【作者】 郑涓；

【导师】 陈璐璐；

【作者基本信息】 华中科技大学 ， 内科学， 2011， 博士

【摘要】 第一部分：SIRT1单核苷酸变异与中国人群超重的相关性研究目的：肥胖是遗传与环境因素相互作用所导致的疾病。随着分子遗传学的快速发展,使得在基因水平上筛选肥胖的易感基因成为可能。SNP是重要的遗传标记,其相关研究有助于理解遗传因素对肥胖症发病的影响。SIRT1是一种烟酰胺腺嘌呤二核苷酸(NAD+)依赖性去乙酰化酶,已有基于白种人的研究证明SIRT1某些的SNP位点变异与能量消耗减少及肥胖相关。因此,我们拟研究SIRT1中国人群常见基因型变异型与超重是否有关。方法：依据连锁不平衡原理,从HapMap数据库SIRT1及其邻近区域常见SNP位点(最小基因型频率≥0.05%)中,按照覆盖绝大多数SIRT1基因的变异性的原则,我们选出四种单倍型标签SNP。然后通过PCR扩增和连接酶检测反应,检测了820名中国人的SIRT1基因型并分析其与超重肥胖的关系。结果：基因型rs10509291AA和rs10823116 GG的频率在超重人群中明显增高,而基因型rs7894483TT的频率明显减低。rs10509291AT携带者超重风险较高,其基因型A符合显性基因模型。两种单倍型的携带者ATAA (rs7894483/rs10823116)和ATAG与AAAG (P<0.01)相比,表现出明显的高超重风险(OR:17.11和5.12)。TTATAA或TTATGG [rs10509291 /rs7894483 /rs10823116]的携带者分别比TTATAA或TTATGG [rs10509291 /rs7894483 /rs10823116]携带者超重风险低,该结果与rs10509291TT基因型携带者超重风险降低一致。结论：本研究结果表明基因型rs10509291,rs7894483,和rs10823116 SNP相互作用,并且与中国人群超重明显相关。第二部分：SIRT1在追赶生长大鼠腹性肥胖和胰岛素抵抗形成中的作用目的：低营养水平者之营养提升过快,即可在一过性的生长抑制后出现快速生长的现象,称之为追赶生长。追赶生长会引起大鼠内脏脂肪沉积,腹型肥胖并导致全身胰岛素抵抗。SIRT1是一种烟酰胺腺嘌呤二核苷酸(NAD+)依赖性去乙酰化酶,它在胰岛素敏感性调控中有重要作用。我们拟观察追赶生长对SIRT1表达水平及其活性的影响,并用白藜芦醇来干预追赶生长大鼠模型,提高SIRT1的活性,从而减少线粒体功能损伤的负面效应。从而了解SIRT1在追赶生长胰岛素抵抗形成中的作用。方法：采用热卡限制4周后予正常饮食8周构建营养快速提升所致成年期追赶生长大鼠模型。我们将8周龄的大鼠分为三组：正常对照组(NC),追赶生长组(CUG)和追赶生长白藜芦醇干预组(CUGE)。在喂养4周和8周以后分别检测每组大鼠的脂肪分布,骨骼肌和全身胰岛素抵抗情况。同时评价骨骼肌线粒体数量和功能、氧化应激水平及抗氧化酶活性。结果：追赶生长可引起SIRT1活性的下降,腹部脂肪量增加,并导致系统及骨骼肌胰岛素抵抗形成,表现为60-120分钟的葡萄糖平均输注速率(30%,P<0.05)和骨骼肌葡萄糖摄取率(42%,P<0.05)明显下降；线粒体柠檬酸合酶活性下降；肌膜下和肌纤维间的线粒体复合体Ⅰ-Ⅳ活性下降20-40%(P<0.05),并且肌膜下的下降得更为明显。在肌膜下和肌纤维间的线粒体中,活性氧和丙二醛的水平在是明显升高的,而抗氧化酶活性是降低的。然而,口服SIRT1激动剂白藜芦醇能增加SIRT1活性、减少腹部脂肪,增加骨骼肌线粒体数量和改善胰岛素敏感性(43%,P<0.05)。白藜芦醇还能降低脂质过氧化反应和活性氧,恢复抗氧化酶的活性。结论：本次实验表明了追赶生长可导致SIRT1表达及活性的降低,SIRT1激动剂白藜芦醇能增加SIRT1的表达及活性,通过增加骨骼肌线粒体复合体活性和加强抗氧化防御体系,来改善追赶生长大鼠的胰岛素敏感性。因此,SIRT1在大鼠腹部脂肪沉积、腹型肥胖胰岛素抵抗的形成中有重要的作用。更多还原

【Abstract】 PART I:Three single nucleotide variants of SIRT1 gene are associated with overweight in a Chinese populationObjective:The prevalence of obesity and metabolic syndrome has fueled research on genetic loci of candidate genes. We herein aimed to explore whether common allelic variations in the SIRT1 gene were associated with overweight in a Chinese cohort.Methods:Four haplotype-tagging single nucleotide polymorphisms (SNPs) were chosen for linkage disequilibrium, prevalence≥2.0%, minor allele frequency≥0.05, and coverage of the SIRT1 variability. They were genotyped in 820 Chinese individuals by PCR amplification and the ligase detection reaction.Results:The rs1050929AA and rs10823116 GG alleles had significantly higher occurrence rates in the overweight group whereas the rs7894483TT alleles had a significantly lower rate. The association of carriers of rs10509291AT with modestly higher risk of overweight was consistent with a dominant model for the A allele. Carriers of two haplotypes, ATAA (rs7894483/rs10823116) and ATAG showed a significantly higher risk (OR:17.11 and 5.12) of overweight compared with carriers of AAAG (P<0.01). In agreement with a reduced risk of overweight in carriers of rs10509291TT genotype, carriers of TTATAA or TTATGG [rs10509291/rs7894483/rs10823116] had a lower risk of overweight than carriers of ATAA(OR:13.88 vs 17.11) and ATGG (OR:1.14 vs 5.12), respectively.Conclusions:The findings of this study suggest that the rs10509291, rs7894483, and rs10823116 SNP alleles interact and have a significant association with excess weight gain in the Chinese population.PARTⅡ:Resveratrol-activated SIRT1 Improves insulin resisitance in Catch-up Growth RatsObjective:Caloric restriction (CR) followed by re-feeding, a phenomenon known as catch-up growth (CUG), affects mitochondrial function and results in systemic insulin resistance (IR). We intended to observe the effect of CUG on SIRTl expression and activity, and to increase SIRT1 activity, we interfered the rats with resveratrol, thus relieving the negative effects of mitochondrial dysfunction.Methods:Rats (8 weeks of age) were divided into three groups:normal chow (NC), CUG, and catch-up growth with RES intervention (CUGE). Fat distribution, skeletal muscle and systemic IR were measured in each group after 4 and 8 weeks. Mitochondrial biogenesis and function, oxidative stress levels, and antioxidant enzyme activity in skeletal muscle were assessed. Results:CUG induced IR resulted in significant reductions in both average glucose infusion rate60-120 (GIR60-120) at euglycemia and skeletal muscle glucose uptake. Mitochondrial citrate synthase (CS) activity was lower and activity of complexesⅠ-Ⅳin the intermyofibrillar (IMF) and subsarcolemmal (SS) mitochondria were reduced by 20-40%, with the decrease being more pronounced in the SS fraction. Reactive oxygen species (ROS) and malondialdehyde (MDA) levels were significantly higher in IMF and SS mitochondria while antioxidant enzyme activity was decreased. However, oral administration of RES increased SIRT1 activity, reduced abdominal fat accumulation, improved mitochondrial number and insulin sensitivity. RES treatment decreased levels of lipid peroxides and ROS, and restored antioxidant enzyme activity.Conclusions:We investigated the potential of RES in CUG to prevent IR by increasing activity of the mitochondrial respiratory chain and antioxidant enzymes in skeletal muscle. This study demonstrates that SIRT1 protects insulin sensitivity by improving the activity of skeletal muscle mitochondrial complexes and the antioxidant defense status in CUG rats. Thus, SIRT1 has therapeutic potential for preventing CUG-related metabolic disorders.更多还原