Increase In Hepatic Steatosis Research Articles

Regulatory Role of Endothelial PHD2 in the Hepatic Steatosis

Background/Aims: Liver disease is a leading cause of high mortality and morbidity worldwide. The aim of the present study is to investigate the regulatory role of prolyl hydroxylase-2 (PHD2)-hypoxia-inducible factor-2a (HIF-2α) axis on nonalcoholic fatty liver disease (NAFLD) and to explore the potential mechanisms by which endothelial (EC)-specific PHD2 deficiency regulates hepatic steatosis and fibrosis. Methods: In the endothelial-specific PHD2 knockout (PHD2ECKO) mouse fed with normal diet or high fat diet (HFD), liver lipid accumulation and fibrosis were measured by Oil Red O and Masson trichrome staining. The fat and body weight (FW/BW) ratio and glucose tolerance were measured. The expression of HIF-2α, atrial natriuretic peptide (ANP), angiopoietin-2 (Ang-2), and transforming growth factor-b (TGF-β) were analyzed by western blot analysis. Results: The steatosis and fibrosis were significantly increased in the PHD2ECKO mice. FW/BW ratio was significantly increased in the PHD2ECKO mice. Moreover, knockout of endothelial PHD2 resulted in an impairment of glucose tolerance in mice. Western blot analysis showed that the expression of HIF-2α in liver tissues was not significantly increased. Interestingly, the expression of ANP was decreased, and Ang-2 and TGF-β levels were significantly increased in the liver of PHD2ECKO mice. The FW/BW ratio was also significantly increased in the PHD2ECKO mice fed with HFD for 16 weeks. Feeding HFD resulted in a significant increase in hepatic steatosis in the control PHD2f/f mice, but did not further enhance hepatic steatosis in the PHD2ECKO mice. Conclusions: We concluded that the endothelial PHD2 plays a critical role in hepatic steatosis and fibrosis, which may be involved in the regulation of ANP and Ang-2/TGF-β signaling pathway, but not the HIF-2α expression.

Genetic variation in the microsomal triglyceride transfer protein (\u2212493G/T) is associated with hepatic steatosis in patients infected with hepatitis C virus

BackgroundIn chronic hepatitis C, the fibrosis progression rates are extremely variable and can be influenced by factors associated with the host, virus and environment. Among the associated metabolic factors, hepatic steatosis is characterized by an accumulation of triglycerides in hepatocytes. In the host, genetic determinants of hepatic steatosis are observed, such as single-nucleotide polymorphisms (SNPs) in the microsomal triglyceride transfer protein (MTTP) gene. The MTTP -493G/T SNP appears to play an important role in the regulation of gene expression and influences the plasma concentration of circulating low-density lipoprotein (LDL). The present study investigated the influence of this SNP in the development of hepatic steatosis in patients with chronic hepatitis C and evaluated the association of hepatic steatosis with certain characteristics of these patients and the hepatitis C virus (HCV).MethodsTwo hundred thirty-nine patients with chronic hepatitis C were genotyped for the MTTP -493G⁄T SNP by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. The association between hepatic steatosis and selected characteristics of the patient and virus was evaluated using bivariate and multivariate analyses.ResultsThe most prevalent MTTP -493G/T genotype was GG (46%) followed by GT (43.5%) and TT (10.5%). Multivariate analysis of the total cohort revealed associations between the presence of hepatic steatosis and inflammatory activity of moderate to high intensity (P < 0.001), advanced age (P = 0.010), elevated gamma glutamyl transpeptidase (GGT) levels (P = 0.010) and low LDL levels (P = 0.022). Hepatic steatosis was also associated with the TT/GT genotype of the MTTP -493G⁄T SNP in patients infected with HCV genotype 3 (P < 0.001).ConclusionsIn chronic hepatitis C patients infected with HCV genotype 3 and with the TT/GT genotype of the MTTP -493G/T SNP, a significant increase in hepatic steatosis was observed, which may indicate that this SNP has a significant influence on the accumulation of triglycerides in hepatocytes. Furthermore, associations were observed between hepatic steatosis and inflammatory activity of moderate to high intensity, advanced age, elevated GGT and low LDL levels.

Glucagon-like peptide-1 analogue prevents nonalcoholic steatohepatitis in non-obese mice.

To investigate whether a glucagon-like peptide-1 (GLP-1) analogue inhibits nonalcoholic steatohepatitis (NASH), which is being increasingly recognized in Asia, in non-obese mice. A methionine-choline-deficient diet (MCD) along with exendin-4 (20 μg/kg per day, ip), a GLP-1 analogue, or saline was administered to male db/db mice (non-obese NASH model). Four or eight weeks after commencement of the diet, the mice were sacrificed and their livers were excised. The excised livers were examined by histochemistry for evidence of hepatic steatosis and inflammation. Hepatic triglyceride (TG) and free fatty acid (FFA) content was measured, and the expression of hepatic fat metabolism- and inflammation-related genes was evaluated. Oxidative stress-related parameters and macrophage recruitment were also examined using immunohistochemistry. Four weeks of MCD feeding induced hepatic steatosis and inflammation and increased the hepatic TG and FFA content. The expression of fatty acid transport protein 4 (FATP4), a hepatic FFA influx-related gene; macrophage recruitment; and the level of malondialdehyde (MDA), an oxidative stress marker, were significantly augmented by a 4-wk MCD. The levels of hepatic sterol regulatory element-binding protein-1c (SREBP-1c) mRNA (lipogenesis-related gene) and acyl-coenzyme A oxidase 1 (ACOX1) mRNA (β-oxidation-related gene) had decreased at 4 wk and further decreased at 8 wk. However, the level of microsomal triglyceride transfer protein mRNA (a lipid excretion-related gene) remained unchanged. The administration of exendin-4 significantly attenuated the MCD-induced increase in hepatic steatosis, hepatic TG and FFA content, and FATP4 expression as well as the MCD-induced augmentation of hepatic inflammation, macrophage recruitment, and MDA levels. Additionally, it further decreased the hepatic SREBP-1c level and alleviated the MCD-mediated inhibition of the ACOX1 mRNA level. These results suggest that GLP-1 inhibits hepatic steatosis and inflammation through the inhibition of hepatic FFA influx and oxidative stress in a non-obese NASH model.

Abstract 19220: Hepatic Steatosis is Independently Associated With an Increased Vascular Stiffness in Overweight Subjects

Introduction: The prevalence of non-alcoholic fatty liver disease (NAFLD) has increased dramatically during the past decades, affecting 50-75 % of the obese subjects. Subjects with NAFLD have an increased risk of cardiovascular diseases. We investigated whether an increase in hepatic steatosis is independently associated with an increase in vascular stiffness. Methods: We recruited a subcohort of the Nijmegen Biomedical Study, a large population based cohort, including 112 subjects, BMI &gt; 27 kg/m2, aged 55-81 years. The amount of liver fat was quantified by MR spectroscopy. Arterial stiffness was assessed by measurement of the carotid-femoral pulse wave velocity (PWV). The association between the hepatic steatosis and vascular stiffness was investigated by linear regression analysis with and without adjustment for the conventional cardiovascular risk factors. Results: In univariate analysis, hepatic steasosis was found to be correlated with PWV (r = 0.267; p = 0.005). After adjustment for age, sex, heart rate, alcohol use, and BMI still a significant positive association was present with a standardized beta of 0,298 (p=0.004). Even after adjustment for conventional cardiovascular risk factors including mean arterial pressure, blood pressure lowering and anti-diabetic drugs, hepatic steatosis remained independently associated with PWV ( standardized beta 0.269, p = 0.019). Conclusions: In a group of overweight subjects hepatic steatosis is associated with arterial stiffness. This association is independent of blood pressure, use of antihypertensives and the other traditional cardiovascular risk factors.

Liver specific knockout of biliverdin reductase‐A (BVRA) enhances high fat diet induced hepatic steatosis and type II diabetes in mice

Biliverdin reductase‐A (BVRA) is the enzyme responsible for the reduction of biliverdin to bilirubin. In order to determine the role of intracellular heptatic bilirubin generation in the development of dietary induced hepatic steatosis, we developed mice with hepatocyte specific deletion of BVRA. Floxed BVRA mice were crossed with mice specifically expressing the Cre recombinase in hepatocytes under the control of the albumin promoter (Alb‐Cre). Liver specific BVRA knockout (KO) mice exhibited a significant decrease in hepatic BVRA protein measured by both Western blot and immunofluorescence. Liver BVRA KO mice also exhibited a significant decrease in hepatic BVR activity as compared to WT mice (4 + 1 vs. 14 + 3 µmoles bilirubin/min/mg protein, P&lt;0.05). Mice were placed on a high fat (60%) diet at 6 weeks of age for 12 weeks. At the end of the 12 week period, liver specific BVRA knockout (KO) mice exhibited significantly greater levels of fat in the liver as compared to control mice measured by echoMRI (21 + 2 vs. 10 + 3 %, P&lt;0.05). A similar increase in hepatic steatosis in the liver of BVRA KO mice was also determined by Oil Red O staining of liver sections (50 + 3 vs. 22 + 5 %, P&lt;0.05). Liver BVRA KO mice also exhibited higher fasting blood glucose levels at the end of the 12 week study as compared to control mice (155 + 3 vs. 120 + 2 mg/dL, P&lt;0.05). These results suggest that intracellular BVRA generation of bilirubin is an important factor to protect the liver against hepatic steatosis and the development of type II diabetes in response to a high fat diet.

Endoplasmic reticulum stress signaling through C/ebp homologous protein is required for adaptation to a high‐fat diet (641.15)

Endoplasmic reticulum (ER) stress leads to activation of the NLRP3 inflammasome and cleavage of pro‐caspase‐1 to its active form, resulting in the secretion of IL‐1B and IL‐18. C/ebp homologous protein (CHOP) is an ER stress signaling protein that amplifies IL‐1B secretion. We hypothesized that CHOP‐mediated ER stress signaling links adipose tissue leukocytosis with metabolic dysfunction during obesity. Male C57BL/6 and CHOP‐/‐ mice were fed a 60% high‐fat for 5 months. CHOP‐deficient mice had increased body weight and body fat, which was explained by a small reduction in energy expenditure. Contrary to our hypothesis, CHOP‐/‐ mice displayed a large increase (P&lt;0.05) of adipose tissue B cells, T cells, and macrophages. In accordance with increased adipose tissue inflammation CHOP‐/‐ mice had decreased glucose and insulin tolerance, which was accompanied by an increase in hepatic steatosis. These results indicate that during obesity CHOP reduces recruitment of leukocytes to adipose tissue, which may be related to its role in promoting apoptosis in response to cell stressors. This work was funded by the Pennington Foundation and RWG is supported by NIH T32 DK064584.

Attenuation of chronic‐binge alcohol‐induced liver injury by Lactobacillus rhamnosus GG culture supernatant via inhibition of intestinal barrier dysfunction (653.12)

Binge drinking after chronic alcohol consumption is one of the important factors contributing to the progression of hepatic steatosis to steatohepatitis. Our previous study showed that Lactobacillus rhamnosus GG (LGG) culture supernatant (LGGs) ameliorated acute alcohol‐induced liver injury. The present study investigated the effects and potential mechanisms of LGGs on chronic‐binge alcohol‐induced liver injury. Mice were fed Lieber DeCarli diet containing 5% alcohol for 4 weeks, then gavaged single dose of alcohol at 5 g/kg and lasted for 6 hours. Chronic‐binge alcohol exposure induced marked increase in hepatic steatosis and injury, which were attenuated by LGGs supplementation. LGGs application prevented chronic‐binge alcohol‐elevated intestinal permeability and circulating endotoxin level, indicating a beneficial effect of LGGs on intestinal barrier integrity. Further studies demonstrated that the effect of LGGs is through increasing intestinal tight junction proteins and proteins involved in the barrier homeostasis, such as ZO‐1, occludin, p‐glycoprotein, HIF‐2a and intestinal trefoil factor. LGGs also inhibited intestinal microRNA 122a expression, which has been shown to decrease occludin protein translation. In summary, LGGs is effective in preventing chronic‐binge alcohol‐induced liver injury by improving intestinal barrier function.Grant Funding Source: Supported by NIH, DOD and Veterans Administration

Activation of Kupffer cell and related signal pathway proteins in the liver of high fat and high fructose diet induced NAFLD mice

To investigate the expression of F4/80, NF-kappaB, p-AKT, AKT in the liver of nonalcoholic fatty liver disease (NAFLD) mice. To determine the role of Kupffer cells (KCs) in the development of NASH (non-alcoholic steatohepatitis), and understand the pathogenic mechanism of NASH. Five C3H/HeN mice fed with normal diet were served as controls, while fifteen fed with high fat, high fructose, high fat combined fructose diet respectively for 16 weeks were as NAFLD mice models. The liver inflammation and hepatic damage were examined, and the expression of F4/80, NF-Kb, p-AKT, AKT and the content of lipid in the liver were also detected. Chronic intake of high fat and 30% fructose solution caused a significant increase in hepatic steatosis in animals in comparison to water controls. Liver F4/80 and NF-kappaB were significantly higher in high fat and high fat combined fructose diet fed mice than that in controls (P < 0.01, P < 0.01), F4/80 protein were higher in high fat diet treated mice than those in fructose and high fat combined fructose groups (P < 0.01, P < 0.01). Markers of insulin resistance (e. g, hepatic phospho-AKT, AKT) were only altered in fructose-fed or high fat combined fructose animals (P < 0.01, P < 0.01). High fat and fructose diet may induce NAFLD in C3H/HeN mice. Kupffer cells and signal pathway proteins were activated, and they may play key roles in the initiation and progression of NASH.

Lipids and HCV

Chronic hepatitis C virus (HCV) infection is associated with an increase in hepatic steatosis and a decrease in serum levels of total cholesterol, low-density lipoprotein cholesterol (LDL) and apolipoprotein B (apoB), the main protein constituent of LDL and very low-density lipoprotein (VLDL). These changes are more marked in HCV genotype 3 infection, and effective treatment results in their reversal. Low lipid levels in HCV infection correlate not only with steatosis and more advanced liver fibrosis but also with non-response to interferon-based therapy. The clinical relevance of disrupted lipid metabolism reflects the fact that lipids play a crucial role in the life cycle of hepatitis C virus. HCV assembly and maturation in hepatocytes depend on microsomal triglyceride transfer protein and apoB in a manner that parallels the formation of VLDL. VLDL production from the liver occurs throughout the day with an estimated 10(18) particles produced every 24 h whilst the estimated hepatitis C virion production rate is 10(12) virions per day. HCV particles in the serum exist as a mixture of complete low-density infectious lipo-viral particles (LVP) and a vast excess of apoB-associated empty nucleocapsid-free sub-viral particles that are complexed with anti-HCV envelope antibodies. Apolipoprotein E (apoE) is also involved in HCV particle morphogenesis and is an essential apolipoprotein for HCV infectivity. ApoE is a critical ligand for the receptor-mediated removal of triglyceride rich lipoprotein (TRL) remnants by the liver. The dynamics of apoB-associated lipoproteins, including HCV-LVP, change post-prandially with an increase in large TRL remnants and very low density HCV-LVP which are rapidly cleared by the liver (at least three HCV receptors are cellular receptors for uptake of TRL remnants). In summary, HCV utilises triglyceride-rich lipoprotein pathways within the liver and the circulation to its advantage.

Lactobacillus casei Shirota protects from fructose-induced liver steatosis: A mouse model

To test the hypothesis that Lactobacillus casei Shirota (Lcs) protects against the onset of non-alcoholic fatty liver disease (NAFLD) in a mouse model of fructose-induced steatosis, C57BL/6J mice were either fed tap water or 30% fructose solution +/- Lcs for 8 weeks. Chronic consumption of 30% fructose solution led to a significant increase in hepatic steatosis as well as plasma alanine-aminotransferase (ALT) levels, which was attenuated by treatment with Lcs. Protein levels of the tight junction protein occludin were found to be markedly lower in both fructose treated groups in the duodenum, whereas microbiota composition in this part of the intestine was not affected. Lcs treatment markedly attenuated the activation of the Toll-like receptor (TLR) 4 signalling cascade found in the livers of mice only treated with fructose. Moreover, in livers of fructose fed mice treated with Lcs peroxisome proliferator-activated receptor (PPAR)-γ activity was markedly higher than in mice only fed fructose. Taken together, the results of the present study suggest that the dietary intake of Lcs protects against the onset of fructose-induced NAFLD through mechanisms involving an attenuation of the TLR-4-signalling cascade in the liver.

Dysregulated phosphorylation and nuclear translocation of cyclic AMP response element binding protein (CREB) in rat liver after chronic ethanol binge

Binge ethanol during chronic ethanol abuse augments liver injury but the underlying mechanism remains unknown. CREB (cyclic AMP response element binding protein) is implicated as a key transcription factor in liver regeneration and hepatic glucose and lipid metabolism. We examined the effects of ethanol on the phosphorylation of CREB in hepatocytes, and in vivo in rat liver after chronic ethanol binge. For in vivo studies, rats were fed ethanol in liquid diet for 4weeks followed by single binge administration of ethanol (intragastric, 5g/kg body weight). Four hours after binge administration, liver samples were collected and analyzed. Treatment of hepatocytes with ethanol caused increased phosphorylation of p38 MAPK (mitogen activated protein kinase), MSK-1 (mitogen and stress activated kinase) and CREB in the nuclear compartment without activation of ERK1/2 (extracellular regulated kinase); whereas angiotensin II induced activation of CREB was accompanied by activation of ERK1/2. In chronic ethanol-binge studies, analysis of the whole cell extracts showed increased phosphorylation of CREB, with no effect on CREB protein levels; increased phospho-ERK1/2, and decreased phospho-p38 MAPK. In contrast, the nuclear levels of phospho-CREB and CREB protein were reduced. Reduction in phospho-CREB and CREB proteins in the nuclear extracts was accompanied by suppression of mRNA levels for CPT-1 (carnitine palmitoyl transferase-1) and increase in hepatic steatosis after binge. It is concluded that binge ethanol causes defect in the nuclear accumulation of CREB protein, phospho-CREB, and an exaggerated hepatic steatosis. These in vivo effects are distinct from the effects of ethanol on hepatocytes in vitro.

Role of the Inducible Nitric Oxide Synthase in the Onset of Fructose-Induced Steatosis in Mice

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.

Lack of SIRT1 (Mammalian Sirtuin 1) Activity Leads to Liver Steatosis in the SIRT1+/− Mice: A Role of Lipid Mobilization and Inflammation

Mammalian sirtuin 1 (SIRT1) may control fatty acid homeostasis in liver. However, this possibility and underlying mechanism remain to be established. In this study, we addressed the issues by examining the metabolic phenotypes of SIRT1 heterozygous knockout (SIRT1(+/-)) mice. The study was conducted in the mice on three different diets including a low-fat diet (5% fat wt/wt), mediate-fat diet (11% fat wt/wt), and high-fat diet (HFD, 36% fat wt/wt). On low-fat diet, the mice did not exhibit any abnormality. On mediate-fat diet, the mice exhibited a significant increase in hepatic steatosis with elevated liver/body ratio, liver size, liver lipid (triglyceride, glycerol, and cholesterol) content, and liver inflammation. The hepatic steatosis was deteriorated in the mice by HFD. In the liver, lipogenesis was increased, fat export was reduced, and beta-oxidation was not significantly changed. Body weight and fat content were increased in response to the dietary fat. Fat was mainly increased in sc adipose tissue and liver. Inflammation was also elevated in epididymal fat. Whole body energy expenditure and substrate utilization were reduced. Food intake, locomotor activity, and fat absorption were not changed. These data suggest that a reduction in the SIRT1 activity increases the risk of fatty liver in response to dietary fat. The liver steatosis may be a result of increased lipogenesis and reduced liver fat export. The inflammation may contribute to the pathogenesis of hepatic steatosis as well. A reduction in lipid mobilization may contribute to the hepatic steatosis and low energy expenditure.

Hepatic insig-1 or -2 overexpression reduces lipogenesis in obese Zucker diabetic fatty rats and in fasted/refed normal rats.

To determine whether the antilipogenic actions of insulin-induced gene 1 (insig-1) demonstrated in cultured preadipocytes also occur in vivo, we infected Zucker diabetic fatty (ZDF) (fa/fa) rats, with recombinant adenovirus containing insig-1 or -2 cDNA. An increase of both proteins appeared in their livers. In control ZDF (fa/fa) rats infected with adenovirus containing the beta-galactosidase (beta-gal) cDNA, triacylglycerols in the liver and plasma rose steeply whereas the insig-infected rats exhibited substantial attenuation of the increase in hepatic steatosis and hyperlipidemia. Insig overexpression was associated with a striking reduction in the elevated level of nuclear sterol regulatory element-binding protein (SREBP)-1c, the activated form of the transcription factor. The mRNA of SREBP-1c lipogenic target enzymes also fell. The mRNA of endogenous insig-1, but not -2a and -2b, was higher in the fatty livers of untreated obese ZDF (fa/fa) rats compared with controls, but the elevation was not sufficient to block the approximately 3-fold increase in SREBP-1c expression and activity. In normal animals, adenovirus-induced overexpression of the insigs reduced the increase in SREBP-1c mRNA and its target enzymes caused by refeeding. The findings demonstrated that both insigs have antilipogenic action when transgenically overexpressed in livers with increased SREBP-1c-mediated lipogenesis. However, the increase in endogenous insig-1 expression associated with augmented lipogenesis may limit it, but is insufficient to prevent it.