Effects of resistance exercise on lipolysis pathway in obese pre and postmenopausal women

Background and objectives: The purpose of study was to examine the effects of regular resistance exercise for 12 weeks on lipolysis pathway in pre- and post- menopausal women with obesity. Methods: Twenty-three pre- and post- menopausal women with body fat percentages of 30% or more divided into pre- menopausal group (n=9) and post- menopausal group (n=14). All subjects participated in resistance exercise training for 12 weeks. Anthropometric and physical tness tests were performed on all participants. Protein analyses were performed with subcutaneous fatty tissue extracted, and the samples were analyzed of relevant protein levels changes by using Western blotting. All serum samples were submitted for enzyme-linked immunosorbent assay measurements of adipocyte factors. Results: After 12 weeks between pre- menopausal and post- menopausal groups adipose triglyceride lipase (ATGL), monoacylglycerol lipase (MGL) and perilipin (PLIN) protein levels were signicantly lower in the post- menopausal group than in the pre- menopausal group. Hormone-sensitive lipase (HSL) protein levels were signicantly higher in the post- menopausal group than in the pre- menopausal group. In addition, leptin concentration was signicantly decreased after resistance exercise in the postmenopausal group. Adiponectin concentration was signicantly increased after resistance exercise in the both groups. Conclusions: This study indicates that regular resistance exercise to change of leptin and adiponectin might be release of reduction of % fat, and driving overall greater change ATGL, HSL, MGL and PLIN levels in subcutaneous fatty tissue in the obese post- menopausal group more than obese premenopausal group.


Introduction
Adipose tissue is recognized as an endocrine organ. A complex interplay between multiple endocrine mediators and the sympathetic nervous system has been shown to govern adipocyte metabolism. In the postprandial state, insulin promotes glucose and fatty acid uptake as well as lipogenesis and suppresses triglyceride lipolysis [1].
Excess body weight results from an imbalance between energy intake and energy expenditure [2], one way to maintain a correct body weight is to stimulate lipid catabolism through increased physical activity.
Properly designed training simulates fat breakdown, that is, hydrolysis of triacylglycerol stored in adipose tissue, releasing free fatty acids into the circulation and causing oxidation in muscles and other tissues [3]. Some studies demonstrated that low-intensity endurance training leads to maximal lipid oxidation, but available evidence in this matter is inconclusive [4]. The post-exercise decrease in triacylglycerols content in adipose tissue is with no doubt a consequence of enhanced lipolysis. The process, initiated by adipose triglyceride lipase (ATGL), is then continued by hormone-sensitive lipase (HSL), upon phosphorylation thereof; eventually, the last free fat chain is hydrolyzed by monoacylglycerol lipase (MGL) [5]. Chronic exercise was shown to normalize the markers of this process, phosphorylated HSL and Page 3/20 ATGL, in mice that have been previously maintained on a high fat diet [5]. In addition, the lipolysis response to exercise is also reduced in elderly subjects, which can be attributed to a decrease in intracellular activation of fat breakdown in fat cells to some extent [6], and a recent study demonstrated that acute resistance exercise contributed to triacylglycerol lipase activity increased 16-fold at 10 min in the obese men's adipose tissue [7]. However, voluntary wheel running for 42 days contributed to a decrease in phosphorylated HSL level in rat [8].
Changes in the metabolism of adipose tissue can greatly contribute to changes in body fat distribution during menopause transition, and the menopause condition might be also affect adipose tissue lipolysis, which in turn can contribute to changes in body composition [9]. Previous study observed that higher lipolytic reactions and sensitivities in the abdomen and breast compared to premenopausal female femoral adipose cells, but no postmenopausal female [10]. However, resistance exercises have been effective in improving body composition for pre-and post-menopausal women [11], acute resistance exercise increases subcutaneous abdominal adipose tissue lipid decomposition and fatty oxidation in women [12].
Thus, physical exercise may stimulate lipolytic activity within adipose tissue. Furthermore, resistance exercise may contribute to more e cient reduction of adipose tissue mass and prevent accumulation thereof. However, there is a lack of research that directly analyzes adipose tissue in humans and longterm resistance exercise intervention.
The purpose of this study was to investigate the modulation of 12-weeks of regular resistance exercise on lipolysis pathway in obese pre and postmenopausal women. In addition, this study aims to provide basic data on the prevention of postmenopausal who may be exposed to obesity or metabolic syndrome. In this study, it was hypothesized that resistance exercise would be effective for lipolysis like endurance exercise in obese human, and regular resistance exercise would be independently associated with menopause.

Participations
Participants were arbitrary recruited from the general population of local communities. Among the 40 women participants enrolled in the study. To be eligible for inclusion, participants had to meet the following inclusion criteria: 1) postmenopausal (absence of a menstrual cycle for at least 1 year and follicle-stimulating hormone >30 IU/L) at least 40 (premenopausal) and 50 (postmenopausal) years of age on the date of the assessment; 2) at least body fat percentages of 30% or more on the date of the assessment; 3) not receiving hormone replacement treatment; and 4) not using drugs such as betablockers, and statins. To achieve a nal sample of 23 women were divided into PRM (pre-menopausal group, n=9) and POM (post-menopausal group, n=14). The sample size of the participations was calculated by using ANOVA a large size of effect size of .90, a signi cance level of .05 and a power of .80 (G*power 3.2.1).
All volunteers underwent medical screening by medical specialist, including a health status interview and physical examination. Written informed consent was obtained from all subjects. The study was approved by Kangwon National University Institutional Review Board (KWNUIRB-2016-04-009-002), and conducted in agreement with the Declaration of Helsinki. were asked to bend their upper body while xing the two legs in plate. For standing long jump, all subjects started with their feet in place and jumped as far as possible with the two feet landing together. For side step, parallel lines were drawn at a distance of 120cm on the oor and subjects stood on both feet, one foot on the left side and the other foot on the right side, from the center line.

Subcutaneous fatty tissue extracted and Western blot
All study participants agreed to an abdominal biopsy.
Plastic surgeon extracts the abdominal fat 30g twice before and after exercise program by Hirsch et al. [13].

Blood collection and analysis
Fasting venous blood samples were collected from all participants at baseline, 6-weeks, and 12-weeks. Fasting was maintained for at least 8 h, and blood samples were collected on the following day. Enough sleep and the radical movement as much as possible to refrain. All samples were taken at 0830 AM from an antecubital vein. Serum samples were obtained after centrifugation and stored at -80°C. Serum levels of leptin and adiponectin were measured using enzyme-linked immunosorbent assay Dueset kits (R&D systems, Minneapolis, MN, USA) according to the manufacturer's instructions, as described previously.

Exercise intervention
Resistance exercise programs were used following experiments performed by Gurudut and Rajan with slight [15], modi cations to t the purpose of our experiment. The goal of the resistance exercise intervention was to moderate intensity exercise 60 min per day, 3 days per week for 12 weeks. At the beginning of each session, there was a 10 min of warm-up. It was followed by 40 min of the main part of the exercise with speci c content and 10 min of cool-down. Warm-up exercises included 5 minutes of stretching, and 5 minutes of power walking at 50% intensity of the maximal heart rate reserve. Among resistance exercises, moderate intensity exercise was de ned as circuit exercise at 55~65% intensity of 1 RM with burn 230~260kcal, 12 repetitions, and 3 sets. The rest period between each category was 30 seconds. The rest period between sets was 1 minute in total resistance exercise time of 60 min.
Participants performed the same ve lower and upper body resistance exercise (upper body resistance exercise: chest press, let pull down, biceps curl, triceps extension, and crunch; lower body resistance exercises: squat, lunge, knee extension, and calf raises). All exercise groups were given a polar (heart rate monitor; M400, Kempele, Finland), a portable exercise intensity setting device, for 60 minutes.
In addition, dietitians and exercise physiologists met regularly with a clinical health psychologist experienced in lifestyle behavior change to discuss participant progress and re ne behavior modi cation goals (pain during exercise, side effects due to weight loss, and encouragement to participate in exercise) according to each participant's needs. Nutritional education, self-management exercise, and behavior change techniques were provided. Furthermore, telephone consultations were scheduled biweekly for monitoring and motivation. Post-hoc analysis using Bonferroni correction with a paired t-test indicated in the PRM group that signi cantly decreased 12-weeks than 6-weeks and baseline in weight, % fat, BMI, WHR, SBP and DBP. Weight, BMI, % fat and WHR were greater at 12-weeks than 6-weeks and baseline in the POM group. Post-hoc analysis using Bonferroni correction with a paired t-test indicated in the PRM group that signi cantly increased 12weeks than 6weeks and baseline in grip strength, sit and reach, sit-up, and side step test. Left grip strength, sit and reach, sit-up, standing long jump, and side step test were greater at 12weeks than 6-weeks and baseline in the POM group. Change of adipokines after exercise The change in adipokines according to each group is shown in Table 5. Results from two-way factor ANOVA showed that no signi cant group × time interaction. Leptin (main effect of time, p<0.01) was signi cantly decreased than baseline values after 12 weeks. Adiponectin (main effect of time, p<0.0001) was signi cantly increased than baseline values after 12 weeks.
Post-hoc analysis using Bonferroni correction with a paired t-test indicated in the POM group that signi cantly decreased 12weeks than 6weeks and baseline in leptin concentration. Adiponectin concentration was greater at 12-weeks than 6-weeks and baseline in the both groups.

ATGL, HSL, MGL and PLIN levels in subcutaneous fatty tissue
As shown in Figure. 1, ATGL levels in subcutaneous fatty tissue two groups at baseline and post 12weeks. Within-group analysis showed that the ATGL protein levels was signi cantly decreased in the post-menopausal group after 12 weeks relative to the baseline values (-37.15%, P<.05), but no signi cant changes was observed in the pre-menopausal group. After 12 weeks between pre-menopausal and postmenopausal groups, ATGL protein levels was 42.51% signi cantly lower in the post-menopausal group than in the pre-menopausal group.
As shown in Figure. 2, HSL levels in subcutaneous fatty tissue two groups at baseline and post 12weeks. After 12 weeks between pre-menopausal and post-menopausal groups, HSL protein levels was 79.27% signi cantly higher in the post-menopausal group than in the pre-menopausal group. However, no signi cant difference in the within-group.
As shown in Figure. 3, MGL levels in subcutaneous fatty tissue two groups at baseline and post 12weeks. Within-group analysis showed that the MGL protein levels was signi cantly decreased in the postmenopausal group after 12 weeks relative to the baseline values (-56.34%, P<.001), but no signi cant changes was observed in the pre-menopausal group. After 12 weeks between pre-menopausal and postmenopausal groups, MGL protein levels was 43.21% signi cantly lower in the post-menopausal group than in the pre-menopausal group.
As shown in Figure. 4, PLIN levels in subcutaneous fatty tissue two groups at baseline and post 12weeks. After 12 weeks between pre-menopausal and post-menopausal groups, PLIN protein levels was 27.50% signi cantly lower in the post-menopausal group than in the pre-menopausal group. However, no signi cant difference in the within-group.

Discussion
In this study, investigate the modulation of regular resistance exercise on the lipolysis pathway in pre-and post-menopausal women. The main nding of this study after 12 weeks between pre-menopausal and post-menopausal groups ATGL, MGL, and PLIN protein levels were lower by 42.51%, 43.21%, and 27.50%, HSL protein levels were higher by 79.27% in the post-menopausal group than in the pre-menopausal group. In addition, leptin concentration was signi cantly decreased after resistance exercise both groups, and adiponectin concentration was signi cantly increased after resistance exercise both groups. In physical tness, grip strength, sit and reach, sit-up and standing long-jump were signi cantly increased after exercise both groups.
The postmenopausal women have higher intramuscular fat and subcutaneous adipose tissue compared to men and premenopausal women, in the activity of key enzymes involved in free fatty acid (FFA) metabolism between pre-and post-menopausal women [17,18]. The relationship of FFA release to the amount of body fat that originating from the release of adipose tissue triglyceride fatty acid represents virtually the only route by which these fat stores can be transported through oxidation to non-adipose tissue for net loss [19]. Triacylglycerol and diacylglycerol are degraded by the lipases ATGL and HSL, and fasting lipolysis, expressed per unit fat mass in obese patients may be reduced [20]. Diacylglycerol is converted to monoacylglycerol and a second fatty acid by the action of HSL, after which MGL hydrolyzes monoacylglycero to producing glycerol and the last fatty acid [21]. Besides protein perilipin (PLIN) which is thought to modulate the accession of HSL to the surface of the fat droplet [22], that plays a major role in lipogenesis by regulating the function of lipases [23]. In this study, changes lipolysis related lipase such as ATGL, HSL, MGL, and PLIN were observed after 12 weeks of resistance exercise in pre-and postmenopausal obese women. As the results that ATGL and MGL levels were 37.15% and 56.34% signi cantly decreased after 12 weeks of resistance exercise in post-menopausal group. Cyclic adenosine monophosphate (cAMP) produced by adenylate cyclase activates protein kinase A (PKA) and PKA phosphorylates and activates two or more substrates: HSL and PLIN [24]. HSL and PLIN phosphorylation leads to the translocation of HSL from the cytosol to the surface of the lipid droplet and insulin can activate protein phosphatases resulting in the subsequent dephosphorylation of HSL at which time, ATGL instead of phosphate and translation [24]. Estrogen and estrogen receptor alpha are known to repress intra-abdominal adipose formation [25]. Wend et al. reported that lipid droplet size (relative area per lipid droplet) and the number of lipid droplets per cell were measured in estrogen receptor alpha knockout (ERαKO) with postmenopausal model mice, as ATGL levels and lipid droplets in ERαKO cells signi cantly decreased compared to wild model mice [26]. It is mean that ERαKO mice develop more adipose tissue in the perirenal, periovarian, and mesenteric/omental regions than wild-type model mice [27]. Walhin et al. reported that HSL was downgraded to positive energy balance period and HSL was raised to negative energy balance period [28]. However, other studies have shown that activation of lipolysis occurred through increase levels of ATGL, HSL, and MGL and glycerol release [29]. The activities of ATGL, HSL, and MGL were signi cantly higher in the diet and exercise mice group than the high-fat diet mice group [30]. In this study results, ATGL and MGL levels were signi cantly decreased after 12 weeks of resistance exercise. HSL and PLIN levels tended to decrease after 12 weeks of resistance exercise in postmenopausal group more than pre-menopausal group which similar to Wend et al. and Walhin et al [26,27]. After 12 weeks of resistance exercise, total adipose tissue may be reduced due to a signi cant decrease in body weight and body % fat. In addition, a greater decrease in lipolysis factors in postmenopausal group seems to have a greater effect on resistance exercise than in the pre-menopausal group.
Moreover, 12weeks were signi cantly reduced than 6 weeks and the baseline of leptin concentration and adiponectin concentration was higher at 12 weeks than 6 weeks, and the baseline in post-menopausal group than pre-menopausal group, and these results supported lipolysis at ATGL, HSL, MGL and PLIN levels in the study.
Adipose tissue releases several polypeptides from adipocyte, as well as free fatty acids, which are products of lipolysis, which are also known as adipocytokines, leptin, and adiponectin [31]. In humans, plasma adiponectin concentrations decrease with increasing obesity, and plasma leptin concentrations are highly correlated with BMI [32]. Adiponectin is an adipocyte-derived hormone that sensitizes insulin and improves energy metabolism of tissues [33]. In this regard, adiponectin is relatively unique as adipokine because it is expressed at highest levels in lean and healthy individuals [34]. and PLIN in adipose tissues that cause in ammation by cholesterol has contributed to the increase in fat decomposition and the increase in cyclic nonesteri ed fatty acids [38]. In this study, regular resistance exercise might be reducing leptin, body weight, % fat, and increase adiponectin levels. This reason why reduced total adipose tissue supported by decreased ATGL, HSL, MGL, and PLIN levels in subcutaneous fatty tissue after 12 weeks' resistance exercise.
Resistance exercise is well-known improve body composition in obese adults [39]. In additional, resistance exercise increasing fat free mass and decreasing body weight in pre-and post-menopausal with obesity [40,41]. In this study observed that body weight, body mass index, % fat, waist-to-hip ratio were signi cantly decreased after 12-weeks than 6-weeks resistance exercise in both groups. Although, did not signi cantly in muscle mass but tended to increased. Even though, signi cantly improvement of grip strength, sit and reach, sit-up, and side step test after 12-weeks than 6-weeks resistance exercise in both groups. Weight gain and obesity largely drives the increased prevalence of metabolic syndrome in pre-and post-menopausal women [42]. Because of the associated sequelae of coronary heart disease, cardiovascular disease, diabetes, and mortality, life style management should be of paramount importance in obese women.
Finally, in this study meaningful to directly analyzes adipose tissue in humans, however the present study has some limitations. The sample size was small, which limits our ability to determine the signi cance of the results. Therefore, additional studies with larger sample sizes and control group are required to determine the effectiveness of regular resistance exercise on lipolysis. Another limitation is that to determine whether there is a gender and age difference in lipolysis factors. Also, we did not control for confounding factors such as food energy intake and habitual activity. Lipolysis reaction and lipid storage for food energy intake and habitual activity are key factors in determining body fat stores, confounding factors should be considered in future studies.

Conclusion
In conclusion, this study indicates that regular resistance exercise to change of lipolysis factors (ATGL, Availability of data and materials The datasets analysed during the current study are available from the corresponding author on reasonable request.
Ethics approval and consent to participate The study was approved by Kangwon National University Institutional Review Board, and conducted in agreement with the Declaration of Helsinki. In advance of their participation, all of the participants were fully informed about the purpose and experimental procedures of the study. All of the participants completed consent forms. The participants were informed that all data collected would be processed anonymously.

Consent for publication
All participants provided consent for publishing their data anonymously. ATGL expression baseline and after exercise 12weeks in adipose tissue * p < 0.05, signi cantly different from baseline in the within group § p < 0.05, signi cantly different from 12weeks in the each groups

Figure 2
Page 19/20 HSL expression baseline and after exercise 12weeks in adipose tissue § p < 0.05, signi cantly different from 12weeks in the each groups MGL expression baseline and after exercise 12weeks in adipose tissue *** p < 0.001, signi cantly different from baseline in the within group § p < 0.05, signi cantly different from 12weeks in the each groups Figure 4