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

https://doi.org/10.1016/j.jsbmb.2017.10.018Get rights and content

Highlights

  • Skeletal muscle cells accumulate tritiated 25(OH)D in a time dependent manner.

  • Three hour pre-incubation with calcitriol increased net uptake of trititated 25(OH)D and intracellular DBP concentrations.

  • Sixteen hour pre-incubation with calcitriol reduced net uptake of trititated 25(OH)D, with unchanged intracellular DBP concentrations.

  • The effects of calcitriol pre-incubation were abolished by DIDS and in muscle fibers from VDR knockout mice.

  • Calcitriol modulates net uptake and retention of 25(OH)D by skeletal muscle.

Abstract

The major circulating metabolite of vitamin D3, 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'-diisothiocyano-2,2'-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.

Introduction

Our knowledge about the role of vitamin D continues to expand beyond its classical roles in calcium and phosphate homeostasis. The concentration of 25-hydroxycholecalciferol (25(OH)D) in blood is usually taken as an index of vitamin D status. However, it gives no indication of the total body pool size of 25(OH)D.

There is now evidence that skeletal muscle cells contain a mobile pool of 25(OH)D which accumulates from and returns to the extracellular environment. We have previously shown in cultures of mature muscle cells that 25(OH)D, is taken up and retained in the cells by binding to vitamin D binding protein (DBP), which had been internalized via membrane megalin and then binds to actin in the cytoplasm [1]. We postulated that if the capacity to hold 25(OH)D out of the circulation in skeletal muscle were high, when vitamin D status was falling in winter, it would be protected from wasteful uptake and destruction in the liver. This would increase the residence time of circulating 25(OH)D and thus would maintain adequate status during the months when vitamin D supply was low. If this muscle retention mechanism for 25(OH)D were important in increasing its half-life in blood, the process may be regulated by calciotropic hormones, such as parathyroid hormone and 1,25-dihydroxyvitamin D3 (calcitriol). We have shown that net muscle uptake or release of 25(OH)D is indeed modulated by parathyroid hormone [2] and have reported a short term effect of calcitriol on muscle uptake of 25(OH)D over short incubation periods [3].

In the current study, we tested whether longer incubations with calcitriol would have similar or different effects on 25(OH)D uptake or release. 4,4′-Diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) was used as an inhibitor of chloride channel opening, which has been used to investigate non-genomic actions of calcitriol [4].

Section snippets

Materials

Reagents were purchased from Sigma Aldrich (MO, USA) unless otherwise indicated. Calcitriol was purchased from Cayman, USA, dissolved in spectroscopic grade ethanol and then diluted in the differentiation medium to concentrations of 10−10, 10−9, and 10−8M. 4,4′-Diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) was purchased from Sigma Aldrich (MO, USA) and dissolved in dimethyl sulfoxide (DMSO). The vitamin D receptor antibody, clone D6, used for immunohistochemistry and western blots and the

Immunohistochemistry: VDR detection in different cell types

Immunofluorescent studies of freshly isolated FDB myofibers showed immunoreactivity to the VDR (D6) antibody (Fig. 1A). Isotype staining was negligible for all cell types (Figs. 1b,d,f,h,). We previously reported that VDR staining was not detected in muscle fibers from VDR −/- mice [3]. VDR staining was detected throughout differentiated C2 myotubes (Fig. 1c). Normal skin fibroblasts showed dense staining distributed along the cell and in the perinuclear regions (Fig. 1e) but no staining was

Discussion

This study examined the concentration- and time-dependent effects of calcitriol on the capacity of muscle cells to take up and release 25(OH)D. We have previously reported that when C2 cells were differentiated into myotubes, the time-dependent uptake of labelled-25(OH)D is 2–3 times higher than in undifferentiated myoblasts and osteoblasts. Our previous experiments also showed that C2 myotubes released only 32% of the previously accumulated 25(OH)D after 4 h compared to 60% for osteoblasts [1].

References (12)

There are more references available in the full text version of this article.

Cited by (0)

View full text