Disruption of gut barrier integrity and host-microbiome interactions underlie MASLD severity in patients with type-2 diabetes mellitus.

Aberration of the "gut-liver axis" contributes to the development and progression of metabolic dysfunction-associated steatotic liver disease (MASLD). Here, we use multi-omics to analyze the gut microbiota composition and metabolic profile of patients with type-2 diabetes mellitus (T2DM). T2DM patients were screened for liver disease by blood tests, ultrasound, and liver stiffness measurements. Stool microbiota was analyzed by 16S rRNA gene sequencing; metabolomic profiling by Nuclear Magnetic Resonance spectroscopy and Ultra-High Performance-Mass Spectrometry. Microbiome and metabolic signatures were analyzed in the whole cohort and in matched subsets to identify signatures specific for steatosis (MASLD±) or fibrosis (Fibrosis±). Gut permeability was assessed in-vitro using monolayers of MDCK cells and trans-epithelial electric resistance (TEER). Cytokine profile was assessed in serum and stools.Overall, 285 patients were enrolled: 255 serum, 252 urine and 97 stool samples were analyzed. Anaeroplasma and Escherichia/Shigella ASVs were higher, while Butyricicoccus ASVs were lower in those with normal liver. In MASLD±, Butyricicoccus ASV was significantly higher in those with steatosis. In the Fibrosis±, Butyricicoccus ASV was significantly lower in those with fibrosis. Glycochenodeoxycholic acid-3-sulfate (G-UDCA-3S) appeared to be higher in MASLD with fibrosis. Fecal water from patients with MASLD and fibrosis caused the greatest drop in the TEER vs those with normal liver; this was reversed with protease inhibitors. Finally, fecal IL-13 was lower in MASLD with fibrosis. We identified microbiome signatures which were specific for steatosis and fibrosis and independent of other metabolic risk factors. Moreover, we conclude that protease-related gut permeability plays a role in those MASLD patients with fibrosis, and that disease progression is linked to a gut-liver axis which is at least partially independent of T2DM.

Aramchol in patients with nonalcoholic steatohepatitis: a randomized, double-blind, placebo-controlled phase 2b trial.

Nonalcoholic steatohepatitis (NASH), a chronic liver disease without an approved therapy, is associated with lipotoxicity and insulin resistance and is a major cause of cirrhosis and hepatocellular carcinoma. Aramchol, a partial inhibitor of hepatic stearoyl-CoA desaturase (SCD1) improved steatohepatitis and fibrosis in rodents and reduced steatosis in an early clinical trial. ARREST, a 52-week, double-blind, placebo-controlled, phase 2b trial randomized 247 patients with NASH (n = 101, n = 98 and n = 48 in the Aramchol 400 mg, 600 mg and placebo arms, respectively; NCT02279524 ). The primary end point was a decrease in hepatic triglycerides by magnetic resonance spectroscopy at 52 weeks with a dose of 600 mg of Aramchol. Key secondary end points included liver histology and alanine aminotransferase (ALT). Aramchol 600 mg produced a placebo-corrected decrease in liver triglycerides without meeting the prespecified significance (-3.1, 95% confidence interval (CI) -6.4 to 0.2, P = 0.066), precluding further formal statistical analysis. NASH resolution without worsening fibrosis was achieved in 16.7% (13 out of 78) of Aramchol 600 mg versus 5% (2 out of 40) of the placebo arm (odds ratio (OR) = 4.74, 95% CI = 0.99 to 22.7) and fibrosis improvement by ≥1 stage without worsening NASH in 29.5% versus 17.5% (OR = 1.88, 95% CI = 0.7 to 5.0), respectively. The placebo-corrected decrease in ALT for 600 mg was -29.1 IU l-1 (95% CI = -41.6 to -16.5). Early termination due to adverse events (AEs) was <5%, and Aramchol 600 and 400 mg were safe, well tolerated and without imbalance in serious or severe AEs between arms. Although the primary end point of a reduction in liver fat did not meet the prespecified significance level with Aramchol 600 mg, the observed safety and changes in liver histology and enzymes provide a rationale for SCD1 modulation as a promising therapy for NASH and fibrosis and are being evaluated in an ongoing phase 3 program.

Current Concepts, Opportunities, and Challenges of Gut Microbiome-Based Personalized Medicine in Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NALFD) is now a leading cause of chronic liver disease worldwide, in part, as a consequence of rapidly rising levels of obesity and metabolic syndrome and is a major risk factor for cirrhosis, hepatocellular carcinoma, and liver-related mortality. From NAFLD stems a myriad of clinical challenges related to both diagnosis and management. A growing body of evidence suggests an intricate linkage between the gut microbiome and the pathogenesis of NAFLD. We highlight how our current knowledge of the gut-liver axis in NAFLD may be leveraged to develop gut microbiome-based personalized approaches for disease management, including its use as a non-invasive biomarker for diagnosis and staging, as a target for therapeutic modulation, and as a marker of drug response. We will also discuss current limitations of these microbiome-based approaches. Ultimately, a better understanding of microbiota-host interactions in NAFLD will inform the development of novel preventative strategies and precise therapeutic targets.

Association between non-alcoholic fatty liver disease and decreased lung function in adults: A systematic review and meta-analysis.

AIM: Recent observational studies assessed the association between non-alcoholic fatty liver disease (NAFLD) and lung function in adults, but the magnitude of this association remains uncertain. We estimated the magnitude of the association between NAFLD and lung function on spirometry (predicted forced expiratory volume in 1 s [FEV1] and forced vital capacity [FVC]). METHODS: We searched publication databases using predefined keywords to identify studies (published up to October 4, 2018), in which NAFLD was diagnosed by imaging or biochemistry (no studies with biopsy-proven NAFLD were available). Data from selected studies were extracted, and meta-analysis was performed using random-effects modelling. RESULTS: Six observational studies (5 cross-sectional and 1 longitudinal) with aggregate data on 133,707 individuals (27.8% with NAFLD) of predominantly Asian ethnicity (74.6%) were included in the final analysis. There were significant differences in predicted FEV1 (n = 5 studies; pooled weighted mean difference [WMD]: -2.43%, 95% CI: -3.28 to -1.58; I2 = 69.7%) and predicted FVC (pooled WMD: -2.96%, 95% CI: -4.75 to -1.17; I2 = 91.7%) between individuals with and without NAFLD. Decreased FEV1 and FVC at baseline were also independently associated with a ∼ 15% increased risk of incident NAFLD (n = 1 study in Korean individuals). Subgroup analyses did not materially modify these findings. CONCLUSIONS: NAFLD is associated with significant reductions of both FEV1 and FVC in Asian and United States adults, and such small, but significant, reductions of lung volumes at baseline may be also associated with increased NAFLD incidence in Asian individuals. Further research is needed to better elucidate the link between NAFLD and impaired lung volumes.

Blood pressure levels and hypertension prevalence in a high selenium environment: results from a cross-sectional study.

BACKGROUND AND AIMS: Recent human and laboratory studies have suggested the possibility that selenium overexposure may increase blood pressure. We sought to ascertain whether adults living in a seleniferous area exhibit an association between selenium exposure and both blood pressure levels as well as prevalence of hypertension. METHODS AND RESULTS: We measured selenium levels in blood (serum), hair and nail samples obtained from 680 adult volunteers (267 men and 413 women), living in seven Punjabi villages in a seleniferous area and related them to health outcomes, including systolic and diastolic blood pressure and presence of hypertension. In a multivariable restricted cubic spline regression model, adjusted for age, sex and history of hypertension, we found a positive association between systolic blood pressure and both serum (P = 0.004) and hair (P = 0.058) selenium levels, but not with nail selenium content. Little association emerged between the three selenium biomarkers and diastolic blood pressure. Hypertension prevalence was positively associated with the three exposure indicators (P < 0.001). The associations we found were generally stronger in women than in men. CONCLUSIONS: Overall, these findings suggest that chronic overexposure to environmental selenium may increase blood pressure, though there were inconsistencies for this association according to the choice of exposure indicator, the study endpoint and the sex.

Clinical and metabolic effects associated with weight changes and obeticholic acid in non-alcoholic steatohepatitis.

BACKGROUND: In a 72-week, randomised controlled trial of obeticholic acid (OCA) in non-alcoholic steatohepatitis (NASH), OCA was superior to placebo in improving serum ALT levels and liver histology. OCA therapy also reduced weight. AIMS: Because weight loss by itself can improve histology, to perform a post hoc analysis of the effects of weight loss and OCA treatment in improving clinical and metabolic features of NASH. METHODS: The analysis was limited to the 200 patients with baseline and end-of-treatment liver biopsies. Weight loss was defined as a relative decline from baseline of 2% or more at treatment end. RESULTS: Weight loss occurred in 44% (45/102) of OCA and 32% (31/98) of placebo-treated patients (P = 0.08). The NAFLD Activity score (NAS) improved more in those with than without weight loss in both the OCA- (-2.4 vs -1.2, P<0.001) and placebo-treated patients (-1.2 vs -0.5, P = 0.03). ALT levels also improved in those with vs without weight loss in OCA- (-43 vs -34 U/L, P = 0.12) and placebo-treated patients (-29 vs -10 U/L, P = 0.02). However, among those who lost weight, OCA was associated with opposite effects from placebo on changes in alkaline phosphatase (+21 vs -12 U/L, P<0.001), total (+13 vs -14 mg/dL, P = 0.02) and LDL cholesterol (+18 vs -12 mg/dL, P = 0.01), and HbA1c (+0.1 vs -0.4%, P = 0.01). CONCLUSIONS: OCA leads to weight loss in up to 44% of patients with NASH, and OCA therapy and weight loss have additive benefits on serum aminotransferases and histology. However, favourable effects of weight loss on alkaline phosphatase, lipids and blood glucose seen in placebo-treated patients were absent or reversed on OCA treatment. These findings stress the importance of assessing concomitant metabolic effects of new therapies of NASH. Clinical trial number: NCT01265498.

Assessment of treatment response in non-alcoholic steatohepatitis using advanced magnetic resonance imaging.

BACKGROUND: Magnetic resonance imaging-derived measures of liver fat and volume are emerging as accurate, non-invasive imaging biomarkers in non-alcoholic steatohepatitis (NASH). Little is known about these measures in relation to histology longitudinally. AIM: To examine any relationship between MRI-derived proton-density fat-fraction (PDFF), total liver volume (TLV), total liver fat index (TLFI), vs. histology in a NASH trial. METHODS: This is a secondary analysis of a 24-week randomised, double-blind, placebo-controlled trial of 50 patients with biopsy-proven NASH randomised to oral ezetimibe 10 mg daily (n = 25) vs. placebo (n = 25). Baseline and post-treatment anthropometrics, biochemical profiling, MRI and biopsies were obtained. RESULTS: Baseline mean PDFF correlated strongly with TLFI (Spearman's ρ = 0.94, n = 45, P < 0.0001) and had good correlation with TLV (ρ = 0.57, n = 45, P < 0.0001). Mean TLV correlated strongly with TLFI (ρ = 0.78, n = 45, P < 0.0001). After 24 weeks, PDFF remained strongly correlated with TLFI (ρ = 0.94, n = 45, P < 0.0001), maintaining good correlation with TLV (ρ = 0.51, n = 45, P = 0.0004). TLV remained strongly correlated with TLFI (ρ = 0.74, n = 45, P < 0.0001). Patients with Grade 1 vs. 3 steatosis had lower PDFF, TLV, and TLFI (P < 0.0001, P = 0.0003, P < 0.0001 respectively). Regression analysis of changes in MRI-PDFF vs. TLV indicates that 10% reduction in MRI-PDFF predicts 257 mL reduction in TLV. CONCLUSIONS: The MRI-PDFF and TLV strongly correlated with TLFI. Decreases in steatosis were associated with an improvement in hepatomegaly. Lower values of these measures reflect lower histologic steatosis grades. MRI-derived measures of liver fat and volume may be used as dynamic and more responsive imaging biomarkers in a NASH trial, than histology.

Magnetic resonance elastography identifies fibrosis in adults with alpha-1 antitrypsin deficiency liver disease: a prospective study.

BACKGROUND: Limited data exist on the clinical presentation and non-invasive detection of liver fibrosis in adults with homozygous Z genotype alpha-1 antitrypsin (AAT) deficiency. AIMS: To compare demographic, biochemical, histological and imaging data of AAT deficient patients to normal-control and biopsy-proven non-alcoholic fatty liver disease (NAFLD) patients, and to assess the diagnostic accuracy of magnetic resonance elastography (MRE) in detecting fibrosis in AAT deficiency. METHODS: Study includes 33 participants, 11 per group, who underwent clinical research evaluation, liver biopsy (AAT and NAFLD groups), and MRE. Histological fibrosis was quantified using a modified Ishak 6-point scale and liver stiffness by MRE. Diagnostic performance of MRE in detecting fibrosis was assessed by receiver operating characteristic (ROC) analysis. RESULTS: Mean (±s.d.) of age and BMI of normal-control, AAT and NAFLD groups was 57 (±19), 57 (±18), and 57 (±13) years, and 22.7 (±2.5), 24.8 (±4.0) and 31.0 (±5.1) kg/m(2) respectively. Serum ALT [mean ± s.d.] was similar within normal-control [16.4 ± 4.0] and AAT groups [23.5 ± 10.8], but was significantly lower in AAT than NAFLD even after adjustment for stage of fibrosis (P < 0.05, P = 0.0172). For fibrosis detection, MRE-estimated stiffness had an area under the ROC curve of 0.90 (P < 0.0001); an MRE threshold of ≥3.0 kPa provided 88.9% accuracy, with 80% sensitivity and 100% specificity to detect presence of any fibrosis (stage ≥1). CONCLUSIONS: This pilot prospective study suggests magnetic resonance elastography may be accurate for identifying fibrosis in patients with alpha-1 antitrypsin deficiency. Larger validation studies are warranted.

Review article: emerging anti-fibrotic therapies in the treatment of non-alcoholic steatohepatitis.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) can lead to non-alcoholic steatohepatitis (NASH) and cirrhosis. Fibrosis predicts worse outcomes and mortality. New treatments targeting fibrosis are being investigated to reverse disease progression. AIM: To review the new pipeline therapeutic agents targeting fibrosis in NASH patients, with particular focus on clinical trials in which reversing fibrosis and portal hypertension are the primary outcomes. METHODS: The literature was searched in PubMed between January 2000 and January 2016 using search terms non-alcoholic fatty liver disease and NASH, with filters of 'English language'. We focused on fibrosis improvement as the key outcome. We also searched the ClinicalTrials.gov for promising agents that target fibrosis in NASH patients. RESULTS: Significant advances have been made on approaches targeting fibrosis in NASH patients. Many therapeutic agents are already in development, some of which have shown promising results in preclinical and phase I studies. Novel therapies have entered phase II and III studies targeting fibrosis reversal and/or improvement in portal hypertension. Innovative studies have also started looking into combining these agents, aiming at different mechanisms to maximise therapeutic outcomes. We found five clinical trials in phase II and one in phase III focusing on fibrosis in NASH patients as key outcomes. One of the phase II trials is using combination therapy to target fibrosis. CONCLUSIONS: Ongoing research studies are already investigating new pathways aimed at reversing fibrosis in NASH patients. Novel therapeutic agents are in development and are expected to offer unique options to NASH patients with advanced fibrosis.

Non-invasive screening of diabetics in primary care for NAFLD and advanced fibrosis by MRI and MRE.

BACKGROUND: Current guidelines do not recommend screening for non-alcoholic fatty liver disease (NAFLD) or advanced fibrosis. Patients with type 2 diabetes mellitus (T2DM) are known to be at increased risk for NAFLD and advanced fibrosis. AIM: To assess the feasibility in diabetics in a primary care setting of screening for NAFLD and advanced fibrosis, by using non-invasive magnetic resonance imaging (MRI) to estimate the hepatic proton density fat fraction (MRI-PDFF) and magnetic resonance elastography (MRE) to estimate hepatic stiffness. METHODS: We performed a cross-sectional analysis of a prospective study that included 100 (53% men) consecutively enrolled diabetics who did not have any other aetiology of liver disease. All patients underwent a standardised research visit, laboratory tests, MRI-PDFF, and MRE. RESULTS: Mean (±s.d.) age and body mass index (BMI) was 59.7 (±11.2) years and 30.8 (±6.5) kg/m(2) , respectively. The prevalence of NAFLD (defined as MRI-PDFF ≥5%) and advanced fibrosis (defined as MRE ≥3.6 kPa) was 65% and 7.1%, respectively. One patient with advanced fibrosis had definite hepatocellular carcinoma. When compared to those without NAFLD, patients with NAFLD were younger (P = 0.028) and had higher mean BMI (P = 0.0008), waist circumference (P < 0.0001) and prevalence of metabolic syndrome (84.6% vs. 40.0%, P < 0.0001). Only 26% of those with NAFLD had elevated alanine aminotransferase. CONCLUSIONS: This proof-of-concept study demonstrates that T2DM has significant rates of both NAFLD and advanced fibrosis. Concomitant screening for NAFLD and advanced fibrosis by using MRI-proton density fat fraction and magnetic resonance elastography in T2DM is feasible and may be considered after validation in a larger cohort.