THE EFFECTS OF POST PUBERTAL FOOD RESTRICTION ON BONE ARCHITECTURE, STRENGTH, AND MEDULLARY ADIPOSE COMPOSITION

Abstract

The purpose of this investigation was to determine the effects of post pubertal caloric restriction on bone architecture, strength, and medullary adipose quantity. A randomized control comparison design was utilized and the study was conducted in a laboratory setting. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Temple University (protocol number 3396). Female Sprague Dawley rats (23days-of-age, n=120) were randomly assigned into seven groups, baseline (BL) (n=18), control (C) (n=17), caloric restriction (FR) (n=17), control recovery (RC) (n=17), caloric restriction recovery (RFR) (n=17), control ovariectomy (COVX) (n=17) and food restricted ovariectomy (FROVX) (n=17). On day 65, a 6 week 30% caloric restriction protocol was administered. Following food restriction, a subset of the control and food restricted groups were sacrificed (n=34) and the remaining animals (n=68) control recovery (RC) and food restricted recovery (RFR) groups had a 10 week recovery with ad lib food. Recovery groups, RC and RFR: were sacrificed after the 10 week recovery period at 183 days of age (n=34). The remaining animals were ovariectomized (OVX) and grouped into control ovariectomy (COVX) and food restricted ovariectomy (FROVX). Six weeks post OVX the animals were sacrificed at 270 days of age. After sacrifice blood was taken by cardiac puncture, bones were harvested, cleaned of soft tissue, fixed and prepared for analysis. Anthropometric measurements were taken including retroperitineal and gonadal fat pad weights as well as adrenal glands, ovaries, uteri, and tricep surae muscle group weights. Main Outcome Measures: The outcome variables for this study were bone mechanical competence, trabecular and cortical bone mass and architecture, marrow adipocyte number as well as serum markers of bone formation and resorption. Insulin - like growth factor - 1 (IGF-1) and C- terminal telopeptide (CTX) was measured to determine bone formation and resorption. Statistical Analysis: One-way Analysis of Variance (ANOVA) was performed to determine differences between all groups. Tukey's honestly significant difference (HSD) post hoc analysis was conducted to determine differences between groups. Student's t - tests were used to detect differences between age groups (acute, recovery, post-OVX) A p value was set at less than or equal to 0.05 for all statistical tests. All statistical analysis was performed using (GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego California USA). Variables were normalized with a linear regression-based correction using body weight. All variables with an R2 level greater than 0 were normalized to avoid choosing an arbitrary R2 value as a cut-off for normalization. Results: Body weight was 18% lower than control animals following caloric restriction. Weight loss was due to fat mass predominately; muscle mass was maintained relative to body weight. Bone length and growth rates were diminished however no differences were found following refeeding. No differences were found in bone strength at any time point. However relative to body weight peak moment and stiffness were significantly higher following caloric restriction. Cortical bones mass and cross sectional moment of inertia were enhanced in the femoral diaphysis with bone mass greater post OVX in the calorically restricted group (FR-OVX). No significant differences were found in ash percent in the femur was found between any groups at any time point however vertebral bone mineral density in acute FR and post OVX time points in FROVX was significantly greater indicating an enhanced bone quality in the restricted. No change in trabecular quantity or quality were observed in the distal femur between groups however vertebral trabecular architecture was enhanced in number and thickness in acute FR and post OVX time points in FROVX. No significant difference in number of marrow adipocytes were found at any time point. Serum CTX decreased significantly in acute in FR and increased at recovery in RFR and post OVX in FROVX. Serum IGF - 1 decreased in the acute FR with IGF - 1 significantly greater after recovery in RFR. Conclusions: Evidence was found to suggest that moderate caloric restriction (nutrient replete) post puberty was positive for bone. Bone quantity was increased with relative cortical area and bone area relative to body weight increased in the FR group. Significant increases in FROVX bone quantity post OVX suggests that bone mass gains during caloric restriction attenuated cortical bone loss at maturity post OVX. Bone quality increases in cross sectional moment of inertia relative to body weight may have accounted for the transient increase in FR bone strength in the femur. Decreases in acute CTX and IGF- 1 levels indicates that bone formation and resorption were decreased during development that may have been the mechanism for bone loss attenuated post OVX in calorically restricted. Growth rate slowing during caloric restriction may have decreased the rate of formation and resorption during a crucial time of peak bone mass accrual and bone modeling. This decrease in one modeling may have been mechanism that preserved bone quantity during acute caloric restriction. Increases in femur quality in polar moment of inertia coupled with a decrease in bone length changed the shape of the bone making it more robust. A shorter bone with a thicker cortex with no change in mineral content may have been the mechanism in the transient increase in bone strength in the femur. Quality changes in mineral density in vertebrae acting as a mineral storage back up as a last resort if quantity and quality changes were not sufficient in maintain bone strength. Moderate caloric restriction transiently increased strength, by increasing bone mass relative to body, altering bone geometry and increased vertebral mineral density.