The Impacts Of Testosterone On Insulin Sensitivity In C57BL/6 Mice, 3T3-L1 Adipocytes And HepG2 Liver Cells And Their Related Molecular Mechanisms

Partâ… The impacts of testosterone on insulin sensitivity in adult C57BL/6 female mice and its related mechanismsObjectives:Insulin resistance(IR) is a common manifestation in patients with polycystic ovarian syndrome(PCOS).Both clinical observations and animal studies have demonstrated that IR could be induced by hyperandrogenemia,which is the chaficteristic of PCOS. Nevertheless,the mechanisms of IR in PCOS are still unclear especially at the molecular level.We conduct this study to ellucidate the effects of androgen on insulin sensitivity in adult C57BL/6 female mice.Methods:Eleven adult female C57BL/6 mice aged 8 weeks were injected daily(i.p.) with testosterone(1.0 mg/100 g body weight)dissolved in sesame oil (experimental group T) for 24 weeks.Ten control mice were injected with sesame oil only(group Con).(1) The changes of body weight and body fat content were detected.(2) Intraperitoneal glucose tolerance tests(IGTT)were performed at 0,2,3 and 16 weeks treatment,blood from tail vein was taken to detect levels of glucose.(3) Intraperitoneal insulin tolerance tests(ITT) were performed at 17 weeks treatment.(4) Both group were sacrificed at 24 weeks treatment,and phosphorylation of GSK3β,down-stream signaling molecule of insulin signaling pathway,was detected by western blot from adipose and liver tissue.Results:(1) No obvious significance of body weight as well as body fat content was detected between both experimental groups.(P＞0.05);(2) 2 weeks treatment with testosterone induced the increase of fasting blood glucose and displayed obvious significance compared with group Con. (P＜0.05)(3) 3 weeks treatment with testosterone induced the increase of area under the curve(AUC) of the blood glucose following IGTT and displayed significance compared with group Con.(P＜0.05).And more obvious significance was detected at 16 weeks treatment.(P＜0.01)(4) 17 weeks treatment with testosterone induced the increase of AUC of the blood glucose following ITT.(P＜0.01)(5) 24 weeks treatment with testosterone decreased phosphorylation of GSK3βin C57BL/6 adipose and liver tissues(P＜0.05).Conclusions:Treatment with testosterone in adult female mice can induce insulin resistance by blocking insulin signal transduction,without influencing body weight and body fat content. Partâ…¡The impacts of testosterone on insulin sensitivity in 3T3-L1 adipocytes and its related molecular mechanismsObjectives:To investigate the regulation of insulin sensitivity by rapid nongenomic and slow genomic androgen signaling.Methods:The response of insulin-stimulated glucose uptake to pretreated testosterone was determined by adding 2-deoxy³[H]glucose to differentiated 3T3-L1 adipocytes. Phosphorylation and protein expression of insulin receptor(InsR) and its downstream signaling molecules(Akt and GSK3β) were analyzed by western blot.Results:Insulin-stimulated glucose uptake decreased gradually in response to the increasing of testosterone following short-time(30 minutes) or long-time(24 hour) treatment with the nadir at 10^-5M testosterone(P＜0.05).Phosphorylation of InsR,Akt and GSK3βwere significantly down-regulated by adding of testosterone at 10^-6M following short-time(3 and 9 minutes) and long-time(3,24,and 48 hours) treatment,or at 10^-7M following long-time(24 and 48 hours) treatment.The protein expression of InsR,Akt,and GSK3β, however,were not significantly affected by testosterone treatment.Conclusion:Rapid nongenomic androgen signaling might contribute to the insulin resistance in adipocytes.The effect of slow genomic androgen signaling will need further elucidation. Partâ…¢The impacts of testosterone on insulin sensitivity in HepG2 liver cells and its related molecular mechanismsObjectives:Our previous study has indicated that injection with Testosterone for 24 weeks decreased phosphorylation of GSK3βin adult C57BL/6 mice,while the underlined molecular mechanisms are unclear.The aim of this part were to investigate the regulation of insulin sensitivity by testosterone in human liver cancer cell lines HepG2.Methods:Phosphorylation and expression of insulin signaling molecules(Akt and GSK3β) were analyzed by western blot.(1)HepG2 were pretreated with different doses of testosterone(10^-9～10^-5M) for 6h, 24h and 36h followed by stimulation with 100nM insulin for 15min.(2)HepG2 were consistently pretreated with 10^-7M testosterone for 3h,12h, 24h,36h and 96h followed by stimulation with 100nM insulin for 15min.(3)HepG2 liver cells were pretreated with 10^-7M testosterone for 36h followed by stimulation with 100nM insulin for 15min,and then restimulated with 100nM insulin for 15 min at 4h,6h and 8h interval respectively.Results:(1) Pretreated with 10^-9～10^-6 M testosterone within 36h obviously increased phosphorylation of Akt and GSK3β(P＜0.05),whereas pretreated with 10^-5 M did not influence phosphorylation of Akt and GSK3β(P＞0.05);(2) Pretreated with 10^-7M testosterone within 36h obviously increased phosphorylation of Akt and GSK3β(P＜0.05),whereas pretreated for 96h did not influence phosphorylation of Akt and GSK3β(P＞0.05);(3) Pretreated with 10^-7M testosterone for 36h followed by insulin stimulation and restimulation after 6h interval obviously decrease phosphorylation of Akt and GSK3β(P＜0.05),whereas restimulation after 4h or 8h did not influence phosphorylation of Akt and GSK3β(P＞0.05)Conclusions:Pretreating within a certain concentration(10^-9～10^-6M) and a certain duration(6～36h),testosterone increase insulin sensitivity in HepG2 cells.But pretreating with a higher concentration(10^-5M) or a longer duration(96h),testosterone has no influence on HepG2 insulin sensitivity.Furthermore,when HepG2 were stimulated by insulin twice at a 6h interval,testosterone even could down regulate insulin sensitivity.