Calpain-10 mRNA levels in human pancreatic islets are influenced by type 2 diabetes (a) and correlates with arginine-stimulated insulin release in non-diabetic islets (b).

<p>SI is the insulin stimulation index i.e. incremental folds above baseline insulin release. Results are expressed as mean±SEM.</p

Insulin secretion in response to glucose, arginine and glibenclamide in human non-diabetic and diabetic pancreatic islets cultured in vitro.

<p>SI is the insulin stimulation index i.e. incremental folds above baseline insulin release. Results are mean±SEM.</p

Association between SNP-43 and SNP-44 of the <i>CAPN10</i> gene and calpain-10 mRNA expression, glucose-, arginine- and glibenclamide-stimulated insulin secretion in human non-diabetic pancreatic islets cultured in vitro.

<p>Basal insulin secretion is analysed when culturing islets in 5.5 mmol/l glucose. Glucose stimulated insulin secretion (GSIS) is analysed after culturing islets in 16.7 mmol/l glucose for 45 min as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006558#s2" target="_blank">method</a> section.</p>1<p>µU/islet/minutes. SI is the insulin stimulation index i.e. incremental folds above baseline insulin release. Results are mean±SEM with number of individuals shown in parentheses.</p>2<p>For a dominant model. <i>P</i> values are not corrected for multiple testing.</p

Additional file 1: Table S1. of 36 h fasting of young men influences adipose tissue DNA methylation of LEP and ADIPOQ in a birth weight-dependent manner

Primers used in amplification of the specific DNA sequences of the ADIPOQ and LEP promoter regions. Table S2. DNA methylation (%) of CpG sites in the LEP promoter in adipose tissue. Table S3. DNA methylation (%) of CpG sites in the ADIPOQ promoter in adipose tissue. Table S4. DNA methylation (%) of CpG sites in the LEP promoter in adipose tissue. Table S5. DNA methylation (%) of CpG sites in the ADIPOQ promoter in adipose tissue. (DOCX 34 kb

Additional file 1: Table S1. of Diabetes medication associates with DNA methylation of metformin transporter genes in the human liver

Clinical characteristics of the 33 type 2 diabetic patients according to drug treatment and of 60 non-diabetic subjects. Table S2. Average and promoter DNA methylation according to diabetes medication, first comparing other oral medication such as Sitagliptin or Glimepiride + metformin to only metformin therapy, and second excluding all subjects that were receiving Sitagliptin or Glimepiride. Table S3. Clinical characteristics of subjects from the Kuopio Obesity Surgery Study. Table S4. DNA sequences of the primers used for pyrosequencing. Figure S1. DNA methylation of SLC22A3 and SLC47A1 in hepatocytes cultured in vitro after 8 h of metformin (0.5 mM), insulin plus metformin, insulin (100 nM) or glucose treatment in Huh-7 cells (n = 4). Means and standard deviations are shown, and paired t test was used for the analysis. Figure S2. Gene expression levels of the three metformin transporter genes in the human liver (A) (n = 42); gene expression of transporter genes according to diabetes medication after adjusting for age, sex and non-alcoholic steatohepatitis (NASH) (B). ns: no significant. (DOCX 213 kb

Distribution of CpG sites significantly associated with one or more mRNA transcripts, separated based on negative or positive correlations.

<p>(<b>A</b>) 20,376 combinations in the region 0–500 kb upstream of transcription start site and (<b>B</b>) 5,718 intragenic combinations. Negative correlations were enriched in the region surrounding the transcription start site, both (<b>C</b>) upstream and (<b>D</b>) downstream. (<b>E</b>) 5,221 combinations 0–100 kb downstream of the gene. Associations corrected for multiple testing using false discovery rate at 5% (Q<0.05).</p

Identified mQTL/eQTL candidate genes <i>GPX7</i>, <i>GSTT1</i> and <i>SNX19</i> affect β-cell number and apoptosis.

<p>Associations identified in the mQTL/eQTL analyses of human pancreatic islets. (<b>A</b>) rs835342 located approximately 5 kb upstream of <i>GPX7</i> associates with DNA methylation of cg18087326 located 406 bp upstream of the <i>GPX7</i> transcription start site (TSS) as well as with mRNA expression of <i>GPX7</i>. (<b>B</b>) rs4822453 located ∼121 kb downstream of <i>GSTT1</i> associates with DNA methylation of cg17005068 located 241 bp upstream of the <i>GSTT1</i> TSS as well as with mRNA expression of <i>GSTT1</i>. (<b>C</b>) rs3751035 located within exon 1 of <i>SNX19</i> associates with DNA methylation of cg08912652 located within the gene body of <i>SNX19</i> as well as with mRNA expression of <i>SNX19</i>. Data are presented as Box and Whisker plots with P-values adjusted for multiple testing. (<b>D</b>) qPCR quantification of siRNA mediated knockdown of <i>Gpx7</i> (siGpx7), <i>Gstt1</i> (siGstt1) and <i>Snx19</i> (siSnx19) compared to negative control siRNA (siNC). * P<0.01, the graphs show the average of four independent knockdown experiments presented as mean ± SEM. (<b>E</b>) Knockdown of <i>Gpx7</i> and <i>Gstt1</i> resulted in increased combined caspase-3/7 activity compared to negative control siRNA under both control (white bars) and lipotoxic (black bars) conditions. * P<0.05, the graph shows the average of three independent knockdown experiments presented as mean ± SEM. (<b>F</b>) Knockdown of <i>Snx19</i> (siSnx19) resulted in increased cell number compared to negative control siRNA (siNC) under both control (white bars) and lipotoxic (black bars) conditions. * P<0.05, the graph shows the average of six independent knockdown experiments presented as mean ± SEM.</p

CIT analysis identifies mQTLs where DNA methylation potentially mediates genetic associations with mRNA expression in human pancreatic islets.

<p>(<b>A</b>) Depiction of possible relationship models between genotype as a causal factor (G), DNA methylation as a potential mediator (M) and islet mRNA expression as a phenotypic outcome (E). Left diagram: The causal or methylation mediated model. Middle diagram: The reactive or methylation-consequential model (reverse causality). Right diagram: The independent model. (<b>B</b>) Illustration of the study approach to identify if DNA methylation of CpG sites potentially mediates the causal association between SNPs and islet mRNA expression. Left: Workflow steps. Middle: Tested relationships between G, M and E in the different steps. Right: Number of identified sites in each step. Bottom: Conditions that must be fulfilled to conclude a mathematical definition of a causal relationship between G, M and E. Significantly called as causal at 5% FDR (causal hypothesis Q<0.05).</p

KEGG pathways with enrichment of genes annotated to CpG sites of significant <i>cis</i>-mQTLs in human pancreatic islets.

<p>P-values have been adjusted for multiple testing using Benjamini-Hochberg.</p><p>KEGG pathways with enrichment of genes annotated to CpG sites of significant <i>cis</i>-mQTLs in human pancreatic islets.</p

Number of significant eQTL results in the human pancreatic islets.

<p>Only SNPs of significant mQTLs are included in the eQTL analysis.</p><p>SNPs of significant <i>cis</i>-mQTLs are regressed against mRNA expression of transcripts located in <i>cis</i> (≤500 kb).</p><p>SNPs of significant <i>trans</i>-mQTLs are regressed against mRNA expression of all transcripts.</p><p>Significance threshold <0.05 after correction for multiple testing.</p><p>Correction value of eQTL analysis for <i>cis</i>-mQTL-SNPs = 692, 616.</p><p>Correction value of eQTL analysis for <i>trans</i>-mQTL-SNPs = 16,982,420.</p><p>LD = linkage disequilibrium.</p><p>Number of significant eQTL results in the human pancreatic islets.</p