Alterations in ethyl alcohol pharmacokinetics during oral consumption of malt liquor beverages in African Americans

Background: Malt liquor (ML) beverages have become increasingly popular among urban minority groups, due partly to their inexpensive price and targeted advertising. We hypothesized that nonfermented by‐products contained in ML beverages will alter the pharmacokinetics (PK) and pharmacodynamic (PD) effects of its ethanol content. In addition, we determined the effect of alcohol dehydrogenase (ADH) genotypes on the PK following consumption of ML beverages. Methods: The study was conducted in 31 healthy adult African‐American social drinkers, mean ± SD age of 22.3 ± 1.3 years, and weight of 70.7 ± 10.9 kg. Participants were administered ethanol, in randomized order, 2‐weeks apart, in the form of oral ML beverage (6%v/v), or isocaloric solution of diet soda–ethanol (DS–Etoh) beverage (6%v/v). During each session the beverage was consumed over 4 minutes and breath alcohol concentrations (BrAC) as well as subjective and behavioral effects of ethanol were evaluated over 180 minutes. Pharmacokinetic parameters of ethanol were calculated using Michaelis–Menten elimination kinetics. The effect of ML and ADH genotype on PK was evaluated using the Wilcoxon rank‐sum test and the Wilcoxon signed rank test, respectively. Results: Results show a slower mean rate of absorption, K_a, (0.12 vs. 0.15 min^?1, p = 0.03) and a longer time to reach maximum concentration, T_max, (28 vs. 23 minute, p < 0.01) for the ML compared with DS–Etoh beverage. The ML beverage resulted in a larger area under the BrAC–time curve compared with DS–Etoh beverage (8.4 vs. 6.8 min g/dl, p = 0.02). There was no difference in the subjective PD effects between the 2 beverages. Conclusion: Results show that exposure to ethanol following the consumption of ML beverages is different compared to that following nonmalt beverages in African‐Americans. These differences may be related to nonfermented by‐products present in commercially available ML products. These PK differences do not appear to result in significant perceived alcohol PD changes, nor are they related to ADH genotype.     

1H and 31P NMR Lipidome of Ethanol‐Induced Fatty Liver

Background: Hepatic steatosis (fatty liver), an early and reversible stage of alcoholic liver disease, is characterized by triglyceride deposition in hepatocytes, which can advance to steatohepatitis, fibrosis, cirrhosis, and ultimately to hepatocellular carcinoma. In the present work, we studied altered plasma and hepatic lipid metabolome (lipidome) to understand the mechanisms and lipid pattern of early‐stage alcohol‐induced‐fatty liver. Methods: Male Fischer 344 rats were fed 5% alcohol in a Lieber‐DeCarli diet. Control rats were pair‐fed an equivalent amount of maltose‐dextrin. After 1 month, animals were killed and plasma collected. Livers were excised for morphological, immunohistochemical, and biochemical studies. The lipids from plasma and livers were extracted with methyl‐tert‐butyl ether and analyzed by 750/800 MHz proton nuclear magnetic resonance (¹H NMR) and phosphorus (³¹P) NMR spectroscopy on a 600 MHz spectrometer. The NMR data were then subjected to multivariate statistical analysis. Results: Hematoxylin and Eosin and Oil Red O stained liver sections showed significant fatty infiltration. Immunohistochemical analysis of liver sections from ethanol‐fed rats showed no inflammation (absence of CD3 positive cells) or oxidative stress (absence of malondialdehyde reactivity or 4‐hydroxynonenal positive staining). Cluster analysis and principal component analysis of ¹H NMR data of lipid extracts of both plasma and livers showed a significant difference in the lipid metabolome of ethanol‐fed versus control rats. ³¹P NMR data of liver lipid extracts showed significant changes in phospholipids similar to ¹H NMR data. ¹H NMR data of plasma and liver reflected several changes, while comparison of ¹H NMR and ³¹P NMR data offered a correlation among the phospholipids. Conclusions: Our results show that alcohol consumption alters metabolism of cholesterol, triglycerides, and phospholipids that could contribute to the development of fatty liver. These studies also indicate that fatty liver precedes oxidative stress and inflammation. The similarities observed in plasma and liver lipid profiles offer a potential methodology for detecting early‐stage alcohol‐induced fatty liver disease by analyzing the plasma lipid profile.     

Genistein improves liver function and attenuates non-alcoholic fatty liver disease in a rat model of insulin resistance

Background: The high fructose‐fed rat is widely used as a model of insulin resistance. Genistein, a soy isoflavone, has been shown to improve insulin sensitivity in this model. The present study investigated whether genistein could prevent fatty liver disease in this model. Methods: Male Wistar rats were fed a diet containing starch (control) or 60% fructose (insulin‐resistant model). Fifteen days later, rats in each dietary group were divided into two groups and were treated with either genistein (1 mg/kg per day) in dimethylsulfoxide (DMSO) or 30% DMSO alone. After 60 days, markers of liver injury, oxidative stress, interleukin (IL)‐6, tumor necrosis factor (TNF)‐α, lipids, lipoprotein profile, nitrite, and nitrosothiol in the plasma and liver were quantified. Liver sections were examined for 3‐nitrotyrosine (3‐NT) expression and pathological lesions. Results: Fructose‐fed rats displayed hyperlipidemia, significant changes in plasma lipoprotein profile, and increases in IL‐6 and TNF‐α levels compared with control. In addition, the accumulation of lipids, liver injury, a decline in liver function, inactivation of the glyoxalase system, depletion of antioxidants, and increased 3‐NT expression were observed in the fructose‐fed group. Administration of genistein to fructose‐fed rats significantly reduced these biochemical and histological abnormalities. Conclusions: Genistein activates the antioxidant profile, decreases IL‐6 and TNF‐α concentrations, prevents oxidative damage, and ameliorates fatty liver in insulin‐resistant rats.     

(Z)2-(5-(4-methoxybenzylidene)-2, 4-dioxothiazolidin-3-yl) acetic acid protects rats from CCl(4) -induced liver injury

Background and Aim: (Z)2‐(5‐(4‐methoxybenzylidene)‐2, 4‐dioxothiazolidin‐3‐yl) acetic acid (MDA) is an aldose reductase (AR) inhibitor. Recent studies suggest that AR contributes to the pathogenesis of inflammation by affecting the nuclear factor κB (NF‐κB)‐dependent expression of cytokines and chemokines and therefore could be a novel therapeutic target for inflammatory pathology. The current study evaluated the in vivo role of MDA in protecting the liver against injury and fibrogenesis caused by CCl₄ in rats, and the underlying mechanisms. Methods: A single injection of CCl₄ induced acute hepatitis, and repeated injections were used to induce hepatic fibrosis in rats. Therapeutic efficacy was assessed by comparison of the severity of hepatic injury and fibrosis in MDA ‐ treated rats versus untreated controls. Results: MDA significantly protected the liver from injury by reducing the activity of serum alanine aminotransferase, and improving the histological architecture of the liver. MDA modulated NF‐κB‐dependent activation of inflammatory cytokines by reducing hepatic mRNA levels of tumor necrosis factor‐α, interleukin‐1β, inducible nitric oxide (NO) synthase and transforming growth factor‐β. In addition, MDA attenuated oxidative stress by increasing the content of hepatic glutathione. These favorable changes were associated with suppressed hepatic NF‐κB activation by MDA. MDA treatment improved liver fibrosis in rats that received repeated CCl₄ injections. In vitro, MDA attenuated phosphorylation of IκB and activation of NF‐κB, and thus prevented biosynthesis of NO in lipopolysaccharide‐activated RAW264.7 cells. Conclusions: The present study suggests that AR is a novel therapeutic anti‐inflammatory target for the treatment of hepatitis and liver fibrosis.     

Nitric Oxide-Mediated Intestinal Injury Is Required for Alcohol-Induced Gut Leakiness and Liver Damage

Background: Alcoholic liver disease (ALD) requires endotoxemia and is commonly associated with intestinal barrier leakiness. Using monolayers of intestinal epithelial cells as an in vitro barrier model, we showed that ethanol‐induced intestinal barrier disruption is mediated by inducible nitric oxide synthase (iNOS) upregulation, nitric oxide (NO) overproduction, and oxidation/nitration of cytoskeletal proteins. We hypothesized that iNOS inhibitors [NG‐nitro‐l ‐arginine methyl ester (l ‐NAME), l ‐N⁶‐(1‐iminoethyl)‐lysine (l ‐NIL)] in vivo will inhibit the above cascade and liver injury in an animal model of alcoholic steatohepatitis (ASH). Methods: Male Sprague–Dawley rats were gavaged daily with alcohol (6 g/kg/d) or dextrose for 10 weeks ± l ‐NAME, l ‐NIL, or vehicle. Systemic and intestinal NO levels were measured by nitrites and nitrates in urine and tissue samples, oxidative damage to the intestinal mucosa by protein carbonyl and nitrotyrosine, intestinal permeability by urinary sugar tests, and liver injury by histological inflammation scores, liver fat, and myeloperoxidase activity. Results: Alcohol caused tissue oxidation, gut leakiness, endotoxemia, and ASH. l ‐NIL and l ‐NAME, but not the d ‐enantiomers, attenuated all steps in the alcohol‐induced cascade including NO overproduction, oxidative tissue damage, gut leakiness, endotoxemia, hepatic inflammation, and liver injury. Conclusions: The mechanism we reported for alcohol‐induced intestinal barrier disruption in vitro — NO overproduction, oxidative tissue damage, leaky gut, endotoxemia, and liver injury — appears to be relevant in vivo in an animal model of alcohol‐induced liver injury. That iNOS inhibitors attenuated all steps of this cascade suggests that prevention of this cascade in alcoholics will protect the liver against the injurious effects of chronic alcohol and that iNOS may be a useful target for prevention of ALD.     

Daidzin, an Antioxidant Isoflavonoid, Decreases Blood Alcohol Levels and Shortens Sleep Time Induced by Ethanol Intoxication

The extract from an edible vine, Pueraha lebata has been reported to be efficacious in lessening alcohol intoxication. In this study, we have tested the efficacy of one of the major components, daidzin, from this plant extract. When ethanol (40% solution, 3 g/kg body weight) was given to fasted rats intragastrically, blood alcohol concentration (BAC) peaked at 30 min after alcohol ingestion and reached 1.77 ± 0.14 mg/ml (mean values ±sd , n = 6). If daidzin (30 mg/kg) was mixed with the ethanol solution and given to animals intragastrically, BAC was found to peak at 90 min after alcohol ingestion and reached only 1.20 ± 0.30 mg/ml (n = 6) (p < 0.05 vs. controls). The ability of daidzin to delay and decrease peak BAC level after ethanol ingestion was also observed in fed animals, in both fasted and fed rats given alcohol without daidzin, BAC quickly declined after reaching its peak at 30 min. By contrast, BAC levels receded more slowly if daidzin was also fed to the animals. Daidzin showed a chronic effect. Rats fed daidzin for 7 days before ethanol challenge, but not on the day of challenge, also produced lower and later peak BAC levels. Interestingly, daidzin, whether fed to rats only once or chronically for 7 days, did not significantly alter activities of either alcohol dehydrogenase or mitochondrial aldehyde dehydrogenase in the liver. Further experiments demonstrated that daidzin shortened sleep time for rats receiving ethanol intragastrically (7 g/ kg) but not intraperitoneally (2 g/kg). To test whether daidzin delayed stomach-emptying, [Clpolyethylene glycol was mixed with ethanol and fed to rats. It was found that, 30 min after intragastric feeding, more ethanol and [¹⁴C]polyethylene glycol remained in the stomach if rats were also given daidzin. Because daidzin is an isoflavonoid glucoside that possesses strong antioxidant activity, two other antioxidants (i.e., vitamin E and thioctic acid) were tested. Similar to daidzin, these two antioxidants also delayed and suppressed peak BAC, as well as shortened sleep time induced by alcohol ingestion. We conclude that: (1) daidzin is effective in countering alcohol intoxication; (2) suppression of BAC by daidzin is due mainly to delay of stomach-emptying, but not to accelerated clearance of ethanol in circulation by liver enzymes; and (3) the effect of daidzin may in part be due to its antioxidant activity.     

Splenectomy reduces fibrosis and preneoplastic lesions with increased triglycerides and essential fatty acids in rat liver cirrhosis induced by a choline‐deficient L‐amino acid‐defined diet

Aim: This study investigated whether splenectomy is of significance in non‐alcoholic steatohepatitis (NASH). Methods: Five‐week‐old Wistar rats were fed a choline‐deficient diet for 8 weeks to create a NASH model. A sham‐operation or splenectomy was then performed, and rats were killed 4 weeks later. Results: Liver fibrosis and liver preneoplastic lesions were significantly reduced in the splenectomy group compared to the sham‐operation group, and α‐smooth muscle actin (SMA) expression was significantly inhibited (liver fibrosis area: sham 8.63 ± 4.09%, splenectomy 5.45 ± 3.69%, P < 0.01; preneoplastic lesion size: sham 6.56 ± 3.68 ×10⁶ µm²/cm², splenectomy 4.63 ± 3.27 ×10⁶ µm²/cm², P < 0.05; the number of preneoplastic lesions: sham 8.33 ± 3.96/cm², splenectomy 5.17 ± 1.80/cm², P < 0.01; α‐smooth muscle actin‐positive area: sham 4.41 ± 2.48%, splenectomy 2.75 ± 1.66%, P < 0.01) On the other hand, liver triglycerides and essential fatty acids were significantly increased in the splenectomy group (liver triglycerides: sham 182 ± 35.0 mg/g, splenectomy 230 ± 35.0 mg/g, P < 0.05; liver linoleic acid: sham 17.2 ± 4.9 mg/g, splenectomy 23.3 ± 6.9 mg/g, P < 0.05; liver α‐linolenic acid: sham 118 ± 36.6 µg/g, splenectomy 162 ± 51.4 µg/g, P < 0.05). In addition, expressions of hepatic fatty acid metabolism‐related genes (e.g. acyl‐CoA oxidase, liver carnitine palmitoyl‐CoA transferase I, cytochrome P450 4A, long‐chain acyl‐CoA dehydrogenase and medium‐chain acyl‐CoA dehydrogenase) were significantly inhibited in the splenectomy group. Conclusion: These findings suggest that spleen plays an important regulatory role in the fibrosis, preneoplastic lesion and lipid metabolism of liver in a rat choline‐deficient L‐amino acid model.     

Oxidative stress and regulation of anti‐oxidant enzymes in cytochrome P4502E1 transgenic mouse model of non‐alcoholic fatty liver

Background and Aim: Reactive oxygen species produced by cytochrome P4502E1 (CYP2E1) are believed to play a role in pathophysiology of non‐alcoholic fatty liver disease (NAFLD). However, little is known about the expression, protein content and activity of anti‐oxidant enzymes and the role of inducible nitric oxide synthase (iNOS), a source of reactive nitrogen species, in NAFLD. In the present study, we evaluate gene expression, protein content and activity of anti‐oxidant enzymes, and iNOS, in a CYP2E1 overexpressing model of non‐alcoholic fatty liver (NAFL). Methods: Non‐transgenic (nTg) and CYP2E1 transgenic (Tg) mice were fed rodent chow for 8 months. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), liver triglycerides, malondialdehyde and protein carbonyls were measured. Gene expression of NF‐E2‐related factor (Nrf2), superoxide dismutase‐1, ‐2 (SOD‐1,2), catalase (CAT), glutathione peroxidase (GPx), heme oxygenase‐1 (HO‐1) and iNOS were determined. Protein content, activity and nitrosylation of the enzymes were also measured. Results: Tg mice had greater CYP2E1 activity and histological liver injury. MDA and protein carbonyls were increased, indicating insufficient anti‐oxidant response. Gene expression of Nrf2, CAT, GPx, HO‐1 and iNOS were significantly increased. Protein content and enzyme activities of most anti‐oxidant enzymes were not correspondingly increased. iNOS activity and nitrosylation of CAT and SOD was greater in Tg mice liver. Conclusion: Hepatocyte‐specific CYP2E1 overexpression results in increased oxidative stress and nitrosative stress. Several anti‐oxidant enzymes are upregulated. Failure of corresponding increase in total protein and activity of anti‐oxidant enzymes suggests modification/degradation, possibly by nitrosylation, due to increased iNOS activity in a CYP2E1 overexpressing NAFL mouse model.     

Total parenteral nutrition modulates hormone release by stimulating expression and activity of inducible nitric oxide synthase in rat pancreatic islets

The expression and activities of constitutive nitric oxide synthase (cNOS) and inducible nitric oxide synthase (iNOS) in relation to insulin and glucagon secretory mechanisms were investigated in islets isolated from rats subjected to total parenteral nutrition (TPN) for 10 d. TPN is known to result in significantly increased levels of plasma lipids during the infusion time. In comparison with islets from freely fed control rats, islets taken from TPN rats at d 10 displayed a marked decrease in glucose-stimulated insulin release (4.65±0.45 ng/[islet·h] vs 10.25±0.65 for controls) (p<0.001) accompanied by a strong iNOS activity (18.3±1.1 pmol of NO/[min·mg of protein]) and a modestly reduced cNOS activity (11.3±3.2 pmol of NO/[min.mg of protein] vs 17.7±1.7 for controls) (p<0.01). Similarly, Western blots showed the expression of iNOS protein as well as a significant reduction in cNOS protein in islets from TPN-treated rats. The enhanced NO production, which is known to inhibit glucose-stimulated insulin release, was manifested as a strong increase in the cyclic guanosine 5′-monophosphate content in the islets of TPN-treated rats (1586±40 amol/islet vs 695±64 [p<0.001] for controls). Moreover, the content of cyclic adenosine monophosphate (cAMP) was greatly increased in the TPN islets (80.4±2.1 fmol/islet vs 42.6±2.6 [p<0.001] for controls). The decrease in glucose-stimulated insulin release was associated with an increase in the activity of the secretory pathway regulated by the cAMP system in the islets of TPN-treated rats, since the release of insulin stimulated by the phosphodiesterase inhibitor isobutylmethylxanthine was greatly increased both in vivo after iv injection and after in vitro incubation of isolated islets. By contrast, the release of glucagon was clearly reduced in islets taken from TPN-treated rats (33.5±1.5 pg/[islet·h] vs 45.5±2.2 for controls) (p<0.01) when islets were incubated at low glucose (1.0 mmol/L). The data show that long-term TPN treatment in rats brings about impairment of glucose-stimulated insulin release, that might be explained by iNOS expression and a marked iNOS-derived NO production in the β-cells. The release of glucagon, on the other hand, is probably decreased by a direct “nutrient effect” of the enhanced plasma lipids. The results also suggest that the islets of TPN-treated rats have developed compensatory insulin secretory mechanisms by increasing the activity of their β-cell cAMP system.     

Utility of a 13C‐methacetin breath test in evaluating hepatic injury in rats

Background and Aim: Methacetin is thought to be a good substrate for the evaluation of different cytochrome P450 enzymatic systems of liver microsomes because of its rapid metabolism and lack of toxicity in small doses. Recent studies indicate that a methacetin breath test may be a non‐invasive alternative for the evaluation of liver function since it correlates well with the severity of liver damage. It may also discriminate between different stages of liver cirrhosis and correlates with the Child–Pugh score. The application of this test in experimental liver damage in animal models has not yet been examined. This study aimed to evaluate the efficacy of the ¹³C‐methacetin breath test in assessing the extent of hepatic injury in models of acute liver failure, liver cirrhosis, and fatty liver in rats. Methods: Absorption of methacetin given per os or intraperitoneally in normal rats was evaluated. The association between liver mass and ¹³C‐methacetin breath test results was assessed in a 70% hepatectomy rat model. Fulminant hepatic failure was induced by three consecutive intraperitoneal injections of thioacetamide, 300 mg/kg, at 24 h intervals. For induction of liver cirrhosis, rats were given intraperitoneal injections of thioacetamide, 200 mg/kg, twice a week for 12 weeks. A methionine‐choline deficient diet was used for the induction of fatty liver. Rats were analyzed for ¹³C‐methacetin by BreathID (MBID) using molecular correlation spectrometry. BreathID continuously sampled the animal's breath for 60 min and displayed the results on the BreathID screen in real‐time. Results: Methacetin was absorbed well irrespective of the administration method in normal rats. Liver mass was associated with peak amplitude, complete percent dose recovery (CPDR) at 30 and 60 min and MBID peak time. A high degree of association was also demonstrated with MBID results in acute hepatitis (peak amplitude, 19.6 ± 3.4 vs 6.3 ± 1.63.4; CPDR30, 6.0 ± 3.3 vs 1.2 ± 0.5; CPDR60, 13.3 ± 4.5 vs 3.2 ± 1.4; and peak time, 31.0 ± 14.9 vs 46.9 ± 10.8 min) and liver cirrhosis (peak amplitude, 24.4 ± 2.3 vs 15.6 ± 6.4; CPDR30, 7.9 ± 1.2 vs 2.7 ± 1.0; CPDR60, 17.8 ± 2.6 vs 8.8 ± 2.1; and peak time, 30.2 ± 1.5 vs 59.6 ± 14.5 min), but not with grade of liver steatosis. Conclusions: Methacetin is well absorbed and exclusively metabolized in the liver. MBID is a sensitive test and may be a useful tool for the evaluation of functional liver mass in animal models of acute liver failure and cirrhosis. However, MBID could not distinguish between fatty liver and normal liver in rats.     

Melatonin ameliorates cerulein‐induced pancreatitis by the modulation of nuclear erythroid 2‐related factor 2 and nuclear factor‐kappaB in rats

Abstract: Melatonin exhibits a wide variety of biological effects, including antioxidant and anti‐inflammatory functions. Its antioxidant role impedes the etiopathogenesis of pancreatitis, but little is known about the signaling pathway of melatonin in the induction of antioxidant enzymes in acute pancreatitis (AP). The aim of this study was to determine whether melatonin could prevent cerulein‐induced AP through nuclear factor erythroid 2‐related factor 2 (Nrf2) and curtail inflammation by inhibition of NF‐κB. AP was induced by two intraperitoneal (i.p.) injections of cerulein at 2 h intervals (50 μg/kg) in Sprague‐Dawley rats. Melatonin (10 or 50 mg/kg/daily, i.p.) was administered 24 h before each injection of cerulein. The rats were killed 12 h after the last injection. Acinar cell degeneration, pancreatic edema, and inflammatory infiltration were significantly different in cerulein‐ and melatonin‐treated rats. Melatonin significantly reduced amylase, lipase, MPO, and MDA levels, and increased antioxidant enzyme activities including SOD and GPx, which were decreased in AP (P < 0.05). Melatonin increased the expression of NQO1, HO‐1, and SOD2 when compared with the cerulein‐induced AP group (P < 0.05). In addition, melatonin increased Nrf2 expression, and reduced expressions of tumor necrosis factor‐alpha, IL‐1β, IL‐6, IL‐8, and iNOS. The elevated nuclear binding of NF‐κB in the cerulein‐induced pancreatitis group was inhibited by melatonin. These results show that melatonin increases antioxidant enzymes and Nrf2 expression, and limits inflammatory mediators in cerulein‐induced AP. It is proposed that melatonin may play an important role in oxidative stress via the Nrf2 pathway in parallel with reduction of inflammation by NF‐κB inhibition.     

Limited therapeutic effect of N-acetylcysteine on hepatic insulin resistance in an experimental model of alcohol-induced steatohepatitis

Background: Alcohol‐related steatohepatitis is associated with increased oxidative stress, DNA damage, lipotoxicity, and insulin resistance in liver. As inflammation and oxidative stress can promote insulin resistance, effective treatment with antioxidants, for example, N‐acetylcysteine (NAC), may restore ethanol‐impaired insulin signaling in the liver. Methods: Adult male Sprague–Dawley rats were fed for 130 days with liquid diets containing 0 or 37% ethanol by caloric content, and simultaneously treated with vehicle or NAC. Chow‐fed controls were studied in parallel. Liver tissues were used for histopathology, cytokine activation, and insulin/IGF‐1 signaling assays. Results: We observed significant positive trends of increasing severity of steatohepatitis (p = 0.016) with accumulation of neutral lipid (p = 0.0002) and triglycerides (p = 0.0004) from chow to control, to the ethanol diet, irrespective of NAC treatment. In ethanol‐fed rats, NAC reduced inflammation, converted the steatosis from a predominantly microvesicular to a mainly macrovesicular histological pattern, reduced pro‐inflammatory cytokine gene expression, ceramide load, and acid sphingomyelinase activity, and increased expression of IGF‐1 receptor and IGF‐2 in liver. However, NAC did not abrogate ethanol‐mediated impairments in signaling through insulin/IGF‐1 receptors, IRS‐1, Akt, GSK‐3β, or p70S6K, nor did it significantly reduce pro‐ceramide or GM3 ganglioside gene expression in liver. Conclusions: Antioxidant treatments reduce the severity of chronic alcohol‐related steatohepatitis, possibly because of the decreased expression of inflammatory mediators and ceramide accumulation, but they do not restore insulin/IGF‐1 signaling in liver, most likely due to persistent elevation of GM3 synthase expression. Effective treatment of alcohol‐related steatohepatitis most likely requires dual targeting of oxidative stress and insulin/IGF resistance.     

Comparative effects of liraglutide 3 mg vs structured lifestyle modification on body weight,liver fat and liver function in obese patients with non‐alcoholic fatty liver disease: A pilot randomized trial

We compared the effects of weight loss induced by the glucagon‐like peptide 1‐agonist liraglutide with a structured lifestyle intervention in obese adults with non‐alcoholic fatty liver disease (NAFLD). Obese (body mass index ≥30 kg/m², mean weight 96.0 ± 16.3 kg) non‐diabetic Asian adults, with NAFLD diagnosed by liver fat fraction (LFF) ≥ 5.5% on magnetic resonance imaging without other causes of hepatic steatosis, were randomized to a supervised program of dieting (restriction by 400 kilocalories/d) plus moderate‐intensity aerobic exercise (~200 min/wk; DE group, n = 12), or liraglutide at the 3 mg daily dose approved for weight loss (LI group, n = 12), for 26 weeks. Both DE and LI groups had significant (P < .01) and similar reductions in weight (?3.5 ± 3.3 vs ?3.5 ± 2.1 kg, respectively, P = .72), LFF (?8.9 ± 13.4 vs ?7.2% ± 7.1%, P = .70), serum alanine aminotransferase (?42 ± 46 vs ?34 ± 27 U/L, P = .52) and aspartate aminotransferase (?23 ± 24 vs ?18 ± 15 U/L, P = .53). In this first randomized study comparing the 2 weight‐loss modalities for improving NAFLD, liraglutide was as effective as structured lifestyle modification.     

The novel sodium glucose transporter 2 inhibitor dapagliflozin sustains pancreatic function and preserves islet morphology in obese, diabetic rats

Aims: To investigate whether glucose lowering with the selective sodium glucose transporter 2 (SGLT2) inhibitor dapagliflozin would prevent or reduce the decline of pancreatic function and disruption of normal islet morphology. Methods: Female Zucker diabetic fatty (ZDF) rats, 7–8 weeks old, were placed on high‐fat diet. Dapagliflozin (1 mg/kg/day, p.o.) was administered for ～33 days either from initiation of high‐fat diet or when rats were moderately hyperglycaemic. Insulin sensitivity and pancreatic function were evaluated using a hyperglycaemic clamp in anaesthetized animals (n = 5–6); β‐cell function was quantified using the disposition index (DI) to account for insulin resistance compensation. Pancreata from a matched subgroup (n = 7–8) were fixed and β‐cell mass and islet morphology investigated using immunohistochemical methods. Results: Dapagliflozin, administered from initiation of high‐fat feeding, reduced the development of hyperglycaemia; after 24 days, blood glucose was 8.6 ± 0.5 vs. 13.3 ± 1.3 mmol/l (p < 0.005 vs. vehicle) and glycated haemoglobin 3.6 ± 0.1 vs. 4.8 ± 0.26% (p < 0.003 vs. vehicle). Dapagliflozin improved insulin sensitivity index: 0.08 ± 0.01 vs. 0.02 ± 0.01 in obese controls (p < 0.03). DI was improved to the level of lean control rats (dapagliflozin 0.29 ± 0.04; obese control 0.15 ± 0.01; lean 0.28 ± 0.01). In dapagliflozin‐treated rats, β‐cell mass was less variable and significant improvement in islet morphology was observed compared to vehicle‐treated rats, although there was no change in mean β‐cell mass with dapagliflozin. Results were similar when dapagliflozin treatment was initiated when animals were already moderately hyperglycaemic. Conclusion: Sustained glucose lowering with dapagliflozin in this model of type 2 diabetes prevented the continued decline in functional adaptation of pancreatic β‐cells.     

Association between changes in body composition and the increasing prevalence of fatty liver in Japanese men

Aim: Prevalence of fatty liver is increasing. In this study, to elucidate the factor that contributes most to recent increases in prevalence of fatty liver, we determined the independent predictors for the onset of fatty liver and compared these predictors between 2000 and 2005. Methods: Japanese persons, aged 30–74 years, who participated in regular health checks at Kagoshima Kouseiren Medical Health Care Center (10 336 persons in 2000 and 11 011 persons in 2005) were enrolled in the study. Diagnosis of fatty liver was performed by ultrasonography. Body fat percentage (BFP) was determined using a bipedal bioimpedance instrument. Results: The prevalence of fatty liver has increased between 2000 and 2005 in men (33.3 vs 38.5% in 2000 vs 2005, respectively, P < 0.0001), but not in women (21.3 vs 21.0%, P = 0.8101). Logistic regression analysis revealed that both body mass index (BMI) and BFP are independent predictors of fatty liver in both men and women. BMI did not change in either men (23.4 ± 2.9 vs 23.8 ± 3.0 kg/m², P = 0.0528) or women (22.8 ± 3.1 vs 22.8 ± 3.3 kg/m², P = 0.9862) during the survey period. In contrast, BFP increased in men (20.6 ± 4.7 vs 22.3 ± 5.0 kg/m², P = 0.0003), but not in women (27.4 ± 5.5 vs 28.4 ± 5.9 kg/m², P = 0.3993). There was no significant change in triglycerides and glucose levels. Conclusion: These results suggest that altered body composition, particularly increased BFP without an increase in BMI, has developed in men and is strongly associated with the increasing prevalence of fatty live amongst Japanese men.     

Inducible nitric oxide synthase is required in alcohol-induced liver injury: studies with knockout mice

BACKGROUND & AIMS : Oxidative stress contributes to early alcohol-induced liver injury, and superoxide (O(2)*-) production from NADPH oxidase plays a key role. However, the production of the free radical nitric oxide (NO*) by inducible nitric oxide synthase (iNOS) could also be involved. METHODS : To test this hypothesis, iNOS knockout (B6.129P2-Nos2 (tm1 Lau)) and wild-type mice were fed high-fat control or ethanol-containing diets for 4 weeks. RESULTS : Mean body weight gains were not significantly different between treatment groups, and average urine ethanol concentrations were similar in wild-type and iNOS knockout mice. After 4 weeks, serum alanine aminotransferase (ALT) levels were increased significantly about 4-fold over control values (29 +/- IU/L) by enteral ethanol (113 +/- 20) in wild-type mice; this effect of ethanol was significantly blunted in iNOS knockout mice (50 +/- 9). Similar protective effects against liver damage were observed if wild-type mice were treated with the iNOS inhibitor N -(3-aminomethyl)benzyl-acetamindine (1400W). Enteral ethanol also caused severe fatty accumulation, mild inflammation, and necrosis in the liver in wild-type mice but had no effect in iNOS knockout mice. The accumulation of 4-hydroxynonenal (lipid peroxidation) and 3-nitrotyrosine (reactive nitrogen species formation) protein adducts caused by alcohol was completely blocked in iNOS knockout mice. CONCLUSIONS : These data strongly support the hypothesis that iNOS is required for the pathogenesis of early alcohol-induced hepatitis by production of nitric oxide-derived pro-oxidants (e.g., peroxynitrite).     

Chronic Alcohol Consumption Disrupted Cholesterol Homeostasis in Rats: Down‐Regulation of Low‐Density Lipoprotein Receptor and Enhancement of Cholesterol Biosynthesis Pathway in the Liver

Background: Chronic alcohol consumption causes alcoholic liver disease, which is associated, or initiated, with dysregulated lipid metabolism. Very recent evidence suggested that dysregulated cholesterol metabolism plays an important role in the pathogenesis of alcoholic fatty liver diseases, however, the effects of chronic alcohol exposure on cholesterol homeostasis have not been well studied and underlying mechanisms behind are still elusive. Methods: Male Sprague–Dawley rats weighing 250 ± 5.5 g (mean ± SEM) divided into 2 groups (8 rats per group) and pair‐fed with liquid diets containing (in percent of energy intake) 18% protein, 35% fat, 12% carbohydrate, and 35% either ethanol (ethanol diet) or an isocaloric maltose‐dextrin mixture (control diet), according to Lieber and De Carli, for 4 weeks. Results: Long‐term excessive alcohol feeding to rats caused fatty liver and liver injury, which was associated with disrupted cholesterol homeostasis, characterized by increased hepatic cholesterol levels and hypercholesterolemia. Hepatic cholesterol increases were concomitant with constantly activated sterol regulatory element‐binding protein‐2 (SREBP‐2) in the liver and increased expression of 3‐hydroxy‐3‐methyl‐glutaryl‐CoA (HMG‐CoA) reductase, a rate‐limiting enzyme for cholesterol de novo synthesis, indicating enhanced cholesterol biosynthesis. Alcohol‐induced hypercholesterolemia was accompanied by decreased LDL receptor (LDLr) levels in the liver. Further investigations revealed that chronic alcohol exposure increased hepatic proprotein convertase subtilisin/kexin type 9 (PCSK9) contents to down‐regulate LDLr via a post‐translational mechanism. Moreover, alcohol feeding suppressed extracellular signal‐regulated kinase (ERK) activation in the liver. In vitro studies showed that inhibition of ERK activation was associated with decreased LDLr expression in HepG2 cells. Conclusions: Our study provides the first evidence that both increased PCSK9 expression and suppressed ERK activation in the liver contributes to alcohol‐induced hypercholesterolemia in rats.     

Expression of hepatic transporters OATP‐C and MRP2 in primary sclerosing cholangitis

Abstract: Background/Aims: In chronic cholestatic liver diseases, biliary excretion of organic anions from blood into bile is impaired. The aim of this study was to identify the underlying mechanism. Methods: Expression of the basolateral organic anion transporting polypeptide OATP‐C (SLC21A6) and the canalicular multidrug resistance protein 2 (MRP2) was studied in patients with primary sclerosing cholangitis (PSC) (n=4), a chronic cholestatic liver disease, and in non‐cholestatic controls (n=4) (two with chronic hepatitis C, one with idiopathic liver cirrhosis and one with fatty liver). Total RNA was isolated from liver tissue, reverse transcribed and subjected to polymerase chain reaction (PCR) amplification using primers specific for OATP‐C, MRP2 and β‐actin. PCR products were quantified densitometrically. Results: When normalized for β‐actin expression, the level of OATP‐C mRNA in liver tissue of patients with PSC was 49% of controls (OATP‐C/β‐actin 1.60±0.25 vs. 3.24±0.69; p<0.05) and the level of MRP2 mRNA was 27% of controls (MRP2/β‐actin 0.70±0.36 vs. 2.54±0.56; p<0.01). Conclusions: Both OATP‐C and MRP2 are decreased as measured by mRNA level in PSC. Downregulation of OATP‐C might be the consequence of impaired canalicular secretion of organic anions and could serve to reduce the organic anion load of cholestatic hepatocytes.     

Steatosis correlates with hepatic expression of death receptors and activation of nuclear factor‐κB in chronic hepatitis C

Background: Steatosis is recognized as a predictor of the severity as well as the progression of fibrosis in chronic hepatitis C. The mechanisms that cause increased hepatocellular injury associated with steatosis remain largely unknown. Methods: We studied the correlation of hepatic expression of death receptors: Fas and tumour necrosis factor‐α receptor 1 (TNF‐R1), and downstream caspase (caspase‐3) with hepatic steatosis by immunohistochemical study in chronic hepatitis C and determined the role of nuclear factor‐κB (NF‐κB). Results: Ninety patients (49 males and 41 females, mean age of 50.5 ± 10.4 years, genotype 1 or 2) with chronic hepatitis C virus infection were recruited. The factors associated with steatosis grade were body mass index (P=0.004) and fibrosis stage (P=0.034). Moderate/severe steatosis was an independent variable associated with advanced fibrosis stage by stepwise logistic regression analysis. The expression of immunoreactivity for Fas, TNF‐R1 and active caspases‐3 in liver tissues was significantly correlated with the steatosis grade (P<0.001, P<0.001 and P<0.001 respectively). The extent of active caspases‐3 correlated significantly with the expression of Fas (r=0.659, P<0.001) and TNF‐R1 (r=0.617, P<0.001). NF‐κB p65 expression correlated significantly with the extent of Fas (r=0.405, P<0.001), TNF‐R1 (r=0.448, P=0.002) and active caspase‐3 (r=0.313, P=0.003), and correlated with steatosis grade (P<0.001) but not with inflammatory and fibrosis scores. Conclusion: Our observations suggest a mechanism whereby steatosis contributes to the progression of liver injury in chronic hepatitis C through upregulation of death receptors and activation of NF‐κB.