Regulation of porcine granulosa cell 3-hydroxy-3-methylglutaryl coenzyme A reductase by insulin and insulin-like growth factor I: synergism with follicle-stimulating hormone or protein kinase A agonist.
In species such as the pig and human, gonadal steroidogenesis is believed to be dependent upon the availability of low density lipoprotein (LDL) cholesterol. However, before ovulation, Graafian follicles are impermeant to lipoproteins in the LDL class. Thus, de novo cholesterol biosynthesis via the rate-determining enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is likely to provide a significant mechanism for generating sterol substrate for steroidogenesis by granulosa cells before follicular rupture. As serum-free monolayer culture of (swine) granulosa cells offers an in vitro model of hormonally responsive HMG-CoA reductase, we generated a (porcine) complementary DNA and homologous complementary RNA to investigate by sensitive and specific ribonuclease protection assay the hormonal regulation of HMG-CoA reductase gene expression in ovarian cells from immature Graafian follicles. Using reverse transcriptase-polymerase chain reaction, we cloned and sequenced a 238-base pair complementary DNA from porcine luteal tissue that encodes the catalytic region of HMG-CoA reductase. GenBank analysis of the DNA sequence homology between the pig and other species showed the greatest concordance with human (88%) and hamster (90%). Solution hybridization/ribonuclease protection analysis of total RNA isolated from serum-free monolayer cultures of porcine granulosa cells revealed that insulin (3 micrograms/ml) increased HMG-CoA messenger RNA (mRNA) concentrations corrected for constitutive 18S ribosomal RNA expression in a time-dependent fashion, with significant effects observed at 12 h and a 6-fold increase by 48 h. Recombinant human insulin-like growth factor I (IGF-I) peptide was able to mimic the action of insulin alone. Neither FSH (100 ng/ml) nor 8-bromo-cAMP (1 mM) had observable effects on HMG-CoA message accumulation at any time point studied. However, the combined action of either FSH and insulin or 8-bromo-cAMP and insulin resulted in synergistic increases in reductase mRNA by 31- and 17-fold, respectively. To assess the possible feedback effects of sterol on HMG-CoA gene expression, granulosa cells were treated with LDL. At physiological concentrations, LDL suppressed basal expression of HMG-CoA mRNA to levels below the control value. In addition, LDL inhibited insulin-stimulated HMG-CoA mRNA accumulation by 84% as well as the synergistic effects of insulin and FSH (by 94%) and of insulin and 8-bromo-cAMP (by 93%). We conclude that insulin alone or in combination with FSH or cAMP augments the accumulation of HMG-CoA reductase mRNA in ovarian (granulosa) cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Maitra
A
A
Department of Internal Medicine, University of Virginia Health Science Center, Charlottesville 22908, USA.
LaVoie
H A
HA
Day
R N
RN
Garmey
J C
JC
Veldhuis
J D
JD
D000818:Animals; D001483:Base Sequence; D017868:Cyclic AMP-Dependent Protein Kinases / Q000819:agonists*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004357:Drug Synergism; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects*; D006107:Granulosa Cells / Q000201:enzymology*; D006801:Humans; D006903:Hydroxymethylglutaryl CoA Reductases / Q000235:genetics*; D007328:Insulin / Q000494:pharmacology*; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D017385:Sequence Homology; D013552:Swine
D018076:DNA, Complementary; D007328:Insulin; D012333:RNA, Messenger; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I; D005640:Follicle Stimulating Hormone; D006903:Hydroxymethylglutaryl CoA Reductases; D017868:Cyclic AMP-Dependent Protein Kinases