DOI 10.1515/hmbci-2013-0070      Horm Mol Biol Clin Invest 2014; 17(1): 39–51

Silvia Migliaccio*, Emanuela A. Greco, Francesca Wannenes, Lorenzo M. Donini and Andrea Lenzi

Adipose, bone and muscle tissues as new endocrine organs: role of reciprocal regulation for osteoporosis and obesity development Abstract: The belief that obesity is protective against osteoporosis has recently been revised. In fact, the latest epidemiologic and clinical studies show that a high level of fat mass, but also reduced muscle mass, might be a risk factor for osteoporosis and fragility fractures. Furthermore, increasing evidence seems to indicate that different components such as myokines, adipokines and growth factors, released by both fat and muscle tissues, could play a key role in the regulation of skeletal health and in low bone mineral density and, thus, in osteoporosis development. This review considers old and recent data in the literature to further evaluate the relationship between fat, bone and muscle tissue. Keywords: obesity; osteoporosis; sarcopenia. *Corresponding author: Dr. Silvia Migliaccio, MD, PhD, Department of Movement, Human and Health Sciences, “Foro Italico” University of Rome, Largo Lauro De Bosis 15, 00195 Rome, Italy, Phone: +390636733395, Fax: +3906490530, E-mail: [email protected] Emanuela A. Greco, Lorenzo M. Donini and Andrea Lenzi: Department of Experimental Medicine, Section of Medical Pathophysiology, Endocrinology and Nutrition, “Sapienza” University of Rome, Italy Francesca Wannenes: Department of Movement, Human and Health Sciences, “Foro Italico” University of Rome, Italy

Introduction Obesity and osteoporosis are two important widespread health problems, which lead to a high prevalence of both mortality and morbidity [1–4]. During the last decades, these pathological conditions have become major health threats around the world [2] for both economic and social costs. Obesity is recognized as a risk factor for metabolic and cardiovascular diseases, however it was considered a protective factor for bone loss and osteoporosis. Agerelated changes in body composition, metabolic factors,

and hormonal levels, accompanied by a decline in physical activity, may all provide mechanisms for the propensity to gain fat mass, lose muscle mass, but also develop bone loss [3, 4]. Thus, age, both in the male and female population, increases the risk of developing osteoporosis and obesity, alterations which affect millions of men and women [3, 5–7]. Obesity is caused by an imbalance in which energy intake exceeds energy expenditure over a prolonged period [2, 7]. In healthy adults, body weight is tightly regulated despite day-to-day variations in food intake and energy expenditure. Several environmental, nutritional, and hormonal factors appear to influence body weight [1–3]. For instance, postmenopausal women often show increased body weight, likely because of a decrease in basal metabolism, alteration of hormonal levels, and reduced physical activity [8]. Moreover, obese postmenopausal women are often affected by hypertension, dyslipidemia, diabetes mellitus, and cardiovascular disease, and have an increased risk of developing some cancers [3, 9, 10]. Conversely, these women have been considered protected against osteoporosis [5, 6, 11]. Osteoporosis is a bone metabolic disease characterized by a decrease in bone strength because of a reduction in both bone quantity and quality, leading to an increased risk of developing spontaneous and traumatic fractures [7]. More than 40% of postmenopausal women, on average, will suffer at least one osteoporosis fracture, often resulting in permanent and severe disabilities, nursing home placement, and even death [11, 12]. The rate of bone loss in adults reflects the interaction between genetic and environmental factors, which also influence the extent of bone acquisition during growth, leading to a peak bone mass [12–14]. Interestingly, fractures in childhood have been associated with alterations in body composition, such as increased adiposity, and bone structure, suggesting that these might be the earliest signs of skeletal insufficiency [14]. Soon after menopause, a process of bone loss begins, due to increased bone resorption, exerted by osteoclasts, which overcomes bone formation acted by osteoblasts [14]. Moreover, osteoblasts activity

40      Migliaccio et al.: Adipose, bone and muscle tissues as new endocrine organs declines with aging, determining the imbalance between bone resorption and bone formation [15]. Traditionally, osteoporosis has been regarded as a condition only associated with fracture and skeletal disability in old age, but recent studies demonstrate that bone mineral density (BMD) appear to be a better long-term predictor of death than blood pressure and cholesterol [16–18]. Further data, published in recent decades, indicated that low BMD is a strong and independent predictor of all-cause mortality, including cardiovascular ones [17, 18]. Interestingly, recent studies describe new pathological entities linked to different factor risks as, for instance, a clinical condition known as sarcopenic obesity, which appears related to the well-known chronic conditions described above. The term sarcopenia was first proposed in 1989 by Irwin Rosenberg and it is derived from the Greek “sarx”, meaning flesh and “penia”, meaning loss [19]. Originally, sarcopenia referred to the loss of muscle mass associated with aging, but since then the significance of this term has been extended to the age-related loss of muscle strength. The skeletal muscle mass index (SMI) allows the quantification of the level of sarcopenia [20] and it is obtained by dividing appendicular skeletal muscle mass (ASM), evaluated by DEXA, by body height squared (ASM/height2). According to this definition, individuals presenting an ASM/height2 ratio between −1 and −2 standard deviations (SD) of the genderspecific mean value of young adults are categorized as having class I sarcopenia. Individuals with an ASM/height2 ratio below −2 SD are categorized as having class II sarcopenia. Another definition of sarcopenia uses percent of the SMI% = total muscle mass/body mass × 100) [21]. Despite the limitation discussed above, most of the literature focuses on the obesity/low muscle mass combination, appropriately identified as sarcopenic obesity, which, like sarcopenia, is defined as a muscle mass index   30) might actually interfere with bone health [39]. In accordance with the data of Zhao, Hsu et al. showed that the matching of Chinese subjects by BMI, across 5-kg strata of body weight, revealed a negative relationship between fat and bone mass, and the risk of osteoporosis and non-spine fractures were significantly higher for subjects with a higher proportion of body fat, independent of body weight [39]. Our group has recently demonstrated that the 37% of a population of 395 adult obese subjects had significant skeletal alterations [40]. In particular, this subpopulation showed a lower BMD at lumbar spine than expected for both young age and high BMI [41]. A further characterization showed that different grades of adiposity could differently affect skeletal health status. In fact, the subdivision of the population into three different groups upon BMI status showed a slightly different BMD pattern among groups: overweight subjects (BMI  > 25   30) had a significant alteration in their BMD levels, with an increased number of individuals with a low bone mass not expected for age and body weight [41]. Evaluation of hormonal, metabolic and lipid profile did not show significant differences among groups. In a following study, our group described, in 340 obese women a significant inverse correlation between trunk fat and lumbar and femur BMD, significant low vitamin D circulating levels, and significant inverse correlation between vitamin D levels, BMI and trunk fat. In addition, a significant inverse correlation between trunk fat, osteocalcin and IGF-1 levels, and a direct correlation between trunk fat and HOMA-index and inflammatory markers such as fibrinogen and eritrocyte sedimentation rate was also observed [42]. A similar situation was

recently also observed in a population of 86 obese male subjects. As expected, trunk fat and HOMA-index were greater for higher BMIs, while testosterone levels were significantly lower in the male population with the higher BMI. Also, an inverse relationship between testosterone levels and trunk fat, but a direct relationship with testosterone and osteocalcin circulating levels, was observed, suggesting again a close link between bone, fat and hormonal regulation. It is noteworthy that osteocalcin levels decreased with the same trend in the groups with a higher BMI, showing an inverse relationship with HOMA-index and trunk fat. Finally, as expected, a direct correlation between testosterone levels and BMD, but an inverse correlation between 17β-estradiol serum levels, both at lumbar and femoral neck sites was observed [43]. Indeed data published by Blum et al., in a cohort of 153 premenopausal women, demonstrated that a high amount of fat mass is negatively associated with bone mass [44]. A recent study by Kim et  al. demonstrated, in a sample of 907 healthy postmenopausal women, that body weight was positively related with BMD and low risk for vertebral fractures, whereas percent of body fat and waist circumference were related to a low BMD and to higher risk for vertebral fractures [45]. Thus, all these data suggest an important role of fat distribution and muscle tissue, as much as total fat mass itself, with the maintenance of bone tissue health. Even racial differences appear to influence fat and bone interaction. Castro et  al. reported that obesity is negatively associated with BMD in Black, but not in White women [46], while Afghani and Goran reported an inverse correlation between subcutaneous abdominal adipose tissue and BMD in White women, but not in Black women. In the same study the authors reported an inverse association between visceral fat and BMD in Black women but not in White women [47]. These conflicting results suggest a complex effect of fat mass on bone tissue related to sample size, ethnicity, sex, study design, methods of statistical analysis and population structure. Nevertheless, several lines of evidence from environmental factors and medical interventions support an inverse correlation between fat and bone mass: physical exercise increases muscle and bone mass while it reduces fat mass [48], supplementation with calcium and vitamin D appears beneficial for the prevention of both osteoporosis and obesity [49], and menopause is also associated with increased fat mass, increased bone loss, and decreased lean mass [50, 51]. It is noteworthy that estrogen replacement therapy in postmenopausal women improves both lean and bone mass and reverses menopause-related weight gain [50,  51]. Whereas estrogens reduce the risk of bone loss and obesity, other pharmacological interventions have been

42      Migliaccio et al.: Adipose, bone and muscle tissues as new endocrine organs shown to increase both osteoporosis and obesity such as the treatment with gonadotropin-releasing hormone agonists and the use of glucocorticoids [52–55]. Recent findings have indicated that some anti-diabetic drugs, which interfere with PPARγ and thus with adipocytes differentiation, can also influence significantly skeletal homeostasis and fracture risk [56]. Longitudinal studies have shown that fat mass increases with age and is higher among later birth cohorts peaking at about age 60 years, whereas muscle mass and strength starts to decline progressively around the age of 30 years with a more accelerated loss after the age of 60 [57–61]. Both visceral and intramuscular fat tend to increase, while subcutaneous fat in other regions of the body declines [62–65]. Furthermore, fat infiltration into muscle is associated with lower muscle strength and leg performance capacity [66]. The increase in body weight and fatness are probably caused by progressive decline in total energy expenditure, stemming from decreased physical activity and reduced basal metabolic rate in the presence of increased or stable caloric intake exceeding basal and activity-related needs [67]. Aging is also

associated with a decline in a variety of neural, hormonal and environmental trophic signals to muscle. Physical inactivity, hormonal changes, pro-inflammatory state, malnutrition, loss of alpha-motor units in the central nervous system, and altered gene expression accelerate the loss of muscle mass and mass-specific strength [68–71]. The age-related changes in body composition, obesity and low muscle mass, or low muscle strength, may coexist in same person, identifying the condition of sarcopenic obesity, an important risk factor for weight gain [72] and for loss of muscle mass and strength. Obese individuals tend to be less physically active and this may contribute to a decrease in muscle strength [73]. Muscle atrophy leads to reduction in metabolic rate both at rest and during physical activity and may further aggravate the sedentary state, all of which can cause weight gain. Two recent studies have shown that weight loss intervention, by combining diet and exercise, among older obese people improves muscle strength and muscle quality in addition to fat loss, confirming the hypothesis of tight connection between adiposity and impaired muscle function (Figure 1) [74, 75].

Cross-talk regulation among adipose & muscle & skeletal tissue

Macrophages

IL-6 TNFa Leptin Adiponectin

Paracrine and autocrine inflammatory signals

Tissue insulin resistance Bone cell homeostasis alteration, Bone resorption, Osteopenia

Myostatin

Figure 1 A complex link between adipose, bone, and muscle cell exists. Several cytokines are secreted by fat tissue and act on bone cells. In particular several pro-inflammatory cytokines (i.e., IL6 and TNFα) act as osteoclastogenetic factors with a potential stimulating mechanism. Additionally, more recent observations have shown that bone-derived factors such as osteocalcin and osteopontin might affect body weight control and glucose homeostasis, suggesting a potential role of bone tissue as an endocrine organ with the presence of a feedback between the skeleton and other endocrine organs. Finally, it has been recently suggested that myostatin not only plays a key role in muscle homeostasis, but also seems to affect fat and bone. Adipocytes are a source of bioactive molecules (adipokines) that act in either a paracrine or endocrine manner to modulate insulin sensitivity locally as well as in the liver and skeletal muscle. Adipose tissue is also a source of inflammatory mediators. Thus, adipose tissue promotes atherosclerosis through a number of pathologic mechanisms.

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Few studies have examined the combined effect of obesity and muscle mass or strength in older persons on physical functioning or disability. Studies based on muscle mass and obesity provides conflicting data. The use of muscle mass as an indicator of sarcopenia, rather than a functional measure such as strength, may possibly explain the reason for which conflicting results are found, although the Baumgartner definition was based on muscle mass and obesity [76]. There are only few studies focusing on the combined effect of obesity and muscle impairment and skeletal alterations [77]. In the InCHIANTI study, Stenholm et  al. showed that older persons with high BMI and low mass strength experience a steeper decline in walking speed and also have a higher probability of mobility disability than those with either poor muscle strength or obesity alone, with also higher risk of falling and, thus, developing fractures [78–80].

Fat, muscle and bone correlation: from basic observations to potential mechanisms of interaction More recently, the relationship between fat, bone and muscle tissues has been investigated [81]. In particular myostatin (GDF-8), a member of the transforming growth factor-beta (TGF-β) superfamily that is highly expressed in skeletal muscle, seems to link the muscle, skeletal and adipose tissues. Myostatin was first described in 1997, and since then it has gained growing attention because of the discovery that its inhibition leads to muscle mass accrual. Actually it is known that myostatin not only plays a key role in muscle homeostasis, but also seems to affect fat and bone. In their elegant review, Buering and Binkley have revised the impact of myostatin, and its inhibition, on muscle mass/function, adipose tissue and bone density/geometry in humans [82]. Although existing data are sparse, myostatin inhibition leads to increased lean mass and one study found a decrease in fat mass and an increase in bone formation. In addition, myostatin levels are increased in sarcopenia, cachexia and after bed rest, whereas they are decreased after resistance training, suggesting physiological regulatory of myostatin. Interestingly, increased myostatin levels have also been found in obese individuals while the levels decrease after weight loss. Knowledge of the relationship of myostatin with bone is largely based on data in experimental animal models, demonstrating that elevated myostatin levels lead

to decreased BMD while myostatin inhibition improved BMD. In summary, myostatin appears to be a key factor in the integrated physiology of muscle, fat, and bone. It is unclear whether myostatin directly affects fat and bone, or indirectly via muscle. Nonetheless myostatin inhibition appears to increase muscle and bone mass and decrease adipose tissue, a combination that truly seems to be a holy grail [82]. Adipose tissues contain two distinct types of fat cells  – white and brown. White fat cells are specialized for the storage of chemical energy as triglycerides, while brown fat cells dissipate chemical energy in the form of heat [83]. Similarities in cell morphology, lipid metabolism and patterns of gene expression between the two fat cell types has led most investigators to assume that they share a common developmental origin but it was demonstrated that it is not so [84]. To elucidate the complex connection between adipose tissue and skeletal tissue, it is necessary to distinguish between visceral white fat and brown fat. Several potential mechanisms have been proposed to explain the complex relationship between visceral white adipose tissue (VAT) and bone tissues. VAT was long viewed as a passive energy reservoir, but since the discovery of leptin and following identification of other adipose tissue-derived hormones and serum mediators, fat has been considered an active endocrine organ that modulates energy homeostasis [85–87]. Adipose tissue also secretes various inflammatory cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) [88, 89], and an altered production of these pro-inflammatory mediators play pivotal roles in the mechanism underlying adverse metabolic and cardiovascular consequences [90–92]. All these molecules (i.e., resistin, leptin, adiponectin, IL-6) affect human energy homeostasis and might well be involved in bone metabolism, contributing to the complex relationship between adipose and bone tissues, but also muscle. Fat tissue is one of the major source of aromatase, an enzyme also expressed in gonads, which synthesizes estrogens by androgen precursors [93]. Estrogens are steroidal hormones that play a pivotal role in the maintenance of skeletal homeostasis, thus protecting against osteoporosis by reducing bone resorption and stimulating bone formation. This extragonadal estrogen synthesis becomes the dominant estrogen source in postmenopausal women, leading to the hypothesis of the protective effect of fat mass on bone [5, 25]. Conversely, studies on either humans lacking aromatase and on estrogen receptor-α or -β knockout mice indicate that estrogens protect against bone loss and support the hypothesis that these hormones might also

44      Migliaccio et al.: Adipose, bone and muscle tissues as new endocrine organs inhibit the development of obesity [93–95], as also suggested by the prevention of menopause-induced fat mass gain [96] and reduction of the incidence of osteoporosis fracture by estrogen replacement therapy [97–99]. In support of this hypothesis, decreased endogenous estrogen levels were shown to be coupled to an increase in adipocyte numbers and decreased osteoblast counts in the bone marrow of postmenopausal women [100]. As mentioned above, several adipokynes are involved in the fat-bone interaction. Leptin is the most important adipocyte-derived hormone, which suppresses appetite, increases energy expenditure and regulates bone remodeling [101]. The effect of leptin on bone is complex: both negative [102–104] and positive actions [105–108] have been reported. Leptin-deficient ob/ob mice and leptin receptor-deficient db/db mice are extremely obese with increased vertebral trabecular bone volume because of increased bone formation, despite hypogonadism and hypercortisolism. Interestingly, the intra-cerebroventricular infusion of leptin in both ob/ob and wild-type mice decreased vertebral trabecular bone mass. In vivo studies indicate that the effect of leptin may depends on its site and mode of action [109–111]. In vitro studies also found that leptin can act directly on bone marrow-derived mesenchymal stem cells (MSC) to enhance their differentiation to osteoblasts and to inhibit their differentiation to adipocytes [110, 111]. Furthermore, Takeda et  al. expanded these observations, demonstrating that the effects of intra-cerebroventricular leptin are mediated by the sympathetic nervous system and that osteoblasts express β-adrenergic receptors through which, most likely, the administration of β-adrenergic agonists decreases trabecular bone volume by inhibiting bone formation [103]. Adiponectin is another adipocyte-derived hormone, which has anti-inflammatory and anti-atherogenic effects, regulating energy homeostasis and bone remodeling [108–110]. In contrast to leptin, adiponectin serum levels are reduced in obese and diabetic subjects [111] and increase after weight loss [112]. Human osteoblasts express adiponectin and its receptors [108], but, as also seen for leptin, both negative and positive correlations between adiponectin and skeleton have been reported [113]. Other in vivo and in vitro studies show that adiponectin increases bone mass by suppressing osteoclastogenesis and activating osteoblastogenesis, indicating that a rise in adiponectin levels because of fat reduction could have a beneficial effect on bone density, further suggesting a link between adipose and bone tissues. Thommesen et  al. showed that resistin may play a role in bone remodeling, indicating that it is expressed in

bone marrow MSCs, osteoblasts, and osteoclasts. Resistin increases osteoblast proliferation and cytokines release, as well as osteoclast differentiation, suggesting that the effect of resistin on bone is still unclear and further studies are needed to better understand its role [114]. Interleukin-6 (IL-6), is a pluripotent inflammatory cytokine, released from adipocytes, adipose tissue matrix, osteoblast and elsewhere [92]. Obese subjects have high circulating levels of this pro-inflammatory cytokine and IL-6 gene polymorphism is associated with obesity. In contrast, administration of IL-6 in the central nervous system increases energy expenditure and decreases body fat in rodents [114]. IL-6 is a well-recognized osteoclastogenesis and resorption-stimulating factor [114] but some data show that IL-6 mRNA is expressed in pre-osteoblasts and osteoblasts [89, 114] and that IL-6 stimulates osteoblast proliferation and differentiation by controlling the production of local factors likely playing a role in bone formation in situations of high bone turnover [87, 89, 114]. In addition to adipocytes, the adipose organ includes various stromal and vascular cells, including fibroblasts, vascular endothelial cells and inflammatory cells. Initially, adipocytes were thought to be the major source of adipose-derived mediators, but more recent studies have shown that macrophages infiltrate adipose tissue, and that these cells, along with others residing in the stroma, also contribute to the production and secretion of humoral mediators, particularly inflammatory cytokines [87, 89, 114]. A paracrine loop, involving free fatty acids and inflammatory cytokines, has been postulated to establish a vicious cycle between adipocytes and macrophages maintaining and propagating the inflammation. Therefore, it is important to define interactions between adipocytes, osteoblasts and stromal cells [115]. In addition to the white adipose tissue, in mammals there exists a thermogenic brown adipose tissue (BAT) that produce heat. In humans and other large mammalian species BAT was traditionally thought to be restricted to the neonatal and early childhood periods [116, 117] in adult humans active BAT is present at discrete anatomical sites, especially in the upper trunk [118–120] even though the physiological significance of adult human brown fat has not yet been fully explored and clarified. It was recently established that the cell lineage of these brown adipocytes present in VAT depots arise from the same Myf5 negative precursor as white adipocytes, indicating that these BAT cells are different from classical and Myf5 expressing brown adipocytes [121]. This new fat tissue is called “beige fat” or “brite fat” (from “brown in white”) and arise from white cells transdifferentiation under particular conditions, such as chronic cold

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exposure, hormonal stimuli, pharmacological treatments [122]. The browning of VAT has been shown to have antiobesity and anti-diabetic effects in rodent models [122] and humans [123], conditions known to be associated with a decrease in bone mass. Furthermore high BAT activity in healthy young women correlates positively with high BMD [124], and there is a positive association between BAT volume, the amount of bone, and cross-sectional size of the femur in children and adolescents [125]. Recently an elegant study has demonstrated that in a mouse model the inducible beige fat (FoxC2AD+/Tg mice) is anabolic for the skeleton [126]. In these mice the trabecular bone mass is higher as compared to age-matched wild-type animals; the phenotype of high bone mass not only persisted but was even more pronounced with aging, although it was accompanied by different structural changes in males and females and with an increased number of differentiated osteoblastic cells, and reduced expression of sclerostin, an inhibitor of osteoblasts proliferation. Furthermore, beige cells expressed IGFBP2 and Wnt10b, two well-known bone anabolic factors, demonstrating that beige adipose tissue possess an endocrine/paracrine anabolic activity on skeletal tissue. These data are not surprising, because adipocytes, osteoblasts and myoblasts originate from a common progenitor: a pluripotential MSC [127], which has an equal propensity for differentiation into either adipocytes or osteoblasts, (or other lines) under the influence of several cell-derived transcription factors. This process is complex, suggesting significant plasticity and multifaceted mechanism(s) of regulation within different cell lineages, among which adipocytes and osteoblasts. Several studies have examined the function of adipocytes in bone marrow. Marrow MSCs isolated from postmenopausal osteoporotic patients express more adipose differentiation markers, than those of subjects with normal bone mass [129]. As mentioned earlier, adipocytes secrete endocrine and paracrine factors that strongly influence bone differentiation and remodeling. Estrogens are among these factors, which explains why an increased body weight in postmenopausal women is associated with slower rates of bone loss [130–132]. The interaction between estrogens and fat appears complex, however. Martin et al. showed a pronounced fatty bone marrow infiltration in rats following oophorectomy, suggesting a pivotal role of estrogen in regulating adipocyte recruitment. Conversely, the presence of aromatase in fat cells allows higher intra-marrow conversion of testosterone into estrogens which, in turn, can restrain bone resorption [128]. The effect of estrogen on bone and adipose tissue formation has long been studied and recognized, also

in experimental animal models. In humans, changes in estrogen status because of advancing age and menopause have been correlated with increased levels of cytokines IL-6 and IL-11, both associated with bone loss [133]. It is interesting to speculate, and needs further accurate characterization, whether the increase in adipogenesis successive to menopause is caused by a relief of repression or an induction of the adipogenic phenotype even though in vitro data suggest that the default ‘switch’ might be adipogenesis, a process that might be normally inhibited in vivo prior to estrogen depletion. Other members of the nuclear hormone receptor family control adipogenic and osteogenic steps. The peroxisome proliferator activated receptor γ (PPARγ) plays a central role in initiating adipogenesis [128]. Mutations of the PPARγ gene are associated with an altered balance between bone and fat formation in bone marrow. The nuclear hormone receptor family of transcriptional regulatory proteins is activated by a range of ligands, including steroid hormones, naturally occurring metabolites, synthetic chemicals, and as yet to be identified endogenous compounds (orphan receptors). Thiazolidinedione and other PPARγ ligands such as rosiglitazone and pioglitazone play a prominent role in the treatment of type 2 diabetic patients. However, in vitro analyses demonstrate that various PPARγ ligands not only induce murine bone marrow stromal cell adipogenesis but also inhibit osteogenesis. In particular, PPARγ-2 is the dominant regulator of adipogenesis, and ligand activation of PPARγ-2 favors differentiation of MSCs into adipocytes rather than osteoblasts [131]. Akune et al. showed that PPARγ insufficiency led to increased osteoblastogenesis in vitro and higher trabecular bone volume in vivo, confirming the key role of MSC lineage allocation for the skeleton [127]. Interestingly, aged mice exhibit fat-bone-marrow infiltration and enhanced expression of PPARγ-2 along with reduced mRNA expression of bone differentiation factors [134]. Mice with premature aging (SAM-P/6 model), also show nearly identical patterns of adipocyte infiltration with impaired osteoblastogenesis [135, 136], indicating that aging, or events that accelerate aging, result in significant bone marrow adiposity and a defect in osteoblastogenesis in mice. The Wnt signaling pathway works in co-ordination with other transmembrane signals, which include multiple ligands, antagonists, receptors, coreceptors, and transcriptional mediators, such as β-catenin [137]. Specific elements of the Wnt signaling pathway have been found to inhibit adipogenesis while promoting osteogenesis; Wnt inhibition of adipogenesis is mediated via β-catenin, which interferes with PPARγ transcriptional activation of

46      Migliaccio et al.: Adipose, bone and muscle tissues as new endocrine organs downstream targets. Following exposure to TGF-β, human bone marrow MSCs increased their expression of various Wnt receptors and ligands [138, 139]. Members of the epidermal growth factor family, such as protein Pref-1, influence adipogenesis and osteogenesis. In vitro analysis in human bone marrow MSCs has shown that Pref-1 overexpression blocks both adipogenesis and osteogenesis. This finding is consistent with the hypothesis that Pref-1 maintains MSCs in a multipotent state [139]. New experimental tools, such as gene microarrays, are being used to document the relationship of classical steroid hormones to bone and fat formation in marrow. One study has examined the skeletal phenotype of mice deficient in both thyroid receptors α and β. These mice showed increased mRNA levels for adipocyte specific genes, increased bone marrow adipocyte numbers, and reduced trabecular and total BMD [136]. The inbred SAM-P/6 murine strain provides a model of accelerated senescence characterized by osteopenia and increased bone marrow fat mass. Recent studies found that 1,25(OH)2 vitamin D treatment inhibited adipogenesis and enhanced osteogenesis in the SAM-P/6 mice with a 50% reduction in PPARγ mRNA and protein levels [138]. Moreover, gene microarray analyses demonstrated a coordinated induction of osteoblastogenic genes and a reduction of adipogenic genes after 1,25(OH)2 vitamin D treatment, which stimulates not only bone formation but also bone resorption based on circulating biomarkers of bone turnover [138]. Overall, these recent findings involving classical steroid receptors support the inverse relationship between adipogenic and osteogenic differentiation in the bone marrow microenvironment. This is mediated, in part, through cross-talk between the pathways activated by steroid receptors, the PPARs, and other cytokines and paracrine factors. Finally, other factors, such as total caloric intake, type of nutrients, alcohol consumption, oxygen tension and cellular redox pathways influence bone marrow adipogenesis despite osteoblastogenesis, showing that the bone marrow MSC may consider multiple differentiation pathways during its lifetime and, indeed, may dedifferentiate and transdifferentiate in response to changes in the microenvironment. As earlier mentioned, myostatin seems to be an important link between fat, bone and muscle homeostasis [82]. In their elegant work, Elkasrawy et al. used a myostatindeficient mice model for studying muscle-bone interactions. Myostatin is a key regulator of MSCs proliferation and differentiation, and mice lacking the myostatin gene show decreased body fat and a generalized increase in

bone density and strength. The increase in bone density is observed in most anatomical regions, including the limbs, spine, and jaw, and myostatin inhibitors have been observed to significantly increase bone formation. Myostatin is also expressed in the early phases of fracture healing, and myostatin deficiency leads to increased fracture callus size and strength. Together, these data suggest that myostatin has direct effects on the proliferation and differentiation of osteoprogenitor cells, and that myostatin antagonists and inhibitors enhance both muscle mass and bone strength [139]. Although the role of myostatin in muscle growth regulation has been widely investigated, its role in regulating bone mass, architecture and regeneration is becoming an area of increased interest. Genetic studies in human populations have shown that myostatin gene polymorphisms are associated with variation in peak bone mineral density [140], and transgenic overexpression of myostatin propeptide, which inhibits myostatin signaling in vivo, increases BMD in mice [141]. Thus, there is evidence from both human studies and animal models to suggest that myostatin is an important regulator of both muscle mass and bone density. The mechanisms by which myostatin regulates bone formation are not well understood, but it is clear that myostatin has direct effects on the proliferation and differentiation of MSCs [142, 143], and that myostatin and its receptor are expressed during bone regeneration [142]. Myostatin therefore appears to be a potent antiosteogenic factor that may function to suppress the proliferation and possibly the survival of osteoand chondroprogenitors.

Conclusions Obesity, sarcopenia and osteoporosis are major global health problems with an increasing prevalence and high impact on mortality and morbidity. Menopause and aging are characterized by increased bone loss, increased fat mass, and decreased lean mass. It is likely that age-related changes in hormone levels, in association with changes in body composition, metabolic factors, and declined physical activity, provide the mechanisms for the propensity to age-related gain of fat mass, thus in body weight, and loss of lean mass. Even though previous data indicate that high body weight and BMI are protective factors against osteoporosis, an increasing number of evidence suggest that obesity might actually interfere with bone health at different ages, increasing the fracture risks [144–146]. In particular, the relationship between obesity and osteoporosis depending on how obesity is defined [147, 148]. Longitudinal studies

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have shown that fat mass increases with age, whereas muscle mass and strength decline progressively, and that fat infiltration into muscle is associated with lower muscle strength and performance capacity, leading to sarcopenia and osteopenia/osteoporosis with the potential presence, in the same individual, of the devil triade: obesity, sarcopenia and osteoporosis. Indeed, the existence of a cross-talk between fat, muscle and skeleton that constitutes a homeostatic feedback system in which adipokines and bone- and muscle-derived molecules represent the link of an active bone-muscle-adipose axis. The mechanism(s) by which all these events occur are not fully understood yet. However, new emerging data indicate clearly that to better understand the correlation between these organs it is necessary distinguish between white and beige fat.

Thus, further studies are needed to build a careful and full knowledge of skeletal, muscle and energy metabolism interaction, as aging might also increase the risks of developing alterations in the homeostasis of these tissues, and thus, development of osteoporosis and obesity. A full characterization of these mechanism(s) will not only help physicians in the holistic clinical approach of the obese patient but will also help the development of specific life style and/or pharmacological treatments to improve health of these subjects. Acknowledgments: FW is supported by an ELI Lilly grant.

Received December 31, 2013; accepted February 14, 2014

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