【作者】 王晓珂；

【导师】 杨年红；

【作者基本信息】 华中科技大学 ， 营养与食品卫生学， 2011， 博士

【摘要】 随着社会经济的发展和人们生活方式的改变,肥胖的发生率逐年上升,已经成为世界范围内的公共卫生问题。肥胖不仅仅是脂肪组织的过度聚集,同时与2型糖尿病、动脉粥样硬化、高血压和脂肪肝等慢性病密切相关。高脂饮食是肥胖发生的重要危险因素,但是高脂饮食条件下,并不是所有的个体都发生肥胖,而一部分发生肥胖,而另外一部分表现出了肥胖抵抗,目前相关的机制还未完全阐明。最近研究发现,高脂饮食与下丘脑炎性反应密切相关。下丘脑炎性因子表达的增加可以导致胰岛素和瘦素抵抗,从而引起机体的摄食紊乱和体重的增加。Toll样受体-4 (Toll like receptor 4, TLR4)作为保守的模式识别受体,在机体的免疫调控中有着重要作用。脑室注入饱和脂肪酸可以显著增加下丘脑TLR4和相关炎性基因的表达,TLR4抗体干预可逆转高脂饮食诱导的下丘脑炎性反应和胰岛素抵抗并降低食物的摄入量。核因子-κB (Nuclear factor-KB, NF-κB)作为TLR4的下游信号分子,在机体的炎性反应中起着关键的作用。最新研究发现NF-κB与下丘脑的胰岛素和瘦素抵抗也密切相关。利用外源性载体激活下丘脑的NF-κB可以引起下丘脑的胰岛素和瘦素敏感性降低,导致小鼠的摄食增加,而同样的方法抑制下丘脑NF-κB的活性后可使肥胖小鼠的摄食量降低,体重减轻。以上研究提示高脂饮食诱导的下丘脑炎性反应在机体的摄食紊乱和肥胖的发生中起着重要的作用。同样高脂饮食条件下,肥胖抵抗大鼠是否可以通过抑制下丘脑的炎性反应从而阻止肥胖的发生,目前还没有相关的报道。饮食诱导的肥胖个体通常伴随着胰岛素抵抗。但是有研究发现不同高脂饮食条件下,肥胖个体的胰岛素敏感性存在差异,富含多不饱和脂肪酸的高脂饮食可以改善肥胖个体的胰岛素敏感性。目前,相关的机制还不是很清楚。近年来,肥胖被认为是一种低度慢性炎症状态。长期低度的炎性反应可以导致外周的胰岛素抵抗,引起糖脂代谢紊乱。脂肪组织在肥胖个体伴随的炎性反应有着极其重要的作用,脂肪细胞与免疫细胞相互作用促进炎性因子分泌的增加,进一步研究发现脂肪组织中的巨噬细胞是炎性因子分泌的主要来源,伴随着巨噬细胞浸润的增加,炎性因子分泌增加。在介导巨噬细胞迁移和聚集的因素中,骨桥蛋白(osteopontin, OPN)因其较强的粘附趋化能力及在细胞迁移过程中的重要作用而备受关注。还有研究发现,高脂饮食条件下,OPN-/-小鼠与野生型小鼠相比体重无显著变化,但是胰岛素敏感性却显著增加,静脉注入OPN抗体可以明显改善饮食诱导肥胖小鼠的胰岛素敏感性。以上研究说明OPN介导的脂肪组织炎性反应在高脂饮食诱导的胰岛素抵抗的发生中起着重要的作用。但OPN介导的脂肪组织炎性反应是否参与了脂肪酸类型不同高脂饮食诱导的肥胖大鼠的胰岛素敏感性差异,目前尚不清楚。为了了解炎性反应和高脂饮食诱导的肥胖易感性及胰岛素敏感性差异的关系,进一步探讨肥胖和胰岛素抵抗发生的机制,我们分别建立了肥胖和肥胖抵抗动物模型以及脂肪酸类型不同的高脂饮食诱导的肥胖动物模型,分析了不同模型中大鼠下丘脑或脂肪组织炎性基因的表达变化,同时观察了膳食干预对炎性基因表达的影响。第一部分下丘脑炎性反应和高脂饮食诱导的肥胖易感性差异关系的研究目的：建立高脂饮食诱导的肥胖和肥胖抵抗动物模型,探讨下丘脑炎性反应与高脂饮食诱导的肥胖易感性差异的关系,同时观察低脂饮食干预对大鼠体重和下丘脑炎性反应的影响。方法：雄性清洁级SD大鼠55只,适应性喂养1周之后,随机分为高脂饲料组(HF,n=45)和基础饲料对照组(CF,n=10)。基础饲料由华中科技大学同济医学院实验动物中心提供；每100g高脂饲料含有基础饲料60g,猪油15g,蛋黄粉13g,干酪素10g,白糖2g。基础饲料总热能3.29kcal/g(碳水化合64.44%、蛋白质21.88%、脂肪13.68%)；高脂饲料总热能4.62kcal/g(碳水化合物30.15%、蛋白质22.00%、脂肪49.85%)。第10周时,高脂实验组大鼠体重基本稳定,参照对照组大鼠的平均体重及标准差,将高脂饲料组大鼠分为饮食诱导肥胖(Dietary induced obesity, DIO)大鼠(体重大于对照组平均体重加1.96倍标准差)和饮食诱导肥胖抵抗大鼠(Dietary induced obesity resistant, DIO-R)大鼠(体重小于对照组平均体重加1倍标准差),体重介于二者之间的大鼠不纳入本次研究。再分别将DIO和DIO-R大鼠随机分为两组,一组继续高脂饲料喂养,一组转为基础饲料喂养,干预8周。各组大鼠自由摄食和饮水,动物房温度(20±2)℃,相对湿度(50+10)%,明暗周期12比12。每天记录进食量,每周记录体重。分别与实验开始、10周和实验结束时过夜禁食12h采集尾血,离心之后保存待测。实验结束时,大鼠断头处死,迅速分离下丘脑并置液氮冻存,24小时后转移至-80℃冰箱保存,以备RNA抽提。同时分离大鼠的肾周和睾周脂肪组织并准确称重。实时定量PCR法(Real-time PCR)检测各组大鼠TLR4、NF-κB、IL-1β和IL-6基因表达水平。结果：1.喂养10周后,高脂饲料组中18只大鼠判定为肥胖易感大鼠(DIO),12只判定为肥胖抵抗大鼠(DIO-R)。DIO组大鼠的能量摄入和能量利用率显著高于DIO-R和CF组,但DIO-R组与CF组相比无统计学差异。DIO组大鼠的TC、TG、LDL-C、FBG、FINS和HOMA-IR显著高于DIO-R和CF组大鼠(P<0.05),DIO-R大鼠的TC、LDL-C、FINS和HOMA-IR与CF组相比显著增高(P<0.05)2. DIO/HF组大鼠下丘脑TLR4、NF-κB、IL-6和IL-1β表达水平显著高于DIO-R/HF和CF组大鼠,DIO-R/HF与CF组相比(P<0.05),下丘脑TLR4、NF-κB、IL-1β和IL-6表达水平没有变化。3.低脂饲料干预后,DIO/CF组大鼠的体重和脂肪蓄积量与DIO/HF组相比显著下降,DIO-R/CF的体重与DIO-R/HF组大鼠相比无变化,但脂肪蓄积量显著降低。DIO/CF和DIO-R/CF与其相应的高脂组相比,能量摄入无显著变化,能量利用率显著降低。DIO/CF组大鼠的TC、FINS和HOMA-IR与相应的高脂组相比显著降低。DIO/CF和DIO-R/CF组大鼠下丘脑TLR4、NF-κB、IL-6和IL-1β的表达水平与其相对应的高脂组相比无显著差异,DIO/CF组大鼠下丘脑炎性基因的表示仍显著高于CF组大鼠。结论：猪油配制的高脂饲料喂养10周可以诱导大鼠肥胖易感性差异的发生,高脂饮食诱导的下丘脑炎性反应可能使肥胖易感大鼠的摄食紊乱,从而导致能量摄入增加和肥胖的发生,而肥胖抵抗大鼠可以通过抑制下丘脑炎性反应保持适宜的摄食和稳定的体重。低脂饮食干预可以显著降低肥胖易感大鼠的体重和脂肪蓄积量,但对肥胖大鼠下丘脑炎性基因的表达没有影响。第二部分脂肪组织炎性反应与脂肪酸类型不同的高脂饮食诱导的肥胖大鼠胰岛素敏感性差异的研究目的：建立富含饱和脂肪酸高脂饮食和富含多不饱和脂肪酸高脂饮食诱导的肥胖大鼠模型,探讨脂肪组织炎性反应与不同类型脂肪酸高脂饮食诱导的肥胖大鼠胰岛素敏感性差异的关系,并观察低脂饮食干预对胰岛素敏感性和脂肪组织炎性基因表达的影响。方法：雄性清洁级SD大鼠100只,适应性喂养1周之后,随机分为猪油高脂饲料组(SF,n=45)、大豆油高脂饲料组(PF,n=45)和基础饲料对照组(CF,n=10)。基础饲料由华中科技大学同济医学院实验动物中心提供；每100g高脂饲料含有基础饲料60g,猪油/大豆油15g,蛋黄粉13g,干酪素10g,白糖2g。基础饲料总热能3.29kcal/g(碳水化合64.44%、蛋白质21.88%、脂肪13.68%、)；高脂饲料总热能4.62kcal/g(碳水化合物30.15%、蛋白质22.00%、脂肪49.85%)。第10周时,高脂实验组大鼠体重基本稳定,参照对照组大鼠的平均体重及标准差,分别筛选出富含饱和脂肪酸高脂饲料诱导的肥胖大鼠(Saturated fat diet induced obesity, SF-DIO)和富含多不饱和脂肪酸诱导的肥胖大鼠(polyunsaturated fat diet induced obesity, PF-DIO)大鼠。再分别将SF-DIO和PF-DIO大鼠随机分为两组,一组继续高脂饲料喂养,一组转为基础饲料喂养,继续喂养8周。实验期间,各组大鼠自由摄食和饮水,动物房温度(20+2)℃,相对湿度(50±10)%,明暗周期12比12。每天记录进食量,每周记录体重。分别与实验开始、10周和实验结束时过夜禁食12h采集尾血,离心之后保存待测。实验结束后,大鼠断头处死,迅速分离出大鼠肾周与睾周脂肪组织,准确称重后置液氮快速冷冻,-80℃冰箱保存待测。酶联免疫法和免疫印记法(Western Blotting)分别检测大鼠血清和脂肪组织OPN蛋白表达；实时定量PCR法(Real-time PCR)检测各组大鼠OPN、NF-κB、IL-6和IL-10基因表达水平。结果：1.猪油高脂饲料和大豆油高脂饲料组大鼠的肥胖发生率分别为42.2%(SF-DIO)和48.9%(PF-DIO),两组大鼠肥胖发生率无显著差异。SF-DIO组大鼠的FBG、FINS和HOMA-IR显著高于PF-DIO组大鼠和CF组大鼠(P<0.05)；PF-DIO组大鼠的FINS和HOMA-IR显著高于CF组,而FBG在两组间的差异无显著性。2. SF-DIO/SF大鼠脂肪组织OPN的基因和蛋白表达水平以及NF-κB和IL-6的基因表达水平显著高于PF-DIO/PF和CF组大鼠,同时,PF-DIO/PF组OPN、NF-κB和IL-6的表达水平也显著高于CF组,SF-DIO/SF和PF-DIO/PF组大鼠脂肪组织IL-10表达无差异,并显著低于CF组大鼠。3.低脂干预以后,SF-DIO/CF和PF-DIO/CF组大鼠的体重和脂肪蓄积量与相应的高脂组相比显著下降,能量摄入无显著变化。同时,SF-DIO/CF的FINS和HOMA-IR与SF-DIO/SF组相比显著降低,PF-DIO/CF的HOMA-IR与PF-DIO/PF组相比显著降低。SF-DIO/CF和PF-DIO/CF组大鼠脂肪组织OPN的基因和蛋白表达以及NF-κB和IL-6的基因表达与其相应的高脂组相比显著降低,但SF-DIO/CF组脂肪组织OPN和NF-κB表达水平仍显著高于PF-DIO/CF与CF组大鼠,PF-DIO/CF与CF组相比无显著差异,SF-DIO/CF和PF-DIO/CF组大鼠脂肪组织的IL-10的表达出现了增加的趋势,但与其相应的高脂组相比,差异无显著意义,仍显著低于基础饲料对照组大鼠。血清OPN含量在各个组间无统计学差异。结论：能量密度相同而脂肪酸类型不同的高脂饲料对SD大鼠具有相同的肥胖诱导作用,但是对肥胖大鼠的胰岛素敏感性具有不同的影响。饱和脂肪酸高脂饮食通过上调OPN的表达,促进脂肪组织炎性反应从而诱导肥胖大鼠的胰岛素抵抗；而多不饱和脂肪酸高脂饮食则可以抑制脂肪组织OPN的表达,降低脂肪组织炎性反应从而改善肥胖大鼠的胰岛素敏感性。低脂饮食干预可以增强肥胖大鼠的胰岛素敏感性并降低脂肪组织炎性基因的表达。创新性：1.首次探讨了高脂饮食诱导的肥胖和肥胖抵抗大鼠下丘脑炎性基因表达的变化并进一步观察了低脂饮食干预对不同肥胖易感性大鼠下丘脑炎性表达的影响,为继续探讨肥胖发生的机制及预防和治疗途径提供了新的思路和方法。2.首次针对性的探讨了脂肪组织炎症与脂肪酸类型不同高脂饮食诱导的肥胖大鼠胰岛素敏感性差异的关系,为预防和改善胰岛素抵抗提供了一条新的途径,同时观察了低脂饮食干预后炎性基因表达的变化,进一步阐明了脂肪酸的含量和类型对肥胖大鼠胰岛素敏感性的影响。更多还原

【Abstract】 The prevalence of obesity is growing rapidly and has become a major public health problem worldwide. In addition to the fat accumulation, obesity also plays a crucial role in the development of metabolic syndrome (MS).Dietary fat has been recognized as a major risk factor for the development of obesity. However, both human beings and rodents appear to be different in developing obesity when they exposed to high fat diet, described as dietary induced obesity (DIO) and dietary induced obesity resistant (DIO-R). The potential mechanism related to the different susceptibility to obesity induced by high fat diet has not been clearly elucidated. Recently, many studies revealed that increased inflammatory response in hypothalamus produces insulin and leptin resistance and contributes to the energy imbalance. TLRs are pattern recognition receptors providing the first line of host defense. Emerging study proposed that TLR4 act as a predominant molecular target for saturated fatty acids in the hypothalamus trigger the inflammatory response and ultimately results to leptin resistance and weight gain. Moreover, pharmacological inhibition designed to disrupt TLR4 signaling could reverse leptin resistance and protect against high fat diet induced food intake. NF-κB was a master switch of innate inmmunity and also associated with diminished hypothalamic insulin and leptin signal transduction. Further, experimental and genetic interventions that block the hypothalamic NF-κB signaling reversed hypothalamic insulin and leptin resistance and was associated with reduced food intake and weight loss. These data collectively implicated that the activation of hypothalamic inflammation is necessary and sufficient for the control of energy intake and likely involved in the mechanism underlying the pathogenesis of obesity during high fat diet feeding. We wonder wether the hypothalamic inflammatory response was involved in the different obesity susceptibility induced by high fat diet.Additionally, high fat diet induced obesity usually was associated with peripheral insulin resistance. Many studies have demonstrated that saturated fat diet and polyunsaturated fat diet had different effect on insulin sensitivity and the related mechanism remain unclear. Recently, obesity is considered to be a low grade chronic inflammatory state and associated with insulin resistance. Adipose tissue is major endocrine tissue promoting the interaction between metabolism and inflammation. Further research showed that the macrophages in adipose tissue are a major source of inflammatory cytokine. Among all the factors associated with migration and aggregation of macrophages, OPN (osteopontin) received more attention for its strong chemotactic ability. Further research found that OPN was also related with insulin resistance. OPN-/- mice could prevent the insulin resistance induced by high fat diet compared with the OPN+/+mice which hadthe same body weight. Moreover, the antibody of OPN could improve the insulin resistance in obese mice. These findings collectively demonstrated that OPN play an important role in the development of insulin resistance. However, it remains unclear wether OPN is involved in the different insulin sesentivity induced by saturated fat diet and polyunsaturated fat diet.Therefore, we established different animal model for investigate the underlying mechanism of different obesity susceptibility and insulin sensitivity induced by high fat diet and observe the influence of low fat diet on inflammation in hypothalamus and adipose tissue. Part IHypothalamic inflammatory response and different obesity susceptibility induced by high fat dietObjective: To investigate the hypothalamic inflammation in dietary induced obesity (DIO) and dietary induced obesity resistant (DIO-R) rats for investigating the potential mechanism related to different susceptibility to obesity and their responses to low fat diet intervention.Methods:Fifty-five six-week-old outbred male SD rats (purchased from Shanghai Sippr-BK lab animal Co. Ltd.) weighing 150-160g were housed individually with regulated temperature (22±5℃) and humidity (50±10%) on a daily cycle of 12 h light and darkness (08:00-20:00 h). All rats were allowed ad libitum access to water and food throughout the experimental period. After a week acclimation, tail blood was collected and serum was stored under-80℃for further assay. Then the rats were randomly divided into two groups:the HF group (n=45) were placed on a high-fat diet containing 4.62kcal/g (49.85% fat,20.00% protein, and 30.15% carbohydrate) and the CF group (n=10) were remained on normal laboratory chow food (purchased from Tongji Medical college lab animal center, Wuhan) containing 3.29kcal/g (13.68% fat,21.88% protein, and 64.44% carbohydrate). Dietary intake was recorded daily and body weight was measured weekly in the morning throughout the study. After 10 weeks of free access to their corresponding diet, rats in HF group with body weights more than x+1.96s of CF group were designated as DIO and those with body weight less than x+1.0s of CF group designated as DIO-R rats. Then one half of the DIO and DIO-R rats were switched to chow diet and the other half of the DIO rats and DIO-R rats were kept on HF diets for the following 8 weeks. All rats were provided water and food ad libitum. Terminally, all animals were killed after 12h fasting. Trunk blood was collected and centrifuged and serum was stored for further use. Perirenal and epididymal white adipose tissue was dissected and weighed. Hypothalamus was located and isolated according to brain coronal plane iconography of rat and related articles Samples of hypothalamic tissues were snap-frozen in liquid nitrogen immediately and stored at-80℃for RNA extraction.Results:1. Rats fed high fat diet had a wide distribution in body weight and weight gain.18 of the 45 were designated as DIO rats and 12 of which were DIO-R rats. The DIO rats had higher energy intake and energy efficiency compared with DIO-R and CF rats. TC、TG、LDL-C、FBG、FINS和HOMA-IR in DIO rats was increased compared with DIO-R and CF rats, TC、LDL-C and FINS in DIO-R rats was higher than that in CF rats.2. At the end of experiment, Hypothalamic TLR4, NF-κB, IL-1βand IL-6 mRNA expression in DIO/HF rats was significantly increased compared to DIO-R/HF and CF rats (P<0.05).3. Switching to chow food from high fat diet reduced the body weight and fat mass in DIO-R rats. The TC、FINS and HOMA-IR in DIO/CF rats was decreased compared with their counterpart on high fat diet. Low fat diet intervention failed to affect hypothalamic TLR4, NF-κB, IL-1βand IL-6 mRNA expression in DIO rats.Conclusion:SD rat fed a high fat diet showed different susceptibility to obesity. Upregulated hypothalamic inflammation may contribute to the overeating and development of obesity susceptibility induced by high fat diet and obesity resistant rats could main the appropriate energy intake and body weight by inhibiting the increased hypothalamic inflammatory response. While reduced body weight in DIO rats by chow food was not through correcting hypothalamic inflammation during the intervention period. PartⅡInflammatory response in adipose tissuce and different insulin sensitivity induced by saturated fat diet and polyunsaturated fat dietObjective:To explore the expression of OPN and related inflammatory factor in adipose tissue for investigating the underlying mechanism of the different insulin sensitivity induced by saturated and polyunsaturated fatty acid and their response to low fat diet.Methods:100 male SD rats weighing 150-160 g were housed individually in cages under controlled conditions. All rats had ad libitum access to their respective food and water throughout the study. After a week acclimation, tail blood was collected and serum was stored under-80℃for further assay. Then the rats were randomly divided into three groups, the SF group (n=45) were placed on a saturated fat diet containing 4.62kcal/g (49.85% fat,20.00% protein, and 30.15% carbohydrate), PF group (n=45) were placed on a polyunsaturated fat diet containing 4.62kcal/g (49.85% fat,20.00% protein, and 30.15% carbohydrate)and the CF group (n=10) remained on normal laboratory chow food (purchased from Tongji Medical college lab animal center, Wuhan) containing 3.29kcal/g (13.68% fat,21.88% protein, and 64.44% carbohydrate).At the end of 10th week, rats that fed high-fat diet with body weights more than x+1.96s of CF group were classified as saturated fat diet induced obesity (SF-DIO) and polyunsaturated fat diet induced obesity (PF-DIO). The SF-DIO and PF-DIO rats were then randomly subdivided into two groups respectively:one subgroup of each was shifted to chow food (SF-DIO/CF and PF-DIO/CF) and the other maintained on their respective high-fat diet (SF-DIO/SF and PF-DIO/PF). After another 8 weeks of feeding, all rats were sacrificed after 12 h fasting and trunk blood was collected and serum samples were stored at-80℃. The epididymal adipose tissue were rapidly separated, immediately frozen in liquid nitrogen, and then stored at-80℃until analysis. OPN, NF-κB, IL-6 and IL-10 mRNA expression in adipose tissue were determined by RT-PCR. Western blotting was used to determine OPN protein expression in adipose tissue and ELISA for serum OPN.Results:1. At the end of the 10th week,19 of SF group (42.2%) and 22 of PF group (48.9%) rats were designated as SF-DIO and PF-DIO respectively according to the body weight as compared with that of CF group. Serum FBG, FINS and HOMA-IR in SF-DIO group were significantly higher than PF-DIO and CF group, while the FINS and HOMA-IR in PF-DIO group were significantly higher than CF group.2. SF-DIO/SF rats had higher OPN, NF-κB and IL-6 expression compared with PF-DIO/PF and CF group, and PF-DIO/PF also had higher OPN, NF-κB and IL-6 expression compared with CF group, IL-10 expression in SF-DIO and PF-DIO rats was significantly reduced compared with CF control.3. Switching to chow food from high fat diet reduced the body weight and fat mass both in SF-DIO and PF-DIO rats without alteration of energy. FINS and HOMA-IR in SF-DIO/CF rats was reduced compared with SF-DIO/SF rats, and HOMA-IR in PF-DIO/CF rats was lower than that in PF-DIO/PF rats. The expression of OPN, NF-κB and IL-6 in SF-DIO/CF and PF-DIO/CF rat was decreased compared with their counterpart on high fat diet, SF-DIO/CF rats remained higher OPN and NF-κB expression than PF-DIO/CF rats. No difference in serum OPN was found among the group.Conclusion:Polyunsaturated fat diet showed improved insulin sensitivity compared with the saturated fat diet although they had the same effect on the development of obesity. The increased expression of OPN and related inflammatory factor in SF-DIO rats might be responsible for the development of insulin resistance, while inhibited expression of OPN and related inflammatory factors in PF-DIO rats compared with SF-DIO rats might be contributed to improved insulin sensitivity. And the improved insulin sensitivity by chow food both in SF-DIO and PF-DIO rats may be through the reduced the inflammatory response in adipose tissue during the intervention period.更多还原