It has now been unequivocally demonstrated that humans possess functional brown adipose tissue (BAT) and that human BAT can be recruited upon chronic cold stimulation. Recruitment of BAT has been postulated as a potential strategy to counteract the current global obesity epidemic. Recently, it was shown in rodents that endurance exercise training could stimulate the recruitment of brown-like adipocytes within white adipose tissue (WAT) via exercise-induced myokines such as irisin (the cleaved circulating product of the type 1 membrane protein FNDC5) and interleukin-6 (IL-6). Our objective was to test whether endurance-trained athletes had increased cold-stimulated BAT activity and browning of subcutaneous WAT compared with lean sedentary males.
Twelve endurance-trained athletes and 12 lean sedentary males were measured during 2 h of mild cold exposure to determine cold-induced BAT activity via [18F]fluorodeoxyglucose-positron emission tomography-computed tomography ([18F]FDG-PET-CT) scanning. Skeletal muscle FNDC5 expression, as well as plasma irisin and IL-6 levels were determined. In addition, a subcutaneous abdominal WAT biopsy was taken to measure gene expression of several markers for browning of WAT.
Cold-induced BAT activity was significantly lower in athletes, and no differences in gene expression of classical brown and beige adipocyte markers were detected in subcutaneous WAT between the groups. As expected, mRNA expression of FNDC5 in skeletal muscle was significantly higher in endurance athletes but plasma irisin and Il-6 levels were similar in both groups.
These results indicate that chronic endurance exercise is not associated with brown and beige adipocyte recruitment; in fact endurance training appears to be linked to lower the metabolic activity of BAT in humans.
The presence of functional brown adipose tissue (BAT) in humans has now been unequivocally demonstrated. Brown adipocytes possess uncoupling protein 1 (UCP1), which uncouples respiration from ATP synthesis thereby dissipating chemical energy as heat. The thermogenic capacity of BAT makes this organ an interesting target to tackle obesity and associated diseases. In adult humans, BAT activity is negatively correlated with adiposity.1, 2, 3 Furthermore, there is strong evidence for a role of human BAT in cold-induced heat production.4, 5, 6 Therefore, enhancing the metabolic capacity of BAT in adult humans would be a promising way to increase energy expenditure.
In addition to classical or constitutional BAT, a distinct type of thermogenic adipocytes has recently been identified within white adipose tissue (WAT), designated as beige7 or brite8 adipocytes. These beige adipocytes typically appear in WAT depots after stimulation (for example, pharmacological or cold stimulation) and have comparable respiratory capacity as classical brown adipocytes.7 Beige adipocytes—next to classical BAT adipocytes—may not only be present and recruited in the neck and supraclavicular area in humans,9, 10, 11 but may also be recruited in subcutaneous WAT, a process nowadays called ‘browning’.
Recently, exercise has been hypothesized to induce browning of WAT in rodents.12 Initially, in the 1980’s, it was hypothesized that exercise could stimulate BAT via increased sympathetic activity.13 However, studies in rodents generally did not show any stimulating effects of exercise on classical BAT.13, 14, 15, 16 In the past 3 years, however, it was demonstrated in rodents that the focus should be more on browning of typical WAT depots and less on classical BAT.12, 17, 18, 19, 20 Interestingly, Boström et al.12 demonstrated in mice that exercise increases the expression of the FNDC5 gene, which codes for a type 1 membrane protein that is cleaved after translation and subsequently released in the plasma as ‘irisin’. It should be mentioned though, that the data on the effect of exercise on FNDC5 expression in humans is not unequivocal.21 In mice, however, this myokine stimulated browning of WAT and increased energy expenditure leading to reduced body weight and improved glucose homeostasis.12 In addition, the cytokine interleukin-6 (IL-6), which is released from skeletal muscle into the circulation in response to exercise, has also been shown to regulate inguinal WAT (iWAT) UCP1 expression upon exercise and cold exposure in mice.22
Whether endurance exercise per se recruits brown and beige adipocytes in humans remains undetermined. We therefore measured cold-induced BAT activity by means of [18F]fluorodeoxyglucose-positron emission tomography-computed tomography ([18F]FDG-PET-CT) scanning, and ‘browning’ by analyzing brown and beige markers in subcutaneous WAT in endurance-trained athletes in comparison with lean sedentary controls.
Subjects and methods
Subjects and study design
Twelve healthy endurance-trained and 12 lean sedentary males aged between 18–35 years were studied between January and September 2013. Endurance-trained (long-distance running, cycling, swimming) athletes were included in the trained group when they performed endurance exercise at least three times a week for the last 2 years, and had a maximal oxygen consumption (VO2max) >55 ml min−1 kg−1. The sedentary males were included in the untrained group if they did not perform more than 1 h of exercise per week for the last 2 years and had a VO2max <45 ml min−1 kg−1. Owing to technical problems we included one untrained individual solely based on his maximal power output, which was <4 Watt kg−1. General exclusion criteria were use of medication, smoking, weight gain/loss of >3 kg in the last 6 months, hypertension, (family history of) diabetes and diabetes-related diseases, and contra indications for magnetic resonance imaging and PET-CT. All subjects signed a written informed consent. The local medical ethical committee approved the study protocol and all subjects were treated according to the declaration of Helsinki principles of 1975, as revised in 1983.
We paired each trained individual with an untrained subject and measured both within 2 weeks to preclude possible seasonal effects (acclimatization) on BAT activity and browning of WAT. Owing to practical reasons, one pair was measured within 3 weeks. All subjects were measured in the morning after an overnight fast and were asked to refrain from exercise the day before the PET-CT scan, and in the 3 days previous to the biopsies to exclude any acute effects on skeletal muscle and adipose tissue. A standardized meal was provided on the evening before the mild cold experiment (55% carbohydrate, 30% fat, 15% protein, 518 kcal). In the morning before the mild cold experiment, body composition was determined by means of dual x-ray absorptiometry (type discovery A, Hologic).
Mild cold experiment
Each subject underwent a mild cold experiment. This experiment started with 1-h baseline measurements during thermoneutral conditions (t=0–60 min). Subsequently, subjects were exposed to 2 hours of mild cold exposure (t=60–180), in which a standardized cooling protocol was used (for details see Vosselman et al.23). To measure BAT activity, the glucose analog [18F]FDG was injected 1 h after the onset of cold exposure (at t=120) followed by the [18F]FDG-PET/CT scan 1 h later (t=180). The experiment was conducted in a specially equipped air-permeable tent (Colorado altitude training, Louisville, CO, USA), in which ambient temperature could be tightly controlled. During the experiment subjects wore standardized clothing (shorts and T-shirt; 0.19 clo). Energy expenditure was continuously measured by a ventilated-hood system (Omnical, Maastricht, The Netherlands; Jaeger, Hoechberg, Germany). Body skin temperatures were measured continuously with iButtons (Maxim integrated, San Jose, CA, USA) at 14 sites (ISO-standard 9886:200). Core temperature (telemetric pill, HT150002; CoreTemp, Palmetto, FL, USA), skin perfusion (Laster Doppler Flowmetry, Perimed, Järfälla, Sweden) and heart rate (Polar T31, Lake Success, NY, USA) were sampled each minute. Blood pressure (Cresta, Taipei, Taiwan) was measured every 15 min, as well as thermal comfort and thermal sensation via Visual Analog Scales. Muscle shivering was monitored by means of EMG (PASAQ4.01, Maastricht Instruments BV, Maastricht, The Netherlands) and Visual Analog Scales scales. Venous blood samples were taken during baseline and 1 h after the onset of cold exposure.
Plasma concentrations of glucose, free glycerol, total glycerol and non-esterified fatty acids were determined on a Cobas FARA centrifugal spectrophotometer (Roche Diagnostics, Basel, Switzerland), as described previously.23 Triacylglycerol levels were calculated as the difference between total and free glycerol. Serum insulin was analyzed with a Human Insulin Specific RIA kit (Millipore, Billerica, MA, USA) on a gamma counter (2470 Automatic Counter Wizard2, PerkinElmer, Waltham, MA, USA). Serum thyroid-stimulating hormone and free thyroxine were analyzed as described previously.23 Plasma irisin (EK-067-29, Phoenix Pharmaceuticals, Burlingame, CA, USA) and IL-6 (D6050, R&D Systems, Minneapolis, MN, US) concentrations were determined with commercially available ELISA kits according to the manufacturer’s instructions.
We used a static PET-CT scanning protocol to determine [18F]FDG uptake. The protocol started with a low-dose CT scan (120 kV, 30 mAs) immediately followed by the PET scan. The uptake of [18F]FDG (2 mCi, 74 MBq) was determined with the PET scan, and the CT scan was used for attenuation and scatter correction of the PET scan and to determine the anatomical location of the [18F]FDG uptake. We calculated BAT activity as SUVtotal, which is the average SUV (SUVmean) per volume of interest × its volume, and SUVmax, which is the maximal [18F]FDG uptake in BAT. Finally, the average SUV was determined in supraclavicular BAT, skeletal muscle, subcutaneous and visceral WAT, the liver and the brain (cerebellum) by selecting cubes as volume of interests.6
Muscle and adipose biopsies
Within 2 weeks from the PET-CT scan, we took an abdominal subcutaneous adipose biopsy (needle) 6–8 cm lateral from the umbilicus under local anesthesia (2% lidocaine) to determine gene expression of several markers for WAT browning. Subsequently, a muscle biopsy from m. vastus lateralis was taken according to the technique of Bergström24 to determine FNDC5 mRNA expression. All subjects were fasted during these biopsies. The tissue was rinsed from blood, and directly frozen in melting isopentane. Subsequently, the biopsies were snap frozen in liquid nitrogen and stored at −80 °C for future analysis.
For skeletal muscle FNDC5, RNA was isolated from muscle tissue, using Trizol according to the method from Chomczynski et al.25 RNA from WAT was extracted using Trizol reagent followed by protocol described in the RNeasy kit from Qiagen (Hildenberg, Germany). Quality and yield of RNA was assessed using a NanoDrop spectrophotometer (Thermo Fisher scientific, Waltham, NA, USA). cDNA was created using the high-capacity RNA-to-cDNA kit from Applied Biosystems (Foster City, CA, USA). Gene expression was determined using a 7900HT Fast Real-Time PCR System from Applied Biosystems. Skeletal muscle gene expression data was normalized against RPLPO using the standard curve method. Comparison of gene expression data for WAT was accomplished using the 2−ΔΔCt method. Gene expression data was normalized against the geometric mean of TBP and RPLPO. Taqman gene expression assays were used for analysis of UCP1 (Hs00222453_m1), CIDEA (Hs00154455_m1), CD137 (Hs00155512_m1) and TMEM26 (Hs00415619_m1).
We used SPSS 20.0 (IBM, Armonk, NY, USA) for statistical analysis. To compare all parameters between baseline and mild cold we took the average of the final 30 min of baseline and 30 min after the injection of the [18F]FDG-tracer. The effects of mild cold on all variables within each group were tested by means of paired-sample t-tests. Note that we used non-parametric testing (Wilcoxon’s test) to measure differences in blood parameters in the untrained group (n=7). There were five missing values due to difficulties with blood collection in this group during the cold (n=7). Independent sample t-tests were used to compare the trained with the untrained group. Furthermore, Pearson correlation was performed to identify relations between variables on whole-group level (n=24), and within each group (n=12). All values are expressed as mean±s.d. Outcomes were regarded as statistically significant if P<0.05.
Both age and adiposity are known to negatively correlate with BAT activity in human adults. We therefore created comparable groups regarding age and body mass index (Table 1). As expected, fat percentage was significantly lower in the trained group (endurance athletes) compared with the untrained group (sedentary males; P<0.05), but was not related to BAT activity neither within each group, nor within the entire study population. Resting heart rate was significantly lower in the trained group (P<0.05), indicating the typical bradycardia found after regular endurance exercise training.26 No significant differences in baseline concentrations of thyroid hormones, free fatty acids and glucose were found. Plasma triglyceride concentration was 36% lower in the trained group, which tended to be significant (P=0.099). Finally, baseline serum insulin concentration was significantly lower in the trained group (P<0.05).
Lower cold-induced BAT activity in endurance athletes
Energy expenditure significantly increased during cold exposure in the trained (P<0.05) and untrained group (P<0.001, Table 2). Mean non-shivering thermogenesis (NST) was slightly lower in the trained group, although not significantly different (8.0±8.9% vs 11.8±6.5%, P=0.25). In line with previous findings,6 cold exposure significantly increased fatty acid oxidation during cold in both groups (P<0.05), whereas the oxidation of carbohydrates significantly decreased in the trained group (P<0.05), and tended to decrease in the untrained group (P=0.058).
Interestingly, total cold-induced BAT activity was significantly lower in the trained group compared with the untrained group (622±801 vs 1837±1308 SUVtotal, P<0.05; Figure 1a), as was maximal BAT activity (6.25±4.09 vs 13.09±8.57 SUVmax, P<0.05). Furthermore, the average [18F]FDG uptake in supraclavicular BAT was significantly lower in the trained group (3.86±3.00 vs 8.68±6.55 SUVmean, P<0.05; Figure 1b). Cold-induced [18F]FDG uptake in skeletal muscle (0.58±0.12 vs 0.58±0.09 SUVmean, P>0.05), subcutaneous WAT (0.29±0.09 vs 0.24±0.07 SUVmean, P>0.05), visceral WAT (0.89±0.43 vs 0.91±0.61 SUVmean, P>0.05), liver (1.65±0.29 vs 2.15±1.49 SUVmean, P>0.05) and brain (6.50±1.44 vs 6.95±0.85 SUVmean, P>0.05) were not significantly different between both groups. It thus appears that regular endurance exercise training is associated with lower cold-induced activity of BAT specifically. In line with our previous findings,6, 27 we found a relationship between BAT activity and NST in the whole study group (r=0.67, P<0.001; Figure 2a), in the trained group (r=0.86, P<0.001; Figure 2b), and a trend in the untrained group (r=0.57, P=0.053; Figure 2c), supporting the prominent role for BAT in NST in humans.
Muscle FDNC5 mRNA expression, plasma myokines and browning of WAT
It has been suggested that endurance exercise stimulates browning of WAT via the hormone irisin, which is processed from the skeletal muscle protein FNDC5.12 Indeed, we found that FNDC5 mRNA expression was higher in the trained group (Figure 3a). However, baseline plasma irisin levels were similar between trained and untrained subjects (11.1±1.5 vs 11.9±1.2 ng ml−1, respectively, non significant). Furthermore, plasma irisin levels remained unchanged upon 1 h of cold exposure and averaged 11.0±1.4 ng ml−1 in trained vs 12.6±0.9 ng ml−1 in untrained subjects, respectively (non significant). Please note that there is currently a debate on whether the antibodies used to measure plasma FNDC5/irisin are valid or not28, 29, 30 and whether irisin in humans is functional, because a mutation in the start codon of the human FNDC5 gene might lead to the release of a truncated form of irisin in plasma.28, 29 Basal plasma levels of the exercise-responsive myokine IL-6, also implicated in browning of WAT, were not statistically different between trained vs untrained individuals. If anything, trained individuals displayed lower plasma IL-6 levels, when compared with untrained subjects (0.73±0.67 vs 2.02±2.58 pg ml−1, respectively, P=0.11).
In WAT, UCP1 mRNA was undetectable in both groups. Furthermore, we did not detect differences in mRNA expression of PGC-1α between both groups (P>0.05; Figure 3b). Also the mRNA expression of the brown adipocyte marker Cidea (Figure 3c) and the two beige markers TMEM26 (Figure 3d) and CD137 (Figure 3e) were similar between the trained and untrained subjects (P>0.05). We did not observe any correlations, neither between the myokines and BAT activity nor between the myokines and the WAT browning markers.
Vasoconstriction and body temperatures during cold exposure
In addition to cold-induced heat production, minimizing heat loss via vasoconstriction is the other important thermoregulatory mechanism to prevent hypothermia. Both groups significantly increased vasoconstriction in the toe, hand and underarm (P<0.001, Table 2), whereas no significant difference was observed in the abdomen during cold exposure (P>0.05). Vasoconstriction in the hand, toe and abdomen was not significantly different between groups (P>0.05). Interestingly, the trained group showed a larger vasoconstriction in the underarm (61.4±14.1% vs 34.3±21.2%, P<0.01) thereby minimizing heat loss in the extremities, which might decrease the need for metabolic heat production. Lower skin temperatures in the extremities confirmed this larger vasoconstriction (Table 2).
The effects of cold on thermal sensation and comfort, blood pressure, heart rate and blood values
During the 2-h cold period we controlled ambient temperature via air conditioning to maximally cool subjects without inducing shivering. The average temperature in the climate tent after the injection of [18F]FDG (when shivering was absent) was not significantly different between both groups (trained: 15.95±1.41 °C vs untrained: 17.07±2.28 °C, P>0.05), indicating comparable cold stress in both groups. The thermal sensation and comfort during the mild cold experiment was comparable at each time point (P>0.05). Both diastolic and systolic blood pressure significantly increased (P<0.001) during mild cold in both groups (Table 2). Heart rate decreased in the trained group (−3±3 b.p.m., P<0.01), whereas it did not change in the untrained group (1±6 b.p.m., P>0.05), and tended to be different between groups (P=0.06). Cold exposure significantly increased plasma free fatty acid, free glycerol and triglycerides concentrations in the trained group (P<0.05), whereas no cold-induced changes were found in the untrained group (P>0.05), except a trend for an increase in plasma free fatty acid (P=0.06). Furthermore, glucose concentrations did not change during cold exposure, although a trend for a decrease was observed in the trained group (P=0.09). Insulin significantly decreased during cold exposure in the trained group (P<0.05), whereas it did not change in the untrained group (P>0.05). Nevertheless, the differences between thermoneutral and cold conditions of these blood parameters were not significantly different between the groups (Table 2).
Health problems associated with obesity warrant strategies to restore energy balance. Stimulating BAT recruitment might be a plausible strategy to enhance the capacity to increase thermogenesis and induce weight loss. Endurance exercise is known for its favorable metabolic effects, and it was recently shown in rodents that one of these effects involves browning of WAT,12, 17, 20 and possibly the stimulation of classical BAT depots.17, 18, 19 However, the effects of chronic endurance exercise on BAT in humans remain unknown. We here show that chronic endurance exercise training is not associated with a higher, but rather lower cold-induced BAT activity, and is not associated with browning of subcutaneous abdominal WAT in young adult males.
We examined cold-induced BAT activity in 12 endurance-trained and twelve lean sedentary males, with comparable age and body mass index. Both total and maximal [18F]-FDG uptake in BAT was significantly lower in athletes. Boström et al.12 demonstrated that browning of subcutaneous WAT in mice was promoted by the exercise-induced myokine irisin. So far, human studies have shown conflicting results on the stimulating effects of exercise on FNDC5 and irisin.28, 31, 32, 33, 34, 35, 36, 37 We here measured mRNA expression of the precursor of irisin, FNDC5, and indeed found 1.6-fold higher mRNA levels in the trained athletes, confirming the stimulating effects of chronic endurance exercise on FNDC5. However, plasma irisin levels were unchanged, although it should be noted that the measurement of circulating irisin in humans is precarious due to a possible specificity problem in commercially available ELISA kits.38 Therefore, the interpretation of the plasma irisin results in the current study should be done with care.
Besides irisin, also the exercise-induced cytokine IL-6 has been suggested as a mediator of WAT browning. Thus, expression of UCP1 in iWAT in mice was increased upon injection of recombinant IL-6.22 Furthermore, exercise training-induced iWAT UCP1 mRNA levels in wild-type but not in IL-6 knockout mice, indicating a requirement for IL-6 in the exercise training-induced changes in iWAT UCP1 expression.22 In the present study, plasma IL-6 levels were not significantly different in trained vs untrained subjects and did not correlate with cold-induced BAT activity.
We then measured several typical brown and beige adipocyte cell markers to determine browning of subcutaneous WAT. We analyzed the expression of UCP1 in biopsies from the abdominal subcutaneous adipose tissue and found that it was mostly undetectable in both groups. Furthermore, the mRNA expression of the beige markers CD137 and TMEM26 were comparable between groups, as well as PGC-1α and the classical brown adipocyte marker Cidea. A previous study by Norheim et al. 32 also showed very low UCP1 expression (mean Ct=36.6±1.8) in abdominal subcutaneous WAT before and after 12 weeks of combined endurance and strength training in healthy and prediabetic subjects. Nevertheless, in that study UCP1 expression significantly increased (1.82-fold) upon training when both groups were combined. In agreement with our study, no exercise effects were found on PRDM16, TBX1, TMEM26 and CD137 expression, neither in healthy nor in prediabetic subjects.32 As further evidence against an effect of exercise training on browning in humans, we found no difference in [18F]-FDG uptake in subcutaneous WAT between trained and untrained subjects. All in all, we here show that exercise per se does not lead to detectable browning of subcutaneous abdominal WAT in humans.
Previous human studies have solely examined the effects of exercise and irisin on adipose browning by measuring specific markers within adipose tissue. The present study also measured the effects of chronic exercise training on cold-induced BAT activity by means of FDG-PET/CT imaging, which reflects the total amount of active BAT in each individual. Recent studies have shown that human BAT from the neck region possesses both classical brown as beige adipocytes.7, 10, 11 Therefore, we hypothesized that if exercise—via irisin—can induce browning, it would result in enhanced BAT activity in both the neck region as in subcutaneous WAT. However, in support with the mRNA expression results on WAT browning, [18F]FDG uptake was not different between athletes and sedentary subjects in both subcutaneous and visceral WAT, and was in fact significantly lower in BAT depots from the neck region in the athletes. These PET-data therefore strengthen the conclusion that chronic endurance training recruits neither brown nor beige adipocytes.
In addition to the lower observed BAT activity, average NST was slightly lower in the endurance athletes (8% vs 12%) as well, although not significant. NST was strongly correlated with BAT activity, supporting the notion that BAT is an important mediator in cold-induced heat production.2, 4, 5, 6 Interestingly, the athletes showed larger vasoconstriction in the arm. It could thus be speculated that the endurance athletes rely more on insulation rather than on BAT-mediated heat production during mild cold exposure. This larger ‘non-fat insulation’, which reflects regulated insulation by means of vasoconstriction, has been observed previously in long-distance runners39 and after 9 weeks of endurance exercise in young men.40 The authors hypothesized that this physical conditioning may promote vascularization of muscle tissue and/or alter tissue sensitivity to circulating catecholamines thereby increasing the capacity for vasoconstriction. Despite increased insulation, there was a trend for lower core temperature during the cold exposure in the athletes, suggesting that athletes might tolerate lower internal body temperatures, which has been demonstrated previously in long-distance runners.39 This was, however, not reflected by differences in thermal sensation and comfort between both groups.
In summary, endurance-trained athletes had lower cold-induced BAT activity compared with lean sedentary individuals. Moreover, despite increased gene expression of FNDC5 in skeletal muscle chronic endurance exercise did not lead to browning of subcutaneous abdominal WAT. Therefore, our data do not support the notion, in contrast to findings in rodents, that chronic endurance exercise trained individuals display an increased thermogenic capacity of BAT.
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We thank Reni van Erp, Jasper Most, Esther Phielix, Jos Stegen, Johanna Jörgensen, Esther Moonen-Kornips, Gert Schaart, Roel Wierts, Loek Wouters, Paul Schoffelen, Nancy Hendrix and Paul Menheere for their (technical) assistance during the study. Moreover, we thank our literature club for the helpful discussions. This work was financed by the Netherlands Organization for Scientific Research (TOP 91209037 to WDvML) and by the EU FP7 project DIABAT (HEALTH-F2-2011-278373).
The authors declare no conflict of interest.
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Vosselman, M., Hoeks, J., Brans, B. et al. Low brown adipose tissue activity in endurance-trained compared with lean sedentary men. Int J Obes 39, 1696–1702 (2015). https://doi.org/10.1038/ijo.2015.130
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