Low bone accrual is associated with osteocyte apoptosis in alcohol-induced osteopenia☆,☆☆
Highlights
► Chronic alcohol consumption decreases bone density through bone remodeling imbalance. ► Little is known about the effect of alcohol on osteocyte. ► We assessed the effects of alcohol on bone density, osteocyte apoptosis, morphology and on marrow fat content. ► We found increased osteocyte apoptosis and marrow fat content, which were inversely correlated to the low BMD. ► Alcohol-induced osteopenia is linked to osteocyte apoptosis and increased marrow fat content.
Introduction
Chronic alcohol consumption has been associated with a lower bone mineral content (BMC) and bone mineral density (BMD) in trabecular and cortical bone [1], enhancing the risk of osteoporotic fracture [2], [3]. This osteopenia is considered as a consequence of an unbalance in bone remodeling [3].
Several data suggest a direct effect of ethanol on osteoblasts and osteoclasts [1], [4]. Bone formation is often decreased and bone resorption is sometimes increased in drinkers versus non drinkers [5], [6] and in animal models [7], [8]. The decrease of bone formation in men has been shown to be due to an inhibition of the osteoblast proliferation and activity [9], [10]. The inhibitory effects of alcohol on bone formation seem to be dose dependent, as well as the effects on bone resorption [3]. In a study by Turner, the bone formation rate (BFR) decreased as the ethanol dose increased, while the number of osteoclasts decreased with low alcohol consumption but not with high doses [3]. These results confirm that excessive alcohol consumption is responsible for an uncoupling process with a reduction of bone formation and in some studies an excess in bone resorption [7], [11].
Osteocyte is the third bone cell type and is the most abundant in bones [12]. It is embedded in the bone matrix and has not been as much studied as osteoblasts and osteoclasts because of its localization. It was thought to be a passive cell but some recent studies attribute important roles to osteocytes such as the mechano-transduction activity [13]. It is now understood that the osteocyte network is connected to bone lining cells on the bone surface and with cells within the bone marrow [14]. One of the roles of the osteocyte is to send signals to prevent bone loss under normal conditions, or to induce bone loss in response to unloading, to conserve energy [15], [16]. Osteocyte apoptosis has been shown to signal the triggering of resorption [17], [18]. It has also been shown that some proteins expressed in osteocytes can target osteoblasts to inhibit bone formation [19]. In patients with different bone pathologies such as osteopenia and osteopetrosis, osteocytes are associated with increased lacunar size and different 3D morphology compared to osteoarthritis osteocytes, which may reflect an adaptation in response to environmental factors such as different external loading conditions [20]. For example, it has been shown that the administration of glucocorticoids increases the lacunar size and induces the apoptosis of osteocytes (revealed by trypan blue, nuclear morphology or caspase 3 activation) [21].
Recently, Vatsa et al. [22] have demonstrated differences in alignment of osteocytes between mouse fibulae and calvaria samples, and have underlined the possible role of multi-directional loading in calvaria versus unidirectional loading in fibula in this different anisotropy. Characterization and location of osteocytes in situ, especially their spatial relationships with the bone matrix, have been particularly difficult because the mineralized matrix of bone was an impediment in the analysis of their fine structural details at the subcellular level in situ. Nevertheless, combination of imaging by confocal microscopy or classic photonic epifluorescent microscopy and recently developed live-cell staining with fluorescent markers have allowed evaluating details of the osteocyte cytoskeleton in situ[23]. It has been demonstrated that CD44, a transmembrane glycoprotein with cell–cell and cell–matrix adhesion functions could be a sensitive marker of the osteoblastic lineage differentiation in humans [24], [25].
Alcohol induces changes in the function of osteoblasts and osteoclasts [3], [8], [26] but little is known about the effects of alcohol on the osteocytes. We performed this study to analyze if chronic alcohol consumption elicits changes in osteocyte morphology and apoptosis in situ, in relation to the changes occurring in bone.
Section snippets
Animals
Thirty four male Wistar rats (Elevage Janvier, Le Genet-St-Isle, France) were acclimatized for 2 weeks and maintained under constant temperature (21 ± 2 °C) and under 12 h/12 h light–dark cycles all along the experiment. The rats were housed two per standard cage and provided with a commercial standard diet (M20, SDS, France).
Alcoholization treatment
The rats were 8 weeks old at baseline. They were not skeletally mature but skeletal maturity occurs late in rats (7 months old) compared to sexual maturity (2 months old) [27]. We
Results
The average daily beverage consumption was lower in A35 versus C (18.2 ± 2.9 versus 26.9 ± 2.4 ml for A35 and C; p = 0.02). The percentage of calories provided by ethanol among the total calories was significantly higher in A35 (38.1 ± 3.1 versus 0.0 ± 0.0% for A35 and C; p = 0.01). As the food consumption was controlled in order to pair-fed the groups in the total amount of calories, we did not observe any difference in the average daily total calories between C and A35 (respectively 84.7 ± 3.3 and 86.4 ± 8.4
Discussion
This study has confirmed that in the rat, chronic alcohol consumption induces low BMD, trabecular and cortical thickness. The major finding of this study was the large increase of osteocyte apoptosis, which appears to be associated with the alcohol-induced osteopenia of these rats.
Alcohol consumption led to lower body weight gain in the A35 group compared to C (Table 1). The lean and fat mass gains were also significantly lower compared to the C group. These observations are consistent with the
Acknowledgments
We would like to thank Eric Dolleans for technical help in dissecting the bones, Carine Martin from the University of Orléans for the formalin preparation and Dr Brigitte Arbeille from the Département des Microscopies, Programme Pluriformation Analyse des Systèmes Biologiques, Université de Tours, Tours, France for the collaboration on the transmission electron microscopy. We also are grateful to the Department of Anatomo-Pathologie in Orleans Regional Hospital directed by Dr Patrick Michenet
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Cited by (0)
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This work was supported by two grants from the IREB (Institut de Recherche sur les Boissons, France) and the Programme interdisciplinaire Longévité et vieillissement, CNRS, France.
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Disclosures: All the authors state that they have no conflicts of interest.
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Both senior authors equally contributed to this work.