Insulinotropic action of
-D-glucose pentaacetate:
functional aspects
Willy J.
Malaisse,
Carmen
Sánchez-Soto,
M. Elena
Larrieta,
Marcia
Hiriart,
Hassan
Jijakli,
Concepción
Viñambres,
María L.
Villanueva-Peñacarrillo,
Isabel
Valverde,
Ole
Kirk,
Marcel M.
Kadiata, and
Abdullah
Sener
Laboratory of Experimental Medicine, Brussels Free University,
B-1070 Brussels, Belgium; Department of Biophysics, Institute of
Cellular Physiology, Universidad Nacional Autónoma de
México, Mexico City, DF-04510 Mexico City, Mexico;
Fundación Jiménez Díaz, 28040 Madrid, Spain;
and Novo Nordisk, DK-2880 Bagsvaerd, Denmark
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ABSTRACT |
The functional determinants of the
insulinotropic action of
-D-glucose pentaacetate were
investigated in rat pancreatic islets. The ester mimicked the effect of
nutrient secretagogues by recruiting individual B cells into an active
secretory state, stimulating proinsulin biosynthesis, inhibiting
86Rb outflow, and augmenting
45Ca efflux from prelabeled
islets. The secretory response to the ester was suppressed in the
absence of Ca2+ and potentiated by
theophylline or cytochalasin B. The generation of acetate from the
ester apparently played a small role in its insulinotropic action. Thus
acetate, methyl acetate, ethyl acetate,
-D-galactose pentaacetate,
and
-D-galactose pentaacetate
all failed to stimulate insulin release. The secretory response to
-D-glucose pentaacetate was
reproduced by
-D-glucose
pentaacetate and, to a lesser extent, by
-L-glucose pentaacetate. It
differed from that evoked by unesterified
D-glucose by its resistance to 3-O-methyl-D-glucose,
D-mannoheptulose, and
2-deoxy-D-glucose. It is
concluded that the insulinotropic action of
-D-glucose pentaacetate,
although linked to the generation of the hexose from its ester, entails
a coupling mechanism that is not identical to that currently implied in
the process of glucose-induced insulin release.
pancreatic islets; insulin release
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INTRODUCTION |
THE PENTAACETATE ESTER of
-D-glucose was recently
reported to stimulate insulin release from rat pancreatic islets and
proposed as a tool for the supply of the sugar to cells by a process
that might not involve the carrier-mediated transport of the hexose across the plasma membrane (9). To provide further evidence to support
this hypothesis and to elucidate the mode of action of
-D-glucose pentaacetate in
islet cells, we have now scrutinized the metabolic, ionic, and
functional aspects of
-D-glucose pentaacetate insulinotropic action. This report deals mainly with the third of these
three themes.
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MATERIALS AND METHODS |
The pentaacetyl esters of
-D-glucose,
-D-glucose, and
-L-glucose, as well as
D-mannoheptulose,
2-deoxy-D-glucose, theophylline, and cytochalasin B, were obtained from Sigma (St. Louis, MO). Acetate
(sodium salt) and
3-O-methyl-D-glucose
were purchased from Merck (Darmstadt, Germany) and Aldrich (Milwaukee,
WI), respectively. The pentaacetyl esters of
-D-galactose and
-D-galactose, methyl acetate,
and ethyl acetate were synthesized by methods described elsewhere (18,
19).
Except if otherwise mentioned, all experiments were conducted in
pancreatic islets isolated by the collagenase method (12) from Wistar
rats.
The procedures used for measuring insulin release from incubated (12)
or perifused (5) islets, for quantification of secretory activity of
isolated B cells in a reverse hemolytic plaque assay (6), to assess
biosynthetic activity in islets exposed to
L-[4-3H]phenylalanine
(4), and to monitor 86Rb (3) and
45Ca (5) efflux from prelabeled
islets were identical to those described in the cited references. In
the chromatographic procedure used to separate tritiated peptides in
islet homogenates, the site of elution of nonhormonal peptides,
proinsulin, and insulin corresponds, respectively, to fractions
3-10, 10-16, and 16-30 (see Fig. 3).
All results are expressed as means ± SE together with the number of
individual observations (n) or
degree of freedom (df). The statistical significance of differences
between mean values was assessed by either analysis of variance or
Student's t-test. Such comparisons
are restricted to data collected in close to equal numbers within the
same experiments. Each individual measurement was made in a separate
group of islets.
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RESULTS |
Insulinotropic action of
-D-glucose
pentaacetate.
At a concentration of 1.7 mM, which is close to the limit of solubility
of the ester,
-D-glucose
pentaacetate caused a three- to fourfold increase of basal
insulin release (Table 1). The secretory
response to the ester was considerably enhanced
(P < 0.001) by either theophylline
(1.4 mM) or cytochalasin B (0.02 mM), although neither the
phosphodiesterase inhibitor nor the mold metabolite affects basal
insulin secretion (2, 13). The insulinotropic action of
-D-glucose pentaacetate was
suppressed in the absence of extracellular
Ca2+, the basal release of insulin
being higher (P < 0.001) in
Ca2+-deprived islets than at
normal extracellular Ca2+
concentration.
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Table 1.
Modulation of secretory response to hexose esters by extracellular
Ca2+ concentration, theophylline, and cytochalasin B
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When
-D-glucose pentaacetate
(1.7 mM) was tested in islets exposed to increasing concentrations of
D-glucose, the measurements of
insulin output were compatible with a shift to the left of the
sigmoidal relationship between secretory rate and hexose concentration, the ester failing to significantly affect
(P > 0.13) the hormonal release at a
high concentration (16.7 mM) of
D-glucose (Fig.
1). As judged from the data recorded in the low range of
hexose concentrations (up to 8.3 mM), the insulinotropic action of 1.7 mM
-D-glucose pentaacetate
was comparable to that of ~5.6 mM
D-glucose.

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Fig. 1.
Effect of increasing concentrations of
D-glucose on insulin release
from islets incubated in absence ( ) or presence of either 1.7 mM
-D-glucose pentaacetate ( )
or 1.7 mM -D-galactose
pentaacetate ( ). Mean values (±SE) refer to 24-71
individual determinations. Solid and dotted lines are drawn in parallel
up to 8.3 mM D-glucose.
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In a reverse hemolytic plaque assay of insulin secretion from
individual B cells,
-D-glucose pentaacetate (1.0 mM) enhanced the percentage of plaque-forming cells by ~80%, doubled
the amount of insulin secreted by the single cells (plaque area), and
caused a fourfold increase of overall insulin secretion from islet
cells incubated in the presence of 5.0 mM
L-leucine (Table
2; P < 0.001 in all
cases). Neither D-glucose nor
-D-galactose pentaacetate, tested at the same concentration (1.0 mM) as
-D-glucose pentaacetate, significantly affected the indexes of secretory activity in the B cells
exposed to the branched-chain amino acid, except for a modest increase
of the insulin secretion index (P < 0.05) in cells exposed to the
D-galactose ester.
In islets perifused at normal Ca2+
concentration in the absence of any other exogenous nutrient,
-D-glucose pentaacetate (1.7 mM) provoked a rapid, sustained, and rapidly reversible decrease in
86Rb outflow from perifused
islets, this coinciding with stimulation of both
45Ca efflux and insulin release
(Fig. 2). The latter two effects, but not the decrease
in 86Rb outflow, were suppressed
when the experiments were conducted in the absence of extracellular
Ca2+ (data not
shown).

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Fig. 2.
Effect of -D-glucose
pentaacetate (1.7 mM), administered from
min 46 to
min 70, on
86Rb
(A) and
45Ca
(B) fractional outflow rate (FOR)
and insulin output (C) from
perifused islets. Mean values (±SE) refer to 8 (A and
B) and 15 (C) individual experiments.
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The D-glucose ester also
stimulated biosynthetic activity in islets exposed to
L-[4-3H]phenylalanine
(Table 3).
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Table 3.
Effect of nutrients and D-mannoheptulose on incorporation
of L-[4-3H]phenylalanine into
TCA-precipitable and TCA-soluble material after 90-min incubation in
the presence of tritiated amino acid (4.0 µM)
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In the absence of D-glucose,
-D-glucose pentaacetate (1.7 mM) slightly increased the incorporation of the tritiated amino acid (4 µM) into trichloroacetic acid (TCA)-precipitable material. D-Mannoheptulose (10.0 mM)
failed to affect the biosynthetic, as well as secretory (see below),
response to
-D-glucose
pentaacetate. The former response achieved statistical significance
(P < 0.05) only when the
results obtained in islets exposed to the ester, whether in the absence
or presence of the heptose, were expressed relative to their paired
value recorded in islets deprived of exogenous nutrient, such a paired
comparison yielding a mean ratio of 125.7 ± 12.4%
(n = 24). In the presence of 4.2 mM
D-glucose, which augmented the
synthesis of tritiated peptides relative to basal value
(P < 0.001),
-D-glucose pentaacetate
further increased (P < 0.001)
biosynthetic activity. The incorporation of
L-[4-3H]phenylalanine
into TCA-precipitable material observed in the presence of both 4.2 mM
D-glucose and 1.7 mM
-D-glucose pentaacetate was
comparable to that otherwise found in the sole presence of 7.0 mM
D-glucose; it remained
significantly lower (P < 0.001), however, than that recorded in the presence of 16.7 mM
D-glucose. L-Leucine (10 mM)
tended to decrease the labeling of islet peptides, but this effect
failed to achieve statistical significance as judged by comparison of
either data collected within each experiment or the overall mean values
listed in Table 3. In the presence of the branched-chain amino acid,
-D-glucose pentaacetate
doubled (P < 0.005) the synthesis of
tritiated peptides. As judged from comparisons made within each
experiment, none of the nutrients or combination of nutrients listed in
Table 3 significantly affected either the pool of TCA-soluble
radioactive material or the paired ratio between TCA-precipitable and
total radioactive content of the islets.
To further characterize the influence of
-D-glucose pentaacetate and
other secretagogues on islet biosynthetic activity, the tritiated
peptides were separated by gel chromatography (Fig. 3).
D-Glucose (4.2 and 16.7 mM)
caused a concentration-related increase
(P < 0.05 or less) of the synthesis
of nonhormonal peptides (void volume), proinsulin, and insulin (Table
4). At the highest concentration of the
hexose, the biosynthesis of proinsulin was preferentially stimulated
(P < 0.01), the
(pro)insulin-to-total paired ratio for the labeling of islet peptides
increasing from 30.4 ± 2.2 to 45.7 ± 4.7%
(n = 8 in both cases). The fractional conversion of proinsulin to insulin was not significantly affected, however, by D-glucose, with mean
insulin-to-(pro)insulin paired ratios of 28.2 ± 2.6 and 35.1 ± 3.0% in the absence and presence of 16.7 mM
D-glucose, respectively.
Likewise, L-leucine (10.0 mM)
preferentially stimulated (P < 0.05)
the labeling of hormonal peptides, while failing to significantly
affect the insulin-to-(pro)insulin ratio. In the presence of 4.2 mM
D-glucose,
-D-glucose pentaacetate augmented (P < 0.005) the total
synthesis of (pro)insulin, whereas the increase in the radioactive
content of the void volume failed to achieve statistical significance.
Nevertheless, the ester did not significantly affect the paired
(pro)insulin-to-total ratio for the tritiation of islet peptides. A
comparable situation prevailed in islets exposed to 10.0 mM
L-leucine. The results of the
two series of experiments pooled, the ratio between the results
obtained in the presence or absence of
-D-glucose pentaacetate
within each experiment yielded mean values of 148.2 ± 23.7 (P < 0.05) and 148.2 ± 24.9%
(P < 0.06) for (pro)insulin and the
nonhormonal peptides, respectively (n = 8 in both cases). Unexpectedly, in the presence of either 4.2 mM
D-glucose or 10.0 mM leucine,
-D-glucose pentaacetate
decreased the fractional conversion of proinsulin to insulin, the
insulin-to-(pro)insulin ratio found in the presence of the ester
averaging 71.6 ± 8.6% (n = 8;
P < 0.03) of the paired value
recorded in its absence.

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Fig. 3.
Elution profiles of tritiated peptides separated by gel chromatography
in extracts from islets incubated with
L-[4-3H]phenylalanine
in absence or presence of
L-leucine,
-D-glucose pentaacetate
( DGlcPA), and D-glucose. Mean
values (±SE) refer to 4 individual determinations.
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Table 4.
Effects of nutrients on L-[4-3H]phenylalanine
incorporation into nonhormonal peptides (void volume), proinsulin,
and insulin in two series of experiments
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Taken as a whole, these findings indicate that
-D-glucose pentaacetate acts
on biosynthetic, cationic, and secretory variables in a manner
comparable, albeit not identical, to that characterizing the response
of islet cells to D-glucose and
other nutrient secretagogues.
Concentration-response relationship for the insulinotropic action of
-D-glucose
pentaacetate.
Figure 4 documents the effect of increasing
concentrations of
-D-glucose
pentaacetate on insulin release by islets incubated either in the
absence of any other exogenous nutrient or in the presence of 8.3 mM
D-glucose. In the former
situation, a sizeable increase in insulin output
(P < 0.001) above basal value was
observed in islets exposed to 1.7 mM
-D-glucose pentaacetate, but
lower concentration of the ester (0.17 to 0.85 mM) failed to stimulate hormonal secretion. In the presence of 8.3 mM
D-glucose, however, as little as
0.17 mM
-D-glucose
pentaacetate significantly increased insulin release
(P < 0.005). The
concentration-response relationship suggested that the concentration of
1.7 mM
-D-glucose
pentaacetate, which was used in most further experiments, yielded, in
the presence of 8.3 mM
D-glucose, a close-to-maximal
secretory response.

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Fig. 4.
Effect of increasing concentrations of
-D-glucose pentaacetate on
insulin release from islets incubated in absence (dotted line) or
presence (solid line) of
D-glucose (8.3 mM). Mean values
(±SE) refer to 24 individual observations in all cases.
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Specificity of the insulinotropic action of
-D-glucose
pentaacetate.
The specificity of the secretory response to
-D-glucose pentaacetate was
explored in a double perspective, attempting to assess the
participation of both the acetate and glucose moieties of the ester in
its insulinotropic action.
The effect of acetate, methylacetate, and ethylacetate on insulin
release was first investigated (Table 5). In the absence of any other exogenous nutrient, both acetate and ethylacetate but not
methylacetate, augmented, slightly but significantly
(P < 0.025 or less), basal insulin
output. The secretory rate recorded in the presence of acetate or
methylacetate remained much lower, however, than that found in islets
exposed to
-D-glucose
pentaacetate (1.7 mM), despite the fact that the former nutrients were
used at molar concentrations equal to or higher than the concentration of the acetate moiety of the glucose ester. A comparable situation prevailed when D-glucose was
present in the incubation medium at a 1.7 mM concentration. At a higher
concentration (8.3 mM) of the hexose, neither acetate nor methylacetate
significantly affected insulin release, whereas ethylacetate inhibited
(P < 0.005) glucose-stimulated
hormonal output. Last, in the presence of
L-leucine (10.0 mM), a modest
inhibition of insulin secretion was caused by both acetate
(P < 0.02) and ethylacetate
(P = 0.06) to, respectively, 84.9 ± 3.9 and 87.4 ± 4.7% of the paired control value (100.0 ± 4.6%) found within the same experiments in the sole presence of the
branched-chain amino acid (n = 30 in
all cases).
The second procedure designed to assess the respective contribution of
the glucosidic and acidic parts of
-D-glucose pentaacetate to
its secretory potential relied on a comparison between the insulinotropic action of several hexose pentaacetate esters.
In this perspective, we have first compared the secretory response to
-D-glucose pentaacetate,
-D-glucose pentaacetate, and
-L-glucose pentaacetate
(Table 6). Whether in the
absence of any other exogenous nutrient or in the presence of either
D-glucose (8.3 mM) or
L-leucine (10.0 mM), all three
esters significantly augmented (P < 0.005 or less) insulin release. The
-anomer of D-glucose pentaacetate appeared
slightly less potent than the
-anomer. Such a difference failed,
however, to achieve statistical significance. The
-anomer of
L-glucose pentaacetate was much less potent (P < 0.001) than the
D-eniantomers. As already
mentioned, it nevertheless augmented insulin release significantly. At
variance with these results, unesterified
L-glucose (5.6-55.6 mM)
failed to affect insulin release evoked by either 8.3 mM
D-glucose or 10.0 mM
L-leucine (data not shown).
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Table 5.
Effect of acetate and its esters on insulin release from islets
incubated in the absence or presence of either D-glucose or
L-leucine
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Table 6.
Effect of pentaacetate esters of glucose enantiomers and isomers on
insulin release from islets incubated in absence of any other exogenous
nutrient and in presence of either D-glucose or
L-leucine
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We then compared the insulinotropic action of
-D-glucose pentaacetate to
that of either
- or
-D-galactose pentaacetate (Table 7). The
-anomer of
D-galactose pentaacetate (1.7 mM) failed to affect insulin release when tested in the absence of any
other exogenous nutrient and even decreased insulin output (P < 0.001) in the presence of
L-leucine (10.0 mM). The
-anomer of D-galactose
pentaacetate (1.7 mM) inhibited both basal
(P < 0.001) and leucine-stimulated
(P < 0.01) insulin secretion. It did
not significantly affect insulin release from islets incubated in the
presence of 4.0 mM D-glucose.
Neither
3-O-methyl-D-glucose (80.0 mM) nor D-mannoheptulose
(10.0 mM) significantly affected insulin output from islets exposed to
-D-galactose pentaacetate (1.7 mM). The latter ester failed to significantly affect insulin release from islets incubated in the absence of extracellular Ca2+ (Table 1).
The third approach used to distinguish between the participation of the
acetate and glucose moieties of
-D-glucose pentaacetate to
its insulinotropic action was based on the use of
D-glucose esters in which the
acidic component might display a metabolic efficiency different from
that of acetate.
For this purpose, several esters of
D-glucose, including
-D-glucose pentaisobutyrate,
-D-glucose
pentamethacrylate,
-D-glucose pentaisovalerate,
and
-D-glucose pentabenzoate,
were synthesized, but they could not be tested because of their low
solubility (<0.1 mM, even in the presence of 1.0% dimethyl
sulfoxide). Only
-D-glucose pentapropionate could be used, this ester being tested at an 0.1 mM
concentration (Table 8). Its effect on
insulin release was examined in the presence of 10.0 mM
L-leucine to optimize the detection of its possible insulinotropic action. Under these
experimental conditions, it significantly augmented insulin release
(P < 0.001). As judged by comparison
of results obtained within the same experiments, the increment in
insulin output attributable to
-D-glucose pentapropionate (21.5 ± 4.5 µU · islet
1 · 90 min
1; df = 54) was even
more marked (P < 0.05) than that
caused by
-D-glucose
pentaacetate (8.3 ± 4.3 µU · islet
1 · 90 min
1; df = 55), which at
the low concentration used in these experiments exerted an
insulinotropic action of borderline statistical significance (P < 0.06). The secretory response
to
-D-glucose pentapropionate was unaffected by
D-mannoheptulose (10 mM) and
contrasted with the failure of unesterified
D-glucose, tested at the same
low concentration (0.1 mM), to significantly affect leucine-stimulated insulin release (Table 8).
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Table 8.
Comparison of the secretory effects of
-D-glucose pentaacetate,
-D-glucose pentapropionate, and
D-glucose, all tested in the presence of 10 mM
L-leucine
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Effect of
3-O-methyl-D-glucose.
In the next series of experiments, we explored the possible
interference of agents affecting
D-glucose transport or
metabolism with the insulinotropic action of
-D-glucose pentaacetate. In such a perspective, the effect of
3-O-methyl-D-glucose
was first investigated.
Both basal insulin release and
-D-glucose
pentaacetate-stimulated insulin output were not significantly affected
by
3-O-methyl-D-glucose, tested at a concentration of 80 mM, which is sufficient to inhibit by
>60% insulin secretion evoked by 16.7 mM
D-glucose (Fig.
5). As indicated in Table
9
(experiment
1), at the concentration of 80 mM,
3-O-methyl-D-glucose
failed to affect insulin release evoked by nonglucidic nutrients such
as 2-ketoisocaproate (10 mM) or the association of
L-leucine and
L-glutamine (both 10 mM).

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Fig. 5.
Effect of
3-O-methyl-D-glucose
(3-OMG; 80 mM; striped columns) on insulin release from islets
incubated in absence (basal) or presence of 1.7 mM
-D-glucose pentaacetate
(n = 43-44).
Inset illustrates effect of increasing
concentrations of 3-OMG on insulin release evoked by 16.7 mM
D-glucose
(n = 20 in each case).
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To increase the secretory response to
-D-glucose pentaacetate, the
experiments illustrated in Fig. 5 were repeated in the presence of
other nutrient secretagogues. As shown in Fig.
6, D-glucose (8.3 mM) increased
(P < 0.001) the secretory response to
-D-glucose pentaacetate
(1.7 mM) from a basal value of 14.1 ± 5.5 to 46.4 ± 7.8 µU · islet
1 · 90 min
1 (df = 21-22). The
insulinotropic action of
D-glucose was decreased (P < 0.001) by 20.2 ± 5.5 µU · islet
1 · 90 min
1 (df = 22) when
3-O-methyl-D-glucose
(80 mM) was also present in the incubation medium (Table 9,
experiment
3). The inhibitor of
D-glucose transport apparently
also decreased (P < 0.06) the increment in insulin output attributable to
-D-glucose pentaacetate, from
46.4 ± 7.8 to 26.0 ± 6.6 µU · islet
1 · 90 min
1 (df = 22 in both
cases). However, as illustrated in Fig. 6, the increment in insulin
release attributable to
-D-glucose pentaacetate (1.7 mM) was virtually proportional to the control secretory rate recorded
in its absence, so that the lower increment observed in the presence,
rather than absence, of
3-O-methyl-D-glucose may well merely reflect the decrease of glucose-stimulated insulin release caused by the hexose analog. The experimental data illustrated in Fig. 6 also document that
-D-glucose pentaacetate
remained fully efficient in stimulating insulin release from islets
exposed to 80 mM
3-O-methyl-D-glucose
and stimulated by the association of
D-glucose (10 mM) and
L-leucine (10 mM).

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Fig. 6.
Relationship between increment in insulin release caused by
-D-glucose pentaacetate (1.7 mM) and control output recorded in its absence in islets incubated in
absence of exogenous nutrient (Ø), sole presence of 8.3 mM
D-glucose (G), concomitant
presence of 8.3 mM D-glucose and
80 mM 3-OMG (G + 3-OMG), or simultaneous presence of 10 mM
L-leucine, 10 mM
D-glucose, and 80 mM 3-OMG (L + G + 3-OMG). Mean values (±SE) refer to 11-24 individual
observations. Regression line (dotted line) is compared with that
defining a rule of proportionality between 2 variables (solid line).
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To further explore the effect of
3-O-methyl-D-glucose
on the secretory response to
-D-glucose pentaacetate, the
last two series of experiments were conducted in the presence of
nutrients other than D-glucose.
In the presence of 2-ketoisocaproate (10 mM),
-D-glucose pentaacetate (1.7 mM) augmented insulin release (P < 0.05) modestly to 114.0 ± 4.9% (n = 18) of the mean control value found within the same experiments in
the sole presence of the branched-chain 2-keto acid (100.0 ± 4.4%; n = 18). No
significant effect (P > 0.1 or more)
of
3-O-methyl-D-glucose
(80 mM) on insulin release was observed, whether in the sole presence
of 2-ketoisocaproate or in the concomitant presence of the 2-keto acid
and
-D-glucose pentaacetate
(Table 9, experiment
3). Indeed, the ester still augmented insulin release significantly
(P < 0.01) in the presence of
3-O-methyl-D-glucose.
Likewise,
3-O-methyl-D-glucose
failed to affect (P > 0.8) the
secretory response to L-leucine
and the much higher secretory rate found in the concomitant presence of
the branched-chain amino acid and
-D-glucose pentaacetate
(Table 9, experiment
4).
Taken as a whole, these data strongly suggest that the insulinotropic
action of
-D-glucose
pentaacetate does not involve the transport of the ester across the
plasma membrane by the carrier system otherwise mediating the entry of
D-glucose into the islet B cell.
It is unlikely that the disparity between the insulinotropic action of
D-glucose and that of its ester,
in terms of the sensitivity toward
3-O-methyl-D-glucose,
can be accounted for by a difference in the concentrations of the two
nutrients, such as that prevailing in the experiments illustrated in
Fig. 5. Indeed, the relative extent of the inhibitory action of
3-O-methyl-D-glucose
on insulin release was little affected by the changes in the
concentrations of D-glucose
(Fig. 7). It cannot be ruled out, however,
that
3-O-methyl-D-glucose is less efficient in inhibiting the transport of
-D-glucose pentaacetate, as
distinct from unesterified
D-glucose, by GLUT-2.

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Fig. 7.
A: effect of increasing concentrations
of D-glucose on insulin release
from islets incubated in absence ( ) or presence ( ) of 80 mM 3-OMG
(n = 19-93).
B: effect of increasing concentrations
of 3-OMG on insulin release from islets incubated in presence of either
8.3 ( ) or 16.7 mM ( )
D-glucose
(n = 20 in all cases).
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Effect of D-mannoheptulose.
The second set of experiments dealing with inhibitors of the
insulinotropic action of
D-glucose concerned the effect
of D-mannoheptulose.
Both basal insulin release and
-D-glucose
pentaacetate-stimulated insulin output were not significantly affected
by D-mannoheptulose tested at a
concentration of 10 mM, which is sufficient to abolish insulin
secretion evoked by 16.7 mM
D-glucose (Fig.
8).

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Fig. 8.
Effect of D-mannoheptulose (MH;
10.0 mM; striped columns) on insulin release from islets incubated in
absence (basal) or presence of 1.7 mM
-D-glucose pentaacetate
(n = 24-69).
Inset illustrates effect of increasing
concentrations of MH on insulin release evoked by 16.7 mM
D-glucose
(n = 10-60).
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Even when the islets were both preincubated for 30 min and incubated
for 90 min at 37°C in the presence of
D-mannoheptulose (10 mM), the
secretory response to
-D-glucose pentaacetate (1.7 mM), which was present in the medium solely during the final
incubation, remained not significantly different from that found in
islets both preincubated and incubated in the absence of the heptose. Thus, during the final incubation, the secretory rate averaged 32.6 ± 1.4 and 31.9 ± 1.4 µU · islet
1 · 90 min
1
(n = 27-28) in control and
mannoheptulose-exposed islets, respectively, compared with a basal
value of 13.1 ± 0.8 µU · islet
1 · 90 min
1
(n = 30).
Likewise, in the presence of
L-leucine (10 mM), which was
used to augment the magnitude of the secretory response to
-D-glucose pentaacetate,
D-mannoheptulose (1.0-10.0
mM) failed to adversely affect the enhancing action of the ester on
insulin release evoked by the branched-chain amino acid (Table
10,
experiments
1 and 2). In these experiments,
D-mannoheptulose also failed to
suppress the insulinotropic action of
L-leucine.
Further experiments provided the following information (Table 10).
First, at the concentration of 10 mM used in the present experiments,
D-mannoheptulose failed to
significantly affect the secretory response to either 2-ketoisocaproate
(10 mM) or the association of
L-leucine and
L-glutamine (10 mM each).
Second, to explore the effect of
D-mannoheptulose on the
secretory response to D-glucose
at low concentrations of the hexose, advantage was taken of its
enhancing action on insulin release evoked by 2-ketoisocaproate, the
branched-chain 2-keto acid being used at a concentration (5 mM) close
to the threshold value for its insulinotropic action (7). Under these
experimental conditions, as little as 1.7 and 2.8 mM
D-glucose indeed caused a
concentration-related increase in insulin output
(P < 0.001). Unexpectedly, however,
D-mannoheptulose (10 mM) failed
to significantly affect the release of insulin evoked by
2-ketoisocaproate in the presence of 1.7 mM
D-glucose (Table 10,
experiment
4). The concentration of the hexose
had to be raised to 2.8 mM to detect a sizeable inhibitory effect (P < 0.001) of
D-mannoheptulose (Table 10,
experiment
5). In the presence of
D-mannoheptulose, the
release of insulin evoked by 2-ketoisocaproate was no more
significantly different at 1.7 and 2.8 mM
D-glucose, whether expressed in
absolute terms (P > 0.18) or
relative to the mean secretory rate recorded within the same experiments in islets exposed to 16.7 mM
D-glucose (19.5 ± 2.7 vs.
16.9 ± 2.5%; n = 20 in both
cases; P > 0.4). In these
experiments, it was duly verified that
D-mannoheptulose abolishes the
insulinotropic action of
D-glucose (16.7 or 27.8 mM).
Actually, in the presence of the heptose, the output of insulin found
in islets exposed to these high concentrations of
D-glucose was lower
(P < 0.001) than that evoked by
2-ketoisocaproate at much lower concentrations of the hexose.
Taken as a whole, the data summarized in Table 10 indicate that
D-mannoheptulose, although a
potent and specific inhibitor of the insulinotropic action of
D-glucose, when the hexose is tested in high concentrations, is much less efficient in suppressing the secretory response to the sugar when it is used in low
concentrations. Therefore, the failure of
D-mannoheptulose to
significantly affect insulin release in islets exposed to
-D-glucose pentaacetate (see
above) does not provide conclusive evidence on the relevance of hexose
phosphorylation to the insulinotropic action of this ester.
The latter consideration motivated a last series of experiments
conducted in the presence of 8.3 mM
D-glucose (Table 10,
experiment 6). As expected,
D-mannoheptulose (10 mM) suppressed (P < 0.001) the
secretory response to the hexose. The heptose failed, however, to
suppress the insulinotropic action of
-D-glucose pentaacetate (1.7 mM). Actually, the increment in insulin output attributable to the
ester was not significantly different
(P > 0.1) in the absence
(+134.7 ±8 17.9 µU · islet
1 · 90 min
1; df = 28) or presence
(+101.3 ± 9.6; df = 28) of
D-mannoheptulose. Moreover, the
secretory rate recorded in the simultaneous presence of
D-glucose (8.3 mM),
D-mannoheptulose (10 mM), and
-D-glucose pentaacetate (1.7 mM) was much higher than that otherwise recorded in the sole presence
of the ester (see above). These findings strongly suggest that the
insulinotropic action of
-D-glucose pentaacetate is
not dependent on the phosphorylation of the hexose moiety of the ester
or that D-mannoheptulose fails
to inhibit such a phosphorylation.
Effect of 2-deoxy-D-glucose. The third inhibitor
of D-glucose insulinotropic
action used in the present study, namely
2-deoxy-D-glucose, when tested
in the 8.3 to 16.7 mM range caused a concentration-related decrease of
the secretory response evoked by 11.1 mM
D-glucose (Fig.
9). At a concentration of 16.7 mM,
2-deoxy-D-glucose, however, caused only a minor decrease of the secretory response to
-D-glucose pentaacetate (Fig.
9). Moreover, when these experiments were repeated in the presence of
10.0 mM L-leucine to increase
the magnitude of the B cell response to the hexose ester, a significant
effect of 2-deoxy-D-glucose on
the release of insulin evoked by
-D-glucose pentaacetate (1.7 mM) was no longer observed (Table 11).

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Fig. 9.
Effect of 2-deoxy-D-glucose
(2-DOG; 16.7 mM; striped columns) on insulin release from islets
incubated in absence (basal) or presence of 1.7 mM
-D-glucose pentaacetate
(n = 45 in each case).
Inset illustrates effect of increasing
concentrations of 2-DOG on insulin release evoked by 11.1 mM
D-glucose
(n = 15 in each case).
|
|
View this table:
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Table 11.
Effect of 2-deoxy-D-glucose on secretory response to
-D-glucose pentaacetate in islets exposed
to 10.0 mM L-leucine
|
|
 |
DISCUSSION |
Several of the present findings are compatible with the view that the
insulinotropic action of
-D-glucose pentaacetate is causally linked to its capacity to act as a fuel in pancreatic islet
cells.
First, like other nutrient secretagogues,
-D-glucose pentaacetate
caused a shift to the left of the sigmoidal curve relating insulin
output to D-glucose
concentration. Likewise, whereas the concentration of
-D-glucose pentaacetate had
to exceed a critical value (>0.85 mM) to stimulate insulin release in
the absence of any other exogenous nutrient, no threshold phenomenon
was observed when the ester was tested in the presence of 8.3 mM
D-glucose.
Second, the secretory response to
-D-glucose pentaacetate
coincided with the recruitment of more individual B cells to an active
functional state, as is also the case with other nutrients (1, 6).
Third,
-D-glucose
pentaacetate stimulated biosynthetic activity in pancreatic islets
incubated in the absence or presence of either
D-glucose or
L-leucine. In this respect, the
present data extend the knowledge that the threshold concentration for stimulation of functional events by nutrients is lower in the case of
biosynthetic activity than insulin release (14). It should be noted,
however, that
-D-glucose
pentaacetate failed to significantly increase the biosynthesis of
proinsulin relative to that of nonhormonal peptides and apparently
decreased the rate of proinsulin conversion to insulin. The latter
finding represents a first indication that
-D-glucose pentaacetate may
affect B cell function not solely through its nutritional value.
Fourth,
-D-glucose
pentaacetate simulated the effect of other nutrient secretagogues in
causing both a decrease of 86Rb
outflow and an increase of 45Ca
efflux from prelabeled islets. The latter phenomenon was abolished in
the absence of extracellular Ca2+,
suggesting that it results from an increased influx of the divalent cation into the islet cells. And, indeed,
-D-glucose pentaacetate stimulates 45Ca net uptake by the
islets (unpublished observation).
Last, the secretory response to
-D-glucose pentaacetate was
suppressed in the absence of extracellular
Ca2+ and potentiated by both
theophylline and cytochalasin B. This suggests that the insulinotropic
action of the ester involves interaction between
Ca2+, adenosine
3',5'-cyclic monophosphate, and the microfilamentous effector system in the same manner as in islets stimulated by unesterified D-glucose.
Taken as a whole, these findings thus suggest that the stimulation of
insulin release by
-D-glucose
pentaacetate involves the same sequence of metabolic, cationic, and
motile events currently thought to be operative in the process of
nutrient-stimulated insulin secretion (11).
Even the observation that
D-mannoheptulose failed to
suppress insulin release evoked by
-D-glucose pentaacetate
remains compatible with such a proposal, since the heptose fails to
inhibit the utilization and oxidation of the ester in isolated islets
(unpublished observation). The modest but significant reduction of the
secretory response to
-D-glucose pentaacetate
caused by 2-deoxy-D-glucose in
islets deprived of any other exogenous nutrient is also consistent with such a view.
If the insulinotropic action of
-D-glucose pentaacetate were
indeed to result from its role as a nutrient in islet cells, the
question must be raised whether it is the hexose or acetate moiety of
the ester that plays such a role.
Several of the present data argue against any major role of the acetate
residues presumably generated by the intracellular hydrolysis of
-D-glucose pentaacetate in
its insulinotropic action.
First, except for a modest positive effect of acetate and ethyl acetate
on insulin release from islets incubated in the absence of any
exogenous nutrient, neither acetate nor its methyl or ethyl ester was
able to augment insulin release from islets incubated in the presence
of D-glucose or
L-leucine, namely under
conditions in which the insulinotropic action of the ester is most
pronounced.
Second, even at the very low concentration of 0.1 mM,
-D-glucose pentapropionate
stimulated insulin release from islets exposed to 10.0 mM
L-leucine, the secretory
response to this ester being significantly higher than that evoked at
the same low concentration by
-D-glucose pentaacetate and
contrasting with the lack of effect, under the same experimental
conditions, of unesterified
D-glucose. Yet, propionate is
very poorly metabolized in islet cells. At a 10 mM concentration, the
oxidation of
[1-14C]propionate or
the generation of 13C-labeled
L-lactate from
[2-13C]propionate does
not exceed ~3
pmol · 103
cells
1 · 90 min
1 (10, 17).
Third, both
- and
-D-galactose pentaacetate
failed to exert any positive effect on insulin release whether in the
absence or presence of D-glucose
or L-leucine.
Although these three series of observations strongly suggest a key role
for the glucose moiety of
-D-glucose pentaacetate in
its insulinotropic action, they do not rule out an interference of the
acetate moiety with selected metabolic, ionic, and functional variables. For instance, the generation of acetic acid from the ester
could account for a decrease in cellular pH in islets exposed to
-D-glucose pentaacetate
(unpublished observation). This, in turn, may explain the fact that
-D-glucose pentaacetate
barely increased insulin output from islets incubated in the presence of 2-ketoisocaproate while considerably augmenting leucine-induced insulin release. It is indeed known that the respective effects of the
branched-chain 2-keto acid and amino acid on intracellular pH affect in
a dissociated manner the coupling between metabolic and secretory
events in nutrient-stimulated islets (8, 15).
Three other sets of observations, however, argue against the view that
the catabolism of D-glucose
generated from
-D-glucose pentaacetate represents an essential, or at least the sole, determinant of its insulinotropic action.
First, and as already mentioned, the ester failed to cause a
preferential stimulation of proinsulin biosynthesis relative to that of
other islet peptides.
Second, in the presence of 10.0 mM
L-leucine, the secretory
response to
-D-glucose
pentaacetate was unaffected by
2-deoxy-D-glucose.
Third, and most importantly,
-L-glucose pentaacetate
caused an obvious stimulation of insulin release whether in the absence of any other exogenous nutrient or in the presence of
D-glucose or
L-leucine. Exogenous
L-glucose failed to reproduce
such an effect.
The secretory response to
-D-glucose pentaacetate also
differed from that evoked by exogenous
D-glucose by its insensitiveness to
3-O-methyl-D-glucose
and D-mannoheptulose. Although
is seems likely that the resistance to
3-O-methyl-D-glucose
may be accounted for by the fact that
-D-glucose pentaacetate
penetrates into the islet cells without requiring the intervention of a
specific hexose transport system, the failure of
D-mannoheptulose to oppose the
utilization and insulinotropic action of the ester suggests heterogeneity in the location and/or regulation of hexose
phosphorylation in islets exposed to
-D-glucose pentaacetate
and/or unesterified D-glucose.
In conclusion, although the hexose moiety generated from
-D-glucose pentaacetate
indeed seems to participate in the insulinotropic action of the ester,
the modality of such a participation may well differ from that
currently implied in the process of glucose-stimulated insulin release.
 |
ACKNOWLEDGEMENTS |
We are grateful to M. Mahy, J. Schoonheydt, and M. Urbain for
technical assistance and C. Demesmaeker for secretarial help.
 |
FOOTNOTES |
This study was supported by a Concerted Research Action of the French
Community of Belgium (94/99-183), Grant IN212194 from Direccion
General de Asuntos del Personal Academico (Universidad Nacional Autónoma de Mexico) and grants from the Spanish Fondo de
Investigaciones Sanitarias (96/1383) and Dirección General de
Investigaciones Científica y Técnica (95/0048) and
Belgian Foundation for Scientific Medical Research (3.4513.94).
Address for reprints requests: W. J. Malaisse, Laboratory of
Experimental Medicine, Brussels Free University, 808 Route de Lennik,
B-1070 Brussels, Belgium.
Received 11 June 1997; accepted in final form 20 August 1997.
 |
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