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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

Brown adipose tissue (BAT) possesses a unique energy expenditure property known as non-shivering thermogenesis (NST), which involves both mitochondrial uncoupling protein 1 (UCP1)-dependent and UCP1-independent mechanisms1,2,3,4,5. These distinctive characteristics implicate BAT in the regulation of systemic metabolism and the pathogenesis of metabolic diseases, including obesity, type 2 diabetes, cardiovascular disease, and cancer cachexia6,7,8. Recent retrospective studies have shown an inverse association between BAT mass and/or its metabolic activity with obesity, hyperglycemia, and cardiometabolic health in humans9,10,11.

Recently, BAT has been proposed as a metabolic sink responsible for maintaining NST, as it requires substantial amounts of circulating nutrients as thermogenic fuel6,7. Furthermore, BAT can generate and release bioactive factors, referred to as brown adipokines or BATokines, which act as endocrine and/or paracrine signals, indicating its active involvement in systems-level metabolic homeostasis12,13,14,15. Therefore, understanding BAT's nutrient metabolism should enhance our understanding of its pathophysiological significance in humans, beyond its conventional role as a thermoregulatory organ.

Metabolomic studies employing stable isotope tracers, in combination with classic nutrient uptake studies using non-metabolizable radiotracers, have significantly improved our understanding of which nutrients are preferentially taken up by BAT and how they are utilized16,17,18,19,20,21,22,23,24,25,26,27. For instance, radioactive tracer studies have demonstrated that cold-activated BAT takes up glucose, lipoprotein-bound fatty acids, and branched-chain amino acids16,17,18,19,20,21,22,23,27. Recent isotope tracing combined with metabolomic studies has allowed us to measure the metabolic fate and flux of these nutrients within tissues and cultured cells24,25,26,28,29,30. However, these analyses primarily focus on the individual utilization of nutrients, leaving us with limited knowledge of BAT's systems-level roles in organ metabolite exchange. Questions regarding the specific series of circulating nutrients consumed by BAT and their quantitative contributions in terms of carbon and nitrogen remain elusive. Additionally, the exploration of whether BAT can generate and release metabolite-derived BATokines (e.g., lipokines) using nutrients is just beginning12,13,14,15,31,32.

Arteriovenous blood analysis is a classic physiological approach used to assess the specific uptake or release of circulating molecules in organs/tissues. This technique has previously been applied to the interscapular BAT of rats to measure oxygen and several metabolites, thereby establishing BAT as the major site of adaptive thermogenesis with its catabolic potential33,34,35,36,37. Recently, an arteriovenous study using rat interscapular BAT was coupled with a trans-omics approach, leading to the identification of undiscovered BATokines released by thermogenically stimulated BAT38.

Recent advances in high-sensitivity gas chromatography- and liquid chromatography-mass spectrometry (GC-MS and LC-MS)-based metabolomics have reignited interest in arteriovenous studies for the quantitative analysis of organ-specific metabolite exchange39,40,41. These techniques, with their high resolving power and mass accuracy, enable the comprehensive analysis of a wide range of metabolites using small sample quantities.

In alignment with these advancements, a recent study successfully adapted arteriovenous metabolomics for studying BAT at the mouse level, enabling the quantitative analysis of metabolite exchange activities in BAT under different conditions42. This article presents a BAT-targeted arteriovenous metabolomics protocol using GC-MS in a C57BL/6J mouse model.

Protocol

All experiments were conducted with the approval of the Sungkyunkwan University Institutional Animal Care and Use Committee (IACUC). Mice were housed in an IACUC-approved animal facility located in a clean room set at 22 Β°C and 45% humidity, following a daily 12 h light/dark cycle. They were kept in ventilated racks and had access to a standard chow diet ad libitum (comprising 60% carbohydrate, 16% protein, and 3% fat). Bedding and nesting materials were changed on a weekly basis. For this study, male C57BL/6J mice aged 12 weeks and weighing between 25 g and 30 g were utilized. These animals were sourced from a commercial supplier (see Table of Materials).

1. Modulation of metabolic activity of the brown adipose tissue through temperature acclimation and pharmacological stimulation

NOTE: Temperature acclimation over several days to weeks or pharmacological stimulation using Ξ²-adrenergic receptor agonists are commonly employed methods for modulating BAT activity1. Therefore, a concise overview of the method is provided below to enable readers to choose the appropriate approach as required. To obtain metabolically inactive (less thermogenic) BAT, a baseline warm temperature, referred to as thermoneutrality (28-30 Β°C), is selected for C57BL/6J mice. This range ensures that the mice do not need to expend extra energy to maintain a constant body temperature. To obtain metabolically modestly or highly active (thermogenic) BAT, mild cold (20-22 Β°C) or severe cold (6 Β°C) temperatures can be chosen, respectively. For the purposes of this experiment, mice were raised under standard housing conditions at 22 Β°C, which, although mildly cold for mice, did not involve any pharmacological stimulations.

  1. Temperature acclimation
    1. Separate the mice to house 1 or 2 mice per cage at least 1 week prior to starting temperature acclimation. Prepare rodent incubators equipped with air ventilation, temperature, and humidity control with the desired conditions.
    2. Move the cages to their respective rodent incubators on appropriate days for the chosen type of temperature acclimation.
    3. Ensure that the distribution of mice numbers are even across all groups, with either 1 or 2 mice per cage. Single housing is preferred as it is more sensitive to temperature-induced physiological changes compared to group housing43. Here are the specific housing conditions for each group:
      1. Thermoneutrality (30 Β°C) group: Keep the mice in this group continuously at a temperature of 30 Β°C for up to four weeks.
      2. Severe cold group: Initially, house the mice in this group at 18 Β°C without any nesting materials. They will experience a gradual weekly temperature decrease, reaching 6 Β°C by the fourth week. The temperature progression is as follows: 18 Β°C β†’ 14 Β°C β†’ 10 Β°C β†’ 6 Β°C.
      3. Mild cold group (20-22 Β°C): House the mice in this group in rodent incubators under the same conditions as standard housing conditions mentioned earlier.
      4. Acute cold challenges: For acute cold challenges, place 1-2 mice per cage without any nesting materials and expose them to a rodent incubator set at 6 Β°C for up to 8 h.
        NOTE: These housing conditions and temperature variations are essential for studying the effects of different temperature environments on BAT activity and metabolism.
    4. Change cages and replenish food and water every week. Pre-acclimate the cages at least 24 h prior to supplementation, at their respective temperature (rodent incubator).
      NOTE: To prevent the disturbance of the appropriate temperature stimulus, it is important not to provide mouse enrichments that could lead to nest building. In response to severe cold, mice expend more energy to maintain body temperature, resulting in increased food intake and higher excretion rates. Therefore, it is crucial to check the cages at least two to three times per week (following local institutional guidelines) to ensure that the mice have an adequate supply of food and water, and that the cages are not excessively wet. This monitoring is essential to maintain the well-being and health of the mice during the study.
  2. Pharmacological stimulation1 using the Ξ²3-adrenergic receptor agonist CL316,243
    1. To maximize the stimulatory effect, pre-house the mice at thermoneutrality (30 Β°C) for 2-4 weeks before injections.
    2. After the acclimation period, intraperitoneally inject 1 mg/kg CL316,243.
      ​NOTE: For chronic stimulation with the Ξ²3-adrenergic receptor agonist (e.g. from 3 days up to weeks1,44,45), daily injections are required due to drug stability. Diluted CL316,243 should be prepared on the day of injection due to stability.

2. Arteriovenous blood sampling

NOTE: Mice over 12-14 weeks are best recommended for arteriovenous blood sampling. Younger mice may not have sufficiently sized Sulzer's veins, a distinct blood vessel that specifically drains venous blood from the interscapular BAT46.

  1. Gently anesthetize using the calibrated vaporizer with 3% isoflurane for induction (in a 205 x 265 x 200 mm sized chamber) and 2% isoflurane for maintenance (with a gas anesthesia mask).
    NOTE: The entire arteriovenous blood sampling procedure should be promptly performed after anesthesia. A water-heated warming pad can be used during anesthesia to maintain core body temperature.
    CAUTION: Isoflurane is highly volatile and toxic when inhaled. Therefore, primary anesthetization must be performed under a fume hood.
  2. Verify the animal's reflexes such as paw retraction to confirm that the anesthetization has reached an appropriate depth.
  3. Collecting venous blood from the Sulzer's vein
    1. Position the mouse to expose the dorsal skin, confirm the depth of anesthesia via a toe-pinch right before making the incision. Dampen the dorsal skin with ample 70% ethanol to prevent hair detachment, and make an incision along the back from the lower part of the thorax all the way up to the neck.
      NOTE: The interscapular BAT is located right under the skin, consisting of two fat pads covered by thin white adipose tissues. It is important to ensure that the mouse remains under anesthesia through the gas anesthesia mask.
    2. Gently lift the interscapular BAT with bent tip forceps and carefully cut the attached tissues, most of which are muscles. Lift up the fat pad toward head, continue to cut the attached tissues and carefully open the region until the Sulzer's vein (a large 'Y'-shaped dark red vessel connected to both fat pads of interscapular BAT) is exposed.
      NOTE: Be careful not to cut the capillary vessels of muscle tissues that are connected with the front- and side-region of interscapular BAT, as doing so will significantly reduce the amount and quality of blood draining from the Sulzer's vein.
    3. Carefully cut the Sulzer's vein, and collect approximately 40 Β΅L of blood using bottom-cut 200 Β΅L pipette tips and a P100 pipette. Store the blood in a blood collection tube and keep the tube on ice until the serum collection process.
      NOTE: It is important to collect blood from just below the Y-shaped division point of Sulzer's vein, to prevent blood contamination from the superior vena cava47. Collecting too much blood will diminish the characteristics of Sulzer's vein. Thus, be sure to collect the minimum amount of blood needed from Sulzer's vein for analysis. Be thoughtful when selecting blood collection tubes, as serum and plasma yield different metabolite profiles48,49,50. For plasma collection, it is necessary to use heparin-coated tubes. In this experiment, clotting activator-coated tubes were chosen to obtain serum. Under no circumstances should EDTA coated tubes be used, as EDTA significantly impacts mass spectrometry signals51,52.
  4. Collecting arterial blood from the left ventricle
    1. Flip the mouse without losing contact to the nose cone, to expose ventral skin. Confirm the depth of anesthesia via a toe-pinch before making the incision. Dampen the ventral skin with ample amount of 70% ethanol to prevent hair detachment, and carefully open the thoracic cavity with scissors to expose the heart without damaging any internal structure.
    2. Accurately puncture the apex area of the left ventricle using a 1 mL syringe with a 29 G (1/2") needle. Insert two-thirds of a 1/2'' needle to 3-5 mm to the right horizontal of the apex of the heart (Figure 1B), and pull the syringe back to collect blood from the left ventricle (50-100 Β΅L). Blood from the left ventricle is oxygenated arterial blood which is bright red. Store the blood in a blood collection tube, and keep the tube on ice until the serum collection process.
      NOTE: A minimal incision should be made in the chest cavity for access to the heart. Excessive incision could lead to local bleeding, which may subsequently result in low blood pressure. This could affect the quality of arterial blood collected from the left ventricle. Avoid rotating or flipping the heart, as it will make it difficult to determine the precise position of the left ventricle.
    3. Perform euthanasia and ensure it with an appropriate method following the guidelines of the local institutions. For example, euthanasia can be performed through cervical dislocation and confirmed by the cessation of heartbeat.
  5. Centrifugate blood samples at 10,000 x g for 10 min at 4 Β°C. Carefully collect the supernatant using a pipette. The supernatant should contain either serum or plasma depending on the type of blood collection tube used. One can stop here and store the samples at -20 Β°C until serum processing for GC-MS analysis.
    ​NOTE: Hemolysis may potentially have occurred if red-colored supernatant is observed. To avoid hemolysis, vortex the sample before clotting is completed. Some red blood cell-enriched metabolites including glutamine and lactate could lead to data misinterpretation, although most metabolites may not be significantly affected by hemolysis. Analyzing the serum/plasma within a week is recommended.

3. Metabolite extraction from serum and chemical derivatization

  1. Preparation for the extraction
    NOTE: All methods including metabolite extraction, derivatization, and data analysis are slightly modified versions of previously described methods53,54.
    1. Prepare the extraction buffer by adding 100 Β΅M solution of DL-norvaline, internal standard (see Table of Materials), to MS-grade methanol.
    2. Ensure that all experimental procedures are conducted on ice.
  2. Transfer 10 Β΅L of mice serum extracted from either the Sulzer's vein or the left ventricle into a 1.5 mL microcentrifuge tube containing 40 Β΅L of extraction buffer.
  3. To remove cell debris and protein, briefly vortex the serum samples, followed by the centrifugation at maximum speed (18,000 x g) for 30 min at 4 Β°C.
  4. After centrifugation, carefully transfer 40 Β΅L of supernatant into a glass vial followed by 3 h of drying in a vacuum centrifuge at 4 Β°C.
    NOTE: A glass insert (see Table of Materials) is recommended instead of plastic tubes due to plastic-reactive chemicals used throughout the subsequent derivatization step.
  5. Subject the dried samples to two consecutive derivatization steps for the GC-MS analysis of the serum metabolites.
    NOTE: The following steps should be performed under a fume hood due to irritation risks of the solvents.
    1. Resuspend the dried serum extracts in 30 Β΅L of 10 mg/mL methoxyamine hydrochloride (see Table of Materials) dissolved in pyridine and incubate it at 37 Β°C for 30 min.
    2. Derivatize samples for silylation of metabolites with 70 Β΅L of N-methyl-N-tert-butyldimethylsilylrifluoroacetamide (MTBSTFA, see Table of Materials) at 70 Β°C for 1 h.
      ​NOTE: Using a glass syringe is recommemded rather than a plastic tip in the following steps due to the derivatization solvents which react with plastic.

4. Metabolomics analysis using GC-MS

NOTE: Single quadruple GC-MS (see Table of Materials) was employed to measure the various serum metabolites including carbohydrates, amino acids, and TCA cycle intermediates in derivatized samples from the Sulzer's vein and the left ventricle. Other columns can alternatively be used, although the experimental settings including the temperature program may vary depending on the types of columns used.

  1. Inject 1 Β΅L of the derivatized sample into the GC in splitless mode at 280 Β°C (inlet temperature), using helium as a carrier gas with a flow rate of 1.500 mL/min (set point).
  2. Set the quadrupole at 200 Β°C with GC-MS interface at 300 Β°C.
    NOTE: The oven program for all metabolites analyses starts at 60 Β°C, is held for 1 min, and is then increased at a rate of 10 Β°C/min until the temperature reaches 320 Β°C.
  3. Collect data by electron ionization (EI) set at 70 eV and acquire the sample data in scan mode (50-550 m/z)53. All metabolites used in this study were previously validated with standards to confirm mass spectra and retention times.
  4. Carry out peak area integration using a commercially available analysis software (see Table of Materials).
  5. Match the compounds with the product ion of each TBMDS derivative. Then, obtain the extract ion chromatogram (EIC) by integrating the m/z value of the fragment ion in the corresponding peak area, and then export it. Fragment ions are shown in Table 1.
    NOTE: The EIC of each compound was normalized by that of DL-norvaline in each sample. The data are represented by Log2 Sulzer's vein to the left ventricle (Log2(SV/LV)) using each normalized EIC value.

Results

Figure 1 illustrates the experimental scheme of BAT-optimized AV metabolomics. As mentioned in the Protocol section, to obtain differentially stimulated brown adipose tissues, mice undergo temperature acclimation using rodent incubators or receive pharmacological administration such as Ξ²-adrenergic receptor agonists. Subsequently, mice are anesthetized, and blood samples are collected for metabolomic analysis (Figure 1A). For blood sampling, venous blood sp...

Discussion

A critical step in understanding the metabolic potential of BAT in whole-body energy balance is to define which nutrients it consumes, how they are metabolically processed, and what metabolites are released into the circulation. This protocol introduces a specialized arteriovenous sampling technique that enables access to the venous vasculature of interscapular BAT and systemic arterial vasculature in C57BL/6J mice, which was recently developed and validated by Park et al42. Below are key points y...

Disclosures

The authors declare that they have no conflicts of interest to report.

Acknowledgements

We thank all members of the Choi and Jung laboratories for methodological discussion. We thank C. Jang and D. Guertin for advice and feedback. We thank M.S. Choi for critical reading of the manuscript. This work was funded by NRF-2022R1C1C1012034 to S.M.J.; NRF-2022R1C1C1007023 to D.W.C; NRF-2022R1A4A3024551 to S.M.J. and D.W.C. This work was supported by Chungnam National University for W.T.K. Figure 1 and Figure 2 were created using BioRender (http://biorender.com/).

Materials

NameCompanyCatalog NumberComments
0.5-20 Β΅L Filter TipsAxygenAX.TF-20-R-S
1 mL Syringe with attached needle - 26 G 5/8"BD Biosciences309597
Agilent 5977B GC/MSD (mass selective detector)AgilentG7077B
Agilent 7693A AutosamplerAgilentG4513A
Agilent 8890 GC SystemAgilentG3542A
Agilent J&W GC column (Capilary column) HP-5MS UIAgilent19091S-433UI
Agilent MassHunter Workstation software_MS Quantitative analysis(Quant-My-way)AgilentG3335-90240
C57BL/6J mouseDBLC57BL/6JBomTac
CentriVap -50 Β°C Cold Trap (with Stainless steel Lid)LABCONCOΒ 7811041
DL-NorvalineSigma-AldrichN7502-25G
Eppendorf centrifuge 5430REppendorf5428000210
Eppendorf Safe-Lock Tubes 1.5 mLEppendorf30120086
Glass insert 250 ΞΌLΒ Agilent5181-1270
Methanol (LC-MS grade)Sigma-AldrichQ34966-1L
Methoxyamine hydrochlorideSigma-Aldrich226904-5G
MicrovetteΒ 200 Serum, 200 Β΅L, cap red, flat baseSarstedt20.1290.100
MTBSTFASigma-Aldrich394882-100ML
Pyridine(anhydrous, 99.8%)Sigma-Aldrich270970-100ML
Refrigerated CentriVap Complete Vaccum ConcentratorsLABCONCOΒ 7310041
Rodent dietSAFESAFE R+40-10
Rodent incubatorPower scientificRIT33SD
Ultra-Fine Pen Needles - 29 G 1/2"BD Biosciences328203
Vial Cap 9 mmAgilent5190-9067
Vial, ambr scrw wrtn 2 mLAgilent5190-9063
Vial, ambr scrw wrtn 2 mL+A2:C40AxygenPCR-02-C

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