Knockout maternal adiponectin increases fetal growth in mice: potential role for trophoblast IGFBP-1

Abstract

Aims/hypothesis: The main objective of this study was to investigate whether maternal adiponectin regulates fetal growth through the endocrine system in the fetal compartment.

Methods: Adiponectin knockout (Adipoq (-/-) ) mice and in vivo adenovirus-mediated reconstitution were used to study the regulatory effect of maternal adiponectin on fetal growth. Primary human trophoblast cells were treated with adiponectin and a specific peroxisome proliferator-activated receptor α (PPARα) agonist or antagonist to study the underlying mechanism through which adiponectin regulates fetal growth.

Results: The body weight of fetuses from Adipoq (-/-) dams was significantly greater than that of wild-type dams at both embryonic day (E)14.5 and E18.5. Adenoviral vector-mediated maternal adiponectin reconstitution attenuated the increased fetal body weight induced by maternal adiponectin deficiency. Significantly increased blood glucose, triacylglycerol and NEFA levels were observed in Adipoq (-/-) dams, suggesting that nutrient supply contributes to maternal adiponectin-regulated fetal growth. Although fetal blood IGF-1 concentrations were comparable in fetuses from Adipoq (-/-) and wild-type dams, remarkably low levels of IGF-binding protein 1 (IGFBP-1) were observed in the serum of fetuses from Adipoq (-/-) dams. IGFBP-1 was identified in the trophoblast cells of human and mouse placentas. Maternal fasting robustly increased IGFBP-1 levels in mouse placentas, while reducing fetal weight. Significantly low IGFBP-1 levels were found in placentas of Adipoq (-/-) dams. Adiponectin treatment increased IGFBP-1 levels in primary cultured human trophoblast cells, while the PPARα antagonist, MK886, abolished this stimulatory effect.

Conclusions/interpretation: These results indicate that, in addition to nutrient supply, maternal adiponectin inhibits fetal growth by increasing IGFBP-1 expression in trophoblast cells.

Keywords: Adiponectin; Fetus; Growth; IGF-binding protein; Placenta.

Fig. 1

Effects of maternal adiponectin on fetal body weight. (a) Schematic diagram showing the production of Adipoq^−/+ fetuses. For this, 10–12 week-old nulliparous Adipoq^−/− and WT female mice were mated with WT or Adipoq^−/− male mice. (b) Maternal body weight. (c) Gestational weight gain was calculated by the difference in body weight between E0.5 and E18.5 (with pups). (d) Maternal body fat, (e) litter size and (f) fetal body weight were determined after removal of fetuses through Caesarean section at E18.5. (g) At E15, Adipoq^−/− dams (n=6) were transduced with purified Ad-gfp or Ad-Adipoq through tail vein injection. Fetal samples were collected and weighed at E18.5. (h,k) Blood glucose, (i) NEFA and (j) TG levels were measured using glucose oxidase or assay kits. (l) Fetal:placental weight ratios were calculated using samples at E18.5. (b, d) Maternal body composition was determined by EchoMRI. (b–e, h–j) n=8–10; (f, g, l) n=45–72; (k) n=12. *p<0.05, **p<0.01, ***p<0.001

Fig. 2

Maternal adiponectin has no effect on blood IGF-1 concentration but increases IGFBP-1 protein levels in fetal blood. (a, b) Blood total IGF-1 concentrations were analysed in (a) fetuses (n=12) and (b) dams (n=8). (c) mRNA levels of Igf2 and Igf2r were determined using qPCR with a relative quantification assay (white bar, Fwtd; filled bar, Fakd). (d) Fetal blood insulin was measured using a mouse diabetes multiplex assay kit (n=12). (e, f) IGFBP-1 protein levels in fetal serum samples from Adipoq^−/− or adiponectin-reconstituted and control dams (n=12) were measured by western blotting with specific antibodies. (g) Levels of phosphorylated or total IGF-1Rβ protein were determined in fetal livers at E18.5 (n=6) by western blotting. RU, relative units. *p<0.05

Fig. 3

IGFBP-1 levels in trophoblast cells and the effect of maternal adiponectin on placental IGFBP-1 content. (a) IGFBP-1 protein levels were determined by western blotting of fetal liver samples (E18.5) from WT or Adipoq^−/− dams obtained under normal feeding conditions (n=12). (b, c) The placental labyrinth of C57BL/6 mice at E18.5 and (d, e) the villous fraction of term human placentas were probed with (b, d; brown colour) an anti-IGFBP-1 antibody or (c, e) normal rabbit serum. Images were captured with magnification ×40. (f–j) Pregnant C57BL/6 mice were fasted overnight or fed before tissue collection at E18.5 (n=8). IGFBP-1 levels were determined by western blotting in (f) fetal blood, (g) placental or (h) fetal liver samples. (i) Overnight fasting reduced fetal weight (n=54–68). (j) IGFBP-1 levels were measured by western blotting in placental tissue samples from WT or Adipoq^−/− dams (n=8). (k) Igfbp1 mRNA levels were measured by qPCR in placenta samples from WT or Adipoq^−/− dams (n=8). (l) IGFBP-1 levels were measured by western blotting in placental tissue samples from Ad-gfp or Ad-Adipoq-transduced Adipoq^−/− dams (n=8). RU, relative units. *p<0.05, **p<0.01

Fig. 4

Adiponectin enhanced the IGFBP-1 expression in trophoblast cells via PPARα. (a) IGFBP-1 levels in primary trophoblast cells treated overnight with a PPARα agonist WY14643 (5 µmol/l) or antagonist MK886 (10 µmol/l) were analysed by western blotting. (b) Adiponectin was ectopically expressed in Ad-Adipoq-transduced Fao cells. Insert wells containing transduced Fao cells were co-cultured overnight with trophoblast cells. MK886 was then added to one group of adiponectin-treated cells (Adipoq). IGFBP-1 protein levels in trophoblast cells were analysed by western blotting (n=6). RU, relative units. *p<0.05 vs control cells (Con)