 |
INTRODUCTION |
In 1997, Scruel et al. (1) first revealed a
glucose-induced positive cooperativity of D-fructose
phosphorylation by human B-cell glucokinase. In 2001, Moukil and Van
Schaftingen (2) analyzed the cooperativity of human B-cell glucokinase
through such a stimulatory effect of D-glucose on
D-fructose phosphorylation. They concluded that the effect
of the aldohexose on D-fructose phosphorylation indeed
reflects the positive cooperativity for D-glucose, as
mediated by its binding to the catalytic site. Further experiments
conducted in isolated rat pancreatic islets have documented that
D-glucose also causes a concentration-related increase in the oxidation of D-[U-14C]fructose (3). A
comparable situation was observed in pancreatic islets prepared from
either Goto-Kakizaki rats or adult rats that had been injected with
streptozotocin during the neonatal period, i.e. in two
animal models of non-insulin-dependent diabetes mellitus (4, 5).
The major aim of the present study was to investigate whether the
stimulatory effect of D-glucose on D-fructose
phosphorylation by human liver glucokinase displays anomeric
specificity. The effects of the two anomers of D-glucose
upon D-[U-14C]fructose conversion to
14CO2 and 14C-labeled acidic
metabolites and upon the cationic and insulin secretory responses to
D-fructose were also examined in isolated rat pancreatic
islets. The experiments were conducted over 10 min of incubation at
25 °C (D-fructose phosphorylation), 60 min of incubation
at 4 °C (D-fructose metabolism in islets) or with D-glucose anomers maintained for 90 min or less at 4 °C
(perifused islets) to minimize the interconversion of the
glucose anomers (6). Under these conditions, the fraction
of
-D-glucose converted to
-D-glucose,
expressed relative to the equilibrium value, is close to 5.4 and 9.0%
after 60 and 90 min of incubation at 4 °C and to 17.8% after 10 min
incubation at 25 °C (6). The mean value for the fractional
conversion of each anomer during the incubation period used for the
measurement of biological variables is close to only half of these percentages.
 |
EXPERIMENTAL PROCEDURES |
Materials--
D-Fructose and the anomers of
D-glucose were purchased from Sigma.
D-[U-14C]fructose was prepared from
D-[U-14C]glucose (PerkinElmer Life Sciences),
and its purity assessed as previously reported (7). Recombinant liver
glucokinase was kindly provided by Prof. E. Van Schaftingen
(Université Catholique de Louvain, Brussels, Belgium).
D-[U-14C]Fructose
Phosphorylation--
The phosphorylation of D-fructose (10 mM; mixed with a tracer amount of
D-[U-14C]fructose) was conducted over 10 min
of incubation at 25 °C in a reaction mixture (0.1 ml) consisting of
a Hepes-NaOH buffer (50 mM, pH 7.5) containing 6 mM MgCl2, 60 mM KCl, 10 mM KH2PO4, 0.2 mg/ml bovine serum
albumin, 5 mM ATP, human liver glucokinase (about 25 µg/ml or 0.3 units/ml) and, when required, freshly dissolved
- or
-D-glucose. Labeled D-fructose 6-phosphate
was then separated from its precursor by ion exchange chromatography
(8). Blank values were measured in the absence of glucokinase.
Metabolism of D-[U-14C]Fructose in
Pancreatic Islets--
Groups of 30 pancreatic islets each, prepared
by the collagenase procedure from fed Wistar rats (9), were incubated
for 60 min at 4 °C in 0.1 ml of a Hepes- and bicarbonate-buffered medium (10) containing bovine serum albumin (5 mg/ml),
D-fructose (5 mM, mixed with a tracer amount of
D-[U-14C]fructose) and, when required,
freshly dissolved
- or
-D-glucose (5.6 mM). The production of 14CO2 and
14C-labeled acidic metabolites was measured as described
elsewhere (11, 12).
45Ca Efflux and Insulin Release from Perifused
Pancreatic Islets--
Groups of 110 pancreatic islets each were
preincubated for 60 min at 37 °C in the same Hepes- and
bicarbonate-buffered medium as mentioned above in the presence of
D-glucose (16.7 mM), this medium being enriched
with a tracer amount of 45CaCl2. The islets
were then placed in a perifusion chamber. The perifusion media
containing either
-D-glucose or
-D-glucose (5.6 mM each) and, when required,
D-fructose (20.0 mM) were maintained at 4 °C
in a container placed on ice and warmed to 37 °C just before
reaching the perifusion chamber. The efflux of 45calcium
(expressed as a fractional outflow rate) and release of insulin were
measured as previously described (13).
Presentation of Results--
All results are presented as mean
values (± S.E.) together with the number of individual observations
(n). The statistical significance of differences between
mean values was assessed by use of Student's t test.
 |
RESULTS |
Enzymatic Data--
Two batches of recombinant human liver
glucokinase were used. Over 10 min of incubation at 25 °C, they
yielded for the phosphorylation of
D-[U-14C]fructose (10.0 mM) mean
reaction velocities of 122 ± 3 (n = 3) and
163 ± 11 (n = 8) nmol/min per mg of protein.
In two pilot experiments, D-glucose, tested at anomeric
equilibrium, caused a concentration-related increase in
D-[U-14C]fructose phosphorylation (Fig.
1). As little as 0.25 mM
D-glucose increased the reaction velocity by 43 ± 1%. The peak value for D-[U-14C]fructose
phosphorylation was reached at a 5.0 mM concentration of
D-glucose and corresponded to a 6-fold increase in reaction velocity. At higher concentrations of D-glucose, the
reaction velocity progressively decreased. The value reached at 20.0 mM D-glucose averaged 63 ± 4% of that
recorded, within the same experiment(s), at 5.0 mM
D-glucose.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 1.
Effect of increasing concentrations of
D-glucose, tested at anomeric equilibrium, upon
the phosphorylation of
D-[U-14C]fructose (10.0 mM) by human liver glucokinase. Mean values
(± range of individual variations) are expressed relative to the mean
reaction velocity recorded in the absence of D-glucose,
after normalization of the data (14) within each of two individual
experiments. The inset refers to the results obtained in the low range
of D-glucose concentrations (0.25 to 2.0 mM).
|
|
In further experiments, the effects of
-D-glucose and
-D-glucose upon
D-[U-14C]fructose phosphorylation were
compared, distinct ranges of the anomer concentrations being explored
in each case. In a first series of five experiments conducted at anomer
concentrations of 0.25-2.0 mM, a progressive increase in
reaction velocity was observed with each anomer. The stimulation of
D-[U-14C]fructose phosphorylation was much
more pronounced, however, with
-D-glucose than
-D-glucose (Fig. 2,
left panel). For instance, at the lowest anomer
concentration tested in these experiments (i.e. 0.25 mM), the reaction velocity, expressed relative to the basal
value (no D-glucose present) recorded within the same
experiment(s), averaged 189 ± 9 and 121 ± 7%
(n = 5 in both cases; p < 0.001) with
-D-glucose and
-D-glucose, respectively.
At an anomer concentration of 2.0 mM, these values were
increased to 557 ± 24 and 271 ± 14% (n = 8 in both cases; p < 0.001) with
-D-glucose and
-D-glucose.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 2.
Effects of increasing concentrations of
- or
-D-glucose upon the
phosphorylation of
D-[U-14C]fructose (10.0 mM) by human liver glucokinase. Mean values
(± S.E.) are expressed relative to the reaction velocity recorded
within the same experiment(s) in the absence of D-glucose
and refer to 5-8 (left panel) or three (right
panel) individual measurements.
|
|
In a second series of four experiments conducted at higher
concentrations of the two anomers (2.0-2.5 to 10.0-20.0
mM), the peak value for
D-[U-14C]fructose phosphorylation was reached
at a concentration of
-D-glucose of 4.7 ± 0.3 mM, as distinct (p < 0.001) from 9.4 ± 0.6 mM in the case of
-D-glucose
(n = 4 in both cases). The peak value reached with the
-anomer was no greater than 68 ± 3% of the peak value
recorded with the
-anomer but at half the concentration within the
same experiments. In these and further experiments, the phosphorylation
of D-[U-14C]fructose was virtually the same
with
- and
-D-glucose when the anomers were tested in
the 10.0-20.0 mM range (Fig. 2, right panel).
In the last set of experiments, the effects of
-D-glucose,
-D-glucose and
D-glucose at anomeric equilibrium upon
D-[U-14C]fructose phosphorylation were
compared at three concentrations of the aldohexose (Table
I). At D-glucose
concentrations of 2.0 and 5.0 mM, the results obtained with
equilibrated D-glucose were always in between those
recorded with the pure anomers. At 10.0 mM, however, the
reaction velocity measured in the presence of equilibrated
D-glucose always exceeded that found with each anomer. The
experimental values found with equilibrated D-glucose were about the same as the corresponding theoretical values calculated from
both the relative abundance of
-D-glucose (36.5%) and
-D-glucose (63.5%) at anomeric equilibrium, and the
reaction velocities measured in the presence of each anomer. In
calculating these theoretical values, allowance was made for the
increase or decrease in reaction velocity found at increasing
concentrations of
-D-glucose (as illustrated in Fig. 2),
the measurements made in the presence of
-D-glucose
being converted to corresponding molar amounts of
-D-glucose. The ratio between theoretical and
experimental values for D-[U-14C]fructose
phosphorylation in the presence of equilibrated D-glucose averaged 103 ± 2% (n = 9) and, as such, was not
significantly different from unity (p > 0.2). Fig.
3 illustrates the comparison between
these theoretical and experimental values. These findings document that
the results recorded with equilibrated D-glucose were not
significantly different from those expected from the combined effects
of each anomer upon D-[U-14C]fructose
phosphorylation.
View this table:
[in this window]
[in a new window]
|
Table I
Effects of - or -D-glucose and equilibrated
D-glucose upon the phosphorylation of
D-[U-14C]fructose (10.0 mM) by human
liver glucokinase
|
|

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 3.
Comparison between theoretical and
experimental values for the phosphorylation of
D-[U-14C]fructose in
the presence of increasing concentrations (2.0, 5.0, and 10.0 mM) of equilibrated
D-glucose. The experimental values were
collected in the experiments summarized in Table I. The
solid and dashed lines correspond, respectively,
to the calculated regression line and a line defining identity between
theoretical and experimental values. All results are expressed relative
to the reaction velocity found in the absence of
D-glucose.
|
|
Metabolic Data--
When groups of 30 isolated pancreatic islets
each were incubated for 60 min at 4 °C,
-D-glucose
(5.6 mM) and, to a lesser extent,
-D-glucose
(also 5.6 mM) increased the mean value for the conversion
of D-[U-14C]fructose (5.0 mM) to
14CO2 (Fig. 4,
upper panel). None of the mean values for
D-[U-14C]fructose oxidation illustrated in
Fig. 4 were significantly different from one another, however. Such was
also the case when groups of 50 islets each were incubated for only 30 min at 25 °C under otherwise the same experimental conditions (data
not shown).

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 4.
Effects of
-D-glucose and
-D-glucose (5.6 mM each) upon the conversion of
D-[U-14C]fructose (5.0 mM) to
14CO2 (upper
panel) and 14C-labeled acidic
metabolites (lower panel) in islets incubated for 60 min at 4 °C. Mean values (± S.E.) are expressed as pmol of
D-[U-14C]fructose equivalent per islet and
refer to the number of individual determinations indicated at the
bottom of each column.
|
|
In the experiments conducted at 4 °C, no significant production of
14C-labeled acidic metabolites could be detected from
islets incubated in the sole presence of
D-[U-14C]fructose (Fig. 4, lower
panel). Once again,
-D-glucose and, to a lesser
extent,
-D-glucose augmented the mean value for the conversion of D-[U-14C]fructose to
radioactive acidic metabolites. Such an increase failed to achieve
statistical significance in the case of
-D-glucose but
was highly significant (p < 0.01) in the case of
-D-glucose. The increment in the net generation of
14C-labeled acid metabolites caused by
-D-glucose represented 37.9 ± 18.2%
(n = 15; p < 0.07) of the mean
corresponding value found within the same experiment(s) in the case of
-D-glucose (100.0 ± 27.6%; n = 14).
Secretory and Cationic Data--
In pilot experiments conducted
over 30 min of incubation at 37 °C, neither equilibrated
D-glucose (5.6 mM) nor D-fructose (20.0 mM), when tested separately from one another,
augmented significantly (p > 0.5 or more) insulin
output above basal value (7.3 ± 1.2 microunit/islet per 30 min;
n = 29). Thus, the secretory rate averaged 7.8 ± 1.5 and 8.3 ± 1.2 microunit/islet per 30 min (n = 27-28) in the presence of 5.6 mM D-glucose and
20.0 mM D-fructose, respectively. In the
concomitant presence of the aldohexose and ketohexose, however, the
output of insulin (12.7 ± 1.5 microunit/islet per 30 min;
n = 29) reached a value significantly higher than the
basal insulin release (p < 0.01) or that recorded in
the sole presence of either D-glucose (p < 0.025) or D-fructose (p < 0.05).
To investigate the functional response of isolated islets to
D-fructose in the presence of either
-D-glucose or
-D-glucose, groups of 110 islets each were first preincubated for 60 min at 37 °C in the
presence of 16.7 mM equilibrated D-glucose in a
medium enriched with 45CaCl2 and then placed in
a perifusion chamber. The perifusion media containing either
-D-glucose or
-D-glucose (5.6 mM each) and, when required, D-fructose (20 mM) were maintained at about 4 °C (in a container placed
in ice) and warmed to 37 °C just before reaching the perifusion chamber.
As documented in the upper panel of Fig.
5, the mean value for 45Ca
fractional output rate before administration of
D-fructose (min 31 to 45) was higher in the islets
exposed to
-D-glucose (0.62 ± 0.09 10
2/min; n = 4) as compared with
-D-glucose (0.49 ± 0.04 10
2/min;
n = 4); this difference failed, however, to achieve
statistical significance. D-fructose exerted opposite
effects upon 45Ca outflow in the islets exposed to either
-D-glucose or
-D-glucose. In the former
case, D-fructose provoked a sustained increase in 45Ca outflow. Thus, the integrated value for
45Ca fractional output rate was, during the period of
D-fructose administration (min 46-70) 814 ± 321 10
6/min higher (n = 4; p < 0.08) than the theoretical values calculated by exponential
extrapolation (see below) between the 15 measurements made before
introduction of the ketohexose (min 31-45) and the five last
measurements made 16-20 min after halting its administration (min
86-90). This corresponded to a 17.0 ± 6.9% increase in effluent radioactivity, relative to the paired theoretical value. On the contrary, in the presence of
-D-glucose, the integrated
value for 45Ca fractional output rate was, over the same
period (min 46-70), 787 ± 174 10
6/min lower
(n = 4; p < 0.025) than the
theoretical values, this corresponding to a 18.5 ± 3.5% relative
decrease in effluent radioactivity. The difference between the response
to D-fructose in the presence of
-D-glucose
versus
-D-glucose was thus highly significant (p < 0.005), whether judged from the absolute or
relative magnitude of the changes in effluent radioactivity.

View larger version (41K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of D-fructose
(20.0 mM) administered from min 46-70 upon
45Ca fractional outflow rate (upper
panel) and insulin release (lower panel)
from islets perifused in the presence of 5.6 mM
-D-glucose (open
circles) or
-D-glucose (closed
circles). Mean values (± S.E.) refer to four
(upper panel) or 12 (lower panel) individual
experiments.
|
|
As illustrated in the lower panel of Fig. 5, the output of
insulin before introduction of D-fructose (min 31-45) was
slightly higher (p < 0.05) in the islets exposed to
-D-glucose (490 ± 83 nanounit/min per
islet; n = 12) rather than
-D-glucose
(291 ± 38 nanounit/min per islet; n = 12). The
increment in insulin output provoked by D-fructose (min
46-70) above the control values (min 42-45) was much higher
(p < 0.01) in the experiments conducted in the
presence of
-D-glucose (607 ± 102 nanounit/min per
islet) rather than
-D-glucose (264 ± 63 nanounit/min per islet). As a matter of fact, D-fructose
provoked, in the presence of
-D-glucose, a typical
biphasic secretory response (early peak followed by a later reascension
in insulin output), this contrasting with a modest and sluggish
increase in insulin release in the presence of
-D-glucose. As already observed in a prior study (14),
the stimulation of insulin release by D-fructose was
apparently not rapidly reversible. Thus, between min 71 and 90, the
output of insulin remained significantly higher (p < 0.001) than the control value (min 42-45), the increment above such a
control value averaging 889 ± 196 and 444 ± 86 nanounit/min
per islet (n = 12 in both cases; p < 0.05) in the case of
- and
-D-glucose, respectively. Incidentally, even when the theoretical control curve for insulin output was calculated according to an exponential equation
(y = b × mx,
equation in which y and x refer to the output of
insulin and time, respectively, and b and m to
constants) and took into account both the eight first measurements made
between min 31-45 and the last four measurements made between min
84-90, the increment in insulin output attributable to the
administration of D-fructose (min 46-70) remained higher
(p < 0.05) in the presence of
-D-glucose (448 ± 119 nanounit/min per islet) than
in the presence of
-D-glucose (152 ± 62 nanounit/min per islet).
 |
DISCUSSION |
The experimental conditions used in this study to assess the
anomeric specificity of the effect of D-glucose upon the
phosphorylation, metabolism, and insulinotropic action of
D-fructose were selected to minimize the interconversion of
the D-glucose anomers (6).
In the absence of D-glucose, the phosphorylation of
D-fructose by glucokinase does not display positive
cooperativity (1), the apparent Km for the
ketohexose being close to 160 mM (15). The present data
extend to human liver glucokinase the knowledge that
D-glucose stimulates the phosphorylation of D-fructose, as previously documented in the case of human
B-cell glucokinase (1, 2). Such a stimulation is much more marked in
the case of
-D-glucose than
-D-glucose.
This anomeric specificity disappeared, however, at high concentrations
of the D-glucose anomers, i.e. when the
glucose-induced increase in D-fructose phosphorylation
became itself progressively less marked.
The metabolic data collected from islets incubated for 60 min at
4 °C are compatible with the view that the anomeric specificity of
the enhancing action of D-glucose upon
D-fructose phosphorylation, as documented in the
experiments conducted in the presence of human liver glucokinase, is
also operative in intact pancreatic islets. More convincingly, the
present results indicate that the effect of D-fructose upon
both 45Ca efflux and insulin release from prelabeled and
perifused islets is different at 37 °C in the islets exposed to
-D-glucose versus
-D-glucose.
In this respect, the cationic and secretory response to
D-fructose recorded in islets exposed to
-D-glucose are comparable to those otherwise observed
when the concentration of D-glucose is raised from a level
close to the threshold value for stimulation of insulin release
(i.e. close to 5 mM) to a much higher
concentration. They indeed consisted in an increase in 45Ca
efflux corresponding to the stimulation of
40Ca2+ influx into the islets and subsequent
increase in 45Ca efflux and a biphasic stimulation of
insulin release. In the islets exposed to
-D-glucose,
however, the prevailing cationic effect of D-fructose
consisted in a decrease in 45Ca efflux, as otherwise
observed in response to the administration of equilibrated
D-glucose in a concentration not exceeding 7 mM (16) and as attributable to the effects of the hexose upon both the
sequestration of Ca2+ by intracellular organelles and
Na+-Ca2+ countertransport at the level of the
B-cell plasma membrane (17, 18). This coincided with a delayed and
quite modest increase in insulin output. This interpretation of the
experiment results is further supported by the fact that, before
introduction of D-fructose, the release of insulin was
somewhat higher in the presence of
-D-glucose rather
than
-D-glucose.
In conclusion, therefore, the present data unambiguously document that
the enhancing effect of D-glucose upon
D-fructose phosphorylation by glucokinase displays anomeric
preference toward
-D-glucose and that such an anomeric
specificity remains operative in intact rat pancreatic islets.