Oral calcium and vitamin D supplements differentially alter exploratory, anxiety-like behaviors and memory in male rats.

Oral calcium and calcium plus vitamin D supplements are commonly prescribed to several groups of patients, e.g., osteoporosis, fracture, and calcium deficiency. Adequate and steady extracellular calcium levels are essential for neuronal activity, whereas certain forms of calcium supplement (e.g., CaCO3) probably interfere with memory function. However, it was unclear whether a long-term use of ionized calcium (calcium chloride in drinking water ad libitum), vitamin D supplement (oral gavage) or the combination of both affected anxiety and memory, the latter of which was probably dependent on the hippocampal neurogenesis. Here, we aimed to determine the effects of calcium and/or vitamin D supplement on the anxiety- and memory-related behaviors and the expression of doublecortin (DCX), an indirect proxy indicator of hippocampal neurogenesis. Eight-week-old male Wistar rats were divided into 4 groups, i.e., control, calcium chloride-, 400 UI/kg vitamin D3-, and calcium chloride plus vitamin D-treated groups. After 4 weeks of treatment, anxiety-, exploration- and recognition memory-related behaviors were evaluated by elevated pulse-maze (EPM), open field test (OFT), and novel object recognition (NOR), respectively. The hippocampi were investigated for the expression of DCX protein by Western blot analysis. We found that oral calcium supplement increased exploratory behavior as evaluated by OFT and the recognition index in NOR test without any effect on anxiety behavior in EPM. On the other hand, vitamin D supplement was found to reduce anxiety-like behaviors. Significant upregulation of DCX protein expression was observed in the hippocampus of both calcium- and vitamin D-treated rats, suggesting their positive effects on neurogenesis. In conclusion, oral calcium and vitamin D supplements positively affected exploratory, anxiety-like behaviors and/or memory in male rats. Thus, they potentially benefit on mood and memory in osteoporotic patients beyond bone metabolism.

Fe3+ opposes the 1,25(OH)2D3-induced calcium transport across intestinal epithelium-like Caco-2 monolayer in the presence or absence of ascorbic acid.

Although iron is an essential element for hemoglobin and cytochrome synthesis, excessive intestinal iron absorption-as seen in dietary iron supplementation and hereditary disease called thalassemia-could interfere with transepithelial transport of calcium across the intestinal mucosa. The underlying cellular mechanism of iron-induced decrease in intestinal calcium absorption remains elusive, but it has been hypothesized that excess iron probably negates the actions of 1,25-dihydroxyvitamin D [1,25(OH)2D3]. Herein, we exposed the 1,25(OH)2D3-treated epithelium-like Caco-2 monolayer to FeCl3 to demonstrate the inhibitory effect of ferric ion on 1,25(OH)2D3-induced transepithelial calcium transport. We found that a 24-h exposure to FeCl3 on the apical side significantly decreased calcium transport, while increasing the transepithelial resistance (TER) in 1,25(OH)2D3-treated monolayer. The inhibitory action of FeCl3 was considered rapid since 60-min exposure was sufficient to block the 1,25(OH)2D3-induced decrease in TER and increase in calcium flux. Interestingly, FeCl3 did not affect the baseline calcium transport in the absence of 1,25(OH)2D3 treatment. Furthermore, although ascorbic acid is often administered to maximize calcium solubility and to enhance intestinal calcium absorption, it apparently had no effect on calcium transport across the FeCl3- and 1,25(OH)2D3-treated Caco-2 monolayer. In conclusion, apical exposure to ferric ion appeared to negate the 1,25(OH)2D3-stimulated calcium transport across the intestinal epithelium. The present finding has, therefore, provided important information for development of calcium and iron supplement products and treatment protocol for specific groups of individuals, such as thalassemia patients and pregnant women.

Activation of calcium-sensing receptor by allosteric agonists cinacalcet and AC-265347 abolishes the 1,25(OH)2D3-induced Ca2+ transport: Evidence that explains how the intestine prevents excessive Ca2+ absorption.

Long-term high-calcium intake and intestinal calcium hyperabsorption are hazardous to the body. It is hypothesized that enterocytes possess mechanisms for preventing superfluous calcium absorption, including secretion of negative regulators of calcium absorption and utilization of calcium-sensing receptor (CaSR) to detect luminal calcium. Herein, Caco-2 monolayers were treated with high doses of 1,25(OH)2D3 to induce calcium hyperabsorption or directly exposed to high apical calcium. The expression of counterregulatory factor of calcium absorption, fibroblast growth factor (FGF)-23, was also investigated in the intestine of lactating rats, which physiologically exhibit calcium hyperabsorption. We found that FGF-23 expression was enhanced in all intestinal segments of lactating rats. In Caco-2 monolayers, high apical calcium and 1,25(OH)2D3 induced FGF-23 secretion into culture media. FGF-23 antagonized 1,25(OH)2D3-induced calcium transport and led to a significant, but small, change in paracellular permeability. Furthermore, high-dose 1,25(OH)2D3 upregulated FGF-23 expression, which was prevented by CaSR inhibitors. Activation of apical CaSR by cinacalcet and AC-265347 abolished 1,25(OH)2D3-induced calcium transport in a dose-dependent manner. In conclusion, the intestinal FGF-23 expression was upregulated in conditions with calcium hyperabsorption, presumably to help protect against excessive calcium absorption, while CaSR probably monitored calcium in the lumen and induced FGF-23 production for preventing superfluous calcium uptake.

Intestinal calcium transport and its regulation in thalassemia: interaction between calcium and iron metabolism.

Osteoporosis and derangement of calcium homeostasis are common complications of thalassemia. Despite being an important process for bone and calcium metabolism, little is known about intestinal calcium transport in thalassemia. Recent reports of decreases in both intestinal calcium transport and bone mineral density in thalassemic patients and animal models suggested that defective calcium absorption might be a cause of thalassemic bone disorder. Herein, the possible mechanisms associated with intestinal calcium malabsorption in thalassemia are discussed. This includes alterations in the calcium transporters and hormonal controls of the transcellular and paracellular intestinal transport systems in thalassemia. In addition, the effects of iron overload on intestinal calcium absorption, and the reciprocal interaction between iron and calcium transport in thalassemia are elaborated. Understanding the mechanisms underlining calcium malabsorption in thalassemia would lead to development of therapeutic agents and mineral supplements that restore calcium absorption as well as prevent osteoporosis in thalassemic patients.

Prolonged exposure to 1,25(OH)2D3 and high ionized calcium induces FGF-23 production in intestinal epithelium-like Caco-2 monolayer: A local negative feedback for preventing excessive calcium transport.

Overdose of oral calcium supplement and excessive intestinal calcium absorption can contribute pathophysiological conditions, e.g., nephrolithiasis, vascular calcification, dementia, and cardiovascular accident. Since our previous investigation has indicated that fibroblast growth factor (FGF)-23 could abolish the 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-enhanced calcium absorption, we further hypothesized that FGF-23 produced locally in the enterocytes might be part of a local negative feedback loop to regulate calcium absorption. Herein, 1,25(OH)2D3 was found to enhance the transcellular calcium transport across the epithelium-like Caco-2 monolayer, and this stimulatory effect was diminished by preceding prolonged exposure to high-dose 1,25(OH)2D3 or high concentration of apical ionized calcium. Pretreatment with a neutralizing antibody for FGF-23 prevented this negative feedback regulation of calcium hyperabsorption induced by 1,25(OH)2D3. FGF-23 exposure completely abolished the 1,25(OH)2D3-enhanced calcium transport. Western blot analysis revealed that FGF-23 expression was upregulated in a dose-dependent manner by 1,25(OH)2D3 or apical calcium exposure. Finally, calcium-sensing receptor (CaSR) inhibitors were found to prevent the apical calcium-induced suppression of calcium transport. In conclusion, prolonged exposure to high apical calcium and calcium hyperabsorption were sensed by CaSR, which, in turn, increased FGF-23 expression to suppress calcium transport. This local negative feedback loop can help prevent unnecessary calcium uptake and its detrimental consequences.

CFTR-mediated anion secretion across intestinal epithelium-like Caco-2 monolayer under PTH stimulation is dependent on intermediate conductance K+ channels.

Parathyroid hormone (PTH), a pleiotropic hormone that maintains mineral homeostasis, is also essential for controlling pH balance and ion transport across renal and intestinal epithelia. Optimization of luminal pH is important for absorption of trace elements, e.g., calcium and phosphorus. We have previously demonstrated that PTH rapidly stimulated electrogenic [Formula: see text] secretion in intestinal epithelial-like Caco-2 monolayers, but the underlying cellular mechanism, contributions of other ions, particularly Cl- and K+, and long-lasting responses are not completely understood. Herein, PTH and forskolin were confirmed to induce anion secretion, which peaked within 1-3 min (early phase), followed by an abrupt decay and plateau that lasted for 60 min (late phase). In both early and late phases, apical membrane capacitance was increased with a decrease in basolateral capacitance after PTH or forskolin exposure. PTH also induced a transient increase in apical conductance with a long-lasting decrease in basolateral conductance. Anion secretion in both phases was reduced under [Formula: see text]-free and/or Cl--free conditions or after exposure to carbonic anhydrase inhibitor (acetazolamide), CFTR inhibitor (CFTRinh-172), Na+/H+ exchanger (NHE)-3 inhibitor (tenapanor), or K+ channel inhibitors (BaCl2, clotrimazole, and TRAM-34; basolateral side), the latter of which suggested that PTH action was dependent on basolateral K+ recycling. Furthermore, early- and late-phase responses to PTH were diminished by inhibitors of PI3K (wortmannin and LY-294002) and PKA (PKI 14-22). In conclusion, PTH requires NHE3 and basolateral K+ channels to induce [Formula: see text] and Cl- secretion, thus explaining how PTH regulated luminal pH balance and pH-dependent absorption of trace minerals.

Site-Specific Onset of Low Bone Density and Correlation of Bone Turnover Markers in Exclusive Breastfeeding Mothers.

PURPOSE: Lactation often affects calcium metabolism and induces bone loss. Calcium supplementation and a high calcium diet are recommended to prevent bone loss, especially during inadequate calcium intake. Our study aimed at determining bone loss in breastfeeding mothers, and if it occurred, whether it was site specific and there were correlations between serum bone turnover markers. MATERIALS AND METHODS: Since the 6-month exclusive breastfeeding is usually recommended in several countries, our study examined bone mineral density (BMD) in early (1-2 month), mid (3-4 month)-, and late (5-6 month) lactation compared with nonpregnant, nonlactating control women. Site-specific bone loss was monitored in lumbar vertebrae and femora. Bone turnover markers, that is, C-terminal telopeptide of type 1 collagen and N-terminal propeptide of type 1 collagen (P1NP), were determined by electrochemiluminescence immunoassays. RESULTS: The onset of bone loss in exclusive breastfeeding mothers was site specific, for example, in the lumbar bone at mid-lactation and in the femoral bone in late lactation. Serum ionized calcium levels in late lactation were lower than the normal levels. In addition, a correlation was found between bone turnover marker, P1NP, and femoral BMD. CONCLUSIONS: The onset of bone loss in exclusive breastfeeding mothers was site specific, and the lumbar bone was a vulnerable and perhaps better representative site for bone loss detection. It was suggested that the optimal starting time for calcium supplementation should be before the mid-lactation when the bone loss was observed. In addition, the biochemical marker that best predicted the onset of bone loss in lactating women was P1NP.

Pre-suckling calcium supplementation effectively prevents lactation-induced osteopenia in rats.

During lactation, osteoclast-mediated bone resorption and intestinal calcium hyperabsorption help provide extra calcium for lactogenesis. Since the suckling-induced surge of pituitary prolactin (PRL) rapidly stimulates calcium absorption in lactating rats, it is hypothesized that pre-suckling oral calcium supplementation should be an efficient regimen to shift the calcium source from bone to diet, thereby slowing lactation-induced osteopenia. Our results showed that 30-min suckling markedly stimulated maternal duodenal calcium transport, which returned to the baseline at 45 min. Lactating rats given 4 mg/kg per dose calcium via a gavage tube at 90 min pre-suckling 4 doses a day for 14 days prevented a decrease in bone mineral density (BMD) of long bones and vertebrae. On the other hand, a single-dose supplementation, despite the same amount of calcium per day, appeared less effective. Because glucose and galactose further stimulated duodenal calcium transport in lactating rats, pre-suckling calcium supplement containing both sugars successfully normalized plasma ionized calcium and led to better bone gain than that with calcium alone. A histomorphometric study revealed that lactating rats given pre-suckling calcium plus monosaccharide supplement manifested greater trabecular bone volume and thickness and exhibited less eroded surface than in vehicle-treated lactating rats. Beneficial effects of the 14-day calcium supplementation persisted until 6 mo postweaning in dams and also elevated the baseline BMD of the offspring. In conclusion, our proof-of-concept study has corroborated that pre-suckling calcium supplements, especially regimens containing monosaccharides, are efficient in preventing osteopenia in lactating rats and could increase bone density in both breastfeeding mothers and neonates.

Two-step stimulation of intestinal Ca(2+) absorption during lactation by long-term prolactin exposure and suckling-induced prolactin surge.

During pregnancy and lactation, the enhanced intestinal Ca(2+) absorption serves to provide Ca(2+) for fetal development and lactogenesis; however, the responsible hormone and its mechanisms remain elusive. We elucidated herein that prolactin (PRL) markedly stimulated the transcellular and paracellular Ca(2+) transport in the duodenum of pregnant and lactating rats as well as in Caco-2 monolayer in a two-step manner. Specifically, a long-term exposure to PRL in pregnancy and lactation induced an adaptation in duodenal cells at genomic levels by upregulating the expression of genes related to transcellular transport, e.g., TRPV5/6 and calbindin-D(9k), and the paracellular transport, e.g., claudin-3, thereby raising Ca(2+) absorption rate to a new "baseline" (Step 1). During suckling, PRL surge further increased Ca(2+) absorption to a higher level (Step 2) in a nongenomic manner to match Ca(2+) loss in milk. PRL-enhanced apical Ca(2+) uptake was responsible for the increased transcellular transport, whereas PRL-enhanced paracellular transport required claudin-15, which regulated epithelial cation selectivity and paracellular Ca(2+) movement. Such nongenomic PRL actions were mediated by phosphoinositide 3-kinase, protein kinase C, and RhoA-associated coiled-coil-forming kinase pathways. In conclusion, two-step stimulation of intestinal Ca(2+) absorption resulted from long-term PRL exposure, which upregulated Ca(2+) transporter genes to elevate the transport baseline, and the suckling-induced transient PRL surge, which further increased Ca(2+) transport to the maximal capacity. The present findings also suggested that Ca(2+) supplementation at 15-30 min prior to breastfeeding may best benefit the lactating mother, since more Ca(2+) could be absorbed as a result of the suckling-induced PRL surge.

Prolactin directly enhances bone turnover by raising osteoblast-expressed receptor activator of nuclear factor kappaB ligand/osteoprotegerin ratio.

Hyperprolactinemia leads to high bone turnover as a result of enhanced bone formation and resorption. Although its osteopenic effect has long been explained as hyperprolactinemia-induced hypogonadism, identified prolactin (PRL) receptors in osteoblasts suggested a possible direct action of PRL on bone. In the present study, we found that hyperprolactinemia induced by anterior pituitary transplantation (AP), with or without ovariectomy (Ovx), had no detectable effect on bone mineral density and content measured by dual-energy X-ray absorptiometry (DXA). However, histomorphometric studies revealed increases in the osteoblast and osteoclast surfaces in the AP rats, but a decrease in the osteoblast surface in the AP+Ovx rats. The resorptive activity was predominant since bone volume and trabecular number were decreased, and the trabecular separation was increased in both groups. Estrogen supplement (E2) fully reversed the effect of estrogen depletion in the Ovx but not in the AP+Ovx rats. In contrast to the typical Ovx rats, bone formation and resorption became uncoupled in the AP+Ovx rats. Therefore, hyperprolactinemia was likely to have some estrogen-independent and/or direct actions on bone turnover. Osteoblast-expressed PRL receptor transcripts and proteins shown in the present study confirmed our hypothesis. Furthermore, we demonstrated that the osteoblast-like cells, MG-63, directly exposed to PRL exhibited lower expression of alkaline phosphatase and osteocalcin mRNA, and a decrease in alkaline phosphatase activity. The ratios of receptor activator of nuclear factor kappaB ligand (RANKL) and osteoprotegerin (OPG) proteins were increased, indicating an increase in the osteoclastic bone resorption. The present data thus demonstrated that hyperprolactinemia could act directly on bone to stimulate bone turnover, with more influence on bone resorption than formation. PRL enhanced bone resorption in part by increasing RANKL and decreasing OPG expressions by osteoblasts.