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Endocannabinoid regulation in white and brown adipose tissue following thermogenic activation

2016; Elsevier BV; Volume: 57; Issue: 3 Linguagem: Inglês

10.1194/jlr.m065227

ISSN

1539-7262

Autores

Lucia M. Krott, Fabiana Piscitelli, Markus Heine, Simona Borrino, Ludger Scheja, Cristoforo Silvestri, Jöerg Heeren, Vincenzo Di Marzo,

Tópico(s)

Lipid metabolism and biosynthesis

Resumo

The endocannabinoids and their main receptor, cannabinoid type-1 (CB1), suppress intracellular cyclic AMP levels and have emerged as key players in the control of energy metabolism. CB1 agonists and blockers have been reported to influence the thermogenic function of white and brown adipose tissue (WAT and BAT), affecting body weight through the inhibition and stimulation of energy expenditure, respectively. The purpose of the current study was to investigate the regulation of the endocannabinoid system in WAT and BAT following exposure to either cold or specific agonism of β3-adrenoceptors using CL316,243 (CL), conditions known to cause BAT activation and WAT browning. To address this question, we performed quantitative PCR-based mRNA profiling of genes important for endocannabinoid synthesis, degradation, and signaling, and determined endocannabinoid levels by LC-MS in WAT and BAT of control, cold-exposed, and CL-treated wild-type mice as well as primary brown adipocytes. Treatment with CL and exposure to cold caused an upregulation of endocannabinoid levels and biosynthetic enzymes in WAT. Acute β3-adrenoceptor activation increased endocannabinoids and a subset of genes of biosynthesis in BAT and primary brown adipocytes. We suggest that the cold-mediated increase in endocannabinoid tone is part of autocrine negative feed-back mechanisms controlling β3-adrenoceptor-induced BAT activation and WAT browning. The endocannabinoids and their main receptor, cannabinoid type-1 (CB1), suppress intracellular cyclic AMP levels and have emerged as key players in the control of energy metabolism. CB1 agonists and blockers have been reported to influence the thermogenic function of white and brown adipose tissue (WAT and BAT), affecting body weight through the inhibition and stimulation of energy expenditure, respectively. The purpose of the current study was to investigate the regulation of the endocannabinoid system in WAT and BAT following exposure to either cold or specific agonism of β3-adrenoceptors using CL316,243 (CL), conditions known to cause BAT activation and WAT browning. To address this question, we performed quantitative PCR-based mRNA profiling of genes important for endocannabinoid synthesis, degradation, and signaling, and determined endocannabinoid levels by LC-MS in WAT and BAT of control, cold-exposed, and CL-treated wild-type mice as well as primary brown adipocytes. Treatment with CL and exposure to cold caused an upregulation of endocannabinoid levels and biosynthetic enzymes in WAT. Acute β3-adrenoceptor activation increased endocannabinoids and a subset of genes of biosynthesis in BAT and primary brown adipocytes. We suggest that the cold-mediated increase in endocannabinoid tone is part of autocrine negative feed-back mechanisms controlling β3-adrenoceptor-induced BAT activation and WAT browning. The recent discovery of active brown adipose tissue (BAT) in adult humans (1Cypess A.M. Lehman S. Williams G. Tal I. Rodman D. Goldfine A.B. Kuo F.C. Palmer E.L. Tseng Y.H. Doria A. et al.Identification and importance of brown adipose tissue in adult humans.N. Engl. J. Med. 2009; 360: 1509-1517Crossref PubMed Scopus (3139) Google Scholar, 2van Marken Lichtenbelt W.D. Vanhommerig J.W. Smulders N.M. Drossaerts J.M. Kemerink G.J. Bouvy N.D. Schrauwen P. Teule G.J. Cold-activated brown adipose tissue in healthy men.N. Engl. J. Med. 2009; 360: 1500-1508Crossref PubMed Scopus (2589) Google Scholar, 3Virtanen K.A. Lidell M.E. Orava J. Heglind M. Westergren R. Niemi T. Taittonen M. Laine J. Savisto N.J. Enerback S. et al.Functional brown adipose tissue in healthy adults.N. Engl. J. Med. 2009; 360: 1518-1525Crossref PubMed Scopus (2316) Google Scholar) is one of the most intriguing findings, as it raises hope for the treatment of obesity and related chronic metabolic diseases. The natural function of BAT is to combust energy from high-caloric nutrients to defend the body against cold environments (4Bartelt A. Heeren J. The holy grail of metabolic disease: brown adipose tissue.Curr. Opin. Lipidol. 2012; 23: 190-195Crossref PubMed Scopus (59) Google Scholar). The ability to burn energy-dense triglycerides as fuels for heat production could enable BAT to diminish hypertrophic white adipose tissue (WAT) depots, a prerequisite for the prevention of metabolic lifestyle diseases (5Eriksson J. Lindstrom J. Tuomilehto J. Potential for the prevention of type 2 diabetes.Br. Med. Bull. 2001; 60: 183-199Crossref PubMed Scopus (44) Google Scholar, 6Knowler W.C. Barrett-Connor E. Fowler S.E. Hamman R.F. Lachin J.M. Walker E.A. Nathan D.M. Diabetes Prevention Program Research Group Reduction in the incidence of type 2 diabetes with lifestyle intervention or metformin.N. Engl. J. Med. 2002; 346: 393-403Crossref PubMed Scopus (14526) Google Scholar). In humans, BAT activity, determined by positron emission tomography-computed tomography (PET-CT), is positively correlated with BAT mass (1Cypess A.M. Lehman S. Williams G. Tal I. Rodman D. Goldfine A.B. Kuo F.C. Palmer E.L. Tseng Y.H. Doria A. et al.Identification and importance of brown adipose tissue in adult humans.N. Engl. J. Med. 2009; 360: 1509-1517Crossref PubMed Scopus (3139) Google Scholar, 2van Marken Lichtenbelt W.D. Vanhommerig J.W. Smulders N.M. Drossaerts J.M. Kemerink G.J. Bouvy N.D. Schrauwen P. Teule G.J. Cold-activated brown adipose tissue in healthy men.N. Engl. J. Med. 2009; 360: 1500-1508Crossref PubMed Scopus (2589) Google Scholar, 3Virtanen K.A. Lidell M.E. Orava J. Heglind M. Westergren R. Niemi T. Taittonen M. Laine J. Savisto N.J. Enerback S. et al.Functional brown adipose tissue in healthy adults.N. Engl. J. Med. 2009; 360: 1518-1525Crossref PubMed Scopus (2316) Google Scholar), BAT activation status (2van Marken Lichtenbelt W.D. Vanhommerig J.W. Smulders N.M. Drossaerts J.M. Kemerink G.J. Bouvy N.D. Schrauwen P. Teule G.J. Cold-activated brown adipose tissue in healthy men.N. Engl. J. Med. 2009; 360: 1500-1508Crossref PubMed Scopus (2589) Google Scholar), and environmental factors such as low temperatures (7Ouellet V. Routhier-Labadie A. Bellemare W. Lakhal-Chaieb L. Turcotte E. Carpentier A.C. Richard D. Outdoor temperature, age, sex, body mass index, and diabetic status determine the prevalence, mass, and glucose-uptake activity of 18F-FDG-detected BAT in humans.J. Clin. Endocrinol. Metab. 2011; 96: 192-199Crossref PubMed Scopus (401) Google Scholar). Repeated cold exposure leads to increased BAT activity (8Yoneshiro T. Aita S. Matsushita M. Kayahara T. Kameya T. Kawai Y. Iwanaga T. Saito M. Recruited brown adipose tissue as an antiobesity agent in humans.J. Clin. Invest. 2013; 123: 3404-3408Crossref PubMed Scopus (668) Google Scholar, 9van der Lans A.A. Hoeks J. Brans B. Vijgen G.H. Visser M.G. Vosselman M.J. Hansen J. Jorgensen J.A. Wu J. Mottaghy F.M. et al.Cold acclimation recruits human brown fat and increases nonshivering thermogenesis.J. Clin. Invest. 2013; 123: 3395-3403Crossref PubMed Scopus (588) Google Scholar), a condition that is associated with a self-reported decrease in sensitivity to cold. The thermogenic process is dependent on the presence of uncoupling protein 1 (UCP1), a protein located in the inner membrane of mitochondria that is able to separate electron transport in the respiratory chain from the production of energy in the form of ATP. The heat generated by this exothermic reaction is transported via the blood circulation system to maintain body temperature (10Cannon B. Nedergaard J. Brown adipose tissue: function and physiological significance.Physiol. Rev. 2004; 84: 277-359Crossref PubMed Scopus (4525) Google Scholar). Low BAT activity in humans correlates with ageing and obesity (2van Marken Lichtenbelt W.D. Vanhommerig J.W. Smulders N.M. Drossaerts J.M. Kemerink G.J. Bouvy N.D. Schrauwen P. Teule G.J. Cold-activated brown adipose tissue in healthy men.N. Engl. J. Med. 2009; 360: 1500-1508Crossref PubMed Scopus (2589) Google Scholar, 11Yoneshiro T. Aita S. Matsushita M. Okamatsu-Ogura Y. Kameya T. Kawai Y. Miyagawa M. Tsujisaki M. Saito M. Age-related decrease in cold-activated brown adipose tissue and accumulation of body fat in healthy humans.Obesity (Silver Spring). 2011; 19: 1755-1760Crossref PubMed Scopus (324) Google Scholar), suggesting a causal link between decreased BAT activity, weight gain, and the development of metabolic diseases. In this context, channeling fatty acids and triglycerides into BAT could attenuate deleterious effects that saturated fatty acids cause by ectopic lipid accumulation in the liver or heart. In fact, up to 90% of energy for heat production is derived from fatty acids that are delivered by triglyceride-rich lipoproteins (4Bartelt A. Heeren J. The holy grail of metabolic disease: brown adipose tissue.Curr. Opin. Lipidol. 2012; 23: 190-195Crossref PubMed Scopus (59) Google Scholar, 10Cannon B. Nedergaard J. Brown adipose tissue: function and physiological significance.Physiol. Rev. 2004; 84: 277-359Crossref PubMed Scopus (4525) Google Scholar, 12Bartelt A. Bruns O.T. Reimer R. Hohenberg H. Ittrich H. Peldschus K. Kaul M.G. Tromsdorf U.I. Weller H. Waurisch C. et al.Brown adipose tissue activity controls triglyceride clearance.Nat. Med. 2011; 17: 200-205Crossref PubMed Scopus (1152) Google Scholar). These latter are hepatic VLDLs and intestinal chylomicrons that are both processed by endothelium-bound lipoprotein lipase to allocate fatty acids to BAT and energy storing WAT, respectively. In response to cold exposure, both brown and white adipocytes are activated via sympathetic neurons that release noradrenalin (10Cannon B. Nedergaard J. Brown adipose tissue: function and physiological significance.Physiol. Rev. 2004; 84: 277-359Crossref PubMed Scopus (4525) Google Scholar). Catecholamine release causes the activation of β3-adrenoceptor signaling stimulating lipolysis of triglycerides stored in lipid droplets, a process mediated by the enzymatic activity of adipose tissue triglyceride lipase (ATGL) and hormone sensitive lipase (HSL) (13Young S.G. Zechner R. Biochemistry and pathophysiology of intravascular and intracellular lipolysis.Genes Dev. 2013; 27: 459-484Crossref PubMed Scopus (243) Google Scholar). In brown adipocytes, fatty acids are transferred to mitochondria for β-oxidation and UCP1-dependent heat production. In white adipocytes, lipid droplet-derived fatty acids are released into the circulation for hepatic VLDL production to maintain energy supply for cold-activated brown adipocytes. Thus, short term activation of β3-adrenoceptors stimulates intracellular lipolysis orchestrating the systemic energy homeostasis. It is not surprising that both WAT and BAT undergo adaptive and dynamic changes in response to sustained β3-adrenoceptor activation or cold exposure. In this context one of the most intensively studied cell types implicated in cold-induced tissue remodeling in WAT (browning) is the so-called beige adipocyte (14Wu J. Cohen P. Spiegelman B.M. Adaptive thermogenesis in adipocytes: is beige the new brown?.Genes Dev. 2013; 27: 234-250Crossref PubMed Scopus (628) Google Scholar). Prolonged cold exposure or pharmacological treatment using β3-adrenoceptor agonists, such as CL316,243 (CL), stimulate the development of these inducible brown-like adipocytes (14Wu J. Cohen P. Spiegelman B.M. Adaptive thermogenesis in adipocytes: is beige the new brown?.Genes Dev. 2013; 27: 234-250Crossref PubMed Scopus (628) Google Scholar, 15Bartelt A. Heeren J. Adipose tissue browning and metabolic health.Nat. Rev. Endocrinol. 2014; 10: 24-36Crossref PubMed Scopus (722) Google Scholar, 16Lowell B.B. Spiegelman B.M. Towards a molecular understanding of adaptive thermogenesis.Nature. 2000; 404: 652-660Crossref PubMed Scopus (1306) Google Scholar, 17Zhao J. Cannon B. Nedergaard J. Thermogenesis is beta3- but not beta1-adrenergically mediated in rat brown fat cells, even after cold acclimation.Am. J. Physiol. 1998; 275: R2002-R2011PubMed Google Scholar). Brown and beige adipocytes are characterized by a large number of mitochondria and numerous small lipid droplets and both cell types are functionally active with regard to adaptive thermogenesis (18Shabalina I.G. Petrovic N. de Jong J.M. Kalinovich A.V. Cannon B. Nedergaard J. UCP1 in brite/beige adipose tissue mitochondria is functionally thermogenic.Cell Reports. 2013; 5: 1196-1203Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar). The endocannabinoid system and, in particular, the G protein-coupled receptor known as cannabinoid type-1 (CB1) and its endogenous agonists, N-arachidonoylethanolamine (AEA, anandamide) and 2-arachidonoylglycerol (2-AG), have emerged as major players in the control of metabolism at both the central and peripheral level (19Silvestri C. Di Marzo V. The endocannabinoid system in energy homeostasis and the etiopathology of metabolic disorders.Cell Metab. 2013; 17: 475-490Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar). Importantly, malfunctioning of this system due to enhanced tissue levels of endocannabinoids and subsequent overactivation of CB1 receptors in the hypothalamus, visceral WAT, liver, muscle, and pancreas, accompanies, and is probably one of the causes of, fat accumulation and insulin resistance in animal models of obesity. High plasmatic levels of endocannabinoids have been associated with increased cardiometabolic risk, visceral WAT accumulation, and type 2 diabetes in obese patients (19Silvestri C. Di Marzo V. The endocannabinoid system in energy homeostasis and the etiopathology of metabolic disorders.Cell Metab. 2013; 17: 475-490Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar). Genetic impairment of CB1 receptors selectively in neurons of the brain and sympathetic nervous system (SNS) enhances energy expenditure by the BAT (20Quarta C. Bellocchio L. Mancini G. Mazza R. Cervino C. Braulke L.J. Fekete C. Latorre R. Nanni C. Bucci M. et al.CB(1) signaling in forebrain and sympathetic neurons is a key determinant of endocannabinoid actions on energy balance.Cell Metab. 2010; 11: 273-285Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar), and very recent evidence suggests an inhibitory role of prejunctional CB1 on brown adipocyte glucose utilization (21Bajzer M. Olivieri M. Haas M.K. Pfluger P.T. Magrisso I.J. Foster M.T. Tschop M.H. Krawczewski-Carhuatanta K.A. Cota D. Obici S. Cannabinoid receptor 1 (CB1) antagonism enhances glucose utilisation and activates brown adipose tissue in diet-induced obese mice.Diabetologia. 2011; 54: 3121-3131Crossref PubMed Scopus (70) Google Scholar), thermogenesis, and lipid droplet formation (22Boon M.R. Kooijman S. van Dam A.D. Pelgrom L.R. Berbee J.F. Visseren C.A. van Aggele R.C. van den Hoek A.M. Sips H.C. Lombes M. et al.Peripheral cannabinoid 1 receptor blockade activates brown adipose tissue and diminishes dyslipidemia and obesity.FASEB J. 2014; 28: 5361-5375Crossref PubMed Scopus (75) Google Scholar). Furthermore, CB1 blockade in Sim1-expressing neurons of the paraventricular nucleus of the hypothalamus enhances energy expenditure during high-fat diet feeding (23Cardinal P. Bellocchio L. Guzman-Quevedo O. Andre C. Clark S. Elie M. Leste-Lasserre T. Gonzales D. Cannich A. Marsicano G. et al.Cannabinoid type 1 (CB1) receptors on Sim1-expressing neurons regulate energy expenditure in male mice.Endocrinology. 2015; 156: 411-418Crossref PubMed Scopus (37) Google Scholar). While mice overexpressing neuropeptide Y on noradrenergic neurons of the brain and SNS exhibit age-dependent elevation of endocannabinoid levels in many target organs, including the WAT (24Vähätalo L.H. Ruohonen S.T. Mäkelä S. Ailanen L. Penttinen A.M. Stormi T. Kauko T. Piscitelli F. Silvestri C. Savontaus E. et al.Role of the endocannabinoid system in obesity induced by neuropeptide Y overexpression in noradrenergic neurons.Nutr. Diabetes. 2015; 5: e151Crossref PubMed Scopus (8) Google Scholar), genetic knockout of CB1 in Sim1-expressing neurons is accompanied by increased mRNA expression of the β3-adrenoceptor and UCP1 in the BAT of high-fat diet-fed mice (23Cardinal P. Bellocchio L. Guzman-Quevedo O. Andre C. Clark S. Elie M. Leste-Lasserre T. Gonzales D. Cannich A. Marsicano G. et al.Cannabinoid type 1 (CB1) receptors on Sim1-expressing neurons regulate energy expenditure in male mice.Endocrinology. 2015; 156: 411-418Crossref PubMed Scopus (37) Google Scholar). These data point to a strong and SNS-mediated association between the endocannabinoid system and the control of BAT and WAT function. While there are several examples of reports [summarized in (19Silvestri C. Di Marzo V. The endocannabinoid system in energy homeostasis and the etiopathology of metabolic disorders.Cell Metab. 2013; 17: 475-490Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar)] pointing to an autocrine role of endocannabinoids and CB1 receptors on white adipocytes, also facilitating, among others, their transformation into beige adipocytes (25Perwitz N. Wenzel J. Wagner I. Buning J. Drenckhan M. Zarse K. Ristow M. Lilienthal W. Lehnert H. Klein J. Cannabinoid type 1 receptor blockade induces transdifferentiation towards a brown fat phenotype in white adipocytes.Diabetes Obes. Metab. 2010; 12: 158-166Crossref PubMed Scopus (81) Google Scholar, 26Wagner I.V. Perwitz N. Drenckhan M. Lehnert H. Klein J. Cannabinoid type 1 receptor mediates depot-specific effects on differentiation, inflammation and oxidative metabolism in inguinal and epididymal white adipocytes.Nutr. Diabetes. 2011; 1: e16Crossref PubMed Scopus (18) Google Scholar), evidence for a similar role in brown adipocytes is still scant. Through the use of a peripherally restricted CB1 antagonist in vivo, and of T37i brown adipocyte-like cells in vitro, Boon et al. (22Boon M.R. Kooijman S. van Dam A.D. Pelgrom L.R. Berbee J.F. Visseren C.A. van Aggele R.C. van den Hoek A.M. Sips H.C. Lombes M. et al.Peripheral cannabinoid 1 receptor blockade activates brown adipose tissue and diminishes dyslipidemia and obesity.FASEB J. 2014; 28: 5361-5375Crossref PubMed Scopus (75) Google Scholar) very recently suggested that some of the above effects of CB1 blockade in the BAT, and in particular the stimulation of uncoupled respiration, may be exerted postjunctionally and directly on brown adipocytes, most likely by enhancing cyclic AMP/PKA signaling induced by β3-adrenoceptor activation. However, the question of whether exposure to cold, via sympathetic stimulation of β3-adrenoceptors in brown adipocytes, enhances endocannabinoid levels in BAT has not been tested so far. In the present study, we addressed this hypothesis by examining the effect of cold exposure and pharmacological β3-adrenoceptor activation in mice using the selective agonist, CL, on: a) the levels of AEA, 2-AG, and nonendocannabinoid N-acylethanolamines (NAEs); and b) mRNA expression levels of genes encoding for CB1 receptors and endocannabinoid metabolic enzymes (Fig. 1). In order to analyze the effects on BAT activity, as well as browning of subcutaneous WAT, these studies were conducted under conditions of acute and chronic cold and β3-adrenergic stimulation. All animal experiments were approved by the Animal Welfare Officers of University Medical Center Hamburg-Eppendorf and Behörde für Gesundheit und Verbraucherschutz Hamburg. Mice were bred and housed in the animal facility of University Medical Center Hamburg-Eppendorf at 22–24°C with a day and night cycle of 12 h. Male age-matched (12–14 weeks) C57BL/6J wild-type mice, housed in single cages and fed a standard chow diet (Lasvendi) with ad libitum access to food and water, were used for the experiments. Cold exposure was performed by housing the mice at 6°C for 1 day or 7 days. The β3-adrenoceptor agonist, CL (0.2 mg CL/ml in 0.9% NaCl (w/v); Tocris) was administered by subcutaneous injection (1 μg per gram body weight per day) either for: a) 7 days without treatment on the day of necropsy (chronic CL); b) only once, 4 h before necropsy (acute CL); or c) chronically for 7 days with an additional injection on day 8 before necropsy (chronic+acute CL). Mock-treated control mice and acute CL mice received corresponding 0.9% NaCl (w/v) injections throughout the treatment period. All tissue and blood collections were performed after 4 h fasting. Mice were anesthetized with a lethal dose (15 μl per gram mouse body weight) of a mix containing ketamine (25 mg/ml)/xylazine (0.2%) in 0.9% NaCl. Blood was withdrawn transcardially and animals were perfused with 5 ml ice-cold PBS containing 10 U/ml heparin. Organs were harvested, immediately frozen, and stored at −80°C. For RNA analysis samples were conserved in TRIzol® reagent (Invitrogen). Total RNA was isolated from inguinal subcutaneous WAT and subscapular BAT using NucleoSpin RNA II kit (Macherey and Nagel). Synthesis of cDNA was performed using SuperScript® III reverse transcriptase (Invitrogen). Quantitative real-time PCR reactions for indicated genes were conducted on a 7900HT sequence detection system (Applied Biosystems) using TaqMan Assay-on-Demand primer sets [Ucp1, Mm00494069_m1; Ppargc1a, Mm00447183_m1; Dio2, Mm00515664_m1; Elovl3, Mm00468164_m1; Cnr1, Mm01212171_s1; Cnr2, Mm02620087_s1; α/β domain containing-4 (Abhd4), Mm00506368_m1; glycerophosphodiester phosphodiesterase-1 (Gde1), Mm00450997_m1; N-acyl phosphatidylethanolamine phospholipase D (Napepld), Mm00724596_m1; fatty acid amide hydrolase (Faah), Mm00515684_m1; diacylglycerol lipase-α (Dagla), Mm00813830_m1; Daglb, Mm00523381_m1; monoglyceride lipase (Mgll), Mm00449274_m1] supplied by Applied Biosystems and selected to recognize RefSeq sequences and a maximum of GenBank expressed sequence tags. Gene of interest cycle thresholds (Cts) were normalized to TATA-box binding protein (Tbp, Mm00446973_m1) house keeper levels by the ΔΔCt method and displayed as normalized copy numbers relative to experimental control groups (fold). Lipids were extracted from inguinal subcutaneous WAT and interscapular BAT of mice, and endocannabinoid purified from lipid extracts, as previously described (27Bartelt A. Orlando P. Mele C. Ligresti A. Toedter K. Scheja L. Heeren J. Di Marzo V. Altered endocannabinoid signalling after a high-fat diet in Apoe (-/-) mice: relevance to adipose tissue inflammation, hepatic steatosis and insulin resistance.Diabetologia. 2011; 54: 2900-2910Crossref PubMed Scopus (55) Google Scholar). Measurement of endocannabinoids (AEA and 2-AG), as well as of nonendocannabinoid NAEs, N-palmitoylethanolamine (PEA), and N-oleoylethanolamine (OEA), was carried out by isotope dilution LC-atmospheric pressure chemical ionization-MS using deuterated standards, as previously described (27Bartelt A. Orlando P. Mele C. Ligresti A. Toedter K. Scheja L. Heeren J. Di Marzo V. Altered endocannabinoid signalling after a high-fat diet in Apoe (-/-) mice: relevance to adipose tissue inflammation, hepatic steatosis and insulin resistance.Diabetologia. 2011; 54: 2900-2910Crossref PubMed Scopus (55) Google Scholar). For the preparation of primary brown adipocytes, 9-week-old C57BL6/J mice were anesthetized, interscapular BAT was removed and digested in isolation buffer [123 mM NaCl, 5 mM KCl, 1.3 mM CaCl2, 5 mM glucose, 100 mM HEPES (pH 7.4)] containing collagenase II (Biochrom). The stromavascular fraction was isolated by filtration of the cell suspension through 100 μm and 40 μm nylon mesh and plated out. The cells (including preadipocytes) were cultured for 10 days and differentiated through addition of 20 nM insulin (Sigma), 1 nM tri-iodothyronine-sodium (Sigma), 0.5 mM 3-isobutyl-1-methylxanthine (Sigma), 1 μM dexamethasone (Sigma). The resulting primary brown adipocytes were treated with and without 1 μM of CL in DMEM containing 0.1% fatty acid-free BSA (GE Healthcare) for 4 h. Supernatants were harvested and nonesterified fatty acids were determined using standard colorimetric assays (Wako). RNA analysis of brown adipocytes was performed as described for the tissue samples. In order to study the endocannabinoid system in cold-activated BAT and WAT, and to differentiate short-term from long-term effects, the following treatments were chosen: cold exposure was applied to mice for either 1 day or for 7 days. To another set of mice, the β3-adrenergic agonist, CL, was administered either once, 4 h before necropsy (acute CL) for seven consecutive days and also on day 8 before necropsy (chronic+acute CL) or for 7 days, but not before necropsy (chronic CL). Stimulation of β3-adrenoceptors with CL and exposure to cold are known to markedly enhance the expression of characteristic markers of uncoupled respiration and thermogenesis via specific transcription factors in WAT and BAT (14Wu J. Cohen P. Spiegelman B.M. Adaptive thermogenesis in adipocytes: is beige the new brown?.Genes Dev. 2013; 27: 234-250Crossref PubMed Scopus (628) Google Scholar, 15Bartelt A. Heeren J. Adipose tissue browning and metabolic health.Nat. Rev. Endocrinol. 2014; 10: 24-36Crossref PubMed Scopus (722) Google Scholar), revealing different degrees of WAT browning and BAT activation, respectively (Fig. 2). In both tissues, activation of the respective signaling pathways induced mRNA levels of Ucp1 (Fig. 2A, E), which encodes the heat generating protein, UCP1, that is located in the inner mitochondrial membrane of thermogenic adipocytes. In comparison to its expression in BAT, the basal Ucp1 mRNA levels are much lower in WAT, leading to a much higher relative induction of Ucp1 mRNA after CL treatment or cold exposure (10Cannon B. Nedergaard J. Brown adipose tissue: function and physiological significance.Physiol. Rev. 2004; 84: 277-359Crossref PubMed Scopus (4525) Google Scholar). The increased expression of the PPAR coactivator 1α (Ppargc1a, Fig. 2B, F) indicates an upregulation of genes important for brown and beige adipocyte differentiation and their functional maintenance (28Puigserver P. Wu Z. Park C.W. Graves R. Wright M. Spiegelman B.M. A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis.Cell. 1998; 92: 829-839Abstract Full Text Full Text PDF PubMed Scopus (3070) Google Scholar). The respective transcriptional networks also rely on enhanced activation of thyroid hormone receptors that is mediated by the induction of type II iodothyronine deiodinase (Dio2; Fig. 2C, G) mRNA (29de Jesus L.A. Carvalho S.D. Ribeiro M.O. Schneider M. Kim S.W. Harney J.W. Larsen P.R. Bianco A.C. The type 2 iodothyronine deiodinase is essential for adaptive thermogenesis in brown adipose tissue.J. Clin. Invest. 2001; 108: 1379-1385Crossref PubMed Scopus (398) Google Scholar), which encodes an enzyme converting inactive tetra-iodothyronine to active tri-iodothyronine. In addition, we observed a profound induction of Elovl3 (Fig. 2D, H), a fatty acid elongase specifically expressed in brown adipocytes during increased fatty acid oxidation (30Jakobsson A. Jörgensen J.A. Jacobsson A. Differential regulation of fatty acid elongation enzymes in brown adipocytes implies a unique role for Elovl3 during increased fatty acid oxidation.Am. J. Physiol. Endocrinol. Metab. 2005; 289: E517-E526Crossref PubMed Scopus (48) Google Scholar). In both tissues, chronic+acute CL treatment, but not any of the other treatments, significantly stimulated the expression of Cnr1, the gene encoding for the CB1 endocannabinoid receptor (Fig. 2I, K). Finally, in BAT, but not in WAT, acute CL, as well as acute+chronic CL treatment, and 1 day exposure to cold, reduced the expression of Cnr2, the cannabinoid type-2 (CB2) receptor (Fig. 2J, L), which was, however, based on total copy numbers less expressed than CB1 in both adipose tissues (data not shown). Both acute and chronic+acute CL treatment of the mice, but not chronic CL treatment per se, elevated the levels of the endocannabinoids, 2-AG and AEA, in BAT. The effect was statistically significant for AEA in both cases, however, for 2-AG only with chronic+acute CL treatment. Cold exposure did not cause the same effect, although a trend was observed for AEA with a 1 day exposure (Fig. 3A, B). Interestingly, acute and chronic+acute CL administration, as well as short-term cold exposure, caused a strong and significant elevation in the levels of the AEA congeners, PEA and OEA, which was also present after 1 day and 7 day cold exposure (Fig. 3C, D, Table 1). Thus, short-term β3-adrenergic stimulation leads to increased levels of both CB1 agonists and nonendocannabinoid NAEs in BAT.TABLE 1Absolute values for endocannabinoids in WAT and BATAEA (pmol/mg)2-AG (pmol/mg)PEA (pmol/mg)OEA (pmol/mg)BAT>Control0.25 ± 0.040.93 ± 0.121.31 ± 0.161.95 ± 0.12Acute CL0.70 ± 0.17aP < 0.05, Student's t-test.1.80 ± 0.433.31 ± 0.55aP < 0.05, Student's t-test.4.16 ± 0.78aP < 0.05, Student's t-test.Chronic CL0.30 ± 0.060.78 ± 0.091.49 ± 0.212.39 ± 0.15aP < 0.05, Student's t-test.Chronic and acute CL0.54 ± 0.09aP < 0.05, Student's t-test.1.78 ± 0.31aP < 0.05, Student's t-test.3.33 ± 0.38cP < 0.001, Student's t-test.5.68 ± 0.49cP < 0.001, Student's t-test.1 day cold0.50 ± 0.130.99 ± 0.063.19 ± 0.50bP < 0.01, Student's t-test.4.35 ± 0.64bP < 0.01, Student's t-test.7 day cold0.32 ± 0.060.70 ± 0.131.96 ± 0.10bP < 0.01, Student's t-test.2.99 ± 0.17cP < 0.001, Student's t-test.WATControl0.03 ± 0.0030.30 ± 0.040.75 ± 0.100.67 ± 0.11Acute CL0.06 ± 0.007bP < 0.01, Student's t-test.0.31 ± 0.061.08 ± 0.10aP < 0.05, Student's t-test.1.06 ± 0.13aP < 0.05, Student's t-test.Chronic CL0.09 ± 0.010cP < 0.001, Student's t-test.0.85 ± 0.07cP < 0.001, Student's t-test.1.12 ± 0.131.37 ± 0.08cP < 0.001, Student's t-test.Chronic and acute CL0.11 ± 0.0013cP < 0.001, Student's t-test.0.94 ± 0.10cP < 0.001, Student's t-test.1.99 ± 0.30aP < 0.05, Student's t-test.2.11 ± 0.23cP < 0.001, Student's t-test.1 day cold0.10 ± 0.0015cP < 0.001, Student's t-test.0.40 ± 0.041.61 ± 0.15cP < 0.001, Student's t-test.1.60 ± 0.08cP < 0.001, Student's t-test

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