Adiponectin regulates glycogen metabolism at the human fetal–maternal interface

in Journal of Molecular Endocrinology
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Throughout the entire first trimester of pregnancy, fetal growth is sustained by endometrial secretions, i.e. histiotrophic nutrition. Endometrial stromal cells (EnSCs) accumulate and secrete a variety of nutritive molecules that are absorbed by trophoblastic cells and transmitted to the fetus. Glycogen appears to have a critical role in the early stages of fetal development, since infertile women have low endometrial glycogen levels. However, the molecular mechanisms underlying glycogen metabolism and trafficking at the fetal–maternal interface have not yet been characterized. Among the various factors acting at the fetal–maternal interface, we focused on adiponectin – an adipocyte-secreted cytokine involved in the control of carbohydrate and lipid homeostasis. Our results clearly demonstrated that adiponectin controls glycogen metabolism in EnSCs by (i) increasing glucose transporter 1 expression, (ii) inhibiting glucose catabolism via a decrease in lactate and ATP productions, (iii) increasing glycogen synthesis, (iv) promoting glycogen accumulation via phosphoinositide-3 kinase activation and (v) enhancing glycogen secretion. Furthermore, our results revealed that adiponectin significantly limits glycogen endocytosis by human villous trophoblasts. Lastly, we demonstrated that once glycogen has been endocytosed into placental cells, it is degraded into glucose molecules in lysosomes. Taken as a whole, the present results demonstrate that adiponectin exerts a dual role at the fetal–maternal interface by promoting glycogen synthesis in the endometrium and conversely reducing trophoblastic glycogen uptake. We conclude that adiponectin may be involved in feeding the conceptus during the first trimester of pregnancy by controlling glycogen metabolism in both the uterus and the placenta.

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  • Supplementary data
  • Figure Suppl. 1 - FITC-dextran co-localizes with a placental endosomal marker
  • Figure Suppl. 2 - FITC-dextran co-localizes with a placental lysosomal marker
  • Figure Suppl. 3 - FITC-dextran is absorbed by endocytosis and directed to lysosomes in placental cells

 

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    Adiponectin increases GLUT 1 expression and glucose uptake by EnSCs throughout decidualization. Human EnSCs were cultured in DMEM/F12 medium supplemented with E2 and P4 and exposed or not to adiponectin (25 ng/mL and 250 ng/mL). (A and B) Total RNA was extracted after 3 (D3), 8 (D8) and 15 days (D15) of cell decidualization. GLUT1 and GLUT3 mRNA expression levels were quantified by RT-qPCR, as described in the ‘Materials and methods’ section. The data are quoted as the mean ± s.e.m. of nine separate experiments. (A) GLUT1 mRNA expression. (B) GLUT3 mRNA expression. (C) Total protein were extracted at D15. GLUT1 Western blot and densitometric analysis were performed as described in ‘Materials and methods’ section. The data are quoted as the mean ± s.e.m. of five separate experiments. The insert figure shows one representative of five separate experiments. (D) The glucose level in the supernatant was measured in supernatant after 3 (D3), 8 (D8) and 15 days (D15) of cell decidualization. The data are quoted as the mean ± s.e.m. of six separate experiments. The control values were 14.31 ± 1.63 mmol/µg protein at D3, 18.40 ± 1.28 mmol/µg protein at D8, and 16.51 ± 1.26 mmol/µg protein at D15. *P < 0.05; **P < 0.01. (a) vs a control experiment in the absence of adiponectin. Wilcoxon test. (b) vs D3. ANOVA test. (c) vs D8. ANOVA test. A full colour version of this figure is available at https://doi.org/10.1530/JME-18-0013.

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    Adiponectin decreases glucose catabolism in EnSCs. Human EnSCs were cultured in DMEM/F12 medium supplemented with E2 and P4, and exposed or not to adiponectin (25 ng/mL and 250 ng/mL) for 15 days (D15) of cell decidualization. (A) Total ATP production was determined at D15. The data are quoted as the mean ± s.e.m. of eight separate experiments. The control value was 80.59 ± 12.17 µM ATP. (B) Lactate production was measured at D15. The data are quoted as the mean ± s.e.m. of six separate experiments. The control value was 2.08 ± 0.66 mmol/µg protein. *P < 0.05. Wilcoxon test. A full colour version of this figure is available at https://doi.org/10.1530/JME-18-0013.

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    Adiponectin promotes glycogenesis in EnSCs. Human EnSCs were cultured in DMEM/F12 medium supplemented with E2 and P4 and exposed or not to adiponectin. (A and B) Human EnSCs were exposed or not to adiponectin (25 ng/mL and 250 ng/mL). Total RNA was extracted after three (D3), eight (D8), and 15 days (D15) of cell decidualization. mRNA expression levels of GS and GP were quantified by RT-qPCR, as described in ‘Materials and methods’ section. The data are quoted as the mean ± s.e.m. of nine separate experiments. (A) GS mRNA expression. (B) GP mRNA expression. (C) Human EnSCs were exposed or not to adiponectin (25, 250, 500 and 1000 ng/mL) for 15 days (D15) of cell decidualization. Intracellular glycogen accumulation was determined at D15. The data are quoted as the mean ± s.e.m. of eight separate experiments. The control value was 0.91 ± 0.08 µg/µL. *P < 0.05. (a) vs a control experiment in the absence of adiponectin. Wilcoxon test. (b) vs D3. ANOVA test. (c) vs D8. ANOVA test. A full colour version of this figure is available at https://doi.org/10.1530/JME-18-0013.

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    Adiponectin regulates glucose metabolism pathways in EnSCs. Human EnSCs were cultured in DMEM/F12 medium supplemented with E2 and P4 and exposed or not to adiponectin (25 ng/mL and 250 ng/mL) for 15 days (D15) of cell decidualization. Total protein were extracted at D15. (A) Phospho-AMPKα (Thr-172) and total-AMPKα Western blot analysis were realized. The figure represents one representative of five separate experiments. (B) Densitometric analysis of the active phospho-AMPKα (Thr-172)/total-AMPKα protein immunoblots was performed as described in the ‘Materials and methods’ section. (C) Phospho-GSK3β (Ser-9) and total-GSK3β Western blot analysis were realized. The figure represents one representative of five separate experiments. (D) Densitometric analysis of the active phospho-GSK3β (Ser-9)/total-GSK3β protein immunoblots was performed as described in the ‘Materials and methods’ section. The data are quoted as the mean ± s.e.m. of five separate experiments. *P < 0.05. Wilcoxon test. A full colour version of this figure is available at https://doi.org/10.1530/JME-18-0013.

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    Adiponectin activates EnSC glycogen accumulation via PI3K pathway. Human EnSCs were cultured in DMEM/F12 medium supplemented with E2 and P4 and exposed or not to adiponectin (250 ng/mL) in presence or in absence of PI3K inhibitor (LY294002, 10−6 M) and AMPK inhibitor (Compound C, 10−6 M) for 15 days (D15) of cell decidualization. Intracellular glycogen accumulation was determined at D15. The data are quoted as the mean ± s.e.m. of six separate experiments. The control value was 0.91 ± 0.08 µg/µL. *P < 0.05. (a) vs control (without inhibitors nor adiponectin). (b) adiponectin + Compound C vs adiponectin alone. (c) adiponectin + LY294002 vs adiponectin alone. Wilcoxon test. A full colour version of this figure is available at https://doi.org/10.1530/JME-18-0013.

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    Adiponectin enhances EnSCs glycogen secretion and VT glycogen accumulation. (A) Human EnSCs were cultured in DMEM/F12 medium supplemented with E2 and P4 and exposed or not to adiponectin (25 ng/mL and 250 ng/mL) for 15 days (D15) of cell decidualization. Extracellular glycogen secretion was measured in cell supernatants at D15. The data are quoted as the mean ± s.e.m. of eight separate experiments. The control value was 0.28 ± 0.03 µg/µL. (B) Human VTs were cultured in the presence of conditioned medium (CM) from fully decidualized EnSCs treated or not with adiponectin (25 ng/mL or 250 ng/mL) for 15 days (D15). As a negative control, VTs were cultured in the presence of CM from non-decidualized EnSCs (i.e. cultured without E2 and P4). After 48 h, intracellular glycogen accumulation in VTs was measured. The data are quoted as the mean ± s.e.m. of ten separate experiments. The control value was 0.77 ± 0.13 µg/µL. *P < 0.05. Wilcoxon test. A full colour version of this figure is available at https://doi.org/10.1530/JME-18-0013.

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    Adiponectin decreases FITC-dextran uptake by VTs. (A) The structure of glycogen. (B) The structure of dextran. (C) VTs were incubated with FITC-dextran as described in the ‘Materials and methods’ section. FITC-dextran absorption was analyzed by confocal microscopy. Magnification ×40. The figure represents one representative of three separate experiments. (a) 30-min incubation. (b) 1-h incubation. (c) 2-h incubation. (d) 4-h incubation. (D) VTs cultured for 3 days and incubated for the two last hours with FITC-dextran. Quantification of FITC-dextran absorption by VTs with or without adiponectin (25 ng/mL and 250 ng/mL) was performed as described in the ‘Materials and methods’ section. The data are quoted as the mean ± s.e.m. of ten separate experiments. *P < 0.05. Wilcoxon test. A full colour version of this figure is available at https://doi.org/10.1530/JME-18-0013.

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    Proposed mechanism by which adiponectin controls glycogen metabolism and trafficking at the fetal–maternal interface. In human EnSCs, adiponectin stimulates glucose uptake by upregulating GLUT1 mRNA expression. Then, adiponectin significantly reduces the catabolic glucose pathways (anaerobic and aerobic glycolysis) and conversely increases both glycogen synthesis (by directly inducing GS mRNA expression) and glycogen accumulation. This last effect is, in part, mediated by PI3K transduction signaling. Glycogen secretion being an apocrine mechanism, adiponectin also improves glycogen secretion. At the feto–maternal interface, adiponectin limits glycogen uptake by VTs. Once endocytosis, glycogen is directed toward lysosomes in order to be degraded into glucose. Glucose could then be used by placental cells for its own metabolic needs, or transmitted to the fetus in order to insure fetal nutrition. GS, glycogen synthase; ↗, increase; ↘, inhibition. A full colour version of this figure is available at https://doi.org/10.1530/JME-18-0013

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