Mfn2表达对高脂诱导的胰岛素抵抗大鼠肝脏糖、脂代谢的影响及机制研究
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摘要
近年来，2型糖尿病的患病率有急剧增加的趋势，据估计到2025年，患糖尿病的人数将超过3亿。胰岛素抵抗是肥胖和2型糖尿病早期重要的发病机制，已经成为重要公众健康问题。胰岛素抵抗状态下存在糖类和脂肪代谢效率降低。线粒体是机体能量代谢和供给的核心细胞器，在代谢性疾病中的作用不容忽视。已有研究表明线粒体功能障碍与胰岛素抵抗和2型糖尿病的发生密切相关，可能是胰岛素抵抗的发生机制之一。线粒体功能异常包括氧化能力的降低及ATP合成的减少，可导致脂质沉积，引起胰岛素抵抗。
线粒体形态结构的维持在调节能量代谢中发挥重要作用。线粒体是一种动态的细胞器，通过不断的融合和断裂过程来维持其网状结构及功能。线粒体融合蛋白（mitofusin，Mfn）促进线粒体融合，调节线粒体生物合成及网状结构的维持。而Mfn2是哺乳动物细胞中介导线粒体融合的关键蛋白之一。体内和体外实验模型表明生理或病理状态可导致Mfn2表达的改变，如糖尿病、肥胖、胰岛素抵抗下调Mfn2表达，而运动和体重减轻则上调Mfn2表达。有研究报道，下调Mfn2表达使线粒体膜电位降低、氧耗及葡萄糖氧化能力降低，而上调Mfn2表达使线粒体膜电位增加，氧耗及葡萄糖氧化能力增强。先前研究表明高脂饮食下调了胰岛素抵抗大鼠骨骼肌Mfn2表达。
越来越多的研究表明Mfn2表达与胰岛素抵抗状态相关，但具体机制仍不清楚。本课题通过高脂饮食喂养大鼠建立胰岛素抵抗模型，同时用饱和脂肪酸孵育HepG2细胞建立胰岛素抵抗细胞模型，采用重组腺病毒介导的基因转染方法上调Mfn2表达，研究Mfn2对胰岛素抵抗大鼠肝脏糖、脂代谢的的影响，分别在动物和细胞水平探讨其改善胰岛素抵抗的具体分子机制。本实验内容主要包括以下四部分：
第一部分高脂诱导胰岛素抵抗发生及对肝脏Mfn2表达的影响
目的：建立高脂诱导的胰岛素抵抗动物模型，观察高脂对大鼠肝脏Mfn2表达的影响。
方法：雄性Sprague-Dawley（SD）大鼠84只，体重60-80g，适应性喂养1周后，随机分为正常对照组（normal diet, ND）12只和高脂组（high-fat diet, HFD）72只。ND组给予基础饲料，热量组成为脂肪10.3%，碳水化合物65.5%，蛋白质24.2%，总热量为348kcal/100g；HFD组给予高脂饲料，其热量组成为：脂肪59.8%，碳水化合物20.1%，蛋白质20.1%，总热量为501kcal/100g。所有动物自由摄食饮水，明暗周期为12h（6am-6pm），室温20℃-23℃。实验第8周末，两组分别随机选出6只进行清醒状态下高胰岛素-正葡萄糖钳夹（hyperinsulinaemic euglycaemic clamp）实验，计算葡萄糖输注率（glucose infusion rate，GIR），评价胰岛素敏感性。判断造模成功后，处死大鼠，留取血清及肝组织进行相关指标检测。血糖（blood glucose，BG）应用快速血糖仪测定。血浆胰岛素（insulin，INS）、游离脂肪酸（free fatty acid，FFA）检测采用ELISA试剂盒测定。血甘油三酯（triglyceride，TG）及总胆固醇（total cholesterin，TC）用全自动生化分析仪测定。肝组织Mfn2mRNA及蛋白水平分别采用Real time PCR及Western blot方法检测。
结果：
1两组大鼠一般指标的比较
各组大鼠体重随时间延长而增加，HFD组大鼠体重（357.0±32.5g）与ND组（355.7±39.0g）相比，差异无统计学意义（P>0.05）。HFD组FBG水平（5.72±0.43mmol/L）高于ND组（5.21±0.38mmol/L），差异有统计学意义（P<0.05）。HFD组INS水平（37.59±7.02mU/L）高于ND组（27.24±4.68mU/L），差异有统计学意义（P<0.05）。HFD组血TG水平（0.32±0.09mmol/L）高于ND组（0.24±0.05mmol/L）；HFD组血TC水平（1.42±0.17mmol/L）较ND组（1.16±0.15mmol/L）高，差异有统计学意义（P<0.05）。HFD组血FFA水平（0.80±0.12mol/L）较ND组（0.60±0.08mol/L）高，差异有统计学意义（P<0.01）。
2两组大鼠葡萄糖输注率的比较
HFD组GIR（13.66±2.54mg/(kg·min)）较ND组（28.14±5.00mg/(kg·min)）显著降低，差异有统计学意义（P<0.01）。
3两组大鼠肝脏Mfn2mRNA及蛋白水平的比较
HFD组大鼠肝脏Mfn2mRNA表达水平（0.23±0.04）较ND组（0.91±0.14）降低，差异有统计学意义（P<0.01）。HFD组Mfn2蛋白含量（0.28±0.05）较ND组（0.87±0.07）低，差异有统计学意义（P<0.01）。
4肝组织形态学的比较
光镜下观察：ND组肝组织结构完整，肝小叶规则，肝细胞排列整齐，在中央静脉周围呈放射状分布，肝细胞中央有大而圆的核，细胞质均匀，未见脂滴和炎症细胞侵润；HFD组大鼠肝小叶结构紊乱，肝细胞肿胀，肝细胞胞浆内可见大量脂滴空泡，以中央静脉周围最为明显，重者胞质疏松呈网状改变，且可见小叶内及汇管区炎症细胞侵润。
透射电镜观察：ND组肝细胞中细胞器结构完好，线粒体、粗面内质网丰富，胞浆内无脂滴及脱颗粒现象，线粒体膜、嵴结构完整，排列规则，粗面内质网排列整齐；HFD组肝细胞内可见大量大小不等的脂滴分布，线粒体有一定程度的肿胀，内外膜部分融合，线粒体嵴变少变短，部分或全部消失，甚至出现空泡化。
结论：
1高脂饮食诱导大鼠产生胰岛素抵抗，具有高血糖、高胰岛素血症、高脂血症等特点。
2高脂饮食导致大鼠肝细胞脂肪变性及线粒体超微结构的异常。
3高脂诱导的胰岛素抵抗大鼠肝脏Mfn2表达降低，Mfn2与胰岛素抵抗的发生相关。
第二部分Mfn2对胰岛素抵抗大鼠肝脏GK、PEPCK活性的影响
目的：观察腺病毒介导的Mfn2转染对胰岛素抵抗大鼠糖代谢相关酶葡萄糖激酶（glucoskinase, GK）及磷酸烯醇式丙酮酸羧激酶（phosphoen-olpyruvate carboxykinase, PEPCK）的活性影响。
方法：实验8周末判断胰岛素抵抗大鼠模型建立成功后，将高脂组剩余60只大鼠随机分为5组，分别为：高脂对照组（Con）12只、空载体对照组（emptyAd adenovirus, Ad）12只、Ad-Mfn2低剂量组（1×10~8v.p/kg）12只、Ad-Mfn2中剂量组（1×10~8v.p/kg）12只、Ad-Mfn2高剂量组（1×10~(10)v.p/kg）12只。以鼠尾静脉注射的方式，Con组给予磷酸盐缓冲液（PBS缓冲液），Ad组给予空载体（1×10~8v.p/kg体重），Ad-Mfn2低中高剂量组分别给予相应剂量Mfn2的重组腺病毒。每周1次，连续3周。给予腺病毒干预3周后，进行清醒状态下高胰岛素-正葡萄糖钳夹实验，计算GIR，处死动物并留取血清及肝组织进行相关指标检测。血糖、血胰岛素及游离脂肪酸测定方法同第一部分。肝脏糖原含量采用肝糖原试剂盒测定。肝GK及PEPCK活性采用酶偶联比色法测定。
结果:
1给予Mfn2重组腺病毒干预3周后，各组大鼠肝脏Mfn2mRNA及蛋白表达的比较
随着Ad-Mfn2剂量增加，大鼠肝脏Mfn2mRNA及蛋白表达水平升高，与Ad组相比差异有统计学意义（P<0.01）。
2给予Mfn2重组腺病毒干预3周后，各组一般指标的比较
随时间延长各组大鼠体重均增加，但差异无统计学意义（P>0.05）。大鼠FBG、INS随着Ad-Mfn2剂量增加而降低，而GIR升高，与Ad组相比差异有统计学意义（P<0.05）。
3各组肝脏糖原含量的比较
8周末：HFD组肝糖原含量（4.29±0.45mg/g）较ND组（5.31±0.63mg/g）低，差异有统计学意义（P<0.05）。
给予Mfn2重组腺病毒干预3周后：肝糖原含量随着Ad-Mfn2剂量增加而升高，Ad-Mfn2中、高剂量组肝糖原含量与Ad组相比差异有统计学意义（P<0.05）。
4各组大鼠肝脏GK、PEPCK活性的比较
8周末：HFD组肝脏GK活性（7.70±1.41mIU/(min·mg protein)）较ND组（13.87±1.72mIU/(min·mg protein)）低，差异有统计学意义（P<0.01）；而PEPCK活性（18.40±2.20mIU/(min·mg protein)）较ND组（10.67±2.50mIU/(min·mg protein)）高，差异有统计学意义（P<0.01）。
给予Mfn2重组腺病毒干预3周后：与Ad组相比，肝GK活性随着Ad-Mfn2剂量的增加而升高，PEPCK活性随着Ad-Mfn2剂量的增加降低，差异有统计学意义（P<0.05）。
5给予Mfn2重组腺病毒干预3周后，大鼠肝脏形态学及超微结构的改变
光镜下观察：Ad组可见肝小叶结构明显紊乱，肝细胞索消失，肝细胞变大，肝细胞胞浆内可见大量圆形脂滴空泡，核居边，且小叶内炎细胞侵润程度增加。Ad-Mfn2不同剂量组可见肝细胞体积较高脂组变小，胞浆内脂滴减少。
透射电镜观察：Ad组肝细胞胞浆中可见大量脂滴，肝细胞核不规整，线粒体膜模糊不清，部分膜破裂，线粒体嵴几乎消失不见，偶有少量残存，有空泡样变；Ad-Mfn2不同剂量组肝细胞胞浆中脂滴较高脂组数量少，线粒体肿胀减轻，内外膜部分融合，线粒体嵴部分或大部分消失。
结论：
1高脂饮食可导致大鼠血糖水平升高，肝糖原含量减少，糖酵解及氧化关键酶GK活性降低、糖异生关键酶PEPCK活性增加，引起糖代谢异常。
2上调Mfn2表达，胰岛素抵抗大鼠肝糖原含量升高，GK活性增加、PEPCK活性降低，并且使肝细胞脂肪变性和线粒体超微结构损伤减轻。Mfn2改善胰岛素抵抗可能与调节GK、PEPCK活性及影响线粒体形态结构有关。
第三部分Mfn2对胰岛素抵抗大鼠肝脏ACCα、CPT-Ⅰα表达的影响
目的:观察腺病毒介导的Mfn2转染对胰岛素抵抗大鼠脂代谢相关酶乙酰辅酶A羧化酶α（acetyl CoA carboxylase α, ACCα）、肉毒碱棕榈酰转移酶-Ⅰα（carnitine palmitoyltransferase-Ⅰ, CPT-Ⅰα）表达的影响。
方法：动物分组及处理同第一、二部分。肝脏TG含量按试剂盒说明测定，Real time PCR测定肝组织ACCα、CPT-Ⅰα mRNA水平，Western blot方法检测ACCα、CPT-Ⅰα蛋白水平。
结果：
1给予Mfn2重组腺病毒干预3周后，各组大鼠血液一般指标的比较
血TG、TC及FFA水平随着Ad-Mfn2剂量的增加而降低，Ad-Mfn2中、高剂量组TC、 FFA水平低于Ad组，差异有统计学意义（P<0.05）；Ad-Mfn2高剂量组TG水平低于Ad组，差异有统计学意义（P<0.05）。
2各组大鼠肝脏TG含量的比较
8周末：HFD组肝脏TG含量（595.35±159.24μmol/L）高于ND组（371.75±105.47μmol/L），差异有统计学意义（P<0.05）。
Mfn2重组腺病毒干预3周后：肝脏TG含量随着Ad-Mfn2剂量的增加而降低，与Ad组相比，差异有统计学意义（P<0.05）。
3各组大鼠肝脏CPT-Ⅰα和ACCα mRNA及蛋白水平的比较
8周末：HFD组肝脏CPT-Ⅰα mRNA表达及蛋白水平较ND组低，差异有统计学意义（P<0.05）。HFD组ACCα mRNA水平较ND组高，但蛋白磷酸化水平低于ND组，差异有统计学意义（P<0.05）。
Mfn2重组腺病毒干预3周后：CPT-Ⅰα mRNA及蛋白表达随Ad-Mfn2剂量的增加而升高，与Ad组相比，以中、高剂量组明显，差异有统计学意义（P<0.05）。与Ad组相比，ACCα mRNA水平随着Ad-Mfn2剂量的增加而降低，而蛋白磷酸化水平则升高，差异有统计学意义（P<0.05）。
结论：
1高脂饮食可导致肝脏脂质沉积，脂肪酸氧化关键酶CPT-Ⅰα表达降低，脂肪酸合成关键酶ACCα磷酸化水平降低。
2上调Mfn2表达，胰岛素抵抗大鼠血脂降低，肝脏脂质沉积减轻，脂代谢相关酶CPT-Ⅰα表达升高，ACCα磷酸化水平升高。Mfn2改善胰岛素抵抗可能与CPT-Ⅰα表达升高及ACCα活性降低，改善脂代谢异常有关。
第四部分Mfn2改善胰岛素抵抗的机制研究
目的：测定胰岛素抵抗大鼠肝脏及HepG2细胞胰岛素信号通路磷脂酰肌醇3激酶（phosphatidylinositol3-kinase，PI3K）/蛋白激酶B（proteinkinase B, PKB/AKT）途径相关分子的表达，探讨Mfn2改善胰岛素抵抗的具体作用机制。
方法：胰岛素抵抗大鼠模型及动物实验同第一、二部分。细胞培养：分别用含0.25mmol/L软脂酸（palmitate，PA）培养基和普通培养基（normalcontrol， NC）培养HepG2细胞24小时后采用葡糖糖氧化酶法测定细胞培养液的葡糖糖含量。判断胰岛素抵抗模型建立成功后，设立软脂酸对照组（Con）、软脂酸空载体组（Ad）、Ad-Mfn2低剂量组（50pfu/cell，Mfn2L）、Ad-Mfn2高剂量组（100pfu/cell, Mfn2H）。Con组给予PBS缓冲液，Ad组按50pfu/cell空载体给予，Ad-Mfn2低剂量组按50pfu/cell给予Ad-Mfn2，Ad-Mfn2高剂量组按100pfu/cell给予Ad-Mfn2。培养24h后，测定各组细胞培养基葡萄糖含量，并采用Real time PCR、Western blot方法分别检测各组Mfn2、INSR、IRS2、PI3K、AKT2、GLUT2mRNA及蛋白水平表达变化。
结果：
1HepG2细胞的胰岛素抵抗体外模型建立
PA组葡萄糖含量（12.16±0.54mmol/(L·mg protein)）较Con组（16.02±0.73mmol/(L·mg protein)）高，差异有统计学意义（P<0.01），表明用软脂酸诱导胰岛素抵抗体外细胞模型成功建立。
2HepG2胰岛素抵抗细胞Mfn2及胰岛素信号通路相关分子的表达
PA组Mfn2mRNA水平（0.48±0.11）较Con组（0.97±0.03）低，差异有统计学意义（P<0.01）。PA组Mfn2的蛋白表达量（0.46±0.06）较Con组（0.90±0.11）低，差异有统计学意义（P<0.01）。PA组INSR、IRS2、GLUT2mRNA及蛋白表达较Con组低，差异有统计学意义（P<0.05）。PA组PI3K、AKT2mRNA及总蛋白水平与Con组相比，差异无统计学意义（P>0.05）。但是PI3K和AKT2蛋白磷酸化水平较Con组显著下降，差异有统计学意义（P<0.05）。
3MTT法检测Ad、Ad-Mfn2对HepG2细胞增殖的影响，Ad组、Mfn2低剂量及高剂量组细胞增殖率无明显差别。
4Mfn2重组腺病毒干预后各组HepG2细胞胰岛素敏感性的比较Mfn2L组及Mfn2H组细胞培养液葡萄糖含量较Ad组降低（P<0.01）。
5Mfn2重组腺病毒干预后各组细胞Mfn2及胰岛素信号通路相关分子的表达
Mfn2L组及Mfn2H组HepG2细胞Mfn2表达较Ad组显著增加（P<0.01），INSR、IRS2、GLUT2mRNA水平及蛋白表达量较Ad组高，差异有统计学意义（P<0.05）。与Ad组相比，PI3K、AKT2mRNA表达水平及总蛋白表达量变化不显著（P>0.05），但其蛋白磷酸化水平明显升高，差异有统计学意义（P<0.05）。
6大鼠肝脏INSR、IRS2、PI3K、AKT2、GLUT2mRNA水平及蛋白表达的比较
实验8周末，HFD组大鼠肝脏INSR、IRS2、GLUT2mRNA表达水平较ND组低，差异有统计学意义（P<0.05）。PI3K、AKT2mRNA及总蛋白水平与ND组相比，差异无统计学意义（P>0.05）。但PI3K和AKT2蛋白磷酸化水平较ND组显著下降，差异有统计学意义（P<0.05），见Fig.5。
Mfn2重组腺病毒干预3周后，转染Ad-Mfn2的胰岛素抵抗大鼠肝脏INSR、IRS2、GLUT2mRNA及蛋白表达水平较Ad组升高，差异有统计学意义（P<0.05）。PI3K和AKT2蛋白磷酸化水平较Ad组升高，差异有统计学意义（P<0.05）。
结论：
1高浓度游离脂肪酸可诱导HepG2细胞产生胰岛素抵抗，伴随Mfn2表达降低，胰岛素信号通路相关的INSR、IRS2、GLUT2表达降低，PI3K、AKT2蛋白磷酸化水平下降。
2高脂诱导大鼠肝胰岛素信号通路相关分子INSR、IRS2、GLUT2表达下降，PI3K、AKT2蛋白磷酸化水平下降。
3上调Mfn2表达，胰岛素抵抗大鼠肝脏及HepG2细胞胰岛素信号通路相关分子INSR、IRS2、GLUT2表达升高及PI3K、AKT2蛋白磷酸化水平升高，说明Mfn2通过调节胰岛素信号传导通路相关分子表达改善胰岛素抵抗。
The incidence of type2diabetes（T2DM） has increased dramatically inrecent years. It is estimated that more than300million individuals worldwidewill be at high risk of developing T2DM by the year2025. Insulin resistanceplays a primary role in the pathogenesis of T2DM, and becomes a majorpublic health issue. Metabolic efficiency of glucose and lipid has decreasedduring insulin resistance. Mitochondria are core organelles that have a centralrole in the energy metabolism, and its role in metabolic diseases can not beignored. Studies have shown that mitochondrial dysfunction is closely relatedto the development of insulin resistance and type2diabetes, andmitochondrial damage may be one of the mechanisms of insulin resistance.Mitochondrial dysfunction characterized by reduced oxidative capacity andATP generation can lead to lipid deposition and induce insulin resistance.
The maintenance of mitochondrial morphology play a pivotal rolein energy control. Mitochondria is a dynamic organelle, maintaining abalance by continuous fusion and division. Mitofusin (Mfn) promotesmitochondrial fusion, and regulates the biosynthesis and maintenance of themitochondrial network structure. Mitofusin2(Mfn2) is a mitochondrial fusionprotein, mainly expressed in mammalian cells. Mitofusin2(Mfn2) is amitochondrial fusion protein, mainly expressed in mammalian cells. Researchin vitro and in vivo experimental models suggest that the physiological orpathological conditions can lead to changes in Mfn2expression, for example,diabetes, obesity and insulin resistance down-regulate expression of Mfn2,exercise and weight loss up-regulate expression of Mfn2. Data sugguest thatdown-regulation of Mfn2expression lead to low mitochondrial membranepotential and reduced oxygen consumption and glucose oxidation, while up-regulation of Mfn2expression lead to high mitochondrial membranepotential and enhanced glucose oxidation. In addition, previous studies haveshown that high-fat diet down-regulated Mfn2expression.
Increasing evidence indicates that expression of Mfn2is related to insulinresistance. However, the specific mechanism remains unclear. To enhance ourunderstanding to these questions, we established a high-fat diet inducedinsulin resistant rat model, at the same time HepG2cells were incubated withpalmitate to establish a insulin resistant HepG2cell model, infected insulinresistant rats with Mfn2expression adenovirus, the effect of Mfn2over-expression on the glucose and lipid metabolism in rats liver wasevaluated. Then, the specific molecular mechanism at the animal and cellularlevels through which Mfn2ameliorates insulin resistance was exploredrespectively.
The paper contains four parts as below:
Part Ⅰ: High-fat diet induces insulin resistance and its effect on theexpression of Mfn2
Objective: To establish an animal model of insulin resistance induced byhigh-fat diet and to determine the effect of high-fat diet on expression ofMfn2.
Methods: Male SD rats, weight about60-80g, were randomly dividedinto2groups: normal control (ND, n=12) and high fat diet group (HFD, n=72).Rats were fed with a regular low fatty acids diet contained10.3%fat,24.2%protein, and65.5%carbohydrate (kcal) or high-fat diet which consists of59.8%fat,20.1%protein, and20.1%carbohydrate (kcal). Rats in every grouphad free access to water and chow. The environment was controlled in termsof light (12:12-h light-dark cycle starting at6:00AM) and humidity. Insulinresistance was evaluated by glucose infusion rate (GIR) of hyperinsulinemiceuglycemic clamp technique at the end of8weeks（six rats in each group).The blood samples were obtained from the abdominal aorta. Fasting bloodglucose (FBG) levels were measured by Accu-chek Active Meter(ACCU-CHEK Active; Roche). Insulin (INS) and free fatty acids (FFA) levels were analyzed by using a Rat insulin or FFA ELISA kit (Crystal Chem.Inc). The liver tissue samples of rats were taken immediately and kept at-70℃after quick frozen in liquid nitrogen. The expression of Mfn2wasdetected by quantitative Real time PCR and Western blot. The morphologicalchanges in the ultrastructure of their hepatic cells were investigated by meansof electron microscopy assay.
Results:
1The body weight between two groups had no significant difference (P>0.05). Compared with ND group, GIR in HFD group was significantlydecreased (P<0.01); while the levels of BG, TG, TC, INS and FFA in HFDgroup were significantly higher than these in the ND group (P <0.05).
2Expression of Mfn2mRNA and protein were significantly decreased inHFD group (P<0.01), compared with ND group.
3Rats liver tissue morphology by histological HE staining: normal liver cellswere regularly aligned, liver lobular architecture was intact, and only a smallnumber of fibers were formed in blood vessel walls. There was diffusesteatosis, especially surrounding central veins, significant cell swelling,massive lipid droplet vacuole and inflammation cellular infiltration in lobulaof liver in HFD group. Electron microscopy assay revealed that the shape andsize of mitochondria in the hepatocytes of the control group were normal andhad few lipid droplets. In contrast, many mitochondria from rats in thehigh-fat diet group were enlarged and swollen, and showed morphologicalchanges, including large lipid droplet, and mitochondrial inner-outermenbrance meromixis. The cristae on the inner membrane of mitochondriawere disappeared or unclear.
Conclusions：
1High-fat diet-fed rats showed high concentrations of glucose,hyperlipidemia, hyperinsulinemia and GIR was lower. High-fat diet inducedinsulin resistance in rats.
2High-fat diet cause hepatic steatosis and ultrastructure damage inhepatocellular mitochondria.
3Expression of Mfn2in the liver tissues of High-fat diet-fed rats wasdown-regulated, implicating that the development of insulin resistance may berelated to low expression of Mfn2.
Part Ⅱ: Effect of Mfn2over-expression on the activity ofglucoskinase and phosphoenolpy ruvate carboxykinase in the liver ofinsulin resistant rats
Objective: To investigate the effects of Mfn2on the insulin sensitivityand the activity of Glucoskinase (GK) and Phosphoenolpyruvatecarboxykinase (PEPCK) in liver of rats fed with high-fat diets.
Methods: After8weeks, rats fed high-fat diets were randomly dividedinto5groups: high-fat diet control group infused with PBS buffer (Con, n=12),high fat diet group infected with empty control adenovirus (Ad, n=12),high-fat diet group were respectively infected with different amount ofAd-Mfn2(108,109or1010v.p/kg body weight) once a week, for3weeks. Afterintervention with adenovirus for3weeks, insulin resistance was evaluated byGIR of hyperinsulinemic euglycemic clamp technique. The blood sampleswere obtained from the abdominal aorta. The liver tissue samples of rats weretaken immediately and kept at-70℃after quick frozen in liquid nitrogen.FBG, TG, TC, INS and FFA were tested by methods as part one. The activityof GK and PEPCK were detected by enzyme-coupled colorimetric assay. Thelevel of hepatic glycogen was detected by using a glycogen assay kit. Theexpression levels of Mfn2were detected by Real time PCR and Western blot.
Results:
1The expression of Mfn2of rats infected with different amount of Ad-Mfn2(10~8,10~9or10~(10)v.p/kg body weight) for3weeks, were increased dramatically(P<0.01).
2After intervention with adenovirus for3weeks, the body weight had nosignificant difference among groups (P>0.05). FBG, INS of rats infected withdifferent amount of Ad-Mfn2(10~8,10~9or10~(10)v.p/kg body weight) for3weeks were decreased (P<0.05), while GIR was significantly increased(P<0.01).
3At the end of8weeks, concentration of hepatic glycogen in rats fed withhigh-fat diet was significantly decreased (P<0.05), compared with the NDgroup. After intervention with adenovirus for3weeks, concentrations ofhepatic glycogen in rats infected with Ad-Mfn2was increased with theincreasing of dose. Compared with the rats fed with high-fat diet, the glycogencontents in rats infected with Ad-Mfn2(109or1010v.p/kg body weight) wereincreased (P<0.05).
4The activity of GK was increased (P<0.01)and PEPCK decreased in theliver of rats fed high-fat diet for8weeks (P<0.01). After intervention withadenovirus for3weeks, with increasing dose of Ad-Mfn2, the activty of GKwas increased (P<0.01), while the activity of PEPCK was decreased (P<0.01).Compared with rats high-fat diet-fed,the activity of GK and PEPCK of liver inrats infected with Ad-Mfn2(109or1010v.p/kg body weight),there weresignificant difference (P<0.05or P<0.01).
5All of the sections in the Con and Ad group exhibited diffuse hepaticsteatosis under a light microscope. Hepatic steatosis was most obvious aroundthe portal area (mostly microvesicular and macrovesicular mixed steatosis)and was accompanied by inflammatory cell infiltration. The liver HE stainingof rat infected with Ad-Mfn2shows less cell volume and fat dropletaccumulation. Electron microscope revealed that mitochondria from rats in theCon and Ad group were enlarged and swollen, and showed morphologicalchanges, including larger lipid droplet, and mitochondrial membrane isunclear, even had vacuolar degeneration. The cristae on the inner membraneof mitochondria were almost disappeared.
Conclusions：
1High-fat diets can lead to abnormal glucose metabolism, accompanied bylower concentration of hepatic glycogen，lower activity of GK and higher ofPEPCK.
2Over-expression of Mfn2improves the glucose metabolism.The activity ofGK was increased， the activity of PEPCK was decreased， and theconcentration of hepatic glycogen was higher. It also alleviates hepatic steatosis and ultrastructure damage in hepatocellular mitochondria.Over-expression of Mfn2ameliorated high-fat diet induced insulin resistance，and maybe related to the increasing activity of GK，decreasing activity ofPEPCK，and alleviating the demage of the liver cell and mitochondria.
Part Ⅲ: Effect of Mfn2on expression of ACCα and CPT-Ⅰα in liverof insulin resistant rats
Objective: To investigate the effects of Mfn2on the expression of AcetylCoA carboxylase α (ACCα) and Carnitine palmitoyltransferase-Ⅰα (CPT-Ⅰα)in liver of rats fed with high-fat diets.
Methods: Animal grouping and treatment was the same as the partⅠand partⅡ. Hepatic triglyceride (TG) content was detected with a triglyceridedetermination kit. The expression levels of ACCα and CPT-Ⅰα were detectedby Real time PCR and Western blot.
Results:
1After intervention with adenovirus for3weeks, the plasma TG, TC and FFAlevels in liver of rats infected with Ad-Mfn2(108,109or1010v.p/kg bodyweight) for3weeks were decreased (P<0.05), compared with Ad group.
2At the end of8weeks, hepatic TG content of rats fed with high-fat diet wassignificantly increased (P<0.05), compared with the ND group. Afterintervention with adenovirus for3weeks, hepatic TG contents of rats infectedwith Ad-Mfn2was increased with the increasing dose of Ad-Mfn2, Comparedwith Ad, hepatic TG contents in rats infected with Ad-Mfn2(109or1010v.p/kg body weight) were decreased (P<0.05or P<0.01).
3The mRNA expression of ACCα was increased and CPT-Ⅰα was decreased(P<0.05). Phosphorylation of ACCα protein was decreased (P<0.05), butCPT-Ⅰα protein was decreased at the end of8weeks. After intervention withadenovirus for3weeks, with increasing dose of Ad-Mfn2, the mRNAexpression of ACCα was decreased (P<0.01) and CPT-Ⅰα was increased(P<0.05). Phosphorylation of ACCα protein was increased (P<0.01), butCPT-Ⅰ α protein expression was increased, compared with Ad group(P<0.05).
Conclusions：
1High-fat diet can lead to abnormal lipid metabolism, accompanied by highhyperlipidemia, high FFA levels, and Lipid deposition. The activity of ACCαwas increased and expression of CPT-Ⅰα was down-regulated in rats fed withhigh-fat diet.
2Over-expression of Mfn2improved the lipid metabolism. Mfn2amelioratedinsulin resistance, probably by decreasing the activity of ACCα and heptic TGcontents, while enhancing expression of CPT-Ⅰα..
Part Ⅳ: Mechanism of over-expression Mfn2ameliorating insulinresistance
Objective: To explore the mechanism of Mfn2over-expressionameliorating insulin resistance by testing the expression of relative moleculesof the insulin signaling pathway.
Methods: The model of IR was established with HepG2cells cultured athigh concentrations of palmitate (PA,0.25mmol/L) for24h. The glucosecontent was measured by glucose assay kit. Insulin resistant HepG2cells weredivided into4groups: HepG2cells cultured with PA (Con) group, HepG2cells cultured with PA infected with empty control adenovirus (Ad) group，HepG2cells cultured with PA infected with Ad-Mfn2(50or100pfu/cell), andcultured for24h. The expression of INSR, IRS2, PI3K, AKT2and GLUT2were detected by Real time PCR and Western blot.
Results:
1The glucose contents of PA group were significantly higher than those incontrol (P<0.01).
2The expression of Mfn2of insulin resistant HepG2cells was decreasedsignificantly (P<0.01). The expression of INSR, IRS2and GLUT2weredown-regulated in HepG2cells cultured with PA (P<0.01). There were nochanges in PI3K and AKT2expression, but their phosphorylation levelsdecreased significantly (P<0.01), compared with Ad group.
3After infected with Ad-Mfn2for24h, the glucose contents of HepG2cellscultured with PA infected with Ad-Mfn250and100pfu/cell were lower thanAd group (P<0.01)。
4The expression of Mfn2in HepG2cells cultured with PA infected withAd-Mfn250or100pfu/cell were increased, compared with Ad group.Over-expression of Mfn2were confirmed (P<0.01). Over-expression of Mfn2up-regulated the expression of INSR, IRS2and GLUT2, and phosphorylationlevels of PI3K and AKT2were increased (P<0.01).
5At the end of8weeks, the expression of INSR, IRS2and GLUT2weredown-regulated markedly by high-fat diets at8weeks (P<0.01). There wereno changes in PI3K and AKT2expression, their phosphorylation levelsdecreased significantly (P<0.01), compared with Ad group.
6After intervention with adenovirus for3weeks, Over-expression of Mfn2up-regulated the expression of INSR, IRS2and GLUT2, phosphorylationlevels of PI3K-P85and AKT2increased (P<0.01).
Conclusions：
1High-fat diets inhibited the expression of insulin signaling pathway. Theexpressions of INSR, IRS2and GLUT2were down-regulated by high-fat dietand protein phosphorylation levels of PI3K and AKT2were decreased.
2High concentrations of palmitate could induce HepG2cells hepatic insulinresistance, and down-regulate the expression of INSR, IRS2and GLUT2andprotein phosphorylation levels of PI3K and AKT2.
3Over-expression of Mfn2improved insulin resistance of rats or HepG2cells,and up-regulated the expression of INSR, IRS2and GLUT2. Proteinphosphorylation levels of PI3K and AKT2were decreased. Our data suggestthat Mfn2might ameliorate insulin resistance by enhancing the expression ofinsulin signaling pathway that are import in glucose and lipid metablism.
线粒体形态结构的维持在调节能量代谢中发挥重要作用。线粒体是一种动态的细胞器，通过不断的融合和断裂过程来维持其网状结构及功能。线粒体融合蛋白（mitofusin，Mfn）促进线粒体融合，调节线粒体生物合成及网状结构的维持。而Mfn2是哺乳动物细胞中介导线粒体融合的关键蛋白之一。体内和体外实验模型表明生理或病理状态可导致Mfn2表达的改变，如糖尿病、肥胖、胰岛素抵抗下调Mfn2表达，而运动和体重减轻则上调Mfn2表达。有研究报道，下调Mfn2表达使线粒体膜电位降低、氧耗及葡萄糖氧化能力降低，而上调Mfn2表达使线粒体膜电位增加，氧耗及葡萄糖氧化能力增强。先前研究表明高脂饮食下调了胰岛素抵抗大鼠骨骼肌Mfn2表达。
越来越多的研究表明Mfn2表达与胰岛素抵抗状态相关，但具体机制仍不清楚。本课题通过高脂饮食喂养大鼠建立胰岛素抵抗模型，同时用饱和脂肪酸孵育HepG2细胞建立胰岛素抵抗细胞模型，采用重组腺病毒介导的基因转染方法上调Mfn2表达，研究Mfn2对胰岛素抵抗大鼠肝脏糖、脂代谢的的影响，分别在动物和细胞水平探讨其改善胰岛素抵抗的具体分子机制。本实验内容主要包括以下四部分：
第一部分高脂诱导胰岛素抵抗发生及对肝脏Mfn2表达的影响
目的：建立高脂诱导的胰岛素抵抗动物模型，观察高脂对大鼠肝脏Mfn2表达的影响。
方法：雄性Sprague-Dawley（SD）大鼠84只，体重60-80g，适应性喂养1周后，随机分为正常对照组（normal diet, ND）12只和高脂组（high-fat diet, HFD）72只。ND组给予基础饲料，热量组成为脂肪10.3%，碳水化合物65.5%，蛋白质24.2%，总热量为348kcal/100g；HFD组给予高脂饲料，其热量组成为：脂肪59.8%，碳水化合物20.1%，蛋白质20.1%，总热量为501kcal/100g。所有动物自由摄食饮水，明暗周期为12h（6am-6pm），室温20℃-23℃。实验第8周末，两组分别随机选出6只进行清醒状态下高胰岛素-正葡萄糖钳夹（hyperinsulinaemic euglycaemic clamp）实验，计算葡萄糖输注率（glucose infusion rate，GIR），评价胰岛素敏感性。判断造模成功后，处死大鼠，留取血清及肝组织进行相关指标检测。血糖（blood glucose，BG）应用快速血糖仪测定。血浆胰岛素（insulin，INS）、游离脂肪酸（free fatty acid，FFA）检测采用ELISA试剂盒测定。血甘油三酯（triglyceride，TG）及总胆固醇（total cholesterin，TC）用全自动生化分析仪测定。肝组织Mfn2mRNA及蛋白水平分别采用Real time PCR及Western blot方法检测。
结果：
1两组大鼠一般指标的比较
各组大鼠体重随时间延长而增加，HFD组大鼠体重（357.0±32.5g）与ND组（355.7±39.0g）相比，差异无统计学意义（P>0.05）。HFD组FBG水平（5.72±0.43mmol/L）高于ND组（5.21±0.38mmol/L），差异有统计学意义（P<0.05）。HFD组INS水平（37.59±7.02mU/L）高于ND组（27.24±4.68mU/L），差异有统计学意义（P<0.05）。HFD组血TG水平（0.32±0.09mmol/L）高于ND组（0.24±0.05mmol/L）；HFD组血TC水平（1.42±0.17mmol/L）较ND组（1.16±0.15mmol/L）高，差异有统计学意义（P<0.05）。HFD组血FFA水平（0.80±0.12mol/L）较ND组（0.60±0.08mol/L）高，差异有统计学意义（P<0.01）。
2两组大鼠葡萄糖输注率的比较
HFD组GIR（13.66±2.54mg/(kg·min)）较ND组（28.14±5.00mg/(kg·min)）显著降低，差异有统计学意义（P<0.01）。
3两组大鼠肝脏Mfn2mRNA及蛋白水平的比较
HFD组大鼠肝脏Mfn2mRNA表达水平（0.23±0.04）较ND组（0.91±0.14）降低，差异有统计学意义（P<0.01）。HFD组Mfn2蛋白含量（0.28±0.05）较ND组（0.87±0.07）低，差异有统计学意义（P<0.01）。
4肝组织形态学的比较
光镜下观察：ND组肝组织结构完整，肝小叶规则，肝细胞排列整齐，在中央静脉周围呈放射状分布，肝细胞中央有大而圆的核，细胞质均匀，未见脂滴和炎症细胞侵润；HFD组大鼠肝小叶结构紊乱，肝细胞肿胀，肝细胞胞浆内可见大量脂滴空泡，以中央静脉周围最为明显，重者胞质疏松呈网状改变，且可见小叶内及汇管区炎症细胞侵润。
透射电镜观察：ND组肝细胞中细胞器结构完好，线粒体、粗面内质网丰富，胞浆内无脂滴及脱颗粒现象，线粒体膜、嵴结构完整，排列规则，粗面内质网排列整齐；HFD组肝细胞内可见大量大小不等的脂滴分布，线粒体有一定程度的肿胀，内外膜部分融合，线粒体嵴变少变短，部分或全部消失，甚至出现空泡化。
结论：
1高脂饮食诱导大鼠产生胰岛素抵抗，具有高血糖、高胰岛素血症、高脂血症等特点。
2高脂饮食导致大鼠肝细胞脂肪变性及线粒体超微结构的异常。
3高脂诱导的胰岛素抵抗大鼠肝脏Mfn2表达降低，Mfn2与胰岛素抵抗的发生相关。
第二部分Mfn2对胰岛素抵抗大鼠肝脏GK、PEPCK活性的影响
目的：观察腺病毒介导的Mfn2转染对胰岛素抵抗大鼠糖代谢相关酶葡萄糖激酶（glucoskinase, GK）及磷酸烯醇式丙酮酸羧激酶（phosphoen-olpyruvate carboxykinase, PEPCK）的活性影响。
方法：实验8周末判断胰岛素抵抗大鼠模型建立成功后，将高脂组剩余60只大鼠随机分为5组，分别为：高脂对照组（Con）12只、空载体对照组（emptyAd adenovirus, Ad）12只、Ad-Mfn2低剂量组（1×10~8v.p/kg）12只、Ad-Mfn2中剂量组（1×10~8v.p/kg）12只、Ad-Mfn2高剂量组（1×10~(10)v.p/kg）12只。以鼠尾静脉注射的方式，Con组给予磷酸盐缓冲液（PBS缓冲液），Ad组给予空载体（1×10~8v.p/kg体重），Ad-Mfn2低中高剂量组分别给予相应剂量Mfn2的重组腺病毒。每周1次，连续3周。给予腺病毒干预3周后，进行清醒状态下高胰岛素-正葡萄糖钳夹实验，计算GIR，处死动物并留取血清及肝组织进行相关指标检测。血糖、血胰岛素及游离脂肪酸测定方法同第一部分。肝脏糖原含量采用肝糖原试剂盒测定。肝GK及PEPCK活性采用酶偶联比色法测定。
结果:
1给予Mfn2重组腺病毒干预3周后，各组大鼠肝脏Mfn2mRNA及蛋白表达的比较
随着Ad-Mfn2剂量增加，大鼠肝脏Mfn2mRNA及蛋白表达水平升高，与Ad组相比差异有统计学意义（P<0.01）。
2给予Mfn2重组腺病毒干预3周后，各组一般指标的比较
随时间延长各组大鼠体重均增加，但差异无统计学意义（P>0.05）。大鼠FBG、INS随着Ad-Mfn2剂量增加而降低，而GIR升高，与Ad组相比差异有统计学意义（P<0.05）。
3各组肝脏糖原含量的比较
8周末：HFD组肝糖原含量（4.29±0.45mg/g）较ND组（5.31±0.63mg/g）低，差异有统计学意义（P<0.05）。
给予Mfn2重组腺病毒干预3周后：肝糖原含量随着Ad-Mfn2剂量增加而升高，Ad-Mfn2中、高剂量组肝糖原含量与Ad组相比差异有统计学意义（P<0.05）。
4各组大鼠肝脏GK、PEPCK活性的比较
8周末：HFD组肝脏GK活性（7.70±1.41mIU/(min·mg protein)）较ND组（13.87±1.72mIU/(min·mg protein)）低，差异有统计学意义（P<0.01）；而PEPCK活性（18.40±2.20mIU/(min·mg protein)）较ND组（10.67±2.50mIU/(min·mg protein)）高，差异有统计学意义（P<0.01）。
给予Mfn2重组腺病毒干预3周后：与Ad组相比，肝GK活性随着Ad-Mfn2剂量的增加而升高，PEPCK活性随着Ad-Mfn2剂量的增加降低，差异有统计学意义（P<0.05）。
5给予Mfn2重组腺病毒干预3周后，大鼠肝脏形态学及超微结构的改变
光镜下观察：Ad组可见肝小叶结构明显紊乱，肝细胞索消失，肝细胞变大，肝细胞胞浆内可见大量圆形脂滴空泡，核居边，且小叶内炎细胞侵润程度增加。Ad-Mfn2不同剂量组可见肝细胞体积较高脂组变小，胞浆内脂滴减少。
透射电镜观察：Ad组肝细胞胞浆中可见大量脂滴，肝细胞核不规整，线粒体膜模糊不清，部分膜破裂，线粒体嵴几乎消失不见，偶有少量残存，有空泡样变；Ad-Mfn2不同剂量组肝细胞胞浆中脂滴较高脂组数量少，线粒体肿胀减轻，内外膜部分融合，线粒体嵴部分或大部分消失。
结论：
1高脂饮食可导致大鼠血糖水平升高，肝糖原含量减少，糖酵解及氧化关键酶GK活性降低、糖异生关键酶PEPCK活性增加，引起糖代谢异常。
2上调Mfn2表达，胰岛素抵抗大鼠肝糖原含量升高，GK活性增加、PEPCK活性降低，并且使肝细胞脂肪变性和线粒体超微结构损伤减轻。Mfn2改善胰岛素抵抗可能与调节GK、PEPCK活性及影响线粒体形态结构有关。
第三部分Mfn2对胰岛素抵抗大鼠肝脏ACCα、CPT-Ⅰα表达的影响
目的:观察腺病毒介导的Mfn2转染对胰岛素抵抗大鼠脂代谢相关酶乙酰辅酶A羧化酶α（acetyl CoA carboxylase α, ACCα）、肉毒碱棕榈酰转移酶-Ⅰα（carnitine palmitoyltransferase-Ⅰ, CPT-Ⅰα）表达的影响。
方法：动物分组及处理同第一、二部分。肝脏TG含量按试剂盒说明测定，Real time PCR测定肝组织ACCα、CPT-Ⅰα mRNA水平，Western blot方法检测ACCα、CPT-Ⅰα蛋白水平。
结果：
1给予Mfn2重组腺病毒干预3周后，各组大鼠血液一般指标的比较
血TG、TC及FFA水平随着Ad-Mfn2剂量的增加而降低，Ad-Mfn2中、高剂量组TC、 FFA水平低于Ad组，差异有统计学意义（P<0.05）；Ad-Mfn2高剂量组TG水平低于Ad组，差异有统计学意义（P<0.05）。
2各组大鼠肝脏TG含量的比较
8周末：HFD组肝脏TG含量（595.35±159.24μmol/L）高于ND组（371.75±105.47μmol/L），差异有统计学意义（P<0.05）。
Mfn2重组腺病毒干预3周后：肝脏TG含量随着Ad-Mfn2剂量的增加而降低，与Ad组相比，差异有统计学意义（P<0.05）。
3各组大鼠肝脏CPT-Ⅰα和ACCα mRNA及蛋白水平的比较
8周末：HFD组肝脏CPT-Ⅰα mRNA表达及蛋白水平较ND组低，差异有统计学意义（P<0.05）。HFD组ACCα mRNA水平较ND组高，但蛋白磷酸化水平低于ND组，差异有统计学意义（P<0.05）。
Mfn2重组腺病毒干预3周后：CPT-Ⅰα mRNA及蛋白表达随Ad-Mfn2剂量的增加而升高，与Ad组相比，以中、高剂量组明显，差异有统计学意义（P<0.05）。与Ad组相比，ACCα mRNA水平随着Ad-Mfn2剂量的增加而降低，而蛋白磷酸化水平则升高，差异有统计学意义（P<0.05）。
结论：
1高脂饮食可导致肝脏脂质沉积，脂肪酸氧化关键酶CPT-Ⅰα表达降低，脂肪酸合成关键酶ACCα磷酸化水平降低。
2上调Mfn2表达，胰岛素抵抗大鼠血脂降低，肝脏脂质沉积减轻，脂代谢相关酶CPT-Ⅰα表达升高，ACCα磷酸化水平升高。Mfn2改善胰岛素抵抗可能与CPT-Ⅰα表达升高及ACCα活性降低，改善脂代谢异常有关。
第四部分Mfn2改善胰岛素抵抗的机制研究
目的：测定胰岛素抵抗大鼠肝脏及HepG2细胞胰岛素信号通路磷脂酰肌醇3激酶（phosphatidylinositol3-kinase，PI3K）/蛋白激酶B（proteinkinase B, PKB/AKT）途径相关分子的表达，探讨Mfn2改善胰岛素抵抗的具体作用机制。
方法：胰岛素抵抗大鼠模型及动物实验同第一、二部分。细胞培养：分别用含0.25mmol/L软脂酸（palmitate，PA）培养基和普通培养基（normalcontrol， NC）培养HepG2细胞24小时后采用葡糖糖氧化酶法测定细胞培养液的葡糖糖含量。判断胰岛素抵抗模型建立成功后，设立软脂酸对照组（Con）、软脂酸空载体组（Ad）、Ad-Mfn2低剂量组（50pfu/cell，Mfn2L）、Ad-Mfn2高剂量组（100pfu/cell, Mfn2H）。Con组给予PBS缓冲液，Ad组按50pfu/cell空载体给予，Ad-Mfn2低剂量组按50pfu/cell给予Ad-Mfn2，Ad-Mfn2高剂量组按100pfu/cell给予Ad-Mfn2。培养24h后，测定各组细胞培养基葡萄糖含量，并采用Real time PCR、Western blot方法分别检测各组Mfn2、INSR、IRS2、PI3K、AKT2、GLUT2mRNA及蛋白水平表达变化。
结果：
1HepG2细胞的胰岛素抵抗体外模型建立
PA组葡萄糖含量（12.16±0.54mmol/(L·mg protein)）较Con组（16.02±0.73mmol/(L·mg protein)）高，差异有统计学意义（P<0.01），表明用软脂酸诱导胰岛素抵抗体外细胞模型成功建立。
2HepG2胰岛素抵抗细胞Mfn2及胰岛素信号通路相关分子的表达
PA组Mfn2mRNA水平（0.48±0.11）较Con组（0.97±0.03）低，差异有统计学意义（P<0.01）。PA组Mfn2的蛋白表达量（0.46±0.06）较Con组（0.90±0.11）低，差异有统计学意义（P<0.01）。PA组INSR、IRS2、GLUT2mRNA及蛋白表达较Con组低，差异有统计学意义（P<0.05）。PA组PI3K、AKT2mRNA及总蛋白水平与Con组相比，差异无统计学意义（P>0.05）。但是PI3K和AKT2蛋白磷酸化水平较Con组显著下降，差异有统计学意义（P<0.05）。
3MTT法检测Ad、Ad-Mfn2对HepG2细胞增殖的影响，Ad组、Mfn2低剂量及高剂量组细胞增殖率无明显差别。
4Mfn2重组腺病毒干预后各组HepG2细胞胰岛素敏感性的比较Mfn2L组及Mfn2H组细胞培养液葡萄糖含量较Ad组降低（P<0.01）。
5Mfn2重组腺病毒干预后各组细胞Mfn2及胰岛素信号通路相关分子的表达
Mfn2L组及Mfn2H组HepG2细胞Mfn2表达较Ad组显著增加（P<0.01），INSR、IRS2、GLUT2mRNA水平及蛋白表达量较Ad组高，差异有统计学意义（P<0.05）。与Ad组相比，PI3K、AKT2mRNA表达水平及总蛋白表达量变化不显著（P>0.05），但其蛋白磷酸化水平明显升高，差异有统计学意义（P<0.05）。
6大鼠肝脏INSR、IRS2、PI3K、AKT2、GLUT2mRNA水平及蛋白表达的比较
实验8周末，HFD组大鼠肝脏INSR、IRS2、GLUT2mRNA表达水平较ND组低，差异有统计学意义（P<0.05）。PI3K、AKT2mRNA及总蛋白水平与ND组相比，差异无统计学意义（P>0.05）。但PI3K和AKT2蛋白磷酸化水平较ND组显著下降，差异有统计学意义（P<0.05），见Fig.5。
Mfn2重组腺病毒干预3周后，转染Ad-Mfn2的胰岛素抵抗大鼠肝脏INSR、IRS2、GLUT2mRNA及蛋白表达水平较Ad组升高，差异有统计学意义（P<0.05）。PI3K和AKT2蛋白磷酸化水平较Ad组升高，差异有统计学意义（P<0.05）。
结论：
1高浓度游离脂肪酸可诱导HepG2细胞产生胰岛素抵抗，伴随Mfn2表达降低，胰岛素信号通路相关的INSR、IRS2、GLUT2表达降低，PI3K、AKT2蛋白磷酸化水平下降。
2高脂诱导大鼠肝胰岛素信号通路相关分子INSR、IRS2、GLUT2表达下降，PI3K、AKT2蛋白磷酸化水平下降。
3上调Mfn2表达，胰岛素抵抗大鼠肝脏及HepG2细胞胰岛素信号通路相关分子INSR、IRS2、GLUT2表达升高及PI3K、AKT2蛋白磷酸化水平升高，说明Mfn2通过调节胰岛素信号传导通路相关分子表达改善胰岛素抵抗。
The incidence of type2diabetes（T2DM） has increased dramatically inrecent years. It is estimated that more than300million individuals worldwidewill be at high risk of developing T2DM by the year2025. Insulin resistanceplays a primary role in the pathogenesis of T2DM, and becomes a majorpublic health issue. Metabolic efficiency of glucose and lipid has decreasedduring insulin resistance. Mitochondria are core organelles that have a centralrole in the energy metabolism, and its role in metabolic diseases can not beignored. Studies have shown that mitochondrial dysfunction is closely relatedto the development of insulin resistance and type2diabetes, andmitochondrial damage may be one of the mechanisms of insulin resistance.Mitochondrial dysfunction characterized by reduced oxidative capacity andATP generation can lead to lipid deposition and induce insulin resistance.
The maintenance of mitochondrial morphology play a pivotal rolein energy control. Mitochondria is a dynamic organelle, maintaining abalance by continuous fusion and division. Mitofusin (Mfn) promotesmitochondrial fusion, and regulates the biosynthesis and maintenance of themitochondrial network structure. Mitofusin2(Mfn2) is a mitochondrial fusionprotein, mainly expressed in mammalian cells. Mitofusin2(Mfn2) is amitochondrial fusion protein, mainly expressed in mammalian cells. Researchin vitro and in vivo experimental models suggest that the physiological orpathological conditions can lead to changes in Mfn2expression, for example,diabetes, obesity and insulin resistance down-regulate expression of Mfn2,exercise and weight loss up-regulate expression of Mfn2. Data sugguest thatdown-regulation of Mfn2expression lead to low mitochondrial membranepotential and reduced oxygen consumption and glucose oxidation, while up-regulation of Mfn2expression lead to high mitochondrial membranepotential and enhanced glucose oxidation. In addition, previous studies haveshown that high-fat diet down-regulated Mfn2expression.
Increasing evidence indicates that expression of Mfn2is related to insulinresistance. However, the specific mechanism remains unclear. To enhance ourunderstanding to these questions, we established a high-fat diet inducedinsulin resistant rat model, at the same time HepG2cells were incubated withpalmitate to establish a insulin resistant HepG2cell model, infected insulinresistant rats with Mfn2expression adenovirus, the effect of Mfn2over-expression on the glucose and lipid metabolism in rats liver wasevaluated. Then, the specific molecular mechanism at the animal and cellularlevels through which Mfn2ameliorates insulin resistance was exploredrespectively.
The paper contains four parts as below:
Part Ⅰ: High-fat diet induces insulin resistance and its effect on theexpression of Mfn2
Objective: To establish an animal model of insulin resistance induced byhigh-fat diet and to determine the effect of high-fat diet on expression ofMfn2.
Methods: Male SD rats, weight about60-80g, were randomly dividedinto2groups: normal control (ND, n=12) and high fat diet group (HFD, n=72).Rats were fed with a regular low fatty acids diet contained10.3%fat,24.2%protein, and65.5%carbohydrate (kcal) or high-fat diet which consists of59.8%fat,20.1%protein, and20.1%carbohydrate (kcal). Rats in every grouphad free access to water and chow. The environment was controlled in termsof light (12:12-h light-dark cycle starting at6:00AM) and humidity. Insulinresistance was evaluated by glucose infusion rate (GIR) of hyperinsulinemiceuglycemic clamp technique at the end of8weeks（six rats in each group).The blood samples were obtained from the abdominal aorta. Fasting bloodglucose (FBG) levels were measured by Accu-chek Active Meter(ACCU-CHEK Active; Roche). Insulin (INS) and free fatty acids (FFA) levels were analyzed by using a Rat insulin or FFA ELISA kit (Crystal Chem.Inc). The liver tissue samples of rats were taken immediately and kept at-70℃after quick frozen in liquid nitrogen. The expression of Mfn2wasdetected by quantitative Real time PCR and Western blot. The morphologicalchanges in the ultrastructure of their hepatic cells were investigated by meansof electron microscopy assay.
Results:
1The body weight between two groups had no significant difference (P>0.05). Compared with ND group, GIR in HFD group was significantlydecreased (P<0.01); while the levels of BG, TG, TC, INS and FFA in HFDgroup were significantly higher than these in the ND group (P <0.05).
2Expression of Mfn2mRNA and protein were significantly decreased inHFD group (P<0.01), compared with ND group.
3Rats liver tissue morphology by histological HE staining: normal liver cellswere regularly aligned, liver lobular architecture was intact, and only a smallnumber of fibers were formed in blood vessel walls. There was diffusesteatosis, especially surrounding central veins, significant cell swelling,massive lipid droplet vacuole and inflammation cellular infiltration in lobulaof liver in HFD group. Electron microscopy assay revealed that the shape andsize of mitochondria in the hepatocytes of the control group were normal andhad few lipid droplets. In contrast, many mitochondria from rats in thehigh-fat diet group were enlarged and swollen, and showed morphologicalchanges, including large lipid droplet, and mitochondrial inner-outermenbrance meromixis. The cristae on the inner membrane of mitochondriawere disappeared or unclear.
Conclusions：
1High-fat diet-fed rats showed high concentrations of glucose,hyperlipidemia, hyperinsulinemia and GIR was lower. High-fat diet inducedinsulin resistance in rats.
2High-fat diet cause hepatic steatosis and ultrastructure damage inhepatocellular mitochondria.
3Expression of Mfn2in the liver tissues of High-fat diet-fed rats wasdown-regulated, implicating that the development of insulin resistance may berelated to low expression of Mfn2.
Part Ⅱ: Effect of Mfn2over-expression on the activity ofglucoskinase and phosphoenolpy ruvate carboxykinase in the liver ofinsulin resistant rats
Objective: To investigate the effects of Mfn2on the insulin sensitivityand the activity of Glucoskinase (GK) and Phosphoenolpyruvatecarboxykinase (PEPCK) in liver of rats fed with high-fat diets.
Methods: After8weeks, rats fed high-fat diets were randomly dividedinto5groups: high-fat diet control group infused with PBS buffer (Con, n=12),high fat diet group infected with empty control adenovirus (Ad, n=12),high-fat diet group were respectively infected with different amount ofAd-Mfn2(108,109or1010v.p/kg body weight) once a week, for3weeks. Afterintervention with adenovirus for3weeks, insulin resistance was evaluated byGIR of hyperinsulinemic euglycemic clamp technique. The blood sampleswere obtained from the abdominal aorta. The liver tissue samples of rats weretaken immediately and kept at-70℃after quick frozen in liquid nitrogen.FBG, TG, TC, INS and FFA were tested by methods as part one. The activityof GK and PEPCK were detected by enzyme-coupled colorimetric assay. Thelevel of hepatic glycogen was detected by using a glycogen assay kit. Theexpression levels of Mfn2were detected by Real time PCR and Western blot.
Results:
1The expression of Mfn2of rats infected with different amount of Ad-Mfn2(10~8,10~9or10~(10)v.p/kg body weight) for3weeks, were increased dramatically(P<0.01).
2After intervention with adenovirus for3weeks, the body weight had nosignificant difference among groups (P>0.05). FBG, INS of rats infected withdifferent amount of Ad-Mfn2(10~8,10~9or10~(10)v.p/kg body weight) for3weeks were decreased (P<0.05), while GIR was significantly increased(P<0.01).
3At the end of8weeks, concentration of hepatic glycogen in rats fed withhigh-fat diet was significantly decreased (P<0.05), compared with the NDgroup. After intervention with adenovirus for3weeks, concentrations ofhepatic glycogen in rats infected with Ad-Mfn2was increased with theincreasing of dose. Compared with the rats fed with high-fat diet, the glycogencontents in rats infected with Ad-Mfn2(109or1010v.p/kg body weight) wereincreased (P<0.05).
4The activity of GK was increased (P<0.01)and PEPCK decreased in theliver of rats fed high-fat diet for8weeks (P<0.01). After intervention withadenovirus for3weeks, with increasing dose of Ad-Mfn2, the activty of GKwas increased (P<0.01), while the activity of PEPCK was decreased (P<0.01).Compared with rats high-fat diet-fed,the activity of GK and PEPCK of liver inrats infected with Ad-Mfn2(109or1010v.p/kg body weight),there weresignificant difference (P<0.05or P<0.01).
5All of the sections in the Con and Ad group exhibited diffuse hepaticsteatosis under a light microscope. Hepatic steatosis was most obvious aroundthe portal area (mostly microvesicular and macrovesicular mixed steatosis)and was accompanied by inflammatory cell infiltration. The liver HE stainingof rat infected with Ad-Mfn2shows less cell volume and fat dropletaccumulation. Electron microscope revealed that mitochondria from rats in theCon and Ad group were enlarged and swollen, and showed morphologicalchanges, including larger lipid droplet, and mitochondrial membrane isunclear, even had vacuolar degeneration. The cristae on the inner membraneof mitochondria were almost disappeared.
Conclusions：
1High-fat diets can lead to abnormal glucose metabolism, accompanied bylower concentration of hepatic glycogen，lower activity of GK and higher ofPEPCK.
2Over-expression of Mfn2improves the glucose metabolism.The activity ofGK was increased， the activity of PEPCK was decreased， and theconcentration of hepatic glycogen was higher. It also alleviates hepatic steatosis and ultrastructure damage in hepatocellular mitochondria.Over-expression of Mfn2ameliorated high-fat diet induced insulin resistance，and maybe related to the increasing activity of GK，decreasing activity ofPEPCK，and alleviating the demage of the liver cell and mitochondria.
Part Ⅲ: Effect of Mfn2on expression of ACCα and CPT-Ⅰα in liverof insulin resistant rats
Objective: To investigate the effects of Mfn2on the expression of AcetylCoA carboxylase α (ACCα) and Carnitine palmitoyltransferase-Ⅰα (CPT-Ⅰα)in liver of rats fed with high-fat diets.
Methods: Animal grouping and treatment was the same as the partⅠand partⅡ. Hepatic triglyceride (TG) content was detected with a triglyceridedetermination kit. The expression levels of ACCα and CPT-Ⅰα were detectedby Real time PCR and Western blot.
Results:
1After intervention with adenovirus for3weeks, the plasma TG, TC and FFAlevels in liver of rats infected with Ad-Mfn2(108,109or1010v.p/kg bodyweight) for3weeks were decreased (P<0.05), compared with Ad group.
2At the end of8weeks, hepatic TG content of rats fed with high-fat diet wassignificantly increased (P<0.05), compared with the ND group. Afterintervention with adenovirus for3weeks, hepatic TG contents of rats infectedwith Ad-Mfn2was increased with the increasing dose of Ad-Mfn2, Comparedwith Ad, hepatic TG contents in rats infected with Ad-Mfn2(109or1010v.p/kg body weight) were decreased (P<0.05or P<0.01).
3The mRNA expression of ACCα was increased and CPT-Ⅰα was decreased(P<0.05). Phosphorylation of ACCα protein was decreased (P<0.05), butCPT-Ⅰα protein was decreased at the end of8weeks. After intervention withadenovirus for3weeks, with increasing dose of Ad-Mfn2, the mRNAexpression of ACCα was decreased (P<0.01) and CPT-Ⅰα was increased(P<0.05). Phosphorylation of ACCα protein was increased (P<0.01), butCPT-Ⅰ α protein expression was increased, compared with Ad group(P<0.05).
Conclusions：
1High-fat diet can lead to abnormal lipid metabolism, accompanied by highhyperlipidemia, high FFA levels, and Lipid deposition. The activity of ACCαwas increased and expression of CPT-Ⅰα was down-regulated in rats fed withhigh-fat diet.
2Over-expression of Mfn2improved the lipid metabolism. Mfn2amelioratedinsulin resistance, probably by decreasing the activity of ACCα and heptic TGcontents, while enhancing expression of CPT-Ⅰα..
Part Ⅳ: Mechanism of over-expression Mfn2ameliorating insulinresistance
Objective: To explore the mechanism of Mfn2over-expressionameliorating insulin resistance by testing the expression of relative moleculesof the insulin signaling pathway.
Methods: The model of IR was established with HepG2cells cultured athigh concentrations of palmitate (PA,0.25mmol/L) for24h. The glucosecontent was measured by glucose assay kit. Insulin resistant HepG2cells weredivided into4groups: HepG2cells cultured with PA (Con) group, HepG2cells cultured with PA infected with empty control adenovirus (Ad) group，HepG2cells cultured with PA infected with Ad-Mfn2(50or100pfu/cell), andcultured for24h. The expression of INSR, IRS2, PI3K, AKT2and GLUT2were detected by Real time PCR and Western blot.
Results:
1The glucose contents of PA group were significantly higher than those incontrol (P<0.01).
2The expression of Mfn2of insulin resistant HepG2cells was decreasedsignificantly (P<0.01). The expression of INSR, IRS2and GLUT2weredown-regulated in HepG2cells cultured with PA (P<0.01). There were nochanges in PI3K and AKT2expression, but their phosphorylation levelsdecreased significantly (P<0.01), compared with Ad group.
3After infected with Ad-Mfn2for24h, the glucose contents of HepG2cellscultured with PA infected with Ad-Mfn250and100pfu/cell were lower thanAd group (P<0.01)。
4The expression of Mfn2in HepG2cells cultured with PA infected withAd-Mfn250or100pfu/cell were increased, compared with Ad group.Over-expression of Mfn2were confirmed (P<0.01). Over-expression of Mfn2up-regulated the expression of INSR, IRS2and GLUT2, and phosphorylationlevels of PI3K and AKT2were increased (P<0.01).
5At the end of8weeks, the expression of INSR, IRS2and GLUT2weredown-regulated markedly by high-fat diets at8weeks (P<0.01). There wereno changes in PI3K and AKT2expression, their phosphorylation levelsdecreased significantly (P<0.01), compared with Ad group.
6After intervention with adenovirus for3weeks, Over-expression of Mfn2up-regulated the expression of INSR, IRS2and GLUT2, phosphorylationlevels of PI3K-P85and AKT2increased (P<0.01).
Conclusions：
1High-fat diets inhibited the expression of insulin signaling pathway. Theexpressions of INSR, IRS2and GLUT2were down-regulated by high-fat dietand protein phosphorylation levels of PI3K and AKT2were decreased.
2High concentrations of palmitate could induce HepG2cells hepatic insulinresistance, and down-regulate the expression of INSR, IRS2and GLUT2andprotein phosphorylation levels of PI3K and AKT2.
3Over-expression of Mfn2improved insulin resistance of rats or HepG2cells,and up-regulated the expression of INSR, IRS2and GLUT2. Proteinphosphorylation levels of PI3K and AKT2were decreased. Our data suggestthat Mfn2might ameliorate insulin resistance by enhancing the expression ofinsulin signaling pathway that are import in glucose and lipid metablism.
引文
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