糖耐量受损期脂联素、脂联素受体表达变化及罗格列酮干预作用的实验研究
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摘要
目的
脂联素(Adiponectin,APN)是脂肪组织特异性分泌的一种脂肪因子,近年来的研究证实脂联素有多种生物学作用,主要参与体内糖、脂、能量代谢,增加胰岛素敏感性、抗炎和抗动脉粥样硬化的生物学效应。脂联素的生物学作用是在脂联素受体(Adiponectin receptor,AR)的介导下发生的。动物及人体内有两种脂联素受体,即脂联素受体1(AR1)和脂联素受体2(AR2),它们在不同的靶器官组织中表达量各不相同,介导脂联素发挥不同的生物学效应。
脂联素受体mRNA及其蛋白的表达受多种因素影响,其表达异常时会对许多疾病的病理生理过程产生影响,如促进胰岛素抵抗,引起血管内皮功能受损及冠状动脉粥样硬化等。
本研究从人体、动物、细胞三个水平进行研究,其目的旨在:
①研究脂联素及其受体表达的变化与糖耐量减低(IGT)之间的关系;
②探讨脂联素及其受体在胰岛素抵抗(Insulin resistancc,IR)中的作用及意义;
③从动物及细胞水平探讨罗格列酮对脂联素及其受体表达的影响;
④通过改善脂联素及其受体的表达而改善胰岛素抵抗、糖脂代谢,从而为IGT早期干预提供新的治疗靶点。
本研究包括以下四部分内容:
①糖耐量受损人群血清脂联素水平的变化及其影响因素分析。
②糖耐量异常Wistar大鼠血清脂联素及肝脏脂联素受体的表达及相关因素分析。
③罗格列酮及二甲双胍对高脂饲养糖耐量异常大鼠肝脏脂联素受体mRNA表达的作用及机制研究。
④罗格列酮对不同浓度脂肪酸培养HepG2细胞脂联素受体mRNA表达的作用及机制研究。
通过研究以上内容,预期获得如下研究结果:
①糖耐量异常时人及大鼠血清脂联素及肝脏脂联素受体表达减少。
②罗格列酮对改善糖耐量减低大鼠肝脏脂联素及其受体的表达有重要的作用,可以上调AR1、AR2的mRNA表达,这是罗格列酮改善胰岛素敏感性、抗胰岛素抵抗的机制之一。
③以脂联素受体为研究切入点,为开发抗糖尿病、抗动脉粥样硬化的新药物提供理论基础。
方法
本研究涉及临床及基础研究两个部分,从方法学角度包括以下内容:
①全自动生化分析仪检测血液生化指标。
②ELISA法测定血液激素及细胞因子水平。
③免疫印迹法(Western blot)检测AR1及AR2蛋白的表达水平。
④逆转录聚合酶链反应(RT-PCR)方法检测AR1及AR2 mRNA的表达水平,其中涉及总RNA提取方法。
⑤细胞培养及对培养细胞药物处理方法。
具体研究方法按照四部分研究内容介绍如下。
第一部分:糖耐量受损人群血清脂联素水平变化及其影响因素的分析
1.研究对象及分组
纳入标准:122例研究对象全部来自于山西医科大学第二医院健康体检中心,年龄35—65岁;按照2007年美国糖尿病协会(ADA)诊断标准,60例空腹血糖(FPG)＜5.6mmol/L,口服葡萄糖耐量试验(OGTT)2小时血糖(OGTT 2hP6)＜7.8 mmol/L者纳入糖耐量正常组(NGT);62例FPG介于5.6～6.9 mmol/L,OGTT2hPG介于7.8～11.0mmol/L之间者纳入糖耐量减低组(IGT)。
排除标准:①曾明确诊断为糖尿病者;②进行过任何方式的血糖干预治疗;③继发性血糖升高;④有影响血糖升高的因素存在;⑤肝肾功能不全者。满足其中一项者即被排除。
2.研究资料的采集及统计学分析
所有研究对象由专职工作人员检测体重(W)、腰围及臀围、血压(收缩压SBP,舒张压DBP),腹部B型超声波,并计算体重指数(BMI)和腰臀比(WHR)。
用ELISA法检测血清脂联素(APN)、胰岛素(INS)、纤溶酶原活化抑制物(PAI-1)、C反应蛋白(CRP)水平。应用全自动生化分析仪检测血脂谱。
应用SPSS13.0统计软件进行统计分析,计量资料使用均数±标准差表示,正态分布资料组间比较采用t检验:计数资料采用频数或百分率表示;多因素分析Logistic回归分析。
第二部分:糖耐量受损Wistar大鼠血清脂联素及肝脏脂联素受体的表达及相关因素分析
1.动物分组及糖耐量减低动物模型建立
40只5-6周龄雄性Wistar大鼠按照随机数字表法随机分为对照组20只,高脂组20只。
20只高脂组Wistar大鼠给予高脂饲料饲养,每日投食饲料量为体重的3%,在温度22℃-25℃,相对湿度30%-70%的动物房中单笼饲养,每天光照12小时,天黑前投食,自由饮水。高脂饲料配方:5.2kcal/g,其中脂肪提供热量占56.0%,主要为饱和脂肪酸的动物脂肪,碳水化合物占37.0%,蛋白质占7.0%。具体饲料配制:基础面料72%,猪油20%,白糖5%,蛋黄粉5%。第8周开始陆续出现IGT动物模型,至第12周时85%造模成功。
对照组大鼠给予标准饲料饲养,标准饲料配方:3.2kcal/g,其中脂肪提供热量占10.2%,蛋白质占23.3%,碳水化合物占66.5%;饲养环境的条件同IGT动物模型组。
纳入标准:对照组大鼠FPG＜5.6mmol/L,OGTT2hPG＜7.8mmol/L;高脂组大鼠FPG介于5.6～6.9 mmol/L,OGTT2hPG介于7.8～11.0mmol/L之间者纳入糖耐量减低组。
OGTT:过夜禁食8 h后,剪尾采血测空腹血糖,以50%葡萄糖2g/kg.wt灌胃,2小时后再次剪尾采血测血糖。
2.检测、分析研究指标
成模后继续饲养8周,分别处死各组1/2数量大鼠,测定血脂谱、血清胰岛素、血清脂联素,计算稳态模型胰岛素抵抗指数(HOMA-IR),评价胰岛素抵抗(IR)程度,用RT-PCR方法检测肝脏脂联素受体1(AR1)、脂联素受体2(AR2)的mRNA的表达量,用Western blot方法检测肝脏AR1、AR2的蛋白表达量,并在光镜下观察对照组及高脂组肝脏的病理变化。其余两组各1/2数量大鼠饲养至第12周时全部处死,操作程序同第8周。
比较高脂组及对照组BMI、内脏脂肪/体重(VF/W)、血脂谱、血清胰岛素、脂联素、HOMA-IR、肝脏AR1、AR2的mRNA的表达量和AR1、AR2的蛋白表达量,并进行相关因素分析。
第三部分:罗格列酮及二甲双胍对高脂饲养糖耐量受损大鼠肝脏脂联素受体mRNA表达的作用及机制研究
1.动物分组
60只雄性Wistar大鼠按照随机数字表法分为对照组10只(C组)及高脂组(HF组)50只。各组饲养至12周,高脂组大鼠成为糖耐量受损模型,之后分组给予罗格列酮和二甲双胍干预治疗。将高脂组随机分为单纯高脂组10只,高脂+罗格列酮组(GF+ROS组)20只:罗格列酮3mg/kg·d~(-1)灌胃;高脂+二甲双胍组(GF+Met组)20只:二甲双胍300mg/kg·d~(-1)灌胃。
2.肝脏AR1、AR2 mRNA表达的检测
干预治疗第8周时,干预组各组处死1/2数量大鼠,同时处死同期对照组和单纯高脂组大鼠各1/2数量,分离肝脏,迅速保存于-70℃冰箱,待测肝脏AR1、AR2 mRNA的表达;继续饲养至第12周时,处死各组所有大鼠,操作程序同第8周。
应用RT-PCR检测AR1、AR2 mRNA在肝脏的表达;比较各组AR1、AR2 mRNA的表达量,探讨PPAR-γ激动剂罗格列酮和AMPK-γ激活剂二甲双胍对高脂诱导的糖耐量受损大鼠肝脏AR1、AR2基因表达以及肝脏病理学的影响,对肝脏胰岛素抵抗可能的调节机制进行分析。
第四部分:罗格列酮对不同浓度脂肪酸培养HepG2细胞脂联素受体mRNA表达的作用及机制研究
1.细胞培养和分组
用含10%胎牛血清(FCS)的高糖DMEM培养液培养HepG2细胞,置于37℃,5%CO_2浓度的CO_2孵箱内孵育48 h。将细胞随机分为8组,每组含3个9cm直径培养皿,分别为对照组,单加罗格列酮10μmol/L的ROS组,3个加入不同浓度软脂酸(PA)(100μmol/L、200μmol/L和400μmol/L)的PA组,以及3个上述各浓度PA加10μmol/L罗格列酮的PA+ROS组。
PA的配制:13.9mg PA加入0.5ml 0.1M的NaOH中,加热至100℃溶解1小时以上,将高糖DMEM培养液预热至45℃,迅速将溶解于NaOH的PA加入培养液中配置成含所需PA浓度的培养液,待培养液冷却至室温可用于实验培养。
用不含PA和ROS、单含PA、含有PA和/或ROS的培养液,分别置换各组原培养液,继续培养24 h。
2.检测不同组别HepG2细胞AR1、AR2 mRNA表达
提取细胞总RNA,RT-PCR检测AR1、AR2 mRNA水平,用凝胶扫描分析系统定量分析RT-PCR电泳图象。以β-actin为内参照,分别测定AR1和AR2的平均积分光密度(integrated density value,IDV),分别以AR1/β-actin、AR2/β-actin的IDV比值表示其mRNA表达的相对水平,各组mRNA的表达量采用均数±标准差表示。分别对不同PA浓度和同PA浓度的各组采用SPSS 13.0软件进行单因素方差分析(one-way检验)。
结果
1.在临床研究中显示,与NGT组比较,IGT组血清脂联素水平显著降低(P＜0.05),高密度脂蛋白胆固醇(HDL)显著降低(P＜0.01);BMI、WHR、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL)、HOMA-IR、CRP、PAI-1均显著升高(均P＜0.05)。人血清脂联素的相关因素分析显示:脂联素与HDL、OGTT2hPG、WHR、BMI显著相关。以脂联素水平为应变量,各可能相关的因素为自变量,Logistic多元回归分析显示HDL和FPG为影响脂联素水平变化的独立危险因素。
2.动物研究结果显示:
①高脂饲料饲养12周可以成功制备IGT大鼠模型,成模率达85%。
②高脂饲料饲养8周、12周时,与同期标准饲料饲养对照组比较,其体重、腹内脂肪量/体重、空腹胰岛素、HOMA-IR、总胆固醇、甘油三酯、低密度脂蛋白胆固醇高于对照组(均P＜0.05),血清脂联素低于对照组(P＜0.05),高密度脂蛋白胆固醇在12周时低于对照组(P＜0.05)。
③IGT组8周、12周时肝脏组织AR1、AR2 mRNA及蛋白表达量均较NGT组降低(P＜0.05),AR1、AR2蛋白的表达与胰岛素抵抗指数呈负相关,与BMI、VF/W、TG呈负相关。
④IGT组大鼠8周、12周时肝脏组织HE染色显示有肝细胞脂肪变性,小叶内有单核细胞等浸润,出现脂肪肝的病理改变;12周时病理改变较8周时严重。
3.罗格列酮干预治疗高脂饲养IGT大鼠8周及12周后,肝脏AR1及AR2 mRNA表达均较同期单纯高脂组增高。干预治疗8周时AR1 mRNA变化与同期单纯高脂组比较无显著性差异(P＞0.05),AR2 mRNA变化有显著性差异(P＜0.05);干预治疗12周时,AR1及AR2 mRNA变化与同期单纯高脂组比较均有显著性差异(均P＜0.05)。
4.分别用浓度为100μmol/L、200μmol/L和400μmol/L的软脂酸(PA)培养HepG2细胞,观察不同浓度PA培养的HepG2细胞AR1及AR2 mRNA表达的变化,结果显示:当PA浓度大于200μmol/L时,AR2 mRNA表达显著减少,而AR1 mRNA表达无显著变化;再经罗格列酮(ROS)10μmol/L处理不同浓度PA培养的HepG2细胞,观察处理前后HepG2细胞AR1及AR2 mRNA表达的变化,结果显示:在ROS处理的各组中,AR2mRNA的表达均较同PA浓度组增高,尤其是在PA浓度大于200μmol/L时。
结论
1.临床研究及动物实验结果均证实,在IGT阶段已经出现脂联素水平下降;体重增加、内脏脂肪堆积,胰岛素抵抗与脂联素水平下降密切相关。
2.IGT阶段随脂联素水平下降,胰岛素抵抗加剧,促进体内脂质代谢紊乱,体内脂肪分布异常,出现中心型肥胖及非酒精性脂肪性肝病。
3.脂联素受体介导脂联素的生物学效应。IGT阶段肝脏AR1、AR2 mRNA及蛋白的表达均下降,降低了胰岛素的敏感性,加重机体的糖脂代谢紊乱。
4.罗格列酮上调AR1、AR2 mRNA表达,其机制可能通过激活过氧化物酶增殖物激活受体(PPAR-γ),直接促进肝脏AR1、AR2 mRNA的转录及翻译,尤其对AR2的调节作用更强,并调节胰岛素受体后信号转导途径,从而改善胰岛素抵抗。
5.脂联素水平下降可以作为IGT早期干预的重要标志之一以及治疗靶点。展望
脂联素作为一个脂肪因子,在调节糖脂代谢、改善胰岛素抵抗、抗炎、抗动脉粥样硬化及保护血管内皮功能等方面发挥着广泛的生物学效应。脂联素受体的发现为更好地研究APN的生物学作用及作用机制奠定了基础。近年未发现脂联素基因位点与2型糖尿病的一个易感基因位点均出现在3q17染色体处,提示APN与2型糖尿病在基因水平存在某种必然联系。深入研究脂联素和脂联素受体的表达、作用机制及调控机制,有助于进一步阐明一些与脂联素有关的疾病如糖尿病、动脉粥样硬化、冠心病等的发病机制,从而对这些疾病从病因角度寻找有效的防治方法,甚至从基因水平对这些疾病进行预防。研究脂联素及脂联素受体的作用,有助于寻找抗糖尿病、抗动脉粥样硬化新的治疗靶点,并针对这些治疗靶点研发新药物,从而对糖尿病及动脉粥样硬化疾病的治疗带来突破性进展。
Objective
①To establish impaired glucose tolerance(IGT) animal model.②To study the function of adiponectin(APN) and adiponectin receprors (AR) in procession of insulin resistance(IR),and the effect of Rosiglitazone(ROS) on AR'S mRNA expression,Humanity,rat model and cultured cells were studied sequently.The study included four parts:(1) The change and affect factors of plasma adiponectin level in people with impaired glucose regulating(IGR).(2) The change and affect factors of plasma adiponectin,adiponetin receptoos mRNA in liver of Wistar rat's with IGT.(3) The effect of fat-fed and Rosiglitazone on mRNA expression of adiponetin with its receptors.(4) The effects of free fat aid(FFA) and Rosiglitazone on the expression of adiponectin receptors in HepG2 cells.
Methods
Total 122 people were recruited,which included 60 people with normal glucose tolerance(NGT) and 62 people with impaired glucose tolerance(IGT).Oral glucose Tolerance test(OGTT) were tested in all people.
1.The body weight,waistline,hipline,blood pressure and B ultrasound in Liver were measured.The body mass index(BMI) and waistline/hipline ratio(WHR) were calculated.Plasma insulin, adiponectin,PAI-1 and c raction protein(CRP) were examined by using ELISA.Assessed the degree of insulin resistance by using homeostasis model assessment of insulin resistance(HOMA-IR). The hepatic pathological changes were observed by suing light microscope.
2.Fourty Wistar male rats were randomly divided into 2 groups: control group and fat-fed(HF) group.Adiponectin receptor were examined by reverse transcription-polymerse chain raction(RT-PCR). AR protein were measure by western blotting.
3.Sixty male Wistar rats were randomly distributed into 2 groups: including control group 10 rats and HF group 50 rats.In HF group, 20 rats were fed with Rosiglitazone(3mg/kg·d) and other 20 rats were fed with Metformin(300mg/kg·d) AR1 and AR2 mRNA of liver were examined by RT-PCR.
4.HepG2 cells were randomly divided into 8 equivalent groups, including control group,Resiglitazone group,3 groups treated with different concentration of palmitic acid(PA) and 3 groups accordingly treated with above concentration PA plus Rosiglitazone. Then incubating 24 hours,extracted total RNA,RT-PCR and half quantitative analysis the expression of AR1 and AR2 mRNA.
Results
1.People with IGT had lower adiponectin compared with people with NGT.HDL and FPG were independent risk factors that influenced plasma adiponectin level.
2.In HF group,weight,visceral fat content / weight ratio(VF/W), FPG,Insulin and HOMA-IR were higher than that in control group. Adiponectin was lower than the control group.AR1 and AR2 mRNA and protein in liver were decreased in HF group.When treated with Rosiglitazone for 8 weeks and 12 weeks,AR1 and AR2 mRNA of liver increased obviously.
3.mRNA expression of AR2 decreased in groups treated only with PA,especially in the groups over 200 umol/L.While in PA+ROS groups,all AR2 mRNA were higher than corresponding PA groups, particularly in groups which PA above 200 umol/L.
Conclusions
1.The decline of plasma adiponectin may play an importaut role in the development of impaired glucose tolerance in people and Wistar rats.
2.The expressions level of adiponectin and its receptors are closely associated with the development of obesity,high plasma free acid and IR.Rosiglitazone can increase the expression of AR mRNA, especially AR2 mRNA.
3.The increased expression of AR2 mRNA play a key role in Rosiglitazone induced insulin sensitivity amelioration.
脂联素(Adiponectin,APN)是脂肪组织特异性分泌的一种脂肪因子,近年来的研究证实脂联素有多种生物学作用,主要参与体内糖、脂、能量代谢,增加胰岛素敏感性、抗炎和抗动脉粥样硬化的生物学效应。脂联素的生物学作用是在脂联素受体(Adiponectin receptor,AR)的介导下发生的。动物及人体内有两种脂联素受体,即脂联素受体1(AR1)和脂联素受体2(AR2),它们在不同的靶器官组织中表达量各不相同,介导脂联素发挥不同的生物学效应。
脂联素受体mRNA及其蛋白的表达受多种因素影响,其表达异常时会对许多疾病的病理生理过程产生影响,如促进胰岛素抵抗,引起血管内皮功能受损及冠状动脉粥样硬化等。
本研究从人体、动物、细胞三个水平进行研究,其目的旨在:
①研究脂联素及其受体表达的变化与糖耐量减低(IGT)之间的关系;
②探讨脂联素及其受体在胰岛素抵抗(Insulin resistancc,IR)中的作用及意义;
③从动物及细胞水平探讨罗格列酮对脂联素及其受体表达的影响;
④通过改善脂联素及其受体的表达而改善胰岛素抵抗、糖脂代谢,从而为IGT早期干预提供新的治疗靶点。
本研究包括以下四部分内容:
①糖耐量受损人群血清脂联素水平的变化及其影响因素分析。
②糖耐量异常Wistar大鼠血清脂联素及肝脏脂联素受体的表达及相关因素分析。
③罗格列酮及二甲双胍对高脂饲养糖耐量异常大鼠肝脏脂联素受体mRNA表达的作用及机制研究。
④罗格列酮对不同浓度脂肪酸培养HepG2细胞脂联素受体mRNA表达的作用及机制研究。
通过研究以上内容,预期获得如下研究结果:
①糖耐量异常时人及大鼠血清脂联素及肝脏脂联素受体表达减少。
②罗格列酮对改善糖耐量减低大鼠肝脏脂联素及其受体的表达有重要的作用,可以上调AR1、AR2的mRNA表达,这是罗格列酮改善胰岛素敏感性、抗胰岛素抵抗的机制之一。
③以脂联素受体为研究切入点,为开发抗糖尿病、抗动脉粥样硬化的新药物提供理论基础。
方法
本研究涉及临床及基础研究两个部分,从方法学角度包括以下内容:
①全自动生化分析仪检测血液生化指标。
②ELISA法测定血液激素及细胞因子水平。
③免疫印迹法(Western blot)检测AR1及AR2蛋白的表达水平。
④逆转录聚合酶链反应(RT-PCR)方法检测AR1及AR2 mRNA的表达水平,其中涉及总RNA提取方法。
⑤细胞培养及对培养细胞药物处理方法。
具体研究方法按照四部分研究内容介绍如下。
第一部分:糖耐量受损人群血清脂联素水平变化及其影响因素的分析
1.研究对象及分组
纳入标准:122例研究对象全部来自于山西医科大学第二医院健康体检中心,年龄35—65岁;按照2007年美国糖尿病协会(ADA)诊断标准,60例空腹血糖(FPG)＜5.6mmol/L,口服葡萄糖耐量试验(OGTT)2小时血糖(OGTT 2hP6)＜7.8 mmol/L者纳入糖耐量正常组(NGT);62例FPG介于5.6～6.9 mmol/L,OGTT2hPG介于7.8～11.0mmol/L之间者纳入糖耐量减低组(IGT)。
排除标准:①曾明确诊断为糖尿病者;②进行过任何方式的血糖干预治疗;③继发性血糖升高;④有影响血糖升高的因素存在;⑤肝肾功能不全者。满足其中一项者即被排除。
2.研究资料的采集及统计学分析
所有研究对象由专职工作人员检测体重(W)、腰围及臀围、血压(收缩压SBP,舒张压DBP),腹部B型超声波,并计算体重指数(BMI)和腰臀比(WHR)。
用ELISA法检测血清脂联素(APN)、胰岛素(INS)、纤溶酶原活化抑制物(PAI-1)、C反应蛋白(CRP)水平。应用全自动生化分析仪检测血脂谱。
应用SPSS13.0统计软件进行统计分析,计量资料使用均数±标准差表示,正态分布资料组间比较采用t检验:计数资料采用频数或百分率表示;多因素分析Logistic回归分析。
第二部分:糖耐量受损Wistar大鼠血清脂联素及肝脏脂联素受体的表达及相关因素分析
1.动物分组及糖耐量减低动物模型建立
40只5-6周龄雄性Wistar大鼠按照随机数字表法随机分为对照组20只,高脂组20只。
20只高脂组Wistar大鼠给予高脂饲料饲养,每日投食饲料量为体重的3%,在温度22℃-25℃,相对湿度30%-70%的动物房中单笼饲养,每天光照12小时,天黑前投食,自由饮水。高脂饲料配方:5.2kcal/g,其中脂肪提供热量占56.0%,主要为饱和脂肪酸的动物脂肪,碳水化合物占37.0%,蛋白质占7.0%。具体饲料配制:基础面料72%,猪油20%,白糖5%,蛋黄粉5%。第8周开始陆续出现IGT动物模型,至第12周时85%造模成功。
对照组大鼠给予标准饲料饲养,标准饲料配方:3.2kcal/g,其中脂肪提供热量占10.2%,蛋白质占23.3%,碳水化合物占66.5%;饲养环境的条件同IGT动物模型组。
纳入标准:对照组大鼠FPG＜5.6mmol/L,OGTT2hPG＜7.8mmol/L;高脂组大鼠FPG介于5.6～6.9 mmol/L,OGTT2hPG介于7.8～11.0mmol/L之间者纳入糖耐量减低组。
OGTT:过夜禁食8 h后,剪尾采血测空腹血糖,以50%葡萄糖2g/kg.wt灌胃,2小时后再次剪尾采血测血糖。
2.检测、分析研究指标
成模后继续饲养8周,分别处死各组1/2数量大鼠,测定血脂谱、血清胰岛素、血清脂联素,计算稳态模型胰岛素抵抗指数(HOMA-IR),评价胰岛素抵抗(IR)程度,用RT-PCR方法检测肝脏脂联素受体1(AR1)、脂联素受体2(AR2)的mRNA的表达量,用Western blot方法检测肝脏AR1、AR2的蛋白表达量,并在光镜下观察对照组及高脂组肝脏的病理变化。其余两组各1/2数量大鼠饲养至第12周时全部处死,操作程序同第8周。
比较高脂组及对照组BMI、内脏脂肪/体重(VF/W)、血脂谱、血清胰岛素、脂联素、HOMA-IR、肝脏AR1、AR2的mRNA的表达量和AR1、AR2的蛋白表达量,并进行相关因素分析。
第三部分:罗格列酮及二甲双胍对高脂饲养糖耐量受损大鼠肝脏脂联素受体mRNA表达的作用及机制研究
1.动物分组
60只雄性Wistar大鼠按照随机数字表法分为对照组10只(C组)及高脂组(HF组)50只。各组饲养至12周,高脂组大鼠成为糖耐量受损模型,之后分组给予罗格列酮和二甲双胍干预治疗。将高脂组随机分为单纯高脂组10只,高脂+罗格列酮组(GF+ROS组)20只:罗格列酮3mg/kg·d~(-1)灌胃;高脂+二甲双胍组(GF+Met组)20只:二甲双胍300mg/kg·d~(-1)灌胃。
2.肝脏AR1、AR2 mRNA表达的检测
干预治疗第8周时,干预组各组处死1/2数量大鼠,同时处死同期对照组和单纯高脂组大鼠各1/2数量,分离肝脏,迅速保存于-70℃冰箱,待测肝脏AR1、AR2 mRNA的表达;继续饲养至第12周时,处死各组所有大鼠,操作程序同第8周。
应用RT-PCR检测AR1、AR2 mRNA在肝脏的表达;比较各组AR1、AR2 mRNA的表达量,探讨PPAR-γ激动剂罗格列酮和AMPK-γ激活剂二甲双胍对高脂诱导的糖耐量受损大鼠肝脏AR1、AR2基因表达以及肝脏病理学的影响,对肝脏胰岛素抵抗可能的调节机制进行分析。
第四部分:罗格列酮对不同浓度脂肪酸培养HepG2细胞脂联素受体mRNA表达的作用及机制研究
1.细胞培养和分组
用含10%胎牛血清(FCS)的高糖DMEM培养液培养HepG2细胞,置于37℃,5%CO_2浓度的CO_2孵箱内孵育48 h。将细胞随机分为8组,每组含3个9cm直径培养皿,分别为对照组,单加罗格列酮10μmol/L的ROS组,3个加入不同浓度软脂酸(PA)(100μmol/L、200μmol/L和400μmol/L)的PA组,以及3个上述各浓度PA加10μmol/L罗格列酮的PA+ROS组。
PA的配制:13.9mg PA加入0.5ml 0.1M的NaOH中,加热至100℃溶解1小时以上,将高糖DMEM培养液预热至45℃,迅速将溶解于NaOH的PA加入培养液中配置成含所需PA浓度的培养液,待培养液冷却至室温可用于实验培养。
用不含PA和ROS、单含PA、含有PA和/或ROS的培养液,分别置换各组原培养液,继续培养24 h。
2.检测不同组别HepG2细胞AR1、AR2 mRNA表达
提取细胞总RNA,RT-PCR检测AR1、AR2 mRNA水平,用凝胶扫描分析系统定量分析RT-PCR电泳图象。以β-actin为内参照,分别测定AR1和AR2的平均积分光密度(integrated density value,IDV),分别以AR1/β-actin、AR2/β-actin的IDV比值表示其mRNA表达的相对水平,各组mRNA的表达量采用均数±标准差表示。分别对不同PA浓度和同PA浓度的各组采用SPSS 13.0软件进行单因素方差分析(one-way检验)。
结果
1.在临床研究中显示,与NGT组比较,IGT组血清脂联素水平显著降低(P＜0.05),高密度脂蛋白胆固醇(HDL)显著降低(P＜0.01);BMI、WHR、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL)、HOMA-IR、CRP、PAI-1均显著升高(均P＜0.05)。人血清脂联素的相关因素分析显示:脂联素与HDL、OGTT2hPG、WHR、BMI显著相关。以脂联素水平为应变量,各可能相关的因素为自变量,Logistic多元回归分析显示HDL和FPG为影响脂联素水平变化的独立危险因素。
2.动物研究结果显示:
①高脂饲料饲养12周可以成功制备IGT大鼠模型,成模率达85%。
②高脂饲料饲养8周、12周时,与同期标准饲料饲养对照组比较,其体重、腹内脂肪量/体重、空腹胰岛素、HOMA-IR、总胆固醇、甘油三酯、低密度脂蛋白胆固醇高于对照组(均P＜0.05),血清脂联素低于对照组(P＜0.05),高密度脂蛋白胆固醇在12周时低于对照组(P＜0.05)。
③IGT组8周、12周时肝脏组织AR1、AR2 mRNA及蛋白表达量均较NGT组降低(P＜0.05),AR1、AR2蛋白的表达与胰岛素抵抗指数呈负相关,与BMI、VF/W、TG呈负相关。
④IGT组大鼠8周、12周时肝脏组织HE染色显示有肝细胞脂肪变性,小叶内有单核细胞等浸润,出现脂肪肝的病理改变;12周时病理改变较8周时严重。
3.罗格列酮干预治疗高脂饲养IGT大鼠8周及12周后,肝脏AR1及AR2 mRNA表达均较同期单纯高脂组增高。干预治疗8周时AR1 mRNA变化与同期单纯高脂组比较无显著性差异(P＞0.05),AR2 mRNA变化有显著性差异(P＜0.05);干预治疗12周时,AR1及AR2 mRNA变化与同期单纯高脂组比较均有显著性差异(均P＜0.05)。
4.分别用浓度为100μmol/L、200μmol/L和400μmol/L的软脂酸(PA)培养HepG2细胞,观察不同浓度PA培养的HepG2细胞AR1及AR2 mRNA表达的变化,结果显示:当PA浓度大于200μmol/L时,AR2 mRNA表达显著减少,而AR1 mRNA表达无显著变化;再经罗格列酮(ROS)10μmol/L处理不同浓度PA培养的HepG2细胞,观察处理前后HepG2细胞AR1及AR2 mRNA表达的变化,结果显示:在ROS处理的各组中,AR2mRNA的表达均较同PA浓度组增高,尤其是在PA浓度大于200μmol/L时。
结论
1.临床研究及动物实验结果均证实,在IGT阶段已经出现脂联素水平下降;体重增加、内脏脂肪堆积,胰岛素抵抗与脂联素水平下降密切相关。
2.IGT阶段随脂联素水平下降,胰岛素抵抗加剧,促进体内脂质代谢紊乱,体内脂肪分布异常,出现中心型肥胖及非酒精性脂肪性肝病。
3.脂联素受体介导脂联素的生物学效应。IGT阶段肝脏AR1、AR2 mRNA及蛋白的表达均下降,降低了胰岛素的敏感性,加重机体的糖脂代谢紊乱。
4.罗格列酮上调AR1、AR2 mRNA表达,其机制可能通过激活过氧化物酶增殖物激活受体(PPAR-γ),直接促进肝脏AR1、AR2 mRNA的转录及翻译,尤其对AR2的调节作用更强,并调节胰岛素受体后信号转导途径,从而改善胰岛素抵抗。
5.脂联素水平下降可以作为IGT早期干预的重要标志之一以及治疗靶点。展望
脂联素作为一个脂肪因子,在调节糖脂代谢、改善胰岛素抵抗、抗炎、抗动脉粥样硬化及保护血管内皮功能等方面发挥着广泛的生物学效应。脂联素受体的发现为更好地研究APN的生物学作用及作用机制奠定了基础。近年未发现脂联素基因位点与2型糖尿病的一个易感基因位点均出现在3q17染色体处,提示APN与2型糖尿病在基因水平存在某种必然联系。深入研究脂联素和脂联素受体的表达、作用机制及调控机制,有助于进一步阐明一些与脂联素有关的疾病如糖尿病、动脉粥样硬化、冠心病等的发病机制,从而对这些疾病从病因角度寻找有效的防治方法,甚至从基因水平对这些疾病进行预防。研究脂联素及脂联素受体的作用,有助于寻找抗糖尿病、抗动脉粥样硬化新的治疗靶点,并针对这些治疗靶点研发新药物,从而对糖尿病及动脉粥样硬化疾病的治疗带来突破性进展。
Objective
①To establish impaired glucose tolerance(IGT) animal model.②To study the function of adiponectin(APN) and adiponectin receprors (AR) in procession of insulin resistance(IR),and the effect of Rosiglitazone(ROS) on AR'S mRNA expression,Humanity,rat model and cultured cells were studied sequently.The study included four parts:(1) The change and affect factors of plasma adiponectin level in people with impaired glucose regulating(IGR).(2) The change and affect factors of plasma adiponectin,adiponetin receptoos mRNA in liver of Wistar rat's with IGT.(3) The effect of fat-fed and Rosiglitazone on mRNA expression of adiponetin with its receptors.(4) The effects of free fat aid(FFA) and Rosiglitazone on the expression of adiponectin receptors in HepG2 cells.
Methods
Total 122 people were recruited,which included 60 people with normal glucose tolerance(NGT) and 62 people with impaired glucose tolerance(IGT).Oral glucose Tolerance test(OGTT) were tested in all people.
1.The body weight,waistline,hipline,blood pressure and B ultrasound in Liver were measured.The body mass index(BMI) and waistline/hipline ratio(WHR) were calculated.Plasma insulin, adiponectin,PAI-1 and c raction protein(CRP) were examined by using ELISA.Assessed the degree of insulin resistance by using homeostasis model assessment of insulin resistance(HOMA-IR). The hepatic pathological changes were observed by suing light microscope.
2.Fourty Wistar male rats were randomly divided into 2 groups: control group and fat-fed(HF) group.Adiponectin receptor were examined by reverse transcription-polymerse chain raction(RT-PCR). AR protein were measure by western blotting.
3.Sixty male Wistar rats were randomly distributed into 2 groups: including control group 10 rats and HF group 50 rats.In HF group, 20 rats were fed with Rosiglitazone(3mg/kg·d) and other 20 rats were fed with Metformin(300mg/kg·d) AR1 and AR2 mRNA of liver were examined by RT-PCR.
4.HepG2 cells were randomly divided into 8 equivalent groups, including control group,Resiglitazone group,3 groups treated with different concentration of palmitic acid(PA) and 3 groups accordingly treated with above concentration PA plus Rosiglitazone. Then incubating 24 hours,extracted total RNA,RT-PCR and half quantitative analysis the expression of AR1 and AR2 mRNA.
Results
1.People with IGT had lower adiponectin compared with people with NGT.HDL and FPG were independent risk factors that influenced plasma adiponectin level.
2.In HF group,weight,visceral fat content / weight ratio(VF/W), FPG,Insulin and HOMA-IR were higher than that in control group. Adiponectin was lower than the control group.AR1 and AR2 mRNA and protein in liver were decreased in HF group.When treated with Rosiglitazone for 8 weeks and 12 weeks,AR1 and AR2 mRNA of liver increased obviously.
3.mRNA expression of AR2 decreased in groups treated only with PA,especially in the groups over 200 umol/L.While in PA+ROS groups,all AR2 mRNA were higher than corresponding PA groups, particularly in groups which PA above 200 umol/L.
Conclusions
1.The decline of plasma adiponectin may play an importaut role in the development of impaired glucose tolerance in people and Wistar rats.
2.The expressions level of adiponectin and its receptors are closely associated with the development of obesity,high plasma free acid and IR.Rosiglitazone can increase the expression of AR mRNA, especially AR2 mRNA.
3.The increased expression of AR2 mRNA play a key role in Rosiglitazone induced insulin sensitivity amelioration.
引文
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