Proadipogenic effect of leptin on rat preadipocytes in vitro: activation of MAPK and STAT3 signaling pathways

F. Machinal-Quélin, M. N. Dieudonné, M. C. Leneveu, R. Pecquery, and Y. Giudicelli

Service de Biochimie, Faculté de Médecine Paris-Ouest, Université Descartes (Paris V), Centre hospitalier de Poissy, 78303 Poissy Cedex, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Because leptin has recently been shown to induce proliferation and/or differentiation of different cell types through different pathways, the aim of the present study was to investigate, in vitro, the influence of leptin on adipogenesis in rat preadipocytes. A prerequisite to this study was to identify leptin receptors (Ob-Ra and Ob-Rb) in preadipocytes from femoral subcutaneous fat. We observed that expressions of Ob-Ra and Ob-Rb increase during adipogenesis. Furthermore, leptin induces an increase of p42/p44 mitogen-activated protein kinase phosphorylated isoforms in both confluent and differentiated preadipocytes and of STAT3 phosphorylation only in confluent preadipocytes. Moreover, exposure to leptin promoted activator protein-1 complex DNA binding activity in confluent preadipocytes. Finally, exposure of primary cultured preadipocytes from the subcutaneous area to leptin (10 nM) resulted in an increased proliferation ([3H]thymidine incorporation and cell counting) and differentiation (glycerol-3-phosphate dehydrogenase activity and mRNA levels of lipoprotein lipase, peroxisome proliferator-activated receptor-gamma 2, and c-fos). Altogether, these results indicate that, in vitro at least, leptin through its functional receptors exerts a proadipogenic action in subcutaneous preadipocytes.

leptin receptors; mitogen-activated protein kinase; adipogenesis


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

LEPTIN, THE RECENTLY IDENTIFIED product of the ob gene, is a 16-kDa protein produced and secreted primarily by adipocytes (61). Leptin acts through functional receptors (Ob-R) on specific regions of the brain to regulate food intake and energy expenditure. These receptors are related to class I cytokine receptors and their different isoforms (Ob-Ra, b, c, d, e, and f) are transcribed from a single gene via alternative splicing (54). These receptors have the same extracellular domain. Each isoform is expressed in a wide variety of tissues and in a tissue-specific manner. The Ob-Ra, which has a short intracellular domain, is expressed predominantly in kidney, lung, intestine, heart, testes, choroid plexus, brain microvessels, and adipose tissue and less in liver, skeletal muscle, adrenal, spleen, and pancreatic beta -cells (29, 54). The Ob-Rb, which has the longest cytoplasmic domain, is highly expressed in the hypothalamus, cerebellum, and pancreatic beta -cells but weakly expressed in spleen, heart, choroid plexus, and kidney (29, 54).

The leptin-bound Ob-Rb stimulates gene transcription via an activation of different pathways. Indeed, Ob-Rb has the capacity to activate the Janus kinase/signal transducers and activators of transcription (Jak/STAT) and the mitogen-activated protein kinase (MAPK) pathways (54) and to increase transcription of suppressor of cytokine signaling 3 (SOCS-3) (6, 19). In the hypothalamus, the Ob-Rb transduces the leptin signal via an activation of STAT3, which results in a negative regulation of food intake and a positive influence on energy expenditure (54). The leptin-bound Ob-Ra activates Jak2 but is unable to induce STAT activation and is thought to play a role in the transport of leptin into the brain (7, 54).

Besides these important metabolic effects in the hypothalamus, leptin elicits various peripheral actions, as recently shown. Indeed leptin intervenes as a modulator of hematopoiesis, thermogenesis, angiogenesis, lipid metabolism, reproduction, immunological responses, and of pancreatic, intestinal, and kidney functions (24, 50, 52, 54). Moreover, the observation that leptin stimulates lipolysis both in vivo and in vitro supports the concept that leptin acts as an autocrine regulatory signal in adipose tissue (21, 22). Cytokines and growth factors other than leptin are also produced and secreted by adipocytes [i.e., tumor necrosis factor-alpha (TNF-alpha ) and insulin-like growth factor I]. These factors play an important role in the regulation of adipogenesis (15, 49) by modulating the STAT and the MAPK pathways (8, 26, 31, 62). Because leptin signaling also involves the STAT and MAPK cascades (7, 54), a role for leptin as a putative autocrine/paracrine regulatory signal controlling body fat mass development at the level of the adipoconversion process is questionable. The purpose of the present study was to test this hypothesis.

We have at first attempted to identify the presence of Ob-Ra and Ob-Rb in primary cultured confluent and differentiated preadipocytes from the stromal vascular fraction of rat subcutaneous adipose tissue. Second, we have studied the influence of leptin on the activation of MAPK and Jak/STAT pathways during the adipoconversion process. After these receptors were shown to be expressed and functional in preadipocytes, we then investigated the direct influence of leptin on the adipoconversion of these cells in vitro. The results presented herein clearly show that leptin stimulates in vitro the proliferation and differentiation capacities of rat subcutaneous preadipocytes.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials

DMEM, DMEM/Ham's F-12 (50:50 mix), penicillin, streptomycin, HEPES, transferrin, triiodothyronine (T3), leupeptin, aprotinin, phenylmethylsulfonyl fluoride (PMSF) and BSA were purchased from Sigma Chemical (St. Louis, MO). Collagenase was from Roche Molecular Biochemicals (Mannheim, Germany). Recombinant murine leptin was from Pepro Tech (London, UK). Superscript II RNase H-RT was from GIBCO BRL (Grand Island, NY). Taq polymerase and RNA guard were provided by Pharmacia Biotechnology (Uppsala, Sweden). The MEK inhibitor, U-0126, and T4 polynucleotide kinase were from Promega (Madison, WI).

Animals

Male Sprague-Dawley rats were kept under controlled lighting conditions (light, 6:00 AM; dark, 8:00 PM) and constant temperature (21°C) with free access to water and food. Animals (250-275 g) were killed by decapitation, and femoral subcutaneous adipose tissue was immediately removed under sterile conditions.

Cell Culture

The stromal vascular fraction was obtained after digestion of subcutaneous adipose tissues by collagenase as previously described (46). The cells were plated in DMEM supplemented with HEPES (20 mM), streptomycin (0.1 mg/ml), penicillin (100 U/ml), and 8% FCS and maintained at 37°C under 5% CO2 atmosphere. After they were plated, the cells were extensively washed and maintained in primary culture as follows.

First, for cell growth experiments, the cells were maintained for 24 h in DMEM supplemented with 8% FCS and then for another 24 h in DMEM with 2% FCS in the absence or presence of recombinant leptin (10 nM). In preliminary experiments, different concentrations of leptin (1, 5, 10, 50, and 100 nM) and different exposure times to leptin (24 and 48 h) were tested. The optimal conditions found were 24 h of exposure to 10 nM leptin.

Second, for differentiation studies, the cells were maintained in DMEM with 8% FCS until 70-80% confluence was reached (1 day after plating), and then in DMEM-F12 (1:1) supplemented with insulin (5 µg/ml), transferrin (10 µg/ml), T3 (2 nM), and antibiotics (0.1 mg/ml streptomycin and 100 U/ml penicillin) (ITT medium) as described in Ref. 14. Two days later, the ITT medium was removed and replaced by fresh ITT medium with or without leptin (10 nM) and the cell culture was extended by 24 or 48 h. Preliminary experiments again revealed that the best leptin effectiveness occurs with 10 nM leptin.

RNA Extraction

To detect Ob-Ra and Ob-Rb mRNA, total RNA was isolated using Trizol reagent (12) from cultures at total confluence (2 days after plating) and at the differentiated state (6 days after plating, i.e., after 4 days in ITT medium). For the study of lipoprotein lipase (LPL) and peroxisome proliferator-activated receptor-gamma 2 (PPARgamma 2) mRNA expressions, total RNA was extracted on the third and fourth day of culture in ITT medium from differentiated preadipocytes exposed or not to leptin for 24 and 48 h. Finally, the effect of leptin on c-fos mRNA expression was shown on the third day after 2 days in ITT medium and an overnight in a DMEM/Ham's F-12 medium. Total RNA was then extracted after 15, 30, and 60 min of exposure to 10 nM leptin. RNA recovery and quality were checked by measuring the 260/280 optical density ratio and by electrophoresis under denaturing conditions on 2% agarose gel.

RT-PCR

As described previously (37), 2.5 µg total RNA (Ob-Ra and Ob-Rb expressions) or 0.5 µg total RNA (LPL, PPARgamma 2, c-fos, cyclophilin, and beta -actin expressions) were submitted to RT-PCR (47). PCR was performed with a thermocycler Gene Amp PCR 2400 (Perkin Elmer) followed by quantification realized with Bio-gene software. To ensure that amplifications of the genes tested were within the exponential range, different numbers of PCR were run. The number of PCR amplification cycles chosen for each studied gene is indicated in Table 1.

                              
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Table 1.   Oligonucleotide primer sequences and PCR conditions

Cell Counting

At day 1 postplating, culture medium was replaced by fresh medium containing 2% FCS, and cells were then exposed or not to recombinant leptin (10 nM). Twenty-four hours later, cells were washed three times with Hanks' buffer (in mM: 136.7 NaCl, 5.36 KCl, 0.42 Na2HPO4, 0.44 KH2PO4, and 4.16 NaHCO3) followed by addition of 0.2% trypsin in Hanks' buffer to dishes for 2-3 min at 37°C while shaking. Cells were then collected in Hanks' supplemented buffer with 10% FCS (vol/vol). Twenty microliters of this suspension were diluted with five microliters of crystal violet, and the number of cells was established using a hemocytometer.

[3H]thymidine Incorporation

In addition to direct cell counts, [3H]thymidine incorporation was used as a measure of DNA synthesis. At day 1 postplating, the culture medium was changed into DMEM containing 2% FCS, [3H]thymidine (1 mCi/ml) and supplemented or not with recombinant leptin (10 nM). Twenty-four hours later, the dishes were washed three times with saline, and cells were treated for 5 min with 1% SDS and then with 10% TCA for 45 min at 4°C. After filtration on GF/C glass fiber filters (Whatman, Clifton), radioactivity was counted.

Glycerol-3-Phosphate Dehydrogenase Activity Assay

After 24 and 48 h of culture in the absence or presence of leptin (10 nM) in ITT medium, differentiation media were discarded and cells were scraped and sonicated [3 blasts for 15 s; VibraCell (Bioblock, Strasbourg, France)] in ice-cold buffer containing (in mM) 50 Tris · HCl (pH 7.4), 0.25 sucrose, 1 EDTA, and 1 beta -mercaptoethanol. The homogenates were centrifuged at 20,000 g for 10 min at 4°C, and the resulting supernatants were used for glycerol-3-phosphate dehydrogenase (GPDH) assays according to Wise and Green (58). Activities are expressed in milliunits per milligram of protein (1 mU being equal to the oxidation of 1 nmol of NADH2/min).

Ob-R Protein Expression

Rat preadipocyte membranes were prepared at the confluent and differentiated states. Cells were scraped on ice with buffer A containing 10 mM Tris, 0.25 M sucrose, 5 mM EDTA, and protease inhibitors (0.57 mM PMSF, 5 µg/ml leupeptin, and 5 µg/ml aprotinin). Next, cells were sonicated for 15 s and centrifuged (21,000 g at 4°C) for 20 min, and the resulting supernatant was collected and stored in Laemmli's buffer (35) (vol/vol) at -20°C.

In parallel experiments, membranes were obtained from rat hypothalamus and brain, following the procedure previously described in Ref. 36, and used as positive controls for Ob-Rb and Ob-Ra. Equal amounts of membrane proteins (100 µg) were subjected to SDS-PAGE (10% acrylamide). Proteins were transferred to polyvinylidene difluoride (PVDF) membrane for 90 min at room temperature. After blocking in buffer A (20 mM Tris · HCl, 137 mM NaCl, and 0.1% Tween 20) containing 2.5% gelatin for 2 h at room temperature, filters were incubated overnight at room temperature with goat polyclonal anti-Ob-R antibody (K-20) (1: 300 dilution; Santa Cruz Biotechnology, Santa Cruz, CA). Blots were then extensively washed with buffer A and incubated with the secondary antiserum (horseradish peroxidase-labeled anti-goat IgG 1:10,000 dilution) for 1 h at room temperature and washed. Finally, an enhanced chemiluminescence kit from Pierce (Interchim) was used for signal detection. Control experiments with various protein amounts (50-200 µg) were performed to ensure that the densitometric signal intensity was proportional to the loaded amount of protein.

Specificity of the immunoreactive Ob-R proteins was verified by loss of the immunoreactivity of samples when incubated with the antiserum neutralized by the corresponding specific peptide.

STAT3 and MAPK Activation

The time course of the STAT3 phosphorylated isoform was investigated as follows: confluent or differentiated preadipocytes were maintained overnight in a serum-free culture medium at day 1 postplating or after 2 days in ITT medium, respectively. After exposure to recombinant leptin (10 nM) for 5, 15, and 30 min, cells were scraped and sonicated on ice in buffer containing 50 mM Tris, 120 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.5 mM desoxycholate, 0.1% SDS, 1 mM sodium vanadate, 0.57 mM PMSF, 30 mM beta -glycerophosphate, 5 µg/ml aprotinin, and 12.5 µg/ml leupeptin. After centrifugation at 100,000 g for 10 min at 4°C, supernatants were diluted in Laemmli's buffer (vol/vol).

Equal amounts (50 µg) of cellular extracts were subjected to SDS-PAGE (7.5%). Proteins were transferred to PVDF membrane and blocked in buffer A with 5% milk for 1 h. Membranes were then incubated overnight at 4°C with rabbit polyclonal phospho-STAT3 (Tyr-705) antibody (1:1,000 dilution; New England Biolabs) in buffer A with 5% milk or with rabbit polyclonal STAT3 antibody (C-20) (1:200 dilution; Santa Cruz Biotechnology) in buffer A with 2.5% gelatin. Blots were then extensively washed with buffer A and incubated with the secondary antiserum (horseradish peroxidase-labeled anti-rabbit IgG 1:5,000 dilution) for 1 h at room temperature and washed. Finally, signal detection was performed as described above.

To study the MAPK pathway, confluent or differentiated preadipocytes were also maintained overnight in serum-free culture medium before in vitro leptin stimulation. The time course of activation of the p42/p44 MAPK phosphorylated form was investigated as follows: cells were exposed for 2, 5, 15, and 30 min to recombinant leptin (10 nM); for 5 min to U-0126 (10 µM) in the presence or absence of leptin (10 nM); or for 5 min to 10% FCS. Cells were then scraped and sonicated on ice in buffer containing 10 mM Tris, 150 mM NaCl, 2 mM EGTA, 0.5 mM dithiothreitol (DTT), 1 mM sodium vanadate, 30 mM beta -glycerophosphate, 0.57 mM PMSF, 5 µg/ml leupeptin, and 5 µg/ml aprotinin. After cells were centrifuged at 100,000 g for 15 min at 4°C, cytosolic extracts were diluted in Laemmli's buffer (vol/vol).

Equal amounts (10 µg) of cytosolic extracts were subjected to SDS-PAGE (12.5%). Proteins were transferred to a PVDF membrane and blocked in buffer A with 2.5% gelatin for 2 h. Membranes were then incubated overnight at room temperature with rabbit polyclonal p42/p44 MAPK phosphorylated antibody (pTEpY, V8031; 1: 7,000 dilution; Promega) or with mouse monoclonal anti-total MAPK antibody (1:500 dilution; Transduction Laboratories, Lexington, KY) in buffer A with 2.5% gelatin. The resulting blots were extensively washed with buffer A and incubated with the secondary antiserum (horseradish peroxidase-labeled anti-rabbit IgG or anti-mouse IgG 1:10,000 dilution) for 1 h at room temperature and washed. Finally, signal detection was performed as described above.

Activator Protein-1 Complex DNA Binding

The influence of leptin on activator protein-1 complex (AP-1) DNA binding activity was tested as follows: confluent or differentiated preadipocytes were maintained overnight in a serum-free culture medium before in vitro stimulation. Activation of AP-1 DNA binding was measured after 15, 30, and 60 min exposure to recombinant leptin (10 nM) or 30 min exposure to 10% FCS. Then, as described in Ref. 30, cells were scraped on ice in buffer A containing 10 mM Tris, 0.15 M NaCl, 1 mM EDTA, 0.6% Nonidet P-40, 1 mM sodium vanadate, 0.57 mM PMSF, 20 mM beta -glycerophosphate, 10 mM NaF, 0.5 mM DTT, 5 µg/ml aprotinin, and 5 µg/ml leupeptin, pH 7.9. The homogenates were centrifuged at 2,400 g for 5 min at 4°C, and the nuclear extracts were prepared as follows: supernatants were removed, and the resulting pellets were resuspended in cold buffer B (10 mM HEPES, 420 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 1.5 mM MgCl2, 25% glycerol, 0.6% Nonidet P-40, 0.57 mM PMSF, 1 mM sodium vanadate, 20 mM beta -glycerophosphate, 10 mM NaF, 0.5 mM DTT, 5 µg/ml aprotinin, and 5 µg/ml leupeptin, pH 7.9). Suspensions were vigorously shaken at 4°C for 20 min followed by centrifugation at 20,000 g for 20 min at 4°C. The resulting supernatants containing the nuclear extracts were then used for electrophoretic mobility shift assay.

Protein-DNA complexes were formed by incubating 2 µg of nuclear extracts in 10 µl binding cocktail (10 mM HEPES, 50 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 50 mM NaCl, 4 mM spermidin, 2 mM DTT, 100 µg/ml albumin, and 35% glycerol, pH 8) in the presence of 2.5 µg of poly(dI-dC) for 15 min at 4°C. Then 100,000 cpm of 32P-labeled double-stranded oligonucleotide containing a binding site for AP-1 (5'-CGC TTG ATG AGT CAG CCG GAA-3') was added, and the incubations were further extended for 15 min at room temperature. The resulting DNA-protein complexes were separated from the unbound probes by electrophoresis on a native 6% polyacrylamide gel in 0.5× Tris-borate-EDTA buffer. Gels were then dried and subjected to autoradiography. In competition experiments, 1, 10, and 100-fold molar excesses of unlabeled AP-1 double-stranded oligonucleotides were included in the binding reaction mixture. The double-stranded oligonucleotides were labeled with [gamma -32P]ATP (3,000 Ci/mmol) using T4 polynucleotide kinase.

Protein concentration was measured according to Bradford (10) with BSA as standard.

Statistical Analysis

All values were expressed as means ± SE of three to four different experiments, and statistical analysis was performed using unpaired Student's t-test.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We chose to investigate the effects of leptin on rat adipogenesis in subcutaneous preadipocytes, a superficial fat deposit that presented lower replication and differentiation capacities than other deep fat localizations (16, 34) and thus is more appropriate to observe a possible modulation of adipogenesis by an effector.

Expression of Leptin Receptors in Rat Subcutaneous Preadipocytes

Using specific primers for Ob-Ra (short isoform) and Ob-Rb (long isoform) (see Table 1), we measured the expression of both Ob-R isoforms in rat subcutaneous preadipocytes at confluence and differentiation states. By this semiquantitative RT-PCR method, we observed that Ob-Ra and Ob-Rb mRNA expressions increased in subcutaneous preadipocytes during the adipoconversion process (Fig. 1A). The same picture was provided by Western blot analysis (Fig. 1B) using an antibody directed against the amino terminus of the rat leptin receptor, which recognized ~120- and ~210-kDa bands, in preadipocytes. These sizes are consistent with the short and long forms of rat leptin receptor (50). As also shown in Fig. 1B, these bands were identical to the positive controls (brain and hypothalamus) (38) and disappeared in peptide-saturating experiments.


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Fig. 1.   Ob-Ra and Ob-Rb mRNA and protein expressions in rat subcutaneous preadipocytes. A: total RNA was extracted from femoral subcutaneous preadipocytes at confluent (Conf) or differentiated (Diff) states and analyzed by RT-PCR with the primers described in MATERIALS AND METHODS. The densitometric analysis of Ob-R/beta actin RT-PCR signals is represented. Results are means ± SE of 3-4 experiments and are normalized as percentages of the value found in confluent subcutaneous preadipocytes. * P < 0.05; ** P < 0.005. B: membrane fractions from rat brain, hypothalamus, and preadipocytes (confluent and differentiated), prepared as described in MATERIALS AND METHODS, were immunoblotted with goat polyclonal anti-Ob-R antibody, and the specificity of the signals was studied with peptide saturation experiments. One representative experiment is shown.

These results indicate that, at both confluent and differentiated states, rat subcutaneous preadipocytes express the two Ob-Ra and Ob-Rb isoforms of the leptin receptor.

Activation of STAT3 by Leptin

Because the phosphorylation-dependent activation of STAT3 is a major transduction pathway for leptin signaling (33, 50, 54), expression of phosphorylated STAT3 was investigated in cellular extracts from preadipocytes during adipogenesis in response to recombinant leptin. As shown in Fig. 2, exposure of confluent subcutaneous preadipocytes to leptin (10 nM) for 15 min resulted in an increase in the STAT3 phosphorylated form (×1.8 ± 0.35), and the magnitude of this effect was decreased after 30 min. In differentiated subcutaneous preadipocytes, however, no effect of leptin on STAT3 activation could be observed.


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Fig. 2.   Tyrosine phosphorylation of STAT3 by leptin in confluent and differentiated subcutaneous preadipocytes. Cells were maintained in a serum-free culture medium overnight before exposure to leptin (10 nM) as described in MATERIALS AND METHODS. At the indicated times, cellular extracts of confluent or differentiated subcutaneous preadipocytes were prepared and immunoblotted with rabbit polyclonal anti-phospho-STAT3 (Tyr-705) antibody or with anti-STAT3. A: Western blot analysis from 1 representative experiment. STAT3P, phospho-STAT3. B: densitometric analysis of phosphorylated STAT3. Values are means ± SE obtained from 3 separate experiments and are expressed as percentages of control value without leptin (0). * P < 0.05; ns, not significant.

Activation of p42/p44 MAPK by Leptin

Because it was previously shown that Ob-Rb and Ob-Ra can also lead to activation of the MAPK pathway (18, 53, 59), the activated form of p42/p44 MAPK was investigated in cytosolic extracts of confluent and differentiated subcutaneous preadipocytes in response to recombinant leptin. As shown in Fig. 3, addition of leptin (10 nM) induced a clear increase in the p42/p44 MAPK phosphorylated isoforms in both confluent (×2.2 ± 0.25) and differentiated (×3.05 ± 0.57) preadipocytes. The level of activation was maximal after 5 min. The same effect was observed after preincubation with 10% FCS (positive control). Preincubation of preadipocytes for 5 min with the two specific MAPK inhibitors U-0126 (20) or PD-98059 (17) (data not shown) blunted the leptin effect, which confirmed that p42/p44 MAPK-activated isoforms were targets of leptin (Fig. 3). Moreover, we observed that total MAPK protein expression was unaltered by the leptin treatment.


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Fig. 3.   Activation of p42/p44 mitogen-activated protein kinase (MAPK) by leptin in confluent and differentiated subcutaneous preadipocytes. Cells were maintained in a serum-free culture medium overnight before exposure to leptin (10 nM), 10% FCS, or U-0126 (U0; 10 µg/ml) in the presence or absence of leptin (lept). At the indicated times, cytosolic extracts were prepared and immunoblotted with either rabbit anti-p42/p44 MAPK or mouse anti-total MAPK antibodies. A: Western blot analysis from 1 representative experiment. B: densitometric analysis of the active p42 MAPK/total MAPK (t MAPK) protein immunoblots. Values are means ± SE obtained from 3 separate experiments and are expressed as percentages of control value without leptin (0). ** P < 0.005.

Because activation of the MAPK signaling pathway by growth factors results in AP-1 activation (for review see Ref. 57), we next investigated whether the AP-1 DNA binding activity in vitro could be a possible target for leptin in preadipocytes.

As shown in Fig. 4, after 15 min exposure to leptin, an increase (×1.5 ± 0.12) in AP-1 DNA binding activity occurred in confluent but not in differentiated preadipocytes (data not shown). Moreover, this band completely disappeared in the presence of 100-fold unlabeled AP-1 probe, and, after 15 min of exposure to 10% FCS, a twofold increase in AP-1 binding activity was observed, thus confirming the validity of our assay.


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Fig. 4.   Influence of leptin on AP-1 DNA binding activity in confluent preadipocytes. Cells were maintained in a serum-free culture medium overnight before exposure to leptin (10 nM) or to 10% FCS. At the indicated times, nuclear extracts were prepared and then incubated with a radiolabeled consensus AP-1 probe in the absence or presence of 1-, 10-, and 100-fold molar excess (×1, ×10, and ×100) of unlabeled AP-1 (competitor DNA) as described in MATERIALS AND METHODS. Representative of 3 independent experiments.

In vitro Effects of Leptin on Adipogenesis

In several reports, leptin was shown to control cell proliferation (24, 50, 56). Conversely, other cytokines like TNF and interleukin-6 have been reported to inhibit cell differentiation (25, 43, 49). In contrast, leptin has been shown to enhance differentiation of human marrow stromal cells into osteoblasts and to inhibit their adipoconversion (55). In another studies, leptin was described as an inductor of differentiation for hematopoietic (24) and germ cells (18). These different observations led us to consider the possibilities that leptin may modulate the proliferation and differentiation capacities of rat subcutaneous preadipocytes.

Cell growth. As shown in Fig. 5, a 24 h exposure to leptin (10 nM) resulted in a significant increase in subcutaneous preadipocyte growth (×1.44 ± 0.07), as measured by [3H]thymidine incorporation into DNA. It should be noted that the same results were obtained whether 2% FCS was present or not in the culture media and also that insulin (1 µM), used as a positive control, enhanced preadipocyte cell growth (data not shown). Moreover, the addition of U-0126 (10 µM), which per se had no effect on cell proliferation, abolished the positive effect of leptin on subcutaneous preadipocyte growth.


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Fig. 5.   Effects of leptin on subcutaneous preadipocyte growth. One day after plating, cells were exposed to recombinant leptin (10 nM), U-0126 (10 µM), and U-0126 + leptin, and [3H]thymidine for 24 h. Results are means ± SE of 3-4 experiments and are normalized as percentages of the control value (without leptin). ** P < 0.005.

Finally, the leptin-stimulated proliferation of subcutaneous preadipocytes was also confirmed by direct cell counting (×1.4 ± 9, n = 3). These results show that leptin increases subcutaneous preadipocyte replication in vitro.

Cell differentiation. As shown in Fig. 6A, exposure to leptin (10 nM) for 48 h results in a clear increase in lipid droplet accumulation in subcutaneous preadipocytes. These morphological observations were confirmed by measurements of GPDH activity, a late marker of adipocyte differentiation that faithfully reflects cellular triacylglycerol content (23). As a matter of fact, as shown in Fig. 6B, exposure to leptin (10 nM) for 48 h resulted in an increase in the level of GPDH activity (×1.5) in subcutaneous preadipocytes. This effect was suppressed in the presence of the specific MEK inhibitor, U-0126 (10 µM), which per se had little effect on GPDH activity (data not shown). No effect of leptin was seen, however, after 24 h of exposure to leptin (data not shown) probably because GPDH expression occurs later.


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Fig. 6.   Effects of leptin on in vitro adipogenesis in rat preadipocytes. A: photomicrographs of differentiated subcutaneous preadipocytes after a 48-h exposure to leptin. Subcutaneous preadipocytes were cultured in ITT medium for 48 h and then exposed to recombinant leptin (10 nM) for 48 h. B: glycerol-3-phosphate dehydrogenase (GPDH) activity in differentiated subcutaneous preadipocytes after a 48-h exposure to leptin. Preadipocytes from subcutaneous fat deposits were cultured in ITT medium for 48 h and then exposed to recombinant leptin (10 nM) for 48 h. Results are means ± SE of 3-4 experiments. * P < 0.05.

Because the LPL gene is expressed early during the adipogenic process (1), we investigated the effect of leptin on LPL mRNA levels in primary cultured subcutaneous preadipocytes. After 2 days of culture in the differentiation medium, the preadipocytes were exposed to leptin (10 nM) for either 24 or 48 h, and then RT-PCR analysis of LPL mRNA was performed. As shown in Fig. 7A, LPL mRNA expression was increased in subcutaneous preadipocytes (×3.07 ± 0.27) after 24 h exposure to leptin. After 48 h, however, leptin failed to elicit any effect on LPL mRNA, which is not surprising considering the early expression of the LPL gene during the adipoconversion process and the observation that LPL mRNA levels increase (×3.5) between 24 and 48 h of culture in control cells (Fig. 7A).


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Fig. 7.   Effects of leptin on lipoprotein lipase (LPL) mRNA and peroxisome proliferator-activated receptor-gamma 2 (PPARgamma 2) mRNA expressions during adipogenesis in rat preadipocytes. Total RNA was extracted from differentiated subcutaneous preadipocytes cultured in ITT medium for 48 h and then exposed to 10 nM recombinant leptin (lept) for 24 or 48 h. Total RNA was subjected to RT-PCR with the primers described in MATERIALS AND METHODS. Densitometric analysis of LPL/beta actin (A) or PPARgamma /beta -actin (B) RT-PCR signal ratios is represented. Results are means ± SE of 3-4 experiments and are normalized as percentages of the respective control (cont) values (without leptin). *** P < 0.0005; ** P < 0.005; * P < 0.05; (a), leptin (24 h) or control (48 h) vs. control (24 h); (b), leptin (48 h) vs. control (48 h).

PPARgamma is a major transactivating factor involved in the adipoconversion process (39). To investigate whether leptin might affect PPARgamma 2 mRNA expression, cells were maintained in the differentiating medium for 2 days before exposure to leptin for either 24 or 48 h (Fig. 7B).

As observed for LPL mRNA expression, the results of RT-PCR analysis show that PPARgamma 2 mRNA levels in subcutaneous preadipocytes were increased after 24 h exposure to leptin (×1.5 ± 0.1), but this effect could no longer be observed after 48 h leptin exposure, probably because in control cells PPARgamma 2 mRNA increased 1.5-fold between 24 and 48 h of culture (Fig. 7B).

These experiments, indicating that leptin modulates in vitro the adipogenic process in subcutaneous preadipocytes, led us to investigate the underlying mechanism(s). AP-1 consists of Fos and Jun protein homo- or heterodimers and binds to regulatory sequences in the promoter of various target genes involved in cell growth, differentiation, and metabolism (45). Indeed, various observations have led to the assignment of an important role to the AP-1 complex in the regulation of the adipocyte differentiation process (27, 60). This complex induces transcription of both the adipocyte intracellular lipid-binding protein P2 (aP2) gene (51) and the LPL gene in Ob1771 preadipose cells (3). These observations led us to investigate the effects of leptin on c-fos mRNA levels in preadipocytes during differentiation (after 2 days in ITT medium). Using RT-PCR analysis, we found that exposure of differentiating preadipocytes to 10 nM leptin resulted in a significant increase in c-fos transcript levels after 15 min (×3.3 ± 1), an effect that was further amplified (×6.5 ± 0.6) after 30 min (Fig. 8).


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Fig. 8.   Effects of leptin on the c-fos mRNA expression in differentiated subcutaneous preadipocytes. Cells were maintained in DMEM/Ham's F-12 medium before exposure to leptin (10 nM). At the indicated times, total RNA was extracted and subjected to RT-PCR to determine c-fos mRNA levels using cyclophilin as an internal standard. A: RT-PCR from 1 representative experiment. B: densitometric analysis of c-fos mRNA RT-PCR. Abundance of c-fos mRNA relative to that of cyclophilin mRNA was expressed as percentages of control value without leptin (0). Values are means ± SE of 3 separate experiments. *** P < 0.0005; * P < 0.05.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Leptin, the ob gene product, is synthesized in adipose tissue and plays an important role in body fat mass homeostasis (28, 42). Leptin acts by binding to specific receptors, the Ob-R receptors, which belong to the class I cytokine receptor family. Recently, the presence of Ob-R in adipose tissue has been reported (29), suggesting that the leptin effects on this tissue such as lipolysis stimulation (21, 22) are mediated through these receptors.

In the present study, Ob-R mRNA and protein were characterized by RT-PCR and immunoblotting in both rat confluent and differentiated subcutaneous preadipocytes. In agreement with a recent report in human differentiated preadipocytes (9), we show here that rat preadipocytes express the two Ob-R isoforms, Ob-Ra and Ob-Rb. Moreover, our experiments indicate that these expressions increase during the adipoconversion process. The latter results strongly suggest that leptin intervenes as an autocrine/paracrine signal controlling preadipocyte replication and/or differentiation, as do other cytokines such as TNF-alpha (49) and the leukemia inhibitory factor (LIF) (2).

The functionality of these Ob-R receptors in preadipocytes at both confluent and differentiated states was evaluated by measuring the activation of the STAT3 and MAPK signaling pathways. Because the concentration of leptin in our in vitro experiments (10 nM) was 40 times higher than the normal rat blood leptin level, the physiological relevance of the present data could be questionable. However, the leptin concentration used in our study is in the lower part of the range of those tested in other in vitro studies (10-120 nM) (40, 11), and, due to the lack of soluble leptin receptor in our culture medium, the stability of the recombinant leptin is certainly lower than in the plasma in vivo. Moreover, a recent in vivo study reported much higher leptin levels in fat interstitial fluid than in blood (41), suggesting that the leptin concentration tested in the present study is physiological.

In the present in vitro study, it was demonstrated that leptin rapidly and transiently activated the MAPK pathway through phosphorylation of the p42 and p44 isoforms in both confluent and differentiated preadipocytes. Activation of MAPK generally results in a phosphorylation-dependent modulation of the transactivation activity of various transcriptional factors including the AP-1. As shown here, exposure to leptin increases AP-1 DNA-binding activity in confluent but not in differentiated preadipocytes, suggesting that, in the former cells, leptin acts like growth factors (for review see Ref. 57) through MAPK activation and AP-1 DNA binding activity stimulation. Moreover, we observed, for the first time, that leptin in vitro increases preadipocyte proliferation and that this positive effect was blocked by the specific MEK inhibitor, U-0126. These results suggest that leptin stimulates the proliferation of rat subcutaneous preadipocytes through activation of both the MAPK cascade and AP-1 DNA binding activity as leptin does in other cell types (48, 53).

In parallel to MAPK pathway activation, we also found that leptin activates the STAT pathway through increased STAT3 phosphorylation in rat confluent preadipocytes. In a recent report, Deng et al. (13) have shown that STAT3 phosphorylation correlates with postconfluent preadipocyte mitotic clonal expansion, suggesting a role for STAT3 in the proliferative phase of adipogenesis. This observation allows postulation that the positive effect of leptin on rat preadipocyte growth may be linked to STAT3 phosphorylation in addition to MAPK pathway activation. Moreover, our in vitro present finding in confluent preadipocytes is consistent with in vivo experiments showing the same leptin effects in white adipose tissue (4, 33, 53).

However, in differentiated preadipocytes leptin was ineffective on STAT3 phosphorylation. Possible explanations to this negative finding are 1) the occurrence of a sustained STAT3 activation state during the course of the adipoconversion process or 2) an increased expression of the suppressor of cytokine signaling, SOCS-3, whose expression is STAT3-dependent and whose role is to prevent leptin-induced STAT3 activation through inhibition of Jak2 tyrosine phosphorylation (6).

Another signaling cascade that could also be involved in the leptin effects on adipose tissue is the phosphatidylinositol 3-kinase (PI3-kinase)-mediated pathway. It is known that in C2C12 myotubes (5) leptin was shown to display various effects of insulin such as stimulation of glucose transport and glycogen synthesis through stimulation of PI3-kinase (32). However, a recent study reported that leptin rapidly activates STAT3 and MAPK in adipose tissue explants ex vivo and in 3T3-L1 adipocytes but has no significant effect on PI3-kinase activity and Akt phosphorylation and activity (33). Although not investigated in the current study, the effects of leptin on the PI3-kinase pathway during preadipocyte-adipocyte conversion remain to be established.

This study describes a proadipogenic effect of leptin on subcutaneous preadipocytes. We observed that this effect was correlated with increased expressions of the early and late markers of differentiation: LPL and GPDH, respectively, in these cells. In the same cells, leptin also stimulated expression of two key adipogenic transcriptional factors c-fos and PPARgamma 2, an effect that was accompanied by an increased fat storage in these cells. Moreover, in the differentiated cells, leptin induced a clear activation of p42/p44 MAPK, which seems to play a pivotal role in the proadipogenic effect of leptin, since, in the presence of the specific MEK inhibitor U-0126, the positive effect of leptin on GPDH activity was abolished. These results are to be compared with those observed with another cytokine, LIF, which also increase the differentiation of the preadipose cells Ob 1771 and 3T3-F442A via the MAPK cascade (2).

The present observation of increased adipogenesis in response to leptin in subcutaneous preadipocytes leads to speculation of a possible physiological role for leptin in vivo in the setting of subcutaneous adipose tissue in rats. Such a role, if any, would be consistent with a recent study reporting that transgenic mice overexpressing leptin showed at first a severe loss of body weight followed by a rebound effect characterized by an increase in body fat mass and number of lipid-filled adipocytes (44).

In conclusion, the present study demonstrates that leptin increases both the proliferation and differentiation of subcutaneous preadipocytes in vitro and suggests that leptin in vivo might be an autocrine/paracrine activator of adipogenesis in rat subcutaneous adipose tissue.


    ACKNOWLEDGEMENTS

This work was supported by the Université Paris V and the Comité des Yvelines de la Ligue Contre le Cancer.


    FOOTNOTES

Address for reprint requests and other correspondence: Y. Giudicelli, Service de Biochimie, Centre Hospitalier, 78303 Poissy Cedex, France (E-mail: biochip{at}wanadoo.fr).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpcell.00331.2001

Received 19 July 2001; accepted in final form 29 October 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Ailhaud, G, Grimaldi P, and Negrel R. Cellular and molecular aspect of adipose tissue development. Annu Rev Nutr 12: 207-233, 1992[ISI][Medline].

2.   Aubert, J, Dessolin S, Belmonte N, Li M, McKenzie FR, Staccini L, Villageois P, Barhanin B, Vernallis A, Smith AG, Ailhaud G, and Dani C. Leukemia inhibitory factor and its receptor promote adipocyte differentiation via the mitogen-activated protein kinase cascade. J Biol Chem 274: 24965-24972, 1999[Abstract/Free Full Text].

3.   Barcellini-Courget, S, Pradines-Figueres A, Roux P, Dani C, and Ailhaud G. The regulation by growth hormone of lipoprotein lipase gene expression is mediated by c-fos protooncogene. Endocrinology 134: 271-276, 1993[Abstract].

4.   Bendinelli, P, Maroni P, Pecori Giraldi F, and Piccoletti R. Leptin activates Stat3, Stat1 and AP-1 in mouse adipose tissue. Mol Cell Endocrinol 168: 11-20, 2000[ISI][Medline].

5.   Berti, L, Kellerer M, Capp E, and Haring HU. Leptin stimulates glucose transport and glycogen synthesis in C2C12 myotubes: evidence for PI3-kinase mediated effect. Diabetologia 40: 606-609, 1997[ISI][Medline].

6.   Bjørbaek, C, El-Haschimi K, Frantz JD, and Flier JS. The role of SOCS-3 in leptin signaling and leptin resistance. J Biol Chem 274: 30059-30065, 1999[Abstract/Free Full Text].

7.   Bjørbaek, C, Shigeo U, da Silva B, and Flier JS. Divergent signaling capacities of the long and short isoforms of the leptin receptor. J Biol Chem 272: 32686-32695, 1997[Abstract/Free Full Text].

8.   Boney, CM, Gruppuso PA, Faris RA, and Frackelton AR. The critical role of Shc in insulin-like-growth factor-I-mediated mitogenesis and differentiation in 3T3-L1 preadipocytes. Mol Endocrinol 14: 805-813, 2000[Abstract/Free Full Text].

9.   Bornstein, SR, Abu-Asab M, Glasow A, Päth G, Hauner H, Tsokos M, Chrousos GP, and Scherbaum WA. Immunohistochemical and ultrastructural localization of leptin and leptin receptor in human white adipose tissue and differentiating human adipose cells in primary culture. Diabetes 49: 532-538, 2000[Abstract].

10.   Bradford, MM. A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72: 248-254, 1976[ISI][Medline].

11.   Caprio, M, Isidori AM, Carta AR, Moretti C, Dufau ML, and Fabbri L. Expression of functional leptin receptors in rodent Leydig cells. Endocrinology 140: 4939-4947, 1999[Abstract/Free Full Text].

12.   Chomczynski, P, and Sacchi N. Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156-159, 1987[ISI][Medline].

13.   Deng, J, Hua K, Lesser SS, and Harp JB. Activation of signal transducer and activator of transcription-3 during proliferative phases of 3T3-L1 adipogenesis. Endocrinology 141: 2370-2376, 2000[Abstract/Free Full Text].

14.   Deslex, S, Negrel R, and Ailhaud G. Development of a chemically defined serum free medium for differentiation of rat adipose precursor cells. Exp Cell Res 168: 15-30, 1987[Medline].

15.   Dieudonné, MN, Pecquery R, Leneveu MC, and Giudicelli Y. Opposite effects of androgens and estrogens on adipogenesis in rat preadipocytes: evidence for sex and site-related specificities and possible involvement of insulin-like growth factor 1 receptor and peroxisome proliferator-activated receptor gamma 2. Endocrinology 141: 649-656, 2000[Abstract/Free Full Text].

16.   Djian, P, Roncari DAK, and Hollenberg CH. Influence of anatomic site and age on the replication and differentiation of rat adipocyte precursors in culture. J Clin Invest 72: 1200-1208, 1983[ISI][Medline].

17.   Dudley, DT, Pang L, Decker SJ, Bridges AJ, and Saltiel AR. A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc Natl Acad Sci USA 92: 7686-7689, 1995[Abstract].

18.   El-Hefnawy, T, Ioffe S, and Dym M. Expression of the leptin receptor during germ cell development in the mouse testis. Endocrinology 141: 2624-2630, 2000[Abstract/Free Full Text].

19.   Emilsson, V, Arch JR, de Groot RP, Lister CA, and Cawthorne MA. Leptin treatment increases suppressors of cytokine signaling in central and peripheral tissues. FEBS Lett 455: 170-174, 1999[ISI][Medline].

20.   Favata, MF, Horiuchi KY, Manos EJ, Daulerio AJ, Stradley DA, Feeser WS, Van Dyk DE, Pitts WJ, Earl RA, Hobbs F, Copeland RA, Magolda RL, Scherle PA, and Trzaskos JM. Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J Biol Chem 273: 18623-18632, 1998[Abstract/Free Full Text].

21.   Frühbeck, G, Aguado M, Gómez-Ambrosi J, and Martínez JA. Lipolytic effect of in vivo leptin administration on adipocytes of lean and ob/ob mice, but not db/db mice. Biochem Biophys Res Commun 250: 99-102, 1998[ISI][Medline].

22.   Frühbeck, G, Aguado M, and Martínez JA. In vitro lipolytic effect of leptin on mouse adipocytes: evidence for a possible autocrine/paracrine role of leptin. Biochem Biophys Res Commun 240: 590-594, 1997[ISI][Medline].

23.   Gaillard, D, Negrel R, Lagarde M, and Ailhaud G. Requirement and role of arachidonic acid in the differentiation of preadipose cells. Biochem J 257: 389-397, 1989[ISI][Medline].

24.   Gainsford, T, Willson TA, Metcalf D, Handman E, McFarlane C, Ng A, Nicola NA, Alexander WS, and Hilton DJ. Leptin can induce proliferation, differentiation, and functional activation of hemapoietic cells. Proc Natl Acad Sci USA 93: 14564-14568, 1996[Abstract/Free Full Text].

25.   Greenberg, AS, Nordan RP, McIntosh J, Calvo JC, Scow RO, and Jablons S. Interleukin 6 reduces lipoprotein lipase activity in adipose tissue of mice in vivo and in 3T3-L1 adipocytes: a possible role for interleukin 6 in cancer cachexia. Cancer Res 52: 4113-4116, 1992[Abstract].

26.   Guo, D, Dunbar JD, Yang CH, Pfeffer LM, and Donner DB. Induction of Jak/STAT by activation of the type 1 TNF receptor. J Immunol 160: 2742-2750, 1998[Abstract/Free Full Text].

27.   Gurland, P, Ashcom G, Cochran BH, and Schwartz J. Rapid events in growth hormone action. Induction of c-fos and c-jun transcription in 3T3-F442A preadipocytes. Endocrinology 127: 3187-3195, 1990[Abstract].

28.   Halaas, JL, Gajiwala KS, Maffei M, Cohen SL, Chait BT, Rabinowitz D, Lallone RL, Burley SK, and Friedman JM. Weight-reducing effects of the plasma protein encoded by the obese gene. Science 269: 546-549, 1995[ISI][Medline].

29.   Hoggard, N, Mercer JG, Rayner DV, Moar K, Trayhurn P, and Williams LM. Localization of leptin receptor mRNA splice variants in murine peripheral tissues by RT-PCR and in situ hybridization. Biochem Biophys Res Commun 232: 383-387, 1997[ISI][Medline].

30.   Hoppe-Seyler, F, Butz K, Rittmuller C, and Von Knebel Doeberitz M. A rapid microscale procedure for the simultaneous preparation of cytoplasmic RNA, nuclear binding proteins and enzymatically active luciferase extracts (Abstract). Nucleic Acids Res 19: 5080, 1991[ISI][Medline].

31.   Jain, RG, Phelps KD, and Pekala PH. Tumor necrosis factor-alpha initiated signal transduction in 3T3-L1 adipocytes. J Cell Physiol 179: 58-66, 1999[ISI][Medline].

32.   Kellerer, M, Koch M, Metzinger E, Mushack J, Capp E, and Haring HU. Leptin activates PI-3 kinase in C2C12 myotubes via janus kinase-2 (JAK-2) and insulin receptor substrate-2 (IRS-2) dependent pathways. Diabetologia 40: 1358-1362, 1997[ISI][Medline].

33.   Kim, YB, Uotani S, Pierroz DD, Flier JS, and Khan BB. In vivo administration of leptin activates signal transduction directly in insulin-sensitive tissues: overlapping but distinct pathways from insulin. Endocrinology 141: 2328-2339, 2000[Abstract/Free Full Text].

34.   Lacasa, D, Agli B, Moynard D, and Giudicelli Y. Evidence for a regional-specific control of rat preadipocyte proliferation and differentiation by the androgenic status. Endocrine 3: 789-793, 1995[ISI].

35.   Laemmli, UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970[ISI][Medline].

36.   Le Jossec, M, Trivalle C, Cloix JF, Pecquery R, Giudicelli Y, and Dausse JP. Differential distribution of alpha 2-adrenoceptor subtypes and messenger RNA expression between renal cortex of salt-sensitive and salt-resistant Sabra rats. J Hypertens 13: 781-790, 1995[ISI][Medline].

37.   Machinal, F, Dieudonne MN, Leneveu MC, Pecquery R, and Giudicelli Y. In vivo and in vitro ob gene expression and leptin secretion in rat adipocytes: evidence for a regional specific regulation by sex steroid hormones. Endocrinology 140: 1567-1574, 1999[Abstract/Free Full Text].

38.   Magni, P, Vettor R, Pagano C, Calcagno A, Beretta E, Messi E, Zanisi M, Martini L, and Motta M. Expression of a leptin receptor in immortalized gonadotropin-releasing hormone-secreting neurons. Endocrinology 140: 1581-1585, 1999[Abstract/Free Full Text].

39.   Mandrup, S, and Lane MD. Regulating adipogenesis. J Biol Chem 272: 5367-5370, 1997[Free Full Text].

40.   Murakami, T, Yamashita T, Iida M, Kuwajima M, and Shima K. A short form of leptin receptor performs signal transduction. Biochem Biophys Res Commun 231: 26-29, 1997[ISI][Medline].

41.   Orban, Z, Remaley AT, Sampson M, Trajanoski Z, and Chrousos GP. The differential effect of food intake and beta -adrenergic stimulation on adipose-derived hormones and cytokines in man. J Clin Endocrinol Metab 84: 2126-2133, 1999[Abstract/Free Full Text].

42.   Pelleymounter, MA, Cullen MJ, Baker MB, Hecht R, Winters D, Boone T, and Collins F. Effect of the obese gene product on body weight regulation in ob/ob mice. Science 269: 540-543, 1995[ISI][Medline].

43.   Petruschke, TH, and Hauner H. Tumor necrosis factor-alpha prevents the differentiation of human adipocyte precursor cells and causes delipidation of newly developed fat cells. J Clin Endocrinol Metab 76: 742-747, 1993[Abstract].

44.   Qiu, J, Ogus S, Lu R, and Chehab FF. Transgenic mice overexpressing leptin accumulate adipose mass at an older, but not younger, age. Endocrinology 142: 348-358, 2001[Abstract/Free Full Text].

45.   Ransone, LJ, and Verma IM. Nuclear proto-oncogenes fos and jun. Annu Rev Cell Biol 6: 539-557, 1990[ISI].

46.   Rodbell, M. The role of hormone receptors and GTP-regulatory proteins in membrane transduction. Nature 284: 17-22, 1980[ISI][Medline].

47.   Saiki, RK, Gelfand DH, Stoffei S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, and Erlich HA. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487-491, 1988[ISI][Medline].

48.   Schneider, R, Bornstein SR, Chrousos GP, Boxberger S, Ehninger G, and Breidert M. Leptin mediates a proliferative response in human gastric mucosa cells with functional receptor. Horm Metab Res 33: 1-6, 2001[ISI][Medline].

49.   Sethi, JK, and Hotamisligil GS. The role of TNFalpha in adipocyte metabolism. Cell Dev Biol 10: 19-29, 1999.

50.   Sierra-Honigmann, MR, Nath AK, Murakami C, Garcia-Cardeña G, Papapetropoulos A, Sessa WC, Madge LA, Schechner JS, Schwabb MB, Polverini PJ, and Flores-Riveros JR. Biological action of leptin as an angiogenic factor. Science 281: 1683-1686, 1998[Abstract/Free Full Text].

51.   Spiegelman, BM, Distel RJ, Ro HS, Rosen BS, and Satterberg B. Fos protooncogene and the regulation of gene expression in adipocyte differentiation. J Cell Biol 107: 829-832, 1988[ISI][Medline].

52.   Stenvinkel, P, Lönnqvist F, and Schalling M. Molecular studies of leptin: implications for renal disease. Nephrol Dial Transplant 14: 1103-1112, 1999[Abstract].

53.   Takahashi, Y, Okimura Y, Mizuno I, Iida K, Takahashi T, Kaji H, Abe H, and Chihara K. Leptin induces mitogen-activated protein kinase-dependent proliferation of C3H10T1/2 cells. J Biol Chem 272: 12897-12900, 1997[Abstract/Free Full Text].

54.   Tartaglia, L. The leptin receptor. J Biol Chem 272: 6093-6096, 1997[Free Full Text].

55.   Thomas, T, Gori F, Khosla S, Jensen MD, Burguera B, and Riggs BL. Leptin acts on human marrow stromal cells to enhance differentiation to osteoblasts and to inhibit differentiation to adipocytes. Endocrinology 140: 1630-1638, 1999[Abstract/Free Full Text].

56.   Umemoto, Y, Tsuji K, Yang FC, Ebihara Y, Kaneko A, Furukawa S, and Nakahata T. Leptin stimulates the proliferation of murine myelocytic and primitive hematopoietic progenitor cells. Blood 90: 3438-3443, 1997[Abstract/Free Full Text].

57.   Wisdom, R. AP-1: one switch for many signals. Exp Cell Res 253: 180-185, 1999[ISI][Medline].

58.   Wise, LS, and Green H. Participation of one isoenzyme of cytosolic glycerophosphate dehydrogenase in adipose conversion of 3T3 cells. J Biol Chem 254: 273-275, 1979[Abstract].

59.   Yamashita, T, Murakami T, Otani S, Kuwajima M, and Shima K. Leptin receptor signal transduction: OBRa and OBRb of fa type. Biochem Biophys Res Commun 246: 752-759, 1998[ISI][Medline].

60.   Zeng, G, Dave JR, and Chiang PK. Induction of proto-oncogenes during 3-deazaadenosine-stimulated differentiation of 3T3-L1 fibroblasts to adipocytes: mimicry of insulin action. Oncol Res 9: 205-211, 1997[ISI][Medline].

61.   Zhang, Y, Proenca R, Maffei M, Barone M, Leopold L, and Friedman JM. Positional cloning of the mouse obese gene and its human homologue. Nature 372: 425-432, 1994[ISI][Medline].

62.   Zhong, CS, Chan J, Levy DE, Horvath C, Sadowski HB, and Wang LH. Mechanism of STAT3 activation by insulin-like growth factor I receptor. J Biol Chem 275: 15099-15105, 2000[Abstract/Free Full Text].


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