【作者】 陈敏；

【导师】 林金芳； 翟琦巍；

【作者基本信息】 复旦大学 ， 妇产科学， 2008， 博士

【摘要】 第一部分睾酮对成年C57BL/6雌鼠胰岛素敏感性的影响目的:多囊卵巢综合征（PCOS）患者普遍存在胰岛素抵抗（IR）,但其IR的形成原因尚不明确。高雄激素血症是PCOS的主要生化特征,有临床与动物实验表明高雄激素可引起IR,但机制不明。本部分拟探讨睾酮对成年C57BL/6近交系雌鼠的胰岛素敏感性的影响及其对肝脏、脂肪组织胰岛素通路信号分子活性的影响。方法:将8周龄C57BL/6雌性小鼠21只随机分为睾酮组（n=11,每天按1.0mg/100g体重腹腔注射睾酮）及对照组（n=10,每天注射等体积sesame oil）,共给药24周:（1）检测体重及体脂含量变化;（2）于处理前、处理后2周、3周及16周行腹腔内注射葡萄糖耐量试验（IGTT）;（3）于处理后第17周行腹腔内注射胰岛素耐受试验（ITT）;（4）于处理后第24周处死雌鼠,western blot检测肝脏及脂肪组织胰岛素通路下游信号分子GSK3β的磷酸化水平。结果:（1）睾酮组与对照组成年雌鼠的体重以及体脂含量无明显差异（P＞0.05）;（2）处理2周睾酮组空腹血糖开始明显高于对照组（P＜0.05）;（3）处理3周睾酮组IGTT曲线下面积开始明显大于对照组（P＜0.05）,至16周后差异更加显著（P＜0.01）;（4）处理17周后睾酮组ITT曲线下面积极显著大于对照组（P＜0.01）;（5）处理24周后睾酮组的肝脏及脂肪组织的GSK3β的磷酸化水平均明显高于对照组（P＜0.05）。结论:睾酮不影响成年C57BL/6雌鼠的体重及体脂含量,但是可以通过影响胰岛素在其主要靶器官肝脏和脂肪组织的信号转导来诱导胰岛素抵抗的形成。第二部分睾酮对3T3-L1脂肪细胞胰岛素敏感性的影响目的:在离体水平探讨雄激素的慢速基因组效应及快速非基因组效应对3T3-L1脂肪细胞胰岛素敏感性的影响及分子机制。方法:将3T3-L1前脂肪细胞诱导成熟,用10^-9～10^-5mol/L睾酮分别预处理0～30min及3h、24h、48h,[³H]-2-脱氧葡萄糖掺入法检测葡萄糖摄取率;免疫印迹检测InsR/Akt/GSK3β的活性及表达。结果:有胰岛素刺激时,睾酮预处理30min的胰岛素促葡萄糖摄取率随睾酮浓度增高而下降,10^-5mol/L睾酮组的葡萄糖摄取率的升高倍数（4.24±0.42）显著低于无睾酮组（5.46±0.4）,P＜0.05;10^-6mol/L睾酮预处理3min-30min的InsR/Akt/GSK3β的磷酸化水平较无睾酮组均显著下降（均P＜0.05）。有胰岛素刺激时,睾酮预处理24h的胰岛素促葡萄糖摄取率也随睾酮浓度增高而下降,10^-5mol/L睾酮组的葡萄糖摄取率的升高倍数（3.97±1.04）显著低于无睾酮组（4.53±1.02）,P＜0.05;10^-7～10^-6mol/L睾酮预处理3h、24h、48h的InsR/Akt/GSK3β的活性均显著下降（均P＜0.05）,但InsR/Akt/GSK3β的表达水平均不受睾酮影响（均P＞0.05）。此外,我们课题组以前的研究结果提示10^-8mol/L～10^-5mol/L睾酮预处理3T3-L1细胞24h时,胰岛素通路另外两个重要的信号分子IRS-1和GLUT4表达随睾酮浓度增加而逐渐下降;而用10^-9mol/L预处理24～48h时,IRS-1、GLUT4的表达水平随睾酮作用时间的延长而逐渐下降。结论:高浓度的睾酮持续作用时,膜受体通路的快速非基因组效应及核受体通路的慢速基因组效应共同作用,从抑制基因转录和抑制激酶活化这两个层次与胰岛素信号通路进行cross-talk,进而分别抑制InsR/Akt/GSK3β的活性和IRS-1/GLUT4的表达,阻遏胰岛素信号的胞内转导,诱导脂肪细胞受体后IR产生。第三部分睾酮对HepG2肝癌细胞胰岛素敏感性的影响目的:探讨睾酮对人肝癌HepG2细胞的胰岛素信号转导通路的影响。方法:HepG2培养至12孔板,做以下处理后细胞提取蛋白,western blot检测胰岛素信号分子Akt、GSK3β的活性和表达的改变（1）用10^-9～10^-5M的睾酮预处理细胞6～36h后100nM胰岛素单次刺激15min;（2）用10^-7M的睾酮持续处理细胞3～96h后100nM胰岛素单次刺激15min;（3）用10^-7M睾酮预处理细胞36h后100nM胰岛素刺激15min,接着分别于4h、6h、8h后加用100nM胰岛素再次刺激15min。结果:（1）胰岛素单次刺激时,10^-9～10^-6M的睾酮预处理细胞6～36h的Akt/GSK3β的磷酸化水平明显高于对照组（P＜0.05）,且随着睾酮作用浓度的升高而升高;但10^-5M的睾酮预处理6～36h的Akt/GSK3β的磷酸化水平与对照组相比无显著差异（P＞0.05）;（2）胰岛素单次刺激时,10^-7M的睾酮预处理细胞12～36h的Akt/GSK3β的磷酸化水平明显高于对照组（P＜0.05）,但10^-7M睾酮预处理96h的Akt/GSK3β的磷酸化水平与对照组相比无显著差异（P＞0.05）;（3）10^-7M睾酮预处理细胞36h后第一次用胰岛素刺激15min,并间隔6h后再用胰岛素再次刺激15min,结果发现此时睾酮组的Akt/GSK3β磷酸化水平明显低于对照组（P＜0.05）。结论:一定浓度(10^-9～10^-6M)的睾酮作用一定时间（6h～36h）能增强HepG2的胰岛素敏感性;但过高浓度(10^-5M)或过长时间（96h）的睾酮预处理不增强人肝癌细胞HepG2的胰岛素敏感性;此外,较高浓度的睾酮(10^-7M)在胰岛素反复刺激的情况下,反而降低了胰岛素敏感性,诱导胰岛素抵抗形成。更多还原

【Abstract】 PartⅠThe 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. PartⅡThe 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. PartⅢThe 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.更多还原