Adrenergic Stimulation and Mitochondrial Respiration

Oxygen consumption was measured using an XF96 oxymeter (Seahorse Biosciences, North Billerica, MA, USA). The hADMSCs were seeded and differentiated in 96-well XF96 assay plates. After recording the baseline OC, the cells received a single bolus of dibutyril-cAMP at 500 μm concentration modeling adrenergic stimulation. Then, stimulated OC was recorded every 30 min. The final reading took place at 5 h post treatment. To exclude that dibutyril-cAMP enhanced lipolysis might provoke artefactual mitochondrial uncoupling, we repeated the OC measurements including 2% BSA in the respiration buffer.^{44 (link)} The adipocytes were treated with 5 μm etomoxir or with 2 mm β-guanidinopropionic acid to block beta-oxidation and creatine-driven substrate cycle.^{45 (link)} Next, proton leak respiration was determined after adding oligomycin at 2 μm concentration to block ATP synthase activity. As a last step, the cells received a single bolus of Antimycin A at 10 μm concentration for baseline correction. The oxygen consumption rate (OCR) was normalized to protein content and normalized readings were displayed. For statistical analysis (n=4), the fold change of OC levels were calculated comparing the basal, cAMP-stimulated and oligomycin-inhibited (both in unstimulated and stimulated cells) OCRs of each sample to the basal OCR of untreated white adipocytes.^{38 (link), 42 (link), 46 (link)}