From the Department of Nutrition, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106
Received for publication, August 30, 2000, and in revised form, November 20, 2000
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ABSTRACT |
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To understand how glucose regulates the
expression of the glucose-6-phosphatase gene, the effect of glucose was
studied in primary cultures of rat hepatocytes. Glucose-6-phosphatase
mRNA levels increased about 10-fold when hepatocytes were incubated with 20 mM glucose. The rate of transcription of the
glucose-6-phosphatase gene increased about 3-fold in hepatocytes
incubated with glucose. The half-life of glucose-6-phosphatase mRNA
was estimated to be 90 min in the absence of glucose and 3 h in
its presence. Inhibition of the oxidative and the nonoxidative branches
of the pentose phosphate pathway blocked the stimulation of
glucose-6-phosphatase expression by glucose but not by xylitol or
carbohydrates that enter the glycolytic/gluconeogenic pathways at the
level of the triose phosphates. These results indicate that (i) the
glucose induction of the mRNA for the catalytic unit of
glucose-6-phosphatase occurs by transcriptional and
post-transcriptional mechanisms and that (ii) xylitol and glucose
increase the expression of this gene through different signaling pathways.
Glc-6-Pase1 (EC 3.1.3.9)
is a multicomponent protein complex comprising catalytic and
transporting entities (1-5). The complex is tightly associated with
the endoplasmic reticulum, and the enzymatic component catalyzes the
hydrolysis of glucose 6-phosphate to glucose, a final common step to
both the pathways of glycogenolysis and gluconeogenesis. Hepatic
Glc-6-Pase activity is effectively regulated by hormonal and
nutritional status. For example, fasting and hormones that increase
cAMP concentration stimulate its gene expression while re-feeding and
insulin decrease it (1, 2, 6-12).
The expression of the genes for several other proteins is regulated by
glucose; these include genes for L-type pyruvate kinase (13, 14),
fatty-acid synthase (15, 16), PEPCK (17, 18), and the type 2 glucose
transporter (GLUT-2) (19, 20). The mechanism by which glucose regulates
the expression of these genes remains largely unknown. Recently, Kahn
and colleagues (21, 22) have proposed a signaling pathway model to
explain the molecular mechanism by which glucose regulates the
expression of the L-type pyruvate kinase. This model consists of the
following details: (i) the presence of glucose is sensed in the cell;
(ii) this information is transduced by intracellular messengers; (iii)
a second messenger, presumably xylulose 5-phosphate, rises and then
modulates the activity of protein kinase and protein phosphatase
involved in a cascade of phosphorylation/dephosphorylation. This
cascade then leads to a modification of the phosphorylation state of
the glucose-responsive complex, followed by an increase in the
transcriptional rate of the target gene. In this model, the presence of
an active glucokinase that phosphorylates glucose to glucose
6-phosphate (Glc-6-P) is paramount.
The molecular mechanism by which glucose regulates the expression of
the Glc-6-Pase gene is currently unknown. Here we show that glucose
regulation of the expression of this gene involves metabolism of
glucose through the glycolytic pathway, transcriptional activation of
the Glc-6-Pase gene promoter, and a decrease in the degradation of
Glc-6-Pase mRNA.
Cell Culture--
Hepatocytes were isolated from male Harlan
Sprague-Dawley rats (200-250 g) according to the two-step procedure of
Berry and Friend (23) as modified by Leffert et al. (24).
Cell viability was estimated by trypan blue dye exclusion, and only
preparations with a viability of 85% or higher were used. The cells
were cultured in 100-mm Petri dishes in RPMI 1640 (Life Technologies,
Inc.) medium, supplemented with penicillin (100 units/ml), streptomycin (100 µg/ml), fetal calf serum (10% v/v), calf serum (5% v/v), 10 µg of insulin/ml (Sigma), and 100 nM dexamethasone
(Sigma). After cell attachment (4 h), the medium was replaced with
fresh medium supplemented with fetal calf serum (5% v/v), penicillin (100 units/ml), streptomycin (100 µg/ml), without insulin or
dexamethasone. The cells were then cultured for additional 16 h,
at which time the cells were washed and fed the basal culture medium
containing 5 mM glucose or other additions, as indicated in
the legends to the figures. At the end of the test period, the cells
were harvested and washed twice with 10 ml of cold phosphate-buffered saline.
Northern Blotting Analysis--
Total RNA was isolated with the
Trizol method according to the manufacturer's protocol (Life
Technologies, Inc.). The isolated RNA was assessed for purity by the
260/280 absorbancy ratio. The RNA (20 µg) was electrophoresed on a
1.2% formaldehyde-denatured agarose gel in 1× MOPS running buffer,
transferred to a Hybond-N+ membrane (Amersham Pharmacia
Biotech), and hybridized with a 1.25-kilobase pair
Eco-HindIII rat Glc-6-Pase cDNA (kindly
provided by Dr. Rebecca Taub, University of Pennsylvania, Philadelphia, PA) or a 1.4-kilobase pair PEPCK cDNA (kindly provided by Dr. Richard W. Hanson, Case Western Reserve University, Cleveland, OH). The
cDNAs were labeled with [ Nuclear Run-on Assay--
The nuclear run-on assay was used to
measure gene transcription rates. The procedure was as described by
Aulak et al.2 In
brief, plates were washed twice in phosphate-buffered saline and
scraped in the same buffer. Cells were pelleted by centrifugation at
600 × g at 4 °C for 5 min and then lysed in buffer
A (10 mM HEPES (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.5% Nonidet P-40) and incubated for
7 min at 4 °C. Nuclei were isolated by centrifugation at 600 × g at 4 °C for 5 min and resuspended in 20 mM
HEPES buffer (pH 8.3), containing 5 mM MgCl2,
0.1 mM EDTA, and 40% glycerol, and stored at G6PDH and Lactate Assays--
G6PDH activity was measured
spectrophotometrically by the rate of production of NADPH at 340 nm
(25). The assay medium contained 50 mM Tris (pH 8.0), 10 mM MgCl2, 2 mM Glc-6-P, and 0.9 mM NADP. Since 6-PGDH, contained in the extract, also
produces NADPH under this assay condition, the assay reflects the total
activity of both dehydrogenases. Lactate was assayed by the rate of
formation of NADH at 340 nm (25).
Statistics--
Results are expressed as means ± S.E.
Statistical analysis was performed with Student's t test.
Glucose Induces Glc-6-Pase mRNA Production in Primary Cultures
of Rat Hepatocytes--
We have previously shown in conscious normal
nondiabetic and diabetic rats that glucose is a major stimulator of
Glc-6-Pase gene expression, independent of insulin or other hormones
(11). To determine the mechanism by which glucose stimulates Glc-6-Pase gene expression, we have studied this effect in primary cultures of rat
hepatocytes because cell cultures provide discrete advantages over
intact animals in dissecting out the contribution of nutrients without
interference from hormonal secretion. Primary cultures provide an
additional advantage in the sense that the cells still display the same
regulation as seen in vivo, as opposed to transformed cell
lines. First we established the optimal conditions to study glucose
regulation of Glc-6-Pase in cultured rat hepatocytes. The addition of
glucose to the culture medium caused a marked dose-dependent increase in the induction of the mRNA
levels for Glc-6-Pase (Fig.
1A) with a maximal induction
at 20 mM glucose. Under the culture conditions used,
glucose decreases the mRNA levels of the phosphoenolpyruvate
carboxykinase (PEPCK) gene (Fig. 1B) as would be expected
(12). These results establish that in this primary culture system, the
hepatocytes maintain the glucose regulation as seen in
vivo.
Fig. 2 shows the time course of
glucose-induced accumulation of Glc-6-Pase mRNA. The increase in
the mRNA levels was already present after 1 h; peak values
were observed at 2-4 h, after which the mRNA level declined
rapidly, returning to near basal values after 8 h. Subsequent
experiments were therefore performed with cells incubated with 20 mM glucose for 4 h.
Role of Glucose Phosphorylation--
In intact animals, metabolism
of glucose beyond glucose 6-phosphate (Glc-6-P) is necessary for the
glucose-induced stimulation of the expression of the Glc-6-Pase gene
(12). This effect is also demonstrable in primary cultures of rat
hepatocytes. When glucosamine (2 mM) was used to inhibit
glucokinase activity, glucose failed to increase Glc-6-Pase mRNA
levels (Fig. 3). Also,
3-ortho-methylglucose, an analog of glucose that is
transported into the cell but not metabolized, was unable to increase
Glc-6-Pase mRNA levels. Similarly, when hepatocytes were cultured
in the presence of 2-deoxyglucose, another glucose analog that is
phosphorylated but not metabolized further, Glc-6-Pase mRNA levels
were not changed. These results mimic the results obtained with intact
animals (12) and provide more evidence that glucose needs to be
metabolized to stimulate the expression of Glc-6-Pase.
To determine whether substrates that enter the glycolytic pathway
downstream of glucose 6-phosphate can stimulate Glc-6-Pase expression,
lactate and pyruvate were tested. When a combination of lactate (10 mM) and pyruvate (1 mM) was provided as
substrate, no increase in Glc-6-Pase mRNA levels was observed (Fig.
3). Two other sugars, fructose and mannose, that enter the glycolytic pathway at the level of triose phosphate were also tested. Both sugars
increased Glc-6-Pase mRNA concentrations in a
dose-dependent manner (Fig.
4). Glycerol (2 mM) was also
able to elicit a small increase in Glc-6-Pase mRNA; however, higher
concentrations (>2 mM) of glycerol had an inhibitory
effect.
Identification of Potential Metabolic Sites Mediating the Effect of
Glucose on Glc-6-Pase Gene Expression--
Doiron et al.
(26) have recently challenged the idea of Glc-6-P (27, 28) as the
signaling molecule that transduces the effect of glucose in regulating
the expression of certain genes. Instead, they propose that xylulose
5-phosphate, a metabolite in the pentose phosphate pathway, may be the
major messenger candidate that transduces the effect of glucose.
Glycolytic intermediates downstream of triose phosphates, such as
phosphoglycerate and phosphoenolpyruvate, have been proposed as well
(29). The infusion of xylitol into intact animals mimics the effect of
glucose on Glc-6-Pase mRNA (12). Because xylitol is metabolized to
xylulose 5-phosphate, we tested whether xylitol can alter Glc-6-Pase
mRNA accumulation. At 5 mM, xylitol caused a much
higher increase in the abundance of Glc-6-Pase mRNA than that
caused by 20 mM glucose (Fig.
5A). In a parallel experiment
(Fig. 5B) xylitol inhibited PEPCK gene expression, a finding
consistent with previous results with intact animals (12).
We then studied the effect of inhibiting the pentose phosphate pathway
at the glucose-6-phosphate dehydrogenase (G6PDH) level, the first
committed enzyme of the pathway, with 6-aminonicotinamide (6-AN). When
hepatocytes were preincubated with 200 µM 6-AN before the
addition of glucose, the usual glucose-induced stimulation of
Glc-6-Pase mRNA was completely prevented (Fig.
6A). To rule out any possible
toxic effect of 6-AN, we measured the release of lactate dehydrogenase
in the medium as an indicator of cell injury. We did not detect any
lactate dehydrogenase activity during the duration of these experiments
(results not shown).
Since the nonoxidative branch of the pentose phosphate pathway is
reversible, intermediates from the glycolytic pathway (i.e. fructose 6-phosphate and glyceraldehyde 3-phosphate) could be re-entering this pathway through the reactions catalyzed by
transaldolase and transketolase. To evaluate the role of the
nonoxidative branch of the pentose phosphate pathway in transducing the
observed glucose signaling, we inhibited this portion of the pathway
with oxythiamine, a transketolase inhibitor (30, 31), the rationale
being that this compound would interfere with interconversions in this
portion of the pathway. At concentrations that completely inhibit
transketolases in vitro (30), oxythiamine did not prevent
the accumulation of Glc-6-Pase mRNA by glucose (Fig.
6A), suggesting that metabolism through the nonoxidative
portion of the pathway may not be required for the response to glucose.
To evaluate the effect of simultaneously inhibiting the oxidative and
nonoxidative branches of the pentose phosphate pathway, we incubated
the hepatocytes with 6-AN and oxythiamine and then re-assessed the
response to glucose and xylitol. The combination of the two inhibitors
had no effect on the stimulation of Glc-6-Pase gene expression by
xylitol (Fig. 6B), but it prevented glucose from inducing an
accumulation of Glc-6-Pase mRNA. Although this effect was not
surprising, since 6-AN alone could prevent the glucose effect, it
established that a different mechanism underlies the xylitol and
glucose induction of this mRNA. When the inhibitor combination was
tested on the inductive effects of fructose or glycerol, the
combination had no effect on the ability of these substrates to induce
Glc-6-Pase mRNA levels (Fig. 6C).
Because it has been reported that 6-AN can act as an inhibitor of the
glycolytic enzyme, phosphoglucoisomerase (32, 33), we assayed lactate
production (as an indication of glycolytic activity) in cells incubated
with 6-AN. Lactate levels were 4-fold higher in cells incubated with 20 mM glucose than in cells incubated with 5 mM
glucose. Preincubation of the cells with 200 µM 6-AN caused a more than 60% inhibition of glycolysis; this inhibition was
evident at the earliest time point (1 h) measured. 6-AN also blocked
glycolysis from mannose (Fig.
7B). Since the metabolism of
mannose involves its isomerization to fructose 6-phosphate by
phosphomannose isomerase, this result suggests that indeed phosphohexoisomerases were inhibited in the presence of 6-AN. Interestingly, mannose-induced accumulation of Glc-6-Pase mRNA was
also inhibited (results not shown). Lactate production from xylitol was
significantly less compared with glucose. These results could be
explained by the well documented blocking effect of xylitol on
glycolysis (34, 35). Xylitol is known to exert an ATP-depleting action
by trapping phosphate inside the cells since the bulk of phosphorylated
intermediates following xylitol metabolism enters the gluconeogenic
pathway instead of glycolysis (34, 35) so the ATP used is not being
regenerated.
Role of Transcriptional and Post-transcriptional Mechanisms in
Mediating the Effects of Glucose on Glc-6-Pase mRNA--
Because
the stimulatory effect of glucose on Glc-6-Pase mRNA level can
result from either an increase in the transcription of the gene or a
stabilization of the mRNA, both of these two possibilities were
tested. The effect of glucose on the transcription rate of the
Glc-6-Pase gene was measured by using nuclear run-on assays. In the
presence of 20 mM glucose, the transcription rate increased
3-fold above the basal value (Fig. 8), a
value less than that for Glc-6-Pase mRNA accumulation. To test the
possibility that glucose might also stabilize Glc-6-Pase mRNA, the
half-life of the Glc-6-Pase mRNA was measured. For this experiment,
the transcription inhibitor actinomycin D (10 µg/ml) was added to the
hepatocytes after 4 h in culture in the presence of 20 mM glucose, and the culture was continued in the presence
or absence of glucose. Total RNA was then isolated at various time
points, and Glc-6-Pase mRNA abundance was quantified by Northern
blot analysis. As shown in Fig. 9, the
level of Glc-6-Pase mRNA decayed at a slower rate in the presence
(t1/2 = 3 h) of glucose than in its absence
(t1/2 = 90 min). Thus, we conclude that
glucose-stimulated transcription of the Glc-6-Pase gene involves both
transcriptional and post-transcriptional mechanisms. To examine whether
de novo protein synthesis is required for the
glucose-induced increase in Glc-6-Pase mRNA, cycloheximide (10 µg/ml) was used to block protein synthesis. We found that glucose-induced Glc-6-Pase mRNA expression was similar in the presence or in the absence of cycloheximide (data not shown), indicating that glucose-induced Glc-6-Pase mRNA accumulation does not require the synthesis of new protein.
The molecular mechanisms underlying glucose-induced regulation of
the Glc-6-Pase gene is not clearly understood. Here we present evidence
that stimulation of Glc-6-Pase gene expression by glucose involves both
transcriptional and post-transcriptional components. The specific DNA
sequence(s) responsible for the transcriptional response remains to be
established. The half-life of Glc-6-Pase mRNA was found to be 90 min. The fact that glucose prolongs this half-life suggests
glucose-induced stabilization of the Glc-6-Pase mRNA. New protein
synthesis does not seem to be necessary for glucose to induce
Glc-6-Pase mRNA. This would argue against a mechanism whereby
glucose stimulated the synthesis of a protein factor(s) that bind to
the mRNA and thereby prevent its degradation. The stabilization of
the Glc-6-Pase mRNA by glucose does not depend on the ability of
this mRNA to be translated, since the 2-fold increase in the level
of Glc-6-Pase protein was much lower than the increase in its mRNA.
It has been extensively documented that mRNAs whose 3'-UTRs contain
an AU-rich element (AURE) and/or an oligonucleotide (U) tend to be
unstable (36). Such AURE in the 3'-UTR had been found in transcripts
such as c-fos and c-myc (37, 38) that are
known to have a short half-life. Their significance in mRNA
turnover has been demonstrated in vivo and in
vitro (38-41). The 3'-UTR of the Glc-6-Pase gene contains a few
AUREs. Whether these AUREs play any role in Glc-6-Pase mRNA
stability is not known.
One unresolved question about the glucose-induced accumulation of
Glc-6-Pase mRNA is whether this effect requires glucose metabolism
through the pentose phosphate pathway. If this pathway is important for
the effect, then suppressing it should be expected to prevent the
accumulation of Glc-6-Pase mRNA. Glucose-induced accumulation of
the mRNA for Glc-6-Pase was indeed prevented when hepatocytes were
incubated with 0.2 mM 6-AN. When metabolized to 6-amino-NAD
and 6-amino-NADP, this nucleotide behaves as a competitive inhibitor of
NAD(P+)-requiring dehydrogenases that would include G6PDH,
6-PGDH, and glutathione reductase (32, 33, 42-46, 48). 6-Amino-NADP is an extremely potent competitive inhibitor of 6-PGDH, and concentrations that do not affect G6PDH may completely block 6-PGDH (43). On the other
hand, no inhibition of NAD-dependent enzymes results from
the use of this compound (48). The inhibition of glycolysis from
glucose by 6-AN makes it difficult to interpret the results using
glucose as an energy source. Nevertheless, this compound is very
valuable to study the effect of substrates such as fructose and
glycerol that enter glycolysis beyond the formation of fructose 6-phosphate.
When both the pentose phosphate pathway and phosphoglucoisomerase were
inhibited with 6-AN and oxythiamine, stimulation of Glc-6-Pase gene
expression by fructose or glycerol was not affected. These two
substrates enter the glycolytic pathway at the level of triose
phosphate(s), and the fact that the nonoxidative branch of the pentose
phosphate pathway was blocked suggests that the pentose phosphate
pathway is not required for the glucose effect on the Glc-6-Pase gene.
In contrast to glucose, xylitol induced Glc-6-Pase mRNA even in the
presence of inhibitors of the pentose phosphate pathway. Taken
together, these results support the idea that xylitol and glucose
signal through different pathways.
The signaling pathway from glucose to the DNA sequences termed
carbohydrate responsive element (ChoRE/GIRE) is not totally characterized. Phosphorylation/dephosphorylation of transcription factors have been implicated in the glucose responsiveness of many
genes. Xylulose 5-phosphate, produced by the pentose phosphate pathway,
has been suggested as a secondary messenger in sensing glucose
concentration in the hepatocyte. This proposal stems from the fact that
xylitol, a precursor of xylulose 5-phosphate, is able to stimulate the
expression of a number of genes, both in vivo (12) and in
cultured cells (26, 28). Xylulose 5-phosphate has been shown to
activate the phosphatase 2A-mediated dephosphorylation (49, 50) of
fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase and to
decrease the activity of protein kinase A. The same phosphatase is also
involved in the dephosphorylation of the transcription factor Sp1 (51,
52). Xylulose 5-phosphate-activated phosphatase is also activated by
glucose or a glucose metabolite (50). Furthermore, glucose-induced
transcription of the acetyl-CoA carboxylase gene is mediated by the
transcription factor Sp1 (52). This stimulation is prevented by okadaic
acid, an inhibitor of protein phosphatase types 1 and 2A (52). On the
other hand, okadaic acid only partially inhibits the action of glucose
and xylitol to stimulate the
fructose-6-phosphate,2-kinase:fructose-2,6-biphosphatase (53). These
results suggest that either dephosphorylation of Sp1 is not universal
in the glucose signaling pathway or that glucose signaling also
involves other transcription factors such as the
upstreamstimulatory factor proteins (54). These upstream stimulatory factor proteins were among the first transcription factors
suggested to link glucose action to the ChoRE/GIRE (54, 55). The
involvement of protein phosphatase(s) in the modulation of
transcription factors involved in glucose signaling is worth exploring further.
The metabolic consequences of increased Glc-6-Pase gene expression by
glucose are not known. More than 25 years ago, Nordlie and colleagues
(47) proposed the controversial idea that Glc-6-Pase, in the presence
of high glucose levels, might act as a phosphotransferase that uses
carbamoyl phosphate as the phosphate donor. Whether Glc-6-Pase plays
any role in glucose phosphorylation is debatable. Nonetheless, the
paradoxical induction of Glc-6-Pase gene by glucose points toward a
potential role of this enzyme in the removal of glucose from the
circulation. Glucose stimulation might also be needed to make sure that
the enzyme does not disappear when the cell shifts from fed to starved
conditions. One speculation is that glucose stimulation of Glc-6-Pase
gene may take place in vivo when glucose cycling is needed
following hyperglycemia. Glc-6-Pase is a very complex protein with
different protein components that are not yet well characterized.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dCTP, using the
random primer labeling system kit (Amersham Pharmacia Biotech).
Pre-hybridization was performed for 4 h at 55 °C in Church
buffer (0.5 M phosphate buffer (pH 7.0), 7% SDS, 1 mM EDTA, and 1% bovine serum albumin). Hybridization with
the 32P-labeled probe was carried out for 16 h in the
same buffer. The membranes were washed twice for 10 min in 2× SSC,
0.1% SDS at room temperature and once in 0.1× SSC, 0.1% SDS for 15 min at 55 °C. The membranes were then exposed to Fuji or Kodak x-ray films for 12-48 h at
80 °C, using intensifying screens. 18 S ribosomal RNA was used to correct for loading irregularities. Quantification was done by scanning densitometry, using the
one-dimensional analysis software from Kodak.
80 °C
until used. Nuclear run-on assays were performed as follows. Frozen
nuclei (2 × 107 nuclei, 100 µl) were added to 100 µl of a buffer containing 20 mM HEPES-KOH (pH 8.0), 25%
glycerol, 10 mM MgCl2, 0.2 mM KCl, 1.2 mM ATP, 0.6 mM CTP, 0.6 mM GTP.
After the addition of 40 units/ml RNase inhibitor and 50 µCi of
[
-32P]UTP, the mixture was incubated at room
temperature for 45 min. The labeled RNA was passed through a
QIAshredder column (Qiagen Inc., Valencia, CA) and purified with the
Qiagen RNeasy kit, according to the manufacturer's protocol. The RNA
was resuspended in 100 µl of diethyl pyrocarbonate-treated water. The
labeled RNA from each sample was denatured and hybridized to dot-blots
containing 2 µg of purified cDNA fragments or rat genomic DNA
immobilized onto nylon filters. Blots were hybridized for 72 h at
55 °C in Church buffer (7% SDS, 0.5 M phosphate buffer
(pH 7.5), 1% bovine serum albumin). The blots were then washed twice
for 10 min in 2× SSC, 0.1% SDS at room temperature and once in 0.1×
SSC, 0.1% SDS for 15 min at 55 °C. The membranes were then exposed
to Fuji or Kodak x-ray films for 12-48 h at
80 °C, using
intensifying screens. Quantification was done by scanning densitometry,
using the one-dimensional analysis software from Kodak. Slots
containing genomic DNA were used to normalize the efficiency of the
nuclear run-on assays.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effect of glucose on Glc-6-Pase mRNA
levels in primary cultures of rat hepatocytes. Freshly
isolated rat hepatocytes were cultured in RPMI 1640 medium for 16 h. The cells were then washed and incubated with various concentrations
of glucose for an additional 4 h in serum-free RPMI 1640 medium.
The relative abundance of Glc-6-Pase mRNA was determined as
described under "Experimental Procedures." Results are expressed as
fold increase in Glc-6-Pase mRNA relative to the level observed in
hepatocytes cultured in the presence of the basal medium containing 5 mM glucose (A) using band intensity for 18 S
rRNA to correct for RNA loading. Values plotted are the means ± S.E. of 6 separate preparations. *, significantly different from cells
incubated with 5 mM glucose (p < 0.01).
Inset A, is a representative Northern blot. B, is
a control experiment showing the effect of glucose (20 mM)
on PEPCK mRNA levels.
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Fig. 2.
Time course of Glc-6-Pase mRNA induction
by glucose. Hepatocytes were prepared as described in the
legend to Fig. 1 and incubated with glucose (20 mM) for the
various times indicated. The cells were washed, and total RNA was
isolated. Northern blot analysis was performed to estimate Glc-6-Pase
mRNA levels. Results are expressed as fold increase of Glc-6-Pase
mRNA over that observed at time 0. *, significantly different from
cells incubated with 5 mM glucose (p < 0.01).
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Fig. 3.
Effect of different glucose analogs on
Glc-6-Pase mRNA levels. Hepatocytes were cultured for
16 h, washed, and then incubated in serum-free RPMI 1640 medium,
as in Fig. 1. Different glucose analogs were then added, and the
relative abundance of Glc-6-Pase mRNA was determined after 4 h
of incubation. Representative Northern blots are shown. The values for
Glc-6-Pase mRNA were normalized with reference to the values for
18 S rRNA, to correct for RNA loading. GlcN, glucosamine (2 mM); Glc, glucose (20 mM),
DOG, 2-deoxyglucose (20 mM); OMG,
3-ortho-methylglucose (20 mM);
Lact/Pyr, lactate (10 mM)/pyruvate (1 mM).
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Fig. 4.
Effect of various sugars and glycerol on
Glc-6-Pase mRNA levels. Hepatocytes from fed rats were
prepared as described in legend to the Fig. 1 and then cultured for
4 h in the presence of different concentrations of glucose and
fructose (A), glycerol (B), and mannose
(C). Northern blot analysis was performed to estimate
Glc-6-Pase mRNA levels, using band intensity for 18 S rRNA to
correct for RNA loading.
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Fig. 5.
Effect of xylitol on Glc-6-Pase
mRNA levels. Hepatocytes from fed rats were prepared as
described in legend to the Fig. 1 and then cultured for 4 h in the
absence or presence of xylitol (5 mM) (A). The
relative abundance of Glc-6-Pase was estimated by Northern blot
analysis using band intensity for 18 S rRNA to correct for RNA
loading. Results are expressed as means ± S.E. of means from five
different experiments. *, significantly different from cells incubated
with 5 mM glucose (p < 0.03). A
is a representative Northern blot. B is a control experiment
showing the effect of xylitol (5 mM) on PEPCK mRNA
levels. Xlt, xylitol.
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Fig. 6.
Inhibition of the pentose phosphate pathway
decreases glucose induction of Glc-6-Pase mRNA levels.
Hepatocytes from fed rats were preincubated for 1 h with or
without 6-aminonicotinamide (200 µM) or oxythiamine (500 µM) and then cultured for 4 h in the absence or
presence of glucose (20 mM) (A), xylitol (5 mM) (B), fructose (10 mM) or
glycerol (2 mM) (C). The relative abundance of
Glc-6-Pase was estimated by Northern blot analysis. The values for
Glc-6-Pase mRNA were normalized with reference to the values for
18 S rRNA, to correct for RNA loading. Representative Northern blots
are shown. 6-AN, 6-aminonicotinamide; Glc,
glucose; Fru, fructose; Gly, glycerol;
Xlt, xylitol.
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Fig. 7.
Effect of 6-aminonicotinamide on
glycolysis. Lactate production was measured as an indicator of
glycolysis. A shows the accumulation of lactate in
hepatocytes (1 × 106 cells) cultured with or without
glucose (20 mM) in the absence ( ) or presence
(
) of 6-aminonicotinamide (200 µM).
B shows the effect of 6-aminonicotinamide on lactate
production from control cells and cells incubated with different
carbohydrates and glycerol. The results are means of two different
experiments and are expressed as percentage of total lactate production
in the presence of glucose.
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Fig. 8.
Transcriptional effect of glucose on the
Glc-6-Pase gene. Nuclei were isolated from hepatocytes
maintained in serum-free medium in the presence or absence of 20 mM glucose. Run-on assays were carried out to determine
transcription initiation rate. Results are means ± S.E. of four
experiments. *, significantly different from cells incubated with 5 mM glucose (p < 0.001).
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Fig. 9.
Decay of Glc-6-Pase mRNA as a function of
time. Isolated hepatocytes were cultured in the presence of
20 mM glucose. Transcription was halted with the
administration of actinomycin D (10 µg/ml), and incubation was
continued either in the absence ( ) or the presence (
) of 20 mM glucose. Total RNA was isolated and analyzed by Northern
blotting analysis at the time points indicated. Inset shows
semi-log plot used to estimate mRNA half-life, assuming first-order
kinetics. Radiolabeled Glc-6-Pase and 18 S rRNA were used to visualize
and quantitate mRNA levels, as determined by the one-dimensional
image analysis software from Kodak. The results are means ± S.E.
of means from four different experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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I thank Dr. Richard W. Hanson for the use of his laboratory during the performance of much of this work. I also thank Frederic Bone for excellent assistance in various phases of this work and Dr. Ifeanyi J. Arinze for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by a Case Western Reserve University Faculty Fellowship from the Mount Sinai Health Care 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.
Recipient of a Case Western Reserve University Faculty Fellowship
from the Mount Sinai Health Care Foundation. To whom correspondence and
reprint requests should be addressed: Dept. of Nutrition, Case Western
Reserve University School of Medicine, 10900 Euclid Ave., Cleveland OH
44106. Tel.: 216-368-2135; Fax: 216-368-6644; E-mail:
dxm71@po.cwru.edu.
Published, JBC Papers in Press, November 21, 2000, DOI 10.1074/jbc.M007939200
2 K. S. Aulak, S. L. Hyatt, Q. A. Albakri, and M. Hatzogloa, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: Glc-6-Pase, glucose-6-phosphatase; 6-AN, 6-aminonicotinamide; Glc-6-P, glucose 6-phosphate; G6PDH, glucose-6-phosphate dehydrogenase; PEPCK, phosphoenolpyruvate carboxykinase; 6-PGDH, phosphogluconate dehydrogenase; MOPS, 4-morpholinepropanesulfonic acid; UTR, untranslated region; AURE, AU-rich element.
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