We would like to thank all the study volunteers for their participation in our study. This work was supported by the Holsworth Research Initiative, La Trobe University, and the Defence Science Institute (DSI, Australia).

All participants (n =&#160;8) provided written informed consent, and all experiments were approved by the University Human Ethics Committee; data were derived from Van Schaik et al.27.
1. Equipment and software installation
<ol>
	<li>Measure the fat mass via dual-energy X-ray absorptiometry (DXA) as per Van Schaik et al.27.</li>
	<li>Estimate the substrate utilization and energy expenditure from expired gas; measure this using a respiratory gas analyzer as per the manufacturer&#39;s guidelines.</li>
	<li>Collect blood samples via finger (capillary) puncture, and determine the blood glucose levels using a glucometer as per the manufacturer&#39;s guidelines.</li>
	<li>Use a non-contact infrared thermometer to determine core body temperature measurements as per the manufacturer&#39;s guidelines (the error of this device is &#177;0.2 &#176;C).</li>
</ol>
2. Procedures prior to the participant visits
<ol>
	<li>Screen all the participants for their health status.</li>
	<li>Set the following exclusion criteria: a body mass index of &#62;30 kg/m2 (due to BAT activity being inversely correlated with adiposity34,35, participants using prescribed medications, and diabetes mellitus.</li>
	<li>Before or after the testing session, ensure that participants undergo a DXA scan to measure their fat mass, as BAT activity is inversely correlated with adiposity34,35.</li>
	<li>For 24 h prior to arriving for the study, ensure that the participants abstain from any strenuous exercise or activity and are water-fasted for 10 h before arriving in the lab.</li>
</ol>
3. Procedures on the study day
<ol>
	<li>Ensure that the room temperature at which the data are collected is set to a constant temperature to minimize external confounds due to differences in room temperature. 
	NOTE: This can result in incorrect thermal or metabolic measurements. For the purposes of this experiment, a temperature-controlled room maintained at 22 &#176;C under thermal neutral conditions was used.</li>
	<li>Ask the participants to arrive at the lab at 08:00 a.m. to account for daily hormone rhythms.</li>
	<li>Measure the participants&#39; height and weight.</li>
	<li>Ask the participants to lie on a plinth for a minimum of 30 min before the baseline measurements are taken.</li>
	<li>Over a 120 min period, measure the participants&#39; IRT, indirect calorimetry, blood glucose, and core temperature every 15 min following the expired O2and CO2sampling (Figure 1).</li>
	<li>Following the baseline measurements, ensure that the participants are carbohydrate-loaded through the consumption of three carbohydrate gels (90 g glucose each) between the time points of 0 min and 15 min.</li>
	<li>Ensure that the participants ingest the treatment 45 min following the carbohydrate load. To follow this protocol, use 100 mg of caffeine capsules as the intervention27. 
	NOTE: A washout period of 7 days between intervention and placebo is required, meaning a period of 7 days is required between caffeine and placebo treatment.</li>
</ol>
4. Indirect calorimetry
<ol>
	<li>Estimate the energy expenditure and substrate utilization values from the expired gas, as measured using a respiratory gas analyzer. Complete the calibration of the respiratory gas analyzer following the manufacturer&#39;s instructions.</li>
	<li>Fit the cold-sterilized silicone mask to the participant to allow delivery of room air and the acquisition of metabolic data. Ensure that the mask is equipped with a presterilized non-rebreathing valve (two-way non-rebreathing valve) and fix it on the participant&#39;s face with a mesh attachment and check for leaks.</li>
	<li>Ensure that the inspiratory and expiratory tubes are connected.</li>
	<li>Export the digital data file in a spreadsheet format.</li>
	<li>Sample the expired O2 and CO2with 5 s averaging. This measures the energy expenditure and respiratory exchange ratio (Figure 1). Remove the face mask to complete the additional measures.</li>
	<li>Calculate the substrate oxidation rates (carbohydrate and lipid oxidation) and total energy expenditure using the non-protein Weir equations 1-331,36: 
	Fat oxidation rate (g/min&#8722;1) = (1.695 VO2)-(1.701 VCO2)&#160; &#160;(1) 
	Carbohydrate oxidation rate (g/min&#8722;1) = (4.585 VCO2) -(3.226 VO2)&#160; &#160;(2) 
	Energy Expenditure (kcal/min) = (3.94 &#215; VO2)+ (1.1 &#215; VCO2)&#160; &#160;(3)</li>
</ol>
5.&#160;Plasma blood glucose measurements
<ol>
	<li>Conduct blood glucose readings via finger prick and a glucometer following each round of expired gas measurements (Figure 2).</li>
</ol>
6. Core temperature
<ol>
	<li>Record the core temperature (Tcore) following each round of expired gas measurements. Ideally, measure the core temperature either rectally or intra-aurally (Figure 2). 
	NOTE: Owing to COVID-19 safe practices, minimize person-to-person contact.</li>
	<li>Ensure that the participants are supine and their head is in a neutral position. Consistently direct the non-contact thermometer toward the center of the participant&#39;s forehead.</li>
</ol>
7. Infrared thermography
<ol>
	<li>Conduct the IRT following each round of expired gas measurements (Figure 2).</li>
	<li>Ask the participants to sit up in an upright posture looking straight ahead, with the chest area to neck region exposed (Figure 3).</li>
	<li>Use a thermal imaging camera to acquire infrared images of the anterior neck and upper chest region.
	<ol>
		<li>Position the camera on a tripod at the level of the neck 1 m from the subject&#39;s face (Figure 4D). Use the following settings: detector type = uncooled microbolometer; detector pitch = 17 &#181;m; camera spectral range = 7.5-14.0 &#181;m; thermal sensitivity = 20 mK at 30 &#176;C; lenses = 36 mm; resolution = 1,024 pixels x 768 pixels.</li>
		<li>Turn the camera on.</li>
		<li>Adjust the focus of the camera by rotating the focus ring. 
		NOTE: It is very important to adjust the focus correctly. Incorrect focus adjustment affects the temperature measurement.</li>
		<li>Point the laser pointer to the midline of the neck of the participant.</li>
		<li>Take the image. 
		NOTE: The image will save automatically if a memory card is used.</li>
	</ol>
	</li>
</ol>
8. Image analysis
<ol>
	<li>Choose three regions of the anterior thorax and neck for the analysis of the surface temperature: bilaterally the skin overlying BAT in the supraclavicular fossa (SCF) and the lateral region of the neck, with the sternal area considered as a control reference point (Tref), as this area does not contain BAT (Figure 4A-C).</li>
	<li>Place triangular regions of interest (ROIs) in the left and right SCF areas and a circular ROI over the sternal region.</li>
	<li>When the required regions have been cross-located, confirm that the software displays the average and standard deviation of the temperature for each selected region.</li>
</ol>
9. Data analysis
<ol>
	<li>Use a double-blind approach for the analysis of the interventions using the techniques described. Have a researcher not involved in the data collection or analysis code the interventions generically.</li>
	<li>Perform the statistical analysis.
	<ol>
		<li>Calculate averages for the IRT, core temperature, and blood glucose data from the measured single time point.</li>
		<li>Calculate averages for the RER, fat oxidation, carbohydrate oxidation, and energy expenditure in 10 min epochs.</li>
		<li>For energy expenditure, sum the rate of energy expenditure for each group, and separate it into pre- and post intervention. 
		NOTE: Refer to Van Shaik et al. for statistical tests to analyze the data27.</li>
	</ol>
	</li>
</ol>

The authors have no conflicts of interest to disclose.

The method we have shown here is a technically simple, safe, and cost-effective protocol for measuring BAT thermogenesis in humans. The protocol addresses concerns related to the reliability of using IRT on its own to distinguish between local warming due to altered skin blood flow and deeper warming due to thermogenesis by correlating IRT with both measures of energy expenditure (EE) and substrate utilization. Since this technique does not use ionizing radiation, it permits repeated-measures analysis, which is not possible with PET imaging techniques. Finally, while PET imaging techniques can identify BAT activation, they do not report on the physiological outcomes (increased temperature and EE) that this protocol measures.
The strength of the protocol described here is that there are four lines of evidence that support the conclusion of evoked BAT thermogenesis: (1) increased measured Tscf, in parallel with unchanged core temperature and stable skin temperature over the adjacent reference region; (2) increased energy expenditure; (3) a change in substrate utilization; and (4) a fall in blood glucose levels. The converging observations are all consistent with the predicted outcomes for BAT thermogenesis. The essential part of the protocol is the carbohydrate loading of the participants to ensure carbohydrate metabolism before intervention. BAT thermogenesis switches substrate metabolism from carbohydrates to free fatty acids, as shown by the fall in RER. While the preferred substrate for BAT thermogenesis is free fatty acids, a significant uptake of glucose into active BAT is well established5,6,7. Therefore, we observe a fall in blood glucose levels concurrent with BAT thermogenesis. It would not be possible to observe the mutual shift in substrate utilization (RER) and the fall in blood glucose levels in a fasted state.
Previous studies have concluded that increased Tscf (measured by IRT) is sufficient to conclude BAT thermogenesis. However, this conclusion is only certain if the Tscf exceeds the core temperature. If the Tscf is less than or equal to the core temperature, then a local change in temperature due to increased skin blood flow cannot be excluded. A systematic review concluded that IRT alone is unable to determine whether increases in supraclavicular skin temperature are due to BAT thermogenesis37. The review noted that the most common method (18F-FDG PET/CT) measures the uptake of glucose into BAT37. However, the preferred substrate for BAT thermogenesis is fatty acids13. This methodological issue prevents any meaningful comparison between PET/CT data in validating IRT data, as either of these measures alone are not a suitable measure of the true metabolic activity of the BAT as it cannot indicate the change in energy expenditure and substrate utilization due to BAT thermogenesis. Nevertheless, with the protocol described here, not only can we quantify the change in temperature, but we can also confirm an increase in energy expenditure-a key physiological outcome of BAT thermogenesis. IRT is a non-contact, non-invasive, and relatively inexpensive method for measuring temperature and temperature changes associated with BAT thermogenesis. In contrast, PET-CT is expensive and exposes individuals to ionizing radiation, thus restricting the applicability of this method to small retrospective analyses of clinical imaging studies. The application of the current protocol to large-scale, randomized clinical trials would be relatively simple and cost-effective.
It is important to note that the decrease in carbohydrate oxidation following caffeine intervention can be explained by the switch in substrate utilization as a result of increased BAT thermogenesis due to the intervention. Measures of insulin signaling would make the results of this study more robust. However, it is not clear based on the results of this study as to whether caffeine would affect insulin signaling via action on the BAT or whether the fall in blood glucose is a result of the BAT taking up more energy substrates.
The 18F-FDG PET/CT method has several inherent limitations when it is used to quantify and measure the physiological activity of BAT, particularly when investigating the influence of nutrients or dietary ingredients on BAT activity. The 18F-FDG PET/CT method requires subjects to be fasted to avoid feeding-induced increases in glucose uptake by the muscular tissue, which can significantly reduce the detection of both the BAT and BAT function38. Furthermore, this technique alone cannot measure the physiological impact or extent of BAT activation. Additionally, the use of ionizing radiation in PET imaging studies is an ethical and health and safety hurdle for designing repeated-measures cross-over studies. In addition, 18F-FDG represents glucose uptake only, which is not the same as measuring glucose metabolism. This method of carbohydrate loading subjects prior to measuring the BAT temperature and combining blood glucose levels with indirect calorimetry allows us to rigorously measure the physiological impact of thermogenesis and changed substrate utilization, which would otherwise not be available in a fasted state.
Strengths and limitations 
This protocol has wider implications than purely studying BAT. By carbohydrate-loading participants prior to intervention, the oscillation of blood glucose levels in response to both carbohydrate loading and the caffeine intervention, as well as changes in substrate utilization, can be observed. Therefore, this technique can be used to improve human indirect calorimetry studies and metabolic measures. It is not yet known whether the results from this study can be replicated following other interventions, such as cold exposure or adrenergic stimulation. However, the results of this study have been replicated following intervention with a different dietary ingredient, namely Capsicum annuum27. Additional rigor and confidence in the results could be obtained using a double-blind approach for the analysis of interventions using the techniques described, and this could be easily implemented27.
The potential confound of varied room temperature is not relevant in this protocol, as the room temperature was kept stable from participant to participant. Additionally, the humidity was taken into account during the calibration of the respiratory gas analyzer. This is inferred in the setup of this piece of equipment, as calibration is completed as per the manufacturer&#39;s instruction.
The time intervals for the measurement and treatment were determined following a small pilot study in which troubleshooting of the protocol was conducted. Essentially, the time intervals for measurement were determined based on the time needed for the researcher to perform the measurements and for the participant&#39;s comfort. The time for the intervention was determined based on the time taken for carbohydrate metabolism to occur following the carbohydrate load to investigate whether the intervention increased free fatty acid oxidation (i.e., BAT thermogenesis) and lowered carbohydrate oxidation.
Notably, there are differences between capillary and venous glucose levels39. However, in the context of out-of-hospital care, the most common way in which blood glucose levels are measured is via a blood sample of capillary origin analyzed by a hand-held, point-of-care glucometer40. Additionally, in healthy individuals (similar to those included in this protocol) in a non-clinical setting, there is a statistically significant, but not clinically significant, difference between capillary and venous blood glucose levels when measured using a point-of-care, capillary-based glucometer41. In this context, capillary sampling would remain the optimal approach due to the fact that most point-of-care glucometers available on the market are engineered to analyze capillary blood samples41. From a clinical perspective, it might be argued that venous blood glucose is the superior method of analysis. However, venous blood sampling is not only expensive and requires specialized equipment (ibid), but it is also invasive. The ethical considerations of increasing the risk of adverse events during the protocol need to be balanced against the reported literature showing the high correlation and reliability of capillary blood glucose as a proxy measure of venous blood glucose42. The key here, of course, is that we have not set out to diagnose diabetes but to measure changes in blood glucose levels, for which capillary blood glucose monitoring is a more than suitable protocol.
Glucose can induce thermogenesis, and single meals can activate the BAT43. However, and rather importantly, the data included in this manuscript show no significant effect of glucose loading in the intervention group or the placebo group. Furthermore, the data included in the manuscript were derived from the results of Van Schaik et al., which included a third intervention (Capsicum annuum), and the glucose load did not produce a significant effect on the measures27.
It should be noted that this protocol has only been used in male participants with low body fat and active BAT (to reduce the number of controllable variables, females were excluded from the study). There is a known inverse correlation between adiposity and BAT mass in humans44. In addition, it is known that previously obese people who have lost weight through diet and exercise have a lower basal metabolic rate and must consume lower-calorie diets to maintain a normal weight45,46. Furthermore, BAT activity can stimulate BAT growth8. The method described here will allow for long-term studies to investigate changes in BAT activity associated with metabolic diseases in a way not afforded by other techniques.
Conclusion 
In conclusion, we demonstrate a measurement approach to quantify human brown adipose tissue activity using IRT and indirect calorimetry following a carbohydrate load. The critical steps include 1) carbohydrate loading the participants that are in a fasted state prior to measuring the BAT temperature whilst combining indirect calorimetry and blood glucose levels to allow the quantification of the physiological extent of BAT thermogenesis and altered substrate utilization; 2) assessing relevant IRT BAT depots and temperatures from a reference point and core temperature to demonstrate any increase in Tscf that would be indicative of BAT activation based on the anatomical location. We believe that these quantitative measurements allow for a more accurate evaluation of the contribution of BAT to adult human energy metabolism and thermoregulation. This thorough approach should be used by researchers to study BAT physiology and serve as a new standard for developing human BAT activation approaches in the future.

Brown adipose tissue (BAT) most notably differs from white adipose tissue (WAT) in its mitochondrial content, sympathetic innervation, multilocular lipid droplets, heat-generating ability, and anatomical distribution. BAT was considered to exist only in infants and small mammals until the confirmation of its presence in human adults in 20091,2,3. Thus, until relatively recently, the role of BAT in human physiology and metabolic homeostasis has been poorly understood. Extensive studies in small animals have shown that during cold exposure, more than half of metabolism is due to the non-shivering thermogenic ability of BAT4. Several studies have demonstrated that upon mild cold exposure (17-18 &#176;C), increases in energy expenditure and glucose uptake into the BAT correlate strongly with BAT thermogenesis in humans5,6,7. Furthermore, BAT thermogenesis can contribute up to 10% of resting energy expenditure in humans during cold exposure (for a review, see Van Schaik et al.8). Studying the physiology and impact of BAT on human health and disease is currently restricted by protocol limitations. It is, therefore, essential to have an accurate method for measuring the true metabolic impact of BAT to better understand the impact of BAT thermogenesis on obesity and its metabolic complications in humans.
The anatomical distribution of human BAT makes obtaining accurate measurements of the BAT challenging. Within humans, the BAT is distributed inside the depots of WAT in the abdomen, thorax, and, most notably, the neck9. Autopsy and cadaveric studies have been used to characterize the BAT anatomically10,11, but these methods cannot provide functional information. It is challenging to distinguish the BAT using conventional imaging techniques because of the similar densities of WAT and BAT8. An additional confounding issue is that beige fat depots are also located within the same narrow layers of fascia or in certain depots with the WAT8, which makes it challenging to distinguish using conventional imaging techniques.
To overcome this issue, the BAT volume is typically measured through combining positron emission tomography (PET) and computed tomography (CT). The radiolabeled glucose analog 18F-fluourodeoxyglucose (18F-FDG) is the most common tracer used for studying BAT12. However, it suffers several limitations, such as exposing subjects to ionizing radiation and being invasive and expensive. Additionally, the greatest limitation of the 18F-FDG tracer is that it measures the uptake of a glucose analog, which is not ideal given that free fatty acids are the preferred substrates for BAT thermogenesis13. The 18F-FDG PET/CT technique does not measure the uptake of free fatty acids as a substrate for thermogenesis and, therefore, does not measure the physiological importance of BAT thermogenesis. There are alternative techniques used to assess human BAT, which include the measurement of the uptake of oxygen-15 labeled water (15O-O2) 14, 11C-acetate15, a long chain fatty acid (18F-fluoro-6-thia-heptadecanoic acid)16, or adenosine17, as well as magnetic resonance spectroscopy18&#160;and magnetic resonance imaging 19, but these are still extremely expensive and expose subjects to ionizing radiation. Therefore, a reliable, inexpensive, and importantly, safe gold standard for the quantification of human BAT is lacking.
Infrared thermography (IRT) is an alternative non-invasive imaging technique20,21&#160;that measures the skin temperature overlaying a known BAT depot. While this infers increased energy expenditure, if the measured temperature does not exceed the core temperature, then it cannot be determined whether the measured change in temperature is simply a consequence of altered blood flow. Further, a measured increase in local temperature does not provide values of altered energy expenditure, which is frequently the desired endpoint. A number of research groups have used IRT to measure an increase in temperature in depots of human BAT following a caffeine intervention or cold stimulus; this depot is the supraclavicular fossa22,23,24,25,26,27.
However, it is not clear whether the action of caffeine on BAT is direct or mediated via neural circuitry. There is evidence that caffeine induces browning features in adipocytes in vitro22, and previous work has demonstrated that caffeine (100 mg) increases heart rate variability, which may be an indicator of an increase in sympathetic nerve drive systemically in the body27. This is in line with evidence in rodents, in which caffeine via the central nervous system increases thermogenesis without an adverse cardio-dynamic impact28.
As the preferred substrate for BAT thermogenesis is free fatty acids derived from triglycerides13, and active BAT sequesters circulating lipids to sustain thermogenesis29, measures of substrate utilization are important in assessing the physiological activation of BAT. The respiratory exchange ratio (RER) is the ratio of the volume of oxygen consumed (V&#775;O2) and carbon dioxide produced (V&#775;CO2)30. An RER of 0.7 is indicative of fatty acid metabolism, and an RER of 1.0 is indicative of carbohydrate metabolism31. Therefore, evidence of a preference for fatty acid utilization over and above an increase in energy expenditure is a key correlate of BAT thermogenesis.
Additionally, given that the uptake of glucose is a known correlate of BAT activity (see above), a fall in blood glucose in parallel with the change in substrate utilization are key correlates of BAT thermogenesis. Previous studies utilizing indirect calorimetry alone, or together with temperature recording in fasted individuals, have reported little to no acute change in substrate utilization32,33. As this is likely masked by the fasted state (where preabsorptive metabolism favors fat utilization), we propose combining IRT and indirect calorimetry with carbohydrate loading.
This article aims to provide a step-by-step approach that clinical researchers can use to reliably and, importantly, safely quantify the physiological importance of BAT in humans by combining IRT, indirect calorimetry, and blood glucose levels. This technique is best used after subjects have been carbohydrate-loaded and exposed to either pharmacological BAT agents or environmental stimuli. The results of this approach can be used to study BAT activity, substrate utilization, and energy expenditure following activation of the BAT in individual study subjects27.

<table><tbody><tr><td>Automated Sphygmomanometer</td><td>Omron SEM-2 advanced, Omron, Kyoto, Japan</td><td></td><td></td></tr><tr><td>Dual-energy X-ray absorptiometry scanner&amp;nbsp;</td><td>Hologic Horizon, Hologic Inc., Bedford, MA, USA</td><td></td><td></td></tr><tr><td>ECG electrodes</td><td>Ambu Blue Sensor R, Malaysia</td><td></td><td></td></tr><tr><td>Five lead ECG</td><td>Medilog AR12 plus; Schiller, Germany</td><td></td><td></td></tr><tr><td>FLIR E60 camera</td><td>FLIR Systems Australia, Melbourne , Australia</td><td></td><td></td></tr><tr><td>FLIR Research Studio Professional Edition</td><td>FLIR Systems Australia, Melbourne , Australia</td><td></td><td></td></tr><tr><td>Freestyle Optium Xceed</td><td>Abbott Diabetes Care, Alameda, Canada</td><td></td><td></td></tr><tr><td>Glucose Gel</td><td>Winners Sports Nutrition, Mt Martha, Victoria, Australia</td><td></td><td></td></tr><tr><td>MaskA cold-sterilized silicone mask</td><td>7400 series Oro-Nasal Mask, Hans Rudolph</td><td></td><td></td></tr><tr><td>Medilog Darwin2 software</td><td>Professional; Schiller, Germany</td><td></td><td></td></tr><tr><td>Non-contact Infrared Thermometer&amp;nbsp;</td><td>Berrcom, JXB-178, Guangdong, China</td><td></td><td></td></tr><tr><td>Optium Glucose Strip Xceed</td><td>Abbott Diabetes Care, Alameda, Canada</td><td></td><td></td></tr><tr><td>ParvoMedics TrueOne 2400 respiratory gas analyser</td><td>ParvoMedics Inc, East Sandy, UT, USA</td><td></td><td></td></tr><tr><td>Pre-sterilized Non-rebreathing Valve</td><td>Two-way non-rebreathing valve T-Shape configuration, 2600 Medium or 2700 Large, Hans Rudolph</td><td></td><td></td></tr></tbody></table>

using a combination of indirect calorimetry, infrared thermography, and blood glucose levels to measure brown adipose tissue thermogenesis in humans

In mammals, brown adipose tissue (BAT) is activated rapidly in response to cold in order to maintain body temperature. Although BAT has been studied greatly in small animals, it is difficult to measure the activity of BAT in humans. Therefore, little is known about the heat-generating capacity and physiological significance of BAT in humans, including the degree to which components of the diet can activate BAT. This is due to the limitations in the currently most used method to assess the activation of BAT-radiolabeled glucose (fluorodeoxyglucose or 18FDG) measured by positron emission tomography-computerized tomography (PET-CT).
This method is usually performed in fasted subjects, as feeding induces glucose uptake by the muscles, which can mask the glucose uptake into the BAT. This paper describes a detailed protocol for quantifying total-body human energy expenditure and substrate utilization from BAT thermogenesis by combining indirect calorimetry, infrared thermography, and blood glucose monitoring in carbohydrate-loaded adult males. To characterize the physiological significance of BAT, measures of the impact of BAT activity on human health are critical. We demonstrate a protocol to achieve this by combining carbohydrate loading and indirect calorimetry with measurements of supraclavicular changes in temperature. This novel approach will help to understand the physiology and pharmacology of BAT thermogenesis in humans.

Figure 1 and Figure 2&#160;present a flowchart of the study design. Images of the protocol&#160;setup are represented in Figure 3. The participant characteristics can be found in Table 1. Representative examples of IRT of the images of a participant, including baseline (Figure 4A), post carbohydrate load (Figure 4B), and 60 min following caffeine supplementation (Figure 4C), with a representative image of the camera setup, are presented in Figure 4D. Notably, Figure 4A-C provides a visual representation of the changes in supraclavicular fossa temperature (Tscf) following the intervention; the differences in temperature are particularly marked between Figure 4B and Figure 4C.
In Figure 5A-C, results from Van Schaik et al. show the Tscf (Figure 5A), the temperature of a reference point (Tref; Figure 5C), and the core temperature (Tcore; Figure 5B) from baseline (0 min) to completion of the data collection (120 min). The data show a caffeine intervention compared to placebo27. The results described in this manuscript are purely representative of this published paper. Additionally, the data on Tscf do not show a group effect. The statistics can be found in the supplemental data of Van Schaik et al.27.
The marked increase in supraclavicular temperature coincides with changes in substrate utilization and rapid lowering of the blood glucose levels following the intervention, as shown in Figure 6. These results, combined with the lack of change in temperature for the Tref and Tcore temperatures (Figure 5B,C) are indicative of BAT thermogenesis. Additionally, as the energy expenditure increases (Figure 6E), RER decreases (Figure 6A), which coincides with fat oxidation increasing (Figure 6B) following the intervention.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64451/64451fig01.jpg" /> 
Figure 1: Schematic of measures with time to complete in each 15 min period. <a href="https://www.jove.com/files/ftp_upload/64451/64451fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64451/64451fig02.jpg" /> 
Figure 2: Flow chart schematic of the study design. Experimental process. Black square = time of carbohydrate load; black circle = time of intervention. Abbreviations: IRT = infrared thermography; BGL = blood glucose levels. <a href="https://www.jove.com/files/ftp_upload/64451/64451fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64451/64451fig03.jpg" /> 
Figure 3: Representative images of the protocol. (A) Setup without the participant present; (B) data collection of the participants at baseline; (C) indirect calorimetry computer; (D) participant consuming the carbohydrate load post baseline measures. <a href="https://www.jove.com/files/ftp_upload/64451/64451fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64451/64451fig04.jpg" /> 
Figure 4: Representative examples of the IRT and camera setup. Thermal images from a participant, at (A) baseline, (B) post carbohydrate load, and (C) 60 min following the intervention of caffeine, with (D) a representative image of the camera setup. Abbreviation: IRT = infrared thermography. <a href="https://www.jove.com/files/ftp_upload/64451/64451fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64451/64451fig05.jpg" /> 
Figure 5: Effects of the intervention on the temperature measures. Baseline raw temperature changes of (A) Tscf, (B) Tcore, and (C) Tref in participants following a carbohydrate load (timepoint = 0) and the administration of a caffeine intervention or a placebo capsule (time = 45 min to 120 min)27. This figure is modified from Van Schaik et al.27. (A-C) Light grey box 1 = time of carbohydrate load; box 2 = pre-intervention; dark grey box 3 = post-intervention; blue circles = caffeine intervention; black triangles = placebo intervention. The data are expressed as the minimum to maximum, with all points shown in the box and whisker plots. The variance is expressed as mean &#177; SD, n = 8 per intervention; * represents the caffeine interaction effect (*p &#60; 0.05). The data values were analyzed using a repeated-measures three-way analysis of variance. Abbreviations: Tscf = temperature in the supraclavicular fossa; Tcore = core temperature; Tref = control reference point. <a href="https://www.jove.com/files/ftp_upload/64451/64451fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64451/64451fig06.jpg" /> 
Figure 6: Effects of the intervention on metabolic measures. Changes in (A) RER, (B) the fat oxidation rate, (C) the carbohydrate oxidation rate, (D) blood glucose levels, and (E) energy expenditure in participants following a carbohydrate load (time = 0) and the administration of a caffeine capsule or a placebo capsule (time = 45 min to 120 min). Light grey box 1 = time of carbohydrate load; box 2= pre-intervention; dark grey box 3 = post-intervention; blue circles = caffeine intervention; black triangles = placebo intervention. The data are expressed as the minimum to maximum, with all points shown in the box and whisker plots. (E) Pre- and post-administration of the interventions; grey bar = placebo intervention; blue bar= caffeine intervention. The variance is expressed as mean &#177; SD, n = 8 per intervention; * represents the caffeine interaction effect (*p &#60; 0.05). The data values were analyzed using a repeated-measures three-way analysis of variance. <a href="https://www.jove.com/files/ftp_upload/64451/64451fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>All participants</td>
		</tr>
		<tr>
			<td>n</td>
			<td>8</td>
		</tr>
		<tr>
			<td>Age, years</td>
			<td>22 &#177; 2</td>
		</tr>
		<tr>
			<td>Height, cm</td>
			<td>176 &#177; 5</td>
		</tr>
		<tr>
			<td>Weight, kg&#160;</td>
			<td>74 &#177; 8</td>
		</tr>
		<tr>
			<td>BMI, kg/m2&#160;</td>
			<td>23 &#177; 2</td>
		</tr>
		<tr>
			<td>Body fat, %&#160;</td>
			<td>20 &#177; 8</td>
		</tr>
	</tbody>
</table>
Table 1: Participant demographics. The values are means &#177; SD unless otherwise indicated.&#160;This table is from Van Schaik et al.27.

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Using a Combination of Indirect Calorimetry, Infrared Thermography, and Blood Glucose Levels to Measure Brown Adipose Tissue Thermogenesis in Humans

Here, we present a protocol to quantify the physiological significance of the impact of brown adipose tissue (BAT) activity on human metabolism. This is achieved by combining carbohydrate loading and indirect calorimetry with measurements of supraclavicular changes in temperature. This novel approach can help develop a pharmacological target for BAT thermogenesis in humans.

In mammals, brown adipose tissue (BAT) is activated rapidly in response to cold in order to maintain body temperature. Although BAT has been studied ...

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Research

JoVE Journal

Medicine

1.3K Views.  La Trobe University. Dit protocol kwantificeert de fysiologische betekenis van menselijke bruine vetweefselactiviteit door koolhydraatbelasting te combineren met indirecte calorimetrie en veranderingen in supraclaviculaire temperatuur. Dit protocol stelt deelnemers niet bloot aan ioniserende straling. Het detecteert veranderingen in de gemeten supraclaviculaire temperatuur, gecombineerd met de kerntemperatuur en de temperatuur van een referentiegebied.Begin op de dag van het onderzoek met het meten van de ...

Video: Using a Combination of Indirect Calorimetry, Infrared Thermography, and Blood Glucose Levels to Measure Brown Adipose Tissue Thermogenesis in Humans

Cerenkov Luminescence Imaging of Interscapular Brown Adipose Tissue

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely related to metabolism and energy expenditure, and its dysfunction is one important contributor for obesity and diabetes. Contrary to previous belief, recent PET/CT imaging studies indicated the BAT depots are still present in human adults. PET imaging clearly shows that BAT has considerably high uptake of 18F-FDG under certain conditions. In this video report, we demonstrate that Cerenkov luminescence imaging (CLI) with 18F-FDG can be used to optically image BAT in small animals. BAT activation is observed after intraperitoneal injection of norepinephrine (NE) and cold treatment, and depression of BAT is induced by long anesthesia. Using multiple-filter Cerenkov luminescence imaging, spectral unmixing and 3D imaging reconstruction are demonstrated. Our results suggest that CLI with 18F-FDG is a practical technique for imaging BAT in small animals, and this technique can be used as a cheap, fast, and alternative imaging tool for BAT research.

In this video report, we show the application of Cerenkov Luminescence Imaging (CLI) for interscapular brown adipose tissue in mice under activated and depressed conditions.

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely ...

Measuring Skeletal Muscle Thermogenesis in Mice and Rats

Skeletal muscle thermogenesis provides a potential avenue for better understanding metabolic homeostasis and the mechanisms underlying energy expenditure. Surprisingly little evidence is available to link the neural, myocellular, and molecular mechanisms of thermogenesis directly to measurable changes in muscle temperature. This paper describes a method in which temperature transponders are utilized to retrieve direct measurements of mouse and rat skeletal muscle temperature.
Remote transponders are surgically implanted within the muscle of mice and rats, and the animals are given time to recover. Mice and rats must then be repeatedly habituated to the testing environment and procedure. Changes in muscle temperature are measured in response to pharmacological or contextual stimuli in the home cage. Muscle temperature can also be measured during prescribed physical activity (i.e., treadmill walking at a constant speed) to factor out changes in activity as contributors to the changes in muscle temperature induced by these stimuli.
This method has been successfully used to elucidate mechanisms underlying muscle thermogenic control at the level of the brain, sympathetic nervous system, and skeletal muscle. Provided are demonstrations of this success using predator odor (PO; ferret odor) as a contextual stimulus and injections of oxytocin (Oxt) as a pharmacological stimulus, where predator odor induces muscle thermogenesis, and Oxt suppresses muscle temperature. Thus, these datasets display the efficacy of this method in detecting rapid changes in muscle temperature.

Mice and rats are surgically implanted with remote temperature transponders and then habituated to the testing environment and procedure. Changes in muscle temperature are measured in response to pharmacological or contextual stimuli in the home cage or during prescribed physical activity (i.e., treadmill walking at a constant speed).

Skeletal muscle thermogenesis provides a potential avenue for better understanding metabolic homeostasis and the mechanisms underlying energy expenditure. ...

Behavior

Human Brown Adipose Tissue Depots Automatically Segmented by Positron Emission Tomography/Computed Tomography and Registered Magnetic Resonance Images

Reliably differentiating brown adipose tissue (BAT) from other tissues using a non-invasive imaging method is an important step toward studying BAT in humans. Detecting BAT is typically confirmed by the uptake of the injected radioactive tracer 18F-Fluorodeoxyglucose (18F-FDG) into adipose tissue depots, as measured by positron emission tomography/computed tomography (PET-CT) scans after exposing the subject to cold stimulus. Fat-water separated magnetic resonance imaging (MRI) has the ability to distinguish BAT without the use of a radioactive tracer. To date, MRI of BAT in adult humans has not been co-registered with cold-activated PET-CT. Therefore, this protocol uses 18F-FDG PET-CT scans to automatically generate a BAT mask, which is then applied to co-registered MRI scans of the same subject. This approach enables measurement of quantitative MRI properties of BAT without manual segmentation. BAT masks are created from two PET-CT scans: after exposure for 2 hr to either thermoneutral (TN) (24 &#176;C) or cold-activated (CA) (17 &#176;C) conditions. The TN and CA PET-CT scans are registered, and the PET standardized uptake and CT Hounsfield values are used to create a mask containing only BAT. CA and TN MRI scans are also acquired on the same subject and registered to the PET-CT scans in order to establish quantitative MRI properties within the automatically defined BAT mask. An advantage of this approach is that the segmentation is completely automated and is based on widely accepted methods for identification of activated BAT (PET-CT). The quantitative MRI properties of BAT established using this protocol can serve as the basis for an MRI-only BAT examination that avoids the radiation associated with PET-CT.

The method presented here uses 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET-CT) and fat-water separated magnetic resonance imaging (MRI), each scanned following 2 hr exposure to thermoneutral (24 &#176;C) and cold conditions (17 &#176;C) in order to map brown adipose tissue (BAT) in adult human subjects.

Reliably differentiating brown adipose tissue (BAT) from other tissues using a non-invasive imaging method is an important step toward studying BAT in ...

Exploring Metabolite Exchange in Brown Adipose Tissue

Arteriovenous Metabolomics to Measure In Vivo Metabolite Exchange in Brown Adipose Tissue

Brown adipose tissue (BAT) plays a crucial role in regulating metabolic homeostasis through a unique energy expenditure process known as non-shivering thermogenesis. To achieve this, BAT utilizes a diverse menu of circulating nutrients to support its high metabolic demand. Additionally, BAT secretes metabolite-derived bioactive factors that can serve as either metabolic fuels or signaling molecules, facilitating BAT-mediated intratissue and/or intertissue communication. This suggests that BAT actively participates in systemic metabolite exchange, an interesting feature that is beginning to be explored. Here, we introduce a protocol for in vivo mouse-level optimized BAT arteriovenous metabolomics. The protocol focuses on relevant methods for thermogenic stimulations and an arteriovenous blood sampling technique using Sulzer's vein, which selectively drains interscapular BAT-derived venous blood and systemic arterial blood. Next, a gas chromatography-based metabolomics protocol using those blood samples is demonstrated. The use of this technique should expand the understanding of BAT-regulated metabolite exchange at the inter-organ level by measuring the net uptake and release of metabolites by BAT.

Author Spotlight: Illuminating New Avenues for Adipose Tissue Metabolism and Disease Prevention 

In this protocol, methods relevant for BAT-optimized arteriovenous metabolomics using GC-MS in a mouse model are outlined. These methods allow for the acquisition of valuable insights into BAT-mediated metabolite exchange at the organismal level.

Brown adipose tissue (BAT) plays a crucial role in regulating metabolic homeostasis through a unique energy expenditure process known as non-shivering ...

Biochemistry

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

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 &beta; 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 &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 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.

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

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 ...

Biology

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to expend energy has made it a potential target for novel therapies to ameliorate obesity and associated metabolic disorders in humans. Though this tissue has been well studied in small animals, BAT&#39;s thermogenic capacity in humans remains largely unknown due to the difficulties of measuring its volume, activity, and distribution. Identifying and quantifying active human BAT is commonly performed using 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography and computed tomography (PET/CT) scans following cold-exposure or pharmacological activation. Here we describe a detailed image-analysis approach to quantify total-body human BAT from 18F-FDG PET/CT scans using an open-source software. We demonstrate the drawing of user-specified regions of interest to identify metabolically active adipose tissue while avoiding common non-BAT tissues, to measure BAT volume and activity, and to further characterize its anatomical distribution. Although this rigorous approach is time-consuming, we believe it will ultimately provide a foundation to develop future automated BAT quantification algorithms.

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

Using free, open-source software, we have developed an analytical approach to quantify total and regional brown adipose tissue (BAT) volume and metabolic activity of BAT using 18F-FDG PET/CT.

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to ...

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.

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 ...

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

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.

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

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 ...

Determining Basal Energy Expenditure and the Capacity of Thermogenic Adipocytes to Expend Energy in Obese Mice

Energy expenditure measurements are necessary to understand how changes in metabolism can lead to obesity. Basal energy expenditure can be determined in mice by measuring whole-body oxygen consumption, CO2 production, and physical activity using metabolic cages. Thermogenic brown/beige adipocytes (BA) contribute significantly to rodent energy expenditure, particularly at low ambient temperatures. Here, measurements of basal energy expenditure and total BA capacity to expend energy in obese mice are described in two detailed protocols: the first explaining how to set up the assay to measure basal energy expenditure using analysis of covariance (ANCOVA), a necessary analysis given that energy expenditure co-varies with body mass. The second protocol describes how to measure BA energy expenditure capacity in vivo in mice. This procedure involves anesthesia, needed to limit expenditure caused by physical activity, followed by the injection of beta3-adrenergic agonist, CL-316,243, which activates energy expenditure in BA. These two protocols and their limitations are described in sufficient detail to allow a successful first experiment.

This manuscript describes a protocol to measure the basal metabolic rate and the oxidative capacity of thermogenic adipocytes in obese mice.

Energy expenditure measurements are necessary to understand how changes in metabolism can lead to obesity. Basal energy expenditure can be determined in ...

Differentiation and Imaging of Brown Adipocytes from the Stromal Vascular Fraction of Interscapular Adipose Tissue from Newborn Mice

Brown adipose tissue (BAT) is only present in mammals and has a thermogenic function. Brown adipocytes are characterized by a multilocular cytoplasm with multiple lipid droplets, a central nucleus, a high mitochondrial content, and the expression of uncoupling protein 1 (UCP1). BAT has been proposed as a potential therapeutic target for obesity and its associated metabolic disorders due to its ability to dissipate metabolic energy as heat. To investigate BAT function and regulation, brown adipocyte culturing is indispensable. The present protocol optimizes tissue processing and cell differentiation for culturing brown adipocytes from newborn mice. Additionally, procedures for the imaging of differentiated adipocytes with both confocal immunofluorescence and transmission electron microscopy are shown. In the brown adipocytes differentiated with the techniques described herein, the major defining features of classical BAT are preserved, including high UCP1 levels, increased mitochondrial mass, and very close physical contact between the lipid droplets and mitochondria, making this method a valuable tool for BAT studies.

Preadipocytes are isolated from the stromal vascular fraction of interscapular brown adipose tissue from newborn mice and differentiated into cells that accumulate lipid droplets, express molecular markers, and show the mitochondrial morphology of mature brown adipocytes. These cells are further analyzed by immunofluorescence and transmission electron microscopy.

Brown adipose tissue (BAT) is only present in mammals and has a thermogenic function. Brown adipocytes are characterized by a multilocular cytoplasm with ...

Using a Combination of Indirect Calorimetry, Infrared Thermography, and Blood Glucose Levels to Measure Brown Adipose Tissue Thermogenesis in Humans

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