/ . Biochem., 80, 1159-1164 (1976) Dedicated to Prof. N. Shimazono on his 70th birthday.

Control of Ketogenesis from Amino Acids TV. Tissue Specificity in Oxidation of Leucine, Tyrosine, and Lysine1 Chiseko NODA and Akira ICHIHARA Institute for Enzyme Research, School of Medicine, Tokushima University, Tokushima, Tokushima 770 Received for publication, March 27, 1976

In vitro and in vivo studies were made on the tissue specificity of oxidation of the ketogenic amino acids, leucine, tyrosine, and lysine. In1 n vitro studies the abilities of slices of various tissues of rats to form 14COi from X4C-amino acids were examined. With liver, but not kidney slices, addition of a-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 "COz was measured. In contrast to results with slices, no difference was found in the respiratory 14COt 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.

It is known that amino acids ingested in excess are metabolized before carbohydrate and fat and that amino acids are an important body fuel, particularly in catabolic states, such as starvation and the diabetic condition (7, 2). Gluconeogenesis from alanine has been studied 1

This work was supported in part by a grant for science research from the Ministry of Education, Science and Culture, of Japan. ..Yol. 80, No. 5, 1976

extensively, but very little attention has been paid to ketogenesis from amino acids. However, it has been reported that on complete oxidation ketogenic amino acids can generate twice as much ATP as glucogenic amino acids (3) and the importance of ketone bodies as a body fuel has been discussed ( 2 ) . Most ketogenic amino acids are essential amino acids and ketone bodies are in fact mainly produced from depot fat. However, these

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ketogenic amino acids are fairly abundant in tissue proteins and as free amino acids in the blood and tissues and when given to animals 20 to 40% of the administered dose is oxidized within a few hours (4, 5). Therefore, the importance of ketogenic amino acids as an energy source should not be underestimated. Most amino acids are metabolized in the liver and liver perfusion experiments and measurements of the plasma concentrations of these amino acids have shown that lysine and tyrosine are metabolized mostly in the liver (6, 7). The enzymes concerned with the metabolisms of these amino acids are also mainly located in the liver, but they are also found in kidney, though in lesser amount (8— 11). However, tissue specificity of metabolism of branched chain amino acids is quite different. Namely, branched chain amino acid transaminase [EC 2.6.1.42] shows highest activity in the stomach and pancreas, followed by the kidney, heart, lactating mammary gland, muscle and brain and its activity is lowest in liver {12-15). Branched chain keto acid dehydrogenase is also found in various tissues (76). Therefore, branched chain amino acids are mainly metabolized extrahepatically, probably in muscle and adipose tissue (6~, 7, 17). However, in vitro studies using tissue slices showed that kidney has the highest activity for oxidation of leucine, followed by liver and muscle (18—21). Recently it was found that pancreas and stomach also have as high activity as that of kidney (12, 22). It was also shown that lactating mammary gland has quite high activity (15, 23). Recent studies have also suggested that leucine is utilized mainly for protein synthesis in liver and for lipogenesis in adipose tissue, but that it is oxidized in muscle (21). In this work we compared the oxidative activities of liver and kidney for the three ketogenic amino acids in vitro and in vivo. Using tissue slices we found that kidney had the highest activity for oxidations of leucine and lysine and some activity for tyrosine, but our in vivo experiments indicated that the kidney, in fact, plays very little role in metabolism of these amino acids, the liver being the main site of oxidation of lysine and

C. NODA and A. ICHIHARA

tyrosine, and both extrahepatic tissues and the liver metabolizing leucine. EXPERIMENTAL PROCEDURE In Vivo Experiments—Male, Wistar straia rats, weighing about 200 g, were fed on laboratory chow ad libitum unless otherwise indicated. In some experiments 75% casein diet, prepared as reported previously (24), was given for one night and then hydrocortisone (5mg/100g body weight) was injected intraperitoneally and rats were killed 6 hr later. For ligation of hepatic or renal blood vessels rats were treated essentially by the method of Featherston et al. (25). Immediately after ligation a tracer dose of the test 14 C-amino acid (0.5 /uCi/100 g body weight) was injected into the vena cava, the abdominal wall was closed with Michel's clips and animals were placed in metabolic cages. Respiratory "CO2 was trapped in 40 ml of ethanolaminemethylcellosolve ( 2 : 1 , . v/v) as described by Jeffay and Alvarez (26) and its radioactivity was measured in an Aloka Liquid Scintillation Spectrometer Model 601. Results are expressed as percentages of the administered dose of radioactivity recovered as "COi in 1 hr. In Vitro Experiments—Rats were fed on laboratory chow ad libitum. Then they were killed and slices of their tissues were prepared. Amino acid oxidation was measured in a Warburg vessel containing 2 ml of KrebsRinger phosphate buffer (pH 7.4), 100 mg of tissue slices and L-[U-14C]amino acid as indicated. a-Ketoglutarate (5 mM) was added when indicated. Filter paper soaked in 0.2 mL of hyamine was placed in the center well and the reaction was started by addition of the test amino acid and carried out with shakings in a closed vessel at 37°J Reactions were terminated by addition of 0.2 ml of 30% perchloric acid and the vessels were shaken for 1 hr further. Then, the filter paper in the center well was transferred to a vial and the center well was washed with 1 ml of methanol. The washing fluid and toluene scintillator (9ml) were added to the vial and radioactivity was measured as described for in vivo experiments. Activity is expressed as dpm 14CO« J. Biochtm.

CONTROL OFKETOGENESIS FROM AMINO ACIDS. IV

iormed/hr/g wet tissue. All L-[14C]amino acids used were obtained irom Japan Radioisotope Association, Tokyo. RESULTS AND DISCUSSION Oxidations of Leudne, Lysine, and Tyrosine •by Tissue Slices of Rats—As shown in Table I, kidney slices had the highest activity for lysine oxidation, followed by liver. Leucine •oxidation was also highest in kidney, but diaphragm showed second highest activity, adipose tissue having almost the same activity as liver. A previous report from this laboratory .showed that stomach and pancreas had almost

1161

as high activity as kidney (12). Tyrosine was oxidized mostly by liver slices, followed by those of kidney, but other tissues showed no activity. During the experiments we found that with liver slices it was necessary to add a-ketoglutarate to obtain maximal activities for oxidations of these amino acids, while with kidney slices addition of a-ketoglutarate was rather inhibitory (Table II). a-Ketoglutarate could be partially replaced by succinate, but not by malate. These results suggest that under the present conditions the supply of endogenous a-ketoglutarate was poor in liver slices. A similar but more pronounced requirement for a-ketoglutarate has been ob-

"TABLE I. Oxidations of L-[U-uC]lysine, L-[U-"C]leucine, and L-[U-"C]tyrosine by slices of various tissues •of rats in the presence of a-ketoglutarate. Concentrations of 5 mM (0.1 /iCi//*mole) and 2 mM (0.125 ftCi/ //mole) of L-[U-1;1C]lysine and 2 mM (0.125 fiCi/pimo\e) of L-[U-"C]leucine or L-[U-14C]tyrosine were used. Tive mM a-ketoglutarate was added in all cases. "COj formed (dpm x lO-'/hr/g tissue) Tissue

Lysine (2 mM)

Lysine (5 mM) Liver Kidney Brain Heart Diaphragm Intestine Adipose tissue

1,108 1,807

852

1,461 6

Leucine (2 mM)

Tyrosine (2 mM)

248

2,122

1, 266

577

895

0

246

0

4 5 15 5

TABLE II. Effects of a-ketoglutarate, malate, and succinate on the oxidations of lysine, leucine, and tyrosine by slices of liver and kidney. The reaction mixture was as for Table I, except that malate or succinate was added in place of a-ketoglutarate as indicated. "CO2 formed (dpm x 10"l/hr/g tissue) Tissue

Addition (5 mM) Lysine (5 mM)

. Liver

Kidney

None a-Ketoglutarate Malate Succinate None a-Ketoglutarate Malate Succinate

•Vol. 80, No. 5, 1976

Lysine (2 mM)

Leucine (2 mM)

Tyrosine (2 mM)

16

16

250

683

1,168

1,108

284

2,122

26 157

3,460 2,312 1,056 1,397

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C. NODA and A. ICHIHARA.

TABEL III. Recovery of respiratory

14

C0, from ["C]lysine, ["C]leucine, and ["C]tyrosine in vivo. Percentage "COj recovered in hr

Diet and treatment

Ligated organ [U-"C]lysine

Laboratory chow

High protein diet +cortisol

5.0±0.5* 0.4±0.0» 6.4±0.6

None Liver Kidney None Liver Kidney

• Means±SE of results in 5 to 10 rats. cant.

b

[U-"C]leucine

[l-l4C]tyrosine

7.6±1.0 3.0±0.3t>

19.0±1.0

6.6±0.5

15.8±0.7

17.0±0.7

0.4±0.0 b 15.4±1.1

10.0±1.2b 13.0±0.5»

0.6 ±0.0" 22.0±2.0

Difference from the value in intact animals statistically signifi-

served in oxidation of leucine (20). a-Ketoglutarate is necessary for the transaminations of leucine and tyrosine and it is also a cosubstrate of lysine-a-ketoglutarate reductase [EC 1.5.1.8]. The Km value of the reductase of rat liver for cr-ketoglutarate is 1.4 mM (Noda, C. & Ichihara, A., unpublished data), and those of tyrosine transaminase [EC 2.6.1.5] and branched chain amino acid transaminase [EC 2.6.1.42] are 0.7mM (27) and 0.07mM (28), respectively. This may be why addition of a-ketoglutarate has more effect on the oxidation of lysine than on those of tyrosine and leucine. Care should be taken in interpretation of results obtained with tissue slices, because 14COi production does not per se represent complete oxidation of tyrosine, but may also reflect the activity of aromatic Lamino acid decarboxylase [EC 4.1.1.28]. On administration at higher concentrations, tyrosine is decarboxylated, rather than transaminated in vivo (29). However, the activities of the decarboxylase in liver and kidney are almost the same (30), whereas in the present work oxidation of tyrosine was much higher in liver slices than in kidney slices, suggesting that the decarboxylase probably does not play a major role in the liver. No significant activity of decarboxylase for leucine and lysine has been reported in mammals. Another enzyme which may participate in the oxidations of these amino acids is L-amino acid oxidase [EC 1.4.3.2], but it has been reported

that this oxidase may have virtually no physiological significance (31). Therefore, in vitrostudies suggest that lysine and tyrosine may be metabolized in the liver and kidney, whereas leucine may be oxidized in the liver and extrahepatic tissues, including kidney. The activities for oxidations of lysine and leucine, respectively in the kidney cortex and medulla were similar (data not shown); In Vivo Oxidation—Table III shows results on recovery of respiratory "COt after intravenous injection of labeled amino acids. The oxidations of lysine and tyrosine were not affected by ligation of the renal blood vessels, but were greatly reduced by ligation of the hepatic blood vessels. Thus, despite the high activities for oxidation of these amino acids found in kidney slices, in vivo experiments suggested that the kidney plays little, if any, part in oxidation of these amino acids; these two amino acids are probably mainly metabolized in the liver. However, the metabolism of leucine was partially reduced by ligation of the blood vessels of either liver or kidney* especially when animals had been given a high protein diet and cortisol. It is known that these treatments stimulate metabolism of these amino acids (20, 24, 32). Therefore, leucine is probably metabolized slightly in the liver and kidney and mostly in extrahepatorenal tissues, such as muscle and adipose tissues. Our results suggest that in vitro experi/. Biochemi

CONTROL OF KETOGENESIS FROM AMINO ACIDS. IV

ments may show the maximal oxidative capacity of a tissue, because the conditions employed were optimal for activity, whereas in in vivo experiments the substrate concentration may have been far too low for maximal activity and, particularly in kidney, the amount of substrate available may actually have been negligible. If the plasma concentrations of these amino acids were raised, it seems very likely that the kidney would participate in their metabolism. It is still controversial whether metabolism is regulated by the amount of enzyme or the substrate concentration (33, 34). We have shown that the activity of tyrosine transaminase in liver controls the rates of oxidation and lipogenesis of tyrosine in vivo (24). Another possibility is that the enzymes in liver have much higher affinity for substrate than those in the kidney. However, in preliminary studies no difference was found in the properties of transaminase for leucine or of lysine-a-ketoglutarate reductase in liver and kidney. Other possible explanations are that the liver may show increased compensatory activity to oxidize these amino acids when the kidney is ligated or that amino acid transport to the kidney may be negligible in vivo. However, both these explanations of the present results seem very unlikely. It might be possible to reconcile values obtained in vitro with those obtained in vivo by correcting the former for the relative weight (18). When corrected by these factors, the values obtained with tissue slices suggest that leucine and tyrosine are mainly oxidized in muscle and liver, respectively and this is consistent with results obtained in vivo. However, the corrected values also suggest that liver oxidizes only twice as much lysine as kidney, whereas results in vivo suggest that liver is a major site of lysine oxidation. Moreover, although corrected in vitro results indicate that the liver and kidney are responsible for only one fortieth of the leucine oxidation of muscle, ligation of either the liver or kidney was found to inhibit leucine oxidation considerably. Thus the reasons for differences between results in vivo and in vitro are unknown. The requirement of ar-ketoglutarate for Vol. 80, No. 5, 1976

1163

oxidation of these amino acids in liver slices suggests that the cells in slices may be damaged so that the observed values in vitro may be underestimates. This may explain some, if not all, of the difference between the activities of liver and kidney in vivo and in vitro. Drastic treatments of rats, such as ligation of the blood vessels, could cause unphysiological blood flow and altered metabolic activity of the tissues. However, Featherston et al. used a method similar to ours to study the tissue specificity of arginine metabolism and showed that the kidney was of major importance (25). It may be argued that measurement of respiratory COt is not per se a direct measure of oxidation of these amino acids. However, in the cases of tyrosine and leucine we have shown that there is no accumulation of intermediate (24, 35). Therefore, the amount of respiratory COi does in fact reflect the extent of oxidation of these amino acids. Our results suggest that for comprehensive understanding of metabolism both in vivo and in vitro experiments are required. REFERENCES 1. Krebs, H.A. (1972) Advances in Enzyme Regulation 10, 397-420 2. Cahill, G.F., Jr. (1970) New Engl. J. Med. 282, 668-675 3. Krebs, H.A. (1964) in Mammalian Protein Metabolism (Munro, H.N. & Allison, J.B., ed.) Vol. I, pp. 125-176, Academic Press, New York 4. Aguilar, T.S., Harper, A.E., & Benevenga, N.J. (1972) / . Nutr. 102, 1199-1208 5. McFarlane, I.G. & von Holt, C. (1969) Biochem. J. I l l , 557-563 6. Elwyn, D.H. (1970) in Mammalian Protein Metabolism (Munro, H.N., ed.) Vol. IV, pp. 523-557, Academic Press, New York 7. Miller, L.L. (1962) in Amino Acid Pools (Holden, J.T., ed.) pp. 708-721, Elsevier, Amsterdam 8. Lin, E.C.C. & Knox, W.E. (1958) / . Biol. Chem. 233, 1186-1189 9. Crandall, D.I. & Halikis, D.N. (1954) /. Biol. Chem. 208, 629-638 10. Rosenberg, L.E., Berman, M., & Segal, S. (1963) Biochim. Biophys. Ada 71, 664-675 11. Hutzler, J. & Dancis, J. (1968) Biochim. Biophys. Ada 158, 62-69 12. Ichihara, A., Noda, C , & Goto, M. (1975) Biochem. Biophys. Res. Commun. 67, 1313-1318

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/ . Biochem.