高脂诱导的细胞嘌呤核苷酸异常代谢和调控的分子机制

Cell damage caused by high-fat-induced cellular stress response is closely associated with cardiovascular and metabolic disease, but how high-fat diet triggers such as oxidative stress, endoplasmic reticulum stress and its really starting signal and transduction mechanisms are unclear. This project will investigate the releasing mechanism of intracellular prine nucleotide to extracellar that cuased by high-fat-induced apotosis and damage of vascular endothelial cells by using cell culture and multiple obese mice models. A stably typical prine extracellular purine nucleotide signal will be clarified and it will be investigated to be as an upstream signal and the mechanism of this signal initiation in high-fat-induced cell stress and. We will analyze purine nucleotide receptors knockout mice and culture cell treating with agonist and antagonist for receptors to explore the mechanism that high-fat-induced extracellular purine nucleotide signal transduct into cells, and demonstrate the control machanism of adenosine transporter on adenosine getting in and out cell. Furtherly, we will investigate the direct effects of extracellular purine nucleotide on the activity of enzymes that closely related to high-fat-induced cellular stress response. These results will illuminate the generation and transduction mechanisms of purine nucleotide signal during high-fat-induced cell stress response, and provide a better understanding of the mechanism of high-fat-induced cell damage-related cardiovascular and metabolic disease.

细胞对高脂诱导的代偿性反应导致的组织细胞损伤与代谢疾病和心血管的疾病密切相关，但是高脂饮食如何触发诸如胰岛素抵抗及代谢异常等生理状态的真正的起始信号及传导机制还不清楚。本课题将采用细胞和多种肥胖的小鼠模型，研究高脂状态下血管内皮细胞的凋亡和破坏造成的胞内高浓度的嘌呤核苷酸向胞外释放的生化机理；确定高脂状态下具有典型标记的胞外嘌呤核苷酸信号以及作为高脂应激上游信号的发生机制；通过分析嘌呤核苷酸受体的基因敲除小鼠以及细胞受体的功能控制产生的效应，探索高脂诱导的胞外嘌呤核苷酸信号传入细胞的机制；研究腺苷通道蛋白对腺苷进出细胞的控制以及在嘌呤核苷酸信号传导中的关键角色；系统地研究胞内嘌呤核苷酸对高脂应激反应中重要代谢酶活性直接的调控功能，结果将阐明细胞高脂诱导的代谢异常中嘌呤核苷酸信号的发生和传导机制，并加深对高脂应激损伤相关疾病发生和发展的认识。

二型糖尿病是威胁人类健康的最重要疾病之一。高脂饮食诱导的代偿性反应导致的细胞损伤与包括二型糖尿病在内的代谢性疾病和心血管疾病相关，但高脂饮食引起代谢异常等生理状态的起始信号仍未知。我们在多种糖尿病模型小鼠以及二型糖尿病患者中发现了血浆中5’-AMP的含量是上升的，血浆中5’-AMP含量的上升是二型糖尿病发生的重要标志。游离脂肪酸引起血管内皮细胞和红细胞释放腺嘌呤核苷酸是胞外5’-AMP的重要来源。另外，肝脏和肌肉中ATP和腺苷酸代谢产物含量上升，这与注射5’-AMP的野生型小鼠的变化类似。向四种腺苷受体敲除小鼠（A1 -/-，A2a -/-，A2b -/-，A3 -/-）注射5’-AMP，其血糖的变化与野生型小鼠基本一致，说明5’-AMP引起高血糖不依赖腺苷受体的作用。小鼠实验结果显示外源5'-AMP使得小鼠的肝糖原明显减少，并增加丙酮酸作为底物的糖异生作用，5’-AMP在肝脏和骨骼肌中能够抑制胰岛素引起的胰岛素受体（IR）自磷酸化以及IR下游蛋白AKT的磷酸化水平，并且呈剂量依赖性。Docking分析显示5’-AMP导致的高浓度腺苷是IR酪氨酸激酶潜在的抑制剂，从而削弱了胰岛素的信号通路。这些结果表明，5’-AMP和胰岛素之间相互影响，共同调控血糖的动态平衡。在给小鼠注射 5'-AMP 后 1h胞内的腺苷含量明显上升，使得S-腺苷-L-蛋氨酸（AdoMet）与S-腺苷高半胱氨酸（AdoHcy）的比例降低，进一步减少了叉头蛋白家族1（Foxo1）、葡萄糖-6-磷酸酶（G6Pase）和磷酸烯醇式丙酮酸羧激酶（PEPCK）基因启动子区域的组蛋白 H3K9 二甲基化（Me2-H3K9）水平，导致 Foxo1、G6Pase 和 PEPCK 基因表达的增加，从而使血糖含量上升。5’-AMP抑制了Foxo1的出核过程，是Foxo1定位于细胞核内，增强了Foxo1的转录活性。二甲双胍可以改善5’-AMP引起的高血糖和胰岛素抵抗。这种作用与胞内pH的变化有关。5’-AMP可以使胞内pH下降而二甲双胍可以使胞内pH上升。pH的变化影响酪氨酸去磷酸化酶的酶活，从而影响胰岛素信号通路。这些数据说明了高脂引起的腺苷酸代谢异常在二型糖尿病高血糖和胰岛素抵抗中的作用。