fphys-09-01931 January 14, 2019 Time: 14:42 # 1 ORIGINAL RESEARCH published: 17 January 2019 doi: 10.3389/fphys.2018.01931 Opposing Actions of Adrenocorticotropic Hormone and Glucocorticoids on UCP1-Mediated Respiration in Brown Adipocytes Katharina Schnabl1,2,3, Julia Westermeier1,2, Yongguo Li1,2 and Martin Klingenspor1,2,3* 1 Chair for Molecular Nutritional Medicine, TUM School of Life Sciences Weihenstephan, Technical University of Munich, Freising, Germany, 2 EKFZ – Else Kröner-Fresenius Zentrum for Nutritional Medicine, Technical University of Munich, Freising, Germany, 3 ZIEL – Institute for Food & Health, Technical University of Munich, Freising, Germany Brown fat is a potential target in the treatment of metabolic disorders as recruitment and activation of this thermogenic organ increases energy expenditure and promotes Edited by: Rita De Matteis, satiation. A large variety of G-protein coupled receptors, known as classical drug Università degli Studi di Urbino targets in pharmacotherapy, is expressed in brown adipocytes. In the present study, Carlo Bo, Italy we analyzed transcriptome data for the expression of these receptors to identify Reviewed by: Alessandro Bartolomucci, potential pathways for the recruitment and activation of thermogenic capacity in University of Minnesota Twin Cities, brown fat. Our analysis revealed 12 Gs-coupled receptors abundantly expressed in United States murine brown fat. We screened ligands for these receptors in brown adipocytes Vicente Lahera, Complutense University of Madrid, for their ability to stimulate UCP1-mediated respiration and Ucp1 gene expression. Spain Adrenocorticotropic hormone (ACTH), a ligand for the melanocortin 2 receptor (MC2R), *Correspondence: turned out to be the most potent activator of UCP1 whereas its capability to stimulate Martin Klingenspor [email protected] Ucp1 gene expression was comparably low. Adrenocorticotropic hormone is the glandotropic hormone of the endocrine hypothalamus–pituitary–adrenal-axis stimulating Specialty section: the release of glucocorticoids in response to stress. In primary brown adipocytes ACTH This article was submitted to Integrative Physiology, acutely increased the cellular respiration rate similar to isoproterenol, a b-adrenergic a section of the journal receptor agonist. The effect of ACTH on brown adipocyte respiration was mediated Frontiers in Physiology via the MC2R as confirmed by using an antagonist. Inhibitor-based studies revealed Received: 08 October 2018 that ACTH-induced respiration was dependent on protein kinase A and lipolysis, Accepted: 21 December 2018 Published: 17 January 2019 compatible with a rise of intracellular cAMP in response to ACTH. Furthermore, it Citation: is dependent on UCP1, as cells from UCP1-knockout mice did not respond. Taken Schnabl K, Westermeier J, Li Y together, ACTH is a non-adrenergic activator of murine brown adipocytes, initiating and Klingenspor M (2019) Opposing Actions of Adrenocorticotropic the canonical adenylyl cyclase–cAMP–protein kinase A-lipolysis-UCP1 pathway, and Hormone and Glucocorticoids on thus a potential target for the recruitment and activation of thermogenic capacity. UCP1-Mediated Respiration in Brown Based on these findings in primary cell culture, the physiological significance might Adipocytes. Front. Physiol. 9:1931. doi: 10.3389/fphys.2018.01931 be that cold-induced ACTH in concert with norepinephrine released from sympathetic Frontiers in Physiology| www.frontiersin.org 1 January 2019| Volume 9| Article 1931 fphys-09-01931 January 14, 2019 Time: 14:42 # 2 Schnabl et al. Non-Adrenergic Modulators of Brown Fat nerves contributes to BAT thermogenesis. Notably, dexamethasone attenuated isoproterenol-induced respiration. This effect increased gradually with the duration of pretreatment. In vivo, glucocorticoid release triggered by ACTH might oppose beta- adrenergic stimulation of metabolic fuel combustion in BAT and limit stress-induced hyperthermia. Keywords: glucocorticoids (GC), brown adipose tissue, non-adrenergic activation, non-shivering thermogenesis, uncoupling protein 1, adrenocorticotropic hormone, obesity INTRODUCTION expenditure through UCP1 dependent energy dissipation. The recruitment and activation of BAT and appears as an attractive We are facing a worldwide epidemic of obesity, a major risk and potentially effective strategy for the prevention and treatment factor in the development of non-communicable diseases such of obesity (Tseng et al., 2010). Importantly, in the attempt to as diabetes mellitus and arteriosclerosis. In 2016, 1.9 billion increase energy expenditure, the recruitment of more brown adults aged 18 and over were overweight, of which 650 million adipocytes with higher UCP1 expression and higher respiration were obese, representing an almost threefold increase in obesity capacity is required, but not sufficient. UCP1 is not constitutively prevalence since 1975 (WHO, 2018). Obesity is the state of active, but rather must be activated to dissipate mitochondrial excessive white adipose tissue (WAT) accumulation caused by proton-motive force as heat. This activation of UCP1 is inevitable prolonged positive energy balance. In mammals there is a second to increase carbohydrate and lipid oxidation. Beyond boosting type of adipose tissue, brown adipose tissue (BAT), which in energy expenditure, we recently demonstrated that meal-induced contrast to WAT generates heat in response to cold exposure activation of BAT thermogenesis also induces satiation which and food consumption (Rosen and Spiegelman, 2006; Vosselman might also be applicable to promote negative energy balance (Li et al., 2013; U Din et al., 2018). It dissipates the chemical energy of et al., 2018). macro-nutrients by uncoupling oxygen consumption from ATP As sympathomimetic drugs exhibit unwanted detrimental synthesis in mitochondria (Klingenspor, 2003). This mechanism, cardiovascular effects, their application as BAT-stimulating known as non-shivering thermogenesis, is dependent on the agents is considered problematic (Cypess et al., 2015). Thus, presence of mitochondrial uncoupling protein 1 (UCP1), which the identification of druggable non-adrenergic regulators of BAT is a unique feature of brown adipocytes. The activation of is one step toward the modulation of the heating organ as a BAT increases energy expenditure and opposes positive energy regulator of energy expenditure and body fat in humans. balance. In addition to the activation of UCP1, it is worth Besides the b-adrenergic receptors mature brown adipocytes noting that cold exposure also induces the thermogenic gene express a variety of around 230 other G protein-coupled expression program and thereby recruits thermogenic capacity in receptors (GPCRs) (Klepac et al., 2016) which are responsible brown fat (Cannon and Nedergaard, 2004). Furthermore, brown- for transferring extracellular signals to the cytosol. GPCRs group like adipocytes, also known as inducible brown fat cells, beige in a large family of seven transmembrane proteins (Lefkowitz, (Ishibashi and Seale, 2010) or “brite” (brown-in-white) (Petrovic 2007; Kobilka, 2011) that regulate important biological processes et al., 2010) adipocytes, can be found interspersed in WAT (Li in diverse tissues including adipose tissues (Wettschureck and et al., 2014a). Similar to brown adipocytes in classical brown fat Offermanns, 2005; Latek et al., 2012). Approximately 30% of depots brite adipocyte abundance can be increased by adrenergic all approved drugs target GPCRs, illustrating their importance stimulation (Galmozzi et al., 2014) and cold exposure (Maurer in disease and therapeutics (Hauser et al., 2017; Santos et al., et al., 2015) in a process coined browning of WAT. Besides 2017). These receptors are coupled to heterotrimeric G proteins activation and recruitment of BAT, the browning of WAT displays which are composed of a, b, and g subunits. Ligand binding therapeutic potential with regard to the development of new and thus activation of GPCRs leads to the dissociation of Ga obesity treatment strategies. from the Gbg dimer, allowing the binding and regulation of For a long time, the occurrence of metabolically active BAT signaling effectors. The downstream signaling of GPCRs is in part was believed to be restricted to hibernators, small mammals determined by their G protein coupling (Neves et al., 2002). There and human newborns. Adult humans, however, also have are four main sub-classes of Ga proteins: Gs,Gi,Gq and G12/13. metabolically active BAT, as demonstrated by the detection of Activation of Gs and Gi leads to the stimulation or inhibition cold-induced uptake of tracers for glucose, fatty acids and acetate the second messenger cyclic adenosine monophosphate (cAMP), with positron emission tomography (Cypess et al., 2009, 2015; respectively, while Gq activates phospholipase C und thus, to van Marken Lichtenbelt et al., 2009; Virtanen et al., 2009; Ouellet an increase of inositol triphosphate. G12=13 activates the small et al., 2011). Furthermore, cold-induced BAT activity is strongly GTPase Rho, a pathway also known to be modulated by Gq reduced in obese (Saito et al., 2009) and diabetic patients and can family proteins (Buhl et al., 1995; Wang et al., 2013). Due to be recovered by cold acclimation (van Marken Lichtenbelt et al., cAMP-PKA activating properties the analysis of BAT GPCRs 2009). has mainly focused on Gs-coupled receptors [for example, On this background BAT displays a focal point of current b-adrenergic (Cannon and Nedergaard, 2004) and adenosine research