【作者】 任瑞珍；

【导师】 赵家军； 高聆；

【作者基本信息】 山东大学 ， 内科学， 2012， 博士

【摘要】 研究背景：脂肪组织传统上被认为是能量储存的场所,并为机体提供缓冲。然而,现在认为脂肪组织是一个重要的内分泌器官。肪组织分为白色脂肪组织和棕色脂肪组织,近年来的研究认为白色脂肪组织可以分泌大量具有生物活性的肽类物质和蛋白质,称为脂肪因子,其中一些脂肪因子参与调节葡萄糖和脂肪代谢及胰岛素抵抗,与肥胖及糖尿病的发生密切相关。Chemerin也称为他扎罗汀诱导基因2及维甲酸受体反应元件,以18-kDa的不具活性的前蛋白的形式分泌,经过丝氨酸蛋白酶水解C末端后转化为16-kDa的具有生物活性的蛋白质。Chemerin在肝脏及白色脂肪组织高表达,在肺脏、肾脏、垂体、胎盘及卵巢也有表达。Chemerin做为一种趋化蛋白参与免疫反应,近年来认为chemerin是一种脂肪因子,chemerin及其受体的表达在前脂肪细胞分化为脂肪细胞的过程中增加,chemerin以自分泌及旁分泌的方式调节脂肪分化。近年来研究表明chemerin也与胰岛素敏感性密切相关。到目前为止,大部分的研究证实chemerin通过不同的作用机制诱导脂肪组织及骨骼肌的胰岛素抵抗。饮酒是一种与2型糖尿病密切相关的生活方式。适量饮酒可以通过改善脂代谢、增加胰岛素敏感性、减少血小板聚集及减少腹部脂肪含量发挥保护作用,而大量饮酒可以影响脂代谢及增加胰岛素抵抗,从而导致糖尿病的发生。目前关于饮酒对脂肪因子的影响的研究甚少,文献报道在慢性酒精中毒患者中瘦素水平以剂量依赖方式升高,而适量酒精摄入明显升高血浆脂联素水平。目前国内外关于饮酒对脂肪因子影响的研究主要是单独观察血清、组织或细胞中脂肪因子水平的变化,但甚少同时研究血清及组织脂肪因子含量的变化,也很少探究血清及组织中脂肪因子的相关性。不同于脂联素、抵抗素及内脂素等绝大部分由脂肪组织产生的脂肪因子,chemerin在脂肪组织和肝脏均高表达。目前国内外尚无关于饮酒对chemerin影响的研究,且近年来其他关于chemerin的研究多单独在血清及脂肪细胞水平进行,而缺少同时对血清及组织chemerin的研究。因此,本研究的目的是观察长期不同剂量酒精摄入对人及大鼠血清、脂肪组织及肝脏chemerin的影响,研究chemerin与人各代谢综合征组分的关系,并探究酒精对血清chemerin的影响与组织chemerin的关系。目的：1.观察长期不同剂量酒精摄入对人血清脂肪因子chemerin水平的影响,并研究人血清chemerin水平与血脂、胰岛素抵抗指数及体脂等代谢参数的关系。3.观察不同剂量酒精摄入对大鼠血清、脂肪组织及肝脏chemerin水平的影响,并研究大鼠血清chemerin和脂肪组织chemerin及肝脏chemerin之间的相关性。方法：1.人体实验1.1实验对象选择从参加山东省立医院糖尿病流行病学调查的人群中选取148名男性健康饮酒者及55名健康非饮酒者参加实验。所有受试者体重指数均小于25kg/m2。每位受试者严格排除糖尿病、急慢性炎症性疾病、肝脏疾病及肝功异常者、高血压、心脑血管疾病、肾脏疾病及甲状腺疾病,所有受试者均未服用影响糖代谢及脂代谢的药物和影响血压及体重的药物。选出的所有受试者年龄均在22-75岁之间,均在本地生活超过5年。1.2研究对象分组所有饮酒者的饮酒时间均超过3年,饮酒频率均等于或超过每周两次。每日酒精摄入量通过每周饮酒次数乘以每次饮酒量除以7计算而得。根据每日饮酒量,饮酒者被分为以下4组：将受试者分为正常对照组(C组)、小剂量饮酒组(L组：酒精量<15g·d-1)、中剂量饮酒组(M组：酒精量15-47.9g·d-1)及大剂量饮酒组(H组：酒精量≥48g·d-1)。1.3人体学测量每位受试者在空腹状态进行一次彻底的体格检查,测量身高、体重、腰围及臀围,计算体重指数及腰臀比。体脂分析仪测定体脂质量、体脂百分数及体脂腰臀比。1.4实验室检测空腹血糖用葡萄糖氧化酶法测定。空腹胰岛素用放免法测定。血脂组分(包括总胆固醇、甘油三酯、低密度脂蛋白胆固醇和高密度脂蛋白胆固醇)用常规酶法测定。用HOMA胰岛素抵抗指数评价胰岛素敏感性,HOMA-IR=空腹胰岛素(mU·L-1)×空腹血糖(mmol·L-1)/22.5.血清chemerin用酶联免疫吸附试验(ELISA)测定。2.动物实验2.1动物分组及喂养27只雄性Wistar大鼠适应性喂养一周后,完全随机分为四组：对照组(C组：蒸馏水5.0g·kg-1·d-1)、小剂量饮酒组(L组：酒精0.5g·kg·d-1)、中剂量饮酒组(M组：酒精2.5g·kg-1·d-1)及大剂量饮酒组(H组：酒精5.0g·kg-1·d-1)。酒精和蒸馏水均每日一次由胃管注入,共喂养22周。2.2标本获取及存放大鼠喂养22周后空腹经下腔静脉取血,分离血清置于-80℃保存；快速分离1肾周脂肪组织和附睾脂肪组织,分别称其重量后置于液氮中保存。2.3空腹血糖和空腹胰岛素测定葡萄糖氧化酶法测定空腹血糖；放免法测定空腹胰岛素；用HOMA胰岛素抵抗指数评价胰岛素敏感性,HOMA-IR=空腹胰岛素(mU·L-1)×空腹血糖(mmol.L-1)/22.5。2.4脂肪组织及肝脏组织总蛋白的提取分别剪取液氮保存的脂肪组织及肝脏200mg,放入200μl磷酸盐缓冲液,超声破碎匀浆,于-80℃冻存过夜,两次冻融后离心,吸取脂肪组织匀浆的中间层及肝脏组织匀浆的上层,检测总蛋白浓度及chemerin含量。2.5大鼠血清、肝脏及脂肪组织chemerin含量测定用ELISA测定大鼠血清、肝脏及脂肪组织Chemerin含量。结果：1.人体实验1.1人体学测量及各代谢指标一般情况校正年龄后大剂量饮酒组体脂质量、体脂腰臀比、体脂百分数、甘油三酯、空腹血糖、空腹胰岛素和HOMA-IR明显高于对照组,差别有统计学意义。1.2慢性酒精摄入升高人血清chemerin水平慢性酒精摄入以剂量依赖方式引起血清chemerin升高。与对照组相比,小剂量饮酒组、中剂量饮酒组及大剂量饮酒组chemerin分别升高9.75%(P=0.265),13.84%(P=0.094)和40.83%(P<0.001)。进一步校正年龄后,大剂量饮酒组chemerin的升高仍有统计学意义(p<0.01)。1.3血清chemerin水平与甘油三酯、空腹血糖、空腹胰岛素及HOMA-IR相关相关分析显示血清chemerin水平与体重指数、体脂质量、甘油三酯、空腹血糖、空腹胰岛素及HOMA-IR正相关,与高密度脂蛋白胆固醇负相关。以血清chemerin作为因变量进行多元线性回归分析,结果显示甘油三酯、空腹血糖、空腹胰岛素及HOMA-IR是血清chemerin的独立影响因素。2.动物实验2.1大剂量酒精摄入引起大鼠体重下降及内脏脂肪与体重的比值增加实验开始时各组大鼠体重无差别,实验结束时,与对照组相比,中剂量饮酒组大鼠体重下降5.28%(p<0.01),大剂量饮酒组大鼠体重下降6.23%(p<0.01)。虽然大鼠的体重随饮酒量增大而下降,但大鼠附睾脂肪组织与体重的比值增加了18.28%(p<0.01),而小剂量及中剂量组大鼠上述指标变化无统计学意义。2.2大剂量酒精摄入增加大鼠空腹胰岛素及HOMA-IR随着酒精剂量增加,空腹胰岛素水平及HOMA-IR均升高,大剂量饮酒组空腹胰岛素增加48.34%(p<0.05),大剂量饮酒组HOMA-IR增加44.04%(p<0.05)。2.3长期酒精摄入升高大鼠血清和内脏脂肪组织中chemerin含量酒精以剂量依赖方式增加大鼠血清和内脏脂肪组织中chemerin水平。与对照组相比,小、中、大剂量饮酒组大鼠血清chemerin分别增加了7.96%(p=0.524)、25.33%(p=0.005)、50.60%(p<0.001)。与对照组相比,小、中、大剂量饮酒组大鼠内脏脂肪组织chemerin水平分别升高了13.59%(p=0.357)、34.84%(p=0.025)、50.17%(p=0.002)。与照组相比,小、中、大剂量饮酒组大鼠肝脏chemerin水平分别升高了5.71%(p=0.493)、9.85%(p=0.242)、16.06%(p=0.063)。2.4血清chemerin水平与内脏脂肪组织中chemerin含量正相关相关分析显示在校正肝脏chemerin含量后,血清chemerin水平和内脏脂肪组织中chemerin水平正相关(r=0.767,p<0.001)。但校正内脏脂肪组织chemerin水平后,血清chemerin水平和肝脏chemerin水平无明显相关(r=0.260,p=0.199)。结论：1.慢性酒精摄入以剂量依赖方式引起人血清、大鼠血清及内脏脂肪组织中chemerin水平升高。酒精引起的大鼠血清chemerin升高主要来源于内脏脂肪组织中chemerin的升高。2.长期大剂量酒精摄入增加人的体脂及大鼠内脏脂肪含量。更多还原

【Abstract】 BackgroundAdipose tissue is classically considered a tissue that stores excess energy and provides insulation for the body; however, it is now considered to be an endocrine organ. The adipose tissue secretes multiple metabolic proteins known as adipokines. Since the discovery of the most notable adipokines, leptin and adiponectin, the member of adipokine extends continuously. Some of these adipokines play important roles in glucose and lipid metabolism, insulin resistance, obesity and type2diabetes.Chemerin, a newly found adipokine, is secreted as an18-kDa inactive proprotein named prochemerin and is converted into the16-kDa active chemerin by a serine protease cleavage of the C-terminal portion of the protein. It is most highly expressed in the white adipose tissue and liver, which are followed by the lung, kidney, pituitary, placenta and ovary. Chemerin is a novel chemoattractant protein that plays roles in adaptive and innate immunity. Recent research found that chemerin participated in the regulation of adipocyte differentiation and had effects on insulin sensitivity. Until now, the majority of studies have demonstrated that chemerin induces insulin resistance in the adipose tissue and skeletal muscle.Ethanol consumption is a lifestyle factor and is relevant to type2diabetes. Moderate alcohol consumption and a high amount of alcohol intake produced different effects on lipid metabolism and insulin sensitivity. Until now, few studies have focused on the effectts of ethanol on adipokines. Our previous study demonstrated that ethanol consumption elevated the leptin, resistin and visfatin levels and decreased the adiponectin concentrations in both the sera and visceral adipose tissues (VAT) of rats. Unlike the other adipokines, chemerin was highly expressed in the adipose and liver tissues. Until now, there was no study on the correlations between chemerin and ethanol. Therefore, this study aimed to observe the effects of a long-term intake of different doses of ethanol on chemerin in humans and rats and to evaluate the relationship of chemerin with metabolic parameters in humans.Objective1To observe the effect of chronic ethanol consumption on human serum chemerin.2To explore correlations between serum chemerin and metabolic paratems in humans.3To observe the effect of different dose of ethanol on chemerin in serum, liver and VAT in rats.4To explore correlations between serum chemerin, liver chemerin and VAT chemerin in rats.Materials and Methods1Human study1.1SubjectsData were obtained from an epidemiological investigation of type2diabetes in the Shandong Provincial Hospital in China. According to the study criteria,148healthy men who consumed alcohol and55healthy men who abstained from alcohol were included in the study. The body mass indexes (BMI) of all of the subjects were less than25kg/m2. The subjects were classified into four groups:a control group, a low-dose group (group L; ethanol consumption<15g·d-1), a middle-dose group (group M; ethanol consumption15-47.9g·d-1), and a high-dose group (group H; ethanol consumption≥48g·d-1).1.2Anthropometric measurementsA complete physical examination was conducted on each individual under the condition of an empty bladder and stomach. The values of height, weight, waist circumference and hip girth were taken and the BMI and the waist-to-hip ratio (WHR) was calculated. The body fat, percentage of body fat and waist-hip ratio of body fat were assessed using the body composition analyzer.1.3Laboratory measurementsThe fasting blood glucose (FBG) was determined by the glucose oxidase method. The fasting serum insulin (FINS) was measured using a radioimmunoassay kit. The plasma concentrations of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were measured using routine enzymatic methods. The insulin sensitivity was estimated using the homeostasis model assessment for insulin resistance (HOMA-IR), which was calculated as fasting insulin (mU·L-1) multiplied by fasting glucose (mmol·L-1) divided by22.5. The chemerin levels of humans was determined by enzyme-linked immunosorbent assay (ELISA).2Animal study2.1Animal protocols and housingTwenty-seven male Wistar rats were divided into four groups and given the following different treatments:control group (group C; distilled water at5.0g·kg-1·d-1), low-dose group (group L; ethanol at0.5g·kg-1·d-1), middle-dose group (group M; ethanol at2.5g·kg-1·d-1), and high-dose group (group H; ethanol at5.0g·kg-1·d-1). Distilled water or edible ethanol was given by a gastric tube every morning for22weeks.2.2Blood and tissue collectionBlood samples from all of the rats were obtained from the inferior vena cava after an overnight fast. The serum samples were separated after centrifugation and immediately stored at-80℃for subsequent analyses. The epididymal and perirenal fat pads and livers were removed and weighted and rapidly frozen in liquid nitrogen for adipokine measurements.2.3Laboratory measurementsThe FBG was determined by the glucose oxidase method. The FINS was measured using a radioimmunoassay kit. The insulin sensitivity was estimated using HOMA-IR, which was calculated as fasting insulin (mU·L-1) multiplied by fasting glucose (mmol·L-1) divided by22.5. Samples of the liver and adipose tissues (200mg) were excised from the frozen specimens. The excised tissues were added to200μL of phosphate-buffered saline (PBS) and homogenized. The tissue homogenates were frozen overnight at-80℃and thawed on ice the following day. After two freeze-thaw cycles, the homogenates were centrifuged. The middle layer of the adipose tissue homogenate and the supernatant of the liver homogenate were isolated and stored at-80℃to determine the total protein level and chemerin concentration.2.4ELISAThe chemerin levels in serum, adipose tissue and liver were determined by commercially available ELISA kits according to the manufacturer’s instructions.Results1Human study1.1Anthropometric and metabolic characteristics of the human participantsThe body fat, percentage of body fat, waist-hip ratio of body fat, TG, FPG, FINS, and HOMA-IR in group H were significantly elevated compared with group C.1.2Chronic ethanol consumption increased serum chemerin in humansChronic ethanol consumption caused a dose-dependent increase of chemerin in human sera. The chemerin levels in group L, group M, and group H increased by9.75%(P=0.265),13.84%(P=0.094), and40.83(P<0.001), respectively, compared to group C. Furthermore, even after the adjustment for age, chemerin remained significantly different among the groups.1.3Serum chemerin levels are associated with TG and HOMA-IR in humansA pearson’s correlation analysis was performed between the serum chemerin concentrations and clinical characteristics. The results showed that the chemerin levels positively correlated with the BMI, body fat, TG, FPG, FINS and HOMA-IR and negatively correlated with the HDL-C. A multivariate linear regression analysis using chemerin as the dependent variable revealed that the TG, FPG, FINS and HOMA-IR were independently associated with the chemerin concentrations.2Animal study 2.1High-doses ethanol intake decreased body weight (BW) and increased epididymal adipose tissue to BW ratio in ratsRats of the four groups had similar body weight at baseline. But after the22-week treatment, the BW of group M and group H decreased by5.28%and6.23%, respectively (both P<0.01). The epididymal adipose tissue to BW ratio increased by18.28%in group H in relation to the control group (P<0.01).2.2High-dose ethanol consumption increased FINS and HOMA-IRThe FINS levels and HOMA-IR of rats increased in ethanol-treated groups compared with controls. High doses of ethanol increased FINS levels by48.34%(P<0.05) and HOMA-IR values by44.04%(P<0.05) compared to the control group respectively.2.3Chronic ethanol treatment increased the chemerin levels in the sera and VAT of the ratsEthanol increased chemerin level in a dose-dependent manner in rats. Serum chemerin levels in group L, group M and group H increased by7.96%(p=0.524),25.33%(p=0.005) and50.60%(p<0.001) in relation to those in group C. Chemerin levels in VAT in group L, group M and group H increased by13.59%(p=0.357),34.84%(p=0.025) and50.17%(p=0.002) compared with those in group C. Chemerin levels in liver in group L, group M and group H increased by5.71(p=0.493),9.85%(p=0.242) and16.06%(p=0.063) compared with those in group C.2.4Positive correlation of chemerin levels between the serum and VATA correlation analysis between the serum and VAT or liver chemerin showed that the serum chemerin concentrations were positively associated with the chemerin in the VAT after adjusting for the liver chemerin. The relationship between the serum and liver chemerin was not statistically significant after adjusting for the chemerin in the VAT.Conclusions1Chronic ethanol consumption increases serum chemerin levels in human in a dose-dependent manner. 2Chronic ethanol consumption increases chemerin levels of serum and VAT in rats in a dose-dependent manner. The increase of serum chemerin is mainly attributed to the elevation of chemerin in VAT after ethanol treatment.3The chronic high-dose ethanol consumption increases body fat in humans and VAT in rats.更多还原