Supplemental Material Details for the biopsy procedure During surgery, both the surgeon (N.B.) and the researcher (G.V.) visually inspected the exposed operative area for presence of small islands (±1-2 cm) of adipose tissue from the BAT region along the sternocleidomastoid muscle and more cranially along the thyroid gland. These adipose tissue depots are typically light brown/beige and have a firm consistency compared to white adipose tissue. When adipose tissue from the BAT region was recognized, a biopsy of 0.5cm 3 was taken via sharp dissection with a scalpel, together with 0.5cm 3 of subcutaneous white adipose tissue. Adipose tissue biopsies showed both univacuolar and multivacuolar adipocytes and were characterized as white adipocytes and brown adipocytes, respectively (Supplemental Figure 1). Details for the tissue processing and sample preparations In the operating room, all tissue biopsies were immediately placed in a tube with warm (37ºC) PBS. One portion each of the adipose tissue from the BAT and the WAT regions (±10% of total biopsy) was snap-frozen and stored at -80ºC for future analyses. Unfortunately, we did not do a quantification of the wet weight of the tissue because we aimed to perform oxygraph measurements immediately following the biopsy. Based on the personal experience of Dr. Sparks from processing >500 biopsies over the past 10 years, it is estimated that the biopsies were ~100mg each. Previous work in our lab demonstrated poor respiration in adipose tissue permeabilized in the oxygraph chamber with digitonin as previously described in the literature (1). We rationalized that a collagenase digestion would allow for a more complete liberation of the cells from the tissue. Accordingly, BAT and WAT tissues were washed two times in a warm PBS solution with 1% antibiotic until all blood vessels and connective tissue had 1
been released. Tissues were then placed in 2mL tubes with Collagenase solution (10mMCaCl 2, 1%BSA and 0.1% Collagenase-type-I), minced with scissors and incubated in a shaking water bath (75RPM) at 37ºC for ~15-30 minutes or when the tissue appeared smooth upon visual inspection. Since prolonged digestion can lead to cell damage, visual inspection of the tissue was necessary. The reaction was terminated sooner if the tissue appeared smooth and fully digested. Samples were then centrifuged for 5 min at 1200RPM at room temperature (RT), manually shaken to disrupt the pellet and centrifuged again for 5 min at RT. We could not know the homogeneity of each preparation such that those brown adipocytes containing much less lipid than the white adipocytes (also present in the sample) would presumably be contained in the stromal vascular fraction after centrifugation. Therefore, we combined the top layer (containing predominately those cells with high lipid content) and the bottom layer (containing the stromal vascular fraction), discarded the interphase that contained the remaining collagenase solution (2) and placed it in 2.3mL tube with MiR05 respiration buffer (OROBOROS, Innsbruck, Austria). These samples were then added to the two separate respiration chambers for simultaneous respiration measurements. Details for respiration measurements Basal respiration was measured once the tissue preparation was added to the respiration chamber without addition of exogenous substrates for about 5-7 minutes. Once stable, mitochondrial respiration uncoupled from ATP synthesis (state 4) was determined via addition of the ATP synthase inhibitor oligomycin (1.0 µl, 2mg/mL) for about 4-5 minutes. In addition, saturating concentrations of the chemical uncoupler FCCP (carbonylcyanide 4-(trifluoromethoxy)-phenylhydrazone) were titrated (0.5 µl, 10mM at a time) in 2
order to maximize oxygen flux, which allows for assessment of maximal mitochondrial capacity (state u respiration). Correction of raw oxygen fluxes All raw oxygen fluxes (expressed as picomoles O 2 consumption x seconds -1 ) for both WAT and AT from the BAT region were normalized for the DNA content measured in the cell suspensions derived from the oxygraph chambers (mg/ml). The DNA contents of these cell suspensions were not different for WAT and AT from BAT region (0.037±0.014 mg/ml vs. 0.043±0.021 mg/ml, P=0.108). Corrected oxygen fluxes are expressed as picomoles O 2 -consumption x seconds -1 x (mg/ml) -1. Calculation of respiratory control ratios For comparison of the relative decrease in oxygen consumption upon the addition of oligomycin, the oligomycin-insensitive leak-state-4 respiration was divided by baseline respiration. This uncoupling ratio (UCR) was not different in AT from the BAT region compared to WAT (AT from the BAT region; 0.83±0.17, WAT; 0.79±0.20, P=0.501). The relative contribution of baseline respiration and state 4 respiration to the maximally uncoupled respiration, as calculated by baseline respiration or state 4 respiration divided by state u, was higher in AT from the BAT region but this did not reach statistical significance (baseline / state u; AT from the BAT region: 0.83±0.21, WAT: 0.70±0.33, P=0.292 and state 4 / state u; AT from the BAT region: 0.68±0.17, WAT: 0.54±0.26, P=0.122). 3
Supplemental Table 1. Sex, weight, plasma free thyroxine (FT4), indications and surgical therapy per included subject. MENIIA-syndrome indicates the Multiple Endocrine Neoplasia syndrome type IIA, as was confirmed by genetic screening in subject families; a preventive, total thyroidectomy is indicated due to the high lifetime risk of developing thyroid gland cancer, mostly of the medullary type. Hyperparathyroidism indicates supraphysiological activity of the parathyroid gland, which is located adjacent to the thyroid gland itself. Parathyroidectomy indicates resection of the parathyroid gland, hemithyroidectomy indicates resection of the left/right half of the thyroid gland and total thyroidectomy indicates a total resection of the thyroid gland as a whole. Subject Sex Weight FT4 Indication Surgery 1 F 80.0 14.1 hyperparathyroidism parathyroidectomy 2 M 91.0 14.1 suspected thyroid gland hemithyroidectomy 3 M 93.0 15.3 suspected thyroid gland hemithyroidectomy 4 F 95.0 12.0 cyst left thyroid gland hemithyroidectomy 5 F 55.0 19.6 MENIIA-syndrome total thyroidectomy 6 M 86.0 15.2 suspected thyroid gland hemithyroidectomy 7 F 80.0 22.4 suspected thyroid gland hemithyroidectomy 8 F 50.0 13.4 MENIIA-syndrome total thyroidectomy 9 F 60.0 11.8 MENIIA-syndrome total thyroidectomy 10 F 75.0 15.6 struma hemithyroidectomy 11 F 60.0 13.0 hyperparathyroidism parathyroidectomy 12 M 80.0 15.4 struma hemithyroidectomy 13 M 74.0 18.0 suspected thyroid gland hemithyroidectomy 4
Supplemental Table 2. Subject characteristics for eight male and five female subjects indicated for thyroid gland surgery. BMI, Body Mass Index; TSH, Thyroid-Stimulating Hormone (normal TSH assay range; 0.4-4.3 mu/l); FT4, plasma free thyroxine (normal FT4 assay range; 4.0-18.0 pmol/l). Parameter Mean ± S.D. Male / Female 8 / 5 Age (years) 43 ± 19 Body (kg) 75.3 ± 14.9 BMI (kg/m 2 ) 25.7 ± 4.9 TSH (mu/l) 2.1 ± 1.4 FT4 (pmol/l) 15.4 ± 3.2 5
Supplemental Figure 1. Histology of adipose tissue taken from the neck region during thyroid gland surgery. A: a representative 18 F-FDG-PET-CT image of a young lean male subject after mild cold exposure (16 C), from our previous study (3). Black areas indicate 18 F-FDG glucose uptake. The red box indicates the anatomical adipose tissue area bordered by the medial edge of the sternocleidomastoid muscle, the lower edge of the thyroid gland and the jugulum. B: Hematoxylin-eosin (HE) section (5µm) of adipose tissue from the SCV region of the subject showing both univacuolar and multivacuolar adipocytes, characterized as white adipose tissue (WAT) and brown adipose tissue (BAT), respectively. C: Immunofluorescence-staining for uncoupling-protein-1 (UCP-1) was positive in the multivacuolar adipocytes, confirming the presence of brown adipocytes. The histology shown was obtained from a peri-operative biopsy taken during a preventive total thyroidectomy for MENIIA-syndrome in a 19-year old male subject with a BMI of 21.9 kg/m 2. BAT indicates brown adipose tissue; WAT indicates white adipose tissue. 6
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References 1. Kraunsoe R, Boushel R, Hansen CN, Schjerling P, Qvortrup K, Stockel M, Mikines KJ, Dela F 2010 Mitochondrial respiration in subcutaneous and visceral adipose tissue from patients with morbid obesity. J Physiol 588:2023-2032 2. Dubois SG, Floyd EZ, Zvonic S, Kilroy G, Wu X, Carling S, Halvorsen YD, Ravussin E, Gimble JM 2008 Isolation of human adipose-derived stem cells from biopsies and liposuction specimens. Methods Mol Biol 449:69-79 3. van Marken Lichtenbelt WD, Vanhommerig JW, Smulders NM, Drossaerts JM, Kemerink GJ, Bouvy ND, Schrauwen P, Teule GJ 2009 Cold-activated brown adipose tissue in healthy men. N Engl J Med 360:1500-1508 8