From the Department of Biochemistry and Molecular
Biology, § Gene Therapy Program and Departments of Medicine
and Pediatrics, Louisiana State University Health Sciences Center,
New Orleans, Louisiana 70112
Received for publication, August 6, 2002, and in revised form, November 25, 2002
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ABSTRACT |
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Glucose exerts powerful effects on hepatocyte
gene transcription by mechanisms that are incompletely understood.
c-Myc regulates hepatic glucose metabolism by increasing glycolytic
enzyme gene transcription while concomitantly decreasing gluconeogenic
and ketogenic enzyme gene expression. However, the molecular mechanisms by which c-Myc exerts these effects is not known. In this study, the
glucose-mediated induction of L-type pyruvate kinase and
glucose-6-phosphatase mRNA levels was diminished by maneuvers
involving recombinant adenoviral vectors that interfere with (i) c-Myc
protein levels by antisense expression or (ii) c-Myc function through a
dominant-negative Max protein. These results were obtained using both
HL1C rat hepatoma cells and primary rat hepatocytes. Furthermore, a
decrease in c-Myc abundance reduced glucose production in HL1C cells,
presumably by decreasing glucose-6-phosphatase activity. The repression
of hormone-activated phosphoenolpyruvate carboxykinase gene
transcription by glucose was not affected by a reduction in c-Myc
levels. The basal mRNA levels for L-pyruvate kinase and
glucose-6-phosphatase were not altered to any significant degree by
adenoviral treatment. Furthermore, adenoviral overexpression of the
c-Myc protein induced glucose-6-phosphatase mRNA in the absence of
glucose stimulation. We conclude that multiple mechanisms exist to
communicate the glucose-derived signal and that c-Myc has a key role in
the hepatic glucose signaling pathway.
Insulin and glucose act jointly to influence glucose homeostasis
by altering hepatic gene expression patterns. Insulin increases glucokinase (GK)1 gene
transcription and protein levels in hepatocytes (1). The
phosphorylation of glucose by GK leads to increased glucose flux and
metabolism, generating signaling metabolites that modify the gene
expression profile of the liver (2). For example, the L-pyruvate kinase
(L-PK) and glucose-6-phosphatase (Glc-6-Pase) genes (encoding
key glycolytic and gluconeogenic enzymes, respectively) are stimulated
by increases in hepatic glucose metabolism (3-5). Conversely, glucose
metabolism can also negatively regulate gene transcription, exemplified
by the repression of hormone-activated phosphoenolpyruvate
carboxykinase (PEPCK) gene promoter activity (6, 7). Although glucose
metabolism modulates the gene expression patterns of key glycolytic and
gluconeogenic enzymes, the signaling mechanisms and coordinating
transcription factors involved are not fully characterized.
One transcription factor that is a candidate for establishing hepatic
glucose-dependent gene expression patterns is the basic, helix-loop-helix leucine-zipper (bHLH-LZ) transcription factor c-Myc
(8, 9). The Myc family of proteins (e.g. c-, N-, L-Myc, USF,
Max, Mad, etc.) participates in control of proliferation, growth,
differentiation, apoptosis, and metabolism (10, 11). c-Myc binds DNA by
interacting with the E box sequence CACGTG (and other related
non-canonical sites) as a heterodimer with Max, another bHLH-LZ
protein. The Myc/Max heterodimer activates or represses transcription
depending on the target gene (11, 12). Sequences very similar to the E
box are present in the carbohydrate-response elements of the L-pyruvate
kinase and spot 14 gene promoters (3, 13, 14).
A 3-fold overexpression of c-Myc in the liver of transgenic mice,
driven by the PEPCK promoter, increases hepatic glucose metabolism and
storage, presumably because of the substantial rise in GK activity (no
increase in hexokinase isoform I activity was detected) (9). The
expression of glucose transporter 2 (GLUT2), L-PK, and
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK2) all
increase, as well as liver glycogen content, indicating that hepatic
glucose metabolism is enhanced. Furthermore, in c-Myc overexpressing
animals treated with streptozotocin, and therefore lacking insulin,
glucose and ketone production decrease (8). These changes are
accompanied by a decrease in mRNA levels for PEPCK and carnitine
palmitoyltransferase I and II. Importantly, this hepatic elevation in
c-Myc protein levels in vivo is sufficient to nearly
normalize fasting serum glucose levels and thus prevent streptozotocin-induced diabetes (8). Taken together, these results are
consistent with c-Myc playing a major role in the control of hepatic
glucose metabolism in vivo. However, the molecular mechanisms mediating these changes have not been elucidated.
In the present study, we decreased the abundance of the c-Myc protein
using a recombinant adenovirus that expresses antisense c-myc RNA (AdCMV-ASmyc) to test whether this
transcription factor is required for glucose-regulated gene expression
in hepatocytes. Cells treated with AdCMV-ASmyc had 50% less
c-Myc protein than cells treated with an equal amount of a control
adenovirus. This reduction in c-Myc was sufficient to block the
glucose-stimulated expression of glucose-responsive genes in both HL1C
rat hepatoma cells and rat primary hepatocytes. Furthermore, a
recombinant adenovirus expressing a dominant-negative Max protein
mimicked the effect of AdCMV-ASmyc. Reducing c-Myc levels
decreased glucose production in HL1C hepatoma cells, presumably by
decreasing Glc-6-Pase activity. In addition, treatment with a
recombinant adenovirus that expresses c-Myc increased Glc-6-Pase gene
expression when cells were incubated in 2 mM glucose,
partially mimicking the effect of 20 mM glucose. Finally,
repression of hormone-activated PEPCK gene promoter activity by glucose
was not alleviated by a reduction in c-Myc protein, suggesting multiple
pathways for glucose signaling.
Cell Culture--
The HL1C rat hepatoma cell line contains the
PEPCK promoter sequence (from Primary Hepatocytes--
Hepatocytes from overnight-fasted or
ad libitum fed male Wistar rats (250-350 g) were isolated
using the collagenase perfusion method as described previously (18).
Cells were allowed to attach for 3 h in hepatocyte medium
(glucose-free Dulbecco's modified Eagle's medium (Invitrogen)
supplemented with 15 mM HEPES, 33 µM biotin,
40 µM phenol red, 1 mM sodium pyruvate, 17 µM pantothenate, 14 mM sodium bicarbonate,
5 × 104 units/liter penicillin, 5 × 104 µg/liter streptomycin, 4 mM
L-glutamine, 50 mg/liter gentamicin, 2.5 mg/liter
fungizone) plus 5.5 mM glucose, 10% FBS, 0.5 µM dexamethasone, and 10 nM insulin.
Recombinant adenovirus was added and allowed to attach to the cells for
1 h. The medium containing adenovirus was aspirated, and cells
were then incubated for 24 h in fresh hepatocyte medium
supplemented with 0.2% bovine serum albumin and 2 mM
glucose. Thus, insulin and FBS were removed from the media for 24 h, and cells were kept in 2 mM glucose for 24 h prior to overnight treatment with 20 mM glucose. Preparation of
hepatocytes from ad libitum fed rats was as described above
for fasted rats, with the exception that the cells were exposed to 10 nM insulin and 5% FBS along with 2 mM glucose
for 24 h after the 3-h attachment period. The cells were then
washed three times with Dulbecco's phosphate-buffered saline and
re-incubated in hepatocyte medium (without insulin or FBS) for
treatment with 2 or 20 mM glucose overnight.
Construction, Preparation, and Use of Recombinant
Adenoviruses--
A 500-bp cDNA generated by RT-PCR, and
corresponding to nucleotides 2169-2569 (the entire exon 1) and
2570-2670 (100 bp of exon 2) of the rat c-myc gene
(GenBankTM accession number Y00396), was subcloned in a
3'-5' orientation into the adenovirus vector pACCMV.pLpA. The insert
was sequenced using the dideoxynucleotide method for verification of
correct orientation and sequence and subsequently was used to generate a recombinant adenovirus (AdCMV-ASmyc) as described
previously (19). The resulting virus was plaque-purified and used to
treat 50% confluent HL1C cells in 10-cm dishes at varying titers.
Primary hepatocytes at 80% confluence were treated in 60-mm
collagen-coated dishes as described in the figure legends. A virus
containing the bacterial RNA Isolation--
Total RNA was isolated from HL1C cells and
primary hepatocytes using TRI-reagent (Molecular Research Center)
according to the manufacturer's protocol.
Semi-quantitative RT-PCR Assay--
The reverse transcription
(RT) reaction contained 1 µg of total RNA, 1 µl of random primer
(50 µM, Applied Biosystems), 1× reverse transcriptase
buffer, 10 units of Moloney murine leukemia virus-reverse transcriptase
(Promega) in a total volume of 20 µl. The RNA and primer were
annealed by heating to 72 °C and slowly cooled to room temperature
before the reverse transcriptase was added, and the reaction was
allowed to proceed at 42 °C for 1 h. The RT reaction was then
diluted to 100 µl with RNase-free water. The PCR contained 1×
Taq polymerase buffer (Promega), 0.5 µl of 20 pmol/µl of
each of the primer pairs, 200 µM dNTPs, 0.25 µl of
HotStart-Taq polymerase, and 2.5 µl of the RT reaction. The primers for each gene were selected using the PRIMER3 program (MIT)
so that each PCR product was ~300 bp and each primer had an annealing
temperature of 60 °C. The primers (upstream and downstream, respectively) used are as follows: for Glc-6-Pase,
5'-GTGGGTCCTGGACACTGACT and 5'-CAATGCCTGACAAGACTCCA; for
pyruvate kinase, 5'-AACCTCCCCACTCAGCTACA and 5'-TGCTCCACTTCTGTCACCAG;
for Preparation of Nuclear Extracts--
Nuclear extracts were
prepared as a modification of the procedure described previously (21).
The protein concentration was determined using the BCA assay (Pierce)
with bovine serum albumin as the standard.
Western Blots--
Twenty µg of nuclear extract were mixed in
sample buffer containing 2% SDS, 100 mM
Transient Transfection and Dual Luciferase
Assay--
Transfections of HL1C rat hepatoma cells were performed
using LipofectAMINE (Invitrogen) according to the manufacturer's
instructions. Cells were first treated with AdCMV- Glucose Production Assay--
HL1C cells at 50% confluence were
treated with AdCMV-GKL and either AdCMV- Statistical Analysis--
All results are expressed as
means ± S.E. Data analysis was performed using the statistics
module of Microsoft Excel version 9.0 (Microsoft Corp.). Statistical
significance in the form of a two-tailed t test was rejected
at p > 0.05.
AdCMV-ASmyc Produces an Antisense c-myc mRNA--
A
recombinant adenovirus (AdCMV-ASmyc) was constructed to
produce an antisense c-myc mRNA. HL1C rat hepatoma cells
were transduced with AdCMV-ASmyc or a control adenovirus,
AdCMV- AdCMV-ASmyc Decreases c-Myc Protein Levels and Transcriptional
Activity--
HL1C cells were transduced with either AdCMV- AdCMV-ASmyc Blunts the Glucose-stimulated Expression of the L-PK
and Glc-6-Pase Genes--
Glucose metabolism generates a signal that
potently affects hepatic gene transcription, and c-Myc has been
implicated in the control of glucose-regulated gene expression (2, 9,
25). Therefore, HL1C cells were transduced with AdCMV-ASmyc
to determine whether the glucose-mediated induction of the L-PK or
Glc-6-Pase genes requires the full complement of endogenous c-Myc
protein expression. HL1C cells do not express GK, a feature shared with many hepatoma cell lines (26). Thus, GK was expressed via a recombinant
adenovirus to increase glucose flux and metabolism. HL1C cells
expressing GK, and treated with 20 mM glucose, display a 2- and 4-fold increase in the mRNAs for the L-pyruvate kinase and
Glc-6-Pase genes, respectively, when compared with control cells (Fig.
3). These observations are consistent
with previous findings wherein glucose decreased PEPCK gene promoter
activity in the same cell system (6). Treatment with
AdCMV-ASmyc blunted the glucose-mediated
increase in the mRNA levels of L-PK and Glc-6-Pase in a
concentration-dependent manner in HL1C cells, whereas
treatment with AdCMV-
After observing these effects in HL1C cells, we carried out similar
experiments in primary hepatocytes isolated from overnight-fasted rats.
In this system, insulin levels, and thus GK levels, are quite low. To
provide an increased glucose flux, GK was expressed by adenoviral
transduction, which allows us to separate effectively the
glucose-signaling pathway from the insulin-signaling pathway. Subsequent addition of 20 mM glucose to these cells
increased Glc-6-Pase gene expression by 4-fold when compared with
incubation in 2 mM glucose. However, treatment with
AdCMV-ASmyc blocked the glucose-mediated induction, whereas
AdCMV- Expression of a Dominant-negative Max Protein Inhibits the
Glucose-mediated Induction of Glc-6-Pase--
We determined whether
disabling the ability of the Myc-Max transcription complex to bind DNA
had the same effect as reducing c-Myc protein levels on the
glucose-mediated induction of the Glc-6-Pase gene. In these
experiments, primary hepatocytes from fed rats were used to take
advantage of high endogenous levels of glucokinase, thereby alleviating
the need for adenoviral replacement of the enzyme. This cell culture
system displays robust glucose-stimulated gene expression similar to
our previous model systems described above and is the predominant
system used in the study of glucose-mediated gene expression (3, 13).
We note that AdCMV-ASmyc also decreases the abundance of
Glc-6-Pase mRNA is this system (data not shown). An adenovirus
expressing a dominant-negative, FLAG-tagged Max protein (AdCMV-AMax),
wherein the basic region of Max is replaced with an acidic domain (27),
was used to evaluate the necessity of the heterodimer complex. The AMax
protein heterodimerizes with c-Myc generating a protein-protein
interaction that is thermodynamically more stable than the association
of c-Myc with endogenous Max, and the AMax-Myc complex is unable to
bind to DNA (27). Adenoviral mediated expression of the AMax protein in
primary rat hepatocytes inhibited the induction of Glc-6-Pase gene
expression by glucose metabolism (Fig.
5). We conclude that a functional Myc-Max
complex is necessary for maximal glucose-stimulated expression of the Glc-6-Pase gene.
AdCMV-ASmyc Blocks Glucose Production in HL1C Rat Hepatoma
Cells--
Because AdCMV-ASmyc blunts the glucose-mediated
expression of the pyruvate kinase and Glc-6-Pase genes, we determined
if treatment with this adenovirus had an effect on cellular metabolism.
We saw no effect on lactate production (data not shown), perhaps because the effect of AdCMV-ASmyc on PK mRNA was
relatively small compared with that of Glc-6-Pase (Figs. 3 and 4).
Glc-6-Pase catalyzes the removal of the phosphate group from glucose
6-phosphate as the terminal step in gluconeogenesis. We sought to
determine whether reducing c-Myc levels would interfere with the
ability of HL1C rat hepatoma cells to produce glucose. HL1C cells
transduced with AdCMV-GKL and either AdCMV- Reducing c-Myc Protein Levels in HL1C Rat Hepatoma Cells Does Not
Affect Unstimulated mRNA Levels of Either the L-PK or Glc-6-Pase
Genes--
One possible mechanism by which AdCMV-ASmyc may
influence gene expression is to alter basal transcription. Thus, we
examined the effect of reducing c-Myc protein levels on L-PK and
Glc-6-Pase mRNA levels in HL1C cells after an overnight culture in
2 mM glucose (Fig. 7). We
found that the mRNA abundance of each of these genes was not
significantly different in cells transduced with AdCMV- Overexpressing c-Myc via a Recombinant Adenovirus Increases the
mRNA Levels of Glc-6-Pase at a Non-stimulating Glucose
Concentration--
Quiescent cells, including hepatocytes, express
very little c-Myc protein. However, c-Myc is clearly involved in the
regulation of glucose metabolism in a variety of cell types (28, 29), and in this study, it is evident that endogenous, unstimulated levels
of c-Myc are required for glucose-induced expression of the L-PK and
Glc-6-Pase genes. To determine whether overexpression of c-Myc was able
to induce the expression of the Glc-6-Pase gene in the presence of 2 mM glucose, the relative levels of Glc-6-Pase mRNA were
assessed after transduction of primary hepatocytes from ad
libitum-fed rats with either AdCMV- Reducing c-Myc Levels Does Not Affect the Glucose-mediated
Repression of the Hormone-activated PEPCK
Promoter--
Glucocorticoids and glucagon, via the cyclic AMP
signaling pathway, activate the PEPCK gene, leading to hepatic glucose
production that maintains plasma euglycemia in the absence of ingested
fuel substrates (30). Glucose metabolism provides a signal that leads to transcriptional repression of the PEPCK gene in HL1C cells (6, 7).
We sought to determine whether diminishing c-Myc protein levels
alleviated the glucose-mediated repression of PEPCK promoter activity.
HL1C cells contain the PEPCK promoter ( Glucose is a strong regulator of hepatic gene transcription (2,
25). Increased glucose metabolism coordinately increases the expression
of glycolytic and lipogenic enzyme genes and decreases the expression
of ketogenic and gluconeogenic enzyme genes. c-Myc regulates hepatic
glucose metabolism in vivo by increasing the expression and
activity of glycolytic enzymes and exerting the reverse
effect on those of glucose and ketone production (8, 9). In this study,
we demonstrate several important observations regarding the role of
c-Myc in hepatic glucose metabolism as follows. 1) Maneuvers that
result in a reduction of c-Myc protein levels were able to blunt the
glucose-stimulated induction of Glc-6-Pase and L-PK mRNAs in both
HL1C rat hepatoma cells and rat primary hepatocytes. 2) Adenoviral
expression of a Max dominant-negative protein had the same effect as
reducing c-Myc levels. 3) The abundance of c-Myc influences glucose
production, presumably by altering Glc-6-Pase activity. 4) The
mRNAs for L-PK or Glc-6-Pase were not affected by treatments
resulting in decreased c-Myc protein levels in unstimulated (culture at
2 mM glucose) cells. 5) c-Myc overexpression leads to an
induction of the Glc-6-Pase gene in cells cultured with 2 mM glucose. 6) The signal for a glucose-mediated repression
of hormone-activated PEPCK is unaffected by diminished c-Myc protein levels.
A 3-fold overexpression of c-Myc driven by the PEPCK promoter in the
liver of transgenic mice increases GK, PFK2, and L-PK gene expression
despite insulin deprivation by streptozotocin treatment (8). These
changes in gene expression are sufficient to increase hepatic glucose
flux and suppress glucose output from the liver, thereby protecting the
animals from overt diabetes. In the present study, a 50% reduction of
c-Myc in rat hepatoma cells and primary hepatocytes blunts the ability
of glucose to stimulate expression of the L-PK and Glc-6-Pase genes
(Figs. 3 and 4). In addition, overexpression of c-Myc partially mimics the glucose effect in primary hepatocytes (Fig. 8). Therefore, our
observations are consistent with the transgenic findings. Together,
these data indicate that c-Myc is important for controlling hepatic
gene expression patterns and is a key factor in the regulation of gene
expression by glucose.
In this study, a dominant-negative Max protein (AdCMV-AMax) was
expressed by adenovirus to test whether a Myc-Max heterodimer is
required for the glucose activation of Glc-6-Pase. Introduction of the
AMax protein, which functions as a dominant-negative by allowing
heterodimerization but not DNA binding, prevented the glucose-induced
rise in Glc-6-Pase mRNA levels (Fig. 5). This observation suggests
that a functional Myc-Max heterodimer is required for the stimulatory
glucose effect. How this complex regulates gene transcription upon
stimulation with glucose is unknown.
Glucose metabolism increases Glc-6-Pase mRNA and protein levels (5,
31, 32). Because reducing c-Myc levels blunted the ability of glucose
to stimulate Glc-6-Pase mRNA levels (Figs. 3 and 4), we determined
whether this affected the ability of the cell to produce and release
glucose. We found that decreasing c-Myc levels diminished the glucose
production of HL1C cells (Fig. 6). Thus, glucose production can be
modified by altering levels of c-Myc.
The physiological significance of glucose exerting a stimulatory effect
on Glc-6-Pase and a repressive effect on PEPCK, both gluconeogenic
enzymes, has never been fully understood (6, 7). There are several
possible explanations for the paradoxical glucose-mediated induction of
Glc-6-Pase. One possibility is that a glucose-mediated elevation in
Glc-6-Pase activity serves as a feedback mechanism to decrease the
signal intensity provided by increased glucose flux and metabolism. By
this view, the glucose-stimulated increase in Glc-6-Pase would decrease
the abundance of glucose 6-phosphate and other potential signaling
metabolites, particularly after the suppressive effect of insulin (33)
has diminished toward the end of the fed state. This would limit the
extent of glucose signaling and potentiate a transition to the fasted
state. One consequence of this arrangement is that an overexpression of
Glc-6-Pase, as seen in type II diabetes, would lead to a decreased ability of c-Myc and other factors to coordinately regulate hepatic metabolic enzyme genes, and may partially explain the dysregulated hepatic glucose metabolism seen in type II diabetes (34).
The glucose-mediated induction of the L-PK gene, and presumably other
glucose-responsive genes, requires GK expression and glucose metabolism
in primary hepatocytes, rat hepatomas, and in
vivo2 (31, 35). Furthermore, the repression of
hormone-activated PEPCK promoter activity is dependent upon the
metabolism of glucose and occurs in the absence of insulin provided GK
is expressed (6). Thus, glucose down-regulates its own production in
the liver via a negative feedback mechanism at the same time that it
promotes glucose utilization. However, it is not known if the coordinating mechanisms for increasing gene transcription are the same
for the repression of gene transcription. In the present study, we
found that reducing c-Myc levels had no effect on the glucose
repression of the PEPCK promoter (Fig. 9), suggesting that multiple
glucose signaling pathways exist.
Glucose regulates gene transcription by acting through specific
promoter elements, designated carbohydrate response elements (ChoREs)
(3, 25). These elements were first described in the context of the L-PK
gene promoter and require a bHLH-LZ family member to fully stimulate
the response of the gene to carbohydrate (3, 36). The L-PK ChoRE
contains two non-canonical E boxes separated by 5 bp. The E boxes
described for the glucose-response element of the L-PK gene promoter
are different from the c-Myc family canonical E box by a single
nucleotide (3, 13). Also, a conserved carbohydrate-response element
consisting of "half-E boxes" (CACG), and documented in the spot 14 gene promoter, is necessary and sufficient to regulate transcription in
response to glucose (37). Establishment of the ChoRE leads to the
detection, by electrophoretic mobility shift assay, of a novel
ChoRE-binding factor that still awaits complete characterization (36,
37).
Another candidate for activation of glucose-responsive genes was
provided by the discovery of a novel transcription factor, termed
carbohydrate-response element-binding protein (ChREBP), that regulates
hepatic L-PK gene expression upon refeeding in rats (38, 39). ChREBP,
another bHLH-L2 protein, is regulated by phosphorylation status, which
controls its nuclear localization and DNA binding capabilities (39). In
the presence of glucose, ChREBP is de-phosphorylated, migrates to the
nucleus, and participates in controlling glucose-dependent
L-PK gene expression, possibly by binding to the E box motif present in
the L-PK promoter (38, 39). Interestingly, the Williams-Beuren syndrome
critical region 14 protein (WBSCR14), which is the human homologue of
rat ChREBP, heterodimerizes with Mlx, a member of the c-Myc family of
transcription factors (40). The association, if any, between c-Myc,
Max, Mlx, and ChREBP/WBSCR14 remains undetermined.
The question that remains unanswered at the present time is whether
c-Myc or the Myc-Max heterodimer is a carbohydrate-response element-binding protein. We found that, unlike pancreatic beta cells
(41), glucose does not increase the abundance of c-myc mRNA or protein in hepatocytes.3 Assuming that the
Myc-Max heterodimer binds directly to ChoREs, there are
several explanations regarding the necessity of c-Myc for
glucose-stimulated gene expression. These include but are not limited
to the following. The signal provided by increased glucose metabolism
in hepatocytes: 1) promotes a stronger Myc-Max heterodimer interaction;
2) strengthens the ability of the complex to bind to DNA; 3)
facilitates contact with co-activators or other constituents of the
transcriptional machinery; and 4) destabilizes non-Myc-Max heterodimer
interactions that would serve to repress the transcriptional activity
of glucose-responsive genes. It cannot be ruled out that combinations
of these possibilities exist. Another explanation is that Myc-Max acts
indirectly, by promoting the expression or activity of other
ChoRE-binding factors.
The work of Osthus et al. (29) shows that c-Myc activates
glycolytic genes in rat fibroblasts and in vivo in
hepatocytes. These investigators demonstrated that mRNA levels for
the glycolytic enzyme genes, GLUT1, phosphofructokinase-1,
glyceraldehyde-3-phosphate dehydrogenase, and In summary, c-Myc depletion inhibits glucose-dependent gene
expression of both the L-PK and Glc-6-Pase genes but does not alleviate
the glucose repression of hormone-activated PEPCK. We interpret these
findings to indicate that multiple signaling pathways exist to
communicate the glucose signal to target genes. Also, expressing a Max
dominant-negative protein has the same effect as depletion of c-Myc,
illustrating the importance of a functional Myc-Max heterodimer for
maximal glucose stimulation of the L-PK and Glc-6-Pase genes.
Furthermore, overexpression of c-Myc mimics the effect of increased
glucose metabolism. Taken together, these observations demonstrate an
important role for c-Myc in hepatic glucose-mediated gene expression.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2100 to +69, relative to the
transcription start site) ligated to the chloramphenicol
acetyltransferase (CAT) reporter gene (15). The culture of HL1C cells
and the assay for measurement of CAT activity have been described (16,
17).
-galactosidase gene (AdCMV-
Gal) was used
as a control (20). After a 60-90-min incubation with adenovirus, the
cells were placed in fresh medium and harvested 40 h later.
-actin, 5'-AACACCCCAGCCATGTACGTAG and 5'-GAACCGCTCATTGCCGATAGT;
and for cyclophilin, 5'-TGGTGGCAAGTCCATCTACG and
5'-AAAATGCCCGCAAGTCAAAG. The number of cycles used in the PCR was
determined empirically for each primer pair to be within the linear
range of amplification. In addition, the amount of RNA used for the RT
reaction (1 µg) was determined to be in the linear response range
when comparing RNA input to PCR product abundance (data not shown).
Furthermore, the sequence of the PCR products was verified by dideoxy
sequencing (Sequenase). The PCR was as follows: 15 min at 95 °C,
followed by 25-30 cycles of 30 s at 95 °C, 30 s at
62 °C, and 30 s at 72 °C, followed by a 5-min extension at
72 °C. PCR products were separated on 1.8% agarose gels and stained
with ethidium bromide. The abundance of the PCR products was
quantitated using a Kodak EDAS 290 gel documentation system and
NIH/Scion image software. We have validated this assay by finding that
the relative abundance of cyclophilin mRNA was linear from 0.1 to
1.0 µg of input RNA (r = 0.995).2 Thus, this semi-quantitative RT-PCR assay has a
high degree of precision and results in a linear response with respect
to input RNA concentrations over at least 1 order of magnitude.
-mercaptoethanol, 60 mM Tris-HCl, pH 7.8, and 0.01%
bromphenol blue. This mixture was boiled in a water bath for 5 min and
loaded onto 8% polyacrylamide gels (iGels, Gradipore). The proteins
were electrophoresed for 90 min at 40 mA and transferred to
polyvinylidene difluoride membrane (Pierce) overnight at 12 V in a
transfer buffer containing 10% ethanol, 25 mM Tris base,
and 192 mM glycine. After blocking with 5% non-fat dry
milk in Tris-buffered saline solution for 1 h, the primary antibody (anti-c-Myc, 1:1000 Research Diagnostics; anti-tubulin, 1:5000, a gift from Dr. Kevin Brown) was incubated at room temperature with the membrane for 4 h with continuous shaking. The membranes were then washed three times for 5 min each with Tris-buffered saline.
After washing, the secondary antibody (anti-rabbit coupled to
horseradish peroxidase, Kirkegaard & Perry Laboratories, Inc.) was
applied at 1:20,000 and allowed to bind for 2 h at room
temperature with continuous shaking. The membranes were again washed
three times for 5 min. After the third wash, the membranes were exposed to the SuperSignal chemiluminescent reagent (Pierce) for 1 min. Equal
protein loading was confirmed by either Ponceau S (Sigma) staining of
the polyvinylidene difluoride membrane after transfer or by
immunoblotting for the abundance of tubulin. Densitometry was performed
using Scion image software (National Institutes of Health).
Gal or
AdCMV-ASmyc at 50 pfu/cell. Twenty four hours after viral
transduction, the cells were transfected using 5 µg of either the
pMyc-TA-Luc or the pTA-Luc vectors (Clontech) and
with 1 µg of pRL-TK as a control for transfection efficiency
(Promega). These cells were harvested after an additional 24-h
incubation, and luciferase assays were performed in cell lysates using
the Dual-luciferase Reporter Assay System (Promega) in a TD-20/20
luminometer (Turner Designs).
Gal or
AdCMV-ASmyc. Twenty four hours after viral transduction, the
cells were treated overnight with 2 or 20 mM glucose. The
media were then replaced with glucose-free Dulbecco's modified
essential medium, and glucose production was measured essentially as
described using a glucose assay kit (Sigma 510-A) (22, 23). The medium
used for measurement of glucose production was modified slightly to
include 1 mM glycerol, 2 mM pyruvate, and 20 mM lactate. The pH of the medium was 7.4 and did not
contain phenol red.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Gal (20), and the effect of increasing the m.o.i. from 12.5 to
50 pfu/cell on mRNA abundance was assessed by RT-PCR (Fig.
1). Elevation of the viral titer
augmented the steady state mRNA levels of antisense c-myc and
-galactosidase.
View larger version (34K):
[in a new window]
Fig. 1.
Treatment with AdCMV-ASmyc
generates antisense c-myc mRNA. HL1C
cells were treated with either AdCMV- Gal or AdCMV-ASmyc at 12.5, 25, or 50 pfu/cell. The medium was replaced, and cells were cultured for an
additional 24 h, and total RNA was collected. The abundance of
mRNA expressed by each virus was detected by RT-PCR using the
specific primers indicated on the left. Inverse exposures
are shown of PCR products separated on agarose gels in the presence of
ethidium bromide. Each column represents a series of PCRs using the
products of a single reverse transcription reaction as the template.
Gal,
-galactosidase.
Gal or
AdCMV-ASmyc at 50 pfu/cell to test whether antisense
c-myc mRNA would decrease the abundance of c-Myc
protein. At 18 h post-transduction, nuclear protein extracts were
harvested, and the relative amount of c-Myc was determined by
immunoblotting (Fig. 2, A and
B). Treatment with AdCMV-ASmyc depleted c-Myc
protein levels by ~50% (n = 3, p < 0.05) relative to cells treated with AdCMV-
Gal. This reduction is
similar to the adenoviral mediated antisense reduction of p300 reported
by Kolli et al. (24). We have subsequently found that AdCMV-ASmyc decreases c-Myc levels by at least 50% in several different culture systems.3
As a control, HL1C cells were treated with either AdCMV-
Gal or
AdCMV-ASmyc for 24 h and were then transiently
transfected with a plasmid bearing a luciferase reporter gene driven by
a minimal promoter (pTA-Luc, Clontech) or a similar
construct with the addition of an E box upstream of the minimal
promoter (pMyc-TA-Luc, Clontech; Fig.
2C). The presence of the E box element, which binds to
c-Myc, gives a measure of c-Myc transcriptional activity. Whereas cells
transfected with pTA-Luc had negligible reporter activity (data not
shown), those transfected with pMyc-TA-Luc yielded robust luciferase
activity. Furthermore, cells treated with AdCMV-ASmyc and
transfected with pMyc-TA-Luc displayed significantly reduced promoter
activity compared with cells treated with AdCMV-
Gal and transfected
with the same reporter plasmid. Together, these results demonstrate
that treatment with AdCMV-ASmyc leads to decreased c-Myc
protein levels and transcriptional activity.
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Fig. 2.
AdCMV-ASmyc decreases c-Myc
protein levels and transcriptional activity in HL1C rat hepatoma
cells. HL1C cells were treated with either AdCMV- Gal or
AdCMV-ASmyc at 50 pfu/cell. A, nuclear extracts
were harvested 18 h post-transduction, and the abundance of c-Myc
was determined by immunoblotting. B, a densitometric
analysis of the autoradiograms was performed wherein the values were
normalized to tubulin. The protein abundance from AdCMV-
Gal-treated
cells was set at 100%. The data are the means ± S.E.
(n = 4, * p < 0.05). C,
HL1C cells were transduced with AdCMV-
Gal or AdCMV-ASmyc
for 24 h, and then cells were transfected with 5 µg of either
pMyc-TA-Luc or the pTA-Luc, and with 1 µg of pRL-TK as a control for
transfection efficiency. The plasmid constructs were allowed to express
for 24 h before harvesting and measurement of luciferase
activities in the cell lysates. RLU, relative light unit,
defined as the ratio between Renilla and firefly luciferase
activities. Data are presented as the means (± S.E.) of three
independent experiments, each performed in duplicate (*
p < 0.05).
Gal,
-galactosidase.
Gal over the same concentration range had no
significant effect on the expression of these genes (Fig. 3).
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Fig. 3.
AdCMV-ASmyc decreases
glucose-stimulated gene expression in HL1C cells. HL1C cells were
treated with AdCMV-GKL in the presence of either 2 or 20 mM
glucose or 20 mM glucose with the addition of various
amounts of either AdCMV-ASmyc or AdCMV- Gal (12, 25, or 50 pfu/cell). RNA was collected, and the relative abundance of pyruvate
kinase (A) or Glc-6-Pase (B) mRNAs was
determined by RT-PCR. The data are the means (± S.E.,
n = 4) of the mRNA abundance normalized to
-actin mRNA levels with the maximal glucose response set at
100% (*, p < 0.05; **, p < 0.01).
Representative pictures of ethidium-bromide stained agarose gels with
the inverse image are shown (insets).
Gal had no significant effect (Fig.
4A). L-PK gene expression was
also repressed in rat primary hepatocytes in the presence of
AdCMV-ASmyc, although not as dramatically as in HL1C
hepatoma cells, as it did not reach statistical significance
(p = 0.07) (Fig. 4B). Taken together, these
data demonstrate that a 50% reduction in c-Myc protein levels is
sufficient to inhibit glucose-regulated gene expression in HL1C rat
hepatoma cells and rat primary hepatocytes.
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Fig. 4.
AdCMV-ASmyc decreases
glucose-stimulated gene expression in primary hepatocytes.
Hepatocytes were isolated from the livers of rats that had been fasted
overnight. The cells were treated with AdCMV-GKL in the presence of
either 2 or 20 mM glucose. In addition, the indicated
groups of cells were treated with various amounts of either
AdCMV-ASmyc or AdCMV- Gal (12, 25, or 50 pfu/cell). RNA
was collected, and the relative abundance of Glc-6-Pase (A)
or pyruvate kinase (B) mRNAs was determined using
RT-PCR. The data are the means (± S.E., n = 3)
normalized to
-actin with the mRNA abundance of the maximal
glucose response set at 100% (*, p < 0.05).
Representative inverse images of the PCR products on agarose gels are
displayed (insets).
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Fig. 5.
AdCMV-AMax blunts the glucose stimulation of
Glc-6-Pase mRNA in rat primary hepatocytes. Primary
hepatocytes isolated from ad libitum fed rats were treated
overnight with various amounts of AdCMV-AMax or AdCMV- Gal (30, 60, or 90 pfu/cell) in the presence of either 2 or 20 mM
glucose. A, representative immunoblot of the FLAG-tagged
AMax protein (90 pfu/cell) is shown. B, shown is a
representative photo of PCR products from an inverted image of an
ethidium bromide-stained agarose gel (inset). The data are
means ± S.E. of four independent experiments with the mRNA
abundance normalized to cyclophilin and the maximal glucose response
set at 100% (*, p < 0.005).
Gal,
-galactosidase.
Gal or
AdCMV-ASmyc were used for this purpose. Glycerol was added
to the medium as a gluconeogenic substrate to bypass the PEPCK reaction
(conversion of oxaloacetate to phosphoenolpyruvate) because the gene
encoding this enzyme is known to be repressed by glucose metabolism (6,
7). Under these conditions, glycerol enters the gluconeogenic pathway
at the triose level, and glucose production should increase with
increasing Glc-6-Pase activity despite a reduction in PEPCK activity.
The cells expressing AdCMV-ASmyc and treated with 20 mM glucose (to stimulate Glc-6-Pase gene expression) produced ~50% less glucose than did cells expressing AdCMV-
Gal and treated with the same concentration of glucose (Fig.
6). Therefore, reducing c-Myc abundance
reduces the glucose production capacity of HL1C cells.
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Fig. 6.
AdCMV-ASmyc represses
glucose production in glucose-stimulated HL1C cells. HL1C cells
transduced with AdCMV-GKL and either AdCMV- Gal or
AdCMV-ASmyc for 24 h were treated with either 2 or 20 mM glucose for an additional 18 h. The cells were
washed three times with phosphate-buffered saline, followed by
incubation in glucose-free Dulbecco's modified Eagle's medium,
supplemented with 1 mM glycerol, 2 mM
pyruvate, and 20 mM lactate for 4 h. The medium was
removed, and glucose was measured as described under "Experimental
Procedures." Data represent the means (± S.E.), normalized to total
protein, for four independent experiments (*, p < 0.01).
Gal when
compared with cells transduced with the same m.o.i. of
AdCMV-ASmyc. These data demonstrate that
reducing c-Myc protein levels does not affect unstimulated mRNA
levels of the L-PK and Glc-6-Pase genes and suggests that c-Myc
participates specifically in the glucose stimulation of gene
transcription.
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[in a new window]
Fig. 7.
AdCMV-ASmyc does not affect
basal mRNA levels of the L-pyruvate kinase or Glc-6-Pase
genes. HL1C cells were transduced with various amounts of
AdCMV-ASmyc or AdCMV- gal (12.5, 25, or 50 pfu/cell) and
then cultured overnight in 2 mM glucose. Cells were
harvested after an additional 24 h in fresh medium containing 2 mM glucose. The data are the means of the mRNA
abundance of L-PK (A) or Glc-6-Pase (B) (± S.E.)
and represent three independent experiments. Representative inverse
images of ethidium-bromide stained agarose gels displaying PCR product
abundance are shown (insets).
Gal,
-galactosidase.
Gal or AdCMV-c-Myc. Under the conditions used, c-Myc protein is undetectable in extracts from
primary hepatocytes treated with AdCMV-
Gal, but quite abundant after
transduction with AdCMV-c-Myc (Fig.
8A). The c-Myc treatment produced a 50% increase in Glc-6-Pase gene expression from cells incubated in 2 mM glucose (Fig. 8B). However,
treatment with AdCMV-
Gal at the same m.o.i. did not alter expression
of the gene. Although the increase in gene expression seen in this
experiment is not as dramatic as that induced by 20 mM
glucose, it is further evidence that c-Myc regulates the expression of
the Glc-6-Pase gene.
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Fig. 8.
Overexpression of c-Myc induces the
expression of the Glc-6-Pase gene. Primary hepatocytes from
ad libitum fed rats were transduced with AdCMV-c-Myc or
AdCMV- Gal (20 or 40 pfu/cell) in the presence of 2 mM
glucose. Also, primary hepatocytes from the same preparation were
cultured with either 2 or 20 mM glucose without adenoviral
transduction. A, representative immunoblot of c-Myc protein
(40 pfu/cell) accumulation is shown. B, the relative
Glc-6-Pase mRNA abundance was determined by RT-PCR. The means (± S.E.) are shown for five independent experiments (*, p < 0.05). An inverse of an agarose gel showing PCR product accumulation
is shown (inset).
Gal,
-galactosidase.
2100 to +69) sequence linked
to the CAT reporter gene (15). Treating HL1C cells with
dexamethasone and 8-(4-chlorophenyl-thio)-cAMP produces a 50-fold
induction of CAT activity. In the presence of adenovirally expressed GK
and 20 mM glucose, this activity is reduced by 80% (6).
Depleting c-Myc levels did not reverse the
glucose-dependent repression of CAT activity, nor did the control virus lessen the glucose-dependent effects (Fig.
9). Surprisingly, the highest m.o.i. of
AdCMV-ASmyc further repressed promoter activity significantly, whereas the same m.o.i. of AdCMV-
Gal did not. These
results demonstrate that c-Myc is not necessary for the glucose-mediated repression of the hormone-activated PEPCK promoter and
suggest the existence of multiple glucose signaling pathways.
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Fig. 9.
AdCMV-ASmyc does not
alleviate the glucose repression of hormone-activated PEPCK promoter
activity. HL1C cells were treated with AdCMV-GKL and various
amounts of either AdCMV-ASmyc or AdCMV- Gal overnight in
serum-free medium. The cells were then incubated overnight in control
medium or medium containing 0.5 µM dexamethasone
(dex) and 100 µM
8-(4-chlorophenyl-thio-)cAMP (cAMP) or
dexamethasone/cAMP supplemented with either 2 or 20 mM
glucose. The cells were harvested, and chloramphenicol
acetyltransferase (CAT) activity in the cell lysates was
measured. Shown are the means ± S.E. of three separate
experiments (*, p < 0.05).
Gal,
-galactosidase.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-enolase, were all
up-regulated in c-Myc-transformed fibroblasts. Furthermore, the same
gene expression profile was observed in hepatocytes after mice were
infused with an adenovirus expressing c-Myc (29). In addition, they
determined which genes are direct c-Myc targets using a chimeric
protein that has c-Myc fused to the estrogen receptor ligand-binding
domain (MycER). The induction of GLUT1, PFK, and enolase mRNAs in
rat fibroblasts increases when the MycER fusion protein is
activated by 4-hydroxytamoxifen. This effect is not blocked by the
protein synthesis inhibitor cycloheximide, demonstrating that these
genes are direct targets of the c-Myc protein (29). c-Myc may then
directly regulate the expression of certain glycolytic enzyme genes and
participate, directly or indirectly, in the stimulation by glucose of
other metabolic enzyme genes, such as L-PK and Glc-6-Pase. Indeed, the results of the present study indicate a role for c-Myc in the glucose-mediated stimulation of the L-PK and Glc-6-Pase genes but not
in the glucose-mediated repression of the hormone-activated PEPCK
promoter, suggesting the existence of c-Myc-dependent and -independent glucose signaling pathways.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Daryl Granner for providing HL1C cells; Dr. Christopher Newgard for the AdCMV-GKL adenovirus; Dr. Kevin Brown for providing anti-tubulin antibody; Dr. Wafik El-Deiry for providing the AdCMV-Myc adenovirus; and Dr. Charles Vinson for providing the AdCMV-AMax adenovirus. We also thank members of the Scott and Claycomb laboratories for critical reading of the manuscript.
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FOOTNOTES |
---|
* This work was supported by an American Diabetes Association Career Development award (to D. K. S.).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, Louisiana State University Health Sciences Center, 1901 Perdido St., New Orleans, LA 70112. Tel.: 504-568-4055; Fax: 504-568-3370; E-mail: dscott3@lsuhsc.edu.
Published, JBC Papers in Press, December 11, 2002, DOI 10.1074/jbc.M208011200
2 D. K. Scott, J. J. Collier, T.-T. T. Doan, A. S. Bunnell, M. C. Daniels, D. T. Eckert and R. M. O'Doherty, manuscript submitted for publication.
3 J. J. Collier and D. K. Scott, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: GK, glucokinase; L-PK, liver-type pyruvate kinase; Glc-6-Pase, glucose-6-phosphatase; PEPCK, phosphoenolpyruvate carboxykinase; bHLH-LZ, basic helix-loop-helix leucine-zipper; GLUT2, glucose transporter isoform 2; PFK2, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; CAT, chloramphenicol acetyltransferase; RT, reverse transcription; pfu, plaque-forming units; ChREBP, carbohydrate-response element-binding protein; FBS, fetal bovine serum; m.o.i., multiplicity of infection; ChoRE, carbohydrate-response elements.
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