Effects of Cardiotrophin on Adipocytes
2004; Elsevier BV; Volume: 279; Issue: 46 Linguagem: Inglês
10.1074/jbc.m403998200
ISSN1083-351X
AutoresSanjin Zvonic, Jessica C. Hogan, Patricia Arbour-Reily, Randall L. Mynatt, Jacqueline M. Stephens,
Tópico(s)Pharmacological Effects of Natural Compounds
ResumoCardiotrophin (CT-1) is a naturally occurring protein member of the interleukin (IL)-6 cytokine family and signals through the gp130/leukemia inhibitory factor receptor (LIFR) heterodimer. The formation of gp130/LIFR complex triggers the auto/trans-phosphorylation of associated Janus kinases, leading to the activation of Janus kinase/STAT and MAPK (ERK1 and -2) signaling pathways. Since adipocytes express both gp130 and LIFR proteins and are responsive to other IL-6 family cytokines, we examined the effects of CT-1 on 3T3-L1 adipocytes. Our studies have shown that CT-1 administration results in a dose- and time-dependent activation and nuclear translocation of STAT1, -3, -5A, and -5B as well as ERK1 and -2. We also confirmed the ability of CT-1 to induce signaling in fat cells in vivo. Our studies revealed that neither CT-1 nor ciliary neurotrophic factor treatment affected adipocyte differentiation. However, acute CT-1 treatment caused an increase in SOCS-3 mRNA in adipocytes and a transient decrease in peroxisome proliferator-activated receptor γ (PPARγ) mRNA that was regulated by the binding of STAT1 to the PPARγ2 promoter. The effects of CT-1 on SOCS-3 and PPARγ mRNA were independent of MAPK activation. Chronic administration of CT-1 to 3T3-L1 adipocytes resulted in a decrease of both fatty acid synthase and insulin receptor substrate-1 protein expression yet did not effect the expression of a variety of other adipocyte proteins. Moreover, chronic CT-1 treatment resulted in the development of insulin resistance as judged by a decrease in insulin-stimulated glucose uptake. In summary, CT-1 is a potent regulator of signaling in adipocytes in vitro and in vivo, and our current efforts are focused on determining the role of this cardioprotective cytokine on adipocyte physiology. Cardiotrophin (CT-1) is a naturally occurring protein member of the interleukin (IL)-6 cytokine family and signals through the gp130/leukemia inhibitory factor receptor (LIFR) heterodimer. The formation of gp130/LIFR complex triggers the auto/trans-phosphorylation of associated Janus kinases, leading to the activation of Janus kinase/STAT and MAPK (ERK1 and -2) signaling pathways. Since adipocytes express both gp130 and LIFR proteins and are responsive to other IL-6 family cytokines, we examined the effects of CT-1 on 3T3-L1 adipocytes. Our studies have shown that CT-1 administration results in a dose- and time-dependent activation and nuclear translocation of STAT1, -3, -5A, and -5B as well as ERK1 and -2. We also confirmed the ability of CT-1 to induce signaling in fat cells in vivo. Our studies revealed that neither CT-1 nor ciliary neurotrophic factor treatment affected adipocyte differentiation. However, acute CT-1 treatment caused an increase in SOCS-3 mRNA in adipocytes and a transient decrease in peroxisome proliferator-activated receptor γ (PPARγ) mRNA that was regulated by the binding of STAT1 to the PPARγ2 promoter. The effects of CT-1 on SOCS-3 and PPARγ mRNA were independent of MAPK activation. Chronic administration of CT-1 to 3T3-L1 adipocytes resulted in a decrease of both fatty acid synthase and insulin receptor substrate-1 protein expression yet did not effect the expression of a variety of other adipocyte proteins. Moreover, chronic CT-1 treatment resulted in the development of insulin resistance as judged by a decrease in insulin-stimulated glucose uptake. In summary, CT-1 is a potent regulator of signaling in adipocytes in vitro and in vivo, and our current efforts are focused on determining the role of this cardioprotective cytokine on adipocyte physiology. Cardiotrophin (CT-1), 1The abbreviations used are: CT-1, cardiotrophin; JAK, Janus kinase; LIF, leukemia inhibitory factor; LIFR, LIF receptor; MAPK, mitogen-activated protein kinase; STAT, signal transducers and activators of transcription; ERK, extracellular signal-regulated kinase; CNTF, ciliary neurotrophic factor; PPAR, peroxisome proliferator-activated receptor; IRS, insulin receptor substrate; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; FAS, fatty acid synthase.1The abbreviations used are: CT-1, cardiotrophin; JAK, Janus kinase; LIF, leukemia inhibitory factor; LIFR, LIF receptor; MAPK, mitogen-activated protein kinase; STAT, signal transducers and activators of transcription; ERK, extracellular signal-regulated kinase; CNTF, ciliary neurotrophic factor; PPAR, peroxisome proliferator-activated receptor; IRS, insulin receptor substrate; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; FAS, fatty acid synthase. first identified in a screen of a cDNA library derived from the mouse embryoid bodies, has been shown to support in vitro cardiomyocyte survival and hypertrophy. CT-1 is a naturally occurring protein, 200 amino acids long, with a molecular mass of ∼21.5 kDa (1Pennica D. Swanson T.A. Shaw K.J. Kuang W.J. Gray C.L. Beatty B.G. Wood W.I. Cytokine. 1996; 8: 183-189Crossref PubMed Scopus (63) Google Scholar). CT-1 mRNA expression has been detected at high levels in the heart, skeletal muscle, prostate, ovaries, and liver, as well as fetal heart, lung, and kidney. Lower amounts have also been detected in the thymus, small intestine, lung, kidney, pancreas, testes, and brain, whereas no expression was observed in the spleen (2Pennica D. King K.L. Shaw K.J. Luis E. Rullamas J. Luoh S.M. Darbonne W.C. Knutzon D.S. Yen R. Chien K.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1142-1146Crossref PubMed Scopus (497) Google Scholar). Sequence analyses and structural considerations have shown that CT-1 is a member of the IL-6 cytokine family (2Pennica D. King K.L. Shaw K.J. Luis E. Rullamas J. Luoh S.M. Darbonne W.C. Knutzon D.S. Yen R. Chien K.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1142-1146Crossref PubMed Scopus (497) Google Scholar). The members of this family do not share a great deal of primary amino acid sequence homology (14–24%) (3Rose T.M. Bruce A.G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8641-8645Crossref PubMed Scopus (339) Google Scholar), but they do share common structural features (4Robinson R.C. Grey L.M. Staunton D. Vankelecom H. Vernallis A.B. Moreau J.F. Stuart D.I. Heath J.K. Jones E.Y. Cell. 1994; 77: 1101-1116Abstract Full Text PDF PubMed Scopus (190) Google Scholar) and utilize gp130 signal transducer protein in their receptor complexes (5Kishimoto T. Akira S. Narazaki M. Taga T. Blood. 1995; 86: 1243-1254Crossref PubMed Google Scholar). Both functional and receptor binding studies in cultured cardiomyocytes have shown that CT-1 signals through the gp130/LIFR heterodimer without the further requirement for the α-subunit (6Wollert K.C. Taga T. Saito M. Narazaki M. Kishimoto T. Glembotski C.C. Vernallis A.B. Heath J.K. Pennica D. Wood W.I. Chien K.R. J. Biol. Chem. 1996; 271: 9535-9545Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 7Pennica D. Shaw K.J. Swanson T.A. Moore M.W. Shelton D.L. Zioncheck K.A. Rosenthal A. Taga T. Paoni N.F. Wood W.I. J. Biol. Chem. 1995; 270: 10915-10922Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar, 8Pennica D. Arce V. Swanson T.A. Vejsada R. Pollock R.A. Armanini M. Dudley K. Phillips H.S. Rosenthal A. Kato A.C. Henderson C.E. Neuron. 1996; 17: 63-74Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). However, CT-1 signaling in neuronal cells may require an additional 80-kDa α-subunit (45 kDa after N-linked deglycosylation) present in the receptor complexes in addition to gp130 and LIFR (6Wollert K.C. Taga T. Saito M. Narazaki M. Kishimoto T. Glembotski C.C. Vernallis A.B. Heath J.K. Pennica D. Wood W.I. Chien K.R. J. Biol. Chem. 1996; 271: 9535-9545Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 9Robledo O. Fourcin M. Chevalier S. Guillet C. Auguste P. Pouplard-Barthelaix A. Pennica D. Gascan H. J. Biol. Chem. 1997; 272: 4855-4863Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). These studies have also shown that CT-1 binds the LIFR with affinity equivalent to that of LIF but fails to bind gp130 alone. However, the addition of gp130 enhances its binding to LIFR. From these observations, it has been deduced that CT-1 initially binds LIFR with a low affinity, followed by the recruitment of gp130 into a high affinity binding complex (7Pennica D. Shaw K.J. Swanson T.A. Moore M.W. Shelton D.L. Zioncheck K.A. Rosenthal A. Taga T. Paoni N.F. Wood W.I. J. Biol. Chem. 1995; 270: 10915-10922Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar). The formation of gp130/LIFR complex triggers the activation and auto/trans-phosphorylation of receptor-associated JAK kinases. The activation of JAK family kinases leads to the phosphorylation of the receptor subunits, which can then recruit STAT1 and STAT3, leading to their phosphorylation, nuclear translocation, and the ability to regulate gene expression. Phosphorylated receptor subunits can also recruit the Src homology domain 2 adapter Shc, leading to a complex formation with Grb2 and SOS (10Kumar G. Gupta S. Wang S. Nel A.E. J. Immunol. 1994; 153: 4436-4447PubMed Google Scholar). Grb2-SOS complexes can then activate p21ras, leading to the subsequent, cell type-specific, activation of raf-1, MEK, and ERK1/2 MAPKs (11Nakafuku M. Satoh T. Kaziro Y. J. Biol. Chem. 1992; 267: 19448-19454Abstract Full Text PDF PubMed Google Scholar, 12Boulton T.G. Stahl N. Yancopoulos G.D. J. Biol. Chem. 1994; 269: 11648-11655Abstract Full Text PDF PubMed Google Scholar). As previously noted CT-1 was first identified as a factor promoting cardiac myocyte hypertrophy in vitro with activity at concentrations (0.1 nm) much lower than other IL-6 family cytokines. Similarly, an in vivo chronic administration of CT-1 to rodents resulted in dose-dependent increases in both heart weight and ventricular weight, whereas the total body weight was unaffected (13Jin H. Yang R. Keller G.A. Ryan A. Ko A. Finkle D. Swanson T.A. Li W. Pennica D. Wood W.I. Paoni N.F. Cytokine. 1996; 8: 920-926Crossref PubMed Scopus (60) Google Scholar). These results mimicked those of chronic in vivo stimulation of gp130 receptor (14Hirota H. Yoshida K. Kishimoto T. Taga T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4862-4866Crossref PubMed Scopus (443) Google Scholar). It has been suggested that this function of CT-1 is governed through its activation of the JAK/STAT pathway (15Sheng Z. Knowlton K. Chen J. Hoshijima M. Brown J.H. Chien K.R. J. Biol. Chem. 1997; 272: 5783-5791Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar). CT-1 has also been shown to reduce the expression of tumor necrosis factor-α in lipopolysaccharide-treated animals (16Benigni F. Sacco S. Pennica D. Ghezzi P. Am. J. Pathol. 1996; 149: 1847-1850PubMed Google Scholar). Elevated tumor necrosis factor-α levels have been correlated with myocardial infarction and chronic heart failure (17Latini R. Bianchi M. Correale E. Dinarello C.A. Fantuzzi G. Fresco C. Maggioni A.P. Mengozzi M. Romano S. Shapiro L. J. Cardiovasc. Pharmacol. 1994; 23: 1-6Crossref PubMed Scopus (134) Google Scholar) as well as the development of insulin resistance in type 2 diabetes (18Stephens J.M. Pekala P.H. J. Biol. Chem. 1991; 266: 21839-21845Abstract Full Text PDF PubMed Google Scholar). Like other IL-6 cytokine family members, CT-1 can induce liver acute phase response (19Peters M. Roeb E. Pennica D. Meyer zum Buschenfelde K.H. Rose-John S. FEBS Lett. 1995; 372: 177-180Crossref PubMed Scopus (33) Google Scholar). In vivo administration of CT-1 has also resulted in hypertrophy of the liver, kidneys, and spleen, atrophy of the thymus, and increasing platelet and red blood cell counts (13Jin H. Yang R. Keller G.A. Ryan A. Ko A. Finkle D. Swanson T.A. Li W. Pennica D. Wood W.I. Paoni N.F. Cytokine. 1996; 8: 920-926Crossref PubMed Scopus (60) Google Scholar). Also, CT-1 supports the long term survival of spinal motor neurons and ciliary ganglion neurons, and the effects of CT-1 on ciliary ganglions mimic those of CNTF (7Pennica D. Shaw K.J. Swanson T.A. Moore M.W. Shelton D.L. Zioncheck K.A. Rosenthal A. Taga T. Paoni N.F. Wood W.I. J. Biol. Chem. 1995; 270: 10915-10922Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar). Previous work from our laboratory has shown that CNTF activates JAK/STAT and MAPK (ERK1 and -2) pathways in adipocytes and has several effects on adipocyte physiology (20Zvonic S. Cornelius P. Stewart W.C. Mynatt R.L. Stephens J.M. J. Biol. Chem. 2003; 278: 2228-2235Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Because of its ability to potently activate the JAK/STAT pathway through the gp130/LIFR dimer, we predicted that CT-1 could affect adipocyte physiology and gene expression. Indeed, we have observed that CT-1 activates the JAK/STAT pathway and ERK signaling pathway in fat cells in vitro and in vivo. Although this gp130 cytokine does not attenuate 3T3-L1 adipocyte differentiation, we have observed that CT-1 decreased fatty acid synthase and IRS-1 protein in mature adipocytes and results in a decrease of insulin-stimulated glucose uptake. In addition, we have shown that CT-1 induces SOCS-3 mRNA and acutely represses PPARγ expression. The CT-1-induced repression of PPARγ correlates with binding of STAT 1 to the PPARγ2 promoter, and mutation of this site indicates that it contributes to the expression of PPARγ2 under basal conditions. We have also observed that these effects of CT-1 are independent of ERK1 and -2. To our knowledge, this is the first study to demonstrate the effects of CT-1 on fat cells. Recently, it has been shown that the circulating levels of CT-1 are increased in the serum of patients with ischemic heart disease and valvular heart disease (21Freed D.H. Moon M.C. Borowiec A.M. Jones S.C. Zahradka P. Dixon I.M. Mol. Cell Biochem. 2003; 254: 247-256Crossref PubMed Scopus (56) Google Scholar). Since the onset of cardiovascular disease can be associated with obesity/type 2 diabetes (22Sowers J.R. Frohlich E.D. Med. Clin. North Am. 2004; 88: 63-82Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 23Reaven G. Abbasi F. McLaughlin T. Recent Prog. Horm. Res. 2004; 59: 207-223Crossref PubMed Scopus (269) Google Scholar), the actions of CT-1 in fat may prove to be a relevant link between these two deadly diseases. Materials—Dulbecco's modified Eagle's medium was purchased from Invitrogen. Bovine and fetal bovine sera were purchased from Sigma and Invitrogen, respectively. Rat recombinant CNTF and human recombinant CT-1 were purchased from Calbiochem. Mouse recombinant LIF was purchased from Chemicon International. Insulin and human recombinant growth hormone were purchased from Sigma. U0126 was purchased from Promega. DNase I and Trizol were purchased from Invitrogen. All STAT antibodies were monoclonal IgGs purchased from Transduction Laboratories or polyclonal IgGs purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The highly phosphospecific polyclonal antibodies for STAT1 (Tyr701), STAT3 (Tyr705), and STAT5 (Tyr694) were IgGs purchased from BD Transduction Laboratories and Upstate Biotechnology, Inc. (Lake Placid, NY). Sterol regulatory element-binding protein-1 antibody was a rabbit polyclonal IgG, whereas PPARγ antibody was a mouse monoclonal IgG, both purchased from Santa Cruz Biotechnology. ERK1/ERK2 antibody was a rabbit polyclonal IgG purchased from Santa Cruz Biotechnology. Active ERK antibody was a rabbit polyclonal IgG purchased from Cell Signaling Technology. Akt and FAS antibodies were rabbit polyclonal IgGs purchased from BD Transduction Laboratories. IRS-1 antibody was a polyclonal IgG purchased from Upstate Biotechnology. Horseradish peroxidase-conjugated streptavidin used for detection of ACC was purchased from Pierce. Horseradish peroxidase-conjugated secondary antibodies were purchased from Jackson Immunoresearch. An enhanced chemiluminescence kit was purchased from Pierce. Nitrocellulose and Zeta Probe-GT membranes were purchased from Bio-Rad. Cell Culture—Murine 3T3-L1 preadipocytes were plated and grown to 2 days postconfluence in Dulbecco's modified Eagle's medium with 10% bovine serum. Medium was changed every 48 h. Cells were induced to differentiate by changing the medium to Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 0.5 mm 3-isobutyl-1-methylxanthine, 1 μm dexamethasone, and 1.7 μm insulin. After 48 h, this medium was replaced with Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, and cells were maintained in this medium until utilized for experimentation. Preparation of Whole Cell Extracts—Monolayers of 3T3-L1 preadipocytes or adipocytes were rinsed with phosphate-buffered saline and then harvested in a nondenaturing buffer containing 150 mm NaCl, 10 mm Tris, pH 7.4, 1 mm EGTA, 1 mm EDTA, 1% Triton X-100, 0.5% Igepal CA-630 (Nonidet P-40), 1 μm phenylmethylsulfonyl fluoride, 1 μm pepstatin, 50 trypsin inhibitory milliunits of aprotinin, 10 μm leupeptin, and 2 mm sodium vanadate. Samples were extracted for 30 min on ice and centrifuged at 15,000 rpm at 4 °C for 15 min. Supernatants containing whole cell extracts were analyzed for protein content using a BCA kit (Pierce) according to the manufacturer's instructions. Preparation of Nuclear and Cytosolic Extracts—Cell monolayers were rinsed with phosphate-buffered saline and then harvested in a nuclear homogenization buffer containing 20 mm Tris (pH 7.4), 10 mm NaCl, and 3 mm MgCl2. Igepal CA-630 (Nonidet P-40) was added to a final concentration of 0.15%, and cells were homogenized with 16 strokes in a Dounce homogenizer. The homogenates were centrifuged at 1500 rpm for 5 min. Supernatants were saved as cytosolic extract, and the nuclear pellets were resuspended in half the volume of nuclear homogenization buffer and were centrifuged as before. The pellet of intact nuclei was resuspended again in half of the original volume of nuclear homogenization buffer and centrifuged again. A small portion of the nuclei was used for trypan blue staining to examine the integrity of the nuclei. The majority of the pellet (intact nuclei) was resuspended in an extraction buffer containing 20 mm HEPES (pH 7.9), 420 mm NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, and 25% glycerol. Nuclei were extracted for 30 min on ice and then placed at room temperature for 10 min. Two hundred units of DNase I was added to each sample, and tubes were inverted and incubated an additional 10 min at room temperature. Finally, the sample was subjected to centrifugation at 15,000 rpm at 4 °C for 30 min. Supernatants containing nuclear extracts were analyzed for protein content, using a BCA protein assay kit (Pierce). Gel Electrophoresis and Western Blot Analysis—Proteins were separated in 5, 7.5, 10, or 12% polyacrylamide (acrylamide from National Diagnostics) gels containing SDS according to Laemmli (24Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar) and transferred to nitrocellulose membrane in 25 mm Tris, 192 mm glycine, and 20% methanol. Following transfer, the membrane was blocked in 4% fat-free milk for 1 h at room temperature. Results were visualized with horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence. RNA Analysis—Total RNA was isolated from cell monolayers with Trizol according to the manufacturer's instructions with minor modifications. For Northern blot analysis, 20 μg of total RNA was denatured in formamide and electrophoresed through a formaldehyde-agarose gel. The RNA was transferred to Zeta Probe-GT, cross-linked, hybridized, and washed as previously described (18Stephens J.M. Pekala P.H. J. Biol. Chem. 1991; 266: 21839-21845Abstract Full Text PDF PubMed Google Scholar). Probes were labeled by random priming using the Klenow fragment and [α-32P]dATP. Rodent Adipose Tissue Isolation—Animals were euthanized by cervical dislocation, and tissues were immediately removed and frozen in liquid nitrogen. Frozen tissues were homogenized in a buffer containing 150 mm NaCl, 10 mm Tris, pH 7.4, 1 mm EGTA, 1 mm EDTA, 1% Triton X-100, 0.5% Igepal CA-630 (Nonidet P-40), 1 μm phenylmethylsulfonyl fluoride, 1 μm pepstatin, 50 trypsin inhibitory milliunits of aprotinin, 10 μm leupeptin, and 2 mm sodium vanadate. Homogenates were centrifuged for 10 min at 5,000 rpm to remove any debris and insoluble material and then analyzed for protein content. All C57Bl/6J mice were obtained from a colony at the Pennington Biomedical Research Center. All animal studies were carried out with protocols that were reviewed and approved by institutional animal care and use committees. Electromobility Shift Assays—Double-stranded oligonucleotides were annealed by heating single-stranded 5′ and 3′ oligonucleotides in a boiling water bath and gradually cooling to room temperature. The 4 μg of double-stranded oligonucleotides were 5′-end-labeled with 20 μCi of [32P]dCTP (400–800 Ci/mmol) and with 1 ml each of 5 mm dATP, dTTP, and dGTP with Klenow fragment. The end-labeling reaction was incubated for 15 min at 30 °C and was stopped by adding 1 μl of 0.5 m EDTA. End-labeled oligonucleotides were purified using a Princeton CENTRISEP column, according to the manufacturer's instructions (Princeton Separations, Inc.). Specific activity of the oligonucleotides was determined by scintillation counting. Nuclear extracts were incubated with the end-labeled oligonucleotides (50,000 cpm/μl) for 30 min on ice. The samples were loaded into a prerun (1 h, 100 V at 4 °C) 6% acrylamide/bisacrylamide TBE gel containing 90 mm Tris, 90 mm boric acid, and 2 mm EDTA, pH 8.0. For supershift analysis, nuclear extracts were preincubated with antibody for 1 h at room temperature. For cold competition, the nuclear extracts were incubated with unlabeled oligonucleotide for 15 min on ice prior to incubation with the labeled probe. The gels were run at 20 mA for ∼2 h, dried at 80 °C for 1 h under a vacuum, and then exposed to Eastman Kodak Co. BioMax MS film with a Kodak BioMax high energy intensifying screen. Constructs—The PPARγ2 promoter (–609 to +52) luciferase construct was a generous gift of Dr. Jeffrey Gimble, (Pennington Biomedical Research Center). The PPARγ2 promoter (–609 to +52) luciferase construct was mutated at positions –217 and –212 within the STAT recognition element using the QuikChange site-directed mutagenesis kit according to the manufacturer's instructions (Stratagene). The following oligonucleotide and corresponding antisense oligonucleotide was used to mutate the STAT recognition element (GAC AAT GTA GCA ACG TTC TCC TCG TAA TGT ACC AAG TC). The mutated bases were altered from T to C and are underlined. The resulting reporter plasmid was termed PPARγ2 m217/212 (–609 to +52). The change in sequence was confirmed by sequencing using a Big Dye Terminator Extension Reaction (ABI Prism). The minimum promoter thymidine kinase Renilla promoter was obtained from Promega. Transfections—3T3-L1 preadipocytes were grown to 60% confluence and were transiently transfected with the PPARγ2 promoter (–609 to +52)/luciferase construct or the PPARγ2 m217/212 (–609 to +52)/luciferase construct and with the thymidine kinase/Renilla vector to control for transfection efficiency, using Polyfect transfection reagent according to the manufacturer's instructions (Qiagen). After 48 h of incubation with the Polyfect-DNA solution, cells were treated and then harvested for analysis for firefly luciferase and Renilla luciferase activity using the Dual Luciferase Reporter Assay System (Promega). Relative light units were determined by dividing firefly luciferase activity by Renilla luciferase activity. Results are given ± S.D. Determination of 3H-Labeled 2-Deoxyglucose Uptake—The assay of 3H-labeled 2-deoxyglucose uptake was performed as previously described (18Stephens J.M. Pekala P.H. J. Biol. Chem. 1991; 266: 21839-21845Abstract Full Text PDF PubMed Google Scholar). Prior to the assay, fully differentiated 3T3-L1 adipocytes were serum-deprived for 4 h. Uptake measurements were performed in triplicate under conditions where hexose uptake was linear. The results were corrected for nonspecific uptake, and absorption was determined by 3H-labeled 2-deoxyglcuose uptake in the presence of 5 μm cytochalasin B. Nonspecific uptake and absorption was always less than 10% of the total uptake. In order to examine the sensitivity of 3T3-L1 cells to CT-1 administration, confluent 3T3-L1 preadipocytes and fully differentiated 3T3-L1 adipocytes were treated with CT-1 (0.20 nm) for the times indicated in Fig. 1. Western blot analysis of cell extracts revealed that both undifferentiated and differentiated 3T3-L1 cells responded to CT-1 treatment in a time-dependent manner. Exposure to CT-1 resulted in a time-dependent activation and tyrosine phosphorylation of STAT1 and -3 as well as activation of MAPK (ERK1 and -2). The magnitudes of the responses were undistinguishable in undifferentiated and differentiated 3T3-L1 cells. However, the tyrosine phosphorylation of STAT3 was sustained for a longer period of time in preadipocytes. The total levels of MAPK are shown as a control for even loading. The subcellular distribution of STAT proteins following CT-1 treatment was assessed by treating fully differentiated 3T3-L1 adipocytes with CT-1 (0.20 nm) for various periods of time, followed by isolation of cytosolic and nuclear extracts. Western blot analysis of these extracts, shown in Fig. 2, clearly demonstrated that CT-1 treatment results in the nuclear translocation of STAT1, -3, -5A, and -5B as well as the activation of MAPK. For each STAT protein examined, nuclear translocation occurred within 15 min and returned to basal level within 2 h following the cytokine treatment. This pattern is consistent with STAT activation by CNTF and other gp130 cytokines in 3T3-L1 adipocytes (20Zvonic S. Cornelius P. Stewart W.C. Mynatt R.L. Stephens J.M. J. Biol. Chem. 2003; 278: 2228-2235Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). To further examine the ability of CT-1 to activate STATs in 3T3-L1 adipocytes in comparison with other gp130 cytokines, we exposed fully differentiated adipocytes to various doses of CT-1, CNTF, and LIF for 15 min. Western blot analysis of whole cell extracts, shown in Fig. 3, indicated that the activation of STAT1, -3, and -5 and MAPK (ERK1 and -2) by CT-1 is dose-dependent. Moreover, the STAT3 activation was achieved at much lower doses of CT-1 than those required for activation of STAT1 and STAT5. However, as previously shown, CNTF does not activate STAT3 in a dose-dependent manner (20Zvonic S. Cornelius P. Stewart W.C. Mynatt R.L. Stephens J.M. J. Biol. Chem. 2003; 278: 2228-2235Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). CNTF treatment also did not result in either STAT1 or STAT5 activation. Treatment with 2.0 nm CNTF resulted in substantially less STAT 3 and MAPK activation when compared with 2.0 nm CT-1. LIF treatment also resulted in a dose-dependent activation of STAT1, -3, and -5 and MAPK in a manner and dose comparable with that of CT-1. The total levels of MAPK are shown as a control for even loading. To determine if CT-1 could affect signaling pathways in adipose tissue in vivo, the effects of this cytokine were examined in 7-week-old C57B1/6J mice given an intraperitoneal injection of CT-1 (0.5 μg/animal) or vehicle (saline) control and sacrificed after 15 min. Western blot analysis of whole tissue extracts isolated from the epididymal fat pads of the animals, shown in Fig. 4, demonstrated that the four mice injected with CT-1 had a significant increase in levels of active MAPK and tyrosine-phosphorylated STAT1 and -3. However, there was no detectable STAT activation in the four saline-injected mice, although some active MAPK was observed. Also, the activation of STAT5 proteins by CT-1 was not observed under these conditions. The total levels of MAPK are shown as a control for even loading, whereas an extract from 3T3-L1 adipocytes treated with growth hormone was used as a positive control for STAT5 activation (25Zvonic S. Story D.J. Stephens J.M. Mynatt R.L. Biochem. Biophys. Res. Commun. 2003; 302: 359-362Crossref PubMed Scopus (13) Google Scholar). The differentiation of 3T3-L1 preadipocytes into mature adipocytes is governed by a variety of cell signals. Hence, we examined the ability of CT-1 to modulate adipogenesis of 3T3-L1 cells. Our results clearly demonstrated that neither CT-1 nor CNTF treatment had any profound effects on adipocyte differentiation, as evident by the unaltered expression levels of various adipocyte proteins including PPARγ, IRS-1, STAT5A, and ACC (data not shown). Since CT-1 did not affect the adipogenesis of 3T3-L1 cells, we examined the effects of this cytokine on the expression of genes in fully differentiated 3T3-L1 adipocytes. Since this cytokine is a potent STAT activator, both in vitro and in vivo (Figs. 2 and 4), we hypothesized that activated STATs would modulate transcription in fat cells. Serum-deprived, fully differentiated 3T3-L1 adipocytes were treated with CT-1 (0.20 nm) or CNTF (0.45 nm) for various times, indicated in Fig. 5. Total RNA and whole cell extracts were collected following the treatment. As shown in Fig. 5A, both CT-1 and CNTF administration resulted in a rapid induction of SOCS-3 mRNA levels. However, this up-regulation was very transient following CNTF treatment, whereas CT-1 treatment, at a dose lower than that of CNTF, resulted in a more sustained induction of SOCS-3 mRNA. Both cytokines also resulted in a transient down-regulation of PPARγ mRNA, which was more evident following a 2-h CT-1 treatment. The levels of aP2 mRNA were unchanged. The efficacy of the cytokines in this experiment was demonstrated by their ability to induce STAT3 phosphorylation (Fig. 5B). Since CT-1 is a potent activ
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