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

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 weeks^1,44,45), 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 profiles^48,49,50. 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 signals^51,52.
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 methods^53,54.
   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.