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Strength training improves plasma parameters, body composition and liver morphology in ovariectomized rats
Science & Sports (2012) 27, 94—100
ORIGINAL ARTICLE
Strength training improves plasma parameters, body composition and liver morphology in ovariectomized rats L’entraînement en force améliore les variables biologiques plasmatiques, la composition corporelle et la morphologie hépatique de rates ovariectomisées M. Ojeika Vasques a, L. Vidal Andreato a,b, F.N. Almeida a,c, J.V. Del Conti Esteves a, R. Fernandes de Souza a, S.M. Franzói de Moraes a,d,∗ a
Exercise Physiology Laboratory, Human Physiology Department, State University of Maringá, Brazil School of Sports and Physical Education, University of São Paulo, Brazil c Institute of Biomedical Sciences, Department of Physiology and Biophisics, University of São Paulo, Brazil d Laboratório de Fisiologia do Esforc¸o, Departamento de Ciências Fisiológicas, Universidade Estadual de Maringá, Avenida Colombo, 5790, Bloco H79, sala 109, Maringá, Paraná, Brazil b
Received 5 February 2011; accepted 6 June 2011 Available online 1st September 2011
KEYWORDS Strength training; Menopause; Non-alcoholic fatty liver disease
∗
Summary Objective. — The aim of this study was to identify the effects of strength training on plasma parameters, body composition and the liver of ovariectomized rats. Methods. — Wistar sedentary (SHAM), ovariectomized (OVX), and ovariectomized trained rats (strength training [OVX-EXE]) of 85% of one maximal repetition (1 RM), three times per week, for 10 weeks, were used on this study. We monitored the body weight and visceral (uterine, mesenteric and retroperitoneal) and subcutaneous adiposity, total cholesterol, triglycerides, HDL, blood glucose and liver morphology to identify the presence of macrovesicular steotosis (haematoxylin and eosin staining). Results. — We observed that strength training changed body weight (SHAM 293.0 ± 14.5 g; OVX 342.6 ± 10.8 g; OVX-EXE 317.7 ± 11.9 g, P < 0.05), visceral and subcutaneous adiposity, glucose (SHAM 111.2 ± 10.0 mg/dL; OVX 147.4 ± 18.8 mg/dL; OVX-EXE 118.5 ± 2.2 mg/dL, P < 0.05), increased HDL (SHAM 82.7 ± 1.4 mg/dL; OVX 64.6 ± 2.8 mg/dL; OVX-EXE 91.4 ± 2.6 mg/dL, P < 0.05) and reduced macrovesicular steatosis in liver tissue.
Corresponding author. Tel.: +55 87020 900. E-mail address: [email protected] (S.M. Franzói de Moraes).
0765-1597/$ – see front matter © 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.scispo.2011.06.009
Strength training improves plasma parameters, body composition and liver morphology
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Conclusions. — Considering the data obtained in this research, we emphasise the use of strength exercise training as a therapeutic means to combat or control the metabolic disturbances associated with menopause, including adiposity, and adverse changes in blood glucose, blood HDL and macrovesicular steatosis. © 2011 Elsevier Masson SAS. All rights reserved.
MOTS CLÉS Entraînement en force ; Ménopause ; Stéatose hépatique non alcoolique
Résumé Objectif. — Le but de cette étude était d’identifier les effets de l’entraînement en force sur les paramètres du plasma, la composition corporelle et le foie des rats ayant subi une ovariectomie. Méthode. — Nous avons comparé des rats Wistar ovariectomisés après entraînement en force (OVX-EX) à raison de 85 % de 1 RM, trois fois par semaine pendant dix semaines à un groupe de rats ovariectomisés (OVX) et SHAM pour l’ovariectomie. Nous avons analysé le poids corporel et l’adiposité viscérale (utérus, mésentériques et rétropéritonéaux), graisse sous-cutanée, le cholestérol total et le cholestérol HDL, les triglycérides, glycémie et de la morphologie du foie pour identifier la présence de la stéatose macrovésiculaire (hématoxyline-éosine). Résultats. — Nous avons observé que l’entraînement en force a entraîné des changements dans le poids corporel (SHAM 293,0 ± 14,5 g ; OVX 342,6 ± 10,8 g ; OVX-EXE 317,7 ± 11,9 g, p < 0,05), l’adiposité viscérale et sous-cutanée, le glucose (SHAM 111,2 ± 10,0 mg/dL ; OVX 147,4 ± 18,8 mg/dL ; OVX-EXE 118,5 ± 2,2 mg/dL, p < 0,05), augmentation du HDL-cholestérol (SHAM 82,7 ± 1,4 mg/dL ; OVX 64,6 ± 2,8 mg/dL ; OVX-EXE 91,4 ± 2,6 mg/dL, p < 0,05) et réduit l’apparition du stéatose macrovésiculaire. Conclusion. — Compte tenu des données obtenues dans cette étude, nous mettons en évidence l’utilisation de l’entraînement en force comme un moyen thérapeutique pour combattre et/ou contrôler les troubles métaboliques associés à la ménopause, y compris l’adiposité et les changements défavorables de la glycémie, HDL sanguine et la stéatose macrovésiculaire. © 2011 Elsevier Masson SAS. Tous droits réservés.
1. Introduction Menopause, which is characterised by loss of ovarian function and consequent reduction in the production of ovarian hormones (estradiol and progesterone), can bring about a set of harmful health effects that are observed in women and in female experimental models [1,2]. It was observed that at postmenopause, or after ovariectomy (experimental model that mimics menopause), there is an increase in body weight and visceral adiposity and a greater risk of developing diseases related to metabolic syndrome [3], as well as an increased incidence of lipid accumulation in the liver [4—8]. Non-alcoholic fatty liver disease (NAFLD) is the accumulation of lipids in liver that can progress to non-alcoholic steatohepatitis (characterised by the presence of lipid associated with inflammatory cells), advanced fibrosis, cirrhosis, and in extreme manifestations, hepatocellular carcinoma [9]. The prevalence of NAFLD has increased rapidly in tandem with the dramatic increase in obesity and diabetes [10,11]. Approximately 20 to 30% of adults in the general population in western countries have non-alcoholic fatty liver disease, and its prevalence increases from 70 to 90% among persons who are obese or have diabetes [12]. However, there are no proven effective pharmacological treatments for NAFLD [13—15]. Yet, exercise has been identified as a key component in the treatment of these diseases [16—18]. Furthermore, the control of risk factors associated with metabolic syndrome is a key factor in preventing the development of NAFLD. In this respect, adopting an active lifestyle has generated significant results for this type of control [19—21]. Several studies have shown the effectiveness of physical exercise as a means of prevention
and treatment of hepatic steatosis [22]. However, although strength training has been suggested as being effective for increasing muscle mass [23,24], muscle strength [25] and bone mineral density [26,27], little is known about the effects of the accumulation of lipids in the liver in postmenopausal women. Thus, to investigate interventionist approaches that can prevent and/or attenuate NAFLD, we examined whether strength training had effects on plasma and morphological parameters related to NAFLD in ovariectomized Wistar rats.
2. Methods 2.1. Animals and experimental procedures The sample consisted of 22 female Wistar rats that were three months old and provided by Central Animal Laboratory, State University of Maringá. They were given diet and water ad libitum, and they were kept under a light/dark cycle of 12/12 hrs at 23 ± 2 ◦ C. The animals were randomly divided into three groups: group 1 consisted of sedentary rats that passed through the surgical stress (SHAM), group 2 consisted of ovariectomized rats (OVX), and group 3 consisted of ovariectomized rats subjected to strength exercise (OVX-EXE).
2.2. Surgical procedures The animals, with 3 months of age, were anesthetised by intramuscular xylazine (0.1 mL/100 g) and ketamine (0.1 mL/100 g) and ovaries surgically removed. The surgical technique was performed according to the description
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by Zarrow et al. [28]. Twenty-four hours before surgery, all rats received 0.1 ml/100 g of intramuscular pentabiotic, and after surgery, they received 0.2 mg/kg of atropine subcutaneously following the procedure, as proposed by Serakides et al. [29].
concentrations of triglycerides, total cholesterol, highdensity lipoprotein (HDL) and glucose were conducted using the colorimetric method. Analyses were performed using a commercial kit (Gold Analisa® , Belo Horizonte, Brazil) and a spectrophotometer (Bioplus® UV-2000, São Paulo, Brazil).
2.3. Strength exercise training
2.6. Histological analysis
For the strength exercise protocol, a training apparatus was developed such as the one adopted by Tamaki et al. [30], which simulated the execution of the squat. The apparatus has an electronic module that generates an electrical current (20 V, 0.3 s duration and 3 s intervals between stimuli). The electrical current is then directly applied to the tail of rats. As a result of these stimuli, the rats, which are attached to a device similar to the squat machine, flex their legs repeatedly, lifting the weight located in the training apparatus. The animals underwent two weeks of adaptation to the apparatus (weight set at 100 g). Subsequently, the 1 RM test was realized to determine the training load (1 RM 85%). The 1 RM was measured fortnightly. The group OVX-EXE held three sets of 10 repetitions with 45 seconds of rest between each set, three times a week starting 60 days after surgery for a period of 10 weeks, as proposed by Barauna et al. [31].
Part of the liver tissue was fixed in Bouin and stored in 70% alcohol until histological analysis. A portion of the stored liver was embedded in paraffin and subjected to microtome (Microtome Leica RM 2245, Wetzlar, Germany) with semiserial histological sections that were 6 m thick. We used haematoxylin and eosin to highlight the possible macrovesicular steatosis. The capture of images was performed by using a high-resolution digital camera Pro-Series Media Cibertecnics coupled to an Olympus B × 40 microscope (Tokyo, Japan), and for reading the images, we used Image Pro-Plus 4.1 (Springfield, United States of America). For morphometric analyses, we obtained the infiltrated area by considering the microscope field area (149886.64 m2 ) and then subtracting the area occupied by the central vein from it.
2.4. Collection of tissues
Statistical evaluation of results was performed by one-way analysis of variance (Anova) with Newman-Keuls multiple comparisons test, prefixing the level of significance at 5% (P < 0.05). Statistical tests were performed using the Prism v.2.1 (GraphPad, USA) and Microsoft Excel® (São Paulo, Brazil) softwares.
After the experimental procedure, the animals were anesthetised with sodium pentobarbital (Hypinol® 3%, 4 mg/100 g bw, ip), and laparotomy was carried out to collect blood (4 mL) from the vena cava. Mesenteric, uterine, retroperitoneal and subcutaneous (inguinal) fat and part of the liver were also removed.
2.5. Biochemical analysis The blood samples were centrifuged at 2,000 rpm (4 ◦ C, 15 minutes) and divided into serum and plasma. The serum and plasma were stored in Eppendorf tubes and frozen at −12 ◦ C until their analysis. The determination of plasma
2.7. Statistical analysis
3. Results Table 1 shows the values of body weight and white adipose tissue pads of several body regions from control (SHAM), ovariectomized (OVX) and exercised (OVX-EXE) rats. We observed that ovariectomy resulted in significant weight gain, as evidenced by the rats in OVX group having higher body weight when compared to rats in the
Table 1 Body weight and cushions of white adipose tissue from many body regions of rats in control (SHAM), ovariectomized (OVX) and exercised (OVX-EXE) groups. SHAM (n = 6) Body weight (g) Uterine fat (g) Retroperitoneal fat (g) Mesenteric fat (g) Subcutaneous fat (g) fat deposits (g) Liver (g) Right adrenal gland (mg) Left adrenal gland (mg)
293.0 6.5 6.2 5.4 3.8 20.0 10.3 0.022 0.025
OVX (n = 8) ± ± ± ± ± ± ± ± ±
Values are represented as mean ± standard deviation. * P < 0.05 compared to SHAM control group. ** P < 0.05 compared to OVX group.
14.5 1.2 0.6 0.8 1.0 3.0 0.8 0.006 0.004
342.6 8.4 8.6 7.2 5.1 29.3 9.5 0.022 0.022
OVX-EXE (n = 8) ± ± ± ± ± ± ± ± ±
10.8* 0.7* 0.8* 0.9* 0.7* 2.2* 0.5 0.008 0.011
317.7 6.9 7.3 5.2 3.6 22.6 9.6 0.021 0.020
± ± ± ± ± ± ± ± ±
11.9** 0.7** 0.7*,** 0.8** 0.7** 2.3** 0.4 0.008 0.006
Strength training improves plasma parameters, body composition and liver morphology Table 2
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Plasma parameters of rats in control (SHAM), ovariectomized (OVX) and exercised (OVX-EXE) groups. SHAM (n = 6)
Total cholesterol (mg/dL) Triglycerides (mg/dL) HDL (mg/dL) Glucose (mg/dL)
85.8 80.5 82.7 111.2
OVX (n = 8) ± ± ± ±
7.1 5.5 1.4 10.0
97.1 91.4 64.6 147.4
OVX-EXE (n = 8) ± ± ± ±
6.1* 6.1* 2.8* 18.8*
100.7 106.6 91.4 118.5
± ± ± ±
6.4* 3.2* 2.6*,** 2.2**
HDL: high-density lipoprotein. Values are represented as mean ± standard deviation. * P < 0.05 compared to SHAM. ** P < 0.05 compared to OVX group.
SHAM group. However, strength training was effective in controlling body weight gain in OVX-EXE group (OVX vs OVXEXE, P < 0.05). Additionally, we noted that ovariectomy also resulted in increased visceral adiposity (as demonstrated by uterine, retroperitoneal and mesenteric fat depots) and subcutaneous fat (SHAM vs OVX, P < 0.05), a factor normalized by the addition of strength training (OVX vs OVX-EXE, P < 0.05). The weight of the liver was not affected. The adrenal gland weight was not different between the three groups, indicating that ovariectomy and strength training did not influence this parameter. Ovariectomy also resulted in changes in all plasma parameters analyzed. The strength training reduces blood glucose and increases cholesterol (HDL) of ovariectomized rats, but had no effect on total cholesterol and triglycerides (Table 2). Histological analysis revealed a marked increase in liver (H/E) macrovesicular steatosis in OVX group, which may have represented a possible high incidence of lipid droplets in liver tissue of those animals. As shown in Fig. 1, the macrovesicular steatosis was 2.5 times higher in OVX rats (8.6 ± 0.2%) as compared with SHAM rats (3.5 ± 0.1%). The strength training led to a reduction of 1.5 times in rats OVXEXE (5.7 ± 0.2%). The presence of macrovesicular steatosis can also be observed in the photomicrograph shown in Fig. 2. We note that the figure representing the OVX group presents more whitish appearances than the other groups, which correspond to the presence of vacuoles. Histologically, there were no other structural changes in liver tissue. The effect of strength training protocol was showed in Fig. 3. An elevation of 144% of strength was observed in OVX-EXE group (first 1 RM test 178.1± 26.6 g and last 1 RM test 436.3 ± 53.6 g).
Figure 1 Percentage area of macrovesicular steatosis in liver tissue from rats in control (SHAM), ovariectomized (OVX) and exercised (OVX-EXE) groups. Values are expressed as mean ± SEM; * P < 0.05 compared to SHAM; # P < 0.05 compared with OVX.
4. Discussion Our data indicate that the strength training protocol used was effective to increase strength, control the accumulation of fat in the visceral and subcutaneous tissues and macrovesicular steatosis in liver tissue. Therefore, this led us to infer that strength training, which has numerous health benefits and has protective effects against many diseases, also appears to be a good intervention to combat NAFLD associated to ovariectomy. The practice of regular physical exercise is very important for women who suffer from the effects of reduced ovarian hormones at menopause [32]. Physical exercise is
Figure 2 Photomicrograph of liver tissue from: (A) control (SHAM), (B) ovariectomized (OVX), and (C) exercised (OVX-EXE) groups. The magnification was × 230 with the Olympus B × 40 microscope.
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Figure 3 Load (g) improvement in response to strength training protocol in OVX-EXE group. The first and last 1 RM test is presented. * P < 0.05.
used as a non-pharmacological intervention to prevent and reverse alterations occurred in association to menopause, and strength training is one of them. Strength training is associated to attenuation or reduction of many alterations involved in this period, including sarcopenia, osteopenia, insulin resistance, inflammatory markers, NAFLD, among others. However, strength training is still not routinely used as a therapeutic intervention [33]. Menopause is critical to women’s health, because there is a decrease in the production of sex hormones and a parallel increase in the incidence of chronic degenerative diseases in postmenopausal women [1—4]. Thus, we sought to develop treatment methods and identify means of prevention of NAFLD in this population. Physical activity is included as a preventive intervention to treat metabolic syndrome, and NAFLD is strongly associated with such syndrome. Therefore, this study sought to verify the effect of strength exercise training on plasma and morphological variables related to chronic degenerative diseases. Strength training was selected for observation because relatively less is known of its effects, given the emphasis on aerobic exercise as the primary intervention used when we have a condition (ovariectomy/menopause) that is related to a metabolic disorder. Our study presents data that positively reflect the use of strength training to treat metabolic disorders. This can be proven by the decrease in fat, improvement in glucose and HDL as well as lower macrovesicular steatosis in liver tissue (which may be associated with a reduced presence of lipid droplets) observed in the group OVX-EXE. Menopause is associated with increased adiposity, particularly visceral adiposity, and a high risk of metabolic diseases [3]. Recent studies have shown that visceral adipose tissue contributes to these metabolic complications of menopause [34], including NAFLD [35]. Thus, the prevention of the increase or reduction in body fat is very important, and this confirms the need for the inclusion of regular physical exercises that makes the body expend more energy or increase its daily energy expenditure [36]. Although, the effects of physical activity have been attributed mostly to aerobic exercise in terms of energy expenditure [37], our study showed a beneficial effect of strength training (OVX vs OVX-EXE) on visceral and subcutaneous adiposity. Diet is also an important component to determine body composition, however, we have not evaluated such parameter in our study. In addition, in a classical work, Wade et al.
M. Ojeika Vasques et al. [38] demonstrated that ovariectomy increases adiposity and body weight without alterations in food intake. In relation to effect of strength training in food intake, in a recent research from our laboratory (not published), we observed that the trained rats were not statistical different from control rats. Several studies demonstrated the effects of strength training on body composition. Twenty-four weeks (3 days/week) of strength training decreased body weight, body mass index and body fat of postmenopausal woman [39]. In addition, Bea et al. [40] observed that strength training is a viable long-term method to prevent weight gain and deleterious changes in body composition in postmenopausal woman. Teixeira et al. [41] demonstrated that strength training during 1 year changed total body composition and regional body fat in postmenopausal woman. Our body fat data associated with these data presented elsewhere highlight the importance of strength training on fat distribution in postmenopausal period. In addition to body composition alterations, menopause is related to abnormal plasma lipids [42]. Our study showed a worsening of total cholesterol, triglycerides, HDL and glucose levels as a result of ovariectomy (SHAM vs OVX). Evidences indicate that a decline in estrogen levels, characteristic of ovariectomy model [43], is associated to an atherogenic lipid profile, due an increase in LDL, triglycerides, total cholesterol and a decrease in HDL [33]. Again, most studies showed satisfactory effects associated with aerobic exercise [44] and with association of aerobic and strength training [45]. However, the results related to strength training alone are contradictory. Brochu et al. [46] did not observe effects of strength training during 6 months on lipid profile of postmenopausal woman. Opposite effects were observed after 14 weeks of strength training that resulted in total and LDL cholesterol reduction without effects on triglycerides and HDL cholesterol [47]. We observed significant response in HDL cholesterol (OVX vs OVX-EXE), demonstrating that strength training may also be suitable as a therapeutic measure to improve lipid profile in ovariectomized rats. The strength training was also effective in reducing blood glucose. But, it is important to highlight that only glycaemia was not enough to indicate glucose profile and metabolism in body in response of the strength training. A limit of our research was not realized: glucose and insulin tolerance test, and in particular, insulinemia to confirm these benefit because insulin is the principal regulator of blood glucose (for more information, [48—50]). However, the result observed in glycaemia was very interesting and important to show the strength training effects in health parameters. The implementation of daily exercise can prevent most chronic diseases and is commonly recommended for individuals diagnosed with NAFLD [51]. As reported, NAFLD is often associated with metabolic syndrome and menopause [22,52]. In our study, we noted the presence of an indicator of increase in lipid droplets (macrovesicular steatosis visualized by H/E staining) in liver tissue of OVX group, confirming the association with menopause. However, to verify the actual presence of these lipid inclusions, it would have been necessary to use a specific stain for this purpose, for example, Sudan III, Sudan Black or Red oil-O.
Strength training improves plasma parameters, body composition and liver morphology With regard to liver lipid inclusions and/or NAFLD, the practice of regular physical exercise was indicated as part of the treatment of NAFLD [16—22], but few studies have investigated the effects of strength physical training on clinical cases. Our data demonstrated that strength training was able to reduce liver macrovesicular steatosis, pointing to a new measure that can be adopted for the treatment of menopause.
5. Conclusion Considering the data obtained here, we emphasize the use of strength exercise training as a therapeutic means to combating and/or controlling the metabolic disturbances associated with menopause, including adiposity, and adverse changes in blood glucose, blood HDL and liver macrovesicular steatosis.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
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in climateric. Ginecología y obstetricia de Mexico 2009;77: 535—46. Rabii J, Ganong WF. Responses of plasma ‘‘estradiol’’ and plasma LH to ovarietomy, ovariectomy plus adrenalectomy and estrogen injection at various age. Neuroendocrinology 1976;20:270—81. Poirier P, Despres JP. Exercise in weight management of obesity. Cardiol clin 2001;19:459—70. Zois CE, Tokmadikis SP, Volaklis KA, Kotsa K, Touvra AM, Douda E, et al. Lipoprotein profile, glycemic control and physical fitness after strength and aerobic training in postmenopausal woman with type 2 diabetes. Eur J Appl Physiol 2009;106:901—7. Brochu M, Malita MF, Messier V, Doucet E, Strychar I, Lavoie J, et al. Resistance training does not contribute toimproving metabolic profile after a 6-month weight loss program in overweight and obese postmenopausal woman. J Clin Endocrinol Metab 2009;94:3226—33. Prabhakaran B, Dowling EA, Branch JD, Swain DP, Leutholtz BC. Effects of 14 weeks of resistance training on lipid profile and body fat percentage in premenopausal woman. Br J Sports Med 1999;67:26—36. Krook A, Wallberg-Henriksson H, Zierath JR. Sending the signal: molecular mechanisms regulating glucose uptake. Med Sci Sports Exerc 2004;36:1212—7. Perseguin G, Petersen K, Shulman GI. Cellular mechanisms of insulin resistance: potential links with inflammation. Int J Obes 2003;27:S6—11. Shulman GI. Cellular mechanisms of insulin resistance in humans. Am J Cardiol 1999;8:3J—10J. Farrell CG, Larter CZ. Non-alcoholic fatty liver disease: from steatosis to cirrhosis. Hepatology 2006;43: 99—112. Suzuki A, Abdelmalek MF. Non-alcoholic fatty liver disease in woman. Women Health 2009;5:191—203.
ORIGINAL ARTICLE
Strength training improves plasma parameters, body composition and liver morphology in ovariectomized rats L’entraînement en force améliore les variables biologiques plasmatiques, la composition corporelle et la morphologie hépatique de rates ovariectomisées M. Ojeika Vasques a, L. Vidal Andreato a,b, F.N. Almeida a,c, J.V. Del Conti Esteves a, R. Fernandes de Souza a, S.M. Franzói de Moraes a,d,∗ a
Exercise Physiology Laboratory, Human Physiology Department, State University of Maringá, Brazil School of Sports and Physical Education, University of São Paulo, Brazil c Institute of Biomedical Sciences, Department of Physiology and Biophisics, University of São Paulo, Brazil d Laboratório de Fisiologia do Esforc¸o, Departamento de Ciências Fisiológicas, Universidade Estadual de Maringá, Avenida Colombo, 5790, Bloco H79, sala 109, Maringá, Paraná, Brazil b
Received 5 February 2011; accepted 6 June 2011 Available online 1st September 2011
KEYWORDS Strength training; Menopause; Non-alcoholic fatty liver disease
∗
Summary Objective. — The aim of this study was to identify the effects of strength training on plasma parameters, body composition and the liver of ovariectomized rats. Methods. — Wistar sedentary (SHAM), ovariectomized (OVX), and ovariectomized trained rats (strength training [OVX-EXE]) of 85% of one maximal repetition (1 RM), three times per week, for 10 weeks, were used on this study. We monitored the body weight and visceral (uterine, mesenteric and retroperitoneal) and subcutaneous adiposity, total cholesterol, triglycerides, HDL, blood glucose and liver morphology to identify the presence of macrovesicular steotosis (haematoxylin and eosin staining). Results. — We observed that strength training changed body weight (SHAM 293.0 ± 14.5 g; OVX 342.6 ± 10.8 g; OVX-EXE 317.7 ± 11.9 g, P < 0.05), visceral and subcutaneous adiposity, glucose (SHAM 111.2 ± 10.0 mg/dL; OVX 147.4 ± 18.8 mg/dL; OVX-EXE 118.5 ± 2.2 mg/dL, P < 0.05), increased HDL (SHAM 82.7 ± 1.4 mg/dL; OVX 64.6 ± 2.8 mg/dL; OVX-EXE 91.4 ± 2.6 mg/dL, P < 0.05) and reduced macrovesicular steatosis in liver tissue.
Corresponding author. Tel.: +55 87020 900. E-mail address: [email protected] (S.M. Franzói de Moraes).
0765-1597/$ – see front matter © 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.scispo.2011.06.009
Strength training improves plasma parameters, body composition and liver morphology
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Conclusions. — Considering the data obtained in this research, we emphasise the use of strength exercise training as a therapeutic means to combat or control the metabolic disturbances associated with menopause, including adiposity, and adverse changes in blood glucose, blood HDL and macrovesicular steatosis. © 2011 Elsevier Masson SAS. All rights reserved.
MOTS CLÉS Entraînement en force ; Ménopause ; Stéatose hépatique non alcoolique
Résumé Objectif. — Le but de cette étude était d’identifier les effets de l’entraînement en force sur les paramètres du plasma, la composition corporelle et le foie des rats ayant subi une ovariectomie. Méthode. — Nous avons comparé des rats Wistar ovariectomisés après entraînement en force (OVX-EX) à raison de 85 % de 1 RM, trois fois par semaine pendant dix semaines à un groupe de rats ovariectomisés (OVX) et SHAM pour l’ovariectomie. Nous avons analysé le poids corporel et l’adiposité viscérale (utérus, mésentériques et rétropéritonéaux), graisse sous-cutanée, le cholestérol total et le cholestérol HDL, les triglycérides, glycémie et de la morphologie du foie pour identifier la présence de la stéatose macrovésiculaire (hématoxyline-éosine). Résultats. — Nous avons observé que l’entraînement en force a entraîné des changements dans le poids corporel (SHAM 293,0 ± 14,5 g ; OVX 342,6 ± 10,8 g ; OVX-EXE 317,7 ± 11,9 g, p < 0,05), l’adiposité viscérale et sous-cutanée, le glucose (SHAM 111,2 ± 10,0 mg/dL ; OVX 147,4 ± 18,8 mg/dL ; OVX-EXE 118,5 ± 2,2 mg/dL, p < 0,05), augmentation du HDL-cholestérol (SHAM 82,7 ± 1,4 mg/dL ; OVX 64,6 ± 2,8 mg/dL ; OVX-EXE 91,4 ± 2,6 mg/dL, p < 0,05) et réduit l’apparition du stéatose macrovésiculaire. Conclusion. — Compte tenu des données obtenues dans cette étude, nous mettons en évidence l’utilisation de l’entraînement en force comme un moyen thérapeutique pour combattre et/ou contrôler les troubles métaboliques associés à la ménopause, y compris l’adiposité et les changements défavorables de la glycémie, HDL sanguine et la stéatose macrovésiculaire. © 2011 Elsevier Masson SAS. Tous droits réservés.
1. Introduction Menopause, which is characterised by loss of ovarian function and consequent reduction in the production of ovarian hormones (estradiol and progesterone), can bring about a set of harmful health effects that are observed in women and in female experimental models [1,2]. It was observed that at postmenopause, or after ovariectomy (experimental model that mimics menopause), there is an increase in body weight and visceral adiposity and a greater risk of developing diseases related to metabolic syndrome [3], as well as an increased incidence of lipid accumulation in the liver [4—8]. Non-alcoholic fatty liver disease (NAFLD) is the accumulation of lipids in liver that can progress to non-alcoholic steatohepatitis (characterised by the presence of lipid associated with inflammatory cells), advanced fibrosis, cirrhosis, and in extreme manifestations, hepatocellular carcinoma [9]. The prevalence of NAFLD has increased rapidly in tandem with the dramatic increase in obesity and diabetes [10,11]. Approximately 20 to 30% of adults in the general population in western countries have non-alcoholic fatty liver disease, and its prevalence increases from 70 to 90% among persons who are obese or have diabetes [12]. However, there are no proven effective pharmacological treatments for NAFLD [13—15]. Yet, exercise has been identified as a key component in the treatment of these diseases [16—18]. Furthermore, the control of risk factors associated with metabolic syndrome is a key factor in preventing the development of NAFLD. In this respect, adopting an active lifestyle has generated significant results for this type of control [19—21]. Several studies have shown the effectiveness of physical exercise as a means of prevention
and treatment of hepatic steatosis [22]. However, although strength training has been suggested as being effective for increasing muscle mass [23,24], muscle strength [25] and bone mineral density [26,27], little is known about the effects of the accumulation of lipids in the liver in postmenopausal women. Thus, to investigate interventionist approaches that can prevent and/or attenuate NAFLD, we examined whether strength training had effects on plasma and morphological parameters related to NAFLD in ovariectomized Wistar rats.
2. Methods 2.1. Animals and experimental procedures The sample consisted of 22 female Wistar rats that were three months old and provided by Central Animal Laboratory, State University of Maringá. They were given diet and water ad libitum, and they were kept under a light/dark cycle of 12/12 hrs at 23 ± 2 ◦ C. The animals were randomly divided into three groups: group 1 consisted of sedentary rats that passed through the surgical stress (SHAM), group 2 consisted of ovariectomized rats (OVX), and group 3 consisted of ovariectomized rats subjected to strength exercise (OVX-EXE).
2.2. Surgical procedures The animals, with 3 months of age, were anesthetised by intramuscular xylazine (0.1 mL/100 g) and ketamine (0.1 mL/100 g) and ovaries surgically removed. The surgical technique was performed according to the description
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by Zarrow et al. [28]. Twenty-four hours before surgery, all rats received 0.1 ml/100 g of intramuscular pentabiotic, and after surgery, they received 0.2 mg/kg of atropine subcutaneously following the procedure, as proposed by Serakides et al. [29].
concentrations of triglycerides, total cholesterol, highdensity lipoprotein (HDL) and glucose were conducted using the colorimetric method. Analyses were performed using a commercial kit (Gold Analisa® , Belo Horizonte, Brazil) and a spectrophotometer (Bioplus® UV-2000, São Paulo, Brazil).
2.3. Strength exercise training
2.6. Histological analysis
For the strength exercise protocol, a training apparatus was developed such as the one adopted by Tamaki et al. [30], which simulated the execution of the squat. The apparatus has an electronic module that generates an electrical current (20 V, 0.3 s duration and 3 s intervals between stimuli). The electrical current is then directly applied to the tail of rats. As a result of these stimuli, the rats, which are attached to a device similar to the squat machine, flex their legs repeatedly, lifting the weight located in the training apparatus. The animals underwent two weeks of adaptation to the apparatus (weight set at 100 g). Subsequently, the 1 RM test was realized to determine the training load (1 RM 85%). The 1 RM was measured fortnightly. The group OVX-EXE held three sets of 10 repetitions with 45 seconds of rest between each set, three times a week starting 60 days after surgery for a period of 10 weeks, as proposed by Barauna et al. [31].
Part of the liver tissue was fixed in Bouin and stored in 70% alcohol until histological analysis. A portion of the stored liver was embedded in paraffin and subjected to microtome (Microtome Leica RM 2245, Wetzlar, Germany) with semiserial histological sections that were 6 m thick. We used haematoxylin and eosin to highlight the possible macrovesicular steatosis. The capture of images was performed by using a high-resolution digital camera Pro-Series Media Cibertecnics coupled to an Olympus B × 40 microscope (Tokyo, Japan), and for reading the images, we used Image Pro-Plus 4.1 (Springfield, United States of America). For morphometric analyses, we obtained the infiltrated area by considering the microscope field area (149886.64 m2 ) and then subtracting the area occupied by the central vein from it.
2.4. Collection of tissues
Statistical evaluation of results was performed by one-way analysis of variance (Anova) with Newman-Keuls multiple comparisons test, prefixing the level of significance at 5% (P < 0.05). Statistical tests were performed using the Prism v.2.1 (GraphPad, USA) and Microsoft Excel® (São Paulo, Brazil) softwares.
After the experimental procedure, the animals were anesthetised with sodium pentobarbital (Hypinol® 3%, 4 mg/100 g bw, ip), and laparotomy was carried out to collect blood (4 mL) from the vena cava. Mesenteric, uterine, retroperitoneal and subcutaneous (inguinal) fat and part of the liver were also removed.
2.5. Biochemical analysis The blood samples were centrifuged at 2,000 rpm (4 ◦ C, 15 minutes) and divided into serum and plasma. The serum and plasma were stored in Eppendorf tubes and frozen at −12 ◦ C until their analysis. The determination of plasma
2.7. Statistical analysis
3. Results Table 1 shows the values of body weight and white adipose tissue pads of several body regions from control (SHAM), ovariectomized (OVX) and exercised (OVX-EXE) rats. We observed that ovariectomy resulted in significant weight gain, as evidenced by the rats in OVX group having higher body weight when compared to rats in the
Table 1 Body weight and cushions of white adipose tissue from many body regions of rats in control (SHAM), ovariectomized (OVX) and exercised (OVX-EXE) groups. SHAM (n = 6) Body weight (g) Uterine fat (g) Retroperitoneal fat (g) Mesenteric fat (g) Subcutaneous fat (g) fat deposits (g) Liver (g) Right adrenal gland (mg) Left adrenal gland (mg)
293.0 6.5 6.2 5.4 3.8 20.0 10.3 0.022 0.025
OVX (n = 8) ± ± ± ± ± ± ± ± ±
Values are represented as mean ± standard deviation. * P < 0.05 compared to SHAM control group. ** P < 0.05 compared to OVX group.
14.5 1.2 0.6 0.8 1.0 3.0 0.8 0.006 0.004
342.6 8.4 8.6 7.2 5.1 29.3 9.5 0.022 0.022
OVX-EXE (n = 8) ± ± ± ± ± ± ± ± ±
10.8* 0.7* 0.8* 0.9* 0.7* 2.2* 0.5 0.008 0.011
317.7 6.9 7.3 5.2 3.6 22.6 9.6 0.021 0.020
± ± ± ± ± ± ± ± ±
11.9** 0.7** 0.7*,** 0.8** 0.7** 2.3** 0.4 0.008 0.006
Strength training improves plasma parameters, body composition and liver morphology Table 2
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Plasma parameters of rats in control (SHAM), ovariectomized (OVX) and exercised (OVX-EXE) groups. SHAM (n = 6)
Total cholesterol (mg/dL) Triglycerides (mg/dL) HDL (mg/dL) Glucose (mg/dL)
85.8 80.5 82.7 111.2
OVX (n = 8) ± ± ± ±
7.1 5.5 1.4 10.0
97.1 91.4 64.6 147.4
OVX-EXE (n = 8) ± ± ± ±
6.1* 6.1* 2.8* 18.8*
100.7 106.6 91.4 118.5
± ± ± ±
6.4* 3.2* 2.6*,** 2.2**
HDL: high-density lipoprotein. Values are represented as mean ± standard deviation. * P < 0.05 compared to SHAM. ** P < 0.05 compared to OVX group.
SHAM group. However, strength training was effective in controlling body weight gain in OVX-EXE group (OVX vs OVXEXE, P < 0.05). Additionally, we noted that ovariectomy also resulted in increased visceral adiposity (as demonstrated by uterine, retroperitoneal and mesenteric fat depots) and subcutaneous fat (SHAM vs OVX, P < 0.05), a factor normalized by the addition of strength training (OVX vs OVX-EXE, P < 0.05). The weight of the liver was not affected. The adrenal gland weight was not different between the three groups, indicating that ovariectomy and strength training did not influence this parameter. Ovariectomy also resulted in changes in all plasma parameters analyzed. The strength training reduces blood glucose and increases cholesterol (HDL) of ovariectomized rats, but had no effect on total cholesterol and triglycerides (Table 2). Histological analysis revealed a marked increase in liver (H/E) macrovesicular steatosis in OVX group, which may have represented a possible high incidence of lipid droplets in liver tissue of those animals. As shown in Fig. 1, the macrovesicular steatosis was 2.5 times higher in OVX rats (8.6 ± 0.2%) as compared with SHAM rats (3.5 ± 0.1%). The strength training led to a reduction of 1.5 times in rats OVXEXE (5.7 ± 0.2%). The presence of macrovesicular steatosis can also be observed in the photomicrograph shown in Fig. 2. We note that the figure representing the OVX group presents more whitish appearances than the other groups, which correspond to the presence of vacuoles. Histologically, there were no other structural changes in liver tissue. The effect of strength training protocol was showed in Fig. 3. An elevation of 144% of strength was observed in OVX-EXE group (first 1 RM test 178.1± 26.6 g and last 1 RM test 436.3 ± 53.6 g).
Figure 1 Percentage area of macrovesicular steatosis in liver tissue from rats in control (SHAM), ovariectomized (OVX) and exercised (OVX-EXE) groups. Values are expressed as mean ± SEM; * P < 0.05 compared to SHAM; # P < 0.05 compared with OVX.
4. Discussion Our data indicate that the strength training protocol used was effective to increase strength, control the accumulation of fat in the visceral and subcutaneous tissues and macrovesicular steatosis in liver tissue. Therefore, this led us to infer that strength training, which has numerous health benefits and has protective effects against many diseases, also appears to be a good intervention to combat NAFLD associated to ovariectomy. The practice of regular physical exercise is very important for women who suffer from the effects of reduced ovarian hormones at menopause [32]. Physical exercise is
Figure 2 Photomicrograph of liver tissue from: (A) control (SHAM), (B) ovariectomized (OVX), and (C) exercised (OVX-EXE) groups. The magnification was × 230 with the Olympus B × 40 microscope.
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Figure 3 Load (g) improvement in response to strength training protocol in OVX-EXE group. The first and last 1 RM test is presented. * P < 0.05.
used as a non-pharmacological intervention to prevent and reverse alterations occurred in association to menopause, and strength training is one of them. Strength training is associated to attenuation or reduction of many alterations involved in this period, including sarcopenia, osteopenia, insulin resistance, inflammatory markers, NAFLD, among others. However, strength training is still not routinely used as a therapeutic intervention [33]. Menopause is critical to women’s health, because there is a decrease in the production of sex hormones and a parallel increase in the incidence of chronic degenerative diseases in postmenopausal women [1—4]. Thus, we sought to develop treatment methods and identify means of prevention of NAFLD in this population. Physical activity is included as a preventive intervention to treat metabolic syndrome, and NAFLD is strongly associated with such syndrome. Therefore, this study sought to verify the effect of strength exercise training on plasma and morphological variables related to chronic degenerative diseases. Strength training was selected for observation because relatively less is known of its effects, given the emphasis on aerobic exercise as the primary intervention used when we have a condition (ovariectomy/menopause) that is related to a metabolic disorder. Our study presents data that positively reflect the use of strength training to treat metabolic disorders. This can be proven by the decrease in fat, improvement in glucose and HDL as well as lower macrovesicular steatosis in liver tissue (which may be associated with a reduced presence of lipid droplets) observed in the group OVX-EXE. Menopause is associated with increased adiposity, particularly visceral adiposity, and a high risk of metabolic diseases [3]. Recent studies have shown that visceral adipose tissue contributes to these metabolic complications of menopause [34], including NAFLD [35]. Thus, the prevention of the increase or reduction in body fat is very important, and this confirms the need for the inclusion of regular physical exercises that makes the body expend more energy or increase its daily energy expenditure [36]. Although, the effects of physical activity have been attributed mostly to aerobic exercise in terms of energy expenditure [37], our study showed a beneficial effect of strength training (OVX vs OVX-EXE) on visceral and subcutaneous adiposity. Diet is also an important component to determine body composition, however, we have not evaluated such parameter in our study. In addition, in a classical work, Wade et al.
M. Ojeika Vasques et al. [38] demonstrated that ovariectomy increases adiposity and body weight without alterations in food intake. In relation to effect of strength training in food intake, in a recent research from our laboratory (not published), we observed that the trained rats were not statistical different from control rats. Several studies demonstrated the effects of strength training on body composition. Twenty-four weeks (3 days/week) of strength training decreased body weight, body mass index and body fat of postmenopausal woman [39]. In addition, Bea et al. [40] observed that strength training is a viable long-term method to prevent weight gain and deleterious changes in body composition in postmenopausal woman. Teixeira et al. [41] demonstrated that strength training during 1 year changed total body composition and regional body fat in postmenopausal woman. Our body fat data associated with these data presented elsewhere highlight the importance of strength training on fat distribution in postmenopausal period. In addition to body composition alterations, menopause is related to abnormal plasma lipids [42]. Our study showed a worsening of total cholesterol, triglycerides, HDL and glucose levels as a result of ovariectomy (SHAM vs OVX). Evidences indicate that a decline in estrogen levels, characteristic of ovariectomy model [43], is associated to an atherogenic lipid profile, due an increase in LDL, triglycerides, total cholesterol and a decrease in HDL [33]. Again, most studies showed satisfactory effects associated with aerobic exercise [44] and with association of aerobic and strength training [45]. However, the results related to strength training alone are contradictory. Brochu et al. [46] did not observe effects of strength training during 6 months on lipid profile of postmenopausal woman. Opposite effects were observed after 14 weeks of strength training that resulted in total and LDL cholesterol reduction without effects on triglycerides and HDL cholesterol [47]. We observed significant response in HDL cholesterol (OVX vs OVX-EXE), demonstrating that strength training may also be suitable as a therapeutic measure to improve lipid profile in ovariectomized rats. The strength training was also effective in reducing blood glucose. But, it is important to highlight that only glycaemia was not enough to indicate glucose profile and metabolism in body in response of the strength training. A limit of our research was not realized: glucose and insulin tolerance test, and in particular, insulinemia to confirm these benefit because insulin is the principal regulator of blood glucose (for more information, [48—50]). However, the result observed in glycaemia was very interesting and important to show the strength training effects in health parameters. The implementation of daily exercise can prevent most chronic diseases and is commonly recommended for individuals diagnosed with NAFLD [51]. As reported, NAFLD is often associated with metabolic syndrome and menopause [22,52]. In our study, we noted the presence of an indicator of increase in lipid droplets (macrovesicular steatosis visualized by H/E staining) in liver tissue of OVX group, confirming the association with menopause. However, to verify the actual presence of these lipid inclusions, it would have been necessary to use a specific stain for this purpose, for example, Sudan III, Sudan Black or Red oil-O.
Strength training improves plasma parameters, body composition and liver morphology With regard to liver lipid inclusions and/or NAFLD, the practice of regular physical exercise was indicated as part of the treatment of NAFLD [16—22], but few studies have investigated the effects of strength physical training on clinical cases. Our data demonstrated that strength training was able to reduce liver macrovesicular steatosis, pointing to a new measure that can be adopted for the treatment of menopause.
5. Conclusion Considering the data obtained here, we emphasize the use of strength exercise training as a therapeutic means to combating and/or controlling the metabolic disturbances associated with menopause, including adiposity, and adverse changes in blood glucose, blood HDL and liver macrovesicular steatosis.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
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