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/).

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 &#176;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 &#946;-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 &#176;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 &#176;C) or severe cold (6 &#176;C) temperatures can be chosen, respectively. For the purposes of this experiment, mice were raised under standard housing conditions at 22 &#176;C, which, although mildly cold for mice, did not involve any pharmacological stimulations.
<ol>
	<li>Temperature acclimation
	<ol>
		<li>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.</li>
		<li>Move the cages to their respective rodent incubators on appropriate days for the chosen type of temperature acclimation.</li>
		<li>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:
		<ol>
			<li>Thermoneutrality (30 &#176;C) group: Keep the mice in this group continuously at a temperature of 30 &#176;C for up to four weeks.</li>
			<li>Severe cold group: Initially, house the mice in this group at 18 &#176;C without any nesting materials. They will experience a gradual weekly temperature decrease, reaching 6 &#176;C by the fourth week. The temperature progression is as follows: 18 &#176;C &#8594; 14 &#176;C &#8594; 10 &#176;C &#8594; 6 &#176;C.</li>
			<li>Mild cold group (20-22 &#176;C): House the mice in this group in rodent incubators under the same conditions as standard housing conditions mentioned earlier.</li>
			<li>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 &#176;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.</li>
		</ol>
		</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Pharmacological stimulation1 using the &#946;3-adrenergic receptor agonist CL316,243
	<ol>
		<li>To maximize the stimulatory effect, pre-house the mice at thermoneutrality (30 &#176;C) for 2-4 weeks before injections.</li>
		<li>After the acclimation period, intraperitoneally inject 1 mg/kg CL316,243. 
		&#8203;NOTE: For chronic stimulation with the &#946;3-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.</li>
	</ol>
	</li>
</ol>
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&#39;s veins, a distinct blood vessel that specifically drains venous blood from the interscapular BAT46.
<ol>
	<li>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.</li>
	<li>Verify the animal&#39;s reflexes such as paw retraction to confirm that the anesthetization has reached an appropriate depth.</li>
	<li>Collecting venous blood from the Sulzer&#39;s vein
	<ol>
		<li>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.</li>
		<li>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&#39;s vein (a large &#39;Y&#39;-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&#39;s vein.</li>
		<li>Carefully cut the Sulzer&#39;s vein, and collect approximately 40 &#181;L of blood using bottom-cut 200 &#181;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&#39;s vein, to prevent blood contamination from the superior vena cava47. Collecting too much blood will diminish the characteristics of Sulzer&#39;s vein. Thus, be sure to collect the minimum amount of blood needed from Sulzer&#39;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.</li>
	</ol>
	</li>
	<li>Collecting arterial blood from the left ventricle
	<ol>
		<li>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.</li>
		<li>Accurately puncture the apex area of the left ventricle using a 1 mL syringe with a 29 G (1/2&#34;) needle. Insert two-thirds of a 1/2&#39;&#39; 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 &#181;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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Centrifugate blood samples at 10,000 x g for 10 min at 4 &#176;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 &#176;C until serum processing for GC-MS analysis. 
	&#8203;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.</li>
</ol>
3. Metabolite extraction from serum and chemical derivatization
<ol>
	<li>Preparation for the extraction 
	NOTE: All methods including metabolite extraction, derivatization, and data analysis are slightly modified versions of previously described methods53,54.
	<ol>
		<li>Prepare the extraction buffer by adding 100 &#181;M solution of DL-norvaline, internal standard (see Table of Materials), to MS-grade methanol.</li>
		<li>Ensure that all experimental procedures are conducted on ice.</li>
	</ol>
	</li>
	<li>Transfer 10 &#181;L of mice serum extracted from either the Sulzer&#39;s vein or the left ventricle into a 1.5 mL microcentrifuge tube containing 40 &#181;L of extraction buffer.</li>
	<li>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 &#176;C.</li>
	<li>After centrifugation, carefully transfer 40 &#181;L of supernatant into a glass vial followed by 3 h of drying in a vacuum centrifuge at 4 &#176;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.</li>
	<li>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.
	<ol>
		<li>Resuspend the dried serum extracts in 30 &#181;L of 10 mg/mL methoxyamine hydrochloride (see Table of Materials) dissolved in pyridine and incubate it at 37 &#176;C for 30 min.</li>
		<li>Derivatize samples for silylation of metabolites with 70 &#181;L of N-methyl-N-tert-butyldimethylsilylrifluoroacetamide (MTBSTFA, see Table of Materials) at 70 &#176;C for 1 h. 
		&#8203;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.</li>
	</ol>
	</li>
</ol>
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&#39;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.
<ol>
	<li>Inject 1 &#181;L of the derivatized sample into the GC in splitless mode at 280 &#176;C (inlet temperature), using helium as a carrier gas with a flow rate of 1.500 mL/min (set point).</li>
	<li>Set the quadrupole at 200 &#176;C with GC-MS interface at 300 &#176;C. 
	NOTE: The oven program for all metabolites analyses starts at 60 &#176;C, is held for 1 min, and is then increased at a rate of 10 &#176;C/min until the temperature reaches 320 &#176;C.</li>
	<li>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.</li>
	<li>Carry out peak area integration using a commercially available analysis software (see Table of Materials).</li>
	<li>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&#39;s vein to the left ventricle (Log2(SV/LV)) using each normalized EIC value.</li>
</ol>

Exploring Metabolite Exchange in Brown Adipose Tissue

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

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...

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&#39;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&#39;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.

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

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.

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 &#946;-adrenergic receptor agonists. Subsequently, mice are anesthetized, and blood samples are collected for metabolomic analysis (Figure 1A). For blood sampling, venous blood sp...

Watch this Scientific Journal Video about Illuminating New Avenues for Adipose Tissue Metabolism and Disease Prevention at JoVE.com

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.

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 ...

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Biochemistry

1.3K Views.  Sungkyunkwan University (SKKU). 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.

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

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug used to treat depression and anxiety by binding to the serotonin transporter with high-affinity, blocking serotonin reuptake. Here we report an efficient procedure and a set of tools to stabilize, express, purify, and crystallize serotonin transporter-antibody complexes bound to S-citalopram and other antidepressants. Mutations which stabilize the serotonin transporter were identified using an S-citalopram binding assay. Serotonin transporter expressed in baculovirus-transduced HEK293S GnTI- cells, was reconstituted into proteoliposomes and used to raise high-affinity antibodies. We have developed a strategy to discover antibodies that are useful for structural studies. A straightforward approach for the expression of antibody fragments in Sf9 cells has also been established. Transporter-antibody complexes purified using this procedure are well-behaved and readily crystallize, producing complexes with S-citalopram that diffract X-rays to 3-4 &#197; resolution. The strategies developed here can be utilized to determine the structure of other challenging membrane proteins.

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

This manuscript describes how to screen for thermostabilizing mutations, purify the human serotonin transporter, generate high affinity antibodies, and crystallize the serotonin transporter-antibody complex bound to the antidepressant drug S-citalopram. This protocol can be adapted to the study of other challenging membrane transporters, receptors, and channels.

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Quantitative Immunofluorescence to Measure Global Localized Translation

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and organogenesis, as well as in multiple diseases. Numerous publications have convincingly shown that specific mechanisms tightly regulate mRNA translation. Increased interest in the translation-induced regulation of protein expression has led to the development of novel methods to study and follow de novo protein synthesis in cellulo. However, most of these methods are complex, making them costly and often limiting the number of mRNA targets that can be studied. This manuscript proposes a method that requires only basic reagents and a confocal fluorescence imaging system to measure and visualize the changes in mRNA translation that occur in any cell line under various conditions. This method was recently used to show localized translation in the subcellular structures of adherent cells over a short period of time, thus offering the possibility of visualizing de novo translation for a short period during a variety of biological processes or of validating changes in translational activity in response to specific stimuli.

This manuscript describes a method to visualize and quantify localized translation events in subcellular compartments. The approach proposed in this manuscript requires a basic confocal imaging system and reagents and is rapid and cost-effective.

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and ...

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 ...

Medicine

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 ...

Analyzing Ex Vivo Metabolic Flux in Splenic and Cardiac Macrophages and Bone Marrow Monocytes

Metabolic reprogramming is a hallmark of monocyte/macrophage activation and polarization between pro- and anti-inflammatory states. For example, pro-inflammatory (i.e., M1-like) monocytes/macrophages display more reliance on anaerobic glycolysis and less reliance on mitochondrial oxidative phosphorylation, whereas anti-inflammatory (M2-like) macrophages display more reliance on glucose and fatty acid oxidation in the mitochondria. Here, we describe in-depth protocols for extracting macrophages from the two major monocyte/macrophage reservoirs in the body, the spleen and bone marrow, as well as injured tissues such as the heart following myocardial infarction.
Macrophages or monocytes are extracted by immunomagnetic sorting by using antibody-tagged microbeads, which easily bind to cells without compromising their phenotypes. The extracted cells are then cultured in 96-well plates, followed by extracellular flux analysis using a metabolic flux analyzer. Both glycolysis and mitochondrial oxidative phosphorylation can be measured simultaneously in small numbers of cells (as little as 2-3 &#215; 105 cells). This method can easily be performed in 1 day and produces reliable and repeatable results. Ultimately, these methods help to enhance our understanding of metabolic changes during immune and inflammatory responses to injury and disease, which could lead to the development of novel therapeutic targets for immunometabolic pathways.

Here, we describe in detail methods for extracting macrophages from the bone marrow, spleen, and infarcted heart, and subsequently assessing metabolic flux in live cells.

Metabolic reprogramming is a hallmark of monocyte/macrophage activation and polarization between pro- and anti-inflammatory states. For example, ...

Immunology and Infection

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.

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 ...

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 ...

Biology

Measuring the Rate of Lipolysis in Ex Vivo Murine Adipose Tissue and Primary Preadipocytes Differentiated In Vitro

Adipocytes store energy in the form of triglycerides in lipid droplets. This energy can be mobilized via lipolysis, where the fatty acid side chains are sequentially cleaved from the glycerol backbone, resulting in the release of free fatty acids and glycerol. Due to the low expression of glycerol kinase in white adipocytes, glycerol re-uptake rates are negligible, while fatty acid re-uptake is dictated by the fatty acid binding capacity of media components such as albumin. Both glycerol and fatty acid release into media can be quantified by colorimetric assays to determine the lipolytic rate. By measuring these factors at multiple time points, one can determine the linear rate of lipolysis with high confidence. Here, we provide a detailed protocol for the measurement of lipolysis in in vitro differentiated adipocytes and ex vivo adipose tissue from mice. This protocol may also be optimized for other preadipocyte cell lines or adipose tissue from other organisms; considerations and optimization parameters are discussed. This protocol is designed to be useful in determining and comparing the rate of adipocyte lipolysis between mouse models and treatments.

Measuring the Rate of Lipolysis in Ex Vivo Murine Adipose Tissue and Primary Preadipocytes Differentiated In Vitro

Triglyceride lipolysis in adipocytes is an important metabolic process resulting in the liberation of free fatty acids and glycerol. Here, we provide a detailed protocol to measure basal and stimulated lipolysis in adipocytes and ex vivo adipose tissue from mice.

Adipocytes store energy in the form of triglycerides in lipid droplets. This energy can be mobilized via lipolysis, where the fatty acid side chains are ...

Measuring In Vivo Adipose Tissue Kinetics in Humans Using the Deuterium (2H)-Labeling Approach

White adipose tissue is a highly plastic organ that is necessary to maintain whole-body energy homeostasis. The adipose tissue mass and changes in the fat mass or distribution are regulated by changes in the synthesis and breakdown (i.e., turnover) of adipose cells and triacylglycerols. Evidence suggests that the manner and magnitude of subcutaneous adipose tissue expansion (i.e., hypertrophy vs. hyperplasia) and turnover can influence metabolic health, as adipogenesis has been implicated in the pathogenesis of obesity and related diseases. Despite the potential role of adipose turnover in human health, there is a lack of knowledge about the in vivo kinetics of adipose cells. This is due, in part, to the slow turnover rate of the cells in adipose tissue and the practical complexity of directly labeling their metabolic precursors in vivo. Herein, we describe methods to measure in vivo adipose kinetics and turnover rates in humans through the consumption of deuterium (2H)-labeled water. The incorporation of 2H into the deoxyribonucleotide moieties of DNA in pre-adipocytes and adipocytes provides an accurate measure of cell formation and death (adipose turnover). Overall, this is an innovative approach to measuring in vivo adipose kinetics and represents a substantive departure from other in vitro assessments.

Measuring In Vivo Adipose Tissue Kinetics in Humans Using the Deuterium 2H-Labeling Approach

Presented here is a protocol to measure in vivo adipose tissue kinetics in humans using the deuterium (2H)-labeling method.

White adipose tissue is a highly plastic organ that is necessary to maintain whole-body energy homeostasis. The adipose tissue mass and changes in the fat ...

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

Summary

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

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

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Measuring the Rate of Lipolysis in Ex Vivo Murine Adipose Tissue and Primary Preadipocytes Differentiated In Vitro

Measuring In Vivo Adipose Tissue Kinetics in Humans Using the Deuterium (²H)-Labeling Approach