Sunlight exposure is just one of the factors which influence Vitamin D status

© The Royal Society of Chemistry and Owner Societies. Studies on the determinants of vitamin D status have tended to concentrate on input-exposure to ultraviolet B radiation and the limited sources in food. Yet, vitamin D status, determined by circulating concentrations of 25-hydroxyvitamin D (25(OH)D), can vary quite markedly in groups of people with apparently similar inputs of vitamin D. There are small effects of polymorphisms in the genes for key proteins involved in vitamin D production and metabolism, including 7-dehydrocholesterol reductase, which converts 7-dehydrocholesterol, the precursor of vitamin D, to cholesterol, CYP2R1, the main 25-hydroxylase of vitamin D, GC, coding for the vitamin D binding protein which transports 25(OH)D and other metabolites in blood and CYP24A1, which 24-hydroxylates both 25(OH)D and the hormone, 1,25-dihydroxyvitamin D. 25(OH)D has a highly variable half-life in blood. There is evidence that the half-life of 25(OH)D is affected by calcium intake and some therapeutic agents. Fat tissue seems to serve as a sink for the parent vitamin D, which is released mainly when there are reductions in adiposity. Some evidence is presented to support the proposal that skeletal muscle provides a substantial site of sequestration of 25(OH)D, protecting this metabolite from degradation by the liver, which may help to explain why exercise, not just outdoors, is usually associated with better vitamin D status

1,25-Dihydroxycholecalciferol (calcitriol) modifies uptake and release of 25-hydroxycholecalciferol in skeletal muscle cells in culture

© 2017 Elsevier Ltd The major circulating metabolite of vitamin D 3 , 25-hydroxycholecalciferol [25(OH)D], has a remarkably long half-life in blood for a (seco)steroid. Data from our studies and others are consistent with the hypothesis that there is a role for skeletal muscle in the maintenance of vitamin D status. Muscle cells internalise vitamin D-binding protein (DBP) from the circulation by means of a megalin/cubilin plasma membrane transport mechanism. The internalised DBP molecules then bind to actin and thus provide an intracellular array of high affinity binding sites for its specific ligand, 25(OH)D. There is evidence that the residence time for DBP in muscle cells is short and that it undergoes proteolytic degradation, releasing bound 25(OH)D. The processes of internalisation of DBP and its intracellular residence time, bound to actin, appear to be regulated. To explore whether 1,25-dihydroxycholecalciferol (calcitriol) has any effect on this process, cell cultures of myotubes and primary skeletal muscle fibers were incubated in a medium containing 10 −10 M calcitriol but with no added DBP. After 3 h pre-incubation with calcitriol, the net uptake of 25(OH)D by these calcitriol-treated cells over a further 4 h was significantly greater than that in vehicle-treated control cells. This was accompanied by a significant increase in intracellular DBP protein. However, after 16 h of pre-incubation with calcitriol, the muscle cells showed a significantly depressed ability to accumulate 25(OH)D compared to control cells over a further 4 or 16 hours. These effects of pre-incubation with calcitriol were abolished in fibers from VDR-knockout mice. The effect was also abolished by the addition of 4,4\u27-diisothiocyano-2,2\u27-stilbenedisulfonic acid (DIDS), which inhibits chloride channel opening. Incubation of C2 myotubes with calcitriol also significantly reduced retention of previously accumulated 25(OH)D after 4 or 8 h. It is concluded from these in vitro studies that calcitriol can modify the DBP-dependent uptake and release of 25(OH)D by skeletal muscle cells in a manner that suggests some inducible change in the function of these cells

The effect of parathyroid hormone on the uptake and retention of 25-hydroxyvitamin D in skeletal muscle cells

© 2017 Elsevier Ltd Data from our studies, and those of others, support the proposal that there is a role for skeletal muscle in the maintenance of vitamin D status. We demonstrated that skeletal muscle is able to internalise extracellular vitamin D binding protein, which then binds to actin in the cytoplasm, to provide high affinity binding sites which accumulate 25-hydroxyvitamin D3 (25(OH)D3) [1]. This study investigated the concentration- and time-dependent effects of parathyroid hormone (PTH) on the capacity of muscle cells to take up and release 3H-25(OH)D3. Uptake and retention studies for 3H-25(OH)D3 were carried out with C2C12 cells differentiated into myotubes and with primary mouse muscle fibers as described [1]. The presence of PTH receptors on mouse muscle fibers was demonstrated by immunohistochemistry and PTH receptors were detected in differentiated myotubes, but not myoblasts, and on muscle fibers by Western blot. Addition of low concentrations of vitamin D binding protein to the incubation media did not alter uptake of 25(OH)D3. Pre-incubation of C2 myotubes or primary mouse muscle fibers with PTH (0.1 to 100 pM) for 3 h resulted in a concentration-dependent decrease in 25(OH)D3 uptake after 4 or 16 h. These effects were significant at 0.1 or 1 pM PTH (p \u3c 0.001) and plateaued at 10 pM, with 25(OH)D3 uptake reduced by over 60% (p \u3c 0.001) in both cell types. In C2 myotubes, retention of 25(OH)D3 was decreased after addition of PTH (0.1 to 100 pM) in a concentration-dependent manner by up to 80% (p \u3c 0.001) compared to non-PTH treated-C2 myotubes. These data show that muscle uptake and retention of 25(OH)D3 are modulated by PTH, a physiological regulator of mineral homeostasis, but the cell culture model may not be a comprehensive reflection of vitamin D homeostatic mechanisms in whole animals

Role of transglutaminase 2 in PAC1 receptor mediated protection against hypoxia-induced cell death and neurite outgrowth in differentiating N2a neuroblastoma cells

The PAC1 receptor and tissue transglutaminase (TG2) play important roles in neurite outgrowth and modulation of neuronal cell survival. In this study, we investigated the regulation of TG2 activity by the PAC1 receptor in retinoic acid-induced differentiating N2a neuroblastoma cells. TG2 transamidase activity was determined using an amine incorporation and a peptide cross linking assay. In situ TG2 activity was assessed by visualising the incorporation of biotin-X-cadaverine using confocal microscopy. TG2 phosphorylation was monitored via immunoprecipitation and Western blotting. The role of TG2 in PAC1 receptor-induced cytoprotection and neurite outgrowth was investigated by monitoring hypoxia-induced cell death and appearance of axonal-like processes, respectively. The amine incorporation and protein crosslinking activity of TG2 increased in a time and concentration-dependent manner following stimulation with pituitary adenylate cyclase-activating polypeptide-27 (PACAP-27). PACAP-27 mediated increases in TG2 activity were abolished by the TG2 inhibitors Z-DON and R283 and by pharmacological inhibition of protein kinase A (KT 5720 and Rp-cAMPs), protein kinase C (Ro 31-8220), MEK1/2 (PD 98059), and removal of extracellular Ca2+. Fluorescence microscopy demonstrated PACAP-27 induced in situ TG2 activity. TG2 inhibition blocked PACAP-27 induced attenuation of hypoxia-induced cell death and outgrowth of axon-like processes. TG2 activation and cytoprotection were also observed in human SH-SY5Y cells. Together, these results demonstrate that TG2 activity was stimulated downstream of the PAC1 receptor via a multi protein kinase dependent pathway. Furthermore, PAC1 receptor-induced cytoprotection and neurite outgrowth are dependent upon TG2. These results highlight the importance of TG2 in the cellular functions of the PAC1 receptor

Rescue of High Glucose Impairment of Cultured Human Osteoblasts Using Cinacalcet and Parathyroid Hormone

Patients with type 2 diabetes mellitus (T2DM) experience a higher risk of fractures despite paradoxically exhibiting normal to high bone mineral density (BMD). This has drawn into question the applicability to T2DM of conventional fracture reduction treatments that aim to retain BMD. In a primary human osteoblast culture system, high glucose levels (25 mM) impaired cell proliferation and matrix mineralization compared to physiological glucose levels (5 mM). Treatment with parathyroid hormone (PTH, 10 nM), a bone anabolic agent, and cinacalcet (CN, 1 µM), a calcimimetic able to target the Ca2+-sensing receptor (CaSR), were tested for their effects on proliferation and differentiation. Strikingly, CN+PTH co-treatment was shown to promote cell growth and matrix mineralization under both physiological and high glucose conditions. CN+PTH reduced apoptosis by 0.9-fold/0.4-fold as measured by Caspase-3 activity assay, increased alkaline phosphatase (ALP) expression by 1.5-fold/twofold, increased the ratio of nuclear factor κ-B ligand (RANKL) to osteoprotegerin (OPG) by 2.1-fold/1.6-fold, and increased CaSR expression by 1.7-fold/4.6-fold (physiological glucose/high glucose). Collectively, these findings indicate a potential for CN+PTH combination therapy as a method to ameliorate the negative impact of chronic high blood glucose on bone remodeling

Vitamin D and Death by Sunshine

Exposure to sunlight is the major cause of skin cancer. Ultraviolet radiation (UV) from the sun causes damage to DNA by direct absorption and can cause skin cell death. UV also causes production of reactive oxygen species that may interact with DNA to indirectly cause oxidative DNA damage. UV increases accumulation of p53 in skin cells, which upregulates repair genes but promotes death of irreparably damaged cells. A benefit of sunlight is vitamin D, which is formed following exposure of 7-dehydrocholesterol in skin cells to UV. The relatively inert vitamin D is metabolized to various biologically active compounds, including 1,25-dihydroxyvitamin D3. Therapeutic use of vitamin D compounds has proven beneficial in several cancer types, but more recently these compounds have been shown to prevent UV-induced cell death and DNA damage in human skin cells. Here, we discuss the effects of vitamin D compounds in skin cells that have been exposed to UV. Specifically, we examine the various signaling pathways involved in the vitamin D-induced protection of skin cells from UV

Sex Differences in Photoprotective Responses to 1,25-Dihydroxyvitamin D3 in Mice Are Modulated by the Estrogen Receptor-β

Susceptibility to photoimmune suppression and photocarcinogenesis is greater in male than in female humans and mice and is exacerbated in female estrogen receptor-beta knockout (ER-β−/−) mice. We previously reported that the active vitamin D hormone, 1,25-dihydroxyvitamin D3 (1,25(OH)2D), applied topically protects against the ultraviolet radiation (UV) induction of cutaneous cyclobutane pyrimidine dimers (CPDs) and the suppression of contact hypersensitivity (CHS) in female mice. Here, we compare these responses in female versus male Skh:hr1 mice, in ER-β−/−/−− versus wild-type C57BL/6 mice, and in female ER-blockaded Skh:hr1 mice. The induction of CPDs was significantly greater in male than female Skh:hr1 mice and was more effectively reduced by 1,25(OH)2D in female Skh:hr1 and C57BL/6 mice than in male Skh:hr1 or ER-β−/− mice, respectively. This correlated with the reduced sunburn inflammation due to 1,25(OH)2D in female but not male Skh:hr1 mice. Furthermore, although 1,25(OH)2D alone dose-dependently suppressed basal CHS responses in male Skh:hr1 and ER-β−/− mice, UV-induced immunosuppression was universally observed. In female Skh:hr1 and C57BL/6 mice, the immunosuppression was decreased by 1,25(OH)2D dose-dependently, but not in male Skh:hr1, ER-β−/−, or ER-blockaded mice. These results reveal a sex bias in genetic, inflammatory, and immune photoprotection by 1,25(OH)2D favoring female mice that is dependent on the presence of ER-β

Distinct Effects of a High Fat Diet on Bone in Skeletally Mature and Developing Male C57BL/6J Mice

Increased risks of skeletal fractures are common in patients with impaired glucose handling and type 2 diabetes mellitus (T2DM). The pathogenesis of skeletal fragility in these patients remains ill-defined as patients present with normal to high bone mineral density. With increasing cases of glucose intolerance and T2DM it is imperative that we develop an accurate rodent model for further investigation. We hypothesized that a high fat diet (60%) administered to developing male C57BL/6J mice that had not reached skeletal maturity would over represent bone microarchitectural implications, and that skeletally mature mice would better represent adult-onset glucose intolerance and the pre-diabetes phenotype. Two groups of developing (8 week) and mature (12 week) male C57BL/6J mice were placed onto either a normal chow (NC) or high fat diet (HFD) for 10 weeks. Oral glucose tolerance tests were performed throughout the study period. Long bones were excised and analysed for ex vivo biomechanical testing, micro-computed tomography, 2D histomorphometry and gene/protein expression analyses. The HFD increased fasting blood glucose and significantly reduced glucose tolerance in both age groups by week 7 of the diets. The HFD reduced biomechanical strength, both cortical and trabecular indices in the developing mice, but only affected cortical outcomes in the mature mice. Similar results were reflected in the 2D histomorphometry. Tibial gene expression revealed decreased bone formation in the HFD mice of both age groups, i.e., decreased osteocalcin expression and increased sclerostin RNA expression. In the mature mice only, while the HFD led to a non-significant reduction in runt-related transcription factor 2 (Runx2) RNA expression, this decrease became significant at the protein level in the femora. Our mature HFD mouse model more accurately represents late-onset impaired glucose tolerance/pre-T2DM cases in humans and can be used to uncover potential insights into reduced bone formation as a mechanism of skeletal fragility in these patients