A hierarchical hepatic de novo lipogenesis substrate supply network utilizing pyruvate, acetate, and ketones.

Hepatic de novo lipogenesis (DNL) is a fundamental physiologic process that is often pathogenically elevated in metabolic disease. Treatment is limited by incomplete understanding of the metabolic pathways supplying cytosolic acetyl-CoA, the obligate precursor to DNL, including their interactions and proportional contributions. Here, we combined extensive 13C tracing with liver-specific knockout of key mitochondrial and cytosolic proteins mediating cytosolic acetyl-CoA production. We show that the mitochondrial pyruvate carrier (MPC) and ATP-citrate lyase (ACLY) gate the major hepatic lipogenic acetyl-CoA production pathway, operating in parallel with acetyl-CoA synthetase 2 (ACSS2). Given persistent DNL after mitochondrial citrate carrier (CiC) and ACSS2 double knockout, we tested the contribution of exogenous and leucine-derived acetoacetate to acetoacetyl-CoA synthetase (AACS)-dependent DNL. CiC knockout increased acetoacetate-supplied hepatic acetyl-CoA production and DNL, indicating that ketones function as mitochondrial-citrate reciprocal DNL precursors. By delineating a mitochondrial-cytosolic DNL substrate supply network, these findings may inform strategies to therapeutically modulate DNL.