Vitamin D metabolite production and function in keratinocytes.
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Animals↗
Biomedical subjects
D D Bikle
Publications and source records attributed to D D Bikle.
Glucocorticoids and inhibition of bone formation induced by skeletal unloading.
Skeletal unloading or loss of normal weight bearing in the growing animal inhibits bone formation and reduces bone calcium. To determine whether the inhibition of bone formation induced by skeletal unloading is a consequence of an increase in plasma glucocorticoids and/or an increase in bone sensitivity to glucocorticoids, we measured plasma corticosterone throughout the day in unloaded and normally loaded rats (hindlimb elevation model) and examined the effect of adrenalectomy on the response of bone to skeletal unloading. Plasma corticosterone levels were similar in normally loaded and unloaded rats at all times. Skeletal unloading in sham-adrenalectomized animals reduced tibial and vertebral calcium by 11.5 and 11.1%, respectively, and in adrenalectomized animals by 15.3 and 20.3%, respectively. Uptake of 45Ca and [3H]proline in the tibia was reduced by 8 and 14%, respectively, in the sham-adrenalectomized animals and by 13 and 19% in the adrenalectomized animals. Bone formation and apposition rates were reduced to the same level in sham- and adrenalectomized animals. These results suggest that the inhibition of bone formation induced by skeletal unloading is not a consequence of increased plasma glucocorticoids or an increase in bone sensitivity to the glucocorticoids but, rather, point to a local mediator in bone that senses mechanical load and transmits that information to the bone-forming cells directly.
Adrenalectomy↗
Vitamin D and epidermal differentiation: evidence for a role of endogenously produced vitamin D metabolites in keratinocyte differentiation.
Vitamin D3 is produced in the skin. It is metabolized primarily in the liver to the major circulating form 25-hydroxy vitamin D3, [25(OH)D3] which is metabolized in the kidney to produce the biologically active form of the hormone, 1,25-dihydroxy vitamin D3, [1,25(OH)2D3]. The skin not only participates in the production of vitamin D3 but also contains receptors for 1,25(OH)2D3 suggesting a role of this hormone in the growth and differentiation of this tissue. 1,25(OH)2D3 appears to play a role in epidermopoiesis and melanin pigmentation. Recently, using cultures of neonatal human foreskin keratinocytes, we have demonstrated that these cells produce abundant quantities of 1,25(OH)2D3 from 25(OH)D3. The production of 1,25(OH)2D3 varies with the degree of differentiation of keratinocytes and is regulated by exogenous 1,25(OH)2D3. 1,25(OH)2D3 induces differentiation possibly because of its ability to increase intracellular free calcium levels. A tentative model is provided to demonstrate potential mechanisms by which 1,25(OH)2D3-induced changes in intracellular calcium could regulate epidermal differentiation.
Animals↗
Serum dihydrotachysterol levels and biological action in normal man.
Dihydrotachysterol (DHT2) has been a safe and effective treatment for hypocalcemic disorders for many years, but few assays for quantitation of DHT2 have been developed. Thus little is known about its pharmacokinetics. The 2-fold purpose of this study was 1) to develop a practical method for quantitating DHT2 after oral dosing in normal subjects, and 2) to assess changes in serum DHT2 levels and calcium and phosphorus metabolism after DHT2 administration for 8 days. Peak serum DHT2 levels in six normal subjects, assayed by high performance liquid chromatography were achieved 4 h after administration of 0.4-0.8 mg DHT2; at 24 h, levels had declined by 70% whether DHT2 had been given for 1 or 8 days. These data indicate that a standard approach is needed to interpret the results of serum DHT2 measurements in treated patients. Interfering substances were detected in lipemic serum. The major biological effects of DHT2 administration were hypercalciuria in two subjects and a fall in serum 1,25-dihydroxyvitamin D[1,25-(OH)2D] levels, including free levels when measured, in all subjects. Possible explanations for this fall in serum 1,25-(OH)2D levels include decreased 1,25-(OH)2D production because of competition for the 1 alpha-hydroxylase enzyme by a metabolite(s) of DHT or increased metabolic clearance of 1,25-(OH)2D.
Adult↗
Effects of alcohol abuse on bone.
The relationship between bone disease and alcohol abuse is well established. A large percentage of individuals whose drinking habits have caused them to seek medical help can be diagnosed, with routine X-rays, as having bone disease. The degree to which bone disease is present in the overall drinking population remains uncertain. The predominant form of bone disease is osteoporosis, and it seems likely that most osteoporosis in the middle-aged man can be attributed to alcohol abuse. The reason alcohol abuse leads to osteoporosis remains unclear. Vitamin D deficiency, hyperparathyroidism, and decreased intestinal calcium absorption may play a role, but a direct inhibitory effect of alcohol on bone remodeling seems a more likely explanation. No effective form of treatment has yet been established. Bone lost may not be regained. The best approach is to stop the alcohol abuse before the bone disease becomes manifest.
Alcoholism↗
The effect of vitamin D status on cutaneous sterologenesis in vivo and in vitro.
Recent studies have shown that cutaneous sterologenesis is autonomous from the influence of circulating sterols, and that the epidermis is an important site of sterologenesis which is regulated by permeability barrier requirements. In addition to barrier function, an additional, important function of the epidermis is to synthesize sterol precursors of vitamin D3. The present study was designed, first, to determine whether vitamin D status and/or circulating levels of 1,25-dihydroxyvitamin D3 might play a role in regulating cutaneous sterol synthesis in vivo and, second, whether 1,25-dihydroxyvitamin D3 modulates sterologenesis in cultured human keratinocytes. Hairless mice were maintained on a vitamin D-deficient diet in the dark and supplemented with various doses of vitamin D3/day. Despite demonstrating serum 25-hydroxyvitamin D3 levels ranging from less than 10 to 343 ng/ml, the incorporation of tritiated water into cholesterol and total nonsaponifiable lipids in both the epidermis and dermis was similar in the four groups of animals. Likewise, administration of various doses of 1,25-dihydroxyvitamin D3 to vitamin D-deficient mice resulted in serum levels of 1,25-dihydroxyvitamin D3 ranging from less than 10 to 85 pg/ml; yet, cholesterol and total nonsaponifiable lipid synthesis was similar in both the dermis and epidermis in all groups of animals. Moreover, administration of 0.6 micrograms/kg per day of 1,25-dihydroxyvitamin D3 to 'normal' vitamin D-replete mice also had no effect on cutaneous sterol synthesis. Furthermore, conversion of 7-dehydrocholesterol to cholesterol in vitamin D-deficient vs. supplemented animals did not differ. Finally, addition of 1,25-dihydroxyvitamin D3 to cultured keratinocytes over a concentration range of 10(-12)-10(-7) M did not affect sterologenesis, except at supraphysiologic doses (10(-7) M). Together, these results suggest that vitamin D status does not influence sterol synthesis in the skin.
Animals↗
Effect of dietary acid and calcium on 25-hydroxyvitamin D metabolism by chick kidney.
Studies of the effects of acidosis and dietary calcium on 25-hydroxyvitamin D3 (25OHD3) metabolism were conducted utilizing assays of mitochondrial 1-hydroxylase and 24-hydroxylase activity following 96 hours of acid loading. The results showed decreased 1-hydroxylase (206 +/- 6 vs. 132 +/- 22 fmol min-1 mg/mitochondrial protein-1, p less than 0.01), augmented 24-hydroxylase (48 +/- 14 vs. 180 +/- 30 fmol min-1 mg-1, p less than 0.001) and increments in plasma calcium and phosphate following acidosis. High calcium diet also increased 24-hydroxylase activity (48 +/- 14 vs. 160 +/- 24 fmol min-1 mg-1, p less than 0.001) and plasma calcium, but decreased plasma phosphate. The extramitochondrial concentrations of calcium, potassium, and phosphate which maximally stimulated the hydroxylases in vitro were not altered by the in vivo dietary perturbations. The increments in 1-hydroxylase and 24-hydroxylase resulting from the presence of calcium, potassium, or phosphate in optimal concentrations (10(-5) M, 100 mM, and 100 mM, respectively) were changed by both acid loading and high calcium diet, decreasing in the case of 1-hydroxylase and increasing in that of 24-hydroxylase. The data indicate that the suppression of 1-hydroxylase and augmentation of 24-hydroxylase following these dietary perturbations may depend on both changed total enzyme capacity and altered enzyme sensitivity to extramitochondrial ions and are consistent with mediation by calcium of the effects of acidosis.
Acidosis↗
The effects of simulated weightlessness on bone maturation.
In earlier studies we showed that elevating the hind limbs of growing rats for up to 2 weeks results in a temporary cessation of bone growth in the hind limbs and a transient fall in the serum levels of 1,25-dihydroxyvitamin D. To determine whether such skeletal unloading also retards the maturation of bone, as seen in vitamin D-deprived animals, we fractionated by density the tibiae from rats whose hind limbs had been elevated for up to 15 days. These fractions were analyzed for dry weight, calcium content, and calcium and proline uptake. The most dense fraction (fraction 4) had the highest degree of mineralization (ratio of calcium to dry weight) and comprised 82% of the total dry weight of the control tibiae. The total incorporation of [3H]proline administered in vivo 24 h before removing the tibiae was evenly distributed among all of the fractions, although it was highest in the least dense fraction (fraction 1) when normalized to dry weight. Total incorporation of 45Ca was highest in fraction 4, although when normalized to dry weight it was highest in fraction 3. With skeletal unloading, the proportions of bone and 45Ca incorporation in fraction 4 decreased, while the proportions in less dense fractions increased. [3H]Proline incorporation fell in all fractions. These effects were maximal after 10 days of unloading and returned toward the control values after that time. We conclude that skeletal unloading transiently reduced bone formation and retarded mineralization in the growing rat, which resulted in a decrease in mature bone.
Aging↗
Neonatal human foreskin keratinocytes produce 1,25-dihydroxyvitamin D3.
Primary cultures of neonatal human foreskin keratinocytes converted 25-hydroxyvitamin D in high yield to a metabolite with the chromatographic behavior of 1,25-dihydroxyvitamin D3. The identity of this metabolite as 1,25-dihydroxyvitamin D3 was confirmed both by its potency in displacing 1,25-dihydroxyvitamin D3 in the chick cytosol receptor assay and by mass spectral analysis. These results suggest that 1,25-dihydroxyvitamin D3 may be formed in the epidermis to regulate vitamin D production by the epidermis and to provide an alternative to 1,25-dihydroxyvitamin D3 production by the kidneys.
Animals↗
Skeletal response to dietary calcium in a rat model simulating weightlessness.
Unweighting the hindlimbs of a rat by tail suspension leads to a decrease in bone in the unweighted hindlimbs, but not in the normally weighted forelimbs. We evaluated whether increments in dietary calcium could prevent this. Growing rats were fed diets ranging in calcium content from 0.1% to 2.4%. After the rats were suspended for two weeks, we found no differences between suspended and control animals fed the same diet with respect to calcium transport or serum levels of calcium, phosphorus, 1,25-dihydroxyvitamin D, and parathyroid hormone. In both groups, increasing dietary calcium reduced active intestinal calcium transport and serum 1,25-dihydroxyvitamin D levels. The calcium content of the tibia and lumbar vertebra (but not the humerus) was reduced in suspended rats compared to control rats fed the same diet. However, increasing dietary calcium increased the calcium content of all bones in both suspended and control animals. The bone formation rate at the tibiofibular junction (measured by double-label tetracycline) was reduced in the suspended animals compared to controls and was not altered by dietary calcium. However, the marrow area of the tibia, an indication of bone resorption, did not differ between suspended and control animals and was equally reduced in both groups when dietary calcium was increased. Our data suggest that the deleterious effects of skeletal unweighting on bone formation cannot be explained by changes in the calciotropic hormones and are not reversed by increments in dietary calcium. However, increasing dietary calcium can increase bone calcium, even in unweighted limbs, by decreasing bone resorption.
Animals↗
Effect of ethanol on intestinal calcium transport in chicks.
To test the hypothesis that chronic ethanol exposure would alter the membrane fluidity of the intestinal brush-border membrane, which would lead to calcium malabsorption, we gave chicks 15% ethanol in their drinking water from hatching to 3 or 4 wk of age. Although such chicks grew less quickly than their hatchmates not ingesting ethanol, their ability to absorb calcium from the duodenum in vivo was unimpaired. However, when calcium accumulation by isolated duodenal brush-border membrane vesicles (BBMVs) was assessed, the BBMVs from chicks ingesting ethanol for 1-3 wk had a lower rate of uptake than the BBMVs from the controls at all calcium concentrations evaluated (0.1-25 mM). This difference could not be explained by differences in membrane fluidity as assessed by fluorescence depolarization, or by changes in intravesicular volume. Glucose uptake was not affected. The acute addition of ethanol (up to 1 M) in vitro to the control BBMVs increased both membrane fluidity and calcium accumulation. No difference in the fluidizing effect of ethanol in BBMVs between ethanol-ingesting chicks and control chicks could be demonstrated, although the acute effect of ethanol on calcium accumulation was blunted in the BBMVs from chicks ingesting ethanol. Increasing the temperature of the incubation medium also increased membrane fluidity and calcium accumulation in BBMVs from control and ethanol-ingesting chicks, with a greater increase in calcium uptake by the control BBMVs despite comparable increases in fluidity in BBMVs from the control and ethanol-ingesting chicks. We conclude that the chronic ingestion of ethanol by chicks, although markedly altering growth rates, has minimal impact on the intestinal absorption of calcium when assessed in vivo. However, chronic ethanol ingestion does appear to alter the intestinal brush-border membrane to make it less permeable to calcium and less susceptible to the stimulation by ethanol of calcium flux across this membrane; this adaptation may prevent increased flux of calcium across the brush-border membrane into the cell in the presence of ethanol.
Animals↗
1,25-Dihydroxyvitamin D3 production by human keratinocytes. Kinetics and regulation.
Human foreskin keratinocytes in vitro metabolize 25-hydroxyvitamin D3 to a number of metabolites, including 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). This metabolite remains mostly within the cell and does not accumulate in the medium under the conditions of these experiments. With time, 1,25(OH)2D3 is catabolized, and more polar metabolites appear in both the cells and the medium. The production of 1,25(OH)2D3 has an apparent Michaelis constant (Km) for 25-hydroxyvitamin D3 of 5.4 X 10(-8) M. The levels of 1,25(OH)2D3 within the cell are increased both by increased production and decreased catabolism when parathyroid hormone(1-34) and isobutylmethylxanthine are added. Exogenously added 1,25(OH)2D3 at concentrations as low as 10(-12) M reduces endogenous 1,25(OH)2D3 production, increases 1,25(OH)2D3 catabolism, and increases 24,25-dihydroxyvitamin D3 production by an actinomycin D-sensitive process. These data indicate that the regulation of 1,25(OH)2D3 production by keratinocytes is similar to, but not identical to the regulation of 1,25(OH)2D3 by the kidney.
1-Methyl-3-isobutylxanthine↗
Chronic 1,25-dihydroxyvitamin D3 administration in the rat reduces the serum concentration of 25-hydroxyvitamin D by increasing metabolic clearance rate.
Administration of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] can lower the serum concentration of 25-hydroxyvitamin (25-OH-D). To determine if 1,25(OH)2D3 lowers serum 25-OH-D by increasing clearance or reducing production, we directly measured the metabolic clearance rate (MCR) of 25-OH-D in rats chronically infused with 1,25(OH)2D3. Chronic 1,25(OH)2D3 administration (0 to 75 pmol/d) reduced, in a time- and dose-dependent fashion, the serum concentrations of 25-OH-D3 and 24,25(OH)2D3 from 18 +/- 2 to 9 +/- 1 ng/ml and from 4.8 +/- 0.7 to 1.3 +/- 0.3 ng/ml, respectively, and increased sevenfold the in vitro conversion of 25-OH-D to 24,25(OH)2D3 by kidney homogenates. The reduction in serum 25-OH-D3 was completely accounted for by an increase in MCR. No change in production occurred. The influence of 1,25(OH)2D3 on serum 25-OH-D3 and 24,25(OH)2D3 was shown not to be dependent on induction of hypercalcemia. These data suggest that chronic 1,25(OH)2D3 administration lowers serum 25-OH-D by increasing the metabolic clearance of 25-OH-D3 and not by decreasing its production.
24,25-Dihydroxyvitamin D 3↗
Free 25-hydroxyvitamin D levels are normal in subjects with liver disease and reduced total 25-hydroxyvitamin D levels.
We determined the free fraction of 25-dihydroxyvitamin D (25OHD) in the serum of subjects with clinical evidence of liver disease and correlated these measurements to the levels of vitamin D binding protein and albumin. These subjects when compared to normal individuals had lower total 25OHD levels, higher percent free 25OHD levels, but equivalent free 25OHD levels. These subjects also had reduced vitamin D binding protein and albumin concentrations. The total concentration of 25OHD correlated positively with both vitamin D binding protein and albumin, whereas the percent free 25OHD correlated negatively with vitamin D binding protein and albumin. The free 25OHD levels did not correlate with either vitamin D binding protein or albumin. We conclude that total vitamin D metabolite measurements may be misleading in the evaluation of the vitamin D status of patients with liver disease, and recommend that free 25OHD levels also be determined before making a diagnosis of vitamin D deficiency.
Adult↗
The villus gradient of brush border membrane calmodulin and the calcium-independent calmodulin-binding protein parallels that of calcium-accumulating ability.
We have recently proposed that calmodulin (CaM) may mediate calcium transport across the intestinal brush border membrane. Since calcium transport across this membrane varies as a function of cellular location on the villus (the highest rates of transport occur across the brush border membrane from cells near the tip), we tested this hypothesis by determining whether CaM and its principal binding protein in the brush border membrane [a 102,000 mol wt (102K) protein] also showed this gradient of activity along the villus. Cells were sequentially eluted from the tip to the base of the villus, brush border membrane vesicles (BBMV) were prepared from the eluted cells, and CaM, CaM binding, and calcium-accumulating ability were determined for each preparation of BBMV. We observed that BBMV prepared from cells originating near the tip of the villus possessed the greatest calcium-accumulating activity, CaM content, and CaM binding by the 102K protein. All three measurements were reduced in parallel in BBMV prepared from cells originating from more basal regions of the villus. Calcium-accumulating ability correlated with CaM content (r = 0.876) and CaM binding to the 102K protein (r = 0.788); likewise, CaM correlated with CaM binding to the 102K protein (r = 0.928). When 1,25-dihydroxyvitamin D was administered to vitamin D-deficient chicks, the binding of CaM to the 102K CaM-binding protein appeared to increase more rapidly in BBMV from cells near the tip of the villus than in cells from more basal regions, comparable to our previously reported data for 1,25-dihydroxyvitamin D-stimulated calcium accumulation by similarly prepared BBMV. These data support the hypothesis that CaM and the 102K CaM-binding protein are involved in the regulation of calcium flux across the intestinal brush border membrane.
Animals↗
The temporal response of bone to unloading.
A model of weightlessness in which the hindlimbs of rats are elevated by their tails at a 40 degrees angle to unload the hindlimbs while maintaining normal weight bearing on the forelimbs has been used to simulate certain conditions of space flight. When we used this model in growing rats, we found that growth in bone weight ceased by 1 week in the hindlimbs and lumbar vertebrae, whereas growth in bone weight in the forelimbs and cervical vertebrae remained unaffected. Within 2 weeks, however, the accretion of bone weight in the hindlimbs and lumbar vertebrae returned to normal despite continued skeletal unloading. Since bone weight in the growing rat is primarily determined by bone formation (bone resorption is modest), we investigated the effects of selective skeletal unloading on bone formation during 2 weeks of hindlimb elevation using radioisotope incorporation (with 45Ca and [3H]proline) and histomorphometry (with tetracycline labeling). The studies using radioisotope incorporation showed that bone formation was inhibited by the fifth day of skeletal unloading. By the 10th to 12th day, bone formation had returned toward normal. In comparison with cortical bone, cancellous bone (lumbar vertebrae and proximal tibiae) incorporated more 45Ca and [3H]proline (indicating greater metabolic activity) and had a greater absolute response to skeletal unloading. The results of these studies were confirmed by histomorphometric measurements of bone formation using triple tetracycline labeling. We conclude that this model of simulated weightlessness results in an initial inhibition of bone formation in the unloaded bones. This temporary cessation of bone formation is followed by a cessation in the accretion of bone weight, which then resumes at a normal rate by 14 days despite continued skeletal unloading. We believe that this cycle of inhibition and resumption of bone formation has profound implications for understanding bone dynamics during space flight, immobilization, or bed rest and offers an opportunity to study the hormonal and mechanical factors that regulate bone formation.
Animals↗
The role of 1,25-dihydroxyvitamin D in the inhibition of bone formation induced by skeletal unloading.
Skeletal unloading results in osteopenia. To examine the involvement of vitamin D in this process, the rear limbs of growing rats were unloaded, and alterations in bone calcium and bone histology were related to changes in serum calcium (Ca), inorganic phosphorus, 25-hydroxyvitamin D, 24,25-dihydroxyvitamin D [24,25-(OH)2D], and 1,25-dihydroxyvitamin D [1,25-(OH)2D]. Acute skeletal unloading induced a transitory inhibition of Ca accumulation in unloaded bones. This was accompanied by a transitory rise in serum Ca, a 21% decrease in longitudinal bone growth (P less than 0.01), a 32% decrease in bone surface lined with osteoblasts (P less than 0.05), no change in bone surface lined with osteoclasts, and a decrease in circulating 1,25-(OH)2D from 130 +/- 10 to 53 +/- 11 pg/ml. No significant changes in the serum concentrations of inorganic phosphorus, 25-hydroxyvitamin D, or 24,25-(OH)2D were observed. After 2 weeks of unloading, bone Ca stabilized at approximately 70% of control values, and serum Ca and 1,25-(OH)2D returned to control values. Maintenance of a constant serum 1,25-(OH)2D concentration by chronic infusion of 1,25-(OH)2D (Alza osmotic minipump) throughout the study period did not prevent the bone changes induced by acute unloading. These results suggest that acute skeletal unloading in the growing rat produces a transitory inhibition of bone formation, which, in turn, produces a transitory hypercalcemia, leading to a temporary decrease in serum 1,25-(OH)2D. No evidence could be found for a direct involvement of 1,25-(OH)2D in the bone changes induced by skeletal unloading.
24,25-Dihydroxyvitamin D 3↗
Stimulation by 1,25-dihydroxyvitamin D3 of adenylate cyclase along the villus of chick duodenum.
Administration of 650 pmol 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] to vitamin D-deficient chicks increased adenylate cyclase activity in the basolateral membrane of duodenal epithelial cells within 24 h. This increase in enzymatic activity was accompanied by an increase in calmodulin content of the basolateral membrane. Although neither exogenously added calmodulin (up to 10 micrograms/ml) nor calcium (from 10(-7)-10(-5) M) stimulated enzyme activity, calmodulin antagonists trifluoperazine, W7, and W13 inhibited it. When calmodulin content, adenylate cyclase activity, and alkaline phosphatase activity were measured in cells sequentially eluted from the tip to the base of the villus, cells from the midregion and base had the highest calmodulin content and adenylate cyclase activity, whereas alkaline phosphatase activity (a brush border membrane enzyme) was highest in cells eluted from the tip. Adenylate cyclase activity was increased by 1,25-(OH)2D3, particularly in cells from the midvillus. Our results indicate that the response of adenylate cyclase activity to 1,25-(OH)2D3 varies along the villus and suggest that calmodulin may be involved.
Adenylyl Cyclases↗