Regulation of pyruvate dehydrogenase activity by altered expression of its kinase and phosphatase

In this study, factors that regulate PDK2 and PDK4 expression were investigated in Morris hepatoma 7800 C1 cells. The results indicate that the mRNA level of PDK4 is markedly increased by the glucocorticoid receptor ligand dexamethasone and the peroxisome proliferator-activated receptor alpha (PPARalpha) ligand WY-14,643. Neither compound affects the half-life of the PDK4 message, suggesting both of them increase PDK4 gene transcription. Fatty acids cause an increase in PDK4 message comparable to that induced by WY-14,643. Insulin prevents and effectively reverses the stimulatory effects of dexamethasone on PDK4 gene expression but is much less effective against the stimulatory effects of WY-14,643 and fatty acids. Insulin also effectively suppresses the mRNA level of PDK2. Changes in PDK4 and PDK2 message levels by these factors correlate with changes in PDK4 and PDK2 protein levels. These findings suggest that decreased level of insulin and increased levels of fatty acids and glucocorticoids promote PDK4 gene expression in starvation and diabetes. Decreased levels of insulin may also be responsible for the increased level of PDK2 in starvation and diabetes.;Further studies indicate that the decreases in PDK4 and PDK2 mRNA level caused by insulin are completely blocked by the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin. The effect of insulin on PDK2 mRNA can also be partially blocked by Akt inhibitor, MEK inhibitor PD98059, and the immunosuppressant rapamycin. The effect of insulin on PDK4 mRNA is insensitive to rapamycin but can be blocked by PD98059, Akt inhibitor, and ceramide. In addition, transfection of HepG2 cells with constitutively active protein kinase B (PKB/Akt) causes a greater decrease in PDK4 promoter activity (measured with a PDK4 promote-luciferase reporter construct) than treatment of cells with insulin. Therefore, the repression of PDK4 gene expression by insulin may involve activation of Pl3K, PKB/Akt, MEK, and transcription factors that remain to be identified. Insulin repression of PDK2 may occur through PI3K, PKB/Akt, MEK, and p70s6k pathway.;Whether altered expression of PDP isoenzymes also contributes to changes in the phosphorylation state of PDC during starvation and diabetes was also investigated. The results indicate that starvation causes significant decreases in PDP2 mRNA and protein in rat heart and kidney as well as PDP1 protein in rat kidney. Re-feeding effectively reverses the effect of starvation. Likewise, streptozotocin-induced diabetes decreases PDP2 mRNA and protein in rat heart and kidney. Insulin treatment completely reverses the effect of diabetes. In addition, PDP activity assay revealed that starvation and diabetes decrease PDP activity in these tissues, whereas re-feeding of starved rats and insulin treatment of diabetic rats restore PDP activity.;The findings of this work provide new insight with respect to regulation of the activity of the pyruvate dehydrogenase complex. Inactivation of this complex is critically important for conservation of gluconeogenic substrates during starvation and contributes to hyperglycemia during diabetes. My studies indicate that concurrent down regulation of PDP expression and up regulation of PDK expression promote hyperphosphorylation and therefore inactivation of the pyruvate dehydrogenase complex in starvation and diabetes. (Abstract shortened by UMI.).