游離脂肪酸致胰島素抵抗的機(jī)制研究
發(fā)布時(shí)間：2019-01-05 14:08
【摘要】：胰島素抵抗(insulin resistance,IR)是大部分2型糖尿病(type II diabetes mellitus,T2DM)的主要代謝紊亂,在全球范圍內(nèi)呈現(xiàn)流行性的發(fā)病趨勢。IR由一系列生理因素的改變導(dǎo)致,其中包括胰島素受體信號(hào)轉(zhuǎn)導(dǎo)缺陷、線粒體功能障礙、微脈管功能障礙和炎癥作用等。這些改變導(dǎo)致肝臟和骨骼肌特異性脂質(zhì)代謝物(甘油二脂和/或神經(jīng)酰胺)的積累,最終損傷胰島素信號(hào)轉(zhuǎn)導(dǎo),導(dǎo)致IR。血清中游離脂肪酸(free fatty acid,FFA)水平升高是脂肪代謝異常的典型表現(xiàn),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的機(jī)制包括了氧化應(yīng)激、炎癥作用、凋亡、線粒體功能障礙、內(nèi)質(zhì)網(wǎng)應(yīng)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(核苷酸結(jié)合寡聚化結(jié)構(gòu)域樣受體蛋白3)等。但大部分的研究都是在細(xì)胞和動(dòng)物模型中進(jìn)行的,同時(shí)涉及的分子數(shù)量有限,信號(hào)通路單一,因此,本研究從前期糖尿病(pre-diabetes mellitus,Pre-DM)和T2DM患者血清中FFA與IR的關(guān)系研究出發(fā),通過對(duì)FFA水平異常升高的IR患者與FFA水平正常的健康體檢人群的血液樣本進(jìn)行胰島素信號(hào)通路基因芯片分析,篩選參與FFA致IR疾病過程的與胰島素信號(hào)轉(zhuǎn)導(dǎo)通路相關(guān)的差異表達(dá)基因,尋找可能的致病分子,從而為早期診斷及疾病治療提供分子靶點(diǎn)和理論依據(jù)。本研究收集在蘭州總醫(yī)院入院治療并診斷為T2DM的患者154例、體檢中心體檢人群中被診斷為Pre-DM的患者39例、以及體檢中心體檢的健康人33例,按相關(guān)診斷標(biāo)準(zhǔn)將T2DM分為T2DM肥胖組(O組)、T2DM非肥胖組(N組),將Pre-DM分為空腹血糖受損組(IFG組)和糖耐量受損組(IGT),健康對(duì)照組為NC組。測定入選者的基本體質(zhì)指標(biāo)、生化指標(biāo)、內(nèi)分泌指標(biāo)和血清FFA水平,計(jì)算胰島素抵抗指數(shù)(HOMA-IR),用t檢驗(yàn)、非參數(shù)秩和檢驗(yàn)、Pearson相關(guān)分析和多元線性回歸分析進(jìn)行統(tǒng)計(jì)學(xué)分析。然后在O組、N組和NC組中分別篩選符合條件的病例共10例,提取外周血中的mRNA,反轉(zhuǎn)錄為cDNA,進(jìn)行基因芯片檢測,篩選差異表達(dá)基因。最后將篩選出的與FFA致IR的相關(guān)差異表達(dá)基因DOK1,在T2DM和Pre-DM中其他符合條件的樣品中進(jìn)行熒光定量PCR檢測,分析表達(dá)情況,驗(yàn)證基因芯片結(jié)果的可靠性。本研究結(jié)果表明:(1)T2DM組、N組以及O組的FFA水平和HOMA-IR均顯著高于NC組;在N組中,HOMA-IR與FFA存在正相關(guān)關(guān)系,隨著FFA的增加,HOMA-IR增大,FFA對(duì)HOMA-IR的影響較大。(2)IFG組、IGT組和Pre-DM組中FFA和HOMA-IR均顯著高于NC組;Pre-DM患者HOMA-IR與FFA正相關(guān)關(guān)系,隨著FFA的增加,HOMA-IR增大,同時(shí)FFA對(duì)HOMA-IR的影響最大。(3)基因芯片分析結(jié)果表明O組與NC組相比,基因FOS(FBJ murine osteosarcoma viral oncogene homolog,FBJ鼠科動(dòng)物骨肉瘤病毒致癌基因同系物)顯著下調(diào),差異倍數(shù)為3.58(p0.05);N組與NC組相比,基因Fos、己糖激酶2(hexokinase 2,HK2)、銜接蛋白1(docking protein 1,DOK1)顯著下調(diào),差異倍數(shù)分別為10.71、2.32、2.22(p0.05);O組與N組相比,基因FOS、絲裂原活化蛋白激酶(mitogen-activated protein kinase1,MAPK1)顯著上調(diào),基因AE結(jié)合蛋白1(AE binding protein1,AEBP1)顯著下調(diào)的,差異倍數(shù)分別為2.72、1.77、1.79(p0.05)。(4)熒光定量PCR分析各實(shí)驗(yàn)組中DOK1表達(dá)水平的結(jié)果表明與NC組相比,N組和Pre-DM組的DOK1表達(dá)顯著降低(z=-2.121、-2.40,p0.05),T2DM-O組的DOK1表達(dá)并無顯著變化(z=-1.633,p0.05)。綜上所述,我們推測,對(duì)于T2DM中的非肥胖患者以及前期糖尿病的患者來說,血清FFA水平的異常升高可能與DOK1的顯著下調(diào)有關(guān),同時(shí)可能通過下調(diào)DOK1的表達(dá),抑制脂肪合成,促進(jìn)脂肪分解,促使或加重IR狀態(tài)。
[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.
【學(xué)位授予單位】：蘭州理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：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.
【學(xué)位授予單位】：蘭州理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R587.1
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