褪黑激素受体MTNR1B基因变异对妊娠血糖代谢和胰岛素作用的影响机制的研究

Gestational maternal metabolism is linked to increasing food intake, energy expenditure, body mass, insulin release and insulin resistance，and usually these adaptive changes subside after postpartum recovery. Genetic evidences of the transiently changed glucose tolerance and up-regulated insulin signaling during pregnancy have been limited so far. Previous studies have suggested that common variants in melatonin receptor 1B (MTNR1B) gene are linked with fasting glucose and early insulin release. Our research in the pregnant Chinese women showed that the MTNR1B variants were also significantly associated with gestational postprandial glucose level and proinsulin to insulin conversion. Melatonin is a circulating hormone from the pineal gland and regulates seasonal/circadian rhythms, MTNR1B was identified as a G protein-coupled melatonin receptor and expressed in pancreatic islets. MTNR1B could lead to the acute inhibitory effect of melatonin on insulin secretion by binding specifically to inhibitory G protein and activating the downstream signals. MTNR1B variants impairing melatonin receptor function have been known to contribute to type 2 diabetes. However, how MTNR1B functioned during pregnancy has not yet been investigated. We took advantage of our sample database of pregnant Southwest Chinese women, and sequenced the 2 exons of MTNR1B from 1,084 pregnant women (age 28.5±4.1 years; BMI 21.4±2.6 kg/m2). Matsuda index of insulin sensitivity (Matsuda ISI), homeostatic model assessment of insulin resistance (HOMA-IR), insulin disposition index, early-phase insulin release, fasting state and 0-120 minute's proinsulin to insulin conversion were used as physiological insulin indices. The primary data showed that MTNR1B variants affected specifically the association with gestational glucose-insulin axis. Hence, considering the very different physiological conditions between pregnancy and outside of pregnancy, we hypothesized that there could exist distinct MTNR1B functional mechanism during pregnancy. Our first purpose is to confirm the association between MTNR1B variants and gestational glucose levels, and between the variants and insulin indexes by sequencing the variants with an increasing sample size and statistical power. Secondly, we will compare the variant with sequence- and structure-based predictions made by Polymorphism Phenotyping software, and characterize the melatonin binding/signaling capabilities of variant by transfection in HEK293T cell and mouse islet cell. Thirdly, we will test the MTNR1B variant function by allo-islet transplantation into pregnant and non-pregnant mice. Finally, we will further investigate the role of MTNR1B variants by contrasing the related clinic data of the pregnant women. We hope the study could provide new insights into the functional link among genetic factors, gestational glucose metabolism and pregnant outcomes.

褪黑激素受体MTNR1B与血糖调控、糖尿病紧密相关。我们在西南地区孕妇群体中发现，与非妊娠期血糖代谢有所不同，MTNR1B基因变异不仅与妊娠期空腹血糖水平和早期胰岛素释放相关，还与餐后血糖水平和前胰岛素转化显著相关。通过对其外显子进行测序分析，我们观察到MTNR1B可能存在特殊地与妊娠血糖代谢相关的变异和机制。因此，我们拟扩大所研究的孕妇群体，对照MTNR1B变异在非妊娠期血糖代谢的功能特征，确定其相关变异与妊娠血糖-胰岛素调控的关联性；并利用基因表达操作技术在细胞中进行MTNR1B基因的定位突变，从常规细胞表达，胰岛细胞表达以及孕期和非孕期小鼠胰岛细胞的移植实验，深入调查MTNR1B在妊娠期血糖-胰岛素调控的机理和分子信号通路；试图从功能机制上说明其与妊娠血糖代谢及妊娠结局/围产期发病情况的可能联系。

在该项目资助下，我们课题组按照研究计划，在前期工作基础上，增加收集了近3000名西南地区孕妇血糖代谢资料和血液细胞，提取制备DNA样本，丰富了中国西南地区孕妇血糖代谢资料库和DNA样本库。. 课题增加测定了近1000名孕妇的MTNR1B基因外显子序列，并对应相关孕妇临床血糖代谢资料以及孕期、围产期临床数据进行分析，分析确定了孕妇群体MTNR1B序列变异与妊娠空腹血糖/餐后血糖，餐前后胰岛素水平，胰岛素转化、处置、释放和胰岛素抗性指数以及围产期妊娠结局的相互关系。. 课题构建了MTNR1B外显子的有益/有害突变表达载体，在不同的胰岛细胞系以及分离纯化的小鼠和人体胰岛中进行表达，检测分析MTNR1B外显子序列的相关变异在孕期血糖-胰岛素调控系统中与可能的分子机理和信号通路。由于小鼠内源性褪黑激素受体的干扰，课题采用了慢病毒载体SiRNA干扰技术构建了Mtnr1b被敲减的MIN6细胞株开展研究。同时，由于实验发现小鼠和人体胰岛分子信号途径的差异，后续研究直接采用人体胰岛来进行。课题采用褪黑激素或/和雌激素处理人体胰岛，对胰岛素释放以及相关分子信号传导，尤其是G蛋白相关的MAPK，MEK1&2，ERK1/2，PKC，PKA和CalmKII的信号通路进行了检测分析。. 课题通过对MTNR1B基因序列变异的功能分析以及孕妇临床数据库的统计分析，发现MTNR1B介导的褪黑激素信号通路，不仅是在体外培养的胰岛细胞中受到妊娠雌激素的影响，雌激素受体和MTNR1B相关信号在细胞膜上的传导还受到特定脂肪酸的干预；同时，孕妇体重指数和脂肪代谢，明显地与孕期胰岛素释放和胰岛素抵抗有关联，而携带MTNR1B突变风险位点的肥胖孕妇，妊娠糖尿病、不良妊娠结局/围产期发病风险更高。课题探讨了在已知孕妇MTNR1B变异的情况下，如何通过改变生活节律调节妊娠血糖代谢状态，争取良好妊娠结局的可能性。. 有关MTNR1B调节妊娠期血糖-胰岛素的分子作用机理的研究，还需在人体胰岛中得到更多的验证，而课题新发现的其它问题还需继续深入地探索。. 课题组培养了1名博士研究生和3名硕士研究生。发表有标注本项目资助编号SCI收录论文5篇。