Closure Of KATP Channels Research Articles

Glucose Inhibits Glucagon Secretion from Pancreatic Alpha-Cells by Closure of K ATP-Channels

Glucose inhibits glucagon secretion but the mechanisms involved are not known. We performed whole-cell perforated patch membrane potential recordings on alpha-cells in intact mouse pancreatic islets. Alpha-cells are electrically active at low glucose concentrations (<1 mM) and generate overshooting action potentials that peak at +10 mV. These action potentials result from activation of voltage-gated Na++ and A-type K+-channels. The most negative interspike membrane potential is approx. −45 mV. Application of 6 mM glucose reversibly depolarized the alpha-cell by approx. 5 mV and reduced peak voltage of the action potentials by up to 15 mV. We estimated the impact this decrease in action potential amplitude on alpha-cell exocytosis by capacitance measurements of depolarization-evoked exocytosis. We found that exocytosis is reduced by approx. 7% for every mV reduction of spike height. Thus, the glucose-induced reduction of spike height is sufficient to account for the 70-80% inhibition of glucagon secretion measured biochemically. We also measured alpha-cell electrical activity elicited by current injection and found that small depolarizations above the normal interspike voltage mimic the effects of glucose on action potential height. In parallel measurements of glucagon secretion, the inhibitory effect of glucose could be mimicked by tolbutamide (a blocker of KATP-channels) and antagonized by diazoxide (an activator of KATP-channels). We conclude that glucose inhibition of glucagon secretion involves closure of plasmalemmal KATP-channels. The resulting membrane depolarization leads to voltage-dependent inactivation of the ion channels involved in action potential firing and a lowered action potential amplitude. This culminates in reduced activation of the N-type Ca2+-channels mediating the Ca2+ entry required to trigger exocytosis of the glucagon-containing secretory vesicles. Thus, alpha-cells provide an example where reduced K+-conductance is associated with lowered rather than increased (as seen in most other cell types) membrane excitability.

Studies of first phase insulin secretion using imposed plasma membrane depolarization

The first phase of glucose-induced insulin secretion is generally regarded to represent the release of a finite pool of secretion-ready granules, triggered by the depolarization-induced influx of Ca2+ through L-type Ca2+ channels. However, the experimental induction of insulin secretion by imposed plasma membrane depolarization may be more complicated than currently appreciated. A comparison of the effects of high K+ concentrations with those of KATP channel closure, which initiates the electrical activity of the beta cell, suggests that 40 mM K+, which is a popular tool to produce a first phase-like secretion, is of supraphysiological strength, whereas the 20 mV depolarization by 15 mM K+ is nearly inefficient. A major conceptual problem consists in the occurrence of action potentials during KATP channel closure, but not during K+ depolarization, which leaves the K+ channel conductance unchanged. Recent observations suggest that the signal function of the endogenously generated depolarization is not homogeneous, but may rather differ between the component mainly determined by KATP channel closure (slow waves) and that mainly determined by Ca2+ influx (action potentials).

Glucose sensing in the pancreatic beta cell: a computational systems analysis

BackgroundPancreatic beta-cells respond to rising blood glucose by increasing oxidative metabolism, leading to an increased ATP/ADP ratio in the cytoplasm. This leads to a closure of KATP channels, depolarization of the plasma membrane, influx of calcium and the eventual secretion of insulin. Such mechanism suggests that beta-cell metabolism should have a functional regulation specific to secretion, as opposed to coupling to contraction. The goal of this work is to uncover contributions of the cytoplasmic and mitochondrial processes in this secretory coupling mechanism using mathematical modeling in a systems biology approach.MethodsWe describe a mathematical model of beta-cell sensitivity to glucose. The cytoplasmic part of the model includes equations describing glucokinase, glycolysis, pyruvate reduction, NADH and ATP production and consumption. The mitochondrial part begins with production of NADH, which is regulated by pyruvate dehydrogenase. NADH is used in the electron transport chain to establish a proton motive force, driving the F1F0 ATPase. Redox shuttles and mitochondrial Ca2+ handling were also modeled.ResultsThe model correctly predicts changes in the ATP/ADP ratio, Ca2+ and other metabolic parameters in response to changes in substrate delivery at steady-state and during cytoplasmic Ca2+ oscillations. Our analysis of the model simulations suggests that the mitochondrial membrane potential should be relatively lower in beta cells compared with other cell types to permit precise mitochondrial regulation of the cytoplasmic ATP/ADP ratio. This key difference may follow from a relative reduction in respiratory activity. The model demonstrates how activity of lactate dehydrogenase, uncoupling proteins and the redox shuttles can regulate beta-cell function in concert; that independent oscillations of cytoplasmic Ca2+ can lead to slow coupled metabolic oscillations; and that the relatively low production rate of reactive oxygen species in beta-cells under physiological conditions is a consequence of the relatively decreased mitochondrial membrane potential.ConclusionThis comprehensive model predicts a special role for mitochondrial control mechanisms in insulin secretion and ROS generation in the beta cell. The model can be used for testing and generating control hypotheses and will help to provide a more complete understanding of beta-cell glucose-sensing central to the physiology and pathology of pancreatic β-cells.

The Gq/G11-mediated signaling pathway is critical for autocrine potentiation of insulin secretion in mice

A variety of neurotransmitters, gastrointestinal hormones, and metabolic signals are known to potentiate insulin secretion through GPCRs. We show here that beta cell-specific inactivation of the genes encoding the G protein alpha-subunits Galphaq and Galpha11 resulted in impaired glucose tolerance and insulin secretion in mice. Interestingly, the defects observed in Galphaq/Galpha11-deficient beta cells were not restricted to loss of muscarinic or metabolic potentiation of insulin release; the response to glucose per se was also diminished. Electrophysiological recordings revealed that glucose-induced depolarization of isolated beta cells was impaired in the absence of Galphaq/Galpha11, and closure of KATP channels was inhibited. We provide evidence that this reduced excitability was due to a loss of beta cell-autonomous potentiation of insulin secretion through factors cosecreted with insulin. We identified as autocrine mediators involved in this process extracellular nucleotides such as uridine diphosphate acting through the Gq/G11-coupled P2Y6 receptor and extracellular calcium acting through the calcium-sensing receptor. Thus, the Gq/G11-mediated signaling pathway potentiates insulin secretion in response to glucose by integrating systemic as well as autocrine/paracrine mediators.

PI3Kinase/Akt activation trigger ROS production in a model of pulmonary ischemia

We reported earlier that ischemia or loss of shear in the lung endothelium causes endothelial membrane depolarization (via KATP channel closure) that leads to NADPH oxidase activation and reactive oxygen species (ROS) production. ROS production was abolished in the presence of PI3Kinase/Akt inhibitor, wortmanin. We thus hypothesized that PI3K/Akt activation is the trigger for ROS production with pulmonary ischemia. We used pulmonary microvascular endothelial cells, either wild type (WT), KATP null (KIR6.2−/−), NADPH oxidase null (gp91phox−/−) or WT infected with dominant negative (DN) PI3K, and isolated WT and Akt‐1 null lungs to study signal transduction with ischemia. In WT lungs, the PI3K inhibitor wortmannin had no effect on ischemia mediated depolarization. Cells with DN PI3K and lungs with Akt1−/− depolarized with ischemia but failed to show NADPH oxidase assembly and ROS generation. Depolarization in WT and Akt−/− lungs was blocked upon addition of cromakalim (KATP channel agonist). In WT cells, Akt phosphorylation that was observed with ischemia and was abolished in KIR6.2−/− but not in gp91phox−/− cells. These results indicate that KATP induced depolarization leads to PI3K/Akt activation that triggers NADPH oxidase assembly and eventual ROS generation. Thus, ROS which has hitherto been shown to activate PI3K/Akt in other systems lies downstream of PI3K/Akt in our model of pulmonary ischemia.

Endothelial mechanotransduction with loss of shear is a signal for angiogenesis

We have reported earlier that ischemia in the pulmonary vasculature results in reactive oxygen species (ROS) generation due to KATP channel closure that leads to NADPH oxidase activation. Here we found that pulmonary endothelial cells respond to ischemia in vitro, by increasing their angiogenic potential as compared to cells in static culture. We also investigated ischemia in the vasculature in vivo by femoral artery ligation. Angiogenesis (neovascularization) was assessed by visualization of fluorescence microbeads in the ischemic tissue following their injection into tail vein of mice. Neovascularization observed in wild type mice at 5 days after ligation was significantly decreased in mice that were null for KATP channel, gp91phox subunit of NADPH oxidase or Akt1. Pulmonary microvascular endothelial cells and lungs isolated from these knockout mice do not produce ROS with ischemia. The ischemic tissues from wild type were analyzed for vascular endothelial growth factor (VEGF). Wild type mice showed increased expression of VEGF with ischemia which was not observed in KATP channel, gp91phox and Akt null mice. We conclude that ROS generated with endothelial mechanotransduction causes angiogenesis via increased VEGF expression. ROS thus represents an attempt at neovascularization to restore the impeded blood flow.

New Uses for Old Drugs: Neonatal Diabetes and Sulphonylureas

The discovery that their neonatal diabetes is caused by gain-of-function mutations in the K(ATP) channel has enabled many patients to switch from insulin to oral sulphonylurea drugs. Here, I review molecular, physiological, and clinical features of this change in therapy.

Electrophysiological identification of glucose-sensing neurons in rat nodose ganglia

The vagal afferent system is strategically positioned to mediate rapid changes in motility and satiety in response to systemic glucose levels. In the present study we aimed to identify glucose-excited and glucose-inhibited neurons in nodose ganglia and characterize their glucose-sensing properties. Whole-cell patch-clamp recordings in vagal afferent neurons isolated from rat nodose ganglia demonstrated that 31/118 (26%) neurons were depolarized after increasing extracellular glucose from 5 to 15 mm; 19/118 (16%) were hyperpolarized, and 68/118 were non-responsive. A higher incidence of excitatory response to glucose occurred in gastric- than in portal vein-projecting neurons, the latter having a higher incidence of inhibitory response. In glucose-excited neurons, elevated glucose evoked membrane depolarization (11 mV) and an increase in membrane input resistance (361 to 437 M). Current reversed at 99 mV. In glucose-inhibited neurons, membrane hyperpolarization (13 mV) was associated with decreased membrane input resistance (383 to 293 M). Current reversed at 97 mV. Superfusion of tolbutamide, a K(ATP) channel sulfonylurea receptor blocker, elicited identical glucose-excitatory but not glucose-inhibitory responses. Kir6.2 shRNA transfection abolished glucose-excited but not glucose-inhibited responses. Phosphatidylinositol bisphosphate (PIP(2)) depletion using wortmannin increased the fraction of glucose-excited neurons from 26% to 80%. These results show that rat nodose ganglia have glucose-excited and glucose-inhibited neurons, differentially distributed among gastric- and portal vein-projecting nodose neurons. In glucose-excited neurons, glucose metabolism leads to K(ATP) channel closure, triggering membrane depolarization, whereas in glucose-inhibited neurons, the inhibitory effect of elevated glucose is mediated by an ATP-independent K(+) channel. The results also show that PIP(2) can determine the excitability of glucose-excited neurons.

ATP-Sensitive K+ Channel Mediates the Zinc Switch-Off Signal for Glucagon Response During Glucose Deprivation

OBJECTIVEThe intraislet insulin hypothesis proposes that glucagon secretion during hypoglycemia is triggered by a decrease in intraislet insulin secretion. A more recent hypothesis based on in vivo data from hypoglycemic rats is that it is the decrease in zinc cosecreted with insulin from β-cells, rather than the decrease in insulin itself, that signals glucagon secretion from α-cells during hypoglycemia. These studies were designed to determine whether closure of the α-cell ATP-sensitive K+ channel (KATP channel) is the mechanism through which the zinc switch-off signal triggers glucagon secretion during glucose deprivation.RESEARCH DESIGN AND METHODSAll studies were performed using perifused isolated islets.RESULTSIn control experiments, the expected glucagon response to an endogenous insulin switch-off signal during glucose deprivation was observed in wild-type mouse islets. In experiments with streptozotocin-treated wild-type islets, a glucagon response to an exogenous zinc switch-off signal was observed during glucose deprivation. However, this glucagon response to the zinc switch-off signal during glucose deprivation was not seen in the presence of nifedipine, diazoxide, or tolbutamide or if KATP channel knockout mouse islets were used. All islets had intact glucagon responses to epinephrine.CONCLUSIONSThese data demonstrate that closure of KATP channels and consequent opening of calcium channels is the mechanism through which the zinc switch-off signal triggers glucagon secretion during glucose deprivation.

Plasma membrane depolarization as a determinant of the first phase of insulin secretion

The role of plasma membrane depolarization as a determinant of the initial phase of insulin secretion was investigated. NMRI mouse islets and beta-cells were used to measure the kinetics of insulin secretion, ATP and ADP content, membrane potential, and cytosolic free Ca(2+) concentration ([Ca(2+)](i)). The depolarization of metabolically intact beta-cells by KCl corresponded closely to the theoretical values. In contrast to physiological (glucose) or pharmacological (tolbutamide) ATP-sensitive K(+) (K(ATP)) channel block, KCl depolarization did not induce action potential spiking. The depolarization by 15 mM K(+) (21 mV) corresponded to the plateau depolarization by 50 or 500 microM tolbutamide; that by 40 mM K(+) (41 mV) corresponded to the action potential peaks. Nifedipine and diazoxide abolished action potentials but not KCl depolarization, suggesting that the depolarizing strength of 15, but not 40 mM K(+) corresponds to that of K(ATP) channel closure. K(+) (40 mM) induced a massive secretory response in the presence of 5 mM glucose, whereas 15 mM K(+), like 50 microM tolbutamide, was only slightly effective, even though a marked increase in [Ca(2+)](i) was produced. Raising glucose from 5 to 10 mM in the continued presence of 15 mM K(+) resulted in a strongly enhanced biphasic response. The depolarization pattern of this combination could be mimicked by combining basal glucose with 15 mM K(+) and 50 microM tolbutamide; however, the secretory response to these nonnutrients was much weaker. In conclusion, the initial secretory response to nutrient secretagogues is largely influenced by signaling mechanisms that do not involve depolarization.

Depolarization is the trigger for PI3K/Akt activation and leads to ROS production in a model of pulmonary ischemia

We reported that ischemia or loss of shear in the lung endothelium causes endothelial plasma membrane depolarization (via KATP channel closure) that leads to NADPH oxidase activation and reactive oxygen species (ROS) production. Both high K+ induced depolarization and ischemia resulted in activation of PI3K; its inhibitor (wortmanin) prevented NADPH oxidase assembly and ROS production with ischemia. We used pulmonary microvascular endothelial cells, either wild type (WT), KATP−/−, gp91phox−/− or WT infected with dominant negative (DN) PI3K, and isolated Akt‐1 null lungs to study signal transduction with ischemia. Cells with DN PI3K and lungs with Akt1−/−, both showed the depolarization response but failed to show NADPH oxidase assembly and ROS generation. In WT cells, Akt activation (as monitored by the expression of p‐Akt) that was observed with ischemia was abolished in KATP−/− but not in gp91phox−/− cells. Blocking the ischemic depolarization by addition of cromakalim (KATP channel agonist) to gp91phox−/− cells prevented Akt phosphorylation. These results indicate that KATP induced depolarization leads to NADPH oxidase assembly and ROS generation and precedes PI3K/Akt activation. Thus, ROS which has hitherto been shown to activate PI3K/Akt in other systems lies downstream of PI3K/Akt in our model of pulmonary ischemia.

Mitochondrial Energy Metabolism and Ca2+ Handling in Pancreatic Beta-cells. A System Analysis Approach

Pancreatic islet beta-cells respond to rising blood glucose by increasing oxidative metabolism, leading to both an increased mitochondrial membrane potential (Ψm) and ATP/ADP ratio in cytoplasm. This leads to a closure of KATP channels, depolarization of the plasma membrane, influx of calcium and the eventual secretion of insulin. Such a signaling mechanism suggests that mitochondrial metabolism and ATP/ADP ratio regulation in beta-cells may be specially coupled in comparison with other cell types.

Expression of an activating mutation in the gene encoding the KATP channel subunit Kir6.2 in mouse pancreatic beta cells recapitulates neonatal diabetes.

Neonatal diabetes is a rare monogenic form of diabetes that usually presents within the first six months of life. It is commonly caused by gain-of-function mutations in the genes encoding the Kir6.2 and SUR1 subunits of the plasmalemmal ATP-sensitive K+ (KATP) channel. To better understand this disease, we generated a mouse expressing a Kir6.2 mutation (V59M) that causes neonatal diabetes in humans and we used Cre-lox technology to express the mutation specifically in pancreatic beta cells. These beta-V59M mice developed severe diabetes soon after birth, and by 5 weeks of age, blood glucose levels were markedly increased and insulin was undetectable. Islets isolated from beta-V59M mice secreted substantially less insulin and showed a smaller increase in intracellular calcium in response to glucose. This was due to a reduced sensitivity of KATP channels in pancreatic beta cells to inhibition by ATP or glucose. In contrast, the sulfonylurea tolbutamide, a specific blocker of KATP channels, closed KATP channels, elevated intracellular calcium levels, and stimulated insulin release in beta-V59M beta cells, indicating that events downstream of KATP channel closure remained intact. Expression of the V59M Kir6.2 mutation in pancreatic beta cells alone is thus sufficient to recapitulate the neonatal diabetes observed in humans. beta-V59M islets also displayed a reduced percentage of beta cells, abnormal morphology, lower insulin content, and decreased expression of Kir6.2, SUR1, and insulin mRNA. All these changes are expected to contribute to the diabetes of beta-V59M mice. Their cause requires further investigation.

Lung Ischemia: A Model for Endothelial Mechanotransduction

Endothelial cells in vivo are constantly exposed to shear associated with blood flow and altered shear stress elicits cellular responses (mechanotransduction). This review describes the role of shear sensors and signal transducers in these events. The major focus is the response to removal of shear as occurs when blood flow is compromised (i.e., ischemia). Pulmonary ischemia studied with the isolated murine lung or flow adapted pulmonary microvascular endothelial cells in vitro results in endothelial generation of reactive oxygen species (ROS) and NO. The response requires caveolae and is initiated by endothelial cell depolarization via K(ATP) channel closure followed by activation of NADPH oxidase (NOX2) and NO synthase (eNOS), signaling through MAP kinases, and endothelial cell proliferation. These physiological mediators can promote vasodilation and angiogenesis as compensation for decreased tissue perfusion.

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Both Gi and Go Heterotrimeric G Proteins Are Required to Exert the Full Effect of Norepinephrine on the β-Cell KATP Channel

The effects of norepinephrine (NE), an inhibitor of insulin secretion, were examined on membrane potential and the ATP-sensitive K+ channel (K ATP) in INS 832/13 cells. Membrane potential was monitored under the whole cell current clamp mode. NE hyperpolarized the cell membrane, an effect that was abolished by tolbutamide. The effect of NE on K ATP channels was investigated in parallel using outside-out single channel recording. This revealed that NE enhanced the open activities of the K ATP channels approximately 2-fold without changing the single channel conductance, demonstrating that NE-induced hyperpolarization was mediated by activation of the K ATP channels. The NE effect was abolished in cells preincubated with pertussis toxin, indicating coupling to heterotrimeric G i/G o proteins. To identify the G proteins involved, antisera raised against alpha and beta subunits (anti-G alpha common, anti-G beta, anti-G alpha i1/2/3, and anti-G alpha o) were used. Anti-G alpha common totally blocked the effects of NE on membrane potential and K ATP channels. Individually, anti-G alpha i1/2/3 and anti-G alpha o only partially inhibited the action of NE on K ATP channels. However, the combination of both completely eliminated the action. Antibodies against G beta had no effects. To confirm these results and to further identify the G protein subunits involved, the blocking effects of peptides containing the sequence of 11 amino acids at the C termini of the alpha subunits were used. The data obtained were similar to those derived from the antibody work with the additional information that G alpha i3 and G alpha o1 were not involved. In conclusion, both G i and G o proteins are required for the full effect of norepinephrine to activate the K ATP channel.

The Mitochondrial Citrate/Isocitrate Carrier Plays a Regulatory Role in Glucose-stimulated Insulin Secretion

Glucose-stimulated insulin secretion (GSIS) is mediated in part by glucose metabolism-driven increases in ATP/ADP ratio, but by-products of mitochondrial glucose metabolism also play an important role. Here we investigate the role of the mitochondrial citrate/isocitrate carrier (CIC) in regulation of GSIS. Inhibition of CIC activity in INS-1-derived 832/13 cells or primary rat islets by the substrate analogue 1,2,3-benzenetricarboxylate (BTC) resulted in potent inhibition of GSIS, involving both first and second phase secretion. A recombinant adenovirus containing a CIC-specific siRNA (Ad-siCIC) dose-dependently reduced CIC expression in 832/13 cells and caused parallel inhibitory effects on citrate accumulation in the cytosol. Ad-siCIC treatment did not affect glucose utilization, glucose oxidation, or ATP/ADP ratio but did inhibit glucose incorporation into fatty acids and glucose-induced increases in NADPH/NADP+ ratio relative to cells treated with a control siRNA virus (Ad-siControl). Ad-siCIC also inhibited GSIS in 832/13 cells, whereas overexpression of CIC enhanced GSIS and raised cytosolic citrate levels. In normal rat islets, Ad-siCIC treatment also suppressed CIC mRNA levels and inhibited GSIS. We conclude that export of citrate and/or isocitrate from the mitochondria to the cytosol is an important step in control of GSIS.

Increased KATP channel current in pancreatic beta cells over expressing fatty acyl CoA synthetase I

Closure of KATP channels links glucose metabolism to enhanced electrical activity and insulin secretion in the pancreatic beta cell. Long chain acyl CoAs can potently activate KATP when perfused directly across excised membrane patches, which could account for impaired glucose-stimulated insulin secretion in obese and type II diabetic individuals via reduced beta cell excitability. However, the effects of chronic elevation of acyl CoAs within beta cells have not been documented. We have enhanced endogenous acyl CoA levels within intact primary mouse beta cells using virally delivered over expression of fatty acyl CoA synthetase-1 (FACS-1), and assessed the effects of this manipulation on beta cell excitability using the perforated whole-cell patch-clamp technique. Infection with AdFACS-1 produced a large increase in FACS-1 protein expression compared to controls (AdGFP-treated), as confirmed by Western blot. Patch-clamp data indicated a significant enhancement (p<0.05, n=7) of basal KATP current measured at low glucose in AdFACS-1 infected cells compared to controls. Furthermore, membrane potential recordings showed a decreased spiking frequency in response to high glucose in AdFACS-1 infected cells. We provide direct evidence for reduced beta cell excitability by a FACS-1 driven cytosolic accumulation of acyl CoAs. These observations suggest a strong and direct link between intracellular fatty esters and KATP activity and may have major implications for our current understanding of the involvement of diet and obesity in the development of type II diabetes. Work is sponsored by CIHR and AHFMR.

The mitochondria and insulin release: Nnt just a passing relationship

Nicotinamide nucleotide transhydrogenase (Nnt) detoxifies reactive oxygen species (ROS), byproducts of mitochondrial metabolism that, when accumulated, can decrease mitochondrial ATP production. In this issue of Cell Metabolism, demonstrate that Nnt in pancreatic beta cells is important for insulin release. Their compelling data highlight the critical roles for ATP generation and subsequent closure of KATP channels for insulin secretion.

Stronger control of ATP/ADP by proton leak in pancreatic β-cells than skeletal muscle mitochondria

Pancreatic beta cells respond to rising blood glucose concentrations by increasing their oxidative metabolism, which leads to an increased ATP/ADP ratio, closure of K(ATP) channels, depolarization of the plasma membrane potential, influx of calcium and the eventual secretion of insulin. Such a signalling mechanism implies that the ATP/ADP ratio is flexible in beta cells (beta-cells), which is in contrast with other cell types (e.g. muscle and liver) that maintain a stable ATP/ADP poise while respiring at widely varying rates. To determine whether this difference in flexibility is accounted for by mitochondrial peculiarities, we performed a top-down metabolic control analysis to quantitatively assess how ATP/ADP is controlled in mitochondria isolated from rat skeletal muscle and cultured beta cells. We show that the ATP/ADP ratio is more strongly controlled (approx. 7.5-fold) by proton leak in beta cells than in muscle. The comparatively high importance of proton leak in beta cell mitochondria (relative to phosphorylation) is evidenced furthermore by its relatively high level of control over membrane potential and overall respiratory activity. Modular-kinetic analysis of oxidative phosphorylation reveals that these control differences can be fully explained by a higher relative leak activity in beta cell mitochondria, which results in a comparatively high contribution of proton leak to the overall respiratory activity in this system.