See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/343230150 Genetic variants in glutamate cysteine ligase confer protection against type 2 diabetes Article in Molecular Biology Reports · August 2020 DOI: 10.1007/s11033-020-05647-5 CITATIONS READS 0 115 5 authors, including: Iuliia Azarova Elena Klyosova Kursk State Medical University Kursk State Medical University 23 PUBLICATIONS 21 CITATIONS 17 PUBLICATIONS 8 CITATIONS SEE PROFILE SEE PROFILE Alexander Konoplya Alexey V Polonikov Kursk State Medical University Kursk State Medical University 9 PUBLICATIONS 12 CITATIONS 212 PUBLICATIONS 2,100 CITATIONS SEE PROFILE SEE PROFILE Some of the authors of this publication are also working on these related projects: Type 2 diabetes mellitus View project All content following this page was uploaded by Iuliia Azarova on 28 July 2020. The user has requested enhancement of the downloaded file. Molecular Biology Reports https://doi.org/10.1007/s11033-020-05647-5 ORIGINAL ARTICLE Genetic variants in glutamate cysteine ligase confer protection against type 2 diabetes Iuliia Azarova1,2 · Elena Klyosova2 · Victor Lazarenko3 · Alexander Konoplya1 · Alexey Polonikov4,5 Received: 19 March 2020 / Accepted: 8 July 2020 © Springer Nature B.V. 2020 Abstract Oxidative stress contributes to the pathogenesis of type 2 diabetes (T2D). This study investigated whether single nucleotide polymorphisms (SNPs) at genes encoding glutamate cysteine ligase catalytic (rs12524494, rs17883901, rs606548, rs636933, rs648595, rs761142 at GCLC) and modifer (rs2301022, rs3827715, rs7517826, rs41303970 at GCLM) subunits are asso- ciated with susceptibility to type 2 diabetes. 2096 unrelated Russian subjects were enrolled for the study. Genotyping was done with the use of the MassArray System. Plasma levels of reactive oxygen species (ROS) and glutathione in the study subjects were analyzed by fuorometric and colorimetric assays, respectively.The present study found, for the frst time, an association of SNP rs41303970 in the GCLM gene with a decreased risk of T2D (P = 0.034, Q = 0.17). Minor alleles such as rs12524494-G GCLC gene (P = 0.026, Q = 0.17) and rs3827715-C GCLM gene (P = 0.03, Q = 0.17) were also associ- ated with reduced risk for T2D. Protective efects of variant alleles such as rs12524494-G at GCLC (P = 0.02, Q = 0.26) and rs41303970-A GCLM (P = 0.013, Q = 0.25) against the risk of T2D were seen solely in nonsmokers. As compared with healthy controls, diabetic patients had markedly increased levels of ROS and decreased levels of total GSH in plasma. Interestingly, fasting blood glucose level positively correlated with oxidized glutathione concentration (rs = 0.208, P = 0.01). Three SNPs rs17883901, rs636933, rs648595 at GCLC and one rs2301022 at GCLM were associated with decreased levels of ROS, while SNPs rs7517826, rs41303970 at GCLM were associated with increased levels of total GSH in plasma. Single nucleotide polymorphisms in genes encoding glutamate cysteine ligase subunits confer protection against type 2 diabetes and their efects are mediated through increased levels of glutathione. Keywords Type 2 diabetes mellitus · Oxidative stress · Reactive oxygen species · Glutathione · Glutamate cysteine ligase · Single nucleotide polymorphism Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s1103 3-020-05647 -5) contains supplementary material, which is available to authorized users. * Iuliia Azarova Kursk State Medical University, 18 Yamskaya St., Kursk, [email protected]; [email protected] Russian Federation 305041 Elena Klyosova 3 Department of Surgical Diseases of Postgraduate Faculty, [email protected] Kursk State Medical University, 3 Karl Marx Street, Kursk, Russian Federation 305041 Victor Lazarenko [email protected] 4 Department of Biology, Medical Genetics and Ecology, Kursk State Medical University, 3 Karl Marx Street, Kursk, Alexander Konoplya Russian Federation 305041 [email protected] 5 Laboratory of Statistical Genetics and Bioinformatics, Alexey Polonikov Research Institute for Genetic and Molecular Epidemiology, [email protected]; [email protected] Kursk State Medical University, 18 Yamskaya St., Kursk, Russian Federation 305041 1 Department of Biological Chemistry, Kursk State Medical University, 3 Karl Marx Street, Kursk, Russian Federation 305041 2 Laboratory of Biochemical Genetics and Metabolomics, Research Institute for Genetic and Molecular Epidemiology, Vol.:(0123456789)1 3 Molecular Biology Reports Introduction Methods Diabetes is one of the fastest growing health challenges Study population of the twenty-frst century, with the number of adults liv- ing with diabetes having more than tripled over the past The study protocol conforms to the ethical guidelines of the 20 years. Today, International Diabetes Federation calculates Declaration of Helsinki and was approved by the Regional that 9.3% of adults aged 20–79 years—a staggering 463 mil- Ethics Review Committee of Kursk State Medical Uni- lion people—sufer from diabetes worldwide, and more than versity. Written informed consent was obtained from each 8 million of them live in Russia [1]. T2D is a multifactorial participant before enrollment in the study. A total of 2096 disease with a strong genetic component [2, 3]. According unrelated Russian individuals were recruited into the study, to the GeneCards database, more than 700 genetic markers including 1032 T2D patients and 1064 age- and sex-matched are associated with T2D susceptibility [4], and a majority healthy individuals. T2D patients were admitted to the Divi- of these genetic variants have been implicated in pancreatic sion of Endocrinology of the Kursk Emergency Hospital β-cell dysfunction and peripheral insulin resistance [5, 6]. from November 2016 to December 2018. T2D was diag- There are many hypotheses regarding pathogenesis of nosed on the basis of WHO criteria [2]: fasting blood glu- T2D, including impaired pro- and antioxidant balance char- cose (FBG) level ≥ 7.0 mmol/L or random blood glucose acterized by increased reactive oxygen species (ROS) pro- level ≥ 11.1 mmol/L and/or glycated hemoglobin HbA1c duction and decreased antioxidant defense [7–9]. The redox level ≥ 6.5%. The control group included healthy volun- homeostasis has been investigated in numerous studies and it teers who presented at the Kursk Blood Transfusion Station has been revealed that patients with T2D have lower plasma within the same time frame as well as healthy individuals GSH levels [10–13] and higher static oxidation reduction recruited in our previous studies [23]. Subjects of the control potential [14] in comparison to healthy subjects. However, group had normal FBG levels and 75-g oral glucose toler- the causes and molecular mechanisms responsible for these ance test results. All study subjects were mainly from the metabolic changes in patients with type 2 diabetes remain Kursk region (Central Russia). Demographic, clinical and unclear. laboratory characteristics of the study groups are shown in Glutathione (GSH) is one of the major cellular antioxi- Table 1. A positive history of diabetes was greater in the dants and can be produced from glutamate, cysteine and case group than in the control group. Most patients with dia- glycine via the GSH cycle or via a regeneration reaction betes sufer from hypertension, and approximately one-third from the oxidized form of GSSG. Glutamate cysteine ligase of them had coronary artery disease (CAD). The number (GCL) is the enzyme catalyzing the frst rate-limiting step of smokers was higher among healthy subjects than among for a de novo biosynthesis of GSH [15]. Catalytic and mod- patients with diabetes. Two-thirds of the patients were ifer subunits of the enzyme are encoded by two distinct females. Most patients (88%) were overweight or obese. genes such as GCLC and GCLM, respectively. Functional Compared with the control group, diabetic patients showed polymorphisms of these genes could affect the enzyme signifcantly increased levels of glycated hemoglobin, FBG, activity and hence the production of GSH. Several stud- triacylglycerols, total cholesterol, low-density lipoproteins, ies reported the associations of rs17883901 at GCLC and and creatinine (Table 1). The levels of high-density lipopro- rs41303970 at GCLM genes with myocardial infarction and teins were higher in healthy subjects than in patients with acute coronary syndrome [16–18]. Other variants of GCL T2D. genes are associated with hemolytic anemia [19], stroke [20], blood cholesterol level, and schizophrenia [21, 22]. The relationship between these genes and T2D susceptibility Genetic analysis has been insufciently investigated. The present study was designed to investigate associa- Ten common tagSNPs at the GCLC (rs12524494, tions of SNPs rs12524494, rs17883901, rs606548, rs636933, rs17883901, rs606548, rs636933, rs648595, rs761142) and rs648595, rs761142 at GCLC and rs2301022, rs3827715, GCLM (rs2301022, rs3827715, rs7517826, rs41303970) rs7517826, and rs41303970 at GCLM with susceptibility to genes were selected for the study (Suppl. Table 1A). Func- T2D and to assess the impact of these gene polymorphisms tional SNPs were selected using a set of web-based SNP on pro- and antioxidant status and metabolic parameters in selection tools available at SNPinfo Web Server (https :// diabetic patients. snpin fo.niehs .nih.gov). The selection of SNPs was based on their predicted functional characteristics,