A minimum of fuel is necessary for tolbutamide to mimic the effects of glucose on electrical activity in pancreatic beta-cells

Glucose stimulation of pancreatic beta-cells triggers electrical activity (slow waves of membrane potential with superimposed spikes) that is best monitored with intracellular microelectrodes. Closure of ATP-sensitive K+ channels underlies the depolarization to the threshold potential and participates in the increase in electrical activity produced by suprathreshold (>7 mM) concentrations of glucose, but it is still unclear whether this is the sole mechanism of control. This was investigated by testing whether blockade of ATP-sensitive K+ channels by low concentrations of tolbutamide is able to mimic the effects of glucose on mouse beta-cell electrical activity even in the absence of the sugar. The response to tolbutamide was influenced by the duration of the perifusion with the low glucose medium. Tolbutamide (25 microM) caused a rapid and sustained depolarization with continuous activity after 6 min of perifusion of the islet with 3 mM glucose, and a progressive depolarization with slow waves of the membrane potential after 20 min. In the absence of glucose, the beta-cell response to tolbutamide was a transient phase of depolarization with rare slow waves (6 min) or a silent, small, but sustained, depolarization (20 min). Readministration of 3 mM glucose was sufficient to restore slow waves, whereas an increase in the glucose concentration to 5 and 7 mM was followed by a lengthening of the slow waves and a shortening of the intervals. In contrast, induction of slow waves by tolbutamide proved very difficult in the absence of glucose, because the beta-cell membrane tended to depolarize from a silent level to the plateau level, at which electrical activity is continuous. Azide, a mitochondrial poison, abrogated the electrical activity induced by tolbutamide in the absence of glucose, which demonstrates the influence of the metabolism of endogenous fuels on the response to the sulfonylurea. The partial repolarization that azide also produced was reversed by increasing the concentration of tolbutamide, but reappearance of the spikes required the addition of glucose. It is concluded that inhibition of ATP-sensitive K+ channels is not the only mechanism by which glucose controls electrical activity in beta-cells.     

Inhibition of the ATP-sensitive potassium channel by class I antiarrhythmic agent, cibenzoline, in rat pancreatic beta-cells

1. Cibenzoline, a class I antiarrhythmic agent, was investigated for its effect on the ATP-sensitive K+ channel of pancreatic beta-cells by the patch clamp technique. 2. In perforated patch clamp experiments, cibenzoline depolarized the membrane of single beta-cells and thereafter, caused firing of action potentials in the presence of 2.8 mM glucose. 3. Cibenzoline inhibited the activity of the ATP-sensitive K+ channel in cell-attached recordings in the presence of 2.8 mM glucose and evoked repetitive fluctuations of the baseline current, apparently reflecting the action potentials of the beta-cell. 4. In whole-cell clamp experiments, time-independent outward current was induced by depleting cytoplasmic ATP with 0.1 mM ATP and 0.1 mM ADP in the solution contained in the pipette. The outward current was inhibited by cibenzoline in a dose-dependent manner in the concentration range of 1 microM to 100 microM and half maximum inhibition occurred at 1.5 microM. 5. Cibenzoline blocked substantially the ATP-sensitive K+ channel current when applied at the inner side of the membrane in isolated inside-out membrane patches. 6. It is concluded that cibenzoline blocks the ATP-sensitive K+ channel of pancreatic beta-cells and, thereby, stimulates insulin secretion at sub-stimulatory levels of glucose.     

Altered bcl-2 and bax expression and intracellular Ca2+ signaling in apoptosis of pancreatic cells and the impairment of glucose-induced insulin secretion

Apoptosis is the process of cellular self-destruction, and genes such as bcl-2 and bax are known to inhibit and promote apoptosis, respectively. In this study, we show that apoptosis can be induced in pancreatic beta-cell lines, and we investigate the apoptotic pathways through the bcl-2 and bax genes and intracellular Ca2+. Serum deprivation induces apoptosis in the MIN6 and RINm5F pancreatic beta-cell lines, and alters the bcl-2 messenger RNA (mRNA) and protein. KCl, BayK, A23187, and ionomycin elicit an elevation of cytosolic/nuclear Ca2+, which, however, is insufficient to evoke apoptosis or to alter bcl-2 or bax mRNA expression in MIN6 cells. The extracellular Ca2+ chelators, EGTA and 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetrapotassium salt, hydrate, evoke apoptosis and also alter the ratio of bcl-2 to bax mRNA and protein concomitantly with the depletion of cytosolic/nuclear Ca2+. This indicates that there are at least two apoptotic pathways in pancreatic beta-cells: through serum deprivation and through a decrease in cytosolic/nuclear Ca2+. MIN6 cells exhibit reduced insulin secretion induced by glucose regardless of the molecular pathway of apoptosis. Apoptosis in pancreatic beta-cells, therefore, may be closely related to the impairment of insulin secretion in certain pathological conditions such as diabetes mellitus.     

Insulin exocytosis and glucose-mediated increase in cytoplasmic free Ca2+ concentration in the pancreatic beta-cell are independent of cyclic ADP-ribose

Stimulation of pancreatic beta-cells by glucose gives rise to an increase in the cytoplasmic free calcium concentration ([Ca2+]i) and exocytosis of insulin. Cyclic adenosine 5'-diphosphate ribose (cADPR), a metabolite of beta-NAD+, has been reported to increase [Ca2+]i in pancreatic beta-cells by releasing Ca2+ from inositol 1,4,5-trisphosphate-insensitive intracellular stores. In the present study, we have examined the role of cADPR in glucose-mediated increases in [Ca2+]i and insulin exocytosis. Dispersed ob/ob mouse beta-cell aggregates were either pressure microinjected with fura-2 salt or loaded with fura-2 acetoxymethyl ester, and [Ca2+]i was monitored by microfluorimetry. Microinjection of beta-NAD+ into fura-2-loaded beta-cells did not increase [Ca2+]i nor did it alter the cells' subsequent [Ca2+]i response to glucose. Cells microinjected with the cADPR antagonist 8NH2-cADPR increased [Ca2+]i in response to glucose equally well as those injected with cADPR. Finally, the ability of cADPR to promote exocytosis of insulin in electropermeabilized beta-cells was investigated. cADPR on its own did not increase insulin secretion nor did it potentiate Ca2+-induced insulin secretion. We conclude that cADPR neither plays a significant role in glucose-mediated increases in [Ca2+]i nor interacts directly with the molecular mechanisms regulating exocytosis of insulin in normal pancreatic beta-cells.     

Microenvironmental factors regulate Ly-6 A/E expression on PyV-transformed BALB/c 3T3 cells

Ouabain-induced changes of the free cytoplasmic Na+ concentration ([Na+]i) were monitored in aggregates of cells prepared from beta-cell-rich pancreatic mouse islets and the results were compared with the total islet content of sodium. The steady-state [Na+]i was lower in 20 mM glucose (11 mM) than in 3 mM glucose (14 mM). In the presence of 3 mM glucose the addition of 1 mM ouabain resulted in a rise in [Na+]i with an initial rate of 1.5 mM/min. However, the increase of total sodium corresponded to 2.8 mM/min, suggesting that rapid binding and/or sequestration of Na+ are prominent features for pancreatic beta-cells. Elevation of the glucose concentration to 20 mM increased the rate of ouabain-dependent rise of [Na+]i. The effect of glucose was mimicked by 1 mM tolbutamide or 100 microM carbachol and was counteracted by 100 nM of the alpha 2-adrenergic agonist clonidine. Glucose also accelerated the lowering of [Na+]i after withdrawal of ouabain. In promoting not only the entry but also the extrusion of Na+, glucose actually enhances the turnover of the ion in pancreatic beta-cells.     

The novel insulinotropic mechanism of pimobendan: direct enhancement of the exocytotic process of insulin secretory granules by increased Ca2+ sensitivity in beta-cells

Pimobendan is a new class of inotropic drug that augments Ca2+ sensitivity and inhibits phosphodiesterase (PDE) activity in cardiomyocytes. To examine the insulinotropic effect of pimobendan in pancreatic beta-cells, which have an intracellular signaling mechanism similar to that of cardiomyocytes, we measured insulin release from rat isolated islets of Langerhans. Pimobendan augmented glucose-induced insulin release in a dose-dependent manner, but did not increase cAMP content in pancreatic islets, indicating that the PDE inhibitory effects may not be important in beta-cells. This agent increased the intracellular Ca2+ concentration ([Ca2+]i) in the presence of 30 mM K+, 16.7 mM glucose, and 200 microM diazoxide, but failed to enhance the 30 mM K+-evoked [Ca2+]i rise in the presence of 3.3 mM glucose. Insulin release evoked by 30 mM K+ in 3.3 mM glucose was augmented. Then, the direct effects of pimobendan on the Ca2+-sensitive exocytotic apparatus were examined using electrically permeabilized islets in which [Ca2+]i can be manipulated. Pimobendan (50 microM) significantly augmented insulin release at 0.32 microM Ca2+, and a lower threshold for Ca2+-induced insulin release was apparent in pimobendan-treated islets. Moreover, 1 microM KN93 (Ca2+/calmodulin-dependent protein kinase II inhibitor) significantly suppressed this augmentation. Pimobendan, therefore, enhances insulin release by directly sensitizing the intracellular Ca2+-sensitive exocytotic mechanism distal to the [Ca2+]i rise. In addition, Ca2+/calmodulin-dependent protein kinase II activation may at least in part be involved in this Ca2+ sensitization for exocytosis of insulin secretory granules.     

Studies on the microsomal cytochrome P450s in the livers of carcinogen-resistant and sensitive rats during long-term administration of 3''-methyl-4-dimethylaminoazobenzene

The fluorescent indicator Fura-2 was used to characterize the store-operated Ca2+ entry in insulin-releasing pancreatic beta-cells. To avoid interference with voltage-dependent Ca2+ entry, the cells were hyperpolarized with 400 microM diazoxide and the channel blocker methoxyverapamil was also present in some experiments. The cytoplasmic Ca2+ concentration ([Ca2+]j) of hyperpolarized mouse beta-cells was strikingly resistant to changes in external Ca2+. In cells exposed to 20 mM glucose, stimulation with 100 microM carbachol induced an initial [Ca2+]j peak followed by a sustained increase due to store-operated influx of the cation. Store-operated influx was also induced by the intracellular Ca(2+)-ATPase inhibitor thapsigargin. In the presence of store-operated influx, [Ca2+]j became markedly sensitive to variations in external Ca2+, but this sensitivity was blocked by La3+. In beta-cells exposed to both Ca2+ and Mn2+ there was slow Mn2+ quenching of the Fura-2 fluorescence, which was accelerated upon stimulation of store-operated influx. This acceleration was reversed by glucose-stimulated filling of the internal Ca2+ stores. The store-operated Ca2+ entry increased markedly during culture of the beta-cells. Activation of protein kinase C by the phorbol ester 12-O-tetradecanoylphorbol-13 acetate, inhibition of serine/threonine phosphatase by okadaic acid and inhibition of tyrosine kinase by genistein had little effect on the store-operated influx of Ca2+. In beta-cells equilibrated in 5 mM Sr2+, carbachol exposure resulted in a pronounced cytoplasmic Sr2+ ([Sr2+]j) peak due to intracellular mobilization, but little or no sustained elevation. Moreover, after activating the store-operated pathway by exposure to thapsigargin, variations in extracellular Sr2+ between 0-2 mM had only marginal effects on [Sr2+]j. Although the store-operated influx apparently accounts for a minor fraction of the Ca2+ entry, its depolarizing influence may under certain conditions be up-regulated with resulting distortion of the beta-cell function.     

Direct effects of diazoxide on mitochondria in pancreatic B-cells and on isolated liver mitochondria

1. The direct effects of diazoxide on mitochondrial membrane potential, Ca2+ transport, oxygen consumption and ATP generation were investigated in mouse pancreatic B-cells and rat liver mitochondria. 2. Diazoxide, at concentrations commonly used to open adenosine 5'-triphosphate (ATP)-dependent K+-channels (K(ATP) channels) in pancreatic B-cells (100 to 1000 microM), decreased mitochondrial membrane potential in mouse intact perifused B-cells, as evidenced by an increase of rhodamine 123 fluorescence. This reversible decrease of membrane potential occurred at non-stimulating (5 mM) and stimulating (20 mM) glucose concentrations. 3. A decrease of mitochondrial membrane potential in perifused B-cells was also caused by pinacidil, but no effect could be seen with levcromakalim (500 microM each). 4. Measurements by a tetraphenylphosphonium-sensitive electrode of the membrane potential of rat isolated liver mitochondria confirmed that diazoxide decreased mitochondrial membrane potential by a direct action. Pretreatment with glibenclamide (2 microM) did not antagonize the effects of diazoxide. 5. In Fura 2-loaded B-cells perifused with the Ca2+ channel blocker, D 600, a moderate, reversible increase of intracellular Ca2+ concentration could be seen in response to 500 microM diazoxide. This intracellular Ca2+ mobilization may be due to mitochondrial Ca2+ release, since the reduction of membrane potential of isolated liver mitochondria by diazoxide was accompanied by an accelerated release of Ca2+ stored in the mitochondria. 6. In the presence of 500 microM diazoxide, ATP content of pancreatic islets incubated in 20 mM glucose for 30 min was significantly decreased by 29%. However, insulin secretion from mouse perifused islets induced by 40 mM K+ in the presence of 10 mM glucose was not inhibited by 500 microM diazoxide, suggesting that the energy-dependent processes of insulin secretion distal to Ca2+ influx were not affected by diazoxide at this concentration. 7. The effects of diazoxide on oxygen consumption and ATP production of liver mitochondria varied depending on the respiratory substrates (5 mM succinate, 10 mM alpha-ketoisocaproic acid, 2 mM tetramethyl phenylenediamine plus 5 mM ascorbic acid), indicating an inhibition of respiratory chain complex II. Pinacidil, but not levcromakalim, inhibited alpha-ketoisocaproic acid-fuelled ATP production. 8. In conclusion, diazoxide directly affects mitochondrial energy metabolism, which may be of relevance for stimulus-secretion coupling in pancreatic B-cells.     

Glucose decreases Na+,K+-ATPase activity in pancreatic beta-cells. An effect mediated via Ca2+-independent phospholipase A2 and protein kinase C-dependent phosphorylation of the alpha-subunit

In the pancreatic beta-cell, glucose-induced membrane depolarization promotes opening of voltage-gated L-type Ca2+ channels, an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i), and exocytosis of insulin. Inhibition of Na+,K+-ATPase activity by ouabain leads to beta-cell membrane depolarization and Ca2+ influx. Because glucose-induced beta-cell membrane depolarization cannot be attributed solely to closure of ATP-regulated K+ channels, we investigated whether glucose regulates other transport proteins, such as the Na+,K+-ATPase. Glucose inhibited Na+,K+-ATPase activity in single pancreatic islets and intact beta-cells. This effect was reversible and required glucose metabolism. The inhibitory action of glucose was blocked by pretreatment of the islets with a selective inhibitor of a Ca2+-independent phospholipase A2. Arachidonic acid, the hydrolytic product of this phospholipase A2, also inhibited Na+, K+-ATPase activity. This effect, like that of glucose, was blocked by nordihydroguaiaretic acid, a selective inhibitor of the lipooxygenase metabolic pathway, but not by inhibitors of the cyclooxygenase or cytochrome P450-monooxygenase pathways. The lipooxygenase product 12(S)-HETE (12-S-hydroxyeicosatetranoic acid) inhibited Na+,K+-ATPase activity, and this effect, as well as that of glucose, was blocked by bisindolylmaleimide, a specific protein kinase C inhibitor. Moreover, glucose increased the state of alpha-subunit phosphorylation by a protein kinase C-dependent process. These results demonstrate that glucose inhibits Na+, K+-ATPase activity in beta-cells by activating a distinct intracellular signaling network. Inhibition of Na+,K+-ATPase activity may thus be part of the mechanisms whereby glucose promotes membrane depolarization, an increase in [Ca2+]i, and thereby insulin secretion in the pancreatic beta-cell.     

Autonomic functions in restrictive cardiomyopathy and constrictive pericarditis: a comparison

1. The glucose-dependence of beta-cell electrical activity and the effects of tolbutamide and diazoxide were studied in anaesthetized mice. 2. In untreated animals there was a direct relationship between glycaemia and the burst pattern of electrical activity. Animals with high glucose concentration showed continuous electrical activity. The application of insulin led to a steady decrease in blood glucose concentration and a transition from continuous to oscillatory activity at 7.7+/-0.1 mM glucose (mean+/-s.d.) and a subsequent transition from oscillatory to silent at 4.7+/-0.6 mM glucose. 3. At physiological blood glucose concentrations the electrical activity was oscillatory. The injection of tolbutamide (1800 mg kg[-1]) transformed this oscillatory pattern into one of continuous electrical activity. The increased electrical activity was associated with a decrease in blood glucose concentration from 7.1+/-0.9 (control) to 5.5+/-1.0 mM (10 min after tolbutamide injection). The effects of tolbutamide are consistent with a direct blocking effect on the K(ATP) channel that leads to membrane depolarization. 4. The injection of diazoxide (6000 mg kg[-1]) hyperpolarized the cells and transformed the oscillatory pattern into a silent one. This is consistent with a direct stimulant effect by diazoxide on the K(ATP) channel. The use of tolbutamide or diazoxide correspondingly led to the lengthening or shortening of the active phase of electrical activity, respectively. This indicates that in vivo, such activity can be modulated by the relative degree of activation or inhibition of the K(ATP) channel. 5. These results indicate that under physiological conditions, tolbutamide and diazoxide have direct and opposite effects on the electrical activity of pancreatic beta-cells, most likely through their action on K(ATP) channels. This is consistent with previous work carried out on in vitro models and explains the drugs hypo- and hyperglycaemic effects.     

Mediation by intracellular calcium-dependent signals of hypoxic hyperpolarization in rat hippocampal CA1 neurons in vitro

In response to oxygen deprivation, CA1 pyramidal neurons show a hyperpolarization (hypoxic hyperpolarization), which is associated with a reduction in neuronal input resistance. The role of extra- and intracellular Ca2+ ions in hypoxic hyperpolarization was investigated. The hypoxic hyperpolarization was significantly depressed by tolbutamide (100 microM); moreover, the response was reversed in its polarity in medium containing tolbutamide (100 microM), low Ca2+ (0.25 mM), and Co2+ (2 mM), suggesting that the hypoxic hyperpolarization is mediated by activation of both ATP-sensitive K+ (KATP) channels and Ca(2+)-dependent K+ channels. The hypoxic depolarization in medium containing tolbutamide, low Ca2+, and Co2+ is probably due to inhibition of the electrogenic Na(+)-K+ pump and concomitant accumulation of interstitial K+. Hypoxic hyperpolarizations were depressed in either low Ca2+ (0.25 or 1.25 mM) or high Ca2+ (5 or 7.5 mM) medium (control: 2.5 mM), indicating that there is an optimal extracellular Ca2+ concentration required to produce the hypoxic hyperpolarization. Bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA)-AM (50-100 microM), procaine (300 microM), or ryanodine (10 microM) significantly depressed the hypoxic hyperpolarization, suggesting that Ca2+ released from intracellular Ca+ stores may have an important role in the generation of hypoxic hyperpolarization. The high-affinity calmodulin inhibitor N-(6-amino-hexyl)-5-chloro-1-naphthalenesulfonomide hydrochloride (W-7) (5 microM) completely blocked, whereas the low-affinity calmodulin inhibitor N-(6-aminohexyl)-1-naphthalenesulfonomide hydrochloride (W-5) (50 microM) did not affect, the hypoxic hyperpolarization. The calmodulin inhibitor trifluoperazine (50 microM) also suppressed the hypoxic hyperpolarization. In addition, calcium/ calmodulin kinase II inhibitor 1-[N,O-bis (1,5-isoquinol-inesulfonyl)-N-methyl-L-tyrosyl]-4-phenyl-pip erazine (KN-62) (10 microM) markedly depressed the amplitude and net outward current of the hypoxic hyperpolarization without affecting the reversal potential. In contrast, neither the myosin light chain kinase inhibitor 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexa-hydro-1,4-diazepin hydrochloride (ML-7) (10 microM) nor the protein kinase A inhibitor N-[2-(p-bromocinnamyl-amino) ethyl]-5-isoquinolinesulfonamide (H-89) (1 microM) significantly altered the hypoxic hyperpolarization. These results suggest that calmodulin kinase II, which is activated by calmodulin, may contribute to the generation of the hypoxic hyperpolarization. In conclusion, the present study indicates that, in the majority of hippocampal CA1 neurons, the hypoxic hyperpolarization is due to activation of both KATP channels and Ca(2+)-dependent K+ channels.     

Cosecretion of islet amyloid polypeptide (IAPP) and insulin from isolated rat pancreatic islets following stimulation or inhibition of beta-cell function

The aim of this work was to simultaneously study the secretion of islet amyloid polypeptide (IAPP) and insulin from isolated rat pancreatic islets in vitro. For examination of stimulated beta-cells, nutrient secretagogues (16.7 mM glucose, 10 mM leucine + 2 mM glutamine), phosphodiesterase inhibition (5 mM theophylline), a sulphonylurea (0.5 microgram/ml glipizide), a non-nutrient amino acid (10 mM arginine), cholinergic stimulation (0.1 mM carbamylcholine) and insulinotropic peptides (0.1 microM vasoactive intestinal polypeptide and 0.1 microM glucagon), were used. For beta-cell suppression glucose phosphorylation inhibition (10 mM mannoheptulose), depletion of extracellular calcium, activation of the ATP-regulated K(+)-channel (0.5 mM diazoxide), adrenoreceptor stimulation (3 microM adrenaline), paracrine modulation (0.1 microM somatostatin), short-term treatment with a selective beta-cytotoxin (1.1 and 2.2 mM streptozotocin) and long-term treatment with a cytokine (25 U/ml interleukin-1 beta), were studied. The compounds with known effects on insulin secretion exerted their expected actions and this was paralleled by similar relative changes, with a possible exception for glucagon, in the IAPP secretion. The ratio of IAPP/insulin released did not change significantly under any of the tested experimental conditions, except for a slight increase following carbamylcholine stimulation. On a molar basis approx. 1% of IAPP was released when compared with insulin. These results are consistent with the hypothesis that the regulation of IAPP secretion from beta-cells of isolated rat pancreatic islets is essentially regulated by the same mechanisms as insulin secretion.     

Jejunal varices as a cause of massive gastrointestinal bleeding--a case report

This study investigated regulatory volume increase (RVI) in rat pancreatic beta-cells. Volume changes in isolated beta-cells were measured by a video-imaging method. Cell shrinkage was induced by exposure to solutions made hypertonic by the addition of 100 mM mannitol. In HEPES-buffered solutions, beta-cells exhibited an RVI which was almost completely abolished by 10 microM bumetanide. These data indicate that Na+-2Cl--K+ cotransporters make a major contribution to RVI in beta-cells. In HCO3--buffered solutions, however, an RVI was observed in the presence of 10 microM bumetanide. This bumetanide-insensitive component of RVI was inhibited by 100 microM amiloride or 100 microM 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS). These data suggest that, in addition to the Na+-2Cl--K+ cotransporter, functionally coupled Na+-H+ exchangers and Cl--HCO3- exchangers may also contribute to RVI in pancreatic beta-cells.     

Human pancreatic beta-cell deoxyribonucleic acid-synthesis in islet grafts decreases with increasing organ donor age but increases in response to glucose stimulation in vitro

Human pancreatic beta-cell proliferation may be crucial for the success of islet transplantation. The aim of this study was to test the hypothesis that adult human beta-cells proliferate in vitro and in vivo and respond with increased rates of replication to factors known to promote rodent islet-cell proliferation, i.e. glucose, human recombinant GH, and FCS. For this purpose, human islets were prepared from a total of 19 adult heart-beating organ donors and cultured for 48 h with or without the additives described above. 3H-thymidine was added to the medium during the last 60 min of culture. After immunohistochemical staining for insulin and autoradiography, the labeling index (LI; i.e. % of labeled beta-cells over total number of beta-cells) was estimated by light microscopy. Islets also were transplanted under the kidney capsule of normal or alloxan-diabetic nude mice. After 2 weeks, 3H-thymidine was injected and the islet grafts prepared for determination of LI, as described above. Islets cultured at 5.6 mM glucose showed an increased beta-cell proliferation compared with islets cultured at 2.8 mM glucose (P < 0.05). However, culture at 11 mM glucose failed to further increase beta-cell proliferation. Addition of GH (1 microg/ml) to the medium, in the presence of 1% FCS and 5.6 mM glucose, did not influence the rate of beta-cell proliferation. In islets transplanted to hyperglycemic nude mice, beta-cell proliferation was similar to that observed in islets grafted into normoglycemic nude mice. Proliferation, however, decreased with increasing organ donor age. This study shows that pancreatic beta-cells from adult man are able to proliferate both in vitro and in vivo. Moreover, beta-cells from adult human donors respond with increased proliferation to glucose in vitro and show a decreased proliferation in vivo with increasing donor age.     

Mass spectrometric evidence that agents that cause loss of Ca2+ from intracellular compartments induce hydrolysis of arachidonic acid from pancreatic islet membrane phospholipids by a mechanism that does not require a rise in cytosolic Ca2+ concentration

Stimulation of pancreatic islets with glucose induces phospholipid hydrolysis and accumulation of nonesterified arachidonic acid, which may amplify the glucose-induced Ca2+ entry into islet beta-cells that triggers insulin secretion. Ca2+ loss from beta-cell intracellular compartments has been proposed to induce both Ca2+ entry and events dependent on arachidonate metabolism. We examine here effects of inducing Ca2+ loss from intracellular sequestration sites with ionophore A23187 and thapsigargin on arachidonate hydrolysis from islet phospholipids. A23187 induces a decline in islet arachidonate-containing phospholipids and release of nonesterified arachidonate. A23187-induced arachidonate release is of similar magnitude when islets are stimulated in Ca2+-replete or in Ca2+-free media or when islets loaded with the intracellular Ca2+ chelator BAPTA are stimulated in Ca2+-free medium, a condition in which A23187 induces no rise in beta-cell cytosolic [Ca2+]. Thapsigargin also induces islet arachidonate release under these conditions. A23187- or thapsigargin-induced arachidonate release is prevented by a bromoenol lactone (BEL) inhibitor of a beta-cell phospholipase A2 (iPLA2), which does not require Ca2+ for catalytic activity and which is negatively modulated by and physically interacts with calmodulin by Ca2+-dependent mechanisms. Agents that cause Ca2+ loss from islet intracellular compartments thus induce arachidonate hydrolysis from phospholipids by a BEL-sensitive mechanism that does not require a rise in cytosolic [Ca2+], and a BEL-sensitive enzyme-like iPLA2 or a related membranous activity may participate in sensing Ca2+ compartment content.     

Cloning and expression of a group IV cytosolic Ca2+-dependent phospholipase A2 from rat pancreatic islets. Comparison of the expressed activity with that of an islet group VI cytosolic Ca2+-independent phospholipase A2

Stimulation of pancreatic islets with glucose induces phospholipid hydrolysis and accumulation of nonesterified arachidonic acid, which may play signaling or effector roles in insulin secretion. Of enzymes that catalyze phospholipid hydrolysis, islet beta-cells express low molecular weight secretory phospholipases A2 (PLA2) and a Group VI, Ca2+-independent PLA2 (iPLA2). Previous studies indicate that islets also express a protein recognized by antibodies against a Group IV, cytosolic, Ca2+-dependent PLA2 (cPLA2). To further examine the possible expression of cPLA2 by islets, we screened a rat islet cDNA library with a probe that recognizes cPLA2 sequence, and isolated a full-length cPLA2 cDNA. The rat islet cPLA2-deduced amino acid sequence is 96% identical to those of human and mouse cPLA2. Transfection of COS-7 cells with cPLA2 cDNA in an expression vector induced expression of Ca2+-dependent PLA2 activity and of a protein recognized by anti-cPLA2 antibody. Comparison of recombinant islet cPLA2 and iPLA2 activities expressed in transfected COS-7 cells indicated that iPLA2 but not cPLA2 is stimulated by ATP. Both activities are similarly sensitive to inhibition by arachidonyltrifluoromethyl ketone, but iPLA2 is more effectively inhibited by a haloenol lactone suicide substrate than cPLA2. RT-PCR experiments with RNA from purified islet beta-cells and from an alpha-cell-enriched population prepared by fluorescence-activated cell-sorting indicated that cPLA2 mRNA is more abundant in the beta-cell population. Immunoblotting analyses indicate that islets express cPLA2-immunoreactive protein, and that interleukin-1 does not affect its expression. The cPLA2 is thus one of at least three classes of PLA2 enzymes with distinct properties expressed in beta-cells.     

A role for Ca2+-sensitive nonselective cation channels in regulating the membrane potential of pancreatic beta-cells

The incretin hormones, glucagon-like peptide 1 and pituitary adenylyl cyclase-activating polypeptide, are proposed to activate a maitotoxin (MTX)-sensitive, Ca2+-dependent nonselective cation current in pancreatic beta-cells and insulinoma cells. This MTX-sensitive current is present in human beta-cells as well as in mouse and rat beta-cells, and is accompanied by a rise in cytosolic Ca2+ in voltage-clamped cells in which the activation of voltage-dependent Ca2+ channels is prevented. Activation of the nonselective cation current is inhibited by reduction of disulfide bonds with intracellular, but not extracellular, dithiothreitol, and is also abolished by intracellular dialysis with trypsin. The nonselective cation channels that carry this current have a conductance of about 30 pS, with Na+ as the major extracellular cation. We estimate that these cation channels are expressed on beta-cells at a density similar to that of ATP-sensitive potassium channels (K(ATP) channels) and exhibit spontaneous activity at basal glucose concentrations. We propose that this spontaneous cation channel activity constitutes at least part of the depolarizing background conductance that permits changes in the activity of K(ATP) channels to regulate the resting potential of beta-cells.     

Cytoplasmic Ca2+ oscillations in pancreatic beta-cells

In the last 15 years it has been a growing interest in the cyclic variations of circulating insulin [46]. After the suggestion that this phenomenon may be due to oscillations of the beta-cell membrane potential [8,39], it was demonstrated that [Ca2+]i oscillates in the glucose-stimulated beta-cell with a similar frequency to that of pulsatile insulin release. The present review describes four types of [Ca2+]i oscillations in the pancreatic beta-cell. The slow sinusoidal oscillations, referred to as type-a, are those which most closely correspond to pulsatile insulin release. Although not affecting the properties of the type-a oscillations in individual beta-cells, the concentration of glucose is a determinant for their generation and further transformation into a sustained increase. Accordingly, cytoplasmic Ca2+ is regulated by sudden transitions between oscillatory and steady-state levels at threshold concentrations of glucose, which are characteristic for the individual beta-cell. This behaviour explains the observation of a gradual recruitment of previously non-secreting cells with increase of the extracellular glucose concentration [44]. However, it still remains to be elucidated how the sudden transitions between these three states translate into the co-ordinated slow oscillations of [Ca2+]i in the intact islet. Cyclic variations of circulating insulin require a synchronization of the [Ca2+]i cycles also among the islets in the pancreas. It is still an open question by which means the millions of islets communicate mutually to establish a pattern of pulsatile insulin release from the whole pancreas. The discovery that the beta-cell is not only the functional unit for insulin synthesis but also generates the [Ca2+]i oscillations required for pulsatile insulin release has both physiological and clinical implications. The fact that minor damage to the beta-cells prevents the type-a oscillations with maintenance of a glucose response in terms of raised [Ca2+]i reinforces previous arguments [54] that loss of insulin oscillations is an early indicator of type-2 diabetes. Further analyses of the [Ca2+]i oscillations in the beta-cells should include not only the mechanisms for their generation and subsequent propagation within or among the islets but also how modulation of their frequency affects the insulin sensitivity of various target cells. The latter approach may be important in the attempts to maintain normoglycemia under conditions minimizing the vascular effects of insulin supposed to precipitate hypertonia and atherosclerosis [70,71,77].     

Intracellular glucose switches between cyclic ADP-ribose and inositol trisphosphate triggering of cytosolic Ca2+ spiking

Cyclic ADP-ribose (cADPR) is a potentially important intracellular Ca2+ releasing messenger [1-5]. In pancreatic acinar cells where intracellular infusion of both inositol trisphosphate (IP3) and cADPR evoke repetitive Ca2+ spiking [6], the cADPR antagonist 8-NH2-cADPR [7], which blocks cADPR-evoked but not IP3-evoked Ca2+ spiking, can abolish Ca2+ spiking induced by physiological levels of the peptide hormone cholecystokinin (CCK) [8]. We have tested the effect of intracellular glucose on the ability of IP3, cADPR and CCK to induce cytosolic Ca2+ spikes in pancreatic acinar cells. In order to gain access to the intracellular cytosol, we used the whole-cell configuration of the patch-clamp technique [9] and monitored cytosolic Ca2+ concentration changes by measuring the Ca(2+)-dependent ionic current [10-13]. Glucose (300 microM to 10 mM) in the patch pipette/intracellular solution prevented cADPR from evoking Ca2+ spiking. The same effect was observed with 2-deoxy-glucose, but not L-glucose. In contrast, glucose potentiated IP3-evoked Ca2+ spiking. CCK evoked Ca2+ spiking irrespective of the presence or absence of intracellular glucose, but the cADPR antagonist 8-NH2-cADPR blocked CCK-evoked Ca2+ spiking only in the absence of intracellular glucose. This suggests that the hormone can evoke Ca2+ spiking via either the IP3 or the cADPR pathway. The intracellular glucose level may control a switch between these two pathways.