Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common reason for elevated liver enzymes in children in Europe, affecting more than 5% of all children. Since the last iteration of this position paper, there have been substantial advances in our understanding of the disease. After a detailed literature review and thorough discussion, we established consensus recommendations for the diagnosis and assessment of MASLD in children. Alanine aminotransferase (ALT) >= 30 IU/L is a suitable screening test for MASLD in children over 10 years of age with obesity (body mass index z-score >=+2), or in children of any age with additional risk factors. All patients with suspected MASLD should be assessed for alternative or concomitant diagnoses, as well as comorbidities. Patients who are not overweight, under 8 years old, or have any other red flags should be promptly referred for specialist assessment. Liver biopsy remains the gold standard for diagnosis and staging of MASLD, but should be reserved for diagnostic uncertainty, to guide treatment decisions, and risk stratification (e.g., prior to transition to adult care). While there is emerging data for non-invasive tests (e.g., transient elastography), it is unclear how to routinely implement these investigations in clinical practice. Combined changes of >= 20% in both ALT and gamma-glutamyl transferase may represent a useful non-invasive tool for monitoring disease severity over time. Most patients are appropriately investigated and managed by non-specialists where the focus is on holistic management of obesity and its complications. Future research should focus on how to use non-invasive tests to risk-stratify children, in particular, how to identify those with advanced fibrosis.

BACKGROUND:Reliable biomarkers are warranted to identify patients with metabolic dysfunction-associated steatotic liver disease (MASLD), including metabolic dysfunction-associated steatohepatitis (MASH), at risk for developing hepatocellular carcinoma (HCC). RESEARCH AND METHODS:We evaluated whether circulating histones can predict this risk. Plasma histones were measured using imaging flow cytometry in patients with MASLD (n = 25), MASH (n = 25), HCC (n = 40), and 30 healthy controls. RESULTS:We detected (p < 0.05), compared to controls: 1) elevated levels of H2A, H3, H2A/H2B/H3/H4, macroH2A1.1, macroH2A1.2 in MASLD/MASH and HCC; 2) decreased levels of macroH2A1.2/H2B/H3/H4 in MASLD/MASH and increased in HCC; 3) reduced H4 levels discriminating between MASH and non-MASH. Machine-learning analysis showed that being diabetic/dyslipidemic, having decreased H2A (p = 0.002) and H4 (p = 0.0156) levels favor MASH. CONCLUSIONS:Our data indicate plasma histones H2A and H4 as new biomarkers of liver disease progression. The identification of histone-based biomarkers using imaging flow cytometry could provide a rapid approach to discriminate between non-MASH and MASH, and to predict the risk of HCC development.

Artificial Intelligence (AI) is transforming medicine, providing unprecedented opportunities to enhance diagnosis, prognosis, and treatment across all areas of hepatology. This Position Paper by the Italian Association for the Study of the Liver (AISF) offers a comprehensive overview of the current and emerging roles of AI in liver diseases, highlighting both its promise and its limitations. The document critically examines methodological, ethical, and educational challenges associated with AI integration in clinical and research settings, and reviews key applications in MASLD/MASH, alcohol-related liver disease, autoimmune and cholestatic liver diseases, viral hepatitis, drug-induced liver injury, hepatocellular carcinoma, cholangiocarcinoma, and liver transplantation.Particular emphasis is placed on data quality, algorithmic fairness, explainability, and reproducibility, as well as on the necessity of robust external validation and adherence to international reporting standards (TRIPOD-AI, CONSORT-AI, DECIDE-AI, CHAMAI and others). The AISF advocates a responsible and evidence-based adoption of AI through multidisciplinary collaboration, professional education, and patient engagement. This Position Paper outlines a national roadmap to align AI innovation with ethics, transparency, and equity, ensuring that AI becomes a genuine ally of hepatology and patient-centered care.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is an increasingly prevalent condition in both adults and children. Dysregulated telomere maintenance has been proposed as a mechanism underlying disease progression, although pediatric evidence remains limited and controversial. This study aimed to investigate the relationship between telomere length (TL) and hepato-metabolic features in children with MASLD. A total of 212 pediatric patients with biopsy-proven MASLD and 40 controls were enrolled. Telomere length in leukocytes (LTL) and liver tissue (HTL) was measured using quantitative polymerase chain reaction, and telomerase reverse transcriptase (TERT) mRNA and protein expression were also evaluated. Associations between TL and clinical, metabolic, and perinatal variables were analyzed. Children with MASLD showed significantly shorter LTL and HTL compared to controls. Shorter LTL was observed in more advanced steatohepatitis (MASH) and was associated with fibrosis severity. TERT expression was reduced in patients. LTL was also associated with perinatal factors, including preterm birth and low birthweight. Multivariable analysis identified MASH, fibrosis, and small-for-gestational-age status as independently associated with shorter LTL. In conclusion, LTL is associated with disease severity in pediatric MASLD, particularly fibrosis. These findings support a potential role of telomere dynamics in disease progression, although causal relationships require confirmation in longitudinal studies.

The increasing prevalence of metabolic dysfunction-associated fatty liver disease (MASLD) in children requires robust, non-invasive biomarkers to enable accurate disease staging and risk stratification. Elevated serum levels of homocysteine (Hcy) have emerged as potential risk factors for cardiometabolic disease in adults, including MASLD. In this observational retrospective study, we investigated the role of serum Hcy levels as a potential biomarker for disease severity and liver fibrosis in a pediatric cohort of 182 children with MASLD. In 89 patients, liver biopsy allowed the classification into metabolic dysfunction-associated steatohepatitis (MASH). Associations between Hcy, metabolic parameters, fibrosis scores, and histological features were examined, and the diagnostic performance of Hcy for liver fibrosis was evaluated using ROC analysis. Multivariate analyses identified elevated Hcy levels as independently associated with HOMA-IR (β = 0.55; p = 0.049), TG/HDL ratio (β = 3.23; p = 0.002), and liver fibrosis (β = 2.59; p = 0.04). Hcy showed a predictive accuracy of 81% for fibrosis. However, the combined diagnostic models of Hcy with non-invasive fibrotic scores (i.e., APRI and FIB-4) or TG/HDL ratio showed only a modest accuracy (AUC = 0.62–0.69). In conclusion, our data suggest that Hcy is associated with fibrosis and cardiometabolic risk. However, these results should be interpreted as exploratory and do not establish homocysteine as a diagnostic biomarker.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common reason for elevated liver enzymes in children in Europe, affecting more than 5% of all children. Since the last iteration of this position paper, there have been substantial advances in our understanding of the disease. After a detailed literature review and thorough discussion, we established consensus recommendations for the diagnosis and assessment of MASLD in children. Alanine aminotransferase (ALT) ≥ 30 IU/L is a suitable screening test for MASLD in children over 10 years of age with obesity (body mass index z-score ≥+2), or in children of any age with additional risk factors. All patients with suspected MASLD should be assessed for alternative or concomitant diagnoses, as well as comorbidities. Patients who are not overweight, under 8 years old, or have any other red flags should be promptly referred for specialist assessment. Liver biopsy remains the gold standard for diagnosis and staging of MASLD, but should be reserved for diagnostic uncertainty, to guide treatment decisions, and risk stratification (e.g., prior to transition to adult care). While there is emerging data for non-invasive tests (e.g., transient elastography), it is unclear how to routinely implement these investigations in clinical practice. Combined changes of ≥20% in both ALT and gamma-glutamyl transferase may represent a useful non-invasive tool for monitoring disease severity over time. Most patients are appropriately investigated and managed by non-specialists where the focus is on holistic management of obesity and its complications. Future research should focus on how to use non-invasive tests to risk-stratify children, in particular, how to identify those with advanced fibrosis.

Background and Aims: Metabolic dysfunction-associated steatotic liver disease (MASLD) and its progressive form, steatohepatitis (MASH), are leading causes of chronic liver disease, driving liver damage, cirrhosis, and hepatocellular carcinoma. So far, the heterogeneous nature of MASLD etiopathogenesis has limited the development of reliable non-invasive tools for early detection of patients susceptible to liver-related complications. Thus, investigating factors linked to MASLD onset and progression may accelerate the identification of biomarkers and therapeutic targets. Here, we explored the interplay between oxidative stress and epigenetic markers in relation to liver disease progression in a murine model of MASLD.Methods: We generated a progressive MASLD mouse model by administering streptozotocin (S) alongside a high-fat (SHF) diet for three different durations (8, 13, and 16 weeks). Control groups (CTL) were fed a normal diet without any treatments. Biochemical and anthropometric parameters were collected at the time of sacrifice. Liver samples underwent histopathological evaluation, gene expression, and telomere length (TL) analysis. Serum samples were used to assess redox homeostasis through total glutathione (GSH) and homocysteine (HCY) quantification using high-performance liquid chromatography with fluorimetric detection (HPLC-FD). Circulating epigenetic hallmarks, including nucleosome, total histone H3, and its modifications (H3K4me3, H3K9me3, and H3K27me3, corresponding to trimethylation at lysines 4, 9, and 27), were measured on plasma samples by commercially available kits.Results: The model of MASLD was confirmed by histological, anthropometric, and hepato-metabolic evaluations. Notably, progressive increase of steatosis, serum levels of ALT, and glucose were observed in MASLD mice across all time points, from 8 to 16 weeks. Fibrosis occurred from week 13, alongside transcriptional up-regulation of COL1A1, COL3A1, and ACTA2 genes. Hepatic TL was significantly lower in 16-week SHF mice than in CTLs and the 8-week SHF group. GSH levels decreased and HCY levels increased in all SHF groups versus CTLs, with HCY showing a significant upward trend alongside liver damage progression. Circulating nucleosome levels were significantly elevated in 16-week SHF mice compared to CTL. Accordingly, we also found elevated concentrations of total histone H3 in all SHF groups compared to CTLs. Interestingly, the plasma levels of H3 modifications, H3K4me3, H3K9me3, and H3K27me3 were consistently higher in all MASLD groups than in CTLs.Conclusions: In conclusion, our murine model of progressive MASLD induced by S and HF diet shows that worsening liver damage parallels alterations in hepatic and circulating redox and epigenetic biomarkers. Further investigation of the interplay between these redox and epigenetic traits is necessary to improve their utility in staging liver injury in MASLD and identifying novel therapeutic targets.

Background/Objectives: We evaluated the efficacy of a good lifestyle intervention on the severity of metabolic dysfunction-associated steatotic liver disease (MASLD) in children with Down syndrome (DS). Methods: This retrospective longitudinal study included 31 children with Down syndrome (DS) who were affected by MASLD and attended nutritional counseling based on a nutritional approach (e.g., Mediterranean diet and antioxidant supplements), as well as physical exercise. Clinical parameters, markers of low-grade systemic inflammation, and hepatic steatosis, as assessed by ultrasound, were evaluated at baseline (T0) and after 6 months (T1). Results: Several anthropometric and biochemical parameters, including body mass index, waist circumference, diastolic and systolic blood pressure, aspartate aminotransferase, basal insulin, insulin resistance, pro-inflammatory interleukin-1β, and anti-inflammatory interleukin-10, showed significant improvement after 6 months of a nutritional approach. This study also found a regression of at least one grade of hepatic steatosis in a significant portion of patients, especially in those who received antioxidant supplements. Conclusions: Our study further supports the hypothesis that a healthy lifestyle intervention, based on adherence to the Mediterranean diet, natural supplements with antioxidant properties, and regular physical activity, can be considered a safe therapeutic approach for reducing the risk and severity of MASLD in children with DS.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common chronic progressive hepatopathy in children, and the identification of non-invasive biomarkers is urgently needed. Growth differentiation factor 15 (GDF15) was associated with MASLD in adults. In this study, we investigated the circulating and hepatic levels of GDF15 and their association with liver damage in pediatric MASLD and in a murine model. This observational study included 158 children with biopsy-proven MASLD. Patients with MASLD were categorized into two groups based on steatohepatitis (MASH) presence and evaluated for GDF15 circulating levels, while GDF15 hepatic levels were assessed only in a subset of patients. Children with MASLD exhibited higher levels of circulating GDF15 compared to the controls. Moreover, the MASH subgroup had significantly higher values of GDF15 compared to the Not-MASH subgroup. The GDF15 levels in the MASH subgroup showed a positive correlation with fibrosis. Finally, the hepatic expression of the GDF15 gene correlated with GDF15 circulating levels and with the hepatic expression of the COL1A1 and COL3A1 genes in 15 children with MASLD. In conclusion, our study demonstrated that GDF15 levels are associated with liver damage, reinforcing the potential role of GDF15 as a biomarker for MASLD-related fibrosis in children.

BACKGROUND:Cell junctions play a pivotal role in the normal functioning of tissues. The alterations in the expression or structure of these junctions are linked to several pathologies including diabetes mellitus and its secondary complications. Therefore, this review discusses the implications of diabetes mellitus on cell junctions across different organs and tissues. METHODOLOGY:A systematic review of PubMed and other relevant search sources was conducted using relevant keywords in accordance with PRISMA guidelines. In addition, the reference lists of all included full texts were meticulously reviewed to find additional reports. RESULTS:Among 646 initially identified articles, 323 were found relevant and have been included in this review. The findings of the review suggested that hyperglycemia induces hyperphosphorylation of cell junctional proteins leading to mis-localization, downregulation and impaired intracellular communication. Although multiple junctions are affected, connexin-based gap junctions appear to be consistently affected across various tissues. This highlights their potential as a therapeutic target for mitigating diabetes mellitus and its associated complications. CONCLUSIONS:Overall, this review highlights the types of cell junctions affected in diabetes mellitus across different tissues. Moreover, integration of cell junction-targeted interventions along with standard glycemic control regimens might provide synergistic benefits to prevent secondary diabetic complications.

Background Metabolic dysfunction-associated steatotic liver disease (MASLD) is an emerging health concern in both children and adults. A few studies have suggested that dysregulation of telomere-maintaining processes may be a molecular mechanism involved in the disease; however, data in paediatric patients are controversial. This study aimed to evaluate the relationship between telomere length (TL) and hepato-metabolic features in a cohort of children with MASLD. Methods In all, 212 paediatric patients with biopsy-proven MASLD and 31 controls were enrolled in the Hepatology Unit of Bambino Gesù Children's Hospital. TL of leukocytes (LTL) and hepatic cells (HTL) was measured by quantitative polymerase chain reaction (qPCR). Telomerase reverse transcriptase (TERT) mRNA and protein levels were evaluated in a subgroup of liver samples using qPCR and immunofluorescence analyses. TL data association with hepato-metabolic and perinatal features was evaluated using different approaches. Results Our results revealed that children with MASLD had significantly lower LTL and HTL than the control children. TL shortening worsened in more advanced phases of the disease (MASH) and was associated with fibrosis grade. TERT expression was lower in the liver of patients than in controls. LTL was significantly associated with preterm birth and birthweight, and a general linear model highlighted the impact of MASH, fibrosis, and being born small for gestational age on LTL decrease. Conclusion In conclusion, our study demonstrated for the first time a strong relationship between TL and pediatric MASLD-related features, mainly fibrosis. Further studies are needed to clarify the causal relationship between MASH, fibrosis, birthweight and TL.

The peroxisome proliferator-activated receptor family of lipid-activated nuclear receptors (PPARs) plays a critical role in the regulation of cellular lipid metabolism. In cardiac muscle, PPARα is highly expressed and regulates genes involved in fatty acid oxidation, but its activity is downregulated in hypertrophic hearts; however, the consequences of chronic PPARα deficiency on the cardiac contractile apparatus remain unclear. This study aimed to investigate the PPARα role in hypertrophic phenotype and to evaluate the potential effects of the antioxidant Ebselen (Ebs) treatment on changes associated with PPARα depletion. We thus generated an in vitro model of cardiac hypertrophy by stable silencing of the PPARA gene in H9c2 rat cardiomyoblasts. We observed that PPARα silencing induces a hypertrophic phenotype, characterized by increased NPPB and decreased FBXO32 expression, mitochondrial dysregulation, impaired lipid metabolism, oxidative stress, and ferroptosis-related alterations. Epigenetically, H3K27ac levels increased while H3K27me3 decreased. Moreover, miR-34a, miR-132, and miR-331 were downregulated, implicating a miRNA-mediated mechanism in PPARα-linked cardiac hypertrophy. Treatment with Ebs, a redox-active compound with inhibitory effects on ferroptosis and epigenetics, reversed hypertrophic phenotype and restored miRNA levels. In conclusion, we found that PPARα depletion promotes oxidative stress and hypertrophic phenotype and that Ebs may act as a potential therapeutic agent.

Background/Objectives: Metabolic dysfunction-associated steatotic liver disease (MASLD) is a growing global health problem and the discovery of drugs is challenging. In this study, we aimed to investigate the effects of intestinal activation of the liver X receptor (LXR)α on MASH. Methods: An intestinal-specific LXRα activation model in mice was established and subjected to MASH development by combining a Western diet and carbon tetrachloride. Lipid metabolism, reverse cholesterol transport (RCT), steatosis, inflammation, and fibrosis were evaluated. In vitro models of steatosis and fibrosis were used to explore the role of scavenger receptor class B type 1 (SRB1). Results: We found that the intestinal activation of LXRα improved several MASLD features, including levels of triglycerides, RCT, steatosis, systemic and hepatic inflammatory profiles, and liver fibrosis. These effects were associated with increased high-density lipoprotein (HDL) levels and hepatic SRB1 expression. In vitro depletion of SRB1 hampered the beneficial effects of HDL on steatosis and fibrogenesis in liver cells by altering the activation of both peroxisome proliferator-activated receptors γ and small mothers against decapentaplegic homolog protein (SMAD)2/3 proteins. Conclusions: Our findings showed that the intestinal activation of LXRα and a parallel induction of hepatic SRB1 are protective against inflammation, steatosis, and advanced liver fibrosis in MASLD.

Metabolic dysfunction-associated steatotic liver disease (MASLD), previously known as non-alcoholic fatty liver disease (NAFLD), comprises a spectrum of liver disorders characterized by excessive accumulation of triglycerides in hepatocytes. There are multiple treatment strategies proposed for pediatric MASLD, with lifestyle modifications and a balanced dietary regimen – particularly the Mediterranean diet in combination with weight loss, when needed – currently representing the cornerstone of clinical management. Behavioral psychological interventions are gaining increasing importance particularly in addressing comorbid eating disorders and supporting long-term adherence to therapeutic regimens. In addition, in daily clinical practice, the use of food supplements with antioxidant and hepatocyte anti-inflammatory effects is indicated in more advanced stages of steatosis, with the aim of limiting disease progression toward high-grade fibrosis and/or cirrhosis. This review aims to summarize current behavioral and nutritional therapeutic approaches for the management of pediatric MASLD.

Background & Aims:Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common cause of chronic liver disease worldwide, paralleling the rising prevalence of obesity. We previously reported that the rs17618244 variant in the Klotho-beta (KLB) gene, which encodes the hepatic obligate co-receptor of fibroblast growth factor receptor 4 (FGFR4), reduces hepatic and circulating KLB levels, leading to more severe MASLD in both children and adults. The present study aimed to evaluate the impact of another KLB variant, the intronic rs12152703 G>T polymorphism, on histological liver damage in patients with MASLD. Methods:The rs12152703 variant was genotyped in 1,311 patients with biopsy-proven MASLD, including 261 children, and its association with the disease spectrum was assessed. We also investigated the relationship between this variant and hepatic and circulating KLB expression. Finally, we evaluated in an in vitro model whether KLB overexpression in HepG2 cells affects lipid homeostasis and inflammation. Results:In multivariate analyses, the KLB rs12152703 variant was associated with lower serum aminotransferase levels and protection against steatosis, lobular inflammation, and steatohepatitis in the overall cohort (p <0.05). Hepatic and circulating KLB levels were increased in both adult and pediatric patients with MASLD carrying the variant (p <0.05). In vitro, KLB overexpression reduced intracellular lipid accumulation in free fatty acid-loaded HepG2 cells by modulating the expression of genes involved in lipid metabolism. Moreover, KLB induction counteracted lipopolysaccharide-induced activation of inflammatory genes and NF-κB (p65) phosphorylation. Conclusions:The KLB rs12152703 variant confers protection against lobular inflammation and is associated with increased hepatic and circulating KLB levels, in contrast to the at-risk rs17618244 variant. Consistently, KLB overexpression ameliorated steatosis and the pro-inflammatory state in lipid-loaded hepatocytes. These findings suggest that KLB may represent a novel druggable target for the treatment of severe MASLD. Impact and implications:This study highlights the protective effect of a genetic variant in the Klotho-beta (KLB) gene in attenuating hepatic inflammation and disease severity in both adults and children with metabolic dysfunction-associated steatotic liver disease. The favorable clinical associations appear to be mediated by increased circulating and hepatic KLB protein levels. Consistently, KLB overexpression alleviates lipid overload in hepatocytes by modulating the expression of genes involved in lipid metabolism. Together, these findings support the FGF19/KLB axis as a promising therapeutic target for the treatment of severe metabolic dysfunction-associated steatotic liver disease.

Background & aims: Mitochondrial (mt-) D-loop and cell-free circulating (ccf-) mtDNA fragments, respectively reflecting mt-mass and tissue damage, are promising metabolic dysfunction-associated steatotic liver disease (MASLD) biomarkers. We previously found that PNPLA3/MBOAT7/TM6SF2 deficiency in HepG2 cells increased mt-mass, D-loop levels, and ccf-COXIII release. We explored mt-biogenesis and mt-biomarkers in patients with MASLD stratified by the number of risk variants (NRV = 3). We exploited GPT-4 to develop and validate new risk scores, predicting MASLD evolution, in two independent cohorts by integrating anthropometric and genetic data with mt-biomarkers. Methods: A cohort of 28 patients with MASLD (Discovery cohort) was consecutively enrolled for hepatic mt-dynamics assessment by transmission electron microscopy and immunohistochemistry. Data were confirmed by quantitative real time-PCR in a retrospective cohort (Hepatic Validation, n = 184). D-loop and ccf-COXIII were retrospectively measured in peripheral blood mononuclear cells and serum samples of biopsied outpatients with MASLD (Serum Validation cohort, n = 824) and individuals with non-invasive MASLD diagnosis (n = 386, Non-invasive cohort). Risk scores were developed using random forest algorithms. Results: In the Discovery and Hepatic Validation cohorts, the PNPLA3/MBOAT7/TM6SF2 variants altered hepatic mt-dynamics, enhancing mt-content and D-loop levels (p <0.05) through the p38/PGC-1α pathway. Furthermore, NRV = 3 patients showed an increase in mt-fragmentation at transmission electron microscopy (TEM) and ccf-COXIII release (p <0.05). In the Serum Validation cohort, circulating D-loop and ccf-COXIII positively correlated with genetics [βD-loop:0.17 (95% CI: 0.04–0.29), p = 0.01; βccf-COXIII:0.33 (95% CI: 0.19–0.46), p <0.0001] and MASLD severity [ORD-loop:1.31 (95% CI: 1.01-1.71), p = 0.03; ORccf-COXIII:2.41 (95% CI: 1.69-3.44), p <0.0001] at multivariate analysis. Random forest allowed prediction models named Mitochondrial, Anthropometric, and Genetic Integration with Computational intelligence for assessing hepatocellular carcinoma risk (MAGIC-H), considering age, BMI genetics, D-loop, and ccf-COXIII. In both Serum and Non-invasive cohorts, the MAGIC-H score reached AUC >85% in identifying HCC cases regardless of cirrhosis, outperforming existing non-invasive tests. Conclusions: Mt-biomarkers have a prognostic significance in genetically-predisposed patients with MASLD. Impact and implications: The study highlights that genetic variants in PNPLA3, MBOAT7, and TM6SF2 genes deeply contribute to metabolic dysfunction-associated steatotic liver disease (MASLD) progression by affecting hepatic mitochondrial adaptability. It also identified two novel biomarkers of mitochondrial origin which are strongly linked to disease severity and genetic background of patients with MASLD. The use of generative artificial intelligence tools, such as GPT-4, can enhance the use of biomarkers and polygenic risk scores for clinical risk stratification. We developed a customized version of GPT-4 (rsGPT-4), which identified a machine-learning approach (random forest) as the best method for creating prediction models for metabolic dysfunction-associated steatohepatitis, fibrosis, and hepatocellular carcinoma. The new scores combined the two mitochondrial biomarkers, genetic data, and anthropometric data and outperformed existing non-invasive tests for monitoring patients with MASLD.