Relationship Between Lifespan And Altered Expression Of Klotho In Insulin Resistance Rats

Objective:To establish an animal modal of insulin resistant in newbornWistar rats induced by streptozotocin(STZ).We seek to investigate the relationship between survival time and altered expression of Klotho in insulin resistance rats. and the effect of Aminoguanidine and tetramethylpyrazine on insulin resistance rats.Methods:1.Animal and group division:The insulin resistant model of newborn Wistar rats was duplicated withstreptozocin(90mg/kg).The rats were raised for 8 weeks, and those whose fasting plasma glucose higher than or equal to 7.0mmol/L or whose 2 hours postprandial blood glucose higher than or equal to 11.1 mmol/L in glucose tolerance test were selected. Rats were divided into 4 groups randomly:normal control(CN)group,insulin resistant(IR) group, and the combination of tetramethylpyrazine and aminoguanidine(TMP+AG)group.2. The measurement of fasting blood glucose(FPG),fasting insulin(FINS) and glucosylated serum protein(GSP) Measurement FPG,FINS and GSP were detected in 8th,24 th,32 thweeks after treatmentfrom the tail vein, insulin resistance index(IRI) was count by calculation formula of FPG xFINS/22.5.3. Measurement of blood biochemical indexAt the end of the experiment, the serum total cholesterol(TC),triglyceride(TG),blood urea nitrogen(BUN),creatinine (Cr) were detected.4.Real-Time Fluorescent Quantitative Polymerase Chain Reaction(RT-PCR)The expression of Klotho and IGF-1R mRNA were measured by RT-PCR in kidney and brian; the expression of advanced glycation end products receptors (RAGE) and IGF-1R mRNA were measured in liver tissue5. Western BlotWestern blot were used to detect the protein expression of klotho,RAGE and IGF-1R in kidney and brian; the protein expression of RAGE and IGF-1R were detected in liver.6.Immunohistochemistry stainingKlotho and phosphated IGF-1R protein were detected by immunohistochemistry staining.Results:1. Survival rate of each groupAt the end of the experiment, the Survival rate in IR group, TMP+AG group and CN group were 35%,85% and 100% respectively. The Survival rate in IR group was significantly lower than that in the CN group (P< 0.05), The Survival rate in TMP+ AG group were higher than IR group (P< 0.05)2.FPG,FINS and GSPThe FPG,FINS,IRI and GSP in modal groups were higher than CN group at the age of 8 weeks (P<0.05); The index above in TMP+AG group were lower than IR group at the age of 16 weeks (P＜0.05), higher than CN group (P＜0.05); At the age of 32 weeks,The FPG and FINS in TMP+AG group were more decreased compared with IR group at the age of 24 weeks (P<0.05), the FPG had no significance compared with CN group, FINS and IRI in TMP+AG group were higher than CN group(P<0.05), there was no significant difference in GSP levels between TMP+AG group and CN group.3.Blood biochemical index ResultThe differences of serum TG, TC and Cr among groups were insignificant, the BUN in TMP+AG group was decreased significantly compared to IR group.4.RT-PCR Analyses(1) The renal expression of RAGE mRNAReal-time RT-PCR amplification revealed a significantly increase in IR group RAGE mRNA compared to the control group mRNA level (P<0.05). The treated IR rats had significantly decrease when compared with the IR group (P＜0.05), There were no differences observed between control and treated IR animals.(2) The expression of IGF-1R mRNAThe expression of IGF-1R mRNA in brain, hepatic tissues in IR group was significantly lower than CN group (P＜0.05),which was increased in brain and hepatic tissues of TMP+AG group than that of IR group (P＜0.05).No statistical differences in the expression of IGF-1R mRNA in renal tissues of CN group and TMP+AG group were found.(3) The expression of Klotho mRNAReal-time RT-PCR demonstrated a significant difference in renal and brain klotho gene expression between the control and IR groups,a significantly decrease in the IR group when compared with the control group (P＜0.01).There were also a significant difference between the IR and in TMP+AG IR group,about a 3-fold increase in TMP+AG group (P＜0.01).5.Western Blot Analysis(1) The renal RAGE protein expressionWestern blot analysis of RAGE revealed a similar trend with RT-PCR, in which RAGE protein expression was significantly increased in IR group when compared with the control group (P＜0.05); The TMP+AG group showed a decrease as compared with the IR group (P＜0.05).(2) The IGF-1R protein expressionThe protein expression of IGF-1R in brain, hepatic and kidney tissues in IR group was significantly lower than CN group (P＜0.05),which was increased in TMP+AG group than IR group (P＜0.05)(3) The Klotho protein expressionWestern blot analysis of expression of renal klotho revealed that klotho protein expression was significantly decreased in IR group when compared with the control group (P＜0.05); The TMP+AG group showed a decrease as compared with the IR group (P＜0.05).The Klotho protein expression in brain tissue did not detect. 6. Immunohistochemistry(1) The Klotho protein expressionThe Klotho expression from IR group was deceased compared with CN group (P<0.05), which was increased in TMP+AG group compared to IR group (P<0.05).(2) The IGF-1R protein expressionThe expression of IGF-1R in the distal convoluted renal tubule and collecting duct in IR group was significantly lower than CN group (P＜0.05), IGF-1R expression in TMP+AG group was higher than IR group (P＜0.05).There was no significant difference between TMP+AG group and CN group.Conclusions:1.The insulin resistant modal of newborn was established with streptozotocin intraperitoneally.The level of FPG,FINS,IRI and GSP were increased with the time going on. Meanwhile the survival rate was decreased.2. Renal klotho mRNA and protein expression were decreased in IR rats, overexpression of the RAGEmRNA and protein was also noted in the kidneys from IR rats, and high mortality, compared with the control group. Our result also suggesed that TMP+AG treated rats have decreased expression of RAGE,and increased expression of klotho. And the mortality rate was significantly decreased. The results indicated that the RAGE have been negatively linked to longevity, yet klotho has an opposite role.The exact mechanism needs to be further explored.3. It was found that the mechanism of TMP+AG for the prolongation of survival period was directly related with regulating the balance of the Ins/IGF-1 signal system functions in multiple organs in IR rats. To maintain normal life functions needs the relative stability of the Ins/IGF-1 signal system.