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

Female Sprague-Dawley (SD) rats (206.19 ± 6.2 g), aged 8 weeks, were obtained from Central Lab Animal (Seoul, Korea). Animals were housed in an air-conditioned room at 23 ± 1℃, 64.1% relative humidity, with a 12-hour light/dark cycle. The ovaries of all rats were removed surgically to induce a postmenopausal status and a 1-week recovery period was given. After the recovery period, the rats were randomly divided into two treatment groups: (1) high-fat diet-sedentary (HFD) and (2) high-fat diet-exercise (HFD+EX). The high-fat diets were prepared daily, for 8 weeks, using premade diets. The experimental diets were purchased from Research Diet Inc. (New Brunswick, NJ, USA). The high-fat diet contained 45% of energy as fat, derived from corn oil and lard (225:1598) (Product# D12451).

After a 1-week recovery period, the animals were acclimatized to running on a treadmill for 15 minutes, 8 m/min, at 0° inclination for one day. After this, the animals were regularly trained 5 times per week, for 8 weeks, and the training started at 1000 hours. From week 1 to week 4, the animals ran on a treadmill for 30 minutes, at 15 m/min with 0° inclination. Subsequently, from week 5 to week 8, the training progressed to 40 minutes and 18 m/min with 0° inclination. The exercise training protocol used a modified version taken from previous studies^{18, 19}. All training rats were restrained from training 24 hours before sacrifice.

The rats were suffocated with CO₂ and the plantaris muscles were freshly dissected, trimmed, dried with filter paper, weighed and the weights recorded using an electronic balance scale (OHAUS, NJ, USA). The muscles were snap-frozen in liquid nitrogen or preserved in 4% phosphate buffered formalin for further studies.

Tissues were lysed and homogenized in ice-cold RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% Sodium deoxycholate, 2 mM EDTA, complete protease inhibitor, and phosphatase inhibitor cocktails). The tissue extracts were then centrifuged at 13,000 rpm for 15 min at 4℃. Subsequently, the protein in the supernatant was quantified using a Bradford protein assay kit (Bio-Rad, Hercules, CA) and 80 μg of total protein were resolved on 6%-10% SDS-PAGE gel (90 V, 25℃, 20 min, after 150 V, 25℃, 1 hour) and transferred to a nitrocellulose membrane (25 V, 25℃, 7 min). All the blots were incubated with Ponceau S (Sigma, St. Louis, MO) to ensure equal loading in all lanes (data not shown). To detect target proteins, the membranes were blocked with 5% skim milk in Tris-buffered saline (TBS) with 0.1% Tween 20 (0.1% TBST) for 1 hour, at room temperature, and subsequently incubated with total-AMPK (#2532), phospho-AMPKα (Thr172) (#2535), total-p70S6K (#9202) (polyclonal rabbit antibody, 1:1000), phospho-p70S6K (Thr389) (#9206) (monoclonal mouse antibody, 1:1000) (Cell Signaling, Beverley, MA), MAFbx/Atrogin-1 (sc- 33782) (polyclonal rabbit antibody, 1:2000) (Santa Cruz Biotechnology, Santa Cruz, CA), MuRF1 (ab77577) (polyclonal rabbit antibody, 1:1000) (Abcam, Cambridge, UK), and actin (A2066) (polyclonal rabbit antibody, 1:5000) (Sigma, St. Louis, MO, USA) antibodies overnight at 4℃, in 3%-5% BSA in 0.1% TBST. Subsequently, the membranes were washed three times for 5 min each in 0.1% TBST, after which they were incubated for 1 hour with anti-mouse or rabbit IgG horse-radish peroxidase-linked secondary antibody (1:2500) (Cell signaling). The membranes were then washed as described above, after which West Femto Stable Peroxide Buffer (Thermo Fisher Scientific, Waltham, MA, USA) was applied, according to the manufacturer’s instructions, to develop a signal that was subsequently detected using the Chemiluminescence imaging system (ATTO, Tokyo, Japan) and quantified using densitometry Image J software (NIH, USA). The target protein levels were then normalized against the actin protein levels.

The plantaris muscles were directly placed in a formalin solution (Sigma, St. Louis, MO). Cross-sections were cut from the mid-belly region of each muscle. Formalin-fixed paraffin-embedded sections (5 μm) were deparaffinized, hydrated and antigen retrieval was performed using xylene. The tissue was permeabilized with 0.02% Triton X-100 in PBS (PBST) for 15 min and blocked with 5% BSA in PBST for 30 min. Subsequently, the slides were washed once with PBS, after which they were probed with TSC2 (#4308) polyclonal rabbit antibody (Cell Signaling) at a dilution of 1:800 overnight at 4℃, in 5% BSA in PBS. The slides were then washed three times for 5 min each in 0.05% Tween 20 in PBS, after which they were incubated with Alexa 488-conjugated goat anti-rabbit IgG secondary antibody (Invitrogen Life Technologies, Carlsbad, CA) diluted 1:200 for 20 min, at room temperature, in PBS that contained 5% BSA. Subsequently, the slides were washed three times with 0.05% Tween 20 in PBS, after which they were mounted with mounting media. Finally, the slides were viewed and photographed using a Nikon Imaging System (Nikion, Tokyo, JAPAN).

Total RNA was extracted from the plantaris muscle 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, USA). Subsequently, cDNA was synthesized from 1 μg of total RNA in the presence of a random primer, 2.5 mM dNTP, RNase inhibitor, and reverse transcriptase (Invitrogen Life Technologies, Carlsbad, CA) in a final volume of 20 μg at 25℃ for 10 min, followed by 42℃ for 60 min, and 95℃ for 5 min. Real-time quantitative polymerase chain reaction (qPCR) was performed using the Step-One-Plus system (Applied Biosystems). Polymerase chain reaction (PCR) was performed in duplicate using the SYBR Green Master Mix (Kapa Biosystems, Wilmington, MA, USA) according to the manufacturer’s instructions. The primer sets for target genes were atrogin-1 (F) 5’- CCATCAGGAGAAGTGGATCTATGTT-3’, (R) 5’- GCTTCCCCCAAAGTGCAGTA-3’; MuRF-1 (F) 5’- TGTCTGGAGGTCGTTTCCG-3’, (R) 5’- ATGCCGGTCCATGATCACTT-3’; GAPDH (F) TGCACCACCAACTGCTTA -3’, (R) 5’-GGATGCAGGGATGATGTTC-3’. The primers were purchased from Cosmo (Cosmo Genetech, KOREA). The expression of these target genes was then normalized to the amount of GAPDH, and the relative expression of all genes was calculated using the comparative CT method.

All values are reported as mean ± SE. Statistical significance was determined with an independent sample t-test using SPSS 22.0. Differences between groups were considered significant at p< 0.05.

The plantaris muscle mass to body weight ratio increased with low intensity aerobic exercise but the difference was not statistically significant (Table. 1).

To test the effect of the high-fat diet and aerobic exercise on total TSC2 protein expression, which inhibits the mammalian target of rapamycin (mTOR) and p70S6K, we performed immunohistochemical staining. As expected, TSC2 (green) was strongly detected in the HFD group. Conversely, aerobic exercise largely eliminated the high-fat-induced increase in TSC2 expression (Fig. 1).

To investigate the effects of aerobic exercise on the ubiquitin ligases, we performed western blot analysis. The muscle content of atrogin-1 was significantly decreased in the HFD+EX group (p< 0.05; Fig. 2A). Although the difference was not statistically significant, MuRF1 also tended to decrease in the exercise group when compared with the HFD group. As shown in Fig. 4A, real-time qPCR analysis revealed that atrogin-1 gene expression was not significantly decreased in the exercise group when compared with the non-exercise group. The MuRF1 mRNA level was also not significantly decreased in the exercise group. (Fig. 4B).

The phosphorylation of AMPK (Thr172) was significantly increased in response to aerobic exercise (p<0.05; Fig. 3A). However, the p-AKT (Ser473) activity was not altered in the exercise group (Fig. 3B). To demonstrate whether AMPK mediates the inhibition of the mTOR signaling pathway, we measured the level of p70S6K protein activity. As shown in Fig. 3C, p70S6K protein phosphorylation was not significantly elevated in the exercise group.