Association of Metabolites with Obesity and Type 2 Diabetes Based on <i>FTO</i> Genotype

<div><p>The single nucleotide polymorphism rs9939609 of the gene <i>FTO</i>, which encodes fat mass and obesity–associated protein, is strongly associated with obesity and type 2 diabetes (T2D) in multiple populations; however, the underlying mechanism of this association is unclear. The present study aimed to investigate <i>FTO</i> genotype–dependent metabolic changes in obesity and T2D. To elucidate metabolic dysregulation associated with disease risk genotype, genomic and metabolomic datasets were recruited from 2,577 participants of the Korean Association REsource (KARE) cohort, including 40 homozygous carriers of the <i>FTO</i> risk allele (AA), 570 heterozygous carriers (AT), and 1,967 participants carrying no risk allele (TT). A total of 134 serum metabolites were quantified using a targeted metabolomics approach. Through comparison of various statistical methods, seven metabolites were identified that are significantly altered in obesity and T2D based on the <i>FTO</i> risk allele (adjusted <i>p</i> < 0.05). These identified metabolites are relevant to phosphatidylcholine metabolic pathway, and previously reported to be metabolic markers of obesity and T2D. In conclusion, using metabolomics with the information from genome-wide association studies revealed significantly altered metabolites depending on the <i>FTO</i> genotype in complex disorders. This study may contribute to a better understanding of the biological mechanisms linking obesity and T2D.</p></div

Functional analysis of the OCRs of multi-target regulators.

<p>(A–B) The average (A) gene density and (B) recombination hotness score (log2 ratio) for the OCRs associated with the multi-target regulators listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003229#pgen-1003229-g003" target="_blank">Figure 3C</a>. Unannotated QTLs were denoted as NA concatenated with the chromosome number (e.g., NA14 is on chromosome XIV). (C) Each spot corresponds to a genomic locus having a score for recombination hotness. Loci with a hotness score >1 located near the <i>RDH54</i>-associated OCRs are highlighted. (D) The dots indicate the OCRs of the multi-target regulators. The <i>CDC13</i> OCRs are colored according to the chromosome they belong to. The <i>CDC13</i> OCRs within 50 kb of telomeres are highlighted.</p

FZC18 reduces cell sensitivity to soluble Wnt3a.

<p>HEK293T cell batches stably expressing FZC18 (1; 4; 5) or empty vector (V) were incubated with either 50% control or Wnt3a conditioned medium (CM) for 16 hr before lysis. CRT (β-catenin-T-Cell factor Regulated Transcription) assays using Super8•Topflash or the negative control Super8•Fopflash reporters are representative of three independent experiments performed in triplicate and normalized to Renilla luciferase activity (mean±SD).</p

Metabolites displaying significant differences between <i>FTO</i> risk allele carriers and non-carriers.

<p>They show the differentiation of the population that is induced by these genetically determined metabotypes. Boxes extend from the first to third quartiles, the median is indicated as a horizontal line, and the number of individuals in each group is indicated (n). The <i>p</i> values are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156612#pone.0156612.t002" target="_blank">Table 2</a>.</p

Characterization of <i>trans</i>-associations.

<p>(A) The number of <i>trans</i>-regulatory loci associated with each chromatin trait (left) and gene expression trait (right). (B) The number of target traits of each <i>trans</i>-regulatory locus was examined for chromatin QTLs and expression QTLs. Annotated QTLs were defined as having at least one known regulator in the vicinity. (C) In this chromatin association map, each dot indicates a linkage between a genetic marker (QTL; y axis) and a trait (OCR; x axis); red or blue indicates that the BY or RM genotype positively regulates the OCR, respectively. The annotation of the 17 QTL hotspots is shown on the right side. The names of the regulators associated with the same genetic marker are separated by a semicolon and those associated with closely located markers by a dot. N/A denotes an unannotated QTL. (D–E) Different regulation architectures of chromatin traits (D) and gene expression traits (E). On the regulator side, most chromatin regulatory loci are responsible for a few traits; however, certain regulatory loci can have upwards of 100 targets. On the target side, individual chromatin traits are usually targeted by less than five loci. The average number of associated loci is three times higher for gene expression traits than for chromatin traits, an indication that the transcription process is responsive to more regulatory inputs or stimuli.</p

Stable expression of FZC18 in HEK293T cells.

<p>(A) Schematic structure showing the variant 3 of collagen XVIII containing DUF-959, FZC18, Tsp-1 (thrombospondin-1) and ES (endostatin) domains and the FZC18 expression vector. <i>Interrupted collagenous</i> indicates multiple triple helices (collagenous sequences) interrupted by globular domains. Thick horizontal lines indicate the antibodies used. <i>SP</i>, signal peptide; <i>CRD</i>, Cysteine-Rich Domain; <i>myc</i>, myc epitope tag. (B) HEK293T cells stably expressing FZC18 (batches 1; 4; 5) or empty vector (V) were fixed, permeabilized and immunostained with anti-myc, followed by peroxidase-conjugated antibodies (brown). Cells were counterstained with hematoxylin (blue). Original magnification: ×100. Images were acquired on an Olympus BX60 microscope. (C) Immunoblot with anti-FZC18 <i>(red)</i> and anti-myc <i>(yellow)</i> antibodies in HEK293T cell batches (1; 4; 5) stably expressing FZC18 or empty vector (V). α-tubulin is a loading standard.</p

Measurement of <i>trans</i>- and <i>cis</i>-variation.

<p>(A) Sequence effects on chromatin regulation. The two peaks (OCR #464 and OCR #465) are shown for strains with the BY genotype and RM genotype, as determined based on the two SNPs found within OCR #464. (B) The two <i>trans</i>-variation measures were obtained as illustrated in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003229#pgen-1003229-g001" target="_blank">Figure 1A</a> and compared with each other. (C) The significance of <i>cis</i>-variation was measured by the t-test for the 1,738 OCRs. (D) Peak density of OCR #464 as a function of its genotype. (E) Anti-correlation between the peak density of OCR #464 and that of OCR #465 across all yeast strains.</p

Partially purified FZC18 inhibits Wnt3a-induced Wnt/β-catenin signaling.

<p>CRT assay using the β-catenin reporters Super8•Topflash and Super8•Fopflash, as indicated. (A) HEK293-EBNA cells incubated for 16 hr with either 50% control CM or 50% Wnt3a CM. Wnt3a induces an 80-fold increase in CRT. (B and C) Partially purified, Fc tagged human FZC18_CRD (hFZC18_CRD-Fc) dose-dependently inhibits Wnt3a-induced CRT in HEK293-EBNA cells, as shown with Super8•Topflash <i>(B)</i> and Super8•Fopflash <i>(C)</i> CRT reporters. Cells were incubated for 16 hr with 50% Wnt3a CM that had been pre-incubated overnight on a rotary wheel at +4°C with the indicated concentrations of hFc tag alone (recombinant human Fc from IgG, negative control) or hFZC18_CRD-Fc. Results are shown as mean±SD of hFZC18_CRD-Fc/hFc tag ratios. R² indicates 2^nd degree polynomial regression coefficient. Curve fitting is shown by a red line. Super8•Fopflash <i>(C)</i> negative control CRT reporters (C) are shown for the highest concentrations of hFZC18_CRD-Fc/hFc. (D) Immunoblots show hFc and hFZC18_CRD-Fc from each sample using anti-Fc tag antibody.</p

Schematic diagram of metabolic pathways relevant to SNP-metabolite associations.

<p><b>Left:</b> PC lipid levels were increased in the rs9939609 risk allele group. Increased PC levels dependent on the <i>FTO</i> variant rs9939609 might promote T2D and obesity via fat accumulation in body and by inflammation caused by ApoB-induced LDL augmentation in the blood. <b>Right:</b> Valine levels were also increased in the rs9939609 risk allele group. Increased valine levels induce activation of the mTOR/S6K1 kinase pathway and phosphorylation of several serine residues in IRS-1, contributing to insulin resistance. In addition, increased valine catabolic flux may contribute to increased gluconeogenesis and glucose intolerance via glutamate transamination to alanine.</p

Baseline characteristics of the KARE S2 cohort sample^a,^b.

<p>Baseline characteristics of the KARE S2 cohort sample<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156612#t001fn001" target="_blank">^a</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156612#t001fn002" target="_blank">^b</a>.</p