Rotenone antimycin

The oxygen consumption rate in adipocytes was measured to evaluate oxidative phosphorylation using the Agilent Seahorse XFe24 Analyzers (Agilent Technologies) following the manufacturer’s operating instructions. In brief, 2–4 × 10⁴ cells were seeded in wells of a 24-well XF Cell Culture Microplate (Agilent Technologies, 100777-004). Cells were grown in growth medium until they reached confluence and then they were differentiated into mature adipocytes. Assay was performed on mature adipocytes. The medium was exchanged to XF DMEM medium pH 7.4 (Agilent Technologies, 103575-100) with addition of following compounds: 25 mM glucose (G8270, Sigma-Aldrich), 2 mM glutamine (G9003, Sigma-Aldrich) and 2 mM sodium-pyruvate (P5280, Sigma-Aldrich). Cells were incubated in this medium for 1 h at 37 °C without CO₂. Oxygen consumption rate measurements were performed with or without CL-316243 (10 µM) (C5976, Sigma-Aldrich) stimulation, which was followed by the sequential addition of 2 µM oligomycin (Complex V inhibitor), 1 µM FCCP and 0.5 µM rotenone/antimycin (Complex I/III inhibitor) (Agilent Technologies, 103015-100). All above experimental procedures were carried out at 37 °C. The basal and uncoupled respiration were determined and the results are expressed in pmol min⁻¹ and normalized to the cell number.

The OCR was measured using the XF24 Extracellular Flux Analyzer (Seahorse Bioscience). SVF cells (5 × 10⁵ cells/well) were plated in an XF24-well cell culture microplate (Seahorse Bioscience) and differentiated into brown adipocytes, followed by OCR measurement at 37 °C according to the manufacturer’s instructions. Briefly, differentiated cells were incubated in pre-warmed assay medium (Seahorse XF base medium (Agilent Technologies) with 25 mM glucose, 1 mM sodium pyruvate, 2 mM L-glutamine) for 30 min without CO₂, and a mitochondrial stress test was conducted. To detect the uncoupled respiration, maximal respiration, and non-mitochondrial respiration, 5 μM oligomycin, 10 μM FCCP, and 0.5 μM rotenone/antimycin (Agilent Technologies) were injected, respectively. The OCR was normalized to the lipid accumulation (OD492 value), and the data are expressed as pmol of O₂/min/OD492.