Beta-cell Electrical Activity Research Articles

A loss-of-function mutation in KCNJ11 causing sulfonylurea-sensitive diabetes in early adult life

Aims/hypothesisThe ATP-sensitive potassium (KATP) channel couples beta cell electrical activity to glucose-stimulated insulin secretion. Loss-of-function mutations in either the pore-forming (inwardly rectifying potassium channel 6.2 [Kir6.2], encoded by KCNJ11) or regulatory (sulfonylurea receptor 1, encoded by ABCC8) subunits result in congenital hyperinsulinism, whereas gain-of-function mutations cause neonatal diabetes. Here, we report a novel loss-of-function mutation (Ser118Leu) in the pore helix of Kir6.2 paradoxically associated with sulfonylurea-sensitive diabetes that presents in early adult life.MethodsA 31-year-old woman was diagnosed with mild hyperglycaemia during an employee screen. After three pregnancies, during which she was diagnosed with gestational diabetes, the patient continued to show elevated blood glucose and was treated with glibenclamide (known as glyburide in the USA and Canada) and metformin. Genetic testing identified a heterozygous mutation (S118L) in the KCNJ11 gene. Neither parent was known to have diabetes. We investigated the functional properties and membrane trafficking of mutant and wild-type KATP channels in Xenopus oocytes and in HEK-293T cells, using patch-clamp, two-electrode voltage-clamp and surface expression assays.ResultsFunctional analysis showed no changes in the ATP sensitivity or metabolic regulation of the mutant channel. However, the Kir6.2-S118L mutation impaired surface expression of the KATP channel by 40%, categorising this as a loss-of-function mutation.Conclusions/interpretationOur data support the increasing evidence that individuals with mild loss-of-function KATP channel mutations may develop insulin deficiency in early adulthood and even frank diabetes in middle age. In this case, the patient may have had hyperinsulinism that escaped detection in early life. Our results support the importance of functional analysis of KATP channel mutations in cases of atypical diabetes.Graphical

Oestrogen receptor β mediates the actions of bisphenol-A on ion channel expression in mouse pancreatic beta cells.

Bisphenol-A (BPA) is a widespread endocrine-disrupting chemical that has been associated with type 2 diabetes development. Low doses of BPA modify pancreatic beta cell function and induce insulin resistance; some of these effects are mediated via activation of oestrogen receptors α (ERα) and β (ERβ). Here we investigated whether low doses of BPA regulate the expression and function of ion channel subunits involved in beta cell function. Microarray gene profiling of isolated islets from vehicle- and BPA-treated (100μg/kg per day for 4days) mice was performed using Affymetrix GeneChip Mouse Genome 430.2 Array. Expression level analysis was performed using the normalisation method based on the processing algorithm 'robust multi-array average'. Whole islets or dispersed islets from C57BL/6J or oestrogen receptor β (ERβ) knockout (Erβ-/-) mice were treated with vehicle or BPA (1nmol/l) for 48h. Whole-cell patch-clamp recordings were used to measure Na+ and K+ currents. mRNA expression was evaluated by quantitative real-time PCR. Microarray analysis showed that BPA modulated the expression of 1440 probe sets (1192 upregulated and 248 downregulated genes). Of these, more than 50 genes, including Scn9a, Kcnb2, Kcnma1 and Kcnip1, encoded important Na+ and K+ channel subunits. These findings were confirmed by quantitative RT-PCR in islets from C57BL/6J BPA-treated mice or whole islets treated ex vivo. Electrophysiological measurements showed a decrease in both Na+ and K+ currents in BPA-treated islets. The pharmacological profile indicated that BPA reduced currents mediated by voltage-activated K+ channels (Kv2.1/2.2 channels) and large-conductance Ca2+-activated K+ channels (KCa1.1 channels), which agrees with BPA's effects on gene expression. Beta cells from ERβ-/- mice did not present BPA-induced changes, suggesting that ERβ mediates BPA's effects in pancreatic islets. Finally, BPA increased burst duration, reduced the amplitude of the action potential and enlarged the action potential half-width, leading to alteration in beta cell electrical activity. Our data suggest that BPA modulates the expression and function of Na+ and K+ channels via ERβ in mouse pancreatic islets. Furthermore, BPA alters beta cell electrical activity. Altogether, these BPA-induced changes in beta cells might play a role in the diabetogenic action of BPA described in animal models.

Human Islets Exhibit Electrical Activity on Microelectrode Arrays (MEA)

This study demonstrates for the first time that the microelectrode array (MEA) technique allows analysis of electrical activity of islets isolated from human biopsies. We have shown before that this method, i.e., measuring beta cell electrical activity with extracellular electrodes, is a powerful tool to assess glucose responsiveness of isolated murine islets. In the present study, human islets were shown to exhibit glucose-dependent oscillatory electrical activity. The glucose responsiveness could be furthermore demonstrated by an increase of insulin secretion in response to glucose. Electrical activity was increased by tolbutamide and inhibited by diazoxide. In human islets bursts of electrical activity were markedly blunted by the Na(+) channel inhibitor tetrodotoxin which does not affect electrical activity in mouse islets. Thus, the MEA technique emerges as a powerful tool to decipher online the unique features of human islets.Additionally, this technique will enable research with human islets even if only a few islets are available and it will allow a fast and easy test of metabolic integrity of islets destined for transplantation.

Ca2+ Microdomains in the Pancreatic Beta-Cell: a Three-Dimensional Modeling Approach

Beta-cells are responsible for secreting insulin as a response to an increase in blood glucose levels. Being electrically excitable, beta-cells exhibit electrical activity in response to a glucose stimulus driven by a well established mechanism involving glucose metabolism, ionic channels and calcium signaling. The purpose of beta-cell electrical activity is to allow the influx of Ca 2+ through ionic channels located in the plasma membrane in order to generate a high Ca 2+ icrodomain, which is the key signal triggering insulin exocytosis. It is known that Ca 2+ channels and insulin granules co-localize, and that they are not evenly distributed over the cell. Accounting for these morphological haracteristics we have developed a three-dimensional model of a beta-cell. By including a mathematical description of the ionic channels, our model reproduces the electrical activity observed experimentally. This allow us to simulate the spatiotemporal distribution of Ca 2+ in the microdomain generated by the electrical activity pattern. Our modeling approach enable us to evaluate the effect of distinct distributions of Ca 2+ channels over the cell membrane. Besides reproducing experimental observations, we also assess the impact of impaired functioning of ionic channels on Ca 2+ microdomains, which could ultimately affect insulin secretion.

Modeling KATP-Dependent Excitability in Pancreatic Islets

In pancreatic beta cells, KATP channels respond to changes in blood glucose to regulate cell excitability and insulin release. Confirming this role, gain or loss of function mutations in KATP are linked to neonatal diabetes or hyperinsulinism, respectively. Compared to neurons or cardiac myocytes, cellular models of beta-cell electrical activity have remained relatively rudimentary, but a recent detailed computational model of single cell pancreatic beta cell excitability (Cha et al., J Gen Physiol. 2011 138:21-37) accurately reproduces the beta cell response to varying glucose concentrations. Our aim was to test whether the model could also reproduce experimentally observed changes in excitability when KATP conductance is altered by genetic manipulation. Since the experiments were conducted in islets, we extended the model from a single cell to a 3D model (10x10x10 cell) islet with 1000 cells. For each cell, the conductances of the major currents were allowed to vary as was the gap junction conductance between cells. Initial simulations showed that the model was unable to reproduce KATP conductance-dependent changes in glucose-responses of excitability. By altering the ATP dependence of the L-type Ca2+ channel and the Na+-K+ pump to better match experiment, appropriate KATP dependence was quantitatively reproduced. In extending and refining the previous model, these simulations highlight the criticality of these parameters and suggest further experiments to improve characterization of beta cell excitability.

Modeling of oscillatory bursting activity of pancreatic beta-cells under regulated glucose stimulation

A mathematical model to describe the oscillatory bursting activity of pancreatic beta-cells is combined with a model of glucose regulation system in this work to study the bursting pattern under regulated extracellular glucose stimulation. The bursting electrical activity in beta-cells is crucial for the release of insulin, which acts to regulate the blood glucose level. Different types of bursting pattern have been observed experimentally in glucose-stimulated islets both in vivo and in vitro, and the variations in these patterns have been linked to changes in glucose level. The combined model in this study enables us to have a deeper understanding on the regime change of bursting pattern when glucose level changes due to hormonal regulation, especially in the postprandial state. This is especially important as the oscillatory components of electrical activity play significant physiological roles in insulin secretion and some components have been found to be lost in type 2 diabetic patients.

Polymorphisms in the gene encoding the voltage-dependent Ca2+ channel CaV2.3 (CACNA1E) are associated with type 2 diabetes and impaired insulin secretion

Glucose-stimulated insulin secretion is dependent on the electrical activity of beta cells; hence, genes encoding beta cell ion channels are potential candidate genes for type 2 diabetes. The gene encoding the voltage-dependent Ca(2+) channel Ca(V)2.3 (CACNA1E), telomeric to a region that has shown suggestive linkage to type 2 diabetes (1q21-q25), has been ascribed a role for second-phase insulin secretion. Based upon the genotyping of 52 haplotype tagging single nucleotide polymorphisms (SNPs) in a type 2 diabetes case-control sample (n = 1,467), we selected five SNPs that were nominally associated with type 2 diabetes and genotyped them in the following groups (1) a new case-control sample of 6,570 individuals from Sweden; (2) 2,293 individuals from the Botnia prospective cohort; and (3) 935 individuals with insulin secretion data from an IVGTT. The rs679931 TT genotype was associated with (1) an increased risk of type 2 diabetes in the Botnia case-control sample [odds ratio (OR) 1.4, 95% CI 1.0-2.0, p = 0.06] and in the replication sample (OR 1.2, 95% CI 1.0-1.5, p = 0.01 one-tailed), with a combined OR of 1.3 (95% CI 1.1-1.5, p = 0.004 two-tailed); (2) reduced insulin secretion [insulinogenic index at 30 min p = 0.02, disposition index (D (I)) p = 0.03] in control participants during an OGTT; (3) reduced second-phase insulin secretion at 30 min (p = 0.04) and 60 min (p = 0.02) during an IVGTT; and (4) reduced D (I) over time in the Botnia prospective cohort (p = 0.05). We conclude that genetic variation in the CACNA1E gene contributes to an increased risk of the development of type 2 diabetes by reducing insulin secretion.

Properties of voltage-gated Ca2+ currents measured from mouse pancreatic β-cells in situ

We used the single-microelectrode voltage-clamp technique to record ionic currents from pancreatic beta-cells within intact mouse islets of Langerhans at 37 degrees C, the typical preparation for studies of glucose-induced "bursting" electrical activity. Cells were impaled with intracellular microelectrodes, and voltage pulses were applied in the presence of tetraethylammonium. Under these conditions, a voltage-dependent Ca2+ current (I(Cav)), containing L-type and non-L-type components, was observed. The current measured in situ was larger than that measured in single cells with whole-cell patch clamping, particularly at membrane potentials corresponding to the action potentials of beta-cell electrical activity. The temperature dependence of I(Cav) was not sufficient to account for the difference in size of the currents recorded with the two methods. During prolonged pulses, the voltage-dependent Ca2+ current measured in situ displayed both rapid and slow components of inactivation. The rapid component was Ca2+-dependent and was inhibited by the membrane-permeable Ca2+ chelator, BAPTA-AM. The effect of BAPTA-AM on beta-cell electrical activity then demonstrated that Ca2+-dependent inactivation of I(Cav) contributes to action potential repolarization and to control of burst frequency. Our results demonstrate the utility of voltage clamping beta-cells in situ for determining the roles of ion channels in electrical activity and insulin secretion.

Current status of the E23K Kir6.2 polymorphism: implications for type-2 diabetes

The ATP-sensitive potassium (KATP) channel couples membrane excitability to cellular metabolism and is a critical mediator in the process of glucose-stimulated insulin secretion. Increasing numbers of KATP channel polymorphisms are being described and linked to altered insulin secretion indicating that genes encoding this ion channel could be susceptibility markers for type-2 diabetes. Genetic variation of KATP channels may result in altered beta-cell electrical activity, glucose homeostasis, and increased susceptibility to type-2 diabetes. Of particular interest is the Kir6.2 E23K polymorphism, which is linked to increased susceptibility to type-2 diabetes in Caucasian populations and may also be associated with weight gain and obesity, both of which are major diabetes risk factors. This association highlights the potential contribution of both genetic and environmental factors to the development and progression of type-2 diabetes. In addition, the common occurrence of the E23K polymorphism in Caucasian populations may have conferred an evolutionary advantage to our ancestors. This review will summarize the current status of the association of KATP channel polymorphisms with type-2 diabetes, focusing on the possible mechanisms by which these polymorphisms alter glucose homeostasis and offering insights into possible evolutionary pressures that may have contributed to the high prevalence of KATP channel polymorphisms in the Caucasian population.

Tolbutamide potentiates the volume-regulated anion channel current in rat pancreatic beta cells

Hypoglycaemic sulphonylureas are thought to stimulate insulin release by binding to a sulphonylurea receptor, closing K(ATP) channels and inducing electrical activity. However, the fact that these drugs stimulate insulin release at high glucose concentrations where K(ATP) channels are closed suggests additional ionic actions. The aim of this study was to test the hypothesis that sulphonylureas influence the current of the glucose- and volume-regulated anion channel. Electrical and ion-channel activity were recorded in isolated rat beta cells using the patch-clamp technique. (86)Rb(+) efflux was measured using intact islets. Beta cell volume was measured using a video-imaging technique. In the absence of glucose, tolbutamide (100 micromol/l) transiently depolarised the cells. In the presence of glucose (5 mmol/l), tolbutamide evoked a sustained period of electrical activity, whilst at 10 mmol/l glucose, the drug evoked a pronounced 'silent' depolarisation. In the absence of glucose, tolbutamide inhibited (86)Rb(+) efflux. However, at 10 mmol/l glucose, tolbutamide induced a transient stimulation of efflux. Tolbutamide potentiated the whole-cell volume-regulated anion conductance in a glucose-dependent manner with an EC(50) of 85 micromol/l. In single channel recordings, tolbutamide increased the channel-open probability. Tolbutamide caused beta cell swelling in the presence of glucose, but not in its absence. Tolbutamide can induce beta cell electrical activity by potentiating the glucose- and volume-regulated anion channel current. This effect is probably not due to a direct effect of the drug on the channel, but could be secondary to a metabolic action in the beta cell.

Long-chain CoA esters activate human pancreatic beta-cell KATP channels: potential role in Type 2 diabetes.

The ATP-regulated potassium (KATP) channel in the pancreatic beta cell couples the metabolic state to electrical activity. The primary regulator of the KATP channel is generally accepted to be changes in ATP/ADP ratio, where ATP inhibits and ADP activates channel activity. Recently, we showed that long-chain CoA (LC-CoA) esters form a new class of potent KATP channel activators in rodents, as studied in inside-out patches. In this study we have investigated the effects of LC-CoA esters in human pancreatic beta cells using the inside-out and whole-cell configurations of the patch clamp technique. Human KATP channels were potently activated by acyl-CoA esters with a chain length exceeding 12 carbons. Activation by LC-CoA esters did not require the presence of Mg2+ or adenine nucleotides. A detailed characterization of the concentration-dependent relationship showed an EC50 of 0.7+/-0.1 micromol/l. Furthermore, in the presence of an ATP/ADP ratio of 10 (1.1 mmol/l total adenine nucleotides), whole-cell KATP channel currents increased approximately six-fold following addition of 1 micro mol/l LC-CoA ester. The presence of 1 micro mol/l LC-CoA in the recording pipette solution increased beta-cell input conductance, from 0.5+/-0.2 nS to 2.5+/-1.3 nS. Taken together, these results show that LC-CoA esters are potent activators of the KATP channel in human pancreatic beta cells. The fact that LC-CoA esters also stimulate KATP channel activity recorded in the whole-cell configuration, points to the ability of these compounds to have an important modulatory role of human beta-cell electrical activity under both physiological and pathophysiological conditions.

Voltage-dependent K(+) channels in pancreatic beta cells: role, regulation and potential as therapeutic targets.

Insulin secretion from pancreatic islet beta cells is acutely regulated by a complex interplay of metabolic and electrogenic events. The electrogenic mechanism regulating insulin secretion from beta cells is commonly referred to as the ATP-sensitive K(+) (K(ATP)) channel dependent pathway. Briefly, an increase in ATP and, perhaps more importantly, a decrease in ADP stimulated by glucose metabolism depolarises the beta cell by closing K(ATP) channels. Membrane depolarisation results in the opening of voltage-dependent Ca(2+) channels, and influx of Ca(2+) is the main trigger for insulin secretion. Repolarisation of pancreatic beta cell action potential is mediated by the activation of voltage-dependent K(+) (Kv) channels. Various Kv channel homologues have been detected in insulin secreting cells, and recent studies have shown a role for specific Kv channels as modulators of insulin secretion. Here we review the evidence supporting a role for Kv channels in the regulation of insulin secretion and discuss the potential and the limitations for beta-cell Kv channels as therapeutic targets. Furthermore, we review recent investigations of mechanisms regulating Kv channels in beta cells, which suggest that Kv channels are active participants in the regulation of beta-cell electrical activity and insulin secretion.

The Ca2+ Dynamics of Isolated Mouse β-Cells and Islets: Implications for Mathematical Models

[Ca(2+)](i) and electrical activity were compared in isolated beta-cells and islets using standard techniques. In islets, raising glucose caused a decrease in [Ca(2+)](i) followed by a plateau and then fast (2-3 min(-1)), slow (0.2-0.8 min(-1)), or a mixture of fast and slow [Ca(2+)](i) oscillations. In beta-cells, glucose transiently decreased and then increased [Ca(2+)](i), but no islet-like oscillations occurred. Simultaneous recordings of [Ca(2+)](i) and electrical activity suggested that differences in [Ca(2+)](i) signaling are due to differences in islet versus beta-cell electrical activity. Whereas islets exhibited bursts of spikes on medium/slow plateaus, isolated beta-cells were depolarized and exhibited spiking, fast-bursting, or spikeless plateaus. These electrical patterns in turn produced distinct [Ca(2+)](i) patterns. Thus, although isolated beta-cells display several key features of islets, their oscillations were faster and more irregular. beta-cells could display islet-like [Ca(2+)](i) oscillations if their electrical activity was converted to a slower islet-like pattern using dynamic clamp. Islet and beta-cell [Ca(2+)](i) changes followed membrane potential, suggesting that electrical activity is mainly responsible for the [Ca(2+)] dynamics of beta-cells and islets. A recent model consisting of two slow feedback processes and passive endoplasmic reticulum Ca(2+) release was able to account for islet [Ca(2+)](i) responses to glucose, islet oscillations, and conversion of single cell to islet-like [Ca(2+)](i) oscillations. With minimal parameter variation, the model could also account for the diverse behaviors of isolated beta-cells, suggesting that these behaviors reflect natural cell heterogeneity. These results support our recent model and point to the important role of beta-cell electrical events in controlling [Ca(2+)](i) over diverse time scales in islets.

Inhibition of Kv2.1 Voltage-dependent K+Channels in Pancreatic β-Cells Enhances Glucose-dependent Insulin Secretion

Voltage-dependent (Kv) outward K(+) currents repolarize beta-cell action potentials during a glucose stimulus to limit Ca(2+) entry and insulin secretion. Dominant-negative "knockout" of Kv2 family channels enhances glucose-stimulated insulin secretion. Here we show that a putative Kv2.1 antagonist (C-1) stimulates insulin secretion from MIN6 insulinoma cells in a glucose- and dose-dependent manner while blocking voltage-dependent outward K(+) currents. C-1-blocked recombinant Kv2.1-mediated currents more specifically than currents mediated by Kv1, -3, and -4 family channels (Kv1.4, 3.1, 4.2). Additionally, C-1 had little effect on currents recorded from MIN6 cells expressing a dominant-negative Kv2.1 alpha-subunit. The insulinotropic effect of acute Kv2.1 inhibition resulted from enhanced membrane depolarization and augmented intracellular Ca(2+) responses to glucose. Immunohistochemical staining of mouse pancreas sections showed that expression of Kv2.1 correlated highly with insulin-containing beta-cells, consistent with the ability of C-1 to block voltage-dependent outward K(+) currents in isolated mouse beta-cells. Antagonism of Kv2.1 in an ex vivo perfused mouse pancreas model enhanced first- and second-phase insulin secretion, whereas glucagon secretion was unaffected. The present study demonstrates that Kv2.1 is an important component of beta-cell stimulus-secretion coupling, and a compound that enhances, but does not initiate, beta-cell electrical activity by acting on Kv2.1 would be a useful antidiabetic agent.

Na/Ca exchange in function, growth, and demise of beta-cells.

Recent knowledge concerning the Na/Ca exchanger (NCX) in the pancreatic beta-cell is reviewed. The beta-cell expresses various NCX1 splice variants in a species-specific pattern (NCX1.3 and 1.7 in the rat; NCX1.2, 1.3, and 1.7 in the mouse) and in variable and different proportions. In the rat beta-cell, the exchanger displays a high capacity, accounts for about 70% of Ca(2+) extrusion, and participates in Ca(2+) inflow during membrane depolarization. In the mouse, however, the contribution of the exchanger to Ca(2+) extrusion is more modest, and to Ca(2+) inflow, less evident. The exchanger has a stoichiometry of 3 Na(+) for 1 Ca(2+), is electrogenic, and displays a reversal potential at -20 mV. Although being of low magnitude, the current generated by the exchanger shapes glucose-induced beta-cell electrical activity and intracellular Ca(2+) oscillations. Intracellular Ca(2+) may also trigger apoptosis. For instance, overexpression of the exchanger increases Ca(2+)-dependent and Ca(2+)-independent beta-cell death by apoptosis, a phenomenon resulting from the depletion of ER Ca(2+) stores with subsequent activation of caspase-12. Na/Ca exchange overexpression also reduces beta-cell growth. Hence, the Na/Ca exchanger is a versatile system that appears to play an important role in the function, growth, and demise of the beta-cell.

Nerve growth factor increases sodium channel expression in pancreatic beta cells: implications for insulin secretion.

The importance of nerve growth factor (NGF) modulation of pancreatic beta cells is demonstrated by the fact that these cells secrete and respond to this trophic factor. Among NGF effects on beta cells is an increase in Na+ and Ca2+ current densities. This study investigates the mechanisms involved in the NGF-induced increase in Na+ current and the implications of this effect for insulin secretion. The following results were obtained in single beta cells cultured with NGF for 5-7 days: 1) A steady-state level of mRNA coding for type III sodium channel alpha subunit increased twofold compared with that for control cells. 2) The increase in Na+ current density was blocked either by cycloheximide or by actinomycin D, indicating that it is mediated by protein synthesis and mRNA transcription. 3) NGF treatment strengthened, by nearly fourfold, the beta-cell electrical activity; this effect is partially related to the increased Na+ current, because tetrodotoxin (TTX) decreased the duration of the depolarized plateau level. 4) Single beta cells secreted nearly two times more insulin in response to 5.6 or 15.6 mM glucose. This effect was inhibited by TTX, indicating that the enhanced Na+ current plays an important role. These data suggest that NGF could preserve an adequate expression of sodium channels and that impairment of NGF modulation could lead to deficient insulin secretion and diabetes mellitus.

Selective inhibition of glucose-stimulated beta-cell activity by an anion channel inhibitor.

4,4'-Dithiocyanatostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of the volume-sensitive anion channel, was used to investigate the role of this channel in the stimulation of rat pancreatic beta-cells by glucose and by tolbutamide. Glucose-stimulated electrical activity in beta-cells was markedly and reversibly inhibited by DIDS. The increase in cytosolic [Ca2+] and stimulated insulin release evoked by glucose were also inhibited by DIDS. In contrast to its inhibitory effect on glucose-induced beta-cell activity, DIDS had no effect on electrical activity, the rise in [Ca2+]i or insulin release induced by tolbutamide. DIDS failed to increase beta-cell input conductance, an index of whole-cell K(ATP) channel activity, or the rate of efflux of 86Rb+ from perifused islets, a measure of net K+ permeability. Furthermore, DIDS had no effect on intracellular pH or on regulatory volume increase following exposure of cells to hypertonic solutions, indicating that the effects of DIDS were not the result of inhibition of Cl- transport systems. It is suggested that the DIDS-induced repolarization is caused by inactivation of the volume-sensitive anion channel. The stimulation of beta-cell electrical and secretory activity by glucose, but not tolbutamide, may therefore involve activation of the anion channel.

Glucose-dependent stimulatory effect of glucagon-like peptide 1(7-36) amide on the electrical activity of pancreatic beta-cells recorded in vivo.

The stimulatory effect of the glucagon-like peptide (GLP)-1(7-36) amide on electrical activity in pancreatic b-cells recorded in vivo was studied. The injection of GLP-1 produces a lengthening of the active phase with respect to the silent phase, leading to a stimulation of insulin release, which produces a secondary decrease in blood glucose concentration and eventually, to the hyperpolarization of the membrane at a blood glucose level of approximately 5 mmol/l. The injection of GLP-1 at a glycemic level <5 mmol/l does not stimulate electrical activity. This is in contrast to the effect of tolbutamide, which stimulates electrical activity at low glucose concentrations. These results demonstrate that in vivo, the stimulatory effect of GLP-1 on insulin secretion is at least partially mediated by its effect on beta-cell electrical activity. Furthermore, the glucose dependence of the effect confers to GLP-1, a security factor that supports its potential use in the treatment of type 2 diabetes.

RINm5f cells express inactivating BK channels whereas HIT cells express noninactivating BK channels.

Large-conductance Ca2+- and voltage-activated BK-type K+ channels are expressed abundantly in normal rat pancreatic islet cells and in the clonal rat insulinoma tumor (RINm5f) and hamster insulinoma tumor (HIT) beta cell lines. Previous work has suggested that the Ca2+ sensitivity of BK channels in RIN cells is substantially less than that in HIT cells, perhaps contributing to differences between the cell lines in responsiveness to glucose in mediating insulin secretion. In both RIN cells and normal pancreatic beta cells, BK channels are thought to play a limited role in responses of beta cells to secretagogues and in the electrical activity of beta cells. Here we examine in detail the properties of BK channels in RIN and HIT cells using inside-out patches and whole cell recordings. BK channels in RIN cells exhibit rapid inactivation that results in an anomalous steady-state Ca2+ dependence of activation. In contrast, BK channels in HIT cells exhibit the more usual noninactivating behavior. When BK inactivation is taken into account, the Ca2+ and voltage dependence of activation of BK channels in RIN and HIT cells is essentially indistinguishable. The properties of BK channel inactivation in RIN cells are similar to those of inactivating BK channels (termed BKi channels) previously identified in rat chromaffin cells. Inactivation involves multiple, trypsin-sensitive cytosolic domains and exhibits a dependence on Ca2+ and voltage that appears to arise from coupling to channel activation. In addition, the rates of inactivation onset and recovery are similar to that of BKi channels in chromaffin cells. The charybdotoxin (CTX) sensitivity of BKi currents is somewhat less than that of the noninactivating BK variant. Action potential voltage-clamp waveforms indicate that BK current is activated only weakly by Ca2+ influx in RIN cells but more strongly activated in HIT cells even when Ca2+ current magnitude is comparable. Concentrations of CTX sufficient to block BKi current in RIN cells have no effect on action potential activity initiated by glucose or DC injection. Despite its abundant expression in RIN cells, BKi current appears to play little role in action potential activity initiated by glucose or DC injection in RIN cells, but BK current may play an important role in action potential repolarization in HIT cells.

Effects of hexose pentaacetates on electrical activity and cytosolic Ca2+ in mouse pancreatic islets.

Electrical activity of beta-cells and cytosolic Ca2+ concentration ([Ca2+]i) were monitored in mouse pancreatic islets exposed to the pentaacetate esters of alpha-D-glucose, beta-D-galactose and beta-L-glucose, all tested at 1.7 mM concentration. In the presence of 5 mM D-glucose, alpha-D-glucose pentaacetate induced electrical activity and increased [Ca2+]i, whilst beta-D-galactose pentaacetate failed to do so. The electrical and cationic response to the D-glucose ester occurred with a delay of between 5 and 10 min, the ester-induced increase in [Ca2+]i being suppressed in the absence of extracellular Ca2+. As a rule, beta-L-glucose pentaacetate also failed to evoke biophysical responses in the islets exposed to 5 mM D-glucose. However, in the presence of 10 mM L-leucine the L-glucose ester induced electrical activity. These findings, which parallel the insulinotropic action of the same esters in rat pancreatic islets reinforce the view that the positive insulinotropic action of selected hexose pentaacetates cannot be attributed to the catabolism of their acetate moiety but, instead, involves a dual mode of action linked to both the metabolism of their carbohydrate moiety and a direct effect of the ester itself upon a yet unidentified receptor system. Furthermore, this study provides the first evidence that the latter direct effect results in the induction of both electrical activity and [Ca2+]i oscillations.