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Notes: —The oxidation to CO2 and the incorporation of [U-14C]glucose and [U-14C]acetate into lipids by cortex slices from rat brain during the postnatal period were investigated. The oxidation of [U-14C]glucose was low in 2-day-old rat brain, and increased by about two-fold during the 2nd and 3rd postnatal weeks. The oxidation of [U-14C]acetate was increased markedly in the second postnatal week, but decreased to rates observed in 2-day-old rat brain at the time of weaning. Both labeled substrates were readily incorporated into non-saponifiable lipids and fatty acids by brain slices from 2-day-old rat. Their rates of incorporation and the days on which maximum rates occurred were different, however, maximum incorporation of [U-14C]glucose and [U-14]acetate into lipid fractions being observed on about the 7th and 12th postanatal days, respectively. The metabolic compartmentation in the utilization of these substrates for lipogenesis is suggested. The activities of glucose-6-phosphate dehydrogenase, cytosolic NADP-malate dehydrogenase, cytosolic NADP-isocitrate dehydrogenase, ATP-citrate lyase and acetyl CoA carboxylase were measured in rat brain during the postnatal period. All enzymes followed somewhat different courses of development; the activity of acetyl CoA carboxylase was, however, the lowest among other key enzymes in the biosynthetic pathway, and its developmental pattern paralleled closely the fatty acid synthesis from [U-14C]glucose. It is suggested that acetyl CoA carboxylase is a rate-limiting step in the synthesis de novo of fatty acids in developing rat brain.

Notes: The activities of a number of mitochondrial enzymes involved in the metabolism of pyruvate during development of the rat brain were investigated. The rates of decarboxylation of [1-14C]pyruvate to 14CO2 via pyruvate dehydrogenase and the fixation of H14CO3− in the presence of pyruvate via pyruvate carboxylase by brain homogenates were very low in newborn rats. These rates increased markedly by about four-fold and 15-fold respectively during 10–35 postnatal days. The rates of the fixation of H14CO3− by cerebral homogenates were supported by the development of the activity of pyruvate carboxylase in rat brain. The activities of citrate synthase, aconitase, NAD-malate dehydrogenase, aspartate aminotransferase, alanine aminotransferase and phosphoenol-pyruvate carboxykinase were very low in the particulate fraction of the newborn rat brain. The activities of all these enzymes increased makedly by about three- to 10-fold during 10–35 days after birth. The activity of mitochondrial phosphoenolpyruvate carboxykinase from rat brain was not precipitated by an antibody prepared against rat liver cytosolic phosphoenolpyruvate carboxykinase suggesting that cerebral mitochondrial enzyme is immunologically different from that of the cytosolic form in hepatocytes. The significance of the development of the cerebral mitochondrial metabolism is discussed in relation to biochemical maturation of the brain.

Notes: To evaluate the relative significance of CO2-fixing enzymes in the metabolism of rat brain, the subcellular distribution of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, NADP-isocitrate dehydrogenase and NADP-malate dehydrogenase, as well as the fixation of H14CO3− by the cytosol and the mitochondria was investigated. Pyruvate carboxylase and phosphoenol-pyruvate carboxykinase are mainly localized in the mitochondria whereas NADP-isocitrate dehydrogenase and NADP-malate dehydrogenase are present in both the cytosol and the mitochondria. In the presence of pyruvate rat brain mitochondria fixed H14CO3− at a rate of about 170 nmol/g of tissue/min whereas these organelles fixed negligible amounts of H14CO3− in the presence of α-ketoglutarate or phosphoenolpyruvate. Rat brain cortex slices fixed H14CO3− at a rate of about 7 nmol/g of tissue/min and it was increased by two-fold when pyruvate was added to the incubation medium. The carboxylation of α-ketoglutarate and pyruvate by the reversal of the cytosolic NADP-isocitrate dehydrogenase and NADP-malate dehydrogenase respectively was very low as compared to that by pyruvate carboxylase. The rate of carboxylation reaction of both NADP-isocitrate dehydrogenase and NADP-malate dehydrogenase was only about 1/10th of that of decarboxylation reaction of the same enzyme. It is suggested that under physiological conditions these two enzymes do not play a significant role in CO2-fixation in the brain. In rat brain cytosol, citrate is largely metabolized to α-ketoglutarate by a sequential action of aconitate hydratase and NADP-isocitrate dehydrogenase. The operation of the citrate-cleavage pathway in rat brain cytosol is demonstrated. The data show that among four CO2-fixing enzymes, pyruvate carboxylase, an anaplerotic enzyme, plays the major role in CO2-fixation in the brain.

Notes: Abstract: Age-dependent changes in the oxidative metabolism in nonsynaptic and synaptic mitochondria from brains of 3, 12, and 24-month-old rats were investigated. When pyruvate and malate were used in conjunction as substrates, a significant reduction in State 3 respiration was observed in both mitochondrial populations from 12-and 24-month-old rats compared with 3-month-old animals. A similar age-dependent reduction in the oxidation of [1-11C]pyruvate was also observed in nonsynaptic and synaptic mitochondria from senescent rats. Pyruvate dehydrogenase complex activity (both active and total) was, however, not decreased in the two mitochondrial populations from brains of 3, 12, and 24-month-old rats. When DL-3-hydroxybutyrate plus malate were used as substrates, a decrease in State 3 respiration was observed only in synaptic mitochondria from 24-month-old rats compared with 3- month-old animals. Similarly, an age-dependent reduction in the oxidation of 3-hydroxy[3-11C]butyrate was also observed only in synaptic mitochondria from 12-and 24-month-old rats. However, a significant reduction in the activities of ketone body-metabolizing enzymes, namely, 3-hydroxybutyrate dehydrogenase, 3-ketoacid CoA transferase, and acetoacetyl-CoA thiolase was observed in both mitochondrlal populations from 12- and 24-month-old rats compared with 3 month-old animals. These findings show that specific alterations in oxidative metabolism occur in nonsynaptic and synaptic mitochondria from aging rats. The data also suggest that in addition to alterations in enzyme activities, permeability of anions (e.g. pyruvate) across the inner mitochondrial membrane may be altered in nonsynaptic and synaptic mitochondria from senescent animals.

Notes: Abstract— The oxidation of l-[U-14C]leucine and l-[l-14C]leucine at varying concentrations from 0.1 to 5mM to CO2 and the incorporation into cerebral lipids and proteins by brain slices from 1-week old rats were markedly stimulated by glucose. Although the addition of S mM-dl-3-hydroxybutyrate had no effect on the metabolism of [U-14C]leucine by brain slices from suckling rats, the stimulatory effects of glucose on the metabolism of l-[U-14C]leucine were markedly reduced in the presence of dl-3-hydroxybutyrate. The stimulatory effect of glucose on leucine oxidation was, however, not observed in adult rat brain. Furthermore, the incorporation of leucine-carbon into cerebral lipids and proteins was also very low in the adult brain. The incorporation of l-[U-14C]leucine into cerebral lipids by cortex slices was higher during the first 2 postnatal weeks, which then declined to the adult level. During this time span, the oxidation of l-[U-14C]leucine to CO2 remained relatively unchanged. The incorporation in vivo of D-3-hydroxy[3-14C]butyrate into cerebral lipids was markedly decreased by acute hyperleucinemia induced by injecting leucine into 9-day old rats. In in vitro experiments, 5 mM-leucine had no effect on the oxidation of [U-14C]glucose to CO2 or its incorporation into lipids by brain slices from 1-week old rats. However, 5 mM-leucine inhibited the oxidation of d-3-hydroxy-[3-14C]butyrate, [3-14C]acetoacetate and [1-14C]acetate to CO2 by brain slices, but their incorporation into cerebral lipids was not affected by leucine. In contrast 2-oxo-4-methylvalerate, a deaminated metabolite of leucine, markedly inhibited both the oxidation to CO2 and the incorporation into lipids of labelled glucose, ketone bodies and acetate by cortex slices from 1-week old rats. These findings suggest that the reduction in the incorporation in vivo of d-3-hydroxy[3-14C]butyrate into cerebral lipids in rats injected with leucine is most likely caused by 2-oxo-4-methylvalerate formed from leucine. Since the concentrations of leucine and 2-oxo-4-methylvalerate in plasma of untreated patients with maple-syrup urine disease are markedly elevated, our findings are compatible with the possibility that an alteration in the metabolism of glucose and ketone bodies in the brain may contribute to the pathophysiology of this disease.

Notes: Cerebral cortex slices from 1-day old rats incorporated actively 3-hydroxy[3-14C] butyrate into lipids. The rate of synthesis increased moderately during the first postnatal week and then decreased rapidly reaching negligible rates by the end of the fourth postnatal week. The incorporation of [3-14C]acetoacetate (5 mM) and 3-hydroxy[3-14C]butyrate (5 mM) at non-physiological concentrations into lipids by cerebral cortex from 1-week old rats was markedly stimulated in the presence of added glucose; however, the addition of glucose had no effect on the oxidation of ketone bodies to 14CO2 At physiological concentrations of acetoacetate (0.5 mM) and 3-hydroxybutyrate (1 mM), glucose stimulated both the oxidation of ketone bodies to 14CO2 and the incorporation of ketone body-carbon into cerebral lipids. In contrast, the oxidation of [U-14C]glucose to CO2 and its incorporation into lipids by brain slices were markedly reduced in the presence of ketone bodies at physiological as well as non-physiological concentrations. However, the total rate of lipid synthesis, as determined by the incorporation of 3H from 3H2O, was maintained under these conditions. This indicated that the decreased contribution of the acetyl moiety from glucose was compensated to a similar extent by acetyl-CoA formed from ketone bodies. The findings show that both acetoacetate and 3-hydroxybutyrate are better precursors than glucose for in vitros lipid synthesis in developing rat brain.