The Study Of Mechanisms For Dynamin-Related Protein 1 Participate In High Glucose Induced Pancreatic Beta-Cell Apoptosis

The beta cell dysfunction is an important characteristic of the late stage ofType 2 diabetes. High glucose induced-beta cell apoptosis played a crucial role in theprocess of beta cell dysfunction. It is important for understanding the pathogenesisof type 2 diabetes development and for its prevention and treatment to furtherinvestigate the molecular mechanism of high glucose-induced isletβcell apoptosis.Dynamin-related protein1 (Drp-1) was a main factor in mitochondrial fission.Mitochondria underwent rapid, extensive fragmentation early in the apoptotic process.Drp-1 gene expression was up-regulated by high glucose. But the relation betweenexpression of Drp-1 gene and high glucose inducedβcell apoptosis was not wellunderstood.Objectives: To explore the effect of Dynamin-related protein 1(Drp-1) geneoverexpression on high glucose induced beta-cell dysfunction.Methods: The inducible wild-type Drp-1(Drp-1WT) and dominant-negative mutant ofDrp-1(Drp-1K38A) stable cell lines were respectively established using Tet-on Systemin INS-1 cells. The expression of Drp-1 induced by doxycycline in the both cell lineswas confirmed by immunofluorescence staining and western blot. The cell viability andinsulin secretion were analyzed when the both cell lines were cultured in standard orhigh glucose conditions with or without doxycycline. The activities of the fourcomplexes of the respiratory chain were assayed using biochemical methods.Theproduction of ATP in Drp-1WT and Drp-1K38A cells was estimated usingbioluminescent methods.The doxycycline induced cell apoptosis was detected byAnnexinⅤ-PI, Hochest33342-PI,TUNEL staining and DNA fragmentation, respectively. The mitochondrial morphology and membrane potential, the release ofcytochrome C, Caspase-3 activity and the reactive oxygen species (ROS) generationwere further investigated for understanding the relevant mechanisms of the cellapoptosis.Results: Drp-1 expression was significantly induced in the both inducible cell lines inresponse to doxycycline. After induction of doxycycline, the cell viability and insulinsecretion were reduced in Drp-1WT cells, but not in Drp-1K38A cells, especially inhigh glucose condition. It was found that the activities of the four complexes of therespiratory chain in the mitochondria of Drp-1WT cells and Drp-1K38A cells werealmost no change after Dox induction in standard glucose or high glucose condition.But the production of ATP in the mitochondria of Drp-1WT cells in high glucosecondition was decreased after Dox induction. The doxycycline induced cell apoptosiswas more in Drp-1WT cells and was less in Drp-1K38A cells in high glucose condition.Furthermore, the mitochondrial fission and the release of cytochrome C were observedin Drp-1WT cells by electron microscopy and confocal microscopy. The mitochondriamembrane potential was decreased and Caspase-3 activity and ROS generation weresignificantly increased in Drp-1WT cells, but were less in Drp-1K38A cells.Conclusions: Drp-1 expression may participate in high glucose induced beta celldysfunction.