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

Utforsker metabolittutveksling i brunt fettvev

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.

Forfatter Spotlight: Belyse nye veier for fettvevsmetabolisme og sykdomsforebygging 

Arteriovenøs metabolomikk for å måle in vivo metabolittutveksling i brunt fettvev

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  

Arteriovenøs blodprøvetakingsteknikk hos mus ved bruk av Sulzers vene og venstre ventrikkel for metabolomisk analyse

For å begynne, plasser musen på et disseksjonsbrett utstyrt med en nesekegle for anestesilevering. Bekreft dyrets reflekser, for eksempel pote tilbaketrekning, for å bekrefte at anestesien har nådd en passende dybde. Plasser deretter musen for å eksponere rygghuden.Fukt huden med rikelig med 70% etanol for å forhindre hårløsrivelse. Lag et snitt langs ryggen, start fra den nedre delen av thoraxen og forleng den opp til nakken. Bruk bøyde spisstanger, løft forsiktig det interskapulære brune fettvevet og kutt forsiktig det vedlagte vevet.Løft fettputen mot hodet og fortsett å kutte det vedlagte vevet for å åpne regionen forsiktig til Sulzer-venen blir eksponert. Kutt nå forsiktig Sulzer-venen. Bruk bunnkutt 200 mikroliter pipettespiss og en P100-pipette, samle ca. 40 mikroliter blod og lagre det oppsamlede blodet i et blodoppsamlingsrør.Hold røret på is til serumoppsamlingsprosessen. Fukt den ventrale huden sjenerøst med 70% etanol for å forhindre hårløsing. Bruk saks, åpne forsiktig thoraxhulen for å avsløre hjertet.Ved hjelp av en milliliter sprøyte utstyrt med en 29-gauge halv tommers nål, nøyaktig punktere apexområdet av venstre ventrikel. Sett inn to tredjedeler av nålen omtrent tre til fem millimeter til høyre horisontalt for hjertespissen, og trekk sprøyten tilbake for å samle blod fra venstre ventrikkel. Deretter sentrifugerer du blodprøvene ved 10.000 G i 10 minutter ved fire grader Celsius.Bruk en pipette til å samle supernatanten og fortsett med metabolittekstraksjon og 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 ...