arteriovenous blood sampling technique in mice using sulzer's vein and left ventricle for metabolomic analysis

探索棕色脂肪组织中的代谢物交换

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.

作者聚焦：阐明脂肪组织代谢和疾病预防的新途径 

动静脉代谢组学测量棕色脂肪组织 中的体内 代谢物交换

Our lab is interested in identifying mechanisms by which signaling pathways and metabolic pathways collaborate to regulate adipose tissue metabolism and physiology. And through that approach, we aim to find a way to prevent or target metabolic diseases caused by energy imbalances such as obesity and diabetes. The major advantage of our protocol is we have shifted from the conventional rat model to using a mouse model for arterial venous blood sampling and metabolomic analysis.We believe that this transition will allow us to validate our findings using the gold standard genetic models more rapidly for brown fat research.

Watch this Scientific Journal Video about Arteriovenous Blood Sampling Technique in Mice Using Sulzer's Vein and Left Ventricle for Metabolomic Analysis at JoVE.com

Arteriovenous Blood Sampling Technique in Mice Using Sulzer&#039;s Vein and Left Ventricle for Metabolomic Analysis  

使用苏尔寿静脉和左心室对小鼠进行动静脉采血技术进行代谢组学分析

首先，将鼠标放置在装有鼻锥的解剖托盘上，以进行麻醉输送。验证动物的反应，例如爪子缩回，以确认麻醉已达到适当的深度。然后，放置鼠标以露出背部皮肤。用充足的70%乙醇润湿皮肤，以防止头发脱落。从胸部下部开始，沿着背部做一个切口，一直延伸到颈部。使用弯曲的尖端镊子，轻轻提起肩胛间棕色脂肪组织并小心地切割附着的组织。将脂肪垫抬向头部并继续切割附着的组织，小心地打开该区域，直到苏尔寿静脉暴露出来。现在，小心地切割苏尔寿的静脉。使用底部切口的 200 微升移液器吸头和 P100 移液器，收集约 40 微升血液并将收集的血液储存在采血管中。将试管放在冰上，直到血清收集过程。用70%乙醇大量润湿腹侧皮肤，以防止头发脱落。用剪刀小心地打开胸腔，露出心脏。使用配备 29 号半英寸针头的一毫升注射器，精确刺穿左心室的顶点区域。将三分之二的针头插入心尖右侧水平约三至五毫米处，然后将注射器向后拉以从左心室收集血液。然后，将血液样本在10,000G下在4摄氏度下离心10分钟。使用移液管收集上清液并进行代谢物提取和分析。

arteriovenous-blood-sampling-technique-mice-using-sulzer-s-vein-left

Research

JoVE Journal

Biochemistry

Arteriovenous Blood Sampling Technique in Mice Using Sulzer's Vein and Left Ventricle for Metabolomic Analysis

194 次观看.  Sungkyunkwan University (SKKU). 首先，将鼠标放置在装有鼻锥的解剖托盘上，以进行麻醉输送。验证动物的反应，例如爪子缩回，以确认麻醉已达到适当的深度。然后，放置鼠标以露出背部皮肤。用充足的70%乙醇润湿皮肤，以防止头发脱落。从胸部下部开始，沿着背部做一个切口，一直延伸到颈部。使用弯曲的尖端镊子，轻轻提起肩胛间棕色脂肪组织并小心地切割附着的组织。将脂肪垫抬向头部...

视频: Arteriovenous Blood Sampling Technique in Mice Using Sulzer's Vein and Left Ventricle for Metabolomic Analysis  

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.

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 ...

科研

生物化学

To begin, position the mouse on a dissection tray equipped with a nose cone for anesthesia delivery. Verify the animal's reflexes, such as paw retraction, to confirm that the anesthesia has reached an appropriate depth. Then, position the mouse to expose the dorsal skin.Dampen the skin with ample 70%ethanol to prevent hair detachment. Make an incision along the back, starting from the lower part of the thorax and extend it up to the neck. Using bent tip forceps, gently lift the interscapular brown adipose tissue and carefully cut the attached tissue.Lift the fat pad toward the head and continue cutting the attached tissues to carefully open the region until the Sulzer's vein is exposed. Now, carefully cut the Sulzer's vein. Using bottom cut 200 microliter pipette tip and a P100 pipette, collect approximately 40 microliters of blood and store the collected blood in a blood collection tube.Keep the tube on ice until the serum collection process. Dampen the ventral skin generously with 70%ethanol to prevent hair detachment. Using scissors, carefully open the thoracic cavity to expose the heart.Using a one milliliter syringe equipped with a 29-gauge half inch needle, precisely puncture the apex area of the left ventricle. Insert two-thirds of the needle approximately three to five millimeters to the right horizontal of the heart apex, and pull the syringe back to collect blood from the left ventricle. Then, centrifuge the blood samples at 10, 000G for 10 minutes at four degrees Celsius.Using a pipette, collect the supernatant and proceed with metabolite extraction and analysis.