Study subjects
Healthy study subjects (n = 7) of both genders with normal oral glucose tolerance test and cardiovascular status were included after written informed consent and screening. Subjects were screened for hypertension, diabetes, and elevated hepatic enzymes (ALAT, AFOS, GT). Whole-body insulin sensitivity (M-value) was also measured using a hyperinsulinemic euglycemic clamp technique [13]. The basic characteristics are given in Table 1. The local ethical review committee of the Southwestern Finland Hospital District reviewed and approved the study.
Table 1 Characteristics of study subjects
Study design
Subjects underwent two PET scanning sessions; one of the scanning sessions was performed at room temperature and the other during acute cold exposure (Fig. 1). Scanning sessions were organised on separate days in a random order with the minimum interval between the sessions being one week. Studies were performed after overnight fasting. All scans were performed at the same time of day in order to minimise any possible effect caused by individual circadian rhythm. Cold exposure was started 2 h prior to the scan using cooling blankets (Blanketrol III, Cincinnati Sub-Zero, Cincinnati, OH, USA), and cooling was continued during the PET scanning. Cooling was initiated with the temperature of the water circulating in the cooling blanket set to 6 °C; this temperature was gradually raised once the subjects were visually observed to be shivering or reported shivering themselves. The skin temperature of the subjects was also monitored during scanning using a digital thermometer (Art.183, Termometerfabriken Viking AB, Eskilstuna, Sweden) while the temperature sensing probe attached to the lateral abdominal skin surface. RT was maintained at approximately 22 °C.
Scanning protocol
Subjects were placed supine in a head first position inside the PET-CT scanner (Discovery 690 PET-CT scanner; General Electric Medical Systems, Milwaukee, WI, USA; PET voxel size = 3.64 x 3.64 x 3.27 mm), while the level of the clavicles was set to be the centre of the axial field of view (AFOV). Comfortable, relaxed position of the subjects was ensured in order to avoid any tension in the neck muscles, and the arms were placed next to the body. The positioning of the subjects within the scanner was kept identical irrespective the cooling protocol utilised. Scanning started with an attenuation correction transmission CT scan followed by three separate dynamic emission PET scans using three different radiotracers, i.e. [15O]O2, [15O]H2O, and [18F]FTHA. In the [15O]O2 scans, the subjects were given radioactive oxygen gas (509 ± 37 MBq) using a plastic mask with a single deep inhalation and scanning was started simultaneously; 20 frames of variable lengths were acquired over a period of 7 min (6 × 5 s, 6 × 15 s, 6 × 30 s, 2 × 60 s). After sufficient radioactive decay of [15O]O2 (approx. 10 min.), radiowater [15O]H2O (493 ± 35 Mbq) was intravenously injected into the left antecubital vein and scanning started immediately using 20 frames with a dynamic acquisition protocol (6 × 5 s, 6 × 15 s, 6 × 30 s, 2 × 60 s). [18F]FTHA scans were performed and quantified as described by Saari et al. [14]. Each subject received an estimated radiation dose of 8.8 mSv from the PET-CT scans during our study. The details of the production of tracers and PET image reconstruction can be found in the supplementary data.
PET image analysis
Carimas 2.8 software (Turku PET Centre, Turku, Finland) was used to analyse all acquired PET-CT images. The volume of interests (VOIs) in BAT were drawn manually on the supra-clavicular fat depots on the fused PET-CT images by taking into account the CT Hounsfield unit (HU) value of the voxels within −50 to −250 HU range (Fig. 2). White adipose tissue VOIs were drawn on the posterior subcutaneous neck area. For skeletal muscle, VOIs were drawn on deltoid, trapezius, levator scapulae, splenius cervicis, and pectoralis major muscles. For all the radiotracers used, arterial input function was determined by drawing a VOI comprising of 70–100 voxels on the arch of the aorta on the fused PET-CT images.
Measurement of blood flow (BF) and metabolic rate of oxygen (MRO2)
Blood flow was calculated from [15O]H2O PET scans by assuming a one-tissue compartmental model as follows,
$$ {C}_T(t)={K_1}^W\cdot {C}_A(t)\otimes {e}^{-K2\cdot t}+{V}_A\cdot {C}_A(t) $$
where C
T is the tissue time activity curve, C
A is the input function and V
A is the arterial blood volume. The K
1
w, k
2, and V
A values were estimated by an optimization procedure (Gauss-Newton method).
The metabolic rate of oxygen was calculated by incorporating the TACs of [15O]O2 PET scans by assuming a one-tissue compartmental model as follows,
$$ {C}_T(t)={K_1}^O\cdot {C_A}^O(t)\otimes {e}^{-K2\cdot t}+{K_1}^W\cdot {C_A}^W(t)\otimes {e}^{-K2\cdot t}+{V}_0\cdot {C_A}^0(t) $$
where C
T
is the tissue TAC, C
A
O and C
A
w are the input functions for oxygen and water, respectively. For the estimation of oxygen and water content, the aorta TAC was separated for each component according to the mathematical model described by Iida et al. [15]. V
0 is the arterial blood volume. The first term in the equation expresses the kinetics of oxygen and the second that of water, namely recirculation water. The K
1
o, and V
0 values were estimated by an optimization procedure (Gauss-Newton method), by inputting the obtained K
1
w and k
2 from the water data [16]. Subsequently, the MRO2 in the specific tissue was calculated as a product of K
1
o and the arterial oxygen concentration aO2 (mLO2/100 mL). The arterial concentration of oxygen was considered to be 19.8 mL per 100 mL of blood volume. MRO2 for muscles was calculated by taking into account the oxygen binding in myoglobin [17, 18].
Indirect calorimetry
In order to measure whole-body EE and substrate utilisation rates, indirect calorimetry (using Deltatrac II, Datex-Ohmeda) was performed simultaneously with PET scans (100–120 min, Fig. 1). From the data set, measurements were excluded from analyses if they deviated more than 1.5 SD from the mean vO2, vCO2, EE or respiratory quotient values, caused by irregular breathing. The first 30 min of the calorimetry data was also excluded. Whole-body EE, substrate utilisation rates, and respiratory quotient were calculated according to Weir equation [19] and the manufacturer’s equations [20] using Matlab (Version: R2011a). Protein oxidation was accounted for in the equations by considering urinary nitrogen to be 13 g/24 h.
Tissues mass calculation
BAT mass in the cervico-upper thoracic region was estimated on fused PET-CT images by first thresholding all CT voxels between a range of −50 to −250 HU at all potential cervico-upper thoracic BAT sites (cervical, supraclavicular, and axillary adipose depots). The acquired voxels underwent further thresholding and all voxels with less than 0.7 μmol/100 g/min NEFA uptake on parametric cold-exposure [18F]FTHA PET images were excluded. Finally, the volume of all these voxels (cm3) was converted into mass by assuming the density of BAT to be 0.92 g/cm3. Muscle mass in cervico-upper thoracic region was calculated from the CT images by thresholding all CT voxels at all muscle sites between 0 to +250 HU. Afterwards, the volume of all these voxels (cm3) was converted into mass by assuming the density of muscle to be 1.06 g/cm3.
Tissue specific DEE calculation
Finally, tissue specific daily energy expenditure (DEE) was calculated from MRO2 according to the formula below,
$$ DEE\; tissue\left( kcal/ day\right)=MR{O}_2\left( mL/100g/ \min \right)\times tissue\; mass(100g)\times 0.0048\left( kcal/ mL\right)\times 1440\left( min/ day\right) $$
The energy (kcal) produced per millilitre of oxygen consumption was assumed to be for respiratory quotient (RQ) of 0.80 (4.801 kcal/ litre O2 consumed) [21].
Statistical analyses
Statistical analyses were performed using IBM SPSS Statistics (version 22). To test for differences in mean values, a two-tailed paired Student t-test and a Wilcoxon rank-sum test were used. Pearson and Spearman’s correlation tests were used to analyse correlations. p-value of ≤ 0.05 was considered to be significant.