P2Y receptor activation enhances insulin release from pancreatic beta-cells by triggering the cyclic AMP/protein kinase A pathway.

Adenine nucleotides stimulate insulin secretion by binding to P2 receptors of the pancreatic beta-cells; the stimulus-secretion coupling is not yet clearly established and may depend on the receptor subtype. The aim of the present study was to further investigate the mechanism whereby P2Y receptor agonists enhance glucose-induced insulin secretion. Experiments were performed in rat pancreatic islets and in the INS-1 secreting cell line in the presence of a slightly stimulating glucose concentration (8.3 mmol/l). In isolated islets, the P2Y receptor agonist ADPbetaS (50 micromol/l) induced a significant fivefold increase in the cyclic AMP (cAMP) content, from 43.4+/-3.7 fmol/10 islets in controls to 210.6+/-12.0; it still induced a 4.5-fold increase in cAMP content in the absence of calcium. In another series of experiments, ADPbetaS (50 micromol/l) significantly increased glucose-induced insulin secretion from 7.7+/-0.6 ng/3 islets in controls to 11.2+/-1.0. The adenylyl cyclase inhibitor SQ 22,536 (9-[tetrahydro-2-furanyl]-9 H-purin-6-amine; 100 micromol/l), which was ineffective alone, completely prevented the stimulating effect of ADPbetaS. In a set of experiments in which ADPbetaS increased glucose-induced insulin secretion from 10.0+/-0.7 ng/3 islets to 12.6+/-0.8, the inhibitor of cAMP-dependent protein kinase, TPCK (tos-phe-chloromethylketone; 3 micromol/l), which was ineffective alone, also prevented the stimulating effect of ADPbetaS. In incubated INS-1 cells, the P2Y receptor ligand ATPalphaS increased significantly both the content of cAMP and the release of insulin, in a concentration-dependent manner in the range of 50-150 micromol/l; the insulin release was significantly correlated with the cAMP content. In conclusion, the present results show that P2Y receptor agonists, ADPbetaS and ATPalphaS, amplify glucose-induced insulin secretion by activating beta-cell adenylyl cyclase and the subsequent cAMP/protein kinase A signaling pathway.

A neuronal isoform of nitric oxide synthase expressed in pancreatic beta-cells controls insulin secretion.

Evidence is presented showing that a neuronal isoform of nitric oxide synthase (NOS) is expressed in rat pancreatic islets and INS-1 cells. Sequencing of the coding region indicated a 99.8% homology with rat neuronal NOS (nNOS) with four mutations, three of them resulting in modifications of the amino acid sequence. Double-immunofluorescence studies demonstrated the presence of nNOS in insulin-secreting beta-cells. Electron microscopy studies showed that nNOS was mainly localized in insulin secretory granules and to a lesser extent in the mitochondria and the nucleus. We also studied the mechanism involved in the dysfunction of the beta-cell response to arginine and glucose after nNOS blockade with N(G)-nitro-L-arginine methyl ester. Our data show that miconazole, an inhibitor of nNOS cytochrome c reductase activity, either alone for the experiments with arginine or combined with sodium nitroprusside for glucose, is able to restore normal secretory patterns in response to the two secretagogues. Furthermore, these results were corroborated by the demonstration of a direct enzyme-substrate interaction between nNOS and cytochrome c, which is strongly reinforced in the presence of the NOS inhibitor. Thus, we provide immunochemical and pharmacological evidence that beta-cell nNOS exerts, like brain nNOS, two catalytic activities: a nitric oxide production and an NOS nonoxidating reductase activity, both of which are essential for normal beta-cell function. In conclusion, we suggest that an imbalance between these activities might be implicated in beta-cell dysregulation involved in certain pathological hyperinsulinic states.

Insulinotropic effect of Citrullus colocynthis fruit extracts.

Infusions of Citrullus colocynthis Schrad. (Cucurbitaceae) fruits are traditionally used as antidiabetic medication in Mediterranean countries, but to our knowledge no studies have been undertaken so far to determine the possible mechanisms involved in the antidiabetic properties of the fruit. The present study was designed to investigate whether these fruits possess insulinotropic effects. For this purpose, different extracts of Citrullus colocynthis seed components were obtained: RN II (crude extract), RN VI (hydro-alcoholic extract), RN X (purified extract) and RN XVII (beta-pyrazol-1-ylalanine), the major free amino acid present in the seeds. The insulin secretory effects of these different extracts were evaluated in vitro in the isolated rat pancreas and isolated rat islets in the presence of 8.3 mM glucose. All tested extracts, when perfused for 20 min at 0.1 mg/ml, immediately and significantly stimulated insulin secretion. This effect was transient. In addition, the purified extract (RN X) provoked a clear dose-dependent increase in insulin release from isolated islets. Moreover, a significant and persistant increase in pancreatic flow rate appeared during RN VI, RN X and RN XVII perfusions. In conclusion, our results show that different Citrullus colocynthis seed extracts have an insulinotropic effect which could at least partially account for the antidiabetic activities of these fruits.

4-Hydroxyisoleucine: effects of synthetic and natural analogues on insulin secretion.

4-Hydroxyisoleucine, a peculiar amino acid extracted from fenugreek seeds and never found in mammalian tissues, exhibits interesting insulinotropic activity. To investigate the structural requirements for this stimulating effect, the insulinotropic activity of the major isomer (2S,3R,4S) of 4-hydroxyisoleucine, in the presence of 8. 3 mM glucose, was compared to that of (1) its minor isomer (2R,3R, 4S) (2) its lactone form, (3) classical structurally related amino acids, and (4) synthetic monomethylated analogues. In the isolated, ex vivo, perfused rat pancreas, only the major isomer of 4-hydroxyisoleucine (200 microM) potentiated insulin release. On incubated isolated rat islets, the threshold concentration for a significant increase (P<0.05) in insulin release was 200 microM for (2S,3R,4S) 4-hydroxyisoleucine, 500 microM for (2S,4R) and (2S,4S) gamma-hydroxynorvalines as well as (2S,3S) and (2S,3R) gamma-hydroxyvalines, and 1 mM or more for other congeners. In conclusion, the insulinotropic properties of 4-hydroxyisoleucine, in the micromolar range, are seen only in the presence of the linear major isoform; they also require carbon alpha in S-configuration, full methylation and carbon gamma-hydroxylation.

4-Hydroxyisoleucine: experimental evidence of its insulinotropic and antidiabetic properties.

We have recently shown in vitro that 4-hydroxyisoleucine (4-OH-Ile), an amino acid extracted from fenugreek seeds, potentiates insulin secretion in a glucose-dependent manner. The present study was designed to investigate whether 4-OH-Ile could exert in vivo insulinotropic and antidiabetic properties. For this purpose, intravenous or oral glucose tolerance tests (IVGTTs and OGTTs, respectively) were performed not only in normal animals but also in a type II diabetes rat model. During IVGTT in normal rats or OGTT in normal dogs, 4-OH-Ile (18 mg/kg) improved glucose tolerance. The lactonic form of 4-OH-Ile was ineffective in normal rats. In non-insulin-dependent diabetic (NIDD) rats, a single intravenous administration of 4-OH-Ile (50 mg/kg) partially restored glucose-induced insulin response without affecting glucose tolerance; a 6-day subchronic administration of 4-OH-Ile (50 mg/kg, daily) reduced basal hyperglycemia, decreased basal insulinemia, and slightly, but significantly, improved glucose tolerance. In vitro, 4-OH-Ile (200 microM) potentiated glucose (16.7 mM)-induced insulin release from NIDD rat-isolated islets. So, the antidiabetic effects of 4-OH-Ile on NIDD rats result, at least in part, from a direct pancreatic B cell stimulation.

Evidence for two different types of P2 receptors stimulating insulin secretion from pancreatic B cell.

Adenine nucleotides have been shown to stimulate insulin secretion by acting on P2 receptors of the P2Y type. Since there have been some discrepancies in the insulin response of different analogues of ATP and ADP, we investigated whether two different types of P2 receptors exist on pancreatic B cells. The effects of alpha,beta-methylene ATP, which is more specific for the P2X subtype, were studied in vitro in pancreatic islets and isolated perfused pancreas from rats, in comparison with the potent P2Y receptor agonist ADPbetaS. In isolated islets, incubated with a slightly stimulating glucose concentration (8.3 mM), alpha,beta-me ATP (200 microM) and ADPbetaS (50 microM) similarly stimulated insulin secretion; by contrast, under a non stimulating glucose concentration (3 mM), alpha,beta-me ATP was still effective whereas ADPbetaS was not. In the same way, in islets perifused with 3 mM glucose, alpha,beta-me ATP but not ADPbetaS induced a partial but significant reduction in the peak 86Rb efflux induced by the ATP-dependent potassium channel opener diazoxide. In the isolated pancreas, perfused with a non stimulating glucose concentration (4.2 mM), ADPbetaS and alpha,beta-me ATP (5-50 microM), administered for 10 min, induced an immediate, transient and concentration-dependent increase in the insulin secretion; their relative potency was not significantly different. In contrast, with a slightly stimulating glucose concentration (8.3 mM), ADPbetaS was previously shown to be 100 fold more potent than alpha,beta-me ATP. Furthermore, at 4.2 mM glucose a second administration of alpha,beta-me ATP was ineffective. In the same way, ADPbetaS was also able to desensitize its own insulin response. At 3 mM glucose, alpha,beta-me ATP as well as ADPbetaS (50 microM) induced a transient stimulation of insulin secretion and down regulated the action of each other. These results give evidence that pancreatic B cells, in addition to P2Y receptors, which potentiate glucose-induced insulin secretion, are provided with P2X receptors, which transiently stimulate insulin release at low non-stimulating glucose concentration and slightly affect the potassium conductance of the membrane.

4-Hydroxyisoleucine: a novel amino acid potentiator of insulin secretion.

We report the characterization of a new insulinotropic compound, 4-hydroxyisoleucine. This amino acid has been extracted and purified from fenugreek seeds, which are known in traditional medicine for their antidiabetic properties. 4-Hydroxyisoleucine increases glucose-induced insulin release, in the concentration range of 100 micromol/l to 1 mmol/l, through a direct effect on isolated islets of Langerhans from both rats and humans. The stimulating effect of 4-hydroxyisoleucine was strictly glucose dependent; indeed, ineffective at low (3 mmol/l) or basal (5 mmol/l) glucose concentrations, the amino acid potentiated the insulin secretion induced by supranormal (6.6-16.7 mmol/l) concentrations of glucose. In addition, in the isolated perfused rat pancreas, we could show 1) that the pattern of insulin secretion induced by 4-hydroxyisoleucine was biphasic, 2) that this effect occurred in the absence of any change in pancreatic alpha- and delta-cell activity, and 3) that the more glucose concentration was increased, the more insulin response was amplified. Moreover, 4-hydroxyisoleucine did not interact with other agonists of insulin secretion (leucine, arginine, tolbutamide, glyceraldehyde). Therefore, we conclude that 4-hydroxyisoleucine insulinotropic activity might, at least in part, account for fenugreek seeds' antidiabetic properties. This secretagogue may be considered as a novel drug with potential interest for the treatment of NIDDM.

Experimental NIDDM: development of a new model in adult rats administered streptozotocin and nicotinamide.

We took advantage of the partial protection exerted by suitable dosages of nicotinamide against the beta-cytotoxic effect of streptozotocin (STZ) to create a new experimental diabetic syndrome in adult rats that appears closer to NIDDM than other available animal models with regard to insulin responsiveness to glucose and sulfonylureas. Among the various dosages of nicotinamide tested in 3-month-old Wistar rats (100-350 mg/kg body wt), the dosage of 230 mg/kg, given intraperitoneally 15 min before STZ administration (65 mg/kg i.v.) yielded a maximum of animals with moderate and stable nonfasting hyperglycemia (155 +/- 3 vs. 121 +/- 3 mg/dl in controls; P < 0.05) and 40% preservation of pancreatic insulin stores. We also evaluated beta-cell function both in vitro and in vivo 4-9 weeks after inducing diabetes. In the isolated perfused pancreas, insulin response to glucose elevation (5-11 mmol/l) was clearly present, although significantly reduced with respect to controls (P < 0.01). Moreover, the insulin response to tolbutamide (0.19 mmol/l) was similar to that observed in normal pancreases. Perfused pancreases from diabetic animals also exhibited a striking hypersensitivity to arginine infusion (7 mmol/l). In rats administered STZ plus nicotinamide, intravenous glucose tolerance tests revealed clear abnormalities in glucose tolerance and insulin responsiveness, which were interestingly reversed by tolbutamide administration (40 mg/kg i.v.). In conclusion, this novel NIDDM syndrome with reduced pancreatic insulin stores, which is similar to human NIDDM in that it has a significant response to glucose (although abnormal in kinetics) and preserved sensitivity to tolbutamide, may provide a particularly advantageous tool for pharmacological investigations of new insulinotropic agents.

Mechanisms involved in the effect of nitric oxide synthase inhibition on L-arginine-induced insulin secretion.

1. A constitutive nitric oxide synthase (NOSc) pathway negatively controls L-arginine-stimulated insulin release by pancreatic beta cells. We investigated the effect of glucose on this mechanism and whether it could be accounted for by nitric oxide production. 2. NOSc was inhibited by N omega-nitro-L-arginine methyl ester (L-NAME), and sodium nitroprusside (SNP) was used as a palliative NO donor to test whether the effects of L-NAME resulted from decreased NO production. 3. In the rat isolated perfused pancreas, L-NAME (5 mM) strongly potentiated L-arginine (5 mM)-induced insulin secretion at 5 mM glucose, but L-arginine and L-NAME exerted only additive effects at 8.3 mM glucose. At 11 mM glucose, L-NAME significantly inhibited L-arginine-induced insulin secretion. Similar data were obtained in rat isolated islets. 4. At high concentrations (3 and 300 microM), SNP increased the potentiation of arginine-induced insulin output by L-NAME, but not at lower concentrations (3 or 30 nM). 5. L-Arginine (5 mM) and L-ornithine (5 mM) in the presence of 5 mM glucose induced monophasic beta cell responses which were both significantly reduced by SNP at 3 nM but not at 30 nM; in contrast, the L-ornithine effect was significantly increased by SNP at 3 microM. 6. Simultaneous treatment with L-ornithine and L-arginine provoked a biphasic insulin response. 7. At 5 mM glucose, L-NAME (5 mM) did not affect the L-ornithine secretory effect, but the amino acid strongly potentiated the alteration by L-NAME of L-arginine-induced insulin secretion. 8. L-Citrulline (5 mM) significantly reduced the second phase of the insulin response to L-NAME (5 mM) + L-arginine (5 mM) and to L-NAME + L-arginine + SNP 3 microM. 9. The intermediate in NO biosynthesis, NG-hydroxy-L-arginine (150-300 microM) strongly counteracted the potentiation by L-NAME of the secretory effect of L-arginine at 5 mM glucose. 10. We conclude that the potentiation of L-arginine-induced insulin secretion resulting from the blockade of NOSc activity in the presence of a basal glucose concentration (1) is strongly modulated by higher glucose concentrations, (2) is not due to decreased NO production but (3) is probably accounted for by decreased levels of NG-hydroxy-L-arginine or L-citrulline, resulting in the attenuation of an inhibitory effect on arginase activity.

Alterations of insulin response to different beta cell secretagogues and pancreatic vascular resistance induced by N omega-nitro-L-arginine methyl ester.

1. We studied a possible interplay of pancreatic NO synthase activity on insulin secretion induced by different beta cell secretagogues and also on pancreatic vascular bed resistance. 2. This study was performed in the isolated perfused pancreas of the rat. Blockage of NO synthase was achieved with Nw-nitro-L-arginine methyl ester (L-NAME); The specificity of the antagonist was checked by using its D-enantiomer as well as by substitutive treatments with sodium nitroprusside (SNP) as a NO donor in studies of glucose-induced insulin secretion. 3. Arginine (5 mM) induced a monophasic response which was, in the presence of L-NAME at equimolar concentration, very strongly potentiated and converted into a 13 times higher biphasic one. D-NAME (5 mM) was only able to induce a 3 times higher response, but provoked a similar vasoconstrictor effect. 4. The small biphasic insulin secretion induced by L-leucine (5 mM) was also strongly enhanced, by 8 times, in the presence of L-NAME (5 mM) vs 2 times in the presence of D-NAME (5 mM). 5. beta cell responses to KCl (5 mM) and tolbutamide (0.185 mM) were only slight increased by L-NAME (5 mM) to values not far from the sum of the effects of L-NAME and of the two drugs alone. D-NAME (5 mM) was totally ineffective on the actions of both secretagogues. 6. L-NAME, infused 15 min before and during a rise in glucose concentration from 5 to 11 mM, was able in the low millimolar range (0.1-0.5 mM) to blunt the classical biphasic pattern of beta cell response to glucose and, at 5 mM, to convert it into a significantly greater monophasic one. In contrast, D-NAME (5 mM) was unable to induce similar effects. 7. SNP alone at 3 microM was ineffective but at 30 microM substantially reduced to second phase of insulin response to glucose; however, at both concentrations the NO donor partly reversed alterations in insulin secretion caused by L-NAME (5 mM) and restored a biphasic response.

Effects of a fenugreek seed extract on feeding behaviour in the rat: metabolic-endocrine correlates.

Fenugreek seeds (Trigonella foenum graecum L.) are assumed to have restorative and nutritive properties. The present work was designed to investigate the effects of a fenugreek seed extract on feeding behaviour. Experiments were performed to determine food consumption and motivation to eat as well as metabolic-endocrine changes in chronically treated animals. Male Wistar rats were given the seed extract orally (10 and 100 mg/day per 300 g body weight), mixed together with food, and control animals were monitored in parallel. The results show that chronic oral administration of the fenugreek extract significantly increases food intake and the motivation to eat. The treatment, however, does not prevent the anorexia nor the decreased motivation to eat induced by d-fenfluramine (2 mg/kg, IP). An increase in plasma insulin and a decrease in total cholesterol and very low-density lipoprotein (VLDL)-low-density lipoprotein (LDL) total cholesterol were also observed. In conclusion, chronic administration of a fenugreek seed extract enhances food consumption and motivation to eat in rats and also induces hyperinsulinemia as well as hypocholesterolemia.

[Research of the mechanism of the inhibition of insulin secretion induced by a peripheral benzodiazepine: 4'-chlordiazepam]. / Recherche du mécanisme de l'inhibition de la sécrétion d'insuline induite par une benzodiazépine périphérique, le 4'-chlordiazépam.

We have previously shown that the selective peripheral-type benzodiazepine agonist 4'-chlordiazepam inhibited glucose-induced insulin secretion from rat pancreatic islets. The present work was designed to further investigate the mechanism of this effect. Since interactions have been described between benzodiazepines and purinergic modulators such as adenosine, we studied the effect of the adenosine receptor antagonist 8-phenyltheophylline. This substance, which on its own had no effect on insulin release, prevented the inhibiting effect of 4'-chlordiazepam. We then performed experiments in the presence of forskolin, which directly stimulates adenylyl-cyclase, and dibutyryl-cyclic AMP, which enhances the intracellular cAMP content. The inhibiting effect of 4'-chlordiazepam was prevented neither by forskolin nor dibutyryl-cAMP, in contrast to the inhibiting effect of adenosine, which has been previously shown to be prevented by dibutyryl-cAMP. In conclusion, the inhibiting effect of 4'-chlordiazepam on glucose-induced insulin secretion is antagonized by 8-phenyltheophylline; the signalling mechanism of this effect appears to be independent of the adenylyl cyclase/cAMP pathway.

Effects of a peripheral-type benzodiazepine on glucose-induced insulin secretion.

Benzodiazepines, besides interacting with central-type receptors which mediate their well-known pharmacological actions, bind to peripheral-type receptors that are distributed in a variety of peripheral tissues including numerous endocrine organs. The present work was designed to investigate the effects of a selective peripheral-type benzodiazepine, 4'-chlordiazepam (Ro 5-4864), on glucose-induced insulin secretion in vitro. In the rat isolated pancreas perfused with a Krebs-bicarbonate buffer containing 8.3 mM glucose, the drug (10(-6) and 10(-5) M) induced a progressive and significant decrease in insulin release. Concomitantly, it induced a vasodilator response of the pancreatic vascular bed. In rat isolated islets incubated for 1 h in the presence of 15 mM glucose, 4'-chlordiazepam (10(-5) and 10(-4) M) induced a significant and dose-dependent inhibition of insulin release. In contrast, the selective central-type benzodiazepine, clonazepam (10(-6) - 10(-4) M), did not significantly modify glucose-induced insulin secretion. In addition, experiments were performed to test the effect of 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline-carboxamid e (PK 11195), a peripheral non-benzodiazepine ligand proposed as a putative antagonist. This substance did not counteract the inhibitory effect of 4'-chlordiazepam but itself (10(-6) and 10(-5) M) elicited a potent inhibitory effect on insulin secretion. These results show that drugs such as 4'-chlordiazepam and PK 11195 which have a high affinity for peripheral-type benzodiazepine receptors, in contrast to a central-type benzodiazepine agonist, inhibit glucose-induced insulin secretion in vitro.

Comparative effects of LN 5330 and diazoxide on insulin release and 86Rb+ permeability in perifused rat islets of Langerhans.

The benzothiadiazine derivative LN 5330 (chloro-7 trifluoromethyl-6 benzothiadiazine-1,2,4-dioxide-1,1) has been shown to inhibit insulin secretion and calcium uptake. The present study was carried out to investigate the effects of LN 5330 on insulin release and 86Rb- efflux from perifused rat pancreatic islets; a comparison was made with the structural analogue diazoxide. In the presence of 8.3 mM glucose, LN 5330 (100 microM) accelerated 86Rb+ efflux while reducing insulin output from the islets. LN 5330 induced a dose-dependent acceleration of 86Rb+ efflux and appeared to be a more potent activator of 86Rb+ efflux than diazoxide. The stimulatory effect of LN 5330 on 86Rb+ efflux persisted in the absence of extracellular calcium. In the absence of glucose, 86Rb+ permeability also increased, LN 5330 being again significantly more efficient than diazoxide at an equimolar concentration. These data indicate that the benzothiadiazine derivative LN 5330 inhibits insulin secretion by increasing the potassium permeability of the plasma membrane. It is suggested that, like diazoxide, this drug could activate the ATP-sensitive K+ channel.

Differential effects of purinergic and cholinergic activation on the hydrolysis of membrane polyphosphoinositides in rat pancreatic islets.

This work was designed to investigate the effects of a P2 purinoreceptor agonist, alpha, beta-methylene ADP, on membrane polyphosphoinositide hydrolysis in relation to insulin release from rat isolated islets of Langerhans. The effects of this stable structural analogue of ADP (10(-4) M) were compared with those of a muscarinic cholinergic agonist, carbachol (10(-4) M). The interaction between alpha, beta-methylene ADP and carbachol was studied on polyphosphoinositide breakdown and insulin secretion. The experiments were performed in presence of a slightly stimulating glucose concentration (8.3 mM). Whereas carbachol-induced insulin release was accompanied by a concomitant increase in inositol phosphates accumulation, alpha, beta-methylene ADP at the same concentration produced a similar insulin secretion without eliciting an accumulation of inositol phosphates. The combined effect of both substances added simultaneously resulted in a significant increase in insulin release as compared with the secretion induced by either substance used separately. By contrast, the accumulation of inositol phosphates induced by both substances was not different from the accumulation induced by carbachol alone. These results seem to rule out the involvement of polyphosphoinositide hydrolysis in the coupling mechanism between P2 purinoreceptor activation and insulin response of the B cell. Moreover, purinergic stimulation appears not to interact with the effect of muscarinic stimulation on polyphosphoinositide breakdown.

ATP and phosphate-modified adenine nucleotide analogues. Effects on insulin secretion and calcium uptake.

Our previous experiments on isolated rat pancreas gave evidence for a P2 purinergic receptor on the insulin-secreting B cell. This work was designed to investigate whether the stimulation of insulin release by phosphorylated adenosine derivatives could also be observed in rat isolated Langerhans islets and whether this stimulation was accompanied by changes in calcium uptake. The results indicate that two structural methylene analogues of ATP and ADP (alpha,beta-methylene ATP and alpha,beta-methylene ADP) display an insulin stimulatory effect comparable to that of ATP, confirming the membrane action of the latter. It was also found that calcium uptake increased concomitantly with insulin release under the effect of alpha,beta-methylene ADP; on the other hand this agent also increased the total exchangeable calcium content of islets at isotopic equilibrium. Verapamil, a blocker of voltage-sensitive calcium channels, counteracted the stimulation of insulin release and also blocked the increase in total exchangeable calcium content. These results demonstrate the involvement of calcium in the stimulus-secretion coupling of insulin release induced by an activator of P2 purinergic receptors and suggest the implication of voltage-sensitive calcium channels.

Enhancement of insulin release and islet cell calcium content by an acyl-amino-alkyl benzoic acid derivative, HB 699.

HB 699, a hypoglycaemic agent which lacks the structural requirements regarded as essential for the insulin releasing action of the sulfonamide group, was studied in isolated rat islets and compared with tolbutamide. In the presence of 8.3 mM glucose for long exposure (24 h) both substances induced an increase of insulin release without altering the islet insulin content. After preloading of the islets to isotopic equilibrium with 45Ca++ (24 h), HB 699 and tolbutamide induced a significant increase in total exchangeable calcium content parallelled by an increase in insulin release. Both effects could be suppressed by verapamil, a blocker of voltage-sensitive Ca++ channels. These results indicate that HB 699, like tolbutamide, stimulates insulin release by increasing Ca++ uptake by the B cells.

Effect of LN 5330 on insulin release and calcium uptake by isolated rat islets of Langerhans. Comparison with diazoxide.

The effect of a new benzothiadiazine derivative, LN 5330, was studied on insulin release and 45Ca++ uptake by isolated rat islets of Langerhans. LN 5330 (0.1 mmol/l) significantly inhibited both insulin release and calcium uptake of islets incubated in MEM (glucose 8.3 mmol/l). This effect was comparable to that of diazoxide. The withdrawal of LN 5330 from the medium resulted in a significant increase of insulin release and calcium uptake by the islets during a subsequent incubation in MEM (glucose 8.3 mmol/l). In contrast, after withdrawal of diazoxide, insulin release and calcium uptake by the islets were not different from the controls. These results suggest a dual action of LN 5330: an effect, similar to that of diazoxide, responsible for the inhibition of insulin release in the presence of the drug; and an effect, specific to LN 5330, which would induce cellular changes, the consequence of which appears only upon withdrawal of the drug.

A different action of hypothermia on insulin release from the isolated, perfused rat pancreas, depending on the stimulating agent.

Two series of experiments were performed in parallel on the isolated perfused rat pancreas. The experimental conditions differed only as pertaining to temperature. In one series the organ and the perfusion liquid were maintained at 37.5 degrees C and in the other at 28 degrees C. The pancreases were perfused from the start of the experiments with a perfusion medium containing 8.3 mmol/l glucose. The effects of various stimulatory agents were studied (glucose 16.6 mmol/l, tolbutamide 0.4 mmol/l, acetylcholine 0.5 micromole/l, glucagon, 2.8 nmol/l, and L-isoprenaline 0.05 micromole/l). At 37.5 degrees C the insulin secretion induced by high glucose or tolbutamide, acetylcholine, and glucagon was biphasic and not statistically different. In all cases the hypothermia (28 degrees C) decreased insulin secretion. However, glucose-induced and tolbutamide-induced insulin secretion was more decreased than the secretion induced by acetylcholine and glucagon. The study of the secretion ratios obtained at 28 degrees C relative to 37.5 degrees C showed that the ratios for the glucose and tolbutamide groups were significantly lower than those obtained for acetylcholine and glucagon groups for both the first and the second phase. The ratios were not significantly different between glucose and tolbutamide on the one hand and acetylcholine and glucagon on the other hand. In all groups the ratios 28 degrees/37.5 degrees for the second phase were lower than those obtained during the first phase. L-isoprenaline induced only a weak increase in insulin secretion and this was not long lasting; this increase was not statistically different at both temperatures.