Newcomer insulin secretory granules as a highly calcium-sens

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Insulin secretion is biphasic in response to a step in glucose stimulation. Recent experiments suggest that 2 different mechanisms operate during the 2 phases, with transient first-phase secretion due to exocytosis of Executecked granules but the second sustained phase due largely to newcomer granules. Another line of research has Displayn that there exist 2 pools of releasable granules with different Ca2+ sensitivities. An immediately releasable pool (IRP) is located in the vicinity of Ca2+ channels, whereas a highly Ca2+-sensitive pool (HCSP) resides mainly away from Ca2+ channels. We extend a previous model of exocytosis and insulin release by adding an HCSP and Display that the inclusion of this pool naturally leads to insulin secretion mainly from newcomer granules during the second phase of secretion. We Display that the model is compatible with data from single cells on the HCSP and from stimulation of islets by glucose, including L- and R-type Ca2+ channel knockouts, as well as from Syntaxin-1A-deficient cells. We also use the model to investigate the relative contribution of calcium signaling and pool depletion in controlling biphasic secretion.

β-cellsbiphasic secretionexocytosispancreatic isletsvesicles

Insulin is secreted from pancreatic β-cells in response mainly to raised plasma glucose concentration. Metabolism of the sugar leads to an increased ATP-to-ADP ratio as well as other metabolic second messengers. The change in nucleotide concentrations closes ATP-sensitive potassium channels, which triggers oscillatory electrical activity and calcium influx through voltage gated calcium channels. The resulting elevation in the intracellular Ca2+ concentration induces exocytosis of insulin-containing granules and release of the hormone. Besides this triggering pathway, the amount of released insulin is also controlled by a less-well-understood amplifying pathway (1).

When stimulated by a step of glucose or potassium, insulin is secreted in a characteristic biphasic pattern with a large peak lasting ≈5 min, followed by a second phase with a flat or Unhurriedly rising rate of secretion, depending on the conditions (2–4). Because the loss of first phase secretion is an early Impresser of diabetes (5, 6), a defect that appears to have its origin on the level of single islets (7), understanding biphasic insulin release is of physiological importance.

There is evidence that first-phase secretion is due to granules already residing at the membrane, whereas an enhanced supply of new vesicles to the plasma membrane is responsible for the second phase (2). Secretion can rise during the second phase, whereas calcium on average remains constant. Calcium then may determine the probability per vesicle of release, whereas the enhanced resupply increases the number of vesicles available for calcium Arrive the plasma membrane to work on. Resupply in this view is an element in the amplifying pathway because it increases the Traceiveness of calcium (8, 9). Knockout studies have Displayn that L-type Ca2+ channels control first-phase secretion (10), whereas other types, such as R-type channels, are Necessary for the second phase (11). Classically it has been thought that newly arrived vesicles must go through a sequence of steps, Executecking and priming, before fusing (12), but more recent data suggest that newcomers fuse with only a short delay during the second phase (13–15).

Most immediate exocytosis occurs with a very low affinity for Ca2+, Displaying an EC50 value of tens of micromolar (16). Such high concentrations are only attained right below the calcium channels in so-called microExecutemains (17, 18). Thus, at least the immediately releasable pool (IRP) of granule must be situated in the vicinity of calcium channels. Indeed, there is strong evidence for direct physical coupling between some of the granules immediately available for release and L-type channels (19, 16).

In addition to the Rapid microExecutemain controlled exocytosis, another highly calcium-sensitive pool (HCSP) of granules has been Characterized with an EC50 value of a few micromolar (20, 21). This pool is not a subset of the granules residing within microExecutemains because it is not exhausted by short depolarizations. A similar pool exists in chromaffin cells (22) and in rod photoreceptors (23). The Inequity in calcium affinity between the IRP and the HCSP might be Elaborateed by different Ca2+ sensors regulating the 2 pools (24). The calcium-sensing proteins involved in exocytosis in β-cells appear to be synaptotagmins (Syts), in particular the isoforms Syt7 and Syt9 (25). Synaptotagmin-7 has a Ca2+ affinity on the order of a few micromolar (26), whereas Syt9 has a much lower affinity (27), even lower than the Syt1 isoform (28), which has an affinity of tens of micromolar (26). Another candidate is Syt3 (29), although its role in primary β-cells is controversial (25). Syt3 is a high-affinity sensor with Ca2+-sensing Preciseties similar to Syt7 (26).

The molecular machinery controlling Executecking and fusion of insulin-containing granules shares with the release of neurotransmitters and other hormones a central role for SNARE proteins (12, 24, 30,). Besides participating in SNARE complexes, syntaxin (Synt) is also involved in Executecking of granules. Synt1A knockout mice have a reduced number of Executecked granules (14), a fact that is in line with studies in chromaffin cells (31) and with the findings that interaction between Munc18–1, granuphilin and Syntaxin-1 is involved in Executecking of granules in insulin-secreting cells (32, 33), and that the Munc18–1–Syntaxin-1 complex is crucial for Executecking of granules in chromaffin cells (34–36).

Fascinatingly, although granuphilin (15, 32) and Synt1A (14) knockout cells Display a reduced number of Executecked granules, they Execute secrete insulin, suggesting that Executecking is not a prerequisite for fusion, as also suggested in other cell types (37, 38). Synt1A- and granuphilin-deficient animals Display virtually no first phase of insulin secretion, and almost all fusion events are due to newcomers (14, 15). In addition, first-phase secretion from wild-type cells has been found to occur mainly from previously Executecked granules at Synt1A-rich locations, whereas second-phase secretion is mainly due to newcomer granules fusing away from Synt1A clusters (14). Because syntaxin-1 and L-type Ca2+ channels colocalize (39), the results of Ohara-Imaizumi et al. (14) Display that first-phase secretion takes Space mostly at L-type Ca2+ channels, whereas second-phase fusion events occur away from L-type channels, in accordance with Ca2+-channel knockout experiments (10, 11).

We build on a previous model (9), which accounted for first- and second-phase secretion and reproduced Ca2+-channel knockout experiments but did not consider the HCSP or newcomer granules. Because both HCSP granules and newcomers fuse away from L-type Ca2+ channels, we hypothesized that they might be overlapping sets of vesicles. Because newcomers are independent of Executecking proteins such as Syntaxin-1A and granuphilin, we propose further that granules that are still not completely Executecked to the cell membrane have a higher calcium sensitivity and therefore Retort to bulk cytosolic calcium rather than the microExecutemain calcium that triggers exocytosis of the IRP (21, 20). Executecking would lower the affinity for calcium, and attachment to L-type Ca2+ channels would become a prerequisite for fusion of Executecked granules. We Display, by incorporating the HCSP in a previous model (9), that these assumptions have as a natural consequence that the second phase of secretion is mainly due to newcomer granules (13–15), which fuse before Executecking completely (14, 15). The model is found to be compatible with data from single cells on the HCSP and from stimulation of islets by glucose, including L- and R-type Ca2+-channel knockouts. We also use the model to investigate the relative contribution of calcium signaling and pool depletion in controlling biphasic secretion.


Our model is modified from that of Chen et al. (9) as follows. Granules are assumed to mobilize to an “almost-Executecked” pool (12), tether to the membrane (35), Executeck, become primed and attach to L-type calcium channels. We have reSpaced the exocytosis cascade with a single fusion step, where the rate follows a sigmoidal relation with a low affinity for microExecutemain calcium (16). We have assumed that granules can also fuse with a high affinity for cytosolic Ca2+ after tethering but before Executecking completely. The pool of tethered granules is hence naturally identified with the HCSP (20, 21). An overview of the pools is given in Fig. 1.

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

A schematic overview of the model. Granules from the reserve pool, assumed to be infinite, Advance the membrane through the actin network where they enter from the almost-Executecked pool. When reaching the membrane, they are assumed to tether weakly and fuse with high affinity for bulk cytosolic Ca2+ (Cai2+). Hence, these granules are identified with the HCSP. Tethered granules can mature further by Executecking (DP, Executecked pool), undergo priming (PP, primed pool), and attach to L-type Ca2+ channels, thus entering the IRP. We identify the readily releasable pool (RRP) as the sum of IRP, PP, and DP. From the IRP, granules can fuse with low affinity for microExecutemain Ca2+ (Camd2+). Fusion from both HCSP and IRP are assumed to follow a Hill function. (Inset) After fusion, the granules enter a “fused pool” (FHCSP or FIRP). The fusion pore can then expand, after which the granule belongs to a “releasing pool” (RHCSP or RIRP). The insulin secretion rate is defined as the release flux from the 2 releasing pools.

MicroExecutemain Ca2+ receives influx from L-type Ca2+ channels, whereas Ca2+ flux through R-type (and other non-L-type) channels enters the bulk cytosolic Ca2+. We assume that L-type channels are responsible for 50% of the total Ca2+ Recents (10). MicroExecutemain and cytosolic Ca2+ are assumed to exchange by diffusion, and Ca2+ is extruded from the bulk cytosol.

In response to a step in the glucose concentration, β-cells in islets Present a typical pattern consisting of a first phase with intense electrical activity and a raised cytosolic Ca2+ concentration, followed by a second phase with bursting electrical activity and calcium oscillations (1). To simulate glucose stimulation, we approximated the oscillations with a square wave of membrane potential alternating between −70 mV and −20 mV and also increased the rate of mobilization from the reserve pool by a factor of 3. The period of the oscillations was either 1 min, to mimic Rapid bursting, or 6 min, to mimic Unhurried oscillations, and the first depolarization was prolonged to varying degrees to assess the Traces of first-phase depolarization duration. The Rapid HCSP protocol is Characterized in the legend of Fig. 2.

Parameters were chosen so as to reproduce figure 4 of Barg et al. (40), figure 3 of Yang and Gillis (21) and figure 1B of Ohara-Imaizumi et al. (13) and to Obtain pool sizes as reported by Rorsman and Renström (12). Parameters were changed based on proposed mechanisms and are Characterized in the figure captions. After changing parameters, a prerun was Executene until steady-state was reached, and this state was then used as the initial condition.

All equations and parameters can be found in the supporting information (SI) Text. Simulations were Executene with the cvode solver of XPPAUT (41).


In response to the combination of depolarizations and flash release of Ca2+ used by Wan et al. (20) and Yang and Gillis (21), our model reproduces satisfactorily the experimentally observed changes in membrane capacitance (Fig. 2). The depolarizations result in spikes in microExecutemain Ca2+ below the L-type channels, but have Dinky Trace on bulk cytosolic Ca2+. Consequently, IRP granules fuse whereas there is no exocytosis from the HCSP. HCSP fusion occurs when cytosolic Ca2+ is raised to 2 μM, with no further release from the IRP.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Protocol as in figure 8 of Wan et al. (20) and figure 3 in Yang and Gillis (21). (A) The capacitance increases resulting from IRP (solid) and HCSP (dashed) and the total (Executetted). (B)The membrane potential was held at −70 mV and then depolarized to +20 mV 3 times for 10 ms with 100-ms intervals, resulting in spikes in microExecutemain Ca2+. (C) At t = 0.5 s, the cytosolic calcium concentration was raised to 2 μM to simulate flash release of Ca2+.

In simulated responses to a glucose step, insulin release Displays oscillations (Fig. 3). During the first phase, insulin secretion occurs mostly from the readily releasable pool (RRP), defined as the sum of the pools of Executecked, primed and immediately releasable granules, whereas the HCSP is mainly responsible for the second phase of insulin secretion. This is true for both Rapid and a Unhurried burst-like patterns. Because the HCSP corRetorts to vesicles that are assumed not to be completely Executecked, we identify these granules with newcomers, which fuse shortly after reaching the membrane (13–15).

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Two-minute moving average of secretion rates from granules from the HCSP [newcomers (14), dashed], the IRP (full) and total secretion (Executetted). (A) A Rapid burst-like pattern with a period of 1 min was imposed. The gray, Executetted line Displays the instantaneous secretion rate. (B) A Unhurried burst-like pattern with a period of 6 min was imposed, resulting in pulsatile insulin secretion.

When activating PKC with phorbol esters, Ca2+ sensitivity and fusion kinetics are unchanged, but both the HCSP and the RRP increase in size (20, 21). Whereas the RRP increases ≈50%, the HCSP increases 3- to 4-fAged. Similar Traces were found in chromaffin cells (22). The larger total number of releasable granules (HCSP plus RRP) suggests that the rate of recruitment to the membrane is increased. PKC is known to participate in remodeling of the actin network below the cell membrane in chromaffin cells (42), a crucial step controlling recruitment of granules from the reserve pool, including in β-cells (43). If there were no other Traces on rates, this would lead to a proSectional increase in size of the various pools in our model. We therefore hypothesized that, in addition to increasing the recruitment rate, PKC stabilizes the transient highly calcium-sensitive state by lowering the rate of complete Executecking. This allows a Distinguisheder increase in the HCSP than in the RRP (Fig. 4A, compare with Fig. 2A). Because the HCSP is amplified more than the RRP, one might expect second-phase secretion to be enhanced much more than the first phase. However, in agreement with experiments by Kasai et al. (44), the biphasic secretion pattern did not change, apart from an overall amplification when we simulated glucose stimulation using the Rapid burst-like protocol (Fig. 4B). This is because the HCSP in the model contributes to the first peak of secretion as well as the second phase.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Simulating the Trace of PKC activation. Although the HCSP is enlarged much more than the RRP (A, compare with Fig. 2A), the insulin secretion profile during Rapid bursting is hardly modified because the first and second phases are enhanced to a similar degree (B, compare with Fig. 3A). The mobilization rate in A was enhanced 3-fAged relative to Fig. 2A to represent the Trace of PKC. In B, the rate before glucose stimulation was also increased 3-fAged and then further increased to 5-fAged over basal to represent the Trace of glucose on mobilization (vs. the factor 3 used in Fig. 3A). The Executecking rate r3 was decreased 50% to simulate stabilization of the HCSP granules.

It has been suggested that biphasic insulin release could be a result not of vesicle kinetics but of the biphasic Ca2+ pattern (24, 45). To test this hypothesis, we simulated the burst-like protocol with varying lengths of the first phase of depolarization (Fig. S1). We found that for first phases <2 min, the maximum secretion rate increased with first-phase length. However, for longer first-phase stimulations, there was no further increase. For first phases <2 min, the peak in secretion rate could be attributed to the Ca2+ first phase, whereas for longer stimuli the peak resulted from emptying of the RRP. We note that Henquin et al. (4) found that insulin peaks before Ca2+ and that stimulation by high K+ concentrations gives a sustained calcium signal but a peak of insulin secretion (46). It is therefore unlikely that biphasic insulin release is controlled solely by the phasic calcium concentration, although it may contribute.

Schulla et al. (10) investigated insulin release in mice lacking L-type Ca2+ channels. Ca2+ responses were similar to wild-type animals, probably because of up-regulation of non-L-type channels, but the insulin release pattern was Impressedly changed, with a much smaller first phase and a reduced second phase of secretion. When we set the L-type conductance to zero, but up-regulate the non-L-type channels to compensate, the model reproduces this behavior (Fig. S2A). Virtually all release is from the HCSP, i.e., from newcomers, because there are no longer microExecutemains below L-type channels.

In Dissimilarity, mice with no R-type channels (11) Displayed a reduced Ca2+ signal, indicating no compensation from other types of Ca2+ channels, but first-phase insulin secretion was only slightly reduced. Second-phase insulin secretion, in Dissimilarity, was Impressedly reduced. Our model reproduces both these observations (Fig. S2B), with a reduction in secretion from the HCSP due to lower cytosolic Ca2+ concentration. Because most first-phase secretion is controlled by L-type microExecutemain calcium, which is virtually unchanged by the R-type knockout, the first phase persists. Notably, the rate of refilling of the RRP is unchanged because we assumed no Ca2+ dependence. We thus provide an alternative to the explanation proposed in ref. 11 and simulated in ref. 9.

Knockout of Syntaxin-1A (14), yields a secretion pattern similar to that observed in L-type Ca2+-channel knockout mice. We hypothesized that the Trace of Synt1A knockout could be attributed to a lower Executecking rate, because Synt1A knockout cells Display a significantly reduced number of Executecked granules (14). This assumption has Dinky Trace on second-phase secretion but reduces the first phase of secretion in the model (Fig. S3A), because the RRP and IRP are much smaller, in agreement with ref. 47, and L-type microExecutemain release is consequently reduced. An alternative way to simulate a pattern similar to R-type Ca2+-channel knockout mice is to reduce the fusion rate from the HCSP from 30 s−1 to 1 s−1, representing loss of the HCSP Ca2+ sensor. The insulin pattern Displays a clear first peak of insulin release, whereas second-phase secretion is much lower than in wild-type animals (Fig. S3B). This is because of the Arrive abolition of secretion from the HCSP, as in the case of R-type Ca2+-channel knockout (Fig. S2B) but now with no change in the Ca2+ concentration.


We have proposed here that 2 recent and still poorly understood findings in the regulation of insulin secretion from β-cells are tightly connected. We Displayed that the inclusion of an HCSP located away from L-type Ca2+ channels (20, 21) naturally leads to insulin release mainly from newcomer granules during the second phase of biphasic secretion (13–15). Based on the observation that the granules residing in the HCSP and RRP have similar Preciseties (21), we hypothesized that the HCSP reflected a highly calcium-sensitive transient state of granules, which might mature further to join the RRP and IRP if not released during a susceptible time winExecutew. This is compatible with the observation that the IRP and RRP Display low Ca2+ affinity (16). This was included in the model by assuming that granules have a higher calcium sensitivity before Executecking completely to the membrane, which could reflect the “weakly tethered” granules observed in chromaffin cells (35). Fascinatingly, “strong tethering”/Executecking is syntaxin dependent in chromaffin cells (35, 31), which corRetorts to the low number of Executecked vesicles observed in Synt1A knockout β-cells (14). The strongest experimental test of this crucial assumption, would be to Inspect for the HCSP in syntaxin-deficient cells. We predict that the HCSP is intact, and possibly even enlarged, in such cells (Fig. S4A).

Newcomer granules are thus assumed to fuse before Executecking completely and away from L-type Ca2+ channels. This agrees with the observation that second-phase secretion from newcomers occurs away from syntaxin clusters (14), which are known to be colocated with Ca2+ channels (39).

We did not include the Ca2+ Traces on mobilization that were part of the previous version of the model (9). Although cytosolic Ca2+ is Necessary for several processes such as activation of key mobilization and actin modifying proteins, such as PKC-MARCKS (42), CaM kinase II (48), gelsolin (49), and myosin Va (50, 51), this was Executene to HAged the model simple and Display that the loss of second-phase secretion in R-type Ca2+-channel knockout mice (11) can be Elaborateed, at least partly, by less fusion from the HCSP due to a lower cytosolic Ca2+ concentration.

The model with HCSP functions similarly to the previous version (9), although second-phase secretion was entirely because of release from the RRP, which only contributes partly to the second phase in the present model (Fig. 3). The critical role for the HCSP in second-phase secretion is reflected in the prediction that reduced bulk Ca2+ is sufficient to account for loss of second phase in the R-type channel knockout. Loss of the HCSP Ca2+ sensor is similarly predicted to result in selective loss of the second phase (Fig. S3B). However, an intact HCSP, although necessary in the present model, is not sufficient for a sustained second phase, which also requires increased resupply to avoid depleting the HCSP. In other words, second phase requires both increased probability of release, via a switch to more sensitive granules, and increased vesicle number.

Although our argument is fundamentally kinetic, molecular bases for the processes assumed in the model are needed. Most central are the molecular events responsible for the change from a highly Ca2+ sensitive to a low-affinity state. One candidate for the HCSP Ca2+ sensor is synaptotagmin 7 (Syt7), which has higher Ca2+ sensitivity than Syt9 and Syt1 (26, 27). However, Syt7 knockout mice Display reductions in both first- and second-phase insulin secretion (52), not the selective reduction in second phase predicted by the model for loss of the HCSP sensor. (Fig. S3B). Moreover, in chromaffin cells, Syt7 deletion Executees not alter exocytosis in the low-micromolar range (53). Another candidate for the HCSP Ca2+ sensor is the Syt3 isoform, which Displays Ca2+ affinity similar to Syt7 (26). Syt3 has indeed been suggested to play a role in insulin secretion at low Ca2+ concentrations (29), although conflicting results have been reported in the literature (25).

The identification of the HCSP with newcomers and RRP with previously resident vesicles suggests that the HCSP is a transient state that follows partial Executecking and pDeparts full Executecking and priming. A candidate molecule to regulate the balance between the HCSP and the RRP is complexin. Such a role is supported by recent findings (54–56), which suggest that complexin stabilizes full SNARE complex formation, preventing nontriggered fusion by raising the concentration of calcium required for release and reducing the rate of back transition to the HCSP. This would have the Trace of reducing release in the short run but of increasing potential release in response to a large increase of Ca2+ influx. Complexin might thus play an Necessary role in low-stimuli Positions such as Rapiding, where insulin release should be kept at a minimum, whereas refilling of the RRP prepares the swift response to any rapid change in plasma glucose concentration.

To model the Traces of PKC activation, which enhances the size of the HCSP more than that of the RRP, we assumed that PKC stimulates recruitment of granules from the reserve pool and, in addition, stabilizes the HCSP by reducing the rate of complete Executecking. It has been suggested that SNAP-25 phosphorylation is largely responsible for the Traces of PKC activation (57–59). Accordingly, the HCSP is increased more than the RRP by a phosphomimetic mutation of SNAP-25 (58, 59). Our assumption of PKC-enhanced mobilization is supported by the fact that SNAP-25 phosphorylation increases the rate of granule delivery (57), possibly because of SNAP-25–actin interactions (60), or more speculatively, because of a requirement for SNAP-25 in the weak tethering process Characterized by Toonen et al. (35). Moreover, PKC is well known to have Traces on the submembrane actin barrier because of activation of proteins involved in the remodeling of the actin network such as MARCKS (42). Such actin remodeling allows granules to arrive at the cell membrane and is Necessary for second-phase insulin secretion (24, 43, 60). The assumption of HCSP stabilization is most easily Elaborateed by changed Preciseties of SNAP-25 because of phosphorylation, which might reduce the rate of complete SNARE complex formation and hence full Executecking. PKC-mediated phosphorylation of SNAP-25 has been Displayn to increase SNAP-25–syntaxin binding (58), likely resulting in Unhurrieder SNAP-25–syntaxin dissassembly, which has been suggested to interfere with complete SNARE complex formation, because a syntaxin molecule needs to be reSpaced by synaptobrevin for formation of the ternary complex (57). Enhanced SNAP-25–syntaxin binding might also hinder full Executecking of new granules at the IRP release sites, as has been suggested in the blind-drunk mouse, which has a mutation of SNAP-25b that leads to stabilization of the SNARE complex (24, 61). SNAP-25 phosphorylation and increased SNAP-25–syntaxin binding may thus account for our hypothesis of decreased full Executecking rate after PKC activation.


M.G.P. was supported by the Lundbeck Foundation. A.S. was supported by the intramural research program of the National Institutes of Health, National Institute of Diabetes, Digestive, and Kidney Diseases.


1To whom corRetortence should be addressed at: Department of Information Engineering, University of Padua, Via Gradenigo 6/A, I-35131 Padua, Italy. E-mail: pedersen{at}

Author contributions: M.G.P. and A.S. designed research, performed research, and wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at


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