Effects of protein kinase C activation on the regulation of the stimulus-secretion coupling in pancreatic β-cells

Effects of protein kinase C (PKC) activation on the insulin-secretory process were investigated, by using β-cell-rich suspensions obtained from pancreatic islets of obese-hyperglycaemic mice. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), which is known to activate PKC directly, the muscarinic-receptor agonist carbamoylcholine and high glucose concentration enhance the phosphorylation of a specific 80 kDa PKC substrate in the β-cells. At a non-stimulatory glucose concentration, 10 nM-TPA increased insulin release, although there were no changes in either the cytoplasmic free Ca²⁺ concentration ([Ca²⁺](i)) or membrane potential, as measured with the fluorescent indicators quin-2 and bisoxonol respectively. At a stimulatory glucose concentration TPA caused a lowering in [Ca²⁺](i), whereas membrane potential was unaffected. Despite the decrease in [Ca²⁺](i), there was a large stimulation of insulin release. Addition of TPA lowered [Ca²⁺](i) also in β-cells stimulated by tolbutamide or high K⁺, although to a lesser extent than in those stimulated by glucose. There was no effect of TPA on either Ca²⁺ buffering or the ability of Ins(1,4,5)P₃ to release Ca²⁺ in permeabilized β-cells. However, the phorbol ester inhibited the rise in [Ca²⁺](i) in response to carbamoylcholine, which stimulates the formation of InsP₃, in intact β-cells. Down-regulation of PKC influenced neither glucose-induced insulin release nor the increase in [Ca²⁺](i). Hence, although PKC activation is of no major importance in glucose-stimulated insulin release, this enzyme can serve as a modulator of the glucose-induced insulin-secretory response. Such a modulation involves mechanisms promoting both amplification of the secretory response and lowering of [Ca²⁺](i).