Autophagy Overexpression Stimulates CTSB-mediated Expression Of NLRP3-dependent IL-1?

Diabetes is a common serious disease and the number of patients is on the rise every year.The current epidemic of type 2 diabetes mellitus is largely associated with wide spread obesity.Among multiple mechanisms,the best-established connection between obesity and diabetes is the elevated or deregulated levels of circulating free fatty acids(FFA).There are two main hot researches that focus on the pathogenesis of T2D.One is the exploration of autophagy.The study of autophagy has undergone great advances in the past few years and it is clear that autophagy plays a critical role in ? cells.Another research focus on the inflammation.Increasing evidences strongly suggest that inflammation is involved in the pathology of T2D.Many inflammatory factors,especially IL-1? can be used to predict diabetes and many therapeutic drugs for inflammation are used to treat diabetes.However,it is not yet known the relationship between autophagy and inflammation in the pathology of obesity-related T2D.Investigation on the relations between autophagy and inflammation will help us better understand the pathophysiology of obesity-related T2D.Thus,we conducted the following experiments:1.Based on previously published papers,our autophagy model was established by use Paltimate(PA)-a major dietary saturated fatty acid in INS-1 cells.We first incubated INS-1 cells with different concentrations of PA to find the optimal concentration that can balance autophagy and apoptosis;Next we processed the PA at different time point to find the best treatment time;Autophagy and apoptosis relationship experiments were performed to test whether the autophagy model of INS-1 was successful.All the results suggested our autophagy model was successfully established and we could further investigate the effect of autophagy overexpression using this model.2.We next proceeded to test the expression of IL-1 ? under autophagy overexpression conditions.We found that autophagy overexpression after Atg7 transfected into INS-1 caused a significant induction of IL-1 ?.Necrosis inhibitor Necrostatin-1 and autophagy inhibitor 3-methyladenine(3-MA)were used to further confirm the increased expression of IL-1 ? was autophagy-mediated inflammation activity in this study.3.CTSB,an important caspain protein in lysosome,is responsible for degradation of various ubiquitinated proteins and organelles in autolysome.CTSB is also associated with NLRP3 inflammasome activation in some cells.To determine whether CTSB was involved in the process of inflammation in this model,levels of IL-1 ? was detected in INS-1 cells by inhibiting CTSB.Results showed that siCTSB suppressed IL-1? expression.This indicated CTSB was a molecular linker between autophagy and the proinflammatory factor IL-1 ?.To further investigate the effects of NLRP3 on autophagy overexpression induced IL-1?,we silenced NLRP3 by transfering siNLRP3 into INS-1,the expression of IL-1? and caspase-1 within cells and in supernatants were assessed by immunoblotting respectively.All datas indicated IL-1? production was mediated by CTSB and it was a NLRP3-dependent expression way in INS-1 cells.In conclusion,we found that autophagy and inflammation were interrelated in INS-1 cells.Overexpression of autophagy could promote inflammatory reactions.Our data suggested a novel pathogenesis mechanism of diabetes,which may yield novel potential therapies for the disease and have great significances for basic research.