SGBS cell differentiation – a model for adipogenesis – studied by quantitative proteomics and metabolomics

Introduction: The Simpson-Golabi-Behmel Syndrom (SGBS) cells are a reliable model to study human adipogenesis. However, the molecular mechanisms that occur during adipocyte differentiation and which are potentially involved in the development of obesity and related sequelae are not fully understood. Therefore the aim of this study was to investigate comprehensively the adipogenesis of SGBS preadipocytes to adipocytes on a molecular level using a global proteomics and a targeted metabolomic approach.

Methods: In this study we used Stable Isotope Labeling in Cell Culture (SILAC) combined with LC-MS/MS for an accurate in-depth quantitative proteome analysis of intracellular and secreted proteins. Half of the preadipocytes were grown in culture media which contained heavy amino acids (¹³C₆ ¹⁵N₄-Arg and ¹³C₆-Lys) for stable isotope labeling of all proteins. The other half was grown in medium supplemented with light amino acids (¹²C₆-Arg and ¹²C₆-Lys) before and during differentiation. After harvesting the cells as well as the cell culture supernatants samples were conditioned by precipitation with TCA. The pellets were dissolved in buffer containing 6M Urea, 0.1M ammonium bicarbonate und 1M DTT and concentrated using a 10kDa cutoff centrifugal filter units. Subsequently adipocyte and preadipocyte protein extracts were mixed equimolar. After further separation by 1D-SDS-PAGE the entire protein gel lanes were cut into gel slices, subjected to in-gel digestion with trypsin and subsequently measured by nano-HPLC/nano-ESI-LTQ-Orbitrap-MS. MS raw data were analyzed and quantified using the MaxQuant software. Differenzially expressed proteins were clustered according their protein functions as well as their participation in metabolic pathways using the functional annotation tool DAVID. For the assessment of 163 metabolites MeOH/water extracted cells and the media were applied to Biocrates Absolute IDQ kit.

Results: In a preliminary analysis we were already able to quantify more than 1100 proteins. 54 proteins were found to be more abundant in adipocytes whereas 378 proteins were of significant higher abundance in preadipocytes. Also in agreement with the whole-genome transcriptomics analysis differenzially expressed proteins play especially an important role in cell structure, translational elongation and protein degradation as well as in metabolic processes. Among the significantly regulated metabolic pathways „fatty acid metabolism“, „valine, leucine and isoleucine degradation“ and „carbohydrate metabolism“ were found to be highly enriched, which could be fully confirmed by metabolome data.

Conclusion: Our preliminary analysis revealed numerous differenzially expressed proteins and metabolites in preadipocytes compared to adipocytes. The next step will be to combine transcriptome, proteome and metabolome data in order to identify a small but reliable set of new markers or even mediators potentially involved in the pathogenesis of obesity and related disease.