Control of ketogenesis from amino acids: III. In vitro and in vivo studies on ketone body formation, lipogenesis and oxidation of tyrosine by rats

Ketone body formation from tyrosine was studied in rat liver in vitro with special references to the activities of tyrosine aminotransferase (EC 2.6.1.5) and p-hydroxyphenylpyruvate hydroxylase (EC 1.14.2.2). Liver was obtained from rats which had been given a high protein diet or cortisol to induce various levels of tyrosine aminotransferase. The enzyme activities of the preparations were plotted against the amounts of ketone body formed from tyrosine. It was found that over a low range of tyrosine aminotransferase activities, activity was proportional to the amount of ketone body formed. However, above this range, ketone body formation ceased to increase and p-hydroxyphenylpyruvate started to accumulate. This inhibition of ketone body formation and accumulation of the p-hydroxyphenylpyruvate could be prevented by addition of ascorbate. These results suggest that the primary factor regulating metabolism of tyrosine in vitro is tyrosine aminotransferase and when the activity of this is high so that it is no longer rate limiting, p-hydroxyphenylpyruvate hydroxylase becomes the rate limiting step because its activity is inhibited by the accumulation of p-hydroxyphenylpyruvate.For in vivo studies rats were given a high protein diet or cortisol to induce various levels of tyrosine aminotransferase and then injected with a tracer dose of [U- or 1-¹⁴ C]tyrosine. Then their respiratory ¹⁴CO₂ and the incorporation of ¹⁴C into total lipids of liver were measured. The amounts of radioactivity in CO₂ and lipids were found to be proportional to the tyrosine aminotransferase activity and were not affected by the free tyrosine concentration in the liver. After injection of [U-¹⁴C] acetate the radioactivities in CO₂ and lipids were not proportional to the tyrosine aminotransferase activity. These results indicate that the enzyme activity also regulates tyrosine metabolism in vivo. In vivo studies gave no evidence of the participation of p-hydroxyphenylpyruvate hydroxylase in regulation of tyrosine metabolism.     

The metabolism of L-phenylalanine and L-tyrosine by liver cells isolated from adrenalectomized rats and from streptozotocin-diabetic rats.

Flux through, and maximal activities of, key enzymes of phenylalanine and tyrosine degradation were measured in liver cells prepared from adrenalectomized rats and from streptozotocin-diabetic rats. Adrenalectomy decreased the phenylalanine hydroxylase flux/activity ratio; this was restored by steroid treatment in vivo. Changes in the phosphorylation state of the hydroxylase may mediate these effects; there was no significant change in the maximal activity of the hydroxylase. Tyrosine metabolism was enhanced by adrenalectomy; this was not related to any change in maximal activity of the aminotransferase. Steroid treatment increased the maximal activity of the aminotransferase. Both acute (3 days) and chronic (10 days) diabetes were associated with increased metabolism of phenylalanine; insulin treatment in vivo did not reverse these changes. Although elevated hydroxylase protein concentration was a major factor, changes in the enzyme phosphorylation state may contribute to differences in phenylalanine degradation in the acute and chronic diabetic states. Tyrosine metabolism, increased by diabetes, was partially restored to normal by insulin treatment in vivo. These changes can, to a large extent, be interpreted in terms of changes in the maximal activity of the aminotransferase.     

Modulation of branched-chain amino acid oxidation in rat hemidiaphragms in vitro by glucose and ketone bodies

Branched-chain amino acid metabolism in hemidiaphragms from 40 h-starved rats is influenced by the provision of glucose as co-substrate. Glucose inhibits 14CO2 production from [l-14C]valine and [U-14C]valine but stimulates 14CO2 production from [l-14C]leucine, [U-14C]leucine and [U-14C]isoleucine. In the presence of glucose, ketone bodies inhibit alanine release and 14CO2 production from [l-14C]valine, [l-14C]leucine and [U-14C]isoleucine, but inhibition is not observed in the absence of glucose as cosubstrate. Glucose-dependent inhibition by ketone bodies of branched-chain amino acid oxidation via inhibition of the branched-chain 2-oxo acid dehydrogenase complex or branched-chain amino acid aminotransferase may account in part for the reported hypoalanaemic action of ketone bodies in vivo.     

Effect of hypobaric stress on enzymes of tryptophan metabolism

1. On exposure of rats to hypobaric stress the tryptophan pyrrolase and tyrosine aminotransferase activities of the liver increased about threefold in 4h. 2. The tryptophan hydroxylase activity increased about 50% on exposure for 24h or more. 3. The increased activities reverted to the basal value on removal of the stress. 4. Treatment with cycloheximide inhibited the increase in the enzyme activities when the time of exposure was short (4h). However, the inhibitor-treated animals showed paradoxically high tyrosine aminotransferase activity on prolonged exposure (24h). 5. The pattern of haematin saturation indicated that the increase in pyrrolase activity under low pressure resembled that obtained with cortisol and not with tryptophan. 6. Repeated administration of cortisol or tryptophan did not have any effect on the activity of tryptophan hydroxylase. 7. The stress-induced increase in hydroxylase activity was not eliminated by the prior administration of 5-hydroxytryptophan to the animals.     

Stabilization of rat liver tyrosine aminotransferase by tetracycline.

Rat liver tyrosine aminotransferase was purified 200-fold and an antiserum raised against it in rabbits. 2. Hepatic tyrosine aminotransferase activity was increased fourfold by tyrosine, twofold by tetracycline, 2.5-fold by cortisone 21-acetate and ninefold by a combination of tyrosine and cortisol administered intraperitoneally to rats. 3. Radioimmunoassay with 14C-labelled tyrosine aminotransferase, in conjunction with rabbit antiserum against the enzyme, revealed that cortisol stimulates the synthesis of the enzyme de novo, but that tetracycline has no such effect. 4. Incubation of rat liver homogenates with purified tyrosine aminotransferase in vitro leads to a rapid inactivation of the enzyme, which tetracycline partially inhibits. 5. The inactivation is brought about by intact lysosomes, and the addition of 10mM-cysteine increases the rate of enzyme inactivation, which is further markedly increased by 10mM-Mg2+ and 10mM-ATP. Here again tetracycline partially inhibits the decay rate, leading to the inference that the increase of tyrosine aminotransferase activity in vivo by tetracycline is brought about by the latter inhibiting the lysosomal catheptic action.     

Liver phenylalanine hydroxylase activity in relation to blood concentrations of tyrosine and phenylalanine in the rat

The plasma concentration of phenylalanine and tyrosine decreases in normal rats during the first few postnatal days; subsequently, the concentration of phenylalanine remains more or less constant, whereas that of tyrosine exhibits a high peak on day 13. The basal concentrations of the two amino acids were not altered by injections of thyroxine or cortisol, except in 13-day-old rats, when an injection of cortisol decreased the concentration of tyrosine. In young rats (13-15 days old), treatment with cortisol increased the activity of phenylalanine hydroxylase in the liver (measured in vitro) and accelerated the metabolism of administered phenylalanine: the rate constant of the disappearance of phenylalanine from plasma and the initial increase in tyrosine in plasma correlated quantitatively with the activity of phenylalanine hydroxylase in the liver. In adult rats, the inhibition of this enzyme (attested by assay in vitro) by p-chlorophenylalanine resulted in a proportionate decrease in tyrosine formation from an injection of phenylalanine. However, the quantitative relationship between liver phenylalanine hydroxylase activity and phenylalanine metabolism within the group of young rats was different from that observed among adult rats.     

Cycloheximide causes increased accumulation of translatable mRNA for tyrosine aminotransferase and tryptophan oxygenase in livers of cortisol-treated rats.

Messenger RNA activities for two cortisol-inducible enzymes, tyrosine aminotransferase and tryptophan oxygenase, have been determined by translation in a wheat germ system. The effects of cycloheximide on the two mRNA activities have been evaluated. Cortisol leads to an increase of the translatable mRNAs for tyrosine aminotransferase and tryptophan oxygenase with a maximum at approximately 6 h. Cycloheximide was administered 4 h after treatment with cortisol; 2 h later, the activities of tyrosine aminotransferase and tryptophan oxygenase mRNA had increased five-fold and two-fold, respectively, compared to the activities reached with cortisol alone. Thereafter the amount of the two translatable mRNAs declined, though 14 h after cortisol administration the mRNA activities were still several fold higher than in control animals. Application of alpha-amanitin together with cycloheximide did not prevent an increased accumulation of specific translatable mRNAs. The increase in tyrosine aminotransferase and tryptophan oxygenase activity by cortisol was immediately blocked by cycloheximide. Whereas tryptophan oxygenase activity rapidly declined after cycloheximide application, tyrosine aminotransferase activity remained at the same level. Approximately 4 h thereafter, both enzyme activities increased again.     

No correlation between binding of glucocorticosteroids to specific cytoplasmic proteins in vivo and enzyme induction in the rat liver.

Time- and dose-dependence of the formation of the different cytoplasmic hormone-protein complexes were studied in the rat liver after administration in vivo of [3H]cortisol or [3H]dexamethasone and compared with the stimulation of RNA polymerase B and induction of tyrosine aminotransferase and tryptophan oxygenase. No correlation could be found between formation in vivo of any of the five cytoplasmic hormone-protein complexes found and stimulation of RNA polymerase B activity or enzyme induction. After administration of [3H]cortisol, different metabolites of cortisol could be demonstrated in the isolated hormone-protein complexes. No time- or dose-dependence of the metabolite patterns could be observed after application of hormone doses that were in the range of the biologically active doses. After administration of [3H]dexamethasone, the same hormone-protein complexes were observed, which contained, however, the injected steroid instead of metabolites. These results seem to indicate that the cytoplasmic binding components present in the rat liver are enzymes involved in the metabolism of the glucocorticosteroids and that dexamethasone binds to these enzymes as a substrate analogue.     

Tyrosine aminotransferase activity in human fetal liver

There are at least two enzymes in adult human liver that transaminate tyrosine: cytoplasmic tyrosine aminotransferase (EC 2.6.1.5) and mitochondrial aspartate aminotransferase (EC 2.6.1.1). Total tyrosine aminotransferase activity in the supernatant fraction of adult human liver was 19.8 nmol of p-hydroxyphenylpyruvate formed per min/mg of protein as compared to 0.53 in fetuses of 12--22 weeks of gestational age and 2.0 in the newborn. The presence of specific tyrosine aminotransferase (EC 2.6.1.5) could be demonstrated by isoelectric focusing techniques in fetal human liver during the first trimester. No specific tyrosine aminotransferase could be detected in the placenta. Total tyrosine aminotransferase activity was elevated by dexamethasone and tyrosine administration to organ cultures of fetal liver.     

Quantification of the importance of individual steps in the control of aromatic amino acid metabolism.

The quantitative importance of the individual steps of aromatic amino acid metabolism in rat liver was determined by calculation of the respective Control Coefficients (Strengths). The Control Coefficient of tryptophan 2,3-dioxygenase for tryptophan degradation was determined in a variety of physiological conditions and with a range of activities of tryptophan 2,3-dioxygenase. The Control Coefficient varied from 0.75 with basal enzyme activity to 0.25 after maximal induction of the enzyme by dexamethasone. The remainder of the control for tryptophan degradation was associated with the transport of the amino acid across the plasma membrane, with only very small contributions from kynureninase and kynurenine hydroxylase. The Control Coefficients of tyrosine aminotransferase for tyrosine degradation were approx. 0.70 and 0.20 with basal and dexamethasone-induced tyrosine aminotransferase activities respectively; the Control Coefficients of the transport of the amino acid into the cell were 0.22 and 0.58 respectively. Phenylalanine hydroxylase was found to have a Control Coefficient for the degradation of phenylalanine of approx. 0.50 under conditions of basal enzyme activity; after maximal activation by glucagon, the Control Coefficient decreased to 0.12. The transport of phenylalanine was responsible for the remaining control in the pathway. These results have important implications, directly for the regulation of aromatic amino acid metabolism in the liver, and indirectly for the regulation of neuroamine synthesis in the brain.     

Control of ketogenesis from amino acids. IV. Tissue specificity in oxidation of leucine, tyrosine, and lysine.

In vitro and in vivo studies were made on the tissue specificity of oxidation of the ketogenic amino acids, leucine, tyrosine, and lysine. In in vitro studies the abilities of slices of various tissues of rats to form 14CO2 from 14C-amino acids were examined. With liver, but not kidney slices, addition of alpha-ketoglutarate was required for the maximum activities with these amino acids. Among the various tissues tested, kidney had the highest activity for lysine oxidation, followed by liver; other tissues showed very low activity. Kidney also had the highest activity for leucine oxidation, followed by diaphragm; liver and adipose tissue had lower activities. Liver had the highest activity for tyrosine oxidation, but kidney also showed considerable activity; other tissues had negligible activity. In in vivo studies the blood flow through the liver or kidney was stopped by ligation of the blood vessels. Then labeled amino acids were injected and recovery of radioactivity in respiratory 14CO2 was measured. In contrast to results with slices, no difference was found in the respiratory 14CO2 when the renal blood vessels were or were not ligated. On the contrary ligation of the hepatic vessels suppressed the oxidations of lysine and tyrosine completely and that of leucine partially. Thus in vivo, lysine and tyrosine seem to be metabolized mainly in the liver, whereas leucine is metabolized mostly in extrahepatic tissues and partly in liver. Use of tissue slices seems to be of only limited value in elucidating the metabolisms of these amino acids.     

New assays for the tyrosine hydroxylase and dopa oxidase activities of tyrosinase.

New assays for the tyrosine hydroxylase and dopa oxidase activities of tyrosinase (EC 1.14.18.1) have been developed. The tyrosine hydroxylase assay uses L-[carboxy-14C]tyrosine as the substrate, 14CO2 is released from the products of the hydroxylation and further metabolism of L-[carboxy-14C]tyrosine by incubation with ferricyanide, and measured radiometrically. D-Dopa is a preferable cofactor to L-dopa for the assay. Dopa oxidase activity is measured spectrophotometrically. Dopaquinone, produced on the oxidation of L-dopa, reacts with Besthorn's hydrazone (3-methyl-2-benzothiazolinone hydrazone) to form a pink pigment with an absorbance maximum at 505 nm. Details of the optimisation of conditions for the assays and their specificities for the two enzyme activities are described.     

Phenylalanine-pyruvate aminotransferase in immature and adult mammalian tissues induction in fetal rat liver

Normal human fetal liver contains little phenylalanine-pyruvate aminotransferase: between the 11th and 22nd week of gestation its activity (per g) is 8.8% of that in adult liver. In rat liver this enzyme begins to rise a few hours before birth. Precocious increases in the phenylalanine-pyruvate aminotransferase activity of fetal rat liver (but not kidney or brain) were evoked by premature delivery and also by the administration of thyroxine or glucagon in utero. These results, Discussed in relation to related observations on other enzymes, suggest that thyroxine secreted by the fetus, and also another factor relaesed at the beginning of labour, may be the natural stimuli for the developmental formation of phenylalanine-pyruvate aminotransferase.The regulation of hepatic phenylalanine-pyruvate aminotransferase and phenylalanine hydroxylase (L-phenylalanine, tetrahydropteridine:oxygen oxidoreductase (4-hydroxylating), EC 1.14.16.1) during fetal development is different: in both man and rat, phenylalanine hydroxylase begins to rise earlier and is unaffected by the treatments which enhanced the formation of phenylalanine-pyruvate aminotransferase. In suckling rats (but not in fetuses and adults), an injection of cortisol increased the levels of both enzymes. Hepatocarcinomas of the adult rat were devoid phenylalanine hydroxylase as well as phenylalanine-pyruvate aminotransferase. However, suppression in vivo by substrate analogues (α-methylphenylalanine and p-chlorophenylalanine) was unique for phenylalanine hydroxylase.     

The influence of starvation and tryptophan administration on the metabolism of phenylalanine, tyrosine and tryptophan in isolated rat liver cells.

Liver cells from fed Sprague-Dawley rats metabolized phenylalanine, tyrosine and tryptophan at rates consistent with the known kinetic properties of the first enzymes of each pathway. Starvation of rats for 48 h did not increase the maximal activities of phenylalanine hydroxylase, tryptophan 2,3-dioxygenase and tyrosine aminotransferase in liver cell extracts, when results were expressed in terms of cellular DNA. Catabolic flux through the first two enzymes was unchanged; that through the aminotransferase was elevated relatively to enzyme activity. This is interpreted in terms of changes in the concentrations of 2-oxoglutarate and glutamate. Cells from tryptophan-treated animals exhibited significant increases in the catabolism of tyrosine and tryptophan, but not of phenylalanine. The activities of tyrosine aminotransferase and tryptophan 2,3-dioxygenase were also increased, although the changes in flux and enzyme activity did not correspond exactly. These results are discussed with reference to the control of aromatic amino acid catabolism in liver; the role of substrate concentration is emphasized.     

Ketone body utilization for energy production and lipid synthesis in isolated rat brain capillaries

Isolated brain capillaries from 2-month-old rats were incubated for 2 h in the presence of [3-14C]acetoacetate, D-3-hydroxy[3-14C]butyrate, [U-14C]glucose, [1-14C]acetate or [1-14C]butyrate. Labelled CO2 was collected as an index of oxidative metabolism and incorporation of label precursors into lipids was determined. The rate of CO2 production from glucose was slightly higher than from the other substrates. Interestingly, acetoacetate was oxidized at nearly the same rate as glucose. This shows that ketone bodies could be used as a source of energy by brain capillaries. Radiolabelled substrates were also used for the synthesis of lipids, which was suppressed by the addition of albumin. The incorporation of [U-14C]glucose in total lipids was 10-times higher than that from other precursors. However, glucose labelled almost exclusively the glycerol backbone of phospholipids, especially of phosphatidylcholine. Ketone bodies as well as glucose were incorporated mainly into phospholipids, whereas acetate and butyrate were mainly incorporated into neutral lipids. The contribution to fatty acid synthesis of various substrates was in the following order: butyrate greater than or equal to acetate greater than ketone bodies greater than or equal to glucose. All precursors except glucose were used for sterol synthesis. Glucose produced almost exclusively the glycerol backbone of phospholipids.     

Stabilization of rat liver tyrosine aminotransferase in vivo by pyridoxine administration.

Administration of pyridoxine stabilizes rat liver tyrosine aminotransferase in vivo, whereas administration of cortisol, cyclic AMP, glucagon, insulin, tryptophan or tyrosine does not. The results of these and other experiments with pyridoxine are discussed in relation to the mechanisms of action of this vitamin on the activity of the enzyme.     

Biphasic effects of translational inhibitors on liver tyrosine aminotransferase.

Low doses of cycloheximide or emetine cause rat liver tyrosine aminotransferase activity to rise up to twice the control levels in 2 h. By contrast, in the same interval no changes, or only a slight decrease, are produced by either drug at high dosage. Adrenalectomised animals display the same pattern of response. High doses of either antibiotic virtually afford a complete inhibition of 14C-labelled amino acid incorporation into liver and plasma proteins, whereas no more than a 30% decrease is observed with low doses. When administered in the course of the induction by cortisol, high doses of inhibitor prevent any further change in tyrosine aminotransferase activity, stabilising it at the level already attained; low doses, while slightly affecting the synthetic phase evoked by cortisol, drastically interfere with the deinduction. Six hours after various doses of either inhibitor the tyrosine aminotransferase activity is markedly increased, this late effect being largely dependent on the presence of adrenals. The amino acid incorporating actitivy of the liver may exceed that of controls, as observed particularly after small doses of emetine.     

Tetrahydrobiopterin and Biogenic Amine Metabolism in the hph-1 Mouse

Abstract: hph-1 mice, which have defective tetrahydrobiopterin biosynthesis due to decreased GTP cyclohydrolase I activity, have been used to investigate the effects of tetrahydrobiopterin deficiency on aromatic l -amino acid monooxygenases and brain monoamine metabolism. Liver tetrahydrobiopterin levels were decreased, and tetrahydrobiopterin deficiency and reduced levels of dopamine, norepinephrine, serotonin, and their metabolites in the brain occurred both pre- and postnatally. Chronic subcutaneous tetrahydrobiopterin elevated brain levels to values higher than those seen in controls but had no effect on monoamine metabolism. In vivo activities of tyrosine hydroxylase and tryptophan hydroxylase were significantly decreased. There was a 30% decrease in the in vitro activity of striatal tyrosine hydroxylase and 50% decrease in liver phenylalanine hydroxylase. Western blotting demonstrated that the lower monooxygenase activities resulted from a reduced absolute amount of tyrosine hydroxylase and phenylalanine hydroxylase protein. The findings suggest involvement of tetrahydrobiopterin in the control of the steady-state concentration of the aromatic l -amino acid monooxygenases. In addition, demonstration of central monoamine changes in the hph-1 mouse make it a possible model system for the investigation of the neuropathological mechanisms in Dopa-responsive dystonia, which has recently been linked with mutations in the gene for GTP cyclohydrolase I.     

Effects of Ammonia and β-Methylene-dl-Aspartate on the Oxidation of Glucose and Pyruvate by Neurons and Astrocytes in Primary Culture

Both ammonia and beta-methylene-DL-aspartate (beta-MA), an irreversible inhibitor of aspartate aminotransferase activity and thus of the malate-aspartate shuttle, were found previously to decrease oxidative metabolism in cerebral cortex slices. In the present work, the possibility that ammonia and beta-MA affect energy metabolism by a common mechanism (i.e., via inhibition of the malate-aspartate shuttle) was investigated using primary cultures of neurons and astrocytes. Incubation of astrocytes for 30 min with 5 mM beta-MA resulted in a decreased production of 14CO2 from [U-14C]glucose, but did not affect 14CO2 production from [2-14C]pyruvate. Conversely, incubation of astrocytes with 3 mM ammonium chloride resulted in decreased 14CO2 production from [2-14C]pyruvate, but 14CO2 production from [U-14C]glucose was not significantly affected. Ammonium chloride had no significant effect on 14CO2 production from either [U-14C]glucose or [2-14]pyruvate by neurons. However, incubation of neurons with beta-MA or beta-MA plus ammonium chloride resulted in a approximately 45% decrease of 14CO2 production from both [U-14C]glucose and [2-14C]pyruvate. A 2-h incubation of astrocytes with beta-MA resulted in no change in ATP levels, but a 35% decrease in phosphocreatine. Similar treatment of neurons resulted in greater than 50% decrease in ATP, but had little effect on phosphocreatine. beta-MA also caused a decrease in glutamate and aspartate content of neurons, but not of astrocytes. The different metabolic responses of neurons and astrocytes towards beta-MA were probably not due to a differential inhibition of aspartate aminotransferase which was inhibited by approximately 45% in astrocytes and by approximately 55% in neurons.     

Functional significance of aromatic amino acid: aromatic keto acid aminotransferases in rat brain and liver: competition for tryptophan between aminotransferases and tryptophan hydroxylase in vitro.

Using low concentrations of substrates and cofactors, a comparison was made of the relative rates by which aminotransferases catalysed transaminations between aromatic amino acids and aromatic or aliphatic keto acids. Tryptophan aminotransferase in homogenates of rat midbrain and liver transaminated phenylpyruvate at a rate 70 to 150-fold greater than the rate with α-ketoglutarate at low concentrations of substrates. Phenylalanine aminotransferase in liver and midbrain also was more active with aromatic keto acids than with aliphatic keto acids. However, tyrosine aminotransferase in dialysed homogenates of midbrain transaminated α-ketoglutarate and phenylpyruvate at approximately equal rates. Fresh homogenates of midbrain contained an inhibitor which markedly decreased tyrosine aminotransferase activity with α-ketoglutarate but not with phenylpyruvate. Tyrosine aminotransferase in homogenates of rat liver transaminated α-ketoglutarate and phenylpyruvate at equal rates below 10 μM keto acid, but above 10 μM, transamination of α-ketoglutarate was favoured. With homogenates of liver, transamination of α-ketoglutarate, but not phenylpyruvate, by tyrosine was increased 650% by exogenous pyridoxal phosphate. Since tryptophan aminotransferase in the brain may compete with tryptophan hydroxylase for available tryptophan, a comparison was made of the relative activities of tryptophan hydroxylase and tryptophan aminotransferase. At concentrations above 7.5 μM phenylpyruvate, transamination was 8 to 17-fold greater than the rate of hydroxylation of 50 μM tryptophan.