Establishment,Functionality And Antidiabetic Utility Of Heterotypic Pseudo-islets Containing Cholinergic Cells

Background:The prevalence of diabetes was 11.6% in the Chinese adult population.The prevalence among men was 12.1% and among women was 11.0%.Among patients with diabetes,only 25.8% received treatment for diabetes,and only 39.7% of those treated had adequate glycemic control.The current treatment of diabetes is limited,and the effect of treatment is not ideal,especially type 1 diabetes(T1DM).People with T1 DM need lifetime insulin injections at present.However,clinical insulin replacement therapy is difficult to maintain the stability of glucose metabolism,can not effectively prevent the development of chronic vascular disease,and the risk of hypoglycemia.The pancreas transplantion patients can regain their ability to regulate blood sugar physiologically,but the technical difficulty and the rate of post-operative complications of this surgery is high.Islet transplantation,by contrast,is easier to operate and has minimally invasion.Since the establishment of Edmonton protocol,the clinical pancreatic islet transplantation has made a great progress.However,patients with poor glycemic control,poor graft implantation and survival and the shortage of organs for transplantation,remain significant barriers to islet transplantation.In addition,immune rejection,revascularization and poor reinnervation are significant obstacles for the survival and function of islet grafts.Under physiological conditions,insulin secretion is controlled by the sympathetic and parasympathetic nervous systems.When the sympathetic nerve is stimulated,it releases norepinephrine,which inhibits insulin secretion by inducing vasoconstriction and suppressing β cell function.In contrast,when the parasympathetic nerve is stimulated,it can promote insulin secretion by releasing acetylcholine,which activates muscarinic receptors in β cells.In animal islet transplantation models,the density of the grafts’ cholinergic innervation when implanted in the liver,spleen or renal capsule was significantly lower than those islets in situ.However excessive sympathetic reinnervation could inhibit the function of islet grafts and cause islet transplantation failure.Consequently,this study attempted to simulate the cholinergic reinnervation of pancreatic islet β cells by co-culture of rat pancreatic β-cell line INS-1 and cholinergic NG108-15 cells,demonstrate that cholinergic cells can enhance pancreatic β-cell functions in vitro and in vivo,contribute important new knowledge to the fields of islet transplantation and β-cell replacement therapy.Methods:1.Cell viability and cell count were measured using CCK-8 kits for optimization of co-culture medium and proliferation assay of co-culture system.2.INS-1 cells with green fluorescence label and NG108-15 cells with red fluorescence label were established using lentiviral transfection.3.The cholinergic reinnervation of beta cells was simulated by 2D and 3D co-culture of INS-1 and NG108-15 cells.3D co-culture was operated using ultra-low attachment flat bottomed tissue culture plates.4.Glucose-stimulated insulin secretion was detected using insulin ELISA kits.5.E-cadherin expression was detected by immunofluorescence and western blot.6.The role of E-cadherin in the formation of heterotypic pseudo-islets was analysed using antibody incubation experiment.7.Proliferation of INS-1 cells was detected by EdU incorporation assay.8.Type 1 Diabetes was induced using streptozotocin(STZ)in nude mice.9.Subcutaneous pseudo-islet transplantation was operated using subcutaneous injection.10.The function of pseudo-islet grafts was assessed blood sugar monitoring and intraperitoneal glucose tolerance test(IPGTT).11.Distribution of heterotypic pseudo-islet grafts was examined using HE and immunohistochemistry staining.Results:1.Aminopterin in HAT was toxic to INS-1 cells;Sudden removal of HAT from media caused a significant decrease in viability in NG108-15 cells;INS-1 cells and NG108-15 cells could be passaged normally in INS-1 medium supplemented with 1×HT.2.INS-1 cells with green fluorescence label and NG108-15 cells with red fluorescence label were established successfully;In 3D co-culture conditions,NG108-15 cells and INS-1 cells adhered to each other and grew into an islet-like spheroid(heterotypic pseudo-islet).3.Carbachol could significantly increase the insulin secretion levels of INS-1 cells under high glucose conditions,but carbachol did not affect their insulin secretion levels under low glucose conditions;In 2D culture conditions,20 mM glucose induced a 1.24-fold increase(p<0.0001)in insulin secretion from co-culture group,while in the 3D culture condition,a 1.78-fold increase(p<0.0001)in insulin secretion from heterotypic pseudoislets group was observed.There were no differences in insulin release between the groups at low glucose(1.1 mM)concentrations.Independent NG108-15 clusters could not increase insulin release of INS-1 PIs.Glucose-stimulated insulin secretion(GSIS)from 2D INS-1 cells showed minimal changes when compared to 3D structures.4.E-cadherin is expressed in INS-1 and NG108-15 cells;Antibody incubation inhibited the formation of heterotypic pseudo-islets.And 3D culture induced the upregulation of E-cadherin expression in both INS-1 and NG108-15 cells.5.Carbachol and NG108-15 cells hardly affected the proliferation of INS-1 cells in vitro.6.Heterotypic pseudo-islets transplantation recipient mice reverted to normoglycaemic levels(<11.1mmol/l)faster(p<0.0001)compared to INS-1 pseudo-islets recipient mice.7.Heterotypic pseudo-islets transplantation recipient mice had a greater blood glucose clearance(p<0.05)compared to INS-1 pseudo-islets recipient mice.8.Pseudo-islets were gathered into a mass rather than diffusely distributed under subcutaneous space after injection.Conclusion:INS-1 medium supplemented with 1×HT was suitable for short-term co-culture of INS-1 cells and NG108-15 cells;INS-1 cells and NG108-15 cells could form heterotypic pseudo-islets under 3D culture condition;Cholinergic NG108-15 cells could enhance the GSIS capacity of INS-1 β-cells;E-cadherin played an important role in the formation of heterotypic pseudo-islets;NG108-15 cells hardly affected the proliferation of INS-1 cells in vitro;Heterotypic pseudo-islets transplantation recipient mice reverted to normoglycaemic levels faster and had a greater blood glucose clearance.