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

Kahverengi Yağ Dokusunda Metabolit Değişiminin Araştırılması

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.

Yazar Spotlight: Yağ Dokusu Metabolizması ve Hastalıkların Önlenmesi için Yeni Yolların Aydınlatılması 

Kahverengi Yağ Dokusunda İn Vivo Metabolit Değişimini Ölçmek için Arteriyovenöz Metabolomikler

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  

Metabolomik Analiz için Sulzer Veni ve Sol Ventrikül Kullanan Farelerde Arteriyovenöz Kan Örnekleme Tekniği

Başlamak için, fareyi anestezi uygulaması için bir burun konisi ile donatılmış bir diseksiyon tepsisine yerleştirin. Anestezinin uygun bir derinliğe ulaştığını doğrulamak için hayvanın pençe retraksiyonu gibi reflekslerini doğrulayın. Ardından, fareyi sırt derisini ortaya çıkaracak şekilde konumlandırın.Tüylerin ayrılmasını önlemek için cildi bol miktarda %70 etanol ile nemlendirin. Göğüs kafesinin alt kısmından başlayarak sırt boyunca bir kesi yapın ve boyuna kadar uzatın. Bükülmüş uçlu forseps kullanarak, interskapular kahverengi yağ dokusunu yavaşça kaldırın ve bağlı dokuyu dikkatlice kesin.Yağ yastığını başa doğru kaldırın ve Sulzer'in damarı açığa çıkana kadar bölgeyi dikkatlice açmak için bağlı dokuları kesmeye devam edin. Şimdi, Sulzer'in damarını dikkatlice kesin. Alttan kesilmiş 200 mikrolitrelik pipet ucu ve P100 pipet kullanarak yaklaşık 40 mikrolitre kan toplayın ve toplanan kanı bir kan alma tüpünde saklayın.Serum toplama işlemine kadar tüpü buz üzerinde tutun. Saç ayrılmasını önlemek için ventral cildi %70 etanol ile cömertçe nemlendirin. Makas kullanarak, kalbi ortaya çıkarmak için göğüs boşluğunu dikkatlice açın.29 gauge yarım inçlik bir iğne ile donatılmış bir mililitrelik bir şırınga kullanarak, sol ventrikülün apeks bölgesini hassas bir şekilde delin. İğnenin üçte ikisini kalp ucunun sağ yatağına yaklaşık üç ila beş milimetre sokun ve sol ventrikülden kan almak için şırıngayı geri çekin. Daha sonra, kan örneklerini 10.000G'de dört santigrat derecede 10 dakika santrifüjleyin.Bir pipet kullanarak süpernatanı toplayın ve metabolit ekstraksiyonu ve analizine devam edin.

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 Görüntüleme.  Sungkyunkwan University (SKKU). Başlamak için, fareyi anestezi uygulaması için bir burun konisi ile donatılmış bir diseksiyon tepsisine yerleştirin. Anestezinin uygun bir derinliğe ulaştığını doğrulamak için hayvanın pençe retraksiyonu gibi reflekslerini doğrulayın. Ardından, fareyi sırt derisini ortaya çıkaracak şekilde konumlandırın.Tüylerin ayrılmasını önlemek için cildi bol miktarda %70 etanol ile nemlendirin. Göğüs kafesinin alt kısmından başlayarak sırt boyunca bir kesi y...

Video: 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 ...

Biyokimya

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.