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Keywords:

  • bone mass;
  • elderly;
  • fracture;
  • nutrition;
  • osteoporosis

Abstract

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

Protein undernutrition is a known factor in the pathogenesis of osteoporotic fracture in the elderly, but the mechanisms of bone loss resulting from this deficiency are still poorly understood. We investigated the effects of four isocaloric diets with varying levels of protein content (15, 7.5, 5, and 2.5% casein) on areal bone mineral density (BMD), bone ultimate strength, histomorphometry, biochemical markers of bone remodeling, plasma IGF-I, and sex hormone status in adult female rats. After 16 weeks on a 2.5% casein diet, BMD was significantly decreased at skeletal sites containing trabecular or cortical bone. Plasma IGF-I was decreased by 29–34% and no estrus sign in vaginal smear was observed. To investigate the roles of estrogen deficiency and protein undernutrition, the same protocol was used in ovariectomized (OVX) or sham-operated (SHAM) rats, pair-fed isocaloric diets containing either 15 or 2.5% casein. Trabecular BMD was decreased by either manipulation, with effects appearing to be additive. Cortical BMD was decreased only in rats on a low-protein diet. This was accompanied by an increased urinary deoxypyridinoline excretion without any change in osteocalcin levels, suggesting an uncoupling between resorption and formation. Isocaloric protein undernutrition decreased bone mineral mass and strength. This effect might be related to decreased plasma IGF-I and/or estrogen deficiency with a consequent imbalance in bone remodeling.


INTRODUCTION

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

Nutritional deficiency has consistently been documented in elderly patients with hip fracture.(1–8) Low-protein intake can be associated with reduced femoral neck bone mineral density (BMD) and a decline in physical fitness.(9) By increasing falling propensity as a result of muscle weakness, impaired movement coordination,(10) and/or by decreasing bone mass, protein undernutrition increases the risk of osteoporotic fractures. Thus, the integrity of the skeleton in the elderly could be affected by an inadequate low-protein intake, apart from an insufficient supply of bone mineral elements and vitamin D.(11–13) However, undernutrition can concern all kinds of nutrients and the specific role of a low-protein intake can be difficult to appraise in the elderly.(9)

In association with the progressive age-dependent decrease in both protein intakes and bone mass, several reports have documented a corresponding decrement in plasma insulin-like growth factor I (IGF-I) levels.(14,15) Experimental and clinical studies suggest that dietary proteins, by influencing both the production and action of growth factors, particularly the growth hormone (GH)-IGF system, could influence bone metabolism.(8,16,17) Dietary proteins are important regulators of the hepatic production and plasma level of this growth factor,(18,19) and their restriction has been shown to reduce IGF-I plasma levels by inducing a resistance to the action of GH at the hepatic level,(20,21) which depends on a decrease in GH hepatic binding sites, on postreceptor defects,(22) and on transcriptional alteration.(23) However, protein restriction can increase IGF-I metabolic clearance rate(24) and render target organs less sensitive to IGF-I, as suggested by a resistance to IGF-I exogenous administration in rats maintained under a low-protein diet.(25) Because IGF-I can increase bone mass and strength in osteoporotic rats,(26,27) the relationship between protein intake, IGF-I levels, and bone homeostasis was worth being assessed.

To address the issue of the specific influence of protein deficiency in the pathogenesis of osteoporosis, we investigated the effect in adult female rats of selective protein deprivation with isocaloric low-protein diets supplemented by identical amounts of minerals, on bone mineral mass, bone strength and remodeling, IGF-I plasma levels, and sex hormone status.

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 treatments

All experimental designs and procedures have received the approval of the Animal Ethics Committee of the Geneva University Faculty of Medicine. Female Sprague-Dawley rats (Novartis Ltd., 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. Isocaloric diets were obtained by the addition of fat and carbohydrate from corn. The animals were also given a daily dose of vitamin D dissolved in peanut oil (100 IU/kg body weight).

Effect of protein undernutrition on areal bone mineral density, bone strength, cortical histomorphometry, and plasma IGF-I

Experiment 1: After 2 weeks of equilibration on a diet containing 15% casein, 5.5-month-old female rats were allocated to four groups of 7 rats each, which were pair-fed isocaloric diets containing 2.5, 5, 7.5, and 15% casein for 16 weeks. Dual energy X-ray absorptiometry (DXA) measurements were performed at the level of the lumbar spine and tibia before nutritional restriction and after 7 and 15 weeks. Femoral neck BMD was also measured ex vivo. Blood samples were collected regularly to determine IGF-I and osteocalcin plasma levels. At the end of the study, sex hormone status was evaluated by determining the presence and regularity of cycles using vaginal smear analysis.(28)

Experiment 2: A similar protocol was used. To study the effects of a long-term low-protein diet, 5-month-old female rats were allocated to two groups of 7 rats each, and 7 were pair-fed isocaloric diets containing 2.5 or 15% casein for 7 months. DXA measurements were performed before nutritional restrictions, and after 4 and 7 months. Blood samples were regularly collected for IGF-I plasma level determination. Bone strength was evaluated at the end of the experiment.

Relative contribution of protein undernutrition and estrogen deficiency

Experiment 3: After 2 weeks of adaptation to a 15% casein diet, 6-month-old rats underwent transabdominal ovariectomy (OVX) (n = 20) or sham operation (SHAM) (n = 20) under anesthesia with intraperitoneal ketamine hydrochloride (100 mg/kg body weight). Effectiveness of OVX was verified at the end of the experiment by visualizing the atrophy of the uterus and the absence of ovarian tissue. The animals were pair-fed isocaloric diets containing either 15 or 2.5% casein (SHAM 15% [n = 10], SHAM 2.5% [n = 10], OVX 15% [n = 10], and OVX 2.5% [n = 10]) for 16 weeks. DXA measurements were performed before dietary manipulation, and after 7 and 30 weeks. At that time, blood was sampled from the tip of the tail and urine collected in metabolic cages over 12 h for the determination of total deoxypyridinoline excretion. At death, lumbar vertebrae, tibia, and femur were collected for mechanical testing.

Bone mineral density measurements

BMD was measured by DXA using a Hologic QDR-1000 instrument adapted to measurement in small animals.(29) An ultra high resolution mode (line spacing 0.254 mm and resolution 0.127 mm) was used with a 0.9-mm-diameter collimator. During the measurements, the animals were anesthetized with ketamine hydrochloride (100 mg/kg body weight). BMD, bone mineral content (BMC), and scanned area were recorded in vivo at the level of lumbar vertebrae, tibia, and femur as previously described.(27) The in vivo reproducibility was evaluated by the coefficient of variation (CV) of repeated measurements with repositioning, and was below 1.8% overall. The stability of the instrument was controlled by scanning a phantom six times a week.

Bone mechanical testing

The lumbar spine, tibia, and femur were excised immediately after death, and frozen at −20°C in plastic bags. During the night before mechanical testing, bones were slowly thawed at 7°C and then maintained at room temperature. Vertebra L4 was isolated from the lumbar spine at the level of the intervertebral discs. The vertebral pedicules were dissected out with care to avoid any damage to the cortical shell. Because the caudal and cranial surfaces of the rat vertebral body are not parallel, 1 mm of the caudal and cranial parts of each vertebral body was embedded in methylmethacrylate cement (Technovit 4071; Heraeus Kulzer GmbH, Wehrheim, Germany), to ensure regular distribution of the compressive forces. Between the different steps of preparation, each specimen was kept immersed in physiological solution. The fibula was removed and the length of tibia (distance from intermalleolar to intercondylar region) was measured, using a caliper with electronic digital display and the middle of the shaft determined. The tibia was then placed in the material testing machine on two supports separated by a distance of 20 mm and load was applied on the middle of the shaft, realizing a three-point bending test. A proximal tibia compression test was also performed using axial compression of the tibia plateau, in which the shaft was fixed in methylmethacrylate cement.(30) Femoral neck testing was performed by maintaining the femur in a vertical position, and embedding the shaft in methylmethacrylate cement up to the minor trochanter before application of a vertical load on the femoral head.(26) The mechanical resistance to failure was tested using a servo-controlled electromechanical system (Instron 1114, Instron Corp., High Wycombe, U.K.) with the actuator displaced at 2 mm/minute. Both displacement and load were recorded. Ultimate strength (maximal load, N) and stiffness (slope of the linear part of the curve, representing the elastic deformation, N/cm) were calculated. Reproducibility for vertebrae, tibia, and femur was between 3.3 and 5.8%, evaluated as the CV of pair sample measurements (left/right, L3/L4).

Bone histomorphometry

In the group that had undergone a long term of protein undernutrition, trabecular bone was markedly reduced and precluded a reliable analysis. Thus, only cortical histomorphometry was performed in animals fed 15 and 2.5% casein. Tibia was fixed, dehydrated, and embedded undecalcified in a methylmethacrylate-based solution, as previously described.(31) One 150-μm-thick mid-diaphysis cross-section was obtained with a low-speed diamond saw (Buehler Ironet, Switzerland). Samples were then fixed using a cyano-acrylic glue and manually ground to a thickness of 15-20 μm. Histomorphometric analysis was performed semiautomatically with a Leica Quantimet Q550 color image processor equipped with a Sony 930 camera coupled to a Leitz DM/RBE microscope. One section per animal was examined. The following parameters were measured and calculated (standard terms and abbreviations are used, according to the American Society for Bone and Mineral Research histomorphometry nomenclature): cortical area (Ct. Ar. expressed in mm2), percentage cortical area (expressed in percentage of total area [T. Ar.] of the bone section), marrow area (Ma. Ar. expressed in mm2), percentage marrow area, endosteal perimeter (E. Pm. expressed in mm), and periosteal perimeter (P. Pm. expressed in mm).

Bone dimensions

External diameter of the femoral neck, midshaft femur, and tibia, were measured in the axis of the applied force using a digital caliper.

Biochemical determinations

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

Determination of cycle duration

Vaginal smear was obtained by five injections and aspirations of 37°C NaCl solution between 10 and 12 a.m. on 14 consecutive days. The cellular material obtained was spread on cover glass, dried, and colored with a toluidine blue solution. The characteristics of the cells (form and staining) allowed the determination of the cycle stage and its duration.(28)

Statistical analysis

All results are expressed as means ± SEM. Significance of difference between groups was evaluated with a one-way analysis of variance (ANOVA), followed by a Fisher test, or a two-way ANOVA for repeated measures.

RESULTS

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

Effect of low-protein isocaloric diets on areal bone mineral density, bone strength, and bone histomorphometry

A decrease in BMD induced by protein undernutrition was already detectable in animals fed 2.5% casein after 7 weeks and was more pronounced after 15 weeks (Fig. 1). At that time, the decrease in BMD (5-16%) was observed in skeletal sites containing both cortical and trabecular bone such as the lumbar spine, proximal tibia, and femoral neck, as well as in skeletal sites containing mainly cortical bone (midshaft tibia and femur). After 7 months on a 2.5% casein isocaloric diet, all the skeletal sites were markedly affected (Table 1). These BMD decreases were associated with significant alterations of bone mechanical properties as evaluated at the level of the lumbar vertebra body, femoral neck, or midshaft tibia (Table 1). A decrease in ultimate strength, stiffness, and energy absorbed in rats fed the lowest protein diet was also observed.

Histomorphometric analysis was performed at the level of the cortical midshaft of the tibia after 16 weeks of low-protein intake. Protein undernutrition resulted in an increase in marrow area and endosteal perimeter, and a decrease in cortical surface without modification of external perimeter and total area (Table 2). These results are compatible with an increased endosteal bone resorption and an absence of marked periosteal bone apposition.

Effect of low-protein isocaloric diets on plasma IGF-I

The low-protein isocaloric diet containing 2.5% casein decreased plasma IGF-I by 30% within 1 week (Fig. 2). The 15, 7.5, or 5% casein isocaloric diets did not influence plasma IGF-I. After 16 weeks, body weight was 249.3 ± 4.7, 253.0 ± 5.2, 248.7 ± 4.6, and 211.4 ± 4.9 g in animals fed with diets containing 15, 7.5, 5, and 2.5% casein, respectively. The same dietary challenge performed in 2-month-old growing rats revealed a greater sensitivity to lowering dietary protein because plasma IGF-I was already decreased with a 5% casein isocaloric diet (588 ± 43 vs. 740 ± 47 ng/ml).

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Figure FIG. 1.. Effect of various protein-containing isocaloric diets on lumbar spine and proximal and midshaft tibia BMD. BMD was measured before dietary manipulation, and after 7 and 15 weeks of isocaloric regimen containing 15, 7.5, 5, and 2.5% casein. Squares represent rats fed 15% casein; circles, 7.5%; open triangles, 5%; and closed triangles, 2.5%. Values expressed as percentage of time control (rats fed a 15% casein diet) are means ± SEM. Using an analysis of variance (ANOVA) for repeated measures, * indicated a significant difference (p < 0.01) from rats fed 15, 7.5, and 5% casein.

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Effect of low-protein isocaloric diets on estrogen status

To test whether long-term protein undernutrition could impair ovarian function, thereby contributing to bone loss, vaginal smear was analyzed and cycle duration determined. Regular cycles of 5.6 ± 0.2, 5.8 ± 0.3, and 6.1 ± 0.3 days were observed in animals fed isocaloric diets containing 15, 7.5, and 5% casein, respectively. In contrast, cycles were totally absent in animals fed a 2.5% casein diet, indicating a state of probable estrogen deficiency or resistance to estrogen action.

Relative contribution of protein undernutrition and estrogen deficiency

Because bone loss induced by protein undernutrition was associated with alterations of the IGF-I system and of sex hormone status, we investigated the relative role played by alterations of these hormonal systems by determining the effects of the 15 or 2.5% casein isocaloric diets in OVX or sham-operated rats. In rats fed a 15% casein diet, OVX led to a decrease of BMD in skeletal sites formed by both cortical and trabecular bone (e.g., lumbar spine, femoral neck, and proximal tibia), but not in predominantly cortical sites such as the midshaft femur or tibia (Fig. 3, Table 3). A low-protein diet caused a similar BMD decrease in vertebrae, proximal tibia, and femoral neck in sham-operated rats. Furthermore, the 2.5% casein diet significantly decreased BMD in the cortical skeletal site. A combination of both OVX and low-protein isocaloric diet produced additive effects on proximal tibia and lumbar spine.

OVX and low-protein isocaloric diet decreased bone strength at each investigated site (Table 3). Interestingly, however, a borderline significant increase of midshaft tibia diameter was observed in OVX rats fed a normal diet. This was accompanied by a slight increase of bone strength. In contrast, in OVX rats fed the 2.5% casein diet, midshaft diameter was unchanged and bone strength was decreased.

As previously shown,(32) OVX rats fed the control diet had increased plasma IGF-I. In contrast, a low-protein isocaloric diet led to a similar decrease in plasma IGF-I in both OVX and sham-operated rats (Table 4). Thus, IGF-I was not affected by OVX in rats receiving the 2.5% casein diet. At the end of the experiment, despite strict pair-feeding of isocaloric diets, the body weights of the different groups were 262.9 ± 3.4 and 286.0 ± 2.5 g in sham-operated or OVX rats fed the 15% casein diet, and 225.0 ± 4.8 and 227.0 ± 4.1 g in sham-operated or OVX rats fed the 2.5% casein diet.

Plasma osteocalcin was increased after OVX in rats fed the 15% casein diet, but not in those on the low-protein diet (Table 4). Bone resorption, as indicated by urinary deoxypyridinoline excretion, was increased after OVX in rats fed the control diet (Table 4). This increase was higher in OVX or sham-operated animals fed the low-protein diet.

DISCUSSION

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

It is recognized that protein deficiency plays an important role in the pathogenesis of osteoporotic fracture in the elderly. Poor intake of various nutrients including mineral, energy, and protein can also contribute to this process.(9,33) In the present study, we investigated the influence of a selective isocaloric protein-restricted diet on bone mineral mass and strength in adult female rats. The results indicate that a low-protein diet induced bone loss and bone fragility at both cortical and trabecular skeletal sites, through mechanisms probably involving an alteration in the IGF-I system and in sex hormone status.

The specific influence of a low-protein diet was determined without interference of the calorie intake because animals were maintained under strict conditions of pair-feeding of isocaloric diets. Deleterious effects of protein undernutrition were observed in rats fed a diet containing less than 5% casein. Thus, a daily intake of at least 5% casein appears to represent the minimal requirement for the adult rat to maintain bone mass. In contrast, growing animals receiving a 5% casein-containing diet have significant growth retardation caused by both a decrease of IGF-I and a resistance to its action.(25) In our model, a 2.5% protein intake represents 50% of the apparent minimal-required protein intake in the adult rat.

Table Table 1.. Effect of a Long-Term Low-Protein Isocaloric Diet in Adult Female Rats
 Dietary casein contentSpineFemoral neckProximal tibiaMidshaft tibia
  1. Values are means ± SEM. BMD and bone strength (ultimate strength, US; stiffness, Stif; energy, E) were determined after 7 months of pair-feeding with isocaloric diets containing 2.5 or 15% casein.

  2. * P < 0.05, P < 0.01, P < 0.001.

BMD (mg/cm2)15.0%221.7 ± 7.2258.8 ± 5.2308.0 ± 5.9269.1 ±5.1
 2.5%201.3 ± 2.4201.0 ± 5.8269.9 ± 4.7254.5 ± 3.7*
US (N)15.0%221.6 ± 19.696.7 ± 7.9163.8 ± 15.4109.4 ± 7.8
 2.5%113.9 ± 12.175.3 ± 4.6*89.68 ± 8.4784.07 ± 3.07*
Stif (N/mm)15.0%821.5 ± 68.1510.9 ± 24.1418.8 ± 45.2295.6 ± 28.8
 2.5%618.7 ± 94.1366.0 ± 23.3367.6 ± 64.5234.8 ± 11.6
E (N × mm)15.0%34.80 ± 3.279.01 ± 1.5657.92 ± 14.3432.65 ± 3.47
 2.5%12.75 ± 2.327.58 ± 0.5519.53 ± 2.6219.95 ± 2.19
Table Table 2.. Effects of Long-Term Low-Protein Isocaloric Diet on Cortical Histomorphometry of the Midshaft Tibia
 15% casein2.5% casein
Dietary casein content (n)77
  1. Results are means ± SEM. Samples were collected at the end of 16 weeks of pair-feeding with 15% or 2.5% casein-containing diets.

  2. * P < 0.05, using a Student's t-test.

Cortical area mm2 %4.24 ± 0.133.72 ± 0.11*
 82.81 ± 0.9673.63 ± 1.68*
Marrow area mm2 %0.88 ± 0.071.33 ± 0.09*
 17.2 ± 1.026.4 ± 1.7*
Total area mm25.12 ± 0.165.05 ± 0.08
Endosteal perimeter mm3.64 ± 0.144.40 ± 0.14*
Periosteal perimeter mm8.60 ± 0.158.54 ± 0.01

A decrease of BMD was observed at skeletal sites containing a significant proportion of trabecular bone (i.e., proximal tibia, lumbar spine, proximal and distal femur), after 8 weeks of a low-protein diet; these effects were maximal after 16 weeks of protein undernutrition. In contrast, at the skeletal site formed by cortical bone, a decrease of BMD was detectable only after 16 weeks of protein undernutrition, suggesting a possible different kinetics of the response to the protein deficiency according to the type of bone. This delay might be attributed to the relatively low bone remodeling rate of cortical bone. Changes in bone mineral mass were associated with alterations of bone strength. Bone stiffness, which reflects bone elasticity, was also altered by the low-protein isocaloric diet, suggesting that the quality of the matrix was also implicated in the modification of bone mechanical properties.

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Figure FIG. 2.. Effect of various protein-containing isocaloric diets on plasma IGF-I. Plasma IGF-I was determined in plasma collected 4 h after food intake in rats pair-fed isocaloric diets containing 15, 7.5, 5, and 2.5% casein. Values expressed in percentage of pretreatment value are means ± SEM. Squares represent rats fed 15%; circles, 7.5%; open triangles, 5%; and closed triangles, 2.5%. Using an analysis of variance (ANOVA) for repeated measures, * indicates a significant difference (p < 0.01) from rats fed 15, 7.5, and 5% casein.

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In agreement with previous studies in young growing rats,(19) alteration of the IGF-I system was found under protein deprivation. After 1 week on the low-protein diet, a decrease in plasma IGF-I concentration was already detected, with a maximal diminution of IGF-I after 2 weeks. In analogy with humans in whom low-protein intakes can be associated with perturbation of sex hormone status, such as anorexia nervosa, leading to a decrease in bone mineral mass, we investigated the influence of a low-protein isocaloric diet on the duration of cycles, using histological examination of vaginal smear. In rats fed a 2.5% protein diet no estrus was detected, suggesting estrogen deficiency and/or resistance to estrogen. Regular cycles were observed under diets containing 15, 7.5, and 5% casein. Interestingly, BMD was not modified in these experimental groups. Thus, besides low IGF-I levels, estrogen deficiency or resistance to estrogen action could be involved in the low-protein isocaloric diet-mediated bone loss. Whether these alterations in both hormone systems are interlinked cannot be firmly established at the present time. However, cortical bone mineral mass was decreased in rats under low-protein diet, when such a decrement was barely detectable in estrogen-deprived rats.(29) This might suggest possible different mechanisms of action on bone of low IGF-I and estrogen deficiency in the frame of protein undernutrition. To further address this issue, we investigated the effects of ovariectomy with or without a low-protein diet. We found that the effects of both manipulations seemed to be additive, at least at the level of the proximal tibia. At the level of midshaft of long bone, ovariectomy slightly increased the external diameter, which might be responsible for the apparent protection of cortical bone strength previously reported after ovariectomy.(27) The increment of plasma IGF-I observed after ovariectomy in rats fed the normal protein diet could play a role in this process. This effect was not found on a low-protein diet (data not shown).

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Figure FIG. 3.. Effect of a low-protein isocaloric diet and/or ovariectomy on lumbar spine and proximal and midshaft tibia BMD. BMD was measured before and after 10 and 15 weeks in sham-operated or ovariectomized rats pair-fed isocaloric diets containing 15 or 2.5% casein. Values expressed as a percentage of time control (rats fed a 15% casein diet) are means ± SEM. Using an analysis of variance (ANOVA) for repeated measures, * indicates a significant difference from sham-operated 15%, ° from ovariectomized 15%, and # from sham-operated 2.5%.

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Bone remodeling was evaluated by the measurement of serum and urinary biochemical markers. In rats fed a 15% casein diet, OVX induced an increment of plasma osteocalcin and of urinary deoxypyridinoline excretion.(30) The modification of osteocalcin was not observed in OVX rats fed a 2.5% casein diet. This might suggest that low IGF-I and estrogen deficiency affect bone through distinct mechanisms. However, when bone resorption was estimated using deoxypyridinoline excretion, a marked increase was observed in SHAM as well as in OVX animals on a low-protein diet. This indicates a substantial contribution of increased bone resorption to bone loss induced by the low-protein diet. This observation was sustained by the histomorphometry analysis, performed after 4 months of protein deprivation, indicating a cortical thinning without an increase in cross-sectional area. In contrast, no appreciable periosteal apposition nor modification of external perimeter or total bone area was observed. These results indicate an increased remodeling with reduced formation relative to resorption during protein undernutrition. This mechanism is different from the increased remodeling commonly asociated with sex hormone deficiency. The mechanisms underlying the elevated bone resorption remain to be elucidated, but they could involve the production and/or action of bone-resorbing cytokines.(34–36)

In conclusion, isocaloric protein undernutrition decreased bone mineral mass and strength at skeletal sites formed by cortical, or by trabecular and cortical bone. This effect might be related to decreased plasma IGF-I and/or to estrogen deficiency. This model may mimic osteoporosis observed in elderly women in whom both cortical and trabecular skeletal sites are affected and in whom both sex hormone status and IGF-I system are altered.

Table Table 3.. Effect of Ovariectomy and/or Low-Protein Isocaloric Diet on BMD and Bone Strength
  15% Casein2.5% Casein
Dietary casein contentSHAMOVXSHAMOVX
  1. Values are means ± SEM. BMD and bone strength (ultimate strength, US; stiffness, Stif; energy, E) were determined 15 weeks after OVX or SHAM operation in rats pair-fed isocaloric 2.5 or 15% casein-containing diets.

  2. Significant difference between the groups: *, versus SHAM 15%, †, versus OVX 15%, ‡, versus SHAM 2.5%.

Femoral neckBMD (mg/cm2)263.3 ± 3.0239.7 ± 2.6*209.7 ± 8.2*204.2 ± 2.6*
 US (N)96.4 ± 3.892.01 ± 4.373.6 ± 4.4*77.0 ± 4.5*
 Stif (N/mm)446.1 ± 33.6397.3 ± 35.8344.3 ± 27.0*304.0 ± 26.1*
 E(NX mm)11.4 ± 0.814.1 ± 1.39.3 ± 1.012.3 ± 1.4
Midshaft femurBMD (mg/cm2)248.3 ± 2.2237.6 ± 2.4*211.3 ± 4.2*204.9 ± 3.1*
 US (N)163.3 ± 4.8150.7 ± 5.8125.0 ± 5.8*123.7 ± 4.2*
 Stif (N/mm)412.1 ± 21.3344.8 ± 18.3*300.1 ± 22.8*301.4 ± 19.1*
 E (N × mm)50.8 ± 3.549.6 ± 2.937.6 ± 2.2*38.0 ± 2.5*
Proximal tibiaBMD (mg/cm2)258.1 ± 3.2225.0 ± 2.6*202.4 ± 6.7*187.8 ± 3.7*†‡
 US (N)131.5 ± 5.689.1 ± 5.1111.6 ± 8.888.7 ± 5.8
 Stif (N/mm)474.1 ± 27.8363.5 ± 31.9392.7 ± 44.3388.4 ± 24.3
 E (N × mm)22.6 ± 2.413.3 ± 1.018.3 ± 2.612.6 ± 1.1
Table Table 4.. Effect of Ovariectomy and/or Low-Protein Isocaloric Diet on Biochemical Markers of Bone Remodeling and on IGF-I Plasma Levels
  15% Casein2.5% Casein
Dietary casein contentWeeksSHAMOVXSHAMOVX
  1. Values are means ± SEM. Plasma osteocalcin, and IGF-I and urinary deoxypyridinoline were measured 12 and/or 17 weeks after OVX or SHAM operation in rats pair-fed isocaloric 2.5 or 15% casein-containing diets.

  2. Significant difference between the groups: *, versus SHAM 15%, , versus OVX 15%.

Osteocalcin (μg/l)1216.79 ± 1.1623.91 ± 1.16*19.69 ± 1.4618.02 ± 1.52
Osteocalcin (μg/l)1716.39 ± 2.1423.31 ± 1.19*15.68 ± 1.2014.74 ± 1.36
Deoxypyridinoline (nmol/24 h)171.63 ± 0.252.80 ± 0.49*3.62 ± 0.42*3.60 ± 0.44*
IGF-I (μg/l)17531.4 ± 22.4686.2 ± 22.5*324.3 ± 17.0*287.5 ± 19.8*

Acknowledgements

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

We thank Mrs I. Badoud, S. Clément, M. Linder, H. Francisco, and C. Godin for their expert technical assistance, and M. Perez for typing the manuscript. We acknowledge the valuable help of Dr. M. Aubert, Ph.D., in the determination of estrus status, and the AETAS and Wilsdorf Foundations for their support. This project was supported by grants from the Swiss National Science Research Foundation (grant no. 32-49757.96) and from Novartis Nutrition Ltd. (Berne, Switzerland).

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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