脂肪组织中肿瘤坏死因子-α与血浆纤溶酶原激活抑制物-1相关性的研究
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摘要
前言
肥胖与2型糖尿病、心血管系统疾病的发生发展密切相关,在全世界的患病率逐年上升。对于其密切关系的分子机制目前还不十分清楚,但研究发现脂肪组织作为内分泌器官,分泌多种脂肪细胞因子,如肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)、C-反应蛋白(CRP)、抵抗素(resistin)、脂联素(adiponectin)等,在肥胖相关的心血管系统疾病的发生发展中发挥了极其重要的作用。
越来越多的证据表明脂肪细胞因子通过促炎症反应的作用直接影响到内皮细胞功能。虽然大部分的数据都是基于体外的研究得来的,但我们可以用脂肪细胞因子来解释肥胖、胰岛素抵抗和内皮细胞功能失调密切相关的原因。脂肪组织的分泌产物增加可促使心血管系统疾病发生的危险性增加,但这并不依赖于脂肪细胞因子导致的胰岛素抵抗和糖尿病所产生的影响。脂肪细胞因子对血管稳态的作用各不相同,近来对于TNF-α在肥胖相关心血管系统疾病的发展中的作用较为关注。
然而动物试验的研究表明TNF-a在动脉粥样硬化等心血管系统疾病的发生发展中的作用结果是不一致的。一方面敲除apoE的小鼠,TNF-α水平下降,动脉粥样硬化斑块减少,另一方面TNF-α受体P55缺失型小鼠却加速了动脉粥样硬化的形成;一方面TNF-α被认为在充血性心衰的发展过程中起到了重要作用,另一方面研究却发现在充血性心衰患者中抗TNF-α的治疗并无益处。正因为这些研究结果互相冲突,所以TNF-α在心血管系统疾病中的作用更加受到关注,抗TNF-α治疗以取得心血管保护效益的研究有待于进一步研究。
纤溶酶原激活抑制物-1(PAI-1)是一种丝氨酸蛋白酶抑制剂,是纤维蛋白溶解系统主要的调节因子。PAI-1是重要的纤溶酶原活化的抑制剂,它的增加可以打乱正常的纤维蛋白清除机制而促进血栓的形成。PAI-1的水平在肥胖患者中显著升高,是心血管系统疾病发生的独立危险因素。在人体中,给予注射TNF-α可以增加PAI-1在血浆中的浓度;体外研究中,TNF-α可以增加PAI-1在内皮细胞以及脂肪细胞中的表达。因而TNF-α可通过升高PAI-1的表达而促进了血管病变的发生。
最初发现早期生长反应基因-1(Egr-1)是生长因子、细胞因子、缺血、物理力量、损伤等各种刺激所引起的快速反应的早期基因,所有的刺激均与血管疾病的进展相关。McCaffrey的研究表明Egr-1在血管损伤后所导致的动脉粥样硬化处表达升高,用Egr-1的反义技术可以通过抑制平滑肌细胞移动和增殖,减少动脉粥样硬化处Egr-1的表达。在巨噬细胞中的研究发现,TNF-α可通过ERK1/2路径引起Egr-1表达的升高。因此本研究将检测在TNF-α作用的内皮细胞中Egr-1的表达,进一步探讨TNF-α所致心血管系统疾病发生的机制。
本研究旨在探讨TNF-α对心血管系统疾病的作用及其机制。本研究检测了受试者网膜脂肪组织和皮下脂肪组织中TNF-α表达、空腹血糖、胰岛素、血脂及血浆中PAI-1的水平等临床参数,探讨TNF-α与胰岛素抵抗、血脂异常症和心血管系统疾病之间的关系。并进行了体外试验,观察TNF-α对内皮细胞中Egr-1蛋白表达的影响,探讨TNF-α引起心血管系统疾病的机制。
材料与方法
1、选取进行腹部外科手术的肥胖患者和正常体重的对照组患者,进行常规指标的测定,包括体重、身高、腰围。
2、采用酶学方法测定血糖、血胆固醇、血高密度脂蛋白胆固醇水平;采用化学发光免疫分析法测定胰岛素、C肽水平。计算胰岛素抵抗指数(HOMA-IR):HOMA-IR=FPG×FINS/22.5。
3、采用免疫组化方法和western blot方法检测受试者腹部皮下脂肪和网膜脂肪中TNF-α的表达。
4、ELISA方法检测受试者血清中PAI-1的水平。
5、采用人脐静脉内皮细胞株培养的方法,TNF-α和/或葡萄糖与内皮细胞共同孵育,收集内皮细胞及细胞培养液。加入MEK抑制剂(PD98059)进行干预,收集内皮细胞及细胞培养液。
6、采用western blot方法检测内皮细胞中Egr-1、ERK1/2蛋白的表达。
7、采用ELISA方法测定各组细胞培养液中PAI-1水平。
结果
1、男性和女性受试者测量和代谢指标的数据表明无论在男性还是女性组,肥胖者BMI、腰围、空腹胰岛素水平、HOMA-IR、甘油三酯、胆固醇和血浆PAI-1水平均高于正常对照组,而HDL-C水平低于正常对照组。
2、肥胖组网膜脂肪和皮下脂肪标本中TNF-α蛋白水平均高于正常对照组。
3、在女性肥胖受试者中两处脂肪细胞的大小与其对应的TNF-α的表达呈正相关(γ=0.779,P＜0.01,网膜脂肪处;γ=0.452,P＜0.05,皮下脂肪处)。在男性肥胖受试者中,仅在网膜脂肪处两者成正相关(γ=0.828,P＜0.01)。
4、在女性肥胖受试者中,网膜脂肪细胞TNF-α的表达与空腹血糖浓度呈正相关(γ=0.541,P＜0.05),与空腹胰岛素呈正相关(γ=0.599,P＜0.01),与胰岛素抵抗指数呈正相关(γ=0.546,P＜0.05),与甘油三酯呈显著正相关(γ=0.469,P＜0.05),与高密度脂蛋白胆固醇呈负相关(γ=-0.759,P＜0.01)。这些参数与皮下脂肪TNF-α表达不相关。在男性肥胖组中,两处脂肪TNF-α的表达与HOMA-IR均无明显相关,但网膜脂肪TNF-α表达与葡萄糖水平正相关(γ=0.762,P＜0.01),与胰岛素水平正相关(γ=0.622,P＜0.05),与甘油三酯水平正相关(γ=0.650,P＜0.05),与高密度脂蛋白胆固醇水平负相关(γ=-0.880,P＜0.01)。男性肥胖受试者中皮下脂肪TNF-α的表达与葡萄糖、胰岛素、甘油三酯和高密度脂蛋白胆固醇水平无明显相关性。
5、在女性和男性肥胖受试者中,可见网膜脂肪TNF-α蛋白的表达水平与血浆PAI-1水平呈显著正相关(女性:n=20,γ=0.763,P＜0.01;男性:n=12,γ=0.760,P＜0.01)。
6、25mmol/1葡萄糖、10ng/ml TNF-α使Egr-1水平的变化如下:TNF-α使Egr-1升高后即回落,高峰在60分钟,葡萄糖使Egr-1表达的高峰亦在60分钟,但Egr-1的表达持续升高直至240分钟。
7、葡萄糖与TNF-α共同孵育的内皮细胞Egr-1表达较葡萄糖和TNF-α单独作用升高分别达2.64倍、2.34倍(P＜0.05),比其单独作用都要高,说明葡萄糖和TNF-α对Egr-1的表达的影响具有协同作用。
8、细胞与PD98059(20μmol/1)孵育后各组Egr-1的表达较空白对照组、葡萄糖孵育组和TNF-α孵育组Egr-1蛋白表达分别降低16.92%、13.99%、63.08%(P＜0.05)。10ng/ml TNF-α可以使25mmol/1葡萄糖孵育的ERK1/2水平升高1.76倍(P＜0.05),25mmol/1葡萄糖加入到10ng/ml TNF-α孵育后的内皮细胞中,葡萄糖不能提高ERK1/2的活性(P＞0.05)。
9、25mmol/1葡萄糖、10ng/ml TNF-α可使内皮细胞培养液中PAI-1水平升高,与PD98059孵育后葡萄糖组和TNF-α组PAI-1水平分别降低了30.43%、66.67%(P＜0.05)。
讨论
肥胖根据脂肪组织分布的位置不同而分为中心型肥胖和外周型肥胖。中心型肥胖即内脏型肥胖,是指脂肪堆积在大网膜和肠系膜的脂肪处。外周型肥胖,通常是指皮下脂肪的堆积。流行病学研究发现,肥胖相关的并发症,如胰岛素抵抗、2型糖尿病、血脂异常症、心血管系统疾病等,与内脏型肥胖的关系更加密切,但其机制目前还不十分清楚。分子水平的研究发现,内脏脂肪表达的IL-6、IL-8、PAI-1、血管紧张素原等均高于皮下脂肪,因而很可能是由于内脏脂肪不同的生物学性质,引起了中心型肥胖相关疾病的发生。
近年来,关于脂肪组织源性TNF-α在肥胖相关胰岛素抵抗中的作用的研究备受瞩目。TNF-α在肥胖的人类和啮齿动物的脂肪组织中均有表达,是研究所发现的第一个与肥胖胰岛素抵抗密切相关的脂肪因子。本研究发现网膜脂肪TNF-α蛋白表达高于皮下脂肪细胞,与Alessi MC等的研究发现网膜脂肪中TNF-αmRNA的表达高于皮下脂肪这一结果相一致,支持内脏脂肪堆积与并发症出现更相关这一观点。
脂肪细胞的大小是细胞因子合成的一个重要的决定因素,Gabor Winkler等报道脂肪细胞的大小是TNF-α产生和胰岛素抵抗的决定因素。本研究测定了两处脂肪细胞的大小,结果显示网膜脂肪细胞小于皮下脂肪细胞。在两处脂肪细胞中,脂肪细胞的大小与TNF-α表达呈正相关,这一结果与受试者的代谢状况或是各种激素、因子的不同无关,因为两处脂肪来源于同一受试者。因而,脂肪细胞大胁皇蔷龆═NF-α表达的唯一决定因素,TNF-α的表达还决定于脂肪组织所处的解剖位置。
网膜脂肪TNF-α蛋白与胰岛素抵抗指数呈正相关,这与Sada KE的报道相一致,说明TNF-α在内脏脂肪堆积引发胰岛素抵抗的过程中起重要作用。本研究还发现网膜脂肪组织表达的TNF-α与甘油三酯、高密度脂蛋白胆固醇密切相关。已有研究发现内脏脂肪比皮下脂肪的脂解能力要强,本研究进一步支持这一观点。网膜脂肪可通过分泌TNF-α而促进脂解,使游离脂肪酸增加,进一步促进了胰岛素抵抗、血脂异常症以及心血管系统疾病的发生。
纤溶酶原激活抑制物-1(PAI-1)是一种丝氨酸蛋白酶抑制剂,是纤维蛋白溶解系统主要的调节因子。PAI-1是重要的纤溶酶原活化的抑制剂,它的增加可以打乱正常的纤维蛋白清除机制而促进血栓的形成。PAI-1在肥胖患者中水平显著升高,是心血管系统疾病发生的独立危险因素。网膜脂肪TNF-α的表达与血浆PAI-1水平的显著相关,表明了网膜脂肪TNF-α在肥胖相关心血管系统疾病的发生中起到了重要的作用。TNF-α可促进脂肪细胞、内皮细胞表达PAI-1,使血浆中PAI-1的水平升高,从而增加了心血管系统疾病发生的危险性。
血糖和TNF-α不同水平对Egr-1表达的影响研究结果表明,TNF-α、高葡萄糖均可增加Egr-1的表达,且二者可协同作用增加Egr-1的表达。TNF-α可活化内皮细胞中ERK1/2和Egr-1的表达,并且TNF-α依赖于ERK1/2途径诱导Egr-1在内皮细胞中的表达。
Egr-1最初被发现是各种刺激包括生长因子、细胞因子、缺血、物理力量、损伤等所快速引起反应的早期基因,所有的刺激均与血管疾病的进展相关。TNF-α依赖于ERK1/2途径调节HUVEC中Egr-1的表达,这一结果与胰岛素通过ERK1/2途径介导鼠肾小球血管内皮细胞Egr-1的表达是相同的。Egr-1是促有丝分裂信号途径重要的介导因子,用Egr-1的反义技术可以通过减少动脉粥样硬化处Egr-1的表达抑制SMC移动和增殖。McCaffrey的研究表明Egr-1在血管损伤后所导致的动脉粥样硬化处表达升高,Egr-1是血管内皮受损后功能变化的重要介导因子。因而在肥胖患者高表达的TNF-α可能通过升高Egr-1在内皮细胞的表达,从而介导了心血管系统疾病的发生。
TNF-α依赖ERK1/2路径介导Egr-1的表达,因而ERK1/2路径在血管病变发生中起到了重要作用,研究表明接受ERK1/2的反义治疗可减少动脉粥样硬化的形成。ERK1/2可以调节细胞的生长分化和增殖,还与动脉粥样硬化的改变密切相关,抑制ERK1/2路径是可以达到保护心血管的效益的,但同时也可能扰乱正常细胞的生长分化和增殖。本实验用到的是MEK抑制剂,目前尚无特异性选择ERK1/2的抑制剂应用于临床。
虽然MEK抑制剂可以抑制葡萄糖诱导的Egr-1的表达,但葡萄糖不能增加TNF-α引起的ERK1/2的活化,数据表明葡萄糖诱导的Egr-1的表达可能并不依赖于ERK1/2的活化。研究显示葡萄糖诱导Egr-1可能是通过PKC路径实现的。由此推测,MEK抑制剂可能抑制了其他信号途径如PKC路径,从而使葡萄糖诱导的Egr-1表达下降。结果表明TNF-α和葡萄糖以不同的方式调节Egr-1表达,葡萄糖通过经典PKC依赖途径,TNF-α通过ERK1/2途径介导Egr-1表达。
因而,TNF-α在内脏脂肪的堆积与胰岛素抵抗、血脂异常症等相关疾病的发生中起到了中心介质的作用。对于其进一步分子机制的研究将有益于中心型肥胖患者并发症的预防和治疗策略的制定。本研究表明了Egr-1在血管并发症中重要的中心环节作用,以及在动脉粥样硬化形成过程中的重要意义。TNF-α和葡萄糖使血管内皮细胞Egr-1上调是糖尿病发生血管并发症的初始事件,对以后在这一环节进行抑制从而达到治疗血管并发症的作用提供了重要的理论支持。
结论
1、肥胖者网膜脂肪处TNF-α蛋白水平的高表达与肥胖相关胰岛素抵抗、心血管系统疾病等并发症的发生密切相关。
2、网膜脂肪可通过分泌TNF-α而促进脂解,使游离脂肪酸增加,进一步促进了胰岛素抵抗、血脂异常症以及心血管系统疾病的发生。TNF-α还可促进脂肪细胞、内皮细胞表达PAI-1,使血浆中PAI-1的水平升高,从而增加了心血管系统疾病发生的危险性。
3、TNF-α可活化内皮细胞中ERK1/2和Egr-1的表达,并且TNF-α部分依赖于ERK1/2途径诱导Egr-1在内皮细胞中的表达。
Introduction
Obesity now present the biggest health challenges worldwide because of its increasing prevalence rate each year. Many epidemiological studies have shown that obesity is associated with a high risk of obesity-related comorbidites, such as type 2 diabetes, cardiovascular disease. However, the underlying mechanism for this is largely unknown. Adipose tissue secretes bioactive peptides, termed 'adipocytokines', which act locally and distally through autocrine, paracrine and endocrine effects. These include tumor necrosis factors (TNF)-α, IL-6, CRP, resistin or adiponectin, which are involved in the pathogenesis of vascular diseases and may represent a link between obesity and atherosclerosis.
Evidence is mounting to suggest that adipocytokines may directly influence endothelial function through their proinflammatory properties. While the majority of the data are based on in vitro studies, they shed light on the molecular links between obesity, insulin resistance, and endothelial dysfunction. The secretary products of adipose tissue contribute to the elevated risks of cardiovascular diseases, and these effects appear to be independent of their effects on insulin resistance and diabetes. The effects of adipocytokines on vascular homeostasis are different. Recently more attention to TNF-αin the development of obesity related cardiovascular diseases are being paid.
The animal studies on TNF-αand development of atherosclerosis have produced mixed results. Although reducing TNF-αlevel in apoE-deficient mice resulted in significant decrease of atherosclerosis lesions, in a wild-type background, it produced no improvements. However, mice deficient for the p55 TNF-αreceptor exhibited accelerated atherosclerosis. Finally, although TNF-αis thought to play a role in the progression of ischemia-related congestive heart failure, anti-TNF-αtherapy has shown no benefits for congestive heart failure progression in patients. Despite these conflicting results, there remains a great interest in testing anti-TNF-αtherapies for cardioprotective effects.
Plasminogen activator inhibitor-1 (PAI-1) is a kind of inhibitor of serine protein enzyme, and a key factor in fibrinolysis. PAI-1 was an important inhibitor of fibrinolytic activity, and it could disturb normal fibrin clearance mechanisms and promote thrombosis. PAI-1 was dramatically increased in obesity, and it was an independent risk factor of the development of cardiovascular diseases. After intravenous injection of TNF-αto human beings, the level of PAI-1 in plasma was increased. In vitro, TNF-αcould increase the expression of PAI-1 in both endothelial cells and adipocytes. Thus, TNF-αmay be contribute to cardiovascular diseases through increasing the expression of PAI-1.
Early growth response gene-1 (egr-1) was originally identified as one of the immediate early genes associated with many kinds of stimulations, include growth factor, cell factor, ischemia, physical energy and trauma, etc. All of these stimulations were associated with vascular diseases. McCaffrey demonstrated Egr-1 expression levels in atherosclerotic lesions after vascular injury were elevated. Anti-Egr-1 may decrease the expression of Egr-1 in the lesion of atherosclerosis through inhibiting smooth muscle cells migration and proliferation. Hence, our study would observed the expression of Egr-1 in endothelial cells affected by TNF-α, to discuss the mechanisms about TNF-αinduced cardiovascular diseases.
Our study is aimed to discuss the effect of adipocytokine TNF-αin obesity related cardiovascular complications and the mechanism. Therefore, we set out to examine the association between the expression of TNF-αprotein in omental and subcutaneous adipose tissue and homeostasis model assessment insulin resistance (HOMA-IR), lipids, and plasma PAI-1 levels in female and male respectively, to discuss the relationship between TNF-αand insulin resistance or cardiovascular diseases. Then in cell culture, we observed the effect of TNF-αon HUVEC, as well as the expression of Egr-1 in HUVEC, to discuss the mechanism of TNF-αin cardiovascular disease.
Materials and methods
1. Lean and central obesity subjects involved in surgery were recruited at surgery department. Participants in a fasting state underwent anthropometric evaluation. Anthropometric measurements included weight, height, waist circumferences.
2. Fasting plasma glucose concentration (glucose oxidase method), serum cholesterol (CHOD-PAP method), serum triglycerides (GPO-PAP method), and high-density lipoprotein cholesterol (IRC method) of all the patients were estimated. Fasting serum insulin concentrations were determined with chemiluminescence method using automated immunoassay system. The homeostasis model assessment insulin resistance (HOMA-IR) index derives an estimate of whole-body insulin sensitivity from fasting glucose and insulin concentrations: HOMA-IR=fasting insulin (mU/l)×fasting plasma glucose (mM)/22.5.
3. The expression of TNF-αprotein in omental and subcutaneous fat was quantified by using immunohistochemistry and western blot method.
4. PAI-1 in human plasma was determined by specific enzyme-linked immuno-sorbent assay (ELISA) method.
5. We used HUVEC culture method, and examined the effect of TNF-αand glucose on cell preincubated. And also examined the effect of PD98059 on cell.
6. The expression of Egr-1 and ERK1/2 protein in HUVEC was quantified by using western blot method.
7. The level of PAI-1 in cell culture solution was quantified by using ELISA method.
Results
1. The basic anthropometric and metabolic characteristics of the subjects enrolled in this study are presented that BMI, waist circumference, fasting plasma insulin, HOMA-IR, triglycerides, total cholesterol, and plasma PAI-1 were higher in obese than in lean subjects in both male and female. HDL-cholesterol were lower in obese than in lean subjects.
2. TNF-αprotein levels were significantly increased in obese compared with lean subjects in both omental and subcutaneous adipose tissue.
3. Within each fat depot, there was a positive correlation between adipose cells size and TNF-αexpression (omental: r=0.779, P<0.01; subcutaneous: r=0.452, P<0.05) in female obese subjects. While in male obese subjects, positive correlation only was found in omental adipose tissue (r=0.828, P<0.01).
4. In female obese subjects, omental TNF-αprotein levels showed a close association with most of the parameters studied: fasting glucose (r=0.541, P<0.05); fasting insulin (r=0.599, P<0.01); HOMA-IR (r=0.546, P<0.05); triglycerides (r=0.469, P<0.05); HDL-cholesterol (r=-0.759, P<0.01). There was not a similar statistically significant relationship between subcutaneous TNF-αprotein levels and these parameters in obese women. In obese men, no significant correlations between TNF-αprotein and HOMA-IR were apparent in either adipose depot. Although we found correlations between omental TNF-αprotein levels and glucose (r=0.762, P<0.01); insulin (r=0.622, P<0.05); triglycerides (r=0.650, P<0.05); HDL-cholesterol (r=-0.880, P<0.01) in the male obese population. There was no correlation between subcutaneous TNF-αprotein levels and glucose, insulin, triglycerides, HDL-cholesterol in men.
5. In both female and male obesity subjects, omental TNF-αprotein levels showed a close association with plasma PAI-1 levels (female, n=20, r=0.763, P<0.01; male, n=12, r=0.760, P<0.01).
6. 25mmol/l glucose and TNF-αincreased Egr-1 protein levels: Both glucose and TNF-αincreased Egr-1 protein levels at 60 minutes, but with different time course. TNF-αinduced Egr-1 expression was transient, peaking at 60 minutes, whereas Egr-1, induced by glucose, remained elevated until 240 minutes.
7. When cells were incubated with both glucose and TNF-α, Egr-1 expression level was increased 2.64 fold and 2.34 fold (P<0.05) respectively, higher than that in cells incubated with either glucose or TNF-αalone. It suggested that glucose and TNF-αhad an additive effect on Egr-1 expression.
8. Cells pretreatment with MEK inhibitors PD98059 (20μmol/l) downregulated control group, glucose group and TNF-αgroup induced Egr-1 expression 16.92%, 13.99% and 63.08% respectively (P<0.05). 10ng/ml TNF-αincreased phosphorylated ERK1/2 levels 1.76 fold (P<0.05) after 25mmol/l glucose pretreatment, but glucose did not enhance ERKl/2 activation after TNF-αtreatment.
9. 25mmol/l glucose or 10ng/ml TNF-αmay increase the levels of PAI-1 in endothelial cells culture solution. When pretreatment with PD98059, the levels of PAI-1 decreased 30.43% and 66.67% respectively (P<0.05) in glucose group and TNF-αgroup.
Discussion
Obesity is a heterogeneous condition with respect to regional distribution of fat tissue; visceral obesity refers to fat accumulation within omental and mesenteric fat depots, whereas peripheral obesity generally refers to subcutaneous fat accumulation. Many epidemiological studies have shown that visceral obesity is associated with a high risk of obesity-related comorbidites, such as insulin resistance, type 2 diabetes, cardiovascular disease, and dyslipidemia, than is peripheral obesity. However, the underlying mechanism for this pathophysiological difference is largely unknown. Visceral adipocytes express higher levels of interleukin-6, interleukin-8, plasminogen activator inhibitor 1 (PAI-1), and angiotensinogen than subcutaneous adipocytes. Presumably, distinctive biological properties of visceral fat contribute to the increased pathogenecity of central obesity.
The expression of tumor necrosis factor-alpha (TNF-α) in adipose tissue is increased in human obesity, and TNF-αis proposed as molecular link between obesity and insulin resistance. Alessi MC et al found that TNF-αmRNA in the omental is higher than in the sc adipose tissue. Our results confirm this data and additionally show that difference by the groups of male and female.
Since a strong correlation has previously been demonstrated between TNF-αexpression and adipocytes size, we were interested to determine whether our data on subcutaneous and omental adipocytes confirmed this. We found that subcutaneous fat cells were larger than omental fat cells in obese female. In obese male, there was no significance difference between the two depots fat cell size. While within each depot, there is a positive relationship was found between TNF-αprotein and fat cell size in the two adipose depots in obesity except subcutaneous fat in obese male, a finding consistent with previous reports. Obesity, characterized by adipocytes hypertrophy, is a major cause of insulin resistance, type 2 diabetes, hyperlipidaemia and hypertension, clustering of risk factors for atherosclerosis. Thus, we indicate that hypertrophic adipocytes with increased expressions of adipokines causing insulin resistance, such as TNF-α, are associated with insulin resistance which may be a central mechanism to cause life style-related disease. However, the use of isolated adipose tissue rather than whole adipose tissue controlled for differences in the ratio of adipocytes to adventitial tissue within the individual biopsies.
The key role of TNF-αin the genesis of insulin resistance has been reported in humans and in animal models especially in relation to obesity associated with insulin resistance. In the fatty tissue of obese animals with insulin resistance and type 2 diabetes, the TNF-αconcentration is elevated. Neutralization of TNF-αin vivo dramatically increased the insulin sensitivity of these obese animals, resulting in increased glucose uptake in peripheral tissues. These observations confirmed the role of TNF-αas a key mediator of insulin resistance in obesity. In our study, omental TNF-αprotein concentration was associated with the serum insulin concentrations in obese group. Moreover, it was significantly associated with insulin resistance in obese women, which manifested as correlation of omental TNF-αlevel with HOMA-IR. From our observations it can be concluded that omental TNF-αprotein plays an important role in the development of insulin resistance in obesity. Four large prospective studies have shown that hyperinsulinemia is a predictor of coronary artery disease. The greatest association of hyperinsulinemia with coronary artery disease has been found in Finland in a population with a very high frequency of coronary artery disease. Results of a prospective investigation of 2103 men clearly showed that high fasting insulin concentrations are an independent predictor of cardiovascular diseases. Additionally, increased TNF-αlevel have been associated with high triglycerides concentrations in metabolic syndrome. In the present study we found that in obesity omental TNF-αprotein was not only positively associated with triglycerides, also negatively with HDL-cholesterol levels, which reflected an increased risk of heart disease. More importantly, our data showed that omental TNF-αprotein was significantly associated with plasma PAI-1 levels. To date, TNF-αis known to be effective for stimulation of PAI-1 release in many cell types, especially adipocytes. PAI-1 could disturb normal fibrin clearance mechanisms and promote thrombosis. The elevated omental TNF-αprotein observed during central obesity and the positive correlation observed between omental TNF-αprotein and PAI-1 levels have led us to characterize omental TNF-αprotein as an important factor in the pathophysiology of obesity-related cardiovascular diseases.
In conclusion, these results indicated that TNF-αmay be the link between the cluster of visceral obesity, insulin resistance, and cardiovascular comorbidities. Further studies should identify the molecular mechanisms underlying such events, which may lead to effective therapeutic strategies designed to protect against atherosclerosis in obese patients.
The effect of different concentrations of glucose and TNF-αon Egr-1 expression suggest that glucose and TNF-αall increased the expression of Egr-1, and the two factors additively increased Egr-1 expression. TNF-αactivate the expression of ERK1/2 and Egr-1 in the HUVEC. TNF-αinduced Egr-1 expression through ERK1/2 activation in HUVEC.
Egr-1 was originally identified as an immediate-early gene that is rapidly induced in response to a variety of stimuli, including growth factors, cytokines, hypoxia, physical forces, and injury, all of which are implicated in the progress of vascular diseases. Recent studies demonstrated that Egr-1 expression is elevated in both atherosclerotic lesions after vascular injury. Interestingly, reduction of Egr-1 expression by Egr-1 antisense technology decreased intimal through inhibiting smooth muscle cell migration and proliferation. Taken together, these findings suggest that Egr-1 is the key mediator in orchestrating the functional characteristics of the vessel wall after injury. And TNF-αmay be increase Egr-1 expression in endothelial cells to contribute the development of cardiovascular diseases in obesity.
The importance of TNF-αand the ERK1/2 pathway in the pathogenesis of vascular alterations, however, is well accepted and underscored by recent studies showing reduced vascular lesion formation in TNF-αdeficient mice receiving ERK1/2 antisense treatment. ERK1/2 serves as a key signaling molecule for migration, proliferation and other cellular responses that contribute to atherosclerotic of the arterial wall. Inhibition of the ERK1/2 pathway, therefore, constitutes a logical pharmacological approach for protecting the vasculature from atherosclerotic damage. To date, no selective ERK1/2 inhibitor is clinically available.
Although MEK inhibitors inhibited glucose-induced Egr-1 expression,we ruled out the involvement of ERK1/2, because glucose did not increase TNF-αrelated ERK1/2 activity. Rukhsana reported that glucose induced Egr-1 expression was mediated by PKC activation. MEK inhibitors may inhibiting other signaling pathways, such as PKC pathway, then inhibited glucose-induced Egr-1 expression. The datas suggest that TNF-αand glucose may regulate Egr-1 expression through different ways, with glucose mediating its effects through one of the classical PKC isoforms and TNF-αacting through the ERK1/2 pathway.
Thus, TNF-αmay play a key role in the development of accumulation of visceral adipose tissue, insulin resistance and dyslipidemia. Further studies should identify the molecular mechanisms underlying such events, which may lead to effective therapeutic strategies designed to protect against atherosclerosis in obese patients. Our studies conclude that Egr-1 play an important role in the development of cardiovascular complications, especially in the process of atherosclerosis. TNF-αand glucose-induced the higher expression of Egr-1 maybe the initial event in the diabetes related cardiovascular complications, and the inhibition of the pathway may provide basic datas for the therapy of cardiovascular complications.
Conclusion
1. The higher expression of TNF-αin omental adipose tissue is associated with the development of obesity related cardiovascular diseases in obesity subjects.
2. Omental adipose tissue may through secret TNF-αto stimulate lipolysis, increase free fatty acids, hence accelerates the development of insulin resistance, hyperlipidemia and cardiovascular diseases. TNF-αalso stimulates the expression of PAI-1 in adipocytes and endothelial cells, increases the level of plasma PAI-1, which increases the risk of cardiovascular diseases.
3. TNF-αmay increase the expression of ERK1/2 and Egr-1 in endothelial cells, and TNF-αmay regulate Egr-1 expression through the ERK1/2 pathway.
肥胖与2型糖尿病、心血管系统疾病的发生发展密切相关,在全世界的患病率逐年上升。对于其密切关系的分子机制目前还不十分清楚,但研究发现脂肪组织作为内分泌器官,分泌多种脂肪细胞因子,如肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)、C-反应蛋白(CRP)、抵抗素(resistin)、脂联素(adiponectin)等,在肥胖相关的心血管系统疾病的发生发展中发挥了极其重要的作用。
越来越多的证据表明脂肪细胞因子通过促炎症反应的作用直接影响到内皮细胞功能。虽然大部分的数据都是基于体外的研究得来的,但我们可以用脂肪细胞因子来解释肥胖、胰岛素抵抗和内皮细胞功能失调密切相关的原因。脂肪组织的分泌产物增加可促使心血管系统疾病发生的危险性增加,但这并不依赖于脂肪细胞因子导致的胰岛素抵抗和糖尿病所产生的影响。脂肪细胞因子对血管稳态的作用各不相同,近来对于TNF-α在肥胖相关心血管系统疾病的发展中的作用较为关注。
然而动物试验的研究表明TNF-a在动脉粥样硬化等心血管系统疾病的发生发展中的作用结果是不一致的。一方面敲除apoE的小鼠,TNF-α水平下降,动脉粥样硬化斑块减少,另一方面TNF-α受体P55缺失型小鼠却加速了动脉粥样硬化的形成;一方面TNF-α被认为在充血性心衰的发展过程中起到了重要作用,另一方面研究却发现在充血性心衰患者中抗TNF-α的治疗并无益处。正因为这些研究结果互相冲突,所以TNF-α在心血管系统疾病中的作用更加受到关注,抗TNF-α治疗以取得心血管保护效益的研究有待于进一步研究。
纤溶酶原激活抑制物-1(PAI-1)是一种丝氨酸蛋白酶抑制剂,是纤维蛋白溶解系统主要的调节因子。PAI-1是重要的纤溶酶原活化的抑制剂,它的增加可以打乱正常的纤维蛋白清除机制而促进血栓的形成。PAI-1的水平在肥胖患者中显著升高,是心血管系统疾病发生的独立危险因素。在人体中,给予注射TNF-α可以增加PAI-1在血浆中的浓度;体外研究中,TNF-α可以增加PAI-1在内皮细胞以及脂肪细胞中的表达。因而TNF-α可通过升高PAI-1的表达而促进了血管病变的发生。
最初发现早期生长反应基因-1(Egr-1)是生长因子、细胞因子、缺血、物理力量、损伤等各种刺激所引起的快速反应的早期基因,所有的刺激均与血管疾病的进展相关。McCaffrey的研究表明Egr-1在血管损伤后所导致的动脉粥样硬化处表达升高,用Egr-1的反义技术可以通过抑制平滑肌细胞移动和增殖,减少动脉粥样硬化处Egr-1的表达。在巨噬细胞中的研究发现,TNF-α可通过ERK1/2路径引起Egr-1表达的升高。因此本研究将检测在TNF-α作用的内皮细胞中Egr-1的表达,进一步探讨TNF-α所致心血管系统疾病发生的机制。
本研究旨在探讨TNF-α对心血管系统疾病的作用及其机制。本研究检测了受试者网膜脂肪组织和皮下脂肪组织中TNF-α表达、空腹血糖、胰岛素、血脂及血浆中PAI-1的水平等临床参数,探讨TNF-α与胰岛素抵抗、血脂异常症和心血管系统疾病之间的关系。并进行了体外试验,观察TNF-α对内皮细胞中Egr-1蛋白表达的影响,探讨TNF-α引起心血管系统疾病的机制。
材料与方法
1、选取进行腹部外科手术的肥胖患者和正常体重的对照组患者,进行常规指标的测定,包括体重、身高、腰围。
2、采用酶学方法测定血糖、血胆固醇、血高密度脂蛋白胆固醇水平;采用化学发光免疫分析法测定胰岛素、C肽水平。计算胰岛素抵抗指数(HOMA-IR):HOMA-IR=FPG×FINS/22.5。
3、采用免疫组化方法和western blot方法检测受试者腹部皮下脂肪和网膜脂肪中TNF-α的表达。
4、ELISA方法检测受试者血清中PAI-1的水平。
5、采用人脐静脉内皮细胞株培养的方法,TNF-α和/或葡萄糖与内皮细胞共同孵育,收集内皮细胞及细胞培养液。加入MEK抑制剂(PD98059)进行干预,收集内皮细胞及细胞培养液。
6、采用western blot方法检测内皮细胞中Egr-1、ERK1/2蛋白的表达。
7、采用ELISA方法测定各组细胞培养液中PAI-1水平。
结果
1、男性和女性受试者测量和代谢指标的数据表明无论在男性还是女性组,肥胖者BMI、腰围、空腹胰岛素水平、HOMA-IR、甘油三酯、胆固醇和血浆PAI-1水平均高于正常对照组,而HDL-C水平低于正常对照组。
2、肥胖组网膜脂肪和皮下脂肪标本中TNF-α蛋白水平均高于正常对照组。
3、在女性肥胖受试者中两处脂肪细胞的大小与其对应的TNF-α的表达呈正相关(γ=0.779,P＜0.01,网膜脂肪处;γ=0.452,P＜0.05,皮下脂肪处)。在男性肥胖受试者中,仅在网膜脂肪处两者成正相关(γ=0.828,P＜0.01)。
4、在女性肥胖受试者中,网膜脂肪细胞TNF-α的表达与空腹血糖浓度呈正相关(γ=0.541,P＜0.05),与空腹胰岛素呈正相关(γ=0.599,P＜0.01),与胰岛素抵抗指数呈正相关(γ=0.546,P＜0.05),与甘油三酯呈显著正相关(γ=0.469,P＜0.05),与高密度脂蛋白胆固醇呈负相关(γ=-0.759,P＜0.01)。这些参数与皮下脂肪TNF-α表达不相关。在男性肥胖组中,两处脂肪TNF-α的表达与HOMA-IR均无明显相关,但网膜脂肪TNF-α表达与葡萄糖水平正相关(γ=0.762,P＜0.01),与胰岛素水平正相关(γ=0.622,P＜0.05),与甘油三酯水平正相关(γ=0.650,P＜0.05),与高密度脂蛋白胆固醇水平负相关(γ=-0.880,P＜0.01)。男性肥胖受试者中皮下脂肪TNF-α的表达与葡萄糖、胰岛素、甘油三酯和高密度脂蛋白胆固醇水平无明显相关性。
5、在女性和男性肥胖受试者中,可见网膜脂肪TNF-α蛋白的表达水平与血浆PAI-1水平呈显著正相关(女性:n=20,γ=0.763,P＜0.01;男性:n=12,γ=0.760,P＜0.01)。
6、25mmol/1葡萄糖、10ng/ml TNF-α使Egr-1水平的变化如下:TNF-α使Egr-1升高后即回落,高峰在60分钟,葡萄糖使Egr-1表达的高峰亦在60分钟,但Egr-1的表达持续升高直至240分钟。
7、葡萄糖与TNF-α共同孵育的内皮细胞Egr-1表达较葡萄糖和TNF-α单独作用升高分别达2.64倍、2.34倍(P＜0.05),比其单独作用都要高,说明葡萄糖和TNF-α对Egr-1的表达的影响具有协同作用。
8、细胞与PD98059(20μmol/1)孵育后各组Egr-1的表达较空白对照组、葡萄糖孵育组和TNF-α孵育组Egr-1蛋白表达分别降低16.92%、13.99%、63.08%(P＜0.05)。10ng/ml TNF-α可以使25mmol/1葡萄糖孵育的ERK1/2水平升高1.76倍(P＜0.05),25mmol/1葡萄糖加入到10ng/ml TNF-α孵育后的内皮细胞中,葡萄糖不能提高ERK1/2的活性(P＞0.05)。
9、25mmol/1葡萄糖、10ng/ml TNF-α可使内皮细胞培养液中PAI-1水平升高,与PD98059孵育后葡萄糖组和TNF-α组PAI-1水平分别降低了30.43%、66.67%(P＜0.05)。
讨论
肥胖根据脂肪组织分布的位置不同而分为中心型肥胖和外周型肥胖。中心型肥胖即内脏型肥胖,是指脂肪堆积在大网膜和肠系膜的脂肪处。外周型肥胖,通常是指皮下脂肪的堆积。流行病学研究发现,肥胖相关的并发症,如胰岛素抵抗、2型糖尿病、血脂异常症、心血管系统疾病等,与内脏型肥胖的关系更加密切,但其机制目前还不十分清楚。分子水平的研究发现,内脏脂肪表达的IL-6、IL-8、PAI-1、血管紧张素原等均高于皮下脂肪,因而很可能是由于内脏脂肪不同的生物学性质,引起了中心型肥胖相关疾病的发生。
近年来,关于脂肪组织源性TNF-α在肥胖相关胰岛素抵抗中的作用的研究备受瞩目。TNF-α在肥胖的人类和啮齿动物的脂肪组织中均有表达,是研究所发现的第一个与肥胖胰岛素抵抗密切相关的脂肪因子。本研究发现网膜脂肪TNF-α蛋白表达高于皮下脂肪细胞,与Alessi MC等的研究发现网膜脂肪中TNF-αmRNA的表达高于皮下脂肪这一结果相一致,支持内脏脂肪堆积与并发症出现更相关这一观点。
脂肪细胞的大小是细胞因子合成的一个重要的决定因素,Gabor Winkler等报道脂肪细胞的大小是TNF-α产生和胰岛素抵抗的决定因素。本研究测定了两处脂肪细胞的大小,结果显示网膜脂肪细胞小于皮下脂肪细胞。在两处脂肪细胞中,脂肪细胞的大小与TNF-α表达呈正相关,这一结果与受试者的代谢状况或是各种激素、因子的不同无关,因为两处脂肪来源于同一受试者。因而,脂肪细胞大胁皇蔷龆═NF-α表达的唯一决定因素,TNF-α的表达还决定于脂肪组织所处的解剖位置。
网膜脂肪TNF-α蛋白与胰岛素抵抗指数呈正相关,这与Sada KE的报道相一致,说明TNF-α在内脏脂肪堆积引发胰岛素抵抗的过程中起重要作用。本研究还发现网膜脂肪组织表达的TNF-α与甘油三酯、高密度脂蛋白胆固醇密切相关。已有研究发现内脏脂肪比皮下脂肪的脂解能力要强,本研究进一步支持这一观点。网膜脂肪可通过分泌TNF-α而促进脂解,使游离脂肪酸增加,进一步促进了胰岛素抵抗、血脂异常症以及心血管系统疾病的发生。
纤溶酶原激活抑制物-1(PAI-1)是一种丝氨酸蛋白酶抑制剂,是纤维蛋白溶解系统主要的调节因子。PAI-1是重要的纤溶酶原活化的抑制剂,它的增加可以打乱正常的纤维蛋白清除机制而促进血栓的形成。PAI-1在肥胖患者中水平显著升高,是心血管系统疾病发生的独立危险因素。网膜脂肪TNF-α的表达与血浆PAI-1水平的显著相关,表明了网膜脂肪TNF-α在肥胖相关心血管系统疾病的发生中起到了重要的作用。TNF-α可促进脂肪细胞、内皮细胞表达PAI-1,使血浆中PAI-1的水平升高,从而增加了心血管系统疾病发生的危险性。
血糖和TNF-α不同水平对Egr-1表达的影响研究结果表明,TNF-α、高葡萄糖均可增加Egr-1的表达,且二者可协同作用增加Egr-1的表达。TNF-α可活化内皮细胞中ERK1/2和Egr-1的表达,并且TNF-α依赖于ERK1/2途径诱导Egr-1在内皮细胞中的表达。
Egr-1最初被发现是各种刺激包括生长因子、细胞因子、缺血、物理力量、损伤等所快速引起反应的早期基因,所有的刺激均与血管疾病的进展相关。TNF-α依赖于ERK1/2途径调节HUVEC中Egr-1的表达,这一结果与胰岛素通过ERK1/2途径介导鼠肾小球血管内皮细胞Egr-1的表达是相同的。Egr-1是促有丝分裂信号途径重要的介导因子,用Egr-1的反义技术可以通过减少动脉粥样硬化处Egr-1的表达抑制SMC移动和增殖。McCaffrey的研究表明Egr-1在血管损伤后所导致的动脉粥样硬化处表达升高,Egr-1是血管内皮受损后功能变化的重要介导因子。因而在肥胖患者高表达的TNF-α可能通过升高Egr-1在内皮细胞的表达,从而介导了心血管系统疾病的发生。
TNF-α依赖ERK1/2路径介导Egr-1的表达,因而ERK1/2路径在血管病变发生中起到了重要作用,研究表明接受ERK1/2的反义治疗可减少动脉粥样硬化的形成。ERK1/2可以调节细胞的生长分化和增殖,还与动脉粥样硬化的改变密切相关,抑制ERK1/2路径是可以达到保护心血管的效益的,但同时也可能扰乱正常细胞的生长分化和增殖。本实验用到的是MEK抑制剂,目前尚无特异性选择ERK1/2的抑制剂应用于临床。
虽然MEK抑制剂可以抑制葡萄糖诱导的Egr-1的表达,但葡萄糖不能增加TNF-α引起的ERK1/2的活化,数据表明葡萄糖诱导的Egr-1的表达可能并不依赖于ERK1/2的活化。研究显示葡萄糖诱导Egr-1可能是通过PKC路径实现的。由此推测,MEK抑制剂可能抑制了其他信号途径如PKC路径,从而使葡萄糖诱导的Egr-1表达下降。结果表明TNF-α和葡萄糖以不同的方式调节Egr-1表达,葡萄糖通过经典PKC依赖途径,TNF-α通过ERK1/2途径介导Egr-1表达。
因而,TNF-α在内脏脂肪的堆积与胰岛素抵抗、血脂异常症等相关疾病的发生中起到了中心介质的作用。对于其进一步分子机制的研究将有益于中心型肥胖患者并发症的预防和治疗策略的制定。本研究表明了Egr-1在血管并发症中重要的中心环节作用,以及在动脉粥样硬化形成过程中的重要意义。TNF-α和葡萄糖使血管内皮细胞Egr-1上调是糖尿病发生血管并发症的初始事件,对以后在这一环节进行抑制从而达到治疗血管并发症的作用提供了重要的理论支持。
结论
1、肥胖者网膜脂肪处TNF-α蛋白水平的高表达与肥胖相关胰岛素抵抗、心血管系统疾病等并发症的发生密切相关。
2、网膜脂肪可通过分泌TNF-α而促进脂解,使游离脂肪酸增加,进一步促进了胰岛素抵抗、血脂异常症以及心血管系统疾病的发生。TNF-α还可促进脂肪细胞、内皮细胞表达PAI-1,使血浆中PAI-1的水平升高,从而增加了心血管系统疾病发生的危险性。
3、TNF-α可活化内皮细胞中ERK1/2和Egr-1的表达,并且TNF-α部分依赖于ERK1/2途径诱导Egr-1在内皮细胞中的表达。
Introduction
Obesity now present the biggest health challenges worldwide because of its increasing prevalence rate each year. Many epidemiological studies have shown that obesity is associated with a high risk of obesity-related comorbidites, such as type 2 diabetes, cardiovascular disease. However, the underlying mechanism for this is largely unknown. Adipose tissue secretes bioactive peptides, termed 'adipocytokines', which act locally and distally through autocrine, paracrine and endocrine effects. These include tumor necrosis factors (TNF)-α, IL-6, CRP, resistin or adiponectin, which are involved in the pathogenesis of vascular diseases and may represent a link between obesity and atherosclerosis.
Evidence is mounting to suggest that adipocytokines may directly influence endothelial function through their proinflammatory properties. While the majority of the data are based on in vitro studies, they shed light on the molecular links between obesity, insulin resistance, and endothelial dysfunction. The secretary products of adipose tissue contribute to the elevated risks of cardiovascular diseases, and these effects appear to be independent of their effects on insulin resistance and diabetes. The effects of adipocytokines on vascular homeostasis are different. Recently more attention to TNF-αin the development of obesity related cardiovascular diseases are being paid.
The animal studies on TNF-αand development of atherosclerosis have produced mixed results. Although reducing TNF-αlevel in apoE-deficient mice resulted in significant decrease of atherosclerosis lesions, in a wild-type background, it produced no improvements. However, mice deficient for the p55 TNF-αreceptor exhibited accelerated atherosclerosis. Finally, although TNF-αis thought to play a role in the progression of ischemia-related congestive heart failure, anti-TNF-αtherapy has shown no benefits for congestive heart failure progression in patients. Despite these conflicting results, there remains a great interest in testing anti-TNF-αtherapies for cardioprotective effects.
Plasminogen activator inhibitor-1 (PAI-1) is a kind of inhibitor of serine protein enzyme, and a key factor in fibrinolysis. PAI-1 was an important inhibitor of fibrinolytic activity, and it could disturb normal fibrin clearance mechanisms and promote thrombosis. PAI-1 was dramatically increased in obesity, and it was an independent risk factor of the development of cardiovascular diseases. After intravenous injection of TNF-αto human beings, the level of PAI-1 in plasma was increased. In vitro, TNF-αcould increase the expression of PAI-1 in both endothelial cells and adipocytes. Thus, TNF-αmay be contribute to cardiovascular diseases through increasing the expression of PAI-1.
Early growth response gene-1 (egr-1) was originally identified as one of the immediate early genes associated with many kinds of stimulations, include growth factor, cell factor, ischemia, physical energy and trauma, etc. All of these stimulations were associated with vascular diseases. McCaffrey demonstrated Egr-1 expression levels in atherosclerotic lesions after vascular injury were elevated. Anti-Egr-1 may decrease the expression of Egr-1 in the lesion of atherosclerosis through inhibiting smooth muscle cells migration and proliferation. Hence, our study would observed the expression of Egr-1 in endothelial cells affected by TNF-α, to discuss the mechanisms about TNF-αinduced cardiovascular diseases.
Our study is aimed to discuss the effect of adipocytokine TNF-αin obesity related cardiovascular complications and the mechanism. Therefore, we set out to examine the association between the expression of TNF-αprotein in omental and subcutaneous adipose tissue and homeostasis model assessment insulin resistance (HOMA-IR), lipids, and plasma PAI-1 levels in female and male respectively, to discuss the relationship between TNF-αand insulin resistance or cardiovascular diseases. Then in cell culture, we observed the effect of TNF-αon HUVEC, as well as the expression of Egr-1 in HUVEC, to discuss the mechanism of TNF-αin cardiovascular disease.
Materials and methods
1. Lean and central obesity subjects involved in surgery were recruited at surgery department. Participants in a fasting state underwent anthropometric evaluation. Anthropometric measurements included weight, height, waist circumferences.
2. Fasting plasma glucose concentration (glucose oxidase method), serum cholesterol (CHOD-PAP method), serum triglycerides (GPO-PAP method), and high-density lipoprotein cholesterol (IRC method) of all the patients were estimated. Fasting serum insulin concentrations were determined with chemiluminescence method using automated immunoassay system. The homeostasis model assessment insulin resistance (HOMA-IR) index derives an estimate of whole-body insulin sensitivity from fasting glucose and insulin concentrations: HOMA-IR=fasting insulin (mU/l)×fasting plasma glucose (mM)/22.5.
3. The expression of TNF-αprotein in omental and subcutaneous fat was quantified by using immunohistochemistry and western blot method.
4. PAI-1 in human plasma was determined by specific enzyme-linked immuno-sorbent assay (ELISA) method.
5. We used HUVEC culture method, and examined the effect of TNF-αand glucose on cell preincubated. And also examined the effect of PD98059 on cell.
6. The expression of Egr-1 and ERK1/2 protein in HUVEC was quantified by using western blot method.
7. The level of PAI-1 in cell culture solution was quantified by using ELISA method.
Results
1. The basic anthropometric and metabolic characteristics of the subjects enrolled in this study are presented that BMI, waist circumference, fasting plasma insulin, HOMA-IR, triglycerides, total cholesterol, and plasma PAI-1 were higher in obese than in lean subjects in both male and female. HDL-cholesterol were lower in obese than in lean subjects.
2. TNF-αprotein levels were significantly increased in obese compared with lean subjects in both omental and subcutaneous adipose tissue.
3. Within each fat depot, there was a positive correlation between adipose cells size and TNF-αexpression (omental: r=0.779, P<0.01; subcutaneous: r=0.452, P<0.05) in female obese subjects. While in male obese subjects, positive correlation only was found in omental adipose tissue (r=0.828, P<0.01).
4. In female obese subjects, omental TNF-αprotein levels showed a close association with most of the parameters studied: fasting glucose (r=0.541, P<0.05); fasting insulin (r=0.599, P<0.01); HOMA-IR (r=0.546, P<0.05); triglycerides (r=0.469, P<0.05); HDL-cholesterol (r=-0.759, P<0.01). There was not a similar statistically significant relationship between subcutaneous TNF-αprotein levels and these parameters in obese women. In obese men, no significant correlations between TNF-αprotein and HOMA-IR were apparent in either adipose depot. Although we found correlations between omental TNF-αprotein levels and glucose (r=0.762, P<0.01); insulin (r=0.622, P<0.05); triglycerides (r=0.650, P<0.05); HDL-cholesterol (r=-0.880, P<0.01) in the male obese population. There was no correlation between subcutaneous TNF-αprotein levels and glucose, insulin, triglycerides, HDL-cholesterol in men.
5. In both female and male obesity subjects, omental TNF-αprotein levels showed a close association with plasma PAI-1 levels (female, n=20, r=0.763, P<0.01; male, n=12, r=0.760, P<0.01).
6. 25mmol/l glucose and TNF-αincreased Egr-1 protein levels: Both glucose and TNF-αincreased Egr-1 protein levels at 60 minutes, but with different time course. TNF-αinduced Egr-1 expression was transient, peaking at 60 minutes, whereas Egr-1, induced by glucose, remained elevated until 240 minutes.
7. When cells were incubated with both glucose and TNF-α, Egr-1 expression level was increased 2.64 fold and 2.34 fold (P<0.05) respectively, higher than that in cells incubated with either glucose or TNF-αalone. It suggested that glucose and TNF-αhad an additive effect on Egr-1 expression.
8. Cells pretreatment with MEK inhibitors PD98059 (20μmol/l) downregulated control group, glucose group and TNF-αgroup induced Egr-1 expression 16.92%, 13.99% and 63.08% respectively (P<0.05). 10ng/ml TNF-αincreased phosphorylated ERK1/2 levels 1.76 fold (P<0.05) after 25mmol/l glucose pretreatment, but glucose did not enhance ERKl/2 activation after TNF-αtreatment.
9. 25mmol/l glucose or 10ng/ml TNF-αmay increase the levels of PAI-1 in endothelial cells culture solution. When pretreatment with PD98059, the levels of PAI-1 decreased 30.43% and 66.67% respectively (P<0.05) in glucose group and TNF-αgroup.
Discussion
Obesity is a heterogeneous condition with respect to regional distribution of fat tissue; visceral obesity refers to fat accumulation within omental and mesenteric fat depots, whereas peripheral obesity generally refers to subcutaneous fat accumulation. Many epidemiological studies have shown that visceral obesity is associated with a high risk of obesity-related comorbidites, such as insulin resistance, type 2 diabetes, cardiovascular disease, and dyslipidemia, than is peripheral obesity. However, the underlying mechanism for this pathophysiological difference is largely unknown. Visceral adipocytes express higher levels of interleukin-6, interleukin-8, plasminogen activator inhibitor 1 (PAI-1), and angiotensinogen than subcutaneous adipocytes. Presumably, distinctive biological properties of visceral fat contribute to the increased pathogenecity of central obesity.
The expression of tumor necrosis factor-alpha (TNF-α) in adipose tissue is increased in human obesity, and TNF-αis proposed as molecular link between obesity and insulin resistance. Alessi MC et al found that TNF-αmRNA in the omental is higher than in the sc adipose tissue. Our results confirm this data and additionally show that difference by the groups of male and female.
Since a strong correlation has previously been demonstrated between TNF-αexpression and adipocytes size, we were interested to determine whether our data on subcutaneous and omental adipocytes confirmed this. We found that subcutaneous fat cells were larger than omental fat cells in obese female. In obese male, there was no significance difference between the two depots fat cell size. While within each depot, there is a positive relationship was found between TNF-αprotein and fat cell size in the two adipose depots in obesity except subcutaneous fat in obese male, a finding consistent with previous reports. Obesity, characterized by adipocytes hypertrophy, is a major cause of insulin resistance, type 2 diabetes, hyperlipidaemia and hypertension, clustering of risk factors for atherosclerosis. Thus, we indicate that hypertrophic adipocytes with increased expressions of adipokines causing insulin resistance, such as TNF-α, are associated with insulin resistance which may be a central mechanism to cause life style-related disease. However, the use of isolated adipose tissue rather than whole adipose tissue controlled for differences in the ratio of adipocytes to adventitial tissue within the individual biopsies.
The key role of TNF-αin the genesis of insulin resistance has been reported in humans and in animal models especially in relation to obesity associated with insulin resistance. In the fatty tissue of obese animals with insulin resistance and type 2 diabetes, the TNF-αconcentration is elevated. Neutralization of TNF-αin vivo dramatically increased the insulin sensitivity of these obese animals, resulting in increased glucose uptake in peripheral tissues. These observations confirmed the role of TNF-αas a key mediator of insulin resistance in obesity. In our study, omental TNF-αprotein concentration was associated with the serum insulin concentrations in obese group. Moreover, it was significantly associated with insulin resistance in obese women, which manifested as correlation of omental TNF-αlevel with HOMA-IR. From our observations it can be concluded that omental TNF-αprotein plays an important role in the development of insulin resistance in obesity. Four large prospective studies have shown that hyperinsulinemia is a predictor of coronary artery disease. The greatest association of hyperinsulinemia with coronary artery disease has been found in Finland in a population with a very high frequency of coronary artery disease. Results of a prospective investigation of 2103 men clearly showed that high fasting insulin concentrations are an independent predictor of cardiovascular diseases. Additionally, increased TNF-αlevel have been associated with high triglycerides concentrations in metabolic syndrome. In the present study we found that in obesity omental TNF-αprotein was not only positively associated with triglycerides, also negatively with HDL-cholesterol levels, which reflected an increased risk of heart disease. More importantly, our data showed that omental TNF-αprotein was significantly associated with plasma PAI-1 levels. To date, TNF-αis known to be effective for stimulation of PAI-1 release in many cell types, especially adipocytes. PAI-1 could disturb normal fibrin clearance mechanisms and promote thrombosis. The elevated omental TNF-αprotein observed during central obesity and the positive correlation observed between omental TNF-αprotein and PAI-1 levels have led us to characterize omental TNF-αprotein as an important factor in the pathophysiology of obesity-related cardiovascular diseases.
In conclusion, these results indicated that TNF-αmay be the link between the cluster of visceral obesity, insulin resistance, and cardiovascular comorbidities. Further studies should identify the molecular mechanisms underlying such events, which may lead to effective therapeutic strategies designed to protect against atherosclerosis in obese patients.
The effect of different concentrations of glucose and TNF-αon Egr-1 expression suggest that glucose and TNF-αall increased the expression of Egr-1, and the two factors additively increased Egr-1 expression. TNF-αactivate the expression of ERK1/2 and Egr-1 in the HUVEC. TNF-αinduced Egr-1 expression through ERK1/2 activation in HUVEC.
Egr-1 was originally identified as an immediate-early gene that is rapidly induced in response to a variety of stimuli, including growth factors, cytokines, hypoxia, physical forces, and injury, all of which are implicated in the progress of vascular diseases. Recent studies demonstrated that Egr-1 expression is elevated in both atherosclerotic lesions after vascular injury. Interestingly, reduction of Egr-1 expression by Egr-1 antisense technology decreased intimal through inhibiting smooth muscle cell migration and proliferation. Taken together, these findings suggest that Egr-1 is the key mediator in orchestrating the functional characteristics of the vessel wall after injury. And TNF-αmay be increase Egr-1 expression in endothelial cells to contribute the development of cardiovascular diseases in obesity.
The importance of TNF-αand the ERK1/2 pathway in the pathogenesis of vascular alterations, however, is well accepted and underscored by recent studies showing reduced vascular lesion formation in TNF-αdeficient mice receiving ERK1/2 antisense treatment. ERK1/2 serves as a key signaling molecule for migration, proliferation and other cellular responses that contribute to atherosclerotic of the arterial wall. Inhibition of the ERK1/2 pathway, therefore, constitutes a logical pharmacological approach for protecting the vasculature from atherosclerotic damage. To date, no selective ERK1/2 inhibitor is clinically available.
Although MEK inhibitors inhibited glucose-induced Egr-1 expression,we ruled out the involvement of ERK1/2, because glucose did not increase TNF-αrelated ERK1/2 activity. Rukhsana reported that glucose induced Egr-1 expression was mediated by PKC activation. MEK inhibitors may inhibiting other signaling pathways, such as PKC pathway, then inhibited glucose-induced Egr-1 expression. The datas suggest that TNF-αand glucose may regulate Egr-1 expression through different ways, with glucose mediating its effects through one of the classical PKC isoforms and TNF-αacting through the ERK1/2 pathway.
Thus, TNF-αmay play a key role in the development of accumulation of visceral adipose tissue, insulin resistance and dyslipidemia. Further studies should identify the molecular mechanisms underlying such events, which may lead to effective therapeutic strategies designed to protect against atherosclerosis in obese patients. Our studies conclude that Egr-1 play an important role in the development of cardiovascular complications, especially in the process of atherosclerosis. TNF-αand glucose-induced the higher expression of Egr-1 maybe the initial event in the diabetes related cardiovascular complications, and the inhibition of the pathway may provide basic datas for the therapy of cardiovascular complications.
Conclusion
1. The higher expression of TNF-αin omental adipose tissue is associated with the development of obesity related cardiovascular diseases in obesity subjects.
2. Omental adipose tissue may through secret TNF-αto stimulate lipolysis, increase free fatty acids, hence accelerates the development of insulin resistance, hyperlipidemia and cardiovascular diseases. TNF-αalso stimulates the expression of PAI-1 in adipocytes and endothelial cells, increases the level of plasma PAI-1, which increases the risk of cardiovascular diseases.
3. TNF-αmay increase the expression of ERK1/2 and Egr-1 in endothelial cells, and TNF-αmay regulate Egr-1 expression through the ERK1/2 pathway.
引文
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