【作者】 曾静波；

【导师】 王姮； 金自孟； 李玉秀； 孙琦；

【作者基本信息】 中国协和医科大学 ， 内分泌代谢学， 2007， 博士

【摘要】 目的磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,P13K)-Akt通路为胰岛素的主要信号传导通路,发生胰岛素抵抗时P13K-Akt通路活性降低。PTEN(phosphatase andtensin homologue deleted on chromosome 10)是一种脂质磷酸酶,最初是作为一种肿瘤抑制因子而被发现,是一个很有效的PI3K-Akt通路的负性调节因子。FoxO蛋白是一种转录因子,是P13K-Akt通路激活后的一个下游因子,FoxO可直接影响PI3K-Akt通路的最后效应即糖代谢。但目前国内外尚无胰岛素抵抗时胰岛素敏感组织PTEN及FoxO3a蛋白表达的报道。因此本研究主要观察胰岛素抵抗KKAy糖尿病小鼠胰岛素敏感组织肝、骨骼肌及脂肪组织胰岛素刺激后Aktser473磷酸化水平、PTEN、FoxO3a蛋白的表达以及罗格列酮及二甲双胍治疗后上述蛋白表达的变化,从而探计PTEN、FoxO3a与PI3K-Akt通路活性在胰岛素抵抗机制中的作用。方法实验动物分为C57BL/6J小鼠普通饲料喂养组、C57BL/6J小鼠高脂饲料喂养组、KKAy糖尿病小鼠未治疗组、KKAy糖尿病小鼠罗格列酮治疗组以及KKAy糖尿病小鼠二甲双胍治疗组。C57BL/6J小鼠普通饲料组普通饲料喂养至16-18周龄,处死取材;C57BL/6J高脂饲料喂养组普通饲料喂养至12周,随后高脂喂养4周后处死取材;KKAy小鼠普通饲料喂养至12周龄,随后高脂饲料喂养4周,尾静脉血测末梢血糖,连续两周随机血糖大于300mg/dl(16.7mmol/L)诊断为糖尿病;KKAy糖尿病小鼠组未治疗组在诊断为糖尿病后即处死取材;KKAy糖尿病小鼠治疗组分别给予罗格列酮(12.5mg/kg/day)以及二甲双胍(3.0g/kg/day,分两次灌胃)治疗4周后处死取材。实验动物均在处死前空腹12h,每组动物分别随机分为未用胰岛素处理组及胰岛素处理组,未用胰岛素处理组小鼠尾静脉血测空腹血糖(fasting blood glucose,FBG),眼内疵静脉取血,留取血清ELISA法测定血胰岛素后处死;胰岛素处理组胰岛素处理前后尾静脉取血测血糖,腹腔注射胰岛素(5mU/g)后10分钟处死小鼠。各组小鼠在处死后即取肝组织、股四头肌及附睾脂肪垫,组织样本加组织裂解液,高速离心取上清-70℃保存,取等量组织蛋白样本进行western blots检测组织PTEN、FoxO3a及磷酸化Akt水平,图像数据用SPSS12.0软件,多组比较采用方差分析,组间比较采用q检验。结果FBG及空腹胰岛素C57BL/6J小鼠高脂饲料喂养组FBG(7.13±1.30mmol/L)及空腹胰岛素(0.60±0.33ng/ml)与普通饲料喂养组FBG(6.06±0.34mmoll/L)及空腹胰岛素(0.64±0.34ng/ml)相比无显著差异(P均＞0.05);KKAy糖尿病小鼠FBG(10.30±2.58mmol/)及空腹胰岛素(6.00±1.79ng/ml),较C57BL/6J小鼠普通及高脂饲料喂养组均明显升高(P均＜0.001);罗格列酮治疗组FBG(6.76±1.39mmol/L)(P＜0.01)及二甲双胍治疗组FBG(5.80±1.50mmol/L)(P＜0.001)与KKAy糖尿病小鼠组相比均明显下降,空腹胰岛素水平罗格列酮治疗组(2.74±1.74ng/ml)(P＜0.05)以及二甲双胍治疗组(2.40±1.1ng/ml)(P＜0.01)与KKAy糖尿病小鼠组相比也均明显下降;罗格列酮治疗组以及二甲双胍治疗组FBG明显下降至C57BL/6J普通饲料及高脂饲料喂养组水平(P均＞0.05)。罗格列酮治疗组以及二甲双胍治疗组空腹胰岛素亦明显下降,与C57BL/6J高脂饲料喂养组无差异(P均＞0.05),但未降至C57BL/6J普通饲料喂养组水平(P均＜0.05)。胰岛素刺激后Aktser473磷酸化水平C57BL/6J小鼠高脂饲料喂养组胰岛素刺激后Aktser473磷酸化升高水平肝(4.57±0.38倍)、骨骼肌(4.74±0.60倍)及脂肪组织(4.56±0.68倍)与C57BL/6J小鼠普通饲料喂养组胰岛素刺激后肝(4.67±0.55倍)、骨骼肌(4.82±0..22倍)及脂肪组织(4.92±0.41)Aktser473磷酸化升高水平无显著差异(P均＞0.05),KKAy糖尿病小鼠胰岛素刺激后Aktser473磷酸化升高水平肝(2.46±0.65倍)、骨骼肌(2.29±0.56倍)及脂肪组织(2.50±0.14倍)较C57BL/6J普通饲料及高脂喂养组喂养组相比均显著降低(P均＜0.001)。KKAy糖尿病小鼠罗格列酮胰岛素刺激后Aktser473磷酸化升高水平肝(2..77±0.26倍)、骨骼肌(2.65±0.40倍)及脂肪组织(2.54±0.60倍)以及二甲双胍治疗后胰岛素刺激后Aktser473磷酸化升高水平肝(2.56±0.18倍)、骨骼肌(2.40±0.31倍)及脂肪组织(1.99±0.44倍)与KKAy糖尿病小鼠相比无显著变化(P均＞0.05)。PTEN蛋白C57BL/6J小鼠高脂喂养组骨骼肌组织PTEN的蛋白水平(0.59±0.26)较C57BL/6J小鼠普通饲料喂养组(0.18±0.19)显著升高(P＜0.05),C57BL/6J小鼠高脂喂养组PTEN蛋白水平肝(6.27±1.10)及脂肪组织(2.85±0.59)与C57BL/6J小鼠普通饲料喂养组肝(5.97±1.23)及脂肪组织(2.55±0.42)相比无显著差异(P均＞0.05);KKAy糖尿病小鼠肝(13.37±0.28)、骨骼肌(1.08±0.14)及脂肪组织(4.92±1.74)PTEN蛋白的表达较C57BL/6J小鼠均显著升高(P均＜0.001);PTEN蛋白水平罗格列酮治疗后肝(15.60±1.06)、骨骼肌(1.13±0.21)及脂肪组织(5.50±0.95)以及二甲双胍治疗后肝(14.97±0.87)、骨骼肌(1.07±0.18)及脂肪组织(5.39±0.72)与KKAy糖尿病小鼠相比无变化(P均＞0.05)。FoxO3a蛋白KKAy糖尿病小鼠脂肪组织FoxO3a蛋白水平(1.76±0.14)明显高于C57BL/6J小鼠普通饲料喂养(1.15±0.10)及高脂饲料喂养组(1.09±0.13)(P均＜0.05),KKAy糖尿病小鼠罗格列酮治疗后(1.43±0.22)和二甲双胍治疗后(1.59±0.18)脂肪组织FoxO3a的蛋白水平KKAy糖尿病小鼠对照相比没有统计学差别(P均＞0.05)。结论KKAy小鼠短期高脂喂养可以较好的模拟人类2型糖尿病发病过程;发生胰岛素抵抗时,胰岛素敏感组织肝、骨骼肌及脂肪组织PTEN的表达增加;脂肪组织FoxO3a的表达增多;高脂摄入可以使骨骼肌组织PTEN的表达增多,但对肝及脂肪组织PTEN的表达无影响;PTEN可能是一种重要的负性调节因子,调节P13K/Akt所介导的信号传导通路,在胰岛素抵抗的发生、发展中起着重要的作用。罗格列酮及二甲双胍的降糖及改善胰岛素抵抗作用与PTEN及FoxO3a可能无关,其降糖作用可能为非P13K-AKT通路。更多还原

【Abstract】 Objective:Signaling through the phosphatidylinositol 3-kinase(PI3K)-Akt pathway is crucial for metabolic responses to insulin,and defects in PI3K-Akt signaling have been demonstrated in insulin resistance.PTEN(phosphatase and tensin homologue deleted on chromosome 10),firstly named after tumor suppressor,is a potent negative regulator of the PI3K-Akt pathway.FoxO family are transcription factors and belong to downstream molecules and then can be activated by PI3K-Akt pathway.Until now there was no report of the expression of PTEN and FoxO in insulin target tissue when insulin resistance occur.The purpose of this study is to detect protein level of PTEN,FoxO3a and insulin-stimulated phosphoserine 473-Akt in live,muscle and fat tissue in KKAy diabetic mice and the changes of these proteins after treatment of rosiglitazone and metformin,to investigate the roles of PTEN,FoxO3a and PI3K-Akt pathway in the mechanism of insulin resistance.Methods:Study group was divided into normal diet C57BL/6J mice group,high fat diet C57BL/6J mice group,KKAy diabetic mcie group,KKAy diabetic mice randomly assigned to rosiglitazone treating group and metformin-treating group;normal diet C57BL/6J mice group were killed utill 16-18 weeks old,high fat diet C57BL/6J mice group and KKAy mcie group were fed with normal diet until week 12,thereafter fed with high fat diet for 4 weeks and then high fat diet C57BL/6J mice group were killed.Blood glucose of KKAy mice were measured from tail vein every week since 15 week old,it would be diagnosed of diabetes if blood glucose more than 300mg/dl(16.7mmol/L) in two consecutive weeks. KKAy diabetic mice will be divided into untreating group and two treating groups. Untreating KKAy diabetic mice were killed afte diabetes were diagnoised.Two treating KKAy diabetic mice were be gaven rosiglitazone 12.5 mg/kg/day and metformin (3.0g/kg/day,divided for two times) for 4 week and then be killed.Food was withdrawn 12h before samples were taken.Each group mice were be divided into insulin-untreating mice and insulin-treating mice.Insulin-treating mice were intraperitoneally injected with insulin(5mU/g body wt) in saline,after 10 min,the mice were killed.Blood glucose were measured from tail before and after insulin treating in insulin-treating KKAy diabetic mice. After the mice were killed,dispatched the mice and took quadriceps of femoris muscle and liver tissue,added tissue lytic solution,measured the protein concentration by Bradford method and detect the PTEN,FoxO3a and phosphoserine 473-AKT protein expression in muscle and liver tissue by Western bolts methods.Results:Fasting blood glucose(FBG) and fasting insulin:Both FBG (10.30±2.58mmol/L)and fasting insulin(6.00±1.79ng/ml) of KKAy diabetic mice were significantly increased compared with normal diet C57BL/6J mice group FBG(6.06±0.34mmol/L) and fasting insulin(0.64±0.34ng/ml);FBG (7.13±1.30mmol/L) and fasting insulin(0.60±0.33ng/ml) of high fat diet C57BL/6J mice group don’t increased compared with normal diet C57BL/6J mice(P＜0.001).High fat diet don’t impact the FBG and fasting serum insulin of C57BL/6J mice.After treating with rosiglitazone,the FBG(6.76±1.39mmol/L) and fasting serum insulin(2.74±1.74ng/ml) of KKAy diabetic mice were decreased(p＜0.01 and p＜0.05),so were FBG(5.80±1.50mmol/L) and fasting insulin(2.40±1.1ng/ml) in KKAy diabetic mice treating with metformin ((p＜0.001 and p＜0.01).Insulin stimulation of phosphoserine 473-Akt:Insulin-induced phosphoserine 473-Akt were significantly decreased in liver(2.46±0.65-fold),muscle(2.29±0.56-fold)and fat (2.50±0.14-fold) tissue of KKAy diabetic mice compared with liver(4.67±0.55-fold),muscle(4.82±0.22-fold)and fat(4.92±0.41-fold)tissue of normal diet C57BL mice(P＜0.001).Insulin-induced phosphoserine 473-AKT were not significantly decreased in liver(4.57±0.38-fold),muscle(4.74±0.60-fold)and fat(4.56±0.68-fold)tissue of high fat diet C57BL/6J mice group compared with normal diet C57BL/6J mice (P＜0.001).High fat don’t impact the insulin-induced phosphoserine 473-Akt in C57BL/6J mice.Insulin-induced phosphoserine 473-AKT in live(2.77±0.26-fold),musle(2.65± 0.40-fold) and fat(2.54±0.60-fold) tissue of rosiglitazone-treated and live(2.56±0.18-fold),musle(2.40±0.31-fold) and fat(1.99±0.44-fold) tissue of metformin-treated KKAy diabetic mice are all not changed compared with those in KKAy diabetic mice(P＞0.05).PTEN protein level:PTEN protein level was significantly elevated in live(13.37±0.28),muscle(1.08±0.14) and fat(4.92±1.74)tissue of KKAy diabetic mice and in muscle tissue(0.59±0.26) of C57BL high fat diet group compared to live(5.97±1.23),muscle((0.18±0.19)and fat(2.55±0.42) of C57BL normal diet groups.(P＜0.001 and P＜0.05).PTEN protein level was not significantly elevated in live(6.27±1.10) and fat(2.85±0.59) tissue of high fat diet C57BL mice compared with C57BL normal diet groups(P＞0.05).PTEN protein level in live(15.60±1.06),musle(1.13±0.21) and fat(5.50±0.95) tissue of rosiglitazone-treated and live(14.97±0.87),musle(1.07±0.18)and fat(5.39±0.72) tissue of metformin-treated KKAy diabetic mice are all not changed compared with those in KKAy diabetic mice(P＞0.05).FoxO3a protein level:FoxO3a protein level(1.76±0.14) was significantly elevated in fat tissue of KKAy diabetic mice compared to that in fat tissue(1.15±0.10) of normal diet C57BL group(p＜0.05).FoxO3a protein level(1.09±0.13) of fat tissue of high fat diet C57BL mice was not significantly elevated compared with C57BL normal diet groups(P＞0.05) FoxO3a protein level in fat tissue of rosiglitazone-treated(1.43±0.22) and metformin-treated KKAy diabetic mice(1.59±0.18) are all not changed compared with those in KKAy diabetic mice.Conclusion:KKAy mice given high fat diet for a short period is a good animal model of type 2 diabetes.The expression of PTEN were increased at insulin-sensitive tissue live,skeletal muscle and fat and FoxO3a was increased at fat tissue of insulin resistance. High fat diet can increase the expression of PTEN at skeletal muscle.PTEN may be a potent negative modulator of the PI3K-Akt pathway and then play an important role in the development of insulin resistance.Rosiglitazone and metformin can reduce blood glucose and improve insulin resistance.But the mechanism may not correlate with the expression of PTEN and FoxO3a in insulin sensitive tissue and then also may not related to the PI3K-Akt pathway.更多还原