Role and Metabolism of Free Leucine in Skeletal Muscle in Protein Sparing Action of Dietary Carbohydrate and Fat

Transcription

1 Agric. Biol. Chem., 41 (2), 229 `234, 1977 Role and Metabolism of Free Leucine in Skeletal Muscle in Protein Sparing Action of Dietary Carbohydrate and Fat Kiwao NAKANO and Tamotsu ISHIKAWA Laboratory of Nutritional Regulation, Institute for Biochemical Regulation, Faculty of Agriculture, Nagoya University, Chikusa, Nagoya 464, Japan Received March 30, 1976 Feeding rats with either a carbohydrate meal or a fat meal to the previously fasted rats caused significant decrease in urinary output of urea and total nitrogen. The content of free leucine in skeletal muscle decreased in the rats fed either a carbohydrate meal or a fat meal. Feeding of either a carbohydrate meal or a fat meal stimulated incorporation of L-leucine-l- 14C into protein fraction of skeletal muscle and reduced its oxidation to 14CO 2. These results suggest that the metabolism of leucine is under nutritional regulation and that the decrease in content of free leucine in skeletal muscle might be caused by enhanced reutilization of leucine into protein by the feeding of a carbohydrate meal or a fat meal. The role of free leucine in skeletal muscle as a regulator of protein turnover in the tissue are discus sed in relation to the metabolism of this branched chain amino acid. Unlike other essential amino acids, the branched chain amino acids are degradated ex trahepatically.1) Although a large number of tissues can oxidize them, skeletal muscle is thought to be the principal site of branched chain amino acid catabolism,2 `4) because of its bulk. Recently, several laboratories re ported that the metabolism of the branched chain amino acids by muscle is under nutri tional and hormonal regulation. Meikle and kl ain2) showed in vivo that the oxidation of L-leucine-1-14Cto14CO2 increased during fasting and decreased during refeeding. Buse et al.5) presented an evidence suggesting that the oxida tion of branched chain amino acid was ac celerated in muscles of uncontrolled diabetes and that insulin treatment inhibited this in crease. They also reported that addition of certain fatty acids stimulated branched chain amino acid oxidation by isolated diaphragm, while it caused no effect on oxidation of other amino acids, including phenylalanine. Insu lin,6) epinephrine and glucagon7) stimulated branched chain amino acid oxidation by iso lated rat diaphragm or heart. In recent years the efforts have been made in our laboratory to evaluate the effects of dietary carbohydrate and fat on amino acid meta bolism in rats.8 `11) In the previous paper, it was shown, in vivo and in vitro, that feeding rats with either a carbohydrate meal or a fat meal stimulated the incorporation of L- phenylalanine-u-14c into protein fraction of skeletal muscle with a concomitant decrease in oxidation to 14C02.11) Phenylalanine is believ ed to be exclusively degraded in the liver but not in muscle.1) It was also shown that the addition of fatty acids to media enhanced the incorporation of L-phenylalanine-U-14C into protein fraction of isolated diaphragm muscle, whereas no influence of glucose by itself was observed on protein synthesis.10) One of the purpose of the present study is to explore the nutritional regulation of the metabolism of leucine. Recently, evidences have been accumulated suggesting that leucine may play a pivotal role in regulation of protein turnover in skeletal muscle. Thus, Fulkes et a1.12) have shown that the addition of these branched chain amino acid together stimulated protein synthesis and inhibited degradation in the isolated diaphragm while remaining plasma amino acids did not affect either process significantly. They have also demonstrated that leucine, by itself, or isoleucine and valine together, also were able

2 230 K. NAKANO and T. ISHIKAWA to inhibit protein degradation and promote protein synthesis. Buse and Reid13) reported that leucine added to media stimulated the incorporation of radioactive amino acids into protein fraction of the isolated rat diaphragm and that more of the other amino acids tested (valine, isoleucine, glutamate, histidine, methio nine, phenylalanine or tryptophan) exerted this effect. They also presented results suggesting that leucine inhibited protein degradation. In considering these results, Buse and Reid13) proposed that the protein turnover in muscle cells may be regulated by the concentration of leucine in this tissue which may be varied under various nutritional and hormonal conditions. Unfortunately they presented no data on the actual change in content of free leucine in any tissues. It was shown in our laboratory11) that feeding of either a carbohydrate meal or a fat meal to the previously fasted rats caused significant reduction in urinary output of urea and total nitrogen, and that the reduction in amino acid degradation might be caused by enhanced reutilization of amino acids by feeding of these meals. The second purpose of the present study is to evaluate, in vivo, the role of free leucine in muscle as a possible regulator of body protein turnover, by examining the possible relation ship between the content of free leucine in muscle cells and rate of protein turnover in rats, in relation to the metabolism of leucine. with 0.5g H2O with the aid of 2.2 mg sodium cholate. The amount of dextrin and lard was adjusted to be isocaloric. Rats of the control group were given 1ml of H20. Each meal was given also at 9 a.m. and 5 p.m. on the second day. Rats were given these meals finally at 9 a.m. on the third day. In this experiment 1, all the 4 feedings were administered to the experimental ani mals. At 5 p.m. on the last day, rats were anesthetized with pentobarbital, and blood was withdrawn from heart. Blood was centrifuged at 600 ~g in the cold and plasma was used for the analysis of urea.8) An aliquot of urine collected was subjected to the analy sis of urea and nitrogen by the methods described previ ously.8) Free leucine in tissues. Rats weighing 120 `130g were used. Diets and feeding schedule were essentially the same as those for experiment 1 as described above, except that the first and the second feedings of the meals were excluded in this experiment. Concentrations of free leucine in blood plasma, liver, gastrocnemius muscle and diaphragm muscle were analyzed by the method described previously.11) Metabolism of L-leucine-1-14C. Rats weighing 120 `130g were used. Feeding schedule was essenti ally the same as that employed in experiment 1, except that the first feeding of the meals in experiment 1 was excluded in this experiment. Four hours after the final feeding rats were injected intraperitoneally with 4 Đci of L-leucine-1-14C (60 mci/mmole, obtained from the Radiochemical Centre Amersham) dissolved in 0.4ml of 0.9% NaCl solution. After injection, rats were housed in metabolism cages and expired CO2 was trap ped in a mixture of ethanolamine and ethyleneglycolmonoethylether (1:2 v/v). Exactly 30 min after the injection, rats were decapitated and both livers and gastrocnemius muscles were removed immediately, weighed, frozen in liquid nitrogen and stored in deep freezer. Preparation of samples and measurement of radioactivity in CO2, and trichloroacetic acid (TCA) EXPERIMENTAL Male albino rats of the Wistar strain were used throughout. soluble and insoluble fractions of liver and muscle were carried out by the method described previously.11) Statistical analysis of results between groups was carried out by using t-test.15) Urinary output of urea and total nitrogen. In the preliminary period of the experiment, animals were allowed to eat the commercial stock diet (CE-2, Japan Clea Co., Ltd., Tokyo, Japan) and water ad libitum. At 9 a.m. on the first day of the experiments, when the body weight of the rats reached to 105 `110g, diet was removed and the rats were fasted. At 3 p.m. rats were divided into three groups. All animals were housed in metabolism cages to collect urine. To the rats of the first group, 3.6ml of 33.3% dextrin solution was tube fed at 4 a.m. The second group of the rats were tube fed 1ml of a fat meal, containing 0.5g lard emulsified RESULTS Urinary output of urea and total nitrogen The first experiment was carried out to evaluate the effect of feeding of a carbohydrate meal and a fat meal on urinary output of urea and total nitrogen. As expected from earlier studies,8 `11) feeding of either of these meals caused marked decrease in urinary output of urea and total nitrogen (Table I). It is likely

3 Role of Free Leucine in Body Protein Metabolism 231 TABLE I. EFFECT OF FEEDING WITH A CARBOHYDRATE AND FAT MEAL ON THE LEVEL OF PLASMA UREA AND URINARY OUTPUT OF UREA AND N 1) mean }SD Significance of difference from the control value: a)p<0.025, b)p<0.001 that feeding of carbohydrate or fat to the pre viously fasted rats caused decrease in amino acid degradation in these rats. Content of free leucine in the tissues The next experiment was done to examine the possible relationship between the content of free leucine in skeletal muscle and the regulation of protein turnover in the tissue. To explore this, effect of feeding either of a carbo hydrate meal or a fat meal on the concentration of free leucine was examined in various tissues at early stages of the experiment. Table II shows that feeding rats with either of these meals caused significant reduction in the con centration of free leucine in blood, diaphragm muscle and gastrocnemius muscle. These results suggest that the decrease in the content of free leucin in these tissues may be caused either by an enhanced reutilization of free leucine into muscle protein or by increase in oxidation of leuince to expired CO2. Metabolism of leucine, in vivo To explore the possibilities described above, fate of free leucine was traced by using L- leucine-l-14c as a tracter. Table III sum marizes the results of analysis of in vivo oxidation of L-leucine-l-14C to 14C02. As shown in this table, feeding rats with either a carbohydrate meal or a fat meal caused signi ficant decrease in oxidation of L-leucine-l-14C to 14CO2. In considering the higher specific activity of free leucine in muscle (dpm/Đmole of free leucine, Table IV, column (H)) of rats fed either of these meals, it is probable that the decreased recovery of radioactivity in ex pired CO2 may result from actual depression of net oxidation of leucine in these animals. Since feeding of either a carbohydrate meal or a fat meal caused decrease in the content of free leucine in the gastrocnemius muscle (Table II), the increase in the incorporation of L-leucine-1-14C into total protein fraction of the muscle (Table V, column N and 0) may be due, in part, to the altered extent of dilution of the isotopic leucine by unlabelled leucine in the tissue. Accordingly, the distribution of14c recovered in expired C02, liver protein TABLE II. EFFECT OF FEEDING OF A CARBOHYDRATE MEAL OR A FAT MEAL TO THE PREVIOUSLY FASTED RATSa) ON CONTENT OF FREE LEUCINE IN BLOOD PLASMA, LIVER AND MUSCLE a) Samples were obtained from 6 rats for each group and pooled for analysis of free leucine content. b) Rats were fed on commercial stock diet.

4 232 K. NAKANO and T. ISHIKAWA TABLE III. EFFECT OF FEEDING RATS WITH A CARBOHYDRATE MEAL OR A FAT MEAL ON OXIDATION OF L-LEUCINE-1-14C TO EXPIRED 14C02 Significance of differences from the control value: a) P<0.001 TABLE IV. EFFECT OF FEEDING OF DEXTRIN OR LARD, ON INCORPORATION OF L-LEUCINE-1-14C INTO LIVER PROTEIN AND ACID SOLUBLE FRACTION Significance of difference from the control value; a) p<0.025, b) p<0.005, c) p< ) Micromoles leucine per mg liver protein; K. Yamashita and K. Ashida (personal communication). TABLE V. EFFECT OF FEEDING OF DEXTRIN AND LARD ON INCORPORATION OF L-LEUCINE-1-14 C INTO GASTROCNEMIUS MUSCLE PROTEIN AND ACID-SOLUBLE FRACTION 2) Micromole leucine per mg muscle protein; K. Yamashita and K. Ashida (personal communication).3 ) (0)=(K) ~0.5772) ~(L) ~(final body weight) ~0.25; The amount of whole body muscle was calculated by assuming 25% of body weight might be muscle weight.14) Significance of difference from the control value: a) p<0.05, b) p<0.025, c) p<0.005, d> p<0.001

5 Role of Free Leucine in Body Protein Metabolism 233 TABLE VI. RELATIVE RECOVERY Y OF RADIOACTIVITY IN RESPIRATORY CO2, LIVER AND MUSCLE PROTEINS AFTER INJECTION OF L-LEUCINE-1-14C Significance of difference from the control value; a) p<0.005, b> p<0.001 TABLE VIL RELATIVE RECOVERY RATIO OF RADIOACTIVITY IN RESPIRATORY C02, LIVER AND MUSCLE PROTEINS AFTER INJECTION OF L-PHENYLALANINE-U-14C Significance of difference from the control value: a) p<0.025, b) p<0.005, c) p<0.001 and muscle protein was then calculated, as suming that the sum of the radioactivities re covered in all of these fractions is 100%.14) As shown in Table VI, recovery % of 14C in expired CO2 is smaller in the rats fed either of a carbohydrate meal or a fat meal compared with that in the starved rats. Feeding either of these meals caused significant increase in recovery % of 14C in whole body muscle protein. These results suggest that feeding of either a carbohydrate meal or a fat meal caused significant decrease in oxidation of free leucine to CO2 and increase in the reutilization of this amino acid into skeletal muscle protein. All of total recovery (Table IV, column F), relative specific activity (column I) and relative recovery % (Table VI) of 14C from L-leucine-l- 14C were higher in the liver of rats fed either of a carbohydrate meal or a fat meal than those in the fasted rats. These results indicate that the incorporation of leucine into liver protein is stimulated by the feeding of these meals. DISCUSSION The relative recovery % of 14C shown in Table VII was newly calculated from the results obtained in the previous study.11) It was carried out to examine the fate of free amino acids in the rats fed either a carbohydrate meal or a fat meal by using L-phenylalanine-U-14C as index. As shown in Table VII, the relative recovery % of 14C in expired CO2 were lower in the rats fed these meals, whereas those in whole body muscle protein were higher in these rats than those in the starved rats. Thus, it is probable that the oxidation of free phenyl alanine was reduced in these rats and that reutilization of the amino acid into muscle protein was stimulated. In considering these results together with those shown in Table I, it is likely that decrease in urinary excretion of urea and total nitrogen in the rats fed either a carbohydrate or a fat meal may be due to the enhanced reutilization of free amino acids to muscle protein. It is also likely that de crease in oxidation of amino acids in these rats may be caused by removal of free amino acids from their pool in the tissue for synthesis of muscle protein. The present study was carried out to examine, in vivo, whether or not the concentration of

6 234 K. NAKANO and T. ISHIKAWA leucine in muscle cells or compartment thereof may play a role in regulating the turnover of muscle protein, in the rats whose muscle protein synthesis was already demonstrated to be enhanced, as mentioned above. Contrary to the expectation, no positive correlation was observed in the present study between the concentration of leucine in the skeletal muscle and the turnover of protein in the tissue. Thus, the content of free leucine decreased in muscles where protein synthesis was stimulated (Table II and Table VI). These results suggest, therefore, that leucine may not act as a regulator of protein metabol ism in muscle in the fashion as proposed by Buse and Reid13) under the conditions described in the present paper. Insulin and/or cyclic AMP may act as a possible regulator of protein metabolism in the rats fed with a carbohydrate meal as proposed previously by us.16) The decrease in the content of free leucine in muscle cells may be caused by enhanced reutilization of this amino acid into protein fraction of both muscle and liver in the rats fed either carbo hydrate meal or a fat meal as shown in the present paper. By the way, it is also noteworthy that the behavior of L-leucine-1-14C is somewhat dif ferent, in part, from that of L-phenylalanine- U-14C. As shown in Tables VI and VII, relative magnitudes of oxidation of L-leucine- 1-14C to 14CO2 were far greater (38 `57%) than those of L-phenylalanine-U-14C (4 `9%). Feeding of either a carbohydrate meal or a fat meal similarly caused significant decrease in oxidation of both of L-leucine-1-14C and L- phenylalanine-u-14c to 14CO2. The results were in opposition concerning the incorpora tion of 14C into liver protein depending on kind of radioactive amino acids used, although the magnitudes of incorporation of L-leucine- 1-14C were similar to those of L-phenylalanine- U-14C in all groups of rats examined. Thus, incorporation of L-leucine-1-14C to liver protein was stimulated by the feeding of either of these meals, whereas those of L-phenylalanine-U- 14C were significantly reduced in these rats Feeding rats with either a carbohydrate meal or a fat meal caused significant increase in incor poration of both L-leucine-1-14C and L-phenyl alanine-u-14c into skeletal muscle protein, although the magnitudes of relative recovery were quite different (11 `19% from L-leucine- 1-14C and 48 `63% from L-phenylalanine-U- 14C) These amino acids are commonly used to estimate, in vivo, and in vitro, the turnover of protein metabolism as reliable indices. These results indicate that some attention should be paid in choosing a kind of radioactive amino acid as a reliable index. REFERENCES 1) L. L. Miller, "Amino Acid Pools," Elsevier Publi shing Co., Amsterdam, 1962, p ) A. W. Meikle and J. G. Klain, Am. J. Physiol., 222, 1246 (1972). 3) R. Odessey and A. C. Goldberg, ibid., 223, 1316 (1972). 4) H. Yamamoto, T. Aikawa, H. Matsutaka, T. Okuda and E. Ishikawa, ibid., 226, 1428 (1974). 5) M. G. Buse, H. F. Herlong and D. A. Weugand, Diabetes, 23, 366 (1974). 6) M. G. Buse, F. Biggers, K. H. Friderici and J. F. Buse, J. Biol. Chem., 247, 8085 (1972). 7) M. G. Buse, J. F. Biggers, C, Drier and J. F. Buse, ibid., 248, 697 (1973). 8) K. Nakano and K. Ashida, J. Nutr., 100, 208 (1970). 9) K. Nakano, M. Katsuzaki, M. Mizutani and K. Ashida, ibid., 102, 283 (1972). 10) K. Nakano and K. Ashida, Nutr. Rep. Int., 10, 69 (1973). 11) K. Nakano, T. Ando and K. Ashida, J. Nutr., 104, 264 (1974). 12) R, M. Fulks, J. B. Li and A. L. Goldberg, J. Biol. Chem., 250, 290 (1975). 13) M. Buse and S. S. Reid, J. Clin. Invest., 56, 1250 (1975). 14) K. Yamashita and K. Ashida, J. Nutr., 99, 267 (1969). 15) G. W. Snedecor and W. G. Cockran, "Statistical Methods," 6th ed, Iowa State Univ. Press, Ames, 1967, p ) K. Nakano and K. Ashida, J. Nutr., 105, 906 (1975)..