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Department of Animal Science, Cornell University, Ithaca, NY 14853
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGMENT
FOOTNOTES
LITERATURE CITED
ABSTRACT 
Experiments were conducted to determine if hepatic threonine dehydrogenase (TDH) activity is influenced by dietary protein or specific amino acid concentrations. In an initial experiment, young chicks were deprived of feed for 60 h or had access for 72 h to a 22% protein basal diet, a protein-free diet or a 51% high protein diet. TDH activity was determined as aminoacetone and glycine accumulation during incubation of liver mitochondria. TDH activity was significantly (P < 0.01) lower in chicks fed the protein-free diet and significantly greater in chicks fed the high protein diet compared with chicks fed the basal diet. Food deprivation had no effect on TDH activity. A second experiment was conducted using the 22 and 51% protein diets, the 22% protein diet plus 1.14 g/100 g diet threonine (equivalent to the free plus protein-bound threonine content of the high protein diet), and the 51% protein diet containing 0.15 g/100 g diet less threonine. TDH was increased in chicks fed either high protein diet (P < 0.05). There were no significant differences in TDH activity, however, between chicks fed the basal diet and the threonine-supplemented diet or between chicks fed the two high protein diets. In two other experiments, the activity of TDH was investigated in chicks fed for 9 d dietary supplements of either serine or glycine (5.5 or 4 g/100 g basal diet, respectively). The supplements were added to the basal diet or the basal diet imbalanced by the addition of 6% branched-chain amino acids. Neither the serine nor the glycine supplement significantly altered TDH activity or the increased activity associated with a branched-chain amino acid-induced threonine imbalance. The results suggest that hepatic TDH activity is influenced by protein level or other amino acids more than by threonine itself.
KEY WORDS: threonine dehydrogenase · chickens · dietary protein · threonine imbalanceINTRODUCTION
The potential pathways of catabolism of the indispensable amino acid L-threonine involve the activities of three enzymes. Two of these enzymes, threonine aldolase (EC 4.1.2.5) and threonine dehydratase (EC 4.2.1.16) are cytosolic, whereas the third enzyme, threonine dehydrogenase (TDH5; EC 1.1.1.103), is a mitochondrial enzyme. It could be questioned, however, if threonine aldolase is actually a functional enzyme of threonine catabolism. Threonine aldolase is the same protein as serine-glycine hydroxymethyltransferase (EC 2.1.2.1), which catalyzes the interconversion of glycine and serine (Schirch and Gross 1968
). Recent studies (Bird and Nunn 1983, Pagani et al. 1991, Yeung 1986) have also shown that previous assays have overestimated the activity of threonine aldolase in rat liver and that this enzyme actually has very little or no activity in catabolizing L-threonine in this species. Threonine dehydratase exists as isozymes differing in relative activities toward L-threonine and L-serine. Catabolism of L-threonine by threonine dehydratase appears to be minimal except under conditions of gluconeogenesis (Ballèvre et al. 1991, Bird and Nunn 1983).
Threonine dehydrogenase is located in the mitochondrial matrix and apparently forms a soluble complex with 2-amino-3-oxobutyrate CoA ligase, which catalyzes the conversion of 2-amino-3-oxobutyrate to acetyl CoA and glycine. Aminoacetone, the only known alternate product of 2-amino-3-oxobutyrate, is formed nonenzymatically. Threonine dehydrogenase is considered to be the primary enzyme initiating L-threonine catabolism. It has been shown to account for 87% of the L-threonine degraded in the livers of normally fed rats (Bird and Nunn 1983) and for about 80% of the L-threonine degraded in pig liver (Ballèvre et al. 1990).
In previous experiments conducted in this laboratory (Davis and Austic 1994), the activity of TDH in isolated chicken hepatic mitochondria was observed to be significantly increased under conditions of dietary threonine imbalance. Supplements of 6% branched-chain amino acids (BCAA)6 or 5.6% indispensable amino acids (IAA)7 to a semipurified basal diet resulted in threonine imbalances and associated increases in the activity of TDH, whereas a 3% serine supplement failed to create an imbalance or an increase in TDH activity. TDH activity was examined at two time points, 24 h and 9 d after the chicks were given access to the supplemented basal diets, and a significant increase in TDH activity was found at both time points in threonine-imbalanced chicks. The increase in TDH activity was not distributed proportionally between the aminoacetone and glycine pathways leading from 2-amino-3-oxobutyrate. There was greater accumulation of glycine than aminoacetone.
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Table 1. Composition of basal diet |
The present investigation was performed to determine the influence of dietary protein and threonine on TDH activity. Because the increase in TDH activity in response to threonine imbalance is large and results primarily in the conversion of threonine to glycine and acetyl CoA (Davis and Austic 1994), it seemed possible that the increased TDH activity reflects a metabolic need for glycine. Therefore, the present investigation was conducted also to determine if the addition of dietary glycine or serine (a glycine precursor) to a threonine-imbalanced diet would prevent the associated increase in TDH activity in chicks consuming the threonine-imbalanced diet.
MATERIALS AND METHODS
) were raised in thermostatically controlled, electrically heated battery brooder cages with raised wire floors. Cages were lighted from 0700 to 2100 h and the chicks had free access to water and a practical chick starter diet. Six to eight days after hatching, the chicks were sorted by weight and those with extreme weights were discarded. The remaining chicks were assigned to experimental groups in such a manner to achieve equal weight distributions among all pens. At the end of experiments, chicks were killed by cervical dislocation and livers were sampled immediately thereafter. All animal procedures were approved by the Institutional Animal Care and Use Committee of Cornell University.
Experiment 1.
The purpose of this experiment was to determine the effects of food deprivation and of feeding a protein-free or a high protein diet on the activity of chicken hepatic TDH. On four consecutive days, 7- to 10-d-old birds were assigned to eight pens of four chicks (2 females and 2 males). Chicks in all of the pens were fed for 5 d the same basal diet which contained a total of 22 g protein/100 g diet. The basal diet contained a total of 0.6 g L-threonine/100 g diet, which was derived from the isolated soybean protein. The composition of the semipurified basal diet (Table 1) was similar to basal Diet B of Davis and Austic (1982a). After the 5-d acclimation period, the eight pens for each consecutive day were randomly split into four groups of two pens of chicks. One group of two pens was maintained for 72 more hours on the basal diet, while a second group of two pens was fed a protein-free diet (Table 2) for 72 h. The third group of two pens was fed for 72 h a high protein diet that contained a total of 51 g protein/100 g diet. This high protein diet contained 1.74 g L-threonine/100 g diet of which 1.59 g/100 g diet was derived from isolated soybean protein, and 0.15 g/100 g diet was from crystalline L-threonine. Thus, the composition of the high protein diet was the same as the basal diet except that crystalline L-threonine was added at 0.15 g/100 g diet, and the amounts of isolated soybean protein, L-glycine and DL-methionine were increased to total amounts of 48.2, 1.02 and 1.2 g/100 g diet, respectively. The addition of threonine was made to ensure that threonine was no longer marginally inadequate as in the basal diet. Methionine and glycine supplements were used in the basal diet to satisfy the requirements for these amino acids (Scott et al. 1976). The additional amounts of glycine and methionine were added to the high protein diets to maintain the same ratio of these amino acids to isolated soybean protein as in the basal diet. The addition of protein and amino acids was at the expense of glucose monohydrate, which was reduced to 30.45 g/100 g diet. The fourth group of two pens was maintained on the basal diet for 12 more hours and then food deprived for 60 h. Thus, after four consecutive days, each treatment had a total of eight replicate pens, with each pen containing four birds. At the end of each 72-h period, total feed consumption and weight change were determined for each pen and whole livers were collected and pooled from all four birds of each pen.
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Table 2. Composition of the protein-free diet |
. It should be noted that when the mitochondria were resuspended in buffer, the integrity of the inner mitochondrial membranes was compromised enough for TDH and 2-amino-3-oxobutyrate-CoA ligase to utilize the exogenous NAD and CoA provided in the TDH assay medium. The mitochondria were not disrupted by sonication or with solubilizing agents because such treatment disrupts the soluble complex between TDH and 2-amino-3-oxobutyrate CoA ligase (Bird et al. 1984). Disruption of this enzyme complex prevents the formation of glycine and acetyl CoA (Tressel et al. 1986).
Threonine dehydrogenase assay and analytical procedures.
The assay procedures used in these experiments were the same as those described previously by Davis and Austic (1994). Aminoacetone was determined using the rapid colorimetric procedure of Urata and Granick (1963). Mitochondrial protein was determined colorimetrically (Lowry et al. 1951). Threonine concentrations of the diets after acid hydrolysis (Krick et al. 1993) and glycine concentrations from the TDH assays were determined by HPLC. The HPLC used in Experiments 1 and 2 consisted of a Beckman (Palo Alto, CA) 110B Pump, a Beckman SP8780XR Autosampler, and an Interaction (Mountain View, CA) ion exchange lithium column (4.6 × 120 mm). Amino acids were separated using lithium elution buffers, separated amino acids were reacted with o-phthaldehyde in a Beckman 230 postcolumn reactor, and amino acid derivatives were quantitated using a Beckman 157 Fluorescence Detector. Amino acid concentrations were determined with a Spectra Physics SP4270 Integration System (San Jose, CA), using Pickering Laboratories standards with norleucine added to the samples as an internal standard. The HPLC apparatus used for Experiments 3 and 4 was described previously by Davis and Austic (1994).
Statistical analyses.
Data from each experiment were subjected to ANOVA according to the General Linear Model procedure using day, treatment, and day × treatment as factors. Single degree of freedom tests were used in all experiments to determine significance of differences between selected dietary groups. In Experiments 2, 3 and 4, the Tukey multiple-comparison procedure (Neter et al. 1990) was used to detect significant differences between all of the diets. All statistical procedures were done with the Minitab statistical software package (Release 8.2, State College, PA), and differences were considered significant when P values were <0.05.
RESULTS
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Table 3. Effects of food deprivation and dietary protein on growth, feed utilization and hepatic threonine dehydrogenase activity of chicks (Experiment 1)1 |
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Table 4. Effects of dietary threonine (Thr) and dietary protein on growth, feed utilization and hepatic threonine dehydrogenase activity of chicks (Experiment 2)1 |
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Table 5. Effect of serine on growth, feed utilization and hepatic threonine dehydrogenase activity in chicks subjected to threonine imbalance (Experiment 3)1 |
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Table 6. Effect of glycine on growth, feed utilization and hepatic threonine dehydrogenase activity in chicks subjected to threonine imbalance (Experiment 3)1 |
DISCUSSION
, which suggested that hepatic TDH activity was increased in chicks fed a 60% casein diet vs. a 20% casein diet. On the basis of in vitro aminoacetone production, pigs fed a diet with 0.52 g/100 g dietary threonine also had a significant increase in hepatic TDH activity when the crude protein level of the diet was raised from 12.6 to 15.6 g per 100 g of diet. This same increase in protein level, however, only slightly raised the activity of TDH of pigs fed a diet containing 0.42 g/100 g dietary threonine (Le Floc'h et al. 1994). In rats, feeding a high protein diet did not increase hepatic TDH activity on the basis of in vitro measurements (Bird and Nunn 1983).
) with young chicks in which no significant change in TDH activity was seen as the threonine level of the diet was increased from 0.6 to 1.8 g per 100 g diet. The apparent insensitivity of chicken hepatic TDH to increasing dietary threonine levels has not been found with porcine hepatic TDH. Le Floc'h and co-workers (1994) found that increasing the dietary threonine concentration in pigs from 0.42 g to a value of 0.52 g per 100 g of diet resulted in a significant increase in pig hepatic TDH activity on the basis of aminoacetone production in vitro. Similarly, using a multitracer infusion method in pigs, Ballèvre and colleagues (1990) determined that there was an increase in threonine flux through the hepatic TDH pathway as the level of dietary threonine increased from a value of 0.68 to 0.81 g per 100 g of diet. The fact that the activity of chicken hepatic TDH was sensitive to dietary protein levels but not to increased dietary levels of threonine in the present research suggests that this enzyme's activity may be regulated by cellular concentrations of other amino acids. This conclusion is supported by the finding in the current and previous research (Davis and Austic 1982b and 1994) that hepatic TDH activity was increased when specific amino acids or a mixture of IAA lacking threonine was added to create threonine imbalances. In addition, increasing the level of glutamic acid in pig diets has been shown to increase hepatic TDH activity (Le Floc'h et al. 1994).
, Rex et al. 1991). Similar regulatory motifs have been found in vertebrates. Marten et al. (1994) observed that amino acid limitation in rat hepatoma cells caused a decreased expression of some genes and increased expression of other genes. Furthermore, Marten et al. (1994) have shown that the expression responses of some of these genes depended on the limitation of one particular amino acid, whereas for other genes, it depended on the limitation of more than one amino acid. Expression of the ornithine decarboxylase gene (Pohjanpelto and Holtta, 1990) and of the asparagine synthetase gene (Guerrini et al. 1993) has also been shown to be influenced by amino acid concentrations.
, Tressel et al. 1986), the unstable product of the TDH reaction seems to be directly transferred to the ligase with subsequent formation of glycine and acetyl CoA. Because the formation of glycine in vitro is dependent on the availability of CoA, it has been assumed that under physiological conditions, the availability of CoA would determine whether glycine or aminoacetone was produced (Bird et al. 1984, Tressel et al. 1986). Bird and colleagues (1984) calculated, on the basis of in vitro studies of intact hepatic mitochondria from rats, that at physiological concentrations of L-threonine, at least 65% of the threonine catabolized in the fed state would be converted to glycine and acetyl CoA.
), the formation of glycine became significantly more predominant, with five to twelve times more glycine produced than aminoacetone. In contrast, when total hepatic TDH activity was decreased in the present experiments by feeding a protein-free diet, there was a proportional decrease in aminoacetone and glycine accumulation.
).
and 1994) and to high protein diets were so striking that it was considered possible that there was a metabolic need for glycine. The results from Experiment 4, however, suggest that at least the predominant formation of glycine seen with feeding a threonine-imbalanced diet does not reflect a metabolic need for glycine. The growth of all of the experimental chicks in Experiment 4 was influenced to some degree by insufficient levels of vitamins A and D in the practical starter diet, and we believe this may have resulted in the failure of the 6% BCAA supplement to cause a threonine imbalance and a significant increase in hepatic TDH activity. Nevertheless, even with this problem, the addition of 4% glycine to the basal diet had no effect on hepatic TDH activity. Furthermore, when compared with the basal diet, the addition of 4% glycine to the 6% BCAA-supplemented diet resulted in a significant increase in TDH activity and the predominance of glycine formation, whereas feeding the 6% BCAA-supplemented diet alone did not. Because serine is readily converted to glycine by serine-glycine hydroxymethyltransferase, the results from Experiment 3 also indicate that the increase in glycine accumulation was not due to an increased metabolic need for glycine.
) if not converted through the action of 2-amino-3-oxobutyrate ligase to glycine and acetyl CoA. Because of its short half-life, all 2-amino-3-oxobutyrate present in the TDH incubation mixture at the end of incubation would convert to aminoacetone before the colorimetric determination of the latter compound is carried out. Aminoacetone was observed not to disappear from chicken liver homogenates during incubation (Green and Elliott 1964). Therefore, aminoacetone can be assumed to be an end product in our TDH assay. Because 2-amino-3-oxobutyrate ligase is present in mitochondrial preparations, it is necessary to measure both aminoacetone and glycine to achieve an indirect measure of TDH activity. Glycine, however, can be degraded by the glycine cleavage system which exists in chickens (Matsui et al. 1993) and mammals (Jois et al. 1990, Petzke and Albrecht 1987). The activity of this system in mammals is increased in response to high protein diets (Ewart et al. 1992). If the system is active in the chickens under the conditions of the TDH assay, then the sum of aminoacetone and glycine may underestimate the actual in vitro activity of TDH. If the glycine cleavage system is affected by dietary protein in chicks as in rats, this underestimate could be greater when chicks have been fed diets containing increased levels of protein.
ACKNOWLEDGMENT
FOOTNOTES
Manuscript received 30 August 1996. Initial reviews completed 9 October 1996. Revision accepted 15 January 1997.
LITERATURE CITED
purification, characterization, and physiological significance.
J. Biol. Chem.
1981;
256:12367-12373
-Amino-
-keto acids. Part II. Rates of decarboxylation of the free acids and the behaviour of derivatives on titration. J. Chem. Soc. 1483-1491.-aminoketones and the metabolism of aminoacetone.
J. Biol. Chem.
1963;
238:811-820
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