Brown Adipose Tissue Response Dynamics: In Vivo Insights with the Voltage Sensor 18F-Fluorobenzyl Triphenyl Phosphonium

Brown adipose tissue (BAT) thermogenesis is an emerging target for prevention and treatment of obesity. Mitochondria are the heat generators of BAT. Yet, there is no noninvasive means to image the temporal dynamics of the mitochondrial activity in BAT in vivo. Here, we report a technology for quantitative monitoring of principal kinetic components of BAT adaptive thermogenesis in the living animal, using the PET imaging voltage sensor 18F-fluorobenzyltriphenylphosphonium (18F-FBnTP). 18F-FBnTP targets the mitochondrial membrane potential (ΔΨm)—the voltage analog of heat produced by mitochondria. Dynamic 18F-FBnTP PET imaging of rat’s BAT was acquired just before and during localized skin cooling or systemic pharmacologic stimulation, with and without administration of propranolol. At ambient temperature, 18F-FBnTP demonstrated rapid uptake and prolonged steady-state retention in BAT. Conversely, cold-induced mitochondrial uncoupling resulted in an immediate washout of 18F-FBnTP from BAT, which was blocked by propranolol. Specific variables of BAT evoked activity were identified and quantified, including response latency, magnitude and kinetics. Cold stimulation resulted in partial washout of 18F-FBnTP (39.1%±14.4% of basal activity). The bulk of 18F-FBnTP washout response occurred within the first minutes of the cold stimulation, while colonic temperature remained nearly intact. Drop of colonic temperature to shivering zone did not have an additive effect. The ß3-adrenergic agonist CL-316,243 elicited 18F-FBnTP washout from BAT of kinetics similar to those caused by cold stimulation. Thus, monitoring ΔΨm in vivo using 18F-FBnTP PET provides insights into the kinetic physiology of BAT. 18F-FBnTP PET depicts BAT as a highly sensitive and rapidly responsive organ, emitting heat in short burst during the first minutes of stimulation, and preceding change in core temperature. 18F-FBnTP PET provides a novel set of quantitative metrics highly important for identifying novel therapeutic targets at the mitochondrial level, for developing means to maximize BAT mass and activity, and assessing intervention efficacy.


Introduction
The recent discovery of metabolically active brown adipose tissue (BAT) depots in human adults [1][2][3][4][5] has opened new avenues for the search of therapeutic approaches to the prevention and treatment of obesity and comorbidities (e.g., diabetes, heart disease). BAT is unique in its capacity to dissipate a huge amount of caloric energy into heat, 300 times more than an equivalent volume of any other tissue [6]. BAT activity can be evoked by mild cold stimulation, in line with its thermo-regulatory role, but also by a high-fat diet (HFD) [7][8][9] and insulin [10]. HFD resulted in concomitant increases in energy expenditure and BAT thermogenesis [11][12][13][14], whereas, fat loss reduced BAT thermogenesis [15][16][17]. The absence of BAT [18][19][20] or UCP1 [21,22] resulted in metabolic inefficiency leading to obesity, hyperphagia and insulin resistance [22]. Fatty acids derived from triglyceride-rich lipoproteins are the major energy carriers for brown adipocytes [23]. Activation of BAT resulted in a significant decrease of triglycerides in blood, which otherwise would be stored in the body as white fat lipids [24].
The current extensive efforts to develop drugs and methods for increasing BAT mass and activity are hindered by our current partial knowledge of the physiology of BAT in vivo, due in part to the absence of tools for dynamic imaging of BAT activity in real-time. FDG PET has been instrumental in advancing out knowledge of BAT in vivo [1][2][3][4][5]. FDG PET is an effective tool for detecting BAT at activation, but not at resting-state [25]. The absence of basal values hampers FDG quantitative and spatial values. Both limitations ultimately lead to a loss of important information. Therefore, a noninvasive tool is needed with better functional resolution than that available to date.
Mitochondria are the heat generators in BAT [26]. The mitochondrial membrane potential (ΔCm) is the standard and most direct quantitative measure of the BAT heat production [27]. In the absence of heat production, the energy released by the electron transfer in the respiratory chain is used to translocate protons against the concentration gradient, thus creating a large voltage difference across the mitochondrial inner-membrane (i.e., ΔCm). At resting-state, protons reenter the matrix via ATPase, providing the energy required for ATP synthesis. At thermogenic-state, protons bypass ATPase and reenter the matrix through UCP1 [28], and the energy stored in the concentration gradient is dissipated as heat [26,29]. Protons reentrance through UCP1 leads to a proportional decline of ΔCm. Thus, monitoring ΔCm provides a direct quantitative measure of the extent of protons flux through UCP1, and thereby of the amount of heat produced by mitochondria.
The PET imaging agent 18 F-fluorobenzyltriphenyl phosphonium ( 18 F-FBnTP) is an indicator of ΔCm [30][31][32][33]. Previous ex vivo studies in rats have demonstrated the capacity of 18 F-FBnTP to detect the collapse of ΔCm in cold-stimulated BAT [33]. A 4-hrs exposure of rats to 4°C environment, either before or after administration of 18 F-FBnTP, resulted in a marked decrease of both uptake and retention of 18 F-FBnTP in BAT, which was blocked by prior treatment with the ß-noradrenergic antagonist propranolol. This finding is consistent with previous observations of the tight linear relationship of 18 F-FBnTP and ΔCm in a spectrum of preparations from cardiomyocyte mitochondria, single cells and up to an intact-heart model [30,31]. Stepwise hyper-polarization of membrane potential in isolated mitochondria and single cells resulted in a linear increase of 18 F-FBnTP uptake over a large range of membrane potentials, whereas selective mitochondrial depolarization, using uncoupling protocols, resulted in tight linear dose-dependent washout of 18 F-FBnTP. Pharmacological manipulations have shown that the large majority of 18 F-FBnTP (>80%) concentrates in the mitochondrial compartment in a ΔCm-dependent manner, and with very low nonspecific binding (~5%). The remainder of activity was found in cytosol [30]. A similar ΔCm-dependent fraction of 18 F-FBnTP was measured by dynamic PET imaging of Guinea Pig's isolated perfused intact heart [31]. Adding the uncoupler FCCP (10 μg) to the perfusion medium resulted in a linear washout of 18 F-FBnTP-75% washout was obtained within 20 min. Uncouplers mimic UCP1 activity by translocating protons across the mitochondrial inner-membrane and into the matrix, leading to selective collapse of ΔCm.
The present work aims at expanding our previous ex vivo studies, and characterizing the in vivo kinetics of mitochondrial uncoupling induced by controlled localized skin cooling and systemic pharmacologic stimulation, using dynamic 18 F-FBnTP PET imaging of the rat's BAT. At ambient temperature, 18 F-FBnTP accumulated rapidly and extensively, generating within several minutes high-contrast images of resting BAT. Conversely, mitochondrial uncoupling induced by skin cooling or systemic administration of the ß3-noradrenergic agonist CL-316,243 resulted in an immediate washout of 18 F-FBnTP from BAT, which was blocked by propranolol. We characterized some key variables of BAT-evoked activity, including response latency, magnitude and temporal kinetics, and demonstrated indications that rat's BAT is a highly sensitive and rapidly responsive organ, which generates heat in an immediate, short burst of several minutes duration, while body's core temperature remains intact.

Animals and materials
Brown Norway rats (6-mo old male; 250-350 g BW, n = 22) were purchased from the NIA colony. 18 F-FBnTP was prepared in our radiochemistry laboratory with a specific activity ranging from 111 to 185 GBq/mmol (12,000-25,000 mCi/mmol), as described elsewhere [34]. Propranolol and CL-316,243 were purchased from Sigma Eldrich. Animals were acclimated at ambient temperature of 23°C for at least one week before the imaging study with ad libitum food and 12/12h light/dark cycle.

PET/CT acquisition
PET data were acquired on a GE eXplore VISTA dual-ring small-animal scanner (61 slices, 0.775-mm slice thickness, 4.8-cm axial FOV, 1.1-mm FWHM). Animals were sedated throughout the PET/CT study by isoflurane (2-3%) inhalation. Sixty to ninety minutes dynamic PET scan was commenced with tail-vein administration of 37 MBq 18 F-FBnTP (1 mCi). Images were corrected for decay, dead times, random count and scatter. Images were reconstructed using the ordered-subsets expectation maximization (OSEM) algorithm (32 subsets, 2 iterations), into a 175 x 175 x 61-pixel matrix and 0.3875 x 0.3875 x 0.775-mm voxel size. CT images were acquired immediately after the completion of the PET scan, using small animal SPECT/CT scanner (X-SPECT; Gamma Medica), which stands next to the microPET. Animals were transferred to the SPECT/CT scanner while restrained to the bed, and sedated by isoflurane inhalation. CT images were obtained at 50 kVp and 0.6 mA. Images were captured for 5 sec per view for 256 views in a 360°rotation. PET-CT image coregistration was carried out using Mirada cd and Analyze cd packages. In all animals, colonic temperature was monitored periodically throughout the imaging study using digital thermometer.

Study protocols
The following PET protocols were employed: 1. 18 F-FBnTP uptake and retention kinetics in BAT at room temperature (RT): Animals were kept warm during the scan using heating lamp (colonic temperature 36°C). Dynamic PET, initiated concurrently with IV administration of 18 F-FBnTP, was carried out for up to 60 min (n = 5). Frame duration increased gradually from 10 to 180 sec.

Validation of 18 F-FBnTP selectivity for BAT:
Dynamic PET was carried out as in protocol I (n = 3). At the completion of the PET scan, animals were quickly euthanized by isoflurane overdose, and BAT was excised surgically. Next, a 10 min static scan was carried out in same bed position, as the pre-excision scan. Time interval between scan 1 and 2 did not exceed 10 min. In a separate group of animals (n = 3), the effect of euthanasia on 18 F-FBnTP uptake in BAT was assessed. Animals were euthanized and scanned as above, but without excision of BAT.
3. Effect of cold stimulation on 18 F-FBnTP uptake kinetics in BAT. Dynamic PET was carried for 90 min, beginning with IV administration of 18 F-FBnTP (n = 6). First 20-30 min of the scan were acquired while the animal was kept warm using heating lamp. Next, cold stimulation was applied by turning off the heating lamp and carefully placing wrapped shredded ice on the caudal back of the animal, which extended out of the scanner gantry, for the remaining duration of the scan. Colonic temperature was kept above 28°C by carefully removing the ice bag, when necessary.
4. Effect of propranolol on 18 F-FBnTP washout response to cold stimulation: Propranolol (5 mg/kg, IP) was administered 30 min before the administration of 18 F-FBnTP and the PET scans were acquired as in protocol III (n = 4).

5.
Effect of the ß3-noradrenergic agonist CL-316,243 on 18 F-FBnTP uptake and retention in BAT. A 90 minutes dynamic PET was initiated concurrently with 18 F-FBnTP IV administration (37MBq); 30 min after the start of the scan CL-316,243 (10 μg/kg) was given IV (n = 4). Both 18 F-FBnTP and CL-316,243 were administered via a tail-vein catheter.
All animal protocols were approved by the Johns Hopkins School of Medicine's Animal Care and Use Committee.

Image and data analysis
Quantification of 18 F-FBnTP uptake was carried out on coronal section of interscapular BAT (iBAT). Images were resampled to cubic voxels (0.775-mm 3 ), and a medial section intersecting iBAT was selected for further analysis. Frames acquired over 10-min period just before stimulus application were summed up, representing 18 F-FBnTP BAT basal activity, and segmented using 50% of maximum activity as cutoff value (Tmax50%). All basal iBAT voxels, visible on the segmented basal PET image and localized to low CT Hounsfield unit area, were delineated using automatic ROI. The ROI template was copied to the temporal images and mean activity was computed. Small cubic ROIs (0.775-mm 3 ) were placed just outside of BAT (background activity) and on the left ventricular (LV) wall. 18 F-FBnTP activity was expressed as [counts/ mCi injected/kg BW]. Image processing was carried out by PMOD ct and Analyze ct packages.

Statistical analysis
Results are expressed as mean±SD. Level of significance between different organs and conditions was calculated using 2-tailed paired t-test. P value 0.05 was considered to indicate statistical significance.

F-FBnTP Strongly Accumulates in BAT at Rest
Dynamic PET was carried out in BN rats (n = 5), as outlined in Protocol I. In all animals, a strong preferential accumulation of 18 F-FBnTP was found in the interscapular area, localized to regions of low Hounsfield units as identified by coregistered CT (Fig 1). To validate that 18 F-FBnTP uptake is restricted to iBAT, PET scan was acquired in same-bed position before and after excision of BAT (Protocol II, n = 3). 18 F-FBnTP uptake in iBAT was observed before, but not after excision (Fig 2). Total time interval between the pre-and post-excision scans was less than 8 min. In a separate group of animals, we examined the effect of euthanasia on 18 F-FBnTP retention in BAT (n = 3). Animals underwent same procedure as above, but without excision of BAT. In all animals, BAT was clearly visible on PET images, albeit 18 F-FBnTP uptake was slightly lower (8.1%±7.5%; P<0.281), compared to that measured in the living animal.
Analysis of time activity profiles revealed a very rapid and extensive accumulation of 18 F-FBnTP in BAT. 18 F-FBnTP peak activity in resting BAT was obtained within 10 to 20 seconds and plateaued within a few minutes (Fig 2F). 18 F-FBnTP maintained prolonged steadystate concentration in BAT for the entire scan duration (Fig 2E). 18 F-FBnTP plateau activity in BAT at ambient temperature ranged between 471 and 635 (522.2±108.3; n = 5). 18 F-FBnTP BAT-to-background ratio was 6:1 to 10:1. 18 F-FBnTP plateau concentration in BAT was similar to that of the heart (Fig 2E). Mean BAT-to-heart ratio was 0.88±0.13 (n = 5).
In large and small animals, heart is a major target organ of 18 F-FBnTP, second only to kidney [32,35]. Cold Stimulation Results in an Immediate-Washout of 18

F-FBnTP from BAT
The effect of localized skin cooling on 18 F-FBnTP retention in BAT was studied using 90-min dynamic PET scan (Protocol III, n = 6). 18 F-FBnTP was administered IV, and the first 20 or 30 min of the dynamic scan were acquired while the animal was kept warm, using heating lamp. Cold stimulation was applied for the remaining scan time. Colonic temperature was monitored throughout scan time.
Contrary to the prolonged steady-state retention observed at room temperature (RT), cold stimulation resulted in a rapid washout of 18 F-FBnTP from BAT. Fig 3 depicts an example of 18 F-FBnTP PET images and related time-activity curve acquired before (RT) and during cold stimulation (COLD). Each image represents summed activity over 3 min, and start-times of each frame is indicated at the upper right corner (Fig 3B). 18 F-FBnTP washout kinetics can be evaluated qualitatively by the PET images (Fig 3B), and quantitatively by the time activity curve (Fig 3C).
Time-to-onset of washout was in the few-minutes range (2.2±1.3 min, n = 6). The duration of the early washout phase ranged from 2 to 8 min (2.71±2.42 min). Extent of cold-induced washout of 18 F-FBnTP from BAT, expressed as percentage of mean basal activity, was 39.1% ±14.4% (n = 6, P <0.007) (Fig 3D). The kinetics of the late response phase varied between animals. Both, slow or no washout, were observed in different animals. An additive but insignificant washout of 11.1%±17.5%, (n = 6; P <0.18) was measured at 50 to 60 min, compared to 20 to 30 min post-administration time interval (Fig 3D).
FBnTP retention in the myocardium was not significantly affected by skin cooling (Fig 3D). A slight washout of 18 F-FBnTP was observed in 2 out of 6 rats. Unlike the abrupt washout observed in BAT, the clearance from heart was linear, if at all. Note the lack of uptake in the interscapular area after BAT excision (D). Images in (A to C) and (D) represent PET scans acquired in the same animal at an interval of 12 min. Chart in (E) represents 18 F-FBnTP time activity curve generated from the same animal in (A to C). Chart in (F) is zooming of the first 240 sec in (E). Yaxis in (F) has same unit value and scale as in (E). Note the strong 18 F-FBnTP uptake in BAT, which is similar to that in the heart, and 8 times greater than background activity (E). 18 F-FBnTP reaches plateau concentration in BAT within less than a minute (F).

The Bulk of 18 F-FBnTP Washout Occurs while Body Core Temperature Remains Intact
Onset of 18 F-FBnTP washout from stimulated BAT occurred before significant change in colonic temperature was observed (Fig 4A and 4B). The cold stimulation protocol employed in the present study induced a typical linear decrease of colonic temperature at rate of 0.17 ±0.05°C/min (n = 6) ( Fig 4C). In all animals, the most of the early steep washout phase was maintained while colonic temperature was 35.8°C (Fig 4D). Decreasing of colonic temperature below 35.8°C and into the shivering range did not elicit an additional washout of 18 F-FBnTP (Fig 4D). 18

F-FBnTP Washout Response Is Mediated by the Noradrenergic System
Two sets of studies were carried out to examine the role of the ß-noradrenergic receptor system in 18 F-FBnTP washout response. First, the effect of the sub-type non-selective ß-noradrenergic antagonist propranolol was studied (Protocol IV, n = 4). Propranolol (5 mg/kg, IP) was administered 30 min before commencement of 18 F-FBnTP PET dynamic scan. First 20 min of the scan were acquired at room temperature, and cold stimulation was employed for the remaining scan time. Administration of propranolol has had two effects. (i) 18 F-FBnTP basal uptake was 17.6% greater in propranolol-treated, compared to non-treated rats (Prop 613.5±121.3; no-Prop 522.2±108.3, P <0.052); (ii) Propranolol significantly reduced 18 F-FBnTP washout response to cold. 18 F-FBnTP washout from BAT, measured as the mean decrease over the time interval of 50-60 min of cold stimulation, was significantly lower in propranolol-treated, compared to non-treated rats (9.6%±11.6% (P<0.21) vs. 39.1±14.4% (P<0.007), respectively) ( Fig 5). 18 F-FBnTP retention in heart was not affected by propranolol treatment.
Second, the effect of the ß3-noradrenergic selective agonist CL-316,243 on 18 F-FBnTP uptake in BAT was documented using 90 min dynamic PET (Protocol V, n = 3). CL-316,243 (100 μg/kg) was administered IV via tail vein 30 min after the commencement of the 90min dynamic 18 F-FBnTP PET. Administration of CL-316,243 resulted in immediate 18 F-FBnTP washout from BAT, compared to baseline activity (Fig 5). Washout response kinetics were similar to those observed during cold stimulation. CL-316,243 had no effect on 18 F-FBnTP retention in heart (Fig 5).

Discussion
Mitochondrial respiration and ΔCm are the two most established interrelated measures of thermogenesis' in vitro. Heat is produced by the protons flux down the concentration gradient, resulting in loss of ΔCm and disengagement of substrate phosphorylation and ATP synthesis. This aberrant condition imposes a hypoxia-like condition and a compensatory increase of the organelle's oxygen utilization. Perfusion studies using O-15 PET documented the increase of oxygen utilization in BAT during thermogenesis, but not the kinetics of the BAT response [36]. In the present study, we demonstrated the advantage of targeting ΔCm using 18 F-FBnTP PET, for dynamic imaging of BAT thermogenesis. 18 F-FBnTP PET provided evidence that BAT is a highly responsive organ in the living animal, and that the bulk of heat (i.e., mitochondrial uncoupling) is generated as a short burst, of few-to-several minutes, immediately upon stimulation.
Three key requirements are essential for an imaging compound to act as a reliable noninvasive indicator of ΔCm and thermogenesis. (i) Linear dose-dependent relationship with ΔCm over a wide range of membrane potentials. This characteristic determines the quality of resting BAT image; the benchmark for measuring alterations of ΔCm during thermogenesis. Importantly, mitochondrial capacity to produce heat is dictated by the extent of proton gradient (i.e., ΔCm). The greater the protons gradient, the greater ΔCm and the potential capacity for heat production. Some potentiometric probes plateau at high ΔCm values, and therefore may not provide a true measure of the tissue's capacity for thermogenesis [26]. (ii) The fraction of the probe molecules concentrating in the mitochondrial compartment should be in a labile form, and readily expelled upon ΔCm decline. This characteristic is crucial for a reliable monitoring of rapid changes in ΔCm, as these occur in BAT during activation. In vitro, mitochondrial uncoupling is a rapid event in the seconds-range. (iii) It has to maintain low nonspecific binding. Once UCP1 are opened, proton reentrance to matrix is expected to be maintained until concentration gradient is completely abolished, leading to collapse of ΔCm to near zero values and complete expulsion of the potentiometric probe. This highlights the need for minimal nonspecific binding. Most potentiometric probes are lipophilic, and nonspecific binding to membrane constituents may mask the decline of ΔCm during thermogenesis. [33].
The results of the present in vivo study, together with previously-obtained in vitro and ex vivo data, suggest that 18 F-FBnTP complies with the above requirements. First, at rest, when ΔCm is intact, 18 F-FBnTP accumulated extensively in BAT. 18 F-FBnTP BAT-to-background contrast was > 6:1. Whole-body PET scans in large and small animals have shown that 18 F-FBnTP is targeting body organs, in proportion to their mitochondrial content, and heart uptake is second only to that of the kidney [30,32]. In the present study, 18 F-FBnTP uptake in BAT was similar to that in heart [30,32]. 18 F-FBnTP avidity for mitochondria is because ΔCm is much greater than the plasma membrane potential (200-240 mV vs. 30-60 mV, respectively [37]). According to Nernst Equation, each 60 mV difference results in 10-fold increase of the potentiometric probe uptake. In carcinoma cells, 18 F-FBnTP concentration in the mitochondrial compartment was approximately 10 4 times that in the cytosol and comprised >80% of total cellular uptake [30]. Under the assumptions of 150 mV for ΔCm and a matrix volume of 1% of total cytoplasm, 75% of Nernstian probe is expected to concentrate in the mitochondria in a ΔCm-dependent manner [38], similar to that observed for 18 F-FBnTP in carcinoma cells [30].
Second, mitochondrial depolarization induced by localized skin cooling and systemic activation of ß3-noradrenergic receptors, resulted in an immediate, abrupt washout of 18 F-FBnTP from BAT. The short latency and rapid washout rate indicate that the fraction of 18 F-FBnTP concentrated in mitochondria is labile, and readily expelled upon decline of ΔCm. Third, in vitro studies of pharmacologic manipulations of cytoplasma and mitochondrial membrane potentials have shown that 18 F-FBnTP maintains very low nonspecific binding (~5%) [30].
The receptor mechanism underlying 18 F-FBnTP evoked response was validated using activation (CL-316243), and suppression (propranolol) of the β-adrenergic receptor (AR) system. The results of both studies supported β-adrenergic mediation of 18 F-FBnTP washout response. The β3-AR-specific agonist CL-316243 elicited 18 F-FBnTP washout from BAT similar to the kinetics observed upon cold stimulation, including short onset time and rapid washout rate. In rodents, the β3-AR is found nearly exclusively on brown adipocytes, and treatment with CL-316243 substantially increases energy expenditure [39][40]. The administration of the adrenergic antagonist propranolol strongly mitigated 18 F-FBnTP washout from stimulated BAT. This further bolsters the linkage of 18 F-FBnTP response to cold-induced mitochondrial uncoupling and heat production.

BAT Response Kinetics
18 F-FBnTP PET has shed light on some key aspects of the physiology of BAT evoked activity.
First, 18 F-FBnTP PET has shown that BAT is a rapidly responsive organ. Both, cold-and ß3-AR stimulation caused a nearly immediate washout of 18 F-FBnTP with a response onset time in the few-minute range. This finding is consistent with in vitro observations in isolated mitochondria and brown adipocytes. Administration of noradrenalin to the incubation medium resulted in an immediate mitochondrial uncoupling, expressed by sharp increase of mitochondrial respiration [26]. Maximum respiration was obtained within 2 min [26]. Similar results were obtained in brown adipocytes [41]. The present study suggests that the rapid kinetics of mitochondrial uncoupling observed in vitro are preserved in the intact animal. The results of the present study are also consistent with whole-body measurement of respiration in rats. Cold [42] and noradrenergic agonists [43] resulted in an early abrupt increase of wholebody oxygen utilization in the few-minutes range.
The present study provides indications, in the intact animal model, that BAT is only partially activated by cold stimulation. The skin cooling protocol employed in the present study resulted in an abrupt drop of approximately one-third of 18 F-FBnTP, compared to basal uptake. Partial clearance of 18 F-FBnTP from BAT was obtained by systemic activation of ß3-AR. Magnitude of 18 F-FBnTP washout may serve as an index of both extent of decline of DY and amount of mitochondria recruited for heat production. However, a rigorous assessment of extent of activation should take into account the distribution kinetics of 18 F-FBnTP once released from uncoupled mitochondria. In the present study, both cold and pharmacological activation of ß3-AR resulted in bimodal washout; an abrupt decline of activity over a short time (2-8 min), which was followed by steady state concentration for the remaining scan time (60-70 min). Dynamic PET of isolated perfused heart has shown that mitochondrial uncoupling induced by 10 μM FCCP resulted in linear, rather than bimodal, washout of 18 F-FBnTP from the LV wall, and the extent of washout was significantly greater than that observed in stimulated BAT-50% to 75% depletion of uptake was obtained within 20 min. This suggests that 18 F-FBnTP washout kinetics observed in the present study are organ specific, and may point to additional players, such as 18 F-FBnTP re-distribution from uncoupled (i.e., thermogenically active) to yet coupled (i.e., inactive) mitochondria. Thus, the slow, late washout may represent the net result of two opposing dynamics, re-uptake of 18 F-FBnTP to mitochondria of yet intact ΔCm, which may mask diffusion of the imaging agent from tissue to the blood pool. Furthermore, carful quantitative assessment of magnitude of BAT activation requires examining the contribution of the increased blood flow to 18 F-FBnTP washout kinetics. Efforts to identify the forces involved in 18 F-FBnTP early-and late-phase dynamics using dose-and duration-dependent protocols of pharmacologic and cold stimulation, respectively, are in progress.
The common school of thought holds that BAT thermo-homeostatic role is in the nonshivering temperature range. Accordingly, we explored the effect of shivering colonic temperature on mitochondria uncoupling in BAT. Our results suggest that mitochondrial depolarization in BAT is indeed confined to non-shivering conditions. Drop of body core temperature to the shivering range has marginal effect, if at all, on BAT mitochondria. This provides an important physiological validation of the non-shivering adaptive role of BAT in the living animal.

Conclusion
Monitoring ΔCm using 18 F-FBnTP PET provided important insights into key aspects of BAT thermogenesis in vivo. 18 F-FBnTP PET depicts rodent's BAT as a highly sensitive and rapidly responsive organ, emitting the bulk of heat in a short-lasting burst, over the first minutes of the cold stimulation. The present study also provide physiological evidence in support the the nonshivering adapative role of BAT. Prolonogation and decrease of core tempearture to shivering range has mariginal additive effect on short-term mitochondrial recrutiment. The capacity of 18 F-FBnTP PET to monitor BAT response kinetics in real-time, allowed us to identify and quantify principal variables of thermogenesis, including response onset time, magnitude and kinetics. As such, 18 F-FBnTP PET provides a powerful research platform for the study of BAT physiology in vivo, as well as a novel set of quantitative metrics, which can be helpful for identifying therapeutic targets at the mitochondrial level, for developing of means to maximize BAT mass and activity, thus enabling sensitive and accurate assessment of their efficacy.