Endocrine Research

259

Bone Structure and Strength are Enhanced in Rats Programmed by Early Overfeeding

Affiliations

Key words

▶ early overfeeding ● ▶ bone ● ▶ programming ●

received 25.08.2013 accepted 29.01.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1368728 Published online: March 13, 2014 Horm Metab Res 2014; 46: 259–268 © Georg Thieme Verlag KG Stuttgart · New York ISSN 0018-5043 Correspondence Prof. E. G. de Moura, PhD Departamento de Ciências Fisiológicas – 5 ° andar Universidade do Estado do Rio de Janeiro Instituto de Biologia Av. 28 de setembro 87 – Rio de Janeiro RJ 20550-030 Brazil Tel.: + 55/21/2587 6434 Fax: + 55/21/2587 6129 [email protected]

L. de Albuquerque Maia1, P. C. Lisboa1, E. de Oliveira1, E. P. S. da Conceição1, I. C. B. Lima2, R. T. Lopes2, L. D. G. Ruffoni3, K. O. Nonaka3, E. G. de Moura1 1

Department of Physiological Sciences, State University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil Nuclear Instrumentation Laboratory, COPPE-PEN, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil 3 Laboratory of Exercise Physiology, Federal University of São Carlos, São Carlos, São Paulo, SP, Brazil 2

Abstract

▼

Childhood obesity is growing in prevalence. Obesity and bone dysfunctions may be related disorders, and therefore our aim was to study the impact of the early overfeeding (EO) in offspring bone health since weaning up to adulthood. To induce EO during lactation, litter size was adjusted to 3 male rats per litter (SL). Litter containing 10 pups per mother was the control (NL). Bone tissue was evaluated by dual-energy X-ray absorptiometry, computed tomography, microcomputed tomography, biomechanical tests, and serum analyses. SL offspring presented higher body weight, fat mass, lean mass from 21 up to 180 days, hyperphagia, and higher visceral fat mass. Bone analysis showed that SL offspring presented higher total bone mineral density (BMD) only at 180 days, and higher total bone mineral content and higher bone area from 21 until 180

Introduction

▼

Obesity is growing in prevalence and is associated with some bone dysfunctions. Adipocytes and osteoblasts have common precursor cell [1], which makes these aforementioned diseases share some common characteristics: both disorders can begin early in life, although the complete phenotypic presentation can take decades to be manifested; both diseases are associated with significant morbidity and mortality. Obesity in childhood is reported to be rising dramatically and it may increase the risk of adult morbidity [2, 3]. Studies have shown that children with overweight are more likely to become obese adults [4, 5]. In animal models, studies have shown that excess of nutrition in perinatal life represents a risk factor for obesity and associated metabolic disturbances in adulthood [6–8]. The association among nutritional, environmen-

days. At 180 days, SL offspring presented higher femur BMD and fourth lumbar vertebra (LV4) BMD, higher femoral head radiodensity and LV4 vertebral body radiodensity, lower trabecular pattern factor and trabecular separation, however with higher trabecular number, higher maximal load, resilience, stiffness and break load, and lower break deformation. SL group had, at 180 days, higher osteocalcin and lower C-terminal cross-linked telopeptide of type I collagen (CTX I). We have shown that the excess of fat mass contributed to an increased bone mass, and hypothesized that this increase could be mediated by the hypothyroidism and previous higher thyroid hormone action and hyperleptinemia at weaning. Furthermore, the increased biomechanical loading due to increased body weight probably help us to understand the protective effects obesity exerts upon bone health.

tal, and hormonal influences during critical periods early in life and changes in metabolism at adulthood is named metabolic programming [9– 11]. The perinatal environment also affects skeletal health. Early life seems to be a critical period for the development and/or programming of metabolic systems, including the skeleton in children [12]. Recently, Devlin et al. [13] have shown that maternal high fat diet induces perinatal programming of offspring bone mass and strength, reinforcing the idea that perinatal environment also affects skeletal health. As emphasized in the NIH Consensus Statement report on osteoporosis, the bone mass attained during growth is a critical determinant of the risk of osteoporosis later in life [14]. Bone mass of an individual in later adult life depends upon the peak attained during skeletal growth and the subsequent rate of bone loss. Approximately 20–30 % of the varia-

de Albuquerque Maia L et al. Early Overfeeding Improves Bone Tissue … Horm Metab Res 2014; 46: 259–268

This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.

Authors

260 Endocrine Research Dual-energy X-ray absorptiometry (DXA) At different ages (21, 90, 120, 150, and 180 days), rats were anesthetized with an intraperitoneal injection of 2:1 solution of ketamine hydrochloride (Cetamin®, 50 mg/ml), and xylazine hydrochloride (Xilazin®, 20 mg/ml), respectively, at a dose of 0.1 ml/100 g and subjected to DXA [24], using a Lunar DEXA 200368 GE instrument (Lunar, Wisconsin, USA) with specific software (encore 2008, version 12.20 GE Healthcare). The evaluation was blind, since the DXA technician did not know the experimental protocol. Body weight (g); body fat mass (g); lean mass (g); total bone mineral density; total BMD (g/cm2); total bone mineral content; total BMC (g), and bone area (cm2) were measured for each rat. At PN180, after DXA procedures and after 12 h of fasting, 1 male rat from each dam was euthanized with a lethal dose of ketamine hydrochloride/xylazine hydrochloride (2:1) and blood was obtained by cardiac puncture. Visceral fat mass was excised (mesenteric, epididymal, and retroperitoneal white adipose tissue) and immediately weighed for evaluation of central adiposity. Right femur and the fourth lumbar vertebra (LV4) were collected, cleaned of soft tissue, and preserved in saline solution (0.9 % of NaCl) at − 2 °C until analyzed. Bone lengths were measured using a caliper with 0.01 mm readability. Femur and LV4 bone mineral density (BMD) were determined by DXA (Lunar, Wisconsin, USA) using a modification of previously described procedure [25]. In order to mimic soft tissue conditions, excised bones were fixed on constant volume of rice in a plastic container.

Single-scan computed tomography (CT) Materials and Methods

▼

The use of the animals according to our experimental design was approved by the Animal Care and Use Committee of the Biology Institute of the State University of Rio de Janeiro (CEA/017/2009), which based its analysis on the principles adopted and promulgated by Brazilian Law (no. 11.794/2008). Experiments were conducted to minimize the number of animals and the suffering caused by the procedures following the ethical doctrine of the 3 ‘R’s’ – reduction, refinement, and replacement [23]. Wistar rats were kept in a temperature-controlled room (23/24 °C) with artificial dark/light cycles (lights on at 07:00 h and lights off at 19:00 h). Virgin female rats, 3 months old, were caged with male rats at a proportion of 3:1. After mating, each female was placed in an individual cage with free access to water and food until delivery. We used only the dams whose litter size was 10–12 pups in order to avoid the influence of litter size.

Experimental models of post-natal early overfeeding To induce EO during lactation, 3 days after birth, the litter size was adjusted to 3 male rats per litter (SL) [7, 16]. Litter containing 10 pups (at least 6 males and completed with females) per mother was used as control (NL). One male rat was randomly chosen from each of the 16 different litters (8 SL litters and 8 NL litters) for subsequent analysis. After postnatal day 21 (PN21) that corresponds to weaning period, both groups had free access to water and standard diet. From PN21 until postnatal day 180 (PN180), offspring’s body weight (g) and food intake (g) were monitored every 7 days.

After DXA, bones were analyzed by a single-scan computed tomography (CT Helicoidally model HISPEED, GE®). The images of femur and LV4 were obtained through axial cuts of thickness of 1 mm. The radiodensity (expressed as Hounsfield units, HU) of femoral head and vertebral body regions (R1 and R2, respec▶ Fig. 1a, b) were measured with a computerized anatively, ● lyzer software system (eFilm Lite, 2.0, 2003, Milwaukee, USA) by manual measurement.

Microcomputed tomography (micro-CT) analysis The micro-CT was performed in a high-resolution setup (Skyscan/Bruker model 1173). The samples were placed at micropositioning arrangement. The system was calibrated in order to operate with energy equal to 80 kV and current of 90 μm and an aluminum filter (1.0 mm of thickness) was used in order to correct beam hardening effects. Each sample was scanned with pixel size of 19.8 μm recorded by a Hamamatsu detector made up of a 2 240 × 2 240 pixels grid with a step of 50 μm. Each scan took about 19 min and generated 600 TIFF projections that were used for the reconstruction that resulted in volumetric data with 11.4 Gb/1074 sections. After the acquisition procedure, the images were reconstructed by using Nrecon® (v.1.6.5.8, SkyScan/Bruker micro-CT, Kartuizerweg 3B 2 550 Kontich, Belgium) and InstaRecon® (v.1.3.8.5, CBR Premium 12-8KTM, InstaRecon, Champaign, IL, USA) software, with algorithm based on Feldkamp work [26]. CTAn® (v1.11.8.0, Skyscan/Bruker micro-CT, Kartuizerweg 3B 2 550 Kontich, Belgium) was used in order to analyze and process the images. At this stage, the goal was to quantify the stereological parameters related to trabecular bone. Since proximal femur is of particular relevance to human osteoporosis and its microarchitecture has a role in the fracture at this region, 3-dimensional

de Albuquerque Maia L et al. Early Overfeeding Improves Bone Tissue … Horm Metab Res 2014; 46: 259–268

This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.

tion in peak of bone mass is determined by environmental factors such as nutrition and may be modified to obtain optimal bone mineral density, and morphological, and mechanical properties of skeletal system [15]. Understanding how nutrition in early life can affect bone health could give important insights into the relationship between bone tissue and obesity. Rats raised in ‘small litters’ (SL) are considered an established animal model to study short- and long-term consequences of childhood obesity. This model of postnatal early overnutrition (EO) was associated with hyperphagia, obesity, hyperinsulinemia, hyperleptinemia since weaning [7, 16–18]. Previously, we have shown the programming for overweight, higher total and visceral fat mass, lower HDL-C, hyperphagia, central leptin resistance, higher oxidative stress, higher glycogen and triglycerides contents in the liver of adult SL rats, suggesting liver microesteatosis as well as thyroid hypofunction in adulthood [7, 8, 19, 20]. Leptin and insulin can have profound effects upon osteogenesis [21, 22]. Thus, the early increase in serum leptin and insulin in EO pups can program their bone development. Since EO model can promote several metabolic and hormonal changes, programming these animals to obesity, we hypothesized that bone tissue also can be programmed early in life. Thus, the aim of the present study was to test this hypothesis, studying the impact of EO in offspring bone health since weaning up to adulthood. Here, we tested the effect of EO on postnatal acquisition of bone mass, microarchitecture, and strength by comparing rats raised in small litters (SL) vs. rats raised in normal litters (NL) during lactation.

Endocrine Research

Biomechanical analysis The femur’s biomechanical properties were measured by the 3-point bending test using a universal test machine (Instron, model 4444, Canton, Massachusetts, USA), a load cell with a capacity of 100 kgf. Bones’ extremities were supported on 2 rollers with 3 mm diameter and at a distance of 21.70 mm. Load was applied in the central region of each bone [28]. At the beginning of the test, a 10 N pre-load was applied in the posterior-anterior direction (perpendicular to the longitudinal axis) to establish the femur. After a 1-min accommodation and stabilization period, a force was applied in the same way at a constant velocity of 0.5 cm/min up to the fracture moment. As a result of the force applied to the femur, the Instron software (series IX) generated a graphic load strain; in this graphic, the main biomechanical properties were obtained: maximal load (the higher load supported by the femur, kN), break load (the load at which

a

the bone fractures, kN), break deformation (mm), resilience (J), tenacity (J), and stiffness (N/mm).

Serum analysis Samples were centrifuged (1 500 × g for 20 min at 4 °C) to obtain serum, which was stored at –20 °C for posterior analysis of ionized calcium, osteocalcin, C-terminal cross-linked telopeptide of type I collagen (CTX I), leptin, and 25-hydroxivitamin D3 [25(OH)D]. Ionized calcium was measured using the ion-selective electrode (ISE) method with a specific kit (AVL 9180 Electrolyte Analyzer; Roche Diagnostics GmbH, Mannheim, Germany) with a range of detection from 0.8 to 20 mg/dl. Leptin was measured with a specific RIA kit (Linco Research, St Charles, MO, USA) with a range of detection from 0.5 to 50 ng/ml; the intra-assay variation was 2.9 %. Total osteocalcin was determined by an enzyme-linked immunosorbent assay kit for rat with intra-assay variation of less than 10 % (USCN Life Science & Technology Co., Ltd., Beijing, China) with range of detection from 0.78 to 50 ng/ml. For determination of CTX-I, an immunoassay kit for rat was used (Wuhan EIAab Science Co., Ltd., Wuhan, China) with a range of detection from 0.156 to 30 ng/ml and an intra-assay variation of less than 10 %. The 25(OH)D was determined through a commercially available electrochemiluminescence immunoassay kit (Elecsys and Cobas immunoassay analyzers, Roche Diagnostics GmbH, Indianapolis, USA) with a detection range from 4.0 to 100 ng/ml and an intra-assay coefficient of variation of 4.8 %.

Statistical analysis Statistical analyses were carried out using the Graph Pad Prism statistical package version 5.00, 2007 (San Diego, CA, USA). Data by were analyzed by unpaired Student’s t-test. All results are expressed as means ± SEM with significance level of 0.05. Fig. 1 a Computed tomography image of femur, the region of interest is shown by R1 (femoral head); b Computed tomography image of lumbar vertebra, the region of interest is shown by R2 (vertebral body); c 3D representative microcomputed tomography image of the cylindrical region of interest (1.86 mm of diameter) at the center of the femoral head showing the region of trabecular bone studied.

b

R1

R2

c

de Albuquerque Maia L et al. Early Overfeeding Improves Bone Tissue … Horm Metab Res 2014; 46: 259–268

This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.

architectural measurements made at femoral head were obtained. This may be particularly important for femoral head and neck fractures, since this region has a relatively thin cortical shell surrounding a larger volume of trabecular bone that is likely responsible for carrying the majority of the loads transmitted across the hip [27]. For that purpose cylindrical region of interest (1.86 mm of diameter) at the center of the femoral head ▶ Fig. was used in order to examine the trabecular bone region (● 1c). The following parameters were determined: bone volume fraction (BV/TV, in %, the ratio of 3D total bone volume to the total tissue volume); trabecular pattern factor (Tb.Pf, per millimeter); trabecular thickness (Tb.Th, in mm); trabecular number (Tb.N, the inverse of the mean distance between the mid-axes of the structures, per millimeter); and trabecular separation (Tb.Sp, the mean distance of the non-bone regions, in mm).

261

262 Endocrine Research

b

500 400

*

*

*

*

300 200

*

100

150 Body fat mass (g)

* *

100

*

*

Fig. 2 a Body weight evolution after weaning until 180 days old; b Body fat mass evolution; c Lean mass evolution evaluated by DXA, after weaning until 180 days of NL (•) and SL (■). * SL mean values were significantly different from those of NL (p < 0.05); n = 8 animals/group.

50

*

0

0 30

60

90 120 150 180 Days c

400

Lean mass (g)

0

300

30

* *

200

60

90 120 150 180 Days

* *

100

* 0 30

60

a

b

20

*

500

* 15

400

VFM (g)

Acumulated Ingestion (g)

90 120 150 180

300 200

10

Fig. 3 a Accumulated ingestion; b Visceral fat mass; c Serum leptin at 180 days of NL and SL offspring. Values are means, with their standard errors represented by vertical bars; n = 8 animals/ group. * Mean values were significantly different from those of NL (p < 0.05).

5

100

0

0 NL

SL

Leptin (ng/ml)

c

NL

SL

10 8 6 4 2 0 NL

SL

Results

▼

▶ Fig. 2, body weight, body fat mass, and lean mass evolution In ● are shown from weaning up to 180 days analyzed by DXA. We can observe that SL offspring presented higher body weight at 21 ( + 32 %), 90 ( + 10.8 %), 120 ( + 11.8 %), 150 ( + 8.7 %), and 180 days ( + 11.7 %). In the same way, body fat mass was higher at these ages: 21 ( + 74 %), 90 ( + 27 %), 120 ( + 29 %), 150 ( + 34 %), and 180 days ( + 52 %). And also lean mass: 21 ( + 16.6 %), 90 ( + 6.4 %), 120 ( + 6.7 %), 150 ( + 6.2 %), and 180 days ( + 8.1 %). As depicted ▶ Fig. 3, in PN180, SL offspring showed hyperphagia, once its in ● accumulated ingestion was higher ( + 8.1 %) as well as higher visceral fat mass ( + 88 %). Leptin serum levels, despite higher in SL group were not statistically different from controls. ▶ Fig. 4, 5. SL offspring presented Bone parameters are shown in ● higher total BMD at 180 days ( + 6.8 %), higher total BMC at 21 days ( + 62 %), 90 (12 %), 120 (12 %), 150 (11 %), and 180 days

( + 12.5 %) and higher bone area also at these ages: 21 ( + 54 %), 90 (8 %), 120 (9 %), 150 (9.6 %), and 180 days ( + 9.6 %). When bones were analyzed individually, SL offspring showed higher femur BMD ( + 9.2 %) and LV4 BMD ( + 8.2 %). In the same way, SL offspring also showed higher femoral head radiodensity ( + 30 %) and LV4 vertebral body radiodensity ( + 27 %). There were no differences in regard to femur and LV4 length between groups (data not shown). Bone structural parameters, including trabecular pattern factor (Tb.Pf), trabecular thickness (Tb.Th), trabecular number (Tb.N), ▶ Fig. 6. The and trabecular separation (Tb.Sp) are shown in ● bone volume fraction (BV/TV) was not significant different between groups (data not shown). SL offspring presented lower Tb.Pf ( − 2.6 times) and Tb.Sp ( − 9.2 %), whereas higher Tb.N ( + 9.3 %). There was no significant difference in Tb.Th between groups.

de Albuquerque Maia L et al. Early Overfeeding Improves Bone Tissue … Horm Metab Res 2014; 46: 259–268

This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.

Body weight (g)

a

a

0.20

* BMD (g/cm2)

0.15

0.10

0.05

0.00 0 b

30

60

90 Days

120

150

180

*

*

150

180

16 14

BMC(g)

12

*

10

*

8 6 4

*

2 0 0 c

30

60

90 Days

120

80

*

Bone Area (cm2)

*

*

*

60

40

* 20

0 0

30

60

90 Days

120

150

180

Fig. 4 a Total BMD; b Total BMC; c Bone Area of NL (•) and SL (■) at different ages (21, 90, 120, 150, and 180 days). * SL mean values were significantly different from those of NL (p < 0.05); n = 8 animals/group.

▶ Fig. 7. SL Bone tissue mechanical properties are presented in ● group presented higher maximal load ( + 14.4 %), resilience ( + 29 %), stiffness ( + 17 %) and break load ( + 22 %), and lower break deformation ( − 31 %). Tenacity was not significant different between groups (data not shown). ▶ Fig. 8. The offspring serum analyses at 180 days are shown in ● We observed SL group had higher osteocalcin ( + 69 %) and lower CTX-I ( − 52 %), but there was no significant difference between groups for the serum concentrations of 25(OH)D and ionized calcium.

Discussion and Conclusions

▼

In the present study, we observed that postnatal EO induced by litter size reduction causes an increase in body mass gain since

lactation up to adulthood, higher body fat mass, and higher lean mass. Furthermore, it programs for hyperphagia and higher visceral fat mass. These findings are in accordance with our group’s earlier studies [7, 8, 20]. Here, we evaluated for the first time, in the EO model, different aspects involved in bone structure and metabolism. We have shown that EO programs the offspring for a higher total BMD at 180 days and higher BMC and bone area since 21 up to 180 days. At 180 days, we also noted higher BMD in femur and vertebra, as well as higher radiodensity in specific bone regions, as the femoral head and vertebral body. We also analyzed bone microarchitecture and SL rats presented lower trabecular pattern factor and trabecular separation, whereas higher trabecular number. SL offspring differ from NL in biomechanical properties of bone tissue, such as higher maximal load, resilience, stiffness, break load, and lower break deformation. These data, put together with the higher osteocalcin and lower CTX-I, demonstrate an increase of bone mass and in its strength in SL group. DXA has become an increasingly important tool for the measurement of soft tissue body composition. Nowadays, many scientists consider this method as the standard for precision and accuracy for body composition measurements, not only to analyze total body composition but also specific regions of the body and it is the most common technique for assessing BMD, providing absolute values of BMD in terms of bone mineral per unit projected area in g/cm2 [24, 29]. Bone characteristics are represented by 2 factors: the calcium accretion reflected by the BMC and the volumetric development, which is reflected by the bone area. The relation between these 2 parameters is the BMD given by DXA. The first factor (BMC) depends on calcium intake, absorption and accretion, while the second factor depends mainly on the protein synthesis needed to build the bone structure [30]. In the present study, SL offspring presented a small increase of total BMD only at 180 days, but the other parameters, BMC and bone area were higher at 21, 90, 120, 150, and 180 days. This could be explained by the fact that BMC and bone area increase in the same proportion, since BMD is a relation of both, its small increase becomes evident only at 180 days due to a stabilization of the temporal increase of BMC and to a small decrease in bone area comparing 180 days to 150 days. Despite the small increase in total BMD at 180 days, if we consider the bones more susceptible to fractures at old age, such as the femur and lumbar vertebrae, when evaluating individually by DXA, we observed higher femur and LV4 BMD for SL group, as well as higher radiodensity of femoral head and LV4 vertebral body obtained by computed tomography, which are in fact the areas of these bones more susceptible to lesions. Studies examining the possible relations between fat mass and bone mass have found a positive association between these 2 tissues, regardless of age [31, 32]. Indeed, available data suggest that increased fat enhances bone mass and may protect against osteoporosis in both children and adults [33, 34]. This positive fatbone relation is credited not only to stress from mechanical loading but also to the metabolic effects of bone-active hormones secreted or regulated by adipocytes [35]. Leptin is a hormone secreted by adipose tissue, which reduces food intake and increases energy expenditure via specific hypothalamic signals, maintaining the body weight homeostasis and that is more secreted by obese individuals [36]. Recently, it has raised the possibility that leptin may also be a possible mediator of the bone protective effect. Consistent with this, some observational studies found that circulating leptin levels were positively associated with

de Albuquerque Maia L et al. Early Overfeeding Improves Bone Tissue … Horm Metab Res 2014; 46: 259–268

263

This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.

Endocrine Research

264 Endocrine Research

0.20 LV4 BMD (g/cm2)

Femur BMD (g/cm2)

* 0.15 0.10 0.05 0.00

LV4 Radiodensity (HU)

Femoral Head Radiodensity (HU)

750 500 250

*

600 400 200 0 NL

SL

b

0.15 Tb.Th (mm)

Tb.Pf (1/mm)

SL

800

SL

0 –2 –4

*

–6 NL

* 0.05

SL

NL d

5 *

4

0.10

3 2 1

Fig. 6 a Trabecular pattern factor; b Trabecular thickness; c Trabecular number; d Trabecular separation at 180 days of NL and SL offspring, evaluated by micro-CT. Values are means, with their standard errors represented by vertical bars; n = 8 animals/ group. * Mean values were significantly different from those of NL (p < 0.05).

0.00

Tb.Sp (mm)

Tb.N (1/mm)

0.05

NL

d

*

1 000

NL

c

0.10

SL

0

a

*

0.15

0.00 NL

c

Fig. 5 a Femur BMD; b LV4 BMD; c Femoral head radiodensity; d LV4 vertebral body radiodensity at 180 days of NL and SL offspring; a and b were evaluated by DXA and c and d were evaluated by CT. Values are means, with their standard errors represented by vertical bars; n = 8 animals/group. * Mean values were significantly different from those of NL (p < 0.05).

b

0.20

SL

0.20 *

0.15 0.10 0.05 0.00

0 NL

SL

NL

bone mass at various sites [37, 38], leptin injection induces to an increase in femoral length, total body bone area, BMC and BMD in leptin-deficient (ob/ob) mice [21], and act as a growth factor on the chondrocytes of skeletal growth centers [39]. SL animals presented hyperleptinemia at weaning [7, 40] and, at least at 21 days, the higher leptin action on the bone plus the mechanical loading caused by the higher body weight may be responsible for the higher BMC and bone area observed in SL rats. The same rational can be attributed to early hyperinsulinemia in SL group, since it was demonstrated an osteogenic insulin effect [22]. During development, the continuous hyperinsulinemia and hyperleptinemia may cause both insulin and leptin resistance. At 180 days, both insulin and leptin serum levels normalize, but their effect early in life upon the bone seems to persist. Thus, it seems more important the hormonal effects early in life than in adulthood. The hypothalamic-pituitary-thyroid axis plays a key role in skeletal development, acquisition of peak bone mass and regulation of adult bone turnover. Studies in mutant mice have demonstrated that thyroid hormones exert anabolic actions during growth but have catabolic effects on the adult skeleton [41]. In adulthood, hyperthyroidism increases bone turnover and

SL

reduces the remodeling cycle time leading to an increased risk of osteoporosis and fractures in overt and subclinical hyperthyroid patients [42, 43]. As for the effect of hypothyroidism on bone, hystomorphometry data indicated that hypothyroidism significantly prolongs the bone remodeling cycle resulting in a reduced bone turnover and a gain in bone mass and mineralization [44]. In a previous study, our group had shown that animals raised in small litters presented higher plasma T3, T4 and TSH at weaning, while the opposite profile was detected at 180 days [7]. Since higher levels of thyroid hormones during development may accelerate bone growth [41] and hypothyroidism may be protective to osteoporosis, we hypothesized that this combination of thyroid changes during development may contribute, at least in part, to the high bone mass observed in the SL adult offspring. Moreover, lean mass seems to have a role in the increase of bone density in obese individuals. Madeira et al. [45] found positive correlations among lean mass, bone density, and bone microstructure in a sample of obese adults with metabolic syndrome, suggesting a relevant relationship between lean mass and bone health. Since our SL animals presented higher lean mass at all ages studied, we should consider an impact of lean mass at bone

de Albuquerque Maia L et al. Early Overfeeding Improves Bone Tissue … Horm Metab Res 2014; 46: 259–268

This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.

a

Endocrine Research

b

0.20

*

0.15

Resilience (J)

0.10 0.05 0.00 NL

*

Break Load (kN)

Stiffness (N/mm)

0.06 0.04 0.02

NL

d 300 200 100

Fig. 7 a Maximal load; b Resilience; c Stiffness; d Break load; e Break deformation at 180 days of NL and SL offspring, evaluated by 3-point bending test. Values are means, with their standard errors represented by vertical bars; n = 8 animals/group. * Mean values were significantly different from those of NL (p < 0.05).

SL

0.20

*

0.15 0.10 0.05 0.00

0

Break Deformation (mm)

0.08

SL

400

NL

SL

NL

SL

2.5 2.0 1.5

*

1.0 0.5 0.0 NL

SL

b

a

*

0.8 0.6 0.4 0.2

Fig. 8 a Osteocalcin; b CTX-I; c 25(OH)D at 180 days of NL and SL offspring. Values are means, with their standard errors represented by vertical bars; n = 8 animals/group. * Mean values were significantly different from those of NL (p < 0.05).

8

1.0 CTX I (ng/ml)

Osteocalcin (ng/ml)

*

0.00

c

e

0.10

6 4

*

2 0

0.0 NL

SL 25(OH)D (ng/ml)

c

NL

SL

15 10 5 0 NL

SL

density. However, when its increase was compared to fat mass increase, we suggest that the impact of body weight on bone density was mainly derived from fat mass, once its increase was higher than lean mass in all ages analyzed. In the same way, Reid et al. [46] found that fat mass alone was a significant predictor of bone mineral density and Iwaniec et al. [47] demonstrate that there is a positive relationship between body mass and bone mass in growing and mature wild-type mice consuming regular mouse chow ad libitum. Bone strength is determined by its material composition, structure, geometry, and microarchitecture. The quantity of bone tis-

sue is measured by calculating BMD with DXA, but bone density does not discriminate between cortical and cancellous bone compartments nor does it permit assessment of bone microarchitecture [48]. The introduction of microcomputed tomography (micro-CT) in biomedical research has made accurate assessment of the bone microstructure. Micro-CT is widely used for observing and analyzing the internal structure of hard tissues because it is quick, reproducible, and nondestructive [49]. Based on different algorithms, the 3D structure and related parameters of bone can be obtained. It describes the properties of bone better than the “golden standard” histomorphometry

de Albuquerque Maia L et al. Early Overfeeding Improves Bone Tissue … Horm Metab Res 2014; 46: 259–268

This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.

Maximal Load (kN)

a

265

based on stereological assumptions, given a real estimate of its morphology [50, 51]. Microarchitecture is in particular one of the contributors to bone strength [52]. According to Xu et. al. [53] there is a strong correlation between BMD and bone strength, but BMD can only explain about 60 % of the bone strength. The rest 40 % is related to trabecular structure, which also plays an important role in bone strength [54]. Concerning the variations in bone mass through trabecular microarchitecture analysis of the femur using micro-CT, we found an improvement of microarchitecture in SL group with higher trabecular number and lower trabecular separation. Analyses of models have shown that Tb.Pf is a very sensitive parameter for the detection of changes in trabecular bone structure [55]. Tb.Pf is used to represent the ratio of the convex to curve surfaces and describes quantitatively the ratio of intertrabecular connectivity. It leads to low values in cases of wellconnected trabecular bone, whereas a lot of isolated trabeculae result in high values of Tb.Pf [56]. We had shown lower values for Tb.Pf in SL offspring. So, this group presented an increased proportion of concave elements relative to convex elements, indicating a well-connected bone pattern. Since Tb.Pf, Tb.N and Tb.Sp are indexes that reflect the status of the trabecular structure, these results revealed that the trabeculae structure were better in SL group. Corroborating with our data, other studies had shown that the obese mice have a more abundant, thicker, and well-connected trabecular structure with higher Tb.Th and Tb.N, and smaller Tb.Sp [50, 57]. In order to complement the bone analyses, we submitted the rats’ femora to the 3-point bending test to assess bone strength. We had shown an improvement of biomechanical strength in SL group. In agreement, a study of bone biomechanics in adult rats also showed significantly greater bone strength in the dietaryinduced obese rats [58]. In the present study, osteocalcin and CTX levels were performed to assess the balance of bone formation and resorption. Osteocalcin is a bone-specific protein secreted by osteoblasts and often used as a bone formation biomarker [59]. Higher serum osteocalcin concentrations indicates increased osteoblast activity, as seen in obese compared to lean male Zucker rats [60] and in our obese SL offspring. CTX is produced during the breakdown of type I collagen, and its serum concentration corresponds to bone resorption [61]. The reduced serum CTX-I in SL offspring corroborates with other authors data in human athletes concerning the inverse correlation between BMD and CTX-I [62, 63]. This bone remodeling profile, in SL group, with higher osteocalcin and lower CTX-I shows a bone turnover favoring bone formation over resorption, and this is confirmed with higher values for BMD, BMC and bone area. It has been reported that the skeletal system also plays a role in the regulation of energy and glucose metabolism [64]. It has been shown in experimental animals that osteocalcin administration increases insulin sensitivity and adiponectin levels [65, 66]. Lee et al. [65] demonstrated that osteocalcin is involved in glucose metabolism by increasing insulin secretion and cell proliferation in pancreatic β-cells and by up regulating the expression of the adiponectin gene in adipocytes, thus improving insulin sensitivity. Studies in adults and children have shown an association between low osteocalcin levels and insulin resistance [67–69]. In this way, it is possible that the higher osteocalcin levels in SL group help us to explain the absence of insulin resistance and the similar levels of adiponectin observed in this same model previously reported by our group [8], in spite of

higher body weight and visceral fat mass, reinforcing the association between the skeletal system and insulin resistance. Vitamin D is well known for their essential role in bone metabolism and calcium homeostasis [70]. It has become increasingly clear that the vitamin D endocrine system is related to obesity in adults. Obesity has been found to be associated with lower levels of serum 25(OH)D [71, 72]. Although 25(OH)D serum concentration was not significantly lower in SL group, there was a clear tendency in being lower than in the NL group. Other programming model also studied by our group, such as early weaning, is associated with undernutrition at weaning and obesity at adulthood [73] we detected higher serum vitamin D at weaning [74] and this continues at adulthood [75]. Despite we did not measure vitamin D at weaning, we suppose that in overnutrition, such in the present model, vitamin D could be lower, due to early obesity. Thus, a normal action of the vitamin D since the weaning could be important to avoid obesity. On the other hand, it is important to consider here that other authors have found controversy results: the increase in adiposity may have the potential to decrease bone mass [76]. Some studies have shown that overweight and obesity are associated with osteoporosis and osteopenia [77–80]. But recent observations corroborate our findings that obesity seems to be protective to bone health [81, 82]. The possible explanation for the discrepancies between these results might be related to populations, research designs, sampling methods, methodological differences, and also due to the fact that bone tissue changes might be sex, age, and bone site-specific. The present study reinforces the idea that EO induces short- and long-term effects not only upon body weight, adiposity and thyroid function but also upon bone metabolism and density. We have shown that the excess of fat mass contributed to an increased bone mass and suggest that this increase could be mediated by the hypothyroidism, and previous higher thyroid hormone action and hyperinsulinemia and hyperleptinemia at weaning, previously reported in this experimental model [7]. Furthermore, the increased biomechanical loading due to increased body weight may probably help us to understand the protective effects obesity exerts upon bone health.

Acknowledgements

▼

All the authors are grateful to José Firmino Nogueira-Neto, Jacqueline Siqueira, and Vania Pinto from Laboratory of Lipids (LabLip, UERJ) for ionized calcium determination, to Marcos Borges from University Hospital Pedro Ernesto (HUPE – UERJ) for 25(OH)D dosage, and to Interdisciplinary Laboratory for Nutritional Assessment (LIAN – UERJ) for DXA analyzes.

Conflict of Interest

▼

The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.

References 1 Rosen CJ, Bouxsein ML. Mechanisms of disease: is osteoporosis the obesity of bone? Nat Clin Pract Rheumatol 2006; 2: 35–43 2 Chinn S, Rona RR. Prevalence and trends in overweight and obesity in three cross-sectional studies of British children 1974–94. BMJ 2001; 322: 24–26

de Albuquerque Maia L et al. Early Overfeeding Improves Bone Tissue … Horm Metab Res 2014; 46: 259–268

This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.

266 Endocrine Research

3 de Onis M, Blössner M, Borghi E. Global prevalence and trends of overweight and obesity among preschool children. Am J Clin Nutr 2010; 92: 1257–1264 4 Whitaker RC, Pepe MS, Wright JA, Seidel KD, Dietz WH. Early adiposity rebound and the risk of adult obesity. Pediatrics 1998; 101: E5 5 Krassas GE, Tzotzas T. Do obese children become obese adults: childhood predictors of adult disease. Pediatr Endocrinol Rev 2004; 3: 455–459 6 Dörner G, Hagen N, Witthuhn W. Early postnatal overfeeding as an etiopathogenetic factor in adult obesity. Acta Biol Med Ger 1976; 35: 799–803 7 Rodrigues AL, de Moura EG, Passos MC, Dutra SC, Lisboa PC. Postnatal early overnutrition changes the leptin signaling pathway in the hypothalamic-pituitary-thyroid axis of young and adult rats. J Physiol 2009; 587: 2647–2661 8 Rodrigues AL, de Moura EG, Passos MC, Trevenzoli IH, da Conceição EP, Bonono IT, Neto JF, Lisboa PC. Postnatal early overfeeding induces hypothalamic higher SOCS3 expression and lower STAT3 activity in adult rats. J Nutr Biochem 2011; 22: 109–117 9 Barker DJ. The developmental origins of adult disease. J Am Coll Nutr 2003; 23: 588S–595S 10 Moura EG, Passos MC. Neonatal programming of body weight regulation and energetic metabolism. Biosci Rep 2005; 25: 251–269 11 de Moura EG, Lisboa PC, Passos MC. Neonatal programming of neuroimmunomodulation – role of adipocytokines and neuropeptides. Neuroimmunomodulation 2008; 15: 176a–188a 12 Jones G. Early life nutrition and bone development in children. Nestle Nutr Workshop Ser Pediatr Program 2011; 68: 227–233 13 Devlin MJ, Grasemann C, Cloutier AM, Louis L, Alm C, Palmert MR, Bouxsein ML. Maternal perinatal diet induces developmental programming of bone architecture. J Endocrinol 2013; 217: 69–81 14 NIH. Osteoporosis prevention, diagnosis, and therapy. NIH Consensus Statement 2000; 17: 1–45 15 Eastell R, Lambert H. Diet and healthy bones. Calcif Tissue Int 2002; 70: 400–404 16 Plagemann A, Heidrich I, Götz F, Rohde W, Dörner G. Obesity and enhanced diabetes and cardiovascular risk in adult rats due to early postnatal overfeeding. Exp Clin Endocrinol 1992; 99: 154–158 17 Davidowa H, Plagemann A. Hypothalamic neurons of postnatally overfed, overweight rats respond differentially to corticotropin-releasing hormones. Neurosci Lett 2004; 371: 64–68 18 Boullu-Ciocca S, Dutour A, Guillaume V, Achard V, Oliver C, Grino M. Postnatal diet-induced obesity in rats upregulates systemic and adipose tissue glucocorticoid metabolism during development and in adulthood: its relationship with the metabolic syndrome. Diabetes 2005; 54: 197–203 19 Conceição EP, Moura EG, Trevenzoli IH, Peixoto-Silva N, Pinheiro CR, Younes-Rapozo V, Oliveira E, Lisboa PC. Neonatal overfeeding causes higher adrenal catecholamine content and basal secretion and liver dysfunction in adult rats. Eur J Nutr 2013; 52: 1393–1404 20 Conceição EP, Franco JG, Oliveira E, Resende AC, Amaral TA, Peixoto-Silva N, Passos MC, Moura EG, Lisboa PC. Oxidative stress programming in a rat model of postnatal early overnutrition – role of insulin resistance. J Nutr Biochem 2013; 24: 81–87 21 Steppan CM, Crawford DT, Chidsey-Frink KL, Ke H, Swick AG. Leptin is a potent stimulator of bone growth in ob/ob mice. Regul Pept 2000; 92: 73–78 22 Thrailkill KM, Lumpkin CK Jr., Bunn RC, Kemp SF, Fowlkes JL. Is insulin an anabolic agent in bone? Dissecting the diabetic bone for clues. Am J Physiol Endocrinol Metab 2005; 289: E735–E745 23 Marques RG, Morales MM, Petroianu A. Brazilian law for scientific use of animals. Acta Cir Bras 2009; 24: 69–74 24 Lukaski HC, Hall CB, Marchello MJ, Sider WA. Validation of dual x-ray absorptiometry for body-composition assessment of rats exposed to dietary stressors. Nutrition 2001; 17: 607–613 25 Sirois I, Cheung AM, Ward WE. Biomechanical bone strength and bone mass in young male and female rats fed a fish oil diet. Prostaglandins Leukot Essent Fatty Acids 2003; 68: 415–442 26 Feldkamp LA, Davis LC, Kress JW. Practical cone beam algorithm. J Opt Soc Am A 1984; 1: 612–619 27 Ciarelli TE, Fyhrie DP, Schaffler MB, Goldstein SA. Variations in threedimensional cancellous bone architecture of the proximal femur in female hip fractures and in controls. J Bone Miner Res 2000; 15: 32–40 28 Trebacz H, Zduneck A. Three-point bending and acoustic emission study of adult rat femora after immobilization and free remobilization. J Biomech 2006; 39: 237–245 29 Haarbo J, Gotfredsen A, Hassager C, Christiansen C. Validation of body composition by dual energy X-ray absorptiometry (DEXA). Clin Physiol 1991; 11: 331–341

30 Lac G, Cavalie H, Ebal E, Michaux O. Effects of a high fat diet on bone of growing rats Correlations between visceral fat, adiponectin and bone mass density. Lipids Health Dis 2008; 7: 1–4 31 Pluijm SM, Visser M, Smit JH, Popp-Snijders C, Roos JC, Lips P. Determinants of bone mineral density in older men and women: body composition as mediator. J Bone Miner Res 2001; 16: 2142–2151 32 Reid IR. Relationships among body mass, its components, and bone. Bone 2002; 31: 547–555 33 Clark EM, Ness AR, Tobias JH. Adipose tissue stimulates bone growth in prepubertal children. J Clin Endocrinol Metab 2006; 91: 2534–2541 34 Albala C, Yanez M, Devoto E, Sostin C, Zeballos L, Santos JL. Obesity as a protective factor for postmenopausal osteoporosis. Int J Obes Relat Metab Disord 1996; 20: 1027–1032 35 Klein KO, Larmore KA, de Lancey E, Brown JM, Considine RV, Hassink SG. Effect of obesity on estradiol level, and its relationship to leptin, bone maturation, and bone mineral density in children. J Clin Endocrinol Metab 1998; 83: 3469–3475 36 Halaas JL, Gajiwala KS, Maffei M, Cohen SL, Chait BT, Rabinowitz D, Lallone RL, Burley SK, Friedman JM. Weight-reducing effects of the plasma protein encoded by the obese gene. Science 1995; 269: 543–546 37 Thomas T, Burguera B, Melton LJ 3rd, Atkinson EJ, O’Fallon WM, Riggs BL, Khosla S. Role of serum leptin, insulin, and estrogen levels as potential mediators of the relationship between fat mass and bone mineral density. Bone 2001; 29: 114–120 38 Blain H, Vuillemin A, Guillemin F, Durant R, Hanesse B, de Talance N, Doucet B, Jeandel C. Serum leptin level is a predictor of bone mineral density in postmenopausal women. J Clin Endocrinol Metab 2002; 87: 1030–1035 39 Maor G, Rochwerger M, Segev Y, Phillip M. Leptin acts as a growth factor on the chondrocytes of skeletal growth centers. J Bone Miner Res 2002; 17: 1034–1043 40 Plagemann A, Harder T, Rake A, Waas T, Melchior K, Ziska T, Rohde W, Dörner G. Observations on the orexigenic hypothalamic neuropeptide Y-system in neonatally overfed weanling rats. J Neuroendocrinol 1999; 11: 541–546 41 Gogakos AI, Duncan Bassett JH, Williams GR. Thyroid and bone. Arch Biochem Biophys 2010; 503: 129–136 42 Ben-Sholmo A, Hagag P, Evans S, Weiss M. Early postmenopausal bone loss in hyperthyroidism. Maturitas 2001; 39: 19–27 43 Murphy E, Gluer CC, Reid DM, Felsenberg D, Roux C, Eastell R, Williams GR. Thyroid function within the upper normal range is associated with reduced bone mineral density and an increased risk of nonvertebral fractures in healty euthyroid postmenopausal women. J Clin Endocrinol Metab 2010; 95: 3173–3181 44 Eriksen EF, Moskelide L, Melsen F. Kinetics of trabecular bone resorption and formation in hypothyroidism: evidence for a positive balance per remodeling cycle. Bone 1986; 7: 101–108 45 Madeira E, Mafort TT, Madeira M, Guedes EP, Moreira RO, de Mendonça LM, Lima IC, de Pinho PR, Lopes AJ, Farias ML. Lean mass as a predictor of bone density and microarchitecture in adult obese individuals with metabolic syndrome. Bone 2014; 59: 89–92 46 Reid IR, Ames R, Evans MC, Sharpe S, Gamble G, France JT, Lim TM, Cundy TF. Determinants of total body and regional bone mineral density in normal postmenopausal women – a key role for fat mass. J Clin Endocrinol Metab 1992; 75: 45–51 47 Iwaniec UT, Dube MG, Boghossian S, Song H, Helferich WG, Turner RT, Kalra SP. Body mass influences cortical bone mass independent of leptin signaling. Bone 2009; 44: 404–412 48 Seeman E, Delmas PD. Bone quality – the material and structural bias of bone strength and fragility. N Engl J Med 2006; 354: 2250–2261 49 Feldkamp LA, Goldstein SA, Parfitt AM, Jesion G, Kleerekoper M. The direct examination of three dimensional bone architecture in vitro by computed tomography. J Bone Miner Res 1989; 4: 3–11 50 Ma H, Turpeinen T, Silvennoinen M, Torvinen S, Rinnankoski-Tuikka R, Kainulainen H, Timonen J, Kujala UM, Rahkila P, Suominen H. Effects of diet-induced obesity and voluntary wheel running on the microstructure of the murine distal femur. Nutr Metab (Lond) 2011; 8: 1–11 51 Hildebrand T, Rüegsegger P. A new method for the model-independent assessment of thickness in three-dimensional images. J Microsc 1997; 185: 67–75 52 Eckstein F, Matsuura M, Kuhn V, Priemel M, Müller R, Link TM, Lochmüller EM. Sex differences of human trabecular bone microstructure in aging are site-dependent. J Bone Miner Res 2007; 22: 817–824 53 Xu Y, Li D, Chen Q, Fan Y. Full supervised learning for osteoporosis diagnosis using micro-CT images. Microsc Res Tech 2013; 76: 333–341 54 Saha PK, Xu Y, Duan H, Heiner A, Liang GY. Volumetric topological analysis: A novel approach for trabecular bone classification on the continuum between plates and rods. IEEE Trans Med Imaging 2010; 29: 1821–1838

de Albuquerque Maia L et al. Early Overfeeding Improves Bone Tissue … Horm Metab Res 2014; 46: 259–268

267

This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.

Endocrine Research

55 Kleerekoper M, Villanueva AR, Stanciu J, Rao DS, Parfitt AM. The role of three-dimensional trabecular microstructure in the pathogenesis of vertebral compression fractures. Calcif Tissue Int 1985; 37: 594–597 56 Hahn M, Vogel M, Kempa MP, Delling G. Trabecular bone pattern factora new parameter for simple quantification of bone microarchitecture. Bone 1992; 13: 327–330 57 Heep H, Wedemeyer C, Wegner A, Hofmeister S, von Knoch M. Differences in trabecular bone of leptin-deficient ob/ob mice in response to biomechanical loading. Int J Biol Sci 2008; 4: 169–175 58 Brahmabhatt V, Rho J, Bernardis L, Gillespie R, Ziv I. The effects of dietary-induced obesity on the biomechanical properties of femora in male rats. Int J Obes Relat Metab Disord 1998; 22: 813–818 59 Lee AJ, Hodges S, Eastell R. Measurement of osteocalcin. Ann Clin Biochem 2000; 37: 432–446 60 Ip TY, Peterson J, Byrner R, Tou JC. Bone responses to body weight and moderate treadmill exercising in growing male obese (fa/fa) and lean Zucker rats. J Musculoskelet Neuronal Interact 2009; 9: 155–166 61 Looker AC, Bauer DC, Chesnut CH III, Gundberg CM, Hochberg MC, Klee G, Kleerekoper M, Watts NB, Bell NH. Clinical use of biochemical markers of bone remodeling: current status and future directions. Osteoporos Int 2000; 11: 467–480 62 Egan E, Reilly T, Giacomoni M, Redmond L, Turner C. Bone mineral density among female sports participants. Bone 2006; 38: 227–233 63 Bréban S, Chappard C, Jaffré C, Benhamou CL. Hypoleptinaemia in extreme body mass models: the case of international rugby players. J Sci Med Sport 2010; 13: 479–484 64 Fukumoto S, Martin TJ. Bone as an endocrine organ. Trends Endocrinol Metab 2009; 20: 230–236 65 Lee NK, Sowa H, Hinoi E, Ferron M, Ahn JD, Confavreux C, Dacquin R, Mee PJ, McKee MD, Jung DY, Zhang Z, Kim JK, Mauvais-Jarvis F, Ducy P, Karsenty G. Endocrine regulation of energy metabolism by the skeleton. Cell 2007; 130: 456–469 66 Lee NK, Karsenty G. Reciprocal regulation of bone and energy metabolism. Trends Endocrinol Metab 2008; 19: 161–166 67 Confavreux CB, Levine RL, Karsenty G. A paradigm of integrative physiology, the crosstalk between bone and energy metabolisms. Mol Cell Endocrinol 2009; 310: 21–29 68 Gravenstein KS, Napora JK, Short RG, Ramachandran R, Carlson OD, Metter EJ, Ferrucci L, Egan JM, Chia CW. Crosssectional evidence of a signaling pathway from bone homeostasis to glucose metabolism. J Clin Endocrinol Metab 2011; 96: E884–E890 69 Kanazawa I, Yamaguchi T, Tada Y, Yamauchi M, Yano S, Sugimoto T. Serum osteocalcin level is positively associated with insulin sensitivity and secretion in patients with type 2 diabetes. Bone 2011; 48: 720–725

70 Reinehr T, de Sousa G, Alexy U, Kersting M, Andler W. Vitamin D status and parathyroid hormone in obese children before and after weight loss. Eur J Endocrinol 2007; 157: 225–232 71 Bell NH, Epstein S, Greene A, Shary J, Oexmann MJ, Shaw S. Evidence for alteration of the vitamin D-endocrine system in obese subjects. J Clin Invest 1985; 76: 370–373 72 Parikh SJ, Edelman M, Uwaifo GI, Freedman RJ, Semega-Janneh M, Reynolds J, Yanovski JA. The relationship between obesity and serum 1,25- dihydroxyvitamin D concentrations in healthy adults. J Clin Endocrinol Metab 2004; 89: 1196–1199 73 Lima N, da S, de Moura EG, Passos MC, Nogueira Neto FJ, Reis AM, de Oliveira E, Lisboa PC. Early weaning causes undernutrition for a short period and programmes some metabolic syndrome components and leptin resistance in adult rat offspring. Br J Nutr 2011; 105: 1405–1413 74 de Albuquerque Maia L, Lisboa PC, de Oliveira E, da Silva Lima N, da Costa CA, de Moura EG. Two Models of Early Weaning Decreases Bone Structure by Different Changes in Hormonal Regulation of Bone Metabolism in Neonate Rat. Horm Metab Res 2013; 45: 332–337 75 Nobre JL, Lisboa PC, Lima Nda S, Franco JG, Nogueira Neto JF, de Moura EG, de Oliveira E. Calcium supplementation prevents obesity, hyperleptinaemia and hyperglycaemia in adult rats programmed by early weaning. Br J Nutr 2012; 107: 979–988 76 Devlin MJ, Bouxsein ML. Influence of pre- and peri-natal nutrition on skeletal acquisition and maintenance. Bone 2012; 50: 444–451 77 Paniagua MA, Malphurs JE, Samos LF. BMI and low bone mass in an elderly male nursing home population. Clin Interv Aging 2006; 1: 283–287 78 Chung W, Lee J, Ryu OH. Is the negative relationship between obesity and bone mineral content greater for older women? J Bone Miner Metab 2013, doi:10.1007/s00774-013-0519-9 79 Gower BA, Casazza K. Divergent effects of obesity on bone health. J Clin Densitom 2013; 16: 450–454 80 Pramojanee SN, Phimphilai M, Kumphune S, Chattipakorn N, Chattipakorn SC. Decreased jaw bone density and osteoblastic insulin signaling in a model of obesity. J Dent Res 2013; 92: 560–565 81 Hage RE, Bachour F, Sebaaly A, Issa M, Zakhem E, Maalouf G. The Influence of Weight Status on Radial Bone Mineral Density in Lebanese Women. Calcif Tissue Int 2013, doi:10.1007/s00223-013-9822-7 82 Salamat MR, Salamat AH, Abedi I, Janghorbani M. Relationship between Weight, Body Mass Index, and Bone Mineral Density in Men Referred for Dual-Energy X-Ray Absorptiometry Scan in Isfahan, Iran. J Osteoporos 2013; 2013: 205963

de Albuquerque Maia L et al. Early Overfeeding Improves Bone Tissue … Horm Metab Res 2014; 46: 259–268

This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.

268 Endocrine Research

Copyright of Hormone & Metabolic Research is the property of Georg Thieme Verlag Stuttgart and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.