Permanent neonatal diabetes: combining sulfonylureas with insulin may be an effective treatment.

BACKGROUND: Permanent neonatal diabetes caused by mutations in the KCNJ11 gene may be managed with high-dose sulfonylureas. Complete transfer to sulfonylureas is not successful in all cases and can result in insulin monotherapy. In such cases, the outcomes of combining sulfonylureas with insulin have not been fully explored. We present the case of a woman with diabetes due to a KCNJ11 mutation, in whom combination therapy led to clinically meaningful improvements. CASE: A 22-year-old woman was found to have a KCNJ11 mutation (G334V) following diagnosis with diabetes at 3 weeks. She was treated with insulin-pump therapy, had hypoglycaemia unawareness and suboptimal glycaemic control. We assessed the in vitro response of the mutant channel to tolbutamide in Xenopus oocytes and undertook sulfonylurea dose-titration with C-peptide assessment and continuous glucose monitoring. In vitro studies predicted the G334V mutation would be sensitive to sulfonylurea therapy [91 ± 2% block (n = 6) with 0.5 mM tolbutamide]. C-peptide increased following a glibenclamide test dose (from 5 to 410 pmol/l). Glibenclamide dose-titration was undertaken: a lower glibenclamide dose did not reduce blood glucose levels, but at 1.2 mg/kg/day insulin delivery was reduced to 0.1 units/h. However, when insulin was stopped, hyperglycaemia ensued. Glibenclamide was further increased (2 mg/kg/day), but once-daily long-acting insulin was still required to maintain glycaemia. This resulted in improved HbA1c of 52 mmol/mol (6.9%), restoration of hypoglycaemia awareness and reduced glycaemic variability. CONCLUSION: In people with KCNJ11 mutations causing permanent neonatal diabetes, and where complete transfer is not possible, consideration should be given to dual insulin and sulfonylurea therapy. This article is protected by copyright. All rights reserved.

ß-Cell dysfunction in diabetes: a crisis of identity?

Type 2 diabetes is characterized by insulin resistance and a progressive loss of ß-cell function induced by a combination of both ß-cell loss and impaired insulin secretion from remaining ß-cells. Here, we review the fate of the ß-cell under chronic hyperglycaemic conditions with regard to ß-cell mass, gene expression, hormone content, secretory capacity and the ability to de- or transdifferentiate into other cell types. We compare data from various in vivo and in vitro models of diabetes with a novel mouse model of inducible, reversible hyperglycaemia (ßV59M mice). We suggest that insulin staining using standard histological methods may not always provide an accurate estimation of ß-cell mass or number. We consider how ß-cell identity is best defined, and whether expression of transcription factors normally found in islet progenitor cells, or in α-cells, implies that mature ß-cells have undergone dedifferentiation or transdifferentiation. We propose that even in long-standing diabetes, ß-cells predominantly remain ß-cells-but not as we know them.

Mice expressing a human K(ATP) channel mutation have altered channel ATP sensitivity but no cardiac abnormalities.

AIMS/HYPOTHESIS: Patients with severe gain-of-function mutations in the Kir6.2 subunit of the ATP-sensitive potassium (K(ATP)) channel, have neonatal diabetes, muscle hypotonia and mental and motor developmental delay-a condition known as iDEND syndrome. However, despite the fact that Kir6.2 forms the pore of the cardiac K(ATP) channel, patients show no obvious cardiac symptoms. The aim of this project was to use a mouse model of iDEND syndrome to determine whether iDEND mutations affect cardiac function and cardiac K(ATP) channel ATP sensitivity. METHODS: We performed patch-clamp and in vivo cine-MRI studies on mice in which the most common iDEND mutation (Kir6.2-V59M) was targeted to cardiac muscle using Cre-lox technology (m-V59M mice). RESULTS: Patch-clamp studies of isolated cardiac myocytes revealed a markedly reduced K(ATP) channel sensitivity to MgATP inhibition in m-V59M mice (IC(50) 62 µmol/l compared with 13 µmol/l for littermate controls). In vivo cine-MRI revealed there were no gross morphological differences and no differences in heart rate, end diastolic volume, end systolic volume, stroke volume, ejection fraction, cardiac output or wall thickening between m-V59M and control hearts, either under resting conditions or under dobutamine stress. CONCLUSIONS/INTERPRETATION: The common iDEND mutation Kir6.2-V59M decreases ATP block of cardiac K(ATP) channels but was without obvious effect on heart function, suggesting that metabolic changes fail to open the mutated channel to an extent that affects function (at least in the absence of ischaemia). This may have implications for the choice of sulfonylurea used to treat neonatal diabetes.

Transporters, channels and human disease.

The past year has seen significant advances in our understanding of the molecular biology of ion channels and transporters and their role in human disease. The star of the show has to be the cystic fibrosis chloride channel about which an enormous amount of information has been accumulated, and the functional effects of some of the mutations found in cystic fibrosis patients have been characterized.

Inwardly rectifying potassium channels.

Inwardly rectifying potassium (Kir) channels regulate the resting membrane potential of the cell and thereby modulate the electrical activity of cardiac and neuronal cells, insulin secretion and epithelial K(+) transport. Considerable progress in understanding the molecular structure of Kir channels and the way in which they are regulated by extracellular and intracellular modulators has been made during the past year.

A mutation in KCNJ11 causing human hyperinsulinism (Y12X) results in a glucose-intolerant phenotype in the mouse.

AIMS/HYPOTHESIS: We identified a mouse with a point mutation (Y12STOP) in the Kcnj11 subunit of the K(ATP) channel. This point mutation is identical to that found in a patient with congenital hyperinsulinism of infancy (HI). We aimed to characterise the phenotype arising from this loss-of-function mutation and to compare it with that of other mouse models and patients with HI. METHODS: We phenotyped an N-ethyl-N-nitrosourea-induced mutation on a C3H/HeH background (Kcnj11 ( Y12STOP )) using intraperitoneal glucose tolerance testing to measure glucose and insulin plasma concentrations. Insulin secretion and response to incretins were measured on isolated islets. RESULTS: Homozygous male and female adult Kcnj11 ( Y12STOP ) mice exhibited impaired glucose tolerance and a defect in insulin secretion as measured in vivo and in vitro. Islets had an impaired incretin response and reduced insulin content. CONCLUSIONS/INTERPRETATION: The phenotype of homozygous Kcnj11 ( Y12STOP ) mice is consistent with that of other Kcnj11-knockout mouse models. In contrast to the patient carrying this mutation homozygously, the mice studied did not have hyperinsulinaemia or hypoglycaemia. It has been reported that HI patients may develop diabetes and our mouse model may reflect this clinical feature. The Kcnj11 ( Y12STOP ) model may thus be useful in further studies of K(ATP) channel function in various cell types and in investigation of the development of hyperglycaemia in HI patients.

The first clinical case of a mutation at residue K185 of Kir6.2 (KCNJ11): a major ATP-binding residue.

BACKGROUND: Closure of the adenosine triphosphate (ATP)-sensitive potassium (K(ATP)) channel plays a key role in insulin secretion from the pancreatic beta-cells. Many mutations in KCNJ11 and ABCC8, which respectively encode the pore-forming (Kir6.2) and regulatory (SUR1) subunits of the K(ATP) channel, cause neonatal diabetes. All such mutations impair the ability of metabolically generated ATP to close the channel. Although lysine 185 is predicted to be a major contributor to the ATP-binding site of Kir6.2, no mutations at this residue have been found to cause neonatal diabetes to date. METHODS: We report a 3-year-old girl with permanent neonatal diabetes (PNDM) caused by a novel heterozygous mutation (K185Q) at residue K185 of KCNJ11. The patient presented with marked hyperglycaemia and ketoacidosis at 70 days after birth, and insulin therapy was commenced. RESULTS: Wild-type and mutant K(ATP) channels were expressed in Xenopus oocytes and the effects of intracellular ATP on macroscopic K(ATP) currents in inside-out membrane patches were measured. In the simulated heterozygous state, the K185Q mutation caused a substantial reduction in the ability of MgATP to inhibit the channel. Heterozygous K185Q channels were still blocked effectively by the sulphonylurea tolbutamide. CONCLUSIONS: We report the first clinical case of a PNDM caused by a mutation at K185. Functional studies indicate that the K185Q mutation causes PNDM by reducing the ATP sensitivity of the K(ATP) channel, probably via a reduction in ATP binding to Kir6.2. Based on the experimental data, the patient was successfully transferred to sulphonylurea therapy.

Calcium dependence of the inactivation of calcium currents in skeletal muscle fibers of an insect.

Calcium currents in skeletal muscle fibers of an insect, Carausius morosus, inactivate under depolarization. This inactivation depends on the current being carried across the membrane by calcium ions, rather than strontium or bariumions.

Variable phenotypic spectrum of diabetes mellitus in a family carrying a novel KCNJ11 gene mutation.

AIMS: Heterozygous activating mutations in KCNJ11, which encodes the Kir6.2 subunit of the pancreatic ATP-sensitive potassium (K(ATP)) channel, cause both permanent and transient neonatal diabetes. Identification of KCNJ11 mutations has important therapeutic implications, as many patients can replace insulin injections with sulphonylurea tablets. The aim was to determine if a KCNJ11 mutation was responsible for a dominantly inherited form of diabetes mellitus, showing variability in age at diagnosis, in an Italian family. METHODS: We sequenced KCNJ11 in members of a three-generation family with variable phenotypes of dominantly inherited diabetes mellitus. One had transient early-onset diabetes, one had impaired glucose tolerance during the second pregnancy, and two had young-onset diabetes. None of the subjects showed permanent neonatal diabetes or neurological symptoms. RESULTS: A novel heterozygous mutation (c. 679C-->G and c. 680A-->T) was identified, resulting in a GAG-->CTG (E227L) substitution in KCNJ11. Functional studies of recombinant heterozygous K(ATP) channels revealed a small reduction in channel inhibition by ATP (IC(50) of 15 micromol/l and 38 micromol/l for wild-type and heterozygous channels, respectively) and an increase in the resting K(ATP) current. This would be expected to impair insulin secretion. The results are in agreement with the mild phenotype of the patients. CONCLUSIONS: Our results broaden the spectrum of diabetes phenotypes resulting from KCNJ11 mutations. They indicate testing for KCNJ11 mutations should be considered not only for neonatal diabetes but also for other forms of dominantly inherited diabetes with later onset, especially where these are associated with a low body mass index and low birth weight.

Correlating structure and function in ATP-sensitive K+ channels.

ATP-sensitive K+ channels couple cell metabolism to electrical activity in nerve, muscle and endocrine cells, and play important roles in these tissues under both physiological and pathological conditions. The KATP channel is an octameric complex of two unrelated types of subunit: a pore-forming subunit (Kir6.2) and a regulatory subunit, the sulphonylurea receptor (SUR). This review focuses on the regulation of KATP channel activity by nucleotides and cell metabolism and considers which regulatory mechanisms are intrinsic to Kir6.2 and which are conferred by association with SUR.

A touching case of channel regulation: the ATP-sensitive K+ channel.

The classical type of KATP channel is an octameric (4:4) complex of two structurally unrelated subunits, Kir6.2 and SUR. The former serves as an ATP-inhibitable pore, while SUR is a regulatory subunit endowing sensitivity to sulphonylurea and K+ channel opener drugs, and the potentiatory action of MgADP. Both subunits are required to form a functional channel.

Behavioral phenotyping of mice lacking the K ATP channel subunit Kir6.2.

ATP-sensitive potassium (K(ATP)) channels are expressed in various tissues and cell-types where they act as so-called metabolic sensors that couple metabolic state to cellular excitability. The pore of most K(ATP) channel types is built by Kir6.2 subunits. Analysis of a general Kir6.2 knockout (KO) mouse has identified a variety of different functional roles for central and peripheral K(ATP) channels in situations of metabolic demand. However, the widespread distribution of these channels suggests that they might influence cellular physiology and animal behavior under metabolic control conditions. As a comprehensive behavioral description of Kir6.2 KO mice under physiological control conditions has not yet been carried out, we subjected Kir6.2 KO and corresponding wild-type (WT) mice to a test battery to assess emotional behavior, motor activity and coordination, species-typical behaviors and cognition. The results indicated that in these test situations Kir6.2 KO mice were less active, had impaired motor coordination, and appeared to differ from controls in their emotional reactivity. Differences between KO and WT mice were generally attenuated in test situations that resembled the home cage environment. Moreover, in their home cages KO mice were more active than WT mice. Thus, our results suggest that loss of Kir6.2-containing K(ATP) channels does affect animal behavior under metabolic control conditions, especially in novel situations. These findings assign novel functional roles to K(ATP) channels beyond those previously described. However, according to the widespread expression of K(ATP) channels, these effects are complex, being dependent on details of test apparatus, procedure and prior experience.

New windows on the mechanism of action of K(ATP) channel openers.

K(ATP) channel openers are a diverse group of drugs with a wide range of potential therapeutic uses. Their molecular targets, the K(ATP) channels, exhibit tissue-specific responses because they possess different types of regulatory sulfonylurea receptor subunits. It is well recognized that complex interactions occur between K(ATP) channel openers and nucleotides, but the cloning of the K(ATP) channel has introduced a new dimension to the study of these events and has furthered our understanding of the molecular basis of the action of K(ATP) channel openers.

Structural basis for the interference between nicorandil and sulfonylurea action.

Nicorandil is a new antianginal agent that potentially may be used to treat the cardiovascular side effects of diabetes. It is both a nitric oxide donor and an opener of ATP-sensitive K(+) (K(ATP)) channels in muscle and thereby causes vasodilation of the coronary vasculature. The aim of this study was to investigate the domains of the K(ATP) channel involved in nicorandil activity and to determine whether nicorandil interacts with hypoglycemic sulfonylureas that target K(ATP) channels in pancreatic beta-cells. K(ATP) channels in muscle and beta-cells share a common pore-forming subunit, Kir6.2, but possess alternative sulfonylurea receptors (SURs; SUR1 in beta-cells, SUR2A in cardiac muscle, and SUR2B in smooth muscle). We expressed recombinant K(ATP) channels in Xenopus oocytes and measured the effects of drugs and nucleotides by recording macroscopic currents in excised membrane patches. Nicorandil activated Kir6.2/SUR2A and Kir6.2/SUR2B but not Kir6.2/SUR1 currents, consistent with its specificity for cardiac and smooth muscle K(ATP) channels. Drug activity depended on the presence of intracellular nucleotides and was impaired when the Walker A lysine residues were mutated in either nucleotide-binding domain of SUR2. Chimeric studies showed that the COOH-terminal group of transmembrane helices (TMs), especially TM 17, is responsible for the specificity of nicorandil for channels containing SUR2. The splice variation between SUR2A and SUR2B altered the off-rate of the nicorandil response. Finally, we showed that nicorandil activity was unaffected by gliclazide, which specifically blocks SUR1-type K(ATP) channels, but was severely impaired by glibenclamide and glimepiride, which target both SUR1 and SUR2-type K(ATP) channels.

Tissue specificity of sulfonylureas: studies on cloned cardiac and beta-cell K(ATP) channels.

Sulfonylureas stimulate insulin secretion from pancreatic beta-cells by closing ATP-sensitive K+ (K(ATP)). The beta-cell and cardiac muscle K(ATP) channels have recently been cloned and shown to possess a common pore-forming subunit (Kir6.2) but different sulfonylurea receptor subunits (SUR1 and SUR2A, respectively). We examined the mechanism underlying the tissue specificity of the sulfonylureas tolbutamide and glibenclamide, and the benzamido-derivative meglitinide, using cloned beta-cell (Kir6.2/SUR1) and cardiac (Kir6.2/SUR2A) K(ATP) channels expressed in Xenopus oocytes. Tolbutamide inhibited Kir6.2/SUR1 (Ki approximately 5 micromol/l), but not Kir6.2/SUR2A, currents with high affinity. Meglitinide produced high-affinity inhibition of both Kir6.2/SUR1 and Kir6.2/SUR2A currents (Kis approximately 0.3 micromol/l and approximately 0.5 micromol/l, respectively). Glibenclamide also blocked Kir6.2/SUR1 and Kir6.2/SUR2A currents with high affinity (Kis approximately 4 nmol/l and approximately 27 nmol/l, respectively); however, only for cardiac-type K(ATP) channels was this block reversible. Physiological concentrations of MgADP (100 micromol/l) enhanced glibenclamide inhibition of Kir6.2/SUR1 currents but reduced that of Kir6.2/SUR2A currents. The results suggest that SUR1 may possess separate high-affinity binding sites for sulfonylurea and benzamido groups. SUR2A, however, either does not possess a binding site for the sulfonylurea group or is unable to translate the binding at this site into channel inhibition. Although MgADP reduces the inhibitory effect of glibenclamide on cardiac-type K(ATP) channels, drugs that bind to the common benzamido site have the potential to cause side effects on the heart.

Identification of the high-affinity tolbutamide site on the SUR1 subunit of the K(ATP) channel.

ATP-sensitive potassium channels (K(ATP)) are formed from four pore-forming Kir6.2 subunits complexed with four regulatory sulfonylurea receptor subunits (SUR1 in pancreatic beta-cells, SUR2A in heart). The sensitivity of the channel to different sulfonylureas depends on the SUR isoform. In particular, Kir6.2-SUR1 but not Kir6.2-SUR2A channels are blocked by tolbutamide with high affinity. We made chimeras between SUR1 and SUR2A to identify the region of the protein involved in high-affinity tolbutamide block. Chimeric SURs were coexpressed with Kir6.2 in Xenopus oocytes, and macroscopic currents were measured in inside-out membrane patches. High-affinity tolbutamide inhibition could be conferred on SUR2A by replacing transmembrane domains (TMs) 14-16 with the corresponding region of SUR1. Conversely, high-affinity tolbutamide inhibition of SUR1 was abolished by replacing TMs 13-16 with the corresponding SUR2A sequence, or by mutating a single serine residue within this region to tyrosine (S1237Y). Binding of [3H]glibenclamide to membranes expressing SUR1 was abolished concomitantly with the loss of high-affinity tolbutamide block. These results suggest that a site in the COOH-terminal set of TMs of the SUR1 subunit of the K(ATP) channel is involved in the binding of tolbutamide and glibenclamide.

Electrophysiological and metabolic characterization of single beta-cells and islets from diabetic GK rats.

We have used the whole-cell recording technique to determine whether ATP-sensitive potassium (K[ATP]) currents, voltage-dependent Ca2+ currents, and exocytosis are different in single beta-cells from pancreatic islets of Goto-Kakizaki (GK) rats, a novel model of NIDDM, and normal rats. In addition, we have also measured the insulin secretory responses, ATP content, and the rate of glucose metabolism in intact islets. Although the glucose sensitivity of the K(ATP) current was similar between GK rats and controls, in the absence of glucose, K(ATP) current density was larger in GK rats, which resulted in a more hyperpolarized membrane potential. Whole-cell Ca2+ currents were similar. By monitoring the cell capacitance with a fixed intracellular solution, no difference was detected in the exocytotic responses of beta-cells from normal and GK rats. In islets from GK rats, the rates of glucose utilization ([3H]H2O production from 5-[3H]glucose) and oxidation ([14C]CO2 production from U-[14C]glucose) were not significantly different from controls. Insulin secretion, however, was impaired (by 50%), and this was paralleled by a smaller increase in ATP content in response to stimulation by 10 mmol/l glucose in islets from GK rats when compared with controls. Under conditions in which K(ATP) channels were held open and the effects of glucose were independent of membrane potential, insulin release was still significantly lower in GK rat islets than in controls. These findings suggest that the impaired insulin secretion in islets from GK rats does not simply result from a failure to close K(ATP) channels, nor does it result from an impairment in calcium secretion coupling.

No evidence for mutations in a putative beta-cell ATP-sensitive K+ channel subunit in MODY, NIDDM, or GDM.

The beta-cell ATP-sensitive K+ (K-ATP) channel has a major role in glucose-induced insulin secretion. Screening the entire coding sequence of the gene for a putative beta-cell K-ATP channel subunit, K-ATP2, with single-strand conformation polymorphism did not show any mutations associated with diabetes in white Caucasian diabetic patients, including five pedigrees with maturity onset diabetes of the young (MODY), 25 patients with noninsulin-dependent diabetes mellitus (NIDDM) selected for marked beta-cell deficiency, 25 selected for mild diabetes presenting before age 50 years with fasting plasma glucose levels < 10 mmol/l, 25 unselected NIDDM patients, and 25 subjects with gestational diabetes mellitus (GDM) and subsequent raised fasting plasma glucose. In five large MODY pedigrees, linkage analysis with simple tandem-repeat polymorphisms (STRPs) near the K-ATP2 gene excluded linkage. In a population association study, no linkage disequilibrium for the STRP was found between 237 unselected white Caucasian NIDDM patients and 104 geographically matched and age-matched white Caucasian nondiabetic subjects. In addition, two silent polymorphisms were found with similar frequency in nondiabetic and diabetic subjects. Mutations in the gene for K-ATP2 are unlikely to be a major cause of MODY, NIDDM, or GDM.