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 (Figure 1B-C). This co-registered imaging data is demonstrated as maximal intensity projection (MIP), where iWAT was clearly delineated to allow quantification of the [18F]FDG uptake. Consistently, the multilocular adipocytes, which are characteristic morphology for beige adipocytes, were more pronounced in iWAT from iBATx mice, compared to the sham operated group (Figure 1D).
To verify whether changes on iBAT and iWAT activities upon this prolonged cold induction can be monitored by micro-PET/MR imaging, imaging studies were performed on the mice exposed to 30 °C and 6 °C and the results between groups were compared. PET/MR imaging also demonstrated that mice subjected to 6 °C have markedly elevated [18F]FDG uptake on iBAT in sham operated mice (Figure 2A), which is consistent with the previous reported literature11. Mice with their iBAT removed (iBATx) prior to cold treatment showed the highest [18F]FDG uptake in the iWAT among the 30 °C and 6 °C group (Figure 2B). PET images were further quantified using an SUV-based approach. In iBAT, cold exposure caused a 7-fold increase in [18F]FDG uptake when compared to the 30 °C group. In iWAT, [18F]FDG uptake was higher in cold-acclimated iBATx mice than the remaining groups (Figure 2C). Removal of iBAT in the cold-induced mice resulted in an 8-fold increase in the uptake of iWAT compared to the thermoneutrality mice, whereas only a modest increase (2-fold) was observed when iBAT was present in mice.
Figure 1: Micro-PET/MR Imaging of inguinal white adipose tissue (iWAT) in mice. Interscapular brown adipose tissue was surgically removed (iBATx). After recovery, the mice were housed at 6 °C for 7 days before analysis. (A) Flow chart for the surgical and the subsequent procedures. (B) Illustration of the position of the mouse and the PET/MR scanner. (C) Maximal intensity projection (MIP) of co-registered PET/MR images. White arrows: Location of iWAT. A: Anterior L: Left. (D) Hematoxylin and Eosin (HE) staining of iWAT in sham and iBATx mice after cold exposure. Scale bar = 100 µm. Please click here to view a larger version of this figure.
Figure 2: Representative in vivo [18F]FDG uptake in brown adipose tissue at the interscapular region (iBAT) and inguinal subcutaneous white adipose tissue (iWAT). Mice housed at thermoneutrality (30 °C), cold-acclimated (6 °C) and cold-acclimated + iBATx were subjected to [18F]FDG PET/MR imaging. (A) Sagittal section of PET/MR images showing iBAT in mice. (B) Axial section of PET/MR images showing bilateral iWAT. (C) Quantitative analysis of [18F]FDG uptake in iBAT (left) and iWAT (right). Yellow arrows: Location of iBAT. White arrows: Location of iWAT. n = 3 for each group. Values of SUVratio are presented as mean ±SD. Please click here to view a larger version of this figure.
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 activated adipocytes as a result of high glucose utilization in an accurate manner. The experimental conditions outlined here highlights the feasibility of using [18F]FDG PET to study up-regulation of iBAT and iWAT in vivo, and is potentially useful for evaluation of the thermogenic impact of new drug candidates. In addition, this protocol can be easily modified into high throughput format by simultaneously imaging multiple mice using a specially designed animal bed, thereby increasing the statistical power and confidence in the imaging data at a reduced cost and time12,13.
Currently, [18F]FDG PET/CT remains the most common approach to visualize BAT in humans and rodents and standard protocols have been well established8,11. In recent years, there are also several studies using [18F]FDG PET/MR imaging to assess BAT in humans14,15,16. In contrast, no detailed description on [18F]FDG PET/MRI for small animals is available. Described here is a detailed protocol that relies on the use of a combined PET and MR imaging system in mice. This method takes advantage of the higher resolution of MRI, especially on the detection of fat tissues, making them easy to identify and segment compared to the commonly used CT method. Therefore, the current approach enables an improved accuracy of PET quantification compared to the PET/CT method, which is of great value for studies in small animals with more delicate adipose depots. When analyzing the results of tissues of interest at their baseline uptake, MRI becomes an essential tool to accurately draw the VOIs to ensure consistency of their volumes between mice and avoid inclusion of neighboring organs. In addition, accurate image processing such as image registration and VOI delineation are important to allow reliable quantification. The anatomical location of the glucose-responsive BAT is distinct between humans and mouse. While the functional BAT locates at the interscapular region, [18F]FDG PET/MR imaging-based analysis mainly identify functional BAT in the supraclavicular region in humans14,15,16.
The fasting or fed status of the mice should also be taken into consideration when performing the [18F]FDG uptake experiment. In some studies, the mice are fasted for several hours or even overnight before the uptake experiment since it is supposed that endogenous glucose will compete with [18F]FDG. In the protocol, the [18F]FDG at fed status was measured and strong uptake signal was still observed in both iBAT and iWAT. This, thus, demonstrates that it is not necessary to put the mice at a fasted status for robust uptake signals, which is less physiologically relevant. Actually, caution should be taken when examining BAT and beige adipocytes in fasted animals since a previous finding has reported that the hypothalamic neuropeptide Y (NPY)-mediated hunger signal acts on the medullary motor systems to inhibit BAT thermogenesis by reducing the sympathetic innervation17. Consistently, in humans, it is suggested that upon high calorigenic diets, the thermogenic adipocytes burn out extra calories so as to maintain energy balance. In contrast, upon nutrient deprivation, counter-regulatory mechanisms are activated to suppress energy waste.
Another consideration for [18F]FDG PET imaging involves the routes for radiotracer administration into mice. Intraperitoneal and intravenous techniques are two common ways to inject [18F]FDG into mice, and both methods result in a relatively similar biodistribution of [18F]FDG in mice 60 min post injection18. While the intraperitoneal method is relatively easy to perform and the injection can be done quickly to avoid unwanted stress imposed onto the mice, direct injection accidentally into the bowel is common and is not immediately identified, leading to unreliable PET results19. Intravenous method is the preferred method and employed in this study. Successful tail vein injection can be determined when a visible blood flashback is observed prior to infusion, indicating that the needle is properly positioned inside the vein for infusion. One limitation to this technique is the difficulty to notice a visible blood flashback, potentially due to low blood pressure and the presence of dark hair on the tails. This can be overcome by warming the tail with a warm washcloth to increase the blood flow, hence improving the visibility of the vein for needle insertion.
An accurate scanner and a relevant equipment are other important factors for reliable PET image quantification. It is imperative to perform routine quality control examinations on PET and MR components of the scanner. MR quality control involves the signal-to-noise ratio assessment on different T1- and T2-weighted sequences, which should be performed on a weekly basis as recommended by the scanner manufacturer. For PET, accuracy of activity must be determined using a syringe containing a known concentration of radioactivity on a weekly basis or before the start of an important study. This quality control test also allows determination of co-registration of PET and MR images. Calibration must be performed if the recovered activity falls outside the recommended range or mis-registration between PET and MR images is found. In addition, the dose calibrator should be regularly calibrated according to the manufacturer guidelines since this is an important tool for quality control of the scanner as well as radioactivity measurement for PET imaging.
This study shows that the activation of adipose depots in both iBAT and iWAT in mice can be visualized and quantified using [18F]FDG PET/MR imaging upon exposure to cold temperature. However, the current study is limited by the fact that [18F]FDG uptake in iWAT was relatively low unless in the absence of iBAT. This indicates that compared to the iBAT that is readily activated by cold stimulus, beige adipocytes are relatively reluctant to be mobilized and act more like a backup thermogenic depot of iBAT in mice. More efficient methods to induce the [18F]FDG signal in the iWAT and/or other adipose depots in normal mice, such as the use of beige-specific activators or stronger cold challenge condition, are to be identified, which is beyond the scope of the current study.
The authors have nothing 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.
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 |