From the Laboratoire de Bioénergétique Fondamentale et Appliquée, Université J. Fourier, Grenoble 38041, France
Received for publication, May 16, 2000, and in revised form, November 30, 2000
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ABSTRACT |
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Mg-ATP infusion in vivo has been
reported to be beneficial both to organ function and survival rate in
various models of shock. Moreover, a large variety of metabolic effects
has been shown to occur in several tissues due to purinergic receptor
activation. In the present work we studied the effects of exogenous
Mg-ATP in rat liver cells perifused with dihydroxyacetone to
investigate simultaneously gluconeogenetic and glycolytic pathways. We
found a significant effect on oxidative phosphorylation as
characterized by a decrease in oxygen consumption rate and in the
cellular ATP-to-ADP ratio associated with an increase in
lactate-to-pyruvate ratio. In addition, exogenous Mg-ATP induced rapid
and reversible inhibition of both gluconeogenesis and glycolysis. The
main effect on gluconeogenesis was located at the level of the fructose
cycle, whereas the decrease in glycolysis was due to a strong
inhibition of pyruvate kinase. Although pyruvate kinase inhibition
induced by exogenous Mg-ATP was allosteric when assessed in
vitro after enzyme extraction, we found a large decrease in the
apparent maximal velocity when kinetics were assessed in
vivo in intact perifused hepatocytes. This newly described
short-term regulation of pyruvate kinase occurs only in the intact cell
and may open new potentials for the pharmacological regulation of
pyruvate kinase in vivo.
Numerous publications have emphasized the beneficial role of
Mg-ATP infusion in various models of shock or severe trauma in animals.
Dysfunction of heart (1), kidney (2), muscle (3), endothelial (4), and
immune cells (5, 6) are all improved by this treatment, which
significantly increases the survival rate. Because such a benefit was
independent of hemodynamic status (4, 7), a metabolic mechanism was
proposed (8). Because the liver plays a central role in the metabolic
response to such severe illnesses, it may represent a major target for
Mg-ATP (9, 10).
It is well known that purinergic receptor activation is responsible for
a large variety of metabolic effects (11-24). ATP or adenosine or
several analogues of purinergic receptors have been noted to affect
liver glucose metabolism: i.e. stimulation of glycogenolysis
(12, 13), increase (19, 25, 26) or decrease (11) of gluconeogenesis,
and decrease of glycolysis (25). These effects have been related to a
cAMP-dependent mechanism (12), a cAMP-independent inositol
3-phosphate/calcium-mediated signaling (13, 14, 16, 18, 21, 23), a
phospholipase C activation (18), or a transcriptional effect (25). It
must be noted, however, that if some effects are shared among the
adenine nucleotide family, others appear to be specific (12, 14, 18), suggesting that different signaling pathways may be involved.
Because the beneficial effects were observed in vivo with
Mg-ATP but not with adenosine (8, 27), we investigated the metabolic
effects of exogenous Mg-ATP in rat liver cells perifused with
DHA.1 Besides a significant
effect on oxidative phosphorylation and on gluconeogenesis, we found
that Mg-ATP was responsible for a large inhibition of pyruvate kinase.
Interestingly, although pyruvate kinase (EC 2.7.1.40) inhibition
induced by exogenous Mg-ATP was allosteric when assessed in
vitro after enzyme extraction, we found a large decrease in the
apparent maximal velocity when kinetics were assessed in
vivo in intact perifused hepatocytes.
Male Wistar rats (200-250 g), fasted for 24 h, were
anesthetized intraperitoneally with sodium thiopental (125 mg/kg).
Hepatocytes were isolated by the method of Berry and Friend (28) as
modified by Groen et al. (29).
Liver cells (200 mg of dry cells in 15 ml) were perifused by the method
of van der Meer and Tager (30) as modified by Groen et al.
(29, 31). Hepatocytes were perifused at 37 °C at a flow rate of 5 ml·min The time course of the effect of exogenous nucleotide addition was
studied in hepatocytes perifused in the presence of a constant DHA
concentration (9.6 mM). After an initial period of 45 min, a first steady state was reached and liver cells were exposed to a
mixture of 100 µM MgCl2 and 100 µM ATP (Mg-ATP) for 30 min. After this period, Mg-ATP
infusion was stopped, leading to a rapid decrease in ATP in the
perifusate (3 min), and cells were further perifused for another 40 min
in the absence of Mg-ATP. Perifusate samples were taken at different
time intervals as indicated.
To study the metabolic effect of exogenous ATP, perifused liver cells
were titrated with DHA and in the presence or absence of exogenous
Mg-ATP (100 µM). After an initial steady state had been
reached (45 min) in the absence of DHA, seven successive steady states
were obtained in the presence of increasing DHA concentrations (0.15, 0.30, 0.60, 1.20, 2.40, 4.80, and 9.60 mM) as indicated.
Each of the successive steady states was obtained after 20 min, then
both perifusate and cell samples were taken for subsequent analysis.
The steady state was always confirmed by stable values of glucose,
lactate, and pyruvate in three successive perifusate samples taken at
1-min intervals. Because these three values were always very close they
have been averaged. Proteins in the perifusate were denatured by
heating the samples (80 °C for 10 min) before centrifugation (36).
Glucose, lactate, and pyruvate were measured in the perifusate and
DHAP; glucose 6-phosphate; fructose 6-phosphate; 3-phosphoglycerate;
and PEP were measured in the cellular fraction as described previously
(31-35). The net fluxes (micromoles/min/g of dry cells) of
gluconeogenesis (Jglucose), glycolysis
(Jlactate + pyruvate), and DHA metabolism (JDHA), were calculated from the total cell
content of the perifusion chamber, the perifusate flow rate, and the
concentration of glucose, lactate, and pyruvate in the perifusate. All
determinations were made by enzymatic procedures (37) with either
spectrophotometric or fluorometric determination of NADH.
The effects of addition of exogenous Mg-ATP on cytosolic and
mitochondrial adenine nucleotide content were studied in similar steady-state conditions as described above for the time course effect
of Mg-ATP. After a first steady state in the presence of DHA (9.6 mM), Mg-ATP was added (100 µM) and, at 5, 10, 15, 20, and 30 min, cells were taken from the chamber for intracellular and mitochondrial nucleotide determinations. Experiments were performed
with or without Mg-ATP. Samples of cell suspension were quickly removed
from the chamber, and cellular content was separated from the
extracellular medium by centrifugation of the cell suspension through a
layer of silicone oil as described previously (21). The mitochondrial
fraction was obtained by liver cell fractionation with digitonin as
described previously (38). Cytosolic adenine nucleotide concentrations
were calculated by subtraction of the mitochondrial from the total
intracellular value. ATP, ADP, and AMP were determined by high pressure
liquid chromatography (32).
After centrifugation of the cell suspension, pyruvate kinase activity
was assessed on cell pellets resuspended in 1.5 ml of a buffer
containing: 20 mM potassium phosphate (pH 7.4); 0.25 M sucrose; 1 mM EDTA; 1 mM
dithiothreitol. After homogenization for 1 min with an Ultraturax, this
homogenate was centrifuged at 30,000 × g for 15 min
(Beckman J 21). Pyruvate kinase activity in the supernatant was
determined in 2 ml of a buffer containing 50 mM Tris-HCl
(pH 7.4), 100 mM KCl, 5 mM MgCl2,
and 10 µl of the supernatant. To obtain partially purified enzyme (L
form), 0.4 ml of the supernatant obtained after homogenization was
washed with 0.3 ml of 100%
(NH4)2SO4 (final concentration
40%) and centrifuged at 30,000 × g for 15 min; the
pellets were suspended in a medium (2 ml) containing 20 mM
potassium phosphate (pH 7.4), 30% glycerol, 1 mM EDTA, 1 mM dithiothreitol, 50 mM NaF, and pyruvate
kinase activity was measured in a buffer containing 50 mM
Tris-HCl (pH 7.4), 20 mM KCl, 5 mM
MgCl2 (39, 40). Enzyme activity was expressed as the ratio
of activity measured at 0.4 mM PEP to that at 4 mM PEP (v/Vmax), because
this expression of the results has been shown to accurately reflect the
phosphorylated state of the enzyme (41). The maximal velocity
(Vmax, µmol/min/mg of proteins) was determined
at 4 mM PEP. The velocity obtained at 4 mM PEP was very close to that obtained from a kinetic analysis of the saturation curve (data not shown). Protein content was determined in
the supernatant by the Biuret method after homogenization. Due to the
limited volume of samples and to the low protein content of the samples
after partial purification by ammonium sulfate denaturation, we have
determined in separate experiments the ratio of the protein content
before and after purification in similar conditions. This ratio was
4.9 ± 0.2 (n = 4), and this value was used as the
correcting factor for the protein content of the purified samples.
The oxygen consumption rate in hepatocytes was determined after
incubation in closed vials with Krebs-Ringer bicarbonate and DHA (20 mM) with or without 100 µM Mg-ATP. After 15 min, cells samples were removed from the vials and placed in an
oxygraph vessel equipped with a Clark electrode at 37 °C for oxygen
determination. Respiratory rate was expressed as micromoles of
O2/min/g of dry cells.
Results are expressed as means ± S.E.; Mg-ATP effect was
assessed either by a one-way analysis of variance (StatView, Abacus Concepts, Inc., Berkley, CA) or by Student's t test.
Kinetics of Changes of DHA Metabolism by Exogenous
Mg-ATP--
Because glycogen storage was almost exhausted after
24 h of fasting, the use of DHA as substrate permitted the
simultaneous investigation of gluconeogenesis and glycolysis in
hepatocytes from 24-h-fasted rats by measuring net glucose and
lactate-plus-pyruvate production. After 45 min of cell perifusion with
9.6 mM DHA, stable glucose and lactate-plus-pyruvate
productions indicated a steady state had been reached (not shown).
Exogenous Mg-ATP was then added resulting in a rapid inhibitory effect
(3 min) on both glucose (Fig.
1A) and lactate-plus-pyruvate
(Fig. 1B) production. The effect on gluconeogenesis was fast
and completely reversible, because inhibition of glucose production was
maximal after 20 min and returned to control values 10 min after
cessation of infusion. Conversely, the inhibitory effect of exogenous
Mg-ATP on lactate-plus-pyruvate production was much less reversible,
because 40 min after cessation of Mg-ATP infusion some inhibition was
still present. It must be noted that inhibition of DHA metabolism was
detected with as little as 10 µM Mg-ATP, whereas the
maximal effect was obtained with 50 µM (data not
shown).
Effect of Exogenous Mg-ATP on Hepatocytes Titrated with
DHA--
The metabolic effects of exogenous ATP were further studied
in more detail by titrating perifused liver cells with DHA. Fig. 2 (A and B) shows
that exogenous Mg-ATP inhibited both gluconeogenic and glycolytic
fluxes. As a result there was a significant decrease in total DHA
metabolism expressed as three-carbon equivalents (glucose × 2 + lactate + pyruvate, Fig. 2C). Gluconeogenesis and glycolysis
are branched pathways in DHA metabolism after its phosphorylation. Hence, the concomitant decrease in both gluconeogenesis and glycolysis could be due to an effect occurring in this first step of DHA phosphorylation. In this case, steady-state DHAP concentration should
be lowered by Mg-ATP. From Fig.
3A it is clear that DHAP concentration is increased by Mg-ATP at each concentration of infused
DHA, indicating that the effect does not actually occur during the
first phosphorylating step but downstream in both
gluconeogenesis and glycolysis. This is confirmed by the data presented
in Fig. 3 (B and C), showing that Mg-ATP was
responsible for a clear inhibition between DHAP and either glucose
(Fig. 3B) or lactate-plus-pyruvate (Fig. 3C)
production.
Inhibitory Effect of Mg-ATP on Gluconeogenesis and
Glycolysis--
From Fig. 4
(A and B), it appears that Mg-ATP had a slight
effect on the relationship between glucose 6-phosphate or fructose 6-phosphate and glucose production. Such an effect probably occurs in
the glucose 6-phosphatase step, because there is no effect on
the phosphoglucoisomerase (EC 5.3.1.9), which is close to equilibrium
in this condition (see Fig. 4C (31)). Fig. 4D
shows the relationships between the cellular concentrations of DHAP and
that of fructose 6-phosphate, permitting the investigation of the
fructose-1,6-bisphosphatase/phosphofructokinase step. It is assumed
that both aldolase- and triose-phosphate isomerase work near
equilibrium (31, 42); therefore, DHAP concentration probably reflects
the fructose 1,6-bisphosphate free concentration. From Fig.
4D, it appears that Mg-ATP affected this step: in both groups the values of fructose 6-phosphate were in the same range (between 50 and 125 nmol/g of dry cells), whereas the corresponding concentrations of DHAP were much higher in the Mg-ATP group compared with controls, except for the two lowest values corresponding to the
first two steady states (0 and 0.15 mM of DHA).
Using the perifusion method, the kinetics of pyruvate kinase can be
directly assessed in intact cells by measuring the relationships between PEP concentration and pyruvate flux at several rates and under
true steady-state conditions. In these conditions, where lactate and
pyruvate are continuously rinsed out, pyruvate kinase flux
(Jpyruvate kinase) can be evaluated by the net
production of lactate plus pyruvate. Although the classic sigmoid shape
appears in control cells (Fig.
5A), the kinetics of pyruvate
kinase were profoundly affected by Mg-ATP, the relationship being no
longer of allosteric type but hyperbolic, the apparent
Vmax being decreased by half (Fig.
5A). In rat liver cells, PEP is present in both cytosol and
mitochondrial matrix, whereas the substrate for pyruvate kinase is the
cytosolic intermediate (43). Determination of 3-phosphoglycerate, a
purely cytosolic intermediate in equilibrium with cytosolic PEP (43,
44), further confirms that Mg-ATP affected the pyruvate kinase (Fig.
5B). Given this large effect of exogenous Mg-ATP observed
in vivo on pyruvate kinase, we determined its activity
in vitro after extraction and partial purification from
liver cells exposed or not to Mg-ATP (Table
I). These results show that Mg-ATP was
responsible for a significant decrease of v/Vmax both in nonpurified and
purified enzyme extracts, whereas Vmax was not
different.
Effect of Mg-ATP on Liver Cell Respiration, Redox State, and
ATP-to-ADP Ratios--
The addition of exogenous Mg-ATP was
responsible for a significant decrease in oxygen consumption rate in
the presence of saturating DHA concentration: 20.9 ± 1.1 versus 15.9 ± 0.4 µmol of O2/min/g of
dry cells, respectively, for controls and Mg-ATP (p < 0.01, n = 6 in each group). The steps located between
DHAP and PEP are believed to be at a near equilibrium state with the cytosolic redox state and ATP-to-ADP ratio (31, 42, 45). Therefore, the
relationship between DHAP and PEP concentrations is dependent on the
potential effect of Mg-ATP on both redox state and cytosolic ATP-to-ADP
ratio. As shown on Fig. 6, this
relationship was affected by Mg-ATP: For a given concentration of PEP,
DHAP increased 2-fold. The change in the ratio of PEP-to-DHAP could be
the consequence of a change in phosphate and/or redox potentials. Fig.
7 shows the relationship between
lactate-to-pyruvate ratio, a metabolic indicator of cytosolic redox
state (46), and the flux through pyruvate kinase. The relationship in
the presence of exogenous Mg-ATP was shifted to the right
(p < 0.01), indicating a more reduced cytosolic redox
state. Infusion of exogenous Mg-ATP may also affect cytosolic phosphate
potential and subsequently the ratio between PEP and DHAP. Table
II shows cytosolic and mitochondrial ATP,
ADP, total nucleotide (ATP + ADP + AMP), and ATP-to-ADP ratio. In the
cytosol, exogenous Mg-ATP was responsible for an increase in total
nucleotide content (p < 0.01) due to an increase in
both ATP (p < 0.02) and ADP (p < 0.01). But ADP increase was larger than that of ATP, leading to a
significant lowering of ATP-to-ADP ratio (p < 0.05).
In the mitochondrial matrix, exogenous Mg-ATP was also responsible for
an increase in the total adenine nucleotide pool (p < 0.01). ADP level increased (p < 0.01) while ATP
content decreased (p < 0.01), resulting in a decrease
in the ATP-to-ADP ratio (p < 0.01).
The clear inhibitory effect of exogenous Mg-ATP addition on DHA
metabolism in intact hepatocytes reported in this work consists of a
decrease of gluconeogenesis associated with a potent inhibition of
lactate-plus-pyruvate production. In addition, there seems to be a new
mechanism for pyruvate kinase regulation after exogenous Mg-ATP
addition occurring only in vivo in intact cells.
By investigating DHA metabolism with successive steady states and
measuring glucose and lactate-plus-pyruvate fluxes simultaneously with
cellular intermediate concentrations, perifusion of liver cells is a
suitable tool to determine the step(s) affected by exogenous Mg-ATP.
Glucose production accurately reflects the rate of gluconeogenesis,
because glycogen synthesis in these conditions is negligible as
compared with the flux of glucose production. Moreover, glycogen
synthesis is inhibited by exogenous ATP (18). The flux of
lactate-plus-pyruvate truly reflects the pyruvate kinase flux
(i.e. glycolysis) only in the absence of significant pyruvate oxidation or transamination. Pyruvate oxidation or
transamination may lead to an underestimate of glycolytic flux, but
this is probably limited because of the very low pyruvate concentration
due to the continuous rinsing of the perifusate.
Moreover, if decrease of lactate-plus-pyruvate production by Mg-ATP had
been the consequence of an increased pyruvate oxidation or
transamination, PEP should not accumulate, in contrast to our findings
(Fig. 5).
Considering the pathway between DHAP and glucose production, it appears
that exogenous Mg-ATP is responsible for a minor effect at the level of
glucose/glucose 6-phosphate cycle (Fig. 4A), whereas the
main effect is located at the fructose 6-phosphate/fructose 1,6-bisphophate step (Fig. 4, C and D). A
cAMP-dependent phosphorylation has been reported to inhibit
6-phosphofructo-1-kinase (47), but this enzyme is not very active in
hepatocytes from fasted rats (48). On the other hand,
fructose-1,6-bisphosphatase can be activated by a
c-AMP-dependent phosphorylation (49). Nevertheless, such
effects of cAMP-related phosphorylation on 6-phosphofructo-1-kinase or
on fructose-1,6-bisphosphatase seems to have a minor functional impact
(48), and it is believed that fructose 2,6-bisphosphate is the main
regulator via changes in the bifunctional enzyme: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (50). Bartrons et al. (12) have reported that adenosine as well as the
adenosine analogue 2-chloroadenosine are responsible for a decrease in
fructose 2,6-bisphosphate due to an activation of
fructose-2,6-bisphosphatase by adenylate cyclase activation and cAMP
rise. Both adenosine and 2-chloroadenosine increase cAMP and decrease
fructose 2,6-bisphosphate, but adenosine is responsible for a decreased
rate of gluconeogenesis, whereas 2-chloroadenosine increases it (12).
Adenosine is responsible for an inhibition of gluconeogenesis (11, 12,
20, 22) at the level of fructose cycling. Hence, our results might
support the view that the effect of exogenous Mg-ATP on gluconeogenesis from DHA are similar to, if not mediated by, the effect of adenosine as
was suggested by Asensi et al. (11).
The most striking finding of this work is a significant decrease of
lactate-plus-pyruvate production resulting from a potent inhibition of
pyruvate kinase. Cytosolic adenine nucleotides are potent regulators of
glycolysis, but given the decrease in cytosolic ATP-to-ADP ratio
following exogenous Mg-ATP, an activation of glycolysis would actually
have been predicted (32, 34, 35). The increased cytosolic NADH/NAD
ratio as shown by the increased lactate-to-pyruvate ratio (Fig. 7)
could theoretically account, at least partly, for such an inhibition of
lactate-plus-pyruvate production. But in the presence of Mg-ATP, the
relationship between PEP and lactate-plus-pyruvate production reached a
plateau indicating a clear saturation of the substrate PEP (Fig.
5A). Hence, the Mg-ATP inhibitory effect of
lactate-plus-pyruvate production cannot be explained by a decrease in
PEP concentration. Thus it can be concluded that acute administration
of exogenous Mg-ATP strongly decreases the apparent
Vmax of pyruvate kinase when determined in
vivo in intact cells. It has already been reported that Mg-ATP was
responsible for a decrease in pyruvate kinase activity in vivo, but this was observed 4 h after exogenous Mg-ATP
administration and was related to a transcriptional effect (25). In our
experiments the effect was observed after a few minutes, indicating
that another mechanism must be involved.
The second striking finding of the present work is related to the fact
that the change in apparent maximal velocity of pyruvate kinase was
found only when determined in vivo but not in
vitro after enzyme extraction and partial purification. This
indicates that the amount of enzyme was not modified by such short-term effect, which is not surprising. Actually, we found an allosteric inhibition of this enzyme when assessed in vitro. An acute
inhibitory effect of adenosine on pyruvate kinase, due to a
cAMP-related phosphorylation, was already described by Bartrons
et al. (12). However, in the present work the allosteric
inhibition was not apparent when enzyme activity was determined in
intact cells. This finding indicates that another mechanism controls
this step in vivo. Because this effect disappears after
enzyme extraction, an intracellular metabolite could be involved.
Indeed, there are several cellular metabolites, such as fructose
1,6-bisphosphate, alanine, ATP, and ADP known to regulate pyruvate
kinase, but these effectors act via an allosteric mechanism (48) and do
not affect the Vmax in contrast to the present
results. In the presence of Mg-ATP, the hypothetical inhibitor should
be noncompetitive with PEP and does not bind abnormally tightly to the
enzyme, because it is lost after extraction. Such a hypothesis opens
the possibility of a new regulatory mechanism of pyruvate kinase, which
can be detected only in vivo in intact cells.
The question of the nature of the cellular effects of exogenous Mg-ATP
is obviously complex. Although some effects of Mg-ATP seem to be
similar to those of adenosine or adenosine agonists, others are
different. Indeed, besides cAMP-related phosphorylation, inositol
1,4,5-trisphosphate, calcium, and phospholipase C signaling, other receptors and signaling pathways are also probably involved, resulting in a very complicated and subtle regulation leading to
multiple metabolic responses as described in this paper (including changes in oxidative phosphorylation, redox state, phosphate potential, glycolysis, gluconeogenesis, etc.).
Given the clear beneficial effect of exogenous Mg-ATP administration
demonstrated by Chaudry (7, 8) in several animal models of shock
on survival and organ or cellular functions, a possible link with the
present finding can be hypothesized. It has been recently demonstrated
that isolated rat hepatocytes can signal to other hepatocytes by the
release of ATP, suggesting a novel paracrine signaling pathway exists
(51). This finding favors a physiological role of exogenous ATP, which
may trigger a protective effect.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 with Krebs bicarbonate buffer (pH 7.4)
continuously saturated with O2/CO2 (19:1) and
containing calcium (1.3 mM) (32-35).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Kinetic of Mg-ATP effect on DHA metabolism in
isolated perifused hepatocytes. Hepatocytes (200 mg of dry cells
in 15 ml), isolated from 24-h-starved Wistar rats were perifused with
9.6 mM DHA. The flow rate of perifusate was 5 ml·min 1 (Krebs-Ringer bicarbonate buffer, pH 7.4)
continuously saturated with 95% O2/5% CO2.
When a steady state was reached (45 min, not shown), 100 µM Mg-ATP was added in one chamber (
) compared with
the control chamber (
). Sequential perifusate samples were taken for
subsequent glucose (A) and lactate-plus-pyruvate
(B) determination to calculate the gluconeogenesis
(Jglucose) and the glycolysis
(Jlactate + pyruvate) rates. A typical
experiment is shown; similar results were obtained in three other
experiments.
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Fig. 2.
Inhibition of DHA metabolism in hepatocytes
perifused with 100 µM exogenous Mg-ATP. Hepatocytes
(200 mg of dry cells in 15 ml) were perifused as described in Fig. 1.
DHA was titrated by infusing increasing concentrations: 0.15, 0.30, 0.60, 1.20, 2.40, 4.80, and 9.60 mM as indicated in the
figure with ( ) or without (
) 100 µM exogenous
Mg-ATP. The rates of gluconeogenesis (Jglucose,
A), glycolysis
(Jlactate + pyruvate, B) and DHA
metabolism (JDHA, C,
JDHA = 2 × [glucose] + [lactate] + [pyruvate]) were calculated from the glucose, lactate, and pyruvate
concentrations in the perifusate. Results are expressed as means ± S.E.; n = 4 in each group.
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Fig. 3.
Effect of Mg-ATP on DHAP concentration:
relationship with glucose and lactate-plus-pyruvate productions.
Hepatocytes were perifused as described in Fig. 2 with ( ) or without
(
) 100 µM exogenous Mg-ATP. At each steady state,
0.5-ml samples of cell suspension were removed from the perifusion
chamber and centrifuged. Intracellular DHAP concentration was measured
in the neutralized cell fractions. A, relationship between
intracellular DHAP concentration and infused DHA concentration. The
rates of gluconeogenesis (Jglucose,
B) and glycolysis
(Jlactate + pyruvate, C) were
calculated from glucose, lactate, and pyruvate concentrations in the
perifusate. Results are expressed as means ± S.E.;
n = 3 in each group.
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Fig. 4.
Effect of Mg-ATP on gluconeogenesis from
DHA. Hepatocytes were perifused as described in Fig. 2 with ( )
or without (
) 100 µM exogenous Mg-ATP. At each steady
state, 0.5-ml samples of cell suspension were removed from the
perifusion chamber and centrifuged. Intracellular glucose 6-phosphate
(A and C), fructose 6-phosphate (B and
C), and DHAP (D) concentrations were measured in
the neutralized cell fractions. Results are expressed as means ± S.E.; n = 3 in each group.
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Fig. 5.
Effect of Mg-ATP on the relationship between
PEP or 3-phosphoglycerate concentrations and glycolytic flux in
isolated hepatocytes perifused with DHA. Hepatocytes were
perifused as described in Fig. 2 with ( ) or without (
) 100 µM exogenous Mg-ATP. At each steady state, 0.5-ml samples
of cell suspension were removed from the perifusion chamber and
centrifuged. Intracellular PEP (A) and 3-phosphoglycerate
(B) concentrations were measured in the neutralized cell
fractions. Results are expressed as means ± S.E.;
n = 3 in each group.
Effect of exogenous Mg-ATP on pyruvate kinase activity determined in
vitro in nonpurified or partially purified enzyme
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Fig. 6.
Effect of Mg-ATP on the relationship between
intracellular PEP and DHAP concentrations in isolated hepatocytes
perifused with DHA. Hepatocytes were perifused as described in
Fig. 2 with ( ) or without (
) 100 µM exogenous
Mg-ATP. At each steady state, 0.5-ml samples of cell suspension were
removed from the perifusion chamber and centrifuged. Intracellular PEP
and DHAP concentrations were measured in the neutralized cell
fractions. Results are expressed as means ± S.E.;
n = 3 in each group.
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Fig. 7.
Effect of Mg-ATP on the relationship between
the lactate-to-pyruvate ratio and glycolytic flux in isolated
hepatocytes perifused with DHA. Hepatocytes were perifused as
described in Fig. 2 with ( ) or without (
) 100 µM
exogenous Mg-ATP. Flux of lactate-plus-pyruvate and lactate-to-pyruvate
ratios were obtained from lactate and pyruvate determinations in the
perifusate. Results are expressed as means ± S.E.;
n = 3 in each group, values of lactate-to-pyruvate
ratio in the presence of Mg-ATP were significantly different from that
of controls (p < 0.01, analysis of variance).
Effect of exogenous Mg-ATP on cytosolic and mitochondrial adenine
nucleotides concentrations and ATP/ADP ratios in isolated perifused
hepatocytes
AN (ATP + ADP + AMP) were
significantly higher in the Mg-ATP group (p < 0.01),
whereas the ATP/ADP was significantly lower in the Mg-ATP group
(p < 0.01); mitochondrial ATP/ADP was significantly
lower in the presence of Mg-ATP (p < 0.01).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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The authors are grateful to Drs. Marian Baker and Dan Veale for precious help in editing the manuscript.
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FOOTNOTES |
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* This work was supported by the Ministère de l'Enseignement, de la Recherche et de la Technologie, and by the Université (GERCBT) and the Centre-Hospitalo-Universitaire, Nice (to C. I.).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.
On leave from the Département
d'Anesthésie-Réanimation CHU de Nice France.
§ On leave from the Departamento de Fisiologia y Farmacologia, Facultad de Farmacia, Universidad de Salamanca.
¶ On leave from the Service de Gastroenterologie, Hépatologie et Nutrition, CHU Côte de Nacre, Caen, France.
To whom correspondence should be addressed: Laboratoire de
Bioénergétique Fondamentale et Appliquée,
Université Joseph Fourier, BP 53X, Grenoble 38041 Cedex, France.
Tel.: 33-4-76-51-43-86; Fax: 33-476-51-43-05; E-mail:
xavier.leverve@ujf-grenoble.fr.
Published, JBC Papers in Press, December 4, 2000, DOI 10.1074/jbc.M004169200
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ABBREVIATIONS |
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The abbreviations used are: DHA, dihydroxyacetone; DHAP, dihydroxyacetone phosphate; PEP, phosphoenolpyruvate.
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