Transcriptional expression changes of glucose metabolism genes after exercise in thoroughbred horses Jeong-An Gim a , Selvam Ayarpadikannan a , Jungwoo Eo a , Yun-Jeong Kwon a , Yuri Choi a , Hak-Kyo Lee b , Kyung-Do Park b , Young Mok Yang c , Byung-Wook Cho d , Heui-Soo Kim a, ⁎ a Department of Biological Sciences, College of Natural Sciences, Pusan National University, Busan 609-735, Republic of Korea b Department of Biotechnology, Hankyong National University, Anseong 456-749, Republic of Korea c Department of Pathology, School of Medicine, and Institute of Biomedical Science and Technology, Konkuk University, Seoul 143-701, Republic of Korea d Department of Animal Science, College of Life Sciences, Pusan National University, Miryang 627-702, Republic of Korea abstract article info Article history: Received 8 March 2014 Received in revised form 15 May 2014 Accepted 23 June 2014 Available online xxxx Keywords: Thoroughbred racehorse Glucose metabolism gene MicroRNA Lactate dehydrogenase Glycogen synthase 1 Physical exercise induces gene expression changes that trigger glucose metabolism pathways in organisms. In the present study, we monitored the expression levels of LDHA (lactate dehydrogenase) and GYS1 (glycogen synthase 1) in the blood, to confirm the roles of these genes in exercise physiology. LDHA and GYS1 are related to glucose metabolism and fatigue recovery, and these processes could elicit economically important traits in racehorses. We collected blood samples from three retired thoroughbred racehorses, pre-exercise and immediately after 30 min of exercise. We extracted total RNA and small RNA (≤200 nucleotide-long) from the blood, and assessed the expression levels of LDHA, GYS1, and microRNAs (miRNAs), by using qRT-PCR. We showed that LDHA and GYS1 were down-regulated, whereas eca-miR-33a and miR-17 were up-regulated, after exercise. We used sequences from the 3′ UTR of LDHA and GYS1, containing eca-miR-33a and miR-17 bind- ing sites, to observe the down-regulation activity of each gene expression. We observed that the two miRNAs, namely, eca-miR-33a and miR-17, inhibited LDHA and GYS1 expression via binding to the 3′ UTR sequences of each gene. Our results indicate that eca-miR-33a and miR-17 play important roles in the glucose metabolism pathway. In addition, our findings provide a basis for further investigation of the exercise metabolism of racehorses. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Thoroughbred horses possess many genetic features related to rac- ing ability, robustness, recovery, and stamina. These traits can be distin- guished by using SNP (single-nucleotide polymorphism) or gene expression (Doan et al., 2012; Hill et al., 2010a,b). Despite the impor- tance of glucose metabolism genes in the selection of superior race- horses (Schroder et al., 2011), few published studies have focused on the characterization of these genes in thoroughbred horses. Physical ex- ercise induces changes in glucose metabolism; therefore, the mainte- nance of blood glucose metabolism in galloping horses is very important for homeostasis and recovery after exercise. In addition, physical exercise changes the physiological state of the body in various species, including the horse. Hence, the genes related to physiological traits have been analyzed in horses (Gu et al., 2009; Petersen et al., 2013; Suontama et al., 2013). Gene expression profiles have frequently been shown to change after exercise in horses (Eivers et al., 2010; Park et al., 2012), humans (Barres et al., 2012; Jemiolo and Trappe, 2004), mice (Handschin et al., 2007), and rats (Jin et al., 2000). Further, exercise-dependent differential expression of genes related to exercise physiology – including glucose transport, glucose metabolism, angiogenesis, myogenesis, mitochondrial biogenesis (Eivers et al., 2010; McGivney et al., 2010), actin and myosin (Jin et al., 2000; McGivney et al., 2010), and the extracellular matrix (Jin et al., 2000; Mienaltowski et al., 2009) – has previously been demonstrated. Physical exercise is classified into aerobic exercise and anaerobic ex- ercise, according to whether or not oxygen is required to exploit energy via metabolic pathways (Hughson et al., 2001; Laursen, 2010). When organisms undergo an intensive activity, aerobic exercise is concurrent with anaerobic exercise, because the energy demands exceed the output energy of the aerobic exercise pathway. In anaerobic exercise, lactate is generated through pyruvate fermentation; the lactate is accu- mulated in muscles, leading to fatigue (Duffield et al., 2005; Laursen, Gene xxx (2014) xxx–xxx Abbreviations: LDHA, lactate dehydrogenase; GYS1, glycogen synthase 1; miRNA, microRNA; M-MLV RT, Moloney-Murine Leukemia Virus Reverse Transcriptase; PBMC, pe- ripheral blood mononuclear cell. ⁎ Corresponding author. E-mail address: khs307@pusan.ac.kr (H.-S. Kim). GENE-39775; No. of pages: 7; 4C: http://dx.doi.org/10.1016/j.gene.2014.06.051 0378-1119/© 2014 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene Please cite this article as: Gim, J.-A., et al., Transcriptional expression changes of glucose metabolism genes after exercise in thoroughbred horses, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.06.051