The Effect Of Defective Endoplasmic Reticulum Export On Insulin Biosynthesis

Objective: Insulin is essential for the metabolism of glucose,as the main hormone to decrease blood glucose.The biosynthesis of insulin begins with the translation of the preproinsulin on the ribosome of the rough endoplasmic reticulum.The proinsulin is translocated into the endoplasmic reticulum to form proinsulin.After the proinsulin is correctly folded in the endoplasmic reticulum,it exits from endoplasmic reticulum into Golgi apparatus.Proinsulin is digested by enzymes into insulin and C peptide in the Golgi apparatus.Mature insulin and C-peptide are stored in secretory granules and secrete extracellular to exert biological effects under glucose stimulation.As a secreted protein,the secretion pathway of insulin is essential for the formation of active insulin.Proinsulin must correctly folded before been transported from ER.Then proinsulin is transported by the COP-? vesicles into the Golgi apparatus.Researches have confirmed that mutant insulin induced diabetes of young(MIDY)is due to the proinsulin cannot to form three disulfide bonds during the folding process,resulting in proinsulin cannot exit from ER.Studies have reported that wild-type proinsulin also form 10%-20% misfolded proinsulin during folding in ER.However,once proinsulin is blocked in ER,the steady state of proinsulin is not very clear.So this study investigate the effect of reducing proinsulin transport from the endoplasmic reticulum on insulin synthesis and proinsulin steady state and the effect on ?-cell endoplasmic reticulum stress.Methods: There are two aspects to reduce proinsulin exit from ER.One is to impair COP-? vesical assemble.The second method is that we found that chloroquine,antimalarial drugs,block proinsulin in the ER.Infection of MIN6 cells or human islets with mutant adenovirus sar1 A H79G and sar1 A T39N and control adenovirus sar1 A WT and GFP,using western blotting and immunofluorescence methods to investigate the dominant negative effect of sar1 A mutant protein on COP-? vesicles assemble,the distribution and the steady state of proinsulin and the endoplasmic reticulum homeostasis of ? cells.The coimmunoprecipitation method explores which proteins are needed for proinsulin folding in ER.In addition to the application of mutant protein to damage the assemble of COP-? vesicles,we also knock down sar1 protein to detect the distribution and the steady state of proinsulin in ER.The biosynthesis of insulin was observed by pulse-chase and western blotting method after chloroquine treatment MIN6 cells or INS-1832/13 cells.The steady state of proinsulin was detected by western blotting under reduced and non-reduced conditions.The distribution of proinsulin was detected by immunofluorescence.Results: After COP-? vesicle assemble impaired,proinsulin converses to insulin decreased.Proinsulin in the mutant group was significantly increased after cycloheximide chase 2 hours.The misfolded proinsulin significantly increased in MIN6 cells and human islets and the ERS marker of p-e-IF-2? and CHOP increased after overexpressing sar-1A mutant protein.However,after knocking down the INS-1/2 gene,the insulin synthesis was reduced and misfolded proinsulin was also reduced.At the same time,the above markers of ERS decreased.Proinsulin misfolding was observed in sar-1A/B protein knocking down group.Proinsulin interacts with BIP and PDI in ER.Chloroquine blocks proinsulin in ER and reduces insulin synthesis.Chloroquine causes proinsulin misfolding,but doesn't impair COP-? vesicle.Chloroquine does not affect the transport of carboxypeptidase.Chloroquine increases the ERS marker,p-e-IF-2?and CHOP.The ERS marker improved when misfolded proinsulin decreased.Conclusions:1.This study suggests that misfolded proinsulin is the main cause of endoplasmic reticulum stress in the ? cells2.This study revealed pathophysiological significance of efficient ER export in maintaining ER homeostasis and native folding of proinsulin.Given the fact that proinsulin misfolding plays an important role in both mutant INS-gene induced diabetes of youth(MIDY)and type 2 diabetes,targeting cellular machinery to enhance ER export may be a potential therapeutic target to prevent/delay ?-cell failure caused by proinsulin folding and ER stress.