Brown Adipose Tissue Bioenergetics: A New Methodological Approach

Abstract

The rediscovery of brown adipose tissue (BAT) in humans and its capacity to oxidize fat and dissipate energy as heat has put the spotlight on its potential as a therapeutic target in the treatment of several metabolic conditions including obesity and diabetes. To date the measurement of bioenergetics parameters has required the use of cultured cells or extracted mitochondria with the corresponding loss of information in the tissue context. Herein, we present a method to quantify mitochondrial bioenergetics directly in BAT. Based on XF Seahorse Technology, we assessed the appropriate weight of the explants, the exact concentration of each inhibitor in the reaction, and the specific incubation time to optimize bioenergetics measurements. Our results show that BAT basal oxygen consumption is mostly due to proton leak. In addition, BAT presents higher basal oxygen consumption than white adipose tissue and a positive response to b-adrenergic stimulation. Considering the whole tissue and not just subcellular populations is a direct approach that provides a realistic view of physiological respiration. In addition, it can be adapted to analyze the effect of potential activators of thermogenesis, or to assess the use of fatty acids or glucose as a source of energy.

Keywords: bioenergetics; brown adipose tissue; mitochondria; oxygen consumption rate; seahorse.

Figure 1

Oxygen consumption rate in brown adipose tissue explants. 9 mg of iBAT from 13 mice were analyzed using the XF24 Islet Capture Microplate. The amount of tissue, concentration of inhibitors, and incubation times were used as described in the Experimental Section. The injection of inhibitors is represented as dashed lines. A) Average OCR trace, depicting basal oxygen consumption, oligomycin resistant respiration (H⁺ leak), and ATP‐linked OCR after the addition of 24 µg mL⁻¹ oligomycin. Maximal respiratory rate and reserve capacity were calculated after the injection of 0.8 × 10⁻⁶ m FCCP. Finally, 5 × 10⁻⁶ m rotenone and 15 × 10⁻⁶ m antimycin A were added to observe nonmitochondrial respiration. B) Bioenergetic parameters were inferred from the OCR traces. The results are represented as mean ± standard error of the mean (SEM). * Shows statistical differences between bioenergetic parameters and basal respiration. N = 13, p < 0.05.

Figure 2

Potential applications of bioenergetics measurement in brown adipose tissue. A) Comparative analysis of basal oxygen consumption in epididymal white adipose tissue (eWAT) and interscapular brown adipose tissue (iBAT). Basal OCR was analyzed in equal amounts of eWAT and iBAT, using the XF24 Islet Capture Microplate and normalized by milligrams of protein. B) OCR trace in iBAT explants after norepinephrine injection. The injection time and the concentrations used for the different compounds are shown. Rot: Rotenone. AA: Antimycin A. Results are represented as mean ± SEM. * Shows statistical differences between eWAT and iBAT. N = 13, p < 0.05.