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* These authors contributed equally
Functional imaging and quantitation of thermogenic adipose depots in mice using a micro-PET/MR imaging-based approach.
Brown and beige adipocytes are now recognized as potential therapeutic targets for obesity and metabolic syndromes. Non-invasive molecular imaging methods are essential to provide critical insights into these thermogenic adipose depots. Here, the protocol presents a PET/MR imaging-based method to evaluate the activity of brown and beige adipocytes in mouse interscapular brown adipose tissue (iBAT) and inguinal subcutaneous white adipose tissue (iWAT). Visualization and quantification of the thermogenic adipose depots were achieved using [18F]FDG, the non-metabolizable glucose analog, as the radiotracer, when combined with the precise anatomical information provided by MR imaging. The PET/MR imaging was conducted 7 days after cold acclimation and quantitation of [18F]FDG signal in different adipose depots was conducted to assess the relative mobilization of thermogenic adipose tissues. Removal of iBAT substantially increased cold-evoked [18F]FDG uptake in iWAT of the mice.
In response to changing nutritional needs, adipose tissue serves as an energy cache to adopt either lipid storage or mobilization mode to meet the needs of the body1. Moreover, adipose tissue also plays a key function in thermoregulation, via a process called non-shivering thermogenesis, also called facultative thermogenesis. This is typically achieved by the brown adipose tissue (BAT), which expresses abundant level of mitochondria membrane protein uncoupling protein 1 (UCP1). As a proton carrier, UCP1 generates heat by uncoupling the proton transport and ATP production2. Upon cold stimulation, thermogenesis in BAT is set in motion by activation of the sympathetic nervous system (SNS), followed by release of norepinephrine (NE). NE binds to the β3 adrenergic receptors and leads to elevation of intracellular cyclic AMP (cAMP). As a consequence, cAMP/PKA-dependent engagement of CREB (cAMP response element-binding protein) stimulates Ucp1 transcription via direct binding on CREB-response elements (CRE)2. In addition to BAT, brown-like adipocytes are also found within white adipose tissue and are therefore named beige or brite (brown-in-white) cells1,3. In response to specific stimuli (such as cold), these otherwise quiescent beige cells are remodeled to exhibit multiple brown-like features, including multilocular lipid droplets, densely-packed mitochondria, and augmented UCP1 expression3,4,5.
Animal studies have demonstrated that brown and beige adipocytes possess multiple metabolic benefits beyond its fat-reducing effect, including insulin-sensitization, lipid-lowering, anti-inflammation, and anti-atherosclerosis6,7. In humans, the amount of beige/brown fat is inversely correlated with age, insulin resistance index, and cardiometabolic disorders8. Moreover, activation of beige/brown adipocytes in humans by either cold acclimation or β3 adrenergic receptor agonist confers protection against a series of metabolic disorders4,9,10. These pieces of evidence collectively indicate that induction of brown and beige adipose tissue is a potential therapeutic strategy for management of obesity and its related medical complications8.
Interestingly, although they share similar function, beige and classical brown adipocytes are derived from different precursors and activated by overlapping but distinct mechanisms1. Therefore, in vivo imaging and quantification of brown and beige adipocytes are essential to achieve a better understanding of the molecular control of these adipose tissues. Currently 18F-fluorodeoxyglucose ([18F]FDG) positron emission tomography (PET) scan combined with computed tomography (CT) remains the gold standard for characterization of thermogenic brown and beige cells in clinical studies8. Magnetic resonance imaging (MRI) uses powerful magnetic fields and radio frequency pulses to produce detailed anatomical structures. Compared to CT scan, MRI generates images of organs and soft tissues with a higher resolution. Provided here is a protocol for visualization and quantification of functional brown and beige adiposes in mouse models after acclimation to cold exposure, a common and most reliable way to induce adipose browning. This method can be applied to characterize the thermogenic adipose depots in small animal models with high precision.
The protocol described below follows the animal care guidelines of The University of Hong Kong. The animals used in the study were 8-week-old C57BL/6J mice.
1. Animal surgical procedures and cold challenge
2. Micro-PET/MR calibrations and workflow setup
NOTE: Micro-PET/MR imaging is performed using a sequential PET/MR system (see Table of Materials). Each mouse is placed on the imaging bed; first scan with the MR for an anatomical reference (scout view) before advancing to the center of the PET field-of-view (FOV) for a static [18F]FDG PET acquisition, followed by MR imaging for anatomic reference. An imaging workflow is created in the scanner-operating software (see Table of Materials) to enable automated, sequential PET/MR scans prior to the imaging session.
3. Injection of [18F]FDG
4. Micro-PET/MR acquisition
5. Post-imaging analysis
Three groups of mice (n = 3 per group) underwent micro-PET/MR imaging in this study, where they were housed at either thermoneutrality (30 °C) or cold (6 °C) for 7 days. One group of mice (n = 3) had their iBAT removed (iBATx) prior to cold treatment (Figure 1A). This method led to an alteration to the white adipose tissue activity in all three mice. In particular, a remarkable increase in [18F]FDG uptake was observed in iWAT using micro-PET/MR imaging (
In this study, a PET/MR -based imaging and quantification of functional brown and beige adipose tissue in small animal was described. This method uses the non-metabolizable glucose analog [18F]FDG as an imaging biomarker so as to identify the adipose tissues with high glucose-demand in a non-invasive manner. MR offers good soft tissue contrast and can better differentiate adipose fat tissues from the neighboring soft tissues and muscle. When combined with PET, this enables imaging and quantifying of the activa...
The authors have no conflicts of interest to disclose.
We thank the support of National Natural Science Foundation of China (NSFC) - Excellent Young Scientists Fund (Hong Kong and Macau) (81922079), Hong Kong Research Grants Council General Research Fund (GRF 17121520 and 17123419), and Hong Kong Research Grants Council Collaborative Research Fund (CRF C7018-14E) for small animal imaging experiments.
Name | Company | Catalog Number | Comments |
0.9% sterile saline | BBraun | 0.9% sodium chloride intravenous infusion, 500 mL | |
5 mL syringe | Terumo | SS05L | 5 mL syringe Luer Lock |
Dose Calibrator | Biodex | Atomlab 500 | |
Eye lubricant | Alcon Duratears | Sterile ocular lubricant ointment, 3.5 g | |
Insulin syringe | Terumo | 10ME2913 | 1 mL insulin syringe with needle |
InterView Fusion software | Mediso | Version 3.03 | Post-processing and image analysis software |
Isoflurane | Chanelle Pharma | Iso-Vet, inhalation anesthetic, 250 mL | |
Ketamine | Alfasan International B.V. | HK-37715 | Ketamine 10% injection solution, 10 mL |
Medical oxygen | Linde HKO | 101-HR | compressed gas, 99.5% purity |
Metacam | Boehringer Ingelheim | 5 mg/mL Meloxicam solution for injection for dogs and cats, 10 mL | |
nanoScan PET/MR Scanner | Mediso | 3 Tesla MR | |
Nucline nanoScan software | Mediso | Version 3.0 | Scanner operating software |
Wound clips | Reflex 7 | 203-100 | 7mm Stainless steel wound clips, 20 clips |
Xylazine | Alfasan International B.V. | HK-56179 | Xylazine 2% injection solution, 30 mL |
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