BACKGROUND:Autoimmune hepatitis (AIH) may be difficult to diagnose and distinguish clinically and biochemically from other chronic liver diseases like metabolic dysfunction-associated steatotic liver disease (MASLD). AIMS:To identify pathways involved in the pathogenesis and identify disease-specific biomarkers of AIH. METHODS:We recruited 19 newly diagnosed patients with AIH, 17 with MASLD, and 19 healthy controls. Liver tissue and plasma were collected, and untargeted mass-spectrometry-based proteomics was performed. For classification of AIH versus MASLD and healthy, machine learning analyses were performed employing logistic regression models on liver and plasma proteome data. Findings were validated using data from the United Kingdom Biobank (UKB). RESULTS:We identified 7632 liver and 556 plasma proteins with 2521 liver and 227 plasma proteins differing between AIH and healthy, including 56 overlapping. Metabolic dysregulation and systemic immune activation characterised the AIH liver and plasma proteome, respectively. Plasma proteome profiling enabled classification of AIH from MASLD and healthy with an area under the receiver operating characteristic curve of 0.91 (0.09 SD). Validation in the UKB was possible for 8 of 20 diagnostic proteins and showed consistent directional changes. Three proteins (C7, ICAM1, cAST) were significantly different between AIH and MASLD/healthy in unadjusted analyses, and 6 of 8 proteins (C7, ICAM1, cAST, IGFBP3, TIMP1, TTR) were significantly different when adjusting for age and sex. CONCLUSIONS:Clinical proteomic analyses of paired liver-plasma samples from patients with AIH enabled high diagnostic potential. Proteomics may constitute a novel non-invasive diagnostic tool for AIH if validated in larger, age- and sex-matched cohorts. CLINICAL TRIAL NUMBER:NCT05335603.

The human liver is dynamic organ with minute to hourly adaptions in response to feeding. Patients with non-alcoholic fatty liver disease (NAFLD) and cirrhosis have altered transcriptomic features compared to controls but how and if food intake affects such is unknown in humans. Our aim was to investigate the hepatic transcriptome at both fasting and postprandial states in patients with NAFLD, cirrhosis, and healthy controls and secondly to develop a browsable resource enabling easy and unrestricted access to such data. We hypothesized that hepatic transcriptome differed between groups, and this was also regulated by food intake. We obtained liver tissue by transjugular liver biopsies from patients with NAFLD (n = 9, mean age 49 (16 SD) y, BMI 35 (5) kg/m2), cirrhosis (n = 9, age 61 (11) y, BMI 32 (5) kg/m2) and healthy controls (n = 10, age 25 (3) y, BMI 23 (3) kg/m2). The hepatic transcriptome was sequenced using NGS and evaluated in bioinformatic analyses to assess differentially expressed genes (DEG) and gene ontology biological processes (GOBP). We identified 553 DEG between healthy controls and patients with NAFLD, 5527 DEG between healthy controls and patients with cirrhosis, and 3898 DEG in NAFLD compared with cirrhosis. A hitherto uncharacterized gene (MET proto-oncogene) was differentially expressed in human NAFLD and cirrhosis. The hepatic transcriptome changed significantly during a standardized meal and these changes were blunted in patients with NAFLD and cirrhosis. GOBP analyses revealed an increase in pro-inflammatory and pro-fibrotic genes in NAFLD and cirrhosis, as well as a decrease in genes related to metabolism. Data were made browsable using two web-based apps. The hepatic transcriptome is differentially regulated by a standardized meal in healthy individuals compared to patients with fatty liver disease.

INTRODUCTION AND OBJECTIVES:Disruptions in one-carbon metabolism (OCM) have been linked to cardiometabolic diseases. We evaluated alterations in OCM metabolites and enzymes and the impact of semaglutide in MASLD. MATERIALS AND METHODS:Using targeted metabolomics and bulk-transcriptomics, we analyzed components of OCM in plasma samples and liver biopsies from MASLD patients (n = 100 with F0-F4 fibrosis, 51 % type 2 diabetes) and healthy controls (n = 50). Untargeted metabolomics and transcriptomics were used to analyze plasma and liver specimens from mice with diet-induced obesity and steatohepatitis (DIO-MASH) treated with vehicle or semaglutide compared with chow for 12 weeks. RESULTS:In patients with MASLD vs healthy controls, changes in the concentrations of folate and pyridoxal 5'-phosphate (vitamin B6), betaine, serine, and glycine suggested alteration to the activity of the transsulfuration pathway as well as the methionine and folate cycles. Increased concentrations of metabolites related to enhanced homocysteine conversion and elevated glutamate were also observed. These findings were supported by corresponding alterations in the activity of key enzymes. Similar patterns were identified in comparisons of patients with significant vs no/mild fibrosis and in DIO-MASH mice vs chow. Treatment with semaglutide led to reductions in body weight, hepatic inflammation, and fibrosis and largely reversed the metabolic and gene expression changes observed in DIO-MASH mice. CONCLUSIONS:MASLD may be linked to alterations in key metabolites and enzymes involved in OCM. The changes in humans were replicated in a MASLD mouse model, and reversal was found after treatment with semaglutide.

Autoimmune liver diseases are associated with an increased risk of diabetes, yet the underlying mechanisms remain unknown. In this cross-sectional study, we investigated the glucose-regulatory disturbances in patients with autoimmune hepatitis (AIH, n = 19), primary biliary cholangitis (PBC, n = 15), and primary sclerosing cholangitis (PSC, n = 6). Healthy individuals (n = 24) and patients with metabolic dysfunction-associated steatotic liver disease (MASLD, n = 18) were included as controls. Blood samples were collected during a 120-min oral glucose tolerance test. We measured the concentrations of glucose, C-peptide, insulin, glucagon, and the two incretin hormones, glucose insulinotropic peptide (GIP) and glucagon-like peptide-1 (GLP-1). We calculated the homeostasis model assessment of insulin resistance (HOMA-IR), whole body insulin resistance (Matsuda index), insulin clearance, and insulinogenic index. All patient groups had increased fasting plasma glucose and impaired glucose responses compared with healthy controls. Beta-cell secretion was increased in AIH, PBC, and MASLD but not in PSC. Patients with AIH and MASLD had hyperglucagonemia and hepatic, as well as peripheral, insulin resistance and decreased insulin clearance, resulting in hyperinsulinemia. Patients with autoimmune liver disease had an increased GIP response, and those with AIH or PBC had an increased GLP-1 response. Our data demonstrate that the mechanism underlying glucose disturbances in patients with autoimmune liver disease differs from that underlying MASLD, including compensatory incretin responses in patients with autoimmune liver disease. Our results suggest that glucose disturbances are present at an early stage of the disease. NEW & NOTEWORTHY Patients with autoimmune liver disease but without overt diabetes display glucose disturbances early on in their disease course. We identified pathophysiological traits specific to these patients including altered incretin responses.

AIM:Increased growth differentiation factor 15 (GDF15) may reflect impaired metabolic health and an inflammatory state in metabolic dysfunction-associated steatotic liver disease (MASLD). We investigated the role of GDF15 in histologically verified MASLD in a meal test (discovery) cohort (n = 20) and a prospective (validation) cohort with 2 years of follow-up (n = 276). METHODS:Participants were evaluated clinically and histologically in both cohorts. Fibrosis severity was classified as no/mild (F0/F1) or significant (F2-4). Plasma GDF15 was measured by enzyme-linked immunosorbent assays and the SOMAScan platform. Hepatic GDF15 mRNA expression was analyzed by RNA in situ hybridization and bulk RNA-sequencing. In addition, we used data from public single-nucleus RNA-sequencing datasets. RESULTS:In both cohorts, plasma GDF15 was increased in MASLD compared with healthy controls (p < 0.0001) with the highest levels in patients with significant fibrosis (area under the curve 0.83; 95% confidence interval [CI], 0.76-0.91). The GDF15 levels were unaffected by a standardized meal and there was no difference in peripheral or hepatic venous concentrations. After 2 years, the increase in GDF15 levels was reduced in patients treated with glucagon-like peptide receptor agonists (GLP-1-RA) compared to patients receiving lifestyle advice (-28%; 95% CI, -44 to -8; p = 0.01). Plasma GDF15 was associated with circulating insulin-like growth factor 1 and related proteins. Hepatic GDF15 mRNA was mainly expressed in hepatocytes and in cholangiocytes in fibrotic areas and was increased in MASLD (p = 0.02) with the highest expression in the group with steatohepatitis (p = 0.009). CONCLUSIONS:Increased hepatic and circulating GDF15 are found in MASLD. Treatment with GLP-1-RA may reduce GDF15, possibly reflecting beneficial metabolic and inflammatory effects.

Introduction and Objectives: Studies on the societal burden of patients with biopsy-confirmed non-alcoholic fatty liver disease (NAFLD) are sparse. This study examined this question, comparing NAFLD with matched reference groups. Materials and Methods: Nationwide Danish healthcare registers were used to include all patients (>= 18 years) diagnosed with biopsy-verified NAFLD (1997-2021). Patients were classified as having simple steatosis or non-alcoholic steatohepatitis (NASH) with or without cirrhosis, and all matched with liver-disease free reference groups. Healthcare costs and labour market outcomes were compared from 5 years before to 11 years after diagnosis. Patients were followed for 25 years to analyse risk of disability insurance and death. Results: 3,712 patients with biopsy-verified NASH (n = 1,030), simple steatosis (n = 1,540) or cirrhosis (n =1,142) were identified. The average total costs in the year leading up to diagnosis was 4.1-fold higher for NASH patients than the reference group (EUR 6,318), 6.2-fold higher for cirrhosis patients and 3.1-fold higher for simple steatosis patients. In NASH, outpatient hospital contacts were responsible for 49 % of the excess costs (EUR 3,121). NASH patients had statistically significantly lower income than their reference group as early as five years before diagnosis until nine years after diagnosis, and markedly higher risk of becoming disability insurance recipients (HR: 4.37; 95 % CI: 3.17-6.02) and of death (HR: 2.42; 95 % CI: 1.80-3.25). Conclusions: NASH, simple steatosis and cirrhosis are all associated with substantial costs for the individual and the society with excess healthcare costs and poorer labour market outcomes. (c) 2024 Published by Elsevier Espana, S.L.U. on behalf of Fundacion Clinica Medica Sur, A.C. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Increased plasma concentrations of glucagon (hyperglucagonemia) are reported in patients with type 2 diabetes (T2D) and are considered a diabetogenic risk factor. Emerging evidence suggests that hepatic steatosis in obesity is causing a condition of resistance toward glucagon's effects on amino acid metabolism, resulting in an amino acid-induced hyperglucagonemia. We investigated the presence of hyperglucagonemia in individuals with biopsy-verified metabolic dysfunction-associated steatotic liver disease (MASLD), and whether body mass index (BMI), T2D, hepatic steatosis, and/or fibrosis contribute to this relationship. To dissect potential mechanisms, we also determined hepatic gene expression related to amino acid transport and catabolism. Individuals with MASLD had hyperglucagonemia {controls (n = 74) vs. MASLD (n = 106); median [Q1, Q3]; 4 [3, 7] vs. 8 [6, 13] pM), P < 0.0001} and were glucagon resistant (assessed by the glucagon-alanine index) {1.3 [0.9, 2.1] vs. 3.3 [2.1, 5.3] pM·mM, P < 0.0001}. These changes were associated with hepatic steatosis (P < 0.001, R2 > 0.25) independently of BMI, sex, age, and T2D. Plasma levels of glucagon were similar in individuals with MASLD when stratified on T2D status {MASLD-T2D (n = 52) vs. MASLD + T2D (n = 54); 8 [6, 11] vs. 8 [6, 13] pM, P = 0.34} and hepatic fibrosis {MASLD + F0 (n = 25) vs. MASLD + F1-F3 (n = 67); 8.4 [7.0, 13.3] vs. 7.9 [5.2, 11.6] pM, P = 0.43}. Obesity (BMI = 30 kg/m2) did not alter glucagon levels (P = 0.65) within groups (control/MASLD). The mRNA expression of proteins involved in amino acid transport and catabolism was downregulated in MASLD. Thus, relative hyperglucagonemia is present in individuals with biopsy-verified MASLD, and hepatic steatosis partially drives hyperglucagonemia and glucagon resistance, irrespective of T2D, BMI, and hepatic fibrosis.NEW & NOTEWORTHY Individuals with metabolic dysfunction-associated steatotic liver disease (MASLD) present with increased plasma levels of glucagon (hyperglucagonemia), irrespective of body mass index (BMI) and type 2 diabetes. Therefore, MASLD and the resultant hyperglucagonemia may act as a diabetogenic risk factor. Notably, hepatic steatosis was a significant contributor to the hyperglucagonemia in MASLD, potentially unveiling a pathway for the hyperglucagonemia in some patients with type 2 diabetes.

Gremlin-1 has been implicated in liver fibrosis in metabolic dysfunction-associated steatohepatitis (MASH) via inhibition of bone-morphogenetic protein (BMP) signalling and has thereby been identified as a potential therapeutic target. Using rat in vivo and human in vitro and ex vivo model systems of MASH fibrosis, we show that neutralisation of Gremlin-1 activity with monoclonal therapeutic antibodies does not reduce liver inflammation or liver fibrosis. Still, Gremlin-1 was upregulated in human and rat MASH fibrosis, but expression was restricted to a small subpopulation of COL3A1/THY1 + myofibroblasts. Lentiviral overexpression of Gremlin-1 in LX-2 cells and primary hepatic stellate cells led to changes in BMP-related gene expression, which did not translate to increased fibrogenesis. Furthermore, we show that Gremlin-1 binds to heparin with high affinity, which prevents Gremlin-1 from entering systemic circulation, prohibiting Gremlin-1-mediated organ crosstalk. Overall, our findings suggest a redundant role for Gremlin-1 in the pathogenesis of liver fibrosis, which is unamenable to therapeutic targeting.

Non-alcoholic steatohepatitis (NASH) is emerging as a major cause of hepatocellular carcinoma (HCC), however, it is not resolved if compounds in late-stage clinical development for NASH may have additional therapeutic benefits in NASH-driven HCC (NASH-HCC). Here, we profiled monotherapy with semaglutide (glucagon-like-receptor-1 receptor agonist) and lanifibranor (pan-peroxisome proliferator-activated receptor agonist) in a diet-induced obese (DIO) mouse model of NASH-HCC. Disease progression was characterized in male C57BL/6 J mice fed the GAN (Gubra Amylin NASH) diet high in fat, fructose and cholesterol for 12–72 weeks (n = 15 per group). Other GAN DIO-NASH-HCC mice fed the GAN diet for 54 weeks and with biopsy-confirmed NASH (NAFLD Activity Score ≥ 5) and advanced fibrosis (stage F3) received vehicle (n = 16), semaglutide (30 nmol/kg, s.c., n = 15), or lanifibranor (30 mg/kg, p.o., n = 15) once daily for 14 weeks. GAN DIO-NASH-HCC mice demonstrated progressive NASH, fibrosis and HCC burden. Tumors presented with histological and molecular signatures of poor prognostic HCC. Consistent with clinical trial outcomes in NASH patients, both lanifibranor and semaglutide improved NASH while only lanifibranor reduced fibrosis in GAN DIO-NASH-HCC mice. Notably, only semaglutide reduced tumor burden in GAN DIO-NASH-HCC mice. In conclusion, the GAN DIO-NASH-HCC mouse is a clinical translational model of NASH-HCC. Semaglutide improves both NASH and tumor burden in GAN DIO-NASH-HCC mice, highlighting the suitability of this preclinical model for profiling novel drug therapies targeting NASH-HCC.

Immunohistochemistry (IHC) has for decades been an integrated method within pathology applied to gain diagnostic, prognostic, and predictive information. However, the multimodality of the analytical phase of IHC is a challenge to ensure the reproducibility of IHC, which has been documented by external quality assessment (EQA) programs for many biomarkers. More than 600 laboratories participate in the Nordic immunohistochemical Quality Control EQA program for IHC. In the period, 2017-2021, 65 different biomarkers were assessed and a total of 31,967 results were evaluated. An overall pass rate of 79% was obtained being an improvement compared with 71% for the period, 2003-2015. The pass rates for established predictive biomarkers (estrogen receptor, progesterone receptor, and HER2) for breast carcinoma were most successful showing mean pass rates of 89% to 92%. Diagnostic IHC biomarkers as PAX8, SOX10, and different cytokeratins showed a wide spectrum of pass rates ranging from 37% to 95%, mean level of 75%, and attributed to central parameters as access to sensitive and specific antibodies but also related to purpose of the IHC test and validation performed accordingly to this. Seven new diagnostic biomarkers were introduced, and all showed inferior pass rates compared with the average level for diagnostic biomarkers emphasizing the challenge to optimize, validate, and implement new IHC biomarkers. Nordic immunohistochemical Quality Control operates by "Fit-For-Purpose" EQA principles and for programmed death-ligand 1, 2 segments are offered aligned to the "3-dimensional" approach-bridging diagnostic tests, drugs to be offered, and diseases addressed. Mean pass rates of 65% and 79% was obtained in the 2 segments for programmed death-ligand 1.