H2S-induced S-sulfhydration of pyruvate carboxylase contributes to gluconeogenesis in liver cells.
BACKGROUND
Cystathionine gamma-lyase (CSE)-derived hydrogen sulfide (H(2)S) possesses diverse roles in the liver, affecting lipoprotein synthesis, insulin sensitivity, and mitochondrial biogenesis. H(2)S S-sulfhydration is now proposed as a major mechanism for H(2)S-mediated signaling. Pyruvate carboxylase (PC) is an important enzyme for gluconeogenesis. S-sulfhydration regulation of PC by H(2)S 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 H(2)S 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 H(2)S-induced gluconeogenesis. H(2)S had no effect on the expressions of PC mRNA and protein, while H(2)S S-sulfhydrated PC in a dithiothreitol-sensitive way. PC S-sulfhydration was significantly strengthened by CSE overexpression but attenuated by CSE knockout, suggesting that H(2)S enhances glucose production through S-sulfhydrating PC. Mutation of cysteine 265 in human PC diminished H(2)S-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/H(2)S pathway plays an important role in the regulation of glucose production through S-sulfhydrating PC in the liver.


CONCLUSIONS
Tissue-specific regulation of CSE/H(2)S pathway might be a promising therapeutic target of diabetes and other metabolic syndromes.
Cardiovascular and Metabolic Research Unit, Lakehead University, Thunder Bay, ON P7B 5E1, Canada.
YoungJun
Y
Ju
Cardiovascular and Metabolic Research Unit, Lakehead University, Thunder Bay, ON P7B 5E1, Canada; Department of Health Science, Lakehead University, Thunder Bay, ON P7B 5E1, Canada.
Ashley
A
Untereiner
Cardiovascular and Metabolic Research Unit, Lakehead University, Thunder Bay, ON P7B 5E1, Canada; Department of Health Science, Lakehead University, Thunder Bay, ON P7B 5E1, Canada.
Lingyun
L
Wu
Cardiovascular and Metabolic Research Unit, Lakehead University, Thunder Bay, ON P7B 5E1, Canada; Department of Chemistry and Biochemistry, Laurentian University, Sudbury, ON P3E 2C6, Canada. Electronic address: gyang2@laurentian.ca.
Guangdong
G
Yang
D000818:Animals; D003545:Cysteine / Q000378:metabolism; D059305:Diet, High-Fat; D005943:Gluconeogenesis; D005947:Glucose / Q000096:biosynthesis; D057809:HEK293 Cells; D056945:Hep G2 Cells; D006801:Humans; D006862:Hydrogen Sulfide / Q000494:pharmacology*; D008099:Liver / Q000378:metabolism*; D008297:Male; D051379:Mice; D011766:Pyruvate Carboxylase / Q000378:metabolism*
D011766:Pyruvate Carboxylase; D005947:Glucose; D003545:Cysteine; D006862:Hydrogen Sulfide
CSE; Gluconeogenesis; H(2)S; PC; S-sulfhydration