ABSTRACT Liver plays a central role in S-adenosylmethionine (SAMe, the principal methyl donor) metabolism, as this is where 50% of methionine intake is catabolized to SAMe. In liver, SAMe biosynthesis is mediated by methionine adenosyltransferase 1A (MAT1A)-encoded enzyme. Patients with advanced non-alcoholic steatohepatitis (NASH) have reduced MAT1A expression. We developed the Mat1a knockout (KO) mouse model, which has chronically low hepatic SAMe level, spontaneous development of NASH and hepatocellular carcinoma (HCC). This grant, currently in its 16th year, has supported 77 original papers plus 27 reviews. During the past funding period we showed how dysregulation of SAMe leads to NASH, defined the metabolomic signatures as a function of hepatic SAMe content and found nearly 50% of non-alcoholic fatty liver disease (NAFLD) patients share the M-subtype metabolomic signature of the Mat1a KO mice. Using this KO model, we have uncovered two novel SAMe targets, aldolase B (ALDOB) hypomethylation and La Ribonucleoprotein Domain Family Member 1 (LARP1) hyperphosphorylation, that we hypothesize are linked to NASH progression during fructose consumption and NASH-HCC, respectively. This renewal application tests this novel hypothesis in two specific aims. Specific Aim 1 examines the role of ALDOB R173 methylation in NAFLD progression. Our data support the hypothesis that ALDOB exists in two forms: one that is unmethylated, enzymatically active and senses fructose 1,6-bisphosphate (FBP) level to regulating AMPK activity; and a second form that is methylated, enzymatically inactive and does not regulate AMPK activity. We further hypothesize that in Mat1a KO mice ALDOB hypomethylation (with higher activity and lower FBP) activates AMPK as an adaptive response to halt further fatty acid accumulation but this will be disrupted by fructose consumption, which will increase FBP content and accelerate NASH progression. We will define 1) how SAMe depletion affect glucose/fructose flux, 2) how ALDOB R173 is methylated and how it affects ALDOB structure, activity, and FBP sensing, 3) whether fructose consumption accelerates NASH progression through dampening AMPK activation, and 4) relevance to human NAFLD. Specific Aim 2 will examine the role of LARP1 in NASH-HCC progression. Our data show LARP1 protein level and phosphorylation at multiple sites (some are novel) are increased under SAMe depletion, leading to enhanced translation of 5’-terminal oligopyrimidine tract mRNAs, including proteins involved in translation. Overexpressing LARP1 enhanced HCC cell growth, migration and invasion. We will define 1) how SAMe regulates LARP1 expression, 2) which kinases are responsible, 3) how phosphorylation affects LARP1 function, 4) whether LARP1 is a driver of NASH-HCC in SAMe deficiency, and 5) relevance to human NAFLD. Successful completion of these proposed aims will provide important insights on how fructose consumption accelerates NASH progression and how LARP1 drive...