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Keywords:

  • bone mass;
  • low protein;
  • resorption;
  • pamidronate;
  • osteoporosis

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Isocaloric dietary protein deficiency is associated with decreased BMD and bone strength as well as depressed somatotroph and gonadotroph axis. Inhibition of increased bone resorption by the bisphosphonate pamidronate in rats fed an isocaloric low-protein diet fully prevents bone loss and alteration of bone strength.

Introduction: Isocaloric dietary protein deficiency is associated with decreased BMD and bone strength as well as depressed somatotroph and gonadotroph axis. This negative bone balance is the consequence of increased bone resorption and decreased bone formation. Whether inhibition of bone resorption could prevent low-protein diet-induced bone loss and alteration of biomechanics is not known.

Materials and Methods: The effect of the bisphosphonate pamidronate was studied in 5.5-month-old female or 6-month-old male rats pair-fed a control (15% casein) or an isocaloric low-protein (2.5% casein) diet for 19 and 26 weeks, respectively. Pamidronate (0.6 mg/kg) was given subcutaneously 5 days/month for 4 months in female rats or for 5 months in male rats. BMD, microarchitecture, and bone strength were measured at the level of the proximal and midshaft tibia. Urinary deoxypyridinoline excretion, serum osteocalcin, and IGF-I were also measured.

Results: The increase in bone resorption in female rats (+100%) and in male rats (+33%) fed a low-protein diet was prevented by pamidronate treatment. The reduced osteocalcin levels observed in rats fed a low-protein diet were further decreased in both female (−34%) and male (−30%) rats treated with pamidronate. The bone turnover decrease induced by pamidronate prevented bone strength reduction, trabecular bone loss, microarchitecture, and BMD alterations induced by the isocaloric low-protein diet. Similar effects were observed at the level of the midshaft tibia. Significant decrease of plasma IGF-I was observed in rats fed a low-protein diet independently of the pamidronate treatment.

Conclusion: In conclusion, inhibition of increased bone resorption in rats fed an isocaloric low-protein diet fully prevents bone loss and alteration of bone strength.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

PREVIOUS STUDIES PERFORMED in adult female or male rats have shown that isocaloric low-protein diet decreases BMD and bone strength at skeletal sites formed by trabecular or cortical bone.(1, 2) These modifications of BMD and mechanical properties induced by an isocaloric low-protein diet are related to a decrease in bone formation and increase in bone resorption with negative bone balance.(1, 2) This leads not only to bone loss, but also to major alterations of the microarchitecture(3) and also of the intrinsic bone tissue quality.(4) The mechanisms underlying these modifications of bone implicate depression of the gonadotroph and somatotroph axis in both genders.(1, 2) The elevated bone resorption could be related to sex hormone deficiency at a later stage and/or the production of bone-resorbing cytokines like TNF.(1, 5, 6)

Bisphosphonates inhibit bone resorption. The benefits of bisphosphonate treatments on fracture risk and bone mechanical properties occur in the presence of a decreased bone formation. Pamidronate is capable of reducing bone turnover, increasing BMC and bone strength in various types of experimental osteoporosis, such as after immobilization,(7) ovariectomy,(8, 9) orchidectomy,(8) administration of corticosteroids,(8) or low calcium diet.(8) However, the influence of bisphosphonates on the bone alterations caused by low-protein diet-induced bone turnover uncoupling is not known.

The aim of this study was to investigate whether inhibition of bone resorption in animals fed an isocaloric low-protein diet could prevent bone loss and alterations of mechanical properties.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Animals and treatment

All experimental designs and procedures received the approval of the Animal Ethics Committee of the Geneva University Faculty of Medicine. Female or male Sprague-Dawley rats (BRL, Basel, Switzerland), housed individually at 25°C with a 12:12-h light-dark cycle, were strictly pair-fed with isocaloric synthetic diets provided by Novartis Nutrition (Berne, Switzerland) containing varying amounts of casein, 0.8% phosphorus, and 1.1% calcium throughout the experimental period. The animals were also given a daily dose of vitamin D dissolved in peanut oil (100 IU/kg body weight).

After 2 weeks of equilibration on a diet containing 15% casein diet, 5.5-month-old female rats were allocated to three groups of 8 rats each; 16 were pair-fed isocaloric diet containing 2.5% casein and 8 others 15% casein for 19 weeks. Among 16 animals pair-fed isocaloric diet containing 2.5% casein, 8 received subcutaneously pamidronate (0.6 mg/kg) at 1, 5, 9, 13, and 17 weeks of the experiment, and 8 other rats were injected with the vehicle at the same time. The dose and schedule of treatment with pamidronate were selected according to previous studies to obtain a control of bone turnover and optimal effect on bone mass and mechanical properties.(10, 11) Blood samples were collected to determine IGF-I and osteocalcin plasma levels. Urine was collected in metabolics cages over 24 hours for the determination of total deoxypyridinoline excretion. DXA measurements were performed at the level of the tibia before nutritional restriction and after 7 and 15 weeks. The same protocol was applied in 6-month-old male rats for 26 weeks. Pamidronate (0.6 mg/kg) was administered at 1, 7, 11, 15, and 22 weeks of the experiment; DXA measurements were done before nutritional restriction and after 8, 16, and 24 weeks.

Biochemical determinations

Plasma osteocalcin and IGF-I were measured by radioimmunoassay, with reagents from Biomedical Technologies (Stoughton, MA, USA) for the former and with a kit from Nichols Institute (San Juan Capistrano, CA, USA) after extraction by acid-ethanol and cryoprecipitation for the latter. Total urinary deoxypyridinoline was determined after acid hydrolysis using a kit from Metra-Biosystems (Mountain View, CA, USA).

Bone mechanical testing

The tibias were excised immediately after death and frozen together with the soft tissues at −20°C in plastic bags. During the night before mechanical testing, bones were slowly thawed at 4°C and maintained at room temperature. The length of tibia was measured using a caliper with electronic digital display, and the middle of the shaft was determined. The tibia midshaft was placed in the material testing machine on two supports separated by 20 mm, and a load was applied on the middle of the shaft, realizing a three-point bending test. Proximal tibia testing was also performed using axial compression of the tibia plateau, the shaft being fixed in methylmethacrylate cement (Technovit 4071; Heraeus Kulzer, Wehrheim, Germany), as previously described.(12) Between the different steps of preparation, each specimen was kept immersed in physiological solution. The mechanical resistance to failure was tested using a servo-controlled electromechanical system (Instron 1114; Instron Corp., High Wycombe, UK) with the actuator displaced at 2 mm/min. Displacement and load were simultaneously recorded every 0.01 s. Ultimate strength (maximal load, N) was directly obtained from the load-deformation curves, the stiffness (slope of the linear part of the curve, representing the elastic deformation, N/mm), and the energy absorbed by the bone tissue (area under the load-deformation curve before the bone breaks, in N × mm) were calculated. Reproducibility was 3.3% and 5.8%, as evaluated by the CV of pair sample measurements (left/right).

BMD measurements

BMD of the whole tibia was measured in vivo using a Hologic QDR-1000 DXA instrument adapted to measurements in small animals as previously described.(11) An ultra-high-resolution mode (line spacing 0.254 mm and resolution 0.127 mm) was used with an 0.9-mm-diameter collimator. During the measurements, the animals were anesthetized with ketamine hydrochloride (100 mg/kg body weight, IP). The in vivo reproducibility of these measurements was evaluated with repositioning, and was <1.8% overall. The stability of the instrument was controlled by scanning a phantom six times a week.

Microtomographic histomorphometry by μCT

Parameters of mass and architecture of the secondary spongiosa of the proximal tibia were studied with a high-resolution μCT system (μCT 40; Scanco Medical, Bassersdorf, Switzerland) as previously described.(13, 14) In summary, 3D images of a proximal tibia were acquired with a voxel size of 20 μm in all spatial directions. The imbedded tibias were secured in a cylindrical sample holder in air. The resulting grayscale images were segmented using a low-pass filter to remove noise and a fixed threshold to extract the mineralized bone phase. The trabecular and cortical parts of the tibia were separated with semi-automatically drawn contours.

From the binarized images, structural indices were assessed. Relative bone volume (BV/TV), trabecular number (Tb.N), thickness (Tb.Th), and separation (Tb.Sp) were calculated by measuring directly the 3D distances(14, 15) in the trabecular network. Connectivity density based on Euler number(16) and the structure model index (SMI) were calculated. The SMI quantifies the plate versus rod characteristics of trabecular bone,(15) where an SMI of 0 indicates a purely plate-shaped bone, an SMI of 3 a rod-like bone, and values in between stand for a mixture of plates and rods.

Statistical analysis

Data are expressed as the mean ± SE for all parameters measured. The significance of difference was evaluated using an ANOVA.p < 0.05 andp < 0.01, using a Fisher test.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Effect of pamidronate on body weight in female or male rats fed an isocaloric low-protein diet

Body weight was lower in female and male rats fed a low-protein diet compared with the 15% casein diet group. In rats treated with pamidronate and fed a low-protein diet, similar body weight decrease was observed in female and male rats, respectively. Thus, an isocaloric low-protein diet resulted in a decreased body weight in female and male rats, independently of the administration of pamidronate.

Effect of pamidronate treatment on markers of bone turnover in female or male rats fed an isocaloric low-protein diet

Osteocalcin was significantly decreased in female rats (−34%) or male rats (−30%) treated with pamidronate (Table 1). Bone resorption, as indicated by urinary deoxypyridinoline excretion, was increased in female rats fed a low-protein diet (+100%). This stimulation was completely prevented by pamidronate administration. Male rats fed an isocaloric protein diet showed a trend toward an increase (+33%) in deoxypyridinoline. Pamidronate induced a decreased (−67%) urinary deoxypyridinoline excretion in male rats fed the 2.5% casein diet.

Table Table 1.. Biochemical Markers of Bone Remodeling in Female or Male Rats Fed a Low-Protein Diet and Treated With Pamidronate
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Effect of pamidronate treatment on plasma IGF-I in female or male rats fed an isocaloric low-protein diet

The low-protein isocaloric diet containing 2.5% casein significantly decreased plasma IGF-I levels in female (−55%) and in male (−34%) rats without any effect of pamidronate (Fig. 1).

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Figure FIG. 1.. IGF-I plasma levels in female or male rats fed a low-protein diet and treated with pamidronate. Values are means ± SE. IGF-I was measured in female or male rats fed a 15%, 2.5%, or 2.5% casein diet and treated with pamidronate. Pamidronate (0.6 mg/kg) was administered 1 week subcutaneously in female rats after 1, 5, 9, 13, and 17 weeks and in male rats after 1, 7, 11, 15, and 22 weeks. Significant differences between the groups (n = 8) using an ANOVA and Fisher test were observed: *vs. 15% casein diet.

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Effect of pamidronate treatment on BMD, microarchitecture, and bone strength in tibia metaphysis of female or male rats fed an isocaloric low-protein diet

Proximal tibia BMD was significantly decreased after 15 weeks in female rats fed a low-protein diet (Fig. 2). This was associated with major alteration of bone microarchitecture as evaluated by μCT. These changes were completely prevented by pamidronate (Table 2). A decrease in BMD induced by protein undernutrition was detectable in male rats fed 2.5% casein after 16 weeks and even more pronounced after 24 weeks (Fig. 3). An alteration of microarchitecture was also observed in male rats (Table 2). These alterations were prevented by pamidronate treatment. Even a significant increase in BMD and in microarchitecture parameters was observed in male rats (Fig. 4).

Table Table 2.. Effect of Administration of Pamidronate on Microarchitecture of the Tibia Secondary Spongiosa in Female or Male Rats Fed a Low-Protein Diet
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Figure FIG. 2.. Proximal tibia metaphysis BMD in female rats fed a low-protein diet and treated with pamidronate. BMD of proximal tibia metaphysis was measured before dietary manipulation and after 7 and 15 weeks of 15%, 2.5%, or 2.5% casein diet and treated with pamidronate. Pamidronate (0.6 mg/kg) was administered 1 week subcutaneously in female rats after 1, 5, 9, 13, and 17 weeks. Results are means ± SE. Significant differences between groups (n = 8) using an ANOVA were observed: *vs. 15% casein diet;vs. 2.5% casein diet.

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Figure FIG. 3.. Proximal tibia metaphysis BMD in male rats fed a low-protein diet and treated with pamidronate. BMD in proximal tibia metaphysis was measured before dietary manipulation, and after 8, 16, and 24 weeks of 15%, 2.5%, or 2.5% casein diet treated with pamidronate. Pamidronate (0.6 mg/kg) was administered 1 week subcutaneously in male rats after 1, 7, 11, 15, and 22 weeks. Results are means ± SE. Significant differences between the groups (n = 8) using an ANOVA were observed: *vs. 15% casein diet;vs. 2.5% casein diet.

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Figure FIG. 4.. Effect of pamidronate on trabecular bone volume of the secondary spongiosa of the proximal tibia of female or male rats fed a low-protein diet. Values are means ± SE. Trabecular bone volume was measured in females rats (after 19 weeks) or in male rats (after 26 weeks) fed 15%, 2.5%, or 2.5% casein diet treated with pamidronate. Pamidronate (0.6 mg/kg) was administered 1 week subcutaneously in female rats after 1, 5, 9, 13, and 17 weeks, and in male rats after 1, 7, 11, 15, and 22 weeks. Significant differences between the groups (n = 8) using an ANOVA and Fisher test were observed: *vs. 15% casein diet;vs. 2.5% casein diet.

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BMD decrease and alteration of microarchitecture in proximal tibia were associated with a significant reduction in ultimate strength (−42% and −27%), stiffness (−32% and −24%), and energy absorbed (−50% and −49%) in female and male rats, respectively, fed the low-protein diet. These changes were fully prevented by pamidronate (Table 3).

Table Table 3.. Effect of Administration of Pamidronate on Mechanical Properties of Midshift Tibia in Female or Male Rats Fed a Low-Protein Diet
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At the level of the midshaft tibia, a significant decrease in ultimate strength (−13%) was prevented by pamidronate in female rats fed a low-protein diet (Table 3; Fig. 5). Stiffness and energy showed a trend toward a decrease. These alterations of bone mechanical properties were also associated with a significant decrease of BMD at this site and were prevented by pamidronate (Table 3). In male rats fed a low-protein diet, although slightly lower, BMD, ultimate strength, stiffness, and energy of the midshaft tibia did not significantly differ from the 15% protein control rats. Diameter of the midshaft tibia remains unchanged with or without pamidronate administration in both sexes.

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Figure FIG. 5.. Effect of pamidronate on strength, stiffness, and absorbed energy on the proximal tibia of female or male rats fed a low-protein diet. Values are means ± SE. Proximal tibia ultimate strength, stiffness, and absorbed energy were measured in females rats (after 19 weeks) or in male rats (after 26 weeks) fed 15%, 2.5%, or 2.5% casein diet treated with pamidronate. Pamidronate (0.6 mg/kg) was administered 1 week subcutaneously in female rats after 1, 5, 9, 13, and 17 weeks, and in male rats after 1, 7, 11, 15, and 22 weeks. Significant differences between the groups (n = 8) using an ANOVA and Fisher test were observed: *vs. 15% casein diet;vs. 2.5% casein diet.

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Effect of pamidronate treatment on bone dimensions in female or male rats fed an isocaloric low-protein diet

Tibias external diameter and length were similar in the three studied groups.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

The inhibition of bone resorption by the bisphosphonate pamidronate fully prevented the decrease in BMD and the alterations of microarchitecture and mechanical properties induced by isocaloric low-protein intake both in male and female rats. By blocking the increased bone resorption induced by protein depletion, pamidronate was thus able to maintain a positive bone balance, despite a low bone formation. This resulted in a preservation of bone mass, bone microarchitecture, and bone strength, and even in an improvement in male rats at the level of the proximal tibia.

Malnutrition, and more specially protein malnutrition, is frequent in the elderly.(17, 18) It is associated with bone loss(19, 20) and increased risk of fracture.(21) The recommended protein intake in elderly is 1–1.2 g/kg of ideal body weight; thus, it was shown that patients with a fracture of the proximal femur and most of the elderly have a mean protein intake of 0.6 g/kg body weight.(17, 18) The decrease of protein intake corresponds to 50% of the minimal requisite. To study the mechanisms involved, we set up a rat model of osteoporosis induced by feeding an isocaloric low-protein diet in male(22) and female(1) rats. In this model, we showed that when decreasing the casein content from 15% to 5%, bone turnover was normal, and no modifications of microarchitecture and BMD were detectable.(1) In contrast, by decreasing the casein intake from 5% to 2.5% (i.e. a 50% reduction), we observed major modifications of bone turnover and a net bone loss. These results are in agreement with previous studies that showed that an isocaloric low-protein diet decreased BMD and altered mechanical resistance at skeletal sites formed by trabecular and cortical bone (proximal tibia) or by cortical bone (midshaft tibia). The effects of the low-protein diet were less pronounced at skeletal sites formed mainly of cortical bone. Because there is a different time-course of protein depletion between male and female rats,(22) with modification of bone mass and quality occurring later in male than in female rats, we applied a different schedule in males or in females. Different response rates to gonadectomy were also described,(23–25) with significant decreases of BMD and bone mechanical properties detectable earlier in females than in males.

Under an isocaloric low-protein diet, a state of partial androgen deficiency(2) was progressively observed in males; in contrast, estrogen deficiency(1) was observed after 6 weeks in females.(1) Previous studies clearly showed an early increased bone turnover after orchidectomy,(23) with an initial rapid increase of bone turnover and the persistence of moderately elevated turnover.(24) In these experimental conditions, the increase of bone resorption was more marked in female than in male rats, as indicated by the increased urinary excretion of deoxypyridinoline. In contrast to the model of castration, the bone formation was not concomitantly increased under an isocaloric low-protein diet as indicated by decreased osteocalcin. Indeed, a depression of the gonadotroph axis is one mechanism underlying the bone loss and alteration of bone strength observed under an isocaloric low-protein diet.(1) The decreased plasma IGF-I observed in this study is in agreement with previous studies and underlines the gonadotroph axis depression under protein restriction. This uncoupling between bone formation and bone resorption results in a negative bone balance. This observation of uncoupling of bone turnover was previously described using histomorphometric analysis and biochemical markers measurement.(1, 22) It could explain the decrease of BMD, alteration of microarchitecture, and bone fragility in animals fed a low-protein diet.

The aim of this study was to investigate whether inhibition of bone resorption in animals fed an isocaloric low-protein diet could prevent bone loss and alterations of microarchitecture and mechanical properties. To prevent any interference between bone growth, dietary changes, and treatment, the study was performed in animals that had achieved their adult size. At the end of the study, the length and external diameter of the tibias were the same in the various groups. The only bone modification was remodeling. The inhibition of bone resorption by pamidronate treatment resulted in a major decrease of the urinary deoxypyridinoline excretion in male (−67%) and female (−84%) rats. The resorption was significantly lower than in control animals. Plasma osteocalcin was already decreased in rats fed a low-protein diet, reflecting the decreased bone formation. Pamidronate reduced bone turnover, leading to a further decrease of osteocalcin. A significant increment of BMD under pamidronate suggests that a positive bone balance was achieved despite a low bone formation. This was confirmed by μCT analysis, showing an increased trabecular volume compared with baseline and control values in male rats and control values in female rats. A transient increment of PTH after pamidronate administration is not excluded and could account for the significantly higher trabecular volume and BMD than in controls. Furthermore, the treatment was started simultaneously with the introduction of the low-protein diet. Thus, at the beginning of the treatment, the microarchitecture was still conserved.

This modification of bone turnover (inhibition of bone resorption) was associated at the level of the proximal tibia with a full prevention of bone loss and alteration of bone mechanical properties in male and female rats; a significant increment of these two parameters, compared with controls, was observed in male rats. A full prevention of the deleterious effects of the low-protein diet was observed in male and female rats at the level of the midshaft tibia. The preservation of bone strength results from the maintenance of bone mass and microarchitecture. An improvement of bone mineralization, as well as a reduced volume of bone remodeling space, was previously suggested to explain the improvement of bone strength under bisphosphonate.(26) Similar effects in animals fed a low-protein diet could be expected. These positive effects of an inhibitor of bone resorption, in protein malnourished rats, contrast with the deleterious effects induced by a stimulator of bone formation in similar experimental conditions.(2) Indeed, treatments with IGF-I or growth hormone were ineffective or even dose-dependently deleterious in rats fed an isocaloric low-protein diet.(2, 27) Our results show that bisphosphonates are efficacious even in protein deficiency.

In conclusion, despite a catabolic status induced by the isocaloric low-protein diet (decreased body weight, muscle weight, and bone strength), the inhibition of bone resorption maintains a positive bone balance. A full prevention of bone strength decrease by pamidronate was obtained in male and female rats and even a significant increment of bone strength in male rats at the level of the proximal tibia. A significant increment of trabecular volume was observed in animals fed an isocaloric low-protein diet and receiving bisphosphonates. Thus, despite depressed bone formation, an inhibition of bone resorption could restore a positive bone balance.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

We thank I Badoud, S Clément, and C Godin for expert technical assistance and M Perez for secretarial assistance. This project was supported by Swiss National Science Foundation Grants 3200B0–100714 and 32–67942.02.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
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