Pancreatic β-Cell Electrical Activity and Insulin Secretion: Of Mice and Men

Abstract

The pancreatic β-cell plays a key role in glucose homeostasis by secreting insulin, the only hormone capable of lowering the blood glucose concentration. Impaired insulin secretion results in the chronic hyperglycemia that characterizes type 2 diabetes (T2DM), which currently afflicts >450 million people worldwide. The healthy β-cell acts as a glucose sensor matching its output to the circulating glucose concentration. It does so via metabolically induced changes in electrical activity, which culminate in an increase in the cytoplasmic Ca2+ concentration and initiation of Ca2+-dependent exocytosis of insulin-containing secretory granules. Here, we review recent advances in our understanding of the β-cell transcriptome, electrical activity, and insulin exocytosis. We highlight salient differences between mouse and human β-cells, provide models of how the different ion channels contribute to their electrical activity and insulin secretion, and conclude by discussing how these processes become perturbed in T2DM.

Copyright © 2018 the American Physiological Society.

Figures

FIGURE 1.

A and B: immunohistochemistry of mouse (A) and human (B) pancreatic islets (red, insulin; green, glucagon; blue, somatostatin). Images provided by Dr A Clark, Oxford. Scale bars: 20 μm. C and D: schematic of mouse (C) and human (D) islets highlighting differences in blood supply, innervation, and islet cell distribution. The α- (green), β- (red), and δ-cells (blue) are indicated. Also illustrated (C, gray) is a pancreatic ganglion cell (613). E and F: electron micrographs of mouse (E) and human (F) β-cells. Scale bars: 500 nm. In F, the β-cell is surrounded by a δ- and an α-cell (granules indicated by α and δ). Electron micrographs provided by Prof. L. Eliasson, Lund (E), and Dr. A. Clark, Oxford (F). m, Mitochondrion; l, lipfuscin body; sg, secretory granule.

FIGURE 2.

A: relationship between glucose concentration and insulin secretion in static incubations of isolated mouse (red) and human (black). Secretory responses have been normalized to secretion at 20 mM glucose. [Modified from Walker et al. (717).] B: relationship between glucose concentration and oxidative metabolism of the sugar (measured as 14CO2 production radiolabeled glucose) in isolated mouse islets (75% β-cells). Glucose oxidation is half-maximal at ~6 mM glucose (arrow). [Modified from Ashcroft et al. (33).] C: relationship between glucose concentration and ATP content (red) and β-cell whole-cell KATP channel conductance (GK,ATP: normalized to conductance at 0 mM glucose) in isolated mouse islets. Effects of glucose on both parameters are half-maximal at ~3 mM glucose (arrowed). [Modified from Ashcroft et al. (34) and Zhang et al. (762).]

FIGURE 3.

Patch-clamp techniques. A: the experiments start with establishment of the cell-attached configuration. In this recording mode, a patch electrode is tightly sealed to the surface of an intact cell, allowing channel activity in the patch of membrane under the electrode tip to be studied under physiological conditions. For example, changes in channel activity in response to glucose metabolism can be measured by adding glucose to the bath solution. The seal between the electrode and the membrane is mechanically very stable, which enables additional configurations to be obtained. B: upon withdrawal of the electrode, the piece of membrane spanning the electrode tip is ripped off, forming an excised membrane patch that has its intracellular surface exposed to the bath solution (an inside-out patch). This is used for testing the effects of cytosolic constituents, such as ATP, on channel function. C: the plasma membrane outside the recording electrode can be permeabilized using detergents [like digitonin or saponin (162)] or the pore-forming peptide α-toxin [from Staphyloccus aureus (674)] to allow exchange of small molecules with a diameter of <1.5 nm (such as ATP) but not larger molecules (like enzymes). This recording configuration is referred to as the open cell-attached. D: the membrane beneath the electrode tip can be destroyed by suction, providing electrical access to the cell interior. This is known as the standard whole-cell configuration as it measures the summed activity of all ion channels in the cell membrane. It allows dialysis of the cell contents with the pipette solution. For example, the intracellular ion concentrations and cytosolic constituents (like ATP) can be manipulated by this route. The whole-cell configuration can also be used to preload the cells with biologically inert precursors of intracellular regulators that can then be photoliberated by a flash of ultraviolet light (‟caged compoundsˮ). E: withdrawal of the pipette from the standard whole-cell configuration produces an outside-out patch, in which the external membrane surface faces the bath solution. This is used to test the effects of extracellular ligands on channel activity. It can also be used as a ‟snifferˮ patch to probe the release of substances from the β-cell, if the membrane patch contains receptors to the compound of interest. F: the perforated patch whole-cell configuration allows measurement of electrical activity or whole-cell currents from a metabolically intact cell (291). In this variant of the whole-cell configuration, a pore-forming antibiotic [such as amphotericin (531)] is incorporated into the membrane below the pipette tip, thereby establishing electrical access to the cell while leaving cellular metabolism and intracellular second messenger systems intact.

FIGURE 4.

A: at low glucose, high KATP channel activity (thick black arrow) keeps the β-cell membrane potential negative, and depolarizing conductances (narrow red arrow) are too small to have a major impact. B: at high glucose, KATP channel activity is strongly reduced, and depolarizing currents (even small ones) will exert a stronger effect on the membrane potential. C: the input resistance (R) of the β-cell membrane determines the ease with which electrical activity can be initiated. When KATP channel activity is high, R is low. Conversely, when the KATP channels are shut, R is high. From Ohm's Law (V=R·I), it is evident that the same magnitude of current (I) will produce a much greater change in membrane potential (ΔV) when R is high (red trace) than when it is low (black trace). At high glucose, a small current may depolarize the β-cell sufficiently to trigger action potential firing (dotted red line). The ‟tug-of-warˮ between repolarizing and depolarizing membrane currents explains why potentiators of insulin secretion such as acetylcholine and arginine, which activate small depolarizing currents, are ineffective in the absence of glucose, when the activity of the KATP channels is high (i.e., R is low), but are able to stimulate electrical activity and insulin secretion at glucose concentrations that shut most KATP channels (i.e., R is high).

FIGURE 5.

A: glucose-induced electrical activity recorded from a β-cell in an acutely isolated intact mouse islet when the glucose concentration was increased from 1 to 10 or 20 mM (as indicated by horizontal bars). Note that 10 mM glucose evokes a biphasic response and that continuous action potential firing is replaced by oscillatory (bursting) electrical activity after the initial 3–4 min (red horizontal line beneath the membrane potential recording). At 20 mM glucose, electrical activity is continuous and there is a time-dependent ~15 mV reduction in the amplitude of the action potentials (recording provided by Dr. Q. Zhang, Oxford). B: bursts of action potentials in a β-cell in a freshly isolated mouse islet exposed to 10 mM glucose shown on an expanded time base. C: changes in cytoplasmic free Ca2+ ([Ca2+]i) in response to a step increase in glucose from 1 to 15 mM glucose. Note the triphasic response to glucose: an initial lowering below basal [Ca2+]i (red dashed line) (1), a rapid increase to a peak (2) followed by a decline to an elevated plateau on which small oscillations are superimposed (3). D: the consensus model of GIIS. Glut2, glucose transporter; KATP channels, ATP-sensitive K+ channels; Ψ, membrane potential; SG, secretory granules. The + and – signs denote stimulation and inhibition, respectively, whereas the arrows (↑,↓) indicate an increase or decrease of the indicated parameter. The red arrow connecting insulin and glucose indicates feedback regulation of insulin secretion via changes in plasma glucose.

FIGURE 6.

A: glucose-induced electrical activity in a β-cell in an intact human pancreatic islet in response to increasing glucose concentrations, as indicated by the staircase above the membrane potential recording. Note that it takes >40 min for β-cell to repolarize following exposure to glucose. B: response to 6 mM glucose (indicated by red rectangle in A) shown on an expanded time base. The action potentials undergo complex time-dependent changes in amplitude and peak voltage (see also FIGURE 13C). At 6 mM glucose, bursts of 3 or 4 action potentials are sometimes observed (red horizontal line under recording: see FIGURE 13B). Recordings in A and B were performed by Dr. E. Rebelato, Oxford. C: changes in [Ca2+]i in response to increasing glucose from 1 to 6 mM in a cell (presumably a β-cell) in an intact human islet. [Modified from Rorsman et al. (569).]

FIGURE 7.

A: topology of Kir6.2 and SUR1, showing two (of 4) Kir6.2 and two (of 4) SUR1 subunits. Kir6.2 has two transmembrane domains and cytosolic NH2 and COOH termini. SUR1 has 17 transmembrane domains arranged as groups of 5, 6, and 6 (TMD0, TMD1, and TMD2) and 2 nucleotide-binding domains (NBD1 and NBD2) that associate to form 2 nucleotide-binding sites at their interface. Binding of ATP (or ADP) to Kir6.2 inhibits channel activity. Binding of MgADP/MgATP to SUR1 stimulates activity. B and C: the KATP channel complex viewed from the side (B) and bottom (C). The Kir6.2 tetramer is surrounded by 4 SUR1 subunits. In B, the front subunit has been removed for clarity. Blue: TMD1, TMD2 of SUR1. Pink: TMD0 of SUR1. Green: NBDs of SUR1. Gray: Kir6.2. Brown: 3rd cytosolic loop of SUR1. The plasma membrane (yellow) is shown behind the channel in B. Figure provided by Dr. M. Puljung, Oxford.

FIGURE 8.

A: schematic of voltage-gated Na+ current (INa; red), Ca2+ current (ICa; green), delayed rectifying K+ current (IK; blue), and total membrane current (Itotal; i.e., INa+ ICa+ IK; black) elicited by a voltage-clamp depolarization (V) from −70 to −10 mV. The dashed line indicates the zero-current level. Downward and upward deflections represent inward (depolarizing) and outward (repolarizing) membrane currents, respectively. B: time-dependent changes in the value of m (dotted red), m3 (continuous red), and h (black) where m and h vary with time after the onset of depolarization (t) according to the expressions m(t)=m∞ [1−exp(−t/τm)] and h(t)=h0·exp(−t/τh). The parameter values for m and h have been normalized to their maximum. The green trace (below) shows the product m3h, which approximates the activation and inactivation of the whole-cell Na+ current. The curve has been inverted to facilitate comparison with the Na+ current. C: time-dependent changes in values of n and n4 where n varies with time after onset of depolarization (t) according to the expression n(t)=n∞[1−exp(t/τn)]. This approximates the time course of the whole-cell K+ current. Note that m3 and n4 result in sigmoidal activation kinetics. [Modified from Hille (282).]

FIGURE 9.

Relationship between membrane potential and the open probability (Popen), the single Ca2+ channel current (i), and the whole-cell Ca2+ current (I; i.e., the product of N·Popen·i) Note the U-shaped current-voltage relationship for the whole-cell current. The whole-cell slope conductance (G = I/V) between 0 and +50 mV (where the Ca2+ channels are maximally active) is ~1 nS (i.e., 50 pA/50 mV). [Modified from Larsson-Nyrén et al. (369).]

FIGURE 10.

Steady-state voltage-dependent inactivation of Na+ channels in mouse and human β-cells. This was analyzed by a two-pulse protocol (inset) in which a test pulse to 0 mV (to maximally activate the Na+ channels) was preceded by a conditioning depolarization (50–100 ms) to various membrane potentials. During the conditioning pulse, the Na+ channels undergo voltage-dependent activation: the more depolarized the conditioning voltage, the fewer the Na+ channels that remain to be activated by the test pulse. This gives rise to a sigmoidal relationship between membrane potential and the peak current during the test pulse that describes the voltage dependence of inactivation. In human β-cells, inactivation of the principal Na+ current component is half-maximal at −45 mV (arrowed). The corresponding value in mouse β-cells is 50–60 mV more negative (about −100 mV, arrowed). [Modified from Braun et al. (90) and Göpel et al. (221).]

FIGURE 11.

A: schematic of electrical coupling between β-cells in an intact islet. The green rectangles indicate gap junctions. Electrically coupled cells are indicated in pink. One β-cell within the islet is voltage-clamped at −70 mV through the recording electrode, so inhibiting electrical activity. Spontaneous electrical activity in neighboring electrically coupled cells (black traces in pink cells) results in an inward current in the voltage-clamped cell that resembles an inverted burst of action potentials. B: membrane potential (top) and membrane current (bottom) recorded from the same β-cell under current- and voltage-clamp conditions, respectively. Assuming that electrical activity recorded in the β-cell connected to the patch electrode before voltage-clamping approximates that of its neighbors, the total gap-junctional conductance (Gj) can be estimated from the equation Gj = ΔI/ΔV, where ΔV (above) and ΔI (below) represent the current and voltage differences between the plateau current/potential and the most repolarized voltage/least negative current. C: an example of bursting electrical activity where low-amplitude action potentials (coming from an adjacent β-cell) precede full-amplitude action potentials (red rectangle). These low-amplitude action potentials probably reflect electrical activity in a neighboring β-cell(s) that ‟leaksˮ into the cell from which the recording is made via gap junctions.

FIGURE 12.

A: schematic of the effects of increasing glucose concentrations (indicated at top) on mouse β-cell membrane potential and electrical activity, ATP/ADP ratio, and whole-cell KATP channel conductance (GKATP). The dashed vertical lines separate phase I, II, III, and IV (see main text). Schematic based on Cha and co-workers (112, 113). Schematic courtesy of Dr. C. Cha, Oxford. B and C: bursts of β-cell action potentials recorded from a β-cell exposed to 10 mM glucose. The numbers 1–6 highlight different phases of β-cell electrical activity: 1) the upstroke of the action potential, 2) action potential repolarization, 3) the plateau potential, 4) the progressive decrease in action potential amplitude during the burst, 5) burst termination, and 6) the pacemaker depolarization between two successive bursts (see sect. VIIIA).

FIGURE 13.

A: stimulus-secretion coupling in a human β-cell. Glucose uptake via GLUT1 and GLUT2 leads to accelerated mitochondrial glucose metabolism, increased ATP production, and closure of the KATP channels (consisting of the pore-forming subunit Kir6.2 and the sulfonylurea-binding protein SUR1). Inwardly rectifying Kir5.1 and Kir7.1 channels also contribute to the resting conductance of the human beta-cells, but their contribution is small. The increased membrane resistance (Rm↑) resulting from closure of the KATP channels allows occasional spontaneous opening of T-type Ca2+-channels (Ca[T]) to depolarize the β-cell (Ψ↓), and this leads to regenerative opening of additional T-type Ca2+ channels and a further membrane depolarization that culminates in rapid activation of L-type Ca2+ channels (Ca[L]) and voltage-gated Na+ channels (NaV) during the upstroke of the action potential. At the peak of the action potential, P/Q-type Ca2+ channels (Ca[P/Q]) open and the associated Ca2+ influx triggers exocytosis of insulin-containing secretory granules (SG). Opening of Ca2+-activated high-conductance K+ channels (BK) underlies action potential repolarization. Interspike membrane potential is influenced by TALK-1/TASK-1, small conductance Ca2+-activated K+ channels (SK), and/or delayed rectifying K+. B: burst of overshooting action potentials recorded from a human beta-cell. Rm↑ and Ψ↓ indicate an increase in membrane resistance and membrane depolarization, respectively. The numbers 1–9 highlight different phases of β-cell electrical activity: 1) the initial depolarization, 2) the upstroke of the action potential, 3) the peak of the action potential, 4) action potential repolarization, 5) the afterhyperpolarization, 6) the plateau/interspike potential, 7) the progressive reduction of action potential amplitude during electrical activity, 8) burst termination, and 9) the pacemaker depolarization between two bursts of action potentials that eventually results in the initiation of a new burst of action potetentials (1) (see sect. VIIIB6). C and D: schematics explaining why the Na+ channel blocker TTX has a weaker inhibitory effect on electrical activity (above) and insulin secretion (below) at high glucose than at low glucose concentrations. GIIS is greater at high glucose (D) than at low glucose (C) despite the reduction of action potential height because glucose exerts an amplifying effect on insulin secretion in addition to triggering electrical activity (see sect. IXD7a).

FIGURE 14.

A: insulin secretion measured in the perfused mouse pancreas preparation when glucose was elevated from 1 to 20 mM (as indicated by horizontal bar). B: same as in A but secretion was elicited by 2 µM glibenclamide (a KATP channel blocker) (horizontal bar). Glucose was subsequently elevated to 20 mM glucose (horizontal bar) in the continued presence of glibenclamide. The dotted line illustrates the insulin response to glucose shown in A. In A and B, data points indicate mean values and the shaded areas the SE (shaded areas). Data provided by N. Rorsman, Oxford. C: geometric mean of plasma insulin concentrations during a hyperglycemic clamp at 15 mM glucose (horizontal bar) in nondiabetic subjects (ND) and in patients with type 2 diabetes mellitus treated by diet (T2D) or the sulfonylurea gliclizide (T2D + SU). [Modified from Hosker et al. (292).]

FIGURE 15.

A: estimated rates of insulin secretion in the perfused mouse pancreas based on the experiment shown in FIGURE 14A and a total pancreatic insulin content of 50 μg [based on 0.008 g pancreas/g body weight, 0.25 μg insulin/g pancreas and a body weight of ~25 g (69)]. B: rates of insulin secretion in vivo estimated by solving Equation 1 using plasma insulin levels reported by (275) and a granule insulin content of 1.6 amol (294)(equivalent to ~8 fg of insulin). C: pulsatile insulin secretion in humans. The red and black traces show a 3-min moving average of the plasma insulin and glucose concentrations of a normal subject upon glucose infusion. Note that increasing glucose by ~0.5 mM more than doubles plasma insulin levels during the first oscillation. [Data modified from Lang et al. (363).] D: pulsatile insulin secretion measured using the perfused mouse pancreas preparation and a perfusion rate of ~0.3 ml/min. Data provided by N. Rorsman, Oxford.

FIGURE 16.

Schematic of the molecular machinery mediating Ca2+-dependent synaptic vesicle release. A: the core fusion machinery comprises the SNARE/SM protein complex. It consists of the vesicular (v-)SNARE protein VAMP-2 (red), the plasma membrane (target, t) SNARE proteins syntaxin-1 (yellow), and SNAP-25 (green), Munc18–1 (gray), and the Ca2+-sensor synaptotagmin-1 (blue, on vesicle). Tethering of the synaptic vesicle to the active zone involves a plasmalemmal voltage-gated Ca2+ channel (VGCC) and an active zone protein complex consisting of RIM (violet), Munc13 (brown), and RIM-BP (purple). RIM binds to the vesicular rab proteins Rab3 and Rab27 (sea green). B: five stages of exocytosis are illustrated: i) tethering; ii) docking and assembly of a loose trans-SNARE complex; iii) the formation of a tight 4-helix or ternary SNARE complex, with one helix coming from syntaxin, two helices from SNAP-25 and one helix from VAMP-2. This process is stabilized by complexin; iv) Ca2+ binding to synaptotagmins results in displacement of complexin, completion of zippering, and fusion pore opening; and v) expansion of the fusion pore and release of the cargo (a dense core vesicle is shown rather than a clear synaptic vesicle). After fusion, the resulting cis-SNARE complexes (cis, in the same membrane; trans, in opposite membrane) are disassembled by the NSF/SNAP ATPases and recycled. Modulators of β-cell exocytosis and where they act are given in green (see sect. IX, A and B). [Modified from Südhof (652).]

FIGURE 23.

A: different modes of insulin granule fusion. Release of previously ‟dockedˮ granules upon stimulation (left), release of granules not originally docked with the plasma membrane but that are recruited to the plasma membrane and undergo exocytosis following a variable period of being docked (‟newcomer,ˮ middle) and granules that arrive at the plasma membrane and are instantly released without being docked (‟crash fusion,ˮ right). [Modified from Shibasaki et al. (617).] B: imaging of secretory granules by TIRF microscopy. Red area indicates the evanescent field. Illumination leads to time-dependent bleaching of fluorescently labeled secretory granules (bright green → dark green). Granules that were initially bright (at t = 0) might thus appear to vanish within a few minutes (t = 2 min) because of loss of fluorescence. When such granules subsequently undergo exocytosis, the dramatic increase in fluorescence as the fluorophore moves further into the evanescent field will result in the reappearance of fluorescence giving the impression that granules released by glucose (at t = 2 min) were not docked with the plasma membrane. With agents that act more quickly (like K+), exocytosis will occur (at t = 0.5 min) before granules have faded enough to make them invisible. C: insulin secretion elicited by sequential stimulation with high extracellular K+ ([K+]o) and glucose. Note that high [K+]o transiently stimulates insulin secretion and that release rates subsequently decline back towards baseline values and that subsequent addition of glucose leads to a slowly developing increase in insulin release without any sign of a 1st phase (compare FIGURE 21A). Values are means (red circles) + SE (gray shaded area). Experiment performed by M. Söderström, Oxford.

FIGURE 17.

A: capacitance measurements of exocytosis. Ca2+ influx triggered by a brief depolarization (ΔV) leads to the fusion of (five) secretory granules with the plasma membrane (gray). The resultant increase in membrane area can be detected as an increase in cell capacitance (ΔC) because cell capacitance (C) is proportionally related to cell surface area (A) [i.e., C = ε*A, where ε is the specific membrane capacitance (10 fF/μm2)]. For technical reasons, the recording is usually interrupted during the depolarization (illustrated schematically by the red trace). The net increase in cell capacitance (ΔC) that occurred during the pulse is shown by the black trace (334). B: schematic of on-cell (cell-attached) single-granule capacitance measurements and the equivalent circuit. Orange, green, and gray lines correspond to the plasma membrane, the granule membrane, and the walls of the recording pipette, respectively (not to scale). Gp, Cv, and Cp are fusion pore conductance, granule capacitance, and patch capacitance, respectively. [Modified from Lindau (397).] C: carbon fiber amperometry. A carbon fiber connected to an amplifier is placed in the vicinity of the cell. Exocytosis can be detected as amperometric current spikes (right) that develop when the substance released (e.g., serotonin) is oxidized by the high voltage (e.g., 0.65 V) applied to the carbon fiber giving rise to a rapid current transient (right). D: electrophysiological detection of ATP release. ATP is co-released with insulin and activates P2X2 receptors (P2X2Rs) in β-cells engineered to express such receptors. ATP release and activation of P2X2Rs result in rapid current transients.

FIGURE 18.

A: imaging of granules in a β-cell using conventional and two-photon confocal microscopy. Note that the multiphoton microscopy allows imaging within a thinner slice of the cell (because two photons must simultaneously excite the fluorophore, which is unlikely to occur outside the focal plane). Insulin or other proteins of interest are visualized by expression of GFP-tagged proteins. B: evanescent wave (TIRF) microscopy. Illumination of the specimen is restricted to a layer above the coverslip, only a few hundred nanometers thick, thus facilitating the study of events taking place in the immediate vicinity of the plasma membrane. C: TIRF imaging of granule exocytosis. Images were captured at 20 Hz (50-ms intervals) in a voltage-clamped β-cell held at −70 mV and depolarized to 0 mV as indicated above the images. Granules close to the membrane are seen as bright spots at −70 mV. Exocytosis on depolarization to 0 mV is seen as a brief flash of light followed by the rapid dissipation of fluorescence as the tagged protein diffuses away from the release site. D: imaging of exocytosis using a fluorescent fluid phase marker (e.g., sulforhodamine; abbreviated SRB). SRB occupies the thin space between adjacent cells (top). Exocytosis results in the fusion of granules with the plasma membrane, enabling SRB to enter the granule lumen, producing fluorescent invaginations that can be visualized by multiphoton confocal imaging (bottom). E: three examples (red arrows) of granules labeled with SRB that have undergone exocytosis in response to high glucose (20 mM). Scale bar: 3 μm. [From Hoppa et al. (289).] F: monitoring fusion pore expansion and exocytosis by using extracellular fluid space markers (e.g., 10 kDa Alexa-conjugated dextran; red dots) with the membrane label FM1–43 (green). Upon membrane fusion, FM1–43 (which has prelabeled the outer leaflet of the plasma membrane; dashed green line) labels the granule membrane via lateral diffusion. Entry of fluorescent dextran will only occur once the fusion pore has expanded sufficiently to accommodate dextran. G: schematic of parallel recordings of FM1–43 fluorescence (green trace) and dextran fluorescence (red trace). Note that FM1–43, measured by two-photon confocal microscopy within a rectangular square (superimposed on the leftmost granule), increases promptly upon exocytosis. Uptake of dextran is delayed relative to FM1–43 uptake and the fluorescence signal (measured within the rectangle) for both FM1–43 and dextran decrease when the granule membrane collapses into the plasma membrane (see Ref. 664).

FIGURE 19.

A: relationship between pulse duration and exocytotic response (measured as depolarization-evoked increases in cell capacitance) in isolated β-cells (red trace) and in β-cells within intact acutely isolated islets (black trace). Measurements were performed in the presence of 0.1 mM intracellular cAMP to potentiate depolarization-evoked exocytosis. [Modified from Eliasson et al. (170) and Göpel et al. (222).] B: example of capacitance increase elicited by a 300-ms depolarization from −70 mV to 0 mV in a β-cell within an intact islet. Note that the capacitance increase is restricted to the depolarization and that there is little sign of exocytosis continuing beyond the depolarization. [Modified from Göpel et al. (222).] C: voltage dependence of exocytosis in β-cells in intact islets. The ∩-shaped voltage dependence mirrors that of the voltage-gated Ca2+ current (cf. FIGURE 9). The gray rectangle indicates the approximate voltage range of the β-cell action potential. Note that exocytosis is steeply voltage-dependent between −20 and 0 mV. D: ‟active zoneˮ of elevated [Ca2+]i produced by Ca2+ channel opening is restricted to the vicinity of the channel. In the resting (hyperpolarized) state, the Ca2+ channels are closed, submembrane [Ca2+]i is low, and exocytosis of secretory granules (SG) cannot proceed. Upon membrane depolarization, Ca2+ channels activate, [Ca2+]i increases to very high levels close to the inner mouth of the Ca2+ channels (red zone), and exocytosis is initiated. When the membrane potential is subsequently repolarized, Ca2+ channels instantly close, the active zone quickly collapses and [Ca2+]i rapidly falls below that required to trigger exocytosis, so insulin release ceases.

FIGURE 20.

Tethering of voltage-gated Ca2+ channels to secretory granules. A: SNAREs bind to the II-III loop of the L-type Ca2+ channel, the synaptic protein interaction (synprint peptide), and thereby tether the granule close to the inner mouth of the channel. Upon Ca2+ channel activation, the exocytotic machinery becomes exposed to a localized increase in [Ca2+]i. These transients are too rapid to be buffered by slow Ca2+ chelators like EGTA, explaining why depolarization-evoked exocytosis is resistant to this Ca2+ buffer. B: following the addition of a large excess of the ‟synprint peptide,ˮ the endogenous synprint peptide (which is part of the Ca2+ channel) is competitively displaced, leading to the disassembly of the granule-channel complex. Although Ca2+ channel activity is unperturbed, the secretory granule is no longer sufficiently close to the inner mouth of the Ca2+ channel (where [Ca2+]i exists at exocytotic levels), leading to the suppression of insulin release. C: the rapid Ca2+ chelator BAPTA binds Ca2+ so quickly that even granules tethered to the inner mouth of the Ca2+ channels are not exposed to Ca2+ concentrations high enough to evoke secretion (right). Figure courtesy of Professor E. Renström, Lund.

FIGURE 21.

A: relationship between exocytosis (measured as the depolarization-evoked increase in membrane capacitance) in isolated human β-cells (solid black line) and isolated mouse β-cells (dashed red line). Responses have been normalized to exocytosis elicited by a 500-ms depolarization. Note that depolarizations <100 ms evoke only small increases in capacitance in human β-cells. In β-cells within intact human islets, there is an additional, rapid component of exocytosis (568). Measurements were performed in the presence of 0.1 mM cAMP. [Modified from Braun et al. (90) and Eliasson et al. (170).] B: example of capacitance increase (ΔCm, red trace) elicited by a 500-ms depolarization in an isolated human β-cell. Note that in human β-cells, unlike mouse β-cells (FIGURE 19B), exocytosis continues for ~500 ms beyond the end of the pulse (gray rectangle). The dashed red line represents a linear extrapolation of the postdepolarization exocytosis to the start of the depolarization. The blue trace shows the decay of depolarization-evoked [Ca2+]i recorded in a β-cell following membrane repolarization. The dashed line superimposed on the capacitance trace is the inverted [Ca2+]i signal. This type of “asynchronous” exocytosis suggests that exocytosis in human β-cells is determined by the bulk rather than the local (near-membrane) [Ca2+]i. [Modified from Braun et al. (90) and Rorsman et al. (563).]

FIGURE 22.

A: TIRF imaging of near-membrane [Ca2+]i increases in a single voltage-clamped β-cell stimulated by a single 50-ms depolarization from −70 to 0 mV. The cell was infused with EGTA (10 mM) to restrict intracellular diffusion of Ca2+. Changes in [Ca2+]i are displayed in pseudocolors with black/blue and yellow/red corresponding to very low and high concentrations, respectively. Scale bar: 2 μm. Note that Ca2+ entry is not uniform but restricted to many ‟hotspotsˮ (red). [Modified from Hoppa et al. (288).] B: schematic showing how spatial [Ca2+]i domains overlap. Resting: the membrane contains four Ca2+ channels (green). Three of these channels sit beneath a secretory granule (the outline of which is indicated by dashed circle). Depolarization leads to localized Ca2+ entry and [Ca2+]i increases within spatially restricted domains (red). If the Ca2+ channels sit close to each other, these domains overlap. C: spatiotemporal domain overlap. Changes in [Ca2+]i at four different locations, as indicated in B. During the depolarization (gray area), the individual Ca2+ channels open and close stochastically and [Ca2+]i (traces 1–3) echoes Ca2+ channel activity. Individually, the [Ca2+]i are too brief to evoke exocytosis, but a sufficiently long elevation is generated by spatiotemporal domain overlap (location/trace 4).

FIGURE 24.

A: schematic of glucose-induced biphasic insulin secretion. The numbers 1, 2, and 3 indicate the onset of 1st phase secretion, the end of 1st phase secretion, and steady-state 2nd phase insulin secretion, respectively. The green and red areas correspond to the release of docked granules and mobilization of granules, respectively. B: at the onset of 1st phase insulin secretion (1), exocytosis principally involves docked granules situated close to voltage-gated Ca2+ channels. The depletion of this granule pool, together with inactivation of voltage-gated Ca2+ channels (from 3 to 1 active channels in the schematic, indicated by the lack of arrows through Ca2+ channels), account for the transient nature of 1st phase insulin secretion (2). The 2nd phase insulin secretion (3) involves the priming of granules already docked with the plasma membrane but that had previously not acquired release competence (going from red to green: i), physical translocation of granules from within the β-cell (ii), and redistribution of the voltage-gated Ca2+ channels within the plasma membrane (iii).

FIGURE 25.

A: single-vesicle capacitance increases (Cm, black trace), fusion pore conductance changes (G, red trace), and patch-clamp measurements of ATP release (green trace). Exocytosis leads to a step increase in membrane capacitance of 4–8 fF and a transient increase in G that reflects the initial opening of the fusion pore. In measurements of ATP release, activation of the purinergic receptors results in a membrane current that can be recorded using the whole-cell patch-clamp technique (see FIGURE 17D). For display, the current responses have been inverted. Arrow indicates the pedestal. B: schematic of full fusion. The fusion pore is initially large enough to accommodate ATP but not bulkier molecules (like insulin). Release of ATP via the fusion pore gives rise to a pedestal (arrow in A), whereas the main spike reflects full fusion and emptying of the entire cargo. ATP and insulin are shown as space-filling models and are inserted to scale. The width of the fusion pore is also to scale but not the dimensions of the rest of the granule. C: as in A but measurements obtained during ‟kiss-and-runˮ exocytosis. In kiss-and-run exocytosis, the increase in capacitance is only transient and is associated with a maintained (for the duration of the capacitance increase) increase in fusion pore conductance. The persistence of the increased fusion pore conductance indicates the failure of the fusion pore to expand. During kiss-and-run exocytosis, a more sustained but lower amplitude ATP-activated current is observed. D: as in B but showing kiss-and-run exocytosis. Slow release of ATP via the fusion pore results in the sustained but smaller ATP-activated current in C. Insulin remains trapped inside the granule lumen. Thus the fusion pore may function as a molecular sieve.

FIGURE 26.

Vicious cycle of hyperglycemia and GIIS initiating another vicious cycle of increased lipolysis/elevated NEFA and reduced GIIS. The combination of these two vicious cycles may account for the progression of T2DM. The slight initial increase in plasma glucose that initiates the vicious cycles may be caused by age-dependent insulin resistance or reduced β-cell metabolism and ATP production that culminates in T2DM (see sect. XA3).

FIGURE 27.

A: effects of increasing glucose concentrations (top) on electrical activity recorded from a β-cell from an organ donor diagnosed with T2DM (male, 51 yr of age, BMI 31 kg/m2, newly diagnosed, HbA1C 6.3). Note the action potential firing at 1 mM glucose (arrow), weak depolarizing effect of 10–20 mM glucose, and low action potential frequency even at the highest glucose concentration (compare FIGURE 6A). B: as in A but the experiment concluded by the addition of the sulfonylurea tolbutamide, which produced strong membrane depolarization, stimulation of action potential firing, and a time-dependent decrease in action potential height. In this cell, 6–10 mM glucose (if anything) appeared to hyperpolarize the β-cell. Data in A and B provided by Dr. M. Shigeto, Oxford, as described in Ref. . Data are representative of a total of 9 experiments on β-cells from one donor. C and D: schematics showing KATP channel activity (top), membrane potential (middle), and insulin secretion (bottom) in a healthy β-cell (C) and a T2DM β-cell (D). In the nondiabetic β-cell (C), KATP channel activity is high at low glucose, keeping the membrane hyperpolarized and inhibiting insulin secretion (1). When glucose is elevated to an insulin-releasing concentration, KATP channels close, the β-cell depolarizes and starts firing action potentials, which leads to stimulation of insulin secretion (2). At steady state, KATP channel activity remains low, electrical activity continues, but the amplitude of the action potentials has declined because of membrane potential-dependent inactivation of voltage-gated Na+ channels, resulting in a reduction of insulin secretion. In the diabetic β-cell (D), KATP channel activity in low glucose is less than that in the nondiabetic β-cell, leading to a slight depolarization and firing of the occasional action potential (1). This may underlie the elevated basal insulin secretion seen in T2DM. Increasing glucose has only a small effect on KATP channel activity so depolarization is limited and the stimulation of action potential firing and insulin secretion is marginal (2).There is no diminution of action potential height and thus no biphasic insulin secretion. Upon addition of sulfonylureas (abbreviated SU; such as glibenclamide), KATP channel activity is strongly reduced, strong membrane depolarization, stimulation of action potential firing, and a time-dependent decrease in action potential height follows, leading to a biphasic stimulation of insulin secretion (3).