Laboratory of Experimental Medicine, Brussels Free University, B-1070 Brussels', Belgium and Novo Nordisk A/S, DK-2880 Bagsvaerd, Denmark

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1 Vol. 43, No. 2, October 1997 Pages CATONC AND SECRETORY EFFECTS OF 6-O-ACETYL-D-GLUCOSE N RAT PANCREATC SLETS Hassan JJAKL, Ole KRK and Willy J. MALASSE Laboratory of Experimental Medicine, Brussels Free University, B-1070 Brussels', Belgium and Novo Nordisk A/S, DK-2880 Bagsvaerd, Denmark Received June 27, 1997 SUMMARY: Selected esters of D-glucose were recently proposed as tools to supply the sugar to cells affected by a defect in the carrier-mediated process of hexose transport across the plasma membrane. The present study extends this knowledge to 6-O-acetyt-D-glucose. At a 2.0 mm concentration, the ester was indeed found to stimulate insulin release from perifused rat pancreatic islets. This coincided with stimulation of calcium influx into the islet cells, as judged from comparison of the ester effects on 45Ca outflow from prelabelled islets perifused in either the absence or presence of extracellulm" Ca ~+. The facilitated inflow of Ca z~ did not appear attributable to a decrease in K + conductance, 6-O-acetyl-D-glucose augmenting 86Rb efflux from islets prelabelled with this radioactive cation. Both the latter phenomenon and the insulinotropic action of 6-O-acetyl-D-glucose failed to be antagonized by D- marmoheptulose. These findings indicate that the cationic events coupling the recognition of D-glucose to the stimulation of insulin release are not identical in islets exposed to the hexose or its ester. NTRODUCTON Several esters of glucose, such as 6-O-octanoyl-D-glucose, 6-O-decanoyl-D-glucose, D-glucose pentaacetate, D-glucose pentapropionate and even L-glucose pentaacetate were recently found to stimulate insulin release in rat pancreatic islets [1-3]. n the case of the ocanomer of D-glucose pentaacetate, the insulinotropic action of the ester coincides with its uptake and hydrolysis, the further metabolism of both its hexose and acetate moieties, stimulation ofproinsulin biosynthesis, decrease in K + conductance and subsequent stimulation of Ca 2+ inflow and net uptake [2, 4]. The secretory response to cz-d-glucose pentaacetate is not affected by 3-O-methyl-D-glucose [1, 2]. The ester is also efficiently metabolized in rat erythrocytes [5]. Taken as a whole, these findings led us to propose that selected esters of D- glucose could be used to prevent glucopenia in cells affected by an impain~aent of carriermediated hexose transport across the plasma membrane /97/020233M /0 Copyright by Academic Press Australia. All rights of reproduction in any form reserved.

2 BOCHEMSTRY(/nd MOLECULAR BOLOGY NTERNATONAL The major aims of the present study were to investigate whether 6-O-acetyt-D-glucose also displays insulinotropic potential and to identify those cationic events possibly involved in the secretory response to this ester. MATERALS AND METHODS The 6-O-acetyl ester of D-glucose was synthesized by a procedure described elsewhere [3]. D-mannoheptulose was purchased from Sigma (St. Louis, MO). All experiments were conducted in islets isolated by the collagenase technique [6] from fed female Wistar rats. The methods used to measure insulin release [7], 86Rb outflow [8], and 45Ca outflow [9] from prelabelled perifused islets were previously described in the cited references. All results are presented as mean values ( SEM) together with the number of individual determinations. The procedure used for the analysis of secretory and cationic data was defined elsewhere [10]. The statistical significance of differences between mean values was assessed by use of Student's t-test. RESULTS n islets deprived of any exogenous nutrient, 6-O-acetyl-D-glucose (2.0 mm) unexpectedly augmented 86Rb efflux from prelabelled islets (Fig. 1, upper panel). Comparable results were recorded when D-mannoheptulose (10.0 ram) was present in the perfusion medium throughout the experiment (Fig. 2, upper panel). Pooling the results of the two series of experiments, the 86Rb fi'actional outflow rate reached after 3 min of exposure to 6-O-acetyl-D-glucose was already rain 1 higher (n = 12; P < 0.05) than the" paired nadir value recorded just before introduction of the ester or immediately thereafter (min ). Between the 48 'h and 53 ~d min of perifusion, the 86Rb fractional outflow rate remained at its high level or further increased, with a paired change averaging rain -1 (n = 12). n the absence of D-mannoheptulose, 6-O-acetyl-D-glucose failed to provoke any major change in 45Ca outflow (Fig. 1, second panel). Nevertheless, there was a less rapid decrease in effluent radioactivity in the presence of the ester than in its absence. Thus, the slope of the regression line defining the time-related exponential fall in 45Ca fractional outflow rate changed from a basal value of min -t (n = 4; P < 0.005) from rain 31 to 45 inclusive to a mean value no more significantly different from zero ( min~; P > 0.6) during the early period of 6-O-acetyl-D-glucose administration (min 47 to 56 inclusive). Thereafter, such a slope resumed its initial value, averaging 13.9 ~: min ~ (P < 0.005) between the 56 th and 70 ~ min of perifusion. 234

3 5,0 A 4,0 [- 6-0-acety-D-glucose (2 mm) r.r. i" 'i 'e- ~" 3,0 b -' t 0 2,0 &J_ e,- 1,0 0~0... r ,4 " i L 1,2 T= 1,0 N bo,8 t.1_ 0,6 0,4 0,2 0,6 i f i< 84 -~ 0,5 L- 0,4 E ::~= 0,3 ~'0,2 O 0,O "rime (rain) Fig. 1. Effect of 6-O-acetyl-D-glucose(2.0 mm) administered from the 46 th to 70 'h rain upon 86Rb fractional outflow rate (FOR), 45Ca fractional outflow rate and insulin output from islets perifused in the absence of any other exogenous nutrient and presence of 1.0 mm Ca 2+. Mean values (+ SEM) refer to 8 (upper panel), 4 (middle panel) and 12 (lower panel) individual experiments. a 235

4 Vol. 43, No O -acety-d-giucose (2 mm) 5,0 4,0 "7,=_ E ~," 3,0 O 2,0 e~ 1, ,8 1,6 ~, 1,4 "7 1,2.E; E. 1,o 0,8 O,G g.~_ 0,4 0,2 1 1 i J Time (min) Fig. 2. Effect of 6-O-acetyl-D-glucose (2.0 mm) administered from the 4@' to 70 th rain upon ~6Rb fractional outflow rate (FOR) and insulin output from islets per in the presence of 10.0 mm D-mannoheptulose and 1.0 mm Ca 2+. Mean values ( SEM) refer to 4 individual experiments. n these experiments, the administration of 6-O-acetyl-D-glucose caused a monophasic and rapidly reversible stimulation of insulin release (Fig. 1, lower panel), the mean secretory rate during the 25 min of exposure to the ester being gu.min -1 per islet higher (n = 12; P < 0.025) than the paired basal insulin output (min 42 to 45 inclusive). The insulinotropie action of 6-O-acetyl-D-glucose was not suppressed by D-mannoheptulose (Fig. 2, lower panel), Relative to the paired basal value, the increment in secretory rate attributable to the ester, as computed over 25 min of stimulation, yielded comparable results (P > 0.8) in the presence and absence of D-mannoheptulose, with an overall mean value of % (n = 16; P < 0.005). 236

5 When the experiments conducted in the absence of D-mannoheptulose were repeated in media deprived of CaCt2 and enriched with EGTA (0.5 mm), both the ester-induced change in the slope of the regression lines characterizing the time-related exponential fall in 45Ca fractional outflow rate and the stimulation of insulin release by 6-O-acetyl-D-glucose were suppressed (Fig. 3). The slope of the above-mentioned regression lines was indeed virtually identical during the control period (min 31 to 45) and either the early (min 47 to 56) or late (min 56 to 70) period of 6-O-acetyl-D-glucose administration, with mean respective values of 8.9 =k 3.4, and min -1 (n = 4 in all cases). As expected from previous observations [11], the basal release of insulin (min 42 to 45) was higher (P < 0.001) in the absence of extracellular Ca 2~ (1.34 a: 0.17 gu.min -1 per islet; n = 4) than in its presence (0.26 :k 0.03 gu.min ~ per islet; n = 12). n the former situation, no significant difference was found between the mean insulin output before and during exposure to the D-glucose ester (paired change : gu.min -~ per islet; n = 4). DSCUSSON The present study reveals that 6-O-acetyl-D-glucose stimulates insulin release when tested at a concentration far below that required to detect, under the same experimental conditions, a positive secretory response to unesterified D-glucose [12]. The metabolic and cationic determinants of 6-O-acetyl-D-glucose insulinotropic action are not easy to identify in the light of the present findings. The comparison between the effects of the ester upon 45Ca outflow from islets perifused in either the absence or presence of extracellular Ca 2+ suggests that 6-O-acetyl-D-glucose provokes a modest increase of Ca 2+ influx into the islet cells, leading to a process of exchange between influent 4~ and effluent 45Ca2+ [11]. Yet, the postulated facilitation of Ca 2+ inflow does not appear attributable to a decrease in K + conductance mad subsequent gating of voltage-sensitive Ca 2+ channels, as currently implied in the stimulation of insulin release by unesterified D-glucose [13]. ndeed, in 10 out of 12 individual experiments, 6-O-acetyl-D-glucose augmented, rather than decreased, 86Rb outflow from prelabelled islets. As an alternative hypothesis, it could be proposed that the stimulation of Ca 2+ influx by the ester results from the gating of voltageinsensitive Ca 2 channels, in a manner comparable to that recently documented in depolarized islets perifused in the presence of both diazoxide and verapamil and suddenly exposed to a 237

6 1,8 1,6 ~, 1,4,r E 1,2 o 1,0,-,. 0,8 o u. 0,6 0,4 0,2 6-O -acety-d-glucose (2 mm) i 1,8 --~._~ 1,6 1,4 v= 1,Z E. 1,0 "~=0,8 ~0,6 '~ 0,4 c 40,2 < 50 Time (min) i Fig. 3. Effect of 6-O-acetyl-D-glucose (2.0 mm) administered from the 46 th to 70 th min upon 45Ca fractional outflow rate (FOR) and insulin output from islets perifused in the absence of CaC12 and presence of 0.5 mm EGTA. Mean values ( SEM) refer to 4 individual experiments. rise in extracellular D-glucose concentration [14]. Such a proposal could also conceivably account for an increase in 86Rb outflow resulting from the activation of Ca2+-responsive K + channels [15]. The fact that D-mannoheptulose failed to suppress the early increase in 86Rb outflow and the stimulation of insulin release caused by 6-O-acetyl-D-glucose does not necessarily imply that the hexose moiety of the ester, liberated by its intracellular hydrolysis, does not participate in the functional response of the islets to this nutrient. Thus, D-mannoheptulose fails to affect both the catabolism of a-d-glucose pentaacetate and its insulinotropic action, 238

7 . although ~-D-galactose pentaacetate, which also undergoes extensive hydrolysis in isolated islets, fails to stimulate insulin release [2, 4]. These findings indeed indicate that the secretory response to these esters is not solely attributable to the generation and catabolism of their acetate moieties and displays specificity towards selected epimers of aldohexose. Moreover, in the present experiments, the increase in 86Rb outflow provoked by 6-O-acetyl-D-glucose appeared less sustained in the presence of D-mannoheptulose than in its absence. For instance, relative to the paired value recorded after 5 rain of exposure to 6-O-acetyl-D-glucose (min 50), the 86Rb fractional outflow rate measured at the 70 'h min averaged in the presence of D-marmoheptulose % (n = 4), as distinct (P < 0.02) from % (n = 8) in the absence of the ketoheptose. Moreover, when the administration of the ester was halted at the 70 th min of perifusion, the mean value for the 86Rb fractional outflow rate was slightly and transiently increased in islets exposed to D-mannoheptulose, whilst the opposite situation prevailed in islets perifused in the absence of the heptose. Likewise, when islets are incubated for 90 min in the presence of 6-O-acetyl-D-glucose, rather than being exposed for only 25 min to the ester, its insulinotropic action is indeed suppressed by D-mannoheptulose [3]. n conclusion, the present study extends to 6-O-acetyl-D-glucose the knowledge that selected esters of D-glucose are able to stimulate insulin release under experimental conditions in which the tmesterified hexose fails to do so. The present findings also reinforce the view that the process of stimulus-secretion coupling involved in the insulinotropic action of these esters differs, in some respect at least, from that currently held responsible for the stimulation of insulin release by unesterified D-glucose. Acknowledgments. This work was supported by a Concerted Research Action of the French Community of Belgium (94/99-183) and a grant from the Belgian Foundation for Scientific Medical Research ( ). The authors are grateful to M. Malay for technical assistance and 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,., Kirk, O., Kadiata, M.M. and Sener, A. (1997) Am. J. Physiol., submitted for publication. Malaisse, W.J. and Kirk, O. (1997) Horm. Metab. Res., submitted for publication. Sener, A., Welsh, N., Malaisse-Lagae, F., Kadiata, M.M. and Malaisse, W.J. (1997) J. Biol. Chem., submitted for publication. 239

8 BOCHEMSTRYand MOLECULAR BOLOGY NTERNATONAL 5. Ladri6re, L., Kadiata, M.M., Kirk, O. and Malaisse, W.J. (1997) J. Biol. Chem., submitted for publication. 6. Malaisse-Lagae, F. and Malaisse, W.J. (1984) in Methods in Diabetes Research (eds. Lamer, J. and Pohl, S.L.) , Wiley, New York. 7. Herchuelz, A. and Malaisse, W.J. (1978) J. Physiol. (London) 283, Carpinelli, A. and Malaisse, W.J. (1980) Mol. Cell. Endocrinol. 17, t Herchuelz, A. and Malaisse, W.J. (1980) Am. J. Physiol. 238, E87-E Jijakli, H., Ulusoy, S. and Mataisse, W.J. (1996) Pharmacol. Res. 34, Herchuelz, A., Couturier, E. and Malaisse, W.J. (1980) Am. J. Physiol. 238, E96- E Brisson, G.R., Malaisse-Lagae, F. and Malaisse, W.J. (1972) J. Clin. nvest. 51, Malaisse, W.J., Herchuelz, A. and Sener, A. (1981) in The islets of Langerhans (eds. Cooperstein, S.J. and Watkins, D.) , Academic Press, New York. 14. Jijakli, H. and Malaisse, W.J. (1997) Cell. Signalling, in press. 15. Carpinelli, A.R. and Malaisse, W.J. (1981) J. Physiol. (London) 315,