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(The FASEB Journal. 2001;15:1357-1366.)
© 2001 FASEB

Insulin and glucocorticoids differentially regulate leptin transcription and secretion in brown adipocytes

MARION BUYSE*, SAY VIENGCHAREUN{dagger}, ANDRÉ BADO* and MARC LOMBÈS{dagger}1

* INSERM U 410 and
{dagger} INSERM U 478, Institut Fédératif de Recherche ‘Cellules épithéliales’ IFR2, Faculté de Médecine Xavier Bichat, Paris cedex 18, France

1Correspondence: INSERM U 478, Institut Fédératif de Recherche ‘Cellules épithéliales’ IFR2, Faculté de Médecine Xavier Bichat, 16, rue Henri Huchard 75870, Paris cedex 18, France. E-mail: mlombes{at}bichat.inserm.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Leptin, the ob gene product, is produced by adipose tissue and is submitted to a complex hormonal and metabolic regulation. Leptin plays a critical role in the balance of body weight. Here we report on secretion and hormonal regulation of leptin by brown adipocytes. Using the recently established T37i cell line, we show that leptin expression and secretion occurred as a function of cell differentiation. In differentiated T37i cells, insulin induced leptin release (~0.25 ng/106 cells/h) in a concentration-dependent manner (EC50=0.1 nM), and this was totally suppressed by ß3-adrenergic ligand, thiazolidinedione, cycloheximide, or actinomycin D. Insulin induced a strong, rapid (within 2 h) but transient fivefold increase in leptin mRNA levels. This transcriptional control of ob gene expression by insulin involved both phosphatidylinositol 3-kinase- and MAP kinase-dependent pathways. Glucocorticoids inhibited both insulin-stimulated leptin secretion and ob gene expression without affecting leptin mRNA stability (t1/2=3h05). Altogether, our results demonstrate that brown adipocytes express and secrete leptin, whose hormonal regulation clearly differs from that described in white adipose tissue. These findings point to tissue-specific molecular mechanisms and suggest that leptin might exert direct effects on energy homeostasis through an autocrine mechanism.—Buyse, M., Viengchareun, S., Bado, A., Lombès, M. Insulin and glucocorticoids differentially regulate leptin transcription and secretion in brown adipocytes.


Key Words: cell line • uncoupling protein • ob gene • signaling pathway • obesity


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Leptin, the ob gene product (1) , is a 16 kDa peptide hormone that was initially reported to be synthesized and secreted exclusively from the adipocytes of white fat. Leptin has also been shown to be produced by the placenta (2) , muscle (3) , stomach (4) , and the pituitary gland (5) . Therefore, its specific white adipocyte protein status is no longer true.

Leptin plays a central role in the regulation of feeding behavior and energy homeostasis via a cross-talk with various neuropeptides in the hypothalamus (6) . The mutation in the ob gene results in massive obesity, hyperphagia, hyperinsulinemia, and sterility characteristics of the ob/ob mice, whose phenotype is reversed by exogenous leptin administration (7 8 9) . Because leptin is mainly produced and secreted from adipocytes, circulating leptin levels correlate directly with body mass index. Plasma leptin levels and ob gene expression increase at night and acutely decrease during fast. Nutrients, hormones, and neurotransmitters also seem to play a major role in the regulation of leptin expression and secretion (10 11 12) .

The hormonal regulation of leptin synthesis and secretion in white adipocytes with regard to insulin and glucocorticoid action has already been well documented, but several contradictory conclusions persist. Although glucocorticoids undoubtedly stimulate ob gene expression and leptin secretion in vivo in humans or rats as well as in vitro in primary culture cells (13 , 14) , the effects of insulin remain unclear. Several studies have reported a stimulatory effect of insulin on leptin synthesis and/or secretion both in vitro and in vivo. Insulin increases ob gene expression and leptin secretion in 3T3 L1 or in 3T3 F442A cell lines (15 , 16) , and hyperinsulinemia also increases plasma leptin levels and gene expression in rodent and human white adipose tissue (17) . Other studies have shown no effect on one or both parameters (18) . Moreover, in isolated human subcutaneous (s.c.) adipocytes, insulin completely blocked dexamethasone-stimulated increase in the ob mRNA and leptin release (13 , 19) .

The majority of studies have focused on the expression and regulation of leptin in white adipocyte and the role and importance of leptin in the brown adipose tissue (BAT) remain unclear.

In contrast to the white adipose tissue (WAT), whose main function is to store energy in the form of lipids, BAT, characterized by its thermogenic activity, dissipates energy and provides heat. This results from the activity of several uncoupling proteins (UCP) that translocate protons through the inner membrane of mitochondria, bypassing the last enzymatic step of the respiratory chain, ATP synthase (20) and thus uncoupling oxidative phosphorylation. Therefore, BAT plays a major role in regulating body temperature especially in hibernal animals and rodents. In human infants, BAT may provide a similar role; however, this tissue atrophies with aging. Therefore, the importance and putative role of BAT in human adults remain unclear.

The specific expression of ob gene in BAT remains to be elucidated. Previous studies reported that leptin mRNA was expressed exclusively in mature white adipocytes, whereas subsequent ones showed a very low level of ob gene expression in BAT (20- to 50-fold lower) as compared with WAT. Because BAT is known to contain some white adipocytes, this low signal might originated from these contaminating cells. Indeed, some authors found that only unilocular, UCP1-negative brown adipocytes express leptin, whereas in typical brown adipocytes characterized by a multilocular morphology and UCP1 expression, leptin mRNA could not be detected (21) . On the other hand, leptin has been reported to be expressed in a human immortalized brown adipocyte cell line (22) as well as in rat or mouse differentiated brown adipocytes (23 24 25) .

We have recently established a transgenic mouse model in which the expression of SV40 large T antigen (TAg) was placed under the control of the proximal promoter of the human mineralocorticoid receptor (hMR) gene (26) . This targeted oncogenesis resulted in precocious development of voluminous malignant liposarcomas originating from the BAT (called hibernomas) in all founder mice. Several cell lines were derived from hibernomas; among them, the T37i cell line was extensively characterized. Indeed, T37i cells remain capable of differentiating into brown adipocytes upon insulin and triiodothyronine treatment and during differentiation express adipogenic genes such as adipocyte-specific fatty acid binding protein 2 (aP2), lipoprotein lipase (LPL), and peroxisome proliferator-activated receptor {gamma} 2 (PPAR{gamma}). More important, expression of UCP1, characteristic of brown adipocytes, but also UCP2 and UCP3 have been also demonstrated in this cell line (27 , 28) . Therefore, the T37i cell line represents an attractive model to study the BAT physiology.

The aim of the present study was to examine whether leptin is expressed and secreted by brown adipocytes and to further investigate hormonal regulation of these two processes. We report that insulin is the major stimulator of ob gene transcription and leptin secretion in T37i cells whereas glucocorticoids are potent inhibitors of leptin synthesis. Thus, leptin is produced by brown adipocytes and its physiological control is clearly different from previously reported for white adipocytes, pointing to tissue-specific molecular mechanisms.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
The T37i cell line was derived from a hibernoma (malignant brown fat tumor) of the transgenic founder mouse 37 carrying a hybrid gene composed of the human mineralocorticoid receptor proximal promoter linked to the SV40 large T antigen (26) . T37i cells were cultured in DMEM:HAM’s F12 medium (Gibco-BRL, Cergy Pontoise, France) supplemented with 10% fetal calf serum, 2 mM glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, 20 mM HEPES and grown at 37°C in a humidified atmosphere with 5% CO2. Differentiation into mature brown adipocytes was achieved under standard conditions by incubating subconfluent undifferentiated T37i cells with 2 nM triiodothyronine (Sigma Chemicals Co., St. Louis, MO) and 20 nM insulin (Gibco-BRL) for 7 days.

Leptin expression was studied under different experimental conditions. Cells were generally incubated for 18 h in medium supplemented with dextran-coated charcoal treated serum before treatment with various hormones and for different periods of time. All the experiments were performed with T37i cells between passage 10 and 20.

Radioimmunoassay (RIA)
Extraction of total proteins from T37i cells was performed at 4°C in RIPA buffer containing 0.1 mg/ml PMSF, 100 µM benzamidine, and 100 mM NaVO3 as protease inhibitors. The lysate was centrifuged at 15,000 g for 20 min. The amount of protein was quantified using the Bradford method (Bio-Rad Protein assay, Bio-Rad laboratories GmbH, Munchen, Germany).

Leptin content in aliquot of culture medium or in cell lysate was quantified using the mouse leptin RIA kit from Linco Research, Inc. (St. Charles, MO) with a detection limit of 0.2 ng/ml.

Immunocytochemical analysis
Immunocytochemical studies were performed on cytospin preparation of undifferentiated and differentiated T37i cells. Immunostaining of leptin was performed by an overnight incubation with a rabbit polyclonal anti-leptin antibody (A20, Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C (1:50 dilution), followed by an incubation with a secondary goat anti-rabbit IgG coupled to horseradish peroxidase (1:100) (Santa Cruz). Peroxidase activity was detected by diaminobenzidine (Sigma). Specificity of the reaction was tested by omitting primary antibody.

Subcloning of the mouse ob cDNA by RT-PCR
Total RNA was extracted from mouse brown adipose tissue using the TRIZOL reagent (Gibco-BRL). Briefly, 2 µg of total RNA were reverse-transcribed with 200 units of reverse transcriptase using the SuperscriptTM II kit (Gibco-BRL) according to the manufacturer’s recommendations. Mouse ob cDNA was then amplified for 30 cycles (95°C for 45 s; 65°C for 45 s, 72°C for 45 s) in a total volume of 25 µl containing 50 mM KCl, 20 mM Tris-HCl, pH 8.4, 200 µM dNTPs, 1.5 mM MgCl2, 10 pmol of sense 5'-CCTGTGGCTTTGGTCCTATCTG-3' and antisense primers 5'-CTGCTCAAAGCCAC CACCTCTG-3', and 0.25 units of Taq polymerase (Gibco-BRL). The mouse ob cDNA was subcloned into the pGEM-T easy cloning plasmid (Promega, Madison, WI). To shorten the mouse ob cDNA for RNase protection assays (RPA), we digested the ob-PGEM-T easy plasmid with PstI and religated the plasmid. The new ob-PGEM-T easy plasmid (ob 260-PGEM-T) contained a cDNA 260 bp long. The sequencing analysis of cDNA confirmed that the PCR product subcloned corresponded to mouse ob (GenBankTM accession number MMU18812).

Analysis of ob transcripts by RPA
In vitro synthesis of ob riboprobes was achieved using the ob 260-PGEM-T plasmid, linearized by NcoI. [{alpha}32P]-labeled antisense riboprobes were synthesized with Sp6 RNA polymerase (Promega). The mouse ß-actin riboprobe, used as an internal control, was synthesized with T7 RNA polymerase (Promega) after digestion of the ß-actin-pGEM3 plasmid (kindly provided by Denise Laouri, INSERM U426, Paris, France) by Bsu 36I.

RPA were performed as described previously (29) . Briefly, 50 µg of total RNA, isolated from differentiated cells using TRIZOL (Gibco-BRL), were hybridized overnight at 50°C in a formamide PIPES hybridization buffer with 4 x 105 cpm ob riboprobe and 5 x 104 cpm ß-actin riboprobe. Nonhybridized RNA was digested at 30°C for 1 h with a ribonulease A and T1 mixture, followed by a 30 min proteinase K and SDS treatment, at 37°C. After phenol-chloroform extraction and ethanol precipitation, protected fragments were electrophoresed on a 6% polyacrylamide urea gel. Gels were thereafter fixed in 10% acetic acid and dried. Radioactivity was counted overnight with an InstantImager (Packard, Meriden, CT), followed by an autoradiography. Results are expressed in arbitrary units and corresponded to the ratio between ob specific counts vs. ß-actin signal. Unprotected ob riboprobe migrated at 305 bases with a protected fragment migrating at 260 bases. Unprotected ß-actin riboprobe migrated at 158 bases with a protected fragment migrating at 137 bases.

Drugs
Wortmannin, brefeldin A, colchicine, dexamethasone, isoproterenol, actinomycin D, and cycloheximide were purchased from Sigma. PD 98059 was purchased from Calbiochem (France Biochem, Meudon, France) and BRL 37344 from Interchim (Paris, France). Thiazolidinedione was a generous gift from R. Negrel (Sophia Antipolis, Nice, France).

Statistical analysis
Data are expressed as the mean ± SE and were analyzed by an ANOVA, followed by Tukey Kramer multiple comparison test (GraphPadInstat Program). The differences were considered significant for P < 0.05. Linear regression slopes were calculated to measure ob mRNA stability using a GraphPad, Prism program.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Leptin is expressed in T37i cells
Expression of leptin mRNA was first studied by RT-PCR in T37i cell line as a function of the differentiation state. After 3–7 day treatment with 20 nM insulin and 2 nM triiodothyronine, T37i cells undergo differentiation into mature brown adipocytes, as assessed by their ability to display multilocular intracytoplasmic lipid droplets and express UCP1 (27) . A 415 bp product 100% identical to the mouse ob gene transcript, as confirmed by cDNA sequencing, was detected in differentiated T37i cells as well as in mouse brown adipose tissue (Fig. 1A ). However, ob transcripts were not detected in undifferentiated T37i cells, suggesting that leptin mRNA was expressed exclusively in mature brown adipocytes. Quantification of leptin mRNA levels by RPA revealed that leptin expression progressively increased during the differentiation process, reaching a maximal level at day 7 (Fig. 1B ). This correlated with the cell morphological changes characteristic of brown adipocytes, as well as with a sequential activation of the adipogenic genes expression (LPL, PPAR{gamma} 2, and aP2) already reported (27) . Therefore, ob gene expression may be considered as a late marker of differentiation, together with UCP1, in the T37i cell line.



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  		Figure 1. Leptin is expressed in differentiated T37i cells. A) RT-PCR analysis of ob gene expression. Lane 1: undifferentiated T37i cells; lane 2: differentiated T37i cells; lane 3: mouse interscapular brown adipose tissue; lane 4: negative control omitting the reverse transcriptase; M: marker ladders. Arrow indicates the expected size (415 bp) of the PCR product. B) Time-dependent expression of ob mRNA in T37i cells during differentiation. At different days of culture (from day 1 to day 7), total RNA was extracted and processed for RPA using specific probes for ob and ß-actin. Signals were quantified by InstantImager and expressed as fold induction of ob expression. C) Time-dependent secretion of leptin in T37i cells during differentiation. An aliquot of culture medium was taken from undifferentiated cells or differentiated cells at day 7 and 10, then leptin content was quantified by RIA. Results represent means ± SE for 4 determinations. ***P < 0.001 vs. undifferentiated cells.

We next measured leptin release over 24 h in culture medium by RIA. Whereas leptin was undetectable in the culture medium of undifferentiated cells, measurable amounts of leptin readily accumulated in the culture medium of differentiated cells on days 7 and 10 (Fig. 1C ). In addition, immunocytochemical studies with an anti-mouse leptin antibody revealed a cytoplasmic staining in differentiated T37i cells (Fig. 2 ).



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  		Figure 2. Immunocytochemical detection of leptin in T37i cells. A, B) Undifferentiated T37i cells; C–D) differentiated T37i cells. A, C) Negative controls by omission of the primary antibody. A cytoplasmic staining of leptin can be seen in differentiated T37i cells. A–D) Magnification x 195; insert; magnification x 320

These results indicate that differentiated but not undifferentiated T37i cells express ob gene transcripts and are able to synthesize and secrete leptin. Thus, this cell line appears to be a suitable model to investigate the hormonal regulation of leptin transcription and secretion in brown adipocytes.

Insulin is the major stimulator of leptin secretion in T37i cells
Insulin treatment of differentiated T37i cells induced a concentration-dependent stimulation of leptin secretion over 24 h (Fig. 3A ). This stimulation was maximal from 0.5 nM to 20 nM concentration, followed by a supramaximal effect at 100 nM. The estimated EC50 was 0.1 nM, a value consistent with an action through the insulin receptor. Since 20 nM insulin was required for the cell differentiation process and for the maximal leptin release, we used this concentration in all the ensuing experiments.



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  		Figure 3. Insulin is the major stimulator of leptin secretion in brown adipocytes. Differentiated T37i cells were grown in culture medium supplemented with 10% charcoal-treated serum. After different treatments, an aliquot of culture medium was taken and leptin content was quantified by RIA. Results represent means ± SE for 3 determinations. *P < 0.05; **P < 0.01; ***P < 0.001 vs. control. A) Insulin stimulates leptin secretion in a dose-dependent manner. T37i cells were incubated for 24 h with various insulin concentrations (0.1 to 100 nM). B) Time-dependent secretion of leptin by T37i in response to insulin. T37i cells were incubated for 0, 2, 4, 6, 8, or 24 h alone or in the presence of insulin (20 nM). C) Hormonal regulation of leptin secretion. T37i cells were incubated for 24 h with insulin (20 nM), triiodothyronine (2 nM), or a combination of both hormones alone or in the presence of isoproterenol (1 µM), BRL 37344 (1 µM), or thiazolidinedione (1 µM). D) Molecular mechanisms of leptin secretion. T37i cells were preincubated in the presence or absence of actinomycin D (0.4 µM) or cycloheximide (5 µg/ml) for 30 min. The conditioned medium was changed and T37i cells were incubated for various periods of time with or without insulin (20 nM).

The time course effects of insulin on leptin secretion showed that leptin steadily accumulated in culture medium in a time-dependent manner, whereas basal secretion of untreated cells remained at a relatively low steady-state level (Fig. 3B ). Leptin secretion was significantly increased 4 h after addition of insulin; after 24 h of treatment, it reached a concentration of 6 ng leptin/ml of medium. Such a value, which corresponds to ~0.25 ng/ml/h or 6 ng leptin/106 cells/24 h, represents a relatively high level of leptin secretion compared with other cellular models.

Insulin induced a dramatic 8- to 12-fold increase over basal values in 24 h leptin secretion (Fig. 3C ). Also, triiodothyronine (T3) produced a significant twofold stimulatory effect on leptin secretion and slightly, but not significantly, increased insulin-induced leptin secretion. In contrast, isoproterenol, a ß-adrenergic agonist, as well as BRL 37344, a selective ß3-adrenoreceptor agonist, completely inhibited insulin T3-stimulated leptin release over 24 h. Similarly, thiazolidinedione (BRL 49653), a specific PPAR{gamma} ligand, completely abolished leptin secretion measured after 24 h treatment with insulin T3.

For a deeper insight into the mechanisms of insulin-induced leptin secretion, T37i cells were preincubated for 30 min with the transcriptional inhibitor actinomycin D or the protein synthesis inhibitor cycloheximide before insulin stimulation. Actinomycin D and cycloheximide completely blocked insulin-stimulated leptin release (Fig. 3D ) without affecting basal leptin secretion (data not shown). These data are consistent with transcriptional and post-transcriptional regulatory mechanisms for insulin stimulation of leptin secretion. To further investigate the secretory pathway of insulin-induced release of leptin, we used brefeldin A, an inhibitor of translocation of secretory proteins from endoplasmic reticulum to the Golgi apparatus and colchicine, which induces a depolymerization of microtubules and then disrupts the translocation of proteins from a preformed cytoplasmic pool. Thus, T37i cells stimulated by insulin were incubated with either inhibitor for 4 h. Treatment of T37i cells with brefeldin blocked secretion of leptin into the medium, leading to a massive intracellular accumulation (Fig. 4 ). On the other hand, colchicine had no significant effect on leptin secretion and storage (Fig. 4) . Taken together, these results indicate that insulin-stimulated leptin secretion requires a transcriptional control of ob gene expression and a neosynthesis of leptin that is dependent on the Golgi apparatus.



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  		Figure 4. Secretory pathway of insulin-induced leptin release. Differentiated T37i cells grown in culture medium supplemented with 10% charcoal-treated serum were incubated for 4 h with insulin (20 nM) in the presence or absence of brefeldin A (5 µM) or colchicine (10 µM). An aliquot of culture medium was taken and leptin content was quantified by RIA. Total proteins content was measured by Bradford method, and intracellular leptin was determined by RIA. Results were expressed as ng leptin/ml culture medium or ng leptin/mg protein and represent means ± SE for 6 determinations.

Ob gene transcription is activated by insulin
As insulin was found to stimulate leptin secretion, we examined whether insulin was able to modulate ob gene expression. To address this question, RPA were performed with total RNA extracted from differentiated T37i cells incubated for various periods of time with insulin. The ob gene specific signal was quantified and normalized by the ß-actin signal.

Insulin induced a four- to fivefold increase in ob mRNA levels after 2 h treatment as compared to control values, indicating that insulin can rapidly regulate ob gene transcription in T37i cells (Fig. 5A ). The time course of insulin action on the steady-state levels of ob transcripts showed a less pronounced increase of leptin mRNA after 4 h insulin treatment whereas no variation in ob mRNA accumulation was detected at 24 h (data not shown). Pretreatment with actinomycin D significantly decreased basal ob mRNA level and completely blocked the insulin-stimulated increase in ob mRNA accumulation (Fig. 5A ). On the other hand, cycloheximide alone slightly increased basal ob mRNA levels, and the addition of insulin still induced an increase in leptin mRNA expression similar to that observed in the absence of cycloheximide. Therefore, these findings strongly suggest a transcriptional regulatory mechanism of insulin on ob mRNA expression as well as on leptin secretion, and indicate that the stimulatory effect of insulin on ob transcription is a direct process not requiring on-going protein synthesis.



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  		Figure 5. Activation of ob gene transcription by insulin. Differentiated T37i cells grown in culture medium supplemented with 10% charcoal-treated serum were preincubated for 30 min in the presence or absence of the specific agents. The conditioned medium was changed and T37i cells were incubated for 120 min with or without insulin (20 nM). Total RNA was extracted and processed for RPA using specific probes for ob and ß-actin. Signals were quantified by InstantImager and expressed as fold induction of ob expression. A) Effect of actinomycin D and cycloheximide on the insulin-stimulated leptin expression. Actinomycin D (0.4 µM) or cycloheximide (5 µg/ml) were used. Results represent means ± SE for 5 determinations. **P < 0.01, ***P < 0.001 vs. basal. B) Effects of wortmannin and PD98059 on ob gene expression. T37i cells were incubated for 30 or 120 min with wortmannin (10 mM) or PD98059 (50 µM). Results represent means ± SE for 9 determinations. *P < 0.05, **P < 0.01, ***P < 0.001 vs. insulin.

To investigate the signaling pathway mediating insulin-stimulated ob gene expression, the effects of wortmannin, phosphatidylinositol (PI) 3-kinase inhibitor, and PD98059, a MEK inhibitor, the mitogen-activated protein kinase/extracellular-signal related kinase, were examined. As shown in Fig. 5B , a 30 min wortmannin treatment induced no modification on either basal ob mRNA level or 2 h insulin-stimulated ob gene expression. However, after a 2 h incubation, wortmannin completely abolished the insulin effect. A 50% inhibition of insulin-stimulated increase in leptin mRNA levels was achieved after 30 min preincubation with PD98059; a longer incubation resulted in a similar inhibition. Our results are consistent with the requirement of PI 3-kinase and MAP-dependent pathways for insulin-stimulated increase in leptin transcription, although the events involved in the signaling cascade might operate with different kinetics.

Glucocorticoids inhibit insulin-stimulated leptin secretion and expression
We showed that insulin stimulated leptin expression and secretion mostly through transcriptional mechanism in brown adipocytes. Since glucocorticoids like insulin are known to promote brown adipocyte differentiation and to stimulate leptin secretion in white adipocytes, we further investigated whether dexamethasone regulates leptin secretion in brown adipocytes. As shown in Fig. 6A , in contrast to insulin, dexamethasone alone had no effect on basal leptin release during 24 h incubation. Dexamethasone (100 nM) reduced insulin-stimulated leptin secretion in T37i cells. Time- and dose-dependent experiments indicated that 1 nM dexamethasone significantly inhibited insulin-stimulated leptin secretion by 50% as early as after 4 h incubation (data not shown). A 2 h pretreatment of adipocytes with dexamethasone also reduced the stimulatory effects of insulin on leptin release.



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  		Figure 6. Glucocorticoids inhibit insulin-stimulated leptin secretion and expression. A) Effects of dexamethasone on insulin-stimulated leptin secretion. T37i cells were incubated for 24 h with or without insulin (20 nM) in the absence or presence of dexamethasone (DXM) (100 nM). An aliquot of culture medium was taken and leptin content was quantified by RIA. Results represent means ± SE for 3 determinations and are expressed as percentage of insulin-stimulated leptin release taken as 100%; ***P < 0.001 vs. control. B) Effects of dexamethasone on insulin-stimulated leptin expression. T37i cells were incubated for 2 h with or without insulin (20 nM) in the presence of various concentrations of dexamethasone (DXM). Total RNA was extracted and processed for RPA using specific probes for ob and ß-actin. Signals were quantified by InstantImager and expressed as fold induction of ob expression. Results represent means ± SE for 3 determinations. **P < 0.01,***P < 0.001 vs. insulin.

Dexamethasone alone induced no (at 24 h) or a slight decrease (at 2 h) in the basal ob mRNA level (data not shown). More important, dexamethasone strongly inhibited in a dose-dependent manner (1 to 100 nM) the insulin-stimulated increase of leptin gene expression (Fig. 6B ). To determine whether insulin and dexamethasone can modify the stability of ob transcripts, we measured the ob mRNA half-life time in the presence and absence of either dexamethasone or insulin after transcription was terminated by addition of actinomycin D. Figure 7 shows that in the absence of insulin or dexamethasone, ob mRNA levels rapidly declined with a calculated half-life time (t1/2) of ~3h05 (r2=0.9763). Insulin or dexamethasone did not affect the half-life time of ob gene mRNA, 3h35 (r2=0.9743) and 3h09 (r2=0.9623), respectively.



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  		Figure 7. Effects of insulin and dexamethasone on the stability of ob transcripts. Differentiated T37i cells grown in culture medium supplemented with 10% charcoal-treated serum were preincubated with insulin (20 nM) for 2 h. The conditioned medium was changed and T37i cells were incubated with actinomycin D (0.4 µM) in the presence or absence of insulin (20 nM) or dexamethasone (100 nM) for 0 to 6 h. Total RNA was extracted and processed for RPA using specific probes for ob and ß-actin. Signals were quantified by InstantImager and expressed as ratio between ob mRNA expression at (T) time and at 0 time taken as 1. Linear regression slopes are represented. Results are expressed in arbitrary units and represent means ± SE for 4 determinations. No statistical difference was detected between equations. Control: y = 1–0.162x (r2=0.9763); insulin: y = 1–0.140x (r2=0.9642); DXM : y = 1–0.159x (r2=0.9623).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we provide evidence for the expression of leptin in differentiated brown adipocyte T37i cells by RT-PCR and RPA. In contrast to other adipocyte models (3T3-L1) (30) , the level of expression of ob mRNA transcripts in T37i cells estimated by RPA is relatively high since it is ~50% of that detected in mouse brown fat. Our results confirm previous studies demonstrating that leptin gene is expressed in rat or mouse differentiated brown adipocytes (23 , 24) as well as in a human immortalized brown adipocyte cell line (22) , consistent with the view that leptin expression initially reported in the brown adipose tissue is not due to a contamination by white adipocytes. Moreover, we have clearly shown the ability of differentiated T37i cells to secrete leptin. The amounts of leptin released in the culture medium (0.25 ng/ml/h/106 cells) was on the same order of magnitude as those previously reported for freshly isolated white adipocytes in rats (18) and humans (31) , but significantly higher than in 3T3L1 culture (32) . As already reported for white adipocytes, we found that leptin is specifically secreted by differentiated cells, which suggests that leptin could be considered as a late marker of brown adipocyte differentiation as the UCP1. Taken together, these findings clearly indicate that the T37i cell line is a suitable system to analyze molecular mechanisms underlying leptin secretion and its hormonal regulation present in brown adipocytes.

Insulin is considered to be a potent regulator of leptin, because plasma insulin concentrations decrease during fast and increase after refeeding in parallel with plasma leptin levels (10) . However, the effect of insulin on ob gene expression and leptin secretion remains controversial. Several in vitro studies using isolated white adipocytes (33 34 35) reported that insulin increased leptin secretion. In addition, insulin was shown to induce ob gene expression in 3T3 L1 and 3T3 F442A cells (15 , 16) , whereas Bradley et al. reported that insulin stimulates leptin secretion without modification in ob gene expression in rat isolated white adipocytes (18) . On the other hand, other authors have shown that insulin completely blocked the glucocorticoid-stimulated increase in leptin release and ob mRNA in isolated human s.c. adipocytes (13 , 19) .

In T37i cells, we demonstrated that insulin clearly stimulates leptin secretion with an EC50 of 0.1 nM, a value consistent with an involvement of insulin receptors. A similar dose-dependent increase in insulin-stimulated glucose transport was observed in the same model (P. Penfornis, M. Lombès, and A. Marette, unpublished observations), which is consistent with the high sensitivity of T37i cells to insulin stimulation.

In T37i cells, insulin-stimulated leptin secretion appears to require both transcriptional and post-transcriptional events. Indeed, insulin induced a rapid (2 h), substantial (five- to sixfold) but transient (less pronounced effect at 4 h) increase in leptin mRNA levels; this effect was completely abolished by actinomycin D pretreatment, supporting the involvement of transcriptional mechanisms. A direct effect of insulin on ob mRNA transcription is further sustained by the fact that cycloheximide did not affect either basal or insulin-stimulated ob mRNA levels. However, the lag time observed between the rapid and transient induction of leptin transcription and the sustained effect of insulin on leptin secretion strongly suggest that in addition to gene activation, insulin also exerts major post-transcriptional actions, including modification of leptin mRNA translation. This is confirmed by the action of cycloheximide, which blocked insulin-induced-leptin secretion.

We further investigated the signaling pathways mediating insulin-stimulated leptin secretion. Tyrosine phosphorylation of IRSs and/or Shc by insulin receptor activates two major signaling pathways: the MAP kinase pathway and the PI3-kinase pathway. Although MAP kinases are involved in proliferation and differentiation processes by regulating the transcriptional activity of the nucleus, the PI3-kinase pathway seems to play a preponderant role in most insulin-regulated metabolic effects (36) . Here we demonstrated that the PI3-kinase inhibitor wortmannin blunted the insulin-stimulated ob gene expression, indicating that the PI3-kinase pathway is necessary for insulin action in T37i cells. The MEK inhibitor (PD98059) also decreased leptin mRNA levels by 50%, in agreement with a partial involvement of MAP kinase in insulin effect. Altogether, these results indicate that both PI3-kinase and MAP kinase pathways are required for insulin action on leptin expression and secretion in brown adipocytes. However, additional experiments are needed to define the underlying molecular events.

Consistent with previous studies of white adipocytes, ß-adrenergic agonist through ß3-adrenoreceptor and antidiabetic thiazolidinedione drastically suppressed leptin secretion (14 , 37 38 39 40 41) , suggesting that with respect to regulation of leptin secretion by ß-adrenoceptor and PPAR{gamma}-dependent mechanisms, there is no difference between BAT and WAT.

In contrast to these drugs, glucocorticoid control of leptin secretion appears to depend on the nature of adipose tissue. Whereas in glucocorticoids WAT stimulate synthesis and secretion of leptin (13 , 14 , 18 , 42 43 44) , dexamethasone had no effect on basal leptin synthesis or secretion and inhibited insulin-stimulated leptin release and expression in T37i cells. These results provide an additional support for a post-transcriptional effect of insulin on leptin secretion. The repressive effects of glucocorticoids on leptin release have been reported in another cellular model, the C6 glioblastoma cells (45) , indicating a strict tissue-specific control of leptin expression. Such a tissue-specific control of gene expression by glucocorticoids has also been described for several genes, including phosphoenolpyruvate carboxykinase, whose transcription is activated in liver and kidney but repressed in the adipose tissue (46) .

Insulin-induced expression of leptin is likely to involve an increase of the rate of ob gene transcription without affecting mRNA stability or requiring an ongoing protein synthesis. The inductive effects of insulin and the repressive effects of dexamethasone on leptin expression could be due at least in part to a regulation of the leptin gene promoter activity. Promoter analysis of the mouse (47) , rat (48) , and human (49) gene has revealed general interesting features. Mason et al. (50) reported that a 109 bp promoter is as effective as the longer promoter in directing leptin transcription. Four important motifs in this proximal promoter contribute to regulation of leptin transcription: the TATA box (at -30), a C/EBP motif (at -53), the LP1 region (at -87), and a Sp1 motif (at -97). A computer scan of the 6.5 kb 5'-flanking region of the mouse leptin gene (accession no. 65742) with the Transfac database (51) disclosed several putative binding sites such as Sp1 sites, half-sites of glucocorticoid response elements, AP-1, and insulin response sequence. Whether these elements are involved in the regulation of leptin transcription in brown adipocyte remains to be established.

Several studies have clearly demonstrated the key role of C/EBP{alpha} in transcriptional activation of the mouse ob gene (52 53 54) . C/EBP{alpha}/{alpha} homodimers appear to activate transcriptional process whereas alternation of the occupation and replacement by other members of the C/EBP family might attenuate gene expression, such as reported for GLUT4 transcription (55) . We propose that insulin in brown adipocytes might potentiate C/EBP {alpha}/{alpha} homodimer formation and/or their transcriptional activity, possibly through modification of phosphorylation status of C/EBP{alpha} (56 , 57) . Similarly, since glucocorticoids were shown to activate the transcription of C/EBP{delta}, which behaved as a transcriptional repressor of the {alpha} isoform (56) , it could be proposed that glucocorticoids modify the composition of the regulatory complex that binds to the C/EBP site of the ob gene promoter, thus explaining the relative insulin resistance in response to glucocorticoids observed in the brown adipocytes. Other transcription factors also seem to regulate leptin expression. For instance, Sp1 was shown to inactivate leptin transcription by binding to an overlapping binding site in the -101 to -83 region of the rat gene that is implicated in insulin-stimulated transcription (48) . Finally, ADD1/SREBP1 considered as a bona fide insulin response factor trans-activated leptin gene probably through interaction with sterol regulatory-like elements present in the leptin promoter (58) .

Since insulin activated leptin transcription independent of ongoing protein synthesis, we propose that besides activation of trans-acting factors such as ADD1 or C/EBP gene expression, insulin modifies their transcriptional activity and/or their nuclear localization.

Several mechanisms could be proposed for glucorticoid repression of insulin-induced leptin expression (for a review, see ref 59 ). The glucocorticoid receptor (GR) could antagonize other transcription factor through direct competitive DNA binding to an overlapping cis-acting element contained in the leptin promoter region. Alternatively, GR could interfere with insulin signal transduction cascade or titrate essential coactivators. Additional studies are required to unravel the exact molecular mechanisms.

The major finding of this study is that leptin is synthesized and secreted by brown adipocytes. Beside the tissue-specific regulation of leptin secretion (brown vs. white adipose tissue), the physiological significance of brown adipocyte-secreted leptin is still open to speculation. Brown adipose tissue seems to be the first adipose tissue to appear during development; at birth, extensive deposits of BAT constitute a predominant factor in heat production by nonshivering thermogenesis in response to cold exposure at birth (60) . Dessolin et al. (25) reported that the ob gene is expressed in BAT of newborn rats immediately after birth; they demonstrated that within the first hours postpartum, plasma leptin levels correlated with the variation of leptin mRNA levels expressed in BAT, suggesting that BAT might be the predominant if not exclusive source of leptin at birth, thus underlying the physiological importance of BAT-secreted leptin and its potential role in the control of food intake immediately after birth.

Recent studies reported that leptin could modulate thermogenesis in BAT not only by increasing sympathetic stimulation of BAT, but also by stimulating UCP1 mRNA and protein expression in BAT (61 , 62) . These data explain the stimulation of energy utilization by increasing thermogenic activity observed after exogenous leptin administration in lean rats or mice (C57BL6) as well as in ob/ob mice (9) . It has been also reported that leptin stimulates glucose utilization and increases expression of malic enzyme and LPL in differentiated brown adipocytes (63) . We have recently shown that glucocorticoids inhibit UCP1 expression in T37i cell line (28) , suggesting that glucocorticoids by inhibiting leptin secretion might also participate to the reduction of thermogenic activity of brown fat. Taken together, we suggest that in addition to the classical leptin action through blood borne molecule, autocrine effects of leptin on brown adipose tissue are likely. Indeed, preliminary experiments (M. Buyse, S. Viengchareun, A. Bado, and M. Lombès, unpublished observations) have shown that T37i cells expressed Ob-Rb transcripts as detected by RT-PCR, and the presence of leptin receptors (Ob-Rb and Ob-Ra) was confirmed by Western blot. An interesting perspective opened by the present study is the possibility that leptin regulates its own expression through a mechanism that remains to be defined.

In summary, we have shown that brown adipocytes express and secrete leptin. The hormonal regulation of brown adipocyte secreted leptin clearly differs from that observed in the white adipose tissue. Insulin is an important stimulator of leptin synthesis and exerts its effects mostly through a strong, rapid, and transient activation of leptin transcription. Glucocorticoids inhibit insulin action in T37i cells, pointing to specific molecular mechanisms that account for the tissue-specificity of leptin gene expression. Our findings also strongly suggest that leptin might exert direct and important effects on brown adipocytes, most notably related to thermoregulation and energy expenditure, through an autocrine pathway.


   ACKNOWLEDGMENTS
 
We thank M. J. M Lewin, and M. C. Zennaro for helpful discussions, support during this work, and critical reading of this manuscript, and J. C Marie for linguistic revision of this manuscript. The assistance of J. P. Laigneau for illustrations is also gratefully acknowledged. M.B. is supported by le Ministère de l’Education et de la Recherche (MRT fellowship).

Received for publication October 12, 2000. Revision received February 8, 2001.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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