From the Departments of Pediatrics and
¶ Biological Chemistry, Johns Hopkins University School of
Medicine, Baltimore, Maryland 21287-3311
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ABSTRACT |
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Prolonged treatment of 3T3-L1 adipocytes
decreases expression of GLUT4, the insulin-responsive glucose
transporter. Expression of promoter-reporter gene constructs that
contained 2900 or 785 base pairs of 5'-flanking region of the murine
GLUT4 gene was down-regulated by insulin (p < 0.0005), whereas expression of constructs that contained 641, 469, or
78 base pairs of 5'-flanking region was not. Nuclear extract from
3T3-L1 adipocytes protected the region from 707 to
681 in the GLUT4
5'-flanking region from DNase I digestion. Using an oligonucleotide
probe that corresponded to this footprinted region, two major
protein-DNA complexes were identified by a gel mobility shift assay.
Southwestern analysis identified four protein bands with molecular
masses from 38 to 46 kDa that bound to the insulin-responsive region
probe. A reporter gene construct in which bases
706 to
676 were
deleted was not repressed by insulin treatment, confirming that this
sequence is necessary for the repression of the GLUT4 promoter by
insulin in 3T3-L1 adipocytes. This sequence does not show homology to previously described insulin response elements and thus represents a
distinct mechanism of gene regulation by insulin.
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INTRODUCTION |
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GLUT4 is a member of the protein family of glucose transporters that mediates facilitated diffusion of glucose across cell membranes. GLUT4, the insulin-responsive glucose transporter, is present in adipose and muscle cells, where it catalyzes the rate-limiting step for glucose uptake and metabolism (1). Insulin increases glucose uptake into these cell types by stimulating the translocation of GLUT4 from an intracellular microsomal compartment to the plasma membrane. Perturbation of GLUT4 expression through overexpression in transgenic mice and underexpression through targeted gene disruption has been shown to increase and decrease insulin-stimulated glucose uptake, respectively, in these animals. In fact, even modest increases in GLUT4 expression have been shown to ameliorate insulin resistance in the db/db mouse (2) and to completely alleviate the insulin resistance that develops in mice fed a high fat diet (3).
Alterations in GLUT4 expression have been found in many models of insulin resistance (4). GLUT4 expression is decreased in adipose tissue in human obesity and type 2 diabetes mellitus (5, 6). The decreased expression of GLUT4 correlates with the decrease in insulin-stimulated glucose uptake in these adipocytes. However, GLUT4 expression is not decreased in muscle tissue in these states, and muscle tissue, not adipose tissue, accounts for the majority of whole body insulin-stimulated glucose uptake. Therefore, decreased expression of GLUT4 in adipocytes is not the primary site of decreased insulin-stimulated glucose uptake in insulin resistant states. Nonetheless, changes in glucose uptake into adipocytes can affect whole body glucose disposal as shown with transgenic mice in which a targeted increase in GLUT4 expression in adipose tissue increased whole body glucose disposal in both the basal and insulin-stimulated state (7). In addition, altered lipid metabolism in the adipocyte could affect glucose uptake into muscle (8, 9), and changes in GLUT4 expression in the adipocyte can alter lipid metabolism in the animal (10). Finally, the level of GLUT4 expression in adipocytes may play a role in the development of obesity by regulating adipose tissue development, because GLUT4-null mice have markedly decreased adipose tissue mass (11), and mice that overexpress GLUT4 in adipocytes have two to three times more adipose tissue (7). In addition, young fa/fa rats show increased expression of GLUT4 in adipocytes at a time when they are rapidly accumulating adipose tissue (12). Because changes in GLUT4 expression are able to alter so many aspects of energy utilization, better understanding of the mechanisms that mediate regulation of GLUT4 expression will be needed as attempts are made to modulate insulin sensitivity and to affect changes in the development of obesity.
A number of hormonal and metabolic stimuli have been found to regulate GLUT4 expression in adipose tissue (13). Expression of GLUT4 in adipocytes is decreased in obese, high fat fed and fasted animals. Furthermore, insulin deficiency in streptozotocin-induced diabetes leads to decreased GLUT4 expression in adipose tissue as well as in muscle tissue. Although insulin acutely stimulates an increase in GLUT4 activity at the plasma membrane, it has been shown to decrease GLUT4 gene expression in the 3T3-L1 adipocyte (14), an in vitro model of tissue adipocytes. This effect suggests that the decreased expression of GLUT4 found in adipose tissue in obesity and type 2 diabetes mellitus might be due to suppression of GLUT4 expression by the hyperinsulinemia that occurs in both of these conditions.
The effect of insulin on GLUT4 expression in 3T3-L1 adipocytes is rapid, with a decrease in GLUT4 mRNA levels occurring within 4 h of insulin treatment and a decrease in GLUT4 gene transcription occurring within 1 h. This effect is most likely mediated through the insulin receptor, because the down-regulation is seen with a relatively low level of insulin and requires a much higher level of IGF-I1 (14). We have investigated the molecular mechanism underlying this action of insulin on GLUT4 expression to identify the cis-acting elements in the GLUT4 gene that mediate this response and the trans-acting factors that interact with these elements.
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EXPERIMENTAL PROCEDURES |
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Cell Culture-- 3T3-L1 cells were cultured in Dulbecco's modified Eagle's medium containing 10% calf serum. The cells were induced to differentiate into the adipocyte phenotype by treating confluent cells with 0.5 mM isobutylmethylxanthine, 1 µM dexamethasone, and 167 nM insulin in 10% fetal bovine serum for 2 days, followed by treatment with 167 nM insulin in 10% fetal bovine serum for 2 days. The cells were then maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and fed every other day. Eight days after induction of differentiation a medium change was made, and on the ninth day, adipocytes were treated with 1 µM insulin. Cells were harvested 4 h after insulin treatment for preparation of nuclear extract or after 8 h for preparation of RNA.
Plasmid Constructs--
Murine GLUT4/chloramphenicol
acetyltransferase (CAT) reporter plasmids contained the 5'-flanking
region of the GLUT4 gene, the GLUT4 transcription initiation site, 171 bp of GLUT4 5'-untranslated sequence, and the coding sequence for the
bacterial CAT gene. The 785,
469, and
78 GLUT4/CAT plasmids were
prepared as described previously (15). The
2,900 GLUT4/CAT plasmid
was constructed by digesting
7000 GLUT4/CAT (15) with
ApaI, filling in the ends using the Klenow fragment of DNA
polymerase I, and re-ligating the plasmid. The
641 GLUT4/CAT plasmid
was constructed by digesting
785 GLUT4/CAT with BssH II
and HindIII, filling in the ends using the Klenow fragment
of DNA polymerase I, and re-ligating the plasmid. The
785
A
GLUT4/CAT plasmid was constructed by digesting the
785 GLUT4/CAT
plasmid with HindIII and SmaI to remove bases
785 to
469 of GLUT4 promoter. This sequence was then replaced with an insert generated by the polymerase chain reaction (PCR) using Taq and Pwo DNA polymerase (Expand, Boehringer Mannheim) to
amplify two fragments encompassing bases
785 to
707 and bases
675
to
469 of the GLUT4 promoter. These fragments were connected at an
EcoRI site within one of the primers for each fragment (the 3'-primer for the
785 to
707 fragment and the 5'-primer for the
675 to
469 fragment). The resulting plasmid was identical to the
785 GLUT4/CAT plasmid except that bases
706 to
676 had been
replaced with the sequence 5'-GAATTC-3'. This plasmid was sequenced to
confirm the ligated ends, the deleted sequence, and the PCR-amplified
sequence.
Stable Transfections-- Transfection was performed using the calcium/phosphate co-precipitation method (16). Subconfluent 3T3-L1 preadipocytes were transfected with 25 µg of the reporter construct and 2.5 µg of the pSV2Neo plasmid. The cells were incubated with the precipitate for 4 h and then shocked with 10% glycerol in phosphate buffered saline for 2 min. Cells were selected and maintained in 300 µg/ml G418 beginning 24 h after transfection. After 7-10 days, resistance foci of clones were pooled and maintained for further study. Two independent pools of 20-50 foci were studied for each construct.
Quantitation of mRNA--
Cells were lysed in 5 M guanidinium isothiocyanate, 0.5% sarcosyl, and 5%
-mercaptoethanol, and total RNA was isolated by centrifugation over
a 5.7 M CsCl cushion. For quantitation of the endogenous
GLUT4 message, 10 µg of total RNA was separated by electrophoresis
through a 1.2% agarose gel containing 6.5% formaldehyde. The RNA was
transferred to a nylon membrane (Hybond-N; Amersham Corp.), fixed by UV
irradiation, and hybridized with a 1.7-kb murine GLUT4 cDNA (2 × 106 cpm/ml) that was labeled by random hexamer priming
(Decaprime; Ambion, Inc.). Hybridization was performed in a solution
containing 50% formamide, 4× SSC, 5× Denhardt's solution, 50 mM phosphate buffer (pH 7.0), 100 µg/ml yeast tRNA, 0.5 mg/ml sodium pyrophosphate, and 1% SDS at 42 °C for 16 h. The
filter was washed at high stringency (0.1× SSC, 0.1% SDS at 65 °C)
for 1 h. Band intensity of the 2.7-kb GLUT4 mRNA was
quantitated on a Fujifilm BAS2000 bio-imaging autoanalyzer.
DNase I Footprinting Analysis--
DNase I footprint analysis
was performed using the technique described by Brenowitz et
al. (17). The HindIII SmaI fragment of the GLUT4 5'-flanking region (bases
785 to
469) was ligated into
the polycloning site of the Bluescript KS plasmid. To label the coding
strand, the plasmid was linearized with HindIII, labeled with a fill-in reaction using the Klenow fragment of DNA polymerase (Megaprime; Ambion Inc.) in the presence of
[
-32P]dATP, and digested from the plasmid with
BamHI. To label the non-coding strand, the plasmid was
linearized with BamHI, labeled with a fill-in reaction, and
digested from the plasmid with HindIII. The probes were
purified on a 6% acrylamide gel, followed by purification using a
NACS-52 column (Life Technologies, Inc.). Nuclear extract was prepared
from ninth day 3T3-L1 adipocytes following the method described by
Lavery and Schibler (18). The following were added to each of the
solutions during purification as inhibitors of protein phosphatase
activity: 30 mM
-glycerophosphate (Sigma), 1 mg/ml
p-nitrophenyl phosphate (Sigma), and 5 mM sodium
vanadate (Fisher). The protein concentration of the nuclear extract was quantitated using the BCA Protein Assay Reagent (Pierce). 20 µl of
1× NUN solution (0.3 M NaCl, 1 M urea, 1%
Nonidet P-40, 25 mM Hepes (pH 7.9), and 1 mM
dithiothreitol) containing the indicated quantity of nuclear protein
was added to 180 µl of binding buffer containing radiolabeled probe
(100,000 cpm), 10 mM Tris-HCl (pH 8.0), 5 mM
MgCl2, 1 mM CaCl2, 2 mM
dithiothreitol, 50 µg/ml bovine serum albumin, 2 µg/ml calf thymus
DNA, 100 mM KCl, and 2 µg of poly[d(I-C)]. The binding
reaction was incubated on ice for 1 h. Probe/nuclear extract
samples were brought to room temperature and 0.25-0.33 units of DNase
I (Pharmacia) was added to the samples. The digestion reaction was
stopped after 3 min by the addition of 200 µl of 1.3% SDS, 27 mM EDTA, 270 mM NaCl, and 50 µg yeast tRNA.
The samples were extracted with phenol/chlorophorm/isoamyl alcohol
(25:24:1) and precipitated in ethanol. Samples were resuspended in
formamide loading dye and electrophoresed on a 6 or 10% acrylamide/8 M urea gel. A sequencing reaction was run concurrently to
correlate protected regions with the corresponding GLUT4 sequence.
Gel Mobility Shift Assay--
A 41-bp double-stranded
oligonucleotide probe (probe a; see Table I), corresponding to bases
710 to
674 of the 5'-flanking region of the GLUT4 gene plus GATC on
the 3' end for labeling, was synthesized and labeled with
[
-32P]dATP by a fill-in reaction (Megaprime; Ambion,
Inc.) The probe for the corresponding region of the human gene was also
synthesized (probe h) and included GATC on the 3' end for labeling; the
sequence encompassed bases 1264-1300 from Buse et al. (19)
(see Table I; bold capital letters indicate bases that differ from the
murine sequence.) Mutant probes (probes m1, m2, and m3), with base
changes from the murine sequence introduced as indicated by bold
capital letters in Table I, were also synthesized. 50-100 × 103 cpm of labeled probe was incubated with 2 µg of
nuclear extract in a 30 µl solution containing 0.33× NUN buffer,
8.3% glycerol, 20 mM Hepes (pH 7.6), 2 mM
dithiothreitol, 10 µg of bovine serum albumin, and 2 µg of
poly[d(I-C)]. The binding reaction was incubated on ice for 1 h
and then separated by electrophoresis on a polyacrylamide gel in 0.5×
TBE (45 mM Tris-borate, 1 mM EDTA). For
competition reactions, 100 ng of unlabeled oligonucleotide
(approximately 100-fold excess over the labeled probe) was added to the
binding reaction. The
710 to
674 GLUT4 double-stranded
oligonucleotide (probe a) was used as a specific competitor, and a
24-bp double-stranded oligonucleotide including the unrelated DNA
binding sequence of the stearoyl-CoA desaturase 2 (SCD2) gene
corresponding to bases
437 to
421 of the SCD2 5'-flanking region
was used as a nonspecific competitor. Nuclear extract was prepared from
adult mouse epididymal adipose tissue using the same technique
described above for 3T3-L1 adipocyte nuclear extract except that the
protein phosphatase inhibitors were not included in the
preparation.
Southwestern Analysis--
50 µg of nuclear extract was boiled
for 10 min in 6% SDS and 60 mM dithiothreitol and then
separated on a polyacrylamide gel. The stacking gel contained 5%
acrylamide, 125 mM Tris (pH 6.8), and 0.1% SDS; the
resolving gel contained 12% acrylamide, 375 mM Tris (pH
8.8), and 0.1% SDS. After separation, the proteins were transferred to
a nylon membrane (Imobilon; Millipore) in 20% methanol, 25 mM Tris, 0.2 M glycine by electrotransfer at 15 V overnight. The filters were washed in denaturing buffer (25 mM Hepes (pH 7.5), 25 mM NaCl, 5 mM
MgCl2, 0.5 mM dithiothreitol) with 6 M guanidine HCl and then renatured by five successive
washings in 2-fold dilutions of the guanidine HCl in denaturing buffer, with a final incubation in denaturing buffer with no guanidine HCl. All
washes were for 10 min at 4 °C. The filters were blocked in 5%
nonfat dry milk in binding buffer (50 mM Tris (pH 7.5), 50 mM NaCl, 1 mM EDTA, 1 mM
dithiothreitol) for 1 h at room temperature and then hybridized
overnight at 4 °C in binding buffer with 0.25% nonfat dry milk and
1 × 106 cpm/ml of labeled probe a. Competition was
performed by adding 1 µg/ml of unlabeled double-stranded
oligonucleotide (approximately 200-fold excess). Filters were washed
with 0.25% nonfat dry milk in binding buffer at 4 °C for 30 min.
Autoradiography was performed at 80 °C with Kodak X-Omat AR film
(Eastman Kodak Co.) and an intensifying screen for the indicated
times.
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RESULTS |
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Identification of an Insulin-responsive Region in the 5'-Flanking Region of the GLUT4 Gene-- Previous studies showed that transcription of the GLUT4 gene is down-regulated by insulin in 3T3-L1 adipocytes (14). To locate the cis-acting elements of the GLUT4 gene responsible for the insulin-induced decrease in GLUT4 expression, a series of GLUT4 5'-flanking region-reporter gene constructs was prepared. These constructs contained various lengths of the murine GLUT4 5'-flanking region, the GLUT4 transcription start site, 171 bp of GLUT4 5'-untranslated region, and the coding sequence for bacterial CAT. The constructs were stably transfected into 3T3-L1 preadipose cells, after which two independent pools of foci for each construct were collected and used for study.
The stable cell lines expressing the GLUT4/CAT reporter constructs were induced to differentiate into cells exhibiting the adipocyte phenotype. On day 9 of the differentiation protocol, cells were treated with 1 µM insulin for 8 h and compared with untreated control cells. Expression of the endogenous GLUT4 gene was quantitated by Northern analysis of total RNA. In all transfected cell lines, insulin treatment reduced GLUT4 mRNA levels by 50-60%. Expression of the GLUT4/CAT reporter genes was quantitated by a competitive RT-PCR assay. Fig. 1 shows representative results of the RT-PCR assay for the reporter constructs that define the insulin-responsive region in the gene. As shown in Fig. 2, reporter genes containing 785 bp or 2.9 kb of GLUT4 5'-flanking region were repressed by insulin to a level similar to that of the endogenous GLUT4 gene. In contrast, reporter genes containing 641 bp or less of 5'-flanking region were not repressed by insulin treatment. Thus, the region between
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Identification of a Sequence Element in the Insulin-responsive
Region Protected from DNase I Digestion--
To identify sites of
nuclear protein-DNA interaction within the 785 to
641 bp region of
the GLUT4 gene, DNase I footprinting analysis was conducted. The
HindIII-SmaI fragment of the GLUT4 5'-flanking
region, containing bases
785 to
469, was labeled on one end with
[
-32P]dATP. The labeled probe was incubated with
3T3-L1 adipocyte nuclear extract and then subjected to digestion with
DNase I. A single protected region was identified when either the
coding or non-coding strand was labeled. Bases
681 to
706 were
protected on the coding strand, and bases
686 to
707 were protected
on the non-coding strand (Fig. 3). To
determine if insulin treatment of 3T3-L1 cells altered the binding of
nuclear proteins detected by the footprinting assay, the reaction was
also carried out using nuclear extract from 3T3-L1 adipocytes that had
been treated with insulin. As seen in Fig. 3, the footprint pattern was
the same when the probe was incubated with nuclear extract prepared
from control 3T3-L1 adipocytes or from cells treated with 1 µM insulin for 4 h.
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Identification of Nuclear Protein-DNA Complexes Binding to the
Insulin-responsive Element--
To further characterize the
interaction of specific nuclear proteins with the DNA sequence
identified by DNase I footprinting, gel mobility shift assays were
performed. A 41-bp double-stranded oligonucleotide was prepared that
corresponded to the sequence from 710 to
674 bp in the 5'-flanking
region of the murine GLUT4 gene. This oligonucleotide was labeled with
[
-32P]dATP and then incubated with 2 µg of 3T3-L1
adipocyte nuclear extract after which the protein-DNA complexes were
resolved by polyacrylamide gel electrophoresis. As shown in Fig.
4A, two major protein-DNA
complexes were identified. Each of these complexes resulted from
specific interactions of nuclear proteins with the labeled
oligonucleotide, as they were competed away with an excess of unlabeled
oligonucleotide but not by an excess of an unlabeled, unrelated
oligonucleotide. As with the DNase I footprint pattern, the gel shift
pattern did not differ when the oligonucleotide was incubated with
nuclear extract from control 3T3-L1 adipocytes compared with that from
cells treated with 1 µM insulin for 4 h.
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Verification of the Identification of an Insulin-responsive
Element--
The importance of the sequence identified by DNase I
footprinting in mediating the response to insulin was assessed by
reporter gene analysis with a mutated GLUT4 promoter-reporter
construct. When the sequence from 676 to
706 bp was deleted from
the
785 GLUT4/CAT reporter, down-regulation by insulin of the
785
bp of GLUT4 5'-flanking region was lost (Figs. 1 and 2). This confirms that bases
676 to
706 are essential for the repression of the GLUT4
promoter by insulin in 3T3-L1 adipocytes and thus constitute an
insulin-responsive element.
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DISCUSSION |
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We have used promoter-reporter gene constructs to identify an insulin-responsive region in the GLUT4 gene. Our results show that, like the endogenous GLUT4 gene (14), expression of promoter-reporter constructs containing at least 785 bp of 5'-flanking region of the GLUT4 gene is decreased by insulin treatment in 3T3-L1 adipocytes, whereas expression of constructs containing 641 bp or less of 5'-flanking region is not altered by treatment with insulin. Previously, this laboratory had been unable to demonstrate repression by insulin of a transfected GLUT4 minigene that included 7-kb of 5'-flanking region (20). In contrast with the present work, those studies were performed with a single clonal cell line that had been specifically selected for a high level of GLUT4 expression and therefore may have selected against regulated expression. The present study was performed on duplicate independently prepared pools of transfected cells to minimize the effect of integration site-specific events.
The sequence from bases 707 to
681 is protected from DNase I
digestion by nuclear proteins from 3T3-L1 adipocytes. Deletion of this
sequence results in loss of reporter gene regulation by insulin. Gel
mobility shift assays using a probe with a block mutation of bases
683 to
678 showed the same pattern as that for the wild-type
sequence, further delineating the DNA-binding sequence to bases
707
to
684. Thus, it appears that bases
707 to
684 in the GLUT4
5'-flanking region contain a sequence element necessary for the
repression of GLUT4 expression by insulin in 3T3-L1 adipocytes. Gel
mobility shift studies showed that the oligonucleotide corresponding to
the insulin-responsive region bound to nuclear proteins from murine
adipose tissue and produced complexes similar to those produced from
3T3-L1 adipocytes, suggesting that this element also functions in
vivo. In addition, an oligonucleotide corresponding to the
homologous human gene sequence was also able to bind to 3T3-L1
adipocyte nuclear proteins, indicating that this element may regulate
GLUT4 expression in human adipose tissue.
As an initial attempt at elucidating the mechanism for the change in expression induced by insulin, we looked for changes in the DNA sequence footprinted by nuclear proteins from insulin-treated cells compared with that from control cells. No difference was observed. Similarly, no difference in the protein-DNA complexes identified by the gel mobility shift assay was seen between nuclear extract prepared from insulin-treated cells compared with control cells. Finally, Southwestern analysis identified the same set of nuclear proteins from control and insulin-treated cells. The lack of an increase in binding detected by the gel mobility shift assay suggests that the small and variable increase in the quantity of the probe bound to nuclear proteins from insulin-treated cells in the Southwestern analysis is not a significant finding. The lack of insulin-induced changes in protein-DNA interactions detected by DNase I footprinting or gel mobility shift assays has been observed with insulin-responsive sequences from other genes. Perhaps the most extensively examined insulin-responsive gene is that for phosphoenolpyruvate carboxykinase (PEPCK). Repression of PEPCK expression is not correlated with changes in gel mobility shift pattern of the insulin-responsive sequence (21, 22). Similarly no change is observed in the gel mobility shift pattern for the insulin-responsive sequences of the IGFBP-1 (22, 23) or glucagon (24) genes, genes that are repressed by insulin, or for the pattern for the insulin-responsive sequence of the amylase gene (25, 26), a gene that is transcriptionally activated by insulin treatment. In contrast, insulin was shown to alter the protein-DNA complexes identified by gel mobility shift in a number of other genes whose expression is stimulated by insulin, including the IGFBP-3 (27), prolactin (28), fatty acid synthase (29), c-fos (30), and glyceraldehyde-3-phosphate dehydrogenase (31) genes.
We previously showed that the repression of GLUT4 expression by insulin in 3T3-L1 adipocytes was not dependent on new protein synthesis, because the effect was not blocked by cycloheximide (15). It is likely, therefore, that the effect is mediated by post-translational modification of an existing transcription factor, with phosphorylation or dephosphorylation being a possible mechanism. The present results that show no change in the patterns on the DNase I footprint analysis or gel mobility shift assay induced by insulin treatment suggest that this modification does not alter the DNA binding of the transcription factor per se but probably affects the interaction of the transcription factor with other proteins involved in transcription. Alternatively, it is possible that DNA binding activity is altered by a change in phosphorylation state in vivo but that the phosphorylation state was not maintained during the nuclear extract preparation, despite the use of protein phosphatase inhibitors.
We have identified four nuclear proteins that show specific interactions with the insulin-responsive sequence of the GLUT4 gene. These proteins have molecular masses ranging from 38 to 47 kDa (Fig. 7). A number of proteins have now been identified that are involved in mediating the regulation of gene expression induced by insulin. HNF-3 and possibly members of the C/EBP family interact with the insulin-responsive sequence of the PEPCK (22, 32) and IGFBP-1 (22) genes. Etsrelated proteins have been implicated in mediating the regulation of the prolactin and somatostatin genes by insulin (33, 34), sterol regulatory element binding proteins mediate low density lipoprotein receptor expression regulation by insulin (35), and upstream stimulatory factors binding to E-box sequence (29, 36) and possibly Sp1 (37) mediate the stimulation of fatty acid synthase by insulin. In addition, a number of uncharacterized proteins have also been identified as mediating insulin-induced changes in gene expression including 90- and 70-kDa (IGFBP-3 (27)), 57-kDa (glyceraldehyde-3-phosphate dehydrogenase (38)), and 70-80-kDa (growth hormone (39)) proteins. The insulin-responsive sequence of the GLUT4 gene does not have significant homology to the PEPCK/IGFBP-1 core insulin response sequence (T(G/A)TTTTG (22)), Ets-related transcription factor response element (CGGA (33)), Sp1 binding site (GGGCGG), sterol regulatory element sequence (CACCCCAC (40)), or E-box sequence (CANNTG (36)). The only similarity between the insulin-responsive sequence of the GLUT4 gene to insulin response sequences from other genes (41) is homology to the insulin/glucose response element of the L-type pyruvate kinase gene and to the insulin response element of the thyrotropin receptor. However, the response of the thyrotropin receptor gene appears to be mediated by the IGF-I receptor, not the insulin receptor, and the regulation of both the thyrotropin receptor and L-type pyruvate kinase genes is dependent on new protein synthesis, which is not required for the regulation of GLUT4 by insulin. Finally, the proteins we have identified by Southwestern analysis do not correspond in size to the other identified proteins. Therefore, the regulation of GLUT4 by insulin appears to involve a unique DNA-binding protein.
Several pieces of evidence suggest that C/EBP- could be a regulator
of GLUT4 expression. C/EBP-
has been shown to have the ability to
transactivate the GLUT4 promoter (42). In addition, C/EBP-
may be
involved in the regulation of PEPCK gene expression by insulin (22),
and Hemati et al. (45) have recently found that repression
of GLUT4 expression by insulin in 3T3-L1 adipocytes correlates with the
dephosphorylation of C/EBP-
. However, as noted above, there is no
sequence with homology to a C/EBP binding site in the
insulin-responsive sequence we have identified. The studies that
demonstrated transactivation of GLUT4 by C/EBP-
found the effect to
be mediated by the proximal 469 bp of 5'-flanking region of the GLUT4
gene and identified a C/EBP binding site at bases
254 to
262.
Nonetheless, we were interested in determining whether C/EBP-
was
present in the protein-DNA complexes identified by the gel mobility
shift assay. C/EBP-
antibody altered the mobility of protein-DNA
complexes from 3T3-L1 adipocyte nuclear extract incubated with a
consensus C/EBP binding site oligonucleotide but did not alter the
mobility of the protein-DNA complexes identified using the
676 to
706 bp oligonucleotide of the insulin-responsive region.2 Therefore, C/EBP-
does not appear to be present in the protein-DNA complexes. Although
these findings show that C/EBP-
does not interact directly with the
insulin-responsive region of the GLUT4 gene, it is still possible that
C/EBP-
is involved in the regulation of GLUT4 by insulin, but this
would have to occur through the interaction of the protein-DNA
complexes at the insulin-responsive region with C/EBP-
that is bound
at a separate site.
We have found that bases 707 to
684 in the GLUT4 5'-flanking region
mediate the regulation of GLUT4 by insulin in 3T3-L1 cells. These bases
are within a region that has a high degree of sequence homology between
the human and murine genes and has been identified as a region that
mediates regulation of GLUT4 by a number of other stimuli. The
regulation of GLUT4 in skeletal muscle by exercise and in skeletal
muscle and white adipose tissue by a high fat diet has been localized
to bases
441 to
1000 of the murine GLUT4 gene using transgenic mice
(43). Olson and Pessin studied the regulation of the human GLUT4
promoter using reporter genes in transgenic mice (44). They found that
1154 bp of the 5'-flanking region was sufficient to confer regulation in muscle and adipose tissue by uncontrolled diabetes. A reporter with
only 730 bp of 5'-flanking region, which by sequence homology would
interrupt the insulin-responsive region of the murine gene at base
698, was not regulated. We have identified only a single DNase I
protected site in the sequence from
785 to
480 using nuclear
extract from 3T3-L1 nuclear extract, raising the possibility that these
different effectors converge to regulate GLUT4 expression through a
common region of the promoter.
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FOOTNOTES |
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* This work was supported by research grants from the NIDDK, National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: CMSC 3-110, 600 North Wolfe St., Baltimore, MD 21287-3311. Tel.: 410-955-6463; Fax: 410-955-9773; E-mail: dcooke{at}welchlink.welch.jhu.edu.
1 The abbreviations used are: IGF-1, insulin-like growth factor-1; CAT, chloramphenicol acetyltransferase; RT, reverse transcriptase; PCR, polymerase chain reaction; SCD2, stearoyl CoA desaturase 2; C/EBP, CCAAT/enhancer-binding protein; kb, kilobase(s); bp, base pair(s); PEPCK, phosphoenolpyruvate carboxykinase; IGFBP, insulin-like growth factor-binding protein.
2 D. W. Cooke, and M. D. Lane, unpublished data.
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REFERENCES |
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