| Objective Previous studies have shown that the activities of kallikrein-kinin system reduces in hypertension and diabetes.That bradykinin potentiates insulin stimulated glucose uptake and enhances insulin signals through the bradykinin BK2 receptor in vivo and in cultured cells.. Our previous study has demonstrated that the intravenous delivery of recombinant adeno-associated viral vector (rAAV) carrying human tissue kallikrein (HK) gene can improve insulin resistance and to prevent renal complications in STZ induced type 2 diabetic rats. But the mechanism is unknown.To further investigate its mechanisms as well as the feasibility that HK gene administration treats at the downstream of insulin receptor and diabetic neuropathy, we studied the regulation of HK on insulin signal molecules in vitro and in vivo , and the possible involved signal transduction pathways.Methods And ConclusionPart 1(in vivo)In previous study ,48 wistar rats were divided into control and experimental groups randomly. Wistar rats of experimental group were injected low dose streptozotocin (25mg/kg) and were fed with diets enriched in both glucose and fat to form type 2 diabetic model,then were divided into 2 groups randomly.And each group had 8 diabetic rats. Recombinant adeno-associated virus expressing HK gene (HK group) or LacZ gene (LacZ group) was injected into diabetic rats through tail vein,then all rats were killed after 12 weeks. In this study,the expression of HK protein in rats and the phosphorylation of insulin receptorβsubunit(IRβ) ,Akt and PI3K were detected by Western blot.And the kidney apoptotic cells were identified by terminal deoxynucleotidyl transferase mediated d-UTP nick end labeling (TUNEL).And the apoptosis associated gene expression of bcl-2/bax was identified by western blot.And the result showed ,the expression of HK protein was detected in rat main organs such as heart, liver and kidney by western-blot in the HK group.The result of western blotting show that the expression of phosphorylated IR,Akt and PI3K in kidney of LacZ group reduced significantly compared with control group,whereas kallikrein gene transfer increasd the P-IR,P-AKT.Whereas total IR,Akt levels were not altered among the three groups.The apoptotic cells of LacZ kidney were more than those in control group.And delivery of rAAV?HK can inhibit apoptosis through increasing bcl-2 and reducing bax protein expression.Part 2(in vivo) Adipocytes are rich of insulin receptors.we induced 3T3-L1 preadipocytes to adipocytes with 0.5mmol/ L IBMX,1.0μmol/ L DEX and 2mg/ L insulin.then incubated adipocyte cell with bradykinin(BK) , BK +HOE-140, BK +MAPK inhibitor(apigenin),BK +PI3K inhibitor(LY294002)and BK +PKC inhibitor(H7)inhibitors to assess the expression of phosphorylation of insulin signal molecules IRβ,AKT,MAPK as well as PI3K by western Blot. Results showed: 1.BK markedly up-regulated the expression of phosphorylation of IRβ,AKT,MAPK as well as PI3K in adipocyte cell. And the specific B2 receptor icatibate HOE140 could block the role.Conclusions Our study demonstrated that the mechanism of the rAAV-mediated HK gene delivery improving insulin resistance and diabetic neuphropathy included: (1)HK can increase the expression of IRβ,AKT phosphorylation and PI3K(2)BK can increase the expression of IRβ,AKT,MAPK phosphorylation and PI3K via BKR2 (3)HK can inhibite the apoptosis of kidney via increasing bcl-2 and reducing bax protein expression on diabetic rats.
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