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Endocrinology Vol. 141, No. 9 3149-3155
Copyright © 2000 by The Endocrine Society

ARTICLES

Dietary Protein Restriction Lowers Plasma Insulin-Like Growth Factor I (IGF-I), Impairs Cortical Bone Formation, and Induces Osteoblastic Resistance to IGF-I in Adult Female Rats1

S. Bourrin, P. Ammann, J. P. Bonjour and R. Rizzoli

Division of Bone Diseases, World Health Organization Collaborating Center for Osteoporosis and Bone Diseases, Department of Internal Medicine, University Hospital, Geneva CH-1211, Switzerland

Address all correspondence and requests for reprints to: Sandrine Bourrin, Ph.D., Division of Bone Diseases, Department of Internal Medicine, University Hospital of Geneva, Rue Micheli-du-Crest, 24, CH- 1211 Geneva 14, Switzerland.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Dietary protein deficiency, common in elderly, is associated with decreased areal bone mineral density and plasma insulin-like growth factor I (IGF-I). To investigate the early adaptation of bone cells to protein restriction, 6-month-old female rats were pair-fed with isocaloric 15% (control) or 2.5% casein diets for 14 days. Animals were then treated daily with rhIGF-I/IGFBP-3 (1:4, 2.5 mg IGF-I/kg BW) or with vehicle for 10 days. After double-labeling, proximal metaphysis and mid-diaphysis of the tibia were analyzed histomorphometrically. Plasma osteocalcin, IGF-I, and urinary deoxypyridinoline were quantified. After 14 days of protein restriction, significant drops in plasma osteocalcin (13%) and IGF-I (37%), in periosteal formation (83%) and mineral apposition (49%) rates are observed, indicating a decreased osteoblast recruitment and activity. In cancellous bone, a significant decrease in active eroded surfaces (27%) and osteoclast number (24%) indicates a transient depression of resorption. In rats fed the 15% casein diet, rhIGF-I/IGFBP-3 increases cancellous (42%) and periosteal (600%) formation rates, indicating an increased osteoblast recruitment. In protein-restricted rats, rhIGF-I/IGFBP-3 fails to increase cancellous or periosteal bone formation and plasma osteocalcin is significantly lower than in 15% casein+rhIGF-I/IGFBP-3 rats. Protein restriction induces osteoblast resistance to rhIGF-I/IGFBP-3 in both bone envelopes. Low plasma IGF-I and osteoblast resistance to IGF-I, may contribute to the impaired periosteal formation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NUTRITIONAL deficiencies may contribute to age-related bone loss, and protein intake is frequently low in the elderly (1, 2). Areal bone mineral density (aBMD) is significantly lower in aged women with low protein intake (1). Conversely, protein supplements decrease complications after hip fracture (2, 3) and attenuate bone loss (4). Low protein intake may contribute not only to increased frailty but also to osteoporosis, both leading to fractures, impairment, of activities and loss of independence (1, 5, 6, 7).

We have previously shown a marked decrease in plasma insulin-like growth factor I (IGF-I) and in the aBMD of lumbar spine, distal and proximal femur, and diaphyseal and proximal tibia in protein-restricted adult female rats (8, 9). Such aBMD changes are associated with a marked decrease in bone strength (8, 9). In a long-term study, biochemical results in female rats fed a low protein diet suggest not only an impairment of bone formation, but also an increase in bone resorption, in association with a complete loss of ovarian cycles (8, 9). Thus, bone loss in adult female rats fed a low protein diet may result from intricate mechanisms involving both a decrease in plasma IGF-I and sex hormone deficiency. However, the early cellular mechanisms involved in the response of bone to a selective dietary protein restriction in adult female rats are still poorly understood.

To study the early cellular events in both cortical and trabecular bone, we submitted adult female rats to a diet with low protein content but isocaloric to the control diet. Histomorphometric and biochemical analyses were performed after short-term protein deficiency, when plasma IGF-I was significantly lower in protein-restricted rats. Thereafter, to investigate the bone cellular response to IGF-I, we administered a pharmacological dose of rhIGF-I/IGFBP-3 to 15% and 2.5% casein-fed rats and evaluated its effects histologically and biochemically.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
The experimental protocol received the approval of the Animal Ethics Committee of the Geneva University Faculty of Medicine. Animals were housed individually at 22 C with a 12-h light, 12-h dark cycle and given demineralized water ad libitum throughout the experiment. Forty-two 6-month-old female Sprague Dawley rats (Ciba-Geigy Ltd., Basel, Switzerland) were pair-fed isocaloric diets (Novartis-Nutrition, Berne, Switzerland) containing either 15% casein or 2.5% casein for 14 days (n = 21 rats per group). Seven rats of each group were killed after this given period of time. The protein content of either diet was chosen according to previous results obtained in female rats (8, 9). Both diets contained 0.8% phosphorus, 1.1% calcium, and 100 IU/kg body weight/day vitamin D3. The 2.5% casein diet was rendered isocaloric to the 15% casein diet by addition of the corresponding amount of calories in the form of corn starch carbohydrates. Before the beginning of the experiment, all rats were pair-fed the diet containing 15% casein for 14 days, to get them accustomed to the diet and to equilibrate the pair-feeding.

After 14 days, rats fed 15% casein or 2.5% casein were sc injected with rhIGF-I/IGFBP-3 complex (ratio IGF-I to IGFBP-3 is 1:4) (kindly supplied by Dr. D. Mascarenhas, Celtrix Pharmaceuticals, Inc., Santa-Clara, CA), at the dose of 2.5 mg of IGF-I/kg BW or with vehicle (50 mM acetate, pH 5.5, 108 mM NaCl) daily for 10 days (n = 7 rats in each group). Administration of IGF-I in combination with IGFBP-3 enhances the growth-promoting activity of IGF-I and protects from its hypoglycemic effects (10).

To estimate the endosteal resorption activity of cortical bone, all rats killed at the very end of the experiment (n = 28) were labeled once with tetracyclin hydrochloride (Sigma, St. Louis, MO, 15 mg/kg) on day 0, so that the label remaining after 24 days reflects the amount of bone not resorbed for this given period of time. To label the mineralizing surfaces, all animals were given ip injections of calcein (Sigma, 10 mg/kg) 9 days before they were killed, and of tetracyclin hydrochloride (Sigma, 15 mg/kg) 1 day before euthanasia by Ketamine hydrochloride overdose.

Blood and urine parameters
Blood samples were taken from the tip of the tail 4 h after feeding, 3–4 days preceding each euthanasia. Blood was also collected by aortic puncture under general anesthesia at the time of euthanasia. Serum IGF-I was quantified with a RIA kit from Nichols Institute Diagnostics (San Juan Capistrano, CA) after extraction by acid-ethanol and cryoprecipitation to remove all the IGF binding proteins. Osteocalcin was quantified by a RIA with reagents from Biomedical Technologies (Stoughton, MA). Urine was collected in metabolic cages for 24 h during the week preceding euthanasia. Total urinary deoxypyridinoline was assayed after acid hydrolysis with a kit from Metra Biosystems Inc. (Mountain View, CA) and urinary deoxypyridinoline excretion rate was calculated.

Bone histology
The proximal metaphyses and diaphyses of the right tibiae were collected, cleaned of soft tissues, and fixed in 10% ice-cold formalin for 24 h at 4 C. Bones were then infiltrated and embedded in a methylmethacrylate-based medium at low temperature (11).

Cortical bone. One mid-diaphysis 150-µm cross-section per animal was sawed with a low-speed diamond-coated saw, fixed to a plastic slide with a cyanoacrylic glue, and ground to a thickness of 15–20 µm for microscopic analysis. The mid-diaphyseal cross-sectional area, and bone area were measured semiautomatically with a Leica Corp. Q550 image analyzer (Leica Corp., Cambridge, UK) at a 25-fold magnification. The labeled perimeters were also measured semiautomatically, under UV illumination, with the apparatus described above, at a 200-fold magnification. The following parameters were measured and calculated:

Bone mass parameters. P. Pm and E. Pm (mm): periosteal and endosteal perimeters; %Ct. Ar (%): cortical area/tissue area1100 is the percent cortical area.

Bone formation parameters. P-%L. Pm and E-%L. Pm (%): periosteal and endosteal percent-labeled perimeter, respectively. The percent-labeled perimeter is calculated as (1/2 single-labeled perimeter + double-labeled perimeter/periosteal or endosteal perimeter)1100; P-BFR and E-BFR (µm/d): periosteal and endosteal bone formation rate surface-based, respectively.

Trabecular bone. 7-µm-thick sections were cut perpendicularly to the sagittal plane of proximal metaphysis with a Leica Corp. Polycut E microtome (Leica Corp. Microsystems AG, Glattbrugg, Switzerland). Five were stained with a modified Goldner’s trichrome (12) and 6 with a tartrate-resistant acid phosphatase histochemical staining (13). Four 12-µm sections were cut and mounted unstained for calcein and tetracyclin fluorescence evaluation. The quantitative study of bone mass was performed in the secondary spongiosa of the proximal tibial metaphysis, as previously described (14). Bone mass parameters were measured on 4 Goldner-stained sections with the Leica Corp. Q550 image analyzer at a x50 magnification. For each animal, the mean of the values obtained for the four sections was taken. To study the general pattern of bone remodeling activity, bone cellular parameters were measured in the secondary spongiosa of the proximal tibial metaphysis, using a semiautomatic system composed of a digitizing tablet (Ultrapad, Wacom Computer systems GmbH, Neuss, Germany) coupled with the Leica Corp. Q550 and connected to a Leitz DM/RBE microscope equipped with a camera lucida. Three TRAP-stained sections per animal were examined for the parameters of bone resorption at a 200-fold magnification, and three sections per animal were examined for the bone formation parameters (static and dynamic) at a 200-fold magnification. Standardized terms were used hereafter, according to the ASBMR histomorphometry nomenclature (15):

Bone mass parameters. BV/TV (%): bone volume/tissue volume corresponding to the part of cancellous space filled with trabeculae; Tb.Th (µm): mean thickness of trabeculae; Tb.N (/mm): mean number of bone trabeculae.

Bone resorption activity parameters. NOc/B.Ar (c/mm2): number of osteoclast profiles/mm2 of trabecular bone, which represents the number of TRAP-positive cells referred to trabecular bone volume; NOc/B.Pm (c/mm): number of osteoclast profiles/mm of trabecular bone, which is the number of osteoclasts per mm of trabecular surfaces; OcS/BS (%): osteoclast surfaces/bone surface, representing the part of total trabecular surfaces with active eroded surfaces (scalloped surfaces filled with TRAP-positive cells).

Bone formation activity parameters. O.Th (µm): mean thickness of the osteoid seams; MAR (µm/day): cancellous mineral apposition rate is provided by dividing the distance between two fluorescent bands (evidenced under UV light) by the number of days between the two labels; sLS/BS and dLS/BS (%): single- and double-labeled surfaces referred to trabecular surfaces. MS/BS (%): mineralizing surfaces, calculated as (1/2 sLS/BS+dLS/BS)1100; BFR/BS (µm3/µm2/d): bone formation rate, surface referent, calculated according to the formulae: (MS/BS1MAR)/100.

Body weight evolution and organ weights
Body weight was recorded every week. Dry weights of adrenal glands and uterus were recorded at 14 and 24 days of experiment.

Statistical analysis
Two-way ANOVAs were performed to determine the effects of time and diet on all parameters. Then, one-way factorial ANOVAs, followed by Fisher’s PLSD posthoc test were used to specify the effects of time on control rats and rats fed the low protein diet. At day 14, a Mann and Whitney-U test was used to evaluate the effects of diet and at day 24, a one-way ANOVA followed by Fisher’s PLSD was performed to evaluate the effects of rhIGF-I/IGFBP-3 and diet treatments. ANOVA for repeated measures were performed for the assessment of the effects of time or diet on the biochemical parameters.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of an isocaloric low protein diet on cortical bone mass, endosteal bone resorption, and periosteal and endosteal bone formation activities
After 14 and 24 days of dietary protein deficiency, the percent cross-sectional area, the periosteal and endosteal perimeters, and the percent marrow area do not significantly differ from the time-control 15% casein group (Tables 1 and 2).

After 14 days of low dietary protein intake, periosteal percent-labeled surfaces and bone formation rate, reflecting formation activity in the periosteum, drop significantly when compared with time controls (Table 1Go). Endosteal bone formation rate also decreases significantly after 14 days of dietary protein deficiency (Table 1Go). After 24 days of protein deficiency, although the difference with time controls does not reach a significant level, mineralizing surfaces (Table 2Go) and bone formation rate (Fig. 1AGo) in the periosteum are markedly decreased by 30% and 67%, respectively.


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  		Table 1. Effects of an isocaloric low protein diet on cortical bone mass, and periosteal and endosteal bone formation drifts

 

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  		Table 2. Effects of rhIGF-I/IGFBP-3 on cortical bone mass, and periosteal and endosteal bone formation drifts

 


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  		Figure 1. A, Cortical (periosteum) bone formation rate: P-BFR, measured in the mid- diaphysis of the tibia. B, Cancellous bone formation rate: BFR/BS, measured in the secondary spongiosa of the proximal tibia, after 24 days of 15% or 2.5% casein isocaloric diets, with or without rhIGF-I/IGFBP-3 treatment during the last 10 days. Results are means ± SE. {circ}, P < 0.05 vs. 15% casein time-control; $, P < 0.03 vs. 15% casein + IGF-I complex.

 
Effect of an isocaloric low protein diet on cancellous bone mass and architecture, cancellous bone resorption, and bone formation activities
In rats fed the low protein diet for 14 and 24 days, bone volume in the remodeling zone is similar to the value obtained for the 15% casein group (Tables 3Go and 4Go).


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  		Table 3. Effects of an isocaloric low protein diet on cancellous bone mass, architecture, bone resorption and formation activities

 

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  		Table 4. Effects of rhIGF-I/IGFBP-3 on cancellous bone mass, architecture, bone resorption and formation activities

 
After 14 days of protein restriction, bone resorption values decrease significantly compared with the 15% casein group (Table 3Go). However, after 24 days, the active eroded surfaces and osteoclasts number per mm2 or mm of cancellous bone increase (+28, +29, +31%, respectively) but are not significantly different from the control rats fed 15% protein (Table 4Go). In rats fed the 2.5% casein diet for 14 and 24 days, parameters reflecting trabecular osteoblast recruitment and activity, such as mineralizing surfaces, mineral apposition rate and osteoid thickness (Tables 3Go and 4Go) and bone formation rate (Table 3Go and Fig. 1BGo), are not significantly changed compared with time controls.

Effects of an isocaloric low protein diet on biochemical parameters
After 14 and 24 days, the urinary deoxypyridinoline excretion rate in the 2.5% casein group slightly decreases but is not significantly different from that of the 15% casein-time controls (after 14 days: 2.82 ± 0.55 vs. 3.18 ± 0.30 nmol/24 h, and after 24 days: Table 5Go).


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  		Table 5. Effects of rhIGF-I/IGFBP-3 treatment on plasma and urinary parameters

 
Plasma osteocalcin decreases by 13% with low protein intake (10.98 ± 0.73 vs. 12.57 ± 0.64 ng/ml, NS), and the osteocalcine drop between day 0 and day 14 in ng/ml is significantly larger than in the control rats fed the 15% casein diet (-4.62 ± 1.03 vs. -1.05 ± 0.74 in 15% casein, P < 0.04). After 24 days, plasma osteocalcin decreases by 14% in rats fed the low protein diet (Table 5Go).

In the 2.5% casein group, plasma IGF-I drops significantly after 14 days (393.1 ± 19.8 vs. 625.8 ± 40.4 ng/ml, P < 0.0001). After 24 days, plasma IGF-I in rats fed the 2.5% casein diet rats is decreased by 38% compared with the 15% casein-fed rats (Table 5Go).

Effect of an isocaloric low protein diet on body weight and organ weights
Body weight significantly decreases with low protein intake after 24 days of experiment (Fig. 2Go). In addition, there is a decrease in the dry weight of adrenal glands (18.5 ± 1.4 vs. 23.5 ± 1.2 mg, P < 0.05) and of uterus (196.0 ± 7.3 vs. 215.3 ± 8.9 mg, P < 0.05) in the 2.5% casein group after 24 days.



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  		Figure 2. Body weight evolution in rats fed 15% or 2.5% casein isocaloric diets for 0, 14, and 24 days, treated or not with rhIGF-I/IGFBP-3 during the last 10 days. Results are means ± SE. *, P < 0.03 vs. day 0, {circ}, P < 0.03 vs. 15% casein diet group.

 
Effect of rhIGF-I/IGFBP-3 on cortical bone mass, endosteal bone resorption, and periosteal and endosteal bone formation activities
IGF-I complex does not significantly change cortical bone mass parameters on any diet (Table 2Go).

As far as endosteal bone resorption is concerned, the IGF-I complex increases the amount of remaining tetracyclin label by 63% (performed on day 0) in the 15% casein group, reflecting a trend to reduced endosteal resorption (15% casein+rhIGF-I/IGFBP-3: 12.77 ± 2.57 vs. 7.83 ± 1.83% in 15% casein diet, NS). The latter is similar in protein-restricted rats treated with rhIGF-I/IGFBP-3 to values in rats fed the 15% casein diet treated with the IGF-I complex (11.55 ± 3.00 vs. 12.77 ± 2.57, NS).

The IGF-I complex significantly increases periosteal percent-labeled surfaces (Table 2Go) and bone formation rate (Fig. 1AGo) in rats fed the 15% casein diet (+407% and +573%, respectively), indicating a marked increase in periosteal formation activity. In contrast, in the 2.5% casein group the IGF-I complex fails to significantly increase periosteal percent-labeled surfaces (Table 2Go) and bone formation rate (Fig. 1AGo).

Effect of rhIGF-I/IGFBP-3 on cancellous bone mass and architecture, cancellous bone resorption, and bone formation activities
Treatment with the IGF-I complex for 10 days does not significantly modify cancellous bone mass parameters in any group (Table 4Go).

The IGF-I complex has no significant effects on cancellous bone resorption activity on any diet (Table 4Go).

As far as cancellous bone formation activity is concerned, the IGF-I complex in 15% casein-fed rats significantly increases parameters reflecting trabecular osteoblast recruitment and activity: mineralizing surfaces (Table 4Go), bone formation rate (Fig. 1BGo) and mineral apposition rate (Table 4Go). In contrast, the IGF-I complex fails to increase mineral apposition rate (Table 4Go), mineralizing surface (Table 4Go) and bone formation rate in the 2.5% casein group (Fig. 1BGo).

Effects of rhIGF-I/IGFBP-3 on biochemical parameters
After 10 days of rhIGF-I/IGFBP-3 treatment, the urinary deoxypyridinoline excretion rate is significantly modified neither in the 15% nor in the 2.5% casein groups (Table 5Go). Treatment with rhIGF-I/IGFBP-3 increases plasma IGF-I by 420% in the 15% casein group and by 472% in the 2.5% casein group. Despite this marked rise, plasma IGF-I is still significantly lower in the protein-restricted group treated with the IGF-I complex than in the 15% casein+rhIGF-I/IGFBP-3 group (Table 5Go). In rats fed the 15% casein diet, the IGF-I complex significantly increases plasma osteocalcin (Table 5Go). However, rhIGF-I/IGFBP-3 fails to significantly increase plasma osteocalcin in rats fed the 2.5% casein diet (Table 5Go). In the 2.5% casein+rhIGF-I/IGFBP-3 group, plasma osteocalcin values are still significantly lower than the ones in the 15% casein+rhIGF-I/IGFBP-3 group (Table 5Go).

Effect of rhIGF-I/IGFBP-3 on body weight and organ weights
In rats fed the 15% casein diet, treatment with rhIGF-I/IGFBP-3 for 10 days does not significantly modify body weight (Fig. 2Go). In addition, rhIGF-I/IGFBP-3 treatment for 10 days in rats fed the 2.5% casein diet fails to restore their body weight to the level of the 15% casein group (Fig. 2Go).

Even though the IGF-I complex does not induce significant changes in organ weights in 15% casein-fed rats, it restores adrenal gland dry weight of rats fed the low protein diet to levels comparable to the 15% casein group values (22.4 ± 1.4 vs. 24.1 ± 1.8 mg, NS). However, IGF-I treatment for 10 days fails to restore uterus dry weight of the rats fed the 2.5% casein diet to levels similar to control rats (74.2 ± 1.7 vs. 105.9 ± 11.6 mg, P < 0.05).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Low dietary protein intake in adult female rats decreases plasma IGF-I and osteocalcin, reduces periosteal bone formation drifts in cortical bone of the tibia mid-diaphysis, transiently decreases bone resorption in cancellous bone of the proximal tibia metaphysis, and induces an osteoblast resistance to the growth-promoting effects of IGF-I in both cortical and cancellous bone. Changes in IGF-I levels and/or in sensitivity of bone cells to IGF-I may be thus responsible for the early decrease in cortical bone formation drifts while other factors may maintain cancellous bone remodeling at levels slightly higher than in controls.

Plasma IGF-I already decreases significantly by 14 days of protein deficiency and levels off between 14 and 24 days, confirming previous observations made in protein/calorie restricted humans and animals (8, 9, 16, 17, 18, 19, 20, 21, 22). Despite strict pair-feeding of isocaloric diets, rats protein-restricted for 24 days significantly loose body weight. Plasma osteocalcin decreases parallel to plasma IGF-I. In previous experiments, plasma osteocalcin also decreased significantly after 1 month of low protein intake in aged male rats (23, 24), and in adult female rats after 7 months of dietary protein restriction (8, 9). The present decrease in plasma osteocalcin with low protein intake is compatible with an early decrease in bone formation activity.

In the periosteum of cortical bone, histomorphometric analysis shows a marked decrease in bone formation rate after 14 and 24 days of protein deficiency, clearly indicating a decrease in osteoblast recruitment and activity. Similarly, protein/energy deficiency impairs very early radial growth (25, 26, 27) and periosteal cortical mineral apposition rate (28, 29) in young rats and monkeys. At the endosteal surface, dietary protein restriction transiently decreases bone formation drifts. The increase in the remaining endosteal labeled-surfaces (labeled day 0) after 24 days on low protein diet suggests that endosteal resorption drifts are also transiently depressed with dietary protein restriction in adult female rats. Previous experiments evidence an enlargement of the medullary cavity and thinning of the cortical shell with protein/energy deficiency in adult (29) and aged male rats (24), in adult female rats (8, 9) and also in young monkeys (28). In humans, similar thinning of the cortices occurs in young malnourished children (30, 31). In the present study, the early decrease in periosteal apposition of bone and an imbalance between formation and resorption drifts at the endosteal surface in rats fed the low protein diet could explain the cortical bone loss due to marrow cavity expansion showed in other studies (8, 9, 23, 24, 28, 29). The duration of the experiment was too short to evidence any significant cortical bone loss.

Surprisingly, cancellous bone formation and mineral apposition rates are not significantly changed with low protein intake, indicating that over the experimental period of time, protein restriction does not modify trabecular osteoblast recruitment and activity. On the other hand, the significant decrease in active eroded surfaces and osteoclast number after 14 days of low protein intake but not after 24 days indicates that low dietary protein intake depresses bone resorption only transiently. This observed transient decrease may explain controversial results obtained in various species concerning the effects of low energy/protein intake on the activity and number of osteoclasts (29, 32, 33). Cancellous bone remodeling and endosteal modeling may slightly rise after 24 days of protein restriction, and such changes could be due to a decrease in estrogenic impregnation. Indeed, uterus dry weight significantly drops after 24 days of low protein intake, which may indicate some estrogen deficiency. Furthermore, in adult female rats, 3 months of protein restriction induce a complete disappearance of estrous cycles (9). We thus cannot exclude the possibility that both estrogen and protein deficiency could influence cancellous and endosteal bone cells activities at 24 days.

Daily injection of rhIGF-I/IGFBP-3 in rats fed 15% and 2.5% casein diets induces a similar rise in plasma IGF-I (5.2-fold vs. 5.7-fold respectively). However, plasma IGF-I remains significantly lower in protein-restricted rats treated with the complex than in treated animals fed the control diet. Such a difference with dietary protein restriction may partly result from an increase in the activity and/or the quantity of serum proteases (34), thereby increasing the clearance of IGF-I from plasma as previously observed in young rats fed a low protein diet (35).

Daily injection of rhIGF-I/IGFBP-3 for 10 days restores the dry weight of adrenal glands in protein-restricted rats to the values observed in the 15% controls but does not correct the dry weight of uterus and body weight, indicating a resistance to rhIGF-I/IGFBP-3. Body weight and organ-specific resistance to the growth-promoting effects of exogenous IGF-I are already demonstrated in protein-restricted young growing rats (34). In humans, the anabolic action of GH disappears in energy-restricted subjects despite high plasma IGF-I, also indicating a resistance to IGF-I (36). The reasons for such a resistance to exogenous IGF-I are unknown but a decrease in receptor binding or number is unlikely because moderate caloric restriction increases IGF-I binding by tissue (37) and increases type-I IGF receptor expression (38) in aged rats.

In 15% casein controls, the IGF-I complex significantly increases plasma osteocalcin. Histomorphometric analysis of the periosteal surface of the cortical bone shows that the bone formation rate significantly increases in the 15% casein+rhIGF-I/IGFBP-3 group, indicating an increase in periosteal osteoblast recruitment and activity. The IGF-I complex does not modify bone formation drifts at the endosteal surface. Several studies in adult or aged rats also report a marked rise in periostal bone formation drifts with IGF-I or rhIGF-I/IGFBP-3 (39, 40, 41), whereas endosteal surface formation drifts are unchanged (41). In contrast, rhIGF-I/IGFBP-3 does not significantly increase plasma osteocalcin in rats fed 2.5% casein, suggesting a failure to stimulate bone formation activity. In the protein-restricted group, histomorphometric analysis at the tibia periosteal surface demonstrates that the IGF-I complex does not increase osteoblast recruitment and activity. We thus show for the first time an osteoblastic resistance to the growth-promoting effects of IGF-I induced by an isocaloric low protein diet. Taken together, the simultaneous decrease in plasma IGF-I and in periosteal bone formation rate and the resistance of periosteal osteoblasts to rhIGF-I/IGFBP-3 strongly suggest that with dietary protein deficiency, plasma IGF-I and sensitivity to IGF-I are significant factors contributing to the impairment of periosteal bone formation drifts.

Effects of IGF-I on cancellous bone are less clear, depending on the age and the hormonal status of the animals or the dose used. Studies in adult ovariectomized or aged intact rats report an increase in trabecular bone formation activity in response to rhIGF-I/IGFBP-3 or IGF-I (40, 42, 43), whereas Tobias et al. (41) observe an impaired bone formation activity. In opposition, studies consistently report that IGF-I slightly but not significantly depresses cancellous bone resorption activity (40, 41, 42, 43). The present experiment shows that urinary deoxypyridinoline excretion rate, cancellous active bone resorption surfaces, and osteoclast number per bone area tend to decrease in 15% casein-fed animals in response to the IGF-I complex. Concurrently, in 15% casein controls, the IGF-I complex significantly increases trabecular mineralized surfaces, bone formation, and mineral apposition rates, demonstrating an increased recruitment and mineralizing activity of cancellous osteoblasts. In opposition, rhIGF-I/IGFBP-3 completely fails to increase bone formation activity in cancellous bone of protein-restricted rats. Thus, low protein intake in adult female rats suppresses the response of osteoblastic cells to IGF-I in both periosteal surfaces and in cancellous bone. However, in trabecular bone, protein deficiency does not decrease bone formation activity, suggesting that low plasma IGF-I and a resistance of osteoblasts to IGF-I do not impair cancellous bone formation in female rats.

In conclusion, we show for the first time that the early response of bone cells activities to isocaloric low protein intake in adult female rats is envelope specific. In cortical bone, dietary protein restriction impairs periosteal bone formation drifts whereas in cancellous bone, bone formation activity remains unchanged. Concurrently, plasma IGF-I and osteocalcin drop. In addition, dietary protein restriction induces an osteoblastic resistance to IGF-I in both envelopes. This may suggest that low plasma IGF-I and/or osteoblast resistance to IGF-I in response to low protein intake could play an important role in the impairment of periosteal osteoblasts. Moreover, these results suggest that therapeutic administration of IGF-I to subjects with a dietary protein deficiency may be ineffective on various organs, including bone.


   Acknowledgments
 
We thank Séverine Clément for her precious help for urine and blood collections, expert animal care, and IGF-I complex injections. We also thank Michaela Linder for biochemical assays, and perfect histologic preparations. Special thanks to Dr. J. Guicheux for helpful discussions and suggestions. We are also grateful to Dr. Gaby Palmer for her careful reading of the manuscript.


   Footnotes
 
1 This work was supported by grants from the Swiss National Research Foundation (Grant No. 32-49753-96) and from Novartis-Nutrition (Berne, Switzerland). Back

Received February 16, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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