Pancreatic ATP-sensitive Potassium Research Articles

Modulatory Effect of Medicinal Plants and Their Active Constituents on ATP-Sensitive Potassium Channels (KATP) in Diabetes.

Hyperglycemia, which is a chronic metabolic condition caused by either a defect in insulin secretion or insulin resistance, is a hallmark of diabetes mellitus (DM). Sustained hyperglycemia leads to the onset and development of many health complications. Despite the number of available antidiabetic medications on the market, there is still a need for novel treatment agents with increased efficacy and fewer adverse effects. Many medicinal plants offer a rich supply of bioactive compounds that have remarkable pharmacological effects with less toxicity and side effects. According to published evidence, natural antidiabetic substances influence pancreatic β-cell development and proliferation, inhibit pancreatic β-cell death, and directly increase insulin output. Pancreatic ATP-sensitive potassium channels play an essential role in coupling glucose metabolism to the secretion of insulin. Although much of the literature is available on the antidiabetic effects of medicinal plants, very limited studies discuss their direct action on pancreatic KATP. The aim of this review is to focus on the modulatory effects of antidiabetic medicinal plants and their active constituents on pancreatic KATP. The KATP channel should be regarded as a key therapeutic milestone in the treatment of diabetes. Therefore, continuous research into the interaction of medicinal plants with the KATP channel is crucial.

The open structure of a neonatal diabetes-causing variant of the pancreatic KATP channel determined using cryo-electron microscopy.

Pancreatic ATP-sensitive potassium (KATP) channels, comprising four Kir6.2 and four SUR1 subunits, control insulin secretion in β-cells by coupling glucose metabolism with membrane excitability. Regulation of KATP channels involves orchestrated interactions of their subunits, Kir6.2 and SUR1, and ligands. ATP and PIP2 are known to inhibit and stimulate channel activity, respectively. Although Kir6.2 harbors the primary binding sites for ATP and PIP2, SUR1 is required to confer high channel sensitivity to both ligands. Mutations in channel proteins that disrupt ATP and PIP2 gating cause insulin secretion disease. Kir6.2-Q52R is a neonatal diabetes mutation that results in channels with extremely high open probability and low ATP sensitivity. Previous studies suggest the Kir6.2-Q52R mutation stabilizes channel opening in a SUR1-dependent manner, but the structural mechanism is unknown. Here, we determined the cryoEM structure of the Kir6.2-Q52R4SUR14 channel in the absence of nucleotides or pharmacological ligands. This 3.3 Å resolution structure reveals that Kir6.2 is open, with its cytoplasmic domain (CTD) corkscrewed up to the membrane. The side-chain of Kir6.2-Q52R is flipped away from the ATP-binding site and embedded into SUR1-TMD0 in a cation-pi interaction with SUR1-W51 at the membrane. The cryo-EM map has clear density showing the first intracellular loop of SUR1-TMD0 (ICL1, 52-60) is ordered and forms main chain interactions between Kir6.2-βA (the CTD β-strand just N-terminal to the interfacial helix) and SUR1-ICL1, tethering the CTD to TMD0 to stabilize the open state. Our study provides structural insights into the mechanism by which the Kir6.2-Q52R mutation causes severe neonatal diabetes and how Kir6.2 and SUR1 interact to stabilize KATP channels in the open state.

Structural Insights into the Inhibitory Mechanism of Insulin Secretagogues on the Pancreatic ATP-Sensitive Potassium Channel.

Sulfonylureas and glinides are commonly used oral insulin secretagogues (ISs) that act on the pancreatic ATP-sensitive potassium (KATP) channel to promote insulin secretion in order to lower the blood glucose level. Physiologically, KATP channels are inhibited by intracellular ATP and activated by Mg-ADP. Therefore, they sense the cellular energy status to regulate the permeability of potassium ions across the plasma membrane. The pancreatic KATP channel is composed of the pore-forming Kir6.2 subunits and the regulatory SUR1 subunits. Previous electrophysiological studies have established that ISs bind to the SUR1 subunit and inhibit the channel activity primarily by two mechanisms. First, ISs prevent Mg-ADP activation. Second, ISs inhibit the channel activity of Kir6.2 directly. Several cryo-EM structures of the pancreatic KATP channel determined recently have provided remarkable structural insights into these two mechanisms.

The Structural Basis for the Binding of Repaglinide to the Pancreatic KATP Channel

Repaglinide (RPG) is a short-acting insulin secretagogue widely prescribed for the treatment of type 2 diabetes. It boosts insulin secretion by inhibiting the pancreatic ATP-sensitive potassium channel (KATP). However, the mechanisms by which RPG binds to the KATP channel are poorly understood. Here, we describe two cryo-EM structures: the pancreatic KATP channel in complex with inhibitory RPG and adenosine-5'-(γ-thio)-triphosphate (ATPγS) at 3.3Å and a medium-resolution structure of a RPG-bound mini SUR1 protein in which the N terminus of the inward-rectifying potassium channel 6.1 (Kir6.1) is fused to the ABC transporter module of the sulfonylurea receptor 1 (SUR1). These structures reveal the binding site of RPG in the SUR1 subunit. Furthermore, the high-resolution structure reveals the complex architecture of the ATP binding site, which is formed by both Kir6.2 and SUR1 subunits, and the domain-domain interaction interfaces.

Effectiveness and safety of long-term treatment with sulfonylureas in patients with neonatal diabetes due to KCNJ11 mutations: an international cohort study

SummaryBackgroundKCNJ11 mutations cause permanent neonatal diabetes through pancreatic ATP-sensitive potassium channel activation. 90% of patients successfully transfer from insulin to oral sulfonylureas with excellent initial glycaemic control; however, whether this control is maintained in the long term is unclear. Sulfonylurea failure is seen in about 44% of people with type 2 diabetes after 5 years of treatment. Therefore, we did a 10-year multicentre follow-up study of a large international cohort of patients with KCNJ11 permanent neonatal diabetes to address the key questions relating to long-term efficacy and safety of sulfonylureas in these patients.MethodsIn this multicentre, international cohort study, all patients diagnosed with KCNJ11 permanent neonatal diabetes at five laboratories in Exeter (UK), Rome (Italy), Bergen (Norway), Paris (France), and Krakow (Poland), who transferred from insulin to oral sulfonylureas before Nov 30, 2006, were eligible for inclusion. Clinicians collected clinical characteristics and annual data relating to glycaemic control, sulfonylurea dose, severe hypoglycaemia, side-effects, diabetes complications, and growth. The main outcomes of interest were sulfonylurea failure, defined as permanent reintroduction of daily insulin, and metabolic control, specifically HbA1c and sulfonylurea dose. Neurological features associated with KCNJ11 permanent neonatal diabetes were also assessed. This study is registered with ClinicalTrials.gov, number NCT02624817.Findings90 patients were identified as being eligible for inclusion and 81 were enrolled in the study and provided long-term (>5·5 years cut-off) outcome data. Median follow-up duration for the whole cohort was 10·2 years (IQR 9·3–10·8). At most recent follow-up (between Dec 1, 2012, and Oct 4, 2016), 75 (93%) of 81 participants remained on sulfonylurea therapy alone. Excellent glycaemic control was maintained for patients for whom we had paired data on HbA1c and sulfonylurea at all time points (ie, pre-transfer [for HbA1c], year 1, and most recent follow-up; n=64)—median HbA1c was 8·1% (IQR 7·2–9·2; 65·0 mmol/mol [55·2–77·1]) before transfer to sulfonylureas, 5·9% (5·4–6·5; 41·0 mmol/mol [35·5–47·5]; p<0·0001 vs pre-transfer) at 1 year, and 6·4% (5·9–7·3; 46·4 mmol/mol [41·0–56·3]; p<0·0001 vs year 1) at most recent follow-up (median 10·3 years [IQR 9·2–10·9]). In the same patients, median sulfonylurea dose at 1 year was 0·30 mg/kg per day (0·14–0·53) and at most recent follow-up visit was 0·23 mg/kg per day (0·12–0·41; p=0·03). No reports of severe hypoglycaemia were recorded in 809 patient-years of follow-up for the whole cohort (n=81). 11 (14%) patients reported mild, transient side-effects, but did not need to stop sulfonylurea therapy. Seven (9%) patients had microvascular complications; these patients had been taking insulin longer than those without complications (median age at transfer to sulfonylureas 20·5 years [IQR 10·5–24·0] vs 4·1 years [1·3–10·2]; p=0·0005). Initial improvement was noted following transfer to sulfonylureas in 18 (47%) of 38 patients with CNS features. After long-term therapy with sulfonylureas, CNS features were seen in 52 (64%) of 81 patients.InterpretationHigh-dose sulfonylurea therapy is an appropriate treatment for patients with KCNJ11 permanent neonatal diabetes from diagnosis. This therapy is safe and highly effective, maintaining excellent glycaemic control for at least 10 years.FundingWellcome Trust, Diabetes UK, Royal Society, European Research Council, Norwegian Research Council, Kristian Gerhard Jebsen Foundation, Western Norway Regional Health Authority, Southern and Eastern Norway Regional Health Authority, Italian Ministry of Health, Aide aux Jeunes Diabetiques, Societe Francophone du Diabete, Ipsen, Slovak Research and Development Agency, and Research and Development Operational Programme funded by the European Regional Development Fund.

Glibenclamide Prevents Hypoglycemia-Induced Fatal Cardiac Arrhythmias in Rats.

Sulfonylureas increase the incidence of severe hypoglycemia in people with type 2 diabetes and might increase the risk of sudden cardiac death. Sulfonylureas stimulate insulin secretion by closing pancreatic ATP-sensitive potassium ion (KATP) channels. To investigate the role of KATP channel modulators on cardiac arrhythmias and mortality in the setting of severe hypoglycemia, adult Sprague-Dawley rats underwent hyperinsulinemic (0.2 U/kg/min) severe hypoglycemic (10 to 15 mg/dL) clamps with continuous electrocardiography. The rats were randomized for treatment with intravenous vehicle (VEH), the sulfonylurea glibenclamide (GLIB; KATP channel blocker; 5 mg/kg/h), or diazoxide (DIAZ; KATP channel opener; 5 mg/kg/h). The results demonstrated that GLIB completely prevented first-degree heart block compared with VEH (0.18 ± 0.09/min) and DIAZ (0.2 ± 0.05/min). Second-degree heart block was significantly reduced with GLIB (0.12 ± 0.1/min) compared with VEH (0.6 ± 0.2/min) and DIAZ (6.9 ± 3/min). The incidence of third-degree heart block was completely prevented by GLIB compared with VEH (67%) and DIAZ (87.5%). Hypoglycemia-induced mortality was completely prevented by GLIB compared with VEH (60%) and DIAZ (82%). In conclusion, although GLIB increases the risk of hypoglycemia by increasing insulin secretion, these results have demonstrated a paradoxical protective role of GLIB against severe hypoglycemia-induced fatal cardiac arrhythmias.

Ligand binding and conformational changes of SUR1 subunit in pancreatic ATP-sensitive potassium channels

ATP-sensitive potassium channels (KATP) are energy sensors on the plasma membrane. By sensing the intracellular ADP/ATP ratio of β-cells, pancreatic KATP channels control insulin release and regulate metabolism at the whole body level. They are implicated in many metabolic disorders and diseases and are therefore important drug targets. Here, we present three structures of pancreatic KATP channels solved by cryo-electron microscopy (cryo-EM), at resolutions ranging from 4.1 to 4.5 Å. These structures depict the binding site of the antidiabetic drug glibenclamide, indicate how Kir6.2 (inward-rectifying potassium channel 6.2) N-terminus participates in the coupling between the peripheral SUR1 (sulfonylurea receptor 1) subunit and the central Kir6.2 channel, reveal the binding mode of activating nucleotides, and suggest the mechanism of how Mg-ADP binding on nucleotide binding domains (NBDs) drives a conformational change of the SUR1 subunit.

Prediabetes is associated with genetic variations in the gene encoding the Kir6.2 subunit of the pancreatic ATP-sensitive potassium channel (KCNJ11): A case-control study in a Han Chinese youth population.

The E23K variant of the potassium voltage-gated channel subfamily J member 11 (KCNJ11) gene has been reported to be associated with type 2 diabetes (T2D) in many populations. However, little is known about the role of E23K in the development of prediabetes in Chinese youth. To investigate the role of E23K in the development of prediabetes, 279 subjects with prediabetes and 240 normal controls (mean [± SD] age 18.1 ± 3.2 and 17.8 ± 4.3 years, respectively) were recruited to the study. Height, weight, and hip and waist circumferences were measured by trained physicians. Genotyping of KCNJ11 polymorphisms and clinical laboratory tests to determine cholesterol, triglyceride (TG), blood glucose, and insulin levels were performed. The carrier rate of K23 allele-containing genotypes was higher for prediabetic than control subjects (P = 0.005). Logistic regression analyses revealed that higher body mass index percentiles (P = 0.013), lower insulin levels at 30 min during an oral glucose tolerance test (P = 0.001), a higher ratio of total cholesterol: high-density lipoprotein cholesterol (P = 0.001), and a K allele-containing genotype (P = 0.019) are independent risk factors for prediabetes in Chinese Han youth. Furthermore, K23 allele-containing genotypes were associated with impaired indices of insulin secretion and β-cell function in female youth with prediabetes. These effects were not seen in male youth with prediabetes. The results confirm that the common E23K polymorphism of KCNJ11 carries a higher susceptibility to the development of prediabetes in the Chinese Han population. The results suggest that E23K may have a greater effect on the development of T2D in female Chinese youth.

Structure of a Pancreatic ATP-Sensitive Potassium Channel

ATP-sensitive potassium channels (KATP) couple intracellular ATP levels with membrane excitability. These channels play crucial roles in many essential physiological processes and have been implicated extensively in a spectrum of metabolic diseases and disorders. To gain insight into the mechanism of KATP, we elucidated the structure of a hetero-octameric pancreatic KATP channel in complex with a non-competitive inhibitor glibenclamide by single-particle cryoelectron microscopy to 5.6-Å resolution. The structure shows that four SUR1 regulatory subunits locate peripherally and dock onto the central Kir6.2 channel tetramer through the SUR1 TMD0-L0 fragment. Glibenclamide-bound SUR1 uses TMD0-L0 fragment to stabilize Kir6.2 channel in a closedconformation. In another structural population, aputative co-purified phosphatidylinositol 4,5-bisphosphate (PIP2) molecule uncouples Kir6.2 from glibenclamide-bound SUR1. These structural observations suggest a molecular mechanism for KATP regulation by anti-diabetic sulfonylurea drugs, intracellular adenosine nucleotide concentrations, and PIP2 lipid.

Molecular genetics, correlation between genotype and phenotype of 65 Vietnames patients with congenital hyperinsulinism

Hyperinsulinemic hypoglycemia (HH) is a consequence of unregulated insulin secretion by pancreatic β-cells. Congenital HH is caused by mutations in genes involved in regulation of insulin secretion (ABCC8, KCNJ11, GLUD1, CGK, HADH, SLC16A1, HNF4A and UCP2). Severe forms of congenital HH are caused by inactivating mutations in ABCC8 and KCNJ11, which encode the two components of the pancreatic β-cell ATP-sensitive potassium channel. Our aim is to identify mutations in the ABCC8 and KCNJ11, HNF4A and GLUD genes, and to describe genotype and phenotype correlations of Vietnamese children with congenital hyperinsulinism. A prospective study was conducted on 65 cases with congenital hyperinsulinism diagnosed and treated at the National Hospital of Pediatric from January 2007 to April 2014. Patients were selected by using inclusion criteria of Hussain K (2008). Mutations were identified in 32 cases (49.2%) including mutations of ABCC8 gene (28 cases; 43.1%), KCNJ11 (3 cases; 4.6%), HNF4A (1 case; 1.5%). 100% of cases with homozygous/compound heterozygous recessive mutations or one paternal dominant mutation of ABCC8 gene did not respond to diazoxide treatment and required 95% pancreatectomy. Molecular analysis using pancreas tissue after surgery from cases with one mutation of ABCC8 gene inherited from father confirmed focal lesion type. Other cases without identified mutations usually responded to diazoxide. In conclusions, children with congenital hyperinsulinism should be performed mutation analysis which helps in making diagnosis and treatment decision. Families of children with congenital hyperinsulinism should be given genetic counseling. Prenatal diagnosis should be performed as well as follow - up and treatment should be given to children with congenital hyperinsulinism immediately after birth.

Incidence of new onset of type-2 diabetes with the use of atenolol for treatment of hypertension in north indian population: No role of irs-1 and kir 6.2 Gene polymorphism

Introduction: Previous research has suggested that β 1 adreno-receptor blockers commonly used for management of hypertension may promote new onset of type-2 diabetes mellitus. The objective of this study is to evaluate the role of insulin receptor substrate-1 (IRS-1) gene and pancreatic ATP-sensitive potassium inward rectifying channel (Kir 6.2) genetic polymorphism in induction of diabetes mellitus with chronic use of β 1 blocker. Materials and Methods: A total of 150 patients with essential hypertension aged between 17 and 65 years who were diagnosed with essential hypertension and prescribed atenolol therapy, were recruited. Of these, only 100 patients responding to atenolol were followed-up for 12 months for monitoring blood glucose level every month. The IRS-1 and pancreatic ATP-sensitive potassium channel Kir 6.2 (E23K) gene polymorphism were genotyped using genomic DNA extracted from the whole blood of the recruited patients by polymerase chain reaction and restriction fragment length polymorphism. Results: This study revealed that among the 100 patients responding to atenolol 27% showed a significant increase in the fasting blood sugar. Genotyping study of the recruited patients revealed a difference in allelic frequencies for IRS-1 (Gly972Arg) and pancreatic ATP-sensitive potassium channel Kir 6.2 (E23K) variants. However, allelic distribution between the hypertensive patients on atenolol showing hyperglycemia and normoglycemia was not significantly different for these genes. Conclusion: Thus, showing no correlation, for incidence of diabetes post atenolol therapy in the studied population with these gene polymorphisms.

Genetic Variations in the Kir6.2 Subunit (KCNJ11) of Pancreatic ATP-Sensitive Potassium Channel Gene Are Associated with Insulin Response to Glucose Loading and Early Onset of Type 2 Diabetes in Childhood and Adolescence in Taiwan.

To investigate the role of E23K polymorphism of the KCNJ11 gene on early onset of type 2 diabetes in school-aged children/adolescents in Taiwan, we recruited 38 subjects with type 2 diabetes (ages 18.6 ± 6.6 years; body mass index percentiles 83.3 ± 15.4) and 69 normal controls (ages 17.3 ± 3.8 years; body mass index percentiles 56.7 ± 29.0) from a national surveillance for childhood/adolescent diabetes in Taiwan. We searched for the E23K polymorphism of the KCNJ11 gene. We found that type 2 diabetic subjects had higher carrier rate of E23K polymorphism of KCNJ11 gene than control subjects (P = 0.044). After adjusting for age, gender, body mass index percentiles, and fasting plasma insulin, the E23K polymorphism contributed to an increased risk for type 2 diabetes (P = 0.047). K23-allele-containing genotypes conferring increased plasma insulin level during OGTT in normal subjects. However, the diabetic subjects with the K23-allele-containing genotypes had lower fasting plasma insulin levels after adjustment of age and BMI percentiles. In conclusion, the E23K variant of the KCNJ11 gene conferred higher susceptibility to type 2 diabetes in children/adolescents. Furthermore, in normal glucose-tolerant children/adolescents, K23 allele carriers had a higher insulin response to oral glucose loading.

A Novel Mutation in ABCC8 Gene in a Newborn with Congenital Hyperinsulinism –A Case Report

Congenital hyperinsulinism (CHI) is the most common cause of persistent hypoglycemia in infancy. The genetic basis of CHI includes a variety of defects in key genes regulating insulin secretion. Mutations in at least seven genes are found in 50% of cases. The most common forms of medically unresponsive CHI, which requires a near-total pancreatectomy are associated with autosomal recessive mutations in the ABCC8 and KCNJ11 genes encoding the two subunits of the pancreatic β-cell ATP-sensitive potassium channel. We report a neonate with CHI and have a novel homozygous splicing mutation in the ABCC8 gene.

Novel mutation c.597_598dup in exon 5 of ABCC8 gene causing congenital hyperinsulinism

Congenital hyperinsulinism (CHI), a clinically and genetically heterogeneous disease, characterized by the unregulated secretion of insulin from pancreatic β-cells, is the most common cause of persistent hypoglycemia in infancy. Early diagnosis and maintenance of normoglycaemia are essential to prevent adverse neurodevelopmental outcomes. The most common and severe forms of CHI are caused by inactivating mutations in ABCC8 and KCNJ11 genes, encoding the two subunits of the pancreatic β-cell ATP sensitive potassium channel (KATP). We report a case of neonatal CHI due to a novel homozygous recessive mutation in the ABCC8 gene.

Syntaxin 1A regulates surface expression of β-cell ATP-sensitive potassium channels

The pancreatic ATP-sensitive potassium (K(ATP)) channel consisting of four inwardly rectifying potassium channel 6.2 (Kir6.2) and four sulfonylurea receptor SUR1 subunits plays a key role in insulin secretion by linking glucose metabolism to membrane excitability. Syntaxin 1A (Syn-1A) is a plasma membrane protein important for membrane fusion during exocytosis of insulin granules. Here, we show that Syn-1A and K(ATP) channels endogenously expressed in the insulin-secreting cell INS-1 interact. Upregulation of Syn-1A by overexpression in INS-1 leads to a decrease, whereas downregulation of Syn-1A by small interfering RNA (siRNA) leads to an increase, in surface expression of K(ATP) channels. Using COSm6 cells as a heterologous expression system for mechanistic investigation, we found that Syn-1A interacts with SUR1 but not Kir6.2. Furthermore, Syn-1A decreases surface expression of K(ATP) channels via two mechanisms. One mechanism involves accelerated endocytosis of surface channels. The other involves decreased biogenesis and processing of channels in the early secretory pathway. This regulation is K(ATP) channel specific as Syn-1A has no effect on another inward rectifier potassium channel Kir3.1/3.4. Our results demonstrate that in addition to a previously documented role in modulating K(ATP) channel gating, Syn-1A also regulates K(ATP) channel expression in β-cells. We propose that physiological or pathological changes in Syn-1A expression may modulate insulin secretion by altering glucose-secretion coupling via changes in K(ATP) channel expression.

Constitutive Endocytic Recycling and Protein Kinase C-mediated Lysosomal Degradation Control KATP Channel Surface Density

Pancreatic ATP-sensitive potassium (K(ATP)) channels control insulin secretion by coupling the excitability of the pancreatic beta-cell to glucose metabolism. Little is currently known about how the plasma membrane density of these channels is regulated. We therefore set out to examine in detail the endocytosis and recycling of these channels and how these processes are regulated. To achieve this goal, we expressed K(ATP) channels bearing an extracellular hemagglutinin epitope in human embryonic kidney cells and followed their fate along the endocytic pathway. Our results show that K(ATP) channels undergo multiple rounds of endocytosis and recycling. Further, activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate significantly decreases K(ATP) channel surface density by reducing channel recycling and diverting the channel to lysosomal degradation. These findings were recapitulated in the model pancreatic beta-cell line INS1e, where activation of PKC leads to a decrease in the surface density of native K(ATP) channels. Because sorting of internalized channels between lysosomal and recycling pathways could have opposite effects on the excitability of pancreatic beta-cells, we propose that PKC-regulated K(ATP) channel trafficking may play a role in the regulation of insulin secretion.

Differential PKC Isoform Regulation of KATP Channel Trafficking and Function

Pancreatic ATP sensitive potassium (KATP) channels play an important role in insulin secretion, linking the metabolic state and excitability of the beta cell. KATP channel function is under complex regulation by protein kinase C (PKC) with both activation and inhibition reported. To address this apparent contradiction we have employed a range of cell biological, electrophysiological and biochemical techniques to elucidate the mechanisms of PKC regulation of pancreatic KATP channels. Acute PKC activation (5 min) led to a large increase in KATP currents recorded from HEK cells stably expressing Kir6.2 and SUR1. This increase in currents was accompanied by increased colocalisation of KATP channels with classical PKC isoforms and was sensitive to inhibition by the classical PKC inhibitor Go6976. Prolonged (1 hour) activation of PKC however led to a significant reduction in KATP channel currents accompanied by a loss of channels from the cell surface and an increase in their lysosomal degradation. Decreased KATP channel surface expression was not sensitive to Go6976 indicating novel PKC isoforms. Both dominant negative PKC epsilon and PKC epsilon inhibiting peptides were able to inhibit the PKC mediated decrease in channel surface density. These data suggest that classical and novel PKC isoforms differentially regulate the function and trafficking of pancreatic KATP channels comprising Kir6.2 and SUR1. Further work is underway to assess the physiological significance of differential PKC isoform activation following pancreatic beta cell stimulation.This work is supported by the Medical Research Council, UK.

Replication of association between polymorphisms of the pancreatic ATP-sensitive potassium channel and susceptibility to type 2 diabetes in two Russian urban populations

AbstractThe KCNJ11 and ABCC8 genes encode components of the pancreatic ATP-sensitive potassium (KATP) channel. Previously, we reported association of the KCNJ11 E23K and ABCC8 R1273R G/A variants with type 2 diabetes (T2D) in a small Russian population sample (n=244). Here we replicated association between these genetic variants and T2D in a larger cohort (588 diabetic and 597 non-diabetic subjects). Using the ANCOVA analysis, Odds Ratios (ORs) and relationships between the carriage of a genotype and biochemical parameters of the patients were assessed and then adjusted for confounders (age, gender, HbA1c, hypertension, and obesity). The KCNJ11 K23 variant and the ABCC8 R1273R allele A showed association with higher risk of T2D (adjusted OR of 1.41 and 2.03, P&lt;0.0001, respectively). Diabetic patients homozygous for K/K had lower 2h insulin (Padjusted=0.044). The ABCC8 A/A variant was associated with increased 2h serum insulin in diabetic and non-diabetic subjects (Padjusted=0.027 and 0.033, respectively). The carriage of the risk variant K/K of KCNJ11 E23K or A/A of ABCC8 G/A R1273R was associated with reduced response to nonsulfonylurea and sulfonylurea blockers of the pancreatic KATP channel. Adjusted attributable population risk was 3.0% (KCNJ11 E23K) and 4.8% (ABCC8 G/A) suggesting for the modest effects of these genetic variants on diabetes susceptibility.

Protein Kinase C Regulation Of KATP Channel Recycling

Pancreatic ATP sensitive potassium (KATP) channels play an important role in insulin secretion, linking the metabolic state of the beta cell to its excitability. Protein kinase C (PKC) has previously been shown to down regulate the cell surface density of KATP channels in cardiac, neuronal and recombinant cells, but there are no studies on pancreatic beta cells. Here we show that activation of PKC results in significant down regulation of KATP channel conductance in the model beta cell line, INS1e. To investigate the underlying mechanism, we expressed a Kir6.2 construct containing an extracellular HA epitope plus SUR1 in INS1e and HEK293 cells and examined the role of PKC in regulation of cell surface density and endosomal trafficking. We found that PKC activation with the phorbol ester drug PMA reduced the surface density of KATP channels by reducing recycling of endocytosed channels, but had no effect on the rate of endocytosis. Endocytosed channels entered the peripheral and perinuclear compartments when PKC was activated, but remained at a peripheral location when PKC was inhibited with chelerythrine. Since pancreatic beta-cells express 9 different isoforms of PKC, we asked which of these isoforms is responsible for the observed effect. Using the dominant negative approach, our results show that PKC epsilon regulates endosomal trafficking of KATP channels. We conclude that that activation of PKC epsilon leads to dramatic changes in the distribution of internalised KATP channels and that the decrease in channel surface density is due to inhibition of channel recycling. The results may have important consequences for the electrical excitability of the pancreatic beta cell and therefore the insulin secretory response.

Mutations in UCP2 in Congenital Hyperinsulinism Reveal a Role for Regulation of Insulin Secretion

Although the most common mechanism underlying congenital hyperinsulinism is dysfunction of the pancreatic ATP-sensitive potassium channel, the pathogenesis and genetic origins of this disease remains largely unexplained in more than half of all patients. UCP2 knockout mice exhibit an hyperinsulinemic hypoglycemia, suggesting an involment of UCP2 in insulin secretion. However, a possible pathogenic role for UCP2 protein in the development of human congenital hyperinsulinism or of any human disease has not yet been investigated. We studied ten children exhibiting congenital hyperinsulinism, without detectable mutations in the known congenital hyperinsulinism-causing genes. Parental-inherited heterozygous UCP2 variants encoding amino-acid changes were found in two unrelated children with congenital hyperinsulinism. Functional assays in yeast and in insulin-secreting cells revealed an impaired activity of UCP2 mutants. Therefore, we report the finding of UCP2 coding variants in human congenital hyperinsulinism, which reveals a role for this gene in the regulation of insulin secretion and glucose metabolism in humans. Our results show for the first time a direct association between UCP2 amino acid alteration and human disease and highlight a role for mitochondria in hormone secretion.