Vascular endothelial PDPK1 plays a pivotal role in the maintenance of pancreatic beta cell mass and function in adult male mice
Vascular endothelial PDPK1 plays a pivotal role in the maintenance of pancreatic beta cell mass and function in adult male mice
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DOI：
10.1007/s00125-019-4878-1
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发表时间：
2019-07-01
期刊：
影响因子：
8.2
通讯作者：
Kaneto, Hideaki
中科院分区：
文献类型：
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作者：
Obata, Atsushi;Kimura, Tomohiko;Kaneto, Hideaki
目的/假说本研究旨在阐明血管内皮细胞中3-磷酸肌醇依赖的蛋白激酶-1(PDPK1)在维持胰岛β细胞质量和功能中的作用。方法采用雄性血管内皮细胞特异性Pdpk1基因敲除小鼠(Tie2(+/-)/Pdpk1(Flox/Flox)小鼠)及其野生型窝仔(Tie2(-/-)/Pdpk1(Flox/Flox)小鼠；对照)。在12周龄时，进行IPGTT和OGTT。麻醉状态下测量胰腺血流量。用微球法测定胰岛血流量。处死小鼠进行胰岛分离和进一步的功能研究，并从胰岛中提取mRNA。结果在IPGTT过程中，FLOX基因敲除小鼠的血糖水平与对照FLOX小鼠相当，而胰岛素水平明显低于FLOX基因敲除小鼠。在OGTT期间，基因敲除小鼠的葡萄糖耐量轻微恶化，并伴随着血清胰岛素水平的下降。在Exendin-4(Ex-4)预处理后的IPGTT中，基因敲除小鼠的葡萄糖耐量显著受损。事实上，与对照FLOX小鼠相比，葡萄糖刺激的基因敲除小鼠胰岛胰岛素分泌显著减少，加入Ex-4显示基因敲除小鼠胰岛对胰岛素激素的敏感性受损。在免疫组织化学分析中，基因敲除小鼠的α和β细胞团都显著减少。此外，CD31阳性面积在基因敲除小鼠的胰岛中显著减少。在基因敲除的小鼠中，匹莫硝唑阳性的胰岛比例显著增加。在基因敲除小鼠的胰岛中，与胰岛素生物合成(Ins1、Ins2、Mafa、Pdx1和Neurod[也称为Neurod1])和β细胞功能(如GCK和SLc2a2)相关的mRNA表达水平显著降低。微球实验显示胰岛血流量显著减少。此外，HIF1(也称为HIF1a)及其下游因子Adm、eno1、Tpi1(也称为Ets1)、Hmox1和Vegfa在基因敲除小鼠的胰岛中的表达水平显著增加，表明基因敲除小鼠的胰岛处于比对照FLOX小鼠更低的缺氧状态。结果，在基因敲除小鼠的胰岛中，与适应性未折叠蛋白反应和内质网应激相关的凋亡基因相关的mRNA表达水平显著升高。此外，基因敲除小鼠的胰岛中炎性细胞因子水平增加。电子显微镜显示基因敲除小鼠胰岛内皮细胞窗口减少，血管内皮细胞基底膜增厚。结论/解释血管内皮细胞PDPK1通过维持胰腺和胰岛的血管，保护其免受缺氧、缺氧相关内质网应激、炎症和毛细血管结构扭曲的影响，在维持胰岛β细胞质量和功能方面发挥重要作用。
Aims/hypothesis The aim of this study was to elucidate the impact of 3-phosphoinositide-dependent protein kinase-1 (PDPK1) in vascular endothelial cells on the maintenance of pancreatic beta cell mass and function.Methods Male vascular endothelial cell-specific Pdpk1-knockout mice (Tie2(+/-)/Pdpk1(flox/flox) mice) and their wild-type littermates (Tie2(-/-)/Pdpk1(flox/flox) mice; control) were used for this study. At 12 weeks of age, an IPGTT and OGTT were conducted. Pancreatic blood flow was measured under anaesthesia. Thereafter, islet blood flow was measured by the microsphere method. Mice were killed for islet isolation and further functional study and mRNA was extracted from islets. Pancreases were sampled for immunohistochemical analyses.Results During the IPGTT, the blood glucose level was comparable between knockout mice and control flox mice, although serum insulin level was significantly lower in knockout mice. During the OGTT, glucose tolerance deteriorated slightly in knockout mice, accompanied by a decreased serum insulin level. During an IPGTT after pre-treatment with exendin-4 (Ex-4), glucose tolerance was significantly impaired in knockout mice. In fact, glucose-stimulated insulin secretion of isolated islets from knockout mice was significantly reduced compared with control flox mice, and addition of Ex-4 revealed impaired sensitivity to incretin hormones in islets of knockout mice. In immunohistochemical analyses, both alpha and beta cell masses were significantly reduced in knockout mice. In addition, the CD31-positive area was significantly decreased in islets of knockout mice. The proportion of pimonidazole-positive islets was significantly increased in knockout mice. mRNA expression levels related to insulin biosynthesis (Ins1, Ins2, Mafa, Pdx1 and Neurod [also known as Neurod1]) and beta cell function (such as Gck and Slc2a2) were significantly decreased in islets of knockout mice. Microsphere experiments revealed remarkably reduced islet blood flow. In addition, mRNA expression levels of Hif1 (also known as Hif1a) and its downstream factors such as Adm, Eno1, Tpi1 (also known as Ets1), Hmox1 and Vegfa, were significantly increased in islets of knockout mice, indicating that islets of knockout mice were in a more hypoxic state than those of control flox mice. As a result, mRNA expression levels related to adaptive unfolded protein response and endoplasmic reticulum stress-related apoptotic genes were significantly elevated in islets of knockout mice. In addition, inflammatory cytokine levels were increased in islets of knockout mice. Electron microscopy revealed reduced endothelial fenestration and thickening of basal membrane of vascular endothelium in islets of knockout mice. Conclusions/interpretation Vascular endothelial PDPK1 plays an important role in the maintenance of pancreatic beta cell mass and function by maintaining vascularity of pancreas and islets and protecting them from hypoxia, hypoxia-related endoplasmic reticulum stress, inflammation and distortion of capillary structure.