SIRT4/NAD+介导的SIRT3活性调节及其在2型糖尿病线粒体功能中的作用研究

Mitochondrial homeostasis disorder is closely associated with aging and aging-related diseases (i.e. type 2 diabetes). Previously we found mitochondrial loss and dysfunction in multiple tissues of type 2 diabetic rat model. However, the mechanism underlying mitochondrial dysfunction is unclear. The mitochondria-located SIRT3 and SIRT4 play critical roles in the development of type 2 diabetes, but how these two molecules interact with each other to regulate mitochondrial function is unknown. SIRT3 activity can be modulated by mitochondrial NAD+ level, whereas SIRT4 has a much higher affinity to NAD+ compared with SIRT3. Our previous study shows that protein level of SIRT4 is increased in liver of NAFLD patients, indicating the possibility that SIRT4 competes for mitochondrial NAD+, leading to inhibited SIRT3 activity, and resultant mitochondrial dysfunction and development of type 2 diabetes. By using Sirt4 single- and Sirt3/Sirt4 double-gene knockout mice as well as mitochondrial-specific NAD+ biosensor transgenic mice, we will make a deep research on mitochondrial NAD+-dependent mechanism underlying regulation of SIRT3 and SIRT4 activity, and its further role in regulation of mitochondrial function. This study will provide potential new targets for the treatment of type 2 diabetes as well as other aging-related diseases.

线粒体稳态失常与衰老及相关疾病（如2型糖尿病）密切相关。我们的前期研究发现，2型糖尿病大鼠模型中线粒体数量和功能均明显降低，但线粒体功能下降的机制不清楚。线粒体SIRT3和SIRT4均参与2型糖尿病病理调节，但两者间存在何种互作机制，进而调控2型糖尿病中线粒体功能不明确。SIRT3活性受线粒体NAD+水平调节，而SIRT4与NAD+的亲和力远高于SIRT3。我们的数据显示脂肪肝患者肝脏组织中SIRT4蛋白水平升高，提示SIRT4可能通过竞争性消耗线粒体内NAD+而抑制SIRT3活性，进而导致线粒体功能下降和2型糖尿病发生。本项目将利用Sirt4基因敲除及Sirt3/Sirt4双基因敲除小鼠，结合线粒体特异的NAD+荧光探针转基因小鼠，深入解析线粒体NAD+水平介导的SIRT3和SIRT4活性调节机制，及其在线粒体功能调控中的作用，为防治2型糖尿病和其他衰老相关疾病提供新的可能靶点和思路。

线粒体稳态失常与衰老及相关疾病（如2型糖尿病）密切相关。线粒体SIRT3和SIRT4均参与2型糖尿病病理调节，但两者间存在何种互作机制，进而调控2型糖尿病中线粒体功能不明确。重点关注2型糖尿病发生发展过程中肝脏的脂肪性病变，本项目拟验证的假设为，SIRT4通过竞争性消耗线粒体NAD+从而抑制SIRT3活性，进而导致肝细胞氧化应激和肝组织病变。.实验发现，高脂饮食小鼠体重及血糖显著高于正常饮食小鼠；其肝脏内SIRT4蛋白水平显著升高，SIRT3及其靶标蛋白SOD2蛋白水平未见变化，但SOD2乙酰化水平显著升高，提示SIRT3去乙酰化活性减低；与此同时，肝组织整体NAD+水平以及线粒体NAD+水平均显著降低。其次以棕榈酸处理小鼠原代肝细胞作为肝脏脂肪性病变细胞模型，发现棕榈酸可浓度依赖性降低肝细胞整体NAD+水平以及线粒体NAD+水平；同时SOD2乙酰化水平呈棕榈酸浓度依赖性升高；但在该细胞模型中，我们观察到SIRT3和SIRT4蛋白表达水平亦有显著降低，可能与棕榈酸诱导了肝细胞脂性凋亡、造成整体蛋白合成减弱降解增加有关，后续可优化棕榈酸浓度等实验条件或考虑采取高糖处理。.为探索SIRT4与线粒体NAD+水平之间的直接关系，我们随后构建了SIRT4过表达载体，并在人肝癌细胞系HepG2细胞进行了SIRT4高表达。过表达SIRT4的HepG2细胞增殖能力显著减弱，细胞耗氧量显著减低，提示线粒体功能损伤。但与预期不符，我们发现SIRT4过表达引起HepG2细胞整体NAD+水平以及线粒体NAD+水平显著升高。.2型糖尿病发生发展伴随着肝脏内整体及线粒体NAD+水平降低、SIRT4蛋白水平升高及SIRT3活性降低；但后续细胞水平实验未能发现SIRT4水平升高与线粒体NAD+水平降低存在直接关系，因而暂未支持项目拟验证的SIRT4通过竞争性消耗线粒体NAD+从而抑制SIRT3活性，进而导致肝细胞氧化应激和肝组织病变的假设。