Roles of leptin in the recovery of muscle and bone by reloading after mechanical unloading in high fat diet-fed obese mice
Authors:
Naoyuki Kawao aff001; Masayoshi Ishida aff001; Hiroshi Kaji aff001
Authors place of work:
Department of Physiology and Regenerative Medicine, Kindai University Faculty of Medicine, Osakasayama, Japan
aff001
Published in the journal:
PLoS ONE 14(10)
Category:
Research Article
doi:
https://doi.org/10.1371/journal.pone.0224403
Summary
Muscle and bone masses are elevated by the increased mechanical stress associated with body weight gain in obesity. However, the mechanisms by which obesity affects muscle and bone remain unclear. We herein investigated the roles of obesity and humoral factors from adipose tissue in the recovery phase after reloading from disuse-induced muscle wasting and bone loss using normal diet (ND)- or high fat diet (HFD)-fed mice with hindlimb unloading (HU) and subsequent reloading. Obesity did not affect decreases in trabecular bone mineral density (BMD), muscle mass in the lower leg, or grip strength in HU mice. Obesity significantly increased trabecular BMD, muscle mass in the lower leg, and grip strength in reloading mice over those in reloading mice fed ND. Among the humoral factors in epididymal and subcutaneous adipose tissue, leptin mRNA levels were significantly higher in reloading mice fed HFD than in mice fed ND. Moreover, circulating leptin levels were significantly higher in reloading mice fed HFD than in mice fed ND. Leptin mRNA levels in epididymal adipose tissue or serum leptin levels positively correlated with the increases in trabecular BMD, total muscle mass, and grip strength in reloading mice fed ND and HFD. The present study is the first to demonstrate that obesity enhances the recovery of bone and muscle masses as well as strength decreased by disuse after reloading in mice. Leptin may contribute to the recovery of muscle and bone enhanced by obesity in mice.
Keywords:
leptin – Body weight – Fats – obesity – Skeletal muscles – Adipose tissue – Legs
Introduction
Increasing evidence suggests that obesity affects bone metabolism and muscle functions [1–3]. Obese individuals have a higher bone mineral density (BMD) than non-obese individuals [4]. De Laet et al. revealed that obesity reduced self-reported overall and hip fracture risks in a meta-analysis [5]. On the other hand, Compston et al. reported that obesity is a risk factor for ankle and upper leg fractures in postmenopausal women, suggesting that obesity differently affects bone metabolism by the sites [6]. Moreover, in obese mice, tibial bone mass is increased by enhancing mechanical stress associated with body weight gain, but subsequently reduced by impairing bone metabolism [7]. Viljakainen et al. revealed that indices of bone metabolism are lower in obese individuals than in non-obese individuals, suggesting that obesity reduces bone turnover [8]. These findings indicate that the effects of obesity on bone metabolism are influenced by bone formation enhanced by mechanical stress and adipose tissue-derived abnormalities in bone metabolism.
Regarding skeletal muscle, obesity increases muscle mass and function in adolescent girls because increased weight-bearing played as a chronic mechanical loading on skeletal muscle [9]. In contrast, obesity reduces muscle mass and function in the elderly with sarcopenia [2]. Moreover, previous studies showed that obesity impairs myogenic differentiation in mice and reduces contractile function in skeletal muscle collected from mice [3,10]. Overall, these findings suggest that muscle mass is regulated by the balance of a training effect associated with body weight gain and a negative effect associated with metabolic abnormalities in obesity. However, the mechanisms by which obesity influences muscle and bone remain unclear.
White adipose tissue (WAT) affects other tissues through the release of humoral factors [11]. Adipocytokines, such as tumor necrosis factor (TNF)-α, plasminogen activator inhibitor (PAI)-1, monocyte chemoattractant protein (MCP)-1, leptin, and adiponectin, play critical roles in obesity-related diseases [11]. Among them, TNF-α, PAI-1, and MCP-1 are primarily recognized as negative regulators of muscle and bone [12–14]. Circulating adiponectin levels were found to be lower in obese individuals than in non-obese individuals [15]. Richards et al. revealed that serum adiponectin levels were negatively associated with BMD in postmenopausal women, even at non-load-bearing sites, suggesting that adiponectin affects bone metabolism through non-mechanical mechanisms [16]. Circulating leptin levels were previously reported to be elevated in obese individuals and positively correlated with BMD in humans [17]. The influences of leptin on bone metabolism are complicated by its enhancing and inhibitory effects on bone mass through peripheral and central actions, respectively, in mice [17]. On the other hand, Bartell et al. showed the contradictory findings that central leptin injection increases bone formation in leptin-deficient ob/ob mice, which is in agreement with previous studies that hypothalamic leptin gene therapy rescues a reduction in BMD in ob/ob mice [18–20]. As for muscle, leptin may exert anabolic effects on skeletal muscle in mice [21].
Muscle atrophy and osteopenia are concomitantly induced by a reduction of mechanical stress, such as long term space flight [22], which are prevented by exercise in astronauts [23], suggesting that mechanical stress is crucial for maintaining muscle and bone homeostasis. However, the effects of obesity on recovery from disuse-induced muscle and bone loss have not yet been elucidated in detail. Moreover, the roles of adipose tissue-derived humoral factors in the recovery of muscle and bone mass after reloading in unloading-induced sarcopenia and osteopenia remain unknown. Therefore, we herein examined the effects of obesity on recovery after reloading from disuse-induced muscle and bone loss using high fat diet (HFD)-fed mice with hindlimb unloading (HU) and subsequent reloading. Moreover, we aimed to investigate the roles of humoral factors from WAT on the increases observed in muscle and bone mass during mechanical reloading in mice fed HFD.
Materials and methods
Ethics statement
Animal experiments were performed according to the guidelines of the National Institutes of Health and the institutional rules for the use and care of laboratory animals at Kindai University. All animal experiments were approved by the Experimental Animal Welfare Committee of Kindai University (Permit number: KAME-27-029). All efforts were made to minimize suffering. Mice were euthanized with excess isoflurane.
Animal experiments
Male C57BL/6J mice were obtained from CLEA Japan (Tokyo, Japan). Four-week-old mice were fed ad libitum with a normal diet (ND) or HFD (57% of calories from fat, CLEA Japan) and water. Mice were fed ND or HFD for 15 weeks, and HU was started at 8 weeks and reloading at 11 weeks after ND or HFD feeding was started. Mice were randomly divided into four groups: ND-fed control (ND/Control, n = 8), HFD-fed control (HFD/Control, n = 8), ND-fed reloading after HU (ND/reload, n = 8) and HFD-fed reloading after HU (HFD/reload, n = 8) groups. To induce HU, the mouse tail was suspended using a tail clip (Yamashita Giken, Tokushima, Japan) for 3 weeks at 8 weeks after ND or HFD feeding was started, as previously described [24]. HU mice were reloaded by removing the tail clip for 4 weeks after HU for 3 weeks. Mice with 6 hours of fasting were anesthetized using 2% isoflurane, and blood samples were collected 4 weeks after reloading. After mice were euthanized with excess isoflurane, epididymal and subcutaneous WAT were removed. In the present study, epididymal and subcutaneous WAT represent visceral and body fat, respectively. The ND/Control and HFD/Control groups were not subjected to HU followed by reloading.
Quantitative computed tomography (QCT) analysis
A QCT analysis was performed using an X-ray CT system in vivo (Latheta LCT-200; Hitachi Aloka Medical, Tokyo, Japan) on mice 3 weeks after HU and then 4 weeks after reloading according to the guidelines of the American Society for Bone and Mineral Research [25], as described previously [24]. After mice were anesthetized with 2% isoflurane, CT images were acquired using the following parameters: 50 kVp tube voltage, 500 μA tube current, 48 mm axial field of view, and 96×192×1008 μm voxel size to analyze total fat and muscle masses, 48×48×192 μm voxel size to analyze fat and muscle masses in the lower leg, and 24 μm isotropic voxel size to analyze tibial BMD. The region of interest for the assessment of total fat and muscle masses was defined as the whole body. Regions of interest were defined as 1680-μm segments 96 μm distal to the end of the proximal growth plate towards the diaphysis for the assessment of trabecular BMD and as 2160-μm segments of the mid-diaphysis of the tibia for the assessment of cortical BMD. The region of interest for the assessment of fat and muscle masses in the lower leg was defined as the segment from the proximal to distal end of the tibia. Fat and muscle masses and BMD were analyzed using LaTheta software (version 3.41).
Measurement of grip strength
The grip strength was measured using a grip strength meter (1027SM, Columbus Instruments, Columbus, OH, USA) 3 weeks after HU and then 4 weeks after reloading, as described previously [24]. Mice were allowed to grip a pull bar attachment by the four limbs. The mouse tail was then continuously pulled at a rate of approximately 2 cm/sec. The grip strength was measured five times and the results obtained were represented as an average for each mouse.
Real-time PCR
Total RNA was isolated from mouse epididymal and subcutaneous WAT using Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA) and purified using an RNeasy Mini Kit (Qiagen, Hilden, Germany), as previously described [24]. The reverse transcription reaction was performed using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster, CA, USA). cDNA was amplified by real-time PCR using ABI PRISM 7900HT (Applied Biosystems) with the Fast SYBR Green Master Mix (Applied Biosystems). Each PCR primer sequence is shown in S1 Table. Relative changes in target gene levels were calculated using the ΔΔCt method, and were normalized with 18S rRNA levels.
Blood chemistry
Serum leptin levels were measured using the Quantikine enzyme-linked immunosorbent assay kit for mouse/rat leptin (R&D systems, Minneapolis, MN, USA, Cat. No. MOB00) [26]. Intra- and inter-assay coefficients of variations were ≤4.3% (n = 20) and ≤7.6% (n = 20), respectively.
Statistical analysis
Data are represented as the mean ± standard error of the mean (SEM). Relative changes (% before reloading) in body weight, calorie intake, fat mass, BMD, muscle mass, and grip strength were calculated by dividing each parameter before reloading by that after reloading for 4 weeks. The significance of differences was evaluated using the Mann-Whitney U test for comparisons of 2 groups. A two-way analysis of variance followed by the Tukey-Kramer test was performed for multiple comparisons. Main effects (unloading and HFD, or reloading and HFD) were reported if there was no interaction effect. Pearson’s test was performed for a simple regression analysis. The significance level was set at P < 0.05. All statistical analyses were performed using GraphPad PRISM 7.00 software.
Results
Effects of HU and reloading on body weight and fat mass in mice fed HFD
There was a significant HFD × HU interaction in body weight (F1,28 = 53.8, P < 0.01). Body weights were significantly higher in mice fed HFD for 11 weeks than in mice fed ND (Fig 1A). HU for 3 weeks reduced body weight elevated by HFD in mice, whereas HU did not affect body weight in mice fed ND (Fig 1A). There was a significant HFD × reloading interaction in body weight (F1,28 = 5.11, P = 0.031). Body weights were significantly higher in mice fed HFD with reloading for 4 weeks than in reloading mice fed ND (Fig 1A). HFD significantly increased calorie intake in control and HU mice (Fig 1B), although there was no significant HFD × HU interaction (F1,28 = 0.024, P = 0.878). There was a significant HFD × reloading interaction in calorie intake (F1,28 = 4.499, P = 0.042, Fig 1B). Neither HU nor reloading affected calorie intake in mice fed ND and HFD (Fig 1B). There were no significant differences in the amount of food intake between control and HU mice fed HFD on day 18 to 21 after HU (Control, 3.7 ± 0.28 g/day; HU, 4.0 ± 0.27 g/day). There were significant interaction effects of HFD × HU on total fat mass (F1,28 = 176.3, P < 0.01), HFD × HU on fat mass of the lower leg (F1,28 = 105.2, P < 0.01), HFD × reloading on total fat mass (F1,28 = 7.173, P = 0.012), and HFD × reloading on fat mass of the lower leg (F1,28 = 53.38, P < 0.01). Fat masses in the whole body and lower leg were significantly higher in mice fed HFD than in mice fed ND, as assessed by the QCT analysis (Fig 1C and 1D). HU significantly reduced fat masses in the whole body and lower leg in mice fed HFD, but not in those fed ND (Fig 1C and 1D). HFD facilitated increases in fat masses in the whole body and lower leg in reloading mice over those in reloading mice fed ND (Fig 1C and 1D).
Effects of HFD feeding on recovery from BMD reduced by HU after reloading in mice
We examined the effects of HU and reloading on BMD in mice fed ND and HFD. There were no significant interaction effects of HFD × HU on trabecular BMD (F1,28 = 0.833, P = 0.369), HFD × reloading on trabecular BMD (F1,28 = 3.021, P = 0.093), HFD × HU on cortical BMD (F1,28 = 0.075, P = 0.786), and HFD × reloading on cortical BMD (F1,28 = 1.678, P = 0.206). HU significantly reduced trabecular BMD in the tibia of mice fed ND and HFD (Fig 2A). Although reloading increased trabecular BMD in the tibia of mice fed ND and HFD after HU, trabecular BMD was significantly higher in mice fed HFD than in mice fed ND (Fig 2A). Neither HU nor reloading affected cortical BMD in mice fed ND and HFD (Fig 2B).
Effects of HFD feeding on recovery from reductions in muscle mass and grip strength by HU after reloading in mice
We examined the effects of HU and reloading on muscle mass and strength in mice fed ND and HFD. There were no significant interaction effects of HFD × HU on total muscle mass (F1,28 = 1.467, P = 0.236), HFD × reloading on total muscle mass (F1,28 = 0.001, P = 0.970), HFD × HU on muscle mass of the lower leg (F1,28 = 0.072, P = 0.821), and HFD × reloading on muscle mass of the lower leg (F1,28 = 2.919, P = 0.099). Muscle mass in the whole body was significantly reduced by HU (F1,28 = 6.907, P = 0.014), but not reloading, in mice fed ND or HFD (Fig 3A). The increase observed in muscle mass in the whole body was significantly higher in reloading mice fed HFD than in mice fed ND (Fig 3A). Muscle mass in the lower leg in mice was significantly reduced and increased by HU (F1,28 = 93.73, P < 0.01) and HFD (F1,28 = 7.475, P = 0.011), respectively (Fig 3B). Muscle mass in the lower leg was higher in reloading mice fed HFD than in mice fed ND (Fig 3B), although it was significantly increased by HFD (F1,28 = 15.02, P < 0.01). No significant differences were observed in the recovery rate of muscle mass in the lower leg between reloading mice fed ND and HFD (Fig 3B). HU significantly reduced grip strength in mice fed ND and HFD (Fig 3C), although there was no significant HFD × HU interaction (F1,28 = 0.176, P = 0.678). There was a significant HFD × reloading interaction in grip strength (F1,28 = 31.22, P < 0.01). HFD feeding significantly increased grip strength and its recovery rate in reloading mice over that in reloading mice fed ND (Fig 3C).
Levels of humoral factors in epididymal and subcutaneous WAT in reloading mice fed ND and HFD
Adipocytokines, such as MCP-1, PAI-1, TNF-α, and leptin, influence bone metabolism [13,14,17,21,27]. We therefore examined the levels of these adipocytokines in the epididymal and subcutaneous WAT of reloading mice fed ND and HFD. MCP-1 mRNA levels were significantly higher in the epididymal, but not subcutaneous, WAT of control and reloading mice fed HFD than in mice fed ND (Fig 4A and 4B), although there was no significant HFD × reloading interaction (F1,28 = 4.134, P = 0.052). PAI-1 mRNA levels were significantly increased by HFD in the subcutaneous WAT of control and reloading mice with no HFD × reloading interaction (Fig 4B). There was a significant HFD × reloading interaction on TNF-α mRNA levels in the epididymal WAT of mice (F1,28 = 11.11, P = 0.002). Although TNF-α mRNA levels were elevated in the epididymal WAT of control mice fed HFD, HFD feeding did not affect TNF-α mRNA levels in reloading mice (Fig 4A). Leptin mRNA levels in epididymal and subcutaneous WAT were significantly higher in mice fed HFD than in mice fed ND (Fig 4A and 4B), although there were no HFD × reloading interaction effects in epididymal (F1,28 = 0.008, P = 0.930) and subcutaneous (F1,28 = 0.748, P = 0.395) adipose tissues. Osteoglycin is a novel circulating humoral factor that affects bone metabolism and glucose homeostasis [28]. Osteoglycin levels are negatively related to fat and bone masses in mice fed HFD [28]. We therefore examined osteoglycin expression in the epididymal and subcutaneous WAT of mice fed ND and HFD. Osteoglycin mRNA levels were significantly lower and higher in epididymal and subcutaneous WAT, respectively, in mice fed HFD than in those fed ND with no HFD × reloading interaction (Fig 4A and 4B). Sclerostin, an inhibitor of Wnt/β-catenin signaling, was previously reported to be up-regulated in bone and suppressed bone formation during mechanical unloading [29]. We therefore investigated sclerostin expression in tibial bone tissues. HFD feeding did not affect sclerostin mRNA levels in the tibia of mice (Fig 4C).
Relationships between the expression of humoral factors in WAT and the recovery rate of trabecular BMD in the tibia of reloading mice fed ND and HFD
We examined the relationships between the expression of humoral factors in WAT and the recovery rate of trabecular BMD in reloading mice using a simple regression analysis to identify which factors are involved in the enhanced recovery from HU-induced bone loss in obese mice. Leptin mRNA levels in epididymal WAT positively correlated with the recovery rate of trabecular BMD in reloading mice fed ND and HFD (Fig 5). Moreover, leptin mRNA levels in epididymal as well as subcutaneous WAT positively correlated with trabecular BMD in control and reloading mice fed ND and HFD (S2 Table). On the other hand, no significant relationships were observed between the recovery rate of trabecular BMD and the mRNA levels of MCP-1, PAI-1, TNF-α, and osteoglycin in the epididymal and subcutaneous WAT of reloading mice fed ND and HFD (Fig 5); however, the mRNA levels of MCP-1 and osteoglycin in epididymal WAT positively and negatively correlated with trabecular BMD in control and reloading mice fed ND and HFD, respectively (S2 Table). Sclerostin mRNA levels in tibial bone tissues did not correlate with the recovery rate of trabecular BMD in reloading mice fed ND and HFD (r = 0.235, P = 0.381).
Relationships between humoral factors in WAT and the recovery rate of muscle mass and strength in reloading mice fed ND and HFD
We examined the relationships between the expression of humoral factors in WAT and the recovery rate of muscle mass and strength in mice fed ND and HFD. Leptin mRNA levels in epididymal and subcutaneous WAT positively correlated with the recovery rate of muscle mass in the whole body in mice fed ND and HFD (S3 Table). MCP-1 and PAI-1 mRNA levels in epididymal WAT positively correlated with the recovery rate of muscle mass in the whole body (S3 Table). On the other hand, osteoglycin mRNA levels negatively correlated with the recovery rate of muscle mass in the whole body (S3 Table). Regarding muscle strength, the recovery rate of grip strength positively correlated with leptin and PAI-1 mRNA levels in the epididymal and subcutaneous WAT of reloading mice fed ND and HFD (S4 Table). Osteoglycin mRNA levels in subcutaneous WAT positively correlated with the recovery rate of grip strength in reloading mice fed ND and HFD (S4 Table). Sclerostin mRNA levels in tibial bone tissues did not correlate with the recovery rate of total muscle mass or grip strength in reloading mice fed ND and HFD (Total muscle mass: r = -0.446, P = 0.083, grip strength: r = -0.396, P = 0.129).
Relationships between serum leptin levels and the recovery rate of trabecular BMD, muscle mass in the whole body, and grip strength in reloading mice
Since leptin mRNA levels in the WAT of reloading mice positively correlated with the recovery rate of trabecular BMD, muscle mass in the whole body, and grip strength, we investigated serum leptin levels in control and reloading mice fed ND and HFD. HFD feeding significantly increased serum leptin levels in control and reloading mice over those in mice fed ND (Fig 6A), although there was a significant HFD × reloading interaction (F1,28 = 8.722, P = 0.006). Serum leptin levels positively correlated with the recovery rate of trabecular BMD, muscle mass in the whole body, and grip strength in reloading mice fed ND and HFD (Fig 6B). Trabecular BMD, muscle mass in the lower leg, and grip strength positively correlated with fat mass in the whole body in control and reloading mice fed ND and HFD (S5 Table). Muscle mass in the whole body and grip strength positively correlated with the rate of increases in whole body fat mass (S5 Table).
Discussion
Extensive clinical evidence has indicated that obese subjects have higher BMD than non-obese subjects, presumably through the increased mechanical stress caused by the higher body weight burden [4,7]. In the present study, obesity elevated trabecular BMD in reloading mice, but did not affect a reduction in trabecular BMD in HU mice. These findings were consistent with the recent evidence that obesity does not affect HU-induced bone loss in leptin-deficient ob/ob mice [30]. Previous studies revealed that the weight bearing resistance exercise using weighted vests increases trabecular bone area and BMD in rats fed ND [31,32]. Song et al. showed that resistance ladder climbing exercise with weight bearing facilitates the recovery from a reduction in BMD by HU after reloading in rats [33]. These findings suggest that HFD facilitates the recovery of bone mass reduced by mechanical unloading after reloading partly through an enhancement of mechanical stress by the increased body weight. As for the skeletal muscles, we showed that HFD elevates muscle mass in the lower leg and grip strength in reloading mice in the present study, but did not affect the reductions in those muscle-related parameters in HU mice. We therefore cannot rule out the possibility that HFD may facilitate the recovery of bone mass reduced by mechanical unloading after reloading partly through a mechanical stimulation by increased muscle mass and strength on bone in mice.
In the present study, HFD increased the recovery rate of grip strength in reloading mice over that in reloading mice fed ND, although the effects of HFD on the recovery rate of muscle mass seemed to be less. These results suggest that the recovery of muscle function is not only attributable to recovery of muscle mass after reloading. It may be due to the greater body weight or muscle loading. Alternatively, a previous study showed that HU impairs neuromuscular axonal excitability in mice [34]. Avraham et al. showed that leptin treatment exerts neruoprotective effects through enhancement of neurogenesis and angiogenesis after stroke in mice [35]. These findings suggest that that improved neural activation may be partly involved in the recovery of muscle function reduced by HU after reloading in mice fed HFD.
Adipocytokines produced from adipose tissues in the obese state play crucial roles in the regulation of bone and muscle in obesity [1,11]. Therefore, some adipocytokines induced by the obese state may be related to the recovery of bone mass, muscle mass, and grip strength reduced by mechanical unloading after reloading in mice. Leptin regulates bone metabolism through central and peripheral actions [17]. Previous studies on mice reported that leptin suppressed and enhanced bone formation and bone resorption, respectively, through central actions [17,36], whereas Bartell et al. showed contradictory findings on the central actions of leptin [18]. On the other hand, previous studies indicate that leptin enhances and suppresses bone formation and resorption, respectively, through peripheral action in vitro [37,38]. Indeed, numerous clinical studies indicated that leptin exerts positive effects on bone in humans [17,39]. Yamauchi et al. revealed that circulating leptin levels were positively and inversely related to BMD at the lumbar spine, femoral neck, and forearm as well as the presence of vertebral fractures, respectively, in postmenopausal women [39]. Roux et al. showed that circulating leptin levels were positively related to BMD at the lumbar spine and femoral neck in healthy postmenopausal women [40]. In the present study, adipose tissue leptin expression and serum leptin levels were elevated in mice fed HFD. Moreover, the recovery rate of trabecular BMD after reloading was positively related to adipose tissue leptin expression and serum leptin levels in mice. Collectively, these findings and the present results suggest that leptin contributes to the obesity-induced increased trabecular bone mass presumably through peripheral action. Regarding the effects of leptin on skeletal muscle, leptin enhanced the proliferation and myogenic differentiation of primary myoblasts [41]. In the study by Sáinz et al., leptin treatment increased muscle mass and reduced the levels of the protein degradation-related factor, muscle RING-finger protein-1, in mice [42]. In the present study, serum leptin levels were elevated in mice fed HFD, and positively correlated with the recovery rates of muscle mass and strength in reloading mice. These findings suggest that elevated circulating leptin levels are involved in the recovery of muscle mass and strength enhanced by obesity in reloading mice. However, although leptin mRNA levels in the epididymal adipose tissues were positively related to the recovery rate of trabecular BMD with statistical significance in the present study, the correlation was not strong, suggesting that leptin is partly responsible for the recovery of BMD enhanced by obesity.
MCP-1, a chemoattractant for monocytes and macrophages, plays a key role in obesity-associated pathological conditions [43]. In the present study, MCP-1 expression was elevated in the epididymal adipose tissue of mice fed HFD. Moreover, MCP-1 expression in epididymal adipose tissues positively correlated with an increase in muscle mass, but not muscle strength, in reloading mice. Wang et al. reported that inflammatory macrophages impair muscle differentiation in obese mice [10]. Moreover, circulating MCP-1 levels were higher in obese individuals with or without sarcopenia than in non-obese individuals [44]. These findings suggest that an elevation in MCP-1 levels in epididymal adipose tissues is not responsible for muscle mass recovery enhanced by obesity in reloading mice. Although PAI-1, a primary inhibitor of plasminogen activators, plays pleiotropic roles as an adipokines and negatively regulates muscle mass and functions [12,14,45], PAI-1 expression in the adipose tissues was positively related to the recovery rates of muscle mass and strength in reloading mice. Therefore, PAI-1 may not be responsible for the facilitation of muscle mass and strength recovery induced by obesity in reloading mice. As for osteoglycin, a class III small leucine-rich proteoglycan, its expression in the epididymal adipose tissues was reduced by HFD and negatively correlated with the recovery rates of muscle mass and strength by reloading in mice in our study, although it may exert positive effects on muscle mass [28,46]. Further studies will be necessary to clarify the roles of osteoglycin in the recovery process of skeletal muscle in an obese state.
The mechanisms by which HU reduced fat mass enhanced by HFD have remained unknown in the present study. HU did not affect calorie intake in mice fed ND and HFD. Moreover, there were no significant differences in the amount of food intake between control and HU mice fed HFD on days 18 to 21 after HU, which is consistent with previous studies in leptin-deficient ob/ob mice [30]. These results suggest that a decrease in fat mass in mice fed HFD is not attributable to amounts of calorie and food intakes during HU. Since HU reduces bone mass through the sympathetic nervous system, which stimulates lipolysis [47,48], an activation of the sympathetic nervous system by HU may decrease fat mass in HFD mice.
There were several limitations in the present study. First, the direct biological relationships between leptin and the recovery of bone and muscle after reloading have still remained unclear, although we showed the significant relationships between serum leptin levels and the increases in trabecular BMD, total muscle mass, and grip strength after reloading in mice using a simple regression analysis. Further studies using an anti-leptin neutralizing antibody for mice, leptin-deficient ob/ob, and leptin receptor-deficient db/db mice will be necessary to clarify these issues. Next, there are contradictory findings due to differences in nutrient composition, sources of fats, and percentage of fats, although high-fat diet-induced obesity in mice is well established as an animal model of obesity [49,50]. HFD used in the present study does not appear to reflect a normal human diet which typically contains <35% of calories from fat [49].
Sarcopenic obesity is a multifactorial condition and characterized by comorbidity of sarcopenia and obesity [51]. Sarcopenia may be exacerbated by the presence of obesity. Although effective treatments for sarcopenia have not been established yet, the exercise therapy may be effective for sarcopenia [52]. The present findings suggest that obesity facilitates the recovery of muscle and bone reduced by HU after reloading in mice. We can therefore speculate that exercise may be more effective in patients with sarcopenic obesity, compared to those with sarcopenia without obesity.
In conclusion, the present results provide novel evidence to show that obesity enhances the recovery of bone and muscle masses as well as strength decreased by mechanical unloading after reloading in mice. Our data suggested that leptin may be related to the recovery of muscle and bone enhanced by obesity in mice; further studies are required to elucidate the mechanisms by which the reloading induces the recovery of muscle and bone in mice fed HFD. Leptin may be a target for the prevention and treatment of immobilization-induced osteoporosis and sarcopenia.
Supporting information
S1 Table [docx]
Primers used in real-time PCR experiments.
S2 Table [docx]
Relationship between trabecular BMD in the tibia and humoral factors in the adipose tissue of mice fed ND or HFD.
S3 Table [docx]
Relationship between total muscle mass and humoral factors in the adipose tissue of mice fed ND or HFD.
S4 Table [docx]
Relationship between grip strength and humoral factors in the adipose tissue of mice fed ND or HFD.
S5 Table [docx]
Relationship between total fat mass and parameters of bone and muscle in mice fed ND and HFD.
Zdroje
1. Savvidis C, Tournis S, Dede AD. Obesity and bone metabolism. Hormones (Athens). 2018;17: 205–217.
2. Batsis JA, Villareal DT. Sarcopenic obesity in older adults: aetiology, epidemiology and treatment strategies. Nat Rev Endocrinol. 2018;14: 513–537. doi: 10.1038/s41574-018-0062-9 30065268
3. Tallis J, Hill C, James RS, Cox VM, Seebacher F. The effect of obesity on the contractile performance of isolated mouse soleus, EDL, and diaphragm muscles. J Appl Physiol (1985). 2017;122: 170–181.
4. Evans AL, Paggiosi MA, Eastell R, Walsh JS. Bone density, microstructure and strength in obese and normal weight men and women in younger and older adulthood. J Bone Miner Res. 2015;30: 920–928. doi: 10.1002/jbmr.2407 25400253
5. De Laet C, Kanis JA, Oden A, Johanson H, Johnell O, Delmas P, et al. Body mass index as a predictor of fracture risk: a meta-analysis. Osteoporos Int. 2005;16: 1330–1338. doi: 10.1007/s00198-005-1863-y 15928804
6. Compston JE, Watts NB, Chapurlat R, Cooper C, Boonen S, Greenspan S, et al. Obesity is not protective against fracture in postmenopausal women: GLOW. Am J Med. 2011;124: 1043–1050. doi: 10.1016/j.amjmed.2011.06.013 22017783
7. Lecka-Czernik B, Stechschulte LA, Czernik PJ, Dowling AR. High bone mass in adult mice with diet-induced obesity results from a combination of initial increase in bone mass followed by attenuation in bone formation; implications for high bone mass and decreased bone quality in obesity. Mol Cell Endocrinol. 2015;410: 35–41. doi: 10.1016/j.mce.2015.01.001 25576855
8. Viljakainen H, Ivaska KK, Paldanius P, Lipsanen-Nyman M, Saukkonen T, Pietilainen KH, et al. Suppressed bone turnover in obesity: a link to energy metabolism? A case-control study. J Clin Endocrinol Metab. 2014;99: 2155–2163. doi: 10.1210/jc.2013-3097 24606073
9. Garcia-Vicencio S, Coudeyre E, Kluka V, Cardenoux C, Jegu AG, Fourot AV, et al. The bigger, the stronger? Insights from muscle architecture and nervous characteristics in obese adolescent girls. Int J Obes (Lond). 2016;40: 245–251.
10. Wang X, Zhao D, Cui Y, Lu S, Gao D, Liu J. Proinflammatory macrophages impair skeletal muscle differentiation in obesity through secretion of tumor necrosis factor-alpha via sustained activation of p38 mitogen-activated protein kinase. J Cell Physiol. 2019;234: 2566–2580. doi: 10.1002/jcp.27012 30264458
11. Tilg H, Moschen AR. Adipocytokines: mediators linking adipose tissue, inflammation and immunity. Nat Rev Immunol. 2006;6: 772–783. doi: 10.1038/nri1937 16998510
12. Tamura Y, Kawao N, Shimoide T, Okada K, Matsuo O, Kaji H. Role of plasminogen activator inhibitor-1 in glucocorticoid-induced muscle change in mice. J Bone Miner Metab. 2018;36: 148–156. doi: 10.1007/s00774-017-0825-8 28321652
13. Roy B, Curtis ME, Fears LS, Nahashon SN, Fentress HM. Molecular mechanisms of obesity-induced osteoporosis and muscle atrophy. Front Physiol. 2016;7: 439. doi: 10.3389/fphys.2016.00439 27746742
14. Kaji H. Adipose tissue-derived plasminogen activator inhibitor-1 function and regulation. Compr Physiol. 2016;6: 1873–1896. doi: 10.1002/cphy.c160004 27783862
15. Naot D, Musson DS, Cornish J. The activity of adiponectin in bone. Calcif Tissue Int. 2017;100: 486–499. doi: 10.1007/s00223-016-0216-5 27928591
16. Richards JB, Valdes AM, Burling K, Perks UC, Spector TD. Serum adiponectin and bone mineral density in women. J Clin Endocrinol Metab. 2007;92: 1517–1523. doi: 10.1210/jc.2006-2097 17264180
17. Chen XX, Yang T. Roles of leptin in bone metabolism and bone diseases. J Bone Miner Metab. 2015;33: 474–485. doi: 10.1007/s00774-014-0569-7 25777984
18. Bartell SM, Rayalam S, Ambati S, Gaddam DR, Hartzell DL, Hamrick M, et al. Central (ICV) leptin injection increases bone formation, bone mineral density, muscle mass, serum IGF-1, and the expression of osteogenic genes in leptin-deficient ob/ob mice. J Bone Miner Res. 2011;26: 1710–1720. doi: 10.1002/jbmr.406 21520275
19. Philbrick KA, Martin SA, Colagiovanni AR, Branscum AJ, Turner RT, Iwaniec UT. Effects of hypothalamic leptin gene therapy on osteopetrosis in leptin-deficient mice. J Endocrinol. 2018;236: 57–68. doi: 10.1530/JOE-17-0524 29191939
20. Kalra SP, Dube MG, Iwaniec UT. Leptin increases osteoblast-specific osteocalcin release through a hypothalamic relay. Peptides. 2009;30: 967–973. doi: 10.1016/j.peptides.2009.01.020 19428775
21. Hamrick MW. Role of the cytokine-like hormone leptin in muscle-bone crosstalk with aging. J Bone Metab. 2017;24: 1–8. doi: 10.11005/jbm.2017.24.1.1 28326295
22. Kawao N, Kaji H. Interactions between muscle tissues and bone metabolism. J Cell Biochem. 2015;116: 687–695. doi: 10.1002/jcb.25040 25521430
23. Stein TP. Weight, muscle and bone loss during space flight: another perspective. Eur J Appl Physiol. 2013;113: 2171–2181. doi: 10.1007/s00421-012-2548-9 23192310
24. Kawao N, Moritake A, Tatsumi K, Kaji H. Roles of irisin in the linkage from muscle to bone during mechanical unloading in mice. Calcif Tissue Int. 2018;103: 24–34. doi: 10.1007/s00223-018-0387-3 29332162
25. Bouxsein ML, Boyd SK, Christiansen BA, Guldberg RE, Jepsen KJ, Muller R. Guidelines for assessment of bone microstructure in rodents using micro-computed tomography. J Bone Miner Res. 2010;25: 1468–1486. doi: 10.1002/jbmr.141 20533309
26. Ravussin Y, Edwin E, Gallop M, Xu L, Bartolome A, Kraakman MJ, et al. Evidence for a non-leptin system that defends against weight gain in overfeeding. Cell Metab. 2018;28: 289–299 e285. doi: 10.1016/j.cmet.2018.05.029 29937378
27. Tamura Y, Kawao N, Yano M, Okada K, Okumoto K, Chiba Y, et al. Role of plasminogen activator inhibitor-1 in glucocorticoid-induced diabetes and osteopenia in mice. Diabetes. 2015;64: 2194–2206. doi: 10.2337/db14-1192 25552599
28. Lee NJ, Ali N, Zhang L, Qi Y, Clarke I, Enriquez RF, et al. Osteoglycin, a novel coordinator of bone and glucose homeostasis. Mol Metab. 2018;13: 30–44. doi: 10.1016/j.molmet.2018.05.004 29799418
29. Ke HZ, Richards WG, Li X, Ominsky MS. Sclerostin and Dickkopf-1 as therapeutic targets in bone diseases. Endocr Rev. 2012;33: 747–783. doi: 10.1210/er.2011-1060 22723594
30. Keune JA, Branscum AJ, Wong CP, Iwaniec UT, Turner RT. Effect of leptin deficiency on the skeletal response to hindlimb unloading in adult male mice. Sci Rep. 2019;9: 9336. doi: 10.1038/s41598-019-45587-0 31249331
31. Westerlind KC, Fluckey JD, Gordon SE, Kraemer WJ, Farrell PA, Turner RT. Effect of resistance exercise training on cortical and cancellous bone in mature male rats. J Appl Physiol (1985). 1998;84: 459–464.
32. Tromp AM, Bravenboer N, Tanck E, Oostlander A, Holzmann PJ, Kostense PJ, et al. Additional weight bearing during exercise and estrogen in the rat: the effect on bone mass, turnover, and structure. Calcif Tissue Int. 2006;79: 404–415. doi: 10.1007/s00223-006-0045-z 17160577
33. Song H, Cho S, Lee HY, Lee H, Song W. The effects of progressive resistance exercise on recovery rate of bone and muscle in a rodent model of hindlimb suspension. Front Physiol. 2018;9: 1085. doi: 10.3389/fphys.2018.01085 30150940
34. Banzrai C, Nodera H, Kawarai T, Higashi S, Okada R, Mori A, et al. Impaired axonal Na+ current by hindlimb unloading: implication for disuse neuromuscular atrophy. Front Physiol. 2016;7: 36. doi: 10.3389/fphys.2016.00036 26909041
35. Avraham Y, Davidi N, Lassri V, Vorobiev L, Kabesa M, Dayan M, et al. Leptin induces neuroprotection neurogenesis and angiogenesis after stroke. Curr Neurovasc Res. 2011;8: 313–322. 22023616
36. Turner RT, Kalra SP, Wong CP, Philbrick KA, Lindenmaier LB, Boghossian S, et al. Peripheral leptin regulates bone formation. J Bone Miner Res. 2013;28: 22–34. doi: 10.1002/jbmr.1734 22887758
37. Gordeladze JO, Drevon CA, Syversen U, Reseland JE. Leptin stimulates human osteoblastic cell proliferation, de novo collagen synthesis, and mineralization: Impact on differentiation markers, apoptosis, and osteoclastic signaling. J Cell Biochem. 2002;85: 825–836. doi: 10.1002/jcb.10156 11968022
38. Holloway WR, Collier FM, Aitken CJ, Myers DE, Hodge JM, Malakellis M, et al. Leptin inhibits osteoclast generation. J Bone Miner Res. 2002;17: 200–209. doi: 10.1359/jbmr.2002.17.2.200 11811550
39. Yamauchi M, Sugimoto T, Yamaguchi T, Nakaoka D, Kanzawa M, Yano S, et al. Plasma leptin concentrations are associated with bone mineral density and the presence of vertebral fractures in postmenopausal women. Clin Endocrinol (Oxf). 2001;55: 341–347.
40. Roux C, Arabi A, Porcher R, Garnero P. Serum leptin as a determinant of bone resorption in healthy postmenopausal women. Bone. 2003;33: 847–852. doi: 10.1016/j.bone.2003.07.008 14623061
41. Arounleut P, Bowser M, Upadhyay S, Shi XM, Fulzele S, Johnson MH, et al. Absence of functional leptin receptor isoforms in the POUND (Leprdb/lb) mouse is associated with muscle atrophy and altered myoblast proliferation and differentiation. PLoS One. 2013;8: e72330. doi: 10.1371/journal.pone.0072330 23967295
42. Sainz N, Rodriguez A, Catalan V, Becerril S, Ramirez B, Gomez-Ambrosi J, et al. Leptin administration favors muscle mass accretion by decreasing FoxO3a and increasing PGC-1α in ob/ob mice. PLoS One. 2009;4: e6808. doi: 10.1371/journal.pone.0006808 19730740
43. McNelis JC, Olefsky JM. Macrophages, immunity, and metabolic disease. Immunity. 2014;41: 36–48. doi: 10.1016/j.immuni.2014.05.010 25035952
44. Lim JP, Leung BP, Ding YY, Tay L, Ismail NH, Yeo A, et al. Monocyte chemoattractant protein-1: a proinflammatory cytokine elevated in sarcopenic obesity. Clin Interv Aging. 2015;10: 605–609. doi: 10.2147/CIA.S78901 25848236
45. Koh TJ, Bryer SC, Pucci AM, Sisson TH. Mice deficient in plasminogen activator inhibitor-1 have improved skeletal muscle regeneration. Am J Physiol Cell Physiol. 2005;289: C217–223. doi: 10.1152/ajpcell.00555.2004 15716324
46. Freire PP, Cury SS, de Oliveira G, Fernandez GJ, Moraes LN, da Silva Duran BO, et al. Osteoglycin inhibition by microRNA miR-155 impairs myogenesis. PLoS One. 2017;12: e0188464. doi: 10.1371/journal.pone.0188464 29161332
47. Fruhbeck G, Mendez-Gimenez L, Fernandez-Formoso JA, Fernandez S, Rodriguez A. Regulation of adipocyte lipolysis. Nutr Res Rev. 2014;27: 63–93. doi: 10.1017/S095442241400002X 24872083
48. Kondo H, Nifuji A, Takeda S, Ezura Y, Rittling SR, Denhardt DT, et al. Unloading induces osteoblastic cell suppression and osteoclastic cell activation to lead to bone loss via sympathetic nervous system. J Biol Chem. 2005;280: 30192–30200. doi: 10.1074/jbc.M504179200 15961387
49. Hariri N, Thibault L. High-fat diet-induced obesity in animal models. Nutr Res Rev. 2010;23: 270–299. doi: 10.1017/S0954422410000168 20977819
50. Fuchs T, Loureiro MP, Macedo LE, Nocca D, Nedelcu M, Costa-Casagrande TA. Animal models in metabolic syndrome. Rev Col Bras Cir. 2018;45: e1975. doi: 10.1590/0100-6991e-20181975 30379216
51. Cauley JA. An overview of sarcopenic obesity. J Clin Densitom. 2015;18: 499–505. doi: 10.1016/j.jocd.2015.04.013 26141163
52. Polyzos SA, Margioris AN. Sarcopenic obesity. Hormones (Athens). 2018;17: 321–331.
Článek vyšel v časopise
PLOS One
2019 Číslo 10
- Řekněte mi, co jíte, a já vám řeknu, jaká je úroveň vaší kognice
- Původní český test ALBA pro odhalení mírných kognitivních deficitů získal certifikaci MZ ČR
- Duševní onemocnění se u teenagerů přenáší v rámci školní třídy
- Od kostiček k lidské kůži. Jak si postavit 3D biotiskárnu?
- Prof. Libor Grubhoffer: Nová varianta mpox je příležitostí pokročit v poznání původce varioly
Nejčtenější v tomto čísle
- Correction: Low dose naltrexone: Effects on medication in rheumatoid and seropositive arthritis. A nationwide register-based controlled quasi-experimental before-after study
- Combining CDK4/6 inhibitors ribociclib and palbociclib with cytotoxic agents does not enhance cytotoxicity
- Risk factors associated with IgA vasculitis with nephritis (Henoch–Schönlein purpura nephritis) progressing to unfavorable outcomes: A meta-analysis
- Prevalence of pectus excavatum (PE), pectus carinatum (PC), tracheal hypoplasia, thoracic spine deformities and lateral heart displacement in thoracic radiographs of screw-tailed brachycephalic dogs