Single Pancreatic Beta-cells Research Articles

Nanoscale Amperometry Reveals Only a Fraction of Vesicular Serotonin Content Is Released during Exocytosis from Beta Cells

Recent work has shown that chemical release during the fundamental cellular process of exocytosis in model cell lines is not all or none. We tested this theory for vesicular release from single pancreatic beta cells. The vesicles in these cells release insulin, but also serotonin, which is detectable with amperometric methods. Traditionally, it is assumed that exocytosis in beta cells is all-or-none. Here we use a multidisciplinary approach involving nanoscale amperometric chemical methods to explore the chemical nature of insulin exocytosis. We amperometrically quantified the number of serotonin molecules stored inside of individual nanoscale vesicles (39317 ± 1611) in the cell cytoplasm before exocytosis and the number of serotonin molecules released from single cells (13310 ± 1127) for each stimulated exocytosis event. Thus, beta cells release only one-third of their granule content, clearly supporting partial release in this system. We discuss these observations in the context of type-2 diabetes. Finally, these data therefore raise the tantalising possibility that pharmacological intervention can be used to increase the fraction released during beta cell exocytosis and lead to new therapies for type-2 diabetes.

Nanoscale Amperometry Reveals that Only a Fraction of Vesicular Serotonin Content is Released During Exocytosis from Beta Cells

AbstractRecent work has shown that chemical release during the fundamental cellular process of exocytosis in model cell lines is not all‐or‐none. We tested this theory for vesicular release from single pancreatic beta cells. The vesicles in these cells release insulin, but also serotonin, which is detectible with amperometric methods. Traditionally, it is assumed that exocytosis in beta cells is all‐or‐none. Here, we use a multidisciplinary approach involving nanoscale amperometric chemical methods to explore the chemical nature of insulin exocytosis. We amperometrically quantified the number of serotonin molecules stored inside of individual nanoscale vesicles (39 317±1611) in the cell cytoplasm before exocytosis and the number of serotonin molecules released from single cells (13 310±1127) for each stimulated exocytosis event. Thus, beta cells release only one‐third of their granule content, clearly supporting partial release in this system. We discuss these observations in the context of type‐2 diabetes.

Nanoscale Amperometry Reveals that Only a Fraction of Vesicular Serotonin Content is Released During Exocytosis from Beta Cells.

Recent work has shown that chemical release during the fundamental cellular process of exocytosis in model cell lines is not all‐or‐none. We tested this theory for vesicular release from single pancreatic beta cells. The vesicles in these cells release insulin, but also serotonin, which is detectible with amperometric methods. Traditionally, it is assumed that exocytosis in beta cells is all‐or‐none. Here, we use a multidisciplinary approach involving nanoscale amperometric chemical methods to explore the chemical nature of insulin exocytosis. We amperometrically quantified the number of serotonin molecules stored inside of individual nanoscale vesicles (39 317±1611) in the cell cytoplasm before exocytosis and the number of serotonin molecules released from single cells (13 310±1127) for each stimulated exocytosis event. Thus, beta cells release only one‐third of their granule content, clearly supporting partial release in this system. We discuss these observations in the context of type‐2 diabetes.

Subcellular glucose exposure biases the spatial distribution of insulin granules in single pancreatic beta cells

In living tissues, a cell is exposed to chemical substances delivered partially to its surface. Such a heterogeneous chemical environment potentially induces cell polarity. To evaluate this effect, we developed a microfluidic device that realizes spatially confined delivery of chemical substances at subcellular resolution. Our microfluidic device allows simple setup and stable operation for over 4 h to deliver chemicals partially to a single cell. Using the device, we showed that subcellular glucose exposure triggers an intracellular [Ca2+] change in the β-cells. In addition, the imaging of a cell expressing GFP-tagged insulin showed that continuous subcellular exposure to glucose biased the spatial distribution of insulin granules toward the site where the glucose was delivered. Our approach illustrates an experimental technique that will be applicable to many biological experiments for imaging the response to subcellular chemical exposure and will also provide new insights about the development of polarity of β-cells.

Isolation of Langerhans islets by dielectrophoresis

The purification of Langerhans islets from fragments of pancreatic exocrine tissue is a critical stage for the further transplantation of insulin secreting islets in patients affected by type I diabetes. Aim of our work was the evaluation of dielectrophoresis as a promising method for pancreatic islets isolation without physical contact in miniaturized lab-on-chip devices. DEP exploits the dielectric properties of particles suspended in a fluid, in a region where the amplitude of the electric field is characterized by a high gradient. Langerhans islets are aggregates of cells and have a minimum diameter of 50 microns. Dielectric models of pancreatic islets as cell aggregates were derived from single pancreatic beta cells model. Numerical simulations were performed to optimize the exact shape and size of the quadrupole microelectrode configuration and to determine the DEP forces acting on islets. A custom electronic setup was developed for the generation of sinusoidal signals with proper voltage and frequency and used to perform DEP experiments with samples of Langerhans islets. Dielectric models were found sufficiently accurate and negative DEP, showing repulsion from the electrodes, was observed for pancreatic islets. The results of this work demonstrate that Langerhans islet can be manipulated without physical contact by dielectrophoresis, a technique that can be applied on cell aggregates in miniaturized lab-on-chip devices.

5′-AMP-activated Protein Kinase Controls Insulin-containing Secretory Vesicle Dynamics

Changes in 5'-AMP-activated protein kinase (AMPK) activity have recently been implicated in the control of insulin secretion by glucose (da Silva Xavier, G., Leclerc, I., Varadi, A., Tsuboi, T., Moule, S. K., and Rutter, G. A. (2003) Biochem. J. 371, 761-774). Here, we examine the possibility that activation of AMPK may regulate distal steps in insulin secretion, including vesicle movement and fusion with the plasma membrane. Vesicle dynamics were imaged in single pancreatic MIN6 beta-cells expressing lumen-targeted pH-insensitive yellow fluorescent protein, neuropeptide Y.Venus, or monomeric red fluorescent protein by total internal reflection fluorescence and Nipkow disc confocal microscopy. Overexpression of a truncated, constitutively active form of AMPK (AMPKalpha1, 1-312, T172D; AMPK CA), inhibited glucose-stimulated (30 versus 3.0 mM) vesicle movements, and decreased the number of vesicles docked or fusing at the plasma membrane, while having no effect on the kinetics of individual secretory events. Expression of the activated form of AMPK also prevented dispersal of the cortical actin network at high glucose concentrations. Monitored in permeabilized cells, where the effects of AMPK CA on glucose metabolism and ATP synthesis were bypassed, AMPK CA inhibited Ca2+ and ATP-induced insulin secretion, and decreased ATP-dependent vesicle movements. These findings suggest that components of the vesicle transport network, including vesicle-associated motor proteins, may be targets of AMPK in beta-cells, dephosphorylation of which is required for vesicle mobilization at elevated glucose concentrations.

Effects of tolbutamide and N-benzoyl-d-phenylalanine (NBDP) on the regulation of [Ca2+]i oscillations in mouse pancreatic islets

The sulfonylurea derivative, tolbutamide, and the phenylalanine derivative, N-benzoyl-d-phenylalanine (NBDP), both of which stimulate insulin secretion through interaction with the sulfonylurea receptor (SUR1), were studied for their ability to increase the [Ca2+]i and to interact with the glucose-induced slow large amplitude [Ca2+]i oscillations in isolated mouse pancreatic islets. Tolbutamide as well as NBDP induced [Ca2+]i oscillations of extremely slow frequency. Both compounds also lowered the threshold for the glucose-induced slow large amplitude [Ca2+]i oscillations and significantly reduced their frequency in intact islets as well as in single pancreatic beta cells. These [Ca2+]i oscillations apparently require a glucokinase-mediated glycolytic flux. This conclusion is supported by the observations that KIC, a mitochondrial fuel, cannot replace glucose in this synergism and that mannoheptulose, an inhibitor of glucokinase and glucose-induced insulin secretion, abolishes these slow [Ca2+]i oscillations. In conclusion, these compounds potentiate the effect of glucose. This additive effect is the likely result of a synergistic closing action upon the ATP-sensitive K+ (KATP) channel, mediated in the case of glucose through an action upon the channel protein itself of ATP generated in glucose catabolism and in the case of tolbutamide and NBDP upon the sulfonylurea receptor (SUR1) associated with this channel.

Detection of secretion from single pancreatic beta-cells using extracellular fluorogenic reactions and confocal fluorescence microscopy.

Confocal microscopy with Zinquin, a fluorogenic Zn(2+)-specific indicator, was used for spatially and temporally resolved measurement of Zn2+ efflux from single pancreatic beta-cells. When cells were incubated in buffer containing Zinquin, application of insulin secretagogues evoked an increase in fluorescence around the surface of the cell, indicative of detection of Zn2+ efflux from the cell. The fluorescence increases corresponded spatially and temporally with measurements of exocytosis obtained simultaneously by amperometry. When images were taken at 266-ms intervals, the detection limit for Zn2+ was approximately 0.5 microM. With this image frequency, it was possible to observe bursts of fluorescence which were interpreted as fluctuations of Zn2+ level due to exocytosis. The average intensity of these fluorescence bursts corresponded to a Zn2+ concentration of approximately 7 microM. Since insulin is co-stored with Zn2+ in secretory vesicles, it was concluded that the Zn2+ efflux corresponded to exocytosis of insulin/Zn(2+)-containing granules from the beta-cell. Exocytosis sites identified by this technique were frequently localized to one portion of the cell, indicative of active areas of release.

Comparison of Amperometric Methods for Detection of Exocytosis from Single Pancreatic β-Cells of Different Species

Two methods for amperometric detection of exocytosis at single pancreatic beta-cells were compared. In the first, direct detection of insulin was accomplished using an insulin-sensitive chemically modified electrode. In the second, 5-hydroxytryptamine (5-HT) that had been allowed to accumulate within the beta-cell secretory vesicles was detected with a bare carbon electrode. The goal of the comparison was to determine whether 5-HT secretion was a valid marker of insulin secretion in single beta-cells. To aid in this comparison, some experiments involved simultaneous measurement of insulin and 5-HT at cells previously allowed to accumulate 5-HT. Upon application of common insulin secretagogues, current spikes resulting from detection of 5-HT, insulin, or both compounds were obtained indicative of secretion via exocytosis. The mean area of current spikes obtained from simultaneous measurements equaled the sum of the mean area of insulin and 5-HT measured independently. Additionally, analyses of the number of spikes obtained for detection of insulin, 5-HT, or both compounds were similar for several common secretagogues. These data support the hypothesis that accumulated 5-HT is released from insulin containing secretory vesicles, exclusively. In addition, measurement of insulin and 5-HT from beta-cells of different species was compared to determine whether a species dependence exists for the two methods compared here. Detection of 5-HT results in a similar number of spikes that are equivalent to insulin in frequency and amplitude in human, porcine, and canine beta-cells; however, in mouse and INS-1 beta-cells, 5-HT is more readily detected than insulin.

Insulin-stimulated Insulin Secretion in Single Pancreatic Beta Cells

Functional insulin receptors are known to occur in pancreatic beta cells; however, except for a positive feedback on insulin synthesis, their physiological effects are unknown. Amperometric measurements at single, primary pancreatic beta cells reveal that application of exogenous insulin in the presence or absence of nonstimulatory concentrations of glucose evokes exocytosis mediated by the beta cell insulin receptor. Insulin also elicits increases in intracellular Ca2+ concentration in beta cells but has minimal effects on membrane potential. Conditions where the insulin receptor is blocked or cell surface concentration of free insulin is reduced during exocytosis diminishes secretion induced by other secretagogues, providing evidence for direct autocrine action of insulin upon secretion from the same cell. These results indicate that the beta cell insulin receptor can mediate positive feedback for insulin secretion. The presence of a positive feedback mechanism for insulin secretion mediated by the insulin receptor provides a potential link between impaired insulin secretion and insulin resistance.

Ca2+ Signaling and the Insulin Secretory Cascade in the Pancreatic Beta-Cell

Recent progress in electrophysiological and microscopic techniques have enabled us to estimate exocytotic and pre-exocytotic events in the secretory machinery in single pancreatic beta-cells. We have been studying mechanisms involved in the regulation of insulin granule movement, which supplies release-ready granules, by direct visualization of granule traffic in living beta-cells and found the movement to be regulated by a mechanism different from that controlling exocytosis. From the obtained findings together with those from electrophysiological approaches, a new understanding of the role of the crucial second messenger Ca2+, and other second messengers, as well as resultant protein phosphorylation has been generated. The aim of this review is to describe a synergistic network for the control of insulin release by second messengers and protein kinases.

Atrial natriuretic peptide and cyclic nucleotides affect glucose-induced Ca2+ responses in single pancreatic islet beta-cells: correlation with (Ca[2+] + Mg2+)-ATPase activity

Atrial natriuretic peptide and cyclic nucleotides affect glucose-induced Ca2+ responses in single pancreatic islet beta-cells: correlation with (Ca[2+] + Mg2+)-ATPase activity

Electrical bursting and luminal calcium oscillation in excitable cell models.

It is shown in this paper that electrical bursting and the oscillations in the intracellular calcium concentration, [Ca2+]i, observed in excitable cells such as pancreatic beta-cells and R-15 cells of the mollusk Aplysia may be driven by a slow oscillation of the calcium concentration in the lumen of the endoplasmic reticulum, [Ca2+]lum. This hypothesis follows from the inclusion of the dynamic changes of [Ca2+]lum in the Chay bursting model. This extended model provides answers to some puzzling phenomena, such as why isolated single pancreatic beta-cells burst with a low frequency while intact beta-cells in an islet burst with a much higher frequency. Verification of the model prediction that [Ca2+]lum is a primary oscillator which drives electrical bursting and [Ca2+]i oscillations in these cells awaits experimental testing. Experiments using fluorescent dyes such as mag-fura-2-AM or aequorin could provide relevant information.

Ca(2+)- and GTP-dependent exocytosis in mouse pancreatic beta-cells involves both common and distinct steps.

1. The effects of GTP and Ca2+ on secretion from single pancreatic beta-cells were studied using capacitance measurements as an indicator of exocytosis. 2. GTP or GTP gamma S produced a concentration-dependent increase in cell capacitance in the absence of intracellular calcium. There was no effect of cyclic AMP or BAPTA an GTP-induced secretion. 3. In the absence of GTP, the relationship between intracellular calcium concentration and the maximum rate of secretion was fitted by the Hill equation with a slope factor of 2.5 and half-maximal activation at 1.6 microM intracellular Ca2+. Similar values were obtained in the presence of GTP gamma S, suggesting GTP does not alter the sensitivity of the secretory machinery to Ca2+. 4. GDP beta S alone had no effect on cell capacitance but caused a dose-dependent inhibition of exocytosis induced by infusion of either GTP gamma S or Ca2+, suggesting both stimuli involve G-protein activation. GDP beta S was without effect on exocytosis evoked by depolarization-mediated Ca2+ entry. 5. The time course of exocytosis following rapid elevation of GTP gamma S by photolysis of a caged precursor was dependent on the intracellular Ca2+ and cyclic AMP concentrations. 6. Our results are interpreted in terms of a model in which the secretory pathways stimulated by Ca2+ and GTP contain both common and separate parts.

Glucose-induced oscillatory insulin secretion in perifused rat pancreatic islets and clonal beta-cells (HIT).

Normal insulin secretion is oscillatory in vivo and from groups of perifused islets. Stimulation of rat islets with different glucose concentrations gave insulin oscillations of similar period (5-8 min) but increasing amplitude. It has been assumed that oscillatory secretion is due to oscillations in intracellular free Ca2+, as seen in single islets and single pancreatic beta-cells. However, when islets were perifused with diazoxide and high KCl to maintain high intracellular free Ca2+, insulin oscillations of similar amplitude and period still occurred on glucose stimulation, although superimposed on elevated basal secretion. Several likely possibilities for a diffusible synchronizing factor were tested, including pyruvate, lactate, ATP, and insulin itself; nevertheless, perifusion with high concentrations of these did not prevent insulin oscillations. Clonal pancreatic beta-cells (HIT) and dissociated islets also exhibited oscillatory insulin secretion, but with the 5- to 8-min period oscillations superimposed on 15- to 20-min period oscillations. These results indicate that the mechanisms for generating and synchronizing insulin oscillations reside in the beta-cell, although the structure of the islet may modulate the oscillation pattern.

Endocytosis of secretory granules in mouse pancreatic beta‐cells evoked by transient elevation of cytosolic calcium.

1. To investigate the mechanisms regulating the reuptake of secretory granule membranes following regulated exocytosis, we have monitored changes in cell capacitance in single pancreatic beta-cells. 2. Membrane retrieval (endocytosis) occurred both in a continuous manner and in abrupt steps, corresponding to the simultaneous retrieval of 50-100 granules. The large endocytotic steps were associated with a conductance change of about 1 nS which we attribute to the formation of a fission pore with a pore radius of approximately 1 nm. 3. In some cells, we observed large amplitude capacitance fluctuations, suggesting that aggregates of granules are connected to the plasma membrane by a single pore and are subsequently retrieved as a single unit. 4. Endocytosis was evoked by elevation of cytosolic [Ca2+]i, but once initiated, a sustained increase in [Ca2+]i was not required for endocytosis to continue. 5. The [Ca2+]i dependence of exo- and endocytosis was studied by photorelease of Ca2+ from the 'caged' precursor Ca(2+)-nitrophenyl-EGTA (Ca(2+)-NP-EGTA). Both exo- and endocytosis were initiated at between 0.5 and 2 microM Cai(2+). The rate of endocytosis saturated above 2 microM Cai(2+), whereas exocytosis continued to increase up to 4 microM Cai(2+). The maximum rate of endocytosis was < 25% of that of exocytosis. 6. Unlike exocytosis, endocytosis proceeded equally well in the presence or absence of Mg-ATP. 7. Our data indicate that in the pancreatic beta-cell, exocytosis and endocytosis are regulated by different mechanisms.

Block of pancreatic ATP-sensitive K+ channels and insulinotrophic action by the antiarrhythmic agent, cibenzoline.

1. We investigated the effect of cibenzoline (a class Ia antiarrhythmic drug) on basal insulin secretory activity of rat pancreatic islets and ATP-sensitive K+ channels (KATP) in single pancreatic beta cells of the same species, using radioimmunoassay and patch clamp techniques. 2. Micromolar cibenzoline had a dose-dependent insulinotrophic action with an EC50 of 94.2 +/- 46.4 microM. The compound inhibited the activity of the KATP channel recorded from a single beta-cell in a concentration-dependent manner. The IC50 was 0.4 microM in the inside-out mode and 5.2 microM in the cell-attached mode, at pH 7.4. 3. In the cell-attached mode, alkalinization of extracellular solution increased the inhibitory action of cibenzoline and the IC50 was reduced from 26.8 microM at pH 6.2 to 0.9 microM at pH 8.4. On the other hand, the action of cibenzoline in the excised inside-out mode was acute in onset with a small IC50, indicating that the drug attains its binding site from the cytoplasmic side of the cell membrane. 4. In the inside-out mode, micromolar ADP reactivated the cibenzoline-blocked KATP channels in a manner similar to that by which ADP restored ATP-dependent block of the channel. 5. The binding of [3H]-glibenclamide to pancreatic islets was inhibited by glibenclamide but not by cibenzoline. In contrast, the [3H]-cibenzoline binding was displaced by unlabelled cibenzoline but not by glibenclamide. It is concluded that cibenzoline blocks pancreatic KATP channels via a binding site distinct from the sulphonylurea receptor.

A monitor of secretion from single pancreatic beta-cells

A monitor of secretion from single pancreatic beta-cells

Expression and role of ionotropic glutamate receptors in pancreatic islet cells

Although the excitatory amino acid glutamate and its receptors play crucial roles in many functions of the central nervous system (CNS), their presence in the peripheral tissues has remained unclear. In the present study, we have identified kainate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), and N-methyl-D-aspartate (NMDA) receptor subtype mRNAs in pancreatic islets, using reverse transcriptase polymerase chain reaction (RT-PCR). Intracellular calcium ([Ca2+]i) measurements and electrophysiological recordings indicate that kainate, AMPA, and NMDA all elicit increases of [Ca2+]i in single pancreatic beta-cells and depolarize them. In addition, kainate and AMPA stimulate insulin secretion from isolated pancreatic islets, whereas NMDA does not. Also, immunocytochemical study shows the presence of intense glutaminase immunoreactivity in pancreatic alpha-cells and intrapancreatic ganglia, a finding compatible with the possibility that glutamate is released from alpha-cells as well as from neurons. Because the inhibitory amino acid gamma-amino butyric acid (GABA) is present in beta-cells as well as in neurons and inhibits glucagon secretion from alpha-cells, the present study suggests that glutamate and GABA are coordinated in the regulation of hormone secretion in pancreatic islets.

A new hypoglycemic agent, A-4166, inhibits ATP-sensitive potassium channels in rat pancreatic beta-cells.

Effects of a new hypoglycemic drug, N-[trans-4-isopropylcyclohexy-carbonyl]-D-phenylalanine (A-4166), on membrane current were investigated using the patch-clamp technique in single pancreatic beta-cells isolated from rats. A-4166, at a concentration of 10 microM, depolarized membrane potential of beta-cells and evoked action potentials in the presence of 2.8 mM glucose. The single ATP-sensitive K+ channel (K-ATP channel) current recorded in cell-attached membrane patches was reversibly inhibited by A-4166 (> 0.1 microM) without a change in the single-channel conductance of the K-ATP channel. Both A-4166 and tolbutamide inhibited the whole cell K-ATP channel current with half-maximum inhibition (IC50) of 0.23 and 12.8 microM, respectively (Hill coefficient = 1). In inside-out membrane patches, the IC50 with A-4166 occurred at 4.5 nM, in contrast to 0.7 microM for tolbutamide. A-4166 did not affect L- and T-type Ca2+ channels or the time-dependent outward current. We conclude that A-4166 specifically blocks the K-ATP channel and that the blockade is more potent than that of tolbutamide. The action of A-4166 underlies the mechanism by which the drug stimulates insulin secretion from beta-cells.