Comparative Proteome Analysis And Preliminary Study Of Partial Adipogenic Relevant Proteins' Function In3T3-L1Adipocyte Differentiation

Chapter1Comparative proteome analysis of3T3-L1adipocyte differentiation using iTRAQ-coupled2D LC-MS/MSObesity 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 proteinsUsing 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-L1adipocytesNeuropeptide 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.