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Biochimie

动静脉代谢组学测量棕色脂肪组织 中的体内 代谢物交换

Published: October 06, 2023
doi:
10.3791/66012
, , , , , , ,
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

Summary

在该方案中，概述了与在小鼠模型中使用GC-MS进行BAT优化的动静脉代谢组学相关的方法。这些方法允许在生物体水平上获得对BAT介导的代谢物交换的宝贵见解。

Abstract

棕色脂肪组织 （BAT） 通过一种称为非颤抖产热的独特能量消耗过程在调节代谢稳态中起着至关重要的作用。为了实现这一目标，英美烟草利用多样化的循环营养素菜单来支持其高代谢需求。此外，BAT 分泌代谢物衍生的生物活性因子，可作为代谢燃料或信号分子，促进 BAT 介导的组织内和/或组织间通讯。这表明BAT积极参与全身代谢物交换，这是一个有趣的特征，正在开始探索。在这里，我们介绍了一种 体内小鼠水平 优化的BAT动静脉代谢组学的方案。该方案侧重于产热刺激的相关方法和使用苏尔寿静脉的动静脉采血技术，该技术选择性地引流肩胛间 BAT 来源的静脉血和全身动脉血。接下来，演示了使用这些血液样本的基于气相色谱的代谢组学方案。该技术的使用应通过测量 BAT 对代谢物的净摄取和释放，在器官间水平上扩展对 BAT 调节的代谢物交换的理解。

Introduction

棕色脂肪组织 （BAT） 具有一种独特的能量消耗特性，称为非颤抖产热 （NST），它涉及线粒体解偶联蛋白 1 （UCP1） 依赖性和 UCP1 非依赖性机制1、2、3、4、5。这些显着特征使 BAT 参与全身代谢的调节和代谢疾病的发病机制，包括肥胖、2 型糖尿病、心血管疾病和癌症恶病质 6,7,8。最近的回顾性研究表明，BAT 质量和/或其代谢活性与人类肥胖、高血糖和心脏代谢健康呈负相关 9,10,11。

最近，BAT被提议作为负责维持NST的代谢汇，因为它需要大量的循环营养物质作为产热燃料6,7。此外，BAT可以产生和释放生物活性因子，称为棕色脂肪因子或BATokines，其充当内分泌和/或旁分泌信号，表明其积极参与系统水平的代谢稳态12,13,14,15。因此，了解BAT的营养代谢应该增强我们对它在人类中的病理生理学意义的理解，超越其作为体温调节器官的传统作用。

使用稳定同位素示踪剂的代谢组学研究，结合使用非代谢放射性示踪剂的经典营养素摄取研究，显著提高了我们对哪些营养素优先被 BAT 吸收以及如何利用的理解 16,17,18,19,20,21,22,23,24,25，26,27.例如，放射性示踪剂研究表明，冷活化的 BAT 会吸收葡萄糖、脂蛋白结合的脂肪酸和支链氨基酸16、17、18、19、20、21、22、23、27.最近的同位素示踪与代谢组学研究相结合，使我们能够测量这些营养物质在组织和培养细胞内的代谢命运和通量24,25,26,28,29,30。然而，这些分析主要集中在营养物质的个体利用上，使我们对BAT在器官代谢物交换中的系统级作用的了解有限。关于英美烟草消耗的特定循环营养物质系列及其在碳和氮方面的定量贡献的问题仍然难以捉摸。此外，对 BAT 是否可以使用营养物质产生和释放代谢物衍生的 BATokines（例如脂因子）的探索才刚刚开始 12,13,14,15,31,32。

动静脉血液分析是一种经典的生理学方法，用于评估器官/组织中循环分子的特异性摄取或释放。该技术以前已应用于大鼠的肩胛间 BAT 以测量氧气和几种代谢物，从而将 BAT 确立为适应性产热的主要位点，具有分解代谢潜力33、34、35、36、37。最近，一项使用大鼠肩胛间 BAT 的动静脉研究与跨组学方法相结合，从而鉴定了由生热刺激的 BAT38 释放的未发现的 BATokines。

基于高灵敏度气相色谱和液相色谱质谱（GC-MS 和 LC-MS）的代谢组学的最新进展重新点燃了人们对动静脉研究的兴趣，该研究用于器官特异性代谢物交换的定量分析 39,40,41。这些技术具有高分辨能力和质量精度，能够使用少量样品对各种代谢物进行全面分析。

与这些进展相一致，最近的一项研究成功地将动静脉代谢组学应用于小鼠水平的 BAT 研究，从而能够定量分析不同条件下 BAT 中的代谢物交换活动42。本文介绍了在 C57BL/6J 小鼠模型中使用 GC-MS 的 BAT 靶向动静脉代谢组学方案。

Protocol

所有实验均在成均馆大学机构动物护理和使用委员会（IACUC）的批准下进行。将小鼠饲养在IACUC批准的动物设施中，该设施位于22°C和45%湿度的洁净室中，每天进行12小时的光/暗循环。他们被放在通风的架子上，可以随意获得标准的食物饮食（包括 60% 的碳水化合物、16% 的蛋白质和 3% 的脂肪）。垫料和筑巢材料每周更换一次。在这项研究中，使用了12周龄且体重在25g至30g之间的雄性C57BL / 6J小鼠。这…

Representative Results

图 1 显示了 BAT 优化房室代谢组学的实验方案。如协议部分所述，为了获得差异刺激的棕色脂肪组织，小鼠使用啮齿动物培养箱进行温度驯化或接受药理学给药，例如β-肾上腺素能受体激动剂。随后，麻醉小鼠，并收集血液样本进行代谢组学分析（图1A）。对于血液采样， 通过 苏尔寿静脉收集从肩胛间 BAT 特异性引流的静脉血，而直接从心脏左?…

Discussion

了解 BAT 在全身能量平衡中的代谢潜力的一个关键步骤是确定它消耗了哪些营养物质，它们是如何代谢处理的，以及哪些代谢物被释放到循环中。该协议引入了一种专门的动静脉取样技术，该技术能够进入 C57BL/6J 小鼠肩胛间 BAT 和全身动脉脉管系统的静脉脉管系统，该技术最近由 Park 等人42 开发和验证。以下是您在遵循协议时应认真考虑的关键点。

对于生热…

Divulgations

The authors have nothing to disclose.

Acknowledgements

我们感谢Choi和Jung实验室的所有成员进行的方法论讨论。我们感谢 C. Jang 和 D. Guertin 提供的建议和反馈。我们感谢 M.S. Choi 对手稿的批判性阅读。这项工作由 NRF-2022R1C1C1012034 资助给 S.M.J.;NRF-2022R1C1C1007023 至 D.W.C;NRF-2022R1A4A3024551 至 S.M.J. 和 D.W.C.这项工作得到了忠南国立大学对WTK的支持，图1和图2是使用BioRender（http://biorender.com/）创建的。

Materials

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
DOWNLOAD MATERIALS LIST

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Arteriovenous MetabolomicsMetabolite ExchangeEnergy ExpenditureThermogenesisMouse ModelGas Chromatography-based Metabolomics
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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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