Name: Arteriovenous Blood Sampling Technique in Mice Using Sulzer's Vein and Left Ventricle for Metabolomic Analysis
Uploaded: 2023-10-06T12:40:00
Description: 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's vein and left ventricle for metabolomic analysis

Explorando el intercambio de metabolitos en el tejido adiposo marrón

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.

Autor destacado: Iluminando nuevas vías para el metabolismo del tejido adiposo y la prevención de enfermedades 

Metabolómica arteriovenosa para medir el intercambio de metabolitos in vivo en tejido adiposo marrón

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's Vein and Left Ventricle for Metabolomic Analysis  

Técnica de toma de muestras de sangre arteriovenosa en ratones utilizando la vena de Sulzer y el ventrículo izquierdo para su análisis metabolómico

Para comenzar, coloque el mouse en una bandeja de disección equipada con un cono nasal para la administración de anestesia. Verifique los reflejos del animal, como la retracción de las patas, para confirmar que la anestesia ha alcanzado una profundidad adecuada. A continuación, coloque el ratón para exponer la piel dorsal.Humedece la piel con una gran cantidad de etanol al 70% para evitar el desprendimiento del cabello. Haz una incisión a lo largo de la espalda, comenzando desde la parte inferior del tórax y extiéndela hasta el cuello. Con unas pinzas de punta doblada, levante suavemente el tejido adiposo marrón interescapular y corte con cuidado el tejido adjunto.Levante la almohadilla de grasa hacia la cabeza y continúe cortando los tejidos adheridos para abrir con cuidado la región hasta que la vena de Sulzer quede expuesta. Ahora, corta con cuidado la vena del Sulzer. Utilizando la punta de pipeta de 200 microlitros y una pipeta P100, recoja aproximadamente 40 microlitros de sangre y almacene la sangre recolectada en un tubo de extracción de sangre.Mantenga el tubo en hielo hasta el proceso de recolección del suero. Humedecer generosamente la piel ventral con etanol al 70% para evitar el desprendimiento del vello. Con unas tijeras, abra con cuidado la cavidad torácica para exponer el corazón.Con una jeringa de un mililitro equipada con una aguja de media pulgada de calibre 29, perfore con precisión el área del ápice del ventrículo izquierdo. Inserte dos tercios de la aguja aproximadamente de tres a cinco milímetros hacia la horizontal derecha del ápice cardíaco y tire de la jeringa hacia atrás para recolectar sangre del ventrículo izquierdo. A continuación, centrifugar las muestras de sangre a 10.000 g durante 10 minutos a cuatro grados centígrados.Con una pipeta, recoja el sobrenadante y proceda a la extracción y análisis de metabolitos.

Research

JoVE Journal

Biochemistry

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 ...