The Protective Role Of The MKP-5-JNK/P38 Pathway In Glucolipotoxicity-induced Islet ?-cell Dysfunction And Apoptosis

BackgroundDiabetes is a chronic metabolic disease,among which type 2 diabetes(T2DM)accounts for more than 90%of the diabetic population.Insulin resistance and islet ?cell damage are the mainly pathophysiological mechanisms of T2DM.Obesity(BMI>35 kg/m2)is a crucial inducer of T2DM and over 80%T2DM patients suffering it.Obesity characterized as adipocytes hypertrophy and secret abundant free fatty acid(FFA),which result in macrophages secreting inflammatory cytokines and chemokines and occurs inflammation,ultimately lead to tissue and systemic insulin resistance.However,the mechanism of islet ? cell damage triggered by FFA is still unclear.Mitogen-activated protein kinase(MAPK)is involved in multi stress response in vivo.Some studies reported that MAPK influenced the pathogenesis of T2DM by regulating variety of signal pathways including endoplasmic reticulum stress(ER-stress),inflammatory response,insulin resistance and insulin secretion,while the role of MAPK in glucolipotoxicity induced islet ? cell damage is not completely understood.MAPK phosphatase(MKP)can negatively regulate the activation of MAPKs by specifically dephosphorylating threonine and tyrosine.MKP-5,which was classified into class III of MKPs,has been reported to aggravate the inflammation in white adipose tissue and liver then resulted in insulin resistance when its expression been knock down.These results indicated a relevance between MKP-5 and the pathogenesis of T2DM.However,whether MKP-5 participate the regulate process of glucolipotoxicity induced islet ? cell disfunction and apoptosis is remind to be researched.PurposesIn this study,the role of MKP-5 in glucolipotoxicity induced islet ? cell damage and its mechanism would be researched by modulating the expression level of MKP-5 in primary islet and MIN6 islet ? cell line.MethodsFed the obese mice model and extracted the pancreatic tissue protein for detecting the expression of MKP-5 by western blot analysis.To up-regulating the expression level of MKP-5,a stably over-expression MKP-5 cell line was constructed by transfecting pcDNA3.1-MKP5 recombinant plasmid to MIN6 cells,which referred to MIN6-MKP5,MIN6 transfected with pcDNA3.1 empty plasmid as control(MIN6-PC).For another,MIN6 cells or primary islet cells affected the adenovirus expressed MKP-5(Ad-MKP5)to overexpressed MKP-5,All the above-mentioned cells were exposed to glucose and palmitic acid (GP),then performed the followed experiments:Annexin-V/PI staining and Hoechst 33258 staining to analysis the apoptosis rate,western blot and qRT-PCR to measure the expression or activation of inflammatory cytokines,islet ? cell function and apoptosis related proteins and MAPKs.Glucose stimulate insulin secretion assay(GSIS)and ELISA to detect the secretion level of insulin and inflammatory cytokines.Interfered the expression of MKP-5 by siRNA in MIN6 cells,then analysis the apoptosis rate of MIN6 cells by flow cytometry,the expression or activation of islet ? cell function and apoptosis related proteins and MAPKs were also measured by western blot analysis.Isolated the primary islet cells and transfected or affected with siRNA or Ad-MKP5 to modulate the expression of MKP-5,then analysis the expression of function and apoptosis related proteins by western blot.MIN6 cells exposed to siRNA and MAPK inhibitors,then stimulated with GP, the activation or expression of MAPKs and the function and apoptosis related proteins were detected by western blot.Results(1)The expression of MKP-5 was down-regulated in pancreatic tissue result from HFD-induced obesity.(2)In MIN6 cells,MKP-5 over-expression can relieve the apoptosis triggered by GP through regulate the activation or expression of factors related to mitochondria apoptosis pathway(BCL-2?BAX?caspase-3,-9 and PARP-1)and ER-stress induced apoptosis(GRP-94?IRE-1??eIF2-??XBP-1s?CHOP and caspase-12).Meanwhile,silencing MKP-5 can exacerbate the GP induced islet ? cell apoptosis by regulating mitochondria apoptosis pathway and ER-stress pathway.However,MKP-5 cannot affect the death receptor pathway during GP induced MIN6 apoptosis as no change was detected in caspase8 activation when MKP-5 overexpressed or silenced.(3)MKP-5 overexpression in MIN6 cells can improve GP triggered disfunction,which characterized as abnormal insulin secretion and function related factors(AKT?GLUT2?PDX-1?GCK)activation or expression decreased.Similarly,inhibited MKP-5 can aggravate this phenomenon.(4)The increased expression and secretion level of pro-inflammatory cytokines(IL-1??TNF-a and IL-6)and chemokine(MCP-1)induced by GP were inhibited when MKP-5 overexpressed in MIN6 cells.(5)Increased the expression level of MKP-5 in primary islet cells can relieve the apoptosis induced by obesity,which presented as down-regulated the expression or activation of factors related to mitochondria apoptosis pathway and ER-stress pathway upon GP stimulation.Otherwise,interfered the expression of MKP-5 can promote the activation of caspase-3 and inhibit the activation of AKT.(6)In MIN6 cells,MKP-5 overexpression can inhibit the activation of MAPKs(P38,JNK and ERK)triggered by GP and there was a time dependence.Silenced MKP-5 presented the contrary tendency.siRNA combined with P38,JNK or ERK inhibitor stimulated MIN6 cells uncovered that MKP-5 participate the glucolipotoxicity triggered MIN6 cells apoptosis and disfunction through regulate the P38 and JNK pathways.Conclusion and significanceMKP-5 plays a role in regulating the apoptosis and functional dysfunction of islet beta cells induced by high glucose and fat through the P38 and JNK MAPK signaling pathways.A complement of this study will afford more theoretical basis for pathogenesis of T2DM,and provide a novel potential target for T2DM therapy.Innovativeness(1)The effect of obesity on MKP-5 expression in pancreatic tissue was studied.(2)The role of MKP-5 in glucolipotoxicity induced MAPKs activation was researched.(3)Explored the role of MKP-5 in glucolipotoxicity triggered disfunction and apoptosis in MIN6 and primary islet cells.(4)Identified which MKP-5-MAPK pathways involved,the glucolipotoxicity induced MIN6 cells disfunction and apoptosis.