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J C Henquin

Publications and source records attributed to J C Henquin.

At least 19 recordsLinked to original sources
PubMed2001

Inhibition of protein synthesis sequentially impairs distinct steps of stimulus-secretion coupling in pancreatic beta cells.

Proteins with a short half-life are potential sites of pancreatic ss cell dysfunction under pathophysiological conditions. In this study, mouse islets were used to establish which step in the regulation of insulin secretion is most sensitive to inhibition of protein synthesis by 10 microM cycloheximide (CHX). Although islet protein synthesis was inhibited approximately 95% after 1 h, the inhibition of insulin secretion was delayed and progressive. After long (18-20 h) CHX-treatment, the strong (80%) inhibition of glucose-, tolbutamide-, and K(+)-induced insulin secretion was not due to lower insulin stores, to any marked impairment of glucose metabolism or to altered function of K(+)-ATP channels (total K(+)-ATP currents were however decreased). It was partly caused by a decreased Ca(2+) influx (whole-cell Ca(2+) current) resulting in a smaller rise in cytosolic Ca(2+) ([Ca(2+)](i)). The situation was very different after short (2-5 h) CHX-treatment. Insulin secretion was 50-60% inhibited although islet glucose metabolism was unaffected and stimulus-induced [Ca(2+)](i) rise was not (2 h) or only marginally (5 h) decreased. The efficiency of Ca(2+) on secretion was thus impaired. The inhibition of insulin secretion by 15 h of CHX treatment was more slowly reversible (>4 h) than that of protein synthesis. This reversibility of secretion was largely attributable to recovery of a normal Ca(2+) efficiency. In conclusion, inhibition of protein synthesis in islets inhibits insulin secretion in two stages: a rapid decrease in the efficiency of Ca(2+) on exocytosis, followed by a decrease in the Ca(2+) signal mediated by a slower loss of functional Ca(2+) channels. Glucose metabolism and the regulation of K(+)-ATP channels are more resistant. Proteins with a short half-life appear to be important to ensure optimal Ca(2+) effects on exocytosis, and are the potential Achille's heel of stimulus-secretion coupling.

Animals↗
PubMed2001

Measurements of cytoplasmic Ca2+ in islet cell clusters show that glucose rapidly recruits beta-cells and gradually increases the individual cell response.

The proportion of isolated single beta-cells developing a metabolic, biosynthetic, or secretory response increases with glucose concentration (recruitment). It is unclear whether recruitment persists in situ when beta-cells are coupled. We therefore measured the cytoplasmic free Ca2+ correction ([Ca2+]i) (the triggering signal of glucose-induced insulin secretion) in mouse islet single cells or clusters cultured for 1-2 days. In single cells, the threshold glucose concentration ranged between 6 and 10 mmol/l, at which concentration a maximum of approximately 65% responsive cells was reached. Only 13% of the cells did not respond to glucose plus tolbutamide. The proportion of clusters showing a [Ca2+]i rise increased from approximately 20 to 95% between 6 and 10 mmol/l glucose, indicating that the threshold sensitivity to glucose differs between clusters. Within responsive clusters, 75% of the cells were active at 6 mmol/l glucose and 95-100% at 8-10 mmol/l glucose, indicating that individual cell recruitment is not prominent within clusters; in clusters responding to glucose, all or almost all cells participated in the response. Independently of cell recruitment, glucose gradually augmented the magnitude of the average [Ca2+]i rise in individual cells, whether isolated or associated in clusters. When insulin secretion was measured simultaneously with [Ca2+]i, a good temporal and quantitative correlation was found between both events. However, beta-cell recruitment was maximal at 10 mmol/l glucose, whereas insulin secretion increased up to 15-20 mmol/l glucose. In conclusion, beta-cell recruitment by glucose can occur at the stage of the [Ca2+]i response. However, this type of recruitment is restricted to a narrow range of glucose concentrations, particularly when beta-cell association decreases the heterogeneity of the responses. Glucose-induced insulin secretion by islets, therefore, cannot entirely be ascribed to recruitment of beta-cells to generate a [Ca2+]i response. Modulation of the amplitude of the [Ca2+]i response and of the action of Ca2+ on exocytosis (amplifying actions of glucose) may be more important.

Animals↗
PubMed2000

The oscillatory behavior of pancreatic islets from mice with mitochondrial glycerol-3-phosphate dehydrogenase knockout.

Glucose stimulation of pancreatic beta cells induces oscillations of the membrane potential, cytosolic Ca(2+) ([Ca(2+)](i)), and insulin secretion. Each of these events depends on glucose metabolism. Both intrinsic oscillations of metabolism and repetitive activation of mitochondrial dehydrogenases by Ca(2+) have been suggested to be decisive for this oscillatory behavior. Among these dehydrogenases, mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH), the key enzyme of the glycerol phosphate NADH shuttle, is activated by cytosolic [Ca(2+)](i). In the present study, we compared different types of oscillations in beta cells from wild-type and mGPDH(-/-) mice. In clusters of 5-30 islet cells and in intact islets, 15 mM glucose induced an initial drop of [Ca(2+)](i), followed by an increase in three phases: a marked initial rise, a partial decrease with rapid oscillations and eventually large and slow oscillations. These changes, in particular the frequency of the oscillations and the magnitude of the [Ca(2+)] rise, were similar in wild-type and mGPDH(-/-) mice. Glucose-induced electrical activity (oscillations of the membrane potential with bursts of action potentials) was not altered in mGPDH(-/-) beta cells. In single islets from either type of mouse, insulin secretion strictly followed the changes in [Ca(2+)](i) during imposed oscillations induced by pulses of high K(+) or glucose and during the biphasic elevation induced by sustained stimulation with glucose. An imposed and controlled rise of [Ca(2+)](i) in beta cells similarly increased NAD(P)H fluorescence in control and mGDPH(-/-) islets. Inhibition of the malate-aspartate NADH shuttle with aminooxyacetate only had minor effects in control islets but abolished the electrical, [Ca(2+)](i) and secretory responses in mGPDH(-/-) islets. The results show that the two distinct NADH shuttles play an important but at least partially redundant role in glucose-induced insulin secretion. The oscillatory behavior of beta cells does not depend on the functioning of mGPDH and on metabolic oscillations that would be generated by cyclic activation of this enzyme by Ca(2+).

Aminooxyacetic Acid↗
PubMed2000

Triggering and amplifying pathways of regulation of insulin secretion by glucose.

Glucose stimulates insulin secretion by generating triggering and amplifying signals in beta-cells. The triggering pathway is well characterized. It involves the following sequence of events: entry of glucose by facilitated diffusion, metabolism of glucose by oxidative glycolysis, rise in the ATP-to-ADP ratio, closure of ATP-sensitive K+ (KATP) channels, membrane depolarization, opening of voltage-operated Ca2+ channels, Ca2+ influx, rise in cytoplasmic free Ca2+ concentration ([Ca2+]i), and activation of the exocytotic machinery. The amplifying pathway can be studied when beta-cell [Ca2+]i is elevated and clamped by a depolarization with either a high concentration of sulfonylurea or a high concentration of K+ in the presence of diazoxide (K(ATP) channels are then respectively blocked or held open). Under these conditions, glucose still increases insulin secretion in a concentration-dependent manner. This increase in secretion is highly sensitive to glucose (produced by as little as 1-6 mmol/l glucose), requires glucose metabolism, is independent of activation of protein kinases A and C, and does not seem to implicate long-chain acyl-CoAs. Changes in adenine nucleotides may be involved. The amplification consists of an increase in efficacy of Ca2+ on exocytosis of insulin granules. There exists a clear hierarchy between both pathways. The triggering pathway predominates over the amplifying pathway, which remains functionally silent as long as [Ca2+]i has not been raised by the first pathway; i.e., as long as glucose has not reached its threshold concentration. The alteration of this hierarchy by long-acting sulfonylureas or genetic inactivation of K(ATP) channels may lead to inappropriate insulin secretion at low glucose. The amplifying pathway serves to optimize the secretory response not only to glucose but also to nonglucose stimuli. It is impaired in beta-cells of animal models of type 2 diabetes, and indirect evidence suggests that it is altered in beta-cells of type 2 diabetic patients. Besides the available drugs that act on K(ATP) channels and increase the triggering signal, novel drugs that correct a deficient amplifying pathway would be useful to restore adequate insulin secretion in type 2 diabetic patients.

Adenosine Triphosphate↗
PubMed1999

Influence of cell number on the characteristics and synchrony of Ca2+ oscillations in clusters of mouse pancreatic islet cells.

1. The cytoplasmic Ca2+ concentration ([Ca2+]i) was measured in single cells and cell clusters of different sizes prepared from mouse pancreatic islets. 2. During stimulation with 15 mM glucose, 20 % of isolated cells were inert, whereas 80 % showed [Ca2+]i oscillations of variable amplitude, duration and frequency. Spectral analysis identified a major frequency of 0.14 min-1 and a less prominent one of 0.27 min-1. 3. In contrast, practically all clusters (2-50 cells) responded to glucose, and no inert cells were identified within the clusters. As compared to single cells, mean [Ca2+]i was more elevated, [Ca2+]i oscillations were more regular and their major frequency was slightly higher (but reached a plateau at approximately 0.25 min-1). In some cells and clusters, faster oscillations occurred on top of the slow ones, between them or randomly. 4. Image analysis revealed that the regular [Ca2+]i oscillations were well synchronized between all cells of the clusters. Even when the Ca2+ response was irregular, slow and fast [Ca2+]i oscillations induced by glucose were also synchronous in all cells. 5. In contrast, [Ca2+]i oscillations resulting from mobilization of intracellular Ca2+ by acetylcholine were restricted to certain cells only and were not synchronized. 6. Heptanol and 18alpha-glycyrrhetinic acid, two agents widely used to block gap junctions, altered glucose-induced Ca2+ oscillations, but control experiments showed that they also exerted effects other than a selective uncoupling of the cells. 7. The results support theoretical models predicting an increased regularity of glucose-dependent oscillatory events in clusters as compared to isolated islet cells, but contradict the proposal that the frequency of the oscillations increases with the number of coupled cells. Islet cell clusters function better as electrical than biochemical syncytia. This may explain the co-ordination of [Ca2+]i oscillations driven by depolarization-dependent Ca2+ influx during glucose stimulation.

Animals↗
PubMed1999

Unbound rather than total concentration and saturation rather than unsaturation determine the potency of fatty acids on insulin secretion.

Isolated mouse islets were used to compare the effects of three saturated (myristate, palmitate and stearate) and three unsaturated (oleate, linoleate and linolenate) long-chain fatty acids on insulin secretion. By varying the concentrations of fatty acid (250-1250 micromol/l) and albumin simultaneously or independently, we also investigated whether the insulinotropic effect is determined by the unbound or total concentration of the fatty acids. Only palmitate and stearate slightly increased basal insulin secretion (3 mmol/l glucose). All tested fatty acids potentiated glucose-induced insulin secretion (10-15 mmol/l), and the following rank order of potency was obtained when they were compared at the same total concentrations: palmitate approximately = stearate > myristate > or = oleate > or = linoleate approximately = linolenate. The effect of a given fatty acid varied with the fatty acid to albumin molar ratio, in a way which indicated that the unbound fraction is the important one for the stimulation of beta cells. When the potentiation of insulin secretion was expressed as a function of the unbound concentrations, the following rank order emerged: palmitate > myristate > stearate approximately = oleate > linoleate approximately = linolenate. In conclusion, the acute and direct effects of long-chain fatty acids on insulin secretion are due to their unbound fraction. They are observed only at fatty acid/albumin ratios higher than those normally occurring in plasma. Saturated fatty acids are stronger insulin secretagogues than unsaturated fatty acids. Unbound palmitate is by far the most potent of the six common long-chain fatty acids.

Animals↗
PubMed1999

Uptake and release of Ca2+ by the endoplasmic reticulum contribute to the oscillations of the cytosolic Ca2+ concentration triggered by Ca2+ influx in the electrically excitable pancreatic B-cell.

The role of intracellular Ca2+ pools in oscillations of the cytosolic Ca2+ concentration ([Ca2+]c) triggered by Ca2+ influx was investigated in mouse pancreatic B-cells. [Ca2+]c oscillations occurring spontaneously during glucose stimulation or repetitively induced by pulses of high K+ (in the presence of diazoxide) were characterized by a descending phase in two components. A rapid decrease in [Ca2+]c coincided with closure of voltage-dependent Ca2+ channels and was followed by a slower phase independent of Ca2+ influx. Blocking the SERCA pump with thapsigargin or cyclopiazonic acid accelerated the rising phase of [Ca2+]c oscillations and increased their amplitude, which suggests that the endoplasmic reticulum (ER) rapidly takes up Ca2+. It also suppressed the slow [Ca2+]c recovery phase, which indicates that this phase corresponds to the slow release of Ca2+ that was taken up by the ER during the upstroke of the [Ca2+]c transient. Glucose promoted the buffering capacity of the ER and amplified the slow [Ca2+]c recovery phase. The slow phase induced by high K+ pulses was not affected by modulators of Ca2+- or inositol 1,4,5-trisphosphate-induced Ca2+ release, did not involve a depolarization-induced Ca2+ release, and was also observed at the end of a rapid rise in [Ca2+]c triggered from caged Ca2+. It is attributed to passive leakage of Ca2+ from the ER. We suggest that the ER displays oscillations of the Ca2+ concentration ([Ca2+]ER) concomitant and parallel to [Ca2+]c. The observation that thapsigargin depolarizes the membrane of B-cells supports the proposal that the degree of Ca2+ filling of the ER modulates the membrane potential. Therefore, [Ca2+]ER oscillations occurring during glucose stimulation are likely to influence the bursting behavior of B-cells and eventually [Ca2+]c oscillations.

Animals↗
PubMed1999

Alterations of insulin secretion from mouse islets treated with sulphonylureas: perturbations of Ca2+ regulation prevail over changes in insulin content.

1. To determine how pretreatment with sulphonylureas alters the beta cell function, mouse islets were cultured (18 - 20 h) without (controls) or with (test) 0.01 microM glibenclamide. Acute responses to glucose were then determined in the absence of glibenclamide. 2. Test islets were insensitive to drugs (sulphonylureas and diazoxide) acting on K+-ATP channels, and their [Ca2+]i was already elevated in the absence of stimulation. 3. Insulin secretion was increased in the absence of glucose, and mainly stimulated between 0 - 10 instead of 7 - 20 mM glucose in controls. The maximum response was halved, but this difference disappeared after correction for the 45% decrease in the islet insulin content. 4. The first phase of glucose-induced insulin secretion was abrogated because of a paradoxical decrease of the high basal [Ca2+]i in beta cells. The second phase was preserved but occurred with little rise of [Ca2+]i. These abnormalities did not result from alterations of glucose metabolism (NADPH fluorescence). 5. In islets cultured with 50 microM tolbutamide, glucose induced biphasic increases in [Ca2+]i and insulin secretion. The decrease in the secretory response was matched by the decrease in insulin content (45%) except at maximal glucose concentrations. Islets pretreated with tolbutamide, however, behaved like those cultured with glibenclamide if tolbutamide was also present during the acute functional tests. 6. In conclusion, treatment with a low glibenclamide concentration causes long-lasting blockade of K+-ATP channels and rise of [Ca2+]i in beta cells. Glucose-induced insulin secretion occurs at lower concentrations, is delayed and is largely mediated by a modulation of Ca2+ action on exocytosis. It is suggested that glucose regulation of insulin secretion mainly depends on a K+-ATP channel-independent pathway during in vivo sulphonylurea treatment.

Adenosine Triphosphate↗
PubMed1999

Glucose regulation of insulin secretion independent of the opening or closure of adenosine triphosphate-sensitive K+ channels in beta cells.

Two major pathways are implicated in the stimulation of insulin secretion by glucose. The K+-ATP channel-dependent pathway involves closure of these channels, depolarization of the beta-cell membrane, acceleration of Ca2+ influx, and a rise in cytosolic free Ca2+ ([Ca2+]i). The K+-ATP channel-independent pathway potentiates the stimulation of exocytosis by high [Ca2+]i. To determine whether this second pathway is influenced by the configuration of the channel, we compared the effects of glucose on [Ca2+]i and insulin secretion in mouse islets under three conditions. First, in the presence of 20, 25, and 30 mM K+, i.e. without pharmacological action on K+-ATP channels, [Ca2+]i and insulin secretion were already elevated at 3 mM glucose. High glucose (20 mM) caused a transient decrease in [Ca2+]i followed by an ascent to slightly above control levels, and rapidly stimulated insulin secretion. Second, opening of K+-ATP channels with diazoxide did not influence [Ca2+]i and insulin secretion at 3 mM glucose and high K+. However, high glucose now caused a sustained lowering of [Ca2+]i accompanied by a slow increase in secretion that augmented with the K+ concentration. Third, when K+-ATP channels were blocked and beta-cells depolarized by high concentrations of tolbutamide or glibenclamide, [Ca2+]i and insulin secretion were elevated even in low glucose. High glucose transiently lowered [Ca2+]i, which then increased to or slightly above control levels, while insulin secretion was rapidly stimulated. Under all conditions the correlation between [Ca2+]i and insulin secretion was excellent at low and high glucose levels, and high glucose increased release at all [Ca2+]i. The potentiation of Ca2+-induced exocytosis by glucose is thus independent of the closed or open state of K+-ATP channels. It is only when the channels are opened by diazoxide that the increase in release is a strict amplification of the action of Ca2+. When the channels are closed (sulfonylureas) or still closable (high K+ alone), the effect of glucose on secretion also comprises a slight increase in [Ca2+]i and, in the latter case, is not strictly K+-ATP channel independent.

Adenosine Triphosphate↗
PubMed1999

Altered cAMP and Ca2+ signaling in mouse pancreatic islets with glucagon-like peptide-1 receptor null phenotype.

1-Cells from rodents and humans express different receptors recognizing hormones of the secretin-glucagon family, which--when activated--synergize with glucose in the control of insulin release. We have recently reported that isolated islets from mice homozygous for a GLP-1 receptor null mutation (GLP-1R(-/-)) exhibit a well-preserved insulin-secretory response to glucose. This observation can be interpreted in two different ways: 1) the presence of GLP-1R is not essential for the secretory response of isolated islets to glucose alone; 2) beta-cells in GLP-1R(-/-) pancreases underwent compensatory changes in response to the null mutation. To explore these possibilities, we studied islets from control GLP-IR(+/+) mice in the absence or presence of 1 pmol/l exendin (9-39)amide, a specific and potent GLP-1R antagonist. Exendin (9-39)amide (15-min exposure) reduced glucose-induced insulin secretion from both perifused and statically incubated GLP-1R(+/+) islets by 50% (P < 0.05), and reduced islet cAMP production in parallel (P < 0.001). Furthermore, GLP-1R(-/-) islets exhibited: 1) reduced cAMP accumulation in the presence of 20 mmol/l glucose (knockout islets versus control islets, 12 +/- 1 vs. 27 +/- 3 fmol x islet(-1) x 15 min(-1); P < 0.001) and exaggerated acceleration of cAMP production by 10 nmol/l glucose-dependent insulinotropic peptide (GIP) (increase over 20 mmol/l glucose by GIP in knockout islets versus control islets: 66 +/- 5 vs. 14 +/- 3 fmol x islet(-1) x 15 min(-1); P < 0.001); 2) increased mean cytosolic [Ca2+] ([Ca2+]c) at 7, 10, and 15 mmol/l glucose in knockout islets versus control islets; and 3) signs of asynchrony of [Ca2+]c oscillations between different islet subregions. In conclusion, disruption of GLP-1R signaling is associated with reduced basal but enhanced GIP-stimulated cAMP production and abnormalities in basal and glucose-stimulated [Ca2+]c. These abnormalities suggest that GLP-1R signaling is an essential upstream component of multiple beta-cell signaling pathways.

Acetylcholine↗
PubMed1999

Oscillations of insulin secretion can be triggered by imposed oscillations of cytoplasmic Ca2+ or metabolism in normal mouse islets.

Glucose-induced insulin secretion depends on an acceleration of glucose metabolism, requires a rise in the cytoplasmic free Ca2+ concentration ([Ca2+]i), and is modulated by activation of protein kinases in beta-cells. Normal mouse islets were used to determine whether oscillations of these three signals are able and necessary to trigger oscillations of insulin secretion. The approach was to minimize or abolish spontaneous oscillations and to compare the impact of forced oscillations of each signal on insulin secretion. In a control medium, repetitive increases in the glucose concentration triggered oscillations in metabolism [NAD(P)H fluorescence], [Ca2+]i (fura-PE3 method), and insulin secretion. In the presence of diazoxide, metabolic oscillations persisted, but [Ca2+]i and insulin oscillations were abolished. When the islets were depolarized with high K+ with or without diazoxide, [Ca2+]i was elevated, and insulin secretion was stimulated. Forced metabolic oscillations transiently decreased or did not affect [Ca2+]i and potentiated insulin secretion with oscillations of small amplitude. These oscillations of secretion followed metabolic oscillations only when [Ca2+]i did not change. When [Ca2+]i fluctuated, these changes prevailed over those of metabolism for timing secretion. Repetitive depolarizations with high K+ in the presence of stable glucose (10 mmol/l) induced synchronous pulses of [Ca2+]i and insulin secretion with only small oscillations of metabolism. Continuous stimulation of protein kinase A (PKA) and protein kinase C (PKC) did not dissociate the [Ca2+]i and insulin pulses from the high K+ pulses. However, the amplitude of the insulin pulses was consistently increased, whereas that of the [Ca2+]i pulses was either increased (PKA) or decreased (PKC). In conclusion, metabolic oscillations can induce oscillations of insulin secretion independently of but with a lesser effectiveness than [Ca2+]i oscillations. Although oscillations in metabolism may cyclically influence secretion through an ATP-sensitive K+ channel (K+-ATP channel)-independent pathway, their regulatory effects are characterized by a hysteresis that makes them unlikely drivers of fast oscillations, unless they also involve [Ca2+]i changes through the K+-ATP channel-dependent pathway.

Animals↗
PubMed1998

The changes in adenine nucleotides measured in glucose-stimulated rodent islets occur in beta cells but not in alpha cells and are also observed in human islets.

Glucose metabolism by pancreatic beta and alpha cells is essential for stimulation of insulin secretion and inhibition of glucagon secretion. Studies using rodent islets have suggested that the ATP/ADP ratio serves as second messenger in beta cells. This study compared the effects of glucose on glucose oxidation ([U-14C]glucose) and adenine nucleotides (luminometric method) in purified rat alpha and beta cells. The rate of glucose oxidation at 1 mM glucose was higher in beta than alpha cells (4.5-fold, i.e. approximately 2-fold after normalization for cell size). It was more strongly stimulated by 10 mM glucose in beta cells (9-fold) than in alpha cells (5-fold). At 1 mM glucose, ATP levels were similar in both cell types, which corresponds to an approximately 2-fold higher concentration in alpha cells ( approximately 6.5 mM) than in beta cells ( approximately 3 mM). In beta cells, glucose dose-dependently increased ATP and decreased ADP levels, causing a rise in the ATP/ADP ratio from 2.4 to 11.6 at 1 and 10 mM, respectively. In alpha cells, glucose did not affect ATP and ADP levels, and the ATP/ADP ratio remained stable around 7.5. In human islets, the ATP/ADP ratio progressively increased between 1 and 10 mM glucose. In duct cells, which often contaminate human islet preparations, an increase in the ATP/ADP ratio sometimes occurred between 1 and 3 mM glucose. In conclusion, the present observations establish that the regulation of glucagon secretion by glucose does not involve changes in alpha cell adenine nucleotides and further support the role of the ATP/ADP ratio in the control of insulin secretion.

Adenosine Diphosphate↗
PubMed1998

Interplay between cytoplasmic Ca2+ and the ATP/ADP ratio: a feedback control mechanism in mouse pancreatic islets.

In pancreatic beta cells, the increase in the ATP/ADP ratio that follows a stimulation by glucose is thought to play an important role in the Ca2+-dependent increase in insulin secretion. Here we have investigated the possible interactions between Ca2+ and adenine nucleotides in mouse islets. Measurements of both parameters in the same single islet showed that the rise in the ATP/ADP ratio precedes any rise in the cytoplasmic free-Ca2+ concentration ([Ca2+]i) and is already present during the initial transient lowering of [Ca2+]i produced by the sugar. Blockade of Ca2+ influx with nimodipine did not prevent the concentration-dependent increase in the ATP/ADP ratio produced by glucose and even augmented the ratio at all glucose concentrations which normally stimulate Ca2+ influx. In contrast, stimulation of Ca2+ influx by 30 mM K+ or 100 microM tolbutamide lowered the ATP/ADP ratio. This lowering was of rapid onset and reversibility, sustained and prevented by nimodipine or omission of extracellular Ca2+. It was, however, not attenuated after blockade of secretion by activation of alpha2-adrenoceptors. The difference in islet ATP/ADP ratio during blockade and stimulation of Ca2+ influx was similar to that observed between threshold and submaximal glucose concentrations. The results suggest that the following feedback loop could control the oscillations of membrane potential and [Ca2+]i in beta cells. Glucose metabolism increases the ATP/ADP ratio in a Ca2+-independent manner, which leads to closure of ATP-sensitive K+ channels, depolarization and stimulation of Ca2+ influx. The resulting increase in [Ca2+]i causes a larger consumption than production of ATP, which induces reopening of ATP-sensitive K+ channels and arrest of Ca2+ influx. Upon lowering of [Ca2+]i the ATP/ADP ratio increases again and a new cycle may start.

Adenosine Diphosphate↗
PubMed1998

Relative contribution of Ca2+-dependent and Ca2+-independent mechanisms to the regulation of insulin secretion by glucose.

Although insulin secretion is usually regarded as a Ca2+-dependent mechanism, recent studies have suggested the existence of a Ca2+-independent pathway of regulation by glucose. Here, mouse islets were used to compare the contribution of Ca2+-dependent and -independent pathways. Glucose increased insulin release in a concentration-dependent manner both in a control medium, when it depolarizes beta cells and raises [Ca2+]i (triggering signal), and in the presence of 30 mM K+ and diazoxide, when it does not further raise [Ca2+]i but increases its efficacy on exocytosis. Both Ca2+-dependent responses were amplified by glucagon-like peptide-1+acetylcholine, and were strongly potentiated by forskolin+PMA. Under conditions of mild or stringent Ca2+ deprivation, glucose had no effect either alone or with GLP-1 and acetylcholine, and was poorly effective even during pharmacological activation of protein kinases A and C. Similar results were obtained with rat islets. It is concluded that physiological regulation of insulin release by glucose is essentially achieved through the two Ca2+-dependent pathways without significant contribution of a Ca2+-independent mechanism.

Animals↗
PubMed1998

Okadaic acid-induced decrease in the magnitude and efficacy of the Ca2+ signal in pancreatic beta cells and inhibition of insulin secretion.

1. Phosphorylation by kinases and dephosphorylation by phosphatases markedly affect the biological activity of proteins involved in stimulus-response coupling. In this study, we have characterized the effects of okadaic acid, an inhibitor of protein phosphatases 1 and 2A, on insulin secretion. Mouse pancreatic islets were preincubated for 60 min in the presence of okadaic acid before their function was studied. 2. Okadaic acid dose-dependently (IC50 approximately 200 nM) inhibited insulin secretion induced by 15 mM glucose. At 0.5 microM, okadaic acid also inhibited insulin secretion induced by tolbutamide, ketoisocaproate and high K+, and its effects were not reversed by activation of protein kinases A or C. 3. The inhibition of insulin secretion did not result from an alteration of glucose metabolism (estimated by the fluorescence of endogenous pyridine nucleotides) or a lowering of the ATP/ADP ratio in the islets. 4. Okadaic acid treatment slightly inhibited voltage-dependent Ca2+ currents in beta cells (perforated patch technique), which diminished the rise in cytoplasmic Ca2+ (fura-2 method) that glucose and high K+ produce in islets. However, this decrease (25%), was insufficient to explain the corresponding inhibition of insulin secretion (90%). Moreover, mobilization of intracellular Ca2+ by acetylcholine was barely affected by okadaic acid, whereas the concomitant insulin response was decreased by 85%. 5. Calyculin A, another inhibitor of protein phosphatases 1 and 2A largely mimicked the effects of okadaic acid, whereas 1-norokadaone, an inactive analogue of okadaic acid on phosphatases, did not alter beta cell function. 6. In conclusion, okadaic acid inhibits insulin secretion by decreasing the magnitude of the Ca2+ signal in beta cells and its efficacy on exocytosis. The results suggest that, contrary to current concepts, both phosphorylation and dephosphorylation of certain beta cell proteins may be involved in the regulation of insulin secretion.

Adenosine Diphosphate↗