Carlos Lamar

Studies on the mechanism of carbohydrate repression in rat liver.

Abstract In earlier published studies we have shown that the amino acid catabolizing enzymes, threonine dehydrase and ornithine transaminase, can be rapidly induced in the livers of protein depleted rats force-fed casein hydrolysate or injected with glucagon. This induction, which is inhibited by the administration of gamma irradiation, actinomycin, puromycin, or fluoroorotic acid, is also inhibited by the forced feeding of glucose or fructose. The kinetic picture of the inhibition by carbohydrate feeding appears to resemble that produced by puromycin administration rather than by actinomycin or fluoroorotic acid administration, suggesting that the repressive effect of carbohydrate is exerted at the stage of protein synthesis involving peptide formation. The feeding of carbohydrates is accompanied by the rapid accumulation of large quantities of glycogen in the liver, and when glucagon is injected into rats receiving dietary glucose the resultant enzyme induction is accompanied by a decrease in liver glycogen concentration. During the course of these studies we have evolved the working hypothesis that the metabolic control phenomena which we have observed (induction and repression) arise through the interaction of enzyme forming systems with intracellular lipoprotein membranes such as the endoplasmic reticulum. Agents which alter the configuration of these membranes and/or their association with the enzyme forming systems (polysomes) might be expected correspondingly to affect induction and repression. For example, when glycogen is deposited in the liver cell after feeding carbohydrate, it appears to become associated with the endoplasmic reticulum. Numerous workers have shown that this endoplasmic reticulum is morphologically quite different (tubular network, no ribosomes attached to membranes) from that not associated with glycogen (lamellar network, ribosomes attached to membranes) and it is possible that such morphological differences may be accompanied by differences in metabolic properties. In the further exploration of this hypothesis we have tested the effects of a variety of hormones on the induction and repression of certain amino acid catabolizing enzymes (serine dehydrase, ornithine transaminase, and tyrosine transaminase), the rationale being that hormones may exert their metabolic action through an effect on membrane structure. We observed that in protein depleted rats which have been pretreated with cortisone (5 mg/day for 3 days) glucose repression of enzyme induction is significantly less than that in rats not given cortisone. We also found, however, that induction by casein hydrolysate without glucose is substantially less in rats treated with cortisone as compared to those not receiving cortisone. The administration of cortisone alone does not produce significant enzyme induction under the conditions of these experiments. These results suggest that cortisone exerts a “protective” effect on the induction process, permitting induction to occur in the face of carbohydrate, but not itself causing induction. Similar effects are produced by triamcinolone, but deoxycorticosterone has no effect on the induction or repression phenomena. Experiments were also conducted to study the possibility that carbohydrate repression may in fact be a consequence of increased insulin production stimulated by the carbohydrate feeding. No suppression of induction occurs when rats receiving casein hydrolysate are injected with insulin at dosages just below lethality.

Effect of Gamma Radiation on Dietary and Hormonal Induction of Enzymes in Rat Liver

The dietary induction of serine dehydrase, produced by the oral intubation of hydrolyzed casein to protein-depleted rats, is markedly inhibited by doses of γ-radiation of 400 roentgens or higher provided the irradiation is given within an hour after the initial dose of casein. If the irradiation is delayed until 7 hours after the initial dose of casein, induction is not inhibited. In contrast, the cortisone induction of tyrosine α-ketoglutarate transaminase is not inhibited by doses of γ-radiation up to 3200 roentgens; in some instances hormonal induction of this enzyme appears to be enhanced by irradiation.

Studies on the mechanism of the stimulation of tyrosine aminotransferase activity in vivo by pyrimidine analogs: the role of enzyme synthesis and degradation.

Abstract The pyrimidine analogs, 5-fluoroorotate and 5-azacytidine, have been shown to stimulate the basal level as well as the cortisone, tryptophan, and casein hydrolysate-induced levels of the rat liver enzyme, tyrosine aminotransferase. This stimulation was most marked in the case of dietary and hormonal induction when the analog was given 4–6 hr prior to the administration of the inducer. When tryptophan induced tyrosine aminotransferase, maximal stimulation by the analog occurred if it were given 2 hr prior to the administration of the amino acid. The optimal stimulatory dose of 5-azacytidine was 5 mg/kg body weight whereas 5-fluoroorotate gave its highest stimulation at a dose of 60 mg/kg. Of several orotic acid analogs tested, only the chloro-analog had an effect similar to the fluoro-congener. Utilizing quantitative immunochemical precipitation and pulse labeling in vivo, it was demonstrated that the administration of 5-fluoroorotate or 5-azacytidine at doses of 60 and 5 mg per kg, respectively, while causing a stimulation in the basal level of tyrosine aminotransferase, did not result in any change in the rate of enzyme synthesis. Furthermore, after cortisone induction of the enzyme, the delayed administration of these analogs caused either a further stimulation in the level of the enzyme or the maintenance of a high level while the enzyme activity decayed in animals not given the analogs. The rates of synthesis either showed no change or a decrease while the amount of enzyme was increasing. Prelabeling of the enzyme in vivo after induction with cortisone and followed by the administration of 5-fluoroorotate resulted in a marked decrease in the t 1 2 of the decay rate of the enzyme measured either by loss of radioactivity or by loss of enzyme activity. These studies suggest that these analogs act in some manner to prevent enzyme turnover by an inhibition of enzyme degradation.

l-Tryptophan inhibition of tyrosine aminotransferase degradation in rat liver in vivo☆

Abstract The administration of l -tryptophan to both intact and adrenalectomized animals results in a marked increase in the activity of tyrosine aminotransferase. Maximal increases in enzyme activity are stimulated by doses of l -tryptophan much lower than those required for maximal stimulation of tryptophan oxygenase activity in vivo . When l -tryptophan was administered to animals that had been given cortisone 5 hr earlier, a further sustained increase in enzyme activity was demonstrated. 5-Hydroxy- dl -tryptophan and indole administration in amounts equimolar to l -tryptophan also result in similar increases in activity whereas α-methyl- dl -tryptophan produces little or no increase. Utilizing pulse-labeling in vivo with quantitative immunochemical precipitation of tyrosine aminotransferase by specific antisera, it was demonstrated that the administration of tryptophan caused an increase in enzyme amount with no concomitant increase in the rate of enzyme synthesis. In animals given cortisone, subsequent injections of tryptophan caused the amount of enzyme to continue to increase while both the amount of enzyme in control animals, as well as the rates of synthesis in both tryptophan-treated and control animals, decreased in a parallel fashion. Prelabeling of tyrosine aminotransferase in vivo after the enzyme had been induced with cortisone demonstrated that the subsequent administration of tryptophan caused a marked inhibition in the decay of the radioactive enzyme, as well as in enzyme activity. These data support the proposal that the amino acid, tryptophan, has a special role both in the maintenance of hepatic protein synthesis and in the regulation of specific enzyme degradation in rat liver.

COMPARATIVE EFFECTS OF ACTINOMYCIN AND IONIZING RADIATION ON ENZYME SYNTHESIS IN MAMMALIAN CELLS IN VIVO.

Studies using actinomycin D or ionizing radiation have done much to elucidate our knowledge of the control of genetic expression in mammalian tissues. However, little effort has been made to correlate the findings with one inhibitor with those obtained with the other. In the regenerating liver system in rats, DNA synthesis as well as the increase in DNA polymerase resulting from the operation is inhibited by actinomycin and ionizing radiation when the inhibitor is administered within the first 15 hours after the operation or, in the case of radiation, sometime before. If these substances are given after this time, no effect is noted on these processes. Studies on the control of enzyme synthesis in mammalian cells have shown that hormonal induction of enzyme synthesis is inhibited by actinomycin, whereas substrate or dietary induction of several enzymes show different or no effects. The induction of serine dehydrase in rat liver is initially sensitive and later resistant to the effects of actinomycin as we...

Studies on a 32S component of nuclear RNA

Abstract An RNA component having a sedimentation constant of 32S has been isolated from nuclei of rat liver and Novikoff hepatoma cells in tissue culture in good yield. Pulselabeling studies in vivo and in vitro indicated that this species of nuclear RNA does not bear a precursor relationship to other ribosomal components, suggesting that it is a relatively stable component of nuclear RNA. This RNA was shown to have slightly higher adenine.+ uridine:guanine + cytosine base ratios than the 28S ribosomal RNA by means of 32 P labeling in vivo . The possible functions of such a relatively stable nuclear RNA component are discussed.

Studies on two glutaminase systems from rat kidney

Abstract An enzyme, glutamohydroxamase, that accomplishes the hydrolysis of γ-glutamohydroxamate and the deamidation of glutamine at similar rates has been described. This enzyme is predominantly present in rat kidney, is absent from liver, and is bound to the microsomal fraction of the cell. It is activated by SO42− and inhibited by HgCl2 and reduced glutathione in a similar manner as is glutaminase, which it resembles in several other ways as well. However, the failure of glutaminase to hydrolyze γ-glutamohydroxamate serves to distinguish the two enzymes. The products of the glutamohydroxamase reaction were shown to be glutamic acid and hydroxylamine and the reaction was found not to be reversible.

The Effect of Gamma Radiation and Actinomycin on Cortisone Induction of Liver Enzymes

Summary Ionizing radiation and actinomycin-D were employed in an attempt to gain further insight into the mechanisms by which cortisone induces hepatic tyrosine transaminase (L-tyrosine: 2-oxoglutarate aminotransferase, EC 2.6.1.5) and tryptophan pyrrolase (L-tryptophan: H2O2 oxidoreductase, EC 1.11.1.4). Administration of the antibiotic at a dose of 200 μg/kg resulted in slight inhibition of the induction of tyrosine transaminase and tryptophan pyrrolase. Concomitant gamma radiation (6000 R) markedly enhanced this inhibition, while irradiation of the animals wthout actinomycin administration had little or no effect on the basal or induced levels of the enzymes studied. N2-di-methylene-actinomycin-C3, an analogue of actinomycin-D which does not bind to DNA, had no effect on enzyme induction. The authors thank Mrs. Annabelle Cutler for her excellent technical assistance.