Laboratory of Experimental Medicine, Brussels Free University, Brussels, Belgium

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1 Vol. 43, No. 6, December 1997 Pages BITTER TASTE OF MONOSACCHARIDE PENTAACETATE ESTERS Willy J. MALAISSE and Francine MALAISSE-LAGAE Laboratory of Experimental Medicine, Brussels Free University, Brussels, Belgium Received October 27, 1997 SUMMARY: The pentaacetate esters of several, but not all, monosaccharides were recently found to stimulate insulin release from rat pancreatic islets. We have now compared the taste of solutions of either these esters or the corresponding unesterified monosaccharides. The sweet taste of the latter monosaccharides (0.22 M) ranged as follows : D-glucose _= D- galactose ~ D-mannoheptulose > L-glucose. None of the esters (1.7 mm) displayed a sweet taste. However, the c~- and B-anomer of D-glucose pentaacetate, the c~-anomer of D-mannose pentaacetate and the B-anomer of L-glucose pentaacetate all displayed a bitter taste, whilst both ~- and B-D-galactose pentaacetate yielded doubtful results. Since such a situation is comparable to that characterizing the islet B-cell response to these esters, it is proposed that the intracellular interaction between the esters or their hydrolytic products and a protein involved in the recognition of bitter taste in taste buds may participate in their insulinotropic action. INTRODUCTION Several esters of monosaccharides, such as ~- and B-D-glucose pentaacetate and c~-dmannose pentaacetate were recently found to stimulate insulin release from isolated rat pancreatic islets [1, 2]. Even B-L-glucose pentaacetate displays insulinotropic efficiency [2]. Other esters, however, such as c~- and B-D-galactose pentaacetate are apparently devoid of positive insulinotropic action. Although the stimulation of insulin release by the esters of D- glucose or D-mannose could be attributable, in part at least, to the metabolism of the corresponding hexose after intracellular hydrolysis of the esters in the pancreatic B-cell, tiffs is most unlikely to be the case with B-L-glucose pentaacetate. In search of an alternative explanation for the insulin-releasing capacity of the latter ester, we were inspired by the observation that sucrose exerts a direct inhibitory effect upon GTPase activity of GJGo proteins purified from bovine brain membranes and reconstituted into phospholipid vesicles [3]. This finding indeed raises the idea that saccharides, like amphiphilic taste substances, may elicit taste tbxough direct effects on G-proteins, provided that they are made available /97/ /0 Copyright by Academic Press Australia. All rights ~?f reproduction in any form reserved

2 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL inside the gustatory cells. The above-mentioned esters of saccharides were tested for their insulinotropic action precisely with the idea that they may enter into islet cells by a process not requiring the intervention of a carrier system for the transport of hexoses across the plasma membrane and, after intracellular hydrolysis, make the unesterified saccharides available for either phosphorylation and further metabolism or interaction with some target, yet unidentified, protein [1]. In the light of these considerations, we have investigated, in the present study, the taste of several hexose pentaacetate esters, and compared it to that of unesterified monosaccharides. MATERIALS AND METHODS D-glucose, L-glucose, D-galactose, D-mannoheptulose, ~- and B-D-glucose pentaacetate, B-L-glucose pentaaeetate, and o~-d-mannose pentaacetate were all purchased from Sigma Chemical Company (St. Louis, MO). The a- and B-anomers of D-galactose pentaacetate were synthesized by methods described elsewhere [4, 5] and kindly donated by Dr. O. Kirk (Novo Nordisk A/S, Bagsvaerd, Denmark). All tests were conducted in healthy adult subjects (24 to 61 years old), members of our laboratory staff and including 7 males and 9 females, who participated in one or more experiment(s). The solutions of monosaccharides (0.22 M) or their pentaacetate esters (1.7 ram) were prepared in mineral water (Vittel, Vittel, France) and maintained at room temperature (approximately 20~ In each individual test, the subjects were asked to taste 1-2 ml of 3 to 6 different solutions. In most experiments, and in all those dealing with the bitter taste of the monosaccharide esters, the subjects took some mineral water between each trial. The results were ranged according to the two following scales. In the experiments concerned with the sweet taste of monosaccharides, 2 arbitrary units were given for the sweetest solution, 1 unit for the less sweet solution, and zero unit for the solutions devoid of sweet taste. In the tests dealing with the bitter taste of the monosaccharide esters, scores of 4, 3 and 2 arbitrary units were used, respectively, for very bitter, bitter or slightly bitter solutions, a score of 1 unit corresponding to doubtful answers and a null score to solutions devoid of bitter taste. All results are presented as mean values (~: SEM), together with the number of individual observations (n). The statistical significance of differences between mean values was assessed by use of Student's unpaired t-test. RESULTS When tested at a concentration of 0.22 M, both D-glucose and L-glucose displayed a sweet taste. Except in one out 9 individual tests, the D-isomer tasted sweeter than the L- isomer (Table 1, exp. 1). At the same concentration (0.22 M), D-mannoheptulose was found to taste as sweet as D-glucose. All 8 subjects examined in the test gave a double score for a solution containing both the hexose and heptose (Table I, exp. 2). 1368

3 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL Table 1. Sweet taste of solution of monosaccharide(s) and one of their ester Exp. Agent Sweet taste score (Nr) (M) (arbitrary units) I. Nil D-glucose (0.22) L-glucose (0.22) (9) 1.9 :~ 0.1 (9) (9) 2. Nil D-glucose (0.22) D-mannoheptulose (0.22) D-glucose (0.22) +D-mannoheptulose (0.22) (8) (8) (8) (8) 3. Nil (8) D-glucose (0.22) (8) D-galactose (0.22) 1.4 :k 0.2 (8) a-d-glucose pentaacetate (0.0017) (8) a "Solution recognized as bitter by 6 out of 8 subjects In the last test in this first series, D-glucose and D-galactose yielded a comparable mean score in terms of their sweetness, whilst a-d-glucose pentaacetate, tested at a much lower concentration (t.7 mm) failed to evoke a sweet taste in all 8 subjects examined for such a purpose (Table 1, exp. 3). Water was not found to display any taste in the 25 individual trials included in this first series of experiments. Both a-d-glucose pentaacetate and g-d-glucose pentaacetate were identified as having a bitter taste, with 9 negative and 3 doubtful answers in a series of 40 individual trials. The mean score obtained with the a- and g-anomer were not significantly different from one another (Table 2), with an overall mean value of arbitrary units (n = 40). Likewise, g-l-glucose pentaacetate tasted bitter in 11 out of 16 trials, with 4 negative an 1 doubtful answers, yielding a mean score of (n = 16). The a-anomer of D-mamlose pentaacetate was also found to display a bitter taste, with only one negative and one doubtful answers among 16 individual trials. The meen score obtained with this ester was not significantly different from that found for the anomers of D- glucose pentaacetate. Neither a-d-galactose pentaacetate nor g-d-galactose pentaacetate yielded a mean score that was significantly higher than the limit of detection for bitter taste (i.e. 1.0 arbitrary unit). The overall mean value found with these two esters ( ; n = 32) was 1369

4 Table 2. Bitter taste of solutions of monosaccharide esters Agent ram) ct-d-glucose pentaacetate (1.7) B-D-glucose pentaacetate (1.7) B-L-glucose pentaacetate (1.7) ct-d-mannose pentaacetate (1.7) ct-d-galactose pentaacetate (1.7) g-d-galactose pentaacetate (1.7) Bitter taste (arbitrary units) (24) (16) (16) 3.12 (16) (16) (t6) significantly lower (P < 0.02 or less) than that found with either the anomers of D-glucose pentaacetate ( ; n = 40) or the ot-anomer of D-mannose pentaacetate ( ; n = 16). It failed, however, to differ significantly from the mean score found with B-L-glucose pentaacetate. None of the esters tested in this study were identified as sweet by any of the subjects who tasted them. In 5 control trials, acetic acid and sodium acetate, each tested at a concentration (8.5 mm) equimolar to that of the acetate residues in the ester solutions, either always evoked a sour taste (acetic acid) or yielded negative to doubtful answers (sodium acetate). In these control trials, water again failed to display any taste. DISCUSSION The present study reveals that several, but not all, pentaacetate esters of aldohexoses display a bitter taste, even when tested at a concentration as low as 1.7 mm, which corresponds to the limit of solubility of these esters. Sucrose octoacetate was already known as a bitter agent, that stimulates inositol t,4,5- triphosphate generation in membrane preparations mad intact taste buds from mouse taste tissue [6]. Since amphiphilic bitter substances may interact directly with G-proteins [7], it is conceivable that, in the pancreatic B-cell, esters such as a-d-glucose pentaacetate, which accumulates inside the islet cells or in their plasma membrane to yield an apparent distribution space representing up to 10 times or more the intracellular H20 space i8], also interact with G- proteins with resulting changes in phospholipase C, adenylate cyclase and/or phosphodiesterase activity. Alternatively, these changes could be due to the monosaccharides liberated inside the islet cells by hydrolysis of the esters. t370

5 In conclusion, therefore, the finding that monosaccharide esters display a bitter taste, with a hierarchy similar to that recently documented in terms of their effects upon insulin release [2], affords a possible explanation for their insulinotropic action, provided that islet cells are equipped with the same G-proteins as those identified in taste buds. Preliminary observations suggest that such is indeed the case (unpublished observation). Acknowledgments. This study was supported by a Concerted Research Action of the French Community of Belgium (94/99-183). We thank C. Demesmaeker for secretarial help., REFERENCES Malaisse, W.J., Jijakli, H., Kadiata, M.M., Sener, A., and Kirk, O. (1997) Biochem. Biophys. Res. Commun. 231, Malaisse, W.J. Sfinchez-Soto, C., Larrietta, M.E., Hiriart, M., Jijakli, H., Vifiambres, C., Villanueva-Pefiacarrillo, M.L., Valverde, I., Kirk, O., Kadiata, M.M., and Sener, A. (1997) Am. J. Physiol., in press. Naim, M., Seifert, R., Niirnberg, B., Grtinbaum, L., and Schultz, G. (1994) Biochem. J. 297, Vogel, A.I. (1956) A Textbook of Practical Organic Chemistry, 3 ra ed., p Longmans, Green mad Co., London. Wolfrom, M.L., and Thompson, A. (1962) In Methods in Carbohydrate Chemistry (R.L. Whistler, and M.L. Wolfrom, Eds.), Vol. I, pp Academic Press, London. Spielman, A.I., Huque, T., Nagai, H., Whitney, G., and Brand, J.G. (1994) Physiol. Behav. 56, Lindemann, B. (1996) Physiol. Rev. 76, Sener, A., Welsh, N., Malaisse-Lagae, F., Kadiata, M.M., and Malaisse, W.J. (1997) Submitted for publication. 1371