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Endocrinology Vol. 142, No. 11 4880-4890
Copyright © 2001 by The Endocrine Society

ARTICLES

PRL Receptor-Mediated Effects in Female Mouse Adipocytes: PRL Induces Suppressors of Cytokine Signaling Expression and Suppresses Insulin-Induced Leptin Production in Adipocytes in Vitro

Charlotte Ling and Håkan Billig

Department of Physiology, Goteborg University, SE 405 30 Göteborg, Sweden

Address all correspondence and requests for reprints to: Dr Håkan Billig, Department of Physiology, Goteborg University, P.O. Box 434, SE 405 30 Goteborg, Sweden. E-mail: hakan.billig{at}fysiologi.gu.se


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PRL has been reported to regulate fat metabolism in several species. We recently reported PRL receptor (PRLR) expression in mouse adipocytes and increased levels of PRLR expression in the adipose tissue of lactating and PRL-transgenic mice compared with controls. These results suggest PRLR-mediated effects in adipose tissue. However, to date most studies have been performed in vivo, and it is unclear whether PRL has direct effects on adipocytes. The PRLR belongs to the cytokine receptor family, and a family of suppressors of cytokine signaling (SOCS) was recently identified. The present study was performed to investigate whether PRL has direct effects on adipocytes. The expression of cytokine-inducible SH2-domain-containing protein (CIS), SOCS-3, and SOCS-2 mRNA and protein was analyzed using ribonuclease protection assay and immunoblotting, respectively. Ovine PRL induced CIS mRNA expression and a combination of oPRL and insulin induced SOCS-3 mRNA expression in adipocytes cultured in vitro for 0–240 min, demonstrating PRLR-mediated direct effects in these cells. Furthermore, CIS, SOCS-3, and SOCS-2 mRNA and protein were all transiently expressed in adipose tissue obtained from female mice stimulated with oPRL (1 µg/g BW) for 0–24 h. In adipose tissue of female mice with endogenously high PRL levels, PRL-transgenic mice, only SOCS-2 expression was increased. The level of SOCS-2 mRNA was also increased in adipose tissue during pregnancy and lactation compared with that in wild-type virgin female mice. A possible reason for increased SOCS-2 expression after prolonged PRL exposure during lactation and in the PRL transgenes could be to restore the sensitivity of adipose tissue to PRL. In addition, the direct effect of PRL on leptin production was investigated in adipocytes cultured in vitro for 6 h. PRL inhibited insulin-induced leptin production in vitro. However, PRL had no effect on leptin production in the absence of insulin. In contrast, serum leptin concentrations were increased in PRL-transgenic females compared with control mice.

In conclusion, our results demonstrate functional PRLRs in mouse adipocytes and suggest a role for CIS, SOCS-3, and SOCS-2 in regulating PRL signal transduction in adipose tissue.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PRL HAS BEEN reported to exert metabolic actions in different species. For example, hyperprolactinemia in both human patients and rodents leads to increased insulin resistance and decreased glucose tolerance (1, 2, 3, 4, 5, 6, 7). We recently reported PRL receptor (PRLR) expression in mouse white adipose tissue and primary isolated adipocytes (8). Furthermore, in vitro, PRL decreases insulin binding to adipocytes from pregnant women and reduces glucose uptake in adipocytes from female rats (9, 10). During lactation, PRL has been suggested to affect the lipoprotein lipase activity in adipose tissue and mammary gland, resulting in transfer of fatty acids from body fat stores to the mammary gland (11, 12, 13, 14, 15, 16). In addition, we demonstrated that PRLR mRNA expression increased in adipose tissue during lactation compared virgin and pregnant mice and in PRL-transgenic mice compared with controls (8). Furthermore, male mice with chronic hyperprolactinemia have decreased weight of epididymal fat bodies and lower serum FFA levels (17). However, PRLR-deficient mice were recently reported to have reduced abdominal fat content and decreased leptin production compared with control mice (18). These studies indicate that PRL might have both lipolytic and lipogenic effects.

PRL mediates its effects by interacting with the PRLR, which homodimerizes upon ligand binding and activates several intracellular signaling pathways (19). The PRLR belongs to the class 1 cytokine receptor superfamily. In the mouse, one long and three short splice forms of the PRLR have been identified (L-, S1-, S2-, and S3-PRLR) (20). All cytokine receptors lack enzymatic activity, including kinase activity, and to transmit signals within a cell, cytokine receptors work together with one or several Janus kinases (JAKs) (19). In the mouse, only the L-PRLR activates the JAK2/signal transducer and activator of transcription 5 (STAT5) pathway and dimerization of L-PRLR leads to transphosphorylation and activation of JAK2, followed by phosphorylation of the PRLR as well as a variety of cellular substrates, such as STAT5 (19). Phosphorylated STAT5 dimerizes, and the dimers translocate to the nucleus, where they affect the transcription of genes with STAT5 recognition sites in their promoter (19). A new family of genes involved in suppressing cytokine signal transduction was recently identified, suppressors of cytokine signaling (SOCS) (21, 22, 23, 24). At present, the gene family consists of eight proteins, SOCS-1 to SOCS-7 and cytokine-inducible SH2-domain-containing protein (CIS) (25). They all contain a central SH-2 domain, and the inhibitory activity results from their different abilities to interact with cytokine receptors, JAK family members, or STATs (25). A wide variety of cytokines and hormones affecting cytokine receptors, including PRL, induce the expression of SOCS-1, SOCS-2, SOCS-3, and CIS both in vitro and in vivo (21, 22, 25, 26, 27, 28, 29, 30, 31). In addition, overexpression of several of these SOCS proteins in cell lines resulted in inhibition of cytokine receptor signaling (25, 29, 30, 31, 32). To date, there is only limited information on the hormonal regulation of SOCS genes in adipose tissue, and it would be relevant to further investigate SOCS expression in adipose tissue during different physiological events.

Based on our recent findings of PRLR expression and regulation in mouse adipose tissue (8), the aim of this study was to investigate whether PRL has any direct effect on mouse adipocytes. We therefore studied changes in CIS, SOCS-3, and SOCS-2 expression in PRL-stimulated adipocytes in vitro, in adipose tissue obtained from female mice stimulated with PRL in vivo, and in adipose tissue of wild-type pregnant, wild-type lactating, and female PRL-transgenic mice. Furthermore, the direct action of PRL on an adipocyte-related parameter, leptin production, was studied, and PRL’s effect on insulin-induced leptin production was analyzed in medium from adipocytes cultured in vitro.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and tissue isolation
Wild-type virgin female, pregnant, and lactating C57BL/6J-CBA-F1 mice (MochB, Ry, Denmark) and female PRL-transgenic mice and control littermates (33) were used in this study. All mice were kept under controlled environmental conditions with free access to water and pelleted food. The animal experiments were approved by the local ethics committee. Parametrial adipose tissues from wild-type virgin female mice, pregnant mice at 16 d of pregnancy, lactating mice at 7 d of lactation, female PRL-transgenic mice, and control littermates were isolated. An additional group of pregnant mice was treated with a PR antagonist, RU 486 (Excelgyn, Paris, France; 150 µg/animal in sesame oil; 1.5 g/liter, sc), or vehicle for 15 h, and mammary glands and parametrial adipose tissues were isolated. All tissues were fresh-frozen in liquid nitrogen and stored at -70 C until the time of RNA and protein preparations.

Adipocyte culture and in vitro hormonal treatment
Virgin female C57BL/6J-CBA-F1 mice were decapitated, and parametrial adipose tissues were removed aseptically. Adipocytes for primary culture were then isolated by collagenase treatment (Sigma, St. Louis, MO) (34). Adipocytes were filtered through a nylon mesh (250-µm pore size) to remove undigested tissue fragments and stroma. The adipocytes, which float to the surface of the medium, were washed three times. DMEM without phenol red (no. 11880-028, Life Technologies, Inc., Gaithersburg, MD) supplemented with 1% albumin (10 g/liter; Immuno AG, Wien, Austria), fungizone (0.5 µg/ml; no. 15290-026, Life Technologies, Inc.), gentamicin (50 µg/ml; no. 15710-031, Life Technologies, Inc.), HEPES (25 mM; 1M, Life Technologies, Inc.), and 1 x L-glutamine (x100, Life Technologies, Inc.), pH 7.4, was used as a culture medium. Ovine PRL (oPRL; a gift from the National Hormone and Pituitary/NIDDK Program, Baltimore, MD) and insulin (Actrapid, Novo Nordisk, Bagsvaerd, Denmark) were used for hormonal treatments. Four different hormonal treatment groups were studied: group A, no hormone; group B, oPRL (300 ng/ml), group C, insulin (20 nM), and group D, oPRL (300 ng/ml) and insulin (20 nM).

SOCS expression experiment. Adipocytes isolated from 700 mg adipose tissue were cultured in 5 ml medium in sterile Cellstar PP-tubes (3.2-cm diameter; Greiner Labortechnik, Frickenhausen, Germany). After preincubating the adipocytes for 2 h, the medium was changed. A control sample was isolated at time zero, and the four different treatment groups, A–D, were cultured for 30, 60, 120, and 240 min before isolating and freezing the adipocytes in liquid nitrogen.

Leptin secretion experiment. Adipocytes isolated from 250 mg adipose tissue were cultured in 2 ml medium in sterile Falcon PP-tubes (1.6-cm diameter; Becton Dickinson and Co., Meylan, France). Cells from the four treatment groups, A–D, were cultured for 6 h before isolating and freezing the medium before leptin analyses. Leptin production in the respective sample was calculated as leptin concentration in medium/total DNA content, and the average leptin production in the control (no hormone) was set at 100%. To isolate total DNA, adipocytes were incubated overnight with proteinase K (final concentration, 0.1 mg/ml; Merck & Co., Inc., Darmstadt, Germany) at 45 C in PBS buffer. The DNA content was measured with a fluorescence spectrophotometer (356 nm excitation and 458 nm emission; F-2000, Hitatchi, KEBO, Goteborg, Sweden) after addition of Hoechst’s dye H33258 (0.2 µg/ml in 2 M NaCl, 1 mM EDTA, and 10 mM Tris, pH 7.4) (35).

In vivo hormonal treatment
Parametrial adipose tissues were obtained immediately after death from virgin female mice that had been treated with oPRL (1 µg/g BW, ip) for different times.

RNA extraction
Total RNA was extracted from frozen tissues using Tri-Reagent according to the manufacturer’s instructions (Sigma).

RNA probes
Mouse CIS, SOCS-3, SOCS-2, L-PRLR, and cyclophilin antisense RNA probes were used in the ribonuclease protection assay (RPA). The CIS, SOCS-3, and SOCS-2 DNA templates were constructed from pEF-FLAG-I vectors containing cDNAs for the entire open reading frame (provided by D. Hilton’s laboratory) (22). A 247-bp mouse CIS cDNA fragment (nucleotides 528–774, pEF-FLAG-I/mCIS plasmid digested using BamHI/XbaI) was subcloned into a pBluescript vector. The construct was verified by DNA sequencing and then linearized using XhoI. A 208-bp mouse SOCS-3 cDNA fragment (nucleotides 471–678, pEF-FLAG-I/mSOCS-3 plasmid digested using SmaI/XbaI) was subcloned into a pBluescript vector. The construct was verified by DNA sequencing and then linearized using XhoI. A 313-bp mouse SOCS-2 cDNA fragment (nucleotides 219–531) generated by RT-PCR using SOCS-2-specific primers (upstream primer, 5'-AGATAGTTCGCATTCAGACT-3'; downstream primer, 5'-CGTACCGGTACATTTGTTA-3') was subcloned into the pCRII-TOPO vector (Invitrogen, Carlsbad, CA). The construct was verified by DNA sequencing and then linearized using XbaI. A 288-bp mouse L-PRLR cDNA fragment (nucleotides 1507–1794) subcloned into a pBluescript vector was used as a template (8). A mouse cyclophilin template (no. 7675, Ambion, Inc., Austin, TX), generating a 103-bp protected fragment (nucleotides 38–140), was used as an internal standard to control the amount of RNA in each sample.

Antisense [32P]CTP-labeled CIS, SOCS-3, L-PRLR, and cyclophilin RNA probes were synthesized using T3 RNA polymerase, and an antisense [32P]CTP-labeled SOCS-2 RNA probe was synthesized using SP6 RNA polymerase according to the manufacturer’s instructions (Ambion, Inc.). Compared with the SOCS probes and the L-PRLR probe, 6.25 times less [32P]CTP was used when synthesizing the cyclophilin probe, making the cyclophilin probe less radiolabeled. Before use, all [32P]CTP-labeled RNA probes were gel-purified, run on a denaturing 8 mmol/liter urea/6% polyacrylamide gel (Novex, San Diego, CA), identified, excised, and eluted, and specific activity was determined in a scintillation counter.

RPA
The RPA was performed using the RPA III ribonuclease protection assay kit (Ambion, Inc.). Before running the samples, the signals from all probes were confirmed to increase linearly with increasing amounts of total RNA from adipose tissue (5–30 µg). When analyzing SOCS or L-PRLR mRNA expression, each sample was hybridized overnight at 42 C with antisense [32P]CTP-labeled SOCS RNA probe or L-PRLR RNA probe (180,000 cpm/sample), respectively, and antisense [32P]CTPlabeled cyclophilin RNA probe (80,000 cpm/sample). The protected fragments were separated on a denaturing 8 mmol/liter urea/6% polyacrylamide gel (Novex) for 1.5 h at 130 V. After drying, the gel was exposed to a Phosphor Imager screen for 2 d and developed in a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA), and quantitative analysis was performed using ImageQuant software (Molecular Dynamics, Inc.). CIS/cyclophilin, SOCS-3/cyclophilin, SOCS-2/cyclophilin, and L-PRLR/cyclophilin ratios were calculated for the respective samples. In the in vitro hormonal treatment experiments, all SOCS/cyclophilin ratios at time zero, the control values, were set at 100%. In the in vivo oPRL treatment experiment, the average ratio for the SOCS/cyclophilin ratios at time zero, the control value, was set at 100%. Furthermore, the SOCS/cyclophilin ratios at the different times, presented as relative SOCS expression (percentage), were related to the control values in the respective experiment. When SOCS expression was measured in the female PRL-transgenic mice, the average ratio for the control mice was set at 100% for CIS, SOCS-3, and SOCS-2, respectively. In addition, when SOCS expression was measured in female virgin, pregnant, and lactating mice, the average ratio for the female virgin mice was set at 100% for CIS, SOCS-3, and SOCS-2, respectively. L-PRLR expression was measured in RU486- and vehicle-treated pregnant mice, and the L-PRLR/cyclophilin ratio was calculated for each sample. The average ratio for the vehicle-treated pregnant mice was set at 100%.

Protein extraction and immunoblotting
Protein extraction from adipose tissue was performed as previously reported (8). Thirty-five micrograms of total protein were loaded in each lane on SDS-polyacrylamide gels (10% NuPAGE Bis-Tris gels, Novex). A prestained standard (SeeBlue, Novex) was used as a weight marker. The proteins were then transferred to polyvinylidene difluoride membranes (Amersham Pharmacia Biotech, Little Chalfont, UK). Thereafter, the membranes were incubated with polyclonal antibodies against CIS (sc-1529), SOCS-3 (sc-7010), or SOCS-2 (sc-9022; dilution, 1:500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Immunoreactive proteins were visualized by chemiluminescence using secondary antigoat antibodies (CIS and SOCS-3; A-8062, dilution, 1:30,000; Sigma) or antirabbit antibodies (SOCS-2; AC31RL, dilution, 1:40,000; Tropix, Bedford, MA) linked to alkaline phosphatase using CDP-Star as substrate (Tropix). The membranes were exposed to ECL films (Amersham Pharmacia Biotech) and subsequently developed.

Leptin analysis
Leptin levels in culture medium and mouse serum were measured by a mouse leptin ELISA assay (Crystal Chemicals, Inc., Chicago, IL) according to the manufacturer’s instructions.

Statistical analysis
Differences in CIS and SOCS-3 mRNA expression (comparing SOCS/cyclophilin ratios) in the in vitro hormonal treatment experiments were analyzed using two-way ANOVA, followed by Student-Newman-Keuls multiple range test. Differences in CIS, SOCS-3, and SOCS-2 mRNA expression (comparing SOCS/cyclophilin ratios) in the in vivo hormonal treatment experiment, the PRL-transgenic mice, and the virgin, pregnant, and lactating mice were analyzed using one-way ANOVA, followed by Student-Newman-Keuls multiple range test. In addition, differences in leptin levels and differences in L-PRLR mRNA expression (comparing L-PRLR/cyclophilin ratios) were analyzed using one-way ANOVA, followed by Student-Newman-Keuls multiple range test. When appropriate, values were transformed to logarithms.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of oPRL and insulin on CIS, SOCS-3, and SOCS-2 mRNA expression in adipocytes cultured in vitro
To investigate whether PRL acts directly on the adipose tissue, isolated adipocytes were cultured in vitro in the presence or absence of oPRL, and mRNA expression of three SOCS family members, CIS, SOCS-3, and SOCS-2 was measured by RPA. A control sample was isolated at time zero, and four different treatment groups, A–D [A, no hormone (baseline); B, 300 ng/ml oPRL; C, 20 nM insulin; and D, 300 ng/ml oPRL and 20 nM insulin], were studied for 30–240 min (Fig. 1Go).



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  		Figure 1. Time-course induction of SOCS mRNAs by oPRL and insulin in mouse adipocytes cultured in vitro. Primary isolated female mouse adipocytes were cultured as described in Materials and Methods. Four different hormonal treatments were studied: baseline ({blacksquare}; no hormone added), PRL (•; 300 ng/ml oPRL), INS ({blacktriangleup}; 20 nM insulin), and PRL plus INS ({blacktriangledown}; 300 ng/ml oPRL and 20 nM insulin), for 0–240 min. Adipocytes were harvested at different time points. RNA was prepared and analyzed for CIS (A), SOCS-3 (B), and SOCS-2 (data not shown) mRNA together with the internal standard cyclophilin using RPA (8 µg RNA/sample). SOCS/cyclophilin ratios were calculated for each sample. All SOCS/cyclophilin ratios at time zero, the control values, were set at 100%. The SOCS/cyclophilin ratios at the different times, presented as relative SOCS expression (percentage), were related to the control values in the respective experiments (n = 4). Results are expressed as the mean ± SEM, and treatment groups with different superscripts are significantly different from each other (P < 0.05), using two-way ANOVA followed by Student-Newman-Keuls multiple range test.

 
Cis mRNA. Stimulation with oPRL increased the level of CIS mRNA expression significantly in the cultured adipocytes compared with baseline levels (Fig. 1AGo). Insulin also increased CIS mRNA expression compared with baseline levels. Furthermore, an additional increase in CIS mRNA expression was detected when oPRL and insulin were present together compared to when oPRL or insulin was used alone (Fig. 1AGo).

SOCS-3 mRNA. oPRL alone did not induce SOCS-3 expression significantly compared with baseline. However, insulin alone increased the level of SOCS-3 mRNA expression significantly compared with baseline. In addition, a combination of oPRL and insulin further increased the expression of SOCS-3 mRNA, with a peak at 120 min (Fig. 1BGo).

SOCS-2 mRNA. The level of adipocyte SOCS-2 mRNA expression was very low, near the limit of detection, in all treatment groups and at all times studied in the in vitro experiments (data not shown).

Effects of oPRL on CIS, SOCS-3, and SOCS-2 mRNA expression and protein levels in parametrial adipose tissue in vivo
To study whether PRL could also affect the adipose tissue in vivo, the levels of CIS, SOCS-3, and SOCS-2 mRNA expression were analyzed by RPA in parametrial adipose tissues obtained from female mice stimulated with oPRL for different times (Fig. 2Go). In addition, protein levels of CIS, SOCS-3, and SOCS-2 were analyzed by immunoblotting of the parametrial adipose tissue from oPRL-stimulated female mice (Fig. 3Go).



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  		Figure 2. Time-course induction of SOCS mRNAs by oPRL in parametrial adipose tissue in vivo. Parametrial adipose tissues were obtained from female mice that had been treated with oPRL (1 µg/g) for different times (eight mice per time). RNA was prepared and analyzed for CIS (A), SOCS-3 (B), and SOCS-2 (C) mRNA together with the internal standard cyclophilin using RPA (15 µg RNA/sample). The SOCS/cyclophilin ratio was calculated for each sample, and the values were the relative SOCS expression (the average value at time zero was set at 100%). Results are expressed as the mean ± SEM, and statistical significance was assessed using one-way ANOVA, followed by Student-Newman-Keuls multiple range test. *, P < 0.05 vs. time zero.

 


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  		Figure 3. Immunoblotting showing the time course of SOCS protein expression in parametrial adipose tissue of female mice stimulated with oPRL in vivo. Parametrial adipose tissues were obtained from female mice that had been treated with oPRL (1 µg/g) for different times. Protein was prepared and analyzed for 37-kDa CIS (A), 28-kDa SOCS-3 (B), and 28-kDa SOCS-2 (C) proteins (35 µg protein/sample). The experiments were repeated at least three times with an adipose tissue lysate from a different animal at each time point.

 
CIS. In agreement with the in vitro data, oPRL stimulated CIS mRNA expression in the adipose tissue in vivo (Fig. 2AGo). The increase in CIS mRNA expression was transient. CIS mRNA was significantly elevated after 1 h, and after 24 h, CIS expression had returned to the level found at time zero. In addition, oPRL stimulation strongly induced CIS protein expression in adipose tissue in vivo, with a peak at 2 h (Fig. 3AGo).

SOCS-3. oPRL treatments in vivo stimulated SOCS-3 mRNA expression in adipose tissue (Fig. 2BGo). SOCS-3 mRNA was transiently expressed over 24 h, and the level of SOCS-3 mRNA peaked after 1 h. SOCS-3 protein expression was also induced in the adipose tissue of oPRL-stimulated female mice (Fig. 3BGo).

SOCS-2. SOCS-2 mRNA expression was detectable in adipose tissue, in contrast to isolated adipocytes cultured in vitro. Furthermore, SOCS-2 mRNA increased significantly after 1 h of oPRL stimulation in vivo (Fig. 2CGo). However, the relative increase in SOCS-2 mRNA expression in vivo was less than the relative increase in CIS and SOCS-3 expression. In addition, SOCS-2 protein expression was transient over 24 h of oPRL stimulation in vivo (Fig. 3CGo).

Regulation of CIS, SOCS-3, and SOCS-2 mRNA expression in parametrial adipose tissue of female PRL-transgenic mice and controls
The effects of chronically high PRL levels on SOCS expression were investigated in the parametrial adipose tissue isolated from female PRL-transgenic mice and controls using RPA (Fig. 4Go). The level of SOCS-2 mRNA expression was significantly increased in adipose tissue of mice with chronically high PRL levels compared with controls (Fig. 4CGo). However, no difference was detected in CIS or SOCS-3 expression in adipose tissue of PRL-transgenics compared with controls (Fig. 4Go, A and B).



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  		Figure 4. Expression of SOCS mRNA in adipose tissue of female PRL-transgenic mice and control littermates. RNA from adipose tissue of individual mice was prepared (8–13 mice/group) and analyzed for CIS (A), SOCS-3 (B), and SOCS-2 (C) mRNA together with the internal standard cyclophilin using RPA (15 µg RNA/sample). The SOCS/cyclophilin ratio was calculated for each sample, and the values were the relative SOCS expression (the average value for the controls was set at 100%). Results are expressed as the mean ± SEM, and statistical significance was assessed using one-way ANOVA, followed by Student-Newman-Keuls multiple range test. *, P < 0.05 vs. control.

 
Regulation of CIS, SOCS-3, and SOCS-2 mRNA expression in parametrial adipose tissue of virgin female, pregnant, and lactating mice
The regulation of CIS, SOCS-3, and SOCS-2 mRNA expression was examined in adipose tissue of wild-type virgin female, pregnant, and lactating mice using RPA (Fig. 5Go). As in the PRL-transgenics, only the level of SOCS-2 mRNA was significantly increased in adipose tissue during pregnancy and lactation compared with the level in adipose tissue from virgin females (Fig. 5CGo). Furthermore, SOCS-2 mRNA expression increased more during pregnancy (3-fold) than during lactation (2-fold). The levels of CIS and SOCS-3 were unchanged (Fig. 5Go, A and B).



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  		Figure 5. Expression of SOCS mRNA in adipose tissue of wild-type virgin female, pregnant, and lactating mice. RNA from adipose tissue of individual mice was prepared and analyzed for CIS (n = 8–10; A), SOCS-3 (n = 5–6; B), and SOCS-2 (n = 5–6; C) mRNA together with the internal standard cyclophilin using RPA (15 µg RNA/sample). The SOCS/cyclophilin ratio was calculated for each sample, and the values were the relative SOCS expression (the average value for the virgin females was set at 100%). Results are expressed as the mean ± SEM, and statistical significance was assessed using one-way ANOVA, followed by Student-Newman-Keuls multiple range test. *, P < 0.05 vs. virgin females.

 
Effects of oPRL on leptin secretion in adipocytes cultured in vitro and serum leptin levels in female PRL-transgenic mice and controls
The direct effect of PRL on adipocyte leptin secretion was investigated in female mouse adipocytes cultured in vitro. Adipocytes were cultured for 6 h, and four different treatment groups were studied: control (no hormone), insulin (20 nM), insulin (20 nM) and oPRL (300 ng/ml), and oPRL (300 ng/ml; Fig. 6AGo). Leptin secreted into the medium was analyzed by ELISA.



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  		Figure 6. Direct effects of PRL on leptin secretion from female mouse adipocytes cultured in vitro for 6 h (A) and serum leptin concentrations in female PRL-transgenic (n = 8) and control (n = 5) mice at 3 months of age (B). The effects of four different treatments [control (no hormone), insulin (20 nM), insulin (20 nM) and oPRL (300 ng/ml), and oPRL (300 ng/ml)] on leptin secretion from adipocytes cultured in vitro was studied (n = 7). Leptin production in the respective sample was calculated as the leptin concentration (nanograms per ml) in medium/total DNA content, and the average leptin production in the control was set at 100%. Results are expressed as the mean ± SEM, and statistical significance assessed using one-way ANOVA, followed by Student-Newman-Keuls multiple range test. *, P < 0.05 vs. control.

 
After culturing adipocytes for 6 h in basal medium without hormones, 6.9 ± 0.25 ng leptin/ml were secreted into the medium. Addition of insulin stimulated leptin secretion by adipocytes and increased leptin secretion 1.5-fold compared with the control level (Fig. 6AGo). Insulin-induced leptin secretion was suppressed by oPRL to levels similar to those in the control (Fig. 6AGo). However, oPRL alone had no effect on basal leptin secretion. In contrast, a significant increase in serum leptin concentration was found in female PRL-transgenic mice compared with control mice (Fig. 6BGo).

Effects of RU 486 on PRLR mRNA expression in adipose tissue and mammary gland of pregnant mice
To determine whether progesterone affects the level of L-PRLR expression in adipose tissue, pregnant mice on d 16 of pregnancy were treated with the PR antagonist RU 486 (150 µg) or vehicle for 15 h. RU 486 treatment induced a significant increase in L-PRLR mRNA expression in the mammary gland (Fig. 7AGo). However, there was no difference in L-PRLR expression in adipose tissue of RU 486-treated compared with vehicle-treated pregnant mice (Fig. 7BGo).



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  		Figure 7. Expression of L-PRLR mRNA in mammary gland (A) and adipose tissue (B) of RU 486-treated or vehicle-treated pregnant mice. RNA from mammary gland and adipose tissue of individual mice was prepared (8–10 mice/group) and analyzed for L-PRLR mRNA together with the internal standard cyclophilin using RPA (20 µg RNA/sample). The L-PRLR/cyclophilin ratio was calculated for each sample, and the values were the relative L-PRLR expression (the average value for the vehicle-treated pregnant mice was set at 100%). Results are expressed as the mean ± SEM, and statistical significance was assessed using one-way ANOVA, followed by Student-Newman-Keuls multiple range test. *, P < 0.05 vs. vehicle-treated pregnant mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrates functional PRLRs in female mouse adipocytes and adipose tissue because PRL stimulates gene transcription of several SOCS family members in both isolated adipocytes in vitro and adipose tissue in vivo. In addition, PRL suppresses insulin-induced leptin secretion in adipocytes cultured in vitro. oPRL induced CIS expression, and a combination of oPRL and insulin induced SOCS-3 expression in adipocytes in vitro, suggesting a role for these SOCS genes in regulating PRL signal transduction in adipocytes. Furthermore, CIS, SOCS-3, and SOCS-2 were transiently expressed in adipose tissue from female mice stimulated with oPRL for 0–24 h. On the other hand, only SOCS-2 increased in adipose tissue of female PRL-transgenic and wild-type pregnant and lactating mice, suggesting different physiological roles for SOCS proteins during transient and prolonged PRL stimulation.

The PRLR belongs to the cytokine receptor family, and signal transduction through activation of the JAK2/STAT5 pathway is well characterized (19). It has also been established that cytokine receptor signaling pathways are negatively regulated (25). However, little is known about how cytokine receptor signaling is terminated (25). A new family of proteins, SOCS, involved in the negative feedback of cytokine receptor signaling was recently cloned (21, 22, 23, 24). The transcripts encoding SOCS-1, SOCS-2, SOCS-3, and CIS are expressed at low levels in many cells. Several cytokines, hormones, and growth factors, including interferon-{gamma}, erythropoietin, IL-2, IL-3, IL-4, IL-6, leptin, GH, and PRL, have been found to induce SOCS expression in both isolated cells in vitro and tissues in vivo (21, 22, 25, 26, 27, 28, 29, 30, 31). Induction of the different SOCS genes seems to depend on the kind of cell line or tissue studied (25). For example, GH induced CIS, SOCS-3, and SOCS-2 expression in the mouse liver, but only CIS and SOCS-2 in the mouse mammary gland (36). In contrast, there is limited information on the expression and regulation of SOCS genes in adipocytes and adipose tissue, and it would be of physiological relevance to investigate SOCS expression further in adipose tissue during different physiological situations (27, 37, 38, 39). PRL has been reported to affect adipose tissue in several in vivo models (1, 2, 3, 4, 5, 6, 7, 11, 12, 13, 14, 15, 16, 17, 18). However, we investigated whether PRL can have direct effects, mediated by functional PRLRs, on adipocytes. In the present study oPRL induced the expression of CIS mRNA in female mouse adipocytes in vitro, demonstrating functional PRLRs in adipocytes. When oPRL was added in combination with insulin, the induced CIS mRNA expression was potentiated. Insulin alone also stimulated CIS mRNA expression in cultured adipocytes. The role of CIS in regulating PRL signal transduction has been investigated in 293 cells and CIS could not inhibit the PRLR, JAK2, or STAT5 (31, 32). Although CIS did not negatively regulate PRL signal transduction and PRLR, JAK2, or STAT5 in 293 cells, CIS has been found to inhibit cytokine signal transduction by competing with STAT5 or other molecules for docking sites on cytokine receptors (21, 40, 41). In addition, female mice overexpressing CIS failed to lactate after parturition because of a failure in terminal differentiation of the mammary glands, suggesting defective PRL signaling (40). Furthermore, in the present study CIS was transiently expressed in the adipose tissue obtained from female mice stimulated with oPRL in vivo, and the CIS mRNA level increased 3-fold after 1 h. This suggests a role for CIS in regulating the PRL signal transduction in adipose tissue.

In adipocytes in vitro, oPRL alone did not induce SOCS-3 mRNA expression, whereas insulin alone increased the expression. However, oPRL potentiated insulin’s effect, and the combination of oPRL and insulin induced SOCS-3 expression 4-fold compared with baseline. Indeed, in a recent study insulin was found to induce SOCS-3 expression, but not that of SOCS-2 or CIS, in cultured 3T3 adipocytes (37). The insulin receptor does not belong to the cytokine receptor family. However, it belongs to the tyrosine kinase receptors, which are potential STAT activators, and STAT5b has been shown to be a direct substrate of the insulin receptor (42). Furthermore, by inhibiting insulin-stimulated Stat5b, SOCS-3 appears to function as a negative regulator of insulin signaling (37). PRL has been reported to reduce glucose uptake in adipocytes in vitro, and hyperprolactinemia increases insulin resistance and decreases glucose tolerance (1, 2, 3, 4, 5, 6, 7, 9, 10). In this study we demonstrate that oPRL induces SOCS-3 expression in adipose tissue in vivo, and oPRL potentiates the insulin-induced SOCS-3 expression in adipocytes in vitro. One interpretation is that PRL-induced SOCS-3 expression can function as a negative regulator of insulin signaling and thereby decrease glucose uptake and increase insulin resistance in adipose tissue. Furthermore, previous studies have shown that constitutive expression of SOCS-3 in 293 cells suppresses PRL-induced STAT5-dependent gene transcription and reduces JAK2 tyrosine kinase activity (31, 32, 43). To further investigate whether PRL can affect adipocytes and insulin signaling, we analyzed the effect of PRL on insulin-induced leptin production in adipocytes cultured in vitro. Insulin is well known to induce leptin production in adipocytes, both in vivo and in vitro (44, 45, 46). In the present study insulin increased the secretion of leptin 1.5-fold in adipocytes cultured in vitro for 6 h. Furthermore, when PRL was added in combination with insulin, PRL suppressed insulininduced leptin secretion to levels similar to those found in the control. This study is the first to investigate the direct effects of PRL on adipocyte leptin production, and it demonstrates that PRL inhibits insulin-induced leptin production in vitro. Other studies have investigated the effects of PRL on leptin production in vivo (18, 47). Serum leptin concentrations were reduced in female PRLR-deficient mice (18). Furthermore, female rats with increased serum PRL levels in vivo, obtained by pituitary graft, had increased serum leptin concentrations (47). In the present study we demonstrate that serum leptin concentrations increased in female PRL transgenic mice compared with those in control mice. In vivo, PRL has been demonstrated to increase pancreatic insulin production and stimulate pancreatic ß-cell proliferation (19). In addition, serum insulin concentrations were reduced in PRLR-deficient mice (48). Therefore, the changes in serum leptin concentrations found in vivo could be indirect effects of PRL regulated by altered insulin production.

The effects of transient cytokine or hormonal treatments on SOCS expression in different tissues have been addressed in several studies (22, 25, 27, 28, 31, 49). However, the effects of prolonged or chronic hormonal exposure on SOCS expression have not been investigated to the same extent, although SOCS expression has been analyzed in tissues from old, compared with younger, rats (27, 39). In addition, in postpartum lactating mouse mammary glands, CIS was highly expressed on d 1–3, whereas SOCS-2 and SOCS-3 expression gradually increased from d 1 to 3 (32, 43), and SOCS-1 mRNA was highly expressed in rat antimesometrial decidua on d 12 and 13 of pseudopregnancy (50). In the present study we compared the effects of transient and prolonged PRL stimulation on SOCS expression in adipose tissue. CIS mRNA, SOCS-3 mRNA, and SOCS-2 mRNA were all significantly increased after 1 h of oPRL stimulation in vivo. However, they had all returned to the baseline level after 24-h stimulation. In contrast, when chronically exposed to PRL, only SOCS-2 was increased in the adipose tissue of PRL-transgenic female mice. Serum levels of placental lactogens are elevated during pregnancy, and serum levels of PRL increase during lactation (51). Both of these physiological conditions could affect the level of SOCS expression in adipose tissue. In adipose tissue of both pregnant and lactating mice compared with that of virgin females, again only SOCS-2 was increased. PRL is the likely candidate to induce SOCS-2 mRNA expression in the adipose tissue of both PRL-transgenic and lactating mice. In addition, placental lactogen is likely to be a regulator of SOCS-2 expression in adipose tissue during pregnancy (51). SOCS-2 has been reported to have dual functions when affecting PRL signal transduction (31). SOCS-2 first partially inhibits PRL-induced signal transduction in 293 cells. However, when high levels of SOCS-2 were constitutively expressed, it could restore the sensitivity of the cells to PRL (31). A possible function for increased SOCS-2 expression in the adipose tissue of PRL-transgenics and lactating mice, both with prolonged high serum PRL levels and increased L-PRLR expression in adipose tissue (8), could be to restore the sensitivity of the adipose tissue to PRL.

We recently found the level of L-PRLR mRNA expression to increase in adipose tissue during lactation compared with that in virgin female and pregnant mice (8). The level of progesterone decreases at the end of pregnancy, and this has been reported to increase PRL secretion and affect lactogenesis (52). In addition, the level of PRLR expression increases in the mammary gland during lactation, and this increase has been suggested to be regulated by the fall in progesterone and the increased PRL level. Furthermore, when rats were injected with the progesterone antagonist RU 486 during late pregnancy, the level of PRLR mRNA increased in the mammary gland, but not in the liver (52). In the present study when pregnant mice were treated with RU 486, a small increase in L-PRLR expression was detected in the mammary gland. However, no increase in L-PRLR expression was found in the adipose tissue of RU 486-treated mice. This result indicates that progesterone does not affect L-PRLR expression in adipose tissue during pregnancy and lactation. Furthermore, the increased L-PRLR mRNA expression found in the mammary gland of RU 486-treated pregnant mice was lower than the increase seen in rats (52). One interpretation of this result is that progesterone is a stronger regulator of L-PRLR expression in the mammary gland of pregnant rats compared with pregnant mice.

Taken together, the results of the present study demonstrate functional PRLRs in mouse adipocytes. PRL did affect gene transcription of CIS and SOCS-3 in adipocytes in vitro. In addition, PRL induced SOCS expression in the adipose tissue in vivo. The levels of CIS, SOCS-3, and SOCS-2 were transiently increased in adipose tissues obtained from female mice stimulated with oPRL for 0–24 h. Furthermore, the PRL-induced SOCS-3 expression could play a role as a negative regulator of insulin signaling in the adipose tissue. In the present study PRL suppressed insulin-induced leptin secretion in vitro. Furthermore, transient and prolonged PRL exposure regulates SOCS expression in adipose tissue differently, with possible physiological implications. The transient increase in CIS, SOCS-3, and SOCS-2 expression seen after 1 h of in vivo PRL stimulation is probably a negative regulator of the PRL signal transduction in adipose tissue. However, only SOCS-2 increased in adipose tissue after prolonged PRL exposure during lactation and in the PRL-transgenics, and the role of SOCS-2 in these physiological situations could be to restore the sensitivity of adipose tissue to PRL.


   Acknowledgments
 
We thank Birgitta Weijdegård, B.Sc. (Department of Obstetrics and Gynecology, Goteborg University, Goteborg, Sweden), for excellent technical assistance with the RT-PCR analysis; Drs. Håkan Wennbo (AstraZeneca) and Maria Gebre-Medhin for kindly providing the PRL-transgenic mice; Drs. T. Willson and D. Hilton, The Walter and Eliza Hall Institute of Medical Research (Melbourne, Australia), for the gift of SOCS-pEF-BOS expression vectors; and National Hormone and Pituitary Program (Baltimore, MD) for kindly providing oPRL.


   Footnotes
 
This work was supported by Grants 10380 and 13550 from the Swedish Medical Research Council and by grants from the Wilhelm and Martina Lundgrens Vetenskaps Fund, the Kungliga och Hvitfeldtska Stipendiestiftelsen, and the Assar Gabrielssons Forsknings Fond.

Abbreviations: CIS, Cytokine-inducible SH2-domain-containing protein; JAK, Janus kinase; oPRL, ovine PRL; PRLR, PRL receptor; RPA, ribonuclease protection assay; SOCS, suppressors of cytokine signaling; STAT, signal transducer and activator of transcription.

Received May 7, 2001.

Accepted for publication August 6, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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