Proc. Natl. Acad. Sci. USA Vol. 88, pp. 1933-1937, March 1991
Biochemistry
Transcriptional repression of the mouse insulin-responsive glucose transporter (GLUT4) gene by cAMP (3T3-L1 adipocytes/adipose tissue/forskolin/erythrocyte/brain glucose transporter)
KLAUS H. KAESTNER, JAIME R. FLORES-RIVEROS, JOHN C. MCLENITHAN, MICHEL JANICOT, M. DANIEL LANE*
AND
Department of Biological Chemistry, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205
Contributed by M. Daniel Lane, December 12, 1990
Glucose uptake by adipose tissue is mediated ABSTRACT by two glucose transporters: GLUT4, which is most abundant, and GLUT1. While GLUT1 is expressed in many tissues, GLUT4 is unique to tissues that exhibit insulin-stimulated glucose uptake (heart and skeletal muscle and adipose tissue). In the diabetic state and during starvation, insulin-stimulated glucose uptake and GLUT4 expression are decreased in tissue adipocytes. Using 3T3-L1 adipocytes in culture, we investigated the possibility that these effects are mediated by elevated cellular cAMP. When 3T3-L1 adipocytes were treated for 16 hr with forskolin or 8-Br-cAMP, GLUT4 mRNA and protein were decreased by approximately 70%, while expression of GLUT1 mRNA and protein was increased 3-fold. These changes were accompanied by an increased basal rate of 2-deoxyglucose uptake and a loss of acute responsiveness of hexose uptake to insulin. The magnitude of GLUT4 mRNA depletion/GLUT1 mRNA accumulation was dependent upon the concentration of 8-Br-cAMP. The decrease of GLUT4 mRNA caused by 8-BrcAMP was the result of a decreased transcription rate, while the half-life of the message was unaffected. The increase in GLUT1 mRNA caused by 8-Br-cAMP was the result of both transient transcriptional activation and mRNA stabilization. We suggest that down-regulation of GLUT4 mRNA in adipose tissue in the diabetic state and during starvation is the result of repression of transcription of the GLUT4 gene caused by cAMP.
The ability to take up glucose is a fundamental property of animal cells and is mediated by a family of cell type-specific glucose transport proteins. Several glucose transporter cDNAs have been isolated and found to be expressed in a tissue-specific manner (1). A cDNA which encodes a glucose transporter-i.e., GLUT4-expressed only in cells that exhibit insulin-stimulated glucose uptake (heart and skeletal muscle and adipose tissue), has been cloned and sequenced in several laboratories (2-6). Compelling evidence has been presented (2, 3, 5) that GLUT4 is an insulin-responsive glucose transporter. Hormonal regulation of this glucose transporter is likely to play a major role in controlling glucose homeostasis under varying physiological conditions. The rate of glucose uptake by peripheral tissues is subject to both short-term and long-term regulation. It has been shown that insulin acutely-i.e., within minutes-increases the number of glucose transporters at the plasma membrane by activating their translocation from an intracellular membrane compartment (7, 8). The translocation process largely accounts for the increased Vmax of glucose uptake caused by insulin (9). This effect does not require the synthesis of new glucose transporters (10) and is of greatest magnitude for GLUT4 (9).
Examples of long-term alterations in the responsiveness of glucose uptake to insulin include the fasted and diabetic states. In adipose tissue, both fasting and streptozotocininduced diabetes cause a decrease in GLUT4 mRNA and protein (11-14). Since it is known that diabetes and fasting lead to elevated cAMP in adipose tissue (15), we tested the possibility that cAMP represses expression of the GLUT4 gene, using 3T3-L1 adipocytes as a model system.
EXPERIMENTAL PROCEDURES Cell Culture. Mouse 3T3-L1 preadipocytes were cultured and induced to differentiate as described (16). Briefly, conversion into adipocytes was induced 2 days after confluence by feeding Dulbecco's modified Eagle's medium (DMEM) containing 10%o (vol/vol) fetal bovine serum, 0.5 mM isobutylmethylxanthine, 0.1 AuM dexamethasone, and 170 nM insulin for 2 days (time 0 to day 2). The same medium without isobutylmethylxanthine and dexamethasone, but containing insulin, was fed from day 2 to day 4. From day 4 to day 9, cells received DMEM supplemented only with 10% fetal bovine serum and were fed every other day. On day 8, additions of forskolin derivatives, 8-Br-cAMP, or hormones were made for 16 hr, or in certain cases, for the times indicated. Isolation and Analysis of RNA. Total cellular RNA was isolated by the guanidine thiocyanate method (17). RNase protection analysis was performed according to Melton et aL (18). Antisense probes for GLUT1, GLUT4, stearoyl-CoA desaturase (SCD)1, and SCD2 (19) were synthesized by using T7 RNA polymerase, linearized plasmid templates, and [a-32P]CTP (NEN). The subclones used as probes for GLUT1 and GLUT4 were obtained from the original cDNAs (2) by exonuclease III digestion and correspond to nucleotides 878-1073 and 1949-2200, respectively. For the estimation of mRNA half-lives, 3T3-L1 cells were treated for various times with actinomycin D at 5 ,ug/ml or 100 ,tM 5,6-dichloro-1-f3D-ribofuranosylbenzimidazole (DRB) (20). Isolation of Nuclei and Run-On Transcription Assays. Nuclei from 3T3-L1 adipocytes were isolated on day 9 as described (21), except that 0.15% Nonidet P-40 and 15 strokes in a Dounce homogenizer were used to break the cells; 107 nuclei were used in each run-on transcription reaction. Autoradiographs were quantitated by densitometry and the signals obtained were normalized to the amount of 32P-labeled transcripts hybridized to total 3T3-L1 genomic DNA. Subcellular Fractionation of 3T3-L1 Adipocytes. Homogenization and subcellular fractionation was conducted essentially as described by Clancy and Czech (22), except that the cells were homogenized in a buffer supplemented with proAbbreviations: GLUT4, adipose tissue/muscle glucose transporter; GLUT1, erythrocyte/brain glucose transporter; SCD, stearoyl-CoA desaturase; DRB, 5,6-dichloro-1-p-D-ribofuranosylbenzimidazole. *To whom reprint requests should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 1933
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Biochemistry: Kaestner et al.
tease inhibitor cocktails I and II (PIC I and PIC II) (23) by forcing the cell suspension through a stainless steel ballbearing homogenizer 12 times (24). Cellular cAMP Level and Hexose Uptake Rate. Cellular cAMP levels were determined as described (25) in ethanol extracts of cell monolayers by using a competitive binding assay with cAMP-binding protein obtained from human erythrocyte membranes (26). Hexose uptake assays were conducted as described previously (27). Immunoblot Analysis of GLUTi and GLUT4 Proteins. Initial Western blot experiments revealed rather diffuse bands of GLUTi and GLUT4 proteins, which made quantitation difficult. The possibility was considered that these diffuse electrophoretic patterns were due to heterogeneity of glycosylation of the transporter proteins. Therefore, membrane samples were routinely pretreated with peptide N-glycosidase F (1 unit per 100 ,ug of protein; Boehringer Mannheim) for 48 hr at 370C and then were mixed with 1/3 vol of 3 x electrophoresis sample buffer [187.5 mM Tris HCl, pH 6.8/6% SDS/15% (vol/vol) glycerol/60 mM EDTA/60 mM dithiothreitol]. Electrophoresis was performed in SDS/615% polyacrylamide gradient gels, after which the proteins were transferred to nitrocellulose filters (Sartorius). For immunological detection of transporter proteins, polyclonal antibodies against a synthetic peptide corresponding to amino acid residues 484-501 of the murine adipocyte GLUT4 (2) were raised in rabbits and immunopurified on a peptide affinity column. Polyclonal antibodies against a peptide corresponding to the C-terminal sequence of GLUTi were generously provided by S. Cushman (National Institutes of Health). Blots were developed by the enhanced chemiluminescence method (ECL; Amersham) employing horseradish peroxidase-conjugated goat anti-rabbit IgG. Quantitation of relative band intensity was performed by laser scanning densitometry.
RESULTS To test the hypothesis that elevation of intracellular cAMP causes down-regulation of GLUT4 mRNA, fully differentiated 3T3-L1 adipocytes were treated for 16 hr with various agents known to increase cellular cAMP concentration. Of the agents tested, forskolin was most effective in increasing cellular cAMP (10- to 20-fold, Fig. lA). Glucagon or isoproterenol alone had only small effects on cAMP level, but in combination with forskolin they had more than an additive effect. Accompanying the increase in cAMP concentration caused by the 16-hr treatment with forskolin, cellular GLUT4 mRNA fell by 70-80%, while the GLUTi mRNA rose 2- to 2.5-fold (Fig. 1C and results not shown). Apparently, as a consequence of the effects of forskolin (and to a lesser extent isoproterenol) on expression of the two glucose transporter proteins, the basal rate of hexose uptake increased and the responsiveness of hexose uptake to insulin was almost completely lost (Fig. 1B). To ascertain whether the effects of forskolin on GLUT4 and GLUTi mRNA levels were mediated by activation of adenylate cyclase or through an indirect mechanism, an analog of forskolin-i.e., 1,9-dideoxyforskolin, which does not activate the cyclase (results not shown)-was employed. In addition, the effect of 8-Br-cAMP was tested. As shown in Fig. 2, the expression of neither GLUT4 nor GLUTi mRNA was affected by exposure of 3T3-L1 adipocytes to 1,9dideoxyforskolin for 2 or 16 hr. Furthermore, 8-Br-cAMP, like forskolin, caused a marked decrease (-67%) in GLUT4 message at 16 hr and an increase (-3.4-fold) in GLUTi message which was maximal at 6 hr (Fig. 2). The dependence of changes in GLUTi and GLUT4 message levels on 8-Br-cAMP concentration was assessed after 16 hr of exposure to the cyclic nucleotide. Both the increase
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Proc. Natl. Acad. Sci. USA 88 (1991)
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FIG. 1. Effects of forskolin, glucagon, and isoproterenol on hexose uptake and cellular glucose transporter mRNA level. 3T3-L1 adipocytes either were not treated (column C) or were treated for 16 hr with 50 ,uM forskolin (F), 100 nM glucagon (G), 4 ,.M isoproterenol (I), forskolin and glucagon (F+G), or forskolin, glucagon, and isoproterenol (F+G+I). (A) cAMP levels from duplicate determinations, + range. (B) Rate of 2-deoxy[14C]glucose (DG) uptake. Open bars, basal rate without insulin; hatched bars, insulin-stimulated rate after 10-min incubation with 1 ,uM insulin. (C) RNase protection assay of 10 ,g of total RNA with GLUT1- and GLUT4-specific antisense RNA probes. An additional control sample (tRNA) gave no
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in GLUT1 mRNA (2-fold) and the decrease in GLUT4 mRNA (74%) were maximal at 1 mM 8-Br-cAMP, with half-maximal responses at about 500 ,M (Fig. 3 A and B). Parallel culture dishes of adipocytes were subjected to hexose uptake assays (Fig. 3C). Consistent with the effect of 8-Br-cAMP on mRNA levels, the basal rate of hexose uptake (without insulin) increased half-maximally at =500 AM 8-BrcAMP (due apparently to increased expression of GLUT1 mRNA and protein), while the insulin-stimulated component of hexose uptake decreased half-maximally at '500 AM 8-Br-cAMP (presumably due primarily to decreased expression of GLUT4 mRNA and protein). The insulin-stimulated component decreased by about 2/3 at the highest concentration of the cAMP derivative (Fig. 3C). To determine whether the observed changes in GLUT4 and GLUT1 message levels C
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Biochemistry: Kaestner et al. tRNA
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Proc. Natl. Acad. Sci. USA 88 (1991)
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FIG. 3. Dependence of cellular glucose transporter mRNA level and hexose uptake rate on 8-Br-cAMP concentration. Parallel dishes of 3T3-L1 adipocytes were treated for 16 hr with the indicated concentrations of 8-Br-cAMP. (A) RNase protection analysis was performed with antisense RNA probes for GLUT1, GLUT4, SCD1, and SCD2 on 10 ,ug of total RNA. (B) The mRNA levels for GLUT4 and GLUTI in A were quantitated by laser scanning densitometry. o, GLUT1; e, GLUT4. (C) Rate of 2-deoxy[14C]glucose uptake. o, Basal rate without insulin; * (+I), insulin-stimulated rate after a 10-min incubation with 1 tLM insulin; A (I - C), difference between basal and stimulated rate. were due to a generalized change in the genetic programming, or "dedifferentiation" of 3T3-L1 adipocytes, two markers of adipocyte differentiation were assayed. As is demonstrated in Fig. 3A, neither the message for SCD1 (28) nor that for SCD2 (19) was significantly affected by treatment of the cells
with 8-Br-cAMP. To verify that the observed changes in cellular mRNA concentration are paralleled by fluctuations in the proteins they encode, the total cellular levels of GLUTi and GLUT4 proteins were measured by quantitative Western blotting. As shown in Fig. 4 A (lanes 1 and 2) and B, exposure of 3T3-L1 adipocytes to 8-Br-cAMP for 16 hr caused a 3-fold decrease in the amount of GLUT4 protein, while the cellular levels of GLUTi protein increased to a comparable extent. Thus, the cyclic nucleotide-induced changes in the cellular levels of the two transporter mRNAs gave rise to corresponding changes in the levels of their respective transporter proteins. While cyclic nucleotide treatment had no detectable effect on the fractional distribution of GLUT4 protein between the plasma membrane and low-density microsomes, it caused a substantial increase in the fraction of GLUTI protein at the plasma membrane (Fig. 4B).
FIG. 4. Effect of 8-Br-cAMP on total cellular level and subcellular distribution ofGLUT4 and GLUTi proteins. 3T3-L1 adipocytes were treated or not with 1 mM 8-Br-cAMP (as indicated) for 16 hr, at which time cells were treated or not for 10 min with 1 ,uM insulin. Total cellular membranes (Total) were prepared from 1/4 of the cell homogenate. Low-density microsomes (LDM) and plasma membranes (PM) were prepared by subcellular fractionation of the remainder of the homogenate. Membrane samples were treated with peptide N-glycosidase F before electrophoresis to remove N-linked oligosaccharide chains. Samples were then applied to an SDS/ polyacrylamide gel, electrophoresed, and transferred to a nitrocellulose membrane. The GLUT4 and GLUTi transporter proteins were immunodetected by using specific polyclonal antibodies, and the blots were developed by enhanced chemiluminescence. A typical Western blot is shown in A. Total cellular membranes (lanes 1 and 2) are from 1.8 x 105 cells. Low-density microsomes (lanes 3-6) and plasma membranes (lanes 7-10) are from 7 x 105 cells. The relative electrophoretic migration of two molecular weight markers (45 and 29 kDa) is shown. To obtain the results for the bar graphs shown in B, films from the Western blot shown in A (lanes 3, 5, 7, and 9; without insulin) were subjected to laser scanning densitometry for quantitation. The results are expressed relative to the maximum for GLUT4 or GLUTi protein.
Of interest is the fact that the capacities of the two glucose transporters to undergo acute insulin-stimulated translocation differ after chronic exposure to 3T3-L1 adipocytes to 8-Br-cAMP. GLUT4 protein retained the ability to translocate rapidly in response to insulin despite the large decrease in total amount of GLUT4 protein (Fig. 4A). Thus, the fold increase in amount of GLUT4 protein in the plasma membrane (Fig. 4A, lanes 7-10) and the fold decrease in amount of GLUT4 protein in the low-density microsomal fraction (Fig. 4A, lanes 3-6) due to acute insulin treatment were the same whether or not the cells had been exposed chronically to 8-Br-cAMP (Fig. 4A). In contrast, both the steady-state level and the fraction of GLUTi protein in the plasma membrane were substantially higher in cells treated with 8-Br-cAMP (Fig. 4B) and increased little upon addition of insulin (Fig. 4A, lanes 9 and 10). Conversely, the amount of GLUTi protein in the low-density microsomal fraction did not decrease nearly so much upon acute insulin treatment in
1936
Proc. Natl. Acad. Sci. USA 88 (1991)
Biochemistry: Kaestner et al.
cells exposed chronically to 8-Br-cAMP (Fig. 4A, lanes 5 and 6) as in cells not exposed to the cyclic nucleotide (Fig. 4A, lanes 3 and 4). These results provide an explanation for the increased basal hexose uptake rate and for the loss of responsiveness of hexose uptake to insulin caused by chronic exposure of 3T3-L1 adipocytes to 8-Br-cAMP (Fig. 3C). The observed changes in GLUT4 and GLUTi mRNA levels caused by chronic exposure of the cells to 8-Br-cAMP could result from changes in the rates of gene transcription or of mRNA degradation or a combination of both. To analyze transcription rates of the GLUT4 and GLUTi genes, nuclei were isolated from 3T3-L1 adipocytes treated with 1 mM 8-Br-cAMP for various periods of time, after which nuclear transcriptional run-on experiments were performed. This method provides a measure of the number of specific RNA transcripts undergoing elongation at the time the nuclei are isolated. As demonstrated in Fig. 5, run-on transcription of the GLUT4 gene was rapidly suppressed by treatment with 8-Br-cAMP (90% reduction after 18 hr), while transcription of the GLUTi gene was induced transiently (7-fold over basal after 2 hr of exposure to 8-Br-cAMP). These changes in specific transcription rate caused by 8-Br-cAMP precede the changes in steady-state mRNA levels (compare with the results in Fig. 2) and are of similar magnitude. To assess possible additional effects of 8-Br-cAMP on the rates of turnover of GLUT4 and GLUTi mRNAs, changes in the half-lives of these messages caused by the cyclic nucleotide were investigated. 3T3-L1 adipocytes were treated or not with 1 mM 8-Br-cAMP for 16 hr prior to the addition of two different inhibitors of RNA polymerase II. RNA was isolated at various time intervals and GLUTi and GLUT4 mRNA levels were determined by RNase protection (Fig. 6). Rate constants for the turnover of the two messages were obtained from regression analyses of the semilogarithmic plots shown in Fig. 6, assuming first-order kinetics. Although there is a minor discrepancy between the half-life values obtained using the two RNA polymerase inhibitors, there is a consistent 2-fold increase in the half-life (a measure of message stability) of GLUTi mRNA caused by 8-Br-cAMP. The stability of the GLUT4 message, however, is not significantly altered by the treatment of 3T3-L1 adipocytes with 8-Br-cAMP.
DISCUSSION In this study we show that when cAMP concentration in 3T3-L1 adipocytes is increased for an extended period-e.g., 16 hr-the basal (minus insulin) hexose uptake rate increases dramatically (7- to 8-fold; Figs. 1B and 3C). Accompanying U) :5
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FIG. 6. Effect of 8-Br-cAMP on the turnover of glucose transporter mRNAs. 3T3-L1 adipocytes were treated for 16 hr with or without 1 mM 8-Br-cAMP. At time 0, cells were treated or not with 100 AuM DRB (A and B) or actinomycin D at 5 Ag/ml (C and D). RNA was isolated at the times indicated, and GLUTi (A and C) or GLUT4 (B and D) mRNA abundance was determined by RNase protection and then quantitated by densitometry. Results are expressed as percent of the initial amount of RNA, and mRNA half-lives (ti/, in hr) were calculated, assuming first-order kinetics. o, Untreatee;*, treatment with 1 mM 8-Br-cAMP.
this rise in basal uptake rate is a nearly complete loss of acute responsiveness of hexose uptake to insulin, in part because the basal rate has risen substantially. In this sense the cells have become insulin resistant, although they exhibit this atypically high basal rate of hexose uptake. Normally, insulin rapidly (within 4-5 min) activates hexose uptake by 10- to 20-fold in 3T3-L1 adipocytes. However, after chronic exposure of the cells to 8-Br-cAMP (Fig. 3C) or forskolin (Fig. 1B), insulin exerts only a small acute activating effect-i.e., -1.3-fold-on hexose uptake rate. The loss of responsiveness of hexose uptake caused by cAMP in 3T3-L1 adipocytes is associated with both a decrease in the cellular concentration of GLUT4 protein and an increase in the concentration of GLUT1 protein (Fig. 4). The effect on GLUT4 protein may be particularly relevant to adipose tissue in vivo, where GLUT4 is the major transporter isoform, making up -90% of the total (29, 30). We suggest, therefore, that the repressed expression of the GLUT4 isoform in 3T3-L1 adipocytes caused by cAMP reflects changes known to occur in adipose tissue per se. It should be noted that both diabetes and prolonged fasting, which promote a rise in cellular cAMP (15), have been shown to cause a decrease in GLUT4 mRNA and protein in tissue adipocytes (11-14). It is of interest that the level of GLUTi protein increases in 3T3-L1 adipocytes exposed to 8-Br-cAMP for 16 hr. Under these conditions, GLUTi is localized primarily in the plasma membrane (Fig. 4B) despite the fact that the cells had not been treated with insulin. Normally, in 3T3-L1 adipocytes not exposed to insulin or cAMP, most GLUTi protein is located intracellularly and is rapidly translocated to the plasma membrane when the cells are treated with insulin. The fact that most GLUTi protein in cells exposed chronically to 8-Br-cAMP is in the plasma membrane is consistent with the finding of Clancy and Czech (22) that cAMP causes the rapid
Biochemistry: Kaestner et al. translocation of GLUTi protein from low-density microsomes to the plasma membrane. The observed increase in the GLUTi isoform, as well as its predominant localization in the plasma membrane, is undoubtedly responsible for the cAMP-induced rise in basal hexose uptake rate in 3T3-L1 adipocytes (Figs. 1B and 3C). This effect is probably not representative of the situation in vivo, since there is little
expression of GLUTi protein in tissue adipocytes (29, 30). The mechanism by which cAMP causes down-regulation of GLUT4 mRNA and protein in 3T3-L1 adipocytes involves repression of transcription of the GLUT4 gene. Thus, 8-BrcAMP caused a dramatic decease in nuclear run-on transcription of the GLUT4 gene (Fig. 5) while having little or no effect on the rate of turnover of GLUT4 mRNA (Fig. 6). In contrast, the increases in cellular GLUTi mRNA and protein caused by 8-Br-cAMP appear to be the result of both an initial increased rate of transcription of the GLUTi gene (determined by nuclear run-on transcription analysis; Fig. 5) and a reduced rate of turnover of GLUTi mRNA (Fig. 6). Our findings regarding the GLUTi gene are similar to those of other investigators who studied the effects of cAMP in fibroblasts (31). The availability of the GLUT4 gene promoter (32) will enable us to investigate the molecular basis for transcriptional repression of the GLUT4 gene by cAMP. Preliminary experiments with a chimeric GLUT4-7000-base-pair promoter/chloramphenicol acetyltransferase (CAT) construct transfected stably into 3T3-L1 adipocytes reveal the nearly complete repression of expression of CAT mRNA when cells are treated with 8-Br-cAMP (results not shown). The genetic element(s) within this 7-kilobase-pair segment of the GLUT4 gene promoter responsible for the cAMP effect have not yet been identified. We thank Ms. Natalie Tumminia for expert secretarial assistance. We are grateful to Dr. Sam Cushman for generously providing an antibody to GLUT1. This work was supported by National Institutes of Health Research Grant NIDDK-38418. J.R.F.-R. was supported by a National Research Service Award from the National Institutes of Health and M.J., by a postdoctoral fellowship from the Juvenile Diabetes Foundation. 1. Gould, G. W. & Bell, G. I. (1990) Trends Biochem. Sci. 15, 18-23. 2. Kaestner, K. H., Christy, R. J., McLenithan, J. C., Braiterman, L. T., Cornelius, P., Pekala, P. H. & Lane, M. D. (1989) Proc. Nati. Acad. Sci. USA 86, 3150-3154. 3. James, D. E., Strube, M. & Mueckler, M. (1989) Nature (London) 338, 83-87. 4. Charron, M. J., Brosius, F. C., Alper, S. L. & Lodish, H. F. (1989) Proc. Nati. Acad. Sci. USA 86, 2535-2539. 5. Birnbaum, M. J. (1989) Cell 57, 305-315. 6. Fukumoto, H., Kayano, T., Buse, J. B., Edwards, Y., Pilch,
Proc. Natl. Acad. Sci. USA 88 (1991)
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P. F., Bell, G. I. & Seino, S. (1989) J. Biol. Chem. 264, 7776-7779. 7. Suzuki, K. & Kono, T. (1980) Proc. Natl. Acad. Sci. USA 77, 2542-2545. 8. Cushman, S. W. & Wardzala, L. J. (1980) J. Biol. Chem. 255, 4758-4762. 9. Calderhead, D. M., Kitagawa, K., Tamer, L. I., Holman, G. D. & Lienhard, G. E. (1990) J. Biol. Chem. 265, 1380013808. 10. Jones, T. L. Z. & Cushman, S. W. (1989) J. Biol. Chem. 264, 7874-7877. 11. Charron, M. J. & Kahn, B. B. (1990) J. Biol. Chem. 265, 7994-8000. 12. Garvey, W. T., Huecksteadt, T. P. & Birnbaum, M. J. (1989) Science 245, 60-63. 13. Berger, J., Biswas, C., Vicario, P. P., Strout, H. V., Saperstein, R. & Pilch, P. F. (1989) Nature (London) 340, 70-72. 14. Sivitz, W. I., DeSautel, S. L., Kayano, T., Bell, G. I. & Pessin, J. E. (1989) Nature (London) 340, 72-74. 15. Selawry, H., Gutman, R., Fink, G. & Recant, L. (1973) Biochem. Biophys. Res. Commun. 51, 198-204. 16. Student, A. K., Hsu, R. & Lane, M. D. (1980) J. Biol. Chem. 255, 4745-4750. 17. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. & Rutter, W. J. (1979) Biochemistry 18, 5294-5299. 18. Melton, D. A., Krieg, P. A., Rebaghiati, M. R., Maniatis, T., Zinn, K. & Green, M. R. (1984) Nucleic Acids Res. 12, 70357056. 19. Kaestner, K. H., Ntambi, J. M., Kelly, T. J. & Lane, M. D. (1989) J. Biol. Chem. 264, 14755-14761. 20. Zandomein, R., Mittleman, B., Bunick, D., Ackerman, S. & Weinmann, R. (1982) Proc. Natl. Acad. Sci. USA 79, 31673170. 21. Cornelius, P., Marlowe, M., Lee, M. D. & Pekala, P. H. (1990) J. Biol. Chem. 265, 20506-20516. 22. Clancy, B. M. & Czech, M. P. (1990) J. Biol. Chem. 265, 12434-12443. 23. Olson, T. S. & Lane, M. D. (1987) J. Biol. Chem. 262, 68166822. 24. Olson, T. S., Bamberger, M. J. & Lane, M. D. (1988) J. Biol. Chem. 263, 7342-7351. 25. Janicot, M., Clot, J.-P. & Desbuquois, B. (1988) Biochem. J. 253, 735-743. 26. Gilman, A. G. (1970) Proc. Natl. Acad. Sci. USA 67, 305-312. 27. Frost, S. C. & Lane, M. D. (1985) J. Biol. Chem. 260, 26462652. 28. Ntambi, J. M., Buhrow, S. A., Kaestner, K. H., Christy, R. J., Sibley, E., Kelly, T. J., Jr., & Lane, M. D. (1988) J. Biol. Chem. 263, 17291-17300. 29. Oka, Y., Asano, T., Yoshikazu, S., Kasuga, M., Kanazawa, Y. & Takaku, F. (1988) J. Biol. Chem. 263, 13432-13439. 30. Zorzano, A., Wilkinson, W., Kotliar, N., Thoiolis, G., Wadzinski, B. E., Ruoho, A. E. & Pilch, P. F. (1989) J. Biol. Chem. 264, 12358-12363. 31. Hiraki, Y., McMorrow, I. M. & Birnbaum, M. J. (1989) Mol. Endocrinol. 3, 1470-1476. 32. Kaestner, K. H., Christy, R. J. & Lane, M. D. (1990) Proc. Natl. Acad. Sci. USA 87, 251-255.
Biochemistry
Transcriptional repression of the mouse insulin-responsive glucose transporter (GLUT4) gene by cAMP (3T3-L1 adipocytes/adipose tissue/forskolin/erythrocyte/brain glucose transporter)
KLAUS H. KAESTNER, JAIME R. FLORES-RIVEROS, JOHN C. MCLENITHAN, MICHEL JANICOT, M. DANIEL LANE*
AND
Department of Biological Chemistry, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205
Contributed by M. Daniel Lane, December 12, 1990
Glucose uptake by adipose tissue is mediated ABSTRACT by two glucose transporters: GLUT4, which is most abundant, and GLUT1. While GLUT1 is expressed in many tissues, GLUT4 is unique to tissues that exhibit insulin-stimulated glucose uptake (heart and skeletal muscle and adipose tissue). In the diabetic state and during starvation, insulin-stimulated glucose uptake and GLUT4 expression are decreased in tissue adipocytes. Using 3T3-L1 adipocytes in culture, we investigated the possibility that these effects are mediated by elevated cellular cAMP. When 3T3-L1 adipocytes were treated for 16 hr with forskolin or 8-Br-cAMP, GLUT4 mRNA and protein were decreased by approximately 70%, while expression of GLUT1 mRNA and protein was increased 3-fold. These changes were accompanied by an increased basal rate of 2-deoxyglucose uptake and a loss of acute responsiveness of hexose uptake to insulin. The magnitude of GLUT4 mRNA depletion/GLUT1 mRNA accumulation was dependent upon the concentration of 8-Br-cAMP. The decrease of GLUT4 mRNA caused by 8-BrcAMP was the result of a decreased transcription rate, while the half-life of the message was unaffected. The increase in GLUT1 mRNA caused by 8-Br-cAMP was the result of both transient transcriptional activation and mRNA stabilization. We suggest that down-regulation of GLUT4 mRNA in adipose tissue in the diabetic state and during starvation is the result of repression of transcription of the GLUT4 gene caused by cAMP.
The ability to take up glucose is a fundamental property of animal cells and is mediated by a family of cell type-specific glucose transport proteins. Several glucose transporter cDNAs have been isolated and found to be expressed in a tissue-specific manner (1). A cDNA which encodes a glucose transporter-i.e., GLUT4-expressed only in cells that exhibit insulin-stimulated glucose uptake (heart and skeletal muscle and adipose tissue), has been cloned and sequenced in several laboratories (2-6). Compelling evidence has been presented (2, 3, 5) that GLUT4 is an insulin-responsive glucose transporter. Hormonal regulation of this glucose transporter is likely to play a major role in controlling glucose homeostasis under varying physiological conditions. The rate of glucose uptake by peripheral tissues is subject to both short-term and long-term regulation. It has been shown that insulin acutely-i.e., within minutes-increases the number of glucose transporters at the plasma membrane by activating their translocation from an intracellular membrane compartment (7, 8). The translocation process largely accounts for the increased Vmax of glucose uptake caused by insulin (9). This effect does not require the synthesis of new glucose transporters (10) and is of greatest magnitude for GLUT4 (9).
Examples of long-term alterations in the responsiveness of glucose uptake to insulin include the fasted and diabetic states. In adipose tissue, both fasting and streptozotocininduced diabetes cause a decrease in GLUT4 mRNA and protein (11-14). Since it is known that diabetes and fasting lead to elevated cAMP in adipose tissue (15), we tested the possibility that cAMP represses expression of the GLUT4 gene, using 3T3-L1 adipocytes as a model system.
EXPERIMENTAL PROCEDURES Cell Culture. Mouse 3T3-L1 preadipocytes were cultured and induced to differentiate as described (16). Briefly, conversion into adipocytes was induced 2 days after confluence by feeding Dulbecco's modified Eagle's medium (DMEM) containing 10%o (vol/vol) fetal bovine serum, 0.5 mM isobutylmethylxanthine, 0.1 AuM dexamethasone, and 170 nM insulin for 2 days (time 0 to day 2). The same medium without isobutylmethylxanthine and dexamethasone, but containing insulin, was fed from day 2 to day 4. From day 4 to day 9, cells received DMEM supplemented only with 10% fetal bovine serum and were fed every other day. On day 8, additions of forskolin derivatives, 8-Br-cAMP, or hormones were made for 16 hr, or in certain cases, for the times indicated. Isolation and Analysis of RNA. Total cellular RNA was isolated by the guanidine thiocyanate method (17). RNase protection analysis was performed according to Melton et aL (18). Antisense probes for GLUT1, GLUT4, stearoyl-CoA desaturase (SCD)1, and SCD2 (19) were synthesized by using T7 RNA polymerase, linearized plasmid templates, and [a-32P]CTP (NEN). The subclones used as probes for GLUT1 and GLUT4 were obtained from the original cDNAs (2) by exonuclease III digestion and correspond to nucleotides 878-1073 and 1949-2200, respectively. For the estimation of mRNA half-lives, 3T3-L1 cells were treated for various times with actinomycin D at 5 ,ug/ml or 100 ,tM 5,6-dichloro-1-f3D-ribofuranosylbenzimidazole (DRB) (20). Isolation of Nuclei and Run-On Transcription Assays. Nuclei from 3T3-L1 adipocytes were isolated on day 9 as described (21), except that 0.15% Nonidet P-40 and 15 strokes in a Dounce homogenizer were used to break the cells; 107 nuclei were used in each run-on transcription reaction. Autoradiographs were quantitated by densitometry and the signals obtained were normalized to the amount of 32P-labeled transcripts hybridized to total 3T3-L1 genomic DNA. Subcellular Fractionation of 3T3-L1 Adipocytes. Homogenization and subcellular fractionation was conducted essentially as described by Clancy and Czech (22), except that the cells were homogenized in a buffer supplemented with proAbbreviations: GLUT4, adipose tissue/muscle glucose transporter; GLUT1, erythrocyte/brain glucose transporter; SCD, stearoyl-CoA desaturase; DRB, 5,6-dichloro-1-p-D-ribofuranosylbenzimidazole. *To whom reprint requests should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 1933
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Biochemistry: Kaestner et al.
tease inhibitor cocktails I and II (PIC I and PIC II) (23) by forcing the cell suspension through a stainless steel ballbearing homogenizer 12 times (24). Cellular cAMP Level and Hexose Uptake Rate. Cellular cAMP levels were determined as described (25) in ethanol extracts of cell monolayers by using a competitive binding assay with cAMP-binding protein obtained from human erythrocyte membranes (26). Hexose uptake assays were conducted as described previously (27). Immunoblot Analysis of GLUTi and GLUT4 Proteins. Initial Western blot experiments revealed rather diffuse bands of GLUTi and GLUT4 proteins, which made quantitation difficult. The possibility was considered that these diffuse electrophoretic patterns were due to heterogeneity of glycosylation of the transporter proteins. Therefore, membrane samples were routinely pretreated with peptide N-glycosidase F (1 unit per 100 ,ug of protein; Boehringer Mannheim) for 48 hr at 370C and then were mixed with 1/3 vol of 3 x electrophoresis sample buffer [187.5 mM Tris HCl, pH 6.8/6% SDS/15% (vol/vol) glycerol/60 mM EDTA/60 mM dithiothreitol]. Electrophoresis was performed in SDS/615% polyacrylamide gradient gels, after which the proteins were transferred to nitrocellulose filters (Sartorius). For immunological detection of transporter proteins, polyclonal antibodies against a synthetic peptide corresponding to amino acid residues 484-501 of the murine adipocyte GLUT4 (2) were raised in rabbits and immunopurified on a peptide affinity column. Polyclonal antibodies against a peptide corresponding to the C-terminal sequence of GLUTi were generously provided by S. Cushman (National Institutes of Health). Blots were developed by the enhanced chemiluminescence method (ECL; Amersham) employing horseradish peroxidase-conjugated goat anti-rabbit IgG. Quantitation of relative band intensity was performed by laser scanning densitometry.
RESULTS To test the hypothesis that elevation of intracellular cAMP causes down-regulation of GLUT4 mRNA, fully differentiated 3T3-L1 adipocytes were treated for 16 hr with various agents known to increase cellular cAMP concentration. Of the agents tested, forskolin was most effective in increasing cellular cAMP (10- to 20-fold, Fig. lA). Glucagon or isoproterenol alone had only small effects on cAMP level, but in combination with forskolin they had more than an additive effect. Accompanying the increase in cAMP concentration caused by the 16-hr treatment with forskolin, cellular GLUT4 mRNA fell by 70-80%, while the GLUTi mRNA rose 2- to 2.5-fold (Fig. 1C and results not shown). Apparently, as a consequence of the effects of forskolin (and to a lesser extent isoproterenol) on expression of the two glucose transporter proteins, the basal rate of hexose uptake increased and the responsiveness of hexose uptake to insulin was almost completely lost (Fig. 1B). To ascertain whether the effects of forskolin on GLUT4 and GLUTi mRNA levels were mediated by activation of adenylate cyclase or through an indirect mechanism, an analog of forskolin-i.e., 1,9-dideoxyforskolin, which does not activate the cyclase (results not shown)-was employed. In addition, the effect of 8-Br-cAMP was tested. As shown in Fig. 2, the expression of neither GLUT4 nor GLUTi mRNA was affected by exposure of 3T3-L1 adipocytes to 1,9dideoxyforskolin for 2 or 16 hr. Furthermore, 8-Br-cAMP, like forskolin, caused a marked decrease (-67%) in GLUT4 message at 16 hr and an increase (-3.4-fold) in GLUTi message which was maximal at 6 hr (Fig. 2). The dependence of changes in GLUTi and GLUT4 message levels on 8-Br-cAMP concentration was assessed after 16 hr of exposure to the cyclic nucleotide. Both the increase
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FIG. 1. Effects of forskolin, glucagon, and isoproterenol on hexose uptake and cellular glucose transporter mRNA level. 3T3-L1 adipocytes either were not treated (column C) or were treated for 16 hr with 50 ,uM forskolin (F), 100 nM glucagon (G), 4 ,.M isoproterenol (I), forskolin and glucagon (F+G), or forskolin, glucagon, and isoproterenol (F+G+I). (A) cAMP levels from duplicate determinations, + range. (B) Rate of 2-deoxy[14C]glucose (DG) uptake. Open bars, basal rate without insulin; hatched bars, insulin-stimulated rate after 10-min incubation with 1 ,uM insulin. (C) RNase protection assay of 10 ,g of total RNA with GLUT1- and GLUT4-specific antisense RNA probes. An additional control sample (tRNA) gave no
signal.
in GLUT1 mRNA (2-fold) and the decrease in GLUT4 mRNA (74%) were maximal at 1 mM 8-Br-cAMP, with half-maximal responses at about 500 ,M (Fig. 3 A and B). Parallel culture dishes of adipocytes were subjected to hexose uptake assays (Fig. 3C). Consistent with the effect of 8-Br-cAMP on mRNA levels, the basal rate of hexose uptake (without insulin) increased half-maximally at =500 AM 8-BrcAMP (due apparently to increased expression of GLUT1 mRNA and protein), while the insulin-stimulated component of hexose uptake decreased half-maximally at '500 AM 8-Br-cAMP (presumably due primarily to decreased expression of GLUT4 mRNA and protein). The insulin-stimulated component decreased by about 2/3 at the highest concentration of the cAMP derivative (Fig. 3C). To determine whether the observed changes in GLUT4 and GLUT1 message levels C
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Biochemistry: Kaestner et al. tRNA
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Proc. Natl. Acad. Sci. USA 88 (1991)
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FIG. 3. Dependence of cellular glucose transporter mRNA level and hexose uptake rate on 8-Br-cAMP concentration. Parallel dishes of 3T3-L1 adipocytes were treated for 16 hr with the indicated concentrations of 8-Br-cAMP. (A) RNase protection analysis was performed with antisense RNA probes for GLUT1, GLUT4, SCD1, and SCD2 on 10 ,ug of total RNA. (B) The mRNA levels for GLUT4 and GLUTI in A were quantitated by laser scanning densitometry. o, GLUT1; e, GLUT4. (C) Rate of 2-deoxy[14C]glucose uptake. o, Basal rate without insulin; * (+I), insulin-stimulated rate after a 10-min incubation with 1 tLM insulin; A (I - C), difference between basal and stimulated rate. were due to a generalized change in the genetic programming, or "dedifferentiation" of 3T3-L1 adipocytes, two markers of adipocyte differentiation were assayed. As is demonstrated in Fig. 3A, neither the message for SCD1 (28) nor that for SCD2 (19) was significantly affected by treatment of the cells
with 8-Br-cAMP. To verify that the observed changes in cellular mRNA concentration are paralleled by fluctuations in the proteins they encode, the total cellular levels of GLUTi and GLUT4 proteins were measured by quantitative Western blotting. As shown in Fig. 4 A (lanes 1 and 2) and B, exposure of 3T3-L1 adipocytes to 8-Br-cAMP for 16 hr caused a 3-fold decrease in the amount of GLUT4 protein, while the cellular levels of GLUTi protein increased to a comparable extent. Thus, the cyclic nucleotide-induced changes in the cellular levels of the two transporter mRNAs gave rise to corresponding changes in the levels of their respective transporter proteins. While cyclic nucleotide treatment had no detectable effect on the fractional distribution of GLUT4 protein between the plasma membrane and low-density microsomes, it caused a substantial increase in the fraction of GLUTI protein at the plasma membrane (Fig. 4B).
FIG. 4. Effect of 8-Br-cAMP on total cellular level and subcellular distribution ofGLUT4 and GLUTi proteins. 3T3-L1 adipocytes were treated or not with 1 mM 8-Br-cAMP (as indicated) for 16 hr, at which time cells were treated or not for 10 min with 1 ,uM insulin. Total cellular membranes (Total) were prepared from 1/4 of the cell homogenate. Low-density microsomes (LDM) and plasma membranes (PM) were prepared by subcellular fractionation of the remainder of the homogenate. Membrane samples were treated with peptide N-glycosidase F before electrophoresis to remove N-linked oligosaccharide chains. Samples were then applied to an SDS/ polyacrylamide gel, electrophoresed, and transferred to a nitrocellulose membrane. The GLUT4 and GLUTi transporter proteins were immunodetected by using specific polyclonal antibodies, and the blots were developed by enhanced chemiluminescence. A typical Western blot is shown in A. Total cellular membranes (lanes 1 and 2) are from 1.8 x 105 cells. Low-density microsomes (lanes 3-6) and plasma membranes (lanes 7-10) are from 7 x 105 cells. The relative electrophoretic migration of two molecular weight markers (45 and 29 kDa) is shown. To obtain the results for the bar graphs shown in B, films from the Western blot shown in A (lanes 3, 5, 7, and 9; without insulin) were subjected to laser scanning densitometry for quantitation. The results are expressed relative to the maximum for GLUT4 or GLUTi protein.
Of interest is the fact that the capacities of the two glucose transporters to undergo acute insulin-stimulated translocation differ after chronic exposure to 3T3-L1 adipocytes to 8-Br-cAMP. GLUT4 protein retained the ability to translocate rapidly in response to insulin despite the large decrease in total amount of GLUT4 protein (Fig. 4A). Thus, the fold increase in amount of GLUT4 protein in the plasma membrane (Fig. 4A, lanes 7-10) and the fold decrease in amount of GLUT4 protein in the low-density microsomal fraction (Fig. 4A, lanes 3-6) due to acute insulin treatment were the same whether or not the cells had been exposed chronically to 8-Br-cAMP (Fig. 4A). In contrast, both the steady-state level and the fraction of GLUTi protein in the plasma membrane were substantially higher in cells treated with 8-Br-cAMP (Fig. 4B) and increased little upon addition of insulin (Fig. 4A, lanes 9 and 10). Conversely, the amount of GLUTi protein in the low-density microsomal fraction did not decrease nearly so much upon acute insulin treatment in
1936
Proc. Natl. Acad. Sci. USA 88 (1991)
Biochemistry: Kaestner et al.
cells exposed chronically to 8-Br-cAMP (Fig. 4A, lanes 5 and 6) as in cells not exposed to the cyclic nucleotide (Fig. 4A, lanes 3 and 4). These results provide an explanation for the increased basal hexose uptake rate and for the loss of responsiveness of hexose uptake to insulin caused by chronic exposure of 3T3-L1 adipocytes to 8-Br-cAMP (Fig. 3C). The observed changes in GLUT4 and GLUTi mRNA levels caused by chronic exposure of the cells to 8-Br-cAMP could result from changes in the rates of gene transcription or of mRNA degradation or a combination of both. To analyze transcription rates of the GLUT4 and GLUTi genes, nuclei were isolated from 3T3-L1 adipocytes treated with 1 mM 8-Br-cAMP for various periods of time, after which nuclear transcriptional run-on experiments were performed. This method provides a measure of the number of specific RNA transcripts undergoing elongation at the time the nuclei are isolated. As demonstrated in Fig. 5, run-on transcription of the GLUT4 gene was rapidly suppressed by treatment with 8-Br-cAMP (90% reduction after 18 hr), while transcription of the GLUTi gene was induced transiently (7-fold over basal after 2 hr of exposure to 8-Br-cAMP). These changes in specific transcription rate caused by 8-Br-cAMP precede the changes in steady-state mRNA levels (compare with the results in Fig. 2) and are of similar magnitude. To assess possible additional effects of 8-Br-cAMP on the rates of turnover of GLUT4 and GLUTi mRNAs, changes in the half-lives of these messages caused by the cyclic nucleotide were investigated. 3T3-L1 adipocytes were treated or not with 1 mM 8-Br-cAMP for 16 hr prior to the addition of two different inhibitors of RNA polymerase II. RNA was isolated at various time intervals and GLUTi and GLUT4 mRNA levels were determined by RNase protection (Fig. 6). Rate constants for the turnover of the two messages were obtained from regression analyses of the semilogarithmic plots shown in Fig. 6, assuming first-order kinetics. Although there is a minor discrepancy between the half-life values obtained using the two RNA polymerase inhibitors, there is a consistent 2-fold increase in the half-life (a measure of message stability) of GLUTi mRNA caused by 8-Br-cAMP. The stability of the GLUT4 message, however, is not significantly altered by the treatment of 3T3-L1 adipocytes with 8-Br-cAMP.
DISCUSSION In this study we show that when cAMP concentration in 3T3-L1 adipocytes is increased for an extended period-e.g., 16 hr-the basal (minus insulin) hexose uptake rate increases dramatically (7- to 8-fold; Figs. 1B and 3C). Accompanying U) :5
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this rise in basal uptake rate is a nearly complete loss of acute responsiveness of hexose uptake to insulin, in part because the basal rate has risen substantially. In this sense the cells have become insulin resistant, although they exhibit this atypically high basal rate of hexose uptake. Normally, insulin rapidly (within 4-5 min) activates hexose uptake by 10- to 20-fold in 3T3-L1 adipocytes. However, after chronic exposure of the cells to 8-Br-cAMP (Fig. 3C) or forskolin (Fig. 1B), insulin exerts only a small acute activating effect-i.e., -1.3-fold-on hexose uptake rate. The loss of responsiveness of hexose uptake caused by cAMP in 3T3-L1 adipocytes is associated with both a decrease in the cellular concentration of GLUT4 protein and an increase in the concentration of GLUT1 protein (Fig. 4). The effect on GLUT4 protein may be particularly relevant to adipose tissue in vivo, where GLUT4 is the major transporter isoform, making up -90% of the total (29, 30). We suggest, therefore, that the repressed expression of the GLUT4 isoform in 3T3-L1 adipocytes caused by cAMP reflects changes known to occur in adipose tissue per se. It should be noted that both diabetes and prolonged fasting, which promote a rise in cellular cAMP (15), have been shown to cause a decrease in GLUT4 mRNA and protein in tissue adipocytes (11-14). It is of interest that the level of GLUTi protein increases in 3T3-L1 adipocytes exposed to 8-Br-cAMP for 16 hr. Under these conditions, GLUTi is localized primarily in the plasma membrane (Fig. 4B) despite the fact that the cells had not been treated with insulin. Normally, in 3T3-L1 adipocytes not exposed to insulin or cAMP, most GLUTi protein is located intracellularly and is rapidly translocated to the plasma membrane when the cells are treated with insulin. The fact that most GLUTi protein in cells exposed chronically to 8-Br-cAMP is in the plasma membrane is consistent with the finding of Clancy and Czech (22) that cAMP causes the rapid
Biochemistry: Kaestner et al. translocation of GLUTi protein from low-density microsomes to the plasma membrane. The observed increase in the GLUTi isoform, as well as its predominant localization in the plasma membrane, is undoubtedly responsible for the cAMP-induced rise in basal hexose uptake rate in 3T3-L1 adipocytes (Figs. 1B and 3C). This effect is probably not representative of the situation in vivo, since there is little
expression of GLUTi protein in tissue adipocytes (29, 30). The mechanism by which cAMP causes down-regulation of GLUT4 mRNA and protein in 3T3-L1 adipocytes involves repression of transcription of the GLUT4 gene. Thus, 8-BrcAMP caused a dramatic decease in nuclear run-on transcription of the GLUT4 gene (Fig. 5) while having little or no effect on the rate of turnover of GLUT4 mRNA (Fig. 6). In contrast, the increases in cellular GLUTi mRNA and protein caused by 8-Br-cAMP appear to be the result of both an initial increased rate of transcription of the GLUTi gene (determined by nuclear run-on transcription analysis; Fig. 5) and a reduced rate of turnover of GLUTi mRNA (Fig. 6). Our findings regarding the GLUTi gene are similar to those of other investigators who studied the effects of cAMP in fibroblasts (31). The availability of the GLUT4 gene promoter (32) will enable us to investigate the molecular basis for transcriptional repression of the GLUT4 gene by cAMP. Preliminary experiments with a chimeric GLUT4-7000-base-pair promoter/chloramphenicol acetyltransferase (CAT) construct transfected stably into 3T3-L1 adipocytes reveal the nearly complete repression of expression of CAT mRNA when cells are treated with 8-Br-cAMP (results not shown). The genetic element(s) within this 7-kilobase-pair segment of the GLUT4 gene promoter responsible for the cAMP effect have not yet been identified. We thank Ms. Natalie Tumminia for expert secretarial assistance. We are grateful to Dr. Sam Cushman for generously providing an antibody to GLUT1. This work was supported by National Institutes of Health Research Grant NIDDK-38418. J.R.F.-R. was supported by a National Research Service Award from the National Institutes of Health and M.J., by a postdoctoral fellowship from the Juvenile Diabetes Foundation. 1. Gould, G. W. & Bell, G. I. (1990) Trends Biochem. Sci. 15, 18-23. 2. Kaestner, K. H., Christy, R. J., McLenithan, J. C., Braiterman, L. T., Cornelius, P., Pekala, P. H. & Lane, M. D. (1989) Proc. Nati. Acad. Sci. USA 86, 3150-3154. 3. James, D. E., Strube, M. & Mueckler, M. (1989) Nature (London) 338, 83-87. 4. Charron, M. J., Brosius, F. C., Alper, S. L. & Lodish, H. F. (1989) Proc. Nati. Acad. Sci. USA 86, 2535-2539. 5. Birnbaum, M. J. (1989) Cell 57, 305-315. 6. Fukumoto, H., Kayano, T., Buse, J. B., Edwards, Y., Pilch,
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