Name: Illuminating New Avenues for Adipose Tissue Metabolism and Disease Prevention (Video) | JoVE
Uploaded: 2023-10-06T12:40:00
Description: Watch this Scientific Journal Video about Illuminating New Avenues for Adipose Tissue Metabolism and Disease Prevention at JoVE.com

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 ...

Exploring Metabolite Exchange in Brown Adipose Tissue

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.

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			<td>1 mL Syringe with attached needle - 26 G 5/8&#34;</td>
			<td>BD Biosciences</td>
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			<td>Agilent 5977B GC/MSD (mass selective detector)</td>
			<td>Agilent</td>
			<td>G7077B</td>
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			<td>Agilent 7693A Autosampler</td>
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			<td>G4513A</td>
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			<td>Agilent 8890 GC System</td>
			<td>Agilent</td>
			<td>G3542A</td>
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			<td>Agilent J&#38;W GC column (Capilary column) HP-5MS UI</td>
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			<td>19091S-433UI</td>
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			<td>Agilent MassHunter Workstation software_MS Quantitative analysis(Quant-My-way)</td>
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			<td>G3335-90240</td>
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			<td>C57BL/6J mouse</td>
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			<td>DL-Norvaline</td>
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			<td>Eppendorf centrifuge 5430R</td>
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			<td>5428000210</td>
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			<td>Eppendorf Safe-Lock Tubes 1.5 mL</td>
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			<td>30120086</td>
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			<td>Glass insert 250 &#956;L&#160;</td>
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			<td>Methanol (LC-MS grade)</td>
			<td>Sigma-Aldrich</td>
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			<td>Methoxyamine hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>226904-5G</td>
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			<td>Microvette&#160;200 Serum, 200 &#181;L, cap red, flat base</td>
			<td>Sarstedt</td>
			<td>20.1290.100</td>
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			<td>MTBSTFA</td>
			<td>Sigma-Aldrich</td>
			<td>394882-100ML</td>
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			<td>Pyridine(anhydrous, 99.8%)</td>
			<td>Sigma-Aldrich</td>
			<td>270970-100ML</td>
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			<td>Rodent incubator</td>
			<td>Power scientific</td>
			<td>RIT33SD</td>
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			<td>Ultra-Fine Pen Needles - 29 G 1/2&#34;</td>
			<td>BD Biosciences</td>
			<td>328203</td>
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			<td>Vial Cap 9 mm</td>
			<td>Agilent</td>
			<td>5190-9067</td>
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			<td>Vial, ambr scrw wrtn 2 mL</td>
			<td>Agilent</td>
			<td>5190-9063</td>
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			<td>Vial, ambr scrw wrtn 2 mL+A2:C40</td>
			<td>Axygen</td>
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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.

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

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

Illuminating New Avenues for Adipose Tissue Metabolism and Disease Prevention (Video) | JoVE

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

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