Concerted regulation of steroidogenic acute regulatory gene expression by luteinizing hormone and insulin (or insulin-like growth factor I) in primary cultures of porcine granulosa-luteal cells

Endocrinology. 2000 Nov;141(11):3983-92. doi: 10.1210/endo.141.11.7763.


The steroidogenic acute regulatory (StAR) protein is indispensable for maximal trophic hormone-stimulated steroidogenesis by the adrenal gland, testis, and ovary. Recently, our laboratory developed an in vitro primary culture system of porcine granulosa-luteal cells that retain responsiveness to LH and show LH and insulin [or insulin-like growth factor (IGF-I)] synergy in stimulating StAR messenger RNA accumulation. Here, we examine the mechanisms subserving this LH-insulin (IGF-I) augmentation. We corroborate LH's amplification of insulin as well as IGF-I-stimulated granulosa-luteal cell progesterone and cAMP accumulation (P < 0.001). Insulin or IGF-I elevated LH receptor transcript accumulation, and LH did not alter this effect. To determine the hormonal responsiveness of StAR promoter, truncated regions of the -1423 to +130 bp upstream sequence of the porcine gene were ligated into a firefly luciferase reporter plasmid. Transient transfection of the StAR plasmid containing the full-length porcine 5'-flanking region of StAR (pStAR1423/luc) showed superadditive stimulation by LH and insulin or IGF-I after 24 h. LH, but not insulin or IGF-I alone, stimulated pStAR1423/luc activity. Deletion of the proximal putative steroidogenic factor-1 (-48 to -41) site abolished hormonally driven StAR promoter activity. A stable cAMP analog, 8-bromo-cAMP (1 mM), and insulin/IGF-I also evoked supraadditive StAR promoter expression. To further explore the role of cAMP in LH-insulin (or IGF-I) actions, we cotransfected a Rous sarcoma virus (RSV)-driven minigene encoding the heat-stable inhibitor of the cAMP-dependent protein kinase (RSV/PKI) or a mutant plasmid (RSV/PKImut) along with the pStAR1423/luc promoter construct. Cotransfection of PKI, but not PKImut, with pStAR1423/luc significantly attenuated LH's stimulation of luciferase activity and also reduced the magnitude of the transcriptional amplification exerted by LH and insulin or IGF-I. In corollary analyses of the protein kinase A (PKA) pathway, cotransfection of full-length pStAR1423/luc and a complementary DNA encoding a constitutively activated PKA catalytic subunit elevated basal and insulin (or IGF-I)-stimulated StAR promoter expression. LH and insulin (or IGF-I) also augmented steady state StAR transcript levels, as assessed by homologous RT-PCR, and StAR protein concentrations, as evaluated by Western blotting. Together, these investigations document a significant role for insulin or IGF-I in enhancing LH-stimulated progesterone and cAMP biosynthesis and endogenous StAR message and protein accumulation and in augmenting cAMP-PKA-dependent transcriptional activation of the exogenous StAR promoter.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 8-Bromo Cyclic Adenosine Monophosphate / pharmacology
  • Animals
  • Cells, Cultured
  • Cyclic AMP / metabolism
  • Cyclic AMP-Dependent Protein Kinases / genetics
  • Cyclic AMP-Dependent Protein Kinases / metabolism
  • Drug Synergism
  • Female
  • Gene Expression Regulation / drug effects*
  • Granulosa Cells / metabolism*
  • Insulin / pharmacology*
  • Insulin-Like Growth Factor I / pharmacology
  • Luciferases / genetics
  • Luteal Cells / metabolism*
  • Luteinizing Hormone / pharmacology*
  • Phosphoproteins / genetics*
  • Progesterone / metabolism
  • Promoter Regions, Genetic
  • RNA, Messenger / metabolism
  • Receptors, LH / genetics
  • Recombinant Fusion Proteins
  • Swine
  • Transfection


  • Insulin
  • Phosphoproteins
  • RNA, Messenger
  • Receptors, LH
  • Recombinant Fusion Proteins
  • steroidogenic acute regulatory protein
  • 8-Bromo Cyclic Adenosine Monophosphate
  • Progesterone
  • Insulin-Like Growth Factor I
  • Luteinizing Hormone
  • Cyclic AMP
  • Luciferases
  • Cyclic AMP-Dependent Protein Kinases