Betatrophin与胰岛素抵抗的相关性及机制研究

发布时间：2018-02-28 17:19

本文关键词： Betatrophin 2型糖尿病胰岛素抵抗关联性分析 Betatrophin 胰岛素抵抗胰岛素 PI3K/Akt　出处：《华中科技大学》2015年博士论文　论文类型：学位论文

【摘要】：研究背景及目的根据国际糖尿病联盟(International Diabetes Federation, IDF)的数据显示,目前全球有382百万人患有糖尿病,预计这个数字到2035年将达到592百万人。迄今研究发现,2型糖尿病(Type2diabetes, T2D)以一系列代谢紊乱为特点,主要包括胰岛素抵抗(Insulin resistance, IR)和胰岛p细胞功能受损。在正常生理条件下,正常血糖的维持依靠胰岛素敏感性和胰岛素分泌之间的平衡。当胰岛素敏感性下降时,胰岛素分泌增加以维持正常血糖。当摄入热量过多而身体缺乏运动引起胰岛素抵抗时,一旦代偿的胰岛素分泌不足够满足机体对胰岛素的需求,胰岛β细胞超负荷工作引起p细胞功能衰竭,最终导致2型糖尿病的发生。以往研究证实,胰岛素抵抗引起的代偿性的β细胞增生和分泌功能增强很可能是通过循环中的某些生长因子来介导的。因此,在过去几十年,已有大量研究旨在识别这些可以调节胰岛β细胞数量和分泌功能的循环因子,希望可以为治疗糖尿病提供新策略。肠道来源的肠促胰岛素,比如胰高血糖素样肽-1(glucogon-like peptide-1,GLP-1)；肌肉来源的肌肉激素,比如白介素-6,巨噬细胞来源的细胞因子和脂肪细胞来源的脂肪因子,比如瘦素(leptin)和脂联素(adiponectin),都被认为是胰岛β细胞再生疗法的候选靶点。然而,由于这些因子作用的非特异性和有限的作用,他们的临床应用受到了限制。最近,哈佛大学干细胞研究所的研究人员利用一种能与胰岛素受体结合的肽来诱导小鼠胰岛素抵抗模型,发现由肝脏、白色脂肪组织以及棕色脂肪组织所分泌的一种肽类激素(Betatrophin)能够特异性使胰岛p细胞快速增殖。因此有望极大推动新型糖尿病治疗药物的研发。尽管betatrophin在动物模型中可发挥显著地抗糖尿病作用,但其生物学效应的具体机制和作用模式并不清楚。而在2型糖尿病患者中,betatrophin体内的变化及可能的作用仍然有待研究。因此,本研究的目的是探讨外周血循环中betatrophin的表达水平在不同糖耐量状态人群中是否具有显著性差异以及是否与胰岛素抵抗程度相关。实验方法我们分别纳入了年龄、性别、体重指数和血脂等相关指标相匹配的血糖正常人群(n=137)、仅有空腹血糖受损人群(n=69)、仅有糖耐量减低人群(n=120)和新诊断的2型糖尿病人群(n=112)进行研究分析[2011-2012年中国2型糖尿病患者肿瘤发生风险的流行病学研究(the Risk Evaluation of cAncers in Chinese diabeTic Individuals:a lONgitudinal study,REACTION)的一部分人群]。所有研究对象血清中betatrophin的水平用ELISA(武汉伊艾博公司)测定。结果 2型糖尿病患者血清中betatrophin的表达水平明显升高,与血糖正常人群、仅有空腹血糖受损以及仅有糖耐量受损人群相比均具有显著性统计学差异(798.6±42.5vs.692.7±29.0, P0.05; vs.682.7±43.0, P0.05; vs.646.8±34.3pg/ml,P0.01)。在所有人群中,校正了年龄,性别,体质指数和血脂水平等相关因素后,betatrophin表达水平与稳态模型胰岛素抵抗指数(HOMA-IR)正相关(偏相关系数r=0.11)；与定量胰岛素敏感性指标(QUICKI)(偏相关系数r=-0.11),Gutt胰岛素敏感指数(ISIG)(偏相关系数r=-0.12)和Matsuda胰岛素敏感指数(ISIM)(偏相关系数r=-0.11)呈负相关。结论血清betatrophin水平在2型糖尿病患者中明显升高,且与胰岛素抵抗指数相关。研究背景及目的 Betatrophin也称为ANGPTL8, TD26, RIFL, C19orf8和Lipasin,作为一个具有潜在促胰岛p细胞再生作用的明星分子,主要在人肝脏中表达。现有研究表明betatrophin的水平与胰岛素抵抗之间存在密切联系。在多种胰岛素抵抗的小鼠模型,比如db/db小鼠,ob/ob小鼠,妊娠期的小鼠以及用胰岛素受体拮抗剂S961诱导的胰岛素抵抗模型中均发现有betatrophin水平的升高。在人体研究中,我们发现血清betatrophin水平在2型糖尿病患者中是明显升高的,与其他研究小组的结论一致。同时,betatrophin水平也在有明显胰岛素抵抗的肥胖人群(成人及儿童和青少年)中是升高的。Betatrophin水平与胰岛素抵抗呈正相关,与胰岛素敏感性呈负相关。因此,大家普遍认为betatrophin水平的升高是胰岛素抵抗引起的一种代偿反应。但是胰岛素抵抗在促betatrophin表达中的作用及其相关机制仍不清楚。因此,在本研究中我们在正常肝细胞系L02中构建了多种胰岛素抵抗模型并检测不同模型中betatrophin的表达,拟探讨胰岛素抵抗促betatrophin表达的作用及其相关机制。实验方法和结果 (1)用肿瘤坏死因子-a(4ng/ml),白介素-1β(20ng/ml),地塞米松(1μM),棕榈酸(500μM),高糖(33mmM)和高胰岛素(10-6M)分别干预正常肝细胞系L02建立6种体外胰岛素抵抗模型。流式检测L02细胞对荧光标记的葡萄糖(2-NBDG)的摄取率;Western Blot检测Akt的磷酸化水平。6种胰岛素抵抗模型均表现出受损的胰岛素反应(胰岛素抵抗)：葡萄糖摄取率降低和Akt磷酸化减弱。 (2) ELISA检测6种模型中L02分泌betatrophin的水平。结果发现betatrophin的水平仅仅在高胰岛素诱导的胰岛素抵抗模型中升高。 (3)不同浓度的胰岛素(0～10-5M)刺激L02细胞时,低浓度的胰岛素并不能刺激betatrophin的产生；只有当胰岛素浓度较高(10～6M以上)时,作用于L02细胞才能引起betatrophin的表达增多。 (4)高浓度胰岛素(10～6M)作用下引起的betatrophin水平的升高可以被胰岛素信号通路PI3K/Akt特异性抑制剂LY294002所抑制。PI3K/Akt通路的激动剂IGF-1可升高betatrophin水平,且升高的betatrophin水平可被LY294002所抑制。 (5)在人体中,我们收集了7例使用了外源性胰岛素(±二甲双胍)的2型糖尿病患者及与之年龄、性别、BMI和血脂水平相匹配的7例未使用外源性胰岛素(±二甲双胍)的2型糖尿病患者。ELISA检测血清中betatrophin的水平。结果发现与未使用外源性胰岛素的患者相比,使用了外源性胰岛素的2型糖尿病患者体内的betatrophin的水平是明显升高的(785.5±117.8vs.240.1±53.0pg/ml,P0.01)。结论基于以上发现,我们认为高胰岛素本身而非高胰岛素诱导的胰岛素抵抗促进了betatrophin的表达。并且胰岛素升betatrophin的作用是通过激活PI3K/Akt通路而调控的。
[Abstract]:Background and purpose of research
According to data from International Diabetes Federation (IDF), 382 million people worldwide are suffering from diabetes now, and the number is expected to reach 592 million by 2035.
So far the study found that type 2 diabetes mellitus (Type2diabetes, T2D) in a series of metabolic disorders, including insulin resistance (Insulin resistance, IR) and islet P cell function damage. Under normal physiological conditions, maintaining normal blood glucose on insulin sensitivity and insulin secretion balance. When the decreased insulin sensitivity, insulin in order to maintain normal blood glucose secretion. When excessive calorie intake and lack of physical exercise induced insulin resistance, compensatory insulin secretion once not enough insulin demand, islet beta cell overload caused by P cell failure, resulting in the incidence of type 2 diabetes.
Previous studies have shown that insulin resistance caused by compensatory beta cell proliferation and secretion increased probably by some growth factors in the circulation mediated. Therefore, in the past few decades, there has been a lot of research aimed at identifying these can adjust the number of beta cells and the secretion function of circulating factor, the hope can provide new strategy for the treatment of diabetes mellitus. Gut derived incretin, such as glucagon like peptide -1 (glucogon-like peptide-1, GLP-1); muscle derived muscle hormones, such as interleukin -6, fat cytokines from adipocytes and macrophages sources, such as leptin (leptin) and adiponectin (adiponectin), are considered is a candidate target for therapy of pancreatic islet beta cell regeneration. However, due to nonspecific factors and the limited role, their clinical application is restricted. Recently, Kazakhstan The Harvard Stem Cell Institute researchers using a combination of peptide and insulin receptor to induce insulin resistance in mice model, found by the liver, white adipose tissue and secretion of brown adipose tissue is a peptide hormone (Betatrophin) can specifically the rapid proliferation of pancreatic island P cell. It is expected to greatly promote the development of new drug treatment for diabetes.
Although betatrophin can play a significant role in the anti diabetic animal model, but the specific mechanism and mode of action of its biological effect is not clear. But in patients with type 2 diabetes, changes in betatrophin and the possible role remains to be studied. Therefore, the purpose of this study is to investigate the expression of betatrophin in peripheral blood in different glucose tolerance status in the crowd is there a significant difference and whether it was related to the severity of insulin resistance.
Experimental method
We included age, gender, body mass index and blood lipid index matched normal blood glucose group (n=137), the only people with impaired fasting blood glucose (n=69), impaired glucose tolerance only (n=120) and newly diagnosed type 2 diabetes (n=112) research the epidemiological study of [2011-2012 Chinese 2 years patients with type 2 diabetes cancer risk (the Risk Evaluation of cAncers in Chinese diabeTic Individuals:a lONgitudinal study, REACTION betatrophin) is a part of the population. All of the subjects in serum by ELISA (Wuhan eiaab company) were determined.
Result
The expression level of betatrophin in serum of patients with type 2 diabetes mellitus was significantly elevated and normal blood glucose, impaired fasting glucose only and only people with impaired glucose tolerance were compared with significant statistical difference (798.6 + 42.5vs.692.7 + 29, P0.05 + 43; vs.682.7, P0.05; vs.646.8 + 34.3pg/ml, P0.01). In all people, correction age, sex, body mass index and related factors of blood lipid levels, betatrophin expression levels and insulin resistance index (HOMA-IR) positive correlation (partial correlation coefficient r=0.11); and quantitative insulin sensitivity index (QUICKI) (partial correlation coefficient r=-0.11, Gutt) and insulin sensitivity index (ISIG) (partial the correlation coefficient r=-0.12) and insulin sensitivity index (ISIM) and Matsuda (partial correlation coefficient r=-0.11) were negatively correlated.
conclusion
The level of serum betatrophin was significantly higher in type 2 diabetic patients and was associated with the insulin resistance index.
Background and purpose of research
Betatrophin also known as ANGPTL8, TD26, RIFL, C19orf8 and Lipasin, as a potential star molecule of islet P cell regeneration promoting, mainly expressed in human liver. The current study shows that there is a close relationship between betatrophin levels and insulin resistance. Insulin resistance in mice model, such as db/db mice, ob/ob mice. Pregnant mice and induced by insulin receptor antagonist S961 and insulin resistance have elevated levels of betatrophin were found in the model. In human studies, we found that the serum betatrophin level in patients with type 2 diabetes is significantly increased, consistent with the results of other research groups. At the same time, the betatrophin level is also obvious insulin resistance obese people (adults and children and adolescents) is elevated levels of.Betatrophin were positively correlated with insulin resistance and insulin sensitivity. Sex is negatively correlated. Therefore, it is generally believed that the rise of betatrophin level is a compensatory response induced by insulin resistance. However, the role of insulin resistance in promoting betatrophin expression is still unknown.
Therefore, in this study, we constructed a variety of insulin resistance models in normal liver cell line L02, and detected the expression of betatrophin in different models. We intend to explore the effect of insulin resistance on betatrophin expression and its related mechanisms.
Experimental methods and results
(1) with tumor necrosis factor -a (4ng/ml), interleukin -1 beta (20ng/ml), dexamethasone (1 M), palmitic acid (500 M), high glucose (33mmM) and insulin (10-6M) were treated normal liver cell line L02 6 in vitro establishment of insulin resistance model. Flow cytometry L02 the cellular fluorescence labeled glucose uptake rate (2-NBDG); Western Blot Akt detection of phosphorylation of.6 in insulin resistance model showed impaired insulin response (insulin resistance): glucose uptake and phosphorylation of Akt decreased.
(2) ELISA detected the level of L02 secreting betatrophin in 6 models. It was found that the level of betatrophin increased only in hyperinsulinemia induced insulin resistance model.
(3) when different concentrations of insulin (0 ~ 10-5M) stimulate L02 cells, low concentration of insulin does not stimulate betatrophin production. Only when the concentration of insulin is higher than 10 ~ 6M, can the expression of betatrophin increase in L02 cells.
(4) the high concentration of insulin (10 ~ 6M) increased under betatrophin level can be caused by the insulin signaling pathway PI3K/Akt specific inhibitor LY294002 inhibited.PI3K/Akt pathway agonist IGF-1 can increase the level of betatrophin, and elevated levels of betatrophin can be inhibited by LY294002.
(5) in the body, we collected 7 cases of the use of exogenous insulin (+ metformin) in patients with type 2 diabetes and gender and age, BMI, and blood lipid level of 7 matched cases without the use of exogenous insulin (+ metformin) in type 2 diabetic patients with.ELISA detected in serum betatrophin levels results compared with no exogenous insulin in patients with type 2 diabetes patients in exogenous insulin betatrophin levels were significantly elevated (785.5 + 117.8vs.240.1 + 53.0pg/ml, P0.01).
conclusion
Based on the above findings, we believe that high insulin itself, rather than hyperinsulinemia induced insulin resistance, promotes the expression of betatrophin. Moreover, the role of insulin in promoting betatrophin is regulated by activating PI3K/Akt pathway.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R587.1

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