Maternal Choline and Betaine Supplementation Modifies the Placental Response to Hyperglycemia in Mice and Human Trophoblasts

Abstract

Gestational diabetes mellitus (GDM) is characterized by excessive placental fat and glucose transport, resulting in fetal overgrowth. Earlier we demonstrated that maternal choline supplementation normalizes fetal growth in GDM mice at mid-gestation. In this study, we further assess how choline and its oxidation product betaine influence determinants of placental nutrient transport in GDM mice and human trophoblasts. C57BL/6J mice were fed a high-fat (HF) diet 4 weeks prior to and during pregnancy to induce GDM or fed a control normal fat (NF) diet. The HF mice also received 25 mM choline, 85 mM betaine, or control drinking water. We observed that GDM mice had an expanded placental junctional zone with an increased area of glycogen cells, while the thickness of the placental labyrinth zone was decreased at E17.5 compared to NF control mice (p < 0.05). Choline and betaine supplementation alleviated these morphological changes in GDM placentas. In parallel, both choline and betaine supplementation significantly reduced glucose accretion (p < 0.05) in in vitro assays where the human choriocarcinoma BeWo cells were cultured in high (35.5 mM) or normal (5.5 mM) glucose conditions. Expression of angiogenic genes was minimally altered by choline or betaine supplementation in either model. In conclusion, both choline and betaine modified some but not all determinants of placental transport in response to hyperglycemia in mouse and in vitro human cell line models.

Keywords: betaine; choline; gestational diabetes; nutrient transport; placental morphology; vasculature.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1

Study design. BS: betaine supplemented; CO: untreated control; CS: choline supplemented; E: embryonic day; HF: high-fat diet; NF: normal-fat diet.

Figure 2

Placental histology at E12.5 and E17.5. (a,b) Relative thickness of placental layers. (c) Histological appearance of representative placentas at E17.5. (d,e) Glycogen cell area in the junctional zone. Results are presented as glycogen cell area/total junctional zone area. (f) Histological images demonstrate representative junctional zones with glycogen cells (arrowheads) at E17.5. (g) Labyrinth blood space. Results are presented as labyrinth blood space/total labyrinth layer area. Each group contained 5 dams. Different diets were fed to dams from 4 weeks before timed-mating to gestational day 12.5 or E17.5. Placentas from one male and one female embryo in each dam were included in the analysis. Values are mean ± standard error of mean (SEM); a, b: any two groups with no overlapping characters have a statistically significant difference (p < 0.05) between them. D, decidua; J, junctional zone; L, labyrinth zone; NF: normal-fat diet; HF: high-fat diet; BS: betaine supplemented; CO: untreated control; CS: choline supplemented; NS: not significant.

Figure 3

mRNA abundance of angiogenic genes in the placenta at E12.5 (a) and E17.5 (b). Different diets were fed to dams from 4 weeks before timed-mating to gestational day 12.5 or 17.5. mRNA abundance was measured by real-time PCR. Each group contained 5 dams. Different diets were fed to dams from 4 weeks before timed-mating to gestational day 12.5 or E17.5. Placentas from one male and one female embryo in each dam were included in the analysis. Values are mean ± standard error of mean (SEM); a, b, c: any two groups with different characters have a statistically significant difference (p < 0.05) between them. Solid bars: NF-CO; shaded bars: HF-CO; open bars: HF-CS; hatched bars: HF-BS. Casp3: caspase 3; Pcna: proliferating cell nuclear antigen; Pgf: placental growth factor; sFLT1: soluble fms-like tyrosine kinase 1; Vegfa: vascular growth factor A. NF: normal-fat diet; HF: high-fat diet; BS: betaine supplemented; CO: untreated control; CS: choline supplemented; NS: not significant.

Figure 4

mRNA abundance in BeWo cells. (a) Proliferative and apoptotic genes; (b) Angiogenic and anti-angiogenic genes; (c) macronutrient transporters. BeWo cells were treated with 1mM choline (CS), 1 mM betaine (BS), or saline control and with 30 mM glucose (HG) or 30 mM mannose (NG) in the Minimum Essential Medium for 48 h. mRNA expression was analyzed with real-time PCR. Each experiment was done in triplicate and repeated 3 times. Values are mean ± standard error of mean (SEM); a, b, c: any two groups with no overlapping characters have a statistically significant difference (p < 0.05) between them. Solid bars: NG-CO; shaded bars: HG-CO; open bars: HG-CS; hatched bars: HG-BS. CASP3: caspase 3; FATP1: fatty acid transporter 1; FATP4: fatty acid transporter 4; GLUT1: glucose transporter 1; GLUT3: glucose transporter 3; PCNA: proliferating cell nuclear antigen; PGF: placental growth factor; sFLT1: soluble fms-like tyrosine kinase 1; SNAT2: Sodium-dependent neutral amino acid transporter-2; VEGFA: vascular growth factor A; NG: normal glucose; HG: high glucose; BS: betaine supplemented; CO: untreated control; CS: choline supplemented; NS: not significant.

Figure 5

Glucose and fatty acid accumulation by BeWo cells after 1-h incubation. (a) The luminescence intensity of labeled glucose that was accumulated in the BeWo cells; (b) The fluorescence intensity of labeled fatty acids that were accumulated in the BeWo cells. BeWo cells were treated with 1 mM choline (CS), 1mM betaine (BS), or saline control and with 30 mM glucose (HG) or 30 mM mannose (NG) in the Minimum Essential Medium for 48 h, followed by 1-h incubation with the labeled glucose or fatty acid probes. Each experiment was done in triplicate and repeated 3 times. Values are mean ± standard error of mean (SEM); a, b, c: any two groups with no overlapping characters have a statistically significant difference (p < 0.05) between them. Solid bars: NG-CO; shaded bars: HG-CO; open bars: HG-CS; hatched bars: HG-BS. NG: normal glucose; HG: high glucose; BS: betaine supplemented; CO: untreated control; CS: choline supplemented.