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.

Hyperbaric oxygen therapy exerts anti-osteoporotic effects in obese and lean D-galactose-induced aged rats.

Obesity accelerates the aging processes, resulting in an aggravation of aging-induced osteoporosis. We investigated the anti-osteoporotic effect of hyperbaric oxygen therapy (HBOT) in obese- and lean-aged rats through measurement of cellular senescence, hypoxia, inflammation, antioxidants, and bone microarchitecture. Obese and lean male Wistar rats were injected with 150 mg/kg/day of D-galactose for 8 weeks to induce aging. Then, all rats were randomly given either sham or HBOT for 14 days. Metabolic parameters were determined. Expression by bone mRNA for cellular senescence, hypoxia, inflammation, antioxidative capacity, and bone remodeling were examined. Micro-computed tomography and atomic absorption spectroscopy were performed to evaluate bone microarchitecture and bone mineral profiles, respectively. We found that HBOT restored the alterations in the mRNA expression level of p16, p21, HIF-1α, TNF-α, IL-6, RANKL, RANK, NFATc1, DC-STAMP, Osx, ALP, and Col1a1 in the bone in obese-and lean- aging rats. In obese-aging rats, HBOT increased the level of expression of Sirt1 and CuZnSOD mRNA and diminished the expression level of HIF-2α and ctsk mRNA to the same levels as the control group. However, HBOT failed to alter catalase and OCN mRNA expression in obese-aged rats. HBOT partially improved the bone microarchitecture in obese-aged rats, but completely restored it in lean-aged rats. Interestingly, HBOT protected against obesity-induced demineralization in obese-aged rats. In summary, HBOT exerts an anti-osteoporotic effect in lean-aged rats and prevents some, but not all the negative effects of obese-aged conditions on bone health. Therefore, HBOT is considered as a potential therapy for aging-induced osteoporosis, regardless of obese status.

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.

Male Lrp5A214V mice maintain high bone mass during dietary calcium restriction by altering the vitamin D endocrine system.

Environmental factors and genetic variation individually impact bone. However, it is not clear how these factors interact to influence peak bone mass accrual. Here we tested whether genetically programmed high bone formation driven by missense mutations in the Lrp5 gene (Lrp5A214V) altered the sensitivity of mice to an environment of inadequate dietary calcium (Ca) intake. Weanling male Lrp5A214V mice and wildtype littermates (control) were fed AIN-93G diets with 0.125%, 0.25%, 0.5% (reference, basal), or 1% Ca from weaning until 12 weeks of age (ie, during bone growth). Urinary Ca, serum Ca, Ca regulatory hormones (PTH, 1,25 dihydroxyvitamin D3 (1,25(OH)2D3)), bone parameters (µCT, ash), and renal/intestinal gene expression were analyzed. As expected, low dietary Ca intake negatively impacted bones and Lrp5A214V mice had higher bone mass and ash content. Although bones of Lrp5A214V mice have more matrix to mineralize, their bones were not more susceptible to low dietary Ca intake. In control mice, low dietary Ca intake exerted expected effects on serum Ca (decreased), PTH (increased), and 1,25(OH)2D3 (increased) as well as their downstream actions (ie, reducing urinary Ca, increasing markers of intestinal Ca absorption). In contrast, Lrp5A214V mice had elevated serum Ca with a normal PTH response but a blunted 1,25(OH)2D3 response to low dietary Ca that was reflected in the renal 1,25(OH)2D3 producing/degrading enzymes, Cyp27b1 and Cyp24a1. Despite elevated serum Ca in Lrp5A214V mice, urinary Ca was not elevated. Despite an abnormal serum 1,25(OH)2D3 response to low dietary Ca, intestinal markers of Ca absorption (Trpv6, S100g mRNA) were elevated in Lrp5A214V mice and responded to low Ca intake. Collectively, our data indicate that the Lrp5A214V mutation induces changes in Ca homeostasis that permit mice to retain more Ca and support their high bone mass phenotype.

Optimizing peak bone mass (PBM) is critical for strong bones and osteoporosis prevention. Both genetics and dietary factors like calcium (Ca) contribute to PBM. The goal of this research study was to determine how dietary Ca intake and genetics interact with each other to impact bone mass. Lowering dietary Ca in control mice causes hormonal changes that increase intestinal Ca absorption and reduce urinary Ca loss to protect bone; but this process fails when dietary Ca becomes too low. However, mice with genetically programmed high bone mass could maintain high bone mass even when challenged with Ca deficient diets. This protection is because the high bone mass mice maintain higher serum Ca, have altered production and utilization of Ca-regulating hormones, and have increased molecular indicators of intestinal Ca absorption and kidney Ca retention. Our findings are important because they demonstrate how a genetic program that increases bone formation can drive improved efficiency of Ca utilization to accommodate the increased need for Ca deposition into bone. We believe that our preclinical study provides important proof-of-principle support for the concept of personalized recommendations for bone health management.

Intestinal Vitamin D Receptor Is Dispensable for Maintaining Adult Bone Mass in Mice With Adequate Calcium Intake.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3)-mediated intestinal calcium (Ca) absorption supplies Ca for proper bone mineralization during growth. We tested whether vitamin D receptor (VDR)-mediated 1,25(OH)2D3 signaling is critical for adult Ca absorption and bone by using mice with inducible Vdr gene knockout in the whole intestine (villin-CreERT2+/- × Vdrf/f, WIK) or in the large intestine (Cdx2-CreERT2+/- ×Vdrf/f, LIK). At 4-month-old, Vdr alleles were recombined (0.05 mg tamoxifen/g BW, intraperitoneally [i.p.], 5 days) and mice were fed diets with either 0.5% (adequate) or 0.2% (low) Ca. Ca absorption was examined after 2 weeks while serum 1,25(OH)2D3, bone mass, and bone microarchitecture were examined after 16 weeks. Intestinal and renal gene expression was measured at both time points (n = 12/genotype/diet/time point). On the 0.5% Ca diet, all phenotypes in WIK and LIK mice were similar to the controls. Control mice adapted to the 0.2% low-Ca diet by increasing renal Cyp27b1 mRNA (3-fold), serum 1,25(OH)2D3 level (1.9-fold), and Ca absorption in the duodenum (Dd, + 131%) and proximal colon (PCo, + 28.9%), which prevented bone loss. In WIK mice, low-Ca diet increased serum 1,25(OH)2D3 (4.4-fold) but Ca absorption remained unaltered in the Dd and PCo. Consequently, significant bone loss occurred in WIK mice (e.g., cortical thickness, Ct.Th, -33.7%). LIK mice adapted to the low-Ca diet in the Dd but not the PCo, and the effect on bone phenotypes was milder (e.g., Ct.Th, -13.1%). Our data suggest intestinal VDR in adult mice prevents bone loss under low Ca intake but is dispensable under adequate calcium intake.

Diet X Gene Interactions Control Femoral Bone Adaptation to Low Dietary Calcium.

Genetics and dietary calcium (Ca) are each critical regulators of peak bone mass but it is unclear how genetics alters the physiologic response of bone to dietary Ca restriction (RCR). Here, we conducted genetic mapping in C57BL/6J × DBA/2J (BXD) recombinant inbred mouse lines to identify environmentally sensitive loci controlling whole-bone mass (bone mineral density [BMD], bone mineral content [BMC]), distal trabecular bone, and cortical bone midshaft of the femur. Mice were fed adequate (basal) or low Ca diets from 4-12 weeks of age. Femurs were then examined by dual-energy X-ray absorptiometry (DXA) and micro-computed tomography (µCT). Body size-corrected residuals were used for statistical analysis, genetic mapping, and to estimate narrow sense heritability (h2). Genetics had a strong impact on femoral traits (eg, bone volume fraction [BV/TV] basal Ca, h2 = 0.60) as well as their RCR (eg, BV/TV, h2 = 0.32). Quantitative trait locus (QTL) mapping identified up to six loci affecting each bone trait. A subset of loci was detected in both diet groups, providing replication of environmentally robust genetic effects. Several loci control multiple bone phenotypes suggesting the existence of genetic pleiotropy. QTL controlling the bone RCR did not overlap with basal diet QTL, demonstrating genetic independence of those traits. Candidate genes underlying select multi-trait loci were prioritized by protein coding effects or gene expression differences in bone cells. These include candidate alleles in Rictor (chromosome [chr] 15) and Egfl7 (chr 2) at loci affecting bone in the basal or low Ca groups and in Msr1 (chr 8), Apc, and Camk4 (chr 18) at loci affecting RCR. By carefully controlling dietary Ca and measuring traits in age-matched mice we identified novel genetic loci determining bone mass/microarchitecture of the distal femur as well as their physiologic adaptation to inadequate dietary Ca intake. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Intestinal Calcium Absorption.

In this article, we focus on mammalian calcium absorption across the intestinal epithelium in normal physiology. Intestinal calcium transport is essential for supplying calcium for metabolism and bone mineralization. Dietary calcium is transported across the mucosal epithelia via saturable transcellular and nonsaturable paracellular pathways, both of which are under the regulation of 1,25-dihydroxyvitamin D3 and several other endocrine and paracrine factors, such as parathyroid hormone, prolactin, 17ß-estradiol, calcitonin, and fibroblast growth factor-23. Calcium absorption occurs in several segments of the small and large intestine with varying rates and capacities. Segmental heterogeneity also includes differential expression of calcium transporters/carriers (e.g., transient receptor potential cation channel and calbindin-D9k ) and the presence of favorable factors (e.g., pH, luminal contents, and gut motility). Other proteins and transporters (e.g., plasma membrane vitamin D receptor and voltage-dependent calcium channels), as well as vesicular calcium transport that probably contributes to intestinal calcium absorption, are also discussed. © 2021 American Physiological Society. Compr Physiol 11:1-27, 2021.

Dietary calcium intake and genetics have site-specific effects on peak trabecular bone mass and microarchitecture in male mice.

Trabecular bone (Tb) is used for rapid exchange of calcium (Ca) in times of physiologic need and the site-specific characteristics of Tb may explain why certain sites are more vulnerable to osteoporosis. We hypothesized that peak trabecular bone mass (PTBM) and Tb microarchitecture are differentially regulated by dietary Ca intake, genetics, or Gene-by-Diet (GxD) interactions at the distal femur and the fifth lumbar (L5) vertebra. Male mice from 62 genetically distinct lines were fed basal (0.5%) or low (0.25%) Ca diets from 4 to 12 wks of age. Afterwards, the right femur and L5 vertebra were removed and trabecular bone was analyzed by µCT. In mice fed the basal diet, bone volume fraction (BV/TV), trabecular number (Tb.N), and connectivity density (Conn.D) were significantly higher in the L5 vertebra than femur. Femur Tb had a weaker, more rod-like structure than the L5 vertebrae while mice fed the low Ca diet developed rod-like structures at both sites. Dietary Ca restriction also caused a greater relative reduction of Tb.N and Conn.D in the femur than L5 vertebra, i.e. it was more harmful to the integrity of Tb microarchitecture in femur. Genetics was a major determinant of Tb at both sites, e.g. heritability of BV/TV on the basal diet = 0.65 (femur) and 0.68 (L5 vertebra). However, while GxD interactions altered the impact of dietary Ca restriction on Tb parameters at both sites, the effect was not uniform, e.g. some lines had site-specific responses to Ca restriction. The significance of our work is that there are site-specific effects of dietary Ca restriction and genetics that work independently and interactively to influence the attainment of PTBM and Tb microarchitecture.