Optimizing the amount of dietary protein to maximally stimulate post-exercise muscle protein synthesis in elderly men.

Aging is accompanied by a progressive decline in skeletal muscle mass. This
age-related loss of muscle mass is attributed to an imbalance between rates of
muscle protein synthesis and breakdown. As rates of basal muscle protein
synthesis does not seem to differ between the young and elderly, most research
has focused on potential impairments in the muscle protein synthetic response
to the main anabolic stimuli, ie food intake and exercise. Skeletal muscle
protein synthesis is highly responsive to food intake in healthy young adults.
Recent data indicate that the muscle protein synthetic response to food intake
may be blunted in the elderly. This proposed anabolic resistance is now being
regarded as a key factor in the etiology of sarcopenia. Effective strategies to
prevent and/or counteract the age related loss of muscle mass include protein
supplementation, preferably in combination with resistance type exercise
training. Recent studies show the efficacy of dietary protein supplementation
to improve muscle strength and function in frail elderly (14) and to further
augment the gains in muscle mass and function when combined with resistance
type exercise training (13). As combining proper nutrition with exercise has
numerous synergistic benefits, nutrition research is warranted to define the
optimal amount, type and timing of protein that needs to be consumed to
maximize post-exercise muscle protein synthesis rates in the older population.

Improvements in protein balance and/or higher muscle protein synthesis rates
have been reported following the ingestion of various types of dietary protein:
whey protein (2), casein protein (2), soy protein (15), casein protein
hydrolysate (8, 9), egg protein (10), and wholemilk and/or fat-free milk (5,
15). It seems obvious to question which source of dietary protein is most
effective to promote muscle protein synthesis. There is only limited research
comparing the efficacy of the ingestion of different proteins sources on the
post-exercise muscle protein synthetic response. As such, it is difficult to
identify a specific protein source that is most potentiating. The amount and
timing of protein administration, the leucine content of the protein, and the
digestion and absorption kinetics of the protein source may all modulate the
post-exercise muscle protein synthetic response. Milk protein and its main
isolated constituents, whey and casein, are the most widely studied dietary
proteins. Casein and whey protein seem to have distinct anabolic properties,
which are attributed to differences in digestion and absorption kinetics (1-4).
Whereas whey protein is a soluble protein that leads to rapid intestinal
absorption, intact casein clots in the stomach delaying its digestion and
absorption and the subsequent release of amino acids in the circulation (7).
The fast, but transient rise in plasma amino acid concentration after whey
protein ingestion can lead to higher protein synthesis and oxidation rates (1,
3, 4). In addition to intrinsic differences in digestion and absorption rate,
it has been suggested that whey protein can more effectively stimulate
(post-exercise) protein synthesis due to its greater leucine content when
compared to casein (11, 12).

In young men it was reported that ingestion of 20 g intact whole egg protein is
sufficient to maximally stimulate MPS after resistance exercise (Moore, 2009).
In older men it was found that the optimal whey protein dose for non-frail
older adults is 20 g in order to increase myofibrillar MPS above fasting rates.
However, resistance exercise increased MPS in the elderly to the greatest
extent with 40 g of whey ingestion (Yang 2012). Another study (Robinson, 2009)
on unilateral resistance exercise in middle-aged men found that ingestion of
170 g of beef protein (36 g protein) is required to stimulate a rise in
myofibrillar MPS above that seen with lower doses. Since ~40g of protein was
the maximal dose in these studies and no plateauing effect occurred, it is
scientifically relevant to test 60 g protein and identify the upper limit to
post-exercise dietary protein ingestion. In addition, these studies lack data
on whole body protein kinetics, which explains how excess protein is utilized
even when muscle sensitivity towards additional protein is no longer being
achieved. Lastly, the efficacy of co-ingesting leucine along with dairy protein
after whole-body resistance exercise has never been investigated.

To define the amount of dietary protein required to optimally stimulate
post-exercise muscle protein synthesis in the older population.

To assess whether co-ingesting 1.5 g of free leucine (contained within 15 g
dairy protein) along with 15 g protein can achieve a maximal muscle protein
synthetic response as opposed to simply increasing the amount of protein
consumed.

Screening Trial:
When volunteers respond to the advertisement, we contact them by e-mail/phone
and briefly explain the study. We will provide them with the information
brochure and the informed consent (which they will bring during the screening).
To assess whether volunteers are eligible to participate in this study, we will
invite them to the University for a screening. They will be screened in a
fasting and rested state, meaning that they are not allowed to eat and drink
(except for water) from 22h00 the night prior to the screening, and that they
have to come to the University by car or public transport. Before we start the
screening, we will explain the entire experimental trial and answer any
potential questions. We will then ask them to read, fill out, and sign the
informed consent form. After signing the informed consent form, we will start
the screening by going through the medical questionnaire to assess their
general health, use of medication, and physical activity. Subsequently, we
place a catheter into the antecubital vein for blood collection. After the
first (basal) blood draw, subjects receive a drink containing 82.5 g of
dextrose monohydrate, and blood glucose will be measured every 30 minutes for 2
hours (2 h oral glucose tolerance test, OGTT). The glucose drinks will be
prepared in the dietary kitchen of the department of human biology. In the
basal blood sample we will also determine HbA1c as a marker of long-term
hyperglycemia. When subjects appear to be glucose intolerant based on these
measurements according to the WHO (16), they cannot participate in this study.
Furthermore, blood pressure will be measured. Subjects with hypertension
(>140/90 mmHg) will also be excluded from participation. After completion of
the 2 h OGTT and providing the subject with a sandwich, we will assess body
composition by performing a dual-energy X-ray absorptiometry (DEXA) scan and
measure body height, body weight, and leg volume. DEXA is a simple and
non-invasive procedure, which will take place at the University. Subjects will
be instructed to lie down on a table and stay motionless for approximately 3
minutes during which the body scan takes place. Performing the above mentioned
tests allow us to characterize the participants. In case of an unexpected
medical finding, it is our duty to inform the subjects. If a participant does
not want to receive this information, he cannot participate in this study.
During the screening visit, subjects will be asked to fill in an exercise
questionnaire to gather information on past and present exercise habits

On a separate day, successfully screened subjects will return to the university
to be familiarized and tested for strength on the exercise machines. However,
prior to any physical exertion, all subjects will have an electrocardiogram
(ECG) measured at rest and during a submaximal workload on an exercise bike,
consisting of 10 min cycling at 70% of estimated heart rate max (220 - age x
0.70). A cardiologist at the University Hospital will analyze all ECG data and
determine if the subject can safely tolerate further exercise testing.
Following approval by the cardiologist, subjects will then be instructed on
proper weight-lifting technique on each exercise machine (chest press, lat
pulldown, leg-press and leg-extension) and complete a standardized testing
protocol to determine a measurement of maximal strength (1RM) on each exercise
machine. The testing protocol requires that the subjects complete sets on each
exercise machine increasing in weight until volitional fatigue occurs, ideally
occurring between 3-6 repetitions on the heaviest weight. The attained
strength data will be compared to previously published data and used to
calculate an estimation of 1RM

Experimental Trial:

After the subjects arrive at the University, we will check if they met all the
requirements described in section 3.2, ask them to put on their shorts, and
assign them to a bed. A catheter will be inserted into the antecubital vein for
basal blood collection (10 mL, t=-120) and subsequent stable isotope amino acid
tracer infusion. A second catheter will be inserted into antecubital vein of
the contralateral arm for blood collection. After a pre-infusion period of 60
minutes (t=-60), the participant will begin to complete the exercise regimen,
which will last 60 minutes. The exercise bout will consist of a 5-minute
warm-up on a cycle ergometer, followed by 2 sets of chest press and lat
pulldown, and 5 sets of leg-press and 4 sets of leg-extension. For each of the
upper body exercises, the workload will be set at 80% of 1-RM (10 repetitions
per set). For each of the lower body exercises, the workload of the first set
will be set at 50% (10 repetitions), then increased to 70% (10 repetitions per
set) for the remaining 4 sets. Resting periods of 2 minutes will be allowed
between all sets and exercises. After completing the exercise, participants
will have a muscle biopsy taken (t=0) from the vastus lateralis muscle of a
randomized leg. The participant will then ingest a randomized dose of dairy
protein in the amount of 0, 15, 30, 45 g or 15 g + 1.5 g free leucine dissolved
in 500mL of vanilla-flavoured water. Following the ingestion of the protein
dose another muscle biopsie, at 6 hours (t=360) will be sampled from the
vastus lateralis of a randomized leg.
The timing of the post-exercise muscle biopsies is intended to most accurately
determine aggregate (0h - 6h) rates of muscle protein synthesis (MPS). It is
generally assumed that peak MPS rates are more meaningful in predicting
long-term phenotypic outcomes (muscle hypertrophy) than MPS calculated over a
longer period. However, taking a muscle biopsy at t=300 allows for the
determination of the stimulatory effect of each separate protein dose during
the respective digestion periods, which is physiologically relevant information
with regard to the anabolic response to feeding. Muscle biopsies will be taken
through separate incisions divided over the two legs to reduce discomfort. A
total of 12 blood samples (10 mL) will be collected throughout the experimental
trial. A background blood sample will be collected immediately prior to the
start of the tracer infusion along with 2 samples at 2 and 1 hour before the
drink. Sampling frequency will increase to every 15min directly after the
ingestion of the drink for a period of one hour. After the first hour, blood
sampling will occur every half hour for the remaining 3 hours (t=60-300)

The exercise bout will consist of a 5-minute warm-up on a cycle ergometer,
followed by 2 sets of chest press and lat pulldown, and 5 sets of leg-press and
4 sets of leg-extension. For each of the upper body exercises, the workload
will be set at 80% of 1-RM (10 repetitions per set). For the leg press, the
workload of the first set will be set at 50% (10 repetitions), then increased
to 70% (10 repetitions per set) for the remaining 4 sets. For the leg
extension, all 4 sets are done at 80% 1RM. Resting periods of 2 minutes will be
allowed between all sets and exercises. After completing the exercise,
participants will have a muscle biopsy taken and will then ingest a randomized
dose of dairy protein in the amount of 0, 15, 30, 45 g or 15 g + 1.5 g free
leucine dissolved in 500mL of vanilla-flavoured water.

The burden and risks associated with participation are small. Insertion of the
catheters is comparable to a blood draw and could result in a small hematoma.
Muscle biopsies will be taken under local anesthesia by an experienced
physician, but may cause some minor discomfort for maximally up to 24 h after
completion. The discomfort is comparable to muscle soreness or the pain one has
after bumping into a table. We will take 5 (6mL) and 12 blood samples (10 mL)
during the screening and experimental trial respectively. The total amount of
blood we draw is less than half the amount of a blood donation and will be
completely restored in approximately 1 month. For both the screening and the
experimental trial, participants have to be fasted, so they are not allowed to
eat and drink (except for water) from 22h00 the evening before. Also, 2 days
prior to the experimental trial participants should keep their diet as constant
as possible, do not perform any type of intense physical exercise, and do not
consume alcohol. Furthermore, we will ask the participants to fill out a
dietary record for 2 days prior to the experimental trial.
The types of protein used in the beverages are commercially available food
products. Therefore, the test beverage does not form any health risks. The
stable isotope amino acids tracers applied in this experiment are not
radioactive and are completely safe. The production of the tracers for
intravenous administration will occur in a sterile environment according to GMP
guidelines.
There is no risk associated with the DEXA scan. The radiation dose emitted
during a DEXA scan is 0.001 mSv. This is a very low exposure compared to the
total background radiation in the Netherlands, which is ~2.5 mSv/year. For
comparison, the radiation dose during a flight higher than 10 km is 0.005
mSV•h-1.
There is no direct benefit for the participants, only their contribution to
scientific knowledge and nutritional strategies that prevent muscle loss in the
elderly, which will be obtained from this study and can be used in the future.