Regulatory Mechanism And Function Study Of Targeted Deletion Of Deptor Gene In Pancreatic β-cells

BacgroundDiabetes mellitus is a metabolic disorder disease characterized as hyperglycemia owing to insulin secretion deficiency and insulin functional disturbance.Thus,a major goal of diabetes research is to reinforce the mass and function of insulin secreting in pancreatic β cells.The functions of DEP-domain containing mTOR-interacting protein(DEPTOR)are diverse in different types of cells.In skeletal myocytes,glucose stimulates insulin-independent AKT channel activation through DEPTOR to maintain postprandial glucose homeostasis.DEPTOR overexpressed in brain tissue can activate Akt/PKB signaling pathway to prevent mice obesity from high-fat diet.Moreover,researches have confirmed mTOR complexes are key proteins regulated pancreatic β cells mass and function while DEPTOR is a common part of them.Whether DEPTOR loss in islet β cells affects insulin-secreting function and cell growth has never been identified.ObjectiveThis study aims to define whether the expression of DEPTOR in islet is correlated with diabetes and assess the alternation of insulin secretion and cell proliferation by silencing DEPTOR gene in pancreatic β cells NIT-1 while explore its possible mechanism.MethodsImmunohistochemical analysis and Double immunofluorescent labeling were used to determine the differential expression of DEPTOR protein in islet between diabetic mice and the control mice.Lipofectamine Mediated DEPTOR mRNA Silencing,cell transfection were set by six groups:(1)Blank group(transfection complexes only contained Lipofectamine);(2)negative control group(lipofectamine mediated transfection with siRNA Negative Control FAM);(3)Positive group(GAPDH);(4)siRNA DEPTOR group1(lipofectamine mediated transfection with siDEPTOR385);(5)siRNA DEPTOR group2(lipofectamine mediated transfection with siDEPTOR766);(6)siRNA DEPTOR group3(lipofectamine mediated transfection with siDEPTOR1275).Transfection effectiveness could be observed by the density of FAM-flourescence signal by means of Fluorescence microscope after 6h-transfection.Total RNA and protein were extracted after 48h-posttransfected cell culture.Quantitive-PCR was performed to detect the relative quantity of DEPTOR mRNA expression.Insulin secretion in cell conditioned medium were determined by Insulin ELISA KIT.Cell viability was measured by CCK-8 assay.Western blotting for assessment of mTORC1 and mTORC2 signaling by phosphatation of s6,4EBP-1 and akt,concomitantly measuring s6,4EBP-1 and akt as respective loading controls.ResultsImmunohistochemical analysis verified the expression of DEPTOR protein in normal mice islet while its quantity of expression is obviously lower than that in diabetic mice.Double immunofluorescent labeling also confirmed this phenomenon and testify the increased DEPTOR expression were mainly distributed in pancreatic beta cells,which showed the relation between diabetes and DEPTOR gene was explicit.Specific green fluorescence accumulated in a punctated pattern under the fluorescence microscope indicated that the effectiveness of transfection is eligible;QPCR showed two(siDEPTOR385 and siDEPTOR766)of the three siDEPTOR sequences could significantly disrupt the expression of DEPTOR mRNA,which have statistical significance between the negative control sequence(p<0.05);The total amount of insulin secretion in effective transfected groups were notably higher than negative control group(p<0.01)and also the blank group(p<0.05);The cell viability was increased in the effective transfected group(p<0.05).Western blotting showed the grey level of p-s6,p-4EBP-1 protein were remarkably elevated in effective transfected groups compared to negative control group,while p-akt of those former was slightly descedent.ConclusionTargeted deletion of DEPTOR in pancreatic β-cells improved β-cells insulin secretion and cell proliferation in a manner of mTORCl activation.