Effects of glucose and fatty acids interactions on glucose metabolism pathways in human trophoblasts

Abstract

Introduction: Obesity, with its associated co-morbidities of diabetes, hyperlipidaemia and low-grade inflammation, has high prevalence in women of child-bearing age. Of further concern, it is associated with increased risk of adverse pregnancy outcomes. Considering the key role of the placenta in fetal development, alterations to its structure and function in response to the adverse metabolic milieu of maternal obesity must be important. The trophoblast is a key cell within the placental maternal-fetal barrier. The research aims were to evaluate the effects of elevated glucose supply, in the absence and presence of non-esterified fatty acids (NEFA), on pathways of glucose metabolism and trophoblast morphology in human trophoblasts in primary culture. Methods: Trophoblasts were established in primary culture from human term placentas of normal pregnancies. The isolation protocol was optimised to improve trophoblast yield and purity. Experiments were performed at both 36 h (short-term) and 5 days (long-term) after trophoblast isolation, when most cells were, respectively, cytotrophoblasts and syncytiotropblasts. Syncytialisation was promoted by adding epidermal growth factor (EGF; 10 ng/ml) to the media. Metabolism experiments were performed over 2 h at diverse glucose concentrations in the absence or presence of NEFA (0.25 mM, palmitate: oleate ratio; 1:1). Pathways of glucose uptake, glycolysis, glucose oxidation, total and partial palmitate oxidation and glycogen synthesis were assessed using radio-labelled tracers. Lactate production and glycogen content were measured enzymatically. The effects of EGF and NEFA on trophoblast morphology, lipid droplet formation and triacylglycerol (TG) content were also assessed. Results: The improved protocol increased the mean (± SD) trophoblast yield to 266 ± 141 x 10⁶ cells, and purity to 98%. In short-term cultures, rates of trophoblast glucose uptake and glycogen synthesis increased linearly with increasing glucose concentrations; whereas, rates of glycolysis, lactate release and glucose oxidation did not increase with glucose concentrations above 5 mM. NEFA enhanced glycogen synthesis, but did not affect other glucose metabolism pathways. Total trophoblast glycogen content increased minimally with increasing glucose supply, which was not altered by NEFA. In long-term compared to short-term culture experiments, glycogen synthesis was lower and the trophoblasts were able to increase glycolysis and glucose oxidation rates with media glucose concentrations above 5 mM. NEFA increased glucose oxidation at the media glucose concentration of 1 mM. Palmitate complete oxidation to CO₂ and partial oxidation to acid soluble products were significant metabolic pathways in both the short and long-term cultured cells. EGF promoted syncytialisation with a lesser effect of NEFA. Neither EGF nor NEFA affected trophoblast lipid droplet or TG accumulation. Conclusion: During acute exposure to high glucose media, human trophoblasts divert glucose uptake to glycogen synthesis rather than glycolysis and glucose oxidation. This diversion, however, is less evident in more differentiated trophoblasts. NEFA have minimal effects on glucose metabolism pathways, but are a significant source of cellular metabolic energy via oxidation to CO₂, but also by partial oxidation to acid soluble products (e.g. ketone bodies, short-chain fatty acids and tricarboxylic acid cycle intermediates). Further analysis of these pathways, particularly in placentas of obese women, are warranted.