游离脂肪酸致胰岛素抵抗的机制研究
发布时间：2019-01-05 14:08
【摘要】：胰岛素抵抗(insulin resistance,IR)是大部分2型糖尿病(type II diabetes mellitus,T2DM)的主要代谢紊乱,在全球范围内呈现流行性的发病趋势。IR由一系列生理因素的改变导致,其中包括胰岛素受体信号转导缺陷、线粒体功能障碍、微脉管功能障碍和炎症作用等。这些改变导致肝脏和骨骼肌特异性脂质代谢物(甘油二脂和/或神经酰胺)的积累,最终损伤胰岛素信号转导,导致IR。血清中游离脂肪酸(free fatty acid,FFA)水平升高是脂肪代谢异常的典型表现,也是肥胖引起的IR的主要因素之一。目前的研究表明FFA致IR的机制包括了氧化应激、炎症作用、凋亡、线粒体功能障碍、内质网应激等,涉及的分子主要包括JNK(Jun N-尾激酶)、ROS(活性氧)、PKCδ(蛋白激酶C-δ)、NLRP3(核苷酸结合寡聚化结构域样受体蛋白3)等。但大部分的研究都是在细胞和动物模型中进行的,同时涉及的分子数量有限,信号通路单一,因此,本研究从前期糖尿病(pre-diabetes mellitus,Pre-DM)和T2DM患者血清中FFA与IR的关系研究出发,通过对FFA水平异常升高的IR患者与FFA水平正常的健康体检人群的血液样本进行胰岛素信号通路基因芯片分析,筛选参与FFA致IR疾病过程的与胰岛素信号转导通路相关的差异表达基因,寻找可能的致病分子,从而为早期诊断及疾病治疗提供分子靶点和理论依据。本研究收集在兰州总医院入院治疗并诊断为T2DM的患者154例、体检中心体检人群中被诊断为Pre-DM的患者39例、以及体检中心体检的健康人33例,按相关诊断标准将T2DM分为T2DM肥胖组(O组)、T2DM非肥胖组(N组),将Pre-DM分为空腹血糖受损组(IFG组)和糖耐量受损组(IGT),健康对照组为NC组。测定入选者的基本体质指标、生化指标、内分泌指标和血清FFA水平,计算胰岛素抵抗指数(HOMA-IR),用t检验、非参数秩和检验、Pearson相关分析和多元线性回归分析进行统计学分析。然后在O组、N组和NC组中分别筛选符合条件的病例共10例,提取外周血中的mRNA,反转录为cDNA,进行基因芯片检测,筛选差异表达基因。最后将筛选出的与FFA致IR的相关差异表达基因DOK1,在T2DM和Pre-DM中其他符合条件的样品中进行荧光定量PCR检测,分析表达情况,验证基因芯片结果的可靠性。本研究结果表明:(1)T2DM组、N组以及O组的FFA水平和HOMA-IR均显著高于NC组;在N组中,HOMA-IR与FFA存在正相关关系,随着FFA的增加,HOMA-IR增大,FFA对HOMA-IR的影响较大。(2)IFG组、IGT组和Pre-DM组中FFA和HOMA-IR均显著高于NC组;Pre-DM患者HOMA-IR与FFA正相关关系,随着FFA的增加,HOMA-IR增大,同时FFA对HOMA-IR的影响最大。(3)基因芯片分析结果表明O组与NC组相比,基因FOS(FBJ murine osteosarcoma viral oncogene homolog,FBJ鼠科动物骨肉瘤病毒致癌基因同系物)显著下调,差异倍数为3.58(p0.05);N组与NC组相比,基因Fos、己糖激酶2(hexokinase 2,HK2)、衔接蛋白1(docking protein 1,DOK1)显著下调,差异倍数分别为10.71、2.32、2.22(p0.05);O组与N组相比,基因FOS、丝裂原活化蛋白激酶(mitogen-activated protein kinase1,MAPK1)显著上调,基因AE结合蛋白1(AE binding protein1,AEBP1)显著下调的,差异倍数分别为2.72、1.77、1.79(p0.05)。(4)荧光定量PCR分析各实验组中DOK1表达水平的结果表明与NC组相比,N组和Pre-DM组的DOK1表达显著降低(z=-2.121、-2.40,p0.05),T2DM-O组的DOK1表达并无显著变化(z=-1.633,p0.05)。综上所述,我们推测,对于T2DM中的非肥胖患者以及前期糖尿病的患者来说,血清FFA水平的异常升高可能与DOK1的显著下调有关,同时可能通过下调DOK1的表达,抑制脂肪合成,促进脂肪分解,促使或加重IR状态。
[Abstract]:Insulin resistance (IR) is a major metabolic disorder in most type II diabetes mellitus (T2DM), which presents the trend of epidemic in the world. IR is caused by a series of physiological factors, including insulin receptor signal transduction, mitochondrial dysfunction, microvessel dysfunction, and inflammation. These changes lead to the accumulation of the liver and skeletal muscle-specific lipid metabolites (diglycerides and/ or neurotransmitters), which ultimately damage the insulin signal transduction, leading to IR. The increase of free fatty acid (FFA) in serum is a typical manifestation of the abnormal fat metabolism, and is one of the main factors of the IR caused by obesity. The present study shows that the mechanism of FFA-induced IR includes oxidative stress, inflammation, apoptosis, mitochondrial dysfunction, endoplasmic reticulum stress, etc. The molecules involved mainly include JNK (Jun N-tail kinase), ROS (active oxygen), and PKC inhibitor (protein kinase C-1). NLRP3 (nucleonic acid binding to the oligomerization domain-like receptor protein 3), and the like. However, most of the studies were performed in cells and animal models, and the number of molecules involved was limited and the signal pathway was single, and therefore, this study was based on the study of the relationship between the FFA and IR in the serum of pre-diabetes mellitus (Pre-DM) and T2DM patients. Through the analysis of the insulin signal pathway gene chip of the blood sample of the healthy physical examination population with the abnormal increase of the FFA level and the normal healthy physical examination population of the FFA level, the differential expression genes associated with the insulin signal transduction pathways involved in the FFA-induced IR disease process are screened, and the possible pathogenic molecules are searched, so as to provide molecular targets and theoretical basis for early diagnosis and disease treatment. The study collected 154 patients with T2DM, 39 patients diagnosed as Pre-DM in the physical examination group and 33 healthy subjects with physical examination, and the T2DM was divided into the obese group (O-group) according to the relevant diagnostic criteria. The non-obese group of T2DM (group N) was divided into the fasting blood glucose-impaired group (IFG group) and the impaired glucose tolerance group (IGT), and the healthy control group was the NC group. The basic body constitution index, the biochemical index, the endocrine index and the serum FFA level were measured, and the insulin resistance index (HOMA-IR) was calculated. The statistical analysis was performed with t-test, non-parametric rank and test, Pearson correlation analysis and multivariate linear regression analysis. In the group of O, N, and NC, 10 of the eligible cases were selected, the mRNA in the peripheral blood was extracted, the reverse transcription was the cDNA, and the gene chip was detected and the differentially expressed genes were selected. and finally, screening out the relevant difference expression gene DOK1 with the FFA-induced IR, carrying out fluorescence quantitative PCR detection on other eligible samples in the T2DM and the Pre-DM, and analyzing the expression condition and verifying the reliability of the gene chip result. The results showed that: (1) FFA and HOMA-IR of T2DM group, N group and O group were significantly higher than that of NC group; in group N, HOMA-IR was positively correlated with FFA, and with the increase of FFA, HOMA-IR increased, and the effect of FFA on HOMA-IR was large. (2) The FFA and HOMA-IR in the IFG group, the IGT group and the Pre-DM group were significantly higher than those in the NC group; the HOMA-IR of the Pre-DM patients was positively related to the FFA, and the HOMA-IR increased with the increase of FFA, while the effect of FFA on the HOMA-IR was the most. (3) The results of gene chip analysis showed that the gene FOS (FOS (FBJ), FOS (FOS), FOS (FOS), FOS (FOS), hexose kinase 2 (HK2) and linking protein (1) were significantly lower than that of NC group. The difference of DOK1 was 10.71, 2.32, 2.22 (p0.05). Compared with the N group, the gene FOS, mitogen-activated protein kinase 1 (MAPK1) increased significantly, and the gene AE binding protein 1 (AEBP1) was down-regulated. The difference was 2.72, 1.77, 1.79 (p0.05). (4) The results showed that the expression of DOK1 in the group of N and Pre-DM decreased significantly (z =-2.121,-2.40, p0.05) compared with the NC group, and the expression of DOK1 in the T2DM-O group did not change significantly (z =-1.633, p0.05). In conclusion, we have speculated that, for patients with non-obese and early-stage diabetes in T2DM, an abnormal increase in serum FFA levels may be associated with a significant down-regulation of DOK1, possibly by lowering the expression of DOK1, inhibiting fat synthesis, promoting lipolysis, to cause or exacerbate the ir state.
【学位授予单位】：兰州理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1
[Abstract]:Insulin resistance (IR) is a major metabolic disorder in most type II diabetes mellitus (T2DM), which presents the trend of epidemic in the world. IR is caused by a series of physiological factors, including insulin receptor signal transduction, mitochondrial dysfunction, microvessel dysfunction, and inflammation. These changes lead to the accumulation of the liver and skeletal muscle-specific lipid metabolites (diglycerides and/ or neurotransmitters), which ultimately damage the insulin signal transduction, leading to IR. The increase of free fatty acid (FFA) in serum is a typical manifestation of the abnormal fat metabolism, and is one of the main factors of the IR caused by obesity. The present study shows that the mechanism of FFA-induced IR includes oxidative stress, inflammation, apoptosis, mitochondrial dysfunction, endoplasmic reticulum stress, etc. The molecules involved mainly include JNK (Jun N-tail kinase), ROS (active oxygen), and PKC inhibitor (protein kinase C-1). NLRP3 (nucleonic acid binding to the oligomerization domain-like receptor protein 3), and the like. However, most of the studies were performed in cells and animal models, and the number of molecules involved was limited and the signal pathway was single, and therefore, this study was based on the study of the relationship between the FFA and IR in the serum of pre-diabetes mellitus (Pre-DM) and T2DM patients. Through the analysis of the insulin signal pathway gene chip of the blood sample of the healthy physical examination population with the abnormal increase of the FFA level and the normal healthy physical examination population of the FFA level, the differential expression genes associated with the insulin signal transduction pathways involved in the FFA-induced IR disease process are screened, and the possible pathogenic molecules are searched, so as to provide molecular targets and theoretical basis for early diagnosis and disease treatment. The study collected 154 patients with T2DM, 39 patients diagnosed as Pre-DM in the physical examination group and 33 healthy subjects with physical examination, and the T2DM was divided into the obese group (O-group) according to the relevant diagnostic criteria. The non-obese group of T2DM (group N) was divided into the fasting blood glucose-impaired group (IFG group) and the impaired glucose tolerance group (IGT), and the healthy control group was the NC group. The basic body constitution index, the biochemical index, the endocrine index and the serum FFA level were measured, and the insulin resistance index (HOMA-IR) was calculated. The statistical analysis was performed with t-test, non-parametric rank and test, Pearson correlation analysis and multivariate linear regression analysis. In the group of O, N, and NC, 10 of the eligible cases were selected, the mRNA in the peripheral blood was extracted, the reverse transcription was the cDNA, and the gene chip was detected and the differentially expressed genes were selected. and finally, screening out the relevant difference expression gene DOK1 with the FFA-induced IR, carrying out fluorescence quantitative PCR detection on other eligible samples in the T2DM and the Pre-DM, and analyzing the expression condition and verifying the reliability of the gene chip result. The results showed that: (1) FFA and HOMA-IR of T2DM group, N group and O group were significantly higher than that of NC group; in group N, HOMA-IR was positively correlated with FFA, and with the increase of FFA, HOMA-IR increased, and the effect of FFA on HOMA-IR was large. (2) The FFA and HOMA-IR in the IFG group, the IGT group and the Pre-DM group were significantly higher than those in the NC group; the HOMA-IR of the Pre-DM patients was positively related to the FFA, and the HOMA-IR increased with the increase of FFA, while the effect of FFA on the HOMA-IR was the most. (3) The results of gene chip analysis showed that the gene FOS (FOS (FBJ), FOS (FOS), FOS (FOS), FOS (FOS), hexose kinase 2 (HK2) and linking protein (1) were significantly lower than that of NC group. The difference of DOK1 was 10.71, 2.32, 2.22 (p0.05). Compared with the N group, the gene FOS, mitogen-activated protein kinase 1 (MAPK1) increased significantly, and the gene AE binding protein 1 (AEBP1) was down-regulated. The difference was 2.72, 1.77, 1.79 (p0.05). (4) The results showed that the expression of DOK1 in the group of N and Pre-DM decreased significantly (z =-2.121,-2.40, p0.05) compared with the NC group, and the expression of DOK1 in the T2DM-O group did not change significantly (z =-1.633, p0.05). In conclusion, we have speculated that, for patients with non-obese and early-stage diabetes in T2DM, an abnormal increase in serum FFA levels may be associated with a significant down-regulation of DOK1, possibly by lowering the expression of DOK1, inhibiting fat synthesis, promoting lipolysis, to cause or exacerbate the ir state.
【学位授予单位】：兰州理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1
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