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Endocrinology Vol. 138, No. 2 548-552
Copyright © 1997 by The Endocrine Society

Articles

Effects of ß-Adrenoceptor Subtype Stimulation on obese Gene Messenger Ribonucleic Acid and on Leptin Secretion in Mouse Brown Adipocytes Differentiated in Culture1

Chengjun Deng, Madelaine Moinat, Logos Curtis, Anne Nadakal, Frederic Preitner, Olivier Boss, Francoise Assimacopoulos-Jeannet, Josiane Seydoux and Jean-Paul Giacobino

Departments of Medical Biochemistry and Physiology (J.S.), University Medical Center, Geneva, Switzerland

Address all correspondence and requests for reprints to: Dr. J.-P. Giacobino, Département de Biochimie Médicale, Centre Médical Universitaire, 1 rue Michel Servet, 1211 Geneva 4, Switzerland. E-mail: Giacobin{at}CMU.Unige.ch


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The ob gene product is known to control food intake and energy expenditure. To determine whether thermogenic agents directly control ob gene expression, the effects of ß-adrenoceptor agonists on the level of the ob gene messenger RNA (mRNA) and on leptin secretion have been studied in mouse brown adipocytes differentiated in culture. These cells highly expressed the ß3-adrenoceptor, the uncoupling protein, and the ob gene mRNAs. The ob gene was expressed in mouse brown adipocytes earlier than in mouse white adipocytes under the same culture conditions and to a similar level. The ß3-, ß1-, and ß2-adrenoceptor agonists BRL 37344, dobutamine, and terbutaline inhibited ob gene expression in mouse brown adipocytes differentiated in culture with EC50 values of 0.3, 1.0, and 85 nM, respectively. Leptin secretion by the cells under basal conditions was 78 ± 10 pg/µg DNA·4 h and was decreased by exposure to the ß-adrenoceptor agonists. The ob gene mRNA half-life was 9.4 h and was decreased to 2.4 h by 1 nM BRL 37344, indicating that the inhibitory effect of the ß3-agonist might be due to destabilization of ob gene mRNA. (Bu)2cAMP (10–100 µM) and forskolin (20 µM) mimicked the effect of the ß-adrenoceptor agonists. FFA (150–800 µM) had only a small inhibitory effect on ob gene mRNA expression. The results suggest the existence in brown adipose tissue of a retroregulatory pathway by which leptin production is inhibited when the sympathetic nervous system is stimulated.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN 1994, Zhang et al. (1) cloned a gene referred to as the obese (ob) gene, whose mutation is responsible for the phenotype of the hereditary obese (ob/ob) mouse. The ob gene codes for a protein that controls body fat mass, presumably by acting as a lipostat. The protein, when administered to ob/ob and diet-induced obese mice, lowers body weight, percent body fat, and food intake and increases energy expenditure, body temperature, and activity (2, 3, 4, 5). Recently, several forms of leptin receptor were cloned, one of which was expressed in the hypothalamus, choroid plexus, lung, and kidney (6).

In rodents, the main effector of cold- and diet-induced thermogenesis is brown adipose tissue (BAT). The thermogenic pathway involves the ventromedial nucleus of the hypothalamus (VMH) and the sympathetic nervous system (7), whose effects are mainly mediated, at the cellular level, by the ß3-adrenoceptor (8). Leptin could, therefore, affect energy expenditure by acting centrally via the VMH to activate the sympathetic nervous system and thereby increase BAT thermogenic activity. It has, in fact, recently been shown that in vivo administration of leptin to mice resulted in an increase in noradrenaline turnover in BAT (9).

In vivo studies, indeed, have shown that the ob gene is expressed not only in white adipose tissue (1, 10), but also in BAT (11, 12, 13, 14), and that conditions known to increase thermogenic activity, such as cold acclimation or the administration of a ß3-adrenoceptor agonist, decreased its expression (12). These findings suggest the existence of a feedback loop between the VMH and brown adipose tissue that would result in an inhibition of ob gene expression when the sympathetic system is activated. In obese rodents with a mutation of their ob gene or an alteration of their leptin-sensing system, increased expression of ob gene messenger RNA (mRNA) in white adipose tissue was reported (1, 10, 11, 14, 15, 16). This suggests by analogy that in obese rodents, the sympathetic tone of white adipose tissue is decreased.

To assess whether the in vivo down-regulation of ob gene expression by ß3-adrenoceptor stimulation (12) is a direct or an indirect effect, studies were performed in vitro on brown adipocytes differentiated in culture. The results show that stimulation of ß3-adrenoceptors induced a dose-dependent down-regulation of ob gene mRNA, mainly due to a decreased half-life, and a similar down-regulation of leptin secretion. ß1- and ß2-adrenoceptor agonists and cAMP also decreased ob gene mRNA expression. Fatty acids may play a minor role in this down-regulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
All organic and inorganic chemicals were of analytical or molecular biology grade. They were purchased from Merck (Darmstadt, Germany), Boehringer Mannheim (Mannheim, Germany), Sigma Chemical Co. (St. Louis, MO), and Fluka (Buchs, Switzerland). Hybond N membranes, [{alpha}-32P]deoxy-CTP (3000 Ci/mmol), and [{gamma}-32P]ATP (1000 Ci/mmol) were obtained from Amersham (Aylesbury, UK). The Micro RNA Isolation Kit and Taq DNA polymerase were obtained from Stratagene (La Jolla, CA). The ß3-adrenoceptor agonist BRL 37344 was a generous gift from SmithKline Beecham Pharmaceuticals (Epsom, UK), the ß1-adrenoceptor agonist dobutamine was purchased from Research Biomedicals International (Natick, MA), and the ß2-adrenoceptor agonist terbutaline was purchased from Sigma. The mouse leptin RIA kit was obtained from Linco Research (St. Charles, MD).

Three- to 4-week-old BALB/c mice (University Medical Center, Geneva, Switzerland) were used. After decapitation, interscapular and cervical BAT (carefully freed from surrounding white adipose tissue) as well as epididymal white adipose tissue were excised. Precursor cells from brown and epididymal adipose tissues were isolated by incubation for 30 min at 37 C in the presence of collagenase and cultured according to the method of Champigny et al. (17). Briefly, dissociated cells were centrifuged, and the pellet of precursor cells was washed twice in an erythrocyte lysing buffer and then in DMEM-Ham’s F-12 medium supplemented with 10% FCS, 20 nM insulin, 2 nM T3, 50 µg/ml garamycin, and 100 µM ascorbic acid. Cells suspended in the medium were plated in 35-mm diameter plastic dishes and allowed to grow at 37 C in a humidified atmosphere of 5% CO2 in air. The culture medium was changed the day after plating and then every third day. Experiments were performed on 7- to 8-day-old fully differentiated brown adipocytes and 7- to 14-day-old white adipocytes. In experiments in which the effect of various agents were tested, the culture medium was changed at time zero, and the drugs were immediately added at the indicated concentrations. After the indicated period at 37 C, the medium was collected, and the cells were washed once with cold phosphate buffer and collected for RNA and/or DNA extraction. Fatty acids were prepared by stirring sodium oleate at 37 C under nitrogen in the presence of fatty acid-free 13% BSA. The pH was adjusted at pH 7.4, and the solution was filtered through a 0.22-µm filter and kept at -20 C. The final concentration of BSA in the culture medium was 0.5%.

To compare the levels of ob gene mRNA in cultured brown adipocytes with those of BAT, both interscapular and cervical BAT depots were excised, immediately frozen in liquid nitrogen, and stored at -70 C until use. Brown adipose tissue was homogenized, cultured white and brown adipocytes were scrapped in 4 M guanidium isothiocyanate, and total RNA was isolated using the Micro RNA Isolation Kit. Ten micrograms of total RNA were electrophoresed in a 1% agarose gel containing formaldehyde as described by Lehrach et al. (18) and transferred to Hybond N membranes by capillary blotting. An ob gene probe of 354 bp was obtained by reverse transcription-PCR of mouse white adipose tissue RNA as previously described (12). The uncoupling protein (UCP) probe used was that described by Bouillaud et al. (19). The probes were labeled by random priming with [{alpha}-32P]deoxy-CTP to a specific radioactivity of approximately 1 x 109 dpm/µg DNA. RNA blots were hybridized for 2 h at 68 C in Quik-Hyb, then washed in a solution of 0.1 x SSC (1 x SSC is 150 mM NaCl and 15 mM sodium citrate, pH 7.0)-0.1% SDS at 56 C for 15 min and exposed 24 h to Kodak X-AR film (Eastman Kodak, Rochester, NY) at -70 C. Size estimates for the RNA species were established by comparison with a RNA ladder. The amount of RNA in the signals on the autoradiograms was quantified by scanning photodensitometry. Hybridization of the blots with a [{gamma}-32P]ATP-labeled synthetic oligonucleotide specific for the 18S ribosomal RNA subunit was performed to assure that equivalent amounts of total RNA were compared. The leptin level in the culture medium was measured using the mouse leptin RIA kit of Linco Research. After the addition of 1 ml 0.9% NaCl, the cells were scrapped and sonicated, and DNA was measured according to the method of Labarca and Paigen (20). The EC50 and half-life values were calculated using the Microcal Origin 3.5 program (Boltzman model and linear regression analysis). Student’s unpaired t test was used to determine statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Brown adipocyte differentiation, as assessed by the presence of numerous lipid droplets, occurred by 7–8 days after plating, and Fig. 1Go shows that ob gene mRNA was highly expressed in differentiated mouse brown adipocytes, whereas it could not be detected in mouse white adipocytes. At 12–14 days in white adipocytes, a strong expression was observed, which reached a level similar to that in brown adipocytes. At this late stage of differentiation, the white adipocytes were so filled with lipid droplets that they tended to detach from the bottom of the dish. The ob gene mRNA signal had the reported size of 4 kilobases (12, 16, 21). The ob gene mRNA level in differentiated brown adipocytes was 4.4 ± 1.0-fold higher (n = 6) than that measured in 3-week-old mouse interscapular BAT. This might be due to the fact that the tissue contains, besides brown adipocytes, stromal vascular cells, in which ob gene is not expressed (11, 14), or that there is in vivo repression of BAT ob gene expression. Measurable amounts of leptin accumulated in the cell culture medium, and after 4 and 24 h, leptin secretion was 78 ± 10 and 594 ± 3 pg/µg DNA (n = 3), respectively, indicating a secretion rate of about 20 pg/µg DNA·h.



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  		Figure 1. Ob gene mRNA levels in differentiated mouse brown adipocytes (BA) 7–8 days after plating, in differentiated mouse epididymal white adipocytes (WA) 7–8 or 12–14 days after plating, or in 3-week-old mouse interscapular brown adipose tissue (BAT). Ten micrograms of total RNA were electrophoresed, transferred to membrane filters, and hybridized with the mouse ob gene probe as described in Materials and Methods. The results are expressed as the percentage ± SEM of the mean control value, taken as 100% (n = 3–6 experiments). The signals were quantified by scanning photodensitometry. Only signals obtained on the same Northern blot were compared. ***, P < 0.001 compared to the control value.

 
The effects of ß-adrenoceptor agonists on the degree of expression of ob gene and on leptin production were tested in cultured brown adipocytes. Figure 2AGo shows that 4-h exposure to the ß3-adrenoceptor agonist BRL 37344 at concentrations as low as 0.5 nM decreased the ob gene mRNA level by 90 ± 4% (P < 0.001). BRL 37344 had, as expected, an opposite effect on the UCP mRNA level in brown adipocytes, inducing an increase of about 12-fold at a concentration of 100 nM (results not shown). As shown in Fig. 3Go, the effect of BRL 37344 on the ob gene mRNA level was associated with a similar effect on leptin production. BRL 37344, at concentrations of 0.1, 1, and 10 nM, decreased the leptin level in the medium by 48.9 ± 1.1%, 93.5 ± 1%, and 97.3 ± 0.4%, respectively.



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  		Figure 2. A, ob gene mRNA levels in differentiated mouse brown adipocytes in the presence of increasing concentrations of the ß3-adrenoceptor ({circ}), ß1-adrenoceptor (•), or ß2-adrenoceptor () agonists BRL 37344, dobutamine, and terbutaline. The medium was changed at time zero, the drug was immediately added, and after 4 h at 37 C, the medium was collected, and the cells were washed once with cold phosphate buffer and collected for RNA extraction (n = 3–4 experiments). For details, see Fig. 1Go. B, Representative ob gene mRNA and 18S ribosomal RNA signals in differentiated mouse brown adipocytes in the presence of increasing concentrations of BRL 37344 (ß3), dobutamine (ß1), or terbutaline (ß2).

 


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  		Figure 3. Leptin recovered in differentiated mouse brown adipocyte culture medium in the presence of increasing concentrations of the same ß-adrenoceptor agonists as those in Fig. 2AGo. The leptin RIA was performed as described in Materials and Methods. The results are calculated as picograms per µg cell DNA/4 h and expressed as the percentage ± SEM of the mean control value, taken as 100% (n = 3–4 experiments).

 
As shown in Figs. 2AGo and 3Go, ob gene mRNA expression and leptin production were also inhibited by the ß1- and ß2-adrenoceptor agonists dobutamine and terbutaline, although with potencies about 10- and 100-fold lower, respectively, than that of BRL 37344. The EC50 values for the inhibition of ob gene mRNA expression were 0.3, 1, and 85 nM for BRL 37344, dobutamine, and terbutaline, respectively.

The effects of ß-adrenergic agonists could be due to changes in gene transcription and/or mRNA degradation. The latter possibility was assessed by incubating the cells in the presence of the transcription inhibitor actinomycin D at a concentration of 2 µg/ml, which was previously shown to fully block transcription in these cells (22), without or with BRL 37344 (1 nM), and by measuring ob gene mRNA after various periods of time (Fig. 4Go). The half-life of ob gene mRNA was 9.4 h (r = 0.74) in control cells and was decreased to 2.4 h (r = 0.99) in the presence of BRL 37344. The BRL 37344 inhibitory effect on ob gene expression observed after 4 h in the presence of actinomycin D was 66 ± 9%, similar to that of 76 ± 2% observed in the absence of actinomycin D (see Fig. 2Go).



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  		Figure 4. ob gene mRNA levels in differentiated mouse brown adipocytes as a function of time in the presence of actinomycin D (2 µg/ml) alone (•) or in combination with BRL 37344 (1 nM; {circ}). The medium was changed at time zero, the drugs were immediately added, and after the indicated times at 37 C, the cells were washed once with cold phosphate buffer and collected for RNA extraction (n = 3–7 experiments). For details, see Fig. 1Go.

 
Additional experiments were performed to test the possibility that the decrease in the ob gene mRNA expression induced by ß-adrenoceptor agonists in brown adipocytes is mediated by the cAMP pathway. Figure 5Go shows that (Bu)2cAMP at concentrations of 10 and 100 µM decreased the ob gene mRNA level in brown adipocytes by 26 ± 7% (P < 0.001) and 89 ± 6% (P < 0.001), respectively. (Bu)2cAMP was also found to stimulate UCP mRNA 3.8 ± 1.7-fold at a concentration of 100 µM. Forskolin (20 µM), a powerful activator of adenylate cyclase, decreased ob gene mRNA expression by 64 ± 9%. The possibility that the decrease in ob gene mRNA expression induced by ß-adrenoceptor agonists in brown adipocytes could also be due to an increase in the cytoplasmic Ca2+ concentration or in intracellular FFA was investigated (Fig. 6Go). Ionomycin, which is known to increase cytoplasmic [Ca2+] had no effect on ob gene mRNA expression. Oleic acid (150 and 800 µM) had small inhibitory effects (15 ± 5% and 31 ± 7% inhibition; P < 0.05 and P < 0.005, respectively) on ob gene mRNA expression.



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  		Figure 5. Ob gene mRNA levels in differentiated mouse brown adipocytes in the presence of increasing concentrations of (Bu)2cAMP and without (Control) or with forskolin (Forsk; 20 µM; inset; n = 3–5 experiments). ***, P < 0.001 compared to control values. For details, see Figs. 1Go and 2Go.

 


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  		Figure 6. Ob gene mRNA level in differentiated mouse brown adipocytes in the presence of BSA (Control), oleic acid (Oleic; 150 and 800 µM) bound to BSA, or ionomycin (Iono; 1 µM; n = 3 experiments). *, P < 0.05; **, P < 0.005 (compared to the control value). For details, see Figs. 1Go and 2Go.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The brown adipocytes differentiated in culture used in this study displayed two brown adipose tissue-specific features, i.e. they expressed UCP and the ß3-adrenoceptor. They responded to ß3-adrenergic stimulation by an increase in UCP mRNA expression similar to that previously observed in the same cell model (17). In brown adipocytes in culture, ob gene mRNA was highly expressed 7–8 days after plating, whereas white adipocytes required 12–14 days of culture until a similar level of expression was reached. In 3T3 adipocytes, ob gene mRNA was also reported to be a late marker of adipocyte differentiation (23, 24). It is worthy of mention that ob gene mRNA is expressed in brown adipocytes differentiated in culture at a much higher level than in 3T3 differentiated adipocytes, in which ob gene mRNA signal is so low that it can be detected only by ribonuclease protection assay (23, 24).

The order of potency of the ß-adrenoceptor agonists tested on ob gene mRNA expression and leptin secretion was BRL 37344 > dobutamine >> terbutaline. The low EC50 values obtained suggest that the observed effects are of biological significance. A maximal inhibition of ob gene mRNA expression was obtained with each of the ß-adrenoceptor agonists tested. The adenylyl cyclase stimulation by catecholamines in rat BAT membranes was reported to be mediated by ß1- and ß3-adrenoceptors proportionally to their respective amounts in the adipocyte membrane (25), i.e. 30% and 70%, respectively (26). The complete inhibition observed with the ß1- or ß2-adrenoceptor agonists indicates that a partial occupancy of the brown adipocyte ß-adrenoceptors is sufficient to induce a full inhibition of ob gene expression.

The ob gene mRNA half-life of 9.4 h found in the present study is longer than that of 2 h reported by Leroy et al. (23) in murine 3T3-F442A white adipocytes. As mentioned above, the latter cells express very little ob gene mRNA and cannot, therefore, be readily compared with brown adipocytes in culture. The observation that BRL 37344 decreased the half-life of ob gene mRNA indicates that the ß3-adrenoceptor agonist destabilized ob gene mRNA. This destabilizing effect seems to account for the inhibitory effect of BRL 37344 observed in the absence of actinomycin D. These results are in agreement with those reported by Hadcock et al. (27), who showed that in DDT1 MF-2 hamster vas deferens cells, the ß-adrenoceptor mRNA down-regulation observed upon exposure to ß-adrenoceptor agonists was mediated by mRNA destabilization. The latter phenomenon might, therefore, be a common mechanism of ß-adrenoceptor-mediated down-regulations.

This study shows for the first time that brown adipocytes differentiated in culture produce levels of leptin comparable to those produced by isolated white adipocytes (28). The inhibition of ob gene mRNA expression by ß-adrenoceptor agonists was associated with a decrease in the amount of leptin secreted in the culture medium. It has been shown in isolated white adipocytes that 24-h exposure to the nonselective ß-adrenoceptor agonist (-)-isoproterenol (10 µM) and to (Bu)2cAMP decreased ob gene mRNA expression and leptin secretion, and that a high concentration of the ß3-adrenoceptor agonist ICI 201,651 (10 µM) was less effective than (-)-isoproterenol to decrease ob gene expression (28).

ß-Adrenoceptor agonists could act on the expression of the ob gene via cAMP, intracellular Ca2+ (29, 30), or a combination of the two. The data clearly show that forskolin and (Bu)2cAMP mimicked the effects of the ß-adrenoceptor agonists. The fact that ionomycin did not modify ob mRNA expression suggests that either the effects of ß-adrenoceptor agonists are not mediated by an increase in cytosolic Ca2+ or that this signal alone is not sufficient to produce a change in ob gene expression.

ß-Adrenergic stimulation of lipolysis induces an increase in intracellular FFA. It is, therefore, tempting to speculate that this increase could modify the rate of gene transcription via the recently described peroxisome proliferator-activated receptors (PPARs) (31). Intracellular FFA could thus reflect the energetic state of the cell and act on food intake and energy store replenishment via leptin. When exogenous fatty acids bound to albumin were added to the medium, a small, not dose-dependent, inhibition of ob gene expression occurred at concentrations of 150 and 800 µM. The highest concentration used, which has been shown to maximally stimulate brown adipocyte respiration (32), only slightly decreased ob gene mRNA expression compared to the effect of BRL 37344. Therefore, intracellular FFA do not appear to mediate the effect of ß-adrenoceptor agonists to decrease ob gene expression. These results do not exclude an involvement of PPARs in the regulation of ob gene expression. The possibility remains that in brown adipocytes, fatty acids are very rapidly oxidized and esterified, with a consequent decrease in their potential role as PPAR ligands or ligand precursors. Indeed, when either 2-bromopalmitate, a nonoxidizable fatty acid, or PGJ2, a precursor of the ligand of PPAR{gamma}, is added to brown adipocytes, a marked inhibition of ob gene mRNA expression is observed (our manuscript in preparation). Rentsch and Chiesi (24) reported an inhibitory effect of 2-bromopalmitate on ob gene mRNA in 3T3 cells, and De Vos et al. (33) found an inhibitory effect of thiazolidinediones on ob gene mRNA expression in primary rat white adipocytes, suggesting an involvement of PPAR activation.

In our study, under conditions of ß3-adrenergic stimulation or fatty acid supply, which are known to maximally stimulate BAT thermogenesis (32, 34), leptin expression was either strongly inhibited or hardly affected. This indicates that leptin expression is not controlled by the cell metabolic activity. The possibility remains that leptin expression is controlled by the cell lipid content.

In summary, we have shown that ob gene mRNA expression and leptin secretion are controlled by the ß-adrenoceptors in brown adipocytes differentiated in culture, indicating the existence of a retroregulatory pathway by which leptin production is inhibited when sympathetic tone and energy expenditure are increased. Thus, the decreased expression of a satiety signal is consistent with the physiological role of BAT, which is stimulated under conditions that require enhanced food intake to sustain higher thermogenic activity.


   Acknowledgments
 
The expert technical assistance of Mrs. Françine Califano, Mrs. Martine Vollenweider, and Ms. Françoise Kuhne is gratefully acknowledged. We are indebted to Dr. Daniel Ricquier (Meudon, France) for the generous gift of the UCP probe.


   Footnotes
 
1 This work was supported by Grants 31–43405.95 and 31–37616.93 from the Swiss National Science Foundation and grants-in-aid from Ciba-Geigy-Jubiläums-Stiftung, the Carlos and Elsie de Reuter, and the Ingeborg Naegeli II Foundations. Back

Received July 1, 1996.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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