Protein kinase C modulates the insulin secretory process by maintaining a proper function of the beta-cell voltage-activated Ca2+ channels.
In the present study an attempt was made to further elucidate the molecular mechanisms whereby protein kinase C (PKC) modulates the beta-cell stimulus-secretion coupling. Regulation of Ca2+ channel activity, [Ca2+]i, and insulin release were investigated in both normal pancreatic mouse beta-cells and in similar beta-cells deprived of PKC activity. [Ca2+]i was measured with the intracellular fluorescent Ca2+ indicator fura-2 and the Ca2+ channel activity was estimated by the whole cell configuration of the patch-clamp technique. To reveal the various isoenzymes of PKC present in the mouse beta-cell, proteins were separated by one-dimensional gel electrophoresis and Western blotting was performed. The production of inositol phosphates was measured by ion-exchange chromatography and insulin release was measured radioimmunologically. Acute stimulation with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate resulted in suppression of both the carbamylcholine-induced increase in [Ca2+]i and production of inositol 1,4,5-trisphosphate. Under these conditions the increase in [Ca2+]i in response to glucose was similar to that found in control cells. When beta-cells were deprived of PKC, by exposure to 200 nM 12-O-tetradecanoylphorbol-13-acetate for 24-48 h, there was an enhanced response to carbamylcholine. This response constituted increases in both the [Ca2+]i signal and production of inositol 1,4,5-trisphosphate. Interestingly, cells with down-regulated PKC activity responded more slowly to glucose stimulation, when comparing the initial increase in [Ca2+]i, than control cells. On the other hand, the maximal increase in [Ca2+]i was similar whether or not PKC was present. Moreover, PKC down-regulated cells exhibited a significant reduction of maximal whole cell Ca2+ currents, a finding that may explain the altered kinetics with regard to the [Ca2+]i increase in response to the sugar. Both the alpha and beta 1 forms of the PKC isoenzymes were present in the mouse beta-cell and were also subjected to PKC down-regulation. Hence, either of these isoenzymes or both may be involved in the modulation of phospholipase C and Ca2+ channel activity. Since insulin release under physiological conditions is critically dependent on Ca(2+)-influx through the voltage-gated L-type Ca2+ channels, the kinetics of hormone release was expected to demonstrate a similar delay as that of the [Ca2+]i increase. Although not as pronounced, such a delay was indeed also observed in the onset of insulin release. There was, however, no effect on the total amounts of hormone released.(ABSTRACT TRUNCATED AT 400 WORDS)
Arkhammar
P
P
Department of Endocrinology, Karolinska Institute, Rolf Luft Center for Diabetes Research, Karolinska Hospital, Stockholm, Sweden.
Juntti-Berggren
L
L
Larsson
O
O
Welsh
M
M
Nånberg
E
E
Sjöholm
A
A
Köhler
M
M
Berggren
P O
PO
D000818:Animals; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology*; D002217:Carbachol / Q000494:pharmacology; D002478:Cells, Cultured; D016257:Fura-2; D005711:Gallopamil / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology; D006706:Homeostasis; D015151:Immunoblotting; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D008820:Mice, Obese; D011493:Protein Kinase C / Q000302:isolation & purification / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D015220:Calcium Channels; D007328:Insulin; D007527:Isoenzymes; D005711:Gallopamil; D015544:Inositol 1,4,5-Trisphosphate; D002217:Carbachol; D011493:Protein Kinase C; D005947:Glucose; D013755:Tetradecanoylphorbol Acetate; D016257:Fura-2