【作者】 刘英华；

【导师】 郭长江； 薛长勇；

【作者基本信息】 中国人民解放军军事医学科学院 ， 营养与食品卫生， 2012， 博士

【摘要】 目的1.证实中链脂肪酸（MCFA）具有效降低高脂肪饲料诱导的肥胖小鼠体重、改善脂代谢的作用。2.探讨MCFA上述作用的可能机制、作用通路及靶点，为进一步研究MCFA调节机体脂代谢的作用提供理论依据。3.探讨辛酸和癸酸单体对高脂肪饲料诱导的肥胖小鼠体重、体脂肪及脂代谢的影响。方法1. C57BL/6J小鼠肥胖模型的建立4-5周龄C57BL/6J雄性小鼠100只，普通饲料适应喂养1周后，按空腹体重随机选择15只小鼠设为对照组(control)，喂饲普通饲料(AIN-96G),其余小鼠喂饲高脂饲料（脂肪占总重量19.42%，产热比40.5%）。喂养4周后，在高脂饲料喂养的小鼠中，选择体重高于对照组平均体重10%的个体，进行体重分布分析，在分布频率峰值范围内随机选取小鼠15只，设为肥胖组（HFD-1）；采用同样方法在剩余小鼠中随机选取15只，设为肥胖抵抗组（HFD-2）。对3组小鼠进行体长、Lee’s指数、BMI、体重增长值、肝脏重、肠系膜周围脂肪垫、附睾周围脂肪垫及肾周脂肪垫重量以及血清葡萄糖、TG、TC、HDL-C和LDL-C测定，计算HDL-C/LDL-C比值，同时取部分附睾周围脂肪组织固定进行HE染色。在喂养4周内，每周测空腹体重一次，每隔1天更换垫料和水，称量饲料消耗量，并计算食物功效比。2. MCFA对C57BL/6J肥胖小鼠脂代谢的调节作用按第一部分实验方法复制肥胖小鼠模型，将复制成功的肥胖小鼠分三部分进行急性灌胃实验、短期灌胃实验和长期喂养实验。（1）急性灌胃实验将36只建模成功的肥胖C57BL/6J小鼠按空腹体重随机分为2组，每组18只，分别灌胃含有MCFA的油脂（MCT）和含有LCFA的油脂（LCT），剂量为2mg/kg，两组分别于灌胃后1小时、2小时、4小时各处死6只，取腹主动脉血测血清中TG、TC、HDL-C、LDL-C，并计算HDL-C/LDL-C比值。（2）短期灌胃实验24只C57BL/6J肥胖小鼠按空腹体重随机分为两组，每组12只，分别灌胃MCT和LCT，剂量为2mg/kg，每日灌胃1次，持续2周。在实验期间以高脂饲料喂饲动物，每隔1日进行饲料消耗量的称量和记录，并计算食物功效比。2周后，称量小鼠空腹体重后，麻醉小鼠，测量小鼠体长，计算Lee’s指数、BMI和体重增长值。取腹主动脉血处死小鼠，测量血清TG、TC、HDL-C和LDL-C，计算HDL-C/LDL-C比值。同时取小鼠肝脏、肠系膜周围脂肪垫、附睾周围脂肪垫及肾周脂肪垫并称重。另取部分肝脏组织制作匀浆，测定蛋白浓度以及TG、TC、ApoA1和ApoB浓度，并计算ApoA1/ApoB比值。（3）长期喂养实验30只C57BL/6J肥胖小鼠按空腹体重随机分为两组，每组15只，分别给予含2%MCT和LCT的高脂饲料喂养，12周后结束实验，测定指标同短期实验。同时，取部分附睾周围脂肪组织固定进行HE染色。3. MCFA调节C57BL/6J肥胖小鼠脂代谢的机制研究在上述长期喂养实验基础上，采用ELISA法测定血清样本中HSL、cAMP、PKA、FFA、GLY、NADR和T3浓度。冻存部分肝脏组织和附睾周围脂肪组织，用0.9%氯化钠注射液按10%浓度制成组织匀浆，采用BCA法测定脂肪组织及肝脏组织蛋白浓度，采用ELISA法测定脂肪组织中HSL、ATGL、cAMP、PKA、LPL、FAS、ACC、Leptin、APN、PPAR-γ、TNF-α水平，肝脏组织中LPL、FAS、ACC、ME、G6PD、Leptin、APN、PPAR-γ、TNF-α水平。采用Real-time PCR法检测脂肪组织中HSL、ATGL、UCP2、β3-AR、Leptin、PPAR-γ、SREBP-1和C/EBP-α的mRNA表达。采用Westernblotting法检测脂肪组织中β3-AR的蛋白表达。另外，参考Fredrikson G和Belfrage P报道的方法测定血清和脂肪组织中HSL的活性。4.辛酸和癸酸对C57BL/6J肥胖小鼠脂代谢调节作用的比较按上述长期实验研究的方法，喂饲C57BL/6J肥胖小鼠含有2%辛酸（C8）、癸酸（C10）和油酸（C18）的高脂饲料，每组12只，8周后结束实验。观察指标包括：小鼠每周空腹体重、饲料消耗量及食物功效比、小鼠体长、Lee’s指数、BMI和体重增长值、小鼠肝脏及脂肪组织重、脂肪细胞形态学观察、小鼠血清TG、TC、HDL-C、LDL-C、HDL-C/LDL-C比值、脂肪组织中HSL、ATGL、cAMP、PKA、Leptin、TNF-α、PPAR-γ水平以及HSL、ATGL、β3-AR的mRNA表达，肝脏组织中ApoA1、ApoB、LPL、FAS、CYP7A1、HMGCoA、TNF-α水平以及CYP7A1和HMGCoA的mRNA表达。结果1. C57BL/6J小鼠肥胖模型的建立高脂饲料喂养4周后，HFD-1肥胖组小鼠体重、体长、Lee’s指数、BMI、体重增长值、肝重及各部分脂肪组织重以及血糖、TC和LDL-C水平均显著高于HFD-2肥胖抵抗组和对照组（P<0.05），HDL-C/LDL-C比值显著低于HFD-2组和对照组（P<0.05）。脂肪细胞形态学观察结果显示HFD-1组细胞的长径和短径均显著大于HFD-2组和对照组（P<0.05），单个视野小型脂肪细胞数量少于HFD-2组和对照组（P<0.05）。HFD-1组的肥胖小鼠模型建立成功。2. MCFA对C57BL/6J肥胖小鼠脂代谢的调节作用急性灌胃实验结果显示小鼠灌胃MCT、LCT后2小时和4小时，MCT组血清TG均明显低于LCT组（P<0.05），其他血脂相关指标未显示统计学差异（P>0.05）。短期灌胃实验结果显示小鼠体重、血脂、肝脏脂代谢指标等，灌胃MCT与LCT2周后比较均无显著性差异（P>0.05）。长期喂养实验结果显示MCT饲料组小鼠体重、体长、Lee’s指数、BMI、体重增长值、肝脏重、肾周脂肪重、附睾周脂肪重、血脂相关指标如血清TG、TC和LDL-C浓度均显著低于LCT组（P<0.05），血清HDL-C水平和HDL-C/LDL-C比值、小鼠肝脏组织匀浆的ApoA1浓度及ApoA1/ApoB比值，均显著高于LCT组（P<0.05）。脂肪细胞形态学观察结果显示，长期喂养MCT饲料的小鼠脂肪细胞长径和短径显著低于LCT组，而单个视野平均细胞数高于LCT组，差异均有统计学意义（P<0.05）。3. MCFA调节C57BL/6J肥胖小鼠脂代谢的机制研究喂饲MCT12周的C57BL/6J小鼠，ELISA法测定结果显示，与脂肪动员相关的各项指标如血清HSL和NADR水平，肝脏组织LPL水平，脂肪组织中ATGL、HSL和cAMP水平显著高于LCT组（P<0.05），其他指标如血清FFA浓度，脂肪组织中FAS、Leptin和TNF-α水平显著低于LCT组（P<0.05），PPAR-γ水平及肝脏中FAS、ACC、Leptin、APN及TNF-α水平两组之间比较均无统计学差异（P>0.05）。mRNA表达测定结果显示：小鼠脂肪组织中ATGL、HSL、UCP2和β3-AR的mRNA表达量均显著高于LCT组（P<0.05），Leptin、SREBP-1和C/EBP-α的mRNA表达量均显著低于LCT组，PPAR-γ mRNA表达两组之间无显著性差异（P>0.05）。Western blotting测定结果显示，MCT组β3-AR蛋白表达显著高于LCT组（P<0.01）。此外，MCT组脂肪组织中HSL活性显著高于LCT组（P<0.01），而血清中HSL活性，两组之间比较无显著性差异（P>0.05）。4. MCFA不同单体对C57BL/6J肥胖小鼠脂代谢调节作用的比较C57BL/6J小鼠喂饲分别含有C8、C10和C18脂肪酸的饲料8周后，C10组小鼠体重、Lee’s指数、BMI、体重增长值、附睾周围脂肪组织重均显著低于C18组（P<0.05），脂肪细胞形态学观察结果显示，C10组脂肪细胞体积明显减小，与C18组比较，细胞长径和短径均显著降低，而单个视野平均细胞数高于C18组（P<0.05），且与甘油三酯代谢相关指标如血清TG、脂肪组织中ATGL、HSL、cAMP水平均显著高于C18组（P<0.05），Leptin和TNF-α水平以及肝脏中FAS水平均显著低于C18组（P<0.05）。此外，脂肪组织中ATGL、HSL和β3-AR的mRNA表达量均显著高于C18组（P<0.05）。C8组与C18组比较，主要体现在与胆固醇代谢相关指标存在显著性差异，如血清TC和LDL-C浓度低于C18组（P<0.05），血HDL-C/LDL-C比值，肝脏组织中ApoA1浓度、ApoA1/ApoB比值和CYP7A1水平均显著高于C18组（P<0.05）。此外，C8组与C10、C18组分别比较，肝脏中CYP7A1的mRNA表达量均显著增高（P<0.05），而HMGCoA的mRNA表达量，三组之间比较无显著性差异（P>0.05）。结论1.MCFA作为天然来源的小分子中链脂肪酸可有效减轻肥胖小鼠体重，减少机体脂肪聚集，改善血甘油三脂、血胆固醇水平。2.以上作用的可能机制为：（1） MCFA由于其独特的代谢途径增加机体能量消耗，进而作用于中枢增加交感神经系统的活性，使外周NADR释放增加，激活脂肪细胞膜上的β3-AR，导致UCP2的表达增加，增强了与甘油三酯代谢相关酶ATGL、HSL的表达活性和水平，使体内脂肪动员加速，进而促进脂肪组织分解。（2）这种激活机体能量代谢的作用不是通过激活Leptin表达实现的。（3） MCFA可能通过下调脂肪组织中TNF-α、SREBP-1及C/EBP-α水平和mRNA表达，抑制脂肪细胞分化途径，减少脂肪细胞聚集，进而改善机体脂代谢紊乱。（4） MCFA对肝脏中脂肪酸合成途径的调节、对肝脏中脂肪因子的调节、以及MCFA对Leptin、APN和PPAR-γ途径的调节作用并不确定。3.MCFA中的辛酸和癸酸均可有效改善肥胖小鼠血甘油三酯和血胆固醇水平，但辛酸主要通过增加肝脏CYP7A1的表达调节小鼠胆固醇代谢，癸酸主要通过增加脂肪组织β3-AR、ATGL、HSL等表达水平调节小鼠甘油三酯代谢。二者的协同作用可能是MCFA降体重、改善血脂水平的机制之一。更多还原

【Abstract】 Objective1.To confirm that medium-chain fatty acids (MCFA) as a natural source of small moleculefatty acids can effectively reduce body weight of obese mice, and improve lipid metabolism.2.To investigate the mechanism and the possible effective pathways and targets of MCFA inbody and offer the incidence for theory of MCFA regulating lipid metabolism.3.To observe the effect of caprylic acid and capric acid on body weight, body fat and lipidmetabolism of obese mice and to confirm the influence of MCFA on triglyceride andcholesterol metabolism.Methods1. Mouse obesity model100C57BL/6J male mice, aged4-5weeks, were used and were fed normal diet to adaptcircumstance one week. According to their fasting weight,15mice were randomly chosen tofeed normal diet (AIN-96G), which was used to be a control group. The other mice were fedhigh fat diet (HFD). The diet contains19.42%fat from the total weight and fat carloie is40.5%from total carloie. After four weeks feeding, the mice fed high-fat, weight higher than10%of individuals in the average body weight of the control group, weight distributionanalysis, randomly selected15mice in the distribution of peak frequency，as the obese group(HFD-1), weight less than control mice10%of individuals at the same time, after weightdistribution analysis, randomly selected15mice in the distribution of peak frequency range,set to the obesity resistance group (HFD-2). The body length, Lee’s index, BMI, and weightgain during the study of mice as well as serum glucose, TG, TC and HDL-C and LDL-C,HDL-C/LDL-C ratio were measured and calculated. The liver, mesenteric fat fads,epididymal fat pads and perirenal fat fads were taken out and weighed. Another slice ofepididymal adipose tissue was HE stained. During the study, the body weights of the miceand diet consumption were measured, and food efficiency ratio (KJ/d) were calculated.2. Regulations of lipid metabolism of MCFA on the C57BL/6J obese miceThe obese model of mice was estabolished according to the methods of the first part ofthe experiment, and the obese mice were divided into three parts to carry out threeexperiments: acute gavage experiment, short-term gavage experiments and long-term feedingexperiments.(1) Acute gavage experiment36obese C57BL/6J male mice were randomly divided into2groups (n=18) according to the fasting weight, and were orally administered the MCT containing MCFA and LCTcontaining LCFA, which was a dose of2mg/kg. After1hour,2hours,4hours, respectively,6mice of each group were sacrificed, then, blood dsampling were taken from the abdominalaortic ateroia and serum TG, TC, HDL-C and LDL-C, and HDL-C/LDL-C ratio weremeasured.(2) short-term gavage experiment24C57BL/6J obese male mice were randomly divided into two groups (n=12) accordingto the fasting weight, and were orally administered MCT and LCT, which was a dose of2mg/kg daily for two weeks. During the study, mice were fed high fat diet, diet consumptionwas recorded and food efficiency ratio (KJ/d) were calculated. After two weeks, body weightwas weighed, and the body length, Lee’s index, BMI, and weight gain were measured. SerumTG, TC and HDL-C and LDL-C, HDL-C/LDL-C ratio were measured and calculate, and liver,mesenteric fat pads, epididymal pads, perirenal fat pads were excised weighed. Another sliceof liver tissue was made into homogenates, and concentration of protein, TG, TC, ApoA1andApoB were determined and ApoA1/ApoB ratio was calculated.(3) Long-term feeding experiment30C57BL/6J obese mice were randomly divided into two groups (n=15) according tothe fasting weight, and were fed high fat diet with2%concentration of MCT or LCT. After12weeks, the same indicators used in the short-term experiment were determinated. At thesame time, the epididymal adipose tissue was kept for HE staining3. Study of mechanism of MCFA regulating lipid metabolism in C57BL/6J obese miceIn the long-term feeding experiment, blood samples were collected after12weeks, anda part of the liver tissue and epididymal adipose tissue were frozen, then, were made of10%concentration of tissue homogenates with0.9%sodium chloride injection. Theconcentrations of HSL, cAMP, PKA, FFA, GLY, NADR and T3in the serum, the levels ofHSL, ATGL, cAMP, PKA, LPL, FAS, ACC, Leptin, APN, PPAR-γ, TNF-α in adipose tissue,and the levels of LPL, FAS, the ACC, ME, G6PD, Leptin, APN, PPAR-γ, TNF-α in livertissue, were measured by ELISA methods. The protein concentration of the adipose tissueand liver tissue homogenates were determinated by BCA method. mRNA expression ofHSL, ATGL, UCP2, β3-AR, leptin, PPAR-γ, SREBP-1and C/EBP-α in adipose tissue weretested by Real-time PCR assay. The protein expression of β3-AR in adipose tissue weretested using Western blotting analysis. In addition, the HSL activities in serum and adiposetissue were determinated according to the reports of Fredrikson G and Belfrage P.4. Regulations of lipid metabolism by different MCFA on C57BL/6J obese miceThe same experiment was done as the long-term feeding experiment, which was usinghigh fat diet containing2%concentration of octanoic acid (C8), decanoic acid (C10) andoleic acid (C18). After eight weeks, according to above methods, index as followed weremeasured. They were fasting body weight weekly, consumption of diets, food efficiency ratio,body length of mice, Lee’s index, BMI and weight gain, the weight of liver and adiposetissue, fat cell morphology, the concentrations of TG, TC, HDL-C, LDL-C, HDL-C/LDL-C in serum, the levels of HSL, ATGL, cAMP, PKA and Leptin, TNF-α, PPAR-γ, and mRNAexpression of HSL, ATGL and β3-AR in adipose tissue, the levels of ApoA1, ApoB, LPL,FAS, CYP7A1, HMGCoA, TNF-α, and mRNA expression of CYP7A1and HMGCoA inliver tissue.Results1. Mouse obesity modelAt the end of study, the body weight of mice, the body length, Lee’s index, BMI, andweight gain, the weight of liver and adipose tissue, the concentrations of serum glucose, TC,LDL-C in the HFD-1group were significantly higher than that of in the HFD-2group and thecontrol group (P<0.05). The ratio HDL-C/LDL-C in the HFD-1group was significantlylower than that of in the HFD-2group and the control group (P<0.05). Results of fat cellmorphology analysis showed that cell diameter, short diameter in the HFD-1group weresignificantly greater, and the number of fat cells of a single field of vision was less, than thatof in the HFD-2group and the control group (P<0.05). Obese mice model in the HFD-1group was successfully established.2. Regulations of lipid metabolism of MCFA on the C57BL/6J obese miceThe results of acute gavage experiment showed that mice fed the MCT or LCT after twohours and four hours, the concentration of serum TG in the MCT group was significantlylower than that of in the LCT group (P<0.05), and other blood lipid-related indicators werenot shown significant differences (P>0.05). The results of short-term gavage experimentshowed that the body weight of mice, the index of blood lipids, the indicators of liverhomogenates were not shown significant differences between MCT group and LCT groupafter two weeks (P>0.05). The results of long-term feeding experiment showed that the bodyweight of mice, body length, Lee’s index, BMI, and weight gain, liver weight, the weight ofperirenal and epididymal adipose tissue, the concentrations of serum TG, TC and LDL-C inthe MCT diet group were significantly lower, while the levels of serum HDL-C andHDL-C/LDL-C ratio, the concentrations of ApoA1of liver homogenates, and ApoA1/ApoBratio, were significantly higher than that of in the LCT diet group (P<0.05). The results of fatcell morphology analysis showed that cell diameter and short diameter of adipose tissue weresignificantly lower, and the number of cells of a single field of vision was significantly higherin the MCT group than that of in the LCT group (P<0.05).3. Study of mechanism of MCFA regulating lipid metabolism in C57BL/6J obese miceThe results of ELISA analysis showed that the levels of serum HSL and NADR, LPLlevel of liver tissue, ATGL, HSL and cAMP levels of adipose tissue of C57BL/6J obese micein MCT group were significantly higher, and serum FFA concentration, the levels of FAS,leptin and TNF-α of adipose tissue in MCT group were significantly lower than that of inLCT group (P<0.05). The indicators of PPAR-γ of adipose tissue, FAS, ACC, Leptin, APNand TNF-α of liver were not shown significant differences between the two groups (P>0.05).mRNA expression of ATGL, HSL, UCP2, β3-AR in adipose tissue were significantly higher, and mRNA expression of leptin, SREBP-1and C/EBP-α in adipose tissue were significantlylower in MCT group than that of in LCT group(P<0.05), and no significant differenceswere shown in PPAR-γ mRNA expression between MCT and LCT group. β3-AR proteinexpression in adipose tissue was significantly higher in MCT group than in LCT group(P<0.01). In addition, the results of HSL activities assay showed that HSL activities inadipose tissue in the MCT group were significantly higher than in the LCT group (P<0.01),while HSL activities in the serum, were not shown significant differences between the twogroups (P>0.05).4. Regulations of lipid metabolism by different MCFA on C57BL/6J obese miceC57BL/6J mice were fed high fat diet containing C8, C10or C18fatty acids after8weeks, in the C10group, the body weight of mice, Lee’s index, BMI, and weight gain, theweight of epididymal adipose tissue were significantly reduced, fat cell volume wassignificantly reduced by analysis of morphology, such as cell length and short diameter weresignificantly decreased, while the average number of cells of a single field of visionsignificantly increased, moreover, the concentrations of serum TG, the levels of ATGL, HSL,cAMP in adipose tissue were significantly increased, the levels of leptin and TNF-α inadipase tissue and FAS level in liver were significantly decreased, compared with the C18group (P<0.05). mRNA expression of ATGL, HSL, and β3-AR of adipose tissue in the C10group were significantly higher than that of in the C18group(P<0.05).In the C8group, the concentrations of serum TC and LDL-C were significantly lower,and HDL-C/LDL-C ratio of serum, ApoA1concentration and ApoA1/ApoB ratio, the level ofCYP7A1in liver tissue were significantly higher than that of in the C18group (P<0.05). Inaddition, mRNA expression of CYP7A1in liver tissue in the C8group were significantlyincreased compared with the C10and C18groups (P<0.05). mRNA expression ofHMGCoA of liver was not shown any significant differences between the three groups(P>0.05).Conclusions1. Medium-chain fatty acids (MCFA) as a natural source of small molecule nutrients caneffectively reduce body weight, reduce body fat accumulation, and improve blood lipids,blood cholesterol levels of obese mice.2. Its mechanism may be:（1） MCFAcould increase body energy consumption due to its unique metabolic pathways,then, it could increase the activities of the sympathetic nervous system, and thus increasedrelease of the peripheral NADR, which activated the expression of β3-AR on the fat cellmembrane, and leaded to increasing of expression of UCP2and enhancing the activities andlevels of enzymes related triglyceride metabolism, such as of ATGL and HSL. These couldaccelerate fat mobilization of body, and contribute to the decomposition of adipose tissue, asresults.（2） The role of activation energy metabolism was not achieved by stimulating leptin. （3） MCFA may inhibit fat cell differentiation pathway, reduce the aggregation of fat cellsby down-regulating the levels and mRNA expression of TNF-α, SREBP-1and C/EBP-α inadipose tissue, thus improve the body’s lipid metabolism disorders.（4） It was uncertain that MCFA should regulate the pathway of fatty acid synthesis andexpression of adipokines in the liver, and regulatory role of leptin, APN, and PPAR-γpathway.3. Octanoic acid (C8) and decanoic acid (C10) both could be effective in improving thelevels of blood lipids and blood cholesterol of obese mice, but octanoic acid was mainlyregulation of cholesterol metabolism in mice by increasing hepatic CYP7A1expression,while, decanoic acid mainly regulated triglyceride metabolism in mice by and increasing theexpression levels of β3-AR, ATGL and HSL in adipose tissue. The synergistic effect ofoctanoic acid and decanoic acid may be one of the mechanisms of the MCFA reducing bodyweight and improving blood lipids.更多还原