【作者】 李萍；

【导师】 宋光耀；

【作者基本信息】 河北医科大学 ， 内科学， 2009， 博士

【摘要】 2型糖尿病(Type 2 diabetes mellitus,T2DM)的患病率呈快速上升趋势,将成为21世纪全球的新灾难之一,预计到2030年全世界糖尿病患病人数将达到3.66亿,其并发症也在呈直线上升,目前迫切需要寻找一种有效的预防和治疗措施。众所周知,胰岛素抵抗(insulin resistance,IR)是2型糖尿病发生的主要机制之一。长期高脂饮食的人群IR和2型糖尿病发病率明显高于正常人群,动物实验也证实,高脂环境可以损害包括骨骼肌、肝脏和脂肪在内的多种胰岛素靶器官的胰岛素敏感性。目前认为高脂环境是诱发IR的独立危险因素之一。但是,饮食中的脂肪酸类型可能影响糖尿病的发病率,推测不同的脂肪酸类型可能对胰岛素敏感性产生不同的影响。动物实验显示多不饱和脂肪酸(polyunsaturated fatty acid,PUFA)和饱和脂肪酸( saturated fatty acid,SFA)可能导致IR,而单不饱和脂肪酸(monounsaturated fatty acid,MUFA)和n-3系PUFA可能改善IR。但是,关于不同类型的脂肪酸饮食对胰岛素敏感性的影响,至今国内外说法不一。随着研究的逐步深入,人们发现胰岛素信号转导障碍、脂肪源性细胞因子分泌异常、氧化应激在IR发生过程中扮演了重要的角色。脂肪组织作为一个内分泌器官,在许多生理过程中起着重要作用,可以分泌肿瘤坏死因子α(tumor necrosis factor alpha, TNF-α)、脂联素(adiponectin,APN)、抵抗素(resistin,RES)、瘦素(leptin,Lep)、白介素-6(interleukin 6,IL-6)、纤维蛋白溶酶原激活物抑制物-1 (plasminogen activator Inhibitor,PAI-1)等因子,以及发现的内脂素(visfatin,VIS)、视黄醇结合蛋白-4(retinol binding protein 4,RBP-4)等多种细胞因子。当前研究显示脂肪细胞因子的分泌紊乱是IR发生、发展过程中的重要机制,脂联素可以改善胰岛素敏感性,而其他的细胞因子可能导致IR。脂肪酸饮食对胰岛素敏感性的影响是否涉及脂肪细胞因子的改变,目前研究不多。氧化应激是指活性氧簇(reactive oxygen species,ROS)的产生与机体内源性抗氧化防御系统对其的消除能力失去平衡,过多的ROS氧化生物大分子,最终导致细胞损伤。目前对氧化应激参与胰岛素抵抗的机制尚不完全清楚,认为可能与氧化应激时被激活的多种应激敏感通路有关,如核因子-κB(nuclear factor-kappaB,NF-κB)、p38丝裂原活化蛋白激酶(ERK mitogen-activated protein kinases,MAPK)及Jun氨基末端激酶(Jun N-Terminal Kinase,JNK)通路等。各种类型的脂肪酸饮食对氧化应激的影响是否相同,至今报道较少。内质网(endoplasmic reticulum,ER)广泛存在于真核细胞中,ER可加工合成各种分泌型蛋白和膜蛋白,故其对应激极为敏感,应激状态下ER功能紊乱可破坏内质网稳态,导致未折叠蛋白堆积,产生内质网应激(endoplasmic reticulum stress,ERS)。近来研究认为ERS与肝和脂肪细胞胰岛素抵抗密切相关,因此,一些学者推测ERS可能是导致IR和T2DM的核心机制。ERS激活JNK通路,导致胰岛素受体底物-1(insulin receptor substrate 1,IRS-1)的丝氨酸磷酸化水平升高,损伤胰岛素信号通路,形成胰岛素抵抗。体外实验发现棕榈酸可以诱导ERS但油酸不可,其确切机制尚待进一步研究。本课题通过对糖耐量正常人群进行不同脂肪酸饮食干预,观察其对胰岛素敏感性的影响,并进一步研究其对氧化应激和脂肪细胞因子的影响,借以探讨其机制。同时,通过对Wistar大鼠进行不同脂肪酸饮食干预,观察其对内质网应激的影响,探讨脂肪酸饮食对胰岛素敏感性的影响机制,为预防IR和T2DM的发生提供理论基础。本研究主要包括以下四部分:第一部分不同脂肪酸类型对糖耐量正常人群胰岛素敏感性的影响目的:探讨饱和脂肪酸、单不饱和脂肪酸、多不饱和脂肪酸饮食对糖耐量正常人群胰岛素敏感性的影响。方法:20例健康受试者分别接受MUFA(M)饮食、PUFA(P)饮食、SFA(S)饮食干预3天,每组饮食干预后行口服葡萄糖耐量试验(oral glucose tolerance test,OGTT),三组饮食间隔期采用标准饮食洗脱1周。三组饮食总热量相同(热量组成:碳水化合物30%,脂肪50%,蛋白质20%),其中MUFA饮食(M组)采用油茶籽油(MUFA:82.3%,PUFA:7.6 %,SFA:9.9%,福临门),PUFA饮食(P组)采用豆油(MUFA:22.3%,PUFA:62.8%,SFA:14.8,福临门),SFA饮食(S组)采用猪油(MUFA:45.6%,PUFA:8.5%,SFA:42.7%,市售),空腹血清测定空腹血糖(FPG)、总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白-胆固醇(LDL-C)、高密度脂蛋白-胆固醇(HDL-C)、极低密度脂蛋白-胆固醇(VLDL)、胰岛素(INS)、C肽,其余各时间点血清分别测定血糖、胰岛素、C肽。结果:1.与S组相比,M组和P组的FPG显著降低,有统计学意义(P<0.05),但M组和P组之间差异无统计学意义(P>0.05)。其余时间点的血糖三组间差异均无统计学意义(P>0.05)。与S组相比,M组和P组的TC、LDL、apoB显著降低,有统计学意义(P<0.05),但M组和P组之间差异无统计学意义(P>0.05)。而三组间TG、HDL、VLDL、apoA差异均无统计学意义(P>0.05)。2.与M组相比,S组和P组的FINS、HOMA-IR、AUCins、QUCKI、WBISI显著升高,有统计学意义(P<0.01),但S组和P组之间差异无统计学意义(P>0.05)。三组间AUCglu、HOMA-B、△Ins/△FPG差异均无统计学意义(P>0.05)。结论:1.短期MUFA和PUFA饮食均可以降低空腹血糖和TC、LDL、apoB,说明UFA饮食较SFA饮食更具有抗动脉粥样硬化作用。2.MUFA饮食较PUFA、SFA饮食可以改善胰岛素敏感性,说明MUFA饮食可能更适合糖尿病高危人群及糖尿病病人。第二部分不同脂肪酸类型对糖耐量正常人群氧化应激的影响目的:探讨饱和脂肪酸、单不饱和脂肪酸、多不饱和脂肪酸饮食对糖耐量正常人群氧化应激的影响。方法:受试者选择及标本留取同第一部分。血清丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-PX)、超氧化物歧化酶(SOD)测定采用化学比色法,8-异前列腺素F2α(8-iso-PG-F2α)测定采用ELISA方法。结果:1.与M组相比,S组和P组的MDA、8-iso-PGF2α显著升高,有统计学意义(P<0.01),但S组和P组之间差异无统计学意义(P>0.05)。2.与M组相比,S组和P组的SOD、GSH-PX显著降低,有统计学意义(P<0.01),但S组和P组之间差异无统计学意义(P>0.05)。3. MDA、8-iso-PGF2α与HOMA-IR呈明显正相关(r=0.415和0.443,P<0.05),GSH-PX、SOD与HOMA-IR呈明显负相关(r=-0.327和-0.439,P<0.05)。HOMA-IR与8-iso-PGF2α(r=0.38,P<0.01)独立相关。结论:1.单不饱和脂肪酸可以改善机体的氧化应激状态。2.尽管多不饱和脂肪酸饮食对空腹血糖和血脂的影响与单不饱和脂肪酸饮食类似,但是可能由于多不饱和脂肪酸容易发生氧化,产生过氧化物,从而导致胰岛素敏感性减低。第三部分不同脂肪酸类型对糖耐量正常人群脂肪细胞因子的影响目的:探讨饱和脂肪酸、单不饱和脂肪酸、多不饱和脂肪酸饮食对糖耐量正常人群脂肪细胞因子的影响。方法:受试者选择及标本留取同第一部分。血清脂联素(APN)、肿瘤坏死因子-α(TNF-α)、C反应蛋白(CRP)、内脂素(VIS)和视黄醇结合蛋白-4 (RBP-4)测定采用ELISA方法。游离脂肪酸( FFA)测定采用化学比色法。结果:1.与M组相比,S组和P组的VIS、APN显著降低,RBP4显著升高,有统计学意义(P<0.05),但S组和P组之间差异无统计学意义(P>0.05)。与S组相比,M组和P组的TNF-α、FFA显著降低,有统计学意义(P<0.05),但M组和P组之间差异无统计学意义(P>0.05)。三组间CRP差异均无统计学意义(P>0.05)。2.TNF-α、RBP4与HOMA-IR呈明显正相关(r=0.423和0.372,P<0.05),VIS、APN与HOMA-IR呈明显负相关(r=-0.458和-0.349,P<0.05)。HOMA-IR与VIS(r=0.34,P<0.01)独立相关。结论:单不饱和脂肪酸饮食可以降低RBP4、TNF-α,升高VIS、APN水平,可能借此来改善胰岛素抵抗。第四部分不同脂肪酸类型对大鼠肝脏eIF2α和XBP-1的影响目的:探讨饱和脂肪酸、单不饱和脂肪酸、多不饱和脂肪酸饮食对大鼠肝脏真核细胞翻译起始子2α(eIF2α)和X盒结合蛋白-1(XBP-1)的影响。方法:48只大鼠适应性喂养1周后随机分为4组,每组12只,正常对照组(normal control group, N组),实验组分为饱和脂肪酸饮食组(saturated fatty acid diets group, S组)、单不饱和脂肪酸饮食组(monounsaturated fatty acid diets group, M组)、多不饱和脂肪酸饮食组(polyunsaturated fatty acid diets group,P组)。正常对照组给予基础饲料,热量组成为碳水化合物占65.5%,脂肪占10.3%,蛋白质占24.2%;饱和脂肪酸饲料是在基础饲料中添加15%猪油(热量组成为脂肪35.4%,碳水化合物47.2%,蛋白质17.4%);单不饱和脂肪酸饲料是在基础饲料中添加15%茶油(热量组成为脂肪35.4%,碳水化合物47.2%,蛋白质17.4%);多不饱和脂肪酸饲料是在基础饲料中添加15%豆油(热量组成为脂肪35.4%,碳水化合物47.2%,蛋白质17.4%)。所有饲料均由河北医科大学实验动物中心提供。两组每日等热量投喂饲料,每周称体重1次。8周后4组各随机选8只大鼠行高胰岛素-正葡萄糖钳夹试验,同时留取空腹状态血清测定血脂、胰岛素等指标。钳夹实验后喂养3天处死。实验结束后颈动脉放血处死动物,立即取出肝脏组织放入液氮中冷冻,然后保存于-70℃低温冰箱。肝脏XBP-1和p-eIF2α蛋白表达分别用Western Blotting法和免疫组化法测定。结果:1.实验第8周末,与P、M、S组相比,N组大鼠进食量有所增加,差异有统计学意义(P>0.05)。除外进食量的影响,与S组相比,N、M、P组血清TC、TG降低,差异有统计学意义(P<0.05),而N、M、P三组间无统计学意义(P>0.05)。与N、M组相比,S、P组FINS、FBG升高,差异有统计学意义(P<0.05),GIR明显下降,差异有统计学意义(P<0.05),但S、P两组间无统计学意义(P>0.05)。与N组相比,M组血清FINS、FBG有升高趋势,差异无统计学意义(P>0.05),GIR下降,差异有统计学意义(P<0.05)。2.电镜显示P组和S组大鼠肝脏出现内质网扩张,脱颗粒改变,而M组和N组类似。3. p-elF2α主要在细胞核中表达, P、S组表达较N组、M组显著增多,有统计学意义(P<0.05),但P、S两组间及N、M两组间比较均无统计学意义(P>0.05)。XBP-1在细胞浆和细胞核均有表达,呈棕黄色或棕褐色染色,N组、M组主要在细胞核表达,与之相比,P、S组在细胞浆和细胞核均有表达,但是四组间无统计学意义(P>0.05);Western blot显示XBP-1蛋白相对表达的量分别是:M组(0.85±0.15)、N组(0.79±0.14)、P组(1.09±0.16)、S组(1.15±0.21)。与N、M组比较,P、S组肝脏XBP-1蛋白表达升高,差异有统计学意义(P<0.05);但P、S两组间及N、M两组间比较均无统计学意义(P>0.05)。结论:1.动物实验中,尽管单不饱和脂肪酸饮食可以减轻胰岛素抵抗,但是与正常对照组相比还是有差异的。说明无论何种脂肪酸饮食,长期高脂饮食可以加重胰岛素抵抗。2.PUFA、SFA组饮食喂养所致大鼠肝脏中XBP-1、p-eIF2α蛋白表达明显高于正常对照和M组,说明ERS可能参与了IR的发生。更多还原

【Abstract】 Type 2 diabetes is rapidly emerging as one of the greatest global health challenges of the 21st century. The World Health Organization estimates that by the year 2030, 366 million people will be afflicted with diabetes. This looming epidemic is also expected to trigger a steep rise in the complications associated with diabetes. Developing better treatments and novel prevention strategies for type 2 diabetes is therefore a matter of great urgency. It is well known that insulin resistance plays a critical role in the development of type 2 diabetes.High-fat diets have been shown to produce insulin resistance,animal experiments also prove that high-fat exposure on impaires insulin sensitivity in several insulin target organs, including skeletal muscles, liver, adipocytes etc. Certain fatty acids may have a more deleterious effect on insulin action than others. In animal models, high intake of saturated and polyunsaturated fats induce severe insulin resistance, whereas monounsaturated fats and n-3 fatty acids are less detrimental. However, the relationship between dietary fat acids and insulin sensitivity is not clear, and there are few studies in this field.With the development of molecular biological technology, many specialists discovered the defects in the insulin signaling pathway, abnormal secretion of adipokines and oxidative stress all contributed to the development of insulin resistance. More and more people realized that adipose tissue was an endocrine organ. Many cytokines secreted by adipocyte such as leptin, adiponectin, tumor necrosis factor alpha (TNF-α), interleukin 6(IL-6), plasminogen activator Inhibitor(PAI-1), resistin, visfatin, and retinol binding protein 4(RBP-4) are all related with the insulin resistant state. The present studies demonstrated that these adipokines performed different actions in IR; adiponectin can improve the insulin sensitivity, while the increase of other cytokines can impair the insulin sensitivity. Oxidative stress is caused by an imbalance between the production of reactive oxygen and a biological system’s ability to readily detoxify the reactive intermediates or easily repair the resulting damage. It is uncertain that ROS indirectly induce damage to tissues by activating a number of cellular stress-sensitive pathways. These pathways include nuclear factor-kappaB(NF-κB), p38 mitogen-activated protein kinase(MAPK), NH2-terminal Jun kinases/stress-activated protein kinases(JNK), hexosamines, and others.Endoplasmic reticulum(ER) is a membranous network that functions in the synthesis and processing of secretory and membrane proteins. Certain pathological stress conditions disrupt ER homeostasis and lead to accumulation of unfolded or misfolded proteins in the ER lumen. A recent study has uncovered a link between insulin resistance and endoplasmic reticulum (ER) stress in liver and adipose cells. Therefore, some learners postulated that ER stress is probably a core mechanism involved in triggering insulin resistance and type 2 diabetes. Induction of ER stress leads to hyperactivation of JNK, reduced insulin receptor signaling, systemic insulin resistance and type 2 diabetes. In vivo, palmitate but not oleate induces significant ER stress, but the precise mechanism needs to be further studied.In the present study, we explore influence of dietary fat acids on insulin sensitivity in healthy adults. Given the link between hepatic cell ER stress and insulin resistance, we examine the effects of chronic FFA treatment on ER stress to provide a new study direction to improve insulin resistance and type 2 diabetes. The paper contains four parts below:Part one: Effects of dietary fat acids on insulin sensitivity in healthy adultsObjective: To investigate the effects of saturated fats diets, polyunsaturated fats diets and monounsaturated fats diets on insulin sensitivity in healthy adults.Methods: 20 subjects were recruited from Hebei General Hospital, all subjects were healthy. Each of the three experimental diets was fed for 3 days with a minimum 1-week washout between diets. All subjects completed each of the diets in order. At the end of each 3-day diet period, subjects returned to the clinic for measurement of serum glucose, lipids and insulin sensitivity. The target nutrient composition for all three diets was 30% carbohydrate, 50% fat, 20% protein. Monounsaturated fat acid diet (M) was provided by tea oil, polyunsaturated fat acid diet (P) was provided by soybean oil, Saturated fat acid diet (S) was provided by lard oil.Results:1. Fasting blood glucose(FBG) in S group adults were higher than that in isocaloric M and P group. Compared with S group, serum total cholesterol(TC), Low-density lipoprotein(LDL) and apolipoprotein B(apoB) were significantly lower in M and P group,but triglyceride(TG),high-density lipoprotein(HDL), very low-density lipoprotein(VLDL) and apolipoprotein A(apoA) in both groups didn’t increase significantly (p>0.05). 2.Compared with M group, Fasting insulin(FINS), Homeostasis Model Assessment insulin resistance index (HOMA-IR), (AUCins) and whole body insulin sensitivity index(QUCKI) were higher in P and S group(p<0.01). There was no detectable difference in AUCglu、HOMA-B、△Ins/△FPG among three groups (p>0.05).Conclusion: 1.Short-term MUFA-rich and PUFA-rich diets decrease fasting blood glucose and TC, LDL and apoB, which demonstrated that UFA diets may have anti-atherosclerosis effects. 2. MUFA-rich diet improves insulin sensitivity, which probably fits for patients at high risk of diabetes or diabetics.Part two: Effects of dietary fat acids on oxidative stress in healthy adultsObjective: To observe the effects of saturated fats diets, polyunsaturated fats diets and monounsaturated fats diets on oxidative stress in healthy adults.Methods: Subjects grouping and the samples acquirement were the same as those in part one. Fasting serum malondialdehyde (MDA),glutathione peroxidase (GSH-PX), and superoxide dismutase (SOD) were measured by Colorimetric method. 8-iso-PG-F2αwas measured by enzyme-linked immunosorbent assay( ELISA). Results : 1.Compared with M group, MDA and 8-iso-PG-F2αwere significantly higher in S and P group(p<0.01). 2.Compared with M group, SOD and GSH-PX were lower in P and S group(p<0.01), but there was no detectable difference in SOD and GSH-PX between P and S groups (p>0.05). 3. All four markers were significantly associated with HOMA-IR, and 8-iso-PG-F2αwas correlated to HOMA-IR independently.Conclusion: The present study indicates that ingestion of PUFA and SFA diets could result in an imbalance between oxidant and antioxidant, and thus induce oxidative stress in healthy adults, but MUFA diets can reduce oxidative stress.Part three: Effects of dietary fat acids on adipocytokines in healthy adultsObjective: To explore the effects of saturated fats diets, polyunsaturated fats diets and monounsaturated fats diets on adipocytokines in healthy adults.Methods: Subjects grouping and the samples acquirement were the same as those in part one. Samples were analyzed for adiponectin (APN), TNF-α, visfatin (VIS), high-sensitivity C-RP (C-RP), FFA and retinol binding protein 4 (RBP-4). Adiponectin, TNF-α, visfatin, hsC-RP and RBP-4 were analyzed by enzyme-linked immunosorbent assay(ELISA). Free fatty acids (FFA) was measured by Colorimetric method .Results:1. Compared with M group, the levels of RBP4 were significantly higher in S and P groups, and VIS and APN were significantly lower in S and P groups(p<0.05). 2. Compared with S group, TNF-αand FFA were lower in P and M groups(p<0.01), but there was no detectable difference in TNF-αand FFA between P and M groups (p>0.05). No significantly difference in CRP was observed among three groups (p>0.05). 3. Serum TNF-αand FFA were positvely correlated with HOMA-IR. Serum VIS and APN was negatively correlated with HOMA-IR. Multiple regression analysis showed that visfatin was independent related factors influencing HOMA-IR.Conclusion: Fasting serum CRP was not detectably affected by alterations in dietary fatty acid profile in healthy adults. MUFA diets improves insulin sensitivity by decreasing RBP4、TNF-αand increasing VIS、APN. Part four: Effects of dietary fat acids on eIF2αand XBP-1 in liver tissues in ratsObjective: To explore the effects of saturated fats diets, polyunsaturated fats diets and monounsaturated fats diets on eukaryotic initiation factor 2alpha (eIF2α) and X-box binding protein 1(XBP-1) in liver tissues in rats.Methods: Forty-eight male Wistar rats were randomly divided into normal control (N) group (n=12), saturated fatty acid diets group(S)(n=12), monounsaturated fatty acid diets group(M) (n=12),and polyunsaturated fatty acid diets group(P) (n=12). The rats in control group were fed with a regular low fatty acids diet containing 10.3% fat, 24.2% protein, and 65.5% carbohydrate as percentage of total calories. The rats in M, P and S group were fed regular diets mixed with 15% tea oil, soybean oil, lard oil respectively, containing 35.4% fat, 17.4% protein and 47.2% carbohydrate as percentage of total calories. The rats in every group were fed equal calories every day. The body weights were determined weekly for 8 weeks. The blood sample was collected by cardiac puncture after rats were anesthetized with diethyl ether for the biochemical analysis, insulin resistance was evaluated by glucose infusion rate (GIR) of hyperinsulinemic euglycemic clamp technique. At the end of 8 week, the rats were killed after anesthetized with phenobarbital sodium after hyperinsulinemic euglycemic clamp test, and liver tissues were taken out freeze-clamped with copper clamps precooled in liquid N2 and were stored in -70℃refrigerator. Liver XBP-1 was measured by Western-blot method. The SP immunohischemistry methods were used to detect the distribution changes of p-eIF2αand XBP-1 in the rats liver.Results:1. Compared with N group, the amount of food intake were significantly lower in S, M and P groups(p<0.05). Adjusted for the amount of food intake, Compared with S group, serum TC, TG were significantly lower in N, M and P groups(p<0.05), but there was no detectable difference in those among N, P and M groups (p>0.05). Compared with N and M groups, FINS and FBG were significantly higher in S and P groups (p<0.05) , GIR was significantly lower in S and P groups (p<0.05). Compared with N group, GIR were significantly lower in M group (p<0.05). 2.The change of liver ultrastructure in P and S groups haved rough endoplasmic reticulum enlargement, taking off grain. The change in M group were nearly the same as those in N group. 3.Immunohistochemistry analysis results showed that cells positive of p-eIF2αwere intense brown staining and localized mainly in cytoblast,cells positive of XBP-1 were intense brown staining and localized both in cytoblast and cytoplasm. Further, western blot for XBP-1 in liver tissues showed that XBP-1 protein levels increased in P and S groups compared with N and M groups(P<0.05).Conclusions: 1.Long-term high fat diet can lead to insulin resistance,and different kinds of acids may have different impact on insulin sensitivity; diets rich in saturated and polyunsaturated,induce more insulin resistance than other kinds of diets. 2.It may be one of the mechanisms of insulin resistance that PUFA and SFA diets can induce higher expression of XBP-1 and p-eIF2αin liver tissues, simultaneously, which suggested that endoplasmic reticulum stress(ERS) maybe a core mechanism involved in triggering insulin resistance.更多还原