This study was funded by the Croatian Science Foundation research grant (IP-2018-01- 7416).

All experimental procedures on laboratory animals were approved by the National Ethical Committee and the Ministry of Agriculture (EP 185/2018). The experiments were conducted in accordance with the Ethical Codex of The Croatian Society for Laboratory Animal Science and ARRIVE guidelines. All procedures performed in studies involving human participants were in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the University of Zagreb, School of Medicine (UP/I-322-01/18-01/56). In this st...

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T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leptin+and+insulin+act+on+POMC+neurons+to+promote+the+browning+of+white+fat.">Leptin and insulin act on POMC neurons to promote the browning of white fat.</a> Cell. 160 (1-2), 88-104 (2015).</li><li>Habek, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+brown+adipose+tissue+in+diet-induced+thermogenesis+is+GC-C+dependent.">Activation of brown adipose tissue in diet-induced thermogenesis is GC-C dependent.</a> Pfl&#252;gers Archiv: European Journal of Physiology. 472 (3), 405-417 (2020).</li><li>Burke, L. 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I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brown+adipose+tissue+regulates+glucose+homeostasis+and+insulin+sensitivity.">Brown adipose tissue regulates glucose homeostasis and insulin sensitivity.</a> Journal of Clinical Investigation. 123 (1), 215-223 (2013).</li><li>Brasil, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+review+on+the+role+of+infrared+thermography+in+the+brown+adipose+tissue+assessment.">A systematic review on the role of infrared thermography in the brown adipose tissue assessment.</a> Reviews in Endocrine and Metabolic Disorders. 21 (1), 37-44 (2020).</li><li>Byers, S. L., Wiles, M. V., Dunn, S. L., Taft, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+estrous+cycle+identification+tool+and+images.">Mouse estrous cycle identification tool and images.</a> PLoS One. 7 (4), 35538 (2012).</li><li>Crane, J. D., Mottillo, E. P., Farncombe, T. H., Morrison, K. M., Steinberg, G. 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The authors have nothing to disclose.

Recent studies present growing evidence regarding the physiological regulation and importance of BAT activity in adult humans and animals in the development of obesity and diabetes mellitus. Furthermore, possible BAT activation by exogenous activators is becoming a target for pharmaceutical companies. To be able to estimate the physiological regulation and pathophysiological importance of BAT in very burdensome diseases, as well as discover a potential therapeutic approach, infrared thermography is becoming the method of...

Brown adipose tissue (BAT), in contrast to white adipose tissue (WAT), does not store but rather spends energy. Upon sympathetic stimulation, BAT utilizes fatty acids and glucose and produces heat by the activation of uncoupling protein 1 (UCP1). The function of UCP1 is to use an H+ gradient between two mitochondrial membranes to produce heat instead of ATP. The function of BAT is to increase heat production under cold conditions, which leads to an increase in energy expenditure1. After cold exposure, sensory inputs from the skin inhibit warm-sensitive neurons in the median preoptic (MnPO) nucleus of the hypothalamic preoptic area (POA), which diminishes the inhibitory effect of POA neurons on the rostral raphe pallidus (rRPa). The activation of rRPa neurons increases sympathetic activity, which is followed by an increase in BAT activity2,3. Cold-induced BAT activation improves insulin sensitivity in humans4, and this activity is decreased in humans with increased body mass index (BMI) and age1,5,6,7.
Apart from its role in cold-induced thermogenesis, after a meal, glucose uptake in the BAT increases in the lean male population, contributing to diet-induced thermogenesis (DIT), which is higher in BAT-positive male subjects8,9. The state-of-the-art technique used for measuring BAT activity is positron emission tomography computed tomography, known as PET-CT. This method determines BAT activity by measuring the accumulation of the radiotracer fluorodeoxyglucose (18F-FDG). However, PET-CT fails as the method of choice for detecting the activation of BAT after a meal. One of the reasons is that, after a meal, 18F-FDG competes with postprandial hyperglycemia for the same glucose transporter, which makes it unsuitable for determining BAT activation after a meal, especially when comparing BAT activity in healthy and diabetic participants with possible differences in blood glucose concentrations. Furthermore, BAT uses fatty acids as a source of energy for heat production which is not visible with PET-CT. 18F-FDG accumulation in BAT after a meal is barely visible10 and is, therefore, interpreted as a negative result in most cases. Unsurprisingly, recently, it was suggested that the activation of BAT is more pronounced in the human population than we had previously thought; therefore, a new approach to detect BAT activity and its involvement in metabolic disorders is necessary7. An attempt to solve this problem is to measure the volume of BAT with magnetic resonance imaging (MRI) in prediabetic patients and patients with diabetes mellitus type 2 (T2DM) with insulin resistance11. However, BAT volume measured by MRI is not a sufficient indicator for estimating the everyday function and usage of glucose and fatty acids by BAT. Therefore, to estimate real differences in BAT activity in healthy versus T2DM patients, a new approach is needed that offers a possibility to find out the pathological mechanism of BAT malfunction in T2DM patients.
To determine the activation of BAT, we performed measurements of BAT heat production before and after a meal using infrared (IR) thermography (Figure 1)12,13. Establishing IR thermography as a method of choice for measuring BAT activity after a meal in healthy and obese individuals or patients with diabetes mellitus will have a huge impact on the field. To this day, IR thermography is used for the determination of cold-induced activation of BAT13,14,15. In recent human history, cold-induced BAT activity is not very pronounced anymore (due to proper heating of habitats, proper clothing), while BAT activation after a meal occurs every day. Furthermore, the physiological regulation of these two BAT functions via the hypothalamus is completely different. After a meal, the activation of proopiomelanocortin (POMC)-expressing neurons in the hypothalamic arcuate nucleus (Arc) leads to an increase in sympathetic nerve activity via rRPa16. Cold-induced activation of BAT measured by IR thermography or PET-CT is improper when used as a measure for everyday BAT activity. Increased BAT activity after a meal is followed by glucose utilization, which is ultimately important for maintaining glucose homeostasis, insulin sensitivity, and the daily regulation of glucose concentration. Postprandial BAT activation leads to an increase in postprandial glucose consumption, followed by an increase in heat production and body temperature (DIT). This was shown to be gender, age, and BMI dependent12. Similar gender differences in BAT activation after a meal are observed in male and female laboratory mice17. These findings correspond to recently discovered gender differences in the regulation of BAT by Burke et al., who showed that the hypothalamic regulation of BAT browning via a subpopulation of POMC neurons differs in male and female mice18. The postprandial activation of BAT is smaller in women, older populations, and obese people. The lack of BAT activation after a meal (decreased glucose utilization) could lead to a higher prevalence of impaired glucose tolerance in women19,20,21,22. Unfortunately, the majority of studies on BAT activation were done only on men. By activating BAT after a meal, glucose uptake increases in the lean male population. It is not surprising that, after BAT activation, DIT is higher in BAT-positive male subjects8,9. Furthermore, BAT transplantation in male mice improves glucose tolerance, increases insulin sensitivity, and decreases body weight and fat mass23.
PET-CT fails as a method of choice for measuring BAT activity, especially after a meal. Therefore, a non-invasive and more sensitive method was developed. IR thermography enables the estimation of BAT activity in different laboratory animals (knock-out mice), as well as human participants, regardless of gender, age, or the effects of different pathological conditions on BAT activity. An additional benefit of this method is the simplicity for participants and laboratory animals, which allows us to estimate the potential benefits of BAT booster therapy. The recent studies using IR thermography for determining the physiological behavior of BAT after cold exposure or a meal are described in the recent publication of Brasil et al.24.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.1% cresyl violet acetate&#160;</td>
			<td>Commonly used chemical</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Device for measuring air temperature and humidity</td>
			<td>Kesterl</td>
			<td>Kestrel 4200</td>
			<td>Certificat of conformity</td>
		</tr>
		<tr>
			<td>External data storage</td>
			<td>Hard Drive with at least 1 TB</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass microscopic slides</td>
			<td>Commonly used</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small cotton tip swab&#160;</td>
			<td>Urethral swabs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Software for analysis</td>
			<td>FLIR Systems, Wilsonville, OR, USA</td>
			<td>FLIR Tools</td>
			<td></td>
		</tr>
		<tr>
			<td>Software for meassurements</td>
			<td>FLIR Systems, Wilsonville, OR, USA</td>
			<td>ResearchIR software</td>
			<td>FLIR ResearchIR Max, version 4.40.12.38 (64-bit)</td>
		</tr>
		<tr>
			<td>Thermac Camera</td>
			<td>FLIR Systems, Wilsonville, OR, USA</td>
			<td>FLIR T-1020</td>
			<td></td>
		</tr>
	</tbody>
</table>

infrared thermography for the detection of changes in brown adipose tissue activity

Measuring brown adipose tissue (BAT) activity by positron emission tomography computed tomography (PET-CT) via the accumulation of 18F-fluorodeoxyglucose (FDG) after a meal or in obese or diabetic patients fails as the method of choice. The main reason is that 18F-FDG competes with the postprandial high glucose plasma concentration for the same glucose transporter on the membrane of BAT cells. In addition, BAT uses fatty acids as a source of energy as well, which is not visible with PET-CT and could be changed along with glucose concentration in obese and diabetic patients. Therefore, to estimate the physiological importance of BAT in animals and humans, a new infrared thermography method used in recent publications is applied.
After overnight fasting, BAT activity was measured by infrared thermography before and after a meal in human volunteers and&#160;female wild-type mice. The camera software calculates the object&#39;s temperature using distance from the object, skin emissivity, reflected room temperature, air temperature, and relative humidity. In mice, the shaved area above the BAT was a region of interest for which average and maximal temperatures were measured. The phase of the estrous cycle in female mice was determined after an experiment by vaginal smears stained with cresyl violet (0.1%) stain solution. In healthy volunteers, two skin areas of the neck were selected: the supraclavicular area (above the collarbone, where BAT cells are present) and the interclavicular area (between the collarbones, where there is no BAT tissue detected). BAT activity is determined by the subtraction of those two values. Also, the average and maximal temperatures of skin areas could be determined in animals and human participants.
Changes in BAT activity after a meal measured by infrared thermography, a non-invasive and more sensitive method, were shown to be sex, age, and phase of the estrous cycle dependent in laboratory animals. As part of diet-induced thermogenesis, BAT activation in humans was also proven to be sex, age, and body mass index dependent. Further determining the pathophysiological changes in BAT activity after a meal will be of great importance for participants with high glucose plasma concentrations (obesity and diabetes mellitus type 2), as well as in different laboratory animals (knock-out mice). This method is also a variable tool for determining possible activating drugs that could rejuvenate BAT activity.

The easiest way to determine BAT activity is to subtract the maximal skin temperature above the BAT before and after a meal in human subjects. A better way to calculate BAT activity is to select two regions of interest: the skin area above the BAT, which is located in the supraclavicular area, and the interclavicular area of the skin where no BAT tissue is found in humans, designated as a reference area (according to PET-CT; Figure 1). BAT activity is then easily determined by the subtractio...

Watch this Scientific Journal Video about Infrared Thermography for the Detection of Changes in Brown Adipose Tissue Activity at JoVE.com

Infrared Thermography for the Detection of Changes in Brown Adipose Tissue Activity

Here, we present a protocol for measuring brown adipose tissue activity after a meal in humans and laboratory animals.

Measuring brown adipose tissue (BAT) activity by positron emission tomography computed tomography (PET-CT) via the accumulation of 18F-fluorodeoxyglucose ...

Infrared thermography enables the estimation of brown adipose tissue activity in laboratory animals and human participants, regardless of gender, age, or the effects of different pathological conditions. PET-CT fails as a method of choice for measuring brown adipose tissue activity, especially after a meal. Therefore, a noninvasive and more sensitive infrared thermography was developed.Using infrared thermography, we can determine that the pathophysiological changes in brown adipose tissue activity or bath activity in people with obesity and diabetes mellitus, and determine drug that can rejuvenate bad activity in these patients. This method could be used to determine the physiological effects of hormones or synthesize therapeutic substances on bad activity in humans and laboratory animals. Ask the participants if they are well-rested and fasted overnight.They will rest for at least 30 minutes before measurements to avoid possible brown adipose tissue activation. Perform the experiments in the morning hours. 15 minutes before the measurement, ask the participants to remove their upper clothing.While the participants are resting, mount the thermal camera on the tripod and position it one meter away from the spot where the participant will be seated. Connect the thermal camera to a computer and software as instructed by the manufacturer. Determine the reflected temperature of the room by recording wrinkled aluminum foil at a focal distance of one meter.To do so, in the camera software, input the distance of zero meters and emissivity of one. Then, enter values of atmospheric pressure and relative humidity. To determine reflected temperature, choose the square shape and select an ROI on the aluminum foil.Then, choose the first icon from the left in the main camera window and read the mean temperature, which is necessary for further analysis. Just before starting the measurements, determine the room air temperature and air humidity. In the software, above the main camera window, choose the third icon from the left.In the pop-up menu, choose Edit record settings, which opens a new window. In Record mode, select Record to disk and set the record for this duration at the desired time. In the Record Options of the same window, limit record rate to five hertz and choose the location where the recordings will be saved.Close the existing window, open Edit in the main menu, and select Preferences. Enter five in the target frame rate. Select Hotkey/Remote Start can stop record.And from the dropdown menu, select In Start/Stop Mode. At the time of measurement, ensure only the participant and the experimenter are present. Avoid air movement and ensure the participant is away from cold draft, sunlight, or any source of heat like light bulbs.Locate the supraclavicular area of the neck above the collarbone, where brown adipose tissue is located. Position the participant such that this area is at one meter focal distance. Record a short movie for 10 to 15 seconds at a frame rate of 5 FPS by pressing the F5 key.Ensure all the participants eat the same meal. After the meal, make a new recording at 30 minutes and then one, two, and three hours by pressing F5 using the same setting values. The day before the experiment, collect the animal's vaginal swab and spread the cells onto a glass slide.Allow it to air dry. Stain the cells with 500 microliters of 0.1%cresyl violet acetate for one minute, and then rinse them thrice with water. View the cells under a light microscope with 100 times magnification and brightfield illumination.Determine the phase of the estrous cycle based on the number of leukocytes and nucleated and quantified epithelial cells observed in the smear. On the morning of the experiment, prepare the thermal camera and recording settings. Carefully place the animal in a clean cage and place the cage under the thermal camera at a focal distance of one meter.Record a movie by pressing F5.Weigh the food pellet before giving it to each animal to calculate the food intake. Allow the animal to eat for 30 minutes in its cage and weigh the food pellet again after the meal. Repeat IR measurements 30 minutes, one hour, and two hours after starting a meal.After the experiment, test the phase of the estrous cycle in female animals, as shown earlier. For each movie, enter the variables into the camera software such as skin emissivity, reflected room temperature, air temperature, relative humidity, and distance to the object. Select the suitable frame from the movie by moving the playhead at the bottom of the screen or by pressing the Pause button.Select the region of interest by choosing the desired shape of the area on the left side of the main window. Choose the shape that best corresponds to the area of skin between the scapulas. Upon choosing the region of interest, the minimal, maximal, and average temperatures will be displayed on the right.In the image, the red triangle represents the point of maximum recorded temperature, and the blue triangle represents the minimum recorded temperature. Repeat this step for several frames to be sure that the measured temperature is stable during a few seconds of the recording. Subtract the maximal temperatures of the skin above the brown adipose tissue before a meal from the maximal temperatures after a meal to determine the increase in postprandial activity.Brown adipose tissue activity was determined by analyzing two skin areas:one above the brown adipose tissue and the second in the inter clavicular area as a reference point. The brown adipose tissue activity was calculated as 1.8 degrees Celsius from the differences between maximal temperatures in both skin areas. But the activity cannot be presented by maximal temperatures of the skin above the brown adipose tissue since there is no decrease in skin temperature even after three hours of the meal.In mice, the changes in activity after a meal were measured in diestrus females by measuring the average temperature of the skin above brown adipose tissue. Significant changes in activity were determined 30 minutes after a meal only when the maximal temperature was measured. Proper selection of matching control subjects is the hardest part of clinical studies since healthy controls and participants with pathological conditions should be as similar as possible and differ only in the disease investigated.In addition to this protocol, blood glucose concentration and body temperature could be measured, which allows the correlation analysis between bad activity and glucose utilization or increase in body temperature. This sensitive and non-invasive technique opens numerous possibilities in determining the physiological and pathophysiological changes of brown adipose tissue activity in other experimental settings and studies.

infrared-thermography-for-detection-changes-brown-adipose-tissue

Research

JoVE Journal

Medicine

1.7K Views.  MKP Ltd.. Infrared thermography enables the estimation of brown adipose tissue activity in laboratory animals and human participants, regardless of gender, age, or the effects of different pathological conditions. PET-CT fails as a method of choice for measuring brown adipose tissue activity, especially after a meal. Therefore, a noninvasive and more sensitive infrared thermography was developed.Using infrared thermography, we can determine that the pathophysiological changes in brown adipose ti...