Kiyoshi Kunika

SNPs in the KCNJ11-ABCC8 gene locus are associated with type 2 diabetes and blood pressure levels in the Japanese population

AbstractMany genetic association studies support a contribution of genetic variants in the KCNJ11-ABCC8 gene locus to type 2 diabetes (T2D) susceptibility in Caucasians. In non-Caucasian populations, however, there have been only a few association studies, and discordant results were obtained. Herein, we selected a total of 31 SNPs covering a 211.3-kb region of the KCNJ11-ABCC8 locus, characterized the patterns of linkage disequilibrium (LD) and haplotype structure, and performed a case-control association study in a Japanese population consisting of 909 T2D patients and 893 control subjects. We found significant associations between eight SNPs, including the KCNJ11 E23K and ABCC8 S1369A variants, and T2D. These disease-associated SNPs were genetically indistinguishable because of the presence of strong LD, as found previously in Caucasians. For the KCNJ11 E23K variant, the most significant association was obtained under a dominant genetic model (OR 1.32, 95% CI 1.09-1.60, P = 0.004). A meta-analysis of East Asian studies, comprising a total of 3,357 T2D patients (77.4% Japanese) and 2,836 control subjects (77.8% Japanese), confirmed the significant role of the KCNJ11 E23K variant in T2D susceptibility. Furthermore, we found evidence suggesting that the KCNJ11 E23K genotype is independently associated with higher blood-pressure levels.

No evidence for association of the ENPP1 (PC-1) K121Q variant with risk of type 2 diabetes in a Japanese population

AbstractEcto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1, also known as PC-1) inhibits insulin signal transduction pathway(s). Previous studies have demonstrated the K121Q variant of the ENPP1 gene to have a significant functional role in determining susceptibility to insulin resistance and type 2 diabetes (T2D). To assess whether the K121Q variant has any impact on T2D in Japanese, we undertook an extensive case-control association study using a total of 911 unrelated Japanese T2D patients and 876 control subjects. No significant difference was observed in either genotype distribution (P=0.95) or allele frequency (P=0.83) between T2D and control groups. Notably, the frequency of the ancestral Q121 allele, which is also present in other primates, was quite high in African-Americans, and showed a marked ethnic variation (77.3% in African-Americans, 16.7% in European Americans, 10.5% in Japanese and 4.2% in Han Chinese). Consequently, the pairwise FST value (a classic measure of genetic distance between pairs of population) showed highly significant differentiations between African-American and non-African-American populations (FST>0.3). Our results indicated that the K121Q variant of the ENPP1 gene has very little, if any, impact on T2D susceptibility in Japanese, but may play a role in the inter-ethnic variability in insulin resistance and T2D.

Aminoguanidine decreases urinary albumin and high-molecular-weight proteins in diabetic rats

The effect of aminoguanidine (AG) on diabetic proteinuria was studied in control rats ([C]), streptozotocin (SZ)-induced diabetic rats ([DM]), control rats treated with AG [( C + AG]), or diabetic rats treated with AG [( DM + AG]). Increased glycation of hemoglobin (HbA1C), and glomerular basement membrane (GBM) type IV collagen (IV-C) at 10 wk of stable diabetes were associated with the appearance of high-molecular-weight (HMW) cross-linked type I collagen and HMW proteinuria of 62 kD, 69 kD albumin and 77 kD proteins to the levels of 362, 381, and 408%, while 9.9, 13.5, 17, 18, and 23 kD proteins were decreased, respectively, to non-detectable, 37, 16, and 13%. AG decreased cross-linkage of type I collagen and significantly decreased urinary 62 kD protein to 54%, 69 kD albumin to 40%, and 77 kD protein to 49% at 10 wk in [DM + AG] compared to [DM] without changing diabetic control. It is suggested that glycation-derived late-stage protein modification is etiologically important for diabetic proteinuria, and that AG can potentially prevent diabetic HMW proteinuria.

Association of elastin glycation and calcium deposit in diabetic rat aorta

The relationship between glycation of the aortic elastin and calcium deposits in the aorta was studied in streptozocin (STZ)-induced diabetic rats. 5-Hydroxymethylfurfural (5-HMF) which was released from aortic elastin by acid, was assayed after STZ treatment as an index of early stage glycation. The amount of released 5-HMF increased at 5 weeks and paradoxically decreased at 10 weeks after STZ treatment, though it remained higher than that of control rats. This paradoxical pattern was reproduced by the in vitro incubation of elastin with glucose and it is presumably due to further advancement of glycation reactions in diabetic rats. The level of 5-HMF did not change significantly in control rats at corresponding time points of 9, 11 and 16 weeks of age. Fluorescence of porcine pancreatic elastase I-digested elastin which served as an index of advanced glycation, increased by 1.6 times at 3 weeks and reached a maximum of 1.9-fold higher than that of control rats at 10 weeks. The calcium content of the aorta at 10 weeks in diabetic rats was significantly increased by 1.4-fold compared with control rats. This study showed that the increased elastin glycation in the aorta even at the early stage of diabetes is associated with calcium deposit in the aorta. These results are consistent with the interpretation that elastin glycation in the aorta is the potential accelerating factor for diabetic macroangiopathy.

Diabetic modifier QTLs identified in F2 intercrosses between Akita and A/J mice

To identify novel genetic modifiers of type 2 diabetes (T2D), we performed quantitative trait loci (QTL) analysis on F2 progeny of hypoinsulinemic diabetic Akita mice, heterozygous for the Ins2 gene Cys96Tyr mutation, and nondiabetic A/J mice. We generated 625 heterozygous (F2-Hetero) and 338 wild-type (F2-Wild) mice with regard to the Ins2 mutation in F2 intercross progeny. We measured quantitative traits, including plasma glucose and insulin concentrations during the intraperitoneal glucose tolerance test (IPGTT), and body weight (BW). We observed three significant QTLs in hypoinsulinemic hyperglycemic male F2-Hetero mice, designated Dbm1, Dbm3, and Dbm4 on Chromosomes 6, 14, and 15, respectively. They showed linkage to plasma glucose concentrations, with significant maximum logarithm of odds (LOD) scores of 4.12, 4.17, and 6.17, respectively, all exceeding threshold values by permutation tests. In normoinsulinemic normoglycemic male F2-Wild mice, Dbm1 on Chromosome 6 showed linkage to both plasma insulin concentrations and BW, and Dbm2 on Chromosome 11 showed linkage to plasma glucose concentrations only, with LOD scores of 4.52 and 6.32, and 5.78, respectively. Based on these results, we concluded that Dbm1, Dbm2, Dbm3, and Dbm4 represent four major modifier QTLs specifically affecting T2D-related traits and that these diabetic modifier QTLs are conditional on the heterozygous Ins2 gene mutation and sex to exert their modifier functions. Identification of the genes responsible for these QTLs would provide new drug development targets for human T2D.

Transgenic expression of a mutated cyclin-dependent kinase 4 (CDK4/R24C) in pancreatic β-cells prevents progression of diabetes in db/db mice

In an attempt to rectify the hyperglycemic state in obese insulin resistant db/db mice, a transgenic line was generated (db/db-CDK4(R24C)) that expresses a constitutively active form of cyclin-dependent kinase 4 (CDK4/R24C) under the control of the insulin promoter. Compared with non-transgenic db/db littermates, adult db/db-CDK4(R24C) mice show near-complete glycemic normalization and improved plasma lipid concentrations, but are also more susceptible to weight gain and have significantly lower plasma adiponection levels. They have striking islet hypertrophy and beta-cell hyperplasia, and retain an insulin secretory response during the glucose tolerance test. We examined the expression of several key regulatory transcription factor genes involved in lipid and glucose metabolism in insulin target tissues of db/db-CDK4(R24C) as well as db/db mice, and found that the expression levels of members of the peroxisome proliferator-activated receptor (PPAR) family are highly associated with metabolic alterations in a gene- and tissue-specific manner. We show for the first time that the Ppar-delta in skeletal muscle and white adipose tissues is transcriptionally down-regulated in db/db mice. The db/db-CDK4(R24C) mice present a novel model of leptin-resistant obesity with compensatory hyperinsulinemia and normalized blood glucose levels, and thus may be useful for future studies that aim to dissect relationships between insulin and leptin signaling.

Inorganic phosphate accelerates hemoglobin A1c synthesis

The effects of inorganic phosphate (Pi), 2,3-diphosphoglycerate (2,3-DPG) and glucose-6-phosphate (G-6-P) on labile and stable hemoglobin A1c (HbA1c) synthesis were studied. After a 75 gram oral glucose administration, the rate of labile or stable HbA1c synthesis decreased in parallel to the decrease in plasma Pi concentrations. In in vitro incubations of red blood cell suspensions or hemoglobin preparations with glucose, Pi proportionally increased the rate of labile or stable HbA1c synthesis. The increase in 2,3-DPG caused by Pi explained only one fiftieth of the increased rate of labile HbA1c synthesis, and G-6-P did not affect HbA1c synthesis. The kinetic analysis of the effect of Pi showed the unchanged rate constant [K1], the decreased rate constant [K-1], and the increased rate constant [K2]. Based on these data it is concluded that Pi in its physiological range directly increases hemoglobin glycation by decreasing labile HbA1c dissociation and accelerating the Amadori rearrangement for stable HbA1c synthesis, and that Pi should be taken into account when using HbA1c to evaluate diabetic control.

Damage of charge-dependent renal tubular reabsorption causes diabetic microproteinuria

More negatively-charged proteins are harder to pass through the glomerular charge barrier (GCB) and to be reabsorbed by renal tubules. Although the glycation of albumin increases its negative charge compared to non-glycated albumin, the glycation of transferrin does not change its charge. This difference enabled us to examine the charge-dependent renal function in diabetic proteinuria. The percentage of urinary glycated transferrin (serum %G-transferrin) positively correlated with serum fructosamine concentrations and the percentage of serum glycated albumin (serum %G-albumin) in all subjects. Urinary concentrations of transferrin and beta 2-microglobulin strongly correlated in diabetic patients with microproteinuria, while no significant correlation was observed in subjects with diabetic macroproteinuria or non-diabetic proteinuria. Urine/serum (U/S) ratio of %G-albumin in the patients with diabetic proteinuria was significantly lower than that in subjects with non-diabetic proteinuria, while no difference of the U/S ratio of %G-transferrin was observed between any groups. Furthermore, U-%G-transferrin/U-%G-albumin ratio was highest in the diabetic patients with microproteinuria. These results lead to the conclusion that the initial damage in diabetic kidney causing microproteinuria starts with the dysfunction of charge-dependent tubular reabsorption prior to a loss of GCB.

Correction of fructosamine value for serum albumin and globulin concentrations

Using the data of 131 patients with non-insulin dependent diabetes mellitus (NIDDM), the correction formulas of fructosamine value ([FRA]) were devised to standardize the uncorrected [FRA] to serum albumin concentrations ([ALB]) of 4 g/dl, globulin concentrations ([GLB]) of 3 g/dl, and total protein concentrations ([TP]) of 7 g/dl. The following formula was derived for its maximum correlation coefficient (r) between corrected [FRA] ([FRAc]) and fasting blood glucose concentration ([G]); [FRAc] = [FRA]x33.3/(7.6 [ALB]+[GLB]). In these 131 diabetic patients, r between uncorrected [FRA] and [G] at 2 weeks ago was 0.562. When corrected by [FRA]x33.3/(7.6 [ALB]+[GLB]), [FRA]x4/[ALB], [FRA]+30 (4-[ALB]), [FRA]+23 (4-[ALB]), [FRA]+30 (7-[TP]), [FRA]x7/[TP], and [FRA]x3/[GLB], r was, respectively, 0.616, 0.612, 0.595, 0.589, 0.582, 0.581 and 0.478. In 24 patients with NIDDM whose [ALB] is either above 4.5 g/dl or below 3.5 g/dl, r between uncorrected [FRA] and [G] was as low as 0.389 without positive correlation. By using our correction formulas of [FRA]x33.3/(7.6 [ALB]+[GLB]) or [FRA] x 4/[ALB], r was statistically increased, respectively, to 0.769 or 0.788 (P less than 0.05 in both cases) in contrast to no significant increase of r by other formulas being at 0.598, 0.556, 0.540, 0.562 and 0.121. Based on these analyses, it is concluded that our correction formula of [FRA] by [FRAc]=[FRA]x33.3/(7.6 [ALB]+[GLB]) accurately reflects [G] in NIDDM even with hypo- or hyper-albuminemia, and [FRAc]=[FRA]x4/[ALB] is useful for practical application for its simplicity.

Common coding variant in the TCF7L2 gene and study of the association with type 2 diabetes in Japanese subjects

AbstractGenetic variants of the transcription factor 7-like 2 (TCF7L2) gene affect the risk of type 2 diabetes in populations with multiple ethnic groups. However, a comprehensive survey of this gene has not been done for a Japanese population. Thus, we conducted this gene-based association study, in which the common genetic variants were analyzed. Using 24 Japanese type 2 diabetic subjects, we first screened a 9.5 kb region, which included the entire coding sequence, to assess potential functional variants of TCF7L2. Sequencing revealed a common coding variant (Pro477Thr) in exon 14 of TCF7L2 that was not enrolled in the public SNP database. Nineteen SNPs and the microsatellite DG10S478 were genotyped across the gene in 2,877 unrelated Japanese subjects. This independent screen identified the previously reported rs7903146 with a strongest association (allele P = 0.0001, odds ratio = 1.59 [95% confidence interval 1.25–2.01]), but there was no significant association between Pro477Thr and type 2 diabetes (allele P = 0.64). Expression of the Pro477Thr variant did not alter TCF7L2 expression in 30 lymphoblast cells. Although a genotypic effect of Pro477Thr on expression of TCF7L2 was not apparent, Pro477Thr was identified as a common variant of TCF7L2 in 2,877 Japanese subjects. Further functional studies are required to determine the possible effect of this coding variant on type 2 diabetes.