Diabetes Research Group, School of Medicine, King's College London, Guy's Campus, London, SE1 1UL, UK.
Diabetologia. 2011 May;54(5):1109-20. doi: 10.1007/s00125-011-2050-7. Epub 2011 Feb 8.
AIMS/HYPOTHESIS: Irs2, which is upregulated by glucose, is important for beta cell plasticity. Cyclic AMP response element-binding protein (CREB) stimulates beta cell Irs2 expression and is a major calcium/calmodulin-dependent kinase (CaMK)(IV) target in neurons. We therefore hypothesised that CaMK(IV) mediates glucose-induced Irs2 expression in beta cells via CREB activation.
The functions of CaMK(IV) and CREB were investigated in MIN6 beta cells and mouse islets using the CaMK inhibitor KN62, the calcium chelator bapta-(AM) and the voltage-dependent calcium channel inhibitor nifedipine. Small interfering RNAs were used to silence endogenous CaMK(IV) production and expression vectors to overproduce constitutively active and dominant negative forms of CaMK(IV) and CREB. Irs1 and Irs2 expression were determined by quantitative PCR and Western blotting, and the role of CREB was also investigated by assessing its phosphorylation on serine 133.
Increasing the glucose concentration from 2.5 to 25 mmol/l stimulated CREB phosphorylation on serine 133 and specifically stimulated Irs2 but not Irs1 expression. Similarly, overproduction of a constitutively active form of CaMK(IV) promoted sustained CREB phosphorylation and a significant increase in Irs2 but not Irs1 expression. In contrast, these stimulatory effects of glucose were all suppressed by overproducing an inactive CaMK(IV) mutant. Inhibition of glucose-induced calcium influx with nifedipine or chelation of intracellular calcium with bapta-(AM), as well as silencing of CaMK(IV) or inhibition of its activity with KN62 resulted in similar observations. Finally, overproduction of a dominant negative form of CREB completely suppressed glucose and CaMK(IV) stimulation of Irs2 expression.
CONCLUSIONS/INTERPRETATION: Our results suggest that the Ca(2+)/CaMK(IV)/CREB cascade plays a critical role in the regulation of Irs2 expression in beta cells.
目的/假设：葡萄糖上调的 Irs2 对于β细胞的可塑性很重要。环磷酸腺苷反应元件结合蛋白（CREB）刺激β细胞 Irs2 的表达，并且是神经元中钙/钙调蛋白依赖性激酶（CaMK）（IV）的主要靶标。因此，我们假设 CaMK（IV）通过 CREB 激活介导葡萄糖诱导的β细胞中 Irs2 的表达。
使用 CaMK 抑制剂 KN62、钙螯合剂 Bapta-（AM）和电压依赖性钙通道抑制剂硝苯地平，在 MIN6β细胞和小鼠胰岛中研究 CaMK（IV）和 CREB 的功能。使用小干扰 RNA 沉默内源性 CaMK（IV）的产生，并用表达载体过表达组成型激活和显性失活形式的 CaMK（IV）和 CREB。通过定量 PCR 和 Western blot 测定 Irs1 和 Irs2 的表达，并通过评估其丝氨酸 133 上的磷酸化来研究 CREB 的作用。
将葡萄糖浓度从 2.5mmol/l 增加到 25mmol/l 刺激 CREB 丝氨酸 133 的磷酸化，并特异性刺激 Irs2 但不刺激 Irs1 的表达。同样，过表达组成型激活形式的 CaMK（IV）促进持续的 CREB 磷酸化和 Irs2 的显著增加，但不增加 Irs1 的表达。相比之下，葡萄糖的这些刺激作用都被过表达无活性的 CaMK（IV）突变体所抑制。用硝苯地平抑制葡萄糖诱导的钙内流或用 Bapta-（AM）螯合细胞内钙，以及沉默 CaMK（IV）或用 KN62 抑制其活性，也得到了类似的观察结果。最后，过表达显性失活形式的 CREB 完全抑制了葡萄糖和 CaMK（IV）对 Irs2 表达的刺激。
结论/解释：我们的结果表明，钙/钙调蛋白依赖性激酶（CaMK）（IV）/CREB 级联在调节β细胞中 Irs2 的表达中起关键作用。
