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INTRODUCTION |
Glucose enters cells through the activity of glucose transporters,
a family of membrane-spanning proteins (for reviews, see Refs. 1 and
2). GLUT4, the insulin-responsive glucose transporter, is present in
adipose and muscle cells, the two tissues that respond to insulin
stimulation with a large and rapid increase in glucose uptake. Insulin
increases glucose uptake in these cells by stimulating the
translocation of GLUT4 from a sequestered site in the intracellular microsomal compartment to the plasma membrane, where it can then transport glucose into the cell. Glucose uptake is the rate-limiting step for glucose metabolism, so the level of GLUT4 protein at the
plasma membrane is the ultimate determinant of glucose utilization in
adipose and muscle cells. Muscle and, to a lesser degree, adipose tissue are the primary sites of glucose disposal after a meal; therefore, alterations in GLUT4 expression affect the ability of the
whole animal to metabolize a glucose load. This has been well
demonstrated in transgenic mice, where overexpression of GLUT4
increases insulin-stimulated glucose uptake and overall glucose
disposal (3, 4). Even modest increases in GLUT4 expression have been
shown to ameliorate insulin resistance in the db/db mouse (5) and to
completely alleviate the insulin resistance that develops in mice fed a
high fat diet (6). Decreased expression of GLUT4 has also been shown to
affect insulin sensitivity, since targeted disruption of the GLUT4 gene
in mice causes a decrease in insulin-stimulated glucose uptake (7)
and mice that are heterozygous for the GLUT4 knockout develop a
diabetic phenotype (8).
GLUT4 expression in adipose tissue is invariably decreased in states of
insulin resistance (9, 10). This decrease in GLUT4 protein and mRNA
levels is closely correlated with the decrease in insulin-stimulated
glucose uptake into these tissues. No decrease in the expression of
GLUT4 in muscle tissue has been observed in insulin-resistant states.
However, a number of mechanisms have been proposed to explain how
adipocyte metabolism could affect the insulin sensitivity of other
tissues (11). Free fatty acids (12) and tumor necrosis factor-
(13)
are both factors that are secreted by adipocytes and can cause insulin
resistance in other tissues. Thus, changes in lipid metabolism or
changes that alter tumor necrosis factor-
production in the
adipocyte may underlie the ability of alterations in adipocyte
metabolism to affect insulin sensitivity of other tissues. Whatever the
mechanism, evidence for the ability of GLUT4 expression levels in the
adipocyte to affect whole body glucose disposal comes from studies with transgenic mice; if GLUT4 is specifically overexpressed only in adipose
tissue, there is an increase in the insulin sensitivity of the whole
animal with an increase in whole body glucose disposal and decreased
basal insulin levels (14).
The 3T3-L1 adipocyte is a reliable in vitro model of tissue
adipocytes. Just as in adipose tissue, insulin acutely increases GLUT4
activity at the plasma membrane in 3T3-L1 adipocytes. However, prolonged treatment with insulin represses GLUT4 gene expression in
3T3-L1 adipocytes (15), with a rapid decrease occurring in the rate of
GLUT4 transcription. Because circulating insulin levels are increased
in obesity and type 2 diabetes mellitus, the repression of adipocyte
GLUT4 expression by insulin may play a role in the insulin resistance
of obesity and type 2 diabetes mellitus.
Using promoter-reporter gene constructs, we previously identified a
cis-acting element in the GLUT4 gene that mediates the repression of
GLUT4 by insulin in 3T3-L1 adipocytes (16). A potential insulin
response element was identified between bases
706 and
676 in the
5'-flanking region by DNase I footprint analysis. 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. In this report, we identify nuclear proteins
that bind to this insulin response element
(IRE)1 as members of the
nuclear factor I (NF1) family of transcription factors.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
3T3-L1 cells were cultured in Dulbecco's
modified Eagle's medium containing 10% calf serum and induced to
differentiate into adipocytes 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. 8 days after induction of differentiation, a
medium change was made, and cells were studied on day 9.
Plasmid Constructs--
The
785-GLUT4/CAT plasmid (wild type)
contained 785 bp of the 5'-flanking region of the murine GLUT4 gene,
the GLUT4 transcription initiation site, 171 bp of GLUT4
5'-untranslated sequence, and the coding sequence for the bacterial
chloramphenicol acetyltransferase (CAT) gene (16). Mutations in the
GLUT4 IRE were produced by digesting the
785-GLUT4/CAT plasmid with
HindIII and SmaI to remove bases
785 to
469 of the GLUT4 promoter. This sequence was then replaced with an
insert generated by the polymerase chain reaction using Taq
and Pwo DNA polymerase (Expand; Roche Molecular Biochemicals) to amplify the insert in a two-step technique as described by Chen and Przybyla (17). In the first reaction, the
following oligonucleotides containing the desired mutations were used
as 5' primers: M1,
5'-CACCTGTCCCTTAAGTCCCCTCCAAGAACCAGTGTAG-3'; M2,
5'-CACCTGTCCCTTGGGTCCCCTTTAAGAACCAGTGTAG-3'; M3,
5'-CACCTGTCCCTTGGGTCTTCTCCAAGAACCAGTGTAG-3'.
These primers, except for the underlined base changes, were
complementary to bases
673 to
710 in the 5'-flanking region of the
GLUT4 gene. 5'-AGCTTGCGAAATTTCTGAAAGAATTG-3', complementary to bases
248 to
269 in the 5'-flanking region of the GLUT4 gene (excluding the underlined bases), was used as the 3' primer. The
785-GLUT4/CAT plasmid was used as the template. The product from the
first amplification reaction was purified from an agarose gel using
Qiaquick (Qiagen) and was used as the 3' primer in the second reaction.
The M13 reverse primer (5'-CAGGAAACAGCTATGAC-3') was used as the 5'
primer, and
785-GLUT4/CAT was again used as the template. This
product was digested with HindIII and SmaI. The
316-bp fragment was purified from an agarose gel using Qiaquick (Qiagen) and was inserted into the
HindIII/SmaI-digested
785-GLUT4/CAT plasmid.
The plasmids were sequenced to confirm the ligated ends, the generation
of the mutation, and the polymerase chain reaction (PCR)-amplified sequence.
Stable Transfections--
Transfection was performed using the
calcium phosphate co-precipitation method (18). 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
calcium phosphate-DNA 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 days, resistant foci of clones were pooled and
maintained for further study. Two or more 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 Pharmacia Biotech),
fixed by UV irradiation, and hybridized with a 1.7-kilobase pair 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 60 °C) for 1 h. Band intensity of the
2.7-kilobase pair GLUT4 mRNA was quantitated on a Fuji BAS2000
bioimaging autoanalyzer. To quantitate the reporter mRNA levels, a
quantitative reverse transcriptase PCR assay was used as described
previously (16), except that the cellular RNA was not digested with
DNase I prior to the reverse transcriptase PCR. Instead, a control was
performed to document that any DNA contamination of the cellular RNA
was quantitatively insignificant compared with the reporter mRNA. To determine this, a known quantity of competitor cRNA was reverse transcribed. The reverse transcriptase was then heat-inactivated before
adding an aliquot of the RNA sample, followed by amplification by PCR
using the same conditions as for the competitive reverse transcriptase
PCR.
Gel Mobility Shift Assay--
Nuclear extract was prepared from
day 9 3T3-L1 adipocytes following the method described by Lavery and
Schibler (19). Except when noted, 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).
A 41-bp double-stranded GLUT4 IRE oligonucleotide probe (probe A),
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). Mutated GLUT4 IRE probes (probes M1, M2, and M3)
were also synthesized. These probes were identical to the wild-type
GLUT4 IRE probe (probe A) except for two base changes introduced as
indicated by the boldface capital letters in Table I. 50-100 × 103 cpm of labeled probe was incubated with 3 µg of
nuclear extract in a 30-µl solution containing 0.33× NUN buffer (1×
NUN: 0.3 M NaCl, 1 M urea, 1% Nonidet P-40, 25 mM Hepes (pH 7.9), and 1 mM dithiothreitol),
8.3% glycerol, 20 mM HEPES (pH 7.6), 2 mM
dithiothreitol, 10 µg of bovine serum albumin, and 2 µg of
poly(dI-dC). The binding reaction was incubated at room temperature for
30 min 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 corresponding to bases
437 to
421 of the
stearoyl-CoA desaturase 2 5'-flanking region was used as a nonspecific
competitor (competitor S). A 27-bp double-stranded oligonucleotide
containing the NF1 binding site from adenovirus 2 (N in Table I) was
also used as a competitor. For the "supershift" assays, 4 µl of
an anti-CCAAT-binding transcription factor (CTF)/NF1 antibody raised
against recombinant CTF-2 (generously provided by N. Tanese, New York
University) or 4 µl of nonimmune rabbit serum was added to the
binding reaction. Autoradiography was performed at
80 °C with
Kodak X-Omat AR film (Eastman Kodak Co.) and an intensifying screen for
the indicated times.
Western Immunoblot--
15 µg of nuclear extract was boiled in
an SDS sample loading buffer (2% SDS, 10% glycerol, 100 mM dithiothreitol, 50 mM Tris-HCl, pH 6.8) for
5 min. The samples were loaded on a denaturing SDS-polyacrylamide gel,
using a 12.5% stacking gel and a 7% resolving gel. After electrophoresis, proteins were transferred to a polyvinylidene difluoride membrane (Immobilon; Millipore Corp.). The filter was blocked in 5% nonfat milk in 0.05% Tween 20, 25 mM
Tris-HCl, 150 mM NaCl (TTBS) and then incubated with
antibody in 1% nonfat milk in TTBS. The filter was washed with TTBS
and then probed with peroxidase-conjugated anti-rabbit IgG (Sigma),
washed with TTBS, and then developed using an enhanced chemiluminescent
reagent (Amersham Pharmacia Biotech). Primary antibodies used include the anti-CTF/NF1 antibody (generously provided by N. Tanese), and an
anti-NF1 antibody raised against the common N terminus of the NF1
isoforms (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
Phosphatase Treatment--
Nuclear extract from 3T3-L1
adipocytes was prepared using the method of Lavery and Schibler (19) as
described above, except that the inhibitors of protein phosphatase
activity were not included. 15 µg of nuclear extract was treated with
alkaline phosphatase (from calf intestine, Roche Molecular
Biochemicals) in 1× phosphatase buffer, 0.3× NUN buffer, and 2 units
of alkaline phosphatase at 37 °C for 3 h. In some samples, 1%
SDS was added to the reaction mixture, as noted. Inactive alkaline
phosphatase (
) was prepared by incubating in 25 mM EDTA
at 70 °C for 4 h. Control nuclear extract was incubated in the
reaction buffer at 37 °C with no enzyme added.
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RESULTS |
DNA Binding Properties of 3T3-L1 Adipocyte Nuclear Proteins That
Bind to the GLUT4 Insulin Response Element--
Our earlier
experiments identified the nucleotide sequence from bases
676 to
706 in the 5'-flanking region of the GLUT4 gene as necessary for
repression of the GLUT4 promoter by insulin in 3T3-L1 adipocytes (16).
Examination of this sequence revealed the presence of the consensus
binding site sequence for the NF1 family of transcription factors,
TGGN7CCA at bases
687 to
699. In order to determine if
the nuclear factors that bind to this region are related to the NF1
family, a series of mutations were made to specifically disrupt the NF1
consensus sequence (Table I). A gel
mobility shift assay was performed using these mutated oligonucleotides
in direct binding experiments and as competitors of the wild-type GLUT4
IRE oligonucleotide (probe A). When incubated with 3T3-L1 adipocyte
nuclear extract, the wild-type GLUT4 IRE produced two major protein-DNA
complexes, bands 1 and 2 (Fig. 1A, lanes
3, 10, and 18). Closer inspection of
the gels, particularly when the gels were run for a longer time (Fig.
1B, lane 1), revealed that band 1 is
actually composed of two protein-DNA complexes, forming bands 1a and
1b. (In some experiments, an additional protein-DNA complex migrating
between bands 1a and 1b is also seen). As seen in Fig. 1, 3T3-L1
nuclear proteins have altered affinities for mutants 1 and 2, which
have base changes that disrupt the 5'- or 3'-half of the consensus NF1
binding sequence. None of the proteins that form the major protein-DNA
complexes identified by the wild-type sequence bind to mutant 1 (Fig.
1A, lane 11, and Fig. 1B,
lane 7), and mutant 1 does not compete with the
wild-type sequence for the binding of any of these proteins (Fig.
1A, lane 7, and Fig. 1B,
lane 4). The protein that forms the fastest
migrating complex (band 2) does not bind to mutant 2 (Fig.
1A, lane 12, and Fig. 1B,
lane 9), and mutant 2 does not compete with the
wild-type sequence for the binding to this protein (Fig. 1A,
lane 8, and Fig. 1B, lane
5). A protein that produces a protein-DNA complex that has a
similar migration to the slower migrating complex identified by the
wild-type sequence does bind to mutant 2. 3T3-L1 adipocyte nuclear
protein(s) binds to mutant 2 and forms a protein-DNA complex with a
mobility at position 1a but does not form a complex with a mobility at
position 1b (Fig. 1A, lane 12, and
Fig. 1B, lane 9). Similarly, mutant 2 competes with the wild-type sequence for the proteins that form band 1a
but does not compete for binding to the proteins that form band 1b
(Fig. 1A, lane 8, and Fig.
1B, lane 5). Mutant 3 has a two-base
substitution in the middle of the NF1 consensus binding sequence that
should not affect binding of NF1 transcription factors. 3T3-L1
adipocyte nuclear proteins bind to mutant 3 and form the same
protein-DNA complexes as the wild-type sequence (Fig. 1A,
lane 13, and Fig. 1B, lane
11), and mutant 3 can compete with the wild-type sequence
for binding to all of the proteins that bind to it (Fig. 1A,
lane 9, and Fig. 1B, lane
6). Finally, an oligonucleotide that contains the adenovirus 2 NF1 binding site (N) was used as a competing
oligonucleotide. The NF1 oligonucleotide competes with the wild-type
GLUT4 IRE for binding to the 3T3-L1 adipocyte nuclear proteins that
form bands 1b and 2 but does not compete with the wild-type sequence for binding to the protein(s) that forms band 1a (Fig. 1A,
lanes 6 and 14, and Fig.
1B, lane 3) Taken together, these
results indicate that the 3T3-L1 adipocyte nuclear proteins that bind
to the GLUT4 IRE include proteins that have similar binding
characteristics as NF1 family members, forming bands 1b and 2. In
addition, there are proteins that bind to this region that have binding
properties different from NF1 family members, forming band 1a.
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Table I
Nucleotide sequence of the wild type and mutated GLUT4 insulin response
element probes
Also shown are the adenovirus 2 NF1 binding sequence probe (N) and the
consensus binding sequence for NF1 proteins.
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Fig. 1.
Analysis of protein binding to the GLUT4
insulin response element. A, 3 µg of nuclear extract
from 3T3-L1 adipocytes was incubated with
[ -32P]dATP-labeled double-stranded oligonucleotide and
separated by electrophoresis on a 6% polyacrylamide gel for 2 h.
The gel was exposed to x-ray film at 80 °C overnight.
Oligonucleotides used as probes were the GLUT4 IRE containing bases
710 to 684 of the 5'-flanking region of the GLUT4 gene (probe A)
and the GLUT4 IRE containing mutations M1, M2, and M3 (see Table I). A
100-fold excess of unlabeled oligonucleotide was added as a competitor,
as indicated. In addition to the GLUT4 IRE and mutant oligonucleotides,
an NF1 binding oligonucleotide (N, see Table I) was used as
a competitor, and an unrelated DNA binding oligonucleotide
(stearoyl-CoA desaturase 2, S) was used as a nonspecific
competitor. 4 µl of anti-NF1 (N) or nonimmune control
(C) antiserum was added as indicated. 1a,
1b, and 2 indicate specific protein-DNA
complexes. B and C, electrophoresis was run on
5% polyacrylamide gels for 4 h.
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Nuclear Proteins in the NF1 Family Bind to the GLUT4 Insulin
Response Element--
Antibody raised against recombinant CTF/NF1
(generously provided by N. Tanese) was used in the gel mobility shift
assay to determine whether the proteins in the 3T3-L1 adipocyte nuclear extract that bind to the GLUT4 IRE contain NF1. As shown in Fig. 1A (lanes 1 and 2) and Fig.
1C, antibody to CTF/NF1 (N), but not preimmune
serum (C), disrupts formation of complexes 1b and 2, and
produces a "super-shifted" band when the 3T3-L1 nuclear proteins are bound to the GLUT4 IRE. The anti-NF1 antibody does not disrupt band
1a. These results confirm those of the mutational analysis, i.e. that bands 1b and 2 contain proteins in the NF1 family
and that band 1a does not contain an NF1 protein.
To confirm which protein classes bind to the mutated GLUT4 insulin
response elements, the NF1 oligonucleotide was used to compete for
binding of 3T3-L1 adipocyte nuclear proteins to the M1, M2, and M3
oligonucleotides in the gel mobility shift assay. The mutant 1 bound
neither the NF1 nor the non-NF1 proteins (Fig. 1A,
lanes 11 and 15, and Fig.
1B, lanes 7 and 8). The
protein-DNA complex formed with mutant 2 (Fig. 1A,
lane 12, and Fig. 1B, lane 9) was not competed away by the NF1 oligonucleotide (Fig.
1A, lane 16, and Fig. 1B,
lane 10), confirming that this mutant does not
bind NF1 proteins. Some of the protein-DNA complexes formed with the
mutant 3 probe (Fig. 1A, lane 13, and
Fig. 1B, lane 11) are competed away by
the NF1 oligonucleotide, but the complex forming band 1a remains (Fig.
1A, lane 17, and Fig. 1B,
lane 12). Table II
summarizes the classes of proteins that bind to the wild type and
mutated GLUT4 insulin response elements based on the results of these
gel mobility shift assays.
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Table II
3T3-L1 adipocyte nuclear proteins bound to wild type and mutated GLUT4
insulin response element sequences
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The NF1 Binding Site in the GLUT4 Promoter Is Necessary for Full
Repression of Reporter Gene Expression by Insulin--
GLUT4
promoter/CAT reporter constructs were produced that contained mutations
1, 2, and 3 in the context of 785 base pairs of 5'-flanking sequence.
The wild type and mutant constructs were stably transfected into 3T3-L1
preadipocytes. After the cells were differentiated into adipocytes, the
responses of the reporter genes were assessed when the cells were
treated with 1 µM insulin for 8 h. Insulin repressed
endogenous GLUT4 expression by 44-54% in all of the stable cell lines
expressing the reporter constructs, similar to the suppression observed
in untransfected 3T3-L1 adipocytes. As shown in Fig.
2, expression of the wild type
785-GLUT4/CAT reporter construct was repressed by insulin to a
similar degree as the endogenous GLUT4 gene, as was seen previously
(16). Expression of the reporter construct containing the M3 mutation,
which does not disrupt any of the protein-DNA interactions detected in
the gel mobility shift assay, was also repressed by insulin to a
similar degree as both the endogenous GLUT4 gene and the wild type
reporter construct. In contrast, expression of the reporter construct
that contains the M1 mutation, which disrupts all of the major
protein-DNA interactions detected in the gel mobility shift assay,
showed no repression by insulin treatment. Finally, expression of the reporter construct containing the M2 mutation, which disrupts NF1
protein binding but does not disrupt binding of the non-NF1 nuclear
protein(s), showed partial repression by insulin treatment. However,
this repression is significantly less than that of the wild type
(p < 0.005) or M3 mutant (p < 0.05)
constructs.

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Fig. 2.
Effect of insulin on endogenous GLUT4 and
GLUT4/CAT reporter gene mRNA levels. Stable 3T3-L1 cell lines
expressing GLUT4/CAT reporter gene constructs containing 785 bp of the
5'-flanking region of the GLUT4 gene with the wild type sequence or
containing mutations M1, M2, and M3 of the IRE (bases 706 to 676)
were prepared. The cells were induced to differentiate into cells
expressing the adipocyte phenotype, and on day 9 of the differentiation
protocol, insulin was added to treated cells to a concentration of 1 µM. Cells were harvested after 8 h, and RNA was
isolated. Endogenous GLUT4 mRNA was analyzed by Northern analysis.
GLUT4/CAT reporter mRNA was quantitated by a competitive reverse
transcriptase PCR technique (see "Experimental Procedures").
Results are expressed as a ratio of the expression level in treated
cells to the expression level in control cells (mean ± S.D. of
six independent experiments.) *, p < 0.005 different
from 1.0; **, p < 0.02 different from 1.0; ,
p < 0.05. The absolute expression level in control
cells for each of the constructs (10 6 pg of reporter/pg
of total RNA) was as follows: wild type, 0.60 ± 0.32; M1,
1.7 ± 0.75; M2, 1.9 ± 0.51; M3, 1.7 ± 0.48.
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Insulin Treatment Causes a Change in the Mobility of the
NF1-containing Protein-DNA Complexes--
3T3-L1 adipocytes were
treated with 1 µM insulin for 0, 10, 20, or 60 min.
Nuclear extracts were prepared from these cells and studied by gel
mobility shift assay using the GLUT4 IRE as the probe. The gels were
run for a longer period of time to allow for better separation of the
bands. While this prolonged electrophoresis resulted in loss of
sharpness of the bands, it revealed that insulin treatment of the cells
caused a decreased mobility of certain of the protein-DNA complexes
(Fig. 3). While this change in mobility is subtle, it is reproducible, having been observed in numerous experiments using four preparations of nuclear extract. This mobility change was seen in nuclear extract prepared from cells treated with
insulin for 10 min. However, by 20 min the mobility change began to
diminish, and by 60 min the mobility of the protein-DNA complexes
reverted to that of cells not treated with insulin.

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Fig. 3.
Binding of nuclear proteins from control or
insulin-treated 3T3-L1 adipocytes to the GLUT4 insulin response
element. 3 µg of nuclear extract from 3T3-L1 adipocytes treated
with 1 µM insulin for the indicated times was incubated
with [ -32P]dATP-labeled double-stranded
oligonucleotide containing bases 710 to 684 of the 5'-flanking
region of the GLUT4 gene (probe A). The protein-DNA mixtures were
separated by electrophoresis on a 5% polyacrylamide gel. The gel was
exposed to x-ray film at 80 °C overnight. These results were
confirmed with four different preparations of nuclear extracts. The
arrows show the positions of protein-DNA complexes from
untreated 3T3-L1 adipocytes, and the dashed
arrows show the position of the same complexes from cells
treated with insulin for 10 min.
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The bands whose mobility was shifted by insulin treatment correspond to
bands 1b and 2, i.e. the bands that contain the NF1 proteins
(see above). Note that the exposure for Fig. 3 was chosen to best
discern band 1b; the mobility shift of band 2 is better seen with other
exposures (not shown). The identity of the shifted bands as containing
NF1 proteins was verified by examining the mobility of the non-NF1
proteins that bind to the GLUT4 IRE. Fig. 4 shows the results of a gel mobility
shift assay with nuclear extracts incubated with the radiolabeled GLUT4
IRE probe (probe A) in the presence of an excess of an unlabeled NF1
consensus binding site oligonucleotide to compete away binding of NF1
proteins. There is no difference in the mobility of the remaining
non-NF1-containing complex (band 1a) that is formed from nuclear
extract from 3T3-L1 adipocytes that were treated with insulin for 0, 10, 20, or 60 min. In addition, incubating the 3T3-L1 nuclear extract
with CTF/NF1 antibody in the gel mobility shift binding reaction
confirmed that the protein-DNA complexes that had altered mobility when prepared from insulin-treated cells contained NF1
proteins.2

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Fig. 4.
Binding of nuclear proteins from control or
insulin-treated 3T3-L1 adipocytes to the GLUT4 insulin response
element. 3 µg of nuclear extract from 3T3-L1 adipocytes treated
with 1 µM insulin for the indicated times was incubated
with [ -32P]dATP-labeled double-stranded
oligonucleotide containing bases 710 to 684 of the 5'-flanking
region of the GLUT4 gene (probe A). A 100-fold excess of unlabeled NF1
consensus binding site oligonucleotide was added as a competitor as
indicated. The protein-DNA mixtures were separated by electrophoresis
on a 5% polyacrylamide gel. The gel was exposed to x-ray film at
80 °C overnight. 1a, 1b, and 2 indicate specific protein-DNA complexes (see "Results").
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Since 1 µM is a supraphysiologic insulin concentration,
the effect of more physiologic concentrations of insulin on the gel mobility shift pattern was examined. Nuclear extract was prepared from
3T3-L1 adipocytes treated with increasing concentrations of insulin for
10 min. As shown in Fig. 5, insulin at a
concentration as low as 10 nM altered the mobility of the
protein complexes that bind to the GLUT4 IRE, and treatment with 25 nM insulin induced a maximal shift in mobility.

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Fig. 5.
Binding of nuclear proteins from control or
insulin-treated 3T3-L1 adipocytes to the GLUT4 insulin response
element. 3 µg of nuclear extract from 3T3-L1 adipocytes treated
with the indicated concentration of insulin for 10 min was incubated
with [ -32P]dATP-labeled double-stranded
oligonucleotide containing bases 710 to 684 of the 5'-flanking
region of the GLUT4 gene (probe A). The protein-DNA mixtures were
separated by electrophoresis on a 5% polyacrylamide gel. The gel was
exposed to x-ray film at 80 °C overnight. These results were
confirmed with four different preparations of nuclear extracts. The
arrows show the position of protein-DNA complexes from
untreated 3T3-L1 adipocytes.
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The change in mobility of the NF1-containing protein-DNA
complexes induced by insulin observed in the gel mobility shift assay could be due to various causes, including 1) insulin treatment causing
new proteins to bind to the DNA probe, either in addition to or in
place of the proteins bound in untreated cells, or 2) insulin treatment
inducing a covalent modification of the proteins that bind to the DNA
probe. To determine whether insulin induces a covalent modification of
the NF1 proteins in 3T3-L1 cells, Western immunoblot analysis was
performed on nuclear proteins isolated from control and insulin-treated
cells. As shown in Fig. 6A,
anti-CTF/NF1 antibody detects three protein isoforms in 3T3-L1
adipocytes. When the adipocytes are treated with insulin, there is a
rapid disappearance of the fastest migrating protein band. This band disappears in nuclear extract from adipocytes treated with insulin for
10 min and reappears in the nuclear extract from cells treated with
insulin for 60 min. When 3T3-L1 adipocytes are treated with increasing
concentrations of insulin for 10 min, the fastest migrating protein
band begins to disappear with 10 nM insulin treatment and
completely disappears with 25 nM insulin treatment.
Concomitant with the disappearance of the fastest migrating protein,
there is an increased amount of the protein band corresponding to the slowest migrating protein (better seen in Fig.
7). Similar results are obtained when
whole cell lysates are prepared from the 3T3-L1 adipocytes instead of
nuclear extract,2 so the change in mobility is not due to a
change in subcellular localization of the proteins. When Western
immunoblot analysis is performed on these same samples using another
anti-NF1 antibody (Santa Cruz Biotechnology), proteins of different
molecular mass than those detected by the CTF/NF1 antibody of Tanese
are detected, presumably representing other NF1 isoforms. The NF1
isoforms detected by this antibody also show a mobility shift in
response to insulin treatment (Fig. 6B), with insulin
treatment inducing the appearance of two new protein isoforms.
Coincident with this appearance is a decrease in the quantity of the
isoforms detected in the basal state. Thus, insulin treatment of
3T3L1 adipocytes induces a modification of multiple NF1 isoforms
that alters their mobility on SDS-polyacrylamide gel
electrophoresis.

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Fig. 6.
Analysis of NF1 proteins from 3T3-L1
adipocytes treated with insulin. 15 µg of nuclear extracts from
3T3-L1 adipocytes treated with insulin for the indicated times and
concentrations were separated on a 7% SDS-polyacrylamide gel. After
transfer to polyvinylidene difluoride membrane, the proteins were
probed with an anti-NF1 antibody and then developed using an enhanced
chemiluminescent assay. The numbers to the right
show the mobility of molecular mass standards. A, antibody
to CTF/NF1 generously provided by N. Tanese was used. The
arrow shows the fastest migrating protein that is recognized
by the anti-NF1 antibody that disappears with insulin treatment.
B, antibody to the N-teminus of NF1 (Santa Cruz
Biotechnology) was used. The arrowheads indicate the protein
isoforms induced upon exposure of the cells to insulin.
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Fig. 7.
Phosphatase treatment of NF1 proteins from
3T3-L1 adipocytes. 3T3-L1 adipocytes were treated with 1 µM insulin for 10 min, and nuclear extract was prepared
from control or insulin-treated (+) cells. 15 µg of nuclear extract
was treated with alkaline phosphatase (+) or heat-inactivated alkaline
phosphatase ( ) in the presence of 1% SDS as indicated. The extracts
were then separated on a 7% SDS-polyacrylamide gel. After transfer to
polyvinylidene difluoride membrane, the proteins were probed with an
anti-CTF/NF1 antibody (generously provided by N. Tanese) and developed
using an enhanced chemiluminescent assay. The arrows show
the fully dephosphorylated proteins recognized by the anti-CTF/NF1
antibody. The numbers to the left show the
mobility of molecular mass standards.
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Insulin Treatment Induces a Change in Phosphorylation of
NF1--
The insulin-induced mobility shift of the NF1 proteins on
SDS-polyacrylamide gel electrophoresis may be due to a change in their
extent of phosphorylation. To investigate this, 3T3-L1 adipocyte nuclear extracts from control and insulin-treated cells were treated with alkaline phosphatase followed by SDS-polyacrylamide gel
electrophoresis and Western immunoblotting using the CTF/NF1 antibody.
As a control, nuclear extract was treated with alkaline phosphatase
that had been heat-inactivated (
, Fig. 7). There was no change in
the mobility of the NF1 proteins that were treated with the
heat-inactivated phosphatase. As shown in Fig. 7, phosphatase treatment
resulted in an increase in the mobility of the proteins identified by
the CTF/NF1 antibody. When 1% SDS was included in the incubations, virtually all of the NF1 proteins were converted to two faster migrating species, presumably representing fully dephosphorylated forms. Moreover, while there was a change in the mobility of the NF1
proteins from insulin-treated cells compared with control cells, this
difference was abolished when the proteins were fully dephosphorylated.
Partial dephosphorylation of nuclear extract from insulin-treated cells
(Fig. 7, lane 4) resulted in the apparent regeneration of the isoform that disappeared with insulin treatment. These results strongly suggest that exposure of 3T3-L1 adipocytes to
insulin causes a rapid phosphorylation of NF1 proteins.
 |
DISCUSSION |
In a previous paper (16), we demonstrated that the region from
bases
676 to
706 in the 5'-flanking sequence of the GLUT4 gene
contains a cis-element, which mediates the repression of the gene by
insulin in 3T3-L1 adipocytes. Several lines of evidence now implicate
isoforms of the NF1 family of transcription factors as trans-acting
factors that mediate this effect. A subset of 3T3-L1 adipocyte nuclear
proteins that bind to the GLUT4 IRE have a sequence specificity that
corresponds to that of NF1 proteins, and their binding is specifically
competed by an NF1 consensus binding site oligonucleotide (Fig. 1).
These same proteins are recognized by a polyclonal anti-NF1 antibody,
as shown by the supershift experiments using the gel mobility shift
assay (Fig. 1). The importance of NF1 proteins in mediating the
down-regulation of the GLUT4 gene by insulin was confirmed by the
reporter gene studies using constructs that carried specific mutations
that disrupt the NF1 binding site (Fig. 2). Full down-regulation by insulin occurred only with the construct that maintained NF1 binding.
Another protein not related to the NF1 family also binds to the GLUT4
IRE. GLUT4 promoter-reporter gene studies suggest that this protein is
also important for the regulation of GLUT4 expression in 3T3-L1
adipocytes by insulin. Expression of a promoter-reporter construct that
contains a mutation that disrupts NF1 binding but maintains binding of
this other protein (the M2 mutation) is still significantly repressed
by insulin treatment (Fig. 2). However, the level of repression of the
M2 construct is significantly less than that of either the wild-type
reporter construct or the construct containing the mutation that does
not disrupt binding of either the NF1 protein(s) or the non-NF1 protein
(mutant M3). Therefore, although NF1 binding is necessary to mediate
the full effect of insulin, the non-NF1 protein is able to mediate a
partial repression of the GLUT4 promoter.
The NF1 family of transcription factors consists of more than a dozen
isoforms. Early studies by Jones et al. (20) found that CTF
was identical to NF-I (now referred to as NF1), which had been
identified as a factor required for the initiation of adenovirus DNA
replication. The nucleotide sequence to which NF1 binds is the same for
its action as a transcription factor or as a DNA replication factor.
Four distinct genes have been identified (NF1/CTF, NF1-L, NF1/Red1, and
NF1/X), with multiple products from each of these genes produced by
alternative splicing. The predicted molecular masses of the isoforms
range from 27 to 62 kDa. Larger isoforms have been identified (20),
most likely due to post-translational modification of these proteins
(21, 22). NF1 family members bind to DNA both as homo- and heterodimers (23), adding another level of diversity to this protein family. There
is nearly complete amino acid sequence identity in the N-terminal regions of the NF1 isoforms, with more diversity in their C-terminal portions. These transcription factors bind through their N-terminal domain to the sequence TGGN7CCA and regulate transcription
through their proline-rich C-terminal domain. The DNA binding domains of the NF1s show no obvious sequence similarity to any of the known
classes of DNA-binding domains such as zinc finger, leucine zipper, or
helix-loop-helix motifs. Most NF1 isoforms appear to be expressed
constitutively, but at least some tissue-specific isoforms appear to
exist, as reported for brain (24) and bone (25). NF1 has been
implicated in adipocyte gene expression, since an NF1 binding site was
shown to be necessary to direct tissue-specific expression mediated by
the 422/aP2 promoter to adipocytes (26). In addition, an NF1 binding
site is necessary for the transcriptional activation of the
stearoyl-CoA desaturase 1 promoter during differentiation of 3T3-L1
cells into adipocytes (27). It is not yet known which isoforms are
expressed by 3T3-L1 adipocytes or in adipose tissue. The antibodies
used in the present study are not isoform-specific; moreover, specific
isoforms cannot be identified based on molecular mass. However, since
Western analysis using two different anti-NF1 antibodies identified
multiple proteins, it appears that several isoforms are expressed by
3T3-L1 adipocytes.
Because of the rather ubiquitous expression of NF1 isoforms, they were
first believed to be basal transcription factors that contribute to the
expression of unregulated "housekeeping" genes. Recently, however,
data has accumulated that demonstrate various roles for NF1 in
mediating regulated gene expression. NF1 functions as an accessory
factor for gene regulation by a number of other transcription factors,
such that mutations that abolish NF1 binding either eliminate or
greatly diminish regulation by the other transcription factors. These
factors have included the cAMP response element-binding protein (28)
and members of the steroid hormone receptor superfamily, including the
glucocorticoid (29), estrogen (30), androgen (31), and vitamin D (25)
receptors. The studies presented here, however, are the first
demonstration that NF1 mediates the regulation of a gene by insulin.
Thus, NF1 joins a list that so far includes HNF-3 (32), Ets-related
proteins (33, 34), sterol-regulatory element-binding proteins (35),
upstream stimulatory factors (36, 37), and possibly Sp1 (38) and
CCAAT/enhancer-binding protein family members (39) as mediators of gene
regulation by insulin. It will be of interest to determine the role NF1
plays in the regulation of other genes by insulin, since a number
insulin-responsive genes have been found to have NF1 binding sites.
Importantly, an NF1 binding site has been identified near the insulin
response element of the fatty acid synthase gene (40). Further studies will be required to determine if NF1 binding to this element is necessary for the stimulation of fatty acid synthase expression by insulin.
Early studies in Tjian's laboratory found that NF1 can undergo
post-translational modification, including O-glycosylation (22) and phosphorylation (21). The existence of such modification raises the possibility that the function of NF1 may be regulated by
post-translational modification. Indeed, Yang et al.
demonstrated that overexpression of Myc resulted in the phosphorylation
of NF1 and suggested that this may be the mechanism through which Myc
represses the expression of a number of genes (41). We have shown that
insulin induces a change in the mobility of NF1 proteins on
SDS-polyacrylamide gel electrophoresis and that this change in mobility
is abolished if the proteins are dephosphorylated by phosphatase
treatment (Fig. 7). This provides compelling evidence that insulin
induces a change in the level of phosphorylation of NF1 and is the
first demonstration of this phenomenon.
Although the results presented in this paper support the importance of
NF1 in mediating the repression of the GLUT4 promoter by insulin, there
is an apparent inconsistency regarding the role that phosphorylation of
NF1 plays in this regulation; i.e. the change in
phosphorylation in response to insulin treatment is transient, while
repression of GLUT4 expression by insulin is more prolonged (15). It is
possible that the loss of the insulin-induced phosphorylation is due to
the difficulty of maintaining the phosphorylation states of proteins
in vitro. Insulin has been shown to activate protein
phosphatases (42, 43). If NF1 is exposed to one of these phosphatases
during the preparation of nuclear extract, the insulin-induced
phosphorylation could be lost in extracts from the later time points
when the phosphatase has been activated. It is also possible that a
transient phosphorylation of NF1 could initiate a cascade that results
in a sustained repression of GLUT4 expression. For example,
phosphorylation of NF1 could facilitate the binding of another
repressive transcription factor that remains bound even if NF1 becomes
dephosphorylated. Future studies will be needed to identify the
phosphorylation sites of NF1 affected by insulin and to demonstrate
that the change in NF1 phosphorylation induced by insulin is necessary
for the repression of GLUT4 expression by insulin in 3T3-L1 adipocytes.
However, the concentration of insulin required to induce a change in
phosphorylation of NF1 (Fig. 6) correlates well with the concentration
of insulin required to suppress GLUT4 transcription (15), supporting a
relationship between the two findings.
In summary, we have found that two classes of nuclear proteins bind to
the insulin response element of the GLUT4 gene. One of these is a
protein(s) related to the NF1 family of transcription factors.
Furthermore, binding of this NF1 protein(s) is necessary for full
repression of the GLUT4 promoter by insulin, since mutations that
specifically abolish binding of NF1 impair the insulin response. Finally, we find that treatment of 3T3-L1 cells with insulin results in
a rapid phosphorylation of NF1 proteins in 3T3-L1 adipocytes. Thus, we
have identified a new role for the NF1 family of transcription factors
in mediating gene regulation by insulin. This regulation appears to
involve insulin inducing a phosphorylation of NF1 proteins, which will
be a new mechanism of acute gene regulation by insulin.