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

استكشاف تبادل الأيض في الأنسجة الدهنية البنية

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.

تسليط الضوء على المؤلف: إلقاء الضوء على طرق جديدة لاستقلاب الأنسجة الدهنية والوقاية من الأمراض 

الأيض الشرياني الوريدي لقياس تبادل الأيض في الجسم الحي في الأنسجة الدهنية البنية

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  

تقنية أخذ عينات الدم الشرياني الوريدي في الفئران باستخدام وريد سولزر والبطين الأيسر لتحليل التمثيل الغذائي

للبدء ، ضع الماوس على صينية تشريح مجهزة بمخروط الأنف لتوصيل التخدير. تحقق من ردود فعل ، مثل تراجع المخلب ، للتأكد من أن التخدير قد وصل إلى عمق مناسب. ثم ضع الماوس لكشف الجلد الظهري.بللي البشرة بكمية وافرة من الإيثانول بنسبة 70٪ لمنع انفصال الشعر. قم بعمل شق على طول الظهر ، بدءا من الجزء السفلي من الصدر وقم بتمديده حتى الرقبة. باستخدام ملقط الطرف المثني ، ارفع الأنسجة الدهنية البنية بين الكتفين برفق واقطع الأنسجة المرفقة بعناية.ارفع وسادة الدهون باتجاه الرأس واستمر في قطع الأنسجة المرفقة لفتح المنطقة بعناية حتى ينكشف وريد سولزر. الآن ، قطع بعناية الوريد سولزر. باستخدام طرف ماصة 200 ميكرولتر وماصة P100 ، اجمع ما يقرب من 40 ميكرولترا من الدم وقم بتخزين الدم الذي تم جمعه في أنبوب جمع الدم.احتفظ بالأنبوب على الثلج حتى عملية جمع المصل. بللي الجلد البطني بسخاء ب 70٪ إيثانول لمنع انفصال الشعر. باستخدام المقص ، افتح التجويف الصدري بعناية لفضح القلب.باستخدام حقنة واحدة ملليلتر مجهزة بإبرة قياس 29 نصف بوصة ، ثقب بدقة منطقة قمة البطين الأيسر. أدخل ثلثي الإبرة حوالي ثلاثة إلى خمسة ملليمترات إلى الأفقي الأيمن لقمة القلب، واسحب المحقنة للخلف لجمع الدم من البطين الأيسر. ثم ، أجهزة الطرد المركزي عينات الدم في 10،000G لمدة 10 دقائق عند أربع درجات مئوية.باستخدام ماصة ، وجمع طاف والمضي قدما في استخراج المستقلب والتحليل.

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 ...