【摘要】：目的線粒體基因突變與線粒體DNA拷貝量減少共同參與線粒體功能障礙的發(fā)生,線粒體DNA拷貝量減少在年齡相關(guān)退行性疾病-2型糖尿病發(fā)病中起到重要的驅(qū)動性作用。本研究將從線粒體單基因突變的線粒體糖尿病家系入手,結(jié)合2型糖尿病的發(fā)病進(jìn)程,探討線粒體功能障礙與糖脂代謝異常的關(guān)系。通過建立線粒體內(nèi)膜調(diào)控氧化磷酸化解偶聯(lián)作用的解偶聯(lián)蛋白2 (Uncoupling Protein 2, UCP2)基因敲除小鼠高脂飼養(yǎng)模型,探索UCP2及其上游調(diào)控基因在糖脂代謝中的作用,并在血糖譜連續(xù)的人群中(健康人群-糖尿病前期-2型糖尿病)分析UCP2及其上游調(diào)控基因氧化物酶體增殖物激活受體y (Peroxisome Proliferator Activated Receptor y, PPAR y)基因多態(tài)性特點。探尋飲食結(jié)構(gòu)對線粒體功能及對位于染色體末端生物學(xué)老化的標(biāo)志物-端粒長度(Telomere Length, TL)的影響,分析飲食在線粒體介導(dǎo)糖脂代謝異常中的作用,從遺傳、環(huán)境因素兩方面探討2型糖尿病的發(fā)病機(jī)制。同時,探討血脂比(TG/HDL-C)能否作為評估胰島素抵抗以及胰島β細(xì)胞分泌功能受損的臨床指標(biāo),尋找預(yù)測2型糖尿病胰島素抵抗、胰島β細(xì)胞功能的簡易標(biāo)志物。方法人群研究對象：①線粒體糖尿病患者來自2007年4月至2015年12月間在北京協(xié)和醫(yī)院內(nèi)分泌科就診的線粒體DNA 3243 AG位點突變的線粒體糖尿病患者16例；②血糖譜連續(xù)人群來自2014年3月至2015年1月間北京農(nóng)村社區(qū)2型糖尿病管理模式的建立項目共599人；③隊列研究基線人群來自2005年4月-2006年4月就診于北京協(xié)和醫(yī)院門診的76例2型糖尿病患者。收集一般人口學(xué)特征、臨床、生化資料、飲食結(jié)構(gòu)問卷。線粒體DNA 3243位點基因突變檢測采用直接測序法(Sanger法),突變位點堿基G／A峰值比定義為=堿基G峰值高度÷堿基A峰值高度。外周血線粒體DNA拷貝量、端粒長度的測定采用熒光實時定量多聚酶式反應(yīng)。基因多態(tài)性分析采用質(zhì)譜檢測平臺,檢測8個UCP2功能區(qū)多態(tài)性位點(rs660339、rs659366.rs649446、rs586773、rs34408426、rs7109266、 rs3019463、rs591758),7個PPARγ功能區(qū)多態(tài)性位點(rs3856806,rs2920502,rs1702900、 rs73021485、 rs73813168、 rs2920503、 rs79310821)。氧化應(yīng)激指標(biāo)(SOD、 GR.8-oxo-dG)、炎癥因子(IL-6、 TNF-a)測定采用酶聯(lián)免疫吸附法測定。動物研究：C57BL/6背景的雄性UCP2-/-小鼠和同周齡C57BL/6背景的UCP2+/+小鼠。高脂飼養(yǎng)16周。肝臟組織基因表達(dá)譜采用全基因組表達(dá)芯片分析(Affymetrix Mouse Gene ST 1.0 array) 。結(jié)果1.線粒體DNA 3243 AG突變糖尿病患者臨床特點及突變異質(zhì)性與疾病譜間的關(guān)系16例線粒體糖尿病患者(起病年齡：35.0±14.6歲)伴明確的母系遺傳家族史、體型偏瘦(BMI：19.5±2.36 kg/m2)、耳聾累及高頻域。線粒體DNA 3243位點AG突變G/A峰值比與發(fā)病年齡呈顯著負(fù)相關(guān)(相關(guān)系數(shù)r=-0.841,P0.001)。2.外周血線粒體DNA拷貝量與葡萄糖刺激后胰島β細(xì)胞分泌功能的關(guān)聯(lián)分析外周血線粒體DNA拷貝量與口服葡萄糖耐量后早相、總體胰島素分泌指數(shù)呈顯著性正相關(guān),與30min、 60min、120min血糖呈顯著性負(fù)相關(guān),與空腹胰島素分泌指數(shù)、血糖、血脂并不存在顯著性相關(guān)。多元線性回歸分析表明,外周血線粒體DNA拷貝量減少可增加葡萄糖刺激后8細(xì)胞功能受損風(fēng)險(DI30:β=0.104, P= 0.019; DI120:P=0.116, P= 0.009)。多元logistic回歸分析表明外周血線粒體DNA拷貝量與2型糖尿病發(fā)生風(fēng)險呈顯著性負(fù)相關(guān)(OR:0.468,95% CI:0.245-0.893, P=0.021).3.UCP2在糖臘代謝中的作用及其代謝通路分析持續(xù)高脂飼養(yǎng)狀態(tài)下,UCP2-/-小鼠胰島β細(xì)胞功能和胰島素敏感性好于UCP2+/+小鼠,UCP2-/-小鼠血清總膽固醇、甘油三酯和游離脂肪酸顯著性低于UCP2+/+小鼠。根據(jù)肝臟基因芯片分析,UCP2-/-組與UCP2+/+組間,"PPAR信號通路”中7種基因表達(dá)顯著性上調(diào),包括PPARγ,Acsl3,Lpl, Mel,Scdl,Fads2. PPAR另外兩種異構(gòu)體—PPARck PPAR8基因表達(dá)量在UCP2-/-組和UCP2+/+組間并不存在顯著性差異。4.人群UCP2-PPARγ多態(tài)性與糖脂代謝異常相關(guān)性研究8個UCP2基因SNP位點等位基因、基因型在不同糖代謝狀態(tài)人群間不存在顯著性差異。PPARγ基因rs2920502位點等位基因堿基G、基因型GG時是糖代謝異常的保護(hù)因素(等位基因OR:0.818,95% CI:0.526-0.969,P=0.042;基因型OR:0.715,95% CI:0.527-0.97,P=0.031), GG基因型血脂TC、TG、LDL-C、 TG/HDL-C低于GC、CC基因。rs3856806位點等位基因堿基T、等位基因TT時是糖代謝異常的危險因素(等位基因OR:1.46,95%CI:1.055-2.017, P=0.022;基因型OR:1.58,95%CI:1.104-2.761,P=0.032).5.線粒體糖尿病與2型糖尿病、正常人群外周血DNA端粒長度比較端粒長度在線粒體糖尿病、2型糖尿病組顯著性低于正常對照組,但線粒體糖尿病和2型糖尿病組間并不存在顯著性差異(線粒體糖尿病vs 2型糖尿病vs正常對照：1.28±0.54 vs 1.14±0.43 vs 1.63±0.61,P=0.000)。外周血DNA端粒長度與線粒體DNA 3243突變位點堿基G/A峰值比并不存在顯著性相關(guān)性(r=-0.156,P=0.646)。6.飲食成分、碳水化合物構(gòu)成比對外周血DNA端粒長度及血糖的影響糖尿病組端粒長度較正常對照組顯著性縮短(log(TL):血糖正常組vs糖尿病前期組vs糖尿病組：2.01±0.03 vs 1.97±0.03 vs 1.89±0.03,P=0.005)。端粒長度與每日飲食總能量攝入及飲食中脂類/碳水化合物比例不存在顯著性相關(guān),多元線性回歸分析表明,豆制品、堅果、魚類、海藻類是端粒長度的保護(hù)因素,甜飲料是其危險因素(豆類：β=0.105,P=0.018；堅果：p=0.110,P=0.011：魚類:β=0.118,P=0.007：海藻類:β=0.116,P=0.009)。飲食中脂類、碳水化合物構(gòu)成比和谷類、肉類與TNF-α呈顯著性正相關(guān)(脂類：r=0.119,P=0.008；碳水化合物：r=0.094,P=0.043；谷類：r=0.091,P=0.048；肉類:r=0.405,P=0.009)。海藻類、奶制品攝入量與8-oxoˉdG呈顯著性負(fù)相關(guān)(海藻類：r=-0.496,P=0.001；奶制品：r=-0.246,P=0.046),蔬菜、水果類攝入與GR呈顯著性正相關(guān)(蔬菜：r=0.101,P=0.034；水果:r=0.125,P=0.045)。7.血脂比TG/HDL-C對胰島素抵抗和胰島β細(xì)胞功能的預(yù)測作用血糖譜連續(xù)的人群中,TG/HDL-C、TG可作為預(yù)測胰島素抵抗的血脂標(biāo)志(TG/HDL-C:ROC曲線下面積(AUROC):0.71,95% CI:0.66-0.75,P=0.000;TG:AUROC:0.71,95% CI:0.65-0.75,P=0.000);診斷胰島素抵抗TG/HDL-C、TG最佳切點值分別為：1.11,1.33mmol/L。 TG/HDL-C與HOMA-β存在顯著性負(fù)相關(guān),ROC曲線下面積均小,不宜作為判斷β細(xì)胞分泌受損的指標(biāo)。2型糖尿病患者6年隊列研究中,用DeltaC肽曲線下面積(Delta CP AUC)表示6年前后β細(xì)胞分泌功能的變化(Delta CP AUC=基線CP AUC-6年后CP AUC),根據(jù)Delta CP AUC二分位將受試者分為p細(xì)胞功能下降較慢組、下降較快組。β細(xì)胞功能下降較快組基線log (TG)/HDL-C值高于下降較慢組(0.103±0.033vs 0.083±0.030,P=0.027)。β細(xì)胞功能下降較快組基線空腹、標(biāo)準(zhǔn)餐后5個時點TG及曲線下面積均高于下降較慢組,TG與Delta CP AUC呈顯著性正相關(guān),且HDL-C低于下降較慢組,與Delta CP AUC呈顯著性負(fù)相關(guān)。結(jié)論1.早發(fā)糖尿病患者伴母系遺傳家族史、BMI正�；蚱荨⒍@可強(qiáng)烈提示線粒體糖尿病的存在。外周血DNA直接測序法對線粒體DNA 3243位點AG突變進(jìn)行鑒定,其突變G/A峰值比對線粒體疾病的起病年齡及疾病嚴(yán)重程度可起到簡單的預(yù)測作用。2.在血糖譜連續(xù)的人群中,本研究首次發(fā)現(xiàn),線粒體DNA拷貝量與葡萄糖刺激后的早相、總體胰島素分泌功能呈顯著性正相關(guān),其對于餐后血糖的影響大于對空腹血糖的影響。3.UCP2缺乏通過PPAR信號通路提高胰島素敏感性和β細(xì)胞功能來改善血糖、血脂,PPARγ多態(tài)位點(rs2920502、rs3856806)與糖脂代謝密切相關(guān),是高脂飲食狀態(tài)下調(diào)控UCP2表達(dá)的重要基因。4.端粒長度縮短可能參與糖尿病的發(fā)病機(jī)制,但并非線粒體糖尿病的特異性指標(biāo)。飲食成分可能通過改變機(jī)體氧化應(yīng)激和炎癥狀態(tài)影響端粒長度。5.在血糖譜連續(xù)的人群中,本研究首次發(fā)現(xiàn),血脂比TG/HDL-C可作為胰島素抵抗的預(yù)測標(biāo)志。TG/HDL-C與空腹β細(xì)胞分泌功能存在顯著性負(fù)相關(guān)。在2型糖尿病病程中,基線時Log (TG)/HDL-C值高預(yù)示胰島β細(xì)胞功能障礙進(jìn)展較快。
[Abstract]:Objective Mitochondrial gene mutation and mitochondrial DNA copy reduction are involved in the occurrence of mitochondrial dysfunction. Mitochondrial DNA copy reduction plays an important role in the pathogenesis of age-related degenerative disease-type 2 diabetes mellitus. To explore the relationship between mitochondrial dysfunction and abnormal glucose and lipid metabolism, a high-fat feeding model of uncoupling protein 2 (UCP2) knockout mice was established to investigate the role of UCP2 and its upstream regulatory genes in glucose and lipid metabolism and to explore the role of UCP2 and its upstream regulatory genes in blood lipid metabolism. Polymorphisms of UCP2 and its upstream regulatory gene, Peroxisome Proliferator Activated Receptor y (PPAR y), were analyzed in a population with continuous glucose profile (healthy subjects - pre-diabetes mellitus - type 2 diabetes mellitus). The effects of telomere length (TL) and diet on mitochondrial-mediated glucose and lipid metabolism were analyzed. The pathogenesis of type 2 diabetes mellitus was discussed from genetic and environmental factors. Methods A total of 16 patients with mitochondrial diabetes mellitus were selected from the Department of Endocrinology, Peking Union Medical College Hospital from April 2007 to December 2015, and 16 patients with mitochondrial DNA 3243 AG mutation were recruited. A cohort of 599 people from rural communities in Beijing from March 2014 to January 2015 were involved in the establishment of type 2 diabetes management model. Direct sequencing (Sanger method) was used to detect gene mutation at 3243 locus. The peak ratio of base G/A was defined as the peak height of base G and the peak height of base A. Seven polymorphic sites (rs3856806, rs2920502, rs1702900, rs73021485, rs73813168, rs2920503, rs79310821) in the PPAR gamma domain were identified. Oxidative stress indices (TND, GR.8-oxo-dG, IL-6, IL-a) were used for the determination of PPAR gamma domain polymorphism. Animal studies: male UCP2-/-mice with C57BL/6 background and UCP2+/+ mice with C57BL/6 background of the same age. High-fat feeding for 16 weeks. Gene expression profiles in liver tissues were analyzed by whole-genome expression microarray (Affymetrix Mouse Gene ST 1.0 array). Results 1. Clinical characteristics of diabetic patients with mitochondrial DNA 3243 AG mutation Relationship between point and mutation heterogeneity and disease spectrum in 16 patients with mitochondrial diabetes mellitus (onset age: 35.0 + 14.6 years old) with a clear maternal genetic family history, leanness (BMI: 19.5 + 2.36 kg / m2), deafness involvement and high frequency domain. The peak G/A ratio of mitochondrial DNA 3243 AG mutation was negatively correlated with onset age (correlation coefficient r = - 0.841, P 0.0). 01). 2. Correlation analysis of mitochondrial DNA copy in peripheral blood and secretory function of islet beta cells stimulated by glucose Multivariate linear regression analysis showed that the decrease of mitochondrial DNA copy in peripheral blood increased the risk of impaired 8 cell function after glucose stimulation (DI30: beta = 0.104, P = 0.019; DI120: P = 0.116, P = 0.009). Significant negative correlation (OR: 0.468, 95% CI: 0.245-0.893, P = 0.021). 3. The role of UCP2 in glucose and wax metabolism and its metabolic pathway analysis in UCP2 - / - mice islet beta cell function and insulin sensitivity were better than those in UCP2 + / + mice, and the serum total cholesterol, triglycerides and free fatty acids in UCP2 - / - mice were significantly lower than those in UCP2 + / free fatty acids. According to the liver microarray analysis, the expression of seven genes in the PPAR signaling pathway was significantly up-regulated between UCP2-/- and UCP2+/+ groups, including PPAR gamma, Acsl3, Lpl, Mel, Scdl, Fads2.PPAR, and the other two isoforms, PPARck PPAR8, were not significantly different between UCP2-/-and UCP2+/+ groups. Allele G of PPAR gamma gene rs2920502 was the protective factor for abnormal glucose metabolism (allele OR: 0.818, 95% CI: 0.526-0.969, P = 0.042; genotype OR: 0.042). 715,95% CI: 0.527-0.97, P = 0.031), GG genotype TC, TG, LDL-C, TG / HDL-C were lower than GC, CC gene. Allele T at locus. rs3856806 was a risk factor for abnormal glucose metabolism (allele OR: 1.46, 95% CI: 1.055-2.017, P = 0.022; genotype OR: 1.58, 95% CI: 1.104-2.761, P = 0.032). The length of telomere in peripheral blood was significantly lower in type 2 diabetes mellitus than in normal control group, but there was no significant difference between type 2 diabetes mellitus and mitochondrial diabetes mellitus (mitochondrial diabetes vs type 2 diabetes vs type 2 diabetes vs normal control: 1.28 + 0.54 vs 1.14 + 0.43 vs 1.63 + 0.61, P = 0.000). There was no significant correlation between the telomere length of peripheral blood DNA and the G/A peak ratio of mitochondrial DNA 3243 mutation site (r = - 0.156, P = 0.646). 6. Dietary composition, carbohydrate composition ratio of peripheral blood DNA telomere length and blood glucose were significantly shorter in diabetes mellitus group than in normal control group (log (TL): vs diabetes mellitus in normal blood glucose group. There was no significant correlation between telomere length and dietary total energy intake or lipid/carbohydrate ratio. Multivariate linear regression analysis showed that soybean products, nuts, fish and algae were protective factors for telomere length, and sweet beverage was a risk factor. Factors (legume: beta = 0.105, P = 0.105, P = 0.018; nut: P = 0.110, P = 0.011: fish: bet = 0.118, P = 0.011: fish: fish: fish: bet = 0.118, P = 0.007: algae: bet = 0.116, P = 0.009). Dielipid, carbohydrate composition ratio and cereal, meat and TNF-alphawere positively correl (lipid: r = 0.119, P = 0.008; carbohydrate: r = 0.094, P = 0.043; gluten: gluten: r = 0.043; gluten: r = 0.043; gluten: r = 0.041, r = 0.091, r = 0.09Meat Algae, dairy intake and 8-oxo-dG were negatively correlated (algae: r = - 0.496, P = 0.001; dairy products: r = - 0.246, P = 0.046). Vegetable and fruit intake were positively correlated with GR (vegetable: r = 0.101, P = 0.034; fruit: r = 0.125, P = 0.045). 7. TG/HDL-C and TG could be used to predict insulin resistance (AUROC: 0.71, 95% CI: 0.66-0.75, P = 0.000; TG: AUROC: 0.71, 95% CI: 0.65-0.75, P = 0.000); TG / HDL-C and TG best cut-off values were 1.11, 1.33 mmol/L. In a 6-year cohort study of type 2 diabetes mellitus, Delta CP AUC (Delta CP AUC = baseline CP AUC - 6 years later CP AUC) was used to represent the changes of beta cell secretory function before and after 6 years (Delta CP AUC = baseline CP AUC - 6 years later CP AUC) and the delta CP AUC binary was used to determine the impairment of beta cell secretion. The subjects were divided into two groups: the group with slower decline in P-cell function and the group with faster decline in beta-cell function. The baseline log (TG)/HDL-C values of the group with faster decline in beta-cell function were higher than those of the group with slower decline (0.103.033 vs 0.083.030, P = 0.027). The baseline fasting of the group with faster decline in beta-cell function was higher than that of the group with slower decline in TG and area under the curve at 5 time points after standard meal. Conclusion 1. Early onset diabetes mellitus patients with maternal genetic family history, normal or thin BMI, deafness can strongly suggest the existence of mitochondrial diabetes mellitus. Direct sequencing of peripheral blood DNA to identify mitochondrial DNA 3243 AG mutation, its mutation G / A Peak ratio can predict the onset age and severity of mitochondrial disease. 2. In the population with continuous blood glucose profile, this study found for the first time that mitochondrial DNA copy was positively correlated with the early phase after glucose stimulation, and the overall insulin secretion function was significantly positively correlated. The effect on postprandial blood glucose was greater than that on fasting blood. 3. UCP2 deficiency through PPAR signaling pathway to improve insulin sensitivity and beta cell function to improve blood glucose, blood lipids, PPAR gamma polymorphism sites (rs2920502, rs3856806) is closely related to glucose and lipid metabolism, is an important gene regulating UCP2 expression under high-fat diet. 4. Telomere length shortening may be involved in the pathogenesis of diabetes, but Dietary components may influence telomere length by altering oxidative stress and inflammation. 5. In a population with a continuous blood glucose profile, TG/HDL-C was found to be a predictor of insulin resistance for the first time. TG/HDL-C was negatively correlated with fasting beta cell secretion. During the course of type 2 diabetes, the high Log (TG) /HDL-C value at baseline indicated that the progression of islet beta cell dysfunction was faster.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R587.1
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本文編號：2207865