Sebastian Barg

Fast insulin secretion reflects exocytosis of docked granules in mouse pancreatic B-cells.

Abstract. A readily releasable pool (RRP) of granules has been proposed to underlie the first phase of insulin secretion. In the present study we combined electron microscopy, insulin secretion measurements and recordings of cell capacitance in an attempt to define this pool ultrastructurally. Mouse pancreatic B-cells contain ~9,000 granules, of which 7% are docked below the plasma membrane. The number of docked granules was reduced by 30% (200 granules) during 10xa0min stimulation with high K+. This stimulus depolarized the cell to –10xa0mV, elevated cytosolic [Ca2+] ([Ca2+]i) from a basal concentration of 130xa0nM to a peak of 1.3xa0µM and released 0.5xa0ng insulin/islet, corresponding to 200–300xa0granules/cell. The Ca2+ transient decayed towards the prestimulatory concentration within ~200xa0s, presumably reflecting Ca2+ channel inactivation. Renewed stimulation with high K+ failed to stimulate insulin secretion when applied in the absence of glucose. The size of the RRP, derived from the insulin measurements, is similar to that estimated from the increase in cell capacitance elicited by photolytic release of caged Ca2+. We propose that the RRP represents a subset of the docked pool of granules and that replenishment of RRP can be accounted for largely by chemical modification of granules already in place or situated close to the plasma membrane.

Fast exocytosis with few Ca(2+) channels in insulin-secreting mouse pancreatic B cells

The association of L-type Ca(2+) channels to the secretory granules and its functional significance to secretion was investigated in mouse pancreatic B cells. Nonstationary fluctuation analysis showed that the B cell is equipped with <500 alpha1(C) L-type Ca(2+) channels, corresponding to a Ca(2+) channel density of 0.9 channels per microm(2). Analysis of the kinetics of exocytosis during voltage-clamp depolarizations revealed an early component that reached a peak rate of 1.1 pFs(-1) (approximately 650 granules/s) 25 ms after onset of the pulse and is completed within approximately 100 ms. This component represents a subset of approximately 60 granules situated in the immediate vicinity of the L-type Ca(2+) channels, corresponding to approximately 10% of the readily releasable pool of granules. Experiments involving photorelease of caged Ca(2+) revealed that the rate of exocytosis was half-maximal at a cytoplasmic Ca(2+) concentration of 17 microM, and concentrations >25 microM are required to attain the rate of exocytosis observed during voltage-clamp depolarizations. The rapid component of exocytosis was not affected by inclusion of millimolar concentrations of the Ca(2+) buffer EGTA but abolished by addition of exogenous L(C753-893), the 140 amino acids of the cytoplasmic loop connecting the 2(nd) and 3(rd) transmembrane region of the alpha1(C) L-type Ca(2+) channel, which has been proposed to tether the Ca(2+) channels to the secretory granules. In keeping with the idea that secretion is determined by Ca(2+) influx through individual Ca(2+) channels, exocytosis triggered by brief (15 ms) depolarizations was enhanced 2.5-fold by the Ca(2+) channel agonist BayK8644 and 3.5-fold by elevating extracellular Ca(2+) from 2.6 to 10 mM. Recordings of single Ca(2+) channel activity revealed that patches predominantly contained no channels or many active channels. We propose that several Ca(2+) channels associate with a single granule thus forming a functional unit. This arrangement is important in a cell with few Ca(2+) channels as it ensures maximum usage of the Ca(2+) entering the cell while minimizing the influence of stochastic variations of the Ca(2+) channel activity.

Regulation of glucagon release in mouse -cells by KATP channels and inactivation of TTX-sensitive Na+ channels.

1 The perforated patch whole‐cell configuration of the patch‐clamp technique was applied to superficial glucagon‐secreting α‐cells in intact mouse pancreatic islets. 2 α‐cells were distinguished from the β‐ and δ‐cells by the presence of a large TTX‐blockable Na+ current, a TEA‐resistant transient K+ current sensitive to 4‐AP (A‐current) and the presence of two kinetically separable Ca2+ current components corresponding to low‐ (T‐type) and high‐threshold (L‐type) Ca2+ channels. 3 The T‐type Ca2+, Na+ and A‐currents were subject to steady‐state voltage‐dependent inactivation, which was half‐maximal at −45, −47 and −68 mV, respectively. 4 Pancreatic α‐cells were equipped with tolbutamide‐sensitive, ATP‐regulated K+ (KATP) channels. Addition of tolbutamide (0·1 mm) evoked a brief period of electrical activity followed by a depolarisation to a plateau of −30 mV with no regenerative electrical activity. 5 Glucagon secretion in the absence of glucose was strongly inhibited by TTX, nifedipine and tolbutamide. When diazoxide was added in the presence of 10 mm glucose, concentrations up to 2 μm stimulated glucagon secretion to the same extent as removal of glucose. 6 We conclude that electrical activity and secretion in the α‐cells is dependent on the generation of Na+‐dependent action potentials. Glucagon secretion depends on low activity of KATP channels to keep the membrane potential sufficiently negative to prevent voltage‐dependent inactivation of voltage‐gated membrane currents. Glucose may inhibit glucagon release by depolarising the α‐cell with resultant inactivation of the ion channels participating in action potential generation.

Voltage-gated and resting membrane currents recorded from B-cells in intact mouse pancreatic islets

1 The perforated patch whole‐cell configuration of the patch‐clamp technique was applied to superficial cells in intact pancreatic islets. Immunostaining in combination with confocal microscopy revealed that the superficial cells consisted of 35 % insulin‐secreting B‐cells and 65 % non‐B‐cells (A‐ and D‐cells). 2 Two types of cell, with distinct electrophysiological properties, could be functionally identified. One of these generated oscillatory electrical activity when the islet was exposed to 10 mm glucose and had the electrophysiological characteristics of isolated B‐cells maintained in tissue culture. 3 The Ca2+ current recorded from B‐cells in situ was 80 % larger than that of isolated B‐cells. It exhibited significant (70 %) inactivation during 100 ms depolarisations. The inactivation was voltage dependent and particularly prominent during depolarisations evoking the largest Ca2+ currents. 4 Voltage‐dependent K+ currents were observed during depolarisations to membrane potentials above −20 mV. These currents inactivated little during a 200 ms depolarisation and were unaffected by varying the holding potential between −90 and −30 mV. 5 The maximum resting conductance in the absence of glucose, which reflects the conductance of ATP‐regulated K+ (KATP) channels, amounted to ≈4 nS. Glucose produced a concentration‐dependent reduction of KATP channel conductance with half‐maximal inhibition observed with 5 mm glucose. 6 Combining voltage‐ and current‐clamp recording allowed the estimation of the gap junction conductance between different B‐cells. These experiments indicated that the input conductance of the B‐cell at stimulatory glucose concentrations (≈1 nS) is almost entirely accounted for by coupling to neighbouring B‐cells.

Delay between fusion pore opening and peptide release from large dense-core vesicles in neuroendocrine cells.

Peptidergic neurotransmission is slow compared to that mediated by classical neurotransmitters. We have studied exocytotic membrane fusion and cargo release by simultaneous capacitance measurements and confocal imaging of single secretory vesicles in neuroendocrine cells. Depletion of the readily releasable pool (RRP) correlated with exocytosis of 10%-20% of the docked vesicles. Some remaining vesicles became releasable after recovery of RRP. Expansion of the fusion pore, seen as an increase in luminal pH, occurred after approximately 0.3 s, and peptide release was delayed by another 1-10 s. We conclude that (1) RRP refilling involves chemical modification of vesicles already in place, (2) the release of large neuropeptides via the fusion pore is negligible and only proceeds after complete fusion, and (3) sluggish peptidergic transmission reflects the time course of vesicle emptying.

Patch-clamp characterisation of somatostatin-secreting δ-cells in intact mouse pancreatic islets

1 The perforated patch whole‐cell configuration of the patch‐clamp technique was applied to superficial cells in intact mouse pancreatic islets. 2 Three types of electrical activity were observed corresponding to α‐, β‐ and δ‐cells. The δ‐cells were electrically active in the presence of glucose but lacked the oscillatory pattern seen in the β‐cells. By contrast, the α‐cells were electrically silent at high glucose concentrations but action potentials could be elicited by removal of the sugar. 3 Both α‐ and β‐cells contained transient voltage‐activated K+ currents. In the δ‐cells, the K+ currents activated above −20 mV and were completely blocked by TEA (20 mm). The α‐cells differed from the δ‐cells in possessing a TEA‐resistant K+ current activating already at −40 mV. 4 Immunocytochemistry revealed the presence of Kv3.4 channels in δ‐cells and TEA‐resistant Kv4.3 channels in α‐cells. Thus the presence of a transient TEA‐resistant current can be used to functionally separate the δ‐ and α‐cells. 5 A TTX‐sensitive Na+ current developed in δ‐cells during depolarisations beyond −30 mV and reached a peak amplitude of 350 pA. Steady‐state inactivation of this current was half‐maximal at −28 mV. The δ‐cells were also equipped with a sustained Ca2+ current that activated above −30 mV and reached a peak of 60 pA when measured at 2·6 mm extracellular Ca2+. 6 A tolbutamide‐sensitive KATP channel conductance was observed in δ‐cells exposed to glucose‐free medium. Addition of tolbutamide (0·1 mm) depolarised the δ‐cell and evoked electrical activity. We propose that the KATP channels in δ‐cells serve the same function as in the β‐cell and couple an elevation of the blood glucose concentration to stimulation of hormone release.

Multisite regulation of insulin secretion by cAMP-increasing agonists: evidence that glucagon-like peptide 1 and glucagon act via distinct receptors

Abstractu2002The mechanisms by which glucagon-like peptide 1(7–36)amide (GLP-1[7–36]amide) potentiates insulin secretion were investigated by measurements of whole-cell K+ and Ca2+ currents, membrane potential, the cytoplasmic Ca2+ concentration ([Ca2+]i) and exocytosis in mouse pancreatic B-cells. GLP-1(7–36)amide (10 nM) stimulated glucose-induced (10 mM) electrical activity in intact pancreatic islets. The effect was manifested as a 34% increase in the duration of the bursts of action potentials and a corresponding 28% shortening of the silent intervals. GLP-1(7–36)amide had no effect on the electrical activity at subthreshold glucose con- centrations (≤6.5 mM). In cultured B-cells, GLP-1(7–36)amide produced a decrease of the whole-cell ATP-sensitive K+ (KATP) conductance remaining at 5 mM glucose by ≈30%. This effect was associated with membrane depolarization and the initiation of electrical activity. GLP-1(7–36)amide produced a protein-kinase-A- (PKA-) and glucose-dependent fourfold potentiation of Ca2+-induced exocytosis whilst only increasing the Ca2+ current marginally. The stimulatory action of GLP-1(7–36)amide on exocytosis was mimicked by the pancreatic hormone glucagon and exendin-4, a GLP-1 receptor agonist. Whereas the stimulatory action of GLP-1(7–36)amide could be antagonized by exendin-(9–39), this peptide did not interfere with the ability of glucagon to stimulate exocytosis. We suggest that GLP-1(7–36)amide and glucagon stimulate insulin secretion by binding to distinct receptors. The GLP-1(7–36)amide-induced stimulation of electrical activity and Ca2+ influx can account for (maximally) a doubling of insulin secretion. The remainder of its stimulatory action results from a cAMP/PKA-dependent potentiation of Ca2+-dependent exocytosis exerted at a stage distal to the elevation of [Ca2+]i.

Tolbutamide stimulates exocytosis of glucagon by inhibition of a mitochondrial-like ATP-sensitive K+ (KATP) conductance in rat pancreatic A-cells

1 Capacitance measurements were used to examine the effects of the sulphonylurea tolbutamide on Ca2+‐dependent exocytosis in isolated glucagon‐secreting rat pancreatic A‐cells. 2 When applied extracellularly, tolbutamide stimulated depolarization‐evoked exocytosis 4.2‐fold without affecting the whole‐cell Ca2+ current. The concentration dependence of the stimulatory action was determined by intracellular application through the recording pipette. Tolbutamide produced a concentration‐dependent increase in cell capacitance. Half‐maximal stimulation was observed at 33 μm and the maximum stimulation corresponded to a 3.4‐fold enhancement of exocytosis. 3 The stimulatory action of tolbutamide was dependent on protein kinase C activity. The action of tolbutamide was mimicked by the general K+ channel blockers TEA (10 mm) and quinine (10 μm). A similar stimulation was elicited by 5‐hydroxydecanoate (5‐HD; 10 μm), an inhibitor of mitochondrial ATP‐sensitive K+ (KATP) channels. 4 Tolbutamide‐stimulated, but not TEA‐induced, exocytosis was antagonized by the K+ channel openers diazoxide, pinacidil and cromakalim. 5 Dissipating the transgranular K+ gradient with nigericin and valinomycin inhibited tolbutamide‐ and Ca2+‐evoked exocytosis. Furthermore, tolbutamide‐ and Ca2+‐induced exocytosis were abolished by the H+ ionophore FCCP or by arresting the vacuolar (V‐type) H+‐ATPase with bafilomycin A1 or DCCD. Finally, ammonium chloride stimulated exocytosis to a similar extent to that obtained with tolbutamide. 6 We propose that during granular maturation, a granular V‐type H+‐ATPase pumps H+ into the secretory granule leading to the generation of a pH gradient across the granular membrane and the development of a positive voltage inside the granules. The pumping of H+ is facilitated by the concomitant exit of K+ through granular K+ channels with pharmacological properties similar to those of mitochondrial KATP channels. Release of granules that have been primed is then facilitated by the addition of K+ channel blockers. The resulting increase in membrane potential promotes exocytosis by unknown mechanisms, possibly involving granular alkalinization.

Characterization of a naturally occurring mutation (L107I) in the HNF1 alpha (MODY3) gene.

Abstractn Aims/hypothesis. Maturity onset diabetes of the young type 3 (MODY3) is a monogenic form of diabetes mellitus caused by mutations in the gene encoding for hepatocyte nuclear factor 1 alpha, HNF1α. In this study we have examined the in vivo and in vitro effects of a mutation (L107I) outside the DNA binding and dimerization domains in the N terminal part of the HNF1α gene.n Methods. Beta-cell function of the affected family members was assessed by an oral glucose tolerance test. Functional tests were carried out to explain the role of the mutation in vitro by transcriptional activity assay, Western blotting, DNA-binding assays and subcellular localization experiments.n Results. Affected family members showed an 86% decreased insulin response to glucose when compared to age-matched healthy control subjects. In vitro the mutation showed a 79% decrease in transcriptional activity as compared to wild type HNF1α in HeLa cells lacking HNF1α. The transcriptional activity was not suppressed when the mutant was co-expressed with wild type HNF1α suggesting that the decreased activity was not mediated by a dominant negative mechanism. The L107I/HNF1α protein showed normal nuclear targeting but impaired binding to an HNF1α consensus sequence.n Conclusion/interpretation. Our results suggest that the L107I substitution represents a MODY3 mutation which impairs beta-cell function by a loss-of-function mechanism.

MiR-335 regulates exocytotic proteins and affects glucose-stimulated insulin secretion through decreased Ca2+-dependent exocytosis in beta cells

Abstracts of 51st EASD Annual Meetings of 51st EASD Annual Meeting OP 01 Insulin analogues: Is newer always better?