体内抑制LRP16基因调控脂联素和胰岛素抵抗的作用及机制研究

Diabetes is increasing at an alarming rate and has become a serious public health problem worldwide. Insulin resistance (IR) is an essential causal factor for the development of type 2 diabetes (T2DM). Therefore,understanding the in vivo mechanisms underlying IR is essential for the treatment and ultimate prevention of T2DM. Leukemia-related protein 16 (LRP16) is a gene first cloned and characterized by our group. LRP16 is tightly associated with estrogen-induced tumors and might also be involved in inflammation and metabolism regulation. We have conducted a series of studies showing that LRP16 contributes to IR in cell lines in vitro, but the mechanism remains unclear. Adiponectin is a well-recognized specific cytokine highly correlated with IR. Our preliminary data indicate that LPR16 negatively regulates adiponectin functions whereas the effects of LRP16 on adiponectin functions and IR in vivo and their underlying mechanisms remain largely unknown. Our proposed study aims to investigate the molecular mechanisms underlying the role of LRP16 in regulating adiponectin functions and IR in vivo. We will study whether suppression of LRP16 in vivo can facilitate adiponectin functions and ameliorate IR using LRP16 conditional knock-out mouse models and adenovirus-mediated suppression of LRP16 expression in mice. Our work will make an important breakthrough in elucidation of IR mechanisms in vivo and seeking potential drug targets to treat IR, thus providing novel targets and directions for T2DM treatment and prevention.

糖尿病已成为日益严峻的公共健康问题，胰岛素抵抗（IR）是大多数T2DM启动、进展要素，研究如何改善体内IR及作用机制对最终预防T2DM 的发生发展甚为重要。白血病相关蛋白16（LRP16）是我们课题组首先克隆鉴定的与雌激素肿瘤紧密相关并逐渐被发现与炎症、代谢也可能相关的基因。我们近来系列研究发现LRP16在细胞中可导致 IR发生，但机制未明。脂联素是公认与IR关系最为紧密的特异性细胞因子，我们前期体外实验结果提示LRP16对脂联素具有负向调节作用，然而LRP16在体内对脂联素和IR的作用及调控机制尚不清楚。本课题主要在体内研究LRP16对脂联素及IR的调节作用及分子机制，通过条件性LRP16基因敲除和腺病毒介导基因抑制模型在体内探讨抑制LRP16增强脂联素功能和缓解IR的作用及机制。为寻找体内有效缓解IR的潜在靶分子并阐明调控机理做出新突破，进一步拓展糖尿病防治的新靶点和新思路。

项目背景：本项目旨在从体外和体内探究LRP16基因对胰岛素、脂联素信号通路的调节作用以及对胰岛素抵抗的影响，并探讨其调控机制。..研究内容： 1. 建立LRP16过表达以及LRP16抑制表达的HepG2稳定细胞系，测定葡萄糖摄取率、Western blot检测LRP16对脂联素和胰岛素信号通路的影响、测定胞内甘油三酯和胆固醇的含量、以及Q-PCR检测相关脂代谢酶，从而在体外观察LRP16基因对脂联素和胰岛素信号转导通路的影响。2. 转录组分析，比较分析差异表达的基因，并进行KEGG通路分析以及GO功能分析，从而探讨LRP16基因体外调控脂联素和胰岛素抵抗的机制。3. 建立肝脏特异性LRP16基因敲除小鼠模型，检测体重、空腹血糖、空腹胰岛素、HOMA-IR、葡萄糖耐量试验、胰岛素耐量试验，以观察肝脏特异性LRP16基因敲除对胰岛素抵抗的影响。4. Western blot检测肝脏胰岛素、脂联素信号通路关键蛋白，Q-PCR检测肝脏关键糖脂代谢酶，从而在体内观察LRP16基因对胰岛素和脂联素信号通路的影响，并探讨其机制。..结果：1. LRP16过表达HepG2细胞与过表达对照组相比，葡萄糖摄取率显著下降；细胞内甘油三酯和胆固醇含量显著升高；胰岛素、脂联素信号通路明显下调；甘油三酯合成酶的mRNA表达显著下降。LRP16抑制表达HepG2细胞中的结果与之相反。2. 转录组分析结果显示LRP16过表达HepG2细胞中胆固醇代谢关键酶、IRS-2的表达显著下降，脂肪合成关键酶表达明显增强。3. 高脂喂养16周的敲除小鼠与野生型小鼠相比，其体重、空腹血糖、空腹胰岛素、HOMA-IR、肝脏甘油三酯与胆固醇含量无明显差异，葡萄糖耐量试验结果显示糖负荷后的血糖显著下降。4. 高脂喂养的敲除小鼠肝脏中胰岛素、脂联素信号通路显著上调，糖酵解、甘油三酯关键酶的mRNA表达显著增强，糖异生关键酶mRNA表达明显降低。..结论：LRP16基因对脂联素功能和胰岛素功能具有负向调控作用，抑制LRP16基因的表达可以一定程度上缓解高脂诱导的胰岛素抵抗，其机制已确定包括上调胰岛素PI3K信号通路以及脂联素AMPK信号通路，进而上调糖酵解途径关键酶的表达，下调糖异生途径关键酶以及甘油三酯合成途径关键酶的表达。