Protein kinase C in beta-cells: expression of multiple isoforms and involvement in cholinergic stimulation of insulin secretion.
The mammalian protein kinase C (PKC) family consists of at least 11 distinct isotypes with marked differences in tissue distribution, localization, cofactor dependence and substrate specificity. Evidence exists for the expression of some of the PKC isoforms in pancreatic beta-cells but no comprehensive analysis of all the known PKC types has been accomplished. To assess the functional relevance of phosphorylation by PKC in the mechanism of insulin secretion we firstly investigated the expression of PKC isoforms in pancreatic beta-cells. The combination of reverse transcription-polymerase chain reaction (RT-PCR), Northern analysis and immunoblotting demonstrated the expression of PKC-alpha, beta II, epsilon, zeta, lambda and mu in MIN6 beta-cells. PKC-mu has not previously been detected in beta-cells. Expression of PKC-delta was also observed at the mRNA level; however, the protein could not be detected by Western blotting in MIN6 cells but was readily observed in RINm5F beta-cells. In short-term incubations, insulin release from MIN6 cells was augmented by 12-0-tetradecanoyl-phorbol-13-acetate (TPA), by carbachol, and by 40 mM K+. Culture of MIN6 cells overnight with TPA resulted in down-regulation of PKC-alpha (totally) and epsilon (partially), without significant change in the other isoforms. In such TPA-treated cells, the secretory response to TPA and to carbachol was abolished but not that elicited by high K+. It is suggested that PKC-alpha and/or epsilon may play a role in cholinergic potentiation of insulin secretion.
Tian
Y M
YM
Nuffield Department of Clinical Biochemistry, John Radcliffe Hospital, Headington, Oxford, UK.
Urquidi
V
V
Ashcroft
S J
SJ
D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D018679:Cholinergic Agonists / Q000494:pharmacology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D007527:Isoenzymes / Q000032:analysis* / Q000235:genetics; D051379:Mice; D011188:Potassium / Q000494:pharmacology; D011493:Protein Kinase C / Q000032:analysis* / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D018679:Cholinergic Agonists; D007328:Insulin; D007527:Isoenzymes; D012333:RNA, Messenger; D002217:Carbachol; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D011188:Potassium