The Effects of Testosterone Therapy Combined with Swimming Exercise on Adipose Tissue and Biochemical Parameters in Male Obese Wistar Rats

Abstract

Context

Exercise and anabolic steroids are anticipated to promote fat mass reduction and so to decrease the number of comorbidities related to excessive weight.

Objective

The aim of this study was to verify the influence of aerobic exercise and the use of steroids on the accumulation of adipose tissue and on the biochemical limitations of Wistar rats nourished by a hypercaloric diet.

Methods

Forty, young male Wistar rats were split into four groups: obese control (n=10), obese under treatment (n=10), obese under aerobic exercise (n=10) and obese under aerobic exercise and treatment (n=10). All animals were fed with a hypercaloric diet and animals under treatment received intramuscular testosterone. Body (weight and visceral fat) and blood (lipidogram, glucose, and liver enzymes) parameters were assessed.

Results

The group treated with aerobic exercise and testosterone revealed a reduction in body weight and visceral, perirenal, retroperitoneal and epididymal fats, accompanied by the blood levels of glucose, lactate, LDL-cholesterol, HDL-cholesterol, and lactate dehydrogenase; following high-intensity physical activity.

Conclusion

The results support the theory that the combination of steroids and physical activity reduces the side-effects of androgenic-anabolic hormones and conveys benefits to some constraints.

Introduction

Excessive weight and obesity have significantly increased globally and few regions have thus far succeeded to stabilize their populations weight gains, recently (1). According to the World Health Organization (W.H.O.), 2.8 million people die each year as a result of excessive weight (2). This condition has been consolidated because of shifting world food consumption habits. In Brazil, data indicates that starting in the mid-1970s to the start of 2000s persons lowered the procurement of basic traditional foods, such as rice and beans, and increased the consumption of commercially processed foods by over 400% (3).

Research suggests that obesity is a disorder that will cause considerable financial damage to national health systems, as it consistently contributes to the development of numerous chronic non-communicable diseases (CNCD), such as type 2 diabetes mellitus, arterial hypertension, cardiovascular diseases and cancer (4-5). Food plays a crucial role in preventing CNCDs, and the consumption of fruits and vegetables is inversely related to all known origins of cancer and the progression of heart disease (6).

Physical exercise similarly contributes to the prevention and treatment of such diseases, as it reduces the fat mass, elevates metabolic rates and contracts the number of comorbid conditions associated with excessive weight (7-8). Global physical inactivity has reached 31.1%, but then the indices registered in the Americas are even higher than the global mean, attaining approximately 43% (43). As stated by Rezende et al. (9), physical inactivity directly influences CNCD-related morbidity and mortality, representing 5.3% of all-cause mortality in the Southeastern region of Brazil. Ribeiro assessed the association between physical inactivity, chronic diseases and drug consumption in the elderly, and determined that these variables are strongly interrelated (44).

The usage of androgens has increased more than three-fold in the United States of America, increasing from 0.81% in 2001 to 2.91% in 2011, mainly amongst males over the age of 40 (10). Handelsman studied the prescription of testosterone in 41 countries and detected that this might be a global trend, after considering the increased use of this medication in all countries (11).

Excessive weight and obesity may cause a decline in the amount of circulating free testosterone, enabling a risk factor for hypogonadism, a condition that plays an important role in the origin of metabolic syndrome (12,13). Androgenic-anabolic steroids (AAS) can be prescribed to enhance athletic performance, muscle mass and strength when combined with high-intensity training and to decrease body fat mass (14). Testosterone (TT) regulates the deposition of triglycerides in abdominal adipose tissue; low TT levels are linked with insulin resistance and higher rates of lipogenesis (15).

Considering the increasingly sedentary lifestyles and their implications on quality of life, the focus of this study was to confirm the effects of the relationship between aerobic exercise and testosterone in body composition and blood tests of male Wistar rats fed on a hypercaloric diet.

Methods

Experimental design

This is an experimental study in which 40 male Wistar rats (90 days old) were provided from the Central Biotery of the Federal University of Acre. The animals were kept in cages, with a mean of four rats per cage, at a room temperature between 22°C and 25°C and a 12-hour cycle of alternating light and dark periods. All animals were fed a hypercaloric diet and given ad libitum access to water. All animals received care according to national and international laws. All procedures carefully followed the resolutions of the Brazilian College of Animal Experimentation (COBEA) and the Animal Use Ethics Committee (CEUA) of the Federal University of Acre with the number 23107.018612/2016.31.

The Wistar rats in this experiment were randomly assigned to one of four groups. The Control Group (CG) animals were not exposed to any physical exercise (n=10). The Treated Control Group (TCG) animals were not subjected to any physical activity, but they were treated with testosterone (n=10). The Exercised Group (EG) animals were exposed to aerobic physical exercise, 40 minutes a day, five days a week, for six weeks (n=10). Animals of the Treated and Exercised Group (TEG) were treated with testosterone and subjected to aerobic physical exercise, 40 minutes a day, five days a week, for six weeks (n=10).

Testosterone administration and physical training program

Rats of the treated groups (TCG and TEG) received intramuscular administration of 10 mg/kg of testosterone cypionate (Deposteron™, Sigma Pharma, Brazil) into deep muscle masses, twice a week for 6 weeks, using 1 mL disposable syringes (BD™, São Paulo, Brazil). Rats of the control groups (CG and EG) were injected with 10% (v/v) of benzyl alcohol in peanut oil. Testosterone doses were based on previous studies (16-18). We used this dose because previous studies from our group showed that this dose was the most effective. The administration of lower doses showed no difference between treated and untreated groups for the studied variables.

All animals were adapted to water before testing to reduce stress. The adaptation consisted of keeping the animals in the water for 10 minutes. Every three days, the duration of the exercises increased until reaching 40 minutes at the end of two weeks in an adapted pool with a depth of 48 cm and water temperature kept between 30 and 36°C (19).

After this adaptation period, the animals belonging to the same group were collectively exposed to moderate-intensity swimming sessions with lead balls matching 5% of body weight tied to their tails, 40 minutes a day, 5 times a week, for 6 weeks, between 14:00hr and 17:00hr (3 hours).

Hypercaloric diet

The hyperlipidic diet was composed of a mixture of hypercaloric foods in the following ratio: 15g of standard food (Nuvilab™, Curitiba, PR, Brazil), 10 g of roasted peanuts, 10 g of milk chocolate and 5 g of cornstarch cookies. These ingredients were ground, blended and given in a pellet form, containing per 100g: 20% protein, 48.0% carbohydrate, 20.0% lipid, 4.0% cellulose, and 0% vitamins and minerals. The energy content of this hyperlipidic diet was 21.40 kJ/g. Diets with protein content between 20-25% provided the required amount of amino acids, consistent with the American Society for Nutrition (20).

Body weight analysis and food efficiency

Food efficiency values were attained by calculating the mean between the ingested food and the total weight of the animals of the same group during the entire experiment. The rats for all groups were weighed using an analytical balance, twice a week (on Tuesdays and Fridays), throughout the training period, aimed at observing possible changes in the animals’ body mass.

High Intensity Test and animals sacrifice

At the end of the six-week training period, the animals were exposed to a high intensity test (HIT) until complete exhaustion. The exhaustion time was determined when the animals could not keep their nostrils out of the water for over 10 seconds. They were then quickly removed from the water and placed on a laboratory workbench. Body and tail were carefully dried with sterile paper towels. After the last high-test session, the animals were anesthetized with xylazine (50 mg/kg i.m.) and ketamine (50 mg/kg i.p.) (Vetec Chemistry, Rio de Janeiro, Brazil), and blood was collected by cardiac puncture and stored awaiting further analysis.

Blood analysis

Blood collection was completed after 6 weeks of treatment before and after the high intensity test (HIT). Before HIT, blood aliquots (25 μL) were collected from the animals’ tails and placed in flasks containing 50 μL of sodium fluoride to inhibit glycolytic activity. After HIT, blood samples (3mL) were collected by cardiac puncture, placed in a vial without any anticoagulant and centrifuged at 720 x g, then the serum was transferred to new vials, sealed and stored in the refrigerator until further biochemical analyses.

The biochemical parameters were studied by applying a colorimetric enzymatic method with commercial kits (Labtest™, Belo Horizonte, MG, Brazil), then the results were read on a Roche Cobas Mira Plus device (Roche Diagnostics, Montclair, NJ, USA). Creatinine kinase (CK), aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol, triglycerides (TG) and high-density lipoprotein cholesterol (HDL-C) were analyzed. Low-density lipoprotein cholesterol (LDL-C) was computed by using the Friedewald formula (LDL-C = total cholesterol – HDL-C - TG/5). Glucose was determined via an Accu-Chek meter (Roche Diagnostics, Montclair, NJ, USA). Serum lactate and lactate dehydrogenase (LDH) were assessed by the electrical-enzymatic method using an automatic analyzer (YSI 1500 Sport L-Lactate, USI, Yellow Springs, Ohio, USA).

Collection and weighing of blood and tissues

After blood collection, the rats were euthanized to allow the visceral fat to be removed. An incision was made around the abdomen to dissect adipose depots and abdominal viscera. The epididymal fat was excised. To eliminate the mesenteric fat, a transverse cut was made in the esophagus just below the diaphragm, and the digestive tract was pulled out of the abdomen. The mesenteric fat was therefore disconnected from the abdominal viscera. Once the mesenteric fat and the digestive tract were removed from the abdomen, it was possible to dissect the remaining retroperitoneal and perirenal fat. Firstly, a cut was completed around the retroperitoneal fat in the caudal-cranial direction. It was split into two parts: perirenal fat, which was proximately around the kidneys, and retroperitoneal, the remaining and more abundant fat. Differentiation was commenced by performing a cut between a thinner layer of fat located in the vicinity of the kidneys and a thicker layer placed directly after the first layer.

Statistical analysis

All data were expressed as mean and standard deviations. To verify statistical differences, the two-way ANOVA and Tukey post-hoc tests were enforced via the GraphPadPrism™ version 5.0 software (GraphPad Software, Inc., San Diego, CA, USA). Differences were considered significant at the level p<0.05 (or <5%). With the intention of determining the magnitude of the significant differences for effect sizes; this was calculated via Cohen’s d. Large effect size was considered for values greater than 0.9 and medium effect size for values between 0.9 and 0.5 (21).

Results

Table 1 provides data regarding body and food consumption parameters. CG and TEG groups had the lowest and highest food intake, respectively, confirming that CG animals gained extra weight even when eating less. The results confirm that the animals that did not physically exercise presented a greater weight gain in comparison with the group that did physically exercise. TCG and EG groups exhibited a minor difference, whereas TEG demonstrated a lower weight gain.

Table 1.

Mean and standard deviations of body and food consumption parameters

Variables	Without training				With training
	CG		TCG		EG		TEG
Food consumption (g/day)	19.89±1.10	a	27.03±0.66	ab	25.19±1.07	abc	32.32±0.99	abc
Body weight (g)	545.08±1.4	a	522.10±2.03	ab	525.71±1.44	ac	491.39±1.6	abc
Total visceral fat (g)	32.27±1.07	a	30.12±0.59	ab	22.24±0.29	abc	18.83±0.39	abc
Perirenal fat (g)	1.66±0.03	a	1.44±0.03	ab	1.26±0.01	ac	0.78±0.08	abc
Mesenteric fat (g)	9.17±0.5	a	8.04±0.36	b	6.06±0.08	ac	4.81±0.11	abc
Retroperitonial fat (g)	14.92±0.32	a	12.30±0.59	b	8.98±0.07	abc	6.26±0.16	abc
Epididymal fat (g)	6.74±0.17	a	5.89±0.12	b	4.57±0.04	ac	3.82±0.16	abc

The letters ^{a, b} and ^c in the TCG, EG and TEG columns represent statistically significant differences (p<0.05) in relation to the sedentary control group (CG), sedentary control group treated with testosterone (TCG) and trained group (EG), respectively. Results were compared through two-way ANOVA and Tukey’s post-hoc test.

EG showed a reduced total visceral fat compared to CG and TEG presented a reduced total visceral fat compared to TCG groups. The evaluated perirenal fat values were lower in EG compared to CG and it was lower in TEG compared to TCG. No significant change was observed between TCG and EG groups (Table 1).

The mesenteric fat observed in the TEG group revealed the lowest value, as expected, while the CG group displayed the highest value, and no differences were found between EG and TCG groups (Table 1).

The retroperitoneal fat did not show any statistical differences between CG and TCG groups; but lower values were identified in the trained groups (TEG and TCG) (Table 1).

Regarding the analysis of epididymal fat, the lowest value was detected in TEG and the higher in CG; but EG presented lower values compared to CG and TEG had lower values compared to TEG (Table 1).

As can be detected from Figures 1A and 1B, the plasma concentrations of triglycerides and total cholesterol were lower in TEG compared to CG, TCG and EG. Concerning LDL-cholesterol, the group that practiced exercise and received treatment (TEG) reached lower levels when compared with the sedentary groups (CG) (Fig. 1C). Figure 1D illustrates that the concentration of HDL-cholesterol in rats subjected to physical training and treated with testosterone (TEG) was higher than in the untrained groups (CG and EG), confirming the beneficial effect of exercise and testosterone on the lipid profile.

Figure 1.

Plasma concentration of triglycerides, total cholesterol, LDL-cholesterol and HDL-cholesterol after the high intensity test. The symbols *, ** and *** represent statistically significant differences (p<0.05) of TCG, EG and TEG groups in relation to the sedentary control group (CG), sedentary control group treated with testosterone (TCG) and the trained control group (EG), respectively. Results were compared through two-way ANOVA and Tukey’s post-hoc test.

Next, the evaluation of glucose levels was split into two phases: before and after the high intensity test (Fig. 2). Prior to the high intensity test, the groups that undertook physical training (TEG and TCG) revealed lower glycemic levels to the untrained ones (CG and EG), confirming that aerobic exercises exert a positive influence on glucose homeostasis. Yet, no statistical differences were achieved between the untrained groups, which suggests that testosterone therapy for untrained individuals does not affect glucose levels. In other ways, the treatment with testosterone alone was incapable of significantly decreasing glycemia. In contrast, amongst the trained groups, TEG presented lower blood glucose levels, suggesting that the combination of testosterone and aerobic physical exercise is more effective in reducing the plasma glucose concentration. Nevertheless, glucose blood levels did not significantly vary between EG and TEG, suggesting that physical exercise rather than the testosterone and exercise combination is that improves blood sugar control. After the high-performance training, the trained groups presented lower blood glucose values than the untrained subjects, suggesting an improved use of the plasma glucose. The connection between testosterone and aerobic training demonstrated to be more effective in this regard, notwithstanding these types of exercise.

Figure 2.

Plasma concentrations of glucose before and after the high intensity test (HIT). The symbols *, ** and *** represent statistically significant differences (p<0.05) of TCG, EG and TEG groups in relation to the sedentary control group (CG), sedentary control group treated with testosterone (TCG) and trained control group (EG), respectively, before HIT. The symbols †, †† and ††† represent statistically significant differences (p<0.05) of TCG, EG and TEG groups in relation to CG, TCG and EG groups, respectively, after HIT. Results were compared through two-way ANOVA and Tukey’s post-hoc test.

Figure 3A illustrates that the trained groups (TEG and TCG) presented lower serum lactate levels following the high intensity test compared to the sedentary groups (CG and EG), indicating that the aerobic exercise was able to improve the physical fitness of the animals. Nonetheless, the TEG group displayed lower serum lactate values after the high-intensity activity compared to EG, highlighting the hypothesis that the association between testosterone and physical exercise is effective to reduce serum lactate levels.

Figure 3.

A) Plasma concentrations of lactate before and after the high intensity test. The symbols †, †† and ††† represent statistically significant differences (p<0.05) of TCG, EG and TEG groups in relation to the sedentary control group (CG), sedentary control group treated with testosterone (TCG) and trained group (EG), respectively. B) Plasma activities of lactate dehydrogenase (LDH) after the high intensity test. The symbols *, ** and *** represent statistically significant differences (p<0.05) of TCG, EG and TEG groups in relation to CG, TCG and EG groups, respectively. Results were compared through two-way ANOVA and Tukey’s post-hoc test.

Figure 3B specifies that the plasma activity of LDH following high intensity test was lower in the trained group treated with testosterone compared to CG and TCG. Both groups subjected to a 6-week aerobic training program (TEG and TCG) established a reduction in LDH values compared with those of the untrained groups (EG and CG). Amongst those not subjected to physical training (CG and TCG), the sedentary group that did not receive testosterone (CG) presented statistically inferior LDH values compared to EG.

As can be distinguished in Figure 4A, the plasma levels of aspartate aminotransferase (AST) in animals that underwent physical activity (EG and TEG) were greater when equated with the sedentary groups (CG and TCG). Figure 4B illustrates that alanine aminotransferase (ALT) values were similar in all study groups.

Figure 4.

A) Plasma concentrations of aspartate aminotransferase (AST) after the high intensity test (HIT). The symbols *, ** and *** represent statistically significant differences (p<0.05) in the AST values of TCG, EG and TEG groups in relation to the sedentary control group (CG), sedentary control group treated with testosterone (TCG) and trained group (EG), respectively, after HIT. B) Plasma concentrations of alanine aminotransferase (ALT) after HIT. Plasma activities of ALT values after HIT were not significantly (p>0.05) different between groups. Results were compared through two-way ANOVA and Tukey’s post-hoc test.

Table 2 presents the effect size calculations regarding all variables investigated.

Table 2.

Effect size through Cohen’s d

Variable	CG vs. TCG	CG vs. EG	CG vs. TEG	TCG vs. EG	TCG vs. TEG	EG vs. TEG
Total visceral fat	2.5	12.8	16.7	15.6	22.6	9.9
Perirenal fat	7.3	17.9	14.5	8	10.9	8.4
Mesenteric fat	2.6	8.7	12	7.6	12.1	13
Epididymal fat	5.8	15.6	17.7	14.7	14.6	6.4
Triglycerides	1.6	5.3	4.1	4.3	4.8	4.4
Total cholesterol	6.5	11.7	15.6	2.3	8.7	6.1
LDL-cholesterol	0.9	2.4	3.4	1.4	2.9	0.9
HDL-cholesterol	0.08	0.6	1.9	0.5	1.4	1.3
LDH	1	0.6	1.5	1.7	2.4	0.9
AST	0.93	2.1	1.1	2.5	1.7	1.5
ALT	0.3	0.04	0.2	0.4	0.14	0.3

Discussion

This study detected that the association of testosterone and aerobic training was able to improve the physical condition of male Wistar rats, irrespective of an increase in food consumption. These results are comparable to those found by Aparicio, who observed a higher food intake in rats which performed resistance training than in their sedentary counterparts, and the results by Coll-Risco, who assessed the effects of exercise on physical and biochemical parameters in rats (22,23). This observation could be partly elucidated by the effects that physical activity has on metabolism; as it increases muscle mass and energy expenditure, leading to hormonal changes (7,24).

Concerning weight, it was noticed that the trained group and those that underwent hormonal therapy were lighter than the sedentary ones. Ismail achieved similar results, recognizing the positive effects of physical exercise for reduction of weight and visceral fat (25). The group that experienced a physical training program and testosterone therapy offered a significant reduction of retroperitoneal, epidydimal, perirenal and mesenteric fats. This reduction in visceral adiposity was anticipated, as testosterone has a profound influence on the adipose tissue and is considered a fat-lowering hormone, decreasing fat deposition by inhibiting the activity of lipoprotein lipase in adipocytes and indirectly decreasing leptin production (26). Reviewing the effects of the steroid testosterone on fat cells, Abdelhamed discovered a reduction in size in a group of animals that received hormone replacement therapy (27). The application of this hormone in males presenting symptoms of testosterone deficiency prevented visceral fat gain linked with the placebo group, confirming the view that testosterone reduces obesity (28). So, according to Singh, testosterone, through androgen receptors signaling, inhibits the differentiation of stem cells into adipocytes and favors myogenesis (29).

One may accept that through physical exercise, isolated or combined with androgen hormones, promoted a reduction of the lipid profile in the studied groups. This result can be credited to the beneficial effect that physical activity exerts on the lipid status. In humans, aerobic exercise enhanced the lipid rates of obese individuals by reducing the amount of free fatty acids in contrast with the placebo group (30). Speretta estimated the effects of two exercise modalities (aerobic and resistance) on obese rats fed a normal and a hyperlipid diet and concluded that total cholesterol and HDL-cholesterol levels revealed preferable results in the trained animals, though they were fed a high-lipid diet (31). This observation reveals the protective role of physical activity on plasma lipoproteins, even when exposed to an obesogenic situation. Undeniably, low levels of plasma testosterone exert negative effects on the lipidogram, with increases in total and LDL-cholesterol and reduction in HDL-cholesterol, in addition to an increase in lipoperoxidation in rats (32).

It is well recognized that one of the principal mechanisms responsible for the increase in glucose uptake after exercise is triggered because of a higher expression or a greater activity of key signaling proteins involved in glucose regulation, such as AMP kinase, glucose transporters – GLUT – and insulin receptors (33). In this study, after 6-weeks training, the groups subjected to regular aerobic exercise exhibited better glycemic control, a result that is consistent with several scientific studies (34-36). Research undertaken by Crespilho et al. confirmed that exercise enhanced the immune system and glycemic control of diabetic rats (37). Silva et al. evaluated elderly diabetic rats under hormonal treatment and a physical training program and detected better glucose uptake, evidencing that testosterone can apply a beneficial effect on glucose homeostasis (16).

This study demonstrates that testosterone performs a synergistic role with physical exercise, resultant in a more effective consumption of oxygen, possibly owing to increased erythropoiesis. A greater number of circulating erythrocytes permits a more efficient transportation of oxygen in the direction of muscle tissues and its consumption via energy metabolism. A study performed by Silva et al. showed that rats with lower lactate levels were able to perform physical activity for a lengthier time (19). The fact that TEG offered lower values of serum lactate after high-performance exercise advocates that the use of anabolic steroids associated with regular aerobic physical exercise can effectively control the physical condition in the animals.

Amongst the untrained groups, it was noticed that the testosterone-treated group had higher values of plasma LDH. Yet, the association of hormone and physical training was able to significantly reduce LDH levels following high-performance exercise. We believe that the testosterone sedentary group presented higher LDH values due to liver and muscle discharge, which was attenuated by exercise. Evaluating the effects of high-intensity interval exercise, Araújo et al. was unable to determine statistically significant changes in LDH values between groups that were subjected to a 6-week training program and the sedentary ones (38). As an alternative, a survey undertaken by Emami, wherein biochemical parameters in rats submitted to a high-calorie diet and aerobic training were evaluated, found, in contradiction to other results, that a fat-rich diet alone was able to raise AST, ALT and LDH values, suggesting that physical training exerted a positive effect on these parameters (39).

Aminotransferases are enzymatic markers of liver damage (40). This current study elevated levels of ALT, while there was no statistical difference between groups. AST levels were higher in trained animals. The elevation of liver enzymes was detected in individuals that underwent intense physical activity. Pettersson et al. exposed 15 moderately trained human males to one hour of high-intensity training and measured some biochemical parameters; and concluded that AST, ALT, creatine kinase and lactate dehydrogenase remained elevated for up to seven days, without posing a threat of damage to liver or kidneys (41). Altered liver enzyme values caused by physical exercise were observed in two case studies, but they were explained after discontinuing or decreasing the practice of high-intensity exercises (42,43). The use of steroids is related with some intercurrences, together with changes in the levels of these enzymes (44,45). Yet, in this study, no change was observed in the treated groups.

One important limitation in our study is that we did not evaluate side-effects. We were unable to confirm if testosterone changed hair growing mechanisms and whether it influenced heart muscle and acne. So, we should be careful when interpreting our data.

In conclusion, the results revealed that the combination of the steroids and physical activity can reduce the side-effects of the androgenic-anabolic hormones and produce positive effects on body statistical parameters, including visceral fat and lipid metabolism reduction, without influencing the markers of liver damage. However, we should be careful with the level of endogen testosterone.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgement

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Financiadora de Estudos e Projetos (FINEP), from Brazil.