鞘磷脂代谢调控胰岛素分泌过程细胞器互作的生理病理机制研究

Insulin secretion is a cellular process involving a cluster of interactions among different organelles occurred over and over times in pancreatic beta cells. This study aims to dissect this complex flux, and to eventually develop a clearer idea about the development of insulin secretion dysfunction. Sphingomyelin is the one of the structural components of plasma membranes in eukaryotic cells. It is also enriched in the specialized micro-domains termed as “lipid rafts” on the biological membranes, which function as harbors for numerous cellular biological processes. Accumulating clinical studies reported serum sphingomyelin concentration often decreased in subjects with diabetes, when compared with those with normal glucose metabolism, and the changes in sphingomyelin levels sometimes parallels the impairments in pancreatic beta cell function. We previously found that glucose stimulated insulin secretion was compromised in the mice having lower sphingomyelin production, namely impaired glucose uptake by primary pancreatic beta cells and decreased GLUT2 expression at both basal and after stimulated levels. Besides, whole-cell clamp demonstrated significantly decreased insulin release pool volume in the sphingomyelin deficient models, which is independent of the above-mentioned changes in GLUT2 expression. This project is designed to elucidate the organelle interactome during insulin secretion in both animal and cell line models with sphingomyelin deficiency. We will take the most advantage of the Hessian-SIM platform to record the dedicate processes of how two organelles interacting with each other. This project mainly focuses on three parts, including the interactions between Glut2 transport vesicles and plasma membrane, between insulin vesicles and plasma membrane, and between insulin vesicles and endosome/lysosome systems.

胰岛素分泌涉及胰岛β细胞内多细胞器互作，包括胰岛素囊泡与细胞膜的融合分泌过程 。鞘磷脂是这些膜结构的重要组份，能与胆固醇形成脂质筏微结构域调控胰岛素分泌。临床研究证实糖尿病患者血清鞘磷脂水平下降，伴随胰岛功能减退。我们前期发现小鼠鞘磷脂减少致胰岛素分泌下降、葡萄糖转运子2（GLUT2）蛋白水平减低，胰岛RNA-seq分析示多种细胞器相关基因表达存在差异。因此我们推测鞘磷脂可通过影响脂质筏及相关蛋白功能参与胰岛素分泌过程的细胞器互作。项目利用鞘磷脂缺失动物和细胞模型，结合细胞器分离、超速密度梯度离心分离膜脂质筏组份等手段，采用活细胞超高分辨率结构光显微镜、全内反射荧光显微镜、活细胞超高Z轴分辨率显微镜等先进显微成像平台，从(1)GLUT2运载囊泡与质膜；(2)胰岛素囊泡与质膜；(3)胰岛素囊泡与内吞体/溶酶体三个方面揭示鞘磷脂代谢相关的细胞器互作在胰岛功能障碍生理病理过程中的作用机制与意义。

鞘磷脂是真核生物细胞膜结构的重要组成部分，是细胞维持正常的生理结构与生命活动的基本要素。一系列研究发现鞘磷脂紊乱与代谢性疾病如糖尿病、脂肪肝以及心血管疾病息息相关。本课题组在前期研究工作中通过测定人群血清学脂质谱测，发现肥胖、2型糖尿病的患者血清鞘磷脂存在异常。此外，通过在体离体糖刺激实验发现鞘磷脂合成酶2缺失小鼠葡萄糖刺激下胰岛素分泌能力下降，并进一步通过NBDG标记的葡萄糖证实鞘磷脂缺失胰岛β细胞葡萄糖摄取能力下降，β细胞膜上葡萄糖转运蛋白2（Glut2）蛋白表达水平下降，分布异常。CHX稳定性实验和泛素化降解实验等进一步揭示鞘磷脂缺失导致的细胞膜稳定性减低可能与Glut2泛素化降解增多有关，胰岛β细胞葡萄糖摄取能力的下降进一步影响了胰岛素囊泡分泌的能力。随后课题组进一步使用单细胞膜片钳技术发现经直接脉冲电刺激促发的胰岛素囊泡与细胞膜融合的表型探索。我们同时借助Hessian SIM、TIRF等超分辨显微镜观察了胰岛素囊泡分泌过程，并将利用此技术进一步确认鞘磷脂对囊泡运动速度、囊泡与细胞膜融合过程的影响，阐明鞘磷脂代谢调控胰岛β细胞Glut2运载囊泡与细胞膜互作影响Glut2表达分布及功能活性的具体机制，明确鞘磷脂稳态如何调控胰岛素囊泡与细胞膜融合及胰岛素的分泌。综上，本项目从细胞膜鞘磷脂的新角度，揭示了膜脂对于胰岛素分泌减少的新机制，提供了细胞膜与胰岛素囊泡的互作的细胞器互作的生理病理机制研究，为临床糖尿病诊断治疗提供了新依据。