Hiroshi Sakura

Cloning and functional expression of the cDNA encoding a novel ATP-sensitive potassium channel subunit expressed in pancreatic beta-cells, brain, heart and skeletal muscle.

A cDNA clone encoding an inwardly‐rectifying potassium channel subunit (Kir6.2) was isolated from an insulinoma cDNA library. The mRNA is strongly expressed in brain, skeletal muscle, cardiac muscle and in insulinoma cells, weakly expressed in lung and kidney and not detectable in spleen, liver or testis. Heterologous expression of Kir6.2 in HEK293 cells was only observed when the cDNA was cotransfected with that of the sulphonylurea receptor (SUR). Whole‐cell Kir6.2/SUR currents were K+‐selective, time‐independent and showed weak inward rectification. They were blocked by external barium (5 mM), tolbutamide (K d = 4.5μM) or quinine (20μM) and by 5 mM intracellular ATP. The single‐channel conductance was 73 pS. Single‐channel activity was voltage‐independent and was blocked by 1 mM intracellular ATP or 0.5 mM tolbutamide. We conclude that the Kir6.2/SUR channel complex comprises the ATP‐sensitive K‐channel.

Electrogenic arginine transport mediates stimulus-secretion coupling in mouse pancreatic beta-cells.

1. We have investigated the mechanism by which L‐arginine stimulates membrane depolarization, an increase of intracellular calcium ([Ca2+]i) and insulin secretion in pancreatic beta‐cells. 2. L‐Arginine failed to affect beta‐cell metabolism, as monitored by NAD(P)H autofluorescence. 3. L‐Arginine produced a dose‐dependent increase in [Ca2+]i, which was dependent on membrane depolarization and extracellular calcium. 4. The cationic amino acids L‐ornithine, L‐lysine, L‐homoarginine (which is not metabolized) and NG‐monomethyl‐L‐arginine (L‐NMMA, a nitric oxide synthase inhibitor) produced [Ca2+]i responses similar to that produced by L‐arginine. The neutral nitric oxide synthase inhibitors NG‐nitro‐L‐arginine (L‐NNA) and N omega‐monomethyl‐L‐arginine (L‐NAME) also increased [Ca2+]i. D‐Arginine was ineffective. 5. L‐Arginine did not affect whole‐cell Ca2+ currents or ATP‐sensitive K+ currents, but produced an inward current that was carried by the amino acid. 6. The reverse transcriptase‐polymerase chain reaction demonstrated the presence of messenger RNA for the murine cationic amino acid transporters mCAT2A and mCAT2B within the beta‐cell. 7. L‐Arginine did not affect beta‐cell exocytosis as assayed by changes in cell capacitance. 8. Our data suggest that L‐arginine elevates [Ca2+]i and stimulates insulin secretion as a consequence of its electrogenic transport into the beta‐cell. This uptake is mediated by the mCAT2A transporter.

Characterization and variation of a human inwardly-rectifying-K-channel gene (KCNJ6): a putative ATP-sensitive K-channel subunit.

The ATP‐sensitive K‐channel plays a central role in insulin release from pancreatic β‐cells. We report here the cloning of the gene (KCNJ6) encoding a putative subunit of a human ATP‐sensitive K‐channel expressed in brain and β‐cells, and characterisation of its exon‐intron structure. Screening of a somatic cell mapping panel and fluorescent in situ hybridization place the gene on chromosome 21 (21q22.1–22.2). Analysis of single‐stranded conformational polymorphisms revealed the presence of two silent polymorphisms (Pro‐149: CC ‐CC and Asp328: GA ‐GA ) with similar frequencies in normal and non‐insulin‐dependent diabetic patients.

Cloning and functional expression of the cDNA encoding an inwardly-rectifying potassium channel expressed in pancreatic β-cells and in the brain

A cDNA clone encoding an inwardly‐rectifying K‐channel (BIR1) was isolated from insulinoma cells. The predicted amino acid sequence shares 72% identity with the cardiac ATP‐sensitive K‐channel rcKATP (KATP‐1; [6]). The mRNA is expressed in the brain and insulinoma cells. Heterologous expression in Xenopus oocytes produced currents which were K+‐selective, time‐independent and showed inward rectification. The currents were blocked by external barium and caesium, but insensitive to tolbutamide and diazoxide. In inside‐out patches, channel activity was not blocked by 1 mM internal ATP. The sequence homology with KATP‐1 suggests that BIR1 is a subunit of a brain and β‐cell KATP channel. However, pharmacological differences and the lack of ATP‐sensitivity, suggest that if, this is the case, heterologous subunits must exert strong modulatory influences on the native channel.

Altered functional properties of KATP channel conferred by a novel splice variant of SUR1

1 ATP‐sensitive potassium (KATP) channels are composed of pore‐forming (Kir6.x) and regulatory sulphonylurea receptor (SURx) subunits. We have isolated a novel SUR variant (SUR1bΔ33) from a hypothalamic cDNA library. This variant lacked exon 33 and introduced a frameshift that produced a truncated protein lacking the second nucleotide binding domain (NBD2). It was expressed at low levels in hypothalamus, midbrain, heart and the insulin‐secreting β‐cell line MIN6. 2 We examined the properties of KATP channels composed of Kir6.2 and SUR1bΔ33 by recording macroscopic currents in membrane patches excised from Xenopus oocytes expressing these subunits. We also investigated the effect of truncating SUR1 at either the start (SUR1bT1) or end (SUR1bT2) of exon 33 on KATP channel properties. 3 Kir6.2/SUR1bΔ33 showed an enhanced open probability (Po= 0.6 at ‐60 mV) and a reduced ATP sensitivity (Ki, 86 μm), when compared with wild‐type channels (Po= 0.3; Ki, 22 μm). However, Kir6.2/SUR1bT1 and Kir6.2/SUR1bT2 resembled the wild‐type channel in their Po and ATP sensitivity. 4 Neither MgADP, nor the K+ channel opener diazoxide, enhanced Kir6.2/SUR1bΔ33, Kir6.2/SUR1bT1 or Kir6.2/SUR1bT2 currents, consistent with the idea that these agents require an intact NBD2 for their action. Sulphonylureas blocked KATP channels containing any of the three SUR variants, but in excised patches the extent of block was less than that for the wild‐type channel. In intact cells, the extent of sulphonylurea block of Kir6.2/SUR1bΔ33 was greater than that in excised patches and was comparable to that found for wild‐type channels. 5 Our results demonstrate that NBD2 is not essential for functional expression or sulphonylurea block, but is required for KATP channel activation by K+ channel openers and nucleotides. Some of the unusual properties of Kir6.2/SUR1bΔ33 resemble those reported for the KATP channel of ventromedial hypothalamic (VMH) neurones, but the fact that this mRNA is expressed at low levels in many other tissues makes it less likely that SUR1bΔ33 serves as the SUR subunit for the VMH KATP channel.

Glucose modulation of ATP-sensitive K-currents in wild-type, homozygous and heterozygous glucokinase knock-out mice.

Summary One type of maturity-onset diabetes of the young (MODY2) is caused by mutations in the glucokinase gene, a key glycolytic enzyme in the beta cell and liver. Glucose fails to stimulate insulin secretion in mice in which the glucokinase gene has been selectively knocked out in the beta cell. We tested the hypothesis that this effect results from defective metabolic regulation of beta cell ATP-sensitive potassium (KATP) channels. Glucose had little effect on KATP currents in homozygous (-/-) mice but inhibited KATP currents in wild-type (+/+) and heterozygous ( + /-) mice with EC50 of 3.2 mM and 5.5 mM, respectively, in newborn animals, and of 4.7 mM and 9.9 mM, respectively, in 1.5-year-old mice. Glucose (20 mmol/l) did not affect the resting membrane potential of -/- beta cells but depolarised wild-type and + /- beta cells and induced electrical activity. In contrast, 20 mmol/l ketoisocaproic acid or 0.5 mmol/l tolbutamide depolarised all three types of beta-cell. These results support the idea that defective glycolytic metabolism, produced by a loss (-/- mice) or reduction ( + /- mice) of glucokinase activity, leads to defective KATP channel regulation and thereby to the selective loss, or reduction, of glucose-induced insulin secretion. [Diabetologia (1998) 41: 654–659]

Sequence Variations of the Glucokinase Gene in Japanese Subjects With NIDDM

Mutations in the glucokinase gene have been identified recently in patients with maturity-onset diabetes of the young, a subtype of NIDDM. The proposed role of glucokinase as a glucose sensor, combined with the low insulin response to glucose found in most Japanese with NIDDM, prompted us to speculate that mutations in the glucokinase gene might be one of the major causes of NIDDM in Japanese subjects. To determine the prevalence of mutations and sequence variations in the glucokinase gene, we screened all 12 exons of the glucokinase gene, including exon/intron junctions, by polymerase chain reaction followed by single-strand conformation polymorphism in 209 Japanese NIDDM subjects. In addition to the mutation in exon 7, which substituted Arg (AGG) for Gly (GGG) at codon 261 (10), a silent mutation of Pro (CCC→CCG) in exon 4 at codon 145 and several new sequence variations in intervening sequences and the 5′-untranslated region of exon 1β (β-cell-specific exon 1) were identified. Because we identified only one subject who had a structurally abnormal glucokinase molecule, we conclude that the prevalence of structural mutations in the glucokinase gene responsible for NIDDM appears to be rare among Japanese patients. To our knowledge, this is the first thorough study describing the ethnic prevalence of mutations and sequence variations in the glucokinase gene in NIDDM.

Seasonal fluctuations of glycated hemoglobin levels in Japanese diabetic patients

AIMSnWe examined whether glycated hemoglobin (HbA(1C)) levels of Japanese diabetic patients showed seasonal fluctuations.nnnMETHODSnSubjects included 2511 diabetic patients who regularly visited a single diabetic outpatient clinic for 10 years. A total of 253,477 HbA(1C) measurements, as well as sex, age, BMI, type of diabetes, and mode of therapy were extracted from a hospital-based database. For the cross-sectional and longitudinal analyses, average monthly HbA(1C) values of subjects and amplitudes of seasonal fluctuations were calculated. For the time-series analysis, seasonal adjustment factors of each subject were classified as complete, incomplete, or no fluctuation.nnnRESULTSnSubjects showed a clear seasonal fluctuation of HbA(1C) levels, with highest levels in March (7.69%) and lowest levels in August (7.46%; p<0.001). The amplitudes of the seasonal fluctuations were associated with the mean HbA(1C) levels. The time-series analysis showed that 78.3% of patients had complete or incomplete seasonal fluctuations. HbA(1C) levels were highest in winter-spring and lowest in summer-autumn in most patients; however, some patients showed a reverse pattern.nnnCONCLUSIONSnSeasonal fluctuations of HbA(1C) levels were recognized in most of the Japanese diabetic patients. Physiological or metabolic factors related to temperature may be the main cause of seasonal fluctuations in HbA(1C) levels.

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

The β-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 β-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 β-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.

The relationship between depressive symptoms and diabetic complications in elderly patients with diabetes: Analysis using the Diabetes Study from the Center of Tokyo Women's Medical University (DIACET)

AIMSnTo investigate the association between likelihood or severity of depression and symptoms associated with diabetic complications in elderly Japanese patients with diabetes.nnnMETHODSnThis single-center cross-sectional study included 4283 patients with diabetes, 65 years and older (mean age was 73 ± 6 years, 38.7% were women, 3.9% had type 1 diabetes). Participants completed a self-administered questionnaire including items on subjective symptoms associated with diabetic microangiopathy, frequency of clinical visits due to vascular diseases (heart diseases, stroke, or gangrene), hospitalization, and the Patient Health Questionnaire-9 (PHQ-9), a simple but reliable measure of depression. The associations between severity of depression and diabetic complications were examined using logistic regression analysis.nnnRESULTSnAccording to the PHQ-9 scores, patients were classified into the following 3 categories: 0-4 points (n=2975); 5-9 points (n=842); and 10 or more points (n=466). Higher PHQ-9 scores were associated with increased odds ratios for retinopathy, symptoms related to peripheral polyneuropathy and autonomic neuropathy, and end-stage renal disease requiring dialysis after adjustment for age, gender, smoking status, and HbA1c (all p<0.05).nnnCONCLUSIONSnSignificant relationships were found between depression severity and chronic diabetic complications among elderly Japanese patients with diabetes.