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Effect of phenylalanine metabolites on the activities of enzymes of ketone-body utilization in brain of suckling rats.

ABSTRACT: 1. The effects of phenylalanine and its metabolites (phenylacetate, phenethylamine, phenyl-lactate, o-hydroxyphenylacetate and phenylpyruvate) on the activity of 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) 3-oxo acid CoA-transferase (EC 2.8.3.5) and acetoacetyl-CoA thiolase (EC 2.3.1.9) in brain of suckling rats were investigated. 2. The 3-hydroxybutyrate dehydrogenase from the brain of suckling rats had a Km for 3-hydroxybutyrate of 1.2 mM. Phenylpyruvate, phenylacetate and o-hydroxyphenylacetate inhibited the enzyme activity with Ki values of 0.5, 1.3 and 4.7 mM respectively. 3. The suckling-rat brain 3-oxo acid CoA-transferase activity had a Km for acetoacetate of 0.665 mM and for succinyl (3-carboxypropionyl)-CoA of 0.038 mM. The enzyme was inhibited with respect to acetoacetate by phenylpyruvate (Ki equals 1.3 mM) and o-hydroxyphenylacetate (Ki equals 4.5 mM). The reaction in the direction of acetoacetate was also inhibited by phenylpyruvate (Ki equals 1.6 mM) and o-hydroxyphenylacetate (Ki equals 4.5 mM). 4. Phenylpyruvate inhibited with respect to acetoacetyl-CoA both the mitochondrial (Ki equals 3.2 mM) and cytoplasmic (Ki equals 5.2 mM) acetoacetyl-CoA thiolase activities. 5. The results suggest that inhibition of 3-hydroxybutyrate dehydrogenase and 3-oxo acid CoA-transferase activities may impair ketone-body utilization and hence lipid synthesis in the developing brain. This suggestion is discussed with reference to the pathogenesis of mental retardation in phenylketonuria.

SUBMITTER: Benavides J

PROVIDER: S-EPMC1164225 | biostudies-other | 1976 Nov

REPOSITORIES: biostudies-other

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Project description:1. Ketone-body utilization in fed and starved adult and suckling rats has been investigated by measuring arterio-venous differences across the brain. Venous blood was collected from the confluence of sinuses and arterial blood from the femoral artery in adult rats and by cardiac puncture in suckling rats. 2. During starvation the arterio-venous difference of ketone bodies increased in proportion to their concentrations in the blood and reached a value of 0.16mm at 48h. At a given concentration of the respective ketone bodies the arterio-venous differences of acetoacetate were about twice those of 3-hydroxybutyrate. 3. Fed rats in which the concentrations of ketone bodies were raised by intravenous infusion of sodium acetoacetate had the same arterio-venous differences as starved rats at corresponding ketone-body concentrations. Thus the ability of the rat brain to utilize ketone bodies is independent of the nutritional state. 4. The concentrations of glucose, acetoacetate and 3-hydroxybutyrate were much lower in the brain than in the arterial blood. The measured (blood concentration)/(brain concentration) ratio was 4.4 for glucose, 4.5 for acetoacetate and 8.1 for 3-hydroxybutyrate in 48h-starved rats. 5. The mean arterio-venous difference of glucose across the brain was 0.51mm in fed rats and 0.43mm in 96h-starved rats. 6. Conversion of glucose into lactate rose from negligible values in the fed state to 0.2mm after 48h starvation and decreased to zero after 96h starvation. 7. In 16-22-day-old suckling rats the arterio-venous differences of ketone bodies across the brain were also proportional to the ketone-body concentration, but they were about 3-4 times greater than in adult rats at the same blood ketone-body concentration. 8. Arterio-venous differences of glucose were about the same in adult and suckling rats. 9. The brain of fed suckling rats formed more lactate from glucose than fed adult rats. 10. The results indicate that ketone bodies are major metabolic fuels of the brain of the suckling rat under normal conditions.

Project description:1. The maximum activity of hexokinase in lymphocytes is similar to that of 6-phosphofructokinase, but considerably greater than that of phosphorylase, suggesting that glucose rather than glycogen is the major carbohydrate fuel for these cells. Starvation increased slightly the activities of some of the glycolytic enzymes. A local immunological challenge in vivo (a graft-versus-host reaction) increased the activities of hexokinase, 6-phosphofructokinase, pyruvate kinase and lactate dehydrogenase, confirming the importance of the glycolytic pathway in cell division. 2. The activities of the ketone-body-utilizing enzymes were lower than those of hexokinase or 6-phosphofructokinase, unlike in muscle and brain, and were not affected by starvation. It is suggested that the ketone bodies will not provide a quantitatively important alternative fuel to glucose in lymphocytes. 3. Of the enzymes of the tricarboxylic acid cycle whose activities were measured, that of oxoglutarate dehydrogenase was the lowest, yet its activity (about 4.0mumol/min per g dry wt. at 37 degrees C) was considerably greater than the flux through the cycle (0.5mumol/min per g calculated from oxygen consumption by incubated lymphocytes). The activity was decreased by starvation, but that of citrate synthase was increased by the local immunological challenge in vivo. It is suggested that the rate of the cycle would increase towards the capacity indicated by oxoglutarate dehydrogenase in proliferating lymphocytes. 4. Enzymes possibly involved in the pathway of glutamine oxidation were measured in lymphocytes, which suggests that an aminotransferase reaction(s) (probably aspartate aminotransferase) is important in the conversion of glutamate into oxoglutarate rather than glutamate dehydrogenase, and that the maximum activity of glutaminase is markedly in excess of the rate of glutamine utilization by incubated lymphocytes. The activity of glutaminase is increased by both starvation and the local immunological challenge in vivo. This last finding suggests that metabolism of glutamine via glutaminase is important in proliferating lymphocytes.

Project description:1. The effects of starvation and diabetes on brain fuel metabolism were examined by measuring arteriovenous differences for glucose, lactate, acetoacetate and 3-hydroxybutyrate across the brains of anaesthetized fed, starved and diabetic rats. 2. In fed animals glucose represented the sole oxidative fuel of the brain. 3. After 48h of starvation, ketone-body concentrations were about 2mm and ketone-body uptake accounted for 25% of the calculated O(2) consumption: the arteriovenous difference for glucose was not diminished, but lactate release was increased, suggesting inhibition of pyruvate oxidation. 4. In severe diabetic ketosis, induced by either streptozotocin or phlorrhizin (total blood ketone bodies >7mm), the uptake of ketone bodies was further increased and accounted for 45% of the brain's oxidative metabolism, and the arteriovenous difference for glucose was decreased by one-third. The arteriovenous difference for lactate was increased significantly in the phlorrhizin-treated rats. 5. Infusion of 3-hydroxybutyrate into starved rats caused marked increases in the arteriovenous differences for lactate and both ketone bodies. 6. To study the mechanisms of these changes, steady-state concentrations of intermediates and co-factors of the glycolytic pathway were determined in freeze-blown brain. 7. Starved rats had increased concentrations of acetyl-CoA. 8. Rats with diabetic ketosis had increased concentrations of fructose 6-phosphate and decreased concentrations of fructose 1,6-diphosphate, indicating an inhibition of phosphofructokinase. 9. The concentrations of acetyl-CoA, glycogen and citrate, a potent inhibitor of phosphofructokinase, were increased in the streptozotocin-treated rats. 10. The data suggest that cerebral glucose uptake is decreased in diabetic ketoacidosis owing to inhibition of phosphofructokinase as a result of the increase in brain citrate. 11. The inhibition of brain pyruvate oxidation in starvation and diabetes can be related to the accelerated rate of ketone-body metabolism; however, we found no correlation between the decrease in glucose uptake in the diabetic state and the arteriovenous difference for ketone bodies. 12. The data also suggest that the rates of acetoacetate and 3-hydroxybutyrate utilization by brain are governed by their concentrations in plasma. 13. The finding of very low concentrations of acetoacetate and 3-hydroxybutyrate in brain compared with plasma suggests that diffusion across the blood-brain barrier may be the rate-limiting step in their metabolism.

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Effect of phenylalanine metabolites on the activities of enzymes of ketone-body utilization in brain of suckling rats.

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