Gene Expression Suggests Mouse and Bovine Pre-Implantation Embryos Utilize the Warburg Effect. (2012)

Metabolism is a crucial determining factor of embryo quality and viability; however, embryo metabolism is usually thought of in terms of traditional somatic cell pathways that do not take into account the metabolic requirements of rapid cell division occurring during pre-implantation development. The objective of this experiment was to investigate components of the Warburg Effect (WE), or aerobic glycolysis, in mouse and bovine pre-implantation embryos. In WE, glucose is metabolized through glycolysis but glycolytic end products do not enter the TCA cycle. Rather, glycolytic intermediates enter the pentose phosphate pathway, in addition to lactate production. Our hypothesis is that early embryos utilize WE to provide building blocks to support rapid cell division, as production of ATP from glucose is not crucial when sufficient fatty acid oxidation (FAO) occurs. Expression of genes involved in WE (HK2, PDK1, PGAM1, PKM2), glycolysis (GLUT1), FAO (ACADL, ACSL3, CPT1B, CPT2) and lactate production (LDHA/B) were examined in mouse and bovine blastocysts produced after in vitro maturation and fertilization. Bovine and mouse embryos were cultured in four treatment groups; 1) low oxygen (O2; 5%; optimal environment), 2) high O2 (20%; suboptimal environment), 3) low O2 with supplementation of 1 mM carnitine (a necessary FAO co-factor), and 4) high O2 with supplementation of 1 mM carnitine. Blastocysts were frozen in groups of two and RNA was extracted. cDNA was synthesized and relative transcript abundance was determined by quantitative PCR (qPCR) followed by REST analysis. Culturing bovine embryos in high O2 down regulates GLUT1, HK2 and PKM2 compared to a low O2 environment, suggesting a decrease in glucose uptake and WE activity in suboptimal conditions. The addition of carnitine in low O2 increased CPT2 and LDHA expression in bovine blastocysts, suggesting an increase in lactate production and FAO in an optimal environment. In the mouse, culture at high compared to low O2 had no effect on expression of the genes analyzed. Addition of carnitine in low O2 decreased LDHB and PKM2, suggesting that in mice, under optimal conditions, carnitine increases FAO thus decreasing the need for tightly controlled glycolytic regulation. ACSL3 was down regulated and GLUT1 and PKM2 up regulated in high O2 when carnitine was added, suggesting a perturbation of FAO, resulting in an increased need for ATP production from pyruvate and an up regulation of WE to control glucose metabolism. Carnitine in high O2 decreased CPT2 and up regulated GLUT1, HK2, PKM2 and PGAM1 expression compared to carnitine in low O2, again suggesting an up regulation of WE mechanisms when FAO is perturbed to regulate glucose metabolism. There were no differences in gene expression of ACADL, CPT1B, or PDK1 in mouse or bovine embryos due to treatment. In conclusion, this gene expression data supports our proposed hypothesis that under conditions optimal for embryo development, glycolysis is regulated by WE while FAO operates to provide ATP, and suggests an inter-dependency of these two metabolic pathways. This metabolic strategy confers a selective growth advantage by allowing accumulation of glycolytic intermediates, which can then be utilized by the embryo to produce building blocks required during the rapid cell divisions that occur during pre-implantation development. This is a novel way of thinking about embryo metabolism.