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Endocrinology 142(11):4880 – 4890
Copyright © 2001 by The Endocrine Society
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
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-domaincontaining 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 ob-
P
RL 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 (1–7). 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–
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, PRLRdeficient mice were recently reported to have reduced abdominal fat content and decreased leptin production comAbbreviations: 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.
tained from female mice stimulated with oPRL (1 ␮g/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. (Endocrinology 142: 4880 – 4890, 2001)
pared 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) (21–24).
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,
4880
Ling and Billig • PRL Receptor-Mediated Effects in Adipocytes
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, 25–31). In addition, overexpression
of several of these SOCS proteins in cell lines resulted in
inhibition of cytokine receptor signaling (25, 29 –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 wildtype 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
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 ␮g/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-␮m 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 ␮g/ml; no. 15290-026, Life Technologies, Inc.), gentamicin (50 ␮g/ml; no. 15710-031, Life Technologies,
Inc.), HEPES (25 mm; 1M, Life Technologies, Inc.), and 1 ⫻ l-glutamine
(⫻100, 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
Endocrinology, November 2001, 142(11):4880 – 4890 4881
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 ␮g/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 ␮g/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 pEFFLAG-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, pEFFLAG-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 ␮g). 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]CTP-
4882
Endocrinology, November 2001, 142(11):4880 – 4890
labeled 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
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
Ling and Billig • PRL Receptor-Mediated Effects in Adipocytes
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. 1).
Cis mRNA. Stimulation with oPRL increased the level of CIS
mRNA expression significantly in the cultured adipocytes
compared with baseline levels (Fig. 1A). 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. 1A).
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. 1B).
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. 2). 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. 3).
CIS. In agreement with the in vitro data, oPRL stimulated CIS
mRNA expression in the adipose tissue in vivo (Fig. 2A). 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. 3A).
SOCS-3. oPRL treatments in vivo stimulated SOCS-3 mRNA
expression in adipose tissue (Fig. 2B). 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. 3B).
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. 2C). 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. 3C).
Ling and Billig • PRL Receptor-Mediated Effects in Adipocytes
Endocrinology, November 2001, 142(11):4880 – 4890 4883
FIG. 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 (f; no hormone
added), PRL (F; 300 ng/ml oPRL), INS (Œ; 20 nM insulin), and PRL plus INS (; 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 ␮g 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.
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. 4). The level of SOCS-2 mRNA expression was
significantly increased in adipose tissue of mice with chronically high PRL levels compared with controls (Fig. 4C).
However, no difference was detected in CIS or SOCS-3 expression in adipose tissue of PRL-transgenics compared with
controls (Fig. 4, A and B).
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. 5). 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. 5C). 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. 5, A and B).
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. 6A). Leptin secreted into the medium was analyzed by ELISA.
After culturing adipocytes for 6 h in basal medium without
4884
Endocrinology, November 2001, 142(11):4880 – 4890
Ling and Billig • PRL Receptor-Mediated Effects in Adipocytes
FIG. 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 ␮g/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 ␮g 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.
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. 6A). Insulin-induced leptin secretion was suppressed by oPRL to levels similar to those in
the control (Fig. 6A). 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. 6B).
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 ␮g) or vehicle for 15 h. RU 486 treatment induced a
significant increase in L-PRLR mRNA expression in the
mammary gland (Fig. 7A). However, there was no difference
Ling and Billig • PRL Receptor-Mediated Effects in Adipocytes
FIG. 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 ␮g/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 ␮g protein/sample).
The experiments were repeated at least three times with an adipose
tissue lysate from a different animal at each time point.
in L-PRLR expression in adipose tissue of RU 486-treated
compared with vehicle-treated pregnant mice (Fig. 7B).
Discussion
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.
Endocrinology, November 2001, 142(11):4880 – 4890 4885
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 (21–24). 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-␥, 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, 25–31). 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, 37–39). PRL has been reported to affect adipose
tissue in several in vivo models (1–7, 11–18). 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
4886
Endocrinology, November 2001, 142(11):4880 – 4890
Ling and Billig • PRL Receptor-Mediated Effects in Adipocytes
FIG. 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 ␮g 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.
(37). PRL has been reported to reduce glucose uptake in
adipocytes in vitro, and hyperprolactinemia increases insulin
resistance and decreases glucose tolerance (1–7, 9, 10). In this
study we demonstrate that oPRL induces SOCS-3 expression
in adipose tissue in vivo, and oPRL potentiates the insulininduced 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 insulininduced leptin production in adipocytes cultured in vitro.
Insulin is well known to induce leptin production in adipocytes, both in vivo and in vitro (44 – 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
Ling and Billig • PRL Receptor-Mediated Effects in Adipocytes
Endocrinology, November 2001, 142(11):4880 – 4890 4887
FIG. 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 ␮g 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.
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 ␤-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
4888
Endocrinology, November 2001, 142(11):4880 – 4890
FIG. 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.
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 lac-
Ling and Billig • PRL Receptor-Mediated Effects in Adipocytes
togens 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 PRLtransgenic 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
Ling and Billig • PRL Receptor-Mediated Effects in Adipocytes
Endocrinology, November 2001, 142(11):4880 – 4890 4889
FIG. 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 ␮g 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.
prolonged PRL exposure during lactation and in the PRLtransgenics, and the role of SOCS-2 in these physiological
situations could be to restore the sensitivity of adipose tissue
to PRL.
7.
Acknowledgments
8.
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 PRLtransgenic 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.
Received May 7, 2001. Accepted August 6, 2001.
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: [email protected].
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.
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