泛素—蛋白酶体系统和细胞自噬对血清饥饿的3T3-L1脂肪细胞胰岛素敏感性的调控作用

发布时间：2018-10-16 15:06

【摘要】：目的：胰岛素抵抗是2型糖尿病(Type 2 diabetes mellitus, T2DM)患者体内的主要标志事件。最近的研究表明,胰岛素抵抗结合胰腺β细胞功能障碍将导致葡萄糖耐量受损、高血糖、2型糖尿病。然而,当前对细胞胰岛素抵抗机制的理解仍然有限。多种因素如氧化应激、炎症、线粒体功能障碍、内质网(Endoplasmic reticulum, ER)应激、缺氧等均被认为可以引发胰岛素抵抗。细胞泛素-蛋白酶体系统(Ubiquitin-proteasome system, UPS)和自噬(Macroautophagy,以下称为自噬)是两个保守的细胞内蛋白降解的主要途径,负责细胞大多数蛋白质的降解。目前,有研究证明细胞泛素-蛋白酶体系统和自噬与胰岛素抵抗和糖尿病的发生发展有密切关系。然而,其中的机制在很大程度上仍未被了解。本文通过诱导3T3-L1前脂肪细胞分化为3T3-L1脂肪细胞,探讨血清饥饿时,细胞泛素-蛋白酶体系统和自噬对3T3-L1脂肪细胞胰岛素敏感性的影响及其可能的机制。方法：3T3-L1前脂肪细胞采用常规方法诱导分化为3T3-L1脂肪细胞。通过检测GFP-LC3小体的形成、测定肽酶活性、免疫印迹法检测泛素-蛋白酶体系统和自噬各种标志性蛋白分子、检测胰岛素刺激的Akt磷酸化水平和Akt靶蛋白GSK3β Ser9位点磷酸化水平,以及3T3-L1脂肪细胞在胰岛素刺激后葡萄糖吸收实验等,研究血清饥饿对3T3-L1脂肪细胞的泛素-蛋白酶体系统、自噬和胰岛素敏感性的影响。结果：1)血清饥饿可以导致3T3-L1脂肪细胞泛素-蛋白酶体系统和自噬的激活,同时伴随着脂肪细胞胰岛素敏感性增强。2)血清饥饿诱导的胰岛素敏感性可以被自噬抑制剂BFA抑制,但是不会被泛素-蛋白酶体系统抑制剂BZM抑制。脂肪细胞泛素-蛋白酶体系统的抑制可以补偿性地诱导激活脂肪细胞自噬,但并不能显著影响脂肪细胞的胰岛素敏感性。BFA和Atg5/7shRNA基因沉默分别抑制脂肪细胞自噬,都可以大大减轻血清饥饿提高的脂肪细胞胰岛素敏感性。3)抑制脂肪细胞泛素-蛋白酶体系统可以导致自噬补偿性的激活。但是脂肪细胞自噬的抑制可以损伤泛素-蛋白酶体系统功能。4)抑制脂肪细胞自噬可以诱导内质网应激和未折叠蛋白反应(Unfolded protein response, UPR)。抑制脂肪细胞泛素-蛋白酶体系统不会明显激活内质网应激和未折叠蛋白反应。但是,脂肪细胞泛素-蛋白酶体系统的抑制会加剧自噬抑制诱导的内质网应激和未折叠蛋白反应。结论：血清饥饿激活的泛素-蛋白酶体系统和自噬对3T3-L1脂肪细胞的胰岛素敏感性具有不同影响。1)泛素-蛋白酶体系统和细胞自噬所形成的蛋白质质量控制作用是保护脂肪细胞胰岛素敏感性所必需的。2)血清饥饿时,脂肪细胞自噬的适应性活化对增强3T3-L1脂肪细胞胰岛素敏感性是必需的。
[Abstract]:Objective: insulin resistance is a major marker event in patients with type 2 diabetes mellitus (Type 2 diabetes mellitus, T2DM). Recent studies have shown that insulin resistance and pancreatic 尾-cell dysfunction can lead to impaired glucose tolerance, hyperglycemia, and type 2 diabetes. However, the current understanding of the mechanism of cell insulin resistance is still limited. Many factors, such as oxidative stress, inflammation, mitochondrial dysfunction, endoplasmic reticulum (Endoplasmic reticulum, ER) stress, hypoxia and so on, are thought to induce insulin resistance. Ubiquitin proteasome system (Ubiquitin-proteasome system, UPS) and autophagy (Macroautophagy, hereafter called autophagy) is the main pathway of protein degradation in two conserved cells and is responsible for the degradation of most proteins in cells. At present, studies have shown that the Ubiquitin-proteasome system and autophagy are closely related to the development of insulin resistance and diabetes mellitus. However, the mechanism remains largely unknown. By inducing preadipocytes from 3T3-L1 to differentiate into 3T3-L1 adipocytes, the effects of ubiquitin proteasome system and autophagy on insulin sensitivity of 3T3-L1 adipocytes during serum starvation and its possible mechanism were investigated. Methods: 3T3-L1 preadipocytes were induced to differentiate into 3T3-L1 adipocytes by routine methods. The formation of GFP-LC3 bodies, peptidase activity, ubiquitin proteasome system and autophagy were detected by Western blotting. The levels of insulin stimulated Akt phosphorylation and GSK3 尾 Ser9 site phosphorylation of Akt target protein were detected. The effects of serum starvation on the ubiquitin proteasome system autophagy and insulin sensitivity of 3T3-L1 adipocytes after insulin stimulation were studied. Results: 1) Serum starvation resulted in the activation of ubiquitin proteasome system and autophagy in 3T3-L1 adipocytes, accompanied by an increase in insulin sensitivity of adipocytes. 2) the insulin sensitivity induced by serum starvation was inhibited by autophagy inhibitor BFA. However, it is not inhibited by BZM, an inhibitor of ubiquitin-proteasome system. The inhibition of adipocyte ubiquitin proteasome system could induce the activation of adipocyte autophagy, but it could not significantly affect the insulin sensitivity of adipocytes. BFA and Atg5/7shRNA gene silencing inhibited adipocyte autophagy, respectively. 3) inhibition of adipocyte ubiquitin proteasome system could lead to compensatory autophagy activation. But the inhibition of adipocyte autophagy can damage the function of ubiquitin proteasome system. 4) inhibition of adipocyte autophagy can induce endoplasmic reticulum stress and unfolded protein response (Unfolded protein response, UPR). Inhibition of adipocyte ubiquitin-proteasome system did not significantly activate endoplasmic reticulum stress and unfolded protein response. However, inhibition of the ubiquitin proteasome system in adipocytes increased endoplasmic reticulum stress and unfolded protein response induced by autophagy inhibition. Conclusion: Ubiquitin proteasome system activated by serum starvation and autophagy have different effects on insulin sensitivity of 3T3-L1 adipocytes. 1) the protein quality control effects of ubiquitin proteasome system and autophagy are as follows: Necessary to protect adipocytes from insulin sensitivity. 2) when the serum is hungry, Adaptive activation of adipocyte autophagy is necessary to enhance insulin sensitivity of 3T3-L1 adipocytes.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R587.1

【相似文献】