Enhancement of intestinal calcium transport by short-chain fatty acids: roles of Na+/H+ exchanger 3 and transient receptor potential vanilloid subfamily 6.

Small organic molecules in the intestinal lumen, particularly short-chain fatty acids (SCFAs) and glucose, have long been postulated to enhance calcium absorption. Here, we used 45Ca radioactive tracer to determine calcium fluxes across the rat intestine after exposure to glucose and SCFAs. Confirming previous reports, glucose was found to increase the apical-to-basolateral calcium flux in the cecum. Under apical glucose-free conditions, SCFAs (e.g., butyrate) stimulated the cecal calcium fluxes by approximately twofold, while having no effect on proximal colon. Since SCFAs could be absorbed into the circulation, we further determined whether basolateral SCFA exposure rendered some positive actions. It was found that exposure of duodenum and cecum on the basolateral side to acetate or butyrate increased calcium fluxes. Under butyrate-rich conditions, cecal calcium transport was partially diminished by Na+/H+ exchanger 3 (NHE3) inhibitor (tenapanor) and nonselective transient receptor potential vanilloid subfamily 6 (TRPV6) inhibitor (miconazole). To confirm the contribution of TRPV6 to SCFA-stimulated calcium transport, we synthesized another TRPV6 inhibitor that was demonstrated by in silico molecular docking and molecular dynamics to occlude TRPV6 pore and diminish the glucose- and butyrate-induced calcium fluxes. Therefore, besides corroborating the importance of luminal molecules in calcium absorption, our findings provided foundation for development of more effective calcium-rich nutraceuticals in combination with various absorptive enhancers, e.g., glucose and SCFAs.NEW & NOTEWORTHY Organic molecules in the intestinal lumen, e.g., glucose and short-chain fatty acids (SCFAs), the latter of which are normally produced by microfloral fermentation, can stimulate calcium absorption dependent on transient receptor potential vanilloid subfamily 6 (TRPV6) and Na+/H+ exchanger 3 (NHE3). A selective TRPV6 inhibitor synthesized and demonstrated by in silico docking and molecular dynamics to specifically bind to the pore domain of TRPV6 was used to confirm a significant contribution of this channel. Our findings corroborate physiological significance of nutrients and SCFAs in enhancing calcium absorption.

Fibroblast growth factor-21 potentiates the stimulatory effects of 1,25-dihydroxyvitamin D3 on transepithelial calcium transport and TRPV6 Ca2+ channel expression.

Fibroblast growth factor (FGF)-21 is a salient liver-derived endocrine regulator for metabolism of glucose and triglyceride as well as bone remodeling. Previously, certain peptides in the FGF family have been shown to modulate calcium absorption across the intestinal epithelia. Since FGF21 receptor, i.e., FGF receptor-1, is abundantly expressed in the enterocytes, there was a possibility that FGF21 might exert direct actions on the intestine. Herein, a large-scale production of recombinant FGF21 at the multi-gram level was developed in order to minimize variations among various batches. In the oral glucose tolerance test, recombinant FGF21 was found to reduce plasma glucose levels in mice fed high-fat diet. A series of experiments applying radioactive tracer 45Ca in Ussing chamber showed that FGF21 potentiated the stimulatory effect of low-dose 1,25-dihydroxyvitamin D3 [10 nM 1,25(OH)2D3] on the transepithelial calcium transport across intestinal epithelium-like Caco-2 monolayer. FGF21 + 1,25(OH)2D3 also decreased transepithelial resistance, but had no effect on epithelial potential difference or short-circuit current. Furthermore, 1,25(OH)2D3 alone upregulated the Caco-2 mRNA expression of the major apical calcium channels, i.e., transient receptor potential vanilloid subfamily member 6 (TRPV6), which was further elevated by a combination of FGF21 and 1,25(OH)2D3, consistent with the upregulated TRPV6 protein expression in enterocytes of FGF21-treated mice. However, FGF21 was without effects on the mRNA expression of voltage-gated calcium channel 1.3, calbindin-D9k, plasma membrane Ca2+-ATPase 1b, claudin-12 or claudin-15. In conclusion, FGF21 did exert a direct action on the intestinal epithelial cells by potentiating the 1,25(OH)2D3-enhanced calcium transport, presumably through the upregulation of TRPV6 expression.

Modulation of fibroblast growth factor-23 expression and transepithelial calcium absorption in Caco-2 monolayer by calcium-sensing receptor and calcineurin under calcium hyperabsorptive state.

Fibroblast growth factor (FGF)-23 and calcium-sensing receptor (CaSR) have previously been postulated to be parts of a negative feedback regulation of the intestinal calcium absorption to prevent excessive calcium uptake and its toxicity. However, the underlying mechanism of this feedback regulation remained elusive, especially whether it required transcription of FGF-23. Herein, we induced calcium hyperabsorptive state (CHS) by exposing intestinal epithelium-like Caco-2 monolayer to 30 mM CaCl2 and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] after which FGF-23 mRNA levels and transepithelial calcium flux were determined. We found that CHS upregulated FGF-23 transcription, which was reverted by CaSR inhibitors (Calhex-231 and NPS2143) but without effect on CaSR transcription. Although 10 nM 1,25(OH)2D3 was capable of enhancing transepithelial calcium flux, the higher-than-normal calcium inundation as in CHS led to a decrease in calcium flux, consistent with an increase in FGF-23 protein expression. Administration of inhibitors (≤10 µM CN585 and cyclosporin A) of calcineurin, a mediator of CaSR action to control transcription and production of its target proteins, was found to partially prevent FGF-23 protein production and the negative effect of CHS on calcium transport, while having no effect on FGF-23 mRNA expression. Direct exposure to FGF-23, but not FGF-23 + PD173074 (FGFR1/3 inhibitor), also completely abolished the 1,25(OH)2D3-enhanced calcium transport in Caco-2 monolayer. Nevertheless, CHS and CaSR inhibitors had no effect on the mRNA levels of calcineurin (PPP3CB) or its targets (i.e., NFATc1-4). In conclusion, exposure to CHS induced by high apical calcium and 1,25(OH)2D3 triggered a negative feedback mechanism to prevent further calcium uptake. CaSR and its downstream mediator, calcineurin, possibly contributed to the regulatory process, in part by enhancing FGF-23 production to inhibit calcium transport. Our study, therefore, corroborated the physiological significance of CaSR-autocrine FGF-23 axis as a local feedback loop for prevention of excessive calcium uptake.

Emerging roles of calcium-sensing receptor in the local regulation of intestinal transport of ions and calcium.

Whether the intestinal mucosal cells are capable of sensing calcium concentration in the lumen and pericellular interstitium remains enigmatic for decades. Most calcium-regulating organs, such as parathyroid gland, kidney, and bone, are capable of using calcium-sensing receptor (CaSR) to detect plasma calcium and trigger appropriate feedback responses to maintain calcium homeostasis. Although both CaSR transcripts and proteins are abundantly expressed in the crypt and villous enterocytes of the small intestine as well as the surface epithelial cells of the large intestine, the studies of CaSR functions have been limited to amino acid sensing and regulation of epithelial fluid secretion. Interestingly, several lines of recent evidence have indicated that the enterocytes use CaSR to monitor luminal and extracellular calcium levels, thereby reducing the activity of transient receptor potential channel, subfamily V, member 6, and inducing paracrine and endocrine feedback responses to restrict calcium absorption. Recent investigations in zebra fish and rodents have also suggested the role of fibroblast growth factor (FGF)-23 as an endocrine and/or paracrine factor participating in the negative control of intestinal calcium transport. In this review article, besides the CaSR-modulated ion transport, we elaborate the possible roles of CaSR and FGF-23 as well as their crosstalk as parts of a negative feedback loop for counterbalancing the seemingly unopposed calciotropic effect of 1,25-dihydroxyvitamin D3 on the intestinal calcium absorption.

Parathyroid hormone increases CFTR expression and function in Caco-2 intestinal epithelial cells.

Parathyroid hormone (PTH) enhances cystic fibrosis transmembrane conductance regulator (CFTR)-mediated anion secretion by the human intestinal epithelial cell line Caco-2. With the patch-clamp and Ussing chamber techniques, we investigated how PTH stimulates CFTR activity in Caco-2 cells. Cell-attached recordings revealed that PTH stimulated the opening of CFTR-like channels, while impedance analysis demonstrated that PTH increased apical membrane capacitance, a measure of membrane surface area. Using ion substitution experiments, the PTH-stimulated increase in short-circuit current (Isc), a measure of transepithelial ion transport, was demonstrated to be Cl-- and HCO3--dependent. However, the PTH-stimulated increase in Isc was unaffected by the carbonic anhydrase inhibitor acetazolamide, but partially blocked by the intermediate-conductance Ca2+-activated K+ channel (IKCa) inhibitor clotrimazole. TRAM-34, a related IKCa inhibitor, failed to directly inhibit CFTR Cl- channels in cell-free membrane patches, excluding its action on CFTR. In conclusion, PTH enhances CFTR-mediated anion secretion by Caco-2 monolayers by increasing the expression and function of CFTR in the apical membrane and IKCa activity in the basolateral membrane.

Anomalous bone changes in ovariectomized type 2 diabetic rats: inappropriately low bone turnover with bone loss in an estrogen-deficient condition.

Estrogen deprivation accelerates bone resorption, leading to imbalance of bone remodeling and osteoporosis in postmenopausal women. In the elderly, type 2 diabetes mellitus (T2DM) frequently coexists as an independent factor of bone loss. However, little is known about the skeletal changes in a combined condition of estrogen deficiency and T2DM. Herein, we performed ovariectomy (OVX) in nonobese Goto-Kakizaki (GK) T2DM rats to examine changes associated with calcium and phosphate metabolism and bone microstructures and strength. As expected, wild-type (WT) rats subjected to ovariectomy (OVX-WT) had low trabecular bone volume and serum calcium with increased dynamic histomorphometric and serum bone markers, consistent with the high turnover state. T2DM in GK rats also led to low trabecular volume and serum calcium. However, the dynamic histomorphometric markers of bone remodeling were unaffected in these GK rats, indicating the distinct mechanism of T2DM-induced bone loss. Interestingly, OVX-GK rats were found to have anomalous and unique changes in bone turnover-related parameters, i.e., decreased osteoblast and osteoclast surfaces with lower COOH-terminal telopeptide of type I collagen levels compared with OVX-WT rats. Furthermore, the levels of calciotropic hormones, i.e., parathyroid hormone and 1,25(OH)2D3, were significantly decreased in OVX-GK rats. Although the OVX-induced bone loss did not further worsen in GK rats, a three-point bending test indicated that OVX-GK bones exhibited a decrease in bone elasticity. In conclusion, T2DM and estrogen deficiency both led to microstructural bone loss, the appearance of which did not differ from each factor alone. Nevertheless, the combination worsened the integrity and suppressed the turnover, which might eventually result in adynamic bone disease.

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.

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.

Ferrous and ferric differentially deteriorate proliferation and differentiation of osteoblast-like UMR-106 cells.

The association between iron overload and osteoporosis has been found in many diseases, such as hemochromatosis, ß-thalassemia and sickle cell anemia with multiple blood transfusion. One of the contributing factors is iron toxicity to osteoblasts. Some studies showed the negative effects of iron on osteoblasts; however, the effects of two biological available iron species, i.e., ferric and ferrous, on osteoblasts are elusive. Since most intracellular ionized iron is ferric, osteoblasts was hypothesized to be more responsive to ferric iron. Herein, ferric ammonium citrate (FAC) and ferrous ammonium sulfate (FAS) were used as ferric and ferrous donors. Our results showed that both iron species suppressed cell survival and proliferation. Both also induced osteoblast cell death consistent with the higher levels of cleaved caspase 3 and caspase 7 in osteoblasts, indicating that iron induced osteoblast apoptosis. Iron treatments led to the elevated intracellular iron in osteoblasts as determined by atomic absorption spectrophotometry, thereby leading to a decreased expression of genes for cellular iron import and increased expression of genes for cellular iron export. Effects of FAC and FAS on osteoblast differentiation were determined by the activity of alkaline phosphatase (ALP). The lower ALP activity from osteoblast with iron exposure was found. In addition, ferric and ferrous differentially induced osteoblastic and osteoblast-derived osteoclastogenic gene expression alterations in osteoblast. Even though both iron species had similar effects on osteoblast cell survival and differentiation, the overall effects were markedly stronger in FAC-treated groups, suggesting that osteoblasts were more sensitive to ferric than ferrous.

Na+/H+ exchanger 3 inhibitor diminishes the amino-acid-enhanced transepithelial calcium transport across the rat duodenum.

Na+/H+ exchanger (NHE)-3 is important for intestinal absorption of nutrients and minerals, including calcium. The previous investigations have shown that the intestinal calcium absorption is also dependent on luminal nutrients, but whether aliphatic amino acids and glucose, which are abundant in the luminal fluid during a meal, similarly enhance calcium transport remains elusive. Herein, we used the in vitro Ussing chamber technique to determine epithelial electrical parameters, i.e., potential difference (PD), short-circuit current (Isc), and transepithelial resistance, as well as 45Ca flux in the rat duodenum directly exposed on the mucosal side to glucose or various amino acids. We found that mucosal glucose exposure led to the enhanced calcium transport, PD, and Isc, all of which were insensitive to NHE3 inhibitor (100 nM tenapanor). In the absence of mucosal glucose, several amino acids (12 mM in the mucosal side), i.e., alanine, isoleucine, leucine, proline, and hydroxyproline, markedly increased the duodenal calcium transport. An inhibitor for NHE3 exposure on the mucosal side completely abolished proline- and leucine-enhanced calcium transport, but not transepithelial transport of both amino acids themselves. In conclusion, glucose and certain amino acids in the mucosal side were potent stimulators of the duodenal calcium absorption, but only amino-acid-enhanced calcium transport was NHE3-dependent.

Na+/H+ exchanger 3 inhibitor diminishes hepcidin-enhanced duodenal calcium transport in hemizygous ß-globin knockout thalassemic mice.

Recent investigation has shown that the liver-derived iron-regulating hormone, hepcidin, can potentiate intestinal calcium absorption in hemizygous ß-globin knockout thalassemic (BKO) mice. Since the upregulation of Fe2+ and H+ cotransporter, divalent metal transporter (DMT)-1, has been shown to correlate with thalassemia-induced intestinal calcium absorption impairment, the inhibition of the apical Na+/H+ exchanger (NHE)-3 that is essential for cytoplasmic pH regulation and transepithelial sodium absorption was hypothesized to negatively affect hepcidin action. Herein, the positive effect of hepcidin on the duodenal calcium transport was evaluated using Ussing chamber technique. The results showed that BKO mice had lower absorptive surface area and duodenal calcium transport than wild-type mice. Besides, paracellular transport of zinc in BKO mice was compromised. Hepcidin administration completely restored calcium transport. Since this hepcidin action was totally abolished by inhibitors of the basolateral calcium transporters, Na+/Ca2+ exchanger (NCX1) and plasma membrane Ca2+-ATPase (PMCA1b), the enhanced calcium flux potentially occurred through the transcellular pathway rather than paracellular pathway. Interestingly, the selective NHE3 inhibitor, 100 nM tenapanor, markedly inhibited hepcidin-enhanced calcium transport. Accordingly, hepcidin is one of the promising therapeutic agents for calcium malabsorption in ß-thalassemia. It mainly stimulates the transcellular calcium transport across the duodenal epithelium in an NHE3-dependent manner.

Intestinal mucosal changes and upregulated calcium transporter and FGF-23 expression during lactation: Contribution of lactogenic hormone prolactin.

As the principal lactogenic hormone, prolactin (PRL) not only induces lactogenesis but also enhances intestinal calcium absorption to supply calcium for milk production. How the intestinal epithelium res-ponses to PRL is poorly understood, but it is hypothesized to increase mucosal absorptive surface area and calcium transporter expression. Herein, lactating rats were found to have greater duodenal, jejunal and ileal villous heights as well as cecal crypt depths than age-matched nulliparous rats. Morphometric analyses in the duodenum and cecum showed that their mucosal adaptations were diminished by bromocriptine, an inhibitor of pituitary PRL release. PRL also upregulated calcium transporter expression (e.g., TRPV6 and PMCA1b) in the duodenum of lactating rats. Since excessive calcium absorption could be detrimental to lactating rats, local negative regulator of calcium absorption, e.g., fibroblast growth factor (FGF)-23, should be increased. Immunohistochemistry confirmed the upregulation of FGF-23 protein expression in the duodenal and cecal mucosae of lactating rats, consistent with the enhanced FGF-23 mRNA expression in Caco-2 cells. Bromocriptine abolished this lactation-induced FGF-23 expression. Additionally, FGF-23 could negate PRL-stimulated calcium transport across Caco-2 monolayer. In conclusion, PRL was responsible for the lactation-induced mucosal adaptations, which were associated with compensatory increase in FGF-23 expression probably to prevent calcium hyperabsorption.

Expression of osteoclastogenic factor transcripts in osteoblast-like UMR-106 cells after exposure to FGF-23 or FGF-23 combined with parathyroid hormone.

As a bone-derived hormone, fibroblast growth factor-23 (FGF-23) negatively regulates phosphate and calcium metabolism, while retaining growth-promoting action for mesenchymal cell differentiation. Elevated FGF-23 levels, together with hyperparathyroidism, are often observed in chronic kidney disease, which is associated with impaired bone mineralization and enhanced bone resorption. Although overexpression of osteoblast-derived osteoclastogenic cytokines might contribute to this metabolic bone disease, whether FGF-23 alone and FGF-23 plus parathyroid hormone (PTH) directly modulated the expression of osteoblast-derived osteoclastogenic genes remained elusive. Herein, we demonstrated the direct effects of FGF-23 on proliferation and mRNA expression of osteoblast-specific differentiation and osteoclastogenic markers in rat osteoblast-like UMR-106 cells in the presence or absence of PTH. FGF-23 was found to suppress UMR-106 cell proliferation, while increasing FGF-23 expression, the latter of which suggested the presence of positive feedback regulation of FGF-23 expression in osteoblasts. FGF-23 also upregulated the mRNA expression of osteoblast differentiation markers (e.g., Runx2, osterix, AJ18, Dlx5, alkaline phosphatase, and osteopontin), osteoclastogenic factors (e.g., MCSF, MCP-1, IL-6, and TNF-α), and bone resorption regulators (RANKL and osteoprotegerin). However, combined PTH and FGF-23 exposure did not alter the levels of FGF-23-induced transcripts, suggesting that both hormones had no additive effect. In conclusion, FGF-23 directly suppressed osteoblast proliferation, while inducing osteoclastogenic gene expression in UMR-106 cells, and the FGF-23-induced transcripts were not altered by long-standing PTH exposure.

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.

Premature chondrocyte apoptosis and compensatory upregulation of chondroregulatory protein expression in the growth plate of Goto-Kakizaki diabetic rats.

Type 2 diabetes mellitus (T2DM) is much more detrimental to bone than previously thought. Specifically, it is associated with aberrant bone remodeling, defective bone microstructure, poor bone quality, and growth retardation. The T2DM-associated impairment of bone elongation may result from a decrease in growth plate function, but the detailed mechanism has been unknown. The present study, therefore, aimed to test hypothesis that T2DM led to premature apoptosis of growth plate chondrocytes in Goto-Kakizaki (GK) type 2 diabetic rats, and thus triggered the compensatory responses to overcome this premature apoptosis, such as overexpression of Runt-related transcription factor (Runx)-2 and vascular endothelial growth factor (VEGF), the essential mediators for bone elongation. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) of epiphyseal sections successfully revealed increases in chondrocyte apoptosis in the hypertrophic zone (HZ) and chondro-osseous junction of GK rats. Quantitative immunohistochemical analysis further confirmed the overexpression of parathyroid hormone-related protein (PTHrP), Runx2 and VEGF, but not Indian hedgehog (Ihh) in the HZ. Analysis of blood chemistry indicated suppression of bone remodeling with a marked decrease in parathyroid hormone level. In conclusion, GK rats manifested a premature increase in chondrocyte apoptosis in the HZ of growth plate, and a compensatory overexpression of chondroregulatory proteins, such as PTHrP, Runx2, and VEGF. Our results, therefore, help explain how T2DM leads to impaired bone elongation and growth retardation.

Expression of transcripts related to intestinal ion and nutrient absorption in pregnant and lactating rats as determined by custom-designed cDNA microarray.

In pregnancy and lactation, maternal adaptation for the enhancement of intestinal ion and nutrient absorption is of paramount importance for fetal development and lactogenesis. This nutrient hyperabsorption has been reported to result from upregulation of transporter gene expression, in part, under control of lactogenic hormone prolactin (PRL). Since a number of gene families are responsible for ion and nutrient transport in the rat small intestine, we herein developed a custom-designed cDNA microarray (CalGeneArray) to determine the transcriptome responses of duodenal epithelial cells during these reproductive periods, which was subsequently validated by quantitative real-time PCR. We thus designed 277 oligonucleotide probes to detect 113 transcripts related to ion/nutrient transport, bone/calcium metabolism, paracrine regulator, and cell metabolism. Pregnancy was found to upregulate the expressions of several duodenal transporters, e.g., Trpm6, Trpm7, Glut5, and Trpv6. Pregnant rats subjected to 7-day injection of bromocriptine, an inhibitor of PRL release, showed the increased levels of some other transcripts, e.g., insulin-2 and Cyp27b1, compared to untreated pregnant rats. Bromocriptine also increased the mRNA levels of insulin-2, glucose transporter-1 (Sglt1), and Cyp27b1, while decreasing those of Fgfr2c, Atp1b2, and Cldn19 in early lactation. During late lactation, the levels of eight studied transcripts (i.e., NaPi-IIb, Cyp27b1, Cldn18, Casr, Atp1b2, Xpnpep, Pept1, and Trpm7) were altered. In conclusion, the CalGeneArray was powerful to help reveal that pregnancy and lactation modulated the expression of genes related to duodenal nutrient transport and cell metabolism. Our findings supported the physiological significance of PRL in regulating nutrient absorption during pregnancy and lactation.

Prolactin stimulates the L-type calcium channel-mediated transepithelial calcium transport in the duodenum of male rats.

Elevated plasma levels of prolactin (PRL) have been reported in several physiological and pathological conditions, such as lactation, prolactinoma, and dopaminergic antipsychotic drug uses. Although PRL is a calcium-regulating hormone that stimulates intestinal calcium absorption in lactating rats, whether PRL is capable of stimulating calcium absorption in male rats has been elusive. Herein, the transepithelial calcium transport and electrical characteristics were determined in ex vivo duodenal tissues of male rats by Ussing chamber technique. We found that PRL receptors were abundantly present in the basolateral membrane of the duodenal epithelial cells. PRL (200-800 ng/mL) markedly increased the active duodenal calcium transport in a dose-dependent fashion without effect on the transepithelial resistance. The PRL-enhanced active duodenal calcium transport was completely abolished by L-type calcium channel blocker (nifedipine) as well as inhibitors of the major basolateral calcium transporters, namely plasma membrane Ca(2+)-ATPase and Na(+)/Ca(2+) exchanger. Several intracellular mediators, such as JAK2, MEK, PI3K and Src kinase, were involved in the PRL-enhanced transcellular calcium transport. Moreover, PRL also stimulated the paracellular calcium transport in the duodenum of male rats in a PI3K-dependent manner. In conclusion, PRL appeared to be a calcium-regulating hormone in male rats by enhancing the L-type calcium channel-mediated transcellular and the paracellular passive duodenal calcium transport. This phenomenon could help restrict or alleviate negative calcium balance and osteoporosis that often accompany hyperprolactinemia in male patients.

Fibroblast growth factor-23 negates 1,25(OH)2D3-induced intestinal calcium transport by reducing the transcellular and paracellular calcium fluxes.

The calciotropic hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] has been known to stimulate intestinal calcium transport via both transcellular and paracellular pathways. Recently, we reported that the 1,25(OH)2D3-enhanced calcium transport in the mouse duodenum could be abolished by fibroblast growth factor (FGF)-23, but the targeted calcium transport pathway has been elusive. Herein, the 1,25(OH)2D3-enhanced calcium transport was markedly inhibited by FGF-23 and inhibitors of the basolateral calcium transporters, NCX1 and PMCA1b, suggesting the negative effect of FGF-23 on the transcellular calcium transport. Similar results could be observed in the intestinal epithelium-like Caco-2 monolayer. Although the Arrhenius plot indicated that FGF-23 decreased the potential barrier (e.g., activation energy) of the paracellular calcium movement, FGF-23 was found to modestly decrease the 1,25(OH)2D3-enhanced paracellular calcium transport and calcium permeability. Moreover, FGF-23 affected the 1,25(OH)2D3-induced change in duodenal water permeability as determined by tritiated water, but both 1,25(OH)2D3 and FGF-23 were without effects on the transepithelial fluxes of paracellular markers, (3)H-mannitol and (14)C-polyethylene glycol. It could be concluded that FGF-23 diminished the 1,25(OH)2D3-enhanced calcium absorption through the transcellular and paracellular pathways. Our findings have thus corroborated the presence of a bone-kidney-intestinal axis of FGF-23/vitamin D system in the regulation of calcium homeostasis.

Bromocriptine modulates the expression of PTHrP receptor, Indian hedgehog, and Runx2 proteins in the growth plate of lactating rats.

In lactating rats, the endochondral bone growth is markedly enhanced, leading to the lengthening of long bone. This lactation-induced bone elongation could be abolished by a dopaminergic D2 receptor agonist bromocriptine, but how bromocriptine altered the expression of major chondroregulatory proteins in the growth plate cartilage was elusive. Here, we performed a quantitative immunohistochemical analysis to determine the expression of various peptides and transcription factors known to control the growth plate chondrocyte proliferation and differentiation [i.e., parathyroid hormone-related protein (PTHrP), PTHrP receptor, Indian hedgehog (Ihh), and runt-related transcription factor 2 (Runx2)], in bromocriptine-treated lactating rats. The results showed that bromocriptine markedly increased Ihh expression in hypertrophic chondrocytes during early and mid-lactation, while the expression of PTHrP receptor, but not its ligand PTHrP, was upregulated in the proliferative and hypertrophic zones during mid and late lactation. In contrast, the expression of Runx2, an important transcription factor for chondrocyte differentiation, was suppressed in the hypertrophic chondrocytes of bromocriptine-treated rats. In conclusion, bromocriptine increased Ihh and PTHrP receptor expressions and decreased Runx2 expression, which might, in turn, enhance chondrocyte proliferation and delay chondrocyte hypertrophy, thereby slowing down endochondral bone growth. This finding could explain how bromocriptine compromised the lactation-induced bone elongation.

Upregulated mRNA levels of SERT, NET, MAOB, and BDNF in various brain regions of ovariectomized rats exposed to chronic aversive stimuli.

Estrogen deficiency increases the risk of anxiety and mood disorders, presumably by deranging metabolism of the monoamine neurotransmitters and the expression of their reuptake transporters in the brain. Although estrogen-deficient individuals were also susceptible to stress, little was known regarding the effect of stress on the levels of transcripts related to brain monoamine metabolism. Herein, we used quantitative real-time PCR to quantify the mRNA levels of serotonin reuptake transporter (SERT), norepinephrine transporter (NET), monoamine oxidase-B (MAOB), tryptophan hydroxylase (TPH), and tyrosine hydroxylase (TH) in various brain regions of ovariectomized (OVX) rats which had been exposed for 4 weeks to chronic aversive stimuli (CAS), such as water deprivation, cage tilt, and illumination. We found that CAS induced stress responses in OVX rats as indicated by increases in the adrenal gland weight and sucrose intake. After CAS exposure, mRNA levels of SERT and NET were upregulated in the frontal cortex, hippocampus, amygdala, and periaqueductal gray. In addition, CAS also increased the mRNA levels of MAOB, an enzyme for dopamine degradation, in the same brain regions. However, CAS did not alter the mRNA levels of TPH or TH, both of which are rate-limiting enzymes for the synthesis of serotonin and norepinephrine in the dorsal raphé and locus coeruleus, respectively. Interestingly, mRNA expression of brain-derived neurotrophic factor precursor was upregulated in the hippocampus of CAS-exposed OVX rats, suggesting a compensatory mechanism which might counteract the stress-induced depression. Therefore, the present data have provided evidence to explain how stress affected brain monoamine metabolism in estrogen-deficient stressed patients.