Se requiere una suscripción a JoVE para ver este contenido. Inicie sesión o comience su prueba gratuita.
Arteriovenous Metabolomics to Measure In Vivo Metabolite Exchange in Brown Adipose Tissue
* These authors contributed equally
In This Article
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,
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 .......
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 sp.......
Discussion
A critical step in understanding the metabolic potential of BAT in whole-body energy balance is to define which nutrients it consumes, how they are metabolically processed, and what metabolites are released into the circulation. This protocol introduces a specialized arteriovenous sampling technique that enables access to the venous vasculature of interscapular BAT and systemic arterial vasculature in C57BL/6J mice, which was recently developed and validated by Park et al42. Below are key points y.......
Acknowledgements
We thank all members of the Choi and Jung laboratories for methodological discussion. We thank C. Jang and D. Guertin for advice and feedback. We thank M.S. Choi for critical reading of the manuscript. This work was funded by NRF-2022R1C1C1012034 to S.M.J.; NRF-2022R1C1C1007023 to D.W.C; NRF-2022R1A4A3024551 to S.M.J. and D.W.C. This work was supported by Chungnam National University for W.T.K. Figure 1 and Figure 2 were created using BioRender (http://biorender.com/).
....Materials
| 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 |
References
- Cannon, B., Nedergaard, J. Brown adipose tissue: function and physiological significance. Physiol Rev. 84 (1), 277-359 (2004).
- Ikeda, K., et al. UCP1-inde....
This article has been published
Video Coming Soon
ACERCA DE JoVE
Copyright © 2025 MyJoVE Corporation. Todos los derechos reservados