Brown adipose tissue (BAT) can be identified by 18F-fluorodeoxyglucose (FDG)-positron emission tomography (PET) combined with X-ray computed tomography (CT) in adult humans. The objective of this study was to clarify the relationship between BAT and adiposity in healthy adult humans, particularly to test the idea that decreased BAT activity may be associated with body fat accumulation with age. One hundred and sixty-two healthy volunteers aged 20–73 years (103 males and 59 females) underwent FDG-PET/CT after 2-h cold exposure at 19 °C with light clothing. Cold-activated BAT was detected in 41% of the subjects (BAT-positive). Compared with the BAT-negative group, the BAT-positive group was younger (P < 0.01) and showed a lower BMI (P < 0.01), body fat content (P < 0.01), and abdominal fat (P < 0.01). The incidence of cold-activated BAT decreased with age (P < 0.01), being more than 50% in the twenties, but less than 10% in the fifties and sixties. The adiposity-related parameters showed some sex differences, but increased with age in the BAT-negative group (P < 0.01), while they remained unchanged from the twenties to forties in the BAT-positive group, in both sexes. These results suggest that decreased BAT activity may be associated with accumulation of body fat with age.
Brown adipose tissue (BAT) is the major site for nonshivering thermogenesis during cold exposure, and probably during spontaneous overfeeding, at least in small rodents such as the mouse, rat, and hamster (1,2). Although the role of BAT in diet-induced thermogenesis is still controversial (3), BAT is believed to contribute to the control of body temperature, energy expenditure, and adiposity. In humans, the existence of metabolically active BAT has been demonstrated by both clinical and experimental studies using fluorodeoxyglucose (FDG)-positron emission tomography (PET) in combination with computed tomography (CT) (4,5). We and two other groups demonstrated in healthy adults that acute cold exposure results in a marked increase in FDG uptake into fat deposits in the supraclavicular and paraspinal regions (6,7,8), where a large number of multilocular adipocytes expressing brown adipocyte-specific uncoupling protein 1 are detected (6,7,8,9,10). Since cold-stimulated glucose utilization in BAT is known in rodents to be secondary to the activation of uncoupling protein 1 (11,12,13), the cold-induced FDG uptake seen in humans would reflect the activation of BAT thermogenesis.
We and van Marken Lichtenbelt et al. reported that cold-activated FDG uptake in BAT was lower in subjects with higher adiposity, showing a negative correlation with the BMI, body fat content, and visceral fat (6,7). These findings suggest that BAT, depending on its thermogenic activity, contributes to the control of adiposity in humans, as has been established in small rodents (1,2). We also found that the mean age was significantly lower in subjects showing detectable BAT than those showing no BAT (6), suggesting that BAT activity decreased with age. On the other hand, it is well known that the aging process produces notable changes in body composition: that is, in general, percent body fat increases while lean mass and bone mineral density decreases (14). Thus, it seems possible that age-related accumulation of body fat is associated with decreased BAT activity. To test this idea, we examined the relationship between adiposity-related parameters and the incidence of cold-activated BAT in 162 healthy volunteer subjects aged 20–73 years, particularly focusing on the effects of aging. Our data suggest a protective role of BAT against the aging-related accumulation of body fat.
Methods and Procedures
Subjects recruited for this study were 162 healthy volunteers (103 males and 59 females) aged 20–73 years, who had been living in Sapporo for more than 3 years (Table 1). Included among the subjects were 56 and 13 subjects previously reported (6,15). Participants were carefully instructed regarding the study and gave informed consent to participate. They underwent FDG-PET/CT after 2-h cold exposure in winter (January, February, and March) from 2007 to 2010. The protocol was approved by the institutional review boards of Tenshi College.
Table 1. Subject profiles
After fasting for 6–12 h, the subjects entered an air-conditioned room at 19 °C with light clothing (usually a T-shirt with underwear), and put their feet on an ice block wrapped in cloth intermittently (usually for 4 min every 5 min). After 1 h under this cold condition, they were given an intravenous injection of 18F-FDG (1.7–5.4 MBq/kg body weight) and maintained under the same cold conditions. One hour after the 18F-FDG injection, whole-body PET/CT scans were performed employing a PET/CT system (Aquiduo; Toshiba Medical Systems, Otawara, Japan) in a room at 24 °C. With the CT parameters of 120 kv and real-exposure control, unenhanced low-dose spiral axial 2-mm collimated images were obtained. They were used for PET attenuation correction as well as anatomic localization. Subsequently, full-ring PET was performed in six incremental table positions, each ∼15 cm in thickness. The total time for these scans was ∼30 min.
PET and CT images were coregistered and analyzed using a VOX-BASE workstation (J-MAC System, Sapporo, Japan). Two experienced, blinded observers assessed the FDG uptake, particularly on both sides of the neck and paravertebral regions, by visually judging the presence of radioactivity greater than that of the background. BAT activity in the neck region was quantified by calculating the maximal standardized uptake value (SUVmax), defined as the radioactivity per milliliter within the region of interest divided by the injected dose in megabecquerels per gram of body weight. For dividing subjects into BAT-positive and negative groups, the cutoff value of 2.0 was applied (16,17), because SUVmax in subjects visually judged as BAT-positive was 2.1–42.7, with mean ± s.d. of 6.8 ± 6.0.
Anthropometric and body fat measurement and blood analysis
The BMI was calculated as the body weight in kilograms divided by the square of the height in meters (kg/m2), and the percent of body fat was estimated by employing the multifrequency bioelectric impedance method (InBody 320 Body Composition Analyzer; Biospace, Seoul, Korea). The visceral and subcutaneous fat areas at the abdominal level of L4–L5 were estimated from the CT images (18). The abdominal fat was calculated as the sum of the visceral and subcutaneous fats. Serum leptin was assayed using an enzyme-linked immunosorbent assay kit (Human leptin ELISA kit; B-Bridge, Mountain view, CA). Other blood parameters were analyzed by employing laboratory testing services (SRL, Tokyo, Japan).
Data are expressed as means ± s.d., and were analyzed using Student's t-test for the difference between the BAT-positive and negative groups. The effect of age on the incidence of BAT and those on adiposity and BAT activity were analyzed by Fisher's exact test and ANOVA, with post hoc testing by Tukey's test, respectively. Correlation analysis was used to test the effect of age on adiposity in the BAT-positive and negative groups. Interaction between age and group was tested by multivariate regression analysis. Values were considered to be significant if P < 0.05.
A total of 162 healthy volunteer subjects aged 20–73 years underwent FDG-PET/CT after 2-h cold exposure at 19 °C with light clothes and intermittently putting their feet on an ice-cooled footrest. Figure 1 shows some typical examples of middle-aged subjects, revealing considerable individual variations among the subjects in cold-activated FDG uptake and abdominal fat. After assessing FDG uptake into the supraclavicular region visually and quantitatively by calculating SUVmax, the subjects were divided into two groups, those with detectable FDG uptake (BAT-positive) and those with undetectable BAT (BAT-negative).
As summarized in Table 1, cold-activated BAT was found in 67 (41%) out of 162 subjects. When compared with the BAT-negative group, the BAT-positive group showed lower values of adiposity-related parameters such as the BMI, body fat content, subcutaneous fat, and abdominal fat. As the mean age of the BAT-positive group was lower, the effects of age on cold-activated BAT and adiposity were examined. As shown in Figure 2a, the incidence of detectable BAT decreased with age (P < 0.01), being more than 50% in the twenties (44/83), but less than 10% in the fifties (1/8) and sixties (0/7). The BAT activity assessed from SUVmax at the supraclavicular region also decreased with age (Figure 2b). On the contrary, adiposity-related parameters, particularly visceral fat, increased with age (Figure 2c-f).
To clarify the effects of BAT on the age-related increase in adiposity, we analyzed the effects of age on adiposity in the BAT-positive and negative groups. As shown in Figure 3a, a positive correlation was observed between BMI and age in the BAT-negative group (P < 0.01), but not in the BAT-positive group. Similar and clearer effects of age were also found in body fat content, and visceral and subcutaneous fat (Figure 3b-d). The effects of BAT on the age-related change in adiposity were also analyzed separately for 103 male and 59 female subjects. Although BMI was comparable in female and male subjects (21.1 ± 2.9 vs. 22.7 ± 2.9 kg/m2), body fat content was higher in females than males (27.3 ± 6.8 vs.19.6 ± 6.5 %, P < 0.001). Moreover, compared with male subjects, female subjects had smaller visceral fat area (39.1 ± 22.5 vs. 48.8 ± 33.3 cm2, P < 0.05) but larger subcutaneous fat area (146.3 ± 77.2 vs. 108.0 ± 73.2 cm2, P < 0.01). Despite the sex difference in adiposity, significant positive correlations were found between age and all the adiposity-related parameters in the BAT-negative, but not in the BAT-positive, group in both sexes (Table 2).
Table 2. Correlation between age and adiposity-related parameters
Thus, as summarized in Table 3, body fat content, and visceral and subcutaneous fat areas of the BAT-negative group were comparable with those of the BAT-positive group in the twenties, but they increased with age, being higher in the forties. BMI tended to be higher in the BAT-negative group than the BAT-positive group in the thirties and forties, but the difference was statistically not significant (P = 0.072 in the forties).
Table 3. Age-related changes in the BAT-positive and negative groups
We also examined the effects of age on some blood parameters in the two groups. In the BAT-negative group, significant positive correlations with age were found for glucose (R = 0.33, P < 0.001), hemoglobin A1c (R = 0.40, P < 0.001), total cholesterol (R = 0.54, P < 0.001), and leptin (R = 0.25, P < 0.05). In contrast, in the BAT-positive group, a positive correlation with age was found only for total cholesterol (R = 0.30, P < 0.05). However, the difference between the two groups did not reach to statistically significant levels even in the forties (Table 3).
The metabolic activity of BAT can be assessed by FDG-PET/CT performed after acute cold exposure (6). In the present study, we applied this method for the detection of BAT in healthy volunteers aged 20–73 years, and detected cold-activated BAT in about 40% of the subjects. The incidence of BAT is much higher than in previous retrospective clinical studies, where it was less than 10% (10,16,17,19,20). This may be due to the difference in the season and condition of FDG-PET examination; that is, in the clinical studies so far reported, examinations were performed without cold exposure at room temperature regardless of the season, while in our study, it was usually performed after 2-h cold exposure and only in winter, when BAT is expected to be maximally activated (1,6,19,20). Recently, Lee et al. reported in a retrospective study that only 13% of patients judged as BAT-positive in one FDG-PET/CT examination were BAT-positive in the other examinations (17), and suggested a low reproducibility of this method for BAT detection in clinical studies. However, under our study condition of 2-h cold exposure, all subjects considered as BAT-positive were judged again as BAT-positive in another examination (data not shown).
Our results clearly showed that the incidence and activity of BAT decreased with age, the incidence being more than 50% in the younger subjects (20–29 years old) but less than 10% in the older subjects (>40 years old). Au-Yong et al. (20) also reported the number and intensity of BAT-positive depots decreased with increasing age, although the age-related change in the incidence of BAT was not shown. A marked impact of age on the activity and mass of BAT was also demonstrated in patients aged 12–83 years old (16).These results seem consistent with an autopsy study of the anatomical distribution of human brown fat from infancy to late adult life, showing an age-related decrease in multilocular brown adipocytes (21). It should be stressed that our results do not necessarily mean that the number of brown adipocytes and/or the mass of BAT decrease with age, because the present FDG-PET method can only detect the activity of brown adipocytes, not brown adipocytes themselves. Therefore, for example, if there is some defect in the cold-sensing system, the method cannot detect BAT, even when there are large amounts of it. In parallel with FDG-PET examinations, histological studies such as those reported by Virtanen et al. (8) and Zingaretti et al. (9) are necessary to determine the presence or absence of BAT.
It has been well documented that as individuals age body fat increases and lean mass decreases, even without concomitant changes in body weight and BMI (14). In the present study, we also confirmed that body and visceral fat was higher in older subjects. On the other hand, the adiposity-related parameters were lower in the BAT-positive group than the BAT-negative group. This seems compatible with a previously reported negative relationship between BAT activity and adiposity (6,7), which supports the idea of a significant contribution of BAT to body fat control. An important point is that the relationship between adiposity and BAT is altered with age, that is, adiposity increased with age in the BAT-negative group, but it did not change in the BAT-positive group. These results seem compatible with our previous report that uncoupling protein 1-deficient mice showed an increased susceptibility to diet-induced obesity with age (22). Collectively, these results suggest that BAT may protect against the age-related accumulation of body fat. This is further supported by recent findings that whole-body energy expenditure in healthy subjects was higher in those with higher BAT activities (7,15), being compatible with the idea that the energy expenditure by BAT may be involved in the regulation of body fat accumulation.
In a previous study in young men (15), we demonstrated that the cold-induced elevation of whole-body energy expenditure was positively correlated with the BAT activity. The same study also indicated a significant role of BAT in the maintenance of body temperature during cold exposure. The present results, together with these previous observations, suggest the possible involvement of BAT in some age-related changes in energy expenditure and thermoregulation. For example, reduced total energy expenditure in the elderly is reported to be due to not only a decrease in the physical activity and basal metabolic rate, but also decreased thermogenesis (23). It is also known that aging is associated with diminished cold-induced thermoregulation (24). These age-related changes may be attributable, at least in part, to the reduction of BAT activity. At present, the physiological mechanisms underlying the age-related reduction of BAT are not known. It is to be noted that the activity of the sympathetic nervous system, which plays a critical role in the regulation of BAT thermogenesis, increased with age (24). Moreover, in aged animals, sympathetic signaling to BAT is greater both at thermoneutrality and during cold exposure (25,26), whereas BAT cell proliferation induced by cold exposure is substantially attenuated (27). These facts imply that age is associated with the decreased sensitivity of BAT to sympathetic stimulation.
Our results thus suggest a protective role of BAT against body fat accumulation with age in humans, and BAT as a target for interventions to prevent and treat obesity- and age-related diseases. Further studies, particularly longitudinal observations for individual subjects, are necessary to confirm the above idea.
This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (15081201). We thank Dr Mise, Sapporo Medical School, Sapporo, Japan for his advice on statistical analysis. A part of this study was presented at the International Congress of Endocrinology, 26–30 March, 2010, Kyoto, Japan, with an abstract that appeared in Endocrine Journal 57: S226, 2010.