RNA-seq Analysis of Gs-linked GPCRs expressed in mouse inguinal white adipocytes (iWAT), epididymal white adipocytes (eWAT) and brown adipose tissues (BAT)

Purpose: The goals of this study are to identify Gs-linked GPCRs that are endogenously expressed by mouse adipose tissue, we subjected RNA prepared from isolated mouse adipocytes (iWAT and eWAT) and BAT tissue to RNA-seq analysis. Methods: Total RNA extracted from mature adipocytes (iWAT and eWAT) and BAT tissue of C57BL/6J mice (16-week old males) maintained on regular chow were used to construct high throughput sequencing libraries. RNAs with RIN >8 (assessed by the Agilent 2100 Bioanalyzer system) were used to prepare transcriptome libraries using the NEBNext Ultra RNA library prep kit (New England Biolabs). High throughput RNA-sequencing was performed using a HiSeq 2500 instrument (Illumina) at the NIDDK Genomic Core Facility (NIH, Bethesda, MD). Raw reads were mapped to the mouse (mm9) genome. GPCRs were extracted from the RNA-seq data using R form. Gs-coupled GPCRs were identified using the IUPHAR/BPS Guide to Pharmacology Database (https://www.guidetopharmacology.org/). Results: Our study demonstrated that mouse adipocytes/BAT tissue express several GPCRs that are selectively coupled to Gs, including the V2 vasopressin receptor, the glucagon receptor, and different melanocortin receptor subtypes. Inguinal white adipocytes (iWAT), epididymal white adipocytes (eWAT), and brown adipose tissues (BAT) were isolated from 16-week-old male C57BL/6J mice consuming regular chow (n=6)研究目的：本研究旨在鉴定小鼠脂肪组织内源性表达的偶联Gs蛋白的G蛋白偶联受体（G protein-coupled receptor, GPCR），我们对分离的小鼠脂肪细胞（腹股沟白色脂肪细胞（inguinal white adipose tissue, iWAT）、附睾白色脂肪细胞（epididymal white adipose tissue, eWAT））及棕色脂肪组织（brown adipose tissue, BAT）提取的RNA开展RNA测序（RNA-seq）分析。 实验方法：选取饲喂常规饲料的16周龄雄性C57BL/6J小鼠，提取其成熟脂肪细胞（iWAT、eWAT）及BAT组织的总RNA，用于构建高通量测序文库。采用安捷伦2100生物分析仪（Agilent 2100 Bioanalyzer system）检测RNA完整性，选取RNA完整性指数（RNA Integrity Number, RIN）>8的样本，使用NEBNext Ultra RNA文库制备试剂盒（New England Biolabs公司）构建转录组文库。随后于美国国立卫生研究院（NIH，马里兰州贝塞斯达）下属NIDDK基因组核心设施，使用Illumina HiSeq 2500测序仪完成高通量RNA测序。将原始测序reads比对至小鼠mm9参考基因组。通过R语言从RNA-seq数据中提取GPCR数据，并依托国际药理学联合会/英国药理学会药理学指南数据库（https://www.guidetopharmacology.org/）鉴定Gs偶联型GPCR。 研究结果：本研究证实，小鼠脂肪细胞及BAT组织可表达多种选择性偶联Gs蛋白的GPCR，包括血管加压素V2受体、胰高血糖素受体以及多种黑皮质素受体亚型。本实验从饲喂常规饲料的16周龄雄性C57BL/6J小鼠中分离得到iWAT、eWAT及BAT组织，每组样本量n=6。