The rates of conversion into 14CO2 of D-(-)-3-hydroxy3-14Cbutyrate, 3-14Cacetoacetate, 6-14Cglucose and U-14Cglutamine were measured in the presence and absence of unlabelled alternative oxidizable ...substrates in whole homogenates from the brains of young and adult rats. The addition of unlabelled glutamine resulted in decreased 14CO2 production from 6-14Cglucose in brain homogenates from both young and adult rats. In contrast, glucose had no effect on U-14Cglutamine oxidation. In suckling animals, both 3-hydroxybutyrate and acetoacetate decreased the rate of oxidation of 6-14Cglucose, but in adults only 3-hydroxybutyrate had an effect, and to a lesser degree. The addition of unlabelled glucose markedly enhanced the rates of oxidation of both ketone bodies in adult brain tissue and had little or no effect in the young. The rate of production of 14CO2 from U-14Cglutamine was increased by the addition of unlabelled ketone bodies in brain homogenates from young, but not from adult rats. In the converse situation, unlabelled glutamine added to 14C-labelled ketone bodies diminished 14CO2 production in young rats, but had no effect in adult animals. These results revealed a complex age-dependent pattern of interaction in which certain substrates apparently competed with each other, whereas an enhanced rate of 14CO2 production was found with others.
The rates of conversion of D-(-)-3-hydroxy3-14Cbutyrate, 3-14Cacetoacetate, 6-14Cglucose and U-14Cglutamine into 14CO2 were measured in the presence and absence of alternative oxidizable substrates ...in intact dissociated cells from the brains of young and adult rats. When unlabelled glutamine was added to 6-14Cglucose or unlabelled glucose was added to U-14Cglutamine, the rate of 14CO2 production was decreased in both young and adult rats. The rate of oxidation of 3-hydroxy3-14Cbutyrate was also decreased by the addition of unlabelled glutamine in both age groups, but in the reverse situation, i.e. unlabelled 3-hydroxybutyrate added to U-14Cglutamine, only the brain cells from young rats were affected. No significant effects were seen when glutamine and acetoacetate were combined. The addition of either of the two ketone bodies to 6-14Cglucose markedly lowered the rate of 14CO2 production in young rats, but in the adult only 3-hydroxybutyrate was effective and the magnitude of decrease in the rate of 6-14Cglucose oxidation was much lower than in young animals. Unlabelled glucose decreased the rate of 3-14Cacetoacetate oxidation to a minor extent in brain cells from both age groups; when added to 3-hydroxy3-14Cbutyrate, glucose had no effect in young rats and greatly enhanced 14CO2 production in adult brain cells. Many of these patterns of substrate interaction in dissociated brain cells differ from those in whole homogenates; they may be a function of the plasma membranes and the role of a carrier-mediated transport system or a reflection of a difference in the population of cell types or subcellular organelles in these two preparations.
Post-mortem T3 levels have been reported to be increased in victims of SIDS. Recent animal studies suggest, however, that elevated T3 in SIDS may be a non-specific post-mortem phenomenon. Therefore, ...we studied the possible effects of post-mortem time on T3 levels in 10- and 20-day-old rats killed by various methods including: Sodium pentobarbital overdose, injection of KCl, cervical dislocation or asphyxia with 100% N, 95% N-5% CO2 or 100% CO2. In both age groups T3 remained unchanged or increased slightly when the animals were killed with Na Pentobarbital or KCl. Greater increases were observed when rats were killed by cervical dislocation or asphyxia (100% N, 95% N-5% CO2 or 100% CO2). T3 levels did not become elevated in asphyxiated adult rats in which the inferior vena cava was ligated immediately following death. By extension to the human infant, the results of this study support the possibility that elevated T3 levels in SIDS victims may result from post-mortem processes. However, these results also suggest that the post-mortem elevation in T3 levels may be directly related to the mode of death.
Dopamine-beta-Hydroxylase (DBH) activity is decreased and tyrosine hydroxylase activity is increased in three brain regions (hypothalamus, putamen, and caudate nucleus) from victims of Sudden Infant ...Death Syndrome (SIDS) when compared to values in the same regions from infants dying of known causes. No stastically significant difference was detected in tyrosine hydroxylase activity in the thalamus and brain stem although DBH was 20% lower in the former region of the SIDS victims. Two other enzymes of biogenic amine metabolism, catechol-o-methyl transferase and Dopa-decarboxylase, were essentially the same in both groups. These data are consistent with the hypothesis that an alteration in the central nervous system may be a factor in the pathophysiology of SIDS.
Thyroid hormone (T3) has a multiplicity of effects on the developing nervous system. We have investigated T3 action using a cholinergic neuroblastoma cell line (S-20Y) as a model. S-20Y contains a ...nuclear receptor for T3 with binding properties similar to those of other T3 target tissues. In addition, these cells can carry out 5'-deiodination, which is necessary to produce active thyroid hormone in vivo. The enzyme involved in this process appears to be a type I deiodinase, based on its reaction kinetics and its susceptibility to inhibition by propylthiouracil. S-20Y cells maintained in T3-depleted medium showed decreased choline acetyltransferase (ChAT) activity. ChAT activity was restored to the control level in a dose-dependent manner by T3 repletion. Neither cell density nor viability was influenced by the hypothyroid state. The presence of a T3 receptor and the enzyme activity for T3 production, together with an effect of T3 on ChAT activity, demonstrate that S-20Y cells are a target for T3 action and suggest that these cells represent an excellent model system for studies of T3 effects on nervous tissues.
The effect of serum on the rate of substrate oxidation by dissociated brain cells in vitro was examined. At a serum protein concentration of approximately 0.55 milligram per milliliter, oxidation of ...6-$^{14}$Cglucose to $^{14}$CO$_{2}$ was decreased more than 50 percent. Oxidation of 3-$^{14}$C-3-hydroxybutyrate and U-$^{14}$Cglutamine was decreased much less. Serum from cows, rats, horses, and humans produced similar effects, as did serum from young and old animals and from both sexes. The effect on 6-$^{14}$Cglucose oxidation was proportional to serum protein concentration, and significant inhibitory activity was obtained with dialyzed serum. Heating (80°C for 10 minutes) significantly reduced the inhibitory activity. These results suggest the presence of a factor in serum that can preferentially decrease glucose oxidation. Such a factor would have profound implications for metabolic regulation in vivo and for studies of cells in vitro in which serum is included in the growth medium.
A study was carried out to determine the effect of trypsin on glucose transport into brain cells. Two suspensions of dissociated cells were prepared from the two brain hemispheres of adult rats--one ...using only mechanical means to dissociate the cells and one using trypsin. The use of trypsin for preparation of dissociated brain cells caused a marked reduction in the rate of transport of 1,2-3H-2-deoxy-D-glucose compared to uptakes of this glucose analog by cells prepared without trypsin. Responses of the two cell preparations to inhibitors of glucose transport (cytochalasin B and phloretin) were similar. Rates of oxidation of 6-14Cglucose to 14CO2 by trypsin-treated cells were nearly double those in cells prepared without trypsin. Electron microscopic examination of the two preparations revealed much less preservation of structural integrity if trypsin was used to prepare the cells. The findings suggest that trypsin alters cell structure and affects receptor-regulated events in brain cells.
Cultured brain cells oxidize glucose and numerous alternate substrates to CO2 for energy production, however, the importance of these observations to the intact brain have not been established. We ...have adapted in vivo brain microdialysis procedures to measure the rate of 14CO2 formation from 14C-glutamate, 14C-glutamine, and 14C-glucose in the hippocampus of awake unanesthetized free-moving rats. Two, 9 and 16 days after surgery (to implant guide cannulae) microdialysis probes were inserted into the guide cannulae and perfused with artificial CSF containing either 14C-glutamate, 14C-glutamine or 14C-glucose. Dialysate fractions were collected during 20 min intervals for determination of 14CO2. The amount of labelled 14CO2 increased for 40 to 60 min and then plateaued and remained relatively constant for at least 6 hours. When the probe was removed from the hippocampus and inserted into a vial containing non-radioactive CSF, 14CO2 production dropped rapidly. The quantity of 14CO2 recovered from glutamate was greater than from glucose or glutamine reflecting pool sizes, uptake characteristics and point of entry into oxidative pathways. The microdialysis system was verified by using model systems with cultured astrocytes suspended in media to simulate the brain. The present results indicate brain microdialysis may be used to study the role of alternate substrates in specific brain regions under varying physiological states.
Skin fibroblasts (CC-69) cultured from a patient with a unique syndrome of ketoacidosis associated with coenzyme A transferase (EC 2.8.3.5) deficiency showed an altered pattern of carbohydrate ...metabolism. These cells used glucose at a rate significantly less than controls (125 against 680 nmol/mg per hr). The oxidation of 6-14Cglucose to14CO2by these cells was also significantly diminished (12 against 350 pmol/mg per hr), but 2-14Cpyruvate and 1,4-14Csuccinate oxidation by these cells did not differ from that by control cells. Measurements of glycolytic intermediates showed a reduction of several intermediates in the CC-69 cells that confirmed an inhibition of glycolysis between fructose-1,6-bisphosphate and pyruvate. The apparent inhibition in these cells could be reversed by an extended incubation of the cells in a buffered glucose solution. After 18 hr of incubation in 2.5 mM glucose, glucose uptake by the CC-69 cells increased 20-fold to 2560 nmol/mg per hr, whereas the rate for control cells remained constant at 640 ± 90. Concomitant with this increase, 6-14Cglucose oxidation rose from 8 to 2261 pmol/mg per hr while controls remained constant at 428 ± 175. This change was not due to new enzyme formation because incubation with puromycin had no effect on the increased use of glucose. Mixing experiments demonstrated no transfer of a permeable inhibitor or activating substances. In view of the deficiency of coenzyme A transferase in these cells, the data suggest an indirect regulatory role for this enzyme in peripheral tissue glycolysis.
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