Insulinotropic action of alpha -D-glucose pentaacetate: functional aspects

Willy J. Malaisse, Carmen Sánchez-Soto, M. Elena Larrieta, Marcia Hiriart, Hassan Jijakli, Concepción Viñambres, María L. Villanueva-Peñacarrillo, Isabel Valverde, Ole Kirk, Marcel M. Kadiata, and Abdullah Sener

Laboratory of Experimental Medicine, Brussels Free University, B-1070 Brussels, Belgium; Department of Biophysics, Institute of Cellular Physiology, Universidad Nacional Autónoma de México, Mexico City, DF-04510 Mexico City, Mexico; Fundación Jiménez Díaz, 28040 Madrid, Spain; and Novo Nordisk, DK-2880 Bagsvaerd, Denmark

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The functional determinants of the insulinotropic action of alpha -D-glucose pentaacetate were investigated in rat pancreatic islets. The ester mimicked the effect of nutrient secretagogues by recruiting individual B cells into an active secretory state, stimulating proinsulin biosynthesis, inhibiting 86Rb outflow, and augmenting 45Ca efflux from prelabeled islets. The secretory response to the ester was suppressed in the absence of Ca2+ and potentiated by theophylline or cytochalasin B. The generation of acetate from the ester apparently played a small role in its insulinotropic action. Thus acetate, methyl acetate, ethyl acetate, alpha -D-galactose pentaacetate, and beta -D-galactose pentaacetate all failed to stimulate insulin release. The secretory response to alpha -D-glucose pentaacetate was reproduced by beta -D-glucose pentaacetate and, to a lesser extent, by beta -L-glucose pentaacetate. It differed from that evoked by unesterified D-glucose by its resistance to 3-O-methyl-D-glucose, D-mannoheptulose, and 2-deoxy-D-glucose. It is concluded that the insulinotropic action of alpha -D-glucose pentaacetate, although linked to the generation of the hexose from its ester, entails a coupling mechanism that is not identical to that currently implied in the process of glucose-induced insulin release.

pancreatic islets; insulin release

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE PENTAACETATE ESTER of alpha -D-glucose was recently reported to stimulate insulin release from rat pancreatic islets and proposed as a tool for the supply of the sugar to cells by a process that might not involve the carrier-mediated transport of the hexose across the plasma membrane (9). To provide further evidence to support this hypothesis and to elucidate the mode of action of alpha -D-glucose pentaacetate in islet cells, we have now scrutinized the metabolic, ionic, and functional aspects of alpha -D-glucose pentaacetate insulinotropic action. This report deals mainly with the third of these three themes.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The pentaacetyl esters of alpha -D-glucose, beta -D-glucose, and beta -L-glucose, as well as D-mannoheptulose, 2-deoxy-D-glucose, theophylline, and cytochalasin B, were obtained from Sigma (St. Louis, MO). Acetate (sodium salt) and 3-O-methyl-D-glucose were purchased from Merck (Darmstadt, Germany) and Aldrich (Milwaukee, WI), respectively. The pentaacetyl esters of alpha -D-galactose and beta -D-galactose, methyl acetate, and ethyl acetate were synthesized by methods described elsewhere (18, 19).

Except if otherwise mentioned, all experiments were conducted in pancreatic islets isolated by the collagenase method (12) from Wistar rats.

The procedures used for measuring insulin release from incubated (12) or perifused (5) islets, for quantification of secretory activity of isolated B cells in a reverse hemolytic plaque assay (6), to assess biosynthetic activity in islets exposed to L-[4-3H]phenylalanine (4), and to monitor 86Rb (3) and 45Ca (5) efflux from prelabeled islets were identical to those described in the cited references. In the chromatographic procedure used to separate tritiated peptides in islet homogenates, the site of elution of nonhormonal peptides, proinsulin, and insulin corresponds, respectively, to fractions 3-10, 10-16, and 16-30 (see Fig. 3).

All results are expressed as means ± SE together with the number of individual observations (n) or degree of freedom (df). The statistical significance of differences between mean values was assessed by either analysis of variance or Student's t-test. Such comparisons are restricted to data collected in close to equal numbers within the same experiments. Each individual measurement was made in a separate group of islets.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Insulinotropic action of alpha -D-glucose pentaacetate. At a concentration of 1.7 mM, which is close to the limit of solubility of the ester, alpha -D-glucose pentaacetate caused a three- to fourfold increase of basal insulin release (Table 1). The secretory response to the ester was considerably enhanced (P < 0.001) by either theophylline (1.4 mM) or cytochalasin B (0.02 mM), although neither the phosphodiesterase inhibitor nor the mold metabolite affects basal insulin secretion (2, 13). The insulinotropic action of alpha -D-glucose pentaacetate was suppressed in the absence of extracellular Ca2+, the basal release of insulin being higher (P < 0.001) in Ca2+-deprived islets than at normal extracellular Ca2+ concentration.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Modulation of secretory response to hexose esters by extracellular Ca2+ concentration, theophylline, and cytochalasin B

When alpha -D-glucose pentaacetate (1.7 mM) was tested in islets exposed to increasing concentrations of D-glucose, the measurements of insulin output were compatible with a shift to the left of the sigmoidal relationship between secretory rate and hexose concentration, the ester failing to significantly affect (P > 0.13) the hormonal release at a high concentration (16.7 mM) of D-glucose (Fig. 1). As judged from the data recorded in the low range of hexose concentrations (up to 8.3 mM), the insulinotropic action of 1.7 mM alpha -D-glucose pentaacetate was comparable to that of ~5.6 mM D-glucose.


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 1.   Effect of increasing concentrations of D-glucose on insulin release from islets incubated in absence (bullet ) or presence of either 1.7 mM alpha -D-glucose pentaacetate (open circle ) or 1.7 mM beta -D-galactose pentaacetate (down-triangle). Mean values (±SE) refer to 24-71 individual determinations. Solid and dotted lines are drawn in parallel up to 8.3 mM D-glucose.

In a reverse hemolytic plaque assay of insulin secretion from individual B cells, alpha -D-glucose pentaacetate (1.0 mM) enhanced the percentage of plaque-forming cells by ~80%, doubled the amount of insulin secreted by the single cells (plaque area), and caused a fourfold increase of overall insulin secretion from islet cells incubated in the presence of 5.0 mM L-leucine (Table 2; P < 0.001 in all cases). Neither D-glucose nor beta -D-galactose pentaacetate, tested at the same concentration (1.0 mM) as alpha -D-glucose pentaacetate, significantly affected the indexes of secretory activity in the B cells exposed to the branched-chain amino acid, except for a modest increase of the insulin secretion index (P < 0.05) in cells exposed to the D-galactose ester.

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Secretory activity in single B cells exposed to various nutrients

In islets perifused at normal Ca2+ concentration in the absence of any other exogenous nutrient, alpha -D-glucose pentaacetate (1.7 mM) provoked a rapid, sustained, and rapidly reversible decrease in 86Rb outflow from perifused islets, this coinciding with stimulation of both 45Ca efflux and insulin release (Fig. 2). The latter two effects, but not the decrease in 86Rb outflow, were suppressed when the experiments were conducted in the absence of extracellular Ca2+ (data not shown).


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 2.   Effect of alpha -D-glucose pentaacetate (1.7 mM), administered from min 46 to min 70, on 86Rb (A) and 45Ca (B) fractional outflow rate (FOR) and insulin output (C) from perifused islets. Mean values (±SE) refer to 8 (A and B) and 15 (C) individual experiments.

The D-glucose ester also stimulated biosynthetic activity in islets exposed to L-[4-3H]phenylalanine (Table 3).

                              
View this table:
[in this window]
[in a new window]
 
Table 3.   Effect of nutrients and D-mannoheptulose on incorporation of L-[4-3H]phenylalanine into TCA-precipitable and TCA-soluble material after 90-min incubation in the presence of tritiated amino acid (4.0 µM)

In the absence of D-glucose, alpha -D-glucose pentaacetate (1.7 mM) slightly increased the incorporation of the tritiated amino acid (4 µM) into trichloroacetic acid (TCA)-precipitable material. D-Mannoheptulose (10.0 mM) failed to affect the biosynthetic, as well as secretory (see below), response to alpha -D-glucose pentaacetate. The former response achieved statistical significance (P < 0.05) only when the results obtained in islets exposed to the ester, whether in the absence or presence of the heptose, were expressed relative to their paired value recorded in islets deprived of exogenous nutrient, such a paired comparison yielding a mean ratio of 125.7 ± 12.4% (n = 24). In the presence of 4.2 mM D-glucose, which augmented the synthesis of tritiated peptides relative to basal value (P < 0.001), alpha -D-glucose pentaacetate further increased (P < 0.001) biosynthetic activity. The incorporation of L-[4-3H]phenylalanine into TCA-precipitable material observed in the presence of both 4.2 mM D-glucose and 1.7 mM alpha -D-glucose pentaacetate was comparable to that otherwise found in the sole presence of 7.0 mM D-glucose; it remained significantly lower (P < 0.001), however, than that recorded in the presence of 16.7 mM D-glucose. L-Leucine (10 mM) tended to decrease the labeling of islet peptides, but this effect failed to achieve statistical significance as judged by comparison of either data collected within each experiment or the overall mean values listed in Table 3. In the presence of the branched-chain amino acid, alpha -D-glucose pentaacetate doubled (P < 0.005) the synthesis of tritiated peptides. As judged from comparisons made within each experiment, none of the nutrients or combination of nutrients listed in Table 3 significantly affected either the pool of TCA-soluble radioactive material or the paired ratio between TCA-precipitable and total radioactive content of the islets.

To further characterize the influence of alpha -D-glucose pentaacetate and other secretagogues on islet biosynthetic activity, the tritiated peptides were separated by gel chromatography (Fig. 3). D-Glucose (4.2 and 16.7 mM) caused a concentration-related increase (P < 0.05 or less) of the synthesis of nonhormonal peptides (void volume), proinsulin, and insulin (Table 4). At the highest concentration of the hexose, the biosynthesis of proinsulin was preferentially stimulated (P < 0.01), the (pro)insulin-to-total paired ratio for the labeling of islet peptides increasing from 30.4 ± 2.2 to 45.7 ± 4.7% (n = 8 in both cases). The fractional conversion of proinsulin to insulin was not significantly affected, however, by D-glucose, with mean insulin-to-(pro)insulin paired ratios of 28.2 ± 2.6 and 35.1 ± 3.0% in the absence and presence of 16.7 mM D-glucose, respectively. Likewise, L-leucine (10.0 mM) preferentially stimulated (P < 0.05) the labeling of hormonal peptides, while failing to significantly affect the insulin-to-(pro)insulin ratio. In the presence of 4.2 mM D-glucose, alpha -D-glucose pentaacetate augmented (P < 0.005) the total synthesis of (pro)insulin, whereas the increase in the radioactive content of the void volume failed to achieve statistical significance. Nevertheless, the ester did not significantly affect the paired (pro)insulin-to-total ratio for the tritiation of islet peptides. A comparable situation prevailed in islets exposed to 10.0 mM L-leucine. The results of the two series of experiments pooled, the ratio between the results obtained in the presence or absence of alpha -D-glucose pentaacetate within each experiment yielded mean values of 148.2 ± 23.7 (P < 0.05) and 148.2 ± 24.9% (P < 0.06) for (pro)insulin and the nonhormonal peptides, respectively (n = 8 in both cases). Unexpectedly, in the presence of either 4.2 mM D-glucose or 10.0 mM leucine, alpha -D-glucose pentaacetate decreased the fractional conversion of proinsulin to insulin, the insulin-to-(pro)insulin ratio found in the presence of the ester averaging 71.6 ± 8.6% (n = 8; P < 0.03) of the paired value recorded in its absence.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 3.   Elution profiles of tritiated peptides separated by gel chromatography in extracts from islets incubated with L-[4-3H]phenylalanine in absence or presence of L-leucine, alpha -D-glucose pentaacetate (alpha DGlcPA), and D-glucose. Mean values (±SE) refer to 4 individual determinations.

                              
View this table:
[in this window]
[in a new window]
 
Table 4.   Effects of nutrients on L-[4-3H]phenylalanine incorporation into nonhormonal peptides (void volume), proinsulin, and insulin in two series of experiments

Taken as a whole, these findings indicate that alpha -D-glucose pentaacetate acts on biosynthetic, cationic, and secretory variables in a manner comparable, albeit not identical, to that characterizing the response of islet cells to D-glucose and other nutrient secretagogues.

Concentration-response relationship for the insulinotropic action of alpha -D-glucose pentaacetate. Figure 4 documents the effect of increasing concentrations of alpha -D-glucose pentaacetate on insulin release by islets incubated either in the absence of any other exogenous nutrient or in the presence of 8.3 mM D-glucose. In the former situation, a sizeable increase in insulin output (P < 0.001) above basal value was observed in islets exposed to 1.7 mM alpha -D-glucose pentaacetate, but lower concentration of the ester (0.17 to 0.85 mM) failed to stimulate hormonal secretion. In the presence of 8.3 mM D-glucose, however, as little as 0.17 mM alpha -D-glucose pentaacetate significantly increased insulin release (P < 0.005). The concentration-response relationship suggested that the concentration of 1.7 mM alpha -D-glucose pentaacetate, which was used in most further experiments, yielded, in the presence of 8.3 mM D-glucose, a close-to-maximal secretory response.


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of increasing concentrations of alpha -D-glucose pentaacetate on insulin release from islets incubated in absence (dotted line) or presence (solid line) of D-glucose (8.3 mM). Mean values (±SE) refer to 24 individual observations in all cases.

Specificity of the insulinotropic action of alpha -D-glucose pentaacetate. The specificity of the secretory response to alpha -D-glucose pentaacetate was explored in a double perspective, attempting to assess the participation of both the acetate and glucose moieties of the ester in its insulinotropic action.

The effect of acetate, methylacetate, and ethylacetate on insulin release was first investigated (Table 5). In the absence of any other exogenous nutrient, both acetate and ethylacetate but not methylacetate, augmented, slightly but significantly (P < 0.025 or less), basal insulin output. The secretory rate recorded in the presence of acetate or methylacetate remained much lower, however, than that found in islets exposed to alpha -D-glucose pentaacetate (1.7 mM), despite the fact that the former nutrients were used at molar concentrations equal to or higher than the concentration of the acetate moiety of the glucose ester. A comparable situation prevailed when D-glucose was present in the incubation medium at a 1.7 mM concentration. At a higher concentration (8.3 mM) of the hexose, neither acetate nor methylacetate significantly affected insulin release, whereas ethylacetate inhibited (P < 0.005) glucose-stimulated hormonal output. Last, in the presence of L-leucine (10.0 mM), a modest inhibition of insulin secretion was caused by both acetate (P < 0.02) and ethylacetate (P = 0.06) to, respectively, 84.9 ± 3.9 and 87.4 ± 4.7% of the paired control value (100.0 ± 4.6%) found within the same experiments in the sole presence of the branched-chain amino acid (n = 30 in all cases).

The second procedure designed to assess the respective contribution of the glucosidic and acidic parts of alpha -D-glucose pentaacetate to its secretory potential relied on a comparison between the insulinotropic action of several hexose pentaacetate esters.

In this perspective, we have first compared the secretory response to alpha -D-glucose pentaacetate, beta -D-glucose pentaacetate, and beta -L-glucose pentaacetate (Table 6). Whether in the absence of any other exogenous nutrient or in the presence of either D-glucose (8.3 mM) or L-leucine (10.0 mM), all three esters significantly augmented (P < 0.005 or less) insulin release. The beta -anomer of D-glucose pentaacetate appeared slightly less potent than the alpha -anomer. Such a difference failed, however, to achieve statistical significance. The beta -anomer of L-glucose pentaacetate was much less potent (P < 0.001) than the D-eniantomers. As already mentioned, it nevertheless augmented insulin release significantly. At variance with these results, unesterified L-glucose (5.6-55.6 mM) failed to affect insulin release evoked by either 8.3 mM D-glucose or 10.0 mM L-leucine (data not shown).

                              
View this table:
[in this window]
[in a new window]
 
Table 5.   Effect of acetate and its esters on insulin release from islets incubated in the absence or presence of either D-glucose or L-leucine

                              
View this table:
[in this window]
[in a new window]
 
Table 6.   Effect of pentaacetate esters of glucose enantiomers and isomers on insulin release from islets incubated in absence of any other exogenous nutrient and in presence of either D-glucose or L-leucine

We then compared the insulinotropic action of alpha -D-glucose pentaacetate to that of either alpha - or beta -D-galactose pentaacetate (Table 7). The alpha -anomer of D-galactose pentaacetate (1.7 mM) failed to affect insulin release when tested in the absence of any other exogenous nutrient and even decreased insulin output (P < 0.001) in the presence of L-leucine (10.0 mM). The beta -anomer of D-galactose pentaacetate (1.7 mM) inhibited both basal (P < 0.001) and leucine-stimulated (P < 0.01) insulin secretion. It did not significantly affect insulin release from islets incubated in the presence of 4.0 mM D-glucose. Neither 3-O-methyl-D-glucose (80.0 mM) nor D-mannoheptulose (10.0 mM) significantly affected insulin output from islets exposed to beta -D-galactose pentaacetate (1.7 mM). The latter ester failed to significantly affect insulin release from islets incubated in the absence of extracellular Ca2+ (Table 1).

                              
View this table:
[in this window]
[in a new window]
 
Table 7.   Effect of alpha - and beta -D-galactose pentaacetate on insulin release

The third approach used to distinguish between the participation of the acetate and glucose moieties of alpha -D-glucose pentaacetate to its insulinotropic action was based on the use of D-glucose esters in which the acidic component might display a metabolic efficiency different from that of acetate.

For this purpose, several esters of D-glucose, including alpha -D-glucose pentaisobutyrate, alpha -D-glucose pentamethacrylate, alpha -D-glucose pentaisovalerate, and alpha -D-glucose pentabenzoate, were synthesized, but they could not be tested because of their low solubility (<0.1 mM, even in the presence of 1.0% dimethyl sulfoxide). Only alpha -D-glucose pentapropionate could be used, this ester being tested at an 0.1 mM concentration (Table 8). Its effect on insulin release was examined in the presence of 10.0 mM L-leucine to optimize the detection of its possible insulinotropic action. Under these experimental conditions, it significantly augmented insulin release (P < 0.001). As judged by comparison of results obtained within the same experiments, the increment in insulin output attributable to alpha -D-glucose pentapropionate (21.5 ± 4.5 µU · islet-1 · 90 min-1; df = 54) was even more marked (P < 0.05) than that caused by alpha -D-glucose pentaacetate (8.3 ± 4.3 µU · islet-1 · 90 min-1; df = 55), which at the low concentration used in these experiments exerted an insulinotropic action of borderline statistical significance (P < 0.06). The secretory response to alpha -D-glucose pentapropionate was unaffected by D-mannoheptulose (10 mM) and contrasted with the failure of unesterified D-glucose, tested at the same low concentration (0.1 mM), to significantly affect leucine-stimulated insulin release (Table 8).

                              
View this table:
[in this window]
[in a new window]
 
Table 8.   Comparison of the secretory effects of alpha -D-glucose pentaacetate, alpha -D-glucose pentapropionate, and D-glucose, all tested in the presence of 10 mM L-leucine

Effect of 3-O-methyl-D-glucose. In the next series of experiments, we explored the possible interference of agents affecting D-glucose transport or metabolism with the insulinotropic action of alpha -D-glucose pentaacetate. In such a perspective, the effect of 3-O-methyl-D-glucose was first investigated.

Both basal insulin release and alpha -D-glucose pentaacetate-stimulated insulin output were not significantly affected by 3-O-methyl-D-glucose, tested at a concentration of 80 mM, which is sufficient to inhibit by >60% insulin secretion evoked by 16.7 mM D-glucose (Fig. 5). As indicated in Table 9 (experiment 1), at the concentration of 80 mM, 3-O-methyl-D-glucose failed to affect insulin release evoked by nonglucidic nutrients such as 2-ketoisocaproate (10 mM) or the association of L-leucine and L-glutamine (both 10 mM).


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of 3-O-methyl-D-glucose (3-OMG; 80 mM; striped columns) on insulin release from islets incubated in absence (basal) or presence of 1.7 mM alpha -D-glucose pentaacetate (n = 43-44). Inset illustrates effect of increasing concentrations of 3-OMG on insulin release evoked by 16.7 mM D-glucose (n = 20 in each case).

                              
View this table:
[in this window]
[in a new window]
 
Table 9.   Effect of 3-OMG on insulin release evoked by various nutrients

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


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 6.   Relationship between increment in insulin release caused by alpha -D-glucose pentaacetate (1.7 mM) and control output recorded in its absence in islets incubated in absence of exogenous nutrient (Ø), sole presence of 8.3 mM D-glucose (G), concomitant presence of 8.3 mM D-glucose and 80 mM 3-OMG (G + 3-OMG), or simultaneous presence of 10 mM L-leucine, 10 mM D-glucose, and 80 mM 3-OMG (L + G + 3-OMG). Mean values (±SE) refer to 11-24 individual observations. Regression line (dotted line) is compared with that defining a rule of proportionality between 2 variables (solid line).

To further explore the effect of 3-O-methyl-D-glucose on the secretory response to alpha -D-glucose pentaacetate, the last two series of experiments were conducted in the presence of nutrients other than D-glucose. In the presence of 2-ketoisocaproate (10 mM), alpha -D-glucose pentaacetate (1.7 mM) augmented insulin release (P < 0.05) modestly to 114.0 ± 4.9% (n = 18) of the mean control value found within the same experiments in the sole presence of the branched-chain 2-keto acid (100.0 ± 4.4%; n = 18). No significant effect (P > 0.1 or more) of 3-O-methyl-D-glucose (80 mM) on insulin release was observed, whether in the sole presence of 2-ketoisocaproate or in the concomitant presence of the 2-keto acid and alpha -D-glucose pentaacetate (Table 9, experiment 3). Indeed, the ester still augmented insulin release significantly (P < 0.01) in the presence of 3-O-methyl-D-glucose.

Likewise, 3-O-methyl-D-glucose failed to affect (P > 0.8) the secretory response to L-leucine and the much higher secretory rate found in the concomitant presence of the branched-chain amino acid and alpha -D-glucose pentaacetate (Table 9, experiment 4).

Taken as a whole, these data strongly suggest that the insulinotropic action of alpha -D-glucose pentaacetate does not involve the transport of the ester across the plasma membrane by the carrier system otherwise mediating the entry of D-glucose into the islet B cell. It is unlikely that the disparity between the insulinotropic action of D-glucose and that of its ester, in terms of the sensitivity toward 3-O-methyl-D-glucose, can be accounted for by a difference in the concentrations of the two nutrients, such as that prevailing in the experiments illustrated in Fig. 5. Indeed, the relative extent of the inhibitory action of 3-O-methyl-D-glucose on insulin release was little affected by the changes in the concentrations of D-glucose (Fig. 7). It cannot be ruled out, however, that 3-O-methyl-D-glucose is less efficient in inhibiting the transport of alpha -D-glucose pentaacetate, as distinct from unesterified D-glucose, by GLUT-2.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 7.   A: effect of increasing concentrations of D-glucose on insulin release from islets incubated in absence (bullet ) or presence (open circle ) of 80 mM 3-OMG (n = 19-93). B: effect of increasing concentrations of 3-OMG on insulin release from islets incubated in presence of either 8.3 (open circle ) or 16.7 mM (bullet ) D-glucose (n = 20 in all cases).

Effect of D-mannoheptulose. The second set of experiments dealing with inhibitors of the insulinotropic action of D-glucose concerned the effect of D-mannoheptulose.

Both basal insulin release and alpha -D-glucose pentaacetate-stimulated insulin output were not significantly affected by D-mannoheptulose tested at a concentration of 10 mM, which is sufficient to abolish insulin secretion evoked by 16.7 mM D-glucose (Fig. 8).


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 8.   Effect of D-mannoheptulose (MH; 10.0 mM; striped columns) on insulin release from islets incubated in absence (basal) or presence of 1.7 mM alpha -D-glucose pentaacetate (n = 24-69). Inset illustrates effect of increasing concentrations of MH on insulin release evoked by 16.7 mM D-glucose (n = 10-60).

Even when the islets were both preincubated for 30 min and incubated for 90 min at 37°C in the presence of D-mannoheptulose (10 mM), the secretory response to alpha -D-glucose pentaacetate (1.7 mM), which was present in the medium solely during the final incubation, remained not significantly different from that found in islets both preincubated and incubated in the absence of the heptose. Thus, during the final incubation, the secretory rate averaged 32.6 ± 1.4 and 31.9 ± 1.4 µU · islet-1 · 90 min-1 (n = 27-28) in control and mannoheptulose-exposed islets, respectively, compared with a basal value of 13.1 ± 0.8 µU · islet-1 · 90 min-1 (n = 30).

Likewise, in the presence of L-leucine (10 mM), which was used to augment the magnitude of the secretory response to alpha -D-glucose pentaacetate, D-mannoheptulose (1.0-10.0 mM) failed to adversely affect the enhancing action of the ester on insulin release evoked by the branched-chain amino acid (Table 10, experiments 1 and 2). In these experiments, D-mannoheptulose also failed to suppress the insulinotropic action of L-leucine.

                              
View this table:
[in this window]
[in a new window]
 
Table 10.   Effect of D-mannoheptulose on insulin release evoked by various nutrients

Further experiments provided the following information (Table 10). First, at the concentration of 10 mM used in the present experiments, D-mannoheptulose failed to significantly affect the secretory response to either 2-ketoisocaproate (10 mM) or the association of L-leucine and L-glutamine (10 mM each). Second, to explore the effect of D-mannoheptulose on the secretory response to D-glucose at low concentrations of the hexose, advantage was taken of its enhancing action on insulin release evoked by 2-ketoisocaproate, the branched-chain 2-keto acid being used at a concentration (5 mM) close to the threshold value for its insulinotropic action (7). Under these experimental conditions, as little as 1.7 and 2.8 mM D-glucose indeed caused a concentration-related increase in insulin output (P < 0.001). Unexpectedly, however, D-mannoheptulose (10 mM) failed to significantly affect the release of insulin evoked by 2-ketoisocaproate in the presence of 1.7 mM D-glucose (Table 10, experiment 4). The concentration of the hexose had to be raised to 2.8 mM to detect a sizeable inhibitory effect (P < 0.001) of D-mannoheptulose (Table 10, experiment 5). In the presence of D-mannoheptulose, the release of insulin evoked by 2-ketoisocaproate was no more significantly different at 1.7 and 2.8 mM D-glucose, whether expressed in absolute terms (P > 0.18) or relative to the mean secretory rate recorded within the same experiments in islets exposed to 16.7 mM D-glucose (19.5 ± 2.7 vs. 16.9 ± 2.5%; n = 20 in both cases; P > 0.4). In these experiments, it was duly verified that D-mannoheptulose abolishes the insulinotropic action of D-glucose (16.7 or 27.8 mM). Actually, in the presence of the heptose, the output of insulin found in islets exposed to these high concentrations of D-glucose was lower (P < 0.001) than that evoked by 2-ketoisocaproate at much lower concentrations of the hexose.

Taken as a whole, the data summarized in Table 10 indicate that D-mannoheptulose, although a potent and specific inhibitor of the insulinotropic action of D-glucose, when the hexose is tested in high concentrations, is much less efficient in suppressing the secretory response to the sugar when it is used in low concentrations. Therefore, the failure of D-mannoheptulose to significantly affect insulin release in islets exposed to alpha -D-glucose pentaacetate (see above) does not provide conclusive evidence on the relevance of hexose phosphorylation to the insulinotropic action of this ester.

The latter consideration motivated a last series of experiments conducted in the presence of 8.3 mM D-glucose (Table 10, experiment 6). As expected, D-mannoheptulose (10 mM) suppressed (P < 0.001) the secretory response to the hexose. The heptose failed, however, to suppress the insulinotropic action of alpha -D-glucose pentaacetate (1.7 mM). Actually, the increment in insulin output attributable to the ester was not significantly different (P > 0.1) in the absence (+134.7 ±8 17.9 µU · islet-1 · 90 min-1; df = 28) or presence (+101.3 ± 9.6; df = 28) of D-mannoheptulose. Moreover, the secretory rate recorded in the simultaneous presence of D-glucose (8.3 mM), D-mannoheptulose (10 mM), and alpha -D-glucose pentaacetate (1.7 mM) was much higher than that otherwise recorded in the sole presence of the ester (see above). These findings strongly suggest that the insulinotropic action of alpha -D-glucose pentaacetate is not dependent on the phosphorylation of the hexose moiety of the ester or that D-mannoheptulose fails to inhibit such a phosphorylation.

Effect of 2-deoxy-D-glucose. The third inhibitor of D-glucose insulinotropic action used in the present study, namely 2-deoxy-D-glucose, when tested in the 8.3 to 16.7 mM range caused a concentration-related decrease of the secretory response evoked by 11.1 mM D-glucose (Fig. 9). At a concentration of 16.7 mM, 2-deoxy-D-glucose, however, caused only a minor decrease of the secretory response to alpha -D-glucose pentaacetate (Fig. 9). Moreover, when these experiments were repeated in the presence of 10.0 mM L-leucine to increase the magnitude of the B cell response to the hexose ester, a significant effect of 2-deoxy-D-glucose on the release of insulin evoked by alpha -D-glucose pentaacetate (1.7 mM) was no longer observed (Table 11).


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 9.   Effect of 2-deoxy-D-glucose (2-DOG; 16.7 mM; striped columns) on insulin release from islets incubated in absence (basal) or presence of 1.7 mM alpha -D-glucose pentaacetate (n = 45 in each case). Inset illustrates effect of increasing concentrations of 2-DOG on insulin release evoked by 11.1 mM D-glucose (n = 15 in each case).

                              
View this table:
[in this window]
[in a new window]
 
Table 11.   Effect of 2-deoxy-D-glucose on secretory response to alpha -D-glucose pentaacetate in islets exposed to 10.0 mM L-leucine

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Several of the present findings are compatible with the view that the insulinotropic action of alpha -D-glucose pentaacetate is causally linked to its capacity to act as a fuel in pancreatic islet cells.

First, like other nutrient secretagogues, alpha -D-glucose pentaacetate caused a shift to the left of the sigmoidal curve relating insulin output to D-glucose concentration. Likewise, whereas the concentration of alpha -D-glucose pentaacetate had to exceed a critical value (>0.85 mM) to stimulate insulin release in the absence of any other exogenous nutrient, no threshold phenomenon was observed when the ester was tested in the presence of 8.3 mM D-glucose.

Second, the secretory response to alpha -D-glucose pentaacetate coincided with the recruitment of more individual B cells to an active functional state, as is also the case with other nutrients (1, 6).

Third, alpha -D-glucose pentaacetate stimulated biosynthetic activity in pancreatic islets incubated in the absence or presence of either D-glucose or L-leucine. In this respect, the present data extend the knowledge that the threshold concentration for stimulation of functional events by nutrients is lower in the case of biosynthetic activity than insulin release (14). It should be noted, however, that alpha -D-glucose pentaacetate failed to significantly increase the biosynthesis of proinsulin relative to that of nonhormonal peptides and apparently decreased the rate of proinsulin conversion to insulin. The latter finding represents a first indication that alpha -D-glucose pentaacetate may affect B cell function not solely through its nutritional value.

Fourth, alpha -D-glucose pentaacetate simulated the effect of other nutrient secretagogues in causing both a decrease of 86Rb outflow and an increase of 45Ca efflux from prelabeled islets. The latter phenomenon was abolished in the absence of extracellular Ca2+, suggesting that it results from an increased influx of the divalent cation into the islet cells. And, indeed, alpha -D-glucose pentaacetate stimulates 45Ca net uptake by the islets (unpublished observation).

Last, the secretory response to alpha -D-glucose pentaacetate was suppressed in the absence of extracellular Ca2+ and potentiated by both theophylline and cytochalasin B. This suggests that the insulinotropic action of the ester involves interaction between Ca2+, adenosine 3',5'-cyclic monophosphate, and the microfilamentous effector system in the same manner as in islets stimulated by unesterified D-glucose.

Taken as a whole, these findings thus suggest that the stimulation of insulin release by alpha -D-glucose pentaacetate involves the same sequence of metabolic, cationic, and motile events currently thought to be operative in the process of nutrient-stimulated insulin secretion (11).

Even the observation that D-mannoheptulose failed to suppress insulin release evoked by alpha -D-glucose pentaacetate remains compatible with such a proposal, since the heptose fails to inhibit the utilization and oxidation of the ester in isolated islets (unpublished observation). The modest but significant reduction of the secretory response to alpha -D-glucose pentaacetate caused by 2-deoxy-D-glucose in islets deprived of any other exogenous nutrient is also consistent with such a view.

If the insulinotropic action of alpha -D-glucose pentaacetate were indeed to result from its role as a nutrient in islet cells, the question must be raised whether it is the hexose or acetate moiety of the ester that plays such a role.

Several of the present data argue against any major role of the acetate residues presumably generated by the intracellular hydrolysis of alpha -D-glucose pentaacetate in its insulinotropic action.

First, except for a modest positive effect of acetate and ethyl acetate on insulin release from islets incubated in the absence of any exogenous nutrient, neither acetate nor its methyl or ethyl ester was able to augment insulin release from islets incubated in the presence of D-glucose or L-leucine, namely under conditions in which the insulinotropic action of the ester is most pronounced.

Second, even at the very low concentration of 0.1 mM, alpha -D-glucose pentapropionate stimulated insulin release from islets exposed to 10.0 mM L-leucine, the secretory response to this ester being significantly higher than that evoked at the same low concentration by alpha -D-glucose pentaacetate and contrasting with the lack of effect, under the same experimental conditions, of unesterified D-glucose. Yet, propionate is very poorly metabolized in islet cells. At a 10 mM concentration, the oxidation of [1-14C]propionate or the generation of 13C-labeled L-lactate from [2-13C]propionate does not exceed ~3 pmol · 103 cells-1 · 90 min-1 (10, 17).

Third, both alpha - and beta -D-galactose pentaacetate failed to exert any positive effect on insulin release whether in the absence or presence of D-glucose or L-leucine.

Although these three series of observations strongly suggest a key role for the glucose moiety of alpha -D-glucose pentaacetate in its insulinotropic action, they do not rule out an interference of the acetate moiety with selected metabolic, ionic, and functional variables. For instance, the generation of acetic acid from the ester could account for a decrease in cellular pH in islets exposed to alpha -D-glucose pentaacetate (unpublished observation). This, in turn, may explain the fact that alpha -D-glucose pentaacetate barely increased insulin output from islets incubated in the presence of 2-ketoisocaproate while considerably augmenting leucine-induced insulin release. It is indeed known that the respective effects of the branched-chain 2-keto acid and amino acid on intracellular pH affect in a dissociated manner the coupling between metabolic and secretory events in nutrient-stimulated islets (8, 15).

Three other sets of observations, however, argue against the view that the catabolism of D-glucose generated from alpha -D-glucose pentaacetate represents an essential, or at least the sole, determinant of its insulinotropic action.

First, and as already mentioned, the ester failed to cause a preferential stimulation of proinsulin biosynthesis relative to that of other islet peptides.

Second, in the presence of 10.0 mM L-leucine, the secretory response to alpha -D-glucose pentaacetate was unaffected by 2-deoxy-D-glucose.

Third, and most importantly, beta -L-glucose pentaacetate caused an obvious stimulation of insulin release whether in the absence of any other exogenous nutrient or in the presence of D-glucose or L-leucine. Exogenous L-glucose failed to reproduce such an effect.

The secretory response to alpha -D-glucose pentaacetate also differed from that evoked by exogenous D-glucose by its insensitiveness to 3-O-methyl-D-glucose and D-mannoheptulose. Although is seems likely that the resistance to 3-O-methyl-D-glucose may be accounted for by the fact that alpha -D-glucose pentaacetate penetrates into the islet cells without requiring the intervention of a specific hexose transport system, the failure of D-mannoheptulose to oppose the utilization and insulinotropic action of the ester suggests heterogeneity in the location and/or regulation of hexose phosphorylation in islets exposed to alpha -D-glucose pentaacetate and/or unesterified D-glucose.

In conclusion, although the hexose moiety generated from alpha -D-glucose pentaacetate indeed seems to participate in the insulinotropic action of the ester, the modality of such a participation may well differ from that currently implied in the process of glucose-stimulated insulin release.

    ACKNOWLEDGEMENTS

We are grateful to M. Mahy, J. Schoonheydt, and M. Urbain for technical assistance and C. Demesmaeker for secretarial help.

    FOOTNOTES

This study was supported by a Concerted Research Action of the French Community of Belgium (94/99-183), Grant IN212194 from Direccion General de Asuntos del Personal Academico (Universidad Nacional Autónoma de Mexico) and grants from the Spanish Fondo de Investigaciones Sanitarias (96/1383) and Dirección General de Investigaciones Científica y Técnica (95/0048) and Belgian Foundation for Scientific Medical Research (3.4513.94).

Address for reprints requests: W. J. Malaisse, Laboratory of Experimental Medicine, Brussels Free University, 808 Route de Lennik, B-1070 Brussels, Belgium.

Received 11 June 1997; accepted in final form 20 August 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Bosco, D., P. Meda, B. Thorens, and W. J. Malaisse. Heterogeneous secretion of individual B cells in response to D-glucose and to nonglucidic nutrient secretagogues. Am. J. Physiol. 268 (Cell Physiol. 37): C611-C618, 1995[Abstract/Free Full Text].

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.   Herchuelz, A., and W. J. Malaisse. Regulation of calcium fluxes in pancreatic islets: dissociation between calcium and insulin release. J. Physiol. (Lond.) 238: 409-424, 1978.

6.   Hiriart, M., M. C. Sanchez-Soto, M. C. Ramirez-Medeles, and W. J. Malaisse. Functional heterogeneity of single pancreatic beta -cells stimulated by L-leucine and the methyl ester of succinic or glutamic acid. Biochem. Mol. Med. 54: 133-137, 1995[Medline].

7.  Hutton, J. C., I. Atwater, and W. J. Malaisse. Fuel and signal function of 2-keto acids in insulin secretion. Horm. Metab. Res. 10, Suppl.: 31-37, 1980.

8.   Hutton, J. C., A. Sener, and W. J. Malaisse. The metabolism of 4-methyl-2-oxopentanoate in rat pancreatic islets. Biochem. J. 184: 291-301, 1979[Medline].

9.   Malaisse, W. J., H. Jijakli, M. M. Kadiata, A. Sener, and O. Kirk. Stimulation of insulin release by alpha -D-glucose pentaacetate. Biochem. Biophys. Res. Commun. 231: 435-436, 1997[Medline].

10.   Malaisse, W. J., L. Ladrière, T.-M. Zhang, I. Verbruggen, and R. Willem. Enzyme-to-enzyme channelling of symmetric Krebs cycle intermediates in pancreatic islet cells. Diabetologia 39: 990-992, 1996[Medline].

11.   Malaisse, W. J., A. Sener, A. Herchuelz, J. C. Hutton, G. Devis, G. Somers, B. Blondel, F. Malaisse-Lagae, and L. Orci. Sequential events in the process of glucose-induced insulin release. In: Diabetes, edited by J. S. Bajaj. Amsterdam: Excerpta Medica, 1977, p. 95-102.

12.   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.

13.   Orci, L., K. H. Gabbay, and W. J. Malaisse. Pancreatic beta-cell web: its possible role in insulin secretion. Science 175: 1128-1130, 1972[Medline].

14.   Pipeleers, D. G., M. Marichal, and W. J. Malaisse. The stimulus-secretion coupling of glucose-induced insulin release. XIV. Glucose regulation of insular biosynthetic activity. Endocrinology 93: 1001-1011, 1973[Medline].

15.   Sener, A., and W. J. Malaisse. The stimulus-secretion coupling of glucose-induced insulin release. II. Sensitivity to K+, NH+4 and H+ of leucine-stimulated islets. Diabetes Metab. 6: 97-101, 1980.

17.   Tekle, S. Y. W. 13C-labelled nutrient catabolism in intact cells (PhD thesis). Brussels: Free Univ. of Brussels, 1993.

18.   Vogel, A. I. A Textbook of Practical Organic Chemistry (3rd ed.). London: Longmans Green, 1956, p. 383.

19.   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.


AJP Endocrinol Metab 273(6):E1090-E1101
0193-1849/97 $5.00 Copyright © 1997 the American Physiological Society