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Aims/hypothesis: The ATP-sensitive potassium (K ATP ) channel couples beta cell electrical activity to glucose-stimulated insulin secretion. Loss-of-function mutations in either the pore-forming (inwardly rectifying potassium channel 6.2 [Kir6.2], encoded by KCNJ11) or regulatory (sulfonylurea receptor 1, encoded by ABCC8) subunits result in congenital hyperinsulinism, whereas gain-of-function mutations cause neonatal diabetes. Here, we report a novel loss-of-function mutation (Ser118Leu) in the pore helix of Kir6.2 paradoxically associated with sulfonylurea-sensitive diabetes that presents in early adult life., Methods: A 31-year-old woman was diagnosed with mild hyperglycaemia during an employee screen. After three pregnancies, during which she was diagnosed with gestational diabetes, the patient continued to show elevated blood glucose and was treated with glibenclamide (known as glyburide in the USA and Canada) and metformin. Genetic testing identified a heterozygous mutation (S118L) in the KCNJ11 gene. Neither parent was known to have diabetes. We investigated the functional properties and membrane trafficking of mutant and wild-type K ATP channels in Xenopus oocytes and in HEK-293T cells, using patch-clamp, two-electrode voltage-clamp and surface expression assays., Results: Functional analysis showed no changes in the ATP sensitivity or metabolic regulation of the mutant channel. However, the Kir6.2-S118L mutation impaired surface expression of the K ATP channel by 40%, categorising this as a loss-of-function mutation., Conclusions/interpretation: Our data support the increasing evidence that individuals with mild loss-of-function K ATP channel mutations may develop insulin deficiency in early adulthood and even frank diabetes in middle age. In this case, the patient may have had hyperinsulinism that escaped detection in early life. Our results support the importance of functional analysis of K ATP channel mutations in cases of atypical diabetes., (© 2024. The Author(s).)

Aims/hypothesis: MODY can be wrongly diagnosed as type 1 diabetes in children. We aimed to find the prevalence of MODY in a nationwide population-based registry of childhood diabetes., Methods: Using next-generation sequencing, we screened the HNF1A, HNF4A, HNF1B, GCK and INS genes in all 469 children (12.1%) negative for both GAD and IA-2 autoantibodies and 469 antibody-positive matched controls selected from the Norwegian Childhood Diabetes Registry (3882 children). Variants were classified using clinical diagnostic criteria for pathogenicity ranging from class 1 (neutral) to class 5 (pathogenic)., Results: We identified 58 rare exonic and splice variants in cases and controls. Among antibody-negative patients, 6.5% had genetic variants of classes 3-5 (vs 2.4% in controls; p = 0.002). For the stricter classification (classes 4 and 5), the corresponding number was 4.1% (vs 0.2% in controls; p = 1.6 × 10 -5 ). HNF1A showed the strongest enrichment of class 3-5 variants, with 3.9% among antibody-negative patients (vs 0.4% in controls; p = 0.0002). Antibody-negative carriers of variants in class 3 had a similar phenotype to those carrying variants in classes 4 and 5., Conclusions/interpretation: This is the first study screening for MODY in all antibody-negative children in a nationwide population-based registry. Our results suggest that the prevalence of MODY in antibody-negative childhood diabetes may reach 6.5%. One-third of these MODY cases had not been recognised by clinicians. Since a precise diagnosis is important for treatment and genetic counselling, molecular screening of all antibody-negative children should be considered in routine diagnostics.

Aims/hypothesis: The pancreatic ATP-sensitive potassium (KATP) channel plays a pivotal role in linking beta cell metabolism to insulin secretion. Mutations in KATP channel genes can result in hypo- or hypersecretion of insulin, as in neonatal diabetes mellitus and congenital hyperinsulinism, respectively. To date, all patients affected by neonatal diabetes due to a mutation in the pore-forming subunit of the channel (Kir6.2, KCNJ11) are heterozygous for the mutation. Here, we report the first clinical case of neonatal diabetes caused by a homozygous KCNJ11 mutation., Methods: A male patient was diagnosed with diabetes shortly after birth. At 5 months of age, genetic testing revealed he carried a homozygous KCNJ11 mutation, G324R, (Kir6.2-G324R) and he was successfully transferred to sulfonylurea therapy (0.2 mg kg(-1) day(-1)). Neither heterozygous parent was affected. Functional properties of wild-type, heterozygous and homozygous mutant KATP channels were examined after heterologous expression in Xenopus oocytes., Results: Functional studies indicated that the Kir6.2-G324R mutation reduces the channel ATP sensitivity but that the difference in ATP inhibition between homozygous and heterozygous channels is remarkably small. Nevertheless, the homozygous patient developed neonatal diabetes, whereas the heterozygous parents were, and remain, unaffected. Kir6.2-G324R channels were fully shut by the sulfonylurea tolbutamide, which explains why the patient's diabetes was well controlled by sulfonylurea therapy., Conclusions/interpretation: The data demonstrate that tiny changes in KATP channel activity can alter beta cell electrical activity and insulin secretion sufficiently to cause diabetes. They also aid our understanding of how the Kir6.2-E23K variant predisposes to type 2 diabetes.

Aims/hypothesis: The finding that patients with diabetes due to potassium channel mutations can transfer from insulin to sulfonylureas has revolutionised the management of patients with permanent neonatal diabetes. The extent to which the in vitro characteristics of the mutation can predict a successful transfer is not known. Our aim was to identify factors associated with successful transfer from insulin to sulfonylureas in patients with permanent neonatal diabetes due to mutations in KCNJ11 (which encodes the inwardly rectifying potassium channel Kir6.2)., Methods: We retrospectively analysed clinical data on 127 patients with neonatal diabetes due to KCNJ11 mutations who attempted to transfer to sulfonylureas. We considered transfer successful when patients completely discontinued insulin whilst on sulfonylureas. All unsuccessful transfers received ≥0.8 mg kg(-1) day(-1) glibenclamide (or the equivalent) for >4 weeks. The in vitro response of mutant Kir6.2/SUR1 channels to tolbutamide was assessed in Xenopus oocytes. For some specific mutations, not all individuals carrying the mutation were able to transfer successfully; we therefore investigated which clinical features could predict a successful transfer., Results: In all, 112 out of 127 (88%) patients successfully transferred to sulfonylureas from insulin with an improvement in HbA1c from 8.2% (66 mmol/mol) on insulin, to 5.9% (41 mmol/mol) on sulphonylureas (p = 0.001). The in vitro response of the mutation to tolbutamide determined the likelihood of transfer: the extent of tolbutamide block was <63% for the p.C166Y, p.I296L, p.L164P or p.T293N mutations, and no patients with these mutations successfully transferred. However, most individuals with mutations for which tolbutamide block was >73% did transfer successfully. The few patients with these mutations who could not transfer had a longer duration of diabetes than those who transferred successfully (18.2 vs 3.4 years, p = 0.032). There was no difference in pre-transfer HbA1c (p = 0.87), weight-for-age z scores (SD score; p = 0.12) or sex (p = 0.17)., Conclusions/interpretation: Transfer from insulin is successful for most KCNJ11 patients and is best predicted by the in vitro response of the specific mutation and the duration of diabetes. Knowledge of the specific mutation and of diabetes duration can help predict whether successful transfer to sulfonylureas is likely. This result supports the early genetic testing and early treatment of patients with neonatal diabetes aged under 6 months.

Aims/hypothesis: Individuals with heterozygous activating mutations of the KCNJ11 gene encoding a subunit of the ATP-sensitive potassium channel (KATP) can usually be treated with oral sulfonylurea (SU) pills in lieu of insulin injections. The aim of this study was to test our hypothesis that younger age at the time of initiation of SU therapy is correlated with lower required doses of SU therapy, shorter transition time and decreased likelihood of requiring additional diabetes medications., Methods: We performed a retrospective cohort study using data on 58 individuals with neonatal diabetes due to KCNJ11 mutations identified through the University of Chicago Monogenic Diabetes Registry ( http://monogenicdiabetes.uchicago.edu/registry ). We assessed the influence of age at initiation of SU therapy on treatment outcomes., Results: HbA1c fell from an average of 8.5% (69 mmol/mol) before transition to 6.2% (44 mmol/mol) after SU therapy (p < 0.001). Age of initiation of SU correlated with the dose (mg kg(-1) day(-1)) of SU required at follow-up (r = 0.80, p < 0.001). Similar associations were observed across mutation subtypes. Ten participants required additional glucose-lowering medications and all had initiated SU at age 13 years or older. No serious adverse events were reported., Conclusions/interpretation: Earlier age at initiation of SU treatment is associated with improved response to SU therapy. Declining sensitivity to SU may be due to loss of beta cell mass over time in those treated with insulin. Our data support the need for early genetic diagnosis and appropriate personalised treatment in all cases of neonatal diabetes.

Aims/hypothesis: More than 90% of Chinese familial early-onset type 2 diabetes mellitus is genetically unexplained. To investigate the molecular aetiology, we identified and characterised whether mutations in the KCNJ11 gene are responsible for these families., Methods: KCNJ11 mutations were screened for 96 familial early-onset type 2 diabetic probands and their families. Functional significance of the identified mutations was confirmed by physiological analysis, molecular modelling and population survey., Results: Three novel KCNJ11 mutations, R27H, R192H and S116F117del, were identified in three families with early-onset type 2 diabetes mellitus. Mutated KCNJ11 with R27H or R192H markedly reduced ATP sensitivity (E23K>R27H>C42R>R192H>R201H), but no ATP-sensitive potassium channel currents were detected in the loss-of-function S116F117del channel in vitro. Molecular modelling indicated that R192H had a larger effect on the channel ATP-binding pocket than R27H, which may qualitatively explain why the ATP sensitivity of the R192H mutation is seven times less than R27H. The shape of the S116F117del channel may be compressed, which may explain why the mutated channel had no currents. Discontinuation of insulin and implementation of sulfonylureas for R27H or R192H carriers and continuation/switch to insulin therapy for S116F117del carriers resulted in good glycaemic control., Conclusions/interpretation: Our results suggest that genetic diagnosis for the KCNJ11 mutations in familial early-onset type 2 diabetes mellitus may help in understanding the molecular aetiology and in providing more personalised treatment for these specific forms of diabetes in Chinese and other Asian patients.

Aims/hypothesis: Monogenic diabetes (MD) might be misdiagnosed as type 1 diabetes. The prevalence of MD among children with apparent type 1 diabetes has not been established. Our aim was to estimate the prevalence of common forms of MD in childhood diabetes., Methods: We investigated 2,756 children aged 0-14 years with newly diagnosed diabetes who had been recruited to the nationwide population-based Norwegian Childhood Diabetes Registry (NCDR), from July 2002 to March 2012. Completeness of ascertainment was 91%. Children diagnosed with diabetes who were under12 months of age were screened for mutations in KCNJ11, ABCC8 and INS. Children without GAD and protein tyrosine phosphatase-like protein antibodies were screened in two ways. Those who had a parent with diabetes were screened for mutations in HNF1A, HNF4A, INS and MT-TL1. Children with HbA1c <7.5% (<58 mmol/mol) and no insulin requirement were screened for mutations in GCK. Finally, we searched the Norwegian MODY Registry for children with genetically verified MD., Results: We identified 15 children harbouring a mutation in HNF1A, nine with one in GCK, four with one in KCNJ11, one child with a mutation in INS and none with a mutation in MT-TL1. The minimum prevalence of MD in the NCDR was therefore 1.1%. By searching the Norwegian MODY Registry, we found 24 children with glucokinase-MODY, 15 of whom were not present in the NCDR. We estimated the minimum prevalence of MD among Norwegian children to be 3.1/100,000., Conclusions/interpretation: This is the first prevalence study of the common forms of MD in a nationwide, population-based registry of childhood diabetes. We found that 1.1% of patients in the Norwegian Childhood Diabetes Registry had MD.

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.

Aims/hypothesis: The ABCC8 gene encodes the sulfonylurea receptor 1 (SUR1) subunit of the pancreatic beta cell ATP-sensitive potassium (K(ATP)) channel. Inactivating mutations cause congenital hyperinsulinism (CHI) and activating mutations cause transient neonatal diabetes (TNDM) or permanent neonatal diabetes (PNDM) that can usually be treated with sulfonylureas. Sulfonylurea sensitivity is also a feature of HNF1A and HNF4A MODY, but patients referred for genetic testing with clinical features of these types of diabetes do not always have mutations in the HNF1A/4A genes. Our aim was to establish whether mutations in the ABCC8 gene cause MODY that is responsive to sulfonylurea therapy., Methods: We sequenced the ABCC8 gene in 85 patients with a BMI <30 kg/m², no family history of neonatal diabetes and who were deemed sensitive to sulfonylureas by the referring clinician or were sulfonylurea-treated. All had tested negative for mutations in the HNF1A and HNF4A genes., Results: ABCC8 mutations were found in seven of the 85 (8%) probands. Four patients were heterozygous for previously reported mutations and four novel mutations, E100K, G214R, Q485R and N1245D, were identified. Only four probands fulfilled MODY criteria, with two diagnosed after 25 years and one patient, who had no family history of diabetes, as a result of a proven de novo mutation., Conclusions/interpretation: ABCC8 mutations can cause MODY in patients whose clinical features are similar to those with HNF1A/4A MODY. Therefore, sequencing of ABCC8 in addition to the known MODY genes should be considered if such features are present, to facilitate optimal clinical management of these patients.

Aims/hypothesis: Dominantly acting loss-of-function mutations in the ABCC8/KCNJ11 genes can cause mild medically responsive hyperinsulinaemic hypoglycaemia (HH). As controversy exists over whether these mutations predispose to diabetes in adulthood we investigated the prevalence of diabetes in families with dominantly inherited ATP-sensitive potassium (K(ATP)) channel mutations causing HH in the proband., Methods: We studied the phenotype of 30 mutation carriers (14 children and 16 adults) from nine families with dominant ABCC8/KCNJ11 mutations. Functional consequences of six novel missense mutations were examined by reconstituting the K(ATP) channel in human embryonic kidney 293 (HEK293) cells and evaluating the effect of drugs and metabolic poisoning on the channels using the (86)Rb flux assay., Results: The mutant channels all showed a lack of (86)Rb efflux on exposure to the channel agonist diazoxide or metabolic inhibition. In the families, dominant ABCC8/KCNJ11 mutations were associated with increased birthweight (median + 1.56 SD score [SDS]). Fourteen children had HH and five adults were reported with HH or hypoglycaemic episodes (63%). Progression from hypoglycaemia to diabetes mellitus occurred in two individuals. Eight adults had a history of gestational diabetes in multiple pregnancies or were diabetic (diagnosed at a median age of 31 years). Within these families, none of the 19 adults who were not carriers of the ABCC8/KCNJ11 mutation was known to be diabetic., Conclusions/interpretation: The phenotype associated with dominant ABCC8/KCNJ11 mutations ranges from asymptomatic macrosomia to persistent HH in childhood. In adults, it may also be an important cause of dominantly inherited early-onset diabetes mellitus.

Aims/hypothesis: The aim of this study was to investigate the genetic aetiology of permanent diabetes mellitus with onset in the first 12 months of age., Methods: We studied 46 probands with permanent, insulin-requiring diabetes with onset within the first 6 months of life (permanent neonatal diabetes mellitus [PNDM]/monogenic diabetes of infancy [MDI]) (group 1) and eight participants with diabetes diagnosed between 7 and 12 months of age (group 2). KCNJ11, INS and ABCC8 genes were sequentially sequenced in all patients. For those who were negative in the initial screening, we examined ERN1, CHGA, CHGB and NKX6-1 genes and, in selected probands, CACNA1C, GCK, FOXP3, NEUROG3 and CDK4. The incidence rate for PNDM/MDI was calculated using a database of Italian patients collected from 1995 to 2009., Results: In group 1 we found mutations in KCNJ11, INS and ABCC8 genes in 23 (50%), 9 (19.5%) and 4 (8.6%) patients respectively, and a single homozygous mutation in GCK (2.1%). In group 2, we identified one incidence of a KCNJ11 mutation. No genetic defects were detected in other loci. The incidence rate of PNDM/MDI in Italy is estimated to be 1:210,287., Conclusions/interpretation: Genetic mutations were identified in ~75% of non-consanguineous probands with PNDM/MDI, using sequential screening of KCNJ11, INS and ABCC8 genes in infants diagnosed within the first 6 months of age. This percentage decreased to 12% in those with diabetes diagnosed between 7 and 12 months. Patients belonging to the latter group may either carry mutations in genes different from those commonly found in PNDM/MDI or have developed an early-onset form of autoimmune diabetes.

Aims/hypothesis: Mutations that render ATP-sensitive potassium (K(ATP)) channels insensitive to ATP inhibition cause neonatal diabetes mellitus. In mice, these mutations cause insulin secretion to be lost initially and, as the disease progresses, beta cell mass and insulin content also disappear. We investigated whether defects in calcium signalling alone are sufficient to explain short-term and long-term islet dysfunction., Methods: We examined the metabolic, electrical and insulin secretion response in islets from mice that become diabetic after induction of ATP-insensitive Kir6.2 expression. To separate direct effects of K(ATP) overactivity on beta cell function from indirect effects of prolonged hyperglycaemia, normal glycaemia was maintained by protective exogenous islet transplantation., Results: In endogenous islets from protected animals, glucose-dependent elevations of intracellular free-calcium activity ([Ca(2+)](i)) were severely blunted. Insulin content of these islets was normal, and sulfonylureas and KCl stimulated increased [Ca(2+)](i). In the absence of transplant protection, [Ca(2+)](i) responses were similar, but glucose metabolism and redox state were dramatically altered; sulfonylurea- and KCl-stimulated insulin secretion was also lost, because of systemic effects induced by long-term hyperglycaemia and/or hypoinsulinaemia. In both cases, [Ca(2+)](i) dynamics were synchronous across the islet. After reduction of gap-junction coupling, glucose-dependent [Ca(2+)](i) and insulin secretion was partially restored, indicating that excitability of weakly expressing cells is suppressed by cells expressing mutants, via gap-junctions., Conclusions/interpretation: The primary defect in K(ATP)-induced neonatal diabetes mellitus is failure of glucose metabolism to elevate [Ca(2+)](i), which suppresses insulin secretion and mildly alters islet glucose metabolism. Loss of insulin content and mitochondrial dysfunction are secondary to the long-term hyperglycaemia and/or hypoinsulinaemia that result from the absence of glucose-dependent insulin secretion.

Aims/hypothesis: Of the confirmed type 2 diabetes susceptibility loci only a few are known to affect insulin sensitivity. We examined the association of indices of hepatic and adipocyte insulin resistance (IR) with 19 confirmed type 2 diabetes risk loci in a large population-based study., Methods: Non-diabetic participants (n = 8,460, age 57.3 ± 7.0 years, BMI 26.8 ± 3.8 kg/m(2); mean ± SD) from a population-based cohort underwent an OGTT. Of them, 6,733 non-diabetic men were genotyped for single nucleotide polymorphisms (SNPs) in or near PPARG2 (also known as PPARG), KCNJ11, TCF7L2, SLC30A8, HHEX, CDKN2B, IGF2BP2, CDKAL1, HNF1B, WFS1, JAZF1, CDC123, TSPAN8, THADA, ADAMTS9, NOTCH2, KCNQ1, MTNR1B and SNP rs7480010. We investigated hepatic IR with a new index of liver IR. The adipocyte IR index was defined as a product of fasting NEFA and plasma insulin levels., Results: Type 2 diabetes risk SNPs in or near KCNJ11 and HHEX were significantly (p < 0.0013), and those in or near CDKN2B, NOTCH2 and MTNR1B were nominally (p < 0.05), associated with decreased liver IR index. The Pro12 allele of PPARG2 was significantly associated with a high adipocyte IR index and nominally associated with high liver IR., Conclusions/interpretation: The Pro12 allele of PPARG2 seems to impair insulin's antilipolytic effect, leading to high NEFA release in the fasting state and IR. In addition, the type 2 diabetes risk alleles of KCNJ11 and HHEX, which are known to impair insulin secretion, were associated with increased hepatic insulin sensitivity.

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.

Aims/hypothesis: Genetic mapping has identified over 20 loci contributing to genetic risk of type 2 diabetes. The next step is to identify the genes and mechanisms regulating the contributions of genetic risk to disease. The goal of this study was to evaluate the effect of age, height, weight and risk alleles on expression of candidate genes in diabetes-associated regions in three relevant human tissues., Methods: We measured transcript abundance for WFS1, KCNJ11, TCF2 (also known as HNF1B), PPARG, HHEX, IDE, CDKAL1, CDKN2A, CDKN2B, IGF2BP2, SLC30A8 and TCF7L2 by quantitative RT-PCR in human pancreas (n = 50), colon (n = 195) and liver (n = 50). Tissue samples were genotyped for single nucleotide polymorphisms (SNPs) associated with type 2 diabetes. The effects of age, height, weight, tissue and SNP on RNA expression were tested by linear modelling., Results: Expression of all genes exhibited tissue bias. Immunohistochemistry confirmed the findings for HHEX, IDE and SLC30A8, which showed strongest tissue-specific mRNA expression bias. Neither age, height nor weight were associated with gene expression. We found no evidence that type 2 diabetes-associated SNPs affect neighbouring gene expression (cis-expression quantitative trait loci) in colon, pancreas and liver., Conclusions/interpretation: This study provides new evidence that tissue-type, but not age, height, weight or SNPs in or near candidate genes associated with increased risk of type 2 diabetes are strong contributors to differential gene expression in the genes and tissues examined.

Aims/hypothesis: We have previously reported that glucose-stimulated insulin secretion (GSIS) is induced by glucagon-like peptide-1 (GLP-1) in mice lacking ATP-sensitive K(+) (K(ATP)) channels (Kir6.2(-/-) mice [up-to-date symbol for Kir6.2 gene is Kcnj11]), in which glucose alone does not trigger insulin secretion. This study aimed to clarify the mechanism involved in the induction of GSIS by GLP-1., Methods: Pancreas perfusion experiments were performed using wild-type (Kir6.2(+/+)) or Kir6.2(-/-) mice. Glucose concentrations were either changed abruptly from 2.8 to 16.7 mmol/l or increased stepwise (1.4 mmol/l per step) from 2.8 to 12.5 mmol/l. Electrophysiological experiments were performed using pancreatic beta cells isolated from Kir6.2(-/-) mice or clonal pancreatic beta cells (MIN6 cells) after pharmacologically inhibiting their K(ATP) channels with glibenclamide., Results: The combination of cyclic AMP plus 16.7 mmol/l glucose evoked insulin secretion in Kir6.2(-/-) pancreases where glucose alone was ineffective as a secretagogue. The secretion was blocked by the application of niflumic acid. In K(ATP) channel-inactivated MIN6 cells, niflumic acid similarly inhibited the membrane depolarisation caused by cAMP plus glucose. Surprisingly, stepwise increases of glucose concentration triggered insulin secretion only in the presence of cAMP or GLP-1 in Kir6.2(+/+), as in Kir6.2(-/-) pancreases., Conclusions/interpretation: Niflumic acid-sensitive ion channels participate in the induction of GSIS by cyclic AMP in Kir6.2(-/-) beta cells. Cyclic AMP thus not only acts as a potentiator of insulin secretion, but appears to be permissive for GSIS via novel, niflumic acid-sensitive ion channels. This mechanism may be physiologically important for triggering insulin secretion when the plasma glucose concentration increases gradually rather than abruptly.

Aims/hypothesis: Heterozygous activating mutations in the pancreatic ATP-sensitive K+ channel cause permanent neonatal diabetes mellitus (PNDM). This results from a decrease in the ability of ATP to close the channel, which thereby suppresses insulin secretion. PNDM mutations that cause a severe reduction in ATP inhibition may produce additional symptoms such as developmental delay and epilepsy. We identified a heterozygous mutation (L164P) in the pore-forming (Kir6.2) subunit of the channel in three unrelated patients and examined its functional effects., Methods: The patients (currently aged 2, 8 and 20 years) developed diabetes shortly after birth. The two younger patients attempted transfer to sulfonylurea therapy but were unsuccessful (up to 1.1 mg kg(-1) day(-1)). They remain insulin dependent. None of the patients displayed neurological symptoms. Functional properties of wild-type and mutant channels were examined by electrophysiology in Xenopus oocytes., Results: Heterozygous (het) and homozygous L164P K(ATP) channels showed a marked reduction in channel inhibition by ATP. Consistent with its predicted location within the pore, L164P enhanced the channel open state, which explains the reduction in ATP sensitivity. HetL164P currents exhibited greatly increased whole-cell currents that were unaffected by sulfonylureas. This explains the inability of sulfonylureas to ameliorate the diabetes of affected patients., Conclusions/interpretation: Our results provide the first demonstration that mutations such as L164P, which produce a severe reduction in ATP sensitivity, do not inevitably cause developmental delay or neurological problems. However, the neonatal diabetes of these patients is unresponsive to sulfonylurea therapy. Functional analysis of PNDM mutations can predict the sulfonylurea response.

Aims/hypothesis: The P12A variant in the PPARG gene and the E23K polymorphism in KCNJ11 are both known to influence individual predisposition to type 2 diabetes. If the effect of these variants on insulin secretion and action were to extend to an influence on early growth (which is largely mediated by insulin), it would offer an explanation for observed associations between low birthweight and subsequent diabetes. Since previous studies of the effects of these variants on early growth have been limited and conflicting, we examined these associations in a large, well-characterised birth cohort., Methods: The P12A and E23K variants were genotyped in (respectively) 5,652 and 5,632 individuals from the Northern Finland Birth Cohort of 1966 and we sought associations with early growth phenotypes., Results: Neither variant was associated with birthweight (P12A, p = 0.42; E23K, p = 0.44, additive models) or other measures of early growth. Although a previous report had suggested that the P12A effect on adult insulin sensitivity was restricted to small babies, we were unable to reproduce this finding (p = 0.40), nor did we confirm a previous report of an association with gestational age (p = 0.23)., Conclusions/interpretation: Despite a larger sample size than previous studies, we were unable to detect any effect of these variants on early growth. These findings do not support the notion that there are shared genetic determinants of low birthweight and adult diabetes.

Aims/hypothesis: Hyperglycaemia can impair beta cell function after islet transplantation. Appropriate glucose-induced insulin secretion is dependent on a unique expression pattern of genes. Here we examined the effects of diabetes on gene expression in transplanted islets., Materials and Methods: Streptozotocin-induced diabetic or control non-diabetic Lewis rats were transplanted under the kidney capsule with an insufficient number (2,000) of syngeneic islets to normalise blood glucose levels in diabetic rats. Eighteen days after transplantation, islet grafts were retrieved and RT-PCR used to assess expression of selected genes critical for beta cell function. Islet grafts from diabetic rats transplanted with a sufficient number of islets (3,000) to normalise hyperglycaemia were used to assess the effects of correcting blood glucose levels. Additionally, gene expression of transplanted islets from non-diabetic rats was compared with freshly isolated islets., Results: In islet grafts from diabetic rats, mRNA levels of several transcription factors important for the maintenance of beta cell differentiation were reduced (pancreatic and duodenal homeobox 1 [Pdx1], neurogenic differentiation 1 [Neurod1], NK6 transcription factor related, locus 1 [Nkx6.1], paired box gene 6 [Pax6]), as were genes implicated in beta cell function (Glut2 [also known as solute carrier family 2 [facilitated glucose transporter], member 2 [Slc2a2], glucokinase, insulin, islet amyloid polypeptide [Iapp]). Conversely, mRNA levels of lactate dehydrogenase, which is normally suppressed in beta cells, were increased. The majority of the changes in gene expression were normalised after correction of hyperglycaemia, indicating that the severe loss of beta cell differentiation correlates with continuous exposure to diabetes. Even islet grafts from non-diabetic rats showed a few alterations in beta cell gene expression in comparison with fresh islets., Conclusions/interpretation: Chronic hyperglycaemia contributes to the deterioration of beta cell differentiation after islet transplantation.

Aims/hypothesis: The pancreatic beta cell ATP-sensitive potassium (K(ATP)) channel, composed of the pore-forming alpha subunit Kir6.2, a member of the inward rectifier K+channel family, and the regulatory beta subunit sulfonylurea receptor 1 (SUR1), a member of the ATP-binding cassette superfamily, couples the metabolic state of the cell to electrical activity. Several endogenous compounds are known to modulate K(ATP) channel activity, including ATP, ADP, phosphatidylinositol diphosphates and long-chain acyl coenzyme A (LC-CoA) esters. LC-CoA esters have been shown to interact with Kir6.2, but the mechanism and binding site(s) have yet to be identified., Materials and Methods: Using multiple sequence alignment of known acyl-CoA ester interacting proteins, we were able to identify four conserved amino acid residues that could potentially serve as an acyl-CoA ester-binding motif. The motif was also recognised in the C-terminal region of Kir6.2 (R311-332) but not in SUR1., Results: Oocytes expressing Kir6.2DeltaC26 K332A repeatedly generated K(+)currents in inside-out membrane patches that were sensitive to ATP, but were only weakly activated by 1 mumol/l palmitoyl-CoA ester. Compared with the control channel (Kir6.2DeltaC26), Kir6.2DeltaC26 K332A displayed unaltered ATP sensitivity but significantly decreased sensitivity to palmitoyl-CoA esters. Coexpression of Kir6.2DeltaC26 K332A and SUR1 revealed slightly increased activation by palmitoyl-CoA ester but significantly decreased activation by the acyl-CoA esters compared with the wild-type K(ATP) channel and Kir6.2DeltaC26+SUR1. Computational modelling, using the crystal structure of KirBac1.1, suggested that K332 is located on the intracellular domain of Kir6.2 and is accessible to intracellular modulators such as LC-CoA esters., Conclusions/interpretation: These results verify that LC-CoA esters interact at the pore-forming subunit Kir6.2, and on the basis of these data we propose an acyl-CoA ester binding motif located in the C-terminal region.

Aims/hypothesis: Activating mutations in the KCNJ11 gene encoding the Kir6.2 subunit of the K(ATP) channels in pancreatic beta cells are a common cause of neonatal diabetes. One-third of patients also have developmental delay, which probably results from mutated K(ATP) channels in muscle, nerve and brain. Sulfonylureas, which bind to the sulfonylurea receptor 1 subunit of the K(ATP) channel, can replace insulin injections in patients with KCNJ11 mutations. The aim of this study was to investigate the long-term outcome and impact on neurological features of sulfonylurea treatment., Methods: We report the response to sulfonylurea treatment in a boy with neonatal diabetes and marked developmental delay resulting from the KCNJ11 mutation V59M., Results: Glibenclamide (glyburide) treatment was started at 23 months and resulted in insulin being discontinued, lower overall glycaemia, reduced glucose fluctuations and reduced hypoglycaemia. Good control (HbA(1c) 6.5%) was maintained 2 years after discontinuing insulin, despite a reduction in the glibenclamide dose (from 0.41 to 0.11 mg.kg(-1).day(-1)). Within 1 month of starting glibenclamide there was marked improvement in motor function, resulting in the patient progressing from being unable to stand unaided to walking independently, but there was no improvement in mental function., Conclusions/interpretation: This 2-year follow-up of a patient highlights that sulfonylurea treatment can result in prolonged, excellent glycaemic control and may improve motor features, but not mental features, associated with KCNJ11 mutations. This suggests that the neurological actions of sulfonylurea are initially principally on peripheral (nerve or muscle) rather than on central (brain) K(ATP) channels. Early molecular diagnosis is important in patients with neonatal diabetes and neurological features.

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

Aims/hypothesis: 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. A minority of patients also have neurological features. The identification of a KCNJ11 mutation has important therapeutic implications, as many patients can replace insulin injections with sulfonylurea tablets. We aimed to determine the age of presentation of patients with KCNJ11 mutations and to examine if there was a relationship between genotype and phenotype., Subjects and Methods: KCNJ11 was sequenced in 239 unrelated patients from 21 countries, who were diagnosed with permanent diabetes before 2 years of age., Results: Thirty-one of the 120 patients (26%) diagnosed in the first 26 weeks of life had a KCNJ11 mutation; no mutations were found in the 119 cases (0%) diagnosed after this age. Fourteen different heterozygous mutations were identified, with the majority resulting from de novo mutations. These include seven novel mutations: H46Y, R50Q, G53D C166Y, K170T, L164P and Y330S. All 11 probands with the most common mutation, R201H, had isolated diabetes. In contrast, developmental delay in addition to diabetes was seen in four of five probands with the V59M mutation and two of four with the R201C mutation. Five patients with developmental delay, epilepsy and neonatal diabetes (DEND) syndrome had unique mutations not associated with other phenotypes., Conclusions/interpretation: KCNJ11 mutations are a common cause of permanent diabetes diagnosed in the first 6 months and all patients diagnosed in this age group should be tested. There is a strong genotype-phenotype relationship with the mutation being an important determinant of associated neurological features.

Aims/hypothesis: Gestational diabetes mellitus (GDM) and type 2 diabetes share a common pathophysiological background, including beta cell dysfunction and insulin resistance. In addition, women with GDM are at increased risk of developing type 2 diabetes later in life. Our aim was to investigate whether, like type 2 diabetes, GDM has a genetic predisposition by studying five common polymorphisms in four candidate genes that have previously been associated with type 2 diabetes., Materials and Methods: We studied 1,777 unrelated Scandinavian women (588 with GDM and 1,189 pregnant non-diabetic controls) for polymorphisms in the genes encoding potassium inwardly rectifying channel subfamily J, member 11 (KCNJ11 E23K), insulin receptor substrate 1 (IRS1 G972R), uncoupling protein 2 (UCP2 -866G-->A) and calpain 10 (CAPN10 SNP43 and SNP44)., Results: The EE, EK and KK genotype frequencies of the KCNJ11 E23K polymorphism differed significantly between GDM and control women (31.5, 52.7 and 15.8% vs 37.3, 48.8 and 13.9%, respectively; p=0.050). In addition, the frequency of the K allele was increased in women with GDM (odds ratio [OR]=1.17, 95% CI 1.02-1.35; p=0.027), and this effect was greater under a dominant model (KK/EK vs EE) (OR=1.3, 95% CI 1.05-1.60; p=0.016). Analysis of the IRS1 G972R polymorphism showed that RR homozygosity was found exclusively in women with GDM (91.0, 8.3 and 0.7% vs 90.7, 9.3 and 0.0% for GG, GR and RR genotypes, respectively; p=0.014). The genotype and allele frequencies of the other polymorphisms studied were not statistically different between the GDM and control women., Conclusions/interpretation: The E23K polymorphism of KCNJ11 seems to predispose to GDM in Scandinavian women.

Aims/hypothesis: Islets or beta cells from Sur1(-/-) mice were used to determine whether changes in plasma membrane potential (V(m)) remain coupled to changes in cytosolic Ca(2+) ([Ca(2+)](i)) in the absence of K(ATP) channels and thus provide a triggering signal for insulin secretion. The study also sought to elucidate whether [Ca(2+)](i) influences oscillations in V(m) in sur1(-/-) beta cells., Methods: Plasma membrane potential and ion currents were measured with microelectrodes and the patch-clamp technique. [Ca(2+)](i) was monitored with the fluorescent dye fura-2. Insulin secretion from isolated islets was determined by static incubations., Results: Membrane depolarisation of Sur1(-/-) islets by arginine or increased extracellular K(+), elevated [Ca(2+)](i) and augmented insulin secretion. Oligomycin completely abolished glucose-stimulated insulin release from Sur1(-/-) islets. Oscillations in V(m) were influenced by [Ca(2+)](i) as follows: (1) elevation of extracellular Ca(2+) lengthened phases of membrane hyperpolarisation; (2) simulating a burst of action potentials induced a Ca(2+)-dependent outward current that was augmented by increased Ca(2+) influx through L-type Ca(2+) channels; (3) Ca(2+) depletion of intracellular stores by cyclopiazonic acid increased the burst frequency in Sur1(-/-) islets, elevating [Ca(2+)](i) and insulin secretion; (4) store depletion activated a Ca(2+) influx that was not inhibitable by the L-type Ca(2+) channel blocker D600., Conclusions/interpretation: Although V(m) is largely uncoupled from glucose metabolism in the absence of K(ATP) channels, increased electrical activity leads to elevations of [Ca(2+)](i) that are sufficient to stimulate insulin secretion. In Sur1(-/-) beta cells, [Ca(2+)](i) exerts feedback mechanisms on V(m) by activating a hyperpolarising outward current and by depolarising V(m) via store-operated ion channels.

Aims/hypothesis: SUR1(ABCC8)(-/-) mice lacking functional K(ATP) channels are an appropriate model to test the significance of K(ATP) channels in beta-cell function. We examined how this gene deletion interferes with stimulus-secretion coupling. We tested the influence of metabolic inhibition and galanin, whose mode of action is controversial., Methods: Plasma membrane potential (Vm) and currents were measured with microelectrodes or the patch-clamp technique; cytosolic Ca(2+) concentrations ([Ca(2+)](c)) and mitochondrial membrane potential (DeltaPsi) were measured using fluorescent dyes., Results: In contrast to the controls, SUR1(-/-) beta cells showed electrical activity even at a low glucose concentration. Continuous spike activity was measured with the patch-clamp technique, but with microelectrodes slow oscillations in Vm consisting of bursts of Ca(2+)-dependent action potentials were detected. [Ca(2+)](c) showed various patterns of oscillations or a sustained increase. Sodium azide did not hyperpolarize SUR1(-/-) beta cells. The depolarization of DeltaPsi evoked by sodium azide was significantly lower in SUR1(-/-) than SUR1(+/+) cells. Galanin transiently decreased action potential frequency and [Ca(2+)](c) in cells from both SUR1(-/-) and SUR1(+/+) mice., Conclusion/interpretation: The strong dependence of Vm and [Ca(2+)](c) on glucose concentration observed in SUR1(+/+) beta cells is disrupted in the knock-out cells. This demonstrates that both parameters oscillate in the absence of functional K(ATP) channels. The lack of effect of metabolic inhibition by sodium azide shows that in SUR1(-/-) beta cells changes in ATP/ADP no longer link glucose metabolism and Vm. The results with galanin suggest that this peptide affects beta cells independently of K(ATP) currents and thus could contribute to the regulation of beta-cell function in SUR1(-/-) animals.

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