René Koopman

Aging, exercise and muscle protein metabolism

Aging is accompanied by a progressive loss of skeletal muscle mass and strength, leading to the loss of functional capacity and an increased risk of developing chronic metabolic disease. The age-related loss of skeletal muscle mass is attributed to a disruption in the regulation of skeletal muscle protein turnover, resulting in an imbalance between muscle protein synthesis and degradation. As basal (fasting) muscle protein synthesis rates do not seem to differ substantially between the young and elderly, many research groups have started to focus on the muscle protein synthetic response to the main anabolic stimuli, i.e., food intake and physical activity. Recent studies suggest that the muscle protein synthetic response to food intake is blunted in the elderly. The latter is now believed to represent a key factor responsible for the age-related decline in skeletal muscle mass. Physical activity and/or exercise stimulate postexercise muscle protein accretion in both the young and elderly. However, the latter largely depends on the timed administration of amino acids and/or protein before, during, and/or after exercise. Prolonged resistance type exercise training represents an effective therapeutic strategy to augment skeletal muscle mass and improve functional performance in the elderly. The latter shows that the ability of the muscle protein synthetic machinery to respond to anabolic stimuli is preserved up to very old age. Research is warranted to elucidate the interaction between nutrition, exercise, and the skeletal muscle adaptive response. The latter is needed to define more effective strategies that will maximize the therapeutic benefits of lifestyle intervention in the elderly.

Ingestion of a protein hydrolysate is accompanied by an accelerated in vivo digestion and absorption rate when compared with its intact protein

BACKGROUND It has been suggested that a protein hydrolysate, as opposed to its intact protein, is more easily digested and absorbed from the gut, which results in greater plasma amino acid availability and a greater muscle protein synthetic response. OBJECTIVE We aimed to compare dietary protein digestion and absorption kinetics and the subsequent muscle protein synthetic response to the ingestion of a single bolus of protein hydrolysate compared with its intact protein in vivo in humans. DESIGN Ten elderly men (mean +/- SEM age: 64 +/- 1 y) were randomly assigned to a crossover experiment that involved 2 treatments in which the subjects consumed a 35-g bolus of specifically produced L-[1-(13)C]phenylalanine-labeled intact casein (CAS) or hydrolyzed casein (CASH). Blood and muscle-tissue samples were collected to assess the appearance rate of dietary protein-derived phenylalanine in the circulation and subsequent muscle protein fractional synthetic rate over a 6-h postprandial period. RESULTS The mean (+/-SEM) exogenous phenylalanine appearance rate was 27 +/- 6% higher after ingestion of CASH than after ingestion of CAS (P < 0.001). Splanchnic extraction was significantly lower in CASH compared with CAS treatment (P < 0.01). Plasma amino acid concentrations increased to a greater extent (25-50%) after the ingestion of CASH than after the ingestion of CAS (P < 0.01). Muscle protein synthesis rates averaged 0.054 +/- 0.004% and 0.068 +/- 0.006%/h in the CAS and CASH treatments, respectively (P = 0.10). CONCLUSIONS Ingestion of a protein hydrolysate, as opposed to its intact protein, accelerates protein digestion and absorption from the gut, augments postprandial amino acid availability, and tends to increase the incorporation rate of dietary amino acids into skeletal muscle protein.

Long-term leucine supplementation does not increase muscle mass or strength in healthy elderly men

BACKGROUND It has been reported that the blunted muscle protein synthetic response to food intake in the elderly can be normalized by increasing the leucine content of a meal. OBJECTIVE The objective was to assess the effect of 3 mo of leucine supplementation on muscle mass and strength in healthy elderly men. DESIGN Thirty healthy elderly men with a mean (+/-SEM) age of 71 +/- 4 y and body mass index (BMI; in kg/m(2)) of 26.1 +/- 0.5 were randomly assigned to either a placebo-supplemented (n = 15) or leucine-supplemented (n = 15) group. Leucine or placebo (2.5 g) was administered with each main meal during a 3-mo intervention period. Whole-body insulin sensitivity, muscle strength (one-repetition maximum), muscle mass (measured by computed tomography and dual-energy X-ray absorptiometry), myosin heavy chain isoform distribution, and plasma amino acid and lipid profiles were assessed before, during, and/or after the intervention period. RESULTS No changes in skeletal muscle mass or strength were observed over time in either the leucine- or placebo-supplemented group. No improvements in indexes of whole-body insulin sensitivity (oral glucose insulin sensitivity index and the homeostasis model assessment of insulin resistance), blood glycated hemoglobin content, or the plasma lipid profile were observed. CONCLUSION Long-term leucine supplementation (7.5 g/d) does not augment skeletal muscle mass or strength and does not improve glycemic control or the blood lipid profile in healthy elderly men. This trial was registered at clinicaltrials.gov as NCT00807508.

Intramyocellular lipids form an important substrate source during moderate intensity exercise in endurance‐trained males in a fasted state

Both stable isotope methodology and fluorescence microscopy were applied to define the use of intramuscular triglyceride (IMTG) stores as a substrate source during exercise on a whole‐body as well as on a fibre type‐specific intramyocellular level in trained male cyclists. Following an overnight fast, eight subjects were studied at rest, during 120 min of moderate intensity exercise (60 % maximal oxygen uptake capacity (V̇O2,max)) and 120 min of post‐exercise recovery. Continuous infusions of [U‐13C]palmitate and [6,6‐2H2]glucose were administered at rest and during subsequent exercise to quantify whole‐body plasma free fatty acid (FFA) and glucose oxidation rates and the contribution of other fat sources (sum of muscle‐ plus lipoprotein‐derived TG) and muscle glycogen to total energy expenditure. Fibre type‐specific intramyocellular lipid content was determined in muscle biopsy samples collected before, immediately after and 2 h after exercise. At rest, fat oxidation provided 66 ± 5 % of total energy expenditure, with FFA and other fat sources contributing 48 ± 6 and 17 ± 3 %, respectively. FFA oxidation rates increased during exercise, and correlated well with the change in plasma FFA concentrations. Both the use of other fat sources and muscle glycogen declined with the duration of exercise, whereas plasma glucose production and utilisation increased (P < 0.001). On average, FFA, other fat sources, plasma glucose and muscle glycogen contributed 28 ± 3, 15 ± 2, 12 ± 1 and 45 ± 4 % to total energy expenditure during exercise, respectively. Fluorescence microscopy revealed a 62 ± 7 % net decline in muscle lipid content following exercise in the type I fibres only, with no subsequent change during recovery. We conclude that IMTG stores form an important substrate source during moderate intensity exercise in endurance‐trained male athletes following an overnight fast, with the oxidation rate of muscle‐ plus lipoprotein‐derived TG being decreased with the duration of exercise.

Exercising before protein intake allows for greater use of dietary protein-derived amino acids for de novo muscle protein synthesis in both young and elderly men.

BACKGROUND Sarcopenia seems to be attributed to a blunted muscle protein synthetic response to food intake and exercise. This blunted response could be the result of impaired protein digestion and absorption kinetics and lead to lower postprandial plasma amino acid availability. OBJECTIVE The objective was to compare in vivo dietary protein digestion and absorption kinetics and subsequent postprandial muscle protein synthesis rates at rest and after exercise between young and elderly men. DESIGN Young and elderly men consumed a 20-g bolus of intrinsically L-[1-(13)C]phenylalanine-labeled protein at rest or after exercise. Continuous infusions with L-[ring-(2)H(5)]phenylalanine were applied, and blood and muscle samples were collected to assess in vivo protein digestion and absorption kinetics and subsequent postprandial muscle protein synthesis rates. RESULTS Exogenous phenylalanine appearance rates expressed over time did not differ between groups. No differences were observed in plasma phenylalanine availability between the young (51 ± 2%) and elderly (51 ± 1%) men or between the rest (52 ± 1%) and exercise (49 ± 1%) conditions. Muscle protein synthesis rates calculated from the oral tracer were 0.0620 ± 0.0065%/h and 0.0560 ± 0.0039%/h for the rest condition and 0.0719 ± 0.0057%/h and 0.0727 ± 0.0040%/h for the exercise condition in young and elderly men, respectively (age effect: P = 0.62; exercise effect: P < 0.05; interaction of age and exercise: P = 0.52). CONCLUSIONS Dietary protein digestion and absorption kinetics are not impaired after exercise or at an older age. Exercising before protein intake allows for a greater use of dietary protein-derived amino acids for de novo muscle protein synthesis in both young and elderly men. This trial was registered at clinicaltrials.gov as NCT00557388.

Whole Body Deletion of AMP-activated Protein Kinase β2 Reduces Muscle AMPK Activity and Exercise Capacity

AMP-activated protein kinase (AMPK) β subunits (β1 and β2) provide scaffolds for binding α and γ subunits and contain a carbohydrate-binding module important for regulating enzyme activity. We generated C57Bl/6 mice with germline deletion of AMPK β2 (β2 KO) and examined AMPK expression and activity, exercise capacity, metabolic control during muscle contractions, aminoimidazole carboxamide ribonucleotide (AICAR) sensitivity, and susceptibility to obesity-induced insulin resistance. We find that β2 KO mice are viable and breed normally. β2 KO mice had a reduction in skeletal muscle AMPK α1 and α2 expression despite up-regulation of the β1 isoform. Heart AMPK α2 expression was also reduced but this did not affect resting AMPK α1 or α2 activities. AMPK α1 and α2 activities were not changed in liver, fat, or hypothalamus. AICAR-stimulated glucose uptake but not fatty acid oxidation was impaired in β2 KO mice. During treadmill running β2 KO mice had reduced maximal and endurance exercise capacity, which was associated with lower muscle and heart AMPK activity and reduced levels of muscle and liver glycogen. Reductions in exercise capacity of β2 KO mice were not due to lower muscle mitochondrial content or defects in contraction-stimulated glucose uptake or fatty acid oxidation. When challenged with a high-fat diet β2 KO mice gained more weight and were more susceptible to the development of hyperinsulinemia and glucose intolerance. In summary these data show that deletion of AMPK β2 reduces AMPK activity in skeletal muscle resulting in impaired exercise capacity and the worsening of diet-induced obesity and glucose intolerance.

Dietary Protein Digestion and Absorption Rates and the Subsequent Postprandial Muscle Protein Synthetic Response Do Not Differ between Young and Elderly Men

Impaired digestion and/or absorption of dietary protein lowers postprandial plasma amino acid availability and, as such, could reduce the postprandial muscle protein synthetic response in the elderly. We aimed to compare in vivo dietary protein digestion and absorption and the subsequent postprandial muscle protein synthetic response between young and elderly men. Ten elderly (64 +/- 1 y) and 10 young (23 +/- 1 y) healthy males consumed a single bolus of 35 g specifically produced, intrinsically l-[1-(13)C]phenylalanine-labeled micellar casein (CAS) protein. Furthermore, primed continuous infusions with l-[ring-(2)H(5)]phenylalanine, l-[1-(13)C]leucine, and l-[ring-(2)H(2)]tyrosine were applied and blood and muscle tissue samples were collected to assess the appearance rate of dietary protein-derived phenylalanine in the circulation and the subsequent muscle protein fractional synthetic rate over a 6-h postprandial period. Protein ingestion resulted in a rapid increase in exogenous phenylalanine appearance in both the young and elderly men. Total exogenous phenylalanine appearance rates (expressed as area under the curve) were 39 +/- 3 mumol.6 h.kg(-1) in the young men and 38 +/- 2 mumol.6 h.kg(-1) in the elderly men (P = 0.73). In accordance, splanchnic amino acid extraction did not differ between young (72 +/- 2%) and elderly (73 +/- 1%) volunteers (P = 0.74). Muscle protein synthesis rates, calculated from the oral tracer, were 0.063 +/- 0.006 and 0.054 +/- 0.004%/h in the young and elderly men, respectively, and did not differ between groups (P = 0.27). We conclude that protein digestion and absorption kinetics and the subsequent muscle protein synthetic response following the ingestion of a large bolus of intact CAS are not substantially impaired in healthy, elderly men.

Co-ingestion of leucine with protein does not further augment post-exercise muscle protein synthesis rates in elderly men

Leucine has been suggested to have the potential to modulate muscle protein metabolism by increasing muscle protein synthesis. The objective of this study was to investigate the surplus value of the co-ingestion of free leucine with protein hydrolysate and carbohydrate following physical activity in elderly men. Eight elderly men (mean age 73 +/- 1 years) were randomly assigned to two cross-over treatments consuming either carbohydrate and protein hydrolysate (CHO+PRO) or carbohydrate, protein hydrolysate with additional leucine (CHO+PRO+leu) after performing 30 min of standardized physical activity. Primed, continuous infusions with L-[ring-(13)C(6)]phenylalanine and L-[ring-(2)H(2)]tyrosine were applied, and blood and muscle samples were collected to assess whole-body protein turnover as well as protein fractional synthetic rate in the vastus lateralis muscle over a 6 h period. Whole-body protein breakdown and synthesis rates were not different between treatments. Phenylalanine oxidation rates were significantly lower in the CHO+PRO+leu v. CHO+PRO treatment. As a result, whole-body protein balance was significantly greater in the CHO+PRO+leu compared to the CHO+PRO treatment (23.8 (SEM 0.3) v. 23.2 (SEM 0.3) micromol/kg per h, respectively; P < 0.05). Mixed muscle fractional synthetic rate averaged 0.081 (SEM 0.003) and 0.082 (SEM 0.006) %/h in the CHO+PRO+leu and CHO+PRO treatment, respectively (NS). Co-ingestion of leucine with carbohydrate and protein following physical activity does not further elevate muscle protein fractional synthetic rate in elderly men when ample protein is ingested.

Antibody-directed myostatin inhibition in 21-mo-old mice reveals novel roles for myostatin signaling in skeletal muscle structure and function

Sarcopenia is the progressive loss of skeletal muscle mass and function with advancing age, leading to reduced mobility and quality of life. We tested the hypothesis that antibody‐directed myostatin inhibition would attenuate the decline in mass and function of muscles of aged mice and that apoptosis would be reduced. Eighteen‐month‐old C57BL/6 mice were treated for 14 wk with a once‐weekly injection of saline (control, n=9) or a mouse chimera of anti‐human myostatin antibody (PF‐354, 10 mg/kg; n=12). PF‐354 prevented the age‐related reduction in body mass and increased soleus, gastrocnemius, and quadriceps muscle mass (P<0.05). PF‐354 increased fiber cross‐sectional area by 12% and enhanced maximum in situ force of tibialis anterior (TA) muscles by 35% (P<0.05). PF‐354 increased the proportion of type IIa fibers by 114% (P<0.01) and enhanced activity of oxidative enzymes (SDH) by 39% (P<0.01). PF‐354 reduced markers of apoptosis in TA muscle cross‐sections by 56% (P<0.03) and reduced caspase3 mRNA by 65% (P<0.04). Antibody‐directed myostatin inhibition attenuated the decline in mass and function of muscles of aging mice, in part, by reducing apoptosis. These observations identify novel roles for myostatin in regulation of muscle mass and highlight the therapeutic potential of antibody‐directed myostatin inhibition for sarcopenia.—Murphy, K. T., Koopman, R., Naim, T., Léger, B., Trieu, J., Ibebunjo, C. Lynch, G. S. Antibody‐directed myostatin inhibition in 21‐mo‐old mice reveals novel roles for myostatin signaling in skeletal muscle structure and function. FASEB J. 24, 4433–4442 (2010). www.fasebj.org

Nutritional interventions to promote post-exercise muscle protein synthesis

Resistance exercise is a powerful stimulus to augment muscle protein anabolism, as it can improve the balance between muscle protein synthesis and breakdown. However, the intake of food during post-exercise recovery is necessary for hypertrophy to occur. Therefore, athletes need to ingest protein following exercise to attain a positive protein balance and maximise their skeletal muscle adaptive response. The interaction between exercise and nutrition is not only important for athletes, but is also of important clinical relevance in the elderly. Exercise interventions combined with specific nutritional modulation provide an effective strategy to counteract or reduce the loss of skeletal muscle mass with aging.