Evidence for physiological importance of calcitonin in the regulation of plasma calcium in rats.

To determine the physiological importance of calcitonin in the regulation of plasma calcium, studies were carried out in fasting animals to (a) assess the acute effects of thyroparathyroidectomy (TPTX) and thyroidectomy (TX) on plasma and urinary calcium; (b) investigate whether the changes in plasma calcium produced by removal of the glands were dependent on the presence of the kidney; and (c) determine if the effect of TPTX on plasma calcium is affected by age. Except where otherwise indicated, all studies were carried out on fasting male Wistar rats weighing over 300 g. The following observations were made. (a) TPTX and TX caused an increase in plasma calcium in nephrectomized animals. (b) This increase was not dependent on nephrectomy since in intact animals bearing autoparathyroid transplants TX also caused a significant rise in the mean plasma calcium level (0.37 mg/100 ml at 1 1/2 h). (c) Urinary calcium increased twofold in the 3-h period immediately after TX. (d) In unnephrectomized immature (50-g) rats, TPTX caused a progressive decrease in plasma calcium in contrast to old (360-g) rats, where a significant fall observed at 6 h was preceded by an increase in plasma calcium (0.5 mg/100 ml at 1 1/2 h). From these observations we conclude that: (a) calcitonin must play an important physiological role in the regulation of plasma calcium since the termination of its basal secretion caused an immediate but transient increase in plasma calcium in old unfed rats; (b) the relative importance of calcitonin and parathyroid hormone in the acute regulation of plasma calcium is age-related; and (c) the action of parathyroid hormone on bone may be modified by changes in ambient calcitonin concentration.

Effects of ageing on the biomechanical properties of rat articular cartilage.

Previous studies have demonstrated that male Sprague Dawley (SD) rats experience age-related bone loss with the same characteristics as that in ageing men. As articular cartilage, like bone, is a critical component of the health and function of the musculoskeletal system, the authors hypothesized that articular cartilage in the untreated male SD rats could be a suitable model for studying the age-related deterioration of articular cartilage in men. To test this hypothesis, male SD rats were killed at between 6 and 27 months. The right femur of each rat was removed. The effects of ageing on the structural integrity of the distal femoral articular cartilage were studied by biomechanical testing with a creep indentation apparatus. The aggregate modulus, Poisson's ratio, permeability, thickness, and percentage recovery of articular cartilage were determined using finite element/non-linear optimization modelling. No significant differences were observed in these biomechanical properties of the distal femoral articular cartilage as a function of age. Therefore, untreated male SD rats appear to be unsuitable for studying the age-related changes of articular cartilage as they occur in men. However, and more intriguingly, it is also possible that ageing does not affect the biomechanical properties of articular cartilage in the absence of cartilage pathology.

Human parathyroid hormone-(1-34) prevents bone loss and augments bone formation in sexually mature ovariectomized rats.

A group of 3-month-old Sprague-Dawley rats were sham operated or ovariectomized and given daily injections of human PTH-(1-34) (8 or 16 micrograms per 100 g body weight) for 5 weeks. At the termination of the study histomorphometric techniques were used to examine changes in cortical and cancellous bone in the diaphysis and proximal metaphysis of the tibia. Ovariectomy resulted in a 50% decrease in cancellous bone that was accompanied by a 41 and 120% increase in osteoclasts and osteoblasts, respectively. In contrast, in the ovariectomized animals treated with PTH, the metaphyseal cancellous bone increased by over 300% to a level in excess of that present in the sham-operated control animals. The increase in cancellous bone induced by PTH was associated with an over 70% increase in osteoblasts and tetracycline-labeled area and an unexpected decrease in trabecular osteoclasts. In the tibial diaphysis PTH also decreased endosteal osteoclasts and at the same time increased osteoblast size and number as well as endosteal and periosteal bone formation; ovariectomy increased only periosteal bone formation. Our findings demonstrate that intermittent administration of PTH prevents ovariectomy-induced bone loss and augments cancellous and cortical bone formation in sexually mature ovariectomized rats. Although the basis of the bone anabolic action of PTH remains elusive, our data indicate that it may involve the uncoupling of bone formation and resorption such that the latter is inhibited as bone formation is enhanced. Our findings are also compatible with the view that intermittent administration of PTH increases bone mass, in part by stimulating the proliferation and differentiation of osteoblast progenitors while inhibiting osteoclast proliferation.

Ovariectomized murine model of postmenopausal calcium malabsorption.

Two experiments were carried out to examine whether the ovariectomized mouse is a potential in vivo model of intestinal calcium malabsorption as occurs in postmenopausal osteoporosis. In the first experiment, we compared the effects of ovariectomy and 17 beta-estradiol (E2) therapy on calcium absorption in C3H/HeJ (C3H) and C57BL/6J (C57BL) mice, which have high and low peak bone mass, respectively. For each strain of mice, three groups were studied: sham operated, ovariectomized, (OVX), and OVX + E2 (60 micrograms/kg of body weight [bw]/day). Therapy was continued for 35 days and calcium absorption measured. In the C3H mice, ovariectomy caused an increase in fecal calcium (17.6%), and a decrease in the amount (12.8%) and percentage (12.5%) of calcium absorbed. The decrease was prevented by E2 therapy, but the differences in the calcium absorption parameters among the groups were not statistically significant. In contrast, in the C57BL mice, ovariectomy caused a marked increase in fecal calcium (84.5%, p < 0.01), and a marked decrease in the amount (55%, p < 0.01) and percentage (34.8%, p < 0.001) of calcium absorbed, and the decrease in the percentage of calcium absorbed was partially prevented by E2 therapy (p < 0.05). In experiment 2, a dose-response study of the effects of E2 therapy on calcium absorption in OVX C57BL mice was carried out. Five groups of mice were studied: Group 1, sham operated; Group 2, OVX; Group 3, OVX + 60 micrograms of E2/kg of bw/day; Group 4, OVX + 120 micrograms of E2/kg of bw/day; Group 5, OVX + 240 micrograms of E2/kg of bw/day. Therapy was continued for 28 days and calcium absorption measured. Ovariectomy caused a marked increase in fecal calcium (50%, p < 0.0001) and urinary calcium (31%, p < 0.0001) and a marked decrease in calcium absorption (44.9%, p < 0.0001), and these changes were prevented by E2 therapy. The highest level of calcium absorption (109%, p < 0.0001, vs. OVX) was observed in the 120 micrograms of E2 group. Ovariectomy and E2 slightly increased plasma 1,25-dihydroxyvitamin D (1,25(OH)2D) levels but there was no significant correlation between 1,25(OH)2D levels and calcium absorption. The findings in the C57BL mice suggest that estrogen is a physiological regulator of calcium absorption in this strain of mice. Furthermore, these findings share many characteristics with intestinal calcium malabsorption and its reversal by estrogen therapy in hypoestrogenic women. We propose that the OVX C57BL mice warrants further characterization as a potential animal model for investigating issues related to calcium malabsorption in postmenopausal women.

Preexisting bone loss associated with ovariectomy in rats is reversed by parathyroid hormone.

Previous studies have demonstrated that when parathyroid hormone (PTH) administration to rats is started immediately following ovariectomy, it prevents bone loss due to ovarian hormone deficiency. In this study, we examined whether bone loss induced by ovariectomy could be reversed by parathyroid hormone if hormone therapy is started after the bone loss had already occurred. In the first experiment, two groups of animals were ovariectomized or sham operated, killed after 40 days, and their bones examined to ensure that bone loss occurred. In the second experiment, three groups of rats were studied. Group 1 rats were sham operated, and rats in groups 2 and 3 were ovariectomized. Each rat in group 3 received a single subcutaneous injection of 8 micrograms parathyroid hormone [hPTH-(1-34); Bachem, CA] per 100 g body weight per day, starting 40 days following ovariectomy. Rats in groups 1 and 2 received solvent vehicle, and all animals were sacrificed on day 60. Ovariectomized rats had lost an appreciable amount of bone 40 days after surgery, as indicated by a significant decrease in femoral and vertebral densities and calcium and an over 55% loss of cancellous bone in the tibial metaphysis. The loss of bone was reversed by intermittent PTH administration. Increased cancellous bone in the parathyroid hormone-treated ovariectomized rats was associated with increased trabecular osteoblasts, decreased trabecular osteoclasts, and increased serum osteocalcin and urinary hydroxyproline. Our findings indicate that parathyroid hormone can substantially augment bone mass after the loss due to ovarian hormone deficiency has already occurred. The hormone caused positive bone balance in vivo in ovarian hormone-deficient animals by increasing bone formation and decreasing bone resorption.

The effects of gonadectomy on bone size, mass, and volumetric density in growing rats are gender-, site-, and growth hormone-specific.

Peak volumetric bone mineral density (BMD) is determined by the growth in bone size relative to the mineral accrued within its periosteal envelope. Thus, reduced peak volumetric BMD may be the result of reduced mineral accrual relative to growth in bone size. Because sex steroids and growth hormone (GH) influence bone size and mass we asked: What are the effects of gonadectomy (Gx) on bone size, bone mineral content (BMC), areal and volumetric BMD in growing male and female rats? Does GH deficiency (GH-) reduce the amount of bone in the (smaller) bone, i.e., reduce volumetric BMD? Does GH- alter the effect of Gx on bone size and mineral accrual? Gx or sham surgery was performed at 6 weeks in GH- and GH replete (GH+) Fisher 344 male and female rats. Changes in bone size, volume, BMC, areal and volumetric BMD, measured using dual X-ray absorptiometry (DPX-L), were expressed as percentage of controls at 8 months (mean +/- SEM). All results shown were significant (p < 0.05 level) unless otherwise stated. In GH+ and GH- males, respectively, Gx was associated with: lower femur volume (24%, 25%), BMC (43%, 45%), areal BMD (21%, 14%), and volumetric BMD (30%, 28%); lower spine (L1-L3) volume (26%, 28%), BMC (26%, 30%), and areal BMD (28%, 12%), but not volumetric BMD. Following Gx, GH+ females had increased femur volume (11%), no effect on BMC, decreased areal BMD (6%) and decreased volumetric BMD (17%); GH- females had no change in femur volume, but decreased femur BMC (24%), areal BMD (10%), and volumetric BMD (25%). In GH+ and GH- females, respectively, Gx was associated with a decrease in spine (L1-L3) BMC (12%, 15%), areal BMD (16%, 15%), and volumetric BMD (10%, 16%) with no change in volume. Deficits in non-Gx GH- relative to non-Gx GH+ (males, females, respectively) were: femur BMC (49%, 37%), areal BMD (23%, 8%), volume (19%, 19%) and volumetric BMD (37%, 22%); spine (L1-L3) BMC (46%, 42%), areal BMD (37%, 43%), volume (10%, 15%), and volumetric BMD (40%, 33%). Testosterone and GH are growth promoting in growing male rats, producing independent effects on bone size and mass; deficiency produced smaller appendicular bones with reduced volumetric BMD because deficits in mass were greater than deficits in size. At the spine, the reduction in size and accrual were proportional, resulting in a smaller bone with normal volumetric BMD. In growing female rats, estrogen was growth limiting at appendicular sites; deficiency resulted in a GH-dependent increase in appendicular size, relatively reduced accrual, and so, reduced volumetric BMD in a bigger bone. At the spine, accrual was reduced while growth in size was normal, thus volumetric BMD was reduced in the normal sized bone. Understanding the pathogenesis of low volumetric BMD requires the study of the differing relative growth in size and mass of the axial and appendicular skeleton in the male and female and the regulators of the growth of these traits.

Evaluation of the pathogenesis of skeletal changes in ovariectomized rats.

Young adult ovariectomized rats were used to investigate the pathogenesis of ovarian hormone deficiency osteopenia, uncomplicated by other forms of age-related bone loss. It was observed that the femurs of ovariectomized rats were less dense with less hydroxyproline and less calcium (Ca) and phosphorus per unit volume of bone than those of age-matched controls. Compared to animals killed at the beginning of the study to serve as baseline controls, ovariectomized rats and their age-matched controls had increased periosteal and cortical areas, but in contrast to the age-matched controls, ovariectomized rats had markedly increased medullary area and the ratio of their cortical area to periosteal area was decreased (P less than 0.02). When the changes in cross-sectional areas were expressed as percent of the baseline levels, the medullary area was increased 32% in ovariectomized rats, and only 14% in the age-matched controls; the net increase in cortical area was 17% for the controls and 11% for the ovariectomized rats and there was only 3% difference between the increases in their periosteal areas. Furthermore, ovariectomy caused a marked decrease in serum calcitonin and Ca levels but not in PTH levels. As expected, PTH administration increased serum Ca in all rats, but the rise was greater in ovariectomized calcitonin-deficient rats than in rats that were only ovariectomized or only thyroidectomized. Whereas these findings support the concept of increased sensitivity of bone to PTH in ovarian hormone deficiency osteopenia, the decrease in serum Ca in ovariectomized rats indicates that other factors may be involved as well.

Further studies on the hypercalcemic effect of acute calcitonin deficiency in rats.

Studies were carried out to determine the origin of the immediate increase in plasma calcium following acute calcitonin deficiency in mature rats. Animals were pre-labelled with 45calcium 24 hours and 4 weeks before thyroparathyroidectomy (TPTX) and 1 hour, 24 hours, 2 and 4 weeks before nephrectomy and TPTX and bled serially over the following 3 hours. In each study the final average weight of the rats was over 300 g. Plasma calcium increased after TPTX. In rats labelled with 45calcium 1 and 24 hours previously, the rise was to small to alter the specific activity of calcium although radiocalcium was unchanged. In contrast, in animals pre-labelled with 45calcium 2 and 4 weeks before TPTX, the increase in stable calcium was associated with a parallel increase in radiocalcium. Consequently, the specific activity of plasma calcium did not differ appreciably from that of controls. These findings confirm the theory that in mature unfed rats acute calcitonin deficiency results in an immediate rise in plasma calcium. Since this increase is due mainly to enhanced transport of calcium from deep bone, our observations are in accord with the view that calcitonin decreases plasma calcium primarily by inhibiting calcium transport from "stable" bone.

Lifelong dietary modulation of calcitonin levels in rats.

Studies were carried out on specific pathogen-free rats to evaluate the effects of aging and dietary manipulation on serum and thyroid calcitonin (CT) levels. Male Fischer 344 rats were randomized at 6 weeks of age to six dietary groups and subsequently maintained on the following dietary regimens. Group 1 rats were fed ad libitum throughout life; group 2 rats were fed 60% of the ad libitum food uptake, but received the same amounts of calcium, phosphorus, and vitamin D; group 3 rats were fed as the group 2 animals until 6 months of age and from then on were fed ad libitum; group 4 rats were fed ad libitum until 6 months of age and then switched to 60% food restriction; group 5 rats were fed ad libitum on food isocaloric with that of group 1 rats, but containing only 60% of the protein. Group 6 rats were killed at 6 weeks of age to serve as baseline controls. Ten rats were killed in each of the remaining five groups 15 h postprandial at 6-month intervals. The following observations were made. Serum CT increased with age similarly in the ad libitum fed group 1 and 5 rats. Food restriction markedly inhibited the increase in serum CT, and the effect was more profound in animals whose food intake was restricted after 6 months of age (group 4) than in animals on lifelong food restriction (group 2). In rats switched from food restriction to ad libitum feeding (group 3) at 6 months of age, serum CT increased with age to levels identical with those of lifelong ad libitum fed group 1 animals. Thyroid CT showed a similar pattern of age-dependent and dietary modulated changes. In contrast, aging and dietary modulation had no appreciable effect on serum calcium levels, except at 27 months of age when the serum calcium level of group 1 animals increased dramatically from the level for 24-month-old animals. There was a weak positive correlation between serum calcium and serum CT (r = 0.627; P = 0.02) and a highly significant positive correlation between serum CT and thyroid CT (r = 0.917; P = 0.001). These findings indicate that elective and therapeutic restriction of food intake might also attenulate CT levels in humans, with potentially adverse implications for skeletal homeostasis.

Calcium regulatory action of endogenous rat calcitonin demonstrated by passive immunization with calcitonin antibodies.

The calcium regulatory role of calcitonin (CT) in murine physiology was evaluated with a combination of immunological techniques, bioassays, and gland ablation. Passive immunization of rats with neutralizing CT antibodies caused an immediate but transient increase in plasma calcium with a time course similar to that observed in thyroparathyroidectomized animals. In animals fasted during the day and fed at night (1700-0700 h), acutely decreasing endogenous CT (< 100 pg/ml) by thyroparathyroidectomy or by passive immunization with CT antibodies resulted in a greater increase in plasma calcium in the preprandial (1600 h) than in the postprandial inverse correlation between immunoreactive CT and plasma calcium, a known secretagogue for CT. In these animals on a restricted feeding schedule, a sharp preprandial (1600 h) rise in immunoreactive and bioactive CT and a circadian rhythm in plasma calcium were also observed. The onset of the increase in circulating CT preceded the onset of feeding and coincided with or shortly preceded the daily nadir of plasma calcium. These findngs establish that CT has a primary calcium regulatory role in murine physiology. More specifically, they demonstrate that 1) the postthyroparathyroidectomy increase in plasma calcium is due specifically to the loss of CT, 2) passive immunization is a feasible technique for evaluating the actions of endogenous CT, 3) the role of CT in calcium regulation cannot be restricted to that of preventing prandial and postprandial increase in blood calcium, and 4) factors other than calcium must be involved in the control of CT secretion in the nonfed state.

Aging and dietary modulation of rat skeleton and parathyroid hormone.

Studies were carried out on SPF F344 male rats to evaluate the effects of aging and life-prolonging food restriction, without malnutrition, on rat skeleton and circulating PTH. Six-week-old F344 rats were divided into five groups. Group 1 rats were fed ad libitum a diet that contained 21% protein. Group 2 rats were fed 60% of the mean food intake of group 1 rats from 6 weeks of age for the rest of their lives. Group 3 rats were fed 60% of the ad libitum food intake until 6 months of age and then switched to ad libitum feeding. Group 4 rats were fed ad libitum until 6 months of age, and then switched to 60% of the ad libitum food intake. Group 5 rats were fed ad libitum a diet that contained only 12.6% protein so that these animals ingested the same amount of protein per day as the group 2 rats. In group 1 animals, bone length, weight, density, and calcium content increased rapidly with age and plateaued at about 12 months of age. There was no evidence of bone loss in these animals until about 24 months of age, but by 27 months, the animals had lost appreciable amounts of bone. The circulating immunoreactive PTH levels of the animals increased with advancing age, with a marked rise at 27 months. The age-related changes in bone and serum PTH levels of rats in groups 3 and 5 were similar to those of group 1 animals, except that a terminal increase in serum PTH did not occur in group 5 rats. In the groups 2 and 4 animals which were food restricted for the longest period, bone growth and maturation were slowed down, but the animals did not experience senile bone loss or marked terminal increase in circulating PTH. The salutary effects of food restriction were, therefore, not due specifically to the restriction of protein intake or to restricting food intake only during the period of rapid growth.

Modulation of age-related hyperparathyroidism and senile bone loss in Fischer rats by soy protein and food restriction.

Studies were carried out to explore the influence of soy protein and food restriction on age-related changes in serum PTH and bone. Three groups of male Fischer 344 rats were studied from 6 weeks of age. Group A rats were fed ad libitum diet A, which has casein as the protein source. Group B rats were fed diet B (with casein as protein source) at 60% of the mean ad libitum food intake. Group C rats were fed ad libitum diet C, which has soy protein as the protein source. The animals were killed at periodic intervals beginning at 6 months of age after an overnight fast. Serum PTH, measured with an intact N-terminal-specific RIA, and immunoreactive calcitonin increased progressively with aging. The increase was markedly suppressed by food restriction, and in the case of PTH by the soy protein diet as well. Serum creatinine started to increase after 18 months of age, and both dietary regimens of groups 2 and 3 retarded the increase. Aging was associated with a fall in serum 25-hydroxyvitamin D, and loss of bone occurred during the terminal part of life in the ad libitum-fed animals. These were prevented by food restriction, while the soy protein diet delayed the onset of bone loss. We conclude from these findings and other data from this study that in the male F344 rats 1) an age-related increase in serum PTH precedes an age-related increase in serum creatinine concentration; 2) an age-related decline in renal function probably contributes to age-related hyperparathyroidism, which, in turn, contributes to senile bone loss; 3) food restriction inhibits age-related hyperparathyroidism and senile bone loss; 4) on the basis of the data from rats fed a soy protein-containing diet, a decline in renal function and progressive hyperparathyroidism are not inevitable consequences of aging in the ad libitum fed rats.

The aged rat model of ovarian hormone deficiency bone loss.

Three studies were carried out. First, the effects of aging on the maturation of the female skeleton were assessed. Second, the hypothesis that has linked ovarian hormone deficiency bone loss to hypercalcemic suppression of the parathyroids leading to a decrease in 1,25-dihydroxyvitamin D synthesis and gut absorption of calcium was examined. Third, the effects of ovariectomy and a combination of ovarian hormone deficiency and low dietary calcium on bone and the calcium-regulating hormones were evaluated. After 6 months, ovariectomy and a low calcium diet independently decreased the density of the ilium, the femur, and the fourth lumbar vertebra as well as the calcium content of the latter two. The effects of the two treatment regimens were additive and more marked in the vertebral bone. Ovariectomy lowered serum calcitonin only in animals fed a normal diet and had no effect on serum PTH and vitamin D metabolites, while a low calcium diet caused a significant increase in serum 1,25-dihydroxyvitamin D. In both dietary regimens ovariectomy resulted in about a 30% decrease in intestinal calcium absorption. A low calcium diet increased morphometric indices of bone formation and bone resorption as did ovariectomy, with resorption exceeding formation. The discussion of our findings led to the conclusion that the aged rat model of ovarian hormone deficiency bone loss qualifies for serious consideration as a practical convenient cost-effective animal model for exploring aspects of the pathogenesis and treatment of postmenopausal bone loss.

Modulation of ovariectomy-related bone loss by parathyroid hormone in rats.

Studies were carried out to examine whether parathyroid hormone (PTH) will prevent the age-related bone loss that results from ovarian hormone deficiency and to explore the mechanism of its action. Ovariectomy caused a significant decrease in bone density in the distal metaphysis and mid-diaphysis, but not in the vertebra and proximal metaphysis. The decrease was prevented by PTH injection and in all the bones examined PTH administration increased bone density and bone calcium content above the levels in sham-operated controls. Similar findings were made in bone hydroxyproline levels. PTH treated ovariectomized animals had lower serum 25(OH)vitamin D and higher 1,25(OH)2 vitamin D levels than ovariectomized and sham operated animals that received solvent vehicle. Compared to the sham operated controls, ovariectomy caused a 4.5-fold increase in the number of tartrate resistant acid phosphatase (TRAP) positive multinuclear cells. This increase did not occur in PTH-treated animals. We conclude that PTH is effective in preventing ovarian hormone deficiency bone loss in rats. PTH may mediate this effect partly by stimulating osteoblastic bone formation and partly by increasing 1,25(OH)2 vitamin D-mediated calcium absorption. The data from TRAP positive multinuclear cells indicate that an etiologic component of ovarian hormone deficiency bone loss is the expansion of a pool of osteoclast progenitors and that the bone anabolic action of PTH involves, in part, a decrease in bone resorption as a result of the suppression of the proliferation of osteoclast progenitors.

Modulation of intestinal vitamin D receptor by ovariectomy, estrogen and growth hormone.

Age-related decline in intestinal calcium (Ca) absorption often occur in postmenopausal osteoporotic women. The impaired Ca absorption can be corrected by estrogen (E2) therapy. Growth hormone (GH) therapy has also been reported to increase intestinal absorption of calcium. Since 1,25-dyhydroxyvitamin D (1,25(OH)2D) is the primary regulator of calcium absorption, we explored whether the mechanisms by which E2 and GH enhance Ca absorption involves the vitamin D endocrine system. We measured serum 1,25(OH)2D concentrations and determined the binding characteristics of intestinal vitamin D receptors (VDRs) in four groups of female rats: sham operated (sham), ovariectomized (ovx), ovx + E2, and ovx + GH. Serum 1,25(OH)2D levels were 42.4 +/- 3.4 and 42.5 +/- 3.2 pg/ml in sham and ovx rats, respectively, and decreased by 63 and 34% (P < 0.001) in ovx + E2 and ovx + GH-treated rats, respectively. The numbers of total, unoccupied and occupied VDRs were 116.9 +/- 2.0, 72.1 +/- 1.1 and 44.8 +/- 1.9 fmol/mg protein, respectively, in sham operated rats, and decreased significantly following ovariectomy by 24, 27 and 19% (P < 0.01), respectively. E2 therapy not only significantly increased total, unoccupied and occupied VDRs above those of ovx rats by 55, 58 and 49% respectively, but it increased the levels above those of sham operated controls as well (P < 0.01). In contrast, GH administration prevented the decrease that occurred in ovx rats in the number of total and unoccupied VDRs (111.2 +/- 3.3; 72.6 +/- 1.4 fmol/mg protein, respectively), but it had no significant effect on the number of occupied VDRs. The dissociation constant (Kd) of intestinal VDRs was unaltered by ovariectomy, E2 and GH. We conclude that down regulation of intestinal VDRs may contribute to the Ca malabsorption that occurs in ovarian hormone deficient states such as postmenopausal osteoporosis, and that the stimulation of Ca absorption by E2 and GH may result, in part, from up regulation of intestinal VDRs.

Effects of aging and food restriction on the trigeminal ganglion: a morphometric study.

A quantitative morphometric study of the rat trigeminal ganglion was conducted to determine the changes that occur with aging. All measurements were tracked from young to old age in two rat groups simultaneously. One group was fed ad libitum, the other was maintained on restricted food intake from 6 weeks on. Immunocytochemical and radioimmunoassay techniques were used to study the neuron group that produces the peptide, CGRP and to compare it with the CGRP-negative neuron group. We observed that in the trigeminal ganglion, soma diameters and nucleus diameters of all neurons, whether CGRP positive or negative, increased modestly with age; so did total ganglion weight. Food restriction delayed, but did not prevent the increases in neuron diameters. No significant changes occurred as a function of age in the total number of neurons per ganglion, the ratio of CGRP positive to CGRP negative neurons and ganglion content of CGRP. Food restriction did not affect the parameters that remained constant with age. These findings are in contrast to the marked inhibitory effect of food restriction on age-related increase in thyroid calcitonin, a hormone that is encoded by the same gene as CGRP.

Evaluation of the molecular and cellular basis for the modulation of thyroid C-cell hormones by aging and food restriction.

Male F344 rats fed ad libitum or maintained on 60% of the ad libitum food intake were sacrificed at 6, 12, 18 and 24 months of age. The thyroids were removed for the analysis of the C-cell hormones, calcitonin, calcitonin gene-related peptide (CGRP) and somatostatin. In the animals fed ad libitum, the peptide content of all three hormones and their mRNA pools increased significantly with age. The increases were markedly suppressed by food restriction. Similarly, the rate of mRNA synthesis of the hormones increased with age and was attenuated by food restriction. Calcitonin and CGRP containing cells increased in number with age in the ad libitum fed animals. In the food restricted animals the numbers of calcitonin positive cells were consistently but not significantly lower than those of ad libitum fed animals at similar ages. In the case of CGRP containing cells, their numbers were significantly lower in the food restricted than in the ad libitum fed animals from 18 months of age. Our findings indicate that aging and food restriction modulate the levels of the thyroidal C-cell hormones at the levels of cell proliferation and possibly gene transcription.

Bone anabolic effects of separate and combined therapy with growth hormone and parathyroid hormone on femoral neck in aged ovariectomized osteopenic rats.

Previous studies have demonstrated that growth hormone (GH) has a marked anabolic effect on cortical bone and parathyroid hormone (PTH) has been shown to increase cancellous bone and cortical bone markedly in ovariectomized (OVX) rats. Most previous combination therapies used the bone anabolic agent (PTH) and the anti-resorptive agents. In this study, two bone anabolic hormones, GH and PTH, were used in rebuilding bone following loss due to ovariectomy in the femoral neck, which contains both cortical and cancellous bones. Twelve-month-old female F344 rats were divided into five groups: Sham+solvent vehicle, OVX+solvent vehicle, OVX+GH (2.5 mg/kg/day), OVX+PTH (80 microg/kg/day), and OVX+GH (2.5 mg/kg/day)+PTH (80 microg/kg/day). Following surgery, the animals were left for 4 months to become osteopenic before the beginning of hormone therapies. Hormone administrations were given 5 days per week for 2 months and the animals sacrificed. The right femurs were removed and the femoral necks were examined by pQCT densitometry and by histomorphometry. There was a 12.3% decrease in total bone mineral content (BMC) (P<0.01), a 6.2% decrease in total bone mineral density (BMD) (P<0.01), a 12.8% decrease in cortical BMC (P<0.05), a 25.9% decrease in cancellous BMC (P<0.0001), a 20.4% decrease in cancellous BMD (P<0.01), and a 34.2% decrease in cancellous bone volume (BV/TV) (P<0.0001) in vehicle-treated OVX rats. Growth hormone, PTH and GH+PTH treatment increased total BMC of the OVX rats by 14.4% (P<0.01), 23.5% (P<0.0001) and 30.6% (P<0.0001), respectively; increased total BMD by 7.0% (P<0.01), 9% (P<0.001) and 14.8% (P<0.0001), respectively; increased cortical BMC by 15.9% (P<0.05), 25.5% (P<0.001) and 29% (P<0.001), respectively; increased cancellous BMC by 40.9% (P<0.0001), 61.9% (P<0.0001) and 86.8% (P<0.0001), respectively; increased cancellous BMD by 31% (P<0.001), 41.8% (P<0.0001) and 61.8% (P<0.0001), respectively; increased cancellous BV/TV by 30.6% (P<0.05), 76.3% (P<0.0001) and 94.9% (P<0.0001), respectively; and increased trabecular thickness by 26.4% (P<0.05), 41.5% (P<0.001) and 43.2% (P<0.001), respectively, compared to the age-matched vehicle-treated OVX controls. In conclusion, both GH and PTH increased cortical and cancellous bone mass at the osteopenic femoral neck. Using two techniques, it was observed that the effects of PTH were mostly more marked than those of GH. Combined therapy with GH+PTH was more effective in rebuilding cortical bone and cancellous bone than either therapy alone in the aged ovariectomized osteopenic rats, which is in line with our hypothesis.

Analysis of the effects of growth hormone, exercise and food restriction on cancellous bone in different bone sites in middle-aged female rats.

The aim of this study is to determine the effects of growth hormone (GH), exercise (EX), GH+EX and food restriction on cancellous bone in middle-aged female rats. Female F344 rats aged 13 months were divided into (1) age-matched controls; (2) GH treated (2.5 mg/kg. 5 day/week); (3) EX (voluntary wheel running); (4) GH+EX; and (5) food restricted (FR) (fed 60% of the ad libitum food intake). The animals were treated for 18 weeks, at the end of which they were sacrificed. Cancellous bone and cortical bone in the fourth lumbar vertebra, proximal tibial metaphysis (PTM), distal femoral metaphysis (DFM) and femoral neck (NF) were analyzed using peripheral quantitative computerized tomography (pQCT) densitometry. Growth hormone increased cancellous bone area, cancellous bone mineral content, cortical bone area and cortical bone mineral content in the vertebra, PTM, DFM and NF. The tibial muscle wet weight was increased significantly after GH treatment. Exercise increased the cancellous bone area in the vertebra, PTM and DFM. Cortical bone area and cortical bone mineral content increased after EX in the vertebra, PTM, DFM and NF. No significant change was seen in the tibial muscle wet weight after EX. Growth hormone+EX increased cancellous bone area in the vertebra PTM and DFM but had no effect in neck of the femur. Cancellous bone mineral content, cortical bone area and cortical bone mineral content increased with GH+EX in the vertebra, PTM, DFM and NF. The tibial muscle wet weight was increased significantly with GH+EX. Food restriction decreased cancellous bone area and cancellous bone mineral content in all the bones studied. The decrease was statistically significant only at the distal femoral metaphysis. The tibial muscle wet weight decreased when compared with the age-matched control, but this decrease was not statistically significant. We conclude that the effect of the dose of GH used and the levels of voluntary wheel running EX used increased cancellous bone in intact rats; the effect of GH is much greater and different bones respond with varying intensities. The effects of combined treatment of GH and EX on cancellous bone are not always significantly higher than those of GH alone. FR at the level studied has a mostly negative effect on cancellous bone.

Lifelong food restriction prevents senile osteopenia and hyperparathyroidism in F344 rats.

Studies were carried out on male F344 rats to examine the influence of aging and life-prolonging food restriction on bone and circulating parathyroid hormone levels. In ad libitum fed animals, the weight, density and calcium content of the femur increased with age and achieved their peak levels by 12 months of age. These levels remained stable until about 24 months and by 27 months of age the ad libitum fed animals had lost appreciable amounts of bone. The maturation of the femurs of the animals maintained on 60% of the ad libitum food intake was delayed and their bones were lighter, less dense and contained less calcium than bones from ad libitum fed rats of corresponding ages. But at 6, 12 and 24 months of age, the femur strength to body weight ratios were very highly significantly greater (P less than 0.0001) for the restricted animals compared to the ad libitum fed controls. Circulating immunoreactive parathyroid hormone increased progressively with aging in the animals fed ad libitum and the animals that experienced bone loss at advanced age also had the highest level of the hormone. In contrast, in the food restricted animals aging was not associated with a marked increase in serum parathyroid hormone or with senile bone loss. The data are discussed in relation to the mechanism of the observed changes.