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The effects of high-intensity interval training on reverse cholesterol transport elements: A way of cardiovascular protection against atherosclerosis
Accepted Manuscript The effects of high-intensity interval training on reverse cholesterol transport elements: A way of cardiovascular protection against atherosclerosis
Saleh Rahmati-Ahmadabad, Hossein Shirvani, Abbass GhanbariNiaki, Fatemeh Rostamkhani PII: DOI: Reference:
S0024-3205(18)30487-9 doi:10.1016/j.lfs.2018.08.036 LFS 15883
To appear in:
Life Sciences
Received date: Revised date: Accepted date:
10 June 2018 11 August 2018 16 August 2018
Please cite this article as: Saleh Rahmati-Ahmadabad, Hossein Shirvani, Abbass Ghanbari-Niaki, Fatemeh Rostamkhani , The effects of high-intensity interval training on reverse cholesterol transport elements: A way of cardiovascular protection against atherosclerosis. Lfs (2018), doi:10.1016/j.lfs.2018.08.036
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ACCEPTED MANUSCRIPT The Effects of High-Intensity Interval Training on Reverse Cholesterol Transport Elements: a Way of Cardiovascular Protection against Atherosclerosis
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Running head: Cardiovascular Protection against Atherosclerosis by HIIT
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Exercise Physiology Research Center, Life Style Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran
Exercise Biochemistry Division, Faculty of Sport Sciences, University of Mazandaran, Babolsar, Mazandaran, Iran Department of Biology, College of Basic Sciences, Yadegar-e-Imam Khomeini (RAH) Shahre Rey Branch, Islamic Azad University, Tehran, Iran
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Department of Physical education, Pardis branch, Islamic Azad University, Pardis, Iran
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Saleh Rahmati-Ahmadabad1 , Hossein Shirvani*2, Abbass Ghanbari-Niaki3, Fatemeh Rostamkhani4
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*Corresponding author: Hossein Shirvani
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Exercise Physiology Research Center, Baqiyatallah University of Medical Sciences, Nosrati alley, Sheikh Bahaei Street, Mollasadra Street, Vanak Square, Tehran, Iran. Post Office Box: 19395-5487 Tel: +98-21-82482395, Fax: +98-21-88600030 Email: [email protected]
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Reverse cholesterol transport (RCT) is a process that prevents atherosclerosis. Studies showed that exercise training for strenghthing cardiac muscle, increasing heart lipid metabolism and its potency against risk factors could protect cardiovascular health. Thus, the present study aims to investigate the effects of high intensity interval training (HIIT) on RCT and its related elements in plasma and tissues (liver and intestine) of rats. Materials and Methods
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Twenty adult male Wistar rats were randomly divided into control (n =10) and trained (n = 10) groups. The trained group undertook HIIT (90%-95% of VO2max, five days/week, for 10 weeks) on a treadmill. The rats were killed five days after the last training session to minimize the effects of the last training session. Key findings
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A higher and significant ABCA1 mRNA was observed in the liver and intestine of trained rats. However, ABCG1 and LXR expressions only increased in the liver following the HIIT. These changes in the expression of the trained rats were accompanied by higher changes in plasma LCAT and HDL levels. Significance
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The responses of ABCA1, as a key player in plasma HDL biogenesis, are similar in liver and intestine tissues after the HIIT program. However, different responses of ABCG1 and LXR in the liver and intestine tissues of the trained rats confirm the main role of the liver than the intestine in HDL biogenes. Therefore, HIIT modality result in cardiovascular protection by increasing the expression of genes involved in RCT and biogenesis of HDL.
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Keywords: Cardioprotection, ATP-binding cassette transporter A1 (ABCA1), Liver X receptor (LXR), lecithin–cholesterol acyltransferase (LCAT), Scavenger receptor class b member 1 (SR-BI), Reverse cholesterol transport (RCT), High intensity interval training (HIIT)
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Graphic abstract
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Abbreviations: ABCA1: ATP-binding cassette transporter A1 ABCG1: ATP-binding cassette transporter G1 Apo A1: apolipoprotein A1 CETP: cholesterol ester transfer protein HIIT: high intensity interval training HDL: high-density lipoprotein LXR: liver X receptor LCAT: lecithin–cholesterol acyltransferase PLTP: phospholipid Transfer Protein PPAR: peroxisome proliferator-activated receptor RCT: reverse cholesterol transport SR-BI: scavenger receptor class b member 1 TC: total cholesterol TG: triglyceride WBC: with blood cell
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1. Introduction Currently, advanced technology and mechanical facilities have paved the way to a better life. However, such advancement has also brought many problems like inactivity. This kind of life often causes diabetes, hypertension, cardiovascular diseases, osteoarthritis, and cancers. Atherosclerosis and coronary heart diseases are the most common side effects, which can be influenced by many variables including body fat, metabolic disorders, metabolic syndrome, diabetes, and sedentary [1, 2]. Unlike sedentary lifestyle, physical training includes cardioprotective actions that leads to increased heat shock protein production, nitric oxide pathway involvement, cardiac antioxidant capacity, ATP-dependent potassium channel function, opioid system activation, reverse cholesterol transport (RCT) process [3-7]. RCT is a pathway that play a critical role regarding the return of excess cholesterol from peripheral tissues to the liver for excretion in the bile and finally the feces [8]; it is regulated strictly by liver X receptor (LXR) [9]. RCT is a critical mechanism of anti-atherogenecity of high-density lipoproteins (HDL); it is consisted of several main elements including ATPbinding cassette transporter A1 and G1 (ABCA1 and ABCG1), apolipoprotein A1 (Apo A-1), lecithin–cholesterol acyltransferase enzyme (LCAT), scavenger receptor class b member 1 (SR-BI) and etc [8]. RCT process involves removal of cholesterol and phospholipids from the Apo A-1 (Apo A-1 is a major protein component of HDL particles in plasma) cells briefly mediated by ABCA1. ABCA1 is a member 1 of transporter sub-family ABCA that has a critical role in cellular cholesterol regulation and phospholipid homeostasis. Deformation of HDL particles through esterification of cholesterol occurs by the LCAT enzyme. LCAT is an enzyme that converts free cholesterol into cholesterol ester [8, 10]. Exchange between HDL and other lipoproteins and lipid, transfer of more cholesterol to growing HDL particles take place by SR-BI and possibly ABCG1 [7]. ABCG1 is mainly mobilized to the cell surface to support cholesterol efflux [11]. Finally, hydrolysis of the HDL is mediated by different lipases, such as lipoprotein lipase, hepatic lipase, and endothelial lipase, and lipid exchange through phospholipid transfer protein (PLTP) and cholesterol ester transfer protein (CETP) [7, 8]. So far, many researchers have reported a connection between increased risks of cardiovascular diseases and increased levels of blood lipids; they have pointed out that such risks could be reduced by an increasing HDL level [12]. It seems that the main function of HDL is to transport cholesterol from peripheral cells to the liver for digestion into the bile [11, 13], thereby reducing the cholesterol deposits on arterial walls [14]. In addition, previous studies have shown that RCT elements gene is expressed in small intestine similar to the liver tissue. Liver is a metabolic tissue and excess cholesterol from peripheral tissues return to the liver for excretion into the bile. Small intestine lumen express several membrane-associated proteins in absorptive epithelial cells lining which mediate the influx of luminal cholesterol and its subsequent efflux [15]. Thus, small intestine, like the liver tissue, can be important in RCT process. Researchers have been trying to detect the components of the RCT process and its mechanisms that are affected by the exercise [16-23]. In the last decade, studies have been conducted on the impact of long term aerobic training on this process [24, 25]; while today, the high-intensity interval training (HIIT) method is very much considered due to the beneficial effects and lack of time to practice. It has been reported that HIIT affects exercise capacity, endocrine hormones, and fat mobilization more than other types of training with 5
ACCEPTED MANUSCRIPT lower time requirement [26]. However, no research has investigated the effects of HIIT on the process of RCT; the present study has focused on this issue for the first time. Overall, it possible that HIIT creates protection against atherosclerosis and some risk factors by increasing the expression of genes involved in the RCT process and facilitating the biogenesis of HDL as long term physical training does with less time. Thus, the present study examines the impact of HIIT on the expressions of ABCA1, ABCG1, SR-BI, and LXR genes as well as on the concentrations of LCAT and plasma lipids in rats. 2. Materials and Methods
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2.1. Animals
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Twenty male Wistar rats (12–14 weeks age, 280–300) were used in this study. The rats were randomly assigned into control (n = 10) and trained (n = 10) groups and housed in cages made up of PVC with a metal cap; the floors of the cages were covered with clean wood chips (five rat per cage); the temperature was kept at 22ºC (±2) and humidity at 45%–50%; a dark-lighting cycle (12 hours light, 12 hours of darkness) was maintained in the animal lab of the Medical Sciences of Baqiyatallah University, Tehran. The animals were fed with a standard chow obtained from Behaprvar Companyin Karaj; they were given free access to purified water in 500-ml bottles. The study was conducted according to the ethic committee rules and principles of the laboratory animal care agreement. The Medical Sciences of Baqiyatallah University, Tehran approved the conduct of this research (Ethical cod#IR.BMSU.REC.1394.421).
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2.2.Measurement of maximum oxygen consumption (VO2max) The method of calculating the maximum oxygen consumption was used (due to the lack of direct access to tools such as the respiratory gas analysis device) in accordance with the recent research of Hoydal et al. [27]. At first, 10 minutes warm-up was performed at a low speed (10 meters per minute). After the warm-up, the test was started with the rats running at 15 meters per minute for two minutes. The treadmill speed was then increased every two minutes at a rate of 0.03 meter per second (1.8–2 meters per minute) and thus the animals were unable to run. The speed in exhaustion (unable to run) stage was considered as 100% of maximum oxygen consumption and the least oxygen consumption was calculated as a percentage of this speed (Table 1). 2.3.Training protocol The familiarization of rats with the HIIT protocol was performed with 10 sessions for two weeks. On the first day of training, the rats were treated with the utmost precision;—they began to exercise with a very low and uniform speed. In later sessions, the rats progressed well and were consistent with the program; the interval exercises with low speed were used for familiarization with the periodic protocol for the rats that get adapted to the type of training and with the protocol. During the two weeks of exercise, time was increased (18 minutes in the main program). After two weeks, without any kind of problem in the protocol and the rat familiarization, the main exercises began and were completed in 10 weeks and five days a week.
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The exercise program protocol [28] had been designed with respect to the designing principles of intensive periodic programs to maximize the performance of the aerobic tract (oxygen uptake and oxidative capacity of skeletal muscles) in intensity nearly VO2max. Each session of HIIT implementation comprised 30 minutes of physical activity (Table 1). At the end of two weeks, the maximum oxygen consumption was measured; the rats began practicing in accordance with the training protocol based on a percentage of the maximum oxygen consumption (converted into meters per minute). At the end of two weeks, the maximum oxygen uptake test was estimated and new exercises were applied next week. The training group carried out HIIT workouts, including running with an intensity of 90%–95% of VO2max on a treadmill, in five sessions per week for 10 weeks that began at 18:00. At the same time, the control group was placed on a treadmill at a speed of two meters per minute for 15 minutes (to integrate the effects of stress with training group). At the time of HIIT, the slope of the treadmill was not changed (zero slope). The training protocol was continued for five days before killing the animals.
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2.4.Measurement of gene expressions RNA was extracted by a special kit based on the manufacturer’s instructions (Total RNA Extraction Kit, YektaTajhizAzma, Cat NO: YT 9065; 100–80 mg of tissues was used. Moreover, 1 microliter of oligo-dT18 and 10 microliter of RNA were pipetted together in the autoclaved tubes and then stored at PCR at 70°C for 5 minutes. After that, the tubes were put on ice and added to the special kit (YektaTajhizAzma, Cat NO: YT 4509); 12 µl of RNAsefree water was added to the mixture. The new tubes were put in a conventional PCR at 42°C for 60 minutes so that cDNA could be made. Next, the temperature was raised to 95°C until the enzyme (reverse transcriptase) could be inactivated. The prepared cDNA was stored at 20°C to be used for real-time PCR reactions. Real-time PCR was performed by the Corbet device. To measure each gene,7.5 µl of SYBR Green Master (SRBR Green Master Mix, YektaTajhizAzma Cat NO: YT 2551) was mixed with 5µl of RNAse-free water ; then 1µl forward primer and 1µl reverse primer with 0.5µl cDNA were added. The tubes were put in the real-time PCR with the program at 95°C for 5 minutes, then 30–45 times in a program at 90°C for 5 seconds, and finally, at 60°C for 10 seconds. The real-time quantity was determined by measuring the increase in fluorescence radiation; thus, the SYBR Green dye, bound to the double-stranded DNA, was received at the end of each amplification cycle. At the end of PCR, the DNA strands were melted by creating the melting curve by heating the samples gradually from 72°C to 95°C. The cycle threshold (CT), where an increase in the recorded fluorescence was more than that at the base line, was determined for each sample. The mRNA levels of ABCA1, ABCG1, LXR, and SR-BI were estimated based on the PCR efficiency and the deviation of CT of an unknown sample compared to the control using the 2 ^ -ΔΔCT method. Beta actin mRNA was used as the reference gene. In this method, the ΔCT value was obtained from the difference between the CT of gees and the CT of Beta-actin. The ΔΔCT values were calculated after the subtraction of ΔCT obtained from the control-saline from the ΔCT of another group. Next, the 2 ^ ΔΔCT value was calculated [29]. Hence, the gene expressions of all groups were expressed relatively, rather than only that of the control group. 7
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2.5. Measurement of plasma variables
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Direct Immuno Method was used to determine plasma HDL-C level (HDL-C Immuno FS, Pars Azmoun, Tehran, Iran); the intra-assay coefficient of variation and sensitivity of the method were 1.2% and 0.03 mmol/l, resectively. Enzymatic (GPO, Glycerol-3-Phosphate Oxidase) colorimetric method was used to determine plasma TG level (Pars Azmoun, Tehran, Iran); intra-assay coefficient of variation and sensitivity of the method were 2.2% and 1 mg/dL, respectively. Enzymatic (CHOD-PAP, Cholesterol Oxidase-Amino Antipyrine) colorimetric method was used to determine plasma TC level (Pars Azmoun, Tehran, Iran); the intra-assay coefficient of variation and sensitivity of the method were 1.9% and 0.08 mmol/L, respectively. Plasma Fluorometric Method (LCAT activity assay kit, Calbiochem, USA) was used to determine plasma LCAT activity. 2.6. Statistical analysis
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Descriptive statistics were used to classify and determine the distribution parameters. The Kolmogorov–Smirnov test was used to detect data distribution. To investigate the changes between the groups, ANOVA test and LSD were used. Correlation was calculated by the Pearson Product Moment correlation. All significant levels were considered as P<0.05 (using the SPSS Software, Version 19). 3. Results As the results show, a higher and significant ABCA1 mRNA expression was observed in the liver and intestine-trained tissues (P=0.001, and P=0.001, respectively) (Fig.1 A and B). However, the ABCG1 and LXR mRNA expressions increased only in the liver (P=0.003 and P=0.013, respectively) (Fig.2 and Fig.3) and not in the trained intestine tissues (P=0.06, and P=0.07) (Table.3) following the HIIT program. Regarding SR-BI, a significant change was observed only in the trained intestine tissues (P=0.005) (Fig.4), but not in the trained liver tissue (P=0.17) (Table.3). Moreover, the changes in the liver ABCA1 and ABCG1 expressions were accompanied by higher and significant levels of plasma HDL (P=0.001) (Fig.5) and LCAT (P=0.001)(Fig.6) in HIIT animals as compared with the control group (Table. 3). Besides, the changes in the plasma TC and TG levels were not significant (Table.4).
4. Discussion It has been reported that physical training induced cardioprotection via several mechanism including the change in coronary circulation, cardiac HSPs, COX II activity, ER stress protein, myocardial antioxidant, autophagy, RCT process and etc [30-34]. The goal of the present study was to investigate the effects of HIIT protocol training whose employed in this study on exercise-induced possible changes on RCT elements towards cardiovascular protection via activating major players which are originating from the main sources of native and nascent high-density lipoprotein (HDL) in liver and intestine tissues. In this regards, the present study focused on the main elements which are involved in HDL biogenesis such as ABCA1, ABCG1, LXR, SR-BI gene expressions as well as the plasma HDL, LCAT, TC, and TG levels. Our results indicated that HIIT protocol employed in the present study had 8
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more pronounced effects on intestine ABCA1 and SR-BI mRNA in contrast to other measured variables. The results also indicate that these changes were accompanied with an increase in plasma HDL and LCAT levels. Ghanbari–Niaki et al. (2007) reported that 6 weeks of treadmill running (60 min/ session, 25m/min, 5days / week) resulted in a significant increase in liver ABCA1 mRNA expression and plasma HDL levels [24]. The same results were also reported by Khabazian et al (2009). In this study, a higher level of plasma pre-betaHDL and LCAT was accompanied by a higher level of ABCA1 mRNA in the rats’ small intestine [35]. A treadmill-running program (at 34m/min,60min/day, 5days/week, and for 12 weeks) resulted in a higher ABCA1 mRNA expression in the rats’ liver and heart tissues—this is accompanied by significant increases in plasma Apo-A and pre-betaHDL levels [18]. Other studies investigated the expression of ABCA1 mRNA in peripheral blood and found a significant increase shortly after with all given exercise intensities (40%, 60%, and 80% of 1RM) [20]. In their research, Hoang et al. have found that more physically active people have more expressions of the ABCA1 gene in white blood cell (WBC) [36]. All studies investigating the effects of endurance training on RCT have shown that the beneficial effects of exercise, but no research has examined the effects of HIIT. The present study is the first of its kind that applies HIIT to the components of the RCT process, and there is no information available on the impact of this type of training. However, the present research is consistent with all studies in this field and saves time using the cardiovascular benefits of long-term training with moderate and light intensity. There is no comprehensive information available about the mechanisms of physical activity on the expression of genes involved in the reverse cholesterol transport process, but there are some possible mechanisms. It has been shown that liver X receptor are involved in the process of reverse cholesterol transport [37]. However, it has been reported that peroxisome proliferator-activated receptors (PPARs) have receptors like LXR that regulate the expression of genes controlling fat. Spangenburg et al. have examined changes in the PPAR gene expressions in skeletal muscles after acute and chronic training in relation to the role of physical activity on the PPARs gene expression and found changes in the PPAR variety in any of the training types [38]. Butcher et al. have examined the effects of eight weeks endurance training on the expression of the LXR gene, thereby showing this gene increased expression. They have suggested that the activation of the PPAR ligand leads to the initial activation of LXR and eventually LXR causes upregulation of ABCA1 and ABCG1, while all these factors will increase the reverse cholesterol transport process [25].Generally, the results of the present study showed that the response of ABCA1 in HDL biogenesis is similar in both the liver and intestine tissues after the HIIT program. However, different responses of ABCG1 and LXR in the trained liver and intestine might confirm the main role of the liver than the intestine in HDL biogenesis after the HIIT program (Fig. 7). The HIIT program-induced increases in the plasma HDL level means that the HIIT could increase the RCT process and may be considered as an effective exercise modality to improve cardiovascular functions and health. Wright (2013) published a review article under the title of ‘recent clinical trials evaluating the benefit of drug therapy for modification of HDL cholesterol’ [39]. In this review, several large clinical trials with CTEP inhibitors showed that HDL could be increased with pharmacologic agents, but the increase in HDL level had no benefit on clinical outcome [39]. It means that cardioprotective effects of HDL depended on CTEP as an element of RCT process. Thus, we suggest that 9
ACCEPTED MANUSCRIPT cardioprotective effects of HDL take place just via changes in RCT elements that need to be further investigated. Thus, the result of present study is important in terms of cardioprotective effects of HIIT via increase in RCT elements. Overall, HIIT promotes cardiovascular protective effects against atherosclerosis by increasing the expression of genes involved in the RCT process and facilitating the biogenesis of HDL.
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5. Conclusions The aim of the present study was to investigate the effects of HIIT protocol on RCT elements towards cardiovascular protection. The responses of ABCA1 are similar in liver and intestine tissues after the HIIT program. However, different responses of ABCG1 and LXR in the liver and intestine tissues of the trained rats confirm the main role of the liver than the intestine in HDL biogenes. HIIT modality result in cardiovascular protection by increasing the expression of genes involved in RCT and biogenesis of HDL (Fig. 7).
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Acknowledgments The study is the result of an approved research project at the Baqiyatallah University of Medical Sciences under the supervision of Hossein Shirvani. The authors appreciate the students participating in the research.
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Compliance with Ethical Standards Conflict of Interest The authors declare that they have no conflict of interest. Research Involving Human Participants and/or Animals Twentymale Wistar rats (12–14 weeks age, 280–300g)were employed in this study. Informed Consent None. Funding None. Ethical Approval The study was conducted on the basis of the ethic committee rules and the principles of the laboratory animal care agreement. Permission for it was obtained from the Medical Sciences of Baqiyatallah University, Tehran (Ethical cod#IR.BMSU.REC.1394.421). Authors’ contributions SRA conceived and designed this study. SRA collected the materials, performed the experiments, analyzed the data and wrote the manuscript. AGN, HS and FR revised and improved the quality of the paper. All authors read and approved the final version of the manuscript.
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[19] Ghanbari-Niaki A, Zare-Kookandeh N, Zare-Kookandeh A. ABCG5 gene responses to treadmill running with or without administration of Pistachio atlantica in female rats, Iranian journal of basic medical sciences. 17(2014):162-71 [20] Ghanbari-Niaki A, Saghebjoo M, Hedayati M. A single session of circuit-resistance exercise effects on human peripheral blood lymphocyte ABCA1 expression and plasma HDL-C level, Regulatory peptides. 166(2011):42-7.10.1016/j.regpep.2010.08.001 [21] Tofighi A, Rahmani F, Jamali Qarakhanlou B, Babaei S. The effect of regular aerobic exercise on reverse cholesterol transport A1 and apo lipoprotein a-I gene expression in inactive women, Iranian Red Crescent medical journal. 17(2015):e26321.10.5812/ircmj.17(4)2015.26321 [22] Rocco DD, Okuda LS, Pinto RS, Ferreira FD, Kubo SK, Nakandakare ER, Quintao EC, Catanozi S, Passarelli M. Aerobic exercise improves reverse cholesterol transport in cholesteryl ester transfer protein transgenic mice, Lipids. 46(2011):617-25.10.1007/s11745-011-3555-z [23] Meissner M, Nijstad N, Kuipers F, Tietge UJ. Voluntary exercise increases cholesterol efflux but not macrophage reverse cholesterol transport in vivo in mice, Nutrition & metabolism. 7(2010):54.10.1186/1743-7075-7-54 [24] Ghanbari-Niaki A, Khabazian BM, Hossaini-Kakhak SA, Rahbarizadeh F, Hedayati M. Treadmill exercise enhances ABCA1 expression in rat liver, Biochemical and biophysical research communications. 361(2007):841-6.10.1016/j.bbrc.2007.07.100 [25] Butcher LR, Thomas A, Backx K, Roberts A, Webb R, Morris K. Low-intensity exercise exerts beneficial effects on plasma lipids via PPARgamma, Medicine and science in sports and exercise. 40(2008):1263-70.10.1249/MSS.0b013e31816c091d [26] Jung ME, Bourne JE, Little JP. Where does HIT fit? An examination of the affective response to high-intensity intervals in comparison to continuous moderate- and continuous vigorous-intensity exercise in the exercise intensity-affect continuum, PloS one. 9(2014):e114541.10.1371/journal.pone.0114541 [27] Hoydal MA, Wisloff U, Kemi OJ, Ellingsen O. Running speed and maximal oxygen uptake in rats and mice: practical implications for exercise training, European journal of cardiovascular prevention and rehabilitation : official journal of the European Society of Cardiology, Working Groups on Epidemiology & Prevention and Cardiac Rehabilitation and Exercise Physiology. 14(2007):753-60.10.1097/HJR.0b013e3281eacef1 [28] Rahmati-Ahmadabad S, Azarbayjani M ,Nasehi M. The Effects of High-Intensity Interval Training with Supplementation of Flaxseed Oil on BDNF mRNA Expression and Pain Feeling in Male Rats, Annals of Applied Sport Science. 0(2017):0[29] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method, Methods (San Diego, Calif). 25(2001):4028.10.1006/meth.2001.1262 [30] Laughlin MH, Bowles DK, Duncker DJ. The coronary circulation in exercise training, American journal of physiology Heart and circulatory physiology. 302(2012):H1023.10.1152/ajpheart.00574.2011 [31] Borges JP, Lessa MA. Mechanisms Involved in Exercise-Induced Cardioprotection: A Systematic Review, Arq Bras Cardiol. 105(2015):71-81.10.5935/abc.20150024 [32] Powers SK, Smuder AJ, Kavazis AN, Quindry JC. Mechanisms of exercise-induced cardioprotection, Physiology (Bethesda). 29(2014):27-38.10.1152/physiol.00030.2013 [33] Golbidi S, Laher I. Molecular mechanisms in exercise-induced cardioprotection, Cardiol Res Pract. 2011(2011):972807.10.4061/2011/972807 [34] Starnes JW, Taylor RP. Exercise-induced cardioprotection: endogenous mechanisms, Medicine and science in sports and exercise. 39(2007):1537-43.10.1249/mss.0b013e3180d099d4 [35] Khabazian BM, Ghanbari-Niaki A, Safarzadeh-Golpordesari A, Ebrahimi M, Rahbarizadeh F, Abednazari H. Endurance training enhances ABCA1 expression in rat small intestine, European journal of applied physiology. 107(2009):351-8.10.1007/s00421-009-1133-3 [36] Hoang A, Tefft C, Duffy SJ, Formosa M, Henstridge DC, Kingwell BA, Sviridov D. ABCA1 expression in humans is associated with physical activity and alcohol consumption, Atherosclerosis. 197(2008):197-203.10.1016/j.atherosclerosis.2007.03.017
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[37] Hoekstra M, Kruijt JK, Van Eck M ,Van Berkel TJ. Specific gene expression of ATP-binding cassette transporters and nuclear hormone receptors in rat liver parenchymal, endothelial, and Kupffer cells, The Journal of biological chemistry. 278(2003):25448-53.10.1074/jbc.M301189200 [38] Spangenburg EE, Brown DA, Johnson MS, Moore RL. Alterations in peroxisome proliferator-activated receptor mRNA expression in skeletal muscle after acute and repeated bouts of exercise, Mol Cell Biochem. 332(2009):225-31.10.1007/s11010-009-0195-1 [39] Wright RS. Recent clinical trials evaluating benefit of drug therapy for modification of HDL cholesterol, Curr Opin Cardiol. 28(2013):389-98.10.1097/HCO.0b013e328362059d
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ACCEPTED MANUSCRIPT Tables and figures:
Table1. high intensity interval training protocol [28] Warm up
Main body of training (3 intervals) High intensity Low intensity 4 minutes 2 minutes
Intensity of training (Vo2max) First & second week (m/min) Third & fourth week (m/min) Fifth & sixth week (m/min) Seventh & Eight week (m/min) Ninth & tenth week (m/min)
50- 60%
90- 100%
50- 60%
50- 60%
17-20
31-34
17-20
17-20
22-26
40-44
22-26
22-26
25-30
45-50
28-37
50-56
31-37
54-62
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** The slope of the treadmill was 0 degrees at all stages of training.
Cool down 6 minutes
45-50
25-30
28-37
28-37
31-37
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6 minutes
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Training steps Training components Time of training
Table2. The specific primers used in Real- time PCR Reverse primer
ABCA1 ABCG1 LXR SR-BI β-actin
GCATCCACCCCACTCTCTTC TCCTCTCGGTCCAAGCCATA TGGAGAACTCAAAGATGGGGTT GCTGCTTGATGAGGGAGGG AGCACTGTGTTGGCATAGAGG
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Forward primer
Base pairs
ACGAGATTGATGACCGCCTC GCCATCCCTGTCTTGCTCTT CGAGGTGATGCTTCTGGAGAC GGCTGCTGTTTGCTGCG TATCGGCAATGAGCGGTTCC
109 143 146 63 145
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I, ABCA1
.425
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L, ABCG1
.324
.433
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I, ABCG1
-.016
.611**
.305
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L, LXR
.559*
.429
.227
-.152
1
I, LXR
.191
.499*
.023
.059
.744**
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L, SRBI
.318
.416
.182
.339
-.127
-.100
1
I, SRBI
.415
.597**
.248
.253
.216
-.044
.231
1
P, TC
.260
.177
.050
-.082
.488*
.468*
-.392
.190
1
P, TG
.393
.079
.016
-.250
.277
.153
-.010
.043
.118
1
P, HDL
.701**
.446*
.664**
.227
.195
-.032
.312
.354
.009
.268
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P, LCAT
.488*
.674**
.589**
.409
.617**
.080
.093
.672**
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-.022
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.135
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.429
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L, ABCA1
Table4. Mean and standard deviation of non-significant variables HIIT
P
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1.22±0.34
0.06
3.967
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1.19±.0.42
0.17
1.960
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Variable Intestine ABCG1 Liver SRBI Intestine LXR Plasma TG Plasma TC
1.52±0.86
0.07
3.738
82.4±13.70
0.68
0.168
87.7±12.45
94.80±11.86
0.20
1.703
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80.2±9.99
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P, LCAT
P, HDL
P, TG
P, TC
I, SRBI
L, SRBI
I, LXR
L, LXR
I, ABCG1
L, ABCG1
I, ABCA1
Variables
L, ABCA1
Table3. Pearson correlation between variabeles. P: Plasma, L: liver, I: Intestine. *Correlation is significant at the 0.05 level.** Correlation is significant at the 0.01 level.
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Intestine ABCA1 gene expression
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Liver ABCA1 gene expression
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*P=0.001 3 2
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Figure 1. Intestine (A) and liver (B) ABCA1/β-actin gene expression in different groups of wild- type male Rats. Data are expressed as means ± SD. There are 10 rats in each group; HIIT: high intensity interval training.
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Liver ABCG1 gene expression
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Liver LXR gene expression
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Figure 2. Liver ABCG1/β-actin gene expression in different groups of wild- type male Rats. Data are expressed as means ± SD. There are 10 rats in each group. HIIT: high intensity interval training.
0
Control
HIIT
Figure 3. Liver LXR/β-actin gene expression in different groups of wild- type male Rats. Data are expressed as means ± SD. There are 10 rats in each group HIIT: high intensity interval training.
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*P=0.005
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Intestine SRBI gene expression
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70 60 50
30 20 10
*P=0.001
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40
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Plasma HDL concentration
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Figure 4. Intestine SRBI/β-actin gene expression in different groups of wild- type male Rats. Data are expressed as means ± SD. There are 10 rats in each group. HIIT: high intensity interval training.
0
Control
HIIT
Figure 5. Plasma HDL-C (mg/dl) level in different groups of wild- type male Rats. Data are expressed as means ± SD. There are 10 rats in each group. HIIT: high intensity interval training.
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Plasma LCAT concentration
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Figure 6. Plasma LCAT (mg/dl) level in different groups of wild- type male Rats. Data are expressed as means ± SD. There are 10 rats in each group. HIIT: high intensity interval training.
Figure 7. HIIT via RCT elements exert cardioprotective effects potentially
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Saleh Rahmati-Ahmadabad, Hossein Shirvani, Abbass GhanbariNiaki, Fatemeh Rostamkhani PII: DOI: Reference:
S0024-3205(18)30487-9 doi:10.1016/j.lfs.2018.08.036 LFS 15883
To appear in:
Life Sciences
Received date: Revised date: Accepted date:
10 June 2018 11 August 2018 16 August 2018
Please cite this article as: Saleh Rahmati-Ahmadabad, Hossein Shirvani, Abbass Ghanbari-Niaki, Fatemeh Rostamkhani , The effects of high-intensity interval training on reverse cholesterol transport elements: A way of cardiovascular protection against atherosclerosis. Lfs (2018), doi:10.1016/j.lfs.2018.08.036
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ACCEPTED MANUSCRIPT The Effects of High-Intensity Interval Training on Reverse Cholesterol Transport Elements: a Way of Cardiovascular Protection against Atherosclerosis
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Running head: Cardiovascular Protection against Atherosclerosis by HIIT
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Exercise Physiology Research Center, Life Style Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran
Exercise Biochemistry Division, Faculty of Sport Sciences, University of Mazandaran, Babolsar, Mazandaran, Iran Department of Biology, College of Basic Sciences, Yadegar-e-Imam Khomeini (RAH) Shahre Rey Branch, Islamic Azad University, Tehran, Iran
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Department of Physical education, Pardis branch, Islamic Azad University, Pardis, Iran
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Saleh Rahmati-Ahmadabad1 , Hossein Shirvani*2, Abbass Ghanbari-Niaki3, Fatemeh Rostamkhani4
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*Corresponding author: Hossein Shirvani
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Exercise Physiology Research Center, Baqiyatallah University of Medical Sciences, Nosrati alley, Sheikh Bahaei Street, Mollasadra Street, Vanak Square, Tehran, Iran. Post Office Box: 19395-5487 Tel: +98-21-82482395, Fax: +98-21-88600030 Email: [email protected]
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Reverse cholesterol transport (RCT) is a process that prevents atherosclerosis. Studies showed that exercise training for strenghthing cardiac muscle, increasing heart lipid metabolism and its potency against risk factors could protect cardiovascular health. Thus, the present study aims to investigate the effects of high intensity interval training (HIIT) on RCT and its related elements in plasma and tissues (liver and intestine) of rats. Materials and Methods
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Twenty adult male Wistar rats were randomly divided into control (n =10) and trained (n = 10) groups. The trained group undertook HIIT (90%-95% of VO2max, five days/week, for 10 weeks) on a treadmill. The rats were killed five days after the last training session to minimize the effects of the last training session. Key findings
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A higher and significant ABCA1 mRNA was observed in the liver and intestine of trained rats. However, ABCG1 and LXR expressions only increased in the liver following the HIIT. These changes in the expression of the trained rats were accompanied by higher changes in plasma LCAT and HDL levels. Significance
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The responses of ABCA1, as a key player in plasma HDL biogenesis, are similar in liver and intestine tissues after the HIIT program. However, different responses of ABCG1 and LXR in the liver and intestine tissues of the trained rats confirm the main role of the liver than the intestine in HDL biogenes. Therefore, HIIT modality result in cardiovascular protection by increasing the expression of genes involved in RCT and biogenesis of HDL.
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Keywords: Cardioprotection, ATP-binding cassette transporter A1 (ABCA1), Liver X receptor (LXR), lecithin–cholesterol acyltransferase (LCAT), Scavenger receptor class b member 1 (SR-BI), Reverse cholesterol transport (RCT), High intensity interval training (HIIT)
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Graphic abstract
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Abbreviations: ABCA1: ATP-binding cassette transporter A1 ABCG1: ATP-binding cassette transporter G1 Apo A1: apolipoprotein A1 CETP: cholesterol ester transfer protein HIIT: high intensity interval training HDL: high-density lipoprotein LXR: liver X receptor LCAT: lecithin–cholesterol acyltransferase PLTP: phospholipid Transfer Protein PPAR: peroxisome proliferator-activated receptor RCT: reverse cholesterol transport SR-BI: scavenger receptor class b member 1 TC: total cholesterol TG: triglyceride WBC: with blood cell
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1. Introduction Currently, advanced technology and mechanical facilities have paved the way to a better life. However, such advancement has also brought many problems like inactivity. This kind of life often causes diabetes, hypertension, cardiovascular diseases, osteoarthritis, and cancers. Atherosclerosis and coronary heart diseases are the most common side effects, which can be influenced by many variables including body fat, metabolic disorders, metabolic syndrome, diabetes, and sedentary [1, 2]. Unlike sedentary lifestyle, physical training includes cardioprotective actions that leads to increased heat shock protein production, nitric oxide pathway involvement, cardiac antioxidant capacity, ATP-dependent potassium channel function, opioid system activation, reverse cholesterol transport (RCT) process [3-7]. RCT is a pathway that play a critical role regarding the return of excess cholesterol from peripheral tissues to the liver for excretion in the bile and finally the feces [8]; it is regulated strictly by liver X receptor (LXR) [9]. RCT is a critical mechanism of anti-atherogenecity of high-density lipoproteins (HDL); it is consisted of several main elements including ATPbinding cassette transporter A1 and G1 (ABCA1 and ABCG1), apolipoprotein A1 (Apo A-1), lecithin–cholesterol acyltransferase enzyme (LCAT), scavenger receptor class b member 1 (SR-BI) and etc [8]. RCT process involves removal of cholesterol and phospholipids from the Apo A-1 (Apo A-1 is a major protein component of HDL particles in plasma) cells briefly mediated by ABCA1. ABCA1 is a member 1 of transporter sub-family ABCA that has a critical role in cellular cholesterol regulation and phospholipid homeostasis. Deformation of HDL particles through esterification of cholesterol occurs by the LCAT enzyme. LCAT is an enzyme that converts free cholesterol into cholesterol ester [8, 10]. Exchange between HDL and other lipoproteins and lipid, transfer of more cholesterol to growing HDL particles take place by SR-BI and possibly ABCG1 [7]. ABCG1 is mainly mobilized to the cell surface to support cholesterol efflux [11]. Finally, hydrolysis of the HDL is mediated by different lipases, such as lipoprotein lipase, hepatic lipase, and endothelial lipase, and lipid exchange through phospholipid transfer protein (PLTP) and cholesterol ester transfer protein (CETP) [7, 8]. So far, many researchers have reported a connection between increased risks of cardiovascular diseases and increased levels of blood lipids; they have pointed out that such risks could be reduced by an increasing HDL level [12]. It seems that the main function of HDL is to transport cholesterol from peripheral cells to the liver for digestion into the bile [11, 13], thereby reducing the cholesterol deposits on arterial walls [14]. In addition, previous studies have shown that RCT elements gene is expressed in small intestine similar to the liver tissue. Liver is a metabolic tissue and excess cholesterol from peripheral tissues return to the liver for excretion into the bile. Small intestine lumen express several membrane-associated proteins in absorptive epithelial cells lining which mediate the influx of luminal cholesterol and its subsequent efflux [15]. Thus, small intestine, like the liver tissue, can be important in RCT process. Researchers have been trying to detect the components of the RCT process and its mechanisms that are affected by the exercise [16-23]. In the last decade, studies have been conducted on the impact of long term aerobic training on this process [24, 25]; while today, the high-intensity interval training (HIIT) method is very much considered due to the beneficial effects and lack of time to practice. It has been reported that HIIT affects exercise capacity, endocrine hormones, and fat mobilization more than other types of training with 5
ACCEPTED MANUSCRIPT lower time requirement [26]. However, no research has investigated the effects of HIIT on the process of RCT; the present study has focused on this issue for the first time. Overall, it possible that HIIT creates protection against atherosclerosis and some risk factors by increasing the expression of genes involved in the RCT process and facilitating the biogenesis of HDL as long term physical training does with less time. Thus, the present study examines the impact of HIIT on the expressions of ABCA1, ABCG1, SR-BI, and LXR genes as well as on the concentrations of LCAT and plasma lipids in rats. 2. Materials and Methods
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Twenty male Wistar rats (12–14 weeks age, 280–300) were used in this study. The rats were randomly assigned into control (n = 10) and trained (n = 10) groups and housed in cages made up of PVC with a metal cap; the floors of the cages were covered with clean wood chips (five rat per cage); the temperature was kept at 22ºC (±2) and humidity at 45%–50%; a dark-lighting cycle (12 hours light, 12 hours of darkness) was maintained in the animal lab of the Medical Sciences of Baqiyatallah University, Tehran. The animals were fed with a standard chow obtained from Behaprvar Companyin Karaj; they were given free access to purified water in 500-ml bottles. The study was conducted according to the ethic committee rules and principles of the laboratory animal care agreement. The Medical Sciences of Baqiyatallah University, Tehran approved the conduct of this research (Ethical cod#IR.BMSU.REC.1394.421).
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2.2.Measurement of maximum oxygen consumption (VO2max) The method of calculating the maximum oxygen consumption was used (due to the lack of direct access to tools such as the respiratory gas analysis device) in accordance with the recent research of Hoydal et al. [27]. At first, 10 minutes warm-up was performed at a low speed (10 meters per minute). After the warm-up, the test was started with the rats running at 15 meters per minute for two minutes. The treadmill speed was then increased every two minutes at a rate of 0.03 meter per second (1.8–2 meters per minute) and thus the animals were unable to run. The speed in exhaustion (unable to run) stage was considered as 100% of maximum oxygen consumption and the least oxygen consumption was calculated as a percentage of this speed (Table 1). 2.3.Training protocol The familiarization of rats with the HIIT protocol was performed with 10 sessions for two weeks. On the first day of training, the rats were treated with the utmost precision;—they began to exercise with a very low and uniform speed. In later sessions, the rats progressed well and were consistent with the program; the interval exercises with low speed were used for familiarization with the periodic protocol for the rats that get adapted to the type of training and with the protocol. During the two weeks of exercise, time was increased (18 minutes in the main program). After two weeks, without any kind of problem in the protocol and the rat familiarization, the main exercises began and were completed in 10 weeks and five days a week.
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The exercise program protocol [28] had been designed with respect to the designing principles of intensive periodic programs to maximize the performance of the aerobic tract (oxygen uptake and oxidative capacity of skeletal muscles) in intensity nearly VO2max. Each session of HIIT implementation comprised 30 minutes of physical activity (Table 1). At the end of two weeks, the maximum oxygen consumption was measured; the rats began practicing in accordance with the training protocol based on a percentage of the maximum oxygen consumption (converted into meters per minute). At the end of two weeks, the maximum oxygen uptake test was estimated and new exercises were applied next week. The training group carried out HIIT workouts, including running with an intensity of 90%–95% of VO2max on a treadmill, in five sessions per week for 10 weeks that began at 18:00. At the same time, the control group was placed on a treadmill at a speed of two meters per minute for 15 minutes (to integrate the effects of stress with training group). At the time of HIIT, the slope of the treadmill was not changed (zero slope). The training protocol was continued for five days before killing the animals.
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2.4.Measurement of gene expressions RNA was extracted by a special kit based on the manufacturer’s instructions (Total RNA Extraction Kit, YektaTajhizAzma, Cat NO: YT 9065; 100–80 mg of tissues was used. Moreover, 1 microliter of oligo-dT18 and 10 microliter of RNA were pipetted together in the autoclaved tubes and then stored at PCR at 70°C for 5 minutes. After that, the tubes were put on ice and added to the special kit (YektaTajhizAzma, Cat NO: YT 4509); 12 µl of RNAsefree water was added to the mixture. The new tubes were put in a conventional PCR at 42°C for 60 minutes so that cDNA could be made. Next, the temperature was raised to 95°C until the enzyme (reverse transcriptase) could be inactivated. The prepared cDNA was stored at 20°C to be used for real-time PCR reactions. Real-time PCR was performed by the Corbet device. To measure each gene,7.5 µl of SYBR Green Master (SRBR Green Master Mix, YektaTajhizAzma Cat NO: YT 2551) was mixed with 5µl of RNAse-free water ; then 1µl forward primer and 1µl reverse primer with 0.5µl cDNA were added. The tubes were put in the real-time PCR with the program at 95°C for 5 minutes, then 30–45 times in a program at 90°C for 5 seconds, and finally, at 60°C for 10 seconds. The real-time quantity was determined by measuring the increase in fluorescence radiation; thus, the SYBR Green dye, bound to the double-stranded DNA, was received at the end of each amplification cycle. At the end of PCR, the DNA strands were melted by creating the melting curve by heating the samples gradually from 72°C to 95°C. The cycle threshold (CT), where an increase in the recorded fluorescence was more than that at the base line, was determined for each sample. The mRNA levels of ABCA1, ABCG1, LXR, and SR-BI were estimated based on the PCR efficiency and the deviation of CT of an unknown sample compared to the control using the 2 ^ -ΔΔCT method. Beta actin mRNA was used as the reference gene. In this method, the ΔCT value was obtained from the difference between the CT of gees and the CT of Beta-actin. The ΔΔCT values were calculated after the subtraction of ΔCT obtained from the control-saline from the ΔCT of another group. Next, the 2 ^ ΔΔCT value was calculated [29]. Hence, the gene expressions of all groups were expressed relatively, rather than only that of the control group. 7
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2.5. Measurement of plasma variables
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Direct Immuno Method was used to determine plasma HDL-C level (HDL-C Immuno FS, Pars Azmoun, Tehran, Iran); the intra-assay coefficient of variation and sensitivity of the method were 1.2% and 0.03 mmol/l, resectively. Enzymatic (GPO, Glycerol-3-Phosphate Oxidase) colorimetric method was used to determine plasma TG level (Pars Azmoun, Tehran, Iran); intra-assay coefficient of variation and sensitivity of the method were 2.2% and 1 mg/dL, respectively. Enzymatic (CHOD-PAP, Cholesterol Oxidase-Amino Antipyrine) colorimetric method was used to determine plasma TC level (Pars Azmoun, Tehran, Iran); the intra-assay coefficient of variation and sensitivity of the method were 1.9% and 0.08 mmol/L, respectively. Plasma Fluorometric Method (LCAT activity assay kit, Calbiochem, USA) was used to determine plasma LCAT activity. 2.6. Statistical analysis
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Descriptive statistics were used to classify and determine the distribution parameters. The Kolmogorov–Smirnov test was used to detect data distribution. To investigate the changes between the groups, ANOVA test and LSD were used. Correlation was calculated by the Pearson Product Moment correlation. All significant levels were considered as P<0.05 (using the SPSS Software, Version 19). 3. Results As the results show, a higher and significant ABCA1 mRNA expression was observed in the liver and intestine-trained tissues (P=0.001, and P=0.001, respectively) (Fig.1 A and B). However, the ABCG1 and LXR mRNA expressions increased only in the liver (P=0.003 and P=0.013, respectively) (Fig.2 and Fig.3) and not in the trained intestine tissues (P=0.06, and P=0.07) (Table.3) following the HIIT program. Regarding SR-BI, a significant change was observed only in the trained intestine tissues (P=0.005) (Fig.4), but not in the trained liver tissue (P=0.17) (Table.3). Moreover, the changes in the liver ABCA1 and ABCG1 expressions were accompanied by higher and significant levels of plasma HDL (P=0.001) (Fig.5) and LCAT (P=0.001)(Fig.6) in HIIT animals as compared with the control group (Table. 3). Besides, the changes in the plasma TC and TG levels were not significant (Table.4).
4. Discussion It has been reported that physical training induced cardioprotection via several mechanism including the change in coronary circulation, cardiac HSPs, COX II activity, ER stress protein, myocardial antioxidant, autophagy, RCT process and etc [30-34]. The goal of the present study was to investigate the effects of HIIT protocol training whose employed in this study on exercise-induced possible changes on RCT elements towards cardiovascular protection via activating major players which are originating from the main sources of native and nascent high-density lipoprotein (HDL) in liver and intestine tissues. In this regards, the present study focused on the main elements which are involved in HDL biogenesis such as ABCA1, ABCG1, LXR, SR-BI gene expressions as well as the plasma HDL, LCAT, TC, and TG levels. Our results indicated that HIIT protocol employed in the present study had 8
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more pronounced effects on intestine ABCA1 and SR-BI mRNA in contrast to other measured variables. The results also indicate that these changes were accompanied with an increase in plasma HDL and LCAT levels. Ghanbari–Niaki et al. (2007) reported that 6 weeks of treadmill running (60 min/ session, 25m/min, 5days / week) resulted in a significant increase in liver ABCA1 mRNA expression and plasma HDL levels [24]. The same results were also reported by Khabazian et al (2009). In this study, a higher level of plasma pre-betaHDL and LCAT was accompanied by a higher level of ABCA1 mRNA in the rats’ small intestine [35]. A treadmill-running program (at 34m/min,60min/day, 5days/week, and for 12 weeks) resulted in a higher ABCA1 mRNA expression in the rats’ liver and heart tissues—this is accompanied by significant increases in plasma Apo-A and pre-betaHDL levels [18]. Other studies investigated the expression of ABCA1 mRNA in peripheral blood and found a significant increase shortly after with all given exercise intensities (40%, 60%, and 80% of 1RM) [20]. In their research, Hoang et al. have found that more physically active people have more expressions of the ABCA1 gene in white blood cell (WBC) [36]. All studies investigating the effects of endurance training on RCT have shown that the beneficial effects of exercise, but no research has examined the effects of HIIT. The present study is the first of its kind that applies HIIT to the components of the RCT process, and there is no information available on the impact of this type of training. However, the present research is consistent with all studies in this field and saves time using the cardiovascular benefits of long-term training with moderate and light intensity. There is no comprehensive information available about the mechanisms of physical activity on the expression of genes involved in the reverse cholesterol transport process, but there are some possible mechanisms. It has been shown that liver X receptor are involved in the process of reverse cholesterol transport [37]. However, it has been reported that peroxisome proliferator-activated receptors (PPARs) have receptors like LXR that regulate the expression of genes controlling fat. Spangenburg et al. have examined changes in the PPAR gene expressions in skeletal muscles after acute and chronic training in relation to the role of physical activity on the PPARs gene expression and found changes in the PPAR variety in any of the training types [38]. Butcher et al. have examined the effects of eight weeks endurance training on the expression of the LXR gene, thereby showing this gene increased expression. They have suggested that the activation of the PPAR ligand leads to the initial activation of LXR and eventually LXR causes upregulation of ABCA1 and ABCG1, while all these factors will increase the reverse cholesterol transport process [25].Generally, the results of the present study showed that the response of ABCA1 in HDL biogenesis is similar in both the liver and intestine tissues after the HIIT program. However, different responses of ABCG1 and LXR in the trained liver and intestine might confirm the main role of the liver than the intestine in HDL biogenesis after the HIIT program (Fig. 7). The HIIT program-induced increases in the plasma HDL level means that the HIIT could increase the RCT process and may be considered as an effective exercise modality to improve cardiovascular functions and health. Wright (2013) published a review article under the title of ‘recent clinical trials evaluating the benefit of drug therapy for modification of HDL cholesterol’ [39]. In this review, several large clinical trials with CTEP inhibitors showed that HDL could be increased with pharmacologic agents, but the increase in HDL level had no benefit on clinical outcome [39]. It means that cardioprotective effects of HDL depended on CTEP as an element of RCT process. Thus, we suggest that 9
ACCEPTED MANUSCRIPT cardioprotective effects of HDL take place just via changes in RCT elements that need to be further investigated. Thus, the result of present study is important in terms of cardioprotective effects of HIIT via increase in RCT elements. Overall, HIIT promotes cardiovascular protective effects against atherosclerosis by increasing the expression of genes involved in the RCT process and facilitating the biogenesis of HDL.
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5. Conclusions The aim of the present study was to investigate the effects of HIIT protocol on RCT elements towards cardiovascular protection. The responses of ABCA1 are similar in liver and intestine tissues after the HIIT program. However, different responses of ABCG1 and LXR in the liver and intestine tissues of the trained rats confirm the main role of the liver than the intestine in HDL biogenes. HIIT modality result in cardiovascular protection by increasing the expression of genes involved in RCT and biogenesis of HDL (Fig. 7).
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Acknowledgments The study is the result of an approved research project at the Baqiyatallah University of Medical Sciences under the supervision of Hossein Shirvani. The authors appreciate the students participating in the research.
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Compliance with Ethical Standards Conflict of Interest The authors declare that they have no conflict of interest. Research Involving Human Participants and/or Animals Twentymale Wistar rats (12–14 weeks age, 280–300g)were employed in this study. Informed Consent None. Funding None. Ethical Approval The study was conducted on the basis of the ethic committee rules and the principles of the laboratory animal care agreement. Permission for it was obtained from the Medical Sciences of Baqiyatallah University, Tehran (Ethical cod#IR.BMSU.REC.1394.421). Authors’ contributions SRA conceived and designed this study. SRA collected the materials, performed the experiments, analyzed the data and wrote the manuscript. AGN, HS and FR revised and improved the quality of the paper. All authors read and approved the final version of the manuscript.
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ACCEPTED MANUSCRIPT Tables and figures:
Table1. high intensity interval training protocol [28] Warm up
Main body of training (3 intervals) High intensity Low intensity 4 minutes 2 minutes
Intensity of training (Vo2max) First & second week (m/min) Third & fourth week (m/min) Fifth & sixth week (m/min) Seventh & Eight week (m/min) Ninth & tenth week (m/min)
50- 60%
90- 100%
50- 60%
50- 60%
17-20
31-34
17-20
17-20
22-26
40-44
22-26
22-26
25-30
45-50
28-37
50-56
31-37
54-62
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** The slope of the treadmill was 0 degrees at all stages of training.
Cool down 6 minutes
45-50
25-30
28-37
28-37
31-37
31-37
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6 minutes
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Training steps Training components Time of training
Table2. The specific primers used in Real- time PCR Reverse primer
ABCA1 ABCG1 LXR SR-BI β-actin
GCATCCACCCCACTCTCTTC TCCTCTCGGTCCAAGCCATA TGGAGAACTCAAAGATGGGGTT GCTGCTTGATGAGGGAGGG AGCACTGTGTTGGCATAGAGG
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Forward primer
Base pairs
ACGAGATTGATGACCGCCTC GCCATCCCTGTCTTGCTCTT CGAGGTGATGCTTCTGGAGAC GGCTGCTGTTTGCTGCG TATCGGCAATGAGCGGTTCC
109 143 146 63 145
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I, ABCA1
.425
1
L, ABCG1
.324
.433
1
I, ABCG1
-.016
.611**
.305
1
L, LXR
.559*
.429
.227
-.152
1
I, LXR
.191
.499*
.023
.059
.744**
1
L, SRBI
.318
.416
.182
.339
-.127
-.100
1
I, SRBI
.415
.597**
.248
.253
.216
-.044
.231
1
P, TC
.260
.177
.050
-.082
.488*
.468*
-.392
.190
1
P, TG
.393
.079
.016
-.250
.277
.153
-.010
.043
.118
1
P, HDL
.701**
.446*
.664**
.227
.195
-.032
.312
.354
.009
.268
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P, LCAT
.488*
.674**
.589**
.409
.617**
.080
.093
.672**
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-.022
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.135
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.429
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L, ABCA1
Table4. Mean and standard deviation of non-significant variables HIIT
P
F
1
1.22±0.34
0.06
3.967
1
1.19±.0.42
0.17
1.960
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Control
PT E
Variable Intestine ABCG1 Liver SRBI Intestine LXR Plasma TG Plasma TC
1.52±0.86
0.07
3.738
82.4±13.70
0.68
0.168
87.7±12.45
94.80±11.86
0.20
1.703
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80.2±9.99
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P, LCAT
P, HDL
P, TG
P, TC
I, SRBI
L, SRBI
I, LXR
L, LXR
I, ABCG1
L, ABCG1
I, ABCA1
Variables
L, ABCA1
Table3. Pearson correlation between variabeles. P: Plasma, L: liver, I: Intestine. *Correlation is significant at the 0.05 level.** Correlation is significant at the 0.01 level.
1
Intestine ABCA1 gene expression
4
Liver ABCA1 gene expression
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*P=0.001 3 2
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HIIT
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Control
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0
3
*P=0.001
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2 1 0
HIIT
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Figure 1. Intestine (A) and liver (B) ABCA1/β-actin gene expression in different groups of wild- type male Rats. Data are expressed as means ± SD. There are 10 rats in each group; HIIT: high intensity interval training.
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*P=0.003 3 2
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Control
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HIIT
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Liver ABCG1 gene expression
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*P=0.01
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Liver LXR gene expression
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Figure 2. Liver ABCG1/β-actin gene expression in different groups of wild- type male Rats. Data are expressed as means ± SD. There are 10 rats in each group. HIIT: high intensity interval training.
0
Control
HIIT
Figure 3. Liver LXR/β-actin gene expression in different groups of wild- type male Rats. Data are expressed as means ± SD. There are 10 rats in each group HIIT: high intensity interval training.
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4 3
*P=0.005
2
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Control
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0
HIIT
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Intestine SRBI gene expression
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70 60 50
30 20 10
*P=0.001
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40
AC
Plasma HDL concentration
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Figure 4. Intestine SRBI/β-actin gene expression in different groups of wild- type male Rats. Data are expressed as means ± SD. There are 10 rats in each group. HIIT: high intensity interval training.
0
Control
HIIT
Figure 5. Plasma HDL-C (mg/dl) level in different groups of wild- type male Rats. Data are expressed as means ± SD. There are 10 rats in each group. HIIT: high intensity interval training.
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70 60
*P=0.001
50 40 30
PT
20 10 0
HIIT
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Plasma LCAT concentration
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Figure 6. Plasma LCAT (mg/dl) level in different groups of wild- type male Rats. Data are expressed as means ± SD. There are 10 rats in each group. HIIT: high intensity interval training.
Figure 7. HIIT via RCT elements exert cardioprotective effects potentially
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