Are ionic fluxes of pancreatic beta cells a target for gastric inhibitory polypeptide?

Gastric inhibitory polypeptide (GIP), an incretin candidate, is suggested to amplify the glucose-induced insulin secretion. To evaluate its mode of action we examined whether GIP affects 86Rb+ efflux, 45Ca2+ uptake or efflux, and intracellularly recorded electrical activity of mouse pancreatic islets. GIP (5 nM) neither inhibited 86Rb+ efflux at 3 mM glucose nor modulated 86Rb+ efflux that was inhibited by 5.6 mM glucose or stimulated by the calcium ionophore A23187. 45Ca2+ uptake was increased by GIP in the presence of 16.7 mM which was not observed at 3 or 11 mM glucose. GIP elevated 45Ca2+ efflux from islets, but did not modify 45Ca2+ efflux when a virtually Ca2+ free medium was used. Electrical activity of beta cells induced by 16.7 mM glucose was significantly increased by 5 nM GIP. It is concluded that the amplification of insulin release by GIP is based on the effect of GIP on Ca2+ uptake.     

Association between 5-hydroxytryptamine release and insulin secretion.

The efflux of radioactivity after loading with trace amounts of tritiated 5-hydroxytryptamine ([3H]5-HT) or 5-hydroxytryptophan ([3H]5-HTP) was studied in perifused beta-cell-rich pancreatic islets from ob/ob mice. Analysis of the effluent revealed that more than 90% of the radioactivity was released as [3H]5-HT after loading with [3H]5-HTP. Increasing the concentration of glucose in the perifusion medium from 3 to 20 mmol/l enhanced the efflux when islets from fed mice were used and this effect was potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Whereas 20 mM-glucose alone did not stimulate the efflux of 5-HT from islets isolated from mice starved for 3 days, a stimulatory effect was observed in the presence of IBMX. Stimulation of the efflux of radioactivity by glucose was inhibited if calcium was omitted from or adrenaline added to the medium. The results are consistent with the concept of exocytotic release of 5-HT occurring in response to stimulation of insulin secretion, although basal non-exocytotic transport must also be occurring across the beta-cell membrane.     

Effect of extracellular phosphate on Ca2+ and K+ fluxes in pancreatic islets

The idea that a lowering in cytosolic Ca2+ concentration may cause a decrease in K+ conductance in the pancreatic B-cell was tested by investigating the effect of a high extracellular phosphate concentration on 45Ca and 86Rb efflux from prelabelled rat pancreatic islets. Whether in the absence or presence of glucose, 20 mM phosphate tended to decrease 45Ca efflux. This effect was not suppressed in the absence of extracellular Ca2+, at least in glucose-deprived islets, suggesting that it may reflect a fall in cytosolic Ca2+ concentration. The administration of phosphate failed, however, to decrease 86Rb efflux from the islets. In the presence of extracellular Ca2+, 20 mM phosphate also failed to stimulate insulin release from islets perifused at low glucose concentration and inhibited insulin release stimulated by a high glucose concentration. These data indicate that the sequestration of Ca2+ in intracellular organelles and concomitant decrease in cytosolic Ca2+ concentration, as presumably provoked by a rise in extracellular phosphate concentration, is not sufficient to simulate the effect of glucose on K+ conductance.     

Modulation of beta-cell ouabain-sensitive 86Rb+ influx (Na+/K+ pump) by D-glucose, glibenclamide or diazoxide

The activity of the beta-cell Na+/K+ pump was studied by using ouabain-sensitive (1mM ouabain) 86Rb+ influx in beta-cell-rich islets of Ume?-ob/ob mice as an indicator of the pump function. The present results show that the stimulatory effect of glucose on ouabain-sensitive 86Rb+ influx reached its approximate maximum at 5mM glucose. Pre-treatment of the islets with 20mM glucose for 60 min strongly reduced the glucose-induced stimulation of the Na+/K+ pump. Pre-treatment (60 or 180 min) of islets at 0 mM glucose, on the other hand, did not affect the magnitude of the glucose-induced stimulation of 86Rb+ influx during the subsequent 5-min incubation. Glibenclamide stimulated the ouabain-sensitive 86Rb+ uptake in the same manner as glucose. The stimulatory effect showed its apparent maximum at 0.5 microM. Pre-treatment (60 min) of islets with 1 microM glibenclamide did not reduce the subsequent stimulation of the ouabain-sensitive 86Rb+ influx. The stimulatory effect of glibenclamide and D-glucose were not additive, suggesting that they may have the same mechanism of action. No direct effect of glibenclamide (0.01-1 microM) was observed on the Na+/K+ ATPase activity in homogenates of islets. Diazoxide (0.4mM) inhibited the Na+/K+ pump. This effect was sustained even after 60 min of pre-treatment of islets with 0.4mM diazoxide. The stimulatory effect of glibenclamide and D-glucose were abolished by diazoxide. It is concluded that nutrient as well as non-nutrient insulin secretagogues activate the Na+/K+ pump, probably as part of the membrane repolarisation process.     

Glyburide and tolbutamide induce desensitization of insulin release in rat pancreatic islets by different mechanisms.

Insulin secretion was studied in rat pancreatic islets after 24-h exposure to various glyburide or tolbutamide concentrations. Glucose-induced insulin release was significantly (P < 0.05) reduced in islets cultured with 0.1 microM glyburide or 100 microM tolbutamide (2098 +/- 187, 832 +/- 93, and 989 +/- 88 pg/islet.h in control, glyburide-exposed, and tolbutamide-exposed islets, respectively). When glyburide-treated islets were stimulated with glyburide or tolbutamide, insulin release was also impaired compared to that in control islets (P < 0.05). In contrast, tolbutamide-exposed islets showed an impaired response to tolbutamide, but a normal response to glyburide. To investigate the mechanism of the sulfonylurea-induced impairment of insulin secretion, we measured insulin release and Rb+ efflux (a marker of the K+ channel activity) in a perifusion system and islet Ca2+ uptake under static conditions. Insulin release in response to 16.7 mM glucose increased in control islets from 9.4 +/- 1.1 to 131 +/- 19 pg/islet.min (first phase secretion peak). Simultaneously, the fractional 86Rb+ efflux declined from 0.015 +/- 0.002% to 0.006 +/- 0.001% (change in decrement, -63.5%). Glucose-induced insulin release in glyburide- and tolbutamide-treated islets was significantly reduced (first phase peak, 22.1 +/- 5 and 39.7 +/- 8 pg/islet.min, respectively; P < 0.05), and the fractional 86Rb+ efflux decrement was -21 +/- 6% for glyburide (P < 0.005 vs. control islets) and -65 +/- 4% (not different from control) for tolbutamide. When glyburide- or tolbutamide-exposed islets were stimulated with the corresponding sulfonylurea, insulin release was impaired compared to that in control islets (P < 0.05), but, again, 86Rb+ efflux was impaired (P < 0.05) only in glyburide-exposed islets. When 45Ca2+ uptake was studied, the increase in glucose concentration from 2.8 to 16.7 mM increased calcium uptake in control islets from 1.76 +/- 0.58 to 7.27 +/- 1.36 pmol/islet.2 min (n = 4). Preexposure to 0.1 microM glyburide did not change calcium uptake at a glucose concentration of 2.8 mM (1.44 +/- 0.45 pmol/islet.2 min) but significantly reduced calcium uptake stimulated by 16.7 mM glucose (3.21 +/- 0.35 pmol/islet.2 min; n = 4; P < 0.005 compared to control islets). In contrast, preexposure to 100 microM tolbutamide did not change either basal or glucose-stimulated calcium uptake (1.44 +/- 0.45 and 6.90 +/- 0.81 pmol/islet.2 min, respectively; n = 4). These data show that in vitro chronic exposure of pancreatic islets to the sulfonylureas glyburide and tolbutamide impairs their ability to respond to a subsequent glucose or sulfonylurea stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Muscarinic control of pancreatic B cell function involves sodium-dependent depolarization and calcium influx

Mouse pancreatic islets were used to investigate the mechanisms and functional significance of the B cell membrane depolarization by acetylcholine (ACh). At low glucose (3mM), ACh (20 microM) increased 22Na+ influx, and slightly depolarized the B cell membrane but did not induce electrical activity or stimulate 45Ca2+ influx. ACh also accelerated 86Rb+ and 45Ca2+ efflux and barely affected basal insulin release. At a stimulatory concentration of glucose (10 mM), ACh stimulated 22Na+ influx, depolarized the B cell membrane, increased glucose-induced electrical activity, and stimulated 45Ca2+ influx. ACh also accelerated 86Rb+ and 45Ca2+ efflux and strongly potentiated insulin release. Omission of extracellular Ca2+ did not impair ACh stimulation of 22Na+ influx or 86Rb+ efflux, slightly modified the acceleration of 45Ca2+ efflux, and almost completely suppressed the increase in insulin release. Na+ omission (with N-methyl-D-glucamine as substitute) prevented the B cell membrane depolarization and the stimulation of 45Ca2+ influx, largely inhibited the acceleration of 86Rb+ efflux and insulin release, and suppressed the late phase of 45Ca2+ efflux otherwise produced by ACh. On the other hand, ACh stimulation of 3H efflux from islets prelabeled with myo-[2-3H]inositol was not affected by Na+ omission. All effects of ACh were blocked by atropine and unaffected by nicotinic antagonists. It is concluded that activation of muscarinic receptors depolarized the B cell membrane by increasing its permeability to Na+. When the membrane is already depolarized by glucose, this further depolarization augments Ca2+ influx and, hence, potentiates insulin release.     

Is there a role for osmotic events in the exocytotic release of insulin?

The possible role of an osmotic lysis of insulin granules during exocytosis has been studied in perifused mouse pancreatic islets. Raising the osmolarity of the extracellular medium by addition of 400 mM sucrose reversibly inhibited glucose-stimulated insulin release. This inhibition was accompanied by a decrease in the rates of 86Rb+ or 45Ca2+ efflux from the islets. Increasing the osmolarity and restoring a normotonic medium in the presence of a nonstimulatory concentration of glucose accelerated 86Rb+ and 45Ca2+ efflux and augmented basal insulin release in both the presence and absence of Ca2+. Hyperosmolarity did not prevent a rise in glucose concentration from decreasing 86Rb+ efflux from islet cells or from inhibiting 45Ca2+ efflux in Ca2+-free medium. However, the stimulation of 45Ca2+ efflux otherwise produced by glucose in the presence of Ca2+ was abolished, and the stimulation of insulin release was almost suppressed. Hyperosmolarity also strongly impaired the release of insulin during stimulation by eight experimental conditions known to act through at least partially different mechanisms. The changes in 45Ca2+ efflux brought about by these different agents were also altered by hyperosmolarity, whether they resulted from direct mobilization of intracellular Ca2+ or were secondary to increased Ca2+ influx. The blockade of insulin release by hyperosmolarity, whatever the mode of action of the stimulus, is compatible with the participation of osmotic events in exocytosis. However, the marked alterations in Ca2+ handling that occur concomitantly and might account for the inhibition of release make it impossible to demonstrate their exact role in intact islet cells.     

Galanin and epinephrine act on distinct receptors to inhibit insulin release by the same mechanisms including an increase in K+ permeability of the B-cell membrane.

The mechanisms by which galanin and epinephrine affect pancreatic B-cell function were studied in normal mouse islets. In the presence of 15 mM glucose and 2.5 mM Ca2+, galanin (50 nM) and epinephrine (100 nM) hyperpolarized the B-cell membrane and suppressed electrical activity only transiently. These changes were accompanied by a decrease in 86Rb+ efflux from islet cells and nearly complete inhibition of insulin release. Both agents also decreased 86Rb+ efflux in the absence of Ca2+. Low concentrations (10-15 microM) of diazoxide, an activator of ATP-sensitive K+ channels, mimicked some effects of galanin and epinephrine. However, insulin release was more markedly inhibited by galanin or epinephrine than by diazoxide when electrical activity was similarly decreased, and diazoxide had no effect on 86Rb+ efflux in the absence of Ca2+. When the permeability to K+ was increased by 100 microM diazoxide and the hyperpolarization reversed by high extracellular K+, galanin and epinephrine still inhibited insulin release, but did not affect the membrane potential or 86Rb+ efflux. Galanin and epinephrine decreased glucose utilization and oxidation in islet cells by about 10%, whereas diazoxide had no effect. Blockade of alpha 2-adrenoceptors by yohimbine suppressed the effects of epinephrine, but not those of galanin. It is concluded that activation of galanin and alpha2-adrenergic receptors inhibits insulin release by the same mechanisms. These may involve an increase in K+ permeability of the B-cell membrane by opening ATP-sensitive K+ channels and an additional effect independent of the membrane potential.     

Modulation of the effect of acetylcholine on insulin release by the membrane potential of B cells

Mouse islets were used to test the hypothesis that the B cell membrane must be depolarized for acetylcholine to increase insulin release. The resting membrane potential of B cells (at 3 mM glucose) was slightly decreased (5 mV) by acetylcholine, but no electrical activity appeared. This depolarization was accompanied by a Ca-independent acceleration of 86Rb and 45Ca efflux but no insulin release. When the B cell membrane was depolarized by a stimulatory concentration of glucose (10 mM), acetylcholine potentiated electrical activity, accelerated 86Rb and 45Ca efflux, and increased insulin release. This latter effect, but not the acceleration of 45Ca efflux, was totally dependent on extracellular Ca. If glucose-induced depolarization of the B cell membrane was prevented by diazoxide, acetylcholine lost all effects but those produced at low glucose. In contrast, when the B cell membrane was depolarized by leucine or tolbutamide (at 3 mM glucose), acetylcholine triggered a further depolarization with appearance of electrical activity, accelerated 86Rb and 45Ca efflux, and stimulated insulin release. Acetylcholine produced similar effects (except for electrical activity) in the presence of high K or arginine which, unlike the above test agents, depolarize the B cell membrane by a mechanism other than a decrease in K+ permeability. Omission of extracellular Ca abolished the releasing effect of acetylcholine under all conditions but only partially decreased the stimulation of 45Ca efflux. The results show thus that acetylcholine stimulation of insulin release does not result from mobilization of cellular Ca but requires that the B cell membrane be sufficiently depolarized to reach the threshold potential where Ca channels are activated. This may explain why acetylcholine alone does not initiate release but becomes active in the presence of a variety of agents.     

Demonstration of glucose inhibition of insulin release in the presence of diazoxide

beta-Cell-rich pancreatic islets from ob/ob mice were taken for measurements of insulin release in response to glucose after culture in RPMI 1640 medium. The stimulatory effect of 20 mmol/l glucose was converted into an inhibition when the medium was supplemented with 400 mumol/l diazoxide. Glucose inhibition of insulin release was observed when the islets had been cultured in the presence of 1 or 20 mmol/l glucose in media either containing or lacking Ca2+. The data provide further evidence for an inhibitory component in the action of glucose on insulin release, suggesting that glucose stimulation of the Ca2+ efflux is essential for the appearance of this inhibition.     

Stimulus-secretion coupling of arginine-induced insulin release: comparison with lysine-induced insulin secretion

L-Lysine, like-L-arginine, L-ornithine, or L-homoarginine, accumulated in rat pancreatic islets and stimulated 86Rb efflux, 45Ca uptake and efflux, and insulin release in islets exposed to D-glucose (7.0 mM). The effect of L-lysine differed from that of the other cationic amino acids by such features as the absence of a threshold concentration for stimulation of insulin release, a much lesser sensitivity of the secretory response to intracellular acidification, and the stimulation of 86Rb net uptake over 60 min of incubation. This coincided with the fact that even in the absence of another exogenous nutrient, L-lysine was well oxidized, augmented NH4+ production, increased both the ATP content and ATP/ADP ratio, caused a time-related decrease in 86Rb fractional outflow, and provoked either a transient (10 mM L-lysine) or sustained (20 mM L-lysine) stimulation of insulin secretion. It is proposed, therefore, that the functional response of the pancreatic B-cell to L-lysine involves not only a biophysical mechanism similar to that responsible for the insulinotropic action of L-homoarginine, but also a significant, albeit modest, metabolic component, which reflects the capacity of L-lysine to act as a fuel in islet cells.     

Further studies on the relationship between insulin release and lanthanum-nondisplaceable 45Ca2+ uptake by pancreatic islets: effects of fructose and starvation.

Relationships between the release of insulin and the incorporation of 45Ca2+ into a lanthanum-nondisplaceable (intracellular) pool were studied in islets microdissected from the pancreatic glands of non-inbred ob/ob mice. In comparison with D-glucose, D-fructose was slowly oxidized and had only marginal effects on insulin release. However, fructose was as effective as glucose in stimulating the lanthanum-nondisplaceable 45Ca2+ uptake. The 45Ca2+ uptake was dose-dependent on the concentration of fructose in the range 0-20 mM; the same dose-dependence was obtained with glucose. Fasting the mice for 3 days caused a total block of the insulin secretory response to 20 mM glucose, but it produced an enhancement of the glucose-induced 45Ca2+ uptake. Both the inhibition of insulin release and the enhancement of 45Ca2+ uptake were counteracted by pretreating the isolated islets with 20-40 mM D-glucose; pretreatment with L-glucose or fructose could not counteract the effects of fasting. Although some functional relationship may exist between the lanthanum-nondisplaceable uptake of 45Ca2+ and the insulin secretory apparatus, it is concluded that the uptake of Ca2+ is not simply the result of stimulated insulin release.     

The ionic, electrical, and secretory effects of protein kinase C activation in mouse pancreatic B-cells: studies with a phorbol ester

The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) was used to study the effects of protein kinase C activation on stimulus-secretion coupling in mouse pancreatic B-cells. At a nonstimulatory concentration of glucose (3 mM), 100 nM TPA, but not 10 nM TPA, slightly and slowly increased insulin release and 45Ca2+ efflux and decreased 86Rb+ efflux, but did not affect the membrane potential of B-cells. At a threshold concentration of glucose (7 mM), 100 nM TPA markedly increased insulin release without triggering electrical activity in B-cells. At a stimulatory concentration of glucose (10 mM), TPA caused a dose-dependent irreversible increase in insulin release, 45Ca2+ efflux, and 86Rb+ efflux and slightly augmented islet cAMP levels. Omission of extracellular Ca2+ abolished the effects of 10 nM TPA and partially inhibited those of 100 nM TPA on insulin release and 45Ca2+ efflux. In contrast, their effect on 86Rb+ efflux was paradoxically augmented. Glucose-induced electrical activity in B-cells was only marginally affected by TPA; the duration of the slow waves with spikes was not modified, but a small shortening of the polarized intervals raised their frequency and slightly increased the overall activity. This increase was significant only with 10 nM TPA, whereas only 100 nM TPA brought about a minute increase in 45Ca2+ influx. These results thus show that TPA induces insulin release or potentiates glucose-induced insulin release without mimicking or amplifying the initial ionic and electrical signals triggered by glucose. They suggest that protein kinase C activation affects stimulus-secretion coupling by modulating intracellular and/or nonelectrogenic membrane events.     

Alloxan-induced alteration of insulin release,rubidium efflux and glucose metabolism in rat islets stimulated by various secretagogues

Summary Insulin release and⁸⁶Rb efflux were studied in perifused rat islets exposedin vitro to alloxan (2 mmol/l) for 5 min. At a low glucose concentration, alloxan transiently increased⁸⁶Rb efflux. Alloxan immediately and completely abolished the secretory response to glucose (15 mmol/l) and markedly delayed the reduction in⁸⁶Rb efflux normally produced by the sugar. 3-O-methylglucose (20 mmol/l) provided complete protection against the alteration of⁸⁶Rb efflux and partial protection against the inhibition of insulin release. Immediately after alloxan treatment, glyceraldehyde,-ketoisocaproic acid and tolbutamide still induced a rapid release of insulin, but the late phase normally stimulated by glyceraldehyde and-ketoisocaproic acid was inhibited. If islets were exposed to glyceraldehyde or tolbutamide 15 min after alloxan treatment, the rapid insulin release was also markedly impaired. Alloxan failed, however, to affect the ability of these three stimuli to reduce⁸⁶Rb efflux from islet cells. Glucose oxidation and utilization were decreased in alloxan-treated islets and 3-O-methylglucose protected against this effect. The results show that the glucose recognition system in B-cells is the most rapidly and severely affected by alloxan. The drug also alters the response to other secretagogues, the insulin releasing properties of which can be impaired without alteration of their ability to reduce⁸⁶Rb efflux.     

Stimulus-secretion coupling of arginine-induced insulin release. Uptake of metabolized and nonmetabolized cationic amino acids by pancreatic islets

In order to assess the possible role of L-arginine accumulation in islet cells as a determinant of its insulinotropic action, the uptake of L-arginine and other cationic amino acids (L-ornithine, L-homoarginine, D,L-alpha-methylornithine, D,L-alpha-difluoromethylornithine) by rat pancreatic islets was compared to the ionic and secretory responses of the islets to the same amino acids. A tight correlation was found between the net uptake of these amino acids and their capacity to stimulate 86Rb efflux, 45Ca uptake and efflux, and insulin release. In the latter respect, there was little difference between metabolized and nonmetabolized amino acids. Thus, although L-homoarginine and 4-amino-1-guanylpiperidine-4-carboxylic acid failed to act as a substrate for either arginase or amino acid aminotransferase in islet homogenates, they both stimulated 86Rb efflux, 45Ca uptake and efflux, and insulin secretion in intact islets. These findings are compatible with the view that the accumulation of these positively charged amino acids in islet cells represents an essential determinant of their secretory action. Hence, the release of insulin evoked by these amino acids could be due to depolarization of the plasma membrane with subsequent gating of voltage-sensitive Ca2+ channels and/or to some other biophysical effect, as suggested by the persistence of a sizeable secretory response to L-arginine or L-ornithine in islets perifused at a high concentrations of extracellular K+ (50 mM).     

Gastrin releasing peptide augments glucose mediated 45Ca2+ uptake, electrical activity, and insulin secretion of mouse pancreatic islets

Gastrin releasing peptide (GRP) has recently been shown to increase glucose-induced insulin secretion in vivo. Being present in pancreatic tissue, the 27-amino acid peptide could play a role in the control of the glucose-induced insulin secretion of islets of Langerhans. In the presence of a stimulatory glucose concentration, GRP augmented insulin secretion of isolated islets in batch incubations. The peptide did not affect 56Rb+ efflux in the presence of 3 or 5.6 mM glucose but reduced the increase of 86Rb+ efflux evoked by the calcium ionophore A23187. 45Ca2+ uptake and intracellular recorded electrical activity induced by glucose were amplified by GRP. It is suggested that GRP plays a role in the regulation of glucose-induced insulin secretion by increasing the uptake of Ca2+ directly or by inhibition of the Ca(2+)-dependent K+ channel activity and reduced repolarization of the cell.     

Defective early phase insulin release in perifused isolated pancreatic islets of spiny mice (acomys cahirinus)

In order to characterize pancreatic beta cell function in Geneva bred spiny mice (acomys cahirinus), the dynamics of immunoreactive insulin release were examined during perifusion of pancreatic islets isolated from normoglycemic acomys. The initial insulin response of acomys was slow: no clear-cut early (1 to 10 min) peak of insulin release was observed when glucose in the perifusion medium was abruptly raised from 2.8 mM to concentrations as high as 56 mM. This was true for islets of either young, or older more obese acomys. However, after 20 to 30 min of perifusion at the high glucose concentrations, the rate of insulin release from acomysislets became similar to that from islets of rats or mice. By contrast, glucose-induced insulin release responses observed with islets of Wistar-derived rats, Swiss albino mice, and inbred C57BL/6J lean or obese (ob/ob) mice, were clearly biphasic. Tolbutamide 1.5 mM, arginine 16 mM, and theophylline 10 mM were ineffective in stimulating insulin release from acomys islets in the presence of a substimulatory glucose concentration (2.8 mM), whereas these agents were effective in rat islets at the same substimulatory concentration of glucose. On the other hand, when these agents, as well as cyclic AMP 10 mM or cytochalasin B 10 mug/ml were applied in the presence of a stimulating concentration of glucose (16.8 mM), the glucose-stimulated insulin release from acomys islets was increased to the same or to a greater extent than from rat islets. It is suggested that the failure of all the agents tested to stimulate an early rapid phase of insulin release from acomys islets may be secondary to the observed initial insensitivity to glucose, which insensitivity may in turn reflect a selective impairment in the recognition of glucose as an insulinogenic signal in this species.     

Cationic determinants of D-fructose insulinotropic action

In the absence of any other exogenous nutrient, D-fructose stimulates insulin release from rat pancreatic islets provided that it is tested at high concentrations in excess of a threshold value close to 80 mM, an optimal secretory response being recorded at 240 mM. In the present study, the cationic determinants of the insulinotropic action of D-fructose, used at the latter high concentration, were explored in perifused rat islets that had been prelabelled with either ⁸⁶Rb or ⁴⁵Ca. The changes in ⁸⁶Rb outflow and ⁴⁵Ca efflux evoked by 240 mM D-fructose were comparable to those caused by 11.1 mM D-glucose in that both hexoses inhibited ⁸⁶Rb and ⁴⁵Ca outflow and, at normal Ca²⁺ concentration, caused a secondary rise in ⁴⁵Ca efflux. These cationic changes coincided wit stimulation of insulin release. The major differences between the two series of experiments consisted, in the islets exposed to D-fructose, in the occurrence of an early and transient increase in ⁴⁵Ca efflux at normal extracellular Ca²⁺ concentration, a secondary reascension in ⁸⁶Rb outflow and a dramatic off-response in both ⁸⁶Rb and ⁴⁵Ca outflow as well as insulin release. These phenomena were also observed in islets exposed to 240 mM 3-O-methyl-D-glucose, suggesting that they may be linked to the massive influx (or efflux) of monosaccharides, as possibly accompanied by Na⁺ inward co-transport, mobilization of Ca²⁺ from intracellular stores and activation of voltage- and/or Ca²⁺-sensitive K⁺ channels. This interpretation was supported by the finding that, at high concentrations (80.0 mM) of D-glucose or D-mannose, the aldohexoses, also provoked a reascension in ⁸⁶Rb outflow and off-response in insulin release. The cationic determinants of the insulinotropic action of D-fructose, in high concentration (240 mM), thus appear similar, if not identical, to those currently incriminated in the stimulation of insulin release by D-glucose. Received: 31 May 1999 / Accepted in revised form: 22 December 1999     

The Pancreatic β-Cell Recognition of Insulin Secretagogues: Does Cyclic AMP Mediate the Effect of Glucose?

Insulin release and the content of cAMP were studied in microdissected pancreatic islets of noninbred ob/ob (obese) mice. In the absence of 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, 20 mM glucose had no effect on cAMP save a very small initial rise detectable by a freeze-stop perifusion technique only. However, combined with this methylxanthine, 20 mM glucose produced significant increases of cAMP both in perifused islets and in islets conventionally incubated in closed vials. Glucose shared this capacity to raise the cAMP level with D-glyceraldehyde and 1,3-dihydroxyacetone. Isobutylmethylxanthine (0.05-1.0 mM) or 5 mug/ml of cholera toxin, an activator of adenylate cyclase, also increased the islet cAMP level; the effects of the methylxanthine, whether or not combined with cholera toxin, were potentiated by glucose. Isobutylmethylxanthine (0.05-1.0 mM) or 5 mug/ml of cholera toxin potentiated insulin release in response to 20 mM glucose. However, only 0.5-1.0 mM isobutylmethylxanthine stimulated insulin release in the presence of 3 mM glucose, whereas 0.05-0.1 mM isobutylmethylxanthine or 5 mug/ml of cholera toxin had no effect on secretion at the low glucose concentration. These discrepancies between cAMP-promoting and insulin-releasing activities suggest that glucose does not initiate insulin release by activating the beta-cell adenylate cyclase. By being metabolized in the beta-cells, glucose may both create a release-initiating signal not identical with cAMP and enhance cAMP formation, leading to potentiation of the effect of the initiator signal.     

D-glucose stimulates the Na+/K+ pump in mouse pancreatic islet cells

To determine the effect of D-glucose on the beta-cell Na+/K+ pump, 86Rb+ influx was studied in isolated, -cell-rich islets of Ume?-ob/ob mice in the absence or presence of 1mM ouabain. D-glucose (20mM) stimulated the ouabain-sensitive portion of 86Rb+ influx by 65%, whereas the ouabain-resistant portion was inhibited by 48%. The Na+/K+ ATPase activity in homogenates of islets of Ume?-ob/ob mice or normal mice was determined to search for direct effects of D-glucose. Thus, ouabain-sensitive ATP hydrolysis in islet homogenates was measured in the presence of different D-glucose concentrations. No effect of D-glucose (3-20mM) was observed in either ob/ob or normal islets at the optimal Na+/K+ ratio for the enzyme (135mM Na+ and 20mM K+). Neither D-glucose (3-20mM) nor L-glucose or 3-O-methyl-D-glucose (20mM) affected the enzyme activity at a high Na+/K+ ratio (175 mM Na+ and 0.7 mM K+). Diphenylhydantoin (150 microM) decreased the enzyme activity at optimal Na+/K+ ratio, whereas 50 microM of the drug had no effect. The results suggest that D-glucose induces a net stimulation the Na+/K+ pump of beta-cells in intact islets and that D-glucose does not exert any direct effect on the Na+/K+ ATPase activity.