Comparative study on the effects of a hypoglycemic 2-substituted-2-imidazoline derivative (DG-5128) and tolbutamide on insulin secretion from and insulin synthesis in the isolated rat pancreatic islets

2[2-(4,5-Dihydro-1H-imidazol-2-yl)-1-phenylethyl]pyridine dihydrochloride sesquihydrate (DG-5128) was found to stimulate the glucose-primed insulin secretion from the isolated rat pancreatic islets throughout the incubation period, unlike tolbutamide which stimulated it only in the initial phase of incubation. The effect of DG-5128 was more pronounced at a higher glucose concentration (5 mg/ml). In the islet perifusion study, DG-5128 was also found to stimulate the glucose-induced insulin secretion in both the first and the second phases of the reaction, in contrast to tolbutamide which stimulated only the first phase of insulin secretion from the perifused islets. DG-5128 gave no significant effect on the glucose-stimulated increase in incorporation of [³H]leucine into the pro-insulin and insulin fractions, while tolbutamide significantly inhibited the incorporation especially at a low glucose concentration (1 mg/ml). These and the previous findings indicate that DG-5128 is a new class of hypoglycemic agent with a unique mode of action different from the known hypoglycemics ever reported.     

Effect of the alpha 2-blocker DG-5128 on insulin and somatostatin release from the isolated perfused rat pancreas

2[2-(4.5-Dihydro-1H-imidazol-2-yl)-1-phenylethyl] pyridine dihydrochloride sesquihydrate (DG-5128) is an alpha 2-specific-adrenergic antagonist. We have studied the effect of DG-5128 on insulin and somatostatin release from the isolated perfused rat pancrease. DG-5128 stimulated somatostatin and insulin release not only at a low glucose concentration but also at a high glucose concentration. These findings suggest that an alpha 2-adrenergic receptor plays an important role in the regulation of insulin and somatostatin secretion.     

Difference in mechanism between glyceraldehyde- and glucose-induced insulin secretion from isolated rat pancreatic islets

The effects of D-glyceraldehyde and glucose on islet function were compared in order to investigate the difference between them in the mechanism by which they induce insulin secretion. The stimulation of insulin secretion from isolated rat islets by 10 mM glyceraldehyde was not completely inhibited by either 150 microM diazoxide (an opener of ATP-sensitive K(+) channels) or 5 microM nitrendipine (an L-type Ca(2+)-channel blocker), whereas the stimulation of insulin secretion by 20 mM glucose was completely inhibited by either drug. The insulin secretion induced by glyceraldehyde was less augmented by 100 microM carbachol (a cholinergic agonist) than that induced by glucose. The stimulation of myo-inositol phosphate production by 100 microM carbachol was more marked in islets incubated with the hexose than with the triose. The content of glyceraldehyde 3-phosphate, a glycolytic intermediate, in islets incubated with glyceraldehyde was far higher than that in islets incubated with glucose, whereas the ATP content in islets incubated with the triose was significantly lower than that in islets incubated with the hexose. These results suggest that glyceraldehyde not only mimics the effect of glucose on insulin secretion but also has the ability to cause the secretion of insulin without the influx of Ca(2+ )through voltage-dependent Ca(2+) channels. The reason for the lower potency of the triose than the hexose in stimulating insulin secretion is also discussed.     

Cell swelling-induced signaling for insulin secretion bypasses steps involving G proteins and PLA2 and is N-ethylmaleimide insensitive.

BACKGROUND: This study was undertaken to examine putative mechanisms of calcium independent signal transduction pathway of cell swelling-induced insulin secretion. METHODS: The role of phospholipase A(2), G proteins, and soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) in insulin secretion induced by 30% hypotonic medium was studied using isolated rat pancreatic islets. RESULTS: In contrast to glucose stimulation, osmotically induced insulin secretion from pancreatic islets was not inhibited by 10 micromol/l bromoenol lactone, an iPLA(2) (Ca(2+) independent phospholipase) inhibitor. Similarly, preincubation of islets for 20 hours with 25 microg/ml mycophenolic acid to inhibit GTP synthesis fully abolished glucose-induced insulin secretion but was without effect on hypotonicity stimulated insulin release. Glucose-induced insulin secretion was prevented by preincubation with 20 nmol/l tetanus toxin (TeTx), a metalloprotease inactivating soluble SNARE. Cell swelling-induced insulin secretion was inhibited by TeTx in the presence of calcium ions but not in calcium depleted medium. The presence of N-ethylmaleimide (NEM, 5 mmol/l, another inhibitor of SNARE proteins) in the medium resulted in high basal insulin secretion and lacking response to glucose stimulation. In contrast, high basal insulin secretion from NEM treated islets further increased after hypotonic stimulation. CONCLUSION: G proteins and iPLA(2) - putative mediators of Ca(2+) independent signaling pathway participate in glucose but not in hypotonicity-induced insulin secretion. Hypotonicity-induced insulin secretion is sensitive to clostridial neurotoxin TeTx but is resistant to NEM.     

Increased glucose sensitivity of both triggering and amplifying pathways of insulin secretion in rat islets cultured for 1 wk in high glucose

Chronic hyperglycemia has been shown to induce either a lack of response or an increased sensitivity to glucose in pancreatic beta-cells. We reinvestigated this controversial issue in a single experimental model by culturing rat islets for 1 wk in 10 or 30 mmol/l glucose (G10, Controls; or G30, High-glucose islets) before testing the effect of stepwise glucose stimulation from G0.5 to G20 on key beta-cell stimulus-secretion coupling events. Compared with Controls, the glucose sensitivity of High-glucose islets was markedly increased, leading to maximal stimulation of oxidative metabolism and both triggering and amplifying pathways of insulin secretion in G6 rather than G20, hence to loss of glucose effect above G6. This enhanced glucose sensitivity occurred despite an approximately twofold increase in islet uncoupling protein 2 mRNA expression. Besides this increased glucose sensitivity, the maximal glucose stimulation of insulin secretion in High-glucose islets was reduced by approximately 50%, proportionally to the reduction of insulin content. In High-glucose islets, changes in (45)Ca(2+) influx induced by glucose and diazoxide were qualitatively similar but quantitatively smaller than in Control islets and, paradoxically, did not lead to detectable changes in the intracellular Ca(2+) concentration measured by microspectrofluorimetry (fura PE 3). In conclusion, after 1 wk of culture in G30, the loss of glucose stimulation of insulin secretion in the physiological range of glucose concentrations (G5-G10) results from the combination of an increased sensitivity to glucose of both triggering and amplifying pathways of insulin secretion and an approximately 50% reduction in the maximal glucose stimulation of insulin secretion.     

Factors influencing insulin and glucagon secretion in lean and genetically obese mice.

The control of insulin and glucagon secretion from isolated pancreatic islets of lean and genetically obese mice has been compared. The enlarged islets of obese mouse pancreas and islets of obese mouse pancreas and islets of obese mice maintained on a restricted diet manifested a greater response to glucose stimulation of insulin secretion than the lean mice islets. The glucagon content of the islets, the secretion of glucagon in a medium containing 150 mg% glucose and the stimulation of glucagon secretion by arginine did not differ significantly in the two groups. Adrenaline stimulated glucagon secretion in vitro from obese mice but not from lean mice. Antinsulin serum injections into obese mice increased the plasma glucagon levels about twofold and had no effect on glucagon levels in lean mice, although the level of hyperglycaemia was the same in both groups. It is suggested that the suppression of glucagon release by glucose requires a higher concentration of insulin in the obese mouse pancreas than in lean mice.     

Central adrenoceptors and basal prolactin release in the rat

The influence of the central adrenergic system on basal prolactin secretion was investigated in the rat. Several selective adrenoceptor blockers were centrally administered and their effects on prolactin secretion were observed. Blockade of beta-1 receptors by practolol, beta-2 receptors by IPS 339 and alpha-2 receptors by DG-5128 did not modify basal prolactin secretion, but alpha-1 adrenoceptor blockade by prazosin strongly enhanced prolactin plasma levels. These findings suggest that noradrenergic pathways in the central nervous system exert inhibitory tone on basal prolactin secretion, and that this effect seems to be mediated by alpha-1 adrenoceptors.     

Dynamics and Regulation of Insulin Secretion in Pancreatic Islets from Normal Young Children

Insulin secretion has only exceptionally been investigated in pancreatic islets from healthy young children. It remains unclear whether those islets behave like adult islets despite substantial differences in cellular composition and higher β-cell replication rates. Islets were isolated from 5 infants/toddlers (11–36 month-old) and perifused to characterize their dynamics of insulin secretion when subjected to various stimuli and inhibitors. Their insulin responses were compared to those previously reported for similarly treated adult islets. Qualitatively, infant islets responded like adult islets to stimulation by glucose, tolbutamide, forskolin (to increase cAMP), arginine and the combination of leucine and glutamine, and to inhibition by diazoxide and CaCl₂ omission. This similarity included the concentration-dependency and biphasic pattern of glucose-induced insulin secretion, the dynamics of the responses to non-glucose stimuli and metabolic amplification of these responses. The insulin content was not different, but fractional insulin secretion rates were lower in infant than adult islets irrespective of the stimulus. However, the stimulation index was similar because basal secretion rates were also lower in infant islets. In conclusion, human β-cells are functionally mature by the age of one year, before expansion of their mass is complete. Their responsiveness (stimulation index) to all stimuli is not smaller than that of adult β-cells. Yet, under basal and stimulated conditions, they secrete smaller proportions of their insulin stores in keeping with smaller in vivo insulin needs during infancy.     

A possible role of adenylate cyclase in the longterm dietary regulation of insulin secretion from rat islets of Langerhans

1. Adenylate cyclase activity and patterns of insulin release in response to various concentrations of glucose were determined in islets of Langerhans isolated from starving, fed, or glucose-loaded rats. 2. Basal and glucagon-stimulated activities of adenylate cyclase were lower in islets from starved than from fed rats. The minimum glucose concentration required for stimulation of insulin secretion was higher, whereas the maximum secretory response to glucose was lower, in islets from starved than from fed rats. 3. Adenylate cyclase activity in islets of Langerhans obtained from fed rats loaded with glucose by intermittent intravenous or intraperitoneal injections over 5h was significantly higher than that seen in islets from normal fed rats. Islets obtained from glucose-loaded rats required a lower glucose concentration for stimulation of insulin secretion and attained a higher maximal response to glucose stimulation than those derived from fed rats. 4. Incubation in vitro of islets isolated from normal fed rats, for periods of 1 to 24h in the presence of high concentrations of glucose resulted in an activation of adenylate cyclase that occurred progressively from 2 to 7h and which was maintained during 24h of incubation. The increase of adenylate cyclase activity in isolated islets incubated for 4h in the presence of glucose was not prevented by addition of cycloheximide or actinomycin D. Galactose or 2-deoxyglucose was ineffective in increasing adenylate cyclase activity, and pyruvate (20mm) was less effective than glucose. 5. It is suggested that glucose or a glucose metabolite may exert long-term effects on islet cell adenylate cyclase.     

Leptin constrains phospholipase C-protein kinase C-induced insulin secretion via a phosphatidylinositol 3-kinase-dependent pathway

Leptin-deficient Lep(ob)/Lep(ob)mice hypersecrete insulin in response to acetylcholine stimulation of the phospholipase C-protein kinase C (PLC-PKC) pathway, and leptin constrains this hypersecretion. Leptin has been reported to activate phosphatidylinositol 3-kinase (PI 3-K) and subsequently phosphodiesterase (PDE) to impair protein kinase A (PKA)-induced insulin secretion from cultured islets of neonatal rats. We determined if PKA-induced insulin secretion was also hyperresponsive in islets from Lep(ob)/Lep(ob)mice, and if leptin impaired this pathway in islets from these mice. Additionally, the possible role for PI 3-K and PDE in leptin-induced control of acetylcholine-induced insulin secretion was examined. Stimulation of insulin secretion with GLP-1, forskolin (an activator of adenylyl cyclase), or IBMX (an inhibitor of PDE) did not cause hypersecretion of insulin from islets of young Lep(ob)/Lep(ob)mice, and leptin did not inhibit GLP-1-induced insulin secretion from islets of these mice. Inhibition of PDE with IBMX also did not block leptin-induced inhibition of acetylcholine-mediated insulin secretion from islets of Lep(ob)/Lep(ob)mice. But, preincubation of islets with wortmannin, an inhibitor of PI 3-K activity, blocked the ability of leptin to constrain acetylcholine-induced insulin secretion from islets of Lep(ob)/Lep(ob)mice. We conclude that the capacity of the PKA pathway to stimulate insulin secretion is not increased in islets from young Lep(ob)/Lep(ob)mice, and that leptin does not regulate this pathway in islets from mice. Leptin may stimulate PI 3-K to constrain PLC-PKC-induced insulin secretion from islets of Lep(ob)/Lep(ob)mice.     

Activation of insulin-secreting cells by pyruvate and halogenated derivatives.

Addition of pyruvate to rat islets perifused in the presence of 5 mM-glucose elicited an immediate pronounced biphasic stimulation of insulin secretion. At lower concentrations of glucose (2.5 mM), only the initial, transient, phase of secretion was observed. Pyruvate inhibited 45Ca2+ efflux from islets at 2.5 mM-glucose and stimulated efflux at 5 mM-glucose. Pyruvate also decreased the rate of efflux of 86Rb+ from perifused islets. A marked stimulation of insulin secretion and 45Ca2+ efflux rate was observed in response to 3-fluoropyruvate and 3-bromopyruvate, compounds which inhibited oxidative metabolism of [14C]glucose and [14C]pyruvate in islets. The stimulatory effects of 3-fluoro- and 3-bromo-pyruvate were associated with enhanced 86Rb+ efflux. Withdrawal of pyruvate or halogenated analogues from the perfusate resulted in a secondary stimulation of insulin release, 45Ca2+ efflux and, to some extent, 86Rb+ efflux rates. Pyruvate, 3-fluoropyruvate and 3-bromopyruvate were all effective in promoting intracellular acidification and a rise in cytosolic Ca2+ concentration, as judged from fluorescence measurements in HIT-T15 cells loaded with 2',7'-biscarboxyethyl-5'(6')-carboxyfluorescein and Quin 2 respectively. It is proposed that oxidative metabolism of pyruvate is not a prerequisite for its stimulatory actions on pancreatic beta-cells. An alternative mechanism of activation by pyruvate and its halogenated derivatives is proposed, based on the possible electrogenic flux of these anions across the cell membrane.     

Viability criteria of islets of Langerhans isolated and purified using Ficoll

Rejection of islet allografts is generally explained by immunologic problems, due to both cellular and antibody mechanisms. But another great problem is in the isolation of intact and viable islets of Langerhans: it is necessary to use a good method of pancreas distention, to determine the optimal concentration of collagenase for digestion, to select an effective technique for purifying the islets. This study correlates the morphology of isolated pancreatic islets of rats and dogs with secretion of insulin. The islets are incubated in a perifusion system and are tested during four periods; the glucose concentrations of the perifusion fluid are: 5.5 mM during the initial 70 min. period, 16.5 mM during the second 60 min. period, 5.5 mM during the third 60 min. period and 16.5 during the fourth 50 min. period. This "double glucose stimulation" is a good test of islet viability. The intact, viable isolated islets showed a significant increase of insulin secretion during the two 16.5 mM glucose periods. Damaged islets with some little morphologic alterations after showed a good insulin release during the first glucose stimulation, but a very poor insulin response to glucose during the second stimulation period.     

Effects of the calcium-channel agonist CGP 28392 on insulin secretion from isolated rat islets of Langerhans.

The rate of insulin secretion from isolated rat islets of Langerhans was affected by a number of dihydropyridine derivatives known to interact with voltage-sensitive Ca2+ channels in excitable cells. The channel antagonists nifedipine and nitrendipine were potent inhibitors of glucose-induced insulin secretion in response to both 8 mM- and 20 mM-glucose, although they did not lower the basal secretion rate observed in the presence of 4 mM-glucose. The Ca2+-channel agonist, CGP 28392, also failed to alter the basal rate of insulin secretion. In the presence of 8 mM-glucose, however, 1 microM-CGP 28392 enhanced the insulin-secretion rate to a value approximately double that with 8 mM-glucose alone. This effect was dose-dependent, with half the maximal response elicited by 0.1 microM-CGP 28392, and full enhancement at 10 microM. The response was rapid in onset, with an increase in insulin secretion evident within 2 min of CGP 28392 infusion in perifused islets. Stimulation of insulin secretion by CGP 28392 was correlated with a rapid enhancement of glucose-stimulated 45Ca2+ uptake into islets cells, and with a transiently increased rate of 45Ca2+ efflux from pre-loaded islets. Stimulation of insulin secretion by CGP 28392 was abolished in the presence of noradrenaline, although under these conditions the rapid stimulation of 45Ca2+ influx induced by CGP 28392 was only partially inhibited. In contrast with these results, when islets were incubated in the presence of 20 mM-glucose, CGP 28392 caused a dose-dependent inhibition of insulin secretion. Half-maximal inhibition required approx. 0.2 microM-CGP 28392, with maximal effects observed at 10 microM. Under these conditions, however, the extent of insulin secretion was still only decreased by about 50%, to a value which was similar to that seen in the presence of 8 mM-glucose and CGP 28392. These results suggest that dihydropyridine derivatives can alter the activity of voltage-dependent Ca2+ channels in islet cells, and are consistent with the possibility that gating of these channels plays an important role in regulating the rate of insulin secretion after glucose stimulation.     

Beta cell contact and insulin release

Insulin secretion by intact islets, dispersed islet cells and dispersed cells allowed to reaggregate was compared in perifusion. Although single cells and aggregates showed basal insulin secretion and a prompt response to glucose challenge, basal secretion, peak insulin secretion and total insulin secretion during a 60 minute stimulation were profoundly less than those activities of intact islets. These results suggest that dispersed beta cells are responsive to glucose as a secretagogue, but the magnitude of the response is greatly diminished and not restored by simple cell contact.     

Detection of secretion from pancreatic islets using chemically modified electrodes

Secretion of insulin from pancreatic islets was monitored indirectly by detecting zinc. Anodic stripping voltammetric measurements of zinc were done on a bismuth-modified electrode. Comparison of the performance of bismuth-modified electrodes and mercury film electrodes showed that bismuth is an appropriate alternative for Zn detection. The bismuth-coated electrode was used to detect zinc in insulin samples and insulin secreted from pancreatic islets upon stimulation with high concentrations of K(+). Detection of zinc released from pancreatic islets was done in the culture medium without any further cleanup. This detection method can be used to monitor secretion from pancreatic islets in their native environment.     

Effect of neonatal streptozotocin and thyrotropin-releasing hormone treatments on insulin secretion in adult rats

Neonatal STZ (nSTZ) treatment results in damage of pancreatic B-cells and in parallel depletion of insulin and TRH in the rat pancreas. The injury of B-cells is followed by spontaneous regeneration but dysregulation of the insulin response to glucose persists for the rest of life. Similar disturbance in insulin secretion was observed in mice with targeted TRH gene disruption. The aim of present study was to determine the role of the absence of pancreatic TRH during the perinatal period in the nSTZ model of impaired insulin secretion. Neonatal rats were injected with STZ (90 microg/g BW i.p.) and the effect of exogenous TRH (10 ng/g BW/day s.c. during the first week of life) on in vitro functions of pancreatic islets was studied at the age 12-14 weeks. RT-PCR was used for determination of prepro-TRH mRNA in isolated islets. Plasma was assayed for glucose and insulin, and isolated islets were used for determination of insulin release in vitro. The expression of prepro-TRH mRNA was only partially reduced in the islets of adult nSTZ rats when compared to controls. nSTZ rats had normal levels of plasma glucose and insulin but the islets of nSTZ rats failed to response by increased insulin secretion to stimulation with 16.7 mmol/l glucose or 50 mmol/l KCl. Perinatal TRH treatment enhanced basal insulin secretion in vitro in nSTZ animals of both sexes and partially restored the insulin response to glucose stimulation in nSTZ females.     

Secretin uncouples glucose inhibition of glucagon-producing cells resulting in a simultaneous stimulation of both glucagon and insulin release

The effect of secretin on glucagon and insulin release and its interaction with glucose has been studied in cultured mouse pancreatic islets by column perifusion. Glucose alone showed the well-known stimulation of insulin release and inhibition of glucagon release. Addition of 10 mM secretin increased glucagon secretion at 3 mM D-glucose by 300% while no change in insulin release could be seen at this low glucose concentration. At maximal stimulation of insulin release by 20 mM D-glucose addition of 10 nM secretin increased insulin release by 30%. Despite this insulin concentration and the high glucose concentration an increase in glucagon secretion of 1800% was found. These effects of secretin were dose-dependent at 10 mM D-glucose with 1 nM secretin being the lowest effective dose.     

Epigallocatechin-3-gallate (EGCG) activates AMPK through the inhibition of glutamate dehydrogenase in muscle and pancreatic ß-cells: A potential beneficial effect in the pre-diabetic state?

Glucose homeostasis is determined by insulin secretion from the ß-cells in pancreatic islets and by glucose uptake in skeletal muscle and other insulin target tissues. While glutamate dehydrogenase (GDH) senses mitochondrial energy supply and regulates insulin secretion, its role in the muscle has not been elucidated. Here we investigated the possible interplay between GDH and the cytosolic energy sensing enzyme 5′-AMP kinase (AMPK), in both isolated islets and myotubes from mice and humans. The green tea polyphenol epigallocatechin-3-gallate (EGCG) was used to inhibit GDH. Insulin secretion was reduced by EGCG upon glucose stimulation and blocked in response to glutamine combined with the allosteric GDH activator BCH (2-aminobicyclo-[2,2,1] heptane-2-carboxylic acid). Insulin secretion was similarly decreased in islets of mice with ß-cell-targeted deletion of GDH (ßGlud1^−/−). EGCG did not further reduce insulin secretion in the mutant islets, validating its specificity. In human islets, EGCG attenuated both basal and nutrient-stimulated insulin secretion. Glutamine/BCH-induced lowering of AMPK phosphorylation did not operate in ßGlud1^−/− islets and was similarly prevented by EGCG in control islets, while high glucose systematically inactivated AMPK. In mouse C2C12 myotubes, like in islets, the inhibition of AMPK following GDH activation with glutamine/BCH was reversed by EGCG. Stimulation of GDH in primary human myotubes caused lowering of insulin-induced 2-deoxy-glucose uptake, partially counteracted by EGCG. Thus, mitochondrial energy provision through anaplerotic input via GDH influences the activity of the cytosolic energy sensor AMPK. EGCG may be useful in obesity by resensitizing insulin-resistant muscle while blunting hypersecretion of insulin in hypermetabolic states.     

Glucagon-like peptide-1 and islet lipolysis.

A role for glucagon-like peptide 1 (GLP-1) has been suggested in stimulating beta-cell lipolysis via elevation of cAMP and activation of protein kinase A, which in turn may activate hormone-sensitive lipase (HSL), thereby contributing to fatty acid generation (FFA) from intracellular triglyceride stores. FFAs may then be metabolized to a lipid signal, which is required for optimal glucose-stimulated insulin secretion. Since HSL is expressed in islet beta-cells, this effect could contribute to the stimulation of insulin secretion by GLP-1, provided that a lipid signal of importance for insulin secretion is generated. To examine this hypothesis, we have studied the acute effect of GLP-1 on isolated mouse islets from normal mice and from mice with high-fat diet induced insulin resistance. We found, however, that although GLP-1 (100 nM) markedly potentiated glucose-stimulated insulin secretion from islets of both feeding groups, the peptide was not able to stimulate islet palmitate oxidation or increase lipolysis measured as glycerol release. This indicates that a lipid signal does not contribute to the acute stimulation of insulin secretion by GLP-1. To test whether lipolysis might be involved in the islet effects of long-term GLP-1 action, mice from the two feeding groups were chronically treated with exendin-4, a peptide that lowers blood glucose by interacting with GLP-1 receptors, in order to stimulate insulin secretion, for 16 days before isolation of the islets. The insulinotropic effects of GLP-1 and forskolin were exaggerated in isolated islets from exendin-4 treated mice given a high-fat diet, with a augmented palmitate oxidation as well as islet lipolysis at high glucose levels in these islets. Exendin-4 treatment had less impact on mice fed a normal diet. From these results we conclude that while GLP-1 does not seem to induce beta-cell lipolysis acutely in mouse islets, the peptide affects beta-cell fat metabolism after long-term adaptation to GLP-1 receptor stimulation.     

Ethanol and urea affect insulin secretion from islets and insulinoma cells by different mechanisms

Secretion of insulin could be stimulated by several ways. Comparison of glucose- and swelling-induced mechanisms in pancreatic islets revealed the involvement of a novel signal transduction pathway with specific features of osmotically stimulated peptide hormone release including Ca²⁺ independence and resistance to noradrenalin (NA) inhibition. Cell swelling can be induced by hypotonicity or small permeant molecules (e.g. ethanol, urea). Our experiments were aimed to compare the effect of these permeants on insulin secretion from natural system — freshly isolated pancreatic islets and rat insulinoma cell lines INS-1 and INS-1E. As expected glucose and both permeants (80 mM ethanol and urea in isosmotic medium) induced insulin release from islets and NA did not inhibit permeant-induced secretion. Although ethanol and urea induced similar swelling of tumor cells, they produced opposite effect on insulin secretion; while exposure to ethanol led to stimulation of insulin secretion, exposure to urea led to suppression in both types of neoplastic cells. Surprisingly, stimulating effect of ethanol was completely suppressed by NA in both tumor cell lines. Ethanol in hyperosmotic medium failed to stimulate and even inhibited insulin release from both tumor cell lines in present study indicating thus involvement of an osmotic component. In conclusion, the opposite effect of ethanol and urea on insulin secretion from insulinoma cells and sensitivity of ethanol stimulation to NA indicate utilization of different cellular signaling pathways in tumor cells as compared to natural β-cells. Participation of permeant effect in the mechanism of ethanol stimulation remains to be clarified.