Factors Involved in GLUT-1 Glucose Transporter Gene Transcription in Cardiac Muscle

Glucose constitutes a major fuel for the heart, and high glucose uptake during fetal development is coincident with the highest level of expression of the glucose transporter GLUT-1 during life. We have previously reported that GLUT-1 is repressed perinatally in rat heart, and GLUT-4, which shows a low level of expression in the fetal stage, becomes the main glucose transporter in the adult. Here, we show that the perinatal expression of GLUT-1 and GLUT-4 glucose transporters in heart is controlled directly at the level of gene transcription. Transient transfection assays show that the −99/−33 fragment of the GLUT-1 gene is sufficient to drive transcriptional activity in rat neonatal cardiomyocytes. Electrophoretic mobility shift assays demonstrate that the transcription factor Sp1, a trans-activator ofGLUT-1 promoter, binds to the −102/−82 region ofGLUT-1 promoter during the fetal state but not during adulthood. Mutation of the Sp1 site in this region demonstrates that Sp1 is essential for maintaining a high transcriptional activity in cardiac myocytes. Sp1 is markedly down-regulated both in heart and in skeletal muscle during neonatal life, suggesting an active role for Sp1 in the regulation of GLUT-1 transcription. In all, these results indicate that the expression of GLUT-1 and GLUT-4 in heart during perinatal development is largely controlled at a transcriptional level by mechanisms that might be related to hyperplasia and that are independent from the signals that trigger cell hypertrophy in the developing heart. Furthermore, our results provide the first functional insight into the mechanisms regulating muscle GLUT-1 gene expression in a live animal. Glucose constitutes a major fuel for the heart, and high glucose uptake during fetal development is coincident with the highest level of expression of the glucose transporter GLUT-1 during life. We have previously reported that GLUT-1 is repressed perinatally in rat heart, and GLUT-4, which shows a low level of expression in the fetal stage, becomes the main glucose transporter in the adult. Here, we show that the perinatal expression of GLUT-1 and GLUT-4 glucose transporters in heart is controlled directly at the level of gene transcription. Transient transfection assays show that the −99/−33 fragment of the GLUT-1 gene is sufficient to drive transcriptional activity in rat neonatal cardiomyocytes. Electrophoretic mobility shift assays demonstrate that the transcription factor Sp1, a trans-activator ofGLUT-1 promoter, binds to the −102/−82 region ofGLUT-1 promoter during the fetal state but not during adulthood. Mutation of the Sp1 site in this region demonstrates that Sp1 is essential for maintaining a high transcriptional activity in cardiac myocytes. Sp1 is markedly down-regulated both in heart and in skeletal muscle during neonatal life, suggesting an active role for Sp1 in the regulation of GLUT-1 transcription. In all, these results indicate that the expression of GLUT-1 and GLUT-4 in heart during perinatal development is largely controlled at a transcriptional level by mechanisms that might be related to hyperplasia and that are independent from the signals that trigger cell hypertrophy in the developing heart. Furthermore, our results provide the first functional insight into the mechanisms regulating muscle GLUT-1 gene expression in a live animal. Facilitative glucose uptake in mammalian cells is mediated by a family of glucose transporter proteins (GLUT-1 to GLUT-5) (1Gould G.W. Seatter M.J. Gould G.W. Facilitative Glucose Transporters. R. G. Landes Co., Austin, TX1997: 1-38Google Scholar). The pattern of expression of these proteins is very complex: GLUT-1 is found in virtually all tissues and seems to be responsible for the basal glucose uptake (2Flier J.S. Mueckler M. McCall A.L. Lodish H.F. J. Clin. Invest. 1987; 79: 657-661Crossref PubMed Scopus (141) Google Scholar), whereas GLUT-4 is mainly expressed in the peripheral insulin-sensitive tissues (skeletal and cardiac muscle and brown and white adipose tissue) (1Gould G.W. Seatter M.J. Gould G.W. Facilitative Glucose Transporters. R. G. Landes Co., Austin, TX1997: 1-38Google Scholar). In insulin-sensitive-tissues, insulin is able to induce a rapid increase in glucose uptake, and this effect is due to the recruitment of GLUT-4 glucose transporters from an intracellular pool to the plasma membrane (3James D.E. Brown R. Navarro J. Pilch P.F. Nature. 1988; 333: 183-185Crossref PubMed Scopus (449) Google Scholar). Insulin-sensitive tissues express both GLUT-4 and GLUT-1, although GLUT-4 is the major glucose transporter isoform. Thus, in the rat adipose tissue, 90% of glucose transporters expressed are GLUT-4 (4Zorzano A. Wilkinson W. Kotliar N. Thoidis G. Wadzinski B.E. Ruoho A.E. Pilch P.F. J. Biol. Chem. 1989; 264: 12358-12363Abstract Full Text PDF PubMed Google Scholar), and a similar percentage has been observed in skeletal muscle (5Marette A. Richardson J.M. Ramlal T. Balon T.W. Vranic M. Pessin J.E. Klip A. Am. J. Physiol. 1992; 263: C443-C452Crossref PubMed Google Scholar). A higher relative expression of GLUT-1 has been observed in rat cardiomyocytes, where GLUT-1 expression accounts for 30% of the total glucose transporters (6Fischer Y. Thomas J. Sevilla L. Muñoz P. Becker C. Holman G. Kozka I. Palacı́n M. Testar X. Kammermeier H. Zorzano A. J. Biol. Chem. 1997; 272: 7085-7092Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). There are several reports (7Wang C. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3621-3625Crossref PubMed Scopus (23) Google Scholar, 8Clark Jr., C.M. Am. J. Physiol. 1971; 220: 583-588Crossref PubMed Scopus (38) Google Scholar) indicating that in the fetal heart, and also in other muscles, glucose consumption is very high during the late fetal stage and that the response to insulin increases postnatally (7Wang C. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3621-3625Crossref PubMed Scopus (23) Google Scholar). Moreover, the glycogen content in fetal heart and other tissues is higher in near-term fetuses than in the mature rat (9Challice C.E. Virágh S. Challice C.E. Virágh S. Ultrastructure in Byological Systems. 6. Academic Press, New York1973: 91-126Google Scholar). This may constitute an adaptive trait that would confer protection against hypoxic stress in the fetus during delivery (10Newsholme E.A. Leech A.R. Biochemistry for the Medical Sciences. John Wiley & Sons, New York1983: 228-229Google Scholar). Given that glucose transport is a rate-limiting step for glycolysis (11Ren J.M. Marshall B.A. Gulve E.A. Gao J. Johnson D.W. Holloszy J.O. Mueckler M. J. Biol. Chem. 1993; 268: 16113-16115Abstract Full Text PDF PubMed Google Scholar), it is feasible that maintaining a high level of expression of GLUT-1 would be crucial for the fetus, because a constitutive high glucose transport rate would be ensured this way and would help to overcome any hypoxic events that may arise during birth. Thus, anaerobic metabolism of glucose would fulfill the ATP demand of the fetus during the hypoxic episode. Furthermore, the relevance of maintaining appropriate expression levels of GLUT-1 in vivo has been recently highlighted by Seidneret al. (12Seidner G. Garcı́a-Alvarez M. Yeh J.I. O'Driscoll K.R. Klepper J. Stump T.S. Wang D. Spinner N.B. Birnbaum M.J. De Vivo D.C. Nat. Genet. 1998; 18: 188-191Crossref PubMed Scopus (306) Google Scholar); they attribute the cause of a severe human brain disorder to the existence of mutations in GLUT-1 gene that reduce the expression of this transporter, which is specially detrimental to the energy metabolism of brain. It has also been pointed out that an increase in glucose availability may be beneficial for reducing the stress during cardiac ischemic episodes (13Paternostro G. Camici P.G. Lammerstma A.A. Marinho N. Baliga R.R. Kooner J.S. Radda G.K. Ferrannini E. J. Clin. Invest. 1996; 98: 2094-2099Crossref PubMed Scopus (147) Google Scholar, 14Oliver M.F. Opie L.H. Lancet. 1994; 343: 155-158Abstract PubMed Scopus (466) Google Scholar, 15Lazar H.L. Am. J. Cardiol. 1997; 80: 90A-93AAbstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The expression of GLUT-1 and GLUT-4 glucose transporters is strongly regulated during the perinatal development of rat heart, skeletal muscle, and brown adipose tissue. We have described (16Santalucı́a T. Camps M. Castelló A. Muñoz P. Nuel A. Testar X. Palacı́n M. Zorzano A. Endocrinology. 1992; 130: 837-846Crossref PubMed Scopus (0) Google Scholar) that around birth and during the first weeks of neonatal life, glucose transporter expression is characterized by a dramatic change in the accumulations of GLUT-1 and GLUT-4, both mRNA and protein, in rat heart, in skeletal muscle, and in brown adipose tissue. Regarding the signals regulating these processes, we have previously shown (17Castello A. Rodriguez-Manzaneque J.C. Camps M. Perez-Castillo A. Testar X. Palacin M. Santos A. Zorzano A. J. Biol. Chem. 1994; 269: 5905-5912Abstract Full Text PDF PubMed Google Scholar) that thyroid hormones have an essential role in the maintenance of the postnatal induction of GLUT-4 and the repression of GLUT-1 in rat heart. Whether this effect is direct or not is still unknown. In further studies performed in the L6E9 skeletal muscle cell line, we have observed that the differentiation of myoblasts into myotubes is associated with the repression of the expression of GLUT-1 and the induction of GLUT-4 (18Viñals F. Fandos C. Santalucia T. Ferre J. Testar X. Palacin M. Zorzano A. J. Biol. Chem. 1997; 272: 12913-12921Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). In these cells, the −99/−33 region of the GLUT-1proximal promoter drives transcriptional activity of GLUT-1and participates in the reduced transcription after muscle differentiation. Furthermore, we have shown that the Sp1 zinc-finger transcription factor is able to bind to a putative binding site in −91/−86 of the GLUT-1 promoter. Sp1 is able totrans-activate the GLUT-1 promoter in L6E9 cells, and its own expression undergoes down-regulation during muscle cell differentiation and in response to overexpression of the myogenic basic helix-loop-helix factor MyoD (18Viñals F. Fandos C. Santalucia T. Ferre J. Testar X. Palacin M. Zorzano A. J. Biol. Chem. 1997; 272: 12913-12921Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Here we show that the changes in GLUT-1 and GLUT-4 expression in rat heart are due, at least in part, to alterations in the transcriptional rate of both genes. Moreover, the −99/−33 region of the GLUT-1 promoter is also essential for the transcription of GLUT-1 in rat neonatal cardiomyocytes in primary culture and that mutation of the Sp1 site at −91/−86 region compromises the transcriptional activity. Furthermore, binding of Sp1 to this site can be detected in fetal heart and skeletal muscle nuclear extracts but not in extracts from adult heart and muscle. We also show that this reduced binding is due to a reduced Sp1 protein abundance in nuclear extracts from heart and muscle in the adult. Together, these findings suggest that Sp1 contributes to the high level of expression of GLUT-1 in the fetal heart. [α-32P]dCTP, [α-32P]UTP, and [γ-32P]ATP were purchased from ICN, NEN Life Science Products, and Amersham Pharmacia Biotech, respectively. Hybond N+ was from Amersham Pharmacia Biotech, and random primed DNA labeling kit was from Roche Molecular Biochemicals. Immobilon was obtained from Millipore. Most commonly used chemicals were from Sigma. Dulbecco's modified Eagle's medium, fetal bovine serum, glutamine, and antibiotics were obtained from Whittaker (Walkersville, MD). Human recombinant Sp1, a double-stranded oligonucleotide containing an Sp1 consensus binding site, and rRNasin were obtained from Promega (Madison, WI). Clones prGT3 (which contains the 2572-bp 1The abbreviations used are: bp, base pair(s); hrSp1, human recombinant Sp1; EMSA, electrophoretic mobility shift assay. EcoRI rat GLUT-1 cDNA insert) and pSM111 (containing the 2470-bpEcoRI rat GLUT-4 cDNA insert) were kindly provided by Dr. Morris Birnbaum (University of Pennsylvania, Philadelphia, PA) (19Birnbaum M.J. Haspel H.C. Rosen O.M. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5784-5788Crossref PubMed Scopus (434) Google Scholar,20Birnbaum M.J. Cell. 1989; 57: 305-315Abstract Full Text PDF PubMed Scopus (458) Google Scholar). The protocol for the isolation of nuclei was adapted from one previously described (21Boheler K. Chassagne C. Martin X. Wisnewsky C. Schwartz K. J. Biol. Chem. 1992; 267: 12979-12985Abstract Full Text PDF PubMed Google Scholar), although we introduced several modifications, described below. Nuclei were isolated from pooled hearts removed from several litters of anesthetized fetuses or after decapitation of neonates and washed in ice-cold saline. 0.25 g of rat ventricular muscle was homogenized in 25 ml of NA buffer (1 mm Tris-Cl, pH 8, 300 mm sucrose (Merck), 2.5 mm magnesium acetate, 3 mm dithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride, 0.25% (v/v) Triton X-100, and 40 units of hrRNasin/ml of buffer), with 15 strokes in a motor-driven Potter-Elvehjem homogenizer in the cold-room. The homogenate was filtered through four layers of cheesecloth and mixed with one volume of NB buffer (1 mm Tris-Cl, pH 8, 2.4m sucrose, 2.5 mm acetate, 3 mmdithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride, 0.1% (v/v) Triton X-100, and 40 units of rRNasin/ml of buffer). This mixture was layered onto 12-ml cushions of NB buffer and ultracentrifuged in a SW28 Beckman rotor at 27,000 rpm for 60 min at 4 °C. The supernatant and the interphase were discarded, and the nuclei pellet was dislodged with a spatula in 20 ml of NA buffer (without Triton X-100). The nuclear suspension was centrifuged in a SS34 Sorvall rotor for 10 min at 2000 × g, and the nuclei resuspended in 1.5 ml of NA buffer (without Triton X-100). Nuclei were counted in a hemocytometer and pelleted at 4000 rpm for 5 min in a microcentrifuge. The nuclei pellet was resuspended in Keller storage buffer (22Konieczny S.F. Emerson C.P. Mol. Cell. Biol. 1985; 5: 2423-2432Crossref PubMed Scopus (37) Google Scholar) containing 200 units of rRNasin/ml, frozen in liquid nitrogen, and stored in 200-μl aliquots of approximately 2 × 107nuclei each at −80 °C until used. Nuclear run-on reactions were carried out by incubating approximately 2 × 107 heart nuclei in a mixture containing 0.625 mm ATP, 0.312 mm CTP, 0.312 mm GTP, 0.625 μm UTP, 0.5 mCi of 800 Ci/mmol [α-32P]UTP, 300 units of hrRNasin/ml, 40 mmTris-Cl, pH 8.3, 150 mm NH4Cl, and 12.5 mm MgCl2, in a final volume of 400 μl, for 20 min at 27 °C. 2-μl samples were removed from the reactions at 0, 5, 10, and 20 min in order to calculate the incorporation of [α-32P]UTP into RNA. DNA was digested after the addition of an additional 80 units of hrRNasin to each reaction, with 75 μl of RQ1 RNase-free DNase (Promega) for 20 min at 27 °C, prior to isolation of RNA. This was isolated by pelleting the nuclei at room temperature for 2 min at low speed. The supernatant was discarded, and the nuclei were resuspended in 500 μl of GuSCN solution (4m guanidinium thiocyanate, 25 mm sodium citrate, 0.5% N-laurylsarcosyne, 0.1 mβ-mercaptoethanol). Next, 50 μl of 2 m sodium acetate were added to the nuclei, prior to extracting RNA with 500 μl of water-equilibrated acid phenol and 100 μl of chloroform:isoamyl alcohol (49:1) solution. RNA was precipitated with 1 volume of isopropanol and centrifuged for 5 min at full speed. The RNA pellet was washed in 70% ethanol and resuspended in GuSCN solution, followed by another precipitation in 100 μl of isopropanol. The final RNA pellet was resuspended in 100 μl of TE, pH 8, and 2 μl was counted by liquid scintillation, in order to calculate the activity of the RNA solution. Detection of GLUT-4 and GLUT-1 newly transcribed RNA was carried out by hybridization of the labeled RNA to membranes onto which the plasmids containing the cDNAs for GLUT-1 and GLUT-4 (10 μg of each per slot) had been previously slot-blotted. Hybridization and washes were performed as described (21Boheler K. Chassagne C. Martin X. Wisnewsky C. Schwartz K. J. Biol. Chem. 1992; 267: 12979-12985Abstract Full Text PDF PubMed Google Scholar). Data are expressed in ppm as described previously (23Muller F.U. Boheler K.R. Eschenhagen T. Schmitz W. Scholz H. Circ. Res. 1993; 72: 696-700Crossref PubMed Google Scholar, 24Koban M.U. Moorman A.F.M. Holtz J. Yacoub M.H. Boheler K.R. Cardiovasc. Res. 1998; 37: 405-423Crossref PubMed Google Scholar) Preparation of enriched rat neonatal cardiomyocyte culture has been described previously (25Bhavsar P.K. Brand N.J. Yacoub M.H. Barton P.J.R. Genomics. 1996; 35: 11-23Crossref PubMed Scopus (67) Google Scholar). Briefly, ventricular myocardium of 1–2-day Sprague-Dawley rat neonates was minced and digested with collagenase and pancreatin. Isolated cells were collected by centrifugation, pooled, and then preplated for 30 min on plastic dishes (Primaria, Becton Dickinson) to remove non-myocytes, which attach rapidly to plastic. The supernatant, which contains >95% cardiomyocytes as determined by immunohistochemistry with an anti-sarcomeric actin antibody (26Sadoshima J. Aoki H. Izumo S. Circ. Res. 1997; 80: 228-241Crossref PubMed Scopus (76) Google Scholar) 2P. Burton, personal communication. was used to seed 0.5 × 106 cells/well in 35-mm gelatin-coated plastic tissue culture dishes in culture medium (10% horse serum, 5% fetal bovine serum in 4:1 Dulbecco's modified Eagle's medium/199 medium plus 1% (v/v) antibiotics (10,000 units/ml penicillin G and 10 mg/ml streptomycin), 2 mm glutamine, 25 mm HEPES, pH 7.4) containing bromodeoxyuridine (100 μm), in order to stop proliferation of non-myocytic cells. A series of CAT reporter constructs containing different 5′ deletions of rat GLUT-1 promoter extending to a common 3′-end point at +134 (18Viñals F. Fandos C. Santalucia T. Ferre J. Testar X. Palacin M. Zorzano A. J. Biol. Chem. 1997; 272: 12913-12921Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) were transfected into cardiomyocytes (10 μg each). Site-directed mutagenesis of the −99/+134 GLUT-1 CAT construct was performed with the QuickChange™ kit from Stratagene, according to the manufacturer's instructions. The oligonucleotide used for generating the mutant construct was 5′-CCTCAGGCCCCGTACCCCGGCCCACC-3′, which contains a two-nucleotide substitution (underlined) in the core of the Sp1 site (see below). The test plasmids were co-transfected with 7.5 μg of β-galactosidase expression plasmid pON239 (27Cherrington J.M. Mocarski E.S. J. Virol. 1989; 63: 1435-1440Crossref PubMed Google Scholar) to normalize for the efficiency of transfection. Cardiomyocytes were transfected on the day following isolation by a calcium phosphate protocol (25Bhavsar P.K. Brand N.J. Yacoub M.H. Barton P.J.R. Genomics. 1996; 35: 11-23Crossref PubMed Scopus (67) Google Scholar, 28Decock J.B. Gillespie-Brown J. Parker P.J. Sugden P.H. Fuller S.J. FEBS Lett. 1994; 356: 275-278Crossref PubMed Scopus (64) Google Scholar) and harvested 2–3 days later for CAT assay (29Gorman C.M. Moffat L.F. Howard B.H. Mol. Cell. Biol. 1982; 2: 1044-1051Crossref PubMed Scopus (5286) Google Scholar). For the harvesting of cardiomyocytes and preparation of cytoplasmic extracts, cells were washed twice in phosphate-buffered saline and then lysed in 300 μl of reporter lysis buffer (Promega) according to the manufacturer's instructions. After centrifugation in a microcentrifuge for 5 min at 4 °C, the supernatants were stored at −80 °C. CAT activity was measured by incubating 75 μl of cytoplasmic extract with 0.1 μCi of [14C]chloramphenicol, 1.3 mm acetyl-CoA, 200 mm Tris-HCl, pH 7.5, for 3.5 h at 37 °C. At the end of the incubation, extraction into ethyl acetate and thin layer chromatography (29Gorman C.M. Moffat L.F. Howard B.H. Mol. Cell. Biol. 1982; 2: 1044-1051Crossref PubMed Scopus (5286) Google Scholar) were performed. The CAT activity was quantitated using an InstantImager (Packard Instrument Co.). β-Galactosidase activity was measured as described (30). Data are expressed as percentage of maximum expressing construct and represent the average of at least three independent rounds of transfection. Nuclei were isolated from adult rat ventricular muscle for the preparation of nuclear extracts essentially as described above (under “Nuclear Run-on”), although a different homogenization procedure was used, given the particular features of the tissue. Hearts were removed from 20 male Wistar rats after cervical dislocation and were washed in ice-cold saline. Atria were removed from the ventricles, and three sets of ventricular tissue of 4 g each were finely minced with scissors in 35 ml of NA buffer. The tissue was then homogenized with three strokes of 12 s each at 3000 rpm in a Polytron homogenizer (Kynematica, Littau, Switzerland). The homogenate was centrifuged at 2600 × g for 10 min, and the pellets were resuspended in another 35 ml of fresh NA buffer. The suspension was further homogenized in a Potter-Elvehjelm homogenizer with eight strokes of the pestle and then filtered through four layers of cheesecloth. The filtered homogenate was centrifuged again at 2600 × gfor 10 min. 25 ml of NA buffer supplemented with Triton X-100 was used in order to resuspend the pellet, and the suspension was centrifuged again at 2600 × g for 10 min. After this last centrifugation, all three pellets were resuspended in the same 35 ml of NB buffer (see the protocol of the nuclear run-on), and this volume was ultracentrifuged as described under “Nuclear Run-on”. The nuclei pellet was saved and processed for the nuclear extraction. Nuclei from fetal hind limb skeletal muscle were isolated as described under “Nuclear Run-on.” However, 2.2 m sucrose was used for making NB buffer. As to adult rat skeletal muscle, nuclei were isolated according to Zahradka et al. (31Zahradka P. Larson D.E. Selle B.H. Exp. Cell. Res. 1989; 185: 8-20Crossref PubMed Scopus (33) Google Scholar) and Neuferet al. (32Neufer P.D. Carey J.O. Dohm G.L. J. Biol. Chem. 1993; 268: 13824-13829Abstract Full Text PDF PubMed Google Scholar). Nuclei from L6E9 myoblasts and myotubes were isolated as described (33). Nuclear extract preparation and EMSAs were performed as described previously (18Viñals F. Fandos C. Santalucia T. Ferre J. Testar X. Palacin M. Zorzano A. J. Biol. Chem. 1997; 272: 12913-12921Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The −102/−37 probe was obtained by digesting withAvaII the −201/+134 CAT construct, purifying the 66-bp fragment, and filling in the cohesive ends with the Klenow fragment of the DNA polymerase and [α32P]dATP. Both a wild type (wtG1Sp1, 5′-CCTCAGGCCCCGCCCCCCG-3′) and a mutated (mutG1Sp1, 5′-CCTCAGGCCCCGTACCCCG-3′; mutation underlined) oligonucleotide encompassing positions −100 to −82 in the rat GLUT-1 proximal promoter were used as competitors in EMSA. When oligonucleotide wtG1Sp1 was used as a probe, 20 pmol of double stranded oligonucleotide was end-labeled with [γ-32P]ATP by using T4 polynucleotide kinase (Promega), and 10,000 cpm of the probe was incubated with 5 μg of the corresponding nuclear extracts as described by Viñals et al. (18Viñals F. Fandos C. Santalucia T. Ferre J. Testar X. Palacin M. Zorzano A. J. Biol. Chem. 1997; 272: 12913-12921Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). All competitor oligonucleotides were used at 100-fold molar excess. Supershift experiments were performed as described (18Viñals F. Fandos C. Santalucia T. Ferre J. Testar X. Palacin M. Zorzano A. J. Biol. Chem. 1997; 272: 12913-12921Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). SDS-polyacrylamide gel electrophoresis was performed in accordance with the method of Laemmli (34Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar). Proteins were transferred to Immobilon as reported (35Camps M. Castello A. Muñoz P. Monfar M. Testar X. Palacin M. Zorzano A. Biochem. J. 1992; 282: 765-772Crossref PubMed Scopus (130) Google Scholar). Transfer was confirmed by Coomassie Blue staining of the gel after the electroblot. An anti-Sp1 affinity-purified rabbit polyclonal antibody (PEP-2, Santa Cruz Biotechnology) was used at a 5 mg/ml dilution in 1% nonfat dry milk, 0.02% sodium azide in phosphate-buffered saline and incubated overnight at 4 °C. Detection of the immune complexes with the rabbit polyclonal antibody was accomplished using the ECL Western blot detection system (Amersham Pharmacia Biotech). Immunoblots were performed under conditions in which autoradiographic detection was in the linear response range. We have previously shown (16Santalucı́a T. Camps M. Castelló A. Muñoz P. Nuel A. Testar X. Palacı́n M. Zorzano A. Endocrinology. 1992; 130: 837-846Crossref PubMed Scopus (0) Google Scholar) that both GLUT-4 and GLUT-1 glucose transporters are strongly regulated during perinatal development in heart, skeletal muscle, and brown adipose tissue. This results in changes to both mRNA and protein accumulation. The above observations led us to consider that the induction of GLUT-4 mRNA and repression of GLUT-1 during perinatal development may be due to transcriptional control. To test this hypothesis, we carried out a series of nuclear run-on experiments. These experiments were performed on nuclei isolated from fetal and neonatal rat heart, and the transcriptional rate of both GLUT-1 and GLUT-4genes was analyzed over a period of time spanning from fetal day 19 to postnatal day 20. The transcriptional run-on reactions were linear throughout the experiment (Fig. 1), indicating that the incubation conditions were appropriate during the reaction, and substrate was not limiting for the elongation of nascent transcripts. The rate of incorporation of labeled UTP into RNA inversely correlated with the age of the animals from which nuclei were prepared. Thus, transcriptional activity was higher in nuclei from fetal heart than in 15- or 20-day-old neonates. Such a postnatal decrease of general transcription in rat ventricle agrees with previous observations (36McCully J.D. Liew C.C. Biochem. J. 1988; 256: 441-445Crossref PubMed Scopus (9) Google Scholar). Fig. 1 B shows an autoradiograph of a run-on reaction performed with nuclei isolated from 19-day fetal heart. The signals corresponding to the hybridization of newly transcribed GLUT-1 and GLUT-4 RNAs were well above that of background (pBluescript and pGEM), thus indicating that transcription of both genes was active at this developmental stage. The data corresponding to the transcriptional activity ofGLUT-1 and GLUT-4 genes during rat heart perinatal development are shown in Fig.2. Transcription of GLUT-1 was maximal in 19-day fetal heart and had decreased by 50% at birth; in 20-day-old neonates, GLUT-1 transcription was lowest and accounted for <25% of the maximal values (Fig. 2). In contrast,GLUT-4 transcription increased markedly between the late fetal stage and 20-day-old neonates (a nearly 4-fold increase).GLUT-4 transcription levels increased very rapidly after birth, and in 5-day-old neonates were nearly 2-fold greater than those in fetal heart nuclei. As a control, total RNA was prepared from hearts saved from the same litters as the ones used in the nuclear run-on experiments and used to detect the mRNA levels of GLUT-1 and GLUT-4 glucose transporters (data not shown). Thus, GLUT-1 mRNA levels in heart were highest in the fetal stage (18-day-old fetuses) but steadily decreased after birth (1- and 20-day-old rats showed 34 and 10% of the level in 18-day-old fetal heart, respectively), which is in contrast to the increase in GLUT-4 mRNA abundance observed over the same period of time (in 17-day-old fetal heart, expression levels accounted for only 35% of those observed in 20-day-old rat heart, whereas 5-day-old rat heart contained mRNA levels similar to those observed in 20-day-old rat heart). Given the similarity observed between the expression profiles of protein, mRNA, and transcriptional activity of GLUT-1 and GLUT-4 transporters in rat ventricle during perinatal development, the regulation of these two genes in development appears to be regulated chiefly at the level of transcription. Our next goal was to identify thecis-elements that may regulate GLUT-1transcription and to understand how GLUT-1 expression was repressed postnatally in heart and skeletal muscle. In a previous report we showed that the −99/−33 fragment in GLUT-1 promoter drives transcriptional activity of the CAT reporter gene in the L6E9 rat muscle cell line (18Viñals F. Fandos C. Santalucia T. Ferre J. Testar X. Palacin M. Zorzano A. J. Biol. Chem. 1997; 272: 12913-12921Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). These findings prompted us to consider that maybe this region would be also important for the transcription of GLUT-1 in rat cardiomyocytes. This hypothesis was tested through the transfection of a series of 5′ deletion constructs of GLUT-1promoter containing various lengths of the proximal and 5′ upstream sequence through to +134 fused to the CAT gene, into primary cultures of rat neonatal cardiomyocytes prepared from 1–2-day-old neonates (Fig. 3). These experiments showed that transcription was maintained to a high level in the constructs containing deletions from −812 to position −99 of the GLUT-1 promoter, but deletion of the region between −99 and −33 relative toGLUT-1 transcription initiation site resulted in an 80% decrease of CAT transcriptional activity relative to the −99/−33 CAT construct. As observed for the skeletal muscle cell lines, this region, which contains putative binding sites for known transcription factors, such as Sp1, AP2, and a CAAT box, (