Fat tissues are regarded as endocrine organs that play important roles in energy balance regulation and homeostasis. Fat tissue can be either white adipose tissue (WAT) or brown adipose tissue (BAT) (Kajimura and Saito, 2014). BAT is involved in body temperature regulation and energy consumption by generating heat without shivering, while WAT stores energy and is spread throughout the body (Yamauchi et al., 2003). However, excessive fat accumulation due to obesity elevates proinflammatory cytokines, such as tumor necrosis factor-[alpha] (TNF-[alpha]), interleukin (IL)-1, and IL-6 (Cao, 2011). They stimulate osteoclast differentiation and activity by regulating receptor activator of NF-[kappa]B (RANK), RANK ligand (RANKL), and osteoprotegerin pathways (Khosla, 2001), while restricting osteoblastogenesis. This has an adverse effect on bone health, causing reduced bone mineral density (BMD) and micro-structural changes (Rosen and Bouxsein, 2006). Physical exercise is the preferred treatment for prevention of obesity-related poor bone health (Cosman et al., 2014), and exerts an anabolic effect on bone either directly through mechanical signals generated by muscle contraction or indirectly via endocrine regulation (Colaianni and Grano, 2015; DiGirolamo et al., 2013). Thus, bone metabolism represents a close interaction between muscle and bone (Robling and Turner, 2009). Weight-bearing exercises such as walking, running, and weight training have been used to improve bone metabolism, and non-weight bearing exercises such as swimming and cycling have been perceived as less effective (Abrahin et al., 2016). However, recent studies have reported that non-weight bearing exercises, such as swimming, are also effective in improving bone metabolism (Hart et al., 2001; Ju et al., 2015; Lu et al., 2016; Oh et al., 2016). In particular, Falcai et al. (2015) reported that 3 weeks of swimming improved BMD and microstructure as effectively as jumping exercises in rats with reduced bone mass by hindlimb suspension. These results suggest that muscle contraction alone, without weight loading, is sufficient to maintain and improve bone health. Mechanisms underlying improvement of bone metabolism by muscle contraction are regulated by gene expression or various hormones released in the bone or skeletal muscle tissues (Nordstrom et al., 2005). The newly identified irisin, an exercise-induced myokine, is cleaved from fibronectin type III domain containing protein 5 (FNDC5) and released into the serum during exercise (Bostrom et al., 2012).
Recent research reported that irisin inhibits osteoclast activity while increasing osteoblast differentiation in bone cells lines (Zhang et al., 2017). Systemic administration of irisin regulates bone anabolism through direct mechanism via [beta]-catenin and indirect mechanism via browning of the WAT (Motyl et al., 2013; Rahman et al., 2013). Also, irisin is effective in reducing body fat and preventing and treating obesity by inducing energy expenditure (Calton et al., 2016). Thus, exercise-induced irisin may reduce osteoclast differentiation by decreased proinflammatory cytokines associated with fat accumulation, and increase anabolic factors such as [beta]-catenin, which induces osteoblast differentiation. However, it has not been investigated whether this effect occurs in vivo as well as in vitro using cell lines.
This study was conducted to investigate the expression of PGC-1[alpha]/FNDC5/irisin following attenuation of bone accrual by high-fat diet and investigated whether swimming exercise could improve attenuating bone accrual through this mechanism.
Animal and diets
This study used 8-week-old male Sprague--Dawley rats (n = 30) obtained from the Damul Science Inc. (Daejeon, South Korea). The rats were raised in a plastic cage, with two rats in the same group per cage, at the Study Animal Center at J University. The temperature and humidity of the laboratory were set to 23-25[degrees]C and 70%-80%, respectively, with a 12-h light--dark cycle (08:00-20:00). This study was approved by the Institutional Animal Care and Use Committee of our university.
The study animals were acclimatized for 1 week and then randomly divided into the control diet (CD, n = 10) group receiving feed containing 3.5% fat and the high-fat diet (HFD) group (n = 20) receiving feed containing 45% fat. The rats were fed their corresponding feed for 8 weeks via free feeding (Table 1).
The HFD group was further divided into the HFD control (HFD, n = 10) and high-fat diet with swimming exercise (HEx, n = 10) groups. Rats continued to receive HFD during the 8 weeks of swimming exercise. The CD group did not undergo any additional treatment and was used as the control group for the HFD group.
The swimming protocol consisted of 8 weeks of low-intensity swimming exercise, which was based on partial revisions to the methods by Terada and Tabata (2004) and Kim et al. (2017). The exercise groups were adapted to a round water tank (70 cm x 70 cm) containing water at 28 [+ or -] 2[degrees]C for 3 days (10-45 min/day). The rats then began swimming at 10 AM, 45 min/day for the first two weeks and 60 min/day for the last six weeks.
After 8 weeks of swimming exercise, the study animals were given an intra-abdominal injection (1 mL/kg body weight) of a 2:1:2 mixture of Zoletil (Virbac Laboratories), Rompun (Bayer Korea), and normal saline. Blood samples were collected from the abdominal aorta to analyze oste-ocalcin, CTX-1 and irisin concentration. The blood samples were left standing at room temperature for 30 min and centrifuged to isolate the serum. Epididymal fat was extracted to measure body fat mass. The sample was stored at -80[degrees]C until use. In addition, to observe morphological changes of the bone, the right tibial and femoral bones were isolated and stored in a 10% formalin tube until measurement. The left femoral bone was used to analyze expression of bone metabolic factors.
Measurement of bone morphological and structural in bone by micro computed tomography (CT)
BMD and micro structure of the tibia and femur were analyzed via micro-CT (SkyScan 1076, Bruker, Kontich, Belgium). An X-ray of 60 kA and 167 [micro]A was irradiated, and the specimen was filtered through a 0.5 mm aluminum filter to produce a micro-CT image. The micro-CT image was then reconstructed to a gray-scale level using NRecon version 1.3 (SkyScan), and the reconstructed two-dimensional image was converted to a three-dimensional model using the CTAn and CTVox...