 |
INTRODUCTION |
Brown adipose tissue is a major site for non-shivering
thermogenesis in mammals. The unique thermogenic capacity of brown adipose tissue results from the expression of the uncoupling protein-1 (UCP-1)1 in the mitochondrial
inner membrane required to address the physiological hypothermia in
newborn mammals (1). UCP-1 uncouples fatty acid oxidation from ATP
synthesis, which allows dissipation of energy from substrate oxidation
as heat (2). In addition, brown adipose tissue is a major site for
lipid metabolism, fatty acids being the main fuel to maintain the
thermogenic capacity of the tissue (for review, see Ref. 3). It is well
known that in rodents brown adipocytes differentiate at the end of the
fetal life on the basis of two programs, an adipogenic program related
to lipid synthesis and the expression of lipogenic enzymes, resulting
in a multilocular fat droplets phenotype (4-6), and a thermogenic program related to heat production and UCP-1 expression (7). Regarding
thermogenic differentiation, the main pathway involved in the
regulation of UCP-1 gene expression is noradrenergic (8-11). However,
because the noradrenergic stimulus, induced by hypothermia after birth,
it is not yet fully developed in brown adipose tissue during late fetal
development (5, 9), other potential candidates involved in the onset of
differentiation-related gene expression have been implicated. Recently,
regulatory elements for triiodothyronine and retinoic acid have been
identified in the UCP-1 promoter, suggesting alternative pathways for
brown fat thermogenesis (12, 13). In addition, during the last years
our laboratory has found that fetal brown adipocytes display high
affinity binding sites for insulin (6). This hormone seems to be the
main signal involved in fetal brown adipogenesis through its ability to
induce the genetic expression of metabolic genes (6, 14) and may as well have a role in thermogenesis by inducing UCP-1 expression (15).
A complex network of intracellular signaling pathways mediates the
pleiotropic effects of insulin on metabolic processes as well as on
gene expression. Insulin initiates its biological effects by binding to
and activating its endogenous tyrosine kinase receptors (16, 17). These
receptors are believed to transduce signals by phosphorylation on
tyrosine residues of several cellular substrates including IRS proteins
(IRS-1, -2, -3, and -4) (18-21). These phosphorylated substrates then
bind proteins containing Src homology 2 domains including the p85
regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase) (22),
growth factor receptor binding protein 2 (Grb-2), which links signaling
via SOS to activation of the Ras complex (23), and protein-tyrosine
phosphatase SHP2 (24), which lead to activation of various downstream
signaling pathways.
Previous work from our laboratory demonstrated that fetal brown
adipocytes express high levels of both IRS-1 and IRS-2, with the PI
3-kinase cascade being the main pathway involved in adipogenic- and
thermogenic-related gene expression (25). More recently, we have
generated immortalized fetal brown adipocyte cell lines from IRS-1
knockout fetuses (26). These cells are new tools in dissecting the
signaling pathways emerging from IRS proteins responsible of the
biological effects of insulin in brown adipose tissue during late fetal
development. In these cells, IRS-1, but not IRS-2, is an essential
requirement for insulin-induced lipid synthesis (26). However, it
remains unclear the exact role of IRS proteins and the cytosolic
signaling pathways emerging downstream of IRSs that mediate
insulin-induced UCP-1 expression as well as the connection with nuclear
factors responsible of the transcription of the UCP-1 gene.
In the present paper we have found a failure of insulin to induce UCP-1
expression in IRS-1-deficient fetal brown adipocytes. This occurs as a
result of a lack of IRS-1-associated/Akt signaling and the loss of
CCAAT/enhancer-binding protein
(C/EBP
) expression and C/EBP
DNA binding activity in response to insulin. Reconstitution with
wild-type IRS-1 (IRS-1wt) and either with the Y895F mutant
(IRS-1Phe-895), which lacks Grb-2 binding but not p85
binding, or with the IRS-1 mutant, which contains the substitution of
Phe for Tyr in 18 potential tyrosine phosphorylation sites except
tyrosines at positions 608, 628, and 658 (IRS-1Tyr-608/Tyr-628/Tyr-658), results in a recovery of
insulin-mediated IRS-1/PI 3-kinase/Akt activation, transactivation of
the UCP1-promoter, and UCP-1 expression. In addition,
overexpression of C/EBP
or PPAR
transcription factors in
IRS-1-deficient brown adipocytes up-regulates transactivation of the
UCP1 promoter and UCP-1 expression bypassing insulin signaling. These
results demonstrate the essential role played by IRS-1 through the PI
3-kinase/Akt-signaling pathway inducing UCP-1 gene expression by insulin.
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EXPERIMENTAL PROCEDURES |
Materials--
Fetal calf serum and culture media were obtained
from Invitrogen. Insulin, hygromycin, and anti-mouse IgG-agarose were
from Sigma. Protein A-agarose was from Roche Molecular Biochemicals. The bleomycin analogue Zeocin was purchased from Invitrogen. The anti-IRS-1 (06-248), anti-
-Akt (06-558), anti-
-Akt (06-606), anti-
-Akt (06-607) polyclonal antibodies were purchased from Upstate Biotechnology (Lake Placid, NY). The anti-Tyr(P) (Py20) (sc-508), anti-C/EBP
(sc-61), anti-UCP-2 (sc-6525) and anti-UCP-3 (sc-7756) antibodies were purchased from Santa Cruz (Santa Cruz Biotechnology, Palo Alto, CA) The anti-phospho Akt (Ser-473 No. 9271)
and anti-Akt (No. 9272) antibodies were purchased from New England
Biolabs (Beverly, MA). The anti-UCP-1 (AB3038) antibody was from
Chemicon (Chemicon International, CA). The anti-PPAR
(SA
206)
antibody was from BioMol (Biomol Research Laboratories, Plymouth
Meeting, PA). [
-32P]ATP (3000 Ci/mmol) and
[
-32P]dCTP (3000 Ci/mmol) were from Amersham
Biosciences. All other reagents used were of the purest grade available.
Cell Culture and Retroviral Infections--
Brown adipocytes
were obtained from interscapular brown adipose tissue of 17.5-18.5
fetuses from 2-3 pregnant mice of normal genotype or from a pool of
tissue of fetuses obtained from 2-3 pregnant mice
IRS-1+/
mated with males IRS-1
/
and
further submitted to collagenase dispersion as previously described
(4). Viral Bosc-23 packaging cells were transfected at 70% confluence
by calcium phosphate coprecipitation with 3 µg/6-cm dish of the
puromycin resistance retroviral vector pBabe encoding SV 40 Large T
antigen (kindly provided by J. de Caprio, Dana Farber Cancer Institute,
Boston, MA). Then brown adipocytes were infected at 60% confluence
with Polybrene (4 µg/ml)-supplemented virus for 48 h and
maintained in culture medium for 72 h before selection with
puromycin (1 µg/ml) for 1 week. Several cell lines IRS-1+/+, IRS-1+/
, and IRS-1
/
were cloned and expanded, and the expression of IRS-1 was assessed by
Western blot. Three clones of wild type and IRS-1
/
respectively, were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and puromycin (1 µg/ml). The
pBabe/hygro C/EBP
and pBabe/Zeo PPAR
viral expression vectors were generous gifts from Dr. B. Spiegelman (Dana Farber Cancer Institute, Boston, MA). Brown adipocyte IRS-1-deficient cells (clone 4)
were infected with those vectors as described above. Selection with 200 µg/ml hygromycin or 250 µg/ml Zeocin was started 48 h after
infection to select stable cell lines.
Transfections--
IRS-1-deficient brown adipocytes (clone 4)
were cultured for 24 h in the presence of 10% fetal calf serum,
and then, when 60-70% confluence was reached, cells were transfected
according to the calcium phosphate-mediated protocol with the plasmid
constructs indicated in each case. For pCMVhisIRS-1wt,
pCMVhisIRS-1Phe-895 (derived from IRS-1 by introducing a
Phe for Tyr substitution at position 895 (27)) and
pCMVhisIRS-1Tyr-608/Tyr-628/Tyr-658 (derived from IRS-1-F18
by adding back Tyr at positions 608, 628, and 658 (28)) constructs, 10 µg of DNA were added to each dish. After 4-6 h of incubation, cells
were shocked with 3 ml of 15% glycerol for 2 min, washed, and then fed
with Dulbecco's modified Eagle's medium, 10% fetal calf serum.
24 h after transfection, histidinol (10 mM) was
added to select stable transfectants.
Immunoprecipitations--
Quiescent cells (20 h serum-starved)
were treated without or with several doses of insulin as indicated and
lysed at 4 °C in 1 ml of a solution containing 10 mM
Tris/HCl, 5 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM NaF, 100 µM Na3VO4, 1% Triton X-100, and
1 mM phenylmethylsulfonyl fluoride, pH 7.6 (lysis buffer).
Lysates were clarified by centrifugation at 15,000 × g
for 10 min. After protein content determination, equal amounts of
protein (500-600 µg) were immunoprecipitated at 4 °C with the corresponding antibodies. The immune complexes were collected on
protein A-agarose or anti-mouse IgG-agarose beads. Immunoprecipitates were washed with lysis buffer and extracted for 5 min at 95 °C in
2× SDS-PAGE sample buffer (200 mM Tris/HCl, 6% SDS, 2 mM EDTA, 4% 2-mercaptoethanol, 10% glycerol, pH 6.8) and
analyzed by SDS-PAGE.
Western Blotting--
After SDS-PAGE, proteins were transferred
to Immobilon membranes and blocked using 5% nonfat dried milk or 3%
bovine serum albumin in 10 mM Tris-HCl, 150 mM
NaCl, pH 7.5, and incubated overnight with several antibodies as
indicated in 0.05% Tween 20, 10 mM Tris-HCl, 150 mM NaCl, pH 7.5. Immunoreactive bands were visualized using
the ECL Western-blotting protocol (Amersham Biosciences).
PI 3-Kinase Activity--
PI 3-kinase activity was measured in
the anti-IRS-1 or anti-Tyr(P) immunoprecipitates by in vitro
phosphorylation of phosphatidylinositol as previously described
(25).
Akt Activity--
For Akt activity, cells were lysed in Buffer A
containing 50 mM Tris-HCl, pH 7.5, 0.1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 50 mM sodium
fluoride, 10 mM sodium
-glycerol phosphate, 5 mM sodium pyrophosphate, 1 mM
Na3VO4, and 0.1%
-mercaptoethanol and
subjected to immunoprecipitation with the anti-
-Akt, anti-
-Akt, or anti-
-Akt polyclonal antibodies as described above. The
immunoprecipitates were washed 3 times with buffer A containing 0.5 M NaCl, 2 times with Buffer B containing 50 mM
Tris-HCl, pH 7.5, 0.03% Brij-35, 0.1 mM EGTA, 0.1%
-mercaptoethanol, and once with Buffer ADB containing 20 mM MOPS, pH 7.2, 25 mM sodium
-glycerol
phosphate, pH 7, 0.1 mM Na3VO4, 1 mM dithiothreitol. The kinase reaction was started by
adding to each sample 10 µl of Buffer ADB, protein kinase A inhibitor
to a final concentration of 17 µM, 10 µl of 30 µM Akt/synthetic peptide of glycogen synthase kinase-3
(SGK) substrate peptide (Upstate Biotechnology, catalog No.
12-340), and 10 µCi of [
-32P]ATP (3000 Ci/mmol).
After 10 min of incubation at 30 °C, the reaction was terminated by
blotting 25 µl of the supernatant fraction on a P81 paper square
(Whatman). Filters were washed 3 times with 0.75% phosphoric acid and
once with acetone, and the radioactivity remaining on the filters was measured.
Isolation of Mitochondrial Protein--
At the end of the
culture time, cells were scraped off in isotonic isolation buffer (1 mM EDTA, 10 mM HEPES, 250 mM
sucrose, pH 7.6), collected by centrifugation at 2500 × g for 5 min at 4 °C, and resuspended in hypotonic
isolation buffer (1 mM EDTA, 10 mM HEPES, 50 mM sucrose, pH 7.6). Then cells were incubated at 37 °C
for 5 min and homogenized under a Teflon pestle (Overhead Stirrer;
Wheaton Instruments, Milville, NJ). Hypertonic isolation buffer (1 mM EDTA, 10 mM HEPES, 450 mM
sucrose, pH 7.6) was added to balance the buffer tonicity. Samples were
centrifuged at 10,000 × g for 10 min, the pellets,
containing the mitochondrial fraction, were resuspended in isotonic
isolation buffer, and mitochondrial protein content was determined.
Protein Determination--
Protein determination was performed
by the Bradford dye method (29) using the Bio-Rad reagent and bovine
serum albumin as the standard.
RNA Extraction and Northern Blot Analysis--
At the end of the
culture time, cells were washed twice in ice-cold phosphate-buffered
saline, and RNA was isolated as described (30). Total cellular RNA (10 µg) was submitted to Northern blot analysis, i.e.
electrophoresed on 0.9% agarose gels containing 0.66 M
formaldehyde, transferred to GeneScreen membranes (PerkinElmer Life
Sciences), and cross-linked to the membranes by ultraviolet light.
Hybridization was performed in 0.25 mM
Na2HPO4, pH 7.2, 0.25 M
NaCl, 100 µg/ml denatured salmon sperm DNA, 7% SDS, and 50%
deionized formamide containing denatured 32P-labeled
cDNA (106 cpm/ml) for 24 h at 42 °C. cDNA
labeling was carried out with [
-32P]dCTP by using a
multiprimer DNA-labeling system. Blots were hybridized with probes for
fatty acid synthase (FAS) (31) and with 18 S ribosomal probe to
normalize. Membranes were subjected to autoradiography, and the
relative densities of the hybridization signals were determined by
densitometric scanning of the autoradiograms.
Extraction of Nuclear Proteins and Gel Mobility Shift
Assays--
Cells were resuspended at 4 °C in 10 mM
HEPES-KOH, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 0,2 mM phenylmethylsulfonyl fluoride, 0.75 µg/ml leupeptin,
0.75 µg/ml aprotinin (Buffer A), allowed to swell on ice for 10 min,
and then vortexed for 10 s. Samples were centrifuged, and the
pellet was resuspended in cold buffer C (20 mM HEPES-KOH,
pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM
MgCl2, 0.2 mM EDTA, 0.5 mM
dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 0.75 µg/ml leupeptin, 0.75 µg/ml aprotinin) and incubated on ice for 20 min for high salt extraction. Cellular debris was removed by
centrifugation for 2 min at 4 °C, and the supernatant fraction was
stored at
70 °C. The gel mobility shift assay was performed
essentially as previously described (32). The double-stranded oligonucleotide used as C/EBP
probe, corresponding to the nuclear factor-interleukin-6 (NF-IL6) site of the COX2 promoter
(5'-GGGTATTATGCAATTGGAAG-3'), was synthesized in a Pharmacia
oligonucleotide synthesizer. Labeling was performed by using Klenow
polymerase and [
-32P]dCTP. The binding reaction
mixture contained 0.5 ng of doubled-stranded oligonucleotide probe, 2 µg of poly(dI)·poly(dC), and 10 µg of protein in Buffer C
supplemented with 35 mM MgCl2. In some lanes a
100-fold excess of unlabeled oligonucleotide was used to compete away
specific complexes. After 20 min of incubation at 4 °C, the mixture
was electrophoresed through a 6% polyacrylamide gel in 0.5× Tris
borate-EDTA running buffer for 2 h. Gels were then dried and
subjected to autoradiography as well as quantified directly with a
radioimaging device. Supershift assays were carried out after
incubation of the nuclear extracts with 0.5 µg of either C/EBP
or
nonspecific (non-immune rabbit serum) antibodies for 1 h at
4 °C.
CAT Assays--
Cells growing in the presence of 10% fetal
serum were transiently transfected according to the calcium
phosphate-mediated protocol as described above. The plasmid constructs
used were 4551 UCP-1-CAT (where the CAT reporter gene is under the
control of a 4551-bp full-length 5'-flanking region of the UCP-1
promoter) (33),
3628/
2283/linked to
141 CAT corresponding with a
deletion mutant of the UCP-1 proximal promoter that does not contain
C/EBP binding sites (34) and pCMV
-galactosidase (gal) (a viral
promoter driving expression of the reporter gene
-gal). Ten
micrograms of DNA-CAT together with 2 µg of DNA-
-gal (to monitor
transfection efficiency) were added to each 10-cm dish. After 4 h
of incubation, cells were fed with serum-free medium for 15 h and
stimulated with various doses of insulin for a further 24 h. Then
cells were harvested, and lysates were prepared for CAT and
-gal
activity assays. CAT activity was determined by incubating 70 µl of
cell extracts with 0.25 µCi of [14C]chloramphenicol and
0.5 mM acetyl coenzyme A in 0.25 mM Tris, pH
7.8, at 37 °C for 15 h, and then samples were submitted to TLC.
The amount of substrate acetylated was directly quantified with a
radioimaging device (Fujifilm BAS-1000, Japan). CAT enzyme activity was
expressed in arbitrary units normalized to the internal control
-gal
(assayed according to the Stratagene protocol).
Cellular Oxygen Consumption--
Confluent cells
(IRS-1+/+ and IRS-1
/
) were cultured for
20 h in serum-free medium and further stimulated with insulin (100 nM) for 24 h. Then cells were trypsinized and
resuspended in 1 ml of serum-free Dulbecco's modified Eagle's medium
supplemented with 1% (w/v) bovine serum albumin. Basal oxygen
consumption rates were measured in a HANSA-TECH oxygen electrode in the
presence of 30 µM palmitate as described (35). Values
were normalized by cell number. The uncoupler carbonyl cyanide
p-trifluoromethoxyphenylhydrazone at 10 µM
totally collapsed the proton gradient and allowed the determination of
the fully uncoupled respiration rate.
 |
RESULTS |
Reconstitution of IRS-1 in IRS-1-deficient Fetal Brown Adipocytes
Restores IRS-1- and Phosphotyrosine-associated PI 3-Kinase
Activity--
We have previously shown that immortalized fetal brown
adipocytes derived from fetuses of IRS-1-deficient mice completely lack
response to 10 nM insulin in activating IRS-1-associated PI
3-kinase activity, and we also showed a 30% reduction in total PI
3-kinase activity in anti-phosphotyrosine (pY) immunoprecipitates (26).
To investigate whether this response could be recovered by
reintroducing wild-type IRS-1 (IRS-1wt), we reconstituted
IRS-1 expression in IRS-1-deficient brown adipocytes. These cells were
transfected with the pCMVhisIRS-1wt cDNA construct, and
stable cell lines were selected in 10 mM histidinol-containing medium. Fig.
1A shows that
IRS-1wt expression in the reconstituted cell lines
represents about 60% that seen in wild-type cells. IRS-1-associated PI
3-kinase activity was recovered in reconstituted brown adipocytes in
parallel to the level of IRS-1wt protein expressed (Fig.
1B). Likewise, insulin-induced
antiphosphotyrosine-associated PI 3-kinase activity, which was reduced
by 30% in IRS-1
/
cells, was totally recovered after
IRS-1wt reconstitution.

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Fig. 1.
Reconstitution of IRS-1 expression and PI
3-kinase activity in IRS-1-deficient brown adipocytes.
A, IRS-1-deficient brown adipocytes (clone 4) were cultured
until 60-70% confluence was reached. Then cells were transfected with
the pCMVhisIRS-1wt construct according to the calcium
phosphate-mediated protocol. 24 h after transfection, histidinol
(10 mM) was added to select stable transfectants. Several
histidinol-resistant cell lines were obtained, and the expression of
IRS-1wt was assessed by Western blot with the anti-IRS-1
antibody. B, quiescent cells (IRS-1+/+,
pCMVhisIRS-1wt and IRS-1 / ) were stimulated
with 10-100 nM insulin for 5 min. Cell lysates were
immunoprecipitated (IP) with anti-IRS-1 or anti-Tyr(P)
(pY) antibodies and immediately used for an in
vitro PI 3-kinase assay. A representative experiment is shown. The
autoradiograms corresponding to three independent experiments were
analyzed by scanning densitometry. Results are expressed as
arbitrary units of PI 3-kinase activity and are the means ± S.E.
PIP, inositol trisphosphate.
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|
Insulin-induced Phosphorylation and Activation of Akt Is Recovered
after IRS-1wt Reconstitution in IRS-1
/
Fetal Brown Adipocytes--
Because the serine/threonine kinase Akt is
one of the major downstream targets of PI 3-kinase, we explored the
phosphorylation of
-Akt and
-Akt isoforms as well as total Akt
phosphorylation by direct Western blot analysis in wild-type , IRS-1
/
, and IRS-1wt-reconstituted brown
adipocytes. In fetal brown adipocytes, the lack of IRS-1 resulted in a
loss of AktSer-473 phosphorylation; only a remnant
AktSer-473 phosphorylation was detected at 100 nM insulin concentration (Fig.
2A). Both
-Akt and
-Akt
isoforms were phosphorylated upon insulin (10-100 nM)
stimulation of wild-type brown adipocytes, as shown by the
electrophoretic mobility shifts in the presence of the hormone, these
effect being precluded in IRS-1
/
cells. The
reintroduction of IRS-1wt in IRS-1
/
brown
adipocytes resulted in a recovery of AktSer-473
phosphorylation as well as
-Akt,
-Akt phosphorylation as shown by
immunodetection with the anti-phospho specific AktSer-473
antibody, or by the mobility shifts observed in insulin-stimulated cells with the anti-
-Akt,
-Akt, and total Akt antibodies. To confirm the results obtained from the phospho-Akt analysis, Akt kinase
assays were performed in insulin-stimulated IRS-1+/+,
IRS-1
/
, and IRS-1wt-reconstituted fetal
brown adipocytes after immunoprecipitation with the specific antibodies
for each Akt isoform. As shown in Fig. 2B,
-Akt kinase
activity was enhanced by 2- or 3-fold at 10 nM or 100 nM insulin concentrations, respectively, in wild-type brown
adipocytes. Activation of
-Akt by insulin was totally blunted in
IRS-1
/
cells, whereas reconstituted IRS-1wt
cells recovered the level of insulin-induced
-Akt activity observed in wild-type cells. A modest increase in
-Akt activity was observed in wild-type cells upon 100 nM insulin stimulation; no
effect on
-Akt activity was detected in IRS-1
/
cells. Reconstituted IRS-1wt cells recovered the level of
insulin-induced
-Akt activity in parallel to the level of
IRS-1wt protein expressed (see Fig. 1A). Insulin
increased
-Akt activity in a dose-dependent manner in
wild-type brown adipocytes, with its maximal effect (3-3.5-fold)
elicited at 100 nM insulin concentration. Likewise, insulin
failed to activate
-Akt isoform in IRS-1
/
brown
adipocytes, whereas IRS-1wt-reconstituted cells recovered
insulin response in activating
-Akt in parallel to the level of
IRS-1wt protein expressed.

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Fig. 2.
Reconstitution of insulin-induced Akt
phosphorylation and Akt activation by IRS-1wt expression in
IRS-1-deficient brown adipocytes. A, quiescent cells were
stimulated with various doses of insulin for 5 min and then lysed.
Equals amount of protein (50 µg) were analyzed by Western blot with
the anti-phospho-Akt (PAkt, Ser-473), anti-total Akt,
anti- -Akt, and anti- -Akt antibodies. A representative experiment
of five is shown. B, quiescent cells were stimulated with
various doses of insulin for 5 min and then lysed. Equals amount of
protein (600-800 µg) were immunoprecipitated with anti- -Akt,
anti- -Akt, and anti- -Akt antibodies and immediately used for an
in vitro kinase assay as described under "Experimental
Procedures." Quantitative data are expressed in cpm and are the
means ± S.E. from 3-4 independent experiments.
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|
Insulin-dependent UCP-1 Expression and Transactivation
of the Full-length UCP-1 Promoter Is Impaired in IRS-1-deficient Brown
Adipocytes--
We have previously shown that either insulin or IGF-I
are novel thermogenic factors in brown adipocyte primary cultures
involved in UCP-1 expression during late fetal development; this effect was precluded by PI-3 kinase inhibitors (15, 25). Accordingly, our next
step was to assess if IRS-1 could be the main docking protein
connecting insulin-induced PI 3-kinase/Akt signaling pathway with UCP-1
expression. UCP-1 protein content was analyzed by direct Western blot
analysis in mitochondrial protein extracts of wild-type, IRS-1
/
, and reconstituted IRS-1wt brown
adipocytes treated for 24 h with various doses of insulin. As
shown in Fig. 3A, insulin
increased UCP-1 expression in wild-type-immortalized fetal brown
adipocytes in a dose-dependent manner as compared with
untreated cells. The lack of IRS-1 totally precluded insulin effect on
UCP-1 expression. However, we did not find changes in the basal
cellular oxygen consumption either in wild-type or IRS-1-deficient cells in response to insulin using palmitate (30 µM) as a
substrate (results not shown), suggesting that the cellular respiratory status is not dependent on the level of UCP-1 content found under our
experimental conditions. UCP-1 expression in response to insulin was
recovered when we added back IRS-1wt to
IRS-1
/
brown adipocytes in parallel to the level of
protein expressed. In addition we tested the effect of ML-9 compound
(an inhibitor of Akt activity) (36) on insulin-induced UCP-1 expression
in wild-type brown adipocytes. Inhibition of Akt resulted in a loss of
insulin response in inducing UCP-1 expression in wild-type brown
adipocytes as well as in IRS-1-reconstituted null cells (results not
shown). However, neither UCP-2 nor UCP-3 expression was up-regulated by
insulin in wild-type and IRS-1-deficient brown adipocytes (Fig.
3B). Another approach was to study the effect of insulin in
transactivating the UCP-1 promoter in the three cell types. Cells were
transiently transfected with the 4551-bp full-length UCP-1 promoter
driving the expression of the CAT reporter gene (33). Upon
transfection, cells were cultured for 24 h in a serum-free medium
either in the absence or presence of 100 nM insulin, and
then CAT activity was determined. As shown in Fig. 3C, the
4551-CAT fusion gene was weakly transcribed in unstimulated wild-type
brown adipocytes, whereas insulin treatment resulted in 2.5-fold
increase in CAT activity. IRS-1
/
brown adipocytes
lacked insulin stimulation of UCP-1 CAT activity; meanwhile,
re-expression of IRS-1wt in these cells resulted in a
recovery of insulin-induced UCP-1 CAT activity in accordance with the
level of reconstitution. As a positive control, 0.5 mM
dibutyryl cAMP stimulation caused a 4-fold increase in UCP-1 CAT
activity.

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Fig. 3.
Insulin-dependent UCP-1 and FAS
expression and transactivation of UCP-1 promoter is impaired in
IRS-1-deficient brown adipocytes. A, wild-type
(IRS-1+/+), IRS-1-deficient (IRS-1 / ), and
IRS-1-reconstituted (pCMVhisIRS-1wt) brown adipocytes (20 h
serum-starved) were cultured for an additional 24 h in
serum-free medium either in the absence or presence of insulin (10-100
nM). Serum-starved wild-type brown adipocytes were cultured
for an additional 24 h in serum-free medium either in the absence
or presence of insulin (10-100 nM) and ML-9 (50 µM). Then cells were harvested, and mitochondrial protein
extracts were analyzed by Western blot with anti-UCP-1 or
anti-cytochrome c antibodies. B, serum-starved
wild-type and IRS-1 / brown adipocytes were cultured for
24 h in serum-free medium either in the absence or presence of
insulin (10-100 nM). Mitochondrial protein extracts were
analyzed by Western blot with anti-UCP-2, anti-UCP-3, or
anti-cytochrome c antibodies. Representative experiments are
shown. C, wild-type, IRS-1 / , and
pCMVhisIRS-1wt brown adipocytes were transiently
transfected with 10 µg of 4551 full-length promoter UCP-1-CAT fusion
gene. Upon transfection, cells were cultured for 24 h in
serum-free medium either in the absence or in the presence of 100 nM insulin or 0.5 mM dibutyryl cAMP
(dbcAMP) and assayed for CAT activity. The relative CAT
activity normalized to -gal activity is represented in the
histogram. Results are the means ± S.E. from three independent
experiments. D, quiescent cells were cultured for 24 h
in serum-free medium either in the absence or presence of insulin
(10-100 nM). At the end of the culture time, total RNA (10 µg) was submitted to Northern blot analysis and hybridized with
labeled FAS and 18 S rRNA cDNAs. Representative autoradiograms are
shown.
|
|
To substantiate the role of IRS-1 in insulin-induced brown adipocyte
differentiation we also tested whether the lack of IRS-1 could affect
the expression of FAS, an enzyme involved in brown adipocyte lipid
synthesis. As shown in Fig. 3D, FAS mRNA expression was
induced in insulin-treated wild-type brown adipocytes; this effect was
precluded in IRS-1
/
cells and was further recovered in
IRS-1wt-reconstituted cells.
Insulin-induced C/EBP
Expression and Its DNA Binding
Activity in an IRS-1-dependent Manner--
It has been
reported that the 5'-flanking region of the UCP-1 gene contains
C/EBPs-regulated sites (37). In fact, previous data from our laboratory
indicated that the three canonical C/EBP isoforms (
,
, and
)
are expressed in brown adipose tissue during late fetal development,
with C/EBP
positively regulated by insulin (5, 14). Based on that
our next goal was to investigate whether IRS-1-mediated insulin
signaling could be involved in the nuclear expression of C/EBP
and
its DNA binding activity, which has also been described in the
promoters of a number of adipogenic genes (38, 39). First, we tested
the expression of C/EBP
in response to insulin in wild-type,
IRS-1
/
, and reconstituted IRS-1wt brown
adipocyte cell lines. As shown in Fig.
4A, insulin (10-100 nM) treatment for 24 h induced p42C/EBP
expression
in wild-type cells, but not in IRS-1-deficient cells; insulin-induced
p42C/EBP
expression was recovered in
IRS-1wt-reconstituted cells in parallel to the level of
protein reconstitution. In all the experiments similar loading of
protein extracts was assessed by Ponceau red staining of the membranes.
Next, electrophoretic mobility shift assays were conducted to determine
whether the insulin effect on C/EBP
protein levels would be
reflected in its binding to the cognate DNA recognition sites. We
isolated nuclear extracts from wild-type, IRS-1
/
, and
reconstituted IRS-1wt brown adipocyte cell lines, which had
been stimulated with insulin as described above. As shown in Fig.
4B, left panel, the gel shift band patterns
revealed the binding of nuclear proteins from wild-type brown
adipocytes to an oligonucleotide probe corresponding to the C/EBP
consensus site in an insulin-dependent manner, in parallel with C/EBP
protein expression. In supershift assays, incubation with
C/EBP
antibody, but not with a nonspecific antibody, totally eliminated the specific C/EBP
binding (Fig. 4B,
middle panel). However, the insulin effect on the C/EBP
binding activity was blunted in cells lacking IRS-1. This effect was
partially recovered after reconstitution with IRS-1wt (Fig.
4B, left panel). Finally, the requirement of
C/EBP
binding sites within the UCP-1 promoter for insulin-induced
transactivation was tested by using a mutant in which the proximal
promoter containing the C/EBP binding sites had been deleted
(
3628/
2283 linked to
141CAT (34)). As shown in Fig. 4B
(right panel), the transactivation of the UCP-1 promoter
induced by insulin in wild-type brown adipocytes was totally abolished
in the absence of C/EBP binding sites.

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Fig. 4.
Insulin-induced C/EBP
expression and its DNA binding activity in an
IRS-1-dependent manner. A, serum-starved
cells were cultured for a further 24 h in serum-free medium either
in the absence or presence of insulin (10-100 nM). Nuclear
protein extracts were analyzed by Western blot with the anti-C/EBP
antibody. A representative experiment is shown. The autoradiograms
corresponding to 4-5 independent experiments were analyzed by scanning
densitometry. Results are expressed as arbitrary units of C/EBP
protein content and are the means ± S.E. B, left
panel, serum-starved cells were stimulated with insulin (10-100
nM) for 24 h. Ten µg of nuclear extracts were
incubated for 20 min with 0.5 ng of 32P-labeled
doubled-stranded oligonucleotide used as C/EBP (see "Experimental
Procedures"). A competition assay using a 100-fold excess of
unlabeled oligonucleotide was performed to obtain nonspecific binding
activity (lane ). Then the mixture was electrophoresed
through a 6% (w/v) polyacrylamide gel. An autoradiogram of a
representative mobility shift assay of three is shown. Middle
panel, wild-type cells (IRS-1+/+) were stimulated with
insulin (Ins) for 24 h, and nuclear extracts (10 µg)
were incubated with 0.5 µg of anti-C/EPP or nonspecific antibodies
for 1 h at 4 °C and then for a further 20 min with 0.5 ng of
32P-labeled probe. An autoradiogram of a representative
supershift assay is shown. Right panel, wild-type brown
adipocytes were transiently transfected with 10 µg of 4551 full-length promoter UCP-1-CAT fusion gene or with 4551 UCP-1( 2283/ 141) CAT construct. Upon transfection, cells were
cultured for 24 h in serum-free medium either in the absence or in
the presence of 100 nM insulin and assayed for CAT
activity. The relative CAT activity normalized to -gal activity is
represented in the histogram. Results are means ± S.E.
from three independent experiments. Ab, antibody.
C/EBPdel, C/EBP deletion.
|
|
Retroviral Expression of C/EBP
or PPAR
Bypasses
Insulin Signaling in Inducing UCP-1--
The fact that C/EBP
and
PPAR
transcription factors have been implicated in adipocyte
differentiation by a number of laboratories prompted us to investigate
whether overexpression of these transcription factors is able to bypass
insulin signaling in inducing UCP-1 expression in the absence of IRS-1.
For this goal, we performed retrovirus-mediated C/EBP
or PPAR
gene transfer into IRS-1
/
brown adipocytes (Fig.
5A). After retroviral
infection, C/EBP
was overexpressed in the IRS-1
/
cell line about 10-fold, reaching higher levels than those observed in
primary brown adipocytes. On the other hand, PPAR
was overexpressed about 4-fold, reaching similar levels than those in primary cells. Both
C/EBP
- and PPAR
- overexpressing IRS-1
/
cells
up-regulated UCP-1 expression as compared with either wild-type or
IRS-1
/
brown adipocytes. To further analyze the insulin
effect on UCP-1 protein levels in C/EBP
- and
PPAR
-IRS-1
/
cells, we performed anti-UCP-1 Western
blot analysis after 24 h of insulin stimulation (Fig.
5B). In both serum-deprived C/EBP
- or
PPAR
-IRS-1
/
cells UCP-1 expression was up-regulated,
and insulin treatment did not further increase UCP-1 protein levels. In
addition, transient transfection of C/EBP
- or
PPAR
-IRS-1
/
cells with the full-length UCP-1
promoter fused to the CAT reporter gene revealed that both
transcription factors induced the transactivation of the UCP-1 promoter
in the absence of insulin; no further increase was observed by 24 h of insulin treatment (Fig. 5C). As an adipogenic marker,
we analyzed FAS mRNA levels in insulin-stimulated C/EBP
- and
PPAR
-IRS-1
/
cells. The Northern blot depicted in
Fig. 5D revealed that FAS mRNA was also up-regulated;
this effect was truly significant in
C/EBP
IRS-1
/
cells. However, insulin treatment
induced a slight further increase in FAS mRNA in
C/EBP
IRS-1
/
cells but not in
PPAR
IRS-1
/
cells.

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Fig. 5.
Differential recovery of UCP-1 and FAS
expression by overexpression of C/EBP and
PPAR transcription factors in IRS-1-deficient
brown adipocytes. A, IRS-1 / fetal brown
adipocyte cell lines overexpressing C/EBP or PPAR were generated
as described under "Experimental Procedures." Nuclear extracts were
prepared from growing cells (10% fetal serum) and analyzed by Western
blot with the anti-C/EBP and anti-PPAR antibodies. To analyze
UCP-1 expression, mitochondrial protein extracts were isolated and
analyzed by Western blot with the corresponding anti-UCP-1 antibody.
Results are representative of at least two independent experiments.
B, IRS-1 / brown adipocytes and
IRS-1 / cells overexpressing C/EBP or PPAR were
serum-starved for 20 h and further cultured for 24 h in the
absence or presence of insulin (10-100 nM). Mitochondrial
protein extracts were analyzed by Western blot with anti-UCP-1 or
anti-cytochrome c antibodies. C, cells were
transiently transfected with 10 µg of 4551 full promoter UCP-1-CAT
fusion gene. Upon transfection, cells were cultured for 24 h in
serum-free medium either in the absence or in the presence of 100 nM insulin and assayed for CAT activity. The relative CAT
activity normalized to -gal activity is represented in the
histograms. Results are the means ± S.E. from three independent
experiments. D, quiescent cells were cultured for 24 h
in serum-free medium either in the absence or presence of insulin
(10-100 nM). At the end of the culture time, total RNA (10 µg) was submitted to Northern blot analysis and hybridized with
labeled FAS and 18 S rRNA cDNAs. Representative autoradiograms are
shown.
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|
Reconstitution of IRS-1-deficient Brown Adipocytes with
IRS-1Phe-895 Mutant Restores IRS-1/Akt Signaling
and Insulin Effect on UCP-1 Expression--
We have recently shown
that IRS-1Phe-895 mutant, which does not bind Grb-2,
overexpressed in IRS-1-deficient brown adipocytes failed to activate
the Ras/mitogen-activated protein kinase signaling pathway and cell
proliferation in response to insulin (40). To test whether
reconstitution of IRS-1
/
brown adipocytes with
IRS-1Phe-895 mutant restores IRS-1/PI 3-kinase/Akt
signaling pathway, we transfected a pCMVhisIRS-1Phe-895
mutant in which the tyrosine in position 895 (which has been shown to
be responsible for Grb-2 association) was replaced by phenylalanine
(27), and histidinol-resistant cell lines were selected for further
experiments. Fig. 6A shows
that IRS-1wt and IRS-1Phe-895 expression in the
reconstituted cell lines represents about 50-60% that seen in
wild-type cells. In addition, we attempted to determine if exogenously
expressed IRS-1wt and IRS-1Phe-895 were
functional in activating PI 3-kinase/Akt signaling in response to
insulin stimulation. IRS-1Phe-895-reconstituted cells
induced IRS-1-associated PI-3 kinase activity and total Akt
phosphorylation; no differences were found with IRS-1wt
cells (Fig. 6B). Next we studied the effect of
IRS-1Phe-895 reconstitution on insulin-induced thermogenic
differentiation monitored by the expression of UCP-1. As shown in Fig.
6C, IRS-1Phe-895 brown adipocytes reconstituted
UCP-1 expression as IRS-1wt cells. Furthermore,
insulin-induced expression of C/EBP
was also recovered by
re-expression of IRS-1Phe-895. Finally,
IRS-1Phe-895 mutant also restored the transactivation of
the UCP-1 promoter in response to insulin, which had been abolished in
IRS-1-deficient cells (Fig. 6D). As a control of
insulin-induced adipogenic differentiation, we determined FAS mRNA
in insulin-stimulated IRS-1
/
brown adipocytes
reconstituted with IRS-1Phe-895. As shown in the
representative Northern blot (Fig. 6E), FAS mRNA levels
in response to insulin in IRS-1Phe-895 brown adipocytes
were similar of those observed in IRS-1wt cells.

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Fig. 6.
IRS-1Phe-895 mutant reconstitutes
PI-3 kinase/Akt signaling and insulin-induced UCP-1 and FAS
expression. A, IRS-1-deficient brown adipocytes (clone
4) were transfected with the pCMVhisIRS-1wt
(IRS-1wt) and pCMVhisIRS-1Phe-895
(IRS-1Phe-895) cDNA constructs. 24 h after
transfection, histidinol (10 mM) was added to select stable
transfectants, and the expression of IRS-1wt and the mutant
IRS-1Phe-895 was assessed by Western blot with the
anti-IRS-1 antibody. B, quiescent cells
(IRS-1+/+, IRS-1wt, and
IRS-1Phe-895 stable transfectants) were stimulated with
various doses of insulin for 5 min, and total protein (inositol
trisphosphate (PIP)) was either immunoprecipitated
(IP) with the anti-IRS-1 antibody followed by an in
vitro PI 3-kinase assay as described above or submitted to Western
blot analysis with anti-phospho-Akt (PAkt)and anti-Akt
antibodies. A representative experiment of three is shown.
C, quiescent cells were cultured for 24 h in the
absence or presence of insulin (10-100 nM). Mitochondrial
extracts were analyzed by Western blot with anti-UCP-1 and
anti-cytochrome c, and nuclear protein extracts were
analyzed by Western blot with anti-C/EBP . Representative
autoradiograms are shown. D, cells were transiently
transfected with 10 µg of 4551 full promoter UCP-1-CAT fusion gene.
Upon transfection, cells were cultured for 24 h in serum-free
medium either in the absence or in the presence of 100 nM
insulin. At the end of the culture period, cells were collected and
assayed for CAT activity. The relative CAT activity normalized to
-gal activity is represented in the histograms. Results are the
means ± S.E. from three independent experiments. E,
quiescent cells were cultured for 24 h in serum-free medium either
in the absence or presence of insulin (10-100 nM). At the
end of the culture time, total RNA (10 µg) was submitted to Northern
blot analysis and hybridized with labeled FAS and 18 S rRNA
cDNAs.
|
|
Binding of Tyrosines 608, 628, and 658 to p85 Is Sufficient to
Restore PI-3 Kinase/Akt Signaling and Insulin-induced UCP-1
and FAS Expression in Brown Adipocytes--
To substantiate the
contribution of PI 3-kinase/Akt signaling pathway to insulin-induced
FAS and UCP-1 expression in fetal brown adipocytes, we transfected the
pCMVhisIRS-1Tyr-608/Tyr-628/Tyr-658 mutant,
which fully restores PI 3-kinase activity in response to
insulin at a level comparable with that seen with wild-type IRS-1 (28).
Fig. 7A shows that
IRS-1wt and IRS-1Tyr-608/Tyr-628/Tyr-658
expression in the reconstituted cell lines represents about 50-60% that seen in wild-type cells. Indeed,
IRS-1Tyr-608/Tyr-628/Tyr-658-reconstituted
IRS-1-deficient brown adipocytes induced IRS-1-associated PI-3 kinase activity and Akt phosphorylation to the same extent as
IRS-1wt cells upon insulin stimulation (Fig.
7B). Furthermore, we studied the effect of
IRS-1Tyr-608/Tyr-628/Tyr-658 reconstitution in
insulin-induced brown adipocyte thermogenic and adipogenic
differentiation, monitored by the expression of UCP-1 (Fig.
7C) and FAS (Fig. 7D), respectively. The mutant
IRS-1Tyr-608/Tyr-628/Tyr-658 brown adipocytes reconstituted
UCP-1 protein expression as well as FAS mRNA expression at a level
comparable with that seen with IRS-1wt cells.

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Fig. 7.
Binding of tyrosines 608, 628, and 658 to p85
is sufficient to restore PI-3 kinase/Akt signaling and insulin-induced
UCP-1 and FAS expression in brown adipocytes. A,
IRS-1-deficient brown adipocytes (clone 4) were transfected with the
pCMVhisIRS-1wt (IRS-1wt) and
pCMVhisIRS-1Tyr-608/Tyr-628/Tyr-658 (IRS-13Y)
cDNA constructs. 24 h after transfection, histidinol (10 mM) was added to select stable transfectants, and the
expression of IRS-1wt and the mutant
IRS-1Tyr-608/Tyr-628/Tyr-658 was assessed by Western blot
analysis with the anti-IRS-1 antibody. B, quiescent cells
(IRS-1+/+ and IRS-1wt and
IRS-1Tyr-608/Tyr-628/Tyr-658 stable transfectants) were
stimulated with various doses of insulin for 5 min, and total protein
(inositol trisphosphate (PIP)) was either immunoprecipitated
(IP) with the anti-IRS-1 antibody followed by an in
vitro PI 3-kinase assay as described above or submitted to Western
blot analysis with anti-phospho-Akt (PAkt) and anti-Akt
antibodies. A representative experiment of three is shown.
C, cells were serum-starved for 20 h and further
cultured for 24 h in the absence or presence of insulin (10-100
nM). Mitochondrial protein extracts were analyzed by
Western blot with anti-UCP-1 or anti-cytochrome c
antibodies. D, quiescent cells were cultured for 24 h
in serum-free medium either in the absence or presence of insulin
(10-100 nM). At the end of the culture time, total RNA (10 µg) was submitted to Northern blot analysis and hybridized with
labeled FAS and 18 S rRNA cDNAs.
|
|
 |
DISCUSSION |
A report from our laboratory demonstrates that
differentiation of brown adipose tissue occurs during late fetal
development and could be monitored by the expression of a set of
adipogenic enzymes (FAS, glycerol 3-phosphate dehydrogenase (G3PD),
malic enzyme, and acetyl-CoA carboxylase (ACC), etc.) as well as
the thermogenic marker UCP-1 (5). Despite the fact that the
noradrenergic stimulus is not yet fully developed at this stage of
development (9), insulin and IGF-I have been identified as signals able to induce the expression of UCP-1 concurrent with the adipogenic genes
(4, 6), based on the fact that brown adipocytes display high number of
receptors and affinity for both molecules (6).
The first part of this paper (Figs. 1-3) is focused on the upstream
elements in the insulin-signaling cascade involved in insulin-induced UCP-1 expression. For this goal, we have used immortalized brown adipocytes derived from the fetuses of IRS-1-deficient mice as well as
from the wild type. These cells maintain the adipogenic and thermogenic
phenotype of brown adipocytes under growing conditions regardless of
the process of immortalization. However, IRS-1-deficient cells showed
lower cytosolic lipid content than wild-type cells when cultured under
serum-free conditions (26). IRS-1 has recently been described as an
essential signaling molecule acting through the PI 3-kinase pathway in
inducing the adipocyte differentiation in in vitro protocols
developed in mouse fibroblasts (41) and brown fat preadipocytes (42).
Accordingly, we explored this pathway in fetal brown adipocytes as a
possible molecular mechanism responsible of insulin-induced UCP-1
expression. Our previous work demonstrated a failure of insulin in
activating Akt Ser473 phosphorylation in
IRS-1
/
brown adipocytes despite the fact of IRS-2
overexpression and enhanced IRS-2-associated PI 3-kinase activity (26).
Here we show that in these cells insulin-induced IRS-1-mediated PI
3-kinase activation can be recovered by reconstitution of
IRS-1wt expression by retroviral gene transfer.
Consequently, total Akt Ser473 phosphorylation as well as
phosphorylation of
-Akt and
-Akt isoforms were recovered. We
further confirmed these results by measuring Akt enzymatic activity. Of
the three isoforms of Akt assessed in this study,
- and
-Akt, and
to a lesser extent,
-Akt, were induced by insulin in wild-type brown
adipocytes. The lack of IRS-1 blunted the phosphorylation of Akt
isoforms in response to insulin; this effect was recovered after
reconstitution with IRS-1wt. These results indicate that
activation of Akt isoforms (
-,
-, and -
) in brown adipocytes
depends on the IRS-1-mediated PI 3-kinase-signaling pathway because
IRS-2 was unable to compensate this response.
Immortalized wild-type brown adipocytes induced UCP-1 expression upon
insulin stimulation as previously shown in primary cells (15). However,
these cells are unable to respond to insulin in inducing the
transactivation of the UCP-1 gene and, subsequently, the expression of
the protein in the absence of IRS-1 or when Akt was inhibited by ML-9
compound. These results support the notion that insulin through the
IRS-1/PI 3-kinase/Akt signaling pathway might activate nuclear
proteins, as discussed below, which can bind to the UCP-1 promoter,
addressing the point of the underlying molecular mechanism involved in
the insulin effect on UCP-1 gene expression. However, insulin did not
regulate UCP-2 nor UCP-3, strengthening the point that only UCP-1, the
tissue-specific marker, is regulated by insulin in an
IRS-1-dependent manner.
The C/EBP and PPAR families of transcription factors have an important
role in the induction of fully differentiated brown adipocyte
phenotype. Regarding thermogenesis, C/EBP
has been shown to
transactivate the UCP-1 gene (37, 43). In fact, C/EBP
expression
increases in rat brown adipose tissue during late fetal development
concurrently with the expression of UCP-1 (5). In addition, both
C/EBP
and UCP-1 mRNAs are up-regulated by insulin in brown
adipocyte primary cultures in a similar fashion (14). Here we show that
in immortalized fetal brown adipocytes p42C/EBP
is up-regulated by
insulin in an IRS-1-dependent manner. Furthermore, insulin
induced DNA binding activity of the newly synthesized C/EBP
protein
in a similar fashion. Consequently, our results demonstrate the
requirement of C/EBP
binding sites within the UCP-1 promoter in
mediating the insulin effect via IRS-1/PI 3-kinase/Akt, indicating that
this signaling during development may be crucial for the expression and
DNA binding activity of transcription factors involved in the onset of
brown fat differentiation. Moreover, the UCP-1 enhancer also contains a
response element for PPAR
(44). In addition, its effect is strongly
increased by binding of the cold-inducible coactivator PGC-1 (PPAR
coactivator-1) through the ligand-dependent way (45, 46).
However, PPAR
expression in brown adipocytes is not regulated by
insulin (14). Fetal brown adipocytes lacking IRS-1 up-regulated UCP-1
expression and transactivated the UCP-1 promoter in the absence of
insulin when C/EBP
or PPAR
were overexpressed, reaching levels
even higher than those observed in wild-type cells. Insulin stimulation of C/EBP
- or PPAR
-IRS-1
/
cells failed to further
increase the transcriptional activity of the UCP-1 promoter and UCP-1
expression. These results indicate that the lack of insulin signaling
through IRS-1/Akt pathway can by bypassed by C/EBP
or PPAR
.
Importantly, FAS mRNA was also up-regulated in both C/EBP
and
PPAR
IRS-1
/
cell lines. However, the effect of
C/EBP
overexpression was more prominent on FAS expression than that
of PPAR
, suggesting that the effect of insulin on adipogenic markers
in fetal brown adipocytes is mainly mediated by the modulation of
C/EBP
. Moreover, insulin further increased FAS mRNA expression
in C/EBP
-IRS-1
/
cells but not in
PPAR
-IRS-1
/
cells. These data clearly indicate that
C/EBP
, but not PPAR
, reconstitutes insulin sensitivity in brown
adipocytes in an IRS-1-independent manner and are entirely consistent
with those previously described by El-Jack et al. (47)
regarding the role of C/EBP
in inducing insulin sensitivity in
glucose transport during the adipocytic differentiation of 3T3L1 fibroblasts.
We have recently shown that insulin stimulates mitogenesis in brown
adipocytes by a mechanism that exclusively involves
IRS-1Tyr895 through its binding to Grb-2 and, subsequently,
mitogen-activated protein kinase activation (40). In this study, we
demonstrated that the reconstitution of IRS-1
/
cells
with the IRS-1Phe-895 mutant, which lacked Grb-2 binding,
failed to activate mitogen-activated protein kinase and mitogenesis in
response to insulin. The results presented in the present paper
demonstrated that IRS-1-deficient brown adipocytes reconstituted with
IRS-1Phe-895 maintained intact PI 3-kinase/Akt signaling in
response to insulin. Consequently,
IRS-1Phe-895-reconstituted cells maintained insulin
sensitivity in inducing C/EBP
and UCP-1 expression and the
transactivation of UCP-1 promoter, regardless the expected lack of
mitogen-activated protein kinase activation. Moreover, FAS expression
was also unaltered in IRS-1Phe-895-reconstituted cells.
Despite the fact that mitogen-activated protein kinase has been
proposed as a negative effector of adipocyte differentiation (48), in
this study we have not found differences in the expression of brown
adipocyte adipogenic and thermogenic differentiation markers after
reconstitution of IRS-1-deficient brown adipocytes with either
IRS-1wt or IRS-1Phe-895. Indeed, Fasshauer
et al. (42) do not find an improvement in the
differentiation capacity of IRS-1
/
brown preadipocytes
upon inhibition of mitogen-activated protein kinase. Finally, in the
present study we have also evaluated the essential role of IRS-1/PI
3-kinase/Akt signaling by reconstituting IRS-1 null brown adipocytes
with the IRS-1Tyr-608/Tyr-628/Tyr-658 mutant, which
contains only three tyrosine residues in YXXM motifs (608, 628, and 658) among the 18 potential tyrosine phosphorylation sites.
This mutant fully restored PI 3-kinase and Akt activation by insulin in
fetal brown adipocytes, leading to a recovery of both UCP-1 and FAS
expression, virtually lost in IRS-1
/
cells. These data
indicate that tyrosines 608, 628, and 658 of IRS-1 are necessary and
sufficient for the induction of UCP-1 expression by insulin.
In conclusion, the current findings provide strong evidence that
insulin regulation of UCP-1 expression is mediated by IRS-1 through the
PI 3-kinase/Akt-signaling pathway in brown adipocytes and that UCP-2
and UCP-3 are not regulated by insulin. Three tyrosine motifs (608, 628, and 658) in the IRS-1 molecule have been identified as critical in
inducing UCP-1 gene expression. Overexpression of either C/EBP
,
whose expression is regulated by insulin in IRS-1-dependent
manner, or PPAR
bypasses IRS-1 signaling in inducing UCP-1 in the
absence of insulin. However, C/EBP
, but not PPAR
, reconstitutes
insulin sensitivity regarding adipogenic markers.