Limits...
Effects of high levels of glucose on the steroidogenesis and the expression of adiponectin receptors in rat ovarian cells.

Chabrolle C, Jeanpierre E, Tosca L, Ramé C, Dupont J - Reprod. Biol. Endocrinol. (2008)

Bottom Line: This was associated with substantial reductions in the amounts of 3beta HSD, p450scc, p450 aromatase and StAR proteins and MAPK ERK1/2 phosphorylation.Streptozotocin treatment did not affect adiponectin receptors in rat ovary but it increased AMPK phosphorylation without affecting MAPK ERK1/2 phosphorylation.However, the mechanism that leads to reduced ovarian steroid production seems to be different.

View Article: PubMed Central - HTML - PubMed

Affiliation: Unité de Physiologie de la Reproduction et des Comportements, Institut National de la Recherche Agronomique, 37380 Nouzilly, France. chabrolle@tours.inra.fr

ABSTRACT

Background: Reproductive dysfunction in the diabetic female rat is associated with altered folliculogenesis and steroidogenesis. However, the molecular mechanisms involved in the reduction of steroid production have not been described. Adiponectin is an adipocytokine that has insulin-sensitizing actions including stimulation of glucose uptake in muscle and suppression of glucose production in liver. Adiponectin acts via two receptor isoforms - AdipoR1 and AdipoR2 - that are regulated by hyperglycaemia and hyperinsulinaemia in liver and muscle. We have recently identified AdipoR1 and AdipoR2 in rat ovary. However, their regulation in ovaries of diabetic female rat remains to be elucidated.

Methods: We incubated rat primary granulosa cells in vitro with high concentrations of glucose (5 or 10 g/l) + or - FSH (10-8 M) or IGF-1 (10-8 M), and we studied the ovaries of streptozotocin-induced diabetic rats (STZ) in vivo. The levels of oestradiol and progesterone in culture medium and serum were measured by RIA. We used immunoblotting to assay key steroidogenesis factors (3beta HSD, p450scc, p450 aromatase, StAR), and adiponectin receptors and various elements of signalling pathways (MAPK ERK1/2 and AMPK) in vivo and in vitro. We also determined cell proliferation by [3H] thymidine incorporation.

Results: Glucose (5 or 10 g/l) impaired the in vitro production in rat granulosa cells of both progesterone and oestradiol in the basal state and in response to FSH and IGF-1 without affecting cell proliferation and viability. This was associated with substantial reductions in the amounts of 3beta HSD, p450scc, p450 aromatase and StAR proteins and MAPK ERK1/2 phosphorylation. In contrast, glucose did not affect the abundance of AdipoR1 or AdipoR2 proteins. In vivo, as expected, STZ treatment of rats caused hyperglycaemia and insulin, adiponectin and resistin deficiencies. Plasma progesterone and oestradiol levels were also reduced in STZ rats. However, the amounts of 3beta HSD and p450 aromatase were the same in STZ rat ovary and controls, and the amounts of StAR and p450scc were higher. Streptozotocin treatment did not affect adiponectin receptors in rat ovary but it increased AMPK phosphorylation without affecting MAPK ERK1/2 phosphorylation.

Conclusion: High levels of glucose decrease progesterone and oestradiol production in primary rat granulosa cells and in STZ-treated rats. However, the mechanism that leads to reduced ovarian steroid production seems to be different. Furthermore, adiponectin receptors in ovarian cells are not regulated by glucose.

Show MeSH

Related in: MedlinePlus

Effect of glucose treatment on the amounts of the 3βHSD, p450scc, StAR and p450 aromatase proteins in rat granulosa cells. Protein extracts from rat granulosa cells cultured for 48 h in the absence or in the presence of 10 g/l glucose ± 10-8 M FSH or 10-8 M IGF-1 were subjected to SDS-PAGE as described in Materials and Methods. The membranes were incubated with antibodies raised against the 3βHSD (A), p450scc (B), StAR (C) and p450 aromatase (D) proteins. Equal protein loading was verified by reprobing membranes with an anti-vinculin antibody. Results are representative of at least three independent experiments. Blots were quantified and the 3βHSD, p450scc, StAR and p450 aromatase to Vinculin ratios are shown. The results are expressed as means ± SE. Bars with different letters are significantly different (p < 0.05). The letter "a" indicates values which are not significantly different from control (without FSH or IGF-1 and glucose).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2277425&req=5

Figure 2: Effect of glucose treatment on the amounts of the 3βHSD, p450scc, StAR and p450 aromatase proteins in rat granulosa cells. Protein extracts from rat granulosa cells cultured for 48 h in the absence or in the presence of 10 g/l glucose ± 10-8 M FSH or 10-8 M IGF-1 were subjected to SDS-PAGE as described in Materials and Methods. The membranes were incubated with antibodies raised against the 3βHSD (A), p450scc (B), StAR (C) and p450 aromatase (D) proteins. Equal protein loading was verified by reprobing membranes with an anti-vinculin antibody. Results are representative of at least three independent experiments. Blots were quantified and the 3βHSD, p450scc, StAR and p450 aromatase to Vinculin ratios are shown. The results are expressed as means ± SE. Bars with different letters are significantly different (p < 0.05). The letter "a" indicates values which are not significantly different from control (without FSH or IGF-1 and glucose).

Mentions: We then investigated whether this inhibitory effect of glucose on the production of both progesterone and oestradiol resulted from the production of smaller amounts of the three key enzymes in steroidogenesis (3βHSD, p450scc and p450 aromatase) and/or of StAR, a major cholesterol carrier. Glucose treatment (10 g/l, 48 h) in the presence of FSH (10-8M) decreased production of 3βHSD (Fig. 2A, p < 0.001) and p450scc (Fig. 2B, p < 0.001) by a factor of about seven, halved the production of StAR (Fig. 2C, 0.05) and reduced by three-fold the production of p450 aromatase (Fig. 2D, p < 0.001), relative to the values in the presence of FSH without glucose. In the presence of IGF-1 (10-8M), glucose decreased the amounts of the three key enzymes in steroidogenesis and StAR by a factor of three relative to IGF-1 treatment without glucose (Fig. 2A to D, p < 0.05). Similar results were obtained with a lower glucose concentration (5 g/l, data not shown).


Effects of high levels of glucose on the steroidogenesis and the expression of adiponectin receptors in rat ovarian cells.

Chabrolle C, Jeanpierre E, Tosca L, Ramé C, Dupont J - Reprod. Biol. Endocrinol. (2008)

Effect of glucose treatment on the amounts of the 3βHSD, p450scc, StAR and p450 aromatase proteins in rat granulosa cells. Protein extracts from rat granulosa cells cultured for 48 h in the absence or in the presence of 10 g/l glucose ± 10-8 M FSH or 10-8 M IGF-1 were subjected to SDS-PAGE as described in Materials and Methods. The membranes were incubated with antibodies raised against the 3βHSD (A), p450scc (B), StAR (C) and p450 aromatase (D) proteins. Equal protein loading was verified by reprobing membranes with an anti-vinculin antibody. Results are representative of at least three independent experiments. Blots were quantified and the 3βHSD, p450scc, StAR and p450 aromatase to Vinculin ratios are shown. The results are expressed as means ± SE. Bars with different letters are significantly different (p < 0.05). The letter "a" indicates values which are not significantly different from control (without FSH or IGF-1 and glucose).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2277425&req=5

Figure 2: Effect of glucose treatment on the amounts of the 3βHSD, p450scc, StAR and p450 aromatase proteins in rat granulosa cells. Protein extracts from rat granulosa cells cultured for 48 h in the absence or in the presence of 10 g/l glucose ± 10-8 M FSH or 10-8 M IGF-1 were subjected to SDS-PAGE as described in Materials and Methods. The membranes were incubated with antibodies raised against the 3βHSD (A), p450scc (B), StAR (C) and p450 aromatase (D) proteins. Equal protein loading was verified by reprobing membranes with an anti-vinculin antibody. Results are representative of at least three independent experiments. Blots were quantified and the 3βHSD, p450scc, StAR and p450 aromatase to Vinculin ratios are shown. The results are expressed as means ± SE. Bars with different letters are significantly different (p < 0.05). The letter "a" indicates values which are not significantly different from control (without FSH or IGF-1 and glucose).
Mentions: We then investigated whether this inhibitory effect of glucose on the production of both progesterone and oestradiol resulted from the production of smaller amounts of the three key enzymes in steroidogenesis (3βHSD, p450scc and p450 aromatase) and/or of StAR, a major cholesterol carrier. Glucose treatment (10 g/l, 48 h) in the presence of FSH (10-8M) decreased production of 3βHSD (Fig. 2A, p < 0.001) and p450scc (Fig. 2B, p < 0.001) by a factor of about seven, halved the production of StAR (Fig. 2C, 0.05) and reduced by three-fold the production of p450 aromatase (Fig. 2D, p < 0.001), relative to the values in the presence of FSH without glucose. In the presence of IGF-1 (10-8M), glucose decreased the amounts of the three key enzymes in steroidogenesis and StAR by a factor of three relative to IGF-1 treatment without glucose (Fig. 2A to D, p < 0.05). Similar results were obtained with a lower glucose concentration (5 g/l, data not shown).

Bottom Line: This was associated with substantial reductions in the amounts of 3beta HSD, p450scc, p450 aromatase and StAR proteins and MAPK ERK1/2 phosphorylation.Streptozotocin treatment did not affect adiponectin receptors in rat ovary but it increased AMPK phosphorylation without affecting MAPK ERK1/2 phosphorylation.However, the mechanism that leads to reduced ovarian steroid production seems to be different.

View Article: PubMed Central - HTML - PubMed

Affiliation: Unité de Physiologie de la Reproduction et des Comportements, Institut National de la Recherche Agronomique, 37380 Nouzilly, France. chabrolle@tours.inra.fr

ABSTRACT

Background: Reproductive dysfunction in the diabetic female rat is associated with altered folliculogenesis and steroidogenesis. However, the molecular mechanisms involved in the reduction of steroid production have not been described. Adiponectin is an adipocytokine that has insulin-sensitizing actions including stimulation of glucose uptake in muscle and suppression of glucose production in liver. Adiponectin acts via two receptor isoforms - AdipoR1 and AdipoR2 - that are regulated by hyperglycaemia and hyperinsulinaemia in liver and muscle. We have recently identified AdipoR1 and AdipoR2 in rat ovary. However, their regulation in ovaries of diabetic female rat remains to be elucidated.

Methods: We incubated rat primary granulosa cells in vitro with high concentrations of glucose (5 or 10 g/l) + or - FSH (10-8 M) or IGF-1 (10-8 M), and we studied the ovaries of streptozotocin-induced diabetic rats (STZ) in vivo. The levels of oestradiol and progesterone in culture medium and serum were measured by RIA. We used immunoblotting to assay key steroidogenesis factors (3beta HSD, p450scc, p450 aromatase, StAR), and adiponectin receptors and various elements of signalling pathways (MAPK ERK1/2 and AMPK) in vivo and in vitro. We also determined cell proliferation by [3H] thymidine incorporation.

Results: Glucose (5 or 10 g/l) impaired the in vitro production in rat granulosa cells of both progesterone and oestradiol in the basal state and in response to FSH and IGF-1 without affecting cell proliferation and viability. This was associated with substantial reductions in the amounts of 3beta HSD, p450scc, p450 aromatase and StAR proteins and MAPK ERK1/2 phosphorylation. In contrast, glucose did not affect the abundance of AdipoR1 or AdipoR2 proteins. In vivo, as expected, STZ treatment of rats caused hyperglycaemia and insulin, adiponectin and resistin deficiencies. Plasma progesterone and oestradiol levels were also reduced in STZ rats. However, the amounts of 3beta HSD and p450 aromatase were the same in STZ rat ovary and controls, and the amounts of StAR and p450scc were higher. Streptozotocin treatment did not affect adiponectin receptors in rat ovary but it increased AMPK phosphorylation without affecting MAPK ERK1/2 phosphorylation.

Conclusion: High levels of glucose decrease progesterone and oestradiol production in primary rat granulosa cells and in STZ-treated rats. However, the mechanism that leads to reduced ovarian steroid production seems to be different. Furthermore, adiponectin receptors in ovarian cells are not regulated by glucose.

Show MeSH
Related in: MedlinePlus