All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of EWHA Womans University, Seoul, Korea. Permit Number: 14-038.

Sixty female Sprague-Dawley (SD) rats (206.19 ± 6.2 g), aged 8weeks, were obtained from Central Lab Animal (Seoul, Korea). Animals were housed in an air-conditioned room at 23 ± 1℃, 64. 1 % relative humidity and a 12 h light/dark cycle. The experimental diets were purchased from Research Diet Inc. (New Brunswick, NJ, USA): the high fat diet contained 45 % of energy as fat from corn oil plus lard (225:1598) (Product# D12451). Ovaries of all rats were removed surgically to induce postmenopausal status and 1 week of recovery period was given. After recovery periods, rats were randomly divided into 6 groups following treatment groups: (1) high fat diet (HFD) + sedentary + vehicle (HSV), (2) HFD + sedentary + PR (complex of pueraria lobata/rehmannia glutinosa by 3 to 1; 400 mg/kg body weight/day) (HSP), (3) HFD + sedentary + 17β-estradiol (0.5 mg/kg body weight/day) (HSH), (4) HFD + exercise + vehicle (HEV), (5) HFD + exercise + PR (HEP), (6) HFD + exercise + 17β-estradiol (HEH). After 1 week of rest period, the animals were acclimated to running on a treadmill for 15 min, 8 m/min, with 0° of inclination for one day. Subsequently, the animals were regularly exercised 5 times per week for 8 weeks and the running started at 10 o’clock in the morning. From 1st week to 4th week, the animals ran on a treadmill form 30 min/day, and 15 m/min with 0° of inclination. Subsequently, from 5th week to 8th week, the running progressed for 40 min/day and 18 m/min with 0° of inclination (Fig. 1).

Total RNA was extracted from soleus and plantaris muscles using a TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The RNA concentration and quality were measured at 260/280 nm using a spectrophotometer (Nanodrop-2000, Thermo Fisher Scientific, Waltham, MA). Next, cDNA was synthesized from 1 μg of total RNA in the presence of random primer, 2.5 mM dNTP, RNase inhibitor, and reverse transcriptase (Invitrogen Life Technologies, Carlsbad, CA) in a final volume of 20 μl at 25℃ for 10 min, followed by 42℃ for 60 min and 95℃ for 5 min. Real-time qPCR was performed in Step-One-Plus Real time-PCR System (Applied Biosystems, Foster City, CA, USA). PCR was performed in duplicate using the SYBR Green PCR master mix (Kappa, USA) according to the manufacturer’s instructions. The primer sets for rat target genes were showed in Table1. The primers were purchased from Macrogen (Macrogen, KOREA). The expression of the target genes was then normalized to the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). All data were expressed as a relative quantity to each control value.

Soleus and plantaris muscles were directly placed in formalin solution (Sigma, St. Louis, MO). Cross-sections were cut from the mid-belly region of each muscle. Formalin-fixed paraffin-embedded sections (5μm) were deparaffinized, hydrated and antigen retrieval was performed by xylene. The tissue was permeabilized with 0.02 % Triton X-100 in PBS (PBST) for 15 min and blocking with 5 % BSA in PBST for 30 min. Next, the slides were washed once with PBS, after which they were probed with FATP1, FAT/CD36 polyclonal rabbit antibody and FABPpm monoclonal mouse antibody (Abcam, Cambridge, UK) at a dilution of 1:200 overnight at 4℃ in 5 % BSA in PBS. The slides were then washed thrice for 5 min each in 0.05 % Tween 20 in PBS, after which they were incubated with Alexa 488-conjugated goat anti-rabbit or Alexa 568-conjugated goat anti-mouse IgG secondary antibody (Invitrogen Life Technologies, Carlsbad, CA) diluted 1:200 in PBS that contained 5 % BSA for 20 min at room temperature. Next, the slides were washed thrice with 0.05 % Tween 20 in PBS, after which they were mounted with mounting media. The slides were then viewed and photographed using a Nikon Imaging System (Nikion, Tokyo, JAPAN).

Results were presented as mean ± standard error of the mean of observations. Statistical analysis was performed using SPSS 22.0. Between-group differences (exercise groups vs. non-exercise groups and treatment groups vs. non-treatment groups) were analysed using two-way analysis of variance (ANOVA). Comparisons between multiple treatment groups were performed using Turkey’s post-hoc tests. In all analyzes, the values were considered significantly different at values of p<0.05.

To determine the effects of PR, 17β-estradiol and exercise on fatty acid transporters gene expression in OVX rat soleus muscles, fatty acid transporters mRNA level was analyzed by using real-time qPCR (Fig. 2). There was no significant alteration in FATP1 mRNA level (Fig. 2A). Whereas, either exercise (p=0.003) or treatment (p=0.02) had main effect on up-regulation of FABPpm gene expression. Post-hoc test results showed that FABPpm mRNA expression was significantly increased in 17β-estradiol treatment groups, as compared with vehicle groups (p=0.025). However, exercise combined with PR or hormone had no additive effects on FABPpm gene expression (Fig. 2B). As shown in Fig. 2C, FAT/CD36 mRNA expression was not significantly altered in this study.

Real-time qPCR analysis revealed that fatty acid transporters gene expression varied in response to treatment with exercise, PR or 17β-estradiol in OVX rats plantaris muscle (Fig. 3). Exercise does not alter FATP1 gene level in plantaris muscles. Unexpectedly, treatment had main effect on down-regulation of FATP1 mRNA level (p=0.005). Post-hoc test results showed that FATP1 mRNA expression was significantly decreased in 17β-estradiol intake groups when compared with vehicle groups (p=0.004). Furthermore, exercise combined with supplement had interaction effects on FATP1 gene suppression (p=0.004) (Fig. 3A). As shown in Fig. 3B, exercise had main effect on up-regulation of FABPpm mRNA level (p=0.000). whereas treatment had main effect on down-regulation of FABPpm mRNA level (p=0.005). Post-hoc test results showed that especially in 17β-estradiol intake groups, FABPpm gene level was significantly decreased, as compared with vehicle groups (p=0.003). Because of these contradictory results, exercise combined with treatments had no additive effect on FABPpm mRNA expression in plantaris muscle (Fig. 3B). On the contrary, FAT/CD36 mRNA expression showed no significant alteration (Fig. 3C).

Immunohistochemistry was conducted to identify the effects of PR and exercise on fatty acid transporters protein expression in OVX rat soleus muscles. Immunohistochemistry (IHC) results showed that in vehicle and PR intake groups, FATP1 proteins were highly expressed and translocated from an intracellular depot to the plasma membrane in exercise groups, as compared with sedentary groups (green, Fig. 4A). In contrast, in hormone intake groups, FATP1 proteins were expressed less in exercise groups, as compared with the sedentary group. In all groups (vehicle, PR, and estradiol intake groups), FABPpm proteins expression was higher and localized at plasma membrane dominantly in exercise groups, as compared with sedentary groups (red, Fig. 4B). Although there was a subtle difference between all groups, FAT/CD36 proteins were more highly expressed in exercise groups than sedentary groups (green, Fig. 4C). These data indicates that in exercise groups compared with sedentary groups, PR and exercise lead fatty acid transporters proteins expression was increased and translocated from cytosol to plasma membrane in OVX rat soleus muscles.

To determine whether PR and exercise-induced increase in fatty acid transporters protein localization was determined by IHC in OVX rats plantaris muscles. In vehicle, PR, and estradiol intake groups, FATP1 proteins were translocated from intracellular depot to plasma membrane and highly expressed in exercise groups than sedentary groups (green, Fig. 5A). Exercise groups represented high FABPpm proteins expression and translocation from intracellular depot to plasma membrane, as compared with sedentary groups (red, Fig. 5B). In addition, the results indicated that FAT/CD36 proteins were highly expressed and localized at plasma membrane in exercise group in comparison with the other groups (green, Fig. 5C). Thus, in exercise groups in comparison with sedentary groups, PR and exercise induce fatty acid transporters expression and translocation to the plasma membrane in OVX rats plantaris muscles.