosiglitazone did not reverse histological lesions of NAFLD or improve MRC activity. On the contrary, rosiglitazone reduced activity of complex I and increased oxidative stress and liver steatosis.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Body Weight; Fatty Liver; Lipid Peroxidation; Liver; Male; Mice; Mice, Inbred C57BL; Mice, Obese; Microscopy, Electron; Mitochondria, Liver; PPAR gamma; Rosiglitazone; Thiazolidinediones; Tyrosine

To investigate the lipotropic action of betaine on plasma lipoproteins and tissue lipids.. Adult mice, wild type (+/+) or heterozygous (+/-) for a disruption of the methylenetetrahydrofolate reductase (Mthfr) gene, were supplemented with betaine for 1 year and compared with mice on control diets. Outcome measures were plasma homocysteine and lipoprotein levels, aortic and liver morphology, and liver staining for 3-nitrotyrosine (oxidative stress marker) and Apolipoprotein A-I (ApoA-I). We also investigated short-term effects of supplemental betaine on plasma lipoproteins in Mthfr +/+ and +/- mice. Both genotypes showed significantly lower plasma homocysteine after long-term betaine supplementation, and lower plasma triglycerides and higher HDL-cholesterol after both short- and long-term betaine. Lipid accumulation in liver and aortic wall tended to be lower in Mthfr+/+ compared to Mthfr+/- mice and in betaine-supplemented compared to unsupplemented mice. Nitrotyrosine staining was higher and ApoA-I staining was lower in livers of Mthfr+/- compared to Mthfr+/+ mice. Betaine did not affect staining of nitrotyrosine but increased ApoA-I staining. A significant negative correlation was observed between plasma homocysteine and liver ApoA-I.. Mild MTHFR deficiency in mice is associated with increased risk for atherosclerotic disease. Betaine has a lipotropic effect, which is associated with a reduction in homocysteine, an increase in ApoA-I and an amelioration of the atherogenic risk profile.

Topics: Animals; Animals, Genetically Modified; Aorta; Apolipoprotein A-I; Betaine; Cholesterol; Disease Models, Animal; Fatty Liver; Homocysteine; Hyperhomocysteinemia; Immunohistochemistry; Lipotropic Agents; Methylenetetrahydrofolate Reductase (NADPH2); Mice; Time; Triglycerides; Tyrosine

Although nonalcoholic fatty liver disease (NAFLD) is associated with the metabolic syndrome, the mechanisms responsible for the development of NAFLD at different stages of the development of insulin resistance are unknown. Diet, adipokines, and nitrosative stress have been linked to both NAFLD and insulin resistance.. We aimed to identify the factors that are specifically associated with NAFLD at different stages in the development of insulin resistance and the metabolic syndrome.. Circulating concentrations of adipokines (ie, tumor necrosis factor-alpha, adiponectin, resistin, leptin, and interleukin-6), markers of nitrosative stress (nitrotyrosine), dietary habits, and MTP -493G/T polymorphism were cross-sectionally related to the presence and severity of insulin resistance (homeostasis model assessment index for insulin resistance: >or=2), the metabolic syndrome, and fatty liver in 64 nonobese nondiabetic patients with NAFLD (33 insulin-sensitive and 31 insulin-resistant subjects) and 74 control subjects without liver disease who were matched for sex, BMI, homeostasis model assessment index for insulin resistance status, and the various features of the metabolic syndrome.. Persons with NAFLD had greater systemic nitrosative stress and a lower intake of vitamins A and E than did control subjects, but the 2 groups did not differ significantly in any other features. Nitrotyrosine and adiponectin concentrations and vitamin A intakes independently predicted alanine aminotransferase concentrations in NAFLD patients and liver histology in a subgroup of 29 subjects with biopsy-proven nonalcoholic steatohepatitis.. Oxidative stress is operating in NAFLD and nonalcoholic steatohepatitis, even in the absence of insulin resistance, the metabolic syndrome, and hypoadiponectinemia, which aggravate liver histology at more severe stages of metabolic disease. The possible pathogenetic role of reduced vitamin A intake in NAFLD warrants further investigation.

Topics: Adipokines; Analysis of Variance; Biomarkers; Carrier Proteins; Case-Control Studies; Fatty Liver; Feeding Behavior; Female; Humans; Insulin Resistance; Liver; Male; Metabolic Syndrome; Middle Aged; Oxidative Stress; Polymorphism, Genetic; Risk Factors; Severity of Illness Index; Tyrosine; Vitamin A

Non-alcoholic steatohepatitis (NASH) and alcoholic liver disease (ALD) are extremely similar in the pathologic findings and pathogenesis. This study aimed to elucidate the difference and similarity between these diseases.. Twenty-six patients with NASH and 26 with ALD including 11 with alcoholic hepatitis underwent clinico-pathologic analysis. The visceral fat area and liver/spleen ratio, an index of the hepatic fat content, were evaluated with computed tomography. The hepatic iron deposit and oxidative stress induced-lipid peroxidation were estimated by Prussian blue staining and 3-nitrotyrosine staining, respectively.. The most prominent difference between NASH and ALD was the nutritional status, although elevation of AST/ALT ratio and gamma-GT is relatively characteristic of ALD. NASH was more frequently associated with diabetes mellitus as compared with ALD. The BMI and serum levels of total cholesterol and cholinesterase were higher in NASH than in ALD. Although the degree and distribution of fibrosis and necro-inflammatory reaction were similar in NASH and ALD, steatosis was more severe in NASH than in ALD. The liver/spleen ratio was lower and the visceral fat area was larger in NASH than in ALD, regardless of the coincidence of alcoholic hepatitis. Interestingly, the visceral fat area positively correlated with ALT and HOMA-IR in NASH, whereas these correlations were not observed in ALD. The hepatic iron deposit was less in NASH than in ALD, whereas lipid peroxidation in NASH was similar to that in ALD with alcoholic hepatitis and more advanced as compared with that in ALD without alcoholic hepatitis.. NASH was characterized with over-nutrition and visceral fat type obesity as compared with ALD. The visceral fat accumulation was associated with hepatic inflammation and insulin resistance in NASH, but not in ALD. The difference in the nutritional status between NASH and ALD is not only reflected in the clinical features but also may closely associate with the mechanisms of hepatocellular damage in these diseases.

Topics: Abdomen; Adipose Tissue; Adult; Data Collection; Diagnosis, Differential; Fatty Liver; Female; Hepatitis, Alcoholic; Humans; Immunohistochemistry; Iron; Lipid Peroxidation; Liver; Male; Middle Aged; Tomography, X-Ray Computed; Tyrosine