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J. Biol. Chem., Vol. 276, Issue 30, 28126-28133, July 27, 2001
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§,
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From the Laboratoire de Bioénergétique
Fondamentale et Appliquée, Université Joseph Fourier,
Grenoble 38041, France and ¶ INSERM U449, Faculté de
Médecine Laennec, Lyon 69372, France
Received for publication, November 8, 2000, and in revised form, May 15, 2001
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Glucagon affects liver glucose metabolism mainly
by activating glycogen breakdown and by inhibiting pyruvate kinase,
whereas a possible effect on glucose-6-phosphatase has also been
suggested. Although such a target is of physiological importance for
liver glucose production it was never proven. By using a model of liver cells, perifused with dihydroxyacetone, we show here that the acute
stimulation of gluconeogenesis by glucagon (10 The metabolic effects of glucagon on liver glucose metabolism have
long been described, mediated by both cAMP- and
calcium-dependent signalings (1-3). Besides a powerful and
well documented effect on glycogen breakdown, glucagon also increases
liver gluconeogenesis mainly by the allosteric inhibition of pyruvate
kinase by its phosphorylation (4, 5). In addition, some effects of
glucagon on the fructose phosphate cycle have been described,
i.e. inhibition of 6-phosphofructo-1-kinase and
activation of fructose-1,6-bisphosphatase (6, 7). Apart from these
indisputable effects, a stimulation of glucose-6-phosphatase, a key
enzyme in glucose production, has been proposed but never proven
(8-10). In view of a potential key role of glucose 6-phosphate as a
cellular intermediate in the transcriptional effects of glucose (11,
12), an effect of glucagon on glucose-6-phosphatase could be of major
importance. Indeed, despite a prominent effect on liver glucose
metabolism, the role of glucagon in the pathogenesis of type II
diabetes is still unclear and a matter of debate (13).
By studying glucose production under steady state conditions in a model
of rat hepatocytes perifused with dihydroxyacetone (DHA)1 as an exogenous source
of carbohydrate, we show in the present work that despite a clear
inhibitory effect of glucagon on pyruvate kinase, this effect was not
responsible for the enhanced glucose production, indicating that this
step is actually not controlling this pathway. Actually, the dramatic
increase in the glucose production after glucagon addition was related
to a substantial activation of glucose 6-phosphate hydrolysis in
glucose and inorganic phosphate. This finding in vitro in
liver cells was confirmed in vivo in postabsorptive rats
depleted in glycogen, where we showed that glucagon increased hepatic
glucose production whereas liver glucose 6-phosphate concentration
decreased. Because we failed to find any change in the
glucose-6-phosphatase activity in perifused hepatocyte with or without
glucagon and only a minor effect in vivo, we hypothesized
the possibility of an effect linked to a membrane vesicle traffic-based
pathway originating from the endoplasmic reticulum, as it was recently
proposed (14, 15). This hypothesis was supported by the finding that
the effect of glucagon on glucose 6-phosphate hydrolysis was
temperature-sensitive, because it was completely abolished at 15 °C,
converse to the effect on pyruvate kinase, which was not affected by
temperature. Therefore we propose glucagon to be involved in hepatic
glucose 6-phosphate concentration by activating its hydrolysis in a
temperature-dependent pathway. Hence this hormone could be
involved in the glucose sensing by liver cells via its
phosphorylated metabolite, glucose 6-phosphate, and in turn in the
transcriptional effects of glucose (11, 12). Such a finding could lead
to reconsider the role of glucagon in the pathogenesis and treatment of
type II diabetes.
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 (16) as
modified by Groen et al. (17). Liver cells (200 mg of dry
cells in 15 ml) were perifused by the method of van der Meer and Tager
(18) as modified by Groen et al. (17). Hepatocytes were
perifused at 37 or 21 °C as indicated at a flow rate of 5 ml·min The effects of glucagon on cytosolic and mitochondrial adenine
nucleotide contents were studied in hepatocytes perifused in the
presence of a constant DHA concentration (9.6 mM). After an initial period of 45 min, liver cells were exposed to glucagon (10 Pyruvate kinase activity was assessed in hepatocytes incubated in
closed vials containing Krebs bicarbonate buffer saturated with
carbogene and 20 mM dihydroxyacetone with or without
glucagon (10 The metabolic effects of glucagon were also studied in vivo.
Rats were deprived of food for 5 h with free access to water before each experiment. After the rats were anesthetized, a
polyethylene catheter was inserted into the right jugular vein for the
infusion of saline or glucagon (60 ng/kg/min), and
[3-3H]glucose (specific activity 370 GBq/mmol;
Isotopchim, Ganagobie, France). A second catheter was placed in the
left carotid artery for blood sampling. Blood glucose was monitored
every 10 min during infusions using a glucometer II (Bayer Diagnostics,
Puteaux, France). Body temperature was maintained at 37.5 °C by a
rectal probe-monitored blanket. After 180 min, a laparotomy was
performed to allow free access to the liver. A liver lobe was frozen
in situ with tongs cooled with liquid nitrogen
( For statistical analysis, results are expressed as the mean ± S.E. The effects of glucagon effect were assessed by ANOVA (Stat View®; Abacus Concepts, Inc., Berkley, CA, 1992) followed by a Student's t test for post-hoc analysis when necessary.
Activation of DHA Metabolism by Glucagon--
Fig.
1A shows that glucagon
activates DHA metabolism, as deduced from glucose and lactate plus
pyruvate production (JDHA = J(glucose × 2) + J(L+P)), with only 0.30 mM of
infused DHA. Above 2 mM of DHA, JDHA
exhibited a plateau with or without glucagon, but
JDHA was 30% higher in the presence of
glucagon. This stimulatory effect on JDHA was
accompanied by a decrease in DHAP concentration at every steady state
of infusion of DHA (Fig. 1B) allowing us to conclude that
glucagon activates the pathway downstream of its first step of
phosphorylation of DHA. This is confirmed by the shift to the left of
the relationship between the cellular concentration of DHAP and
JDHA (Fig. 1C); for a given cellular
concentration of DHAP, JDHA was higher in the
presence of glucagon, confirming the downstream activation of DHAP.
Inhibitory Effect of Glucagon on (L+P) Production from
DHA--
Glucagon inhibited J(L+P) from DHA,
and the extent of inhibition was dependent upon DHA concentration;
J(L+P) was almost completely inhibited by
glucagon at 0.3 mM DHA (2.9 ± 0.5 versus
0.3 ± 0.03 µmol/min/g of dry cells for controls or glucagon). At 0.6 and 1.2 mM DHA the inhibitory effect was 76 and
63%, respectively, whereas at 9.6 mM the inhibition was
only 32%. Linear double-reciprocal plots were obtained, as would
classically be expected of systems following Michaelis-Menten
hyperbolic kinetics (Fig. 2B).
This allows the extrapolation to an apparent maximal velocity of 10 µmol/min/g of dry cells, which is unaffected by glucagon, whereas the
apparent affinity clearly differed, 0.5 mM
versus 4 mM of DHA for controls and glucagon,
respectively. Hence, glycolytic rate was only slightly affected by
glucagon at high substrate concentrations.
With the model of perifused hepatocytes, it is possible to assess the
kinetics of pyruvate kinase directly in intact cells by determining the
relationship between phosphoenolpyruvate concentration and the pyruvate
flux under steady state conditions. Indeed under this condition of
continuous perifusate rinsing, the pyruvate kinase flux
(JPK) can be assumed to be close to
J(L+P). As shown in Fig.
3A, the classical allosteric
inhibition of pyruvate kinase by glucagon was found in intact cells.
The maximal inhibition by glucagon was observed between 500 and 1000 nmol P-enolpyruvate/g of dry cells, whereas above this value the fluxes
reached almost a similar maximal rate in both groups indicating that
the Vmax was not affected by glucagon. In rat
liver cells, phosphoenolpyruvate is present in both the cytosol and the
mitochondria, whereas the substrate for pyruvate kinase is in the
cytoplasm. The 3-phosphoglycerate (Fig. 3B), a purely
cytosolic metabolite, is an indicator of cytosolic P-enolpyruvate
concentration, because it is in equilibrium with cytosolic
P-enolpyruvate. A similar relationship as that reported for
P-enolpyruvate confirmed the data presented in Fig. 3A. In fact, the linear relationship between 3-phosphoglycerate and
P-enolpyruvate in both controls and glucagon-treated cells (Fig.
3C) permits us to exclude a significant effect of glucagon
on the subcellular distribution of P-enolpyruvate.
When the cellular concentration of DHAP was plotted against
J(L+P), to investigate the pathway from DHAP to
(L+P) production, it appears that a unique relationship was found with
or without glucagon (Fig. 4A).
This excludes any net effect of the hormone on this part of the
pathway, which, however, involves pyruvate kinase. Hence, whereas
glucagon inhibited the pathway from P-enolpyruvate to (L+P) production
(Fig. 3), this effect was no longer present when the pathway was
considered from DHAP (Fig. 4A). Fig. 4B shows the
relationship between the steady state concentrations of DHAP and
P-enolpyruvate, which was downshifted in the presence of glucagon; whereas P-enolpyruvate was in a similar range of concentration in both
groups, DHAP was lowered by glucagon.
Effect of Glucagon on Redox State and on ATP to ADP Ratio--
The
steps located between DHAP and P-enolpyruvate are believed to work at
near equilibrium under similar experimental conditions (34); therefore
the relationship between DHAP and P-enolpyruvate is expected to depend
on the cytosolic ATP to ADP ratio (at the phosphoglycerate kinase
level; EC 2.7.2.3) and on the redox state (at the glyceraldehyde
dehydrogenase level; EC 1.2.1.12). There was no major effect of
glucagon on the lactate to pyruvate ratio; therefore a change in
cytosolic redox state is unlikely (Fig. 4C). Table
I shows ATP, ADP, and total nucleotide
(ATP + ADP + AMP) concentrations, as well as ATP to ADP ratios
in the cytosol and the mitochondrial matrix, with or without glucagon. Although no significant effect was found in the mitochondrial matrix,
glucagon significantly lowered cytosolic ADP (p < 0.01), leading to a marked increase in the ATP to ADP ratio
(p < 0.01). Because, converse to the present results
(Fig. 4B), this effect would increase the ratio of DHAP to
P-enolpyruvate, it indicates that this ratio of cytosolic intermediates
might not be close to equilibrium with the cytosolic concentrations of
ATP and ADP in these conditions. Taken together these results show that
the inhibition of pyruvate kinase by glucagon does not explain the inhibition of (L+P) production, because pyruvate kinase is probably not
a controlling step of the glycolytic pathway between DHAP and
J(L+P).
Effect of Glucagon on Gluconeogenesis from
Dihydroxyacetone--
Fig. 5A
shows that glucagon activated the pathway between DHA and glucose
production resulting in nearly a 2-fold stimulation in
Jglucose. This effect was because of an
activation downstream of DHAP as evidenced by the double relationship
between DHAP and Jglucose with or without
glucagon (Fig. 5B). Phosphoglucoisomerase works at near
equilibrium under these conditions (Fig. 5C), and the
dramatic activation of gluconeogenesis by glucagon was because of a
stimulatory effect located downstream of glucose 6-phosphate (Fig.
5D). Glucose-6-phosphatase activity was determined in our conditions with and without glucagon, and we failed to find any difference according to the presence of the hormone;
glucose-6-phosphatase activity was 1654 ± 70 µmol/min/g of dry
cell and 1620 ± 74 µmol/min/g of dry cell with or without
glucagon, respectively (n = 6; not significant).
Glucose 6-phosphate hydrolysis is located in the lumen of the
endoplasmic reticulum (35, 36), requiring glucose 6-phosphate to be
transported from the cytosol into the endoplasmic reticulum and the
glucose to be transported back into the cytosol (37). Because
glucose-6-phosphatase per se is not involved in the effect
of glucagon, another step must be involved. In a recent work based on
GLUT2-deficient mice, an alternative mechanism for hepatic glucose
output other than GLUT2-facilitated diffusion was proposed based on a
membrane-traffic mechanism, which was temperature-sensitive (14, 15).
We have investigated this possibility, and the data presented in Table
II show that the glucagon-related
activation of glucose production was inhibited at low temperature in
hepatocytes incubated in closed vials. Conversely, the allosteric
inhibition of pyruvate kinase by glucagon was not effected by low
temperature as assessed by the ratio of v 0.4/v 4 mM P-enolpyruvate (see "Material and Methods"), which
was almost identical at 15, 21, and 37 °C. The finding of a low
temperature sensitivity of the effect of glucagon on glucose production
was confirmed in hepatocytes perifused at 21 °C. (Fig.
6A). In this condition the
activation of glucose production by glucagon was still present at
21 °C, albeit to a less extent as compared with the effect observed
at 37 °C (see Fig. 5). This activation was located downstream of
dihydroxyacetone phosphate, as evidenced by the shift to the left of
the relationship between DHAP and Jglucose in
the presence of glucagon (Fig. 6B). Interestingly the shape
of the relationship between fructose 6-phosphate (Fig. 6C)
or glucose 6-phosphate (Fig. 6D) and
Jglucose was not affected by glucagon, converse
to the results obtained at 37 °C (see Fig. 5D). This
finding indicates that the effect of glucagon on glucose 6-phosphate
hydrolysis and glucose release was temperature-sensitive, because it
was present at 37 °C but not at low temperature (21 °C).
Conversely, as already shown in Table II, the effect of glucagon on
(L+P) production was not affected by the temperature, because it was
still present at 21 °C, even at subsaturating DHA concentration (Fig. 6E).
Effect of Glucagon on Gluconeogenesis in Vivo--
The data from
liver cells perifused with dihydroxyacetone are rather far from the
actual physiological conditions. Hence, we have undertaken in
vivo experiments to determine whether glucagon also affects
glucose-6-phosphatase under these conditions. Table III shows the results from postabsorptive
rats receiving a continuous infusion of glucagon for 180 min. A large
and significant increase in blood glucagon concentration was seen,
whereas blood glucose did not differ between saline and glucagon groups
after 3 h of infusion. Hepatic glucose production was increased
2-fold in the glucagon group, indicating a marked increased in the rate
of gluconeogenesis, because liver glycogen was depleted at the time.
Although glucokinase was unaffected by glucagon under these conditions,
glucose-6-phosphatase activity was significantly increased, the most
striking resultant effect being a large decrease in glucose 6-phosphate
concentration. Hence the increase in glucose-6-phosphatase flux, as
demonstrated by the increased hepatic glucose production, associated
with a decreased glucose 6-phosphate concentration, supports the
finding of an activation of glucose 6-phosphate hydrolysis by
glucagon in vivo.
In the present work we report that the acute stimulation of liver
gluconeogenesis by glucagon was related to a potent activation of
glucose 6-phosphate hydrolysis by a low temperature-sensitive pathway,
whereas glucose-6-phosphatase activity was activated to a minor extent
and only in vivo after 3 h of glucagon infusion. Although glucagon affected pyruvate kinase and ATP-to-ADP ratios in our
conditions in vitro, as expected, the acute activation of
glucose 6-phosphate hydrolysis was responsible for both gluconeogenesis activation and glycolysis inhibition at 37 °C. The dramatic
activation by glucagon of the gluconeogenic pathway downstream of
glucose 6-phosphate was also found in vivo, because the
activation of gluconeogenesis by this hormone was accompanied by a
2-fold decrease in glucose 6-phosphate concentration.
As it has long been known, glucagon is responsible for an allosteric
inhibition of pyruvate kinase by a cAMP-dependent mechanism (4, 5), which was evidenced in the present work in both intact cells
and protein extracts, purified or not (see Fig. 3A and Table
II). It is generally believed that the stimulatory effect of glucagon
on glucose production from DHA is mainly the consequence of the
inhibition of the glycolytic flux compensating for the increased
glucose production in such a way that the total flux (JDHA) is almost unaffected (21, 38, 39). The
present finding of a 30% increase of JDHA at
37 °C in the presence of glucagon is not in agreement with this
view. From the comparison between Fig. 2A and Fig.
4A it appears that glucagon was responsible for the
inhibition of (L+P) production from DHA (Fig. 2A) but not from DHAP (Fig. 4A). This indicates that the inhibitory
effect of glucagon on (L+P) production was because of a lowering of
DHAP rather than of its effect on pyruvate kinase. Indeed glucagon was
responsible for an activation of the DHA pathway downstream of DHAP
i.e. between glucose 6-phosphate and glucose
(Fig. 5D). Hence the effect of glucagon on either glucose or
(L+P) fluxes is not because of pyruvate kinase inhibition. The role of
pyruvate kinase inhibition by glucagon as a mechanism of explaining the stimulation of gluconeogenesis has been questioned on the basis of a
re-evaluation of isotopic tracer studies (40). Using a similar model of
perifused hepatocytes, Groen et al. (41) have suggested that
the stimulating effect of glucagon on gluconeogenesis is almost
entirely because of its effect on pyruvate kinase, and this effect is
proportional to the flux through pyruvate kinase (41). Conversely, we
have found in the present study that the inhibition of pyruvate kinase
by glucagon is lessened at high flux and is not responsible for the
increased glucose production. The main difference between these two
studies relies on the nature of the substrates, lactate/pyruvate in the
work by Groen et al. (41) and DHA in our study. With
lactate/pyruvate as a substrate, the cycling between P-enolpyruvate and
pyruvate should likely play a major role in the control of the entire
pathway. In contrast, with DHA as a substrate, this cycling is probably
very limited because of the low pyruvate concentration achieved by
continuous medium rinsing. In the presence of exogenous
lactate/pyruvate, cytosolic P-enolpyruvate is provided from cytosolic
oxaloacetate by P-enolpyruvate carboxykinase, and its concentration is
dependent on a negative feedback mechanism on its own synthesis
(i.e. its elasticity) (34). This is not likely
the case with DHA where P-enolpyruvate is provided from DHAP by a
cascade of near equilibrium reactions (34). It is clear that
P-enolpyruvate reaches much higher concentrations with DHA (1500 nmol/g
of dry cells; see Fig. 3A) as compared with the values
obtained by Groen et al. (41) with lactate/pyruvate (600 nmol/g of dry cells; see Ref. 41). Therefore both views may be
reconciled, because (i) the inhibitory effect of glucagon increases
whereas P-enolpyruvate concentration rises from 0 to 700 nmol/g of dry
cells, and (ii) the lessening of this effect occurs above these values,
i.e. above the highest concentration reported by
Groen et al. (41) with lactate/pyruvate (Fig.
3A). Nevertheless, if the inhibitory effect of glucagon on
pyruvate kinase is not responsible for the decreased (L+P) production
then another mechanism must be. Glucagon decreased the steady state
concentration of DHAP (Fig. 1C), such a decrease being
responsible for the inhibition of (L+P) production, as it is evidenced
by the unique relationship between DHAP and
J(L+P) with or without glucagon (Fig.
4A). This indicates that, under our conditions, the hormone
had no net effect on this part of the pathway.
Glucagon affects the fructose phosphate cycle by inhibiting
6-phosphofructo-1-kinase and activating fructose-1,6-bisphosphatase (6,
7). From the data at 37 °C it is not possible to exclude an acute
role of glucagon on this cycle, the presumed effect being more likely
on fructose-1,6-bisphosphatase than on 6-phosphofructo-1-kinase, because the latter is probably inhibited in fasting conditions (42).
The marked effect of glucagon on glucose 6-phosphate concentration at
37 °C (Fig. 5D) could hide an effect at the fructose
phosphate cycling. Indeed, it is quite interesting to note that at
21 °C, a condition where the activation downstream of glucose
6-phosphate is abolished, the activation by glucagon of the
gluconeogenic pathway downstream of DHAP remains, evidencing the effect
of the hormone at the level of the frucose phosphate cycling (Fig.
6).
The main effect of glucagon is a dramatic activation of glucose release
from glucose 6-phosphate. Glucose production is a good reflection of
the rate of gluconeogenesis, because glycogen synthesis is almost
negligible in comparison to the flux of glucose reported here (43). The
lack of effect of glucagon on glucose production in the absence of DHA
(Fig. 5A) confirms the classical depletion of liver glycogen
after 24 h of fasting. The flux through glucokinase is most likely
very low under these experimental conditions because of the continuous
washout of glucose; the highest glucose concentration in the perifusate
was 0.25 mM, whereas the Km of
glucokinase for glucose is 10 mM (44). Hence, the main
effect of glucagon on DHA metabolism appears to be because of an
activation of glucose 6-phosphate hydrolysis. This result has been
confirmed in vivo in postabsorptive state after 3 h of
glucagon infusion, because the increased hepatic glucose production was
accompanied by a dramatic decrease in the glucose 6-phosphate. The last
step in glucose production, hydrolysis of glucose 6-phosphate to
glucose and phosphate, is catalyzed by the glucose-6-phosphatase in the lumen of the endoplasmic reticulum (35, 36). Considering the modest
activation (15%) of glucose-6-phosphatase activity in vivo after 3 h of glucagon infusion and the lack of change in
vitro, it is not likely that glucagon has any acute effect on
glucose-6-phosphatase activity. The topological organization of
glucose-6-phosphatase in the lumen of the endoplasmic reticulum
requires glucose 6-phosphate, produced in the cytosol, to be
transported into the endoplasmic reticulum for hydrolysis. It is then
assumed that glucose is transported back into the cytosol for its
release out of the liver cells by facilitated diffusion through the
plasma membrane glucose transporter GLUT2 (37). On GLUT2-deficient mice
Guillam et al. (14, 15) have shown that hepatic glucose
production does not need the presence of GLUT2 in the plasma membrane
nor of any other facilitated diffusion mechanism for glucose. This
finding led them to propose an alternative mechanism for hepatic
glucose output based on a membrane-traffic mechanism. Indeed this group
has proposed the existence of two pathways for glucose release, one
temperature sensitive and one relying on diffusion of glucose through
the plasma membrane transporter GLUT2. On the basis of the effects of
glucagon presented in the present work, substantial activation of
gluconeogenesis downstream of glucose 6-phosphate, lack of effect on
glucose-6-phosphatase, and suppression of the activation at low
temperature (contrasting with the lack of effect of temperature on
pyruvate kinase inhibition), we would like to propose that glucagon
activates the temperature-sensitive pathway. As proposed by Guillam
et al. (15) this pathway could be a direct membrane-traffic
mechanism from the endoplasmic reticulum to the plasma membrane. It is
of interest to note that the GLUT2-deficient mice are hyperglycemic
with markedly elevated plasma glucagon levels (15), a hormonal change
that could be viewed as an adaptive mechanism permitting to compensate
for the impaired glucose release because of the lack of GLUT2.
Furthermore in Fanconi-Bickel disease, where GLUT2 is not functional
because of mutations in both alleles of this gene (45), the patients
increase both glycemia (46) and glycosuria (47) in response to
glucagon, indicating that despite the lack of functional GLUT2, hepatic
glucose production can be stimulated by this hormone. It could be noted
that our data do not exclude other hypothetical mechanisms. For
instance a subtle and fragile activational effect by glucagon on the
catalytic site unit of the glucose-6-phosphatase (48) may occur. But it must be noted that such a hypothetical activation seems not to be
present in vitro, because we failed to find any activation even in permeabilized cells (data not shown), and as judged from our
in vivo data, its extent seems limited. Moreover this effect must also be temperature-dependent. Glucagon may also
affect the auxiliary component of the glucose 6-phosphate system
(auxiliary protein or T1 or glucose 6-phosphate transporter; see Refs.
35, 36, 49, and 50), although the effect of glucagon on glucose production was already observed after a few minutes (data not shown).
It is important to consider that besides a clear effect on flux
(i.e. gluconeogenesis and glycolysis), glucagon
markedly affects the cellular concentration of some metabolites. Hence,
except at very low and very high J(L+P),
i.e. where glucagon has almost no effect on
pyruvate kinase, phosphoenolpyruvate concentration is 2-fold increased
by glucagon for a similar J(L+P) (Fig. 3A). In addition, glucagon was responsible for a 60%
decrease in glucose 6-phosphate concentration in vivo
whereas hepatic glucose production was increased 2-fold, and glucose
concentration was identical. Therefore, we may consider that one of the
major roles of glucagon could be related to its effect on intracellular
signaling molecules such as glucose 6-phosphate (51), as it was
proposed for glucose-6-phosphatase (52). Indeed this metabolite could play a key role as a signaling molecule to transmit the effect of
glucose on gene regulation (12, 53). We have also suggested recently
that glucose 6-phosphate could be a crucial signaling metabolite in the
short term inhibition of hepatic glucose production under the action of
insulin and hyperglycemia (54). In this view, glucagon may act as a
modulator of the liver glucose-sensing mechanism. It has been shown
that liver glucose-6-phosphatase overexpression is responsible for
several features classically associated with diabetes, glucose
intolerance, hyperinsulinemia, and decreased liver glycogen (55).
Glucagon is also known to be significantly increased in diabetes (56,
57). Hence the results herein connecting glucagon and glucose
6-phosphate hydrolysis could represent an important clue in the
understanding and the treatment of the illness.
7
M) was not related to the significant inhibition of
pyruvate kinase but to a dramatic activation of the hydrolysis of
glucose 6-phosphate. We failed to find an acute change in
glucose-6-phosphatase activity by glucagon, but the increase in glucose
6-phosphate hydrolysis was abolished at 21 °C; conversely the effect
on pyruvate kinase was not affected by temperature. The activation of
glucose 6-phosphate hydrolysis by glucagon was confirmed in
vivo, in postabsorptive rats receiving a constant infusion of
glucagon, by the combination of a 2-fold increase in hepatic glucose
production and a 60% decrease in liver glucose 6-phosphate
concentration. Besides the description of a novel effect of glucagon on
glucose 6-phosphate hydrolysis by a temperature-sensitive mechanism,
this finding could represent an important breakthrough in the
understanding of type II diabetes, because glucose 6-phosphate is
proposed to be a key molecule in the transcriptional effect of glucose.
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) (19, 20). To study the
metabolic effect of glucagon, perifused liver cells were titrated with
DHA in the presence or absence of glucagon (107
M). After a first steady state was 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 steady state
was obtained within 20 min after which both perifusate and cell samples
were taken for subsequent analysis. Proteins in the perifusate were denatured by heating the samples (80 °C for 10 min) before
centrifugation (21). Glucose, lactate, and pyruvate were measured in
the perifusate, and DHAP, glucose 6-phosphate, fructose 6-phosphate,
3-phosphoglycerate, and P-enolpyruvate were measured in the
cellular fraction as described previously (20, 22, 23). The net fluxes
(µmol/min/g dry cells) of gluconeogenesis
(Jglucose), glycolysis
(J(L+P)), 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 (24) with either
spectrophotometric or fluorometric determination of NADH.
7 M), and samples were taken from the
chamber after 5, 10, 15, 20, and 30 min. The samples of cell suspension
were quickly removed from the chamber, and cellular content was
separated from the extracellular medium by centrifugation through a
layer of silicone oil as described previously (25). The mitochondrial
fraction was obtained by liver cell fractionation with digitonin as
described (26). Cytosolic adenine nucleotides were calculated by
subtraction of the mitochondrial from the total intracellular value.
ATP, ADP, and AMP were determined by HPLC (25).
7 M) and at 15, 21, or 37 °C
as indicated. After 30 min, 500 µl of cell samples were taken from
the vials, and pyruvate kinase activity was assessed on cell pellets
resuspended in 1.5 ml of a buffer containing 20 mmol/liter potassium
phosphate (pH 7.4), 0.25 mol/liter sucrose, 1 mmol/liter EDTA, 1 mmol/liter dithiothreitol. After homogenization for 1 min with an
Ultraturax, the sample was centrifuged at 30,000 × g
for 15 min (Beckman J 21). Pyruvate kinase activity in the supernatant
was determined in a solution of 2 ml of buffer containing 50 mM TRIS-HCl (pH 7.4), 20 mM KCl, 5 mM MgCl2, and 10 µl of the
supernatant. Partially purified enzyme (L form) was obtained from 0.4 ml of the supernatant, 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 mmol/liter
potassium phosphate, pH 7.4, 30% glycerol, 1 mmol/liter EDTA, 1 mmol/liter dithiothreitol, 50 mmol/liter NaF, and pyruvate kinase
activity was measured in a buffer containing 50 mM TRIS-HCl
(pH 7.4), 20 mM KCl, 5 mM MgCl2
(27, 28). Enzyme activity was expressed as the ratio of activity
measured in 0.4 mmol/liter P-enolpyruvate to that of 4 mmol/liter
P-enolpyruvate (v/Vmax), because this ratio has
been shown to accurately reflect the phosphorylated state of the enzyme
(29). Glucose-6-phosphatase was determined in similar experimental
conditions of liver cells incubation as described for pyruvate kinase
activity at 37 °C. Glucose-6-phosphatase activity was determined
after hypo-osmotic shock and cell disruption by sonication and assayed
as described previously (30). The contribution of nonspecific
phosphatase activity was estimated via the hydrolysis of
-glycerophosphate under the same assay conditions and subtracted
from all measurements.
196 °C), weighed, and stored at 80 °C. Blood was collected
for the determination of plasma glucose and glucagon concentration and
the specific activity of glucose. A local ethics committee for animal
experimentation approved this protocol. Glucose-6-phosphatase activity
was assayed at 30 °C and pH 7.3 in liver homogenates obtained from
freeze-clamped samples as described previously (30). Glucokinase was
assayed in 12,000-g supernatants of liver homogenates at 30 °C and
pH 7.3, as described by Bontemps et al. (31). Glucose
6-phosphate concentration was determined as described by Lang and
Michal (32) and glycogen content as described by Keppler and Decker
(33). Plasma glucose concentration was determined as indicated above.
Hepatic glucose production was assessed from
[3-3H]glucose dilution. A bolus (88.8 kBq) was infused
during the first minute, and [3-3H]glucose was then
infused at 8.88 kBq/min. Plasma [3-3H]glucose
radioactivity was measured in triplicate in the supernatants after
ZnSO4 and BaOH2 treatment and evaporation to
eliminate 3H2O. A steady state glucose-specific
activity was obtained during the final 60 min of the experiment in rats
infused with either saline or glucagon. The rate of disappearance of
glucose, which equals the rate of glucose appearance in a steady state,
was calculated by dividing the [3-3H]glucose infusion
rate by the isotopic enrichment of plasma glucose. Hepatic glucose
production was obtained from the rate of glucose appearance.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effect of glucagon 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 at
37 °C with 9.6 mM DHA. Flow rate of perifusate was 5 ml·min 1 (Krebs-Ringer bicarbonate buffer, pH 7.4)
continuously gassed with 95% O2 5% CO2.
Glucagon (107 M) was continuously infused in
one chamber (
) compared with a control chamber (
). After a first
steady state has been reached in absence of exogenous substrate,
corresponding to the endogenous rates, 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. DHA metabolism
(JDHA; A and C) was
calculated from the glucose, lactate, and pyruvate concentrations in
the perifusate; JDHA = flow rate × ([glucose] × 2 + [lactate] + [pyruvate]). At each steady state,
0.5-ml samples of cell suspension were removed from the chamber and
centrifuged. Intracellular DHAP concentration (B and
C) was measured in the neutralized cell fraction.
Panel A, effect of glucagon on DHA metabolism; panel
B, effect of glucagon on steady state concentration of DHAP, first
intermediate of the pathway; panel C, effect of glucagon on
DHA metabolism downstream of DHAP. Results are expressed as the
mean ± S.E.; n = 4 per group.
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Fig. 2.
Inhibition of glycolysis from DHA by glucagon
in hepatocytes perifused. Hepatocytes (200 mg of dry cells in 15 ml) were perifused as described for Fig. 1 with ( ) or without ()
glucagon (107 M). Panel A,
relationship between the rates of glycolysis
(J(L+P)) calculated from lactate and pyruvate
concentrations in the perifusate and steady state concentrations of
infused DHA. Results are expressed as the mean ± S.E.;
n = 4 per group. Panel B, Lineweaver-Burk
(double-reciprocal) representation of the relationship between DHA
concentration and the flux through the glycolytic pathway.
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Fig. 3.
Effect of glucagon on pyruvate kinase in
intact perifused liver cells. Hepatocytes (200 mg of dry cells in
15 ml) were perifused as described for Fig. 1 with ( ) or without
() glucagon (107 M). Panel A,
relationship between the rates of glycolysis
(J(L+P)) and intracellular phosphoenolpyruvate
concentrations; panel B, relationship between the rates of
glycolysis (J(L+P)) and 3-phosphoglycerate
concentrations; panel C, relationship between
phosphoenolpyruvate and 3-phosphoglycerate concentrations.
Intracellular phosphoenolpyruvate and 3-phosphoglycerate concentrations
were measured in the neutralized cell fractions. Results are expressed
as the mean ± S.E.; n = 4 per group.
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Fig. 4.
Lack of effect of glucagon on glycolytic
metabolism of DHAP and on the lactate to pyruvate ratio.
Hepatocytes (200 mg of dry cells in 15 ml) were perifused as described
for Fig. 1 with ( ) or without () glucagon (107
M). The rates of glycolysis (J(L+P))
and the lactate to pyruvate ratios (lactate/pyruvate) were calculated
from lactate and pyruvate concentrations in the perifusate. Panel
A, relationship between rates glycolysis
(J(L+P)) and intracellular DHAP concentrations;
panel B, relationship between intracellular concentrations
of P-enolpyruvate and DHAP investigating the effect of glucagon on the
pathway between these two intermediates; panel C,
relationship between lactate to pyruvate ratios (as indicator of
cytosolic redox state) and the rates of glycolysis. Intracellular DHAP
and P-enolpyruvate concentrations were measured in the neutralized cell
fractions. Results are expressed as the mean ± S.E.;
n = 4 in each group.
Effect of glucagon on adenine nucleotides in isolated perifused
hepatocytes
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Fig. 5.
Effect of glucagon on gluconeogenesis in
isolated hepatocytes perifused with DHA. Hepatocytes (200 mg of
dry cells in 15 ml) were perifused as described for Fig. 1 with ( )
or without () glucagon (107 M).
Panel A, effect of glucagon on the gluconeogenesis from DHA.
The rate of gluconeogenesis (Jglucose) was
calculated from glucose concentration in the perifusate; panel
B, effect of glucagon on gluconeogenesis downstream of DHAP;
panel C, linear relationship between fructose 6-phosphate
and glucose 6-phosphate; panel D, effect of glucagon on
glucose 6-phosphate hydrolysis. Intracellular dihydroxyacetone
phosphate, fructose 6-phosphate, and glucose 6-phosphate were measured
in the neutralized cell fractions. Results are expressed as the
means ± S.E.; n = 4 per group.
Effect of glucagon on glucose and (L+P) production from DHA and on
pyruvate kinase allosteric inhibition at different temperatures
7 M). Temperature of
the incubation was 15, 21, or 37 °C as indicated. After 30 min, 500 µl of cell samples were taken from the vials and treated for pyruvate
kinase activity as described under "Materials and Methods." At the
same time, the rates of glycolysis (J(L+P)) and
gluconeogenesis (Jglucose) were calculated from
lactate, pyruvate, and glucose accumulations in the cell suspensions.
Results are expressed as the mean ± S.E. Statistical comparisons
between controls and glucagon were made using a Student's t
test; *, p < 0.05.
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Fig. 6.
Effect of glucagon on gluconeogenesis at low
temperature. Hepatocytes (200 mg of dry cells in 15 ml) were
perifused as described for Fig. 1 with ( ) or without () glucagon
(107 M) except for the temperature of the
perifusate, which was 21 °C. Panel A, effect of glucagon
on the gluconeogenesis from DHA; panel B, effect of glucagon
on gluconeogenesis downstream of DHAP; panel C, effect of
glucagon on the relationship between fructose 6-phosphate and glucose
production; panel D, effect of glucagon on the relationship
between glucose 6-phosphate and glucose production; panel E,
effect of glucagon on the rate of glycolysis
(J(L+P)). Intracellular dihydroxyacetone
phosphate, fructose 6-phosphate, and glucose 6-phosphate were measured
in the neutralized cell fractions. Results are expressed as the
means ± S.E.; n = 3 per group.
In vivo effects of glucagon on rat liver glucose metabolism
80 °C. Glycogen, glucose 6-phosphate (G6P), glucose-6-phosphatase
(G6Pase), and glukokinase were assayed in 12,000-g supernatants of
centrifuged liver homogenates. The results are expressed as enzymatic
units; 1 unit is the amount of enzyme that converts 1 µmol of
substrate per min under the conditions of the assay. Statistical
analysis was performed by analysis of variance, and two-tailed unpaired
Student's t test was used for post-hoc analysis.
NS, not significant.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
|---|
* This work was supported in part by the Ministère de l'Enseignement, de la Recherche et de la Technologie and by the University and Centre Hospitalier Universitaire (CHU) of Nice (to C. I.) and by a grant from Merck-Lipha (to B. G.).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 06006 Nice, France.
On leave from the Departamento de Fisiologia y Farmacologia,
Facultad de Farmacia, Universidad de Salamanca, Salamanca E-37007, Spain.
** To whom correspondence should be addressed: Laboratoire de Bioénergétique Fondamentale et Appliquée, Université Joseph Fourier, BP 53X, 38041 Grenoble, Cedex, France. Tel.: 33-4-76-51-43-86; Fax: 33-4-76-51-42-18; E-mail: Xavier.Leverve@ujf-grenoble.fr.
Published, JBC Papers in Press, May 22, 2001, DOI 10.1074/jbc.M010186200
| |
ABBREVIATIONS |
|---|
The abbreviations used are: DHA, dihydroxyacetone; J, metabolic flux (µmol/min/g of dry cells); (L+P), lactate plus pyruvate; DHAP, dihydroxyacetone phosphate; P-enolpyruvate, phosphoenolpyruvate; phosphoglycerate kinase, EC 2.7.2.3; enolase, EC 4.2.1.11; glucose-6-phosphatase, EC 3.1.3.9; glucose-6-phosphate 1-dehydrogenase, EC 1.1.1.49; glyceraldehyde 3-phosphate dehydrogenase, EC 1.2.1.12; glycerokinase, EC 2.7.1.30; glycerol-3-phosphate dehydrogenase, EC 1.1.1.8; L-lactate dehydrogenase, EC 1.1.1.27; phosphoenolpyruvate carboxykinase, EC 4.1.1.32; phosphoglucoisomerase, EC 5.3.1.9; phosphoglycerate mutase, EC 5.4.2.1; pyruvate kinase, EC 2.7.1.40; HPLC, high pressure liquid chromatography; ANOVA, analysis of variance.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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