Mechanisms of regulation of ovarian sterol metabolism by insulin-like growth factor type II: in vitro studies with swine granulosa cells.
The present studies were designed to investigate the nature of the actions of insulin-like growth factor-II (IGF-II) on granulosa cell steroidogenesis and assess the potential facilitative interactions between IGF-II and other major regulators of ovarian sterol metabolism, viz. estrogen, FSH, and low density lipoprotein (LDL). In serum-free first passage monolayer cultures of swine granulosa cells, human recombinant IGF-II stimulated progesterone production with a half-maximally effective concentration of 4.6 +/- 1.2 ng/ml (0.61 +/- 0.16 nM) between 0-48 h of culture and 27 +/- 5.7 ng/ml (3.6 +/- 0.76 nM) between 48-96 h. Maximal progesterone accumulation increased 12-fold over that in untreated cultures (48-96 h). Over the latter interval, IGF-I stimulated progesterone production approximately 10-fold, with a significantly lower ED50 of 6.1 +/- 0.70 ng/ml (0.78 +/- 0.09 nM; P < 0.01 vs. IGF-II effect). IGF-II (100 ng/ml) enhanced progesterone biosynthesis approximately 2-fold in the presence of 25-hydroxycholesterol, suggesting that IGF-II increases the effective activity of the mitochondrial cholesterol side-chain cleavage enzyme. IGF-II (100 ng/ml) augmented human LDL-promoted progesterone production approximately 18-fold between 0-48 h of culture and approximately 6-fold between 48-96 h. In addition, IGF-II showed time-dependent stimulatory effects on the rates of [125I]iodo-LDL internalization, and the amounts of cell-associated and degraded lipoprotein. IGF-II increased by approximately 10-fold the number of specific high affinity LDL receptors on granulosa cells, with no apparent change in their binding affinity, as assessed in equilibrium competition studies. Coadministration of IGF-II and FSH (100 ng/ml) or estradiol (E2; 1 microgram/ml) for 2 days increased progesterone production synergistically. Cotreatment with FSH or E2 for 4 days decreased the ED50 of IGF-II's stimulation of progesterone accumulation by 61% and 50%, respectively (P < 0.01). Synergistic interactions also existed between IGF-II and 8-bromo-cAMP, which indicates that IGF-II can act in part at cellular loci distal to cAMP generation. Northern blot analysis of total RNA isolated from granulosa cells treated with IGF-II (100 ng/ml), FSH (100 ng/ml), or IGF-II plus FSH for 2 days revealed 5-, 7-, or 8-fold increases, respectively, in the amount of cytochrome P450 cholesterol side-chain cleavage enzyme mRNA. The same treatments produced 6-fold increases in the level of LDL receptor mRNA, as determined by solution hybridization/RNase protection assays.(ABSTRACT TRUNCATED AT 400 WORDS)
Garmey
J C
JC
Department of Internal Medicine, University of Virginia Health Sciences Center, Charlottesville 22908.
Day
R N
RN
Day
K H
KH
Veldhuis
J D
JD
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D004958:Estradiol / Q000494:pharmacology; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology; D006107:Granulosa Cells / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D007335:Insulin-Like Growth Factor II / Q000494:pharmacology*; D007700:Kinetics; D008077:Lipoproteins, LDL / Q000494:pharmacology; D008969:Molecular Sequence Data; D010053:Ovary / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011374:Progesterone / Q000096:biosynthesis*; D011973:Receptors, LDL / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013552:Swine
D008077:Lipoproteins, LDL; D011973:Receptors, LDL; D015124:8-Bromo Cyclic Adenosine Monophosphate; D011374:Progesterone; D004958:Estradiol; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II; D005640:Follicle Stimulating Hormone