GC / MS-based metabolomics strategy to analyze the effection of Exercise Intervention on Diabetic Rats

Metabolomics was used to explore the effection of exercise intervention on type 2 diabetes. The rat model of type 2 diabetes was induced by an injection of streptozocin (30 mg /kg), after fed with 8-week high-fat diet. The rats were divided into three groups, the control group, the diabetic model group (DM) and the diabetes + exercise group (DME). After exercise for 10 week, blood samples were collected to test biomedical indexes, and 24 h urine samples were collected for the metabolomics experiment. In the DME group, fasting blood glucose (FBG), both total cholesterol (TC) and total plasma triglycerides (TG) were decreased significantly, compared with those in the DM group. Based on gas chromatography-mass spectrometry (GC/MS), a urinary metabolomics method was used to study the mechanism of exercise intervention on diabetes mellitus. Based on the principal component analysis (PCA), it was found that the DM group and control group were separated into two different clusters. The DME group was located between the DM group and the control group, closer to the control group. Twelve significantly changed metabolites of diabetes mellitus were detected and identified , including Glycolate, 4-Methyl phenol, Benzoic acid, 1H-Indole, Arabinitol, Threitol, Ribonic acid, Malic acid, 2,3-Dihydroxy-butanoic, Aminomalonic acid, L-Ascorbic acid and 3-Hydroxy hexanedioic acid. After exercise, 7 metabolites were significantly changed,Compared with the control group, the relative contents of benzoic acid, aminomalonic acid, tetrabutyl alcohol and ribonucleic acid in the diabetic exercise group decreased significantly. The relative contents of 2,3-dihydroxybutyric acid, L-ascorbic acid and 3-hydroxy adipic acid increased significantly. L-ascorbic acid and aminomalonic acid which related with the oxidative stress were significantly regulated to normal. The results showed that exercise could display anti-hyperglycemic and anti-hyperlipidemic effects. The exercise had antioxidation function in preventing the occurrence of complications with diabetes mellitus to some extent. The work illustrates that the metabolomics method is a useful tool to study the mechanism of exercise treatment. Introduction The prevalence of type 2 diabetes (T2D) continues to increase. Within the next 20 years, the number of people affected by this disease is expected to reach almost 600 million in worldwide[1]. T2D is accompanied by a host of risk factors including dyslipidemia, hypertension and cardiovascular disease[2], thus putting a severe burden on our global health care systems. Exercise plays a crucial role in the prevention and treatment of several chronic diseases, including glucose intolerance states, type 2 diabetes[3,4] and diseases of the cardiovascular system[5,6]. Moreover, it has been demonstrated that exercise improves the quality of life in the general population[7]. Nevertheless, T2D patients are encouraged to exercise regularly as a means of improving social integration and cardiovascular health[8]. Thus, a better understanding of the effects of exercise on the metabolic response in T2D patients will allow clinicians to prescribe exercise to their patients with greater clarity. Urine is mainly composed of small hydrophilic molecules such as sugar, organic acids, amino acids, soluble lipids, organic amines and inorganic salts small enough to successfully pass through the human reticular endothelial filtration system. On the other hand, the non-invasive collection of urine is conducive to many metabolomic applications. Metabolomics enables the systematic assessment of the abundant changes of low molecular weight compounds present in biological Page 2 of 15 Accepted Manuscript published as EC-19-0012.R2. Accepted for publication: 30-Apr-2019 Copyright © 2019 the authors Downloaded from Bioscientifica.com at 05/04/2019 11:04:36PM via free access samples, using high-throughput sample analysis techniques and computer-assisted multivariate pattern-recognition techniques[9]. Metabolomics is enriching our current understanding of both the physiologic and physiopathologic processes underlying diabetes mellitus[10-12]. Moreover, recent metabolomic-based studies have described the first metabolic signatures of exercise in human plasma[13-15]. For example, Lewis et al[13] described the metabolic changes in tricarboxylic acid cycle, fatty acid oxidation and lypolisis in the plasma of healthy subjects exposed to different intensities and durations of exercise. The aim of this study is to analyze the metabolic changes induced by a 10 weeks of aerobic exercise performed by T2D rats. A comprehensive GC-MS untargeted metabolomics approach was applied to urine samples taken from all rats. Materials and methods Animals and Induction of diabetes Induction of diabetes 30 male Sprague Dawley rats, weighing 200-220 g were bred in the Center of Experimental Animal, Xuzhou Medical University, where an SPF level laboratory (24 ± 1 °C; humidity of 45% to 55%, 12:12 dark/light cycle) was founded, as authorized by the Jiangsu province government. Animals were allowed food and water ad libitum before and during the experiment. One week after feeding adaptation, the rats were randomly divided into control group and diabetic model (DM), and were given standard diet and high fat diet for 8 weeks, respectively. The rats were fasted for 12 hr and subjected to a single intraperitoneal injection of 30 mg/kg STZ, which was freshly dissolved in 100 mmol/l sodium citrate buffer (pH 4.5). Normal rats received sodium citrate buffer only. On the third day of STZ administration, the fasting blood glucose (FBG) value was determined by a reagent kit. Rats with FBG levels higher than 13.9 mmol/l were considered to be diabetic. All animal procedures were performed in accordance with the guidelines provided by the Experimental Animal Laboratory and approved by the Animal Care and Use Committee of the Chaohu University (Anhui China). Treadmill exercise protocol Before beginning the formal 10 weeks exercise protocol, animals were familiarized to treadmill running (5-20 min/day) for 5 consecutive days. After this period of habituation, the exercised animals performed 5 days of consecutive treadmill exercise (60 min/day) with 22 m/min speeds[16]. At the beginning of 60 min exercise, to warm up the rats, treadmill speed had been set at 5 m/min and progressively increased to 22 m/min. At the final of 60 minute exercise, the speed progressively decreased to 5 m/min to cool down. Mild electrical shock was used the negligible amount to motivate animals to run. Control animals did not carry out treadmill exercise but were placed on a nonmoving treadmill for 60 min/day for 5 days a week. Exercised animals were studied 24 h after their last exercise session.