An experimental model to study synthesis of
cholesterol and
pregnenolone from the precursor
mevalonolactone (MVA) was developed in C6-2B
glioma cells. The steroidogenic capability of this cell line and the regulation of
pregnenolone production by
4'-chlorodiazepam (4'CD), a specific
ligand for the mitochondrial
diazepam binding inhibitor (
DBI) receptor (MDR), were investigated. Cells maintained in
serum-free media were incubated with
lovastatin (20 microM) and two inhibitors of
pregnenolone metabolism,
trilostane (25 microM) and 1,2,3,4-tetrahydro-4-oxo-7-chloro-2-naphthylpyridine (10 microM). Under these conditions the incorporation of [3H]MVA into
cholesterol and
pregnenolone formation was biphasic, with an initial rapid phase (within 1 min) followed by a slower phase.
Cholesterol and
pregnenolone were identified by coelution with authentic
steroids from a Si 60 Lichrosorb column and gas chromatography/mass spectrometry.
Pregnenolone synthesis in intact C6-2B
glioma cells was stimulated by nanomolar concentrations of 4'CD after 5 min of incubation with MVA. The stimulatory effect was dependent on
drug concentration and the maximal effect was achieved
at 10 nM. The time course showed that the incorporation of MVA into
pregnenolone is accelerated by the MDR
ligand.
Cholesterol synthesis is only slightly and not significantly affected by 4'CD. These results support the view that
steroid synthesis occurs in a
glioma cell line. Moreover, we provide evidence for a rapid
steroid synthesis in C6-2B
glioma cells, which in turn appears to be accelerated by 1-100 nM 4'CD, a MDR
ligand.