Unveiling the dynamics of acetylation and phosphorylation in SGBS and 3T3-L1 adipogenesis.

Obesity, characterized by enlarged and dysfunctional adipose tissue, is among today's most pressing global public health challenges with continuously increasing prevalence. Despite the importance of post-translational protein modifications (PTMs) in cellular signaling, knowledge of their impact on adipogenesis remains limited. Here, we studied the temporal dynamics of transcriptome, proteome, central carbon metabolites, and the acetyl- and phosphoproteome during adipogenesis using LC-MS/MS combined with PTM enrichment strategies on human (SGBS) and mouse (3T3-L1) adipocyte models. Both cell lines exhibited unique PTM profiles during adipogenesis, with acetylated proteins being enriched for central energy metabolism, while phosphorylated proteins related to insulin signaling and organization of cellular structures. As candidates with strong correlation to the adipogenesis timeline we identified CD44 and the acetylation sites FASN_K673 and IDH_K272. While results generally aligned between SGBS and 3T3-L1 cells, details appeared cell line specific. Our datasets on SGBS and 3T3-L1 adipogenesis dynamics are accessible for further mining.

Metabolomics in human SGBS cells as new approach method for studying adipogenic effects: Analysis of the effects of DINCH and MINCH on central carbon metabolism.

Growing evidence suggests that exposure to certain metabolism-disrupting chemicals (MDCs), such as the phthalate plasticizer DEHP, might promote obesity in humans, contributing to the spread of this global health problem. Due to the restriction on the use of phthalates, there has been a shift to safer declared substitutes, including the plasticizer diisononyl-cyclohexane-1,2-dicarboxylate (DINCH). Notwithstanding, recent studies suggest that the primary metabolite monoisononyl-cyclohexane-1,2-dicarboxylic acid ester (MINCH), induces differentiation of human adipocytes and affects enzyme levels of key metabolic pathways. Given the lack of methods for assessing metabolism-disrupting effects of chemicals on adipose tissue, we used metabolomics to analyze human SGSB cells exposed to DINCH or MINCH. Concentration analysis of DINCH and MINCH revealed that uptake of MINCH in preadipocytes was associated with increased lipid accumulation during adipogenesis. Although we also observed intracellular uptake for DINCH, the solubility of DINCH in cell culture medium was limited, hampering the analysis of possible effects in the µM concentration range. Metabolomics revealed that MINCH induces lipid accumulation similar to peroxisome proliferator-activated receptor gamma (PPARG)-agonist rosiglitazone through upregulation of the pyruvate cycle, which was recently identified as a key driver of de novo lipogenesis. Analysis of the metabolome in the presence of the PPARG-inhibitor GW9662 indicated that the effect of MINCH on metabolism was mediated at least partly by a PPARG-independent mechanism. However, all effects of MINCH were only observed at high concentrations of 10 µM, which are three orders of magnitudes higher than the current concentrations of plasticizers in human serum. Overall, the assessment of the effects of DINCH and MINCH on SGBS cells by metabolomics revealed no adipogenic potential at physiologically relevant concentrations. This finding aligns with previous in vivo studies and supports the potential of our method as a New Approach Method (NAM) for the assessment of adipogenic effects of environmental chemicals.