Additional file 1: of Physical activity and FTO genotype by physical activity interactive influences on obesity

Intake questionnaire. (DOC 51 kb

Transcriptomic Identification of <i>ADH1B</i> as a Novel Candidate Gene for Obesity and Insulin Resistance in Human Adipose Tissue in Mexican Americans from the Veterans Administration Genetic Epidemiology Study (VAGES)

<div><p>Type 2 diabetes (T2D) is a complex metabolic disease that is more prevalent in ethnic groups such as Mexican Americans, and is strongly associated with the risk factors obesity and insulin resistance. The goal of this study was to perform whole genome gene expression profiling in adipose tissue to detect common patterns of gene regulation associated with obesity and insulin resistance. We used phenotypic and genotypic data from 308 Mexican American participants from the Veterans Administration Genetic Epidemiology Study (VAGES). Basal fasting RNA was extracted from adipose tissue biopsies from a subset of 75 unrelated individuals, and gene expression data generated on the Illumina BeadArray platform. The number of gene probes with significant expression above baseline was approximately 31,000. We performed multiple regression analysis of all probes with 15 metabolic traits. Adipose tissue had 3,012 genes significantly associated with the traits of interest (false discovery rate, FDR ≤ 0.05). The significance of gene expression changes was used to select 52 genes with significant (FDR ≤ 10^-4) gene expression changes across multiple traits. Gene sets/Pathways analysis identified one gene, alcohol dehydrogenase 1B (<i>ADH1B</i>) that was significantly enriched (P < 10^-60) as a prime candidate for involvement in multiple relevant metabolic pathways. Illumina BeadChip derived <i>ADH1B</i> expression data was consistent with quantitative real time PCR data. We observed significant inverse correlations with waist circumference (2.8 x 10^-9), BMI (5.4 x 10^-6), and fasting plasma insulin (P < 0.001). These findings are consistent with a central role for <i>ADH1B</i> in obesity and insulin resistance and provide evidence for a novel genetic regulatory mechanism for human metabolic diseases related to these traits.</p></div

Immunoblot with Adipose Protein Samples from Subjects with High or Low BMI.

<p>Quantitation of adipose tissue protein obtained from biopsy lysates from subjects with high BMI (N = 6, BMI = 42.4 ± 7.3 kg/m²) and subjects with low BMI (N = 6, BMI = 27.5 ± 2.5 kg/m²) was performed by immunoblotting. Protein bands were corrected for relative loading using the Ponceau staining method. Lanes 1–6 BMIs: 22.5, 27.6, 28.0, 28.8, 28.9, 29.2. Lanes 7–12 BMIs: 34.3, 36.5, 36.9, 47.4, 47.7, 51.5. Mean relative signal ± SD: samples 1–6 = 0.37 ± 0.34, samples 7–12 = 0.08 ± 0.05 (P < 0.05, t-test, 1-tailed). Ratio of mean fluorescence intensity, low:high BMI = 4.6).</p

Counts of Probes with Significant Gene Expression Differences by Examined Traits.

<p>Traits are shown in the first column. Gene counts for False Discovery Rate (FDR) ≤ 0.05 are shown in column 3. Multiple regression analysis was conducted on individual probes, while including age, sex, and batch number (2 indicator variables) as variables in the model.</p><p>Counts of Probes with Significant Gene Expression Differences by Examined Traits.</p

Characteristics of VAGES Gene Expression Study Participants for Selected Metabolic Syndrome (MS)-Related Traits.

<p>ND = Non diabetics; BMI = body mass index; WC = waist circumference; FPG = fasting plasma glucose; HbA1c = glycated hemoglobin; FPI = fasting plasma insulin; HOMA-IR = homeostasis model assessment of insulin resistance; Matsuda-ISI = Matsuda insulin sensitivity index; Adipo-IR = adipocyte insulin resistance index; TC = total cholesterol; HDL = high density lipoprotein cholesterol; TG = triglycerides; SBP = systolic blood pressure; DBP = diastolic blood pressure.</p><p>Characteristics of VAGES Gene Expression Study Participants for Selected Metabolic Syndrome (MS)-Related Traits.</p

Candidate Gene List.

<p>Shown are the genes whose expression level (based on one or more probes) was significantly correlated with one or more clinical traits at FDR ≤ E-04. Genes (N = 29), indicated by their official HGNC symbol in the first column, are ordered from top to bottom according to the FDR value in the second column and secondly by the number of shared traits shown in the third column.</p><p>Candidate Gene List.</p

Correlation of <i>ADH1B</i> Expression with Key Traits (II).

<p>Log₂ transformed gene expression measurements of <i>ADH1B</i> mRNA obtained by Illumina BeadArray were analyzed for correlation with <b>A</b> lnHOMA-IR, <b>B</b> lnFFA, <b>C</b> lnFPG. Variables were log transformed to minimize the issue of non-normality.</p

Comparisons of Gene Expression Methods (A) and Correlations between Three Key Obesity/Insulin Resistance Trait Pairs (B-D).

<p>Panel <b>A</b> shows the correlation of <i>ADH1B</i> expression measurements made separately with Illumina Beadarrays and qRT-PCR. Panels <b>B-D</b> show correlations between respectively, lnBMI vs. lnWC, lnWC vs. lnFPI, and lnFPI vs. lnBMI. Data were log transformed to minimize the issue of non-normality. qRT-PCR = quantitative real time polymerase chain reaction.</p