Alanine Modulates Lactyl-CoA Abundance in HepG2 Cells.

The acyl-coenzyme A (CoA) thioester lactyl-CoA has been described as an acyl-donor for histone post-translational lactylation. Understanding the biology and functional significance of lactylation is hindered by a lack of knowledge of contexts that modulate lactyl-CoA abundance and direct evidence of potential substrates that generate lactyl-CoA. Using liquid chromatography- high resolution mass spectrometry and isotope tracing, we found that alanine provides carbons for the acyl-group of lactyl-CoA. Alanine supplementation increased lactyl-CoA in a dose responsive manner. However, to our surprise, this effect was dependent on glucose availability as treatment with exogenous alanine decreased cellular lactyl-CoA abundance after glucose withdrawal. This alanine induced decrease was rescued by cyclosporine, an inhibitor of alanine to pyruvate conversion by alanine transaminase, but not by an inhibitor of mitochondrial pyruvate import. Together, these findings provide a rationale to examine the function of lactyl-CoA in settings where alanine, lactate, and pyruvate metabolism converge such as gluconeogenesis.