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Bone metabolism, osteoprotegerin, receptor activator of nuclear factor-κB ligand and selected adipose tissue hormones in girls with anorexia nervosa
Metabolizm kostny, osteoprotegeryna i ligand receptora aktywatora czynnika jądrowego-κB a wybrane hormony tkanki tłuszczowej u dziewcząt z jadłowstrętem psychicznym
1Clinical Biochemistry Division, Department of Biochemistry, Medical University of Silesia, Zabrze, Poland
2Department of Paediatrics, Medical University of Silesia, Zabrze, Poland
3Department of Pathophysiology and Endocrinology, Medical University of Silesia, Zabrze, Poland
Zofia Ostrowska M.D., Clinical Biochemistry Division, Department of Biochemistry, Medical University of Silesia, Jordana St. 19, 41–808 Zabrze, Poland, e-mail: email@example.com
Introduction: The aim of this study was to determine whether girls with anorexia nervosa (AN) exhibited any relationships between serum levels of LP, ADIPO, RES, VISF, APE-36, APE-12, and bone markers, OPG and sRANKL.
Material and methods: Serum levels of selected adipose tissue hormones, OC, CTx, OPG and sRANKL were assessed using ELISA in 86 study participants suffering from AN and 21 healthy controls, all aged 13 to 18 years.
Results: Girls with AN showed a significant reduction in body mass, BMI, serum concentrations of LP, RES, VISF, APE-36, APE-12, OC, CTx and increased ADIPO concentration. These changes were associated with significant increases in OPG and sRANKL and a decrease in the OPG/sRANKL ratio. Significant positive correlations were revealed between BMI and LP, APE-36, CTx, OPG/sRANKL ratio; OC and VISF; OPG and ADIPO; OPG/sRANKL ratio and LP, APE-36, APE-12. Significant negative correlations were revealed between CTx, sRANKL and RES, APE-36; OPG and APE-36, APE-12; OPG/sRANKL ratio and ADIPO. VISF was shown to be an independent predictor of OC. APE-36 and RES turned out to be independent predictors of CTx, and sRANKL, APE-36 and ADIPO were independent predictors of OPG while APE-36, LP and ADIPO were independent predictors of the OPG/sRANKL ratio.
Conclusions: Changes in bone markers, OPG, sRANKL and/or the OPG/sRANKL ratio exhibited by girls with AN have been found to be associated with changes in the levels of the selected adipose tissue hormones. Abnormal relationships between bone metabolism and LP, ADIPO, RES, VISF and APE might adversely affect the balance of the OPG/sRANKL system and thus potentially compromise the mechanism which compensates for bone remodelling disturbances. (Endokrynol Pol 2014; 65 (1): 33–39)
Key words: anorexia nervosa; girls; adipose tissue hormones; bone metabolism; OPG; sRANKL
Wstęp: Celem pracy było wykazanie, czy u dziewcząt z jadłowstrętem psychicznym istnieje związek między LP, ADIPO, RES, VISF, APE-36 i APE-12 a markerami kostnymi, OPG i sRANKL.
Materiał i metody: U 86 dziewcząt z AN i 21 zdrowych w wieku 13–18 lat oceniono stężenia wybranych hormonów tkanki tłuszczowej, OC, CTx, OPG i sRANKL w surowicy metodą ELISA.
Wyniki: U dziewcząt z AN wykazano istotne zmniejszenie masy ciała i BMI, obniżenie stężeń LP, RES, VISF, APE-36, APE-12 i markerów kostnych (OC i CTx) oraz wzrost stężenia ADIPO. Zmianom tym towarzyszył istotny wzrost stężeń OPG i sRANKL przy obniżonym wskaźniku OPG/sRANKL. Wykazano znamienną dodatnią korelację między BMI a LP, APE-36, CTx i wskaźnikiem OPG/sRANKL; OC a VISF; OPG a ADIPO; wskaźnikiem OPG/sRANKL a LP, APE-36, APE-12. Istotną, ujemną korelację stwierdzono między CTx, sRANKL a RES i APE-36; OPG a APE-36 i APE-12; wskaźnikiem OPG/sRANKL a ADIPO. Wykazano, że niezależnymi predykatorami są: dla OC – VISF, dla CTx i sRANKL – APE-36 i RES, dla OPG – APE-36 i ADIPO, a dla wskaźnika OPG/sRANKL – APE-36, LP i ADIPO.
Wnioski: Obserwowanym u dziewcząt z AN zmianom w stężeniach markerów kostnych, OPG, sRANKL i/lub wskaźniku OPG/sRANKL towarzyszą zmiany w stężeniach badanych hormonów tkanki tłuszczowej. Nieprawidłowości w powiązaniach między metabolizmem kostnym a LP, ADIPO, RES, VISF oraz APE u dziewcząt z AN mogą prowadzić do naruszenia równowagi układu OPG/sRANKL, co może skutkować upośledzeniem mechanizmu kompensującego zaburzenia w przebudowie kośćca. (Endokrynol Pol 2014; 65 (1): 33–39)
Słowa kluczowe: jadłowstręt psychiczny; dziewczęta; hormony tkanki tłuszczowej; metabolizm kostny; OPG; sRANKL
Anorexia nervosa (AN) is an increasingly common eating disorder, a psychosomatic disease characterised by the pursuit of a ‘perfect’ silhouette through strictly limiting food intake, excessive exercise, self-provoked vomiting or engagement in other purging behaviours including the use of laxatives or diuretics. The disease leads to significant reduction of adipose tissue mass resulting in drastic slimness or even physical wasting (cachexia) [1, 2]. Patients with AN not only exhibit somatic and psychic complications resulting from dramatic weight loss but also, quite frequently, imbalance of hormones – also adipose tissue hormones [3–22].
It has been widely acknowledged that white adipose tissue, both subcutaneous and visceral, serves as a reservoir of several active peptides, commonly referred to as adipokines, which have both local (autocrine and paracrine) and systemic (endocrine) actions . It has been documented that adipokines are important regulators of appetite and numerous metabolic, endocrine and immune functions of the organism. Several adipokines are mainly produced in subcutaneous white adipose tissue (leptin – LP, adiponectin – ADIPO) while others (resistin – RES, visfatin –VISF) are predominantly secreted in the visceral adipose tissue. The least known of these hormones, apelin (APE), is expressed not only by adipocytes but has also been identified in vascular endothelium and the cells of several other tissues (lung, heart, brain) .
Osteoporosis is among the most commonly observed complications of AN. Osteoporosis has a multifactorial aetiology. Disturbances in the production, release and action of osteotropic agents, mainly hormones (including adipose tissue hormones, i.e. LP and maybe also ADIPO, RES, VISF and APE), might lead to bone mineral density (BMD) decrease or, alternatively, absence of the BMD increase expected in adolescence [9, 10, 23] resulting from excessive bone resorption and inadequate formation of new bone. The system of receptor activator of nuclear factor-κB ligand/receptor activator of nuclear factor-κB/osteoprotegerin (RANKL/RANK/OPG) might play an important role in this process [24, 25]. According to a convergence hypothesis, the effect of several local and systemic agents on bone resorption consists of the regulation of OPG and/or RANKL expression [24, 25].
In vitro studies indicate that LP stimulates rat bone marrow stromal cells, thus enhancing differentiation to osteoblasts. It also causes an increase in the expression of bone matrix proteins mRNA; the effect depending on the dose and time of administration. Leptin enhances bone mineralisation [26–28] and inhibits osteoclast generation in cultures of murine bone marrow stromal cells [26–28], but does not affect mature osteoclast differentiation . It also increases OPG expression in stromal cells and osteoblasts [29, 30] and/or decreases the expression of RANKL [27, 30, 31]. Leptin acts to enhance in vitro differentiation of human bone marrow stem cells to osteoblasts, stimulates de novo collagen synthesis and bone matrix mineralisation [32–34] and inhibits adipogenesis . It also inhibits osteclastogenesis in cultures of human peripheral blood mononuclear cells and spleen cells relative to OPG increase and RANKL decrease [32, 35]. Kanazawa et al.  demonstrated that ADIPO and AMP kinase activator stimulated the proliferation, differentiation, and mineralisation of MC3T3-E1 cells, while Luo et al. [37, 38] concluded that ADIPO stimulated human osteoblasts proliferation and differentiation and inhibited OPG expression in human osteoblasts via the MAPK signalling pathway. Thommesen et al.  found enhanced RES expression in murine osteoclast and osteoblasts precursor cells (RAW 264.7, MC3T3-E1) and in primary human bone marrow stem cells. The expression of resistin mRNA in RAW 264.7, increased during differentiation, seemed to be regulated through PKCand PKA-dependent mechanisms. Recombinant resistin increased the number of differentiated osteoclasts and stimulated NF-κB promoter activity, indicating a significant role of this adipokine in osteoclastogenesis. Resistin also enhanced the proliferation of MC3T3-E1 cells in a PKA and PKCdependent manner, but only weakly interfered with genes known to be upregulated during differentiation of MC3T3-E1 into osteoblasts .
Other investigations have shown that VISF increases type I collagen production in human osteoblast cultures  while APE stimulates proliferation and suppresses apoptosis of mouse osteoblastic cell line MC3T3-E1 . The latter also has a protective effect on cultured rat bone marrow mesenchymal stem cells against apoptosis . All these findings seem to suggest that the above mentioned adipokines might regulate bone remodelling, most probably through the cytokines of the RANKL/RANK/OPG system, mainly produced in osteoblasts and bone marrow stromal cells [24, 25, 27, 30–32, 35, 37–39].
Several investigations involving healthy men and women have demonstrated a negative correlation between osteocalcin (OC), OPG and LP concentrations as well as between RANKL and ADIPO levels. OC concentrations were also positively correlated with ADIPO [43–46]. OPG concentrations were higher in a premenopausal than in a postmenopausal population [47–49]. The OPG/RANKL ratio correlated negatively with LP and positively with ADIPO independent of age, BMI and the time elapsed from menopause . Investigations of relationships between serum LP, ADIPO, RES, VISF, APE and BMD have revealed that only ADIPO was an independent BMD predictor in postmenopausal women and middle-aged men [47–50]. Other authors found that both LP and ADIPO were determinants of BMD; RES was negatively correlated with BMD, but the correlation did not reach statistical significance (p = 0.062) .
Little data is available on the relationship between adipokines and bone status in patients with AN, and especially girls [3–10, 11, 23, 52–58]. The literature reports available are mainly concerned with the relation between LP, ADIPO and bone metabolism and/or BMD [8–10, 12, 23, 53, 54, 56–59]. Girls with AN exhibit considerable suppression of bone metabolism markers, i.e. bone-specific alkaline phosphatase, OC, C-terminal cross-linked telopeptide of type I collagen α chain (CTx), N-terminal cross-linked telopeptide of type-I collagen type I collagen α chain (NTx) and/or deoxypyridinoline [9, 10, 21–23, 60, 61] correlating with a decrease in BMD and LP levels [9, 10, 23]. Several authors have determined OPG concentrations in girls with AN and found that, similar to young females with AN, the girls had elevated serum levels of this cytokine [4, 8, 21, 22, 61]. Our earlier investigations revealed that serum sRANKL was increased while the OPG/sRANKL ratio was significantly decreased [21, 22, 61]. Similar results were obtained by Munoz-Calvo et al. . However, no complex research studies have been carried out so far in girls with AN concerning the relationships between LP, ADIPO, RES, VISF, APE-36, APE-12 and bone metabolism, OPG and its soluble ligand sRANKL.
Therefore, we decided to thoroughly investigate these relationships. Based on the scarce literature data available, we hypothesised that a BMD decrease or, alternatively, absence of the BMD increase expected in adolescence, might not only be caused by several already well-established factors, but also by the effect of changes in the concentrations of the above mentioned adipokines (or some of them) on the balance of the OPG/sRANKL system.
The aim of this study was: 1) to determine whether girls with AN exhibited any relationships between LP, ADIPO, RES, VISF, APE-36, APE-12 and bone metabolism (as assessed based on serum OC and CTx levels); and 2) to establish whether OPG and its soluble ligand sRANKL might affect such relationships.
Material and methods
The study involved 86 girls aged 13 to 18 years, hospitalised at the Paediatric Endocrinology Division of the Paediatric Department in Zabrze Medical University of Silesia in Katowice (Poland), who, following paediatric examination and psychiatric consultation, had been diagnosed with anorexia nervosa according to Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) published by the American Psychiatric Association in 1994. The average disease duration was 12.1 months (range 3–36 months). All examined girls were at Tanner puberty stages IV and V. They had normal liver and kidney functions; no severe somatic complications or psychiatric disorders were observed. On recruitment, no patients were taking medications known to affect the nutritional and bone status including calcium or vitamin D supplements. During hospitalisation, patients were placed at bed rest, which is the standard care. The control group comprised 21 age-matched healthy regularly menstruating adolescent females with no endocrine or other disorders that might possibly influence adipose and bone tissue metabolism. During the threemonth period before the study, the control participants did not take calcium or vitamin D supplements.
The height (stadiometer) and body mass (electronic scale) of all participants were measured, and their body mass index (BMI) calculated. Blood samples (8 mL) for determination of LP, ADIPO, RES, VISF, APE-36, APE-12, OC and CTx as well as OPG and sRANKL were collected between 08.00 and 09.00 hours following a 12-hour fast. Centrifuged serum was frozen and stored at –75°C until assayed for the above mentioned adipose tissue hormones, bone markers, OPG and sRANKL.
Determinations of the concentrations of adipose tissue hormones, OC, CTx, OPG and sRANKL were performed by ELISA using the following kits: LP and ADIPO (Bio-Vendor, LLC, USA), RES (Mediagnost, Germany), VISF, APE-36 and APE-12 (Phoenix Pharmaceuticals Inc., Burlingame, CA, USA), OC (DSL Inc., USA), CTx (Nornic Bioscience Diagnostics A/S, Denmark), OPG and sRANKL (Biomedica, Austria). The respective sensitivity, intraand inter-assay errors were: 0.5 ng/mL, 7.5 and 9.2% for LP; 0.7 ng/mL, 7 and 8.2% for ADIPO; 0.3 ng/mL, 5 and 6.8% for RES; 0.55 ng/mL, 5 and 14% for VISF; 0.09 ng/mL, 5 and 14% for APE-36; 0.07 ng/mL, 5 and 14% for APE-12; 0.05 µmol/L, 5.8 and 7.3% for OC; 0.08 nmol/L, 5.2 and 6.7% for CTx; 0.14 pmol/L, 7 and 7.5% for OPG; 0.04 pmol/L, 5 and 7% for sRANKL.
The database was prepared using Excel 2000 (Microsoft Corporation). Statistical analysis was carried out with Statistica 10 for Windows (StatSoft Inc., USA). The t-test was used to determine the significance of intergroup differences (normal distribution of variables). In the case of non-normal distribution, the significance was tested using the Mann-Whitney U test. The relationships between LP, ADIPO, RES, VISF, APE-36, APE-12, OC, CTx, OPG, sRANKL and the OPG/sRANKL ratio were analysed by Spearman’s correlation. The level of significance was set at p £ 0.05.
Stepwise regression was used to determine whether any, and if so which, of the adipose tissue hormones under investigation were independent predictors of bone markers (OC and CTx), cytokines of the RANKL/RANK/OPG system and the OPG/sRANKL ratio (model entry was set at p = 0.05, and model exit at p = 0.05).
The study was approved by the Regional Bioethics Committee of the Medical University of Silesia in Katowice (KNW/0022/KB1/105/09).
Our female adolescents with AN showed a significant reduction in body mass and BMI compared to the control group. A significant decrease in mean serum LP, RES, VISF, APE-36 and APE-12 concentrations, and a significant increase in mean serum ADIPO, were also found. These changes were associated with a significant decrease in mean OC and CTx levels, marked increases in OPG and sRANKL, and a significant decrease in the OPG/sRANKL ratio compared to the controls (Table I).
Table I. Mean values of age, height, body mass, body mass index (BMI), mean serum levels of chosen adipose tissue hormones, osteocalcin (OC), collagen type I crosslinked carboxyterminal telopeptide (CTx), osteoprotegerin (OPG), soluble receptor activator of nuclear factor-κB ligand (sRANKL) and OPG/sRANKL ratio values in girls with anorexia nervosa and the control group
Tabela I. Średni wiek, masa ciała, wskaźnik masy ciała (BMI), średnie stężenia wybranych hormonów tkanki tłuszczowej, osteokalcyny (OC), karboksyterminalnego usieciowanego telopeptydu łańcucha α1 kolagenu typu I (CTx), osteoprotegeryny (OPG), rozpuszczalnego ligandu receptora aktywatora czynnika jądrowego-κB (sRANKL) i wartości wskaźnika OPG/sRANKL u dziewcząt z jadłowstrętem psychicznym i w grupie kontrolnej
(n = 86)
(n = 21)
|Age (years)||15.45 ± 1.32||15.35 ± 1.72|
|Height [m]||1.62 ± 0.04||1.63 ± 0.05|
|Body mass [kg]||39.50 ± 5.24*||52.33 ± 5.56|
|BMI [kg/m2]||14.79 ± 1.62*||19.71 ± 1.26|
|Leptin (LP) [ng/mL]||4.96 ± 1.01*||14.62 ±1.13|
|Adiponectin (ADIPO) [µg/mL]||43.67 ± 5.21*||24.35 ± 2.52|
|Resistin (RES) [ng/mL]||2.86 ± 0.45*||4.20 ± 0.39|
|Visfatin (VISF) [ng/mL]||1.00 ± 0.26*||2.49 ± 0.38|
|Apelin-36 (APE-36) [pg/mL]||98.11 ± 8.51*||129.15 ± 14.32|
|Apelin-12 (APE-12) [pg/mL]||96.81 ± 8.90*||130.02 ± 16.61|
|OC [µmol/L]||0.91 ± 20.80*||3.12 ± 1.5|
|CTx [nmol/L]||7.22 ± 0.92*||7.99 ± 0.91|
|OPG [pmol/L]||5.21 ± 0.78*||3.41 ± 0.69|
|sRANKL [pmol/L]||0.45 ± 0.14*||0.28 ± 0.12|
|OPG/sRANKL ratio||9.72 ± 0.83*||13.98 ± 1.08|
Girls with AN exhibited significant positive correlations between BMI and LP, APE-36, CTx and the OPG/sRANKL ratio (Table II). CTx and sRANKL were negatively and significantly correlated with RES and APE-36; OPG was negatively and significantly correlated with APE-36 and APE-12, while the OPG/sRANKL ratio correlated negatively and significantly with ADIPO. Positive and significant correlations were observed between OC and VIFS; OPG and ADIPO as well as between the OPG/sRANKL and LP, APE-36 and APE-12 (Table III).
Table II. Correlation between body mass index (BMI), chosen adipose tissue hormones, osteocalcin (OC), collagen type I crosslinked carboxyterminal telopeptide (CTx), osteoprotegerin (OPG), soluble receptor activator of nuclear factor-κB ligand (sRANKL) and OPG/sRANKL ratio in girls with anorexia nervosa (n = 86)
Tabela II. Korelacja między wskaźnikiem masy ciała (BMI), wybranymi hormonami tkanki tłuszczowej, osteokalcyną (OC), karboksyterminalnym usieciowanym telopeptydem łańcucha α1 kolagenu typu I (CTx), osteoprotegeryną (OPG), rozpuszczalnym ligandem receptora aktywatora czynnika jądrowego-κB (sRANKL) i wartościami wskaźnika OPG/sRANKL u dziewcząt z jadłowstrętem psychicznym (n = 86)
|Variables||Values of correlation coefficients – R|
|BMI [kg/m2]||Leptin (LP) [ng/mL]||0.271*|
|Adiponectin (ADIPO) [µg/mL]||NS|
|Resistin (RES) [ng/mL]||NS|
|Visfatin (VISF) [ng/mL]||NS|
|Apelin-36 (APE-36) [pg/mL]||0.214*|
|Apelin-12 (APE-12) [pg/mL]||NS|
VISF was shown to be an independent predictor of OC (R2 = 0.071, p = 0.020). APE-36 and RES turned out to be independent predictors of CTx and sRANKL (R2 = 0.062, p = 0.015 and R2 = 0.082, p = 0.041, respectively). APE-36 and ADIPO were independent predictors of OPG (R2 = 0.173, p = 0.008), while APE-36, LP and ADIPO were independent predictors of the OPG/sRANKL ratio (R2 = 0.264, p = 0.005).
Table III. Correlationa between chosen adipose tissue hormones, osteocalcin (OC), collagen type I crosslinked carboxyterminal telopeptide (CTx), osteoprotegerin (OPG), soluble receptor activator of nuclear factor-κB ligand (sRANKL) and OPG/sRANKL ratio in girls with anorexia nervosa (n = 86)
Tabela III. Korelacjaa między wybranymi hormonami tkanki tłuszczowej, osteokalcyną (OC), karboksyterminalnym usieciowanym telopeptydem łańcucha α1 kolagenu typu I (CTx), osteoprotegeryną (OPG), rozpuszczalnym ligandem receptora aktywatora czynnika jądrowego-κB (sRANKL) i wartościami wskaźnika OPG/sRANKL u dziewcząt z jadłowstrętem psychicznym (n = 86)
|Variables||Bone markers||Cytokines of RANKL/RANK/OPG system||OPG/sRANKL ratio|
|OC [μmol/L]||CTx [nmol/L]||OPG [pmol/L]||sRANKL [pmol/L]|
|Leptin (LP) [ng/mL]||NS||NS||NS||NS||0.252*|
|Adiponectin (ADIPO) [µg/mL]||NS||NS||0.234*||NS||–0.245*|
|Resistin (RES) [ng/mL]||NS||–0.215*||NS||–0.236*||NS|
|Visfatin (VISF) [ng/mL]||0.266*||NS||NS||NS||NS|
|Apelin-36 (APE-36) [pg/mL]||NS||–0.212*||–0.269*||–0.240*||0.443*|
|Apelin-12 (APE-12) [pg/mL]||NS||NS||–0.361*||NS||0.225*|
|Stepwise regression modelb||VISF||APE-36
|APE-36 RES||APE-36 LP ADIPO|
Girls with AN suffer from a considerable decrease of bone mass [8–10, 12, 23, 54, 60, 62, 63] and bone metabolism disturbances [3–11, 21–23, 53, 54, 57, 61, 62]. The majority of authors emphasise the suppression of bone formation and bone resorption markers in these patients which appears correlated with BMI and/or BMD [4, 5, 12, 21, 22, 52, 53, 61, 63].
Our results are consistent with these findings. However, only few of the above mentioned authors determined OPG concentrations in girls with AN [4, 8, 21, 22, 57, 61]. It was found that, similar to young females with AN , girls with AN exhibited a considerable increase of serum OPG. Misra et al.  suggested that decreased levels of bone resorption markers in girls with AN might indicate a suppressive effect of elevated serum OPG on osteoclast activity and differentiation. However, it should be emphasised that Misra et al.  did not investigate the levels of RANKL and the values of the OPG/sRANKL ratio. Munoz-Calvo et al.  and Ostrowska et al. [21, 22, 61] determined serum OPG and sRANKL in female adolescents with AN and revealed significant suppression of the OPG/sRANKL ratio in patients with normal  or significantly increased OPG levels [21, 22, 61] and significantly increased sRANKL [21, 22, 57, 61].
Low values of the OPG/sRANKL ratio with a parallel increase in serum OPG and sRANKL demonstrated in our previous studies [21, 22, 61] and confirmed in the present investigations, along with some degree of desynchronisation of the relationships between the above mentioned cytokines and bone markers [21, 22], seem to suggest that girls with AN might exhibit a compromise in the mechanism which controls bone remodelling and/or mechanism compensating for enhanced bone turnover. Munoz-Calvo et al.  also reported a significant decrease in the OPG/RANKL ratio correlated with an increase in serum RANKL as well as a positive and significant correlation between the OPG/RANKL ratio and BMD. However, they did not observe a significant OPG increase in serum of girls with AN, or any relationships between OPG, RANKL and BMD. According to Munoz-Calvo et al. , the decrease in the OPG/RANKL ratio in girls with AN only partly explains increased bone loss observed in these patients. It has been well established that 17β-oestradiol is the major regulator of bone metabolism and BMD in both women and girls suffering from AN. However, it is also known that cytokines under investigation, in particular OPG, are modified not only by 17β-oestradiol, but also by other hormones, cytokines and mesenchymal transcription factors [24, 25] whose effect on cytokines concentrations is opposite to that of 17β-oestradiol. Therefore, the ultimate effect of all these actions is difficult to predict.
In vitro and in vivo studies have indicated that, apart from several well-known hormones, bone metabolism may also be modified by some adipose tissue hormones [8, 26–28, 30–34, 40, 42–50, 56–58, 65–68]. It has also been a suggested that the latter might regulate bone remodelling both directly and indirectly, i.e. via affecting OPG and/or RANKL expression [24, 25, 31, 38, 45, 56].
The findings concerning serum LP, ADIPO, RES and VISF concentrations in patients with AN, and especially in adolescent females suffering from the disease, are not unanimous. Our previous [12, 13–19] and present studies in girls with AN revealed suppression of LP, RES and VISF levels while ADIPO concentrations were increased. The results of other authors’ investigations are similar to ours [5–8, 53–56, 60, 66, 69–73]. Only a few of these studies have also assessed the relationships between the above mentioned adipose tissue hormones (or some of them) and BMD and/or bone metabolism in patients with AN [3–8, 53–55, 56, 66]. The same regards the relationships between LP and/or ADIPO, RES, VISF and bone status with a focus on possible interaction of cytokines of the RANKL/RANK/OPG system [4, 8, 55, 56, 66]. Several findings [4, 8, 56, 66] indicate a relationship between LP and/or ADIPO and RES and bone status. In vitro studies [4, 31, 38, 45] seem to suggest that the above mentioned adipokines, and especially LP and ADIPO, modify bone metabolism, most probably via their effect on RANKL and/or OPG expression.
Our investigations of girls with AN demonstrated that serum LP, RES, VISF, APE-36 and APE-12 suppression and ADIPO elevation were associated with a decrease in bone markers concentrations, low values of the OPG/sRANKL ratio with a parallel increase in serum OPG and sRANKL. CTx and sRANKL correlated negatively and significantly with RES and APE-36; OPG correlated negatively and significantly with APE-36 and APE-12, while the OPG/sRANKL ratio was negatively and significantly correlated with ADIPO. However, significant positive correlations were revealed between OC and VISF; OPG and ADIPO; the OPG/sRANKL ratio and LP, APE-36 and APE-12. Our findings confirm in vivo results of other authors [3–11, 23, 52, 53–59, 66], who hypothesised a relationship between bone status and some of the above mentioned adipose tissue hormones (i.e. LP, RES and ADIPO) in adolescent females with AN. We would also like to emphasise the role of VISF and APE in the regulation of bone remodelling in these patients. In vitro investigations into the effect of LP [26, 27, 31–35, 74], ADIPO [36–38, 74], RES , VISF  and APE [41, 42] on bone tissue cells are consistent with the above hypothesis. Stepwise regression analysis carried out in our patients revealed that VISF was an independent predictor of OC. APE-36 and RES turned out to be independent predictors of CTx and sRANKL; APE-36 and ADIPO were independent predictors of OPG, while APE-36, LP and ADIPO were independent predictors of the OPG/sRANKL ratio. Thus, it can be hypothesised that decreased LP, RES, VISF and APE production and increased ADIPO secretion observed in girls with AN might have an adverse effect on bone tissue, probably via a shift in the OPG/sRANKL ratio toward a functional excess of sRANKL.
Changes in bone markers, OPG, sRANKL and/or the OPG/sRANKL ratio exhibited by girls with anorexia nervosa have been found to be associated with changes in the levels of the selected adipose tissue hormones.
Abnormal relationships between bone metabolism and leptin, adiponectin, resistin, visfatin and apelin observed in girls suffering from anorexia nervosa might adversely affect the balance of the OPG/sRANKL system and potentially compromise the mechanism which compensates for bone remodelling disturbances.
- Klein DA, Walsh BT. Eating disorders: clinical features and pathophysiology. Physiol Behav 2004; 81: 359–374.
- Nogal P, Lewiński A. Jadłowstręt psychiczny (anorexia nervosa). Endokrynol Pol 2008; 59: 148–155.
- Misra M, Miller KK, Bjornson J et al. Alterations in growth hormone secretory dynamics in adolescent girls with anorexia nervosa and effects on bone metabolism. J Clin Endocrinol Metab 2003; 88: 5615–5623.
- Misra M, Soyka LA, Miller KK et al. Serum osteoprotegerin in adolescent girls with anorexia nervosa. J Clin Endocrinol Metab, 2003; 88: 3916–3822.
- Misra M, Aggarwal A, Miller KK et al. Effects of anorexia nervosa on clinical, hematologic, biochemical, and bone density parameters in community-dwelling adolescent girls. Pediatrics 2004; 114: 1574–1583.
- Misra M, Miller KK, Almazan C et al. Alterations in cortisol secretory dynamics in adolescent girls with anorexia nervosa and effects on bone metabolism. J Clin Endocrinol Metab 2004; 89: 4972–4980.
- Misra M, Miller KK, Herzog DB et al. Growth hormone and ghrelin responses to an oral glucose load in adolescent girls with anorexia nervosa and controls. J Clin Endocrinol Metab 2004; 89: 1605–1612.
- Misra M, Miller KK, Cord J et al. Relationships between serum adipokines, insulin levels and bone density in girls with anorexia nervosa. J Clin Endocrinol Metab 2007; 92: 2046–2052.
- Misra M, Klibanski A. Neuroendocrine consequence of anorexia nervosa in adolescents. Endocr Dev 2010; 17: 197–214.
- Misra M, Klibanski A. The neuroendocrine basis of anorexia nervosa and its impact on bone metabolism. Neuroendocrinoogy 2011; 93: 65–73.
- Galusca B, Bossu C, Germain N et al. Age-related differences in hormonal and nutritional impact on lean anorexia nervosa bone turnover uncoupling. Osteopros Int 2006; 17: 888–896.
- Oświęcimska J, Ziora K, Pluskiewicz W et al. Skeletal status and laboratory investigations in adolescent girls with anorexia nervosa. Bone 2007; 41: 103–110.
- Ziora K, Oświęcimska J, Świętochowska E et al. Analiza zależności pomiędzy stężeniem wybranych hormonów tkanki tłuszczowej (adiponektyny i leptyny) a stężeniami hormonów tarczycy, nadnerczy i gonad w surowicy krwi u dziewcząt z jadłowstrętem psychicznym. Endokrynol Pediatr 2010; 9: 9–22.
- Ziora K, Oświęcimska J, Świętochowska E et al. Ocena stężenia adiponektyny – hormonu tkanki tłuszczowej w surowicy krwi u dziewcząt z jadłowstrętem psychicznym. Endokrynol Pediatr 2010; 9: 31–41.
- Ziora K, Oświęcimska J, Świętochowska E et al. Ocena stężenia leptyny – hormonu tkanki tłuszczowej w surowicy krwi dziewcząt z jadłowstrętem psychicznyn. Endokrynol Pediatr 2010; 9: 43–53.
- Ziora K, Oświęcimska J, Świętochowska E et al. Assessment of serum apelin levels in girls with anorexia nervosa. J Clin Metab 2010; 95: 2935–2941.
- Ziora K, Oświęcimska J, Świętochowska E et al. Assessment of serum levels resistin in girls with anorexia nervosa. Part II. Relationship between resistin and body mass index. Neuro Endocrinol Lett 2011; 32: 691–696.
- Ziora K, Oświęcimska J, Świętochowska E et al. Assessment of serum levels resistin in girls with anorexia nervosa. Part II. Relationship between serum levels of resistin and thyroid, adrenal and gonadal hormones. Neuro Endocrinol Lett 2011; 32: 697–703.
- Ziora K, Oświęcimska J, Świętochowska E et al. Assessment of serum visfatin levels in girls with anorexia nervosa. Clin. Endocrinol 2012; 76: 514–519.
- Warren M. Endocrine manifestations of eating disorders. J Clin Endocrinol Metab 2011; 96: 333–343.
- Ostrowska Z, Ziora K, Kos-Kudła B et al. Melatonin, the RANKL/RANK/OPG system, and bone metabolism in girls with anorexia nervosa. Endokrynol Pol 2011; 61: 117–123.
- Ostrowska Z, Ziora K, Oświęcimska J et al. Dehydroepiandrosterone sulfate, osteoprotegerin and its soluble ligand sRANKL and bone metabolism in girls with anorexia nervosa. Postepy Hig Med Dośw 2012; 66: 655–662.
- Misra M, Klibanski A. Anorexia nervosa and osteoporosis. Rev Endocr Disord 2006; 7: 91–99.
- Hadjidakis DJ, Androulakis TT. Bone remodeling. Ann N Y Acad Sci 2006; 1092: 385–396.
- Vega D, Maalouf NM, Sakhaee K. The role of receptor activator of nuclear factor-κB (RANK)/RANK ligand/osteoprotegerin: clinical implications. J Clin Endocrinol Metab 2007; 92 4514–4521.
- Cornish J, Callon KE, Bava U et al. Leptin directly regulates bone cell function in vitro and reduces bone fragility in vivo. J Endocrinol 2002; 175: 405–415.
- Holloway WR, Collier FM, Aitken CJ et al. Leptin inhibits osteoclast generation. J Bone Miner Res 2002; 17: 200–209.
- Whipple T, Sharkey N, Demers L. Leptin and the skeleton. Clin Endocrinol 2002; 57: 701–711.
- Whitfield JF, Morley P, Wilick GE. The control of bone growth by parathyroid hormone, leptin and statins. Crit Rev Eucaryot Exp 2002; 12: 23–51.
- Martin A, Vittoris R, David V et al. Leptin modulates both resorption and formation while preventing disuse-induced bone loss in tail-suspend female rats. Endocrinology 2005; 146: 3652–3659.
- Lamghari M, Tavares L, Camboa N et al. Leptin effect on RANKL and OPG expression in MC3T3-E1 osteoblasts. J Cell Biochem 2006; 98: 1123–1129.
- Thomas T, Gori F, Khosla S et al. Leptin acts on human marrow stromal cells to enhance differentiation to osteoblasts and inhibit differentiation to adipocytes. Endocrinology 1999; 140: 1630–1638.
- Reseland JE, Syversen U, Bakke et al. Leptin is expressed in and secreted from primary cultures of human osteoblasts and promotes bone mineralization. J Bone Miner Res 2001; 16: 1426–1433.
- Gordeladze JO, Drevson CA, Syversen U et al. Leptin stimulates human osteoblastic cell proliferation, de novo collagen synthesis, and mineralization: impact on differentiation markers, apoptosis, and osteoclastic signaling. J Cell Biochem 2002; 85: 825–836.
- Thomas T. The complex effects of leptin on bone metabolism through multiple pathways. Current Opinion Pharmacol 2004; 4: 295–300.
- Kanazawa I, Yamaguchi T, Yano S et al. Adiponectin and AMP kinase activator stimulate proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells. Cell Biol 2007; 29: 51–61.
- Luo XH, Guo LJ, Yuan LQ et al. Adiponectin stimulates human osteoblasts proliferation and differentiation via the MAPK signaling pathway. Exp Cell Res 2005; 309: 99–109.
- Luo XH, Guo LJ, Yuan LQ et al. Adiponectin stimulates RANKL and inhibits OPG expression in human osteoblasts through the MAPK signaling pathway. J Bone Miner Res 2006; 1648–1656.
- Thommesen L, Stunes AK, Monjo M et al. Expression and regulation of resistin in osteoblasts and osteoclasts indicate a role in bone metabolism. J Cell Biochem 2006; 99: 824–834.
- Xie H, Tang SY, Luo XH et al. Insulin-like effects of visfatin on human osteoblasts. Calcif Tissue 2007; 80: 201–210.
- Tang SY, Xie H, Yuan LQ et al. Apelin stimulates proliferation and suppresses apoptosis of mouse osteoblastic cell line MC3T3-E1 via JNK and PI3-K/Akt signaling pathways. Peptides 2007; 28: 708–718.
- Zeng X, Yu SP, Taylor T et al. Protective effect of apelin on cultured rat bone marrow mesenchymal stem cells against apoptosis. Stem Cell Research 2012; 8: 357–367.
- Gonelli S, Cafarelli C, Del Santo K et al. The relationship of grelin and adiponectin with bone mineral density and bone turnover markers in elderly men. Calcif Tissue Int 2008; 83: 55–60.
- Peng XD, Xie H, Zhao Q et al. Relationship between serum adiponectin, leptin, resistin, visfatin levels and bone mineral density, and bone biochemical markers in Chinese men. Clin Chim Acta 2008; 387: 31–35.
- Tenta R, Panagiotakos DB, Fragopoulou E et al. Osteoprotegerin and nuclear factor-κB ligand are associated with leptin and adiponectin levels in apparently healthy women. J Musculoskelet Neuronal Interact 2010; 10: 174–179.
- Wu N, Wang QP, Li H et al. Relationship between serum adiponectin, leptin concentrations and bone mineral density, and bone biochemical markers in Chinese women. Clin Chim Acta 2010; 411: 771–775.
- Mazzali G, Francesco VD, Zoico E et al. Interrelations between fat distribution, muscle lipid content, adipocytokines, and insulin resistance: effect of moderate weight loss in older women. Am J Clin Nutr 2006; 84: 1193–1199.
- Jurimae J, Jurimae T, Leppik A et al. The influence of ghrelin, adiponectin, and leptin on bone mineral density in healthy postmenopausal women. J Bone Miner Metab 2008; 26: 618–623.
- Zhang H, Xie H, Zhao Q et al. Relationship between serum adiponectin, apelin, leptin, resistin, visfatin levels and bone mineral density, and bone biochemical markers in postmenopausal Chinese women. J Endocrinol Invest 2010; 33: 707–711.
- Oh KW, Lee WY, Rhee EJ et al. The relationship between serum resistin, leptin, adiponectin, ghrelin levels and bone mineral density in middleaged men. Clin Endocrinol (Oxf) 2005; 53: 131–136.
- Zoico E, Zamboni M, Di Francesco V et al. Relation between adiponectin and bone mineral density in elderly post-menopausal women: role of body composition, leptin, insulin resistance, and dehydroepiandrosterone sulfate. J Endocrinol 2008; 31: 297–302.
- Grinspoon S, Miller K, Coyle C et al. Severity of osteopenia in estrogendeficient women with anorexia nervosa and hypothalamic amenorrhea. J Clin Endocrinol Metab 1999; 84: 2049–2055.
- Soyka LA, Grinspoon S, Levitsky LL et al. The effect of anorexia nervosa on bone metabolism in female adolescents. J Clin Endocrinol Metab 1999; 84:4489–4496.
- Soyka LA, Misra M, Frenchman A et al. Abnormal bone mineral accrual in adolescent girls with anorexia nervosa. J Clin Endocrinol Metab 2002; 87: 4177–4185.
- Heer M, Mika C, Grzella I et al. Bone turnover during inpatient nutrition therapy and outpatient follow-up in patients with anorexia nervosa compared with that in healthy control subjects. Am J Clin Nutr 2004; 80: 774–781.
- Dostalova I, Smitka K, Papezova H et al. Increased insulin sensitivity in patients with anorexia nervosa: the role of adipocytokines. Physiol Res 2007; 56: 587–594.
- Munoz-Calvo MT, Barrios V, Garcia de Alvaro MT et al. Maintained malnutrition produces a progressive decrease in (OPG)/RANKL ratio and leptin levels in patients with anorexia nervosa. Scand J Clin Lab Invest 2007; 67: 387–393.
- Legroux-Gerot I, Vignau J, Biver E et al. Anorexia nervosa, osteoporosis and circulating leptin: the missing link. Osteoporos Int 2010; 21: 1715–1722.
- Weinbrenner T, Zittermann A, Gouni-Berthold I et al. Body mass index and disease duration are predictors of disturbed bone turnover in anorexia nervosa. A case-control study. Eur J Clin Nutr 2003; 57: 1262–1267.
- Mika C, Holtkamp K, Heer M et al. A 2-year prospective study of bone metabolism and bone mineral density in adolescents with anorexia nervosa. J Neural Transm 2007; 114: 1611–1618.
- Ostrowska Z, Ziora K, Oświęcimska J et al. RANKL/RANK/OPG system and bone status in females with anorexia nervosa. Bone 2012; 50: 156–160.
- Audi L, Vargas DM, Gussinyee M et al. Clinical and biochemical determinants of bone metabolism and bone mass in adolescent female patients with anorexia nervosa. Pediatr Res 2002; 51: 497–504.
- Munoz MT, Argente J. Anorexia nervosa in female adolescents: endocrine and bone mineral density disturbances. Eur Psychiat 2005; 20: 173–178.
- Ohwada R, Hotta M, Sato K et al. The relationship between serum levels of estradiol and osteoprotegerin in patients with anorexia nervosa. Endocr J 2007; 54: 953–959.
- Takeda S. Hormones and osteoporosis update. Regulation of bone remodeling by adipokines. Clin Calcium 2009; 19: 1009–1014.
- Zofkova I. Relationship of hormones of adipose tissue and ghrelin to bone metabolism. Vnitr Lek 2009; 55: 560–564.
- Saleem U, Mosley TH Jr, Kullo IJ. Serum osteocalcin is associated with measures of insulin resistance, adipokine levels, and the presence of metabolic syndrome. Arterioscler Thromb Vasc Biol 2010; 30: 1474–1478.
- Zillikens MC, Uitterlinden AG, van Leeuwen PTM et al. The role of body mass index, insulin, and adiponectin in the relation between fat distribution and bone mineral density. Calcif Tissue Int 2010; 86; 116–125.
- Delporte ML, Brichard SM, Hermans et al. Hyperadiponectinemia in anorexia nervosa. Clin Endocrinol (Oxf) 2003; 58: 22–29.
- Pannacciulli N, Vetter R, Milan G et al. Anorexia nervosa is characterized by increased adiponectin plasma levels and reduced nonoxidative glucose metabolism. J Clin Endocrinol Metab 2003; 88: 1748–1752.
- Chan JL, Mantzoros CS. Role of leptin in energy-deprivation states: normal human physiology and clinical implications for hypothalamic amenorrhoea and anorexia nervosa. Lancet 2005; 366: 74–85.
- Housova J, Anderlova K, Krizova J et al. Serum adiponectin and resistin concentrations in patients with restrictive and binge/purge form anorexia nervosa and bulimia nervosa. J Clin Endocrinol Metab 2005; 90: 1366–1370.
- Modan-Moses D, Stein D, Pariente C et al. Modulation of adiponectin and leptin during refeeding of female anorexia nervosa patients. J Clin Endocrinol Metab 2007; 92: 1843–1847.
- Magni P, Dozio E, Galliera E et al. Molecular aspects of adipokine-bone interactions. Curr Mol Med 2010; 10: 522–532.