Increase In Steatosis Research Articles

MicroRNA-26b as novel regulator of cardio-metabolic diseases

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): IZKF Increasing evidence has shown that microRNAs (miRs) are fundamental players in cardio-metabolic diseases. MiR-26b has been associated with the development of metabolic dysfunction-associated steatohepatitis (MASH) and atherosclerosis, although causality has not been demonstrated yet. Therefore, we aimed to determine the causal role of miR-26b in MASH and atherosclerosis. Apoe-/-Mir-26b-/- mice were fed a 4- or 12-week western type diet (WTD). Plasma and liver samples were subjected to various assays to investigate the effects on lipid metabolism, inflammation and fibrosis. Additionally, miR-26b mimic-loaded lipid nanoparticles (LNPs) were used to rescue the phenotype. A 1.5-fold increase in hepatic lipid levels was observed in Apoe-/-Mir26b-/- mice, while plasma cholesterol/triglyceride levels remained unchanged. These results were further confirmed by an increase in steatosis. Additionally, the proinflammatory cytokines TNF-α, IL-6 and CXCL1 were significantly elevated, which coincided with increased macrophage infiltration into the liver. Moreover, loss of miR-26b resulted in more hepatic fibrosis. Finally, a treatment with miR-26b mimic-loaded LNPs reduced hepatic lipid levels, rescuing the observed phenotype. Additionally, Apoe-/-Mir26b-/- mice demonstrated a striking 10-fold increase in atherosclerotic lesion size. Consistent with a more advanced plaque phenotype, collagen content and relative necrotic core area were significantly increased in plaques from mice lacking miR-26b. Intriguingly, this increased atherosclerotic lesion size could also be rescued by repetitive injections of miR-26b mimic-loaded LNPs. Overall, our results demonstrate a protective role of miR-26b in cardio-metabolic diseases, thereby opening doors to future research and potential new treatment options using LNP technology.

Polyploidisation pleiotropically buffers ageing in hepatocytes

Polyploidy in hepatocytes has been proposed as a genetic mechanism to buffer against transcriptional dysregulation. Here, we aim to demonstrate the role of polyploidy in modulating gene regulatory networks in hepatocytes during ageing. We performed single-nucleus RNA sequencing in hepatocyte nuclei of different ploidy levels isolated from young and old wild-type mice. Changes in the gene expression and regulatory network were compared to three independent strains that were haploinsufficient for HNF4A, CEBPA or CTCF, representing non-deleterious perturbations. Phenotypic characteristics of the liver section were additionally evaluated histologically, whereas the genomic allele composition of hepatocytes was analysed by BaseScope. We observed that ageing in wild-type mice results in nuclei polyploidy and a marked increase in steatosis. Haploinsufficiency of liver-specific master regulators (HFN4A or CEBPA) results in the enrichment of hepatocytes with tetraploid nuclei at a young age, affecting the genomic regulatory network, and dramatically suppressing ageing-related steatosis tissue wide. Notably, these phenotypes are not the result of subtle disruption to liver-specific transcriptional networks, since haploinsufficiency in the CTCF insulator protein resulted in the same phenotype. Further quantification of genotypes of tetraploid hepatocytes in young and old HFN4A-haploinsufficient mice revealed that during ageing, tetraploid hepatocytes lead to the selection of wild-type alleles, restoring non-deleterious genetic perturbations. Our results suggest a model whereby polyploidisation leads to fundamentally different cell states. Polyploid conversion enables pleiotropic buffering against age-related decline via non-random allelic segregation to restore a wild-type genome. The functional role of hepatocyte polyploidisation during ageing is poorly understood. Using single-nucleus RNA sequencing and BaseScope approaches, we have studied ploidy dynamics during ageing in murine livers with non-deleterious genetic perturbations. We have identified that hepatocytes present different cellular states and the ability to buffer ageing-associated dysfunctions. Tetraploid nuclei exhibit robust transcriptional networks and are better adapted to genomically overcome perturbations. Novel therapeutic interventions aimed at attenuating age-related changes in tissue function could be exploited by manipulation of ploidy dynamics during chronic liver conditions.

Abstract 6458: Maternal and paternal alcohol exposures program higher incidence of hepatocellular carcinoma in offspring

Abstract Environmental exposures during development are known to influence adult health and disease. Specifically, early developmental exposures have been shown to increase susceptibility to cancer in adults through epigenetic mechanisms. Alcohol is a potent environmental carcinogen and teratogen, causing a wide range of health problems across the life course of an individual. Current literature examining early life exposures solely focuses on maternal exposures despite recent efforts to recognize paternal epigenetic contributions. Thus, we investigated the influence of preconception paternal, maternal, and dual parental alcohol exposures on offspring’s predisposition to develop liver disease and hepatocellular carcinoma (HCC). We hypothesized that dual-parental alcohol exposure would exacerbate the incidence of liver cancer in adult offspring. Using an established mouse model, we exposed male and female adult C57BL/6J mice to control (0% EtOH) or alcohol (10% EtOH) preconception treatments. Next, we randomly assigned pups from each treatment to either a single injection of Diethylnitrosamine (DEN; 25mg/kg) to promote liver tumors, or saline control. Finally, we sacrificed offspring on postnatal day 182 and collected organs and blood for enzyme analysis. DEN-treated males display an increased liver-to-body weight ratio compared to saline males while also exhibiting lower body weight. The male progeny from the dual-parental treatment group exhibited a significant increase in tumor incidence and burden in DEN-treated males, exceeding those measured for the maternal and paternal treatments. Further, offspring from the dual-parental group had high levels of alanine transaminase (ALT) and aspartate transaminase (AST) compared to the other treatment groups. Oil Red-O and Sirius Red staining of liver sections revealed increased hepatic steatosis and fibrosis in DEN males compared to saline males. Again, the dual-parental exposed offspring exhibited the most aberrant phenotype. Blind scoring of H&E-stained liver sections identified increases in steatosis, fibrosis, and HCC lesions, with the highest scores emerging in DEN-treated dual-parental offspring. These findings demonstrate that preconception paternal and gestational maternal alcohol exposures program a predisposition to adult-onset chronic diseases including alcoholic fatty liver disease, hepatic fibrosis, and HCC. These experiments not only confirm our hypothesis that dual-parental exposed offspring exhibit an exacerbated hepatic abnormality phenotype, but also highlight the importance of both maternal and paternal environmental exposures on disease risk in offspring. While the negative effects of maternal alcohol consumption have been well established, our novel finding that paternal alcohol exposure also promotes hepatocellular carcinoma in offspring has potentially profound clinical implications. Citation Format: Alison Basel, Sanat S. Bhadsavle, Kara N. Thomas, Katherine N. Zimmel, Alexis N. Roach, Matthew Gaytan, Grace Parkey, Michael C. Golding. Maternal and paternal alcohol exposures program higher incidence of hepatocellular carcinoma in offspring. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6458.

Histological evaluation of the liver of mice with sarcoma-180 treated with salazinic acid.

Many of the drugs used to fight cancer cells induce various damage causing hepatotoxic effects which are characterized by tissue changes. The aim of the study is to know the possible effects of salazinic acid on livers of mice exposed to Sacoma-180. The tumor was grown in the animals in ascitic form and inoculated subcutaneously in the axillary region of the mouse developing the solid tumor. Treatment with salazinic acid (25 and 50 mg/kg) and 5-Fluorouracil (20 mg/kg) started 24-hours after inoculation and was performed for 7 days. To verify these effects, the qualitative method of histological criteria investigated in liver tissue was used. It was observed that all treated groups showed an increase of pyknotic nuclei in relation to the negative control. There was an increase in steatosis in all groups compared to the negative control but there was a decrease in the groups treated with salazinic acid in the 5-Fluorouracil. There was no necrosis in the salazinic acid treated groups. However, this effect was seen in 20% of the positive control group. Therefore, it can be concluded that salazinic acid did not show hepatoprotective action on mice but demonstrated a decrease in steatosis and absence of tissue necrosis.

Chronic and Binge Alcohol Ingestion Increases Truncated Oxidized Phosphatidylcholines in Mice Lungs Due to Increased Oxidative Stress.

Heavy alcohol drinking has negative health effects in multiple organs. It predisposes lungs to inflammatory conditions associated with acute lung injury and increased incidence of pneumonia and sepsis, which may lead to death due to acute respiratory distress syndrome in some individuals with alcohol use disorder (AUD). In general, rodent models of alcohol exposure either do not recapitulate multiple organ injuries as seen in humans or require longer duration to establish tissue injury and inflammation. The recently introduced NIAAA model of alcohol-induced liver injury, characterized by a marked increase in steatosis and liver damage with 10 days of a liquid diet containing 5% ethanol followed by a single ethanol binge (5 g/kg). Therefore, we employed this model to explore the status of surfactant phospholipids, oxidative stress, tissue injury markers and inflammatory cytokines in lungs. In lungs of C57BL/6J mice, the alcohol feeding significantly increased levels of the surfactant phospholipid dipalmitoyl phosphatidylcholine (DPPC) as well as the truncated oxidized phosphatidylcholines palmitoyl oxovaleryl phosphatidyl-choline (POVPC), palmitoyl glutaryl phosphatidyl-choline (PGPC), palmitoyl oxo-nonanoyl phosphatidyl-choline (ALDO-PC), and palmitoyl azelaoyl phosphatidyl-choline (PAzePC) at 9 h post-binge. Additionally, gene expression of the enzymes catalyzing lipid oxidation, such as arachidonate 15-lipoxygenase (Alox15), prostaglandin synthase 2 (Ptgs2), Cytochrome P450 2E1 (Cyp2E1) and NADPH oxidase 1 (Nox1) were significantly increased. Furthermore, ethanol increased levels of the inflammatory cytokine Interleukin-17 in bronchoalveolar lavage fluid. In conclusion, the NIAAA alcohol feeding model might be suitable to study alcohol-induced lung injury and inflammation.

Cholic acid supplementation accelerates the progression of nonalcoholic fatty liver disease to the procarcinogenic state in mice fed a high-fat and high-cholesterol diet

Nonalcoholic fatty liver disease (NAFLD) is one of the major causes of hepatocellular carcinoma (HCC). Although the intracellular cholesterol accumulation has been demonstrated to regulate the gene expression responsible for steatohepatitis, the role played by cholesterol in the development of NAFLD-associated HCC has not been fully elucidated. In this study, using microarray analysis, we investigated the molecular mechanisms governing cholesterol-mediated progression of NAFLD. To ensure hepatic cholesterol accumulation, either a high-fat and high-cholesterol (HFHC) diet or a high-fat and high-cholesterol with cholic acid (HFHCCA) diet was fed to diethylnitrosamine (DEN)-injected C57BL/6J mice for 10 weeks. While an HFHC diet increased hepatic triglyceride levels, an HFHCCA diet induced hepatic cholesterol accumulation by reducing bile acid biosynthesis in DEN-injected mice. Livers from both HFHC and HFHCCA groups exhibited increases in steatosis and necrosis; however, histological features of HCC were not observed in any of the experimental groups. Hepatic gene expression profile of the HFHCCA group was different from those of other groups. Functional analysis showed that cholic acid supplementation upregulated differentially expressed genes (DEGs) associated with inflammation, proliferation, apoptosis, chemical drug response, and cancer signaling pathway. Downregulated DEGs were associated with steroid metabolism, mitochondrial function, and oxidative phosphorylation pathway. Furthermore, hepatic cholesterol accumulation lowered the expression of DEGs associated with energy and macronutrient metabolism, especially amino acid metabolism. In this study, the results of a global gene expression profile demonstrated that feeding the HFHCCA diet to DEN-injected mice accelerated the carcinogenic progression of NAFLD, implicating the critical role played by hepatic accumulation of cholesterol.

Comparison of liver biopsies before and after direct-acting antiviral therapy for hepatitis C and correlation with clinical outcome

Direct-acting antivirals (DAA) have replaced interferon (IFN)-based therapies for hepatitis C virus. In this retrospective clinical study, we examined differences in histopathologic features in paired liver biopsies collected from the same patient before and after DAA and correlated these findings with clinical outcome. Biopsies (n = 19) were evaluated by quantitative imaging analysis to measure steatosis and fibrosis. Most patients had decreased steatosis in their post-treatment, follow-up biopsies. However, one patient had a striking increase in steatosis (from 0.86 to 6.32%) and later developed decompensated cirrhosis and hepatocellular carcinoma (HCC). This patient had a marked increase in fibrosis between biopsies, with a CPA of 6.74 to 32.02. Another patient, who already had bridging fibrosis at the time of her pre-treatment biopsy, developed cholangiocarcinoma after DAA. Even though the overall inflammatory activity in the post-treatment biopsies significantly decreased after treatment, 60% of patients had persistent portal lymphocytic inflammation. In summary, DAAs decreased steatosis and hepatic inflammation in most patients, although some may have persistence of lymphocytic portal inflammation. Patients known to have advanced fibrosis at treatment initiation and who have other risk factors for ongoing liver injury, such as steatosis, should be followed closely for the development of adverse outcomes, such as portal hypertension and primary liver cancers.

Association Between Anxiety and Depression and Nonalcoholic Fatty Liver Disease.

Backgrounds: Depression and anxiety disorder are frequently seen in patients with nonalcoholic fatty liver disease (NAFLD). However, the associations between mood disorders and NAFLD have not been fully evaluated. In this study, we investigated the relationship between NAFLD and depression or anxiety in a Korean population.Methods: We conducted a retrospective cross-sectional study that included subjects who underwent abdominal ultrasonography and completed a symptom questionnaire for a routine health check-up. NAFLD was diagnosed and graded according to the ultrasonography findings. Depression and anxiety were assessed using the Beck Depression Inventory and State-Trait Anxiety Inventory, respectively.Results: Among the total of 25,333 subjects, the mean age was 47 years (men, 56.2%), and the prevalence rate of NAFLD was 30.9%. In the multivariate analysis, NAFLD showed a significant association with depression [adjusted odds ratio (OR) 1.43 and 95% confidence interval (CI) 1.14–1.80, p = 0.002] in women. Severe NAFLD significantly correlated with state anxiety and trait anxiety (adjusted OR 1.84 and 95% CI 1.01–3.37, p = 0.047 and adjusted OR 2.45 and 95% CI 1.08–4.85, p = 0.018, respectively) in women.Conclusions: There was a higher tendency of women with NAFLD to suffer from depression with increase in steatosis, and severe stage of steatosis was significantly associated with anxiety in the female compared to non-NAFLD. Understanding the association between NAFLD and mood disorders may have clinical implications for reducing the prevalence of comorbidities.

Inhibition of vascular adhesion protein-1 modifies hepatic steatosis in vitro and in vivo.

BACKGROUNDNon-alcoholic fatty liver disease (NAFLD) is associated with obesity, insulin resistance and dyslipidaemia and currently is estimated to affect up to a third of all individuals in developed countries. Current standard of care for patients varies according to disease stage, but includes lifestyle interventions common insulin sensitizers, antioxidants and lipid modifiers. However, to date specific therapies have shown little histological or fibrosis stage improvement in large clinical trials, and there is still no licensed therapy for NAFLD. Given the high prevalence, limited treatment options and significant screening costs for the general population, new treatments are urgently required.AIMTo assess the potential for inhibition of the amine oxidase enzyme vascular adhesion protein-1 (VAP-1) to modify hepatic lipid accumulation in NAFLD.METHODSWe have used immunochemical and qPCR analysis to document expression of VAP-1 and key functional proteins and transporters across the NAFLD spectrum. We then utilised hepatocytes in culture and human precision cut liver slices in concert with selective enzyme activity inhibitors to test the effects of activating the semicarbazide-sensitive amine oxidase activity of VAP-1 on hepatic lipid uptake and triglyceride export. A murine model of NAFLD was also used to determine the consequences of VAP-1 knockout and gene expression arrays were used to quantify the effects of VAP-1 activity on key lipid modifying and proinflammatory gene expression.RESULTSWe confirmed that increasing severity of NAFLD and progression to cirrhosis was associated with a significant increase in hepatocellular VAP-1 expression. Hepatocytes in vitro exposed to recombinant VAP-1 and its substrate methylamine showed increased lipid accumulation as determined by quantification of Oil Red O uptake. This was recapitulated using hydrogen peroxide, and lipid accumulation was accompanied by changes in expression of the lipid transporter molecules FABP3, FATP6, insulin receptor subunits and PPARα. Human liver tissue exposed to recombinant VAP-1 or substrates for endo/exogenous VAP-1 produced less triglyceride than untreated tissue and demonstrated an increase in steatosis. This response could be inhibited by using bromoethylamine to inhibit the SSAO activity of VAP-1, and mice deficient in VAP-1/AOC3 also demonstrated reduced steatosis on high fat diet. Exposure of human liver tissue to methylamine to activate VAP-1 resulted in increased expression of FABP2 and 4, FATP3-5, caveolin-1, VLDLR, PPARGC1 and genes associated with the inflammatory response.CONCLUSIONOur data confirm that the elevations in hepatic VAP-1 expression reported in nonalcoholic steatohepatitis can contribute to steatosis, metabolic disturbance and inflammation. This suggests that targeting the semicarbazide sensitive amine oxidase capacity of VAP-1 may represent a useful adjunct to other therapeutic strategies in NAFLD.

Impact of chronic HCV treatment on quality of life of patients with metabolic disorders in context of immunological disturbances

Chronic viral hepatitis C (CHC) and its complications have a negative effect on patient’s quality of life. We evaluated the impact of a successful interferon-free treatment on the quality of life of patients with obesity and metabolic disorders in the context of immunological disturbances. Twenty overweight or obese (BMI > 25) patients with CHC were tested before the therapy and after a successful treatment regimen. After the therapy, patient’s emotional well-being improved (p = 0.02), while physical well-being remained unchanged. There was a decrease of patient’s liver fibrosis and an increase of steatosis along with body mass. Among HCV-infected individuals, the expression of toll-like receptor 3 (TLR3) on lymphocytes was higher than in the control group (p = 0.03), but it decreased (p = 0.001) after the treatment. There was also a decrease of the intensity of immunofluorescence of FoxP3+ after the treatment (p = 0.04). Our study showed an improvement in mental aspects of patient’s quality of life after the treatment. Unfortunately, probably due to rapid immunological changes, patient’s BMI, serum cholesterol levels and hepatic steatosis have a tendency to increase and may lead to cardiovascular and other complications, like hepatocellular carcinoma.

371-OR: Reduced Hepatic Expression of the Mitochondrial Quality Control Factor PARKIN Hastens the Progression of NAFLD in Mice and Is Associated with NASH in Patients

Nonalcoholic fatty liver disease (NAFLD) encompasses a spectrum of pathologies, including steatosis, steatohepatitis (NASH) and fibrosis and is strongly associated with type 2 diabetes. Changes in mitochondrial function are implicated in the pathogenesis of NAFLD. Mitophagy is a mitochondrial quality control mechanism that allows for selective removal of damaged mitochondria from the cell. Whether changes in mitophagy occur during and contribute to NAFLD is unknown. PARKIN is a ubiquitin E3 ligase that regulates mitophagy by generating a ubiquitin signaling motif on the outer membrane of damaged mitochondria, marking them for degradation. We undertook the studies described to determine effects of genetic inhibition of mitophagy via conditional deletion of PARKIN in liver on progression of NAFLD. Twelve-week old male liver-specific PARKIN knockout (LKO) and wild type (WT) mice were fed a diet previously demonstrated to induce NAFLD that consisted of high-fat (45%), cholesterol (0.15%) and high-fructose corn syrup drinking water (42g/L) for 8 weeks. There was no difference in body weight or composition between groups. Plasma ALT and AST levels were increased 2-fold (p=0.18) and 1.7-fold (p<0.05), respectively, in LKO compared with WT mice, while plasma cholesterol and insulin levels were not different and plasma fatty acid levels were reduced by 27% (p<0.05). NAFLD activity score was increased 2-fold (p<0.05) in LKO mice reflecting an increase in steatosis, ballooning and inflammation. Gene expression markers of NAFLD progression including markers of inflammation (Ccl2, Cd68, Il-1b, ll-6) and fibrosis (Col1a1, Col3a1) were significantly increased in LKO mice. Consistent with these mouse data, we found PARKIN protein expression to be reduced in liver biopsies from patients with NASH compared with healthy controls. These data suggest that loss of PARKIN-mediated mitophagy may contribute to the progression of NAFLD. Disclosure R. Undamatla: None. L.R. Edmunds: None. B. Xie: None. A. Mills: None. I.J. Sipula: None. S.P. Monga: None. M.J. Jurczak: None. Funding National Institutes of Health (DK114012)

Unanticipated increases in hepatic steatosis among human immunodeficiency virus patients receiving mineralocorticoid receptor antagonist eplerenone for non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease is common in human immunodeficiency virus, but there are no approved therapies. The aim of this open-label proof-of-concept study was to determine the effect of the mineralocorticoid receptor antagonist eplerenone on hepatic fat in human immunodeficiency virus-infected patients with hepatic fat ≥5% by magnetic resonance spectroscopy. Five subjects received eplerenone (25mg daily×1week followed by 50mg daily×23weeks). Laboratory tests were done at each visit, and the primary endpoint, change in hepatic fat content, was determined by MRI spectroscopy at baseline and week 24. The study was stopped early after observing unexpected significant increases in hepatic fat at week 24 (mean increase 13.0±7.3%, P=.02). The increases in steatosis were accompanied by a tendency for transaminase values to decrease (alanine aminotransferase mean change -14±16IU/L, P=.14). There were no consistent changes in other metabolic parameters or blood pressure. Repeat assessment of hepatic steatosis 1-2months after stopping study medication revealed improvements in steatosis towards baseline values. The unexpected observation of increased hepatic steatosis with the administration of eplerenone led to early termination of the investigation. While limited because of the small number of participants and the open-label design, this study provides data to suggest that mineralocorticoid receptor antagonism with eplerenone may not be an effective approach to treat hepatic steatosis in human immunodeficiency virus or the general population. Additional research is needed to determine the pathophysiological mechanism behind these unanticipated observations.

Experimental model of hepatic steatosis by fructose in adult zebrafish: A pilot study

Introduction: The consumption of fructose has been questioned, since its increase has led to an associated increase in steatosis caused by nonalcoholic fatty liver disease. Despite the advantages presented by the zebrafish as an animal model, at present there are no models of steatosis by fructose in adult zebrafish. The aim of this study is to establish a model of hepatic steatosis by fructose in adult zebrafish. Methods: Firstly, adult zebrafish were daily exposed to 4% or 6% fructose. Then, animals were exposed to 6% fructose every 2 days. The hepatic lipid accumulation was analyzed by Nile Red and Oil Red O staining. Results: The daily exposure to 6% fructose showed increased accumulation of hepatic lipids when compared to 4% and control groups, but the same concentration showed no difference when the exposure happened every 2 days. Conclusion: We can suggest the daily exposure to a concentration of 6% fructose can be considered as a new experimental model of adult zebrafish. Keywords: Fatty liver; fructose; zebrafish

Factors Associated With Persistent Increase in Level of Alanine Aminotransferase in Patients With Chronic Hepatitis B Receiving Oral Antiviral Therapy.

Despite complete suppression of viral DNA with antiviral agents, in some patients with chronic hepatitis B (CHB), serum levels of alanine aminotransferase (ALT) do not normalize. We investigated factors associated with persistent increases in ALT level in patients with CHB given long-term tenofovir disoproxil fumarate. We analyzed data from 471 hepatitis B e antigen (HBeAg)-positive and HBeAg-negative patients with CHB participating in 2 phase 3 trials. We identified patients with an increased level of ALT (above the upper limit of normal range) after 5 years (240 weeks) of tenofovir disoproxil fumarate therapy. We analyzed findings from liver biopsy specimens collected from 467 patients (99%) at baseline and 339 patients (72%) at year 5 of treatment; biopsy specimens were evaluated by an independent pathologist. We performed stepwise, forward, multivariate regression analyses of specified baseline characteristics and on-treatment response parameters to identify factors associated with persistent increases in ALT level. Of the 471 patients, 87 (18%) still had an increased ALT level at year 5 of treatment. Factors associated significantly with a persistent increase in ALT level were a steatosis score of 5% or greater (grade 1 or more) at baseline (odds ratio [OR], 2.236; 95% confidence interval [CI], 1.031-4.852; P= .042) and at year 5 (OR, 3.392; 95% CI, 1.560 ≥ 7.375; P= .002), HBeAg seropositivity at baseline (OR, 3.297; 95% CI, 1.653-6.576; P < .001), and age 40 years or older (OR, 2.099; 95% CI, 1.014-4.342; P= .046). Of the 42 HBeAg-positive patients with steatosis at baseline, 21 (50%) had an increased ALT level at year 5 of treatment. Patients with persistent increases in ALT level were more likely to have an increase in steatosis at year 5 than those with a normal ALT level. HBeAg seropositivity and hepatic steatosis contribute to persistent increases in ALT level in patients with CHB receiving suppressive antiviral treatment. ClinicalTrials.gov registration numbers: NCT00117676 and NCT00116805.

Global Deletion of Glutathione S-Transferase A4 Exacerbates Developmental Nonalcoholic Steatohepatitis

We established a mouse model of developmental nonalcoholic steatohepatitis (NASH) by feeding a high polyunsaturated fat liquid diet to female glutathione-S-transferase 4-4 (Gsta4-/-)/peroxisome proliferator activated receptor α (Ppara-/-) double knockout 129/SvJ mice for 12 weeks from weaning. We used it to probe the importance of lipid peroxidation in progression of NASH beyond simple steatosis. Feeding Gsta4-/-/Ppara-/- double-knockout (dKO) mice liquid diets containing corn oil resulted in a percentage fat-dependent increase in steatosis and necroinflammatory injury (P<0.05). Increasing fat to 70% from 35% resulted in increases in formation of 4-hydroxynonenal protein adducts accompanied by evidence of stellate cell activation, matrix remodeling, and fibrosis (P<0.05). Comparison of dKO mice with wild-type (Wt) and single knockout mice revealed additive effects of Gsta4-/-and Ppara-/- silencing on steatosis, 4-hydroxynonenal adduct formation, oxidative stress, serum alanine amino transferase, expression of tumor necrosis factor alpha, Il6, interferon mRNA, and liver pathology (P<0.05). Induction of Cyp2e1 protein by high-fat diet was suppressed in Gsta4-/- and dKO groups (P<0.05). The dKO mice had similar levels of markers of stellate cell activation and matrix remodeling as Ppara-/- single KO mice. These data suggest that lipid peroxidation products play a role in progression of liver injury to steatohepatitis in NASH produced by high-fat feeding during development but appear less important in development of fibrosis.

Liver steatosis induced by small bowel resection is prevented by oral vancomycin

Intestinal failure-associated liver disease causes significant mortality in patients with short bowel syndrome. Steatosis, a major component of intestinal failure-associated liver disease has been shown to persist even after weaning from parenteral nutrition. We sought to determine whether steatosis occurs in our murine model of short bowel syndrome and whether steatosis was affected by manipulation of the intestinal microbiome. Male C57BL6 mice underwent 50% small bowel resection and orogastric gavage with vancomycin or vehicle for 10weeks. DNA was extracted from stool samples then sequenced using 16s rRNA. Liver lipid content was analyzed. Bile acids were measured in liver and stool. Compared with unoperated mice, small bowel resection resulted in significant changes in the fecal microbiome and was associated with a >25-fold increase in steatosis. Oral vancomycin profoundly altered the gut microbiome and was associated with a 15-fold reduction in hepatic lipid content after resection. There was a 17-fold reduction in fecal secondary bile acids after vancomycin treatment. Massive small bowel resection in mice is associated with development of steatosis and prevented by oral vancomycin. These findings implicate a critical role for gut bacteria in intestinal failure-associated liver disease pathogenesis and illuminate a novel, operative model for future investigation into this important morbidity.

Activation of the Nrf2-ARE pathway in hepatocytes protects against steatosis in nutritionally induced non-alcoholic steatohepatitis in mice.

Oxidative stress is implicated in the development of non-alcoholic steatohepatitis (NASH). The Nrf2-antioxidant response element pathway protects cells from oxidative stress. Studies have shown that global Nrf2 deficiency hastens the progression of NASH. The purpose of this study was to determine whether long-term hepatocyte-specific activation of Nrf2 mitigates NASH progression. Transgenic mice expressing a constitutively active Nrf2 construct in hepatocytes (AlbCre+/caNrf2+) and littermate controls were generated. These mice were fed standard or methionine-choline-deficient (MCD) diet, a diet used to induce NASH development in rodents. After 28 days of MCD dietary feeding, mice developed significant increases in steatosis, inflammation, oxidative stress, and HSC activation compared with those mice on standard diet. AlbCre+/caNrf2+ animals had significantly decreased serum transaminases and reduced steatosis when compared with the AlbCre+/caNrf2- animals. This significant reduction in steatosis was associated with increased expression of genes involved in triglyceride export (MTTP) and β-oxidation (CPT2). However, there were no differences in the increased oxidative stress, inflammation, and HSC activation from MCD diet administration between the AlbCre+/caNrf2- and AlbCre+/caNrf2+ animals. We conclude that hepatocyte-specific activation of Nrf2-mediated gene expression decreased hepatocellular damage and steatosis in a dietary model of NASH. However, hepatocyte-specific induction of Nrf2-mediated gene expression alone is insufficient to mitigate inflammation, oxidative stress, and HSC activation in this nutritional NASH model.

HCC development is associated to peripheral insulin resistance in a mouse model of NASH.

NAFLD is the most common liver disease worldwide but it is the potential evolution to NASH and eventually to hepatocellular carcinoma (HCC), even in the absence of cirrhosis, that makes NAFLD of such clinical importance. Aim: we aimed to create a mouse model reproducing the pathological spectrum of NAFLD and to investigate the role of possible co-factors in promoting HCC. Methods: mice were treated with a choline-deficient L-amino-acid-defined-diet (CDAA) or its control (CSAA diet) and subjected to a low-dose i.p. injection of CCl4 or vehicle. Insulin resistance was measured by the euglycemic-hyperinsulinemic clamp method. Steatosis, fibrosis and HCC were evaluated by histological and molecular analysis. Results: CDAA-treated mice showed peripheral insulin resistance at 1 month. At 1–3 months, extensive steatosis and fibrosis were observed in CDAA and CDAA+CCl4 groups. At 6 months, equal increase in steatosis and fibrosis was observed between the two groups, together with the appearance of tumor. At 9 months of treatment, the 100% of CDAA+CCl4 treated mice revealed tumor versus 40% of CDAA mice. Insulin-like Growth Factor-2 (IGF-2) and Osteopontin (SPP-1) were increased in CDAA mice versus CSAA. Furthermore, Immunostaining for p-AKT, p-c-Myc and Glypican-3 revealed increased positivity in the tumors. Conclusions: the CDAA model promotes the development of HCC from NAFLD-NASH in the presence of insulin resistance but in the absence of cirrhosis. Since this condition is increasingly recognized in humans, our study provides a model that may help understanding mechanisms of carcinogenesis in NAFLD.

Dualistic evolution of liver damage in mice triggered by a single sublethal exposure to Microcystin-LR

Microcystins (MCYST) are the most frequently reported cyanotoxins in human poisoning incidents. Despite the well-described mechanism of acute and lethal injury, the sublethal effects of this toxin require further investigation. The aim of this study was to contribute to the knowledge of the variant MCYST-LR effects at sublethal doses by investigating biochemical changes and tissue damage in a murine model. For this purpose, mice were intraperitoneally injected with 45 μg of MCYST-LR/kg body weight. Their organs were collected at 2, 8, 24, 48 or 96 h after injection. Control animals received saline solution. We detected oxidative imbalance in the liver, particularly at 8 h after exposure. Furthermore, biomarkers of liver injury were detected in high concentration in the serum of the exposed animals. Stereological analyses of the liver indicated two different phases in the intoxication process: an initial phase characterized by an increase in steatosis was followed by a second, later phase characterized by increased inflammation and hepatocyte binucleation. Formation of areas of necrosis and increased blood vessel diameter were observed throughout the experimental period. The number of hepatocytes per area unit also decreased. However, these parameters recovered over the period of exposure. MCYST accumulated in liver and was detectable until the end of the monitoring period. These results confirm the necessity for further studies of processes involved in sublethal exposure to MCYST.

Mouse Liver Dispersion for the Diagnosis of Early-Stage Fatty Liver Disease: A 70-Sample Study

The accumulation of fat droplets within the liver is an important marker of liver disease. This study assesses gradations of steatosis in mouse livers using crawling waves, which are interfering patterns of shear waves introduced into the liver by external sources. The crawling waves are detected by Doppler ultrasound imaging techniques, and these are analyzed to estimate the shear wave speed as a function of frequency between 200 and 360 Hz. In a study of 70 mice with progressive increases in steatosis from 0% to >60%, increases in steatosis are found to increase the dispersion, or frequency dependence, of shear wave speed. This finding confirms an earlier, smaller study and points to the potential of a scoring system for steatosis based on shear wave dispersion.