胰島β細胞釋放之外泌體對胰島素分泌功能之調節角色

Abstract

糖尿病(diabetes mellitus)是現今最嚴重的代謝性疾病，其主要成因是胰島素分泌不足或胰島素阻抗，而病人體內的高血糖、高血脂、高糖化終產物(advanced glycation end products, AGEs)所引起的胰島素釋放量下降以及血管功能受損，進而提升其罹患糖尿病併發症之風險。葡萄糖代謝恆定是由多重器官組織共同調控，因此器官間訊息傳遞的重要性可想而知。外泌體(exosomes)是一種直徑約50-200奈米的細胞外囊泡，其透過運輸mRNA、microRNA、蛋白質及核酸來調控細胞間訊息傳遞。文獻指出在糖尿病狀態下外泌體參與了葡萄糖及胰島素的調控。然而，外泌體是否參與在高血糖、高血脂及高AGEs所誘導的胰島beta 細胞功能受損目前仍尚未明瞭。因此本研究欲探討AGEs、高糖、高脂是否透過胰島beta細胞所釋放的外泌體進而調節胰島素釋放量。在細胞實驗中我們首先利用MTT試驗法及胰島素ELISA kit證實AGEs、CML、高糖、棕櫚酸在不影響胰島beta細胞存活率的同時仍會干擾其葡萄糖誘導之胰島素分泌，但不論在有無處理AGEs、CML、高糖、棕櫚酸的情況下，來自胰島beta細胞之外泌體皆顯著影響beta細胞之胰島素釋放量。在動物實驗中我們也發現外源性給予beta細胞釋放之外泌體至小鼠體內會顯著影響其血清胰島素含量。此外我們透過西方墨點法證實外泌體顯著降低磷酸化Akt、CaMKII及GLUT2的表現量及顯著提升GSK3、磷酸化NFB、Cox-2表現量。而通過蛋白質體學及次世代定序相關之生物資訊分析，我們推測外泌體可能是透過FAT1(相對豐富度1.71%)以及miR-1224, -122-5p, -133a-3p, -10b-5p, -423-5p, -409a-3p來影響beta細胞之胰島素釋放。總結而言，本研究前所未有的發現正常生理條件下胰島beta細胞所釋放之外泌體具有調節胰島素分泌之能力，而AGEs、CML、高糖、棕櫚酸在外泌體調節胰島素分泌之過程中並無協同作用。

Diabetes mellitus (DM) is the most serious metabolic disease around the world, which is mainly caused by insufficient insulin secretion or insulin resistance. In DM patients, the hyperglycemia, hyperlipidemia, and advanced glycation end products (AGEs) can reduce the insulin secretion levels and deteriorate the vascular function, leading to an increasing risk of diabetic complications. Glucose homeostasis is controlled by several organs and tissues, thus, the importance of message transmission between organs and tissues is conceivable. Exosomes are extracellular vesicles (about 50-200 nm in diameter) that regulate intercellular communication by transporting mRNAs, microRNAs, proteins, and nucleic acids. It has been indicated that exosomes are involved in the regulation of glucose homeostasis and insulin secretion in the diabetic status. However, it is still unclear whether exosomes play roles in impaired function of islet beta cells caused by hyperglycemia, hyperlipidemia or AGEs. Therefore, the purpose of this study was to investigate whether AGEs, high glucose, and fatty acid can regulate the insulin release via exosomes derived from islet beta cells. In the in vitro studies, we first confirmed that AGEs, CML, high glucose, and palmitic acid interfered the glucose-stimulated insulin secretion (GSIS) without affecting the cell viability of islet beta cells by using MTT assay and insulin ELISA kit. However, exosomes from islet beta cells significantly affected the GSIS regardless of treating with AGEs, CML, high glucose, and palmitic acid or not. In vivo experiments also showed that exogenously administered exosomes derived from islet beta cells into ICR mice significantly decreased their serum insulin levels. In addition, exosomes significantly reduced the protein expression of phosphorylated Akt, CaMKII and GLUT2, and increased the protein expression of GSK3, phosphorylated NFB, and Cox-2 by using western blot analysis. Through bioinformatics analysis such as protein identification and Next-generation sequencing, we speculated that exosomes may transfer FAT1 (relative abundance: 1.71%) and miR-1224, -122-5p, -133a-3p, -10b-5p, -423-5p, -409a-3p to affect GSIS in beta cells. In summary, this study has unprecedentedly found that exosomes released from islet beta cells under normal physiological conditions can regulate insulin release, whereas AGEs, CML, high glucose, and palmitic acid have no synergistic effect on the process of regulating insulin secretion by exosomes.