Evidence for a role of protein kinase C in the activation of the pyruvate dehydrogenase complex by insulin in Zajdela hepatoma cells.
The signal transduction pathway involved in the activation of pyruvate dehydrogenase (PDH) by insulin is still unknown. In this study, we have examined the possible involvement of protein kinase C (PKC) in the process. In addressing this question, we examined (1) the insulin-like effects of the PKC activator 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) on the PDH complex, (2) the effects of various PKC inhibitors on the PDH activation by insulin, and (3) the response of PKC-depleted cells to insulin. We used as an experimental model Zajdela hepatoma cultured (ZHC) cells, which have been demonstrated to be responsive to physiological doses of insulin. Half-maximal and maximal stimulations of the PDH complex by insulin were observed at 0.05 and 5 nmol/L, respectively. Stimulation of PDH activity by insulin (5 nmol/L) occurred within 5 minutes of incubation and was maximal (+70%) at 7.5 minutes. In the presence of PMA (162 nmol/L), enzyme activity increased within 30 seconds, was maximal (+90%) at 5 minutes, and was no longer detectable after 10 minutes. Total PDH activity was unchanged by insulin or PMA treatment. The effects of PMA and insulin on basal PDH activity were not additive. Moreover, various inhibitors of PKC--staurosporine, sphingosine, acridine orange--completely blocked the stimulation of PDH activity induced by insulin or PMA. A 17-hour treatment of ZHC cells with 500 nmol/L PMA efficiently downregulated PKC, as attested by the marked decrease in the enzyme activity and the loss of phorbol 12,13-dibutyrate binding to intact cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Benelli
C
C
INSERM U30, Hôpital des Enfants-Malades, Paris.
Caron
M
M
de Gallé
B
B
Fouque
F
F
Cherqui
C
C
Clot
J P
JP
D000704:Analysis of Variance; D000818:Animals; D015536:Down-Regulation; D004789:Enzyme Activation / Q000187:drug effects; D007328:Insulin / Q000494:pharmacology*; D008114:Liver Neoplasms, Experimental / Q000201:enzymology*; D011493:Protein Kinase C / Q000502:physiology*; D011768:Pyruvate Dehydrogenase Complex / Q000187:drug effects* / Q000378:metabolism; D051381:Rats; D014407:Tumor Cells, Cultured
D007328:Insulin; D011768:Pyruvate Dehydrogenase Complex; D011493:Protein Kinase C