MTORC1 Is Required For Maintaining Pancreatic ? Cell Identity

Background and Object As recently reported,the cells in Langerhans islets can transdifferentiate into other cells under specific circumstance.The lateset research reveals a new pathogenesis of type 2 diabetes.Under consistent metabolic stress,pancreatic ? cells lose identity,dedifferentiate to a progenitor cell then transdifferentiate into other endocrine cells.m TORC1 is an atypical serine/threonine protein kinase pathway,which integrate inputs from at least five major intracellular and extracellular cues-growth factors,stress,energy status,oxygen,and amino acids-to control many major processes,including protein and lipid synthesis and autophagy.In order to explore whether m TORC1 participate in ? cells identity maintenance,we use? cells specific Raptor knockout linear tracing mice to observe the function of m TORC1 and its mechanism involved in the pathogenesis of diabetes.Materials and Methods We use Cre-Lox P and Rosa26 system to achieve our goal.Random blood glucose and body weight were continuouslly observed from 2 to 12 weeks every two weeks.At the age of 8 weeks,we performed IPGTT and tested plasm insulin and glucagon levels to make the glucose metabolism phenotype clear.We can get the purified ? cells through FACS.We performed immunofluorescence and immunohistory to observe the islet morphology.With exogenous insulin treatment,we can make the?Rap KOGFP mice under the normal blood glucose levels thus avoiding the influence of hyperglycemia on transdifferentiation.Western blot,RT-PCR and luciferase were employed to study important transcriptional factors in transdifferentiation.Results 1??Rap KOGFP mice began to show elevated random and 6h fasted blood glucose levels.At the age of 8 weeks,plasm insulin levels decreased while plasm glucagon levels were comparable with the WT mice.Impaired glucose tolerance were observed in 8-week-old ?Rap KOGFP mice.2?? cell mass significantly decreased and insulin content decreased after raptor knockout.On the contrary,? cell exact number increased,the ratio of ?cell to ? cell raised and ? cell mass increased.Furher study demonstrated that the proliferation of ? cell was the same between WT and ?Rap KOGFP mice.3?There existed 6.63% GFP and glucagon coexpressing cells in 8-week?Rap KOGFP mice.Meanwhile,we found glucagon positive cells expressing PDX1 and Glut2.4?The electron microscopy showed ? cell lost their outer “halo”,getting the characters of ? cell.5?We observed elevated MAFB and ARX m RNA in Raptor-deficient ? cells getting from flow cytometer.6?Maf B and LAP(an isoform of C/EBP?)expression increased in?Rap KOGFP mice.After the treatment of rapamycin in INS-1 cells,the transcription and translation of LAP increased.With LAP overexpressed,the transcription of Maf B increased.With LIP overexpressed,which competed with LAP,the transcription of Maf B decreased.7?During our observation period,there existed no dedifferentiation.8?With exogenous insulin treatment,there still existed 1.32% GFP+/glucagon+double positive cells which was much higher than age-matched WT mice(0.1%).Under the control of blood glucose,islet morphology and Maf A obviously rectified.Conclusions m TORC1 is required for ? cell identity maintenance.After the ablation of Raptor,? cell mass significantly decreased,? cell mass increased and ? cells transdifferentiated into ?cells.m TORC1 led to the expression of Maf B increasing via regulating the proportion of LAP and LIP.It may provide a new therapy target by modulating m TORC1 activities in type 2 diabetes,and may help induce stem cells to differentiate into mature ? cells.