2486-P: Hepatic Knockdown of ANT2 Promotes Increased Hepatic Fat Oxidation by Upregulation of Uncoupling Protein 3

Introduction and Objective: Mitochondrial ADP/ATP carrier 2 (ANT2) has traditionally been assumed to support oxidative phosphorylation by exchanging cytosolic ADP for mitochondrial ATP, yet its role in hepatic energy metabolism remains unresolved. The objective of this study was to determine whether ANT2 suppression alters mitochondrial oxidative metabolism and to define the underlying mechanism. Methods: Here, we used in vivo fluxomics (Q-Flux) to quantify hepatic mitochondrial oxidative and gluconeogenic fluxes across four independent rodent models (liver-targeted ANT2 GalNAc ASO, liver-specific ANT2 KO, whole-body and liver-targeted ANT2 siRNA in rats or mice). Results: We identified ANT2 as a molecular modifier of hepatic oxidative metabolism. Across all models, hepatic ANT2 suppression markedly increased rates of hepatic mitochondrial oxidation (VIDH, VOGDH, and VSDH), anaplerosis (VGLS), and mitochondrial gluconeogenesis (VPEPCK, VPC) without altering whole-body energy expenditure or inducing ATP depletion. Transcriptomic analysis revealed an unexpected and robust induction of the normally muscle-enriched uncoupling protein 3 (UCP3) in ANT2-deficient livers. Liver-specific UCP3 knockdown abrogated the enhanced hepatic oxidative and gluconeogenic fluxes, establishing UCP3 as an essential mediator of the ANT2 knockdown metabolic phenotype. In contrast, chemical mitochondrial uncoupling by 2,4-dinitrophenol activated hepatic mitochondrial oxidation and gluconeogenesis fluxes independently of hepatic ANT2 expression, indicating that ANT2-UCP3 remodeling represents a mechanistically distinct pathway from classical protonophore-mediated mitochondrial uncoupling. Conclusion: These findings uncover an endogenous ANT2-UCP3 circuit that rewires mitochondrial metabolism to enhance fat oxidation thus highlighting a potential new strategy to treat MASLD and T2D. Disclosure J. Zheng: None. R. Calais Gaspar: None. H.N. Morgan: None. S. Parikh: None. M. Perelis: Employee; Current; Ionis Pharmaceuticals. V. Samuel: None. K. Petersen: Research Support; Current; Merck & Co., Inc. Advisory Panel; Current; Village S.S.D.r.l. G. Shulman: Consultant; Current; Novo Nordisk A/S. Research Support; Current; Novo Nordisk A/S. Other - collaboration; Current; Ionis Pharmaceuticals. Advisory Panel; Current; ESPERION Therapeutics, Inc. Research Support; Current; Novo Nordisk Foundation. Advisory Panel; Current; Orsobio. Research Support; Current; Orsobio. Advisory Panel; Current; Village S.S.D.