Does DNA Methylation of PPARGC1A Influence Insulin Action in First Degree Relatives of Patients with Type 2 Diabetes? Linn Gillberg 1,2 *, Stine Jacobsen 1,2 , Rasmus Ribel-Madsen 3 , Anette Prior Gjesing 3 , Trine W. Boesgaard 2,4 , Charlotte Ling 5 , Oluf Pedersen 3,4,6 , Torben Hansen 3,7 , Allan Vaag 1,2,8 1 Department of Endocrinology, Rigshospitalet, Copenhagen, Denmark, 2 Steno Diabetes Center, Gentofte, Denmark, 3 Section of Metabolic Genetics, The Novo Nordisk Foundation Center for Basic Metabolic Research, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark, 4 Hagedorn Research Institute, Gentofte, Denmark, 5 Department of Clinical Sciences, Lund University, Malmoe, Sweden, 6 Faculty of Health Sciences, University of Aarhus, Aarhus, Denmark, 7 Faculty of Health Sciences, University of Southern Denmark, Odense, Denmark, 8 Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark Abstract Epigenetics may play a role in the pathophysiology of type 2 diabetes (T2D), and increased DNA methylation of the metabolic master regulator peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A) has been reported in muscle and pancreatic islets from T2D patients and in muscle from individuals at risk of T2D. This study aimed to investigate DNA promoter methylation and gene expression of PPARGC1A in skeletal muscle from first degree relatives (FDR) of T2D patients, and to determine the association with insulin action as well as the influence of family relation. We included 124 Danish FDR of T2D patients from 46 different families. Skeletal muscle biopsies were excised from vastus lateralis and insulin action was assessed by oral glucose tolerance tests. DNA methylation and mRNA expression levels were measured using bisulfite sequencing and quantitative real-time PCR, respectively. The average PPARGC1A methylation at four CpG sites situated 867-624 bp from the transcription start was associated with whole-body insulin sensitivity in a paradoxical positive manner (b = 0.12, P = 0.03), supported by a borderline significant inverse correlation with fasting insulin levels (b = 20.88, P= 0.06). Excluding individuals with prediabetes and overt diabetes did not affect the overall result. DNA promoter methylation was not associated with PPARGC1A gene expression. The familiality estimate of PPARGC1A gene expression was high (h 2 = 79627% (h 2 6SE), P = 0.002), suggesting genetic regulation to play a role. No significant effect of familiality on DNA methylation was found. Taken together, increased DNA methylation of the PPARGC1A promoter is unlikely to play a major causal role for the development of insulin resistance in FDR of patients with T2D. Citation: Gillberg L, Jacobsen S, Ribel-Madsen R, Gjesing AP, Boesgaard TW, et al. (2013) Does DNA Methylation of PPARGC1A Influence Insulin Action in First Degree Relatives of Patients with Type 2 Diabetes? PLoS ONE 8(3): e58384. doi:10.1371/journal.pone.0058384 Editor: Anita Magdalena Hennige, University of Tu ¨ bingen, Germany Received September 20, 2012; Accepted February 4, 2013; Published March 7, 2013 Copyright: ß 2013 Gillberg et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by The Danish Strategic Research Council, The Danish Council for Independent Research, EASD, Exgenesis, Rigshospitalets forskningsudvalg and Steno Diabetes Center. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: linn.gillberg@rh.regionh.dk Introduction Type 2 diabetes (T2D) is a multifactorial and slowly progressing multiple organ disease where metabolic abnormalities eventually leading to hyperglycemia are established long before the overt T2D diagnosis has become manifest [1]. Both genetic and non- genetic factors influence the development of T2D, and it has been estimated that the susceptibility to develop T2D in individuals with a T2D parent is 3–4 times higher compared to the background population [2]. Indeed, significant defects of peripheral (muscle) and hepatic insulin action, as well as of pancreatic beta cell function, have been reported to be present decades before first degree relatives (FDR) of patients with T2D are supposed to develop the disease [3,4]. Genetic variants currently associated with susceptibility to T2D only explain up to 10% of the putative ‘‘primary’’ contribution to T2D risk [5] and it has therefore been debated whether the increased diabetes risk among FDR could be linked to epigenetic traits and not to classic genetic traits defined as alterations of the DNA sequence. However, little is known about the influence of heritability on epigenetic variation. DNA methylation represents the most studied epigenetic trait and it is generally believed that increased methylation in the promoter region of tissue specific genes may confer transcriptional repression [6,7]. This putative association has however been difficult to establish in several human studies [8–10]. Both DNA methylation and gene expression of the master metabolic regulator peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A) has been extensively studied in relation to T2D. PPARGC1A upregulates transcription of genes involved in mitochondrial oxidative metabolism and biogenesis as well as skeletal muscle glucose transport [11,12], and because mitochon- drial defects have been associated with peripheral insulin re- sistance in healthy subjects [13,14] it has been suggested that reduced PPARGC1A expression in skeletal muscle may be a primary feature of insulin resistance [11,15]. Furthermore, PPARGC1A may be involved in biological functions with implica- PLOS ONE | www.plosone.org 1 March 2013 | Volume 8 | Issue 3 | e58384