Lund University Diabetes Centre, Department of Clinical Sciences, Clinical Research Centre, Lund University, CRC 91-11, UMAS entrance 72, SE-20502 Malmö, Sweden.
Diabetologia. 2010 Apr;53(4):717-29. doi: 10.1007/s00125-009-1635-x. Epub 2009 Dec 18.
AIMS/HYPOTHESIS: Protein kinase C (PKC) regulates exocytosis in various secretory cells. Here we studied intracellular translocation of the PKC isoenzymes PKCalpha and PKCdelta, and investigated how activation of PKC influences glucagon secretion in mouse and human pancreatic alpha cells.
Glucagon release from intact islets was measured in static incubations, and the amounts released were determined by RIA. Exocytosis was monitored as increases in membrane capacitance using the patch-clamp technique. The expression of genes encoding PKC isoforms was analysed by real-time PCR. Intracellular PKC distribution was assessed by confocal microscopy.
The PKC activator phorbol 12-myristate 13-acetate (PMA) stimulated glucagon secretion from mouse and human islets about fivefold (p < 0.01). This stimulation was abolished by the PKC inhibitor bisindolylmaleimide (BIM). Whereas PMA potentiated exocytosis more than threefold (p < 0.001), BIM inhibited alpha cell exocytosis by 60% (p < 0.05). In mouse islets, the PKC isoenzymes, PKCalpha and PKCbeta1, were highly abundant, while in human islets PKCeta, PKCepsilon and PKCzeta were the dominant variants. PMA stimulation of human alpha cells correlated with the translocation of PKCalpha and PKCdelta from the cytosol to the cell periphery. In the mouse alpha cells, PKCdelta was similarly affected by PMA, whereas PKCalpha was already present at the cell membrane in the absence of PMA. This association of PKCalpha in alpha cells was principally dependent on Ca(2+) influx through the L-type Ca(2+) channel.
CONCLUSIONS/INTERPRETATION: PKC activation augments glucagon secretion in mouse and human alpha cells. This effect involves translocation of PKCalpha and PKCdelta to the plasma membrane, culminating in increased Ca(2+)-dependent exocytosis. In addition, we demonstrated that PKCalpha translocation and exocytosis exhibit differential Ca(2+) channel dependence.
目的/假设：蛋白激酶 C（PKC）调节各种分泌细胞的胞吐作用。在这里，我们研究了 PKC 同工酶 PKCalpha 和 PKCdelta 的细胞内易位，并研究了 PKC 的激活如何影响小鼠和人胰岛α细胞中的胰高血糖素分泌。
在静态孵育中测量完整胰岛中胰高血糖素的释放，并通过 RIA 测定释放的量。使用膜片钳技术监测胞吐作用作为膜电容的增加。通过实时 PCR 分析编码 PKC 同工型的基因的表达。通过共焦显微镜评估细胞内 PKC 分布。
PKC 激活剂佛波醇 12-肉豆蔻酸 13-乙酸酯（PMA）刺激小鼠和人胰岛的胰高血糖素分泌约五倍（p <0.01）。这种刺激被 PKC 抑制剂双吲哚基马来酰亚胺（BIM）消除。虽然 PMA 使胞吐作用增强了三倍以上（p <0.001），但 BIM 抑制α细胞胞吐作用 60%（p <0.05）。在小鼠胰岛中，PKC 同工酶 PKCalpha 和 PKCbeta1 高度丰富，而在人胰岛中，PKCeta、PKCepsilon 和 PKCzeta 是主要变体。PMA 刺激人胰岛α细胞与 PKCalpha 和 PKCdelta 从细胞质向细胞外周的易位相关。在小鼠α细胞中，PMA 也同样影响 PKCdelta，而 PKCalpha 在没有 PMA 的情况下已经存在于细胞膜上。这种 PKCalpha 在α细胞中的关联主要取决于通过 L 型钙通道的钙内流。
结论/解释：PKC 激活增强了小鼠和人胰岛α细胞中的胰高血糖素分泌。这种作用涉及 PKCalpha 和 PKCdelta 向质膜的易位，最终导致 Ca2+依赖性胞吐作用增加。此外，我们证明了 PKCalpha 易位和胞吐作用表现出不同的 Ca2+通道依赖性。
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