Essential Role of Insulin-Like Growth Factor I Receptor in Insulin-Induced Fetal Brown Adipocyte Differentiation
Abstract{k, http://www.100md.com
To define the specific role of IGF-I receptor (IGF-IR) in adipogenic and thermogenic differentiation of brown adipocytes during late fetal life, we have established immortalized brown adipocyte cell lines from fetuses of IGF-IR-deficient mice (IGF-IR-/-) as well as from wild-type mice (IGF-IR+/+). IGF-IR-/- cells showed an increased insulin sensitivity regarding insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation despite a substantial reduction in IRS-1 protein content. Furthermore, insulin-induced total and IRS-1-associated phosphatidylinositol 3-kinase activities were augmented in IGF-IR-deficient cells compared with wild-type cells. Downstream phosphatidylinositol 3-kinase activation of Akt, but not p70s6 kinase, were elicited at lower doses of insulin in IGF-IR-/- brown adipocytes. Activation of protein kinase C{zeta} by insulin was similar in both cell types as was insulin-induced glucose uptake. Treatment of wild-type brown adipocytes with insulin for 12 h up-regulated fatty acid synthase (FAS) and adipocyte determination and differentiation (ADD1/SREBP) mRNAs; this effect was impaired in the absence of IGF-IR. At the protein level, insulin increased FAS content and the amount of the mature form of adipocyte determination and differentiation (ADD1/SREBP) in the nucleus in wild-type cells, but not in IGF-IR-/- cells. Furthermore, 24 h of insulin stimulation induced the expression of both uncoupling protein-1 and CCAAT/enhancer-binding protein (C/EBP) in wild-type brown adipocytes; these effects were abolished in IGF-I-R-/- cells. Retrovirus-mediated reexpression of peroxisomal proliferator-activated receptor (PPAR) in IGF-IR-/- brown adipocytes could overcome FAS mRNA impairment, bypassing insulin signaling. However, insulin further increased FAS mRNA expression in C/EBP{alpha} -IGF-IR-/- cells, but not in PPAR-IGF-IR-/- cells. In addition, fetal brown adipocytes lacking IGF-IR up-regulated uncoupling protein-1 expression in the absence of insulin when PPAR, but not C/EBP{alpha} , was overexpressed. These data provide strong evidence for a critical role of IGF-IR in he differentiation of the brown adipocyte phenotype in fetal life; this effect is mimicked by PPAR{gamma} in an insulin-independent manner.
Introductionq5, http://www.100md.com
ADIPOCYTE differentiation is a complex process that requires communication between extracellular stimuli in a coordinated network of receptors and transcription factors in the nucleus. Although most studies of adipogenesis have focused on the development of white adipose tissue, which is the primary site of storage of triglycerides and release of fatty acids in response to changing energy needs (1), mammals have a second terminally differentiated adipose cell type that composes brown adipose tissue (BAT). BAT is a major site for nonshivering thermogenesis in mammals. The unique thermogenic capacity of BAT results from the expression of uncoupling protein-1 (UCP-1) in the mitochondrial inner membrane, which is required to address physiological hypothermia in newborn mammals (2). By uncoupling the production of ATP from the movement of protons down their concentration gradient, heat is generated (3). In addition, BAT is a major site for lipid metabolism, as fatty acids are the main fuel that maintains the thermogenic capacity of the tissue (for review, see Ref. 4). 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, and a thermogenic program related to UCP-1 expression and heat production (5, 6, 7). During the last years our laboratory has been investigating which hormones/growth factors are the main signals involved in the onset of adipogenic and thermogenic differentiation of BAT during late fetal development. In previous work we demonstrated that fetal brown adipocytes display binding sites of high affinity for both insulin and IGF-I (5, 7). Both molecules are involved in fetal brown adipogenesis through their ability to induce the genetic expression of metabolic genes (5, 6, 8), and they might have a role in thermogenesis by inducing UCP-1 expression (9, 10), as the noradrenergic stimulus, induced by hypothermia after birth, it is not yet fully developed in BAT at birth (6, 11).
The upstream signals regulating the induction and expression of transcription factors involved in activating metabolic genes during brown adipocyte differentiation are poorly understood. Insulin/IGF-I action is mediated by the insulin receptor (IR) and the IGF-I receptor (IGF-IR), respectively, which have very similar heterodimeric {alpha} 2ß2 structure and belong to the family of receptor tyrosine kinases (12). Upon ligand binding, insulin/IGF-I receptor undergoes autophosphorylation on tyrosine residues, which activates the intracellular tyrosine kinase of the ß-subunit (13). This, in turn, stimulates the phosphorylation of cytosolic proteins, including the insulin receptor substrate (IRS) family (IRS-1, -2, -3, and -4) and SHC (14, 15, 16, 17, 18). Then, these phosphorylated IRS bind proteins containing Src homology 2 domains such as the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase) (19); growth factor receptor-binding protein-2 (Grb-2), which links signaling via SOS to activation of the Ras complex (20); and protein tyrosine phosphatase SHP-2 (21) that lead to activation of various downstream signaling pathways. Recent studies suggest that the Ras/MAPK pathway mediates fetal brown adipocyte proliferation (22), whereas the PI 3-kinase pathway is activated during differentiation (9, 10).
The purpose of the present paper is to further study the role of IGF-IR and its signaling in the onset of BAT differentiation. For this goal, we have recently generated immortalized cell lines derived from fetal BAT of IGF-IR-deficient and wild-type mice. These cells showed an increased insulin sensitivity of the MAPK signaling pathway and mitogenesis, as a result of a compensatory mechanism (23). Now, we found that IR and its signaling through IRS-1/PI 3-kinase are unable to compensate for the lack of IGF-IR during fetal development regarding brown adipocyte adipogenic- and thermogenic-related gene expression. However, we can bypass insulin signaling and up-regulate both fatty acid synthase (FAS) and UCP-1 expression by overexpression of peroxisomal proliferator-activated receptor (PPAR) transcription factor, but not CCAAT/enhancer-binding protein {alpha} (C/EBP), in IGF-IR-deficient brown adipocytes. These results demonstrate the essential role played by IGF-IR in initiating and/or maintaining the differentiation of brown adipocytes during late fetal life.
Materials and Methods^!\?, http://www.100md.com
Materials^!\?, http://www.100md.com
Fetal calf serum (FS) and culture media were obtained from Life Technologies, Inc. (Gaithersburg, MD). Insulin, hygromycin, and antimouse IgG-agarose were obtained from Sigma-Aldrich (St. Louis, MO). Protein A-agarose was purchased from Roche Molecular Biochemicals (Mannheim, Germany). The bleomycin analog zeozin was purchased from Invitrogen (Carlsbad, CA) The anti-IRS-1 (06-248), anti-Tyr(P) (clone 4G10; 05-321), anti-Akt (06-558), and anti-Akt ß (06-606) antibodies were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). The anti-TyrP (Py20; sc-508), anti-C/EBP{alpha} (sc-61), antiadipocyte determination and differentiation (anti-ADD1/SREBP-1c; sc-8984, raised against the NH2 terminus of the molecule), and anti-protein kinase C (anti-PKC; c-20; sc-216) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The anti-phospho-Akt (Ser473 9271), anti-phospho-p70s6k (Ser424/Thr421 9204S), anti-Akt (9272), anti-p70s6k (9202), and anti-phospho-PKC (Thr410 9378s) antibodies were purchased from Cell Signaling (Beverly, MA). The anti-UCP-1 (AB3038) and anti-GLUT-4 (AB1346) antibodies were obtained from Chemicon (Temecula, CA). The anti PPAR (SA-206) antibody was purchased from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). [-32P]ATP (3000 Ci/mmol), [{alpha} 32P]deoxy-CTP, and 2-deoxy-D-[1-3H]glucose (11.0 Ci/mmol) were obtained from Amersham International (Little Chalfont, UK). All other reagents used were of the purest grade available.
Cell culture and retroviral infections|]?r), http://www.100md.com
Brown adipocytes were obtained from interscapular BAT of 17.5–18.5 fetuses from 2 or 3 pregnant mice (MF1 and C57BL/6J mouse strains) of normal genotype (IGF-IR+/+) or from a pool of tissue of fetuses with body weight less than 0.7 g (IGF-IR-/-) from 2 or 3 pregnant mice IGF-IR+/- mated with IGF-IR+/- males (MF1 and C57BL/6J mouse strains) (24). Brown adipocyte primary cultures were performed as previously described (5). 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 simian virus 40 large T antigen (provided by J. de Caprio, Dana Farber Cancer Institute, Boston, MA). Then, brown adipocytes (wild-type and IGF-IR-/-) 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 wk. These pools of stable cells were further cultured in DMEM supplemented with 10% FS and puromycin. The pBabe/hygro C/EBP{alpha} and pBabe/zeozin PPAR{gamma} viral expression vectors were generous gifts from Dr. B. Spiegelman (Dana Farber Cancer Institute). Brown adipocyte IGF-IR-deficient cells were infected with those vectors described above. Selection with 200 µg/ml hygromycin or 250 µg/ml zeozin was started 48 h after infection to select stable cell lines.
Immunoprecipitations-yalmkp, http://www.100md.com
Quiescent cells 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 phenylmethylsulfonylfluoride, pH 7.6 (lysis buffer). Lysates were clarified by centrifugation at 15,000 x 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 antimouse IgG-agarose beads. Immunoprecipitates were washed with lysis buffer, were extracted for 5 min at 95 C in 2x SDS-PAGE sample buffer [200 mM Tris-HCl, 6% sodium dodecyl sulfate (SDS), 2 mM EDTA, 4% 2-mercaptoethanol, and 10% glycerol, pH 6.8], and analyzed by SDS-PAGE.-yalmkp, http://www.100md.com
Western blotting-yalmkp, http://www.100md.com
After SDS-PAGE, proteins were transferred to Immobilon membranes, blocked using 5% nonfat dried milk or 3% BSA in 10 mM Tris-HCl and 150 mM NaCl (pH 7.5), and incubated overnight with several antibodies, as indicated, in 0.05% Tween-20, 10 mM Tris-HCl, and 150 mM NaCl (pH 7.5). Immunoreactive bands were visualized using the enhanced chemiluminescence Western blotting protocol (Amersham International).
PI 3-kinase activity*, 百拇医药
PI 3-kinase activity was measured in the anti-IRS-1 or anti-Tyr(P) immunoprecipitates by in vitro phosphorylation of PI as previously described (9).*, 百拇医药
Akt activity*, 百拇医药
Akt activity was measured in vitro using a nonradioactive technique. Quiescent cells were treated for 5 min with or without several doses of insulin and were subsequently lysed in the lysis buffer described above. One milligram of each cell lysate was subjected to immunoprecipitation with anti-Akt{alpha} or anti-Aktß antibodies. The resulting immune complexes were washed in lysis buffer twice and once in kinase buffer containing 25 mM Tris-HCl (pH 7.5), 5 mM ß-glycerol-phosphate, 10 mM MgCl2, 2 mM dithiothreitol, and 100 µM sodium orthovanadate. The activity of Akt in the immunoprecipitates was determined in the presence of a synthetic peptide corresponding to the Akt phosphorylation sites of GSK-3{alpha} ß (Ser9/Ser21) fused to paramyosin and 200 µM ATP for 30 min at 30 C. The reaction products were eluted with Laemmli buffer for 5 min at 100 C, resolved on 10% SDS-PAGE gels, transferred to nylon membranes, and visualized by immunoblotting with antiphospho-GSK-3{alpha} ß (Ser9/Ser21) antibody.
PKC activitykn, http://www.100md.com
Quiescent cells were treated for 10 min without or with several doses of insulin and subsequently lysed. PKC{zeta} activity was measured in anti-PKC immunoprecipitates as previously described (9).kn, http://www.100md.com
Extraction of nuclear proteinskn, http://www.100md.com
Cells were resuspended at 4 C in 10 mM HEPES-KOH (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 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 sec. 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 DTT, 0.2 mM PMSF, 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.
Isolation of mitochondrial protein:, 百拇医药
At the end of the culture, cells were scrapped off in isotonic isolation buffer (1 mM EDTA, 10 mM HEPES, and 250 mM sucrose, pH 7.6), collected by centrifugation at 2500 x g for 5 min at 4 C, and resuspended in hypotonic isolation buffer (1 mM EDTA, 10 mM HEPES, and 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, and 450 mM sucrose, pH 7.6) was added to balance the buffer’s tonicity. Samples were centrifuged at 10,000 x g for 10 min, the pellets, containing the mitochondrial fraction, were resuspended in isotonic isolation buffer, and mitochondrial protein content was determined.:, 百拇医药
Protein determination:, 百拇医药
Protein determination was performed by the Bradford dye method (25), using the Bio-Rad Laboratories, Inc., reagent and BSA as the standard.
RNA extraction and Northern blot analysis$c, http://www.100md.com
At the end of the culture, cells were washed twice in ice-cold PBS, and RNA was isolated as previously described (26). 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 (NEN Life Science Products, Boston, MA), and cross-linked to the membranes by UV light. Hybridization was performed in 0.25 mM NaHPO4 (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]deoxy-CTP by using a multiprimer DNA labeling system. Blots were hybridized with probes for FAS (27) and ADD1/SREBP-1c (28) and with 18S ribosomal probe to normalize RNA loading. Membranes were subjected to autoradiography, and the relative densities of the hybridization signals were determined by densitometric scanning of the autoradiograms.
Glucose uptake8p9.q:, 百拇医药
2-Deoxyglucose transport was measured as previously described (29). After culture, quiescent brown adipocytes were washed three times with Krebs-Ringer phosphate buffer (KRP) containing 136 mM NaCl, 4.7 mM KCl, 1 mM CaCl2, 1 mM MgSO4, 5 mM sodium pyrophosphate, 20 mM HEPES, and 1% BSA and then were incubated with 1 ml KRP in the presence or absence of insulin for 20 min at 37 C. 2-Deoxyglucose-D-[1-3H] was added to this solution to a final concentration of 0.1 mM (250 nCi/ml), and the incubation was continued for 10 min at 37 C. The cells were then washed three times with ice-cold KRP buffer and solubilized in 1% SDS. The radioactivity of a 200-µl aliquot was determined in a scintillation counter.8p9.q:, 百拇医药
Results8p9.q:, 百拇医药
IRS-1 protein content and its tyrosine phosphorylation in insulin-stimulated wild-type and IGF-IR-deficient brown adipocytes8p9.q:, 百拇医药
We have recently shown an enhanced insulin-induced tyrosine phosphorylation of the IR ß-chain in immortalized brown adipocytes derived from fetuses of IGF-IR-deficient mice compared with wild-type cells. In addition, in previous experiments we found a significant reduction of IRS-1 protein content in IGF-IR-/- brown adipocytes despite a marked increase in its tyrosine phosphorylation upon 10 nM insulin stimulation (23). In the dose-response experiment depicted in Fig. 1 we show that IRS-1 tyrosine phosphorylation in IGF-IR-/- brown adipocytes could be detected at a 0.01-nM insulin concentration, with the maximal effect being elicited at 0.1 nM. In wild-type cells, IRS-1 tyrosine phosphorylation was barely detected at 0.01 nM insulin, with the maximal effect elicited at 10 nM. Considering that IRS-1 protein content is severely reduced in IGF-IR-/- brown adipocytes, its tyrosine phosphorylation in these cells is about 7-fold higher than that of wild-type cells (Fig. 1, lower panel). Conversely, neither IRS-2 protein content nor its tyrosine phosphorylation significantly differ between the cell types (results not shown).
fig.ommitteedl, http://www.100md.com
Figure 1. Insulin effect on IRS-1 tyrosine phosphorylation in wild-type and IGF-IR-deficient brown adipocytes. Wild-type (IGF-IR+/+) and IGF-I-deficient brown adipocytes (IGF-IR-/-) were cultured to confluence under growing conditions (10% FS) and then serum-starved for 20 h. Quiescent cells were stimulated with various doses of insulin (0.01–10 nM) for 5 min, and control cells were cultured in the absence of the hormone. At the end of the culture, cells were lysed, and 600 µg total protein were immunoprecipitated with anti-IRS-1 antibody. The resulting immune complexes were analyzed by immunoblotting with anti-Tyr(P) or anti-IRS-1 antibodies. The autoradiograms shown are representative of three experiments. Results are expressed as arbitrary units or IRS-1 tyrosine phosphorylation (normalized to the amount of total IRS-1) and are the mean ± SE.l, http://www.100md.com
Insulin-induced IRS-1- and Tyr(P)-associated PI 3-kinase activity in wild-type and IGF-IR-deficient brown adipocytesl, http://www.100md.com
Our next purpose was to study whether the enhanced insulin sensitivity of IGF-IR-deficient brown adipocytes emerging from the IR signals through the IRS-1/PI 3-kinase pathway. For this goal, serum-starved cells were stimulated with various doses of insulin (0.01–10 nM) for 5 min, and PI 3-kinase activity was measured in both anti-IRS-1 and anti-Tyr(P) immunoprecipitates. As shown in Fig. 2 (left panel), IRS-1-associated PI 3-kinase activity was increased by insulin in wild-type brown adipocytes in a dose-dependent manner; the maximal effect was elicited at 10 nM insulin. In contrast, IGF-IR-/- brown adipocytes showed a significant activation of IRS-1-associated PI 3-kinase at a low insulin dose (0.01 nM); the insulin response curve showed the maximal effect at a 0.1-nM concentration. Again, because of the dramatic reduction of IRS-1 protein content in IGF-IR-deficient cells, IRS-1-associated PI 3-kinase activity was about 6-fold higher than that of wild-type cells. In contrast, IRS-2-associated PI 3-kinase activity does not differ significantly among the cell types (data not shown). Thus, total PI 3-kinase associated with anti-Tyr(P) immunoprecipitates was increased in IGF-IR-/- brown adipocytes (Fig. 2, right panel); this effect was truly significant at low doses of insulin (0.01–1 nM).
fig.ommitteed9ng@x, 百拇医药
Figure 2. Insulin effect on IRS-1- and Tyr(P)-associated PI 3-kinsase activity in wild-type and IGF-IR-deficient brown adipocytes. IGF-IR+/+ and IGF-IR-/- brown adipocytes were cultured to confluence under growing conditions (10% FS) and then serum-starved for 20 h. Quiescent cells were stimulated with insulin (0.01–10 nM) for 5 min or were cultured in the absence of the hormone. At the end of the culture, cells were lysed, and 600 µg total protein were immunoprecipitated with anti-IRS-1 (left) or anti-Tyr(P) (right) antibodies. The resulting immune complexes were washed and immediately used for an in vitro PI 3-kinase assay. The conversion of phosphatidylinositol to phosphatidylinositol phosphate in the presence of [{gamma} -32P]ATP was analyzed by TLC. Representative autoradiograms are shown. The autoradiograms corresponding to three independent experiments were quantitated by scanning densitometry. Results are expressed as arbitrary units of PI 3-kinase (normalized to the amount of total IRS-1 protein content in the left panel) and are the mean ± SE.9ng@x, 百拇医药
Akt and p70s6k phosphorylation in wild-type and IGF-IR-/- brown adipocytes($?du.%, 百拇医药
As the serine/threonine kinase Akt is one of the major downstream targets of PI 3-kinase, and it has been implicated in adipogenic differentiation (30, 31, 32), we explored its phosphorylation by direct Western blot analysis in wild-type and IGF-IR-/- brown adipocytes. Insulin stimulation of both cell types resulted in AktSer473 phosphorylation without changes in the total Akt protein content (Fig. 3, upper panel). In wild-type cells the maximal effect was elicited at 100 nM insulin, whereas the dose-response curve of IGF-IR-deficient brown adipocytes was shifted to the left (maximal effect was elicited at 1 nM insulin), indicating increased insulin sensitivity in these cells. To confirm the results obtained from the phospho-Akt analysis, Akt kinase assays were performed in insulin-stimulated IGF-IR+/+ and IGF-IR-/- fetal brown adipocytes after immunoprecipitation with the specific antibodies for Akt{alpha} and Aktß isoforms. In wild-type brown adipocytes (Fig. 3, lower panel) {alpha} -Akt kinase activity was enhanced by insulin in a dose-dependent manner, reaching the maximal value (2- to 3-fold increase) at 10 nM insulin. Under the same experimental conditions {alpha} Akt was activated by 10-fold in IGF-IR-deficient cells; no differences were observed in ßAkt activity between the cell types. In contrast, phosphorylation of p70s6 kinase, which has also been involved in brown adipocyte differentiation (9), did not show differences between the cell types (Fig. 4).
fig.ommitteed-|, 百拇医药
Figure 3. Activation of Akt in wild-type and IGF-IR-deficient brown adipocytes. Upper panel, Quiescent (20-h serum-starved) wild-type (IGF-IR+/+) and IGF-IR-deficient (IGF-IR-/-) brown adipocytes were stimulated with insulin (0.01–100 nM) for 5 min or were maintained in the absence of the hormone. Cells were lysed, and total protein (50 µg) was submitted to SDS-PAGE and analyzed by immunoblotting with the corresponding antibodies against phospho-Akt (Ser473) and total Akt. The autoradiograms corresponding to four independent experiments were quantitated by scanning densitometry. Results are expressed as arbitrary units of Akt phosphorylation (normalized to the amount of total Akt) and are the mean ± SE. Lower panel, Quiescent cells were stimulated with various doses of insulin for 5 min. Control cells were incubated in the absence of the hormone. Cells were lysed, and equals amount of protein (600–800 µg) were immunoprecipitated with the anti-{alpha} Akt or anti-ßAkt antibodies and immediately used for an in vitro kinase assay as described in Materials and Methods. Quantitative data are expressed as the fold increase in Akt stimulation and are the mean ± SE from three or four independent experiments.
fig.ommitteed4;t%%#(, http://www.100md.com
Figure 4. Insulin effect on p70s6 kinase phosphorylation in wild-type and IGF-IR-deficient brown adipocytes. Quiescent (20-h serum-starved) wild-type (IGF-IR+/+) and IGF-IR-deficient (IGF-IR-/-) brown adipocytes were stimulated with insulin (0.01–10 nM) for 5 min or were maintained in the absence of the hormone. Cells were lysed, and total protein (50 µg) was submitted to SDS-PAGE and analyzed by immunoblotting with the corresponding antibodies against phospho-p70s6k and total p70s6k. The autoradiograms corresponding to three independent experiments were quantitated by scanning densitometry. Results are expressed as arbitrary units of p70s6k phosphorylation (normalized to the amount of total p70s6k) and are the mean ± SE.4;t%%#(, http://www.100md.com
The lack of IGF-IR in brown adipocytes did not affect insulin-induced glucose uptake4;t%%#(, http://www.100md.com
It is well known that activation of PI 3-kinase and its downstream targets is required for the movement of glucose transporters to the cell membrane in BAT (33). In fetal brown adipocytes glucose transport increased significantly upn stimulation with physiological doses of insulin/IGF-I, and this effect was dependent on the PI 3-kinase/PKC signaling pathway (9, 34). Accordingly, we investigated whether the enhanced PI 3-kinase activity observed in IGF-IR-/- brown adipocytes could mediate the insulin effect on glucose uptake. For this goal, brown adipocytes were stimulated for 10 min with various doses of insulin (1–100 nM) and then were incubated for an additional 10 min in the presence of 2-deoxy-D-[1-3H]glucose. As shown in Fig. 5A (left panel), there was no significant difference in basal glucose transport activity in cells from both genotypes. Insulin stimulation resulted in a 2-fold increase in glucose transport in wild-type and IGF-IR-/- brown adipocytes; in both cases the maximal effect was elicited at 10 nM insulin. In addition, the GLUT4 protein content was similar in the two cell types (Fig. 5A, right panel). To further confirm these data we measured PKC activity, which has been shown to mediate insulin-induced glucose uptake in brown adipocytes (34), in both cell types upon insulin stimulation. Quiescent cells were incubated with insulin (10–100 nM) for 10 min, and PKC activity was determined by an in vitro protein kinase assay (Fig. 5B, left panel) or by the phosphorylation of threonine 410 residue by direct Western blot analysis with the anti-phospho-Thr 410 antibody (Fig. 5B, right panel). We obtained similar results using both approaches, indicating that despite enhanced PI 3-kinase activity in IGF-IR-/- brown adipocytes upon insulin stimulation compared with wild-type cells, phosphorylation and PKC{zeta} activation were similar in the two cell types in parallel with the insulin effect on these cell lines regarding glucose uptake.
fig.ommitteed2hhz, 百拇医药
Figure 5. PKCfig.ommitteedfig.ommitteed activity and glucose uptake in insulin-stimulated wild-type and IGF-IR-deficient brown adipocytes. A, Left panel, IGF-IR+/+ and IGF-IR-/- brown adipocytes were cultured for 30 min in KRP buffer in either the absence or presence of insulin (1–100 nM). Glucose uptake was determined by measuring 2-deoxyglucose-D-[1-3H] (250 nCi/ml) incorporation over the last 10 min of culture. Cells were then washed three times with ice-cold KRP buffer and solubilized in 1% SDS. The radioactivity of a 200-µl aliquot was determined in a scintillation counter. Results are expressed as the fold increase in glucose uptake and are the mean ± SE from six independent experiments, each one performed in duplicate. Right panel, IGF-IR+/+ and IGF-IR-/- brown adipocytes were cultured to confluence under growing conditions (10% FS) and then lysed. Equal amounts of total protein were submitted to SDS-PAGE and analyzed by immunoblotting with the antibody against GLUT4. B, Left panel, Quiescent (20 h serum-starved) wild-type (IGF-IR+/+) and IGF-IR-deficient (IGF-IR-/-) brown adipocytes were stimulated with insulin (10–100 nM) for 10 min. Cells were lysed, and PKC activity was determined as described in Materials and Methods. The autoradiograms corresponding to three independent experiments were quantitated by scanning densitometry. Results are expressed as arbitrary units of MBP phosphorylation and are the mean ± SE. Right panel, Quiescent (20-h serum-starved) wild-type (IGF-IR+/+) and IGF-IR-deficient (IGF-IR-/-) brown adipocytes were stimulated with insulin (10–100 nM) for 5 min or were maintained in the absence of the hormone. Cells were lysed, and total protein (50 µg) was submitted to SDS-PAGE and analyzed by immunoblotting with the corresponding antibodies against phospho-PKC (Thr410) and PKC. The autoradiograms corresponding to three independent experiments were quantitated by scanning densitometry. Results are expressed as arbitrary units of phospho-PKC (normalized to the amount of total PKC) and are the mean ± SE.
IGF-IR-deficient brown adipocytes failed to respond to insulin in inducing FAS and ADD1/SREBP-1c expression1ja;, 百拇医药
An insulin-induced adipogenic differentiation program in brown adipocytes can be monitored by the expression of a set of lipogenic enzymes including FAS (6). In addition, insulin or IGF-I-induced-FAS mRNA expression was inhibited in the presence of PI 3-kinase inhibitors in primary cultures of fetal brown adipocytes (9, 10). Accordingly, we investigated whether the enhanced IRS-1/PI 3-kinase/Akt signaling in IGF-IR-/- brown adipocytes could affect lipogenic gene expression. Cells of both genotypes were cultured for 12 or 24 h in the presence of various doses of insulin. Then, FAS mRNA and protein content were assessed by Northern and Western blot analyses, respectively. As shown in Fig. 6A, insulin treatment for 12 h induced FAS mRNA in wild-type brown adipocytes in a dose-dependent manner; the maximal effect was elicited at 10 nM insulin. This effect was abolished in IGF-IR-/- cells. At the protein level, IGF-IR-deficient cells did not respond to insulin in inducing FAS. As FAS expression has been shown to be dependent on ADD1/SREBP-1c transcription factor (35), we analyzed ADD1/SREBP-1c expression in the presence of insulin in both cell types. As shown in Fig. 6B, both ADD1/SREBP-1c mRNA and protein content (the nuclear mature form of ADD1/SREBP-1c) were increased after 12 or 24 h of insulin treatment, respectively, in wild-type cells, but not in IGF-IR-/- cells. These results indicate that IGF-IR-/- brown adipocytes fail to respond to insulin in inducing FAS expression as a result of a loss of ADD1/SREBP-1c expression.
fig.ommitteedv!;|s*d, http://www.100md.com
Figure 6. Up-regulation of FAS and ADD1/SREBP-1c expression by insulin in wild-type, but not in IGF-IR-deficient brown adipocytes. A, Upper panel, Quiescent (20 h serum-starved) wild-type (IGF-IR+/+) and IGF-IR-deficient (IGF-IR-/-) brown adipocytes were stimulated with insulin (1–100 nM) for 12 h. Control cells were cultured in the absence of the hormone. Total RNA (10 µg) was isolated, submitted to Northern blot analysis, and hybridized with FAS and 18S RNA cDNA probes. Results are expressed as arbitrary units of FAS mRNA and are the mean ± SE of three independent experiments. Lower panel, Cells were stimulated with insulin (1–100 nM) for 24 h. At the end of the culture, cells were lysed, and equals amount of total protein were submitted to SDS-PAGE, followed by immunoblotting with the anti-FAS antibody. A representative experiment is shown. The autoradiograms corresponding to three independent experiments were quantitated by scanning densitometry. Results are expressed as arbitrary units of FAS protein content normalized to the amount of ß-actin and are the mean ± SE. B, Upper panel, Quiescent cells were stimulated with insulin as described in A. Total RNA (10 µg) was isolated, submitted to Northern blot analysis, and hybridized with ADD1/SREBP-1c and 18S RNA cDNA probes. Results are expressed as arbitrary units of ADD1/SREBP-1c mRNA and are the mean ± SE of three independent experiments. Lower panel, Quiescent cells were stimulated with insulin (10–100 nM) for 24 h. At the end of the culture, cells were lysed, and equals amount of nuclear protein were submitted to SDS-PAGE, followed by immunoblotting with the anti-ADD1/SREBP-1c antibody. A representative experiment is shown. The autoradiograms corresponding to three independent experiments were quantitated by scanning densitometry. Results are expressed as arbitrary units of ADD1/SREBP-1c protein content and are the mean ± SE.
Insulin induced thermogenic differentiation is impaired in IGF-IR-deficient brown adipocytes8^3%7t[, 百拇医药
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 in a PI 3-kinase-dependent manner (5, 9, 10). Accordingly, the UCP-1 protein content was analyzed by direct Western blot analysis in mitochondrial protein extracts of both cell types upon insulin stimulation. As shown in Fig. 7A, UCP-1 protein expression was up-regulated in response to 24-h insulin treatment in immortalized brown adipocytes derived from the wild-type mice, whereas in IGF-IR-deficient cells this response was completely abolished. It has been reported that the 5'-flanking region of the UCP-1 gene contains C/EBP-regulated sites (36). In fact, previous data from our laboratory indicated that the three canonical C/EBP isoforms ({alpha} , ß, and ) are expressed in BAT during late fetal development, with C/EBP protein content being positively regulated by insulin (6, 37). Based on that, we performed anti-C/EBP Western blot analysis in nuclear extracts from control and insulin-stimulated brown adipocytes. As shown in Fig. 7B, the expression of C/EBP{alpha} was significantly induced by 24 h of insulin treatment in wild-type cells, but not in IGF-IR-/- cells. In all experiments similar loading of protein extracts was assessed by Ponceau Red staining of the membranes. These results indicate that the lack of IGF-IR during fetal development of brown adipocytes leads to a loss of thermogenic-related gene expression.
fig.ommitteed'!2({, http://www.100md.com
Figure 7. Insulin effect on UCP-1 and p42C/EBP expression in wild-type and IGF-IR-deficient brown adipocytes. A, Quiescent IGF-IR+/+ and IGF-I -/- were stimulated with insulin (10–100 nM) for 24 h. Control cells were cultured in the absence of hormone. At the end of the culture, mitochondrial proteins were extracted, and equals amount of protein were submitted to Western blot analysis with anti-UCP-1 and anticytochrome c antibodies. Representative experiments are shown. The autoradiograms corresponding to three independent experiments were quantitated by scanning densitometry. Results are expressed as arbitrary units of UCP-1 normalized to the amount of cytochrome c and are the mean ± SE. B, Quiescent cells were stimulated with insulin (10–100 nM) for 24 h. Control cells were cultured in the absence of the hormone. Cell were then lysed, and equals amount of nuclear protein were submitted to SDS-PAGE, followed by immunoblotting with the anti-C/EBP antibody. A representative experiment is shown. The autoradiograms corresponding to three independent experiments were quantitated by scanning densitometry. Results are expressed as arbitrary units of C/EBP and are the mean ± SE.
Effect of PPAR or C/EBP overexpression in the adipogenic and thermogenic differentiation capacity of IGF-IR-deficient brown adipocytesw|, 百拇医药
C/EBP and PPAR transcription factors are induced during brown adipogenesis in a similar fashion as white cell differentiation. Accordingly, our next goal was to investigate whether overexpression of these transcription factors is able to rescue UCP-1 and FAS expression in IGF-IR-/- brown adipocytes, bypassing insulin signaling. For this goal, we performed retrovirus-mediated C/EBP or PPAR gene transfer into IGF-IR-/- brown adipocytes (Fig. 8A). After retroviral infection, C/EBP was overexpressed in IGF-IR-/- brown adipocytes about 5-fold, whereas PPAR was overexpressed about 10-fold. Interestingly, PPAR expression was slightly induced in deficient cells overexpressing C/EBP. Next, we analyzed FAS expression, an adipogenic marker, in insulin-stimulated C/EBP and PPAR-IGF-IR-/- cells. The Northern blot depicted in Fig. 8B (upper panel) revealed that FAS mRNA was up-regulated in PPAR-IGF-IR-/- cells, and insulin treatment did not further increase FAS mRNA expression. However, insulin treatment slightly increased FAS mRNA in C/EBP-IGF-IR-/- cells, with the overexpression of C/EBP restoring the insulin sensitivity lost in IGF-IR-/- brown adipocytes. To further analyze the effect of insulin on UCP-1 protein content in C/EBP and PPAR-IGF-IR-/- cells, we performed anti-UCP-1 Western blot analysis after 24 h of insulin stimulation (Fig. 8B, lower panel). Overexpression of PPAR, but not C/EBP{alpha} , up-regulated UCP-1 expression in IGF-IR-deficient cells in an insulin-independent manner.
fig.ommitteed@v, http://www.100md.com
Figure 8. Effect of PPAR or C/EBP overexpression in the adipogenic and thermogenic differentiation capacity of IGF-IR-deficient brown adipocytes. A, IGF-IR-/- fetal brown adipocyte cell lines overexpressing C/EBP or PPAR were generated as described in Materials and Methods. Nuclear extracts were prepared from growing cells (10% FS) and submitted to SDS-PAGE, followed by immunodetection with anti-C/EBP and PPAR antibodies. B, Upper panel, IGF-IR-/- brown adipocytes and IGF-IR-/- cells overexpressing C/EBP or PPAR were serum-starved for 20 h. Quiescent cells were cultured for 12 h in serum-free medium in either the absence or presence of insulin (10–100 nM). At the end of the culture, total RNA (10 µg) was submitted to Northern blot analysis and hybridized with labeled FAS and 18S rRNA cDNAs. Representative autoradiograms are shown. Results are expressed as arbitrary units of FAS mRNA and are the mean ± SE of three independent experiments. Lower panel, IGF-IR-/- brown adipocytes and IGF-IR-/- 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). At the end of the culture, cells were harvested, and mitochondrial protein extracts were submitted to SDS-PAGE and immunodetected with anti-UCP-1 or anticytochrome c antibodies. The autoradiograms corresponding to three independent experiments were quantitated by scanning densitometry. Results are expressed as arbitrary units of UCP-1 normalized to the amount of cytochrome c and are the mean ± SE.
Discussion;foc|*, 百拇医药
Fetal brown adipocytes differentiate at the end of fetal life on the basis of two programs: an adipogenic program related to the expression of a set of lipogenic enzymes resulting in the appearance of a multilocular fat droplets phenotype (6) and a thermogenic program of UCP-1 expression to prevent newborn hypothermia through the nonshivering thermogenesis mechanism (reviewed in Ref. 4). Among the complex of signals involved in the onset of fetal brown adipocyte differentiation, our laboratory has reported an essential role of insulin and IGF-I in triggering this process (5, 6, 7). To further investigate the molecular mechanisms by which insulin/IGF-I elicit their actions in fetal brown adipocytes, we have extensively studied the signal transduction pathways leading to the expression of metabolic genes as well as the tissue-specific thermogenic marker UCP-1. However, the fact that primary brown adipocytes display high affinity binding sites for both insulin and IGF-I (6) makes it difficult to dissect the relative contributions of the two receptors. To address this point, we recently generated immortalized fetal brown adipocytes from the fetuses of IGF-IR-deficient mice (IGF-IR-/-). Under growing conditions, these cells maintained the constitutive expression of adipogenic genes as well as UCP-1 compared with immortalized wild-type brown adipocytes (IGF-IR+/+). Surprisingly, the lack of IGF-IR was compensated for by an increased insulin signaling and mitogenic response to the hormone through the IRS-1/Grb-2/MAPK pathway. This occurs because PTP1B enzymatic activity and its association with either IR or IRS-1 is severely impaired in insulin-stimulated IGF-IR-deficient cells (23).
Based on our previous findings suggesting that IGF-I might have a role in vivo leading BAT to differentiate before birth (6), in the present paper we have investigated the consequences of the lack of IGF-IR and its signaling regarding this process. As we have demonstrated an essential role of IRS-1 via PI 3-kinase in inducing adipogenic and thermogenic gene expression upon insulin or IGF-I stimulation of primary brown adipocytes (9, 10, 30), we here have further explored this signaling pathway in immortalized IGF-IR-deficient brown adipocytes. As summarized in Fig. 9, IGF-IR-/- brown adipocytes showed increased insulin sensitivity regarding IRS-1 tyrosine phosphorylation and IRS-1-associated PI 3-kinase activity compared with wild-type cells despite a marked decrease in its IRS-1 protein content. These results indicate that in addition to the IRS-1/Grb-2/Ras/MAPK pathway (23), the IRS-1/PI 3-kinase pathway is overstimulated in IGF-IR-deficient brown adipocytes upon insulin treatment. Downstream from PI 3-kinase, activation of Akt/PKB, but not p70s6 kinase or PKC{zeta} , was significantly increased in IGF-IR-deficient cells. Recently, PKC has been implicated by a number of laboratories, including ours, as a key mediator of glucose transport during insulin action (34, 38, 39, 40). Thus, the results presented here confirm that activation of PKC by insulin parallels glucose uptake in fetal brown adipocytes. Interestingly, neither GLUT4 protein content nor insulin-induced glucose uptake is augmented in IGF-IR-deficient brown adipocytes compared with the wild type despite the significant increase in Akt/PKB activation.
fig.ommitteed!4#ea\, 百拇医药
Figure 9. Differential insulin signaling between wild-type and IGF-IR-/- fetal brown adipocytes. IGF-IR-/- cells showed an increased insulin sensitivity regarding IRS-1 tyrosine phosphorylation and IRS-1-associated PI 3-kinase activity. Downstream PI 3-kinase activation of Akt was elicited at lower doses of insulin in IGF-IR-/- brown adipocytes. Activation of PKC or p70s6 kinase by insulin was similar in both cell types. Insulin up-regulated adipogenic (ADD1/SREBP-1c and FAS) and thermogenic (C/EBP and UCP-1) gene expression in wild-type fetal brown adipocytes; this effect was impaired in the absence of IGF-IR.!4#ea\, 百拇医药
Differentiation of fetal brown adipocytes in response to insulin or IGF-I can be monitored by the expression of lipogenic enzymes. Immortalized brown adipocytes respond to insulin in inducing FAS mRNA and protein expression as previously reported in parental cells (6). It was surprising to find that insulin was not able to induce FAS expression in IGF-IR-/- cells despite an enhancement of IRS-1/PI 3-kinase/Akt signaling. Recently, the effect of insulin on FAS gene expression in the liver has been reported to be dependent on the transcription factor ADD1/SREBP-1c (41, 42, 43), which also controls adipocyte differentiation (44). In our cell system, insulin stimulation resulted in a parallel increase in ADD1/SREBP-1c mRNA in wild-type brown adipocytes, but not in IGF-IR-deficient cells. In addition, the amount of the nuclear mature form of ADD1/SREBP-1c was increased by insulin only in brown adipocytes expressing IGF-IR. Conversely, insulin-induced glucose transport, which is another requirement for the effect of insulin on FAS gene expression in white adipose tissue, is not impaired in IGF-IR-deficient brown adipocytes. However, this result can be explained on basis of previous data from our laboratory indicating that in fetal brown adipocyte primary cultures glucose does not affect FAS mRNA induction by insulin (7). Taken together, these results indicate that in fetal brown adipocytes insulin action on FAS expression requires ADD1/SREPB-1c as well as the intact IGF-IR signaling machinery. However, we cannot conclude whether insulin has a direct effect on the proteolysis of the endoplasmic reticulum-derived precursor in wild-type brown adipocytes or is a rapid constitutive process, as recently suggested by Azzout-Marniche et al. (45).
Regarding transcriptional regulation of thermogenesis, C/EBP{alpha} has been shown to directly trans-activate the UCP-1 gene through specific binding sites in its promoter (36, 46). In addition, C/EBP expression increases gradually in rat BAT during late fetal life (6), its mRNA being up-regulated by insulin in BAT adipocyte primary cultures (37). Here, we show that in immortalized fetal brown adipocytes p42C/EBP expression is also up-regulated by insulin treatment. However, when these cells lack IGF-IR, they are unable to induce both C/EBP{alpha} and UCP-1 upon insulin stimulation. Although the differences in C/EBP{alpha} expression between wild-type and IGF-IR littermates in vivo in BAT are not known, these results have two important implications. Firstly, signaling through IGF-IR during fetal development is essential for insulin action regarding C/EBP{alpha} expression, and secondly, binding of newly synthesized C/EBP{alpha} to UCP-1 promoter seems to be necessary for the induction of UCP-1 expression by insulin. In this regard, Kulkarni et al. (47) in a recent report show that the lack of IGF-IR in ß-cells negatively influences the expression of early transcription factors controlling differentiation. These results support our idea that IGF-IR participates in the control of differentiated function of responsive cells.
The fact that C/EBP and PPAR families of transcription factors play an important role in the induction of fully differentiated brown adipocyte phenotype prompted us to restore differentiation-related gene expression in IGF-IR-deficient cells by overexpressing C/EBP{alpha} or PPAR{gamma} by retroviral infection. Our results indicate that only PPAR{gamma} could overcome FAS mRNA impairment in an insulin-independent manner. However, insulin increased FAS mRNA expression in C/EBP-IGF-IR-/- cells, but not in PPAR{gamma} -IGF-IR-/- cells, in a dose-dependent manner. These data clearly indicate that C/EBP{alpha} , but not PPAR{gamma} , reconstitutes insulin sensitivity regarding FAS expression in brown adipocytes and are entirely consistent with the results previously described in the differentiation protocol of 3T3 L1 adipocytes. In these cells, C/EBP{alpha} is required for establishment of insulin-sensitive glucose transport (48). Conversely, fetal brown adipocytes lacking IGF-IR up-regulated UCP-1 expression in the absence of insulin when PPAR{gamma} , but not C/EBP{alpha} , was overexpressed. These results indicate that despite the presence of binding sites for both transcription factors within the UCP-1 promoter, only PPAR{gamma} can restore UCP-1 expression in the absence of IGF-IR and its signaling through development. A possible explanation for these results is based on the fact that during fetal development of BAT, C/EBP{alpha} expression concurs with IGF-IR expression (6). The lack of IGF-IR in brown adipocytes (IGF-IR-/-cells) might suppress the expression of additional molecules (i.e. coactivators) involved in C/EBP{alpha} induction and UCP-1 trans-activation.
In summary, we have shown that the lack of IGF-IR results in a severe impairment of adipogenic and thermogenic differentiation-related gene expression in response to insulin despite the enhancement of IRS-1/PI 3-kinase/Akt signaling. These data demonstrated a critical role for IGF-IR in fetal brown adipocyte differentiation. The effect of IGF-IR is mimicked by exogenous expression of PPAR in an insulin-independent manner. Further work will be needed to define the link between IGF-IR signaling and the expression of various adipogenic and thermogenic transcription factors during fetal development of BAT.+1+(, 百拇医药
Acknowledgments+1+(, 百拇医药
We thankfully acknowledge Bruce S. Spiegelman (Dana Farber Cancer Institute, Boston, MA) for the pBabe hygro C/EBP and pBabe bleo PPAR{gamma} retroviral constructs.+1+(, 百拇医药
Received August 7, 2002.+1+(, 百拇医药
Accepted for publication October 23, 2002.+1+(, 百拇医药
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To define the specific role of IGF-I receptor (IGF-IR) in adipogenic and thermogenic differentiation of brown adipocytes during late fetal life, we have established immortalized brown adipocyte cell lines from fetuses of IGF-IR-deficient mice (IGF-IR-/-) as well as from wild-type mice (IGF-IR+/+). IGF-IR-/- cells showed an increased insulin sensitivity regarding insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation despite a substantial reduction in IRS-1 protein content. Furthermore, insulin-induced total and IRS-1-associated phosphatidylinositol 3-kinase activities were augmented in IGF-IR-deficient cells compared with wild-type cells. Downstream phosphatidylinositol 3-kinase activation of Akt, but not p70s6 kinase, were elicited at lower doses of insulin in IGF-IR-/- brown adipocytes. Activation of protein kinase C{zeta} by insulin was similar in both cell types as was insulin-induced glucose uptake. Treatment of wild-type brown adipocytes with insulin for 12 h up-regulated fatty acid synthase (FAS) and adipocyte determination and differentiation (ADD1/SREBP) mRNAs; this effect was impaired in the absence of IGF-IR. At the protein level, insulin increased FAS content and the amount of the mature form of adipocyte determination and differentiation (ADD1/SREBP) in the nucleus in wild-type cells, but not in IGF-IR-/- cells. Furthermore, 24 h of insulin stimulation induced the expression of both uncoupling protein-1 and CCAAT/enhancer-binding protein (C/EBP) in wild-type brown adipocytes; these effects were abolished in IGF-I-R-/- cells. Retrovirus-mediated reexpression of peroxisomal proliferator-activated receptor (PPAR) in IGF-IR-/- brown adipocytes could overcome FAS mRNA impairment, bypassing insulin signaling. However, insulin further increased FAS mRNA expression in C/EBP{alpha} -IGF-IR-/- cells, but not in PPAR-IGF-IR-/- cells. In addition, fetal brown adipocytes lacking IGF-IR up-regulated uncoupling protein-1 expression in the absence of insulin when PPAR, but not C/EBP{alpha} , was overexpressed. These data provide strong evidence for a critical role of IGF-IR in he differentiation of the brown adipocyte phenotype in fetal life; this effect is mimicked by PPAR{gamma} in an insulin-independent manner.
Introductionq5, http://www.100md.com
ADIPOCYTE differentiation is a complex process that requires communication between extracellular stimuli in a coordinated network of receptors and transcription factors in the nucleus. Although most studies of adipogenesis have focused on the development of white adipose tissue, which is the primary site of storage of triglycerides and release of fatty acids in response to changing energy needs (1), mammals have a second terminally differentiated adipose cell type that composes brown adipose tissue (BAT). BAT is a major site for nonshivering thermogenesis in mammals. The unique thermogenic capacity of BAT results from the expression of uncoupling protein-1 (UCP-1) in the mitochondrial inner membrane, which is required to address physiological hypothermia in newborn mammals (2). By uncoupling the production of ATP from the movement of protons down their concentration gradient, heat is generated (3). In addition, BAT is a major site for lipid metabolism, as fatty acids are the main fuel that maintains the thermogenic capacity of the tissue (for review, see Ref. 4). 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, and a thermogenic program related to UCP-1 expression and heat production (5, 6, 7). During the last years our laboratory has been investigating which hormones/growth factors are the main signals involved in the onset of adipogenic and thermogenic differentiation of BAT during late fetal development. In previous work we demonstrated that fetal brown adipocytes display binding sites of high affinity for both insulin and IGF-I (5, 7). Both molecules are involved in fetal brown adipogenesis through their ability to induce the genetic expression of metabolic genes (5, 6, 8), and they might have a role in thermogenesis by inducing UCP-1 expression (9, 10), as the noradrenergic stimulus, induced by hypothermia after birth, it is not yet fully developed in BAT at birth (6, 11).
The upstream signals regulating the induction and expression of transcription factors involved in activating metabolic genes during brown adipocyte differentiation are poorly understood. Insulin/IGF-I action is mediated by the insulin receptor (IR) and the IGF-I receptor (IGF-IR), respectively, which have very similar heterodimeric {alpha} 2ß2 structure and belong to the family of receptor tyrosine kinases (12). Upon ligand binding, insulin/IGF-I receptor undergoes autophosphorylation on tyrosine residues, which activates the intracellular tyrosine kinase of the ß-subunit (13). This, in turn, stimulates the phosphorylation of cytosolic proteins, including the insulin receptor substrate (IRS) family (IRS-1, -2, -3, and -4) and SHC (14, 15, 16, 17, 18). Then, these phosphorylated IRS bind proteins containing Src homology 2 domains such as the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase) (19); growth factor receptor-binding protein-2 (Grb-2), which links signaling via SOS to activation of the Ras complex (20); and protein tyrosine phosphatase SHP-2 (21) that lead to activation of various downstream signaling pathways. Recent studies suggest that the Ras/MAPK pathway mediates fetal brown adipocyte proliferation (22), whereas the PI 3-kinase pathway is activated during differentiation (9, 10).
The purpose of the present paper is to further study the role of IGF-IR and its signaling in the onset of BAT differentiation. For this goal, we have recently generated immortalized cell lines derived from fetal BAT of IGF-IR-deficient and wild-type mice. These cells showed an increased insulin sensitivity of the MAPK signaling pathway and mitogenesis, as a result of a compensatory mechanism (23). Now, we found that IR and its signaling through IRS-1/PI 3-kinase are unable to compensate for the lack of IGF-IR during fetal development regarding brown adipocyte adipogenic- and thermogenic-related gene expression. However, we can bypass insulin signaling and up-regulate both fatty acid synthase (FAS) and UCP-1 expression by overexpression of peroxisomal proliferator-activated receptor (PPAR) transcription factor, but not CCAAT/enhancer-binding protein {alpha} (C/EBP), in IGF-IR-deficient brown adipocytes. These results demonstrate the essential role played by IGF-IR in initiating and/or maintaining the differentiation of brown adipocytes during late fetal life.
Materials and Methods^!\?, http://www.100md.com
Materials^!\?, http://www.100md.com
Fetal calf serum (FS) and culture media were obtained from Life Technologies, Inc. (Gaithersburg, MD). Insulin, hygromycin, and antimouse IgG-agarose were obtained from Sigma-Aldrich (St. Louis, MO). Protein A-agarose was purchased from Roche Molecular Biochemicals (Mannheim, Germany). The bleomycin analog zeozin was purchased from Invitrogen (Carlsbad, CA) The anti-IRS-1 (06-248), anti-Tyr(P) (clone 4G10; 05-321), anti-Akt (06-558), and anti-Akt ß (06-606) antibodies were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). The anti-TyrP (Py20; sc-508), anti-C/EBP{alpha} (sc-61), antiadipocyte determination and differentiation (anti-ADD1/SREBP-1c; sc-8984, raised against the NH2 terminus of the molecule), and anti-protein kinase C (anti-PKC; c-20; sc-216) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The anti-phospho-Akt (Ser473 9271), anti-phospho-p70s6k (Ser424/Thr421 9204S), anti-Akt (9272), anti-p70s6k (9202), and anti-phospho-PKC (Thr410 9378s) antibodies were purchased from Cell Signaling (Beverly, MA). The anti-UCP-1 (AB3038) and anti-GLUT-4 (AB1346) antibodies were obtained from Chemicon (Temecula, CA). The anti PPAR (SA-206) antibody was purchased from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). [-32P]ATP (3000 Ci/mmol), [{alpha} 32P]deoxy-CTP, and 2-deoxy-D-[1-3H]glucose (11.0 Ci/mmol) were obtained from Amersham International (Little Chalfont, UK). All other reagents used were of the purest grade available.
Cell culture and retroviral infections|]?r), http://www.100md.com
Brown adipocytes were obtained from interscapular BAT of 17.5–18.5 fetuses from 2 or 3 pregnant mice (MF1 and C57BL/6J mouse strains) of normal genotype (IGF-IR+/+) or from a pool of tissue of fetuses with body weight less than 0.7 g (IGF-IR-/-) from 2 or 3 pregnant mice IGF-IR+/- mated with IGF-IR+/- males (MF1 and C57BL/6J mouse strains) (24). Brown adipocyte primary cultures were performed as previously described (5). 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 simian virus 40 large T antigen (provided by J. de Caprio, Dana Farber Cancer Institute, Boston, MA). Then, brown adipocytes (wild-type and IGF-IR-/-) 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 wk. These pools of stable cells were further cultured in DMEM supplemented with 10% FS and puromycin. The pBabe/hygro C/EBP{alpha} and pBabe/zeozin PPAR{gamma} viral expression vectors were generous gifts from Dr. B. Spiegelman (Dana Farber Cancer Institute). Brown adipocyte IGF-IR-deficient cells were infected with those vectors described above. Selection with 200 µg/ml hygromycin or 250 µg/ml zeozin was started 48 h after infection to select stable cell lines.
Immunoprecipitations-yalmkp, http://www.100md.com
Quiescent cells 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 phenylmethylsulfonylfluoride, pH 7.6 (lysis buffer). Lysates were clarified by centrifugation at 15,000 x 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 antimouse IgG-agarose beads. Immunoprecipitates were washed with lysis buffer, were extracted for 5 min at 95 C in 2x SDS-PAGE sample buffer [200 mM Tris-HCl, 6% sodium dodecyl sulfate (SDS), 2 mM EDTA, 4% 2-mercaptoethanol, and 10% glycerol, pH 6.8], and analyzed by SDS-PAGE.-yalmkp, http://www.100md.com
Western blotting-yalmkp, http://www.100md.com
After SDS-PAGE, proteins were transferred to Immobilon membranes, blocked using 5% nonfat dried milk or 3% BSA in 10 mM Tris-HCl and 150 mM NaCl (pH 7.5), and incubated overnight with several antibodies, as indicated, in 0.05% Tween-20, 10 mM Tris-HCl, and 150 mM NaCl (pH 7.5). Immunoreactive bands were visualized using the enhanced chemiluminescence Western blotting protocol (Amersham International).
PI 3-kinase activity*, 百拇医药
PI 3-kinase activity was measured in the anti-IRS-1 or anti-Tyr(P) immunoprecipitates by in vitro phosphorylation of PI as previously described (9).*, 百拇医药
Akt activity*, 百拇医药
Akt activity was measured in vitro using a nonradioactive technique. Quiescent cells were treated for 5 min with or without several doses of insulin and were subsequently lysed in the lysis buffer described above. One milligram of each cell lysate was subjected to immunoprecipitation with anti-Akt{alpha} or anti-Aktß antibodies. The resulting immune complexes were washed in lysis buffer twice and once in kinase buffer containing 25 mM Tris-HCl (pH 7.5), 5 mM ß-glycerol-phosphate, 10 mM MgCl2, 2 mM dithiothreitol, and 100 µM sodium orthovanadate. The activity of Akt in the immunoprecipitates was determined in the presence of a synthetic peptide corresponding to the Akt phosphorylation sites of GSK-3{alpha} ß (Ser9/Ser21) fused to paramyosin and 200 µM ATP for 30 min at 30 C. The reaction products were eluted with Laemmli buffer for 5 min at 100 C, resolved on 10% SDS-PAGE gels, transferred to nylon membranes, and visualized by immunoblotting with antiphospho-GSK-3{alpha} ß (Ser9/Ser21) antibody.
PKC activitykn, http://www.100md.com
Quiescent cells were treated for 10 min without or with several doses of insulin and subsequently lysed. PKC{zeta} activity was measured in anti-PKC immunoprecipitates as previously described (9).kn, http://www.100md.com
Extraction of nuclear proteinskn, http://www.100md.com
Cells were resuspended at 4 C in 10 mM HEPES-KOH (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 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 sec. 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 DTT, 0.2 mM PMSF, 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.
Isolation of mitochondrial protein:, 百拇医药
At the end of the culture, cells were scrapped off in isotonic isolation buffer (1 mM EDTA, 10 mM HEPES, and 250 mM sucrose, pH 7.6), collected by centrifugation at 2500 x g for 5 min at 4 C, and resuspended in hypotonic isolation buffer (1 mM EDTA, 10 mM HEPES, and 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, and 450 mM sucrose, pH 7.6) was added to balance the buffer’s tonicity. Samples were centrifuged at 10,000 x g for 10 min, the pellets, containing the mitochondrial fraction, were resuspended in isotonic isolation buffer, and mitochondrial protein content was determined.:, 百拇医药
Protein determination:, 百拇医药
Protein determination was performed by the Bradford dye method (25), using the Bio-Rad Laboratories, Inc., reagent and BSA as the standard.
RNA extraction and Northern blot analysis$c, http://www.100md.com
At the end of the culture, cells were washed twice in ice-cold PBS, and RNA was isolated as previously described (26). 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 (NEN Life Science Products, Boston, MA), and cross-linked to the membranes by UV light. Hybridization was performed in 0.25 mM NaHPO4 (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]deoxy-CTP by using a multiprimer DNA labeling system. Blots were hybridized with probes for FAS (27) and ADD1/SREBP-1c (28) and with 18S ribosomal probe to normalize RNA loading. Membranes were subjected to autoradiography, and the relative densities of the hybridization signals were determined by densitometric scanning of the autoradiograms.
Glucose uptake8p9.q:, 百拇医药
2-Deoxyglucose transport was measured as previously described (29). After culture, quiescent brown adipocytes were washed three times with Krebs-Ringer phosphate buffer (KRP) containing 136 mM NaCl, 4.7 mM KCl, 1 mM CaCl2, 1 mM MgSO4, 5 mM sodium pyrophosphate, 20 mM HEPES, and 1% BSA and then were incubated with 1 ml KRP in the presence or absence of insulin for 20 min at 37 C. 2-Deoxyglucose-D-[1-3H] was added to this solution to a final concentration of 0.1 mM (250 nCi/ml), and the incubation was continued for 10 min at 37 C. The cells were then washed three times with ice-cold KRP buffer and solubilized in 1% SDS. The radioactivity of a 200-µl aliquot was determined in a scintillation counter.8p9.q:, 百拇医药
Results8p9.q:, 百拇医药
IRS-1 protein content and its tyrosine phosphorylation in insulin-stimulated wild-type and IGF-IR-deficient brown adipocytes8p9.q:, 百拇医药
We have recently shown an enhanced insulin-induced tyrosine phosphorylation of the IR ß-chain in immortalized brown adipocytes derived from fetuses of IGF-IR-deficient mice compared with wild-type cells. In addition, in previous experiments we found a significant reduction of IRS-1 protein content in IGF-IR-/- brown adipocytes despite a marked increase in its tyrosine phosphorylation upon 10 nM insulin stimulation (23). In the dose-response experiment depicted in Fig. 1 we show that IRS-1 tyrosine phosphorylation in IGF-IR-/- brown adipocytes could be detected at a 0.01-nM insulin concentration, with the maximal effect being elicited at 0.1 nM. In wild-type cells, IRS-1 tyrosine phosphorylation was barely detected at 0.01 nM insulin, with the maximal effect elicited at 10 nM. Considering that IRS-1 protein content is severely reduced in IGF-IR-/- brown adipocytes, its tyrosine phosphorylation in these cells is about 7-fold higher than that of wild-type cells (Fig. 1, lower panel). Conversely, neither IRS-2 protein content nor its tyrosine phosphorylation significantly differ between the cell types (results not shown).
fig.ommitteedl, http://www.100md.com
Figure 1. Insulin effect on IRS-1 tyrosine phosphorylation in wild-type and IGF-IR-deficient brown adipocytes. Wild-type (IGF-IR+/+) and IGF-I-deficient brown adipocytes (IGF-IR-/-) were cultured to confluence under growing conditions (10% FS) and then serum-starved for 20 h. Quiescent cells were stimulated with various doses of insulin (0.01–10 nM) for 5 min, and control cells were cultured in the absence of the hormone. At the end of the culture, cells were lysed, and 600 µg total protein were immunoprecipitated with anti-IRS-1 antibody. The resulting immune complexes were analyzed by immunoblotting with anti-Tyr(P) or anti-IRS-1 antibodies. The autoradiograms shown are representative of three experiments. Results are expressed as arbitrary units or IRS-1 tyrosine phosphorylation (normalized to the amount of total IRS-1) and are the mean ± SE.l, http://www.100md.com
Insulin-induced IRS-1- and Tyr(P)-associated PI 3-kinase activity in wild-type and IGF-IR-deficient brown adipocytesl, http://www.100md.com
Our next purpose was to study whether the enhanced insulin sensitivity of IGF-IR-deficient brown adipocytes emerging from the IR signals through the IRS-1/PI 3-kinase pathway. For this goal, serum-starved cells were stimulated with various doses of insulin (0.01–10 nM) for 5 min, and PI 3-kinase activity was measured in both anti-IRS-1 and anti-Tyr(P) immunoprecipitates. As shown in Fig. 2 (left panel), IRS-1-associated PI 3-kinase activity was increased by insulin in wild-type brown adipocytes in a dose-dependent manner; the maximal effect was elicited at 10 nM insulin. In contrast, IGF-IR-/- brown adipocytes showed a significant activation of IRS-1-associated PI 3-kinase at a low insulin dose (0.01 nM); the insulin response curve showed the maximal effect at a 0.1-nM concentration. Again, because of the dramatic reduction of IRS-1 protein content in IGF-IR-deficient cells, IRS-1-associated PI 3-kinase activity was about 6-fold higher than that of wild-type cells. In contrast, IRS-2-associated PI 3-kinase activity does not differ significantly among the cell types (data not shown). Thus, total PI 3-kinase associated with anti-Tyr(P) immunoprecipitates was increased in IGF-IR-/- brown adipocytes (Fig. 2, right panel); this effect was truly significant at low doses of insulin (0.01–1 nM).
fig.ommitteed9ng@x, 百拇医药
Figure 2. Insulin effect on IRS-1- and Tyr(P)-associated PI 3-kinsase activity in wild-type and IGF-IR-deficient brown adipocytes. IGF-IR+/+ and IGF-IR-/- brown adipocytes were cultured to confluence under growing conditions (10% FS) and then serum-starved for 20 h. Quiescent cells were stimulated with insulin (0.01–10 nM) for 5 min or were cultured in the absence of the hormone. At the end of the culture, cells were lysed, and 600 µg total protein were immunoprecipitated with anti-IRS-1 (left) or anti-Tyr(P) (right) antibodies. The resulting immune complexes were washed and immediately used for an in vitro PI 3-kinase assay. The conversion of phosphatidylinositol to phosphatidylinositol phosphate in the presence of [{gamma} -32P]ATP was analyzed by TLC. Representative autoradiograms are shown. The autoradiograms corresponding to three independent experiments were quantitated by scanning densitometry. Results are expressed as arbitrary units of PI 3-kinase (normalized to the amount of total IRS-1 protein content in the left panel) and are the mean ± SE.9ng@x, 百拇医药
Akt and p70s6k phosphorylation in wild-type and IGF-IR-/- brown adipocytes($?du.%, 百拇医药
As the serine/threonine kinase Akt is one of the major downstream targets of PI 3-kinase, and it has been implicated in adipogenic differentiation (30, 31, 32), we explored its phosphorylation by direct Western blot analysis in wild-type and IGF-IR-/- brown adipocytes. Insulin stimulation of both cell types resulted in AktSer473 phosphorylation without changes in the total Akt protein content (Fig. 3, upper panel). In wild-type cells the maximal effect was elicited at 100 nM insulin, whereas the dose-response curve of IGF-IR-deficient brown adipocytes was shifted to the left (maximal effect was elicited at 1 nM insulin), indicating increased insulin sensitivity in these cells. To confirm the results obtained from the phospho-Akt analysis, Akt kinase assays were performed in insulin-stimulated IGF-IR+/+ and IGF-IR-/- fetal brown adipocytes after immunoprecipitation with the specific antibodies for Akt{alpha} and Aktß isoforms. In wild-type brown adipocytes (Fig. 3, lower panel) {alpha} -Akt kinase activity was enhanced by insulin in a dose-dependent manner, reaching the maximal value (2- to 3-fold increase) at 10 nM insulin. Under the same experimental conditions {alpha} Akt was activated by 10-fold in IGF-IR-deficient cells; no differences were observed in ßAkt activity between the cell types. In contrast, phosphorylation of p70s6 kinase, which has also been involved in brown adipocyte differentiation (9), did not show differences between the cell types (Fig. 4).
fig.ommitteed-|, 百拇医药
Figure 3. Activation of Akt in wild-type and IGF-IR-deficient brown adipocytes. Upper panel, Quiescent (20-h serum-starved) wild-type (IGF-IR+/+) and IGF-IR-deficient (IGF-IR-/-) brown adipocytes were stimulated with insulin (0.01–100 nM) for 5 min or were maintained in the absence of the hormone. Cells were lysed, and total protein (50 µg) was submitted to SDS-PAGE and analyzed by immunoblotting with the corresponding antibodies against phospho-Akt (Ser473) and total Akt. The autoradiograms corresponding to four independent experiments were quantitated by scanning densitometry. Results are expressed as arbitrary units of Akt phosphorylation (normalized to the amount of total Akt) and are the mean ± SE. Lower panel, Quiescent cells were stimulated with various doses of insulin for 5 min. Control cells were incubated in the absence of the hormone. Cells were lysed, and equals amount of protein (600–800 µg) were immunoprecipitated with the anti-{alpha} Akt or anti-ßAkt antibodies and immediately used for an in vitro kinase assay as described in Materials and Methods. Quantitative data are expressed as the fold increase in Akt stimulation and are the mean ± SE from three or four independent experiments.
fig.ommitteed4;t%%#(, http://www.100md.com
Figure 4. Insulin effect on p70s6 kinase phosphorylation in wild-type and IGF-IR-deficient brown adipocytes. Quiescent (20-h serum-starved) wild-type (IGF-IR+/+) and IGF-IR-deficient (IGF-IR-/-) brown adipocytes were stimulated with insulin (0.01–10 nM) for 5 min or were maintained in the absence of the hormone. Cells were lysed, and total protein (50 µg) was submitted to SDS-PAGE and analyzed by immunoblotting with the corresponding antibodies against phospho-p70s6k and total p70s6k. The autoradiograms corresponding to three independent experiments were quantitated by scanning densitometry. Results are expressed as arbitrary units of p70s6k phosphorylation (normalized to the amount of total p70s6k) and are the mean ± SE.4;t%%#(, http://www.100md.com
The lack of IGF-IR in brown adipocytes did not affect insulin-induced glucose uptake4;t%%#(, http://www.100md.com
It is well known that activation of PI 3-kinase and its downstream targets is required for the movement of glucose transporters to the cell membrane in BAT (33). In fetal brown adipocytes glucose transport increased significantly upn stimulation with physiological doses of insulin/IGF-I, and this effect was dependent on the PI 3-kinase/PKC signaling pathway (9, 34). Accordingly, we investigated whether the enhanced PI 3-kinase activity observed in IGF-IR-/- brown adipocytes could mediate the insulin effect on glucose uptake. For this goal, brown adipocytes were stimulated for 10 min with various doses of insulin (1–100 nM) and then were incubated for an additional 10 min in the presence of 2-deoxy-D-[1-3H]glucose. As shown in Fig. 5A (left panel), there was no significant difference in basal glucose transport activity in cells from both genotypes. Insulin stimulation resulted in a 2-fold increase in glucose transport in wild-type and IGF-IR-/- brown adipocytes; in both cases the maximal effect was elicited at 10 nM insulin. In addition, the GLUT4 protein content was similar in the two cell types (Fig. 5A, right panel). To further confirm these data we measured PKC activity, which has been shown to mediate insulin-induced glucose uptake in brown adipocytes (34), in both cell types upon insulin stimulation. Quiescent cells were incubated with insulin (10–100 nM) for 10 min, and PKC activity was determined by an in vitro protein kinase assay (Fig. 5B, left panel) or by the phosphorylation of threonine 410 residue by direct Western blot analysis with the anti-phospho-Thr 410 antibody (Fig. 5B, right panel). We obtained similar results using both approaches, indicating that despite enhanced PI 3-kinase activity in IGF-IR-/- brown adipocytes upon insulin stimulation compared with wild-type cells, phosphorylation and PKC{zeta} activation were similar in the two cell types in parallel with the insulin effect on these cell lines regarding glucose uptake.
fig.ommitteed2hhz, 百拇医药
Figure 5. PKCfig.ommitteedfig.ommitteed activity and glucose uptake in insulin-stimulated wild-type and IGF-IR-deficient brown adipocytes. A, Left panel, IGF-IR+/+ and IGF-IR-/- brown adipocytes were cultured for 30 min in KRP buffer in either the absence or presence of insulin (1–100 nM). Glucose uptake was determined by measuring 2-deoxyglucose-D-[1-3H] (250 nCi/ml) incorporation over the last 10 min of culture. Cells were then washed three times with ice-cold KRP buffer and solubilized in 1% SDS. The radioactivity of a 200-µl aliquot was determined in a scintillation counter. Results are expressed as the fold increase in glucose uptake and are the mean ± SE from six independent experiments, each one performed in duplicate. Right panel, IGF-IR+/+ and IGF-IR-/- brown adipocytes were cultured to confluence under growing conditions (10% FS) and then lysed. Equal amounts of total protein were submitted to SDS-PAGE and analyzed by immunoblotting with the antibody against GLUT4. B, Left panel, Quiescent (20 h serum-starved) wild-type (IGF-IR+/+) and IGF-IR-deficient (IGF-IR-/-) brown adipocytes were stimulated with insulin (10–100 nM) for 10 min. Cells were lysed, and PKC activity was determined as described in Materials and Methods. The autoradiograms corresponding to three independent experiments were quantitated by scanning densitometry. Results are expressed as arbitrary units of MBP phosphorylation and are the mean ± SE. Right panel, Quiescent (20-h serum-starved) wild-type (IGF-IR+/+) and IGF-IR-deficient (IGF-IR-/-) brown adipocytes were stimulated with insulin (10–100 nM) for 5 min or were maintained in the absence of the hormone. Cells were lysed, and total protein (50 µg) was submitted to SDS-PAGE and analyzed by immunoblotting with the corresponding antibodies against phospho-PKC (Thr410) and PKC. The autoradiograms corresponding to three independent experiments were quantitated by scanning densitometry. Results are expressed as arbitrary units of phospho-PKC (normalized to the amount of total PKC) and are the mean ± SE.
IGF-IR-deficient brown adipocytes failed to respond to insulin in inducing FAS and ADD1/SREBP-1c expression1ja;, 百拇医药
An insulin-induced adipogenic differentiation program in brown adipocytes can be monitored by the expression of a set of lipogenic enzymes including FAS (6). In addition, insulin or IGF-I-induced-FAS mRNA expression was inhibited in the presence of PI 3-kinase inhibitors in primary cultures of fetal brown adipocytes (9, 10). Accordingly, we investigated whether the enhanced IRS-1/PI 3-kinase/Akt signaling in IGF-IR-/- brown adipocytes could affect lipogenic gene expression. Cells of both genotypes were cultured for 12 or 24 h in the presence of various doses of insulin. Then, FAS mRNA and protein content were assessed by Northern and Western blot analyses, respectively. As shown in Fig. 6A, insulin treatment for 12 h induced FAS mRNA in wild-type brown adipocytes in a dose-dependent manner; the maximal effect was elicited at 10 nM insulin. This effect was abolished in IGF-IR-/- cells. At the protein level, IGF-IR-deficient cells did not respond to insulin in inducing FAS. As FAS expression has been shown to be dependent on ADD1/SREBP-1c transcription factor (35), we analyzed ADD1/SREBP-1c expression in the presence of insulin in both cell types. As shown in Fig. 6B, both ADD1/SREBP-1c mRNA and protein content (the nuclear mature form of ADD1/SREBP-1c) were increased after 12 or 24 h of insulin treatment, respectively, in wild-type cells, but not in IGF-IR-/- cells. These results indicate that IGF-IR-/- brown adipocytes fail to respond to insulin in inducing FAS expression as a result of a loss of ADD1/SREBP-1c expression.
fig.ommitteedv!;|s*d, http://www.100md.com
Figure 6. Up-regulation of FAS and ADD1/SREBP-1c expression by insulin in wild-type, but not in IGF-IR-deficient brown adipocytes. A, Upper panel, Quiescent (20 h serum-starved) wild-type (IGF-IR+/+) and IGF-IR-deficient (IGF-IR-/-) brown adipocytes were stimulated with insulin (1–100 nM) for 12 h. Control cells were cultured in the absence of the hormone. Total RNA (10 µg) was isolated, submitted to Northern blot analysis, and hybridized with FAS and 18S RNA cDNA probes. Results are expressed as arbitrary units of FAS mRNA and are the mean ± SE of three independent experiments. Lower panel, Cells were stimulated with insulin (1–100 nM) for 24 h. At the end of the culture, cells were lysed, and equals amount of total protein were submitted to SDS-PAGE, followed by immunoblotting with the anti-FAS antibody. A representative experiment is shown. The autoradiograms corresponding to three independent experiments were quantitated by scanning densitometry. Results are expressed as arbitrary units of FAS protein content normalized to the amount of ß-actin and are the mean ± SE. B, Upper panel, Quiescent cells were stimulated with insulin as described in A. Total RNA (10 µg) was isolated, submitted to Northern blot analysis, and hybridized with ADD1/SREBP-1c and 18S RNA cDNA probes. Results are expressed as arbitrary units of ADD1/SREBP-1c mRNA and are the mean ± SE of three independent experiments. Lower panel, Quiescent cells were stimulated with insulin (10–100 nM) for 24 h. At the end of the culture, cells were lysed, and equals amount of nuclear protein were submitted to SDS-PAGE, followed by immunoblotting with the anti-ADD1/SREBP-1c antibody. A representative experiment is shown. The autoradiograms corresponding to three independent experiments were quantitated by scanning densitometry. Results are expressed as arbitrary units of ADD1/SREBP-1c protein content and are the mean ± SE.
Insulin induced thermogenic differentiation is impaired in IGF-IR-deficient brown adipocytes8^3%7t[, 百拇医药
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 in a PI 3-kinase-dependent manner (5, 9, 10). Accordingly, the UCP-1 protein content was analyzed by direct Western blot analysis in mitochondrial protein extracts of both cell types upon insulin stimulation. As shown in Fig. 7A, UCP-1 protein expression was up-regulated in response to 24-h insulin treatment in immortalized brown adipocytes derived from the wild-type mice, whereas in IGF-IR-deficient cells this response was completely abolished. It has been reported that the 5'-flanking region of the UCP-1 gene contains C/EBP-regulated sites (36). In fact, previous data from our laboratory indicated that the three canonical C/EBP isoforms ({alpha} , ß, and ) are expressed in BAT during late fetal development, with C/EBP protein content being positively regulated by insulin (6, 37). Based on that, we performed anti-C/EBP Western blot analysis in nuclear extracts from control and insulin-stimulated brown adipocytes. As shown in Fig. 7B, the expression of C/EBP{alpha} was significantly induced by 24 h of insulin treatment in wild-type cells, but not in IGF-IR-/- cells. In all experiments similar loading of protein extracts was assessed by Ponceau Red staining of the membranes. These results indicate that the lack of IGF-IR during fetal development of brown adipocytes leads to a loss of thermogenic-related gene expression.
fig.ommitteed'!2({, http://www.100md.com
Figure 7. Insulin effect on UCP-1 and p42C/EBP expression in wild-type and IGF-IR-deficient brown adipocytes. A, Quiescent IGF-IR+/+ and IGF-I -/- were stimulated with insulin (10–100 nM) for 24 h. Control cells were cultured in the absence of hormone. At the end of the culture, mitochondrial proteins were extracted, and equals amount of protein were submitted to Western blot analysis with anti-UCP-1 and anticytochrome c antibodies. Representative experiments are shown. The autoradiograms corresponding to three independent experiments were quantitated by scanning densitometry. Results are expressed as arbitrary units of UCP-1 normalized to the amount of cytochrome c and are the mean ± SE. B, Quiescent cells were stimulated with insulin (10–100 nM) for 24 h. Control cells were cultured in the absence of the hormone. Cell were then lysed, and equals amount of nuclear protein were submitted to SDS-PAGE, followed by immunoblotting with the anti-C/EBP antibody. A representative experiment is shown. The autoradiograms corresponding to three independent experiments were quantitated by scanning densitometry. Results are expressed as arbitrary units of C/EBP and are the mean ± SE.
Effect of PPAR or C/EBP overexpression in the adipogenic and thermogenic differentiation capacity of IGF-IR-deficient brown adipocytesw|, 百拇医药
C/EBP and PPAR transcription factors are induced during brown adipogenesis in a similar fashion as white cell differentiation. Accordingly, our next goal was to investigate whether overexpression of these transcription factors is able to rescue UCP-1 and FAS expression in IGF-IR-/- brown adipocytes, bypassing insulin signaling. For this goal, we performed retrovirus-mediated C/EBP or PPAR gene transfer into IGF-IR-/- brown adipocytes (Fig. 8A). After retroviral infection, C/EBP was overexpressed in IGF-IR-/- brown adipocytes about 5-fold, whereas PPAR was overexpressed about 10-fold. Interestingly, PPAR expression was slightly induced in deficient cells overexpressing C/EBP. Next, we analyzed FAS expression, an adipogenic marker, in insulin-stimulated C/EBP and PPAR-IGF-IR-/- cells. The Northern blot depicted in Fig. 8B (upper panel) revealed that FAS mRNA was up-regulated in PPAR-IGF-IR-/- cells, and insulin treatment did not further increase FAS mRNA expression. However, insulin treatment slightly increased FAS mRNA in C/EBP-IGF-IR-/- cells, with the overexpression of C/EBP restoring the insulin sensitivity lost in IGF-IR-/- brown adipocytes. To further analyze the effect of insulin on UCP-1 protein content in C/EBP and PPAR-IGF-IR-/- cells, we performed anti-UCP-1 Western blot analysis after 24 h of insulin stimulation (Fig. 8B, lower panel). Overexpression of PPAR, but not C/EBP{alpha} , up-regulated UCP-1 expression in IGF-IR-deficient cells in an insulin-independent manner.
fig.ommitteed@v, http://www.100md.com
Figure 8. Effect of PPAR or C/EBP overexpression in the adipogenic and thermogenic differentiation capacity of IGF-IR-deficient brown adipocytes. A, IGF-IR-/- fetal brown adipocyte cell lines overexpressing C/EBP or PPAR were generated as described in Materials and Methods. Nuclear extracts were prepared from growing cells (10% FS) and submitted to SDS-PAGE, followed by immunodetection with anti-C/EBP and PPAR antibodies. B, Upper panel, IGF-IR-/- brown adipocytes and IGF-IR-/- cells overexpressing C/EBP or PPAR were serum-starved for 20 h. Quiescent cells were cultured for 12 h in serum-free medium in either the absence or presence of insulin (10–100 nM). At the end of the culture, total RNA (10 µg) was submitted to Northern blot analysis and hybridized with labeled FAS and 18S rRNA cDNAs. Representative autoradiograms are shown. Results are expressed as arbitrary units of FAS mRNA and are the mean ± SE of three independent experiments. Lower panel, IGF-IR-/- brown adipocytes and IGF-IR-/- 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). At the end of the culture, cells were harvested, and mitochondrial protein extracts were submitted to SDS-PAGE and immunodetected with anti-UCP-1 or anticytochrome c antibodies. The autoradiograms corresponding to three independent experiments were quantitated by scanning densitometry. Results are expressed as arbitrary units of UCP-1 normalized to the amount of cytochrome c and are the mean ± SE.
Discussion;foc|*, 百拇医药
Fetal brown adipocytes differentiate at the end of fetal life on the basis of two programs: an adipogenic program related to the expression of a set of lipogenic enzymes resulting in the appearance of a multilocular fat droplets phenotype (6) and a thermogenic program of UCP-1 expression to prevent newborn hypothermia through the nonshivering thermogenesis mechanism (reviewed in Ref. 4). Among the complex of signals involved in the onset of fetal brown adipocyte differentiation, our laboratory has reported an essential role of insulin and IGF-I in triggering this process (5, 6, 7). To further investigate the molecular mechanisms by which insulin/IGF-I elicit their actions in fetal brown adipocytes, we have extensively studied the signal transduction pathways leading to the expression of metabolic genes as well as the tissue-specific thermogenic marker UCP-1. However, the fact that primary brown adipocytes display high affinity binding sites for both insulin and IGF-I (6) makes it difficult to dissect the relative contributions of the two receptors. To address this point, we recently generated immortalized fetal brown adipocytes from the fetuses of IGF-IR-deficient mice (IGF-IR-/-). Under growing conditions, these cells maintained the constitutive expression of adipogenic genes as well as UCP-1 compared with immortalized wild-type brown adipocytes (IGF-IR+/+). Surprisingly, the lack of IGF-IR was compensated for by an increased insulin signaling and mitogenic response to the hormone through the IRS-1/Grb-2/MAPK pathway. This occurs because PTP1B enzymatic activity and its association with either IR or IRS-1 is severely impaired in insulin-stimulated IGF-IR-deficient cells (23).
Based on our previous findings suggesting that IGF-I might have a role in vivo leading BAT to differentiate before birth (6), in the present paper we have investigated the consequences of the lack of IGF-IR and its signaling regarding this process. As we have demonstrated an essential role of IRS-1 via PI 3-kinase in inducing adipogenic and thermogenic gene expression upon insulin or IGF-I stimulation of primary brown adipocytes (9, 10, 30), we here have further explored this signaling pathway in immortalized IGF-IR-deficient brown adipocytes. As summarized in Fig. 9, IGF-IR-/- brown adipocytes showed increased insulin sensitivity regarding IRS-1 tyrosine phosphorylation and IRS-1-associated PI 3-kinase activity compared with wild-type cells despite a marked decrease in its IRS-1 protein content. These results indicate that in addition to the IRS-1/Grb-2/Ras/MAPK pathway (23), the IRS-1/PI 3-kinase pathway is overstimulated in IGF-IR-deficient brown adipocytes upon insulin treatment. Downstream from PI 3-kinase, activation of Akt/PKB, but not p70s6 kinase or PKC{zeta} , was significantly increased in IGF-IR-deficient cells. Recently, PKC has been implicated by a number of laboratories, including ours, as a key mediator of glucose transport during insulin action (34, 38, 39, 40). Thus, the results presented here confirm that activation of PKC by insulin parallels glucose uptake in fetal brown adipocytes. Interestingly, neither GLUT4 protein content nor insulin-induced glucose uptake is augmented in IGF-IR-deficient brown adipocytes compared with the wild type despite the significant increase in Akt/PKB activation.
fig.ommitteed!4#ea\, 百拇医药
Figure 9. Differential insulin signaling between wild-type and IGF-IR-/- fetal brown adipocytes. IGF-IR-/- cells showed an increased insulin sensitivity regarding IRS-1 tyrosine phosphorylation and IRS-1-associated PI 3-kinase activity. Downstream PI 3-kinase activation of Akt was elicited at lower doses of insulin in IGF-IR-/- brown adipocytes. Activation of PKC or p70s6 kinase by insulin was similar in both cell types. Insulin up-regulated adipogenic (ADD1/SREBP-1c and FAS) and thermogenic (C/EBP and UCP-1) gene expression in wild-type fetal brown adipocytes; this effect was impaired in the absence of IGF-IR.!4#ea\, 百拇医药
Differentiation of fetal brown adipocytes in response to insulin or IGF-I can be monitored by the expression of lipogenic enzymes. Immortalized brown adipocytes respond to insulin in inducing FAS mRNA and protein expression as previously reported in parental cells (6). It was surprising to find that insulin was not able to induce FAS expression in IGF-IR-/- cells despite an enhancement of IRS-1/PI 3-kinase/Akt signaling. Recently, the effect of insulin on FAS gene expression in the liver has been reported to be dependent on the transcription factor ADD1/SREBP-1c (41, 42, 43), which also controls adipocyte differentiation (44). In our cell system, insulin stimulation resulted in a parallel increase in ADD1/SREBP-1c mRNA in wild-type brown adipocytes, but not in IGF-IR-deficient cells. In addition, the amount of the nuclear mature form of ADD1/SREBP-1c was increased by insulin only in brown adipocytes expressing IGF-IR. Conversely, insulin-induced glucose transport, which is another requirement for the effect of insulin on FAS gene expression in white adipose tissue, is not impaired in IGF-IR-deficient brown adipocytes. However, this result can be explained on basis of previous data from our laboratory indicating that in fetal brown adipocyte primary cultures glucose does not affect FAS mRNA induction by insulin (7). Taken together, these results indicate that in fetal brown adipocytes insulin action on FAS expression requires ADD1/SREPB-1c as well as the intact IGF-IR signaling machinery. However, we cannot conclude whether insulin has a direct effect on the proteolysis of the endoplasmic reticulum-derived precursor in wild-type brown adipocytes or is a rapid constitutive process, as recently suggested by Azzout-Marniche et al. (45).
Regarding transcriptional regulation of thermogenesis, C/EBP{alpha} has been shown to directly trans-activate the UCP-1 gene through specific binding sites in its promoter (36, 46). In addition, C/EBP expression increases gradually in rat BAT during late fetal life (6), its mRNA being up-regulated by insulin in BAT adipocyte primary cultures (37). Here, we show that in immortalized fetal brown adipocytes p42C/EBP expression is also up-regulated by insulin treatment. However, when these cells lack IGF-IR, they are unable to induce both C/EBP{alpha} and UCP-1 upon insulin stimulation. Although the differences in C/EBP{alpha} expression between wild-type and IGF-IR littermates in vivo in BAT are not known, these results have two important implications. Firstly, signaling through IGF-IR during fetal development is essential for insulin action regarding C/EBP{alpha} expression, and secondly, binding of newly synthesized C/EBP{alpha} to UCP-1 promoter seems to be necessary for the induction of UCP-1 expression by insulin. In this regard, Kulkarni et al. (47) in a recent report show that the lack of IGF-IR in ß-cells negatively influences the expression of early transcription factors controlling differentiation. These results support our idea that IGF-IR participates in the control of differentiated function of responsive cells.
The fact that C/EBP and PPAR families of transcription factors play an important role in the induction of fully differentiated brown adipocyte phenotype prompted us to restore differentiation-related gene expression in IGF-IR-deficient cells by overexpressing C/EBP{alpha} or PPAR{gamma} by retroviral infection. Our results indicate that only PPAR{gamma} could overcome FAS mRNA impairment in an insulin-independent manner. However, insulin increased FAS mRNA expression in C/EBP-IGF-IR-/- cells, but not in PPAR{gamma} -IGF-IR-/- cells, in a dose-dependent manner. These data clearly indicate that C/EBP{alpha} , but not PPAR{gamma} , reconstitutes insulin sensitivity regarding FAS expression in brown adipocytes and are entirely consistent with the results previously described in the differentiation protocol of 3T3 L1 adipocytes. In these cells, C/EBP{alpha} is required for establishment of insulin-sensitive glucose transport (48). Conversely, fetal brown adipocytes lacking IGF-IR up-regulated UCP-1 expression in the absence of insulin when PPAR{gamma} , but not C/EBP{alpha} , was overexpressed. These results indicate that despite the presence of binding sites for both transcription factors within the UCP-1 promoter, only PPAR{gamma} can restore UCP-1 expression in the absence of IGF-IR and its signaling through development. A possible explanation for these results is based on the fact that during fetal development of BAT, C/EBP{alpha} expression concurs with IGF-IR expression (6). The lack of IGF-IR in brown adipocytes (IGF-IR-/-cells) might suppress the expression of additional molecules (i.e. coactivators) involved in C/EBP{alpha} induction and UCP-1 trans-activation.
In summary, we have shown that the lack of IGF-IR results in a severe impairment of adipogenic and thermogenic differentiation-related gene expression in response to insulin despite the enhancement of IRS-1/PI 3-kinase/Akt signaling. These data demonstrated a critical role for IGF-IR in fetal brown adipocyte differentiation. The effect of IGF-IR is mimicked by exogenous expression of PPAR in an insulin-independent manner. Further work will be needed to define the link between IGF-IR signaling and the expression of various adipogenic and thermogenic transcription factors during fetal development of BAT.+1+(, 百拇医药
Acknowledgments+1+(, 百拇医药
We thankfully acknowledge Bruce S. Spiegelman (Dana Farber Cancer Institute, Boston, MA) for the pBabe hygro C/EBP and pBabe bleo PPAR{gamma} retroviral constructs.+1+(, 百拇医药
Received August 7, 2002.+1+(, 百拇医药
Accepted for publication October 23, 2002.+1+(, 百拇医药
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