组织特异性敲除GHR基因在β细胞功能中的作用机制研究

β cell dysfunction is the major cause of diabetes. The growth hormone (GH)-insulin-like growth factor-1 (IGF1) axis and insulin signaling pathways are known to play important roles in the regulation of β cell growth and function. However, the molecular mechanisms of GH action in the β cell remain largely unknown. We used Cre-loxP system to generate a mouse model with the GH receptor specifically disrupted in β cells (βGHRKO). βGHRKO mice fed high-fat diet (HFD) exhibited impaired glucose-stimulated insulin secretion, decreased β cell proliferation and reduced β cell mass (JCI, 2011;121(6):2422-6). The goal of this project is to explore the molecular mechanisms/signaling pathways that are responsible for GHR deficiency-caused impairment of insulin secretion and β cell proliferation, thereby identifying novel genes that regulate insulin secretion and β cell proliferation. We propose four aims: 1) To identify candidate genes that might be responsible for β cell dysfunction in βGHRKO by microarrays and proteomics, 2) To determine the effect of βGHRKO on insulin signaling pathway activity such as PI3K/AKT, IRS2, Foxo1, Stat5, GSK3/LKB1, GCK, and mTOR pathway in liver, adipose tissues and muscles under HFD conditions, 3) Using CRISPR/Cas9 technology to characterize and verify candidate genes involved in β cell proliferation and insulin secretion in cell lines, 4) Ultimately generate transgenic mouse models with candidate genes for phenotype rescue to determine which gene is the direct target of GHR in β cell growth and function. The combination of our molecular, biochemical and genetic studies will shed insight into how GH action is linked to β cell function and help to identify new targets for the development of novel pharmaceutical drugs for diabetes.

β细胞功能异常是糖尿病发生的主要机制和研究新热点。GH-IGF1轴对β细胞的生长和功能具重要调节作用，但分子机制仍不清楚。本课题组前期建立了β细胞特异性生长激素受体敲除小鼠（βGHRKO）。喂食高脂食物后，βGHRKO小鼠胰岛素分泌下降，β细胞增殖降低，β细胞团减少（JCI,2011;121(6)）。本研究拟在此基础上利用肝胆管显微注射胶原酶技术分离具生物活性的胰岛，然后通过基因组学、蛋白质组学研究肥胖条件下由GHR介导的β细胞团增殖的调控分子网络和高脂诱导肥胖条件下βGHRKO小鼠肝脏、脂肪和肌肉组织的胰岛素代谢变化；利用已掌握的CRISPR/Cas9技术敲除胰岛细胞系中候选基因并进行功能验证；构建候选基因转基因小鼠，并和βGHRKO小鼠杂交，进行β细胞团和胰岛素分泌的表型重建。本研究将有助于揭示GH-IGF1轴在胰岛素分泌和β细胞团生长中的作用机制，为糖尿病有效预防和治疗奠定理论基础。

β细胞功能异常是糖尿病发生的主要机制和研究热点，GH-IGF1 轴对β细胞的生长和功能具重要调节作用，但分子机制仍不清楚。本课题应用Cre/LoxP系统新构建的在细胞特异性敲除GHR基因（βGHRKO）小鼠动物模型，给予高脂饮食诱导后，βGHRKO小鼠胰岛素分泌下降，胰岛素抵抗增加，β细胞团减少，α细胞所占胰岛的比例增加。进一步利用肝胆管显微注射胶原酶技术分离具生物活性的胰岛，然后通过蛋白质组学挖掘了肥胖条件下由GHR调控的β细胞内蛋白表达差异网络，其中发现SNARE家族中重要的成员VAMP2、stx3表达下调较明显，并且其对胰岛素分泌（胞吐过程）的影响已被证实，因此我们并对其进行了qPCR及蛋白水平的功能验证，结果表明GHR能够调控VAMP2、stx3的表达，从而影响了胰岛素分泌的过程。本研究将有助于揭示 GH-IGF1 轴在胰岛素分泌和β细胞团生长中的作用机制，为糖尿病有效预防和治疗奠定理论基础。