Robert R. Wolfe

Postexercise net protein synthesis in human muscle from orally administered amino acids

We examined the response of net muscle protein synthesis to ingestion of amino acids after a bout of resistance exercise. A primed, constant infusion of L-[ring-2H5]phenylalanine was used to measure net muscle protein balance in three male and three female volunteers on three occasions. Subjects consumed in random order 1 liter of 1) a mixed amino acid (40 g) solution (MAA), 2) an essential amino acid (40 g) solution (EAA), and 3) a placebo solution (PLA). Arterial amino acid concentrations increased approximately 150-640% above baseline during ingestion of MAA and EAA. Net muscle protein balance was significantly increased from negative during PLA ingestion (-50 +/- 23 nmol. min-1. 100 ml leg volume-1) to positive during MAA ingestion (17 +/- 13 nmol. min-1. 100 ml leg volume-1) and EAA (29 +/- 14 nmol. min-1. 100 ml leg volume-1; P < 0.05). Because net balance was similar for MAA and EAA, it does not appear necessary to include nonessential amino acids in a formulation designed to elicit an anabolic response from muscle after exercise. We concluded that ingestion of oral essential amino acids results in a change from net muscle protein degradation to net muscle protein synthesis after heavy resistance exercise in humans similar to that seen when the amino acids were infused.We examined the response of net muscle protein synthesis to ingestion of amino acids after a bout of resistance exercise. A primed, constant infusion ofl-[ ring-2H5]phenylalanine was used to measure net muscle protein balance in three male and three female volunteers on three occasions. Subjects consumed in random order 1 liter of 1) a mixed amino acid (40 g) solution (MAA), 2) an essential amino acid (40 g) solution (EAA), and 3) a placebo solution (PLA). Arterial amino acid concentrations increased ∼150-640% above baseline during ingestion of MAA and EAA. Net muscle protein balance was significantly increased from negative during PLA ingestion (-50 ± 23 nmol ⋅ min-1 ⋅ 100 ml leg volume-1) to positive during MAA ingestion (17 ± 13 nmol ⋅ min-1 ⋅ 100 ml leg volume-1) and EAA (29 ± 14 nmol ⋅ min-1 ⋅ 100 ml leg volume-1; P < 0.05). Because net balance was similar for MAA and EAA, it does not appear necessary to include nonessential amino acids in a formulation designed to elicit an anabolic response from muscle after exercise. We concluded that ingestion of oral essential amino acids results in a change from net muscle protein degradation to net muscle protein synthesis after heavy resistance exercise in humans similar to that seen when the amino acids were infused.

Effect of severe burn injury on substrate cycling by glucose and fatty acids

Increases in metabolic rate and core temperature are common responses to severe injury. We have investigated the hypothesis that these responses are due to increases in substrate cycling. A substrate cycle exists when opposing, nonequilibrium reactions catalyzed by different enzymes are operating simultaneously. At least one of the reactions must involve the hydrolysis of ATP. Thus, a substrate cycle both liberates heat and increases energy expenditure, yet there is not net conversion of substrate to product. In studies in volunteers (n = 18) and in patients with severe burns who were in a hypermetabolic state (n = 18), we used stable-isotope tracers to quantify substrate cycling in the pathways of glycolysis and gluconeogenesis and a cycle involving the simultaneous breakdown and synthesis of stored triglyceride (triglyceride-fatty acid cycle). The total rates of triglyceride-fatty acid and glycolytic-gluconeogenic cycling were elevated in the patients by 450 and 250 percent, respectively (P less than 0.01). An infusion of propranolol in the patients greatly reduced triglyceride-fatty acid cycling but did not affect gluconeogenic-glycolytic cycling. We conclude that increased substrate cycling contributes to the increased thermogenesis and energy expenditure following severe burns and that the increased triglyceride-fatty acid cycling is due to beta-adrenergic stimulation.

Results of the first year of the new liver allocation plan

Liver allocation policy in the U.S. was recently changed to a continuous disease severity scale with minimal weight given to time waiting in an effort to better prioritize deceased donor liver transplant candidates. We compared rates of waiting list registrations, removals, transplants, and deaths during the year prior to implementation of the new liver allocation policy (2/27/01–2/26/02, Era 1) with the first years experience (2/27/02–2/26/03, Era 2) under this new policy. Rates were adjusted for 1,000 patient years on the waiting list and compared using z‐tests. A 1‐sided test was used to compare death rates; 2‐sided tests were used to compare transplant rates. Overall and subgroup analyses were performed for demographic, geographic, and medical strata. In Era 2, we observed a 12% reduction in new liver transplant waiting list registrations, with the largest reductions seen in new registrants with low MELD/PELD scores. In Era 2, there was a 3.5% reduction in waiting list death rate (P = .076) and a 10.2% increase in cadaveric transplants (P < .001). The reduction in waiting list mortality and increase in transplantation rates were evenly distributed across all demographic and medical strata, with some variation across geographic variables. Early patient and graft survival after deceased donor liver transplantation remains unchanged. In conclusion, by eliminating the categorical waiting list prioritization system that emphasized time waiting, the new system has been associated with reduced registrations and improved transplantation rates without increased mortality rates for individual groups of waiting candidates or changes in early transplant survival rates. (Liver Transpl 2004;10:7–15.)

Exogenous amino acids stimulate net muscle protein synthesis in the elderly.

We have investigated the response of amino acid transport and protein synthesis in healthy elderly individuals (age 71+/-2 yr) to the stimulatory effect of increased amino acid availability. Muscle protein synthesis and breakdown, and amino acid transport were measured in the postabsorptive state and during the intravenous infusion of an amino acid mixture. Muscle-free amino acid kinetics were calculated by means of a three compartment model using data obtained by femoral arterio-venous catheterization and muscle biopsies from the vastus lateralis during the infusion of stable isotope tracers of amino acids. In addition, muscle protein fractional synthetic rate (FSR) was measured. Peripheral amino acid infusion significantly increased amino acid delivery to the leg, amino acid transport, and muscle protein synthesis when measured either with the three compartment model (P < 0.05) or with the traditional precursor-product approach (FSR increased from 0. 0474+/-0.0054 to 0.0940+/-0.0143%/h, P < 0.05). Because protein breakdown did not change during amino acid infusion, a positive net balance of amino acids across the muscle was achieved. We conclude that, although muscle mass is decreased in the elderly, muscle protein anabolism can nonetheless be stimulated by increased amino acid availability. We thus hypothesize that muscle mass could be better maintained with an increased intake of protein or amino acids.

Human Muscle Protein Synthesis is Modulated by Extracellular, Not Intramuscular Amino Acid Availability: A Dose‐Response Study

To test the hypothesis that muscle protein synthesis (MPS) is regulated by the concentration of extracellular amino acids, we investigated the dose‐response relationship between the rate of human MPS and the concentrations of blood and intramuscular amino acids. We increased blood mixed amino acid concentrations by up to 240 % above basal levels by infusion of mixed amino acids (Aminosyn 15, 44‐261 mg kg−1 h−1) in 21 healthy subjects, (11 men 10 women, aged 29 ± 2 years) and measured the rate of incorporation of D5‐phenylalanine or D3‐leucine into muscle protein and blood and intramuscular amino acid concentrations. The relationship between the fold increase in MPS and blood essential amino acid concentration ([EAA], mM) was hyperbolic and fitted the equation MPS = (2.68 ×[EAA])/(1.51 +[EAA]) (P < 0.01). The pattern of stimulation of myofibrillar, sarcoplasmic and mitochondrial protein was similar. There was no clear relationship between the rate of MPS and the concentration of intramuscular EAAs; indeed, when MPS was increasing most rapidly, the concentration of intramuscular EAAs was below basal levels. We conclude that the rates of synthesis of all classes of muscle proteins are acutely regulated by the blood [EAA] over their normal diurnal range, but become saturated at high concentrations. We propose that the stimulation of protein synthesis depends on the sensing of the concentration of extracellular, rather than intramuscular EAAs.

Physiologic hyperinsulinemia stimulates protein synthesis and enhances transport of selected amino acids in human skeletal muscle.

We have investigated the mechanisms of the anabolic effect of insulin on muscle protein metabolism in healthy volunteers, using stable isotopic tracers of amino acids. Calculations of muscle protein synthesis, breakdown, and amino acid transport were based on data obtained with the leg arteriovenous catheterization and muscle biopsy. Insulin was infused (0.15 mU/min per 100 ml leg) into the femoral artery to increase femoral venous insulin concentration (from 10 +/- 2 to 77 +/- 9 microU/ml) with minimal systemic perturbations. Tissue concentrations of free essential amino acids decreased (P < 0.05) after insulin. The fractional synthesis rate of muscle protein (precursor-product approach) increased (P < 0.01) after insulin from 0.0401 +/- 0.0072 to 0.0677 +/- 0.0101%/h. Consistent with this observation, rates of utilization for protein synthesis of intracellular phenylalanine and lysine (arteriovenous balance approach) also increased from 40 +/- 8 to 59 +/- 8 (P < 0.05) and from 219 +/- 21 to 298 +/- 37 (P < 0.08) nmol/min per 100 ml leg, respectively. Release from protein breakdown of phenylalanine, leucine, and lysine was not significantly modified by insulin. Local hyperinsulinemia increased (P < 0.05) the rates of inward transport of leucine, lysine, and alanine, from 164 +/- 22 to 200 +/- 25, from 126 +/- 11 to 221 +/- 30, and from 403 +/- 64 to 595 +/- 106 nmol/min per 100 ml leg, respectively. Transport of phenylalanine did not change significantly. We conclude that insulin promoted muscle anabolism, primarily by stimulating protein synthesis independently of any effect on transmembrane transport.

Latency and duration of stimulation of human muscle protein synthesis during continuous infusion of amino acids.

1 The aim of this study was to describe the time course of the response of human muscle protein synthesis (MPS) to a square wave increase in availability of amino acids (AAs) in plasma. We investigated the responses of quadriceps MPS to a ≈1.7‐fold increase in plasma AA concentrations using an intravenous infusion of 162 mg (kg body weight)−1 h−1 of mixed AAs. MPS was estimated from D3‐leucine labelling in protein after a primed, constant intravenous infusion of D3‐ketoisocaproate, increased appropriately during AA infusion. 2 Muscle was separated into myofibrillar, sarcoplasmic and mitochondrial fractions. MPS, both of mixed muscle and of fractions, was estimated during a basal period (2.5 h) and at 0.5‐4 h intervals for 6 h of AA infusion. 3 Rates of mixed MPS were not significantly different from basal (0.076 ± 0.008 % h−1) in the first 0.5 h of AA infusion but then rose rapidly to a peak after 2 h of ≈2.8 times the basal value. Thereafter, rates declined rapidly to the basal value. All muscle fractions showed a similar pattern. 4 The results suggest that MPS responds rapidly to increased availability of AAs but is then inhibited, despite continued AA availability. These results suggest that the fed state accretion of muscle protein may be limited by a metabolic mechanism whenever the requirement for substrate for protein synthesis is exceeded.

Oral amino acids stimulate muscle protein anabolism in the elderly despite higher first-pass splanchnic extraction

Muscle protein synthesis and breakdown and amino acid transport were measured in 7 healthy young (30 +/- 2 yr) and 8 healthy elderly (71 +/- 2 yr) volunteers in the postabsorptive state and during the oral administration of an amino acid mixture with L-[ring-(2)H(5)]phenylalanine infusion, femoral artery and vein catheterization, and muscle biopsies. Phenylalanine first-pass splanchnic extraction was measured by adding L-[ring-(13)C(6)]phenylalanine to the mixture. In the postabsorptive state, no differences in muscle amino acid kinetics were detected between young and elderly volunteers. Phenylalanine first-pass splanchnic extraction was significantly higher in the elderly (P < 0. 003) during ingestion of amino acids, but the delivery to the leg increased to the same extent in both groups. Phenylalanine transport into the muscle, muscle protein synthesis, and net balance increased significantly (P < 0.01) and similarly in both the young and the elderly. We conclude that, despite an increased splanchnic first-pass extraction, muscle protein anabolism can be stimulated by oral amino acids in the elderly as well as in the young.

Mechanisms of insulin resistance following injury.

To assess the mechanisms of insulin resistance following injury, we examined the relationship between insulin levels and glucose disposal in nine nonseptic, multiple trauma patients (average age 32 years, Injury Severity Score 22) five to 13 days postinjury. Fourteen age-matched normals served as controls. Using a modification of the euglycemic insulin clamp technique, insulin was infused in 35 two-hour studies using at least one of four infusion rates (0.5, 1.0, 2.0 or 5.0 mU/kg min). Basal glucose levels were maintained by a variable infusion of 20% dextrose using bedside glucose monitoring and a servo-control algorithm. The amount of glucose infused reflected glucose disposal (M, mg/kg.min). Tracer doses of (6,6,2D2) glucose were administered in selected subjects to determine endogenous glucose production. At plasma insulin concentrations less than 100 μU/ml, responses in both groups were similar. However, maximal glucose disposal rates were significantly less in the patients than in the controls (9.17 ± 0.87 mg/kg.min vs. 14.3 ± 0.78, mean SEM, p <0.01). Insulin clearance rates in the patients were almost twice that seen in controls. To further characterize this decrease in insulin responsiveness, we studied six additional patients and 12 controls following the acute elevation of glucose 125 mg/dl above basal (hyperglycemic glucose clamp). In spite of exaggerated endogenous insulin production in the patients (80–200 μU/ml vs. 30–70 in controls), M was significantly lower (6.23 ± 0.59 vs. 9.46 ± 0.79, p < 0.02). In conclusion, this study demonstrated that (1) the maximal rate of glucose disposal is reduced in trauma patients; (2) the metabolic clearance rate of insulin in the injured patients is almost twice normal and; (3) insulin resistance following injury appears to occur in peripheral tissues, probably skeletal muscle, and is consistent with a postreceptor defect.

Glucose metabolism in man: Responses to intravenous glucose infusion

We have determined the effect of unlabeled glucose infusions, with and without added insulin, on glucose metabolism in normal male volunteers by means of the simultaneous primed-constant infusion of 6-3H and U-13C-glucose. Glucose kinetics were measured after 90 min of infusion. When steady state had been reached, endogenous glucose production (2.53 +/- .058 mg/kg . min, X +/- SEM) was suppressed at all rates of exogenous glucose tested (1, 2, and 4 mg/kg . min). The absolute degree of suppression was most marked (75%) at the highest rate of infusion, but the greatest degree of suppression, relative to infusion rate, was at the lowest infusion rate. The control of plasma glucose concentration during the glucose infusion was achieved primarily through regulation of endogenous Ra. The rate of uptake of glucose only increased during the 4 mg/kg . min infusion, even though there were significant elevations in the plasma glucose and insulin concentrations during the 2 mg/kg . min infusion as well. The glucose clearance rate increased only when sufficient insulin was infused with the 4 mg/kg . min glucose infusion to control the hyperglycemia that developed if no insulin was administered. Approximately 43% of the infused glucose was directly oxidized when the infusion rate was 1 or 2 mg/kg . min. That value fell to 32% when the infusion rate was increased to 4 mg/kg . min, regardless of whether insulin was infused or not.