Duodenal calbindin-D9k Research Articles

Soy isoflavones increase bone mineral density without altering markers of whole body vitamin D or calcium metabolism in mice

The soy isoflavone genistein enhances the biological activity of the vitamin D (VD) metabolite 1,25 dihydroxyvitamin D (1,25D) in cultured cells by inhibiting CYP24‐mediated degradation of the hormone. We examined if similar effects can be recapitulated in vivo to improve calcium and bone metabolism. Weanling male B6D2F1 mice were fed AIN‐93G diets containing 50 or 400 IU VD with, or without, Novasoy® 400 soy isoflavone concentrate (added to provide 250 mg genistein/kg diet) until 13 weeks of age. Bone mineral density (BMD) was measured by DEXA. Serum VD metabolites were measured by RIA. Gene expression in duodenum and kidney was measured by RT‐PCR. Higher dietary VD intake increased serum 25 hydroxyvitamin D (25D) from levels viewed as adequate (61 ± 6 nM) to optimal (164 ± 10 nM) for human health but this had no impact on either BMD or molecular indices of calcium metabolism. In contrast, isoflavones increased both serum 25D and femur BMD by 15% (p < 0.01). However, neither serum 1,25D nor tissue gene expression (e.g. duodenal calbindin D9K, TRPV6; renal calbindin D9K or D28K, TRPV5) was influenced by isoflavones. Our data show that the beneficial effect of soy isoflavones on bone that we observed is independent of their ability to modulate whole body VD or calcium metabolism and suggest a direct effect of isoflavones on bone formation or turnover. We thank ADM for Novasoy® 400. Funding: Purdue‐UAB Botanicals Center Pilot Grant.Grant Funding Source: Purdue‐UAB Botanicals Center Pilot Grant

Serum Metabolite Profiles and Target Tissue Gene Expression Define the Effect of Cholecalciferol Intake on Calcium Metabolism in Rats and Mice

We studied the effect of cholecalciferol (VD3) intake on VD3 status and markers of calcium (Ca) homeostasis in mice and rats. Serum 25 hydroxycholecalciferol (25OH-VD3) concentrations were increased in animals fed diets containing 400–20,000 international units (IU) VD3/kg (37 nmol·L−1·1000 IU VD3 −1), but body weight, serum Ca, and duodenal gene expression were not altered. High-VD3 intake decreased serum 1, 25-dihydroxycholecalciferol [1,25(OH)2-VD3] and renal 25 hydroxycholecalciferol-1 αhydroxylase (CYP27B1) mRNA, suggesting that rodents tolerate high-VD3 intake by suppressing the activity of the VD3 endocrine system. Serum 25OH-VD3 declined when animals were fed diets containing 1000 to 25 IU VD3/kg (9–11 wk, inflection at 200 IU/kg, 4-fold steeper slope below this). Neither body weight nor serum Ca were influenced by low-VD3 intake. However, mice fed the 25-IU/kg diet had lower serum 1,25(OH)2-VD3, duodenal calbindin D9k mRNA, bone mineral density, and renal 25 hydroxycholecalciferol-24 hydroxylase mRNA, whereas renal CYP27B1 mRNA was elevated when rodents were fed <200 IU VD3/kg. These data reveal a stress on VD3 and Ca metabolism at low dietary VD3 intake. Dietary Ca restriction (0.25 vs. 0.5%, 9 wk) increased serum 1,25(OH)2-VD3 and was 30% greater in rats fed a 10,000-IU VD3/kg diet. High-VD3 intake did not prevent Ca restriction-induced bone loss. Our data show that modeling human VD3 status requires lower intake than the current NRC rodent requirement (1000-IU/kg diet). Also, although rodents are very tolerant of high-VD3 intake, it cannot compensate for moderate Ca restriction.

Ingestion of difructose anhydride III partially restores calcium absorption impaired by vitamin D and estrogen deficiency in rats

Vitamin D (VD) and estrogen deficiencies impair Ca absorption and bone mineralization, and the relevance of the interaction between these factors has not been fully understood. The aim of the present study was to clarify the effects of a nondigestible saccharide, difructose anhydride III (DFAIII), on the interaction of VD and estrogen deficiencies involved in Ca malabsorption by assessing changes in intestinal Ca absorption and bone mineralization by feeding of DFAIII in rats with VD or estrogen deficiency or with a combined loss of VD and estrogen. Three-week-old female Sprague-Dawley rats were divided into four groups: two groups were ovariectomized (OVX) and two were laparotomized (sham). One group each of OVX and sham rats were fed an AIN93G-based normal diet, and the other groups were fed a VD-deficient diet for 8-weeks. Rats from the four groups were divided into two subgroups and fed the normal or VD-deficient diet with or without DFAIII for next 4-weeks. VD deficiency decreased Ca absorption and bone mineralization with reductions in duodenal calbindin D9k mRNA and serum Ca levels. There were no additional reductions in these parameters in the OVX. The reductions in Ca absorption and femoral Ca were restored partially or fully by DFAIII. Recovery of Ca absorption rate by DFAIII was greater in the OVX than in the sham showing an interaction between OVX and VD deficiency in, at least, the DFAIII-fed groups. The cecal pH was lower and the level of short-chain fatty acids in the cecal contents was higher in all the DFAIII groups than those in the control groups. VD deficiency impaired Ca absorption and bone mineralization, and feeding DFAIII partially restored Ca malabsorption and fully recovered bone Ca in VD-deficient rats. No additional reductions in these parameters with a combination of VD deficiency and OVX were noted. However, interactions were found between these factors in the DFAIII-induced increase in Ca absorption.

Aluminium-induced impairment of transcellular calcium absorption in the small intestine: Calcium uptake and glutathione influence

Aluminium (Al) has been recognised as a cause of bone tissue disorders. The aims of this work were to investigate: (i) whether Al affects calcium (Ca) entry into enterocyte, and (ii) the possibility that the Al effect upon calbindin-D-related Ca transport would be influenced by intestinal glutathione (GSH) levels. In isolated chicken duodenal enterocytes, 100 μM Al lactate produced a decrease in both, the maximum uptake rate and the affinity constant of 45Ca uptake (CaUPT). This effect of Al on CaUPT was concentration-dependent in the micromolar range, showing an inhibitory saturation type phenomenon which appeared to be higher at pH 6.5 than at pH 7.4, and was not modified by the Ca channel activators A23187 and capsaicin. The simultaneous administration of Al (50 mg elemental Al/kg body weight, as AlCl 3) and GSH (10 mmol/kg body weight) to rats during 7 days, prevented the inhibitory effects of Al on Ca transport. The protective effect of GSH was accompanied by an increased duodenal calbindin-D 9k expression. Experimental depletion of intestinal GSH by means of d,l-buthionin-[S,R] sulfoximine, a γ-glutamylcystein-synthase inhibitor, given as a single i.p. dose of 2 mmol/kg body weight, enhanced the degree of reduction of Ca absorption ascribed to Al. Our results suggest that Al might interfere Ca uptake by enterocytes through a general effect on cell membrane, and that an oxidative stress state induced by Al would reduce intestine GSH level affecting calbindin-D function and/or synthesis, thus leading to a reduced transcellular Ca absorption in the small intestine.

Pathogenesis of fetal hypomineralization in diabetic rats: evidence for delayed bone maturation.

There is some evidence that fetuses of diabetic rats (FDR) are hypomineralized. To explore the pathogenic role of decreased maternal duodenal Ca absorption, fetal hypotrophy, and decreased placental calbindin-D9K, respectively, spontaneously diabetic rats fed a 1.0% Ca diet were compared with diabetic rats treated with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] (15 ng/ 100 g) during week 3 of pregnancy, which restored duodenal calbindin-D9K concentrations to normal; with nondiabetic rats semistarved during week 3, which resulted in similar fetal hypotrophy; and with nondiabetic rats fed high cation diets (1.5% Ca-1.5% Sr and 1.5% Ca-3.5% Sr) during week 3, the latter of which repressed duodenal and placental calbindin-D9K toward concentrations measured in diabetic rats. In addition, fetal tibiae were studied histologically. Ca content was lower in 21.5-d-old FDR than in control fetuses. FDR had lower plasma osteocalcin (OC) levels and, on histomorphometry, increased hypertrophic cartilage width, indicating retarded bone maturation. Maternal 1,25(OH)2D3 treatment did not change Ca content and hypertrophic cartilage width in FDR. Fetuses of semistarved rats had plasma OC levels and hypertrophic cartilage width comparable to those of control fetuses. Fetuses of rats fed the 1.5% Ca-3.5% Sr diet were more severely hypomineralized than FDR but had higher plasma OC than both FDR and control fetuses, compatible with fetal Ca deficiency. Whereas diabetic placentas showed weak but homogeneous staining of calbindin-D9K in the labyrinth on immunohistology, degenerative zones were present in placentas of rats fed the 1.5% Ca-3.5% Sr diet. Thus, there is no mineralization defect in FDR caused by disturbed maternal duodenal Ca absorption or transplacental Ca transport, but a delay in bone maturation that is unexplained by their lower body weight.

Intestinal calcium absorption in the aged rat: evidence of intestinal resistance to 1,25(OH)2 vitamin D.

We investigated the role of circulating 1,25-dihydroxycholecalciferol (1,25(OH)2D) and intestinal resistance to 1,25(OH)2D in the diminished intestinal calcium absorption capacity of the senescent rat. We measured plasma 1,25(OH)2D, total and unoccupied duodenal vitamin D receptor, duodenal calbindin D9k protein (calbindin D), and net dietary calcium absorption in rats at several ages. As expected, circulating 1,25(OH)2D, calbindin D, and net calcium absorption decreased with age. However, no age-related changes were evident in intestinal vitamin D receptor levels. We then measured duodenal calcium absorption from in situ intestinal loops after continuous s.c. infusion of 1,25(OH)2D for up to 6 days and found that despite a marked elevation of plasma 1,25(OH)2D duodenal calcium absorption was significantly lower in old compared with young rats. To assess calcium absorption over a wide physiological range of plasma 1,25(OH)2D, in a dose-response study we altered plasma 1,25(OH)2D by continuous infusion of 1,25(OH)2D (at 0, 4, or 14 ng/100 g BW/day) for 9 days. We found that the slope of the linear regression between plasma 1,25(OH)2D and duodenal Ca transport in old rats was only 46% of that observed in young rats, suggesting an age-related resistance of the duodenal calcium transport process to the hormonal action of 1,25(OH)2D. Collectively, our observations suggest a dual defect in vitamin D metabolism in old animals: one defect related to the low circulating levels of 1,25(OH)2D and a second defect related to a relative intestinal resistance to the action of 1,25(OH)2D, which is apparently not due to a reduction in intestinal vitamin D receptor levels.

Age and gender effects on 1,25-dihydroxyvitamin D3-regulated gene expression.

Several factors involved in regulation of bone mineral metabolism were compared in male and female Fischer 344 rats of different ages (1, 2.5, 6, and 18 months). Plasma 1,25-(OH)2D3 concentrations decreased with age in rats of both genders. Abundance of calbindin-D28K and its mRNA in kidney and calbindin-D9K and its mRNA in duodenum also decreased with age in both male and female rats. Renal 24-hydroxylase activity and 24-hydroxylase mRNA content were elevated significantly in 18-month-old males and females, compared with younger ages. These data suggest that increased renal catabolism of 1,25-(OH)2D3 may be responsible for low plasma 1,25-(OH)2D3 concentrations observed in older animals. Plasma PTH and 1,25-(OH)2D3 concentrations, renal 24-hydroxylase enzyme activity and 24-hydroxylase mRNA content, duodenal 24-hydroxylase mRNA abundance, and duodenal calbindin-D9K and calbindin-D9K mRNA content were greater in males than in females at 2.5 months of age. Lower plasma 1,25-(OH)2D3 concentrations in females seem to explain observed gender differences in expression of 1,25-(OH)2D3-stimulated genes. The combined effects of these gender differences at ages when peak bone density is being developed may contribute to the greater incidence of osteoporosis in females than in males.

Prevention of type I diabetes in NOD mice by nonhypercalcemic doses of a new structural analog of 1,25-dihydroxyvitamin D3, KH1060.

Pharmacological amounts of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] have potent immunoregulatory activity, but with marked effects on calcium and bone metabolism. In this study we demonstrate that nonhypercalcemia-inducing nondemineralizing doses of an analog of 1,25-(OH)2D3, 1 alpha,25-(OH)2-20-epi-22-oxa-24,26,27-trishomo-vitamin D (KH1060), can prevent type I diabetes. Female NOD mice received 1,25-(OH)2D3 (5 micrograms/kg), KH1060 (0.4 or 0.2 micrograms/kg), or the treatment vehicle ip every 2 days from 21-200 days of age. The incidence of diabetes in controls was 17 of 31 (55%), whereas 7 of 38 (18%) 1,25-(OH)2D3-treated mice, 3 of 27 (11%) KH1060 (0.4 micrograms/kg)-treated mice, and 6 of 27 (22%) KH1060 (0.2 micrograms/kg)-treated mice developed diabetes (P < 0.025 vs. controls). Protection was achieved with the low KH1060 dose without effects on calcium or bone metabolism, as evaluated by serum calcium (9.5 +/- 0.4 vs. 9.4 +/- 0.3 mg/dl in controls; P = NS), serum osteocalcin (82 +/- 17 vs. 83 +/- 20 ng/ml; P = NS), bone calcium content (6.8 +/- 0.7 vs. 6.4 +/- 0.5 mg/tibia; P = NS), urinary calcium (21 +/- 4 vs. 21 +/- 4 mg/dl; P = NS), pyridinoline excretion, and duodenal calbindin-D9K concentration. The proposed mechanism of action is a restoration of suppressor cell activity, as demonstrated in vitro (suppressor cell assay) and in vivo (cell transfer experiments). This study demonstrates that an analog of 1,25-(OH)2D3 prevents type I diabetes in NOD mice without significant effects on calcium or bone metabolism.

In vivo effect of calcitriol on calcium transport and calcium binding proteins in the spontaneously hypertensive rat.

The abnormal intestinal Ca2+ transport reported in spontaneously hypertensive rats (SHR) has been attributed to decreased responsiveness to calcitriol. We reexamined this hypothesis by studying the calcitriol regulation of SHR duodenal calbindin-D9K and calmodulin and the relation of calcitriol to Ca2+ uptake by isolated enterocytes. SHR and normotensive Wistar-Kyoto (WKY) rats were injected with either 50 ng/d calcitriol (vit-D) or vehicle alone (control) for 3 days. Decreased calbindin-D9K (P < .001) and cellular Ca2+ flux (P < .001) were observed in control SHR. Calcitriol increased total cell and brush border calbindin-D9K (P < .0001); this variation paralleled plasma calcitriol levels in both strains. In contrast, Ca2+ flux, which increased in vit-D animals, remained lower in SHR for plasma calcitriol levels similar to those in WKY rats. Immunoreactive calmodulin was similar in both strains whether assayed in total cell or brush border membranes. In contrast, when measured by ligand blotting (45Ca), calmodulin was lower in SHR than in WKY rats (P < .01), suggesting the existence of a calmodulin pool with reduced Ca2+ binding capacity in the hypertensive strain. Calcitriol had no effect on calmodulin in either strain. In conclusion, Ca2+ binding protein regulation by calcitriol is normal in the SHR, and decreased hormone responsiveness cannot account for the defective duodenal calcium transport of this experimental model of hypertension.

Growth hormone and parathyroid hormone stimulate intestinal calcium absorption in aged female rats.

Aged (16-month-old) female rats (n = 8/treatment) were injected for 12 days with GH (100 micrograms/100 g x day), PTH (8 micrograms/100 g x day), GH plus PTH, or vehicle (V) in an experiment designed to determine the effects of these hormones on intestinal mineral absorption in senescent rats. PTH and GH increased fractional net calcium absorption to a similar extent (PTH, 1.6-fold; GH, 1.4-fold) even though PTH increased serum 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] 3.7-fold, and GH had no significant effect. GH plus PTH caused no further increase in serum 1,25-(OH)2D3 above that caused by PTH alone, but resulted in an additive effect on net calcium absorption (2.3-fold increase). PTH and GH also had statistically independent effects on phosphate absorption; magnesium absorption was elevated only by PTH. Duodenal calbindin-D9k levels were increased by GH (from 3.79 +/- 0.72 to 6.98 +/- 0.73 micrograms/mg protein) and PTH (from 3.23 +/- 0.46 to 7.55 +/- 0.75 micrograms/mg protein); PTH plus GH treatment resulted in an additive effect on calbindin-D9k levels. Additional in vitro transport studies in the human intestinal cell line Caco-2 showed that 72 h of pretreatment with the local mediator of GH action, insulin-like growth factor-I (at 10 and 100 ng/ml), stimulated transcellular calcium transport (22% and 44%, respectively) regardless of concomitant 1 nM 1,25-(OH)2D3 pretreatment (80% increase). Our findings suggest a 1,25-(OH)2D3-mediated mechanism for PTH-induced changes in calcium and phosphorus absorption. In contrast, the effects of GH in the senescent rat are independent of changes in circulating 1,25-(OH)2D3 and our data suggest that these effects may be mediated by insulin-like growth factor-I.

Duodenal calcium binding protein and active calcium transport in rats: are they functionally related?

The effects of calcitriol and a novel calcitriol analogue, 22-oxacalcitriol (OCT) on duodenal Ca transport, calbindin-D9k mRNA, and calbindin-D9k content were studied in two animal models reflecting common human pathologies, namely arterial hypertension and chronic renal failure, as well as in normal rats. The hormone or its analogue were administered intraperitoneally to vitamin-D-replete rats. Active Ca transport was increased in both spontaneously hypertensive rats (SHR) and in normotensive control WKY rats 5 h after calcitriol dosing of either 60 and 600 ng per rat. In WKY, calbindin-D9k content was slightly increased after the injection of 60 ng calcitriol, but not of 600 ng calcitriol whereas calbindin-D9k mRNA stayed essentially unchanged. In contrast, active Ca transport was significantly stimulated after the higher dose of 600 ng calcitriol. In SHR, while both doses of calcitriol increased active Ca transport, they had no stimulatory effect on calbindin-D9k mRNA or protein. In chronically uraemic rats, active Ca transport, duodenal calbindin-D9k and calbindin-D9k mRNA were stimulated after the injection of two subsequent doses of 300 ng calcitriol per rat. OCT treatment at same dosage led to a similar stimulation of calbindin-D9k and calbindin-D9k mRNA, but failed to induce an increase in active Ca transport. These results show that the stimulation of intestinal active Ca transport and calbindin-D9k can be entirely dissociated at the protein synthesis and the mRNA expression level (1) after calcitriol administration to normal and hypertensive rats, and (2) after OCT administration to uraemic rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of 22-oxa-calcitriol on calcium metabolism in rats with severe secondary hyperparathyroidism

The purpose of the present study was to examine the effect of a two day and a five day administration of 22-oxa-calcitriol (OCT) on calcium metabolism in rats with advanced chronic renal failure and severe secondary hyperparathyroidism. A first series of 27 uremic rats received either placebo, OCT or calcitriol (0.3 microgram i.p./rat) 48 and 24 hours before sacrifice. A second series of 18 uremic rats received either placebo, OCT (0.3 microgram i.p./rat) or calcitriol (0.05 microgram i.p./rat) for five days. We found that after 48 hours (series 1) both calcitriol and OCT increased blood ionized calcium (Ca2+) as compared to vehicle (1.23 +/- 0.04 and 1.10 +/- 0.02 mM, P < 0.01 and P < 0.05, respectively vs. control, 1.02 +/- 0.03 mM). Duodenal Ca transport (S/M) using the everted gut sac technique was not stimulated by OCT, even though it increased from 2.8 +/- 0.4 to 7.0 +/- 0.6 (P < 0.01) with calcitriol. In contrast, duodenal calbindin-D9k mRNA expression and protein content increased to a similar extent with OCT and calcitriol. Calcitriol was more potent in reducing plasma iPTH1-34 levels than OCT: 344 +/- 75 pg/ml (calcitriol) versus 632 +/- 46 pg/ml (OCT) compared with 897 +/- 74 pg/ml (control), P < 0.01. In the second series of rats, the injection of OCT (0.3 microgram i.p./rat) over five days was less effective than the lower dose of calcitriol (0.05 microgram i.p./rat) in reducing circulating iPTH: 110 +/- 26 (calcitriol) and 281 +/- 64 (OCT) versus 624 +/- 135 pg/ml (control), P < 0.01.(ABSTRACT TRUNCATED AT 250 WORDS)

Abnormal intestinal regulation of calbindin-D9K and calmodulin by dietary calcium in genetic hypertension

Using isolated duodenal cells from spontaneously hypertensive rats (SHR) and their normotensive controls, Wistar-Kyoto rats (WKY), we previously showed that cellular calcium flux was decreased in SHR and that increasing dietary calcium (from 1 to 2%) eliminated strain differences in Ca2+ fluxes. The present study was carried out to investigate the role of calbindin-D9K and calmodulin in the flux difference and dietary calcium effects. Calbindin-D9K and calmodulin were separated by sodium dodecyl sulfate (SDS) gel electrophoresis in duodenal protein extracts of SHR and WKY (12-14 and 24-26 wk old) fed either a 1 or 2% calcium diet and measured by a ligand blotting (45Ca) technique. Young SHR had a significantly lower calbindin-D9K (P less than 0.001) than did WKY on either diet. Calmodulin was significantly lower in young SHR than in WKY (P less than 0.002). There was no strain difference in calmodulin in older rats fed the normal calcium diet. Calbindin-D9K was significantly decreased by the high-calcium diet in both strains at both ages. There was a significant correlation between duodenal calbindin-D9K and plasma levels of calcitriol (r = +0.80, P less than 0.001) in WKY but not in SHR. Calmodulin was significantly decreased by dietary calcium in mature WKY (4.8 +/- 0.2 vs. 3.7 +/- 0.4 micrograms/mg cell protein, P less than 0.03), demonstrating a potential regulation by dietary calcium of this protein. Finally, there was a significant correlation between calbindin-D9K and calmodulin (r = 0.59, P less than 0.001) in WKY but not in SHR.(ABSTRACT TRUNCATED AT 250 WORDS)

Epidermal Growth Factor Increases Intestinal Calbindin-D9kand 1,25-Dihydroxyvitamin D Receptors in Neonatal Rats*

Epidermal growth factor (EGF) has been reported to increase intestinal calcium absorption in suckling rats. The mechanism of this effect is unknown, as are the roles of vitamin D-dependent and independent pathways. The present studies were undertaken to investigate the ability of EGF to accelerate the postnatal induction of the vitamin D-dependent intestinal calcium-binding protein, calbindin-D9k. Subcutaneous administration of EGF increased duodenal calbindin-D9k in suckling rats by more than 100% (P less than 0.001). The effect of EGF was not seen in older weaned animals or when EGF was given to suckling rats by gavage. Administration of EGF simulated the changes of normal development. 1) It increased calbindin-D9k, and the effect was greater in proximal than distal duodenum. 2) EGF increased alkaline phosphatase activity to the same extent in proximal and distal duodenum. 3) EGF increased sucrase more markedly in distal than in proximal epithelium. Maximal and half-maximal effects of EGF on each of these proteins were observed at twice daily doses of 0.1 and 0.04 microgram/g BW, respectively. 4) EGF at the maximally effective dose produced a small (30%) but statistically significant (P less than 0.005) increase in serum 1,25-dihydroxyvitamin D. 5) Most importantly, EGF treatment resulted in a 2-fold increase in intestinal 1,25-dihydroxyvitamin D receptors (VDR) in the proximal segments of the small intestine (P less than 0.001). EGF effects on calbindin-D9k and VDR were specific for the intestine, as EGF did not change kidney calbindin-D9k or kidney VDR. Thus, EGF was able to prematurely initiate a complex series of molecular changes that occur during normal development. The mechanism of EGF's action to stimulate calcium absorption appears to involve a maturation effect on the vitamin D-dependent pathway.