【作者】 张瑞宁；

【导师】 李拥军；

【作者基本信息】 河北医科大学 ， 内科学， 2014， 博士

【摘要】 糖尿病（diabetes mellitus, DM）是一组以高血糖引起胰岛素抵抗为主要标志的代谢紊乱综合征，可导致多种并发症，比如糖尿病肾病,糖尿病视网膜病变,糖尿病心肌病。脂肪组织是一种内分泌器官，可分泌多种脂肪因子，如脂联素、瘦素、内脂素、IL-6等。大量的实验证据表明，脂肪因子影响多种组织的葡萄糖代谢。Chemerin又被称为维甲酸受体反应元件2（retinoicacid receptor responder protein2, RARRES2）或者他扎罗汀诱导基因2（Tazarotene-induced gene2, TIG2），是最近发现的一种脂肪因子,与炎症,脂肪代谢和胰岛素抵抗等具有相关性。已有研究证明，Chemerin及其受体—趋化因子样受体1（chemokine-like receptor1,CMKLR1或ChemR23）在许多组织中表达，特别是在白色脂肪组织、肝脏和肾脏中表达较。目前的研究显示，人类血浆chemerin的高表达是炎症和代谢综合症的标志物之一。因此，Chemerin可能参与了胰岛素抵抗和糖尿病的发生发展。然而，在不同的细胞中，chemerin在胰岛素抵抗中发挥的作用尚存争议。最近的一项研究发现人类骨骼肌细胞虽然不表达chemerin，但是表达chemerin的受体CMKL1。Chemerin通过破坏胰岛素信号，可诱导骨骼肌细胞产生胰岛素抵抗。但是，在体外3T3-L1细胞的研究中尚存争议。Takahashi等发现chemerin能够促进胰岛素刺激下的3T3-L1细胞对葡萄糖的摄取，而Kralisch S等发现chemerin抑制胰岛素刺激下3T3-L1细胞对葡萄糖的摄取率的增加。这种相反结果的产生可能与chemerin同时参与胰岛素作用的靶组织对胰岛素敏感性的负性调节有关。国外已有研究证实大鼠心脏组织表达chemerin及其受体CMKLR1，但是尚无体外实验报道心肌细胞表达chemerin以及chemerin在心肌细胞胰岛素抵抗中的作用。糖尿病心肌病是糖尿病的一个重要并发症，因此我们从细胞水平研究在心肌细胞中chemerin/ChemR23系统与胰岛素抵抗的关系。本实验体外培养SD乳鼠原代心肌细胞和心脏成纤维细胞，检测心肌细胞和心脏成纤维细胞是否表达chemerin，并探讨心肌细胞chemerin的表达与胰岛素抵抗的关系及其分子学机制。本研究主要包括以下四部分：第一部分Chemerin在乳鼠心肌细胞和心脏成纤维细胞中的表达目的：购买出生2~3天的SD乳鼠，成功提取并培养SD乳鼠原代心肌细胞和心脏成纤维细胞，观察其是否表达chemerin，为下一步研究奠定基础。方法：购买出生2~3天的SD乳鼠，提取并培养原代心肌细胞和心脏成纤维细胞，应用Real-Time-PCR、Western-Blot技术观察心肌细胞和心脏成纤维细胞是否表达chemerin。结果：Real-Time-PCR结果示乳鼠心肌细胞和心脏成纤维细胞均有chemerin mRNA的表达。Western-Blot结果示乳鼠心肌细胞和心脏成纤维细胞均有chemerin蛋白的表达结论：SD乳鼠心肌细胞和心脏成纤维细胞正常情况下均能够表达chemerin。第二部分高糖和炎症环境下乳鼠心肌细胞chemerin表达的变化目的：观察高糖和炎症因子TNF-α对乳鼠心肌细胞chemerin mRNA表达的影响方法：购买出生2~3天的SD乳鼠，提取并培养原代心肌细胞，培养48小时后换为无血清的低糖DMEM培养基（葡萄糖浓度为5.5mmol/L）继续培养24小时，然后分组后继续培养：1观察葡萄糖干预心肌细胞后chemerin mRNA表达的变化：（1）不同浓度的葡萄糖干预24小时：对照组(5.5mmol/L)组、10mmol/L组、20mmol/L组、30mmol/L、40mmol/L组和高渗组(5.5mmol/L葡萄糖+34.5mmol/L甘露醇）。应用Real-Time PCR技术检测各组心肌细胞chemerin mRNA的表达。（2）30mmol/L的葡萄糖干预不同时间：0小时、6小时、12小时、24小时和48小时。应用Real-Time PCR技术检测各组心肌细胞chemerin mRNA的表达。2观察TNF-α干预心肌细胞后chemerin mRNA表达的变化：（1）不同浓度的TNF-α干预24小时：空白对照组、5ng/ml组、10ng/ml组和20ng/ml组。应用Real-Time PCR技术检测各组心肌细胞chemerin mRNA的表达。（2）20ng/ml的TNF-α干预不同时间：0小时、6小时、12小时、24小时和48小时。应用Real-Time PCR技术检测各组心肌细胞chemerin mRNA的表达。结果：1高糖可诱导乳鼠心肌细胞chemerin mRNA的表达增加。（1）随着葡萄糖干预浓度的增加，心肌细胞chemerin mRNA的表达随之增加，在葡萄糖浓度30mmol/L组达到峰值，差异与对照组比较有统计学意义（P<0.05）；40mmol/L组较30mmol/L组表达降低，但差异无统计学差异（P>0.05）；与对照组相比，高渗组chemerin mRNA的表达并没有明显的变化，差异无统计学意义（P>0.05）。（2）随着葡萄糖干预时间的延长，心肌细胞chemerin mRNA的表达水平随之升高，在24小时达到峰值，差异有统计学意义（P<0.05）；48小时组较24小时表达降低，差异有统计学意义（P<0.05）。2TNF-α也可诱导乳鼠心肌细胞chemerin mRNA的表达增加。（1）随着TNF-α干预浓度的增加，心肌细胞chemerin mRNA的表达随之增加，在TNF-α浓度20ng/ml组达到最高值。5ng/ml组与对照组比较差异无统计学意义（P>0.05），10ng/ml组、20ng/ml组与对照组比较差异有统计学意义（P<0.05)。（2）随着TNF-α干预时间的延长，心肌细胞chemerin mRNA的表达水平随之升高，在24小时达到峰值，差异有统计学意义（P<0.05）；48小时组较24小时组表达略降低，差异有统计学意义（P<0.05）。结论：高糖环境中和炎症状态下乳鼠心肌细胞chemerin mRNA的表达均上调。第三部分Chemerin诱导心肌细胞产生胰岛素抵抗目的：探讨chemerin是否破坏胰岛素信号，引起心肌细胞的胰岛素抵抗。方法：购买出生2~3天的SD乳鼠，提取并培养原代心肌细胞，培养48h后换为无血清的培养基继续培养24h，然后进行分组。首先以不同浓度的chemerin（对照组、10ng/ml组、100ng/ml组）干预24小时，然后以10-7mol/l胰岛素刺激30分钟。应用Western-Blot技术检测心肌细胞Akt、IRS-1和AMPKα的磷酸化水平，应用荧光酶标仪测定心肌细胞的葡萄糖摄取率（glucose uptake）。结果：无胰岛素刺激组和胰岛素刺激组，随着chemerin浓度的升高，Akt的磷酸化水平均随之降低，差异具有统计学意义（P<0.05）；无胰岛素刺激组和胰岛素刺激组，随着chemerin浓度的升高，IRS-1的磷酸化水平均随之升高，差异具有统计学意义（P<0.05）。无胰岛素刺激组和胰岛素刺激组，随着chemerin浓度的升高，葡萄糖摄取率与AMPKα(Thr172)的磷酸化水平也随之降低，差异具有统计学意义（P<0.05）。结论：Chemerin诱导心肌细胞产生胰岛素抵抗。第四部分Chemerin诱导心肌胰岛素抵抗的分子机制目的：探讨chemerin诱导心肌细胞产生胰岛素抵抗的分子机制方法：购买出生2~3天的SD乳鼠，提取并培养原代心肌细胞，培养48h后换为无血清的培养基继续培养24h，然后进行分组。1探讨chemerin可激活哪些信号通路：首先以0ng/ml或者100ng/mlchemerin干预心肌细胞24小时，然后以10-7mol/l胰岛素刺激30分钟。应用Western-Blot技术检测心肌细胞p38MAPK、ERK-1/2和JNK的总蛋白水平和磷酸化水平。2ERK1/2信号通路在chemerin诱导心肌细胞产生胰岛素抵抗中的作用：应用ERK阻滞剂PD98059（50umol/l）预干预15分钟，之后Chemerin（100ng/ml）干预24小时，最后应用胰岛素（10-7mol/l）刺激30分钟，具体分组如下：①空白对照组；②胰岛素组；③Chemerin组；④Chemerin+胰岛素组；⑤PD98059+chemerin组；⑥PD98059+chemerin+胰岛素组。应用Western-Blot技术检测心肌细胞Akt和AMPKα的磷酸化水平；应用荧光酶标仪测定心肌细胞的葡萄糖摄取率。结果：1胰岛素刺激后，chemerin可激活ERK1/2与p38MAPK信号通路：无胰岛素刺激组，chemerin干预后， p38MAPK的磷酸化水平较对照组无明显升高，差异无统计学意义(P>0.05)；但胰岛素刺激组，p38MAPK磷酸化水平较对照组升高，差异有统计学意义(P<0.05)。无胰岛素刺激组与胰岛素刺激组，chemerin干预后，ERK-1/2的磷酸化水平均较对照组升高，差异有统计学意义(P<0.05)。无胰岛素刺激组与胰岛素刺激组，chemerin干预后，JNK的磷酸化水平均较对照组无明显变化，差异无统计学意义(P>0.05)。2ERK1/2阻滞剂可部分逆转chemerin诱导的心肌细胞胰岛素抵抗：胰岛素刺激组，chemerin和PD98059干预后，Akt的磷酸化水平较单纯chemerin干预组升高，但仍较无chemerin干预的对照组降低，差异具有统计学意义(P<0.05)；AMPKα的磷酸化水平和葡萄糖摄取率也有类似的变化，差异具有统计学意义(P<0.05)结论：胰岛素刺激后，chemerin可激活ERK1/2和p38MAPK信号转导通路；chemerin可部分通过激活ERK1/2信号传导通路诱导心肌细胞产生胰岛素抵抗。更多还原

【Abstract】 Diabetes mellitus (DM) is a metabolic disorder characterized by elevatedblood glucose secondary to insulin resistance that results in many seriouscomplications，such as diabetic nephropathy, diabetic retinopathy and diabeticcardiomyopathy. A large body of experimental evidence supports the notionthat adipokines have a significant influence on glucose metabolism in varioustissues. As an endocrine organ，adipose tissue could secrete a variety of fatfactors, such as adiponectin, leptin, visfatin, IL-6, and so on. Chemerin (alsoknown as retinoicacid receptor responder protein2and tazarotene-inducedgene2) is a recently discovered adipokine that is associated with inflammation,adipogenesis, and insulin resisitance. Studies have previously shown thatchemerin and its receptor, chemokine-like receptor1(CMKLR1, or ChemR23)are expressed in many tissues and particularly highly expressed in whiteadipose tissue, liver and kidney. A high level of circulating chemerin inhumans is considered to be a marker of inflammation and metabolic syndrome.These observations suggest that chemerin may be involved in insulinresistance and the development of DM.Although the evidence described above demonstrates the influence ofchemerin on glucose homeostasis, at present the precise role and significanceof chemerin is unclear in different cell types. A previous study showed thathuman skeletal muscle cells do not express chemerin but do express CMKLR1,and chemerin impairs insulin signaling and induces insulin resistance inskeletal muscle cells. However, there were conflicting results in3T3-L1adipocytes. One study showed that chemerin induces insulin resistance,whereas another study showed that high levels of chemerin enhance insulinsignaling and glucose uptake in3T3-L1adipocytes.Chemerin and CMKLR1have been shown to be expressed in rat heart tissue as well. However, no studies to date have described the presence ofchemerin in rat cardiomyocytes in vitro or the role of chemerin in insulinresistance. Diabetic cardiomyopathy is one of the serious cardiovascularcomplications of long-term DM. Therefore, we investigated the possibleinterplay between the chemerin/ChemR23system and insulin resistance in ratcardiomyocytes in vitro. The objective of this study was to clarify the role andmolecular biological mechanisms of chemerin on insulin resistance in ratcardiomyocytes. It follows by four parts:Part1The expression of chemerin in rat cardiomyocytes and cardiacfibroblastsObjectives: To explore whether cardiomyocytes and cardiac fibroblastscan express chemerin, lay the foundation for the further study.Methods: Primary cardiomyocytes and cardiac fibroblasts were isolatedfrom the ventricles of three-day-old neonatal Sprague-Dawley rats. Chemerinexpression in cardiomyocytes and cardiac fibroblasts were measured byreal-time PCR and Western-Blot.Results: The expression of chemerin mRNA were detected incardiomyocytes and cardiac fibroblasts. The expression of chemerin proteinwere detected in cardiomyocytes and cardiac fibroblasts.Conclusions: Rat cardiomyocytes and cardiac fibroblasts can expresschemerin.Part2Changes of chemerin expression in rat cardiomyocytes in highglucose and inflammatory environmentObjectives: To observe the effects of high glucose and inflammatoryfactor TNF-α on chemerin mRNA expression in rat cardiomyocytes.Methods: Ventricular cardiomyocytes were dissociated from the2to3-day old neonatal SD rats.48hours after seeding, the cardiomyocytes werecultured with the serum-free culture medium for another24hours.1To test the variation of chemerin mRNA expression after glucosestimulation in cardiomyocytes:(1)The cells were treated with increasingconcentrations of D-glucose (5.5,10,20,30, and40mmol/L) and hypertonic control (5.5mmol/L D-glucose+34.5mmol/L mannitol) for24hours. Theexpression of chemerin mRNA were measured by real-time PCR.(2)Thecardiomyocytes were then treated with high glucose (30mmol/L) for differentdurations (0,6,12,24, and48hours). The expression of chemerin mRNAwere measured by real-time PCR.2To test the variation of chemerin mRNA expression afteradministration of TNF-α:(1)The cardiomyocytes were treated with TNF-α (0,5,10,20ng/ml) for24hours. The expression of chemerin mRNA weremeasured by real-time PCR.(2)The cardiomyocytes were then treated withTNF-α (20ng/ml) for different durations (0,6,12,24, and48hours). Theexpression of chemerin mRNA were measured by real-time PCR.Results:1Chemerin mRNA expression was upregulated by administration ofhigh glucose.(1)The expression of chemerin mRNA increased in adose-dependent manner up to30mmol/L glucose (P<0.05), and chemerinmRNA expression was slightly, but not significantly, lower with40mmol/Lglucose (P>0.05). Interestingly, compared with that the control (treated with5.5mmol/L D-glucose), chemerin expression was not significantly increasedin the hypertonic control group (P>0.05).(2)Chemerin mRNA levelsincreased in a time-dependent manner peaking at24hours of treatment andthen significantly decreasing by48hours (P<0.05).2Chemerin mRNA expression was upregulated by administration ofTNF-α.(1)The expression of chemerin mRNA increased in a dose-dependentmanner compared with the control, and the increases were significantly in thegroups of10g/ml and20ng/ml (P<0.05).(2)Chemerin mRNA levels increasedin a time-dependent manner peaking at24hours of treatment and thendecreasing by48hours (P<0.05).Conclusions: Chemerin mRNA expression was upregulated by highglucose and inflammatory environment in rat cardiomyocytes.Part3Chemerin induced insulin resistance in cardiomyocytesObjectives: To study whether chemerin can impaire insulin signaling and induce insulin resistance in cardiomyocytesMethods: Ventricular cardiomyocytes were dissociated from the2to3-day old neonatal SD rats.48hours after seeding, the cardiomyocytes werecultured with the serum-free culture medium for another24hours. Afterthat,cardiomyocytes were cultured with recombinant rat chemerin (0,10, and100ng/ml) for24hours, with chemerin still in the media, cardiomyocytes wereexposed insulin (10-7mol/L) for30min. The phosphorylation of Akt, IRS-1and AMPKα were measured by Western blot analysis. Glucose uptake wasevaluated using a fluorescence microplate reader.Results: The cardiomyocytes showed a marked and dose-dependentdecrease both in basal and insulin-stimulated phosphorylation of Akt uponadministration of chemerin (P<0.05). Upstream of Akt, chemerin significantlyand dose-dependently increased the basal and insulin-stimulated serinephosphorylation of IRS-1(P<0.05).Glucose uptake and phosphorylation of AMPKα (Thr172) weresignificantly decreased compared with basal levels and that of theinsulin-stimulated control (P<0.05).Conclusions: Chemerin induced insulin resistance in cardiomyocytes.Part4The possible molecular mechanism of chemerin on insulinresistance in cardiomyocytes.Objectives: Investigating the possible molecular mechanism of chemerinon insulin resistance in cardiomyocytes.Methods: Ventricular cardiomyocytes were dissociated from the2to3-day old neonatal SD rats.48hours after seeding, the cardiomyocytes werecultured with the serum-free culture medium for another24hours.1To investigate which intracellular signaling pathways are important inchemerin-mediated insulin resistance, cardiomyocytes were pretreated with orwithout chemerin (100ng/ml) for24hours before acute stimulation withinsulin (10-7mol/l,30min). The phosphorylation of p38MAPK, ERK-1/2andJNK were measured by Western blot analysis.2To analyze the role of the ERK1/2pathway in the impairment of insulin signaling by chemerin, cardiomyocytes were pre-cultured with thespecific ERK inhibitor PD98059(50umol/l) for15min before startingadministration of chemerin (100ng/ml) for24hours. The groups were asfollows:①Blank control group;②10-7mol/l insulin stimulation for30min;③100ng/ml chemerin stimulation for24hours;④100ng/ml chemerinstimulation for24hours, then10-7mol/l insulin stimulation for30min;⑤PD98059(50umol/l) pre-stimulation for15min,100ng/ml chemerinstimulation for24hours;⑥PD98059(50umol/l) pre-stimulation for15min,100ng/ml chemerin stimulation for24hours, then10-7mol/l insulin stimulationfor30min. The phosphorylation of Akt and AMPKα were measured byWestern blot analysis. Glucose uptake was evaluated using a fluorescencemicroplate reader.Results:1Chemerin activated p38MAPK and ERK1/2signaling pathways ininsulin-stimulated cardiomyocytes:p38MAPK phosphorylation was not significantly increased in the basalstate (P>0.05) but still significantly increased in the insulin-stimulated state(P<0.05). However, chemerin increased both the basal and insulin-stimulatedphosphorylation of ERK1/2(P<0.05). Interestingly, chemerin had no effect onJNK activity (P>0.05).2ERK1/2inhibition partially restored insulin sensitivity inchemerin-treated cardiomyocytes:The phosphorylation of Akt was significantly increased compared with thosein the insulin-stimulated control with chemerin, but still significantlydecreased compared with those in the insulin-stimulated control withoutchemerin (P<0.05). Meanwhile, the phosphorylation of AMPKα and glucoseuptake had the similar changes.Conclusions: Chemerin activated p38MAPK and ERK1/2signalingpathways in insulin-stimulated cardiomyocytes, and chemerin induced insulinresistance part of by activating ERK1/2signaling pathway in cardiomyocytes.更多还原