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

Onderzoek naar metabolietuitwisseling in bruin vetweefsel

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.

Auteur in de schijnwerpers: nieuwe wegen belichten voor het metabolisme van vetweefsel en ziektepreventie 

Arterioveneuze metabolomics om in vivo metabolietuitwisseling in bruin vetweefsel te meten

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  

Arterioveneuze bloedafnametechniek bij muizen met behulp van de ader en linkerventrikel van Sulzer voor metabolomische analyse

Plaats om te beginnen de muis op een dissectiebak die is uitgerust met een neuskegel voor anesthesietoediening. Controleer de reflexen van het dier, zoals het intrekken van de poten, om te bevestigen dat de anesthesie de juiste diepte heeft bereikt. Plaats vervolgens de muis om de dorsale huid bloot te leggen.Bevochtig de huid met voldoende 70% ethanol om loslating van het haar te voorkomen. Maak een incisie langs de rug, beginnend bij het onderste deel van de thorax en verleng deze tot aan de nek. Til met een pincet met gebogen punt voorzichtig het interscapulaire bruine vetweefsel op en snijd voorzichtig het aangehechte weefsel door.Til het vetkussen naar het hoofd en ga door met het snijden van de aangehechte weefsels om het gebied voorzichtig te openen totdat de ader van Sulzer bloot komt te liggen. Snijd nu voorzichtig de ader van Sulzer door. Verzamel met behulp van een pipetpunt van 200 microliter en een P100-pipet ongeveer 40 microliter bloed en bewaar het verzamelde bloed in een bloedafnamebuisje.Houd de tube op ijs tot het serumverzamelproces. Bevochtig de ventrale huid royaal met 70% ethanol om loslating van het haar te voorkomen. Open met een schaar voorzichtig de borstholte om het hart bloot te leggen.Gebruik een spuit van één milliliter die is uitgerust met een naald van 29 gauge van een halve inch om precies het topgebied van de linker hartkamer te doorboren. Steek tweederde van de naald ongeveer drie tot vijf millimeter naar rechts horizontaal van de harttop en trek de spuit terug om bloed uit de linker hartkamer te verzamelen. Centrifugeer vervolgens de bloedmonsters bij 10.000 G gedurende 10 minuten bij vier graden Celsius.Verzamel met behulp van een pipet het supernatans en ga verder met metabolietextractie en -analyse.

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 ...