利用基因剔除小鼠探讨sidt2在糖尿病骨骼肌肌病的作用

Sidt2 was identified by mass spectrometry in the lysosomal membrane fraction with an unknown function. It was also the subject of our previous grant from National Natural Science Foundation of China, which was completed in the last year. In that project, we developed a global sidt2 knockout mouse, which had an impaired glucose tolerance. Both of in vivo and in vitro insulin stimulating assays showed a decreased insulin secretion, which was supported by less empty vesicles of insulin secreting granules of islet under electronic microscopy. The skeletal muscles of these knockout mice showed apparent atrophy and mitochondrial abnormality, and an elevated expression of GAPDH, the rate-limiting enzyme of glycolysis, resembling the myopathy of human diabetes mellitus. In first part of this study, we plan to check which step of insulin secretion was damaged by sidt2 deletion. To answer the question whether sidt2 deletion has direct impact on the skeletal muscle or the myopathy of the global sidt2 knockout mice was induced by the impaired glucose tolerance, we plan to raise skeletal muscle-specific sidt2 knockout mice by breeding the sidt2 Flox/Flox with mice carring the Cre recombinase gene driven by the muscle creatine kinase promoter. The skeletal muscle from these skeletal muscle-specific sidt2 knockout mice would be investigated to see any signs of myopathy and the whole animal would be evaluated by glucose tolerance test. Affymetrix RNA chips would be applied to check the abundance changes at the RNA level of muscle proteins, which would be verified at the protein level by Western blot, with the focus on proteins of insulin transduction in muscle, glucose transporter 4, and proteins involved with mitochondrial biogenesis, fission and fusion. Through this study, we expect to discover the underling mechanism of myopathy in the global sidt2 knockout mice. In conclusion, sidt2 may be a new therapeutic target of diabetes mellitus and its related myopathy.

Sidt2是申请者在小鼠肝脏溶酶体膜利用蛋白组学发现的功能未知蛋白，也是2011年底结题的一项国家自然科学基金青年项目的研究主体。该项目进行过程中，我们培育了sidt2全身剔除的小鼠，该鼠糖耐量受损，体内及体外胰岛素刺激实验显示胰岛素分泌减少，电镜下胰岛素空泡型分泌颗粒减少，提示胰岛素分泌障碍。并且该鼠骨骼肌有肌萎缩和线粒体异常，糖酵解的关键酶GAPDH表达增加，提示该鼠模拟了人类糖尿病肌病发生。本项目拟行胰岛培养，研究sidt2缺陷影响胰岛素分泌的具体机制。在已有的sidt2 Flox小鼠的基础上，拟培育仅肌肉sidt2剔除的小鼠，观察这种小鼠是否与sidt2全身剔除的小鼠一样有明显的骨骼肌改变和糖耐量变化，以研究糖尿病与肌病的因果关系。并拟用RNA表达芯片及蛋白印迹等技术研究sidt2缺陷引起肌病的机制。通过该项目的研究，预期能发现sidt2是进行糖尿病及其肌病干预治疗的新靶点。

为研究溶酶体膜蛋白Sidt2蛋白的功能，本项目课题组前期成功建立了Sidt2基因全身敲除的小鼠模型，通过后续表型及相关研究发现，该小鼠模型存在糖耐量受损，与糖尿病患者的前期生化特征十分类似。分离的胰岛组织体外刺激试验显示胰岛素分泌障碍，但其胰岛素合成未见明显异常。另一方面，Sidt2基因全身敲除小鼠从第 8 周开始骨骼肌病理显示肌肉明显损伤性改变。骨骼肌与糖代谢之间联系紧密且复杂。本项目检测野生及Sidt2-/-胰岛β细胞在高剂量葡萄糖刺激下胰岛素分泌通路中NADPH，KATP、KV2.1电流及胞内游离钙离子浓度，膜电位和胰岛素分泌颗粒pH。发现Sidt2-/-小鼠胰岛β细胞而钙离子信号反应受损。在酸性细胞器第二信使NAADP孵育条件下Sidt2-/-胰岛β细胞Ca2+对高剂量葡萄糖反应正常。提示Sidt2基因缺陷导致胰岛 β 细胞NAADP信号不足，酸性细胞器钙库释放障碍，最终导致胞内Ca2+信号受损。证明了Sidt2蛋白参与了胰岛β细胞胞内NAADP信号的调控。同时，成功繁育骨骼肌特异性敲除Sidt2基因小鼠.骨骼肌特异性敲除Sidt2基因小鼠，追踪至6个月龄模型小鼠空腹血糖及糖耐量均正常。骨骼肌冰冻切片HE染色显示骨骼肌特异性敲除Sidt2基因小鼠主要病理改变为肌纤维机构紊乱，肌纤维大小不一，出现大量的中央核肌纤维，部分肌纤维内可见颗粒状物质沉积，肌纤维周围还可见炎症细胞浸润，结合肌肉电镜结果提示骨骼肌特异性敲除Sidt2基因小鼠有显著的肌病表现。WB及免疫荧光结果显示结果显示模型小鼠肌纤维自噬相关蛋白有显著变化。基因表达谱芯片分析共发现显著性表达差异基因共590 条。上调通路主要包括：吞噬、溶酶体和肌动蛋白调控等；下调通路主要涉及代谢通路和钙离子信号通路。提示骨骼肌特异性敲除Sidt2造成的肌病表现与自噬显著相关。Sidt2可能为人类未知肌病的一个候选基因。可能通过调控Sidt2蛋白改善人类血糖的稳态及肌病。