Fatty Liver Disease Activity Score Research Articles

The selective peroxisome proliferator-activated receptor-delta agonist seladelpar reverses nonalcoholic steatohepatitis pathology by abrogating lipotoxicity in diabetic obese mice.

Lipotoxicity associated with insulin resistance is central to nonalcoholic steatohepatitis (NASH) pathogenesis. To date, only weight loss fully reverses NASH pathology, but mixed peroxisome proliferator–activated receptor‐alpha/delta (PPAR‐α/δ) agonists show some efficacy. Seladelpar (MBX‐8025), a selective PPAR‐δ agonist, improves atherogenic dyslipidemia. We therefore used this agent to test whether selective PPAR‐δ activation can reverse hepatic lipotoxicity and NASH in an obese, dyslipidemic, and diabetic mouse model. From weaning, female Alms1 mutant (foz/foz) mice and wild‐type littermates were fed an atherogenic diet for 16 weeks; groups (n = 8‐12) were then randomized to receive MBX‐8025 (10 mg/kg) or vehicle (1% methylcellulose) by gavage for 8 weeks. Despite minimally altering body weight, MBX‐8025 normalized hyperglycemia, hyperinsulinemia, and glucose disposal in foz/foz mice. Serum alanine aminotransferase ranged 300‐600 U/L in vehicle‐treated foz/foz mice; MBX‐8025 reduced alanine aminotransferase by 50%. In addition, MBX‐8025 normalized serum lipids and hepatic levels of free cholesterol and other lipotoxic lipids that were increased in vehicle‐treated foz/foz versus wild‐type mice. This abolished hepatocyte ballooning and apoptosis, substantially reduced steatosis and liver inflammation, and improved liver fibrosis. In vehicle‐treated foz/foz mice, the mean nonalcoholic fatty liver disease activity score was 6.9, indicating NASH; MBX‐8025 reversed NASH in all foz/foz mice (nonalcoholic fatty liver disease activity score 3.13). Conclusion: Seladelpar improves insulin sensitivity and reverses dyslipidemia and hepatic storage of lipotoxic lipids to improve NASH pathology in atherogenic diet–fed obese diabetic mice. Selective PPAR‐δ agonists act independently of weight reduction, but counter lipotoxicity related to insulin resistance, thereby providing a novel therapy for NASH. (Hepatology Communications 2017;1:663–674)

A liver-specific long noncoding RNA with a role in cell viability is elevated in human nonalcoholic steatohepatitis.

We identified an lncRNA that can play an important regulatory role in liver function and provide new insights into the regulation of hepatocyte viability in NASH. (Hepatology 2017;66:794-808).

Effect of age on the development of chemotherapy-associated liver injury in colorectal cancer liver metastasis.

With the prolongation of the mean lifespan, the number of elderly individuals undergoing liver resection for colorectal cancer liver metastasis (CRLM) following chemotherapy has increased. However, the effect of age on the development of chemotherapy-associated liver injury (CALI) in CRLM remains unclear. The aim of the present study was to elucidate the effect of age on the development of CALI in CRLM. A total of 64 patients undergoing liver resection for CRLM following oxaliplatin-based chemotherapy (OBC) were investigated. The patients were divided into three groups (group A: <65 years, group B: 65-74 years and group C: >75 years) according to age at surgery, and the development rate of CALI was compared between the groups. The patients underwent pathological assessments to determine the degree of histopathological injury of the non-cancerous liver parenchyma. Group A included 37 cases, group B 17 cases and group C 10 cases. There were no significant differences among the groups regarding the number of OBC cycles and duration of OBC cessation. Sinusoidal injury was observed in 27.0, 29.4 and 30.0% of the cases in groups A, B and C, respectively; the differences were not statistically significant (P=0.479). Steatohepatitis was observed in 35.1, 35.3 and 40.0% of the cases in groups A, B and C, respectively; the differences were not statistically significant (P=0.958). There was no significant correlation between age and sinusoidal pathological score (r=-0.102, P=0.423) or non-alcoholic fatty liver disease activity score (r≤0.001, P=0.997). Therefore, the development of CALI following OBC treatment in CRLM was not found to differ by age.

Sivelestat alleviates nonalcoholic steatohepatitis in mice through inhibiting activation of Kupffer cells

Objective: To investigate the role of neutrophil elastase inhibitor, sivelestat, in preventing and treating nonalcoholic steatohepatitis (NASH) and its underling mechanisms. Methods: A total of forty 4-week-old male C57BL/6J ApoE-/-mice were equally divided into the following four groups: standard chow (SC)+isotonic saline; SC+sivelestat; high-fat, high-cholesterol (HFHC) diet+isotonic saline; and HFHC+sivelestat. These mice were treated with above methods for 12 weeks. Blood and liver tissue samples were collected to measure biochemical parameters, hepatic steatosis and non-alcoholic fatty liver disease (NAFLD) activity score (inflammation) were evaluated by oil red O staining and HE staining, respectively. The mRNA and protein expression levels of hepatic inflammatory cytokines, CD68, and F4/80 were determined by quantitative RT-PCR and immunohistochemistry, respectively. Comparison of means between the four groups was made by one-way analysis of variance, and comparison between any two groups was made by the LSD or SNK method (for data with homogeneity of variance) or the Tamhane or Dunnett method (for data with heterogeneity of variance). Results: Mice fed with an HFHC diet for 12 weeks developed typical pathological features of NASH compared with those fed with SC. Compared with mice fed with HFHC diet without sivelestat, those treated with HFHC and sivelestat exhibited the following features: (1) significantly reduced fast blood glucose, blood cholesterol, and hepatic biochemical parameters, as well as increased insulin sensitivity; (2) significantly reduced NAFLD activity score (5.71±1.11 vs 3.16±1.16, P < 0.05); (3) reduced monocyte chemoattractant protein-1 and tumor necrosis factor -α; (4) significantly reduced mRNA levels of CD68 and F4/80; and (5) reduced expression of CD68 in the liver. Conclusion: Sivelestat alleviates the hepatic steatosis and inflammation of NASH in mice by inhibiting the activation of Kupffer cells.

Population Pharmacokinetics of Morphine in Patients With Nonalcoholic Steatohepatitis (NASH) and Healthy Adults.

Altered expression and function of transporters in nonalcoholic steatohepatitis (NASH) patients may affect the pharmacokinetics (PK), efficacy, and safety of substrate drugs. A population pharmacokinetic (PopPK) analysis was performed to assess differences in morphine and morphine‐3‐glucuronide (M3G) disposition in NASH and healthy subjects. A total of 315 serum and 42 urine samples from 21 subjects (14 healthy; 7 NASH) were analyzed using NONMEM. Morphine and M3G PK were described by three‐ and one‐compartment models, respectively. After accounting for the effect of total body weight on all clearance and volume of distribution parameters using an allometric scaling approach, NASH severity score (NASF; combination of fibrosis and nonalcoholic fatty liver disease activity scores) was the most significant predictor of differences in M3G exposure. The model predicted a linear decrease in the clearance of M3G with increasing NASF scores on a natural logarithmic scale. These results may provide some insight into the potential effect of NASH on the disposition of hepatic transporter substrates.

Preventive effects of the sodium glucose cotransporter 2 inhibitor tofogliflozin on diethylnitrosamine-induced liver tumorigenesis in obese and diabetic mice.

Sodium glucose cotransporter 2 inhibitors are expected to ameliorate the abnormalities associated with metabolic syndrome including non-alcoholic fatty liver disease. In this study, we investigated the effects of the sodium glucose cotransporter 2 inhibitor tofogliflozin on the development of non-alcoholic fatty liver disease-related liver tumorigenesis in C57BL/KsJ-+Leprdb/+Leprdb obese and diabetic mice. The direct effects of tofogliflozin on human liver cancer cell proliferation were also evaluated. Mice were administered diethylnitrosamine-containing water for 2 weeks and were treated with tofogliflozin throughout the experiment. In mice treated with tofogliflozin, the development of hepatic preneoplastic lesions was markedly suppressed, and hepatic steatosis and inflammation significantly reduced, as evaluated using the non-alcoholic fatty liver disease activity score, in comparison with the control mice. Serum levels of glucose and free fatty acid and mRNA expression levels of pro-inflammatory markers in the liver were reduced by tofogliflozin treatment. Conversely, the proliferation of sodium glucose cotransporter 2 protein-expressing liver cancer cells was not inhibited by this agent. These findings suggest that tofogliflozin suppressed the early phase of obesity- and non-alcoholic fatty liver disease-related hepatocarcinogenesis by attenuating chronic inflammation and hepatic steatosis. Therefore, sodium glucose cotransporter 2 inhibitors may have a chemopreventive effect on obesity-related hepatocellular carcinoma.

The association of nonalcoholic fatty liver disease with genetic polymorphisms: a multicenter study.

Growing evidence suggests that multiple factors, such as insulin resistance, nutritional factors, gut microbiota, and hormones released from the adipose tissue, act together on genetically predisposed individuals. We aimed to investigate whether various single-nucleotide polymorphisms (SNPs) play a role in the development of nonalcoholic fatty liver disease (NAFLD) and severity of liver damage in the Anatolian population. Two hundred and sixteen patients with biopsy-proven NAFLD and 150 control participants, aged 18-70 years, were consecutively enrolled in this multicenter study. Blood samples were genotyped for the PNPLA3 (rs738409), IL28B (rs12979860, rs12980275, rs8099917), PPAR-α 227 ALA, PPAR-γ pro 12 ALA, SOD2 C47T, and LOX-1 IVS4-14 polymorphisms using the custom-made LightSNiP assays on a LightCycler 480 II instrument. Genotypic distributions of PNPLA3 rs738409 SNPs were different between NAFLD and control participants, but not for other SNPs. The PNPLA3 rs738409 GG polymorphism was associated with a 27-fold increased risk of development of NAFLD (odds ratio=27.8, 95% confidence interval: 3.5-218.4; P=0.002). Patients with the PNPLA3 GG genotype had higher nonalcoholic fatty liver disease activity score levels compared with patients with the PNPLA3 CC genotype (P<0.005). NAFLD patients without fibrosis had a higher frequency of IL28B rs12979860 TT and rs12980275 GG genotypes compared with NAFLD patients with fibrosis (P<0.005). The present study proposes that polymorphisms in the PNPLA3 gene have highly predictive value in the development of NAFLD and are independently associated with the severity of liver histology in patients with NAFLD. The results of this study suggest that IL28B rs12979860 TT or rs12980275 GG may play an important protective role against the development of advanced fibrosis and even cirrhosis.

Multimaterial Decomposition Algorithm for the Quantification of Liver Fat Content by Using Fast-Kilovolt-Peak Switching Dual-Energy CT: Clinical Evaluation.

Purpose To assess the clinical accuracy and reproducibility of liver fat quantification with the multimaterial decomposition (MMD) algorithm, comparing the performance of MMD with that of magnetic resonance (MR) spectroscopy by using liver biopsy as the reference standard. Materials and Methods This prospective study was approved by the institutional ethics committee, and patients provided written informed consent. Thirty-three patients suspected of having hepatic steatosis underwent non-contrast material-enhanced and triple-phase dynamic contrast-enhanced dual-energy computed tomography (CT) (80 and 140 kVp) and single-voxel proton MR spectroscopy within 30 days before liver biopsy. Percentage fat volume fraction (FVF) images were generated by using the MMD algorithm on dual-energy CT data to measure hepatic fat content. FVFs determined by using dual-energy CT and percentage fat fractions (FFs) determined by using MR spectroscopy were compared with histologic steatosis grade (0-3, as defined by the nonalcoholic fatty liver disease activity score system) by using Jonckheere-Terpstra trend tests and were compared with each other by using Bland-Altman analysis. Real non-contrast-enhanced FVFs were compared with triple-phase contrast-enhanced FVFs to determine the reproducibility of MMD by using Bland-Altman analyses. Results Both dual-energy CT FVF and MR spectroscopy FF increased with increasing histologic steatosis grade (trend test, P < .001 for each). The Bland-Altman plot of dual-energy CT FVF and MR spectroscopy FF revealed a proportional bias, as indicated by the significant positive slope of the line regressing the difference on the average (P < .001). The 95% limits of agreement for the differences between real non-contrast-enhanced and contrast-enhanced FVFs were not greater than about 2%. Conclusion The MMD algorithm quantifying hepatic fat in dual-energy CT images is accurate and reproducible across imaging phases. © RSNA, 2017 Online supplemental material is available for this article.

Nonalcoholic steatohepatitis recurrence and rate of fibrosis progression following liver transplantation.

Nonalcoholic steatohepatitis (NASH) is known to recur following liver transplantation (LT). Metabolic risk factors increase with immunosuppression. However, the rate of fibrosis progression following LT for NASH while on immunosuppression is less clear. The incidences of steatosis, NASH, and fibrosis following LT for NASH were quantified and compared with those transplanted for alcoholic liver disease (ALD). Records of all NASH patients and 1 : 2 match with ALD transplant recipients between 2001 and 2006 were reviewed retrospectively. Patients without liver biopsies beyond 2 months following LT were excluded. NASH patients (n=77) were older (P=0.0006) and less likely male (P<0.001) than ALD patients (n=108). The incidence of steatosis, NASH, and fibrosis stage increased at 1, 3, and 5 years in both groups. Although steatosis and nonalcoholic fatty liver disease activity scores were higher, fibrosis was lower in NASH compared with ALD (0.43 vs. 1.0 stage/year; P=0.0045). The incremental increase in the rate of fibrosis was faster in the first year compared with 4-5 years (0.8 vs. 0.04 stage/year) following LT. The rate of fibrosis progression during 4-5 years was decreased in NASH compared with ALD recipients (0.04 vs. 0.33 stage/year; P=0.015). NASH etiology was associated with reduced rate of fibrosis progression (odds ratio=0.67) on multivariate analysis. Despite having more steatosis and inflammation, progression of fibrosis was slower in NASH compared with ALD recipients. Fibrosis progression slows with time following LT on immunosuppression and approximates the pretransplant progression rate by year 5.

Serum microRNA-34a is potential biomarker for inflammation in nonalcoholic fatty liver disease

Abstract Background MicroRNA-34a (miR-34a) contributes to liver injury through an apoptosis pathway. Objective To determine the correlation between serum miR-34a and liver inflammation as assessed by nonalcoholic fatty liver disease (NAFLD) activity score (NAS). Method We included a cross-selectional study of 50 patients with NAFLD in this observational study and confirmed diagnosis by liver biopsy, with NAS grading. A control group comprised 23 healthy individuals without chronic liver disease. Serum miR-34a was assayed using a real-time quantitative PCR (Applied Biosystems). Result The mean age of NAFLD patients was 46.0 ± 13.7 years, and 52% were female. Metabolic syndrome was found in 76%. Liver histopathology showed that 54% of patients had NAS ≥4 and significant fibrosis (≥2) was found in 22%. Serum levels of miR-34a were significantly correlated with NAS (r = 0.39, P = 0.005), and the degree of steatosis (r = 0.28, P = 0.049), ballooning (r = 0.30, P = 0.034), and fibrosis (r = 0.39, P = 0.005). Serum miR-34a in patients with NAS ≥4 was significantly higher than in those with NAS &lt;4 (P = 0.011) and controls (P &lt; 0.001). There was no significant correlation between serum miR-34a and other variables. The area under receiver operating characteristic curve for serum miR-34a comparing patients with NAS ≥4 and with NAS &lt;4 was 0.67 (95% CI 0.52, 0.82). Conclusion Serum level of miR-34a has a significant fair to good correlation with NAS and may serve as a biomarker of liver inflammation and fibrosis in patients with NAFLD.

TRIF Differentially Regulates Hepatic Steatosis and Inflammation/Fibrosis in Mice.

Background & AimsToll-like receptor 4 (TLR4) signaling is activated through 2 adaptor proteins: MyD88 and TIR-domain containing adaptor-inducing interferon-β (TRIF). TLR4 and MyD88 are crucial in nonalcoholic steatohepatitis (NASH) and fibrosis. However, the role of TRIF in TLR4-mediated NASH and fibrosis has been elusive. This study investigated the differential roles of TRIF in hepatic steatosis and inflammation/fibrosis.MethodsA choline-deficient amino acid defined (CDAA) diet was used for the mouse NASH model. On this diet, the mice develop hepatic steatosis, inflammation, and fibrosis. TLR4 wild-type and TLR4-/- bone marrow chimeric mice and TRIF-/- mice were fed CDAA or a control diet for 22 weeks. Hepatic steatosis, inflammation, and fibrosis were examined.ResultsIn the CDAA diet–induced NASH, the mice with wild-type bone marrow had higher alanine aminotransferase and hepatic tumor necrosis factor levels than the mice with TLR4-/- bone marrow. The nonalcoholic fatty liver disease activity score showed that both wild-type and TLR4-/- bone marrow chimeras had reduced hepatic steatosis, and that both types of chimeras had similar levels of inflammation and hepatocyte ballooning to whole-body wild-type mice. Notably, wild-type recipients showed more liver fibrosis than TLR4-/- recipients. Although TRIF-/- mice showed reduced hepatic steatosis, these mice showed more liver injury, inflammation, and fibrosis than wild-type mice. TRIF-/- stellate cells and hepatocytes produced more C-X-C motif chemokine ligand 1 (CXCL1) and C-C motif chemokine ligand than wild-type cells in response to lipopolysaccharide. Consistently, TRIF-/- mice showed increased CXCL1 and CCL3 expression along with neutrophil and macrophage infiltration, which promotes liver inflammation and injury.ConclusionsIn TLR4-mediated NASH, different liver cells have distinct roles in hepatic steatosis, inflammation, and fibrosis. TRIF promotes hepatic steatosis but it inhibits injury, inflammation, and fibrosis.

The significance of 8-hydroxy-deoxyguanosine acid in the diagnosis of nonalcoholic steatohepatitis

Objective: To evaluate the significance of serum 8-hydroxy-deoxyguanosine acid(8-OHdG) in the diagnosis of nonalcoholic steatohepatitis (NASH). Methods: Patients or healthy subjects were enrolled at the Second Hospital of Tianjin Medical University and the Second People's Hospital of Tianjin from May 2013 to December 2015. A total of 41 patients with nonalcoholic fatty liver disease were enrolled in the study, including 20 nonalcoholic simple fatty liver (NAFL) patients and 21 NASH patients whose diagnosis were proven by liver biopsy. The other 32 healthy subjects were studied as controls. Serum 8-OHdG, ALT, AST and GGT were tested. Nonalcoholic fatty liver disease activity score (NAS) and expression of 8-OHdG in liver was investigated between NAFL patients and NASH patients. The correlations between serum 8-OHdG and serum ALT, AST, GGT, and 8-OHdG in liver tissue in NASH group were investigated. In addition, the receiver operating characteristic (ROC) curve analyses for ALT and 8-OHdG levels were performed in NAFL patients and NASH patients, and the cut-off value was determined. Results: Serum 8-OHdG values in healthy controls, NAFL and NASH patients were (0.19±0.16) μg/L, (0.22±0.16) μg/L, (0.42±0.21) μg/L respectively. The serum 8-OHdG and serum ALT, GGT and 8-OHdG in liver tissue were all positively correlated in NASH group with respective correlation coefficient r values as 0.454 7, 0.382 9, and 0.497 6. AUC of 8-OHdG was 0.901 with cut-off value 0.39 μg/L. Its sensitivity was 88.3% and specificity was 81.5%, which were higher than those of ALT. Conclusion: The value of serum 8-OHdG would be used as a marker for the diagnosis of NASH.

PS-024 - YH25724, a novel long-acting GLP-1/FGF21 dual agonist lowers both non-alcoholic fatty liver disease activity score and fibrosis stage in a diet-induced obese mouse model of biopsy-confirmed non-alcoholic steatohepatitis

PS-024 - YH25724, a novel long-acting GLP-1/FGF21 dual agonist lowers both non-alcoholic fatty liver disease activity score and fibrosis stage in a diet-induced obese mouse model of biopsy-confirmed non-alcoholic steatohepatitis

Anti-steatotic and anti-fibrotic effects of the KCa3.1 channel inhibitor, Senicapoc, in non-alcoholic liver disease.

AIMTo evaluate a calcium activated potassium channel (KCa3.1) inhibitor attenuates liver disease in models of non-alcoholic fatty liver disease (NAFLD).METHODSWe have performed a series of in vitro and in vivo studies using the KCa3.1 channel inhibitor, Senicapoc. Efficacy studies of Senicapoc were conducted in toxin-, thioacetamide (TAA) and high fat diet (HFD)-induced models of liver fibrosis in rats. Efficacy and pharmacodynamic effects of Senicapoc was determined through biomarkers of apoptosis, inflammation, steatosis and fibrosis.RESULTSUpregulation of KCa3.1 expression was recorded in TAA-induced and high fat diet-induced liver disease. Treatment with Senicapoc decreased palmitic acid-driven HepG2 cell death. (P < 0.05 vs control) supporting the finding that Senicapoc reduces lipid-driven apoptosis in HepG2 cell cultures. In animals fed a HFD for 6 wk, co-treatment with Senicapoc, (1) reduced non-alcoholic fatty liver disease (NAFLD) activity score (NAS) (0-8 scale), (2) decreased steatosis and (3) decreased hepatic lipid content (Oil Red O, P < 0.05 vs vehicle). Randomization of TAA animals and HFD fed animals to Senicapoc was associated with a decrease in liver fibrosis as evidenced by hydroxyproline and Masson’s trichrome staining (P < 0.05 vs vehicle). These results demonstrated that Senicapoc mitigates both steatosis and fibrosis in liver fibrosis models.CONCLUSIONThese data suggest that Senicapoc interrupts more than one node in progressive fatty liver disease by its anti-steatotic and anti-fibrotic activities, serving as a double-edged therapeutic sword.

FRI-363 - EDP-305, a novel and highly potent farnesoid X receptor agonist, improves liver steatosis, ballooning and non-alcoholic fatty liver disease (NAFLD) activity score (NAS) in a diet-induced murine model of non-alcoholic steatohepatitis

FRI-363 - EDP-305, a novel and highly potent farnesoid X receptor agonist, improves liver steatosis, ballooning and non-alcoholic fatty liver disease (NAFLD) activity score (NAS) in a diet-induced murine model of non-alcoholic steatohepatitis

Combination curcumin and vitamin E treatment attenuates diet-induced steatosis in Hfe-/- mice.

AIMTo investigate the synergistic hepato-protective properties of curcumin and vitamin E in an Hfe-/- high calorie diet model of steatohepatitis.METHODSHfe-/- C57BL/6J mice were fed either a high calorie diet or a high calorie diet with 1 mg/g curcumin; 1.5 mg/g vitamin E; or combination of 1 mg/g curcumin + 1.5 mg/g vitamin E for 20 wk. Serum and liver tissue were collected at the completion of the experiment. Liver histology was graded by a pathologist for steatosis, inflammation and fibrosis. RNA and protein was extracted from liver tissue to examine gene and protein expression associated with fatty acid oxidation, mitochondrial biogenesis and oxidative stress pathways.RESULTSHfe-/- mice fed the high calorie diet developed steatohepatitis and pericentral fibrosis. Combination treatment with curcumin and vitamin E resulted in a greater reduction of percent steatosis than either vitamin E or curcumin therapy alone. Serum alanine aminotransferase and non-alcoholic fatty liver disease (NAFLD) activity score were decreased following combination therapy with curcumin and vitamin E compared with high calorie diet alone. No changes were observed in inflammatory or fibrosis markers following treatment. Epididymal fat pad weights were significantly reduced following combination therapy, however total body weight and liver weight were unchanged. Combination therapy increased the mRNA expression of AdipoR2, Ppar-α, Cpt1a, Nrf-1 and Tfb2m suggesting enhanced fatty acid oxidation and mitochondrial biogenesis. In addition, combination treatment resulted in increased catalase activity in Hfe-/- mice.CONCLUSIONCombination curcumin and vitamin E treatment decreases liver injury in this steatohepatitis model, indicating that combination therapy may be of value in NAFLD.

Wheat-bran autolytic peptides containing a branched-chain amino acid attenuate non-alcoholic steatohepatitis via the suppression of oxidative stress and the upregulation of AMPK/ACC in high-fat diet-fed mice.

Whole-wheat intake is known to reduce the risk of metabolic syndrome. However, the active component remains unclear. Recently, we identified bioactive peptides [leucine-arginine-proline (LRP) and leucine-glutamine‑proline(LQP)] from wheat bran autolytic hydrolysate. The present study aimed to investigate the effects of LRP and LQP on non-alcoholic steatohepatitis (NASH) in a mouse model. We also evaluated the effects of these peptides on oxidative stress and on the AMP-activated protein kinase (AMPK) signaling pathway, two major pathogenic factors of NASH. Seven‑week-old male C57BL/6 mice were fed a high-fat diet for 10weeks and administered water supplemented with 0.05%LRP, 0.20%LRP, 0.05%LQP, or 0.20%LQP (each n=5) or distilled water (control; n=5) adlibitum. Oxidative stress was evaluated by measuring the serum levels of diacron reactive oxygen metabolite (d-ROM) and biological antioxidant potential (BAP). Hepatic expression of phosphorylated AMPK and phosphorylated acetyl-CoA carboxylase (ACC) were evaluated by immunoblotting. The result showed that non‑alcoholic fatty liver disease activity score was significantly decreased in all types of treatment. Serum d-ROM levels were significantly decreased in the 0.20% LRP group, but not in the 0.05% LRP, 0.05% LQP, and 0.20% LQP groups. Serum BAP levels were significantly increased in the 0.05% LRP and 0.20% LRP groups, but not in the 0.05% LQP and 0.20% LQP groups. Immunoblotting analysis revealed that the expression of phospho-AMPK was increased whereas that of phospho-ACC was decreased in the 0.20% LQP group. In conclusion, we demonstrated that both LRP and LQP alleviated the severity of NASH in a high-fat diet-induced NASH mouse model. In addition, we showed that LRP and LQP modulated oxidative stress and upregulated AMPK/ACC, respectively. Thus, LRP and LQP may constitute clinically applicable therapeutic agents for NASH.

Hepatic fat quantification using the two-point Dixon method and fat color maps based on non-alcoholic fatty liver disease activity score.

The two-point Dixon method for magnetic resonance imaging (MRI) is commonly used to non-invasively measure fat deposition in the liver. The aim of the present study was to assess the usefulness of MRI-fat fraction (MRI-FF) using the two-point Dixon method based on the non-alcoholic fatty liver disease activity score. This retrospective study included 106 patients who underwent liver MRI and MR spectroscopy, and 201 patients who underwent liver MRI and histological assessment. The relationship between MRI-FF and MR spectroscopy-fat fraction was used to estimate the corrected MRI-FF for hepatic multi-peaks of fat. Then, a color FF map was generated with the corrected MRI-FF based on the non-alcoholic fatty liver disease activity score. We defined FF variability as the standard deviation of FF in regions of interest. Uniformity of hepatic fat was visually graded on a three-point scale using both gray-scale and color FF maps. Confounding effects of histology (iron, inflammation and fibrosis) on corrected MRI-FF were assessed by multiple linear regression. The linear correlations between MRI-FF and MR spectroscopy-fat fraction, and between corrected MRI-FF and histological steatosis were strong (R2 = 0.90 and R2 = 0.88, respectively). Liver fat variability significantly increased with visual fat uniformity grade using both of the maps (ρ = 0.67-0.69, both P <0.001). Hepatic iron, inflammation and fibrosis had no significant confounding effects on the corrected MRI-FF (all P >0.05). The two-point Dixon method and the gray-scale or color FF maps based on the non-alcoholic fatty liver disease activity score were useful for fat quantification in the liver of patients without severe iron deposition.

Empagliflozin (an SGLT2 inhibitor), alone or in combination with linagliptin (a DPP-4 inhibitor), prevents steatohepatitis in a novel mouse model of non-alcoholic steatohepatitis and diabetes.

BackgroundSodium-glucose co-transporter-2 (SGLT2) inhibitors are new oral antidiabetic drugs that reduce hyperglycemia by promoting urinary glucose excretion. Glycosuria produced by SGLT2 inhibitors is associated with weight loss, mainly due to reduced fat volume. We investigated the effects of empagliflozin (selective SGLT2 inhibitor) and linagliptin (DPP-4 inhibitor) on steatohepatitis and fibrosis in a mouse model of non-alcoholic steatohepatitis (NASH) with diabetes.MethodsA novel NASH model was generated by administration of streptozotocin to C57BL/6J mice at 2 days old, with a high-fat diet from 4 weeks. NASH mice aged 6 weeks were divided into four groups of 6 animals: vehicle, linagliptin (10 mg/kg), empagliflozin (10 mg/kg), and linagliptin + empagliflozin. The histological non-alcoholic fatty liver disease activity score was significantly lower in the empagliflozin and linagliptin + empagliflozin groups than in the vehicle or linagliptin groups. Hepatic expression of inflammatory genes (tumor necrosis factor-α, interleukin-6, and monocyte chemoattractant protein-1) was decreased in the empagliflozin and linagliptin + empagliflozin groups compared with the vehicle group. The collagen deposition with Sirius red staining was significantly reduced in the linagliptin + empagliflozin group compared with the linagliptin or the empagliflozin group. Immunohistochemistry showed that expression of α-smooth muscle actin, a marker of myofibroblasts (fibrosis), was reduced in the linagliptin + empagliflozin group compared with the vehicle group, as was expression of type 1 and 3 collagen mRNA. Linagliptin + empagliflozin decreased expression of mRNAs for genes related to fatty acid synthesis, but did not increase mRNAs for β-oxidation-related genes.ConclusionsWhile empagliflozin alone attenuates development of NASH showing anti-steatotic and anti-inflammatory effects, combined administration of empagliflozin and linagliptin can synergistically ameliorates NASH with stronger anti-fibrotic effects.

Association of noninvasive quantitative decline in liver fat content on MRI with histologic response in nonalcoholic steatohepatitis.

Magnetic resonance imaging-estimated proton-density-fat-fraction (MRI-PDFF) has been shown to be a noninvasive, accurate and reproducible imaging-based biomarker for assessing steatosis and treatment response in nonalcoholic steatohepatitis (NASH) clinical trials. However, there are no data on the magnitude of MRI-PDFF reduction corresponding to histologic response in the setting of a NASH clinical trial. The aim of this study was to quantitatively compare the magnitude of MRI-PDFF reduction between histologic responders versus histologic nonresponders in NASH patients. This study is a secondary analysis of the MOZART trial, which included 50 patients with biopsy-proven NASH randomized to ezetimibe 10 mg/day orally or placebo for 24 weeks. The primary aim was to perform a head-to-head comparative analysis of histologic responders [defined as a ⩾2-point reduction in the nonalcoholic fatty liver disease (NAFLD) Activity Score (NAS) without worsening fibrosis] versus nonresponders, and the corresponding quantitative change in liver fat content measured via MRI-PDFF. Of the 35 patients who underwent paired liver biopsy and MRI-PDFF assessment at the beginning and end of treatment, 10 demonstrated a histologic response. Compared with histologic nonresponders, histologic responders had a statistically significant reduction in MRI-PDFF of -4.1% ± 4.9 versus -0.6 ± 4.1 (p < 0.04) with a mean relative percent change of -29.3% ± 33.0 versus +2.0% ± 24.0 (p < 0.004), respectively. Utilizing paired MRI-PDFF and liver histology data, we demonstrate that a relative reduction of 29% in liver fat on MRI-PDFF is associated with a histologic response in NASH. After external validation by independent research groups, these results can be incorporated into designing future NASH clinical trials, especially those utilizing change in hepatic fat quantified by MRI-PDFF, as a treatment endpoint.