Mcl-1 And MTOR Mediates High-fat Induced Apoptosis And Functional Damage Of Pancreatic Islet ? Cells

Objective: This project aims to use high-fat-diet fed C57 mice and palmitate-induced islet ? cells to investigate the molecular mechanism that results in ? cell apoptosis and functional impairment.The implementation of the work may not only help us to further understand the pathogenesis of ? cell apoptosis caused by lipid oversupply,but also provide promising therapeutic targets for drug development and intervention.Methods: Male C57 mice were fed with normal-chew-diet(NCD)and high-fat-diet(HFD).Blood lipid profile,body weight,oral glucose tolerance,insulin tolerance and insulin secretion were measured at different feeding time.After cells were treated with different concentration of palmitate(PA)for different time,the proliferation and apoptosis of the cell and function of insulin secretion were detected by MTS,Hoechst-Propidium Iodide staining and ELISA respectively.After genes knockdown or overexpression,related proteins expression and kinases activity were examined in the absence or presence of PA by immunoprecipitation and/or western blot.Results: Compare with NCD group mice,the free fatty acid(FFA),triglyceride(TG)and total cholesterol(TC)increased significantly in the HFD group mice after four-month feeding.And body weight of HFD group was higher than the NCD group.At the same time,oral glucose tolerance and insulin tolerance were impaired in the HFD group.After HFD feeding for five to six months,the size of islets reduced and number of ? cells decreased.Consistently,glucose induced insulin secretion(GIIS)dropped.PA-induced ? cell apoptosis and functional impairment were related to PA concentration and incubation time positively.Treatment with high concentration(500?M)of PA for 24 h could reduce cell viability,increase cell apoptosis and inhibit GIIS via down regulating Mcl-1 expression and inhibiting mTOR activation.Cells were more vulnerable to low concentration(200?M)PA,which would induce cell apoptosis when Mcl-1 or mTOR was knocked down respectively.However,cells showed more resistance to the cytotoxicity of 500?M PA when Mcl-1 or mTOR was overexpressed.The experiments of Raptor or Rictor knockdown demonstrated that mTOR complex 1(mTORC1)was involved in PA-induced cell apoptosis and functional impairment.Study of PA-induced signal transduction revealed that AMPK was activated upon PA stimulation.The activated AMPK inhibited ERK activation,which resulted in lower phosphorylation of Thr163 on Mcl-1.At the same time,AMPK blocked Akt activity,which released the inhibition of Akt on GSK3?.Then the Ser159 on Mcl-1 was phosphorylated by GSK3?.The context of phosphorylation of Mcl-1 finally led to its ubiquitous degradation.On the other hand,activated GSK3? could inhibit the activity of mTOR resulting in suppression of Mcl-1 expression.Conclusion: PA stimulation could block Mcl-1 expression and inhibit mTOR activation.The dual regulation of Mcl-1 and mTOR by PA led to islet ? cell apoptosis and impairment of insulin secretion.