NEURAL PLATE MICROVILLUS LENGTHENING IN RAT EMBRYOS GROWN IN VARIOUS CONCENTRATIONS OF GLUCOSE AND FURTHER-STUDIES OF THE MECHANISM

Glucose is an important cellular nutrient, and in the early embryo, wh ich is dependent mostly on anaerobic glycolysis, it is even more essen tial. Based on tissue culture cells in which glucose utilization has b ecome membrane-limited, a concept has been developed that the tip of t he microvilli is the entrance compartment for glucose and that the sha ft sets vp a diffusion barrier. An increase in length of the microvill us is associated with decreased entry of phosphorylated hexose into th e cells. Our previous findings of lengthening of the microvilli of the neural plate cells after 40 min exposure to glucose at room temperatu re have been extended to a 17 hr whole embryo culture system. In cultu res where the final concentration of glucose was 20 and 24 mg/dl there was embryonic death. In those cultures ending with 29-137 mg/dl of gl ucose the embryos developed normally. Those grown in dialyzed serum su pplemented with B vitamins and glucose grew equally as well as those i n whole rat serum. Somite numbers attained did not change with increas ing glucose concentration but a modest increase in micromoles of gluco se used per embryo was found, suggesting the presence of another sourc e of energy at lower glucose concentrations. The average glucose utili zation per gram of protein per hour was 844 mu mol in these day 9.5-10 embryos and this compares to 733 mu mol previously found using unifor mly labeled C-14 glucose on day 10.3. Lactate production averaged 85% of the glucose utilized. Pyruvate did not support growth in the absenc e of glucose. Lengthening of the microvilli was studied using scanning electron microscopy (SEM) and an association between increasing gluco se concentration and lengthening (with matting) of the microvilli was shown after whole embryo culture and after 40 min exposure at room tem perature. In whole culture the microvilli were short in cultures endin g with glucose concentrations below 30 mg/dl, partially matted at 36-8 1 mg/dl, and fully matted at 78-137 mg/dl. Elongation of microvilli wa s found in the mouse during the early somite neural plate stage, but t he microvilli were more sparse and the cells contained a single cilium . These later two differences might explain the increased sensitivity to hypoglycemia in the mouse. These studies were done after exposure t o glucose at 150 mg/dl for 40 min at room temperature. Also at room te mperature, day 9.5 rat neural epithelial microvilli lengthened when ex posed to glucose. Studies for 40 min at room temperature were carried out to investigate the mechanism of lengthening. L-glucose, fructose, galactose, and pyruvate, which are not transported and phosphorylated, did not cause microvillar lengthening. 2-Deoxyglucose, which is trans ported and phosphorylated but does not enter the glycolytic pathway, c aused microvillar lengthening. Cytochalasin D, which interferes with a ctin polymerization, caused marked shortening and ballooning of the mi crovilli. The elongation of microvilli exposed to glucose at room temp erature was reversed by a 40 min exposure to glucose-free Hanks' balan ced salt solution. (C) 1994 Wiley-Liss, Inc.