Amino acid absorption and subsequent muscle protein accretion following graded intakes of whey protein in elderly men

Whey protein ingestion has been shown to effectively stimulate postprandial muscle protein accretion in older adults. However, the impact of the amount of whey protein ingested on protein digestion and absorption kinetics, whole body protein balance, and postprandial muscle protein accretion remains to be established. We aimed to fill this gap by including 33 healthy, older men (73 ± 2 yr) who were randomly assigned to ingest 10, 20, or 35 g of intrinsically l-[1-13C]phenylalanine-labeled whey protein (n = 11/treatment). Ingestion of labeled whey protein was combined with continuous intravenous l-[ring-2H?]phenylalanine and l-[ring-2H?]tyrosine infusion to assess the metabolic fate of whey protein-derived amino acids. Dietary protein digestion and absorption rapidly increased following ingestion of 10, 20, and 35 g whey protein, with the lowest and highest (peak) values observed following 10 and 35 g, respectively (P < 0.05). Whole body net protein balance was positive in all groups (19 ± 1, 37 ± 2, and 58 ± 2 μmol/kg), with the lowest and highest values observed following ingestion of 10 and 35 g, respectively (P < 0.05). Postprandial muscle protein accretion, assessed by l-[1-13C]phenylalanine incorporation in muscle protein, was higher following ingestion of 35 g when compared with 10 (P < 0.01) or 20 (P < 0.05) g. We conclude that ingestion of 35 g whey protein results in greater amino acid absorption and subsequent stimulation of de novo muscle protein synthesis compared with the ingestion of 10 or 20 g whey protein in healthy, older men.     

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.     

Protein coingestion stimulates muscle protein synthesis during resistance-type exercise

In contrast to the effect of nutritional intervention on postexercise muscle protein synthesis, little is known about the potential to modulate protein synthesis during exercise. This study investigates the effect of protein coingestion with carbohydrate on muscle protein synthesis during resistance-type exercise. Ten healthy males were studied in the evening after they consumed a standardized diet throughout the day. Subjects participated in two experiments in which they ingested either carbohydrate or carbohydrate with protein during a 2-h resistance exercise session. Subjects received a bolus of test drink before and every 15 min during exercise, providing 0.15 g x kg(-1) x h(-1) carbohydrate with (CHO + PRO) or without (CHO) 0.15 g x kg(-1) x h(-1) protein hydrolysate. 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 and muscle protein synthesis rates during exercise. Protein coingestion lowered whole body protein breakdown rates by 8.4 +/- 3.6% (P = 0.066), compared with the ingestion of carbohydrate only, and augmented protein oxidation and synthesis rates by 77 +/- 17 and 33 +/- 3%, respectively (P < 0.01). As a consequence, whole body net protein balance was negative in CHO, whereas a positive net balance was achieved after the CHO + PRO treatment (-4.4 +/- 0.3 vs. 16.3 +/- 0.4 micromol phenylalanine x kg(-1) x h(-1), respectively; P < 0.01). In accordance, mixed muscle protein fractional synthetic rate was 49 +/- 22% higher after protein coingestion (0.088 +/- 0.012 and 0.060 +/- 0.004%/h in CHO + PRO vs. CHO treatment, respectively; P < 0.05). We conclude that, even in a fed state, protein coingestion stimulates whole body and muscle protein synthesis rates during resistance-type exercise.     

Neuromuscular electrical stimulation increases muscle protein synthesis in elderly type 2 diabetic men

Physical activity is required to attenuate the loss of skeletal muscle mass with aging. Short periods of muscle disuse, due to sickness or hospitalization, reduce muscle protein synthesis rates, resulting in rapid muscle loss. The present study investigates the capacity of neuromuscular electrical stimulation (NMES) to increase in vivo skeletal muscle protein synthesis rates in older type 2 diabetes patients. Six elderly type 2 diabetic men (70 ± 2 yr) were subjected to 60 min of one-legged NMES. Continuous infusions with l-[ring-(13)C(6)]phenylalanine were applied, with blood and muscle samples being collected regularly to assess muscle protein synthesis rates in both the stimulated (STIM) and nonstimulated control (CON) leg during 4 h of recovery after NMES. Furthermore, mRNA expression of key genes implicated in the regulation of muscle mass were measured over time in the STIM and CON leg. Muscle protein synthesis rates were greater in the STIM compared with the CON leg during recovery from NMES (0.057 ± 0.008 vs. 0.045 ± 0.008%/h, respectively, P < 0.01). Skeletal muscle myostatin mRNA expression in the STIM leg tended to increase immediately following NMES compared with the CON leg (1.63- vs. 1.00-fold, respectively, P = 0.07) but strongly declined after 2 and 4 h of recovery in the STIM leg only. In conclusion, this is the first study to show that NMES directly stimulates skeletal muscle protein synthesis rates in vivo in humans. NMES likely represents an effective interventional strategy to attenuate muscle loss in elderly individuals during bed rest and/or in other disuse states.     

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.     

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.     

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.     

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.     

Postexercise protein metabolism in older and younger men following moderate-intensity aerobic exercise

Regular aerobic exercise strongly influences muscle metabolism in elderly and young; however, the acute effects of aerobic exercise on protein metabolism are not fully understood. We investigated the effect of a single bout of moderate walking (45 min at approximately 40% of peak O2 consumption) on postexercise (POST-EX) muscle metabolism and synthesis of plasma proteins [albumin (ALB) and fibrinogen (FIB)] in untrained older (n = 6) and younger (n = 6) men. We measured muscle phenylalanine (Phe) kinetics before (REST) and POST-EX (10, 60, and 180 min) using l-[ring-2H5]phenylalanine infusion, femoral arteriovenous blood samples, and muscle biopsies. All data are presented as the difference from REST (at 10, 60, and 180 min POST-EX). Mixed muscle fractional synthesis rate (FSR) increased significantly at 10 min POST-EX in both the younger (0.0363%/h) and older men (0.0830%/h), with the younger men staying elevated through 60 min POST-EX (0.0253%/h). ALB FSR increased at 10 min POST-EX in the younger men only (2.30%/day), whereas FIB FSR was elevated in both groups through 180 min POST-EX (younger men = 4.149, older men = 4.107%/day). Muscle protein turnover was also increased, with increases in synthesis and breakdown in younger and older men. Phe rate of disappearance (synthesis) was increased in both groups at 10 min POST-EX and remained elevated through 60 min POST-EX in the older men. A bout of moderate-intensity aerobic exercise induces short-term increases in muscle and plasma protein synthesis in both younger and older men. Aging per se does not diminish the protein metabolic capacity of the elderly to respond to acute aerobic exercise.     

The single biopsy approach is reliable for the measurement of muscle protein synthesis rates in vivo in older men

We aimed to assess the reliability of the single biopsy approach for calculating muscle protein synthesis rates compared with the well described sequential muscle biopsy approach following a primed continuous infusion of L-[ring-(2)H(5)]phenylalanine and GC-MS analysis in older men. Two separate experimental infusion protocols, with differing stable isotope amino acid incorporation times, were employed consisting of n = 27 (experiment 1) or n = 9 (experiment 2). Specifically, mixed muscle protein FSR were calculated from baseline plasma protein enrichments and muscle protein enrichments obtained at 90 min or 50 min (1BX SHORT), 210 min or 170 min (1BX LONG), and between the muscle protein enrichments obtained at 90 and 210 min or 50 min and 170 min (2BX) of the infusion for experiments 1 and 2, respectively. In experiment 2, we also assessed the error that is introduced to the single muscle biopsy approach when nontracer naive subjects are recruited for participation in a primed continuous infusion of isotope-labeled amino acids. In experiment 1, applying the individual plasma protein enrichment values to the single muscle biopsy approach resulted in no differences in muscle protein FSR between the 1BX SHORT (0.031 ± 0.003%·h(-1)), 1BX LONG (0.032 ± 0.002%·h(-1)), or the 2BX approach (0.034 ± 0.002%·h(-1)). A significant correlation in muscle protein FSR was observed only between the 1BX LONG and 2BX approach (r = 0.8; P < 0.001). Similar results were observed in experiment 2. In addition, using the single biopsy approach in nontracer na?ve state results in a muscle protein FSR that is negative for both the 1BX SHORT (-0.67 ± 0.051%·h(-1)) and 1BX LONG (-0.19 ± 0.051%·h(-1)) approaches. This is the first study to demonstrate that the single biopsy approach, coupled with the background enrichment of L-[ring-(2)H(5)]-phenylalanine of mixed plasma proteins, generates data that are similar to using the sequential muscle biopsy approach in the elderly population.     

Myofibrillar protein synthesis in myostatin-deficient mice

Either increased protein synthesis or prolonged protein half-life is necessary to support the excessive muscle growth and maintenance of enlarged muscles in myostatin-deficient mice. This issue was addressed by determining in vivo rates of myofibrillar protein synthesis in mice with constitutive myostatin deficiency (Mstn(DeltaE3/DeltaE3)) or normal myostatin expression (Mstn(+/+)) by measuring tracer incorporation after a systemic flooding dose of l-[ring-(2)H(5)]phenylalanine. At 5-6 wk of age, Mstn(DeltaE3/DeltaE3) mice had increased muscle mass (40%), fractional rates of myofibrillar synthesis (14%), and protein synthesis per whole muscle (60%) relative to Mstn(+/+) mice. With maturation, fractional rates of synthesis declined >50% in parallel with decreased DNA and RNA [total, 28S rRNA, and poly(A) RNA] concentrations in muscle. At 6 mo of age, Mstn(DeltaE3/DeltaE3) mice had even greater increases in muscle mass (90%) and myofibrillar synthesis per muscle (85%) relative to Mstn(+/+) mice, but the fractional rate of synthesis was normal. Estimated myofibrillar protein half-life was not affected by myostatin deficiency. Muscle DNA concentrations were reduced in both young and mature Mstn(DeltaE3/DeltaE3) mice, whereas RNA concentrations were normal, so the ratio of RNA to DNA was approximately 30% greater than normal in Mstn(DeltaE3/DeltaE3) mice. Thus the increased protein synthesis and RNA content per muscle in myostatin-deficient mice cannot be explained entirely by an increased number of myonuclei.     

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.     

Low muscle glycogen concentration does not suppress the anabolic response to resistance exercise

We determined the effect of muscle glycogen concentration and postexercise nutrition on anabolic signaling and rates of myofibrillar protein synthesis after resistance exercise (REX). Sixteen young, healthy men matched for age, body mass, peak oxygen uptake (Vo(2peak)) and strength (one repetition maximum; 1RM) were randomly assigned to either a nutrient or placebo group. After 48 h diet and exercise control, subjects undertook a glycogen-depletion protocol consisting of one-leg cycling to fatigue (LOW), whereas the other leg rested (NORM). The next morning following an overnight fast, a primed, constant infusion of l-[ring-(13)C(6)] phenylalanine was commenced and subjects completed 8 sets of 5 unilateral leg press repetitions at 80% 1RM. Immediately after REX and 2 h later, subjects consumed a 500 ml bolus of a protein/CHO (20 g whey + 40 g maltodextrin) or placebo beverage. Muscle biopsies from the vastus lateralis of both legs were taken at rest and 1 and 4 h after REX. Muscle glycogen concentration was higher in the NORM than LOW at all time points in both nutrient and placebo groups (P < 0.05). Postexercise Akt-p70S6K-rpS6 phosphorylation increased in both groups with no differences between legs (P < 0.05). mTOR(Ser2448) phosphorylation in placebo increased 1 h after exercise in NORM (P < 0.05), whereas mTOR increased ~4-fold in LOW (P < 0.01) and ~11 fold in NORM with nutrient (P < 0.01; different between legs P < 0.05). Post-exercise rates of MPS were not different between NORM and LOW in nutrient (0.070 ± 0.022 vs. 0.068 ± 0.018 %/h) or placebo (0.045 ± 0.021 vs. 0.049 ± 0.017 %/h). We conclude that commencing high-intensity REX with low muscle glycogen availability does not compromise the anabolic signal and subsequent rates of MPS, at least during the early (4 h) postexercise recovery period.     

Coordinated increase in albumin, fibrinogen, and muscle protein synthesis during hemodialysis: role of cytokines

Serum albumin, fibrinogen levels, and lean body mass are important predictors of outcome in end-stage renal disease (ESRD). We estimated the fractional synthesis rates of albumin (FSR-A), fibrinogen (FSR-F), and muscle protein (FSR-M) in nine ESRD patients and eight controls, using primed constant infusion of l-[ring-(13)C(6)]phenylalanine. Cytokine profile and arteriovenous balance of amino acids were also measured. ESRD patients were studied before (Pre-HD) and during hemodialysis (HD). Plasma IL-6, IL-10, and C-reactive protein increased significantly during HD. Despite a decrease in the delivery of amino acids to the leg, the outflow of the amino acids increased during HD. The net balance of amino acids became more negative during HD, indicating release from the muscle. HD increased leg muscle protein synthesis (45%) and catabolism (108%) but decreased whole body proteolysis (15%). FSR-A during HD (9.7 +/- 0.9%/day) was higher than pre-HD (6.5 +/- 0.9%/day) and controls (5.8 +/- 0.5%/day, P < 0.01). FSR-F increased during HD (19.7 +/- 2.6%/day vs. 11.8 +/- 0.6%/day, P < 0.01), but it was not significantly different from that of controls (14.4 +/- 1.4%/day). FSR-M intradialysis (1.77 +/- 0.19%/day) was higher than pre-HD (1.21 +/- 0.25%/day) and controls (1.30 +/- 0.32%/day, P < 0.001). Pre-HD FSR-A, FSR-F, and FSR-M values were comparable to those of controls. There was a significant and positive correlation between plasma IL-6 and the FSRs. Thus, in ESRD patients without metabolic acidosis, the fractional synthesis rates of albumin, fibrinogen, and muscle protein are not decreased pre-HD. However, HD increases the synthesis of albumin, fibrinogen, and muscle protein. The coordinated increase in the FSRs is facilitated by constant delivery of amino acids derived from the muscle catabolism and intradialytic increase in IL-6.     

Coingestion of whey protein and casein in a mixed meal: demonstration of a more sustained anabolic effect of casein

When consumed separately, whey protein (WP) is more rapidly absorbed into circulation than casein (Cas), which prompted the concept of rapid and slow dietary protein. It is unclear whether these proteins have similar metabolic fates when coingested as in milk. We determined the rate of appearance across the splanchnic bed and the rate of disappearance across the leg of phenylalanine (Phe) from coingested, intrinsically labeled WP and Cas. Either [1?N]Phe or [13C-ring C?]Phe was infused in lactating cows, and the labeled WP and Cas from their milk were collected. To determine the fate of Phe derived from different protein sources, 18 healthy participants were studied after ingestion of one of the following: 1) [1?N]WP, [13C]Cas, and lactose; 2) [13C]WP, [1?N]Cas, and lactose; 3) lactose alone. At 80-120 min, the rates of appearance (R(a)) across the splanchnic bed of Phe from WP and Cas were similar [0.068 ± 0.010 vs. 0.070 ± 0.009%/min; not significant (ns)]. At time 220-260 min, Phe appearance from WP had slowed (0.039 ± 0.008%/min, P < 0.05) whereas Phe appearance from Cas was sustained (0.068 ± 0.013%/min). Similarly, accretion rates across the leg of Phe absorbed from WP and Cas were not different at 80-120 min (0.011 ± 0.002 vs. 0.012 ± 0.003%/min; ns), but they were significantly lower for WP (0.007 ± 0.002%/min) at 220-260 min than for Cas (0.013 ± 0.002%/min) at 220-260 min. Early after meal ingestion, amino acid absorption and retention across the leg were similar for WP and Cas, but as rates for WP waned, absorption and assimilation into skeletal muscle were better retained for Cas.     

Leg glucose and protein metabolism during an acute bout of resistance exercise in humans.

The present study investigated the responses of leg glucose and protein metabolism during an acute bout of resistance exercise. Seven subjects (5 men, 2 women) were studied at rest and during a strenuous lower body resistance exercise regimen consisting of approximately 8 sets of 10 repetitions of leg press at approximately 75% 1 repetition maximum and 8 sets of 8 repetitions of knee extensions at approximately 80% 1 repetition maximum. L-[ring-2H5]phenylalanine was infused throughout the study for measurement of phenylalanine rates of appearance, disappearance, protein synthesis, and protein breakdown across the leg. Femoral arterial and venous blood samples were collected at rest and during exercise for determination of leg blood flow, concentrations of glucose, lactate, alanine, glutamine, glutamate, leucine, and phenylalanine, and phenylalanine enrichments. Muscle biopsies were obtained at rest and immediately after exercise. Leg blood flow was nearly three times (P <0.009) higher and glucose uptake more than five times higher (P=0.009) during exercise than at rest. Leg lactate release was 86 times higher than rest during the exercise bout. Although whole body phenylalanine rate of appearance, an indicator of whole body protein breakdown, was reduced during exercise; leg phenylalanine rate of appearance, rate of disappearance, protein synthesis, and protein breakdown did not change. Arterial and venous alanine concentrations and glutamate uptake were significantly higher during exercise than at rest. We conclude that lower body resistance exercise potently stimulates leg glucose uptake and lactate release. In addition, muscle protein synthesis is not elevated during a bout of resistance exercise.     

Insulin and amino acids independently stimulate skeletal muscle protein synthesis in neonatal pigs

Infusion of physiological levels of insulin and/or amino acids reproduces the feeding-induced stimulation of muscle protein synthesis in neonates. To determine whether insulin and amino acids independently stimulate skeletal muscle protein synthesis in neonates, insulin secretion was blocked with somatostatin in fasted 7-day-old pigs (n = 8-12/group) while glucose and glucagon were maintained at fasting levels and insulin was infused to simulate either less than fasting, fasting, intermediate, or fed insulin levels. At each dose of insulin, amino acids were clamped at either the fasting or fed level; at the highest insulin dose, amino acids were also reduced to less than fasting levels. Skeletal muscle protein synthesis was measured using a flooding dose of l-[4-(3)H]phenylalanine. Hyperinsulinemia increased protein synthesis in skeletal muscle during hypoaminoacidemia and euaminoacidemia. Hyperaminoacidemia increased muscle protein synthesis during hypoinsulinemia and euinsulinemia. There was a dose-response effect of both insulin and amino acids on muscle protein synthesis. At each insulin dose, hyperaminoacidemia increased muscle protein synthesis. The effects of insulin and amino acids on muscle protein synthesis were largely additive until maximal rates of protein synthesis were achieved. Amino acids enhanced basal protein synthesis rates but did not enhance the sensitivity or responsiveness of muscle protein synthesis to insulin. The results suggest that insulin and amino acids independently stimulate protein synthesis in skeletal muscle of the neonate.     

Measurement of hepatic protein fractional synthetic rate with stable isotope labeling technique in thapsigargin stressed HepG2 cells

Severe burn-induced liver damage and dysfunction is associated with endoplasmic reticulum (ER) stress. ER stress has been shown to regulate global protein synthesis. In the current study, we induced ER stress in vitro and estimated the effect of ER stress on hepatic protein synthesis. The aim was two-fold: (1) to establish an in vitro model to isotopically measure hepatic protein synthesis and (2) to evaluate protein fractional synthetic rate (FSR) in response to ER stress. Human hepatocellular carcinoma cells (HepG2) were cultured in medium supplemented with stable isotopes 1,2-(13)C(2)-glycine and L-[ring-(13)C(6)]phenylalanine. ER stress was induced by exposing the cells to 100 nM of thapsigargin (TG). Cell content was collected from day 0 to 14. Alterations in cytosolic calcium were measured by calcium imaging and ER stress markers were confirmed by Western blotting. The precursor and product enrichments were detected by GC-MS analysis for FSR calculation. We found that the hepatic protein FSR were 0.97 ± 0.02 and 0.99 ± 0.05%/hr calculated from 1,2-(13)C(2)-glycine and L-[ring-(13)C(6)]phenylalanine, respectively. TG depleted ER calcium stores and induced ER stress by upregulating p-IRE-1 and Bip. FSR dramatically decreased to 0.68 ± 0.03 and 0.60 ± 0.06%/hr in the TG treatment group (p<0.05, vs. control). TG-induced ER stress inhibited hepatic protein synthesis. The stable isotope tracer incorporation technique is a useful method for studying the effects of ER stress on hepatic protein synthesis.