The deoxyglucose method adapted for studies of glucose metabolism in the early chick embryo

14C-2-deoxyglucose (DG), currently employed in in vivo studies of brain glucose metabolism, has been used for determination of glucose consumption in the in vitro developing chick embryo. DG, presented in traces, accumulates in the embryo in proportion with incubation time. Analysis of tissue homogenates shows that the accumulated radioactivity is due to both phosphorylated (DGP) and nonphosphorylated DG. As it is only the radioactivity originating from the DGP that is proportional to glucose utilization, the nonphosphorylated DG must be washed out. The washout shows two distinct kinetics: a fast one corresponding to DG that has entered the cells but has not yet been phosphorylated and a slow one that is probably due to a dephosphorylated DGP coming from a different cellular compartment. On the basis of these results the optimal experimental conditions have been defined, allowing quantitative studies of glucose metabolism during the first day of development of the chicken embryo. From 18 to 24 hr of incubation (end of gastrulation), total glucose consumption increases from 50 nmol X h-1 at stage 3-4 to 90 nmol X h-1 at stage 6-7. This increase mainly reflects the growth of the blastodisc. Comparison with the values of O2 uptake measured at the same period of development suggests that only a fraction of the glucose consumed is oxidized, the major part being converted aerobically to lactate.     

Kynurenine metabolism in the development chick embryo

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1. In the embryonic chick liver during the midperiod of development (8–12 days), kynureninase is the primary enzyme involved in the catabolism of kynurenine. Kynurenine aminotransferase was not found.     

Increased response to insulin of glucose metabolism in the 6-day unloaded rat soleus muscle

Hind leg muscles of female rats (85-99 g) were unloaded by tail cast suspension for 6 days. In the fresh-frozen unloaded soleus, the significantly greater concentration of glycogen correlated with a lower activity ratio of glycogen phosphorylase (p less than 0.02). The activity ratio of glycogen synthase also was lower (p less than 0.001), possibly due to the higher concentration of glycogen. In isolated unloaded soleus, insulin (0.1 milliunit/ml) increased the oxidation of D-[U-14C]glucose, release of lactate and pyruvate, incorporation of D-[U-14C]glucose into glycogen, and the concentration of glucose 6-phosphate more (p less than 0.05) than in the weight-bearing soleus. At physiological doses of insulin, the percent of maximal uptake of 2-deoxy-D-[1,2-3H]glucose/muscle also was greater in the unloaded soleus. Unloading of the soleus increased by 50% the concentration of insulin receptors, due to no decrease in total receptor number during muscle atrophy. This increase may account for the greater response of glucose metabolism to insulin in this muscle. The extensor digitorum longus, which generally shows little response to unloading, displayed no differential response of glucose metabolism to insulin.     

Glycogen metabolism in the prelaid chick embryo.

Glycogen metabolism has been studied during the development of the early chick embryo, at the cytochemical and ultrastructural levels. Two waves of glycogen synthesis and breakdown have been found. In the first, free clusters of glycogen particles are synthesized at late oogenesis. These clusters are found later in invaginations of the membrane of vesicles containing a floccular material (FLOV). The glycogen clusters are degraded there during ovulation and the first hours in the oviduct. The second wave of glycogen synthesis begins before cleavage, reaching a maximum at mid-uterine age. This second wave occurs in another type of vesicle (GLYV), which eventually disintegrates releasing free clusters of glycogen granules. This glycogen is degraded in membranous structures containing a floccular material, as in the first wave of degradation. The degradation ends at the late uterine stages, and at the same time numerous ribosomes are formed. This period corresponds to area pellucida formation, which probably depends on the energy liberated during the second wave of glycogen degradation.     

Effect of spinal cord stimulation on the metabolism of developing latissimus dorsii muscles in chick embryo

Influence of chronic spinal cord stimulation upon some characteristic enzyme activities of energy metabolism was investigated in slow anterior (ALD) and fast posterior (PLD) latissimus dorsii muscles of the chick embryo. During embryonic life, oxidative metabolism (as evaluated by the activity of malate dehydrogenase (MDH] represents the main energetic pathway in both slow and fast muscles. At the end of embryonic life, an increase in anaerobic (as evaluated by the activity of lactate dehydrogenase (LDH] and creatine phosphokinase (CPK) activities occurs in PLD muscle. Chronic spinal cord stimulation at a low frequency was performed from the 10th day to the 16th day of embryonic development. In ALD, the enzyme activities were unaffected, while in PLD a concomitant decrease in LDH and CPK activities was observed.     

ESR measurement of dissolved oxygen in 4-day chick embryo blood

The magnetic interactions between dissolved oxygen molecules and nitroxide radical spin probes lead to broadening of electron spin resonance (ESR) lines. Based on this property we described an ESR methodology to measure PO2 values in 4-day chick embryo and adult human blood. The total blood volume required for the measurement was only 9 microliter. Using this method PO2 for adult human mixed venous blood was found to be 36.7 mmHg. This is within the established clinical range of 25-40 mmHg for mixed venous blood PO2. The range of mean PO2 values for 4-day chick embryo blood was 27.3- 35.0 mmHg. This is the first time that PO2 values have been reported for individual chick embryos at such an early stage.     

Regulation of glucose utilization in chick embryo fibroblasts by bicarbonate ion

The amount of glucose consumed by chick embryo fibroblasts in primary culture is strongly influenced by the presence of bicarbonate ion in the culture medmum. Cells grown on glucose at physiologic concentration (5.5 mm) and in the absence of bicarbonate ion have a reduced rate of glucose utilization when compared to their counterparts cultivated in medium containing the usual 25 mM bicarbonate. The presence or absence of bicarbonate is without effect on chick embryo fibroblast proliferation over a 6-day growth period. Both lactic acid accumulation per mole of glucose consumed and the utilization of glutamine increase as a function of bicarbonate ion in the growth medium.     

RNA metabolism in chick embryo skin]

Explants from 7, 8, 9, 11, 13-day chick embryonic skin incorporating (3H) Uridine for different periods 1 hr, 3 or 4 hr and a chase with actinomycin) are studied with respect to free (F) or membrane bound (B) cytoplasmic polysomes and to RNA extracted from them. Polysome specific activity decreases at older stages but the amount of polysomes increases due to increased protein synthesis. At each stage B polysomes are less abundant but more radioactive than F polysomes. RNA extracted from each kind is analysed on sucrose gradients: one half of each fraction is precipitated by TCA to estimate total radioactivity, the other is retained on millipore at high salt concentration to estimate radioactivity in messenger-like RNAs due to their poly-A sequences. The pattern of the labelling of the different fractions of RNA changes with the length of incorporation, the stages of explants and the kind of polysomes (F or B); at 11-13 days the incorporation is slow, radioactivity is low and distributed among several peaks of poly-A RNA; at 7-8 days the incorportion is rapid, dispersed throughout the gradient; at 9 days, a midway stage, incorporation is particularly high into 12S and 24S fractions from B RNA. In the 5 studied stages the labelling of this 12S occurs early, remains for a longer time and cannot be chased. These observations suggest stability of the 12S RNA. Since, in 14-day chick embryos, feather keratin m RNA has been shown to sediment at 12S and although our experiments have been done with total skin because this differentiating tissue is the site of extensive interactions between dermis and epidermis, they suggest that this 12S RNA is the actual keratin m RNA and might be synthesised some days before the onset of keratin synthesis. Its template ability will be investigated at earlier stages.