Imre W. K. Kouw

The use of doubly labeled milk protein to measure postprandial muscle protein synthesis rates in vivo in humans

We aimed to determine the impact of precursor pool dilution on the assessment of postprandial myofibrillar protein synthesis rates (MPS). A Holstein dairy cow was infused with large amounts of L-[1-(13)C]phenylalanine and L-[1-(13)C]leucine, and the milk was collected and fractionated. The enrichment levels in the casein were 38.7 and 9.3 mole percent excess, respectively. In a subsequent human experiment, 11 older men (age: 71 ± 1 y, body mass index: 26 ± 0.1 kg·m(-2)) received a primed constant infusion of L-[ring-(2)H5]phenylalanine and L-[1-(13)C]leucine. Blood and muscle samples were collected before and after the ingestion of 20-g doubly labeled casein to assess postprandial MPS based on the 1) constant tracer infusion of L-[ring-(2)H5]phenylalanine, 2) ingestion of intrinsically L-[1-(13)C]phenylalanine-labeled casein, and 3) constant infusion of L-[1-(13)C]leucine in combination with the ingestion of intrinsically L-[1-(13)C]leucine-labeled casein. Postprandial MPS was increased (P < 0.05) after protein ingestion (∼70% above postabsorptive values) based on the L-[1-(13)C]leucine tracer. There was no significant stimulation of postprandial MPS (∼27% above postabsorptive values) when the calculated fractional synthesis rate was based on the L-[ring-(2)H5]phenylalanine (P = 0.2). Comparisons of postprandial MPS based on the primed continuous infusion of L-[1-(13)C]leucine or the ingestion of intrinsically L-[1-(13)C]phenylalanine-labeled casein protein demonstrated differences compared with the primed continuous infusion of L-[ring-(2)H5]phenylalanine (P > 0.05). Our findings confirm that the postprandial MPS assessed using the primed continuous tracer infusion approach may differ if tracer steady-state conditions in the precursor pools are perturbed. The use of intrinsically doubly labeled protein provides a method to study the metabolic fate of the ingested protein and the subsequent postprandial MPS response.

Impact of the Macronutrient Composition of a Nutritional Supplement on Muscle Protein Synthesis Rates in Older Men: A Randomized, Double Blind, Controlled Trial

CONTEXT An impaired muscle protein synthetic response to feeding likely contributes to muscle loss with aging. There are few data available on the effect of the macronutrient composition of clinical supplements on the postprandial muscle protein synthetic response in older subjects. OBJECTIVE The objective of the study was to determine the impact of the macronutrient composition of a nutritional supplement on the postprandial muscle protein synthetic response in older men. METHODS A total of 45 nonsarcopenic older men (aged 69 ± 1 y; body mass index 25.7 ± 0.3 kg/m(2)) were randomly assigned to ingest 21 g of leucine-enriched whey protein with carbohydrate (9 g) and fat (3 g) (Pro-En), an isonitrogenous amount of 21 g of leucine-enriched whey protein without carbohydrate and fat (Pro), or an isocaloric mixture (628 kJ) containing carbohydrate and fat only (En). Stable isotope tracer methodology was applied to assess the basal as well as the postprandial muscle protein synthesis rates in the three groups. RESULTS Ingestion of protein in the Pro-En and Pro groups significantly increased muscle protein synthesis rates when compared with the basal rates (from 0.032 ± 0.003%/h to 0.05%/h 3 ± 0.004%/h and 0.040%/h ± 0.003%/h to 0.049%/h ± 0.003%/h, respectively; P < .05), whereas ingestion of carbohydrate and fat did not increase muscle protein synthesis rates in the En group (from 0.039%/h ± 0.004%/h to 0.040%/h ± 0.003%/h; P = .60). Despite the greater postprandial rise in circulating insulin concentration in the Pro-En group, no significant differences were observed in postprandial muscle protein synthesis rates between the Pro-En and Pro groups (P = .32). Postprandial muscle protein synthesis rates were higher in the Pro-En vs En group (P = .01). CONCLUSION The ingestion of a nutritional supplement containing 21 g of leucine-enriched whey protein significantly raises muscle protein synthesis rates in nonsarcopenic older men, but coingestion of carbohydrate and fat does not modulate the postprandial muscle protein synthetic response to protein ingestion in older men.

A single dose of sodium nitrate does not improve oral glucose tolerance in patients with type 2 diabetes mellitus

Dietary nitrate (NO3(-)) supplementation has been proposed as an emerging treatment strategy for type 2 diabetes. We hypothesized that ingestion of a single bolus of dietary NO3(-) ingestion improves oral glucose tolerance in patients with type 2 diabetes. Seventeen men with type 2 diabetes (glycated hemoglobin, 7.3% ± 0.2%) participated in a randomized crossover experiment. The subjects ingested a glucose beverage 2.5 hours after consumption of either sodium NO3(-) (0.15 mmol NaNO3(-) · kg(-1)) or a placebo solution. Venous blood samples were collected before ingestion of the glucose beverage and every 30 minutes thereafter during a 2-hour period to assess postprandial plasma glucose and insulin concentrations. The results show that plasma NO3(-) and nitrite levels were increased after NaNO3(-) as opposed to placebo ingestion (treatment-effect, P = .001). Despite the elevated plasma NO3(-) and nitrite levels, ingestion of NaNO3(-) did not attenuate the postprandial rise in plasma glucose and insulin concentrations (time × treatment interaction, P = .41 for glucose, P = .93 for insulin). Despite the lack of effect on oral glucose tolerance, basal plasma glucose concentrations measured 2.5 hours after NaNO3(-) ingestion were lower when compared with the placebo treatment (7.5 ± 0.4 vs 8.3 ± 0.4 mmol/L, respectively; P = .04). We conclude that ingestion of a single dose of dietary NO3(-) does not improve subsequent oral glucose tolerance in patients with type 2 diabetes.

Physical Activity Performed in the Evening Increases the Overnight Muscle Protein Synthetic Response to Presleep Protein Ingestion in Older Men

BACKGROUND The age-related decline in skeletal muscle mass is partly attributed to anabolic resistance to food intake. Dietary protein ingestion before sleep could be used as a nutritional strategy to compensate for anabolic resistance. OBJECTIVE The present study assessed whether physical activity performed in the evening can augment the overnight muscle protein synthetic response to presleep protein ingestion in older men. METHODS In a parallel group design, 23 healthy older men (mean ± SEM age: 71 ± 1 y) were randomly assigned to ingest 40 g protein intrinsically labeled with l-[1-(13)C]-phenylalanine and l-[1-(13)C]-leucine before going to sleep with (PRO+EX) or without (PRO) performing physical activity earlier in the evening. Overnight protein digestion and absorption kinetics and myofibrillar protein synthesis rates were assessed by combining primed, continuous infusions of l-[ring-(2)H5]-phenylalanine, l-[1-(13)C]-leucine, and l-[ring-(2)H2]-tyrosine with the ingestion of intrinsically labeled casein protein. Muscle and blood samples were collected throughout overnight sleep. RESULTS Protein ingested before sleep was normally digested and absorbed, with 54% ± 2% of the protein-derived amino acids appearing in the circulation throughout overnight sleep. Overnight myofibrillar protein synthesis rates were 31% (0.058% ± 0.002%/h compared with 0.044% ± 0.003%/h; P < 0.01; based on l-[ring-(2)H5]-phenylalanine) and 27% (0.074% ± 0.004%/h compared with 0.058% ± 0.003%/h; P < 0.01; based on l-[1-(13)C]-leucine) higher in the PRO+EX than in the PRO treatment. More dietary protein-derived amino acids were incorporated into de novo myofibrillar protein during overnight sleep in PRO+EX than in PRO treatment (0.042 ± 0.002 compared with 0.033 ± 0.002 mole percent excess; P < 0.05). CONCLUSIONS Physical activity performed in the evening augments the overnight muscle protein synthetic response to presleep protein ingestion and allows more of the ingested protein-derived amino acids to be used for de novo muscle protein synthesis during overnight sleep in older men. This trial was registered at Nederlands Trial Register as NTR3885.

Resistance Exercise Augments Postprandial Overnight Muscle Protein Synthesis Rates

INTRODUCTION We have previously shown that protein ingestion before sleep increases overnight muscle protein synthesis rates. Whether prior exercise further augments the muscle protein synthetic response to presleep protein ingestion remains to be established. OBJECTIVE This study aimed to assess whether resistance-type exercise performed in the evening increases the overnight muscle protein synthetic response to presleep protein ingestion. METHODS Twenty-four healthy young men were randomly assigned to ingest 30 g intrinsically L-[1-C]-phenylalanine and L-[1-C]-leucine-labeled casein protein before going to sleep with (PRO + EX, n = 12) or without (PRO, n = 12) prior resistance-type exercise performed in the evening. Continuous intravenous L-[ring-H5]-phenylalanine, L-[1-C]-leucine, and L-[ring-H2]-tyrosine infusions were applied. Blood and muscle tissue samples were collected to assess whole-body protein balance, myofibrillar protein synthesis rates, and overnight incorporation of dietary protein-derived amino acids into de novo myofibrillar protein. RESULTS A total of 57% ± 1% of the ingested protein-derived phenylalanine appeared in the circulation during overnight sleep. Overnight myofibrillar protein synthesis rates were 37% (0.055%·h ± 0.002%·h vs. 0.040%·h ± 0.003%·h, P < 0.001, based on L-[ring- H5]-phenylalanine) and 31% (0.073%·h ± 0.004%·h vs. 0.055%·h ± 0.006%·h, P = 0.024, based on L-[1-C]-leucine) higher in PRO + EX compared with PRO. Substantially more of the dietary protein-derived amino acids were incorporated into de novo myofibrillar protein during overnight sleep in PRO + EX compared with PRO (0.026 ± 0.003 vs. 0.015 ± 0.003 molar percent excess, P = 0.012). CONCLUSIONS Resistance-type exercise performed in the evening augments the overnight muscle protein synthetic response to presleep protein ingestion and allows more of the ingested protein-derived amino acids to be used for de novo myofibrillar protein synthesis during overnight sleep.

Habituation to low or high protein intake does not modulate basal or postprandial muscle protein synthesis rates: a randomized trial

BACKGROUND Muscle mass maintenance is largely regulated by basal muscle protein synthesis rates and the ability to increase muscle protein synthesis after protein ingestion. To our knowledge, no previous studies have evaluated the impact of habituation to either low protein intake (LOW PRO) or high protein intake (HIGH PRO) on the postprandial muscle protein synthetic response. OBJECTIVE We assessed the impact of LOW PRO compared with HIGH PRO on basal and postprandial muscle protein synthesis rates after the ingestion of 25 g whey protein. DESIGN Twenty-four healthy, older men [age: 62 ± 1 y; body mass index (in kg/m2): 25.9 ± 0.4 (mean ± SEM)] participated in a parallel-group randomized trial in which they adapted to either a LOW PRO diet (0.7 g · kg-1 · d-1; n = 12) or a HIGH PRO diet (1.5 g · kg-1 · d-1; n = 12) for 14 d. On day 15, participants received primed continuous l-[ring-2H5]-phenylalanine and l-[1-13C]-leucine infusions and ingested 25 g intrinsically l-[1-13C]-phenylalanine- and l-[1-13C]-leucine-labeled whey protein. Muscle biopsies and blood samples were collected to assess muscle protein synthesis rates as well as dietary protein digestion and absorption kinetics. RESULTS Plasma leucine concentrations and exogenous phenylalanine appearance rates increased after protein ingestion (P < 0.01) with no differences between treatments (P > 0.05). Plasma exogenous phenylalanine availability over the 5-h postprandial period was greater after LOW PRO than after HIGH PRO (61% ± 1% compared with 56% ± 2%, respectively; P < 0.05). Muscle protein synthesis rates increased from 0.031% ± 0.004% compared with 0.039% ± 0.007%/h in the fasted state to 0.062% ± 0.005% compared with 0.057% ± 0.005%/h in the postprandial state after LOW PRO compared with HIGH PRO, respectively (P < 0.01), with no differences between treatments (P = 0.25). CONCLUSION Habituation to LOW PRO (0.7 g · kg-1 · d-1) compared with HIGH PRO (1.5 g · kg-1 · d-1) augments the postprandial availability of dietary protein-derived amino acids in the circulation and does not lower basal muscle protein synthesis rates or increase postprandial muscle protein synthesis rates after ingestion of 25 g protein in older men. This trial was registered at clinicaltrials.gov as NCT01986842.

Postprandial Protein Handling Is Not Impaired in Type 2 Diabetes Patients When Compared With Normoglycemic Controls

CONTEXT The progressive loss of muscle mass with aging is accelerated in type 2 diabetes patients. It has been suggested that this is attributed to a blunted muscle protein synthetic response to food intake. OBJECTIVE The objective of the study was to test the hypothesis that the muscle protein synthetic response to protein ingestion is impaired in older type 2 diabetes patients when compared with healthy, normoglycemic controls. DESIGN A clinical intervention study with two parallel groups was conducted between August 2011 and July 2012. SETTING The study was conducted at the research unit of Maastricht University, The Netherlands. Intervention, Participants, and Main Outcome Measures: Eleven older type 2 diabetes males [diabetes; age 71 ± 1 y, body mass index (BMI) 26.2 ± 0.5 kg/m(2)] and 12 age- and BMI-matched normoglycemic controls (control; age 74 ± 1 y, BMI 24.8 ± 1.1 kg/m(2)) participated in an experiment in which they ingested 20 g intrinsically L-[1-(13)C]phenylalanine-labeled protein. Continuous iv L-[ring-(2)H5]phenylalanine infusion was applied, and blood and muscle samples were obtained to assess amino acid kinetics and muscle protein synthesis rates in the postabsorptive and postprandial state. RESULTS Plasma insulin concentrations increased after protein ingestion in both groups, with a greater rise in the diabetes group. Postabsorptive and postprandial muscle protein synthesis rates did not differ between groups and averaged 0.029 ± 0.003 vs 0.029 ± 0.003%/h(1) and 0.031 ± 0.002 vs 0.033 ± 0.002%/h(1) in the diabetes versus control group, respectively. Postprandial L-[1-(13)C]phenylalanine incorporation into muscle protein did not differ between groups (0.018 ± 0.001 vs 0.019 ± 0.002 mole percent excess, respectively). CONCLUSIONS Postabsorptive muscle protein synthesis and postprandial protein handling is not impaired in older individuals with type 2 diabetes when compared with age-matched, normoglycemic controls.

Pre-sleep dietary protein-derived amino acids are incorporated in myofibrillar protein during post-exercise overnight recovery

The purpose of this study was to determine the impact of ingesting 30 g casein protein with and without 2 g free leucine before sleep on myofibrillar protein synthesis rates during postexercise overnight recovery. Thirty-six healthy young men performed a single bout of resistance-type exercise in the evening (1945) after a full day of dietary standardization. Thirty minutes before sleep (2330), subjects ingested 30 g intrinsically l-[1-13C]phenylalanine-labeled protein with (PRO+leu, n = 12) or without (PRO, n = 12) 2 g free leucine, or a noncaloric placebo (PLA, n = 12). Continuous intravenous l-[ ring-2H5]phenylalanine, l-[1-13C]leucine, and l-[ ring-2H2]tyrosine infusions were applied. Blood and muscle tissue samples were collected to assess whole body protein net balance, myofibrillar protein synthesis rates, and overnight incorporation of dietary protein-derived amino acids into myofibrillar protein. Protein ingestion before sleep improved overnight whole body protein net balance ( P < 0.001). Myofibrillar protein synthesis rates did not differ significantly between treatments as assessed by l-[ ring-2H5]phenylalanine (0.057 ± 0.002, 0.055 ± 0.002, and 0.055 ± 0.004%/h for PLA, PRO, and PRO+leu, respectively; means ± SE; P = 0.850) or l-[1-13C]leucine (0.080 ± 0.004, 0.073 ± 0.004, and 0.083 ± 0.006%/h, respectively; P = 0.328). Myofibrillar l-[1-13C]phenylalanine enrichments increased following protein ingestion but did not differ between the PRO and PRO+leu treatments. In conclusion, protein ingestion before sleep improves whole body protein net balance and provides amino acids that are incorporated into myofibrillar protein during sleep. However, the ingestion of 30 g casein protein with or without additional free leucine before sleep does not increase muscle protein synthesis rates during postexercise overnight recovery.

Protein Ingestion before Sleep Increases Overnight Muscle Protein Synthesis Rates in Healthy Older Men: A Randomized Controlled Trial

Background: The loss of skeletal muscle mass with aging has been attributed to the blunted anabolic response to protein intake. Presleep protein ingestion has been suggested as an effective strategy to compensate for such anabolic resistance.Objective: We assessed the efficacy of presleep protein ingestion on dietary protein digestion and absorption kinetics and overnight muscle protein synthesis rates in older men.Methods: In a randomized, double-blind, parallel design, 48 older men (mean ± SEM age: 72 ± 1 y) ingested 40 g casein (PRO40), 20 g casein (PRO20), 20 g casein plus 1.5 g leucine (PRO20+LEU), or a placebo before sleep. Ingestion of intrinsically l-[1-13C]-phenylalanine- and l-[1-13C]-leucine-labeled protein was combined with intravenous l-[ring-2H5]-phenylalanine and l-[1-13C]-leucine infusions during sleep. Muscle and blood samples were collected throughout overnight sleep.Results: Exogenous phenylalanine appearance rates increased after protein ingestion, but to a greater extent in PRO40 than in PRO20 and PRO20+LEU (P < 0.05). Overnight myofibrillar protein synthesis rates (based on l-[ring-2H5]-phenylalanine) were 0.033% ± 0.002%/h, 0.037% ± 0.003%/h, 0.039% ± 0.002%/h, and 0.044% ± 0.003%/h in placebo, PRO20, PRO20+LEU, and PRO40, respectively, and were higher in PRO40 than in placebo (P = 0.02). Observations were similar based on l-[1-13C]-leucine tracer (placebo: 0.047% ± 0.004%/h and PRO40: 0.058% ± 0.003%/h, P = 0.08). More protein-derived amino acids (l-[1-13C]-phenylalanine) were incorporated into myofibrillar protein in PRO40 than in PRO20 (0.033 ± 0.002 and 0.019 ± 0.002 MPE, respectively, P < 0.001) and tended to be higher than in PRO20+LEU (0.025 ± 0.002 MPE, P = 0.06).Conclusions: Protein ingested before sleep is properly digested and absorbed throughout the night, providing precursors for myofibrillar protein synthesis during sleep in healthy older men. Ingestion of 40 g protein before sleep increases myofibrillar protein synthesis rates during overnight sleep. These findings provide the scientific basis for a novel nutritional strategy to support muscle mass preservation in aging and disease. This trial was registered at www.trialregister.nl as NTR3885.

Sodium nitrate co-ingestion with protein does not augment postprandial muscle protein synthesis rates in older, type 2 diabetes patients.

The age-related anabolic resistance to protein ingestion is suggested to be associated with impairments in insulin-mediated capillary recruitment and postprandial muscle tissue perfusion. The present study investigated whether dietary nitrate co-ingestion with protein improves muscle protein synthesis in older, type 2 diabetes patients. Twenty-four men with type 2 diabetes (72 ± 1 yr, 26.7 ± 1.4 m/kg(2) body mass index, 7.3 ± 0.4% HbA1C) received a primed continuous infusion of l-[ring-(2)H5]phenylalanine and l-[1-(13)C]leucine and ingested 20 g of intrinsically l-[1-(13)C]phenylalanine- and l-[1-(13)C]leucine-labeled protein with (PRONO3) or without (PRO) sodium nitrate (0.15 mmol/kg). Blood and muscle samples were collected to assess protein digestion and absorption kinetics and postprandial muscle protein synthesis rates. Upon protein ingestion, exogenous phenylalanine appearance rates increased in both groups (P < 0.001), resulting in 55 ± 2% and 53 ± 2% of dietary protein-derived amino acids becoming available in the circulation over the 5h postprandial period in the PRO and PRONO3 groups, respectively. Postprandial myofibrillar protein synthesis rates based on l-[ring-(2)H5]phenylalanine did not differ between groups (0.025 ± 0.004 and 0.021 ± 0.007%/h over 0-2 h and 0.032 ± 0.004 and 0.030 ± 0.003%/h over 2-5 h in PRO and PRONO3, respectively, P = 0.7). No differences in incorporation of dietary protein-derived l-[1-(13)C]phenylalanine into de novo myofibrillar protein were observed at 5 h (0.016 ± 0.002 and 0.014 ± 0.002 mole percent excess in PRO and PRONO3, respectively, P = 0.8). Dietary nitrate co-ingestion with protein does not modulate protein digestion and absorption kinetics, nor does it further increase postprandial muscle protein synthesis rates or the incorporation of dietary protein-derived amino acids into de novo myofibrillar protein in older, type 2 diabetes patients.