2483-P: 2H-palmitate Kinetics Uncover Active and Inert Triglyceride Pools in Metabolic Dysfunction–Associated Steatotic Liver Disease and Pyruvate Carboxylase Control of Hepatic De Novo Lipogenesis

Introduction and Objective: About 70% of individuals with Type 2 Diabetes have metabolic dysfunction-associated steatotic liver disease (MASLD), reflecting dysregulated glucose and lipid metabolism. Stable isotope tracer studies have linked MASLD to elevated de novo lipogenesis (DNL) and increased TCA cycle flux. Pyruvate carboxylase (PC) is positioned to facilitate both pathways by preventing loss of TCA cycle intermediates to biosynthesis, including provision of mitochondrial citrate exported for DNL. Together, TCA cycle metabolism and DNL contribute to hepatic triglyceride (TG) turnover, yet their significance is unclear since these fluxes are challenging to quantify in vivo. Methods: We combined high resolution Orbitrap mass spectrometry with 2H2O tracing, temporal 2H-palmitate enrichment, and kinetic modeling to estimate hepatic fluxes through DNL, endogenous fatty acid (FA) re-esterification, and TG turnover to uncover PC-mediated hepatic lipid dynamics. Results: Mouse livers after a 6-month MASLD-inducing Western Diet (WD) increased hepatic TG turnover, DNL and FA re-esterification into TGs. Metabolic modeling indicated that chronic WD feeding also resulted in the formation of an inert hepatic lipid pool. Liver specific PC knockout (LPCKO) markedly reduced DNL and TG turnover without altering endogenous FA flux or total hepatic palmitate. LPCKO mice retained an inert hepatic lipid pool on WD, but the total TG content in this pool was reduced compared to controls. LPCKO livers exhibited lower levels of metabolites required for DNL, specifically malonyl-CoA, NADPH and TCA cycle intermediates, redirecting acetyl-CoA towards ketogenesis. Conclusion: These data demonstrate that WD-mediated MASLD in mice is marked by two pools of TG, one that turns over rapidly and one that is remarkably inert. Liver PC loss attenuates WD-induced MASLD by lowering hepatic DNL flux, consistent with PC’s role in coupling acetyl-CoA to the citrate shuttle for DNL. Disclosure M.R. Inigo-Vollmer: None. M. Mcleod: None. B. Kucejova: None. J. Fletcher: None. S. Deja: None. X. Fu: None. S.C. Burgess: None. Funding National Institutes of Health (R01DK121497, R01DK078184, P30DK127984)