PLoS One. 2015 Jun 8;10(6):e0129627

Unraveling the physiology of brown adipose tissue thermogenesis in vivo using dynamic voltage-dependent imaging

Madar I, Naor E, Holt D, Ravert H, Dannals R, Wahl R.

Division of Nuclear Medicine, The Russell H. Morgan Department of Radiology, The Johns Hopkins Medical Institutions, Baltimore, MD, USA



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 bursts during the first minutes of stimulation, and preceding the 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.

PMID: 26053485



Brown adipocyte mitochondria are equipped with a unique mechanism for combusting a huge amount of energy intake as heat. In rodents, brown adipose tissue (BAT) dissipates caloric energy 300 times more than an equivalent volume of any other organ. The recent re-discovery of metabolically active BAT depots in human adults triggered extensive efforts to harness this energy expenditure mechanism as a tool to combat obesity. This potentially high impact field of research is hampered by our current limited knowledge of the real-time physiology of BAT thermogenesis in the living animal, in part due to absence of established non-invasive means for dynamic imaging of mitochondrial bioenergetics. Mitochondria generate heat by the dissipation of the organelle’s membrane potential (ΔΨm) (1). In non-thermogenic state, the energy produced by electron transfer in the respiratory chain is used to pump out protons from the mitochondrial matrix and across the inner membrane, thus creating a large trans-membrane voltage difference (i.e. ΔΨm). In the thermogenic mode, opening of inner-membrane channels results in reentrance of protons into the matrix and the release of the energy stored in the concentration gradient as heat, expressed by proportional decline of ΔΨm. Thus, monitoring ΔΨm provides a direct voltage analog of heat produced by BAT mitochondria. Accordingly, we hypothesized that the voltage sensor 18F-fluorobenzyl triphenylphopshonium (FBnTP) would permit (i) imaging BAT at high contrast when ΔΨm is intact and (ii) monitoring the kinetics of DY dissipation during activation of BAT. The excellent performance of FBnTP as ΔΨm-indicator was tested and validated in several in vitro (2) and in vivo (3) models and specifically in BAT ex vivo (4). In the present study (5), we characterized the kinetics of mitochondrial heat production, using dynamic PET imaging of FBnTP in living rats. FBnTP PET was acquired just before and during local skin cooling. The results of this study validated the above hypothesis: FBnTP accumulated strongly in BAT at rest, whereas mitochondrial uncoupling induced by local skin cooling resulted in a selective washout of FBnTP from BAT. FBnTP provided a new insight, in vivo, into the mechanism of BAT heat production. FBnTP suggests that in rodents, mild cold stimulation results in an immediate synchronized activation, encompassing about one-third of BAT mitochondria population, leading to emission of an abrupt bulk of heat in the first minutes of cold exposure (Fig. 1); and doing so while colonic temperature remains largely intact (Fig. 2), consistent with the postulated adaptive protective role of BAT. The physiology of the later effect of cold on BAT mitochondria is under current investigation. The linkage of FBnTP washout response and cold-induced ΔΨm dissipation in BAT was validated using selective pharmacological means for beta3-adrenergic stimulation (CL-316,243), and beta-adrenergic suppression (propranolol) (Fig. 3). In summary, FBnTP PET permits to detect and accurately quantify, by normalization to baseline activity, key physiological indices of BAT adaptive response, including response latency, magnitude and evolution kinetics, for the first time in the intact animal.


Fig 1. Cold-induced Mitochondrial Uncoupling Elicits an Immediate 18F-FBnTP Washout from BAT. (A) Coronal PET/CT images of BAT acquired at room temperature. (B) 18F-FBnTP PET images of BAT before and during cold stimulation. Images are segmented using max50% cutoff value. Each image represents summed activity over 3 min. Beginning of acquisition time of each frame is indicated in upper right corner. Cold stimulation started at the 30 min point of the scan. (C) 18F-FBnTP time activity curve generated from same animal depicted in (B). (D) 18F-FBnTP mean uptake measured on 10-min image frame, acquired just before (baseline) and 20 and 50 min after the start of cold stimulation (mean±SD, n = 6). Note the immediate sharp decrease of 18F-FBnTP uptake upon application of cold stimulation (B and C), and small, but insignificant washout at later time points (D), as well as lack of effect on uptake in heart (D).



Fig 2. 18F-FBnTP Washout Response Precedes Change in Body Core Temperature. Colonic temperature (A) and 18F-FBnTP uptake kinetics (B) monitored in same animal before and during skin cooling. (C) Mean change in colonic temperature induced by cold stimulation (mean±SD, n = 6). (D) Extent of 18F-FBnTP washout response correlated with colonic temperature (n = 6). Note the sharp washout of 18F-FBnTP, which nearly completed before significant change in colonic temperature was attained (D).



Fig 3. Noradrenergic Mediation of 18F-FBnTP Washout Response. (A) Effect of propranolol 18F-FBnTP-washout response to cold stimulation. Propranolol blocked cold-induced 18F-FBnTP washout from BAT. A slight but insignificant decrease (9.6%±11.6%; P<0.21) was measured. (B) Effect of the ß3-AR agonist CL-316,243 on 18F-FBnTP retention in BAT. Administration of CL-316,243 (100 μg, IV) resulted in 18F-FBnTP washout from BAT with kinetics similar to those observed during cold stimulation. (C) 18F-FBnTP PET imaged of BAT before and after administration of CL-316,243. Both PET images in (C) and time activity curve in (B) were taken form same rat’s BAT. Note the CL-316,243-induced washout kinetics, which are similar to those observed during localized skin cooling. (D) CL-316,243 hardly affected 18F-FBnTP retention in heart muscle.


The importance of this study is multi-fold. Real-time monitoring of ΔΨm in vivo using dynamic PET imaging of FBnTP permits (1) a direct approach for exploring mechanisms of BAT activation in the intact animal and maybe in humans, (2) a novel set of physiological indices, at the level of the mitochondrion, for identifying molecular targets for drugs and methods to potentiate BAT energy expenditure, and (3) a quantitative high-resolution metrics for assessing pharmacokinetics and efficacy of interventions. (4) FBnTP indication of BAT rapid response kinetics opens new avenues for design of BAT activation protocols, such as repetitive, short-duration exposure to physiological and pharmacological stimuli.



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  2. Madar I, Ravert H, Nelkin B, Abro M, Pomper M, Dannals R, and Frost JJ. Characterization of Membrane potential-dependent uptake of the novel PET tracer 18F-fluorobenzyl triphenylphosphonium cation. Eur J Nuc Med Mol Imaging 2007:34:2057-65.
  3. Madar I, Huang Y, Ravert H, Dalrymple S, Davidson NE, Isaacs JT, Dannals RF, Frost JJ. Detection and Quantification of the Evolution Dynamics of Apoptosis Using the PET Voltage Sensor 18F-Fluorobenzyl Triphenyl Phosphonium. J. Nuc Med 2009; 50:774-780.
  4. Madar I, Isoda T, Finley P. Angel J, Wahl R. Fluorobenzyl triphenyl phosphonum (FBnTP): A noninvasive sensor sensor of brown adipose tissue thermogenesis. J Nuc Med. 2011;52:808-14.


Acknowledgment: This work was supported by NIH-NIDDK grant DK201822



Igal Madar PhD

Division of Nuclear Medicine

The Russell H. Morgan Department of Radiology

Johns Hopkins Medical Institutions, Baltimore, MD, USA.




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