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The Cell Physiology of Biphasic Insulin Secretion

Patrik Rorsman, Lena Eliasson, Erik Renström, Jesper Gromada, Sebastian Barg and Sven Göpel

P. Rorsman, L. Elaisson, E. Renström, S. Barg, and S. Göpel are in the Department of Physiological Sciences, Division of Molecular and Cellular Physiology, Lund University, Sölvegatan 19, S-223 62 Lund, Sweden. J. Gromada is at Islet Cell Physiology, Building 1KS18, Novo Nordisk A/S, DK-2880 Bagsvaerd, Denmark.

	Abstract

 
Glucose-stimulated insulin secretion consists of a transient first phase followed by a sustained second phase. Diabetes (type II) is associated with abnormalities in this release pattern. Here we review the evidence that biphasic insulin secretion reflects exocytosis of two functional subsets of secretory granules and the implications for diabetes.

	Introduction

Top Introduction Insulin secretion is controlled... Dissection of the cellular... Nutrients induce a biphasic... Exocytosis triggered by train... A step increase in... What is meant by... Significance to diabetes? References

 
Insulin plays a central role in the control of the body's metabolism. It does so by accelerating glucose uptake into a number of tissues while simultaneously suppressing glucose production and lipolysis. Lack of insulin leads to the serious metabolic disturbances that characterize diabetes mellitus. Insulin is produced and secreted by the ß-cells of the islets of Langerhans, the endocrine part of the pancreas, so named after their discoverer, Paul Langerhans. In his thesis, which he published at the age of 22 in 1869, Langerhans described the cells within the pancreatic islets as "small irregular structures with homogenous contents and with diameters ranging between 10 and 12 micrometers." He also remarked that the cells associate to form clusters with a diameter of 0.1–0.24 mm and that they are distributed throughout the glandular parenchyma. As to the function of the islets, Langerhans wrote "...I frankly acknowledge that I have no explanation [for the function of these structures]." It was to take another 20 years until Oscar Minkowski discovered their relationship to the pancreas and 50 years before the discovery of insulin was made.

	Insulin secretion is controlled by ß-cell electrical activity

Top Introduction Insulin secretion is controlled... Dissection of the cellular... Nutrients induce a biphasic... Exocytosis triggered by train... A step increase in... What is meant by... Significance to diabetes? References

 
As in neurons and many other endocrine cells, electrical signals play a central role in the regulation of secretion. In 1968, Dean and Matthews demonstrated that the ß-cell is electrically excitable (2). When the extracellular glucose concentration is elevated from the basal level of 5 mM (at which little insulin secretion is observed) to the insulin-releasing concentration of 10 mM, the ß-cell undergoes a slow depolarization from the resting potential (–70 mV) up to a threshold from which regenerative electrical activity is elicited. This electrical activity (Fig. 1B) consists of oscillations in the membrane potential between depolarized plateaus, from which Ca²⁺-dependent action potentials originate, which are separated by electrically silent (repolarized) intervals. The induction of electrical activity is a central part of the cascade of events that leads to the initiation of insulin secretion. Very recently, it has been possible to demonstrate that the periods of electrical activity coincide with pulsatile release of insulin (3).

View larger version (27K): [in this window] [in a new window]   FIGURE 1. A: stimulus-secretion coupling of pancreatic ß-cells. SUR, sulphonylurea receptor; KATP, ATP-regulated K+ channel. B: glucose-stimulated electrical activity recorded from a ß-cell in an intact islet when glucose concentration was increased from 5 to 10 mM as indicated by staircase above membrane potential trace. C: profile of glucose-induced insulin secretion. Glucose was elevated to 11 mM as indicated by horizontal bar. Note presence of a rapid first phase (lasting ~10 min) and a slower second phase. Data redrawn from Ref. 10. D: possible interpretation of release pattern. Whereas first phase insulin secretion can be envisaged to reflect exocytosis of a readily releasable pool of granules (RRP), second phase is due to release of granules that may be located further away from release site(s) (reserve pool). Mobilization of granules from reserve pool into RRP involves one or several ATP-dependent reactions (7, 11).

From a pathophysiological point of view, it is of great interest that the hypoglycemic sulphonylureas, which have been used clinically in the treatment of diabetes for more than 40 years, lead to the closure of the K_ATP channels in a way that is independent of the cell's metabolic state (2). The effect is mediated by binding to a sulphonylurea receptor (SUR), which, together with the inwardly rectifying K⁺ channel protein Kir6.2, form functional K_ATP channels (8). Sulphonylureas thus produce membrane depolarization, the opening of the Ca²⁺ channels, and initiation of insulin secretion as outlined above.

	Dissection of the cellular and molecular mechanisms of insulin secretion

Top Introduction Insulin secretion is controlled... Dissection of the cellular... Nutrients induce a biphasic... Exocytosis triggered by train... A step increase in... What is meant by... Significance to diabetes? References

 
The objective of ß-cell electrical activity is to generate the signal that initiates insulin secretion, i.e., the increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i). The recent development of techniques that allow the detection of secretion in single cells [capacitance measurements and carbon fiber amperometry (1, 3)], combined with the tools of molecular biology, have resulted in a dramatic increase in the knowledge of the processes involved. Many of the proteins that are involved in the regulation of neurotransmitter release have recently been identified also in the pancreatic ß-cells. These include the SNARE proteins synaptobrevin/VAMP, SNAP-25, syntaxin, -SNAP, and the putative Ca²⁺-sensing proteins synaptotagmin I and II (for review, see Ref. 15).

In this review, we focus on the characterization of the functional properties of exocytosis in the ß-cells. The experiments we describe were conducted by a combination of the whole cell configuration of the patch-clamp technique with capacitance measurements as a single-cell indicator of insulin secretion (1, 2). This experimental approach enables us to study the properties of secretion in single voltage-clamped ß-cells. Exocytosis can thereby be determined independently of any spontaneous changes of the membrane potential, which would (via modulation of voltage-dependent Ca²⁺ influx) influence the rate of Ca²⁺-induced secretion. Additional advantages of capacitance measurements over more traditional approaches to detecting insulin secretion include 1) that the measurements can be conducted on individual cells and 2) the high (1–10 ms) temporal resolution.

	Nutrients induce a biphasic stimulation of insulin secretion

Top Introduction Insulin secretion is controlled... Dissection of the cellular... Nutrients induce a biphasic... Exocytosis triggered by train... A step increase in... What is meant by... Significance to diabetes? References

 
Glucose-stimulated insulin secretion in vivo typically follows a biphasic time course (Fig. 1C) (3–5, 10). Shortly after elevation of the glucose concentration, a transient stimulation of insulin secretion is observed, referred to as "first phase secretion," which at later times is followed by a gradually developing secondary stimulation, "second phase secretion." Only fuel secretagogues are capable of eliciting the second phase, and, when insulin secretion is evoked by nonmetabolizable stimuli, only the first phase is observed. This suggests that second phase insulin secretion is an energy-dependent process. Interestingly, type II diabetes [non-insulin-dependent diabetes mellitus (NIDDM)] is associated with disturbances in the release pattern manifested as the selective loss of first phase secretion, which precedes other manifestations of the disease (4).

Biochemical and electrophysiological experiments in other endocrine cells (11) have suggested that the secretory granules exist in different pools, which vary with regard to releasability (Fig. 1D). Most of the granules (>95%) belong to the reserve pool and need to be chemically modified, or even physically translocated, to become immediately available for release. The latter subset of granules is referred to as the readily releasable pool (RRP) and characteristically contains <5% of the total granule number. The process in which the granules proceed from the reserve pool into the RRP has been termed mobilization and involves one or several ATP-dependent reactions (11).

	Exocytosis triggered by train of voltage-clamp depolarizations

Top Introduction Insulin secretion is controlled... Dissection of the cellular... Nutrients induce a biphasic... Exocytosis triggered by train... A step increase in... What is meant by... Significance to diabetes? References

 
Insulin secretion is elicited by bursts of Ca²⁺-dependent action potentials. Experimentally, it is more convenient to trigger exocytosis by application of voltage-clamp depolarization. Figure 2A shows an experiment in which a train of 14 depolarizations was applied to a ß-cell under control conditions (5 mM glucose alone). The first depolarization of the train evoked a capacitance increase of 50 fF. Subsequently, the exocytotic responses became progressively smaller during the train. Eventually (in this experiment after the sixth depolarization), when the total capacitance increase amounted to 125 fF, no further increase in cell capacitance was observed despite continued stimulation.

View larger version (26K): [in this window] [in a new window]   FIGURE 2. Repetitive and high-frequency stimulation can be used to estimate size of RRP. Trains of depolarizations were applied both under control conditions (A, top) and 6 min after addition of 2 µM forskolin (to activate PKA; B, top). Note larger exocytotic response in presence of forskolin. In this particular cell, maximum increase in cell capacitance amounted to 125 fF under control conditions and 500 fF in presence of forskolin. A, bottom: schematic representation of situation before (left) and after (right) depletion of RRP. Ca2+ channels continue to open after RRP depletion, but no granules remain to be released, resulting in cessation of exocytosis. B, bottom: fourfold increase in size of RRP in presence of PKA. Compare with A (bottom left). For technical reasons, it is not possible to monitor cell capacitance during depolarizations. Step increase in cell capacitance following each depolarization reflects insertion of granular membranes that occurred during voltage pulse. With a conversion factor of 2 fF/granule, capacitance changes can be converted into exocytosis of granules. An increase in cell capacitance of 50 fF accordingly corresponds to exocytosis of 25 secretory granules.

	A step increase in [Ca2+]i elicits a biphasic stimulation of exocytosis

Top Introduction Insulin secretion is controlled... Dissection of the cellular... Nutrients induce a biphasic... Exocytosis triggered by train... A step increase in... What is meant by... Significance to diabetes? References

 
It could be argued that an increase in [Ca²⁺]_i resulting from Ca²⁺ entry through the voltage-gated Ca²⁺ channels only accesses a fraction of the total number of granules that are immediately releasable and that the trains of depolarizations underestimate the true size of the RRP (Fig. 3D). A way to circumvent this problem is to use photorelease of caged Ca²⁺. In these experiments, Ca²⁺ is provided as a complex with o-nitrophenyl EGTA (NP-EGTA), a photolabile form of the Ca²⁺ chelator EGTA (7). Once loading has been completed, Ca²⁺ is liberated by a flash of ultraviolet light. This cleaves the photosensitive bonds within the NP-EGTA molecule and results in a 10,000-fold decrease in the affinity for Ca²⁺, which consequently is released to exert its biological actions. In this way, an instant and uniform increase of [Ca²⁺]_i is obtained and the spatial arrangement in terms of relationship between the Ca²⁺ channels, the secretory granules, and the release sites does not interfere with the measurements (Fig. 3E). Another important feature of these measurements is that Ca²⁺ has not been present at exocytotic levels before it was released. This means that any granules that resided in RRP at the time the whole cell configuration was established remain there until Ca²⁺ subsequently is elevated to exocytotic levels. Figure 3B shows the exocytotic responses observed when [Ca²⁺]_i was elevated to 30 µM by photoliberation of caged Ca²⁺. It is evident that the secretory response is biphasic and consists of a rapid phase followed by a slower sustained phase of capacitance increase.

View larger version (23K): [in this window] [in a new window]   FIGURE 3. Photorelease of Ca2+ from caged precursor Ca2+ o-nitrophenyl EGTA (NP-EGTA) elicits a biphasic stimulation of exocytosis. A: increase in cytoplasmic Ca2+ ([Ca2+]i) by liberation of Ca2+ from the caged precursor by ultraviolet light effected at time indicated by vertical dotted line. B: associated changes of membrane capacitance (Cm). A double exponential has been approximated to data points and is superimposed on capacitance trace (gray dashed curve). C: time derivative (dCm/dt) of function approximated to capacitance trace. Note biphasic response. Data is from Ref. 7 but change of [Ca2+]i has been reestimated using a dissociation constant for the indicator dye BTC of 70 µM as determined from parallel measurements of fluorescence and free Ca2+ concentration using a Ca2+-sensitive electrode. D–E: comparison of changes of intracellular Ca2+ concentration ([Ca2+]i) obtained when exocytosis is elicited by voltage-clamp depolarization (D) or photorelease of the caged precursor (E). Although there is an instantaneous and uniform increase in [Ca2+]i in E, only granules in immediate vicinity of Ca2+ channels are exposed to elevated [Ca2+]i (D). h, Ultraviolet light.

First phase insulin secretion typically lasts 10 min and reaches a peak rate of secretion of 80 pg • min^–1 • islet^–1 (Fig. 1). If we approximate first phase insulin secretion to a triangle, we can calculate that the first phase insulin secretion corresponds to a total of 400 pg/islet. A secretory granule contains up to 2 fg of insulin (7). Thus, during the entire first phase of insulin secretion, the number of granules released can be estimated to be 200,000 per islet, which corresponds to ~100 granules per ß-cell in an islet containing 2,000 cells. This value is not vastly different from the size of the RRP estimated from the depletion experiments (60 granules; Fig 2A). This argues that a substantial fraction of first phase insulin secretion is attributable to the release of granules belonging to the RRP. However, it must be acknowledged that the two processes take place over vastly different time periods: a few hundred milliseconds compared with a few minutes. Several explanations can be put forward to account for this discrepancy. They include lack of synchronization of exocytosis in the individual cells within the islets and the intensity of stimulation. It also seems likely that the stimulus (several tens of micromoles of [Ca²⁺]_i) is far stronger than anything experienced by the ß-cells in vivo.

In this scenario, the second phase reflects the time- and ATP-dependent mobilization of granules from the reserve pool into the RRP. The value for the mobilization we observe under control conditions (>0.5 granule • s^–1 • ß-cell^–1) corresponds to 120 pg/islet • min. This is not too different from the rates of second phase insulin secretion being observed experimentally [30 pg/islet • min in mouse ß-cells (Fig. 1) and 140 pg/islet • min in rat ß-cells (10)].

Further support of the idea that release of distinct subsets of secretory granules underlies the biphasic insulin release pattern comes from the observation that first and second phase glucose-induced insulin secretion are stimulated four- and sixfold in the presence of forskolin, respectively (5). These effects closely echo the increase in the size of the RRP of granules (Fig. 2) and the acceleration of granule mobilization obtained in response to activation of PKA as determined from measurements of cell capacitance (11).

	What is meant by mobilization?

Top Introduction Insulin secretion is controlled... Dissection of the cellular... Nutrients induce a biphasic... Exocytosis triggered by train... A step increase in... What is meant by... Significance to diabetes? References

 
The possibility that the different phases of insulin secretion reflect the release of distinct subsets of the secretory granules raises the question of whether it is possible to distinguish the RRP and reserve pool by ultrastructural means. Electron microscopy on ß-cells within intact islets suggest that as many as 10% of the granules are docked to the plasma membrane and up to 30% of the granules are situated within one granule diameter from the plasma membrane (L. Eliasson, unpublished observations). In a cell with 13,000 secretory granules, these values correspond to 1,300 and 4,000 granules, respectively. If these granules undergo exocytosis they would give rise to a capacitance increase of 2–8 pF. These values are close to the increases in cell capacitance that can be elicited by infusion of Ca²⁺ through the recording electrode (Fig. 4C), a condition associated with strong stimulation of granule mobilization. These considerations indicate that only a fraction of the docked granules functionally belongs to the RRP and that the mobilization of new granules for release does not require extensive physical translocation within the ß-cells. From these considerations, we conclude that the RRP does not correspond to an ultrastructurally definable subset of granules. This is consistent with recent optical measurements in chromaffin cells using evanescent wave microscopy, showing that the pool of docked granules turns over with a much slower time course than the functional replenishment of the RRP (6 min vs. 10 s) (13).

View larger version (20K): [in this window] [in a new window]   FIGURE 4. Does type II diabetes result from defective granule mobilization and incomplete refilling of RRP? A: insulin secretion when glucose concentration is elevated to 18 mM in healthy individuals and in patients with mild manifest type II diabetes. Data redrawn from Ref. 4. B: example of substrate cycle of glucose metabolism, which becomes activated in type II diabetes. C: increase in cell capacitance (Cm) elicited by protracted stimulation of exocytosis obtained by permanent elevation of [Ca2+]i to 2 µM by wash-in through recording electrode. Note that increase in cell capacitance under control conditions amounts to 6 pF over first 3.5 min after establishment of whole cell configuration. This equates to release of >3,000 secretory granules, which is 10 times larger than RRP under these experimental conditions (500 fF; shaded area), suggesting that >90% of capacitance increase reflects mobilization of granules from reserve pool into RRP. A much smaller increase in cell capacitance was obtained when 5 mM ADP was included in pipette solution dialyzing cell interior. D: lowering of ATP/ADP ratio interferes with granule mobilization and refilling of RRP. When Ca2+ is elevated, only a few granules are present in RRP, indicated by their dashed outlines, accounting for absence of first phase insulin secretion, and mobilization is slow, probably explaining reduced second phase.

	Significance to diabetes?

Top Introduction Insulin secretion is controlled... Dissection of the cellular... Nutrients induce a biphasic... Exocytosis triggered by train... A step increase in... What is meant by... Significance to diabetes? References

	Acknowledgments

 
We thank Drs. Carina Ämmälä, Krister Bokvist, Frances Ashcroft, and Peter Proks for participating in the early experiments leading to this work and thank Dr. Frank Thevenod for stimulating discussions related to the actions of sulphonylureas on exocytosis.

This work was supported by The Novo-Nordisk Foundation, the Swedish Diabetes Association, the Juvenile Diabetes Foundation International, the Knut and Alice Wallenberg Foundation, the Swedish Medical Research Council (grant no 8647), and the Medical Faculty of Lund University.

	References

Top Introduction Insulin secretion is controlled... Dissection of the cellular... Nutrients induce a biphasic... Exocytosis triggered by train... A step increase in... What is meant by... Significance to diabetes? References

 

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4. Cerasi, E. Aetiology of type II diabetes. In: Insulin–-Molecular Biology to Pathology, edited by F. M. Ashcroft and S. J. H. Ashcroft. Oxford, UK: IRL,1992, p. 352–392.
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				D. C. Thurmond, C. Gonelle-Gispert, M. Furukawa, P. A. Halban, and J. E. Pessin Glucose-Stimulated Insulin Secretion Is Coupled to the Interaction of Actin with the t-SNARE (Target Membrane Soluble N-Ethylmaleimide-Sensitive Factor Attachment Protein Receptor Protein) Complex Mol. Endocrinol., April 1, 2003; 17(4): 732 - 742. [Abstract] [Full Text] [PDF]

				K. Yaekura, R. Julyan, B. L. Wicksteed, L. B. Hays, C. Alarcon, S. Sommers, V. Poitout, D. G. Baskin, Y. Wang, L. H. Philipson, et al. Insulin Secretory Deficiency and Glucose Intolerance in Rab3A Null Mice J. Biol. Chem., March 7, 2003; 278(11): 9715 - 9721. [Abstract] [Full Text] [PDF]

				K. Fujimoto, T. Shibasaki, N. Yokoi, Y. Kashima, M. Matsumoto, T. Sasaki, N. Tajima, T. Iwanaga, and S. Seino Piccolo, a Ca2+ Sensor in Pancreatic beta -Cells. INVOLVEMENT OF cAMP-GEFII{middle dot}Rim2{middle dot}PICCOLO COMPLEX IN cAMP-DEPENDENT EXOCYTOSIS J. Biol. Chem., December 20, 2002; 277(52): 50497 - 50502. [Abstract] [Full Text] [PDF]

F. Thevenod Ion channels in secretory granules of the pancreas and their role in exocytosis and release of secretory proteins Am J Physiol Cell Physiol, September 1, 2002; 283(3): C651 - C672. [Abstract] [Full Text] [PDF]