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• W2241599947 abstract "In light of the pivotal role that PPARγ2 plays in the expression of fat specific genes (e.g., A-FABP), we have examined the hypothesis that a rise in PPARγ2 protein is required for the expression of A-FABP, and that the acceleration of fat cell differentiation by the thiazolidinedione agent, pioglitazone (PIOG), reflects an increase in the abundance of PPARγ2 mRNA and protein. Western analyses surprisingly revealed that undifferentiated 3T3-L1 fibroblasts contained significant levels of PPARγ2 protein; that the amount of total cellular PPARγ2 only increased 2-fold during differentiation; and that the levels of PPARγ2 protein and mRNA were not increased by PIOG even though fat cell differentiation was accelerated by PIOG as revealed by a 20-fold increase in A-FABP expression. Cell fractionation studies revealed that PPARγ2 was evenly distributed between the cytosolic and nuclear compartments in both undifferentiated and differentiating 3T3-L1 cells. Immunocytochemical studies with a PPARγ2-specific antibody indicated that PPARγ2 was diffusely distributed throughout the cytosol of undifferentiated 3T3-L1 cells, but as the differentiation progressed, the PPARγ2 became focused around the developing lipid droplets. In contrast to PPARγ2, undifferentiated 3T3-L1 cells contained no measurable quantities of RXRα, but once fat cell differentiation was initiated by treatment with IBMX and dexamethasone, the cellular content of RXRα increased several fold. The rise in RXRα content paralleled the induction of A-FABP, but the expression of RXRα was not enhanced by PIOG. Although the amount of PPARγ2 and RXRα was unaffected by PIOG, gel shift assays revealed that PIOG stimulated PPARγ2/RXRα binding to the adipose response element of A-FABP by 5-fold in less than 12 h. Apparently, RXRα rather than PPARγ2 is the pivotal trans-factor essential for the initiation of terminal fat cell differentiation. However, the high cytsolic content of PPARγ2 and its association with the lipid droplet of differentiating 3T3-L1 cells suggests PPARγ2 may possess a cytosolic function in the developing fat cell.—Thuillier, P., R. Baillie, X. Sha, and S. D. Clarke. Cytosolic and nuclear distribution of PPARγ2 in differentiating 3T3-L1 preadipocytes. J. Lipid Res. 1998. 39: 2329–2338. In light of the pivotal role that PPARγ2 plays in the expression of fat specific genes (e.g., A-FABP), we have examined the hypothesis that a rise in PPARγ2 protein is required for the expression of A-FABP, and that the acceleration of fat cell differentiation by the thiazolidinedione agent, pioglitazone (PIOG), reflects an increase in the abundance of PPARγ2 mRNA and protein. Western analyses surprisingly revealed that undifferentiated 3T3-L1 fibroblasts contained significant levels of PPARγ2 protein; that the amount of total cellular PPARγ2 only increased 2-fold during differentiation; and that the levels of PPARγ2 protein and mRNA were not increased by PIOG even though fat cell differentiation was accelerated by PIOG as revealed by a 20-fold increase in A-FABP expression. Cell fractionation studies revealed that PPARγ2 was evenly distributed between the cytosolic and nuclear compartments in both undifferentiated and differentiating 3T3-L1 cells. Immunocytochemical studies with a PPARγ2-specific antibody indicated that PPARγ2 was diffusely distributed throughout the cytosol of undifferentiated 3T3-L1 cells, but as the differentiation progressed, the PPARγ2 became focused around the developing lipid droplets. In contrast to PPARγ2, undifferentiated 3T3-L1 cells contained no measurable quantities of RXRα, but once fat cell differentiation was initiated by treatment with IBMX and dexamethasone, the cellular content of RXRα increased several fold. The rise in RXRα content paralleled the induction of A-FABP, but the expression of RXRα was not enhanced by PIOG. Although the amount of PPARγ2 and RXRα was unaffected by PIOG, gel shift assays revealed that PIOG stimulated PPARγ2/RXRα binding to the adipose response element of A-FABP by 5-fold in less than 12 h. Apparently, RXRα rather than PPARγ2 is the pivotal trans-factor essential for the initiation of terminal fat cell differentiation. However, the high cytsolic content of PPARγ2 and its association with the lipid droplet of differentiating 3T3-L1 cells suggests PPARγ2 may possess a cytosolic function in the developing fat cell. —Thuillier, P., R. Baillie, X. Sha, and S. D. Clarke. Cytosolic and nuclear distribution of PPARγ2 in differentiating 3T3-L1 preadipocytes. J. Lipid Res. 1998. 39: 2329–2338. Adipocytes first appear late in fetal development in preparation for postnatal life when a substantial energy reserve is needed to survive periods of fasting. However, excessive development of adipose tissue affects over 30% of all adults in the United States and represents a significant risk factor for non-insulin-dependent diabetes mellitus (NIDDM), coronary artery disease, and hypertension. Considerable progress has been made during the past few years in our understanding of the molecular control of adipogenesis and adipocyte-specific gene expression. Using the adipocyte-specific fatty acid binding protein (A-FABP) gene as a model, several cis-acting elements and trans-acting factors have been identified that act cooperatively to trigger the terminal differentiation program of adipocytes (1Ross S.R. Graves R.A. Greenstein A. Platt K.A. Shyu H.L. Mellovitz B. Spiegelman B.M. A fat specific enhancer is the primary determinant of gene expression for adipocyte AP-2 in vivo.Proc. Natl. Acad. Sci. USA. 1990; 87: 9590-9594Google Scholar, 2Cook J.S. Lucas J.J. Sibley E. Bolanowski M.A. Christy R.J. Kelley T.J. Lane M.D. Expression of the differentiation-induced gene for fatty acid-binding protein is activated by glucocorticoid and cyclic AMP.Proc. Natl. Acad. Sci. USA. 1988; 85: 2949-2953Google Scholar, 3Cheneval D. Christy R.J. Geiman D. Cornelius P. Lane M.D. Cell free transcription directed by the 422 adipose P2 gene promoter: activation by the CCAAT-enhancer binding protein.Proc. Natl. Acad. Sci. USA. 1991; 88: 8465-8469Google Scholar, 4MacDougald O.A. Cornelius P. Lin F.T. Chen S.S. Lane M.D. Glucocorticoids reciprocally regulate expression of the CCAAT/enhancer-binding protein α and γ genes in 3T3-L1 adipocytes and white adipose tissue.J. Biol. Chem. 1994; 269: 19041-19047Google Scholar, 5Tontonoz P. Hu E. Graves R.A. Budavari A.I. Spiegelman B.M. mPPARγ2: tissue-specific regulator of an adipocyte enhancer.Genes Dev. 1994; 8: 1224-1234Google Scholar). Particular attention has focused on the fat-specific region of the A-FABP gene which is located from −5400 to −4900 bp. This region of the gene contains the adipose response elements (AREs) that instill adipose tissue-specific expression of A-FABP. The AREs are similar to the direct repeat (DR-1) response elements that bind members of the peroxisome proliferator-activated receptor (PPAR) family of transcription factors (5Tontonoz P. Hu E. Graves R.A. Budavari A.I. Spiegelman B.M. mPPARγ2: tissue-specific regulator of an adipocyte enhancer.Genes Dev. 1994; 8: 1224-1234Google Scholar, 6Graves R.A. Tontonoz P. Spiegelman B.M. Analysis of a tissue-specific enhancer: ARF6 regulates adipogenic gene expression.Mol. Cell. Biol. 1992; 12: 1202-1208Google Scholar, 7Isseman I. Green S. Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators.Nature. 1990; 347: 645-650Google Scholar, 8Kliewer S.A. Forman B.M. Blumberg B. Ong E.S. Borgmeyer U. Mangelsdorf D.J. Umesono K. Evans R.M. Differential expression and activation of a family of murine peroxisome proliferator-activated receptors.Proc. Natl. Acad. Sci. USA. 1994; 91: 7355-7359Google Scholar, 9Tontonoz P. Hu E. Spiegelman B.M. Stimulation of adipogenesis in fibroblasts by PPARγ2, a lipid-activated transcription factor.Cell. 1994; 79: 1147-1156Google Scholar, 10Tontonoz P. Hu E. Devine J. Beale E.G. Spiegelman B.M. PPARγ2 regulates adipose expression of the phosphoenolpyruvate carboxykinase gene.Mol. Cell. Biol. 1995; 15: 351-357Google Scholar, 11Forman B.M. Tontonoz P. Chen J. Brun R.P. Spiegelman B.M. Evans R.M. 15-Deoxy-d12,14-prostaglandin J2 is a ligand for the adipocyte determination factor PPARγ2.Cell. 1995; 83: 803-812Google Scholar, 12Kliewer S.A. Lenhard J.M. Willson T.M. Patel I. Morris D.C. Lehmann J.M. A prostaglandin J2 metabolite binds peroxisome proliferator-activated receptor γ and promotes adipocyte differentiation.Cell. 1995; 83: 813-819Google Scholar, 13Vidal-Puig A.J. Considine R.V. Jimenez-Linan M. Werman A. Pories W.J. Caro J.F. Flier J.S. Peroxisome proliferator-activated receptor gene expression in human tissues.J. Clin. Invest. 1997; 99: 2416-2422Google Scholar, 14Yanase T. Yashiro T. Takitani K. Kato S. Taniguchi S. Takayanagi R. Nawata H. Differential expression of PPAR gamma 1 and gamma 2 isoforms in human adipose tissue.Biochem. Biophys. Res. Commun. 1997; 233: 320-324Google Scholar). The identification of the ARE (DR-1) element led to the cloning and identification of a new member of the PPAR family, PPARγ2, which is found in high abundance in adipose tissue and appears to be responsible for regulating the expression of adipose specific genes such as A-FABP (5Tontonoz P. Hu E. Graves R.A. Budavari A.I. Spiegelman B.M. mPPARγ2: tissue-specific regulator of an adipocyte enhancer.Genes Dev. 1994; 8: 1224-1234Google Scholar, 15Rousseau V. Becker D.J. Ongemba L.N. Rahier J. Henquin J.C. Brichard S.M. Developmental and nutritional changes of ob and PPAR gamma 2 gene expression in rat white adipose tissue.Biochem. J. 1997; 321: 451-456Google Scholar). The induction of fat-specific genes such as A-FABP by PPARγ2 requires that PPARγ2 bind to the ARE (DR-1) sequence as a heterodimer complex with the retinoid X receptor alpha (RXRα) (5Tontonoz P. Hu E. Graves R.A. Budavari A.I. Spiegelman B.M. mPPARγ2: tissue-specific regulator of an adipocyte enhancer.Genes Dev. 1994; 8: 1224-1234Google Scholar, 6Graves R.A. Tontonoz P. Spiegelman B.M. Analysis of a tissue-specific enhancer: ARF6 regulates adipogenic gene expression.Mol. Cell. Biol. 1992; 12: 1202-1208Google Scholar). Moreover, interaction of the PPARγ2 with the ARE (DR-1) appears to be dependent upon its ability to bind a collection of ligand activators including eicosanoids (11Forman B.M. Tontonoz P. Chen J. Brun R.P. Spiegelman B.M. Evans R.M. 15-Deoxy-d12,14-prostaglandin J2 is a ligand for the adipocyte determination factor PPARγ2.Cell. 1995; 83: 803-812Google Scholar, 12Kliewer S.A. Lenhard J.M. Willson T.M. Patel I. Morris D.C. Lehmann J.M. A prostaglandin J2 metabolite binds peroxisome proliferator-activated receptor γ and promotes adipocyte differentiation.Cell. 1995; 83: 813-819Google Scholar, 13Vidal-Puig A.J. Considine R.V. Jimenez-Linan M. Werman A. Pories W.J. Caro J.F. Flier J.S. Peroxisome proliferator-activated receptor gene expression in human tissues.J. Clin. Invest. 1997; 99: 2416-2422Google Scholar, 14Yanase T. Yashiro T. Takitani K. Kato S. Taniguchi S. Takayanagi R. Nawata H. Differential expression of PPAR gamma 1 and gamma 2 isoforms in human adipose tissue.Biochem. Biophys. Res. Commun. 1997; 233: 320-324Google Scholar, 15Rousseau V. Becker D.J. Ongemba L.N. Rahier J. Henquin J.C. Brichard S.M. Developmental and nutritional changes of ob and PPAR gamma 2 gene expression in rat white adipose tissue.Biochem. J. 1997; 321: 451-456Google Scholar, 16Yu K. Bayona W. Kallen C.B. Harding H.P. Ravera C.P. McMahon G. Brown M. Lazar M.A. Differential activation of peroxisome proliferator-activated receptors by eicosanoids.J. Biol. Chem. 1995; 270: 23975-23983Google Scholar, 17Wilson T.M. Lehmann J.M. Kliewer S.A. Discovery of ligands for the nuclear peroxisome proliferator-activated receptors.Ann. NY Acad. Sci. 1996; 804: 276-283Google Scholar, 18Forman B.M. Chen J. Evans R.M. The peroxisome proliferator-activated receptors: ligands and activators.Ann. NY Acad. Sci. 1996; 804: 266-275Google Scholar, 19Berger J. Bailey P. Biswas C. Cullinan C.A. Doebber T. Hayes N.S. Saperstein R. Smith R.G. Leibowitz M.D. Thiazolidinediones produce a conformational change in peroxisomal proliferator-activated receptor-γ: binding and activation correlate with antidiabetic actions in db/db mice.Endocrinology. 1996; 137: 4189-4195Google Scholar, 20Lambe K.G. Tugwood J.D. A human peroxisome-proliferator-activated receptor-γ is activated by inducers of adipogenesis including thiazolidinedione drugs.Eur. J. Biochem. 1996; 239: 1-7Google Scholar), fatty acids, and insulin-sensitizing drugs such as pioglitazone (21Kletzien R.F. Clarke S.D. Ulrich R.G. Enhancement of adipocyte differentiation by an insulin-sensitizing agent.Mol. Pharmacol. 1991; 41: 393-398Google Scholar, 22Lehmann J.M. Moore L.B. Smith-Oliver T.A. Wilkison W.O. Wilson T.M. Kliewer S.A. An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor γ (PPARγ).J. Biol. Chem. 1995; 270: 12953-12956Google Scholar, 23Kletzien R.F. Foellmi L.A. Harris P.K. Wyse B.M. Clarke S.D. Adipocyte fatty acid-binding protein: regulation of gene expression in vivo and in vitro by an insulin-sensitizing agent.Mol. Pharmacol. 1992; 42: 558-562Google Scholar, 24Harris K.W. Kletzien R.F. Localization of a pioglitazone response element in the adipocyte fatty acid-binding protein gene.Mol. Pharmacol. 1994; 45: 439-445Google Scholar). Although these observations demonstrate the importance of PPARγ2 in fat cell differentiation, it is unknown whether the abundance of PPARγ2 protein is the limiting determinant for terminal fat cell differentiation. Thus, we have hypothesized that an increase in PPARγ2 protein is required for the conversion of preadipocytes to mature fat cells, and that activating ligands of PPARγ2 such as the thiazolidinedione, pioglitazone, accelerate fat cell differentiation by increasing the abundance of PPARγ2 protein. In fulfilling our objective we have made two interesting discoveries: a) nearly 50% of the total cellular content of PPARγ2 is located in the cytosol compartment of both preadipocytes and mature fat cells; and b) early events of fat cell differentiation appear to be dependent upon the induction of RXRα expression rather than upon the expression of PPARγ2. 3T3-L1 preadipocytes (ATCC #F8979) were grown in Dulbecco's modified Eagle's medium (Gibco BRL) that contained 5% fetal calf serum. When the cells were 70% confluent they were staged to differentiate by changing the medium to one containing 5% newborn calf serum plus 10 μm IBMX and 1 μm dexamethasone. The IBMX and dexamethasone were removed after 48 h (designated as time 0 h), and the medium was changed to one containing either no hormones, 1 μm insulin, or insulin plus 10 μm pioglitazone. To address our hypothesis that an increase in PPARγ2 is a crucial step in terminal fat cell differentiation, antibodies to two domains of PPARγ2 were prepared: a) amino acid residues 2–16 (p2–16) which are specific for PPARγ2; and b) amino acid residues 32–54 (p32–56) which is a domain shared by both PPARγ2 (56 kDa) and PPARγ1 (52 kDa). The anti-serum to mPPARγ2 was raised in rabbits injected with synthetic peptide antigen corresponding to either p2–16 or p32–56 of PPARγ2. Antisera were harvested every 2 weeks and their antigenicity against the respective peptides was determined. Antiserum collected at day 70 was the most antigenic and was subsequently harvested for antibody purification. Affinity-purified anti-PPARγ2 was prepared from serum using the respective peptides linked to sulfo-link affinity columns (Pierce). As can be seen in Fig. 1, Western blot analysis of total protein extracts from undifferentiated and differentiated 3T3-L1 cells revealed that the PPARγ2 specific antibody (p2–16) detected a single band at 56 kDa (lanes 3 and 4); and that this band co-migrated with a protein produced from an in vitro transcription–translation reaction programmed with a vector containing the PPARγ2 open-reading frame (Fig. 1, lane 5). As expected, antibody prepared to the p32–54 domain of PPARγ2 detected both the larger PPARγ2 (56 kDa) and the smaller PPARγ1 (52 kDa) (Fig. 1, lanes 1 and 2). Clearly, the dominant protein in both undifferentiated and differentiated 3T3-L1 cells was the PPARγ2 isoform. The abundance of A-FABP, PPARγ2, and RXRα mRNA was determined by Northern analysis at the times indicated in the variousfigures. RNA isolation and Northern blotting were performed as described previously (25Kim S. Wilson J.J. Allen K.G.D. Clarke S.D. Suppression of renal γ-glutamylcysteine synthetase expression in dietary copper deficiency.Biochim. Biophys. Acta. 1996; 1313: 89-94Google Scholar). Twenty μg of total RNA was loaded on a 1% formaldehyde–agarose gel as described (26Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) and transferred to a Zeta probe membrane (Bio-Rad). The membranes were hybridized with cDNA probes labeled with α-32P-dCTP (DuPont NEN). The full-length cDNA for A-FABP was provided by D. Bernlohr, University of Minnesota (27Bernlohr D.A. Bolanowski M.A. Kelly T.J. Lane M.D. Evidence for an increase in transcription of specific mRNAs during differentiation of 3T3-L1 preadipocytes.J. Biol. Chem. 1985; 260: 5563-5567Google Scholar). PPARγ2 cDNA was a generous gift from B. M. Spiegelman, Dana-Farber Cancer Institute, and RXRα cDNA was provided by T. Neuman, Colorado State University. Autoradiographs were digitally captured and quantified using the Optical Integrator and Image software from Ambis. Total cellular protein extracts were prepared from undifferentiated and differentiated 3T3-L1 cells grown in 100-mm petri dishes. The plates were first washed twice with PBS, and the cells were subsequently scraped from the plates using 0.6 ml of RIPA buffer (PBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 0.25 mm PMSF, 5 mg/ml aprotinin, 1 mm Na orthovanadate). A cell lysate was prepared by passing the cells through a syringe fitted with a 21-gauge needle; the lysate was transferred to a microfuge tube, incubated on ice for 60 min, and subsequently was microfuged at 10,000 g for 20 min at 4°C. The supernatant containing the total cell lysate was saved and stored at −80°C until use. Cytosolic and nuclear protein extracts were prepared according to the method of Sanchez et al. (28Sanchez E.R. Hu J.L. Zhong S. Shen P. Greene M.J. Housley P.R. Potentiation of glucocorticoid receptor-mediated gene expression by heat and chemical shock.Mol. Endocrinol. 1994; 8: 408-421Google Scholar). Briefly, 3T3-L1 cells grown in a 100-mm dish were rinsed with ice-cold PBS and scraped into a microfuge tube with 1.0 ml PBS. The cells were pelleted by centrifugation (1000 g, 10 min). The resulting cell pellet was resuspended in 250 ml of 10 mm HEPES, 1 mm EDTA, 0.5 mm DTT, 0.25 mm PMSF, 50 mm NaF, 2 mm Na metavanadate, and 5 mg/ml leupeptin and pepstatin (buffer A), and homogenized in a dounce homogenizer with 20 strokes. This homogenate was centrifuged at 1000 g for 10 min. The resulting low speed nuclear pellet was utilized for protein extraction as described below, and the supernatant (S1) was processed as follows: 4 m NaCl was added to the S1 fraction to make a final concentration of 0.5 m NaCl and the mixture was incubated on ice for 1 h with occasional vortexing. The S1 fraction was centrifuged at 100,000 g for 1 h and the supernatant was saved as cytosolic extract. Extracts were aliquoted and stored in liquid nitrogen until use. The low speed nuclear pellet was washed twice by resuspension in 250 ml buffer A containing 250 mm sucrose and pelleted at 1000 g for 10 min. The pellet was then resuspended in 100 ml buffer A and 100 ml 1 m NaCl was added to make a final concentration of 0.5 m NaCl. The pellet was there incubated on ice for 1 h with occasional vortexing. After salt extraction the nuclear pellet was centrifuged at 8000 g for 30 min; the supernatant was saved as nuclear extract. Extracts were aliquoted and stored in liquid nitrogen until use. For Western analysis, proteins were separated by SDS-PAGE, transferred, and immobilized on a nitrocellulose membrane at 4°C. The membrane was blocked by incubation with 5% non-fat dry milk in phosphate-buffered saline (PBS) at 4°C overnight. The membrane was then washed briefly in PBS and hybridized with rabbit antibody raised against rat PPARγ2 diluted 1/15,000, or against rat RXRα (Santa Cruz, CA) diluted 1:500 in PBS containing 0.1% Tween 20 (PBS-T). Incubation with antibodies and detection of the antigen–antibody complex were performed using the ECL kit (Amersham) according to the manufacturer's instructions, except that washes after secondary antibody incubation consisted of six changes of buffer over 1 h. Undifferentiated and differentiated 3T3-L1 cells were grown in 12-well plates and induced to differentiate as described previously. At each time indicated in the figure, the cells were fixed in formallin for 15 min. Subsequently, each well was rinsed twice with PBS and treated with 0.03% H2O2 in methanol. After a 20-min incubation, the cells were washed three times with PBS containing 0.1% bovine serum albumin. Subsequently, the cells were incubated with 10% goat serum for 20 min, and then treated for 45 min with a 1:500 dilution of affinity-purified anti-PPARγ2 (p2–16). After antibody incubation the cells were washed with PBS containing bovine serum albumin. Subsequently, the cells were treated for 30 min with anti-rabbit IgG (Vector Lab, Inc.). After three washes with PBS-albumin, antibody staining was performed using vectastain as described by the manufacturer (Vector Lab, Inc.). Nuclei were visualized by 1 min incubation with Harris's hematoxylin. Five μg of nuclear protein extract (29Dignam J.D. Lebowitz R.M. Roeder R.G. Accurate transcription initiation by RNA polymerase I in a soluble extract from isolated mammalian nuclei.Nucleic Acids Res. 1983; 11: 1475-1489Google Scholar) was mixed with 4X Superdex buffer (100 mm KCl, 0.1% NP40, 10% glycerol, 10 mm HEPES, 5 mm MgCl2, 2.5 mm ZnSO4, 0.8 mm DTT), 1 μg of poly dA-dT, and 1 ng of γ-32P-ATP-labeled DNA probe (20,000 dpm). The 20 ml reaction was incubated on ice for 20 min. For cold competition, 200 ng of unlabeled ARE, PPRE, or GRE was added to the binding reaction and kept on ice for 20 min. For supershift, 2 ml of anti-PPARγ2 (p32–54) was added to the tubes at the end of the binding reaction, and incubated on ice for 30 min. Samples were then loaded onto a 4.5% acrylamide gel containing 0.1% NP40. Electrophoresis was run at 200 v for 3 h in a 40 mm Tris and 380 mm glycine buffer (pH 8.3) containing 0.1% NP40. Gels were dried and quantified by radioimagizing. Ablation of the PPARγ2 gel shift by anti-PPARγ2 (p2–16) was achieved by incubating overnight 5 ml of affinity-purified antibody with 5 μg nuclear protein extract in 1X Superdex buffer. After the overnight incubation at 4°C, radiolabeled ARE (40,000 dpm) was added to the reaction mixture and the incubation was continued on ice for an additional 25 min. After incubation the mixture was separated by electrophoresis as described above. The double-stranded oligonucleotides used were (only one strand is shown): ARE: 5′-CAGAAATGCACATTTCACCCAGAGAGAAGGG-3′ GRE: 5′-AGTTTTTGGTTACAAACTGTTCTTAAAACG AGG-3′ PPRE: 5′-GATCTGTGACCTTTGTCCTAGTAAG-3′ When 3T3-L1 preadipocytes undergo terminal differentiation to mature fat cells, the level of PPARγ2 mRNA increases significantly (9Tontonoz P. Hu E. Spiegelman B.M. Stimulation of adipogenesis in fibroblasts by PPARγ2, a lipid-activated transcription factor.Cell. 1994; 79: 1147-1156Google Scholar, 10Tontonoz P. Hu E. Devine J. Beale E.G. Spiegelman B.M. PPARγ2 regulates adipose expression of the phosphoenolpyruvate carboxykinase gene.Mol. Cell. Biol. 1995; 15: 351-357Google Scholar). However, the relationship between the level of PPARγ2 mRNA and PPARγ2 protein is unknown. This is particularly true for the early stages of 3T3-L1 differentiation. Using a two-stage differentiation protocol (see Experimental Procedures), we discovered that total protein extracts from undifferentiated 3T3-L1 cells contained a significant quantity of PPARγ2 protein (see −48 h in Fig. 2). In fact, the level of immunoreactive PPARγ2 in preadipocytes was approximately 30% of that found in mature fat cells. Similarly, the 3T3-L1 preadipocytes contained a level of PPARγ2 mRNA that was comparable to the amount of PPARγ2 protein (Fig. 3). As expected, the level of PPARγ2 mRNA increased significantly in response to differentiation stimuli. However, by applying our two-step differentiation protocol, we discovered that IBMX and dexamethasone were largely responsible for the increased abundance of PPARγ2 mRNA (Fig. 3). Interestingly, the rise in PPARγ2 mRNA resulting from IBMX and dexamethasone treatment was not accompanied by an increase in PPARγ2 protein (Fig. 2). However, when the medium containing IBMX and dexamethasonewas removed and replaced by one that contained insulin, the amount of PPARγ2 protein increased 2- to 3-fold within 2–4 h after insulin exposure (Fig. 2). This increase in PPARγ2 protein was not enhanced by the pioglitazone, a potent enhancer of fat cell differentiation.Fig. 3Increases in PPARγ2 and A-FABP mRNA abundance during the early stages of preadipocyte differentiation. Undifferentiated 3T3-L1 cells were grown to confluence (−48 h) and staged to differentiate as described in Methods. After a 48-h staging period with IBMX and dexamethasone (0 h), the cells were induced to differentiate by changing the media to one containing either 1 μm insulin (designated with − symbol), or insulin plus 10 μm pioglitazone (designated with + symbol). Total RNA was extracted at the hours indicated in the figure, and the Northern blot was sequentially hybridized with cDNAs for mPPARγ2, A-FABP, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The molecular sizes of the PPARγ2 and A-FABP transcripts are noted at the right.View Large Image Figure ViewerDownload (PPT) Western analysis of cytosolic and nuclear protein extracts from undifferentiated 3T3-L1 cells indicated that PPARγ2 protein was distributed evenly between the cytosol and nucleus (Fig. 4 and Fig. 5). When the undifferentiated 3T3-L1 cells were treated with insulin, the 2- to 3-fold increase in total cellular PPARγ2 protein that was noted previously (Fig. 2) appeared to be restricted to the cytosolic fraction, while the nuclear fraction of PPARγ2 remained relatively constant in undifferentiated and differentiated 3T3-L1 cells (Figs. 4 and 5). Even when the cells were treated with pioglitazone, which is a potent ligand activator for PPARγ2 (8Kliewer S.A. Forman B.M. Blumberg B. Ong E.S. Borgmeyer U. Mangelsdorf D.J. Umesono K. Evans R.M. Differential expression and activation of a family of murine peroxisome proliferator-activated receptors.Proc. Natl. Acad. Sci. USA. 1994; 91: 7355-7359Google Scholar, 19Berger J. Bailey P. Biswas C. Cullinan C.A. Doebber T. Hayes N.S. Saperstein R. Smith R.G. Leibowitz M.D. Thiazolidinediones produce a conformational change in peroxisomal proliferator-activated receptor-γ: binding and activation correlate with antidiabetic actions in db/db mice.Endocrinology. 1996; 137: 4189-4195Google Scholar), the amount of immuno-reactive PPARγ2 in the nucleus did not appear to increase (Fig. 4). However, one interesting observation associated with the pioglitazone-treatment of 3T3-L1 cells was that the nuclear protein extracts from pioglitazone-treated cells did not appear to contain the lower molecular weight PPARγ1, while nuclear extracts from cells treated with insulin contained the predominate PPARγ2 protein, and a small quantity of immunoreactive PPARγ1 (Fig. 4, lanes 1 and 3).Fig. 5Intracellular distribution and abundance of PPARγ2 protein in undifferentiated and differentiated 3T3-L1 cells. Undifferentiated 3T3-L1 cells were grown to confluence (−2 days) and staged to differentiate as described in Methods. After a 48-h staging period with IBMX and dexamethasone (0 days), the cells were induced to differentiate by changing the media to one containing 1 μm insulin plus 10 μm pioglitazone. At −2, 0, 5, and 10 days of treatment, the cellular localization of PPARγ2 protein (red) was determined by immunocytochemical staining using PPARγ2 specific anti-PPARγ24–26 (A). Preincubating the anti-PPARγ2 with its specific peptide completely blocked PPARγ2 protein staining (B). Nuclei were detected by hematoxyllin staining (pale blue) and are marked by the large arrows. Lipid vacuoles were detected by oil red O staining and are noted by the thin arrows.View Large Image Figure ViewerDownload (PPT) The cellular fractionation studies indicated that a significant quantity of PPARγ2 was located in the cytosol. To verify this observation, an antibody specific for the N-terminal peptide domain of PPARγ2 (p2–16) was used in a collection of immunocytochemical studies. These studies supported our conclusion that undifferentiated 3T3-L1 preadipocytes contained both cytosolic and nuclear PPARγ2 (Fig. 5). The staining pattern for immunoreactive PPARγ2 indicated that PPARγ2 protein was scattered diffusely throughout the cytosol (Fig. 5). However, as the preadipoctyes underwent differentiation to mature fat cells, the cytosolic content of PPARγ2 increased, and the cytosolic PPARγ2 appeared to be concentrated around the lipid vacuoles of the maturing fat cell (Fig. 5). The concentrating of the PPARγ2 around the lipid vacuoles is particularly noticeable at day 5 and day 10 (Fig. 5). Because undifferentiated 3T3-L1 fibroblasts expressed significant levels of PPARγ2 mRNA and protein (i.e., 30% of fully di" @default.
• W2241599947 created "2016-06-24" @default.
• W2241599947 creator A5032636719 @default.
• W2241599947 creator A5033963884 @default.
• W2241599947 creator A5049259865 @default.
• W2241599947 creator A5076438436 @default.
• W2241599947 date "1998-12-01" @default.
• W2241599947 modified "2023-10-12" @default.
• W2241599947 title "Cytosolic and nuclear distribution of PPARγ2 in differentiating 3T3-L1 preadipocytes" @default.
• W2241599947 cites W1595843384 @default.
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