The Influence of Exogenous Free Cholesterol on Steroid Synthesis in Cultured Adrenal Cells*

Abstract

Previous studies showed that Y-1 cells in which the concentration of cholesterol in plasma membranes (expressed relative to phospholipid as a molar ratio, C/P) was raised by incubation with cholesterol produced more 20 alpha-dihydroprogesterone than cells with normal or low plasma membrane C/P values. This difference was not associated with any difference in the production of cAMP. The cells respond to (Bu)2cAMP in the order high greater than normal greater than low C/P. High C/P is associated with higher levels of cholesterol in the cytosol and higher concentrations of this steroid in the inner mitochondrial membrane. The rate of transport of cholesterol to the inner membrane differed in the three groups of cells: high greater than normal greater than low C/P. Production of pregnenolone by mitoplasts was no different in the three groups, and the same applies to the production of pregnenolone by mitochondria from cells incubated with and those incubated without (Bu)2cAMP. 25-Hydroxycholesterol increased the production of 20 alpha-dihydroprogesterone by cells showing normal or low C/P values, but not those with high values. It is concluded that the transport of cholesterol to the inner mitochondrial membrane is regulated by at least two processes, namely the supply of substrate (cholesterol), which can saturate this transport mechanism, and some mechanism(s) that is stimulated by cAMP and is capable of stimulating transport above maximal levels produced by high concentrations of cholesterol alone. The second mechanism requires the synthesis of a new protein(s).

Peripheral-Type Benzodiazepine/Diazepam Binding Inhibitor Receptor: Biological Role in Steroidogenic Cell Function*

“…In this model, much of the stored cholesterol comes from free cellular cholesterol, mainly from the plasma membrane (134). Increases in the plasma membrane cholesterol concentration also resulted in increased steroid synthesis in cultured adrenal cells (136). Increases in the plasma membrane cholesterol concentration also resulted in increased steroid synthesis in cultured adrenal cells (136).…”

Molecular Mechanisms of Thyroid Hormone-stimulated Steroidogenesis in Mouse Leydig Tumor Cells

“…This is a rate-limiting and regulated step in steroidogenesis and is dependent on de novo protein synthesis in steroidogenic cells (1,2). Conversion of cholesterol to pregnenolone is the first enzymatic step in steroidogenesis which is catalyzed by the cholesterol side chain cleavage cytochrome P450 enzyme, located in the inner mitochondrial membrane (3)(4)(5). Several factors have been proposed as essential mediators for cholesterol delivery into this site critical for the initiation of steroidogenesis (6).…”

The mitochondrial environment is required for activity of the cholesterol side-chain cleavage enzyme, cytochrome P450scc.

“…5). However, the subcellular localization of enzymes has important consequences: the conversion of cholesterol to pregnenolone by mitochondrial P450scc appears to be the rate-limiting reaction in steroidogenesis because transport of cholesterol substrate into the mitochondria is slow, rather than because of inherent inefficiency of P450scc (14)(15)(16) (23). This was done using upstream oligonucleotide 13 (5'-GGGTACCATGGAGCCCAGTATCTTG-3') and downstream oligonucleotide 14 (S'-GACTAAGAGTAA-CAAGAAGCC-3') to prepare a 69-bp fragment encoding the ER-targeting sequence (the first 23 residues) ofrat P450IIB1.…”

“…Upstream oligonucleotide 15 (5'-ATCTCCACCCGCAGTCCTCGC-3') generated a bluntended cDNA fragment beginning at the codon for amino acid 40 of P450scc (i.e., the first residue of the processed mature intramitochondrial protein), and downstream oligonucleotide 16 (5'-TTGGGGCCCTCGGACTTAAAG-3') extended to the Apa I site at codon 140. The two sequences were then ligated together and subcloned into vector pUC-SF (20).…”

“…After transfections were done on cultures at 60% confluency for 16 hr at 37°C in 5% CO2, the medium was replaced with fresh Dulbecco's modified Eagle's medium-H21 (GIBCO) containing glucose (4.5 g/liter), 10% fetal calf serum, and gentamicin (50 ,ug/ml). After 48 hr of transfection, the medium was removed from the cells and replaced with a depleted medium containing only 0.5% fetal calf serum but supplemented with 5 x 10-6 M (22R)-hydroxycholesterol.…”