Diabetes Volume 64, September 2015 3135 Sini Heinonen,1 Jana Buzkova,2 Maheswary Muniandy,1 Risto Kaksonen,1,3 Miina Ollikainen,4 Khadeeja Ismail,4 Antti Hakkarainen,5 Jesse Lundbom,5,6 Nina Lundbom,5 Katriina Vuolteenaho,7 Eeva Moilanen,7 Jaakko Kaprio,8,9,10 Aila Rissanen,1,11 Anu Suomalainen,2,12 and Kirsi H. Pietiläinen1,8,13 Impaired Mitochondrial Biogenesis in Adipose Tissue in Acquired Obesity Diabetes 2015;64:3135–3145 | DOI: 10.2337/db14-1937 Low mitochondrial number and activity have been sug- and OXPHOS proteins in SAT are downregulated in ac- gested as underlying factors in obesity, type 2 diabetes, quired obesity, and are associated with metabolic distur- and metabolic syndrome. However, the stage at which bances already at the preclinical stage. mitochondrial dysfunction manifests in adipose tissue after the onset of obesity remains unknown. Here we examined subcutaneous adipose tissue (SAT) samples Adipocytes are important contributors to energy balance – from healthy monozygotic twin pairs, 22.8 36.2 years of and metabolic homeostasis. Within these highly dynamic D > 2 age, who were discordant ( BMI 3kg/m, mean length cells, mitochondria are at the center of energy metabo- OBESITY STUDIES 6 n of discordance 6.3 0.3 years, = 26) and concordant lism, using carbohydrates, lipids, and proteins to produce D < 2 n ( BMI 3kg/m, = 14) for body weight, and assessed ATP and metabolites for growth, as well as contributing to their detailed mitochondrial metabolic characteristics: adipocyte differentiation and maturation (1). Mitochon- mitochondrial-related transcriptomes with dysregulated dria possess their own multicopy genome, a 16.6-kb cir- pathways, mitochondrial DNA (mtDNA) amount, mtDNA- cular mitochondrial DNA (mtDNA) that encodes two encoded transcripts, and mitochondrial oxidative phos- phorylation (OXPHOS) protein levels. We report global ribosomal RNAs (12S and 16S), 22 transfer RNAs, and expressional downregulation of mitochondrial oxidative 13 polypeptides (2). These proteins are the core catalytic pathways with concomitant downregulation of mtDNA components of electron transport chain complexes I, III, amount, mtDNA-dependent translation system, and pro- and IV, and ATP synthase (3). Together, these complexes tein levels of the OXPHOS machinery in the obese com- form the oxidative phosphorylation (OXPHOS) system in pared with the lean co-twins. Pathway analysis indicated the inner mitochondrial membrane. mtDNA-encoded pro- downshifting of fatty acid oxidation, ketone body produc- teins are translated on mitochondrial ribosomes that, to- tion and breakdown, and the tricarboxylic acid cycle, which gether with OXPHOS complexes, are the only cellular inversely correlated with adiposity, insulin resistance, entities encoded by both nuclear and mitochondrial and inflammatory cytokines. Our results suggest that genomes. However, ;1,500 other nuclear-encoded mitochondrial biogenesis, oxidative metabolic pathways, proteins—8% of all nuclear genes—encode mitochondrial 1Obesity Research Unit, Research Programs Unit, Diabetes and Obesity, Univer- 11Department of Psychiatry, Helsinki University Central Hospital, Helsinki, sity of Helsinki, Helsinki, Finland Finland 2Research Programs Unit, Molecular Neurology, Biomedicum-Helsinki, University 12Department of Neurology, Helsinki University Central Hospital, Helsinki, of Helsinki, Helsinki, Finland Finland 3Siluetti Hospital, Helsinki, Finland 13Abdominal Center, Endocrinology, Helsinki University Central Hospital and 4Department of Public Health, University of Helsinki, Helsinki, Finland University of Helsinki, Helsinki, Finland 5 Helsinki Medical Imaging Center, University of Helsinki, Helsinki, Finland Corresponding author: Kirsi H. Pietiläinen, kirsi.pietilainen@helsinki.fi. 6Institute for Clinical Diabetology, German Diabetes Center, Leibniz Center for Received 23 December 2014 and accepted 8 May 2015. Diabetes Research, Heinrich Heine University, Düsseldorf, Germany 7The Immunopharmacology Research Group, University of Tampere School of This article contains Supplementary Data online at http://diabetes Medicine and Tampere University Hospital, Tampere, Finland .diabetesjournals.org/lookup/suppl/doi:10.2337/db14-1937/-/DC1. 8Institute for Molecular Medicine Finland, University of Helsinki, Helsinki, S.H. and J.B. share first authorship. Finland © 2015 by the American Diabetes Association. Readers may use this article as 9Finnish Twin Cohort Study, Department of Public Health, Hjelt Institute, long as the work is properly cited, the use is educational and not for profit, and University of Helsinki, Helsinki, Finland the work is not altered. 10National Institute for Health and Welfare, Department of Mental Health and Substance Abuse Services, Helsinki, Finland 3136 Impaired Mitochondrial Function in Obesity Diabetes Volume 64, September 2015 targeted proteins, emphasizing the central role of mito- imager (Avanto; Siemens, Erlangen, Germany) (13). Physi- chondria in cellular metabolism (4). cal activity was assessed by the Baecke questionnaire and Evidence from various study systems suggests that its three subcompartments (sport, leisure time, and work mitochondrial function and biogenesis are compromised indexes). Parental weight, height, and age were asked at the in subcutaneous adipose tissue (SAT) in type 2 diabetes twin age of 16 years. (5), morbid obesity (6), and insulin resistance (7). Re- cently, a reduction of total oxygen consumption rates, Analytical Measurements but not mtDNA content, was shown to occur in visceral The concentrations of fasting plasma glucose, leptin, and subcutaneous adipocytes of obese adults (8), as well adiponectin, and adipsin, and serum hs-CRP were measured, as after b-adrenergic stimulation (9), suggesting a decrease and HOMA insulin resistance and Matsuda insulin sensi- in mitochondrial oxidative activity. We have previously tivity indexes were calculated, as previously described (16). shown in SAT of monozygotic (MZ) weight-discordant SAT Specimens and Analyses twin pairs that mtDNA depletion together with the down- Surgical biopsy samples of abdominal SAT under the regulation of mitochondrial branched-chain amino acid umbilicus were obtained under local anesthesia and were (BCAA) catabolism characterize obesity, insulin resistance, snap-frozen in liquid nitrogen. Based on the availability of fi fatty liver (10), and poor tness (11). However, we still sample material, the frozen SAT specimens were used for lack a thorough understanding of how mitochondrial bio- transcriptomics analyses (all 26 discordant and 14 concor- genesis and function in human adipose tissue changes in dant twin pairs), determination of the amount of mtDNA, obesity, at which stage this process begins, and whether it mtDNA-encoded transcript levels (15 discordant twin contributes to the early development of metabolic distur- pairs), and an OXPHOS protein analysis (seven discordant bances. Moreover, it is not known whether the possible twin pairs). Twenty-four discordant and 11 concordant link between mitochondrial dysfunction and obesity- twin pairs were available for DNA methylation analyses. related metabolic disorders is genetic or acquired. Clinical characteristics of the selected and unselected twin MZ twins discordant for obesity are, despite their dif- pairs were similar. Part of the fresh SAT sample was ferent phenotype, completely matched for genetic variants, digested with collagenase, and was used for the measure- age, and sex. This allows the research of acquired obesity ment and calculation of adipocyte volume and number in without interference of genetic and familial factors that all 26 discordant and 14 concordant twin pairs (16). The confound studies comparing groups of unrelated lean and discordant twin pairs were divided into two groups with obese individuals. Here, we focus on rare weight-discordant, regard to hyperplastic and hypertrophic obesity (16). healthy, young adult MZ twins to investigate in detail the mitochondrial pathways in SAT and their association with Transcriptomics and Pathway Analyses whole-body metabolism in acquired obesity. Total RNA was extracted from SAT by the RNeasy Lipid Tissue Mini Kit (Qiagen) and treated with DNase I RESEARCH DESIGN AND METHODS (Qiagen). Transcriptomics analyses were performed using Subjects the Affymetrix U133 Plus 2.0 array and were validated as The current study included 26 rare, healthy MZ pairs in the study by Naukkarinen et al. (14). Preprocessing of discordant for obesity (within-pair difference, DBMI $3 the expression data was performed using BioConductor kg/m2, n = 9 males, n = 17 females, mean age 29.9 6 0.6 and the GC-RMA algorithm. We first analyzed the differ- years), identified from two population-based twin entially expressed transcripts genome wide between the cohorts, FinnTwin16 (n = 2,839 pairs) and FinnTwin12 obese and the lean co-twins in the discordant pairs by (n = 2,578 pairs) (12). In addition, 14 MZ twin pairs paired moderated t tests (17), and then determined concordant for BMI (DBMI ,3 kg/m2, n = 9 males, n = whether they were found in MitoCarta (4), an online atlas 5 females, mean age 31.6 6 0.6 years) were included as of 1,013 human proteins with mitochondrial localization. control pairs and to provide a wider range of BMI values Forty-nine of the 1,013 MitoCarta protein transcripts for transcriptomics analyses. A detailed description of the were not detectable by the Affymetrix probes, including twin material has been published previously (13,14). the 13 mtDNA-encoded