Metformin improves ovarian insulin signaling alterations caused by fetal programming

in Journal of Endocrinology
Correspondence should be addressed to M F Heber or A B Motta: florencia.heber@gmail.com or aliciabmotta@yahoo.com.ar
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Insulin resistance is the decreased ability of insulin to mediate metabolic actions. In the ovary, insulin controls ovulation and oocyte quality. Alterations in ovarian insulin signaling pathway could compromise ovarian physiology. Here, we aimed to investigate the effects of fetal programming on ovarian insulin signaling and evaluate the effect of metformin treatment. Pregnant rats were hyperandrogenized with testosterone and female offspring born to those dams were employed; at adulthood, prenatally hyperandrogenized (PH) offspring presented two phenotypes: irregular ovulatory (PHiov) and anovulatory (PHanov). Half of each group was orally treated with metformin. Metformin treatment improved the estrous cyclicity in both PH groups. Both PH groups showed low mRNA levels of Ir, Irs1 and Glut4. Irs2 was decreased only in PHanov. Metformin upregulated the mRNA levels of some of the mediators studied. Protein expression of IR, IRS1/2 and GLUT4 was decreased in both PH groups. In PHiov, metformin restored the expression of all the mediators, whereas in PHanov, metformin restored only that of IR and IRS1/2. IRS1 phosphorylation was measured in tyrosine residues, which activates the pathway, and in serine residues, which impairs insulin action. PHiov presented high IRS1 phosphorylation on tyrosine and serine residues, whereas PHanov showed high serine phosphorylation and low tyrosine phosphorylation. Metformin treatment lowered serine phosphorylation only in PHanov rats. Our results suggest that PHanov rats have a defective insulin action, partially restored with metformin. PHiov rats had less severe alterations, and metformin treatment was more effective in this phenotype.

 

      Society for Endocrinology

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    Schematic figure of the model used. Pregnant Sprague–Dawley rats were injected with 1 mg of testosterone or vehicle from day 16 to 19 of gestation; birth occurs in day 22. Female offspring are separated from males, from day 45 to 60 of age the reproductive phenotype was established through analysis of the estrous cycle. Once the phenotype was established, half of each group received a 50 mg/kg dose of metformin administrated orally from day 70 until killing. During treatment, the estrous cycle was controlled to determine the reproductive phenotype at the time of killing.

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    Hormonal profile (A) Proportion of rats corresponding to each reproductive phenotype before and after metformin treatment. (B) Serum testosterone levels of control (CTL) and PH groups (irregular ovulatory (PHiov) and anovulatory (Phanov)); (C) Serum estradiol levels of control (CTL) and PH groups (irregular ovulatory (PHiov) and anovulatory (Phanov)); (D) Estradiol to testosterone ratio of control (CTL) and PH groups (irregular ovulatory (PHiov) and anovulatory (Phanov)); (E) Serum progesterone levels of control (CTL) and PH groups (irregular ovulatory (PHiov) and anovulatory (Phanov)); gray bars: groups without metformin treatment, black bars: groups with metformin treatment. Each column represents the mean + s.e.m. from seven animals per group. Data were analyzed by two-way ANOVA, with post hoc Tukey’s test. a vs b; P < 0.05.

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    Metformin treatment: ratio between AMPKpT172 and AMPK of control (CTL) and PH groups (irregular ovulatory (PHiov) and anovulatory (PHanov)); gray bars: groups without metformin treatment, black bars: groups with metformin treatment. Each column represents the mean + s.e.m., from six animals per group. Data were analyzed by two-way ANOVA, with post hoc Tukey’s test. a vs b; P < 0.05. For the Western blot analysis, representative images for all groups are shown; all the bands for each picture come always from the same gel, but they may be spliced for clarity.

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    Gene and protein expression of insulin receptor (IR). The graphs correspond to (A) mRNA abundance of the IR gene relative to L32 mRNA levels; (B) protein levels of IR relative to B-tubulin of control (CTL) and PH groups (irregular ovulatory (PHiov) and anovulatory (PHanov)); gray bars: groups without metformin treatment, black bars groups with metformin treatment. Each column represents the mean + s.e.m.; from ten animals per group for qPCR analysis and six animals per group for western blot. Data were analyzed by two-way ANOVA, with post hoc Tukey’s test. a vs b; P < 0.05. For the Western blot analysis, representative images for all groups are shown; all the bands for each picture come always from the same gel, but they may be spliced for clarity.

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    Graphs correspond to (A) mRNA abundance of the IRS1 gene relative to L32 mRNA levels; (B) mRNA abundance of the IRS2 gene relative to L32 mRNA levels; (C) protein levels of IRS1/2 relative to B-actin; (D) ratio between pIRS1Y612 and IRS1; (E) ratio between pIRS1S307 and IRS1 gray bars: groups without metformin treatment, black bars: groups with metformin treatment. A representative image of the media of bands obtained from each group is shown. Each column represents the mean + s.e.m.; from ten animals per group for qPCR analysis and six animals per group for western blot. Data were analyzed by two-way ANOVA, with post hoc Tukey’s test. a vs b; P < 0.05. For the Western blot analysis, representative images for all groups are shown; all the bands for each picture come always from the same gel, but they may be spliced for clarity.

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    Glucose transporter. The graphs correspond to (A) mRNA abundance of Glut4 relative to L32 mRNA levels; (B) protein levels of GLUT4 relative to B-tubulin of control and PH groups (irregular ovulatory (PHiov) and anovulatory (PHanov)); a representative image of the media of bands obtained from each group is shown; gray bars: groups without metformin treatment, black bars: groups with metformin treatment. Each column represents the mean + s.e.m.; from six animals per group for western blot. Data were analyzed by two-way ANOVA, with post hoc Tukey’s test. a vs b; P < 0.05. For the Western blot analysis, representative images for all groups are shown; all the bands for each picture come always from the same gel, but they may be spliced for clarity.

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    AKT phosphorylation. The graph correspond to ratio of AKTpS473 and total AKT of control (CTL) and prenatally hyperandrogenized (PH) groups (irregular ovulatory (PHiov) and anovulatory (PHanov)); a representative image of the media of bands obtained from each group is shown; gray bars: groups without metformin treatment, black bars: groups with metformin treatment. Each column represents the mean + s.e.m.; from animals six animals per group. Data were analyzed by two-way ANOVA, with post hoc Tukey’s test. a vs b; P < 0.05. For the Western blot analysis, representative images for all groups are shown; all the bands for each picture come always from the same gel, but they may be spliced for clarity.

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    Insulin pathway and ovarian function. Insulin binding to its receptor promotes the autophosphorylation of the insulin receptor (IR) which phosphorylates intracellular substrates (IRS), initiating signal transduction pathways mediating the pleiotropic actions of insulin. The major pathway for the metabolic actions of insulin is mediated through activation of PI3-K and Akt, resulting in the translocation for the insulin-responsive glucose transporter, GLUT4, from intracellular vesicles to the plasma membrane. Glucose is essential for oocyte development and ovulation. The Akt pathway also promotes the expression of steroidogenic enzymes, thus stimulating steroidogenesis and follicular development. The action of insulin on steroidogenesis is enhanced by the binding of LH and FSH to its receptors.

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