Patrick Ammann

Protein intake, IGF-1 and osteoporosis

Deficiency in nutritional elements could play an important role in the pathogenesis of osteoporotic fracture in the elderly. Results of several studies indicate that calcium supplementation reduces bone loss and fracture incidence in vitamin D replete elderly subjects [1]. Other investigations have suggested that the level of protein intake could influence either calcium phosphate metabolism, bone mass or the risk of osteoporotic fracture [2]. Apparently contradictory information suggests that either a deficient or an excessive protein supplement could negatively affect the balance of calcium and the arnount of bony tissue contained in the skeleton [2,3]. Several reasons can be evoked with respect to the present uncertainty regarding the effect of protein supplementation on calcium balance and bone mass. The outcomes may differ because the protein supplementation was: (a) prescribed either to wellnourished people or to subjects exhibiting signs of malnutrition; (b) given in various forms: natural food products (i.e. ingested in complex forms), purified extracts or mixtures of amino acids; (c) of either animal (e.g. casein) or vegetable (e.g. soya) origin; (d) evaluated over either the short term or the long term; (e) assessed in the presence or absence of modifications in other nutritional factors such as energy and/or calcium. The recommended daily allowance (RDA) for protein varies from 2.0 g/kg body weight in children to 1.0 g/kg in adolescents, and 0.75 g/kg in adults [4]. Protein intake below the RDA could be particularly detrimental for both the acquisition of bone mass and the conservation of bone integrity with aging. Protein undemutrition during childhood and adolescence results in a reduction of height, weight and overall body protein [5] and, therefore, can be expected to affect peak bone mass. A sufficient protein intake is also mandatory for the maintenance of bone homeostasis during adulthood. In the elderly, malnutrition can be considered as a risk factor for hip fracture because it can accelerate age-

The new selective estrogen receptor modulator MDL 103,323 increases bone mineral density and bone strength in adult ovariectomized rats.

Abstract: Selective estrogen receptor modulators (SERMs) can prevent the bone loss induced by ovariectomy (OVX), but it is not established whether they can increase bone mass and strength in a curative protocol in ovariectomized osteopenic animals. We investigated the influence of a SERM of the new generation, MDL 103,323, on areal bone mineral density (BMD), as measured by dual-energy X-ray absorptiometry, bone strength and remodeling in OVX osteopenic rats. Nine weeks after OVX, 8-month-old rats were divided into six groups of 10 animals. MDL 103,323 was given by gavage at doses of 0.01, 0.1 or 0.6 mg/kg body weight, 5 days a week. The effect of MDL 103,323 was compared with that of the bisphosphonate pamidronate (APD), which was injected subcutaneously at a dose of 1.6 mmol/kg body weight for 5 days every 4 weeks. Lumbar spine (LS), femoral neck (FN), proximal tibia (PT) and midshaft tibia (MT) BMD, bone strength, and proximal tibia histomorphometry, serum osteocalcin, urinary total deoxypyridinoline and serum insulin-like growth factor I (IGF-I) were measured. After 16 weeks of treatment, BMD changes (means ± SEM) were −11.4 ± 2.2, +4.0 ± 2.1 and +6.4 ± 1.0% respectively in OVX controls, in rats treated with 0.1 mg/kg MDL 103,323 (p<0.05) and in APD-treated rats (p<0.02) at the level of LS; −0.4 ± 1.1, +6.7 ± 1.4, +7.2 ± 1.8% (p<0.01 and NS) at the level of FN; and −2.6 ± 1.2%, +5.8 ± 1.2, +6.9 ± 1.4% (p<0.03 and 0.01) at the level of PT. MDL 103,323-treated animals had a higher trabecular bone volume, a higher number of trabeculae and smaller intertrabecular spaces compared with OVX controls. Vertebral body ultimate strength was 186 ± 13, 292 ± 16, 249 ± 23 N (p<0.05) in OVX controls, MDL 103,323-treated rats and APD-treated rats, respectively. The administration of 0.6 mg/kg of MDL 103,323 did not further increase BMD or bone strength, indicating a bell-shaped dose–response curve. MDL 103,323 lowered plasma osteocalcin concentration and urinary deoxypyridinoline excretion. In rats treated with 0.1 mg/kg MDL 103,323, plasma IGF-I was increased as compared with OVX controls (664 ± 36 ng/ml vs 527 ± 39 ng/ml, p<0.05). In conclusion, these results indicate that this new SERM positively influences BMD and lumbar spine bone strength in estrogen-deficient rats.

Protein Intake and Osteoporosis

The amount of bone present at a given age is determined by the mass acquired during growth (the so-called peak bone mass) and by the subsequent rate of bone loss. The attainment of peak bone mass appears to be highly affected by the state of protein supply and intake during childhood and adolescence. Indeed, in childhood and adolescence, the need for protein is increased to meet the demand required by body growth. For instance, in animal studies, rats treated with growth hormone spontaneously select a high protein diet (1). Thus, during growth, the adaptation to lower protein intake should be much more difficult than later on in life. Protein undernutrition results in a reduction of height, weight, and overall body protein (2). The recommended daily allowance for protein varies between 2.0 in children to 1.0 in adolescents, and 0.75 g/kg per day in adults (3). On the other hand, a sufficient protein intake is also mandatory for the maintenance of bone homeostasis in the elderly. Indeed, malnutrition can be considered as a risk factor for hip fracture because it can be expected to accelerate age-dependent bone loss, to increase the propensity to fall by impairing movement coordination, to affect protective mechanisms, such as reaction time and muscle strength, and thus to reduce the energy required to fracture an osteoporotic proximal femur.

The increase in vertebral bone mass induced in intact rats by long-term administration of the strontium salt S-12911 is directly correlated with vertebral bone strength

THE INCREASE IN VERTEBRAL BONE MASS INDUCED IN INTACT RATS BY LONG-TERM ADMINISTRATION OF THE STRONTIUM SALT S-12911 IS DIRECTLY CORRELATED WITH VERTEBRAL BONE STRENGTH, p. Ammann, R. Rizzoli, P. Deloffre, Y. Tsouderos, J.M. Meyer and J.P. Bonjour. Die. of Clinical Pathophysiology, Dept. of Internal Medicine, and School of Dentistry, University Hospital, Geneva, Switzerland, and IRI Servier, Courbevoie, France. Strontium salt (S-12911) has been shown to increase bone mass in ovariectomizad rats by inhibition of bone resorption and stimulation of bone formation. Similar effects were demonstrated in osteoporotic postmenopausal women after one year of treatment. However, long-term effects of S-1291t on bone mass and bone mechanical properties in rat were unknown. Seven-week old intact male and female rats were fed ad libitum a diet containing S-12911 (625 mg/kg day) for two years. Ex vivo vertebral body L4 bone mineral density (BMD) was measured using Hologic QDR 1000. BMD was corrected for Strontium content measured in ashed bone. Ultimate strength, stitfnass and energy were evaluated using a compression test of the intact vertebral body L4. External volume of the vertebral body was evaluated using Archimedes law. In male, S-12911 significantly increased vertebral body BMD (0.2116+0.0053, x• n=19, vs 0.1917+_0.0169 g/cm2, n=22, p=0.003), without modification of vertebral body volume (0.107+-0.002 vs 0.105:L-0.002 cm3). S-12911 also increased ultimate strength (391+-20 vs 332+-13 N, p=0.018) and energy (80.1+_7.0 vs 57.5+_4.3 N*mm, p=0.009) but not stiffness (1256+_81 vs 1161+_49 N/mm). Ultimate strength and energy were significantly correlated to BMD (r=0.720, p=0.0001, and r=0.597, p=0.0001, raspectively). In female, S-12911 increased vertebral body BMD (0.2031• n=14, vs 0.1853i-0.0030 g/cm2, n=22, p=0.02). Vertebral body volume was not significantly affected (0.073_+0.002 vs 0.069+-0.002 cm3). Increments of ultimate strength (310-• vs 273+_15 N) and energy (64.6+_7.3 vs 51.2~:3.5 N*mm) did not reach a level of statistical significance but these values were significantly correlated to BMD (r=0.519, p=0.O05, and r=0.481, p=0.009, respectively). In conclusion, Iong-tarr~ administration of S-12911 for 2 years in intact rats increased vertebral bone mass and bone strength. This positive relationship between bone strength and BMD could further support the use of S-12gll in the treatment of ostaoporosis and bone fragility. 9 TUESDAY MAY 21, 1996

Nutrition and Insulin Growth Factor-I in Relation to Bone Health and Disease

At the skeletal level, IGF-I exerts a positive effect on bone mineral mass by a direct action on osteogenic cells. At the kidney level, IGF-I enhances both the renal reabsorption of inorganic phosphate (Pi) and the production of calcitriol, the hormonal form of vitamin D that stimulates the intestinal absorption of calcium and Pi, the two main bone mineral elements. Protein undernutrition reduces IGF-I production, decreases skeletal acquisition during growth, and accelerates bone loss during adulthood. The stimulation of bone formation in response to IGF-I is impaired in presence of an inadequately low intake of proteins. Protein undernutrition, probably by influencing negatively IGF-I production and action, contributes to the pathogenesis of osteoporotic fractures in elderly.

Fluoride potentiates the osteogenic effects of IGF-I in aged ovariectomized rat

The molecular mechanisms whereby fluoride stimulates osteogenic cell proliferation are not clearly established. However, fluoride has been shown to enhance the protein tyrosine phosphorylation of various constituents of intracellular signaling cascades in osteoblastic cells following stimulation of growth factor receptors such as the insulin-like growth factor-I (IGF-I) receptor. Such in vitro findings provided the rationale for testing whether the administration of fluoride could enhance IGF-I effects on bone mass in vivo. Adult ovariectomized osteopenic rats were treated with sodium fluoride at a dose of 6 mg/kg per day in drinking water for 8 weeks in association with IGF-I either at a dose of 2 mg/kg per day, which is capable of increasing bone mass, or at a lower dose without detectable skeletal effects. Bone mineral density (BMD) and content (BMC) were evaluated by dual-energy X-ray absorptiometry at the levels of the lumbar spine and proximal, midshaft, and total tibia before and after 8 weeks of treatment. During this period, fluoride alone did not significantly influence BMD/BMC at any skeletal site. However, it potentiated the effect of the higher dose of IGF-I on bone mass at the level of the proximal tibia. When administered in combination with the lower dose of IGF-I, which per se did not modify bone mass, it appeared to sensitize tibial bone to the effects of IGF-I. These changes were associated with a concomitant increase in osteocalcin, taken as a reflection of bone formation. These results indicate that fluoride could potentiate the osteogenic effects of IGF-I on bone in adult ovariectomized rats.

Aluminum sensitizes ovariectomized rats to fluoride therapy

THE INCREASE IN VERTEBRAL BONE MASS INDUCED IN INTACT RATS BY LONG-TERM ADMINISTRATION OF THE STRONTIUM SALT S-12911 IS DIRECTLY CORRELATED WITH VERTEBRAL BONE STRENGTH, p. Ammann, R. Rizzoli, P. Deloffre, Y. Tsouderos, J.M. Meyer and J.P. Bonjour. Die. of Clinical Pathophysiology, Dept. of Internal Medicine, and School of Dentistry, University Hospital, Geneva, Switzerland, and IRI Servier, Courbevoie, France. Strontium salt (S-12911) has been shown to increase bone mass in ovariectomizad rats by inhibition of bone resorption and stimulation of bone formation. Similar effects were demonstrated in osteoporotic postmenopausal women after one year of treatment. However, long-term effects of S-1291t on bone mass and bone mechanical properties in rat were unknown. Seven-week old intact male and female rats were fed ad libitum a diet containing S-12911 (625 mg/kg day) for two years. Ex vivo vertebral body L4 bone mineral density (BMD) was measured using Hologic QDR 1000. BMD was corrected for Strontium content measured in ashed bone. Ultimate strength, stitfnass and energy were evaluated using a compression test of the intact vertebral body L4. External volume of the vertebral body was evaluated using Archimedes law. In male, S-12911 significantly increased vertebral body BMD (0.2116+0.0053, x• n=19, vs 0.1917+_0.0169 g/cm2, n=22, p=0.003), without modification of vertebral body volume (0.107+-0.002 vs 0.105:L-0.002 cm3). S-12911 also increased ultimate strength (391+-20 vs 332+-13 N, p=0.018) and energy (80.1+_7.0 vs 57.5+_4.3 N*mm, p=0.009) but not stiffness (1256+_81 vs 1161+_49 N/mm). Ultimate strength and energy were significantly correlated to BMD (r=0.720, p=0.0001, and r=0.597, p=0.0001, raspectively). In female, S-12911 increased vertebral body BMD (0.2031• n=14, vs 0.1853i-0.0030 g/cm2, n=22, p=0.02). Vertebral body volume was not significantly affected (0.073_+0.002 vs 0.069+-0.002 cm3). Increments of ultimate strength (310-• vs 273+_15 N) and energy (64.6+_7.3 vs 51.2~:3.5 N*mm) did not reach a level of statistical significance but these values were significantly correlated to BMD (r=0.519, p=0.O05, and r=0.481, p=0.009, respectively). In conclusion, Iong-tarr~ administration of S-12911 for 2 years in intact rats increased vertebral bone mass and bone strength. This positive relationship between bone strength and BMD could further support the use of S-12gll in the treatment of ostaoporosis and bone fragility. 9 TUESDAY MAY 21, 1996

Calcium Absorption in the Rat Colon Measured in Vivo

Calcium absorption in the large intestine was studied under physiological conditions in the rat. When 45Ca was administered directly into the caecum, the isotope was significantly absorbed. This absorption was modulated by dietary Ca and Pi as well as by the 1,25(OH) D3 status. On the other hand, calcium coming from the food, either as 45Ca or as 40Ca, was not absorbed to a significant amount. When the absorption of 45Ca from the caecal content was assessed in duodenal loops, less 45Ca was absorbed when the isotope was administered orally than when it was added to the caecal content. Thus, the fact that oral Ca is not absorbed in the colon despite the efficient Ca transport system of this part of the intestine may be due to the transformation in the digestive tract of Ca from an absorbable into a non-absorbable form.