Diabetes-associated Alterations in the Gut Microbiota-metabolite Profile of NAFLD Under Complex Clinical Conditions.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) frequently coexists with obesity and type 2 diabetes (T2D), and the presence of these metabolic conditions contributes to substantial heterogeneity in disease progression and metabolic dysfunction. Gut microbiota and their derived metabolites are increasingly recognized as important mediators linking NAFLD with systemic metabolic disorders; however, how overweight and T2D differentially shape gut microbiota-metabolite alterations in NAFLD under complex clinical conditions remains unclear. METHODS: In this study, 66 patients with NAFLD (including 27 with T2D) and 26 healthy controls were enrolled. All participants underwent liver ultrasound-based assessment, biochemical testing, and fecal sample collection on the same day. Gut microbiota composition was analyzed using 16S ribosomal RNA gene sequencing, and fecal metabolites, including short-chain fatty acids and bile acids, were quantified using light chromatography-tandem mass spectrometry. Participants were stratified according to overweight and diabetes status to compare microbiota and metabolite profiles across metabolic subgroups. RESULTS: Distinct alterations in gut microbiota and metabolites were observed among NAFLD patients with different metabolic conditions. Patients with T2D showed a butyrate-related pattern in exploratory analysis, including nominally lower butyrate and isobutyrate levels and lower abundance of several butyrate-producing genera, particularly Lachnospira, Roseburia, and Anaerostipes, compared with non-diabetic NAFLD patients. Faecalibacterium was lower in NAFLD patients than in healthy controls but did not show a clear diabetes-specific decrease. Bile acid-related metabolites and associated bacterial taxa also showed exploratory patterns in diabetic NAFLD patients. Overall, diabetes was associated with more evident gut microbiota differences and exploratory metabolite patterns than overweight alone within the NAFLD population. CONCLUSION: Under complex clinical conditions, overweight and T2D were associated with different gut microbiota profiles and exploratory metabolite patterns in patients with NAFLD, with diabetes showing a stronger association than overweight alone. These exploratory findings highlight the importance of considering metabolic comorbidities when interpreting gut microbiota-metabolite changes in imaging-defined NAFLD and provide preliminary insight into microbial-metabolic patterns underlying disease heterogeneity.