We thank the Editor for inviting us to write a response to the letter by Drs. Dela and Helge (1) concerning our recent article in Diabetes (2). The authors draw our attention to an incorrect reference used to describe mitochondrial respiration measurements. The correct reference is an article that Dr. Dela’s group published in 2010 (3). We apologize for any confusion caused. We hope this alleviates the authors’ concerns on the methodology used for the mitochondrial respiration measurements; nevertheless, we provide further information here for clarity. Biopsies were immediately submerged in preservation buffer (BIOPS as described in ref. 3) and immediately (within 30 min) brought to the laboratory. Samples were weighed and suspended in 2 mL of buffer (MIR05 [3]) for respirometry measurements. Measurements were made at 37°C, and the O2 concentration was kept above 100 nmol/mL. As we report one respiratory measurement, we did not think it necessary to report the sequence of additions; however, substrates and ADP were added prior to oligomycin.
Dela and Helge (1) use the term “uncoupled” to refer to respiration following the titration of an uncoupler such as carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP). We use this term more broadly to describe respiration uncoupled from ADP phosphorylation. In our study (2), this was achieved by the addition of substrates and ADP, followed by oligomycin. While this measurement could also have been termed “leak respiration” or “state 4O respiration,” we used the term “uncoupled respiration.”
While we agree that uncoupled respiration determined after the titration of FCCP would be blunted in the presence of oligomycin, this is a moot point as we did not use FCCP (2). Moreover, since we clearly state that uncoupled respiration was determined following titration of substrates, ADP, and oligomycin, our experiments can be replicated by others.
Dela and Helge are correct that no conclusions on intrinsic mitochondrial function can be made from respiratory capacity measurements alone. However, in our view, using biochemical surrogates of mitochondrial density to calculate intrinsic mitochondrial function is not ideal. Indeed, with regards to brown fat, titration of the uncoupling protein 1 (UCP1) inhibitor guanosine diphosphate (GDP) would be a better indicator of intrinsic mitochondrial function (4).
With regard to the gene expression data, we would like to clarify that Fig. 6F and G refer to the biopsy samples collected from all subjects under cold exposure. In Fig. 6H and I and Supplementary Figs. 3–6, subjects #1, #2, and #4 are subjects assigned to the “BAT+” group because they had 134, 41, and 50 mL of active BAT, respectively. Subject #3 was assigned to the “BAT−”group, as this subject had only minimal amount of BAT (11 mL). With regard to the “control BAT” sample, we used a previously established supraclavicular BAT sample as a positive control for BAT (5) and report the gene expression levels in our samples as a fold to that in the control sample. Similar to Virtanen et al. (5), the results in Fig. 6H and I and Supplementary Figs. 3–6 represent technical replicates. A paired t test was used to compare gene expression data during cold exposure and thermoneutral conditions.
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Duality of Interest. No potential conflicts of interest relevant to this article were reported.
