PROJECT SUMMARY Non-alcoholic fatty liver disease (NAFLD) affects >35% of older adults in the US. It is now the primary etiology of chronic liver disease and liver cancer, and the driver of the recent upward trend in these lethal diseases. A dysbiotic gut microbiome has been associated with NAFLD, but mostly in small-scale, cross-sectional or clinic- based studies. Population-based longitudinal studies are needed to provide evidence for the temporal relationship of the gut microbiome with the progression of NAFLD. In the cross-sectional Multiethnic Cohort (MEC) Adiposity Phenotype Study (APS; P01 CA168530), we showed a significant difference in gut microbial composition and inferred microbial function by NAFLD status, including enrichment of Fusobacterium and endotoxin-producing bacteria and altered microbial pathways for bile acid and simple carbohydrate metabolism in NAFLD. We now hypothesize that specific gut bacterial features (genera, metabolic pathways and blood endotoxin biomarker lipopolysaccharide binding protein (LBP)) are associated with increase in liver fat and liver fibrosis over time. We also hypothesize that several dietary factors are associated with NAFLD progression and that fibrosis-promoting gut bacterial features mediate these associations. We propose a longitudinal investigation by efficiently adding a ~10-year follow-up assessment among 300 of the MEC-APS participants, aged 60-77 years at baseline and of three racial/ethnic groups (Japanese American, Native Hawaiian or White), across a wide range of baseline liver fat. We will re-assess liver fat using MRI and measure liver stiffness using MR elastography, gold-standard methods for non-invasive quantification of liver fat and liver stiffness, respectively, and perform 16S rRNA gene sequencing (follow-up stool samples) and metagenomic sequencing (baseline and follow-up stool samples). Specific Aims are to: 1) evaluate the change in specific gut bacterial features (abundance of genera, metabolic pathways, and LBP) over time in relation to the change in liver fat; 2) evaluate the change in gut bacterial features over time in relation to (2a) liver stiffness at follow-up and (2b) change in a blood biomarker panel for liver fibrosis (Enhanced Liver Fibrosis (ELF) score), and (2c) validate the top two bacterial features associated with liver stiffness using ddPCR; and 3) assess the association of diet (3 dietary pattern scores for overall diet quality and 7 key components) with ELF-based change in liver fibrosis and explore the mediation by fibrosis-promoting gut bacterial features from Aim 2, stratified by liver fat level. Our results will identify gut microbial features associated with early NAFLD progression, while specifically addressing the needs of understudied Asian Americans and Native Hawaiians (NOT-HL-23-001), two high-risk populations for NAFLD and liver cancer. The strengths of the proposed longitudinal design and rigorous imaging and laboratory methods will aid in u...