Plasma mitochondrial DNA is elevated in obese type 2 diabetes mellitus patients and correlates positively with insulin resistance

RESEARCH ARTICLE Plasma mitochondrial DNA is elevated in obese type 2 diabetes mellitus patients and correlates positively with insulin resistance Larysa V. Yuzefovych1, Viktor M. Pastukh1, Mykhaylo V. Ruchko1, Jon D. Simmons1,2, William O. Richards2, Lyudmila I. Rachek1* 1 Department of Pharmacology, College of Medicine, University of South Alabama, Mobile, Alabama, United States of America, 2 Department of Surgery, College of Medicine, University of South Alabama, Mobile, Alabama, United States of America a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Yuzefovych LV, Pastukh VM, Ruchko MV, Simmons JD, Richards WO, Rachek LI (2019) Plasma mitochondrial DNA is elevated in obese type 2 diabetes mellitus patients and correlates positively with insulin resistance. PLoS ONE 14(10): e0222278. https://doi.org/10.1371/journal. pone.0222278 Editor: Jonathan M Peterson, East Tennessee State University, UNITED STATES Received: February 19, 2019 Accepted: August 26, 2019 Published: October 10, 2019 Copyright: © 2019 Yuzefovych 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. Data Availability Statement: All relevant data are within the manuscript and Supporting Information files. Funding: This study was supported in part by the George H. A. Clowes Memorial Research Career Development Award from the American College of Surgeons (https://www.facs.org/member-services/ scholarships/research/acsclowes) to JDS. The funder had no role in study design, data collection * Abstract Cells damaged by mechanical or infectious injury release proinflammatory mitochondrial DNA (mtDNA) fragments into the circulation. We evaluated the relation between plasma levels of mtDNA fragments in obese type 2 diabetes mellitus (T2DM) patients and measures of chronic inflammation and insulin resistance. In 10 obese T2DM patients and 12 healthy control (HC) subjects, we measured levels of plasma cell-free mtDNA with quantitative real-time polymerase chain reaction, and mtDNA damage in skeletal muscle with quantitative alkaline Southern blot. Also, markers of systemic inflammation and oxidative stress in skeletal muscle were measured. Plasma levels of mtDNA fragments, mtDNA damage in skeletal muscle and plasma tumor necrosis factor α levels were greater in obese T2DM patients than HC subjects. Also, the abundance of plasma mtDNA fragments in obese T2DM patients levels positively correlated with insulin resistance. To the best of our knowledge, this is the first published evidence that elevated level of plasma mtDNA fragments is associated with mtDNA damage and oxidative stress in skeletal muscle and correlates with insulin resistance in obese T2DM patients. Plasma mtDNA may be a useful biomarker for predicting and monitoring insulin resistance in obese patients. Introduction Insulin resistance in obese patients and the associated disease cluster of type 2 diabetes mellitus (T2DM), hyperlipidemia, and hypertension are major global health problems. Obesity is associated with chronic, low-grade inflammation, known as metabolic inflammation or metaflammation [1], which is considered a pivotal point in the initiation and progression of insulin resistance and T2DM. Mitochondrial dysfunction induced by oxidative stress contributes to obesity-related insulin resistance [2–4], but the relationship between mitochondrial dysfunction and the pathogenesis of insulin resistance is unknown. Damage to mitochondrial DNA (mtDNA) may disrupt transcription of proteins encoded by mtDNA that are essential for energy metabolism, initiate apoptotic cell death, and alter mitochondrial redox signaling PLOS ONE | https://doi.org/10.1371/journal.pone.0222278 October 10, 2019 1 / 14 Circulating mitochondrial DNA in diabetes and obesity and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. [5–9]. In support of the concept that oxidative mtDNA damage contributes to T2DM, we previously showed that damage to mtDNA increases mitochondrial oxidative stress and insulin resistance in skeletal muscle cell [10,11]. Moreover, in a mouse model of insulin resistance induced by a high-fat diet, we showed that mtDNA damage is associated with mitochondrial dysfunction and increased oxidative stress in skeletal muscle and liver [12]. Fragments of mtDNA known as mtDNA damage-associated molecular patterns (DAMPs) may be intercellular mediators of inflammation [13,14]. Such mtDNA fragments are released into the circulation after injury or sepsis and are believed to propagate damage from the initial site of injury or infection to distant organs [15,16]. Inflammation may be propagated by mtDNA DAMPs via activation of one or more pro-inflammatory nucleic acid receptors, including the toll-like receptor 9 (TLR9), NLRP3 inflammasome, and cyclic guanosine monophosphate–adenosine monophosphate synthase–stimulator of interferon genes (cGAS-STING) [13–16]. Since obesity is associated with metainflammation the major goal of the current study was to determine whether obese T2DM patients display elevated contents of plasma mtDNA and whether plasma mtDNA correlates with insulin resistance. Our results comprise the first preliminary evidence in a small group of obese, predominantly women patients, that increased levels of plasma mtDNA fragments correlate with the degree of insulin resistance in obese T2DM patients. Furthermore, obese T2DM patients have significantly increased mtDNA damage and oxidative stress markers in skeletal muscle, which was accompanied with increased systemic inflammation. This study suggests there may be novel therapeutic strategies for reducing insulin resistance and for the design of new biomarkers to measure insulin resistance in humans. Methods Subjects We recruited 10 obese (body mass index >35 kg/m2) T2DM patients who had hemoglobin A1C levels > 6.5% and a diagnosis of T2DM based on fasting plasma glucose level > 126 mg/ dL or current treatment with any oral hypoglycemic drug. De-identified obese diabetic patients were participants in an ongoing research project conducted by WOR in the Department of Surgery, University of South Alabama College of Medicine. We recruited 12 volunteer healthy control (HC) subjects without obesity (body mass index < 30 kg/m2) or T2DM from the general community. All subjects were sedentary. All human studies including the source study for recruited T2DM patients were conducted according to the principles of the Declaration of Helsinki and approved by the Institutional Review Board (protocols #10–131, 11–150) of the University of South Alabama. All human subjects gave informed written consent. Metabolic parameters and muscle biopsy Each subject had a medical history, physical exami (...truncated)