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Flaxseed Lignans Promoted the Growth of Skeletal Muscle in Male Rats and Its Possible Mechanism
Agricultural Sciences in China
December 2009
2009, 8(12): 1511-1516
Flaxseed Lignans Promoted the Growth of Skeletal Muscle in Male Rats and Its Possible Mechanism ZHOU Wei, WANG Guo-jie and HAN Zheng-kang Key Laboratory of Animal Physiology and Biochemistry, Ministry of Agriculture/Nanjing Agricultural University, Nanjing 210095, P.R.China
Abstract This study was aimed to determine whether flaxseed lignans could affect the growth of skeletal muscle in male animals and its possible mechanisms. The impact of flaxseed lignans on the skeletal muscle in male rats was determined in vivo. Flaxseed lignans (50 ppm) and daidzein (5 ppm) were added into the basal diets, respectively. The concentrations of serum lignans and daidzein were measured by high performance liquid chromatography (HPLC), and the serum growth hormone and testosterone (T) levels were analyzed by radioimmunoassay (RIA), and the expression of estrogen receptor β (ER β) in the soleus muscle and hypothalamus were determined by reverse-transcription polymerase chain reaction (RT-PCR). Flaxseed lignans and daidzein could significantly improve the feed efficiency and facilitate the weight gain of the femoral muscle in male rats. The ratio of RNA to DNA in the muscles and serum T levels was remarkably increased, whereas, the urea nitrogen concentrations were significantly decreased by flaxseed lignan and/or its metabolites and daidzein. Meanwhile, the expression of ER β in soleus muscle and hypothalamus were both upgraded by the two phytoestrogens. Flaxseed lignan promoted the growth of male rats, and it might be by regulating serum T levels by binding to ER β in the hypothalamus. In turn, it depressed the catabolism of protein and promoted the hypertrophy of skeletal muscle cells. Key words: phytoestrogens, flaxseed lignans, daidzein, mammalian lignans, skeletal muscle growth
INTRODUCTION Phytoestrogens are estrogen-like compounds, ubiquitous in many plants. They comprise of two major families, namely, isoflavones and lignans. Flaxseed is the richest edible source of lignan, especially secoisolariciresinol diglucoside (SDG), SDG content varies between 2-13 mg g-1, and is nearly 75-80 times that of the others. The average yield of flaxseed in China is about 40 000 t per year, however it is mainly used for oil production (Chen 2004). The development of active ingredient in flaxseed may have large eco-
nomic returns. Lignans can be converted to mammalian lignans by the intestinal flora presented in humans and rats (Wang 2002). Mammalian lignans, primarily enterolactone (ENL) and enterodiol (END), were identified after 1980, and have received a great deal of attention because they may help prevent cancers (Adlercreutz 2003) and atherosclerosis (Prasad 1999), and may affect the activities of some enzymes (Adlercreutz et al. 1993) and the neuroendocrine system in humans and animals (Martin et al. 1996; Zhou et al. 2007). However, the effect of SDG on the growth of animals has not been studied thus far. Previous studies have shown that daidzein (Da), a major isoflavone,
This paper is translated from its Chinese version in Scientia Agricultura Sinica. ZHOU Wei, Ph D, E-mail: [email protected]; Correspondence HAN Zheng-kang, Professor, Tel: +86-25-84396224, E-mail: [email protected]
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(08)60366-1
1512
could facilitate the growth of skeletal muscle in male animals, and this effect may be mediated via the neuroendocrine pathway (Han 1999). Since both isoflavones and lignans have, as their common denominator, a phenolic group that they share with estrogenic steroids, they may have similar physiological functions. Using Da as a reference compound in a Sprague-Dawley (SD) rat model, this study was aimed to elucidate the effect of lignans on skeletal muscle growth in male animals and investigate the possible mechanisms.
MATERIALS AND METHODS Materials Isolation of SDG SDG was isolated from defatted flaxseed meal (DFFS) as described by Andersson et al. (2002). Briefly, DFFS was further defatted with petroleum ether and chloroform and then extracted with dioxane:ethanol (1:1, v/v) for 48 h. The extract was dried and degraded with 1.6% sodium ethoxide in anhydrous methanol. Next, it was concentrated and then acidified to pH 3.0 with 2 mol L-1 sulfuric acid and applied to a silica gel column (0.063 -0.200 mm, Kieselgel 60; MAKALL, Qingdao). It was then eluted with the lower phase of a chloroform: methanol:water (65:35:10, v/v/v) solvent mixture. The crude SDG was purified on a second silica gel column and eluted using chloroform with increasing amount of methanol (10-30%). Its purity was 95.2% as determined by HPLC with an authentic standard (Chromadex, USA). Animals Male Sprague-Dawley (SD) rats (36 animals; 75-85 g BW; 28-d-old) were purchased from SLAC Laboratory Animal Co. Ltd., Shanghai, China.
Methods Experiment design and sampling Rats were assigned randomly to three groups, and maintained in individual stainless steel cages. They had free access to fresh water and basal diet (BD). After adaptation for 1 wk, the control group was fed BD and the lignans group was provided purified SDG by diet (50 mg kg-1 BD),
ZHOU Wei et al.
whereas, Da (99.9%) was offered to the Da group (5 mg kg-1 BD). Diet was provided to the rats each day, and their dietary intake was monitored throughout the study period. The body weights of the rats were recorded every week after a 12-h overnight fast. All the rats were slaughtered at the end of 5 wk. Blood samples (collected from the jugular vein) were centrifuged at 1 000 × g for 15 min to obtain the sera, which were stored at -20°C until further analysis. The total weight of the left femoral muscle was recorded. The gastrocnemius muscle, soleus muscle, and flexor digitalis pedis superficialis (FDPS) of the right leg were weighed and washed with diethylpyrocarboneate (DEPC) buffer and then stored at -70°C until further analysis, for gene expression. After removal, the hypothalamus was washed in the same manner and stored at -70°C for measuring ER β expression. BD contents (%): corn 35, wheat 35, soybean flour 20, fish meal 5, bone meal 1, cod liver oil 1, yeast powder 1, salt 1, and vegetable oil 1. Analysis Serum urea nitrogen and hormones: The blood urea nitrogen (BUN) concentration was measured by using a commercially available kit (Jiancheng Bioengineering Institute, Nanjing, China). The concentrations of growth hormones (GH) and testosterone (T) in the serum were measured by radioimmunoassay (RIA) using commercial kits purchased from the North Institute of Biological Technology (Beijing, China). Nucleic acids: A muscle aliquot (1.0 g) from the same part of the left leg was minced and homogenized in a glass tube containing 1 mL of physiological saline. The nucleic acids present in the supernatant liquid were quantified by a modified (Schmidt-ThannhauserSchncider) STS method, as described previously (Cai and Yuan 1982). RT-PCR: Expression of ER β mRNA in the hypothalamus and soleus muscle was measured by semiquantitative RT-PCR, using glucose 6-phosphate dehydrogenase (G6PDH) as an internal standard. Based on the complete sequence of ER β cDNA in GenBank (AF042059), the primers of ER β and G6PDH were designed by Primer Premier 5.0, and were synthesized by Saibaisheng Ltd., Shanghai, China. The PCR primers for ER β were as follows (Petersen et al. 1998): forward 5´-TGT CCA GCC ACG AAT CAG-3´ and reverse 5´-CCT ACC TCC ACG ATT ACC-3´. The
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd.
Flaxseed Lignans Promoted the Growth of Skeletal Muscle in Male Rats and Its Possible Mechanism
G6PDH gene was used as the control, and the primers used were as follows (Shirwalkar et al. 2007): forward 5´-CTT CCG TGC GAG CAC TGC-3´ and reverse 5´-CAT TAT GGC GTG TGC AG-3´. The PCR reaction system consisted of: 2.5 L 10 × PCR buffer, 1.5 L 1.5 mmol L-1 MgCl2, 2 L 0.2 mmol L-1 dNTP, 2 L cDNA, 1 L primers of target gene for both forward and reverse, 0.5 L primers of G6PDH for both forward and reverse, and 13.8 L ddH2O, 5 U Taq DNA polymerase. The PCR conditions were: denaturized at 95°C for 5 min; 94°C 45 s, 54.5°C 30 s, 72°C 45 s (32 cycles for the soleus muscle and 28 cycles for the hypothalamus); 72°C 10 min. Serum phytoestrogens and mammalian lignans: 1 mL dioxane was added into 1 mL serum, after extraction overnight in a shaking bath (45°C). The mixture was centrifuged (12 000 × g, 30 min, 4°C) to obtain the supernatant. The residuum was extracted again. Nitrogen fumes were used to dry the mixed supernatant and then 200 L mobile phases were added. These were used for the measuring the SDG by reversed phase high performance liquid chromatography (RPHPLC). The analysis of END and ENL were similar to that of SDG, but included a hydrolytic step by NaOH (1 mol L-1, 20°C, 48 h) (Zhou et al. 2007). According to the method of Rickard and Thompson (1998), the condition of RP-HPLC was modified: the Waters HPLC column RP C18 (4.6 mm i.d. × 150 mm, 5 m); the mobile phase consisted of two solvents (A) acetonitrile and (B) deionized water, and the separation was performed using the following gradient of A-B, that is, 0 min 20 min (10.0% A 90.0% B) at a flow-1 rate of 1 mL min ; wave length 280 nm (UV); halfwidth 8 nm; temperature 25°C; and injection volume 20 L. The concentration of serum Da was measured by RP-HPLC as described by Bowey et al. (2003).
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RESULTS Concentrations of serum Da, SDG, END, and ENL in rats As shown in Table 1, the concentrations of Da, SDG, END, and ENL were below the detection levels in rats of the control group. In the SDG treatment group, there were low levels of serum SDG and mammalian lignans in rats, among which the SDG concentration was the highest, whereas, the END level was lower than that of ENL. This might indicate that SDG could be converted to mammalian lignans in male rats. The serum Da concentration was 0.010 mg mL -1 in Datreated rats, but the lignans levels were below the detection levels.
Effects of SDG on the growth of skeletal muscle and feed efficiency in male rats The feed efficiency was significantly increased by both SDG (12.44%, P < 0.05) and Da (20.44%, P < 0.01) as compared to the control group (Table 2). In the SDG treatment and Da treatment groups, the total weight of the femoral muscle was increased by 19.79% (P < 0.05) and 31.35% (P < 0.05), respectively. Simultaneously, the soleus muscle weight in SDG or Da-treated rats tended to be higher (P > 0.05). The DNA content in SDG- or Da-treated rats seemed to be higher than that of the control group, and the Table 1 The concentrations of serum lignans and daidzein mg mL-1 SDG END ENL Da
Control
SDG treatment
Da treatment
UD UD UD UD
0.072 ± 0.018 0.019 ± 0.006 0.030 ± 0.007 UD
UD UD UD 0.010 ± 0.001
UD, under the detection level.
Statistical analyses Statistical analyses were carried out using SPSS (SPSS Inc.) software. Data were presented as mean ± SD. Differences in continuous variables, such as growth factors among groups, were determined by one-way ANOVA, and t-test was used to determine the difference between the different groups.
Table 2 Effects of daidzein and SDG on the feed efficiency and skeletal muscle growth parameters Control Feed efficiency (g g-1) Weight of femoral muscle (g) Soleus muscle weight/Body weight (g kg-1) RNA (mg g-1) DNA (mg g-1) RNA/DNA
SDG treatment
Da treatment
0.225 ± 0.006 a 0.253 ± 0.007 b 0.271 ± 0.009 c 7.87 ± 0.61 b 8.63 ± 0.89 b 6.57 ± 0.63 a 0.417 ± 0.034 a 0.432 ± 0.027 a 0.432 ± 0.029 a 0.529 ± 0.041 a 0.909 ± 0.062 b 1.014 ± 0.044 b 0.461 ± 0.080 a 0.576 ± 0.122 a 0.600 ± 0.068 a 1.147 ± 0.355 a 1.578 ± 0.284 b 1.692 ± 0.362 b
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RNA concentrations were significantly increased by these two phytoestrogens. Moreover, the RNA to DNA ratio was increased by SDG (37.58%, P < 0.05) and Da (47.52%, P < 0.05). This might indicate that the SDG diet could induce the infusion of sarcoblasts and myotubule.
Impacts of SDG on the concentrations of serum blood urea nitrogen (BUN) and related metabolic hormones As show in Fig.1, serum T level was remarkably increased by SDG (263.36%, P < 0.01) and Da (101.80%,
ZHOU Wei et al.
P < 0.05) in male rats. Meanwhile, in the SDG and Da treatment groups, the serum BUN concentrations were significantly reduced by 26.24% (P < 0.01) and 35.34% (P < 0.01), respectively. However, the GH levels were not significantly affected by SDG or Da.
Effects of SDG on ER β expression in the soleus muscle and hypothalamus The expressions of ER β mRNA were detectable both in the soleus muscle and hypothalamus of 70-d-old rats. Fig.2 summarizes the impacts of SDG and Da on the expression of ER β mRNA in the soleus muscle and hypothalamus of rats. SDG could upregulate the expression of ER β mRNA both in the muscle (P < 0.05) and hypothalamus (P > 0.05). Moreover, the expression was remarkably increased by Da in the soleus (P < 0.01) and hypothalamus (P < 0.05).
DISCUSSION
Fig. 1 Impact of SDG and daidzein on serum BUN, testosterone, and growth hormone levels in male rats. Compared with control, * means P < 0.05 and ** P < 0.01.
Thus far, the metabolic fate of SDG in nonruminant animals is still not well defined. Clavel et al. (2005) carried out an in vitro biotransformation of SDG by human intestinal bacteria, after anaerobic incubation with human fecal suspension: first, SDG was metabolized to secoisolariciresinol (SECO), and then after
Fig. 2 Effect of SDG and Da on the expression of ER β mRNA in the soleus muscle and hypothalamus. A, M, DNA molecular weight marker pUC19; lanes 1-3 represent the control, SDG, and Da, respectively. B, the expression levels of SDG and Da compared with control. * P < 0.05; ** P < 0.01.
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd.
Flaxseed Lignans Promoted the Growth of Skeletal Muscle in Male Rats and Its Possible Mechanism
dehydroxylation and demethylation by facultative bacteria, SECO was converted to END (Zhou et al. 2007). Next, ENL was produced from END through oxidation by facultative bacteria (Clavel et al. 2005). It was reviewed and suggested that mammalian lignan production depended on the presence of bacteria in the intestinal tract (Wang 2002). Previous studies in the laboratory had demonstrated that rats fed on a diet with SDG and antibiotics could significantly reduce the production of END and ENL. The presented END and ENL in the serum, in this study, could be produced by intestinal bacteria from SDG. Furthermore, the concentration of serum ENL was higher than that of END. Bowey et al. (2003) and Kunst et al. (2006) had demonstrated that when SDG was offered to human or human flora-associated rats, a larger amount of ENL was formed, whereas, Kah et al. (2004) found that the urinary END concentration in rats during the gestation period was nearly 10 times than that of ENL. Lignan profiles might therefore be dependent on the composition of the dominant intestinal microorganisms in different animals. Meanwhile, SDG and the two mammalian lignans were found in serum, which could indicate that both SDG and its metabolites (END and ENL) might be absorbed into the blood circulation. It was reported that isoflavones could affect the growth and metabolism in animals. Wang et al. (1994) found Da (3 mg kg-1 BD) could promote the growth of chest and femoral muscle and significantly increase the RNA to DNA ratio in male broilers. Ma et al. (2005) demonstrated that Da or ipriflavone (3 mg kg-1 BD) could facilitate the skeletal muscle growth. The current study had confirmed the functions of Da on the growth of skeletal muscle, and found that SDG supplementation could significantly improve the feed efficiency and increase the weight gain of skeletal muscles in male rats. The positive physiological effect of SDG on the growth of skeletal muscles could be similar to that of isoflavones. The myofibrillar formation included following steps (Yang 2000; Wei et al. 2002): satellite cells differentiated to sarcoblast, magnanimous sarcoblasts conjugated to form a myotubule, myoneme transformed to myofilament, and the muscle fiber formaed. In this experiment, SDG or Da could increase the content of nucleic acids (RNA and DNA) and the RNA to DNA ratio in skeletal muscles, suggest-
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ing that both SDG and Da could induce cell fusion of sarcoblasts, and thereby promote hypertrophy of muscle fiber. Previous studies had demonstrated that isoflavones could increase serum T level in male animals by affecting the hypothalamus-pituitary-gonadal axis, and thereby facilitating protein synthesis and the accretion of nitrogen in muscle cells (Han 1999). In this study, SDG or Da could significantly increase the serum T concentration and remarkably reduce the serum BUN level. These indicated that lignans might possess a positive effect on the growth of skeletal muscle cells. As the ratelimiting enzyme in the synthesis of estradiol (E2), aromatase could transform T to E2, thus the changes in the activity of aromatase could induce the serum gonadal hormones levels increase or decrease. Da and the two mammalian lignans, especially ENL, are inhibitors of aromatase (Adlercreutz et al. 1993; Thomas et al. 2006), therefore the increased serum T level might be partly caused by the inhibition of biotransformation of T to E2. It is important to note that E2 has similar binding affinities for the estrogen receptor α (ER α) and ER β, but the binding potency of isoflavones was greater to ER β than to ER α (Kuiper et al. 1998; Patisaul et al. 2002). The expression of ER β mRNA in the soleus muscle and hypothalamus was significantly upregulated in vivo, therefore the positive effects of Da, SDG and/ or mammalian lignans might be mediated through their binding to ER β, and direct or indirect (hormone-related) mechanisms might be involved. However, there are many kinds of subtypes of estrogen receptor β (ER β), such as ER-β1, ER-β2, ER-β5 (Zhang and Cai 2001), and ER-βδ (Petersen et al. 1998). Thus, in order to identify whether ER β is the essential condition of the positive effects of phytoestrogens on skeletal muscle growth in rats, further studies using ER β gene knock-out rats or high performance blocking agent are needed.
CONCLUSION SDG supplemented in diet could significantly increase the serum concentrations of SDG, END, and ENL, and promote the growth of skeletal muscle in male rats. Also, Da might affect the hypothalamus-pituitary-go-
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd.
1516
nadal axis by binding to ER β, and could upgrade the serum T level, inhibit the catabolism of protein, and induce the infusion of satellite cells and myocytes, and thereby promote the growth of the skeletal muscle.
Acknowledgements This work was supported by the National Natural Science Foundation of China (39970534).
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Kuiper G G, Lemmen J G, Carlsson B, Corton J C, Safe S H, Van (Edited by ZHANG Juan)
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd.
December 2009
2009, 8(12): 1511-1516
Flaxseed Lignans Promoted the Growth of Skeletal Muscle in Male Rats and Its Possible Mechanism ZHOU Wei, WANG Guo-jie and HAN Zheng-kang Key Laboratory of Animal Physiology and Biochemistry, Ministry of Agriculture/Nanjing Agricultural University, Nanjing 210095, P.R.China
Abstract This study was aimed to determine whether flaxseed lignans could affect the growth of skeletal muscle in male animals and its possible mechanisms. The impact of flaxseed lignans on the skeletal muscle in male rats was determined in vivo. Flaxseed lignans (50 ppm) and daidzein (5 ppm) were added into the basal diets, respectively. The concentrations of serum lignans and daidzein were measured by high performance liquid chromatography (HPLC), and the serum growth hormone and testosterone (T) levels were analyzed by radioimmunoassay (RIA), and the expression of estrogen receptor β (ER β) in the soleus muscle and hypothalamus were determined by reverse-transcription polymerase chain reaction (RT-PCR). Flaxseed lignans and daidzein could significantly improve the feed efficiency and facilitate the weight gain of the femoral muscle in male rats. The ratio of RNA to DNA in the muscles and serum T levels was remarkably increased, whereas, the urea nitrogen concentrations were significantly decreased by flaxseed lignan and/or its metabolites and daidzein. Meanwhile, the expression of ER β in soleus muscle and hypothalamus were both upgraded by the two phytoestrogens. Flaxseed lignan promoted the growth of male rats, and it might be by regulating serum T levels by binding to ER β in the hypothalamus. In turn, it depressed the catabolism of protein and promoted the hypertrophy of skeletal muscle cells. Key words: phytoestrogens, flaxseed lignans, daidzein, mammalian lignans, skeletal muscle growth
INTRODUCTION Phytoestrogens are estrogen-like compounds, ubiquitous in many plants. They comprise of two major families, namely, isoflavones and lignans. Flaxseed is the richest edible source of lignan, especially secoisolariciresinol diglucoside (SDG), SDG content varies between 2-13 mg g-1, and is nearly 75-80 times that of the others. The average yield of flaxseed in China is about 40 000 t per year, however it is mainly used for oil production (Chen 2004). The development of active ingredient in flaxseed may have large eco-
nomic returns. Lignans can be converted to mammalian lignans by the intestinal flora presented in humans and rats (Wang 2002). Mammalian lignans, primarily enterolactone (ENL) and enterodiol (END), were identified after 1980, and have received a great deal of attention because they may help prevent cancers (Adlercreutz 2003) and atherosclerosis (Prasad 1999), and may affect the activities of some enzymes (Adlercreutz et al. 1993) and the neuroendocrine system in humans and animals (Martin et al. 1996; Zhou et al. 2007). However, the effect of SDG on the growth of animals has not been studied thus far. Previous studies have shown that daidzein (Da), a major isoflavone,
This paper is translated from its Chinese version in Scientia Agricultura Sinica. ZHOU Wei, Ph D, E-mail: [email protected]; Correspondence HAN Zheng-kang, Professor, Tel: +86-25-84396224, E-mail: [email protected]
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(08)60366-1
1512
could facilitate the growth of skeletal muscle in male animals, and this effect may be mediated via the neuroendocrine pathway (Han 1999). Since both isoflavones and lignans have, as their common denominator, a phenolic group that they share with estrogenic steroids, they may have similar physiological functions. Using Da as a reference compound in a Sprague-Dawley (SD) rat model, this study was aimed to elucidate the effect of lignans on skeletal muscle growth in male animals and investigate the possible mechanisms.
MATERIALS AND METHODS Materials Isolation of SDG SDG was isolated from defatted flaxseed meal (DFFS) as described by Andersson et al. (2002). Briefly, DFFS was further defatted with petroleum ether and chloroform and then extracted with dioxane:ethanol (1:1, v/v) for 48 h. The extract was dried and degraded with 1.6% sodium ethoxide in anhydrous methanol. Next, it was concentrated and then acidified to pH 3.0 with 2 mol L-1 sulfuric acid and applied to a silica gel column (0.063 -0.200 mm, Kieselgel 60; MAKALL, Qingdao). It was then eluted with the lower phase of a chloroform: methanol:water (65:35:10, v/v/v) solvent mixture. The crude SDG was purified on a second silica gel column and eluted using chloroform with increasing amount of methanol (10-30%). Its purity was 95.2% as determined by HPLC with an authentic standard (Chromadex, USA). Animals Male Sprague-Dawley (SD) rats (36 animals; 75-85 g BW; 28-d-old) were purchased from SLAC Laboratory Animal Co. Ltd., Shanghai, China.
Methods Experiment design and sampling Rats were assigned randomly to three groups, and maintained in individual stainless steel cages. They had free access to fresh water and basal diet (BD). After adaptation for 1 wk, the control group was fed BD and the lignans group was provided purified SDG by diet (50 mg kg-1 BD),
ZHOU Wei et al.
whereas, Da (99.9%) was offered to the Da group (5 mg kg-1 BD). Diet was provided to the rats each day, and their dietary intake was monitored throughout the study period. The body weights of the rats were recorded every week after a 12-h overnight fast. All the rats were slaughtered at the end of 5 wk. Blood samples (collected from the jugular vein) were centrifuged at 1 000 × g for 15 min to obtain the sera, which were stored at -20°C until further analysis. The total weight of the left femoral muscle was recorded. The gastrocnemius muscle, soleus muscle, and flexor digitalis pedis superficialis (FDPS) of the right leg were weighed and washed with diethylpyrocarboneate (DEPC) buffer and then stored at -70°C until further analysis, for gene expression. After removal, the hypothalamus was washed in the same manner and stored at -70°C for measuring ER β expression. BD contents (%): corn 35, wheat 35, soybean flour 20, fish meal 5, bone meal 1, cod liver oil 1, yeast powder 1, salt 1, and vegetable oil 1. Analysis Serum urea nitrogen and hormones: The blood urea nitrogen (BUN) concentration was measured by using a commercially available kit (Jiancheng Bioengineering Institute, Nanjing, China). The concentrations of growth hormones (GH) and testosterone (T) in the serum were measured by radioimmunoassay (RIA) using commercial kits purchased from the North Institute of Biological Technology (Beijing, China). Nucleic acids: A muscle aliquot (1.0 g) from the same part of the left leg was minced and homogenized in a glass tube containing 1 mL of physiological saline. The nucleic acids present in the supernatant liquid were quantified by a modified (Schmidt-ThannhauserSchncider) STS method, as described previously (Cai and Yuan 1982). RT-PCR: Expression of ER β mRNA in the hypothalamus and soleus muscle was measured by semiquantitative RT-PCR, using glucose 6-phosphate dehydrogenase (G6PDH) as an internal standard. Based on the complete sequence of ER β cDNA in GenBank (AF042059), the primers of ER β and G6PDH were designed by Primer Premier 5.0, and were synthesized by Saibaisheng Ltd., Shanghai, China. The PCR primers for ER β were as follows (Petersen et al. 1998): forward 5´-TGT CCA GCC ACG AAT CAG-3´ and reverse 5´-CCT ACC TCC ACG ATT ACC-3´. The
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd.
Flaxseed Lignans Promoted the Growth of Skeletal Muscle in Male Rats and Its Possible Mechanism
G6PDH gene was used as the control, and the primers used were as follows (Shirwalkar et al. 2007): forward 5´-CTT CCG TGC GAG CAC TGC-3´ and reverse 5´-CAT TAT GGC GTG TGC AG-3´. The PCR reaction system consisted of: 2.5 L 10 × PCR buffer, 1.5 L 1.5 mmol L-1 MgCl2, 2 L 0.2 mmol L-1 dNTP, 2 L cDNA, 1 L primers of target gene for both forward and reverse, 0.5 L primers of G6PDH for both forward and reverse, and 13.8 L ddH2O, 5 U Taq DNA polymerase. The PCR conditions were: denaturized at 95°C for 5 min; 94°C 45 s, 54.5°C 30 s, 72°C 45 s (32 cycles for the soleus muscle and 28 cycles for the hypothalamus); 72°C 10 min. Serum phytoestrogens and mammalian lignans: 1 mL dioxane was added into 1 mL serum, after extraction overnight in a shaking bath (45°C). The mixture was centrifuged (12 000 × g, 30 min, 4°C) to obtain the supernatant. The residuum was extracted again. Nitrogen fumes were used to dry the mixed supernatant and then 200 L mobile phases were added. These were used for the measuring the SDG by reversed phase high performance liquid chromatography (RPHPLC). The analysis of END and ENL were similar to that of SDG, but included a hydrolytic step by NaOH (1 mol L-1, 20°C, 48 h) (Zhou et al. 2007). According to the method of Rickard and Thompson (1998), the condition of RP-HPLC was modified: the Waters HPLC column RP C18 (4.6 mm i.d. × 150 mm, 5 m); the mobile phase consisted of two solvents (A) acetonitrile and (B) deionized water, and the separation was performed using the following gradient of A-B, that is, 0 min 20 min (10.0% A 90.0% B) at a flow-1 rate of 1 mL min ; wave length 280 nm (UV); halfwidth 8 nm; temperature 25°C; and injection volume 20 L. The concentration of serum Da was measured by RP-HPLC as described by Bowey et al. (2003).
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RESULTS Concentrations of serum Da, SDG, END, and ENL in rats As shown in Table 1, the concentrations of Da, SDG, END, and ENL were below the detection levels in rats of the control group. In the SDG treatment group, there were low levels of serum SDG and mammalian lignans in rats, among which the SDG concentration was the highest, whereas, the END level was lower than that of ENL. This might indicate that SDG could be converted to mammalian lignans in male rats. The serum Da concentration was 0.010 mg mL -1 in Datreated rats, but the lignans levels were below the detection levels.
Effects of SDG on the growth of skeletal muscle and feed efficiency in male rats The feed efficiency was significantly increased by both SDG (12.44%, P < 0.05) and Da (20.44%, P < 0.01) as compared to the control group (Table 2). In the SDG treatment and Da treatment groups, the total weight of the femoral muscle was increased by 19.79% (P < 0.05) and 31.35% (P < 0.05), respectively. Simultaneously, the soleus muscle weight in SDG or Da-treated rats tended to be higher (P > 0.05). The DNA content in SDG- or Da-treated rats seemed to be higher than that of the control group, and the Table 1 The concentrations of serum lignans and daidzein mg mL-1 SDG END ENL Da
Control
SDG treatment
Da treatment
UD UD UD UD
0.072 ± 0.018 0.019 ± 0.006 0.030 ± 0.007 UD
UD UD UD 0.010 ± 0.001
UD, under the detection level.
Statistical analyses Statistical analyses were carried out using SPSS (SPSS Inc.) software. Data were presented as mean ± SD. Differences in continuous variables, such as growth factors among groups, were determined by one-way ANOVA, and t-test was used to determine the difference between the different groups.
Table 2 Effects of daidzein and SDG on the feed efficiency and skeletal muscle growth parameters Control Feed efficiency (g g-1) Weight of femoral muscle (g) Soleus muscle weight/Body weight (g kg-1) RNA (mg g-1) DNA (mg g-1) RNA/DNA
SDG treatment
Da treatment
0.225 ± 0.006 a 0.253 ± 0.007 b 0.271 ± 0.009 c 7.87 ± 0.61 b 8.63 ± 0.89 b 6.57 ± 0.63 a 0.417 ± 0.034 a 0.432 ± 0.027 a 0.432 ± 0.029 a 0.529 ± 0.041 a 0.909 ± 0.062 b 1.014 ± 0.044 b 0.461 ± 0.080 a 0.576 ± 0.122 a 0.600 ± 0.068 a 1.147 ± 0.355 a 1.578 ± 0.284 b 1.692 ± 0.362 b
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RNA concentrations were significantly increased by these two phytoestrogens. Moreover, the RNA to DNA ratio was increased by SDG (37.58%, P < 0.05) and Da (47.52%, P < 0.05). This might indicate that the SDG diet could induce the infusion of sarcoblasts and myotubule.
Impacts of SDG on the concentrations of serum blood urea nitrogen (BUN) and related metabolic hormones As show in Fig.1, serum T level was remarkably increased by SDG (263.36%, P < 0.01) and Da (101.80%,
ZHOU Wei et al.
P < 0.05) in male rats. Meanwhile, in the SDG and Da treatment groups, the serum BUN concentrations were significantly reduced by 26.24% (P < 0.01) and 35.34% (P < 0.01), respectively. However, the GH levels were not significantly affected by SDG or Da.
Effects of SDG on ER β expression in the soleus muscle and hypothalamus The expressions of ER β mRNA were detectable both in the soleus muscle and hypothalamus of 70-d-old rats. Fig.2 summarizes the impacts of SDG and Da on the expression of ER β mRNA in the soleus muscle and hypothalamus of rats. SDG could upregulate the expression of ER β mRNA both in the muscle (P < 0.05) and hypothalamus (P > 0.05). Moreover, the expression was remarkably increased by Da in the soleus (P < 0.01) and hypothalamus (P < 0.05).
DISCUSSION
Fig. 1 Impact of SDG and daidzein on serum BUN, testosterone, and growth hormone levels in male rats. Compared with control, * means P < 0.05 and ** P < 0.01.
Thus far, the metabolic fate of SDG in nonruminant animals is still not well defined. Clavel et al. (2005) carried out an in vitro biotransformation of SDG by human intestinal bacteria, after anaerobic incubation with human fecal suspension: first, SDG was metabolized to secoisolariciresinol (SECO), and then after
Fig. 2 Effect of SDG and Da on the expression of ER β mRNA in the soleus muscle and hypothalamus. A, M, DNA molecular weight marker pUC19; lanes 1-3 represent the control, SDG, and Da, respectively. B, the expression levels of SDG and Da compared with control. * P < 0.05; ** P < 0.01.
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd.
Flaxseed Lignans Promoted the Growth of Skeletal Muscle in Male Rats and Its Possible Mechanism
dehydroxylation and demethylation by facultative bacteria, SECO was converted to END (Zhou et al. 2007). Next, ENL was produced from END through oxidation by facultative bacteria (Clavel et al. 2005). It was reviewed and suggested that mammalian lignan production depended on the presence of bacteria in the intestinal tract (Wang 2002). Previous studies in the laboratory had demonstrated that rats fed on a diet with SDG and antibiotics could significantly reduce the production of END and ENL. The presented END and ENL in the serum, in this study, could be produced by intestinal bacteria from SDG. Furthermore, the concentration of serum ENL was higher than that of END. Bowey et al. (2003) and Kunst et al. (2006) had demonstrated that when SDG was offered to human or human flora-associated rats, a larger amount of ENL was formed, whereas, Kah et al. (2004) found that the urinary END concentration in rats during the gestation period was nearly 10 times than that of ENL. Lignan profiles might therefore be dependent on the composition of the dominant intestinal microorganisms in different animals. Meanwhile, SDG and the two mammalian lignans were found in serum, which could indicate that both SDG and its metabolites (END and ENL) might be absorbed into the blood circulation. It was reported that isoflavones could affect the growth and metabolism in animals. Wang et al. (1994) found Da (3 mg kg-1 BD) could promote the growth of chest and femoral muscle and significantly increase the RNA to DNA ratio in male broilers. Ma et al. (2005) demonstrated that Da or ipriflavone (3 mg kg-1 BD) could facilitate the skeletal muscle growth. The current study had confirmed the functions of Da on the growth of skeletal muscle, and found that SDG supplementation could significantly improve the feed efficiency and increase the weight gain of skeletal muscles in male rats. The positive physiological effect of SDG on the growth of skeletal muscles could be similar to that of isoflavones. The myofibrillar formation included following steps (Yang 2000; Wei et al. 2002): satellite cells differentiated to sarcoblast, magnanimous sarcoblasts conjugated to form a myotubule, myoneme transformed to myofilament, and the muscle fiber formaed. In this experiment, SDG or Da could increase the content of nucleic acids (RNA and DNA) and the RNA to DNA ratio in skeletal muscles, suggest-
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ing that both SDG and Da could induce cell fusion of sarcoblasts, and thereby promote hypertrophy of muscle fiber. Previous studies had demonstrated that isoflavones could increase serum T level in male animals by affecting the hypothalamus-pituitary-gonadal axis, and thereby facilitating protein synthesis and the accretion of nitrogen in muscle cells (Han 1999). In this study, SDG or Da could significantly increase the serum T concentration and remarkably reduce the serum BUN level. These indicated that lignans might possess a positive effect on the growth of skeletal muscle cells. As the ratelimiting enzyme in the synthesis of estradiol (E2), aromatase could transform T to E2, thus the changes in the activity of aromatase could induce the serum gonadal hormones levels increase or decrease. Da and the two mammalian lignans, especially ENL, are inhibitors of aromatase (Adlercreutz et al. 1993; Thomas et al. 2006), therefore the increased serum T level might be partly caused by the inhibition of biotransformation of T to E2. It is important to note that E2 has similar binding affinities for the estrogen receptor α (ER α) and ER β, but the binding potency of isoflavones was greater to ER β than to ER α (Kuiper et al. 1998; Patisaul et al. 2002). The expression of ER β mRNA in the soleus muscle and hypothalamus was significantly upregulated in vivo, therefore the positive effects of Da, SDG and/ or mammalian lignans might be mediated through their binding to ER β, and direct or indirect (hormone-related) mechanisms might be involved. However, there are many kinds of subtypes of estrogen receptor β (ER β), such as ER-β1, ER-β2, ER-β5 (Zhang and Cai 2001), and ER-βδ (Petersen et al. 1998). Thus, in order to identify whether ER β is the essential condition of the positive effects of phytoestrogens on skeletal muscle growth in rats, further studies using ER β gene knock-out rats or high performance blocking agent are needed.
CONCLUSION SDG supplemented in diet could significantly increase the serum concentrations of SDG, END, and ENL, and promote the growth of skeletal muscle in male rats. Also, Da might affect the hypothalamus-pituitary-go-
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nadal axis by binding to ER β, and could upgrade the serum T level, inhibit the catabolism of protein, and induce the infusion of satellite cells and myocytes, and thereby promote the growth of the skeletal muscle.
Acknowledgements This work was supported by the National Natural Science Foundation of China (39970534).
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