Division of Endocrinology and Metabolism, Department of Internal Medicine, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan.
Diabetes Obes Metab. 2009 Oct;11(10):939-46. doi: 10.1111/j.1463-1326.2009.01067.x. Epub 2009 May 22.
Glucokinase (GK) in pancreatic beta cells is thought to be involved in insulin secretion and glucose homeostasis. This study investigates whether the long-acting agonist of the glucagon-like peptide 1, namely exendin-4, mediates stimulatory effects on GK gene expression through the Ca(2+)/calmodulin (CaM)-dependent protein kinase (CaMK) cascade.
GK expression was examined by real-time PCR, western blot analysis and reporter gene assay in rat insulin-secreting INS-1 cells incubated with exendin-4. CaMKIV activity was assessed by detection of activation loop phosphorylation (Thr(196)) of CaMKIV. We investigated the effect of the constitutively active form (CaMKIVc) of CaMKIV on GK promoter activity.
Increased expression level of GK protein was noted in response to rising concentrations of exendin-4 with maximum induction at 10 nM. Real-time PCR analysis showed a significant increase in the amount of GK mRNA in response to rising concentrations of exendin-4. Exendin-4 also stimulated GK promoter activity but failed to do so in the presence of STO-609, a CaMKK inhibitor. This result is consistent with the observations that the upregulation of CaMKIV phosphorylation (at Thr(196)) peaked after 15 min of exposure to exendin-4 and that CaMKIVc enhanced or upregulated GK promoter activity in INS-1 cells. Furthermore, STO-609 significantly suppressed the exendin-4 - upregulated the expression of the GK protein.
Activation of the CaMKK/CaMKIV cascade might be required for exendin-4-induced GK gene transcription, indicating that exendin-4 plays an important role in insulin secretion in pancreatic beta cells.
胰β细胞中的葡萄糖激酶（GK）被认为参与胰岛素分泌和葡萄糖稳态。本研究旨在探讨胰高血糖素样肽 1 的长效激动剂——艾塞那肽是否通过钙/钙调蛋白（CaM）依赖性蛋白激酶（CaMK）级联来介导对 GK 基因表达的刺激作用。
通过实时 PCR、Western blot 分析和报告基因检测，在孵育艾塞那肽的大鼠胰岛素分泌细胞 INS-1 中检测 GK 的表达。通过检测 CaMKIV 激活环磷酸化（Thr196）来评估 CaMKIV 活性。我们研究了 CaMKIV 的组成激活形式（CaMKIVc）对 GK 启动子活性的影响。
随着艾塞那肽浓度的升高，GK 蛋白的表达水平增加，在 10 nM 时达到最大诱导。实时 PCR 分析显示，GK mRNA 的量随着艾塞那肽浓度的升高而显著增加。艾塞那肽也刺激 GK 启动子活性，但在 CaMKK 抑制剂 STO-609 存在的情况下则不然。这一结果与以下观察结果一致：暴露于艾塞那肽 15 分钟后，CaMKIV 磷酸化（在 Thr196 处）上调达到峰值，并且 CaMKIVc 在 INS-1 细胞中增强或上调 GK 启动子活性。此外，STO-609 显著抑制了艾塞那肽上调的 GK 蛋白表达。
CaMKK/CaMKIV 级联的激活可能是艾塞那肽诱导 GK 基因转录所必需的，这表明艾塞那肽在胰腺β细胞胰岛素分泌中起重要作用。
