Aims/hypothesis

Protein kinase CK2 acts as a negative regulator of insulin expression in pancreatic beta cells. This action is mainly mediated by phosphorylation of the transcription factor pancreatic and duodenal homeobox protein 1 (PDX1). In pancreatic alpha cells, PDX1 acts in a reciprocal fashion on glucagon (GCG) expression. Therefore, we hypothesised that CK2 might positively regulate GCG expression in pancreatic alpha cells.

Methods

We suppressed CK2 kinase activity in αTC1 cells by two pharmacological inhibitors and by the CRISPR/Cas9 technique. Subsequently, we analysed GCG expression and secretion by real-time quantitative RT-PCR, western blot, luciferase assay, ELISA and DNA pull-down assays. We additionally studied paracrine effects on GCG secretion in pseudoislets, isolated murine islets and human islets. In vivo, we examined the effect of CK2 inhibition on blood glucose levels by systemic and alpha cell-specific CK2 inhibition.

Results

We found that CK2 downregulation reduces GCG secretion in the murine alpha cell line αTC1 (e.g. from 1094±124 ng/l to 459±110 ng/l) by the use of the CK2-inhibitor SGC-CK2-1. This was due to a marked decrease in Gcg gene expression through alteration of the binding of paired box protein 6 (PAX6) and transcription factor MafB to the Gcg promoter. The analysis of the underlying mechanisms revealed that both transcription factors are displaced by PDX1. Ex vivo experiments in isolated murine islets and pseudoislets further demonstrated that CK2-mediated reduction in GCG secretion was only slightly affected by the higher insulin secretion after CK2 inhibition. The kidney capsule transplantation model showed the significance of CK2 for GCG expression and secretion in vivo. Finally, CK2 downregulation also reduced the GCG secretion in islets isolated from humans.

Conclusions/interpretation

These novel findings not only indicate an important function of protein kinase CK2 for proper GCG expression but also demonstrate that CK2 may be a promising target for the development of novel glucose-lowering drugs.

Graphical Abstract

中文翻译：

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CK2 活性对于胰高血糖素的正常表达至关重要

目标/假设

蛋白激酶 CK2 充当胰腺 β 细胞中胰岛素表达的负调节因子。该作用主要由转录因子胰腺和十二指肠同源框蛋白 1 (PDX1) 的磷酸化介导。在胰腺 α 细胞中，PDX1 以相反的方式作用于胰高血糖素 (GCG) 的表达。因此，我们假设CK2可能正向调节胰腺α细胞中GCG的表达。

方法

我们通过两种药理学抑制剂和 CRISPR/Cas9 技术抑制 αTC1 细胞中的 CK2 激酶活性。随后，我们通过实时定量 RT-PCR、蛋白质印迹、荧光素酶测定、ELISA 和 DNA Pull-down 测定分析了 GCG 表达和分泌。我们还研究了旁分泌对伪胰岛、分离的鼠胰岛和人胰岛中 GCG 分泌的影响。在体内，我们通过全身和α细胞特异性CK2抑制来检查CK2抑制对血糖水平的影响。

结果

我们发现，通过使用CK2抑制剂SGC-CK2-1，CK2下调可减少鼠α细胞系αTC1中GCG的分泌（例如从1094±124ng/l至459±110ng/l）。这是由于配对盒蛋白 6 (PAX6) 和转录因子 MafB 与Gcg启动子的结合发生改变，导致Gcg基因表达显着降低。对潜在机制的分析表明，这两个转录因子都被 PDX1 取代。在分离的小鼠胰岛和假胰岛中进行的离体实验进一步表明，CK2 介导的 GCG 分泌减少仅受到 CK2 抑制后较高的胰岛素分泌的轻微影响。肾被膜移植模型显示CK2对体内GCG表达和分泌的重要性。最后，CK2 下调还减少了从人类分离的胰岛中的 GCG 分泌。

结论/解释

这些新发现不仅表明蛋白激酶 CK2 对于正确的 GCG 表达具有重要功能，而且还表明 CK2 可能是开发新型降糖药物的有希望的靶标。

图形概要