Changes In The Expression Of Substance P In Diabetic Rats And Its Effects On Islet

Diabetes mellitus(DM)has emerged as a major threat to health all over the world,including retinopathy,nephropathy,coronary artery disease and stroke.Of note,DM is a well-recognized heterogeneous group of metabolic disorders,which has elevated levels of glucose in blood and lack of insulin.Insulin is one kind of hormone synthesized in β cell of pancreas to mediate blood glucose.Besides storage of glucose,insulin could also inhibit glucagon secretion.SP is a member of the family of tachykinins,which is also an 11 amino acid peptide.Usually,SP interacts directly or indirectly with neurokinin-1(NK-1)receptor on nerves,while in inflammatory cells to execute its function.Interestingly,SP is also discovered as a pain-sensing neurotransmitter in the central nervous system(CNS)and act as an injury messenger and immune modulator in peripheral tissues.There are studies showed that after injected SP into non-obese diabetic(NOD)mice,insulin resistance,chronically inflamed islets,and diabetic symptoms were reversed.In our lab,we have found that the pancreas had much more SP expressed left than the celiac ganglia after surgical denervation of major extrinsic source,which impaired that there might be other extrinsic SP sources innervating the pancreas.Previous studies have demonstrated that α cell could produce GLP-1 to improve the function of β cell,which is considered to be physiological response to the self-protection of pancreas during the development of DM.However,little is known about the factors which affect the secretion of GLP-1 from α cell.By combined with GLP-1 receptors in various organs and tissues of the whole body,GLP-1 plays an essential role in promoting insulin synthesis and secretion,inhibiting β cell apoptosis,improving cell proliferation and regeneration,reducing glucagon secretion,decreasing food intake,delaying gastric emptying,enhancing glucose utilization in peripheral tissues and reducing glycogen output.The hypoglycemic characteristics of GLP-1 is that under higher blood glucose condition,it promotes insulin secretion;while under normal condition,it does not work.GLP-1could significantly improve the function of islet β cell,at the same time decreasing blood glucose safely and effectively.Therefore,it is important to find a safe and effective method to promote the secretion of GLP-1.In this present study,we examined the potential functions of SP in islet clusters of rats.Moreover,these data also showed that pancreatic nervous system connects with pancreas islet and α cell had NK-1 receptor around islet cells.In summary,SP might be a promising therapeutic strategy for treating diabetes mellitus in patients.Method: Part one: Changes in the expression of SP in diabetic ratsMale Sprague-Dawley(SD)rats weighing 200-250 g were obtained from China Medical University.They were housed in a temperature-and humidity-controlled environment under a 12-h light/dark cycle with food and water ad libitum.STZ was given at 1% concentration in citric acid buffer(pH 4.5)to rats once.Seven days later,rat pancreas were used for further test.The rats were anesthetized by 5ml·kg-1 urethane(Sigma,USA)intraperitoneally before any experiments.The co-expression of PGP9.5 and SP of isolated pancreatic ganglia were investigated by immunohistochemical staining.The pancreas were collected for or western blot and sp elisa.Part two: The effects of substance P on isletThe islets were cultured in RPMI 1640 supplemented with 5.5 mmol·L-1 glucose and 10% FBS at 37°C in 5% CO2 and humidified air.Islet survival was assessed by double staining with membrane exclusion dyes,FDA/PI staining.Each group contained 20 to 30 islets.Purified islet cell clusters were divided into 4 groups: control,SP(10-3 μM)group,L703606(NK-1 receptor inhibitor,1 μM),and SP(10-3 μM)& L703606(1 μM)group for detecting the excretion function of islet.Islets were cultured with or without SP for 3 d.NK-1 receptor antagonist,L703606 was added for 2 h before SP.The supernatant was collected at the 3 d.Insulin kit(Millipore,USA),GLP-1(Millipore,USA)and glucagon kit(R&D,USA)secretion from cultured islets were measured with enzyme-linked immunosorbent assay(ELISA).Immunofluorescent staining was used to detect β-cell by insulin,α-cell by glucagon and NK-1 receptor.Each group contained 30 to 50 islets.Result: Part one: Changes in the expression of SP in diabetic ratsThe immunoreactivity to SP was found in 21.3±1.7%(n=5,p<0.05)of PGP 9.5-positive intrapancreatic neurons in DM rats was decreased compared to control group(34.0±1.2%).Densitometry of the bands corresponding showed that the expression of SP in DM rats 76.7±11.6%(n=5,p<0.05)was decreased compared to control group 103±10.7%.The expression of SP in DM rats 1903.3±125.2 pg/ml(n=5,p<0.05)was decreased compared to control group 2989.72±101.3 pg/ml with elisa.Part two: The effects of substance P on isletIslets were then cultured for 3 days with or without SP(10-9 M)then collected the supernatant and measured with ELISA.The insulin release in SP group(3.6 ± 0.2 μIU/IEQ)was significantly increased(p < 0.05),compared to control group(2.8 ± 0.3 μIU/IEQ).The GLP-1 release of SP group(6.3 ± 0.4 pM/IEQ)was increased(p < 0.05),compared to control group(4.4 ± 0.3 pM/IEQ).The glucagon release of SP group(31.2 ± 9.4 pM/IEQ)was not significantly decreased(p > 0.05),compared to control group(43.9 ± 11.7 pM/IEQ).After adding the antagonist of NK-1 receptor,L703,606,the insulin release in L-703,606 group(2.6±0.1 μIU/IEQ)was significantly decreased(p < 0.05),compared to SP group(3.6±0.2 μIU/IEQ).The GLP-1 release of L-703,606 group(4.2±0.5 pM/IEQ)was significantly decreased(p < 0.05),compared to SP group(6.3±0.4 pM/IEQ).The glucagon release of L-703,606 group(22.8±6.4 pM/IEQ)was not significantly decreased(p > 0.05),compared to SP group(31.2±9.3 pM/IEQ).Then low and high doses of glucose(2.7 and 16.7 mM)were given to islet cell clusters for 24 h to evaluate the secretion of GLP-1.The GLP-1 release of high doses of glucose group(11.7 ± 2.1 pM/IEQ)was increase(p < 0.05),compared to low doses of glucose group(2.6 ± 0.7 pM/IEQ).Moreover,in 24 h,the secretion increased with time.Furthermore,the results of immunofluorescence showed pancreatic nervous system connected closely to pancreas islet and α cell had NK-1 receptor around the islet cells.Conclusion : In summary,in the development of diabetes,substance p decrease SP is increased and insulin release through the action on NK-1 receptors in α cells stimulating GLP-1 release in cultured rat islets.