A novel high-affinity inhibitor against the human ATP-sensitive Kir6.2 channel

The adenosine triphosphate (ATP)-sensitive (KATP) channels in pancreatic β cells couple the blood glucose level to insulin secretion. KATP channels in pancreatic β cells comprise the pore-forming Kir6.2 and the modulatory sulfonylurea receptor 1 (SUR1) subunits. Currently, there is no high-affinity and relatively specific inhibitor for the Kir6.2 pore. The importance of developing such inhibitors is twofold. First, in many cases, the lack of such an inhibitor precludes an unambiguous determination of the Kir6.2's role in certain physiological and pathological processes. This problem is exacerbated because Kir6.2 knockout mice do not yield the expected phenotypes of hyperinsulinemia and hypoglycemia, which in part, may reflect developmental adaptation. Second, mutations in Kir6.2 or SUR1 that increase the KATP current cause permanent neonatal diabetes mellitus (PNDM). Many patients who have PNDM have been successfully treated with sulphonylureas, a common class of antidiabetic drugs that bind to SUR1 and indirectly inhibit Kir6.2, thereby promoting insulin secretion. However, some PNDM-causing mutations render KATP channels insensitive to sulphonylureas. Conceptually, because these mutations are located intracellularly, an inhibitor blocking the Kir6.2 pore from the extracellular side might provide another approach to this problem. Here, by screening the venoms from >200 animals against human Kir6.2 coexpressed with SUR1, we discovered a small protein of 54 residues (SpTx-1) that inhibits the KATP channel from the extracellular side. It inhibits the channel with a dissociation constant value of 15 nM in a relatively specific manner and with an apparent one-to-one stoichiometry. SpTx-1 evidently inhibits the channel by primarily targeting Kir6.2 rather than SUR1; it inhibits not only wild-type Kir6.2 coexpressed with SUR1 but also a Kir6.2 mutant expressed without SUR1. Importantly, SpTx-1 suppresses both sulfonylurea-sensitive and -insensitive, PNDM-causing Kir6.2 mutants. Thus, it will be a valuable tool to investigate the channel's physiological and biophysical properties and to test a new strategy for treating sulfonylurea-resistant PNDM.

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Diabetes induced by gain-of-function mutations in the Kir6.1 subunit of the KATP channel

The Journal of General Physiology ◽

10.1085/jgp.201611653 ◽

2016 ◽

Vol 149 (1) ◽

pp. 75-84 ◽

Cited By ~ 4

Author(s):

Maria S. Remedi ◽

Jonathan B. Friedman ◽

Colin G. Nichols

Keyword(s):

Insulin Secretion ◽

Katp Channel ◽

Vascular System ◽

Wild Type Mouse ◽

Neonatal Diabetes ◽

Katp Channels ◽

Β Cells ◽

Pancreatic Β Cells ◽

Gain Of Function ◽

Insulin Promoter

Gain-of-function (GOF) mutations in the pore-forming (Kir6.2) and regulatory (SUR1) subunits of KATP channels have been identified as the most common cause of human neonatal diabetes mellitus. The critical effect of these mutations is confirmed in mice expressing Kir6.2-GOF mutations in pancreatic β cells. A second KATP channel pore-forming subunit, Kir6.1, was originally cloned from the pancreas. Although the prominence of this subunit in the vascular system is well documented, a potential role in pancreatic β cells has not been considered. Here, we show that mice expressing Kir6.1-GOF mutations (Kir6.1[G343D] or Kir6.1[G343D,Q53R]) in pancreatic β cells (under rat-insulin-promoter [Rip] control) develop glucose intolerance and diabetes caused by reduced insulin secretion. We also generated transgenic mice in which a bacterial artificial chromosome (BAC) containing Kir6.1[G343D] is incorporated such that the transgene is only expressed in tissues where Kir6.1 is normally present. Strikingly, BAC-Kir6.1[G343D] mice also show impaired glucose tolerance, as well as reduced glucose- and sulfonylurea-dependent insulin secretion. However, the response to K+ depolarization is intact in Kir6.1-GOF mice compared with control islets. The presence of native Kir6.1 transcripts was demonstrated in both human and wild-type mouse islets using quantitative real-time PCR. Together, these results implicate the incorporation of native Kir6.1 subunits into pancreatic KATP channels and a contributory role for these subunits in the control of insulin secretion.

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Fructose-1,6-Bisphosphatase Regulates Glucose-Stimulated Insulin Secretion of Mouse Pancreatic β-Cells

Endocrinology ◽

10.1210/en.2009-1185 ◽

2010 ◽

Vol 151 (10) ◽

pp. 4688-4695 ◽

Cited By ~ 16

Author(s):

Ye Zhang ◽

Zhifang Xie ◽

Guangdi Zhou ◽

Hai Zhang ◽

Jian Lu ◽

...

Keyword(s):

Insulin Secretion ◽

Specific Inhibitor ◽

Physiological Role ◽

Glucose Sensing ◽

Β Cells ◽

Pancreatic Β Cells ◽

Min6 Cells ◽

Fructose 1,6 Bisphosphatase ◽

Glucose Stimulated Insulin Secretion

Pancreatic β-cells can precisely sense glucose stimulation and accordingly adjust their insulin secretion. Fructose-1,6-bisphosphatase (FBPase) is a gluconeogenic enzyme, but its physiological significance in β-cells is not established. Here we determined its physiological role in regulating glucose sensing and insulin secretion of β-cells. Considerable FBPase mRNA was detected in normal mouse islets and β-cell lines, although their protein levels appeared to be quite low. Down-regulation of FBP1 in MIN6 cells by small interfering RNA could enhance the glucose-stimulated insulin secretion (GSIS), whereas FBP1-overexpressing MIN6 cells exhibited decreased GSIS. Inhibition of FBPase activity in islet β-cells by its specific inhibitor MB05032 led to significant increase of their glucose utilization and cellular ATP to ADP ratios and consequently enhanced GSIS in vitro. Pretreatment of mice with the MB05032 prodrug MB06322 could potentiate GSIS in vivo and improve their glucose tolerance. Therefore, FBPase plays an important role in regulating glucose sensing and insulin secretion of β-cells and serves a promising target for diabetes treatment.

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Somatostatin Inhibits Oxidative Respiration in Pancreatic β-Cells

Endocrinology ◽

10.1210/en.2005-0873 ◽

2006 ◽

Vol 147 (3) ◽

pp. 1527-1535 ◽

Cited By ~ 34

Author(s):

Mathew Daunt ◽

Oliver Dale ◽

Paul A. Smith

Keyword(s):

Insulin Secretion ◽

Glucose Metabolism ◽

Somatostatin Receptor ◽

Katp Channels ◽

Β Cells ◽

Pancreatic Β Cells ◽

K Channels ◽

Min6 Cells ◽

Voltage Gated ◽

Mouse Islets

Somatostatin potently inhibits insulin secretion from pancreatic β-cells. It does so via activation of ATP-sensitive K+-channels (KATP) and G protein-regulated inwardly rectifying K+-channels, which act to decrease voltage-gated Ca2+-influx, a process central to exocytosis. Because KATP channels, and indeed insulin secretion, is controlled by glucose oxidation, we investigated whether somatostatin inhibits insulin secretion by direct effects on glucose metabolism. Oxidative metabolism in β-cells was monitored by measuring changes in the O2 consumption (ΔO2) of isolated mouse islets and MIN6 cells, a murine-derived β-cell line. In both models, glucose-stimulated ΔO2, an effect closely associated with inhibition of KATP channel activity and induction of electrical activity (r > 0.98). At 100 nm, somatostatin abolished glucose-stimulated ΔO2 in mouse islets (n = 5, P < 0.05) and inhibited it by 80 ± 28% (n = 17, P < 0.01) in MIN6 cells. Removal of extracellular Ca2+, 5 mm Co2+, or 20 μm nifedipine, conditions that inhibit voltage-gated Ca2+ influx, did not mimic but either blocked or reduced the effect of the peptide on ΔO2. The nutrient secretagogues, methylpyruvate (10 mm) and α-ketoisocaproate (20 mm), also stimulated ΔO2, but this was unaffected by somatostatin. Somatostatin also reversed glucose-induced hyperpolarization of the mitochondrial membrane potential monitored using rhodamine-123. Application of somatostatin receptor selective agonists demonstrated that the peptide worked through activation of the type 5 somatostatin receptor. In conclusion, somatostatin inhibits glucose metabolism in murine β-cells by an unidentified Ca2+-dependent mechanism. This represents a new signaling pathway by which somatostatin can inhibit cellular functions regulated by glucose metabolism.

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Trafficking of ATP-sensitive potassium channels in health and disease

Biochemical Society Transactions ◽

10.1042/bst0351055 ◽

2007 ◽

Vol 35 (5) ◽

pp. 1055-1059 ◽

Cited By ~ 10

Author(s):

A. Sivaprasadarao ◽

T.K. Taneja ◽

J. Mankouri ◽

A.J. Smith

Keyword(s):

Insulin Secretion ◽

Potassium Channels ◽

Cell Surface ◽

Genetic Diseases ◽

Neonatal Diabetes ◽

Katp Channels ◽

Severe Form ◽

Coated Vesicle ◽

Congenital Hyperinsulinism ◽

Sulfonylurea Receptor

KATP channels (ATP-sensitive potassium channels), comprising four subunits each of Kir6.2 (inwardly rectifying potassium channel 6.2) and the SUR1 (sulfonylurea receptor 1), play a central role in glucose-stimulated insulin secretion by the pancreatic β-cell. Changes in the number of channels at the cell surface are associated with genetic diseases of aberrant insulin secretion, including CHI (congenital hyperinsulinism) and NDM (neonatal diabetes mellitus). The present review summarizes advances in our understanding of the vesicular trafficking of normal KATP channels and how genetic mutations in Kir6.2 interfere with such trafficking. A mutation, E282K, causing CHI, was found to disrupt a DXE [di-acidic ER (endoplasmic reticulum)-exit signal], thereby preventing its assembly into COPII (coatamer protein II)-coated vesicles and subsequent ER exit. The resultant decrease in the cell-surface density of the channel could explain the disease phenotype. Two mutations, Y330C and F333I, reported in patients with NDM, disrupted an endocytic traffic signal, thereby impairing CCV (clathrin-coated vesicle) formation and endocytosis. The consequent increase in the density of KATP channels, together with an attenuated sensitivity to ATP reported previously, may account for the severe form of NDM.

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Both Triggering and Amplifying Pathways Contribute to Fuel-induced Insulin Secretion in the Absence of Sulfonylurea Receptor-1 in Pancreatic β-Cells

Journal of Biological Chemistry ◽

10.1074/jbc.m402076200 ◽

2004 ◽

Vol 279 (31) ◽

pp. 32316-32324 ◽

Cited By ~ 85

Author(s):

Myriam Nenquin ◽

Andras Szollosi ◽

Lydia Aguilar-Bryan ◽

Joseph Bryan ◽

Jean-Claude Henquin

Keyword(s):

Insulin Secretion ◽

Sulfonylurea Receptor ◽

Β Cells ◽

Pancreatic Β Cells ◽

Sulfonylurea Receptor 1

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Epinephrine-induced hyperpolarization of islet cells without KATP channels

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00365.2003 ◽

2004 ◽

Vol 286 (3) ◽

pp. E463-E471 ◽

Cited By ~ 30

Author(s):

Andrea Sieg ◽

Jiping Su ◽

Alvaro Muñoz ◽

Michael Buchenau ◽

Mitsuhiro Nakazaki ◽

...

Keyword(s):

Insulin Secretion ◽

Pertussis Toxin ◽

Action Potentials ◽

Islet Cells ◽

Katp Channels ◽

Sulfonylurea Receptor ◽

Β Cells ◽

Whole Cell ◽

Adrenoceptor Agonist ◽

Spontaneous Action

This study examines the effect of epinephrine, a known physiological inhibitor of insulin secretion, on the membrane potential of pancreatic islet cells from sulfonylurea receptor-1 (ABCC8)-null mice (Sur1KO), which lack functional ATP-sensitive K+ (KATP) channels. These channels have been argued to be activated by catecholamines, but epinephrine effectively inhibits insulin secretion in both Sur1KO and wild-type islets and in mice. Isolated Sur1KO β-cells are depolarized in both low (2.8 mmol/l) and high (16.7 mmol/l) glucose and exhibit Ca2+-dependent action potentials. Epinephrine hyperpolarizes Sur1KO β-cells, inhibiting their spontaneous action potentials. This effect, observed in standard whole cell patches, is abolished by pertussis toxin and blocked by BaCl2. The epinephrine effect is mimicked by clonidine, a selective α2-adrenoceptor agonist and inhibited by α-yohimbine, an α2-antagonist. A selection of K+ channel inhibitors, tetraethylammonium, apamin, dendrotoxin, iberiotoxin, E-4130, chromanol 293B, and tertiapin did not block the epinephrine-induced hyperpolarization. Analysis of whole cell currents revealed an inward conductance of 0.11 ± 0.04 nS/pF ( n = 7) and a TEA-sensitive outward conductance of 0.55 ± 0.08 nS/pF ( n = 7) at -60 and 0 mV, respectively. Guanosine 5′- O-(3-thiotriphosphate) (100 μM) in the patch pipette did not significantly alter these currents or activate novel inward-rectifying K+ currents. We conclude that epinephrine can hyperpolarize β-cells in the absence of KATP channels via activation of low-conductance BaCl2-sensitive K+ channels that are regulated by pertussis toxin-sensitive G proteins.

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Dextromethorphan stimulates insulin secretion from pancreatic β cells—a new role for an old drug?

Nature Reviews Endocrinology ◽

10.1038/nrendo.2015.49 ◽

2015 ◽

Vol 11 (5) ◽

pp. 253-253

Author(s):

Jennifer Sargent

Keyword(s):

Insulin Secretion ◽

Β Cells ◽

Pancreatic Β Cells

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Congenital hyperinsulinism due to compound heterozygous mutations in ABCC8 responsive to diazoxide therapy

Journal of Pediatric Endocrinology and Metabolism ◽

10.1515/jpem-2019-0457 ◽

2020 ◽

Vol 33 (5) ◽

pp. 671-674

Author(s):

Tashunka Taylor-Miller ◽

Jayne Houghton ◽

Paul Munyard ◽

Yadlapalli Kumar ◽

Clinda Puvirajasinghe ◽

...

Keyword(s):

Insulin Secretion ◽

Compound Heterozygous ◽

Congenital Hyperinsulinism ◽

Β Cells ◽

Pancreatic Β Cells ◽

Newborn Period ◽

Case Presentation ◽

Male Baby ◽

Common Causes ◽

Compound Heterozygous Mutations

AbstractBackgroundCongenital hyperinsulinism (CHI), a condition characterized by dysregulation of insulin secretion from the pancreatic β cells, remains one of the most common causes of hyperinsulinemic, hypoketotic hypoglycemia in the newborn period. Mutations in ABCC8 and KCNJ11 constitute the majority of genetic forms of CHI.Case presentationA term macrosomic male baby, birth weight 4.81 kg, born to non-consanguineous parents, presented on day 1 of life with severe and persistent hypoglycemia. The biochemical investigations confirmed a diagnosis of CHI. Diazoxide was started and progressively increased to 15 mg/kg/day to maintain normoglycemia. Sequence analysis identified compound heterozygous mutations in ABCC8 c.4076C>T and c.4119+1G>A inherited from the unaffected father and mother, respectively. The mutations are reported pathogenic. The patient is currently 7 months old with a sustained response to diazoxide.ConclusionsBiallelic ABCC8 mutations are known to result in severe, diffuse, diazoxide-unresponsive hypoglycemia. We report a rare patient with CHI due to compound heterozygous mutations in ABCC8 responsive to diazoxide.

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