2.1.

Subjects

In Experiment 1, three healthy males (24- to 30-years old) were recruited by direct contact. In an independent Experiment 2, 10 healthy male and 10 healthy female college students were recruited by advertising on posters or by direct contact (Table 1). The participants were randomly allocated to the capsinoids or placebo group by a third party who did not participate in this study.

Table 1

Anthropometric parameters and blood pressure before and after the 8-week treatment, and after 8 weeks of follow-up period.

2.2.

Capsinoids

Capsinoids were extracted from CH-19 Sweet (Capsicum annuum L); consisted of capsiate, dihydrocapsiate, and nordihydrocapsiate in a $7:2:1$ ratio; and were provided by Ajinomoto Co., Inc. (Tokyo, Japan). Each capsule contained 0 or 1.5 mg of capsinoids and 199 mg of a mixture of rapeseed oil and medium-chain triglycerides.^13,19 Participants were instructed to take three capsules in each morning and evening of each day¹³ for 6 weeks (Experiment 1) or 8 weeks (Experiment 2).

2.3.

Study Design

In Experiment 1, three male subjects were given six capsules containing 1.5 mg of capsinoids each day for 6 weeks. Before and after the treatment, their BAT activity was assessed by FDG-PET/CT.

In Experiment 2, 10 male and 10 female subjects were given either capsinoid ($9\mathrm{mg}/\text{day}$) or placebo capsules each day for 8 weeks in a double-blind design. Before and after the 8-week treatment, their anthropometric and circulatory parameters were measured. In addition, BAT density was measured every 2 weeks by ${\mathrm{NIR}}_{\mathrm{TRS}}$ (Fig. 1). These parameters were measured again 8 weeks after stopping the capsinoid intake (follow-up period). The subjects were instructed to maintain their usual dietary intake and physical activity during the experimental period, and to record a dietary diary during the 16-week period.

Fig. 1

Schematic illustration of the protocol and the timing of the measurements of Experiment 2.

2.4.

Outcomes

The primary endpoint was the change in BAT density evaluated by [total-Hb] using ${\mathrm{NIR}}_{\mathrm{TRS}}$ after 8 weeks of capsinoid-treatment, and the secondary one was that after cessation of the treatment. An additional endpoint was the capsinoid-induced increase in BAT activity assessed by FDG-PET/CT.

2.5.

${}^{18}\mathrm{F}$-fluorodeoxyglucose Positron Emission Tomography Combined with Computed Tomography

FDG-PET/CT was performed as described previously.² Briefly, after overnight fasting for $\sim 12\mathrm{h}$, the subjects were exposed to cold by being kept in an air-conditioned room at 19°C with standardized light clothing (a patient’s gown), and intermittently placed their feet on an ice block wrapped in cloth for $\sim 4\min$ every 5 min to avoid cooling-associated pain. After 1 h, under these cold conditions, each was given an intravenous injection of ${}^{18}\mathrm{F}$-FDG (1.66 to 5.18 megaBecquerel (${10}^6\mathrm{Bq}$) (MBq)/kg body weight) and kept under the same cold conditions. One hour after the ${}^{18}\mathrm{F}$-FDG injection, FDG-PET/CT scans were performed by using a PET/CT system (Aquiduo, Toshiba Medical Systems, Otawara, Japan). BAT activity in the supraclavicular fat deposits was quantified by calculating the maximal standardized uptake value of FDG (${\mathrm{SUV}}_{\max }$), defined as the radioactivity per milliliter within the region of interest divided by the injected dose in MBq/g of body weight.

2.6.

Near-Infrared Time-Resolved Spectroscopy

The [total-Hb] was measured using ${\mathrm{NIR}}_{\mathrm{TRS}}$ (TRS-20; Hamamatsu Photonics K.K., Hamamatsu, Japan) for 5 min at 27°C by placing the probes on the skin of the supraclavicular region potentially containing BAT deposits; and, as a reference, also in the deltoid muscle region, which is separated from the BAT deposits in the right side. The distance between the emitter and detector was set at 30 mm.¹⁶

The tissue was illuminated with a $200\text{-}\mu \mathrm{m}$ core diameter optical fiber using pulsed light generated from picosecond light pulsers at 760, 800, and 830 nm with 100 ps full width at half-maximum, a 5-MHz repetition rate, and an $80\text{-}\mu \mathrm{W}$ average power of each wavelength. The emitted photons penetrated the tissue and were reflected to a 3-mm diameter optical bundle fiber, through which they were sent to a photomultiplier tube for single-photon detection and a signal processing circuit for time-resolved measurement. Using the nonlinear least-squares method, the digitized temporal profile data from an in vitro sample or tissue were fitted with a theoretical temporal profile derived from the analytical solution of photon diffusion theory with a semi-infinite homogeneous model in reflectance mode. After convolution with the instrumental response function such that the time response of the instrument itself could be compensated, values for absorption coefficient and reduced scattering coefficient at 760, 800, and 830 nm were obtained. Then the absolute concentrations of [total-Hb] were determined using a least-squares fitting method.¹⁸ The ${\mathrm{NIR}}_{\mathrm{TRS}}$ system provided data every 10 s. The coefficient of variation for repeated measurements of [total-Hb] was 4.9%.¹⁶

2.7.

Anthropometric and Circulatory Parameter Measurements

The body mass, fat mass, percent body fat, lean body mass, and bone mass were evaluated by a dual-energy x-ray absorptiometry scan (DXA, Lunar Prodigy; GE Healthcare, Buckinghamshire, UK). The visceral fat area (VFA) and subcutaneous fat area (SFA) at the abdominal level of L4–L5 were estimated using 1.5-T MRI (Signa HDxt; GE Healthcare, Buckinghamshire, UK). During DXA measurements, subjects maintained a supine position. Then a series of transaxial MRI scans of abdominal sections were acquired ($\text{field of view}=420\times 420\mathrm{mm}$, $\text{slice thickness}=10\mathrm{mm}$, $\text{echo time}=7.3\mathrm{ms}$, $\text{repetition time}=\text{one respiration}$). The images were exported and analyzed by the same investigator using an image analysis software program (SliceOmatic 4.3; Tomovision Inc., Magog, Canada). Subcutaneous fat thickness was measured by B-mode ultrasonography (SSD-3500SV; Hitachi Aloka Medical Co., Ltd, Tokyo, Japan) at the supraclavicular region potentially containing BAT and the deltoid muscle region, which is separated from BAT deposits. During ultrasonographic measurements, subjects maintained the same posture as during the ${\mathrm{NIR}}_{\mathrm{TRS}}$ measurement. Systolic blood pressure (SBP), diastolic blood pressure (DBP), and heart rate (HR) were measured using an automated sphygmomanometer (HBP-9020; Omron Corp., Kyoto, Japan) after resting for 10 min.

2.8.

Dietary Diary and Records of Intakes

Dietary habits during the preceding month were assessed using a validated, brief, self-administered diet history questionnaire that contained questions about the consumption frequency of 56 foods and beverages and nine dishes that are commonly consumed in the general Japanese population. Daily intakes of energy, protein, fat, and carbohydrate were calculated²⁰ before and after the 8-week treatment period, and after the 8-week follow-up period.

Daily steps and activity energy expenditure were estimated using pedometers (Omron Health Counter HJ-710IT; Omron Healthcare, Kyoto, Japan), and the mean for 7 days was evaluated before and after the 8-week treatment period, and after the 8-week follow-up period.

2.9.

Statistical Analyses

Data are expressed as $\text{mean}\pm \text{standard deviation}$. In Experiment 1, to compare the ${\mathrm{SUV}}_{\max }$ before and after the 6-week period, Wilcoxon signed-rank testing was conducted after results from the Shapiro–Wilk test proved significant. In Experiment 2, a two-way analysis of variance with repeated measures was used to test the interaction ($\text{group}\times \text{time}$) and the main effects (group and time). If there was a significant interaction or main effect, the time or group differences of the variables were analyzed using the paired or unpaired $t$-test, respectively. Values were considered to be statistically significant if $P$ was $<0.05$. All statistical analyses were performed using SPSS version 19 (Chicago, Illinois).

3.1.

Experiment 1

Three male subjects were given 9 mg of capsinoids every day for 6 weeks, and their BAT activity was assessed by FDG-PET/CT. Figure 2 shows a typical FDG-PET/CT image in the supraclavicular region before and after the 6-week treatment. The calculated ${\mathrm{SUV}}_{\max }$ in both sides was increased by 48.8% ($2.2\pm 0.3$ versus $3.3\pm 1.3$, $P<0.05$) by the treatment, being consistent with our previous results¹³ that the daily ingestion of capsinoids recruits BAT.

Fig. 2

Typical FDG-PET/CT images in (a) the supraclavicular region and (b) ${\mathrm{SUV}}_{\max }$ before and after the 6-week capsinoids treatment.

3.2.

Experiment 2

Twenty subjects were randomly divided into two groups, and given either 9 mg of capsinoids or placebo for 8 weeks. Before and after the 8-week period, there were no significant changes, either in the anthropometric or circulatory parameters (Table 1) or for the physical activity levels (steps and physical activity, energy expenditure) or dietary intake (energy, fat, protein, and carbohydrate intake) (data not shown). No apparent harmful incidents were observed in any individuals in the present study.

Figure 3 shows the [total-Hb] assessed by ${\mathrm{NIR}}_{\mathrm{TRS}}$. There was a significant main effect of group on [total-Hb] in the supraclavicular region close to BAT deposits [Fig. 3(a)], but not in the deltoid muscle region separated from BAT deposits [Fig. 3(b)]. In the supraclavicular region, [total-Hb] increased by 46.4% after the 8-week capsinoid treatment ($70.4\pm 14.8$ versus $102.2\pm 27.2\mu \mathrm{M}$; $P<0.01$), despite large interindividual variations [Fig. 3(c)], while it did not change after the placebo treatment ($71.3\pm 18.1$ versus $81.9\pm 22.0\mu \mathrm{M}$; $P=0.13$) [Fig. 3(d)]. In contrast, the individual data of the deltoid muscle region separated from BAT deposits were stable in both the capsinoid [Fig. 3(e)] and placebo groups [Fig. 3(f)]. In the supraclavicular region, the change in [total-Hb] during the 8-week period was significantly greater in the capsinoid group than in the placebo group [Fig. 3(g)]. After stopping the capsinoid treatment, [total-Hb] in the supraclavicular region tended to decrease by 12.5%. The change in [total-Hb] during the 8-week follow-up period was insignificantly larger ($P=0.07$) in the capsinoids group than in the placebo group [Fig. 3(h)].

Fig. 3

Total hemoglobin concentration [total-Hb] in (a) the supraclavicular region potentially containing brown adipose tissue and (b) the deltoid muscle region separated from brown adipose tissue deposits. [total-Hb] in the supraclavicular region of individual subjects treated with (c) capsinoids and (d) placebo. [total-Hb] in the deltoid muscle region of individual subjects treated with (e) capsinoids and (f) placebo. (g) Changes in [total-Hb] after the 8-week treatment. (h) Changes in [total-Hb] after stopping the treatment.