Patrick Gilon

Mechanisms by which glucose can control insulin release independently from its action on adenosine triphosphate-sensitive K+ channels in mouse B cells.

Glucose stimulation of insulin release involves closure of ATP-sensitive K+ channels (K(+)-ATP channels), depolarization, and Ca2+ influx in B cells. However, by using diazoxide to open K(+)-ATP channels, and 30 mM K to depolarize the membrane, we could demonstrate that another mechanism exists, by which glucose can control insulin release independently from changes in K(+)-ATP channel activity and in membrane potential (Gembal et al. 1992. J. Clin. Invest. 89:1288-1295). A similar approach was followed here to investigate, with mouse islets, the nature of this newly identified mechanism. The membrane potential-independent increase in insulin release produced by glucose required metabolism of the sugar and was mimicked by other metabolized secretagogues. It also required elevated levels of cytoplasmic Cai2+, but was not due to further changes in Cai2+. It could not be ascribed to acceleration of phosphoinositide metabolism, or to activation of protein kinases A or C. Thus, glucose did not increase inositol phosphate levels and hardly affected cAMP levels. Moreover, increasing inositol phosphates by vasopressin or cAMP by forskolin, and activating protein kinase C by phorbol esters did not mimic the action of glucose on release, and down-regulation of protein kinase C did not prevent these effects. On the other hand, it correlated with an increase in the ATP/ADP ratio in islet cells. We suggest that the membrane potential-independent control of insulin release exerted by glucose involves changes in the energy state of B cells.

Mechanisms of the stimulation of insulin release by saturated fatty acids. A study of palmitate effects in mouse beta-cells

The mechanisms by which fatty acids increase insulin release are not known. In this study, mouse islets were used as a model and palmitate as a reference compound to study in vitro how saturated fatty acids influence pancreatic β-cells. Palmitate (625 μM) was bound to albumin. It did not affect basal insulin release (3 mM glucose) but increased the release induced by 10–15 mM glucose. This effect was dependent on the concentration of free rather than total palmitate. It was reversible and abolished by epinephrine, diazoxide, nimodipine, or omission of extracellular Ca. Bromopalmitate and methyl palmoxirate, two inhibitors of fatty acid oxidation, were ineffective alone, and only bromopalmitate partially inhibited the effects of palmitate on insulin release. The increase in insulin release produced by palmitate could not be ascribed to a blockade of ATP-sensitive K+-channels because the fatty acid only barely decreased 86Rb efflux and did not depolarize β-cells in 3 mM glucose. The small effect on 86Rb efflux might be attributed to a slight rise in the ATP/ADP ratio. No such rise occurred when palmitate was tested in 15 mM glucose, and the fatty acid consistently accelerated 86Rb efflux under these conditions. Measurements of β-cell membrane potential (intracellular microelectrodes) and of free cytoplasmic calcium (Cai 2+) in β-cells (Fura 2 technique) showed that palmitate increases Ca2+ influx; it also caused a very small mobilization of intracellular Ca. The persistence of this stimulation of Ca2+ influx in the presence of diazoxide and high K+ suggests that palmitate might act on Ca2+ channels. The rise in Ca12+ produced by palmitate was accompanied by an increase in insulin release only if the concentration of glucose was sufficiently high. The β-cell response to palmitate thus differs from the responses to glucose and other metabolized nutrients in several respects. Saturated fatty acids appear to potentiate insulin release through an increase in Ca12+ and another, yet unidentified, fuel-dependent mechanism.

Hierarchy of the beta-cell signals controlling insulin secretion

The main function of pancreatic β cells is to synthesize and secrete insulin at appropriate rates to limit blood glucose fluctuations within a narrow range. Any alteration in β -cell functioning has a profound impact on glucose homeostasis: excessive secretion of insulin causes hypoglycaemia, and insufficient secretion leads to diabetes. It is therefore not surprising that insulin secretion is subject to very tight control. This control is primarily ensured by glucose itself but also involves an array of metabolic, neural, hormonal and sometimes pharmacological factors (Fig. 1). To integrate all these stimulatory and inhibitory influences, β cells rely on an astonishingly complex stimulus-secretion coupling. This review discusses how the hierarchy between two intracellular pathways, producing triggering and amplifying signals [1], optimizes adequate insulin secretion to changes in blood glucose concentration and enables the β cell to grade the numerous extracellular messages that it receives.

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.

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 ≈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 18α‐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.

Temporal and quantitative correlations between insulin secretion and stably elevated or oscillatory cytoplasmic Ca2+ in mouse pancreatic beta-cells

An increase in cytoplasmic Ca2+ in β-cells is a key step in glucose-induced insulin secretion. However, whether changes in cytoplasmic free Ca2+ ([Ca2+]i) directly regulate secretion remains disputed. This question was addressed by investigating the temporal and quantitative relationships between [Ca2+]i and insulin secretion. Both events were measured simultaneously in single mouse islets loaded with fura-PE3 and perifused with a medium containing diazoxide (to prevent any effect of glucose on the membrane potential) and either 4.8 or 30 mmol/l K+. Continuous depolarization with 30 mmol/l K+ in the presence of 15 mmol/l glucose induced a sustained rise in [Ca2+]i and insulin release. No oscillations of secretion were detected even after mathematical analysis of the data (pulse, spectral and sample distribution analysis). In contrast, alternating between 30 and 4.8 mmol/l K+ (1 min/2 min or 2.5 min/5 min) triggered synchronous [Ca2+]i and insulin oscillations of regular amplitude in each islet. A good correlation was found between [Ca2+]i and insulin secretion, and it was independent of the presence or absence of oscillations. This quantitative correlation between [Ca2+]i and insulin secretion was confirmed by experiments in which extracellular Ca2+ was increased or decreased (0.1–2.5 mmol/l) stepwise in the presence of 30 mmol/l K+. This resulted in parallel stepwise increases or decreases in [Ca2+]i and insulin secretion. However, while the successive [Ca2+]i levels were unaffected by glucose, each plateau of secretion was much higher in 20 than in 3 mmol/l glucose. In conclusion, in our preparation of normal mouse islets, insulin secretion oscillates only when [Ca2+]i oscillates in β-cells. This close temporal relationship between insulin secretion and [Ca2+]i changes attests of the regulatory role of Ca2+. There also exists a quantitative relationship that is markedly influenced by the concentration of glucose.

Emptying of intracellular Ca2+ stores stimulates Ca2+ entry in mouse pancreatic beta-cells by both direct and indirect mechanisms.

1 In non‐excitable cells, the depletion of intracellular Ca2+ stores triggers Ca2+ influx by a process called capacitative Ca2+ entry. In the present study, we have investigated how the emptying of these stores by thapsigargin (1 μM) influences Ca2+ influx in electrically excitable pancreatic β‐cells. The cytoplasmic Ca2+ concentration ([Ca2+]i) was monitored in clusters of mouse β‐cells or in whole islets loaded with fura‐2. 2 The membrane was first held hyperpolarized by diazoxide, an opener of ATP‐sensitive K+(KATP) channels, in the presence of 4.8 mM K+. Alternating between Ca2+‐free medium and medium containing 2.5 mM Ca2+ caused a minor rise in [Ca2+]i (∼14 nM) in clusters of β‐cells. A larger rise (∼65 nM), resistant to the blockade of voltage‐dependent Ca2+ channels by D600, occurred when extracellular Ca2+ was readmitted after emptying intracellular Ca2+ stores with thapsigargin or acetylcholine. Thus there exists a small capacitative Ca2+ entry in β‐cells. 3 When the membrane potential was clamped at depolarized levels with 10, 20 or 45 mM K+ in the presence of diazoxide, [Ca2+]i increased to different plateau levels ranging between 100 and 900 nM. Thapsigargin consistently caused a further transient rise in [Ca2+]i, but had little (at 10 mM K+) or no effect on the plateau level. This confirms that the capacitative Ca2+ entry is small. 4 In clusters of cells whose membrane potential was not clamped with diazoxide, 15 mM glucose (in 4.8 mM K+) induced [Ca2+]i oscillations by promoting Ca2+ influx through voltage‐dependent Ca2+ channels. The application of thapsigargin accelerated these oscillations and increased their amplitude, sometimes causing a sustained elevation of [Ca2+]i. Similar results were obtained from whole islets perifused with a medium containing ≥ 6 mM glucose. The effect of thapsigargin was always much larger than expected from the capacitative Ca2+ entry, probably because of a potentiation of Ca2+ influx through voltage‐dependent Ca2+ channels. 5 This potentiating effect of thapsigargin did not result from an acceleration of cell metabolism since the drug did not affect glucose‐induced changes in NAD(P)H fluorescence. It is also unlikely to involve the inhibition of KATP channels because thapsigargin steadily elevated [Ca2+]i in cells in which [Ca2+]i oscillations persisted in the presence of a maximally effective concentration of tolbutamide. 6 In conclusion, the emptying of intracellular Ca2+ stores in β‐cells induces a small capacitative Ca2+ entry and activates a depolarizing current which potentiates glucose‐induced Ca2+ influx through voltage‐dependent Ca2+ channels.

Multiple effects and stimulation of insulin secretion by the tyrosine kinase inhibitor genistein in normal mouse islets

1 Islets from normal mice were used to test the acute effects of genistein, a potent tyrosine kinase inhibitor, on stimulus‐secretion coupling in pancreatic β‐cells. 2 Genistein produced a concentration‐dependent (10–100 μm), reversible, increase of insulin release. This effect was marginal on basal release or in the presence of non‐metabolized secretagogues, and much larger in the presence of glucose or other nutrients. The increase in insulin release caused by 100 μm genistein was abolished by adrenaline or omission of extracellular Ca2+. It was not accompanied by any rise of cyclic AMP, inositol phosphate or adenine nucleotide levels. 3 Although genistein slightly inhibited ATP‐sensitive K+ channels, as shown by 86Rb efflux and patch‐clamp experiments, this effect could not explain the action of the drug on insulin release because the latter persisted when ATP‐sensitive K+ channels were all blocked by maximally effective concentrations of glucose and tolbutamide. Genistein was also effective when ATP‐sensitive K+ channels were opened by diazoxide and the β‐cell membrane depolarized by 30 mm K, but ineffective in the presence of diazoxide and normal extracellular K. 4 Genistein paradoxically decreased Ca2+ influx in β‐cells, as shown by the inhibition of glucose‐induced electrical activity, by the inhibition of Ca2+ currents (perforated patches) and by the lowering of cytosolic [Ca2+]i (fura‐2 technique). Genistein thus increases insulin release in spite of a lowering of [Ca2+]i in β‐cells. 5 Daidzein, an analogue of genistein reported not to affect tyrosine kinases, was slightly less potent than genistein on K+ and Ca2+ channels, but increased insulin secretion in a similar way. Three other tyrosine kinase inhibitors, tyrphostin A47, herbimycin A and an analogue of erbstatin variably affected insulin secretion. 6 Genistein exerts a number of heretofore unrecognized effects. The unusual mechanisms, by which genistein increases insulin release in spite of a decrease in β‐cell [Ca2+]i and without activating known signalling pathways, do not seem to result from an inhibition of tyrosine kinases.

Culture duration and conditions affect the oscillations of cytoplasmic calcium concentration induced by glucose in mouse pancreatic islets

SummaryThe pattern of the increase in cytoplasmic Cai2+ that glucose produces in beta cells has been reported to be highly variable. Here, we evaluated the influence of the culture duration (1–4 days) and conditions (5–10 mmol/l glucose) on Cai2+ in normal mouse islets stimulated by glucose. After 1 day of culture in 10 mmol/l glucose, a rise of the glucose concentration from 3 to 15 mmol/l induced a triphasic change of Cai2+ in the islets. A small initial decrease was followed by a large peak increase and then by regular fast oscillations (∼2.5/min). When the culture was prolonged to 2, 3 and 4 days, the initial decrease became inconsistent and the peak occurred earlier, whereas the oscillations decreased in frequency, increased in duration and eventually disappeared; on day 4 the Cai2+ rise was sustained. After culture in 5 mmol/l glucose, the pattern of Cai2+ changes induced by 15 mmol/l glucose was different. The initial decrease was very pronounced, the first peak was delayed and clearly separated from the subsequent oscillations. These were of a mixed type (fast Ca2+ transients on top of slow ones) after 1 day, and of a slow type only after 4 days. These alterations in the Cai2+ oscillations triggered by glucose could not be ascribed to desynchronization of the signal between different regions of the islets. In conclusion, culturing normal mouse islets in 5 or 10 mmol/l glucose for 1–4 days, markedly alters the characteristics of the changes in Cai2+ produced by glucose. This pitfall must be borne in mind when studying stimulus-secretion coupling in beta cells from normal or diabetic animals, or from human islets.

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

To determine how pretreatment with sulphonylureas alters the β cell function, mouse islets were cultured (18–20 h) without (controls) or with (test) 0.01 μM glibenclamide. Acute responses to glucose were then determined in the absence of glibenclamide. 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. 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. The first phase of glucose‐induced insulin secretion was abrogated because of a paradoxical decrease of the high basal [Ca2+]i in β 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). In islets cultured with 50 μM 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. In conclusion, treatment with a low glibenclamide concentration causes long‐lasting blockade of K+‐ATP channels and rise of [Ca2+]i in β 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.