Regulation of Steroid Hormone Metabolism Requires L-Ascorbic Acid R. GORALCZYK, U. K. MOSER, U. MATTER, AND H. WEISER F . Hoffmann-La Roche Ltd. Basel, Switzerland
INTRODUCTION The high concentration of L-ascorbic acid (L-AA) in the cortex of the adrenal glands implies that vitamin C plays a critical role in intracellular functions, perhaps by participating in the hydroxylation reactions of the cortisol synthesis pathway. L-AA is also necessary for bone formation because it acts as a cofactor in the hydroxylation steps of collagen synthesis. Furthermore, L-AA has been shown to interact with la- and 24-hydroxylation steps of 25(OH)D3 in the kidney, that is, the biosynthesis of another steroid hormone, and to improve 1,25(OH),D3dependent bone parameters. We were interested in the influence of L-AA on tissuespecific steroid metabolism of both the adrenal glands and bone, and investigated
Cortlsol
nmol/lx60mln 200
Number of ACTH-etlmulatlone 176
0
1
m
2
9
150
126
loo 76
60
26
0
0
4
100 20 L-AA conc. In modlum (JIM)
500
FIGURE 1. Dose-dependent stimulation of repetitive cortisol release by L-AA. 349
ANNALS NEW YORK ACADEMY OF SCIENCES
350
(a) the effect of L-AA on the cortisol release in ACTH-stimulated adrenocortical cells, and (b) the effect of L-AA on the osteoblastic secretion of osteocalcin (OC), a noncollagenous bone protein, which is directly dependent on the presence of the active vitamin D metabolite 1 ,25(OH),D3.
METHODS Adrenocortical Cell Culture and Cortisol Determination Poscine adrenocortical cells were prepared from adrenal glands and collected from the slaughterhouse. After removing the adrenal medulla, the cortex was
%
25(OH)D, conc.
stlmulatlon 0
Kq
10‘M
10’ M
80
70 60 50
40
30
0
25
50 100 L-AA conc. In medium (JIM)
200
FIGURE 2. Osteocalcin secretion after three days of stimulation with L-AAand 25(OH)D,.
minced and digested with collagenase. Cells were collected by centrifugation, seeded in fibronectin-coated wells, and incubated in Dulbecco’s Modified Eagle Medium (D’MEM), 10% FCS, containing L-AA at concentrations indicated in Results. At 48, 72, and 96 h postseeding, cells were stimulated by 0.1 nM (first stimulation) or 10 nM (second and third stimulation) ACTH for the times indicated. Between each stimulation step, medium was collected, cells were washed three times, and L-AA incubation was repeated. Cortisol was determined in cell supernatant by RIA.
GORALCZYK et al.: STEROID HORMONE METABOLISM
351
Bone Cell Culture and OC Determination
Primary rat osteoblasts were established by fractionated dissociation of 20day-old fetal rat calvaria with collagenaseIhyaluronidase. Cells of fraction three were plated at a density of lo4 cells/cm2and cultured in D'MEM, 10% FCS, and 10 mM P-glycerophosphate for three weeks prior to the experiment. The cells were stimulated on three consecutive days with L-AA, in combination with 1,25(OH),D3 or 25(OH)D,. OC secreted into the medium within 24 h after the last stimulation was determined by RIA. RESULTS AND CONCLUSIONS
In adrenocortical cells, multiple stimulations with ACTH led to a decreased release of cortisol. This decrease was fully restored by the addition of 4 pM L-AA, whereas 20 to 100 pM L-AA induced even more elevated cortisol levels after ACTH stimulation (FIG.1). In bone cells, L-AA alone induced secretion of OC, a bone cell product that is transcriptionally regulated by 1,25(OH)&. In addition, L-AA enhanced the effect of 1,25(OH),D3and 25(OH)D3on OC secretion (FIG.2 ) . The nature of these mechanisms is not known at present. L-AA may be directly involved in posttranslational modifications or secretion of OC. The initiation of matrix mineralization, induced by L-AA-dependent collagen processing and deposition, might also be involved. Hence, the similar increase of OC secretion by M 25(OH)D, plus L-AA in comparison to M 1 ,B(OH),D, without L-AA points to a regulatory effect of L-AA on bone cell-specific vitamin D-metabolizing enzymes. These data show that L-AA is essential for optimal steroid hormone functions and suggests an involvement of L-AA in steroid synthesis mechanisms.
INTRODUCTION The high concentration of L-ascorbic acid (L-AA) in the cortex of the adrenal glands implies that vitamin C plays a critical role in intracellular functions, perhaps by participating in the hydroxylation reactions of the cortisol synthesis pathway. L-AA is also necessary for bone formation because it acts as a cofactor in the hydroxylation steps of collagen synthesis. Furthermore, L-AA has been shown to interact with la- and 24-hydroxylation steps of 25(OH)D3 in the kidney, that is, the biosynthesis of another steroid hormone, and to improve 1,25(OH),D3dependent bone parameters. We were interested in the influence of L-AA on tissuespecific steroid metabolism of both the adrenal glands and bone, and investigated
Cortlsol
nmol/lx60mln 200
Number of ACTH-etlmulatlone 176
0
1
m
2
9
150
126
loo 76
60
26
0
0
4
100 20 L-AA conc. In modlum (JIM)
500
FIGURE 1. Dose-dependent stimulation of repetitive cortisol release by L-AA. 349
ANNALS NEW YORK ACADEMY OF SCIENCES
350
(a) the effect of L-AA on the cortisol release in ACTH-stimulated adrenocortical cells, and (b) the effect of L-AA on the osteoblastic secretion of osteocalcin (OC), a noncollagenous bone protein, which is directly dependent on the presence of the active vitamin D metabolite 1 ,25(OH),D3.
METHODS Adrenocortical Cell Culture and Cortisol Determination Poscine adrenocortical cells were prepared from adrenal glands and collected from the slaughterhouse. After removing the adrenal medulla, the cortex was
%
25(OH)D, conc.
stlmulatlon 0
Kq
10‘M
10’ M
80
70 60 50
40
30
0
25
50 100 L-AA conc. In medium (JIM)
200
FIGURE 2. Osteocalcin secretion after three days of stimulation with L-AAand 25(OH)D,.
minced and digested with collagenase. Cells were collected by centrifugation, seeded in fibronectin-coated wells, and incubated in Dulbecco’s Modified Eagle Medium (D’MEM), 10% FCS, containing L-AA at concentrations indicated in Results. At 48, 72, and 96 h postseeding, cells were stimulated by 0.1 nM (first stimulation) or 10 nM (second and third stimulation) ACTH for the times indicated. Between each stimulation step, medium was collected, cells were washed three times, and L-AA incubation was repeated. Cortisol was determined in cell supernatant by RIA.
GORALCZYK et al.: STEROID HORMONE METABOLISM
351
Bone Cell Culture and OC Determination
Primary rat osteoblasts were established by fractionated dissociation of 20day-old fetal rat calvaria with collagenaseIhyaluronidase. Cells of fraction three were plated at a density of lo4 cells/cm2and cultured in D'MEM, 10% FCS, and 10 mM P-glycerophosphate for three weeks prior to the experiment. The cells were stimulated on three consecutive days with L-AA, in combination with 1,25(OH),D3 or 25(OH)D,. OC secreted into the medium within 24 h after the last stimulation was determined by RIA. RESULTS AND CONCLUSIONS
In adrenocortical cells, multiple stimulations with ACTH led to a decreased release of cortisol. This decrease was fully restored by the addition of 4 pM L-AA, whereas 20 to 100 pM L-AA induced even more elevated cortisol levels after ACTH stimulation (FIG.1). In bone cells, L-AA alone induced secretion of OC, a bone cell product that is transcriptionally regulated by 1,25(OH)&. In addition, L-AA enhanced the effect of 1,25(OH),D3and 25(OH)D3on OC secretion (FIG.2 ) . The nature of these mechanisms is not known at present. L-AA may be directly involved in posttranslational modifications or secretion of OC. The initiation of matrix mineralization, induced by L-AA-dependent collagen processing and deposition, might also be involved. Hence, the similar increase of OC secretion by M 25(OH)D, plus L-AA in comparison to M 1 ,B(OH),D, without L-AA points to a regulatory effect of L-AA on bone cell-specific vitamin D-metabolizing enzymes. These data show that L-AA is essential for optimal steroid hormone functions and suggests an involvement of L-AA in steroid synthesis mechanisms.
