Insulin-induced Up-regulated Uncoupling Protein-1 Expression Is Mediated by Insulin Receptor Substrate 1 through the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway in Fetal Brown Adipocytes*

Angela M. ValverdeDagger , Mónica ArribasDagger , Cecilia MurDagger , Paloma NavarroDagger , Sebastián Pons§, Anne-Marie Cassard-Doulcier, C. Ronald Kahn§, and Manuel BenitoDagger ||

From the Dagger  Departamento de Bioquímica y Biología Molecular, Centro Mixto Consejo Superior de Investigaciones Científicas/Universidad Complutense de Madrid, Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain, § Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts 02215, and  Centre de Recherches sur l'Endocrinologie Moléculaire et le Dévelopment, CNRS, 92190 Meudon, France

Received for publication, September 12, 2002, and in revised form, January 7, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To investigate the role of insulin receptor substrate-1 (IRS-1) and its downstream signaling in insulin-induced thermogenic differentiation of brown adipocytes, we have reconstituted IRS-1-deficient fetal brown adipocytes (IRS-1-/-) with wild-type IRS-1 (IRS-1wt). The lack of IRS-1 resulted in the inability of insulin to induce IRS-1-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity and Akt phosphorylation in IRS-1-/- brown adipocytes. In addition, these cells showed an impairment in activating alpha -Akt, beta -Akt, and gamma -Akt isoforms upon insulin stimulation. Reconstitution of IRS-1-/- brown adipocytes with IRS-1wt restored the IRS-1/PI 3-kinase/Akt signaling pathway. Treatment of wild-type brown adipocytes with insulin for 24 h up-regulated uncoupling protein-1 (UCP-1) expression and transactivated the UCP-1 promoter; this effect was abolished in the absence of IRS-1 or in the presence of an Akt inhibitor and further recovered after IRS-1wt reconstitution. Neither UCP-2 nor UCP-3 was up-regulated by insulin in wild-type and IRS-1-deficient brown adipocytes. Insulin stimulated the expression of CCAAT/enhancer-binding protein alpha  (C/EBPalpha ) and its DNA binding activity in wild-type brown adipocytes but not in IRS-1-/- cells. However, insulin stimulation of both C/EBPalpha expression and binding activity was restored after IRS-1wt reconstitution of deficient cells. Retrovirus-mediated expression of C/EBPalpha and peroxisome proliferator-activated receptor gamma  in IRS-1-/- brown adipocytes up-regulated UCP-1 protein content and transactivated UCP-1 promoter regardless of insulin stimulation. Both C/EBPalpha and peroxisome proliferator-activated receptor gamma  reconstituted FAS mRNA expression, but only C/EBPalpha restored insulin sensitivity in the absence of IRS-1. Finally, reconstitution of IRS-1-/- brown adipocytes with the IRS-1 mutants IRS-1Phe-895, which lacks IRS-1/growth factor receptor binding protein 2 binding but not IRS-1/p85-PI 3-kinase binding, or with IRS-1Tyr-608/Tyr-628/Tyr-658, which only binds p85-PI 3-kinase, induced UCP-1 expression and transactivated the UCP-1 promoter. These data provide strong evidence for an essential role of IRS-1 through the PI 3-kinase/Akt signaling pathway inducing UCP-1 gene expression by insulin.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha  (C/EBPalpha ) expression and C/EBPalpha 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/EBPalpha or PPARgamma 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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-alpha -Akt (06-558), anti-beta -Akt (06-606), anti-gamma -Akt (06-607) polyclonal antibodies were purchased from Upstate Biotechnology (Lake Placid, NY). The anti-Tyr(P) (Py20) (sc-508), anti-C/EBPalpha (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-PPARgamma (SA-206) antibody was from BioMol (Biomol Research Laboratories, Plymouth Meeting, PA). [gamma -32P]ATP (3000 Ci/mmol) and [alpha -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/EBPalpha and pBabe/Zeo PPARgamma 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 beta -glycerol phosphate, 5 mM sodium pyrophosphate, 1 mM Na3VO4, and 0.1% beta -mercaptoethanol and subjected to immunoprecipitation with the anti-alpha -Akt, anti-beta -Akt, or anti-gamma -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% beta -mercaptoethanol, and once with Buffer ADB containing 20 mM MOPS, pH 7.2, 25 mM sodium beta -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 [gamma -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 [alpha -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/EBPalpha 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 [alpha -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/EBPalpha 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 beta -galactosidase (gal) (a viral promoter driving expression of the reporter gene beta -gal). Ten micrograms of DNA-CAT together with 2 µg of DNA-beta -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 beta -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 beta -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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

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 alpha -Akt and beta -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 alpha -Akt and beta -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 alpha -Akt, beta -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-alpha -Akt, beta -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, alpha -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 alpha -Akt by insulin was totally blunted in IRS-1-/- cells, whereas reconstituted IRS-1wt cells recovered the level of insulin-induced alpha -Akt activity observed in wild-type cells. A modest increase in beta -Akt activity was observed in wild-type cells upon 100 nM insulin stimulation; no effect on beta -Akt activity was detected in IRS-1-/- cells. Reconstituted IRS-1wt cells recovered the level of insulin-induced beta -Akt activity in parallel to the level of IRS-1wt protein expressed (see Fig. 1A). Insulin increased gamma -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 gamma -Akt isoform in IRS-1-/- brown adipocytes, whereas IRS-1wt-reconstituted cells recovered insulin response in activating gamma -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-alpha -Akt, and anti-beta -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-alpha -Akt, anti-beta -Akt, and anti-gamma -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.

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 beta -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/EBPalpha 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 (alpha , beta , and delta ) are expressed in brown adipose tissue during late fetal development, with C/EBPalpha 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/EBPalpha 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/EBPalpha 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/EBPalpha expression in wild-type cells, but not in IRS-1-deficient cells; insulin-induced p42C/EBPalpha 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/EBPalpha 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/EBPalpha protein expression. In supershift assays, incubation with C/EBPalpha antibody, but not with a nonspecific antibody, totally eliminated the specific C/EBPalpha 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/EBPalpha 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/EBPalpha 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/EBPalpha 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/EBPalpha 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/EBPalpha (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/EPPalpha 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 beta -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/EBPalpha or PPARgamma Bypasses Insulin Signaling in Inducing UCP-1-- The fact that C/EBPalpha and PPARgamma 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/EBPalpha or PPARgamma gene transfer into IRS-1-/- brown adipocytes (Fig. 5A). After retroviral infection, C/EBPalpha 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, PPARgamma was overexpressed about 4-fold, reaching similar levels than those in primary cells. Both C/EBPalpha - and PPARgamma - 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/EBPalpha - and PPARgamma -IRS-1-/- cells, we performed anti-UCP-1 Western blot analysis after 24 h of insulin stimulation (Fig. 5B). In both serum-deprived C/EBPalpha - or PPARgamma -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/EBPalpha - or PPARgamma -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/EBPalpha - and PPARgamma -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/EBPalpha -IRS-1-/-cells. However, insulin treatment induced a slight further increase in FAS mRNA in C/EBPalpha -IRS-1-/-cells but not in PPARgamma -IRS-1-/-cells.


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Fig. 5.   Differential recovery of UCP-1 and FAS expression by overexpression of C/EBPalpha and PPARgamma transcription factors in IRS-1-deficient brown adipocytes. A, IRS-1-/- fetal brown adipocyte cell lines overexpressing C/EBPalpha or PPARgamma 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/EBPalpha and anti-PPARgamma 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/EBPalpha or PPARgamma 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 beta -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.

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/EBPalpha 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/EBPalpha . 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 beta -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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -Akt and beta -Akt isoforms were recovered. We further confirmed these results by measuring Akt enzymatic activity. Of the three isoforms of Akt assessed in this study, alpha - and gamma -Akt, and to a lesser extent, beta -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 (alpha -, beta -, and -gamma ) 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/EBPalpha has been shown to transactivate the UCP-1 gene (37, 43). In fact, C/EBPalpha expression increases in rat brown adipose tissue during late fetal development concurrently with the expression of UCP-1 (5). In addition, both C/EBPalpha 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/EBPalpha is up-regulated by insulin in an IRS-1-dependent manner. Furthermore, insulin induced DNA binding activity of the newly synthesized C/EBPalpha protein in a similar fashion. Consequently, our results demonstrate the requirement of C/EBPalpha 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 PPARgamma (44). In addition, its effect is strongly increased by binding of the cold-inducible coactivator PGC-1 (PPARgamma coactivator-1) through the ligand-dependent way (45, 46). However, PPARgamma 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/EBPalpha or PPARgamma were overexpressed, reaching levels even higher than those observed in wild-type cells. Insulin stimulation of C/EBPalpha - or PPARgamma -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/EBPalpha or PPARgamma . Importantly, FAS mRNA was also up-regulated in both C/EBPalpha and PPARgamma IRS-1-/- cell lines. However, the effect of C/EBPalpha overexpression was more prominent on FAS expression than that of PPARgamma , suggesting that the effect of insulin on adipogenic markers in fetal brown adipocytes is mainly mediated by the modulation of C/EBPalpha . Moreover, insulin further increased FAS mRNA expression in C/EBPalpha -IRS-1-/- cells but not in PPARgamma -IRS-1-/- cells. These data clearly indicate that C/EBPalpha , but not PPARgamma , 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/EBPalpha 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/EBPalpha 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/EBPalpha , whose expression is regulated by insulin in IRS-1-dependent manner, or PPARgamma bypasses IRS-1 signaling in inducing UCP-1 in the absence of insulin. However, C/EBPalpha , but not PPARgamma , reconstitutes insulin sensitivity regarding adipogenic markers.

    ACKNOWLEDGEMENTS

We thankfully acknowledge Morris F. White (Joslin Diabetes Center, Boston, MA) for the pCMVhisIRS-1wt, pCMVhisIRS-1Tyr-608/Tyr-628/Tyr-658, and pCMVhisIRS-1Phe-895 constructs, Bruce S. Spiegelman (Dana Farber Cancer Institute, Boston, MA) for the pBabe hygro C/EBPalpha and pBabe bleo PPARgamma retrovitral constructs, D. Ricquier (CNRS, Meudon, France) for the UCP-1 CAT 4551 full-length promoter, F. Villarroya (Universidad de Barcelona, Spain) for advice with the UCP-1 CAT constructs, and E. Rial (Consejo Superior de Investigaciones Científicas, Spain) for advice with the cellular oxygen consumption experiments.

    FOOTNOTES

* This work was supported by Ministerio de Educación y Cultura, Spain Grants PM 97-0050 and PM 98-0087.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed. Tel.: 34-91-3941777; Fax: 34-91-3941779; E- mail: benito{at}farm.ucm.es.

Published, JBC Papers in Press, January 13, 2003, DOI 10.1074/jbc.M209363200

    ABBREVIATIONS

The abbreviations used are: UCP-1, uncoupling protein-1; IRS-1, insulin receptor substrate-1; PI 3-kinase, phosphatidylinositol 3-kinase; FAS, fatty acid synthase; Grb-2, growth factor receptor binding protein 2; C/EBPalpha , enhancer-binding protein alpha ; wt, wild type; PPAR, peroxisome proliferator-activated receptor; MOPS, 4-morpholinepropanesulfonic acid; CAT, chloramphenicol acetyltransferase; beta -gal, beta -galactosidase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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