From the Department of Biochemistry and Molecular
Biology, the University of Oklahoma Health Sciences Center,
Oklahoma City, Oklahoma 73190 and the § Department of
Physiology and Biophysics, the University of Iowa,
Iowa City, Iowa 52242
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ABSTRACT |
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We have previously demonstrated that important
regulatory elements responsible for regulated expression of the human
GLUT4 promoter are located between 1154 and
412
relative to transcription initiation (Olson, A. L., and Pessin,
J. E. (1995) J. Biol. Chem. 270, 23491-23495).
Through further analysis of this promoter regulatory region, we have
identified a perfectly conserved myocyte enhancer factor 2 (MEF2)-binding domain (-CTAAAAATAG-) that is necessary, but not
sufficient, to support tissue-specific expression of a chloramphenicol
acetyltransferase reporter gene in transgenic mice. Biochemical
analysis of this DNA element demonstrated the formation of a specific
DNA-protein complex using nuclear extracts isolated from heart,
hindquarter skeletal muscle, and adipose tissue but not from liver. DNA
binding studies indicated that this element functionally interacted
with the MEF2A and/or MEF2C MADS family of DNA binding transcription
factors. MEF2 DNA binding activity was substantially reduced in nuclear
extracts isolated from both heart and skeletal muscle of diabetic mice,
which correlated with decreased transcription rate of the
GLUT4 gene. MEF2 binding activity completely recovered to
control levels following insulin treatment. Together these data
demonstrated that MEF2 binding activity is necessary for regulation of
the GLUT4 gene promoter in muscle and adipose tissue.
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INTRODUCTION |
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To promote the storage of metabolic energy in the form of glycogen and triglycerides, insulin increases glucose uptake in skeletal muscle, heart, and adipose tissue. The GLUT41 facilitative glucose transporter protein is the main glucose transporter in fat and muscle tissue (1-6). GLUT4 protein is localized to the interior of the cell and moves to the plasma membrane in response to insulin receptor activation (7, 8). Several lines of evidence have shown that the effect of insulin on glucose uptake in these tissues results directly from the recruitment of GLUT4 from an intracellular vesicle pool to the plasma membrane (for recent reviews see Refs. 9 and 10).
Insulin-mediated glucose homeostasis is extremely sensitive to the overall GLUT4 protein pool size in the major insulin responsive tissues that include heart, skeletal muscle, and adipose tissue. In rodent models of insulin deficiency (fasting- or streptozotocin (STZ)-induced diabetes), the expression of GLUT4 protein and mRNA is markedly reduced and accounts for the insulin resistance of glucose transport under these conditions (11-17). Genetic studies strengthen the relationship between insulin-mediated glucose homeostasis and the cellular levels of GLUT4 protein. Mice engineered to express only one allele of the GLUT4 gene were shown to have diminished GLUT4 protein levels resulting in a progressive diabetic phenotype characterized by impaired glucose homeostasis (18). On the other hand, an increase in GLUT4 protein pool size enhances insulin-sensitive glucose uptake. Transgenic mice expressing the human GLUT4 gene specifically in adipose tissue or in both adipose and muscle tissues displayed a marked increase in basal glucose disposal and insulin-sensitive glucose uptake (19-25). In STZ diabetic mice, overexpression of GLUT4 in either fat or skeletal muscle under the control of heterologous tissue-specific promoters improves glucose homeostasis (26, 27). Finally, expression of the human GLUT4 gene in the genetically diabetic db/db strain of mice improved glycemic control and insulin sensitivity (28). The close association of GLUT4 protein pool size with physiologic changes in glucose homeostasis makes physiologic manipulation of GLUT4 gene expression an attractive target for therapeutic interventions designed to treat the insulin resistance associated with diabetes.
To achieve this goal, a detailed molecular understanding of the regulation of GLUT4 gene expression in a natural physiologic context is required. By using transgenic mice, we have previously reported that regions regulating expression of the human GLUT4 gene are found within 1154 bp of DNA located 5' to the major transcription initiation site (29-31). The 1154-bp fragment regulatory DNA supported a pattern of gene expression that mimics the mouse GLUT4 gene both in terms of tissue-specific and regulated expression in both fasting and STZ-induced models of insulin deficiency (31). In the current study, we have determined an essential role for an MEF2 DNA-binding domain in regulation of the human GLUT4 gene promoter. In addition, we have begun to characterize the nuclear proteins that interact with this binding domain in the major GLUT4 expressing tissues.
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MATERIALS AND METHODS |
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Preparation of Transgenic Mice--
The cDNA constructs used
to generate transgenic mice were derived from the plasmid
hGLUT4(2.4)CAT containing 2400 bp of GLUT4 5'-flanking DNA (29). This
plasmid was used to generate 895-hG4-CAT by digestion of the parental
plasmid with AvrII and SacI. An internal deletion
in hGLUT4(2.4)CAT was made by digestion with RsrII and BssHII, followed by treatment with T4 DNA polymerase in the
presence of nucleotides to fill in the remaining nucleotide overhangs. The plasmid was then religated to form hGLUT4(2.4RB)CAT. This plasmid was used to generate 1154
730/412-hG4-CAT by digestion with
BclI and HindIII and 895
730/412-hG4-CAT by
digestion with AvrII and HindIII. A third plasmid
was generated in which the MEF2-binding site of hGLUT4(2.4)CAT
(CTAAAAATAG) was mutated to form an ApaI site (CTGGGCCCAG).
This plasmid hGLUT4(2.4apa)CAT was used to generate 895APA-hG4-CAT by
digestion with AvrII and HindIII. The DNA
fragments were isolated by agarose gel electrophoresis and injected
into the pronucleus of fertilized mouse embryos at the University of
Iowa Transgenic Animal Facility (Iowa City, IA). Transgenic animals
carrying the appropriate constructs were identified by slot blot
analysis of isolated tail DNA using the 4.6-kilobase pair
SacI/HindIII fragment of hGLUT4(2.4)CAT as a probe.
STZ-induced Diabetes--
Insulin-deficient diabetes was induced
by a single intraperitoneal injection of STZ (200 mg/kg body weight)
following an overnight fast as described previously (32). Seventy-two
hours after injection, tail vein blood samples were assayed for glucose
concentration using chemstrips (Boehringer Mannheim). Animals with
blood glucose levels greater than 400 mg/dl were considered diabetic.
The diabetic animals were either left untreated or treated with 1 unit
of regular insulin per day for 2 days. The mice were killed 5 days
after STZ injection, and the tissues were snap-frozen in liquid
nitrogen and stored at 70 °C until prepared for analysis.
RNA Isolation--
Total cellular RNA was isolated from
snap-frozen tissues using the guanidinium isothiocyanate extraction
followed by purification on a CsCl gradient (33) as described
previously (34). RNA was quantified spectrophotometrically by
absorbance at 260 nm and stored as an ethanol precipitate at
70 °C.
RNase Protection Assay--
An 890-bp
SalI/EcoRI fragment of the mouse GLUT4-CAT
plasmid, p469GLUT4.CAT (obtained from Dr. M. D. Lane, Johns
Hopkins Medical School, Baltimore, MD) (35), was subcloned into the
SalI/EcoRI site of pIBI30 (IBI). This plasmid was
linearized with Bsu36I and was used as a template to
generate a 616-nucleotide antisense RNA probe that is able to anneal to
either the 5' end of mouse GLUT4 sequences or the 5' end of CAT
mRNA. The antisense RNA was labeled with [-32P]UTP
using T3 polymerase in an in vitro transcription assay
(Promega). Ten µg of total RNA was hybridized with 5 × 105 cpm of labeled probe in 30 µl of hybridization buffer
(80% deionized formamide, 0.4 M NaCl, 40 mM
Pipes, pH 6.4, and 1 mM EDTA). Hybridization was carried
out overnight at 50 °C, and the non-hybridized RNA was digested for
30 min at room temperature in 10 mM Tris-HCl, pH 7.5, 200 mM sodium acetate, and 5 mM EDTA containing 50 units of RNase T1/ml (Life Technologies, Inc.). The protected fragments were analyzed using 6% acrylamide, 7.5 M urea gel
electrophoresis exposed to Hyperfilm MP film (Amersham Pharmacia
Biotech) at
70 °C.
Northern Blot Analysis--
Ten µg of total RNA was
fractionated by 1% agarose-formaldehyde gel electrophoresis. Following
electrophoresis, RNA was transferred to nylon filters (Zeta-Probe GT,
Bio-Rad), and filters were prehybridized with a solution of 50%
deionized formamide, 0.12 M
Na2HPO4, pH 7.2, 0.25 M NaCl, and
7% SDS for 1 h at 50 °C. Blots were then hybridized overnight
in fresh prehybridization buffer containing 2 × 106
cpm/ml random primed cDNA probes corresponding to full-length MEF2C and MEF2A mRNA (kindly provided by Dr.
Eric N. Olson, University of Texas, Southwestern Medical Center,
Dallas). The filters were washed according to manufacturer's
specifications including a high stringency, low salt wash at 65 °C
for 15 min. The washed filters were exposed to Hyperfilm MP film
(Amersham Pharmacia Biotech) at 70 °C.
Western Blot Analysis--
Aliquots of 30 µg of total nuclear
extract protein were solubilized in an equal volume of 2× Laemmli
sample buffer (120 mM Tris-Cl, pH 6.8, 4% SDS, 20%
glycerol, and 200 mM DTT) and were fractionated by SDS-PAGE
using 7.5% acrylamide gels. The fractionated proteins were transferred
to polyvinylidene difluoride membranes (Millipore) in buffer (25 mM Tris and 190 mM glycine, pH 8.5) overnight
at 0.2 A at 4 °C. The membranes were blocked 1 h at room
temperature with a solution of Tris-buffered saline (TBS, 20 mM Hepes, pH 7.5, 150 mM NaCl) containing 0.2%
Tween 20 and 7% dried milk (Carnation). The membranes were probed with
the MEF2A rabbit polyclonal antibody (Santa Cruz) diluted 1:1000 in
TBS containing 0.1% Tween 20 and 2% bovine serum albumin (Sigma) for
1 h at room temperature. Immunoreactive proteins were visualized by enhanced chemiluminescence (SuperSignal, Pierce) following incubation of the blot with a goat anti-rabbit secondary antibody conjugated with horseradish peroxidase (Pierce).
Preparation of Nuclear Extracts--
Tissues were harvested and
snap-frozen in liquid nitrogen. The frozen tissues were pulverized in
liquid nitrogen and homogenized in 10 volumes of homogenization buffer
A (250 mM sucrose, 10 mM Hepes, pH 7.6, 25 mM KCl, 1 mM EDTA, 10% glycerol, 0.15 mM spermine, 0.5 mM spermidine, 0.1 mM PMSF, 2 µg/ml each aprotinin, leupeptin, and pepstatin
A, and 6 µg/ml each L-1-tosylamido-2-phenylethyl chloromethyl ketone and 1-chloro-3-tosylamido-7-amino-2-heptanone) with
10 strokes of a Teflon pestle. The homogenate was centrifuged 10 min at
3900 × g at 4 °C. The pellet was resuspended in 10 ml of homogenization buffer A and homogenized with 10 strokes of a
Dounce homogenizer with tight-fitting pestle. The homogenate was
layered over one-half volume of buffer B (1 M sucrose, 10 mM Hepes, pH 7.6, 25 mM KCl, 1 mM
EDTA, 10% glycerol, 0.15 mM spermine, 0.5 mM
spermidine, 0.1 mM PMSF, 2 µg/ml each aprotinin, leupeptin, and pepstatin A, and 6 µg/ml each
L-1-tosylamido-2-phenylethyl chloromethyl ketone and
1-chloro-3-tosylamido-7-amino-2-heptanone) and centrifuged at 3900 × g for 10 min at 4 °C. The pellet was resuspended in
buffer A/glycerol (9:1, w/w) and layered over one-third volume of
buffer B/glycerol (9:1, w/w). The gradient was centrifuged at
48,000 × g for 30 min at 4 °C. The semi-purified
nuclear pellet was resuspended in 1 volume of nuclear extraction buffer
(10 mM Hepes, pH 7.6, 400 mM KCl, 3 mM MgCl2, 0.1 mM EDTA, 10%
glycerol, 1 mM DTT, 0.1 mM PMSF) and 1/10
volume of 4 M KCl. Nuclear proteins were then extracted on
ice for 30 min, and the particulate material was removed by
centrifugation at 13,000 × g in a microcentrifuge for
10 min at 4 °C. The supernatant was dialyzed against buffer C (25 mM Hepes, pH 7.6, 100 mM KCl, 0.1 mM EDTA, 10% glycerol, 1 mM DTT, 0.1 mM PMSF, 2 µg/ml each aprotinin, leupeptin, and pepstatin
A, and 6 µg/ml each L-1-tosylamido-2-phenylethyl
chloromethyl ketone and 1-chloro-3-tosylamido-7-amino-2-heptanone) for
3 to 4 h. The dialysate was assayed for total protein (Bradford)
and stored at 70 °C.
In Vitro Translation--
To generate in vitro
translation products, cDNAs corresponding to human MEF2A and mouse
MEF2C isoforms were subcloned into expression vectors, linearized,
in vitro transcribed, and quantitated on a 1%
agarose-formaldehyde gel. Equivalent amounts of RNA were used to
program rabbit reticulocyte lysates in the presence and absence of
radiolabeled [35S]methionine. The radiolabeled in
vitro translation products were analyzed and quantitated by
SDS-PAGE followed by autoradiography and by immunoblotting with the
MEF2A antibody (data not shown).
Electrophoretic Mobility Shift Assays--
The human GLUT4 MEF2
DNA-binding site double-stranded oligonucleotide
(GGGAGCTAAAAATAGCAG) was made from custom-synthesized primers
(Life Technologies, Inc.). The MCK MEF2 and OCT1 DNA-binding site
oligonucleotides were commercially prepared (Santa Cruz). The
oligonucleotides were end-labeled with T4 polynucleotide kinase. The
probes (0.5 ng) were incubated with up to 14 µg of nuclear extracts
in a 10-µl reaction containing 2 µg poly(dI-dC), 40 mM KCl, 5 mM MgCl2, 15 mM Hepes, pH
7.9, 1 mM EDTA, 0.5 mM DTT, and 5% glycerol
for 20 min at room temperature. For competition studies, the extracts
were preincubated with various concentrations of unlabeled
oligonucleotides for 5 min before addition of the radiolabeled probe.
Similarly, electrophoretic gel supershift studies were carried out by
preincubating the nuclear extracts with 1 µg of either the MEF2
antibody (Santa Cruz) or rabbit IgG (Pierce) for 1 h on ice before
addition of the radiolabeled probe. The samples were then analyzed on a
non-denaturing 6% polyacrylamide (29:1 acrylamide/bisacrylamide) gel
buffered with Tris borate/EDTA (TBE, 22 mM Tris, 22 mM boric acid, and 0.5 mM EDTA) and run at 300 V for 2 h 4 °C. The dried gels were then exposed to Hyperfilm MP at
70 °C.
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RESULTS |
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Tissue-specific Expression of the Human GLUT4 Gene in Transgenic Mice-- To identify the DNA sequences responsible for tissue-specific expression of the human GLUT4 gene, we have generated multiple lines of transgenic mice carrying CAT reporter gene constructs containing various deletions in the 5'-flanking DNA. In a previous report, we demonstrated that the smallest construct capable of supporting full promoter function required 1154 bp of the human GLUT4 5'-flanking DNA (31). To localize further functional elements within this region, we created transgenic mice carrying 895 bp of DNA fused to the CAT reporter gene (895-hG4-CAT). RNase protection analysis of these mice displayed a pattern of CAT expression that paralleled the expression of the endogenous murine GLUT4 mRNA (Fig. 1, A and B). The transgenic CAT and endogenous murine GLUT4 mRNAs were expressed at highest levels in brown adipose tissue and skeletal muscle with slightly lower levels in cardiac muscle followed by white adipose tissue. This protein is identical to transgene expression observed for larger segments of regulatory DNA (30). Importantly, there was no measurable expression of the 895-hG4-CAT transgenic mRNA in liver or brain, tissues which normally do not express GLUT4. Since expression of reporter genes in transgenic animals may be affected by the location of genomic integration and copy number, we analyzed five independent founder lines with essentially identical results (Table I). From these data we conclude that at least 895 bp of 5'-flanking DNA are required for expression of the GLUT4 gene.
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Regulated Expression of 895-hG4-CAT in Insulin-deficient Diabetic
Mice--
It is well established that in both rats and mice
GLUT4 gene expression is down-regulated in insulin-deficient
states due to a decrease in transcription rate of the gene (30, 36). This is an insulin-specific response since the decrease in
transcription and loss of GLUT4 mRNA are fully restored
following insulin therapy. To determine if 895-hG4-CAT was regulated by
insulin deficiency in an analogous manner to the endogenous
GLUT4 gene, we compared the expression of the mouse
GLUT4 and transgenic CAT mRNAs in STZ-induced
diabetes. Expression of CAT mRNA in white and brown adipose tissue, heart, and hindquarter skeletal muscle was reduced in
diabetic mice in a manner that paralleled the down-regulation of the
mouse GLUT4 gene (Fig. 2).
Treatment of these diabetic mice with insulin restored both
CAT mRNA and GLUT4 mRNA levels to normal
or above normal levels (Fig. 2). These data, in conjunction with the 5'
and internal deletion analysis, suggest that the regulatory and
tissue-specific elements are located downstream of nucleotide 895 and
that sequences upstream of this position do not contribute to the
function of the GLUT4 promoter with respect to
tissue-specific expression and regulated expression during insulin
deficiency.
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Tissue-specific Expression of the MEF2A and MEF2C Isoforms-- The MEF2-binding site in the human GLUT4 promoter is perfectly conserved in both the mouse and rat genes (37). This type of domain has been reported to specifically bind MADS box proteins of the MEF2 family of transcription factors (38). Although first described in nuclear extracts from cultured myotubes, skeletal muscle, heart, and brain, some MEF2 isoforms such as MEF2A are more broadly expressed (39). The presence of MEF2 isoform mRNAs and immunoreactive proteins in adipose tissue has not been reported. Therefore, Northern blot analysis was performed using probes specific for either MEF2A or MEF2C to determine the tissue distribution of these genes with respect to the major GLUT4-expressing tissues. MEF2C mRNA was detected at highest levels in skeletal muscle and brain, with much lower levels in white and brown adipose tissue and heart (Fig. 3A). On the other hand, MEF2A mRNA was highly expressed in brown and white adipose tissue, heart, and skeletal muscle as well as in brain, whereas a much lower level of mRNA was detected in liver (Fig. 3B). To determine the levels of MEF2A protein, nuclear extracts prepared from heart, skeletal muscle, and brown adipose tissue were also subjected to Western blot analysis using an antibody against MEF2A. Western blot analysis confirmed the presence of MEF2A immunoreactive material in brown adipose tissue, heart, and skeletal muscle but not in liver (Fig. 3C). Although the MEF2A antibody can cross-react with MEF2C (see below), the presence of both MEF2A mRNA and protein in the major GLUT4 expressing tissues coupled with the functional requirement of the MEF2-binding site strongly suggests that this isoform is likely to play a role in GLUT4 gene expression.
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Specificity of Nuclear Proteins That Bind the GLUT4 MEF2 Site-- To characterize proteins that bind to the human GLUT4 promoter MEF2-binding site, heart nuclear extracts were incubated with a radiolabeled 18-bp oligonucleotide corresponding to the GLUT4 MEF2 site. Electrophoretic mobility shift assay (EMSA) revealed the presence of a diffuse band that was not present in the absence of the heart nuclear extract (Fig. 4A, lanes 1 and 2). Incubation of the radiolabeled MEF2 oligonucleotide with a 5- or 30-fold excess of unlabeled oligonucleotide completely inhibited the formation of this complex (Fig. 4A, lanes 3 and 4). Competition of the labeled GLUT4 MEF2 oligonucleotide with a 5- or 30-fold molar excess of the unlabeled oligonucleotide corresponding to the MEF2-binding site of the muscle creatinine kinase (MCK MEF2) was not as efficient compared with the competition of the labeled GLUT4 MEF2 site with itself (Fig. 4A, compare lanes 5-7 with lanes 2-4). These data indicated that the GLUT4 MEF2-binding site has a slightly higher affinity for this complex compared with the MCK MEF2-binding site. As a control, an oligonucleotide corresponding to the unrelated consensus OCT1-binding site, which binds the ubiquitously expressed family of OCT1 transcription factors, was unable to compete for the heart nuclear extract binding to the radiolabeled GLUT4 MEF2 oligonucleotide (Fig. 4A, lanes 8-10). These data confirm that this nuclear binding activity was specific for the MEF2 DNA-binding domain.
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Biochemical Analysis of Nuclear Proteins That Bind the GLUT4 MEF2
Site--
To identify the nature of this protein-DNA complex, we next
preincubated the heart nuclear extracts with the MEF2A antibody or a
control IgG (Fig. 5). As observed in Fig.
4, incubation of heart nuclear extracts with the 18-bp MEF2
oligonucleotide resulted in the formation of a DNA-protein complex that
was not present in the absence of the nuclear extract (Fig. 5,
lanes 1 and 2). Pretreatment of this complex with
the MEF2A antibody resulted in the supershift of only a small fraction
of the DNA-protein complex (Fig. 5, lanes 2 and
3). The formation of this complex was increased in direct
proportion to the concentration of heart nuclear extract incubated with
the GLUT4 MEF2 oligonucleotide (Fig. 5, lanes 3, 5, 7, and 9). Although the
MEF2A antibody was capable
of inducing a supershift at all these concentrations of extract, only a
fraction of this complex was affected (Fig. 5, lanes 2, 4, 6, and 8). Thus, it appears that although the formation of this complex was, in part, due to the binding of an MEF2
transcription factor, an unidentified complex can form independently of
MEF2A immunoreactive protein.
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STZ Diabetes Decreases MEF2 Binding Activity-- Since the MEF2-binding site appeared to be critical for GLUT4 promoter function, we postulated that this binding site may be necessary for regulated expression during states of insulin deficiency. Nuclear extracts were prepared from heart and skeletal muscle obtained from control, STZ diabetic mice, and diabetic mice that were subsequently treated with insulin (Fig. 8). Electrophoretic mobility shift of the GLUT4 MEF2 oligonucleotide demonstrated a reduction in binding activity from the diabetic nuclear extracts compared with the control extracts (Fig. 8A, compare lanes 2 with 3 and lanes 5 with 6). The loss of DNA binding activity was specific for the insulin-deficient state as insulin treatment completely restored the MEF2 binding activity to control levels (Fig. 8A, lanes 4 and 7). To determine if the decrease in DNA binding was specific for MEF2 or was a general phenomenon for all transcription factors, we also examined the effect of insulin deficiency on OCT1 binding (Fig. 8B). OCT1 binding was not affected by STZ diabetes or by subsequent insulin treatment (Fig. 8B, lanes 2-7). These data demonstrate that the reduced formation of this DNA-binding complex was specific for the GLUT4 MEF2-binding site rather than a generalized decrease in all transcription factors.
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DISCUSSION |
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Previously, we examined the tissue-specific and hormonal/metabolic
regulation of GLUT4 gene expression by generating transgenic mice carrying various 5' GLUT4 promoter CAT
reporter gene constructs (31). In that study, we determined that a
fragment consisting of 1154 bp of the GLUT4 5'-flanking DNA
was sufficient to support the full pattern of gene expression observed
for the endogenous gene. Although a construct containing 730 bp of
5'-flanking data was able to support expression of the reporter gene in
hindquarter skeletal muscle, the levels of expression were low in heart
and adipose tissue, and expression was observed in tissues that do not
normally express high levels of GLUT4 mRNA. Furthermore,
we also noted that a deletion of the DNA upstream of position 412 relative to the site of transcription initiation functioned as a basal
promoter that was not tissue-specific.
To define more precisely the functional GLUT4 regulatory
sequences, we have now demonstrated that 895 bp of 5'-flanking DNA of
the human GLUT4 gene is sufficient to support expression of a CAT reporter gene that mirrors the expression of the mouse
GLUT4 gene. Having established a more proximal 5' boundary,
internal deletions between positions 730 and
412 resulted in a
complete loss of detectable GLUT4 promoter activity. Within
this region, the GLUT4 gene contains a highly conserved
10-bp MEF2-binding sequence (CTA(A/T)4TA(G/A)) that is
completely conserved in the GLUT4 gene (CTAAAAATAG) from
humans, rats, and mice (37). Furthermore, in the context of the
functional 895-bp GLUT4 promoter, a 6-bp substitution of the
core MEF2-binding sequence (CTGGGCCCAG) resulted in a complete
abrogation of promoter function. Together, these data provide
compelling in vivo data demonstrating that the MEF2-binding site within the GLUT4 promoter provides a necessary function
for the tissue-specific and hormonal/metabolic regulation of
GLUT4 expression.
The functional significance of the MEF2-binding site in GLUT4 gene expression prompted us to determine the nature of DNA-protein binding complexes in the major GLUT4-expressing tissues, adipose and muscle. Electrophoretic mobility gel shift assays demonstrated the formation of a specific DNA-protein complex between the GLUT4 MEF2-specific oligonucleotide and nuclear extracts from heart, skeletal muscle, and brown adipose tissue. Although there was no apparent qualitative or quantitative difference in the shifted complexes obtained with these nuclear extracts, this complex was completely absent from liver, a tissue that does not express the GLUT4 gene.
Although the precise composition of this binding complex has not been
fully elucidated, our data support a role for the MEF2A and/or MEF2C
isoforms in both the binding and expression of the GLUT4
gene. Northern blot analysis demonstrated that MEF2A was highly
expressed in muscle and adipose tissue, whereas the MEF2C isoform was
only abundant in muscle and is therefore unlikely to contribute to
GLUT4 expression in adipose tissue. However, since the MEF2A
antibody was found to cross-react with the MEF2C isoform, the relative
levels of protein expression in these tissues could not be determined.
Nevertheless, the ability of MEF2A and MEF2C to specifically interact
with the GLUT4 MEF2-binding sequence was also substantiated
by in vitro translation of these transcription factors.
In vitro translated MEF2A and MEF2C were both fully capable of inducing specific binding to the GLUT4 MEF2-binding
sequence and underwent a complete mobility shift upon incubation with
the MEF antibody (MEF2).
Consistent with these observations, heart, skeletal muscle, and adipose
tissue nuclear extracts also demonstrated an MEF2 antibody-induced
supershift. Surprisingly, however, only a fraction of the endogenous
nuclear extract complex displayed reduced mobility suggesting the
presence of other unknown factors and/or additional complexes. At
present, we have not ruled out the two other MEF2 isoforms (MEF2B and
MEF2D), which have significantly different mobilities than either MEF2A
and MEF2C in gel shift assays (40, 41). It is also unlikely that these
complexes contain the serum response factor (SRF) since the upstream
and downstream nucleotides flanking the core (AT)6 sequence
of the GLUT4 MEF2-binding domain are incompatible for SRF binding (42).
In addition, supershift binding with an SRF antibody was completely
unable to induce a supershift of these complexes (data not shown).
Further study will be required to determine the precise nature of the
DNA-protein complex identity of this DNA-binding protein.
In any case, further support for the involvement of MEF2A and/or MEF2C was obtained by examining the regulation of GLUT4 gene expression in insulin-deficient diabetic mice. It has been well established that in states of insulin deficiency, there is marked reduction in GLUT4 expression due to a decrease in GLUT4 gene transcription which can be restored upon insulin treatment (31, 36). In parallel to the inhibition of GLUT4 transcription, we have also observed that the formation of the GLUT4 MEF2 binding activity was decreased and also recovered following insulin therapy. Concomitant with the changes in binding activity, the protein levels of MEF2 were decreased in the diabetic state and recovered following insulin treatment. Since adipose tissue and heart do not express the MEF2C isoform, these changes appear to be specific for MEF2A. Based on these data, we hypothesize that the level of GLUT4 gene expression may be modulated, at least in part, by the level of MEF2A binding activity in adipose tissue and probably in skeletal muscle as well.
In addition to the complexity of regulation at the GLUT4
MEF2 binding consequence, it is clear that other elements of the GLUT4 promoter have significant impact on expression. For
example, expression of the CAT reporter gene containing 730 bp of GLUT4 5'-flanking DNA resulted in normal expression in
skeletal muscle but not in heart or adipose tissue (31). This construct
also failed to display hormonal/metabolic regulation in the cases of STZ-induced diabetes or fasting. Because this construct contains the
intact MEF2-binding site, this strongly suggests that the MEF2 site
alone is not sufficient to support a full program of GLUT4
gene expression. Thus the presence of upstream regulatory elements
located between 895 and
730 appears required for both heart and
adipose tissue-specific expression as well hormonal/metabolic regulation. Taken together, these data support a model in which GLUT4 promoter activity is regulated by multiple regulatory
elements that may synergize for full promoter function. A model for
such complex regulation is not unexpected for genes regulated in part by an MEF2-binding site. Functional interactions between MEF2 isoforms
and other myogenic transcription factors have been previously described
(43). Likewise, distinct regulatory elements including an MEF2-binding
site are required for complex expression of a myosin light chain gene
in transgenic mice (44).
In summary, the data presented in this study demonstrate through functional analysis of the GLUT4 promoter that a conserved MEF2 binding is necessary but not sufficient to support expression of the GLUT4 gene. Nuclear extracts from the major GLUT4-expressing tissues have significant MEF2 binding activity which appears to be qualitatively similar. This binding activity is absent in liver, a tissue which does not express the GLUT4 gene. MEF2 binding activity is specifically decreased in extracts obtained from STZ diabetic mice but is fully restored following insulin treatment. This decrease and subsequent recovery in binding activity is attributable to changes in MEF2A and/or MEF2C nuclear protein levels. Taken together, the functional and biochemical analyses of the GLUT4 MEF2-binding site support a role for this domain in the GLUT4 gene expression in skeletal muscle, heart, and adipose tissue.
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FOOTNOTES |
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* This work was supported by Research Grants DK47894, DK44612, and DK42452 from the National Institutes of Health and Award 196085 from the Juvenile Diabetes Foundation.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: Dept. of Biochemistry and Molecular Biology, The University of Oklahoma Health Sciences Center, P. O. Box 26901, Rm. 853-BMSB, Oklahoma City, Oklahoma 73190. Tel.: 405-271-2227; Fax: 405-271-3092.
1 The abbreviations used are: GLUT4, adipose/muscle-specific glucose transporter; STZ, streptozotocin; bp base pair; SRF, serum response factor; EMSA, electrophoretic mobility shift assay; PAGE, polyacrylamide gel electrophoresis, MCK, muscle creatinine kinase; MEF2, myocyte enhancer factor 2; Pipes, 1,4-piperazinediethanesulfonic acid; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride.
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REFERENCES |
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