Role of Hypoxia Inducing Factor-1β in Alcohol-Induced Autophagy,Steatosis and Liver Injury in Mice

Chronic alcohol causes liver hypoxia and steatosis, which eventually develops into alcoholic liver disease (ALD). While it has been known that alcohol consumption activates hepatic hypoxia inducing factor-1α (HIF-1α), conflicting results regarding the role of HIF-1α in alcohol-induced liver injury and steatosis in mice have been reported. In the present study, we aimed to use hepatocyte-specific HIF-1β knockout mice to eliminate the possible compensatory effects of the single knockout of the 1α subunit of HIF to study the role of HIFs in ALD. C57BL/6 wild type mice were treated with acute ethanol to mimic human binge drinking. Matched wild-type and hepatocyte specific HIF-1β knockout mice were also subjected to a recently established Gao-binge alcohol model to mimic chronic plus binge conditions, which is quite common in human alcoholics. We found that acute alcohol treatment increased BNIP3 and BNIP3L/NIX expression in primary cultured hepatocytes and in mouse livers, suggesting that HIF may be activated in these models. We further found that hepatocyte-specific HIF-1β knockout mice developed less steatosis and liver injury following the Gao-binge model or acute ethanol treatment compared with their matched wild type mice. Mechanistically, protection against Gao-binge treatment-induced steatosis and liver injury was likely associated with increased FoxO3a activation and subsequent induction of autophagy in hepatocyte-specific HIF-1β knockout mice.     

The role of MLKL in Hepatic Ischemia-Reperfusion Injury of Alcoholic Steatotic Livers

Alcohol-related liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD) are the primary causes of chronic liver disease in western countries. Liver transplantation is currently one of the most efficient approaches to save patients with liver failure, which is often associated with hepatic ischemia-reperfusion (IR) injury. IR injury is exacerbated by hepatic steatosis, yet the mechanism remains elusive. Necroptosis is a form of regulated cell death mediated by receptor-interacting protein kinase 1 (RIP1), RIP3 and mixed lineage kinase domain-like (MLKL) protein, which has been implicated in the pathogenesis of ALD and NAFLD. Though necroptosis plays an important role in IR injury of high fat diet - induced steatotic livers, the role of necroptosis in IR injury of ethanol - induced steototic livers has not been investigated. In the present study, we used chronic plus binge alcohol (Gao-binge) feeding followed by IR surgery to investigate IR liver injury with ethanol-associated steatosis. We found that the levels of key necroptotic proteins MLKL and RIP3 increased in alcohol-fed mouse livers. Moreover, we observed increased liver injury after IR in control diet-fed mice, which was further exacerbated by alcohol feeding based on serum alanine aminotransferase (ALT) levels and TUNEL staining of necrotic cells. Hepatic neutrophil infiltration also increased in alcohol-fed mice after IR surgery. However, deletion of Mlkl did not protect against IR liver injury in alcohol-fed mice compared with matched wild-type mice. In conclusion, alcoholic steatosis promotes IR injury, which seems to be independent of MLKL-mediated necroptosis.     

Molecular insights on intracellular Wnt/β-catenin signaling in alcoholic liver disease

Alcoholic liver disease (ALD) is one of the most common health problems worldwide, especially in developing countries caused by chronic consumption of alcohol on a daily basis. The ALD spectrum is initiated with the early stages of alcoholic fatty liver (steatosis), progressing to alcoholic steatohepatitis, followed by the later stages of fibrosis and in some cases, cirrhosis and hepatocellular carcinoma (HCC). The Wnt/β-catenin signaling required for healthy liver development, function, and regeneration is found to be aberrated in ALD, attributed to its progression. This review is to elucidate the association of Wnt/β-catenin signaling with various stages of ALD progression. Alcohol causes downregulation of Wnt/β-catenin signaling components and thereby suppressing the pathway. Reports have been published that aberrated Wnt/β-catenin signaling, especially the absence of β-catenin, results in decreased alcohol metabolism, causing steatosis followed by steatohepatitis via lipid accumulation, lipid peroxidation, liver injury, increased oxidative stress and apoptosis of hepatocytes, contributing to the advancement of ALD. Contrastingly, the progression of later stages of ALD like fibrosis and HCC depends on the increased activation of Wnt/β-catenin signaling and its components. Existing studies reveal the varied expression of Wnt/β-catenin signaling in ALD. However, the dual role of the Wnt/β-catenin pathway in earlier and later stages of ALD is not clear. Therefore, studies on the Wnt/β-catenin pathway and its components in various manifestations of ALD might provide insight in targeting the Wnt/β-catenin pathway in ALD treatment.     

The effects of Insulin Pre-Administration in Mice Exposed to Ethanol: Alleviating Hepatic Oxidative Injury through Anti-Oxidative,Anti-Apoptotic Activities and Deteriorating Hepatic Steatosis through SRBEP-1c Activation

Alcoholic liver disease (ALD) has become an important liver disease hazard to public and personal health. Oxidative stress is believed to be responsible for the pathological changes in ALD. Previous studies have showed that insulin, a classic regulator of glucose metabolism, has significant anti-oxidative function and plays an important role in maintaining the redox balance. For addressing the effects and mechanisms of insulin pre-administration on ethanol-induced liver oxidative injury, we investigated histopathology, inflammatory factors, apoptosis, mitochondrial dysfunction, oxidative stress, antioxidant defense system, ethanol metabolic enzymes and lipid disorder in liver of ethanol-exposed mice pretreatment with insulin or not. There are several novel findings in our study. First, we found insulin pre-administration alleviated acute ethanol exposure-induced liver injury and inflammation reflected by the decrease of serum AST and ALT activities, the improvement of pathological alteration and the inhibition of TNF-α and IL-6 expressions. Second, insulin pre-administration could significantly reduce apoptosis and ameliorate mitochondrial dysfunction in liver of mice exposed to ethanol, supporting by decreasing caspases-3 activities and the ratio of Bax/Bcl-2, increasing mitochondrial viability and mitochondrial oxygen consumption, inhibition of the decline of ATP levels and mitochondrial ROS accumulation. Third, insulin pre-administration prevented ethanol-mediated oxidative stress and enhance antioxidant defense system, which is evaluated by the decline of MDA levels and the rise of GSH/GSSG, the up-regulations of antioxidant enzymes CAT, SOD, GR through Nrf-2 dependent pathway. Forth, the modification of ethanol metabolism pathway such as the inhibition of CYP2E1, the activation of ALDH might be involved in the anti-oxidative and protective effects exerted by insulin pre-administration against acute ethanol exposure in mice. Finally, insulin pre-administration deteriorated hepatic steatosis in mice exposed to ethanol might be through SRBEP-1c activation. In summary, these results indicated that insulin pre-administration effectively alleviated liver oxidative injury through anti-inflammatory, anti-oxidative and anti-apoptotic activities but also deteriorated hepatic steatosis through SRBEP-1c activation in mice exposed to ethanol. Our study provided novel insight about the effects and mechanisms of insulin on ethanol-induced liver injury.     

S-adenosylmethionine (SAMe) protects against acute alcohol induced hepatotoxicity in mice small star, filled

Although S-Adenosylmethionine (SAMe) has beneficial effects in many hepatic disorders, the effects of SAMe on acute alcohol-induced liver injury are unknown. In the present study, we investigated effects of SAMe on liver injury in mice induced by acute alcohol administration. Male C57BL/6 mice received ethanol (5 g/kg BW) by gavage every 12 hrs for a total of 3 doses. SAMe (5 mg/kg BW) was administrated i.p. once a day for three days before ethanol administration. Subsequent serum ALT level, hepatic lipid peroxidation, enzymatic activity of CYP2E1 and hepatic mitochondrial glutathione levels were measured colorimetrically. Intracellular SAMe concentration was measured by high-performance liquid chromatography (HPLC). Histopathological changes were assessed by H&E staining. Our results showed that acute ethanol administration caused prominent microvesicular steatosis with mild necrosis and an elevation of serum ALT activity. SAMe treatment significantly attenuated the liver injury. In association with the hepatocyte injury, acute alcohol administration induced significant decreases in both hepatic SAMe and mitochondrial GSH levels along with enhanced lipid peroxidation. SAMe treatment attenuated hepatic SAMe and mitochondrial GSH depletion and lipid peroxidation following acute alcohol exposure. These results demonstrate that SAMe protects against the liver injury and attenuates the mitochondrial GSH depletion caused by acute alcohol administration. SAMe may prove to be an effective therapeutic agent in many toxin-induced liver injuries including those induced by alcohol.     

Peripheral Reduction of FGFR4 with Antisense Oligonucleotides Increases Metabolic Rate and Lowers Adiposity in Diet-Induced Obese Mice

Obesity is a primary risk factor for multiple metabolic disorders. Many drugs for the treatment of obesity, which mainly act through CNS as appetite suppressants, have failed during development or been removed from the market due to unacceptable adverse effects. Thus, there are very few efficacious drugs available and remains a great unmet medical need for anti-obesity drugs that increase energy expenditure by acting on peripheral tissues without severe side effects. Here, we report a novel approach involving antisense inhibition of fibroblast growth factor receptor 4 (FGFR4) in peripheral tissues. Treatment of diet-induce obese (DIO) mice with FGFR4 antisense oligonucleotides (ASO) specifically reduced liver FGFR4 expression that not only resulted in decrease in body weight (BW) and adiposity in free-feeding conditions, but also lowered BW and adiposity under caloric restriction. In addition, combination treatment with FGFR4 ASO and rimonabant showed additive reduction in BW and adiposity. FGFR4 ASO treatment increased basal metabolic rate during free-feeding conditions and, more importantly, prevented adaptive decreases of metabolic rate induced by caloric restriction. The treatment increased fatty acid oxidation while decreased lipogenesis in both liver and fat. Mechanistic studies indicated that anti-obesity effect of FGFR4 ASO was mediated at least in part through an induction of plasma FGF15 level resulted from reduction of hepatic FGFR4 expression. The anti-obesity effect was accompanied by improvement in plasma glycemia, whole body insulin sensitivity, plasma lipid levels and liver steatosis. Therefore, FGFR4 could be a potential novel target and antisense reduction of hepatic FGFR4 expression could be an efficacious therapy as an adjunct to diet restriction or to an appetite suppressant for the treatment of obesity and related metabolic disorders.     

Modulation of hepatic redox status and mitochondrial metabolism by exercise: Therapeutic strategy for liver diseases

Liver steatosis (non-alcoholic fatty liver disease, NAFLD) is deemed as the hepatic face of the metabolic syndrome, with both physical inactivity and hypercaloric/unbalanced diet, together with increasing age playing a role as predisposing factors. Consequently, one of the most effective strategies used to counteract this scenario is physical exercise.Given the importance of redox signaling in cellular remodeling, in which mitochondria are closely implicated along with important roles on substrate oxidation, here we briefly review the effects of both acute and chronic forms of physical exercise on the modulation of hepatic redox state, highlighting the relevance of mitochondrial metabolism and function in the induction of liver phenotypes that antagonize metabolic alterations associated with liver metabolic diseases.     

Depdc5 deficiency exacerbates alcohol-induced hepatic steatosis via suppression of PPARα pathway

Alcohol-related liver disease (ALD), a condition caused by alcohol overconsumption, occurs in three stages of liver injury including steatosis, hepatitis, and cirrhosis. DEP domain-containing protein 5 (DEPDC5), a component of GAP activities towards Rags 1 (GATOR1) complex, is a repressor of amino acid-sensing branch of the mammalian target of rapamycin complex 1 (mTORC1) pathway. In the current study, we found that aberrant activation of mTORC1 was likely attributed to the reduction of DEPDC5 in the livers of ethanol-fed mice or ALD patients. To further define the in vivo role of DEPDC5 in ALD development, we generated Depdc5 hepatocyte-specific knockout mouse model (Depdc5-LKO) in which mTORC1 pathway was constitutively activated through loss of the inhibitory effect of GATOR1. Hepatic Depdc5 ablation leads to mild hepatomegaly and liver injury and protects against diet-induced liver steatosis. In contrast, ethanol-fed Depdc5-LKO mice developed severe hepatic steatosis and inflammation. Pharmacological intervention with Torin 1 suppressed mTORC1 activity and remarkably ameliorated ethanol-induced hepatic steatosis and inflammation in both control and Depdc5-LKO mice. The pathological effect of sustained mTORC1 activity in ALD may be attributed to the suppression of peroxisome proliferator activated receptor α (PPARα), the master regulator of fatty acid oxidation in hepatocytes, because fenofibrate (PPARα agonist) treatment reverses ethanol-induced liver steatosis and inflammation in Depdc5-LKO mice. These findings provide novel insights into the in vivo role of hepatic DEPDC5 in the development of ALD.Subject terms: Alcoholic liver disease, Experimental models of disease     

Effect of dry tomato peel supplementation on glucose tolerance,insulin resistance,and hepatic markers in mice fed high-saturated-fat/high-cholesterol diets

Many studies have investigated the effect of crude tomato peel in vivo, but no studies have determined the dose-effect of dry tomato peel (DTP) on glucose intolerance, insulin resistance, and atherogenic dyslipidemia induced by a high-saturated-fat (HSF) diet in vivo. The aim of this study was to investigate the effects of different doses of DTP on the levels of oxidative stress in mice fed an HSF and cholesterol-rich diet for 12 weeks. The main outcomes are glucose and insulin tolerance, plasma lipids, and hepatic steatosis and inflammation. BALB/c male mice (n=40) (8 weeks old, weighing 22.2±1.0 g) were divided into four treatment groups (10 mice/group): (a) high-fat control diet (HF Ctrl), which contains sunflower oil as a sole source of fat; (b) HSF/high-cholesterol (HC) diet; (c) HSF/HC diet supplemented with 9% DTP and (d) HSF/HC diet supplemented with 17% DTP. The HSF/HC diet significantly increased body weight gain, adipose tissue weight, fasting plasma glucose, fasting plasma insulin and lipid peroxidation and caused the development of liver steatosis and inflammation. Supplementation with DTP increased plasma lycopene concentration and reduced the development of indicators of metabolic syndrome, with no consistent effect of the DTP dose. Hepatic steatosis and inflammation were not reversed with DTP supplementation. Among mice fed the HSF/HC diet, DTP supplementation appears to have a beneficial effect on insulin resistance, which confirms the antiatherogenic effect of DTP.     

Chronic high-carbohydrate, high-fat feeding in rats induces reversible metabolic, cardiovascular, and liver changes

Age-related physiological changes develop at the same time as the increase in metabolic syndrome in humans after young adulthood. There is a paucity of data in models mimicking chronic diet-induced changes in human middle age and interventions to reverse these changes. This study measured the changes during chronic consumption of a high-carbohydrate (as cornstarch), low-fat (C) diet and a high-carbohydrate (as fructose and sucrose), high-fat (H) diet in rats for 32 wk. C diet feeding induced changes without metabolic syndrome, such as disproportionate increases in total body lean and fat mass, reduced bone mineral content, cardiovascular remodeling with increased systolic blood pressure, left ventricular and arterial stiffness, and increased plasma markers of liver injury. H diet feeding induced visceral adiposity with reduced lean mass, increased lipid infiltration in the skeletal muscle, impaired glucose and insulin tolerance, cardiovascular remodeling, hepatic steatosis, and increased infiltration of inflammatory cells in the heart and the liver. Chia seed supplementation for 24 wk attenuated most structural and functional modifications induced by age or H diet, including increased whole body lean mass and lipid redistribution from the abdominal area, and normalized the chronic low-grade inflammation induced by H diet feeding; these effects may be mediated by increased metabolism of anti-inflammatory n-3 fatty acids from chia seed. These results suggest that chronic H diet feeding for 32 wk mimics the diet-induced cardiovascular and metabolic changes in middle age and that chia seed may serve as an alternative dietary strategy in the management of these changes.     

High dietary fat exacerbates arsenic-induced liver fibrosis in mice

Many factors could potentially affect the process of arsenic-induced liver fibrosis. The present study was undertaken to examine the effect of high fat diet on arsenic-induced liver fibrosis and preneoplastic changes. Mice were given sodium arsenite (As3+, 200 ppm) or sodium arsenate (As5+, 200 ppm) in the drinking water for 10 months, and provided a normal diet or a diet containing 20% added fat. Serum aspartate aminotransferase (AST), indicative of liver injury, was elevated in both arsenite and arsenate groups, and a high fat diet further increased these levels. Histopathology (H&E and Masson stain) showed that liver inflammation, steatosis (fatty liver), hepatocyte degeneration, and fibrosis occurred with arsenic alone, but their severity was markedly increased with the high fat diet. Total liver RNA was isolated for real-time RT-PCR analysis. Arsenic exposure increased the expression of inflammation genes, such as TNF-alpha, IL-6, iNOS, chemokines, and macrophage inflammatory protein-2. The expression of the stress-related gene heme oxygenase-1 was increased, while metallothionein-1 and GSH S-transferase-pi were decreased when arsenic was combined with the high fat diet. Expression of genes related to liver fibrosis, such as procollagen-1 and -3, SM-actin and TGF-beta, were synergistically increased in the arsenic plus high fat diet group. The expression of genes encoding matrix metalloproteinases (MMP2, MMP9) and tissue inhibitors of metalloproteinases (TIMP1, TIMP2) was also enhanced, suggestive of early oncogenic events. In general, arsenite produced more pronounced effects than arsenate. In summary, chronic inorganic arsenic exposure in mice produces liver injury, and a high fat diet markedly increases arsenic-induced hepatofibrogenesis.     

Ethanol and tobacco smoke increase hepatic steatosis and hypoxia in the hypercholesterolemic apoE−/− mouse: Implications for a “multihit” hypothesis of fatty liver disease

Although epidemiologic studies indicate that combined exposure to cigarette smoke and alcohol increase the risk and severity of liver diseases, the molecular mechanisms responsible for hepatotoxicity are unknown. Similarly, emerging evidence indicates a linkage among hepatic steatosis and cardiovascular disease. Herein, we hypothesize that combined exposure to alcohol and environmental tobacco smoke (ETS) on a hypercholesterolemic background increases liver injury through oxidative/nitrative stress, hypoxia, and mitochondrial damage. To test this, male apoE^?/? mice were exposed to an ethanol-containing diet, ETS alone, or a combination of the two, and histology and functional endpoints were compared to filtered-air-exposed, ethanol-naïve controls. Whereas ethanol consumption induced a mild steatosis, combined exposure to ethanol + ETS resulted in increased hepatic steatosis, inflammation, α-smooth muscle actin, and collagen. Exposure to ethanol + ETS induced the largest increase in CYP2E1 and iNOS protein, as well as increased 3-nitrotyrosine, mtDNA damage, and decreased cytochrome c oxidase protein, compared to all other groups. Similarly, the largest increase in HIF1α expression was observed in the ethanol + ETS group, indicating enhanced hypoxia. These studies demonstrate that ETS increases alcohol-dependent steatosis and hypoxic stress. Therefore, ETS may be a key environmental “hit” that accelerates and exacerbates alcoholic liver disease in hypercholesterolemic apoE^?/? mice.     

β‐Aminoisobutyric Acid Prevents Diet‐induced Obesity in Mice With Partial Leptin Deficiency

β‐Aminoisobutyric acid (BAIBA), a thymine catabolite, increases fatty acid oxidation (FAO) in liver and reduces the gain of body fat mass in Swiss (lean) mice fed a standard chow. We determined whether BAIBA could prevent obesity and related metabolic disorders in different murine models. To this end, BAIBA (100 or 500 mg/kg/day) was administered for 4 months in mice totally deficient in leptin (ob/ob). BAIBA (100 mg/kg/day) was also given for 4 months in wild‐type (+/+) mice and mice partially deficient in leptin (ob/+) fed a high‐calorie (HC) diet. BAIBA did not limit obesity and hepatic steatosis in ob/ob mice, but reduced liver cytolysis and inflammation. In ob/+ mice fed the HC diet, BAIBA fully prevented, or limited, the gain of body fat, steatosis and necroinflammation, glucose intolerance, and hypertriglyceridemia. Plasma β‐hydroxybutyrate was increased, whereas expression of carnitine palmitoyltransferase‐1 was augmented in liver and white adipose tissue. Acetyl‐CoA carboxylase was more phosphorylated, and de novo lipogenesis was less induced in liver. These favorable effects of BAIBA in ob/+ mice were associated with a restoration of plasma leptin levels. The reduction of body adiposity afforded by BAIBA was less marked in +/+ mice. Finally, BAIBA significantly stimulated the secretion of leptin in isolated ob/+ adipose cells, but not in +/+ cells. Thus, BAIBA could limit triglyceride accretion in tissues through a leptin‐dependent stimulation of FAO. As partial leptin deficiency is not uncommon in the general population, supplementation with BAIBA may help to prevent diet‐induced obesity and related metabolic disorders in low leptin secretors.     

Sake yeast suppresses acute alcohol-induced liver injury in mice

Brewer's and baker's yeasts appear to have components that protect from liver injury. Whether sake yeast, Saccharomyces cerevisiae Kyokai no. 9, also has a hepatoprotective effect has not been examined. Here we show that sake yeast suppresses acute alcoholic liver injury in mice. Male C57BL/6 mice that had been fed a diet containing 1% sake yeast for two weeks received three doses of ethanol (5 g/kg BW). In the mice fed sake yeast, ethanol-induced increases in triglyceride (TG) and glutamate pyruvate transaminase (GPT) were significantly attenuated and hepatic steatosis was improved. In addition, sake yeast-fed mice showed a smaller decrease in hepatic S-adenosylmethionine (SAM) level and a smaller increase in plasma homocysteine (Hcy) level after ethanol treatment than the control mice, suggesting that a disorder of methionine metabolism in the liver caused by ethanol was relieved by sake yeast. These results indicate that sake yeast protects against alcoholic liver injury through maintenance of methionine metabolism in the liver.     

Oxidative stress induced by corticosterone administration in broiler chickens (Gallus gallus domesticus) 1. Chronic exposure

Two trials were conducted to evaluate the effects of short-term administration of corticosterone (CORT) on the induction of oxidative injury in broiler chickens (Gallus gallus domesticus). Twelve broiler chickens of 30 and of 40 days of age were respectively employed in Trial 1 and 2. Half of the chickens were administered subcutaneously with CORT (4 mg/kg body weight [BW] in corn oil), while another half served as controls (corn oil) in each trail. In Trial 1, a blood sample was obtained from each chicken immediately before administration and at 1 and 3 h after injection. In Trial 2, the liver and heart were obtained after 3 h of CORT exposure. Short-term administration of CORT resulted in enhanced proteolysis and gluconeogenesis. There were no obvious changes in lipid peroxidation status of the heart and liver, whereas a decrease in lipid peroxidation in the plasma was observed after acute CORT exposure. The significantly increased plasma nonenzymatic antioxidants (uric acid [UA] and total antioxidant capacity) in concert with the enhanced enzymatic antioxidant activity (SOD in heart) during short-term CORT administration indicate preventive changes to counteract the oxidative injury, and these may be tissue specific.     

Neonatal androgenization exacerbates alcohol-induced liver injury in adult rats, an effect abrogated by estrogen

Alcoholic liver disease (ALD) affects millions of people worldwide and is a major cause of morbidity and mortality. However, fewer than 10% of heavy drinkers progress to later stages of injury, suggesting other factors in ALD development, including environmental exposures and genetics. Females display greater susceptibility to the early damaging effects of ethanol. Estrogen (E2) and ethanol metabolizing enzymes (cytochrome P450, CYP450) are implicated in sex differences of ALD. Sex steroid hormones are developmentally regulated by the hypothalamic-pituitary-gonadal (HPG) axis, which controls sex-specific cycling of gonadal steroid production and expression of hepatic enzymes. The aim of this study was to determine if early postnatal inhibition of adult cyclic E2 alters ethanol metabolizing enzyme expression contributing to the development of ALD in adulthood. An androgenized rat model was used to inhibit cyclic E2 production. Control females (Ctrl), androgenized females (Andro) and Andro females with E2 implants were administered either an ethanol or isocalorically-matched control Lieber-DeCarli diet for four weeks and liver injury and CYP450 expression assessed. Androgenization exacerbated the deleterious effects of ethanol demonstrated by increased steatosis, lipid peroxidation, profibrotic gene expression and decreased antioxidant defenses compared to Ctrl. Additionally, CYP2E1 expression was down-regulated in Andro animals on both diets. No change was observed in CYP1A2 protein expression. Further, continuous exogenous administration of E2 to Andro in adulthood attenuated these effects, suggesting that E2 has protective effects in the androgenized animal. Therefore, early postnatal inhibition of cyclic E2 modulates development and progression of ALD in adulthood.