Paul R. Lehr

Kinetics of utilization in vivo of glucose carbon in the chick cerebral hemispheres during postnatal growth

Abstract 1. 1. The incorporation of glucose carbon in vivo into glucose, amino acids and carboxylic acids in the cerebral hemispheres was studied during post-hatching growth of the chick after subcutaneous injection of [U-14C]glucose, the animals being decapitated at different times after the injection. 2. 2. The highest rate of incorporation of glucose carbon into the tissue free amino acids occurs between 10 hr and 4 days after hatching. 3. 3. At hatching time, glutamic acid as well as neutral amino acids account for 40% of the whole radioactivity in the amino acid fraction. On the 30th day of postnatal growth, the % of radioactivity in glutamic acid (56%) is higher than that in the neutral amino acids (23%), which is a characteristic of the metabolism in cerebral hemispheres of adult chicks. 4. 4. Studies of the evolution of cerebral glucose concentration during the postnatal growth show an increasing rate between 10 and 48 hr after hatching. 5. 5. On the 2nd, 4th and 30th days of postnatal growth aspartate and glutamate have approximately the same specific radioactivity. On the other hand, at 10 hr after hatching, the specific radioactivity of cerebral aspartate is always higher than that of glutamate. 6. 6. At 10 hr after hatching, the specific radioactivity of cerebral α-alanine is always considerably higher than that of other amino acids. On the 30th day of postnatal growth, and at 10 min after the injection of [U-14C]glucose, the specific radioactivity of α-alanine is higher than that of other amino acids; afterwards, from 10 to 60 min after the injection of labelled glucose, the specific radioactivity of α-alanine decreases and its value tends towards zero.

Variations of 3-hydroxybutyrate dehydrogenase activity in brain and liver mitochondria of the developing chick

1. The 3-hydroxybutyrate dehydrogenase activity was estimated in the crude mitochondrial fraction isolated from the cerebral hemispheres, the optic lobes, the cerebellum and the liver of the chick between the 20th day of embryonic life and the 30th day of postnatal maturation. 2. The optimal conditions of liberation and of determination of 3-hydroxybutyrate dehydrogenase activity were studied in the mitochondrial fraction isolated from chick cerebral hemispheres and liver. 3. The subcellular distribution of the enzyme in the chick brain and liver is very different from that in the rat. 3-Hydroxybutyrate dehydrogenase is completely mitochondrial in the rat brain and liver whereas in the chick brain and liver, it is located in mitochondrial and microsomal fractions; moreover, a third component can even be found in the soluble fraction of chick liver. 4. The 3-hydroxybutyrate dehydrogenase activity reaches the same value in the three areas of 20-day-old chick embryo brain. Between this stage and the 4th day after hatching, it increases to reach the same peak in the three areas. This peak however, appears at different stages according to the considered brain area. At 30 days after hatching, the enzyme activity is higher in the cerebellum than in the cerebral hemispheres and optic lobes. 5. The activity of hepatic 3-hydroxybutyrate dehydrogenase is 10 to 20 times lower than in the brain. It does not significantly change between 1 day before and 4 days after hatching and increases 2-fold between 4 and 30 days after hatching. 6. The variations of 3-hydroxybutyrate dehydrogenase activity in chick brain indicate correlations of this enzyme activity with development, particularly related to the nutritional state of the chicks. The fairly important differences in the activity of 3-hydroxybutyrate dehydrogenase in the liver of the chick and the rat enable us to come to a better understanding of the regulation of the concentration of the different ketone bodies in the blood of the chick and the rat. Moreover, the presence of the microsomal component of 3-hydroxybutyrate dehydrogenase in chick brain probably originates in the low concentration of acetoacetate in chick blood.

Activity of acetoacetyl-CoA thiolase and regulation of ketone body metabolism in the brain of the developing chick

The acetoacetyl-CoA thiolase activity was estimated in subcellular fractions isolated from the cerebral hemispheres, the optic lobes and the cerebellum of the chick between the 20th day of embryonic life and the 30th day of postnatal maturation. Acetoacetyl-CoA thiolase is located both in mitochondria and microsomes of the chick brain. Mitochondrial enzyme activity remains high between the 20th day of embryonic life and the 2nd or the 4th day after hatching, depending on the considered brain area. It then decreases until 30 days after hatching. Cytoplasmic thiolase activity remains unchanged during pre- and postnatal development in the cerebral hemispheres; it increases during the same time in the optic lobes. In the cerebellum, cytoplasmic thiolase activity develops in the same way as in mitochondria. The regulation of ketone body utilization by the developing chick brain widely differs from that by the mammalian brain. In the chick, 3-hydroxybutyrate is nearly the single ketone body utilized by the brain. It is converted into acetyl-CoA in the mitochondria and cytoplasm of the chick brain through two exactly parallel pathways involving the participation of the same enzymes.

Activities of 3-oxo acid-CoA transferase and acetoacetyl-CoA synthetase in brain and liver of the developing chick

The 3-oxo acid-CoA transferase and acetoacetyl-CoA synthetase activities were estimated in subcellular fractions isolated from the cerebral hemispheres, the optic lobes, the cerebellum and the liver of the chick between the twentieth day of embryonic life and the thirtieth day of postnatal development. In the 3 areas of chick brain, the transferase activity increases during the whole postnatal development. Transferase is located both in mitochondria and microsomes unlike in the rat brain where this enzyme is specifically mitochondrial. There is no detectable activity of synthetase in the chick brain. These differences in enzyme localization imply that acetoacetate is converted into acetoacetyl-CoA only by 3-oxo acid-CoA transferase in the mitochondria and cytoplasm of the chick brain, whereas, in the rat brain, this reaction is performed by 3-oxo acid-CoA transferase in mitochondria and by acetoacetyl-CoA synthetase in the cytosol. There is no detectable activity either for transferase or for synthetase in the chick liver.

Glucose and amino acid metabolism in chick telencephalon slices: Changes with incubation conditions and animals' development

Glucose and amino acid metabolism in 1- and 30-day-old chick telencephalon slices was studied in two incubation media in the presence or in the absence of a continuous oxygenation. Medium 1 has a composition and a tonicity similar to cerebrospinal fluid, medium 2 is hypertonic and does not contain any K+ ions. The incorporation of glucose carbon into amino acids and the distribution of radioactivity between the different amino acids are close to the ones observed in the chick brain in vivo only when the slices are incubated in medium 1, with oxygen at 30 days and without oxygen for the 1-day-old chick. It also appears that if oxygenation is necessary for incubation of mature brain tissue in vitro, the absence of the medium oxygenation is more suitable for the study of glucose metabolism in 1-day-old chick brain slices.

Glucose and 3-hydroxybutyrate utilization by chick telencephalon during postnatal development

The cerebral arteriovenous difference in glucose content remains constant during the whole postnatal development of the chick, whereas that of 3-hydroxybutyrate is 6–9 times as high in the 1-day-old chick as in the 2–30-day-old chick.

Utilization In vivo of glucose carbon in the optic lobes and the cerebellum of the chick during postnatal growth

1. The incorporation of glucose carbon in vivo into amino acids was studied in the chick optic lobes and cerebellum during postnatal growth after subcutaneous injection of [U-14C]glucose. 2. The rapid incorporation of glucose carbon into free amino acids appears between the 1st and the 2nd day of postnatal growth in the optic lobes and between the 1st and the 4th day after hatching in the cerebellum. 3. The period during which the properties of mature brain metabolism are obtained is characterized in both structures during the first 48 hr of postnatal growth by changes in the specific radioactivity of some amino acids such as aspartate and alpha-alanine, and also by transient increases of glucose and glutamine concentrations. 4. The gamma-aminobutyrate content in the optic lobes is very high; the cerebellum on the contrary is characterized by its low gamma-aminobutyrate concentration linked to a very fast metabolism of this amino acid.

Utilization of acetate by chick brain during postnatal maturation

1. The study of the compartmentation of glutamate metabolism has been performed in the chick brain in vivo and in vitro in the presence of [U-14C]acetate between day 1 and day 30 of postnatal maturation. 2. The compartmentation of glutamate metabolism in vivo appears between day 1 and day 4 after hatching in the cerebral hemispheres and optic lobes. It is however more precocious in the optic lobes. In the cerebellum, it appears later, at about day 4 after hatching. The compartmentation of glutamate metabolism appears at the same time as the rapid incorporation of glucose into amino acids takes place in the cerebral hemispheres and the optic lobes. 3. In the chick telencephalon in vitro, the compartmentation of glutamate metabolism is visible from day 1 after hatching onwards. This difference is undoubtedly linked to the absence of an interference of glucose metabolism with acetate metabolism in vitro, and to the presence of a third compartment in the cerebral slices.

Ultrastructural and biochemical studies of the swelling of developing chick telencephalic slices

SummaryAn ultrastructural and biochemical study of the importance and localization of tissue swelling was performed on telencephalic slices of 1- and 30-day-old chicks incubated in an oxygenated or a non-oxygenated physiological medium. The swelling of slices is greater for 30-day-old chick material than for that from 1-day-old chicks. It also reaches higher values in the non-oxygenated than in the oxygenated medium. When the 30-day-old chick telencephalic slices are incubated in an oxygenated medium, swelling mainly affects astrocytes, and especially the astrocytic endfeet. When they are incubated in a non-oxygenated medium, the astrocytes and astrocytic endfeet are very swollen and in addition the swelling also affects the neurons and their organelles. Extracellular space is increased. When 1-day-old chick telencephalic slices are incubated in a non-oxygenated medium, the tissue structures are well preserved. Swelling predominantly affects astrocytes and astrocytic endfeet. Neurons are not affected and the extracellular space is reduced. However, when they are incubated in an oxygenated medium, tissue structures are greatly affected showing a high degree of disorganization. Extracellular space is greatly increased. This study thus indicates that the best incubation conditions are an oxygenated medium for 30-day-old chick telencephalic slices which are characterized by an aerobic metabolism, and a non-oxygenated medium for 1-day-old chick telencephalic slices which have a predominantly anaerobic metabolism.