A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.
Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via β adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of β-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by β3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.
1. Animal Preparation and Cold Treatment
2. Setup Micro-PET/CT Imaging Workflow
In this protocol micro-PET/CT imaging is achieved with the Siemens Inveon Dedicated PET (dPET) System and Inveon Multimodality (MM) System (CT/SPECT) in the docked mode. The animal is placed from the MM entrance, first scanned with the CT for anatomical references, and then slid to the center of the dPET for a static F18 PET acquisition. In order to enable the host computer to carry out these sequential tasks automatically, the following “workflow” is programmed with the Inveon Acquisition Workplace (IAW) software prior to the actual imaging session.
3. Injection of FDG
4. Micro-PET/CT Imaging
5. Post-imaging Analysis
An example of micro-PET/CT imaging of mouse BAT is shown in Figure 1. While the CT imaging provides anatomical information, the PET imaging encodes the distribution and quantity of 18F-FDG uptake throughout the whole body. These imaging data can be viewed separately ( Figure 1A and 1B), fused (Figure 1C), or demonstrated with a 3D feature such as maximal intensity projection (MIP, 1D). With the help of a 3D imaging tool, a volume of interest (VOI), here the interscapular BAT region (indicated by arrows in Figure 1), is drawn over the PET images and the total signals within the VOI can be converted into %ID/g, representing the percentage injected dose (%ID) per gram of tissue. In the mouse demonstrated, FDG uptake in the BAT is 19 %ID/g. In order to verify if this cold-induction and imaging protocol is sensitive enough to detect an altered BAT activity, in either case of up-regulation or down-regulation, we used β adrenoceptor antagonist propranolol to suppress the BAT activation 15, and β3 agonist BRL37344 to enhance BAT induction 16, respectively. These pharmacological interventions were applied during the cold treatment and precisely, at 30 min before the injection of FDG. PET/CT imaging (Figure 2A) and the analysis (Figure 2B) showed that the propranolol treatment significantly reduced the FDG uptake in BAT, whereas BRL37344 markedly elevated the uptake, as compared with the vehicle control.
Figure 1. Micro-PET/CT imaging of BAT in a mouse after 5 hr cold-treatment. (A) A coronal section of CT image. (B) A coronal section of PET image co-registered with the CT in “A”. (C) A fused PET/CT image resulted from the superimposing of “A” and “B”. (D) Maximal intensity projection (MIP) presentation of the fused PET/CT images. Yellow arrows: the area corresponding to interscapular brown adipose tissue.
Figure 2. Micro-PET/CT demonstration of BAT activity alteration by adrenoceptor drugs. (A) 18F-FDG PET/CT imaging of mice treated with different drugs. a) PBS (control). b) Propranolol (β antagonist, 5mg/kg, i.p.). Note a marked reduction of FDG uptake in BAT area. c) BRL37344 (β3 agonist, 5 mg/kg, i.p.). Note a significant increase in FDG accumulation in BAT. Yellow arrows: the area corresponding to interscapular brown adipose tissue. (B) Quantitative analysis of the FDG uptake in BAT. Values of %ID/g (the percentage injected dose per gram of tissue) are presented (mean ± SD). n=10 for the PBS group; n=5 for both Propranolol and BRL37344 groups. *, p < 0.05; **, p < 0.005, as compared with the PBS group.
In this study a micro-PET/CT-based imaging method has been developed for detecting BAT activities in adult mice which simply requires a cold treatment and one injection of commercially available 18F-FDG. The whole procedure can be done in one day following a treatment and imaging sequence which starts every 30 minutes until all animals are treated and imaged. Under the experimental conditions outlined, a total of 10 mice (or 2 groups of 5 mice) can be tested on the same day with a single imaging system. The limitation to this type of throughput can be lifted if 2 or more animals can be scanned simultaneously on a specially designed animal bed as it has been previously reported 17. To complete the study we rely on the use of a combined micro-PET/CT imaging system which takes advantage of the detailed anatomical information provided by the CT. However, a standalone micro-PET is also able to fulfill the task when a 57Co transmission scan is added to the workflow prior to the F18 emission data acquisition. The 57Co transmission data can be used for attenuation correction during PET image reconstruction and can also be reconstructed for anatomical localization.
A critical step of this protocol is to optimize the duration of the cold treatment (e.g., 5 hours in this study). A shorter duration time or the elimination of cold exposure may produce an activity close to the background and the method can be insensitive to any down-regulation of BAT (the floor effect). In contrast, an elongated cold exposure (such as overnight, or 24 hours) may maximize the induction and the method can become saturated masking any differences in the up-regulation of BAT (the ceiling effect). The optimized conditions described in this protocol have been validated through the propranolol suppression and β3 agonist BRL37344 stimulation tests (Figure 2), suggesting that this method is considerably sensitive and consistent in detecting alterations of BAT activities in mice. Applications of this method will include various basic studies using mouse models towards a better understanding of BAT differentiation and regulation, as well as preclinical research aiming to the discovery of safe BAT-stimulating drugs that may benefit the treatment of obesity and diabetes.
The authors have nothing to disclose.
The authors would like to thank Laura Diaz, Kevin Phillips, Willa A. Hsueh, and King C. Li for their helpful comments and technical support in developing this method.
Name of the reagent | Company | Catalogue number | Comments (optional) |
Micro-PET/CT Imaging System | Siemens Medical Solutions USA, Inc. | Inveon Dedicated PET System and Inveon Multimodality CT/SPECT System (docked) | |
Propranolol | Sigma | P0884 | |
BRL 37344 | Sigma | B169 | |
18F-FDG | Cyclotope Inc. | ||
C57BL/6J Male Mice | Jackson Laboratory | 000664 | 3-4 months old |