Mitochondrial function and respiration

Adipose tissue fragments or cultured adipocytes were assayed for oxygen consumption rate (OCR) using an XF24 Extracellular Flux Analyzer (Seahorse Bioscience, MA). Assays of mitochondrial function and respiration rates in whole adipose tissues or cells were performed as previously described (Shao et al., 2016). In brief, adipose tissue was cut into 5–10 mg pieces and locked into an XF24 islet-capture Microplate (Seahorse Bioscience). Adipose tissues or cultured adipocytes were equilibrated for 1 hr at 37°C in a CO₂-free incubator in XF Assay Medium (Modified DMEM, 0 mM Glucose; Seahorse Bioscience) (pH 7.4), supplemented with 1 mM sodium pyruvate, 1 mM L- Glutamine and 7 mM glucose. Tissues or cells were subjected to a 10 min equilibration period and three assay cycles to measure the basal rate, comprising a 3 min mix, a 2 min wait and a 3 min measure period each. For cells, compounds were then added by automatic pneumatic injection followed by assay cycles after each, comprising of 3 min mix, 2 min wait and a 3 min measure period. OCR measurements were obtained following sequential additions of oligomyin (3 µM final concentration), FCCP (9 µM) and antimycinA/rotenone (3 µM/30 nM). OCR measurements were recorded at set interval time points. All compounds and materials above were obtained from Sigma-Aldrich.