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INTRODUCTION |
Myocardial cells can use a wide variety of substrates for energy
production, including free fatty acids, glucose, lactate, and ketone
bodies. Substrate selection by cardiac myocytes is developmentally
regulated. During the perinatal period, substrate metabolism shifts
from predominant non-oxidative glucose utilization to predominant fatty
acid oxidation (1). This shift is associated with a shift in the
expression of several regulatory proteins involved in glucose and fatty
acid metabolism (2-7), including GLUT (glucose
transport) proteins. Specifically, the ubiquitous glucose
transporter GLUT1 is replaced by the insulin-regulated, muscle- and
fat-specific isoform GLUT4 (2, 3, 6).
Myocardial hypertrophy is a pathological condition triggered by excess
workload or cellular stress and is characterized by a number of
phenotypic changes, including activation of immediate/early, fetal, and
contractile protein genes, e.g. c-fos, atrial
natriuretic factor, and myosin light chain-2. In addition, the
hypertrophied heart exhibits a pattern of substrate metabolism similar
to that of the fetal/neonatal heart with increased glycolytic flux and reduced fatty acid oxidation (8-10). Such alterations of the metabolic behavior could be explained by a resumption of the fetal expression pattern of proteins involved in glucose and fatty acid metabolism. Two
preliminary reports have described increased expression of the
Glut1 isoform mRNA in myocardial hypertrophy induced in
adult rats by pressure overload (11) or a large infarct of the left ventricle (12).
In response to treatment with a variety of different agonists, primary
cultures of ventricular myocytes isolated from neonatal rat hearts
display many of the features associated with hypertrophy in
vivo and provide a useful model to study this nonproliferative growth response (13). We have used this model to investigate the
regulation of GLUT1 expression in cardiac myocytes. Cardiac myocytes
were transfected with a reporter construct encoding luciferase under
the control of the Glut1 promoter. The expression of this reporter in response to the hypertrophic agonists
12-O-tetradecanoylphorbol-13-acetate (TPA)1 and phenylephrine was
assessed, and the signaling pathways responsible for increased
expression of GLUT1 were investigated.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
Primary ventricular myocytes were isolated
from 1-day-old rats by collagenase digestion and maintained in
Dulbecco's modified Eagle's medium/medium 199 (4:1) supplemented with
penicillin and streptomycin (maintenance medium) as described
previously (14). For transfection experiments, cells were plated at a
density of 2.5 × 105/3.5-cm dish. For RT-PCR
experiments or ERK activation assay, 2 × 106 cells
were plated in 6-cm dishes. For activated Ras assays, 2 × 106 cells were plated in 10-cm dishes.
Cardiac fibroblasts cultures were prepared by two passages of the cells
adherent to the culture dish during the pre-plating procedure. Cells
were maintained in maintenance medium supplemented with 10% fetal calf
serum. For transfection experiments, cells were plated at a density of
2.5 × 105/3.5-cm dish and grown for 24 h in
maintenance medium with FCS before transfection. For activated Ras
assays, 2 × 106 cells were plated in 10-cm dishes and
grown in maintenance medium with FCS to subconfluence.
Cell Morphology--
Cells for morphological analysis were
plated on glass coverslips coated with gelatin and laminin. Cells were
treated for 48 h and then fixed and stained with fluorescein
isothiocyanate-conjugated phalloidin to show filamentous actin.
ERK Activation Assay--
Cells were pretreated with or without
PD98059 for 30 min and then treated with either TPA or phenylephrine
for 10 min. Cells were then harvested in 500 µl of Laemmli buffer,
and 50 µl of cell lysate were submitted to SDS-polyacrylamide gel
electrophoresis and blotted onto polyvinylidene difluoride membrane.
Total ERKs and doubly phosphorylated
(Thr202/Tyr204) ERKs were detected by Western
blotting using the K-23 rabbit polyclonal antibody (Santa Cruz
Biotechnology) and the E10 monoclonal antibody (New England Biolabs
Inc.), respectively.
Plasmids--
The plasmid containing 1.4 kilobase pairs of the
mouse Glut1 promoter along with enhancer 1 (0.6 kilobase
pair) and enhancer 2 (1.3 kilobase pair) was a kind gift from Dr.
Takashi Murakami (15). The Glut1 promoter along with
enhancers 1 and 2 was subcloned into the pGL3basic multicloning site
(Promega) to generate the luciferase reporter plasmid pLuc-GT1/E1/E2.
The luciferase reporter was cotransfected with a Rous sarcoma virus
(RSV)-
-galactosidase reporter plasmid (provided by Michael
Kapiloff). The estrogen-regulated Raf-1 expression vector
(pCEP4-
Raf-1:ER) expresses the kinase domain of Raf1 fused to the
steroid-binding domain of the human estrogen receptor (16). The
expression plasmids for the Ras mutants V12Ras (constitutively active)
and A15Ras (dominant-negative) were constructed by subcloning a
650-base pair cDNA into an elongation factor 1
-driven plasmid.
The expression plasmid for the MAP kinase phosphatase MKP-3 was
constructed by cloning a 1.4-kilobase pair cDNA that was amplified
from a rat cardiac cDNA library into an elongation factor
1
-driven plasmid. CL100 in the expression vector pSG5 was provided
by Steve Keyse. The cytomegalovirus-driven
N3/S218E/S222D MEK1
construct (17) was provided by Nathalie Ahn. The expression plasmid
pGEX-RBD, encoding the Ras-binding domain (RBD) of c-Raf-1 fused to GST
(GST-RBD) (18), was donated by Stephen J. Taylor.
Gene Expression Assays--
Transient transfections of myocytes
were performed using the calcium phosphate precipitation method as
described previously (14, 19, 20), using the amounts of plasmid DNA
indicated in the figure legends. Transfection of fibroblasts was
achieved using the LipofectAMINE Plus reagent (Life Technologies, Inc.) following the supplier's instruction. Luciferase and
-galactosidase assays were performed with reagents from Promega or Tropix Inc., respectively, as described by the manufacturer.
Expression of Endogenous Glucose Transporters--
To evaluate
the relative expression of the endogenous glucose transporter genes
Glut1 and Glut4 by RT-PCR, we took advantage of
regions of structural similarity and differences between the two
isoforms (21). Untreated cells or cells stimulated with 1 µM TPA or 100 µM phenylephrine for 48 h were harvested in 750 µl of TRIZOL reagent (Life Technologies,
Inc.), and total RNA was isolated following the manufacturer's
instructions. Total RNA was used for reverse transcription and
subsequent polymerase chain reaction using the Titan One Tube RT-PCR
system from Boehringer Mannheim. Primers capable of amplifying both
Glut1 and Glut4 cDNAs such that their
respective products could be resolved on the basis of a 12-base pair
size difference were used (21). PCR products were labeled by adding
0.05 µCi/µl [
-32P]dCTP to the reaction mixture and
subsequently resolved by electrophoresis on 15% polyacrylamide gels in
1× Tris borate/EDTA buffer. The gels were dried and exposed to storage
phosphor screens. Band intensity was determined using a Molecular
Dynamics PhosphorImager with ImageQuant software.
Activated Ras Detection Assay--
Detection of Ras-GTP in cells
extract was performed as described (18). Briefly, cell lysates were
incubated with GST-RBD pre-bound to glutathione-Sepharose (Amersham
Pharmacia Biotech) for 30 min at 4 °C. Bound proteins were eluted
with SDS-polyacrylamide gel electrophoresis sample buffer, resolved on
15% polyacrylamide gels, and subjected to Western blotting. Blots were
probed using a rabbit anti-Ha-Ras polyclonal antibody (Santa Cruz
sc-520).
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RESULTS |
TPA and Phenylephrine Induce Hypertrophy of Cardiac
Myocytes--
Fig. 1 shows phalloidin
staining of rat neonatal ventricular myocytes treated with either TPA
or phenylephrine. As described previously (13, 22-25), cells treated
with either agonist for 48 h showed a dramatic increase in size,
together with increased organization of myofibrils, two hallmarks of
hypertrophy of ventricular myocytes.

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Fig. 1.
TPA and phenylephrine induce hypertrophy of
rat neonatal ventricular myocytes. Cells were plated onto glass
coverslips coated with gelatin and laminin and left untreated or
treated with TPA or PE for 48 h. Cells were then stained with
fluorescein isothiocyanate-labeled phalloidin to show filamentous
actin.
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Hypertrophic Agonists Stimulate the Expression of Endogenous
GLUT1--
The effect of the hypertrophic agonists TPA and PE on the
relative expression of the glucose transporter isoforms
Glut1 and Glut4 is shown in Fig.
2. Untreated myocytes had a
Glut1/Glut4 expression ratio close to 1 (1.2 ± 0.2), typical for neonatal cardiac myocytes (2, 3, 26). Following
48 h of treatment with either TPA or PE, the ratio of
Glut1/Glut4 mRNAs was markedly increased, to
5.1 ± 1.4 and 3.7 ± 1.7, respectively. This increase was
mainly achieved by overexpression of Glut1 and to a minor extent by a decrease in Glut4 expression.

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Fig. 2.
TPA and phenylephrine stimulate expression of
the Glut1 gene. A, RT-PCR products
obtained in a representative experiment. Myocytes (2 × 106 in 6-cm dishes) were untreated (control (C))
or treated with TPA or PE for 48 h. RT-PCR was performed with 1.25 µg of total RNA. B, PhosphorImager quantitation of RT-PCR
experiments. Results are expressed as the
Glut1/Glut4 expression ratio and are means ± S.E. of six separate experiments.
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Hypertrophic Agonists Induce Transcription from the Glut1
Promoter--
To determine whether the increased Glut1
mRNA level in response to hypertrophic stimuli was caused by
increased transcription from the Glut1 promoter, we
performed transient transfection experiments with the pLuc-GT1/E1/E2
construct. As shown in Fig.
3A, both TPA and PE stimulated
transcription from the GT1/E1/E2 promoter construct in cardiac
myocytes. TPA also induced transcription in cardiac fibroblasts, albeit
to a lesser extent, whereas PE did not affect transcription in these
cells. Fig. 3B shows the time course of induction of the
Glut1 promoter in cardiac myocytes. Induction by TPA was
already detectable after 6 h of treatment and reached a plateau by
12 h, whereas induction by phenylephrine was slower, being
detectable after 12 h and reaching a maximum by 48 h only. Therefore, an incubation time of 48 h was selected for subsequent experiments.

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Fig. 3.
TPA and phenylephrine increase transcription
from the Glut1 promoter. A, cardiac
myocytes and fibroblasts were transfected with 1 µg of pLuc-GT1/E1/E2
and 0.7 µg of RSV- -galactosidase ( -gal) and treated
with 1 µM TPA (black bars) or 100 µM PE (white bars) for 48 h (myocytes) or
24 h (fibroblasts). Results are expressed as means ± S.E. of
at least three experiments, each performed in triplicate. B,
myocytes were transfected with 1 µg of pLuc-GT1/E1/E2 and 0.7 µg of
RSV- -galactosidase and treated with 1 µM TPA
(black bars) or 100 µM PE (white
bars) for 6-48 h. Results are expressed as means ± S.E. of
at least three experiments, each performed in triplicate.
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MAP Kinase Pathways Transduce the Hypertrophic Signal to the Glut1
Promoter--
Stimulation of the MAP kinase pathways plays an
important role in the development of hypertrophy of myocardial cells.
We therefore assessed the involvement of MAP kinase pathways in the
overexpression of Glut1. The MEK1 inhibitor PD98059
partially inhibited activation of ERK1 and ERK2 in response to either
TPA or phenylephrine (Fig. 4A). Treatment with PD98059
did not affect base-line expression of either the Glut1 or
Glut4 endogenous gene. However, treatment with PD98059
markedly reduced expression of the Glut1 gene induced by
either TPA or phenylephrine, without affecting expression of the
Glut4 gene (Fig. 4, B and C). We then
assessed the involvement of MAP kinase pathways in the induction of the
Glut1 promoter by TPA and phenylephrine. Cotransfection of
the cells with the broad specificity MAP kinase phosphatase CL100
significantly reduced induction of the Glut1 promoter by
either TPA or phenylephrine (Fig. 4D). PD98059 also
inhibited the response to both hypertrophic agonists, confirming
participation of the ERK pathway. In contrast, the p38 inhibitor
SB203580 did not affect induction of the Glut1 promoter by
TPA or phenylephrine (data not shown).

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Fig. 4.
MAP kinase inhibitors block induction of the
Glut1 promoter. A, the MEK inhibitor
PD98059 reduces ERK activation by TPA or PE. Myocytes (2 × 106 in 6-cm dishes) were untreated (control (C))
or treated with TPA or PE in the presence or absence of 20 µM PD98059 for 10 min. Western blotting using an
anti-doubly phosphorylated ERK antibody or an anti-ERK antibody was
performed. B, RT-PCR products obtained in a representative
experiment. Myocytes (2 × 106 in 6-cm dishes) were
untreated or treated with TPA or PE in the presence or absence of 20 µM PD98059 for 48 h. RT-PCR was performed with 1.25 µg of total RNA. C, PhosphorImager quantitation of RT-PCR
experiments. Cells were untreated or treated with TPA or PE in the
presence (white bars) or absence (black bars) of
20 µM PD98059 for 48 h. Results are expressed as
means ± S.E. of two to three experiments. D, myocytes
were transfected with 1 µg of pLuc-GT1/E1/E2, 0.7 µg of
RSV- -galactosidase ( -gal), and 2 µg of either CL100
or empty vector plasmid. Cells were then stimulated with 1 µM TPA (black bars) or 100 µM PE
(white bars) for 48 h in the presence or absence of 20 µM PD98059. Results are expressed as means ± S.E.
of at least three experiments, each performed in triplicate.
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To further confirm involvement of the ERK pathway in Glut1
promoter induction, cells were cotransfected with increasing amounts of
a plasmid expressing the ERK-specific phosphatase MKP-3 (Fig. 5) (27, 28). As expected, MKP-3 inhibited
induction of the Glut1 promoter by both TPA and
phenylephrine.

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Fig. 5.
ERK activation is required for induction of
the Glut1 gene. Myocytes were transfected with 1 µg of pLuc-GT1/E1/E2, 0.7 µg of RSV- -galactosidase
( -gal), and increasing amounts of MKP-3, balanced with
empty vector. Cells were then stimulated with 1 µM TPA
(black bars) or 100 µM PE (white
bars) for 48 h. Results are expressed as means ± S.E.
of four experiments, each performed in triplicate.
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We next reasoned that if the ERK pathway was important for induction of
the Glut1 promoter, then introduction into cells of constitutively active versions of the proteins of the Ras/Raf/MEK/ERK cascade should mimic the effect of TPA and phenylephrine and induce the
promoter. The results shown in Fig. 6
demonstrate that expression of a constitutively active mutant of Ras
(V12Ras), an estrogen-inducible version of Raf-1 (
Raf-1:ER), or a
constitutively active mutant of MEK1 (
N3/S218E/S222D MEK1) resulted
in increased expression from the Glut1 promoter.
Furthermore, expression induced by all of these agonists was inhibited
by cotransfection with the broad specificity MAP kinase phosphatase
CL100.

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Fig. 6.
Constitutively active mutants of the ERK
pathway induce the Glut1 promoter. Myocytes were
transfected with 1 µg of pLuc-GT1/E1/E2, 0.7 µg of
RSV- -galactosidase ( -gal), and 2 µg of V12Ras or
empty vector, 2 µg of estrogen-inducible Raf-1:ER, or 1 µg of
N3/S218E/S222D MEK1 or wild-type MEK1. Raf-1:ER-transfected cells
were then treated with either 0.8 mM estradiol or the
ethanol vehicle. Cells were also cotransfected with 2 µg of CL100
(white bars) or empty vector (black bars).
Results are expressed as means ± S.E. of three to four
experiments, each performed in triplicate.
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Ras Activity Is Required for the Hypertrophic Response--
Ras
activation is required for phenylephrine-induced hypertrophy and is
sufficient to induce both morphological and genetic markers of
hypertrophy (19, 29, 30). We therefore tested whether Ras was also
required for the Glut1 response to TPA and phenylephrine.
Fig. 7A shows that
cotransfection of cardiac myocytes with increasing amounts of the
dominant-negative Ras mutant A15Ras (31) strongly inhibited induction
of the Glut1 promoter by both TPA and phenylephrine. This
result suggests that Ras activation is required for transduction of the
signal elicited by both TPA and phenylephrine in cardiac myocytes. In
contrast, expression of A15Ras in cardiac fibroblasts did not affect
the Glut1 promoter induction in response to TPA (Fig.
7B), whereas it markedly blunted induction of the
Glut1 promoter by serum (Fig. 7C). These results suggest that Ras does not participate in signal transduction activated by TPA in cardiac fibroblasts, although it does participate in induction by TPA in cardiac myocytes.

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Fig. 7.
Ras activity is required for induction of the
Glut1 promoter in myocytes. A,
myocytes were transfected with 1 µg of pLuc-GT1/E1/E2, 0.7 µg of
RSV- -galactosidase ( -gal), and increasing amounts of
A15Ras, balanced with empty vector. Cells were then stimulated with 1 µM TPA (black bars) or 100 µM PE
(white bars) for 48 h. Results are expressed as
means ± S.E. of four experiments, each performed in triplicate.
B, fibroblasts were transfected with 1 µg of
pLuc-GT1/E1/E2, 0.7 µg of RSV- -galactosidase, and increasing
amounts of A15Ras, balanced with empty vector. Cells were then
stimulated with 1 µM TPA for 24 h. Results are
expressed as means ± S.E. of four experiments, each performed in
triplicate. C, fibroblasts were transfected with 1 µg of
pLuc-GT1/E1/E2, 0.7 µg of RSV- -galactosidase, and 3 µg of either
A15Ras or empty vector. Cells were then stimulated with 10% FCS for
24 h.
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TPA Activates Ras in Myocytes, but Not in Fibroblasts--
To
further investigate the difference between cardiac myocytes and
fibroblasts regarding the requirement for Ras for TPA-induced GLUT1
expression, we performed Ras-GTP loading assays in both cell types. As
shown in Fig. 8, treatment of myocytes
with 1 µM TPA or 10% FCS induced GTP loading of Ras. In
contrast, TPA was unable to elicit activation of Ras in fibroblasts,
although the cells responded to FCS stimulation. Therefore, TPA can
induce Ras in muscle cells, but not in fibroblasts, thus explaining the Ras requirement only in muscle cells.

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Fig. 8.
Stimulation of Ras-GTP loading by TPA in
myocytes. Cells (2 × 106 in 10-cm dishes) were
serum-starved for 24 h and then stimulated for 1 h with
either 1 µM TPA or 10% FCS. Cells were lysed, and
GTP-bound Ras was affinity-precipitated using GST-RBD and detected by
Western blotting. C, control.
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Involvement of the Phosphatidylinositol 3-Kinase Pathway in Glut1
Induction--
In addition to the Ras/Raf/MEK/ERK pathway, GTP loading
of Ras can trigger activation of other signaling pathways, including phosphatidylinositol 3-kinase (PI3K) (32). We therefore investigated whether activation of the PI3K pathway was required for induction of
the Glut1 promoter by hypertrophic agonists. As shown in
Fig. 9, the selective PI3K inhibitor
LY294002 (33) reduced base-line expression of both the Glut1
and Glut4 endogenous genes, but did not affect the
Glut1/Glut4 expression ratio in non-hypertrophic cells. However, treatment with LY294002 reduced the increase in the
Glut1/Glut4 ratio observed upon treatment with
TPA or phenylephrine. In addition, LY294002 slightly, but significantly
inhibited induction of the Glut1 promoter by both TPA and
phenylephrine (Fig. 9C). LY294002 did not inhibit induction
of the Glut1 promoter by V12Ras. These results suggest that
PI3K activation contributes to the induction of the Glut1
promoter in response to TPA and phenylephrine and that it acts upstream
of Ras.

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Fig. 9.
Inhibition of the phosphatidylinositol
3-kinase pathway reduces hypertrophic Glut1 induction.
A, shown are RT-PCR products obtained in a representative
experiment. Myocytes (2 × 106 in 6-cm dishes) were
untreated (control (C)) or treated with TPA or PE in the
presence or absence of 50 µM LY294002 for 48 h.
RT-PCR was performed with 1.25 µg of total RNA. B, RT-PCR
experiments were subjected to PhosphorImager quantitation. Cells were
untreated or treated with TPA or PE in the presence (white
bars) or absence (black bars) of 50 µM
LY294002 for 48 h. Results are expressed as means ± S.E. of
two experiments. C, myocytes were transfected with 1 µg of
pLuc-GT1/E1/E2 and 0.7 µg of RSV- -galactosidase and stimulated
with 1 µM TPA or 100 µM PE for 48 h.
Alternatively, cells were cotransfected with 2 µg of V12Ras. Cells
were incubated in the presence (white bars) or absence
(black bars) of the selective phosphatidylinositol 3-kinase
inhibitor LY294002 (50 µM). Results are expressed as
means ± S.E. of three to four experiments, each performed in
triplicate.
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DISCUSSION |
Myocardial hypertrophy is characterized by expression of
immediate/early, fetal, and contractile genes. In vivo,
hypertrophic hearts have a pattern of substrate metabolism resembling
that observed in fetal hearts, with increased reliance on glycolysis for energy production and reduced oxidation of fatty acids (8-10). In
this study, we observed that hypertrophy of rat neonatal ventricular myocytes is associated with increased expression of the glucose transporter Glut1 isoform mRNA. GLUT1 is the principal
isoform expressed in the fetal heart, and its expression is
down-regulated following birth in normal myocardium (2, 3, 6),
concomitantly with the shift from glycolytic to oxidative metabolism
(1).
Our results suggest that regulation of Glut1 expression
during hypertrophy is primarily achieved at the transcriptional level. Transient transfection experiments with a luciferase reporter under the
control of the mouse Glut1 promoter indicated that treatment of myocytes with hypertrophic agonists resulted in increased
transcription from the Glut1 promoter occurring between 6 and 48 h following addition of the agonist. The two agonists we
used (TPA and phenylephrine) activated the promoter with different
kinetics, with TPA acting more rapidly and efficiently than
phenylephrine. This is probably related to TPA being a better activator
of the ERK mitogen-activated protein kinases compared with PE (Fig.
3A). The mode of action of these agonists is different.
Phenylephrine is an
1-adrenergic agonist whose receptor
is coupled to a G
q-containing heterotrimeric G protein.
Activation of Gq-containing heterotrimeric protein in
cardiac myocytes leads to Ras activation via the tyrosine
kinase/Shc/Grb2/Sos pathway (34). Ras activation, in turn, can trigger
a variety of downstream signaling pathways, including Raf and
phosphatidylinositol 3-kinase (for a review, see Ref. 35). TPA belongs
to the phorbol ester family, a class of compounds able to directly
stimulate the classical and novel protein kinase C (PKC) isoforms.
Stimulation of PKC activity can cause activation of Raf and MAP kinases
independently of Ras (36). Thus, the modes of action of both agonists
may converge onto Raf. In some cell types, TPA-induced activation of
MAP kinases involves Ras (37-39). Our data indicate that primary ventricular myocytes fall into this class (see below).
GLUT1 is a ubiquitous isoform of the glucose transporter, expressed at
a significant level in virtually every tissue of the body. Therefore,
it was a potential concern that the effect of TPA, a
non-tissue-specific protein kinase C agonist, could be due to increased
expression of the Glut1 promoter in contaminating non-myocyte cardiac cells, mainly cardiac fibroblasts. We are confident, however, that this is not the case, for the following reasons. 1) Phenylephrine, which activates common signaling pathways with TPA, but through
1-adrenergic receptor stimulation,
induced a similar response from the Glut1 promoter in
myocyte culture. It failed, however, to induce the Glut1
promoter in cultures of non-myocyte cardiac cells, consistent with the
presence of the
1-adrenergic receptor on myocytes only
(40). 2) TPA-induced activation of the Glut1 promoter was
less in cultures of cardiac fibroblasts than in cultures of myocytes.
The converse would be expected if the effect observed in myocytes was
due to contaminating fibroblasts, which represent at most only 5% of
the cells in myocyte cultures. 3) TPA-induced activation of the
Glut1 promoter was almost totally abolished by
cotransfection with dominant-negative Ras in myocytes, as was
phenylephrine-induced activation of Glut1; it was, however,
unaffected in non-myocytes. This observation is corroborated by the
finding that TPA treatment resulted in activation of Ras in myocytes,
but not in fibroblasts (see below).
Activation of the MAP kinase pathways is involved in the process of
myocardial hypertrophy. Previous results have shown that Ras or Raf-1
activity is required for expression of the c-fos, atrial
natriuretic factor, and myosin light chain-2 promoters in
phenylephrine-induced hypertrophy (14, 19). In addition, active MAP
kinase is required for induction of the c-fos and atrial natriuretic factor promoters by phenylephrine (41, 42), and expression
of constitutively active MEK1 results in overexpression of hypertrophic
genes (43). In this study, we found that activation of the ERK
mitogen-activated protein kinase pathway is required for activation of
the Glut1 promoter during myocardial hypertrophy. PD98059, a
specific inhibitor of the ERK kinase MEK1 (44), partially inhibited
both overexpression of the endogenous Glut1 mRNA and activation of the Glut1 promoter in response to hypertrophic
agonists. Inhibition was only partial, however, as was inhibition of
ERK activation, possibly because MEK2, which is much less sensitive to
PD98059 than MEK1, is the predominant ERK kinase in cardiac myocytes.
Cotransfection of the cells with the MAP kinase phosphatases CL100 (45)
and MKP-3 (27, 28) also significantly reduced induction of the
Glut1 promoter by TPA and phenylephrine. Because of the low
transfection efficiency in primary myocytes, it was not possible to
assess the effect of transfection with these molecules on expression of
endogenous Glut1 mRNA. Although MKP-1, the mouse homologue of CL100, was initially thought to be specific for the ERKs,
we have previously shown that, in our cells, CL100 could inhibit MEK
kinase-induced JNK activity as well as ERK activity (25). Therefore, to
further restrict involvement of the MAP kinase pathways to the ERKs, we
also used the MAP kinase phosphatase MKP-3, which is highly specific
for ERK (28). Together, the results obtained with PD98059, CL100, and
MKP-3 suggest that activation of the ERK pathway plays a major role in
transducing hypertrophic signals to the Glut1 promoter.
Furthermore, transfection of cardiac myocytes with constitutively
active or estrogen-inducible mutants of proteins of the Ras/Raf/MEK/ERK
cascade also leads to increased expression from the Glut1
promoter. Conversely, cotransfection of the cells with a
dominant-negative version of Ras (A15Ras) strongly inhibits induction
of the Glut1 promoter by either TPA or phenylephrine,
suggesting that activation of Ras is not only sufficient, but also
necessary for induction of the Glut1 promoter. The finding
that A15Ras strongly inhibits TPA-induced activation was somewhat
unexpected in view of the ability of active PKC to stimulate Raf
independently of Ras in COS and NIH3T3 cells (36). However, Marais
et al. (39) recently showed that TPA activation of ERK
required active Ras in COS cells. Furthermore, TPA increased the amount
of Ras-GTP in these cells. In our experiments, TPA increased Ras-GTP in
myocytes, but not in fibroblasts, a finding consistent with inhibition
of the TPA effect by A15Ras in myocytes only. These results therefore
suggest that Ras-mediated Raf activation, rather than PKC-mediated Raf
phosphorylation, is the main pathway leading to stimulation of ERK
activity in myocytes treated with phorbol esters. The reason why TPA
activates Ras in myocytes but not in fibroblasts remains unknown, but
could be related to expression of different PKC isoforms.
We have also shown that PI3K activation is involved, at least in part,
in the transduction of the signal from hypertrophic agonists to the
Glut1 promoter. The exact position of PI3K in these signal
transduction pathways remains somewhat controversial. Although an
initial report described PI3K as being a direct target of Ras (32),
other studies have suggested that PI3K could act upstream of Ras (46,
47). The present study does not allow us to draw firm conclusions on
this issue. However, the fact that the PI3K inhibitor LY294002 does not
inhibit induction of Glut1 by V12Ras suggests that PI3K does
not act downstream of Ras in this model. Recently, two reports showed
that PI3K activity was required for activation of some PKC isoforms,
including the TPA-activated isoform nPKC
(48, 49). This would place
PI3K upstream of Ras in cardiac myocytes in which Ras is activated as a
result of PKC activation.
In addition to the ERK pathway, phenylephrine has been shown to
potently activate the JNK and p38 MAP kinase pathways (50, 51).
Experiments using the specific p38 inhibitor SB203580 (52, 53) or
cotransfection with the JNK inhibitory protein JIP (data not shown)
have shown that the p38 pathway was not significantly involved and the
JNK pathway contributed very little to induction of Glut1
transcription during myocardial hypertrophy.
Although this study used pharmacological agonists to study the in
vitro hypertrophic response of glucose transporters, it has
implications for the understanding of glucose metabolism in pathophysiological situations. Physiological agonists that, like phenylephrine, signal through G
q-coupled receptors and
activate ERKs, such as endothelin (54, 55) and angiotensin II (56), have been implicated in the pathogenesis of myocardial hypertrophy in vivo. In addition, a specific inhibition of
G
q-dependent signaling blocks pressure
overload-induced hypertrophy in transgenic mice (57). Other stimuli,
such as oxidative stress (58) or hypoxia/reoxygenation (59), that lead
to activation of the Ras/Raf/MEK/ERK pathway or ischemia/reperfusion
leading to translocation and activation of PKC isoforms (60, 61) could
also potentially lead to increased Glut1 transcription. In
fact, overexpression of Glut1 has recently been reported in
an in vivo model of ischemia/reperfusion (62), a situation
in which PKC activation and oxidative stress take place.
In conclusion, our data indicate that Glut1 expression in
hypertrophied myocytes can be explained primarily through activation of
the Ras/Raf/MEK/ERK pathway. Interestingly the signaling pathways that
are used in different cell types in the heart to activate the ERK
molecules are different.