Introduction
Brown adipose tissue (BAT) provides non-shivering thermogenesis (NST) because of the presence of the mitochondrial uncoupling protein-1(UCP-1) [
1] and BAT activity remains present in humans in adulthood [
2‐
4]. Cold exposure stimulates the sympathetic nervous system (SNS), which triggers BAT into heat production [
5].
Previous studies showing metabolically active BAT under cold stress are based on high symmetrical bilateral [
18F]FDG uptake in supraclavicular adipose tissue regions [
6‐
9], while active BAT is considered to rely primarily on fatty acids as the major fuel for thermogenesis [
10]. The study by Muzik et al. [
11] has shown that during cold stress higher [
18F]FDG standardised uptake values (SUVs) correlate with higher oxygen consumption in BAT. However, a proportional glucose contribution as a BAT substrate could not be suggested in this study due to the static [
18F]FDG PET measurements. Moreover, BAT-specific energy expenditure (EE) during cold is unclear [
12], while the study by Muzik et al. [
11] suggests BAT to be a minor contributor to cold induced thermogenesis. Therefore, it was critical to confirm BAT-specific EE using direct oxygen consumption measurements by using [
15O]O
2 positron-emission tomography (PET) imaging. Moreover, it has also not yet been established how much of the BAT circulatory NEFA uptake is associated with BAT-specific thermogenesis.
In the present study, we hypothesized that increased oxygen consumption by brown adipocytes under cold stimulation can be identified using in vivo [15O]O2 PET imaging in human supraclavicular BAT depots, and we further aimed to determine whether BAT-associated EE correlates with whole-body EE and additionally with BAT blood flow and NEFA uptake in room temperature (RT) conditions and during cold stimulus.
Discussion
The present study addresses BAT-specific contribution to cold-induced thermogenesis (CIT), previously speculated to be primarily a BAT response in a number of reports [
3,
7,
9,
22]. In addition, BAT substrate oxidative metabolism with particular reference to circulatory NEFAs is also addressed. Our study is one of the few attempts to evaluate oxygen consumption of BAT in humans using [
15O]O
2 PET imaging. A previous study by Muzik et al. [
11] has shown an association between BAT oxygen consumption and semi-quantitative glucose uptake measure, SUVs. However, it has been suggested that the main substrate of activated BAT is fatty acid [
10], and glucose is used as a secondary substrate. Therefore, it was critical to establish how much of the fatty acid uptake is associated with oxidative metabolism of BAT both at RT and during cold stimulus. One of the advantages of measuring oxygen consumption in BAT using [
15O]O
2 PET imaging is that it is a direct and non-invasive technique, which is not influenced by substrate availability and utilisation rate as might be the case with [
18F]FDG and [
18F]FTHA radiotracers [
23]. This technique shows the overall oxidative metabolic rate of BAT, which also includes oxidation of all known possible substrates for BAT consumption, e.g. glucose, fatty acid, ketone bodies, or amino acids.
Based on our data, we estimated that under mild cold stimulation the BAT-associated DEE of the cervico-upper thoracic depot is approximately 10 ± 5 kcal/day which is close to the values reported earlier by Muzik et al. [
11]. Whole-body EE increased significantly during cold stress; however, we did not observe any significant linear relationship between BAT DEE and whole-body EE which is indicative that the cervico-upper thoracic BAT does not contribute as a single and linear factor to whole-body EE. Change in BAT-associated DEE (4 ± 3 kcal/day) accounted for only 1 % of the total whole-body CIT (351 ± 372 kcal/day), while once DEE of cervico-thoracic muscles (in our FOV) was also taken into account they contributed 86 ± 68 kcal/day. However, the rest of the CIT could not be accounted for due to limited FOV of our PET scanner. The study by Blondin et al. [
24] supports these findings, where it has been shown that the cold exposed skeletal muscles manifest more than 50 % of the total systemic glucose uptake compared to 1 % in BAT.
Acute cold stress in humans results in autonomic responses of cutaneous vasoconstriction, in order to limit heat loss, and activation of SNS [
25]. Cutaneous vasoconstriction decreases skin temperature (Table
2), and elevation of circulatory catecholamine, as a consequence of SNS activation, triggers lipolysis in adipocytes. Increased lipolysis raises the plasma triglyceride levels (Table
2) likely to fuel NST [
26]. Heat loss exceeding NST leads to an increase in shivering thermogenesis [
25]. Therefore, in order to keep the subjects in the NST-zone, in our cooling protocol we increased the temperature of the cooling blanket once there were visual signs of shivering or the subject verbally reported shivering; however, based on our data it appears that shivering got activated on a microscale level in certain muscles before being observable or perceived by the individual. MRO
2 in the deltoid and trapezius muscle did not increase significantly in our study, which suggests that shivering was not taking place in the appendicular skeletal muscles, and therefore, we deduce that possibly shivering perception in humans is only associated with shivering in the muscles of the appendages. Another explanation, other than the shivering for the increase in MRO
2 in deep muscles, could also be the presence of mitochondrial uncoupling proteins as thermogenic agents in response to cold. These agents have been previously identified in rodent and human skeletal muscles [
27‐
29]. Irrespective of the underlying mechanism of thermogenesis in skeletal muscles, our data suggests that the deep, centrally located cervico-thoracic muscles contribute to cold-induced thermogenesis along with BAT, and that they are a rather major contributor to CIT. Although, interestingly, the BAT mass correlated with CIT (Fig.
5), consistent with earlier findings [
22], it appears that BAT possibly has an endocrine role in CIT, while the muscles surrounding BAT are the major contributor. Further studies that unravel secretory factors of BAT during cold stimulus, in conjunction with BAT oxygen consumption, are needed to draw concrete conclusions.
The strong positive correlation between BAT DEE and blood flow signifies the interdependence of both processes (Fig.
6). This may indicate that either UCP-1 mediated heat produced in BAT is distributed via circulation, or that the perfusion increases to provide oxygen and substrates for active BAT (Fig.
6). We further found that BAT-associated DEE is in correlation with its NEFA uptake both during RT and cold stress (Fig.
7). This suggests that BAT is also oxidizing substrates at RT although in a modest quantity compared to cold conditions, which signifies that BAT could already have ongoing thermogenesis at RT; however, it is not established how much of the UCP-1 protein in BAT mitochondria are uncoupled at RT. We also estimated that if the calculated fatty acids taken up via the blood stream in BAT, during cold stimulus, undergo complete oxidation they will amount to an average of 4.4 kcal/100 g/day (range 1.3–13.2 kcal/100 g/day). Comparing the values of both energy consumption following fatty acids complete oxidation and BAT-associated DEE, we observed a highly variable proportional contribution of circulatory NEFA towards the total BAT-associated DEE (14 % to 146 %, Supplementary Table
1). This variation was marginally linked to the quantity of stored triglycerides within BAT depots (measured as CT radiodensity in Hounsfield units), where less radiodense BAT depots (possessing more intracellular lipids) were found to require less contribution of NEFA from blood circulation (
r = 0.72,
p = 0.068, Supplementary Fig.
1). Other factors that may play a role in determining the quality and quantity of oxidised substrates could not be established with the current methods. Although it has been indicated that human BAT relies on the endogenous fatty acids following intracellular triglyceride breakdown [
30]; there is currently no available tool that has the ability to measure the quantity, as well as the proportional contribution of these consumed lipids in vivo as a result of cold exposure. In our study we also could not determine whether circulatory NEFA taken up by BAT are directly consumed as a substrate for thermogenic respiratory chain reactions, or whether the NEFA replenish the intracellular triglyceride stores subsequent to endogenous production of fatty acids. Nevertheless, it is worth mentioning that energy content of fully oxidised NEFA taken up by BAT, reported here and previously [
30], and glucose uptake [
4,
7,
31] is of the same order of magnitude, although their proportional contribution during cold stimulus is yet to be determined.
The other limitations of our study include small number of study subjects which is due to the complexity of the radioactive oxygen PET scans. The complexity includes the production of the [
15O]O
2, the inhalation protocol, the PET image acquisition, and the kinetic modelling of this very rapidly defusing and decaying radiotracer. Moreover, relatively shorter half-life of
15O and the limited FOV of our scanner restricted assessment of possible BAT depots in further locations [
32]; therefore, the present results may underestimate BAT contribution to whole body EE. Additionally, due to our aforementioned limitations, the relationship of metabolism in other organs to BAT oxidative metabolism could not be established.