脂肪因子VASPIN血清水平、表达调控及功能研究
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摘要
研究背景：
近年研究表明,脂肪组织不仅仅是一个能量储存器官,也是一个重要的内分泌器官,可分泌多种细胞因子,包括瘦素、抵抗素、脂联素、内脂素、肿瘤坏死因子、纤溶酶原激活物抑制因子和白介素-6等。新发现的脂肪因子正在不断被报道。脂肪因子的发现使得脂肪组织已由过去单纯作为能量储存的器官成为一个极其重要的内分泌系统。脂肪因子的表达或功能紊乱参与肥胖、胰岛素抵抗、糖尿病及动脉粥样硬化等多种代谢性疾病的发生发展过程。
内脏脂肪聚集是腹型肥胖的重要特征。与皮下脂肪组织相比,内脏脂肪组织具有更活跃的内分泌功能,其分泌的脂肪因子直接影响胰岛素敏感性,损伤内皮细胞功能,参与炎症反应及纤溶活动,与胰岛素抵抗、2型糖尿病及心血管疾病密切相关。因此深入研究内脏脂肪因子的功能及调控有助于揭示肥胖及相关代谢异常的发生机制,为代谢性疾病的诊断和治疗提供新的靶点。
腹腔脂肪型丝氨酸蛋白酶抑制剂(Vaspin)是新发现的一种内脏脂肪因子,由腹腔脂肪组织合成和分泌,属于丝氨酸蛋白酶抑制剂超家族,研究显示该蛋白具有增加胰岛素敏感性,调节脂代谢,参与体内炎症反应等多种生物学活性,是代谢综合征治疗的新靶点。尽管目前研究表明Vaspin水平变化可能是代谢紊乱性疾病病情变化的敏感指标,但研究结果仍存在争议,肥胖和2型糖尿病患者血清Vaspin水平变化及影响因素尚不清楚,其变化与糖尿病大血管并发症的发生发展的关系及脂肪细胞Vaspin表达调控的分子机制有待于进一步研究。因此本课题主要研究血清Vaspin水平与2型糖尿病及其并发症的关系,并初步探讨脂肪细胞Vaspin表达调控的影响因素及其潜在生理功能,从而为肥胖及相关代谢性疾病提供新的分子机制,为代谢综合征的治疗干预奠定理论基础。
第一部分
血清VASPIN水平与2型糖尿病患者颈动脉粥样硬化的关系研究
目的：应用ELISA方法检测2型糖尿病(T2DM)患者血清vaspin水平,探讨：(1)血清vaspin水平与2型糖尿病合并早期颈动脉粥样硬化的关系。(2)血清vaspin水平与空腹血糖(FPG)、餐后2h血糖(2HPG)、糖化血红蛋白(HbAlc)血脂及胰岛素抵抗等代谢因素的关系。
方法：
1.病例选择选择2007年1月至2009年8月3日自愿参加山东大学齐鲁医院内分泌科“2型糖尿病大血管并发症干预措施与控制模式研究”项目的2型糖尿病患者61例,根据有无颈动脉斑块分为糖尿病非斑块组(男性15例,女性13例)和糖尿病合并斑块组(男性18例,女性15例),另外选择年龄、性别相匹配的健康查体者为对照组(男性15例,女性11例)。
2.标本留取所有研究对象均进行人体测量,体格检查及颈动脉超声检查。空腹12h后次晨抽取抗凝血3ml用于HbAlc等测定,同时抽取自凝血5ml,分离血清用于生化指标及vaspin水平的测定。
3.血清vaspin水平的测定采用ELISA方法测定血清vaspin水平。
结果：
校正年龄、性别后,T2DM非斑块组的血清vaspin水平显著高于对照组(0.65±0.24vs 0.46±0.12 ng/ml, p< 0.01),而T2DM斑块组血清vaspin水平低于非斑块组(0.52±0.23 vs 0.65±0.24 ng/ml, p<0.05)。在对所有入选者进行的简单相关分析中,调整年龄、性别及体质指数(BMI)后偏相关分析,血清vaspin与腰围(WC)呈正相关(r=0.269,p=0.041),与胰岛素抵抗指数(HOMA-IR)负相关(r=-0.262,p=0.047)。糖尿病患者的相关分析表明,vaspin水平与WC正相关(r=0.371,p=0.003),与HOMA-IR和低密度脂蛋白(LDL)负相关(r=-0.272,p=0.034；r=-0.254,p=0.048)。以糖尿病组为整体,行Logistic回归分析,结果显示收缩压(SBP)和Vaspin水平影响糖尿病合并颈动脉硬化的发生,提示血清Vaspin水平下降可能是2型糖尿病动脉粥样硬化早期发生的标志物。
结论：血清vaspin水平可能参与胰岛素抵抗和糖尿病的发生,在以胰岛素抵抗为特征的代谢综合征的发病中发挥重要作用。同时血清Vaspin水平下降可能是2型糖尿病动脉粥样硬化早期发生的标志物,尽早检测血清Vaspin水平,可以预测糖尿病动脉粥样硬化等大血管并发症的发生与发展。
第二部分
血清VASPIN水平与2型糖尿病视网膜病变的关系研究
目的：应用ELISA方法检测2型糖尿病患者血清vaspin水平,探讨：(1)血清vaspin水平与2型糖尿病视网膜病变及其严重程度的关系。(2)血清vaspin水平与年龄、病程、FPG.2HPG.HbAlc、血脂、尿白蛋白排泄率(UAER)及其它脂肪因子的关系。
方法：
1.病例选择选择2009年2月至2009年6月间山东大学齐鲁医院内分泌科门诊和住院的2型糖尿病患者94例,根据有无视网膜病变及严重程度,分为无视网膜病变组(40例)、单纯型糖尿病视网膜病变组(38例)和增殖型糖尿病视网膜病变组(16例)。
2.标本留取所有对象均进行人体测量及体格检查。所有个体空腹12h后次晨抽静脉血,进行生化指标的测定。采取静脉血,分离血清后-80℃保存,集中测定血清vaspin和视黄醇结合蛋白4(RBP4)水平。
3.血清vaspin和RBP4水平的测定采用ELISA方法测定血清vaspin和RBP4水平。
结果：
在糖尿病患者中,调整年龄和病程后,单纯性视网膜病变的患者vaspin血清水平显著低于无视网膜病变患者(0.45±0.13 versus 0.53±0.22 ng/ml,p=0.023),有增殖性视网膜病变的患者vaspin血清水平较无视网膜病变患者更低(0.36±0.06versus 0.53±0.22 ng/ml,p=0.001),增殖性视网膜病变患者vaspin血清水平低于单纯性视网膜患者,但无显著性。简单相关分析示血清vaspin与年龄、病程、UAER及RBP4呈负相关(r=-0.259,p=0.012；r=-0.277,p=0.007；r=-0.322, p=0.002；r=-0.234,p=0.023).偏相关分析显示控制年龄和BMI后,vaspin与UAER的相关性仍然存在(r=-0.281,p=0.007).多元逐步回归分析表明年龄、UAER是血清vaspin水平的独立影响因素。
结论：2型糖尿病患者血清vaspin水平与年龄、UAER呈明显相关。血清vaspin水平在2型糖尿病视网膜病变,尤其是合并增殖性视网膜病变者中显著降低,提示血清vaspin水平与糖尿病视网膜病变有关,可能影响糖尿病微血管病变的发生发展。
第三部分3T3-L1脂肪细胞VASPIN表达的影响因素研究
目的：
Vaspin作为一种新发现的具有胰岛素增敏作用的脂肪因子,主要由内脏脂肪组织分泌。但是参与调节vaspin表达的因素尚未完全清楚。本研究旨在探讨3T3-L1脂肪细胞分化过程中vaspin表达的变化,并研究在不同代谢因素下vaspin表达的调节机制及影响因素。
方法：
1.体外培养3T3-L1脂肪前体细胞并诱导分化为成熟脂肪细胞,分别于85%融合(-4d)、融合(-2d)、融合后2天(0d)、5天(3d)、8天(6d)、10天(8d)提取细胞总RNA。用RT-PCR方法检测vaspin mRNA的表达,ELISA检测细胞上清液vaspin水平。
2.分别以不同浓度地塞米松(1,100μmol/L)、胰岛素(10,100nmol/L)、葡萄糖(10,25mmol/L)、棕榈酸(100,1000μmol/L)、TNF-a(10,20ng/ml)、吡格列酮(10,100μmol/L)、非诺贝特(10,100μmol/L)和硫辛酸(50,100,500,1000μmol/L)干预分化成熟的3T3-L1细胞24h,留取细胞上清液,提取细胞总RNA,RT-PCR检测vaspin mRNA的表达水平。
3.以对照组、TNF-α组(20ng/ml TNF-α), TNF-α(20ng/ml)+吡格列酮(10μmol/L),和TNF-a(20ng/ml)+吡格列酮(100μmol/L),分别培养48h,留取细胞上清液,收集脂肪细胞,提取总RNA,用RT-PCR检测vaspin mRNA的表达；以对照组、TNF-α组(20ng/ml)、TNF-α(20ng/ml)+非诺贝特(10μmol/L)、和TNF-a(20ng/ml)+非诺贝特(100μmol/L),分别培养48h,留取细胞上清液,收集脂肪细胞,提取总RNA并测定vaspin mRNA的表达；以对照组、TNF-α组(20ng/ml)、TNF-α(20ng/ml)+硫辛酸(100μmol/L)、和TNF-α(20ng/ml)+硫辛酸(500μmol/L),分别培养48h,留取细胞上清液,收集脂肪细胞,提取总RNA并测定vaspin mRNA的表达。
结果：
在3T3-L1脂肪前体细胞分化过程中,vaspin mRNA表达明显增加并呈时间依赖性。在80%融合到融合后两天,vaspin表达增加到起始的1.7倍。至分化后第8天增加至起始6倍。地塞米松(100μM,1.6倍),高糖(25mM,6.6倍),吡格列酮(100μM,1.6倍)、非诺贝特(100μM,1.5倍)和硫辛酸(500μM,1.6倍)能显著诱导3T3-L1脂肪细胞中vaspin mRNA的表达。而其它处理因素10-100nM胰岛素,1mM棕榈酸,10-20 ng/ml TNF-a则对其1mRNA表达有下调作用,较基线值分别下降(37-53)%,48%和(15-40)%。同时吡格列酮和硫辛酸可逆转TNF-a所致的vaspin mRNA表达的降低。
同时在3T3-L1脂肪细胞诱导分化中,vaspin蛋白分泌亦逐渐增加,并呈时间依赖性。开始后的前5天vaspin蛋白水平增加迅速,6天以后增加明显变慢。地塞米松、葡萄糖显著增加vaspin的分泌,胰岛素、TNF-a明显降低vaspin的分泌,而棕榈酸无明显影响。吡格列酮和硫辛酸明显改善TNF-a所致的vaspin分泌的降低。
结论：在脂肪细胞分化过程中vaspin表达水平与细胞分化程度明显相关,可能参与脂肪前体细胞向成熟脂肪细胞的分化。同时成熟脂肪细胞vaspin的分泌受多种激素和代谢因素的调节,可能与代谢紊乱的严重程度有关。但其调控的确切机制有待进一步阐明。
Background:
Recently, increasing evidence have suggested that adipose tissue is not only an energy storage organ, but also a key endocrine organ which could secrete various adipokines such as leptin, resistin, adiponectin, visfatin, tumor necrosis factor, plasminogen activator inhibitor, and interleukin-6,etc. Epidemiological and animal studies have shown that visceral obesity is associated with increased prevalence of insulin resistance, type 2 diabetes and cardiovascular diseases. Adipokines secreted by visceral fat tissues play an important role in the occurrence and development of insulin resistance, type 2 diabetes and metabolism syndrome. Vaspin, a newly identified adipokine, is secreted by visceral adipose tissue with predominantly insulin-sensitizing effects,However, its physiological role is largely unknown. Although current studies have shown plasma vaspin level has direct effects on obesity, the result is still in debate. In the present study, we investigated 1) the relationship between serum vaspin level and macrovascular and retinopathy complications in diabetic patients,2) the regulatory mechanism of vaspin expression,3) the potential physiological function of vaspin. We first examined whether serum vaspin levels are associated with the presence of carotid plaque in early stage of type 2 diabetes. Next, we analysed the relationship between serum vaspin levels and diabetic retinopathy. Finally, we explored in vitro the effects of high glucose, insulin, dexamethasone, palmitate, TNF-a, pioglitazone, fenofibrate and lipoic acid on vaspin expression in 3T3-L1 cell and investigated the potential molecular mechanism involved.
PartⅠ
SERUM VASPIN LEVELS IN TYPE 2 DIABETIC PATIENTS WITH CAROTID PLAQUE
Objective:1) To explore whether serum vaspin levels are associated with the presence of carotid plaque in early stage of type 2 diabetes; 2) To explore the association between serum vaspin levels and FPG,2HPG, HbAlc, serum lipid and insulin resistance.
Materials and Methods:Sixty-one type 2 diabetic patients within 1 years's diabetes duration were divided into two groups by the presence or absence of carotid plaque. Twenty-six age-matched apparently healthy controls were recruited as well. Serum vaspin was measured by enzyme-linked immunosorbent assay.
Results:After controlling age and gender, fasting serum vaspin levels were significantly higher in the diabetic patients without carotid plaque than controls (0.65±0.24 vs.0.46±0.12 ng/ml, p<0.01). However, circulating vaspin level was much lower in the diabetic subjects with carotid plaque than those without (0.52±0.23 vs. 0.65±0.24 ng/ml, p<0.05).Partial correlations demonstrated that vaspin level was positively correlated with waist circumference (WC) (r=0.269, p=0.041), negatively correlated with HOMA-IR(r=-0.262, p=0.047) in all subjects after controlling age, gender and BMI. Logistic regression analysis showed that SBP and fasting serum vaspin levels were significantly associated with the presence of carotid plaque in type 2 diabetes.
Conclusion:Serum vaspin levels were significantly elevated in type 2 diabetic patients while its level was negatively associated with the presence of carotid plaque in type 2 diabetic patients,indicating that vaspin might play a compensative role in the early course of diabetes while exert protective effects on atherosclerosis in early stage of diabetes.
Part II
VASPIN SERUM CONCENTRATIONS IN TYPE 2 DIABETIC PATIENTS WITH DIABETIC RETINOPATHY
Objective:1) To explore the relationship between serum vaspin level and type 2 diabetic retinopathy and its severity.2) To explore the association between vaspin level and age, duration, FPG,2HPG, HbAlc, serum lipid, urine albumin excretion rate (UAER) and other adipokines
Materials and Methods:94 patients with type 2 diabetes were recruited through in-patients or clinic service from February to June 2009 at Qilu Hospital of Shandong University, Jinan, China. Based on ophthalmologic examination,94 patients with type 2 diabetes were divided into three subgroups:group 1 without diabetic retinopathy (NDR) (n=40), group 2 with simple diabetic retinopathy (SDR) (n=38) and group 3 with proliferative diabetic retinopathy (PDR) (n=16). Serum vaspin was determined by enzyme-linked immunosorbent assay.
Results:In the diabetic patients, subjects with simple diabetic retinopathy had significantly lower vaspin levels than subjects without diabetic retinopathy (0.45+ 0.13 versus 0.53+0.22 ng/ml, p= 0.023).Moreover, subjects with proliferative diabetic retinopathy had much lower vaspin levels as compared with subjects with simple diabetic retinopathy. Age, disease duration, UAER and retinol binding protein-4 (RBP4) were negatively correlated with vaspin level in correlation analysis (r=-0.259, p= 0.012; r=-0.277, p= 0.007; r=-0.322, p=0.002; r=-0.234, p=0.023 respectively). Partial correlations demonstrated that UAER was still negatively correlated with vaspin levels (r=-0.281, p= 0.007) after controlling age and BMI, A stepwise linear multiple regression model revealed that age and UAER were significant determinants of serum vaspin levels.
Conclusion:Tanken together, these results suggest that the reduction of vaspin level may be involved in the process of diabetic retinopathy and thereby provides a novel biomarker for evaluating its severity in diabetic patients.
PartⅢ
STUDY ON THE REGULATORY MECHANISM OF VASPIN EXPRESSION IN 3T3-L1 CELL
Objective:To explore the effect of metabolic factors on vaspin expression in 3T3-L1 cell and the regulatory mechanism involved.
Methods:
1.3T3-L1 preadipocytes were cultured in DMEM medium supplementd with 10% FBS and differentiated into adipocytes. The total RNA was extracted in the preconfluent, confluent and 2-day,5-day,8-day,10-day confluent cells respectively. The expression of vaspin mRNA was determined by quantitative real-time polymerase chain reaction and vaspin level in cell supernatant was determined by ELISA.
2. Mature 3T3-L1 adipocytes was treated with indicated concentrations of dexamethasone, insulin, glucose, palmitate, TNF-a, pioglitazone, fenofibrate and lipoic acid for 24 or 48 h.The cells were starved for 6 h in DMEM with 0.5% bovine serum albumin (BSA) before the study.
3. TNF-a was added to the cell culture medium alone or with the indicated "concentrations of pioglitazone, fenofibrate or lipoic acid respectively for 48 h. During the treatment period, fresh TNF-a, pioglitazone, fenofibrate or lipoic acid was added every 24 h.
Results:A marked increase in vaspin expression was observed during 3T3-L1 differentiation, with a 1.7-fold increase on 2-day confluent as compared with preconfluent. A further 3.8-fold increase was induced from day 0 to day 8 of differentiation (overall six fold). Overnight incubation with dexamethasone increased vaspin expression in adipocytes (100μM-1.6 fold). High glucose increased vaspin secretion by 6.6-fold at 25 mM, pioglitazone by 1.6-fold at 100μM, fenofibrate by 1.5-fold at 100μM, and lipoic acid by 1.6 fold at 500μM. While 10-100 nM insulin, 1mM palmitate and 10-20 ng/ml TNF-a decreased vaspin expression (.37-53%,48%, 15-40% respectively). Futhermore, pioglitazone and lipoic acid reversed the reduction of vaspin by TNF-α.
Vaspin protein level in cell supernatant increased gradually during differentiation, rapidly in beginning 5 days after differentiation and slowly after 6 days. Dexamethasone and glucose promoted vaspin secretion in mature adipocytes while insulin and TNF-a inhibited vaspin level. Pioglitazone, fenofibrate and lipoic acid reversed the reduction of vaspin induced by TNF-a.
Conclusion:Taken together, vaspin levels are associated with the extent of the differentiation and its level is regulated by multiple hormones and metabolic factors which could be associated with severity of metabolic disorders. The underlying mechanism of vaspin action remains to be elucidated futher.
近年研究表明,脂肪组织不仅仅是一个能量储存器官,也是一个重要的内分泌器官,可分泌多种细胞因子,包括瘦素、抵抗素、脂联素、内脂素、肿瘤坏死因子、纤溶酶原激活物抑制因子和白介素-6等。新发现的脂肪因子正在不断被报道。脂肪因子的发现使得脂肪组织已由过去单纯作为能量储存的器官成为一个极其重要的内分泌系统。脂肪因子的表达或功能紊乱参与肥胖、胰岛素抵抗、糖尿病及动脉粥样硬化等多种代谢性疾病的发生发展过程。
内脏脂肪聚集是腹型肥胖的重要特征。与皮下脂肪组织相比,内脏脂肪组织具有更活跃的内分泌功能,其分泌的脂肪因子直接影响胰岛素敏感性,损伤内皮细胞功能,参与炎症反应及纤溶活动,与胰岛素抵抗、2型糖尿病及心血管疾病密切相关。因此深入研究内脏脂肪因子的功能及调控有助于揭示肥胖及相关代谢异常的发生机制,为代谢性疾病的诊断和治疗提供新的靶点。
腹腔脂肪型丝氨酸蛋白酶抑制剂(Vaspin)是新发现的一种内脏脂肪因子,由腹腔脂肪组织合成和分泌,属于丝氨酸蛋白酶抑制剂超家族,研究显示该蛋白具有增加胰岛素敏感性,调节脂代谢,参与体内炎症反应等多种生物学活性,是代谢综合征治疗的新靶点。尽管目前研究表明Vaspin水平变化可能是代谢紊乱性疾病病情变化的敏感指标,但研究结果仍存在争议,肥胖和2型糖尿病患者血清Vaspin水平变化及影响因素尚不清楚,其变化与糖尿病大血管并发症的发生发展的关系及脂肪细胞Vaspin表达调控的分子机制有待于进一步研究。因此本课题主要研究血清Vaspin水平与2型糖尿病及其并发症的关系,并初步探讨脂肪细胞Vaspin表达调控的影响因素及其潜在生理功能,从而为肥胖及相关代谢性疾病提供新的分子机制,为代谢综合征的治疗干预奠定理论基础。
第一部分
血清VASPIN水平与2型糖尿病患者颈动脉粥样硬化的关系研究
目的：应用ELISA方法检测2型糖尿病(T2DM)患者血清vaspin水平,探讨：(1)血清vaspin水平与2型糖尿病合并早期颈动脉粥样硬化的关系。(2)血清vaspin水平与空腹血糖(FPG)、餐后2h血糖(2HPG)、糖化血红蛋白(HbAlc)血脂及胰岛素抵抗等代谢因素的关系。
方法：
1.病例选择选择2007年1月至2009年8月3日自愿参加山东大学齐鲁医院内分泌科“2型糖尿病大血管并发症干预措施与控制模式研究”项目的2型糖尿病患者61例,根据有无颈动脉斑块分为糖尿病非斑块组(男性15例,女性13例)和糖尿病合并斑块组(男性18例,女性15例),另外选择年龄、性别相匹配的健康查体者为对照组(男性15例,女性11例)。
2.标本留取所有研究对象均进行人体测量,体格检查及颈动脉超声检查。空腹12h后次晨抽取抗凝血3ml用于HbAlc等测定,同时抽取自凝血5ml,分离血清用于生化指标及vaspin水平的测定。
3.血清vaspin水平的测定采用ELISA方法测定血清vaspin水平。
结果：
校正年龄、性别后,T2DM非斑块组的血清vaspin水平显著高于对照组(0.65±0.24vs 0.46±0.12 ng/ml, p< 0.01),而T2DM斑块组血清vaspin水平低于非斑块组(0.52±0.23 vs 0.65±0.24 ng/ml, p<0.05)。在对所有入选者进行的简单相关分析中,调整年龄、性别及体质指数(BMI)后偏相关分析,血清vaspin与腰围(WC)呈正相关(r=0.269,p=0.041),与胰岛素抵抗指数(HOMA-IR)负相关(r=-0.262,p=0.047)。糖尿病患者的相关分析表明,vaspin水平与WC正相关(r=0.371,p=0.003),与HOMA-IR和低密度脂蛋白(LDL)负相关(r=-0.272,p=0.034；r=-0.254,p=0.048)。以糖尿病组为整体,行Logistic回归分析,结果显示收缩压(SBP)和Vaspin水平影响糖尿病合并颈动脉硬化的发生,提示血清Vaspin水平下降可能是2型糖尿病动脉粥样硬化早期发生的标志物。
结论：血清vaspin水平可能参与胰岛素抵抗和糖尿病的发生,在以胰岛素抵抗为特征的代谢综合征的发病中发挥重要作用。同时血清Vaspin水平下降可能是2型糖尿病动脉粥样硬化早期发生的标志物,尽早检测血清Vaspin水平,可以预测糖尿病动脉粥样硬化等大血管并发症的发生与发展。
第二部分
血清VASPIN水平与2型糖尿病视网膜病变的关系研究
目的：应用ELISA方法检测2型糖尿病患者血清vaspin水平,探讨：(1)血清vaspin水平与2型糖尿病视网膜病变及其严重程度的关系。(2)血清vaspin水平与年龄、病程、FPG.2HPG.HbAlc、血脂、尿白蛋白排泄率(UAER)及其它脂肪因子的关系。
方法：
1.病例选择选择2009年2月至2009年6月间山东大学齐鲁医院内分泌科门诊和住院的2型糖尿病患者94例,根据有无视网膜病变及严重程度,分为无视网膜病变组(40例)、单纯型糖尿病视网膜病变组(38例)和增殖型糖尿病视网膜病变组(16例)。
2.标本留取所有对象均进行人体测量及体格检查。所有个体空腹12h后次晨抽静脉血,进行生化指标的测定。采取静脉血,分离血清后-80℃保存,集中测定血清vaspin和视黄醇结合蛋白4(RBP4)水平。
3.血清vaspin和RBP4水平的测定采用ELISA方法测定血清vaspin和RBP4水平。
结果：
在糖尿病患者中,调整年龄和病程后,单纯性视网膜病变的患者vaspin血清水平显著低于无视网膜病变患者(0.45±0.13 versus 0.53±0.22 ng/ml,p=0.023),有增殖性视网膜病变的患者vaspin血清水平较无视网膜病变患者更低(0.36±0.06versus 0.53±0.22 ng/ml,p=0.001),增殖性视网膜病变患者vaspin血清水平低于单纯性视网膜患者,但无显著性。简单相关分析示血清vaspin与年龄、病程、UAER及RBP4呈负相关(r=-0.259,p=0.012；r=-0.277,p=0.007；r=-0.322, p=0.002；r=-0.234,p=0.023).偏相关分析显示控制年龄和BMI后,vaspin与UAER的相关性仍然存在(r=-0.281,p=0.007).多元逐步回归分析表明年龄、UAER是血清vaspin水平的独立影响因素。
结论：2型糖尿病患者血清vaspin水平与年龄、UAER呈明显相关。血清vaspin水平在2型糖尿病视网膜病变,尤其是合并增殖性视网膜病变者中显著降低,提示血清vaspin水平与糖尿病视网膜病变有关,可能影响糖尿病微血管病变的发生发展。
第三部分3T3-L1脂肪细胞VASPIN表达的影响因素研究
目的：
Vaspin作为一种新发现的具有胰岛素增敏作用的脂肪因子,主要由内脏脂肪组织分泌。但是参与调节vaspin表达的因素尚未完全清楚。本研究旨在探讨3T3-L1脂肪细胞分化过程中vaspin表达的变化,并研究在不同代谢因素下vaspin表达的调节机制及影响因素。
方法：
1.体外培养3T3-L1脂肪前体细胞并诱导分化为成熟脂肪细胞,分别于85%融合(-4d)、融合(-2d)、融合后2天(0d)、5天(3d)、8天(6d)、10天(8d)提取细胞总RNA。用RT-PCR方法检测vaspin mRNA的表达,ELISA检测细胞上清液vaspin水平。
2.分别以不同浓度地塞米松(1,100μmol/L)、胰岛素(10,100nmol/L)、葡萄糖(10,25mmol/L)、棕榈酸(100,1000μmol/L)、TNF-a(10,20ng/ml)、吡格列酮(10,100μmol/L)、非诺贝特(10,100μmol/L)和硫辛酸(50,100,500,1000μmol/L)干预分化成熟的3T3-L1细胞24h,留取细胞上清液,提取细胞总RNA,RT-PCR检测vaspin mRNA的表达水平。
3.以对照组、TNF-α组(20ng/ml TNF-α), TNF-α(20ng/ml)+吡格列酮(10μmol/L),和TNF-a(20ng/ml)+吡格列酮(100μmol/L),分别培养48h,留取细胞上清液,收集脂肪细胞,提取总RNA,用RT-PCR检测vaspin mRNA的表达；以对照组、TNF-α组(20ng/ml)、TNF-α(20ng/ml)+非诺贝特(10μmol/L)、和TNF-a(20ng/ml)+非诺贝特(100μmol/L),分别培养48h,留取细胞上清液,收集脂肪细胞,提取总RNA并测定vaspin mRNA的表达；以对照组、TNF-α组(20ng/ml)、TNF-α(20ng/ml)+硫辛酸(100μmol/L)、和TNF-α(20ng/ml)+硫辛酸(500μmol/L),分别培养48h,留取细胞上清液,收集脂肪细胞,提取总RNA并测定vaspin mRNA的表达。
结果：
在3T3-L1脂肪前体细胞分化过程中,vaspin mRNA表达明显增加并呈时间依赖性。在80%融合到融合后两天,vaspin表达增加到起始的1.7倍。至分化后第8天增加至起始6倍。地塞米松(100μM,1.6倍),高糖(25mM,6.6倍),吡格列酮(100μM,1.6倍)、非诺贝特(100μM,1.5倍)和硫辛酸(500μM,1.6倍)能显著诱导3T3-L1脂肪细胞中vaspin mRNA的表达。而其它处理因素10-100nM胰岛素,1mM棕榈酸,10-20 ng/ml TNF-a则对其1mRNA表达有下调作用,较基线值分别下降(37-53)%,48%和(15-40)%。同时吡格列酮和硫辛酸可逆转TNF-a所致的vaspin mRNA表达的降低。
同时在3T3-L1脂肪细胞诱导分化中,vaspin蛋白分泌亦逐渐增加,并呈时间依赖性。开始后的前5天vaspin蛋白水平增加迅速,6天以后增加明显变慢。地塞米松、葡萄糖显著增加vaspin的分泌,胰岛素、TNF-a明显降低vaspin的分泌,而棕榈酸无明显影响。吡格列酮和硫辛酸明显改善TNF-a所致的vaspin分泌的降低。
结论：在脂肪细胞分化过程中vaspin表达水平与细胞分化程度明显相关,可能参与脂肪前体细胞向成熟脂肪细胞的分化。同时成熟脂肪细胞vaspin的分泌受多种激素和代谢因素的调节,可能与代谢紊乱的严重程度有关。但其调控的确切机制有待进一步阐明。
Background:
Recently, increasing evidence have suggested that adipose tissue is not only an energy storage organ, but also a key endocrine organ which could secrete various adipokines such as leptin, resistin, adiponectin, visfatin, tumor necrosis factor, plasminogen activator inhibitor, and interleukin-6,etc. Epidemiological and animal studies have shown that visceral obesity is associated with increased prevalence of insulin resistance, type 2 diabetes and cardiovascular diseases. Adipokines secreted by visceral fat tissues play an important role in the occurrence and development of insulin resistance, type 2 diabetes and metabolism syndrome. Vaspin, a newly identified adipokine, is secreted by visceral adipose tissue with predominantly insulin-sensitizing effects,However, its physiological role is largely unknown. Although current studies have shown plasma vaspin level has direct effects on obesity, the result is still in debate. In the present study, we investigated 1) the relationship between serum vaspin level and macrovascular and retinopathy complications in diabetic patients,2) the regulatory mechanism of vaspin expression,3) the potential physiological function of vaspin. We first examined whether serum vaspin levels are associated with the presence of carotid plaque in early stage of type 2 diabetes. Next, we analysed the relationship between serum vaspin levels and diabetic retinopathy. Finally, we explored in vitro the effects of high glucose, insulin, dexamethasone, palmitate, TNF-a, pioglitazone, fenofibrate and lipoic acid on vaspin expression in 3T3-L1 cell and investigated the potential molecular mechanism involved.
PartⅠ
SERUM VASPIN LEVELS IN TYPE 2 DIABETIC PATIENTS WITH CAROTID PLAQUE
Objective:1) To explore whether serum vaspin levels are associated with the presence of carotid plaque in early stage of type 2 diabetes; 2) To explore the association between serum vaspin levels and FPG,2HPG, HbAlc, serum lipid and insulin resistance.
Materials and Methods:Sixty-one type 2 diabetic patients within 1 years's diabetes duration were divided into two groups by the presence or absence of carotid plaque. Twenty-six age-matched apparently healthy controls were recruited as well. Serum vaspin was measured by enzyme-linked immunosorbent assay.
Results:After controlling age and gender, fasting serum vaspin levels were significantly higher in the diabetic patients without carotid plaque than controls (0.65±0.24 vs.0.46±0.12 ng/ml, p<0.01). However, circulating vaspin level was much lower in the diabetic subjects with carotid plaque than those without (0.52±0.23 vs. 0.65±0.24 ng/ml, p<0.05).Partial correlations demonstrated that vaspin level was positively correlated with waist circumference (WC) (r=0.269, p=0.041), negatively correlated with HOMA-IR(r=-0.262, p=0.047) in all subjects after controlling age, gender and BMI. Logistic regression analysis showed that SBP and fasting serum vaspin levels were significantly associated with the presence of carotid plaque in type 2 diabetes.
Conclusion:Serum vaspin levels were significantly elevated in type 2 diabetic patients while its level was negatively associated with the presence of carotid plaque in type 2 diabetic patients,indicating that vaspin might play a compensative role in the early course of diabetes while exert protective effects on atherosclerosis in early stage of diabetes.
Part II
VASPIN SERUM CONCENTRATIONS IN TYPE 2 DIABETIC PATIENTS WITH DIABETIC RETINOPATHY
Objective:1) To explore the relationship between serum vaspin level and type 2 diabetic retinopathy and its severity.2) To explore the association between vaspin level and age, duration, FPG,2HPG, HbAlc, serum lipid, urine albumin excretion rate (UAER) and other adipokines
Materials and Methods:94 patients with type 2 diabetes were recruited through in-patients or clinic service from February to June 2009 at Qilu Hospital of Shandong University, Jinan, China. Based on ophthalmologic examination,94 patients with type 2 diabetes were divided into three subgroups:group 1 without diabetic retinopathy (NDR) (n=40), group 2 with simple diabetic retinopathy (SDR) (n=38) and group 3 with proliferative diabetic retinopathy (PDR) (n=16). Serum vaspin was determined by enzyme-linked immunosorbent assay.
Results:In the diabetic patients, subjects with simple diabetic retinopathy had significantly lower vaspin levels than subjects without diabetic retinopathy (0.45+ 0.13 versus 0.53+0.22 ng/ml, p= 0.023).Moreover, subjects with proliferative diabetic retinopathy had much lower vaspin levels as compared with subjects with simple diabetic retinopathy. Age, disease duration, UAER and retinol binding protein-4 (RBP4) were negatively correlated with vaspin level in correlation analysis (r=-0.259, p= 0.012; r=-0.277, p= 0.007; r=-0.322, p=0.002; r=-0.234, p=0.023 respectively). Partial correlations demonstrated that UAER was still negatively correlated with vaspin levels (r=-0.281, p= 0.007) after controlling age and BMI, A stepwise linear multiple regression model revealed that age and UAER were significant determinants of serum vaspin levels.
Conclusion:Tanken together, these results suggest that the reduction of vaspin level may be involved in the process of diabetic retinopathy and thereby provides a novel biomarker for evaluating its severity in diabetic patients.
PartⅢ
STUDY ON THE REGULATORY MECHANISM OF VASPIN EXPRESSION IN 3T3-L1 CELL
Objective:To explore the effect of metabolic factors on vaspin expression in 3T3-L1 cell and the regulatory mechanism involved.
Methods:
1.3T3-L1 preadipocytes were cultured in DMEM medium supplementd with 10% FBS and differentiated into adipocytes. The total RNA was extracted in the preconfluent, confluent and 2-day,5-day,8-day,10-day confluent cells respectively. The expression of vaspin mRNA was determined by quantitative real-time polymerase chain reaction and vaspin level in cell supernatant was determined by ELISA.
2. Mature 3T3-L1 adipocytes was treated with indicated concentrations of dexamethasone, insulin, glucose, palmitate, TNF-a, pioglitazone, fenofibrate and lipoic acid for 24 or 48 h.The cells were starved for 6 h in DMEM with 0.5% bovine serum albumin (BSA) before the study.
3. TNF-a was added to the cell culture medium alone or with the indicated "concentrations of pioglitazone, fenofibrate or lipoic acid respectively for 48 h. During the treatment period, fresh TNF-a, pioglitazone, fenofibrate or lipoic acid was added every 24 h.
Results:A marked increase in vaspin expression was observed during 3T3-L1 differentiation, with a 1.7-fold increase on 2-day confluent as compared with preconfluent. A further 3.8-fold increase was induced from day 0 to day 8 of differentiation (overall six fold). Overnight incubation with dexamethasone increased vaspin expression in adipocytes (100μM-1.6 fold). High glucose increased vaspin secretion by 6.6-fold at 25 mM, pioglitazone by 1.6-fold at 100μM, fenofibrate by 1.5-fold at 100μM, and lipoic acid by 1.6 fold at 500μM. While 10-100 nM insulin, 1mM palmitate and 10-20 ng/ml TNF-a decreased vaspin expression (.37-53%,48%, 15-40% respectively). Futhermore, pioglitazone and lipoic acid reversed the reduction of vaspin by TNF-α.
Vaspin protein level in cell supernatant increased gradually during differentiation, rapidly in beginning 5 days after differentiation and slowly after 6 days. Dexamethasone and glucose promoted vaspin secretion in mature adipocytes while insulin and TNF-a inhibited vaspin level. Pioglitazone, fenofibrate and lipoic acid reversed the reduction of vaspin induced by TNF-a.
Conclusion:Taken together, vaspin levels are associated with the extent of the differentiation and its level is regulated by multiple hormones and metabolic factors which could be associated with severity of metabolic disorders. The underlying mechanism of vaspin action remains to be elucidated futher.
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