Development of a Fit-for-Purpose Assay
Development of a Fit-for-Purpose Assay for Glucocorticoid Receptor-Mediated Adipogenesis
Since 1980, obesity rates worldwide have doubled, with over 600 million people classified as obese in 2014. While diet and lack of exercise are major contributors to obesity, other environmental factors may enhance susceptibility by leading to the disruption of normal hormone homeostasis and metabolic processes. Chemical exposure early in development may enhance adipocyte differentiation, thereby increasing the number of adipocytes in adipose tissue and establishing a foundation for obesity and metabolic disease later in life (i.e., the obesogen hypothesis). The identification of endocrine disrupting chemicals (EDCs) has historically been restricted to androgen, estrogen, and thyroid mediated pathways, with a negligible focus on other relevant biological pathways that alter human physiology. We previously developed a novel human primary preadipocyte assay that can evaluate the effect of EDCs on PPARγ-dependent adipocyte differentiation. Here, we modified the assay to evaluate the effects of suspected EDCs on the glucocorticoid receptor (GR) pathway, which is a key signaling mechanism in adipocyte differentiation. The differentiation cocktail was reformulated by removing the synthetic glucocorticoid agonist dexamethasone (DEX) from the complete medium. In the absence of DEX, the adipocyte progenitors do not completely differentiate. When progenitor cells were treated with a 10-point concentration-response of 20 prioritized compounds previously identified by ToxPi as having GR-dependent bioactivity, 5 induced a 2-fold or greater increase in RLA. Orthogonal assays confirmed that these compounds caused terminal adipocyte differentiation in a GR-dependent manner. To determine whether any of the weak agonists or negative compounds were GR-antagonists (as predicted by Tox21), we modified the assay to include the test compounds in the presence of the complete differentiation cocktail. After a series of data analyses of the results in comparison to ToxCast/Tox21 ToxPi scores, we found that 9/10 of the hypothesized antagonists showed decreasing RLA, including all 4 of the weak agonists. Taken together, these data support the use of a modified human adipose-derived stem cell differentiation assay to test the potential of EDCs to alter human adipogenesis through the GR pathway.