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

Anurupa Maitra, Holly A. Lavoie, Richard Day, James C. Garmey, Johannes D. Veldhuis

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

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 nova 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 μg/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. Given the ability of IGF-I to mimic insulin action in the present studies and the putative intrafollicular role of IGF-I, we postulate that synergistic stimulation of the expression of HMG-CoA reductase gene transcript levels in vivo simultaneously by insulinomimetic peptides and activators of the cAMP signal transduction pathway can help meet the demand for increased sterol substrate by differentiating granulosa cells. The down-regulatory actions of LDL may provide a negative feedback mechanism to inhibit de novo cholesterol synthesis when granulosa cells are replete with sterol substrate.

Original languageEnglish (US)
Pages (from-to)5111-5117
Number of pages7
JournalEndocrinology
Volume136
Issue number11
StatePublished - Nov 1995
Externally publishedYes

Fingerprint

Granulosa Cells
Coenzymes
Follicle Stimulating Hormone
Coenzyme A
Insulin-Like Growth Factor I
Protein Kinases
Oxidoreductases
Swine
Insulin
LDL Lipoproteins
Sterols
8-Bromo Cyclic Adenosine Monophosphate
Messenger RNA
Ovarian Follicle
Ribonucleases
Complementary DNA
18S Ribosomal RNA
Cholesterol
Gene Expression
Complementary RNA

ASJC Scopus subject areas

  • Endocrinology
  • Endocrinology, Diabetes and Metabolism

Cite this

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. / Maitra, Anurupa; Lavoie, Holly A.; Day, Richard; Garmey, James C.; Veldhuis, Johannes D.

In: Endocrinology, Vol. 136, No. 11, 11.1995, p. 5111-5117.

Research output: Contribution to journalArticle

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abstract = "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 nova 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 μg/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. Given the ability of IGF-I to mimic insulin action in the present studies and the putative intrafollicular role of IGF-I, we postulate that synergistic stimulation of the expression of HMG-CoA reductase gene transcript levels in vivo simultaneously by insulinomimetic peptides and activators of the cAMP signal transduction pathway can help meet the demand for increased sterol substrate by differentiating granulosa cells. The down-regulatory actions of LDL may provide a negative feedback mechanism to inhibit de novo cholesterol synthesis when granulosa cells are replete with sterol substrate.",
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T1 - Regulation of porcine granulosa cell 3-hydroxy-3-methylglutaryl coenzyme a reductase by insulin and insulin-like growth factor I

T2 - Synergism with follicle-stimulating hormone or protein kinase a agonist

AU - Maitra, Anurupa

AU - Lavoie, Holly A.

AU - Day, Richard

AU - Garmey, James C.

AU - Veldhuis, Johannes D.

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N2 - 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 nova 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 μg/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. Given the ability of IGF-I to mimic insulin action in the present studies and the putative intrafollicular role of IGF-I, we postulate that synergistic stimulation of the expression of HMG-CoA reductase gene transcript levels in vivo simultaneously by insulinomimetic peptides and activators of the cAMP signal transduction pathway can help meet the demand for increased sterol substrate by differentiating granulosa cells. The down-regulatory actions of LDL may provide a negative feedback mechanism to inhibit de novo cholesterol synthesis when granulosa cells are replete with sterol substrate.

AB - 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 nova 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 μg/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. Given the ability of IGF-I to mimic insulin action in the present studies and the putative intrafollicular role of IGF-I, we postulate that synergistic stimulation of the expression of HMG-CoA reductase gene transcript levels in vivo simultaneously by insulinomimetic peptides and activators of the cAMP signal transduction pathway can help meet the demand for increased sterol substrate by differentiating granulosa cells. The down-regulatory actions of LDL may provide a negative feedback mechanism to inhibit de novo cholesterol synthesis when granulosa cells are replete with sterol substrate.

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