鞘氨醇激酶1乙酰基化修饰调控糖脂代谢的作用研究

Lysine acetylation is one of the most important formation of protein post-translational modification. Recently, the tight correlation between protein acetylation and cellular energy metabolism was uncovered, as most key enzymes in glucose and lipid metabolism were regulated by acetylation modification. Sphingosine kinase 1 (SPK1) is an important lipid kinase that catalyzing sphingosine to sphingosine 1-phosphate, which serve as both intra- and extra-cellular signaling molecule. Our previous study showed that SPK1 participates in insulin signaling and plays an important role in the regulation of glucose metabolism. More recently, we demonstrated that SPK1 could be acetylated both in vitro and in vivo by p300 acetyltransferase. The acetylatd SPK1 lowered blood glucose and improved glucose tolerance markedly in KK/Ay type 2 diabetic mice. The structure and function of fatty liver of diabetic animals were also improved dramatically. Importantly, the number of pancreatic islets was increased and the size of pancreatic islets was enlarged significantly in acetylated SPK1-treated mice. Based on these observations, we will set up experiments in present study to investigate the effect of SPK1 acetylation on glucose and lipid metabolism (disorder) by using acetylated SPK1 gene modified cells, acetylated SPK1 knock-in mice, diabetic mice and clinical samples. We will also explore the roles of SPK1 acetylation in pancreatic islet cell's proliferaton, insulin secretion, and in the regulation of insulin sensitivity of peripheral tissues or organs, such as liver, muscle, and fat.

最近研究发现，蛋白的乙酰化修饰与细胞的能量代谢密切相关，参与细胞能量代谢的许多关键酶都受乙酰基化修饰调控。鞘氨醇激酶1（SPK1）是鞘磷脂代谢中的一个关键酶，催化鞘氨醇产生1-磷酸鞘氨醇，参与了多种细胞生命活动。我们前期研究结果表明，SPK1直接参与胰岛素信号通路，是调控糖脂代谢的一个关键分子。最近，我们发现SPK1是一个乙酰基化修饰蛋白。初步结果表明，乙酰化形式的SPK1能显著降低2型糖尿病动物的血糖水平，改善糖耐量和肝脏脂肪变性，并能明显刺激胰岛再生。本课题拟在此基础上，从乙酰化SPK1稳定表达细胞系、乙酰化SPK1基因敲入动物模型、糖尿病动物和临床标本等多层次分析SPK1乙酰化修饰与糖脂代谢（异常）的关系。并从两个方面：①乙酰化SPK1刺激胰岛增生的作用及机制；②乙酰化SPK1改善外周靶组织器官（肝脏、肌肉和脂肪）胰岛素敏感性的作用及机制，系统评价SPK1调控糖脂代谢（异常）的机理。

乙酰基化是蛋白质翻译后修饰的一种重要方式。最近研究发现，蛋白的乙酰化修饰与细胞的能量代谢密切相关，参与细胞能量代谢的许多关键酶都受乙酰基化修饰调控。鞘氨醇激酶1（SPK1）是鞘磷脂代谢中的一个关键酶，催化鞘氨醇产生1-磷酸鞘氨醇，参与了多种细胞生命活动。我们前期研究结果表明，SPK1直接参与胰岛素信号通路，是调控糖脂代谢的一个关键分子。最近，我们发现SPK1是一个乙酰基化修饰蛋白。初步结果表明，乙酰化修饰的SPK1能显著降低2型糖尿病动的血糖水平，改善糖耐量和肝脏脂肪变性，并能明显刺激胰岛再生。本课题在此基础上，取得了如下成果：1、通过W-SPK1、R-SPK1和Q-SPK1稳定表达细胞系和基因瞬时转染表达的手段，发现SPK1的乙酰基化可以促进其细胞外分泌，这为Q-SPK1蛋白细胞外发挥降糖降脂作用提供了实验基础；2、通过共聚焦分析显示，Q-SPK1在细胞内的分布发生了明显改变，有弥散状分布变成了点状分布；3、获得了Q-SPK1基因敲入纯合子和杂合子小鼠，并对其进行了基因水平鉴定。对其大体生长情况、各组织器官进行了组织病理学分析，没有发现任何异常，揭示Q-SPK1基因敲入不影响动物的正常生长和组织器官功能；通过喂养高糖高脂饮食发现，与野生型小鼠相比，Q-SPK1基因敲入小鼠体重增长缓慢，血糖血脂更趋向正常，揭示SPK1乙酰化对血糖血脂代谢具有一定影响；4、获得了Q-SPK1与人IgG4 Fc片段的融合蛋白Q-SPK1-Fc，初步动物实验数据表明，Q-SPK1-Fc具有显著地降糖效果，为研发新型降糖药物奠定了基础。