ZPR1-PPARγ反馈环路在2型糖尿病脂肪细胞胰岛素抵抗中的作用及机制研究

According to our latest Genome-wide association studies, we proved that rs2075290 SNP of zinc finger protein ZPR1 is significantly correlated with blood glucose balance (SCI REP, 2016) and thus the susceptibility of T2DM. However, its specific mechanism has not been clarified. Our preliminary experiments revealed that the expression of ZPR1 was obviously decreased in adipocytes in T2DM mice and ZPR1 inhibition significantly strengthened insulin resistance in adipocytes. Meanwhile, PPARγ, a key transcriptional factor in lipid and glucose metabolism, transcriptionally activates the expression of ZPR1, which in turn forms co-transcript complex with PPARγ and thus plays a role in the regulation of lipid and glucose metabolism related genes expression. Based on the above findings, we plan to reveal the role of ZPR1 in adipocytes in T2DM on the molecular level, and validate the ZPR1-PPARγ reciprocal loop in the regulation of lipid and glucose metabolism signaling network, as well as blood glucose balancing. In addition, using CRISPR/Cas9 gene editing system to clarify the function of rs2075290 SNP in adipocytes insulin resistance in T2DM. This research will for the first time presents a novel role of ZPR1-PPARγ loop mediated regulation of lipid and glucose metabolism. The deep exploration of the function of ZPR1 in T2DM may help us gain a profound knowledge of T2DM, which will potentially provide new strategies and clinical treatment targets for this disease.

我们前期通过GWAS研究发现锌指蛋白ZPR1是2型糖尿病易感基因，其rs2075290 SNP与胰岛素抵抗显著关联(SCI REP, 2016)，但功能机制未知。预实验结果显示在2型糖尿病小鼠脂肪细胞中ZPR1表达明显降低，敲降ZPR1可显著增强脂肪细胞胰岛素抵抗，且ZPR1表达可能受糖脂代谢关键转录因子PPARγ的转录激活，同时ZPR1可能与PPARγ形成共转录复合物参与糖脂代谢相关分子的表达调控。本课题拟在此基础上，利用在体/离体胰岛素抵抗模型明确ZPR1在2型糖尿病脂肪细胞胰岛素抵抗中的作用；阐明PPARγ与ZPR1形成的调控环路及其在血糖调节中的分子机制，利用CRISPR/Cas9系统明确rs2075290在2型糖尿病脂肪细胞胰岛素抵抗中的作用机制。本研究有望发现糖脂代谢调控新模式，即PPARγ-ZPR1环路，并阐明其在2型糖尿病中的重要功能，为临床治疗提供新的策略和药物靶点。

课题组前期基于全基因组关联研究（genome-wide association study，GWAS）技术，对照研究了本地区汉族人群中3505名T2DM患者和 6911 名健康无关个体，分析了编码锌指蛋白1（Zinc Finger Protein1，ZPR1）的ZPR1基因上分布的24个SNPs，发现rs964184位点的GG基因型与空腹血糖值和糖化血红蛋白水平呈正相关，是T2DM患病易感位点。然而当前关于ZPR1基因与糖脂代谢相关疾病的研究均未深入至分子机制层面，因此本研究通过构建高脂饮食诱导胰岛素抵抗小鼠模型及诱导小鼠前脂肪细胞3T3-L1成脂分化模型，对ZPR1参与T2DM发生发展的关键分子机制进行解析，并得到以下结果：.① 证实ZPR1参与调控2型糖尿病脂肪细胞胰岛素抵抗。通过建立高脂饮食诱导胰岛素抵抗小鼠模型及胰岛素抵抗细胞模型，发现内脏脂肪ZPR1表达显著降低，且可能受到转录后调控。验证了脂肪细胞中ZPR1与胰岛素抵抗的相关性。.② 证明了ZPR1的rs964184 SNP位点导致ZPR1的3’UTR不易形成多重发卡结构，并促进miR-4286对Zpr1的转录后调控，Zpr1的低表达抑制3T3-L1前脂肪细胞的分化及胰岛素信号通路。.③ 明确了ZPR1蛋白可以与转录因子PPARγ形成复合物，而锌指蛋白ZPR1作为转录辅因子，协同PPARγ转录调控下游与胰岛素敏感性和糖脂代谢相关基因表达。.④ 在人骨髓来源间充质干细胞Lonza细胞系中，通过CRISPR/dCas9点突变慢病毒系统，构建rs964184位点GG基因型稳转细胞系，并在此基础上诱导细胞向成熟脂肪细胞分化，验证rs964184位点GG基因型对脂肪细胞分化及胰岛素信号通路的影响。（此阶段实验尚在进行中，以完成rs964184位点GG基因型Lonza稳转细胞系构建及测序工作）.本研究为深入认识ZPR1基因促进T2DM发生发展的功能机制提供了新的研究方向及理论支撑，并为突破当前T2DM治疗瓶颈提供了新的思路。