Low energy availability reduces bone mass and gonadal function in male mice

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Low energy availability reduces bone mass and gonadal function in male mice

https://doi.org/10.21203/rs.3.rs-2006996/v1

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In women, the female athlete triad, marked by low energy availability, functional hypothalamic amenorrhea and osteoporosis, is a recognized risk for stress fractures. Stress injuries also occur in men, but by contrast risks and mechanisms underlying them are less characterized. Here we propose that low energy availability puts men at risk for stress injuries as well. Male low energy availability model mice, established by limiting food intake, exhibited significantly reduced testicle weight, serum testosterone levels and bone mass. Such losses in bone mass were enhanced by exercise in food-restricted (FR) male mice. Histological analysis revealed that both bone-resorbing and -forming activities were significantly reduced in FR or FR plus exercise (FR+ex) mice, mimicking a state of low bone turnover. Significantly reduced bone mass in FR or FR+ex male mice was significantly rescued by treatment with 1,25(OH)₂D₃ (1.25) or ED71 (ED), both active vitamin D analogues, with significant restoration of osteoblastic activities. Serum levels of insulin-like growth factor I (IGF-I), which activates osteoblastic activities and is critical for bone remodeling, were significantly lower in FR versus control male mice, but restored by administration of either of vitamin D analogue. However, administration of recombinant IGF-I did not rescue reduced bone mass in FR male mice, and administration of either active vitamin D analogue could increase bone mass even in IGF-I conditional knockout FR male mice. Taken together, conditions comparable to the female athlete triad are also a risk for male stress injuries, and low energy availability is upstream of gonadal dysfunction and osteoporosis in males. Active vitamin D analogues could serve as therapeutic or preventive options for stress injuries in men.

food restriction

stress fractures

male athletes

vitamin D

testicle

IGF-I

In athletes, stress fractures are frequently seen in bones of the lower extremity such as metatarsal bones and are often repeated. Stress fractures hamper an athlete’s career and can limit activities of daily living and quality of life ¹, making their prevention mandatory. Stress injury to bone has been considered caused by repetitive micro damage rather than a single, traumatic high impact event ². Recently, the female athlete triad, which includes low energy availability, functional hypothalamic amenorrhea and osteoporosis, was proposed as a risk for stress injuries ³. In general, exercise is considered to have positive effects on bone ^4-6; however, excessive exercise is a known risk factor for stress fractures ^7,8. To date, mechanisms underlying stress fractures in female athletes have been intensively investigated given that onset of amenorrhea signals a risk for stress fractures ^3,9,10. However, since male athletes do not exhibit such overt warning signs, mechanisms underlying stress fractures in male athletes are not as well characterized.

Bone homeostasis and remodeling are maintained as a delicate balance between bone resorption by osteoclasts and bone formation by osteoblasts ^11,12. Reduced bone mineral density can be caused by either elevated osteoclast differentiation or activity, or decreased osteoblast differentiation or function ¹³. Various bone remodeling factors such as transforming growth factor beta (TGFβ) and insulin-like growth factor I (IGF-I) are secreted from osteoblasts, stored in bone matrix proteins and then become activated upon osteoclastic bone resorption to stimulate following bone formation by osteoblasts ^14-16. Vitamin D3 promotes calcium absorption from intestine, and its deficiency is a known risk factor for stress injuries ^17,18 and for fragility fractures in the elderly ^19,20. Active vitamin D3 agents are now clinically approved to treat osteoporosis patients ²¹ and are not listed among drugs prohibited by the World Anti-Doping Agency (WADA).

In menopausal women, estrogen deficiency promotes osteoclast activation, leading to reduced bone mass in postmenopausal osteoporosis patients ^22,23. Given that amenorrhea is the most serious risk for stress injuries in young female athletes ⁹, normal gonadal function is clearly required to maintain bone homeostasis, even in young female athletes.

Among characteristics of the female athlete triad, low energy availability is the most upstream ^3,24. In fact, low energy availability model female mice show significant uterine atrophy and decreased bone mass and strength ²⁵. Functional hypothalamic amenorrhea is also reportedly upstream of osteoporosis in humans ²⁶, and female functional hypothalamic amenorrhea model mice exhibit reduced bone mass ²⁷. It is not yet clear whether gonadal dysfunction underlies stress injuries in men. However, support for this idea comes from observations that gonadal dysfunction in prostate cancer patients treated with hormone-depleting therapy is known to promote osteoporosis development ²⁸, and men who have undergone exercise training reportedly exhibit lower resting testosterone levels ²⁹. However, studies of mechanisms underlying stress injuries in men are limited, and the potential effects of low energy availability on male physiology or metabolic function are not fully understood.

Here, we establish male low energy availability model mice by limiting food intake. We then show that food-restriction (FR) promotes gonadal dysfunction and reduced bone mass in these animals. Exercise under low energy availability conditions further deteriorated bone mass in male mice. Bones in FR male mice exhibited a low turnover state, with bone-resorption and -formation significantly reduced. Serum IGF-I levels significantly decreased in FR mice, but administration of either 1,25(OH)₂D₃ (1.25) or ED71 (ED), both active vitamin D analogues, partially but significantly increased bone mass and serum IGF-I levels in FR male mice. Interestingly, IGF-I administration was not effective in improving bone mass in FR male mice. We conclude that low energy availability causes bone fragility and gonadal dysfunction in male mice, suggesting that active vitamin D analogues could be considered potential therapeutic options for this condition in humans.

Food restriction reduces bone mass in male mice

First, to analyze the impact of low energy availability on male bones, we limited food intake in wild-type male mice to 60% of average intake (food-restriction, FR) starting at five weeks of age (Figs. 1 and S1). In these experiments some mice were given access to an exercise wheel in their cages to allow by voluntary exercise (ex) activity, which was quantified by counting wheel rotations (Fig. S1). Interestingly, in the exercise groups, FR+ex mice showed significantly higher exercise activity than those fed ad libitum (ad+ex) (Fig. S1a). Five weeks after dietary changes were made, we observed that body weight was significantly lower in FR compared with ad mice (Fig. S1b). Body weight was also significantly lower in FR + exercise (FR+ex) than in ad+ex mice, and exercise further lowered body weight of FR mice (Fig. S1b). Testicle weight and serum testosterone levels were significantly lower in FR than in ad mice (Fig. S1c). Moreover, both parameters were also significantly lower in FR+ex than in ad+ex mice (Fig. S1c), suggesting that low energy availability may promote gonadal dysfunction in male mice. Micro CT analysis in femoral bones indicated that bone parameters, namely, TMD, Ct.Th, Ct.Ar and BMC, were all significantly lower in FR than in ad mice (Figs. 1a and b). Moreover, TMD, Ct.Th, Ct.Ar and BMC were also significantly lower in FR+ex compared to ad+ex groups (Figs. 1a and b). Interestingly, exercise did not alter bone parameters in ad mice; however, relative to FR only conditions, some bone parameters, such as TMD, Ct.Th and Ct.Ar, were worsened by exercise (FR+ex) (Figs. 1a and b).

Bone morphometric analysis revealed that although Oc.N/BS was equivalent in ad, FR FR+ex and ad+ex mice, ES/BS was significantly lower in FR than in ad mice and also in FR+ex compared to ad+ex mice, suggesting that bone resorbing activity decreases in low energy availability conditions (Fig. 1c). Similarly, bone forming parameters including Ob.S/BS, BFR/BS and MAR were all significantly lower in FR compared to ad mice and also in FR+ex compared to ad+ex animals, suggesting that bones enter a low turnover state in low energy availability conditions (Fig. 1d).

Active vitamin D analogues antagonize cortical bone loss by a FR

Next, we administered active vitamin D analogues, which are currently used for osteoporosis therapy, to male FR mice for five weeks starting at five weeks of age (Fig. 2). Specifically, for this analysis, we treated mice with either the native active vitamin D analogue 1,25(OH)₂D₃ (1.25), the synthetic active vitamin D analogue ED71 (ED), or vehicle only (ethanol). Significantly reduced body weight seen in FR mice was not restored by administration of either analogue (Fig. S2a). In contrast, reduced testicle weight seen in FR mice was partially but reversed by treatment with either active vitamin D analogue (Fig. S2b). Moreover, phenotypes seen in FR mice, such as significantly reduced TMD, Ct.Th, Ct.Ar and BMC, were all partially but significantly rescued by treatment with either analogue (Fig. 2a and b).

Bone morphometric analysis of male FR mice demonstrated that Oc.N/BS was also significantly reduced by treatment with either analogue, and ES/BS was significantly inhibited by ED but not 1.25 treatment (Fig. 2c). In addition, bone forming activity, as analyzed by Ob.S/BS and BFR/BS, was significantly reduced by FR but significantly rescued by administration of either vitamin D analogue (Fig. 2d). MAR also appears to be reduced by FR and was significantly recovered by administration of 1.25. (Fig. 2d).

When we treated FR+ex mice with active vitamin D analogues (Figs. 3 and S3a and b), body weight, which was reduced in these mice, was not rescued, while testicle weight was and significantly so (Figs. S3a and b). Reduced bone mass seen in FR+ex mice was also significantly rescued by treatment with either vitamin D analogue, and TMD, Ct.Th, Ct.Ar and BMC were all partially but significantly rescued by either treatment (Fig. 3a and b).

Oc.N/BS but not ES/BS was significantly reduced by treatment with either vitamin D analogue (Fig. 3c). In contrast, Ob.S/BS, and MAR were significantly rescued by treatment with either 1.25 or ED (Figs. 3d).

Serum IGF-I levels significantly decrease in FR or FR+ex conditions but are significantly rescued by treatment with active vitamin D analogues

Levels of IGF-I, which plays a pivotal role in regulating bone remodeling ³⁰, are regulated by nutrient intake ³¹. Serum IGF-I levels in FR mice were significantly lower than in ad animals and lower still in FR+ex mice (Fig. 4a). Interestingly, decreased serum IGF-I levels seen in FR or FR+ex mice were partially restored by administration of either 1.25 or ED (Figs. 4b).

To further assess consequences of decreased serum IGF-I levels, we generated IGF-I conditional knockout adult mice (IGF-I cKO) by crossing Mx1 Cre with IGF-I-flox mice to yield Mx1 Cre; IGF-I^flox/flox mice (Fig. 5). We then injected 5-week-old IGF-I cKO and control male mice, intraperitoneally with polyIpolyC to delete the IGF-I gene and at the same time placed some animals in FR or FR+ex conditions for another five weeks until mice were 10 weeks of age. We then measured serum IGF-I levels by ELISA (Figs. 5a). Serum IGF-I levels were significantly lower in male IGF-I cKO ad mice than in control ad mice (Figs. 5a), indicating successful IGF-I knockout. Body weight was comparable in IGF-I cKO and control mice in the ad condition (Figs. 5b). In contrast, we observed significantly reduced testicle weight in male IGF-I cKO mice relative to controls in ad conditions (Figs. 5c). TMD, Ct.Th, Ct.Ar and BMC were all significantly lower in IGF-I cKO than control male mice in the ad condition (Figs. 5d and e). FR in IGF-I cKO did not further decrease bone mass in male mice (Figs. 5d and e).

To determine if reduced bone mass seen following FR was due to decreased serum IGF-I, we administered recombinant IGF-I to male FR mice starting at five weeks of age for five days a week until mice reached 10 weeks of age (Fig. 6). At that time point, in males, body weight and testicle weight reduction seen following FR were not rescued by an IGF-I administration (Figs. 6a and b), nor was reduced TMD, Ct.Th, Ct.Ar and BMC seen in FR male mice restored by an IGF-I administration (Figs. 6c and d). However, reductions in TMD and Ct.Th, seen in male FR IGF-I cKO mice were all partially but significantly restored by treatment with either active vitamin D analogue, although administration of active vitamin D did not increase serum IGF-I levels (Figs. 7a, b and c).

Currently, our understanding of mechanisms underlying stress fractures in male athletes is not as complete as it is in female athletes. Based on this study, which focuses primarily on male mice, we propose that, like the female athlete triad, there exists a male athlete triad marked by low energy availability, the most upstream factor, and followed by gonadal dysfunction and osteoporosis, which may increase risk of bone fragility in male athletes. Unlike the situation in females, in which amenorrhea alerts women to risk for stress injuries, male athletes under comparable risk do not exhibit overt signs of this condition and remain unaware of their risk. Here, we show that relatively reduced serum IGF-I levels could serve as a potential biomarker for stress injuries in male athletes. Note, however, that we also find that, at least in FR mice, administration of recombinant IGF-I does not increase bone mass. Nonetheless, monitoring the balance between energy intake and consumption and avoiding low energy availability are required to avoid stress injuries in male athletes, and our data indicates that administration of an active vitamin D analogue may prevent such injuries.

In women, osteoclast bone-resorbing activities increase after the menopause, reducing bone mass and leading to osteoporosis development. In premenopausal estrogen-sufficient conditions, activity of hypoxia inducible factor 1 alpha (HIF1α) in osteoclasts is continuously suppressed by estrogen ³². However, in postmenopausal estrogen-deficient conditions, HIF1α accumulates in osteoclasts, activating osteoclast bone resorption ³². Similarly, androgen-deficient conditions in orchiectomized male mice have also been demonstrated to promote HIF1α accumulation in osteoclasts, leading to increased bone resorbing activity ³³. Treatment with ED but not 1.25 has been shown to antagonize HIF1α activity in osteoclasts ³⁴, and ED is a therapeutic agent used for osteoporosis therapy in both male and female patients ^33,34. Here, we show that osteoclast bone-resorbing activity is significantly inhibited in FR male mice, although those mice exhibit gonadal dysfunction. We also demonstrate that either ED or 1.25 treatment increases bone mass in FR mice. In addition, serum IGF-I levels increased along with increased bone mass following treatment with either of the vitamin D analogues tested, and this treatment increased bone mass even in IGF-I cKO FR mice. Further studies are needed to define mechanisms underlying these outcomes.

It is mandatory that athletes avoid use of drugs prohibited by the WADA. IGF-I is one of the agents listed as prohibited by WADA, as are selective estrogen receptor modulators (SERMs), which antagonize HIF1α activity in osteoclasts ³⁵ and are considered potential therapeutic agents or preventatives against stress injuries. By contrast, active vitamin D analogues are not prohibited by the WADA. Since female athletes experience stress injuries under vitamin D suboptimal conditions ⁹, levels of vitamin D higher than suboptimal are required to prevent stress injuries in females. Thus, taking active vitamin D analogues should be considered as a means to prevent, at least in part, stress injuries.

Mice

Four-week-old wild-type C57/B6 (WT) male mice were purchased from Sankyo Labo Service (Tokyo, Japan). During the first week of environmental acclimation, mice were fed a standard diet and water ad libitum. Mx1 Cre/Igf-I^flox/flox mice (IGF-I cKO) were established previously ³⁶. Igf-I^flox/flox mice served as controls. Five-week-old IGF-I cKO and control mice were injected intraperitoneally with 10 μg/g polyIpolyC (P0913, Sigma-Aldrich Co., St Louis, MO) for 3 days, and once after 2 weeks. All mice were fed ad libitum with an experimental diet of CE-2 purchased from CLEA (Tokyo, Japan) until five-weeks-old.

Mice were maintained on a 12-h light/dark cycle under specific pathogen-free conditions in animal facilities certified by the Keio University Institutional Animal Care and Use Committee, and animal protocols were approved by the committee. All animal experiments were conducted in accordance with the Institutional Guidelines on Animal Experimentation at Keio University.

In FR experiments, mice housed in individual cages were randomly divided into four groups. As in a previous experiment ²⁵, the “ad” group consumed the CE-2 diet ad libitum; the “FR” group consumed 60% of that based on the average intake of the ad libitum group; the “ad+ex” group consumed food ad libitum and did running exercise; and the “FR+ex” group was 60% food-restricted and performed voluntary running exercise. Both “ad+ex” and “FR+ex” groups had free access to a running wheel (circumference 0.45m, width 60, φ140 mm) in their cages for voluntary running exercise, while “ad” and “FR” groups did not. The number of wheel rotations was counted using the Actmaster system (Melquest Ltd., Toyama, Japan).

Some WT mice were administered rhIGF-I (Orphan Pacific Inc., Tokyo, Japan) at a dosage of 2mg/kg body weight (IGF-I group) or saline at 1ml/kg body weight (saline group) five days a week for five weeks.

After five weeks of experimentation, all mice were sacrificed at 10 weeks of age. This study is reported in accordance with ARRIVE guidelines.

Treatment with active vitamin D analogues

Mice were administered eldecalcitol (ED71) (provided by Chugai Pharmaceutical Co., Ltd, Tokyo, Japan) or 1.25(OH)₂D₃(1.25) (Wako Pure Chemicals Industries, Osaka, Japan), five days a week for five weeks. In both cases, the dose (0.05 μg/kg/day) was administered orally in a 0.5% methylcellulose solution (Wako Pure Chemicals Industries, Osaka, Japan). Control groups were administered vehicle (ethanol: 0.05μl/kg/day).

Analysis of skeletal morphology

Mice were sacrificed and their hind limbs were removed, fixed in 70% ethanol, and analyzed for bone histomorphometry. Microstructural changes in femoral bone were evaluated using a micro-computed tomography (micro-CT) system (CosmoScan GX; Rigaku corporation, Tokyo, Japan). The distal and diaphyseal femur were scanned at 90kV, 160μA, and a voxel size of 20 × 20 × 20μm. Three-dimensional reconstruction using the TRI-BONE system (Ratoc system Engineering, Co., Ltd., Tokyo, Japan) was performed. For bone morphometric analysis we evaluated tissue mineral density of cortical bone (TMD, mg/cm³), cortical thickness (Ct.Th, μm), cortical bone area (Ct.Ar, mm²), and BMC (mg) as parameters. TMD was evaluated in cortical bone of the metaphyseal femur, and Ct.Th, Ct.Ar and BMC were evaluated in cortical bone of the diaphyseal femur. The metaphysis was measured from 200μm below the growth plate and to 4000μm distal, and the diaphyseal was 500μm wide.

Histopathological and fluorescent immunohistochemical analysis

Calcein (Sigma-Aldrich, Tokyo, Japan) labels were injected intraperitoneally into mice at a dose of 16μg/g first at 5 days before sacrifice and then again 1 day before sacrifice. Mouse tibiae were dissected, fixed in 70% ethanol, and undecalcified. Following ASBMR bone histomorphometry guidelines ³⁷, osteoclast number per bone surface (Oc. N/BS), eroded surface per bone surface (ES/BS), bone formation rate (BFR) and osteoblast surface per bone surface (Ob.S/BS) were calculated in trabecular bone in the region of 200-500μm from the growth plate using a BioRevo microscope and corresponding software (Keyence, Tokyo, Japan). Mineral apposition rate (MAR) was measured in cortical bone of the diaphysis from four representative fields per sample using a BioRevo microscope.

Serum analysis

Serum samples were collected from ten-week-old mice before sacrifice. Serum IGF-I Enzyme-linked immunosorbent (ELISA) assays were performed using a multiple plate analyzer (POWERSCAN HT, DS Pharma Biomedical, Osaka, Japan) according to the manufacturer’s instructions (R&D systems, Minneapolis, MN, USA). Serum testosterone was analyzed by liquid chromatography and mass spectrometry (ASKA Pharma Medical Co., Ltd., Kanagawa, Japan).

Statistical analysis

All data are shown as means ± standard error. Statistical analyses were performed using a one-way analysis of variance (ANOVA) of each group (*p<0.05; **p<0.01; NS, not significant, throughout the paper).

Data availability statement:

The data that support the findings of this study are available from the corresponding author on reasonable request.

Acknowledgment:

T. Miyamoto was supported by a grant-in-aid for Scientific Research in Japan and a grant from the Japan Agency for Medical Research and Development. Y. Sato and K. Miyamoto were supported by a grant-in-aid for Scientific Research in Japan.

Author contributions:

EI and TM (Miyamoto) conceived the study. EI conducted the study. EI, YS and TK provided experimental mice. EI collected and analyzed data. EI, TS, TM (Matsumoto), AK, KM and TM (Miyamoto) interpreted data. HM, MM, MN, KS and TM (Miyamoto) supervised the study. EI and TM (Miyamoto) wrote the manuscript. All authors approved the manuscript. EI takes responsibility for the integrity of the data analysis.

Competing interest:

The authors declare that they have no conflicts of interest with the contents of this article.

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No competing interests reported.

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Low energy availability reduces bone mass and gonadal function in male mice

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