One of the focuses of non-alcoholic fatty liver disease (NAFLD) treatment is exercise. Randomized controlled trials investigating the effects of exercise without dietary changes on NAFLD-related clinical parameters (liver parameters, lipid metabolism, glucose metabolism, gut microbiota, and metabolites) were screened using the PubMed, Scopus, Web of Science, and Cochrane databases on 13 February 2020. Meta-analyses were performed on 10 studies with 316 individuals who had NAFLD across three exercise regimens: aerobic exercise, resistance training, and a combination of both. No studies investigating the role of gut microbiota and exercise in NAFLD were found. A quality assessment via the (RoB)2 tool was conducted and potential publication bias, statistical outliers, and influential cases were identified. Overall, exercise without significant weight loss significantly reduced the intrahepatic lipid (IHL) content (SMD: −0.76, 95% CI: −1.04, −0.48) and concentrations of alanine aminotransaminase (ALT) (SMD: −0.52, 95% CI: −0.90, −0.14), aspartate aminotransaminase (AST) (SMD: −0.68, 95% CI: −1.21, −0.15), low-density lipoprotein cholesterol (SMD: −0.34, 95% CI: −0.66, −0.02), and triglycerides (TG) (SMD: −0.59, 95% CI: −1.16, −0.02). The concentrations of high-density lipoprotein cholesterol, total cholesterol (TC), fasting glucose, fasting insulin, and glycated hemoglobin were non-significantly altered. Aerobic exercise alone significantly reduced IHL, ALT, and AST; resistance training alone significantly reduced TC and TG; a combination of both exercise types significantly reduced IHL. To conclude, exercise overall likely had a beneficial effect on alleviating NAFLD without significant weight loss. The study was registered at PROSPERO: CRD42020221168 and funded by the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement no. 813781.

Metabolic dysfunction-associated steatotic liver disease (MASLD) can lead to liver fibrosis, cirrhosis, and hepatocellular carcinoma. Still, most patients with MASLD die from cardiovascular diseases indicating metabolic alterations related to both liver and cardiovascular pathology. The aim of this study was to assess biologic pathways behind MASLD progression from steatosis to metabolic dysfunction-associated steatohepatitis (MASH) using non-targeted liquid chromatography-mass spectrometry analysis in 106 severely obese individuals (78 women, mean age 47.7 7 ± 9.2 years, body mass index 41.8 ± 4.3 kg/m²) undergoing laparoscopic Roux-en-Y gastric bypass. We identified several metabolites that are associated with MASLD progression. Most importantly, we observed a decrease of lysophosphatidylcholines LPC(18:2), LPC(18:3), and LPC(20:3) and increase of xanthine when comparing those with steatosis to those with MASH. We found that indole propionic acid and threonine were negatively correlated to fibrosis, but not with the metabolic disturbances associated with cardiovascular risk. Xanthine, ketoleucine, and tryptophan were positively correlated to lobular inflammation and ballooning but also with insulin resistance, and dyslipidemia, respectively. The results did not change when taking into account the most important genetic risk factors of MASLD. Our findings suggest that there are several separate biological pathways, some of them independent of insulin resistance and dyslipidemia, associating with MASLD.

Lean metabolic dysfunction-associated steatotic liver disease (lean MASLD) challenges longstanding views that link hepatic steatosis primarily to obesity. Emerging as a distinct and under-recognized clinical entity, lean MASLD affects individuals with a normal body mass index (BMI), yet carries risks of cardiovascular disease, hepatocellular carcinoma, and liver-related mortality comparable to obesity-associated MASLD. The absence of overt metabolic dysfunction complicates diagnosis, revealing critical limitations in current screening frameworks centered on BMI. This review synthesizes evolving clinical insights and epidemiological trends in lean MASLD, and delineates its unique pathophysiological mechanisms. Recent advances in metabolomics have uncovered disease-specific disruptions in lipid and amino acid metabolism, bile acid signaling, and gut microbiota-derived metabolites. By integrating evidence from metabolic, genetic, and epigenetic domains, we identified promising biomarkers, and therapeutic targets that may support earlier detection and precision-guided treatment strategies.

Sterols, bile acids, and acylcarnitines are key players in human metabolism. Precise annotations of these metabolites with mass spectrometry analytics are challenging because of the presence of several isomers and stereoisomers, variability in ionization, and their relatively low concentrations in biological samples. Herein, we present a sensitive and simple qualitative LC–MS/MS (liquid chromatography with tandem mass spectrometry) method by utilizing a set of pure chemical standards to facilitate the identification and distribution of sterols, bile acids, and acylcarnitines in biological samples including human stool and plasma; mouse ileum, cecum, jejunum content, duodenum content, and liver; and pig bile, proximal colon, cecum, heart, stool, and liver. With this method, we detected 24 sterol, 32 bile acid, and 27 acylcarnitine standards in one analysis that were separated within 13 min by reversed-phase chromatography. Further, we observed different sterol, bile acid, and acylcarnitine profiles for the different biological samples across the different species. The simultaneous detection and annotation of sterols, bile acids, and acylcarnitines from reference standards and biological samples with high precision represents a valuable tool for screening these metabolites in routine scientific research.

Non-alcoholic fatty liver disease (NAFLD) pathogenesis remains poorly understood due to the complex metabolic and inflammatory changes in the liver. This study aimed to elucidate hepatic events related to inflammation and lipid metabolism and their linkage with metabolic alterations during NAFLD in American lifestyle-induced obesity syndrome (ALIOS) diet-fed mice. Forty-eight C57BL/6J male mice were fed with ALIOS diet (n=24) or control chow diet (n=24) for 8, 12, and 16 weeks. At the end of each timepoint, eight mice were sacrificed where plasma and liver were collected. Hepatic fat accumulation was followed using magnetic resonance imaging and confirmed with histology. Further, targeted gene expression and non-targeted metabolomics analysis were conducted. Our results showed higher hepatic steatosis, body weight, energy consumption, and liver mass in ALIOS diet-fed mice compared to control mice. ALIOS diet altered expression of genes related to inflammation (Tnfa and IL-6) and lipid metabolism (Cd36, Fasn, Scd1, Cpt1a, and Ppara). Metabolomics analysis indicated decrease of lipids containing polyunsaturated fatty acids such as LPE(20:5) and LPC(20:5) with increase of other lipid species such as LPI(16:0) and LPC(16:2) and peptides such as alanyl-phenylalanine and glutamyl-arginine. We further observed novel correlations between different metabolites including sphingolipid, lysophospholipids, peptides, and bile acid with inflammation, lipid uptake and synthesis. Together with the reduction of antioxidant metabolites and gut microbiota-derived metabolites contribute to NAFLD development and progression. The combination of non-targeted metabolomics with gene expression in future studies can further identify key metabolic routes during NAFLD which could be the targets of potential novel therapeutics.

Exercise is a key component of lifestyle management in patients with metabolic dysfunction-associated steatotic liver disease (MASLD), but neither its therapeutic effect on the active stage of the disease, that is metabolic dysfunction-associated steatohepatitis (MASH) nor the mediating mechanisms have been characterized. Therefore, we performed multi-omic phenotyping of patients with MASLD-MASH on an exercise program. Fifteen patients with MASLD conducted high-intensity interval training (HIIT) combined with home-based training for 12 weeks. MASLD was evaluated using histology, transient elastography, and multiparametric magnetic resonance imaging (MRI) before and after the intervention. Change in maximal oxygen consumption (VO_2max) and MRI-determined liver fat were compared with a control group of patients with MASLD (n = 22). RNA sequencing was performed on liver, muscle, and fat biopsies of patients in the exercise group. Stool was analyzed by shotgun metagenomics and untargeted metabolomics was performed on plasma, urine, adipose, and stool. HIIT increased VO_2max by 10.1% and improved mitochondrial metabolism in skeletal muscle, indicating improved cardiorespiratory fitness and adherence. VO_2max increased significantly in the exercise group compared with controls. Histologically, no reduction in steatosis, MASH, or liver fibrosis was observed; however, transient elastography tended to improve. MRI-determined liver fat did not change in the exercise group compared with controls. HIIT induced changes in mRNA expression of genes related to beiging of adipose tissue and fibrogenesis in liver. In addition, specific gut microbial taxa and metabolites changed. HIIT increased cardiorespiratory fitness and induced beneficial gene expression changes in muscle, adipose tissue, and liver, but without translation into histological improvement of MASLD. Longer exercise intervention trials are warranted to validate or refute current recommendations for exercise as a cornerstone treatment for MASLD-MASH. Despite exercise being considered as a key component of lifestyle management for steatotic liver disease, neither the clinical effects nor the mechanisms involved are completely understood. We show that a high-intensity interval training (HIIT) program in 15 patients with metabolic dysfunction-associated steatotic liver disease (MASLD) improved cardiorespiratory fitness, compared with 22 control patients with MASLD who did not participate in an exercise program, however, it did not improve MASLD. HIIT induced a positive effect on fat tissue and muscle metabolism which was accompanied with changes in certain gut bacteria and metabolites in blood and urine. These findings improve our understanding of the effects of exercise on the whole-body metabolism in relation to steatotic liver disease. As such, this study provides a basis for future exercise interventions in patients with MASLD, required to thoroughly test current guideline advice for exercise as a cornerstone treatment for MASLD of all stages. Dutch Trial Register (registration number NL7932).