TY - JOUR
T1 - Mechanisms underlying the steroidogenic synergy of insulin and luteinizing hormone in porcine granulosa cells
T2 - Joint amplification of pivotal sterol-regulatory genes encoding the low-density lipoprotein (LDL) receptor, steroidogenic acute regulatory (StAR) protein and cytochrome P450 side-chain cleavage (P450scc) enzyme
AU - Sekar, N.
AU - Garmey, James C.
AU - Veldhuis, Johannes D.
N1 - Funding Information:
Supported by NIH grants NICHD No. R01 HD 16393, R01 HD 16806, RCDA No. K04 HD 00634 and NIH U54 HD 96008 Specialized Cooperative Centers in Reproductive Research.
PY - 2000/1/25
Y1 - 2000/1/25
N2 - Growth of ovarian Graafian follicles and cytodifferentiation of granulosa and theca cells are regulated by gonadotropins, sex steroids and peptidyl growth factors. For example insulin and intraovarian insulin-like growth factor type I (IGF-I) may amplify the actions of both follicle stimulating hormone (FSH) and luteinizing hormone (LH) in promoting biochemical luteinization and enhancing steroidogenesis. To explore further the notion of interactions between insulinomimetic peptides and LH and to examine the associated mechanisms, we have established porcine granulosa cells in monolayer culture for 48 h in 3% serum with insulin (1 μg/ml), estradiol (0.5 μg/ml), and follicle stimulating hormone (FSH, 5 ng/ml) to allow cell anchorage, facilitate in vitro cytodifferentiation and confer LH responsiveness. To limit any carry-over effects of serum, granulosa cells were stabilized overnight in serum-free medium. Studies were then initiated to assess the impact of insulin on the dose-responsive actions of LH. A maximally effective concentration of insulin (1 μg/ml) synergistically augmented LH's dose-dependent ampilification of progesterone and cAMP accumulation; viz. by ≃twofold (progesterone) and ≃2.5-fold (cAMP) above that observed in maximally LH-stimulated cultures (P < 0.001). Mechanistically, insulin significantly enhanced the sensitivity of granulosa cells to LH's drive of cAMP accumulation [ED50 for LH 61 ± 14 ng/ml (control) vs. 10 ± 1.0 ng/ml (insulin) (P < 0.01)]. Insulin also augmented the maximal stimulatory effect of LH; i.e. LH efficacy rose from 6.5 ± 0.4 to 17 ± 1.4 (pmole cAMP/μg DNA/48 h; P < 0.001). Insulin dose-response analysis showed that insulin alone minimally elevated basal, but significantly heightened LH's stimulation of progesterone and cAMP accumulation at (insulin) concentrations as low as 3-10 ng/ml. The molecular mechanisms underlying insulin and LH's synergy were assessed by RNase protection assays with (porcine) cRNA probes encoding the low density lipoprotein receptor (LDL-R), Steroidogenic Acute Regulatory Protein (StAR), P450 cholesterol sidechain cleavage enzyme (P450scc) and (as a possible negative control) Sterol Carrier Protein 2 (SCP-2) [data normalized to constitutive 18S rRNA]. Non linear least-squares analysis was applied to confirm or refute an hypothesis of interactive synergy between LH and insulin on gene expression. LH and insulin alone exerted no effect on StAR message accumulation, and LH alone minimally stimulated P450scc and LDL-R mRNA's accumulation at 48 h. In contrast, insulin in combination with LH augmented StAR mRNA concentrations by ≃5-10-fold and stimulated LDL-R message levels by threefold above the respective maximally LH-driven values (P < 0.01). Maximal P450scc mRNA expression was enhanced twofold by cotreatment with LH and insulin compared with maximal LH-treated cultures. In contrast SCP-2 mRNA accumulation remained unaffected by any treatment. In summary, we have used a serum-free, in vitro differentiated porcine granulosa cell culture system to assess regulatory interactions between the disparate first messengers, LH and insulin. We observe marked LH-insulin steroidogenic synergy after 48 h of joint hormonal stimulation, and further clarify that the mechanism(s) of synergy include augmentation of cAMP production and increased steady-state concentrations of transcripts of key sterol-regulatory genes; namely, LDL-R, StAR, and P450scc, but not SCP-2. Since the encoded products of these genes variously control sterol substrate uptake, delivery to and utilization in mitochondrial steroidogenesis, we speculate that the concerted actions of insulin-like peptides and LH may contribute to steroidogenic differentiation during the later stages of follicular maturation and the granulosa-luteal cell transition.
AB - Growth of ovarian Graafian follicles and cytodifferentiation of granulosa and theca cells are regulated by gonadotropins, sex steroids and peptidyl growth factors. For example insulin and intraovarian insulin-like growth factor type I (IGF-I) may amplify the actions of both follicle stimulating hormone (FSH) and luteinizing hormone (LH) in promoting biochemical luteinization and enhancing steroidogenesis. To explore further the notion of interactions between insulinomimetic peptides and LH and to examine the associated mechanisms, we have established porcine granulosa cells in monolayer culture for 48 h in 3% serum with insulin (1 μg/ml), estradiol (0.5 μg/ml), and follicle stimulating hormone (FSH, 5 ng/ml) to allow cell anchorage, facilitate in vitro cytodifferentiation and confer LH responsiveness. To limit any carry-over effects of serum, granulosa cells were stabilized overnight in serum-free medium. Studies were then initiated to assess the impact of insulin on the dose-responsive actions of LH. A maximally effective concentration of insulin (1 μg/ml) synergistically augmented LH's dose-dependent ampilification of progesterone and cAMP accumulation; viz. by ≃twofold (progesterone) and ≃2.5-fold (cAMP) above that observed in maximally LH-stimulated cultures (P < 0.001). Mechanistically, insulin significantly enhanced the sensitivity of granulosa cells to LH's drive of cAMP accumulation [ED50 for LH 61 ± 14 ng/ml (control) vs. 10 ± 1.0 ng/ml (insulin) (P < 0.01)]. Insulin also augmented the maximal stimulatory effect of LH; i.e. LH efficacy rose from 6.5 ± 0.4 to 17 ± 1.4 (pmole cAMP/μg DNA/48 h; P < 0.001). Insulin dose-response analysis showed that insulin alone minimally elevated basal, but significantly heightened LH's stimulation of progesterone and cAMP accumulation at (insulin) concentrations as low as 3-10 ng/ml. The molecular mechanisms underlying insulin and LH's synergy were assessed by RNase protection assays with (porcine) cRNA probes encoding the low density lipoprotein receptor (LDL-R), Steroidogenic Acute Regulatory Protein (StAR), P450 cholesterol sidechain cleavage enzyme (P450scc) and (as a possible negative control) Sterol Carrier Protein 2 (SCP-2) [data normalized to constitutive 18S rRNA]. Non linear least-squares analysis was applied to confirm or refute an hypothesis of interactive synergy between LH and insulin on gene expression. LH and insulin alone exerted no effect on StAR message accumulation, and LH alone minimally stimulated P450scc and LDL-R mRNA's accumulation at 48 h. In contrast, insulin in combination with LH augmented StAR mRNA concentrations by ≃5-10-fold and stimulated LDL-R message levels by threefold above the respective maximally LH-driven values (P < 0.01). Maximal P450scc mRNA expression was enhanced twofold by cotreatment with LH and insulin compared with maximal LH-treated cultures. In contrast SCP-2 mRNA accumulation remained unaffected by any treatment. In summary, we have used a serum-free, in vitro differentiated porcine granulosa cell culture system to assess regulatory interactions between the disparate first messengers, LH and insulin. We observe marked LH-insulin steroidogenic synergy after 48 h of joint hormonal stimulation, and further clarify that the mechanism(s) of synergy include augmentation of cAMP production and increased steady-state concentrations of transcripts of key sterol-regulatory genes; namely, LDL-R, StAR, and P450scc, but not SCP-2. Since the encoded products of these genes variously control sterol substrate uptake, delivery to and utilization in mitochondrial steroidogenesis, we speculate that the concerted actions of insulin-like peptides and LH may contribute to steroidogenic differentiation during the later stages of follicular maturation and the granulosa-luteal cell transition.
KW - Cytochrome P450scc
KW - Gonadotropin
KW - LDL-R
KW - Ovary
KW - StAR
KW - Trophic
UR - http://www.scopus.com/inward/record.url?scp=0033978294&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0033978294&partnerID=8YFLogxK
U2 - 10.1016/S0303-7207(99)00203-8
DO - 10.1016/S0303-7207(99)00203-8
M3 - Article
C2 - 10687849
AN - SCOPUS:0033978294
SN - 0303-7207
VL - 159
SP - 25
EP - 35
JO - Molecular and Cellular Endocrinology
JF - Molecular and Cellular Endocrinology
IS - 1-2
ER -