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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.

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Effect of glucose treatment on MAPK ERK1/2 phosphorylation in rat granulosa cells. Granulosa cell lysates were prepared from cells incubated with 10 g/l glucose for various times: 0, 5, 10, 30, 60 or 120 min (A) or with 10 g/l glucose for 48 h in the presence or absence of FSH (10-8M) or IGF-1 (10-8M). Lysates (50 μg) were resolved by SDS-PAGE, transferred to nitrocellulose membrane, and probed with anti-phospho-MAPK ERK1/2 and then with anti-ERK2 antibodies. Representative blots from three independent experiments are shown. Blots were quantified and the phosphorylated MAPK ERK1/2/ERK2 protein ratio is shown. The results are represented 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).
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Figure 3: Effect of glucose treatment on MAPK ERK1/2 phosphorylation in rat granulosa cells. Granulosa cell lysates were prepared from cells incubated with 10 g/l glucose for various times: 0, 5, 10, 30, 60 or 120 min (A) or with 10 g/l glucose for 48 h in the presence or absence of FSH (10-8M) or IGF-1 (10-8M). Lysates (50 μg) were resolved by SDS-PAGE, transferred to nitrocellulose membrane, and probed with anti-phospho-MAPK ERK1/2 and then with anti-ERK2 antibodies. Representative blots from three independent experiments are shown. Blots were quantified and the phosphorylated MAPK ERK1/2/ERK2 protein ratio is shown. The results are represented 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 examined whether the inhibitory effect of glucose on progesterone and oestradiol production was associated with a variation in the phosphorylation of MAPK ERK1/2 and AMPK. These kinases have been implicated in the regulation of steroidogenesis [11,13]. We analysed the pattern of MAPK ERK1/2 phosphorylation in cells incubated with 10 g/l glucose for various times (0, 5, 10, 30, 60, 120 min, Fig. 3A), or with 10 g/l glucose for 48 h in the presence or absence of FSH (10-8M) or IGF-1 (10-8M, Fig. 3B). MAPK ERK1/2 phosphorylation was significantly reduced after 120 min of treatment with 10 g/l glucose (p < 0.05, Fig. 3A). It was also decreased in the basal and FSH-or IGF-1-stimulated conditions used for assaying progesterone and oestradiol production (48 h of treatment with 10 g/l glucose in serum-free medium, p < 0.05, Fig. 3B). Similar results were obtained with a lower glucose concentration (5 g/l, data not shown). We also determined phosphorylation of Thr172 in AMPK in similar conditions (effect of glucose in the short and long term); glucose (5 or 10 g/l) had no effect on AMPK phosphorylation (data not shown). We also investigated the effect of a high concentration of glucose (10 g/l) on the amounts of AdipoR1 and AdipoR2 (the adiponectin receptors) in rat granulosa cells and we detected no significant effect (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 MAPK ERK1/2 phosphorylation in rat granulosa cells. Granulosa cell lysates were prepared from cells incubated with 10 g/l glucose for various times: 0, 5, 10, 30, 60 or 120 min (A) or with 10 g/l glucose for 48 h in the presence or absence of FSH (10-8M) or IGF-1 (10-8M). Lysates (50 μg) were resolved by SDS-PAGE, transferred to nitrocellulose membrane, and probed with anti-phospho-MAPK ERK1/2 and then with anti-ERK2 antibodies. Representative blots from three independent experiments are shown. Blots were quantified and the phosphorylated MAPK ERK1/2/ERK2 protein ratio is shown. The results are represented 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 3: Effect of glucose treatment on MAPK ERK1/2 phosphorylation in rat granulosa cells. Granulosa cell lysates were prepared from cells incubated with 10 g/l glucose for various times: 0, 5, 10, 30, 60 or 120 min (A) or with 10 g/l glucose for 48 h in the presence or absence of FSH (10-8M) or IGF-1 (10-8M). Lysates (50 μg) were resolved by SDS-PAGE, transferred to nitrocellulose membrane, and probed with anti-phospho-MAPK ERK1/2 and then with anti-ERK2 antibodies. Representative blots from three independent experiments are shown. Blots were quantified and the phosphorylated MAPK ERK1/2/ERK2 protein ratio is shown. The results are represented 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 examined whether the inhibitory effect of glucose on progesterone and oestradiol production was associated with a variation in the phosphorylation of MAPK ERK1/2 and AMPK. These kinases have been implicated in the regulation of steroidogenesis [11,13]. We analysed the pattern of MAPK ERK1/2 phosphorylation in cells incubated with 10 g/l glucose for various times (0, 5, 10, 30, 60, 120 min, Fig. 3A), or with 10 g/l glucose for 48 h in the presence or absence of FSH (10-8M) or IGF-1 (10-8M, Fig. 3B). MAPK ERK1/2 phosphorylation was significantly reduced after 120 min of treatment with 10 g/l glucose (p < 0.05, Fig. 3A). It was also decreased in the basal and FSH-or IGF-1-stimulated conditions used for assaying progesterone and oestradiol production (48 h of treatment with 10 g/l glucose in serum-free medium, p < 0.05, Fig. 3B). Similar results were obtained with a lower glucose concentration (5 g/l, data not shown). We also determined phosphorylation of Thr172 in AMPK in similar conditions (effect of glucose in the short and long term); glucose (5 or 10 g/l) had no effect on AMPK phosphorylation (data not shown). We also investigated the effect of a high concentration of glucose (10 g/l) on the amounts of AdipoR1 and AdipoR2 (the adiponectin receptors) in rat granulosa cells and we detected no significant effect (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