Kannikar Wongdee

Osteoporosis in diabetes mellitus: Possible cellular and molecular mechanisms

Osteoporosis, a global age-related health problem in both male and female elderly, insidiously deteriorates the microstructure of bone, particularly at trabecular sites, such as vertebrae, ribs and hips, culminating in fragility fractures, pain and disability. Although osteoporosis is normally associated with senescence and estrogen deficiency, diabetes mellitus (DM), especially type 1 DM, also contributes to and/or aggravates bone loss in osteoporotic patients. This topic highlight article focuses on DM-induced osteoporosis and DM/osteoporosis comorbidity, covering alterations in bone metabolism as well as factors regulating bone growth under diabetic conditions including, insulin, insulin-like growth factor-1 and angiogenesis. Cellular and molecular mechanisms of DM-related bone loss are also discussed. This information provides a foundation for the better understanding of diabetic complications and for development of early screening and prevention of osteoporosis in diabetic patients.

Is prolactin the cardinal calciotropic maternal hormone

To produce offspring, mothers require a large amount of calcium for fetal growth and milk production. Increased calcium demand leads to enhanced intestinal calcium absorption and stockpiling of bone calcium in pregnancy prior to demineralization in lactation. These coordinated events must be carefully organized by calciotropic hormone(s), but the classical hormones, namely 1,25-dihydroxyvitamin D(3), parathyroid hormone and calcitonin, do not appear to be responsible. Plasma prolactin (PRL) levels are elevated during pregnancy and, in view of the presence of PRL receptors in gut, bone and mammary glands, as well as recent evidence of the stimulatory effects of PRL on intestinal calcium transport, bone resorption and mammary calcium secretion, we postulate that PRL is the cardinal calciotropic hormone during pregnancy and lactation.

Upregulation of osteoblastic differentiation marker mRNA expression in osteoblast-like UMR106 cells by puerarin and phytoestrogens from Pueraria mirifica

Phytoestrogens have attracted attention for their potential in the prevention of postmenopausal osteoporosis. Recently, phytoestrogen-rich herb Pueraria mirifica has been demonstrated to possess an osteogenic effect on bone in ovariectomized rats, but its underlying cellular mechanism was not known. Here, we investigated the effects of P. mirifica extract and its major isoflavone compound, puerarin, on cell viability, cell proliferation and the expression of differentiation markers in rat osteoblast-like UMR106 cells. After exposure to 17β-estradiol (E2), genistein, P. mirifica extract and puerarin, proliferation but not viability of UMR106 cells was markedly decreased. Quantitative real-time PCR revealed that P. mirifica extract and puerarin significantly increased the mRNA expression of alkaline phosphatase (ALP) and osteoprotegerin, but not Runx2, osterix or osteocalcin. Puerarin also decreased the mRNA expression of receptor activator of nuclear factor-κB ligand, an osteoclastogenic factor, suggesting that it could induce bone gain by enhancing osteoblast differentiation and suppressing osteoclast function. Furthermore, after an exposure to high affinity estrogen receptor (ER) antagonist (ICI182780), the E2-, genistein-, P. mirifica extract- and puerarin-induced upregulation of ALP expressions were completely abolished. It could be concluded that P. mirifica extract and puerarin induced osteoblast differentiation rather than osteoblast proliferation in an ER-dependent manner. The present findings, therefore, corroborated the potential benefit of P. mirifica extract and puerarin in the prevention and treatment of postmenopausal osteoporosis.

Bone modeling in bromocriptine-treated pregnant and lactating rats: possible osteoregulatory role of prolactin in lactation

The lactogenic hormone prolactin (PRL) directly regulates osteoblast functions in vitro and modulates bone remodeling in nulliparous rats, but its osteoregulatory roles in pregnant and lactating rats with physiological hyperprolactinemia remained unclear. Herein, bone changes were investigated in rats treated with bromocriptine (Bromo), an inhibitor of pituitary PRL release, or Bromo+PRL at different reproductive phases, from mid-pregnancy to late lactation. PRL receptors were strongly expressed in osteoblasts lining bone trabeculae, indicating bone as a target of PRL actions. By using dual energy X-ray absorptiometry, we found a significant increase in bone mineral density in the femora and vertebrae of pregnant rats. Such pregnancy-induced bone gain was, however, PRL independent and may have resulted from the increased cortical thickness. Bone trabeculae were modestly changed during pregnancy as evaluated by bone histomorphometry. On the other hand, lactating rats, especially in late lactation, showed massive bone loss in bone trabeculae but not in cortical shells. Further study in Bromo- and Bromo+PRL-treated rats suggested that PRL contributed to decreases in trabecular bone volume and number and increases in trabecular separation and eroded surface, as well as a paradoxical increase in bone formation rate in late lactation. Uncoupling of trabecular bone formation and resorption was evident in lactating rats, with the latter being predominant. In conclusion, pregnancy mainly induced cortical bone gain, whereas lactation led to trabecular bone loss in both long bones and vertebrae. Although PRL was not responsible for the pregnancy-induced bone gain, it was an important regulator of bone modeling during lactation.

Endurance swimming stimulates transepithelial calcium transport and alters the expression of genes related to calcium absorption in the intestine of rats

Endurance impact exercise, e.g., running, is known to enhance the intestinal calcium absorption. However, nonimpact exercise, e.g., swimming, is more appropriate for osteoporotic patients with cardiovascular diseases or disorders of bone and joint, but the effect of swimming on the intestinal calcium transport was unknown. This study, therefore, aimed to investigate the transepithelial calcium transport and the expression of related genes in the intestine of rats trained to swim nonstop 1 h/day, 5 days/wk for 2 wk. We found that endurance swimming stimulated calcium transport in the duodenum, proximal jejunum, and cecum, while decreasing that in the proximal colon. Swimming affected neither the transepithelial potential difference nor resistance. As demonstrated by real-time PCR, the small intestine, especially the duodenum, responded to swimming by upregulating a number of genes related to the transcellular calcium transport, i.e., TRPV5, TRPV6, calbindin-D9k, PMCA1b, and NCX1, and the paracellular calcium transport, i.e., ZO-1, ZO-2, ZO-3, cingulin, occludin, and claudins, as well as nuclear receptor of 1,25(OH)2D3. In contrast, swimming downregulated those genes in the colon. Microarray analysis showed that swimming also altered the expression of duodenal genes related to the transport of several ions and nutrients, e.g., Na+, K+, Cl-, glucose, and amino acids. In conclusion, endurance swimming enhanced intestinal calcium absorption, in part, by upregulating the calcium transporter genes. The present microarray study also provided relevant information for further investigations into the intestinal nutrient and electrolyte transport during nonimpact exercise.

Fibroblast growth factor-23 abolishes 1,25-dihydroxyvitamin D3-enhanced duodenal calcium transport in male mice

Despite being widely recognized as the important bone-derived phosphaturic hormone, whether fibroblast growth factor (FGF)-23 modulated intestinal calcium absorption remained elusive. Since FGF-23 could reduce the circulating level of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], FGF-23 probably compromised the 1,25(OH)₂D₃-induced intestinal calcium absorption. FGF-23 may also exert an inhibitory action directly through FGF receptors (FGFR) in the intestinal cells. Herein, we demonstrated by Ussing chamber technique that male mice administered 1 μg/kg 1,25(OH)₂D₃ sc daily for 3 days exhibited increased duodenal calcium absorption, which was abolished by concurrent intravenous injection of recombinant mouse FGF-23. This FGF-23 administration had no effect on the background epithelial electrical properties, i.e., short-circuit current, transepithelial potential difference, and resistance. Immunohistochemical evidence of protein expressions of FGFR isoforms 1-4 in mouse duodenal epithelial cells suggested a possible direct effect of FGF-23 on the intestine. This was supported by the findings that FGF-23 directly added to the serosal compartment of the Ussing chamber and completely abolished the 1,25(OH)₂D₃-induced calcium absorption in the duodenal tissues taken from the 1,25(OH)₂D₃-treated mice. However, direct FGF-23 exposure did not decrease the duodenal calcium absorption without 1,25(OH)₂D₃ preinjection. The observed FGF-23 action was mediated by MAPK/ERK, p38 MAPK, and PKC. Quantitative real-time PCR further showed that FGF-23 diminished the 1,25(OH)₂D₃-induced upregulation of TRPV5, TRPV6, and calbindin-D(9k), but not PMCA(1b) expression in the duodenal epithelial cells. In conclusion, besides being a phosphatonin, FGF-23 was shown to be a novel calcium-regulating hormone that acted directly on the mouse intestine, thereby compromising the 1,25(OH)₂D₃-induced calcium absorption.

Transcriptome responses of duodenal epithelial cells to prolactin in pituitary-grafted rats.

Chronic prolactin (PRL) exposure can affect several functions of duodenal epithelia, especially those associated with fluid and electrolyte transport. However, little is known regarding its molecular mechanism. To identify PRL-regulated genes, microarray analysis was performed on RNA samples from duodenal epithelial cells of anterior pituitary (AP)-grafted hyperprolactinemic rats. Herein, we identified 321 transcripts upregulated and 241 transcripts downregulated after 4 weeks of AP transplantation. Results from real-time PCR analyses of 15 selected genes were consistent with the microarray results. Gene ontology analysis demonstrated pleiotropic effects of PRL on several cellular processes, including cellular metabolic process, cell communication and cell adhesion. Interestingly, 17 upregulated transcripts and 12 downregulated transcripts are involved in the transport of ions and nutrients, e.g., Ca(2+), Na(+), K(+), Cl(-) and glucose, thus agreeing with the established action of PRL on electrolyte homeostasis. The present results provided fundamental information for further investigations on mechanism of PRL actions in the intestine.

Update on type 2 diabetes-related osteoporosis

It was previously understood that body weight gain and obesity observed in type 2 diabetes mellitus (T2DM) could be beneficial since body weight increase elevated bone mineral density and thus helped maintain the skeletal framework. However, a number of recent findings in humans and rodents have revealed that T2DM is not only associated with trabecular defects but also increases cortical porosity, and compromised bone cell function and bone mechanical properties. Hyperglycemia and insulin resistance in T2DM may further induce osteoblast apoptosis and uncoupling bone turnover. Prolonged accumulation of advanced glycation end products and diminished activity of lysyl oxidase, an essential enzyme for collagen cross-link, can lead to structural abnormalities of bone collagen fibrils, brittle matrix, and fragility fractures. Our studies in T2DM rats showed that dyslipidemia, which often occurs in T2DM, could obscure the T2DM-associated changes in bone microstructure and osteopenia. Longitudinal bone growth regulated by the growth plate chondrocytes is also impaired by T2DM since differentiation of growth plate chondrocytes is arrested and retained in the resting state while only a small number of cells undergo hypertrophic differentiation. Such a delayed chondrocyte differentiation may have also resulted from premature apoptosis of the growth plate chondrocytes. Nevertheless, the underlying cellular and molecular mechanisms of insulin resistance in osteoblasts, osteoclasts, osteocytes, and growth plate chondrocytes remain to be investigated.

Regulation of epithelial calcium transport by prolactin: From fish to mammals

Among the reported ∼300 biological actions, the established role of prolactin (PRL) is to act as a vertebrate hypercalcemic hormone that regulates epithelial calcium transport in several organs, such as the gills, intestine, and kidney. In fish, PRL stimulates the branchial calcium transport by increasing the activity of Ca(2+)-ATPase. Although this calciotropic hormone also induces hypercalcemia in amphibians, reptiles and birds, little has been known regarding the underlying mechanism. In contrast, the effects of PRL on the epithelial calcium transport in mammals are well documented. In rodents, PRL has been shown to stimulate the renal tubular calcium reabsorption and intestinal calcium absorption, the latter of which is mediated by the PRL-induced upregulation of calcium transporter gene expression and activities. Recently, we demonstrated that the duodenal calcium absorption in lactating rats was markedly enhanced by the suckling-induced PRL surge, presumably to provide calcium for milk production. The cellular and molecular mechanisms of the PRL-stimulated calcium transport in mammals have been elaborated in this review.

Endochondral bone growth, bone calcium accretion, and bone mineral density: how are they related?

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)2D3, 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.