Nonalcoholic Steatohepatitis Score Research Articles

Environmental toxicants modulate disease severity in pediatric metabolic dysfunction-associated steatohepatitis.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is common in children. We hypothesized environmental toxins could drive progression to metabolic dysfunction-associated steatohepatitis (MASH), and assayed serum toxins and metabolites in children with histologically characterized MASLD/MASH. Environmental chemicals, common in household items, perfluoroalkyl substances (PFAS), polybrominated flame retardants (polybrominated diphenyl ethers [PBDEs]), and metabolic profiles were assayed in children enrolled in the multicenter NASH Clinical Research Network Pediatric Database 2. Mixture models, using repeated holdout weighted quantile sum regression (WQSrh) were run in addition to single chemical/metabolite logistic regression. For metabolomic analyses, random subset version of WQSrh was used for the large number of predictors versus participants. Nominal and false discovery rate (FDR) p-values (two-sided) were computed. Four hundred and thirty-five children distributed across MASH (n = 293) and MASLD (n = 142), with 304 (69.9%) males. Mean (standard deviation) for Nonalcoholic Steatohepatitis Score (NAS) and alanine aminotransferase (ALT) for MASLD were 3.1 (1.0), 67.9 (43.4), and for MASH 4.2 (1.4), 144 (121). There was an inverse association between PFAS/PBDE mixture and MASH versus MASLD, lobular inflammation (p = 0.026), NAS (p = 0.009, FDR p = 0.04), and log-transformed ALT (p = 0.005, FDR p = 0.025) driven by perfluorohexane sulfonate (PFHXS). Metabolites from positive hydrophilic interaction liquid chromatography mode, biliverdin (p = 0.002) and 1-methylhistidine (associated with meat ingestion, p = 0.02) and reverse phase negative mode, hippuric acid (solvent exposure, p = 0.022) significantly associated with MASH. Significant negative PFAS/PBDE mixture effect and odds of MASH were dominated by PHFXS. Several metabolites are significantly associated with MASH which inform mechanistic pathways and could drive key therapeutic and diagnostic strategies in children.

HP inulin-MCT dietary fiber improves lipid metabolism and prevents non-alcoholic steatohepatitis in obese mice

IntroductionRegular intake of dietary fiber (DF) can counteract the negative impacts of obesity on physical health and chronic disease risk. DF mitigates obesity by promoting fullness, reducing calories, slowing nutrient absorption, stabilizing glucose, preventing fat storage, and influencing gut microbiota. High-performance (HP) inulin, a highly fermentable water-soluble DF, shows promise, but its palatability is limited in powder form. Combining HP inulin with beneficial triglycerides like Medium Chain Triglycerides (MCT) could overcome this issue. Aim of studyThis study investigates the effects of HP inulin-MCT on lipid metabolism and immune modulation in high-fat high-sucrose (HFHS) diet-induced obese mice. MethodsObese mice were orally administered with 20 % (w/v) HP inulin-MCT ad-libitum for 30 days. We measured their peripheral blood lipid profile, including LDL, HDL, and triglycerides. Additionally, the atherogenic coefficient, a parameter for assessing cardiovascular disease risk, was determined by calculating the ratio of non-HDL-C to HDL-C. Flow cytometry was used to identify splenic regulatory CD4 + and CD8 + T cells (Tregs). The Non-Alcoholic Steatohepatitis Score was assessed through histological examination. ResultsSignificant reduced body mass index, improved lipid profiles, and reduced cardiovascular disease risk were observed in the obese mice treated with HP inulin-MCT. Moreover, HP inulin-MCT appeared to ameliorate non-alcoholic steatohepatitis, a liver condition associated with obesity. These health improvements were linked to immune system modulation, associated with an increase of CD4+ T-regulatory cells after HP inulin-MCT supplementation. ConclusionHP inulin-MCT is a promising and palatable dietary intervention to combat obesity-related noncommunicable diseases, offering the potential for better overall health outcomes.

Relationship between the changes in hepatokine levels and metabolic effects after laparoscopic sleeve gastrectomy in severely obese patients

PurposeTo clarify the relationships between the changes in hepatokines and weight loss, and between these changes and the metabolic effects, and the roles played by these changes, after laparoscopic sleeve gastrectomy (LSG).MethodsWe recruited 25 Japanese patients with severe obesity, who underwent LSG. We measured two hepatokines: selenoprotein P (SeP) and leukocyte cell-derived chemotaxin 2 (LECT2), at the baseline, and then 6 months and 1 year after LSG. Finally, we compared the changes in the hepatokines with the parameters of type 2 diabetes (T2D) and non-alcoholic steatohepatitis (NASH).ResultsChanges in LECT2 were correlated with the percentage of total weight loss (ρ = − 0.499, P = 0.024) and the decrease in total fat area (ρ = 0.559, P = 0.003). The changes in SeP were correlated with those in hemoglobin A1c (ρ = 0.526, P = 0.043) and the insulinogenic index (ρ = 0.638, P = 0.010) in T2D patients. In patients with NASH, the LECT2 levels were correlated with liver steatosis (ρ = 0.601).ConclusionsSeP levels decrease in association with HbA1c reduction, whereas LECT2 levels are associated with reductions in fat mass and NASH scores after LSG. Hepatokines may be involved in the pathology of obesity and its complications.

LEARN algorithm: a novel option for predicting non-alcoholic steatohepatitis.

There is an unmet need for accurate non-invasive methods to diagnose non-alcoholic steatohepatitis (NASH). Since impedance-based measurements of body composition are simple, repeatable and have a strong association with non-alcoholic fatty liver disease (NAFLD) severity, we aimed to develop a novel and fully automatic machine learning algorithm, consisting of a deep neural network based on impedance-based measurements of body composition to identify NASH [the bioeLectrical impEdance Analysis foR Nash (LEARN) algorithm]. A total of 1,259 consecutive subjects with suspected NAFLD were screened from six medical centers across China, of which 766 patients with biopsy-proven NAFLD were included in final analysis. These patients were randomly subdivided into the training and validation groups, in a ratio of 4:1. The LEARN algorithm was developed in the training group to identify NASH, and subsequently, tested in the validation group. The LEARN algorithm utilizing impedance-based measurements of body composition along with age, sex, pre-existing hypertension and diabetes, was able to predict the likelihood of having NASH. This algorithm showed good discriminatory ability for identifying NASH in both the training and validation groups [area under the receiver operating characteristics (AUROC): 0.81, 95% CI: 0.77-0.84 and AUROC: 0.80, 95% CI: 0.73-0.87, respectively]. This algorithm also performed better than serum cytokeratin-18 neoepitope M30 (CK-18 M30) level or other non-invasive NASH scores (including HAIR, ION, NICE) for identifying NASH (P value <0.001). Additionally, the LEARN algorithm performed well in identifying NASH in different patient subgroups, as well as in subjects with partial missing body composition data. The LEARN algorithm, utilizing simple easily obtained measures, provides a fully automated, simple, non-invasive method for identifying NASH.

Role of Oxidative Stress and Lipid Peroxidation in the Pathophysiology of NAFLD.

Non-alcoholic fatty liver disease (NAFLD) is characterised by an excess of hepatic fat that can progress to steatohepatitis, fibrosis, cirrhosis and hepatocarcinoma. The imbalance between lipid uptake/lipogenesis and lipid oxidation/secretion in the liver is a major feature of NAFLD. Given the lack of a non-invasive and reliable methods for the diagnosis of non-alcoholic steatohepatitis (NASH), it is important to find serum markers that are capable of discriminating or defining patients with this stage of NASH. Blood samples were obtained from 152 Caucasian subjects with biopsy-proven NAFLD due to persistently elevated liver enzyme levels. Metabolites representative of oxidative stress were assessed. The findings derived from this work revealed that NAFLD patients with a NASH score of ≥ 4 showed significantly higher levels of lipid peroxidation (LPO). Indeed, LPO levels above the optimal operating point (OOP) of 315.39 μM are an independent risk factor for presenting a NASH score of ≥ 4 (OR: 4.71; 95% CI: 1.68−13.19; p = 0.003). The area under the curve (AUC = 0.81, 95% CI = 0.73−0.89, p < 0.001) shows a good discrimination ability of the model. Therefore, understanding the molecular mechanisms underlying the basal inflammation present in these patients is postulated as a possible source of biomarkers and therapeutic targets in NASH.

Effects of probiotics and high fructose diet on De Ritis ratio, TGF-B levels, and liver histopathology of Sprague Dawley rats

Background: Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Histopathological diagnosis has been the gold standard for diagnosing and determining the severity of non-alcoholic steatohepatitis (NASH). However, this method is invasive, especially for diagnosing NASH in children. This research aimed to study the effect of probiotics on the De Ritis ratio, TGF-β levels, and histopathological changes in rats given High Fat, High Fructose (HFHFr) diet to look at the correlation between the De Ritis ratio and liver histopathology in diagnosing NAFLD. Method: Twenty-one male Sprague Dawley rats were included and divided into three groups. In the first group, as control only received a normal chow diet for eight weeks. The second group received an HFHFr diet for eight weeks, and the last group received HFHFr for eight weeks and a combination of HFHFr and probiotics for another eight weeks. After decapitation, we took 3 ml of serum from each rat to measure AST, ALT, and TGF-β. A 4 μm thick liver tissue slide was taken and stained with Hematoxylin – Eosin Stain for histopathological analysis. We used the Non-Alcoholic Steatohepatitis (NAS) Score for measuring liver damage progression. Results: De Ritis ratio and TGF-β level did not significantly differ between probiotic and non-probiotic groups (p = 0.064 and 0.383), but there was a significant NAS score difference (p = 0.001) in probiotic and non-probiotic groups. This was followed by a significant correlation between the De Ritis ratio and the NAS score (r = 0.613, p = 0.003). Conclusion: Probiotic supplementation alleviated liver damage caused by the HFHFr diet but did not successfully reduce the De Ritis ratio or improve TGF-β.

MRE-based NASH score for diagnosis of nonalcoholic steatohepatitis in patients with nonalcoholic fatty liver disease.

As the prevalence of nonalcoholic fatty liver disease (NAFLD) is approximately 30% in the general population, it is important to develop a non-invasive biomarker for the diagnosis of nonalcoholic steatohepatitis (NASH). This prospective cross-sectional study aimed to develop a scoring system for NASH diagnosis through multiparametric magnetic resonance (MR) and clinical indicators. Medical history, laboratory tests, and MR parameters of patients with NAFLD were assessed. A scoring system was developed using a logistic regression model. In total, 127 patients (58 with nonalcoholic fatty liver [NAFL] and 69 with NASH) were enrolled. After evaluating 23 clinical characteristics of the patients (4 categorical and 19 numeric variables) for the NASH diagnostic model, an equation for MR elastography (MRE)-based NASH score was obtained using 3 demographic factors, 2 laboratory variables, and MRE. The MRE-based NASH score showed a satisfactory accuracy for NASH diagnosis (c-statistics, 0.841; 95% CI 0.772-0.910). At a cut-off MRE-based NASH score of 0.68 for NASH diagnosis, its sensitivity was 0.68 and specificity was 0.91. When an MRE-based NASH score of 0.37 was used as a cut-off for NASH exclusion, the sensitivity was 0.91 and specificity was 0.55. Overall, 35% (44/127) of patients were in the gray zone (between 0.37 and 0.68). Internal validation via bootstrapping also indicated the satisfactory accuracy of NASH diagnosis (optimism-corrected statistics, 0.811). MRE-based NASH score is a useful and accurate non-invasive biomarker for diagnosis of NASH in patients with NAFLD.

SHOULD ROUTINE LIVER BIOPSY BE CONSIDERED IN BARIATRIC SURGICAL PRACTICE? AN ANALYSIS OF THE LIMITATIONS OF NON-INVASIVE NAFLD MARKERS.

Non-invasive markers are useful and practical tools for assessing non-alcoholic fatty liver disease (NAFLD), but liver biopsy remains the gold-standard method. Liver biopsy can be easily obtained on individuals undergoing bariatric surgery, but there is no ultimate evidence on the relationship between costs, risks and benefits of its systematic performance. To compare the diagnostic accuracy of non-invasive methods with liver biopsy for detection and staging of NAFLD in obese individuals undergoing bariatric surgery. This is a cross-sectional, observational and descriptive study which enrolled individuals who underwent bariatric surgery from 2018 through 2019 at a public tertiary university hospital. Ultrasound scan, hepatic steatosis index, Clinical Non-Alcoholic Steatohepatitis Score (C-NASH), hypertension, alanine aminotransferase (ALT) and insulin resistance (HAIR), aspartate aminotransferase (AST) to Platelet Ratio Index (APRI), NAFLD Fibrosis Score (NFS) and body mass index, AST/ALT ratio, and diabetes (BARD) were the methods compared with the histopathological examination of wedge liver biopsies collected during surgery. Of 104 individuals analyzed, 91 (87.5%) were female. The mean age was 34.9±9.7 years. There was no biopsy-related morbidity. The respective overall accuracies of each marker analyzed were: ultrasound scan (79.81% for steatosis), hepatic steatosis index (79.81% for steatosis), HAIR (40.23% for steatohepatitis), C-NASH (22.99% for steatohepatitis), APRI (94.23% for advanced fibrosis), NFS (94.23% for advanced fibrosis), and BARD (16.35% for advanced fibrosis). Given the high prevalence of liver disease within this population, even the most accurate markers did not present enough discretionary power to detect and/or rule out the NAFLD aspects they were designed to assess in comparison with liver biopsy, which is safe and easy to obtain in these patients. Wedge liver biopsy during bariatric surgery helps to diagnose and stage NAFLD, presents low risks and acceptable costs; given the limitations of non-invasive methods, it is justifiable and should be considered in bariatric routine.

Isolation and co-culture of primary hepatocytes and primary Kupffer cells from rats with nonalcoholic steatohepatitis

Objective: By isolating and purifying primary hepatocytes and primary Kupffer cells from rats with nonalcoholic steatohepatitis (NASH), and establishing the primary cell model of NASH in vitro, to provide reliable technical support for cell experiment in the study of NASH. Methods: Forty SD rats were selected and randomly divided into the control group and the NASH group. The rats in the control group were fed with common feed, and the rats in the NASH group were fed with a high-fat diet (88% basal feed + 10% lard + 2% cholesterol). After 6-8 weeks, using the NASH score table, the liver tissue section steatosis + intralobular inflammation + ballooning degeneration score ≥ 4 points under pathological observation, indicating that the rat NASH model was successfully established. And the primary hepatocytes of NASH rats were isolated and purified by collagenase in situ perfusion. Cells were identified by CK-18 and CD68 immunofluorescence and ink swallowing test. The lipid accumulation was tested by Oil red O staining, and the contents of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined to evaluate the liver function in primary hepatocytes of NASH rats. The expressions of inflammatory factors of primary Kupffer cells were detected by Western blot. Finally, primary hepatocytes and primary Kupffer cells were co cultured at the ratio of 6:1 and observed under microscope. Results: NASH primary hepatocytes and primary Kupffer cells were successfully isolated and purified. Compared with the control group, Oil red O staining showed that the primary hepatocytes of the NASH group had obvious fat deposition, and the AST and ALT levels in the primary hepatocytes of the NASH group were significantly higher than those of the control group, indicating obvious liver damage (P＜ 0.05). The Western blot result showed that the levels of TNF-α, IL-1β and MCP-1 in primary Kupffer cells was significantly higher than the control group (P＜0.05). Conclusion: The primary hepatocytes and primary Kupffer cells of NASH rats were isolated successfully by collagenase in situ perfusion. At the same time, a proportional co-culture rat in vitro primary cell NASH model was successfully established.

Multiparametric US for Identifying Patients with High-Risk NASH: A Derivation and Validation Study.

Background Nonalcoholic fatty liver disease (NAFLD) is common in the general population but identifying patients with high-risk nonalcoholic steatohepatitis (NASH) who are candidates for pharmacologic therapy remains a challenge. Purpose To develop a score to identify patients with high-risk NASH, defined as NASH with an NAFLD activity score (NAS) of 4 or greater and clinically significant fibrosis (stage 2 [F2] or higher). Materials and Methods This was a cross-sectional secondary analysis of data prospectively collected between April 2017 and March 2019 for a group of patients with NAFLD in Japan (Japan NAFLD, the derivation data set) with contemporaneous two-dimensional shear-wave elastography and biopsy-proven NAFLD (age range, 20-89 years). Three US markers (liver stiffness [LS, measured in kilopascals], attenuation coefficient [AC, measured in decibels per centimeter per megahertz], and dispersion slope [DS, measured in meters per second per kilohertz]) were determined, together with aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels and the AST-to-ALT ratio. The best-fit multivariate logistic regression model for identifying patients with high-risk NASH was determined. Diagnostic performance was assessed by using the area under the receiver operating characteristic curve (AUC). The findings were validated in an independent data set (Korea NAFLD; age range, 20-78 years). Results The Japan NAFLD data set included 111 patients (mean age, 53 years ± 18 [standard deviation]; 57 men), 84 (76%) with NASH. The Korea NAFLD data set included 102 patients (mean age, 48 years ± 18; 43 men), 55 (36%) with NASH. The most predictive model (LAD NASH score) combined LS, AC, and DS. Performance was satisfactory in both the derivation sample (AUC, 0.86; 95% CI: 0.79, 0.93) and the validation sample (AUC, 0.88; 95% CI: 0.80, 0.95). The LAD NASH score showed a positive predictive value of 86.5% and a negative predictive value of 87.5% for high-risk NASH in the derivation sample. Conclusion A score combining three US markers may be useful for noninvasive identification of patients with high-risk nonalcoholic steatohepatitis for inclusion in clinical trials and pharmacologic therapy. © RSNA, 2021 Online supplemental material is available for this article. See also the editorial by Lockhart in this issue.

Porphyromonas gingivalis exacerbates the progression of fatty liver disease via CD36-PPARγ pathway

Periodontal diseases have been reported to have a multidirectional association with metabolic disorders. We sought to investigate the correlation between periodontitis and diabetes or fatty liver disease using HFD-fed obese mice inoculated with P. gingivalis. Body weight, alveolar bone loss, serological biochemistry, and glucose level were determined to evaluate the pathophysiology of periodontitis and diabetes. For the evaluation of fatty liver disease, hepatic nonalcoholic steatohepatitis (NASH) was assessed by scoring steatosis, inflammation, hepatocyte ballooning and the crucial signaling pathways involved in liver metabolism were analyzed. The C-reactive protein (CRP) level and NASH score in P. gingivalis-infected obese mice were significantly elevated. Particularly, the extensive lobular inflammation was observed in the liver of obese mice infected with P. gingivalis. Moreover, the expression of metabolic regulatory factors, including peroxisome proliferator-activated receptor γ (Pparγ) and the fatty acid transporter Cd36, was up-regulated in the liver of P. gingivalis-infected obese mice. However, inoculation of P. gingivalis had no significant influence on glucose homeostasis, insulin resistance, and hepatic mTOR/AMPK signaling. In conclusion, our results indicate that P. gingivalis can induce the progression of fatty liver disease in HFD-fed mice through the upregulation of CD36-PPARγ axis.

Metreleptin therapy for nonalcoholic steatohepatitis: Open-label therapy interventions in two different clinical settings

Recombinant leptin therapy reverses nonalcoholic steatohepatitis (NASH) in leptin-deficient lipodystrophy. We inquired if leptin therapy would improve nonalcoholic steatohepatitis in more common forms of this heterogeneous condition. Nine male patients with relative leptin deficiency (level < 25th percentile of body mass index- and gender-matched United States population) and biopsy-proven NASH and 23 patients with partial lipodystrophy and NASH were recruited for two distinctive open-label trials. Participants received leptin therapy in the form of metreleptin for 12 months. The primary endpoints were the global nonalcoholic steatohepatitis scores from paired liver biopsies scored blindly. Of 9 participants recruited in the relative leptin deficiency treatment study, 7 completed 12-months of therapy. Mean global NASH scores were reduced from 8 ± 3 to 5 ± 2 (range: from 1 to 6, P = 0.004). In the partial lipodystrophy study, 19 of 22 subjects completed 12 months of treatment, and 18 completed a second liver biopsy. Global NASH scores also reduced significantly from 6 ± 2 to 5 ± 2 (range: from -2 to 4, P = 0.008). In both studies, the predominant changes were in steatosis and hepatic injury scores. Our findings show that patients with NASH associated with both relative leptin deficiency and partial lipodystrophy have reductions in hepatic steatosis and injury in response to exogenous leptin therapy. Moreover, leptin deficiency may have regulatory effects in mediating fat deposition and ensuing injury in the liver.TRIAL REGISTRATION. ClinicalTrials.gov NCT00596934 and NCT01679197.

Acute Phase Inflammatory Proteins, Gut Microbiota Dysbiosis and Severity of Nonalcoholic Fatty Liver Disease in Patients with Type 2 Diabetes Mellitus

Since obesity and type 2 diabetes mellitus (DM) became almost pandemic, incidence and prevalence of associated nonalcoholic fatty liver disease (NAFLD) rose accordingly. Aim of the study was assessing whether there is a link between circulating C-reactive protein(CRP), gut microbiota dysbiosis and severity of liver disease in patients with NAFLD and confirmed type 2 DM. 50 patients diagnosed with type 2 DM were consecutively enrolled in this cross-sectional, pilot study, being evenly divided in two groups, as matched pairs, on the basis of presence or absence of NAFLD confirmed by abdominal and CT exam, after ruling out a lot of diseases and conditions. Patients underwent measurements of waist circumference, blood pressure (BP), body mass index (BMI) and thoroughly physical examination. Complete blood count (CBC), liver tests, CRP, fasting plasma glucose (FPG), low density lipoprotein(LDL) and high-density lipoprotein(HDL) cholesterol, triglycerides, creatinin and uric acid, micro proteinuria as well as stool microbiology were run, using standardized methods. Fatty liver index (FLI) was also calculated. Fibromax (BioPredictive) was performed in NAFLD (+) patients. NAFLD (+) group displayed significant differences of age and BMI, as well as lab biochemistry: ALT, GGT, LDL-cholesterol and triglycerides, CRP, microprotienuria and stool microbiology.. Strong positive correlation of CRP with gut dysbiosis (DB) severity, microproteinuria, as well as with nonalcoholic steatohepatitis (NASH) score, were observed. DB range correlated positively in NAFLD (+) group with age, BMI, BP, dyspepsia as well as with NASH scores. In conclusion patients with NAFLD and associated type 2 DM displayed a specific profile with significant differences of age, BMI, FLI, diabetic treatment, biochemistry and gut microbiological charts. Strong positive correlations between CRP, gut DB and NASH score suggest the possibility of link between inflammation, gut microbiota DB and severity of liver disease.

OR17-02 Changes in Hepatokines and Apolipoproteins Are Associated with Metabolic Response to Metreleptin in Partial Lipodystrophy

Introduction Metreleptin treatment may improve the metabolic aspects of partial lipodystrophy; however, the treatment response is heterogeneous. This study aimed to explore changes in circulating apolipoprotein concentrations, as well as ANGPLT3, ANGPLT4, and IGF-1 levels in patients treated with Metreleptin as part of a clinical study investigating the efficacy of Metreleptin in nonalcoholic steatohepatitis (NASH) associated with partial lipodystrophy (ClinicalTrials.gov identifier: NCT01679197). Methods Serum samples of 18 patients with partial lipodystrophy who underwent a full metabolic evaluation and paired liver biopsies before and after Metreleptin were studied. Patients were tested at baseline, month (M) 3, M6, and M12. Glycemic response was defined as “more than 1% HbA1c reduction from baseline”. Lipid response was defined as “more than 30% decrease in triglycerides from baseline”. The hepatic response was defined as “a decrease of 2 points or more from baseline in NASH score, without an increase in fibrosis”. Patients with “any 2 of 3 above” at M12 were defined as metabolic responders. Results Metreleptin treatment resulted in significant reductions in triglycerides (346 mg/dL vs. 253 mg/dL; F: 8.474; p < 0.001), apo B (145.24 mg/dL vs. 111.09 mg/dL; F: 9.266: p < 0.001), apo CII (18.65 mg/dL vs. 15.95 mg/dL; F: 6.663: p = 0.001), apo CIII (62.95 mg/dL vs. 49.33 mg/dL; F: 5.640, p = 0.002), apo E (8.16 mg/dL vs. 6.52 mg/dL; F: 11.056, p < 0.001), and ANGPLT3 (14.36 ng/mL vs. 12.00 mg/dL; F: 4.348; p = 0.008) over time. IGF-1 levels significantly increased at M3 (134 ng/mL vs. 139 ng/mL; p = 0.001), however the difference was not significant over time. Metabolic responders had lower baseline leptin (12.4 ng/mL vs. 27.8 ng/mL; p = 0.024) and IGF-1 (95 ng/ml vs. 151 ng/mL; p = 0.008), and higher apo CII (39.06 mg/dL vs. 17.90 mg/dL; p = 0.011), apo CIII (173.57 mg/dL vs. 51.51 mg/dL; p = 0.015), apo E (18.41 mg/dL vs. 5.89 mg/dL; p = 0.002), and ANGPLT3 (17.33 ng/mL vs. 10.06 ng/mL; p = 0.04). Metabolic responders had a significant increase in IGF-1 (95 ng/mL vs. 134 ng/mL; p = 0.019), which was statistically distinguished from non-responders (p = 0.004). Responders also had a greater reduction in apo CII (20.51 mg/dL vs. -1.84 mg/dL; p = 0.001), apo CIII (32.59 mg/dL vs. -7.83 mg/dL; p = 0.007), apo E (8.17 mg/dL vs. 0.22 mg/dL; p = 0.001), and ANGPLT3 (6.08 ng/mL vs. -0.16 ng/mL; p = 0.005) early after treatment at M3. Conclusions Metreleptin treatment lowers levels of apolipoproteins associated with triglyceride metabolism as well as ANGPLT3 in patients with partial lipodystrophy. Metabolic response to Metreleptin appears to be correlated with early changes in these factors and an increase in IGF-1 levels.

334 OBETICHOLIC ACID IMPROVES EXPERIMENTAL NON-INVASIVE MARKERS OF NON-ALCOHOLIC STEATOHEPATITIS AND ADVANCED FIBROSIS; RESULTS OF A SECONDARY ANALYSIS FROM THE MONTH-18 INTERIM ANALYSIS OF THE REGENERATE STUDY

In the REGENERATE 18-month interim analysis, obeticholic acid (OCA) improved surrogate endpoints of liver fibrosis in patients with non-alcoholic steatohepatitis (NASH). New biomarker indices are being developed, including FibroMeter (FM), which is designed to predict fibrosis stage ≥2 using age, gender, alpha-2-macroglobulin, international normalized ratio, platelets, urea, and gamma-glutamyltransferase. FM Vibration-Controlled Transient Elastography (VCTE) uses the same biomarkers (excluding urea) with liver stiffness (LS). The FibroScan AST (FAST™) score uses LS by VCTE, Controlled Attenuation Parameter score, and aspartate aminotransferase to identify patients with NASH and NAFLD Activity Score (NAS) ≥4 and fibrosis stage ≥2. NASH patients with fibrosis stages 2 and 3 were randomized (1:1:1) to placebo (N=311), OCA 10 mg (N=312), or OCA 25 mg (N=308) once daily. Changes in FM (N=604), FM VCTE (N=604), and FAST (N=391) were analyzed using a mixed-effect repeated measures model (MRMM), with factors of treatment, baseline, visit, visit-by-treatment interaction, and stratification. Least square means and p-values were based on MMRM. At baseline, no significant differences were observed in scores across treatment groups (figure 1). Patients with stage 3 fibrosis at baseline had higher scores than those with stage 2 fibrosis. OCA-treated patients experienced improvements in FM, FM VCTE, and FAST at Month 6 through Month 18. No improvements were observed with placebo (figure 1). OCA treatment resulted in early and sustained improvements in non-invasive assessments of fibrosis in NASH. Improvements in FM and FM VCTE are consistent with OCA’s anti-fibrotic effect, while improvements in FAST are consistent with amelioration of NASH inflammation and fibrosis.

Development and Validation of a Scoring System, Based on Genetic and Clinical Factors, to Determine Risk of Steatohepatitis in Asian Patients with Nonalcoholic Fatty Liver Disease

There are no biomarkers of nonalcoholic steatohepatitis (NASH) that are ready for routine clinical use. We investigated whether an analysis of PNPLA3 and TM6SF2 genotypes (rs738409 and rs58542926) can be used to identify patients with nonalcoholic fatty liver disease (NAFLD), with and without diabetes, who also have NASH. We collected data from the Boramae registry in Korea on 453 patients with biopsy-proven NAFLD with sufficient clinical data for calculating scores. Patients enrolled from February 2014 through March 2016 were assigned to cohort 1 (n= 302; discovery cohort) and patients enrolled thereafter were assigned to cohort 2 (n= 151; validation cohort). DNA samples were obtained from all participants and analyzed for the PNPLA3 rs738409 C>G, TM6SF2 rs58542926 C>T, SREBF2 rs133291 C>T, MBOAT7-TMC4 rs641738 C>T, and HSD17B13 rs72613567 adenine insertion (A-INS) polymorphisms. We used multivariable logistic regression analyses with stepwise backward selection to build a model to determine patients' risk for NASH (NASH PT) using the genotype and clinical data from cohort 1 and tested its accuracy in cohort 2. We used the receiver operating characteristic (ROC) curve to compare the diagnostic performances of the NASH PT and the NASH scoring systems. We developed a NASH PT scoring system based on PNPLA3 and TM6SF2 genotypes, diabetes status, insulin resistance, and levels of aspartate aminotransferase and high-sensitivity C-reactive protein. NASH PT scores identified patients with NASH with an area under the ROC (AUROC) of 0.859 (95%CI, 0.817-0.901) in cohort 1. In cohort 2, NASH PT scores identified patients with NASH with an AUROC of 0.787 (95% CI, 0.715-0.860), which was significantly higher than the AUROC of the NASH score (AUROC, 0.729; 95% CI, 0.647-0.812; P= .007). The AUROC of the NASH PT score for detecting NASH in patients with NAFLD with diabetes was 0.835 (95% CI, 0.776-0.895) and in patients without diabetes was 0.809 (95% CI, 0.757-0.861). The negative predictive value of the NASH PT score <-0.785 for NASH in patients with NAFLD with diabetes reached 0.905. We developed a scoring system, based on polymorphisms in PNPLA3 and TM6SF2 and clinical factors that identifies patients with NAFLD, with or without diabetes, who have NASH, with an AUROC value of 0.787. This system might help clinicians better identify NAFLD patients at risk for NASH.

Alterations in the Liver Fat Fraction Features Examined by Magnetic Resonance Imaging Following Bariatric Surgery: a Self-Controlled Observational Study.

Obesity is a worldwide epidemic leading to non-alcoholic fatty liver disease. Alterations in the liver fat fraction (LFF) assessed by MRI following bariatric surgery is a promising feature; however, few studies have been fully elucidated. To determine the alterations in the LFF features following surgery using MRI, to determine the correlation with the clinical non-alcoholic steatohepatitis score (C-NASH score), and to identify the predictive factors for postoperative score changes. Patients (n = 69) underwent MRI to measure the LFF at baseline and 3months postoperatively. Paired sample t tests were applied to investigate the alterations in the major parameters. Univariate analyses were performed to evaluate the factors predicting C-NASH score changes after surgery. Compared with the baseline levels, the LFF significantly decreased 3months postoperatively (P < 0.001). Significant positive correlations were detected between the C-NASH score and LFF levels (P < 0.001). Among the ROC curves for C-NASH score change, the AUC for the ROC curve of LFF was 0.812 (95% CI 0.707, 0.916) and the cut-off value was 6.16%. Weight at baseline was a significant predictive factor for postoperative changes when the C-NASH score was ≥ 3 (P < 0.001). The AUC for the ROC curve of weight was 0.897 (95% CI 0.782, 1.000) and 117kg was the cut-off value. LFF decreased following bariatric surgery, which predicted C-NASH score changes after surgery. For patients with a higher risk of NASH (score ≥ 3) at baseline and lower preoperative body weight, we noted significantly greater effects of surgery on score change value.

PNPLA3 polymorphism influences the association between high-normal TSH level and NASH in euthyroid adults with biopsy-proven NAFLD

AimWe aimed to evaluate the association between serum thyroid stimulating hormone (TSH) levels, within the reference range, and the histological severity of nonalcoholic fatty liver disease (NAFLD), and whether this association was modulated by the patatin-like phospholipase domain-containing 3 (PNPLA3) rs738409 polymorphism. Materials and methodsWe enrolled 327 euthyroid individuals with biopsy-proven NAFLD, who were subdivided into two groups, i.e., a ‘strict-normal’ TSH group (TSH level 0.4 to 2.5mIU/L; n=283) and a ‘high-normal’ TSH group (TSH level 2.5 to 5.3mIU/L with normal thyroid hormones; n=44). Logistic regression analyses were performed to assess the association between TSH status and presence of nonalcoholic steatohepatitis (NASH) after stratifying subjects by PNPLA3 genotypes. ResultsCompared to strict-normal TSH group, patients with high-normal TSH levels were younger and had a greater prevalence of NASH and higher histologic NAFLD activity score. After stratifying by PNPLA3 genotypes, the significant association between high-normal TSH levels and presence of NASH was restricted only to carriers of the PNPLA3 G risk allele and remained significant even after adjustment for potential confounding factors (adjusted-odds ratio: 3.279; 95% CI: 1.298–8.284; P=0.012). ConclusionIn euthyroid individuals with biopsy-proven NAFLD, we found a significant association between high-normal TSH levels and NASH. After stratifying by PNPLA3 rs738409 genotypes, this association was observed only among carriers of the PNPLA3 G risk allele.

Gadoxetic acid enhanced T1ρ and intravoxel incoherent motion MRI in quantitatively assessing the stages of nonalcoholic steatohepatitis

Objective To explore the values of metrics on intravoxel incoherent motion (IVIM) and gadoxetic acid enhanced T1ρ imaging for staging of non-alcoholic fatty liver disease activity scores (NAS) and inflammation in nonalcoholic steatohepatitis (NASH) rabbits model. Methods NASH rabbits model was established by feeding with a varied duration (4, 8, 12 weeks) of high-fat, high-cholesterol diet. IVIM and gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid (Gd-EOB-DTPA) enhanced T1ρ images were performed by a 3.0 T MR scanner. The inter-class correlation coefficients (ICC) and Bland-Altman analysis were applied to evaluate the reproducibility of the IVIM and Gd-EOB-DTPA enhanced T1ρ mapping measurers. Spearman correlation analysis were used to assess the correlation between MR metrics, including ADC, D, D*, f, T1ρ, T1ρ (hepatobiliary phase, HBP), and NAS score and inflammation grades respectively with reference to histopathology. ROC curve analysis was used to evaluate the diagnostic performance of T1ρ and IVIM parameters for NASH, inflammation grade, and hepatic fibrosis. Multiple linear regression equations were used to analyze the independent influence factors of T1ρ (HBP). Results The f value was negatively correlated with the NAS score (r=-0.530, P<0.01). The f value of the fibrosis S1-2 was significantly lower than that of the S0 (P<0.01). There was no statistical difference in D, D*, ADC among NASH score, inflammation, and fibrosis stage. T1ρ and T1ρ (HBP) values were positively correlated with NAS scores and inflammation grades. The area under curve (AUC) for the diagnosis of NASH for T1ρ, T1ρ(HBP), ADC, D, D*, and f values were 0.849, 0.949, 0.728, 0.596, 0.522, and 0.871, respectively. The AUCs of T1ρ (HBP)+f in the diagnosis of NASH, G2-3 inflammation, and F1-2 fibrosis were 0.971, 0.935, and 0.903, respectively. Fibrosis (R2=0.624, P=0.002) and inflammation (R2=0.746, P=0.002) were major independent factors of T1ρ (HBP). Conclusion Gd-EOB-DTPA enhanced T1ρ imaging can reflect the severity of NASH and degree of inflammation. IVIM measurements are not accurate enough to stage liver inflammatory activity of NASH. T1ρ (HBP)+f might be a superior noninvasive imaging biomarker than either non-enhanced T1ρ or IVIM for NASH activity and inflammation assessments. Key words: Magnetic resonance imaging; Nonalcoholic steatohepatitis; Intravoxel incoherent motion; Gadoxetate acid disodium

Liver ASK1 protects from non‐alcoholic fatty liver disease and fibrosis

Non‐alcoholic fatty liver disease (NAFLD) is strongly associated with obesity and may progress to non‐alcoholic steatohepatitis (NASH) and liver fibrosis. The deficit of pharmacological therapies for the latter mainly results from an incomplete understanding of involved pathological mechanisms. Herein, we identify apoptosis signal‐regulating kinase 1 (ASK1) as a suppressor of NASH and fibrosis formation. High‐fat diet‐fed and aged chow‐fed liver‐specific ASK1‐knockout mice develop a higher degree of hepatic steatosis, inflammation, and fibrosis compared to controls. In addition, pharmacological inhibition of ASK1 increased hepatic lipid accumulation in wild‐type mice. In line, liver‐specific ASK1 overexpression protected mice from the development of high‐fat diet‐induced hepatic steatosis and carbon tetrachloride‐induced fibrosis. Mechanistically, ASK1 depletion blunts autophagy, thereby enhancing lipid droplet accumulation and liver fibrosis. In human livers of lean and obese subjects, ASK1 expression correlated negatively with liver fat content and NASH scores, but positively with markers for autophagy. Taken together, ASK1 may be a novel therapeutic target to tackle NAFLD and liver fibrosis.