【作者】 刘师伟；

【导师】 赵宝珍；

【作者基本信息】 山西医科大学 ， 内分泌学， 2009， 博士

【摘要】 研究目的胰岛素抵抗是代谢综合征、糖尿病和动脉粥样硬化的共同病理生理改变。糖尿病和代谢综合征的关系是复杂的,肥胖是中心环节。与肥胖有关的脂肪细胞因子通过调节免疫和炎症反应等途径参与胰岛素抵抗及其相关疾病如代谢综合征、2型糖尿病的发生、发展。脂肪细胞因子和胰岛素抵抗关系的确切机制还需深入阐明。Vaspin是一种新识别出来的内脏脂肪细胞因子,具有胰岛素增敏效应,最近的一些研究报道了用重组vaspin干预2型糖尿病鼠后明显改善了胰岛素抵抗和血糖水平,也抑制了一些将会促进胰岛素抵抗进展的基因表达。然而关于人体血清中vaspin怎样被调节以及与肥胖、胰岛素抵抗的关系还不清楚,有待于进一步研究。以NF-кB为网络中心的内皮细胞活化及炎症基因诱导途径参与2型糖尿病发病机制以及血管并发症和胰岛素抵抗相关疾病的发生发展是当今研究的热点。最近有报道,过度活化的NF-кB炎症通路是胰岛素抵抗的可能机制。深入研究NF-кB和胰岛素抵抗及其相关疾病的关系将有着重要的意义。如能早期诊断和预测肥胖个体发生代谢综合征的危险性将对临床治疗有很大的推动作用。通过2型糖尿病人群作为研究靶点,将观察脂联素、vaspin和代谢综合征临床表型各组分(国际糖尿病联盟IDF关于代谢综合征的诊断标准)的关系及脂联素、vaspin、脂联素与vaspin的比值(A/V)是否可以作为评估代谢综合征有用的指标。研究脂肪细胞因子,肥胖,胰岛素抵抗和代谢综合征的相互关系,进一步探索代谢综合征发生的危险性。肥胖,心血管疾病及与之相关的2型糖尿病和代谢综合征与系统性炎症有密切的关系,本次研究中也将深入观察在人类脐静脉内皮细胞中新发现的脂肪细胞因子vaspin将怎样作用血管内皮细胞NF-кB,和怎样影响NF-кB下游因子如细胞间黏附分子ICAM-1、血管细胞黏附分子VCAM-1和单核细胞趋化蛋白MCP-1,探讨vaspin是否通过影响炎症状态的平衡在胰岛素抵抗发生机制中产生重要作用,从而观察vaspin和胰岛素抵抗的关系。研究方法通过260例初诊2型糖尿病患者(126名男性、134名女性)研究脂联素、vaspin、A/V和肥胖、胰岛素抵抗和代谢综合征表型的关系。了解代谢综合征阴性和代谢综合征阳性患者的临床测量及代谢指标的变化。绘制ROC曲线分析脂联素、vaspin在代谢综合征中的诊断价值。以年龄、体重指数、腰围、腰臀比、收缩压、舒张压、胆固醇、甘油三酯、高密度脂蛋白胆固醇、低密度脂蛋白胆固醇、空腹血糖、空腹胰岛素、糖化血红蛋白、稳态模型评估指数(HOMA-R)、脂联素、vaspin、A/V为自变量,是否代谢综合征为应变量,进行多因素Logistic回归分析,观察上述变量对代谢综合征的影响。非参数Spearman相关和多元回归分析观察脂联素、vaspin、A/V和胰岛素抵抗、肥胖、代谢变量的关系。瞬时转染原代脐静脉内皮细胞(HUVEC),以pNF-кB-1uc质粒和内参照β-gal质粒共转染,GFP质粒同时转染。β-gal活力测定为转染效率内参照,以β-gal的活性值校正相应细胞裂解液中NF-κB的化学发光强度,在此基础上建立一套高效率、稳定的脐静脉内皮细胞转染方法。从而准确的通过发光仪测定NF-κB荧光酶活力,即相对发光值(RLU)。pNF-кB-1uc报告质粒稳定转染脐静脉内皮细胞株EA.hy926(脐静脉内皮细胞和上皮癌细胞杂交的体细胞株),因转染用的质粒载体在哺乳动物细胞内表达hph基因,稳定转染用Hygromycin B压力筛选得到稳定表达pNF-кB-1uc质粒的EA.hy926同源细胞克隆。进而测定NF-κB的活力。NF-κB刺激因子—肿瘤坏死因子-α(TNF-α),激活NF-кB报告基因表达荧光素酶,再加入Bay11-7082(一种常用的NF-кB抑制剂,最终抑制依赖于NF-κB的基因转录),这种激活可被Bay11-7082所抑制。利用此方法检测不同浓度(0-3200ng/ml)vaspin是否能够激活NF-кB报告基因表达荧光素酶,及NF-кB的下游靶基因ICAM-1、VCAM-1和MCP-1。并且这种激活是否能被Bay11-7082所抑制。vaspin预培养后TNF-α干预人脐静脉内皮细胞,观察vaspin是否能够影响其它细胞因子对NF-κB激活。实时荧光定量聚合酶链反应(Real time-PCR)和免疫蛋白印迹(Western blot)观察vaspin作用后NF-κB下游因子ICAM-1、VCAM-1及MCP-1的mRNA和蛋白表达情况。同时在NF-кB抑制剂Bay11-7082参与下,观察NF-κB下游基因表达水平。每组实验均执行三次且为了保证实验的一致排除可能的干扰因素,在做干预实验前均换成无血清培养基后准备实验。实验结果运用SPSS16、Sigma Stat统计软件进行分析,数据描述采用均数±标准差,采用方差分析,T-test和LSD法两两比较。部分图表由Sigma plot绘制。研究结果2型糖尿病人群与正常人群相比血清脂联素水平是降低的,血清vaspin呈现升高的趋势(P<0.05),A/V减少(P<0.05)。血清vaspin在女性中蛋白表达水平增高。代谢综合征阳性病人相对于代谢综合征阴性病人有更多的致动脉粥样硬化形成因素,有较高的胰岛素抵抗和升高的Vaspin水平,降低的脂联素水平和降低的A/V。随着代谢综合征组分增多,血清vaspin水平逐渐增高,A/V及血脂联素水平逐步减少,当具备5个代谢综合征组分时vaspin最高,A/V和脂联素水平最低。ROC曲线分析vaspin,A/V对代谢综合征的诊断价值较高。vaspin与体重指数、腰围、腰臀比、甘油三酯、HOMA-IR、高密度脂蛋白胆固醇和空腹胰岛素有明显的相关性(P<0.01),脂联素除了和收缩压、舒张压、空腹血糖无明显相关外,与其它参数都有相关性(P<0.01)(P<0.05),A/V与所有研究参数都有明显的相关性(P<0.01)(P<0.05)。是否代谢综合征为应变量的多元Logistic回归分析中,自变量vaspin的OR:0.469(95%CI,0.252-0.873;P=0.017),A/V的OR:0.368(95%CI,0.171-0.791;P=0.010),脂联素的OR:0.279(95%CI,0.097-0.804;P=0018)。脂联素、vaspin、及它们的比值作为因变量,性别、是否肥胖、是否敏感、腰围、甘油三酯、高密度脂蛋白为自变量的多元回归分析中,当控制其它变量影响后,超重(肥胖)BMI≥25较非肥胖BMI<25的患者有较高的vaspin,和较低的脂联素和A/V。胰岛素敏感(HOMA<2)相对于胰岛素抵抗(HOMA≥2)时,有较低的vaspin水平,较高的脂联素水平和A/V。A/V或HOMA-R为因变量的多元回归分析中,A/V较HOMA-R与更多的研究变量有明显相关性(P<0.05)。A/V为因变量,性别、BMI、WC、TG、HDL为自变量的模型(校正的R2=0.692)有更好的拟和效果。经pNF-κB-Luc报告质粒稳定转染的EA.hy926细胞珠,0-3200 ng/ml的vaspin作用2小时,Vaspin激活了NF-κB的转录活性(P<0.05)。效应呈剂量依赖性(不同剂量之间的刺激活性相比有统计学意义即高浓度组较低浓度组刺激活性增高P<0.05)。同样观察到vaspin呈剂量依赖性激活了原代HUVEC中NF-κB转录活性(P<0.05)。0-3200ng/ml的vaspin预培养由pNF-κB-Luc报告质粒稳定转染EA.hy926细胞系不同的时间段,之后用10ng/ml TNFα作用2小时,TNFα介导的NF-κB转录活性被vaspin明显的抑制(P<0.05),呈剂量依赖性的抑制(P<0.05),预培养延长到24小时时,抑制TNFα介导NF-κB转录活性最强。0-3200ng/ml的vaspin作用人脐静脉内皮细胞株后,黏附分子ICAM-1,VCAM-1和趋化因子MCP-1的基因水平逐渐增强;先加入10μM Bay11-7082 1小时后再加入3200ng/ml的vaspin 4小时,ICAM-1,VCAM-1,MCP-1增高的mRNA被明显抑制,相比3200ng/ml的vaspin作用EA.hy926细胞珠,差异有统计学意义(p<0.05)。不同浓度vaspin作用EA.hy926细胞株和原代HUVEC,ICAM-1、VCAM-1和MCP-1的蛋白表达随着vaspin浓度的递增依次增高。10μM的Bay11-7082预培养后,Bay11-7082明显降低了TNFα介导的黏附分子ICAM-1,VCAM-1和趋化因子MCP-1的蛋白表达,也明显降低了vaspin介导的ICAM-1,VCAM-1和MCP-1蛋白表达。结论该研究从脂联素和vaspin与胰岛素抵抗和2型糖尿病患者代谢综合征临床表型的关系和vaspin对脐静脉内皮细胞NF-κB及下游基因影响两方面探讨了脂肪细胞因子在胰岛素抵抗中的可能作用。研究结果揭示脂联素、vaspin及脂联素与vaspin的比值(A/V)和肥胖、胰岛素抵抗密切相关,也与代谢综合征有紧密的联系,vaspin参与了胰岛素抵抗的发生;在此基础上采用瞬时转染、稳定转染、RT-PCR和Western blot等手段,从分子生物学层面观察并阐明vaspin在原代脐静脉内皮细胞和EA.hy926细胞珠体外培养过程中激活了NF-κB及其下游炎性因子,提示脂肪细胞因子是调节炎症反应的新靶点,vaspin激活NF-κB在胰岛素抵抗中发挥重要作用;同时研究也观察到vaspin抑制了TNF-α介导的NF-κB活性,显示其作用的复杂性,为今后进一步的理论和临床实践提供了新的思路。更多还原

【Abstract】 Objective:Insulin resistance is a common pathophysiological condition of type 2 diabetes, metabolic syndrome and atherosclerosis. There are numerous mechanisms by which obesity can adversely affect the vasculature changes in diabetes and metabolic syndrome. White adipose tissue, the main energy store of the body, is also a source of factors that modulate the immune/inflammatory response and promote atherosclerosis, vascular dysfunction, insulin resistance, metabolic syndrome and type 2 diabetes. Therefore, investigations focused on the identification of adipose tissue derived molecules will provide further understanding of mechanisms for insulin resistance.Vaspin, a serine protease inhibitor derived from visceral adipose tissue, has been identified as an adipokine with insulin-sensitizing effects in a rat model of type 2 diabetes. The study by Hida et al. showed that the administration of recombinant human vaspin significantly improved insulin sensitivity and glucose tolerance, and reversed the expression of genes that may promote insulin resistance in diet-induced obese mice. However, the regulation of vaspin serum concentrations in human insulin resistance, and relationship between vaspin and insulin resistance related disorders is unclear. The present study focused on the role of vaspin in obesity-associated disorders.NF-κB is a major transcription factor in inflammatory responses, regulating a plethora of genes, activating endothelial cells, playing a vital role in the initiation, progression of type 2 diabetes, vessel complication and insulin resistance related disorders. Recent research has suggested that over-activation of NF-κB may be a mechanism of insulin resistance. It is important to investigate the relationship between NF-κB and diseases associated with insulin resistance.We postulated that adipokines, such as adiponectin and vaspin, can be included in the criteria used for the identification of subjects with metabolic syndrome. Therefore, our aim was to evaluate the determinants and associations of vaspin and adiponectin in relation with the number of clinical and metabolic abnormalities constituting the metabolic syndrome as defined by the International Diabetes Federal (IDF) diagnostic criteria. We also evaluated the performance characteristics of adiponectin, vaspin and the adiponectin/vaspin ratio (A/V) as markers for the identification of patients with the metabolic syndrome. For further exploring the risk of metabolic syndrome, the study was performed to evaluate the associations of adiponectin, vaspin, and their ratio (adiponectin/vaspin) and obesity, insulin resistance, and the metabolic syndrome in patients with type 2 diabetes.Adipocytes within adipose tissue directly augment systemic inflammation. Increased systemic inflammation is associated with higher prevalence of obesity, insulin resistance, type 2 diabetes, metabolic syndrome, and the risk of cardiovascular disease. With the aforementioned in mind, we sought to investigate whether Vaspin activates NF-κB inducing the intercellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1), and monocyte chemoattractant protein (MCP-1) in the vascular endothelium. Vaspin could affect the equilibration inflammatory state, which in turn may have a significant effect in the insulin resistance.Research Design and Methods:260 patients including 126 male and 134 female with newly diagnosed type 2 diabetes were recruited in this study. Patients were classified based on the degree of adiposity, insulin resistance (IR) (homeostasis model assessment of insulin resistance (HOMA-IR)) and the number of IDF criteria of the metabolic syndrome. The relationship between adiponectin, vaspin, A/V and the metabolic syndrome,degree of adiposity, IR, and the correlation with metabolic variables were investigated.Spearman’s correlation coefficients (r) and multiple regression analysis were used to describe the association between serum vaspin, adiponectin or A/V and other continuous variables of interest. Multiple regression analysis was used to analyze the relationship between vaspin, adiponectin, A/V and age, gender, BMI (Body mass index), HOMA-R, WC (Waist circle), TG (Triglyceride), HDL (High-density lipoprotein-cholesterol). Logistic regression analysis was used to ascertain the association of vaspin and adiponectin with the metabolic syndrome as dependent variable. Potential confounding variables such as BMI, WC, WHR (Waist-to-hip ratio), TC, TG, HDL, LDL, FPG (Fasting plasma glucose), FIRI (Fasting plasma insulin level), HbA1c (GlycohemoglobinA1c), HOMA-IR and A/V ratio were included. To assess the diagnostic accuracy of the adiponectin, vaspin, A/V ratio in predicting the metabolic syndrome, receiver operating characteristic (ROC) curve was also constructed.To study NF-κB activation, HUVEC cells were transient transfected with a cis-reporter plasmid (Promega, E849A, #25236701). The pNF-κB-Luc plasmid was transfected together withβ-gal plasmid (Aldevron, #5002) into HUVEC cells using a QIAGEN transfection reagent, GFP plasmid was transfected simultaneously. Intra-reference transfection efficiency was measured byβ-gal activity, which may revise NF-κB chemistry luminous intensity in corresponding cell disruption. With the aforementioned in mind, a set of high efficiency and stable transfection method was established in HUVEC cells. The relative light unit (RLU) of NF-κB was measured using Promega GLOMAX Multi Detection System. Since most Hygromycin-based vectors used in gene transfer experiments harbor the E. coli Hygromycin B phosphotransferase (Hph) gene in mammals, cells were cultured in the presence of Hygromycin B (Catalog # ant-hg-5,InvovoGen) at a concentration of 200μg/ml and the medium was replaced every two to three days. Approximately three weeks after transfection, Hygromycin B-resistant clones were isolated using a cloning cylinder and analyzed individually for the expression of luciferase activity. We obtained the EA.hy926 cell clones with stable expression of pNF-кB-1uc. Multiple clones were also selected for analysis of NF-κB activation. Luciferase activity was measured using a luciferase assay kit (Luciferase Assay system, Promega, # E1501).Tumor necrosis factor (TNF-α), a strong inducer of NF-κB activity, activates cis-reporter plasmid containing luciferase reporter gene linked to five repeats of NF-κB binding sites. Addition of Bay11-7082, an irreversible inhibitor, results in the inactivation of NF-κB and NF-κB-dependent transcriptional expression.EA.hy926 cells or HUVEC cells transfected with pNF-κB-Luc were treated with various concentrations (0-3200 ng/ml) of Vaspin, After two hours, cells were harvested and lysed, and luciferase activity was measured based on NF-κB-dependent transcriptional activity as mentioned above. The effects of Vaspin on ICAM-1,VCAM-1 and MCP-1 activity was performed in EA.hy926 cells or HUVEC cells. In addition, cells pre-incubated with Vaspin were then treated with TNF-α(10 ng/ml), the effect of Vaspin on TNF-αinduced NF-κB activation was then evaluated using the same method. The expression of ICAM-1, VCAM-1 and MCP-1 were investigated using quantitative real time PCR (mRNA level), western blot analysis (protein level) following treatment with Vaspin and also the NF-κB inhibitor, Bay 11-7082.All data in the present study are expressed as mean±SD. Differences between two groups were assessed using the t-test. Comparisons among groups were made by ANOVA and LSD. A p<0.05 was considered statistically significant. All statistical analysis was performed using SPSS 16 and Sigma plot. Serum-starved HUVECs transfected with pNF-κB-Luciferase were treated with or without vaspin. Each experiment was carried out in triplicates. Results:Compared to healthy adult, the patients with type 2 diabetes had lower adiponectin (P<0.05) and A/V (P<0.05), but higher Vaspin level (P<0.05). Serum Vaspin was elevated in females (P<0.05). In general, patients who were metabolic syndrome positive had more atherogenic lipid profile, exhibited higher insulin resistant, lower adiponectin and A/V, and had higher Vaspin level than patients who were negative of metabolic syndrome.Adiponectin and A/V ratio showed stepwise decrease with increasing number of the criteria for diagnosis of the metabolic syndrome, while Vaspin showed the opposite trend. Vaspin was the highest while five constituents of the metabolic syndrome were present. Receiver operating characteristic analysis showed that vaspin and A/V ratio had significantly larger area under the curve (AUC) compared with adiponectin, TG and HDL for the detection of the metabolic syndrome, suggesting the importance of Vaspin and A/V in the diagnosis of this syndrome. Vaspin, A/V and adiponectin were shown to be protector factors of the metabolic syndrome by multiple logistic regression analysis. The odds ratios (OR) are 0.469(95%CI,0.252-0.873;P=0.017), 0.368(95%CI,0.171-0.791;P=0.010), and 0.279(95%CI,0.097-0.804;P=0018)respectively.Vaspin levels were significantly correlated with BMI, WC, WHR, TG, HDL, FIRI, and HOMA-IR (P<0.01). Adiponectin levels were significantly correlated with other parameters in an opposite manner except for SBP, DBP, and FBG (P<0.01) (P<0.05). On the other hand, A/V ratio was significantly correlated with these parameters (P<0.01) (P<0.05). Multiple regression analysis was used to analyze the relationship between vaspin, adiponectin, A/V and gender, BMI, HOMA-R, WC, TG, HDL. Overweight obese patients had significantly lower adiponectin, A/V ratio levels and higher vaspin comparing with non-obese patients. Insulin-sensitive patients had significantly higher adiponectin, A/V ratio levels and lower vaspin than those with IR when adjusted by BMI, WC and other variable.The association between A/V ratio or HOMA-R and objective variables were investigated by multiple regression analysis. Significant correlations between these parameters and A/V ratio were observed (P<0.05). Furthermore, multiple linear regression analysis with A/V ratio or HOMA-R as dependent variables and gender, BMI, WC, TG and HDL as independent variables revealed that the A/V ratio (R2=0.692) was higher than HOMA-R (R2=0.433). This model suggested that A/V ratio might be a more accurate surrogate index for determining insulin resistance in patients with type 2 diabetes.Vaspin (0-3200 ng/ml) treatment for 2 hours induced a significant dose dependent increase in NF-κB mediated transcriptional activity in pNF-κB-Luc transfected EA.hy926 cells (P<0.05). Similar results were obtained in transiently transfected HUVEC (P<0.05). When cells were pretreated with Vaspin (0-3200 ng/ml) for varies time points and then subjected to TNF-α(10ng/ml) treatment for 2 hours. A significant inhibition of TNF-αinduced NF-κB transcriptional activity in human ECs was observed (P<0.05), a greater inhibition was observed in cells with a longer period of incubation with Vaspin (24 hours).Vaspin significantly increased ICAM-1, VCAM-1 and MCP-1 mRNA expression and protein levels in HUVEC cells. Pretreatment of HUVEC cells with Bay 11-7082 (10μM), a NF-κB inhibitor, for 1 hour, the mRNA levels of ICAM-1, VCAM-1 and MCP-1 induced by Vaspin (3200 ng/ml, 4 hour) was significantly reduced (P<0.05). Similarly, in Vaspin/TNF-αtreated human ECs, ICAM-1 and VCAM-1 and MCP-1 protein levels were also significantly decreased by pre-incubation with Bay 11-7082 (10μM).Conclusions:The present study investigated the relationship between adipokines and insulin resistance by examining the Adiponectin, Vaspin, insulin resistance and clinical manifestations of the metabolic syndrome in patients with Type 2 diabetes, and the effects of Vaspin on NF-κB activation and its downstream gene expression in Human vascular endothelial cells. The results revealed that adiponectin, vaspin, and A/V ratio were associated with obesity, insulin resistance, and the clinical manifestations of the metabolic syndrome. Vaspin participated in the process of insulin resistance. Further investigation using human ECs endothelial cells transient or stable transfected with pNF-κB-Luc plasmid revealed that Vaspin increased NF-κB transcriptional activity, significantly enhanced ICAM-1, VCAM-1 and MCP-1 mRNA expression and protein levels. We reaffirmed the emerging role of adipokines as mediators of inflammatory responses. Vaspin activated NF-κB, which may play an important role in developing insulin resistance. At the same time we observed that Vaspin induced NF-κB activation that led to the suppression of cytokine-induced NF-κB activation in Human vascular endothelial cells. Therefore, the complexity of vaspin offers the advantage of further exploring insulin resistance, and in consequence might benefit the treatment specifically targeting insulin resistance.更多还原