Β-cell KATP Channel Research Articles

Glucose Regulation of β-Cell KATP Channels: Is a New Model Needed?

The canonical model of glucose-induced increase in insulin secretion involves the metabolism of glucose via glycolysis and the citrate cycle, resulting in increased ATP synthesis by the respiratory chain and the closure of ATP-sensitive K+ (KATP) channels. The resulting plasma membrane depolarization, followed by Ca2+ influx through L-type Ca2+ channels, then induces insulin granule fusion. Merrins and colleagues have recently proposed an alternative model whereby KATP channels are controlled by pyruvate kinase, using glycolytic and mitochondrial phosphoenolpyruvate (PEP) to generate microdomains of high ATP/ADP immediately adjacent to KATP channels. This model presents several challenges. First, how mitochondrially generated PEP, but not ATP produced abundantly by the mitochondrial F1F0-ATP synthase, can gain access to the proposed microdomains is unclear. Second, ATP/ADP fluctuations imaged immediately beneath the plasma membrane closely resemble those in the bulk cytosol. Third, ADP privation of the respiratory chain at high glucose, suggested to drive alternating, phased-locked generation by mitochondria of ATP or PEP, has yet to be directly demonstrated. Finally, the approaches used to explore these questions may be complicated by off-target effects. We suggest instead that Ca2+ changes, well known to affect both ATP generation and consumption, likely drive cytosolic ATP/ADP oscillations that in turn regulate KATP channels and membrane potential. Thus, it remains to be demonstrated that a new model is required to replace the existing, mitochondrial bioenergetics-based model.

Eugenosedin-A improves obesity-related hyperglycemia by regulating ATP-sensitive K+ channels and insulin secretion in pancreatic β cells

Eugenosedin-A (Eu-A) has been shown to protect against hyperglycemia- and hyperlipidemia-induced metabolic syndrome. We investigated the relationship of KATP channel activities and insulin secretion by Eu-A in vitro in pancreatic β-cells, and examined the effect of Eu-A on streptozotocin (STZ)/nicotinamide (NA)-induced type 2 diabetes mellitus (T2DM) in vivo. We isolated pancreatic islets from adult male Wistar rats (250-350g) and identified pancreatic β-cells by the cell size, capacitance and membrane potential. Perforated patch-clamp and inside-out recordings were used to monitor the membrane potential (current-clamp mode) and channel activity (voltage-clamp mode) of β-cells. The membrane potential of β-cells was raised by Eu-A and reversed by the KATP channel activator diazoxide. Eu-A inhibited the KATP channel activity measured at -60mV and increased the intracellular calcium concentration ([Ca2+]i), resulting in enhanced insulin secretion. Eu-A also reduced Kir6.2 protein on the cell membrane and scattered in the cytosol under normal glucose conditions (5.6mM). In our animal study, rats were divided into normal and STZ/NA-induced T2DM groups. Normal rats fed with regular chow were divided into control and control+Eu-A (5mg/kg/day, i.p.) groups. The STZ/NA-induced diabetic rats fed with a high-fat diet (HFD) were divided into three groups: T2DM, T2DM+Eu-A (5mg/kg/day, i.p.), and T2DM+glibenclamide (0.5mg/kg/day, i.p.; a KATP channel inhibitor). Both Eu-A and glibenclamide decreased the rats' blood glucose, prevented weight gain, and enhanced insulin secretion. We found that Eu-A blocked pancreatic β-cell KATP channels, caused membrane potential depolarization, and stimulated Ca2+ influx, thus increasing insulin secretion. Furthermore, Eu-A decreased blood glucose and increased insulin levels in T2DM rats. These results suggested that Eu-A might have clinical benefits for the control of T2DM and its complications.

Decreased KATP Channel Activity Contributes to the Low Glucose Threshold for Insulin Secretion of Rat Neonatal Islets.

Transitional hypoglycemia in normal newborns occurs in the first 3 days of life and has clinical features consistent with hyperinsulinism. We found a lower threshold for glucose-stimulated insulin secretion from freshly isolated embryonic day (E) 22 rat islets, which persisted into the first postnatal days. The threshold reached the adult level by postnatal day (P) 14. Culturing P14 islets also decreased the glucose threshold. Freshly isolated P1 rat islets had a lower threshold for insulin secretion in response to 2-aminobicyclo-(2, 2, 1)-heptane-2-carboxylic acid, a nonmetabolizable leucine analog, and diminished insulin release in response to tolbutamide, an inhibitor of β-cell KATP channels. These findings suggested that decreased KATP channel function could be responsible for the lower glucose threshold for insulin secretion. Single-cell transcriptomic analysis did not reveal a lower expression of KATP subunit genes in E22 compared with P14 β cells. The investigation of electrophysiological characteristics of dispersed β cells showed that early neonatal and cultured cells had fewer functional KATP channels per unit membrane area. Our findings suggest that decreased surface density of KATP channels may contribute to the observed differences in glucose threshold for insulin release.

1182-P: A Selective Nonpeptide Somatostatin Receptor 5 (sst5) Agonist Effectively Decreases Insulin Secretion in the KATPHI Mouse Model

Inactivating mutations of β-cell KATP channels cause the most common and severe form of congenital hyperinsulinism (HI), a β-cell disorder that causes dysregulated insulin secretion and persistent hypoglycemia. Children with KATPHI are typically unresponsive to diazoxide, the only FDA-approved drug for HI. Octreotide, a sst2 peptide agonist that inhibits insulin secretion, is used as second line therapy, but poor efficacy and sst2-mediated side effects, such as glucagon suppression and necrotizing enterocolitis, limit its use in infants. Thus, most infants with KATPHI require pancreatectomy for intractable hypoglycemia. To address the unmet need for effective and safe therapies, we examined the ability of a selective sst5 nonpeptide agonist CRN02481 (Crinetics Pharmaceuticals) to suppress insulin secretion and prevent fasting hypoglycemia in the SUR1-/- mouse model of KATPHI. SUR1-/- mouse islets were treated with CRN02481 (100 nM) or vehicle and then stimulated with a physiologic amino acid mixture (4 mM). CRN02481 significantly decreased baseline (1.5 ± 0.2 vs. 2.4 ± 0.5 ng/uL, p≤0.01) and amino acid-stimulated (2.8 ± 1.5 vs. 4.4 ± 1.1 ng/uL, p≤0.05) insulin secretion. SUR1-/- mice were gavaged with CRN02481 (10 mg/kg/day) or PBS for 7 days (n=7/group) and fasting glucose, glucose tolerance and insulin tolerance tests were assessed. CRN02481 significantly increased fasting glucose and prevented fasting hypoglycemia (98 ± 18 vs. 44 ± 6 mg/dL, p<0.00001). During a glucose tolerance test CRN02481 increased post-glucose load glucose concentrations significantly by 60 and 90 minutes. CRN02481 significantly decreased the slope of decline of glucose concentrations after insulin administration (1 u/kg): -2.8 ± 2.3 vs. -5.7 ± 2.1, p=0.03). Collectively these data show that the sst5 agonist CRN02481 effectively suppresses insulin secretion and prevents fasting hypoglycemia in the KATPHI mouse model and is a potentially novel therapy for children with HI. Disclosure C. Juliana: Research Support; Self; Crinetics. J. Chai: None. E.F. Rico-Bautista: Employee; Self; Crinetics Pharmaceuticals. Stock/Shareholder; Self; Crinetics Pharmaceuticals. S.F. Betz: Employee; Self; Crinetics Pharmaceuticals. Stock/Shareholder; Self; Crinetics Pharmaceuticals. D.D. De Leon: Consultant; Self; Crinetics, Novartis Pharmaceuticals Corporation, ProSciento, Soleno Therapeutics, Zealand Pharma A/S. Employee; Spouse/Partner; Merck & Co., Inc. Research Support; Self; Crinetics, Dexcom, Inc., Zealand Pharma A/S. Stock/Shareholder; Self; Merck & Co., Inc. Funding Crinetics Pharmaceuticals (to D.D.D.)

Human Genetics of Obesity and Type 2 Diabetes Mellitus: Past, Present, and Future.

Type 2 diabetes mellitus (T2D) and obesity already represent 2 of the most prominent risk factors for cardiovascular disease, and are destined to increase in importance given the global changes in lifestyle. Ten years have passed since the first round of genome-wide association studies for T2D and obesity. During this decade, we have witnessed remarkable developments in human genetics. We have graduated from the despair of candidate gene-based studies that generated few consistently replicated genotype-phenotype associations, to the excitement of an exponential harvest of loci robustly associated with medical outcomes through ever larger genome-wide association study meta-analyses. As well as discovering hundreds of loci, genome-wide association studies have provided transformative insights into the genetic architecture of T2D and other complex traits, highlighting the extent of polygenicity and the tiny effect sizes of many common risk alleles. Genome-wide association studies have also provided a critical starting point for discovering new biology relevant to these traits. Expectations are high that these discoveries will foster development of more effective strategies for intervention, through optimization of precision medicine approaches. In this article, we review current knowledge and provide suggestions for the next steps in genetic research for T2D and obesity. We focus on four areas relevant to precision medicine: genetic architecture, pharmacogenetics and other gene-environment interactions, mechanistic inference, and drug development. As we describe, the genetic architecture of complex traits has major implications for the prospects of precision medicine, rendering some anticipated approaches decidedly unrealistic. We highlight obstacles to the translation of human genetic findings into mechanism inference but are optimistic that, as these are overcome, there is untapped potential for novel drugs and more effective strategies for treating and preventing T2D and obesity.

Chronic β-Cell Depolarization Impairs β-Cell Identity by Disrupting a Network of Ca2+-Regulated Genes.

We used mice lacking Abcc8, a key component of the β-cell KATP-channel, to analyze the effects of a sustained elevation in the intracellular Ca2+ concentration ([Ca2+]i) on β-cell identity and gene expression. Lineage tracing analysis revealed the conversion of β-cells lacking Abcc8 into pancreatic polypeptide cells but not to α- or δ-cells. RNA-sequencing analysis of FACS-purified Abcc8−/− β-cells confirmed an increase in Ppy gene expression and revealed altered expression of more than 4,200 genes, many of which are involved in Ca2+ signaling, the maintenance of β-cell identity, and cell adhesion. The expression of S100a6 and S100a4, two highly upregulated genes, is closely correlated with membrane depolarization, suggesting their use as markers for an increase in [Ca2+]i. Moreover, a bioinformatics analysis predicts that many of the dysregulated genes are regulated by common transcription factors, one of which, Ascl1, was confirmed to be directly controlled by Ca2+ influx in β-cells. Interestingly, among the upregulated genes is Aldh1a3, a putative marker of β-cell dedifferentiation, and other genes associated with β-cell failure. Taken together, our results suggest that chronically elevated β-cell [Ca2+]i in Abcc8−/− islets contributes to the alteration of β-cell identity, islet cell numbers and morphology, and gene expression by disrupting a network of Ca2+-regulated genes.

Functional and Metabolomic Consequences of KATP Channel Inactivation in Human Islets.

Loss-of-function mutations of β-cell KATP channels cause the most severe form of congenital hyperinsulinism (KATPHI). KATPHI is characterized by fasting and protein-induced hypoglycemia that is unresponsive to medical therapy. For a better understanding of the pathophysiology of KATPHI, we examined cytosolic calcium ([Ca2+] i ), insulin secretion, oxygen consumption, and [U-13C]glucose metabolism in islets isolated from the pancreases of children with KATPHI who required pancreatectomy. Basal [Ca2+] i and insulin secretion were higher in KATPHI islets compared with controls. Unlike controls, insulin secretion in KATPHI islets increased in response to amino acids but not to glucose. KATPHI islets have an increased basal rate of oxygen consumption and mitochondrial mass. [U-13C]glucose metabolism showed a twofold increase in alanine levels and sixfold increase in 13C enrichment of alanine in KATPHI islets, suggesting increased rates of glycolysis. KATPHI islets also exhibited increased serine/glycine and glutamine biosynthesis. In contrast, KATPHI islets had low γ-aminobutyric acid (GABA) levels and lacked 13C incorporation into GABA in response to glucose stimulation. The expression of key genes involved in these metabolic pathways was significantly different in KATPHI β-cells compared with control, providing a mechanism for the observed changes. These findings demonstrate that the pathophysiology of KATPHI is complex, and they provide a framework for the identification of new potential therapeutic targets for this devastating condition.

Expression and function of ATP-dependent potassium channels in zebrafish islet β-cells.

ATP-sensitive potassium channels (KATP channels) are critical nutrient sensors in many mammalian tissues. In the pancreas, KATP channels are essential for coupling glucose metabolism to insulin secretion. While orthologous genes for many components of metabolism–secretion coupling in mammals are present in lower vertebrates, their expression, functionality and ultimate impact on body glucose homeostasis are unclear. In this paper, we demonstrate that zebrafish islet β-cells express functional KATP channels of similar subunit composition, structure and metabolic sensitivity to their mammalian counterparts. We further show that pharmacological activation of native zebrafish KATP using diazoxide, a specific KATP channel opener, is sufficient to disturb glucose tolerance in adult zebrafish. That β-cell KATP channel expression and function are conserved between zebrafish and mammals illustrates the evolutionary conservation of islet metabolic sensing from fish to humans, and lends relevance to the use of zebrafish to model islet glucose sensing and diseases of membrane excitability such as neonatal diabetes.

Silymarin Activates c-AMP Phosphodiesterase and Stimulates Insulin Secretion in a Glucose-Dependent Manner in HIT-T15 Cells.

Silymarin (SIL) is a flavonoid extracted from milk thistle seed that has been reported to decrease hyperglycemia in people with type 2 diabetes (T2D). However, it is not known whether SIL has direct secretory effects on β-cells. Using the β-cell line HIT-T15, SIL was shown to decrease intracellular peroxide levels and to augment glucose-stimulated insulin secretion (GSIS). However, the latter was observed using a concentration range of 25–100 µM, which was too low to affect endogenous peroxide levels. The stimulatory effect of SIL dissipated at higher concentrations (100–200 µM), and mild apoptosis was observed. The smaller concentrations of SIL also decreased cAMP phosphodiesterase activity in a Ca2+/calmodulin-dependent manner. The stimulatory effects of SIL on GSIS were inhibited by three different inhibitors of exocytosis, indicating that SIL’s mechanism of stimulating GSIS operated via closing β-cell K-ATP channels, and perhaps more distal sites of action involving calcium influx and G-proteins. We concluded that augmentation of GSIS by SIL can be observed at concentrations that also inhibit cAMP phosphodiesterase without concomitant lowering of intracellular peroxides.

Dissecting the Etiology of Type 2 Diabetes in the Pima Indian Population.

The Pima Indian population of Arizona has one of the highest prevalence of diabetes of any population in the world, and the Pima Indians of the Gila River Indian Community have probably been the most studied group for the causes and consequences of diabetes. They develop what would be considered to be classic type 2 diabetes, characterized by obesity and insulin resistance with progression to diabetes characterized by progressive β-cell dysfunction (1). Given the low degree of admixture and high heritability of type 2 diabetes in the Pima Indians (2), the population should also represent a honeypot for genetic discovery. While in recent years there has been progress in identifying type 2 diabetes risk variants in this population (3,4), the understanding of the genetic contribution to diabetes risk remains far from complete. Like many initiatives in genetics, whole-genome/exome sequencing is now being applied in the hope that rare/low-frequency variants with large effects on diabetes risk will be found. In this issue of Diabetes , Baier et al. (5) report on the results of a targeted sequencing project focusing on the genes encoding the two subunits of the β-cell KATP channel. Variants identified by whole-genome sequencing in ABCC8 and KCNJ11 in 335 Pima Indians are genotyped in 7,710 individuals of full Pima Indian heritage or mixed American Indian heritage (∼4/8th Pima Indian). They identify an R1420H variant present in 3.3% of the population that is associated with …

ABCC8 R1420H Loss-of-Function Variant in a Southwest American Indian Community: Association With Increased Birth Weight and Doubled Risk of Type 2 Diabetes.

Missense variants in KCNJ11 and ABCC8, which encode the KIR6.2 and SUR1 subunits of the β-cell KATP channel, have previously been implicated in type 2 diabetes, neonatal diabetes, and hyperinsulinemic hypoglycemia of infancy (HHI). To determine whether variation in these genes affects risk for type 2 diabetes or increased birth weight as a consequence of fetal hyperinsulinemia in Pima Indians, missense and common noncoding variants were analyzed in individuals living in the Gila River Indian Community. A R1420H variant in SUR1 (ABCC8) was identified in 3.3% of the population (N = 7,710). R1420H carriers had higher mean birth weights and a twofold increased risk for type 2 diabetes with a 7-year earlier onset age despite being leaner than noncarriers. One individual homozygous for R1420H was identified; retrospective review of his medical records was consistent with HHI and a diagnosis of diabetes at age 3.5 years. In vitro studies showed that the R1420H substitution decreases KATP channel activity. Identification of this loss-of-function variant in ABCC8 with a carrier frequency of 3.3% affects clinical care as homozygous inheritance and potential HHI will occur in 1/3,600 births in this American Indian population.

Sulfonylureas suppress the stimulatory action of Mg-nucleotides on Kir6.2/SUR1 but not Kir6.2/SUR2A KATP channels: a mechanistic study.

Sulfonylureas, which stimulate insulin secretion from pancreatic β-cells, are widely used to treat both type 2 diabetes and neonatal diabetes. These drugs mediate their effects by binding to the sulfonylurea receptor subunit (SUR) of the ATP-sensitive K(+) (KATP) channel and inducing channel closure. The mechanism of channel inhibition is unusually complex. First, sulfonylureas act as partial antagonists of channel activity, and second, their effect is modulated by MgADP. We analyzed the molecular basis of the interactions between the sulfonylurea gliclazide and Mg-nucleotides on β-cell and cardiac types of KATP channel (Kir6.2/SUR1 and Kir6.2/SUR2A, respectively) heterologously expressed in Xenopus laevis oocytes. The SUR2A-Y1206S mutation was used to confer gliclazide sensitivity on SUR2A. We found that both MgATP and MgADP increased gliclazide inhibition of Kir6.2/SUR1 channels and reduced inhibition of Kir6.2/SUR2A-Y1206S. The latter effect can be attributed to stabilization of the cardiac channel open state by Mg-nucleotides. Using a Kir6.2 mutation that renders the KATP channel insensitive to nucleotide inhibition (Kir6.2-G334D), we showed that gliclazide abolishes the stimulatory effects of MgADP and MgATP on β-cell KATP channels. Detailed analysis suggests that the drug both reduces nucleotide binding to SUR1 and impairs the efficacy with which nucleotide binding is translated into pore opening. Mutation of one (or both) of the Walker A lysines in the catalytic site of the nucleotide-binding domains of SUR1 may have a similar effect to gliclazide on MgADP binding and transduction, but it does not appear to impair MgATP binding. Our results have implications for the therapeutic use of sulfonylureas.

Phosphatidylinositol 4,5-Biphosphate (PIP2) Modulates Interaction of Syntaxin-1A with Sulfonylurea Receptor 1 to Regulate Pancreatic β-Cell ATP-sensitive Potassium Channels

In β-cells, syntaxin (Syn)-1A interacts with SUR1 to inhibit ATP-sensitive potassium channels (KATP channels). PIP2 binds the Kir6.2 subunit to open KATP channels. PIP2 also modifies Syn-1A clustering in plasma membrane (PM) that may alter Syn-1A actions on PM proteins like SUR1. Here, we assessed whether the actions of PIP2 on activating KATP channels is contributed by sequestering Syn-1A from binding SUR1. In vitro binding showed that PIP2 dose-dependently disrupted Syn-1A·SUR1 complexes, corroborated by an in vivo Forster resonance energy transfer assay showing disruption of SUR1(-EGFP)/Syn-1A(-mCherry) interaction along with increased Syn-1A cluster formation. Electrophysiological studies of rat β-cells, INS-1, and SUR1/Kir6.2-expressing HEK293 cells showed that PIP2 dose-dependent activation of KATP currents was uniformly reduced by Syn-1A. To unequivocally distinguish between PIP2 actions on Syn-1A and Kir6.2, we employed several strategies. First, we showed that PIP2-insensitive Syn-1A-5RK/A mutant complex with SUR1 could not be disrupted by PIP2, consequently reducing PIP2 activation of KATP channels. Next, Syn-1A·SUR1 complex modulation of KATP channels could be observed at a physiologically low PIP2 concentration that did not disrupt the Syn-1A·SUR1 complex, compared with higher PIP2 concentrations acting directly on Kir6.2. These effects were specific to PIP2 and not observed with physiologic concentrations of other phospholipids. Finally, depleting endogenous PIP2 with polyphosphoinositide phosphatase synaptojanin-1, known to disperse Syn-1A clusters, freed Syn-1A from Syn-1A clusters to bind SUR1, causing inhibition of KATP channels that could no longer be further inhibited by exogenous Syn-1A. These results taken together indicate that PIP2 affects islet β-cell KATP channels not only by its actions on Kir6.2 but also by sequestering Syn-1A to modulate Syn-1A availability and its interactions with SUR1 on PM.

Paternally inherited ABCC8 mutation causing diffuse congenital hyperinsulinism

SummaryBackground: Congenital hyperinsulinism (CHI) is a rare genetic disorder characterised by inappropriate insulin secretion in the face of severe hypoglycaemia. There are two histological subtypes of CHI namely diffuse and focal. Diffuse CHI is most common due to recessive mutations in ABCC8/KCNJ11 (which encode the SUR/KIR6.2 components of the pancreatic β-cell KATP channel) whereas focal CHI is due to a paternally inherited ABCC8/KCNJ11 mutation and somatic loss of heterozygosity for the 11p allele inside the focal lesion. Fluorine-18-l-dihydroxyphenylalanine positron emission tomography/computed tomography (18F-DOPA-PET/CT) is used in the pre-operative localisation of focal lesions prior to surgery. Diffuse CHI if medically unresponsive will require a near total pancreatectomy whereas focal CHI will only require a limited lesionectomy, thus curing the patient from the hypoglycaemia.Aims: To report the first case of genetically confirmed CHI in Singapore from a heterozygous paternally inherited ABCC8 mutation.Methods/Results: A term male infant presented with severe hyperinsulinaemic hypoglycaemia (HH) after birth and failed medical treatment with diazoxide and octreotide. Genetic testing (paternally inherited mutation in ABCC8/p.D1472N) suggested focal disease, but due to the unavailability of 18F-DOPA-PET/CT to confirm focal disease, a partial pancreatectomy was performed. Interestingly, histology of the resected pancreatic tissue showed changes typical of diffuse disease.Conclusion: Heterozygous paternally inherited ABCC8/KCNJ11 mutations can lead to diffuse or focal CHI.Learning points HH is a cause of severe hypoglycaemia in the newborn period.Paternal mutations in ABCC8/KCNJ11 can lead to diffuse or focal disease. 18F-DOPA-PET/CT scan is the current imaging of choice for localising focal lesions.Gallium-68 tetra-aza-cyclododecane-N N′N″N-‴-tetra-acetate octreotate PET scan is not a useful imaging tool for localising focal lesions.The molecular mechanism by which a heterozygous ABCC8 mutation leads to diffuse disease is currently unclear.Focal lesions are curable by lesionectomy and so genetic studies in patients with HH must be followed by imaging using 18F-DOPA-PET/CT scan.

ATP and Sulfonylurea Linkage in the KATP Channel Solves a Diabetes Puzzler

At 30,000 feet, ATP-sensitive potassium (KATP) channels are seen coupling the metabolic status of the cell to its electrical excitability. But down at the molecular level of the β-cell, there is a lot more coupling going on in exactly how the KATP channel does this. Coupling is developed in the conceptual framework of linkage (1), which thermodynamically quantifies how conformational states of high and low affinity in regulatory ligand binding sites reciprocally link with additional sites, and active and inactive conformations of proteins. It is also at the heart of a molecular medicine puzzler with a satisfying solution originating from a study by Proks et al. (2) reported in this issue. The main gate of the KATP channel opens and shuts to regulate β-cell electrical excitability and insulin secretion. Only a small number of the channels need be open for outflow of potassium ions to keep the β-cell electrically at rest, thus preventing insulin secretion. In a high glucose setting, however, β-cell metabolism generates ATP at the expense of ADP, which effectively shuts all KATP channels to stop potassium ion outflow. This leads to activation of voltage-dependent calcium channels and calcium influx, which triggers secretory granule exocytosis mediating insulin secretion. Regulatory ligand binding sites link the main gate of the KATP channel to glucose metabolism via ATP, MgATP, and MgADP (3). The β-cell KATP channel is a hetero-octameric protein of four inner pore-forming Kir6.2 and four outer sulfonylurea receptor 1 (SUR1) subunits (Fig. 1). The gate is formed by concerted action of all four Kir6.2 subunits (4). Each Kir6.2 subunit also has an independent ATP site whose occupancy effectively can shut the gate and can keep it shut (5), referred to as inhibition. Pharmacological ligands also inhibit the KATP channel. Each SUR1 subunit …

Cellular impedance assay for prediction of QT prolongation induced by hERG and non-hERG channel modulators using human stem cell derived cardiomyocytes

Torsades de pointes (TdP) arrhythmia is a potentially fatal form of ventricular arrhythmia that occurs under conditions where cardiac repolarization is delayed (as indicated by prolonged QT intervals from electrocardiographic recordings). A likely mechanism for QT interval prolongation and TdP arrhythmias is blockade of the rapid component of the cardiac delayed rectifier K+ current (IKr), which is encoded by human ether-a-go-go-related gene (HERG). Over 100 non-cardiovascular drugs have the potential to induce QT interval prolongations in the electrocardiogram (ECG) or TdP arrhythmias. The binding site of most HERG channel blockers is located inside the central cavity of the channel. An evaluation of possible effects on HERG channels during the development of novel drugs is recommended by international guidelines. During cardiac ischaemia activation of ATP-sensitive K+ (KATP) channels contributes to action potential (AP) shortening which is either cardiotoxic by inducing re-entrant ventricular arrhythmias or cardioprotective by inducing energy-sparing effects or ischaemic preconditioning (IPC). KATP channels are formed by an inward-rectifier K+ channel (Kir6.0) and a sulfonylurea receptor (SUR) subunit: Kir6.2 and SUR2A in cardiac myocytes, Kir6.2 and SUR1 in pancreatic β-cells. Sulfonylureas and glinides stimulate insulin secretion via blockade of the pancreatic β-cell KATP channel. Clinical studies about cardiotoxic effects of sulfonylureas are contradictory. Sulfonylureas and glinides differ in their selectivity for pancreatic over cardiovascular KATP channels, being either selective (tolbutamide, glibenclamide) or non-selective (repaglinide). The possibility exists that non-selective KATP channel inhibitors might have cardiovascular side effects. Blockers of the pore-forming Kir6.2 subunit are insulin secretagogues and might have cardioprotective or cardiotoxic effects during cardiac ischaemia.

Cephalic phase insulin secretion is KATP channel independent

Glucose-induced insulin secretion from pancreatic β-cells critically depends on the activity of ATP-sensitive K⁺ channels (KATP channel). We previously generated mice lacking Kir6.2, the pore subunit of the β-cell KATP channel (Kir6.2(-/-)), that show almost no insulin secretion in response to glucose in vitro. In this study, we compared insulin secretion by voluntary feeding (self-motivated, oral nutrient ingestion) and by forced feeding (intra-gastric nutrient injection via gavage) in wild-type (Kir6.2(+/+) and Kir6.2(-/-) mice. Under ad libitum feeding or during voluntary feeding of standard chow, blood glucose levels and plasma insulin levels were similar in Kir6.2(+/+) and Kir6.2(-/-) mice. By voluntary feeding of carbohydrate alone, insulin secretion was induced significantly in Kir6.2(-/-) mice but was markedly attenuated compared with that in Kir6.2(+/+) mice. On forced feeding of standard chow or carbohydrate alone, the insulin secretory response was markedly impaired or completely absent in Kir6.2(-/-) mice. Pretreatment with a muscarine receptor antagonist, atropine methyl nitrate, which does not cross the blood-brain barrier, almost completely blocked insulin secretion induced by voluntary feeding of standard chow or carbohydrate in Kir6.2(-/-) mice. Substantial glucose-induced insulin secretion was induced in the pancreas perfusion study of Kir6.2(-/-) mice only in the presence of carbamylcholine. These results suggest that a KATP channel-independent mechanism mediated by the vagal nerve plays a critical role in insulin secretion in response to nutrients in vivo.

Exploring the Promise of Resveratrol: Where Do We Go From Here?

Resveratrol (3,5,4’-trihydroxystilbene), a plant-derived polyphenol and activator of the mammalian sirtuin, SIRT1, has demonstrated promising effects on glucose metabolism in rodent models (1). However, despite the widespread use of resveratrol as a nutritional supplement and the many health claims made on its behalf, data from human studies are extremely limited. Here, we explore why, in contrast to many studies in mammalian models, the article by Poulsen et al. (2) in this issue of Diabetes reports that chronic high doses of resveratrol have no demonstrable metabolic effects. Interest in resveratrol has skyrocketed in recent years, initially from its association with the health benefits of red wine (the “French paradox”) and its in vitro anticancer activity (3). Subsequent reports demonstrated that it activates sirtuins and extends the life span of lower organisms (“calorie restriction mimetic”), including rodents (4). Studies in vitro show that resveratrol enhances insulin-stimulated glucose uptake in skeletal muscle, liver, and adipocytes (5,6) and stimulates insulin secretion via inhibition of β-cell KATP channels (7). These observations have been confirmed in vivo in several animal models, including aging, diet-induced obesity, and diabetic ( db / db ) mice (4,8–10). Importantly, key metabolic effects of resveratrol can be monitored in relevant tissues (muscle, fat, and liver), thus providing critical insight into mechanisms. These effects include increased mitochondrial biogenesis and oxidative …

Pharmacological rescue of trafficking-impaired ATP-sensitive potassium channels

ATP-sensitive potassium (KATP) channels link cell metabolism to membrane excitability and are involved in a wide range of physiological processes including hormone secretion, control of vascular tone, and protection of cardiac and neuronal cells against ischemic injuries. In pancreatic β-cells, KATP channels play a key role in glucose-stimulated insulin secretion, and gain or loss of channel function results in neonatal diabetes or congenital hyperinsulinism, respectively. The β-cell KATP channel is formed by co-assembly of four Kir6.2 inwardly rectifying potassium channel subunits encoded by KCNJ11 and four sulfonylurea receptor 1 subunits encoded by ABCC8. Many mutations in ABCC8 or KCNJ11 cause loss of channel function, thus, congenital hyperinsulinism by hampering channel biogenesis and hence trafficking to the cell surface. The trafficking defects caused by a subset of these mutations can be corrected by sulfonylureas, KATP channel antagonists that have long been used to treat type 2 diabetes. More recently, carbamazepine, an anticonvulsant that is thought to target primarily voltage-gated sodium channels has been shown to correct KATP channel trafficking defects. This article reviews studies to date aimed at understanding the mechanisms by which mutations impair channel biogenesis and trafficking and the mechanisms by which pharmacological ligands overcome channel trafficking defects. Insight into channel structure-function relationships and therapeutic implications from these studies are discussed.

SAR studies on β-cell KATP channel openers

Recently Balaban has introduced a J-type G index for modeling those properties/activities where not only the "shape" but also size of the graph influences the property/activity of organic compounds acting as drugs. Here, for the first time G index has been used for modeling the pancreatic β-cell K(ATP) channel openers viz R/S-3,4-dihydro-2,2-dimethyl-6-halo-4-(substituted phenylaminocarbonylamino)-2H-1-benzopyrans. Modeling technology used in this paper has established that the referred activity is excellently modeled by using G as one of the correlating descriptors. The results are critically discussed using Ridge statistics.