Pulsatile Basal Insulin Secretion Is Driven by Glycolytic Oscillations.

In fasted and fed states, blood insulin levels are oscillatory. While this phenomenon is well studied at high glucose levels, comparatively little is known about its origin under basal conditions. We propose a possible mechanism for basal insulin oscillations based on oscillations in glycolysis, demonstrated using an established mathematical model. At high glucose, this is superseded by a calcium-dependent mechanism.

Deletion of Ia-2 and/or Ia-2ß in mice decreases insulin secretion by reducing the number of dense core vesicles.

AIMS/HYPOTHESIS: Islet antigen 2 (IA-2) and IA-2ß are dense core vesicle (DCV) transmembrane proteins and major autoantigens in type 1 diabetes. The present experiments were initiated to test the hypothesis that the knockout of the genes encoding these proteins impairs the secretion of insulin by reducing the number of DCV. METHODS: Insulin secretion, content and DCV number were evaluated in islets from single knockout (Ia-2 [also known as Ptprn] KO, Ia-2ß [also known as Ptprn2] KO) and double knockout (DKO) mice by a variety of techniques including electron and two-photon microscopy, membrane capacitance, Ca(2+) currents, DCV half-life, lysosome number and size and autophagy. RESULTS: Islets from single and DKO mice all showed a significant decrease in insulin content, insulin secretion and the number and half-life of DCV (p < 0.05 to 0.001). Exocytosis as evaluated by two-photon microscopy, membrane capacitance and Ca(2+) currents supports these findings. Electron microscopy of islets from KO mice revealed a marked increase (p < 0.05 to 0.001) in the number and size of lysosomes and enzymatic studies showed an increase in cathepsin D activity (p < 0.01). LC3 protein, an indicator of autophagy, also was increased in islets of KO compared with wild-type mice (p < 0.05 to 0.01) suggesting that autophagy might be involved in the deletion of DCV. CONCLUSIONS/INTERPRETATION: We conclude that the decrease in insulin content and secretion, resulting from the deletion of Ia-2 and/or Ia-2ß, is due to a decrease in the number of DCV.

Niflumic acid-sensitive ion channels play an important role in the induction of glucose-stimulated insulin secretion by cyclic AMP in mice.

AIMS/HYPOTHESIS: We have previously reported that glucose-stimulated insulin secretion (GSIS) is induced by glucagon-like peptide-1 (GLP-1) in mice lacking ATP-sensitive K(+) (K(ATP)) channels (Kir6.2(-/-) mice [up-to-date symbol for Kir6.2 gene is Kcnj11]), in which glucose alone does not trigger insulin secretion. This study aimed to clarify the mechanism involved in the induction of GSIS by GLP-1. METHODS: Pancreas perfusion experiments were performed using wild-type (Kir6.2(+/+)) or Kir6.2(-/-) mice. Glucose concentrations were either changed abruptly from 2.8 to 16.7 mmol/l or increased stepwise (1.4 mmol/l per step) from 2.8 to 12.5 mmol/l. Electrophysiological experiments were performed using pancreatic beta cells isolated from Kir6.2(-/-) mice or clonal pancreatic beta cells (MIN6 cells) after pharmacologically inhibiting their K(ATP) channels with glibenclamide. RESULTS: The combination of cyclic AMP plus 16.7 mmol/l glucose evoked insulin secretion in Kir6.2(-/-) pancreases where glucose alone was ineffective as a secretagogue. The secretion was blocked by the application of niflumic acid. In K(ATP) channel-inactivated MIN6 cells, niflumic acid similarly inhibited the membrane depolarisation caused by cAMP plus glucose. Surprisingly, stepwise increases of glucose concentration triggered insulin secretion only in the presence of cAMP or GLP-1 in Kir6.2(+/+), as in Kir6.2(-/-) pancreases. CONCLUSIONS/INTERPRETATION: Niflumic acid-sensitive ion channels participate in the induction of GSIS by cyclic AMP in Kir6.2(-/-) beta cells. Cyclic AMP thus not only acts as a potentiator of insulin secretion, but appears to be permissive for GSIS via novel, niflumic acid-sensitive ion channels. This mechanism may be physiologically important for triggering insulin secretion when the plasma glucose concentration increases gradually rather than abruptly.

Reduction of voltage-dependent Mg2+ blockade of NMDA current in mechanically injured neurons.

Activation of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors is implicated in the pathophysiology of traumatic brain injury. Here, the effects of mechanical injury on the voltage-dependent magnesium (Mg2+) block of NMDA currents in cultured rat cortical neurons were examined. Stretch-induced injury was found to reduce the Mg2+ blockade, resulting in significantly larger ionic currents and increases in intracellular free calcium (Ca2+) concentration after NMDA stimulation of injured neurons. The Mg2+ blockade was partially restored by increased extracellular Mg2+ concentration or by pretreatment with the protein kinase C inhibitor calphostin C. These findings could account for the secondary pathological changes associated with traumatic brain injury.

Pancreatic B cells are bursting, but how?

Insulin secretogogues have long been known to stimulate and modulate bursting electrical activity in pancreatic islet B cells and thereby supply extracellular Ca2+ for the exocytosis of insulin. Recent results have ruled out a long-held hypothesis for the mechanism of burst formation that postulated key roles for intracellular Ca2+ accumulation and activation of Ca(2+)-activated K+ channels. Here, we present an alternative hypotheses based on a persistent Ca2+ conductance and, possibly, phasic activation of ATP-sensitive K+ channels. These hypotheses are compared with mechanisms of bursting proposed for invertebrate and mammalian neurons.

Enhancement of AMPA-mediated current after traumatic injury in cortical neurons.

Overactivation of ionotropic glutamate receptors has been implicated in the pathophysiology of traumatic brain injury. Using an in vitro cell injury model, we examined the effects of stretch-induced traumatic injury on the AMPA subtype of ionotropic glutamate receptors in cultured neonatal cortical neurons. Recordings made using the whole-cell patch-clamp technique revealed that a subpopulation of injured neurons exhibited an increased current in response to AMPA. The current-voltage relationship of these injured neurons showed an increased slope conductance but no change in reversal potential compared with uninjured neurons. Additionally, the EC(50) values of uninjured and injured neurons were nearly identical. Thus, current potentiation was not caused by changes in the voltage-dependence, ion selectivity, or apparent agonist affinity of the AMPA channel. AMPA-elicited current could also be fully inhibited by the application of selective AMPA receptor antagonists, thereby excluding the possibility that current potentiation in injured neurons was caused by the activation of other, nondesensitizing receptors. The difference in current densities between control and injured neurons was abolished when AMPA receptor desensitization was inhibited by the coapplication of AMPA and cyclothiazide or by the use of kainate as an agonist, suggesting that mechanical injury alters AMPA receptor desensitization. Reduction of AMPA receptor desensitization after brain injury would be expected to further exacerbate the effects of increased postinjury extracellular glutamate and contribute to trauma-related cell loss and dysfunctional synaptic information processing.

An ATP-sensitive Cl- channel current that is activated by cell swelling, cAMP, and glyburide in insulin-secreting cells.

Although chloride ions are known to modulate insulin release and islet electrical activity, the mechanism or mechanisms mediating these effects are unclear. However, numerous studies of islet Cl- fluxes have suggested that Cl- movements and glucose and sulfonylurea sensitive and are blocked by stilbene-derivative Cl- channel blockers. We now show for the first time that insulin-secreting cells have a Cl- channel current, which we term ICl,islet. The current is activated by hypotonic conditions, 1-10 mumol/l glyburide and 0.5 mmol/l 8-bromoadenosine 3':5'-cyclic monophosphate sodium. ICl,islet is mediated by Cl- channels, since replacing [Cl-]o with less permeant aspartate reduces current amplitude and depolarizes its reversal potential. In addition, 100 mumol/l 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) or glyburide, which blocks the Cl- channels of other cell types, block ICl,islet. Reducing [ATP]i reduces the amplitude of the current, suggesting that it may be under metabolic control. The current is time-independent and shows strong outward-rectification beyond approximately 0 mV. At potentials associated with the silent phase of islet electrical activity (approximately -65 mV), ICl,islet mediates a large inward current, which would be expected to depolarize islet membrane potential. Thus, activation of this novel current by increased intracellular cAMP, sulfonylureas, or ATP may contribute to the well-known depolarizing effects of these agents.

ATP-sensitive K+ channels in pancreatic beta-cells. Spare-channel hypothesis.

Since their discovery in pancreatic beta-cells, ATP-sensitive K+ channels in the cell membrane have been thought to mediate glucose-induced beta-cell depolarization, which is required for triggering the voltage-dependent Ca2+ uptake subserving insulin release. The theory is that metabolism of glucose (and other fuel molecules) increases intracellular ATP or possibly other metabolites that diffuse to the membrane and inhibit the opening of ATP-sensitive K+ channels. This slows the efflux of positively charged K+ and depolarizes the cell. A recurrent source of confusion regarding this idea stems from the early observation that these channels are so exquisitely sensitive to intracellular ATP that channel opening is predicted to be approximately 99% inhibited under physiological conditions. To account for this apparent discrepancy, various mechanisms have been proposed that might render the channels less sensitive to intracellular ATP. We use a simple mathematical model to demonstrate that there is no major discrepancy and that, in fact, given the electrophysiological mechanisms existing in the beta-cell, the extreme sensitivity of the channels to ATP is appropriate and even mandatory for their physiological function.

Insulin activates ATP-sensitive K(+) channels in pancreatic beta-cells through a phosphatidylinositol 3-kinase-dependent pathway.

Insulin is known to regulate pancreatic beta-cell function through the activation of cell surface insulin receptors, phosphorylation of insulin receptor substrate (IRS)-1 and -2, and activation of phosphatidylinositol (PI) 3-kinase. However, an acute effect of insulin in modulating beta-cell electrical activity and its underlying ionic currents has not been reported. Using the perforated patch clamp technique, we found that insulin (1-600 nmol/l) but not IGF-1 (100 nmol/l) reversibly hyperpolarized single mouse beta-cells and inhibited their electrical activity. The dose-response relationship for insulin yielded a maximal change (mean +/- SE) in membrane potential of -13.6 +/- 2.0 mV (P < 0.001) and a 50% effective dose of 25.9 +/- 0.1 nmol/l (n = 63). Exposing patched beta-cells within intact islets to 200 nmol/l insulin produced similar results, hyperpolarizing islets from -47.7 +/- 3.3 to -65.6 +/- 3.7 mV (P < 0.0001, n = 11). In single cells, insulin-induced hyperpolarization was associated with a threefold increase in whole-cell conductance from 0.6 +/- 0.1 to 1.7 +/- 0.2 nS (P < 0.001, n = 10) and a shift in the current reversal potential from -25.7 +/- 2.5 to -63.7 +/- 1.0 mV (P < 0.001 vs. control, n = 9; calculated K(+) equilibrium potential = -90 mV). The effects of insulin were reversed by tolbutamide, which decreased cell conductance to 0.5 +/- 0.1 nS and shifted the current reversal potential to -25.2 +/- 2.3 mV. Insulin-induced beta-cell hyperpolarization was sufficient to abolish intracellular calcium concentration ([Ca(2+)](i)) oscillations measured in pancreatic islets exposed to 10 mmol/l glucose. The application of 100 nmol/l wortmannin to inactivate PI 3-kinase, a key enzyme in insulin signaling, was found to reverse the effects of 100 nmol/l insulin. In cell-attached patches, single ATP-sensitive K(+) (K(ATP)) channels were activated by bath-applied insulin and subsequently inhibited by wortmannin. Our data thus demonstrate that insulin activates the K(ATP) channels of single mouse pancreatic beta-cells and islets, resulting in membrane hyperpolarization, an inhibition of electrical activity, and the abolition of [Ca(2+)](i) oscillations. We thus propose that locally released insulin might serve as a negative feedback signal within the islet under physiological conditions.

Chloride channels regulate HIT cell volume but cannot fully account for swelling-induced insulin secretion.

Insulin-secreting pancreatic islet beta-cells possess anion-permeable Cl- channels (I(Cl,islet)) that are swelling-activated, but the role of these channels in the cells is unclear. The Cl- channel blockers 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and niflumic acid were evaluated for their ability to inhibit I(Cl,islet) in clonal beta-cells (HIT cells). Both drugs blocked the channel, but the blockade due to niflumic acid was less voltage-dependent than the blockade due to DIDS. HIT cell volume initially increased in hypotonic solution and was followed by a regulatory volume decrease (RVD). The addition of niflumic acid and, to a lesser extent, DIDS to the hypotonic solution potentiated swelling and blocked the RVD. In isotonic solution, niflumic acid produced swelling, suggesting that islet Cl- channels are activated under basal conditions. The channel blockers glyburide, gadolinium, or tetraethylammonium-Cl did not alter hypotonic-induced swelling or volume regulation. The Na/K/2Cl transport blocker furosemide produced cell shrinkage in isotonic solution and blocked cell swelling normally induced by hypotonic solution. Perifused HIT cells secreted insulin when challenged with hypotonic solutions. However, this could not be completely attributed to I(Cl,islet)-mediated depolarization, because secretion persisted even when Cl- channels were fully blocked. To test whether blocker-resistant secretion occurred via a distal pathway, distal secretion was isolated using 50 mmol/l potassium and diazoxide. Under these conditions, glucose-dependent secretion was blunted, but hypotonically induced secretion persisted, even with Cl- channel blockers present. These results suggest that beta-cell swelling stimulates insulin secretion primarily via a distal I(Cl,islet)-independent mechanism, as has been proposed for K(ATP)-independent glucose- and sulfonylurea-stimulated insulin secretion. Reverse transcriptase-polymerase chain reaction of HIT cell mRNA identified a CLC-3 transcript in HIT cells. In other systems, CLC-3 is believed to mediate swelling-induced outwardly rectifying Cl- channels. This suggests that the proximal effects of swelling to regulate cell volume may be mediated by CLC-3 or a closely related Cl- channel.

Calcium-activated K+ channels of mouse beta-cells are controlled by both store and cytoplasmic Ca2+: experimental and theoretical studies.

A novel calcium-dependent potassium current (K(slow)) that slowly activates in response to a simulated islet burst was identified recently in mouse pancreatic beta-cells (Göpel, S.O., T. Kanno, S. Barg, L. Eliasson, J. Galvanovskis, E. Renström, and P. Rorsman. 1999. J. Gen. Physiol. 114:759-769). K(slow) activation may help terminate the cyclic bursts of Ca(2+)-dependent action potentials that drive Ca(2+) influx and insulin secretion in beta-cells. Here, we report that when [Ca(2+)](i) handling was disrupted by blocking Ca(2+) uptake into the ER with two separate agents reported to block the sarco/endoplasmic calcium ATPase (SERCA), thapsigargin (1-5 microM) or insulin (200 nM), K(slow) was transiently potentiated and then inhibited. K(slow) amplitude could also be inhibited by increasing extracellular glucose concentration from 5 to 10 mM. The biphasic modulation of K(slow) by SERCA blockers could not be explained by a minimal mathematical model in which [Ca(2+)](i) is divided between two compartments, the cytosol and the ER, and K(slow) activation mirrors changes in cytosolic calcium induced by the burst protocol. However, the experimental findings were reproduced by a model in which K(slow) activation is mediated by a localized pool of [Ca(2+)] in a subspace located between the ER and the plasma membrane. In this model, the subspace [Ca(2+)] follows changes in cytosolic [Ca(2+)] but with a gradient that reflects Ca(2+) efflux from the ER. Slow modulation of this gradient as the ER empties and fills may enhance the role of K(slow) and [Ca(2+)] handling in influencing beta-cell electrical activity and insulin secretion.

Temperature modulates the Ca2+ current of HIT-T15 and mouse pancreatic beta-cells.

The effects of raising temperature on the Ca2+ currents of insulin-secreting HIT and mouse pancreatic beta-cells were studied. Currents were measured in 3 mM Ca2+ containing solutions using standard whole-cell techniques. Increasing temperature from 22 degrees C to 35 degrees C increased peak Ca2+ current amplitude, percent (fast) inactivation and decreased the time-to-peak of the current. Ca2+ currents in HIT and mouse beta-cells responded in the same manner to an imposed physiological burstwave with test-pulses: (i) application of the burstwave inactivated the test-pulse Ca2+ current at both high and low temperatures; (ii) Ca2+ current inactivation leveled off during the plateau phase at 20-22 degrees C whereas there was an apparent continual decay at 33-35 degrees C; and (iii) recovery from inactivation occurred during the interburst period at both temperatures. Application of a physiological burstwave without test-pulses to mouse beta-cells before and after addition of 0.2 mM Cd2+ resulted in a Ca2+ difference current. This current activated during the hyperpolarized interburst phase, activated, inactivated and deactivated rapidly and continually during the plateau phase, and recovered from inactivation during the interburst. Although raising temperature strongly modified HIT and mouse beta-cells Ca2+ current, our work suggests that other channels, in addition to Ca2+ channels, are likely to be involved in the control of islet bursts, particularly at different temperatures. In addition, the effect of temperature on islet cell Ca2+ current may be partly responsible for the well-known temperature dependence of glucose-dependent secretion.

Contribution of L- and non-L-type calcium channels to voltage-gated calcium current and glucose-dependent insulin secretion in HIT-T15 cells.

The pharmacological properties of voltage-gated Ca current and glucose-dependent insulin secretion were determined using the HIT insulinoma line to understand the role of Ca channels in stimulus-secretion coupling. The L-type Ca channel antagonist nimodipine inhibited a maximum of 50-55% of the peak Ca current, suggesting that L- and non-L-type channels contribute to Ca current. The L-agonist BAY K 8644 increased Ca current by 155%, whereas the N-channel blocker omega-conotoxin MVIIA reversibly blocked 35% of the peak Ca current. Total block with nimodipine and MVIIA was additive. Conotoxin MVIIC did not affect HIT Ca current. Prolonged depolarizations elicited rapidly and slowly inactivating Ca currents. Nimodipine partially inhibited transient current, but fully inhibited slowly inactivating current, suggesting that the former is mediated by L- and N-channels, and the latter is mediated by L-channels. Like slowly inactivating Ca current, glucose-dependent insulin secretion was fully inhibited by nimodipine and insensitive to MVIIA. BAY K potentiated secretion and antagonized nimodipine block. These results suggest that persistent Ca current is mediated by L-channels and is strongly coupled to insulin secretion, whereas transient Ca current is mediated by L- and N-channels and is weakly coupled. Sustained Ca influx may be preferentially coupled because glucose persistently depolarizes HIT cells and inactivates more transient Ca channel pathways.

Spontaneous miniature outward currents in cultured bullfrog neurons.

Spontaneous miniature hyperpolarizations were observed in cultured bullfrog neurons. Depolarization increased the frequency and amplitude of the events. Under voltage-clamp, these events were manifested as spontaneous miniature outward currents of SMOCs which were usually less than 2 nA, had a rapid rising phase and a slower voltage-dependent exponential decay. Analysis of inter-event intervals suggested that SMOCs occurred randomly, while analysis of their amplitudes yielded exponential amplitude distributions. Mean SMOC amplitudes and SMOC frequency increased with depolarization, even with 100 microM CdCl2 present. Time constants of SMOC decay resembled time constants obtained from voltage-jump experiments on Ca2+-loaded cells, and together with the sensitivity of SMOCs to tetraethyl ammonium (TEA), suggested that SMOCs are due to activation of fast Ca2+-gated potassium channels. We propose that a SMOC occurs when 10-5000 of these channels are activated by punctate intracellular Ca2+ release.

An integrated instrument for rapidly deforming living cells using rapid pressure pulses and simultaneously monitoring applied strain in near real time.

Because many types of living cells are sensitive to applied strain, different in vitro models have been designed to elucidate the cellular and subcellular processes that respond to mechanical deformation at both the cell and tissue level. Our focus was to improve upon an already established strain system to make it capable of independently monitoring the deflection and applied pressure delivered to specific wells of a commercially available, deformable multiwell culture plate. To accomplish this, we devised a custom frame that was capable of mounting deformable 6 or 24 well plates, a pressurization system that could load wells within the plates, and a camera-based imaging system which was capable of capturing strain responses at a sufficiently high frame rate. The system used a user defined program constructed in Labview(®) to trigger plate pressurization while simultaneously allowing the deflection of the silicone elastomeric plate bottoms to be imaged in near real time. With this system, up to six wells could be pulsed simultaneously using compressed air or nitrogen. Digital image capture allowed near-real time monitoring of applied strain, strain rate, and the cell loading profiles. Although our ultimate goal is to determine how different strain rates applied to neurons modulates their intrinsic biochemical cascades, the same platform technology could be readily applied to other systems. Combining commercially available, deformable multiwell plates with a simple instrument having the monitoring capabilities described here should permit near real time calculations of stretch-induced membrane strain in multiple wells in real time for a wide variety of applications, including high throughput drug screening.

Localized calcium influx in pancreatic beta-cells: its significance for Ca2+-dependent insulin secretion from the islets of Langerhans.

Ca2+ influx through voltage-dependent Ca2+ channels plays a crucial role in stimulus-secretion coupling in pancreatic islet beta-cells. Molecular and physiologic studies have identified multiple Ca2+ channel subtypes in rodent islets and insulin-secreting cell lines. The differential targeting of Ca2+ channel subtypes to the vicinity of the insulin secretory apparatus is likely to account for their selective coupling to glucose-dependent insulin secretion. In this article, I review these studies. In addition, I discuss temporal and spatial aspects of Ca2+ signaling in beta-cells, the former involving the oscillatory activation of Ca2+ channels during glucose-induced electrical bursting, and the latter involving [Ca2+]i elevation in restricted microscopic "domains," as well as direct interactions between Ca2+ channels and secretory SNARE proteins. Finally, I review the evidence supporting a possible role for Ca2+ release from the endoplasmic reticulum in glucose-dependent insulin secretion, and evidence to support the existence of novel Ca2+ entry pathways. I also show that the beta-cell has an elaborate and complex set of [Ca2+]i signaling mechanisms that are capable of generating diverse and extremely precise [Ca2+]i patterns. These signals, in turn, are exquisitely coupled in space and time to the beta-cell secretory machinery to produce the precise minute-to-minute control of insulin secretion necessary for body energy homeostasis.

New mechanisms for sulfonylurea control of insulin secretion.

Oral antidiabetic sulfonylureas like tolbutamide and glyburide have been used to treat patients with noninsulin dependent diabetes mellitus. These agents lower blood glucose by stimulating insulin secretion from the pancreatic islets of Langerhans. A major component of this stimulation is sulfonylurea-mediated closure of the ATP-inhibited potassium channels (K(ATP) channels) of islet ß-cells. Closure of these channels leads to cell depolarization, calcium uptake, and insulin exocytosis. Progress leading up to the recent cloning of the high-affinity sulfonylurea receptor and reconstitution of the K(ATP) channel is reviewed in this article together with new data showing that sulfonylureas may control secretion by activating a novel chloride ion channel, inhibiting an islet Na/K/ATPase or via distal stimulation of granule exocytosis by a kinase C dependent mechanism.