The mechanisms of action of chloromercuribenzene-p-sulphonic acid as insulin secretagogue: fluxes of calcium, sodium and rubidium in islets exposed to mercurial and a membrane-active antagonist.

Chloromercuribenzene-p-sulphonic acid (CMBS) is known to markedly stimulate insulin release and to enhance formation of adenosine 3':5'-cyclic monophosphate (cyclic AMP) and monovalent cation permeability in the pancreatic islet cells. The effects on insulin release and cyclic AMP can be inhibited with 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS). To elucidate the role of cationic fluxes in CMBS- induced insulin release, uptake of 22Na+ and 45Ca2+ as well as efflux of 86Rb+ were studied in islets exposed to 0-1 mM CMBS or 1-0 mM SITS or both. 2. The enhancing effect of CMBS on Na+ permeability, and probably also that on Rb+ permeability, was inhibited by SITS. 3. CMBS stimulated the rate of 45Ca2+ uptakes when the islets were incubated in a poly-anionic bicarbonate buffer but not when they were incubated in Tris buffer containing only Cl- as anion. In bicarbonate buffer, the enhancement of 45Ca2+ flux was observed both with the lanthanum method for measuring intracellular 45Ca2+ uptake and with a method estimating the total islet uptake. SITS had no significant effect on the CMBS-induced 45Ca2+ uptake. 4. Chromatography on Sephadex G-15 did not reveal any significant chemical interaction between 0-1 mM CMBS and 1 mM SITS. 5. The following hypothesis for the recognition of CMBS as insulin secretagogue is suggested: by increasing Na+ permeability more than K+ permeability, CMBS depolarizes the beta-cell, leading to initiation of insulin release by an ionic mechanism which may or may not involve a change in transmembrane Ca2+ fluxes. The marked intensity of the secretory response is due to the fact that CMBS also enhances cyclic AMP formation, potentiating the effect of the ionic mechanisms on the insulin discharge apparatus.     

Glucose-stimulated and La3+-nondisplaceable Ca2+ pool in pancreatic islets.

To study intracellular pools of calcium tissue specimens from noninbred ob/ob mice were labeled with 45Ca2+ and subsequently washed with La3+. D-glucose, 20 mM, enhanced the labeling of the La3+-nondisplaceable calcium in pancreatic islets but not in pieces of exocrine pancreas or liver. The disappearance of 45Ca2+ from labeled islets was accelerated by the ionophore, X-537A, but not by dibutyryl cyclic AMP, theophylline, or pentobarbital. On fractionation of 45Ca2+-labeled islets, the greatest radioactivity per unit of protein occured in a fraction rich in insulin secretory granules. The radioactivity of this fraction was higher after the islets had been loaded with 45Ca2+ in the presence of 20 mM glucose as compared to 3 mM glucose. It is concluded that the secretory granules make up a considerable part of the glucose-sensitive calcium store in the beta-cells.     

Different effects of the ionophore A-23187 and D-glucose on 45Ca2+ fluxes in isolated islets of ob/ob-mice.

Fluxes of 45Ca2+ were studied in beta-cell rich islets of non-inbred ob/ob-mice, using LaCl3 to wash out extra-cellular and superficially bound 45Ca2+. The ionophore A-23187 (10 microM) increased the 45Ca2+ uptake in islets both at 3 and 20 mM D-glucose, the effect being more pronounced after 10 min than after 120 min of incubation. In incubations for 120 min, 20 mM D-glucose induced a higher uptake of 45Ca2+ than did A-23187. The ionophore enhanced the unidirectional efflux of 45Ca2+ from preloaded islets. Pretreatment of islets with 20 mM D-glucose in non-radioactive medium inhibited the subsequent D-glucose-induced 45Ca2+ uptake. Similar pretreatment with A-23187 increased the subsequent ionophore-induced 45Ca2+ uptake. The results suggest that A-23187 acts by catalyzing Ca2+ fluxes across the beta-cell plasma membrane. The different effects of D-glucose and A-23187 on 45Ca2+ fluxes suggest that the two agents act through different mechanisms in the beta-cells.     

Effects of alpha- and beta-adrenoceptor stimulation on 45Ca2+ efflux and insulin secretion from perfused rat islets

Exposure to the beta 2-adrenoceptor agonist terbutaline resulted in a transient stimulation of 45Ca2+ efflux from 45Ca2+ preloaded rat islets perfused in 2 mM Ca2+ and 8.3 mM glucose. Concomitantly, an increase in insulin secretion occurred. Under the same experimental conditions, the alpha-adrenoceptor agonist noradrenaline promptly inhibited insulin release without any apparent influence on 45Ca2+ efflux. In contrast, in a medium containing 2 mM Ca2+ and a low glucose concentration (2.8 mM), terbutaline stimulated insulin secretion without any apparent effects on 45Ca2+ efflux. Noradrenaline had no effect on insulin secretion or 45Ca2+ efflux in this medium. When islets were perfused with 8.3 mM glucose in a Ca2+ deficient medium, with or without addition of the chelating agent EGTA, terbutaline induced a marginal stimulation of insulin secretion and a negligible stimulation of 45Ca2+ efflux. On the contrary, noradrenaline stimulated to an immediate and notable 45Ca2+ efflux in these Ca2+ deficient media. Noradrenaline also clearly inhibited insulin secretion, though less markedly and with a slower onset than in islets perfused in 2 mM Ca2+. When the islets were perfused in a Ca2+ deficient medium with 2.8 mM glucose, terbutaline had a slight insulin releasing effect, but stimulated 45Ca2+ efflux potently. Noradrenaline had no influence on insulin secretion but a weak stimulatory effect on 45Ca2+ efflux. The data suggest that the beta 2-adrenoceptor agonist terbutaline has the ability to stimulate insulin secretion in perfused rat islets, requiring extracellular Ca2+ for the full expression of its effects. These effects may be exerted through a Ca2+-Ca2+ exchange over the cell membrane and/or through cAMP and intracellular Ca2+ perturbations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vitamin D3 stimulates calcium-45 uptake by isolated mouse islets in vitro

Islets isolated from ob/ob mice which had been fed a vitamin D-deficient diet released significantly less insulin in response to glucose than did vitamin D-replete islets but showed normal net 45Ca2+ uptake. To determine whether vitamin D3 has a direct effect on the pancreatic B cell, islets from ob/ob mice on a normal diet were exposed to vitamin D3 in vitro for 1 week or only 3 h, and then glucose-stimulated 45Ca2+ uptake and insulin release were measured. Exposure to 1 nM or 1 microM vitamin D3 for 1 week stimulated 45Ca2+ uptake in the presence of 3 mM, but not 20 mM glucose, and did not affect insulin release. Exposure to vitamin D3 for 3 h did not significantly increase net 45Ca2+ uptake although there was a tendency to such an effect (P = 0.10). In conclusion, vitamin D-deficiency in vivo suppressed subsequent glucose-stimulated insulin release in vitro and this effect may be due to a direct effect of the sterol (or one of its metabolites) on calcium handling by the B cell.     

Different effects of glibenclamide and the structural analogue HB 699 on the 45Ca2+ uptake by ob/ob-mouse islets

The effects on 45Ca2+ uptake of HB 699, an acyl-amino-alkyl benzoic acid derivative, was compared to those of glibenclamide in incubations using the La3+ wash technique. HB 699 enhanced the 45Ca2+ net uptake in a concentration range (10-200 microM) where insulin release was also stimulated. Glibenclamide showed maximum stimulation of 45Ca2+ net uptake already at 1 microM. HB 699 did not clearly stimulate the short-term 45Ca2+ uptake whether or not the islets were preincubated with the drug. It is suggested that HB 699-induced insulin release is mediated, at least partly, by increased mobility of beta-cell Ca2+.     

Pancreatic acinar cells: effects of lanthanum ions on amylase release and calcium ion fluxes

1. The effect of La(3+) on amylase release and Ca(2+) fluxes in mouse pancreatic fragments in vitro was studied.2. Amylase release was increased by 0.1 mM-La(3+) and progressively inhibited by 1.0-10 mM-La(3+). Non-stimulated and bethanecol stimulated secretion were altered in an identical manner. Inhibition of amylase release was rapid and reversible.3. Uptake of (45)Ca(2+) was multiphasic with equilibrium with stable Ca(2+) still not complete after 2 hr. La(3+) (10 mM) limited uptake of (45)Ca(2+) to the extracellular space and slightly decreased total Ca(2+) content. Lower concentrations of La(3+) affected (45)Ca(2+) uptake and total Ca(2+) content in a biphasic manner which paralleled effects on amylase release.4. La(3+) restricted washout of (45)Ca(2+) to isotope in the extracellular space and abolished the bethanecol-stimulated increase in (45)Ca(2+) efflux.5. Uptake of (45)Ca(2+) into intracellular space, as measured by the ;lanthanum' method, was not affected by bethanecol.6. Tissue ultrastructure and Na(+) and K(+) content were not affected by La(3+).7. It is concluded that an influx of extracellular Ca(2+) is not important for triggering of secretion and that La(3+) may inhibit amylase release by acting on the release process rather than on Ca(2+) influx.     

Different effects of the ionophore A-23187 and D-glucose on 45Ca2+ fluxes in isolated islets of ob/ob-mice

Fluxes of ⁴⁵Ca²⁺ were studied in β-cell rich islets of non-inbred ob/ob-mice, using LaCl₃ to wash out extracellular and superficially bound ⁴⁵Ca²⁺. The ionophore A-23187 (10,μM) increased the ⁴⁵Ca²⁺ uptake in islets both at 3 and 20 mM D-glucose, the effect being more pronounced after 10 min than after 120 min of incubation. In incubations for 120 min, 20 mM D-glucose induced a higher uptake of ⁴⁵Ca²⁺ than did A-23187. The ionophore enhanced the unidirectional efflux of ⁴⁵Ca^a+ from preloaded islets. Pretreatment of islets with 20 mM D-glucose in non-radioactive medium inhibited the subsequent D-glucose-induced ⁴⁵Ca²⁺ uptake. Similar pretreatment with A-23187 increased the subsequent ionophore-induced ⁴⁵Ca²⁺ uptake. The results suggest that A-23187 acts by catalyzing Ca²⁺ fluxes across the β-cell plasma membrane. The different effects of D-glucose and A-23187 on ⁴⁵Ca²⁺ fluxes suggest that the two agents act through different mechanisms in the β-cells.     

Interactions of Ca2+, Mg2+, and Na+ in regulation of insulin release from rat islets

The effects of glucose and ionic modifications on unidirectional Ca2+ efflux and insulin release has been studied. Rat pancreatic islets were isotopically equilibrated with 45Ca2+ for 2 days and then perifused at 10(-8) M Ca2+ to allow for strict interpretation of 45Ca2+ efflux. Under these conditions 16.7 mM glucose inhibited Ca2+ efflux but did not stimulate insulin release. Removal of Mg2+ from the buffer markedly stimulated Ca2+ efflux that was counteracted by glucose. The omission of Na+ decreased basal Ca2+ efflux by 30% at 10(-8) M Ca2+, thus demonstrating the importance of Na-Ca countertransport for Ca2+ extrusion. Like glucose, Na+ omission or the addition of ouabain attenuated Ca2+ efflux stimulated by Mg2+ removal. Glucose may interfere with Na-Ca countertransport because the actions of 16.7 mM glucose and Na+ omission were not additive. At 10(-8) M Ca2+, glucose elicited insulin release only when both 1) loss of cellular calcium was minimized by prior inhibition of Ca2+ efflux (Na+ omission or ouabain), and 2) Ca2+ mobilization was favored by Mg2+ removal. Under these conditions (in contrast to normal Ca2+), insulin release was not accompanied by increased Ca2+ efflux. Thus, unidirectional Ca2+ measurements do not permit the detection of Ca2+ mobilization in intact islets because glucose may concomitantly inhibit Ca2+ extrusion.     

Effects of Ca2+ channel agonist-antagonist enantiomers of dihydropyridine 202791 on insulin release, 45Ca uptake and electrical activity in isolated pancreatic islets

This is the first study using the selective agonist/antagonist stereoisomers of dihydropyridine 202791 to investigate stimulus-secretion coupling in pancreatic islet cells. We studied effects of the (+)(Ca2+ channel agonist) and (-)(Ca2+ channel antagonist) forms of the dihydropyridine, on 45calcium net uptake, insulin secretion, and membrane potential measured in rodent islets. The antagonist partially inhibited glucose-induced insulin secretion and Ca2+ uptake; however, the potassium-induced Ca2+ uptake was completely inhibited. The antagonist did not completely block glucose-evoked spike activity. Addition of the agonist enhanced insulin release and Ca2+ uptake in the presence of 5.6 mM-glucose, but did not increase insulin release or Ca2+ uptake in 16.7 mM-glucose. In the presence of tetraethylammonium (TEA), (+)202791 increased and (-)202791 decreased the duration of glucose-induced action potentials. The results again confirm the presence of a dihydropyridine-sensitive Ca2+ channel in pancreatic B-cells. In addition these data suggest that in these cells there is activation of a dihydropyridine-insensitive Ca2+ entry in the presence of glucose.     

The functional significance of sodium channels in pancreatic beta-cell membranes

1. The existence and functional significance of Na channels in pancreatic beta-cell membranes were investigated by studying the effects of the plant alkaloid veratridine on the temporal release of insulin from perfused isolated rat islets of Langerhans.2. 100 muM veratridine evoked a sustained threefold increase in insulin release which was almost completely inhibited by 3 muM tetrodotoxin (TTX). This action of TTX was rapidly reversible.3. The simultaneous presence of 100 muM propranolol, 100 muM phenoxy-benzamine and 10 muM atropine did not alter the magnitude of the response to 100 muM veratridine, indicating that the action of veratridine on the beta-cells was direct and was not mediated via the release of neurotrans-mitters from nerve endings within the islets.4. (45)Ca uptake by isolated islets in static incubation was increased almost threefold by 100 muM veratridine. This increase was completely inhibited by the simultaneous presence of 3 muM TTX.5. Replacement of Na(o) by choline caused a transient fourfold increase in insulin release which was associated with an increase in the uptake of (45)Ca from the extracellular space of similar magnitude. Subsequent exposure of islets to 100 muM veratridine still evoked some insulin release but this only achieved 32% of that secreted by islets exposed to veratridine in medium of normal [Na](o).6. The addition of 2.5 mM CoCl(2) to the medium caused a 62.5% inhibition of veratridine-mediated insulin release.7. In Ca-free medium supplemented with 1 mM EGTA, 100 muM veratridine evoked insulin release of equal magnitude and of similar temporal relationship to that obtained in the presence of normal [Ca](o).8. A twofold increase in insulin release that occurred in the 15 min period immediately following exposure to 1 mM ouabain was completely independent of [Ca](o). Subsequent ouabain-evoked release became increasingly dependent on [Ca](o).9. Tetrodotoxin (3 muM) inhibited the first phase of insulin release evoked by 16.7 mMd-glucose by 37% and the second phase by 20%.10. Both Na and Ca appear capable of entering through Na channels opened in the beta-cell membrane by veratridine. The increase in [Na](i), resulting from the veratridine mediated increase in P(Na+), causes depolarization of the beta-cell membrane with a consequent opening of voltage-sensitive, Co(2+)-blockable channels for additional Ca entry. An increase in [Na](i) also increases [Ca](i) by altering the equilibria of intracellular Ca-sequestering mechanisms. The small but significant reduction of glucose-mediated insulin release by TTX indicates that glucose has a rather weak action on the Na channel and a more pronounced effect on the voltage-dependent Co(2+)-blockable Ca channel.     

Insulinotropic action of amino acids at their physiological concentrations: I. Experiments in incubated islets

In order to simulate physiological conditions, the influence of a mixture of 22 amino acids together with taurine, all tested at their normal concentration in plasma, upon insulin release, D-glucose metabolism and (45)Ca net uptake was investigated in isolated rat pancreatic islets. The amino acid mixture had little effect upon insulin release at low concentrations of D-glucose but augmented, by up to 50%, the release of insulin provoked by higher concentrations of D-glucose. The effects of glibenclamide, forskolin, theophylline and cytochalasin B upon insulin release evoked by D-glucose in the absence or presence of the amino acid mixture and the changes in insulin output evoked by the omission from the amino mixture or addition to media containing only D-glucose of selected amino acid(s), as well as the influence of the amino acid mixture upon D-glucose metabolism and (45)Ca net uptake, were considered as compatible with both the role of certain amino acids as nutrients and the accumulation of other amino acids as positively charged molecules in the islet cells.     

Effects of cytochalasin B and D upon insulin release and pancreatic islet cell metabolism.

Cytochalasin B is known to enhance insulin release evoked by nutrient and non-nutrient secretagogues, including D-glucose, despite inhibiting D-glucose uptake and metabolism in pancreatic islets. In the present study, cytochalasin D, which failed to affect D-glucose uptake and metabolism by isolated islets, also augmented glucose-stimulated insulin release, but unexpectedly to a lesser extent than cytochalasin B. Such was not the case, however, in islets stimulated by non-glucidic nutrients such as 2-ketoisocaproate or the association of L-leucine and L-glutamine. This situation coincided with the fact that cytochalasin B inhibited more severely D-glucose metabolism in non-B, as distinct from B, islet cells and, in the former case, caused a relatively greater inhibition of hexose catabolism at 2.8 mM than at 16.7 mM D-glucose. Nevertheless, even in the presence of forskolin, cytochalasin B was more efficient than cytochalasin D in augmenting glucose-stimulated insulin secretion. Thus, although these data document that non-B islet cells are more sensitive than purified islet B cells to cytochalasin B, at least in terms of inhibition of D-glucose catabolism, such a difference and its possible consequence upon the release of glucagon and other non-insulinic hormones by non-B islet cells do not appear sufficient to account for the greater enhancing action of cytochalasin B, as distinct from cytochalasin D, upon glucose-stimulated insulin output. Likewise, the latter difference does not appear attributable to a greater efficiency of cytochalasin B, as compared to cytochalasin D, upon the mechanical events involved in nutrient-stimulated exocytosis of insulin granules. Hence, the present findings suggest a so-far-unidentified interference of cytochalasin B with the B-cell glucose-sensing device.     

Interactions between magnesium and calcium in beta-cell-rich pancreatic islets

Calcium-magnesium interactions, total amounts of intracellular magnesium, and insulin release were studied in beta-cell-rich pancreatic islets from ob/ob mice. Mg2+ inhibited the uptake of intracellular 45Ca and insulin release induced by glucose or high concentrations of potassium. Omission of Mg2+ from a Ca2+-deficient medium resulted in an increased efflux of 45Ca, whereas the characteristic glucose inhibition of the efflux was diminished. After addition of Mg2+ to a Mg2+-depleted medium, the glucose-stimulated 45Ca efflux was markedly reduced. Mg2+ inhibited the basal efflux of 45Ca, and this effect was preceded by a transient stimulation. Ca2+ but not Mg2+ stimulated 45Ca efflux in a medium depleted of Ca2+, Mg2+, and Na+. The data indicate that Mg2+ interferes with Ca2+ entry through voltage-dependent Ca2+ channels. Mg2+ may also inhibit the outward transport of Ca2+ from the cells at a site different from the Na+-Ca2+ countertransport mechanism. The total amount of intracellular magnesium remained unaffected by glucose and was not changed unless the ionic composition of the mediums were changed grossly. Under physiological conditions it is therefore unlikely that fluctuations in the intracellular Mg2+ concentration are part of the mechanism by which the functionally important Ca2+ is regulated.     

Effects of glass ionomers and dental resin composites on viability of beta-cells and insulin release in isolated islets of Langerhans

Information on the biocompatibility of glass ionomers and resin composites is sparse. To extend the scale of biological testing we evaluated the influence of those materials on insulin secretion at whole organ level in vitro. The effects on insulin secretion of three glass ionomers and two resin composites, aged for 1 week, were studied in isolated mouse islets of Langerhans at basal (5.5mM) and at stimulatory (11.1mM) D-glucose concentrations. In addition, viability of single mouse beta-cells was evaluated. The effect of glass ionomer specimens aged for 1 and 4 months on insulin secretion at 11.1mM D-glucose was also studied. None of the materials affected the viability of the beta-cells. At 5.5mM D-glucose none of the materials affected the insulin secretion. At 11.1mM D-glucose, the glass ionomers only decreased the secretion and glass ionomers aged for 1 month still decreased insulin release whereas after 4 months ageing only one of the glass ionomers affected the release. The result shows a dynamic effect on insulin release of the elements and/or compounds released from the specimens.     

Suppression of cellular injury during the calcium paradox in rat heart by factors which reduce calcium uptake by mitochondria

Isolated Langendorff perfused rat hearts were used to study changes in the Ca, Na and K content, contractile force and the loss of cellular material during the Ca paradox. Five minutes perfusion with Ca-free solution containing 1 mM EGTA, followed by 10 min of reperfusion in 1.8 mM Ca causes irreversible contracture, K loss, increase in Na and Ca and a massive release of myoglobin and other cellular material into the perfusate (the calcium paradox). During the Ca-free perfusion the ventricles gain Na but the K content decreases slightly. The size of the Na gain appears to depend upon the buffer used and is larger in bicarbonate than in Tris. When HCO3- or H2PO4- ions are omitted from the bathing solution (in Tris, HEPES, or TES buffered salines) the adverse effects of Ca readmission are reduced. Tris buffer gives the best protection. Metabolic inhibition with FCCP (5 X 10(-7) M), or with CN-(2 X 10(-3) M) together with iodoacetic acid (2 X 10(-3) M), decreases Ca uptake during the Ca paradox and inhibits the release of cellular material. In both cases a contracture is observed. Ruthenium red (10(-4) M) does not inhibit the Ca readmission contracture but reduces the release of cellular material and the gain of Ca and Na. The results suggest that the loss of cellular constituents during the calcium paradox, is related to an active uptake of Ca by the mitochondria and may lead to massive changes in the cellular ion concentration, during Ca-repletion.     

Polysaccharide isolated from Triticum aestivum stimulates insulin release from pancreatic cells via the ATP-sensitive K+ channel

Traditional natural plants have been used throughout the world for their antidiabetic effects. The aim of the present study was to investigate the stimulating activity of a polysaccharide extract derived from T.?aestivum sprout (TASP) on insulin secretion in?vitro using the RIN-5F pancreatic β-cell line and rat pancreatic islets. In these experiments, TASP (0.1 to 2?mg/ml) augmented glucose-stimulated insulin secretion in a dose-dependent manner in the presence of a stimulatory glucose concentration (16.7?mM), but not of a basal concentration (1.1?mM). Although TASP failed to enhance the high K+-induced insulin secretion, the insulinotropic effect of TASP was significantly inhibited by diazoxide, an opener of ATP-sensitive K+ channel blocking insulin release. TASP potentiated the insulin secretion induced by other secretagogues, such as IBMX and tolbutamide. Moreover, glucose-derived blood insulin levels were significantly elevated by oral administration of TASP to mice, similarly to antidiabetic drugs. We also demonstrated that TASP significantly increased glucose-induced 45Ca2+ uptake and proinsulin mRNA expression in rat islets. Overall, our results suggest that TASP has a stimulating effect on insulin secretion and production in pancreatic β-cells via K+ channel closure and calcium influx. These results suggest that TASP may be useful as a candidate for the therapy of diabetes mellitus.     

On the repetitive discharge in myotonic muscle fibres

1. Fluxes of (86)Rb(+) and (22)Na(+) were measured in pancreatic islets of ob/ob-mice. The islets, which contain more than 90% beta-cells, were incubated at 37 degrees C in Krebs-Ringer bicarbonate buffer with modifications known to influence insulin release.2. In the presence of Na(+), the islets vigorously accumulated Rb(+). The Rb(+) uptake was inhibited by depletion of islet Na(+) or by 1 mm ouabain or 0.1 mm chloromercuribenzene-p-sulphonic acid. Rb(+) uptake was stimulated by 1 mm-5,5'-dithiobis (2-nitrobenzoic acid) or by depletion of islet Ca(2+), while 20 mm glucose, 5 mm theophylline, 0.1 mm iodoacetamide, or 1 mm-6,6'-dithionicotinic acid had no significant effects.3. The efflux of Rb(+) from preloaded islets followed exponential kinetics with a half-life of about 16 min. The rate of efflux was enhanced by 0.1 mm chloromercuribenzene-p-sulphonic acid and inhibited by 20 mm glucose. Omission of Na(+), K(+) or Ca(2+) from the incubation medium had no significant effects.4. The efflux of (22)Na(+) from islets preloaded with this isotope was enhanced by 0.1 mm chloromercuribenzene-p-sulphonic acid or by Ca(2+) deficiency. It was inhibited by 1 mm ouabain, 0.1 mm-2,4-dinitrophenol, or by omission of Na(+) from the incubation medium. Omission of K(+) or the addition of 20 mm glucose had no significant effects.5. It is concluded that the beta-cells are permeable to Na(+) and Rb(+) and expel Na(+) by an active mechanism similar to, or identical with, the Na(+)/K(+)-pump in other cells. The mechanisms of active and passive cation movements are discussed in relation to current hypotheses of stimulus-secretion coupling in the beta-cells depending on interactions between Na(+) and Ca(2+). In particular, the results support the hypotheses of insulin release being stimulated by ouabain through inhibition of the Na(+)/K(+)-pump and by organic mercurials through enhancement of membrane permeability to cations.     

Insulin secretion in the isolated islets of single-, regular-fasted and fed rats

To investigate the alterations in insulin secretion induced by habituation to single daily meal, adult rats were trained on a regular-fasting scheme (2-hr feeding/22-hr fasting) for 4 weeks. Insulin secretion induced by nutrient secretagogues (D-glucose and L-leucine) and the rates of 45Ca2+ outflow and 14C-glucose oxidation were studied in isolated islets obtained from these animals and in fed and 22-hr single-fasted rats. As expected, in the 22-hr fasted group, insulin secretion was drastically decreased although not abolished while 45Ca2+ outflow and 14C-glucose oxidation rates were only partially reduced. However, the regular-fasted rats did not secrete insulin in response to the nutrient secretagogues; calcium entry was not detected although a partial reduction in 14C-glucose oxidation rate was observed. These results suggest that regular fasting induces alterations in pancreatic B-cell glycolytic pathways leading to impairment of calcium efflux and insulin secretion. Such impairment is more pronounced than that induced by a single 22-hr fast.     

Insulin secretion to glucose stimulus in pancreatic islets isolated from rats fed unbalanced diets.

To verify the effect of different energetic sources on insulin secretion, just-weaned male Wistar rats were divided into four groups fed as follows: high carbohydrate (HC), high protein (HP), high lipid (HL) and balanced (C) diets during five weeks. Body weight gain and daily food intake were similar among the four groups. Insulin content of the isolated islets was not different; however, insulin release to a high glucose concentration (16.7 mM) stimulus was clearly higher in islets isolated from rats fed a balanced diet as compared to the other groups (HC, HP and HL). The rates of insulin secretion in islet perfusates from rats fed unbalanced diets were also decreased, although 45Ca2+ outflow rate (FOR) was similar in all groups. Since the rate of U-14C-glucose oxidation was decreased in islets isolated from rats fed unbalanced diets, this could be one of the mechanisms for the reduced rates of insulin release observed. Therefore, the increased supply of specific fuels provoke metabolic alterations in B-cell leading to changes in insulin secretion.