Female Sprague-Dawley rats (5 weeks old, female, n=30) were maintained on a high-fat diet (carbohydrates 20 Kcal%, protein 20 Kcal%, fat 60 Kcal% feed; D12492, Rodent Diet Inc., NJ, USA) for 10 weeks to in-duce obesity. The rats were considered obese if their body weight increased by 20% or more compared to the body weight of rats that were fed and water ad libitum the general diet (Commercial rat Chow, Samy-ang Co., Korea) [15]. The experimental rats were randomly divided into five groups: the obesity control group (OCG, n=6), aerobic-resistance exercise in the morning group (ARMG, n=6), resistance-aerobic exercise in the morning group (RAMG, n=6), aerobic-resistance exercise in the evening group (AREG, n=6), and resistance-aerobic exercise in the evening group (RAEG, n=6). Animals were maintained at a constant room temperature of 22-24° C and 60% of humidity under 12/12-hour light-dark cycle. This experiment obtained approval by the Ethics Com-mittee of Jeju National University (2021-0052).

To adapt the treadmill walking, all rats in this study performed low intensity treadmill exercise for a week before the study began. The animals in the exercise groups were subjected to endurance running at a speed of 14-16 m/min for 30 minutes, three a week with no inclination by using Treadmill device (Jeungdo Bio & Plant). Also, there was the resistance exercise (ladder-climbing), the rats climbed a ladder with a length of 1 m, interval of 2 cm, and angle of 80°, three a week. The initial load was started at 50% of the animal body weight and gradually increased to 75%, 90%, and 100%. The rest time was set to 1 minute, and the exercise was continued until 8 climbs were completed or the entire length of the ladder could not be climbed. The weight was fixed to the proximal end of the rat tail using a swivel and tape. The exercise intervention was applied according to the exercise sequence and circadian rhythms in the rat (07:00 AM, 07:00 PM) for 8 weeks. The endurance and resistance exercise protocols were applied based on Zhang et al. [16], Cassilhas et al. [17] and Kraemer et al. [18] studies.

The dissected flexor pollicis longus and soleus muscles tissues were rinsed with phosphate-buffered saline (PBS) and lysed in Triton lysis buffer. Being denatured proteins were separated on sodium dodecyl sul-phate-polyacrylamide gel and then transferred onto polyvinylidene difluoride membrane on ice at 200 mA for 2 hours. The membranes were blocked with 5% skim milk, 0.1% Tween 20 in tris buffered saline for 30 minutes at room temperature. Then, the membranes were incubated overnight with primary antibodies at 4° C. Protein (20 μg) was used for Western blot analysis using anti-Insulin like growth factor 1 (IGF-1) mouse polyclonal antibody (1:1,000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-PI3K mouse polyclonal antibody (1:1,000, Cell Signaling Biotechnology, Danvers, MA, USA), anti-phosphorylated mTOR rabbit polyclonal antibody (1:1,000, Cell Signaling Biotechnology), anti-phosphorylated Akt rabbit polyclonal antibody (1:1,000, Cell Signaling Biotechnology), anti-GAPDH mouse monoclonal antibody (1:1,000, Santa Cruz Biotechnology), anti-phosphorylated AMPK mouse polyclonal antibody (1:1,000, Cell Signaling Biotechnology), anti-PGC-1α mouse monoclonal antibody (1:1,000, Cell Signaling Biotechnology), anti-phosphorylated cAMP-response element binding protein (CREB) mouse monoclonal antibody (1:1,000, Cell Signaling Biotechnology), anti-β-Actin mouse polyclonal antibody (1:1,000, Cell Signaling Biotechnology). For the secondary antibody, horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG antibodies (1:1,000, GeneTex Inc., Irvine, CA, USA) were used. The blotting proteins were detected by using We-star ECL substrates (Cyanagen, Bologna, Italy). Detected band intensity was analyzed using Chemidoc (Bio-Rad, Hercules, CA, USA).

Flexor pollicis longus muscle tissues were embedded and frozen at 20°C. Transverse (10 μm thick) were cut on a cryostat and mounted on positively charged slides (Fisher Scientific, Pittsburgh, PA, USA). For immunofluorescence staining sections were fixed with 4% paraformal-dehyde and 4% sucrose in PBS at room temperature for 40 minutes, per-meabilized with 0.5% Nonidet P-40 in PBS, and blocked with 2.5% horse serum and 2.5% bovine serum albumin for 4 hours at room temperature. The sections were incubated with anti-neurofilament-200 (NF200) rabbit polyclonal antibody (1:700, Sigma-Aldrich, St. Louis, MO, USA). Then they were incubated with rhodamine-goat anti-rabbit secondary antibody (1:400, Molecular Probes, Eugene, OR, USA) and Hoechst (1:500, Sigma-Aldrich) for 1 hour at room temperature, and cover-slipped with gelatin mount medium. In cases when the nonspecific sig-nals were high, which occurred in 1-2 cases out of 10 independent ex-periments. The stained samples were viewed with a fluorescence micro-scope (Nikon model E-600, Nikon, Kawasaki, Japan), and the images were captured with a digital camera, and analyzed using Adobe Photoshop Software (version CS6, San Jose, CA, USA).

All the data is presented as a mean±standard error. Statistical analysis was performed using one-way ANOVA followed by Duncan post hoc test. The significance level was set at p <.05. All graphs were drawn using Prism 6 (GraphPad, La Jolla, CA, USA).

Body weight has been known as a to diagnosing obesity index in the rat and they are closely related to the occurrence of various diseases. To determine the difference in body weight according to the exercise sequence and circadian rhythms exercise intervention, we analyzed body weight after exercise intervention. As shown in Fig. 1A, body weight was significantly decreased in the ARMG, RAMG, AREG, and RAEG groups compared to the OCG group, suggesting that exercise application according to regardless of the exercise sequence and circadian rhythms after exercise intervention may be positive effects for improve obesity.

Obesity and muscle size were closely related to muscle torque and power [19]. To examine the effect of exercise sequence and circadian rhythms on Type II skeletal muscle fiber cross section area in obese rat after exercise intervention. As shown in Fig. 2A and B, Type II skeletal muscle fiber cross section area was significantly increased in the AREG groups compared to the OCG, ARMG, RAMG, RAEG group after intervention, suggesting that resistance exercise after aerobic exercise in the evening may increase Type II muscle fiber.

Aerobic and anaerobic exercise have been known as participating in regulating the muscle hypertrophy signaling pathway. To determine the effect of circadian rhythms and exercise sequence on muscle hypertrophy signaling pathway in obese rat after exercise intervention. we analyzed IGF-1, PI3K, p-Akt, p-mTOR at the flexor pollicis longus muscle. As shown in Fig. 3A and B, AREG group was significantly upregulated in IGF-1 expression level compared to the OCG, ARMG, RAMG groups. PI3K was also significantly increased in AREG and RAEG groups than the OCG, ARMG, and RAMG group. p-Akt and p-mTOR were significantly upregulated in AREG compared to all groups after intervention, suggesting that intra-session exercise sequence (aerobic to resistance exercise) in the evening may increase muscle hypertrophy.

Mitochondrial dysfunctions such as low number and activity have been suggested as specific factors in obesity, type 2 diabetes, and metabolic syndrome [20]. To confirm changes in mitochondrial biogenesis related proteins expression after intervention, we analyzed p-AMPK, CaMK, PGC-1α, p-CREB at the soleus muscle. As shown in Fig. 4A and B, RAMG group was significantly upregulated in p-AMPK and CaMK expression levels compared to all groups. PGC-1α was also significantly increased in RAMG and AREG groups than the OCG, ARMG, and RAEG groups. RAMG and AREG groups were significantly upregulated in p-CREB expression levels compared to the OCG, ARMG, and RAEG groups after intervention, suggesting that intra-session exercise sequence (resistance to aerobic exercise) in the morning may increase mitochondrial biogenesis.