Collagen synthesis in human musculoskeletal tissues and skin

We have developed a direct method for the measurement of human musculoskeletal collagen synthesis on the basis of the incorporation of stable isotope-labeled proline or leucine into protein and have used it to measure the rate of synthesis of collagen in tendon, ligament, muscle, and skin. In postabsorptive, healthy young men (28 +/- 6 yr) synthetic rates for tendon, ligament, muscle, and skin collagen were 0.046 +/- 0.005, 0.040 +/- 0.006, 0.016 +/- 0.002, and 0.037 +/- 0.003%/h, respectively (means +/- SD). In postabsorptive, healthy elderly men (70 +/- 6 yr) the rate of skeletal muscle collagen synthesis is greater than in the young (0.023 +/- 0.002%/h, P < 0.05 vs. young). The rates of synthesis of tendon and ligament collagen are similar to those of mixed skeletal muscle protein in the postabsorptive state, whereas the rate for muscle collagen synthesis is much lower in both young and elderly men. After nutrient provision, collagen synthesis was unaltered in tendon and skeletal muscle, remaining at postabsorptive values (young: tendon, 0.045 +/- 0.008%/h; muscle, 0.016 +/- 0.003%/h; elderly: muscle, 0.024 +/- 0.003%/h). These results demonstrate that the rate of human musculoskeletal tissue collagen synthesis can be directly and robustly measured using stable isotope methodology.     

Measurement of human mixed muscle protein fractional synthesis rate depends on the choice of amino acid tracer

The goal of this study was to discover whether using different tracers affects the measured rate of muscle protein synthesis in human muscle. We therefore measured the mixed muscle protein fractional synthesis rate (FSR) in the quadriceps of older adults during basal, postabsorptive conditions and mixed meal feeding (70 mg protein x kg fat-free mass(-1) x h(-1) x 2.5 h) by simultaneous intravenous infusions of [5,5,5-(2)H(3)]leucine and either [ring-(13)C(6)]phenylalanine or [ring-(2)H(5)]phenylalanine and analysis of muscle tissue samples by gas chromatography-mass spectrometry. Both the basal FSR and the FSR during feeding were approximately 20% greater (P < 0.001) when calculated from the leucine labeling in muscle tissue fluid and proteins (fasted: 0.063 +/- 0.005%/h; fed: 0.080 +/- 0.007%/h) than when calculated from the phenylalanine enrichment data (0.051 +/- 0.004 and 0.066 +/- 0.005%/h, respectively). The feeding-induced increase in the FSR ( approximately 20%; P = 0.011) was not different with leucine and phenylalanine tracers (P = 0.69). Furthermore, the difference between the leucine- and phenylalanine-derived FSRs was independent of the phenylalanine isotopomer used (P = 0.92). We conclude that when using stable isotope-labeled tracers and the classic precursor product model to measure the rate of muscle protein synthesis, absolute rates of muscle protein FSR differ significantly depending on the tracer amino acid used; however, the anabolic response to feeding is independent of the tracer used. Thus different precursor amino acid tracers cannot be used interchangeably for the evaluation of muscle protein synthesis, and data from studies using different tracer amino acids can be compared qualitatively but not quantitatively.     

A high proportion of leucine is required for optimal stimulation of the rate of muscle protein synthesis by essential amino acids in the elderly

This study was designed to evaluate the effects of enriching an essential amino acid (EAA) mixture with leucine on muscle protein metabolism in elderly and young individuals. Four (2 elderly and 2 young) groups were studied before and after ingestion of 6.7 g of EAAs. EAAs were based on the composition of whey protein [26% leucine (26% Leu)] or were enriched in leucine [41% leucine (41% Leu)]. A primed, continuous infusion of L-[ring-2H5]phenylalanine was used together with vastus lateralis muscle biopsies and leg arteriovenous blood samples for the determinations of fractional synthetic rate (FSR) and balance of muscle protein. FSR increased following amino acid ingestion in both the 26% (basal: 0.048 +/- 0.005%/h; post-EAA: 0.063 +/- 0.007%/h) and the 41% (basal: 0.036 +/- 0.004%/h; post-EAA: 0.051 +/- 0.007%/h) Leu young groups (P < 0.05). In contrast, in the elderly, FSR did not increase following ingestion of 26% Leu EAA (basal: 0.044 +/- 0.003%/h; post-EAA: 0.049 +/- 0.006%/h; P > 0.05) but did increase following ingestion of 41% Leu EAA (basal: 0.038 +/- 0.007%/h; post-EAA: 0.056 +/- 0.008%/h; P < 0.05). Similar to the FSR responses, the mean response of muscle phenylalanine net balance, a reflection of muscle protein balance, was improved (P < 0.05) in all groups, with the exception of the 26% Leu elderly group. We conclude that increasing the proportion of leucine in a mixture of EAA can reverse an attenuated response of muscle protein synthesis in elderly but does not result in further stimulation of muscle protein synthesis in young subjects.     

Smoking impairs muscle protein synthesis and increases the expression of myostatin and MAFbx in muscle

Smoking causes multiple organ dysfunction. The effect of smoking on skeletal muscle protein metabolism is unknown. We hypothesized that the rate of skeletal muscle protein synthesis is depressed in smokers compared with non-smokers. We studied eight smokers (> or =20 cigarettes/day for > or =20 years) and eight non-smokers matched for sex (4 men and 4 women per group), age (65 +/- 3 and 63 +/- 3 yr, respectively; means +/- SEM) and body mass index (25.9 +/- 0.9 and 25.1 +/- 1.2 kg/m(2), respectively). Each subject underwent an intravenous infusion of stable isotope-labeled leucine in conjunction with blood and muscle tissue sampling to measure the mixed muscle protein fractional synthesis rate (FSR) and whole body leucine rate of appearance (Ra) in plasma (an index of whole body proteolysis), the expression of genes involved in the regulation of muscle mass (myostatin, a muscle growth inhibitor, and MAFBx and MuRF-1, which encode E3 ubiquitin ligases in the proteasome proteolytic pathway) and that for the inflammatory cytokine TNF-alpha in muscle, and the concentration of inflammatory markers in plasma (C-reactive protein, TNF-alpha, interleukin-6) which are associated with muscle wasting in other conditions. There were no differences between nonsmokers and smokers in plasma leucine concentration, leucine rate of appearance, and plasma concentrations of inflammatory markers, or TNF-alpha mRNA in muscle, but muscle protein FSR was much less (0.037 +/- 0.005 vs. 0.059 +/- 0.005%/h, respectively, P = 0.004), and myostatin and MAFBx (but not MuRF-1) expression were much greater (by approximately 33 and 45%, respectivley, P < 0.05) in the muscle of smokers than of nonsmokers. We conclude that smoking impairs the muscle protein synthesis process and increases the expression of genes associated with impaired muscle maintenance; smoking therefore likely increases the risk of sarcopenia.     

Combined ingestion of protein and free leucine with carbohydrate increases postexercise muscle protein synthesis in vivo in male subjects

The present study was designed to determine postexercise muscle protein synthesis and whole body protein balance following the combined ingestion of carbohydrate with or without protein and/or free leucine. Eight male subjects were randomly assigned to three trials in which they consumed drinks containing either carbohydrate (CHO), carbohydrate and protein (CHO+PRO), or carbohydrate, protein, and free leucine (CHO+PRO+Leu) following 45 min of resistance exercise. A primed, continuous infusion of L-[ring-13C6]phenylalanine was applied, with blood samples and muscle biopsies collected to assess fractional synthetic rate (FSR) in the vastus lateralis muscle as well as whole body protein turnover during 6 h of postexercise recovery. Plasma insulin response was higher in the CHO+PRO+Leu compared with the CHO and CHO+PRO trials (+240 +/- 19% and +77 +/- 11%, respectively, P < 0.05). Whole body protein breakdown rates were lower, and whole body protein synthesis rates were higher, in the CHO+PRO and CHO+PRO+Leu trials compared with the CHO trial (P < 0.05). Addition of leucine in the CHO+PRO+Leu trial resulted in a lower protein oxidation rate compared with the CHO+PRO trial. Protein balance was negative during recovery in the CHO trial but positive in the CHO+PRO and CHO+PRO+Leu trials. In the CHO+PRO+Leu trial, whole body net protein balance was significantly greater compared with values observed in the CHO+PRO and CHO trials (P < 0.05). Mixed muscle FSR, measured over a 6-h period of postexercise recovery, was significantly greater in the CHO+PRO+Leu trial compared with the CHO trial (0.095 +/- 0.006 vs. 0.061 +/- 0.008%/h, respectively, P < 0.05), with intermediate values observed in the CHO+PRO trial (0.0820 +/- 0.0104%/h). We conclude that coingestion of protein and leucine stimulates muscle protein synthesis and optimizes whole body protein balance compared with the intake of carbohydrate only.     

Determination in vitro of the rate of protein synthesis and degradation in human-skeletal-muscle tissue.

The present studies were aimed to evaluate the possibility to use a system for estimation in vitro of the biosynthesis and degradation rates of human skeletal muscle protein. A previously characterized human skeletal muscle preparation was used. Amino acids and insulin stimulated significantly the incorporation rate of leucine into proteins. The effect of amino acids was more pronounced than that of insulin. The stimulatory effect of insulin could be decreased by amino acids. Insulin did not influence the tissue uptake or the oxidation rate of leucine. The release of [14C]leucine deriving from degradation of prelabelled skeletal muscle fibre proteins was linear for at least 2.5 h of incubation and optimal with leucine at concentrations beyond 12.5 mmol/1 or in the presence of puromycin in the incubation medium. The rate of the release of radioactivity was significantly inhibited by amino acids and at borderline significance by insulin but not by puromycin. The specific radioactivity in prelabelled proteins decreased significantly in the presence of puromycin suggesting that leucine derived from protein degradation was reutilized in vitro. This reutilization was found to be 9 +/- 1% of leucine released from degradation of proteins in 30 subjects. A statistically significant positive correlation between the cathepsin D activity in human skeletal muscle tissue and the degradative rate of prelabelled muscle proteins in vitro was observed. The results indicate that biosynthesis and degradation of skeletal muscle proteins in this system in vitro were subjected to control mechanisms. It is suggested that the release of radioactivity from prelabelled muscle fibre proteins during incubation probably only reflects the degradation of some rapidly-turning-over proteins.     

Effect of exercise on protein metabolism in humans as explored with stable isotopes

Exercising for 3.75 h on a treadmill at 50% VO2 max in the fed state induced an increased excretion of 71 mg nitrogen/kg over the 18 h after exercise. However, measurements of the time course of changes in 13CO2 excretion from ingested [1-13C]leucine indicated that all of this increased nitrogen production occurs during the exercise period. Because of the reduced renal clearance and slow turnover of the urea pool, urea excretion lags behind urea production. Measurements of nitrogen flux from the plateau labeling of urinary ammonia achieved by repeated oral doses of 15N-labeled glycine indicated that the nitrogen loss resulted from an increase in protein degradation and a decrease in protein synthesis. Further studies with [1-13C]leucine indicated that a 2-h treadmill exercise induced an increase in the nitrogen loss from 5.4 to 16 mg . kg-1 . h-1 measured with a primed constant infusion of [1-13C]leucine. This resulted from a fall in whole-body protein synthesis. Glucose given at the rate of 0.88 g . kg-1 . h-1 depressed the rate of whole-body protein degradation and appeared to suppress the exercise-induced increase in nitrogen excretion. When leucine oxidation rates were measured at increasing work rates, a linear relationship between percentage of VO2 max and leucine oxidation was observed up to 89% VO2 max when 54% of the flux of leucine was oxidized. These changes may involve nonmuscle as well as muscle tissue. Thus the source of the increased nitrogen losses is probably liver. In muscle, protein degradation is actually decreased judged by methylhistidine excretion, whereas in liver, protein degradation may be increased. Also the fall in whole-body protein synthesis may reflect changes in nonmuscle tissues because in running rats protein synthesis in muscle is maintained. As far as leucine metabolism is concerned, because the increase in leucine oxidation occurs when leucine and its keto acid concentration falls, exercise must specifically activate the 2-oxoacid dehydrogenase.     

Regulation of muscle protein degradation,not synthesis,by dietary leucine in rats fed a protein-deficient diet

The aim of this study was to elucidate the effects of long-term intake of leucine in dietary protein malnutrition on muscle protein synthesis and degradation. A reduction in muscle mass was suppressed by leucine-supplementation (1.5% leucine) in rats fed protein-free diet for 7 days. Furthermore, the rate of muscle protein degradation was decreased without an increase in muscle protein synthesis. In addition, to elucidate the mechanism involved in the suppressive effect of leucine, we measured the activities of degradation systems in muscle. Proteinase activity (calpain and proteasome) and ubiquitin ligase mRNA (Atrogin-1 and MuRF1) expression were not suppressed in animals fed a leucine-supplemented diet, whereas the autophagy marker, protein light chain 3 active form (LC3-II), expression was significantly decreased. These results suggest that the protein-free diet supplemented with leucine suppresses muscle protein degradation through inhibition of autophagy rather than protein synthesis.     

Fed-state clamp stimulates cellular mechanisms of muscle protein anabolism and modulates glucose disposal in normal men

Since maximum anabolism occurs postprandially, we developed a simulated fed state with clamped hyperinsulinemia, physiological hyperglycemia, and hyperaminoacidemia (Hyper-3) and explored muscle cellular mechanisms. Whole body [1-(13)C]leucine and [3-(3)H]glucose kinetics in healthy men were compared between hyperinsulinemic, euglycemic, isoaminoacidemic (Hyper-1, n = 10) and Hyper-3 (n = 9) clamps. In Hyper-3 vs. Hyper-1, nonoxidative leucine R(d) [rate of disappearance (synthesis)] was stimulated more (45 +/- 4 vs. 24 +/- 4 micromol/min, P < 0.01) and endogenous R(a) [rate of appearance (breakdown)] was inhibited similarly; hence net balance increased more (86 +/- 6 vs. 49 +/- 2 micromol/min, P < 0.001). Glucose R(d) was similar; thus Hyper-3 metabolic clearance rate (331 +/- 23 vs. 557 +/- 41 ml/min, P < 0.0005) and R(d)/insulin (M, 0.65 +/- 0.10 vs. 1.25 +/- 0.10 mg.min(-1).pmol(-1).l, P < 0.001) were less, despite higher insulin (798 +/- 74 vs. 450 +/- 24 pmol/l, P < 0.005). In vastus lateralis muscle biopsies, phosphorylation of Akt (P = 0.025), mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase (p70(S6K1); P = 0.008), S6 (P = 0.049), and 4E-binding protein 1 (4E-BP1; P = 0.001) increased. With decreased eukaryotic initiation factor-4E (eIF4E).4E-BP1 complex (P = 0.01), these are consistent with increased mTOR complex 1 (mTORC1) signaling and translation initiation of protein synthesis. Although mRNA expression of ubiquitin, MAFbx 1, and MuRF-1 was unchanged, total ubiquitinated proteins decreased 20% (P < 0.01), consistent with proteolysis suppression. The Hyper-3 clamp increases whole body protein synthesis, net anabolism, and muscle protein translation initiation pathways and decreases protein ubiquitination. The main contribution of hyperaminoacidemia is stimulation of synthesis rather than inhibition of proteolysis, and it attenuates the expected increment of glucose disposal.     

Protein turnover and proliferation. Turnover kinetics associated with the elevation of 3T3-cell acid-proteinase activity and cessation of net protein gain.

1. At least 95% of the total protein of A31-3T3 cell cultures undergoes turnover. 2. First-order exponential kinetics were used to provide a crude approximation of averaged protein synthesis, Ks, degradation, Kd, and net accumulation, Ka, as cells ceased growth at near-confluent density in unchanged Dulbecco's medium containing 10% serum. The values of the relationship Ka = Ks - Kd were : 5%/h = 6%/h - 1%/h in growing cells, and 0%/h = 3%/h - 3%/h in steady-state resting cells. 3. As determined by comparison of the progress of protein synthesis and net protein accumulation, the time course of increase in protein degradation coincided with the onset of an increase in lysosomal proteinase activity and decrease in thymidine incorporation after approx. 2 days of exponential growth. 4. After acute serum deprivation, rapid increases in protein degradation of less than 1%/h could be superimposed on the prevailing degradation rate in either growing or resting cells. The results indicate that two proteolytic mechanisms can be distinguished on the basis of the kinetics of their alterations. A slow mechanism changes in relation to proliferative status and lysosomal enzyme elevation. A prompt mechanism, previously described by others, changes before changes in cell-cycle distribution or lysosomal proteinase activity. 5. When the serum concentration of growing cultures was decreased to 1% or 0.25%, then cessation of growth was accompanied by a lower steady-state protein turnover rate of 2.0%/h or 1.5%/h respectively. When growth ceased under conditions of overcrowded cultures, or severe nutrient insufficiency, protein turnover did not attain a final steady state, but declined continually into the death of the culture.     

Reduced amino acid availability inhibits muscle protein synthesis and decreases activity of initiation factor eIF2B

We have examined the effect of a hemodialysis-induced 40% reduction in plasma amino acid concentrations on rates of muscle protein synthesis and breakdown in normal swine. Muscle protein kinetics were measured by tracer methodology using [(2)H(5)]phenylalanine and [1-(13)C]leucine and analysis of femoral arterial and venous samples and tissue biopsies. Net amino acid release by muscle was accelerated during dialysis. Phenylalanine utilization for muscle protein synthesis was reduced from the basal value of 45 +/- 8 to 25 +/- 6 nmol x min(-1) x 100 ml leg(-1) between 30 and 60 min after start of dialysis and was stimulated when amino acids were replaced while dialysis continued. Muscle protein breakdown was unchanged. The signal for changes in synthesis appeared to be changes in plasma amino acid concentrations, as intramuscular concentrations remained constant throughout. The changes in muscle protein synthesis were accompanied by a reduction or stimulation, respectively, in the guanine nucleotide exchange activity of eukaryotic initiation factor (eIF)2B following hypoaminoacidemia vs. amino acid replacement. We conclude that a reduction in plasma amino acid concentrations below the normal basal value signals an inhibition of muscle protein synthesis and that corresponding changes in eIF2B activity suggest a possible role in mediating the response.     

Protein synthesis is increased in heart failure induced by low dose adriamycin in rabbits

Congestive heart failure was induced in rabbits by a chronic treatment with a low dose of adriamycin (0.75 mg/kg intravenously 3 times per week for 11 weeks). Twenty-four to 48 h after the last injection, adriamycin-treated rabbits had a three-fold increase in plasma norepinephrine, a seven-fold increase in plasma epinephrine, a 19 +/- 8% increase in heart rate, and a 54 +/- 10% decrease in the total tension generated by their isolated papillary muscles, when compared with normal age-matched controls. This demonstrated the occurrence of the cardiomyopathy and heart failure. The effect of adriamycin on myocardial and diaphragmatic protein synthesis was examined in vivo after a 1-h infusion with [3H]leucine and in vitro after a 2-h incubation of right ventricular papillary muscle with [3H]leucine. The rate of in vivo [3H]leucine incorporation into total protein was increased in the heart of the adriamycin-treated rabbits. The increases were 60 +/- 16% in the left ventricle, 49 +/- 18% in the septum, 32 +/- 18% in the right ventricle, and 66 +/- 16% in the atria. A similar increase was observed when measuring the rate of [3H]leucine incorporation into myosin, a myofibrillar protein, and when the rate of [3H]leucine incorporation into total protein was measured in vitro in papillary muscle. In contrast, the rate of [3H]leucine incorporation into total protein of the diaphragm was not significantly changed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Failure of IGF-1 to affect protein turnover in muscle from growth-retarded neonatal rats

To investigate the response of the growth retarded neonatal rat to insulin-like growth factor-I (IGF-I) we have measured the effect of IGF-I on in vitro muscle protein synthesis and degradation rates in growth retarded and control neonatal rat pups. The growth retarded pups were growth retarded in utero by ligation of the uterine blood supply at day 17 of gestation. Basal levels of muscle protein synthesis in vitro were significantly lower in growth retarded pups compared with controls. Protein degradation rate were not different in muscles taken from the two groups. IGF-I stimulated protein synthesis in muscle from control pups by 12% and 15% at 20 ng/ml and 200ng/ml respectively. Net protein degradation was inhibited by 20% in the presence of 20ng/ml IGF-I. IGF-I had no effect on net protein synthesis or degradation in muscle from growth retarded pups. Neither Multiplication Stimulating Activity (at 20ng/ml or 200ng/ml) nor insulin (at 40ng/ml or 800ng/ml) was able to increase synthesis or decrease degradation of protein. Specific receptors for IGF-I are present on muscle membranes from both groups. Unlabelled IGF-I was more effective than MSA or insulin in competing with 125I-IGF-I for binding to the receptor. The relative affinities are consistent with type I IGF receptors. The affinity of these receptors for IGF-I was similar (Kd approximately 5nM) in both groups and the receptor concentration in both cases was approximately 250 fmol/mg protein. The refractility of tissue from growth retarded pups to IGF-I may be partially responsible for the lack of catch up growth in growth retarded neonates.     

Studies on turnover rates of rat gamma-glutamyltranspeptidase after chronic ethanol administration in vivo

Chronic ethanol administration to rats was shown to result in a significant increase of hepatic and serum GGT activities, contrasting to the decreased levels observed in pancreas, intestine, brain, and kidney by the new alcock regimen method. The kinetics of rat GGT synthesis and degradation in vivo among the different sources after chronic ethanol administration has been studied by use of acivicin, which irreversibly inactivates GGT. The comparison of kinetics of GGT return after acivicin injection showed that the kidney and serum GGT exhibits biphasic half-lives in contrast to liver, pancreatic, intestinal, and brain GGT half-lives in chronic ethanol-administered rats. The present studies on kinetics of GGT synthesis (Ks) and degradation (Kd) in vivo would seem to indicate the existence of three types of systems. That is, Ks rather than Kd may be preferential in liver and serum whereas Kd is apparently increased in kidney and intestine without noticeable change in Ks. The reverse phenomenon is also observed for pancreas and brain. These findings suggest that the contributions of alterations in the rates of GGT synthesis and degradation to changing levels of GGT have been evaluated as a mechanism for enzyme adaptation in animal tissues as a change from the control diet to the ethanol diet.     

Effects of insulin, glucose, and amino acids on protein turnover in rat diaphragm.

A simple method is described for measuring rates of protein synthesis and degradation in isolated rat diaphragm. Muscles incubated in Krebs-Ringer bicarbonate buffer showed a linear rate of synthesis for 3 hours. At the same time, the muscle released tyrosine and ninhydrin-positive material, primarily amino acids, at a linear rate. This release was not a nonspecific leakage of material from the intracellular pools, but reflected net protein degradation. Tyrosine was chosen for studies of protein turnover, since it rapidly equilibrates between intracellular pools and the medium, it can be measured fluorometrically, and it is neither synthesized nor degraded by this tissue. To follow protein degradation independently of synthesis, muscles were incubated in the presence of cycloheximide. Under these conditions, the amount of tyrosine in the intracellular pools was constant, while the muscle released tyrosine at a linear rate. This tyrosine release was used as a measure of degradation. This preparation was used to study the influence of various factors known to be important for muscle growth on protein synthesis and degradation. Similar effects were obtained with diaphragms of normal and fasted rats although the latter showed decreased synthesis and increased protein degradation. Insulin by itself not only stimulated synthesis but also inhibited protein degradation (even in the presence of cycloheximide). These two effects served to reduce the net release of tyrosine from muscle protein to comparable extents. Effects of insulin on synthesis and degradation were greater when glucose was also present in the medium. Glucose by itself inhibited protein degradation but in the absence of insulin glucose had no significant effect on synthesis. Nevertheless, glucose stimulated incorporation of radioactivive tyrosine into protein, but this effect was due to an increased intracellular specific activity. Unlike glucose neither beta-hydroxybutyrate or octanoic acid had any demonstrable effects on protein degradion. The addition of amino acids at plasma concentrations both promoted protein synthesis and inhibited degradation in the diaphragm. Five times normal plasma concentrations of the amino acids had larger effects. The three branched chain amino acids together stimulated synthesis and reduced degradation, while the remaining plasma amino acids did not affect either process significantly. Thus leucine, isoleucine, and valine appear responsible for the effects of plasma amino or isoleucine and valine together, also were able to inhibit protein degradation and promote synthesis.     

Protein metabolism in glucocorticoid excess: study in Cushing's syndrome and the effect of treatment

How protein metabolism is perturbed during chronic glucocorticoid excess is poorly understood. The aims were to investigate the impact of chronic glucocorticoid excess and restoration of eucortisolemia in Cushing's syndrome (CS) on whole body protein metabolism. Eighteen subjects with CS and 18 normal subjects (NS) underwent assessment of body composition using DEXA and whole body protein turnover with a 3-h constant infusion of l-[(13)C]leucine, allowing calculation of rates of leucine appearance (leucine R(a)), leucine oxidation (L(ox)), and leucine incorporation into protein (LIP). Ten subjects with CS were restudied after restoration of eucortisolemia. Percentage FM was greater (43.9 +/- 1.6 vs. 33.8 +/- 2.4%, P = 0.002) and LBM lower (52.7 +/- 1.6 vs. 62.1 +/- 2.3%, P = 0.002) in CS. LBM was significantly correlated (r(2) > 0.44, P < 0.005) to leuceine R(a), L(ox), and LIP in both groups. After correcting for LBM, leucine R(a) (133 +/- 5 vs. 116 +/- 5 micromol/min, P = 0.02) and L(ox) (29 +/- 1 vs. 24 +/- 1 micromol/min, P = 0.01) were greater in CS. FM significantly correlated (r(2) = 0.23, P < 0.05) with leucine R(a) and LIP, but not L(ox) in CS. In multiple regression, LBM was an independent determinant of all three indexes of leucine turnover, FM of leucine R(a), and LIP and CS of L(ox). Following restoration of eucortisolemia, L(ox) was reduced (Delta-7.5 +/- 2.6 micromol/min, P = 0.02) and LIP increased (Delta+15.2 +/- 6.2 micromol/min, P = 0.04). In summary, whole body protein metabolism in CS is influenced by changes in body composition and glucocorticoid excess per se, which increases protein oxidation. Enhanced protein oxidation is a likely explanation for the reduced protein mass in CS. Successful treatment of CS reduces protein oxidation and increases protein synthesis to prevent ongoing protein loss.     

Effect of testosterone on muscle mass and muscle protein synthesis

We have studied the effect of a pharmacological dose of testosterone enanthate (3 mg.kg-1.wk-1 for 12 wk) on muscle mass and total-body potassium and on whole-body and muscle protein synthesis in normal male subjects. Muscle mass estimated by creatinine excretion increased in all nine subjects (20% mean increase, P less than 0.02); total body potassium mass estimated by 40K counting increased in all subjects (12% mean increase, P less than 0.0001). In four subjects, a primed continuous infusion protocol with L-[1-13C]leucine was used to determine whole-body leucine flux and oxidation. Whole-body protein synthesis was estimated from nonoxidative flux. Muscle protein synthesis rate was determined by measuring [13C]leucine incorporation into muscle samples obtained by needle biopsy. Testosterone increased muscle protein synthesis in all subjects (27% mean increase, P less than 0.05). Leucine oxidation decreased slightly (17% mean decrease, P less than 0.01), but whole-body protein synthesis did not change significantly. Muscle morphometry showed no significant increase in muscle fiber diameter. These studies suggest that testosterone increases muscle mass by increasing muscle protein synthesis.     

Studies concerning the specificity of the effect of leucine on the turnover of proteins in muscles of control and diabetic rats.

The protein anabolic effect of branched chain amino acids was studied in isolated quarter diaphragms of rats. Protein synthesis was estimated by measuring tyrosine incorporation into muscle proteins in vitro. Tyrosine release during incubation with cycloheximide served as an index of protein degradation. In muscles from normal rats the addition of 0.5 mM leucine stimulated protein synthesis 36--38% (P less than 0.01), while equimolar isoleucine or valine, singly or in combination were ineffective. The three branched chain amino acids together stimulated no more than leucine alone. The product of leucine transamination, alpha-keto-isocaproate, did not stmino norborane-2-carboxylic acid (a leucine analogue) were ineffective. Leucine and isoleucine stimulated protein synthesis in muscles from diabetic rats.Leucine, isoleucine, valine and the norbornane amino acid but not alpha-ketoisocaproate or beta-hydroxybutyrate decreased the concentration of free tyrosine in tissues during incubation with cycloheximide; tyrosine release into the medium did not decrease significantly. Leucine caused a small decrease in total tyrosine release, (measured as the sum of free tyrosine in tissues and media), suggesting inhibition of protein degradation. The data suggest that leucine may be rate limiting for protein synthesis in muscles. The branched chain amino acids may exert a restraining effect on muscle protein catabolism during prolonged fasting and diabetes.     

ACTH receptors in nervous tissue. High affinity binding-sequestration of [125I]Phe2,Nle4]ACTH 1-24 in homogenates and slices from rat brain

We have demonstrated specific, high affinity binding of a biologically active Tyr23-monoiodinated derivative of ACTH, [125I][Phe2,Nle4]ACTH 1-24, in rat brain homogenates. Similarly, in metabolically inhibited and noninhibited rat whole brain slices there is a specific "binding-sequestration" process that is dependent on time, protein concentration, and pH. In homogenates, binding curves were best described by a two-site model and provided the following parameters: Kd1 = 0.65 +/- 0.47 nM, Bmax1 = 21 +/- 41 fmol/mg protein; Kd2 = 97 +/- 48 nM, Bmax2 = 3.5 +/- 1.8 pmol/mg protein. In metabolically viable brain slices, concentration-competition curves of [125I][Phe2,Nle4]ACTH 1-24 binding-sequestration can be described by three components (Kd1 = 14 +/- 24 nM, Bmax1 = 50 +/- 95 fmol/mg protein; Kd2 = 2.4 +/- 1.9 microM, Bmax2 = 44 +/- 49 pmol/mg protein; Kd3 = 0.16 +/- 1.0 mM, Bmax3 = 5.3 +/- 54 nmol/mg protein). Metabolic inhibition, by removal of glucose and addition of 100 microM ouabain, abolishes the lowest affinity, highest capacity binding-sequestrian component only (Kd1 = 7.1 +/- 14 nM, Bmax1 = 8.7 +/- 16 fmol/mg protein; Kd2 = 7.4 +/- 4.49 microM, Bmax2 = 37 +/- 27 pmol/mg protein). The two binding-sequestration parameter estimates obtained from metabolically inhibited tissue slices are not significantly different from those of the two higher affinity components obtained with noninhibited tissue. Thus, metabolic inhibition permits demonstration of ACTH receptor binding only, unconfounded by sequestration or internalization of ligand:receptor complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Leucine catabolism and incorporation into tissue proteins in thyroparathyroidectomized rats

We investigated parameters of leucine metabolism in thyroparathyroidectomized (TPX) and pair-fed control rats using a technique of continuous infusion of [l-14C]leucine. The rate of leucine turnover was significantly smaller in TPX than in control rats (42.5 +/- 2.6 vs 35.1 +/- 1.9 mumole/hr/100 g, mean +/- SEM, six rats). There was no significant difference between rates of alpha-decarboxylation of leucine by the two groups of rats. The protein incorporation of leucine was significantly smaller in the muscle of TPX than control rats (39 +/- 5 vs 24 +/- 4 pmole/mg protein, mean +/- SEM, six rats) but in liver it was not significantly different. Thyroparathyroidectomy also had no significant effect on concentration of either leucine or its ketoacid (alpha-ketoisocaproate) in plasma, liver, and muscle. We conclude that hypothyroidism does not alter catabolism of leucine but reduces its incorporation into muscle protein.