Keizo Ohnaka

Meta-analysis of genome-wide association studies identifies eight new loci for type 2 diabetes in east Asians.

We conducted a three-stage genetic study to identify susceptibility loci for type 2 diabetes (T2D) in east Asian populations. We followed our stage 1 meta-analysis of eight T2D genome-wide association studies (6,952 cases with T2D and 11,865 controls) with a stage 2 in silico replication analysis (5,843 cases and 4,574 controls) and a stage 3 de novo replication analysis (12,284 cases and 13,172 controls). The combined analysis identified eight new T2D loci reaching genome-wide significance, which mapped in or near GLIS3, PEPD, FITM2-R3HDML-HNF4A, KCNK16, MAEA, GCC1-PAX4, PSMD6 and ZFAND3. GLIS3, which is involved in pancreatic beta cell development and insulin gene expression, is known for its association with fasting glucose levels. The evidence of an association with T2D for PEPD and HNF4A has been shown in previous studies. KCNK16 may regulate glucose-dependent insulin secretion in the pancreas. These findings, derived from an east Asian population, provide new perspectives on the etiology of T2D.

Meta-analysis of genome-wide association studies identifies common variants associated with blood pressure variation in East Asians

We conducted a meta-analysis of genome-wide association studies of systolic (SBP) and diastolic (DBP) blood pressure in 19,608 subjects of east Asian ancestry from the AGEN-BP consortium followed up with de novo genotyping (n = 10,518) and further replication (n = 20,247) in east Asian samples. We identified genome-wide significant (P < 5 × 10−8) associations with SBP or DBP, which included variants at four new loci (ST7L-CAPZA1, FIGN-GRB14, ENPEP and NPR3) and a newly discovered variant near TBX3. Among the five newly discovered variants, we obtained significant replication in the independent samples for all of these loci except NPR3. We also confirmed seven loci previously identified in populations of European descent. Moreover, at 12q24.13 near ALDH2, we observed strong association signals (P = 7.9 × 10−31 and P = 1.3 × 10−35 for SBP and DBP, respectively) with ethnic specificity. These findings provide new insights into blood pressure regulation and potential targets for intervention.

Presence of non-selective type of endothelin receptor on vascular endothelium and its linkage to vasodilation

We studied the role of non‐selective type (ET14) of endothelin (ET) receptor in the vasculature, using a ligand specific to the ET14 receptor, [Glu9]‐sarafotoxin S6b ([Glu9]SRTb). Endothelium‐containing rat thoracic aorta possessed specific binding sites for 125I‐[Glu9]SRTb, which were almost eliminated by removal of the endothelium, while ET‐3‐specific binding sites were not detected in the endothelium‐intact rat aorta. Only ET14 receptor was detected in the membranes from the endothelium of porcine thoracic aorta. [Glu9]SRTb exerted only vasodilation in rat aortic ring. These findings indicate that ET14 receptors are located on vascular endothelium and linked to vasodilation.

Confirmation of Multiple Risk Loci and Genetic Impacts by a Genome-Wide Association Study of Type 2 Diabetes in the Japanese Population

OBJECTIVE To identify novel type 2 diabetes gene variants and confirm previously identified ones, a three-staged genome-wide association study was performed in the Japanese population. RESEARCH DESIGN AND METHODS In the stage 1 scan, we genotyped 519 case and 503 control subjects with 482,625 single nucleotide polymorphism (SNP) markers; in the stage 2 panel comprising 1,110 case subjects and 1,014 control subjects, we assessed 1,456 SNPs (P < 0.0025, stage 1); additionally to direct genotyping, 964 healthy control subjects formed the in silico control panel. Along with genome-wide exploration, we aimed to replicate the disease association of 17 SNPs from 16 candidate loci previously identified in Europeans. The associated and/or replicated loci (23 SNPs; P < 7 × 10–5 for genome-wide exploration and P < 0.05 for replication) were examined in the stage 3 panel comprising 4,000 case subjects and 12,569 population-based samples, from which 4,889 nondiabetic control subjects were preselected. The 12,569 subjects were used for overall risk assessment in the general population. RESULTS Four loci—1 novel with suggestive evidence (PEPD on 19q13, P = 1.4 × 10–5) and three previously reported—were identified; the association of CDKAL1, CDKN2A/CDKN2B, and KCNQ1 were confirmed (P < 10–19). Moreover, significant associations were replicated in five other candidate loci: TCF7L2, IGF2BP2, SLC30A8, HHEX, and KCNJ11. There was substantial overlap of type 2 diabetes susceptibility genes between the two populations, whereas effect size and explained variance tended to be higher in the Japanese population. CONCLUSIONS The strength of association was more prominent in the Japanese population than in Europeans for more than half of the confirmed type 2 diabetes loci.

Enhanced secretion of endothelin-1 by elevated glucose levels from cultured bovine aortic endothelial cells

We have investigated the effect of glucose on the release of endothelin‐1‐like immunoreactivity (ET‐l‐LI) from cultured bovine aortic endothelial cells. Elevation of glucose concentrations in cultured media from 5.5 to 11.1 or 22.2 mM significantly stimulated ET‐1‐LI release from cultured endothelial cells. An aldose reductase inhibitor did not affect the high glucose‐induced ET‐1‐LI release, These findings suggest the possibility that hyperglycemia in diabetic patients enhances ET‐1‐LI release at the local site of vascular endothelium. which might be involved in the developments of vascular complications and atherosclerosis.

Blood Pressure and Hypertension Are Associated With 7 Loci in the Japanese Population

Background— Two consortium-based genome-wide association studies have recently identified robust and significant associations of common variants with systolic and diastolic blood pressures in populations of European descent, warranting further investigation in populations of non-European descent. Methods and Results— We examined the associations at 27 loci reported by the genome-wide association studies on Europeans in a screening panel of Japanese subjects (n=1526) and chose 11 loci showing association signals (1-tailed test in the screening, P<0.3) for an extensive replication study with a follow-up panel of 3 Japanese general-population cohorts (n ≤24 300). Significant associations were replicated for 7 loci—CASZ1, MTHFR, ITGA9, FGF5, CYP17A1-CNNM2, ATP2B1, and CSK-ULK3—with any or all of these 3 traits: systolic blood pressure (P=1.4×10−14 to 0.05), diastolic blood pressure (P=1.9×10−12 to 0.05), and hypertension (P=2.0×10−14 to 0.006; odds ratio, 1.10 to 1.29). The strongest association was observed for FGF5. In the whole study panel, the variance (R2) for blood pressure explained by the 7 single-nucleotide polymorphism loci was calculated to be R2=0.003 for male and 0.006 for female participants. Stratified analysis implied the potential presence of a gene-age-sex interaction, although it did not reach a conclusive level of statistical significance after adjustment for multiple testing. Conclusions— We have confirmed 7 loci associated with blood pressure and/or hypertension in the Japanese. These loci can guide fine-mapping efforts to pinpoint causal variants and causal genes with the integration of multiethnic results.

Induction of Cyclooxygenase-2 by Angiotensin II in Cultured Rat Vascular Smooth Muscle Cells

Angiotensin II (Ang II) stimulates the release of prostaglandins (PGs) in various cells and tissues. Recently, cyclooxygenase-2 (COX-2) emerged as a new key regulator for PG synthesis. In the present study, we investigated whether Ang II regulates COX-2 expression in cultured rat vascular smooth muscle cells (VSMCs). Ang II markedly increased the expression of COX-2 mRNA in a time- and dose-dependent manner. This effect was completely blocked by the Ang II type 1 receptor antagonist losartan but not by the Ang II type 2 receptor antagonist PD123319. The p42/44 mitogen-activated protein kinase (MAPK) kinase-1 inhibitor PD98059 and the p38 MAPK inhibitor SB203580 significantly suppressed Ang II-induced COX-2 mRNA and protein expression. Ang II did not increase transcription of the COX-2 gene, as examined with a COX-2 promoter/luciferase chimeric plasmid construct. Instead, it suppressed the degradation of COX-2 mRNA. PD98059 and SB203580 markedly enhanced the decay of COX-2 mRNA induced by Ang II, implying that p42/44 and p38 MAPK activated by Ang II play a role in the regulation of COX-2 through stabilization of its mRNA. The COX-2-specific inhibitor NS-398 attenuated Ang II-stimulated DNA and protein synthesis, as well as PGE(2) production by VSMCs. These results suggest that Ang II regulates COX-2 expression and PG production and modulates cell proliferation through MAPK-mediated signaling pathways in rat VSMCs.

Molecular cloning, sequence analysis and expression of a cDNA encoding human type-1 angiotensin II receptor

We isolated a cDNA encoding type-1 angiotensin II receptor from a human liver cDNA library. The cDNA had an open reading frame encoding a protein of 359 amino acid residues with a relative Mr of 41,060. The deduced amino acid sequence of the human angiotensin II (Ang II) receptor was 95.3% and 94.2% identical to those of bovine and rat type-1 Ang II receptors, respectively, and had a significant similarity with the G protein-coupled receptor. The rank order of the binding to the receptor expressed in COS-7 cells was Ang II greater than Ang III greater than Ang I. The expression of the Ang II receptor mRNA was detected in human liver, lung, adrenal and adrenocortical adenomas but not in adrenomedullary tumor, pheochromocytoma, by Northern blot analysis.

Genome-wide association study of coronary artery disease in the Japanese.

A new understanding of the genetic basis of coronary artery disease (CAD) has recently emerged from genome-wide association (GWA) studies of common single-nucleotide polymorphisms (SNPs), thus far performed mostly in European-descent populations. To identify novel susceptibility gene variants for CAD and confirm those previously identified mostly in populations of European descent, a multistage GWA study was performed in the Japanese. In the discovery phase, we first genotyped 806 cases and 1337 controls with 451 382 SNP markers and subsequently assessed 34 selected SNPs with direct genotyping (541 additional cases) and in silico comparison (964 healthy controls). In the replication phase, involving 3052 cases and 6335 controls, 12 SNPs were tested; CAD association was replicated and/or verified for 4 (of 12) SNPs from 3 loci: near BRAP and ALDH2 on 12q24 (P=1.6 × 10−34), HLA-DQB1 on 6p21 (P=4.7 × 10−7), and CDKN2A/B on 9p21 (P=6.1 × 10−16). On 12q24, we identified the strongest association signal with the strength of association substantially pronounced for a subgroup of myocardial infarction cases (P=1.4 × 10−40). On 6p21, an HLA allele, DQB1*0604, could show one of the most prominent association signals in an ∼8-Mb interval that encompasses the LTA gene, where an association with myocardial infarction had been reported in another Japanese study. CAD association was also identified at CDKN2A/B, as previously reported in different populations of European descent and Asians. Thus, three loci confirmed in the Japanese GWA study highlight the likely presence of risk alleles with two types of genetic effects – population specific and common – on susceptibility to CAD.

Identification and characterization of endothelin converting activity in cultured bovine endothelial cells

Using a specific and sensitive radioimmunoassay (RIA) for the carboxyl terminal tail of endothelin (ET) (His16-Trp21), we have confirmed the presence of the converting activity from synthetic human big ET-1 to ET-1 in the homogenate of cultured bovine aortic endothelial cells. The optimal pHs for the converting activities were found at pH 3.0 and pH 7.0. The activity at pH 3.0 was completely inhibited by pepstatin A, whereas the activity at pH 7.0 was not affected by known various protease inhibitors except EDTA and EGTA. When the products from big ET-1 were analyzed on an ODS and a CN columns, only ET-1 was detected at pH 7.0, but various ET-like immunoreactivities other than ET-1 were detected at pH 3.0. These findings strongly suggest that mature ET-1 is formed from big ET-1 in the endothelial cells by a metal-dependent neutral protease.