Insulinotropic action of beta -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

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The metabolism of beta -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 beta -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

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE POLYACETATE ESTERS of several monosaccharides were recently proposed as tools to increase the metabolic efficiency of the corresponding carbohydrates. For instance, alpha -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 alpha -D-glucose pentaacetate in pancreatic islet cells, the pentaacetate ester of beta -L-glucose was unexpectedly found also to stimulate insulin release, albeit to a lesser extent than alpha -D-glucose pentaacetate (21). The insulin-releasing capacity of beta -L-glucose pentaacetate does not seem to be attributable to the catabolism of its acetate moiety, since beta -D-galactose pentaacetate, which is as efficiently taken up and hydrolyzed in isolated islets as alpha -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 beta -L-glucose pentaacetate on several variables of islet metabolism and function, in search of a possible explanation for its insulinotropic action.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 beta -L-[1-14C]glucose pentaacetate or beta -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.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 ± 0.08per thousand 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 ± 0.09per thousand (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 beta -L-glucose pentaacetate in islet homogenates. In islet homogenates, the rate of acetate formation from alpha -D-glucose pentaacetate and beta -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 beta -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 beta -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 beta -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 beta -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).

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 beta -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.

Metabolism of beta -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 beta -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 alpha -D-[U-14C]glucose pentaacetate (31).

                              
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Table 1.   Metabolism of L-glucose, D-glucose, and beta -L-glucose pentaacetate

To characterize the metabolic fate of the acetyl moiety of beta -L-glucose pentaacetate, the production of 14C-labeled metabolites was measured in islets exposed to beta -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., beta -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 beta -L-glucose pentaacetate with the catabolism of other nutrients. Two series of experiments aimed at investigating the possible interference of beta -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 beta -L-glucose pentaacetate, alpha -D-mannose pentaacetate, or beta -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 beta -L-glucose pentaacetate, alpha -D-mannose pentaacetate, and beta -D-fructose pentaacetate on production of 14CO2 by islets prelabeled with L-[U-14C]glutamine or [U-14C]palmitate

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 alpha -D-mannose pentaacetate than beta -L-glucose pentaacetate, although such was not the case when the results obtained with the latter ester and beta -D-fructose pentaacetate were compared.

In the second series of experiments, the effects of beta -L-glucose pentaacetate or beta -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 beta -L-glucose pentaacetate but was increased less (P < 0.001) by beta -D-galactose pentaacetate.

                              
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Table 3.   Effect of beta -L-glucose pentaacetate and beta -D-galactose pentaacetate on metabolism of unesterified D-glucose (8.3 mM)

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 beta -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 beta -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 beta -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

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 beta -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 beta -L-glucose pentaacetate.

Secretory data. The insulinotropic action of beta -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 beta -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 beta -L-glucose pentaacetate on secretory response of rat pancreatic islet to Ca2+ deprivation, theophylline, and repaglinide

In the presence of 4.0 mM D-glucose, alpha -D-glucose pentaacetate (1.7 mM), but not beta -L-glucose pentaacetate (also 1.7 mM), significantly augmented insulin output (Table 5). Surprisingly, the increment in insulin release caused by alpha -D-glucose pentaacetate was lower (P < 0.001) in the presence of beta -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 alpha -D-glucose pentaacetate and beta -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 alpha -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 beta -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 beta -L-glucose pentaacetate somehow impairs the insulinotropic action of alpha -D-glucose pentaacetate.

Figure 3 illustrates the concentration-response relationship for the insulinotropic action of beta -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 beta -L-glucose pentaacetate.


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Fig. 3.   Effect of increasing concentrations of beta -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.

The secretory data presented so far also document the modulation of beta -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 beta -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 beta -L-glucose pentaacetate or alpha -D-glucose pentaacetate in the presence of 5 mM L-leucine

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 alpha -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 alpha -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 beta -L-glucose pentaacetate and alpha -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 beta -L-glucose pentaacetate.

To explore the possible participation of the adenylate cyclase-protein kinase A axis in the insulinotropic action of beta -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 beta -L-glucose, alpha -D-glucose, and alpha -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 beta -L-glucose pentaacetate, alpha -D-glucose pentaacetate, and alpha -D-galactose pentaacetate on adenylate cyclase activity in a membrane-rich islet subcellular fraction

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 alpha -D-glucose, but not that of beta -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 alpha -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, beta -L-glucose pentaacetate, and alpha -D-glucose pentaacetate on net production of cAMP by islets incubated for 60 min in the presence of 1.0 mM isobutyl methylxanthine

Phosphoinositide hydrolysis. At a 1.7 mM concentration, alpha -D-glucose pentaacetate and beta -D-glucose pentaacetate, but not beta -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, beta -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 beta -D-galactose pentaacetate was less marked (P < 0.05) than that of alpha -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 beta -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

This coincided with the fact that, in islets perifused for 70 min in the presence of 10.0 mM L-leucine, alpha -D-glucose pentaacetate, like beta -D-glucose pentaacetate, administered from minute 31 onward enhanced insulin release, whereas beta -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 alpha - and beta -D-glucose pentaacetate, which is distinct (P < 0.01) from -0.19 ± 0.22 in the case of beta -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 alpha -D-glucose pentaacetate (A), beta -D-glucose pentaacetate (B), beta -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.

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 alpha -D-glucose pentaacetate or beta -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 beta -L-glucose pentaacetate than alpha -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 beta -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 alpha -D-glucose pentaacetate (1.7 mM; A) and beta -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.

Thereafter, alpha -D-glucose pentaacetate, but not beta -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 alpha -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 beta -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 beta -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 beta -L-glucose pentaacetate, alpha -D-glucose pentaacetate, and beta -D-galactose pentaacetate on 45Ca uptake in absence or presence of D-glucose or L-leucine

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 beta -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, beta -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, alpha -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 beta -L-glucose pentaacetate rather than alpha -D-glucose pentaacetate.

The possible effect of beta -L-glucose pentaacetate (1.7 mM) on cationic fluxes was further investigated in perifused islets prelabeled with either 86Rb or 45Ca.

The effect of beta -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 beta -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 beta -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 beta -L-glucose pentaacetate (1.7 mM; A) and beta -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.

In control experiments, beta -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, beta -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 beta -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, alpha -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 beta -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 alpha -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 alpha -D-glucose pentaacetate (D-GPA) followed by 16 mM D-glucose. B and C: effects of 1.7 mM beta -L-glucose pentaacetate (L-GPA) followed by 1.7 mM alpha -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.

In the presence of 10.0 mM L-leucine but absence of glucose, alpha -D-glucose pentaacetate (1.7 mM) again caused a marked depolarization of the membrane potential in all seven experiments (Fig. 8A). Similarly, beta -L-glucose pentaacetate (1.7 mM) also caused a depolarization, although, again, this effect was consistently less pronounced than that seen with the alpha -D-glucose ester (Fig. 8B). In contrast, beta -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 alpha -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 alpha -D-glucose pentaacetate (D-GPA). B: recordings from 2 different cells showing the effects of 1.7 mM beta -L-glucose pentaacetate (L-GPA). C: recordings from 2 different cells showing effects of 1.7 mM beta -D-galactose pentaacetate (D-GALPA) followed by 1.7 mM alpha -D-glucose pentaacetate. Recordings are representative of those from 2-7 separate experiments.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The present data confirm that beta -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 beta -L-glucose pentaacetate was sufficient to augment insulin output significantly. This finding is consistent with the recent observation that beta -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).

beta -L-Glucose pentaacetate was efficiently taken up and hydrolyzed in rat pancreatic islets. The equilibrium value for the islet content of beta -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 beta -L-glucose pentaacetate. The generation of L-[14C]glucose from beta -L-[1-14C]glucose pentaacetate, as well as that of 14CO2 and radioactive acidic metabolites and amino acids from beta -L-glucose penta[1-14C]acetate, were also documented in intact islets.

The insulinotropic action of beta -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 beta -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 beta -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 beta -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 beta -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 beta -L-glucose, like that of alpha -D-mannose or beta -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 beta -L-glucose pentaacetate. Incidentally, the latter sparing action was less pronounced (P < 0.05) than that found in islets exposed to beta -D-fructose pentaacetate or alpha -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 beta -L-glucose pentaacetate and beta -D-fructose pentaacetate indeed averaged, respectively, 56.6 ± 13.5 and 93.2 ± 18.1% of that of alpha -D-mannose pentaacetate (100.0 ± 10.3%; n = 20 in all cases).

The pentaacetate ester of beta -L-glucose and beta -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 beta -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 beta -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 beta -L-glucose pentaacetate differed from that normally evoked by nutrient secretagogues, including alpha -D-glucose pentaacetate. First, beta -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, beta -L-glucose pentaacetate failed to augment above basal values the cAMP content of islets incubated in the presence of isobutyl methylxanthine. Third, beta -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, beta -L-glucose pentaacetate failed to cause intracellular alkalinization in dispersed islet cells. Fifth, beta -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, beta -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 alpha -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 alpha -D-glucose pentaacetate, beta -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 beta -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 beta -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 beta -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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Am J Physiol Endocrinol Metab 275(6):E993-E1006
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