Coronary artery disease susceptibility loci extensively tested in the present Japanese study.

<p>A cororary artery disease (CAD) association study comprises two-tiered sample; the tier-1 was done in 1347 cases and 1.337 controls and the tier-2 done in 3,052 cases and 6.335 controls. Association results from the two tiers were combined by pooling the genotype counts.</p>a<p>rs735396 was genotyped in replacement for rs2259816 in the GWA study panel (<i>r2</i> = 1.000 to rs735396 in HapMap JPT+CHB). Rs735396 is in LD with rs1169300 (<i>r2</i> = 0.783 in HapMap JPT+CHB), which was tested for LDL-C association. The direction of association with CAD risk appears to be opposite to that for increased LDL-C in the <i>HNF1A</i> locus.</p>b<p>Following the previous meta-analysis of CAD association with ApoE genotype (Benett et al. JAMA 2007, ref. 35), CAD risk was compared between E3/E3 individuals and E2 carriers (excluding E2/E4).</p>c<p>Alleles are nominated as those in dbSNP Build 130 mapped on the strand of Human Genome Build 36.3.</p>d<p>Allele frequencies in the Japanese general population from GeMDBJ (<i>n</i> = 964) or HapMap JPT (<i>n</i> = 90; rs662799, rs2259816, rs2303790, and rs7412) or Amagasaki Study panel (rs429358).</p

Cross-population comparison of per-allele effect of SNPs associated with LDL-C (a), HDL-C (b), and TG (c) between the Japanese and European-descent populations.

<p>Effect alleles are defined as those that increase LDL-C or TG or that decrease HDL-C. The effects of each variant on lipid traits are shown by squares, colored in red (Japanese) and blue (Europeans). The gray dotted lines between the red and blue squares represent an identical locus. See details about the individual SNP loci in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046385#pone.0046385.s004" target="_blank">Table S3</a>.</p

Meta-analysis of CAD association with selected SNPs or variants, including the current and previously reported studies.

<p>Effect sizes of <i>SORT1</i> and <i>APOE</i> variants were heterogeneous between the current study and those previously reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046385#pone.0046385-Schunkert1" target="_blank">[29]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046385#pone.0046385-Bennet1" target="_blank">[35]</a>: <i>p</i> =  6.8×10⁻⁴ for <i>SORT1</i>, <i>p</i> = 1.7×10⁻³ for <i>APOE</i> (E2 carriers vs. E3/E3) and <i>p</i> = 0.041 for <i>APOE</i> (E4 carriers <i>vs.</i> E3/E3) by Woolf's test.</p

Correlation of effect sizes for CAD risk and 3 lipid traits–LDL-C (a), HDL-C (b), and TG (c)–at SNPs tested for replication in the current study.

<p>Genetic impacts on lipid level (β in <i>x</i>-axis) and CAD risk (OR in <i>y</i>-axis) are compared for the SNPs that were previously reported to associate with the corresponding (lead) lipid trait in Europeans: 18 SNPs for LDL-C (a), 20 SNPs for HDL-C (b), and 12 SNPs for TG (c), where 3 SNPs at <i>LPL</i> are included in both (b) and (c). The names of SNPs that were genotyped in the tier-2 CAD case-control study panel are denoted in the plots. For the purpose of readability, error bars are not shown at the individual SNP loci in the figure. See details about the individual SNP loci in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046385#pone.0046385.s003" target="_blank">Table S2</a>.</p

Baseline characteristics of participants in the present study.

<p>Plus–minus values are means ± SD.</p><p>Diabetes, hypertension, and dysipidemia were identified as risk factors on the basis of the meeting of diagnostic criteria or the receipt of treatment for these conditions (Note S1).</p><p>In the GWA-scanned panel, 414 individuals were from the Amagasaki Study panel; only the latter panel was included and analyzed in the current replication study.</p

Deletion of CDKAL1 Affects High-Fat Diet–Induced Fat Accumulation and Glucose-Stimulated Insulin Secretion in Mice, Indicating Relevance to Diabetes

<div><h3>Background/Objective</h3><p>The <em>CDKAL1</em> gene is among the best-replicated susceptibility loci for type 2 diabetes, originally identified by genome-wide association studies in humans. To clarify a physiological importance of CDKAL1, we examined effects of a global <em>Cdkal1</em>-null mutation in mice and also evaluated the influence of a <em>CDKAL1</em> risk allele on body mass index (BMI) in Japanese subjects.</p> <h3>Methods</h3><p>In <em>Cdkal1</em>-deficient (<em>Cdkal1</em>^−/−) mice, we performed oral glucose tolerance test, insulin tolerance test, and perfusion experiments with and without high-fat feeding. Based on the findings in mice, we tested genetic association of <em>CDKAL1</em> variants with BMI, as a measure of adiposity, and type 2 diabetes in Japanese.</p> <h3>Principal Findings</h3><p>On a standard diet, <em>Cdkal1</em>^−/− mice were modestly lighter in weight than wild-type littermates without major alterations in glucose metabolism. On a high fat diet, <em>Cdkal1</em>^−/− mice showed significant reduction in fat accumulation (17% reduction in %intraabdominal fat, <em>P</em> = 0.023 vs. wild-type littermates) with less impaired insulin sensitivity at an early stage. High fat feeding did not potentiate insulin secretion in <em>Cdkal1</em>^−/− mice (1.0-fold), contrary to the results in wild-type littermates (1.6-fold, <em>P</em><0.01). Inversely, at a later stage, <em>Cdkal1</em>^−/− mice showed more prominent impairment of insulin sensitivity and glucose tolerance. mRNA expression analysis indicated that <em>Scd1</em> might function as a critical mediator of the altered metabolism in <em>Cdkal1</em>^−/− mice. In accordance with the findings in mice, a nominally significant (<em>P</em><0.05) association between <em>CDKAL1</em> rs4712523 and BMI was replicated in 2 Japanese general populations comprising 5,695 and 12,569 samples; the risk allele for type 2 diabetes was also associated with decreased BMI.</p> <h3>Conclusions</h3><p><em>Cdkal1</em> gene deletion is accompanied by modestly impaired insulin secretion and longitudinal fluctuations in insulin sensitivity during high-fat feeding in mice. CDKAL1 may affect such compensatory mechanisms regulating glucose homeostasis through interaction with diet.</p> </div

Pancreatic histology and insulin content in <i>CDKAL1</i> KO mice.

<p><b>A</b>. <i>CDKAL1</i> KO mice have normal islet architecture. Pancreatic sections were peroxidase stained for insulin. Scale bar: 100 µm. <b>B</b>. Relative area occupied by β cells (percentage of total pancreatic area). Random sections of the entire pancreas from WT and <i>CDKAL1</i> KO mice were immunostained (as shown in <b>A</b>) and analyzed (60 sections from each of three mice per group). <b>C–F</b>. Electron micrographs of pancreatic tissue sections. <b>C</b> Representative sections (scale bar: 5 µm), <b>D</b> β cell size, <b>E</b> total number of granules per cell section, and <b>F</b> mean granule diameter in ultra-thin sections (100 nm) (n = 20 cells per group) of <i>CDKAL1</i> KO and WT β cells. <b>G–I</b>. Insulin content in <i>CDKAL1</i> KO mice. <b>G</b> Pancreatic insulin content measured in acid-ethanol extracts from WT and KO mice by ELISA (n = 6 per group). <b>H</b> DNA content per islet and <b>I</b> islet insulin content per DNA from WT and KO mice (n = 6 per group). Results are means±SEM.</p

Effects of <i>CDKAL1</i> KO on changes in intracellular free calcium ([Ca²⁺]_i).

<p><b>A</b> 22 mM glucose- and <b>B</b> 40 mM high K⁺-induced changes in [Ca²⁺]_i in WT and <i>CDKAL1</i> KO β cells. Changes in [Ca²⁺]_i were measured by Fura-2 acetoxymethyl (2 µM). Time 0 indicates when the stimulants were added. The fluorescence ratio (340/360) at time 0 was taken as 1. Results are means±SEM (n = 12 cells per group).</p

<i>CDKAL1</i> KO did not affect the number of morphologically docked granules or granule fusion kinetics.

<p><b>A</b>. Total internal reflection fluorescence (TIRF) microscopy of insulin granules morphologically docked to the plasma membrane. (top) Typical TIRF images of docked insulin granules in WT and <i>CDKAL1</i> KO β cells. The surrounding lines represent the outline of cells attached to the cover glass. Scale bar: 5 µm. Pancreatic β cells were prepared from WT and KO mice, fixed, and immunostained for insulin. (bottom) Number of insulin granules morphologically docked to the plasma membrane. Individual fluorescent spots shown in the TIRF images were manually counted per 200 µm² in 15 cells per group. <b>B</b>. Electron micrograph of β cell sections. (top) Typical images of the plasma membrane area facing the blood capillary (C) of WT and KO β cells (B). Bar: 500 nm. (bottom) Number of morphologically docked insulin granules per 10 µm of the plasma membrane. Granules at their shortest distance of <10 nm from the plasma membrane were defined as morphologically docked granules (red arrowheads). Results are means±SEM. <b>C</b>. Expression of SNARE proteins in wild-type (WT) and KO islets by immunoblotting. Equal amounts of islet protein (30 µg) were separated by SDS-PAGE and immunoblotted. β-actin was used as a loading control.</p

Effects of <i>CDKAL1</i> KO on glucose-induced biphasic insulin exocytosis.

<p><b>A</b>. Insulin release (for 30 min) in batch-incubated WT and <i>CDKAL1</i> KO β cells in the presence of 2.2 mM or 16.7 mM glucose (n = 8 per group). <b>B</b>. Histogram showing the number of fusion events from GFP-tagged granules in wild-type (WT) and <i>CDKAL1</i> KO β cells (per 200 µm²) at 1-min intervals after stimulation with 22 mM glucose and measured by TIRF microscopy. Data are mean±SEM (WT, n = 16 cells; KO, n = 14 cells). Time 0 indicates the addition of 22 mM glucose. The red column shows fusion events from previously docked granules, and the green column shows those from newcomers. <b>C</b>. Histogram showing the number of fusion events in WT and KO β cells (per 200 µm²) at 1-min intervals after 40 mM high K⁺ stimulation measured by TIRF microscopy (n = 8 cells per group).</p