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. Author manuscript; available in PMC: 2014 Jul 6.
Published in final edited form as: J Nucl Med. 2011 Sep 13;52(10):1616–1620. doi: 10.2967/jnumed.111.090175

Association of Heat Production with FDG Accumulation by Murine Brown Adipose Tissue (BAT) After Stress

Edward A Carter 2,4,5, Ali A Bonab 3,4,5, Kasie Paul 4, John Yerxa 1,4, Ronald G Tompkins 1,4,5, Alan J Fischman 4,5
PMCID: PMC4082963  NIHMSID: NIHMS596238  PMID: 21914754

Abstract

Previous studies have demonstrated that cold stress results in increased accumulation of 18FDG in brown adipose tissue (BAT). Although it has been assumed that this effect is associated with increased thermogenesis by BAT, direct measurements of this phenomenon have not been reported. In the current investigation we evaluated the relationship between stimulation of 18FDG accumulation in BAT by three stressors and heat production measured in vivo by thermal imaging. Male SKH-1 hairless mice were subjected to full-thickness thermal injury (30% total body surface area), cold stress (4°C for 24 hours), or cutaneous wounds. Groups of 6 animals with each treatment were fasted over night and injected with 18FDG. Sixty minutes after injection the mice were sacrificed and biodistribution was measured. Other groups of six animals subjected to the three stressors were studied by thermal imaging and the difference in temperature between BAT and adjacent tissue was recorded (ΔT). Additional groups of 6 animals were studied by both thermal imaging and 18FDG biodistribution in the same animals. Accumulation of 18FDG by BAT was significantly (p <0.0001) increased by all 3 treatments (burn ~5 fold, cold: ~15 fold, and cutaneous wound ~15 fold) whereas accumulation by adjacent white adipose tissue (WAT) was unchanged. Compared with sham control mice; ΔTs in animals exposed to all three stressors showed significant (p<0.001) increases in temperature between BAT and adjacent tissue. The difference in ΔT between stressor groups was not significant, however, there was a highly significant linear correlation (r2=0.835, p<0.0001) between the ΔT measured in BAT vs. adjacent tissue and 18FDG accumulation. These results establish, for the first time, that changes in BAT temperature determined in vivo by thermal imaging parallel increases in 18FDG accumulation.

INTRODUCTION

Brown adipose tissue (BAT) and white adipose tissue (WAT) are both present in mammals. The primary function of WAT is lipid storage whereas BAT is intimately involved in energy metabolism. BAT is especially abundant in newborns and hibernating mammals (1) where its primary function is to generate body heat by “non-shivering thermogenesis”. The mechanism for this process is believed to be related to uncoupling of substrate utilization and ATP production in mitochondria with resulting dissipation of metabolic energy as heat (2).

Until recently, the interpretations of 18FDG-PET studies were confounded by the presence of focal areas of increased tracer accumulation, in the supraclavicuar region, intercostal region, peri-adrenal region, axilla and around the great vessels which were erroneously ascribed to nodal disease. With the introduction of PET/CT, these findings were confirmed to represent focal areas of BAT. Accumulation of 18FDG by BAT has been shown to be particularly prominent in lean females during the cold months (3,4,5,6,7).

Cold stress has also been shown to activate 18FDG accumulation by BAT in rodents (8). BAT has also been considered to be important in insulin resistance which is a condition where there is inability of insulin to lower glucose levels (9). In previous reports, we studied the effects of cold stress, burn injury and cutaneous wounds on BAT at the macroscopic, microscopic and the metabolic levels (10,11,12) in mice. The findings of these studies indicated that all 3 stressors have significant effects on BAT at the structural and functional levels. In a subsequent study, we demonstrated that there is a temporal and size relationship between the stressors and stimulation of 18FDG accumulation by BAT (13). In the present investigation, we examined whether the stimulation of 18FDG accumulation by BAT is associated with increased heat production in vivo by thermal imaging.

Although it has been assumed that increased FDG accumulation in BAT is associated with increased thermogenesis, direct measurements of this phenomenon have not been reported. The findings of the current study establish, for the first time, that changes in BAT temperature determined in vivo by thermal imaging parallel increases in 18FDG accumulation.

MATERIALS and METHODS

MATERIALS

18F labeled FDG prepared by routine methods (14) was purchased from PetNet (Boston, MA).

ANIMAL PREPARATION

Male CD-1 or SKH-1 hairless mice (28–30 grams, Charles River, Wilmington MA) were use in these studies. After delivery, the animals were acclimatizing to the MGH animal facility for at least five days in a room where the temperature was maintained at 20 ± 1°C with a 12–12 light-dark cycle. After acclimatizing, the animals were treated as described in the following sections. A time-line for the basic procedures is shown in Figure 1.

Figure 1. Time-line for the studies.

Figure 1

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Stress indicates cold stress, burn injury or cutaneous wound. Temperature measurements by thermal imaging were performed at 55 minutes after 18FDG injection and were followed by biodistribution measurements or μPET. Six animals were studied with each treatment.

Effect of Shaving

CD-1 mice were anesthetized with ether and their dorsum shaven. The mice were allowed to recover for 24 hrs in mesh bottom cages without food but with water ab libitum prior to thermal imaging.

Burn Injury

Full-thickness, non-lethal thermal injury was produced as described previously (15). Briefly, under ether anesthesia, the mice were placed in molds exposing 30% of total body surface area (TBSA) on the lower dorsum to water at 90°C for 9 sec. The animals were immediately resuscitated with saline (15 ml/kg) by intraperitoneal injection. Sham control animals were treated similarly with the exception that the water was at room temperature. After the procedure, the animals were caged individually in mesh bottom cages and water was provided ab libitum. The mice were fasted overnight at room temperature prior to radiopharmaceutical administration.

Cold Stress

To produce cold stress, the mice were placed in a cold room at 4°C for 24 hours with overnight fasting. The mice were housed three to a cage in mesh bottom cages and radiopharmaceutical was administered on the following morning.

Cutaneous Wounds

For this procedure, the mice were anesthetized and a 1 cm2 section of skin was removed to the level of the fascia to produce a full thickness wound. After the procedure, the mice were housed individually in wire mesh bottom cages and fasted over night at room temperature prior to radiopharmaceutical administration.

Animal Care Approval

Animal care was provided in accordance with the procedures outlined by the National Institutes of Health. Guide for Care and Use of Laboratory Animals (Department of Health and Human Services Publication 85-23), Bethesda, MD, National Institutes of Health, 1996). The study was approved by the Subcommittee on Research Animal Care of the Massachusetts General Hospital. All animals survived the procedures, consumed water and moved freely in their cages without apparent distress.

Biodistribution studies

The fasted animals were injected (without anesthesia) via tail vein with 18FDG (50.0 µCi). One hour after tracer administration, the animals were sacrificed, selected tissues were excised, weighed and biodistribution was measured. Tissue radioactivity was measured with a Wizard Gamma counter. Radioactivity in aliquots of the injected doses was measured in parallel with the tissue samples to correct for radioactive decay. All results were expressed as % injected dose per gram of tissue (%ID/g, mean ± SEM).

Thermal Imaging

Mice (unanesthetized or anesthetized) were imaged with a thermal imaging camera (CTI-TIP, 1719 W. 2800 South Ogden, UT 84401, sensitivity 0.001°C). The pixel size was 0.3 × 0.3 mm. The mice were placed on a metal grid in a plastic box 30 cm below the camera. The tail of each mouse was held gently to partially immobilize the animal for the 5 seconds needed to record the temperature of the dorsum. The temperature of the BAT and adjacent tissue was determined by placing the cursor on the area to be tested and manually recording the temperature displayed by the computer. Since the temperature was quite uniform over the interscapular region and the color scale represents 1°C increments in temperature, the BAT temperature was assigned to the value at the central pixel. The outer boundary of the region was defined as an area with 1° C lower temperature. The areas tested were confirmed as brown or white adipose tissue at necropsy. The difference in temperature between BAT and adjacent tissue was recorded (ΔT). The results were expressed as ΔT (mean±sem).

Thermal imaging and 18FDG biodistribution in the same animals

These studies were performed in additional groups of six animals exposed to the same stressors as described above. For these studies, the mice were injected with FDG as described above and ΔT was determined immediately prior to sacrifice and measurement of biodistribution. The %DPG of 18FDG vs. % ΔT was plotted for each animal and linear regression analysis was performed.

μPET studies

To further evaluate glucose metabolism in major organs, 18FDG μPET was conducted in a sub-set of the animals described above. Imaging was performed with a Concord P4 μPET device. One hour after intravenous injection of 18FDG (~0.5 µCi) the mice were anesthetized, stabilized in the gantry of the camera and a 10 min. image was acquired in list mode. The primary imaging characteristics of the P4 camera are average intrinsic spatial resolution of ~2 mm FWHM, 63 contiguous slices of 1.21 mm separation and a sensitivity of ~650 cps/µCi. The data were reconstructed using an iterative algorithm, maximum a posteriori (MAP) in a 256×256 matrix with zoom 4. Data for attenuation correction was measured with a rotating point source containing 57Co. All projection data were corrected for non-uniformity of detector response, dead time, random coincidences, and scattered radiation. The PET camera was cross-calibrated to a well scintillation counter by comparing the camera response from a uniform distribution of an 18F solution in a 5.0 cm diameter cylindrical phantom with the response of the well counter to an aliquot of the same solution.

Statistical Analysis

Statistical analysis was performed by one-way analysis of variance (ANOVA) or linear regression. Individual means were compared by Duncan’s multiple range test Results with p-values of less than 0.05 were considered to be statistically significant.

RESULTS

Thermal Imaging of SKH-1 Mice

Figure 2 illustrates typical thermal images an unanesthetized hairless mouse with burn injury and a sham treated control. Higher temperatures are in the purple color range and the lower temperatures are in green. Mice with burn injury consistently exhibited an area on the upper dorsum with higher Δ temperature compared with sham controls. The area with the highest recorded Δ temperature was in the interscapular region where BAT is most prominent, as confirmed by surgical examination. The area adjacent to the BAT showed a lower temperature, and was surgically confirmed to be WAT.

Figure 2. Thermal images of a sham unanthesetized fasted SKH-1 hairless mouse with burn injury and a sham treated control.

Figure 2

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The mice were all imaged at the same time after injury. [Temperature Scale: ~30°C–39°C (Green-Purple)]. The arrow indicates the region of interscapular BAT in the burned mouse.

Thermal images of shaven CD-1 mice demonstrated diffusely increased Δ temperature in the dorsum (data not shown); most probably due irritation produced by shaving. Thus, SKH-1 mice were used in the comparative stressor studies.

Effect of Cold stress, Burn Injury and Cutaneous Wound on 18FDG Accumulation by BAT in SKH-1 Hairless Mice

As can be seen in Figure 3, application of cold stress, burn injury and cutaneous wound 24 hrs previously resulted in increased 18FDG accumulation by BAT. Analysis of variance showed a significant main effect of treatment, F3,23 = 149.08; p<0.0001. All 3 stressors produced significant increases in BAT accumulation of 18FDG compared with sham control animals; cold and cutaneous wound (p<0.0001), burn injury (p<0.001). The increases in 18FDG associated with cold and cutaneous wounds were significantly greater than the increase associated with burn injury (p<0.0001). Overall, cold and cutaneous wounds produced approximately 15-fold increases in 18FDG compared with sham control animals, whereas burn injury produced an approximately 5 fold increase.

Figure 3. Effect of cold stress, cutaneous wound and burn injury on 18FDG accumulation by BAT in hairless Mice.

Figure 3

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The mice were treated as described in methods. There were six mice in each group. Values are expressed as % Injected Dose per gram tissue, mean ± SEM. *p< 0.0001 vs. sham treated control mice, **p<0.001 vs. sham treated control mice, ***p<0.0001 vs. mice with burn injury.

μPET studies

Representative 18FDG μPET images (sagittal slices) of a sham control hairless mouse and a hairless mouse subjected to cold-stress are illustrated in Figure 4. 18FDG μPET imaging demonstrated intense focal uptake at sites of BAT after cold stress. Uptake in BAT was so intense that it was associated with significant reductions in uptake by all other tissues, including brain.

Figure 4. Representative 18FDG-μPET images of sham treated and cold stressed mice.

Figure 4

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Uptake in BAT was so intense that it was associated with significant reductions in uptake by all other tissues, including brain.

Thermal Imaging of the Effect of Cold stress, Burn Injury, or Cutaneous Wound on Heat Production by BAT In Vivo

As illustrated in Figure 5, application of cold stress, burn injury and cutaneous wound 24 hrs previously resulted in increased BAT temperature. Analysis of variance showed a significant main effect of treatment, F3,23=8.71; p<0.001. All 3 stressors produced significant increases in the temperature of BAT compared with sham control animals; cutaneous wound (p<0.0001), cold and burn injury (p<0.01). However, the effects of the treatments were not significantly different. As illustrated in Figure 6, regression analysis demonstrated a highly significant linear relationship between 18FDG accumulation by BAT determined by biodistribution measurements and ΔT determined by thermal imaging (r2=0.835, p<0.0001).

Figure 5. Effect of cold stress, cutaneous wound and burn injury on Δ temperature of BAT.

Figure 5

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The mice were treated as described in methods. There were six mice in each group. The values are expressed as difference in temperature between BAT and adjacent area (Δ temperature, mean ± SEM). *p<0.01 vs. sham treated control mice, **p< 0.001 vs. sham treated control mice.

Figure 6. Linear regression Analysis of Temperature and 18FDG Accumulation by BAT of Hairless Mice.

Figure 6

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The mice were treated as described in methods. The values are expressed as difference in temperature between BAT and adjacent area vs. the %FDG accumulation /gram tissue. There was a significant linear correlation (r2=0.835, p<0.0001) between the ΔT measured in BAT vs. the adjacent tissue in vivo and 18FDG accumulation determined by biodistribution measurements.

DISCUSSION

Although non-shivering thermo genesis is usually considered to be important in hibernating animals and to some extent in children, recent 18FDGPET studies have demonstrated significant BAT activity in adults, particularly during cold months (3, 4, 6, 7, 8). In this report, we studied the relationship between 18FDG accumulation by BAT determined by biodistribution and the associated temperature changes in BAT compared to adjacent tissue as determined in vivo by thermal imaging.

In the present study, we used SKH-H1 hairless mice to determine if the three stresses had an effect on BAT temperature compared to adjacent tissues in vivo by thermal imaging. The use of hairless mice in skin research has been reviewed recently (16). We chose to use the hairless mouse for our studies because it eliminates variability in the removal of hair over the BAT area. Mice with hair could have been treated with a chemical hair remover after shaving to produce a “hairless mouse”. However, we have found that this treatment increases mortality after burn injury since histological examination of tissue from these animals shows signs of injury (this laboratory, unpublished observations). It should be pointed out that studies using thermal imaging cameras rely on measurement of the temperature of the area in question and a reference point; yielding a Δ in temperature between the two areas. For our studies we used the point where BAT is expected to be present in large amounts (interscapular region) and the adjacent area.

We first confirmed that 18FDG accumulation by BAT was activated by cold, burn injury, or cutaneous wound. We then used thermal imaging to noninvasively measure brown fat temperature compared to adjacent tissue. Finally we determined if there was a relationship between the Δ change in BAT temperature compared adjacent tissue and 18FDG accumulation by BAT after activation by cold, burn injury, or cutaneous wound.

One of the primary functions of BAT is to produce heat (17). The law of constant heat summation formulated by Hess states that the decrease in enthalpy of a reaction sequence (the heat evolved at constant pressure) depends only on the initial reactants and final products of the sequence and is independent of the intervening reaction steps. Consequently the heat evolved by the oxidation of a substrate such as glucose by BAT will be the same for a direct chemical combustion as when a brown adipocyte catalyzes the same overall reaction through a cascade of some 30 enzymatic steps (15).

Nagashima (18) and Inokuma (19) have demonstrated increased heat production by BAT under the influence of norepinephrine. These studies involved insertion of a temperature probe directly into the BAT. The disadvantage of this approach is that the animal must be anesthetized and it has been demonstrated that anesthesia, especially with volatile anesthetics can alter BAT function (20). In addition, there will be some surgical manipulation of the area. Furthermore, insertion of the temperature probe may introduce a sampling error unless the area is surgically manipulated to document the exact location of BAT vs. WAT. An alternative approach would be to insert devices to monitor BAT temperature continuously. However, this procedure also requires surgical manipulations that could introduce sampling errors.

In summary, our results indicate that activation of 18FDG accumulation by BAT after cold stress, burn injury and cutaneous wound, is correlated with an increase in BAT temperature compared to adjacent tissue as measured by thermal imaging. Clearly, thermal imaging of hairless mice represents an attractive experimental model for screening drugs for their effects on BAT activation as treatments for conditions such as Diabetes Mellitus.

Acknowledgments

Supported in part by grants from the National Institutes of Health (2P50 GM 021700-27A) and Shriners Hospitals for Children (grant # 8470).

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