Effects of increased hypothalamic leptin gene expression on ovariectomy-induced bone loss in rats.

Estrogen deficiency results in accelerated bone turnover with a net increase in bone resorption. Subcutaneous administration of leptin attenuates bone loss in ovariectomized (ovx) rats by reducing bone resorption. However, in addition to its direct beneficial effects, leptin has been reported to have indirect (central nervous system-mediated) antiosteogenic effects on bone, which may limit the efficacy of elevated serum leptin to prevent estrogen deficiency-associated bone loss. The present study evaluated the long-term effects of increased hypothalamic leptin transgene expression, using recombinant adeno-associated virus-leptin (rAAV-Lep) gene therapy, on bone mass, architecture, and cellular endpoints in sexually mature ovx Sprague-Dawley rats. Ovx rats were implanted with cannulae in the 3rd ventricle of the hypothalamus and injected with either rAAV-Lep or rAAV-GFP (control vector encoding green fluorescent protein) and maintained for 10 weeks. Additional controls consisted of ovary-intact rats and ovx rats pair-fed to rAAV-Lep rats. Lumbar vertebrae were analyzed by micro-computed tomography and tibiae by histomorphometry. Cancellous bone volume was lower and osteoclast perimeter, osteoblast perimeter, and bone marrow adipocyte density were greater in ovx rats compared to ovary-intact controls. In contrast, differences among ovx groups were not detected for any endpoint evaluated. In conclusion, whereas estrogen deficiency resulted in marked cancellous osteopenia, increased bone turnover and marrow adiposity, increasing hypothalamic leptin transgene expression in ovx rats had neither detrimental nor beneficial effects on bone mass, architecture, or cellular endpoints. These findings demonstrate that the antiresorptive effects of subcutaneous leptin administration in ovx rats are mediated through leptin targets in the periphery.

Vitamin E provides protection for bone in mature hindlimb unloaded male rats.

The deleterious effects of skeletal unloading on bone mass and strength may, in part, result from increased production of oxygen-derived free radicals and proinflammatory cytokines. This study was designed to evaluate the ability of vitamin E (alpha-tocopherol), a free-radical scavenger with antiinflammatory properties, to protect against bone loss caused by skeletal unloading in mature male Sprague-Dawley rats. A 2 x 3 factorial design was used with either hindlimb unloading (HU) or normal loading (ambulatory; AMB), and low-dose (LD; 15 IU/kg diet), adequate-dose (AD; 75 IU/kg diet), or high-dose (HD; 500 IU/kg diet) vitamin E (DL-alpha-tocopherol acetate). To optimize the effects of vitamin E on bone, dietary treatments were initiated 9 weeks prior to unloading and continued during the 4-week unloading period, at which time animals were euthanized and blood and tissue samples were collected. Serum vitamin E was dose-dependently increased, confirming the vitamin E status of animals. The HD treatment improved oxidation parameters, as indicated by elevated serum ferric-reducing ability and a trend toward reducing tissue lipid peroxidation. Histomorphometric analysis of the distal femur revealed significant reductions in trabecular thickness (TbTh), double-labeled surface (dLS/BS), and rate of bone formation to bone volume (BFR/BV) due by HU. AMB animals on the HD diet and HU animals on the LD diet had reduced bone surface normalized to tissue volume (BS/TV) and trabecular number (TbN); however, the HD vitamin E protected against these changes in the HU animals. Our findings suggest that vitamin E supplementation provides modest bone protective effects during skeletal unloading.

The catechol estrogen, 4-hydroxyestrone, has tissue-specific estrogen actions.

Recent data indicate that the catechol estrogen, 2-hydroxyestrone (2-OHE(1)), has no effect on any target tissue including bone, whereas 16 alpha-hydroxyestrone (16 alpha-OHE(1)) exerts tissue-selective estrogen agonist activity. The effect of the catechol estrogen, 4-hydroxyestrone (4-OHE(1)), putatively associated with tumorigenesis, has not been studied in the skeleton. The purpose of this study was to assess the effect of 4-OHE(1) on tibia, uterine and mammary gland histology and blood cholesterol in ovariectomized (OVX'd) growing rats. Ten-week-old female Sprague-Dawley rats were injected subcutaneously with 200 microg/kg BW per day with 4-OHE(1), 17 beta-estradiol (E(2)) or vehicle for three weeks. OVX resulted in uterine atrophy, increased body weight, radial bone growth and cancellous bone turnover, and hypercholesterolemia. E(2) prevented these changes with the expected exception that the subcutaneous infusion of this high dose of estrogen did not prevent the hypercholesterolemia. 4-OHE(1) prevented the increase in blood cholesterol and the increase in body weight. 4-OHE(1) appeared to have partial estrogen activity in the uterus; uterine weight and epithelial cell height were significantly greater than the OVX rats but significantly less (twofold) than the E(2) animals. Analysis of variance indicated that 4-OHE(1) slightly decreased the periosteal mineral apposition rate (P<0.05) compared with vehicle-treated rats but had no effect on double-labeled perimeter or bone formation rate. Similarly, 4-OHE(1) was a partial estrogen agonist on cancellous bone turnover. The data suggest that the catechol estrogen, 4-OHE(1), unlike 2-OHE(1), has estrogen activity. Furthermore, the profile of activity differs from that of 16 alpha-OHE(1). Our results suggest that estrogen metabolites may selectively influence estrogen-target tissues and, concomitantly, modulate estrogen-associated disease risk.

Is resveratrol an estrogen agonist in growing rats?

Trans-3,4,5-trihydroxystilbene (resveratrol), a polyphenolic compound found in juice and wine from dark-skinned grape cultivars, was recently shown to bind to estrogen receptors in vitro, where it activated transcription of estrogen-responsive reporter genes. The purpose of this 6-day study in weanling rats was to determine the dose response (1, 4, 10, 40, and 100 microg/day) effects of orally administered resveratrol on estrogen target tissues. The solvent (10% ethanol) had no significant effect on any measurement or derived value. 17Beta-estradiol treatment (100 microg/day) decreased the growth rate, final body weight, serum cholesterol, and radial bone growth (periosteal bone formation and mineral apposition rates) at the tibia-fibula synostosis. In the uterus, 17beta-estradiol treatment increased wet weight, epithelial cell height, and steady state messenger RNA levels for insulin-like growth factor I. In contrast, resveratrol treatment had no significant effect on body weight, serum cholesterol, radial bone growth, epithelial cell height, or messenger RNA levels for insulin-like growth factor I. Resveratrol treatment resulted in slight increases in uterine wet weight, but significance was achieved at the 10-microg dose only. A second experiment was performed to determine whether a high dose of resveratrol (1000 microg/day) antagonizes the ability of estrogen to lower serum cholesterol. As was shown for the lower doses, resveratrol had no effect on body weight, uterine wet weight, uterine epithelial cell height, cortical bone histomorphometry, or serum cholesterol. 17Beta-estradiol significantly lowered serum cholesterol, and this response was antagonized by cotreatment with resveratrol. These in vivo results suggest, in contrast to prior in vitro studies, that resveratrol has little or no estrogen agonism on reproductive and nonreproductive estrogen target tissues and may be an estrogen antagonist.

Dietary ethanol does not accelerate bone loss in ovariectomized rats.

The abuse of alcohol is a behavior that can significantly compromise skeletal health. Because postmenopausal women are already at risk for low bone mass and osteoporotic fracture, this investigation sought to determine whether high concentrations of dietary ethanol exacerbate the bone loss associated with ovariectomy in rats, an animal model of human postmenopause. Six-month-old Sprague-Dawley rats were ovariectomized or sham-operated and randomly divided into groups fed a modified Lieber-DeCarli liquid diet isocalorically supplemented with 0%, 13%, or 35% ethanol (by daily caloric intake), for a period of 2 months. All animals were injected with fluorochromes at the start, 2 weeks, and 2 days before sacrifice to label mineralizing bone surfaces. At sacrifice, blood, uterus, and tibiae were harvested. No differences in serum calcium or cholesterol were found. Serum creatinine was also found to be unvaried, indicating this level of alcohol consumption did not compromise liver function. Dietary alcohol consumption at 35% of daily caloric intake was determined to increase tibial cortical medullary area and endocortical perimeter, while not affecting cortical area and periosteal perimeter. Ovariectomy significantly increased indices of bone turnover and resulted in cancellous bone loss, whereas alcohol consumption had no additional detrimental effects. This was a consistent pattern for other indices of proximal tibial architecture. In summary, this investigation has found that chronic ingestion of high concentrations of alcohol does not accentuate bone loss in ovarian hormone-deficient adult female rats.

Effects of growth hormone and low dose estrogen on bone growth and turnover in long bones of hypophysectomized rats.

Pituitary hormones are recognized as critical to longitudinal growth, but their role in the radial growth of bone and in maintaining cancellous bone balance are less clear. This investigation examines the histomorphometric effects of hypophysectomy (Hx) and ovariectomy (OVX) and the subsequent replacement of growth hormone (GH) and estrogen (E), in order to determine the effects and possible interactions between these two hormones on cortical and cancellous bone growth and turnover. The replacement of estrogen is of interest since Hx results in both pituitary and gonadal hormone insufficiencies, with the latter being caused by the Hx-associated reduction in follicle stimulating hormone (FSH). All hypophysectomized animals received daily supplements of hydrocortisone (500 microg/kg) and L-thyroxine (10 microg/kg), whereas intact animals received daily saline injections. One week following surgery, hypophysectomized animals received either daily injections of low-dose 17 beta-estradiol (4.8 microg/kg s.c.), 3 X/d recombinant human GH (2 U/kg s.c.), both, or saline for a period of two weeks. Flurochromes were administered at weekly intervals to label bone matrix undergoing mineralization. Whereas Hx resulted in reductions in body weight, uterine weight, and tibial length, OVX significantly increased body weight and tibial length, while reducing uterine weight. The combination of OVX and Hx resulted in values similar to Hx alone. Treatment with GH normalized body weight and bone length, while not affecting uterine weight in hypophysectomized animals. Estrogen increased uterine weight, while not impacting longitudinal bone growth and reduced body weight. Hypophysectomy diminished tibial cortical bone area through reductions in both mineral appositional rate (MAR) and bone formation rate (BFR). While E had no effect, GH increased both MAR and BFR, though not to sham-operated (control) levels. Hypophysectomy reduced proximal tibial trabecular number and cancellous bone area, and increased trabecular separation. Both GH and E reduced cancellous osteopenia, although employing different mechanisms. GH reduced the decrease in trabecular thickness, whereas E reduced the decrease in trabecular number and the increase in trabecular separation. Hypophysectomy reduced both Tb.MAR and Tb.BFR while treatment with GH enhanced them. This investigation has shown that Hx and GH have a dramatic impact on selected static and dynamic indices of rat cortical and cancellous histomorphometry. Furthermore, the mechanisms of action of GH and E differ, and suggest that some of the skeletal changes associated with Hx are caused by deficiencies in estrogen as well as deficiencies in growth hormone.

Short-term effects of high dose estrogen on tibiae of growing male rats.

The short-term effects of estrogen at a single high dose (4 mg/kg body weight/day for 14 days) were determined on tibiae in the normal (noncastrate) growing male rat. In cortical periosteal bone, at a middiaphyseal site devoid of resorbing activity, estrogen suppressed periosteal bone formation and apposition rates, resulting in a smaller cross-sectional area. In middiaphyseal endocortical bone, estrogen had no effect on apposition and formation rates and, because medullary area was unchanged, probably had no effect on endocortical bone resorption. In the proximal tibial metaphysis, estrogen greatly suppressed longitudinal growth rate. In a site within the metaphysis adjusted for the effects of growth, cancellous mineral apposition was greatly reduced by the hormone. Estrogen-treated rats retained more of a fluorochrome label deposited in cancellous bone at the beginning of the study than vehicle-treated animals, indicating a reduced net bone loss. As a result of the lowered resorption induced by estrogen, cancellous bone mass (area and perimeter) were both significantly higher in estrogen-treated rats. No evidence was found for an anabolic action of the hormone in the male rat; indeed, estrogen reduced indices of bone formation.

Inhibition by human interleukin-1 alpha of parathyroid hormone-related peptide effects on renal calcium and phosphorus metabolism in the rat.

Humoral hypercalcemia of malignancy (HHM) is at least partly caused by tumor secretion of PTH-related peptide (PTHrP), but there is growing evidence for cosecretion with PTHrP of other bone-resorbing peptides, such as the cytokine interleukin-1 alpha (IL-1 alpha). Administration of PTHrP in vivo and in vitro generally mimics the actions of PTH itself, with increases in both resorption and formation of bone. However, bone in HHM is characterized by uncoupling of bone turnover, with increased resorption and decreased formation. We performed experiments to determine whether IL-1 alpha might alter the effects of PTHrP and produce uncoupling. Thus, we administered to 100-g male rats by sc osmotic minipumps synthetic PTHrP-(1-34) alone (2 micrograms/100 g/day), recombinant IL-1 alpha alone (1.5 micrograms/100 g/day), both peptides together at the previous doses, or vehicle only. We infused 5 groups of 12 rats each (PTHrP, IL-1 alpha, PTHrP plus IL-1 alpha, ad libitum fed control, and controls pair-fed to the PTHrP plus IL-1 alpha group) for 14 days. At the end of the study, blood and urine were taken for chemical measurements, and tibias and femurs were harvested for histomorphometry and extraction of RNA from periosteal cells. As expected, PTHrP induced hypercalcemia, relative hypophosphatemia, phosphaturia, and reduced bone mass. Osteoblast number was increased, but osteoclast number was not. Indices of bone formation were unchanged or reduced. The dose of IL-1 alpha chosen had no statistically significant effect, except for reduced longitudinal bone growth, but when combined with PTHrP, IL-1 alpha reduced hypercalcemia, hypophosphatemia, and phosphaturia. In contrast to the blood and urine effects, IL-1 alpha did not interact significantly with PTHrP's effect on bone measurements. Northern analysis of periosteal cell mRNA showed that PTHrP reduced expression of osteocalcin, but not glyceraldehyde-3-phosphate dehydrogenase; IL-1 alpha had no additional effect. These data suggest that 1) continuously administered PTHrP alone may induce uncoupled bone turnover with decreased cortical bone formation; 2) IL-1 alpha appears to inhibit strongly the renal effects of PTHrP and weakly (if at all) its actions on bone and, thus, to decrease its hypercalcemic, phosphaturic, and hypophosphatemic actions; and 3) cosecretion of IL-1 alpha, and possibly other peptide cytokines, with PTHrP may modify the clinical expression of HHM.

Effects of gallium on bone in the rat.

Gallium nitrate lowers the serum calcium in patients with hypercalcemia caused by malignancy and is available for clinical use. The mechanism for the hypocalcemic action is unknown, however. The present studies were undertaken to determine the effects of gallium on bone metabolism. Normal male rats were implanted subcutaneously with mineralized allogeneic bone matrix. Histomorphometry of the implants and of tibiae was determined after three doses of tetracycline administered at intervals of 1 week. Gallium as nitrate was administered daily by intraperitoneal injection at doses of 0.9, 1.8, and 3.6 mg elemental gallium per kg body weight for 21 days in one study and at 3.5 mg/kg for 33 days in a second study. All the gallium-treated rats gained weight. Rats given gallium at doses of 3.5 mg/kg or more grew at a lower rate than untreated controls (-7 and -10% at doses of 3.5 and 3.6 mg/kg, respectively; p less than 0.05). At a dose of 0.9 mg/kg, gallium did not inhibit bone resorption or lower serum calcium but inhibited bone formation by 32% and bone apposition by 36% at the endosteal surface of the tibia. At a dose of 1.8 mg/kg, gallium produced modest hypocalcemia, prevented a rise in circulating 1,25-dihydroxyvitamin D [1,25-(OH)2D], inhibited bone resorption in implants, and inhibited bone formation by 19% and bone apposition by 18%. At a dose of 3.5 mg/kg, gallium lowered the serum calcium and serum 1,25-(OH)2D, inhibited growth, and accentuated the antiresorptive and antiformative effects seen at the two lower doses.(ABSTRACT TRUNCATED AT 250 WORDS)

Abnormal calcium metabolism caused by increased circulating 1,25-dihydroxyvitamin D in a patient with rheumatoid arthritis.

A 35-year-old white male with rheumatoid arthritis who had developed hypercalcemia, hypercalciuria, and nephrolithiasis was found to be abnormally sensitive to vitamin D as a result of lack of regulation of circulating 1,25-dihydroxyvitamin D (1,25-(OH)2D). An increase in daily intake of vitamin D from 10 micrograms (400 units) per day to 50 micrograms (2000 units) per day produced an abnormal elevation in serum 1,25-(OH)2D, hypercalcemia, and hypercalciuria which were corrected by prednisone. Serum 25-hydroxyvitamin D initially was abnormally low, and increased with vitamin D to values which were in the low normal range. There were significant positive correlations between serum 1,25-(OH)2D (p less than .05) and serum calcium and between serum 1,25-(OH)2D and urinary calcium (p less than .05). Serum immunoreactive parathyroid hormone, initially in the lower range of normal, decreased further during hypercalcemia. A radiograph of the chest, gallium scan, and serum angiotensin-converting enzyme activity were normal. No granulomas or evidence of lymphoma were found in biopsies of the liver and of several lymph nodes. It is concluded that the abnormal calcium metabolism in this patient resulted from increased circulating 1,25-(OH)2D and that the defect in vitamin D metabolism was not related to sarcoidosis, other granulomatous disease, Hodgkin's disease, or lymphoma. The relationship, if any, of the abnormal metabolism of vitamin D and calcium to rheumatoid arthritis remains to be established.

Hypercalcemia associated with increased circulating 1,25 dihydroxyvitamin D in a patient with pulmonary tuberculosis.

Studies are described in a 53-year-old man with far-advanced pulmonary tuberculosis who developed transient increases in circulating 1,25 dihydroxyvitamin D (1,25(OH)2D) and hypercalcemia while on antituberculous treatment. Serial dilution of an extract of the patient's serum obtained while he was hypercalcemic displaced [3H]-1,25(OH)2D3 from chick intestinal receptor in a manner identical to authentic 1,25(OH)2D3. Serum 25-hydroxyvitamin D (25OHD) was suppressed during the abnormal elevation of serum 1,25(OH)2D. It is concluded that tuberculosis is another chronic granulomatous disease in which hypercalcemia may result from abnormal metabolism of vitamin D.

Evidence that 1,25-dihydroxyvitamin D3 inhibits the hepatic production of 25-hydroxyvitamin D in man.

Previous in vitro studies in rachitic rat liver suggested that 1,25-dihydroxyvitamin D inhibits the hepatic production of 25-hydroxyvitamin D (25-OHD). An investigation therefore was carried out in eight normal subjects to determine whether concomitant administration of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] would alter the response of serum 25-OHD to challenge with vitamin D. In control studies, vitamin D, 100,000 U/d for 4 d, significantly increased mean serum 25-OHD, from 26.3 +/- 2.9 to 66.7 +/- 12.6 ng/ml (P less than 0.01). In contrast, 1,25(OH)2D3, 2 micrograms/d for 4 d, completely prevented an increase in serum 25-OHD in response to the same dose of vitamin D in the same individuals (25.1 +/- 2.2 vs. 27.4 +/- 5.3 ng/ml, NS). In a post-control study in seven of the normal subjects, vitamin D again significantly increased mean serum 25-OHD, from 18.2 +/- 3.1 to 42.8 +/- 4.7 ng/ml (P less than 0.001). In each of the three studies, mean serum calcium, phosphorus, and creatinine did not change and remained within the normal range. Whereas mean urinary calcium did not change in response to vitamin D alone during the 4 d of the two control studies, it increased significantly in the study in which vitamin D and 1,25(OH)2D3 were given together. A dose-response inhibition of the response of serum 25-OHD to vitamin D by 1,25(OH)2D3 was demonstrated in two of the normal subjects. The results provide evidence that 1,25(OH)2D3 inhibits the hepatic synthesis of its precursor 25-OHD in man.

Evidence for abnormal regulation of circulating 1 alpha, 25-dihydroxyvitamin D in patients with pulmonary tuberculosis and normal calcium metabolism.

Available evidence indicates that hypercalcemia in pulmonary tuberculosis results from increases in circulating 1 alpha, 25-dihydroxyvitamin D [1 alpha, 25(OH)2D]. To further characterize vitamin D metabolism in this disorder, the effects of vitamin D, 100,000 units a day for 4 days, were compared in 25 normal subjects and 11 patients with active pulmonary tuberculosis who were normocalcemic and had not had hypercalcemia. Serum calcium, phosphorus, 25-hydroxyvitamin D (25-OHD) and 1 alpha, 25(OH)2D were measured. Whereas vitamin D increased mean serum 25-OHD from 20 +/- 2 (+/- SE) to 40 +/- 5 ng/ml (P less than 0.001) and did not change mean serum 1 alpha, 25(OH)2D in the normals (33 +/- 2 vs. 31 +/- 2 pg/ml), it increased mean serum 25-OHD from 21 +/- 4 to 55 +/- 13 ng/ml (P less than 0.05) and mean serum 1 alpha, 25(OH)2D from 28 +/- 2 to 35 +/- 3 pg/ml (P less than 0.05) in the patients. Serum calcium was normal and remained within the normal range in all subjects and patients. The findings indicate that there is a modest but significant abnormality in the regulation of circulating 1 alpha, 25(OH)2D in normocalcemic patients with pulmonary tuberculosis. The results are similar to those previously reported by us in normocalcemic patients with sarcoidosis.

Evidence for extrarenal production of 1 alpha ,25-dihydroxyvitamin D in man.

Recent studies provide evidence for extrarenal production of 1 alpha ,25-dihydroxyvitamin D [1 alpha ,25(OH)2D]. To investigate this possibility, serum vitamin D, 25-hydroxyvitamin D (25-OHD), 24,25-dihydroxyvitamin D [24,25(OH)2D], and 1 alpha ,25(OH)2D were measured in eight adult anephric subjects. All were undergoing hemodialysis and three of them were receiving vitamin D, 50,000 or 100,000 U/d. Serum vitamin D was elevated in two of the patients given vitamin D and was abnormally low in the others. Mean serum 25-OHD was increased in patients given vitamin D (94.0 +/- 7.6 ng/ml) and was normal in the others (16.4 +/- 0.9 ng/ml, P less than 0.001). Mean serum 24,25(OH)2D was normal in patients given vitamin D (1.38 +/- 0.27 ng/ml) and was low in the others (0.25 +/- 0.08 ng/ml, P less than 0.001). Serum 24,25(OH)2D correlated significantly with serum 25-OHD (r = 0.848, P less than 0.01). Mean serum 1 alpha ,25(OH)2D determined by receptor assay was 5.8 +/- 1.9 pg/ml in patients who were not given vitamin D and was 14.1 +/- 0.6 in those who were given vitamin D (P less than 0.001). Serum 1 alpha ,25(OH)2D correlated significantly with serum 25-OHD (r = 0.911, P less than 0.01). Mean serum 1 alpha ,25(OH)2D, measured by bioassay, was 8.3 +/- 1.9 pg/ml in patients who were given vitamin D and was 15.9 +/- 2.4 pg/ml in those who were given vitamin D (P less than 0.05). There was a significant correlation between the values for serum 1 alpha ,25(OH)2D obtained with the two methods (r = 0.728, P less than 0.01). The results (a) provide evidence in man for extrarenal production of both 24,25(OH)2D and, by two independent assays, of 1 alpha , 25(OH)2D, and (b) indicate that serum values of the two dihydroxy metabolites of vitamin D in anephric subjects vary with the serum concentration of the precursor 25-OHD.

Bone mineralization and metabolism of 3H-25-hydroxyvitamin D3 in thyroparathyroidectomized rats treated with parathyroid extract.

Thyroparathyroidectomized rats fed a diet containing 1.2% calcium, 0.55% phosphorus maintain normal serum levels of these ions. Treatment of such rats with parathyroid extract (PTE; 20 U/100 g twice daily; 10 days) has no statistically significant effect on rates of bone formation, matrix apposition, or osteoid maturation. Significant decreases in osteoid width and mineralizing front width, as well as a 60% increase in the rate of initial mineralization were observed in the PTE-treated group. Conversion of 3H-25-hydroxyvitamin D3 to 3H-1,25-dihydroxyvitamin D3 was 4-fold higher in the PTE-treated group than in the untreated animals. Increased formation of 1,25-dihydroxyvitamin D3 in response to treatment with PTE may play a major role in correcting the mineralizing defect resulting from thyroparathyroidectomy.