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Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide. It occurs with a prevalence of up to 25%, of which 10-20% cases progress to nonalcoholic steatohepatitis (NASH), cirrhosis, and liver cancer. The histopathology of NASH is characterized by neutrophilic infiltration, and endotoxins from gram-negative rods have been postulated as a contributing factor. Elevations in endotoxin levels in the blood can be classified as intestinal and hepatic factors. In recent years, leaky gut syndrome, which is characterized by impaired intestinal barrier function, has become a significant issue. A leaky gut may prompt intestinal bacteria dysbiosis and increase the amount of endotoxin that enters the liver from the portal vein. These contribute to persistent chronic inflammation and progressive liver damage. In addition, hepatic factors suggest that liver damage can be induced by low-dose endotoxins, which does not occur in healthy individuals. In particular, increased expression of CD14, an endotoxin co-receptor in the liver, may result in leptin-induced endotoxin hyper-responsiveness in obese individuals. Thus, elevated blood endotoxin levels contribute to the progression of NASH. The current therapeutic targets for NASH treat steatosis and liver inflammation and fibrosis. While many clinical trials are underway, no studies have been performed on therapeutic agents that target the intestinal barrier. Recently, a randomized placebo-controlled trial examined the role of the intestinal barrier in patients with NAFLD. To our knowledge, this study was the first of its kind and study suggested that the intestinal barrier may be a novel target in the future treatment of NAFLD., Competing Interests: ANa receives grants and research support from Gilead, Mylan EPD, EA Pharma, Kowa, Taisho, and Biofermin. ANa is also a consultant for Gilead, Boehringer Ingelheim, Bristol Myers Squibb, Kowa, Astellas, EA Pharma, and Mylan EPD. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kessoku, Kobayashi, Imajo, Tanaka, Yamamoto, Takahashi, Kasai, Ozaki, Iwaki, Nogami, Honda, Ogawa, Kato, Higurashi, Hosono, Yoneda, Okamoto, Usuda, Wada, Kobayashi, Saito and Nakajima.)

The liver directly accepts blood from the gut and is, therefore, exposed to intestinal bacteria. Recent studies have demonstrated a relationship between gut bacteria and nonalcoholic fatty liver disease (NAFLD). Approximately 10-20% of NAFLD patients develop nonalcoholic steatohepatitis (NASH), and endotoxins produced by Gram-negative bacilli may be involved in NAFLD pathogenesis. NAFLD hyperendotoxicemia has intestinal and hepatic factors. The intestinal factors include impaired intestinal barrier function (leaky gut syndrome) and dysbiosis due to increased abundance of ethanol-producing bacteria, which can change endogenous alcohol concentrations. The hepatic factors include hyperleptinemia, which is associated with an excessive response to endotoxins, leading to intrahepatic inflammation and fibrosis. Clinically, the relationship between gut bacteria and NAFLD has been targeted in some randomized controlled trials of probiotics and other agents, but the results have been inconsistent. A recent randomized, placebo-controlled study explored the utility of lubiprostone, a treatment for constipation, in restoring intestinal barrier function and improving the outcomes of NAFLD patients, marking a new phase in the development of novel therapies targeting the intestinal barrier. This review summarizes recent data from studies in animal models and randomized clinical trials on the role of the gut-liver axis in NAFLD pathogenesis and progression.

Background: The laxative drug lubiprostone improves intestinal permeability in healthy volunteers. We aimed to assess efficacy and safety of lubiprostone in patients with non-alcoholic fatty liver disease (NAFLD) with constipation via attenuation of intestinal permeability., Methods: This randomised, double-blind, placebo-controlled, phase 2a study in Yokohama City University Hospital, Japan, recruited patients (aged 20-85 years) with NAFLD and constipation, alanine aminotransferase (ALT) at least 40 U/L, liver stiffness (≤6·7 kPa), and hepatic fat fraction at least 5·2% when assessed by MRI-proton density fat fraction. Eligible patients were randomly assigned (11:10:9) by a computer-based system and stratified by age and sex to receive 24 μg lubiprostone, 12 μg lubiprostone, or placebo, orally, once per day for 12 weeks. The primary endpoint was the absolute changes in ALT at 12 weeks. Efficacy analysis was done by intention to treat. Safety was assessed in all treated patients. This trial was registered with University Hospital Medical Information Network Clinical Trials Registry (UMIN000026635)., Findings: Between March 24, 2017, and April 3, 2018, we screened 288 patients, of whom 150 (52%) were randomly assigned to treatment: 55 patients were assigned to receive 24 μg lubiprostone, 50 to receive 12 μg lubiprostone, and 45 to receive placebo. A greater decrease in the absolute ALT levels from baseline to 12 weeks was seen in the 24 μg lubiprostone group (mean -13 U/L [SD 19]) than in the placebo group (1 U/L [24]; mean difference -15 U/L [95% CI -23 to -6], p=0·0007) and in the 12 μg lubiprostone group (-12 U/L [21]) than in the placebo group (mean difference -13 U/L [-22 to -5], p=0·0023). 18 (33%) of 55 patients in the 24 μg group had at least one adverse event, as did three (6%) of 47 patients in the 12 μg group and three (7%) of 43 in the placebo group. The most common adverse event was diarrhoea (17 [31%] of patients in the 24 μg group, three [6%] in the 12 μg group, none in the placebo group). No life-threatening events or treatment-related deaths occurred., Interpretation: Lubiprostone was well tolerated and reduced the levels of liver enzymes in patients with NAFLD and constipation. Further studies are necessary to better define the efficacy and tolerability of lubiprostone in patients with NAFLD without constipation., Funding: Mylan EPD G.K., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background and Aim: The fibrosis stage of liver is associated with the long-term outcomes in patients with non-alcoholic fatty liver disease (NAFLD). However, significant fibrosis, defined as fibrosis stages 2-4, is associated with an elevated risk of progression to severe liver disease; there have been scant reports about diagnosing significant fibrosis. We compare the noninvasive method and aim to identify appropriate liver fibrosis markers for detecting significant fibrosis in NAFLD patients., Methods: We compared the usefulness of liver fibrosis markers (Wisteria floribunda agglutinin-positive Mac-2-binding protein [WFA + -M2BP], type 4 collagen 7S, etc.), clinical scoring systems, and liver stiffness measurement obtained using vibration-controlled transient elastography and magnetic resonance imaging-based magnetic resonance elastography in the same individuals and identified the most appropriate noninvasive method for detecting significant fibrosis in 165 patients with liver biopsy-diagnosed NAFLD., Results: The area under the receiver operating characteristic curve based on the serum cutoff index values of WFA + -M2BP/the serum levels of type IV collagen 7S for the diagnosis of significant fibrosis was 0.832 (95% confidence interval: 0.771-0.894)/0.837 (95% confidence interval: 0.778-0.898). "WFA + -M2BP (cutoff index) ≥ 0.83 or type IV collagen 7S ≥ 5.2 ng/mL" showed a high sensitivity (91.4%) and negative predictive value (87.9%) for the diagnosis of significant fibrosis., Conclusions: We showed that serum WFA + -M2BP or type IV collagen 7S levels serve as useful independent markers for detecting significant fibrosis and that use of both WFA + -M2BP and type IV collagen 7S together increased the sensitivity and negative predictive value for the diagnosis of liver fibrosis. These results need to be validated in larger populations from multiple clinical centers., (© 2018 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

Background & Aims: Noninvasive methods have been evaluated for the assessment of liver fibrosis and steatosis in patients with nonalcoholic fatty liver disease (NAFLD). We compared the ability of transient elastography (TE) with the M-probe, and magnetic resonance elastography (MRE) to assess liver fibrosis. Findings from magnetic resonance imaging (MRI)-based proton density fat fraction (PDFF) measurements were compared with those from TE-based controlled attenuation parameter (CAP) measurements to assess steatosis., Methods: We performed a cross-sectional study of 142 patients with NAFLD (identified by liver biopsy; mean body mass index, 28.1 kg/m(2)) in Japan from July 2013 through April 2015. Our study also included 10 comparable subjects without NAFLD (controls). All study subjects were evaluated by TE (including CAP measurements), MRI using the MRE and PDFF techniques., Results: TE identified patients with fibrosis stage ≥2 with an area under the receiver operating characteristic (AUROC) curve value of 0.82 (95% confidence interval [CI]: 0.74-0.89), whereas MRE identified these patients with an AUROC curve value of 0.91 (95% CI: 0.86-0.96; P = .001). TE-based CAP measurements identified patients with hepatic steatosis grade ≥2 with an AUROC curve value of 0.73 (95% CI: 0.64-0.81) and PDFF methods identified them with an AUROC curve value of 0.90 (95% CI: 0.82-0.97; P < .001). Measurement of serum keratin 18 fragments or alanine aminotransferase did not add value to TE or MRI for identifying nonalcoholic steatohepatitis., Conclusions: MRE and PDFF methods have higher diagnostic performance in noninvasive detection of liver fibrosis and steatosis in patients with NAFLD than TE and CAP methods. MRI-based noninvasive assessment of liver fibrosis and steatosis is a potential alternative to liver biopsy in clinical practice. UMIN Clinical Trials Registry No. UMIN000012757., (Copyright © 2016 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background & Aims: The pathogenesis of non-alcoholic fatty liver disease (NAFLD) is affected by epigenetic factors as well as by genetic variation., Methods: We performed targeted-bisulfite sequencing to determine the levels of DNA methylation of 4 CpG islands (CpG99, CpG71, CpG26, and CpG101) in the regulatory regions of PNPLA3, SAMM50, PARVB variant 1, and PARVB variant 2, respectively. We compared the levels of methylation of DNA in the livers of the first and second sets of patients with mild (fibrosis stages 0 and 1) or advanced (fibrosis stages 2 to 4) NAFLD and in those of patients with mild (F0 to F2) or advanced (F3 and F4) chronic hepatitis C infection. The hepatic mRNA levels of PNPLA3, SAMM50, and PARVB were measured using qPCR., Results: CpG26, which resides in the regulatory region of PARVB variant 1, was markedly hypomethylated in the livers of patients with advanced NAFLD. Conversely, CpG99 in the regulatory region of PNPLA3 was substantially hypermethylated in these patients. These differences in DNA methylation were replicated in a second set of patients with NAFLD or chronic hepatitis C. PNPLA3 mRNA levels in the liver of the same section of a biopsy specimen used for genomic DNA preparation were lower in patients with advanced NAFLD compared with those with mild NAFLD and correlated inversely with CpG99 methylation in liver DNA. Moreover, the levels of CpG99 methylation and PNPLA3 mRNA were affected by the rs738409 genotype., Conclusions: Hypomethylation of CpG26 and hypermethylation of CpG99 may contribute to the severity of fibrosis in patients with NAFLD or chronic hepatitis C infection., (Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background: Although many of the factors and molecules closely associated with non-alcoholic steatohepatitis (NASH) have been reported, the role of inducible nitric oxide synthase (iNOS)-derived nitric oxide (NO) on the progression of NASH remains unclear. We therefore investigated the role of iNOS-derived NO in NASH pathogenesis with a long-term follow-up study using systemic iNOS-knockout mice under high-fat diet (HFD) conditions., Methods: iNOS-knockout and wild-type mice were fed a basal or HFD for 10 or 48 weeks. Lipid accumulation, fibrosis, and inflammation were evaluated, and various factors and molecules closely associated with NASH were analyzed., Results: Marked fibrosis and inflammation (indicators of NASH) were observed in the livers of iNOS-knockout mice compared to wild-type mice after 48 weeks of a HFD; however, lipid accumulation in iNOS-knockout mice livers was less than in the wild-type. Increased expressions of various cytokines that are transcriptionally controlled by NF-kB in iNOS-deficient mice livers were observed during HFD conditions., Conclusions: iNOS-derived NO may play a protective role against the progression to NASH during an HFD by preventing fibrosis and inflammation, which are mediated by NF-kB activation in Kupffer cells. A lack of iNOS-derived NO accelerates progression to NASH without excessive lipid accumulation.

Background: Although therapeutic intervention for nonalcoholic steatohepatitis (NASH) at an early stage is important owing to the progressive nature of the disease, diagnosis using noninvasive methods remains difficult. We previously demonstrated NASH specific impairment of choline metabolism and the use of fasting plasma free choline (fCh) levels for NASH diagnosis. Here, we investigated the utility of an oral choline tolerance test (OCTT), based on disordered choline metabolism, as a novel noninvasive method for NASH diagnosis., Methods: Sixty-five patients with biopsy proven nonalcoholic fatty liver disease (NAFLD) and 17 healthy controls were enrolled. Blood samples were obtained from all subjects five times during the OCTT (before and 1, 2, 3, and 4 h after oral loading with 260 mg choline)., Results: Four-hour fCh levels after oral loading choline were markedly increased in NASH patients, compared with non-NASH subjects. For detecting NASH, compared with non-NASH subjects, the area under the curve for 4-h fCh levels was 0.829 on receiver operating characteristic (ROC) analysis. The cut-off level for NASH diagnosis was ≥0.16 mg/dL, and the sensitivity, specificity, positive predictive value, and negative predictive value were 80.1, 82.6, 78.4, and 84.4 %, respectively. Moreover, 4-h fCh levels were significantly associated with the disease activity based on NAFLD activity score in patients with NAFLD., Conclusions: Four-hour fCh levels obtained by an OCTT reflect a NASH specific disorder of choline metabolism, suggesting that the OCTT is a novel and useful noninvasive method for diagnosing NASH at an early stage with sufficient accuracy for clinical practice.

Background Aims: Liver inflammation is a risk factor for the progression of nonalcoholic fatty liver disease (NAFLD). However, the diagnosis of liver inflammation is very difficult and invasive liver biopsy is still the only method to reliably detect liver inflammation. We previously reported that overexpression of CD14 in Kupffer cells may trigger the progression to nonalcoholic steatohepatitis (NASH) via liver inflammation following hyper-reactivity to low-dose lipopolysaccharide. Therefore, the aim of this study was to investigate the relationship between soluble type of CD14 (sCD14) and histological features in patients with NAFLD., Methods: Our cohort consisted of 113 patients with liver biopsy-confirmed NAFLD and 21 age-matched healthy controls. Serum sCD14 levels were measured by an enzyme-linked immunosorbent assay., Results: Serum sCD14 levels were significantly associated with diagnosis of NASH and the area under the receiver operator characteristic curve (AUROC) to distinguish between not NASH and NASH was 0.802. Moreover, serum sCD14 levels were significantly associated with the disease activity based on NAFLD activity score and hepatic CD14 mRNA expression, which is correlated with membrane CD14 (mCD14) expression, in patients with NAFLD. In multiple regression analysis, the serum sCD14 levels were independently associated with liver inflammation. The AUROC to distinguish between mild and severe liver inflammation in patients with NAFLD was 0.752., Conclusions: We found that serum sCD14 levels increased significantly with increasing liver inflammation grade in patients with NAFLD, reflecting increased hepatic CD14 expression. Serum sCD14 is a promising tool to predict the worsening of liver inflammation, and may offer a potential biomarker for evaluation of therapeutic effects in NAFLD.

Aim: The Enhanced Liver Fibrosis (ELF) test comprises a logarithmic algorithm combining three serum markers of hepatic extracellular matrix metabolism. We aimed to evaluate the performance of ELF for the diagnosis of liver fibrosis and to compare it with that of liver stiffness measurement (LSM) by FibroScan in non‐alcoholic fatty liver disease. Methods: ELF cut‐off values for the diagnosis of advanced fibrosis were obtained using receiver operating characteristic analysis in patients with biopsy‐confirmed non‐alcoholic fatty liver disease (training set; n = 200). Diagnostic performance was analyzed in the training set and in a validation set (n = 166), and compared with that of LSM in the FibroScan cohort (n = 224). Results: The area under receiver operating characteristic curve was 0.81 for the diagnosis of advanced fibrosis, and the ELF cut‐off values were 9.34 with 90.4% sensitivity and 10.83 with 90.6% specificity in the training set, and 89.8% sensitivity and 85.5% specificity in the validation set. There was no significant difference in the area under the receiver operating characteristic curve between ELF and LSM (0.812 and 0.839). A combination of ELF (cut‐off 10.83) and LSM (cut‐off 11.45) increased the specificity to 97.9% and the positive predictive value, versus ELF alone. Sequential use of the Fibrosis‐4 index (cut‐off 2.67) and ELF (cut‐off 9.34) increased the sensitivity to 95.9%. Conclusions: ELF can identify advanced liver fibrosis in non‐alcoholic fatty liver disease, and its diagnostic accuracy is comparable to that of FibroScan. According to the clinical setting, combinations or sequential procedures using other non‐invasive tests complement the diagnostic performance of ELF for the identification of advanced fibrosis. [ABSTRACT FROM AUTHOR]

Background: Few studies have evaluated both liver fibrosis and steatosis in patients with nonalcoholic fatty liver disease (NAFLD) using both FibroScan® M and XL probes. This study was performed to investigate the accuracy of both FibroScan® probes to diagnose liver fibrosis and steatosis in patients with NAFLD.Methods: We prospectively enrolled 137 consecutive patients with clinically suspected NAFLD in our joint-research facilities. Liver biopsies, liver stiffness measurements (LSMs), and controlled attenuation parameter (CAP) measurements were performed, and 122 patients with NAFLD diagnosed pathologically by central pathologists were included in the final analysis.Results: Reliable LSM results were obtained in 85.2% (M) and 89.3% (XL) of patients, and CAP was reliable in 90.2% (M) and 90.2% (XL). The median LSM was significantly lower with the XL than M probe, and CAP was significantly higher with the XL than M probe. The optimal cut-off values for diagnosing the fibrosis stage were lower for LSM with the XL than M probe (stage ≥ 2, 6.7 vs. 7.0; stage ≥ 3, 8.2 vs. 10.8; stage 4, 14.3 vs. 16.8, respectively), whereas those of CAP were higher for the XL than M probe (score of ≥ 2, 273 vs. 267; score of 3, 302 vs. 286, respectively). There were no significant differences in accuracy of the LSM and CAP between the probes.Conclusions: Liver fibrosis and steatosis could be equally evaluated with FibroScan® M and XL probes in patients with NAFLD. There was no significant difference in diagnostic accuracy between the two probes using probe-specific cut-off values. [ABSTRACT FROM AUTHOR]

Aim: Although liver biopsy is the gold standard for the diagnosis and staging of non‐alcoholic fatty liver disease (NAFLD), repeated assessment of patients' liver tissue conditions are impractical. We assessed the 10‐year changes in liver stiffness measurements (LSM) utilizing vibration‐controlled transient elastography in NAFLD patients. Methods: From January 2006 to September 2007, LSM was carried out for 97 biopsy‐proven NAFLD patients. Of these, 34 patients underwent 10‐year LSM reassessments (14 of them with paired biopsies). Results: We evaluated the changes in the fibrosis stage as estimated using LSM (FS‐LSM). Over a 10‐year period, 32.4% had FS‐LSM progression, 50% had static disease, and 17.6% had FS‐LSM improvement. From among the initially diagnosed non‐alcoholic steatohepatitis patients, 18% had progressed to considerable stage 4 (cirrhosis) 10 years later. In this cohort, none of the patients who had been initially diagnosed as FS‐LSM stage 0 had progressed to cirrhosis 10 years later. The changes in LSM were correlated with the change in the histological fibrosis stage, the NAFLD activity score, and the change in the sum of the steatosis, activity, and fibrosis score. Improving more than 1 body mass index (kg/m2) and having a higher initial aspartate aminotransferase, alanine aminotransferase (ALT), or ALT responder (>30% improvement or reduction to less than 40 IU/L) were factors contributing to LSM improvements (≥2 kPa). Conclusions: Vibration‐controlled transient elastography is likely to become a more clinically important tool for the long‐term monitoring of NAFLD patients. [ABSTRACT FROM AUTHOR]

Background and Aim: In the condition of high prevalence of non‐alcoholic fatty liver disease (NAFLD), a new diagnostic algorithm to efficiently identify NAFLD patients with significant fibrosis is urgently required. We evaluated the predictive ability of the fibrosis‐4 index (FIB‐4 index) for significant liver fibrosis (F ≥ 2) in a cohort of Japanese patients with NAFLD. Methods: We prospectively calculated the FIB‐4 index in patients who were incidentally diagnosed as fatty liver in medical checkups and then conducted liver stiffness measurement by vibration‐controlled transient elastography (VCTE) only in patients in whom the FIB‐4 index was more than the low cut‐off index (> 1.45). Results: Of the 5929 people who underwent medical checkups, a total of 1374 people were identified as having fatty liver. Among these, we performed VCTE in 106 patients in whom the FIB‐4 index was higher than 1.45. The distribution of the fibrosis stage as estimated by VCTE in the patients was as follows: F0, 52.8%; F1, 10.3%; F2, 21.6%; F3, 11.3%; and F4, 3.7%. The positive predictive value of the FIB‐4 index for detecting NAFLD with significant fibrosis was 36.6%. The minimum value of the FIB‐4 index was constant for each estimated fibrosis stage. Conclusions: This is the first prospective study to evaluate the usefulness of the FIB‐4 index as the first step to screen NAFLD patients with significant fibrosis. In Japan, addition of one further step that combined with the FIB‐4 index is necessary to meaningfully reduce the number of patients needing liver stiffness measurement or liver biopsy. [ABSTRACT FROM AUTHOR]

Nonalcoholic fatty liver disease (NAFLD) is an important cause of chronic liver injury in many countries. The incidence of NAFLD is rising rapidly in both adults and children, because of the currently ongoing epidemics of obesity and type 2 diabetes. Notably, histological liver fibrosis is recognized as the main predictive factor for the overall long-term outcome of NAFLD, including cardiovascular disease and liver-related mortality. Thus, staging of liver fibrosis is essential in determining the prognosis and optimal treatment for patients with NAFLD and in guiding surveillance for the development of hepatocellular carcinoma (HCC). Whereas liver biopsy remains the gold standard for staging liver fibrosis, it is impossible to enforce liver biopsy in all patients with NAFLD. Noninvasive biological markers, scoring systems and noninvasive modalities are increasingly being developed and investigated to evaluate fibrosis stage of NAFLD patients. This review will highlight recent studies on the diagnosis and staging of NAFLD based on invasive (liver biopsy) or noninvasive (biomarker, scoring systems, US-based elastography and MR elastography) methods. [ABSTRACT FROM AUTHOR]

Background: Although many factors and molecules that are closely associated with non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH) have been reported, the role of endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) in the pathogenesis of NAFLD/NASH remains unclear. We therefore investigated the role of eNOS-derived NO in NAFLD pathogenesis using systemic eNOS-knockout mice fed a high-fat diet.Methods: eNOS-knockout and wild-type mice were fed a basal diet or a high-fat diet for 12 weeks. Lipid accumulation and inflammation were evaluated in the liver, and various factors that are closely associated with NAFLD/NASH and hepatic tissue blood flow were analyzed.Results: Lipid accumulation and inflammation were more extensive in the liver and lipid accumulation was less extensive in the visceral fat tissue in eNOS-knockout mice, compared with wild-type mice, after 12 weeks of being fed a high-fat diet. While systemic insulin resistance was comparable between the eNOS-knockout and wild-type mice fed a high-fat diet, hepatic tissue blood flow was significantly suppressed in the eNOS-knockout mice, compared with the wild-type mice, in mice fed a high-fat diet. The microsomal triglyceride transfer protein activity was down-regulated in eNOS-knockout mice, compared with wild-type mice, in mice fed a high-fat diet.Conclusions: A deficiency of eNOS-derived NO may exacerbate the early-stage of NASH pathogenesis by changing the fat distribution in a mouse model via the regulation of hepatic tissue blood flow. [ABSTRACT FROM AUTHOR]