Label-free dynamic mass redistribution analysis of endogenous adrenergic receptor signaling in primary preadipocytes and differentiated adipocytes

Abstract

IntroductionAdipose tissues release adipokines, which regulate energy intake and expenditure. G protein-coupled receptors (GPCRs) and associated signaling pathways in adipocytes are potentially important drug targets for conditions with disturbed energy metabolism. MethodsThe aim of the current study was to compare signaling of endogenously expressed GPCRs between primary preadipocytes and differentiated adipocytes using a novel state-of-the-art unbiased method that measures dynamic mass redistribution (DMR) in real-time. Adrenergic agonists were chosen since they control adipocyte functions such as lipolysis and glycogenolysis. ResultsIsoprenaline (ISO) and phenylephrine (PE) elicited concentration-dependent responses in preadipocytes and differentiated adipocytes. The effect of ISO was cholera toxin (CTX)-sensitive, indicating it is Gs-dependent. The effect could also be blocked by propranolol proving the signal is mediated through β-adrenergic receptors. The signaling resulting from PE stimulation was completely abolished by the Gq/11-selective inhibitor FR900359 and CTX in preadipocytes but surprisingly became FR900359-insensitive but remained CTX-sensitive in differentiated adipocytes. The use of prazosin and propranolol revealed that the PE-response in differentiated adipocytes had a β-adrenergic receptor component to it. In addition, we tested the bone-derived peptide osteocalcin, which did not result in DMR changes in preadipocytes or differentiated adipocytes. DiscussionIn conclusion, this study for the first time demonstrates that DMR assays can be used to assess signaling in differentiated adipocytes. This platform can serve as a tool for future drug screening in primary adipocytes. Furthermore, this study illustrates that PE-induced effects on adipocytes vary by developmental stage and are not as selective as originally thought.