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

Utforskar metabolitutbyte i brun fettvävnad

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.

Författare i fokus: Belyser nya vägar för fettvävsmetabolism och förebyggande av sjukdomar 

Arteriovenös metabolomik för att mäta in vivo metabolitutbyte i brun fettvävnad

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  

Arteriovenös blodprovstagningsteknik på möss som använder Sulzers ven och vänster kammare för metabolomisk analys

Börja med att placera musen på en dissektionsbricka utrustad med en näskon för anestesitillförsel. Kontrollera djurets reflexer, t.ex. indragning av tassen, för att bekräfta att bedövningen har nått ett lämpligt djup. Placera sedan musen för att exponera rygghuden.Fukta huden med rikligt med 70 % etanol för att förhindra håravlossning. Gör ett snitt längs ryggen, börja från den nedre delen av bröstkorgen och förläng den upp till halsen. Använd en böjd pincett, lyft försiktigt den interscapulära bruna fettvävnaden och skär försiktigt av den bifogade vävnaden.Lyft fettkudden mot huvudet och fortsätt att skära av de bifogade vävnaderna för att försiktigt öppna området tills Sulzers ven är exponerad. Skär nu försiktigt av Sulzers ven. Använd en 200 mikroliters pipettspets och en P100-pipett, samla upp cirka 40 mikroliter blod och förvara det uppsamlade blodet i ett blodprovsrör.Håll tuben på is tills serumuppsamlingsprocessen. Fukta den ventrala huden generöst med 70% etanol för att förhindra håravlossning. Använd en sax och öppna försiktigt brösthålan för att exponera hjärtat.Använd en enmilliliters spruta utrustad med en 29-gauge halv tums nål, punktera exakt spetsområdet på vänster kammare. För in två tredjedelar av nålen cirka tre till fem millimeter till höger horisontellt om hjärtspetsen och dra tillbaka sprutan för att samla upp blod från vänster kammare. Centrifugera sedan blodproverna vid 10 000 G i 10 minuter vid fyra grader Celsius.Använd en pipett, samla upp supernatanten och fortsätt med extraktion och analys av metaboliten.

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 ...