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00004872-201402000-0002500004872_2014_32_389_bordicchia_adrenergic_2article< 127_0_22_4 >Journal of Hypertension© 2014 Wolters Kluwer Health | Lippincott Williams & WilkinsVolume 32(2)February 2014p 389–396Nebivolol induces, via β3 adrenergic receptor, lipolysis, uncoupling protein 1, and reduction of lipid droplet size in human adipocytes[ORIGINAL PAPERS: Obesity]Bordicchia, Maricaa; Pocognoli, Antonellaa; D’Anzeo, Marcoa; Siquini, Walterb; Minardi, Danielec; Muzzonigro, Giovannic; Dessì-Fulgheri, Paoloa; Sarzani, RiccardoaaInternal Medicine and Geriatrics, Department of Clinical and Molecular Sciences, Ospedale ‘U. Sestilli’, Italian National Research Center on Aging IRCCS-INRCA and University ‘Politecnica delle Marche’ AnconabDepartment of Surgery, University Hospital of AnconacDepartment of Urology, University Hospital of Ancona, Ancona, ItalyCorrespondence to Professor Riccardo Sarzani, MD, Ph.D, Internal Medicine and Geriatrics, Department of Clinical and Molecular Sciences, University ‘Politecnica delle Marche’, Via Tronto 10/A, Ancona 60126, Italy. Tel: +39 071 5964595; fax: +39 071 889232; e-mail: Abbreviations: β3AR, β3 adrenergic receptor; CYCS, cytochrome c; PGC-1α, PPARγ coactivator-1α; SVF, stromal vascular fraction; UCP1, uncoupling protein 1Received 18 April, 2013Revised 2 August, 2013Accepted 12 September, 2013Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Website ( ).AbstractObjectives: Most β-blockers may induce weight gain, dysglycemia, and dyslipidemia. Nebivolol is a third-generation β1-blocker with vasodilating properties mediated by β3 adrenergic receptors (β3AR). We investigated whether nebivolol is able to induce β3AR-mediated lipolysis, uncoupling protein 1 (UCP1), and size-reduction in human adipocytes.Methods: Human visceral (n = 28) and subcutaneous adipose tissue (n = 26) samples were used to obtain differentiated subcutaneous and visceral preadipocytes. Adipocytes were used to verify the effects of nebivolol onlipolysis, uncoupling protein 1 (UCP1) and other genes of the thermogenic program.Results: Lipolysis was induced by isoproterenol and specific β3AR agonist, as expected,and also by nebivolol at 100 nmol/l and by its L-enantiomer at 10 nmol/l (P < 0.01). Nebivolol-mediated lipolysis was blocked by SR59230A, a specific β3AR antagonist, suggesting that nebivolol acts through β3AR in human adipocytes. Interestingly, in human adipocytes, nebivolol activated UCP1, PPARγ coactivator-1α (PGC-1α) and cytochrome c (CYCS) gene expression in a p38 MAPK–dependent manner. Using propranolol (β1 and β2 antagonist) together with nebivolol we showed that the induction of these genes was still present suggesting again β3AR activation. Moreover, nebivolol significantly reduced the diameter of lipid droplets in cultured adipocytes.Conclusion: In summary, nebivolol, through β3AR, is able to induce lipolysis and promote thermogenic and mitochondrial genes. The induction of lipolysis and the thermogenic program could explain the reduction of lipid droplets size. In conclusion, the lower dysmetabolic effects of nebivolol in humans may depend on its β3 agonist activity and the consequent induction of thermogenic program in human adipocytes.INTRODUCTIONHypertension guidelines and clinical studies suggest that the more selective β1 blocker nebivolol may be the β blocker of choice because favorable hemodynamic properties (including vasodilation) and metabolic effects without weight gain [1–4].Nebivolol is a racemic mixture of D- and L-enantiomers. D-nebivolol mainly acts as a selective β1 blocker whereas the L-enantiomer behaves as a β3 adrenergic receptor (β3AR) agonist [5,6]. β3AR are present in coronary endothelium where they are involved in the induction of nitric oxide (NO) production [7–9]. In human myocardium β3AR can couple both to Gs and Gi/o to generate NO and cGMP via Gi/NOS with the final effect of reduced contractility [10,11].Despite these important cardiovascular aspects, β3AR have usually been studied in adipocytes where it is coupled to Gi to control fat metabolism [12]. β3AR expression and β3AR-mediated lipolysis have been documented in human visceral adipocytes [13–17]. In ‘brown’ adipocytes the free fatty acid produced by β3AR-mediated lipolysis are used up for thermogenesis via the uncoupling protein 1 (UCP1) [16,18]. Recently, many studies have demonstrated that brown adipose tissue is active in humans with antiobesity and antidysmetabolic properties regulating fat metabolism and energy expenditure [19–23]. Moreover, white human adipocytes might acquire the brown phenotype by the activation of β3AR/UCP1 pathway, suggesting novel ways to treat obesity and the associated metabolic and cardiovascular complications [24–26].In the present study, we investigated whether the lower dysmetabolic side effects of nebivolol may be related to its β3AR agonist activity on human subcutaneous and visceral adipocytes. Therefore, the aims of this work were to study whether nebivolol was able to: induce β3AR-mediated lipolysis in human subcutaneous and visceral adipocytes; induce the thermogenic program and mitochondrialgenes: UCP1, PGC-1α and CYCS; decrease the size of adipocytes lipid droplets.METHODSReagents and antibodiesIsoproterenol (Iso), propranolol, insulin, tri-iodothyronine, and β3AR-selective agonist BRL37344 were obtained from Sigma–Aldrich (St. Louis, Missouri, USA). Nebivolol, L-nebivolol and D-nebivolol were a kind gift from Menarini Spa (Florence, Italy). Other reagents included a proteinase inhibitor cocktail (complete Mini) from Roche (Roche Diagnostics, Indianapolis, Indiana, USA). Antisera used were anti-GAPDH (Santa Cruz Biotech; California, USA), anti-UCP1 (Abcam, Cambridge, Massachusetts, USA), antip38αMAPK and antiphospho-p38 MAPK (Cell Signaling, Danver, Massachusetts, USA). Monoclonal antiserum against β3AR was a kind gift from Dr Trevor Wattam [27]. Secondary antibodies antirabbit (cat #SC2054), and antimouse (cat #SC-2005) were from Santa Cruz Biotech and antirat (cat #31470) was from Pierce. Super signal West Femto Maximum Sensitivity Substrate was from Thermo scientific, Rockford, Illinois, USA.PatientsHuman visceral (n = 28) and subcutaneous adipose tissue (n = 26) samples were obtained from patients during abdominal surgery for nonneoplastic conditions. Visceral adipose samples were taken from 16 men and 12 women, with average age of 66.5 ± 11.2 years and BMI of 26.4 ± 4.7 kg/m2, while subcutaneous adipose tissue from 15 men and 11 women, with average age of 67 ± 13.3 years and BMI of 25.8 ± 4.0 kg/m2. All women were in menopause. The local Ethics Committee approved the study protocol, and all patients gave written informed consent for the collection of clinical data and tissue samples.Primary adipocytes cultures from human visceral and subcutaneous adipose tissueVisceral and subcutaneous adipose tissue samples (2–3 g) were cut into small pieces and digested with collagenase Type I to obtain the stromal vascular fraction (SVF). Adipocyte differentiation was obtained as previously described [28].LipolysisIsoproterenol (nonselective βAR agonist), BRL 37344 (β3AR agonist), SR59230A (β3AR antagonist), metoprolol (β1AR antagonist), carvedilol (nonselective β3AR antagonist and α1 agonist), nebivolol, L-nebivolol, and D-nebivolol were added to cell-culture media. Pharmacological studies reported that nebivolol plasma concentration could be between 4.6 and 7.14 ng/ml after a single dose of 20 mg nebivolol in extensive metabolizer patients. Thus, seven different concentrations, ranging from physiological to supra-therapeutic concentrations (from 10–9 to 10–6 mol/l) were used for each ligand. Control and treated adipocytes from each patient were tested with different ligand concentrations in duplicate. Released glycerol was measured after 6 h by the Free Glycerol Determination Kit (Sigma–Aldrich Corp., St. Louis, Missouri, USA) and glycerol concentrations were determined by comparison with a standard curve. Intra- and inter-assay coefficient of variation were less than 6% and less than 9.1%, respectively.RNA isolation and gene expression analysisTotal RNA was extracted using Trizol (Invitrogen) and reverse transcription was performed with high-Capacity cDNA reverse Transcription Kit with RNase Inhibitor (Applied Biosystems, Warrington, UK). Gene expression of leptin, adiponectin, β3AR, UCP1 CYCS and PGC1α in visceral and subcutaneous adipose tissues were analyzed with TaqMan Gene Expression Assay (Applied Biosystems Darmstadt, Germany). All gene expression experiments were performed in triplicate. Differences in total RNA or different efficiency of cDNA synthesis among samples were normalized using human GAPDH expression.Western blottingTreated cells or tissues were lysed and sonicated in an appropriate buffer as previously described [26]. Protein concentrations were determined using the Bradford Assay (Biorad) and 50 μg of total proteins was resolved in 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a PVDF membrane (Immobilon P, Millipore), and probed overnight at 4°C with specific primary antibodies. Secondary antisera against rabbit, rat or mouse IgG conjugated with peroxidase was used for specific protein detection. β3AR, UCP1, phospho-p38 MAPK protein were visualized using an enhanced chemiluminescent substrate (SuperSignal West Femto Maximum Sensitivity Substrate, Pierce) and were measured in comparison with GAPDH (Santa Cruz Biotech) or total p38 MAPK. Image acquisition was performed on a Chemidoc (Biorad) and analyzed using Quantity One software. In some cases membranes were ‘stripped’ by incubation in a buffer (0.76 g Tris, 2 g SDS, 700 μl β-mercaptoethanol in 100 ml) at 37°C for 45 min in order to be subsequently probed with additional antibodies.Microscopy, image analysis, and basic morphometryA NIKON ECLIPSE Ti-S microscope was used for image analysis. Images were captured with a NIKON digital sight Model C-SHG1 camera. Ten random fields for each sample were analyzed in each experiment to verify the effect of the L-nebivolol (100 nmol/l) treatment. LUCIA Images software (20X S Plan Flour ELWD 20X/0.45 Ph1 ADM magnification) was used to analyze adipocytes size [29]. The method used to measure lipid droplets was also carried out using LUCIA Images software and was based on the following criteria: identification of the 10 larger lipid droplets of 10 adjacent cells repeated for another four different fields to reach at least 500 lipid droplets. The analysis of differences between lipid droplets size of control and the treated adipocytes was performed with paired t-test.Statistical analysisResults are presented as mean ± SEM unless otherwise indicated. Data were analyzed using two-tailed Student's t-test or one-way ANOVA, followed by posthoc Newman-Keuls tests when F was significant. The effects of increasing drugs concentrations on lipolysis were analyzed by a nonparametric test for multiple related samples (Friedman's test). A nonparametric test for two related samples (Wilcoxon's signed ranks test) was used to identify differences between each treated group and controls. Differences in gene expression levels among tissues were evaluated using one-way analysis of variance (ANOVA) test followed by Bonferroni's post hoc-test. Differences between the diameter of controls and the treated (L-nebivolol, 100 nmol/l) adipocytes were performed with paired t-test. SPSS 11.0 software was used for statistical analysis (SPSS Inc., Chicago, Illinois, USA) and a P < 0.05 was considered significant.RESULTSβ3AR expression in human subcutaneous and visceral adipose tissueProtein analysis showed that the levels of β3AR in both subcutaneous and visceral adipose tissues are highly variable among patients (Fig. 1a and b). This variability was lower in visceral adipose tissue (Fig. 1b), may be because this fat depot, taken near the renal arteries, is characterized by higher density of capillaries and noradrenergic fibres with a higher number of brown adipocytes [22].FIGURE 1. Variability of β3AR levels in representative samples of human subctunaeous and visceral adipose tissue included in this study – Protein levels of β3AR of representative samples of human subctunaeous (a) and visceral adipose tissue (b). c and d) β3AR gene expression in human subcutaneous and visceral adipose tissue after overnight incubation at room temperature (RT), 4°C, or 15°C in DMEM-F12, respectively. e and f) Effect of cold exposure on β3AR protein levels in subcutaneous and visceral adipocytes, respectively. Exposure at 15°C induce β3AR protein in both adipose depots. Results are mean ± SEM. *P < 0.05 versus snap-frozen samples ** P < 0.01 versus snap-frozen samples.It is well known that in-vivo β3AR is stimulated by cold exposure in adipose tissue [23], but novel mechanisms suggest that this could happen also in ex-vivo samples [30–32]. We directly tested if cold temperature incubation of ex-vivo tissue samples could induce β3AR expression with the aim to reduce its high variability in our samples.Adipose tissue samples were subdivided in three portions: one portion, after an overnight incubation at room temperature, was ‘snap’ frozen in liquid nitrogen (control). The other two portions were incubated overnight at 15°C or 4°C in DMEM-F12 medium in a refrigerated shacking (40 rpm) water bath. Gene expression analysis showed that subcutaneous and visceral adipose tissue responded to cold temperature. Incubation at 15°C significantly increased β3AR gene expression that was also observed, at lower levels, at 4°C (Fig. 1c and d). Similarly, western blot revealed that cold exposure increased β3AR protein levels in ex-vivo adipose tissue, as shown in Fig. 1e and f. Cold exposure did not alter the expression of other adipose gene markers such as adiponectin and leptin (Suppl. Figure S1, ). Therefore, after this relevant finding, all samples were preincubated overnight at 15°C before cutting and digesting to obtain SVF for adipocyte cultures.β3AR-mediated lipolysis and thermogenic genes expressionSubcutaneous and visceral adipocytes obtained after 10–12 days of differentiation were used to test lipolysis after treatment with β-agonists and antagonists at different concentrations from physiological to supra-therapeutic concentration (Fig. 2a and b). A low concentration of L-nebivolol (10 nmol/l) induced significant lipolysis in both subcutaneous and visceral adipocytes similarly to BRL37344, a specific β3AR agonist. D-nebivolol induced significant lipolysis only at very high concentration (1 μmol/l) and in visceral adipocytes only (Fig. 2B). Treatment with metoprolol, carvedilol and SR59230A did not induce lipolysis.FIGURE 2. Nebivolol and β3 agonist increase lipolysis in human subcutaneous and visceral adipocytes – All experiments were performed in duplicate and cells were treated for 2 h with different concentrations (from 1 nmol/l to 1 μmol/l) of the ligands: nebivolol (Nebi); L-nebivolol (L-Nebi); D-nebivolol (D-Nebi); BRL37344 (BRL) a β3AR agonist; SR59230A (SR) a β3AR antagonist; metoprolol (METO) a β1 antagonist; or carvedilol (CARV) a nonselective β receptors antagonist and α1AR antagonist; combination of SR59230A and L-nebivolol (SR+L-Nebi) and compared with untreated control cells. a) nebivolol and its L-enantiomer induced lipolysis in subcutaneous adipocytesat 100 and 10 nmol/l, respectively. D-nebivolol did not induced lipolysis as well as all the other β receptors antagonists tested. L-nebivolol mediated lipolysis is blocked by SR, suggesting that this effect was β3AR mediated. c) Nebivolol-induced lipolysis also in visceral adipocytes as in subcutaneous adipocytes with the difference that D-nebivolol was able to induce glycerol released at 1 μmol/l. Results are mean ± SEM. *P < 0.05 versus untreated controls, § P < 0.001 versus untreated controls.Of note, lipolysis induced by BRL37344 as well as by nebivolol was blocked by a pretreatment with the β3 antagonist SR59230A (Fig. 2a and b), supporting the idea that lipolysis was β3AR-mediated.Treatment with L-nebivolol and nebivolol at 100 nmol/l not only activated lipolysis in vitro but also induced the expression of brown adipocytes genes UCP1, PGC-1α and the mitochondrial gene CYCS (Fig. 3 a and b, c and d, and e and f, respectively). Treatment of cultured human adipocytes with propranolol, a β1/β2 antagonist, only partially blocked L-nebivolol effect on target genes, suggesting again that the residual activity was mediated by β3AR (Fig. 3 a and b, c and d, and e and f, respectively). Western blotting showed that UCP1 protein was also induced by nebivolol and its enantiomer L-nebivolol in both subcutaneous and visceral adipocytes (Fig. 3g and h). White adipocyte gene markers (adiponectin, leptin) were not induced by β 3AR agonists or by L-nebivolol (data not shown).FIGURE 3. Nebivolol and its L-enantiomer induce brown adipocyte markers in subcutaneous and visceral adipocytes – Cells were treated with 100 nmol/l of nebivolol (Nebi) or L-nebivolol (L-Nebi), 1 μmol/l of isoproterenol (ISO) and were also pretreated or not with 0.5 μmol/l of propranolol (Prop) a β1 and β2 blockers, for 30 min before added L-nebivolol. mRNA levels for UCP1, PGC-1α and Cytochrome c (CYCS) were measured in subcutaneous (a, c, e) and visceral (b, d, f) adipocytes. Subcutaneous (g) and visceral (h) adipocytes were treated as described above and samples were analyzed for protein levels by western blotting. Results are mean ± SEM. *P < 0.05 versus untreated controls, **P < 0.01 versus untreated controls,***P < 0.001 versus untreated controls.In a second series of experiments, to confirm that nebivolol was able to induce brown adipocyte marker genes through β3AR, we examined weather nebivolol used the p-38 MAPK pathway, well known to be involved in Ucp1 gene transcription [26,33,34]. As shown in Supplementary Figure S2, , nebivolol increased p38 MAPK activity in human adipocytes, as indicated by the phosphorylation of p38 MAPK. This was suppressed by a pretreatment with the β3 antagonist SR59230A indicating that nebivolol induced p38 MAPK pathway through β3AR.Morphological analysisMorphologic analysis of subcutaneous adipocytes treated with 500 nmol/l of L-nebivolol showed a 31% reduction of lipid droplets average size compared with untreated cells (21.8 ± 4.4 μm of control versus 15.0 ± 3.9 μm of treated cells; P < 0.001. Figure 4a). The same analysis in visceral adipocytes (Fig. 4b) showed a 20% reduction of lipid droplets size (14.3 ± 3.8 μm of control cells versus 11.4 ± 3.6 μm of treated cells; P < 0.001) as shown in the representative Fig. 4c and d.FIGURE 4. L-nebivolol reduce lipid droplets diameters of subcutaneous and visceral adipocytes – Comparison of lipid droplets diameters of untreated and L-Nebivolol treated (100 nmol/l) subcutaneous (a) and visceral (c) adipocytes. b and d) Representative phase-contrast images of subcutaneous and visceral adipocytes before and after treatment with L-nebivolol, respectively. Original magnification ×20.SummaryThe full datasets suggested that nebivolol, trough β3AR, activates lipolysis as well as the machinery to induce the thermogenic program leading to ’fat burning’ and adipocyte lipid droplets size reduction, supporting the hypothesis that nebivolol has more favorable metabolic effects than previous generations of β-blockers.DISCUSSIONThe main finding of this study was that nebivolol, through its enantiomer L-nebivolol, behaves as a β3AR agonist on human adipocytes at nanomolar concentrations. Nebivolol through β3AR is able to induce lipolysis and thermogenic/mitochondrial genes, leading to adipocyte lipid droplets size reduction as a final effect.β3AR is one of adipocyte key receptors, although its expression is low and variable in human adipose tissue and generated contradictory results that raised questions about its role in humans [24]. Even if the reason of this variability is still unknown, our data confirmed the high variability of β3AR gene expression and protein level among adipose samples.Based on the data that cold temperature increased sympathetic activity, catecholamines and β3AR expression [23], we found that β3AR gene expression was significantly enhanced especially after 15°C incubation. The cooler temperature-dependent increase of β3AR mRNA levels in ex-vivo adipose tissue samples is a novel and potentially important finding suggesting a direct effect of temperature on adipose organ culture. Recently it was suggested that alternative activation of resident macrophages and endothelial cells led to the synthesis and secretion of catecholamines in adipose tissue followed by the induction of the thermogenic program [30,31]. Moreover recently it was demonstrated that the cold-sensing receptor potential melastatin 8 (TRPM8) is expressed in adipose tissue and could be activated in vitro by its agonist [32]. Thus, our results suggest that overnight incubation of adipose tissue at 15°C induce higher β3AR expression increasing gene transcription and/or reducing mRNA degradation may be through local tissue factors like cathecolamines or cold-sensing receptors.Many studies reported that nebivolol is the most selective β1AR blocker but it is also a β3AR agonist [7–9,35]. Our results showed that treatment with 10 nmol/l of L-nebivolol induced lipolysis and lipid volume reduction in human adipocytes, whereas much higher concentrations of nebivolol and D-nebivolol were needed to obtain similar results. Nebivolol was also able to induce lipolysis as well as a size reduction of freshly isolated unilocular floating adipocytes from human adipose tissue samples (Supplementary Figure S3, ).β3AR agonists induce UCP1 expression in mice but data regarding human adipocytes are lacking [36–38]. We showed that isoproterenol as well as L-nebivolol and nebivolol induced UCP1 gene expression and protein in human subcutaneous and visceral adipocytes. Pretreatment with propranolol or SR59230A also suggest that the activity of L-nebivolol is β3AR mediated (Figs. 2 and 3). These results support the concept that free fatty acids released through nebivolol-induced lipolysis can be used up for thermogenesis in human visceral adipocytes because of the β3AR-mediated thermogenic program. In further sets of experiments, we also demonstrated that nebivolol act through β3AR with the activation of p38 MAPK as a key element for the transcription of UCP1 and of mitochondrial and thermogenic genes [33,34]. Microscopy image analysis and basic morphometry showed a significant size reduction of lipid droplets after treatment with L-nebivolol (Fig. 4). The reduction of lipid droplets diameter may result from the combination of lipolysis together with the β3AR/UCP1-mediated fat-burning pathway.The main limitation of our study is that our results are obtained from in-vitro analysis, leaving unaddressed whether nebivolol can exert clinically significant β3AR agonist in human in vivo. But, going up to 40 mg a day (maximum dosage approved by FDA for clinical use) and considering the improved metabolic profile observed in clinical studies with nebivolol suggest that its β3ARagonist may be present in vivo as well [1,2,39,40]. We hypothesized that the lower dysmetabolic effects of nebivolol may depend on its β3AR agonist activity on human adipose tissue as well as on ‘brown’ adipocytes enriched depots that have recently demonstrated to be functional in adult humans [19–23].Moreover, the difficulties in recovering human samples from patients undergoing surgery and the limited total amounts of tissue (also because experiments were carried out in triplicate) did not allow us to perform in-vitro thermogenesis and energy expenditure studies. However, we can reasonably speculate that the final effect of the UCP1 activation is the induction of thermogenesis [41,42].In conclusion, our results suggest that human adipocytes express functional β3AR that can be stimulated to induce lipolysis and UCP1, the key protein for ‘fat-burning’ thermogenesis in a p38 MAPK-dependent manner. A recent trial in humans with novel β3AR agonist (TAK-677) did not show significant effects on metabolic profile (weight, fat depots, fasting insulin or glucose concentration) but its scarce bioavailability and the tachycardia induced by this novel β3AR agonist (without the β1AR blocking activity of nebivolol) may explain the lack of efficacy [43].Randomized clinical trials focused on hypertensive patients with central obesity and dysmetabolism are needed to confirm nebivolol as an ‘eu-metabolic’ or even ‘pro-metabolic’ β1 blocker.ACKNOWLEDGEMENTSWe thank Dr Stefano Evangelista (Menarini) for the gift of nebivolol, Prof. Saverio Cinti for the use of its microscopy facilities, Dr Trevor Wattam and Dr John Arch for the kind gift of β3AR antibody.Sources of Funding: this work was supported by research grants from the Fondazione CARIVERONA (Italy) and from University Politecnicadelle Marche (Ricerca di Ateneo to R.S. and P.D.F.).Conflicts of interestThere are no conflicts of interest.Reviewer's Summary Evaluation Reviewer 2The study by Bordicchia et al. demonstrates that the β1-AR blocker nebivolol can induce lipolysis and the upregulation of the genes involved in thermogenesis via β3-ARs in human adipocytes. 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Siquini, Walter; Minardi, Daniele; Muzzonigro, Giovanni; Dessì-Fulgheri, Paolo; Sarzani, RiccardoORIGINAL PAPERS: Obesity232