Decreased labeling of amino acids by inhibition of the utilization of [3H,14C]glucose via the hexosemonophosphate shunt in rat brain in vivo

Treatment of rats with 6-aminonicotinamide showed a small but significant decrease in the labeling of amino acids in the brain after injection of [³H]acetate. The results of these experiments also gave evidence of the presence of [³H]glucose and [³H]lactate, and an increase in [³H]glucose content in the brain of 6-aminonicotinamide treated rats. To apportion the contribution of [³H]glucose formed by gluconeogenesis from [³H]acetate to the labeling of amino acids a method was formulated based on the measurement of radioactivity of amino acids, lactate and free sugars in brain after injection of [6-³H]glucose or [1-³H]glucose relative to that after co-injection of [U-¹⁴C]glucose or [2-¹⁴C]glucose. In contrast to the expected formation of [1, 6-³H]glucose by gluconeogenesis from [³H]acetate,³H-labeled glucose isolated from brain, blood and liver showed the presence of [6-³H]glucose only. The values corrected for the presence of [6-³H]glucose showed that treatment with 6-aminonicotinamide had no effect on the labeling of amino acids by oxidation of [³H]acetate. These findings indicated that a significant decrease in the labeling of amino acids from [U-¹⁴C]glucose reported previously and again confirmed using [1-³H], [6-³H], [2-¹⁴C] or [U-¹⁴C]glucose in the present investigation was not due to the inhibition of the activities of enzymes of the citric acid cycle. These results support the postulated role of the hexosemonophosphate shunt for the utilization of glucose in providing neurotransmitter amino acids glutamate and -aminobutyrate.Dedicated to Professor K. A. C. Elliott on his 80th birthday.     

Metabolism of glucose into glutamate via the hexose monophosphate shunt and its inhibition by 6-aminonicotinamide in rat brain in vivo

The treatment of rats for 4 h with 6-aminonicotinamide (60 mg kg-1) resulted in an 180-fold increase in the concentration of 6-phosphogluconate in their brains; glucose increased 2.6-fold and glucose 6-phosphate, 1.7-fold. Moreover, lactate decreased by 20%, glutamate by 8% and gamma-aminobutyrate by 12%, and aspartate increased by 10%. No significant changes were found in glutamine and citrate. In blood, 6-phosphogluconate increased 5-fold; glucose, 1.4-fold and glucose 6-phosphate, 1.8-fold. The metabolism of glucose in the rat brain, via both the Embden-Meyerhof pathway and the hexose monophosphate shunt, was investigated by injecting [U-14C]glucose or [2-14C]glucose, and that via the hexose monophosphate shunt alone by injecting [3,4-14C]glucose. The total radioactive yield of amino acids in the rat brain was 5.63 mumol at 20 min after injection of [U-14C]glucose, or 5.82 mumol after injection of [2-14C]glucose; by contrast, it was 0.62 mumol after injection of [3,4-14C]glucose. The treatment of rats with 6-aminonicotinamide showed significant decreases in these values, owing to decreases in the radioactive yields of glutamate, glutamine, aspartate, gamma-aminobutyrate, and alanine+glycine+serine. Glutamate isolated from the brain contained approximately 43% of its radioactivity in carbon 1 after injection of [3,4-14C]glucose, in contrast to 13% and 18% after injection of [U-14C]glucose and [2-14C]glucose, respectively, in both the control and treated rats. The calculations based on these findings showed that approximately 69% of the 14C-labelled glutamate was formed from [14C]acetyl coenzyme A (acetyl CoA) and the residual 31% by 14CO2 fixation of pyruvate after injection of [3,4-14C]glucose in both control and treated rats. The results gave direct evidence that glutamate and gamma-aminobutyrate in the brain were formed by metabolism of glucose via the hexose monophosphate shunt as well as via the Embden-Meyerhof pathway. From the radioactive yields of glutamate formed via [14C]acetyl CoA it was estimated that approximately 7.8% of the total glucose utilized was channelled via the hexose monophosphate shunt. Assuming that [14C]glutamate formed by carbon-dioxide fixation of pyruvate was also dependent on the metabolism of glucose through the hexose monophosphate shunt, the estimated value was approximately 9.5% of the total glucose converted into glutamate. The results of the present investigation, taken in conjunction with other findings, suggest that the utilization of glucose via the hexose monophosphate shunt is functionally important in the rat brain.     

Dirunal rhythms of hexosemonophosphate shunt and phosphorylase in rat pineal gland

Hexosemonophosphate shunt and phosphorylase activities determined in the pineal glands of rats during mid-light and mid-darkness periods were found to undergo diurnal rhythms. A high active hexosemonophosphate shunt -- indicated by the more ready conversion of CO₂ of the glucose carbon in position 1 than of that in position 6 -- was found to correlate to the function of endocrine activity of night time/darkness. Phosphorylase activity, indicating glycogenolysis, measured at the same times, was found to be increased during the period of mid-light when basal metabolism is high compared to that during mid-darkness. The significance of these findings is discussed.     

Selective inhibition of glucose oxidation by triethyltin in rat brain in vivo

Results are reported of a comparative study in vivo of the metabolism of [2-(14)C]-glucose and [1-(14)C]acetate in brains of rats intoxicated with triethyltin sulphate. The incorporation of (14)C from glucose into glutamate, glutamine, gamma-aminobutyrate and aspartate was greatly decreased. The incorporation of (14)C from acetate into these amino acids was unaffected. The experimental data indicated that the main action of triethyltin was to decrease the rate at which pyruvate formed from glucose is oxidized. Glycolysis was not inhibited. Changes in glucose metabolism in the brain are shown not to be directly due to hypothermia. Some of the advantages of measuring the labelling of intermediates at very short time intervals after the injection of the labelled glucose are demonstrated.     

Lithium's effects on rat liver glucose metabolism in vivo

Oral administration of lithium carbonate to fed-healthy rats strongly decreased liver glycogen content, despite the simultaneous activation of glycogen synthase and the inactivation of glycogen phosphorylase. The effect seemed to be related to a decrease in glucose 6-phosphate concentration and to a decrease in glucokinase activity. Moreover, in these animals lithium markedly decreased liver fructose 2,6-bisphosphate, which could be a consequence of the fall in glucose 6-phosphate and of the inactivation of 6-phosphofructo-2-kinase. Liver pyruvate kinase activity and blood insulin also decreased after lithium administration. Lower doses of lithium carbonate had less intense effects. Lithium administration to starved-healthy and fed-streptozotocin-diabetic rats caused a slight increase in blood insulin, which was simultaneous with increases in liver glycogen, glucose 6-phosphate, and fructose 2, 6-phosphate. Glucokinase, 6-phosphofructo-2-kinase, and pyruvate kinase activities also increased after lithium administration in starved-healthy and fed-diabetic rats. Lithium treatment activated glycogen synthase and inactivated glycogen phosphorylase in a manner similar to that observed in fed-healthy rats. Glycemia was not modified in any group of animals. These results indicate that lithium acts on liver glycogen metabolism in vivo in at least two different ways: one related to changes in insulinemia, and the other related to the direct action of lithium on the activity of some key enzymes of liver glucose metabolism.     

The acute action of ammonia on rat brain metabolism in vivo

1. Acute NH(4) (+) toxicity was studied by using a new apparatus that removes and freezes the brains of conscious rats within 1s. 2. Brains were removed and frozen 5min after intraperitoneal injection of ammonium acetate (2-3min before the onset of convulsions). Arterial [NH(4) (+)] rose from less than 0.01 to 1.74mm at 4-5min. The concentrations of all glycolytic intermediates measured, except glucose 6-phosphate, were increased by the indicated percentage above the control value as follows: glucose (by 41%), fructose 1,6-diphosphate (by 133%), dihydroxyacetone phosphate (by 164%), alpha-glycerophosphate (by 45%), phosphoenolpyruvate (by 67%) and pyruvate (by 26%). 4. Citrate and alpha-oxoglutarate concentrations were unchanged and that of malate was increased (by 17%). 5. Adenine nucleotides and P(i) concentrations were unchanged but the concentration of creatine phosphate decreased slightly (by 6%). 6. Brain [NH(4) (+)] increased from 0.2 to 1.53mm. Net glutamine synthesis occurred at an average rate of 0.33mumol/min per g. 7. The rate of brain glucose utilization was measured in vivo as 0.62mumol/min per g in controls and 0.81mumol/min per g after NH(4) (+) injection. 8. The arteriovenous difference of glucose and O(2) increased by 35%. 9. No significant arteriovenous differences of glutamate or glutamine were detected. Thus, although much NH(4) (+) was incorporated into glutamine the latter was not rapidly released from the brain to the circulation. 10. Plasma [K(+)] increased from 3.3 to 5.4mm. 11. The results indicate that NH(4) (+) stimulates oxidative metabolism but does not interfere with brain energy balance. The increased rate of oxidative metabolism could not be accounted for only on the basis of glutamine synthesis. We suggest that increased extracellular [NH(4) (+)] and [K(+)] decreased the resting transmembrane potential and stimulated Na(+),K(+)-stimulated adenosine triphosphatase activity thus accounting for the increased metabolic rate.     

The effect of hypercapnia in vivo on rat brain gangliosides

An investigation on the effect of hypercapnia in vivo on the rat brain gangliosides is reported. In contradiction to the results reported by Lowden and Wolfe (1964) for the cat, no diminution in total brain ganglioside concentration could be demonstrated. The ganglioside pattern in test and control animals was the same.     

The effect of phenylpyruvate on pyruvate metabolism in rat brain

1. The effect of phenylalanine and phenylpyruvate on the metabolism of pyruvate by isolated mitochondria from rat brain was investigated. 2. Phenylpyruvate inhibited the fixation of H(14)CO(3) (-) in the presence of pyruvate by intact rat brain mitochondria, whereas phenylalanine and other metabolites of this amino acid had no inhibitory effect on this process. 3. Pyruvate carboxylase activity in freeze-dried rat brain mitochondrial preparations was also inhibited only by phenylpyruvate, and a ;mixed type' inhibition was observed. 4. The K(m) for pyruvate of rat brain pyruvate carboxylase was about 0.2mm. 5. The concentration of phenylpyruvate required for a 50% inhibition of H(14)CO(3) (-) fixation by the intact mitochondria and of pyruvate carboxylase activity was dependent on the concentration of pyruvate used in the incubation medium. 6. The possible significance of inhibition of pyruvate carboxylase activity by phenylpyruvate in the brains of phenylketonuric patients is discussed.     

The effect of L-3,4-dihydroxyphenylalanine administration on glucose metabolism in brain

The effect of the administration of l -3,4-dihydroxyphenylalanine (l -DOPA) on the metabolism of glucose in brain was studied by administering [U-¹⁴C]glucose to three groups of rats: (1) those injected previously with l -DOPA, 100 mg/kg; (2) those fed 1 % (w/w) l -DOPA in their diet for several months and also injected 15 min before the administration of glucose with l -DOPA, 100 mg/kg; and (3) appropriate controls. Chronic treatment with l -DOPA caused a decrease in the flux of carbon from glucose in plasma to those amino acids in brain that are in equilibrium with the tricarboxylic acid cycle intermediates but not to lactate and alanine. Similar differences from controls, but of smaller magnitude, were observed in rats given a single injection of l -DOPA. Concentrations of glucose in plasma and in brain were increased after acute or chronic treatment with l -DOPA. A single injection of l -DOPA did not cause changes in the levels of the most abundant amino acids in brain, but after chronic treatment with l -DOPA modest changes were noted in the brain levels of some ninhydrin-reacting substances; the contents of taurine and aspartate were lower and those of threonine, serine, glutamine, and glycine were higher.     

The effect of irradiation on dopamine level and metabolism in the rat brain

In experiments on rats it was shown that after 20 Gy irradiation dopamine and homovanilic acid content increases in the caudate nucleus and limbic structures of the forebrain: dopamine disappears more readily when its biosynthesis is blocked. The rate of the mediator degradation in the brain increases by 1.5-2 times, and the rate of the synthesis, by 3-5 times at early times after irradiation.     

In vivo and in vitro effects of beta-endorphin on glucose metabolism in the rat

The effects of beta-endorphin (beta-Ep) on plasma glucose levels in rats and on glucose metabolism in isolated rat liver cells were examined. Intravenous injection of beta-Ep (5 micrograms/100 g BW) into ether-anaesthetized rats resulted in prompt and sustained hyperglycaemia with increases in the plasma glucagon and somatostatin levels and decrease in the plasma insulin level. When liver cells isolated from fed rats were incubated in the presence of beta-Ep at concentrations of 6 X 10(-8) M to 6 X 10(-7) M, glucose release into the medium increased within 15 min in a dose-related manner. Time course experiments showed that beta-Ep increased the level of cyclic AMP within 3 min. Significant increase in gluconeogenesis in liver cells isolated from fasted rats was also observed on addition of 10(-7) M beta-Ep in the presence of 10 mM L-lactate. These results suggest that the hyperglycaemia induced by beta-Ep may be caused, at least in part, by the effects of beta-Ep on releases of pancreatic hormones and glucose production in liver cells.     

The effect of vanadate on glucose transport and metabolism in rat small intestine

The effect of sodium orthovanadate on the absorption, transmural transport and metabolism of glucose was studied by perfusion of isolated loops of rat jejunum in vitro. The presence of 1 mM vanadate in the serosal medium diminished absorption from 539 +/- 19 (n = 12) to 246 +/- 19 (P less than 0.001) mumol/h per g dry weight and transmural transport from 333 +/- 17 to 14 +/- 19 (P less than 0.001) mumol/h per g dry weight, whereas glucose utilisation was unaffected. The rate of release of lactate into the serosal medium was also diminished from 168 +/- 14 to 75 +/- 5 mumol/h per g dry weight (P less than 0.001). The observed rates were linear with respect to time and vanadate was effective within 5 min. In contrast, the rate of release of lactate into the luminal perfusate was strongly enhanced. Moreover, the progress curve showed a positive transient with an apparent lag time of 18.0 +/- 0.3 min, during which the rate increased to a value 9.2-times that of the control. Under the final steady-state conditions, the ratio of mucosal to serosal lactate production was 5.2 +/- 0.2 compared with 0.25 +/- 0.06 for the control, so that the effect of vanadate was to reverse the vectorial disposition of lactate. The concentration dependence of the effect of vanadate on absorption and metabolism was similar to that observed for the inhibition by vanadate of Na+/K+-ATPase activity in mucosal homogenates. The results are discussed in terms of the dissipation of transmembrane Na+ gradients as a result of the inhibition of the Na+/K+-ATPase.     

Ex vivo analysis of lactate and glucose metabolism in the rat brain under different states of depressed activity

Brain metabolism of glucose and lactate was analyzed by ex vivo NMR spectroscopy in rats presenting different cerebral activities induced after the administration of pentobarbital, alpha-chloralose, or morphine. The animals were infused with a solution of either [1-(13)C]glucose plus lactate or glucose plus [3-(13)C]lactate for 20 min. Brain metabolite contents and enrichments were determined from analyses of brain tissue perchloric acid extracts according to their post-mortem evolution kinetics. When amino acid enrichments were compared, both the brain metabolic activity and the contribution of blood glucose relative to that of blood lactate to brain metabolism were linked with cerebral activity. The data also indicated the production in the brain of lactate from glycolysis in a compartment other than the neurons, presumably the astrocytes, and its subsequent oxidative metabolism in neurons. Therefore, a brain electrical activity-dependent increase in the relative contribution of blood glucose to brain metabolism occurred via the increase in the metabolism of lactate generated from brain glycolysis at the expense of that of blood lactate. This result strengthens the hypothesis that brain lactate is involved in the coupling between neuronal activation and metabolism.