Effect Of Chloroquine On Proinsulin Folding In β Cells

Objective:In the face of continuous metabolic demands,the human body needs to store a lot of insulin for energy metabolism.Insulin is produced by the processing and shearing of insulin precursor(pre-proinsulin,proinsulin).Pre-proinsulin is translocated into the endoplasmic reticulum(ER)through transmembrane transfer,which is oxidized and folded in ER to form disulfide bonds and mature proinsulin.Because ER is the only place to synthesize proinsulin,it is easy to form misfolded proinsulin under high pressure.Excessive misfolded proinsulin accumulates in the endoplasmic reticulum,which will lead to the failure of β cells function and apoptosis.There are two outcomes of misfolded proinsulin: refolding to form correct proinsulin or degraded by endoplasmic reticulum degradation pathways.Endoplasmic reticulum associated degradation(ERAD)is a classical degradation pathway of misfolded proinsulin.However,in recent years,some misfolded proinsulin will also be degraded by autophagy,so we studied the autophagy degradation pathway.During the study,chloroquine(CQ)was used as an autophagy inhibitor to observe the effect of autophagy pathway on proinsulin degradation.In vitro experiments showed that CQ could cause a large increase of misfolded proinsulin,ER stress and redox environment changes in β cells,and finally lead to a decrease of insulin production.Not only in the cells,mice which injected chloroquine for one month,the oral glucose tolerance test(OGTT)showed that the blood glucose of CQ group was higher than that of the control group at 30 minutes and 60 minutes.Compared with the other two autophagy inhibitors,the decrease of autophagy cannot fully explain the increase of misfolded proinsulin caused by CQ.When the endoplasmic reticulum environment changes,the function of proinsulin disulfide isomerase(PDI)is very easy to change,PDI plays important roles in the oxidative folding of proinsulin.It has been proved that PDI can reduce the oxidative folding of proinsulin and increase the yield of proinsulin by forming natural disulfide bonds.At the same time,there are two forms of PDI in endoplasmic reticulum: oxidation and reduction.When it works,PDI structure is always in cyclic change,which is easily affected by the environment of endoplasmic network.So,whether CQ can cause the increase of misfolded proinsulin in ER through the interaction between PDI and proinsulin is also our focus.In fact,there are many reports about CQ’s effect on blood glucose and insulin metabolism: it can reduce insulin biosynthesis;improve glucose tolerance in noninsulin-dependent diabetes mellitus patients;affect the interaction between insulin and receptor through p H value,etc.CQ also has been playing an important role in the treatment of new coronavirus pneumonia in early 2020.At the same time,it has been used to treat various diseases such as cancer,rheumatism,malaria and so on.Therefore,it is very important whether the clinical use of CQ will affect the function of β cells,which is also one of the important purposes of designing experiments.Method: 1.Western blot was used to verify the changes of proinsulin and insulin secretion in CQ-treated HEK293 T transfected human proinsulin cells and INS1 E cells,and also verify the changes of misfolded proinsulin in mouse islets and Min6 cells.2.QRT-PCR was used to observe the changes of transcriptional level of the ER stress markers and oxidase in ER in INS1 E cells.3.CQ was injected into mice to observe the change of blood glucose.4.Western Blot was used to observe min6 cells treated with different autophagy inhibitors to observe whether chloroquine caused the increase of misfolded proteins in ER by inhibiting autophagy.5.Immunoprecipitation was used to observe the changes of the interaction between proinsulin and PDI in ins1 e cells,which were treated with CQ.Result and Conclusion:CQ can not only cause the increase of misfolded proinsulin and the decrease of insulin synthesis in β cells,but also cause the stress of endoplasmic reticulum and the change of oxidation environment of endoplasmic reticulum.The changes of glucose tolerance were observed in the mice injected with chloroquine,which might not be caused by CQ through inhibition of autophagy degradation pathway.It can influence the interaction between PDI and proinsulin is reduced.PDI has been proved to be helpful to increase proinsulin production through the formation of natural disulfide bonds.Therefore,the decrease of interaction between proinsulin and PDI may affect the correct folding and the formation of disulfide bonds.