Incorporation of Leucine into Human Skeletal Muscle Proteins: A Study of Tissue Amino Acid Pools and their Role in Protein Biosynthesis

The in vitro incorporation of labelled leucine into human skeletal muscle proteins was studied with the aim to elucidate the relationship between the amino acid tissue pools and protein biosynthesis. The distribution volumes of leucine and cycloleucine in skeletal muscle tissue were similar but the equilibration time was shorter for leucine than for cycloleucine. The cellular uptake of leucine and cycloleucine was competitively inhibited by increased concentration of amino acids in the medium indicating an active transport. Optimal stimulation for incorporation of leucine into proteins was obtained at an amino acid concentration in the medium corresponding to 10 times that of normal human plasma. The incorporation of ¹⁴C-leucine into skeletal muscle proteins was linear before the total pool of free intracellular ¹⁴C-leucin had reached an equilibrium. The regression coefficients for the semilogarithmic plots of the labelling rate of proteins with ¹⁴C-leucine and the incorporation rate of leucine calculated from the specific activity in the medium versus the amino acid concentration in the medium were different in the same experiment indicating a re-utilization of amino acids released at protein degradation. The results are compatible with the hypothesis that the proteolytically released amino acids have a competitive advantage for incorporation as compared with extra- and intracellular free amino acids. It is concluded that the amino acid pool which is in the immediate continuity with the protein biosynthesis sites equilibrates rapidly with the extracellular amino acid pool.     

Tissue Amino Acid Flux After Exposure of Rats to Diplococcus pneumoniae

The concentration of 21 individual free amino acids in serum, liver, and skeletal muscle was determined in rats during the incubation, acute illness, and terminal stages of experimental infection with Diplococcus pneumoniae. By 4 hr after subcutaneous inoculation with bacteria, the concentration of total and many individual free amino acids in serum, liver, and muscle was significantly decreased in comparison to findings in noninfected, pair-fed control rats. By use of a nonmetabolizable amino acid analogue (cycloleucine), it was possible to detect a flux of amino acids from muscle to liver in the infected rat. The endogenous amino acids which moved to the liver were rapidly utilized for the synthesis of serum proteins.     

Role of leucine in protein metabolism during exercise and recovery.

Exercise produces changes in protein and amino acid metabolism. These changes include degradation of the branched-chain amino acids, production of alanine and glutamine, and changes in protein turnover. One of the amino acid most affected by exercise is the branched-chain amino acid leucine. Recently, there has been an increased understanding of the role of leucine in metabolic regulations and remarkable new findings about the role of leucine in intracellular signaling. Leucine appears to exert a synergistic role with insulin as a regulatory factor in the insulin/ phosphatidylinositol-3 kinase (PI3-K) signal cascade. Insulin serves to activate the signal pathway, while leucine is essential to enhance or amplify the signal for protein synthesis at the level of peptide initiation. Studies feeding amino acids or leucine soon after exercise suggest that post-exercise consumption of amino acids stimulates recovery of muscle protein synthesis via translation regulations. This review focuses on the unique roles of leucine in amino acid metabolism in skeletal muscle during and after exercise.     

Changes of protein synthesis in the hypertrophying rat heart

Summary Myocardial protein synthesis was studied in rats in vivo during the first five days of the development of cardiac hypertrophy induced by aortic constriction. Using l-¹⁴C-glycine or l-¹⁴C-leucine as precursor amino acids, rates of protein synthesis were determined from the total radioactivity of proteins and the mean radioactivity of the intracellular amino acid precursor pool and the leucine pool, respectively. During the first 5 h after aortic constriction the radioactivity of proteins did not change remarkably, whereas the radioactivity of the amino acid precursor pool was significantly elevated. Myocardial protein synthesis proved therefore to be diminished in this initial phase. After 24 h protein synthesis reached the range of sham-operated controls and thereafter increased almost parallel with the elevation of the ratio heart weight/body weight. The inhibition of protein synthesis was accompanied by a moderate decrease of ATP and creatine phosphate levels. A diminution of the high energy phosphate compounds, a decreased RNA synthesis or the action of inhibitory metabolites are considered possible factors involved in the decline of protein synthesis during the early phase of cardiac hypertrophy.Preliminary reports of this investigation were presented at the 36th Meeting of the German Physiological Society, Mainz, September 1969 [42] and at the XXV International Congress of Physiological Sciences, Munich, July 1971 [43].Supported by a grant from the Deutsche Forschungsgemeinschaft (Ge 129/7,8).     

Aromatic amino acid incorporation into proteins in rat brain slices: influences of other amino acids affecting transport

Incorporation of [³H]tryptophan, [³H]tyrosine and [³H]phenylalanine into proteins has been studied in cerebral coronal slices from adult and 1-day-old rats. Unlabelled amino acids greatly increased (histidine) or decreased (leucine, phenylalanine, tyrosine, tryptophan) intracellular levels of the [³H]amino acids, but only slightly decreased their incorporation rates. Effects on uptake were similar in experiments with adult and newborn rats, but patterns of protein synthesis inhibition seemed to differ. Amino acid uptake and subsequent incorporation into proteins were thus not closely associated. Incorporation in slices from adult rats was much slower than in those from newborn rats, and moreover, one half of it was cycloheximide-insensitive. The different inhibition patterns by other amino acids may thus be explained by multiple mechanisms in cerebral amino acid incorporation into proteins.     

Insulin action in pancreatic acini from streptozotocin-treated rats. I. Stimulation of protein synthesis

Pancreatic acini were prepared from rats rendered diabetic with streptozotocin. In this tissue, insulin stimulated [3H]leucine incorporation into protein. The full effects of insulin on this function were not immediate but increased linearly with time for up to 2 h of incubation. Insulin had a detectable effect on l eucine incorporation at 50 pM, a half-maximal effect at 0.7 nM, and a maximal effect at 30 nM. Desdipeptide proinsulin was only 10% as potent as native insulin in stimulating [3H]leucine incorporation, whereas proinsulin and desoctapeptide insulin were only 1% as potent. Insulin also increased the incorporation of [3H]valine and [35S]methionine into protein but did not increase the influx of either [14C]cycloleucine or alpha-[3H]aminoisobutyric acid. These observations suggested that the increased incorporation of labeled amino acid into protein reflected stimulation of protein synthesis rather than stimulation of amino acid transport. Furthermore, insulin at 1.67 nM significantly increased the acinar cell concentration of amylase. The present findings are consistent therefore with the concept that insulin regulates pancreatic exocrine functions, including protein and enzyme synthesis.     

Protein synthesis with special reference to S-100 protein in brain slices from rats receiving a restricted protein supply.

The synthesis of S-100 protein and that of soluble and total proteins was investigated using cerebral slices from rats fed a 20% or 3% protein containing diet for 6 days. Incorporation of radioactive amino acids into S-100 protein was significantly higher when rats were fed a diet containing 20% protein. No significant differences were obtained in the radioactivity incorporated into total or soluble proteins between the 2 dietary groups. 14C-leucine of aspecific radioactivity of 55 mCi/mmol or 3.2 mCi/mmol incorporated with time into total protein was similar for the 2 dietary groups. The time-dependent uptake of 14C-leucine by the slices and theinulin space remained unaffected by the dietary conditions used; and amino acid analyser estimates of the free amino acid pool showed no significant differences. Brain wet weight was 1.54+/-0.02 g and1.39+/-0.02 g for protein-fed and protein-restricted rats respectively. The corresponding body weight increased by 7.8 g/day or fell by 0.5 g/day. Although the differences observed in total protein synthesis were small the synthesis of a nervous tissue specific protein S-100 was markedly affected by short-term protein restriction.     

Endocrine activation and altered muscle metabolism after hemorrhagic shock.

Studies were conducted to examine glucose and amino acid metabolism by skeletal muscle isolated from rhesus monkeys before and sequentially after an episode of resuscitated hemorrhagic shock. After shock and reinfusion, the tissue exhibited decreased effect of insulin on glucose utilization increased leucine oxidation, and a reduced rate of leucine incorporation into protein. These changes were observed 15 min after reinfusion and persisted in part for at least 3 days. All of the observed abnormalities were more pronounced 24 h after shock and reinfusion than 15 min after and returned to normal by 2-4 wk. The shock-induced metabolic abnormalities in skeletal muscle occurred in spite of prevention of shock-induced adrenal steroid and catecholamine secretion and of changes in blood insulin concentration using adrenalectomized-streptozotocin diabetic monkeys receiving replacement cortisol and insulin infusions. This study thus demonstrated that hemorrhagic shock in rhesus monkeys was followed by insulin resistance plus abnormalities of glucose and amino acid metabolism by skeletal muscle that were not dependent on the concurrent changes in plasma levels of adrenal steroids or catecholamines or on altered circulating insulin levels associated with shock.     

Protein metabolism in guanethidine-treated rats

The aim of the present study was to evaluate the effect of short-term adrenergic blockade on the rate of whole-body protein turnover and leucine oxidation, and on protein synthesis in specific tissues in male rats. Adrenergic blockade was induced by guanethidine (100 mg/kg body weight subcutaneously). The control group was treated with saline. On the second day, the parameters of whole-body protein and leucine metabolism were evaluated using a primed constant intravenous infusion of L-[1-(14)C]leucine. Protein synthesis in tissues was determined on the basis of L-[1-(14)C]leucine incorporation. Guanethidine treatment caused a decrease in norepinephrine in skeletal muscle. Whole-body leucine oxidation and leucine oxidized fraction were higher in guanethidine-treated rats. There was an insignificant effect of guanethidine on whole-body proteolysis, protein synthesis and leucine clearance. However, protein balance was negative due to the larger difference between protein synthesis and proteolysis in guanethidine-treated animals compared to controls. In guanethidine-treated rats, protein synthesis was higher in the gastrocnemius muscle and in the kidneys and lower in liver and spleen. Changes in the small intestine and colon were insignificant. In addition, a marked decrease in concentration of several amino acids has been observed in the liver, the kidneys and the spleen. It is concluded that adrenergic blockade induced by guanethidine is associated with significant changes in protein metabolism, leucine oxidation and amino acid concentrations in several tissues. The most important consequences of treatment are considered to be a negative effect on protein balance, increased protein turnover in skeletal muscle and kidneys and decreased protein synthesis in the liver and spleen. These changes may also be induced by administration of other sympathetic blocking agents, e.g. in treatment of hypertension.     

Protein Synthesis with Special Reference to S-100 Protein in Brain Slices from Rats Receiving a Restricted Protein Supply

The synthesis of S-100 protein and that of soluble and total proteins was investigated using cerebral slices from rats fed a 20% or 3% protein containing diet for 6 days. Incorporation of radioactive amino acids into S-100 protein was significantly higher when rats were fed a diet containing 20% protein. No significant differences were obtained in the radioactivity incorporated into total or soluble proteins between the 2 dietary groups. ¹⁴C-leucine of a specific radioactivity of 55 mCi/mmol or 3.2 mCi/mmol incorporated with time into total protein was similar for the 2 dietary groups. The time-dependent uptake of ¹⁴C-leucine by the slices and the inulin space remained unaffected by the dietary conditions used; and amino acid analyser estimates of the free amino acid pool showed no significant differences. Brain wet weight was 1.54 ± 0.02 g and 1.39 ± 0.02 g for protein-fed and protein-restricted rats respectively. The corresponding body weight increased by 7.8 g/day or fell by 0.5 g/day. Although the differences observed in total protein synthesis were small the synthesis of a nervous tissue specific protein S-100 was markedly affected by short-term protein restriction.     

Lack of specific prostaglandin antagonistic effects of 7-oxa-13-prostynoic acid on ovarian metabolism in vitro.

Prostaglandins (PGs) have been proposed to function as obligatory intermediates in the action of luteinizing hormone (LH) (Kuehl et al. 1970). This based on experiments on isolated mouse ovaries where the substance 7-oxa-13-prostynoic acid (7-oxa-13-PA) was found to block the effect of both PGE1 and LH on ovarian cyclic AMP formation and progesterone synthesis. We have studied whether 7-oxa-13-PA acts as a PG antagonist also on the isolated rat ovary, where PGs of the E type have many effects. Prepubertal rat ovaries were incubated up to 4 h in modified Krebs bicarbonate buffer containing glucose with PGE1 and 7-oxa-13-PA, alone and in combination. PGE1 stimulated the uptake of the non-utilizable amino acids alpha-aminoisobutyric acid (AIB) and cycloleucine in an equally wide dose-range as has earlier been found for the stimulation of lactic acid and cyclic AMP production as well as protein synthesis. 7-oxa-13-PA alone, in concentrations exceeding 25 microgram/ml, stimulated lactic acid production, but inhibited the incorporation of labelled leucine and the uptake of AIB. The uptake of cycloleucine was slightly stimulated. 7-oxa-13-PA did not antagonize the PGE1 effect on lactic acid production. 7-oxa-13-PA diminished the PGE1 effect on the uptake of amino acids and the incorporation into protein, but only in concentrations where it in itself inhibited the protein synthesis. These results show that 7-oxa-13-PA does not act as a PG-antagonist in the prepubertal rat ovary, since it cannot block the effects of PGs without having marked inherent effects.     

In Vitro Work Load and Rat Heart Metabolism: II. Effect on amino acid transport

In vitro pressure overload was found to accelerate protein synthesis in the isolated working rat heart (Hjalmarson and Isaksson 1972 a). Since membrane transport of amino acids is considered to be one rate-limiting step in protein synthesis, the amino acid transport in the isolated rat heart was investigated using the non-utilizable amino acids α-aminoisobutyric acid (AIB) and 1-aminocyclopentane carboxylic acid (cycloleucine). Increased pressure load (afterload) accelerated amino acid uptake after a perfusion period of 15 ruin, and a 50—100% increase in the intracellular to extracellular distribution ratio of the amino acids was seen after 60 min of perfusion. This accelerated uptake was not inhibited by cycloheximide, suggesting that the work load effect was not dependent upon a continuous synthesis of proteins. The accumulation rate continued to be stimulated for some time after normalization of the work load and coronary flow, indicating that the work load effect was not directly linked to coronary flow. Increased preload did not stimulate amino acid uptake. Hearts from hypophysectomized rats showed a decreased concentrative uptake but the amino acid uptake was still accelerated by pressure overload. It is suggested that an increased uptake of amino acids could be of physiological significance in relation to the increased protein synthesis under these conditions.     

Effects of liver ischemia on hepatic protein synthesis in vitro and in vivo

When an in vitro system is used to study the influence of ischemia on hepatic protein synthesis, an important question is whether alterations observed in vitro reflect changes in vivo. In the present study the effects of liver ischemia on protein synthesis were investigated in rats both in vitro and in vivo. Liver ischemia was induced by hepatic artery ligation. Protein synthesis in vitro was determined from leucine incorporation into proteins in liver slices incubated in a medium containing ¹⁴C-leucine (0.5 mmol/l) and in vivo from leucine incorporation into hepatic proteins after intraportal injection of a tracer dose of ¹⁴C-leucine. Leucine incorporation rate in non-ischemic liver was 0.16 pmol * g pror¹ h-¹ in vitro and 19.6 μmol g prot-¹. h^-1 in vivo. After hepatic artery ligation protein synthesis in vitro was reduced by about 60% and in vivo by about 80%. Thus, the relative changes were of the same magnitude in vitro and in vivo. This indicates that an in vitro system can be used to evaluate the effects of liver ischemia on hepatic protein synthesis.     

Effect of fasting on the metabolism in rat aorta

The oxidation of 14C-labelled glucose, beta-hydroxybutyrate and palmitate to CO2 and the incorporation of 14C-labelled amino acid into alkali-soluble protein were studied in aorta from fed and fasted male Sprague-Dawley rats, weighting about 200 g. Substrate oxidation and amino acid incorporation were measured during incubation of rat aorta in vitro for 2-3 h. After fasting for 6 h there was a slight but significant increase in the plasma concentration of beta-hydroxybutyrate. Blood glucose was lowered after 12 h while an increase in the plasma concentration of free fatty acids was found after 24 h. A decrease in the oxidation of glucose in rat aorta was found after fasting for 12 h and with prolonged fasting a further decrease in the aortic glucose oxidation was found. After fasting for 4 days the oxidation of beta-hydroxybutyrate and the incorporation of 14C-leucine and 14C-phenylalanine into protein were reduced in rat aorta while the oxidation of palmitate was not altered. The effects of fasting on substrate oxidation and amino acid incorporation in rat aorta, found in this study are similar to the known effects of diabetes on vascular metabolism.     

Protein synthesis in rats force-fed for one day purified diets containing complete,threonine-devoid or no amino acids and adequate or low carbohydrate

Young female rats were force-fed purified diets containing complete (C), threonine-devoid (TD) or no (AAD) amino acids and adequate or low carbohydrates for 1 day (three feedings) and were killed the following morning. Protein synthesis (in vivo¹⁴C-leucine incorporation into proteins) was measured in liver, plasma, heart, gastrocnemius muscle, and spleen. The results revealed that protein synthesis was increased in the liver, plasma, and heart of rats force-fed the TD diet and was increased, but less marked, in the liver and plasma of rats force-fed the AAD diet. Protein synthesis in the gastrocnemius muscle and spleen was decreased in both experimental groups. Hepatic polyribosomes revealed greater aggregation and there was increased in vitro incorporation of ¹⁴C-leucine into proteins of livers of rats force-fed the TD and the AAD (less marked than with TD) diets in comparison with those of rats force-fed the C diet. Rats force-fed the experimental diets containing low carbohydrates did not reveal similar changes to those of rats force-fed the experimental diets containing adequate carbohydrates.     

Lack of specific prostaglandin antagonistic effects of 7-oxa-13-prostynoic acid on ovarian metabolism in vitro

Prostaglandins (PGs) have been proposed to function as obligatory intermediates in the action of luteinizing hormone (LH) (Kuehl et al. 1970). This was based on experiments on isolated mouse ovaries where the substance 7-oxa-13-prostynoic acid (7-oxa-13-PA) was found to block the effect of both PGE₁ and LH on ovarian cyclic AMP formation and progesterone synthesis. We have studied whether 7-oxa-13-PA acts as a PG antagonist also on the isolated rat ovary, where PGs of the E type have many effects. Prepubertal rat ovaries were incubated up to 4 h in modified Krebs bicarbonate buffer containing glucose with PGE₁ and 7-oxa-13-PA, alone and in combination. PGEj stimulated the uptake of the non-utilizable amino acids a-aminoisobutyric acid (AIB) and cycloleucine in an equally wide dose-range as has earlier been found for the stimulation of lactic acid and cyclic AMP production as well as protein synthesis. 7-oxa-13-PA alone, in concentrations exceeding 25 μMg/ml, stimulated lactic acid production, but inhibited the incorporation of labelled leucine and the uptake of AIB. The uptake of cycloleucine was slightly stimulated. 7-oxa-13-PA did not antagonize the PGE₁ effect on lactic acid production. 7-oxa-13-PA diminished the PGE₁ effect on the uptake of amino acids and the incorporation into protein, but only in concentrations where it in itself inhibited the protein synthesis. These results show that 7-oxa-13-PA does not act as a PG-antagonist in the prepubertal rat ovary, since it cannot block the effects of PGs without having marked inherent effects.     

Comparison of the use of isotopic proline vs leucine to measure protein synthesis in cultured fibroblasts

Compartmentation of the amino acid precursor pools for protein synthesis in cultured cells can substantially complicate measurements of synthesis rates. This is particularly true for nonessential amino acids such as proline, an amino acid often used in isotopic form to measure collagen synthesis. We have made a comparative study of this problem in cultured IMR-90 fibroblasts using isotopic proline and leucine to measure total protein and collagen synthesis. 3H-leucine in the extracellular (EC) medium equilibrates with tRNA-leucine at an EC concentration of 0.4 mM in both dividing and stationary cells. Thus, under these experimental conditions there is no complicating compartmentation of leucine for protein synthesis. Equilibration of EC and tRNA-bound 3H-proline, however, does not occur even when the EC concentration is in the mM range, based upon simultaneous measurements of synthesis rates using 3H-proline and 3H-leucine together. Furthermore, significant changes in EC proline concentration and specific activity occur over short time intervals (2 hr) if the initial EC proline concentration is below 0.2 mM. Thus, the use of isotopic proline to measure protein synthesis introduces substantial interpretive problems. Serum deprivation causes changes in both total collagen synthesis and the percent of protein synthesis devoted to collagen when measured with either 14C-leucine or 3H-proline. At the same time, isotopic proline remains the better choice for measuring percent collagen synthesis.     

Short-term starvation decreases skeletal muscle protein synthesis rate in man.

The rate of protein synthesis in skeletal muscle was determined in the post-absorptive state and after 3 days of starvation in healthy volunteers. The flooding dose technique employing intravenous injection of (1-13C)leucine (0.05 g kg-1) was used and incorporation of isotope into muscle protein was measured by taking percutaneous biopsies at 0 and 90 min. Blood samples were taken during the incorporation period for assessment of the enrichment of the free amino acid precursor of protein synthesis. The median (25,75 quartiles) rate of muscle protein synthesis after an overnight fast was 2.03 (2.00,2.23) % days-1 when the precursor enrichment was obtained by measurement of the plasma alpha-ketoisocaproate, taken to be representative of muscle free leucine. Repeat measurements in the same subjects after 3 days of total starvation showed a decrease to 1.82 (1.57,2.05) % days-1. Rates calculated on the basis of the plasma leucine as precursor were 5% lower at both times. An interindividual variation in response to starvation was observed, but the median decrease of 13% in the rate of protein synthesis was statistically significant (P less than 0.01).     

Effect of alloxan-diabetes on the metabolism of rabbit colon smooth muscle.

The metabolism of colon smooth muscle from normal and alloxan-diabetic rabbits was studied in vitro. Glucose uptake, incorporation of glucose minus14-C into glycogen and incorporation of leucine minus-14C into protein were determined. These three parameters were all depressed in diabetic smooth muscle incubated in a medium containing 5.6 mM glucose. Raising the glucose concentration in the medium to 22.2 mM almost doubled the glucose uptake both in diabetic and normal smooth muscle and at this glucose concentration no significant difference in this parameter was found. Insulin (0.1 U/ml) added in vitro stimulated glucose uptake, incorporation of glucose minus14-C into glycogen and incorporation of leucine minus14-C into protein in diabetic colon smooth muscle. Insulin stimulated glucose uptake to the same degree in normal and diabetic colon smooth muscle, while its effect on glucose minus14-C and leucine minus14-C incorporation tended to be somewhat more pronounced in diabetic colon muscle.     

Biosynthesis of vitamin B12 by Propionibacterium shermanii IV. Vitamin B12 and porphyrin synthesis in relation to the metabolic activities of the bacterium

The bacterial cell pool contains 16 amino acids. No qualitative change appeared in the amino acid pool at different stages of growth. No amino acids were liberated from the cell pool into the culture medium. Pyruvic and α-ketoglutaric acids were detected in the culture medium and cell pool of Propionibacterium shermanii. Bacterial growth, vitamin B₁₂, and coproporphyrin III synthesis increased with increasing age of the culture. A part of the synthesized porphyrin is released into the culture medium. Glucose, the total nitrogen content of the culture medium, ammonia nitrogen, amino acid nitrogen, methionine and glycine decreased gradually with the increase in the age and the bacterial dry weight of the culture. Succinic acid appeared on the 4^th day and increased with age. Acetic and propionic acids were the only detectable volatile acids. The ratio of propionic to acetic acid was about 3:1 (w/w).