GLP-1对3T3-L1前脂肪细胞分化的影响及其调控机制研究

  本文关键词：GLP-1对3T3-L1前脂肪细胞分化的影响及其调控机制研究，由笔耕文化传播整理发布。

        研究背景：目前,肥胖及2型糖尿病已经成为我国面临的严重公共卫生问题。作为代谢异常性疾病二者存在相近的发病基础,脂质异常沉积与胰岛素抵抗是其共同土壤[1]。尽管多种遗传因素引起肥胖及胰岛素抵抗,但环境因素中最主要的是长期能量摄入超过消耗,导致体内脂肪过多蓄积,引起肥胖及脂质代谢紊乱。肥胖在细胞层面上主要表现为已存在的脂肪细胞体积的肥大和由前脂肪细胞分化而来的脂肪细胞数量的增多。研究表明,脂肪细胞体积的肥大以及由前脂肪细胞分化而来的脂肪细胞数量的增多在成人肥胖的发生发展过程中均发挥着重要作用。然而,这两种变化对脂质及糖代谢的作用有着显著的差别。脂肪细胞体积的肥大与脂质代谢紊乱及胰岛素抵抗密切相关；而脂肪细胞数量的增多(小体积脂肪细胞)则有助于改善高脂血症,增加胰岛素敏感性。胰高糖素肽-1(glucagon-like peptide-1, GLP-1)是由肠L细胞产生的目前己知作用最强的肠促胰岛素分泌肽,除了调控血糖之外,还具有调节食欲、血压、血脂,改善心功能、保护血管内皮功能等多重生物学功能。研究证明,GLP-1能够上调脂肪细胞表面胰岛素受体(IR-β)及胞内胰岛素底物(IRS-1)的数量,促进葡萄糖转运子4(GLUT-4)的表达,抑制脂肪细胞中炎症通路激活等改善脂肪细胞体积的肥大,进而改善胰岛素抵抗；然而,GLP-1对前脂肪细胞的分化的影响尚有争议,其能否促进小体积脂肪细胞的形成尚缺乏足够研究。本实验以3T3-L1前脂肪细胞为实验对象,在其分化过程中给予重组人GLP-1进行干预,研究GLP-1对前脂肪细胞分化的影响及其调控机制,以期为肥胖的防治提供新的作用靶点。研究目的：1.观察GLP-1对3T3-L1前脂肪细胞分化过程中脂肪标志性蛋白LPL、 aP2及转录因子PPAR-γ、C/EBPa表达的影响。2.观察GLP-1对3T3-L1前脂肪细胞分化过程中脂质聚集以及脂肪细胞形态的影响。3.初步探讨GLP-1发挥上述作用的分子机制。研究方法：1.3T3-L1前脂肪细胞的培养与分化：在无菌条件下培养3T3-L1前脂肪细胞,采用传统“鸡尾酒”法来诱导前脂肪细胞分化。2.给予3T3-L1前脂肪细胞不同浓度的GLP-1进行干预,用MTT法检测其对细胞活力的影响,选取合适的浓度范围。3.RT-PCR及Western-blot检测脂肪细胞脂肪标志性蛋白LPL、aP2及转录因子PPAR-γ、C/EBPa的表达。4.油红O染色检测3T3-L1前脂肪细胞诱导分化第8天细胞内脂质聚集,用Image Pro plus5.02软件分析脂肪细胞体积和数量的变化。5. RT-PCR检测诱导分化第8天时解偶联蛋白-1(UCP-1)和肉碱脂酰转移酶-1(CPT-1A)的mRNA表达。6.Western-blot检测Akt、P38、ERK1/2信号通路的变化。研究结果：1.GLP-1显著促进脂肪细胞标志性蛋白LPL、aP2和转录因子PPAR-γ、 C/EBPa的mRNA水平,呈浓度依赖性及时间依赖性(P<0.05)。2.与对照组相比,GLP-1显著促进转录因子PPAR-γ和脂肪细胞标志性蛋白aP2的蛋白表达,呈浓度依赖性及时间依赖性(P<0.05)。3.与对照组相比,GLP-1组且pAkt的蛋白表达水平显著增加(P<0.05),而pP38和pERK1/2的蛋白水平未见显著变化。4.油红O染色结果显示,100nM GLP-1显著增加小体积脂肪细胞的形成(P<0.05)。5.与对照组和其他浓度组相比,100nM GLP-1组脂肪细胞中CPT-1A的mRNA表达水平显著增加(P<0.05),而UCP-1的mRNA表达无显著变化。研究结论：1.在3T3-L1前脂肪细胞分化过程中,GLP-1呈剂量及时间依赖性的促进PPAR-γ、C/EBPα、LPL和aP2的表达。2.Akt信号通路可能参与GLP-1的上述作用过程。3.100nM GLP-1能促进更多小体积脂肪细胞的形成,该细胞中CPT-1A的mRNA表达显著增加。

    Background:Nowadays, obesity and type2diabetes had been the serious public health problems in China. They are both metabolic diseases that have the similar basic reasons. Abnormal lipid deposition and insulin resistance are closely related to them [1]. Although a lot of genetic reasons contribute to the outcomes of obesity and insulin resistance, the most important factor in the environment is that the imbalance between energy intake and expenditure, thus causing too much fat accumulation in the body and causing obesity and lipid metabolism disorders[2.3]. The cellular mechanisms for obesity include the expansion of white adipose tissue via the hypertrophy of preexisting adipocytes and hyperplasia resulting from the adipogenesis of preadipocytes. Recent studies have shown that both the hypertrophy and hyperplasia have played the role in the pathology of obesity in human adults. However, there are significant differences in lipid and glucose metabolism between adipocyte hypertrophy and hyperplasia. Adipocyte hypertrophy is negatively correlated with dyslipidema and insulin resistance, independent of body composition. Interestingly, hyperplasia, which is characterized by an increased number of small subcutaneous adipocytes, may have a positive effect on lipid metabolism and insulin sensitivity through preadipocyte differentiation[4-6]. Glucagon-like peptide1(GLP-1) is the most effective incretin until now, which is secreted from intestinal L-cells and exerts multiple biological effects. Not only can it regulate the blood glucose, appetite, blood pressure and blood lipid; but also improve the cardiac and vascular endothelial function[7] Recent studies have revealed that GLP-1can significantly reduce fat mass and adipocyte hypertrophy, improve insulin sensitivity in adipocyte by up-regulating the expression of insulin receptor, insulin receptor substrate and Glut-4, reducing macrophage infiltration and inhibiting inflammatory adipocytes [8-10]. However, the effect of GLP-1on adipogenesis is less clear; and if GLP-1can improve the information of small adipocyte is also don’t have enough research. In this study, using3T3-L1preadipocyte, we examined the effect of GLP-1on preadipocyte differentiation and the mechanisms involved.Objective:1. To observe the effect of GLP-1on the adipocyte-specific proteins LPL, aP2and the transcription factors PPAR-y, CEBP/a during the process of3T3-L1preadipocyte differentiation.2. To observe the Effect of GLP-1on lipid accumulation and the cytomorphology of adipocyte.3. To investigate the possible mechanism involved in the GLP-1induced effect.Methods:1.3T3-L1preadipocytes culture and differentiation:3T3-L1preadipocytes were cultured in aseptic condition and the traditional "Cocktail method" was adopted to induce the3T3-L1preadipocyte differentiation.2. GLP-1was added to the medium of3T3-L1cells at different concentrations, MTT assay was used to test the cell viability.3. RT-PCR and Western-blot were used to exam the express levels of the adipocyte-specific markers.4. Oil Red O staining to examine the effect of GLP-1on lipid droplet accumulation at the8th day of differentiation, and the Image Pro plus5.02was used to analyse the size and number of lipid droplet.5. RT-PCR was used to exam the express level of Uncoupling protein-1(UCP-1) and Carnitine palmitoyltransferase1(CPT-1A) at the8th day of differentiation.6. Under the treatment of GLP-1, we investigated the levels of Akt, P38and ERK1/2 signaling pathway.Results:1. At varying times during differentiation, GLP-1enhanced the mRNA levels of the transcription factors PPAR-γ,CEBP/α and the adipocyte-specific markers LPL, aP2significantly in a dose-and time-dependent way, as compared to the control group(P<0.05).2. GLP-1enhanced the protein levels of the PPAR-γ and aP2significantly in a dose-and time-dependent way, as compared to the control group(P<0.05).3. During the first24h of differentiation, Akt, P38and ERK1/2signaling way were activated in different degrees. Compared to the control group, the protein level of p Akt in GLP-1group was increased markedly (P<0.05), while the protein levels of pP38and pERK1/2were not observed any significantly changes.4. Oil Red O staining results were shown that the lipid accumulation in experimental group were not increased as the enhanced concentrations of GLP-1. As compared to the control and other concentration groups, the cytomorphology of adipocyte in100nM GLP-1group was shown there were increased numbers of small adipocyte (P <0.05)5. Under the treatment of100nM GLP-1, the mRNA level of CPT-1A was increased significantly, while the mRNA level of UCP-1was not changed as compared to the control and other concentration groups (P<0.05).Conclusion:1. GLP-1enhanced the express of PPAR-γ, CEBP/α, LPL and aP2significantly in a dose-and time-dependent way during the period of3T3-L1preadipocyte differentiation,2. Akt signaling way may be involved in the above GLP-1-induced effects.3.100nM GLP-1can promote the formation of small adipocytes, which have more mRNA level of CPT-1A.

GLP-1对3T3-L1前脂肪细胞分化的影响及其调控机制研究

中文摘要6-9ABSTRACT9-11符号说明12-15前言15-17材料与方法17-33结果33-36讨论36-42创新点42局限性42-43结论43-44附图44-53参考文献53-59致谢59-60攻读硕士学位期间发表的学术论文60-61附件61

  本文关键词：GLP-1对3T3-L1前脂肪细胞分化的影响及其调控机制研究，由笔耕文化传播整理发布。