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. 2019 Jan 17;9:1931. doi: 10.3389/fphys.2018.01931

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Copyright © 2019 Schnabl, Westermeier, Li and Klingenspor.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Screening approach of G_sPCR ligands expressed in brown adipose tissue concerning their ability to activate and recruit Ucp1. (A) Gene expression of G_s-protein coupled receptors (G_sPCRs) obtained from RNA sequencing of murine brown adipose tissue. Data are shown as RPKM (reads per kilobase per million mapped reads). The respective ligands of the receptors are shown in italics above the bars. G_sPCRs with a lower expression have been neglected for the analysis (n = 4). (B) Microplate-based respirometry of primary brown adipocytes following the subsequent protocol. After assessment of basal oxygen consumption oligo (oligomycin) was injected to determine basal leak respiration. Next, compound to be tested was added to investigate uncoupling protein 1-dependant respiration. By the addition of FCCP maximal leak respiration was determined. Lastly, non-mitochondrial oxygen consumption was assessed by injecting anti A. Shown is a time course of oxygen consumption in percentage of basal respiration of primary brown adipocytes (129S6 mice) in response to different stimuli (effectors). (C) Stimulated respiration of primary brown adipocytes as fold increase of basal leak respiration (n = 5). (D) Overnight stimulation of primary brown adipocytes from Ucp1^ki/ki mice expressing the firefly luciferase driven by the gene regulatory elements of endogenous Ucp1 gene. Measurement of luciferase activity as surrogate for Ucp1 expression (n = 3). (E) Plot of Ucp1 enhancing properties against UCP1 activating properties of screened ligands relative to control. Dashed lines indicate control levels (=1), continuous gray line indicates line of equality. Data are presented as means ± SD. Pth1r, parathyroid hormone 1 receptor; Adora2a, adenosine receptor; Sctr, secretin receptor; Gpr4, GPCR4; Tshr, thyroid stimulating hormone receptor; Calcrl, calcitonin receptor-like receptor; Mc5r, melanocortin receptor 5, Ptger4, prostaglandin E receptor 4; Mc2r, melanocortin receptor 2; Gpr64, GPCR 64; Gpr133, GPCR 133; Adcyap1r1, adenylate cyclase activating polypeptide 1 receptor; TSH, thyroid stimulating hormone; CGS21680, adenosine A2A receptor agonist; CGRP, calcitonin gene-related peptide; α-MSH, melanocyte-stimulating hormone; ACTH, adrenocorticotropic hormone; PGE₂, prostaglandin E2; PTHrP, parathyroid hormone related peptide; PTH, parathyroid hormone; ISO, isoproterenol; oligo, oligomycin; FCCP, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; anti A, antimycin A.