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Institut National de la Santé et de la Recherche Médicale (INSERM) E0366 (A.M., R.d.V., V.D., M.-H.L.-P., C.A., L.V., T.T.), University Hospital, 42055 St-Etienne, France; and INSERM Unité 418 (R.M., M.B.), 69005 Lyon, France
Address all correspondence and requests for reprints to: Prof. Thierry Thomas, Institut National de la Santé et de la Recherche Médicale E0366, University Hospital, Boulevard Pasteur, 42055 Saint-Etienne Cedex2, France. E-mail: thierry.thomas{at}univ-st-etienne.fr.
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Abstract |
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B-ligand (RANKL)/osteoprotegerin (OPG) pathway since we observed using direct RT-PCR, a suspension-induced increase in RANKL gene expression in proximal tibia at d 3, which was counterbalanced by leptin administration with a similar 3-fold increase in OPG expression and a RANKL to OPG ratio close to nonsuspended conditions. In addition, leptin prevented the decrease in bone formation rate induced by tail-suspension at d 14. The latter could be related to the role of leptin in mediating the reciprocal differentiation between adipocytes and osteoblasts, because leptin concurrently blunted the disuse-induced increase in bone marrow adipogenesis. In summary, these data suggest that peripheral administration of leptin could prevent disuse-induced bone loss through, first, a major inhibitory effect on bone resorption and, second, a delayed effect preventing the decrease in bone formation. |
Introduction |
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Thomas et al. (3), using a human stromal cell line, and others (4, 5) demonstrated that the cells of the osteoblastic lineage also were targets for leptin action because they expressed both short and long forms of leptin receptors, with the ability to transduce cell signaling as shown by signal transducer and activator of transcription 3 phosphorylation after activation (6). In vitro studies showed that leptin induced both MAPK-dependent cell proliferation of C3H10T1/2 cell line, a multipotential stromal murine cell line (7), as well as a more mature osteoblastic cell line (5) and an increase in osteoblastic differentiation of human marrow stromal cells, hMS2–12, leading to an increase in the mineralization of the extracellular matrix (3). Similar results were observed in other cell models (4, 8, 9). Actually, leptin could be one of several factors that modulate the reciprocal differentiation of stromal cells between osteoblastic and adipocytic pathways, because it also has the ability to inhibit adipogenesis in a negative feedback loop (3, 10).
Similarly, in vivo studies in ob/ob mice deficient in leptin demonstrated a stimulatory effect of ip leptin administration on bone, with a dramatic increase in cortical bone formation (11) and the reverse of defective bone growth and osteopenia, compared with their controls, despite a 40% decrease in food intake and a 14% decrease in body weight (12). Furthermore, systemic daily administration of leptin to sexually mature male mice significantly increased bone strength by more than 20% (5).
In addition to direct positive effects on the osteoblastic differentiation of stromal cells, leptin may also modulate bone remodeling. Indeed, it has been shown recently in human stromal cells that leptin inhibited the expression of receptor activator of nuclear factor B ligand (RANKL), the major downstream cytokine controlling osteoclastogenesis (13). Indeed, the interaction between receptor activator of nuclear factor B (RANK) expressed by preosteoclastic cells and RANKL expressed by stromal and osteoblastic cells, is considered as the central pathway in the control of osteoclastogenesis, promoting differentiation, stimulating cell activity, and reducing osteoclast apoptosis, all events leading to increased bone resorption. Osteoprotegerin (OPG), also expressed by stromal and osteoblastic cells in the bone environment, is a soluble decoy receptor for RANKL that blocks osteoclast formation by preventing RANKL to bind to RANK. It has also been demonstrated that leptin stimulated the expression of OPG by stromal (13) and mononuclear cells (14). These data are consistent with the partial prevention of ovariectomy-induced bone loss in rats treated with continuous sc administration of leptin (13).
Overall, these data strongly suggest that leptin is able to directly alter bone remodeling by modulating both osteoblast and osteoclast activities. Therefore, we proposed the hypothesis that peripheral administration of leptin may prevent disuse-induced bone loss through uncoupled effects on both resorption and formation activities. To test this hypothesis, we took advantage of the well-defined tail-suspended rat model, characterized by a rapid disuse-induced bone loss related to a specific pattern of bone cell activities, namely a transient increase in bone resorption followed by a sustained decrease in bone formation (15, 16).
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Materials and Methods |
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Animals
This animal interventional study was in accordance with the Declaration of Helsinki principles and was approved by the authors institutional review board (Ministère de l’Agriculture, France; authorization number 04827). One hundred and thirty 12-wk-old female Wistar rats (Iffa Credo, L’Arbresle, France), 240 ± 10 g mean body weight, were acclimatized for 1 wk with standard conditions of temperature (23 ± 1 C) and light/dark (11 h/13 h). Animals were individually housed and provided with food (standard diet with UAR A03) and water ad libitum. The rats were randomly assigned in 13 groups of 10 animals each, depending on whether they were tail-suspended (S) or nonsuspended (NS), and treated by human recombinant leptin [(+)L] or its vehicle [(–)L], for 3, 7, or 14 d. A control group was killed at baseline (B). The suspension procedure was performed according to recommendations by Wronski and Morey-Holton (17). Leptin was continuously administered using ip osmotic pumps (Alzet) at 0.35 mg/kg·d, a dose previously shown to alter bone metabolism without significantly modifying body weight (13). Fluorochrome double bone labeling was performed 4 and 1 d before the animals were killed, with 15 mg/kg tetracycline ip injections. Baseline rats were not labeled with respect to acclimatization period. Animals were killed with a high dose of nesdonal (Specia, Paris, France).
Serum leptin and corticosterone measurement
Blood was collected in heparinized tubes for measurement of serum leptin and corticosterone levels. For leptin assay, the quantikine mouse leptin ELISA immunoassay (R&D Systems, Minneapolis, MN) was used because it exhibits 80% cross-reactivity with human leptin. For corticosterone measurement, the OCTEIA corticosterone enzyme immunoassay (ImmunoDiagnostic Systems, Bolton, UK) was used.
Dual energy x-ray absorptiometry (DXA)
A dual energy x-ray PIXImus densitometer (LUNAR Corp., Madison, WI) with specific software for small animals was used for measuring bone mineral density (BMD). Before measurement was performed on d 0, 7, and 14, rats were anesthetized with ip administration of 0.1–0.3 mg/kg ketamine-xylasine solution. The tibia metaphyseal region of interest consisted of a 8.55 x 6.84 mm rectangle positioned over the longitudinal axis of the proximal tibia, which corresponds to the most responsive bone site to unloading induced by the suspension.
Histomorphometry
After the animals were killed, the left tibiae were immediately excised, fixed, dehydrated in absolute acetone, and embedded in methylmetacrylate at low temperature. Longitudinal frontal slices were cut from the embedded bones with a microtome (Reichert-Jung, Polycut-S). Six nonserial sections, 8 µm thick, were used for modified Goldner staining. Twelve-micrometer-thick sections were used to determine the dynamic indices of bone formation [dLS/BS (double-labeled mineralizing surfaces), MAR (mineral apposition rate), BFR/BS (bone formation rate)]. MAR was derived from fluorochrome interlabel distances. BFR were subsequently obtained from the product of dLS/BS and MAR. Six-micrometer-thick sections were used for tartrate-resistant acid phosphatase staining allowing determination of the osteoclastic parameters. For each section, data were collected in 2.6 mm height region of interest (ROI) within the secondary spongiosa. Bone volume and parameters reflecting trabecular structure were measured using an automatic image analysis system (Biocom, Lyon, France). Bone cellular and macroscopic parameters were measured with a semiautomatic device including a digitizing tablet (Summasketch; Summagraphics, Paris, France) connected to a MacIntosh microcomputer (Apple Computers, Cupertino, CA) with software designed in our laboratory. In addition, relative volume of fat in the marrow cavity (Ad.V/MV) as well as adipocyte number, was measured on Goldner staining sections using a manual counter and a hundred-point grid (coefficient of variation = 6.6%).
RNA extraction and RT
RNA extraction was performed on four animals per group at three different time points: 3, 7, and 14 d of suspension. Total RNA was extracted from the right proximal tibia according to the method of Chomczynski and Sacchi. Bones were ground and then treated by guanidium isothiocyanate solution. The purity of mRNA prepared was monitored by the ratio of absorbance at 260 and 280 nm, which was generally between 1.7 and 2. Integrity of RNA was checked by loading 1 µg/lane on 1% agarose-1x Tris-acetate EDTA gel, separated by electrophoresis, and after ethidium bromide staining. RNAs were reverse-transcribed into single-stranded cDNA using a first strand cDNA synthesis kit for RT-PCR (AMV) from 2 µg total RNA in a 20-µl reaction mix containing 2 µl of 10x reaction, 5 mM MgCl2, 20 mmol each of dNTP, 50 pmol oligo-p(dT)15 primer, 50 U RNase inhibitor, and 20 U AMV reverse transcriptase. The reaction was incubated 60 min at 42 C.
Real-time PCR
Fluorescent signals are proportional to the concentration of the product and can be measured once per cycle and immediately displayed on a computer screen, permitting real-time monitoring of the PCR. After amplification, the temperature is slowly elevated above the melting temperature of the PCR product allowing monitoring of the melting curve. This enables identification of specific transcripts, because specific and nonspecific products have different melting temperatures, depending on their nucleotide composition. This temperature was 82.5 C for OPG, 84.2 C for RANKL, 90.1 C for osteocalcin (OC), 90.8 C for type I collagen (Col I), 91.4 C for glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
The single-strand cDNA was diluted 1:20, and 8 µl were amplified in 20 µl PCR mixture containing 2 µl of Light Cycler-FastStart DNA Master SYBR Green I, 3 mM MgCl2, 0.5 µM of 5' and 3' oligo primers and water. Amplifications were performed in a Light Cycler Instrument for the following cDNAs: GAPDH, Col I, OC, OPG, and RANKL. A typical protocol took approximately 60 min to complete and included a denaturation step at 95 C for 480 sec followed by 30 cycles with 95 C for 15 sec, melting temperature annealing for 10 sec, and 72 C extension for 18 sec. Detection of the fluorescence product was performed at the end of the 72 C extension period by a 95 C step for 0 sec, a 65 C step for 15 sec, and a final 95 C denaturation for 0 sec. Because GAPDH gene is not altered by suspension, the housekeeping gene GAPDH was also amplified as a control.
The sequence of primers were: GAPDH forward 5'-ACCACAGTCCATGCCATCAC, reverse 5'-TCCACCACCCTGTTGCTGTA (452 bp); Col I forward 5'-CAACAAATCCCCACACAC, reverse 5'-CACACAAAGACAAGAACGAG (197 bp); OC forward 5'-ATGAGGACCCTCTCTCTGCTC, reverse 5'-GTGGTGCCATAGATGCGCTTG (292 bp); OPG forward 5'-CCTCTTTCTTTCTGCCTCTGATAGTC, reverse 5'-CCAAGTCTGCAACTCGAATCAAAT (149 bp); RANKL forward 5'-GGTGAGGAAATTAGCGTCCA, reverse 5'-TCGAGAGAGGACCGTGAGTT (233 bp).
Quantified data were analyzed with the Light Cycler analysis software. Results were analyzed following the manufacturer’s instructions: 1) checking the PCR products specificity and 2) calculating the variation in PCR products concentration between experimental groups, expressed as percentage of NS(–)L group mean values.
Statistical analysis
The statistical analyses were performed using commercially available statistical software (STATISTICA; StatSoft Inc., Tulsa, OK). Differences between NS(+)L and S(–)L groups were not tested, because of their irrelevance to our working hypothesis. For densitometry and body composition data, differences between groups were analyzed initially by three-way ANOVA, with two between-groups factor (tail-suspended or not, treated or not) and a repeated measures factor (within subjects factor). When F values for a given variable were found to be significant, the post hoc Scheffé test was subsequently used. Results were considered significantly different at P < 0.05.
Three-way ANOVA were performed on histomorphometric data, to determine the influence of both suspension and treatment factors on the structural and cellular parameters. When F values for a given variable were found to be significant, post hoc Scheffé test were performed, and the results were considered to be significantly different at P < 0.05. According to the lower sampling of RNA extraction, nonparametric Mann-Whitney U tests were performed on molecular biology data. Results were considered to be significantly different at P < 0.05.
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Results |
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). Of note, all values remained in the physiological range.
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Leptin prevented suspension-induced bone loss measured at the tibia metaphysis
The S(–)L group had a 6.4% significant decrease in tibia-metaphysis BMD at d 7 compared with the NS(–)L group killed after 1 wk (P < 0.01). In the 2-wk experiment, a similar and time-dependent difference in tibia-metaphysis BMD was observed between S(–)L and NS(–)L groups, with a –7.3% and –12.5% decrease at d 7 and 14, respectively (P < 0.01) (Fig. 1 and Table 2). Leptin treatment prevented suspension-induced bone loss at each time point as no significant difference was observed when comparing S(+)L to NS groups. No difference was observed between NS groups, regardless of leptin administration.
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) with a trend in the same direction when comparing structural parameters in S(–)L and S(+)L groups. In addition, a decrease in trabecular bone volume was observed at d 14 in the sole S(–)L group compared with NS(–)L group (–41%, P < 0.01). As previously reported (15, 16), this decrease in trabecular bone mass with suspension was related to a decrease in both Tb.Th and Tb.N (–23% and –26% compared with NS(–)L group, respectively; P < 0.05). No change was observed at d 7 yet. Again, leptin treatment prevented these changes with no significant difference observed between S(+)L and NS(–)L or NS(+)L groups (Fig. 2 and Table 3).
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). Interestingly, we noted only at d 7, a 24% increase in BFR/BS in NS(+)L group compared with NS(–)L (0.49 ± 0.06 vs. 0.40 ± 0.14, respectively; P < 0.05), as a transient positive effect of leptin administration on bone formation (Table 3).
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), suggesting that most of the effects of both disuse and leptin treatment were secondary to variations in osteoblast recruitment rather than cell activity. This hypothesis was supported by real-time PCR data evaluating Col I and OC gene expression in osteoblasts from proximal tibia area. Indeed, no significant difference was observed both at d 7 and 14 when comparing S(–)L to S(+)L or NS(–)L groups (data not shown).
Leptin exerted both direct and indirect positive effects on osteoblast lineage
We previously showed that leptin was able to modulate in vitro the reciprocal differentiation of stromal cells toward osteoblast and adipocyte lineages (3). On the other hand, it has been shown that tail suspension could enhance adipocyte differentiation in 4-wk-old rats (18). Therefore, we next evaluated whether leptin administration could impair adipocyte lineage in the bone marrow of tibial diaphysis, concurrently to its positive effects on osteoblast lineage. We first confirmed that tail suspension progressively induced an increase in Ad.V/MV in S(–)L group compared with NS(–)L group, with a nonsignificant trend at d 7 (+30%, NS) followed by a 196% increase at d 14 (P < 0.05). We then demonstrated that this increase was blunted by leptin administration in S(+)L group compared with S(–)L group (3.1 ± 2.0% vs. 4.9 ± 2.1%, respectively, P < 0.05), even though Ad.V/MV remained higher than in NS(+)L group (1.7 ± 0.7%, P < 0.05) (Fig. 4). These changes were mainly related to a significant increase in adipocyte number in the S(–)L group compared with both S(+)L and NS(–)L groups at d 14 (99.8 ± 39.7 mm–2 vs. 36.5 ± 29.3 mm–2 and 34.1 ± 15.1 mm–2, respectively, P < 0.05).
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Leptin inhibited the increase in bone resorption and improved OPG/RANKL balance
Based on our data, leptin effects on bone formation could at least in part explain its prevention of disuse-induced bone loss at d 14 but not at d 7. The pattern of bone cellular activities in tail-suspended rat model is characterized by an early and transient increase in bone resorption (15, 16). In fact, we observed at d 7 a dramatic 51% increase in osteoclast number per bone area in S(–)L group compared with NS(–)L group (P < 0.01) (Fig. 5A). Leptin administration in suspended group was able to completely prevent this increase (P < 0.01) (Fig. 5, A and B). No difference among the four groups was observed at d 14.
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; P < 0.05). However, leptin administration in the suspended group concurrently induced a similar increase in OPG expression (P < 0.05) (Fig. 6B), which counterbalanced the increased RANKL expression. Therefore, when looking at RANKL/OPG, we observed at d 3, a much higher ratio in the S(–)L group than in the S(+)L group (2.12 vs. 1.12, respectively, P < 0.05), the latter being not significantly different from the RANKL to OPG ratio in NS(–)L group (Fig. 6C). These changes were transient because RANKL to OPG ratios were then comparable among groups at d 7 and 14.
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Discussion |
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Although several in vitro studies have yielded consistent data on the direct effects of leptin on bone cells, very little information was drawn from in vivo studies. Thanks to the specific pattern of cellular activities changes induced by tail-suspension, we thought this model would be useful to better understand the effects of systemic leptin administration. Indeed, we observed after 2 wk of suspension that leptin was able to prevent the decrease in bone formation rate induced by suspension. Both histomorphometry and mRNA expression evaluation suggest that these effects were more related to an increase in active osteoblast number than to changes in cell activity, as reflected by an increase in mineralizing surfaces and no difference in osteoblast markers expression such as Col I and OC. In addition, we demonstrated that leptin also partially prevented the progressive and concurrent increase in the relative volume of fat in the marrow cavity induced by suspension. These differences were mainly related to changes in adipocyte number. Therefore, these results suggest that leptin modulation of the reciprocal differentiation of stromal cells between adipocyte and osteoblast lineages could, at least in part, explain the positive effects of leptin on bone formation. In this regard, the leptin-induced activation of the MAPK cascade may be critical because this biochemical event stimulates both osteoblastic differentiation (23) and phosphorylation of PPAR
, demonstrated as inhibitory for adipogenesis (24). Eventually, this reciprocal differentiation of stromal cells toward the osteoblastic and adipocytic lineages could serve as a paracrine and regulatory mechanism within the bone marrow because marrow adipocytes produce leptin (25).
Intriguingly, leptin effects on bone were essentially observed when bone formation was impaired by disuse condition. An early increase in bone formation rate was also observed in the NS(+)L group but it was not sufficient or long enough to increase morphometric parameters. A similar lack of effect has been previously reported in other animal models when bone metabolism and leptin secretion were normal (11, 13). The low dose of leptin we used with no decrease in body weight measured over 2 wk, as expected (13, 26), could be an issue, and we cannot rule out that a higher dose of leptin would alter bone metabolism, but certainly with the putative bias of decreased food intake and weight loss. Actually, the decrease in leptin secretion observed with suspension might have unmasked the effects of leptin, which may not appear in a normal situation. Likewise, Goldstone reported that leptin was able to prevent the fall in plasma OC, a marker of bone formation, during starvation in male mice (27). The decrease in leptin observed with this animal model of disuse is also of great interest in the field of microgravity-related disuse because a similar decrease has been reported during space flight in humans (28).
In addition to its direct effects on osteoblast lineage cells, leptin could modulate formation activity by altering hormonal or cytokine changes induced by tail-suspension. In fact, we observed a more rapid decline in corticosterone level, as soon as d 3 in the groups treated with leptin as compared with vehicle administration. It has been largely demonstrated that leptin controls the glucocorticosteroid secretion by the hypothalamo-adrenal axis (29, 30, 31), including in response to stress conditions (32), suggesting a role for leptin as an anti-stress hormone (33). The lack of decrease in weight we noted in the S(+)L group at d 3 may be then related to this mechanism. However, the impact of leptin administration on bone formation in S(+)L group was only observed at d 14, which suggests that long-term mechanisms such as increase in osteoblastic surfaces were more prominent than these early hormonal alterations.
Because no change in bone formation occurred under leptin during the first week of suspension, we postulated that leptin prevention of disuse-induced bone loss already observed at d 7 was related to modulation of osteoclast activity. We measured a dramatic increase in osteoclast number at d 7 as assessed by specific tartrate-positive histoenzymatic technique (34). Again, this burst in resorption was only transient as no difference was measured at d 14, in accordance with previous studies (15, 16). Leptin administration was able to completely prevent this disuse-induced increase in osteoclast number. This observation is in accordance with several human studies, which demonstrated negative correlations between serum leptin levels and bone resorption markers (35, 36, 37). Because the RANKL/OPG pathway is the main and central regulatory system for osteoclastogenesis and because previous studies demonstrated that leptin in vitro could modulate its activity (13, 14), we then evaluated the mRNA expression of these two proteins in the different conditions. In fact, we observed an increase in RANKL expression with suspension at d 3 before the increase in osteoclast number observed later on. However, leptin administration in suspended animals also stimulated OPG expression in a similar magnitude, leaving the RANKL to OPG ratio close to those of nonsuspended conditions. This is of great importance because it has been shown that it is definitely the ratio between these two factors produced by the osteoblast lineage, which eventually determines the effects of different stimuli on osteoclast differentiation and activity (38). Hence, these data suggest a direct causal relationship between the two phenomena. Thereafter, no difference in RANKL to OPG ratios was observed among the different conditions, in accordance with the transient feature of this alteration in bone resorption. Of note, other mechanisms could participate in these effects of leptin on bone resorption because leptin has the ability to induce the expression and secretion of the IL-1 receptor antagonist by human monocytes (39) and to blunt the inflammatory cytokine responses, including TNF-
and IL-6 (40), all factors involved in the regulation of osteoclastogenesis and potential mediators of suspension-induced bone loss (41).
Because several lines of evidences suggest that leptin exerts stimulatory effects on skeletal growth and endochondral ossification (12, 42, 43, 44) as well as on growth hormone axes (45), we selected rats with a growth curve close to plateau, to limit such potential effects of leptin through bone growth. Actually, neither suspension nor leptin had effect on both bone area measured by DXA and bone length (data not shown). Likewise, we observed no difference in cortical thickness. The latter is in opposition to the recent study from Hamrick et al. (46) showing cortical thinning in ob/ob mice compared with lean littermates. This difference between the two studies could be related to the fact that the ob/ob mice obviously lacked leptin over their entire period of rapid growth. Therefore, these data together with previous literature (11, 12, 47) suggest that leptin effects on bone modeling in the cortical envelope are important during bone growth. In more mature bone, it appears to mainly modulate bone remodeling in the trabecular envelope as most of the endocrine factors affecting bone metabolism. This is of great interest because the variety of leptin effects on bone depending on bone envelope, growth status, and bone sites may explain the heterogeneity of the literature published so far, as proposed by Reid et al. (48).
In summary, we demonstrated that peripheral administration of leptin was able to prevent disuse-induced bone loss over 2 wk in tail-suspended female rats. These effects were a combination of the prevention of both the transient and early increase in bone resorption and the late and sustained decrease in bone formation. Whereas the former may be related to leptin modulation of the RANKL/OPG pathway, the latter could be associated, at least in part, with the effects of leptin on the reciprocal differentiation between adipocytes and osteoblasts. Further studies are now needed to clarify the putative dual leptin effects on bone depending on its central brain-mediated or on its direct peripheral effects.
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Acknowledgments |
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Footnotes |
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Conflict of interest: The authors have declared that no conflict of interest exists.
First Published Online April 21, 2005
Abbreviations: AMV, Avian myeloblastosis virus; B, baseline group; BFR/BS, bone formation rate; BMD, bone mineral density; Col I, type I collagen; dLS/BS, double-labeled mineralizing surfaces; DXA, dual energy x-ray absorptiometry; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; (+)L, leptin-treated group; (–)L, vehicle-treated group; MAR, mineral apposition rate; NS, nonsuspended group; OC, osteocalcin; OPG, osteoprotegerin; RANK, receptor activator of nuclear factor B; RANKL, RANK ligand; ROI, region of interest; S, tail-suspended group; TRAcP, tartrate-resistant acid phosphatase.
Received November 22, 2004.
Accepted for publication April 14, 2005.
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), NS(+)L (
), S(–)L (
), and S(+)L (
) groups, respectively]. Note the progressive decrease in BMD observed in the S(–)L group only. Values represent the percent of changes compared with baseline values and are mean ± SEM of 10 female rats per group. Comparisons were performed using three-way ANOVA and post hoc Scheffé test. *, P < 0.01 vs. baseline; **, P < 0.01.
