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Metabolic response of liver lysine α-ketoglutarate reductase activity in rats fed lysine limiting or lysine excessive diets
NUTRITION RESEARCH, Vol. 13, pp. 1433-1443, 1993 0271-5317/93 $6.00 + .00 Printed in the USA. Copyright (c) 1993 Pergamon Press Ltd. All rights reserved.
METABOLIC RESPONSE OF LIVER LYSINE (x-KETOGLUTARATE REDUCTASE ACTIVITY IN RATS FED LYSINE LIMITING OR LYSINE EXCESSIVE DIETS Andrew R Foster, Ph.D.*, Piotr W D Scislowski, Ph.D., CIan Harris, Ph.D. & Malcolm F Fuller, Ph.D. Department of Physiology, Rowett Research Institute, Aberdeen, Scotland, AB2 9SB, UK
ABSTRACT
The effects of feeding isonitrogenous diets (at a restricted ration size), containing limiting or excessive amounts of lysine on various biochemical parameters of rats were investigated. Lysine cx-ketoglutarate reductase (LKGR) and citrate synthase (CS) activities, hepatic portal vein (HPV) blood and liver free amino acid concentrations were measured in rats fed the experimental diets for up to 8 days. LKGR and CS activities remained constant in rats adapted to the lysine limiting diet. In contrast, LKGR activity rapidly increased (after 2 days) in rats fed the lysine excess diet and remained at a high level for the remainder of the experiment. The increase in liver LKGR activity in response to the lysine excess diet was associated with significant increases in HPV and liver free lysine concentrations (4.5- and 2fold, respectively). The significance of these data are discussed with respect to the metabolic response of rats challenged with an increased lysine supply mad its effects on free lysine concentrations and liver LKGR activity. Keywords: Lysine, Lysine c~-ketoglutarate reductase, Citrate synthase.
INTRODUCTION
Although it is well documented that the feeding of high protein diets increases the activities of many amino acid degrading enzymes, inconsistencies exist between studies (1, 2). Lysine tx-ketoglutarate reductase (LKGR, EC 1.5.1.8) is the regulatory enzyme of lysine oxidation in mammals and birds and its in vivo activity is sensitive to both the dietary protein and lysine content (3, 4, 5). However, in most feeding experiments on the effects of dietary protein and/or lysine on LKGR activity animals have been fed ad libitum. As a result, differences in food intake between treatment groups and any consequent effects on enzyme activity may have been ignored. The sensitivity of LKGR activity to both lysine and protein supply therefore prompted us to re-evaluate the in vivo metabolic response of LKGR to lysine, when differences in food intake were precluded. LKGR activity is located principally in the liver, but is also observed in other organs and tissues (3). Within the cell, LKGR is exclusively located in the mitochondrial matrix (6) where it catalyses the condensation of lysine and c~-ketoglutarate to form saccharopine. Whilst the half-life of LKGR is unknown the half-lives of some better characterised mitochondrial
* To whom correspondence should be addressed 1433
1434
A.R. FOSTER et al.
enzymes are known to range between 0.5 h and 8 days (7). These temporal differences in mitochondrial enzyme half-life raise the possibility of adaptive increases in mitochondrial enzyme (mitochondrial protein) resulting in non-specific increases in enzyme activity during relatively long-term (days-weeks) feeding experiments. To circumvent the above problems in the present study we fed rats isonitrogenous diets (at a restricted ration size) which contained a limiting or excessive amount of lysine. The activities of LKGR and citrate synthase (CS) in mitochondrial preparations were measured to enable us to describe the metabolic response of liver LKGR (i.e. LKGR per unit mitochondria, effect on CS activity and the amount of mitochondrial protein/g liver) when the rats were challenged with an increased supply of lysine.
MATERIALS AND METHODS
Animals, diets and feeding. Thirty two 80-85 g male rats (Hooded Lister, Rowett strain) were used in this experiment which was performed with four replicates of eight rats. After weaning, all animals were fed a stock commercial diet (Labsure CRM Diet, Labsure Lavender Mill, Manea, Cambridge) until they reached a minimum weight of 80 g. Each replicate of eight animals was pre-adapted to the lysine limiting diet (LL) which was fed ad libitum for five days. Both experimental diets were formulated using the following ingredients (g/kg diet): casein 72.4, glucose 234.0, sucrose 147.8, cellulose 61.7, corn oil 40.3, mineral and vitamin premix (8) 114.6. The experimental diets were supplemented to provide the following essential amino acids (g/kg) in the concentrations required for growth (9); threonine 1.7, valine 2.1, cystine 1.7, methionine 0.7, isoleucine 2.2, leucine 2.4, ~tyrosine 0.5, phenylalanine 1.8, histidine 0.7, arginine-HC1 3.2, tryptophan 0.7. The LL diet was further supplemented with 18.1 g aspartate, 21.9 g monosodium glutamate, and 0.1 g lysine-HC1/kg diet to give a 10% crude protein diet containing 5.2 g lysine/16 g N. This lysine content accounted for approximately 87% of the growing rat's lysine requirement (9). The lysine excess diet (LE) was identical to the LL diet except that the LE diet was supplemented with 24.6 g lysine-HCl/kg diet to provide 22.8 g lysine/16 g N (approximately 4.4-fold the rat's lysine requirement). Aspartate and monosodium glutamate were omitted from the LE diet to maintain a constant N concentration. After the five day pre-experimental period the animals were individually housed in perspex cages fitted with continuous feeders. The feeders consisted of a circular trough cut into a perspex disc (100 mm diameter) to which the rat only had access to an arc of 10 mm. The trough completed one complete revolution every 24 h. A weighed amount of diet was pressed into the trough daily. The animals were maintained in a temperature-controlled room at 23 + I~ with a 12 h light-12 h dark cycle and given water ad libitum. All the rats were offered their diet at a rate of 10 g per 100 g average body weight at the start of the experiment. Uneaten feed was collected daily from the feeder dish and spilled food from absorbent paper placed beneath each cage. Daily food intake was calculated as the difference between feed offered and feed remaining or spilled after 24 h. Using the above regime, the animals were adapted to the feeder cages for a further five days before the start of the experiment.
LYSINE ec-KETOGLUTARATE REDUCTASE
1435
Experimental protocol. The sixth day that the animals were in the feeder cages was designated as day 0 and was assumed to represent the full adaptation of the animals to the LL diet. The eight rats in each replicate were randomly allocated to either a control group which was offered the LL diet as before, or a treatment group which was offered the LE diet at the same ration size (mean weights of the LL and LE rats on day 0 were 89.1 + 1.2, 86.7 + 1.3 g, respectively). On day 0 two rats were killed as described below. One animal from the LL diet and one from the LE diet was also killed on days 2, 4 and 8 of the experiment.
Dissections and tissue preparation. On each day of sampling, the animals were weighed and killed by cervical dislocation. The livers were rapidly excised and rinsed in ice-cold homogenisation buffer 1 (220 mM mannitol, 70 mM sucrose, 2 mM HEPES and 1 mM EGTA, pH 7.8). A sub-sample of liver was immediately freeze-clamped and stored at -7&C for amino acid analysis. Another sample of liver, was used to make a 10% (w/v) homogenate in homogenisation buffer 1 from which mitochondria were prepared. The tissue was homogenised by hand in a 30 ml glass homogeniser using a teflon pestle. The liver homogenate was differentially centrifuged to isolate mitochondria (10). All procedures were performed at 4~ unless otherwise stated. A further sample of liver (300-400 mg) was used to make a 10% (w/v) whole liver homogenate in homogenisation buffer 2 (identical to homogenisation buffer 1 with the addition of 0.2% (v/v) Nonidet P-40"*). The liver was homogenised by hand in a 10 ml glass homogeniser using a teflon pestle and the homogenate was spun at 13,000 g at 4~ for 15 min. The resulting supernatant was used to determine the citrate synthase activity in the whole liver homogenate.
Enzyme assays. Enzyme activities were measured by initial rate measurements in a Cecil recording spectrophotometer temperature-controlled at 25~ The LKGR assay contained 7.5 mM txketoglutarate, 124 I.tM NADPH and 0.4 mg mitochondrial protein in 0.1 M HEPES containing 0.2% Nonidet P-40, pH 7.8. The assay substrates (except lysine) were mixed in the cuvette and the reaction was initiated by the addition of 25 mM lysine. The decrease in absorbance at 340 nm was read against a blank in which the volume of lysine was replaced by the equivalent volume of assay buffer (0.1 M HEPES pH 7.8). Before use, the mitochondrial fractions were diluted to 20 mg/ml for the LKGR assay; this was further diluted tenfold for the citrate synthase (CS) assay. The whole liver homogenates were diluted fivefold before the measurement of CS (11), with two slight modifications. In our CS assay, the pH of the assay buffer (0.1 M HEPES) was 7.8 and the detergent used was Nonidet P40 (0.2%, v/v). The final assay volume for both enzyme assays was 1000 gtl and the molar extinction coefficients used were 13600 (CS) and 6220 (LKGR), respectively. Enzyme activities were expressed as nmol/min.mg mitochondrial protein, or nmol/min.g tissue in the case of the whole liver homogenates. Protein determinations were performed using the Biuret method (12). Using the
**Nonidet P-40 is a nonionic detergent (octylphenoxypolyethoxyethanol, Sigma, Dorset, UK)
1436
A.R. FOSTER et al.
above methodologies, the amount (rag) of mitochondrial protein/g rat liver was calculated by dividing the total activity of CS activity extracted from 1 g of wet fiver by the CS activity present in 1 mg mitochondrial protein (13).
Blood sampling. The animals killed on day 8 were anaesthetized with an intraperitoneal injection of sodium pentobarbitone (75 lal/100 g rat body weight). Blood was sampled from the hepatic portal vein and immediately added to 25% sulphosalicyclic acid (SSA, ratio blood:SSA, 1:2) in preparation for amino acid analysis as described below.
Amino acid and proximate analyses. For amino acid analysis, 10 % (w/v) liver homogenates were made in 1 M perchloric acid using an Ultraturrax homogenizer (2 x 10 sec) and hepatic portal blood samples were added to 25% SSA; both precipitants contained L-Norleucine and e-amino caproic acid as internal standards. Separation of acidic and neutral amino acids was performed by ion exchange chromatography using lithium citrate buffers (14). Basic amino acids were separated by an accelerated version of the system for physiological fluids (15). The samples for proximate analyses were prepared and assayed according to standard methods (16).
Statistics. Differences between means were tested using a paired t-test (17). The data from days 2, 4 and 8 were subjected to randomised block, two way ANOVA and tested for treatment and time effects (Genstat). Significance was taken at p < 0.05.
RESULTS
Growth, feed conversion, liver size and body composition. Growth rates (g/rat/day), food conversion efficiencies (gain:food consumed) and liver size (g/100 g final body weight) were unaffected by switching rats from the LL to the LE diet (Table 1) although the values tended to be higher in the LE fed animals. Despite the 5 day pre-adaptation period used in the present study, the animals did show a small increase in food intake during the course of the experiment. This occurred in both treatment groups and was presumably the result of further adaptation to the cages or the semi-synthetic diets. The feeding of the LL and LE diets for 8 days had no significant effect on the whole carcass protein or lipid content (lysine limiting, 73.2 + 1.7% protein, 21.6 + 1.1% lipid; lysine excess, 71.5 + 3.0% protein, 26.2 + 2.3% lipid) when expressed as % dry matter.
-H
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4-1
4-1
r"-
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,5= oo
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4-1
+1
oo
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~q. oo II
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+1
+1
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d 4-1 t~
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4-1
E
N~ >.
1437
1438
A.R. FOSTER et al.
o Q.
.~ 1o
8 @
0
2
4
6
8
Time (days) FIG 1. The time course of lysine ct-ketoglutarate reductase activity (nmol/min.mg mitochondrial protein) in rats fed the lysine limiting ( 0 - 0 ) and lysine excess ( A - A ) diets for up to 8 days. The values represent the mean + SEM and the sample sizes were: day 0, n = 8; remaining time points n = 4
0.160
0
0.120
, m
L_
03
0.080
rr" .-1
0.040
0.000
i
,
I
,
,
0
2
4
6
8
Time (days} FIG 2. The ratio of lysine ct-ketoglutarate reductase activity/citrate synthase activity (LKGR/CS) in rats fed the lysine limiting (O-Q) and lysine excess ( A - A ) diets. Values represent the mean + SEM and the sample sizes were the same as in FIG 1.
LYSINEcr
REDUCTASE
1439
2500
2000
1500
"~ 1000
500
0
2
,
,
,
4
6
8
Time (days} FIG 3. The time course of changes in liver free lysine concentration (nmol lysine/g liver) in rats fed the lysine limiting (O-O) and lysine excess (&-&) diets for up to 8 days. The values represent the mean -_. SEM and the sample sizes were as follows: day 0, n = 8; remaining time points n = 4.
Activities of lysine tx-ketoglutarate reductase and citrate synthase. The time-course of in vivo liver LKGR activity in rats fed the LL and LE diets is shown in Figure 1. LKGR activity (nmol/min.mg mitochondrial protein) significantly increased after 2 days when rats were switched to the LE diet. After day 2, the LKGR activity in the LE fed rats remained at a higher level compared with the control group for the duration of the experiment. Citrate synthase activities in the whole liver homogenate and the mitochondrial preparations were unaffected by the dietary manipulation (overall mean + SEM, 8,894 + 202 nmol/min.g liver; mitochondrial CS, 80.0 + 5.6 nmol/min.mg mitochondrial protein). The ratio of LKGR activity:citrate synthase activity (i.e. LKGR per unit mitochondria) was also increased by feeding the LE diet, the difference from the control group reaching statistical significance by day 4 (Figure 2). The calculated amount of mitochondrial protein (mg/g rat liver) was unaffected by feeding either diet for up to 8 days (overall mean + SEM, 101.5 + 5.3 mg mitochondrial proteirgg rat liver.
1440
A.R. FOSTER et al. TABLE 2
Whole Hepatic Portal Vein Blood Amino Acid Concentrations (nmol/ml) In Rats Fed Lysine Limiting (LL) Or Lysine Excess (LE) Diets For 8 Days.
Amino Acid
LL*
LE
LYS
429.3
+
32.2
1931.0
+
420.0""
ORN
67.4
+
6.9
68.3
+
17.5
HIS
70.4
+
7.3
111.7
+
20.7
ARG
79.6
+
8.3
126.3
+
23.4
ASP
226.5
+
15.1
160.9
+
23.6 #
THR
405.6
+
36.7
531.8
+
26.2#
SER
367.4
+
11.4
462.4
+
86.9~
ASN
416.7
+
57.7
444.0
+
25.5
GLU
252.2
+
9.5
265.0
+
69.4#
GLN
651.8
+
57.9
817.3
+
161.7#
GLY
343.3
+
10.4
404.9
+
66.3
ALA
730.7
+
104.1
811.7
+
86.5
CIT
88.3
+
5.1
105.8
+
13.0
VAL
161.9
+
31.9
201.3
+
30.3
ILE
80.5
+
13.4
101.1
+
14.4
LEU
109.9
+
15.6
133.8
+
16.3
TYR
87.9
+
11.2
106.9
+
6.2
PHE
65.7
+
12.6
96.0
+
21.5
PRO
201.8
+
27.9
284.7
+
63.0
* Values represent the mean + SEM where the sample size was n = 4 unless indicated otherwise. Denotes where the sample size was n = 2-3. ** Significant difference between the LL and LE diets at (p < 0.05).
LYSINE (z-KETOGLUTARATE REDUCTASE
1441
Whole hepatic portal vein blood and liver free amino acid concentrations. The whole hepatic portal vein (HPV) blood amino acid profiles of the rats fed the LL or LE diets are summarised in Table 2. The HPV blood lysine concentration of animals fed the LE diet increased approximately 4.5-fold compared with the LL fed group. No significant differences in the whole HPV blood concentrations were observed for any other of the remaining amino acids, although the HPV concentrations of threonine, serine, glutamine and alanine tended to be higher in the animals fed the LE diet. In contrast, the HPV aspartate concentration tended to be lower in animals fed the LE diet. The liver free lysine concentrations of animals fed the LE diet for 2 days was increased approximately 2-fold (p < 0.05, Figure 3). After day 2 the liver free lysine concentration decreased and by day 8 was not significantly different (p = 0.064) from that measured in the control group. Within the liver there were no significant differences in the concentrations of any other amino acids (data not shown).
DISCUSSION
To our knowledge this is the first report of the simultaneous measurement of LKGR activity and the amount of mitochondrial protein in rats challenged with an increased lysine supply. Our rationale for using citrate synthase as a marker of non-specific changes in mitochondrial protein was that by measuring the activity of this mitochondrial matrix enzyme during the time course of the experiment we could determine whether the LE diet gave rise to a specific increase in LKGR activity or a non-specific increase in total mitochondrial enzyme activity or protein content. Our data indicate that liver LKGR activity (nmol/min.mg mitochondrial protein) was increased 2-3 fold in animals fed the LE diet. This increase in LKGR activity occurred when the lysine intake of the rats was increased from 54 to 213 mg/d. This observed increase in LKGR activity was similar to that described by others (3) and appears to represent a lysine-specific effect as there was no significant change in CS activity in either the liver homogenates, or mitochondrial preparations during the present experiment. We also calculate that the amount (mg) of mitochondriat protein/g liver of rats was unaffected by feeding either diet for 8 days. Thus, in the present study, the increased LKGR activity in animals fed the LE diet was the result of increased LKGR activity per unit mitochondria (as confirmed by the ratio of LKGR:CS activity, Figure 2) and not the result of a general increase in liver mitochondrial protein content. Increased enzyme activity with dietary manipulation is conventionally assumed to be evidence of substrate-induction of that enzyme although this may not be strictly true. The present data support the possibility of induction of LKGR by lysine in vivo although antibody precipitation of LKGR protein and quantitation is needed for the unequivocal demonstration of substrate-induction. In vitro studies with rat hepatocytes (18) indicate that in vitro LKGR activity is also sensitive to glucocorticoid and insulin. However, the role of these hormones (if any) in the in vivo regulation of LKGR by lysine warrants further investigation. Switching rats from the LL to the LE diet for 8 days increased the whole HPV blood lysine concentration approximately 4.5-fold compared with animals on the LL diet: this change was similar to the 4.4-fold difference in the lysine content between the LL and LE diets. In contrast, the liver free lysine concentrations of rats fed the LE diet peaked on day
1442
A.R. FOSTER et al.
2 and steadily decreased after this, presumably as a result of the increased liver LKGR activity and lysine oxidation. Thus, at the end of the experiment although there was a 4.5-fold difference in the HPV blood lysine concentration (Table 2), the liver free lysine concentration was not statistically different from the lysine concentration in the LL fed controls (Figure 3). To maintain equal nitrogen contents in the LL and LE diets, aspartate and glutamate were removed from the LE diet. This resulted in a small decrease in HPV aspartate concentration in the LE animals which was not statistically significant (data not shown). At the same time, the HPV glutamate concentration was similar between rats fed the LL and LE diets despite the fact that glutamate was omitted from the LE diet. The HPV glutamine concentration also tended to be higher in the LE fed rats. In the liver, small increases in the free alanine and glutamine concentrations were also observed in animals fed the LE diet (data not shown), but none of these differences was significant. These data suggest that the activity of liver transaminases was probably increased in animals fed the LE diet to maintain the pools of non-essential amino acids for other (anaplerotic) reactions. In conclusion, our data support the hypothesis that liver LKGR activity is specifically increased by dietary lysine and that this phenomenon appears to be the result of increased LKGR synthesis per mitochondfia. However, further work is needed to describe the mechanism(s) by which lysine regulates LKGR activity both in vitro and in vivo.
ACKNOWLEDGEMENT Special thanks to Dr M Franklin, Messrs R Smart and I Grant for assistance during this experiment. We gratefully acknowledge SOAFD for their financial support of this project.
REFERENCES
1.
Ohno T, Tasaki I. Relation between liver amino acid catabolizing enzymes and free amino acids of liver and plasma in adult cockerels fed diets containing graded levels of protein. J Nutr 1977; 107:829-833.
2.
Wang S, Nesheim MC. Degradation of lysine in chicks. J Nutr 1972; 102:583-596.
3.
Chu SH, Hegsted DM. Adaptive response of lysine and threonine degrading enzymes in adult rats. J Nutr 1976; 106:1089-1096.
4.
Muramatsu K, Takada R, Uwa K. Adaptive responses of liver and kidney lysine-ketoglutarate reductase and lysine oxidation in rats fed graded levels of dietary lysine and casein. Agric Biol Chem 1984; 48:703-711.
5.
Foster AR, Beckett PR, Rees WD, Fuller MF. A sensitive isotopic assay for the measurement of lysine ct-ketoglutarate reductase in cultured rat hepatocytes. J Nutr Biochem 1992; 3:554-559.
.
Higashino K, Fujioka M, Yamamura Y. The conversion of L-Lysine to Saccharopine and tx-Amino adipate in Mouse. Arch Biochem Biophys 1971; 142:606-614.
LYSINE o~-KETOGLUTARATE REDUCTASE 7.
.
Vargas JL, Roche E, Knecht E, Grisolia S. Differences in the half-lives of some mitochondrial rat liver enzymes may derive partially from hepatocyte heterogeneity. FEBS Lett 1985; 1:182-186. Pullar JD, Webster AJF. The energy cost of fat and protein deposition in the rat. Br J Nutr 1977; 37:355-363.
9.
Coates ME, O'Donoghue PN, Payne PR, Ward RJ. Dietary standards for laboratory rats and mice. A report to the Council of the Laboratory Animal Science Association, Laboratory Animal Handbook 2, London, Laboratory Animals Limited, 1969: 13-15.
10.
Johnson D, Lardy HA. Isolation of liver or kidney mitochondria. In: RW Estabrook, ME Pullman. eds. Methods In Enzymology, Vol. 10 New York: Academic Press, 1967: 94-96.
11.
Srere PA. Citrate synthase. In: JM Lowenstein ed. Methods In Enzymology, Vol. 13 New York: Academic Press, 1969: 3-5.
12.
Gomell AG, Bardawill CA, David MM. Determination of serum proteins by means of the biuret reaction. J Biol Chem 1949; 177:751-756.
13.
Swierczynski J, Scislowski PWD, Aleksandrowicz Z, Zydowo MM. IntraceUular distribution of fumarase in rat skeletal muscle. Biochim Biophys Acta 1983; 756:271-278.
14.
Benson JV, Gordon MJ, Patterson JA. Accelerated chromatographic analysis of amino acids in physiological fluids containing glutamic acid and asparagine. Anal Biochem 1967; 18:228-240.
15.
Spackman DH, Stem WA, Moore S. Automatic recording apparatus for use in the chromatography of amino acids. Anal Chem 1958; 30:1190-1206
16.
AOAC. Official Methods of Analysis. Association of Official Analytical Chemists. 1965; Ed. 10, 327, Washington, D.C.
17.
Zar JH. Biostatistical analysis. Englewood Cliffs. New Jersey: Prentice-Hall, 1974.
18.
Shinno H, Noda C, Tanaka K, Ichihara A. Induction of L-lysine-2-oxoglutarate reductase by glucagon and glucocorticoid in developing and adult rats. Biochim Biophys Acta 1980; 633:310-316.
Accepted for Publication September 3, 1993
1443
METABOLIC RESPONSE OF LIVER LYSINE (x-KETOGLUTARATE REDUCTASE ACTIVITY IN RATS FED LYSINE LIMITING OR LYSINE EXCESSIVE DIETS Andrew R Foster, Ph.D.*, Piotr W D Scislowski, Ph.D., CIan Harris, Ph.D. & Malcolm F Fuller, Ph.D. Department of Physiology, Rowett Research Institute, Aberdeen, Scotland, AB2 9SB, UK
ABSTRACT
The effects of feeding isonitrogenous diets (at a restricted ration size), containing limiting or excessive amounts of lysine on various biochemical parameters of rats were investigated. Lysine cx-ketoglutarate reductase (LKGR) and citrate synthase (CS) activities, hepatic portal vein (HPV) blood and liver free amino acid concentrations were measured in rats fed the experimental diets for up to 8 days. LKGR and CS activities remained constant in rats adapted to the lysine limiting diet. In contrast, LKGR activity rapidly increased (after 2 days) in rats fed the lysine excess diet and remained at a high level for the remainder of the experiment. The increase in liver LKGR activity in response to the lysine excess diet was associated with significant increases in HPV and liver free lysine concentrations (4.5- and 2fold, respectively). The significance of these data are discussed with respect to the metabolic response of rats challenged with an increased lysine supply mad its effects on free lysine concentrations and liver LKGR activity. Keywords: Lysine, Lysine c~-ketoglutarate reductase, Citrate synthase.
INTRODUCTION
Although it is well documented that the feeding of high protein diets increases the activities of many amino acid degrading enzymes, inconsistencies exist between studies (1, 2). Lysine tx-ketoglutarate reductase (LKGR, EC 1.5.1.8) is the regulatory enzyme of lysine oxidation in mammals and birds and its in vivo activity is sensitive to both the dietary protein and lysine content (3, 4, 5). However, in most feeding experiments on the effects of dietary protein and/or lysine on LKGR activity animals have been fed ad libitum. As a result, differences in food intake between treatment groups and any consequent effects on enzyme activity may have been ignored. The sensitivity of LKGR activity to both lysine and protein supply therefore prompted us to re-evaluate the in vivo metabolic response of LKGR to lysine, when differences in food intake were precluded. LKGR activity is located principally in the liver, but is also observed in other organs and tissues (3). Within the cell, LKGR is exclusively located in the mitochondrial matrix (6) where it catalyses the condensation of lysine and c~-ketoglutarate to form saccharopine. Whilst the half-life of LKGR is unknown the half-lives of some better characterised mitochondrial
* To whom correspondence should be addressed 1433
1434
A.R. FOSTER et al.
enzymes are known to range between 0.5 h and 8 days (7). These temporal differences in mitochondrial enzyme half-life raise the possibility of adaptive increases in mitochondrial enzyme (mitochondrial protein) resulting in non-specific increases in enzyme activity during relatively long-term (days-weeks) feeding experiments. To circumvent the above problems in the present study we fed rats isonitrogenous diets (at a restricted ration size) which contained a limiting or excessive amount of lysine. The activities of LKGR and citrate synthase (CS) in mitochondrial preparations were measured to enable us to describe the metabolic response of liver LKGR (i.e. LKGR per unit mitochondria, effect on CS activity and the amount of mitochondrial protein/g liver) when the rats were challenged with an increased supply of lysine.
MATERIALS AND METHODS
Animals, diets and feeding. Thirty two 80-85 g male rats (Hooded Lister, Rowett strain) were used in this experiment which was performed with four replicates of eight rats. After weaning, all animals were fed a stock commercial diet (Labsure CRM Diet, Labsure Lavender Mill, Manea, Cambridge) until they reached a minimum weight of 80 g. Each replicate of eight animals was pre-adapted to the lysine limiting diet (LL) which was fed ad libitum for five days. Both experimental diets were formulated using the following ingredients (g/kg diet): casein 72.4, glucose 234.0, sucrose 147.8, cellulose 61.7, corn oil 40.3, mineral and vitamin premix (8) 114.6. The experimental diets were supplemented to provide the following essential amino acids (g/kg) in the concentrations required for growth (9); threonine 1.7, valine 2.1, cystine 1.7, methionine 0.7, isoleucine 2.2, leucine 2.4, ~tyrosine 0.5, phenylalanine 1.8, histidine 0.7, arginine-HC1 3.2, tryptophan 0.7. The LL diet was further supplemented with 18.1 g aspartate, 21.9 g monosodium glutamate, and 0.1 g lysine-HC1/kg diet to give a 10% crude protein diet containing 5.2 g lysine/16 g N. This lysine content accounted for approximately 87% of the growing rat's lysine requirement (9). The lysine excess diet (LE) was identical to the LL diet except that the LE diet was supplemented with 24.6 g lysine-HCl/kg diet to provide 22.8 g lysine/16 g N (approximately 4.4-fold the rat's lysine requirement). Aspartate and monosodium glutamate were omitted from the LE diet to maintain a constant N concentration. After the five day pre-experimental period the animals were individually housed in perspex cages fitted with continuous feeders. The feeders consisted of a circular trough cut into a perspex disc (100 mm diameter) to which the rat only had access to an arc of 10 mm. The trough completed one complete revolution every 24 h. A weighed amount of diet was pressed into the trough daily. The animals were maintained in a temperature-controlled room at 23 + I~ with a 12 h light-12 h dark cycle and given water ad libitum. All the rats were offered their diet at a rate of 10 g per 100 g average body weight at the start of the experiment. Uneaten feed was collected daily from the feeder dish and spilled food from absorbent paper placed beneath each cage. Daily food intake was calculated as the difference between feed offered and feed remaining or spilled after 24 h. Using the above regime, the animals were adapted to the feeder cages for a further five days before the start of the experiment.
LYSINE ec-KETOGLUTARATE REDUCTASE
1435
Experimental protocol. The sixth day that the animals were in the feeder cages was designated as day 0 and was assumed to represent the full adaptation of the animals to the LL diet. The eight rats in each replicate were randomly allocated to either a control group which was offered the LL diet as before, or a treatment group which was offered the LE diet at the same ration size (mean weights of the LL and LE rats on day 0 were 89.1 + 1.2, 86.7 + 1.3 g, respectively). On day 0 two rats were killed as described below. One animal from the LL diet and one from the LE diet was also killed on days 2, 4 and 8 of the experiment.
Dissections and tissue preparation. On each day of sampling, the animals were weighed and killed by cervical dislocation. The livers were rapidly excised and rinsed in ice-cold homogenisation buffer 1 (220 mM mannitol, 70 mM sucrose, 2 mM HEPES and 1 mM EGTA, pH 7.8). A sub-sample of liver was immediately freeze-clamped and stored at -7&C for amino acid analysis. Another sample of liver, was used to make a 10% (w/v) homogenate in homogenisation buffer 1 from which mitochondria were prepared. The tissue was homogenised by hand in a 30 ml glass homogeniser using a teflon pestle. The liver homogenate was differentially centrifuged to isolate mitochondria (10). All procedures were performed at 4~ unless otherwise stated. A further sample of liver (300-400 mg) was used to make a 10% (w/v) whole liver homogenate in homogenisation buffer 2 (identical to homogenisation buffer 1 with the addition of 0.2% (v/v) Nonidet P-40"*). The liver was homogenised by hand in a 10 ml glass homogeniser using a teflon pestle and the homogenate was spun at 13,000 g at 4~ for 15 min. The resulting supernatant was used to determine the citrate synthase activity in the whole liver homogenate.
Enzyme assays. Enzyme activities were measured by initial rate measurements in a Cecil recording spectrophotometer temperature-controlled at 25~ The LKGR assay contained 7.5 mM txketoglutarate, 124 I.tM NADPH and 0.4 mg mitochondrial protein in 0.1 M HEPES containing 0.2% Nonidet P-40, pH 7.8. The assay substrates (except lysine) were mixed in the cuvette and the reaction was initiated by the addition of 25 mM lysine. The decrease in absorbance at 340 nm was read against a blank in which the volume of lysine was replaced by the equivalent volume of assay buffer (0.1 M HEPES pH 7.8). Before use, the mitochondrial fractions were diluted to 20 mg/ml for the LKGR assay; this was further diluted tenfold for the citrate synthase (CS) assay. The whole liver homogenates were diluted fivefold before the measurement of CS (11), with two slight modifications. In our CS assay, the pH of the assay buffer (0.1 M HEPES) was 7.8 and the detergent used was Nonidet P40 (0.2%, v/v). The final assay volume for both enzyme assays was 1000 gtl and the molar extinction coefficients used were 13600 (CS) and 6220 (LKGR), respectively. Enzyme activities were expressed as nmol/min.mg mitochondrial protein, or nmol/min.g tissue in the case of the whole liver homogenates. Protein determinations were performed using the Biuret method (12). Using the
**Nonidet P-40 is a nonionic detergent (octylphenoxypolyethoxyethanol, Sigma, Dorset, UK)
1436
A.R. FOSTER et al.
above methodologies, the amount (rag) of mitochondrial protein/g rat liver was calculated by dividing the total activity of CS activity extracted from 1 g of wet fiver by the CS activity present in 1 mg mitochondrial protein (13).
Blood sampling. The animals killed on day 8 were anaesthetized with an intraperitoneal injection of sodium pentobarbitone (75 lal/100 g rat body weight). Blood was sampled from the hepatic portal vein and immediately added to 25% sulphosalicyclic acid (SSA, ratio blood:SSA, 1:2) in preparation for amino acid analysis as described below.
Amino acid and proximate analyses. For amino acid analysis, 10 % (w/v) liver homogenates were made in 1 M perchloric acid using an Ultraturrax homogenizer (2 x 10 sec) and hepatic portal blood samples were added to 25% SSA; both precipitants contained L-Norleucine and e-amino caproic acid as internal standards. Separation of acidic and neutral amino acids was performed by ion exchange chromatography using lithium citrate buffers (14). Basic amino acids were separated by an accelerated version of the system for physiological fluids (15). The samples for proximate analyses were prepared and assayed according to standard methods (16).
Statistics. Differences between means were tested using a paired t-test (17). The data from days 2, 4 and 8 were subjected to randomised block, two way ANOVA and tested for treatment and time effects (Genstat). Significance was taken at p < 0.05.
RESULTS
Growth, feed conversion, liver size and body composition. Growth rates (g/rat/day), food conversion efficiencies (gain:food consumed) and liver size (g/100 g final body weight) were unaffected by switching rats from the LL to the LE diet (Table 1) although the values tended to be higher in the LE fed animals. Despite the 5 day pre-adaptation period used in the present study, the animals did show a small increase in food intake during the course of the experiment. This occurred in both treatment groups and was presumably the result of further adaptation to the cages or the semi-synthetic diets. The feeding of the LL and LE diets for 8 days had no significant effect on the whole carcass protein or lipid content (lysine limiting, 73.2 + 1.7% protein, 21.6 + 1.1% lipid; lysine excess, 71.5 + 3.0% protein, 26.2 + 2.3% lipid) when expressed as % dry matter.
-H
4-1
4-1
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d
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4-1
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4-1
4-1
4-1
r"-
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v
II
,5= oo
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4-1
+1
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~q. oo II
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+1
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,
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1437
1438
A.R. FOSTER et al.
o Q.
.~ 1o
8 @
0
2
4
6
8
Time (days) FIG 1. The time course of lysine ct-ketoglutarate reductase activity (nmol/min.mg mitochondrial protein) in rats fed the lysine limiting ( 0 - 0 ) and lysine excess ( A - A ) diets for up to 8 days. The values represent the mean + SEM and the sample sizes were: day 0, n = 8; remaining time points n = 4
0.160
0
0.120
, m
L_
03
0.080
rr" .-1
0.040
0.000
i
,
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,
,
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2
4
6
8
Time (days} FIG 2. The ratio of lysine ct-ketoglutarate reductase activity/citrate synthase activity (LKGR/CS) in rats fed the lysine limiting (O-Q) and lysine excess ( A - A ) diets. Values represent the mean + SEM and the sample sizes were the same as in FIG 1.
LYSINEcr
REDUCTASE
1439
2500
2000
1500
"~ 1000
500
0
2
,
,
,
4
6
8
Time (days} FIG 3. The time course of changes in liver free lysine concentration (nmol lysine/g liver) in rats fed the lysine limiting (O-O) and lysine excess (&-&) diets for up to 8 days. The values represent the mean -_. SEM and the sample sizes were as follows: day 0, n = 8; remaining time points n = 4.
Activities of lysine tx-ketoglutarate reductase and citrate synthase. The time-course of in vivo liver LKGR activity in rats fed the LL and LE diets is shown in Figure 1. LKGR activity (nmol/min.mg mitochondrial protein) significantly increased after 2 days when rats were switched to the LE diet. After day 2, the LKGR activity in the LE fed rats remained at a higher level compared with the control group for the duration of the experiment. Citrate synthase activities in the whole liver homogenate and the mitochondrial preparations were unaffected by the dietary manipulation (overall mean + SEM, 8,894 + 202 nmol/min.g liver; mitochondrial CS, 80.0 + 5.6 nmol/min.mg mitochondrial protein). The ratio of LKGR activity:citrate synthase activity (i.e. LKGR per unit mitochondria) was also increased by feeding the LE diet, the difference from the control group reaching statistical significance by day 4 (Figure 2). The calculated amount of mitochondrial protein (mg/g rat liver) was unaffected by feeding either diet for up to 8 days (overall mean + SEM, 101.5 + 5.3 mg mitochondrial proteirgg rat liver.
1440
A.R. FOSTER et al. TABLE 2
Whole Hepatic Portal Vein Blood Amino Acid Concentrations (nmol/ml) In Rats Fed Lysine Limiting (LL) Or Lysine Excess (LE) Diets For 8 Days.
Amino Acid
LL*
LE
LYS
429.3
+
32.2
1931.0
+
420.0""
ORN
67.4
+
6.9
68.3
+
17.5
HIS
70.4
+
7.3
111.7
+
20.7
ARG
79.6
+
8.3
126.3
+
23.4
ASP
226.5
+
15.1
160.9
+
23.6 #
THR
405.6
+
36.7
531.8
+
26.2#
SER
367.4
+
11.4
462.4
+
86.9~
ASN
416.7
+
57.7
444.0
+
25.5
GLU
252.2
+
9.5
265.0
+
69.4#
GLN
651.8
+
57.9
817.3
+
161.7#
GLY
343.3
+
10.4
404.9
+
66.3
ALA
730.7
+
104.1
811.7
+
86.5
CIT
88.3
+
5.1
105.8
+
13.0
VAL
161.9
+
31.9
201.3
+
30.3
ILE
80.5
+
13.4
101.1
+
14.4
LEU
109.9
+
15.6
133.8
+
16.3
TYR
87.9
+
11.2
106.9
+
6.2
PHE
65.7
+
12.6
96.0
+
21.5
PRO
201.8
+
27.9
284.7
+
63.0
* Values represent the mean + SEM where the sample size was n = 4 unless indicated otherwise. Denotes where the sample size was n = 2-3. ** Significant difference between the LL and LE diets at (p < 0.05).
LYSINE (z-KETOGLUTARATE REDUCTASE
1441
Whole hepatic portal vein blood and liver free amino acid concentrations. The whole hepatic portal vein (HPV) blood amino acid profiles of the rats fed the LL or LE diets are summarised in Table 2. The HPV blood lysine concentration of animals fed the LE diet increased approximately 4.5-fold compared with the LL fed group. No significant differences in the whole HPV blood concentrations were observed for any other of the remaining amino acids, although the HPV concentrations of threonine, serine, glutamine and alanine tended to be higher in the animals fed the LE diet. In contrast, the HPV aspartate concentration tended to be lower in animals fed the LE diet. The liver free lysine concentrations of animals fed the LE diet for 2 days was increased approximately 2-fold (p < 0.05, Figure 3). After day 2 the liver free lysine concentration decreased and by day 8 was not significantly different (p = 0.064) from that measured in the control group. Within the liver there were no significant differences in the concentrations of any other amino acids (data not shown).
DISCUSSION
To our knowledge this is the first report of the simultaneous measurement of LKGR activity and the amount of mitochondrial protein in rats challenged with an increased lysine supply. Our rationale for using citrate synthase as a marker of non-specific changes in mitochondrial protein was that by measuring the activity of this mitochondrial matrix enzyme during the time course of the experiment we could determine whether the LE diet gave rise to a specific increase in LKGR activity or a non-specific increase in total mitochondrial enzyme activity or protein content. Our data indicate that liver LKGR activity (nmol/min.mg mitochondrial protein) was increased 2-3 fold in animals fed the LE diet. This increase in LKGR activity occurred when the lysine intake of the rats was increased from 54 to 213 mg/d. This observed increase in LKGR activity was similar to that described by others (3) and appears to represent a lysine-specific effect as there was no significant change in CS activity in either the liver homogenates, or mitochondrial preparations during the present experiment. We also calculate that the amount (mg) of mitochondriat protein/g liver of rats was unaffected by feeding either diet for 8 days. Thus, in the present study, the increased LKGR activity in animals fed the LE diet was the result of increased LKGR activity per unit mitochondria (as confirmed by the ratio of LKGR:CS activity, Figure 2) and not the result of a general increase in liver mitochondrial protein content. Increased enzyme activity with dietary manipulation is conventionally assumed to be evidence of substrate-induction of that enzyme although this may not be strictly true. The present data support the possibility of induction of LKGR by lysine in vivo although antibody precipitation of LKGR protein and quantitation is needed for the unequivocal demonstration of substrate-induction. In vitro studies with rat hepatocytes (18) indicate that in vitro LKGR activity is also sensitive to glucocorticoid and insulin. However, the role of these hormones (if any) in the in vivo regulation of LKGR by lysine warrants further investigation. Switching rats from the LL to the LE diet for 8 days increased the whole HPV blood lysine concentration approximately 4.5-fold compared with animals on the LL diet: this change was similar to the 4.4-fold difference in the lysine content between the LL and LE diets. In contrast, the liver free lysine concentrations of rats fed the LE diet peaked on day
1442
A.R. FOSTER et al.
2 and steadily decreased after this, presumably as a result of the increased liver LKGR activity and lysine oxidation. Thus, at the end of the experiment although there was a 4.5-fold difference in the HPV blood lysine concentration (Table 2), the liver free lysine concentration was not statistically different from the lysine concentration in the LL fed controls (Figure 3). To maintain equal nitrogen contents in the LL and LE diets, aspartate and glutamate were removed from the LE diet. This resulted in a small decrease in HPV aspartate concentration in the LE animals which was not statistically significant (data not shown). At the same time, the HPV glutamate concentration was similar between rats fed the LL and LE diets despite the fact that glutamate was omitted from the LE diet. The HPV glutamine concentration also tended to be higher in the LE fed rats. In the liver, small increases in the free alanine and glutamine concentrations were also observed in animals fed the LE diet (data not shown), but none of these differences was significant. These data suggest that the activity of liver transaminases was probably increased in animals fed the LE diet to maintain the pools of non-essential amino acids for other (anaplerotic) reactions. In conclusion, our data support the hypothesis that liver LKGR activity is specifically increased by dietary lysine and that this phenomenon appears to be the result of increased LKGR synthesis per mitochondfia. However, further work is needed to describe the mechanism(s) by which lysine regulates LKGR activity both in vitro and in vivo.
ACKNOWLEDGEMENT Special thanks to Dr M Franklin, Messrs R Smart and I Grant for assistance during this experiment. We gratefully acknowledge SOAFD for their financial support of this project.
REFERENCES
1.
Ohno T, Tasaki I. Relation between liver amino acid catabolizing enzymes and free amino acids of liver and plasma in adult cockerels fed diets containing graded levels of protein. J Nutr 1977; 107:829-833.
2.
Wang S, Nesheim MC. Degradation of lysine in chicks. J Nutr 1972; 102:583-596.
3.
Chu SH, Hegsted DM. Adaptive response of lysine and threonine degrading enzymes in adult rats. J Nutr 1976; 106:1089-1096.
4.
Muramatsu K, Takada R, Uwa K. Adaptive responses of liver and kidney lysine-ketoglutarate reductase and lysine oxidation in rats fed graded levels of dietary lysine and casein. Agric Biol Chem 1984; 48:703-711.
5.
Foster AR, Beckett PR, Rees WD, Fuller MF. A sensitive isotopic assay for the measurement of lysine ct-ketoglutarate reductase in cultured rat hepatocytes. J Nutr Biochem 1992; 3:554-559.
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Higashino K, Fujioka M, Yamamura Y. The conversion of L-Lysine to Saccharopine and tx-Amino adipate in Mouse. Arch Biochem Biophys 1971; 142:606-614.
LYSINE o~-KETOGLUTARATE REDUCTASE 7.
.
Vargas JL, Roche E, Knecht E, Grisolia S. Differences in the half-lives of some mitochondrial rat liver enzymes may derive partially from hepatocyte heterogeneity. FEBS Lett 1985; 1:182-186. Pullar JD, Webster AJF. The energy cost of fat and protein deposition in the rat. Br J Nutr 1977; 37:355-363.
9.
Coates ME, O'Donoghue PN, Payne PR, Ward RJ. Dietary standards for laboratory rats and mice. A report to the Council of the Laboratory Animal Science Association, Laboratory Animal Handbook 2, London, Laboratory Animals Limited, 1969: 13-15.
10.
Johnson D, Lardy HA. Isolation of liver or kidney mitochondria. In: RW Estabrook, ME Pullman. eds. Methods In Enzymology, Vol. 10 New York: Academic Press, 1967: 94-96.
11.
Srere PA. Citrate synthase. In: JM Lowenstein ed. Methods In Enzymology, Vol. 13 New York: Academic Press, 1969: 3-5.
12.
Gomell AG, Bardawill CA, David MM. Determination of serum proteins by means of the biuret reaction. J Biol Chem 1949; 177:751-756.
13.
Swierczynski J, Scislowski PWD, Aleksandrowicz Z, Zydowo MM. IntraceUular distribution of fumarase in rat skeletal muscle. Biochim Biophys Acta 1983; 756:271-278.
14.
Benson JV, Gordon MJ, Patterson JA. Accelerated chromatographic analysis of amino acids in physiological fluids containing glutamic acid and asparagine. Anal Biochem 1967; 18:228-240.
15.
Spackman DH, Stem WA, Moore S. Automatic recording apparatus for use in the chromatography of amino acids. Anal Chem 1958; 30:1190-1206
16.
AOAC. Official Methods of Analysis. Association of Official Analytical Chemists. 1965; Ed. 10, 327, Washington, D.C.
17.
Zar JH. Biostatistical analysis. Englewood Cliffs. New Jersey: Prentice-Hall, 1974.
18.
Shinno H, Noda C, Tanaka K, Ichihara A. Induction of L-lysine-2-oxoglutarate reductase by glucagon and glucocorticoid in developing and adult rats. Biochim Biophys Acta 1980; 633:310-316.
Accepted for Publication September 3, 1993
1443