Laboratory of Experimental Medicine, Brussels Free University, B-1070 Brussels, Belgium
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ABSTRACT |
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The effects of - and
-2-deoxy-D-glucose
tetraacetate (1.7 and 8.5 mM) on insulin, somatostatin, and glucagon
secretion from isolated rat pancreases perfused in the presence of 8.3 mM D-glucose were compared with
those of unesterified
2-deoxy-D-glucose tested at the
same two concentrations. The unesterified glucose analog caused, in a
concentration-related manner, inhibition of glucose-induced insulin and
somatostatin release and augmentation of glucagon secretion. The two
anomers of 2-deoxy-D-glucose
tetraacetate, however, increased the secretion rate of all three
hormones; this effect was also related to the concentration of the
esters. No obvious anomeric specificity of the secretory response to
2-deoxy-D-glucose tetraacetate
was observed. These findings indicate that the insulinotropic action of
hexose esters cannot be accounted for solely by the metabolic effect of
their glucidic moieties. They suggest that the A, B, and D
cells of the endocrine pancreas are each equipped with a receptor
system responsible for the direct recognition of monosaccharide esters
as secretagogues. They further support the view that a paracrine effect
of insulin on glucagon-producing cells does not represent a major
component in the regulation of their secretory activity.
insulin secretion; glucagon secretion; somatostatin secretion; rat pancreas perfusion
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INTRODUCTION |
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THE RECENT INTRODUCTION of selected esters of
monosaccharides in biomedical research has allowed an increase in the
nutritional value or metabolic efficiency of several hexoses and their
antimetabolic analogs (10). For instance,
-D-glucose pentaacetate
augments glycolytic flux and insulin release in rat pancreatic islets
to a larger extent than unesterified
D-glucose tested at the same concentration as its ester (21, 27). Likewise,
D-mannoheptulose hexaacetate
inhibits D-glucose
phosphorylation in different cell types otherwise resistant to the
unesterified heptose (4, 17). Last,
2-deoxy-D-glucose tetraacetate
is more efficient than the unesterified
D-glucose analog in inhibiting
cell growth in various lines of tumoral cells (1, 12, 13, 25). These
situations appear attributable to the fact that the monosaccharide
esters penetrate into intact cells without requiring the intervention of a specific carrier system and then undergo enzymatic hydrolysis inside the cells, so that large amounts of their carbohydrate moiety
then become available as either nutrient or metabolic inhibitor.
In the course of these investigations, however, esters of either
nonmetabolized hexoses, e.g.,
-L-glucose pentaacetate (21), or metabolic inhibitors, e.g.,
2-deoxy-D-glucose tetraacetate (14), were unexpectedly found to also display positive insulinotropic action. This cannot be attributed to the catabolism of their acetate moieties, because other hexose esters, e.g.,
D-galactose pentaacetate, which
are as efficiently taken up and hydrolyzed in pancreatic islets (27,
29), fail to stimulate, and may even inhibit, insulin secretion (15,
21). It was speculated however that the paradoxical stimulation of
insulin release by the esters of nonmetabolized hexoses or metabolic
inhibitors might result from the direct interaction of the ester with a
receptor system, possibly displaying analogy with that involved in the
recognition of bitter compounds by taste buds (19). It was also
proposed that advantage could be taken of the positive or negative
insulinotropic action of esters of nonmetabolized or poorly metabolized
hexoses, such as
-L-glucose
pentaacetate or
-D-galactose
pentaacetate, in the treatment of non-insulin-dependent diabetes
mellitus (16) or persistent hyperinsulinemic hypoglycemia (15).
To gain further insight into the postulated direct action of monosaccharide esters on a specific receptor system, we have now compared the effects of the two anomers of 2-deoxy-D-glucose tetraacetate and of unesterified 2-deoxy-D-glucose on the secretion of insulin, somatostatin, and glucagon by the isolated perfused rat pancreas. These experiments were conducted in the presence of 8.3 mM D-glucose to facilitate the detection of either an enhancing or inhibitory action of the tested agents on insulin release. Both unesterified 2-deoxy-D-glucose and its tetraacetate esters were used at either a 1.7 or 8.5 mM concentration to explore a possible dual effect, both positive and negative, of these agents on hormonal secretion by the endocrine pancreas (14).
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MATERIALS AND METHODS |
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The - and
-tetraacetate esters of
2-deoxy-D-glucose were
synthesized by a method adapted from that described elsewhere (30). 2-Deoxy-D-glucose was purchased
from Sigma (St. Louis, MO).
Two female (B & K Universal, Hull, UK) and 10 male (Iffa Credo,
L'Arbresle, France) fed Wistar rats were used in the present study
(Table 1). The animals were anesthetized
with an intraperitoneal injection of pentobarbital sodium (46 mg/kg),
and the pancreas was perfused without recirculation through both the
celiac and superior mesenteric arteries, as described elsewhere (18). A slight modification of this procedure was introduced. The duodenum was
not excluded during the surgical procedure, and the
secretions from the intestine and exocrine pancreas were diverted
through a plastic tubing that was secured in the upper part of the
duodenum.
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The basal salt-balanced solution (18) contained D-glucose (8.3 mM), dextran (clinical grade; 40 g/l; Sigma), and bovine serum albumin (RIA grade; 5 g/l; Sigma). It was supplemented, as required, with 2-deoxy-D-glucose or its tetraacetate esters (1.7 and 8.5 mM), with separate reservoirs. All solutions were continuously gassed (95% O2-5% CO2), with a resulting pH of 7.4. They were directed to the pancreas-duodenum preparation at a temperature of 37°C with a peristaltic pump (Minipuls 3, Gilson, Villiers-le-Bel, France).
The techniques used for the measurement of plasma glucose and insulin concentrations, perfusion pressure, pancreatic insulin, glucagon, and somatostatin content and release were identical to those detailed previously (6, 9).
The oscillatory pattern of hormonal release was assessed by a procedure reported previously (5). The mean hormonal output in each individual experiment over a given period of time was computed by planimetry from all measurements made over that period.
The vertical dotted lines on Figs. 1-4 were corrected for the dead space of the perfusion device (7). To allow for a better comparison of the responses of insulin, somatostatin, and glucagon to 2-deoxy-D-glucose and its tetraacetate esters, the secretory rates recorded for each individual hormone were expressed with an identical scale (see Figs. 1-3). However, because the hormonal responses to 2-deoxy-D-glucose were in a lower range than those otherwise recorded, the results obtained with this glucose analog were additionally drawn with an extended scale (see Fig. 4).
The results (see Table 1 and Figs. 1-4) are presented as means ± SE together with the number of individual observations (n = 4 in each series of perfusions). The statistical significance of differences between mean values was assessed by use of Student's two-tailed paired or unpaired t-test or one-way analysis of variance (ANOVA) as appropriate (Instat 2, Graphpad Software, San Diego, CA). The null hypothesis was rejected for P values < 0.05.
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RESULTS |
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Characteristics of the rats and perfusion parameters. None of the variables studied differed statistically among the three series of experiments. Their overall mean value (n = 12) averaged 376 ± 19 g for body weight; 4.9 ± 0.1 mM and 72.2 ± 6.9 µU/ml for the plasma glucose and insulin concentrations, respectively, measured before anesthesia; 1.14 ± 0.07 g for the pancreatic wet weight; 136.8 ± 10.4, 0.38 ± 0.03, and 5.8 ± 0.3 µg for the pancreatic content in insulin, somatostatin, and glucagon, respectively; 1.50 ± 0.03 ml/min for the flow rate; and 20.5 ± 0.9 and 20.4 ± 0.7 mmHg for the perfusion pressure at minutes 20 and 105, respectively.
Basal insulin, somatostatin, and glucagon release. During the basal period, the output of insulin in the presence of 8.3 mM D-glucose amounted to 9 ± 1, 8 ± 2, and 8 ± 2 ng/min in the
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Effects of
-2-deoxy-D-glucose
tetraacetate,
-2-deoxy-D-glucose
tetraacetate, and 2-deoxy-D-glucose
on insulin release.
Both the
- and
-2-deoxy-D-glucose
tetraacetate esters stimulated, in a concentration-dependent
manner, the release of insulin (Fig. 1,
top and
middle; Table 1).
Effects of
-2-deoxy-D-glucose
tetraacetate,
-2-deoxy-D-glucose
tetraacetate, and 2-deoxy-D-glucose
on somatostatin release.
The
- and
-2-deoxy-D-glucose
tetraacetate esters concentration dependently stimulated the
release of somatostatin (Fig. 2, top
and middle; Table 1).
Effects of
-2-deoxy-D-glucose
tetraacetate,
-2-deoxy-D-glucose
tetraacetate, and 2-deoxy-D-glucose
on glucagon release.
Both
- and
-2-deoxy-D-glucose
tetraacetate stimulated the secretion of glucagon, but such a
stimulation was only prominent in the presence of the higher 8.5 mM
concentration of the esters (Fig. 3,
top and
middle; Table 1).
Effects of
-2-deoxy-D-glucose
tetraacetate,
-2-deoxy-D-glucose
tetraacetate, and 2-deoxy-D-glucose
on perfusion pressure.
- And
-2-deoxy-D-glucose
tetraacetate at 1.7 mM and
2-deoxy-D-glucose did not modify
the perfusion pressure (data not shown). At the concentration of 8.5 mM,
- and
-2-deoxy-D-glucose
tetraacetate induced a transient increase in perfusion pressure. The
increase was comparable for the
- and
-esters and, at its peak
value at minute
74, did not exceed 1.4 ± 0.3 and
1.1 ± 0.6 mmHg, respectively.
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DISCUSSION |
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The present results confirm prior observations on a concentration-related inhibitory action of 2-deoxy-D-glucose on glucose-stimulated insulin secretion (20, 21). This suppressing action coincides with and is probably attributable to inhibition of D-glucose metabolism in pancreatic islet cells (27). Our results also document that unesterified 2-deoxy-D-glucose inhibits somatostatin secretion and enhances glucagon release by isolated perfused rat pancreases exposed to 8.3 mM D-glucose, and these effects are concentration related. A partial relief by 2-deoxy-D-glucose from the inhibitory action of D-glucose on glucagon release was already reported in either rat pancreatic islets exposed to 10 mM L-arginine (3) or the isolated perfused rat pancreas (31). To our knowledge, no information is available on the effect of 2-deoxy-D-glucose on somatostatin release. The present results are compatible with the view that D-glucose metabolism stimulates hormonal release from both B and D cells, while it inhibits glucagon secretion from A cells (2, 22, 23).
Both the - and
-anomer of
2-deoxy-D-glucose tetraacetate,
however, augmented insulin, somatostatin, and glucagon secretion from
the pancreases exposed to 8.3 mM
D-glucose. This positive tropic
action of the tetraacetate esters failed to display any obvious
anomeric specificity and was, in all cases, more pronounced at a high
(8.5 mM), rather than a low (1.7 mM), concentration of the esters.
Under vastly different experimental conditions, namely in isolated
pancreatic islets concomitantly, but not sequentially, exposed
throughout a prolonged incubation of 90 min to 8.3 mM D-glucose and
2-deoxy-D-glucose tetraacetate,
the ester was found to enhance insulin release, when tested at a low
concentration of 1.7 mM, and to inhibit glucose-stimulated insulin
output, when tested at a much higher concentration of 10.0 mM, both the
enhancing and inhibitory action of the ester displaying
-anomeric
preference (14).
The apparent disparity between the two series of experiments is
probably accounted for, in part at least, by the fact that the rate of
2-deoxy-D-glucose
tetraacetate hydrolysis in islet homogenates, which indeed displays
preference for the -anomer, requires prolonged exposure of intact
islet cells to the ester to generate an amount of the unesterified
D-glucose analog sufficient to
inhibit glycolysis (24). The present results are likely, therefore, to
refer mainly to the postulated direct action of the esters themselves
on hormonal release, thought to be mediated by activation of a specific
receptor system.
If so, the present findings suggest that the three major cell types of the endocrine pancreas, despite their different responsiveness to unesterified D-glucose, are all equipped with the previously mentioned receptors for 2-deoxy-D-glucose tetraacetate. Because the latter ester augments glucagon secretion, as well as insulin and somatostatin output, the second messenger(s) generated by such receptors should, at the first glance, belong to those few coupling factors that may exert a comparable positive secretory effect in A, B, and D cells. Alternatively, the binding of 2-deoxy-D-glucose tetraacetate to its receptor could conceivably lead to the production of distinct messengers in these three cell types.
Whatever the identity of such messengers, the present comparison between the effects of 2-deoxy-D-glucose and its ester on insulin, somatostatin, and glucagon release convincingly documents, in our opinion, that the functional response of the endocrine pancreas to monosaccharide esters cannot be fully accounted for by the metabolism of or metabolic response to their carbohydrate moiety. Moreover, the finding that 2-deoxy-D-glucose tetraacetate augmented glucagon release, despite concomitant stimulation of insulin release, provides further support to our contention that the paracrine effect of insulin plays little, if any, role in the regulation of glucagon secretion (8, 28).
The receptor system postulated to be activated by the esters of
2-deoxy-D-glucose remains to be
identified. The effects of these esters on insulin, somatostatin, and
glucagon release are comparable with those of
-L-glucose pentaacetate in
the perfused rat pancreas (7). In the latter case, it was proposed that the stimulation of insulin release may result from the direct interaction of the ester itself with a receptor system similar to that
involved in the recognition of bitter compounds by taste buds. Indeed,
-L-glucose pentaacetate
displays a bitter taste (19). It causes depolarization of the plasma
membrane and resulting spike activity in single isolated rat B cells,
in a manner comparable with that documented in taste buds exposed to
bitter compounds (11). Moreover, purified islet B cells were recently
found to express the
-gustducin G protein involved in the
recognition of such bitter compounds by taste buds (J. Rasschaert and W. J. Malaisse, unpublished observations). The
-anomer of L-glucose also
increases cytosolic Ca2+
concentration in mouse islets (26). The sequence of cationic and
secretory events evoked by
-L-glucose pentaacetate, and
presumably other esters of nonmetabolized monosaccharides, in islet B
cells is thus reminiscent of that operative in response to stimulation by D-glucose. This analogy could
conceivably account for the present finding that the anomers of
2-deoxy-D-glucose tetraacetate
did not suppress the oscillatory pattern of insulin release previously documented in rat pancreases perfused in the sole presence of 8.3 mM
D-glucose (5).
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ACKNOWLEDGEMENTS |
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We are grateful to J. Marchand for technical assistance and C. Demesmaeker for secretarial help.
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FOOTNOTES |
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This study was supported by a Concerted Research Action (94/99-183) of the French Community of Belgium.
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 correspondence and reprint requests: W. J. Malaisse, Laboratory of Experimental Medicine, Brussels Free University, 808 Route de Lennik, B-1070 Brussels, Belgium (E-mail: malaisse{at}med.ulb.ac.be).
Received 12 August 1998; accepted in final form 15 December 1998.
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