Insulin granule dynamics in pancreatic beta cells
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Glucose-induced insulin secretion in response to a step increase in blood glucose concentrations follows a biphasic time course consisting of a rapid and transient first phase followed by a slowly developing and sustained second phase. Because Type 2 diabetes involves defects of insulin secretion, manifested as a loss of first phase and a reduction of second phase, it is important to understand the cellular mechanisms underlying biphasic insulin secretion. Insulin release involves the packaging of insulin in small (diameter ≈0.3 µm) secretory granules, the trafficking of these granules to the plasma membrane, the exocytotic fusion of the granules with the plasma membrane and eventually the retrieval of the secreted membranes by endocytosis. Until recently, studies on insulin secretion have been confined to the appearance of insulin in the extracellular space and the cellular events preceding exocytosis have been inaccessible to more detailed analysis. Evidence from a variety of secretory tissues, including pancreatic islet cells suggests, however, that the secretory granules can be functionally divided into distinct pools that are distinguished by their release competence and/or proximity to the plasma membrane. The introduction of fluorescent proteins that can be targeted to the secretory granules, in combination with the advent of new techniques that allow real-time imaging of granule trafficking in living cells (granule dynamics), has led to an explosion of our knowledge of the pre-exocytotic and post-exocytotic processes in the beta cell. Here we discuss these observations in relation to previous functional and ultra-structural data as well as the secretory defects of Type 2 diabetes.
KeywordsPancreatic islets insulin secretory granules exocytosis diabetes confocal microscopy
Free cytoplasmic Ca2+-concentration
green fluorescent protein
large dense-core vesicle
readily releasable pool of granules
soluble N-ethylmaleimide-sensitive factor attachment protein receptors
Insulin is the body's only blood glucose-lowering hormone and is secreted by the pancreatic beta cells of the islets of Langerhans. After its synthesis in the endoplasmic reticulum, insulin is processed to its biologically active form and stored in the secretory granules pending its release. Ultra-structural studies have shown that a single beta cell contains more than 10 000 secretory granules [1, 2]. The quantitatively most important route for the release of insulin into the islet interstitium is by regulated Ca2+-dependent exocytosis of the secretory granules [3, 4, 5, 6]. Traditionally, insulin secretion has been measured biochemically using, for example, radioimmunoassays. Such experiments report the amount of insulin being secreted but the temporal resolution is fairly low and the regulation of the steps that precede release cannot be elucidated. During the last decade, several new techniques have emerged to study insulin secretion that allow the release and the pre-exocytotic as well as post-exocytotic events to be monitored at high temporal resolution down to the single-granule [7, 8, 9, 10]. We attempt to summarize the insights that have been derived by applying these new methods to the pancreatic beta cell and discuss these data in relation to systemic regulation of insulin secretion and diabetes.
Electrical activity couples increase in the blood glucose concentration to insulin secretion
Patch-clamp experiments have established that although beta cells contain 10 to 20 different ion channel proteins (each present in 100–5000 copies per beta cell) , two types of ion channels are particularly important for the initiation of insulin secretion: ATP-regulated K+-channels (KATP-channels) and voltage-gated Ca2+-channels (Fig. 1A). The KATP-channels are spontaneously active at low glucose and the efflux of positively charged K+ through these channels generates an excess of negative charges inside the cell and thereby accounts for the negative membrane potential of the unstimulated beta cell (Fig. 1B). Glucose enters the beta cell via the glut2 transporter and the ensuing metabolic breakdown of the sugar leads to the generation of ATP at the expense of ADP. This in turn results in closure of the KATP-channels, membrane depolarisation and initiation of electrical activity. The effect of glucose on KATP-channel activity is concentration-dependent with an EC50-value in intact islets of approximately 5 mmol/l . At insulin-releasing glucose concentrations, the KATP-channels are almost completely inhibited. Action potential firing in the beta cell is dependent on the opening of voltage-gated Ca2+-channels and the resulting increase in [Ca2+]i then triggers exocytosis of the insulin granules [5, 18]. Pancreatic beta cells contain three to four distinct types of Ca2+-channels. However, the L-type Ca2+-channels are particularly important for exocytosis of the insulin-containing granules [19, 20].
Quantitative aspects on insulin secretion
Novel methods to study release
Traditional biochemical assays of exocytosis [such as radioimmunoassay (RIA) or enzyme linked immunosorbent assay (ELISA)] are characterized by a limited temporal and quantitative resolution. At best, secretion can be monitored in single islets (containing ≈1000 cells) with a sample interval longer than1 s . Although this as such represents a considerable accomplishment, the elucidation of the cell biology of islet-hormone secretion requires an even higher temporal resolution.
Carbon fibre amperometry affords the possibility to determine the release of granule content after membrane fusion. Certain oxidizable substances, like catecholamines and serotonin, can be detected electrochemically by carbon microfibres, thus allowing the release of individual secretory vesicles to be resolved at high temporal resolution . A release event typically gives rise to a transient current spike (Fig. 3B). This technique has been successfully applied to beta cells in which the granules have preloaded with serotonin [33, 34, 35, 36]. The carbon fibre technique has subsequently been improved to detect insulin itself . Analysis of the amperometric currents suggests that a single secretory granule in a human beta cell contains 1.7 amol of insulin, which equates to an intragranular insulin concentration of 118 mmol/l. The corresponding values in rat granules were 1.6 amol and 74 mmol/l. Results obtained with serotonin and insulin amperometry are in general agreement but the insulin spikes are somewhat broader than those representing serotonin release. Interestingly, the release of serotonin could be dissociated from that of insulin by lowering extracellular pH from 7.4 to 6.4; whereas serotonin release was still detectable at the lower pH, insulin secretion was abolished . Taken together the latter observations suggest that although membrane fusion can occur at pH 6.4, a pH gradient is necessary to drive release and that dissociation of the Zn-insulin complex limits the rate of hormone release. This scenario is suggested by analogy to the release of catecholamines from chromaffin cells [39, 40].
All the methods discussed above share the weakness of more traditional secretion assays in only reporting that exocytosis has occurred but provide no information about the pre- and post-exocytotic events. This type of information can only be gained by the optical monitoring of individual secretory vesicles before, during and after exocytosis. Despite the small size of the granules, this is in fact possible by engineering chimaeras between green fluorescent protein (GFP) and proteins normally sorted to the secretory granules. The granules rendered fluorescent by transfection of cells with the GFP-constructs can then be visualized using confocal microscopy to study both the release process as well as pre- and post-exocytotic granule movements . Confocal microscopy (Fig. 3C) allows fluorescence in thin sections of the cell (~0.5 µm) to be imaged with little contribution from neighbouring planes. Another possibility is to view the granules using evanescent wave microscopy . The advantage of the latter technique is that only the fluorophore within a few 100 nm from the upper surface of the coverslip is reached by the excitation light. This means that the emitted fluorescence exclusively reflects the dynamics of granules situated within the immediate vicinity of the plasma membrane, avoiding signals from, in this context, irrelevant locations deeper within the cell. Several variants of GFP with distinct spectral properties are now available and it is thus possible to label the granule membrane and the cargo with different constructs and to simultaneously monitor the fluorescence of both compartments in the same vesicle. This provides the means to study the subsequent retrieval of the secreted membranes by endocytosis that must occur following exocytosis.
Molecular machinery of exocytosis in beta cells
Exocytosis in beta cells conforms to the general picture of SNARE-regulated exocytosis. Insulin-secreting cell lines possess the full complement of SNARE proteins that are either very similar or even identical to those participating in synaptic vesicle release [48, 49] and fulfil the same functions. The SNARE proteins also seem to be important for exocytosis in primary beta cells. Using permeabilised mouse beta cells, intracellular application of a monoclonal antibody directed against syntaxin inhibited insulin secretion [50, 51]. There remains uncertainty about the identity of the Ca2+-sensor for exocytosis in beta cells. Although synaptotagmins I and II were initially identified in insulin-secreting cell lines and participate in Ca2+-dependent exocytosis in these cells , primary beta cells do not express either of these synaptotagmins. Synaptotagmin III could play a role  but doubts have been raised as to the specificity of the antibody used in these experiments  and the most recent evidence rather implicates synaptotagmins V or VII . The involvement of a synaptotagmin with a higher Ca2+-affinity would account for the observation that intracellular dialysis with Ca2+-EGTA buffers with free Ca2+-concentrations as low as ≥0.2 µmol/l is capable of evoking exocytosis [55, 56].
A family of small GTP-binding Rab proteins is also involved in the molecular control of regulated exocytosis. Several isoforms of the Rab proteins have been described but the most extensively studied member of this family is Rab3A . This protein cycles between a vesicle-associated GTP-binding form and a cytoplasmic state after exocytosis and hydrolysis of GTP into GDP. In neurones, Rab3a exerts an inhibitory action on neurotransmission by limiting the number of synaptic vesicles released in response to an increase in synaptic [Ca2+]i . In beta cells, Rab3A locates to the cytoplasmic face of the secretory granules. Its functional role in insulin secretion is controversial but most reports are consistent with the idea that it functions as a "brake", thus limiting the release of insulin [59, 60] in a way similar to its role in the synaptic transmission. Recently it was reported, however, that Rab3A null mice exhibit glucose intolerance and decreased first-phase glucose-evoked insulin secretion , an effect that was attributed to impaired replenishment of release-competent pool of granules (RRP). These seemingly contradictory findings might reflect that several regulatory proteins interact with Rab3A, each exerting a variety of distinct actions on the beta cell processes linked to the control of insulin release . For example, both Rim (Rab3-interacting molecule) and its isoform Rim2 are present in beta cells and are involved in glucose-stimulated insulin release . RIM interacts with a plethora of proteins and seems to function as a protein scaffold [63, 64]. For example, binding of Rim2 to the cAMP-sensor cAMP-GEFII transmits the PKA-independent portion of cAMP-mediated stimulation of insulin secretion . Surprisingly, this effect is lost in SUR1 null mice (i.e. mice lacking the sulphonylurea receptor protein SUR1, one of the subunits of the KATP-channel) indicating that the role of the latter protein extends beyond being a KATP-channel subunit and is also involved in the control of secretion . Clearly, the role of Rab3A and its interaction with various effector proteins remain poorly understood and merits further studies.
Cell biological models for phasic hormone release
It is tempting to explain biphasic insulin secretion in terms of functionally distinct pools of granules. Indeed, this possibility was considered more than 30 years ago by Grodsky who proposed an insulin storage-limited mathematical model to describe the kinetics of secretion in beta cells [69, 70]. By analogy to the situation in chromaffin cells, beta cell exocytosis elicited by a step increase in [Ca2+]i is biphasic and consists of a rapid component (reflecting release of RRP) and a subsequent much slower sustained component. In beta cells, RRP has been estimated to contain 20 to 100 granules (0.2–1%) depending on the experimental conditions [71, 72]. The size of RRP thus estimated is in reasonable agreement with the ~40 granules that can be estimated to undergo exocytosis per beta cell during first-phase insulin secretion (Fig. 5A) . Therefore a substantial part of first-phase insulin secretion could be attributable to exocytosis of RRP-granules. We explain the fact that first-phase insulin secretion is only transient and that the secretory rate returns towards the baseline as the use-dependent depletion of RRP. Once this pool of granules has been emptied, exocytosis proceeds at a (usually) much lower rate, presumably reflecting the low rate at which new granules are supplied for release by priming of reserve granules (Fig. 5A,B).
The hypothesis that the different phases of glucose-induced insulin secretion can be understood in terms of release of functionally distinct populations of secretory granules is underpinned by the differential metabolic requirements of first- and second-phase insulin secretion. Accordingly, capacitance measurements have shown that exocytosis of RRP granules does not require ATP-hydrolysis, whereas its refilling is highly ATP-dependent. These metabolic requirements resemble those of first- and second-phase insulin secretion (Fig. 2A,B). Moreover, agents that stimulate granule mobilization and increase the size of RRP exert similar effects of first- and second-phase insulin secretion. For example, compounds that increase cyclic AMP (e.g. the adenylate cyclase activator forskolin and the incretin hormone GLP-1) accelerate granule mobilization and increase the size of RRP in mouse beta cells fivefold (see Fig. 4 in ). These effects nicely correlate with reported fourfold to sixfold enhancement of first- and second-phase insulin secretion in mouse islets .
SNARE proteins direct Ca2+-entry to RRP granules
The SNARE proteins not only play a role in the fusion of the granules with the plasma membrane, they also ensure that Ca2+-entry is restricted to the areas of the plasma membrane in close contact with the secretory granules. The intracellular loop connecting the second and the third homologous domains of the L-type Ca2+-channel (L-loop) binds to syntaxin, SNAP-25 and synaptotagmin and tethers the Ca2+-channel to the secretory granule. The length of the L-loop suggests that the distance between the inner mouth of the Ca2+-channel and the secretory granules is less than 10 nm . Thanks to this arrangement, the release-competent granules (=RRP) are exposed to the high (exocytotic) levels of Ca2+ occurring just beneath the inner mouth of the Ca2+-channel. Exocytosis will therefore proceed in an essentially all-or-none fashion depending on whether the Ca2+-channel is open or not. We point out that the beta-cell action potential is brief (only 50 ms) and that glucose-induced beta-cell electrical activity increases sigmoidally with increasing glucose concentrations .
Ultrastructural correlates of biphasic insulin secretion
The idea that first phase insulin secretion principally reflects release of docked granules was tested by subjecting the beta cell to intense stimulation using 75 mmol/l extracellular K+ in the absence of glucose for 15 min . This stimulation, which is believed to elicit a first-phase-like secretory response, was associated with a 30% reduction in the number of docked granules but had no effect on granule distribution in the remainder of the cell. A subsequent second period of stimulation with high K+ failed to evoke insulin secretion when the experiment was done in the absence of glucose. However, when glucose was present at 5 mmol/l, the secretory response to the second period of stimulation was comparable to that elicited by the first stimulation . The depletion of the docked pool (i.e. a release of 200 granules) established by electron microscopy is larger than that expected to occur if only granules belonging to RRP, as defined by the capacitance measurements, were released. It is possible that this discrepancy reflects a pool of granules that are "nearly RRP", i.e. granules that can attain release competence without access to extra metabolic energy. Indeed, published measurements of K+-induced insulin secretion reveal a tail of insulin secretion after the initial first phase that might reflect such a process (Fig. 4B in ).
Three important conclusions can be drawn from these experiments: (i) first-phase insulin secretion involves exocytosis of RRP, which constitutes a subset of the docked pool of granules; (ii) RRP comprises a distinct and limited population of granules and once this pool has been depleted, secretion stops; and (iii) metabolic energy is required for the refilling of RRP from the reserve pool. These conclusions reinforce those previously reached by others using alternative techniques . Because of the accumulation of granules below the plasma membrane, replenishment of RRP can be envisaged to occur without any (extensive) movements of granules within the cell. The docked granules alone (650 per mouse beta cell) are sufficient for 2 h of glucose-stimulated insulin secretion (the initial first phase of 50 granules + 120 min of secretion at 5 granules/min) . Needless to say, the fact that recruitment is not required for sustained secretion does not mean that it does not occur. Recent data obtained using real-time single-granule imaging in Min6-cells indicate that late secretion is due to exocytosis of granules that have just arrived at the plasma membrane . Also of note, electron microscopy only provides a snapshot of the situation in the beta cell at the time of fixation and it is therefore not possible to conclude with certainty that the "docked" granules are really physically attached to the plasma membrane and not merely situated just beneath the plasma membrane because of random granule movements.
Capacitance measurements on isolated beta cells suggest very high rates of secretion
Capacitance measurements on isolated cells have indicted that exocytosis in the beta cell can proceed at much higher rates than suggested by insulin release measurements [19, 75]. Thus, the peak of exocytosis attained within 20 milliseconds (ms) after onset of depolarisation corresponds to a rate of 18 000 granules per min. The latter rate is approximately 1000-fold higher than that indicated by the insulin release measurements (Fig. 2B). An important difference between the two methods to monitor secretion is that whereas capacitance measurements have so far exclusively been done on isolated beta cells, insulin release is traditionally determined using intact pancreatic islets. When capacitance measurements are instead applied to beta cells in the intact islets, the maximum rate of exocytosis is reduced by 85% (Göpel and Rorsman, unpublished). The reason for the abnormally high rate of exocytosis in single beta cells is not clear but it might be the consequence of the extensive rearrangement of cell architecture likely to take place after cell isolation, plating on a non-biological surface and tissue culture. Alternatively, the lower rates of capacitance increase seen in the intact islet are a consequence of inhibitory paracrine mechanisms. Such an effect can be exerted, for example, by the inhibitory hormone somatostatin that is secreted by neighbouring δ-cells within the intact pancreatic islet and reduces Ca2+-dependent exocytosis by about 80% . However, even if allowance is made for this, there remains a ~150-fold discrepancy between the speed of exocytosis suggested by capacitance recordings and insulin secretion measurements.
Emptying of granules is much slower than membrane fusion
The recent advent of fluorescent proteins that can be tagged to cellular proteins destined for exocytotic release has provided valuable insight into the parameters determining the release kinetics of the peptide cargo. Using this approach, both insulin itself or other granule proteins have been tagged with various variants of GFP [41, 79, 86, 87, 88, 89]. Although the molecular weight of GFP is 4 to 5 times larger than that of insulin, the physical dimensions of the proteins are not vastly different (5.1 nm×3.7 nm×3.5 nm for EGFP vs 3.2 nm×3 nm×2.5 nm for the insulin monomer) . Thus, the disappearance of EGFP from the granules can be expected to approximate the exit of insulin into the extracellular space.
We have monitored insulin granule dynamics in Ins1-cells using enhanced GFP (EGFP) tagged to granular protein islet amyloid poylypeptide (IAPP) . This construct targets correctly to the lumen of secretory granules and, applying confocal imaging, it is possible to monitor the emptying of individual secretory granules in real time as well as the events preceding exocytosis. Gratifyingly, many of the concepts based on electron microscopy and the functional studies could then be verified. For example, the existence of a docked pool of immobile granules in direct contact with the plasma membrane in living cells could be directly shown. Whereas most granules approaching the membrane and all the granules in the cytoplasm undergo quite extensive movements (≥1 µm/10 s) [88, 90], about 25% of the granules situated within the first 1 µm beneath the plasma membrane were essentially immobile . By the combination of single-vesicle imaging and patch-clamp recordings, it was possible to compare the temporal relationship between membrane fusion (monitored as an increase in cell capacitance) and cargo release (disappearance of EGFP fluorescence). Consistent with the observations made by amperometry, there was a long delay (up to 12 s!) between membrane fusion and cargo release with an average of about 2 s. A similar latency between the establishment of the fusion pore and release of insulin was recently derived by measurements of fusion pore dynamics in mouse beta cells using extracellular markers with different molecular dimensions . The slow release of the peptide cargo could result from its slow dissociation from the intragranular matrix, the properties of which in the insulin granule remain poorly defined. Such a scenario is suggested by analogy to the release of catecholamines from chromaffin cells, where ion exchange via the fusion pore leads to matrix swelling that provides the tension which eventually leads to the opening of an aperture between the granule lumen and the extracellular space large enough to allow complete granule emptying [40, 91]. Irrespective of the underlying reason, these observations suggest that release of high molecular weight substances (such as insulin) is about 25-fold slower than the time course of capacitance increase . This delay probably accounts for much of the 150-fold discrepancy between the release rates suggested by capacitance measurements and insulin release measurements that persists even when allowance is made for the different release kinetics of isolated beta cells and cells in the intact islet. The remaining tenfold difference we attribute to the voltage-dependence of exocytosis which is only 10% at –30 mV (the membrane potential attained in the presence of 30 mmol/l K+) of that attained at zero mV (the voltage during the voltage-clamp depolarisations used to trigger exocytosis in the capacitance measurements) .
As membrane fusion can be temporally separated from cargo release it provides the beta cell with an interesting mechanism for secretory plasticity. Recent data obtained using Min6-cells transfected with chimaeric constructs of both synaptotagmin/pHluorin and NPY/venus (to report the pH change that occurs after opening of the fusion pore and cargo release, respectively) suggest that only a fraction of the exocytotic events (detected as granular pH equilibration and increase in pHluorin fluorescence) is associated with cargo release (seen as the disappearance of venus) . It is well established that the granules contain many substances in addition to insulin. Some of these are of low molecular weight. Examples of the latter group include Zn2+, ATP, Ca2+, glutamate, serotonin and dopamine [82, 93, 94, 95, 96]. It is conceivable that these substances can pass through the fusion pore before complete fusion. If the fusion pore can open transiently without proceeding into complete fusion, low molecular weight substances could be released independently of insulin from the granules and possibly exert a regulatory function within the islet (Fig. 7B).
Recovery of RRP does not require granule translocation
Following emptying of RRP (failure of continued stimulation to elicit secretion), the exocytotic capacity gradually recovers but about 1.5 min is required for complete replenishment of RRP . Thus, refilling of RRP is in full operation already during first-phase insulin secretion and this could account for the fact that glucose-induced insulin secretion never returns to the baseline but remains stimulated with respect to the prestimulatory level throughout the glucose challenge. The restoration of RRP requires access to metabolic energy [2, 97]. The EGFP-tagged granules allowed us to determine whether the recovery of RRP requires physical translocation of the granules or if modification of granules already in place is sufficient (compare Fig. 6). Simultaneous confocal single-granule imaging and capacitance measurements in Ins1 cells showed that at the time RRP was functionally depleted by intense stimulation, only 13% of the docked (immobile) granules had undergone exocytosis . This reinforces the concept, based on comparison of electron microscopy with capacitance measurements made in primary mouse beta cells , that RRP represents a subset of the docked pool (20–100 granules depending on the experimental conditions; i.e. 3–15% of the 600 docked granules) and that docking precedes the final preparation of the granules for release (priming). The docked granules that were not released during the first train (i.e. >85%) clearly represent a large reserve pool of granules. The exocytotic capacity of the cell recovered almost completely within less than 2 min without the arrival of "new" granules at the plasma membrane. Rather, renewed stimulation of secretion was associated with the release of granules that were present already during the initial period of stimulation but then failed to undergo exocytosis. These observations suggest that: (i) rapid release (first-phase insulin secretion?) is principally attributable to exocytosis of the docked and primed secretory granules; and (ii) late secretion (second phase?) can, at least in the short run and in Ins1 cells, be accounted for by priming of granules already situated below the plasma membrane.
Intracellular pre-exocytotic granule movements
How do granules become available for release?
The evidence summarized in the preceding sections suggests that many of the granules (85–97%) situated at the plasma membrane are not immediately available for release but that they can quickly gain release competence in a process that does not require any extensive movements of the granules. In this section we consider mechanisms that influence the capacity of a granule to undergo exocytosis.
Once granules have undergone exocytosis, the secreted granular membrane must be recaptured by endocytosis. Measurements of cell capacitance have indicated the presence of a rapid (time constant 10 s) and slow (time constant 100 s) type of endocytosis . Whereas the fast type of endocytosis appeared particularly important after weak stimulation, the slow type predominated after large exocytotic increases in membrane surface area resulting from intense stimulation. In most cases, endocytosis exactly compensated for the increase in cell surface area that occurs during stimulation of exocytosis.
Three types of endocytosis have been postulated to take place after exocytosis [111, 112]. In the first type, popularly referred to as "kiss-and-run", the granule content is released through the fusion pore that transiently and reversibly opens during exocytosis. In this mode of exo-/endocytosis, the fusion pore could close even before emptying of the granule interior has been completed and without mixing of the granule membrane with the plasma membrane (Fig. 11A). Alternatively, the granule membrane is integrated into the plasma membrane (complete fusion) and the extra membrane is subsequently recaptured by conventional clathrin-mediated endocytosis (Fig. 11C). There is an intermediate "kiss-and-run"-like mode of exo-/endocytosis ("semifusion"; Fig. 11B) that involves the establishment of a large opening between the granule lumen and the extracellular space but where the granule remains structurally intact . Similar observations have been made in Min6-cells . Our own measurements in Ins1-cells indicate that "semifusion" accounts for 90% of the release events. Granules undergoing this form of exocytosis are subsequently retrieved within less than 10 s. Complete fusion occurs in only 10% of the cases (Obermüller, Lindqvist, Jovasiene, Rorsman and Barg, submitted). By analogy with findings in retinal bipolar neurones , perhaps the slow and rapid components of capacitance-increase described above represent kiss-and-run and conventional endocytosis, respectively.
Significance to diabetes
The selective loss of first phase insulin secretion is an early feature of Type 2 diabetes . The possible relationship between first- and second-phase insulin secretion and distinct, functionally defined, populations of secretory granules makes it tempting to speculate on the cell physiological mechanisms that underlie the secretory defect of Type 2 diabetes. It should be emphasized that the number of release-competent granules is not a fixed entity and the size of RRP can vary considerably within minutes (or even seconds) due to changes in, for example, the metabolic state and presence of stimulatory or inhibitory hormones and neurotransmitters [71, 80, 97, 116]. Consequently, Type 2 diabetes might not be associated with any gross abnormalities of the intracellular granule distribution but rather result from defects in the preparation of granules for release. As we have attempted to illustrate here, reciprocal changes in the cytoplasmic concentrations of ATP and ADP affect a number of steps pertinent to the initiation and modulation of insulin secretion. Of particular relevance is the ability of ADP to control the release competence of the secretory granules. Type 2 diabetes associates with disturbances of glucose metabolism that impair the generation of ATP at the expense of ADP. These include defects of glycolysis manifested as substrate cycles of glucose metabolism  as well as impaired oxidative metabolism due to age-dependent accumulation of mitochondrial mutations . Obesity can also reduce insulin secretion via higher circulating concentrations of non-esterified fatty acids (NEFA). Chronic exposure to NEFA can be envisaged to impair ATP generation  by up-regulated expression of the mitochondrial uncoupling protein UCP2 synthesis  with resultant reduction in glucose-induced closure of the KATP-channel. Irrespective of whether beta-cell metabolism is impaired as a consequence of age, obesity or the combination of both, it is clear that failure of the beta cell to lower cytoplasmic ADP will affect secretion by interfering with both the triggering and amplifying actions of glucose on insulin secretion. In addition, there is evidence from animal models of human Type 2 diabetes (the GK-rat) that the impaired insulin secretory capacity correlates with reduced expression of proteins involved in the exocytotic process [121, 122]. Our observation that the assembly of a complex between the Ca2+-channels and the secretory granules is required for fast exocytosis [19, 75] further suggest that polymorphisms of genes encoding both the Ca2+-channel itself and the exocytotic proteins participating in the generation of the exocytotic core should be considered as potential diabetes genes. Indeed, there is evidence that a single nucleotide polymorphism in the syntaxin 1A gene is correlated with age at onset and insulin requirement in Type 2 diabetic patients . The functional effects of a given polymorphism are likely to be subtle and insignificant in the healthy beta cell. However, in a beta cell in which the metabolism is slightly compromised leading to decreased granule priming, they might exert more profound effects. Of course, the number of defects may not be limited to two but the greater number of such small defects that we combine, the graver the functional consequences become until eventually insulin secretion is insufficient to maintain euglycaemia. This model is in keeping with the idea that most cases of Type 2 diabetes do not result from a single mutation but rather from an unfortunate combination of genetic traits that individually are of little consequence [124, 125, 126].
The maintenance of insulin secretory capacity requires that the beta cell contains a sufficient number of release-competent granules (RRP) and that exocytosis is continuously balanced by the supply of new granules. It is not difficult to see how changes of the nature described above can account for the secretory abnormalities of Type 2 diabetes , which include the loss of first-phase (decrease in RRP) and reduction of second-phase insulin release (slow replenishment of new granules for release by mobilization of granules from the reserve pool). The concept that loss of functional pool of secretory granules contributes to the secretion defects associated with diabetes might seem at variance with the well-documented ability of arginine to elicit first phase insulin secretion in Type 2 diabetic patients. However, when the acute insulin responses to arginine were compared in diabetic and non-diabetic subjects, it became evident that they were reduced by more than 80% at all tested glucose concentrations . This reduction can be accounted for by either reduced granule priming or the failure of the granules to assemble into a function complex with the Ca2+-channels. The small responses to arginine that remained observable in the diabetic patients, we attribute to a slow rate of granule priming taking place despite the impairment of glucose metabolism or granules that happened to be correctly situated in the vicinity of a Ca2+-channel. If this is so, then the question arises as to why first-phase glucose-induced insulin secretion is more severely affected than that elicited by arginine. This we attribute to the different modes by which glucose and arginine initiate electrical activity. Whereas glucose-induced electrical activity is secondary to accelerated beta-cell metabolism, arginine exerts a direct stimulatory effect via its electrogenic entry . Accordingly, the impairment of glucose-induced insulin secretion results from the loss of triggering and amplifying actions, whereas only the latter is affected in the case of arginine. The concept that reduced granule priming contributes to the insulin secretory defect of Type 2 diabetes indicates that pharmacological agents that promote beta cell granule priming would have beneficial therapeutic effects. Proof of concept comes from the well-documented insulinotropic action of glucagon-like peptide 1 (GLP-1), which at least in mouse beta cells seems principally attributable to PKA-dependent stimulation of granule mobilisation and increase in the size of RRP . A drug that mimics the action of GLP-1 on granule priming in the beta cell would therefore most likely represent a valuable addition to the spectrum of anti-diabetic drugs available.
The review is based on the relevant literature published in the English language during the period 1990–2003, and seminal prior contributions. The sources available to the authors were integrated with sources identified through PubMed searches for "capacitance, exocytosis and insulin", "ion channels and insulin secretion" and "insulin and granule dynamics".
We thank F. Ashcroft for invaluable discussions on several topics covered by this review. We likewise gratefully acknowledge the contributions of our colleagues in Lund. Financial support was obtained from the Juvenile Diabetes Research Foundation, the Swedish Diabetes Association, the Novo Nordisk Foundation, the European Community and the Swedish Research Council.
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