Measurement of skin protein breakdown in a rat model

Whereas skin protein synthesis can be measured with different approaches, no method potentially applicable in humans is available for measurement of skin protein breakdown. To that end, we measured mixed skin fractional protein breakdown (FBR) in a rat model by use of a stable isotope method (tracee release method) originally developed to measure muscle protein breakdown. Skin mixed protein and collagen fractional synthesis rates (FSR) were also measured. A primed continuous infusion of L-[ring-(2)H(5)]phenylalanine and alpha-[5,5,5-(2)H(3)]ketoisocaproate (KIC) was given for 6 h. Arterial and skin phenylalanine and leucine free enrichments were measured at plateau (5-6 h) and during the decay that followed after the infusion was stopped. Skin FBR (%/h) was 0.260 +/- 0.011 with phenylalanine and 0.201 +/- 0.032 with KIC/leucine [P = not significant (NS)]. Mixed skin FSR (%/h) was 0.169 +/- 0.055 with phenylalanine and 0.146 +/- 0.020 with KIC/leucine (P = NS). Collagen FSR was 0.124 +/- 0.023%/h (P = NS vs. mixed protein FSR). The tracee release method is a sensitive method for measurement of skin protein breakdown; however, given the high intersubject variability of FSR, the calculation of skin net balance is not advisable.     

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.     

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.     

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.     

Sequential muscle biopsies during a 6-h tracer infusion do not affect human mixed muscle protein synthesis and muscle phenylalanine kinetics

Stable isotope tracer experiments of human muscle amino acid and protein kinetics often involve a sequential design, with the same subject studied at baseline and during an intervention. However, prolonged fasting and sequential muscle biopsies from the same area could theoretically affect muscle protein metabolism. The purpose of this study was to determine if sequential muscle biopsies and extended fasting significantly affect parameters of muscle protein and amino acid kinetics in six human subjects. After a 12-h overnight fast, a primed continuous infusion of L-[ring-(2)H(5)]phenylalanine was started. After 120 min, we took the first of a series of five hourly muscle biopsies from the same vastus lateralis to measure mixed muscle protein fractional synthetic rate. Furthermore, between 150-180, 210-240, and 330-360 min, we measured leg phenylalanine kinetics using the two-pool and the three-pool arteriovenous balance models. Tracer enrichments were at steady state, and muscle protein FSR and phenylalanine kinetics did not change throughout the experiment (P=not significant). We conclude that a 6-h tracer infusion during extended fasting (up to 18 h) with five sequential muscle biopsies from the same muscle do not affect basal mixed muscle protein synthesis and muscle phenylalanine kinetics in human subjects. Thus, when using a sequential study design over this period of time, it is unnecessary to include a saline only control group to account for these variables.     

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.     

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.     

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.     

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.     

Short-term insulin and nutritional energy provision do not stimulate muscle protein synthesis if blood amino acid availability decreases

Muscle protein synthesis requires energy and amino acids to proceed and can be stimulated by insulin under certain circumstances. We hypothesized that short-term provision of insulin and nutritional energy would stimulate muscle protein synthesis in healthy subjects only if amino acid availability did not decrease. Using stable isotope techniques, we compared the effects on muscle phenylalanine kinetics across the leg of an amino acid-lowering, high-energy (HE, n = 6, 162 +/- 20 kcal/h) hyperglycemic hyperlipidemic hyperinsulinemic clamp with systemic insulin infusion to a low-energy (LE, n = 6, 35 +/- 3 kcal/h, P < 0.05 vs. HE) euglycemic hyperinsulinemic clamp with local insulin infusion in the femoral artery. Basal blood phenylalanine concentrations and phenylalanine net balance, muscle protein breakdown, and synthesis (nmol.min(-1).100 g leg muscle(-1)) were not different between groups. During insulin infusion, femoral insulinemia increased to a similar extent between groups and blood phenylalanine concentration decreased 27 +/- 3% in the HE group but only 9 +/- 2% in the LE group (P < 0.01 HE vs. LE). Phenylalanine net balance increased in both groups, but the change was greater (P < 0.05) in the LE group. Muscle protein breakdown decreased in the HE group (58 +/- 12 to 35 +/- 7 nmol.min(-1).100 g leg muscle(-1)) and did not change in the LE group. Muscle protein synthesis was unchanged in the HE group (39 +/- 6 to 30 +/- 7 nmol.min(-1).100 g leg muscle(-1)) and increased (P < 0.05) in the LE group (41 +/- 9 to 114 +/- 26 nmol.min(-1).100 g leg muscle(-1)). We conclude that amino acid availability is an important factor in the regulation of muscle protein synthesis in response to insulin, as decreased blood amino acid concentrations override the positive effect of insulin on muscle protein synthesis even if excess energy is provided.     

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.     

Effect of insulin on human skeletal muscle protein synthesis is modulated by insulin-induced changes in muscle blood flow and amino acid availability

Insulin promotes muscle anabolism, but it is still unclear whether it stimulates muscle protein synthesis in humans. We hypothesized that insulin can increase muscle protein synthesis only if it increases muscle amino acid availability. We measured muscle protein and amino acid metabolism using stable-isotope methodologies in 19 young healthy subjects at baseline and during insulin infusion in one leg at low (LD, 0.05), intermediate (ID, 0.15), or high (HD, 0.30 mUxmin(-1)x100 ml(-1)) doses. Insulin was infused locally to induce muscle hyperinsulinemia within the physiological range while minimizing the systemic effects. Protein and amino acid kinetics across the leg were assessed using stable isotopes and muscle biopsies. The LD did not affect phenylalanine delivery to the muscle (-9 +/- 18% change over baseline), muscle protein synthesis (16 +/- 26%), breakdown, or net balance. The ID increased (P < 0.05) phenylalanine delivery (+63 +/- 38%), muscle protein synthesis (+157 +/- 54%), and net protein balance, with no change in breakdown. The HD did not change phenylalanine delivery (+12 +/- 11%) or muscle protein synthesis (+9 +/- 19%), and reduced muscle protein breakdown (-17 +/- 15%), thus improving net muscle protein balance but to a lesser degree than the ID. Changes in muscle protein synthesis were strongly associated with changes in muscle blood flow and phenylalanine delivery and availability. In conclusion, physiological hyperinsulinemia promotes muscle protein synthesis as long as it concomitantly increases muscle blood flow, amino acid delivery and availability.     

Exogenous amino acids stimulate human muscle anabolism without interfering with the response to mixed meal ingestion

We sought to determine whether ingestion of a between-meal supplement containing 30 g of carbohydrate and 15 g of essential amino acids (CAA) altered the metabolic response to a nutritionally mixed meal in healthy, recreationally active male volunteers. A control group (CON; n = 6, 38 +/- 8 yr, 86 +/- 10 kg, 179 +/- 3 cm) received a liquid mixed meal [protein, 23.4 +/- 1.0 g (essential amino acids, 14.7 +/- 0.7 g); carbohydrate, 126.6 +/- 4.0 g; fat, 30.3 +/- 2.8 g] every 5 h (0830, 1330, 1830). The experimental group (SUP; n = 7, 36 +/- 10 yr, 87 +/- 12 kg, 180 +/- 3 cm) consumed the same meals but, in addition, were given CAA supplements (1100, 1600, 2100). Net phenylalanine balance (NB) and fractional synthetic rate (FSR) were calculated during a 16-h primed constant infusion of L-[ring-2H5]phenylalanine. Ingestion of a combination of CAA supplements and meals resulted in a greater mixed muscle FSR than ingestion of the meals alone (SUP, 0.099 +/- 0.008; CON, 0.076 +/- 0.005%/h; P < 0.05). Both groups experienced an improvement in NB after the morning (SUP, -2.2 +/- 3.3; CON, -1.5 +/- 3.5 nmol x min(-1) x 100 ml leg volume(-1)) and evening meals (SUP, -9.7 +/- 4.3; CON, -6.7 +/- 4.1 nmol x min(-1) x 100 ml leg volume(-1)). NB after CAA ingestion was significantly greater than after the meals, with values of 40.2 +/- 8.5 nmol x min(-1) x 100 ml leg volume(-1). These data indicate that CAA supplementation produces a greater anabolic effect than ingestion of intact protein but does not interfere with the normal metabolic response to a meal.     

Protein turnover and amino acid transport kinetics in end-stage renal disease

Protein and amino acid metabolism is abnormal in end-stage renal disease (ESRD). Protein turnover is influenced by transmembrane amino acid transport. The effect of ESRD and hemodialysis (HD) on intracellular amino acid transport kinetics is unknown. We studied intracellular amino acid transport kinetics and protein turnover by use of stable isotopes of phenylalanine, leucine, lysine, alanine, and glutamine before and during HD in six ESRD patients. Data obtained from amino acid concentrations and enrichment in the artery, vein, and muscle compartments were used to calculate intracellular amino acid transport and muscle protein synthesis and catabolism. Fractional muscle protein synthesis (FSR) was estimated by the precursor product approach. Despite a significant decrease in the plasma concentrations of amino acids in the artery and vein during HD, the intracellular concentrations remained stable. Outward transport of the amino acids was significantly higher than the inward transport during HD. FSR increased during HD (0.0521 +/- 0.0043 vs. 0.0772 +/- 0.0055%/h, P < 0.01). Results derived from compartmental modeling indicated that both protein synthesis (118.3 +/- 20.6 vs. 146.5 +/- 20.6 nmol.min-1.100 ml leg-1, P < 0.01) and catabolism (119.8 +/- 18.0 vs. 174.0 +/- 14.2 nmol.min-1.100 ml leg-1, P < 0.01) increased during HD. However, the intradialytic increase in catabolism exceeded that of synthesis (57.8 +/- 13.8 vs. 28.0 +/- 8.5%, P < 0.05). Thus HD alters amino acid transport kinetics and increases protein turnover, with net increase in protein catabolism.     

No effect of creatine supplementation on human myofibrillar and sarcoplasmic protein synthesis after resistance exercise

Muscle hypertrophy during resistance training is reportedly increased by creatine supplementation. Having previously failed to find an anabolic effect on muscle protein turnover at rest, either fed or fasted, we have now examined the possibility of a stimulatory effect of creatine in conjunction with acute resistance exercise. Seven healthy men (body mass index, 23 +/- 2 kg/m2, 21 +/- 1 yr, means +/- SE) performed 20 x 10 repetitions of leg extension-flexion at 75% one-repetition maximum in one leg, on two occasions, 4 wk apart, before and after ingesting 21 g/day creatine for 5 days. The subjects ate approximately 21 g maltodextrin + 6 g protein/h for 3 h postexercise. We measured incorporation of [1-13C]leucine into quadriceps muscle proteins in the rested and exercised legs. Leg protein breakdown (as dilution of [2H5]phenylalanine) was also assessed in the exercised and rested leg postexercise. Creatine supplementation increased muscle total creatine by approximately 21% (P < 0.01). Exercise increased the synthetic rates of myofibrillar and sarcoplasmic proteins by two- to threefold (P < 0.05), and leg phenylalanine balance became more positive, but creatine was without any anabolic effect.     

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.     

Acute effect of insulin on nitric oxide synthesis in humans: a precursor-product isotopic study

Nitric oxide (NO) is a key regulatory molecule with wide vascular, cellular, and metabolic effects. Insulin affects NO synthesis in vitro. No data exist on the acute effect of insulin on NO kinetics in vivo. By employing a precursor-product tracer method in humans, we have directly estimated the acute effect of insulin on intravascular NO(x) (i.e., the NO oxidation products) fractional (FSR) and absolute (ASR) synthesis rates in vivo. Nine healthy male volunteers were infused iv with L-[(15)N(2)-guanidino]arginine ([(15)N(2)]arginine) for 6 h. Timed measurements of (15)NO(x) and [(15)N(2)]arginine enrichments in whole blood were performed in the first 3 h in the fasting state and then following a 3-h euglycemic-hyperinsulinemic clamp (with plasma insulin raised to approximately 1,000 pmol/l). In the last 60 min of each experimental period, at approximately steady-state arginine enrichment, a linear increase of (15)NO(x) enrichment (mean r = 0.9) was detected in both experimental periods. In the fasting state, NO(x) FSR was 27.4 +/- 4.3%/day, whereas ASR was 0.97 +/- 0.36 mmol/day, accounting for 0.69 +/- 0.27% of arginine flux. Following hyperinsulinemia, both FSR and ASR of NO(x) increased (FSR by approximately 50%, to 42.4 +/- 6.7%/day, P < 0.005; ASR by approximately 25%, to 1.22 +/- 0.41 mmol/day, P = 0.002), despite a approximately 20-30% decrease of arginine flux and concentration. The fraction of arginine flux used for NO(x) synthesis was doubled, to 1.13 +/- 0.35% (P < 0.003). In conclusion, whole body NO(x) synthesis can be directly measured over a short observation time with stable isotope methods in humans. Insulin acutely stimulates NO(x) synthesis from arginine.     

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.     

Contributions of working muscle to whole body lipid metabolism are altered by exercise intensity and training

To evaluate the contribution of working muscle to whole body lipid oxidation, we examined the effects of exercise intensity and endurance training (9 wk, 5 days/wk, 1 h, 75% Vo(2 peak)) on whole body and leg free fatty acid (FFA) kinetics in eight male subjects (26 +/- 1 yr, means +/- SE). Two pretraining trials [45 and 65% Vo(2 max) (45UT, 65UT)] and two posttraining trials [65% of pretraining Vo(2 peak) (ABT), and 65% of posttraining Vo(2 peak) (RLT)] were performed using [1-(13)C]palmitate infusion and femoral arteriovenous sampling. Training increased Vo(2 peak) by 15% (45.2 +/- 1.2 to 52.0 +/- 1.8 ml.kg(-1).min(-1), P < 0.05). Muscle FFA fractional extraction was lower during exercise (EX) compared with rest regardless of workload or training status ( approximately 20 vs. 48%, P < 0.05). Two-leg net FFA balance increased from net release at rest ( approximately -36 micromol/min) to net uptake during EX for 45UT (179 +/- 75), ABT (236 +/- 63), and RLT (136 +/- 110) (P < 0.05), but not 65UT (51 +/- 127). Leg FFA tracer measured uptake was higher during EX than rest for all trials and greater during posttraining in RLT (716 +/- 173 micromol/min) compared with pretraining (45UT 450 +/- 80, 65UT 461 +/- 72, P < 0.05). Leg muscle lipid oxidation increased with training in ABT (730 +/- 163 micromol/min) vs. 65UT (187 +/- 94, P < 0.05). Leg muscle lipid oxidation represented approximately 62 and 30% of whole body lipid oxidation at lower and higher relative intensities, respectively. In summary, training can increase working muscle tracer measured FFA uptake and lipid oxidation for a given power output, but both before and after training the association between whole body and leg lipid metabolism is reduced as exercise intensity increases.     

Effect of carbohydrate intake on net muscle protein synthesis during recovery from resistance exercise.

The purpose of this study was to determine the effect of ingestion of 100 g of carbohydrates on net muscle protein balance (protein synthesis minus protein breakdown) after resistance exercise. Two groups of eight subjects performed a resistance exercise bout (10 sets of 8 repetitions of leg presses at 80% of 1-repetition maximum) before they rested in bed for 4 h. One group (CHO) received a drink consisting of 100 g of carbohydrates 1 h postexercise. The other group (Pla) received a noncaloric placebo drink. Leg amino acid metabolism was determined by infusion of 2H5- or 13C6-labeled phenylalanine, sampling from femoral artery and vein, and muscle biopsies from vastus lateralis. Drink intake did not affect arterial insulin concentration in Pla, whereas insulin increased several times after the drink in CHO (P < 0.05 vs. Pla). Arterial phenylalanine concentration fell slightly after the drink in CHO. Net muscle protein balance between synthesis and breakdown did not change in Pla, whereas it improved in CHO from -17 +/- 3 nmol.ml(-1).100 ml leg(-1) before drink to an average of -4 +/- 4 and 0 +/- 3 nmol.ml(-1).100 ml leg(-1) during the second and third hour after the drink, respectively (P < 0.05 vs. Pla during last hour). The improved net balance in CHO was due primarily to a progressive decrease in muscle protein breakdown. We conclude that ingestion of carbohydrates improved net leg protein balance after resistance exercise. However, the effect was minor and delayed compared with the previously reported effect of ingestion of amino acids.