Insulinotropic action of
-L-glucose
pentaacetate
Willy J.
Malaisse1,
Leonard C.
Best2,
André
Herchuelz3,
Marcia
Hiriart4,
Hassan
Jijakli1,
Marcel M.
Kadiata1,
Elena
Larrieta-Carasco4,
Aouatif
Laghmich1,
Karim
Louchami1,
Dany
Mercan1,
Elizabeth
Olivares1,
Carmen
Sànchez-Soto4,
Olivier
Scruel1,
Abdullah
Sener1,
Isabel
Valverde5,
María L.
Villanueva-Peñacarrillo5,
Concepción
Viñambres5, and
Walter S.
Zawalich6
Laboratories of 1 Experimental
Medicine and 3 Pharmacology,
Brussels Free University, B-1070 Brussels, Belgium;
2 Department of Medicine,
University of Manchester, M13 9WL Manchester, United Kingdom;
4 Department of Biophysics,
Institute of Cellular Physiology, Universidad Nacional
Autónoma de México, Mexico City, DF-04510 Mexico City,
Mexico; 5 Fundación
Jiménez Díaz, 28040 Madrid, Spain; and
6 Yale University School of
Nursing, New Haven, Connecticut 06536
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ABSTRACT |
The metabolism of
-L-glucose
pentaacetate and its interference with the catabolism of
L-[U-14C]glutamine,
[U-14C]palmitate,
D-[U-14C]glucose,
and
D-[5-3H]glucose
were examined in rat pancreatic islets. Likewise, attention was paid to
the effects of this ester on the biosynthesis of islet peptides, the
release of insulin from incubated or perifused islets, the functional
behavior of individual B cells examined in a reverse hemolytic plaque
assay of insulin secretion, adenylate cyclase activity in a
membrane-enriched islet subcellular fraction, cAMP production by intact
islets, tritiated inositol phosphate production by islets preincubated
with
myo-[2-3H]inositol,
islet cell intracellular pH, 86Rb
and 45Ca efflux from prelabeled
perifused islets, and electrical activity in single isolated B cells.
The results of these experiments were interpreted to indicate that the
insulinotropic action of
-L-glucose pentaacetate is not
attributable to any nutritional value of the ester but, instead,
appears to result from a direct effect of the ester itself on a yet
unidentified receptor system, resulting in a decrease in
K+ conductance, plasma membrane
depolarization, and induction of electrical activity.
pancreatic islets; insulin release
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INTRODUCTION |
THE POLYACETATE ESTERS of several monosaccharides were
recently proposed as tools to increase the metabolic efficiency of the
corresponding carbohydrates. For instance,
-D-glucose
pentaacetate was found to be metabolized and to stimulate insulin
release at higher rates than unesterified D-glucose in rat
pancreatic islets (21, 31). Likewise, D-mannoheptulose
hexaacetate was reported to inhibit D-glucose metabolism in
parotid cells and erythrocytes, which are otherwise resistant to the
unesterified heptose (11, 19). Last, 2-deoxy-D-glucose
tetraacetate is more potent than unesterified
2-deoxy-D-glucose as an inhibitor of both
D-glucose metabolism and insulinotropic action in
pancreatic islets (33) and as a cytotoxic agent in various lines of
tumoral cells (5, 14, 15, 27).
In experiments dealing with the mode of action of
-D-glucose pentaacetate in pancreatic islet cells, the
pentaacetate ester of
-L-glucose was unexpectedly found
also to stimulate insulin release, albeit to a lesser extent than
-D-glucose pentaacetate (21). The insulin-releasing
capacity of
-L-glucose pentaacetate does not seem to be
attributable to the catabolism of its acetate moiety, since
-D-galactose pentaacetate, which is as efficiently taken
up and hydrolyzed in isolated islets as
-D-glucose
pentaacetate (34), fails to display any positive effect on insulin
secretion (21).
The major aim of the present study was to further characterize the
effect of
-L-glucose pentaacetate on several variables of islet metabolism and function, in search of a possible explanation for its insulinotropic action.
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MATERIALS AND METHODS |
The pentaacetyl esters of monosaccharides were purchased from Sigma
(St. Louis, MO) or synthesized by a method described elsewhere (35,
36). The same procedure was used to prepare
-L-[1-14C]glucose
pentaacetate or
-L-glucose
penta-[1-14C]acetate.
Except if otherwise mentioned, all experiments were conducted in
pancreatic islets isolated by the collagenase method (25) from Wistar rats.
The procedures used to measure the phosphorylation of
14C-labeled hexoses (7) and
hydrolysis of hexose pentaacetates (26) in islet homogenates, the net
uptake of 14C-labeled esters (31),
the generation of 3HOH,
14CO2,
and radioactive amino acids or acidic metabolites by islets exposed to
tritiated or 14C-labeled nutrients
(16, 22, 30), and the output of
14CO2
from islets prelabeled with either
L-[U-14C]glutamine
(23) or
[U-14C]palmitate (29)
were identical to those described in the cited references.
The separation of
L-[1-14C]glucose
and its esters was achieved by HPLC in a 5 µm Partisphere C18 column (Whatman
International, Maidstone, UK), using as mobile phase a 35:65 (vol/vol)
methanol-H2O mixture delivered at
a flow of 1.0 ml/min.
The procedures used to assess biosynthetic activity in islets exposed
to
L-[4-3H]phenylalanine
(4), to measure insulin release from incubated (25) or perifused (9,
37) islets, secretory activity of isolated B cells in a reverse
hemolytic plaque assay (10), formation of tritiated inositol phosphates
by islets preincubated with
myo-[2-3H]inositol
(37), 45Ca net uptake (24), to
monitor 86Rb (3) and
45Ca (9) efflux from prelabeled
islets, and to collect electrophysiological data (17) were previously
reported in the cited references.
For measurement of adenylate cyclase activity, groups of ~800 islets
each were homogenized at 4°C in Potter-Elvehjem tubes (10 strokes)
with 0.1 ml of a Tris · HCl buffer (25 mM, pH 7.5) containing 5 mM MgCl2, 0.6 mM
EGTA, and 1.1 mM dithiothreitol. After centrifugation for 1 min at
4°C and 100 g, aliquots (40 µl)
of the supernatant extract, containing 1.4-1.9 µg protein (13),
were mixed with 35 µl of a reaction mixture (32) containing unlabeled
ATP (final concentration 70 µM). After 60 min of incubation at
37°C, the reaction was stopped with 300 µl ethanol (final
concentration 65%). After centrifugation for 15 min at 4°C and
1,700 g, aliquots (365 µl) of the
supernatant solution were vacuum-dried and redissolved in 120 µl of a
cAMP assay buffer for measurement of the cyclic nucleotide by
radioimmunoassay (RIANEN
cAMP[125I]RIA Kits; Du
Pont, Brussels, Belgium).
For measurement of cAMP formation by intact islets, groups of five
islets each were incubated for 60 min at 37°C in 0.1 ml of a
bicarbonate-buffered medium (25) containing 5 mg/ml bovine serum
albumin and 1.0 mM isobutyl methylxanthine. The incubation was halted
by addition of 200 µl ethanol (final concentration 65%). Each sample
was then sonicated for 15 s and eventually centrifuged and examined for
its cAMP content as described above.
For measurement of intracellular pH, dispersed islet cells (8) were
suspended in RPMI medium supplemented with 10% (vol/vol) newborn calf
serum, 2.0 mM L-glutamine, 10 µg/ml gentamicin, 100 U/ml
penicillin, and 100 µg/ml streptomycin, placed on round glass coverslips pretreated with poly-L-lysine, and incubated
overnight at 37°C in a
O2-CO2
(95:5, vol/vol) incubator. The cells were loaded with 1.0 µM
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (Molecular Probes, Eugene, OR) during 60 min of incubation at 37°C
in a bicarbonate-buffered medium (25) containing, when required, 8.3 mM
D-glucose or 10.0 mM L-leucine and equilibrated (pH 7.4) against a mixture of
O2-CO2
(95:5, vol/vol). The coverslips were then transferred to a tissue
chamber mounted onto an inverted fluorescence microscope (Diaphot TDM;
Nikon, Tokyo, Japan) and perifused (~1.0 ml/min) with the same
bicarbonate-buffered medium. The intracellular pH of single cells was
determined, at 11-s intervals, by ratiometric dual-excitation (460/490
nm) fluorimetry (emission wavelength 510 nm) using a camera-based image
analysis system (Joice-Loebl, Gateshead, UK).
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 usually restricted to data collected in close to equal numbers
within the same experiments. Each individual measurement was made in
separate cells or groups of islets.
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RESULTS |
Phosphorylation of
L-glucose. To assess the possible
phosphorylation of L-glucose, its effect on the
phosphorylation of D-glucose was examined first. In
pancreatic islet homogenates, the rate of phosphorylation of
D-glucose increased from 1.33 ± 0.16 to 2.20 ± 0.14 pmol · islet
1 · min
1
as the concentration of the hexose was raised from 0.2 to 17.0 mM
(n = 3 in both cases). At the low
concentration of D-glucose (0.2 mM), the phosphorylation
rate was not affected significantly (P > 0.3) by the presence of L-glucose (1.7-17.0 mM) in
the reaction medium; it averaged 1.22 ± 0.03 pmol · islet
1 · min
1
(n = 6). In these experiments, a
tracer amount of
D-[U-14C]glucose
(37 µM) was mixed with unlabeled D-glucose to reach the
above-mentioned total concentrations of the hexose.
When a tracer amount of
L-[1-14C]glucose
(0.1 mM) was mixed with increasing amounts of unlabeled
L-glucose (0.17-17.0 mM), the radioactive content of
acidic molecules was barely higher after incubation in the presence, as
distinct from absence, of the islet homogenates and, in the former
case, unaffected by the amount of unlabeled L-glucose.
After correction for the blank value (no islet), it corresponded to
only 0.31 ± 0.08
of the total radioactive content of the
incubation medium or to an apparent phosphorylation rate of
L-[1-14C]glucose
not exceeding 0.57 ± 0.16 fmol · islet
1 · min
1
(df = 16 in both cases). Moreover, when unlabeled D-glucose
(1.7-17.0 mM) was added to a medium containing 0.1 mM
L-[1-14C]glucose
and 0.17 mM unlabeled L-glucose, the apparent
phosphorylation of the radioactive hexose was totally suppressed. Thus
the amount of 14C-labeled acidic
metabolites recovered after incubation in the presence of the islet
homogenates became virtually identical
(P > 0.9) to the blank value (no
islet), with a mean negative difference between the two sets of
measurements corresponding to 0.01 ± 0.09
(df = 13) of the
total radioactive content of the incubation medium.
These findings indicate that L-glucose cannot act as a
substrate for phosphorylation in the reactions catalyzed by the islet hexokinase isoenzymes.
Hydrolysis of
-L-glucose pentaacetate
in islet homogenates. In islet homogenates, the rate of
acetate formation from
-D-glucose pentaacetate and
-L-glucose pentaacetate (each 0.25 mM) averaged, respectively, 1.27 ± 0.12 and 0.33 ± 0.08 nmol · islet
1 · 60 min
1
(n = 4 in both cases;
P < 0.001).
Uptake of
-L-[1-14C]glucose
pentaacetate by intact islets.
Groups of 20 islets each were incubated for 3-20 min in the
presence of 3HOH or
L-[1-14C]glucose.
The distribution space of 3HOH was
not significantly different after 3, 10, or 20 min of incubation,
averaging, respectively, 2.81 ± 0.24 nl/islet
(n = 6), 2.62 ± 0.20 nl/islet
(n = 12), and 2.81 ± 0.24 nl/islet (n = 6). After 10 min of
incubation, the distribution space of
L-[1-14C]glucose
(2.0 mM) averaged 1.71 ± 0.14 nl/islet, representing 59.5 ± 2.1% (n = 6) of the paired
3HOH space. The apparent
distribution space of
-L-[1-14C]glucose
pentaacetate (1.7 mM) largely exceeded the
3HOH space, with a paired ratio
between these two spaces of 356.2 ± 18.9%
(n = 5), 562.0 ± 29.9%
(n = 6), and 660.0 ± 36.5%
(n = 6) after 3, 10, and 20 min of
incubation, respectively (Fig. 1). Exponential extrapolation of the latter data yielded a theoretical equilibrium value of 670%, corresponding, after correction for extracellular contamination (as judged from the
L-[1-14C]glucose
space), to a net uptake of
-L-[1-14C]glucose
pentaacetate close to 28 pmol/islet.

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Fig. 1.
Time course for
L-[1-14C]glucose
pentaacetate net uptake by isolated islets. Distribution space of
L-[1-14C]glucose
(2.0 mM; open circle and broken line) and
-L-[1-14C]glucose
pentaacetate (1.7 mM; filled circles and solid line) is expressed
relative to the paired 3HOH space.
Mean values (±SE) are derived from 5-6 individual
observations. Broken line on top
refers to a theoretical equilibrium value.
Inset: time course for the difference
between the theoretical equilibrium value and the experimental data,
with the time 0 reading being
corrected for the mean
L-[1-14C]glucose
distribution space (semilogarithmic coordinates).
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The generation of unesterified
L-[1-14C]glucose
from the labeled ester was also investigated in these experiments.
Although the HPLC procedure used for such a purpose allowed distinction between
L-[1-14C]glucose
pentaacetate and its metabolites, the precise quantification of
unesterified
L-[1-14C]glucose
was hampered by the presence of radioactive molecules, presumably
partially esterified
L-[1-14C]glucose
(e.g.,
L-[1-14C]glucose
monoacetate), eluting together with or in the vicinity of the
unesterified hexose (Fig. 2). Already after
3 min of incubation, the amount of these hydrolytic products was higher
in the presence than absence of islets, coinciding with a fall
(P < 0.001) in the amount of
extracellular
L-[1-14C]glucose
pentaacetate (Fig. 2, C and
D). After 20 min of incubation, the
apparent amount of extracellular unesterified
L-[1-14C]glucose
generated by the islets, as judged from the radioactive material
eluting with the hexose, averaged, after correction for the blank value
found in the absence of islets, 1.34 ± 0.25 nmol/islet (df = 8).
This value overestimates, however, the true amount of unesterified
L-glucose, since it is not corrected for contamination by
co-eluting esters.

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Fig. 2.
Representative elution profiles of
L-[1-14C]glucose
(A) and
-L-[1-14C]glucose
pentaacetate before (B) or after
(D) 3 min of incubation in the
absence of islets and after 3 min of incubation in the presence of
islets (C). Incubation media were
obtained from the experiments depicted in Fig. 1. All results are
expressed as percentage of total radioactive content of each sample.
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Metabolism of
-L-glucose
pentaacetate. In sharp contrast to the results obtained
with
D-[1-14C]glucose
or
D-[U-14C]glucose,
no significant production of
14CO2
or 14C-labeled acidic metabolites
and amino acids could be detected in islets exposed to either
L-[1-14C]glucose
or
-L-[1-14C]glucose
pentaacetate, with all nutrients being tested at a 1.7 mM concentration
(Table 1). In the case of the
L-glucose ester, the readings concerning the
14C-labeled acidic metabolites and
amino acids were even slightly lower after incubation in the presence,
rather than absence, of islets. This situation is reminiscent of that
previously encountered in the study of
-D-[U-14C]glucose
pentaacetate (31).
To characterize the metabolic fate of the acetyl moiety of
-L-glucose pentaacetate, the production of
14C-labeled metabolites was
measured in islets exposed to
-L-glucose penta-[1-14C]acetate
(also 1.7 mM). The oxidation of the ester was comparable to that found
in islets exposed to other hexose esters, e.g.,
-D-galactose
penta-[1-14C]acetate
(31). As in the latter case, the production of
14C-labeled acidic metabolites and
amino acids barely exceeded the blank value found in the absence of islets.
Interference of
-L-glucose pentaacetate
with the catabolism of other nutrients. Two series of
experiments aimed at investigating the possible interference of
-L-glucose pentaacetate with the catabolism of other nutrients.
First, the effect of the ester on the metabolism of endogenous
nutrients was examined in islets prelabeled with either
L-[U-14C]glutamine
or [U-14C]palmitate
(Table 2). After 30 min of preincubation of
the islets in the presence of 1.0 mM
L-[U-14C]glutamine
and 8.3 mM D-glucose, their radioactive content averaged, when expressed as glutamine equivalent, 15.68 ± 0.31 pmol/islet (n = 99). Relative to such a content,
the production of
14CO2
over 30 min of incubation in the absence of any exogenous nutrient
averaged 15.7 ± 0.6% (n = 20),
distinct from only 4.2 ± 0.3% (n = 20) in islets exposed to 5.0 mM KCN, 10 µM antimycin A, and 10 µM
rotenone. After correction for such a blank value, the paired ratio
between
14CO2
output and 14C content was not
significantly different in islets incubated in the presence of
-L-glucose pentaacetate,
-D-mannose
pentaacetate, or
-D-fructose pentaacetate (1.7 mM) and
in the control islets incubated in the absence of any exogenous
nutrient.
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Table 2.
Effect of -L-glucose pentaacetate,
-D-mannose pentaacetate, and
-D-fructose pentaacetate on production of
14CO2 by islets prelabeled with
L-[U-14C]glutamine or
[U-14C]palmitate
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After 120 min of preincubation in the presence of 0.31 mM
[U-14C]palmitate and
8.3 mM D-glucose, the islet radioactive content, expressed
as palmitate equivalent, averaged 1.87 ± 0.36 pmol/islet (n = 96). Relative to the paired
14C content, the output of
14CO2
over 120 min of incubation in the absence of exogenous nutrient averaged 6.8 ± 0.3% (n = 18)
compared with 1.5 ± 0.2% (n = 18) in islets exposed to the metabolic poisons (see preceding
paragraph). After subtraction of this blank value, the
14CO2
output-to-14C content ratio was
lower (P < 0.005 or less) in islets
incubated in the presence of a hexose ester than in the control islets
(Table 2). The relative extent of such a sparing action was
significantly higher (P < 0.03) in
the case of
-D-mannose pentaacetate than
-L-glucose pentaacetate, although such was not the case
when the results obtained with the latter ester and
-D-fructose pentaacetate were compared.
In the second series of experiments, the effects of
-L-glucose pentaacetate or
-D-galactose
pentaacetate (1.7 mM each) on the metabolism of unesterified
D-glucose (8.3 mM) were investigated in islets exposed for
90 min to the latter hexose (Table 3). Both
esters significantly decreased (P < 0.005 or less) the conversion of
D-[5-3H]glucose
to 3HOH and that of
D-[U-14C]glucose
to both 14C-labeled acidic
metabolites and amino acids while failing to affect the oxidation of
D-[U-14C]glucose.
The paired ratio between
D-[U-14C]glucose
oxidation and
D-[5-3H]glucose
utilization was markedly increased (P < 0.001) by
-L-glucose pentaacetate but was increased
less (P < 0.001) by
-D-galactose pentaacetate.
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Table 3.
Effect of -L-glucose pentaacetate and
-D-galactose pentaacetate on metabolism
of unesterified D-glucose (8.3 mM)
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Biosynthetic data. Over 90 min of
incubation, D-glucose (4.2 and 16.7 mM) stimulated, in a
concentration-related manner, the incorporation of
L-[4-3H]phenylalanine
into TCA-precipitable material (Table 4).
L-Leucine (10.0 mM) also increased islet biosynthetic
activity (P < 0.005). The
pentaacetate ester of
-L-glucose (1.7 mM), however,
inhibited (P < 0.02 or less) the
tritiation of islet peptides in islets exposed to either 4.2 mM
D-glucose or 10.0 mM L-leucine. This contrasts
with the fact that the insulinotropic action of
-L-glucose pentaacetate is more marked in the presence
of D-glucose or L-leucine than in the absence
of exogenous nutrient (21).
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Table 4.
Effect of -L-glucose pentaacetate on
incorporation of L-[4-3H]phenylalanine (3.4 µM) in TCA-precipitable and TCA-soluble material
over 90 min of incubation
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The inhibitory action of the ester on islet biosynthetic activity could
not be attributed to a decrease in the size of the intracellular pool
of free
L-[4-3H]phenylalanine.
Indeed, the radioactive content of the TCA-soluble material was not
significantly lower (P > 0.4) in
islets exposed to both D-glucose and
-L-glucose pentaacetate than in islets incubated in the
sole presence of the unesterified hexose. In islets exposed to
L-leucine, the pool of radioactive TCA-soluble material was
even slightly higher (P < 0.05) in
the presence than absence of
-L-glucose pentaacetate.
Secretory data. The insulinotropic
action of
-L-glucose pentaacetate was previously
explored in islets incubated either in the absence of any exogenous
nutrient (21) or in the presence of 8.3 mM D-glucose, 10.0 mM L-leucine (21), or 10.0 mM succinic acid dimethyl ester
(18). In the absence of another exogenous nutrient or at a
noninsulinotropic concentration of D-glucose (4.0 mM), the
increase in insulin output caused by
-L-glucose pentaacetate is quite modest and, on occasion, fails to achieve statistical significance. Such was the case, for instance, in the
experiments illustrated in Table 5. The
ester indeed failed to affect insulin output significantly whether in
islets incubated at normal Ca2+
concentration (1.0 mM) or in the absence of extracellular
Ca2+, in which case the output of
insulin was, as expected (23), significantly increased
(P < 0.001). Nevertheless, in these
experiments, the presence of the ester in the incubation medium allowed
either theophylline (1.4 mM) or repaglinide (0.01 mM) to augment
insulin output significantly (P < 0.05 or less) relative to the basal value, whereas the latter two
agents fail to affect basal in- sulin output (1, 2) in islets deprived
of any exogenous nutrient.
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Table 5.
Effect of -L-glucose pentaacetate on
secretory response of rat pancreatic islet to Ca2+
deprivation, theophylline, and repaglinide
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In the presence of 4.0 mM D-glucose,
-D-glucose pentaacetate (1.7 mM), but not
-L-glucose pentaacetate (also 1.7 mM), significantly augmented insulin output (Table 5). Surprisingly, the increment in
insulin release caused by
-D-glucose pentaacetate was
lower (P < 0.001) in the presence of
-L-glucose pentaacetate (49.2 ± 3.0 µU · islet
1 · 90 min
1; df = 58) than in its
absence (83.2 ± 5.2; df = 57). When the concentration of
D-glucose was raised to 7.0 mM D-glucose, both
-D-glucose pentaacetate and
-L-glucose
pentaacetate increased insulin secretion
(P < 0.001), with the secretory
response to the latter ester remaining significantly lower
(P < 0.001) than that to the former.
Once again, the release of insulin was lower (P < 0.005) in the concomitant
presence of the two esters than in the sole presence of
-D-glucose pentaacetate, with the increment in secretory
rate attributable to the latter ester being decreased from 102.5 ± 9.8 µU · islet
1 · 90 min
1 (df = 28) in the
absence of
-L-glucose pentaacetate to a negligible value
of 9.2 ± 8.7 µU · islet
1 · 90 min
1 (df = 28) in its
presence. These converging findings indicate that
-L-glucose pentaacetate somehow impairs the
insulinotropic action of
-D-glucose pentaacetate.
Figure 3 illustrates the
concentration-response relationship for the insulinotropic action of
-L-glucose pentaacetate in islets incubated in the
presence of 7.0 mM D-glucose. As little as 0.34 mM, but not
0.17 mM, of the ester was sufficient to increase (P < 0.02) glucose-stimulated
insulin release significantly. A close-to-maximal response was observed
at a 0.85 mM concentration of the ester. At 1.7 mM, the ester still
increased insulin output by 13.7 ± 7.0 µU · islet
1 · 90 min
1 (df = 44;
P < 0.06), but the release of
insulin was significantly lower (P < 0.05) than that recorded at 0.85 mM
-L-glucose
pentaacetate.

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Fig. 3.
Effect of increasing concentrations of -L-glucose
pentaacetate on insulin release from islets incubated in the presence
of 7.0 mM D-glucose. Mean values (±SE) refer to
23-24 individual determinations. Horizontal broken line refers to
the mean control value found in the absence of the ester.
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The secretory data presented so far also document the modulation of
-L-glucose pentaacetate insulinotropic action by
D-glucose. Thus, pooling all available data presented in
this report, the increment in secretory rate
(µU · islet
1 · 90 min
1) attributable to the
ester (1.7 mM) averaged 2.4 ± 2.1 (df = 95) in the absence of the
hexose, 3.5 ± 2.2 (df = 58) in the presence of
D-glucose (4.0 mM), and 31.5 ± 5.7 (df = 72) in the
presence of 7.0 mM D-glucose.
Further documentation of the insulinotropic action of
-L-glucose pentaacetate was obtained in experiments
conducted in a reverse hemolytic plaque assay of insulin secretion from
individual B cells (Table 6).
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Table 6.
Secretory activity in single B cells exposed to
-L-glucose pentaacetate or
-D-glucose pentaacetate in the presence
of 5 mM L-leucine
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Over 60 min of incubation in the presence of 5 mM
L-leucine, the ester augmented significantly the percentage
of plaque-forming cells, the plaque area, and the insulin secretion
index, which represents the product of the former two variables. Thus,
for these three parameters, the results recorded in the presence of the
ester averaged, respectively, 136.6 ± 5.0%
(P < 0.001), 127.9 ± 10.4% (P < 0.05), and 175.7 ± 18.8% (P < 0.005) of the mean control values found within the same experiment(s) in its absence, i.e., 100.0 ± 2.6, 100.0 ± 6.9, and 100.0 ± 7.9%
(n = 8 in all cases). Relative to such
control values, the mean percentage of plaque-forming cells (177.2 ± 12.1%), plaque area (139.2 ± 8.6%), and insulin secretion
index (248.3 ± 27.0%) was even higher in cells exposed to
-D-glucose pentaacetate. The difference between the two
esters achieved statistical significance in the case of the percentage
of plaque-forming cells (P < 0.01)
and insulin secretion index (P < 0.05) but not in the case of the plaque area. When the results were
expressed relative to the mean value found within the same experiments
in the presence of both L-leucine and
-D-glucose pentaacetate, the ester-induced increment in
the insulin secretion index above the control value (41.9 ± 4.7%; n = 14) recorded in the sole presence
of L-leucine averaged 29.7 ± 7.7 and 58.1 ± 8.7%
(df = 14 in both cases) with
-L-glucose pentaacetate and
-D-glucose pentaacetate, respectively. This indicates
that the latter ester was about two times as efficient (P < 0.025) as the former in
augmenting insulin release, with this difference appearing to be mainly
attributable to a greater recruitment of cells in an active secretory
state rather than to an increase in insulin output from each activated cell.
Adenylate cyclase activity and cAMP
formation. Further experiments aimed at identifying
possible second messengers participating in the secretory response of
the islets to
-L-glucose pentaacetate.
To explore the possible participation of the adenylate cyclase-protein
kinase A axis in the insulinotropic action of
-L-glucose pentaacetate, the effect of this and other esters on adenylate cyclase
activity was first investigated in a membrane-enriched islet
subcellular fraction (Table 7). GTP (10 µM) and NaF (10 mM) increased (P < 0.001) the reaction velocity from a basal value of 58.0 ± 4.0 fmol · µg
protein
1 · 60 min
1 to 108.8 ± 11.3 and 220.3 ± 19.1 fmol · µg
protein
1 · 60 min
1, respectively. The
pentaacetate esters of
-L-glucose,
-D-glucose, and
-D-galactose (all 1.7 mM)
all failed to affect basal adenylate cyclase activity. In the presence
of GTP (10 µM), the mean reaction velocity was somewhat lower with
than without each ester; such a difference failed, however, to achieve
statistical significance (P > 0.1 or
more).
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Table 7.
Effect of -L-glucose pentaacetate,
-D-glucose pentaacetate, and
-D-galactose pentaacetate on adenylate
cyclase activity in a membrane-rich islet subcellular fraction
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In intact islets incubated for 60 min in the presence of 1.0 mM
isobutyl methylxanthine, the net production of cAMP was significantly increased (P < 0.05) above the basal
value by 10 mM L-leucine and was increased to an even
greater extent (P < 0.02) by 16.7 mM
D-glucose (Table 8). The
pentaacetate ester of
-D-glucose, but not that of
-L-glucose (1.7 mM each), also increased
(P < 0.05) the net generation of
cAMP above the basal value. In islets exposed to 10 mM
L-leucine, the mean value for cAMP production was slightly
higher in the presence of the glucose esters than in their absence
(Table 8). However, even in the case of
-D-glucose pentaacetate, such a difference failed to achieve statistical significance (P < 0.07).
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Table 8.
Effect of D-glucose, L-leucine,
-L-glucose pentaacetate, and
-D-glucose pentaacetate on net production
of cAMP by islets incubated for 60 min in the presence of 1.0 mM
isobutyl methylxanthine
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Phosphoinositide hydrolysis. At a 1.7 mM concentration,
-D-glucose pentaacetate and
-D-glucose pentaacetate, but not
-L-glucose pentaacetate, augmented significantly
(P < 0.001), over 30 min of
incubation in the presence of 10 mM LiCl and 10 mM
L-leucine, the net production of tritiated inositol
phosphates in islets that had been preincubated for 180 min at 5.0 mM
D-glucose in the presence of
myo-[2-3H]inositol
(40 µCi/ml; ~2.0 µM). Unexpectedly,
-D-galactose pentaacetate also augmented significantly
(P < 0.001) the production of
tritiated inositol phosphates (Table 9).
Relative to the paired control value recorded in the sole presence of
L-leucine, however, the effect of
-D-galactose pentaacetate was less marked
(P < 0.05) than that of
-D-glucose pentaacetate. Unesterified
D-glucose, also tested at a 1.7 mM concentration, increased
the mean value for the generation of radioactive inositol phosphates
(P < 0.01). The response to the
unesterified hexose failed, however, to achieve statistical
significance when judged from paired comparison within each experiment.
Incidentally, even the response to
-D-glucose pentaacetate (1.7 mM), which yielded the highest mean value among the
several esters, remained significantly lower
(P < 0.02) than that evoked by
unesterified D-glucose when tested at a high concentration (20.0 mM) known to provoke a close-to-maximal functional response.
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Table 9.
Effects of D-glucose and monosaccharide esters on tritiated
inositol phosphate production by islets incubated in the presence of
10.0 mM L-leucine
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This coincided with the fact that, in islets perifused for 70 min in
the presence of 10.0 mM L-leucine,
-D-glucose pentaacetate, like
-D-glucose
pentaacetate, administered from minute
31 onward enhanced insulin release, whereas
-L-glucose pentaacetate failed to do so (Fig.
4). Unesterified D-glucose,
tested at the same concentration as the ester (1.7 mM), first modestly
augmented leucine-induced insulin release, but this effect faded out
after ~20 min of exposure of the islets to the hexose (Fig.
4C). The difference between the
integrated release of insulin over the 40 min of administration of
D-glucose or the monosaccharide esters and the paired
reference value (min 30) corresponded to a mean change in insulin
output (expressed as
µU · islet
1 · min
1)
of 0.84 ± 0.31 and 0.50 ± 0.18 in the case of
- and
-D-glucose pentaacetate, which is distinct
(P < 0.01) from
0.19 ± 0.22 in the case of
-L-glucose pentaacetate and 0.02 ± 0.13 in the case of unesterified
D-glucose. Over the first 10 min of exposure
to the unesterified hexose, however, such a paired change in insulin output averaged 0.24 ± 0.12 µU · islet
1 · min
1.

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Fig. 4.
Time course for secretory response to -D-glucose
pentaacetate (A),
-D-glucose pentaacetate
(B), -L-glucose
pentaacetate (C), and unesterified
D-glucose (D), all
administered at a 1.7 mM concentration from the 31st min onward in
islets perifused for 70 min in the presence of 10.0 mM
L-leucine. Mean values (±SE or range of individual
variation when n = 2) refer to 2 (B and
C) or 4 (A and
D) individual experiments.
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Intracellular pH. The initial pH
(time 0) averaged 6.38 ± 0.04 (n = 58) in the absence of exogenous
nutrient, which is distinct (P < 0.001) from 6.85 ± 0.03 (n = 68)
and 6.86 ± 0.03 (n = 67) in the
presence of either 8.3 mM D-glucose or 10.0 mM
L-leucine.
The administration of either
-D-glucose pentaacetate or
-L-glucose pentaacetate (1.7 mM each) provoked an
initial and modest fall in pH (Fig. 5).
Such a fall persisted somewhat longer in the case of
-L-glucose pentaacetate than
-D-glucose
pentaacetate. In the latter case, the nadir value was reached 2-3
min after addition of the ester and was 0.05 ± 0.01 (n = 26) and 0.04 ± 0.01 (n = 18) lower than the paired control
value recorded just before introduction of the ester in the experiments
conducted in the presence of D-glucose and
L-leucine, respectively. In the case of
-L-glucose pentaacetate, the nadir value was reached ~5 min after addition of the ester, with the paired fall in pH relative to the value recorded at the 5th minute just before
administration of the ester averaging 0.08 ± 0.01 (n = 42) in the cells exposed to
D-glucose and 0.05 ± 0.01 (n = 49) in the cells exposed to L-leucine.

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Fig. 5.
Changes in intracellular pH evoked by -D-glucose
pentaacetate (1.7 mM; A) and
-L-glucose pentaacetate (1.7 mM;
B) administered for 30 min (301st to
2,100th s; first 2 vertical arrows) in dispersed islet cells perifused
throughout the experiment in the presence of 8.3 mM
D-glucose. Last 2 vertical arrows indicate the time at
which NH4Cl (20 mM) and sodium
acetate (10 mM) were added to the perifusate. Mean values (±SE)
refer to changes in pH relative to the paired initial reading
(time 0) and are derived from 26 (A) and 42 (B) individual measurements made in
distinct islet cells.
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|
Thereafter,
-D-glucose pentaacetate, but not
-L-glucose pentaacetate, provoked a progressive increase
in pH (Fig. 5). After 30 min of administration of these esters, the
paired difference from the basal value (minute
5) averaged, in the case of
-D-glucose pentaacetate, 0.27 ± 0.04 (n = 26)
and 0.53 ± 0.05 (n = 18) in cells
exposed to D-glucose and L-leucine,
respectively, compared (P < 0.001)
with 0.01 ± 0.02 (n = 42) and
0.02 ± 0.03 (n = 49) in the
case of
-L-glucose pentaacetate.
At the end of the experiments, the addition of
NH4Cl (20 mM) and sodium acetate
(10 mM) to the incubation medium provoked the expected alkalinization
and acidification of the islet cells. For instance, in the experiments
illustrated in Fig. 5, the changes in pH over periods of 5 min each
amounted to 0.25 ± 0.01 and
0.36 ± 0.02 after addition
of NH4Cl and sodium acetate,
respectively (n = 68 in both cases).
Cationic data. The participation of
cations in the secretory response to
-L-glucose
pentaacetate was also considered. For such a purpose, the effect of
this and other esters on 45Ca net
uptake was first examined in islets incubated for 90 min in the absence
or presence of either D-glucose (8.3 mM) or
L-leucine (10.0 mM). Both the hexose and amino acid
significantly increased (P < 0.001)
the net uptake of 45Ca above the
basal value (Table 10).
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Table 10.
Effects of -L-glucose pentaacetate,
-D-glucose pentaacetate, and
-D-galactose pentaacetate on
45Ca uptake in absence or presence of
D-glucose or L-leucine
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As judged by comparison of data recorded within the same experiments,
the mean value for 45Ca net uptake
was slightly lower in the presence of
-L-glucose pentaacetate (1.7 mM) than in its absence, with such a difference only
achieving statistical significance (P < 0.001) in the presence of L-leucine. Likewise,
-D-galactose pentaacetate (1.7 mM) failed to affect
significantly net uptake in the absence or presence of
D-glucose (8.3 mM) while decreasing
(P < 0.01) leucine-stimulated 45Ca uptake.
On the contrary,
-D-glucose pentaacetate (1.7 mM)
significantly increased 45Ca net
uptake (P < 0.01 or less) whether in
the absence or presence of D-glucose (8.3 mM) and only
failed to do so in the presence of L-leucine (10.0 mM).
Under all experimental conditions, the net uptake of
45Ca was significantly lower
(P < 0.001) in islets
exposed to
-L-glucose pentaacetate rather than
-D-glucose pentaacetate.
The possible effect of
-L-glucose pentaacetate (1.7 mM)
on cationic fluxes was further investigated in perifused islets
prelabeled with either 86Rb or
45Ca.
The effect of
-L-glucose pentaacetate (1.7 mM) on
86Rb outflow,
45Ca outflow, and insulin release
from prelabeled islets perifused in the absence of any other exogenous
nutrient is illustrated in Fig.
6A, top.
Within 3 min of exposure to the ester, the
86Rb fractional outflow rate was
0.50 ± 0.14 × 10
2/min lower
(n = 4;
P < 0.05) than the paired
theoretical value calculated by linear extrapolation of the
measurements made between minutes 31 and 45 (Fig.
6A,
top). Likewise, 5 min after removal of
-L-glucose pentaacetate, the
86Rb fractional outflow rate was
0.60 ± 0.03 × 10
2/min higher
(n = 4;
P < 0.001) than the paired value
calculated by linear extrapolation of the measurements made during the
last 15 min of exposure to the ester (minutes
56-70). This rapid,
sustained, and rapidly reversible decrease in
K+ conductance was not associated
with any obvious change in 45Ca
fractional outflow rate (Fig. 6A,
middle). Nevertheless, the administration of
-L-glucose pentaacetate caused a rapid
increase in insulin output (Fig. 6A,
bottom). Within 2 min of exposure to
the ester, the secretory rate was already 30 ± 12 nU · islet
1 · min
1
higher (n = 16;
P < 0.025) than the paired basal
value (minutes 42-45 inclusive).
After this initial increase (minute
46-54), a secondary rise in insulin release was
recorded from the 57th minute onward. This late and progressive
ascension in secretory rate continued for at least 20 min after removal
of the ester from the perifusate. Thus the value reached at the 90th
minute was 132 ± 58 nU · islet
1 · min
1
higher (n = 16;
P < 0.05) than the paired basal
value.

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Fig. 6.
Effect of -L-glucose pentaacetate (1.7 mM;
A) and -D-galactose
pentaacetate (1.7 mM; B),
administered from minutes 46 to
70 inclusive, on
86Rb fractional outflow rate
(FOR), 45Ca FOR, and insulin
release from islets perifused in the absence of any other exogenous
nutrient. Mean values (±SE) refer to 4 (top), 7-12
(middle), and 12-16
(bottom) individual experiments.
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In control experiments,
-D-galactose pentaacetate (1.7 mM) apparently also caused a decrease in
86Rb fractional outflow rate that
reached, after 3 min of exposure to the ester, a value 0.25 ± 0.15 × 10
2/min lower
(n = 4) than the paired theoretical
value calculated by linear extrapolation of the measurements made
between minutes 31 and
45 (Fig.
6B,
top). This initial decrease in
86Rb outflow failed, however, to
achieve statistical significance (P < 0.2). Likewise,
-D-galactose pentaacetate failed to
affect 45Ca fractional outflow
rate significantly. The value reached at minute
48 was only 0.06 ± 0.09 × 10
2/min
(n = 7;
P > 0.5) higher than the paired
theoretical value calculated by linear extrapolation of the data
collected between minutes 31 and
45 (Fig.
6B,
middle). No obvious effect of
-D-galactose pentaacetate on insulin release could be
detected (Fig. 6B,
bottom). Even at
minute 48, the secretory rate was not
significantly higher than the basal value (minutes
42-45 inclusive), with a paired difference of 0.04 ± 0.06 µU · islet
1 · min
1
(n = 12).
Electrophysiological data. In the
presence of 4 mM D-glucose,
-D-glucose
pentaacetate (1.7 mM) caused depolarization resulting in the generation
of spiking electrical activity in all five separate experiments (Fig.
7A).
Removal of the ester resulted in a gradual repolarization. Subsequent
exposure of the cell to a stimulatory concentration of
D-glucose (16 mM) resulted in intense electrical activity.
Likewise, in the presence of 4 mM D-glucose, application of
-L-glucose pentaacetate (1.7 mM) caused depolarization
and induced electrical activity (Fig.
7B). In general, the intensity of
electrical activity provoked by this ester was less than that observed
in response to the
-D-glucose ester (Fig.
7C).

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Fig. 7.
Perforated patch recordings of membrane potential from single isolated
rat B cells in the presence of 4 mM D-glucose.
A: effects of 1.7 mM
-D-glucose pentaacetate (D-GPA) followed by
16 mM D-glucose. B and
C: effects of 1.7 mM
-L-glucose pentaacetate (L-GPA) followed by
1.7 mM -D-glucose pentaacetate. Recording in
C was obtained from the same cell as
B, after a period of ~3 min.
Recordings are representative of those from 2-5 separate
experiments.
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In the presence of 10.0 mM L-leucine but absence of
glucose,
-D-glucose pentaacetate (1.7 mM) again caused a
marked depolarization of the membrane potential in all seven
experiments (Fig.
8A). Similarly,
-L-glucose pentaacetate (1.7 mM) also caused
a depolarization, although, again, this effect was consistently less
pronounced than that seen with the
-D-glucose ester
(Fig. 8B). In contrast,
-D-galactose pentaacetate (1.7 mM) failed to cause
depolarization in all three experiments conducted in the presence of
10.0 mM L-leucine (Fig.
8C). After exposure to the
D-galactose ester, the cells still responded to a
subsequent administration of
-D-glucose pentaacetate. It
should be noted that the spiking pattern of electrical activity
observed in response to the esters in the presence of 4 mM
D-glucose was seldom seen when the experiments were carried out in the presence of 10.0 mM L-leucine and absence of
glucose. In the latter case, the response to the esters was often a
relatively "silent" depolarization (see, for example, Fig.
8A). The reasons underlying this
difference are unclear.

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Fig. 8.
Perforated patch recordings of membrane potential from single isolated
rat B cells in the presence of 10.0 mM L-leucine and
absence of glucose. A: recordings from
4 different cells showing effects of 1.7 mM -D-glucose
pentaacetate (D-GPA). B:
recordings from 2 different cells showing the effects of 1.7 mM
-L-glucose pentaacetate (L-GPA).
C: recordings from 2 different cells
showing effects of 1.7 mM -D-galactose pentaacetate
(D-GALPA) followed by 1.7 mM -D-glucose
pentaacetate. Recordings are representative of those from 2-7
separate experiments.
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|
 |
DISCUSSION |
The present data confirm that
-L-glucose pentaacetate
displays, under suitable experimental conditions, a far-from-negligible insulinotropic action. For instance, it unmasked the secretory potential of both theophylline and repaglinide in islets incubated in
the absence of any exogenous nutrient. It also increased, in isolated B
cells exposed to L-leucine, the percentage of
plaque-forming cells and plaque area in a reverse hemolytic plaque
assay for insulin release. Within the limits of the present study, the
secretory response to the ester was most marked in the presence of a
stimulatory concentration of D-glucose, in which case as
little as 0.34 mM
-L-glucose pentaacetate was sufficient
to augment insulin output significantly. This finding is consistent
with the recent observation that
-L-glucose pentaacetate
increases the plasma insulin concentration when administered
intravenously to anesthetized rats in an amount not exceeding 8.8 nmol/g body wt (6).
-L-Glucose pentaacetate was efficiently taken up and
hydrolyzed in rat pancreatic islets. The equilibrium value for the
islet content of
-L-[1-14C]glucose
pentaacetate and its radioactive metabolites yielded a net uptake close
to 28 pmol/islet. This would correspond to an intracellular
concentration of ~25.5 mM, which is distinct from 1.7 mM for the
extracellular concentration of the ester. A large fraction of the ester
may be located, however, in the lipid phase of the islets. Islet
homogenates catalyzed the hydrolysis of
-L-glucose
pentaacetate. The generation of
L-[14C]glucose
from
-L-[1-14C]glucose
pentaacetate, as well as that of
14CO2
and radioactive acidic metabolites and amino acids from
-L-glucose penta[1-14C]acetate,
were also documented in intact islets.
The insulinotropic action of
-L-glucose pentaacetate
cannot be ascribed, however, to the nutritional value of this ester. Thus no significant phosphorylation of
L-[1-14C]glucose
could be detected in islet homogenates, and, in intact islets, neither
L-[1-14C]glucose
nor
-L-[1-14C]glucose
pentaacetate generated any sizable amount of
14CO2
or 14C-labeled acidic metabolites
or amino acids. The modest oxidation of the acetate residues liberated
by intracellular hydrolysis of
-L-glucose
penta-[1-14C]acetate
was obviously not sufficient to account for the stimulation of insulin
release by the L-glucose ester. When expressed as
14CO2
equivalent and assuming equal oxidation of the C-1 and C-2 of the
acetate residues, the oxidation of
-L-glucose
penta-[1-14C]acetate
(1.7 mM) averaged no more than 97.5 ± 30.8 fmol · islet
1 · min
1.
This value is negligible when compared with the generation of 14CO2
from
D-[U-14C]glucose
(8.3 mM), i.e., 1506.7 ± 60.0 fmol · islet
1 · min
1.
Yet, in the presence of 8.3 mM D-glucose, the enhancing
action of
-L-glucose pentaacetate on insulin release is
close to that otherwise provoked by a rise in hexose concentration of
~2.2 mM (see Table 6 and Fig. 1 in Ref. 21).
Moreover, the pentaacetate ester of
-L-glucose, like
that of
-D-mannose or
-D-fructose,
decreased the generation of
14CO2
by islets prelabeled with
[U-14C]palmitate. This
sparing action is probably attributable to the generation of
acetyl-coenzyme A from the acetate residues produced by intracellular
hydrolysis of
-L-glucose pentaacetate. Incidentally, the
latter sparing action was less pronounced
(P < 0.05) than that found in islets
exposed to
-D-fructose pentaacetate or
-D-mannose pentaacetate, as expected from the different
nutritional value of the hexose moiety of each ester. As judged by
comparison of data recorded within each individual experiment, the
sparing action of
-L-glucose pentaacetate and
-D-fructose pentaacetate indeed averaged, respectively,
56.6 ± 13.5 and 93.2 ± 18.1% of that of
-D-mannose pentaacetate (100.0 ± 10.3%;
n = 20 in all cases).
The pentaacetate ester of
-L-glucose and
-D-galactose also decreased the conversion of exogenous
D-[5-3H]glucose
to 3HOH and that of
D-[U-14C]glucose
to 14C-labeled acidic metabolites
and amino acids. Such a decrease in glycolytic flux could be due to
inhibition of phosphofructokinase, as a result of both a modest fall
in cytosolic pH and the production of citrate from the
acetate residues generated by hydrolysis of these esters. The sole
positive effect of
-L-glucose pentaacetate on the
catabolism of nutrients consisted of an increase of the paired ratio
between
D-[U-14C]glucose
oxidation and
D-[5-3H]glucose
utilization. Because such an effect was barely detectable in islets
exposed to
-D-galactose pentaacetate, it may somehow reflect an increase in ATP requirement linked to the stimulation of
secretory activity by the former, but not latter, ester.
In fair agreement with the metabolic data so far considered, several
aspects of the islet functional response to
-L-glucose pentaacetate differed from that normally evoked by nutrient
secretagogues, including
-D-glucose pentaacetate. First,
-L-glucose pentaacetate failed to stimulate biosynthetic
activity and even slightly decreased the tritiation of peptides in
islets stimulated by D-glucose or L-leucine in
the presence of
L-[4-3H]phenylalanine.
Second,
-L-glucose pentaacetate failed to augment above
basal values the cAMP content of islets incubated in the presence of
isobutyl methylxanthine. Third,
-L-glucose pentaacetate failed to increase significantly the production of tritiated inositol phosphate in islets prelabeled with
myo-[2-3H]inositol
and incubated in the presence of L-leucine and LiCl. Fourth,
-L-glucose pentaacetate failed to cause
intracellular alkalinization in dispersed islet cells. Fifth,
-L-glucose pentaacetate failed to enhance, and actually
tended to decrease, the net uptake of
45Ca by islets incubated in the
absence or presence of D-glucose or L-leucine.
Last,
-L-glucose pentaacetate failed to cause any sizable increase in 45Ca efflux
from prelabeled islets perifused in the absence of any exogenous
nutrient. This contrasts with the results obtained with
-D-glucose pentaacetate, which reproduces the effect of
unesterified D-glucose on these six functional variables
(21). It was also reported that, at variance with
-D-glucose pentaacetate,
-L-glucose pentaacetate does not display the potential of inhibiting glucagon release from isolated pancreases perfused in the presence of
L-leucine (12).
The present data are compatible, however, with the recent proposal that
-L-glucose pentaacetate itself may act directly on a
receptor system presenting some analogy with that involved in the
recognition of bitter compounds by taste buds (20). Under suitable
experimental conditions, the ester indeed decreased
86Rb efflux from prelabeled
islets, suggesting a decrease in
K+ conductance, and caused both
depolarization of the plasma membrane and induction of bioelectrical
activity in isolated rat B cells. A comparable situation was also
observed in mouse pancreatic islets in which
-L-glucose
pentaacetate may also provoke oscillations of cytosolic
Ca2+ (28). Incidentally, a direct
interaction of the ester with K+
channels is not ruled out by these observations.
In conclusion, therefore, it is proposed, as a working hypothesis, that
the insulinotropic action of
-L-glucose pentaacetate involves the direct activation by the ester itself of a yet
unidentified receptor system in the islet B cell.
 |
ACKNOWLEDGEMENTS |
We are grateful to M. Mahy, J. Schoonheydt, M. Urbain, and G. Vandenbroeck 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 IN208295 from Dirección General de Asuntos del Personal Académico
(Universidad Nacional Autónoma de México), grants from the
Spanish Fondo de Investigaciones Sanitarias (96/1383) and
Dirreción Generral de Investigaciones Científica y
Técnica (95/0048), Belgian Foundation for Scientific Medical
Research (3.4513.94), National Institute of Diabetes and Digestive and
Kidney Diseases Grant DK-41230, and the Wellcome Trust.
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. §1734 solely to indicate this fact.
Address for reprint requests: W. J. Malaisse, Laboratory of
Experimental Medicine, Brussels Free University, 808 Route de Lennik,
B-1070 Brussels, Belgium.
Received 5 June 1998; accepted in final form 28 August 1998.
 |
REFERENCES |
1.
Bakkali Nadi, A.,
F. Malaisse-Lagae,
and
W. J. Malaisse.
Insulinotropic action of meglitinide analogs: concentration-response relationship and nutrient dependency.
Diabetes Res. Clin. Pract.
27:
81-87,
1994.
2.
Brisson, G. R.,
F. Malaisse-Lagae,
and
W. J. Malaisse.
The stimulus-secretion coupling of glucose-induced insulin release. VII. A proposed site of action for adenosine-3',5'-cyclic monophosphate.
J. Clin. Invest.
51:
232-241,
1972[Medline].
3.
Carpinelli, A.,
and
W. J. Malaisse.
Regulation of 86Rb outflow from pancreatic islets. I. Reciprocal changes in the response to glucose, tetraethylammonium and quinine.
Mol. Cell. Endocrinol.
17:
103-110,
1980[Medline].
4.
Delgado, E.,
M. L. Villanueva-Peñacarrillo,
I. Valverde,
and
W. J. Malaisse.
Stimulation of protein biosynthesis in pancreatic islets by quinine.
Med. Sci. Res.
19:
439-440,
1991.
5.
Delvaux, A.,
M. M. Kadiata,
and
W. J. Malaisse.
Cytotoxicity of 2-deoxy-D-glucose and its tetraacetate ester in tumoral cell lines.
Oncol. Res.
4:
1295-1299,
1997.
6.
García-Martínez, J. A.,
J. Cancelas,
M. L. Villanueva-Peñacarrillo,
I. Valverde,
and
W. J. Malaisse.
Insulinotropic action of
-L-glucose pentaacetate in vivo (Abstract).
Endocrinologie
45:
275,
1998.
7.
Giroix, M.-H.,
A. Sener,
D. G. Pipeleers,
and
W. J. Malaisse.
Hexose metabolism in pancreatic islets. Inhibition of hexokinase.
Biochem. J.
223:
447-453,
1984[Medline].
8.
Gobbe, P.,
and
A. Herchuelz.
Does glucose decrease cytosolic free calcium in normal pancreatic islet cells?
Res. Commun. Chem. Pathol. Pharmacol.
63:
231-247,
1989[Medline].
9.
Herchuelz, A.,
and
W. J. Malaisse.
Regulation of calcium fluxes in pancreatic islets. Dissociation between calcium and insulin release.
J. Physiol. (Lond.)
283:
409-424,
1978[Abstract].
10.
Hiriart, M.,
M. C. Sanchez-Soto,
M. C. Ramirez-Medeles,
and
W. J. Malaisse.
Functional heterogeneity of single pancreatic B-cells stimulated by L-leucine and the methyl ester of succinic or glutamic acid.
Biochem. Mol. Med.
54:
133-137,
1995[Medline].
11.
Ladrière, L.,
M. M. Kadiata,
and
W. J. Malaisse.
Comparison between the effects of D-mannoheptulose and its hexaacetate ester upon D-glucose metabolism in rat erythrocytes.
Horm. Metab. Res.
30:
244-245,
1998[Medline].
12.
Leclercq-Meyer, V.,
and
W. J. Malaisse.
Dual mode of action of glucose pentaacetates upon hormonal secretion from the isolated perfused rat pancreas.
Am. J. Physiol.
275 (Endocrinol. Metab. 38):
E610-E617,
1998[Abstract/Free Full Text].
13.
Lowry, O. H.,
N. R. Rosebrough,
A. L. Farr,
and
R. J. Randall.
Protein measurement with the Folin phenol reagent.
J. Biol. Chem.
193:
265-275,
1951[Free Full Text].
14.
Malaisse, W. J.,
and
A. Delvaux.
Cytostatic effect of 2-deoxy-D-glucose and its tetraacetate ester in transformed mouse fibroblasts.
Med. Sci. Res.
25:
727-728,
1997.
15.
Malaisse, W. J.,
A. Delvaux,
J. Rasschaert,
and
M. M. Kadiata.
Cytotoxic action of 2-deoxy-D-glucose tetraacetate in tumoral pancreatic islet cells.
Cancer Lett.
125:
45-49,
1998[Medline].
16.
Malaisse, W. J.,
M.-H. Giroix,
F. Malaisse-Lagae,
and
A. Sener.
3-O-methyl-D-glucose transport in tumoral insulin-producing cells.
Am. J. Physiol.
251 (Cell Physiol. 20):
C841-C846,
1986[Abstract/Free Full Text].
17.
Malaisse, W. J.,
H. Jijakli,
S. Ulusoy,
L. Cook,
L. Best,
C. Viñambres,
M. L. Villanueva-Peñacarrillo,
I. Valverde,
and
A. Sener.
Insulinotropic action of methyl pyruvate. Secretory, cationic and biosynthetic aspects.
Arch. Biochem. Biophys.
335:
229-244,
1996[Medline].
18.
Malaisse, W. J.,
and
M. M. Kadiata.
Insulinotropic action of the polyacetate esters of two non-nutrient monosaccharides in normal and diabetic rats.
Int. J. Mol. Med.
2:
95-98,
1998.[Medline]
19.
Malaisse, W. J.,
M. M. Kadiata,
O. Scruel,
and
A. Sener.
Esterification of D-mannoheptulose confers to the heptose inhibitory action on D-glucose metabolism in parotid cells.
Biochem. Mol. Biol. Int.
44:
625-633,
1998[Medline].
20.
Malaisse, W. J.,
and
F. Malaisse-Lagae.
Bitter taste of monosaccharide pentaacetate esters.
Biochem. Mol. Biol. Int.
43:
1367-1371,
1997[Medline].
21.
Malaisse, W. J.,
C. Sánchez-Soto,
M. E. Larrieta,
M. Hiriart,
H. Jijakli,
C. Viñambres,
M. L. Villanueva-Peñacarrillo,
I. Valverde,
O. Kirk,
M. M. Kadiata,
and
A. Sener.
Insulinotropic action of
-D-glucose pentaacetate: functional aspects.
Am. J. Physiol.
273 (Endocrinol. Metab. 36):
E1090-E1101,
1997[Abstract/Free Full Text].
22.
Malaisse, W. J.,
and
A. Sener.
Hexose metabolism in pancreatic islets. Feedback control of D-glucose oxidation by functional events.
Biochim. Biophys. Acta
971:
246-254,
1988[Medline].
23.
Malaisse, W. J.,
A. Sener,
F. Malaisse-Lagae,
J. C. Hutton,
and
J. Christophe.
The stimulus-secretion coupling of amino acid-induced insulin release. VI. Metabolic interaction of L-glutamine and 2-ketoisocaproate in pancreatic islets.
Biochim. Biophys. Acta
677:
39-49,
1981[Medline].
24.
Malaisse-Lagae, F.,
and
W. J. Malaisse.
The stimulus-secretion coupling of glucose-induced insulin release. III. Uptake of 45Calcium by isolated islets of Langerhans.
Endocrinology
88:
72-80,
1971[Medline].
25.
Malaisse-Lagae, F.,
and
W. J. Malaisse.
Insulin release by pancreatic islets.
In: Methods in Diabetes Research, edited by J. Larner,
and S. L. Pohl. New York: Wiley, 1984, p. 147-152.
26.
Olivares, E.,
S. Picton,
L. E. Flores,
M. M. Kadiata,
and
W. J. Malaisse.
Failure of streptozotocin tetraacetate to undergo extensive hydrolysis and to inhibit D-glucose metabolism and insulinotropic action in rat pancreatic islets.
Cell Biochem. Funct.
16:
233-237,
1998[Medline].
27.
Reinhold, U.,
and
W. J. Malaisse.
Cytotoxic action of 2-deoxy-D-glucose tetraacetate upon human lymphocytes, fibroblasts and melanoma cells.
Int. J. Mol. Med.
1:
427-430,
1998.[Medline]
28.
Ropero, A. B., R. Pomares, J. V. Sanchez-Solo, A. Nadal, B. Soria, and W. J. Malaisse. Both
-D-glucose and
-L-glucose pentaacetates potentiate electrical activity
and cytosolic Ca2+ in mouse pancreatic islets.
Diabetologia 41, Suppl. 1: A143, 1998.
29.
Sener, A.,
S. Kawazu,
J. C. Hutton,
A. C. Boschero,
G. Devis,
G. Somers,
A. Herchuelz,
and
W. J. Malaisse.
The stimulus-secretion coupling of glucose-induced insulin release. XXXIII. Effect of exogenous pyruvate on islet function.
Biochem. J.
176:
217-232,
1978[Medline].
30.
Sener, A.,
and
W. J. Malaisse.
Stimulation by D-glucose of mitochondrial oxidative events in islet cells.
Biochem. J.
246:
89-95,
1987[Medline].
31.
Sener, A.,
N. Welsh,
F. Malaisse-Lagae,
M. M. Kadiata,
and
W. J. Malaisse.
Insulinotropic action of
-D-glucose pentaacetate: metabolic aspects.
Mol. Gen. Metab.
64:
135-147,
1998[Medline].
32.
Valverde, I.,
A. Vandermeers,
R. Anjaneyulu,
and
W. J. Malaisse.
Calmodulin activation of adenylate cyclase in pancreatic islets.
Science
206:
225-227,
1979[Medline].
33.
Vanhoutte, C.,
M. M. Kadiata,
A. Sener,
and
W. J. Malaisse.
Potentiation by its esterification of the inhibitory action of 2-deoxy-D-glucose on D-glucose metabolism and insulinotropic action.
Biochem. Mol. Biol. Int.
43:
189-195,
1997[Medline].
34.
Vanhoutte, C.,
A. Sener,
and
W. J. Malaisse.
Hydrolysis of hexose pentaacetate esters in rat pancreatic islets.
Biochim. Biophys. Acta
1405:
78-84,
1998[Medline].
35.
Vogel, A. I.
Textbook of Practical Organic Chemistry (3rd ed.). London: Longmans Green, 1956, p. 383.
36.
Wolfrom, M. L.,
and
A. Thompson.
Monosaccharides. D-Galactose.
In: Methods in Carbohydrate Chemistry, edited by R. L. Whistler,
and M. L. Wolfrom. London: Academic, 1962, vol. I, p. 120-122.
37.
Zawalich, W. S.,
M. Bonnet-Eymard,
and
W. J. Malaisse.
1,2,3-Tri(methylsuccinyl)glycerol ester stimulates inositol phosphate accumulation, insulin secretion and induces time-dependent potentiation of release.
Res. Commun. Pharmacol. Toxicol.
3:
11-22,
1998.
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