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JoVE Journal Biochemistry

Biochemistry

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

Published: October 6, 2023 doi: 10.3791/66012
Automatic Translation
*1, *2,3, *1, 1, 3, 2, 3, 1
1Department of Biological Sciences, Sungkyunkwan University (SKKU), 2Department of Biochemistry, College of Natural Sciences, Chungnam National University, 3Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University
* These authors contributed equally

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.

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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 °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 β-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 °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 °C) or severe cold (6 °C) temperatures can be chosen, respectively. For the purposes of this experiment, mice were raised under standard housing conditions at 22 °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 °C) group: Keep the mice in this group continuously at a temperature of 30 °C for up to four weeks.
      2. Severe cold group: Initially, house the mice in this group at 18 °C without any nesting materials. They will experience a gradual weekly temperature decrease, reaching 6 °C by the fourth week. The temperature progression is as follows: 18 °C → 14 °C → 10 °C → 6 °C.
      3. Mild cold group (20-22 °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 °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 β3-adrenergic receptor agonist CL316,243
    1. To maximize the stimulatory effect, pre-house the mice at thermoneutrality (30 °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 β3-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 µL of blood using bottom-cut 200 µL 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 µL). 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 °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 °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 µM 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 µL of mice serum extracted from either the Sulzer's vein or the left ventricle into a 1.5 mL microcentrifuge tube containing 40 µL 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 °C.
  4. After centrifugation, carefully transfer 40 µL of supernatant into a glass vial followed by 3 h of drying in a vacuum centrifuge at 4 °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 µL of 10 mg/mL methoxyamine hydrochloride (see Table of Materials) dissolved in pyridine and incubate it at 37 °C for 30 min.
    2. Derivatize samples for silylation of metabolites with 70 µL of N-methyl-N-tert-butyldimethylsilylrifluoroacetamide (MTBSTFA, see Table of Materials) at 70 °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 µL of the derivatized sample into the GC in splitless mode at 280 °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 °C with GC-MS interface at 300 °C.
    NOTE: The oven program for all metabolites analyses starts at 60 °C, is held for 1 min, and is then increased at a rate of 10 °C/min until the temperature reaches 320 °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.

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Representative 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 β-adrenergic receptor agonists. Subsequently, mice are anesthetized, and blood samples are collected for metabolomic analysis (Figure 1A). For blood sampling, venous blood specifically draining from interscapular BAT is collected via the Sulzer's vein, while arterial blood is directly collected from the left ventricle of the heart (Figure 1B).

Figure 2 represents the schematics of serum metabolomics using GC-MS (Figure 2). Briefly, the methanol solution is added to serum samples followed by centrifugation to remove serum proteins. The supernatant containing the metabolites was dried in a SpeedVac and the dried samples were subjected to two-step derivatization using methoxyamine (MOX) and MTBSTFA (N-tert-Butyldimethylsilyl-N-methyltrifluoroacetamide). The derivatization step aims for the substitution of polar functional groups of the metabolites with TBDMS ester, allowing for GC-MS detection of metabolites as TBDMS derivatives. The derivatized samples were analyzed using single quadruple GC-MS. Metabolites are separated in the column according to their physiochemical properties. Electron ionization (EI) is employed to break the metabolites down into unique fragment ions detected by a mass detector. Compound identification is carried out through the detection of compound-specific fragment ions. The m/z value and retention time of the compound-specific fragment ions in the experiment are shown in the table (Table 1). An extracted ion chromatogram (EIC) is used to integrate the signal of a compound-specific fragment ion in the peak area of the metabolite, which defines the abundance of metabolites.

To determine whether BAT releases or uptakes a series of metabolites, Log2 ratios of ion counts between the Sulzer's vein and left ventricle (Log2(SV/LV)) can be calculated (Figure 3). If the Log2(SV/LV) value for a particular metabolite is >0, it indicates that the metabolite is more abundant in the SV than LV, suggesting that interscapular BAT net releases the metabolite. Conversely, if the Log2(SV/LV) value for a certain metabolite is <0, it suggests that the metabolite is taken up by the interscapular BAT.

Using this approach, it was found that mild cold-stimulated BAT significantly consumes glucose, lactate, 3-hydroxybutyrate (3-HB), BCAAs, aspartate, lysine, and tryptophan, while BAT significantly releases succinate and palmitate (Figure 3A). Most of these phenomena were observed in our previous studies on chronic cold BAT42, suggesting that mild cold BAT metabolically resembles chronic cold BAT, albeit to a lesser extent. To provide a quick visual overview of the data, a heat map displaying Log2(SV/LV) values is depicted (Figure 3B). It is essential to interpret the data with caution, as the heatmap represents Z-scores within the group.

Figure 1
Figure 1: Experimental scheme of BAT-optimized arteriovenous (AV) metabolomics. (A) Diagram showing the workflow of AV metabolomics. (1) To induce different versions of metabolically stimulated brown adipose tissue (BAT), mice are acclimated to different temperatures or receive pharmacological drug injections. (2) Subsequently, the arterial and venous blood samples are collected from the left ventricle and the Sulzer's vein of the mice, respectively. (3) The obtained serum samples are subjected to metabolite extraction, followed by metabolomic analysis. (B) Representative images providing guidance for blood sampling procedures. Arterial blood is collected from the left ventricle, while venous blood is obtained from the Sulzer's vein as it specifically drains blood from BAT. Please click here to view a larger version of this figure.

Figure 2
Figure 2: GC/MS-based targeted metabolomics. Experimental scheme of GC/MS-based targeted metabolomics. TBDMS: tert-butyldimethylsilyl, EI: Electron ionization, EIC: Extract ion chromatogram. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Representative AV metabolomics data. (A) The uptake and subsequent release of selected prevalent and highly active circulating fuel metabolites by BAT, classified according to their respective groups. The data show Log2 Sulzer's vein to left ventricle (SV/LV) ratios. Bars indicating metabolites with mean Log2 (SV/LV) <0 are colored red, and Log2 (SV/LV) >0 are colored blue. Individual data points represent each mouse; n = 11. Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. BCAA; branched-chain amino acid; EAA; essential amino acid; NEAA; non-essential amino acid. (B) A heat map illustrating the differential abundance of metabolites between Sulzer's vein (SV) and left ventricle (LV) blood. Each column shows a Z-score of average values within the group; n = 11. Please click here to view a larger version of this figure.

Compound m/z of Fragment ions Retention time Formular TBDMS derivative formular
Pyruvate 174.1 8.72 C3H4O3 C15H33O3NSi2
Aspartate 418.2 18.45 C4H7NO4 C22H49NO4Si3
α-Ketoglutarate 346.2 17.27 C5H6O5 C18H37NO5Si2
C16:0(Palmitate) 313.3 19.72 C16H32O2 C22H46O2Si
Lactate 261.2 11.66 C3H6O3 C15H34O3Si2
Leucine 200.2 14 C6H13NO2 C18H41NO2Si2
Isoleucine 200.2 14.34 C6H13NO2 C18H41NO2Si2
Valine 186.2 13.53 C5H11NO2 C17H39NO2Si2
Alanine 260.2 12.17 C3H7NO2 C15H35NO2Si2
Serine 390.2 17.04 C3H7NO3 C21H49NO3Si3
Glycine 218.1 12.43 C2H5NO2 C14H33NO2Si2
Lysine 300.2 20.48 C6H14N2O2 C24H56N2O2Si3
Succinate 289.1 14.65 C4H6O4 C16H34O4Si2
Proline 258.2 14.73 C5H9NO2 C17H37NO2Si2
Phenylalanine 336.2 18 C9H11NO2 C21H39NO2Si2
Methionine 320.2 16.84 C5H11NO2S C17H39NO2SSi2
Cysteine 406.2 19 C3H7NO2S C21H49NO2SSi3
Asparagine 417.2 19.86 C4H8N2O3 C22H50N2O3Si3
3-Hydroxybutyrate(3HB) 275.2 12.81 C4H8O3 C16H36O3Si2
Tryptophan 375.2 22.97 C11H12N2O2 C23H40N2O2Si2
Malate 419.2 18.19 C4H6O5 C22H48O5Si3
Glutamate 432.3 19.59 C5H9NO4 C23H51NO4Si3
Glutamine 431.3 20.85 C5H10N2O3 C23H52N2O3Si3
Citrate 459.2 22.38 C6H8O7 C30H64O7Si4
Glucose 476.3 22.7 C6H12O6 C30H68O6Si4

Table 1: GC/MS compound fragment ions used for integration. This table contains a list of 25 serum metabolites identified in the present method, with the retention time and fragment ions corresponding to each compound.

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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 you should critically consider when following the protocol.

For thermogenic stimulation, a key consideration is choosing the most appropriate type of thermogenic stimulation for your experimental setting. For example, when using an experimental group (vs. control) and aiming to observe differences in the metabolic adaptation capacity (crucial for its maximal non-shivering thermogenic potential) of BAT, chronic cold acclimation, or β-adrenergic receptor stimulation (lasting more than 3 days to 4 weeks) is suitable1,45. When BAT is exposed to acute cold exposure (a few hours less than 24 h), rather than metabolically adapting, BAT metabolizes its intrinsic stored fuels and communicates metabolically with shivering muscles for optimal thermogenesis1. A β-adrenergic receptor agonist represents a neuronal response to trigger BAT thermogenesis1,14, and there are other endocrine factors that enhance the thermogenic program55,56,57,58,59,60. Whenever performing thermogenic stimulation as mentioned above, having a baseline control at thermoneutrality (30 °C) should be useful to confirm whether the stimulation worked appropriately.

As mentioned in the protocol section, obtaining arteriovenous blood of the highest quality is key to determining successful establishment of the experiment. To achieve this, it is essential to prevent hemolysis (see step 2.5) and collect a consistent amount of blood to avoid dilution of the distinct characteristics of venous blood from the Sulzer's vein (less than 40 µL from the Sulzer's vein). To this end, during the initial set-up, the authors strongly recommend performing a pilot experiment for metabolite measurement using not only blood from Sulzer's vein, but also blood from non-BAT draining venous blood (e.g. inferior vena cava). This will help confirm whether the serum of the Sulzer's vein reveals distinct metabolite profiling compared to the non-BAT venous vessels. Regarding arterial blood collection from the left ventricle, it is important to consistently puncture the same area on the left ventricle to minimize variations in cardiokine levels61,62. Such variations could yield confusing results in arteriovenous-metabolomics or -proteomics.

Chromatography coupled with mass spectrometry is one of the most powerful tools for metabolomics, one method being GC-MS63. Indeed, the measurement of biologically relevant metabolites using GC-MS was considered challenging due to the coverage issue; GC-MS application is usually limited to the analysis of metabolites with highly volatile and non-polar properties as opposed to the numerous polar metabolites in tissues and biological fluids. However, the development of various extraction and derivatization methods, together with the upsides of GC-MS including excellent peak resolution, reproducibility, and extensive mass spectral libraries allowing for handy peak identification, has led to such a platform as an attractive option for analyzing biologically relevant metabolites64,65.

In this protocol, the GC-MS platform is employed for the analysis of 25 metabolites in serum collected using the aforementioned technique. To this end, metabolite extraction was carried out using 80% methanol solution. Of note, during this step, using certain types of tubes including Eppendorf tubes is recommended to achieve the lowest levels of organic substance contamination in the samples. Careful separation of supernatant from pellets following extraction is also needed to remove protein debris from the serum, which is critical for the maintenance of MS-ion source performance.

The derivatization step can be diversified based on the types of derivatizations including aryl derivatives, silylation, and acylation66. The authors employed two-step derivatization with N-methyl-N-tert-butyldimethylsilylrifluoroacetamide (MTBSTFA) for metabolite sialylation which is a commonly used method with the advantage of stable detection of biologically relevant polar metabolites including sugars, but with a drawback regarding the occasional loss of the N-trimethylsilyl group of amines and amino acids during the analysis step64. Of note, samples should be completely dried prior to the derivatization step, due to the moisture sensitivity of the reagents and derivatives.

One of the main limitations of the GC analysis, in general, still comes down to the range of detectable metabolites despite the aforementioned derivatization steps which improve GC-MS capacity regarding polar metabolite detection, especially as compared to LC-MS-based analysis. Quantitative metabolomics using LC-MS can greatly help gain better insight into the metabolic spectrum of BATs with respect to systemic metabolite exchange24,42.

Although the calculation used in the protocol-measurement of metabolite concentration gradient between the arterial vs. venous blood; Log2(SV/LV)-quantifies fractional absorption and release change by BAT, caution is needed for data interpretation. In particular, for those metabolites whose fractional net exchange value is '0' (e.g. citrate, αKG, malate, methionine, alanine, glutamine), the result could be attributed to either no uptake/release or equally active catabolism and synthesis (where both uptake and release are high). To verify which of the two possibilities is correct, additional metabolic flux experiments using isotope tracing in vivo with the taken-up metabolite and/or its substrate are necessary39.

The limitation of the fractional calculation is that it does not provide the quantitative landscape of the uptake and release for those metabolites42,67. For instance, although the Log2(SV/LV) value indicates a comparable relative net uptake between glucose and 3-hydroxybutyrate (3HB), the actual contribution of glucose to the carbon sources should be much higher than 3-HB due to the fact that the blood concentration of glucose is much higher than that of 3HB, and because glucose contains four more carbons (glucose has six carbons; 3HB has two carbons). If required, a comprehensive analysis incorporating additional parameters, such as blood flow rate, actual blood concentrations of each metabolite, and their chemical equations should inform us of the quantitative contributions of the metabolites taken-up/released and their contributions in total carbon or nitrogen flux42,67.

A major advantage of this miniaturized methodology for arteriovenous blood sampling and metabolomic analysis at the mouse level is its synergistic combination with various genetic models to study brown adipose tissue metabolism and physiology (e.g. UCP1-KO, UCP1-Cre driven BAT-specific loss-of-function models, transgenic models). Recent metabolomic analysis which requires only trace amounts of serum for metabolomic profiling makes this approach more feasible with smaller organisms (see protocol 3)39. However, proteomic analysis, which can provide better insights when considered together with metabolomics, may require larger amounts of samples. In this regard, conventional arteriovenous sampling using rats may be more suitable. We refer the readers to the most recent protocol for arteriovenous blood sampling for BAT in rats, written by Mestres-Arenas and colleagues47.

Although the current protocol adopts the anesthesia procedure using isoflurane as described in Park et al.42, this approach may negatively impact the thermogenic metabolism of brown adipocytes and/or brown adipose tissue in vivo68,69,70,71. Therefore, future arteriovenous metabolomics utilizing pentobarbital warrant investigation.

In summary, this protocol provides a foundational methodology to measure BAT-specific net metabolic activity (consumption vs. production) upon various thermogenic stimulations. This should give us valuable insights into the role of BAT as a systemic nutrient sink as well as provider by quantitatively listing key metabolic fuels as well as secreted metabolites. Moreover, this can also be useful for identifying previously unstudied metabolite-derived brown adipokines, especially when operated with discovery-based metabolomic platforms.

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Disclosures

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

Acknowledgments

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

Name Company Catalog Number Comments
0.5-20 µL Filter Tips Axygen AX.TF-20-R-S
1 mL Syringe with attached needle - 26 G 5/8" BD Biosciences 309597
Agilent 5977B GC/MSD (mass selective detector) Agilent G7077B
Agilent 7693A Autosampler Agilent G4513A
Agilent 8890 GC System Agilent G3542A
Agilent J&W GC column (Capilary column) HP-5MS UI Agilent 19091S-433UI
Agilent MassHunter Workstation software_MS Quantitative analysis(Quant-My-way) Agilent G3335-90240
C57BL/6J mouse DBL C57BL/6JBomTac
CentriVap -50 °C Cold Trap (with Stainless steel Lid) LABCONCO  7811041
DL-Norvaline Sigma-Aldrich N7502-25G
Eppendorf centrifuge 5430R Eppendorf 5428000210
Eppendorf Safe-Lock Tubes 1.5 mL Eppendorf 30120086
Glass insert 250 μL  Agilent 5181-1270
Methanol (LC-MS grade) Sigma-Aldrich Q34966-1L
Methoxyamine hydrochloride Sigma-Aldrich 226904-5G
Microvette 200 Serum, 200 µL, cap red, flat base Sarstedt 20.1290.100
MTBSTFA Sigma-Aldrich 394882-100ML
Pyridine(anhydrous, 99.8%) Sigma-Aldrich 270970-100ML
Refrigerated CentriVap Complete Vaccum Concentrators LABCONCO  7310041
Rodent diet SAFE SAFE R+40-10
Rodent incubator Power scientific RIT33SD
Ultra-Fine Pen Needles - 29 G 1/2" BD Biosciences 328203
Vial Cap 9 mm Agilent 5190-9067
Vial, ambr scrw wrtn 2 mL Agilent 5190-9063
Vial, ambr scrw wrtn 2 mL+A2:C40 Axygen PCR-02-C

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References

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Tags

Brown Adipose Tissue BAT Non-shivering Thermogenesis Metabolic Homeostasis Circulating Nutrients Energy Expenditure Process Bioactive Factors Metabolic Fuels Signaling Molecules Intratissue Communication Intertissue Communication Mouse-level Optimized BAT Arteriovenous Metabolomics Thermogenic Stimulations Arteriovenous Blood Sampling Technique Sulzer's Vein Gas Chromatography-based Metabolomics Protocol Inter-organ Level Metabolite Exchange
Arteriovenous Metabolomics to Measure <em>In Vivo</em> Metabolite Exchange in Brown Adipose Tissue
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Cite this Article

Lee, S., Lim, G., Kim, S., Kim, H.,… More

Lee, S., Lim, G., Kim, S., Kim, H., Roh, Y. J., Kim, W., Choi, D. W., Jung, S. M. Arteriovenous Metabolomics to Measure In Vivo Metabolite Exchange in Brown Adipose Tissue. J. Vis. Exp. (200), e66012, doi:10.3791/66012 (2023).

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