Subplasmalemmal Ca2+ measurements in mouse pancreatic beta cells support the existence of an amplifying effect of glucose on insulin secretion
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Glucose-induced insulin secretion is attributed to a rise of beta cell cytosolic free [Ca2+] ([Ca2+]c) (triggering pathway) and amplification of the action of Ca2+. This concept of amplification rests on observations that glucose can increase Ca2+-induced insulin secretion without further elevating an imposed already high [Ca2+]c. However, it remains possible that this amplification results from an increase in [Ca2+] just under the plasma membrane ([Ca2+]SM), which escaped detection by previous measurements of global [Ca2+]c. This was the hypothesis that we tested here by measuring [Ca2+]SM.
The genetically encoded Ca2+ indicators D3-cpv (untargeted) and LynD3-cpv (targeted to plasma membrane) were expressed in clusters of mouse beta cells. LynD3-cpv was also expressed in beta cells within intact islets. [Ca2+]SM changes were monitored using total internal reflection fluorescence microscopy. Insulin secretion was measured in parallel.
Beta cells expressing D3cpv or LynD3cpv displayed normal [Ca2+] changes and insulin secretion in response to glucose. Distinct [Ca2+]SM fluctuations were detected during repetitive variations of KCl between 30 and 32–35 mmol/l, attesting to the adequate sensitivity of our system. When the amplifying pathway was evaluated (high KCl + diazoxide), increasing glucose from 3 to 15 mmol/l consistently lowered [Ca2+]SM while stimulating insulin secretion approximately two fold. Blocking Ca2+ uptake by the endoplasmic reticulum largely attenuated the [Ca2+]SM decrease produced by high glucose but did not unmask localised [Ca2+]SM increases.
Glucose can increase Ca2+-induced insulin secretion without causing further elevation of beta cell [Ca2+]SM. The phenomenon is therefore a true amplification of the triggering action of Ca2+.
KeywordsAmplifying pathway Beta cell Cytosolic calcium Insulin secretion Islets Plasma membrane Stimulus–secretion coupling Total internal reflection fluorescence microscopy
Cytosolic free Ca2+ concentration
Design 3 with circular permuted venus
Total internal reflection fluorescence
Glucose-induced insulin secretion by beta cells is mediated by two major pathways (reviewed in ). In the triggering pathway, glucose metabolism leads to closure of ATP-sensitive potassium (KATP) channels in the plasma membrane, which permits depolarisation and Ca2+ influx via L-type Ca2+ channels. The subsequent increase in the cytosolic free Ca2+ concentration ([Ca2+]c) triggers exocytosis of insulin-containing granules [2, 3]. In parallel, glucose metabolism activates an amplifying pathway (previously known as the KATP channel-independent pathway) that augments the secretory response to triggering Ca2+ [4, 5, 6, 7]. The cellular mechanisms of this amplification are still incompletely understood, but the effect of glucose is generally thought not to involve a further increase in [Ca2+]c [1, 4, 7]. This concept of a dual regulation of glucose-induced insulin secretion was originally based on measurements of global [Ca2+]c in intact islets with the classical Ca2+ probe fura2 . However, such measurements of global [Ca2+]c did not rule out the possibility that glucose produces an increase in subplasmalemmal [Ca2+] ([Ca2+]SM) that escaped detection. A localised increase in [Ca2+]SM would imply that the increase in secretion is not a true amplification, and the model would require a major revision.
Localised changes in the triggering Ca2+ signal could result from glucose actions that are independent from the KATP channel-mediated control of membrane potential. Glucose metabolism could increase the activity of L-type Ca2+ channels [8, 9], influence Ca2+ handling by intracellular Ca2+ stores  and modulate the rate of Ca2+ extrusion through the plasma membrane . As yet undetected [Ca2+] changes confined to subplasma membrane domains where Ca2+-dependent exocytosis takes place [12, 13, 14, 15, 16, 17] might therefore account for the apparent amplification phenomenon. Studies using capacitance measurements in single, dialysed beta cells also suggested that exocytosis of insulin granules requires higher than average [Ca2+]c measured in intact cells or whole islets .
Mainly because of previous limitations in imaging technology, only a few groups have attempted to measure [Ca2+]SM in beta cells using conventional fluorescent probes [12, 19, 20]. To our knowledge, direct dynamic measurements of [Ca2+]SM were only achieved in three studies [17, 21, 22], but none of these looked for specific [Ca2+]SM changes during amplification of insulin secretion by glucose.
In the present study we therefore tested the hypothesis that the apparent amplifying effect of glucose on insulin secretion is, in fact, attributable to an increase in [Ca2+]SM. To this end, the genetically encoded ratiometric Ca2+ indicator D3cpv (design 3 with circular permuted venus) , a probe based on fluorescence resonance energy transfer, was adenovirally targeted in clusters of mouse beta cells. [Ca2+]SM changes were then monitored by total internal reflection fluorescence (TIRF)  microscopy in parallel with insulin secretion. We also used LynD3cpv, a variant of the Ca2+ probe that is addressed to the plasma membrane , and specifically expressed it in beta cells within clusters or intact islets.
Vectors and adenoviruses generation
To measure [Ca2+]SM, adenoviruses ensuring production of D3cpv or LynD3cpv were constructed (see Electronic supplementary material [ESM 1] for description of method). D3cpv was produced under the control of the cytomegalovirus promoter, while LynD3cpv was specifically produced in beta cells by a dual recombinant adenovirus strategy, using Rip-Cre and LoxStopLox-LynD3cpv adenoviruses. The Rip-Cre adenovirus, permitting production of the Cre recombinase under control of the rat insulin promoter, was kindly provided by H. Ishihara (Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan) .
Preparations and solutions
The study was approved by our Commission d’Ethique d’Expérimentation Animale. Islets from 4–6-month-old female NMRI mice were obtained by collagenase digestion of the pancreas. Clusters of cells were prepared by dispersion of isolated islets with trypsin and plated on glass coverslips (n = 1.53) in RPMI-1640 medium containing 10 mmol/l glucose. After overnight culture, clusters were infected during 4 h with the D3cpv or LynD3cpv and Rip-Cre adenoviruses, and the experiments were performed 1 day after (i.e. after 2 days of culture). Intact islets were infected for 4 h with both LynD3cpv and Rip-Cre adenoviruses immediately after isolation, and the experiments were performed 1 day after (i.e. after 1 day of culture).
The medium used for islet isolation was a bicarbonate-buffered solution containing (mmol/l): NaCl 120, KCl 4.8, CaCl2 2.5, MgCl2 1.2 and NaHCO3 24. It was gassed with O2:CO2 (94:6) to maintain pH 7.4 and was supplemented with 1 mg/ml bovine serum albumin and 10 mmol/l glucose. A similar medium was used for the experiments. Increases in KCl concentration to 30, 32, 35 or 100 mmol/l were compensated for by decreases in NaCl.
A ×100 lens with a 1.49 numerical aperture was used. For the experiments with D3cpv or LynD3cpv, a 405 nm laser was used as excitation source (Roper Scientific, Evry, France) and the emitted light was filtered at both 475 nm (cyan fluorescent protein) and 540 nm (circularly permuted venus [cpv]) (see ESM 1 for details). The experiments were performed in a temperature-controlled perifusion chamber, and islets and clusters of cells were constantly perifused at 37°C.
Cells were fixed for 3 h with 4% paraformaldehyde and immunostained for insulin with a mouse monoclonal antibody (Abcam, Cambridge, UK), or for glucagon with a rabbit polyclonal antibody (Novocastra, Zaventem, Belgium), and subsequently revealed with either an Alexa Fluor 594 goat anti-mouse IgG or goat anti-rabbit IgG (Molecular Probes, Eugene, OR, USA), respectively.
Images were captured on a Nikon Eclipse TE2000-E inverted microscope equipped with a confocal QLC100 spinning disk (Visitech International, Sunderland, UK), using a ×100 oil immersion objective.
In some [Ca2+]SM experiments effluent fractions were collected every 2 min and saved for insulin assay using rat insulin as a standard. Changes in insulin secretion are expressed as a percentage of basal values. [Ca2+]SM measured in a field containing up to six cells of the same coverslip is therefore representative of the changes occurring in the ∼8,000 cells from which insulin secretion was measured in parallel.
Data are shown as representative traces or means ± SE of results obtained with the indicated number of cells and experiments. The statistical significance of differences between means was assessed by paired or unpaired Student’s t test, as appropriate.
The cytosolic Ca2+ indicator D3cpv is preferentially expressed in beta cells
TIRF microscopy detects [Ca2+]SM and D3cpv can be used as a reporter of [Ca2+]SM changes in clusters of beta cells
To ascertain that our TIRF measurements detect [Ca2+]SM, we looked for [Ca2+]SM ‘hot spots’ elicited by opening of voltage-dependent Ca2+ channels. Using a fast Ca2+ probe, Fluo-4, a very fast acquisition rate (100–150 Hz) and intracellular EGTA to inhibit Ca2+ wave diffusion , Ca2+ ‘hot spots’ were detected at the onset of depolarisation with KCl (ESM Video clip, ESM Fig. 1). Such events were not seen by global [Ca2+]c measurements. While this detection establishes the adequacy of our TIRF system to measure [Ca2+]SM, the necessary experimental tricks (cytosolic Ca2+ buffering with EGTA, strong laser illumination) and the use of a non-ratiometric probe are incompatible with the long recordings requested to study metabolic amplification by glucose. We therefore used the ratiometric probe D3cpv and TIRF microscopy  (Fig. 1e, middle panel). Because the evanescent field excites D3cpv over a very thin section close to the coverslip, the technique visualises regions of contact between the plasma membrane and the coverslip, which cannot be seen in epifluorescence mode (Fig. 1e middle, vs Fig. 1e left and right panels). As the plasma membrane does not sit flat on the glass coverslip, some protrusions out of the TIRF field were also visible.
The amplifying pathway does not involve an increase in [Ca2+]SM in beta cells
To verify that the avid uptake of Ca2+ by the endoplasmic reticulum did not mask a slight increase in [Ca2+]SM, clusters of beta cells were pretreated with thapsigargin, a blocker of sarco(endo)plasmic reticulum Ca2+-ATPases. Thapsigargin markedly attenuated the initial decrease in [Ca2+]SM produced by high glucose (p < 0.01), and [Ca2+]SM returned to prestimulatory values after ∼3 min of glucose stimulation in nearly all cells (21/25 cells) (Fig. 4a, thin trace). The four remaining cells showed a marginal [Ca2+]SM increase above baseline (3.8 ± 0.3%) after the transient decrease. At the same time, insulin secretion was stimulated slightly more than in cells not treated with thapsigargin (Fig. 4b, white circles). To ascertain that no effect of glucose on [Ca2+]SM escaped detection because of localised ‘hot spots’ within patches of plasma membrane, we randomly analysed regions of interest within cells and increased the frequency of acquisition (every 800 ms instead of every 5 s). This approach did not reveal any localised increase in [Ca2+]SM (not shown).
When the amplifying pathway was studied by increasing the glucose concentration from 3 to 15 mmol/l in the presence of 30 mmol/l KCl and diazoxide, a transient decrease in [Ca2+]SM occurred in most cells (9/13), followed by a return to prestimulatory values (Fig. 5f), and no cell showed an increase. Pretreatment with thapsigargin abrogated the initial decrease in 50% of cells (6/12) while [Ca2+]SM returned to prestimulatory values (0.934 ± 0.005 vs 0.944 ± 0.006, ns) in the majority of cells (8/12 cells) (Fig. 5 g). Only four cells showed a marginal [Ca2+]SM increase above baseline (2.2 ± 0.3%) after the transient decrease. The detection of a thapsigargin-inhibitable transient drop in [Ca2+]SM by LynD3cpv is attributed to the close proximity of the endoplasmic reticulum to the plasma membrane [32, 33]. Control experiments further showed that the sustained elevation of [Ca2+]SM achieved under these conditions is entirely dependent on a continuous influx of extracellular Ca2+ (Fig. 5h) via L-type Ca2+ channels blocked by nimodipine (Fig. 5i), both at low and high glucose, i.e. when amplification of insulin secretion does not or does operate.
Glucose-induced insulin secretion by beta cells is mediated by a triggering pathway that leads to an indispensable elevation of [Ca2+]c via KATP channel-dependent depolarisation of the plasma membrane, and by an amplifying pathway that increases the exocytotic response [1, 7]. Here we show that this second effect of glucose does not involve a further increase of [Ca2+]SM in mouse beta cells. Our observations therefore support the proposal that this pathway is a true amplification of the action of Ca2+ rather than an increase in the triggering Ca2+ signal via a KATP channel-independent mechanism.
The method of [Ca2+]SM measurement
In several cell types, the TIRF technique has been used to measure [Ca2+]SM changes with classical Ca2+ probes (Kd 1–14 µmol/l) [34, 35, 36, 37]. To perform similar measurements in beta cells we initially tested several Ca2+ indicators with high (Fluo3, Fluo4, Calcium green 1) or low (Fluo-5N, Mag-Fluo4) affinities for Ca2+, but found it unreliable to employ a non-ratiometric probe to monitor small changes in [Ca2+]SM over long periods (M.A.R. and P.G. unpublished data). We therefore used the genetically encoded ratiometric Ca2+ indicator D3cpv, a cameleon protein that has been modified to suppress interactions with endogenous calmodulin  and that was used to detect Ca2+ changes evoked by single action potential in cortical neurons . Although the in vitro Kd for Ca2+ of D3cpv (∼0.76 µmol/l)  is lower than the estimated [Ca2+]SM (2–20 µmol/l) [12, 15, 20, 21, 39], stepwise increases of KCl (30, 32, 35 or 100 mmol/l) clearly established that the probe was not saturated by the high [Ca2+]SM imposed by 30 mmol/l KCl. The reported in vitro Kd of the indicator  might be modified by the intracellular environment and be higher in vivo.
TIRF microscopy is better able to resolve event signals at or just beneath the plasma membrane than confocal microscopy because the technique restricts the fluorescence excitation to a thin section above the coverslip (∼65 nm thick in our experimental conditions, see ESM 1). A remote pitfall would be that localised increases of [Ca2+] beneath the plasma membrane still escaped detection because they occurred in an even thinner and confined region in the z-axis. We could refute this possibility by using another technique, in which the probe was targeted to the plasma membrane (LynD3cpv) , preferentially into cholesterol-rich membrane regions (membrane rafts)  where exocytotic proteins (such as SNAP-25 and syntaxin1A) and Cav1.2L-Type Ca2+ channels are concentrated in beta cells [3, 31]. Indeed, both approaches (D3cpv and LynD3cpv) yielded similar results.
In dispersed islet cells, adenoviral production of D3cpv was under control of the cytomegalovirus promoter. The probe was mainly produced in beta cells rather than non-beta cells, and the level of expression in alpha cells was too low for TIRF measurements. This preferential expression could be the result of a lower capacity of alpha cells to be infected by the adenovirus or that the cytomegalovirus is not a strong promoter in alpha cells. We are therefore confident that the cells that we imaged were beta cells, even more so because they all displayed the characteristic triphasic changes in [Ca2+] when stimulated by high glucose. Moreover, similar results were obtained when production of the probe was controlled by the rat insulin promoter and therefore was specific to beta cells.
Amplification of insulin secretion by glucose does not involve an increase in [Ca2+]SM
The ability of glucose and other metabolised secretagogues to increase insulin secretion by a mechanism that is distinct from their action on KATP channels has now been observed in many laboratories, using different preparations, including human islets [40, 41]. This property has most often been studied under conditions where KATP channels were held closed by a high concentration of sulfonylurea [42, 43] or held opened by diazoxide in the presence of KCl [4, 5]. Under these conditions, which stabilise [Ca2+]c at an elevated level, glucose is able to increase insulin secretion with no further increase in global [Ca2+]c (KCl + diazoxide)  or only a minor one (sulfonylureas) . Comparisons of the spontaneous increases in [Ca2+]c and insulin secretion produced by various glucose concentrations in mouse islets  or dispersed islet cells  also indicate that Ca2+ is not the only signal produced by glucose to control the beta cell secretory response. Because this second pathway of glucose stimulation manifests itself only when [Ca2+]c is high, it would seem appropriate to define it as an amplification of the effect of the triggering Ca2+ signal on secretion . Although the increase in insulin secretion produced by glucose during depolarisation with KCl does not involve an increase in global [Ca2+]c, the possibility that a local increase in [Ca2+] is occurring beneath the plasma membrane has not previously been addressed. If this were the case, the second pathway of glucose action would not be a true amplification but a further change in the triggering signal. The results of the present study refute this hypothesis. Therefore, in the presence of 30 mmol/l KCl + diazoxide, no transient or steady-state increase in [Ca2+]SM was observed in any of the tested cells whereas insulin secretion was doubled. Inhibition of Ca2+ uptake in the endoplasmic reticulum, by blockade of sarco(endo)plasmic reticulum Ca2+-ATPases with thapsigargin, attenuated the lowering of [Ca2+]SM that glucose produces in depolarised beta cells without unmasking an increase. That changes in Ca2+ uptake by the endoplasmic reticulum are detected during measurements of [Ca2+]SM is not surprising owing to the close contact between the plasma membrane and the endoplasmic reticulum [32, 33]. The thapsigargin-resistant component of the transient fall in [Ca2+]SM induced by 15 mmol/l glucose could reflect Ca2+ uptake by PMR1  or in other organelles also in close proximity to the plasma membrane , but cannot be attributed to outward transport of Ca2+ across the plasma because no acceleration of 45Ca2+ efflux occurs under these conditions .
The D3cpv fluorescence appeared uneven beneath the plasma membrane when monitored at a single wavelength (Fig. 1e, middle), but this heterogeneity disappeared when the ratio of both emission wavelength (540/475 nm) was performed, indicating uneven distribution of the probe rather than spatial heterogeneity in [Ca2+]SM. Under conditions testing the amplifying pathway, no ‘hot spots’ of [Ca2+] were detected beneath the plasma membrane. Indeed, detection of ‘hot spots’ of [Ca2+] requires special tricks such as very fast image acquisition, abrupt depolarisation and restriction of Ca2+ diffusion by introduction of high concentrations of Ca2+ buffers intracellularly [17, 34, 35, 37]. By using these tricks, we could detect Ca2+ ‘hot spots’ during the initial response to high K+ (ESM Video clip, ESM Fig. S1), but [Ca2+]SM was diffusely high, and ‘hot spots’ of Ca2+ were already no longer visible after 20 s of depolarisation. None of these technical tricks could be used in our long experiments in which clusters of cells or islets were stably depolarised with 30 mmol/l KCl + diazoxide. It is noteworthy that, under these conditions, no action potentials occurred in either low or high glucose concentrations . Even if we failed to detect glucose-induced increases in [Ca2+]SM because these were minimal, smaller or more localised than those elicited by a rise from 30 to 32 mmol/l KCl, it is not plausible that such tiny changes are sufficient to double the rate of insulin secretion. On the other hand, we do not exclude the possibility that ‘hot spots’ of [Ca2+]SM [34, 35, 36, 37], occurring at the mouth of Ca2+ channels [12, 13, 15], play a role during glucose-induced electrical activity under control conditions.
In conclusion, the present results validate our proposal that glucose metabolism amplifies insulin secretion by an action that requires high [Ca2+]c but is independent of a further increase in [Ca2+]SM [1, 4, 7]. This is of major importance not only for our understanding of beta cell stimulus–secretion coupling, but also for the identification of the functional defects in beta cells from type 2 diabetic patients (and perhaps for the identification of novel drug targets). Other approaches using single beta cells with clamped [Ca2+]c have also established the existence of regulatory sites distal to the increase in the triggering Ca2+ signal [48, 49, 50]. Further studies should focus on the molecular and cellular mechanisms of this amplification that substantially contributes to glucose-induced insulin secretion .
This work was supported by the Fonds de la Recherche Scientifique in Brussels (grants FNRS 3.4530.08 and 3.4569.06), the Belgian Science Policy (PAI 6/40), the Direction de la Recherche Scientifique of the French Community of Belgium (ARC 05/10-328), and a research grant to M. A. Ravier from the ALFEDIAM (Paris). During this study, M. A. Ravier was ‘Chargée de Recherches’ of the FNRS, Brussels and, from March 2009, CR1 of the INSERM, Paris. P. G. Gilon is ‘Directeur de Recherches’ of the FNRS, Brussels. We thank J. Carpent, F. Knockaert and L. Roma for technical assistance. We are grateful to H. Ishihara and P. L. Herrera for the gift of the Rip-Cre adenovirus and pBS302 plasmid, respectively, and to I. Parker for advice related to the detection of ‘hot spots’ of Ca2+.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.