Phosphorylation reactions in islets of Langerhans.

The possible significance of phosphorylation reaction in islets of Langerhans in relation to the secretion of insulin is discussed. The secretagogues, glucose and its metabolites, cAMP and Ca++ and their influences on protein-kinase activity are given particular attention. The data obtained by the authors, as well as by other groups, are in agreement that cAMP is a potent stimulator of protein kinase activity. Glucose and its metabolites influenced protein kinase activity in one instance. Ca++ in supraphysiological amounts inhibited protein phosphorylation. The links between phosphorylation reactions and insulin secretion are, at the present time, conjectural.     

Calcium signaling in plant cell organelles delimited by a double membrane

Increases in the concentration of free calcium in the cytosol are one of the general events that relay an external stimulus to the internal cellular machinery and allow eukaryotic organisms, including plants, to mount a specific biological response. Different lines of evidence have shown that other intracellular organelles contribute to the regulation of free calcium homeostasis in the cytosol. The vacuoles, the endoplasmic reticulum and the cell wall constitute storage compartments for mobilizable calcium. In contrast, the role of organelles surrounded by a double membrane (e.g. mitochondria, chloroplasts and nuclei) is more complex. Here, we review experimental data showing that these organelles harbor calcium-dependent biological processes. Mitochondria, chloroplasts as well as nuclei are equipped to generate calcium signal on their own. Changes in free calcium in a given organelle may also favor the relocalization of proteins and regulatory components and therefore have a profound influence on the integrated functioning of the cell. Studying, in time and space, the dynamics of different components of calcium signaling pathway will certainly give clues to understand the extraordinary flexibility of plants to respond to stimuli and mount adaptive responses. The availability of technical and biological resources should allow breaking new grounds by unveiling the contribution of signaling networks in integrative plant biology.     

Insulin and glucagon secretion from isolated islets of Langerhans. The effects of calcium ionophores.

The role of Ca2+ in the secretion of insulin and glucagon was investigated by studying the effects of Ca2+ ionophores on hormone secretion from isolated perifused islets of Langerhans. Ionophore X537A (100 muM), which binds alkaline earth cations and also complexes some univalent cations, caused a rapid transient increase in insulin and glucagon secretion which was not dependent on the presence of Ca2+ in the perifusion medium. Ionophore A23187 (100 muM), which specifically binds bivalent cations at neutral pH values, similarly increased insulin secretion in complete and Ca2+-free medium, but only stimulated glucagon release in the presence of extracellular Ca2+. Since the stimulatory effects of both ionophores were associated with an increased Ca2+ flux in the islets, these experiments support the hypothesis that Ca2+ may trigger the release of insulin and suggest that it is also involved in the secretion of glucagon. The basal rate of both insulin and glucagon release was significantly increased when Ca2+ was omitted from the perifusion medium, but it is proposed that this finding may be due to adverse effects on cell-membrane function under these conditions.     

Glucose diffusion in pancreatic islets of Langerhans.

We investigate the time required for glucose to diffuse through an isolated pancreatic islet of Langerhans and reach an equilibrium. This question is relevant in the context of in vitro electrophysiological studies of the response of an islet to step changes in the bath glucose concentration. Islet cells are electrically coupled by gap junctions, so nonuniformities in islet glucose concentration may be reflected in the activity of cells on the islet periphery, where electrical recordings are made. Using a mathematical model of hindered glucose diffusion, we investigate the effects of the islet porosity and the permeability of a surrounding layer of acinar cells. A major factor in the determination of the equilibrium time is the transport of glucose into islet beta-cells, which removes glucose from the interstitial spaces where diffusion occurs. This transport is incorporated by using a model of the GLUT-2 glucose transporter. We find that several minutes are required for the islet to equilibrate to a 10 mM change in bath glucose, a typical protocol in islet experiments. It is therefore likely that in electrophysiological islet experiments the glucose distribution is nonuniform for several minutes after a step change in bath glucose. The delay in glucose penetration to the inner portions of the islet may be a major contributing factor to the 1-2-min delay in islet electrical activity typically observed after bath application of a stimulatory concentration of glucose.     

Dissociation between intracellular calcium mobilization and insulin secretion in isolated rat islets of Langerhans

The neuropeptide bombesin provoked a dose-dependent stimulation of 45Ca2+ efflux from pre-loaded islets of Langerhans. This response occurred rapidly, was not sustained and did not depend on the presence of extracellular calcium, suggesting that it resulted from the mobilization of intracellular calcium stores. Under conditions when large increases in 45Ca2+ efflux were observed, bombesin completely failed to stimulate the rate of insulin secretion. Similar results were also obtained with the muscarinic cholinergic agonist, carbachol. The data suggest that the release of calcium from intracellular pools is not sufficient to induce an increase in insulin secretion in normal islet cells.     

Light and electron microscopic study of crocodile islets of Langerhans

The tissue of the islets of Langerhans was studied in Alligator mississippiensis and Caiman niger. The distribution of the insular tissue in the pancreas was described from the picture in the light microscope, together with the incidence of characteristic islets, which frequently surround the duct tress, and of scattered insular cells. Five types of endocrine cells, distinguishable by their staining properties and typical electron microscopic image, were described in the insular tissue. In addition to A-, B- and D-cells, others which very probably correspond to EC- and PP-cells are described. Mixed cells were also found. The pancreatic interstitial connective tissue contains large numbers of nerve fibre bundles; isolated nerve fibres may penetrate to the insular cells. At the site of contact of a nerve ending with an endocrine cell, characteristic thickening of the membranes is sometimes found.     

Calcium behavior in endotoxin-poisoned mice: especially calcium accumulation in mitochondria

A possible role of intracellular Ca2+ and participation of calmodulin in cellular metabolism in endotoxin-poisoned mice were investigated. The levels of calcium in liver cytosol and liver mitochondria fractions in poisoned mice were markedly higher 18-48 hr after endotoxin injection than in the control mice. On the other hand, the levels of serum calcium in the poisoned mice were about 20% lower at 18 hr than in the controls. The serum calcium levels in mice injected with 50 and 100 micrograms of endotoxin showed no dose-response effect, but a dose-response effect was observed at a dose of 200-400 micrograms. The serum Ca2+ levels in endotoxin-tolerant mice were similar to those in the control mice. The levels in mice injected with glucocorticoid-antagonizing factor mice were about 14% lower at 3 hr than in the controls. The mice fed a vitamin D3- and calcium-free diet showed a higher mortality rate in the early stage (12-18 hr) of endotoxication than that of the mice fed a normal diet. The lipid peroxide levels and Ca2+-ATPase activity in the liver mitochondria fraction in endotoxin-poisoned mice showed a higher level than those of the control mice. There was little or no difference in the levels of serum glucose between the mice injected with calmodulin antagonist (trifluoperazine, TFP) plus endotoxin and those given endotoxin alone. However, the liver glycogen levels in TFP plus endotoxin-treated mice were markedly higher than that in mice given endotoxin alone. Furthermore, calcium antagonist (verapamil) plus endotoxin-treated mice had about a 40% higher survival rate after 72 hr than those given endotoxin alone. The findings suggest that there is a possibility of participation of the Ca2+-calmodulin system in carbohydrate metabolic disorders during endotoxemia and that the changes in intracellular Ca2+ may result in various metabolic disorders.     

Phosphatidylinositol hydrolysis in isolated guinea-pig islets of Langerhans.

Previous studies have reported an increased turnover of phospholipid in isolated islets of Langerhans in response to raised glucose concentrations. The present investigation was thus undertaken to determine the nature of any phospholipases that may be implicated in this phenomenon by employing various radiolabelled exogenous phospholipids. Hydrolysis of 1-acyl-2-[14C]arachidonoylglycerophosphoinositol by a sonicated preparation of islets optimally released radiolabelled lysophosphatidylinositol, arachidonic acid and 1,2-diacylglycerol at pH 5,7 and 9 respectively. This indicates the presence of a phospholipase A1 and a phospholipase C. However, the lack of any labelled lysophosphatidylinositol production when 2-acyl-1-[14C]stearoylglycerophosphoinositol was hydrolysed argues against a role for phospholipase A2 in the release of arachidonic acid. Phospholipase C activity as measured by phosphatidyl-myo-[3H]inositol hydrolysis was optimal around pH8, required Ca2+ for activity and was predominantly cytosolic in origin. The time course of phosphatidylinositol hydrolysis at pH 6 indicated a precursor-product relationship for 1,2-diacylglycerol and arachidonic acid respectively. The release of these two products when phosphatidylinositol was hydrolysed by either islet or acinar tissue was similar. However, phospholipase A1 activity was 20-fold higher in acinar tissue. Substrate specificity studies with islet tissue revealed that arachidonic acid release from phosphatidylethanolamine and phosphatidylcholine was only 8% and 2.5% respectively of that from phosphatidylinositol. Diacylglycerol lipase was also demonstrated in islet tissue being predominantly membrane bound and stimulated by Ca2+. The availability of non-esterified arachidonic acid in islet cells could be regulated by changes in the activity of a phosphatidylinositol-specific phospholipase C acting in concert with a diacylglycerol lipase.     

Unmasking of arachidonate-induced insulin release by removal of extracellular calcium. Arachidonic acid mobilizes cellular calcium in rat islets of Langerhans

Exogenous arachidonic acid does not stimulate insulin release in Ca++-containing medium, but a potent effect was unmasked by extracellular Ca++ depletion. This secretion met several criteria of exocytotic release. It did not require the oxygenation of arachidonate or its esterification into islet membranes, but was potentiated by the presence of 16.7 mM glucose such that 33 microM arachidonate could reverse the inhibitory effects of extracellular Ca++ removal on glucose-induced insulin secretion. Arachidonic acid alone stimulated a rise in intracellular Ca++ concentrations in dispersed islet cells (measured by the fura-2 technique) equal to that induced by 16.7 mM glucose in normal medium. Arachidonic acid may be a critical coupling signal in normal islets.     

Regulation of calcium fluxes in rat pancreatic islets: Calcium extrusion by sodium-calcium countertransport

Summary The mechanisms by which glucose regulates calcium fluxes in pancreatic endocrine cells were investigated by monitoring the efflux of⁴⁵Ca from prelabeled and perifused rat pancreatic islets. In the absence of both extracellular calcium and glucose, partial or total removal of extracellular sodium decreases the efflux of⁴⁵Ca from prelabeled islets. Glucose also reduces the efflux of⁴⁵Ca from islets perifused in the absence of extracellular calcium. This inhibitory effect of glucose on⁴⁵Ca efflux is decreased by half when the extracellular concentration of sodium is lowered to 24mm. In the absence of extracellular calcium but presence of glucose, partial or even total removal of extracellular sodium fails to decrease the efflux of⁴⁵Ca. At normal extracellular calcium concentration (1mm) partial removal of extracellular sodium dramatically increases⁴⁵Ca efflux from pancreatic islets. This increase in⁴⁵Ca efflux is partially but not totally suppressed by either 16.7mm glucose or cobalt. It is totally suppressed by 4.4mm glucose or by the combination of 16.7mm glucose and cobalt. At normal extracellular calcium concentration, glucose initially reduces and subsequently increases⁴⁵Ca efflux. The initial fall is unaffected by tetrodotoxin but decreased by 50% at low extracellular sodium concentration (24mm). The present results suggest the existence in pancreatic endocrine cells of a glucose-sensitive process of sodium-calcium counter-transport. By inhibiting such a process, glucose may decrease the efflux of calcium from islet cells. The effect of glucose is not mediated by an increase in intracellular sodium concentration. It could contribute to the intracellular accumulation of calcium which is thought to trigger insulin release.This paper is the IVth in a series.     

Magnetic field effects on calcium efflux and insulin secretion in isolated rabbit islets of Langerhans

Rabbit islets of Langerhans were exposed at 37 °C for 18 h to a low-frequency-pulsed magnetic field, generated in paired Helmholtz coils. Exposed islets showed a reduction of 26.1 ± 4.3% in ⁴⁵Ca²⁺ content (P < .004). a reduction of 25.1 ± 6.3% in ⁴⁵Ca²⁺ efflux (P < .006), and a reduction of 35.0 ± 8.7% (P < .002) in insulin released during glucose stimulation when compared with appropriate controls.     

Phosphatidylinositol kinase in rat pancreatic islets: subcellular distribution and sensitivity to calcium

Plasma membrane of pancreatic islets contains a calcium sensitive phosphatidylinositol kinase. This enzyme catalyzes the first reaction in the pathway leading to the production of inositol trisphosphate, which is believed to cause a redistribution of intracellular calcium. Since the activity of this enzyme is inhibited by calcium (K0.5 = 10 microM), a loss of calcium from plasma membrane (the site of PI kinase) may be necessary for activation of the enzyme in vivo.     

Identification and metabolism of polyphosphoinositides in isolated islets of Langerhans.

The effect of pH variation on the exchangeability with deuterium of protons strongly coupled to Mo(V) in the active and desulpho forms of xanthine oxidase was studied by e.p.r. and rapid freezing, in extension of the work of Gutteridge, Tanner & Bray [Biochem. J. (1978) 175, 887-897]. Above neutrality, exchange rates increased with increasing pH. Detailed studies were made on the desulpho enzyme under a variety of conditions, and exchange rate constants at 22 degrees C ranged from 0.16s -1 at pH 6.6 to 1.6s -1 at pH 11.3. The mechanism of proton exchange in the enzyme is discussed. The interpretation by the above workers that the strongly coupled proton of the active enzyme is on sulphur and that of the desulpho enzyme is on oxygen remains valid (and is in agreement with other work), as do their proposals for the structures of the protonated and deprotonated species. However, pK values cannot be calculated from the exchange data. It is likely that the relatively low rates of exchange observed are due to the difference of structure between the protonated and the deprotonated forms. In the case of the desulpho enzyme, an exchange mechanism, which involves the proton exchanging both as such and along with oxygen in the form of a hydroxyl ion, is discussed.     

Substrate effects on oscillations in metabolism, calcium and secretion in single mouse islets of Langerhans

Glucose induces complex patterns of oscillations in intracellular Ca2+ concentration ([Ca2+]i), metabolism and secretion in islets of Langerhans including "slow" and "fast" pulses with period of 2-5 min and 10-20 s respectively. In an effort to elucidate the origin of slow oscillations, individual mouse islets were exposed to different fuels including glyceraldehyde, pyruvate, methyl pyruvate and alpha-ketoisocaproate (KIC), all of which bypass key steps of glycolytic metabolism, while monitoring [Ca2+]i, oxygen consumption and secretion. Glyceraldehyde gave rise to slow oscillations only when substimulatory glucose was also added to the media. Glucosamine, an inhibitor of glucokinase, blocked these slow oscillations. KIC, pyruvate, and methyl pyruvate did not give rise to slow oscillations alone or with glucose present. The addition of glucose to islets bathed in nutrient-rich cell culture media accelerated metabolism and initiated slow oscillations while glyceraldehyde did not. It is concluded that glucose has a special role in accelerating metabolism and generating slow oscillations in isolated islets of Langerhans from mice. Combined with previous observations of Ca2+ dependency for all oscillations in islets, we propose that interactions between Ca2+ influx and glycolysis are responsible for the slow oscillations. In contrast, fast oscillations can occur independent of glycolytic flux.     

Proinsulin biosynthesis in broken-cell preparations of islets of Langerhans.

1. Rabbit islets of Langerhans were disrupted by ultrasonic methods and the sonicated preparations were used to study proinsulin biosynthesis. 2. When [3h]leucine is incubated in such preparations, incorporation takes place into proinsulin, as evidenced by characterization on polyacrylamide gels, and by the conversion of this labelled material into insulin, by using trypsin. 3. The labelled proinsulin may also be purified by antiinsulin antibody bound to Sepharose. 4. With the broken-cell preparation it was shown that incorporation of leucine is accelerated by increasing the glucose content of the medium from 2mM to 16mM. However, 16mM-galactose or -sucrose did not stimulate incorporation significantly from basal values. This effect of glucose was abolished by cycloheximide. 5. The significance of these findings in relation to the mechanism of glucose stimulation of proinsulin biosynthesis is discussed.     

Regulation of actin polymerizaton in rat islets of Langerhans.

The rate of protein synthesis was assessed in liver, stomach, small and large intestine and in the whole body of rats by injection of 100 mumol of [14C]leucine/100 g body wt. In each of the tissues turnover was very rapid, so that taken together they accounted for 43% of the protein synthesized by the whole animal.     

Insulin secretion and protein phosphorylation in PKC-depleted islets of Langerhans.

Protein kinase C (PKC)-dependent phosphorylation of endogenous substrates was measured in electrically permeabilised rat islets of Langerhans. The PKC-activating phorbol ester, 4 beta-phorbol myristate acetate (PMA), caused a slow but prolonged increase in insulin secretion from permeabilised islets, which was accompanied by increased 32P incorporation into several islet proteins of apparent M.W. 30-50 kDa. Depletion of islet PKC by prolonged exposure to PMA abolished subsequent secretory and phosphorylating responses to the phorbol ester. However, PKC-depleted islets did not show diminished responses to glucose, suggesting that PKC-mediated phosphorylation of these proteins is not essential for nutrient-induced insulin secretion.