VERIFY THE ADJUSTABLE THRESHOLD HYPOTHESIS BY MEASURING THE CELL RESPONSE THRESHOLD TO INSULIN

通过测定细胞对胰岛素响应阈值验证可调门限假说

袁月洋

11849446

硕士

生物学

王冠宇

2021-05-18

2021-06-01

哈尔滨工业大学

深圳

Diabetes has a large number of patients and serious complications. It is the world's third largest non-communicable chronic disease and poses a serious health threat worldwide. Diabetes is divided into type 1 diabetes and type 2 diabetes. The pathogenesis of type 2 diabetes is complex and diverse, and individual differences are large. Environmental factors and genetic factors can lead to the occurrence of type 2 diabetes. Insulin receptor or post-receptor defects can also lead to type 2 diabetes. Insulin controls key energy metabolism. After insulin binds to the insulin receptor on the target cell membrane, it activates enzyme activities related to amino acid and fat metabolism through the second messenger in the cell. Thereby promoting the normal metabolism of amino acids, fats and glucose in target cells. The number of insulin receptors on the target cell membrane is regulated by the concentration of insulin in the blood, and the change in the number of receptors is inversely proportional to the level of insulin in the blood. Although the insulin secretion of obese patients is normal, diabetes will gradually develop. This is due to the decrease of insulin receptors on the target cell membrane and the lack of sufficient second messenger stimulation, which leads to the dysfunction of intracellular fat and sugar metabolism, which in turn stimulates The pancreatic islets secrete insulin, which forms feedback regulation on the insulin receptor, reducing the number of receptors.Insulin resistance refers to a pathological state in which the body's biological response to a certain concentration of insulin is lower than normal, and an excessive amount of insulin is required to respond to a normal amount. Different from the general thought that the body's response to insulin is a gradual pattern, this article explores the possibility of an “all-or-none” threshold-type response of cells to insulin. The gradual change at the body level is achieved through different types of cell thresholds and different mechanisms. Type 2 diabetes and insulin resistance are closely related, and the exploration of the entrance from insulin resistance can bring hope to solving the threat of diabetes. Insulin resistance refers to a pathological state in which the body's biological response to a certain concentration of insulin is lower than normal, and an excessive amount of insulin is required to respond to a normal amount. Clinically, the typical characteristic of insulin resistance is the coexistence of refractory hyperglycemia and hyperinsulinemia. Modern research shows that insulin resistance is widespread in diabetic patients, and it runs through the entire process of the occurrence and development of type 2 diabetes. Eliminating insulin resistance and increasing insulin sensitivity have become effective measures to prevent and treat diabetes. The relationship between the mechanism of the occurrence and development of type 2 diabetes and insulin resistance is summarized as follows: body obesity, polyphagia, etc. sustainably stimulate pancreatic β-cells to promote hyperinsulinemia in the body, negative regulation of target cell receptors, target cells The sensitivity to insulin gradually declines, which triggers insulin resistance. The patient's blood sugar rises, the pancreatic β-cell load becomes larger, and finally the pancreatic β-cells fail, the defect of the target cell receptor becomes more obvious, and the blood sugar of diabeticpatients further rises.Insulin resistance is the result of a combination of genetic factors and environmental factors, among which environmental factors include obesity, sedentary and aging. Most insulin resistance is the result of a disorder in the process of insulin signaling after insulin binds to its receptor. Insulin resistance is a state of defective insulin effect caused by multiple reasons. It is the main mechanism of type 2 diabetes. The body does not respond to insulin or responds slowly, making it difficult for the body to absorb glucose and can only stay in the blood. . Insulin effect defects mainly include decreased insulin receptor tyrosine kinase activity, abnormal insulin signal transduction, decreased glucose transport, glucose phosphorylation, and weakened glycogen synthase activity.Different from the generally thought that the body's response to insulin is a gradual pattern, this article explores the possibility of an "all-or-none" threshold-type response of cells to insulin. The gradual change at the body level is achieved through different types of cell thresholds and different mechanisms. From the perspective of the whole body, the dynamic balance of glucose and insulin in the blood is regulated by a variety of organs and tissues. Mathematical modeling and computational analysis of the system are carried out to put forward a new scientific hypothesis called the adjustable threshold hypothesis on the insulin response of normal cells. In this hypothesis, the cell response to insulin has four characteristics. First, all or nothing. The cell's response to insulin has two different thresholds, Ion and Ioff, corresponding to two states: all or nothing. Nothing means that the cells do not extract blood sugar, and all means that the cells extract blood sugar at the maximum rate. Second, the reversibility of lag. In the hypothesis, cells with low insulin concentration in the initial state do not respond. When the insulin concentration continues to increase beyond the threshold, the cells switch to the maximum response state, and then the insulin concentration continues to decrease. The cells do not switch to nothing at the original Ion, but switch to nothing at another threshold Ioff. Third, threshold heterogeneity. Threshold heterogeneity. Different types of cells have big differences in their thresholds. Even if they belong to the same type, the threshold values of the cells are different. Fourth, the threshold is adjustable. The cell threshold is adjustable. Through the biomolecular network insulin signal pathway to change the external or internal environment to adjust the threshold, so that the cell and the entire body are adaptable to the environment.This article focuses on C2C12 mouse skeletal muscle cells and HepG2 human liver cancer cells, using fluorescence resonance energy transfer (FRET) and Western blot experiments to observe insulin from the single-cell level and the cell population level, respectively. Response mode to verify the adjustability threshold hypothesis. The cells were starved overnight, then the cells were treated with increasing different concentration gradients, and after reaching the peak, the cells were treated with decreasing different concentration gradients. The results are as follows: The immunofluorescence energy resonance transfer experiment was performed on C2C12, and the activity of the key pathway protein Akt was used to observe whether the cells responded to insulin. The experimentally measured insulin response curve of C2C12 accords with the hypothesis of adjustability threshold. Ion is the concentration of insulin at which cells begin to respond to insulin, Ion ≈ 300pM. Ioff is the insulin concentration when the cell's response to insulin is significantly reduced, Ioff ≈100pM. HepG2 liver cancer cells have no obvious threshold. This may be a feature of cancer cells that are different from normal cells and can be developed as a standard for cancer screening based on this. The results of the cell population experiment are consistent with the results of the single-cell experiment.There is indeed a threshold response to insulin in normal cells. The response patterns of cancerous cells and normal cells are not completely consistent. The adjustable threshold hypothesis provides new ideas for the treatment and intervention of diabetes.

糖尿病患病人数多，并发症严重，是世界第三大非传染性慢性疾病，在世界范围带来了严重的健康威胁。糖尿病分为 1 型糖尿病和 2 型糖尿病。2 型糖尿病的发病因素复杂多样，个体差异大。环境因素、遗传因素都会导致 2 型糖尿病的发生。胰岛素受体或受体后缺陷也会导致 2 型糖尿病。胰岛素控制关键的能量代谢，胰岛素和靶细胞膜上的胰岛素受体结合后，通过细胞内的第二信使激活与氨基酸和脂肪代谢相关的酶活性。从而促进靶细胞胞内的氨基酸、脂肪和葡萄糖正常代谢过程。靶细胞膜上胰岛素的受体数目受到血液中胰岛素浓度的调节，且受体数目的改变与血液中胰岛素水平成反比。肥胖病人虽然胰岛素分泌量正常，但是也会慢慢发生糖尿病，这是由于靶细胞膜上胰岛素受体的减少，缺乏足够第二信使刺激，导致胞内脂肪、糖等代谢的功能紊乱，进而又刺激胰岛分泌胰岛素，对胰岛素受体构成了反馈调节，减少了受体的数目。2 型糖尿病和胰岛素抵抗关系密切，由胰岛素抵抗为探索入口可为解决糖尿病威胁带来希望。胰岛素抵抗指机体对一定浓度的胰岛素的生物反应性低于正常，需要超常量的胰岛素才能正常量反应的一种病理状态。临床上，胰岛素抵抗典型的特征是顽固性高血糖与高胰岛素血症并存。现代研究表明：胰岛素抵抗在糖尿病患者中广泛存在，贯穿于 2 型糖尿病发生、发展的整个过程。消除胰岛素抵抗和增加胰岛素的敏感性，已经成为预防和治疗糖尿病的有效措施。2 型糖尿病发生、发展的作用机理与胰岛素抵抗的关系，总结如下：机体肥胖、多食等可持续性地刺激胰岛 β 细胞，促使机体产生高胰岛素血症，靶细胞受体负调节，靶细胞对胰岛素的敏感性逐渐下降，从而引发胰岛素抵抗，患者血糖升高，胰岛 β 细胞负荷变大，最后胰岛 β 细胞发生衰竭，靶细胞受体后缺陷愈加明显，糖尿病患者血糖进一步升高。胰岛素抵抗是遗传因素和环境因素共同作用而导致的结果，其中环境因素包括肥胖、久坐和衰老等。大部分胰岛素抵抗是胰岛素和其受体结合后，胰岛素信号传导过程发生障碍的结果。胰岛素抵抗是由多个原因共同作用导致的胰岛素效应缺陷的状态，是 2 型糖尿病发病的主要机制，机体对胰岛素不反应或反应迟缓使得葡萄糖很难被机体组织所吸收而只能滞留在血液中。胰岛素效应缺陷主要包括胰岛素受体的酪氨酸激酶活性下降、胰岛素信号传导异常、葡萄糖转运减少、葡萄糖磷酸化和糖原合成酶活性减弱等。不同于通常认为的机体对胰岛素的响应是一个渐变的模式，本文探索了细胞对胰岛素是“全或无”门阈型响应的可能性。机体层面的渐变现象是通过不同种类的细胞阈值不同的机制来达成的。从整个机体层面来看，血液中葡萄糖和胰岛素的动态平衡受多种器官组织的调节，对该系统进行数学建模和计算分析，就正常细胞的胰岛素响应提出一个新的科学假说，称为可调门限假说。在该假说中细胞对胰岛素应答具有四个特征。第一，全有或全无。细胞对胰岛素的响应有两个不同的阈值 Ion 和 Ioff，分别对应着两个状态：全有或全无。全无指的是细胞不提取血糖，全有指的是细胞以最大速率提取血糖。第二，迟滞逆转性。在假说中，初始状态胰岛素浓度低细胞无响应。当胰岛素浓度持续增加超过门限值后，细胞切换至最大响应状态，接下来使胰岛素浓度持续降低，细胞并不是在原来的 Ion时切换至全无，而是在另一门限 Ioff 切换到全无。第三，门限异质性。门限异质性。不同类型的细胞，其门限值有较大的差异。即使属于同一种类型，细胞的门限值也不同。其四，门限可调性。细胞的门限值是可调的。通过生物分子网络胰岛素信号通路改变外部或内在环境来调整门限值，使得细胞乃至整个机体对环境具有适应性。本文主要的技术手段是免疫荧光能量共振转移技术(Fluorescence resonance energy transfer，FRET)。目前基于 FRET 的生物传感器现在可用于测量活体中许多高度动态生物分子的活性。FRET 过程涉及通过一个非辐射能量转移将能量从激发态的一个荧光团转移到第二荧光团。 FRET 实验中的荧光蛋白依赖于在激发供体如青色荧光蛋白(CFP)之后测量受体蛋如 Venus 一种黄色荧光蛋白(YFP)的变异体的放射量。CFP 被激发之后，发射光会是青色的；然而，如果能量转移到受体蛋白如 Venus 上，那么即使黄色蛋白质没有被激光或灯光直接激发也会观察到黄色荧光。为了高效地发生 FRET，供体荧光发射光谱和受体荧光激发光谱之间必须有大量的重叠。FRET 测量通常被称为分子标尺，因为 FRET 仅在两个荧光基团在 2-10 nM 之内相互作用时才有效。FRET 通常用于检查分子间两种不同蛋白质结合成配体之间的相互作用，其中一个结合配体包含供体而另一个配体包含受体。必须考虑到每个细胞中供体和受体的差异表达，因为蛋白的变异体水平可能会改变 FRET 结果。FRET 还可用作观察激活后蛋白质结构构象变化的工具，构象变化可能是翻译后修饰的结果，如磷酸化或蛋白质或膜的结合。本文使用 Akind 作为分子内 FRET 探针来检测活细胞中的 Akt 磷酸化水平。当 Akt 报告蛋白被磷酸化后，其构象的改变可引发所连接的荧光蛋白发生荧光能量共振转移，而荧光变化可通过荧光显微镜记录。因此，FRET 技术是检测 Akt 蛋白磷酸化最直观和快速的手段之一。可以凭借活细胞荧光能量共振转移的检测，分析细胞中 Akt 蛋白即时的磷酸化状态，以解析单细胞对胰岛素的动态响应。本文针对 C2C12 小鼠骨骼肌细胞和 HepG2 人来源肝癌细胞，采用免疫荧光能量共振转移技术和蛋白质免疫印迹(Western blot)实验分别从单细胞层面和细胞群体层面观察胰岛素响应模式，验证可调节性阈值假说。先将细胞饥饿处理过夜，然后用上升的不同浓度梯度的胰岛素处理细胞，到达峰值后再用下降的不同浓度梯度的胰岛素处理细胞。结果如下：对 C2C12 进行免疫荧光能量共振转移实验，通过关键通路蛋白 Akt 的活性来观察细胞是否对胰岛素产生反应。实验测得 C2C12 的胰岛素响应曲线符合可调节性阈值假说，Ion 即细胞开始对胰岛素有响应时的胰岛素浓度，Ion ≈ 300pM；Ioff 是细胞对胰岛素的响应显著降低时的胰岛素浓度，Ioff ≈ 100pM。HepG2 肝癌细胞没有明显的阈值，这可能是癌症细胞不同于正常细胞的特征，可以据此发展作为癌症筛查的标准。细胞群体实验得到的结果与单细胞实验结果相符合。正常细胞中确实存在对胰岛素的响应阈值。癌变细胞与正常细胞的响应模式不完全一致。可调节性阈值假说为糖尿病的治疗和干预提出新思路。

Diabetes Insulin resistance Adjustable threshold hypothesis FRET

糖尿病 胰岛素抵抗 可调节性阈值假说 免疫荧光能量共振转移技术

英语

联合培养

南方科技大学

Yuan YY. VERIFY THE ADJUSTABLE THRESHOLD HYPOTHESIS BY MEASURING THE CELL RESPONSE THRESHOLD TO INSULIN[D]. 深圳. 哈尔滨工业大学,2021.