Contribution of protein kinase Cα in the stimulation of insulin by the down-regulation of Cavβ subunits

Abstract

Voltage-gated calcium (Cav) channels and protein kinase C (PKC) isozymes are involved in insulin secretion. In addition, Cavβ, one of the auxiliary subunits of Cav channels, also regulates the secretion of insulin as knockout of Cavβ3 (β3(-/-)) subunits in mice led to efficient glucose homeostasis and increased insulin levels. We examined whether other types of Cavβ subunits also have similar properties. In this regard, we used small interfering RNA (siRNA) of these subunits (20 μg each) to down-regulate them and examined blood glucose, serum insulin and PKC translocation in isolated pancreatic β cells of mice. While the down-regulation of Cavβ2 and β3 subunits increased serum insulin levels and caused efficient glucose homeostasis, the down-regulation of Cavβ1 and β4 subunits failed to affect both these parameters. Examination of PKC isozymes in the pancreatic β-cells of Cavβ2- or β3 siRNA-injected mice showed that three PKC isozymes, viz., PKC α, βII and θ, translocated to the membrane. This suggests that when present, Cavβ2 and β3 subunits inhibited PKC activation. Among these three isozymes, only PKCα siRNA inhibited insulin and increased glucose concentrations. It is possible that the activation of PKCs βII and θ is not sufficient for the release of insulin and PKCα is the mediator of insulin secretion under the control of Cavβ subunits. Since Cavβ subunits are present intracellularly, it is possible that they (1) inhibited the translocation of PKC isozymes to the membrane and (2) decreased the interaction between Cav channels and PKC isozymes and thus the secretion of insulin.

Fig. 1

Western blot (1 A) showing the down regulation of Ca_vβ subunits and the blood glucose and serum insulin levels (1 B) after the administration of Ca_vβ subunit siRNAs. Western blotting and GTT were conducted 24 hours after the administration of the siRNAs. Pancreatic islet protein extracts (100 μg/lane) from the scrambled or Ca_vβ siRNA injected mice were used for the Western blots. Specific antibodies for Ca_vβ1 - β4 subunits and GAPDH were used for test and control respectively. GTT was conducted after 12 hours of food deprivation and with i.p., glucose challenge. ^d<0.05, ^c<0.02, ^a<0.001 compared to respective scrambled siRNA using t-test; n = 4–7. CF, cytoplasmic fraction; MF, membrane fraction; scr., scrambled.

Fig. 2

Insulin determination in intact pancreatic islets (A) and in cultured pancreatic islet cells (B) after challenging with two concentrations [3 mM (basal) and 16.7 mM] of glucose. A. Three equal-sized islets each from the scrambled or Ca_vβ siRNA injected mice were used in this study. B. Cultured pancreatic islet cells from naïve mice were transfected with scrambled or Ca_vβ siRNA as described under the methods. These cultures were challenged with 3 mM or 16.7 mM glucose and insulin release was determined after one hour. ^d<0.05, ^b<0.01, ^a<0.001 compared to respective scrambled siRNA using t-test; n = 3. scr., scrambled.

Fig. 3

Confocal microscopy of cultured pancreatic islet cells showing translocation of PKC isozymes. Only the isozymes that were translocated by Ca_vβ siRNA are shown in the figure. Cultured pancreatic islet cells from naïve mice were cotransfected with scrambled or Ca_vβ2 or β3 siRNA (2.5 μg each) and selected PKC isozyme cDNA (2.5 μg each) subcloned into GFP containing vector. The translocation of the PKC isozymes was studied by immunocytochemistry as described under the methods. Bar, 10 μM. scr., scrambled.

Fig. 4

Blood glucose (top panel) and serum insulin levels (bottom panel) after the administration of PKC isozyme siRNA. GTT was conducted 24 hours after the administration of the siRNAs. The GTT was conducted with i.p., glucose challenge in mice deprived of food for 12 h. ^d<0.05, ^b<0.01 compared to respective scrambled siRNA using t-test; n = 3. scr., scrambled.

Fig. 5

Confocal microscopy of cultured pancreatic islet cells showing the down regulation of PKC isozymes. Cultured pancreatic islet cells from the naive mice were cotransfected with scrambled or PKC α, βII or θ siRNA and PKC α, γ or ε cDNA (top panel), PKC βII, γ or θ cDNA (middle panel) and PKCθ, ε or βII cDNA (bottom panel). The cDNA for PKC isozymes are subcloned into GFP containing vector. 2.5 μg each of PKC isozyme siRNA and PKC isozyme cDNA were used for the cotransfection. The cotransfection and immunohistochemistry were conducted as described under the methods. Bar, 10 μM. scr., scrambled.