Defective insulin secretion and enhanced insulin action in KATP channel-deficient mice

ATP-sensitive K⁺ (K_ATP) channels regulate many cellular functions by linking cell metabolism to membrane potential. We have generated K_ATP channel-deficient mice by genetic disruption of Kir6.2, which forms the K⁺ ion-selective pore of the channel. The homozygous mice (Kir6.2^−/−) lack K_ATP channel activity. Although the resting membrane potential and basal intracellular calcium concentrations ([Ca²⁺]_i) of pancreatic beta cells in Kir6.2^−/− are significantly higher than those in control mice (Kir6.2^+/+), neither glucose at high concentrations nor the sulfonylurea tolbutamide elicits a rise in [Ca²⁺]_i, and no significant insulin secretion in response to either glucose or tolbutamide is found in Kir6.2^−/−, as assessed by perifusion and batch incubation of pancreatic islets. Despite the defect in glucose-induced insulin secretion, Kir6.2^−/− show only mild impairment in glucose tolerance. The glucose-lowering effect of insulin, as assessed by an insulin tolerance test, is increased significantly in Kir6.2^−/−, which could protect Kir6.2^−/− from developing hyperglycemia. Our data indicate that the K_ATP channel in pancreatic beta cells is a key regulator of both glucose- and sulfonylurea-induced insulin secretion and suggest also that the K_ATP channel in skeletal muscle might be involved in insulin action.     

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.     

Abnormalities of pancreatic islets by targeted expression of a dominant-negative KATP channel

ATP-sensitive K⁺ (K_ATP) channels are known to play important roles in various cellular functions, but the direct consequences of disruption of K_ATP channel function are largely unknown. We have generated transgenic mice expressing a dominant-negative form of the K_ATP channel subunit Kir6.2 (Kir6.2G132S, substitution of glycine with serine at position 132) in pancreatic beta cells. Kir6.2G132S transgenic mice develop hypoglycemia with hyperinsulinemia in neonates and hyperglycemia with hypoinsulinemia and decreased beta cell population in adults. K_ATP channel function is found to be impaired in the beta cells of transgenic mice with hyperglycemia. In addition, both resting membrane potential and basal calcium concentrations are shown to be significantly elevated in the beta cells of transgenic mice. We also found a high frequency of apoptotic beta cells before the appearance of hyperglycemia in the transgenic mice, suggesting that the K_ATP channel might play a significant role in beta cell survival in addition to its role in the regulation of insulin secretion.     

Potent and selective activation of the pancreatic beta-cell type KATP channel by two novel diazoxide analogues

Aims/hypothesis We investigated the pharmacological properties of two novel ATP sensitive potassium (K_ATP) channel openers, 6-Chloro-3-isopropylamino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide (NNC 55-0118) and 6-chloro-3-(1-methylcyclopropyl)amino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide (NN414), on the cloned cardiac (Kir6.2/SUR2A), smooth muscle (Kir6.2/SUR2B) and pancreatic beta cell (Kir6.2/SUR1) types of K_ATP channel.Methods We studied the effects of these compounds on whole-cell currents through cloned K_ATP channels expressed in Xenopus oocytes or mammalian cells (HEK293). We also used inside-out macropatches excised from Xenopus oocytes.Results In HEK 293 cells, NNC 55-0118 and NN414 activated Kir6.2/SUR1 currents with EC₅₀ values of 0.33 µmol/l and 0.45 µmol/l, respectively, compared with that of 31 µmol/l for diazoxide. Neither compound activated Kir6.2/SUR2A or Kir6.2/SUR2B channels expressed in oocytes, nor did they activate Kir6.2 expressed in the absence of SUR. Current activation was dependent on the presence of intracellular MgATP, but was not supported by MgADP.Conclusion/interpretation Both NNC 55-0118 and NN414 selectively stimulate the pancreatic beta-cell type of K_ATP channel with a higher potency than diazoxide, by interaction with the SUR1 subunit. The high selectivity and efficacy of the compounds could prove useful for treatment of disease states where inhibition of insulin secretion is beneficial.Abbreviations K_ATP channel ATP-sensitive potassium channel - SUR sulphonylurea receptor - KCO K⁺ channel opener - Kir inwardly rectifying K⁺ channel - TEVC two electrode voltage clamp - HEK293 cell Human Embryonic Kidney 293 cell     

Phentolamine block of KATP channels is mediated by Kir6.2

The ATP-sensitive K⁺-channel (K_ATP channel) plays a key role in insulin secretion from pancreatic β cells. It is closed both by glucose metabolism and the sulfonylurea drugs that are used in the treatment of noninsulin-dependent diabetes mellitus, thereby initiating a membrane depolarization that activates voltage-dependent Ca²⁺ entry and insulin release. The β cell K_ATP channel is a complex of two proteins: Kir6.2 and SUR1. The former is an ATP-sensitive K⁺-selective pore, whereas SUR1 is a channel regulator that endows Kir6.2 with sensitivity to sulfonylureas. A number of drugs containing an imidazoline moiety, such as phentolamine, also act as potent stimulators of insulin secretion, but their mechanism of action is unknown. We have used a truncated form of Kir6.2, which expresses independently of SUR1, to show that phentolamine does not inhibit K_ATP channels by interacting with SUR1. Instead, our results argue that phentolamine may interact directly with Kir6.2 to produce a voltage-independent reduction in channel activity. The single-channel conductance is unaffected. Although the ATP molecule also contains an imidazoline group, the site at which phentolamine blocks is not identical to the ATP-inhibitory site, because phentolamine block of an ATP-insensitive mutant (K185Q) is normal. K_ATP channels also are found in the heart where they are involved in the response to cardiac ischemia: they also are blocked by phentolamine. Our results suggest that this may be because Kir6.2, which is expressed in the heart, forms the pore of the cardiac K_ATP channel.     

Hyperinsulinism in mice with heterozygous loss of KATP channels

Aims/hypothesis ATP-sensitive K⁺ (K_ATP) channels couple glucose metabolism to insulin secretion in pancreatic beta cells. In humans, loss-of-function mutations of beta cell K_ATP subunits (SUR1, encoded by the gene ABCC8, or Kir6.2, encoded by the gene KCNJ11) cause congenital hyperinsulinaemia. Mice with dominant-negative reduction of beta cell K_ATP (Kir6.2[AAA]) exhibit hyperinsulinism, whereas mice with zero K_ATP (Kir6.2^−/−) show transient hyperinsulinaemia as neonates, but are glucose-intolerant as adults. Thus, we propose that partial loss of beta cell K_ATP in vivo causes insulin hypersecretion, but complete absence may cause insulin secretory failure. Materials and methods Heterozygous Kir6.2^+/− and SUR1^+/− animals were generated by backcrossing from knockout animals. Glucose tolerance in intact animals was determined following i.p. loading. Glucose-stimulated insulin secretion (GSIS), islet K_ATP conductance and glucose dependence of intracellular Ca²⁺ were assessed in isolated islets. Results In both of the mechanistically distinct models of reduced K_ATP (Kir6.2^+/− and SUR1^+/−), K_ATP density is reduced by ∼60%. While both Kir6.2^−/− and SUR1^−/− mice are glucose-intolerant and have reduced glucose-stimulated insulin secretion, heterozygous Kir6.2^+/− and SUR1^+/− mice show enhanced glucose tolerance and increased GSIS, paralleled by a left-shift in glucose dependence of intracellular Ca²⁺ oscillations. Conclusions/interpretation The results confirm that incomplete loss of beta cell K_ATP in vivo underlies a hyperinsulinaemic phenotype, whereas complete loss of K_ATP underlies eventual secretory failure.     

HMR 1098 is not an SUR isotype specific inhibitor of heterologous or sarcolemmal K ATP channels

Murine ventricular and atrial ATP-sensitive potassium (K_ATP) channels contain different sulfonylurea receptors (ventricular K_ATP channels are Kir6.2/SUR2A complexes, while atrial K_ATP channels are Kir6.2/SUR1 complexes). HMR 1098, the sodium salt of HMR 1883 {1-[[5-[2-(5-chloro-o-anisamido)ethyl]-2-methoxyphenyl]sulfonyl]-3-methylthiourea}, has been considered as a selective sarcolemmal (i.e. SUR2A-dependent) K_ATP channel inhibitor. However, it is not clear whether HMR 1098 would preferentially inhibit ventricular K_ATP channels over atrial K_ATP channels. To test this, we used whole-cell patch clamp techniques on mouse atrial and ventricular myocytes as well as ⁸⁶Rb⁺ efflux assays and excised inside-out patch clamp techniques on Kir6.2/SUR1 and Kir6.2/SUR2A channels heterologously expressed in COSm6 cells. In mouse atrial myocytes, both spontaneously activated and diazoxide-activated K_ATP currents were effectively inhibited by 10 μM HMR 1098. By contrast, in ventricular myocytes, pinacidil-activated K_ATP currents were inhibited by HMR 1098 at a high concentration (100 μM) but not at a low concentration (10 μM). Consistent with this finding, HMR 1098 inhibits ⁸⁶Rb⁺ effluxes through Kir6.2/SUR1 more effectively than Kir6.2/SUR2A channels in COSm6 cells. In excised inside-out patches, HMR 1098 inhibited Kir6.2/SUR1 channels more effectively, particularly in the presence of MgADP and MgATP (mimicking physiological stimulation). Finally, dose-dependent enhancement of insulin secretion from pancreatic islets and decrease of blood glucose level confirm that HMR 1098 is an inhibitor of Kir6.2/SUR1-composed K_ATP channels.     

Characterisation of new K<Subscript>ATP</Subscript>-channel mutations associated with congenital hyperinsulinism in the Finnish population

Aims/hypothesis ATP-sensitive potassium (K_ATP) channels are crucial for the regulation of insulin secretion from pancreatic beta cells and mutations in either the Kir6.2 or SUR1 subunit of this channel can cause congenital hyperinsulinism (CHI). The aim of this study was to analyse the functional consequences of four CHI mutations (A1457T, V1550D and L1551V in SUR1, and K67N in Kir6.2) recently identified in the Finnish population.Methods Wild type or mutant Kir6.2 and SUR1 subunits were coexpressed in Xenopus oocytes. The functional properties of the channels were examined by measuring currents in intact oocytes or giant inside-out membrane patches. Surface expression was measured by enzyme-linked immunosorbance assay, using HA-epitope-tagged subunits.Results Two mutations (A1457T and V1550D) prevented trafficking of the channel to the plasma membrane. The L1551V mutation reduced surface expression 40-fold, and caused loss of MgADP and diazoxide activation. Both these factors will contribute to the lack of K_ATP current activation observed in response to metabolic inhibition in intact oocytes. The L1551V mutation also increased the channel open probability, thereby producing a reduction in ATP-sensitivity (from 10 µmol/l to 120 µmol/l). The fourth mutation (K67N mutation in Kir6.2) did not affect surface expression nor alter the properties of K_ATP channels in excised patches, but resulted in a reduced K_ATP current amplitude in intact cells on metabolic inhibition, through an unidentified mechanism.Conclusion/interpretation The four CHI mutations disrupted K_ATP channel activity by different mechanisms. Our results are discussed in relation to the CHI phenotype observed in patients with these mutations.Abbreviations CHI Congenital hyperinsulinism - HA haemagluttinin - K_ATP channel ATP-sensitive potassium channel - Po open probability - PIP₂ phosphatidyl inositol bis-phosphate - RLU relative light units - SUR sulphonylurea receptor - UTR untranslated region An erratum to this article can be found at     

Pancreatic β-cell K<Subscript>ATP</Subscript> channels: Hypoglycaemia and hyperglycaemia

The pancreatic β-cell ATP-sensitive K⁺ channel (K_ATP channel) plays a critical role in glucose homeostasis by linking glucose metabolism to electrical excitability and insulin secretion. Changes in the intracellular ratio of ATP/ADP mediate the metabolic regulation of channel activity. The β-cell K_ATP channel is a hetero-octameric complex composed of two types of subunits: four inward-rectifying potassium channel pore-forming (Kir6.2) subunits and four high-affinity sulfonylurea receptor 1 (SUR1) subunits. Kir6.2 and SUR1 are encoded by the genes KCNJ11 and ABCC8, respectively. Mutations in these genes can result in congenital hyperinsulinism and permanent neonatal diabetes. This review highlights the important role of the β-cell K_ATP channel in glucose physiology and provides an introduction to some of the other review articles in this special edition of the Reviews in Endocrine and Metabolic Disorders.     

Serine-385 phosphorylation of inwardly rectifying K+ channel subunit (Kir6.2) by AMP-dependent protein kinase plays a key role in rosiglitazone-induced closure of the KATP channel and insulin secretion in rats

Aims/hypothesis  Rosiglitazone, an insulin sensitiser, not only improves insulin sensitivity but also enhances insulin secretory capacity by ameliorating gluco- and lipotoxicity in beta cells. Rosiglitazone can stimulate insulin secretion at basal and high glucose levels via a phosphatidylinositol 3-kinase (PI3K)-dependent pathway. We hypothesised that regulation of phosphorylation of the ATP-sensitive potassium (K_ATP) channel might serve as a key step in the regulation of insulin secretion. Methods  Insulin secretory responses were studied in an isolated pancreas perfusion system, cultured rat islets and MIN6 and RINm5F beta cells. Signal transduction pathways downstream of PI3K were explored to link rosiglitazone to K_ATP channel conductance with patch clamp techniques and insulin secretion measured by ELISA. Results  Rosiglitazone stimulated AMP-activated protein kinase (AMPK) activity and induced inhibition of the K_ATP channel conductance in islet beta cells; both effects were blocked by the PI3K inhibitor LY294002. Following stimulation of AMPK by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a pharmacological activator, both AICAR-stimulated insulin secretion and inhibition of K_ATP channel conductance were unaffected by LY294002, indicating that AMPK activation occurs at a site downstream of PI3K activity. The serine residue at amino acid position 385 of Kir6.2 was found to be the substrate phosphorylation site of AMPK when activated by rosiglitazone or AICAR. Conclusions/interpretation  Our data indicate that PI3K-dependent activation of AMPK is required for rosiglitazone-stimulated insulin secretion in pancreatic beta cells. Phosphorylation of the Ser³⁸⁵ residue of the Kir6.2 subunit of the K_ATP channel by AMPK may play a role in insulin secretion. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorised users. T.-J. Chang and W.-P. Chen contributed equally to this study.     

MgATP activates the β cell KATP channel by interaction with its SUR1 subunit

ATP-sensitive potassium (K_ATP) channels in the pancreatic β cell membrane mediate insulin release in response to elevation of plasma glucose levels. They are open at rest but close in response to glucose metabolism, producing a depolarization that stimulates Ca²⁺ influx and exocytosis. Metabolic regulation of K_ATP channel activity currently is believed to be mediated by changes in the intracellular concentrations of ATP and MgADP, which inhibit and activate the channel, respectively. The β cell K_ATP channel is a complex of four Kir6.2 pore-forming subunits and four SUR1 regulatory subunits: Kir6.2 mediates channel inhibition by ATP, whereas the potentiatory action of MgADP involves the nucleotide-binding domains (NBDs) of SUR1. We show here that MgATP (like MgADP) is able to stimulate K_ATP channel activity, but that this effect normally is masked by the potent inhibitory effect of the nucleotide. Mg²⁺ caused an apparent reduction in the inhibitory action of ATP on wild-type K_ATP channels, and MgATP actually activated K_ATP channels containing a mutation in the Kir6.2 subunit that impairs nucleotide inhibition (R50G). Both of these effects were abolished when mutations were made in the NBDs of SUR1 that are predicted to abolish MgATP binding and/or hydrolysis (D853N, D1505N, K719A, or K1384M). These results suggest that, like MgADP, MgATP stimulates K_ATP channel activity by interaction with the NBDs of SUR1. Further support for this idea is that the ATP sensitivity of a truncated form of Kir6.2, which shows functional expression in the absence of SUR1, is unaffected by Mg²⁺.     

Permanent neonatal diabetes due to activating mutations in <Emphasis Type="Italic">ABCC8</Emphasis> and <Emphasis Type="Italic">KCNJ11</Emphasis>

The ATP-sensitive potassium (K_ATP) channel is composed of two subunits SUR1 and Kir6.2. The channel is key for glucose stimulated insulin release from the pancreatic beta cell. Activating mutations have been identified in the genes encoding these subunits, ABCC8 and KCNJ11, and account for approximately 40% of permanent neonatal diabetes cases. The majority of patients with a K_ATP mutation present with isolated diabetes however some have presented with the Developmental delay, Epilepsy and Neonatal Diabetes syndrome. This review focuses on mutations in the K_ATP channel which result in permanent neonatal diabetes, we review the clinical and functional effects as well as the implications for treatment.     

Intracellular ATP-sensitive K+ channels in mouse pancreatic beta cells: against a role in organelle cation homeostasis

Aims/hypothesis ATP-sensitive K⁺ (K_ATP) channels located on the beta cell plasma membrane play a critical role in regulating insulin secretion and are targets for the sulfonylurea class of antihyperglycaemic drugs. Recent reports suggest that these channels may also reside on insulin-containing dense-core vesicles and mitochondria. The aim of this study was to explore these possibilities and to test the hypothesis that vesicle-resident channels play a role in the control of organellar Ca²⁺ concentration or pH.Methods To quantify the subcellular distribution of the pore-forming subunit Kir6.2 and the sulfonylurea binding subunit SUR1 in isolated mouse islets and clonal pancreatic MIN6 beta cells, we used four complementary techniques: immunoelectron microscopy, density gradient fractionation, vesicle immunopurification and fluorescence-activated vesicle isolation. Intravesicular and mitochondrial concentrations of free Ca²⁺ were measured in intact or digitonin-permeabilised MIN6 cells using recombinant, targeted aequorins, and intravesicular pH was measured with the recombinant fluorescent probe pHluorin.Results SUR1 and Kir6.2 immunoreactivity were concentrated on dense-core vesicles and on vesicles plus the endoplasmic reticulum/Golgi network, respectively, in both islets and MIN6 cells. Reactivity to neither subunit was detected on mitochondria. Glibenclamide, tolbutamide and diazoxide all failed to affect Ca²⁺ uptake into mitochondria, and K_ATP channel regulators had no significant effect on intravesicular free Ca²⁺ concentrations or vesicular pH.Conclusions/Interpretation A significant proportion of Kir6.2 and SUR1 subunits reside on insulin-secretory vesicles and the distal secretory pathway in mouse beta cells but do not influence intravesicular ion homeostasis. We propose that dense-core vesicles may serve instead as sorting stations for the delivery of channels to the plasma membrane.Electronic Supplementary Material Supplementary material is available for this article at     

Leptin promotes KATP channel trafficking by AMPK signaling in pancreatic β-cells

Leptin is a pivotal regulator of energy and glucose homeostasis, and defects in leptin signaling result in obesity and diabetes. The ATP-sensitive potassium (K_ATP) channels couple glucose metabolism to insulin secretion in pancreatic β-cells. In this study, we provide evidence that leptin modulates pancreatic β-cell functions by promoting K_ATP channel translocation to the plasma membrane via AMP-activated protein kinase (AMPK) signaling. K_ATP channels were localized mostly to intracellular compartments of pancreatic β-cells in the fed state and translocated to the plasma membrane in the fasted state. This process was defective in leptin-deficient ob/ob mice, but restored by leptin treatment. We discovered that the molecular mechanism of leptin-induced AMPK activation involves canonical transient receptor potential 4 and calcium/calmodulin-dependent protein kinase kinase β. AMPK activation was dependent on both leptin and glucose concentrations, so at optimal concentrations of leptin, AMPK was activated sufficiently to induce K_ATP channel trafficking and hyperpolarization of pancreatic β-cells in a physiological range of fasting glucose levels. There was a close correlation between phospho-AMPK levels and β-cell membrane potentials, suggesting that AMPK-dependent K_ATP channel trafficking is a key mechanism for regulating β-cell membrane potentials. Our results present a signaling pathway whereby leptin regulates glucose homeostasis by modulating β-cell excitability.The K_ATP channel, an inwardly rectifying K⁺ channel that consists of pore-forming Kir6.2 and regulatory sulfonylurea receptor 1 (SUR1) subunits (1), functions as an energy sensor: its gating is regulated mainly by the intracellular concentrations of ATP and ADP. In pancreatic β-cells, K_ATP channels are inhibited or activated in response to the rise or fall in blood glucose levels, leading to changes in membrane excitability and insulin secretion (2, 3). Thus, K_ATP channel gating has been considered an important mechanism in coupling blood glucose levels to insulin secretion. Recently, trafficking of K_ATP channels to the plasma membrane was highlighted as another important mechanism for regulating K_ATP channel activity (4–6).AMP-activated protein kinase (AMPK) is a key enzyme regulating energy homeostasis (7). We recently demonstrated that K_ATP channels are recruited to the plasma membrane in glucose-deprived conditions via AMPK signaling in pancreatic β-cells (6). Inhibition of AMPK signaling significantly reduces K_ATP currents, even after complete wash-out of intracellular ATP (6). Given these results, we proposed a model that recruitment of K_ATP channels to the plasma membrane via AMPK signaling is crucial for K_ATP channel activation in low-glucose conditions. However, the physiological relevance of this model remains unclear because pancreatic β-cells had to be incubated in media containing less than 3 mM glucose to recruit a sufficient number of K_ATP channels to the plasma membrane (6). We thus hypothesized that there should be an endogenous ligand in vivo that promotes AMPK-dependent K_ATP channel trafficking sufficiently to stabilize pancreatic β-cells at physiological fasting glucose levels.Leptin is an adipocyte-derived hormone that regulates food intake, body weight, and glucose homeostasis (8, 9). In addition to its central action, leptin regulates the release of insulin and glucagon, the key hormones regulating glucose homeostasis, by direct actions on β- and α-cells of pancreatic islets, respectively (10–12). It thus was proposed that the adipoinsular axis is crucial for maintaining nutrient balance and that dysregulation of this axis contributes to obesity and diabetes (12). However, intracellular signaling mechanisms underlying leptin effects are largely unknown. Leptin was shown to increase K_ATP currents in pancreatic β-cells (13, 14), but the possibility that K_ATP channel trafficking mediates leptin-induced K_ATP channel activation has not been explored.In the present study, we demonstrate that the surface levels of K_ATP channels increase in pancreatic β-cells under fasting conditions in vivo. Translocation of K_ATP channels to the plasma membrane in fasting was absent in pancreatic β-cells from ob/ob mice, but restored by treatment with leptin, suggesting a role for leptin in K_ATP channel trafficking in vivo. We further show that leptin-induced AMPK activation, which is essential for K_ATP channel trafficking to the plasma membrane, is mediated by activation of canonical transient receptor potential 4 (TRPC4) and calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ). Our results highlight the importance of trafficking regulation in K_ATP channel activation and provide insights into the action of leptin on glucose homeostasis.     

Suppressed Ca2+/CaM/CaMKII-dependent KATP channel activity in primary afferent neurons mediates hyperalgesia after axotomy

Painful axotomy decreases K_ATP channel current (IK_ATP) in primary afferent neurons. Because cytosolic Ca²⁺ signaling is depressed in injured dorsal root ganglia (DRG) neurons, we investigated whether Ca²⁺–calmodulin (CaM)–Ca²⁺/CaM-dependent kinase II (CaMKII) regulates IK_ATP in large DRG neurons. Immunohistochemistry identified the presence of K_ATP channel subunits SUR1, SUR2, and Kir6.2 but not Kir6.1, and pCaMKII in neurofilament 200–positive DRG somata. Single-channel recordings from cell-attached patches revealed that basal and evoked IK_ATP by ionomycin, a Ca²⁺ ionophore, is activated by CaMKII. In axotomized neurons from rats made hyperalgesic by spinal nerve ligation (SNL), basal K_ATP channel activity was decreased, and sensitivity to ionomycin was abolished. Basal and Ca²⁺-evoked K_ATP channel activity correlated inversely with the degree of hyperalgesia induced by SNL in the rats from which the neurons were isolated. Inhibition of IK_ATP by glybenclamide, a selective K_ATP channel inhibitor, depolarized resting membrane potential (RMP) recorded in perforated whole-cell patches and enhanced neurotransmitter release measured by amperometry. The selective K_ATP channel opener diazoxide hyperpolarized the RMP and attenuated neurotransmitter release. Axotomized neurons from rats made hyperalgesic by SNL lost sensitivity to the myristoylated form of autocamtide-2-related inhibitory peptide (AIPm), a pseudosubstrate blocker of CaMKII, whereas axotomized neurons from SNL animals that failed to develop hyperalgesia showed normal IK_ATP inhibition by AIPm. AIPm also depolarized RMP in control neurons via K_ATP channel inhibition. Unitary current conductance and sensitivity of K_ATP channels to cytosolic ATP and ligands were preserved even after painful nerve injury, thus providing opportunities for selective therapeutic targeting against neuropathic pain.     

Leptin-stimulated KATP channel trafficking

Insulin secretion from pancreatic β-cells is initiated by the closure of ATP-sensitive K⁺ channels (K_ATP) in response to high concentrations of glucose, and this action of glucose is counteracted by the hormone leptin, an adipokine that signals through the Ob-R_b receptor to increase K_ATP channel activity. Despite intensive investigations, the molecular basis for K_ATP channel regulation remains uncertain, particularly from the standpoint of whether fluctuations in plasma membrane K_ATP channel content underlie alterations of K_ATP channel activity in response to glucose or leptin. Surprisingly, newly published findings reveal that leptin stimulates AMP-activated protein kinase (AMPK) in order to promote trafficking of K_ATP channels from cytosolic vesicles to the plasma membrane of β-cells. This action of leptin is mimicked by low concentrations of glucose that also activate AMPK and that inhibit insulin secretion. Thus, a new paradigm for β-cell stimulus-secretion coupling is suggested in which leptin exerts a tonic inhibitory effect on β-cell excitability by virtue of its ability to increase plasma membrane K_ATP channel density and whole-cell K_ATP channel current. One important issue that remains unresolved is whether high concentrations of glucose suppress AMPK activity in order to shift the balance of membrane cycling so that K_ATP channel endocytosis predominates over vesicular K_ATP channel insertion into the plasma membrane. If so, high concentrations of glucose might transiently reduce K_ATP channel density/current, thereby favoring β-cell depolarization and insulin secretion. Such an AMPK-dependent action of glucose would complement its established ability to generate an increase of ATP/ADP concentration ratio that directly closes K_ATP channels in the plasma membrane.     

Cardiac sarcolemmal KATP channels: Latest twists in a questing tale!

Reconstitution of K_ATP channel activity from coexpression of members of the pore-forming inward rectifier gene family (Kir6.1, KCNJ8, and Kir6.2 KCNJ11) with sulfonylurea receptors (SUR1, ABCC8, and SUR2, ABCC9) of the ABCC protein sub-family, has led to the elucidation of many details of channel gating and pore properties, as well as the essential roles of Kir6.2 and SUR2 subunits in generating cardiac ventricular K_ATP. However, despite this extensive body of knowledge, there remain significant holes in our understanding of the physiological role of the cardiac K_ATP channel, and surprising new findings keep emerging. Recent findings from genetically modified animals include the apparent insensitivity of cardiac sarcolemmal channels to nucleotide levels, and unenvisioned complexities of the subunit make-up of the cardiac channels. This topical review focuses on these new findings and considers their implications.     

Gastric inhibitory polypeptide is the major insulinotropic factor in K(ATP) null mice

OBJECTIVE: ATP-sensitive K(+) (K(ATP)) channels in pancreatic beta-cells are crucial in the regulation of glucose-induced insulin secretion. Recently, K(ATP) channel-deficient mice were generated by genetic disruption of Kir6.2, the pore-forming component of K(ATP) channels, but the mice still showed a significant insulin response after oral glucose loading in vivo. Gastric inhibitory polypeptide (GIP) is a physiological incretin that stimulates insulin release upon ingestion of nutrients. To determine if GIP is the insulinotropic factor in insulin secretion in K(ATP) channel-deficient mice, we generated double-knockout Kir6.2 and GIP receptor null mice and compared them with Kir6.2 knockout mice. METHODS: Double-knockout mice were generated by intercrossing Kir6.2-knockout mice with GIP receptor-knockout mice. An oral glucose tolerance test, insulin tolerance test and batch incubation study of pancreatic islets were performed on double-knockout mice and Kir6.2-knockout mice. RESULTS: Fasting glucose and insulin levels were similar in both groups. After oral glucose loading, blood glucose levels of double-knockout mice became elevated compared with Kir6.2-knockout mice, especially at 15 min (345+/-10 mg/dl vs 294+/-20 mg/dl, P<0.05) and 30 min (453+/-20 mg/dl vs 381+/-26 mg/dl, P<0.05). The insulin response was almost completely lost in double-knockout mice, although insulin secretion from isolated islets was stimulated by another incretin, glucagon-like peptide-1 in the double-knockout mice. Double-knockout mice and Kir6.2-knockout mice were similarly insulin sensitive as assessed by the insulin tolerance test. CONCLUSION: GIP is the major insulinotropic factor in the secretion of insulin in response to glucose load in K(ATP) channel-deficient mice.     

Effect of repaglinide on cloned beta cell, cardiac and smooth muscle types of ATP-sensitive potassium channels

Aims/hypothesis. The carbamoylbenzoic acid derivative repaglinide is a potent short-acting insulin secretagogue that acts by closing ATP-sensitive potassium (K_ATP) channels in the plasma membrane of the pancreatic beta cell. In this paper we investigated the specificity of repaglinide for three types of cloned (K_ATP) channel composed of the inwardly rectifying potassium channel Kir6.2 and either the sulphonylurea receptor SUR1, SUR2A or SUR2B, corresponding to the beta cell, cardiac and either smooth muscle types of K_ATP channel, respectively. Methods. The action of the drug was studied by whole-cell current recordings of K_ATP channels expressed either in Xenopus oocytes or mammalian cells (HEK293). We also used inside-out macropatches excised from Xenopus oocytes for detailed analysis of repaglinide action. Results. The drug blocked all three types of K_ATP channel with similar potency, by interacting with a low-affinity site on the pore-forming subunit of the channel (Kir6.2: half-maximal inhibition 230 μmol/l) and with a high-affinity site on the regulatory subunit, the sulphonylurea receptor (SUR: half-maximal inhibition 2–8 nmol/l). There was no difference in potency between channels containing SUR1, SUR2A or SUR2B. MgADP potentiated the inhibitory effect of repaglinide on Kir6.2/SUR1 and (to a lesser extent) Kir6.2/SUR2B, but not on Kir6.2/SUR2A. Conclusion/interpretation. Repaglinide interacts with a site common to all three types of sulphonylurea receptor leading to inhibition of the K_ATP channel. The fact that MgADP potentiated this effect in the case of the beta cell, but not cardiac, type of channel could help explain why the drug shows no adverse cardiovascular side-effects in vivo. [Diabetologia (2001) 44: 747–756] Received: 13 December 2000 and in revised form: 14 February 2001     

Nutrient-dependent secretion of glucose-dependent insulinotropic polypeptide from primary murine K cells

Aims/hypothesis  Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone with anti-apoptotic effects on the pancreatic beta cell. The aim of this study was to generate transgenic mice with fluorescently labelled GIP-secreting K cells and to use these to investigate pathways by which K cells detect nutrients. Methods  Transgenic mice were generated in which the GIP promoter drives the expression of the yellow fluorescent protein Venus. Fluorescent cells were purified by flow cytometry and analysed by quantitative RT-PCR. GIP secretion was assayed in primary cultures of small intestine. Results  Expression of Venus in transgenic mice was restricted to K cells, as assessed by immunofluorescence and measurements of the Gip mRNA and GIP protein contents of purified cells. K cells expressed high levels of mRNA for Kir6.2 (also known as Kcnj11), Sur1 (also known as Abcc8), Sglt1 (also known as Slc5a1), and of the G-protein-coupled lipid receptors Gpr40 (also known as Ffar1), Gpr119 and Gpr120. In primary cultures, GIP release was stimulated by glucose, glutamine and linoleic acid, and potentiated by forskolin plus 3-isobutyl-1-methylxanthine (IBMX), but was unaffected by the artificial sweetener sucralose. Secretion was half-maximal at 0.6 mmol/l glucose and partially mimicked by α-methylglucopyranoside, suggesting the involvement of SGLT1. Tolbutamide triggered secretion under basal conditions, whereas diazoxide suppressed responses in forskolin/IBMX. Conclusions/interpretation  These transgenic mice and primary culture techniques provide novel opportunities to interrogate the mechanisms of GIP secretion. Glucose-triggered GIP secretion was SGLT1-dependent and modulated by K_ATP channel activity but not determined by sweet taste receptors. Synergistic stimulation by elevated cAMP and glucose suggests that targeting appropriate G-protein-coupled receptors may provide opportunities to modulate GIP release in vivo. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorised users. H. E. Parker and A. M. Habib contributed equally to this study.