Glucose transporter (GLUT) 8 is an insulin-responsive facilitative glucose transporter expressed predominantly in the murine blastocyst. To determine the physiologic role of GLUT8, two-cell embryos were cultured to a blastocyst stage in antisense or sense oligonucleotides to GLUT8. Apoptosis was assessed using the TUNEL techniques and recorded as the percentage of TUNEL-positive nuclei/total nuclei. Embryos cultured in GLUT8 antisense experienced increased TUNEL-positive nuclei, whereas sense embryos did not. Embryos cultured in a control AS oligonucleotide, specific for heat shock protein 70-2, showed a rate of apoptosis similar to sense. To determine the outcome of these apoptotic embryos, blastocysts exposed to sense vs. antisense were transferred back into foster mice and the pregnancy continued until Day 14.5, at which time the uteri were examined for normal gestational sacs and resorptions. Embryos exposed to GLUT8 antisense experienced higher rates of resorptions and lower normal pregnancy rates compared to embryos cultured in GLUT8 sense. To examine the insulin growth factor (IGF)-1/insulin intracellular signaling pathways involved in GLUT8 translocation, IGF-1 receptor (IGF-1R) expression was decreased in the blastocysts with antisense oligonucleotides. Using confocal immunofluorescent microscopy, GLUT8 translocation in response to insulin was observed. Exposure to insulin in the embryos exposed to IGF-1R sense induced translocation of GLUT8 from intracellular compartments to the plasma membrane. Blastocysts exposed to IGF-1R antisense, however, failed to demonstrate any change in the intracellular location of GLUT8 with insulin treatment. The IGF-1R antisense embryos also displayed significantly greater TUNEL staining compared to sense embryos. These data suggest that GLUT8 expression and translocation in response to insulin are critical for blastocyst survival.

Metabolic changes in the glucose-induced apoptotic blastocyst suggest alterations in mitochondrial physiology. Am J Physiol Endocrinol Metab 283: E226-E232, 2002. First published March 27, 2002 10.1152/ajpendo.00046.2002Mammalian preimplantation embryos experience a critical switch from an oxidative to a predominantly glycolytic metabolism. In this study, the change in nutrient metabolism between the 2-cell and blastocyst stages was followed by measuring single embryo concentrations of tricarboxylic acid (TCA) cycle and glycolytic metabolites with microfluorometric enzymatic cycling assays. When the normal values were established, further changes that occur as a result of the induction of apoptosis by exposure to high-glucose conditions were examined. From the 2-cell to the blastocyst stage, the embryos experienced an increase in TCA metabolites and a dramatic increase in fructose 1,6-bisphosphate (FBP). The high TCA metabolites may result from accumulation of substrate due to a slowing of TCA cycle metabolism as glycolysis predominates. Embryos exposed to elevated glucose conditions experienced significantly lower FBP, suggesting decreased glycolysis, significantly higher pyruvate, suggesting increased pyruvate uptake by the embryos in response to decreased glycolysis, and increased TCA metabolites, suggesting an inability to oxidize the pyruvate and a slowing of the TCA cycle. We speculate that the glycolytic changes lead to dysfunction of the outer mitochondrial membrane that results in the abnormal TCA metabolite pattern and triggers the apoptotic event.tricarboxylic acid cycle; glycolysis; preimplantation embryo; programmed cell death GLUCOSE TRANSPORT AND METABOLISM are critical for mammalian blastocyst formation and further development (8,20). At this stage, the switch occurs from oxidation of pyruvate via the tricarboxylic acid (TCA) cycle to the use of glucose as the main substrate via glycolysis (7,14). As a result, the blastocyst exhibits extreme sensitivity to glucose deprivation. We have previously shown that any decrease in glucose transport, basal or insulin stimulated, results in enhanced apoptosis at this stage, which manifests later in pregnancy as a malformation or miscarriage (3,4,21,22). This decrease in blastocyst glucose transport and resulting apoptosis occur in conditions of maternal hyperglycemia and hyperinsulinemia.The blastocyst stage marks a new peak in cellular proliferation and growth as the first epithelial layer, the trophectoderm, is formed. These changes create new biosynthetic demands on the embryo. Maintenance of a high rate of glycolysis is thought to be important for providing a "dynamic buffer" of metabolic intermediates for the biosynthesis of macromolecules (23). For example, glucose 6-phosphate is used in the formation of the ribose 5-phosphate required for DNA and RNA synthesis. Another reason for the increased glucose demand may be that increasing amounts of glucose are converted to lactate at the blastocyst stage in humans and rodents (14). In these species in part...

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