Functional differences between aggregated and dispersed insulin-producing cells.

AIMS/HYPOTHESIS: Beta cells situated in the islet of Langerhans respond more vigorously to glucose than do dissociated beta cells. Mechanisms for this discrepancy were studied by comparing insulin-producing MIN6 cells aggregated into pseudoislets with MIN6 monolayer cells and mouse and human islets. METHODS: MIN6 monolayers, pseudoislets and mouse and human islets were exposed to glucose, α-ketoisocaproic acid (KIC), pyruvate, KIC plus glutamine and the phosphatidylinositol 3-kinase (PI3K) inhibitors LY294002 or wortmannin. Insulin secretion (ELISA), cytoplasmic Ca(2+) concentration ([Ca(2+)]c; microfluorometry), glucose oxidation (radiolabelling), the expression of genes involved in mitochondrial metabolism (PCR) and the phosphorylation of insulin receptor signalling proteins (western blotting) were measured. RESULTS: Insulin secretory responses to glucose, pyruvate, KIC and glutamine were higher in pseudoislets than monolayers and comparable to those of human islets. Glucose oxidation and genes for mitochondrial metabolism were upregulated in pseudoislets compared with single cells and monolayers, respectively. Phosphorylation at the inhibitory S636/639 site of IRS-1 was significantly higher in monolayers and dispersed human and mouse cells than pseudoislets and intact human and mouse islets. PI3K inhibition only slightly attenuated glucose-stimulated insulin secretion from monolayers, but substantially reduced that from pseudoislets and human and mouse islets without suppressing the glucose-induced [Ca(2+)]c response. CONCLUSIONS/INTERPRETATION: We propose that islet architecture is critical for proper beta cell mitochondrial metabolism and IRS-1 signalling, and that PI3K regulates insulin secretion at a step distal to the elevation of [Ca(2+)]c.

Imatinib mesilate-induced phosphatidylinositol 3-kinase signalling and improved survival in insulin-producing cells: role of Src homology 2-containing inositol 5'-phosphatase interaction with c-Abl.

AIMS/HYPOTHESIS: It is not clear how small tyrosine kinase inhibitors, such as imatinib mesilate, protect against diabetes and beta cell death. The aim of this study was to determine whether imatinib, as compared with the non-cAbl-inhibitor sunitinib, affects pro-survival signalling events in the phosphatidylinositol 3-kinase (PI3K) pathway. METHODS: Human EndoC-ßH1 cells, murine beta TC-6 cells and human pancreatic islets were used for immunoblot analysis of insulin receptor substrate (IRS)-1, Akt and extracellular signal-regulated kinase (ERK) phosphorylation. Phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] plasma membrane concentrations were assessed in EndoC-ßH1 and MIN6 cells using evanescent wave microscopy. Src homology 2-containing inositol 5'-phosphatase 2 (SHIP2) tyrosine phosphorylation and phosphatase and tensin homologue deleted on chromosome 10 (PTEN) serine phosphorylation, as well as c-Abl co-localisation with SHIP2, were studied in HEK293 and EndoC-ßH1 cells by immunoprecipitation and immunoblot analysis. Gene expression was assessed using RT-PCR. Cell viability was measured using vital staining. RESULTS: Imatinib stimulated ERK(thr202/tyr204) phosphorylation in a c-Abl-dependent manner. Imatinib, but not sunitinib, also stimulated IRS-1(tyr612), Akt(ser473) and Akt(thr308) phosphorylation. This effect was paralleled by oscillatory bursts in plasma membrane PI(3,4,5)P3 levels. Wortmannin induced a decrease in PI(3,4,5)P3 levels, which was slower in imatinib-treated cells than in control cells, indicating an effect on PI(3,4,5)P3-degrading enzymes. In line with this, imatinib decreased the phosphorylation of SHIP2 but not of PTEN. c-Abl co-immunoprecipitated with SHIP2 and its binding to SHIP2 was largely reduced by imatinib but not by sunitinib. Imatinib increased total ß-catenin levels and cell viability, whereas sunitinib exerted negative effects on cell viability. CONCLUSIONS/INTERPRETATION: Imatinib inhibition of c-Abl in beta cells decreases SHIP2 activity, which results in enhanced signalling downstream of PI3 kinase.

Oscillations of sub-membrane ATP in glucose-stimulated beta cells depend on negative feedback from Ca(2+).

AIMS/HYPOTHESIS: ATP links changes in glucose metabolism to electrical activity, Ca(2+) signalling and insulin secretion in pancreatic beta cells. There is evidence that beta cell metabolism oscillates, but little is known about ATP dynamics at the plasma membrane, where regulation of ion channels and exocytosis occur. METHODS: The sub-plasma-membrane ATP concentration ([ATP]pm) was recorded in beta cells in intact mouse and human islets using total internal reflection microscopy and the fluorescent reporter Perceval. RESULTS: Glucose dose-dependently increased [ATP]pm with half-maximal and maximal effects at 5.2 and 9 mmol/l, respectively. Additional elevations of glucose to 11 to 20 mmol/l promoted pronounced [ATP]pm oscillations that were synchronised between neighbouring beta cells. [ATP]pm increased further and the oscillations disappeared when voltage-dependent Ca(2+) influx was prevented. In contrast, K(+)-depolarisation induced prompt lowering of [ATP]pm. Simultaneous recordings of [ATP]pm and the sub-plasma-membrane Ca(2+) concentration ([Ca(2+)]pm) during the early glucose-induced response revealed that the initial [ATP]pm elevation preceded, and was temporarily interrupted by the rise of [Ca(2+)]pm. During subsequent glucose-induced oscillations, the increases of [Ca(2+)]pm correlated with lowering of [ATP]pm. CONCLUSIONS/INTERPRETATION: In beta cells, glucose promotes pronounced oscillations of [ATP]pm, which depend on negative feedback from Ca(2+) . The bidirectional interplay between these messengers in the sub-membrane space generates the metabolic and ionic oscillations that underlie pulsatile insulin secretion.

cAMP oscillations restrict protein kinase A redistribution in insulin-secreting cells.

Activation of hormone receptors was recently found to evoke oscillations of the cAMP concentration ([cAMP]) beneath the plasma membrane of insulin-secreting cells. Here we investigate how different time courses of cAMP signals influence the generation of cytoplasmic Ca(2+) signals and nuclear translocation of the PKA (protein kinase A) catalytic subunit in individual INS-1 beta-cells. [cAMP] was measured with a fluorescent translocation biosensor and ratiometric evanescent wave microscopy. Analysis of PKA nuclear translocation was performed with epifluorescence microscopy and FlAsH (fluorescein arsenical helix binder) labelling of tetracysteine-tagged PKA-Calpha subunit. Both oscillatory and stable elevations of [cAMP] induced by intermittent or constant inhibition of phosphodiesterases with isobutylmethylxanthine evoked Ca(2+) spiking. During [cAMP] oscillations, the Ca(2+) spiking was restricted to the periods of elevated [cAMP]. In contrast, only stable [cAMP] elevation induced nuclear entry of FlAsH-labelled PKA-Calpha. These results indicate that oscillations of [cAMP] lead to selective target activation by restricting the spatial redistribution of PKA.

The endoplasmic reticulum is a glucose-modulated high-affinity sink for Ca2+ in mouse pancreatic beta-cells.

The regulation of organelle free Ca2+ was analysed in individual mouse pancreatic beta-cells loaded with the fluorescent low-affinity indicator furaptra. Removal of the cytoplasmic indicator by controlled digitonin permeabilization of the plasma membrane resulted in a sudden increase of the 340 nm/380 nm fluorescence excitation ratio followed by a gradual decay, reflecting the emptying of Ca2+ from organelle pools. Subsequent introduction of 3 mM ATP caused rapid refilling of a Ca2+ pool, which represented the endoplasmic reticulum (ER) in being mobilized with inositol 1,4,5-trisphosphate (IP3) and the sarco(endo)plasmic reticulum Ca2+-ATPase inhibitor thapsigargin. The concentration of Ca2+ in the ER observed immediately after permeabilization depended on the glucose concentration in a hyperbolic fashion with half-maximal filling at about 6 mM of the sugar. Glucose promotion of Ca2+ sequestration in the ER involved a high-affinity mechanism not requiring but accelerated by a rise of the cytoplasmic Ca2+ concentration. Glucose also exerted a long-term action on the ER storage of Ca2+, maintaining the set-point for its maximal concentration and preserving the response to IP3. The results indicate that the ER has an important role in the glucose-stimulated beta-cell by serving as a high-affinity sink for Ca2+, irrespective of the prevailing concentration of cytoplasmic Ca2+.

Mobilization of Ca2+ stores in individual pancreatic beta-cells permeabilized or not with digitonin or alpha-toxin.

The concentration of free Ca2+ in the cytoplasm and organelles of individual mouse pancreatic beta-cells was estimated with dual wavelength microfluorometry and the indicators Fura-2 and furaptra. Measuring the increase of cytoplasmic Ca2+ resulting from intracellular mobilization of the ion in ob/ob mouse beta-cells, most organelle calcium (92%) was found in acidic compartments released when combining the Ca2+ ionophore Br-A23187 with a protonophore. Only 3-4% of organelle calcium was recovered from a pool sensitive to the Ca(2+)-ATPase inhibitor thapsigargin. Organelle Ca2+ was also measured directly in furaptra-loaded beta-cells after controlled plasma membrane permeabilization. The permeabilizing agent alpha-toxin was superior to digitonin in preserving the integrity of intracellular membranes, but digitonin provided more reproducible access to intracellular sites. After permeabilization, the thapsigargin-sensitive fraction of Ca2+ detected by furaptra was as high as 90%, suggesting that the indicator essentially measures Ca2+ in endoplasmic reticulum (ER). Both alpha-toxin- and digitonin-permeabilized cells exhibited ATP-dependent uptake of Ca2+ into thapsigargin-sensitive stores with half-maximal and maximal filling at 6-11 microM and 1 mM ATP respectively. Most of the thapsigargin-sensitive Ca2+ was mobilized by inositol 1,4,5-trisphosphate (IP3), whereas caffeine, ryanodine, cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate lacked effects both in beta-cells from ob/ob mice and normal NMRI mice. Mobilization of organelle Ca2+ by 4-chloro-3-methylphenol was attributed to interference with the integrity of the ER rather than to activation of ryanodine receptors. The observations emphasize the importance of IP3 for Ca2+ mobilization in pancreatic beta-cells, but question a role for ryanodine receptor agonists.

Glucose regulation of free Ca(2+) in the endoplasmic reticulum of mouse pancreatic beta cells.

Free Ca(2+) was measured in organelles of individual mouse pancreatic beta cells loaded with the low affinity indicator furaptra. After removal of cytoplasmic indicator by controlled digitonin permeabilization the organelle Ca(2+) was located essentially in the endoplasmic reticulum (ER), >90% being sensitive to inhibition of sarco(endo)plasmic reticulum Ca(2+)-ATPases. The Ca(2+) accumulation in the ER of intact beta cells depended in a hyperbolic fashion on the glucose concentration with half-maximal and maximal filling at 5.5 and >20 mM, respectively. Also elevation of cytoplasmic Ca(2+) by K(+) depolarization significantly enhanced the Ca(2+) accumulation. In permeabilized beta cells 1-3 mM ATP caused rapid Ca(2+) filling of the ER reaching almost 500 microM. At 50 nM, Ca(2+) ER became half-maximally filled at 45 microM ATP, whereas only 3.5 microM ATP was required at 200 nM Ca(2+). Inositol 1,4,5-trisphosphate induced a rapid release of about 65% of the ER Ca(2+), and its precursor phosphatidylinositol 4,5-bisphosphate was found to slowly mobilize 75% by another mechanism. It is concluded that glucose is an efficient stimulator of Ca(2+) uptake in the ER of pancreatic beta cells both by increasing ATP and cytoplasmic Ca(2+). Because physiological concentrations of cytoplasmic ATP are in the mM range, Ca(2+) sequestration can be anticipated to be modulated by factors reducing its ATP sensitivity.

Cell interactions with collagen matrices in vivo and in vitro depend on phosphatidylinositol 3-kinase and free cytoplasmic calcium.

We have investigated the role of phosphatidylinositol 3-kinase (PI3-kinase) in cellular interactions with collagenous matrices. Platelet-derived growth factor-BB (PDGF-BB) elicited a mobilization of intracellular Ca2+ in pig aortic endothelial (PAE) cells transfected with wild type PDGF beta-receptor. This response was greatly reduced in PAE cells transfected with PDGF beta-receptors mutated at positions Y740 and Y751 to prevent PI3-kinase binding. The experimental drug 1D-myo-inositol 1,2,6-trisphosphate (alpha-trinositol) induced a rapid increase and subsequent oscillations of the cytoplasmic Ca2+ concentration in cultured fibroblasts. This response was not due to an effect of alpha-trinositol on inositol 1,4,5-trisphosphate (IP3) receptors. alpha-Trinositol did not influence PDGF-BB elicited chemotaxis through collagen-coated membranes of PAE cells transfected with the wild-type PDGF beta-receptor, but restored PDGF-BB elicited chemotaxis of PAE cells transfected with the PI3-kinase binding-site mutated PDGF beta-receptor. Collagen gel contraction has been suggested to serve as a model for cellular control of interstitial fluid pressure (PIF) in dermis. The PI3-kinase inhibitors wortmannin (50 nM) and LY294002 (5 microM) inhibited the stimulation of fibroblast-mediated collagen gel contraction by 0.4 nM PDGF-BB. Injection of wortmannin in rat paw skin induced a lowering of PIF, and this effect was abolished in animals pre-treated with alpha-trinositol. Pretreatment of rats with alpha-trinositol abolished the decrease in PIF induced by injecting monoclonal anti-rat alpha 2 beta 1 integrin IgG in rat paw skin. Taken together our data indicate that cell-collagen interactions in vivo and in vitro depend on PI3-kinase, and that this dependence can be bypassed by a drug eliciting intracellular Ca2+ mobilization.

Origin of slow and fast oscillations of Ca2+ in mouse pancreatic islets.

1. Pancreatic islets exposed to 11 mM glucose exhibited complex variations of cytoplasmic Ca2+ concentration ([Ca2+]i) with slow (0.3-0.9 min-1) or fast (2-7 min-1) oscillations or with a mixed pattern. 2. Using digital imaging and confocal microscopy we demonstrated that the mixed pattern with slow and superimposed fast oscillations was due to separate cell populations with the respective responses. 3. In islets with mixed [Ca2+]i oscillations, exposure to the sarcoplasmic-endoplasmic reticulum Ca2+-ATPase inhibitors thapsigargin or 2,5-di-tert-butylhydroquinone (DTBHQ) resulted in a selective disappearance of the fast pattern and amplification of the slow pattern. 4. In addition, the protein kinase A inhibitor RP-cyclic adenosine 3',5'-monophosphorothioate sodium salt transformed the mixed [Ca2+]i oscillations into slow oscillations with larger amplitude. 5. Islets exhibiting only slow oscillations reacted to low concentrations of glucagon with induction of the fast or the mixed pattern. In this case the fast oscillations were also counteracted by DTBHQ. 6. The spontaneously occurring fast oscillations seemed to require the presence of cAMP-elevating glucagon, since they were more common in large islets and suppressed during culture. 7. Image analysis revealed [Ca2+]i spikes occurring irregularly in time and space within an islet. These spikes were preferentially observed together with fast [Ca2+]i oscillations, and they became more common after exposure to glucagon. 8. Both the slow and fast oscillations of [Ca2+]i in pancreatic islets rely on periodic entry of Ca2+. However, the fast oscillations also depend in some way on paracrine factors promoting mobilization of Ca2+ from intracellular stores. It is proposed that such a mobilization in different cells within a tightly coupled islet syncytium generates spikes which co-ordinate the regular bursts of action potentials underlying the fast oscillations.

Oscillatory signaling and insulin release in human pancreatic beta-cells exposed to strontium.

Oscillatory signaling and insulin release were studied in isolated pancreatic islets and beta-cells obtained from human cadaveric organ donors. Taking advantage of Sr2+ as an analog for Ca2+, it was possible to demonstrate glucose-induced rhythmic activity in individual beta-cells identified by immunostaining. Glucose-induced slow oscillations of Sr2+ (frequency, 0.1-1.0/min) were sometimes seen at a sugar concentration as low as 3 mM. Addition of 20 nM glucagon resulted in a broadening of the oscillations or in their transformation into sustained elevation. Moreover, the presence of glucagon resulted in the appearance of short transients of Sr2+, which disappeared after exposure to the intracellular Ca2+-adenosine triphosphatase inhibitor thapsigargin. Digital image analyses indicated that slow oscillations can be synchronized among cells in small aggregates and intact islets. The rhythmic activity in the glucose-stimulated beta-cell had its counterpart in pulsatile insulin release when single islets were perifused with a Sr2+-containing medium. It is concluded that the human beta-cell has oscillatory signaling for insulin release similar to that observed in experimental animals.

Glucose induces oscillatory Ca2+ signalling and insulin release in human pancreatic beta cells.

Mechanisms of pulsatile insulin release in man were explored by studying the induction of oscillatory Ca2+ signals in individual beta cells and islets isolated from the human pancreas. Evidence was provided for a glucose-induced closure of ATP-regulated K+ channels, resulting in voltage-dependent entry of Ca2+. The observation of step-wise increases of capacitance in response to depolarizing pulses suggests that an enhanced influx of Ca2+ is an effective means of stimulating the secretory activity of the isolated human beta cell. Activation of muscarinic receptors (1-10 mumol/l carbachol) and of purinergic P2 receptors (0.01-1 mumol/l ATP) resulted in repetitive transients followed by sustained elevation of the cytoplasmic Ca2+ concentration ([Ca2+]i). Periodic mobilisation of intracellular calcium was seen also when injecting 100 mumol/l GTP-gamma-S into beta cells hyperpolarized to -70 mV. Individual beta cells responded to glucose and tolbutamide with increases of [Ca2+]i, manifested either as large amplitude oscillations (frequency 0.1-0.5/min) or as a sustained elevation. Glucose regulation was based on sudden transitions between the basal and the two alternative states of raised [Ca2+]i at threshold concentrations of the sugar characteristic for the individual beta cells. The oscillatory characteristics of coupled cells were determined collectively rather than by particular pacemaker cells. In intact pancreatic islets the glucose induction of well-synchronized [Ca2+]i oscillations had its counterpart in 2-5 min pulses of insulin. Each of these pulses could be resolved into regularly occurring short insulin transients. It is concluded that glucose stimulation of insulin release in man is determined by the number of beta cells entering into a state with Ca(2+)-induced secretory pulses.

Synchronous oscillations of cytoplasmic Ca2+ and insulin release in glucose-stimulated pancreatic islets.

The cytoplasmic Ca2+ concentration ([Ca2+]i) was measured in single pancreatic mouse islets superfused in a system allowing concomitant recordings of insulin release. When glucose was raised from 3 to 11 mM, [Ca2+]i responded by a transient lowering followed by a rise to an average level of 192 +/- 11 nM. In 77% of the islets the rise was associated with the gradual appearance of oscillations, which were either fast (2-7/min), slow (0.3-0.9/min), or a combination of both types. The characteristics of the fast [Ca2+]i oscillations were those expected from a relationship with the electrical burst activity in islets. Accordingly, in most cases the fast oscillations were remarkably regular. The slow [Ca2+]i oscillations had characteristics similar to the large amplitude ones in individual beta-cells. Whereas glucagon and dibutyryl cAMP could transform slow islet oscillations into fast ones, the alpha 2-adrenergic agonist clonidine had the opposite effect. The rapid islet oscillations were also facilitated by elevated concentrations of extracellular Ca2+. Reinforcing the arguments for [Ca2+]i oscillations as responsible for a pulsatile insulin secretion it was possible to demonstrate that the release of the hormone from single islets is synchronized with the slow [Ca2+]i oscillations.