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INSERM U478, Institut Fédératif de Recherche Claude Bernard, Faculté de Médecine Xavier Bichat, 16 rue Henri Huchard, 75018 Paris, France
1 INSERM U584, Faculté de Médecine Necker-Enfants Malades, 156 rue de Vaugirard, 75730 Paris Cedex 15, France
2 INSERM U410, Institut Fédératif de Recherche Claude Bernard, Faculté de Médecine Xavier Bichat, 16 rue Henri Huchard, 75018 Paris, France
(Requests for offprints should be addressed to N Binart; Email: binart{at}necker.fr)
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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BAT is a highly specialized tissue, which clearly differs from the energy-storing white adipose tissue. Indeed, BAT is characterized by its thermogenic function because it has the ability to dissipate energy and to provide heat. This results from the activity of the uncoupling protein 1 (UCP1) which translocates protons through the inner membrane of mitochondria, uncouples oxidative phosphorylation and bypasses the last enzymatic step of the respiratory chain, ATP synthase (Ricquier & Bouillaud 2000). BAT plays therefore a major role in the regulation of body temperature, especially in small animals such as mice or rats. Moreover, we have recently shown that brown adipocytes are also endocrine cells able to secrete leptin (Buyse et al. 2001) and other adipocytokines such as resistin and adiponectin, two adipocyte-specific secretory factors involved in glucose and lipid metabolism (Viengchareun et al. 2002). Despite high levels of PRLR transcripts in BAT, the precise role of PRL in this tissue is still unclear. Indeed, a recent study reported that infusion of PRL to pregnant rats induced an increase in UCP1 protein expression in the BAT of fetuses (Budge et al. 2002), while the same authors reported that PRL caused a reduction in UCP1 abundance only in females and not in males (Pearce et al. 2003). These observations suggested a potential involvement of PRL in thermoregulation.
With the aim of better understanding the specific role of PRL in BAT, we used both the brown adipocyte T37i cell line (Zennaro et al. 1998) and the PRLR knockout mouse model (Ormandy et al. 1997). T37i cells were derived from a hibernoma developed in a transgenic mouse in which the expression of SV40 large T antigen was placed under the control of the proximal promoter of the human mineralocorticoid receptor gene. These cells are capable of differentiating into mature brown adipocytes upon insulin and triiodothyronine (T3) exposure as assessed by their ability to display multilocular intracytoplasmic lipid droplets and by the occurrence of specific adipogenic gene activation (Penfornis et al. 2000). Importantly, expression of UCP1, but also of leptin, resistin and adiponectin were demonstrated in this cell line (Zennaro et al. 1998, Buyse et al. 2001, Viengchareun et al. 2002).
Null mutation of the PRLR has mainly highlighted its essential role in reproduction and mammary gland development and suggested that most of its other reported target tissues are presumably modulated by, rather than strictly dependent on PRL. Among the metabolic actions of PRL (Bole-Feysot et al. 1998), its role in lipid metabolism remains poorly understood. Progressive reduction in body weight associated with a reduction in total abdominal fat mass and in leptin concentrations was observed in null females (Freemark et al. 2001); this is in agreement with the increase of serum leptin concentrations observed in PRL transgenic females (Ling & Billig 2001). The T37i cell line and the PRLR knockout model therefore represent suitable experimental systems to explore the function of PRL in BAT.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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T37i cells were cultured in a complete medium composed of DMEM/Ham’s F12, 10% fetal calf serum, 2 mM glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin and 20 mM HEPES and were grown at 37 °C in a humidified atmosphere with 5% CO2. All products were purchased from Invitrogen. Differentiation into mature brown adipocytes was achieved by incubating subconfluent undifferentiated T37i cells with complete medium supplemented with 2 nM T3 (Sigma) and 20 nM insulin (Invitrogen) for 7 days (Zennaro et al. 1998). Before each hormonal treatment, T37i cells were subsequently cultured for 18 h in the same medium without any serum named ‘minimum medium’. Human PRL was produced by recombinant technology then purified as described previously (Goffin et al. 1996) and kindly provided by Dr V Griffin (INSERM U584, Paris, France). All experiments were performed with T37i cells between passages 10 and 20. Actinomycin D, all-trans retinoic acid and isoproterenol were purchased from Sigma.
PRLR knockout mice
All experimental designs and procedures were in agreement with the guidelines of the animal ethics committee of the Ministère de l’Agriculture (France). Wild-type, PRLR+/– and PRLR–/–mice on a pure 129 genetic background were generated by heterozygous crossbreeding (Ormandy et al. 1997). PCR analysis of tail DNA determined the genotypes of the offspring as described previously (Binart et al. 2000).
Ribonuclease protection assay (RPA)
PRLR cDNA was digested with HindIII and BamHI to generate a 390 bp fragment corresponding to exon 10 of the long-form PRLR gene, which was thereafter ligated into pBluescript KS. Ob-PGEMT easy plasmid was generated as previously described (Buyse et al. 2001). In vitro synthesis of riboprobes was achieved using the PRLR-pBluescript KS or the Ob-PGEMT easy plasmids linearized by HindIII and NcoI respectively. PRLR 
-32P-labeled antisense riboprobe was synthesized with T7 RNA polymerase (Promega) whereas Ob antisense riboprobe was synthesized with Sp6. The mouse ß-actin riboprobe, used as an internal control, was synthesized with T7 RNA polymerase after digestion of the ß-actin-pGEM3 plasmid by Bsu36I. RPAs were performed as previously described (Buyse et al. 2001). Fifty micrograms of total RNA, isolated from T37i cells or BAT using TRIZOL reagent (Invitrogen), were hybridized with 4 x 105 c.p.m. PRLR riboprobe or 4x 105 c.p.m. Ob riboprobe, together with 4 x 104 c.p.m. ß-actin riboprobe. Protected fragments were electrophoresed on a 6% polyacrylamide/urea gel. Gels were dried and fixed in 10% acetic acid. Radioactivity was counted overnight with an InstantImager (Packard, Meriden, CT, USA) followed by an autoradiography. Results are expressed in arbitrary units and correspond to the ratio between PRLR- or Ob-specific counts vs ß-actin signal. Unprotected PRLR riboprobe migrated at 450 bases with a protected fragment migrating at 390 bases whereas 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.
Northern blot analysis
Fifteen micrograms of total RNA were analyzed by Northern blotting as previously described (Maniatis et al. 1982). -32P-labeled probes were synthesized by random priming (Megaprime; Amersham Biotech) of cDNA encoding UCP1 and GAPDH. Membranes were subjected to autoradiography and specific hybridization signals were quantified using an InstantImager. Results are expressed in arbitrary units corresponding to the ratio of specific counts for UCP1 vs GAPDH signal.
Immunoprecipitation and Western blot analysis
COS cells were transiently transfected with a mouse PRLR cDNA (kindly provided by D Linzer, Northwestern University, Evanston, IL, USA) using lipofectamine plus reagent (Life Technologies, Groningen, The Netherlands). Cells were subsequently lysed at 4 °C in lysis buffer (see below). Lysates were clarified by centrifugation at 15 000 g for 30 min and the supernatants were transferred to a fresh tube. After protein content determination (Bradford Method; Bio-Rad), equal amounts of proteins were immunoprecipitated at 4 °C with the anti-PRLR1 antibody (kindly provided by F Talamantes, University of California, Santa Cruz, CA, USA), and the immune complexes were collected on Protein A Sepharose. After washing, immunoprecipitates were reduced for 5 min at 95 °C in Laemmli buffer and analyzed by SDS-PAGE.
Studies of the activation of JAK/STAT, MAP kinase and insulin signaling pathways
Differentiated cells starved for 18 h in minimum medium, were stimulated with 100 ng/ml PRL, 20 nM insulin or with both during 0–60 min; cells were washed twice with ice-cold PBS and lysed in ice with 10 mM Tris–HCl pH 7.6, 5 mM EDTA, 150 mM NaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 10% glycerol and 2% Nonidet P40 (NP40) supplemented, just before use, with 1 µM pepstatin A, 50 µM leupeptin, 1 µM aprotinin and 1 mM phenylmethylsulfonylfluoride (PMSF). Lysates were sonicated and cleared from insoluble material by centrifugation. Protein concentrations were determined by the modified Bradford method and 500 µg total protein were immunoprecipitated overnight at 4 °C with Protein A Sepharose (Amersham) and the respective antibodies: rabbit polyclonal anti-JAK2 (06–255) from Upstate Biotechnology (Lake Placid, NY, USA), rabbit polyclonal anti-STAT5 (C17), rabbit polyclonal anti-STAT3 (C-20) from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA), anti-insulin receptor substrate proteins (anti-IRS)-1 (Upstate Biotechnology), anti-IRS-2 (Upstate Biotechnology), or anti-phosphatidylinositol 3-kinase (PI 3-kinase) (p85 antibodies; Upstate Biotechnology). The immunoprecipitate was reduced in Laemmli buffer and an 8% acrylamide SDS-PAGE was performed. After electrophoretic transfer of proteins to nitrocellulose membranes, they were blocked (TBS/0.1% Tween 20/5% BSA) then incubated with monoclonal anti-phosphotyrosine (clone 4 G10) from Upstate Biotechnology diluted 1/5000 in TBS/0.1% Tween 20/1% BSA, washed with TBS/0.1% Tween 20, then incubated with the secondary antibody, anti-mouse IgG peroxidase-linked (Amersham), washed, developed with ECL Western blotting detection reagent (Amersham) and exposed to film (X-ray film AX; Konica, Roissy, France). Blots were stripped by 30 min incubation at 50 °C in 62.5 mM Tris pH 6.8, 2% SDS, 100 mM 2-mercaptoethanol, washed with TBS/0.1% Tween 20 and incubated with anti-JAK2, anti-STAT5 or anti-STAT3 respectively and anti-rabbit IgG peroxidase-linked (Amersham) as secondary antibody. Direct Western blots were performed on 40 µg total protein extracts revealed with polyclonal anti-MAP kinase (ERK1/2) and monoclonal anti-MAP kinase phosphotyrosines (ERK1/2-pY) clone 12D4 (Upstate Biotechnology). IRS-1 (anti-IRS-1), IRS-2 (anti-IRS-2), PI 3-kinase (anti-PI 3-kinase p85 antibodies) were from Upstate Biotechnology. Quantifications were performed using an EDAS-120 system (Kodak digital imaging system) and software (Grosdemouge et al. 2003). The intensity of phosphorylated proteins was normalized to that of the unphosphorylated form of the same protein. Results are expressed in arbitrary units.
Leptin RIA
Extraction of total proteins from T37i cells was performed at 4 °C in RIPA buffer (50 mM Tris, 1% Triton X-100, 0.5% deoxycholic acid, 0.1% SDS, 50 mM NaF, 150 mM NaCl pH 7.4) containing 0.1 mg/ml PMSF, 100 µM benzamidine and 100 mM sodium orthovanadate as protease inhibitors. The lysate was centrifuged at 15 000 g for 20 min. The amount of protein was quantified using the Bradford method. Leptin content in aliquots of culture medium or in cell lysate was quantified using the mouse leptin RIA kit from Linco Research, Inc. (St Charles, MO, USA) with a detection limit of 0.2 ng/ml. BAT from 2-week-old wild-type and PRLR knockout mice was removed and weighed. Tissues were homogenized using a glass–Teflon homogenizer in PBS buffer containing 0.1 mg/ml PMSF and 200 IU aprotinin. The homogenates were centrifuged at 10 000 g for 10 min and the supernatants were collected and used for leptin determination by RIA.
Statistical analysis
Data are expressed as means±S.E.M. and ±S.D. One-way ANOVA, followed by the Newman–Keuls multiple range test or t-test was used to determine significant differences among groups, they were performed using the computer software InStat version 2.01 for Macintosh (GraphPad Software, San Diego, CA, USA). Statistical significance are indicated at P values <0.05, <0.01 and <0.001.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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We first examined the presence of PRLR mRNA in the T37i cells as a function of differentiation state (Fig. 1A and B
). RPA analyses indicated that PRLR expression progressively increased during brown adipocyte differentiation and reached a maximal level at day 6 then decreased afterwards. Importantly, PRLR transcripts were not detected by RT-PCR in undifferentiated T37i cells (data not shown), suggesting that PRLR expression occurs only during the process of adipocyte differentiation. We next examined the stability of PRLR transcripts by RPA (Fig. 1C) and showed that PRLR transcript levels rapidly declined after actinomycin D incubation with a calculated half-life of approximately 5.5 h (Fig. 1D), indicating that PRLR expression is presumably under a strict transcriptional control. To further investigate PRLR expression, differentiated T37i cells were treated with insulin or T3, two hormones important in brown adipocyte differentiation (Fig. 1E). Quantitative results indicated that the level of PRLR transcripts was increased 2- to 3-fold by insulin or T3 (Fig. 1F), suggesting that both hormones could stimulate PRLR gene transcription.
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). As shown in Fig. 2B, differentiated T37i cells expressed PRLR in contrast to undifferentiated cells, confirming the results obtained at the mRNA level.
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Because the phosphorylation-dependent activation of JAK/STAT is a major transduction pathway for PRL signaling, we investigated the JAK2 and STAT phosphorylation in cellular extracts of differentiated cells in response to PRL. As shown in Fig. 3, the phosphorylated form of JAK2 was maximal after 5 min of PRL stimulation and decreased thereafter. PRL treatment also induced strong tyrosine phosphorylation of STAT5, which declined after 15 min. Interestingly, PRL also led to the phosphorylation of STAT3, indicating that in this cell model, PRL is able to transduce hormonal signal through activation of both the STAT5 and the STAT3 pathways.
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). A clear increase in phosphorylated ERK1/2 kinases was detected in PRL-stimulated cells with a maximal activation level after 5 min, which decreased afterwards. Taken together, these results demonstrate that PRL is able to activate both JAK/STAT and MAP kinase pathways, indicating that PRLRs are functional in brown adipocytes but it questions on the target genes of PRL in BAT. Effects of PRL on brown adipocyte gene expression
Because brown adipocytes are mainly involved in thermoregulation via modulation of UCP1 expression, we investigated the effect of PRL on the expression of UCP1 transcripts in differentiated T37i cells. Figure 4 shows that PRL alone did not modulate UCP1 gene expression nor alter the isoproterenol-stimulated UCP1 transcript levels in differentiated T37i cells. Similar results were obtained after retinoic acid stimulation (data not shown). These results indicate that UCP1 transcription is presumably not directly regulated by PRL.
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), consistent with a synergistic mechanism. We also examined by RIA the leptin concentration in the supernatant of differentiated T37i cells in response to various hormonal treatments (Fig. 5B). Overnight incubation with insulin strongly increased leptin secretion from a basal level of 0.22±0.03 to 1.38±0.18 ng/ml. PRL alone had no effect on basal leptin secretion (0.30±0.14 ng/ml). However, when combined with insulin, the leptin concentration in the medium reached 1.98±0.15 ng/ml, a value significantly higher than that achieved by insulin exposure, indicating that PRL potentiates the stimulatory effect of insulin on leptin release. It is worth noting that pretreatment of differentiated T37i cells with PRL before insulin incubation did not result in a cooperative increase in leptin content (data not shown), suggesting that potentiation only occurs when brown adipocytes are simultaneously exposed to insulin and PRL.
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Insulin stimulates tyrosine phosphorylation of proteins that are composed of IRS-1, -2 and -3. To determine the molecular events underlying this potentiating mechanism, we studied the activation of IRS in differentiated T37i cells in response to PRL, insulin or both (Fig. 6). Equal amounts of cell lysate proteins were immunoprecipitated with either the anti-IRS-1 or the anti-IRS-2 antibodies, and analyzed by Western blotting with the anti-phospho Tyr (pY) antibody. Figure 6A and B show the tyrosine phosphorylation of the IRS proteins induced by insulin, whereas PRL alone fails to activate phosphorylation of IRS-1 or -2. Interestingly, the combination of PRL and insulin resulted in an enhanced activation of IRS-1 and -2. Beside the increase in tyrosine-phosphorylated IRS-1 and -2, an important molecular mass shift of IRS proteins was observed after insulin and PRL treatment as compared with insulin alone. This finding could be due to an increase in IRS phosphorylation status on tyrosine and possibly on serine/threonine residues. Altogether, these results suggest that both IRS-1 and -2 are involved in the insulin signaling in T37i cells and that PRL potentiates insulin action. In addition, the membranes were stripped and incubated with a PI 3-kinase antibody confirming that tyrosine phosphorylated IRS-1 and -2 are associated with the p85 subunit of PI 3-kinase and that the combination of both hormones enhanced the binding of p85 to both IRS-1 and -2, greater than did insulin alone (data not shown). To further demonstrate the balance in the association between IRS-1/2 and p85 subunit of PI 3-kinase in these cells after stimulation by the hormones, lysates were immunoprecipitated with the anti-p85 antibody, then immune complexes were subjected to Western blot analysis. As shown in Fig. 6C, we observed a substantial increase in the amount of both IRS-1 and -2 in the anti-p85 immunoprecipitates upon combined treatment with insulin and PRL. Moreover, not only IRS-1 but also IRS-3 was shown to play a major role in insulin-induced activation of PI 3-kinase in adipocytes. Indeed, we observed that the combined treatment of both insulin and PRL was also able to induce tyrosine phosphorylation of IRS-3 and its association with PI 3-kinase (Fig. 6C).
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As a first attempt to understand the physiological relevance of PRL actions in BAT, we investigated UCP1 expression in the BAT of 2-week-old PRLR knockout animals at both messenger and protein level. No differences were observed among the PRLR genotypes (+/+, +/–, –/–) suggesting that the null mutation of PRLR did not directly affect the expression of UCP1 transcripts and protein in vivo (data not shown).
Since PRL was shown to potentiate the stimulatory effect of insulin on leptin expression and release from differentiated T37i cells (see Fig. 5), we analyzed Ob gene expression in the BAT of wild-type, PRLR+/– and PRLR-null mice and showed that the level of Ob transcripts was not significantly different in any of the three genotypes, suggesting that Ob gene expression was not altered by the null mutation of PRLRs (Fig. 7A). However, leptin content measured by RIA was 2.7-fold higher in the BAT of PRLR–/–pups than in wild-type animals (Fig. 7B). Accordingly, we observed a heterozygous phenotype since leptin content was intermediate in the BAT of PRLR+/– mice, this level of leptin content being significantly 2-fold higher than that measured in BAT of control animals. Altogether, our findings provide additional support for the involvement of PRL signaling pathway in the biosynthesis, the secretion and/or the release of leptin by the BAT in vivo.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Here, we provide the first evidence that PRLRs are present at both mRNA and protein levels and functional in differentiated T37i cells and in BAT of newborn mice. Interestingly, we demonstrated that PRLR transcripts exhibit a short half-life and are up-regulated by insulin and T3, consistent with their appearance during the brown adipocyte differentiation program. This is in accord with what has already been shown in the white adipocyte 3T3-L1 cell line during preadipocyte differentiation (McAveney et al. 1996) and with the presence of PRLRs in mouse white adipose tissue (Ling et al. 2000).
We further demonstrated that PRL activates STAT5 in brown adipocytes, a result consistent with previous results, which reported the involvement of PRLRs in the white adipocyte differentiation program via STAT5 (Nanbu-Wakao et al. 2000, 2002). In addition to activation of the JAK2/STAT5 pathway, we showed that STAT3 is also a downstream effector of PRL in brown adipocytes, as already described in T47D cells (Llovera et al. 2000) and in mouse mammary epithelial cells (DaSilva et al. 1996, Chapman et al. 1999). These results suggest that PRL-mediated STAT3 activation may modulate the transcription of a subset of tissue-specific genes that remains to be determined.
To investigate the biological action of PRL in brown adipocytes, we studied the signal transduction pathways leading to expression of UCP1 and leptin. We first showed that PRL alone was not able to induce UCP1 expression in T37i cells nor was it able to modulate catecholamine- and retinoic acid-stimulated UCP1 expression. In accord with these findings, we were unable to detect any significant change in UCP1 content in the BAT of PRLR knockout mice, indicating that UCP1 is not a direct target of PRL but rather should be regulated mostly by a sympathetic pathway. However, we cannot exclude that in vivo PRL might modulate UCP1 expression, which also depends on the hormonal and metabolic environment in a developmental-stage manner (Pearce et al. 2003, Xiao et al. 2004).
We have previously shown that insulin is an important stimulator of leptin synthesis in brown adipocytes and exerts its effect through a strong, rapid and transient activation of transcription (Buyse et al. 2001). We have now demonstrated that Ob gene expression as well as leptin release from differentiated T37i cells is greatly potentiated by the addition of PRL only in the presence of insulin, indicating the necessity for a concomitant activation of both hormone signaling pathways. We next addressed the question of the molecular mechanisms by which PRL potentiates insulin effects. On the one hand, PRL alone failed to induce tyrosine phosphorylation of IRS in the brown adipocyte T37i cell model, a result which contrasts with those obtained in PRLR-overexpressing cells in which IRS-1, -2 and -3 were tyrosine phosphorylated by PRL (Berlanga et al. 1997, Yamauchi et al. 1998). This indicated that in vitro PRL responsiveness largely depends on the level of PRLR expression and/or its downstream effectors. On the other hand, we clearly demonstrated that the IRS/PI 3-kinase pathway is overstimulated after PRL incubation. Indeed, we showed that PRL potentiates insulin-stimulated phosphorylation of IRS-1, -2 and -3 and their association with p85 PI 3-kinase. These results confirm the essential role played by IRS-1 through PI 3-kinase signaling pathway in inducing adipogenic and thermogenic gene expression upon insulin or insulin-like growth factor stimulation, already demonstrated in primary brown adipocytes (Valverde et al. 1997, 1999, Teruel et al. 1998, Mur et al. 2003). The role of IRS-1, -2 and -3 in PRL signaling is to be phosphorylated by JAK2, thereby providing docking sites for p85 PI 3-kinase and activating PI 3-kinase (Yamauchi et al. 1998). It has also been shown that the expression of dominant negative JAK2 almost completely abolished PRL-induced phosphorylation of IRS-1 and -2, and their association with PI 3-kinase (Yamauchi et al. 1998). Finally, we suggest that PRL acts as an insulin sensitizer in brown fat, consistent with previous work performed in white adipocytes which showed that PRL, in combination with insulin, induces CIS and SOCS 3 mRNA expression, two known PRL targets (Ling & Billig 2001).
This work, which demonstrates that BAT is a novel PRL target tissue, constitutes the initial step of a detailed in vivo study of the PRL signaling pathway. PRL is best known as a lactogenic hormone responsible for the development of the mammary gland, but it also plays a crucial role in reproduction, although many other functions, most notably affecting adipose tissues, are reported (Bole-Feysot et al. 1998, Flint et al. 2003). Thus, the availability of PRLR knockout model allowed us to analyze brown adipocyte gene expression. In accord with the potentiating effect of PRL on insulin-stimulated leptin secretion observed in the T37i cell model, we demonstrated that the absence of PRL signaling curtailed leptin release from brown adipocytes as evidenced by the significant increase in leptin content in BAT of null animals. To the best of our knowledge, this is the first evidence that PRL can affect leptin production from brown fat. There are, however, several conflicting reports about the effect of PRL on leptin release from white adipose tissue. Although it has been recently proposed that PRL suppressed insulin-induced leptin secretion from cultured white adipocytes (Ling & Billig 2001), most studies indicated that in vivo PRL stimulates leptin release notably in transgenic female mice overexpressing PRL (Ling & Billig 2001) but also in rats treated with exogenous PRL (Gualillo et al. 1999) or with lipopolysaccharide, leading to an endogenous PRL secretion (Mastronardi et al. 2001). Furthermore, old PRLR-deficient female mice exhibited a progressive decrease in white adipose tissue mass (Freemark et al. 2001), concomitant with a reduction of circulating leptin levels. The significance of this PRL effect on leptin secretion remains to be elucidated but it is clear from our results that, since no change in Ob mRNA steady state levels was observed in mouse models, PRL is not directly involved in transcription rate, but more likely in the secretion or release process of leptin in vivo. Altogether, these data point toward a stimulatory action of PRL on leptin secretion in BAT. Of interest, it has been proposed that white adipose tissue is not functional at birth, suggesting that brown adipocyte-secreted leptin may represent the predominant if not exclusive source of leptin during this critical perinatal period (Dessolin et al. 1997). Therefore, we propose that leptin secreted by brown adipocytes may have an important physiological role in terms of food intake control, thermogenesis and metabolic characteristics (Yuen et al. 2003), presumably through endocrine and/or autocrine mechanisms that remain to be established.
In summary, we have shown that brown adipocytes express functional PRLRs. We have provided evidence for a cross-talk between PRL and the insulin signaling pathway in brown adipocytes and further demonstrated that PRL potentiates insulin-stimulated secretion and/or release of leptin by BAT. Our findings provide the basis to further investigate the role of PRL and its receptor in brown adipocyte differentiation and function.
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Acknowledgements |
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Received 9 June 2004
Accepted 27 August 2004
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