Measurement of muscle protein fractional synthesis and breakdown rates from a pulse tracer injection

We have developed a new method to determine the fractional synthesis rate (FSR) and breakdown rate (FBR) of muscle protein. This method involves a pulse tracer injection and measurement of enrichment in the arterial blood and muscle at three time points. The calculations of FSR and FBR are based on the precursor-product principle. To test this method, we gave a pulse injection of L-[ring-(13)C(6)]phenylalanine of 4-6 mg/kg in five rabbits. The measured FBR value (0.233 +/- 0.060%/h) was almost identical (P = 0.35) to that (0.217 +/- 0.078%/h) estimated from a leg arteriovenous balance model (Biolo G, Chinkes D, Zhang X-J, and Wolfe RR. J Parenter Enteral Nutr 16: 305-315, 1992). The measured FSR value tended to be lower than that estimated from the leg model (0.125 +/- 0.036 vs. 0.185 +/- 0.086%/h; P = 0.14), possibly because the new method measures only muscle FSR, whereas the leg balance model also includes skin and bone contributions. The pulse tracer injection did not affect muscle protein kinetics as measured by leucine and phenylalanine kinetics in the leg. In another five rabbits, we demonstrated that sampling could be reduced to either one or two muscle biopsies when multiple pulse injections were used. This method can be completed in 1 h with one muscle biopsy and has technical advantages over currently used methods.     

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.     

Amino Acid Availability Regulates the Effect of Hyperinsulinemia on Skin Protein Metabolism in Pigs

The effects of amino acid supply and insulin infusion on skin protein kinetics (fractional synthesis rate (FSR), fractional breakdown rate (FBR), and net balance (NB)) in pigs were investigated. Four-month-old pigs were divided into four groups as follows: control, insulin (INS), amino acid (AA), and INS + AA groups based on the nutritional and hormonal conditions. l-[ring-¹³C₆]Phenylalanine was infused. FBR was estimated from the enrichment ratio of arterial phenylalanine to intracellular free phenylalanine. Plasma INS was increased (p < 0.05) in the INS and INS + AA groups. Plasma glucose was maintained by infusion of glucose in the groups receiving INS. The interventions did not change the NB of skin protein. However, the interventions affected the FSR and FBR differently. An infusion of INS significantly increased both FSR and FBR, although AA infusion did not. When an AA infusion was added to the infusion of insulin (INS + AA group), FSR and FBR were both lower when compared with the INS group. Our data demonstrate that in anesthetized pigs INS infusion did not exert an anabolic effect, but rather it increased AA cycling into and out of skin protein. Because co-infusion of AAs with INS ameliorated this effect, it is likely that the increased AA cycling during INS infusion was related to AA supply. Although protein kinetics were affected by both INS and AAs, none of the interventions affected the skin protein deposition. Thus, skin protein content is closely regulated under normal circumstances and is not subject to transient changes in AAs or hormonal concentrations.     

Resistance-training-induced adaptations in skeletal muscle protein turnover in the fed state

Resistance training changes the balance of muscle protein turnover, leading to gains in muscle mass. A longitudinal design was employed to assess the effect that resistance training had on muscle protein turnover in the fed state. A secondary goal was investigation of the potential interactive effects of creatine (Cr) monohydrate supplementation on resistance-training-induced adaptations. Young (N = 19, 23.7 +/- 3.2 year), untrained (UT), healthy male subjects completed an 8-week resistance-training program (6 d/week). Supplementation with Cr had no impact on any of the variables studied; hence, all subsequent data were pooled. In the UT and trained (T) state, subjects performed an acute bout of resistance exercise with a single leg (exercised, EX), while their contralateral leg acted as a nonexercised (NE) control. Following exercise, subjects were fed while receiving a primed constant infusion of [d5]- and [15N]-phenylalanine to determine the fractional synthetic and breakdown rates (FSR and FBR), respectively, of skeletal muscle proteins. Acute exercise increased FSR (UT-NE, 0.065 +/- 0.025 %/h; UT-EX, 0.088 +/- 0.032 %/h; P < 0.01) and FBR (UT-NE, 0.047 +/- 0.023 %/h; UT-EX, 0.058 +/- 0.026 %/h; P < 0.05). Net balance (BAL = FSR - FBR) was positive in both legs (P < 0.05) but was significantly greater (+65%) in the EX versus the NE leg (P < 0.05). Muscle protein FSR and FBR were greater at rest following T (FSR for T-NE vs. UT-NE, +46%, P < 0.01; FBR for T-NE vs. UT-NE, +81%, P < 0.05). Resistance training attenuated the acute exercise-induced rise in FSR (T-NE vs. T-EX, +20%, P = 0.65). The present results demonstrate that resistance training resulted in an elevated resting muscle protein turnover but an attenuation of the acute response of muscle protein turnover to a single bout of resistance exercise.     

Increased concentration of tracee affects estimates of muscle protein synthesis

Muscle protein synthesis was measured by infusion of L-[2H(5)]phenylalanine in two groups of anesthetized dogs, before and during infusion of insulin with euaminoacidemia, and with differing concentrations of unlabeled phenylalanine (tracee). With the infusion of insulin, muscle protein synthesis increased 39 +/- 12% based on phenylalanyl-tRNA. Calculation with plasma phenylalanine enrichment overestimated insulin stimulation by 40% (56 +/- 12 vs. 39 +/- 12%). Raising the concentration of plasma phenylalanine twofold during infusion of insulin further increased the apparent stimulation of muscle protein synthesis based on plasma relative to phenylalanyl-tRNA by 225% (65 +/- 19 vs. 20 +/- 14%, P < 0.001). In both experiments, the stimulation of synthesis rates calculated from phenylalanine enrichment within the muscle was closer to that from phenylalanyl-tRNA (48 +/- 19%, experiment 1; 30 +/- 14%, experiment 2). Results indicate that the enrichment of a labeled amino acid within plasma and tissue amino acid pools is affected by the concentration of tracee infused. Increasing the concentration of tracee overestimates the insulin-mediated stimulation of muscle protein synthesis when amino acid pools other than aminoacyl-tRNA are used as the precursor enrichment.     

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.     

Effects of enteral glutamine on gut mucosal protein synthesis in healthy humans receiving glucocorticoids

In hypercatabolic patients, the beneficial effects of glutamine on gut mucosa could be partly due to a stimulation of protein synthesis. The fractional synthesis rate (FSR) of gut mucosal protein was measured in four groups of healthy volunteers treated with glucocorticoids for 2 days. Two groups were studied in the postabsorptive state while receiving glutamine or a nitrogen equivalent (control) and two groups in the fed state with or without glutamine, using a 5-h intravenous infusion of [(13)C]leucine, [(2)H(5)]phenylalanine, and cortisone. After nutrient and tracer infusion, duodenal biopsies were taken. In the postabsorptive state, FSR of gut mucosal protein were 87 and 76%/day in the control group and 130% (P = 0.058 vs. control) and 104% (P = 0.17 vs. control)/day in the glutamine group, with leucine and phenylalanine as tracers, respectively. During feeding, FSR did not increase and no significant difference was observed between glutamine and control groups. Overall, FSR of the four groups were two- to threefold higher than those obtained previously in healthy humans, suggesting that glucocorticoids may increase gut mucosal protein synthesis. However, in this situation, a moderate enteral glutamine supply failed to demonstrate a significant effect on gut mucosal protein synthesis in the postabsorptive state and during feeding.     

Resistance training alters the response of fed state mixed muscle protein synthesis in young men

Ten healthy young men (21.0 +/- 1.5 yr, 1.79 +/- 0.1 m, 82.7 +/- 14.7 kg, means +/- SD) participated in 8 wk of intense unilateral resistance training (knee extension exercise) such that one leg was trained (T) and the other acted as an untrained (UT) control. After the 8 wk of unilateral training, infusions of L-[ring-d(5)]phenylalanine, L-[ring-(13)C(6)]phenylalanine, and d(3)-alpha-ketoisocaproic acid were used to measure mixed muscle protein synthesis in the T and UT legs by the direct incorporation method [fractional synthetic rate (FSR)]. Protein synthesis was determined at rest as well as 4 h and 28 h after an acute bout of resistance exercise performed at the same intensity relative to the gain in single repetition maximum before and after training. Training increased mean muscle fiber cross-sectional area only in the T leg (type I: 16 +/- 10%; type II: 20 +/- 19%, P < 0.05). Acute resistance exercise increased muscle protein FSR in both legs at 4 h (T: 162 +/- 76%; UT: 108 +/- 62%, P < 0.01 vs. rest) with the increase in the T leg being significantly higher than in the UT leg at this time (P < 0.01). At 28 h postexercise, FSR in the T leg had returned to resting levels; however, the rate of protein synthesis in the UT leg remained elevated above resting (70 +/- 49%, P < 0.01). We conclude that resistance training attenuates the protein synthetic response to acute resistance exercise, despite higher initial increases in FSR, by shortening the duration for which protein synthesis is elevated.     

Effects of fatty acids on hepatic amino acid catabolism and fibrinogen synthesis in young healthy volunteers

Increased synthesis rate of fibrinogen, an independent risk factor for cardiovascular disease, was recently reported in obese insulin-resistant female adolescents with chronic elevated nonesterified fatty acids (NEFA). It is unknown whether a short-term change of NEFA concentrations controls hepatic fibrinogen synthesis. Therefore, 10 healthy male volunteers (24.5 +/- 3.3 yr, body mass index 23.5 +/- 2.9 kg/m2) were investigated in random order under basal and elevated NEFA for 8 h. Leucine metabolism, the fractional synthesis rates (FSR) of plasma fibrinogen, and endogenous urea production rates were measured during primed, continuous infusion of [1-13C]leucine and [15N2]urea, respectively. Plasma alpha-[13C]ketoisocaproic acid and [15N2]urea enrichment values were measured with GC-MS. Plasma fibrinogen was isolated with the beta-alanine method, and fibrinogen-related [13C]leucine enrichment was analyzed by GC-CIRMS. Lipofundin infusion and subcutaneous heparin tripled NEFA and triglycerides in the tests. Plasma glucose, circulating insulin, human C-peptide, and plasma glucagon were not changed by the study procedure. Fibrinogen FSR were significantly lower in tests with NEFA elevation (18.44 +/- 4.67%) than in control tests (21.48 +/- 4.32%; P < 0.05). Plasma fibrinogen concentrations measured were not significantly different (NEFA test subjects: 1.85 +/- 0.33, controls: 1.97 +/- 0.54 g/l). Parameters of leucine metabolism, such as leucine rate of appearance, leucine oxidation, and nonoxidative leucine disposal, were not influenced by NEFA elevation, and endogenous urea production remained unchanged. NEFA contributes to short-term regulation of fibrinogen FSR in healthy volunteers under unchanged hormonal status, leucine metabolism, and overall amino acid catabolism. Its contribution might be of relevance at least after fat-rich meals, counteracting by reduction of FSR the blood viscosity increase implied by hyperlipidemia.     

Protein synthesis rates of human PBMC and PMN can be determined simultaneously in vivo by using small blood samples

Immune cell functions can be evaluated in vivo by measuring their specific protein fractional synthesis rates (FSR). Using stable isotope dilution techniques, we describe a new method allowing simultaneous in vivo assessment of FSR in two leukocyte populations in healthy human subjects, using small blood samples. Peripheral blood mononuclear cell (PBMC) and polymorphonuclear neutrophil (PMN) FSR were measured during primed continuous intravenous infusion of L-[1-¹³C]leucine. Immune cells from 6 ml of whole blood were isolated by density gradient centrifugation. In a first study, we calculated the FSR using plasma [¹³C]leucine or -[¹³C]ketoisocaproate (KIC) enrichments as precursor pools. In a second study, we compared protein FSR in leukocytes, using enrichments of either intracellular or plasma free [¹³C]leucine as immediate precursor pools. The present approach showed a steady-state enrichment of plasma and circulating immune cell free [¹³C]leucine precursor pools. The linearity of labeled amino acid incorporation rate within mixed PBMC and PMN proteins also was verified. Postabsorptive protein FSR was 4.09 ± 0.39%/day in PBMC and 1.44 ± 0.08%/day in PMN when plasma [¹³C]KIC was the precursor pool. The difference between PBMC and PMN FSR was statistically significant, whatever the precursor pool used, suggesting large differences in their synthetic activities and functions. Use of the plasma [¹³C]KIC pool led to an underestimation of leukocyte FSR compared with the intracellular pool (PBMC: 6.04 ± 0.94%/day; PMN: 2.98 ± 0.30%/day). Hence, the intracellular free amino acid pool must be used as precursor to obtain reliable results. In conclusion, it is possible to assess immune cell metabolism in vivo in humans by using small blood samples to directly indicate their metabolic activity in various clinical situations and in response to regulating factors. peripheral blood mononuclear cells; polymorphonuclear neutrophils; protein metabolism; stable isotopes; leucine     

Acute responses of muscle protein metabolism to reduced blood flow reflect metabolic priorities for homeostasis

The present experiment was designed to measure the synthetic and breakdown rates of muscle protein in the hindlimb of rabbits with or without clamping the femoral artery. l-[ring-(13)C(6)]phenylalanine was infused as a tracer for measurement of muscle protein kinetics by means of an arteriovenous model, tracer incorporation, and tracee release methods. The ultrasonic flowmeter, dye dilution, and microsphere methods were used to determine the flow rates in the femoral artery, in the leg, and in muscle capillary, respectively. The femoral artery flow accounted for 65% of leg flow. A 50% reduction in the femoral artery flow reduced leg flow by 28% and nutritive flow by 26%, which did not change protein synthetic or breakdown rate in leg muscle. Full clamp of the femoral artery reduced leg flow by 42% and nutritive flow by 59%, which decreased (P < 0.05) both the fractional synthetic rate from 0.19 +/- 0.05 to 0.14 +/- 0.03%/day and fractional breakdown rate from 0.28 +/- 0.07 to 0.23 +/- 0.09%/day of muscle protein. Neither the partial nor full clamp reduced (P = 0.27-0.39) the intracellular phenylalanine concentration or net protein balance in leg muscle. We conclude that the flow threshold to cause a fall of protein turnover rate in leg muscle was a reduction of 30-40% of the leg flow. The acute responses of muscle protein kinetics to the reductions in blood flow reflected the metabolic priorities to maintain muscle homeostasis. These findings cannot be extrapolated to more chronic conditions without experimental validation.     

Pioglitazone improves insulin sensitivity through reduction in muscle lipid and redistribution of lipid into adipose tissue

Hemorrhagic coagulopathy is involved in the morbidity and mortality of trauma patients. Nonetheless, many aspects of the mechanisms underlying this disorder are poorly understood. We have therefore investigated changes in fibrinogen metabolism and coagulation function after a moderate hemorrhagic shock, using a new stable isotope approach. Twelve pigs were randomly divided into the control (C) and hemorrhage (H) groups. Hemorrhage was induced by bleeding 35% total blood volume over a 30-min period. A primed constant infusion of [1-(13)C]phenylalanine (Phe), d5-phenylalanine, and alpha-[1-(13)C]-ketoisocaproate (KIC) was given to quantify fibrinogen synthesis and breakdown, together with measurements of circulating liver enzyme activities and coagulation function. Mean arterial pressure was decreased by hemorrhage from 89 +/- 4 mmHg in C to 47 +/- 4 mmHg in H (P < 0.05), followed by a rebound to 68 +/- 5 mmHg afterward. Fibrinogen fractional synthesis rate increased from 2.7 +/- 0.2%/h in C to 4.2 +/- 0.4%/h in H by Phe (P < 0.05) and from 3.1 +/- 0.4%/h in C to 4.4 +/- 0.5%/h in H by KIC (P < 0.05). Fibrinogen fractional breakdown rate increased from 3.6 +/- 1.0%/h in C to 12.9 +/- 1.8%/h in H (P < 0.05). The absolute breakdown rate accelerated from 3.0 +/- 0.4 mg x kg(-1) x h(-1) in C to 5.4 +/- 0.6 mg x kg(-1) x h(-1) in H (P < 0.05), but the absolute synthesis rate remained unchanged. These metabolic changes were accompanied by a reduction in blood clotting time to 92.7 +/- 1.6% of the baseline value by hemorrhage (P < 0.05). No changes were found in liver enzyme activities. We conclude that the observed changes in coagulation after hemorrhagic shock are mechanistically related to the acute acceleration of fibrinogen degradation.     

Whole body protein kinetics in women: effect of pregnancy and IDDM during anabolic stimulation

The effects of pregnancy and type 1 diabetes [insulin-dependent diabetes mellitus (IDDM)] on protein metabolism are still uncertain. Therefore, six normal and five IDDM women were studied during and after pregnancy, using [(13)C]leucine and [(2)H(5)]phenylalanine with a hyperinsulinemic-euglycemic clamp and amino acid infusion. Fasting total plasma amino acids were lower in pregnancy in normal but not IDDM women (2,631 +/- 427 vs. 2,057 +/- 471 and 2,523 +/- 430 vs. 2,500 +/- 440 micromol/l, respectively). Whole body protein breakdown (leucine) increased in pregnancy [change in normal (delta N) and IDDM women (delta D) 0.59 +/- 0.40 and 0.48 +/- 0.26 g. kg(-1). day(-1), both P < 0.001], whereas reductions in protein breakdown due to insulin/amino acids (delta N -0.57 +/- 0.19, delta D -0.58 +/- 0.20 g. kg(-1). day(-1), both P < 0.001) were unaffected by pregnancy. Protein breakdown in IDDM women was not higher than normal, and neither pregnancy nor type 1 diabetes altered the insulin sensitivity of amino acid turnover. Nonoxidized leucine disposal (protein synthesis) increased in pregnancy (delta N 0.67 +/- 0.45, delta D 0.64 +/- 0.34 g. kg(-1). day(-1), both P < 0.001). Pregnancy reduced the response of phenylalanine hydroxylation to insulin/amino acids in both groups (delta N -1.14 +/- 0.74, delta D -1. 12 +/- 0.77 g. kg(-1). day(-1), both P < 0.05). These alterations may enable amino acid conservation for protein synthesis and accretion in late pregnancy. Well-controlled type 1 diabetes caused no abnormalities in the regulation of basal or stimulated protein metabolism.     

Methysergide reduces nonnutritive blood flow in normal and scalded skin

Methysergide is a serotonin antagonist and has been demonstrated to reduce wound blood flow and edema formation. We have determined the effect of methysergide on protein kinetics in normal and scalded skin of anesthetized rabbits. L-[ring-(13)C(6)]- or L-[ring-(2)H(5)]phenylalanine was used to reflect skin protein kinetics by use of an ear model, and L-[1-(13)C]leucine was used to reflect whole body protein kinetics. The results were that infusion of methysergide (2-3 mg. kg(-1). h(-1)) reduced the blood flow rate in normal skin by 50% without changing skin or whole body protein kinetics. After scald injury on the ear, administration of methysergide for 48 h reduced the weight of scalded ears (43 +/- 4 vs. 30 +/- 5 g, P < 0.01) and ear blood flow rate (42.6 +/- 4.9 vs. 5.8 +/- 1.0 ml. 100 g(-1). min(-1), P < 0.0001) and did not change wound protein kinetics. Methysergide reduced arteriovenous shunting and maintained inward phenylalanine transport from the blood to the skin pool. Using the microsphere technique, we found that the infusion of methysergide decreased blood perfusion by 33-36% in both normal and scalded ear skin. We conclude that methysergide administration reduces nonnutritive, as opposed to nutritive, blood flow in normal and scalded skin.     

Amino acid ingestion improves muscle protein synthesis in the young and elderly

We recently demonstrated that muscle protein synthesis was stimulated to a similar extent in young and elderly subjects during a 3-h amino acid infusion. We sought to determine if a more practical bolus oral ingestion would also produce a similar response in young (34 +/- 4 yr) and elderly (67 +/- 2 yr) individuals. Arteriovenous blood samples and muscle biopsies were obtained during a primed (2.0 micromol/kg) constant infusion (0.05 micromol.kg(-1).min(-1)) of L-[ring-2H5]phenylalanine. Muscle protein kinetics and mixed muscle fractional synthetic rate (FSR) were calculated before and after the bolus ingestion of 15 g of essential amino acids (EAA) in young (n = 6) and elderly (n = 7) subjects. After EAA ingestion, the rate of increase in femoral artery phenylalanine concentration was slower in elderly subjects but remained elevated for a longer period. EAA ingestion increased FSR in both age groups by approximately 0.04%/h (P < 0.05). However, muscle intracellular (IC) phenylalanine concentration remained significantly higher in elderly subjects at the completion of the study (young: 115.6 +/- 5.4 nmol/ml; elderly: 150.2 +/- 19.4 nmol/ml). Correction for the free phenylalanine retained in the muscle IC pool resulted in similar net phenylalanine uptake values in the young and elderly. EAA ingestion increased plasma insulin levels in young (6.1 +/- 1.2 to 21.3 +/- 3.1 microIU/ml) but not in elderly subjects (3.0 +/- 0.6 to 4.3 +/- 0.4 microIU/ml). Despite differences in the time course of plasma phenylalanine kinetics and a greater residual IC phenylalanine concentration, amino acid supplementation acutely stimulated muscle protein synthesis in both young and elderly individuals.     

Coingestion of carbohydrate with protein does not further augment postexercise muscle protein synthesis

The present study was designed to assess the impact of coingestion of various amounts of carbohydrate combined with an ample amount of protein intake on postexercise muscle protein synthesis rates. Ten healthy, fit men (20 +/- 0.3 yr) were randomly assigned to three crossover experiments. After 60 min of resistance exercise, subjects consumed 0.3 g x kg(-1) x h(-1) protein hydrolysate with 0, 0.15, or 0.6 g x kg(-1) x h(-1) carbohydrate during a 6-h recovery period (PRO, PRO + LCHO, and PRO + HCHO, respectively). Primed, continuous infusions with L-[ring-(13)C(6)]phenylalanine, L-[ring-(2)H(2)]tyrosine, and [6,6-(2)H(2)]glucose were applied, and blood and muscle samples were collected to assess whole body protein turnover and glucose kinetics as well as protein fractional synthesis rate (FSR) in the vastus lateralis muscle over 6 h of postexercise recovery. Plasma insulin responses were significantly greater in PRO + HCHO compared with PRO + LCHO and PRO (18.4 +/- 2.9 vs. 3.7 +/- 0.5 and 1.5 +/- 0.2 U.6 h(-1) x l(-1), respectively, P < 0.001). Plasma glucose rate of appearance (R(a)) and disappearance (R(d)) increased over time in PRO + HCHO and PRO + LCHO, but not in PRO. Plasma glucose R(a) and R(d) were substantially greater in PRO + HCHO vs. both PRO and PRO + LCHO (P < 0.01). Whole body protein breakdown, synthesis, and oxidation rates, as well as whole body protein balance, did not differ between experiments. Mixed muscle protein FSR did not differ between treatments and averaged 0.10 +/- 0.01, 0.10 +/- 0.01, and 0.11 +/- 0.01%/h in the PRO, PRO + LCHO, and PRO + HCHO experiments, respectively. In conclusion, coingestion of carbohydrate during recovery does not further stimulate postexercise muscle protein synthesis when ample protein is ingested.     

Mechanism of insulin's anabolic effect on muscle: measurements of muscle protein synthesis and breakdown using aminoacyl-tRNA and other surrogate measures

Despite being an anabolic hormone in skeletal muscle, insulin's anticatabolic mechanism in humans remains controversial, with contradictory reports showing either stimulation of protein synthesis (PS) or inhibition of protein breakdown (PB) by insulin. Earlier measurements of muscle PS and PB in humans have relied on different surrogate measures of aminoacyl-tRNA and intracellular pools. We report that insulin's effect on muscle protein turnover using aminoacyl-tRNA as the precursor of PS and PB is calculated by mass balance of tracee amino acid (AA). We compared the results calculated from various surrogate measures. To determine the physiological role of insulin on muscle protein metabolism, we infused tracers of leucine and phenylalanine into 18 healthy subjects, and after 3 h, 10 subjects received a 4-h femoral arterial infusion of insulin (0.125 mUxkg(-1)xmin(-1)), while eight subjects continued with saline. Tracer-to-tracee ratios of leucine, phenylalanine, and ketoisocaproate were measured in the arterial and venous plasma, muscle tissue fluid, and AA-tRNA to calculate muscle PB and PS. Insulin infusion, unlike saline, significantly reduced the efflux of leucine and phenylalanine from muscle bed, based on various surrogate measures which agreed with those based on leucyl-tRNA (-28%), indicating a reduction in muscle PB (P < 0.02) without any significant effect on muscle PS. In conclusion, using AA-tRNA as the precursor pool, it is demonstrated that, in healthy humans in the postabsorptive state, insulin does not stimulate muscle protein synthesis and confirmed that insulin achieves muscle protein anabolism by inhibition of muscle protein breakdown.     

Impact of protein coingestion on muscle protein synthesis during continuous endurance type exercise

This study investigates the impact of protein coingestion with carbohydrate on muscle protein synthesis during endurance type exercise. Twelve healthy male cyclists were studied during 2 h of fasted rest followed by 2 h of continuous cycling at 55% W(max). During exercise, subjects received either 1.0 g·kg(-1)·h(-1) carbohydrate (CHO) or 0.8 g·kg(-1)·h(-1) carbohydrate with 0.2 g·kg(-1)·h(-1) protein hydrolysate (CHO+PRO). Continuous intravenous infusions with l-[ring-(13)C(6)]phenylalanine and l-[ring-(2)H(2)]tyrosine were applied, and blood and muscle biopsies were collected to assess whole body protein turnover and muscle protein synthesis rates at rest and during exercise conditions. Protein coingestion stimulated whole body protein synthesis and oxidation rates during exercise by 22 ± 3 and 70 ± 17%, respectively (P < 0.01). Whole body protein breakdown rates did not differ between experiments. As a consequence, whole body net protein balance was slightly negative in CHO and positive in the CHO+PRO treatment (-4.9 ± 0.3 vs. 8.0 ± 0.3 μmol Phe·kg(-1)·h(-1), respectively, P < 0.01). Mixed muscle protein fractional synthetic rates (FSR) were higher during exercise compared with resting conditions (0.058 ± 0.006 vs. 0.035 ± 0.006%/h in CHO and 0.070 ± 0.011 vs. 0.038 ± 0.005%/h in the CHO+PRO treatment, respectively, P < 0.05). FSR during exercise did not differ between experiments (P = 0.46). We conclude that muscle protein synthesis is stimulated during continuous endurance type exercise activities when carbohydrate with or without protein is ingested. Protein coingestion does not further increase muscle protein synthesis rates during continuous endurance type exercise.     

Acute response of net muscle protein balance reflects 24-h balance after exercise and amino acid ingestion

The purpose of this study was to determine if the acute anabolic muscle response to resistance exercise and essential amino acids (EAA) reflects the response over 24 h. Seven subjects participated in the following two 24-h studies: 1) resting (REST) and 2) rest plus resistance exercise and consumption of EAA (ES). Net balance (NB) across the leg was determined for four amino acids. [(13)C(6)]phenylalanine was infused to determine mixed muscle fractional synthetic rate (FSR). Twenty-four-hour FSR was significantly greater for ES than for REST (P = 0.003). Exchange of phenylalanine across the leg was -194 +/- 74 (SE) mg for ES and -371 +/- 88 mg for REST (P = 0.07) over 24 h and 229 +/- 42 mg (ES) and 28 +/- 15 mg (REST; P < 0.01) over 3 h corresponding to exercise and EAA consumption for ES. The difference in phenylalanine exchange between REST and ES was not different for measurements over 24 and 3 h. Increases in NB during ES were primarily the result of increases in protein synthesis. Results for other amino acids were similar. The acute anabolic response of muscle to EAA intake and exercise is additive to the response at rest and thus reflects the 24-h response.