H₂S-induced S-sulfhydration of pyruvate carboxylase contributes to gluconeogenesis in liver cells

Background Cystathionine gamma-lyase (CSE)-derived hydrogen sulfide (H₂S) possesses diverse roles in the liver, affecting lipoprotein synthesis, insulin sensitivity, and mitochondrial biogenesis. H₂S S-sulfhydration is now proposed as a major mechanism for H₂S-mediated signaling. Pyruvate carboxylase (PC) is an important enzyme for gluconeogenesis. S-sulfhydration regulation of PC by H₂S and its implication in gluconeogenesis in the liver have been unknown. Methods Gene expressions were analyzed by real-time PCR and western blotting, and protein S-sulfhydration was assessed by both modified biotin switch assay and tag switch assay. Glucose production and PC activity was measured with coupled enzyme assays, respectively. Results Exogenously applied H₂S stimulates PC activity and gluconeogenesis in both HepG2 cells and mouse primary liver cells. CSE overexpression enhanced but CSE knockout reduced PC activity and gluconeogenesis in liver cells, and blockage of PC activity abolished H₂S-induced gluconeogenesis. H₂S had no effect on the expressions of PC mRNA and protein, while H₂S S-sulfhydrated PC in a dithiothreitol-sensitive way. PC S-sulfhydration was significantly strengthened by CSE overexpression but attenuated by CSE knockout, suggesting that H₂S enhances glucose production through S-sulfhydrating PC. Mutation of cysteine 265 in human PC diminished H₂S-induced PC S-sulfhydration and activity. In addition, high-fat diet feeding of mice decreased both CSE expression and PC S-sulfhydration in the liver, while glucose deprivation of HepG2 cells stimulated CSE expression. Conclusions CSE/H₂S pathway plays an important role in the regulation of glucose production through S-sulfhydrating PC in the liver. General significance Tissue-specific regulation of CSE/H₂S pathway might be a promising therapeutic target of diabetes and other metabolic syndromes.