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

Journal of pharmacological and toxicological methods (2019-04-05)
Christinna V Jørgensen, Hong Zhou, Markus J Seibel, Hans Bräuner-Osborne

Adipose 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. The 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. Isoprenaline (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. In 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.

Product Number
Product Description

Insulin solution human, sterile-filtered, BioXtra, suitable for cell culture
Dulbecco′s Phosphate Buffered Saline, With MgCl2 and CaCl2, liquid, sterile-filtered, suitable for cell culture
Collagenase from Clostridium histolyticum, suitable for release of physiologically active rat epididymal adipocytes, Type II, 0.5-5.0 FALGPA units/mg solid, ≥125 CDU/mg solid
Dexamethasone-Water Soluble, BioReagent, suitable for cell culture
Rosiglitazone, ≥98% (HPLC)
(±)-Propranolol hydrochloride, ≥99% (TLC), powder
(R)-(−)-Phenylephrine hydrochloride, powder
Prazosin hydrochloride, ≥99.0% (HPLC)
(−)-Isoproterenol (+)-bitartrate salt, powder
Corning® cell strainer, pore size 100 μm, yellow, sterile, pkg of (individually wrapped), pack of 50 ea