Skeletal Muscle Atrophy is Exacerbated By Steatotic and Fibrotic Liver-Derived TNF-Alpha in Senescence-Accelerated Mice

Although liver diseases, including non-alcoholic steatohepatitis (NASH), are associated with skeletal muscle atrophy, the mechanism behind their association has not been fully elucidated. In this study, the effects of aging and NASH on the skeletal muscle and the interaction between the liver and muscle were investigated using a diet-induced NASH model in senescence-accelerated mice (SAM). A total of four groups of SAM and its control mice were fed either an NASH-inducing or control diet. In the SAM/NASH group, the histopathology of NASH and markers of oxidative stress were significant. Skeletal muscles were also markedly atrophied. The expression of the ubiquitin ligase Murf1 in the muscle was significantly increased with muscle atrophy, while that of Tnfa was not significantly different. In contrast, the hepatic Tnfa expression and serum TNF-α levels were significantly increased in the SAM/NASH group. These results suggest that liver-derived TNF-α might promote muscle atrophy associated with steatohepatitis and aging through Murf-1. The metabolomic analysis of skeletal muscle indicated higher spermidine and lower tryptophan levels in the NASH-diet group. The findings of this study revealed an aspect of liver-muscle interaction, which might be important in developing treatments for sarcopenia associated with liver diseases.

Oxidative Stress Linked Organ Lipid Hydroperoxidation and Dysregulation in Mouse Model of Nonalcoholic Steatohepatitis: Revealed by Lipidomic Profiling of Liver and Kidney

Nonalcoholic steatohepatitis (NASH) is a prevalent disease related to lipid metabolism disorder and oxidative stress. Lipid hydroperoxidation is known to be a critical driving force of various disorders and diseases. However, the combination of both intact and hydroperoxidized lipids in NASH has not yet been studied. In this work, the liver and kidney samples from NASH-model mice were comprehensively investigated by using the LC/MS-based lipidomic analysis. As a result, triglycerides showed the amount accumulation and the profile alteration for the intact lipids in the NASH group, while phosphatidylethanolamines, lysophosphatidylethanolamines, plasmalogens, and cardiolipins largely depleted, suggesting biomembrane damage and mitochondria dysfunction. Notably, the lipid hydroperoxide species of triglyceride and phosphatidylcholine exhibited a significant elevation in both the liver and the kidney of the NASH group and showed considerable diagnostic ability. Furthermore, the relationship was revealed between the lipid metabolism disturbance and the lipid hydroperoxide accumulation, which played a key role in the vicious circle of NASH. The present study suggested that the omics approach to the lipid hydroperoxide profile might be the potential diagnostic marker of NASH and other oxidative stress-related diseases, as well as the evaluative treatment index of antioxidants.

Adiponutrin and Adiponectin Gene Variants in Indonesian Patients with Non-Alcoholic Fatty Liver Disease: a Preliminary Study

BackgroundVariants of adiponutrin (PNPLA3) and adiponectin (ADIPOQ) genes were considered to be associated with non-alcoholic fatty liver disease (NAFLD). Although the prevalence of NAFLD is increasing, there are limited numbers of studies about the association in Indonesian population.ObjectiveTo confirm whether specific variants of adiponutrin (PNPLA3) and adiponectin (ADIPOQ) genes are associated with NAFLD in Indonesian patients.MethodsData and DNA of 152 participants were obtained from a previous study in Dr. Kariadi Hospital, Semarang, Indonesia. PCR-RFLP analysis was performed for detection of PNPLA3 rs738409 and ADIPOQ rs2241767 variants. The diagnosis and severity of NAFLD were assessed according to NAFLD activity score (NAS) based on histopathology assessment of liverbiopsy.ResultsAllele G of PNPLA3rs738409 was associated with NAFLD in both bivariate (p=0.009, OR 2.52, CI 95% 1.25–5.07) and multivariate (p=0.008, OR 2.62, CI 95% 1.29%–5.32%) analysis, while ADIPOQ rs2241767 had no significant association. In NAFLD participants, both genotypes showed allele G was higher in the group of possible non-alcoholic steatohepatitis (NASH) – NASH (NAS >2) than in the simple steatosis group (NAS <2) i.e. 40.0% vs. 3.75% for the rs2241767 variant and 23.75% vs. 1.25% for the rs738409 variant, without significant association.ConclusionVariant PNPLA3 rs738409 was associated with NAFLD incidence in studied population. Among NAFLD participants, the frequency of both variants were found higher in the possible NASH – NASH group, yet needs to be confirmed with more participants and a multicenter study.Data and DNA of 152 participants were obtained from a previous study in Dr. Kariadi Hospital, Semarang, Indonesia. PCR-RFLP analysis was performed for detection of PNPLA3 rs738409 and ADIPOQ rs2241767 variants. The diagnosis and severity of NAFLD were assessed according to NAFLD activity score (NAS) based on histopathology assessment of liverbiopsy.

Crosstalk Between Plasma Cytokines, Inflammation, and Liver Damage as a New Strategy to Monitoring NAFLD Progression

Cytokines are involved in the immunopathogenesis of nonalcoholic fatty liver disease (NAFLD), but the relationship between them and clinical parameters of NAFLD progression is still unknown. Using flow cytometry, we evaluated the plasma levels of IL-1β, IL-6, IL-12, TNF and IL-10 and their association with clinical and biochemical parameters of liver function during simple steatosis (NAFL) and nonalcoholic steatohepatitis (NASH) in biopsy-proven patients. The NASH patients showed higher levels of IL-6 associated with a lower IL-10/IL-6 ratio. Besides heatmaps were similar in the NAFL and NASH groups, the same did not occur in signature curves, the NASH patients were high producers to IL-12 and IL-6 while the NAFL patients were not high producers of any cytokines evaluated. Integrative biomarker network analysis revealed that cytokines are differently correlated with clinical parameters, while IL-12, IL-10 presented moderate and negative correlations with glycemic and lipid profile in the NAFL group. The NASH group IL-12 and TNF revealed stronger and positive correlations with transient elastography parameters and NAFLD liver fibrosis score. These data suggest that IL-6 and IL-10 might act in chronic inflammation and insulin resistance whereas IL-12 and TNF may be involved in promoting liver damage and NAFLD progression. Plasma concentration analysis of these molecules and their association with clinical parameters can be used as new biomarkers to monitoring NAFLD progression and to reflect NASH development.

Gut Microbiota and Host Cyp450s Co-contribute to Pharmacokinetic Variability in Mice With Non-Alcoholic Steatohepatitis: Vary From Drug to Drug

Background: Pharmacokinetic variability in disease state is common in clinical practice, but the underlying mechanism remains unclear. We aim to investigate the effects of gut microbiota and host Cyp450s on pharmacokinetic variability in mice with non-alcoholic steatohepatitis (NASH).Methods: The pharmacokinetic variability of mice with NASH was explored under intragastric and intravenous administration of a cocktail mixture of omeprazole, phenacetin, midazolam, tolbutamide, chlorzoxazone, and metoprolol compared with the control group. The pharmacokinetic variability of the drugs and its relation with changes of gut microbiota and host Cyp450s were compared and analyzed.Results: The exposure of all drugs, except metoprolol, significantly increased in the NASH group under intragastric administration. However, no significant increase in the exposure of all drugs, except tolbutamide, was observed in the NASH group under intravenous administration. The pharmacokinetic variabilities of phenacetin, midazolam, omeprazole, and chlorzoxazone were mainly associated with decreased elimination activity of the gut microbiota. By contrast, the pharmacokinetic variability of tolbutamide was mainly related to the change in host Cyp2c65. However, gut microbiota and host Cyp450s exerted minimal effect on the pharmacokinetic variability of metoprolol.Conclusions: Gut microbiota and host Cyp450s co-contribute the pharmacokinetic variability in mice with NASH, and the degree of contribution varies from drug to drug.

Circulating miRNA is a useful diagnostic biomarker for nonalcoholic steatohepatitis in nonalcoholic fatty liver disease

Nonalcoholic steatohepatitis (NASH) is considered as a progressive form of nonalcoholic fatty liver disease (NAFLD). To distinguish NASH from nonalcoholic fatty liver (NAFL), we evaluated the diagnostic value of circulating miRNAs. Small RNA sequencing was performed on 12 NAFL patients and 12 NASH patients, and the miRNA expression was compared. After selecting miRNAs for the diagnosis of NASH, we analyzed the diagnostic accuracy of each miRNA and the combination of miRNAs. External validation was performed using quantitative reverse transcription PCR. Among the 2,588 miRNAs, 26 miRNAs significantly increased in the NASH group than in the NAFL group. Among the 26 elevated miRNAs in the NASH group, 8 miRNAs were selected, and in silico analysis was performed. Only four miRNAs (miR-21-5p, miR-151a-3p, miR-192-5p, and miR-4449) showed significant area under the receiver operating characteristic curve (AUC) values for NASH diagnosis. The combination of the four miRNAs showed satisfactory diagnostic accuracy for NASH (AUC 0.875; 95% CI 0.676–0.973). External validation revealed similar diagnostic accuracy for NASH (AUC 0.874; 95% CI 0.724–0.960). NASH represents significantly distinct miRNA expression profile compared with NAFL. The combination of serum circulating miRNAs can be used as a novel biomarker for the NASH diagnosis in NAFLD.

Betatrophin Levels Are Related to the Early Histological Findings in Nonalcoholic Fatty Liver Disease

Betatrophin, a liver hormone, regulates glucose and lipid metabolism. We investigated the betatrophin levels in nonalcoholic fatty liver disease (NAFLD) and searched for any relationship with histological severity and metabolic parameters. Fifty males with NAFLD [Nonalcoholic Steatohepatitis (NASH) (n = 32); non-NASH (n = 18)] and 30 healthy controls were included. Plasma betatrophin was measured by ELISA method. Insulin sensitivity was assessed by HOMA-IR index. Histological features were scored by the semi quantitative classification and combined as the NAFLD activity score (NAS). Betatrophin levels in the non-NASH group were significantly higher than the controls. Betatrophin was positively correlated to the age, waist circumference, total cholesterol, triglycerides, LDL cholesterol, glucose, insulin, HOMA-IR index and gamma glutamyl transpeptidase levels, and negatively correlated to the steatosis and NAS. In the stepwise linear regression analysis, the triglyceride (β = 0.457, p < 0.001), glucose (β = 0.281, p = 0.02) and NAS (β = −0.260, p = 0.03) were the independent determinants of betatrophin. Betatrophin levels are higher in the early stages of NAFLD and tend to decrease when the disease progresses. This could be an important preliminary mechanistic finding to explain the increased frequency of glucose intolerance during the course of NAFLD.

Dietary Patterns in Patients With Different Forms of Non-alcoholic Fatty Liver Disease

Objectives The aim of the study was to compare food patterns in patients with simple steatosis (SS) and non-alcoholic steatohepatitis (NASH). Methods Prospective study was approved by LEC and enrolled subjects with confirmed non-alcoholic fatty liver disease (SS or NASH group). Nutrilogic software (Nutrilogic, Russia) was used for diet assessment. Dietary patterns were assessed according to the Healthy Eating Index (HEI): amounts of the major groups of foods and food products (grains, fruits, vegetables, dairy products, meats, fats and confectioneries) consumption were compared to the levels described in the HEI, and individual deviation rates were obtained. Nonparametric statistics (Mann-Whitney U test) was used to compare deviation rates found in subjects of SS and NASH groups. Results Subjects in NASH group (n = 22) were younger (Mean ± SD: 48.6 ± 13.4 y.o.) than those in SS group (n = 156; 56.5 ± 12.3 y.o., P = 0.008). Main macronutrients consumption did not differ between the groups. Although dietary patterns of major groups of foods consumption did not differ between SS and NASH groups, analysis of the foods subgroups revealed dissimilarity in the structure of vegetables and fats consumption. Patients with NASH consumed larger amounts of potatoes (0.14 ± 0.08 vs 0.11 ± 0.15, P = 0.006), and lower – of onions (0.02 ± 0.03 vs 0.07 ± 0.1, P = 0.006); they also consumed lower amounts of dairy butter (0.14 ± 0.44 vs 0.15 ± 0.21, P = 0.009) compared to subjects with simple steatosis. No other difference in the structure of vegetables (beans, root crops, leafy and other vegetables), fats (animal fats, vegetable oils, margarines) and other major groups of foods consumption was revealed. Conclusions Dietary patterns of patients with non-alcoholic steatohepatitis and simple steatosis differ. The obtained results may help in diet modification in patients with NAFLD in case of confirmation in larger multicenter trials. Funding Sources Russian Science Foundation, grant #1976-30014.

Bajos niveles de actividad de la lipasa ácida lisosomal y su relación con el desarrollo de la cirrosis de origen criptogénica/NASH: un estudio de cohorte

Introduction and objective. The deficiency of the Lysosomal acid lipase (LAL) activity has been related to cirrhosis due to non-alcoholic steatohepatitis (NASH). Many of the cirrhosis classified as cryptogenic are the evolution of liver disease due to fatty liver. The objective of the present study was to evaluate the lysosomal acid lipase (LAL) activity in the patients with liver cirrhosis of any etiology and establish whether low levels correlate with cryptogenic cirrhosis of origin cryptogenic or NASH. Methods. Was an analytical cohort study including 96 patients with cirrhosis of any etiology for which LAL activity was measured. Results. Fifty-five patients (58%) with cryptogenic cirrhosis or NASH were included and 41 with other etiologies. The fifty-three percent of the total population were women. The severity scores of liver disease were significantly higher in the patients with the cryptogenic cirrhosis or NASH: MELD (11.72 ± 7.3 vs 4,34 ± 5.7; p = 0.001) and Child-Pugh (7.26 ± 3.8 vs 7.26 ± 3.8; p = 0.004). LAL activity was significant lower (202.40 ± 98.8 vs 242.55 ± 121.9; p = 0.04) in the cirrhosis cryptogenic or NASH group. In the multivariate analysis, low LAL activity (< 150 nmol/spot/hour), was correlated with the presence of cryptogenic cirrhosis or NASH. Conclusions. The patients with cryptogenic cirrhosis or NASH have lower levels of LAL activity than those with cirrhosis of other etiologies. LAL activity below 150 nmol/spot/hour is predictive of the cryptogenic cirrhosis or NASH.

Clinical characteristics and risk factors of nonalcoholic fatty liver disease in children with obesity

Background With the increasing number of children with obesity worldwide, nonalcoholic fatty liver disease (NAFLD) has become the most common liver disease among children. It is necessary to recognize the risk factors of NAFLD for prevention in childhood since NAFLD is asymptomatic in the early stage. Objectives. The objective of this study was to investigate possible risk factors of NAFLD in children with obesity, providing evidence for monitoring and prevention strategies at an early stage for obese children with NAFLD. Methods Data were collected from 428 children and adolescents aged 6-16 years recruited from the Children’s Hospital at Nanjing Medical University from September 2015 to April 2018 and analyzed. Based on a combination of ultrasound results and alanine transaminase levels, subjects were divided into three groups: simple obesity (SOB), simple steatosis (SS), and nonalcoholic fatty hepatitis (NASH). Blood biochemical examination included glucose, insulin, uric acid, lipid profile and liver enzymes. Results Among 428 children with obesity, 235 (54.9%) had SS and 45 (10.5%) had NASH. Body mass index, body mass index standard deviation score (BMI-SDS), waist circumference, body fat, liver enzymes, uric acid and HOMA-IR level were significantly higher in the NASH group than in the SS and SOB groups (p < 0.001). 53.3% of the SS group and 49.8% of the NASH group had metabolic syndrome, significantly more than in the SOB group (19.6%, p < 0.001). After adjustment for confounding factors, logistic regression models revealed that NASH was associated with BMI-SDS ≥ 3, gender, hyperuricemia and insulin resistance. Conclusions The prevalence of NASH in children with obesity is closely related to high BMI-SDS, gender, insulin resistance and hyperuricemia. These findings provide evidence that monitoring risk factors of childhood obesity can assist in developing prevention strategies for liver disease at an early stage.