摘要
第一章鉴定3T3-L1脂肪细胞分化相关的蛋白质
肥胖是最常见的疾病之一,2005年世界卫生组织估计全世界肥胖患者超过10亿。大量研究表明肥胖与2型糖尿病、心血管疾病和癌症有着密切联系。肥胖正成为全球一个严重的健康问题。近十多年来,人们对脂肪组织的认识发生了革命性的改变。脂肪组织不仅仅是一个惰性的剩余代谢能量贮藏器官,同时它也是一个活跃的内分泌器官,能分泌多种脂肪激素,调节机体代谢、能量平衡、心血管等多种功能。目前的研究认为脂肪组织在肥胖和2型糖尿病的发生发展中起着重要作用,因此,调节脂肪细胞分化成为了治疗上述疾病的可能策略之一。然而,调控脂肪细胞分化的分子基础仍不清楚。
同位素标记相对和绝对定量(iTRAQ)技术是近年来新开发的一种蛋白质组学定量研究技术。具有高通量、高灵敏度、高重复性、定量准确等多种优点。激素诱导3T3-L1成纤维细胞分化为脂肪细胞是一个成熟的体外脂肪细胞分化模型,但是迄今未见iTRAQ技术用于其研究的文献报导。在本部分研究中,我们运用iTRAQ-coupled2D LC-MS/MS对3T3-L1成纤维细胞诱导分化为脂肪细胞的机制进行研究。共定量了近1000种蛋白质,其中106个蛋白质在脂肪细胞分化过程中有明显改变。这些蛋白质主要涉及代谢酶、结构蛋白及信号转导相关蛋白。部分差异蛋白包括C/EBPs、cAMP反应元件结合蛋白(CREB)和脂蛋白脂肪酶(LPL)等和以前报道的类似。除此之外,我们新发现了HEXB、DPP7、PTTG1IP、PRDX5、EPDR1、SPNB2、 STEAP3、NIBAN、ACADM、RPS19、NEDD4、KANK2、LOC10045699、 EG432502、NUCB1、MBC2、FBN1、CALU、CAPG、TPP1等这些涉及脂肪分化的蛋白质。我们的结果为肥胖与2型糖尿病的治疗提供了新的方向。
第二章部分蛋白质分子差异表达水平的验证及功能研究
在上述研究中,通过iTRAQ技术我们发现了106个差异表达蛋白。为了确保筛选到真正的差异蛋白,我们通过western blot和/或real-time PCR对12个蛋白(Destrirn、Nucleolin、Zyx protein、Transgelin2、VDAC2、VDAC3、Cytochrome b5、PCX、Annexin A1、Ribosomal protein SA、EEF1A1、β-actin)的质谱结果进行了验证。PCX是一个存在于线粒体中的代谢酶。PCX在糖异生、脂肪生成、胰岛素分泌和神经递质的合成中均起着作用。VDAC2是线粒体内外膜的线粒体通透性转运孔复合体的组成部分。VDAC以两种方式调控线粒体介导的细胞死亡,一是作为线粒体通透性转运孔复合体的主要组成调控细胞死亡,另一是与Bcl-2家族蛋白质相互作用调控细胞死亡。文献报道凋亡与脂肪细胞分化存在一定关系。因此,我们运用siRNA技术进一步探讨了PCX和VDAC2在脂肪分化中的作用。结果表明,调节PCX或VDAC2均有助于抑制脂肪细胞分化过程,为肥胖和2型糖尿病的治疗提供了新的药物靶点。
第三章3T3-L1脂肪细胞分泌神经肽Y及其调控的研究
神经肽Y(NPY)是一个含有36个氨基酸的多肽,参与调节代谢平衡。NPY主要在中央和外周神经系统合成。最近研究发现,脂肪组织也能合成和分泌NPY。然而脂肪组织分泌NPY的途径和调节均不清楚。在这项工作中,我们运用全内反射成像技术、荧光测钙、共聚焦成像技术等技术,研究了3T3-L1脂肪细胞的NPY分泌囊泡的转运和分泌,特别是囊泡的定位及其他细胞因子对脂源性NPY的合成和分泌的调节作用,并通过western blot和ELISA验证了我们的观察。结果发现瘦素、TNF-α和肾上腺素参与调节脂肪细胞源性NPY的分泌。NPY与脂联素的分泌囊泡很大程度是相同的,而与抵抗素和瘦素,仅小部分共定位在同一分泌囊泡。此外,通过给予一些不同的信号通路抑制剂,我们发现TNF-α或瘦素经由JNK和NF-KB信号通路调节脂源性NPY的分泌。最后,我们证实Ca2+信号调控脂肪细胞源性NPY的调节性分泌,但不影响其组成性分泌。这些结果表明脂肪细胞源性NPY是一种新的脂肪激素。它的分泌受到机体不同来源的信号分子及脂肪细胞自分泌的调节。脂肪细胞源性NPY与其他脂肪激素可能构成了一个复杂的网络,协同调节机体的摄食及能量代谢等多种功能。
Chapter1Comparative proteome analysis of3T3-L1adipocyte differentiation using iTRAQ-coupled2D LC-MS/MS
Obesity is one of the most frequent physiological disorders that is associated with a wide variety of conditions including type Ⅱ diabetes, cardiovascular diseases and cancer. It is characterized by excess of body fat mass, which is mostly stored in adipose tissue. There are indications that obesity is fast becoming a serious health problem worldwide. Accumulating evidence has indicated that adipocyte differentiation, an increase in fat cell number, plays an important role in obesity, and adipose tissue is a highly active endocrine organ capable of secreting a number of signal molecules called adipokines (including leptin, adiponectin, resistin, etc.). In summary, adipose tissue is critical in obesity and type Ⅱ diabetes. Blocking of adipocyte differentiation is one of the anti-obesity strategies targeting on strong rise in fat storage and secretion of adipokine(s). However, the molecular basis of adipocyte differentiation and its regulation remains obscure. Recently, there has been great progress in comparative proteomic approaches, including DIGE, iTRAQ,18O, ICAT, and SILAC. Among these methods, iTRAQ technology has gained great popularity in quantitative proteomics applications due to its high sensitivity and accurate quantitation and reproducibility. Therefore, we exposed3T3-L1cell line to appropriate hormonal inducers as adipocyte differentiation model. To our best knowledge, there is no report that profiled proteome during3T3-L1adipocyte differentiation using iTRAQ-coupled2D LC-MS/MS. Using iTRAQ-coupled2D LC-MS/MS, a successfully exploited high-through proteomic technology, we nearly quantitated1000protein species and found106significantly altered proteins during adipocyte differentiation. The great majority of differentially expressed proteins were related to metabolism enzymes, structural molecules, and proteins involved in signal transduction. In addition to previously reported differentially expressed molecules including CCAAT/Enhancer Binding Proteins (C/EBPs), cAMP response element-binding protein (CREB), and lipoprotein lipase (LPL), we firstly revealed previously unknown altered proteins during adipogenic process (e.g., HEXB, DPP7, PTTG1IP, PRDX5, EPDR1, SPNB2, STEAP3, NIBAN, ACADM, RPS19, NEDD4, KANK2, LOC10045699, EG432502, NUCB1, MBC2, FBN1, CALU, CAPG, and TPP1etc.). Our data provide valuable information for further understanding of adipogenesis.
Chapter2The validation and function of the partially differential proteins
Using iTRAQ-coupled2D LC-MS/MS, a successfully exploited high-throughput proteomic technology, we found106significantly altered proteins during adipocyte differentiation. Twelve proteins (Destrin; Nucleolin; Zyx protein; Transgelin2; VDAC2; VDAC3; Cytochrome b5; PCX; Annexin A1; Ribosomal protein SA; EEF1A1and β-actin) were validated by western blot and/or real-time PCR analysis. Furthermore, the association of PCX and VDAC2, two altered proteins, with adipocyte conversion was analyzed using siRNA method. PCX is a regulatory metabolic enzyme that provides acetyl-CoA and NADPH for the de novo biosynthesis of fatty acids. PCX plays a role in lipogenesis and gluconeogenesis, in glucose-induced insulin secretion by pancreatic islets, and in the biosynthesis of neurotransmitters. VDAC2is a component of the permeability transition pore complex (PTPC) of the mitochondrial membranes. The subsequent dissipation of mitochondrial inner membrane potential and release of cytochrome c through the outer mitochondrial membrane are critical events in the early stages of apoptosis. Furthermore, VDAC2significantly inhibited apoptosis mediated by BAK. In additional, ligands to VDACs induce non-apoptotic cell death selectively in some tumour cells harbouring activating mutations in the RAS-RAF-MEK pathway. Our results showed that both PCX and VDAC2could contribute considerably to adipogenesis. These data provide new targets for future investigation studies with respect to obesity and type Ⅱ diabetes.
Chapter3Neuropeptide Y, a new adipokine:localization, synthesis, secretion and regulation in3T3-L1adipocytes
Neuropeptide Y (NPY) is a36-amino-acid peptide involved in the regulation of metabolic homeostasis. NPY is mainly synthesized in the central and peripheral nervous systems. Recently, it was found that adipose tissue synthesizes and secretes NPY. Adipose tissue has been recognized as a highly active endocrine organ secreting a variety of signaling molecules called adipokines. Nevertheless, the regulation, secretory pathways and potential endocine effects of NPY in adipose tissue are poorly elucidated. In this work, we have studied several key aspects of NPY from3T3-L1adipocytes, specifically the localization of NPY vesicles, the effect of adipokine on NPY secretion and the influence of TNF-a and epinephrine (E) on adipocyte-derived NPY in vitro. It was found that NPY and adiponectin molecules are compartmentalized into same secretory vesicles. NPY, leptin and resistin molecules are compartmentalized into different secretory vesicles. The synthesis, vesicles trafficking, and the secretion of of NPY are increased by TNF-a or leptin via JNK and NF-κB pathways. Finally, we demonstrated that TNF-a increased NPY secretion by an additional50%. Acute NPY secretion is Ca2+dependent. In contrast, basal NPY secretion is calcium independent. Taken together, these results suggest that adipose-derived NPY is highly regulated by different signal molecules and may have implications for central feedback of adiposity signals.
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