A Preliminary Study On The Regulation And Mechanism Of Insulin In Type2Diabetic Rats With Injection Of Cocaine And Amphetamine Regulated Transcript Via Lateral Ventricle

1. Background and objectiveType2diabetes is a metabolic disease, the pathological process is caused by insulinresistance with relative insulin deficiency becoming insulin secretion defect associated withinsulin resistance, so the body can not use of the insulin effectively and the insulin inpatient is deficient; one of the main reasons is that insulin secretion disorder；typicalsymptoms are polyuria, polydipsia, polyphagia and weight loss；the main feature is theincreased chronic blood glucose levels；long-term high blood glucose levels are toxiceffects on islet β cells, cause the disorder of lipid metabolism, hurt the heart, kidney, liverand nervous, leading to cardiovascular complications, diabetic nephropathy, liver disease,neuropathy and peripheral vascular disease and other complications. Therefore to promoteinsulin secretion and reduce the blood glucose levels become an important treatment fortype2diabetes.Cocaine and amphetamine regulated transcript (CART) is an important endogenousneuropeptide, which is widely distributed in the brain and gastrointestinal systems andinvolved in regulating a variety of physiological functions such as food intake, body weight,sensory perception, central nervous system, autonomic nervous system and endocrine.Studies have shown that human CART gene is located on chromosome5q13-14which hasbeen proved to be a susceptibility locus for obesity. The finding is further proof of CART isinvolved in feeding regulation and energy balance in human obesity. Human CART genemutation makes lower metabolic rate and a higher incidence of type2diabetes. In CARTknockout mice have found that β cell morphology have changed, glucose-stimulated insulinsecretion (GSIS) function is suppressed, significant weight gain, insulin dysfunction, mayhave prompted CART maintaining normal islet function. CART expression except in thepancreatic endocrine cells, while the nerve fibers in the pancreas and pancreatic ganglion cell body also have a widely expressed. Rat pancreatic tissue parasympathetic can stimulateinsulin secretion and therefore CART expression in the pancreas parasympathetic promptsCART not only can regulate islet function which is control by endocrine cell, but alsothe one controlled by parasympathetic nerve. CART as a new type of neuropeptide drugs,has the advantages of high activity, small dosage, low toxicity and good tolerance. Becauseof the characteristics of control of islet cells and regulation of insulin secretion in thetreatment of type2diabetes, it becomes a hot research drugs. Lateral ventricle connectswith various important nucleuses which is an important hub of cerebrospinal fluidcirculation. Clinical applications and trials in animals in the lateral ventricle administeredas a common route of administration. Therefore, in this study we chose the route ofadministration as the lateral ventricle administration to study its effect on the regulation ofinsulin in the pancreas after injection CART, the mechanisms were studied also.2. Content2.1Part one：The effects of CART injection via lateral ventricle on the blood TC, TG,HDLC, LDLC and HbA1C in type2diabetic rats.2.2Part two：The effects of injection of CART via lateral ventricle on FBG、bloodglucose AUC、serum insulin AUC and tissue insulin in type2diabetic rats.2.3Part three：The effects of injection of CART via lateral ventricle on INS mRNAexpression of pancreas in type2diabetic rats.2.4Part four： Location of CART and INS in rat pancreatic tissue and CARTintervention via lateral ventricle affect on the expression of CART and INS in pancreatictissue.2.5Part five：CART impact on INS-1cell insulin secretion in type2diabetic rats3. Method3.1Animal grouping and establishment of model of type2diabetic rat Healthymale SD rats were fasted for12hours and measured the blood from tails. The rats whoseFBG is less than10mmol/L were divided into six groups randomly：⑴Control group；⑵Control/LV injection of ACSF group；⑶Control/LV injection of CART group；⑷Type2diabetes group；⑸Type2diabetes/LV injection of ACSF group；⑹Type2diabetes/LVinjection of ACSF group. The type2diabetes group、the type2diabetes/LV injection ofACSF group and the type2diabetes/LV injection of CART group were fed with a high sugar, high fat diet for4weeks. After they were fasted for12hours, to establish model oftype2diabetic rats with40mg/kg streptozotocin (Streptozotocin, STZ) via intraperitonealinjection. Mesured the blood from tails72hours later and the rats whose FBG were morethan16.7mmol/L were type2diabetes.3.2The lateral ventricle injection of ACSF/CART and detection of blood TC, TG,HDLC, LDLC and HbA1C. To determine the successful model24hours later, the fastingcontrol/LV injection of ACSF group、the fasting control/LV injection of CART group、thefasting type2diabetes/LV injection of ACSF group and the fasting type2diabetes/LVinjection of ACSF group were injected anesthesia. We used stereotaxic apparatus with ratbrain atlas to locate the lateral ventricles, drilled in the skull and injected into0.2nmolACSF/CART. We took abdominal aortic blood24hours later and measured TC, TG,HDLC, LDLC and HbA1C in blood.3.3The effects of the intervention of injection of CART via lateral ventricle on bloodglucose AUC and serum insulin AUC. We detected the blood glucose and serum insulinand compared the change of blood glucose AUC and serum insulin AUC in each group.3.4The effects of the intervention of injection of CART via lateral ventricle on thepancreatic insulin in rats. To measure the changes of insulin in pancreas intervened byCART with ELISA.3.5The effects of injection of CART via lateral ventricle on CART mRNA expressionin rat pancreas. To observe the changes intervened by CART of INS mRNA expression inpancreas by real-time fluorescence quantitative PCR.3.6To locate CART and INS in rat pancreas and the effect intervened by CART onCART and INS. We located CART and INS in rat pancreas with immunofluorescence andcompare the change of the expression of them.3.7We observed the change of insulin of INS-1cells intervened by CART and GLP-1.INS-1cells were cultured and the insulin intervened by CART and GLP-1was measured viaELISA.4. Result4.1Detect the TC, TG, HDLC, LDLC and HbA1C in blood intervened by CARTinjection via lateral ventricle. Compare with control, the blood TC、TG、LDLC andHbA1C in type2diabetic rats were increased significantly (P<0.05)；The blood TC in type2diabetic rats intervened by CART was significantly decreased than type2diabeticrats (P<0.05).4.2The effects of CART injection via lateral ventricle on FBG、fasting serum insulin、blood glucose AUC and serum insulin AUC. We compared the FBG、fasting seruminsulin、blood glucose AUC and serum insulin AUC which were intervened by CART viathe OGTT test, it showed that FBG intervened by CART was significantly decreased(P<0.05), the fasting insulin were significantly increased (P<0.05); But in type2diabeticrats which were without intervention, the FBG was significantly increased than the control(P<0.05), the fasting insulin were significantly decreased (P<0.05); blood glucose AUC andserum insulin AUC which were intervened by CART were without obvious changes. Itsuggested that the CART intervention within24h had changed FBG and promoted thesecretion of insulin, but the properties of diabetes had not been changed.4.3The effects of CART injection via lateral ventricle on insulin in rat pancreas. Wemeasured and compared the insulin levels which were intervened by CART via ELISA, itshowed that the insulin levels in type2diabetic rats pancreas which were intervened byCART were significantly increased than that in type2diabetic rats (P<0.05).Especially theinsulin levels in control rat intervened by CART were significantly increased (P<0.05), ithinted that CART can increase pancreatic insulin levels.4.5The location of CART and INS in pancreas of rats and the effects of CARTinjection via lateral ventricle on the expression of them in pancreas. We located CARTand INS in pancreas of rats by immunofluorescence analysis and detected the expression ofthem intervened by CART. It showed that CART and INS were expressed in pancreatic βcells; both of their expression had been enhanced by the CART intervention. Thisphenomenon suggested that CART injection via lateral ventricle may enhance CART inpancreas and promote insulin secretion.4.6CART impact on type2diabetic rat insulin secretion. The insulin frompancreatic INS-1cells intervened by CART and GLP-1was measured via ELISA. Theresult showed CART and GLP-1could significantly stimulated INS-1cells (P <0.05),GLP-1effect is more significant (P <0.05).5. Conclusion5.1In this study, high sugar and high fat diet/high dose STZ intraperitoneal injection induced rise in fasting blood glucose and lipid metabolism, to establish the model of type2diabetes. The blood TC and FBG in type2diabetic rats which were taken CART injectionvia lateral ventricle24hours later were significantly decreased, the fasting serum insulinwere significantly increased, but the blood glucose AUC and serum insulin AUC werewithout significant change. This phenomenon suggested that CART can promote thesecretion of insulin and improve the metabolism of TC, but the properties of diabetes hadnot been changed.5.2The insulin and INS expression in pancreas which were intervened by CARTinjection via lateral ventricle were significantly increased. It suggested that CART canpromote the secretion of insulin in β cells. The effects were more significant in the case of βcells were undamaged.5.3Immunofluorescence analysis showed CART and INS both were in pancreatic βcells, the expression of them intervened by CART were significantly enhanced. It showedthat the CART injection via lateral ventricle can raise the CART expression and furtherpromote the secretion of insulin. This phenomenon suggested that CART may play a role ofpromoting insulin secretion.5.4Islet INS-1cells were intervened by CART and GLP-1can increase insulinsecretion, the effect of GLP-1is more significant than CART.