Search

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, 17β-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.

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.

Skeletal muscle exhibits enormous plasticity throughout life, however, less is known regarding how the stages of growth regulate its local vitamin D system. Herein, we investigated serum 25(OH)D3 and Ca2+ levels along with the vitamin D system in skeletal muscle and resident myogenic stem cells of male C57BL/6 mice during development, maturation, and ageing. Compared with development, significant increases in vitamin D receptor (VDR) protein expression in mature and aged muscles were associated with increased serum 25(OH)D3 and centronucleated fibres, respectively. The substantial increase in VDR protein expression in aged muscle was also related to reduced downstream mTOR signalling protein expression which was more pronounced in fast-glycolytic compared to slow-oxidative muscles. Intriguingly, serum Ca2+ and vitamin D-metabolising enzyme (CYP27B1 and CYP24A1) levels in skeletal muscle were not different across age. In primary cell culture, nuclear VDR protein was expressed in undifferentiated skeletal muscle stem cells (SMSC) after 1α,25(OH)2D3 treatment. Additionally, a diminished response to 1α,25(OH)2D3 was observed with age as there was a rapid commitment of SMSC towards differentiation under growth-stimulating conditions. Collectively, understanding the local vitamin D system in skeletal muscle could help develop effective interventions for vitamin D supplementation to improve skeletal muscle mass and function during ageing.

We aimed to compare the time-course effect of D-galactose (D-gal)-induced aging, obesity, and their combined effects on bone homeostasis. Male Wistar rats were fed with either a normal diet (ND; n = 24) or a high-fat diet (HFD; n = 24) for 12 weeks. All rats were then injected with either vehicle or 150 mg/kg/day of D-gal for 4 or 8 weeks. Blood was collected to measure metabolic, aging, oxidative stress, and bone turnover parameters. Bone oxidative stress and inflammatory markers, as well as bone histomorphometry were also evaluated. Additionally, RAW 264.7 cells were incubated with either D-gal, insulin, or D-gal plus insulin to identify osteoclast differentiation capacity under the stimulation of receptor activator of nuclear factor κB ligand. At week 4, D-gal-induced aging significantly elevated serum malondialdehyde level and decreased trabecular thickness in ND- and HFD-fed rats, when compared to the control group. At week 8, D-gal-induced aging further elevated advanced glycation end products, increased bone inflammation and resorption, and significantly impaired bone microarchitecture in HFD-fed rats. The osteoclast number in vitro were increased in the D-gal, insulin, and combined groups to a similar extent. These findings suggest that aging aggravates bone dyshomeostasis in the obese condition in a time-dependent manner.

To investigate the effects of hyperbaric oxygen therapy (HBOT) on metabolic disturbance, aging and bone remodeling in D-galactose-induced aging rats with and without obesity by determining the metabolic parameters, aging and oxidative stress markers, bone turnover markers, bone microarchitecture, and bone biomechanical strength.Male Wistar rats were fed either a normal diet (ND; n = 18) or a HFD (n = 12) for 22 weeks. At week 13, vehicle (0.9% NaCl) was injected into ND-fed rats (NDV; n = 6), while 150 mg/kg/day of D-galactose was injected into 12 ND-fed rats (NDD) and 12 HFD-fed rats (HFDD) for 10 weeks. At week 21, rats were treated with either sham (NDVS, NDDS, or HFDDS; n = 6/ group) or HBOT (NDDH, or HFDDH; n = 6/group) for 14 days. Rats were then euthanized. Blood samples, femora, and tibiae were collected.Both NDD and HFDD groups developed aging as indicated by increased AGE level, increased inflammation and oxidative stress as shown by raised serum TNF-α and MDA levels, impaired bone remodeling as indicated by an increase in levels of CTX-1, TRACP-5b, and impaired bone structure/strength, when compared with those of the NDVS group. HFD aggravated these indicators of bone dyshomeostasis in D-galactose-treated rats. HBOT restored bone remodeling and bone structure/strength in the NDD group, however HBOT ameliorated bone dyshomeostasis in the HFDD group.HBOT is a potential intervention to decrease the risk of osteoporosis and bone fracture in aging with or without obesity.

Thalassemia causes anemia, ineffective erythropoiesis, bone loss and iron accumulation in several tissues, e.g., liver, bone and heart, the last of which leads to lethal cardiomyopathy and arrhythmia. Although exercise reportedly improves bone density in thalassemic mice, exercise performance is compromised and might pose risk of cardiovascular accident in thalassemic patients. Therefore, we sought to explore whether mild-intensity physical activity (MPA) with 30–50% of maximal oxygen consumption was sufficient to benefit the heart and bone. Herein, male hemizygous β-globin knockout (BKO) mice and wild-type littermates were subjected to voluntary wheel running 1 h/day, 5 days/week for 3 months (MPA group) or kept sedentary (SDN; control). As determined by atomic absorption spectroscopy, BKO-MPA mice had less iron accumulation in heart and bone tissues compared with BKO-SDN mice. Meanwhile, the circulating level of fibroblast growth factor-23—a factor known to reduce serum iron and intestinal calcium absorption—was increased early in young BKO-MPA mice. Nevertheless, MPA did not affect duodenal calcium transport or body calcium retention. Although MPA restored the aberrant bone calcium-phosphorus ratio to normal range, it did not change vertebral calcium content or femoral mechanical properties. Microstructural porosity in tibia of BKO-MPA mice remained unaltered as determined by synchrotron radiation X-ray tomographic microscopy. In conclusion, MPA prevents cardiac and bone iron accumulation, which is beneficial to thalassemic patients with limited physical fitness or deteriorated cardiac performance. However, in contrast to moderate-intensity exercise, MPA does not improve bone mechanical properties or reduce bone porosity.

The chronic consumption of a high-fat diet (HFD) induces obese–insulin resistance and impairs jawbone health via gut dysbiosis-stimulated inflammatory process. Our previous studies demonstrated that the probiotic Lactobacillus paracasei HII01, prebiotic xylooligosaccharide (XOS), and synbiotics improved several vital organ functions by reducing gut dysbiosis in HFD-induced obese rats. However, the impacts on the cellular level of jawbone microarchitecture have not been examined. Here, we hypothesized that the supplementation of L. paracasei HII01, XOS, and synbiotics ameliorated the bone microarchitectural pathology in HFD-fed rats by reducing systemic inflammation and other metabolic parameters. The dietary regimes (normal or high-fat diet) were provided to 48 male Wistar rats throughout 24-week experiment. After week 12, rats were given either a vehicle, pro-, pre-, or synbiotic for an additional 12 weeks before being killed. Then, blood analyses and bone histomorphometry of the jawbones were performed. The HFD-fed rats developed obese–insulin resistance with significantly elevated systemic inflammation. Bone histomorphometry of these rats showed a decrease in trabecular thickness with increased osteoclasts and active erosion surfaces. Mineral apposition and bone-formation rates were also remarkably diminished. The treatment with pro-, pre-, and synbiotics equally improved metabolic disturbance, reduced systemic inflammation, increased trabecular thickness, decreased osteoclasts and active erosion surfaces and restored mineral apposition and bone-formation rates. The probiotic L. paracasei HII01, prebiotic XOS, and the synbiotics had similarly beneficial effects to improve jawbone microarchitecture in HFD-fed rats by possibly ameliorating osteoclast-related bone resorption and potentiating bone-formation activities.

Excessive salt intake has been associated with the development of non-communicable diseases, including hypertension with several cardiovascular consequences. Although the detrimental effects of high salt on the skeleton have been reported, longitudinal assessment of calcium balance together with changes in bone microarchitecture and strength under salt loading has not been fully demonstrated. To address these unanswered issues, male Sprague–Dawley rats were fed normal salt diet (NSD; 0.8% NaCl) or high salt diet (HSD; 8% NaCl) for 5 months. Elevation of blood pressure, cardiac hypertrophy and glomerular deterioration were observed in HSD, thus validating the model. The balance studies were performed to monitor calcium input and output upon HSD challenge. The HSD-induced increase in calcium losses in urine and feces together with reduced fractional calcium absorption led to a decrease in calcium retention. With these calcium imbalances, we therefore examined microstructural changes of long bones of the hind limbs. Using the synchrotron radiation x-ray tomographic microscopy, we showed that trabecular structure of tibia and femur of HSD displayed a marked increase in porosity. Consistently, the volumetric micro-computed tomography also demonstrated a significant decrease in trabecular bone mineral density with expansion of endosteal perimeter in the tibia. Interestingly, bone histomorphometric analyses indicated that salt loading caused an increase in osteoclast number together with decreases in osteoblast number and osteoid volume. This uncoupling process of bone remodeling in HSD might underlie an accelerated bone loss and bone structural changes. In conclusion, long-term excessive salt consumption leads to impairment of skeletal mass and integrity possibly through negative calcium balance.

The gasotransmitter carbon monoxide (CO) regulates fluid and electrolyte movements across epithelial tissues. However, its action on anion channels is incompletely understood. Here, we investigate the direct action of CO on the cystic fibrosis transmembrane conductance regulator (CFTR) by applying CO-releasing molecules (CO-RMs) to the intracellular side of excised inside-out membrane patches from cells heterologously expressing wild-type human CFTR. Addition of increasing concentrations of tricarbonyldichlororuthenium(II) dimer (CORM-2) (1–300 μM) inhibited CFTR channel activity, whereas the control RuCl3(100 μM) was without effect. CORM-2 predominantly inhibited CFTR by decreasing the frequency of channel openings and, hence, open probability ( Po). But, it also reduced current flow through open channels with very fast kinetics, particularly at elevated concentrations. By contrast, the chemically distinct CO-releasing molecule CORM-3 inhibited CFTR by decreasing Powithout altering current flow through open channels. Neither depolarizing the membrane voltage nor raising the ATP concentration on the intracellular side of the membrane affected CFTR inhibition by CORM-2. Interestingly, CFTR inhibition by CORM-2, but not by CFTRinh-172, was prevented by prior enhancement of channel activity by the clinically approved CFTR potentiator ivacaftor. Similarly, when added after CORM-2, ivacaftor completely relieved CFTR inhibition. In conclusion, CORM-2 has complex effects on wild-type human CFTR consistent with allosteric inhibition and open-channel blockade. Inhibition of CFTR by CO-releasing molecules suggests that CO regulates CFTR activity and that the gasotransmitter has tissue-specific effects on epithelial ion transport. The action of ivacaftor on CFTR Cl−channels inhibited by CO potentially expands the drug’s clinical utility.

One of the potential contributing factors for iron overload-induced osteoporosis is the iron toxicity on bone forming cells, osteoblasts. In this study, the comparative effects of Fe3+ and Fe2+ on osteoblast differentiation and mineralization were studied in UMR-106 osteoblast cells by using ferric ammonium citrate and ferrous ammonium sulfate as Fe3+ and Fe2+ donors, respectively. Effects of 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] and iron chelator deferiprone on iron uptake ability of osteoblasts were examined, and the potential protective ability of 1,25(OH)2D3, deferiprone and extracellular calcium treatment in osteoblast cell survival under iron overload was also elucidated. The differential effects of Fe3+ and Fe2+ on reactive oxygen species (ROS) production in osteoblasts were also compared. Our results showed that both iron species suppressed alkaline phosphatase gene expression and mineralization with the stronger effects from Fe3+ than Fe2+. 1,25(OH)2D3 significantly increased the intracellular iron but minimally affected osteoblast cell survival under iron overload. Deferiprone markedly decreased intracellular iron in osteoblasts, but it could not recover iron-induced osteoblast cell death. Interestingly, extracellular calcium was able to rescue osteoblasts from iron-induced osteoblast cell death. Additionally, both iron species could induce ROS production and G0/G1 cell cycle arrest in osteoblasts with the stronger effects from Fe3+. In conclusions, Fe3+ and Fe2+ differentially compromised the osteoblast functions and viability, which can be alleviated by an increase in extracellular ionized calcium, but not 1,25(OH)2D3 or iron chelator deferiprone. This study has provided the invaluable information for therapeutic design targeting specific iron specie(s) in iron overload-induced osteoporosis. Moreover, an increase in extracellular calcium could be beneficial for this group of patients.

The association between iron overload and osteoporosis has been found in many diseases, such as hemochromatosis, β-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.

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.

Composite materials having mechanical and biological properties similar to those of human bones are needed for bone regeneration and repair. In the present study, composites were made by incorporating bioactive glass (BG) into polycaprolactone (PCL)-polyvinyl alcohol (PVA) (PCLPVA) matrix. Composites with different BG contents of 10, 25 and 50wt% were fabricated by an in-situ blending method. Physicochemical properties measurements found that the composite with 50wt% BG in the PCLPVA organic matrix exhibited the best mechanical properties (compressive strength and compressive young's modulus up to 32.26MPa and 530.91MPa, respectively). We investigated the effects of the BG content on cell adhesion, proliferation and osteogenic activity of UMR-106 cells grown on the scaffolds using in vitro cell culture assay. The composite scaffolds having 25wt% BG showed a significant increase in their cell adhesion capability and a faster cell proliferation. They also exhibited cell adhesion and spreading morphology after only 5days of culturing. For these reasons, we chose to attach the bone morphogenetic protein (BMP)-2 to this composite. The resulting composite (labeled BMP-2-loaded PCLPVABG25) showed significant improvement in the UMR-106 cells adhesion, in the enhancement in osteogenic differentiation and osteoinductivity of this composite.

Several investigations have shown that pregnancy and lactation are able to induce elongation of long bone by altering epiphyseal cartilage function in a prolactin-dependent manner. Since the transcription factor Sox9 is of utmost importance for chondrocyte proliferation and differentiation and since bromocriptine, a dopaminergic D2 agonist widely used to suppress milk production, is known to disrupt the production and release of prolactin, we herein aimed to investigate whether pregnancy and lactation as well as bromocriptine could alter the expression of Sox9. Our immunohistochemical analysis showed that the Sox9 expression levels were markedly upregulated in the tibial proliferative zone of day 21 pregnant rats. In day 8 (early) and day 14 (mid) lactating rats, the Sox9 expression was enhanced only in the proliferative zone, but not in the resting and hypertrophic zones. There was no change in Sox9 expression in day 21 (late) lactating rats. Postweaning rats manifested a decreased Sox9 expression in the hypertrophic zone. Bromocriptine had no effect on Sox9 expression in the proliferative zone of day 21 pregnant rats; however, it completely prevented the Sox9 upregulation in those of early and mid-lactating rats. A differential response was observed in the proliferative and hypertrophic zones of late lactating rats, in which bromocriptine enhanced Sox9 expression. Further investigation of cartilaginous matrix revealed no change in proteoglycans accumulation in lactating rats. In conclusion, the upregulated Sox9 expression predominantly occurred in the proliferative zone during late pregnancy and early and mid-lactation, while the bromocriptine effects depended on the periods and epiphyseal zones.

Hypertension and osteoporosis are the major non-communicable diseases in the elderly worldwide. Although clinical studies reported that hypertensive patients experienced significant bone loss and likelihood of fracture, the causal relationship between hypertension and osteoporosis has been elusive due to other confounding factors associated with these diseases. In this study, spontaneously hypertensive rats (SHR) were used to address this relationship and further explored the biophysical properties and the underlying mechanisms. Long bones of the hind limbs from 18-week-old female SHR were subjected to determination of bone mineral density (BMD) and their mechanical properties. Using synchrotron radiation X-ray tomographic microscopy (SRXTM), femoral heads of SHR displayed marked increase in porosity within trabecular area together with decrease in cortical thickness. The volumetric micro-computed tomography also demonstrated significant decreases in trabecular BMD, cortical thickness and total cross-sectional area of the long bones. These changes also led to susceptibility of the long bones to fracture indicated by marked decreases in yield load, stiffness and maximum load using three-point bending tests. At the cellular mechanism, an increase in the expression of osteoclastogenic markers with decrease in the expression of alkaline phosphatase was found in primary osteoblast-enriched cultures isolated from long bones of these SHR suggesting an imbalance in bone remodeling. Taken together, defective bone mass and strength in hypertensive rats were likely due to excessive bone resorption. Development of novel therapeutic interventions that concomitantly target hypertension and osteoporosis should be helpful in reduction of unwanted outcomes, such as bone fractures, in elderly patients.

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.

β-thalassemia is often associated with hyperglycemia, osteoporosis and increased fracture risk. However, the underlying mechanisms of the thalassemia-associated bone loss remain unclear. It might result from abnormal activities of osteoblasts and osteoclasts, and perhaps prolonged exposure to high extracellular glucose. Herein, we determined the rate of duodenal calcium transport in hemizygous β-globin knockout thalassemic (BKO) mice. Their bones were collected for primary osteoblast and osteoclast culture. We found that BKO mice had lower calcium absorption than their wild-type (WT) littermates. Osteoblasts from BKO mice showed aberrant expression of osteoblast-specific genes, e.g., Runx2, alkaline phosphatase and osteocalcin, which could be partially restored by 1,25(OH)2D3 treatment. However, the mRNA expression levels of RANK, calcitonin receptor (Calcr), c-Fos, NFATc1, cathepsin K and DMT1 were similar in both BKO and WT groups. Exposure to high extracellular glucose modestly but significantly affected the expression of osteoclast-specific markers in WT osteoclasts with no significant effect on osteoblast-specific genes in WT osteoblasts. Thus, high glucose alone was unable to convert WT bone cells to BKO-like bone cells. In conclusion, the impaired calcium absorption and mutation-related aberrant bone cell function rather than exposure to high blood glucose were likely to be the principal causes of thalassemic bone loss.

Besides the two canonical calciotropic hormones, namely parathyroid hormone and 1,25-dihydroxyvitamin D [1,25(OH)2D3], there are several other endocrine and paracrine factors, such as prolactin, estrogen, and insulin-like growth factor that have been known to directly stimulate intestinal calcium absorption. Generally, to maintain an optimal plasma calcium level, these positive regulators enhance calcium absorption, which is indirectly counterbalanced by a long-loop negative feedback mechanism, i.e., through calcium-sensing receptor in the parathyroid chief cells. However, several lines of recent evidence have revealed the presence of calcium absorption inhibitors present in the intestinal lumen and extracellular fluid in close vicinity to enterocytes, which could also directly compromise calcium absorption. For example, luminal iron, circulating fibroblast growth factor (FGF)-23, and stanniocalcin can decrease calcium absorption, thereby preventing excessive calcium uptake under certain conditions. Interestingly, the intestinal epithelial cells themselves could lower their rate of calcium uptake after exposure to high luminal calcium concentration, suggesting a presence of an ultra-short negative feedback loop independent of systemic hormones. The existence of neural regulation is also plausible but this requires more supporting evidence. In the present review, we elaborate on the physiological significance of these negative feedback regulators of calcium absorption, and provide evidence to show how our body can efficiently restrict a flood of calcium influx in order to maintain calcium homeostasis.

Since the in vitro and in vivo anti-osteoporotic effects of Pueraria mirifica (PM) in rodents have been verified, its activity in menopausal monkeys was evaluated as required before it can be applicable for human use. In this study, postmenopausal osteoporotic monkeys were divided into two groups (five per group), and fed daily with standard diet alone (PMP0 group) or diet mixed with 1000 mg/kg body weight (BW) of PM powder (PMP1000 group) for 16 months. Every 2 months, the bone mineral density (BMD), bone mineral content (BMC) and bone geometry parameters (cortical area and thickness and periosteal and endosteal circumference) at the distal radius and proximal tibia were determined using peripheral quantitative computed tomography together with plasma and urinary bone markers. Compared with the baseline (month 0) values, the cortical, but not trabecular, BMDs and BMCs and the cortical area and thickness at the metaphysis and diaphysis of the radius and tibia of the PMP0 group continuously decreased during the 16-month study period. In contrast, PMP1000 treatment ameliorated the bone loss mainly at the cortical diaphysis by decreasing bone turnover, as indicated by the lowered plasma bone-specific alkaline phosphatase and osteocalcin levels. Generally, changes in the cortical bone geometry were in the opposite direction to the cortical bone mass after PMP1000 treatment. This study indicated that postmenopausal monkeys continuously lose their cortical bone compartment, and they have a higher possibility for long bone fractures. Oral PMP treatment could improve both the bone quantity (BMC and BMD) and quality (bone geometry).

Fibroblast growth factor (FGF)-21 is a potent endocrine factor that improves insulin resistance and obesity-associated metabolic disorders. However, concomitant activation of peroxisome proliferator-activated receptor-γ by FGF-21 makes bone susceptible to osteopenia and fragility fracture. Since an increase in body weight often induced adaptive change in bone by making it resistant to fracture, it was unclear whether FGF-21 would still induce bone defects in overweight rats. Therefore, the present study aimed to investigate bone microstructure and its mechanical properties in high fat diet (HF)-fed rats treated with 0.1 mg/kg/day FGF-21. Eighteen male rats were divided into two groups to receive either a normal diet or HF for 12 weeks. HF rats were then divided into two subgroups to receive either vehicle or FGF-21 for 4 weeks. The results showed that HF led to obesity, dyslipidemia and insulin resistance, as indicated by hyperinsulinemia with euglycemia. In HF rats, there was an increase in tibial yield displacement (an indicator of ability to be deformed without losing toughness, as determined by 3-point bending) without changes in tibial trabecular volumetric bone mineral density (vBMD) or cortical bone parameters, e.g., cortical thickness and bone area. FGF-21 treatment strongly improved the metabolic parameters and increased insulin sensitivity in HF rats. However, FGF-21-treated HF rats showed lower yield displacement, trabecular vBMD, trabecular bone volume, trabecular thickness, and osteoblast surface compared with vehicle-treated HF rats. These findings suggest that, despite being a potent antagonist of insulin resistance and visceral fat accumulation, FGF-21 is associated with bone defects in HF rats.

New bioactive scaffolds with improved mechanical properties, biocompatibility and providing structural support for bone tissue are being developed for use in the treatment of bone defects. In this study, we have synthesized bioactive scaffolds consisting of biphasic calcium phosphate (BCP) and zirconia-Mullite (2ZrO2·[3Al2O3 ·2 SiO2] (ZAS)) (BCPZAS) combined with polymers matrix of polycaprolactone (PCL)-alginate (Alg)-chitosan (Chi) (Chi/Alg-PCL) (BCPZAS@Chi/Alg-PCL). The composite material scaffolds were prepared by a blending technique. The microstructure, mechanical, bioactivity and in vitro biological properties with different ratios of BCP to ZAS of 1:0, 3:1, 1:1, 1:3 and 0:1 wt% in polymer matrix were analyzed. Microstructure analysis showed a successful incorporation of the BCPZAS particles with an even distribution of them within the polymer matrix. The mechanical properties were found to gradually decrease with increasing the ratio of ZAS particles in the scaffolds. The highest compressive strength was 42.96 ± 1.01MPa for the 3:1 wt% BCP to ZAS mixing. Bioactivity test, the BCPZAS@Chi/Alg-PCL composite could induce apatite formation in simulate body fluid (SBF). In-vitro experiment using UMR-106 osteoblast-like cells on BCPZAS@Chi/Alg-PCL composite scaffold showed that there is cell attachment to the scaffolds with proliferation. These experimental results demonstrate that the BCPZAS@Chi/Alg-PCL composite especially for the BCP:ZAS at 3:1 wt% could be utilized as a scaffold for bone tissue engineering applications.

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)

Chronic high-fat diet (HFD) consumption results in gut dysbiosis, systemic inflammation, obese-insulin resistance, and osteoporosis of the jawbones. The probiotics, prebiotics or synbiotics alleviated gut dysbiosis and the metabolic disturbance in HFD-induced obesity. However, the effects on jawbone properties have not been investigated. This study aimed to investigate the effects of probiotic Lactobacillus paracasei HII01, prebiotic xylooligosaccharide (XOS), and synbiotics on the jawbone properties along with metabolic parameters, gut and systemic inflammation in HFD-fed rats. Forty-eight male Wistar rats were fed with either a HFD or normal diet for 12 weeks. Rats in each group were subdivided into four subgroups to be treated with either vehicle, probiotics, prebiotics, or synbiotics for the additional 12 weeks. Blood samples, gut, bone marrows, and jawbones were collected to determine metabolic parameters, inflammation, and bone properties. The HFD-fed rats developed obese-insulin resistance, as indicated by increased body weight, dyslipidemia and decreased insulin sensitivity. Serum lipopolysaccharide levels and interleukin-6 mRNA expression in the ileum and bone marrows were elevated. Altered bone metabolism and the impaired jawbone properties were evident as indicated by decreased bone mineral density with increased trabecular separation. Reduced ultimate load and stiffness were observed in HFD-fed rats. Treatments with probiotics, prebiotics or synbiotics in HFD-fed rats improved metabolic parameters and reduced inflammation. However, no alterations in jawbone properties were found in all treatments. The osteoporosis of the jawbone occurred in obese-insulin resistance, and treatments with probiotics, prebiotics and synbiotics were not sufficient to improve the jawbone properties.

Cyperenoic acid is a terpenoid isolated from the root of a medicinal plant Croton crassifolius with a wide range of biological activities. In this study, the effects of cyperenoic acid on osteoclast differentiation were investigated both in vitro and in vivo using receptor activator of nuclear factor-κB ligand (RANKL)-induced bone marrow-derived osteoclasts and senescence-accelerated mouse prone 6 (SAMP6). Cyperenoic acid significantly suppressed RANKL-induced osteoclast differentiation at the concentrations with no apparent cytotoxicity. The half maximum inhibitory concentration (IC50) for osteoclast differentiation was 36.69 μM ± 1.02. Cyperenoic acid treatment evidently reduced the expression of two key transcription factors in osteoclast differentiation, NFATc1 and c-Fos. Detailed signaling analysis revealed that cyperenoic acid did not affect MAPK pathways and canonical NF-κB pathway but impaired activation of p100/p52 in the non-canonical NF-κB pathway upon RANKL stimulation. Moreover, the expression of osteoclast-related genes, nfatc1, ctsk, irf8, acp5 and cfos were disrupted by cyperenoic acid treatment. The bone resorption activity by cyperenoic acid-treated osteoclasts were impaired. In a senile osteoporosis mouse model SAMP6, mice fed on diet supplemented with cyperenoic acid showed delay in bone loss, compared to the control. Taken together, plant-derived cyperenoic acid shows great potential as therapeutic agent for osteoporosis.

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.

NaCl-rich rock salt dissolved in natural water source leads to salinity fluctuation that profoundly affects freshwater ecosystem and aquatic fauna. The snakehead (Channa striata) can live in saline water, but the osmoregulatory mechanisms underlying this ability remain unclear. Herein, we found that exposure to salinities ≥ 10‰ NaCl markedly elevated plasma cortisol and glucose levels, and caused muscle dehydration. In a study of time-dependent response after being transferred from fresh water (0‰ NaCl, FW) to salt-dissolved brackish water (10‰ NaCl, SW), FW-SW, cortisol increased rapidly along with elevations of plasma glucose and lactate. Interestingly, plasma cortisol returned to baseline after prolonged exposure, followed by a second peak that probably enhanced the branchial Na(+)/K(+)-ATPase activity. Under SW-FW condition, Na(+)/K(+)-ATPase activity was not altered as compared to SW-adapted fish. In conclusion, salinity change, especially FW-SW, induced a stress response and hence cortisol release in C. striata, which might increase plasma glucose and lactate to energize the branchial Na(+)/K(+)-ATPase.

Several severe stressful situations, e.g., natural disaster, infectious disease out break, and mass casualty, are known to cause anxiety, depression and cognitive impairment, and preventive intervention for these stress complications is worth exploring. We have previously reported that the serotonin-norepinephrine-dopamine reuptake inhibitor, venlafaxine, as well as voluntary wheel running are effective in the treatment of anxiety- and depression-like behaviors in stressed rats. But whether they are able to prevent deleterious consequences of restraint stress in rats, such as anxiety/depression-like behaviors and memory impairment that occur afterward, was not known. Herein, male Wistar rats were pre-treated for 4 weeks with anti-anxiety/anti-depressive drugs, agomelatine and venlafaxine, or voluntary wheel running, followed by 4 weeks of restraint-induced stress. During the stress period, rats received neither drug nor exercise intervention. Our results showed that restraint stress induced mixed anxiety- and depression-like behaviors, and memory impairment as determined by elevated plus-maze, elevated T-maze, open field test (OFT), forced swimming test (FST), and Morris water maze (MWM). Both pharmacological pre-treatments and running successfully prevented the anxiety-like behavior, especially learned fear, in stressed rats. MWM test suggested that agomelatine, venlafaxine, and running could prevent stress-induced memory impairment, but only pharmacological treatments led to better novel object recognition behavior and positive outcome in FST. Moreover, western blot analysis demonstrated that venlafaxine and running exercise upregulated brain-derived neurotrophic factor (BDNF) expression in the hippocampus. In conclusion, agomelatine, venlafaxine as well as voluntary wheel running had beneficial effects, i.e., preventing the restraint stress-induced anxiety/depression-like behaviors and memory impairment.

Both Type 2 diabetes mellitus (T2DM) and estrogen deprivation have been shown to be associated with the development of cardiovascular disease and adverse cardiac remodeling. However, the role of estrogen deprivation on adverse cardiac remodeling in nonobese T2DM rats has not been clearly elucidated. We hypothesized that estrogen-deprivation aggravates adverse cardiac remodeling in Goto–Kakizaki (GK) rats. Wild-type (WT) and GK rats at the age of 9 months old were divided into two subgroups to have either a sham operation (WTS, GKS) or a bilateral ovariectomy (WTO, GKO) (n = 6/subgroup). Four months after the operation, the rats were killed, and the heart was excised rapidly. Metabolic parameters, cardiomyocytes hypertrophy, cardiac fibrosis, and biochemical parameters were determined. GK rats had hyperglycemia with hypoinsulinemia, and estrogen deprivation did not increase the severity of T2DM. Cardiac hypertrophy, cardiac oxidative stress, and phosphor-antinuclear factor κB were higher in WTO and GKS rats than WTS rats, and they markedly increased in GKO rats compared with GKS rats. Furthermore, cardiac fibrosis, transforming growth factor-β, Bax, phosphor-p38, and peroxisome proliferator- activated receptor γ coactivator-1α expression were increased in GKS and GKO rats compared with the lean rats. However, mitochondrial dynamics proteins including dynamin-related protein 1 and mitofusin-2 were not altered by T2DM and estrogen deprivation. Although estrogen deprivation did not aggravate T2DM in GK rats, it increased the severity of cardiac hypertrophy by provoking cardiac inflammation and oxidative stress in nonobese GK rats.

Breastfeeding leads to bone calcium loss for milk production, resulting in progressive maternal osteopenia. Calcium supplement from natural sources has been postulated to be more beneficial to bone health than purified CaCOBoth forms of calcium supplement, i.e. tuna bone powder and CaCOCalcium supplement from tuna bone powder was effective in preventing maternal osteopenia. Tuna bone, which is a readily available fishing industrial waste, is a good alternative source of calcium supplement that increases BMD in both lactating mothers and the neonates. © 2017 Society of Chemical Industry.

Objective Obese insulin resistance and type 2 diabetes mellitus profoundly impair bone mechanical properties and bone quality. However, because several antidiabetes drugs, especially thiazolidinediones, further aggravate bone loss in individuals with diabetes, diabetic osteopathy should not be treated by using simply any glucose-lowering agents. Recently, incretins have been reported to affect osteoblast function positively. The present study aimed to investigate the effects of vildagliptin, an inhibitor of dipeptidyl peptidase-4, on bone of rats with high-fat-diet-induced prediabetes. Methods Male rats were fed a high-fat diet for 12 weeks to induce obese insulin resistance and then treated with vildagliptin for 4 weeks. The effects of the drug on bone were determined by microcomputed tomography and bone histomorphometry. Results Vildagliptin markedly improved insulin resistance in these obese insulin-resistant rats. It also significantly increased volumetric bone mineral density. Specifically, vildagliptin-treated obese insulin-resistant rats exhibited higher trabecular volumetric bone mineral density than vehicle-treated obese insulin-resistant rats, whereas cortical volumetric bone mineral density, cortical thickness and area were not changed. Bone histomorphometric analysis in a trabecular-rich area (i.e. tibial metaphysis) revealed greater trabecular bone volume and number and less trabecular separation without change in trabecular thickness, osteocyte lacunar area or cortical thickness in the vildagliptin-treated group. Conclusions Vildagliptin had a beneficial effect on the bone of obese insulin-resistant rats with prediabetes, particularly at the trabecular site. Such benefit probably results from enhanced bone formation rather than from suppressed bone resorption.

Although both type 2 diabetes mellitus (T2DM) and aging are related with Alzheimer's disease (AD), the effects of aging on the Alzheimer's proteins and the synaptic markers in T2DM have not been investigated. This study, we hypothesized that T2DM rats with advanced-age, aggravates the reduction of synaptic proteins and an increase in the Alzheimer's protein markers. Goto-Kakizaki rats (GK) were used as a T2DM group and wild-type rats (WT) were used as a control group. Rats in each group were categorized by age into young-adult (7 months) and advanced-age rats (12.5 months). Blood was collected in all rats to determine plasma glucose and insulin levels. The brains were used for determining the level of Alzheimer's and synaptic proteins. Our data demonstrated that GK rats had a decreased body weight and increased blood glucose levels, compared to their age-matched WT. p-Tau was increased in both advanced-age WT and GK, compared to their young-adult rats. Moreover, amyloid-beta (Aβ) level was higher in advanced-age GK than their age-matched WT. The synaptic proteins were decreased in advanced-age GK, compared to young-adult GK rats. However, no difference in the level of Alzheimer's proteins and synaptic proteins in the brains of young-adult GK compared to age-matched WT was found. Our data suggested that aging contributes to the pathogenesis of AD and the reduction of synaptic proteins to greater extent in a diabetic than in a healthy condition.

In type 2 diabetes mellitus (T2DM), the decreased bone strength is often associated with hyperglycemia and bone cell insulin resistance. Since T2DM is increasingly reported in young adults, it is not known whether the effect of T2DM on bone would be different in young adolescents and aging adults. Here, we found shorter femoral and tibial lengths in 7-month, but not 13-month, Goto-Kakizaki (GK) T2DM rats as compared to wild-type rats. Bone µCT analysis showed long-lasting impairment of both cortical and trabecular bones in GK rats. Although insulin treatment effectively improved hyperglycemia, it was not able to rescue trabecular BMD and cortical thickness in young adult GK rats. In conclusion, insulin treatment and alleviation of hyperglycemia did not increase BMD of osteopenic GK rats. It is likely that early prevention of insulin resistance should prevail over treatment of full-blown T2DM-related osteopathy.

Phytoestrogen-rich Pueraria mirifica (PM) tuberous extract is a promising candidate for the development of anti-osteoporosis drugs for postmenopausal women, but its action has never been validated in humans or in non-human primates, which are more closely related to humans than rodents. In vitro study of non-human primate osteoblasts is thus fundamental to prepare for in vivo studies of phytoestrogen effects on primate bone. This study aimed to establish a culture system of baboon primary osteoblasts and to investigate the effects of PM extract and its phytoestrogens on these cells. Primary osteoblasts from adult baboon fibulae exhibited osteoblast characteristics in regard to proliferation, differentiation, mineralization, and estrogen receptor expression. They responded to 17β-estradiol by increased proliferation rate and mRNA levels of alkaline phosphatase (ALP), type I collagen, and osteocalcin. After being exposed for 48 h to 100 μg/ml PM extract, 1000 nM genistein, or 1000 nM puerarin, primary baboon osteoblasts markedly increased the rate of proliferation and mRNA levels of ALP and type I collagen without changes in Runx2, osterix, or osteocalcin expression. PM extract, genistein, and puerarin also decreased the RANKL/OPG ratio, suggesting that they could decrease osteoclast-mediated bone resorption. However, neither PM extract nor its phytoestrogens altered calcium deposition in osteoblast culture. In conclusion, we have established baboon primary osteoblast culture, which is a new tool for bone research and drug discovery. Furthermore, the present results provide substantial support for the potential of PM extract and its phytoestrogens to be developed as therapeutic agents against bone fragility.

Type 2 diabetes mellitus (T2DM) often occurs concurrently with high blood cholesterol or dyslipidemia. Although T2DM has been hypothesized to impair bone microstructure, several investigations showed that, when compared to age-matched healthy individuals, T2DM patients had normal or relatively high bone mineral density (BMD). Since cholesterol and lipids profoundly affect the function of osteoblasts and osteoclasts, it might be cholesterol that obscured the changes in BMD and bone microstructure in T2DM. The present study, therefore, aimed to determine bone elongation, epiphyseal histology, and bone microstructure in non-obese T2DM Goto-Kakizaki rats treated with normal (GK-ND) and high cholesterol diet. We found that volumetric BMD was lower in GK-ND rats than the age-matched wild-type controls. In histomorphometric study of tibial metaphysis, T2DM evidently suppressed osteoblast function as indicated by decreases in osteoblast surface, mineral apposition rate, and bone formation rate in GK-ND rats. Meanwhile, the osteoclast surface and eroded surface were increased in GK-ND rats, thus suggesting an activation of bone resorption. T2DM also impaired bone elongation, presumably by retaining the chondrogenic precursor cells in the epiphyseal resting zone. Interestingly, several bone changes in GK rats (e.g., increased osteoclast surface) disappeared after high cholesterol treatment as compared to wild-type rats fed high cholesterol diet. In conclusion, high cholesterol diet was capable of masking the T2DM-induced osteopenia and changes in several histomorphometric parameters that indicated bone microstructural defect. Cholesterol thus explained, in part, why a decrease in BMD was not observed in T2DM, and hence delayed diagnosis of the T2DM-associated bone disease.

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.

In estrogen-deficient rodents with osteopenia, repetitive exposure to mild-to-moderate stress, which mimics the chronic aversive stimuli (CAS) of the modern urban lifestyle in postmenopausal women, has been hypothesized to cause the bone microstructure to further deteriorate. Recently, we have provided evidence in rats that voluntary impact exercise, e.g., wheel running, is as effective as pharmacological treatments for stress-induced anxiety and depression. The present study, therefore, aims to investigate whether a 4-week CAS exposure aggravates trabecular bone loss in ovariectomized (Ovx) rats, and whether CAS-induced bone loss can be rescued by voluntary wheel running. CAS was found to elevate the serum levels of corticosterone, a stress hormone from the adrenal gland. Dual energy X-ray absorptiometry revealed a decrease in bone mineral content (BMC) in the tibiae of CAS-exposed Ovx rats as compared to the CAS-free Ovx rats (control), while having no detectable effect on bone mineral density (BMD). Bone histomorphometric analysis of the proximal tibial metaphysis showed that CAS decreased trabecular bone volume and increased trabecular separation, which were completely restored to the baseline values of Ovx rats by voluntary wheel running. This CAS-induced trabecular bone loss in Ovx rats was probably due to an enhancement of osteoclast-mediated bone resorption, as indicated by increases in osteoclast surface and active erosion surface. Moreover, wheel running as well as non-impact exercise (endurance swimming) effectively increased the tibial BMD and BMC of CAS-exposed Ovx rats. It can be concluded that exercise is an effective intervention in mitigating CAS-induced bone loss in estrogen-deficient rats.

In the present study, composite scaffolds made with different weight ratios (0.5:1, 1:1 and 2:1) of bioactive glass (15Ca:80Si:5P) (BG)/polyvinyl alcohol (PVA) (PVABG) and chitosan (Chi)/collagen (Col) (ChiCol) were prepared by three mechanical freeze–thaw followed by freeze-drying to obtain the porous scaffolds. The mechanical properties and the in vitro biocompatibility of the composite scaffolds to simulated body fluid (SBF) and to rat osteoblast-like UMR-106 cells were investigated. The results from the studies indicated that the porosity and compressive strength were controlled by the weight ratio of PVABG:ChiCol. The highest compressive modulus of the composites made was 214.64 MPa which was for the 1:1 weight ratio PVABG:ChiCol. Mineralization study in SBF showed the formation of apatite crystals on the PVABG:ChiCol surface after 7 days of incubation. In vitro cell availability and proliferation tests confirmed the osteoblast attachment and growth on the PVABG:ChiCol surface. MTT and ALP tests on the 1:1 weight ratio PVABG:ChiCol composite indicated that the UMR-106 cells were viable. Alkaline phosphatase activity was found to increase with increasing culturing time. In addition, we showed the potential of PVABG:ChiCol drug delivery through PBS solution studies. 81.14% of BSA loading had been achieved and controlled release for over four weeks was observed. Our results indicated that the PVABG:ChiCol composites, especially the 1:1 weight ratio composite exhibited significantly improved mechanical, mineral deposition, biological properties and controlled release. This made them potential candidates for bone tissue engineering applications.

Dragon fruit oligosaccharide (DFO) has a prebiotic property which improves gut health by selectively stimulating the colonic microbiota. Altering microbiota composition may affect intestinal motility. However, no study has been done to understand the DFO effects on gut motor functions. This research thus aimed to investigate the DFO effects on mice colons compared to the prebiotic fructo-oligosaccharide (FOS) and probiotic bifidobacteria. The mice in this study received distilled water; 100, 500, and 1000 mg/kg DFO; 1000 mg/kg FOS; or 109 CFU Bifidobacterium animalis daily for 1 week and some treatments for 2 weeks. Gastrointestinal transits were analysed and motility patterns, smooth muscle (SM) contractions and morphological structures of the colons were assessed. Administration of FOS, 500 and 1000 mg/kg DFO significantly increased fecal output when compared to the control group. In mice treated with FOS and bifidobacteria, gut transit time was reduced, while upper gut transit was increased in comparison to DFO groups. Spatiotemporal maps of colonic wall motions showed that DFO increased the number of colonic non-propagation contractions and fecal pellet velocity, consistent with the results from groups treated with FOS and bifidobacteria. DFO also increased the amplitude and duration of colonic SM contractions. Histological stains showed normal epithelia, crypts, goblet cells, and SM thickness in all groups. In conclusion, DFO increased colonic SM contractions without morphological change and acted as a bulk-forming and stimulant laxative to increase fecal output and intestinal motility. Thus, DFO as a dietary supplement may promote gut health and correct gastrointestinal motility disorders.

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.

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.

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.

Rodents exposed to mild but repetitive stress may develop anxiety- and depression-like behaviors. Whether this stress response could be alleviated by pharmacological treatments or exercise interventions, such as wheel running, was unknown. Herein, we determined anxiety- and depression-like behaviors in restraint stressed rats (2h/day, 5 days/week for 4 weeks) subjected to acute diazepam treatment (30min prior to behavioral test), chronic treatment with fluoxetine, reboxetine or venlafaxine (10mg/kg/day for 4 weeks), and/or 4-week voluntary wheel running. In elevated plus-maze (EPM) and forced swimming tests (FST), stressed rats spent less time in the open arms and had less swimming duration than the control rats, respectively, indicating the presence of anxiety- and depression-like behaviors. Stressed rats also developed learned fear as evaluated by elevated T-maze test (ETM). Although wheel running could reduce anxiety-like behaviors in both EPM and ETM, only diazepam was effective in the EPM, while fluoxetine, reboxetine, and venlafaxine were effective in the ETM. Fluoxetine, reboxetine, and wheel running, but not diazepam and venlafaxine, also reduced depression-like behavior in FST. Combined pharmacological treatment and exercise did not further reduce anxiety-like behavior in stressed rats. However, stressed rats treated with wheel running plus reboxetine or venlafaxine showed an increase in climbing duration in FST. In conclusion, regular exercise (voluntary wheel running) and pharmacological treatments, especially fluoxetine and reboxetine, could alleviate anxiety- and depression-like behaviors in stressed 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.

Recent investigation has shown that the liver-derived iron-regulating hormone, hepcidin, can potentiate intestinal calcium absorption in hemizygous β-globin knockout thalassemic (BKO) mice. Since the upregulation of Fe

Na

Both types 1 and 2 diabetes mellitus (T1DM and T2DM) are associated with profound deterioration of calcium and bone metabolism, partly from impaired intestinal calcium absorption, leading to a reduction in calcium uptake into the body. T1DM is associated with low bone mineral density (BMD) and osteoporosis, whereas the skeletal changes in T2DM are variable, ranging from normal to increased and to decreased BMD. However, both types of DM eventually compromise bone quality through production of advanced glycation end products and misalignment of collagen fibrils (so-called matrix failure), thereby culminating in a reduction of bone strength. The underlying cellular mechanisms (cellular failure) are related to suppression of osteoblast-induced bone formation and bone calcium accretion, as well as to enhancement of osteoclast-induced bone resorption. Several other T2DM-related pathophysiological changes, e.g., osteoblast insulin resistance, impaired productions of osteogenic growth factors (particularly insulin-like growth factor 1 and bone morphogenetic proteins), overproduction of pro-inflammatory cytokines, hyperglycemia, and dyslipidemia, also aggravate diabetic osteopathy. In the kidney, DM and the resultant hyperglycemia lead to calciuresis and hypercalciuria in both humans and rodents. Furthermore, DM causes deranged functions of endocrine factors related to mineral metabolism, e.g., parathyroid hormone, 1,25-dihydroxyvitamin D3, and fibroblast growth factor-23. Despite the wealth of information regarding impaired bone remodeling in DM, the long-lasting effects of DM on calcium metabolism in young growing individuals, pregnant women, and neonates born to women with gestational DM have received scant attention, and their underlying mechanisms are almost unknown and worth exploring.

Chronic stress has been reported to decrease bone density and intestinal calcium absorption, but its underlying mechanism remains elusive. Since long-term exposure to glucocorticoids, major stress hormones from adrenal gland, is known to downregulate the mRNA expression of intestinal calcium transporter TRPV6, the present study aimed to demonstrate whether decreases in mRNA expressions of duodenal calcium transporter genes were observed in male rats subjected to restraint stress for 4 weeks. The results from quantitative real-time PCR showed that restraint stress significantly downregulated the mRNA expressions of apical calcium channels (TRPV6 and Ca(v)1.3), cytoplasmic calcium-binding protein (calbindin-D(9k)), and basolateral calcium pump (PMCA(1b)), but not the expression of TRPV5 or NCX1. The mRNA expressions of paracellular genes, ZO-1, occludin, and claudin-3, were not altered by restraint stress. Since several antidepressant or anxiolytic drugs effectively alleviate stress-induced depressive and anxiety symptoms, we further hypothesized that these drugs may also enhance calcium transporter gene expression in stressed rats. As expected, 4-week daily administration of 10 mg/kg fluoxetine, 10 mg/kg reboxetine, or 10 mg/kg venlafaxine differentially increased calcium transporter mRNA expression in stressed rats, whereas 2 mg/kg diazepam had no such effect. It could, therefore, be concluded that 4-week restraint stress downregulated some important calcium transporter mRNA expression in the duodenal epithelial cells of male rats, which could be prevented by oral administration of fluoxetine, reboxetine, and venlafaxine. The present findings may be applied to help alleviate the stress-induced bone loss and osteoporosis by restoring intestinal calcium absorption to provide calcium for bone formation.

Endochondral bone growth in young growing mammals or adult mammals with persistent growth plates progresses from proliferation, maturation and hypertrophy of growth plate chondrocytes to mineralization of cartilaginous matrix to form an osseous tissue. This complex process is tightly regulated by a number of factors with different impacts, such as genetics, endocrine/paracrine factors [e.g., PTHrP, 1,25(OH)(2)D(3), IGF-1, FGFs, and prolactin], and nutritional status (e.g., dietary calcium and vitamin D). Despite a strong link between growth plate function and elongation of the long bone, little is known whether endochondral bone growth indeed determines bone calcium accretion, bone mineral density (BMD), and/or peak bone mass. Since the process ends with cartilaginous matrix calcification, an increase in endochondral bone growth typically leads to more calcium accretion in the primary spongiosa and thus higher BMD. However, in lactating rats with enhanced trabecular bone resorption, bone elongation is inversely correlated with BMD. Although BMD can be increased by factors that enhance endochondral bone growth, the endochondral bone growth itself is unlikely to be an important determinant of peak bone mass since it is strongly determined by genetics. Therefore, endochondral bone growth and bone elongation are associated with calcium accretion only in a particular subregion of the long bone, but do not necessarily predict BMD and peak bone mass.

Despite being potent anxiolytic agents, benzodiazepines (BDZ) sometimes show reduced therapeutic efficacy in stressed rodents. However, the effectiveness of norepinephrine reuptake inhibitors (NRI) and serotonin-norepinephrine reuptake inhibitors (SNRI) or other anxiolytic interventions, e.g., exercise, remained elusive. Here, we demonstrated that male rats subjected to restraint stress for 4 weeks showed decreases in percent open arm time and open arm entry, as determined by elevated plus-maze test (EPM). Increases in inhibitory avoidance trial 2 and outer zone time were also observed in elevated T-maze (ETM) and open field test (OFT), respectively. To evaluate the anxiolytic-like actions of exercise and anxiolytic drugs, stressed rats were subjected for 4 weeks to swimming or daily gavage with 2mg/kg diazepam (BDZ), or 10mg/kg fluoxetine (selective serotonin reuptake inhibitor), reboxetine (NRI), or venlafaxine (SNRI). In EPM, the open arm activity was higher in the swimming, reboxetine-treated and venlafaxine-treated groups as compared to age-matched controls, while diazepam and fluoxetine were without effect. In ETM, a reduction in avoidance latency was observed only in swimming and venlafaxine-treated groups. However, the combined swimming and pharmacological treatment showed no additive anxiolytic-like effect. It could be concluded that restraint stress induced anxiety-like behaviors, which were not responsive to diazepam or fluoxetine, whereas reboxetine, venlafaxine and swimming showed anxiolytic-like actions in stressed rats.

An earlier study reported that cecal calcium absorption contributes less than 10% of total calcium absorbed by the intestine, although the cecum has the highest calcium transport rate compared with other intestinal segments. Thus, the physiological significance of the cecum pertaining to body calcium metabolism remains elusive. Herein, a 4-wk calcium balance study in cecectomized rats revealed an increase in fecal calcium loss with marked decreases in fractional calcium absorption and urinary calcium excretion only in the early days post-operation, suggesting the presence of a compensatory mechanism to minimize intestinal calcium wasting. Further investigation in cecectomized rats showed that active calcium transport was enhanced in the proximal colon but not in the small intestine, whereas passive calcium transport along the whole intestine was unaltered. Since apical exposure to calcium-sensing receptor (CaSR) agonists similarly increased proximal colonic calcium transport, activation of apical CaSR in colonic epithelial cells could have been involved in this hyperabsorption. Calcium transporter genes, i.e., TRPV6 and calbindin-D9k, were also upregulated in proximal colonic epithelial cells. Surprisingly, elevated serum parathyroid hormone levels and hyperphosphatemia were evident in cecectomized rats despite normal plasma calcium levels, suggesting that colonic compensation alone might be insufficient to maintain normocalcemia. Thus, massive bone loss occurred in both cortical and trabecular sites, including lumbar vertebrae, femora, and tibiae. The presence of compensatory colonic calcium hyperabsorption with pervasive osteopenia in cecectomized rats therefore corroborates that the cecum is extremely crucial for body calcium homeostasis.