The diurnal rhythm of bone resorption in the rat. Effect of feeding habits and pharmacological inhibitors.

Prevention of low bone mass is important to reducing the incidence of osteoporotic fractures. This paper shows that, in rats, bone mass can be increased by feeding habits per se. Using six-hourly urinary excretion of [3H]tetracycline from prelabeled rats to monitor bone resorption, we previously found a peak of bone resorption following food administration. We now demonstrate that dividing the solid and liquid intake into portions blunts this peak and leads to a decrease in 24-h bone resorption to the level observed in thyroparathyroidectomized animals. Calcium balance increases and, when such feeding schedules are imposed for 30 d, bone mass increases. Dividing the intake is not effective in thyroparathyroidectomized animals, indicating the importance of PTH and/or calcitonin. Administration of calcitonin inhibits practically only the peak of bone resorption, suggesting that it is osteoclast mediated. In contrast, treatment with a bisphosphonate reduces basal bone resorption without a specific effect on the peak, indicating a fundamentally different mechanism of action. This is also supported by the finding that their combined effects are additive. Whether bone mass in humans is also under the control of dietary habits is not known. If so, an increased meal frequency may be used to prevent osteoporosis.

Apoptosis during castration-induced regression of the prostate is Fos dependent.

Apoptotic cell death was shown to be accompanied or preceded by an elevated expression of the c-fos protooncogene and DNA binding activity of transcription factor AP-1. We used Fos-deficient mice to study the role of c-Fos during programmed cell death in the prostate. In normal mice apoptosis is induced in the prostate within 2-4 days after castration. Histological features of reduced secretory activity and morphological signs of programmed cell death become obvious. No apparent decrease in secretory activity and no epithelial cell death were observed in Fos-deficient animals after castration. Fragmentation of nuclear DNA was measured by in situ terminal transferase reaction. DNA fragmentation was observed in the prostate epithelium of control mice after castration whereas no similar fragmentation was found in Fos-deficient animals. After castration an AP-1 complex accumulated in the prostate of Fos deficient mice which mainly consists of FosB, Fra-2 and JunD whereas in control animals the AP-1 complex in addition contained c-Fos. Our data strongly suggest that c-Fos is required for programmed cell death of prostate epithelial cells.

Food fractionation is a powerful tool to increase bone mass in growing rats and to decrease bone loss in aged rats: modulation of the effect by dietary phosphate.

The incidence of osteoporotic fractures has been associated with low bone mass. To reduce this incidence, it is therefore important to try to prevent the development of low bone mass by either increasing bone mass built up during adolescence and/or preventing bone loss in later life. It has been shown that food fractionation, a procedure that prevents the diurnal rhythm of bone resorption, increases bone mass in growing rats fed a high calcium (Ca), high phosphate (Pi) diet. In this paper, data are presented that show that providing growing rats with the same daily amount of a high Ca, low Pi diet (a Pi content similar to that of a human diet) in portions every 6 h instead of one meal increases total bone mineral content, trabecular bone mineral density, and cortical thickness, and markedly reduces the decrease in these parameters in aged rats. The effect is smaller when a high Ca, high Pi diet is fractionated. This could be the consequence of the transient postprandial increase in parathyroid hormone (PTH) following one large meal of a high Ca, high Pi diet, since the effect is similar to that reported after PTH injections which are anabolic for bone. Thus, a high dietary Pi must be considered as a confounding factor when treatments affecting bone mass are investigated in rats. The present data show that feeding habits have a profound effect on bone mass in the rat, independent of age. Whether bone mass in humans is also under the control of dietary habits is not yet clear. If so, frequent small meals of appropriate composition may help to prevent osteoporosis.

Tumor necrosis factor alpha and interleukin-1 stimulate bone resorption in vivo as measured by urinary [3H]tetracycline excretion from prelabeled mice.

Tumor necrosis factor alpha (TNF-alpha) and interleukin-1 (IL-1) have been shown to stimulate bone resorption in vitro. We have now investigated whether these cytokines also cause a similar action when administered in vivo. This was made possible by the adaptation of a newly developed technique that enables the continual assessment of bone resorption in vivo in mice by measuring urinary excretion of 3H from [3H]tetracycline-prelabeled animals. Experiments using maneuvers known to influence bone resorption, such as a change in dietary calcium or administration of parathyroid hormone or dichloromethylenebisphosphonate, indicate that the technique is reliable and sensitive in mice. Daily intravenous administration of either recombinant human or recombinant murine TNF-alpha, as well as subcutaneous administration of recombinant human IL-1 alpha, were found to stimulate bone resorption in a dose-dependent manner. The effect was maximal within 2 days. Thus, exogenous TNF-alpha and IL-1 alpha can stimulate bone resorption in vivo, suggesting that these cytokines may also exert a systemic effect on bone.

Evaluation of urinary pyridinium crosslink excretion as a marker of bone resorption in the rat.

The aim of this study was to evaluate the value of the urinary excretion of the pyridinium crosslinks, pyridinoline (Pyr) and deoxypyridinoline (D-Pyr), as markers of bone resorption in the rat. The excretion of the crosslinks was compared with that of urinary [3H]tetracycline ([3H]TC) excretion from chronically [3H]TC-prelabeled animals, a technique established to monitor bone resorption in the rat. Bone resorption was modulated by Ca restriction, infusion of PTH, thyroparathyroidectomy, and administration of different bisphosphonates. Furthermore, the urinary crosslinks were assessed in three different osteopetrotic mutations in the rat. We found a delayed response of Pyr and D-Pyr excretion to acute changes in bone resorption compared with [3H]TC excretion. This delay was 1 day after Ca restriction and longer after other treatments, such as PTH administration or bisphosphonate treatment, with which it was more than 3 weeks. In contrast, chronic states with stimulation or inhibition of bone resorption showed similar changes in excretion of the urinary crosslinks and [3H]TC, except after PTH administration. The excretion of the crosslinks was greatly reduced in osteopetrotic rats (op/op, tl/tl, and ia/ia) and increased to normal levels in tl/tl rats after stimulation of bone resorption by M-CSF administration. These results suggest that, in rats, urinary excretion of the pyridinium crosslinks reflects bone resorption in chronic but not always in acute conditions. The cause of this discrepancy is still unclear.

BM 21.0955, a potent new bisphosphonate to inhibit bone resorption.

A total of 300 new bisphosphonates were screened for their effect on bone resorption in the rat. Among these, 1-hydroxy-3-(methylpentylamino)propylidenebisphosphonate (BM 21.0955) was selected for detailed investigation. It inhibited arotinoid-stimulated bone resorption as assessed by calcemia in thyroparathyroidectomized rats at a SC dose as low as 0.001 mg P (0.016 mumol) per kg body weight per day. The compound was thus about 2, 10, 50, and 500 times more potent than risedronate, alendronate, pamidronate, and clodronate, respectively. Intravenous administration was as effective as subcutaneous, and oral administration was 100 times less effective. The effect after one administration decreased with time but was still measurable after 2 weeks. Nonstimulated bone resorption assayed by the urinary excretion of radiolabeled tetracycline from lifelong prelabeled animals was also inhibited. This effect started 3 days after a single dose and was still maximal after 7 days. Histomorphometric analysis of the tibial metaphysis in growing intact rats also showed an inhibition of bone resorption along with an increase in bone mass. The number of osteoclasts increased in animals treated with 0.01 and 0.1 mg P per kg (0.16 and 1.6 mumol/kg) body weight SC but decreased in animals given 1 mg P per kg (16.1 mumol/kg), showing that the inhibition of bone resorption was not due to an inhibition of osteoclast recruitment. No inhibition of mineralization occurred. This new bisphosphonate appears to have great potential for use in human bone disease.

Evidence for heterogeneity of the osteoblastic phenotype determined with clonal rat bone cells established from transforming growth factor-beta-induced cell colonies grown anchorage independently in semisolid medium.

Numerous reports have appeared in the literature indicating phenotypic heterogeneity among cells of the osteoblastic lineage. This diversity may be due to either certain stages of differentiation or a subspecialization of already terminally differentiated osteoblasts. To obtain answers to this question, we report on studies undertaken to clone bone cell populations from 1 day postnatal rat calvaria which express well defined differences in phenotype. To achieve this goal, we have used the soft agarose cloning technique which previously has almost exclusively been applied to clone cells of neoplastic origin. The reason for being able to employ this method is based on the fact that bone cells can be induced by transforming growth factor-beta to reversibly acquire the transformed phenotype, an event expressed by anchorage-dependent bone cells to form progressively growing colonies in soft agarose. Individual colonies, harvested from agarose, were expanded to clonal bone cell populations. Characterizing 48 cell clones by detection of osteoblastic cell markers such as alkaline phosphatase activity, PTH- and prostaglandin-E2-induced adenylate cyclase activity, osteocalcin mRNA synthesis, as well as collagen synthesis, 7 subsets of osteoblastic cell types were identified. Each subset was found to express a distinct phenotype, indicated by the absence or presence of osteoblastic cell markers. Some clones, previously found not to exhibit any osteoblastic traits, developed PTH responsiveness when treated with insulin-like growth factor-I/transforming growth factor-beta, suggesting that these clones may originate from the osteoprogenitor cell pool. While most clonal cell populations were characterized as fully functional osteoblastic cells, some clones expressed merely 1, 2, or 3 osteoblastic markers, which suggests that they may represent stages of differentiation along the osteogenic pathway. In addition, other subclones displayed the capacity to synthesize osteocalcin and showed PTH and prostaglandin-E2 responsiveness, but were found to be devoid of alkaline phosphatase activity. Others expressed all osteoblastic cell markers except PTH responsiveness. The phenotypic constellation of the latter suggests that these cell clones may represent mature osteoblast-like cells, which, perhaps due to environmental circumstances present at the time of isolation, have become altered in accordance with the physiological requirements of the tissue.

The food-induced stimulation of bone resorption in the rat, assessed by the urinary [3H]-tetracycline excretion, is mediated by parathyroid hormone.

Using the urinary excretion of [3H]-tetracycline from prelabeled rats to monitor bone resorption, we have previously shown that food intake is associated with a rapid and large increase in bone resorption. This increase is blunted when the daily intake is fractionated into 4 portions instead of being given at once. Food fractionation also leads to a large increase in bone mass. In order to establish whether the thyroparathyroid gland is involved in this effect, thyroparathyroidectomized (TPTX) rats were studied. The food-induced increase in bone resorption was absent in TPTX rats. Therefore, the pattern of parathyroid hormone (PTH) was investigated during the development of the food-induced bone resorption, and under food fractionation. Rats were trained to eat their daily high Ca food in less than two hours. Thereafter they were given food portions of 5 or 20 grams, respectively. PTH in serum was measured at 0, 1, 2, 3, and 4 hours after food intake. In rats given the large food portions, a conspicuous increase of PTH was found. In contrast, serum PTH of rats fed the small food portion did only change to an insignificant extent. This study in rats shows that the ingestion of a large meal induces a transient increase of PTH. The present results can therefore explain the formerly observed acute increase in bone resorption following food intake and its blunting by food fractionation. It is not known, whether such a mechanism occurs also in humans. If so, these results would provide the rational basis to decrease bone resorption by food fractionation.

Common herbs, essential oils, and monoterpenes potently modulate bone metabolism.

During our survey of herbs looking for activity on bone metabolism, we found that the dried leaves of sage strongly inhibit bone resorption. Therefore, we investigated several common herbs rich in essential oils (sage, rosemary, and thyme) and essential oils extracted from these herbs and other plants (oils of sage, rosemary, juniper, pine, dwarf pine, turpentine, and eucalyptus) as well as their monoterpene components (thujone, eucalyptol, camphor, borneol, thymol, alpha-pinene, beta-pinene, bornylacetate as well as menthol) and found that they inhibit bone resorption when added to the food of rats. Pine oil, used as a representative essential oil, protects an osteoporosis model, the aged ovariectomized rat, from bone loss. The monoterpenes borneol, thymol, and camphor are directly inhibitory in the osteoclast resorption pit assay. Nonpolar monoterpenes may require metabolism to be active in vitro, for example, cis-verbenol, a metabolite of alpha-pinene occurring in human urine, inhibits osteoclast activity in contrast to the parent compound. Within 30 min borneol inhibits the formation of actin rings, a characteristic of resorbing osteoclasts indicating cell polarization. Both the in vitro and the in vivo effects of borneol are reversible. Our study demonstrates for the first time that essential oils and monoterpenes are efficient inhibitors of bone resorption in the rat.

Frequency of food intake and natural dietary components are potent modulators of bone resorption and bone mass in rats.

Prevention of low bone mass is important to reduce the incidence of osteoporotic fractures. In this report evidence is provided that feeding habits per se, that is, increased frequency of food intake as well as a diet containing soy and other raw components, decrease bone resorption and increase bone mass in growing rats. Interim results after 6 weeks indicate that food fractionation and natural dietary components are both capable of inhibiting trabecular bone loss in aged rats. These interim results indicate that the effect of both dietary interventions are additive and together are capable of nearly completely blunting the age-dependent loss of trabecular bone mineral density. These dietary manipulations are, however, only partially effective in inhibiting the strongly increased loss of trabecular bone mineral density induced by estrogen deprivation. The fact that the natural dietary components are not more effective in ovariectomized rats as compared to intact females confirms our contention that these components may not operate by mimicking the effect of estradiol. Whether bone mass in humans is also under the control of dietary habits is not known. If so, an increased frequency of meals of appropriate composition may be used to prevent osteoporosis.

Some vegetables (commonly consumed by humans) efficiently modulate bone metabolism.

We have hypothesized that some vegetables which are part of the regular human diet may contain modulators of bone metabolism. To mimic a typical Western diet with large proportions of refined components, rats were pair-fed a semi-purified diet to which, in the treated animals, the dried material under investigation was added. Effects are expressed as % of untreated control. Bone parameters in rats were assessed in the proximal tibia by pQCT. Bone resorption (BR) was assessed by the urinary excretion of [3H]-tetracycline from prelabeled rats. Daily administration of 1 g of onion during 4 weeks increased total bone mineral content by 17.4% (p<0.05), trabecular bone mineral density by 13.6% (p<0.05). One g of onion/day administered to male rats blunted BR by 23-/+5% (p<0.05). Daily administration of onion to ovariectomized rats inhibited BR in a dose-dependent manner. At the highest dose (1.5 g of onion) BR was inhibited by 26-/+4% (p<0.01) as compared to 24-/+3% (p<0.001) for estradiol (27microg/kg/day). An additional 13 vegetables displayed significant effects on BR at the dose of 1g/day. Interestingly, 1g/day of soy did not inhibit BR in this model. Also, skimmed milk, meat and egg (all 1 g/day) were ineffective. Thus, common vegetables consumed by humans potently modulate bone metabolism in the rat. This opens the possibility to develop the basis for a low-cost, safe and effective nutritional approach to osteoporosis.

Comparison of knee joint cartilage thickness in triathletes and physically inactive volunteers based on magnetic resonance imaging and three-dimensional analysis.

The objective of this study was to employ quantitative magnetic resonance imaging for the analysis of knee joint cartilage thickness in triathletes and physically inactive volunteers. The right knee joints of nine male triathletes (10 hours training per week for at least 3 years) and nine inactive male volunteers (<1 hour of physical activity per week throughout life) were imaged with a previously validated fat-suppressed gradient echo sequence. The cartilage plates were reconstructed three-dimensionally, and the cartilage thickness was computed independently of the original section orientation with a three-dimensional Euclidian distance transformation. There was a high interindividual variability of the mean and the maximal cartilage thickness values in all surfaces, both in the triathletes and in the inactive volunteers. In the patella, the femoral trochlea, and the lateral femoral condyle, the mean and maximal cartilage thickness values were slightly higher in the triathletes, but they were somewhat lower in the medial femoral condyle, and in the medial and lateral tibial plateau. However, the differences did not attain statistical significance. These results are unexpected in view of the functional adaptation observed in other musculoskeletal tissues, such as muscle and bone, in which a more obvious relationship with the magnitude of the applied mechanical stress has been observed.

[Sex-specific analysis of cartilage volume in the knee joint--a quantitative MRI-based study]. / Geschlechtsspezifische Analyse der Knorpelvolumina des Kniegelenks--eine quantitative MRT-basierte Studie.

The objective of the present study was to determine differences in the normal knee joint cartilage volume of males and females, the analysis of the percentage distribution of the cartilage tissue onto the various joint surfaces, and the determination of the relationship between the cartilage volume, the body weight, and the tibial head diameter. We examined the knee joints of nine healthy men and nine women with a low level of physical activity. The cartilage volume was assessed with magnetic resonance imaging, applying a fat-suppressed gradient-echo sequence with a resolution of 2 x 0.31 x 0.31 mm3 and 3D image reconstruction. In the men, the absolute volumes of the femur and tibia, but not those of the patella, were significantly higher than in the women. The differences between the sexes were considerably lower after normalisation to the body weight and the tibial head diameter and were no more statistically significant. The interindividual variability was reduced after normalisation to these two parameters, the body weight being more effective. We did not observe sex-specific differences in the percentage of the total cartilage volume taken up by the various joint surfaces. Our results suggest that, in young individuals without cartilage lesions, there exist sex-specific differences of the cartilage volume in the knee joint. However, these can be explained in terms of general differences in body constitution (body weight and bone size), without further significant influences of the sex. The knowledge of the normal, sex-specific cartilage volume is relevant when attempting to estimate the amount of tissue loss at the time at which symptoms occur in a patient with degenerative joint disease.

Pharmacological effects of secretin and somatostatin on gastric and renal function in man.

In this randomized, double-blind, placebo-controlled study therapeutic doses of synthetic secretin (0.5 CU/kg/h) and somatostatin (3.5 micrograms/kg/h) given an intravenous infusion suppressed significantly peptone-stimulated gastric acid output per 3 h in nine male healthy subjects from 59.2 +/- 7.4 mmol H+ (placebo) to 16.9 +/- 3.1 and 6.6 +/- 1.5 mmol H+, respectively. Peptone-stimulated gastrin release was reduced to basal concentrations by both hormones. In five subjects secretin raised serum lipase to pathological concentrations. Somatostatin diminished significantly mean blood glucose concentrations. Both had opposite renal effects; secretin showed diuretic and somatostatin antidiuretic properties. Secretin and somatostatin were well tolerated. It is concluded from these data that therapeutic doses of secretin and somatostatin are comparably effective on exocrine and endocrine gastric functions, but have opposite effects on renal functions in man.