The effects of protein type and added leucine on post-exercise muscle protein synthesis

Nutrition plays a key role in facilitating the skeletal muscle adaptive
response to exercise training, thereby modulating muscle reconditioning. A
single bout of exercise stimulates both muscle protein synthesis and, to a
lesser extent, muscle protein breakdown. However, post-exercise protein balance
will remain negative in the absence of food intake. Dietary protein ingestion
stimulates skeletal muscle protein synthesis, inhibits protein breakdown and,
as such, stimulates muscle protein accretion following both resistance as well
as endurance type exercise. This facilitates the skeletal muscle adaptive
response to each successive exercise bout, resulting in more effective muscle
reconditioning.
Though it has been well established that dietary protein ingestion effectively
stimulates muscle protein synthesis rates both at rest and following exercise,
there is much less information on the amount and type of dietary protein that
should be ingested to maximize post-exercise muscle protein synthesis rates.
Moore et al. reported that post-exercise muscle protein synthesis rates
increase with the ingestion of greater amounts of protein, reaching maximal
stimulation after ingesting 20 g (egg) protein. This has led to the general
advice to take at least 20 g of a high quality protein after a workout.
Improvements in post-exercise protein balance and/or greater muscle protein
synthesis rates have been reported following the ingestion of various types of
protein, including whey protein, casein protein, soy protein, casein protein
hydrolysate, egg protein, and whole-milk and/or fat-free milk. To date, only a
few studies have directly compared the post-exercise muscle protein synthetic
responses to the ingestion of different types of protein. For example, Tang and
colleagues compared the muscle protein synthetic response between whey, casein,
and soy protein after resistance exercise in young men and demonstrated that
whey stimulated greater muscle protein synthesis rates than both casein and
soy. Soy was intermediate in that it was less effective than whey protein but
more effective than casein at stimulating muscle protein synthesis after
resistance exercise. Previous work from our own lab corroborated the findings
of Tang and colleagues by demonstrating that whey protein stimulated greater
muscle protein synthesis rates in older men than did casein or casein
hydrolysate. However, bovine milk protein (which is 80% casein and 20% whey
protein) seems to offer an anabolic advantage over soy protein after resistance
exercise. To date, no studies have directly compared milk protein to its
isolated constituent proteins whey and casein. The differences in the muscle
protein synthetic response to the ingestion of various protein sources can be
attributed to differences in protein digestion and absorption kinetics as well
as amino acid composition. Both protein digestion and absorption kinetics as
well as leucine content seem to be instrumental to stimulate muscle protein
synthesis, but are certainly not the only factors responsible for maximizing
post-exercise muscle protein synthesis rates. Whereas casein and whey protein
have been applied in research to investigate the impact of slowly versus more
rapidly digestible proteins, this work is certainly not representative of the
digestion and absorption kinetics of milk protein. Furthermore, recent work in
our group has extended on previous work by showing that the anabolic response
to casein ingestion can be increased by co-ingesting free leucine. We
hypothesize that milk protein is as effective as whey protein at promoting
muscle protein synthesis during recovery from exercise. Furthermore, we
speculate that co-ingestion of free leucine with a typical plant based protein
(soy protein) may further augment post-exercise muscle protein synthesis rates
making soy protein equally effective as milk protein to stimulate post-exercise
muscle protein synthesis.
Besides rehydration, a rapid restoration of depleted muscle glycogen stores is
an important target following completion of a single bout of exercise.
Therefore, recovery drinks generally contain both carbohydrate and protein.
Interestingly, the combined ingestion of protein and carbohydrate have been
shown to accelerate muscle glycogen repletion when less than optimum amounts of
carbohydrate are ingested during the first few hours of post-exercise recovery.
Furthermore, though carbohydrate ingestion does not modulate post-exercise
muscle protein synthesis rates they may attenuate the post-exercise rise in
muscle protein breakdown rates, thereby further improving protein balance.
It is evident that more applied work is needed to define the post-exercise
anabolic properties of various protein sources when ingested in combination
with carbohydrate, as the combined ingestion will modulate both digestion and
absorption kinetics of the various protein sources and the associated
post-prandial hormonal response. This project compares the muscle protein
synthetic response to the ingestion of various dairy protein sources (whey,
casein, and milk protein) as well as a plant-based protein (soy protein) when
ingested with carbohydrate during recovery from an intense bout of resistance
type exercise in young males. Furthermore, we will assess the impact of adding
free leucine to a soy protein plus carbohydrate beverage as a strategy to
further augment the properties of soy protein to increase post-exercise muscle
protein synthesis rates. The findings from the proposed work will yield new
insights within the field of applied exercise physiology by defining the best
protein source to consume with carbohydrate to maximize the skeletal muscle
adaptive response to exercise training.

Overall Objective: To define the properties of whey, casein, milk protein, as
well as soy protein with and without additional leucine to augment
post-exercise muscle protein synthesis when co-ingested with a carbohydrate
containing recovery drink.

Study arm 1
Objective: To compare the anabolic properties of a carbohydrate placebo vs.
whey, casein, and milk protein when co-ingested with a carbohydrate containing
beverage on post-exercise muscle protein synthesis rates in young males.

Study arm 2
Objective: To compare the anabolic properties of whey protein, soy protein, and
soy protein with additional free leucine to match that found in whey protein
when ingested with a carbohydrate containing beverage on post-exercise muscle
protein synthesis rates in young males.

STUDY DESIGN
The present study employs a parallel group design. In total, 96 (6 groups of 12
subjects spread over 2 study arms) healthy young male subjects will be included
in the study. Subjects will be randomly assigned (using permuted blocks) to
consume either carbohydrate, or carbohydrate with whey, casein, milk protein,
soy protein, or soy protein and additional leucine. During the test day,
subjects will perform a bout of concurrent training involving resistance
exercise (press, extensionl) and endurance exercise (cycle ergometry) and
immediately afterwards consume their respective nutritional treatment. Study
arm 2 will be performed in parallel with study arm 1 to allow the whey protein
group in Study arm 1 to act as a positive control group in Study arm 2.

Screening & pre-testing
Subjects will participate in one screening session in which leg volume, blood
pressure, body weight and composition (DEXA) will be assessed. Subjects will be
asked to fill in a medical questionnaire inquiring about their general health,
medical history, use of medication and sports activities. Additionally, all
subjects will participate in an orientation session for familiarization with
the exercise equipment. Following the orientation subjects will undergo
strength testing to determine their single repetition maximum on each exercise
followed by a test of maximum wattage on a cycle ergometer.
Body weight and height will be assessed, as well as body fat composition
(percentage) via a Dual Energy X-ray Absorptiometry (DEXA) scan. In the event
of an unexpected medical finding during the screening, subjects will always be
notified. If a subject does not want to receive this notification he cannot
participate in the study. Following the DEXA, subjects will undergo a blood
pressure reading which involves placing an inflatable cuff around the upper
the. Blood pressure will be taken consecutaively 3 times and the average will
be recorded. During the familiarization session with the exercise equipment,
proper lifting technique will be demonstrated for knee extension exercise.
Guided-motion exercise machines will be used to promote proper form and for the
subject*s personal safety. Prior to the determination of the subjects* one
repetition maximum (1RM), they will perform 2 sets of each respective exercise
for 10 repetitions on the exercise machine at a light load. Thereafter, the
load will be increased after each successful lift until failure. A 2 minute
rest period will be allowed between attempts. A repetition will be considered
valid if the subject uses proper form and is able to complete the entire lift
in a controlled manner without assistance. Maximum wattage will determined
during an incremental test to volitional fatigue. Subjects will commence
cycling at a workload equivalent to 2 W/kg for 150 sec. Thereafter, the
workload will be increased by 25 W every 150 sec until volitional fatigue,
defined as the inability to maintain a cadence > 70 revolutions/min.

Diet and activity prior to testing
All subjects will consume a standardized dinner the evening before the test
day. This standardized dinner is an *Aviko maaltijdpannetje* and will be
purchased at a regular supermarket in Maastricht. The expiration date from the
manufacturer will be checked. Meanwhile, the meals will be stored in an
appropriate freezer of the *dietary-kitchen* at the department of Human
Biology. The subjects will receive the meal after the screening test in a
thermal bag. The precise composition and preparation methods are described on
the label of the product (Appendix D4.1). The subjects will be instructed to
store the meal in a freezer until preparation and to prepare the meal
themselves according to the instructions on the label. All subjects will be
instructed to refrain from any sort of heavy physical exercise and to keep
their diet as constant as possible 3 d before the test day. In addition,
subjects will be asked to record their food intake for 48 h before the start of
the test day in a food diary that will be provided during the screening.

Experimental test day
Each subject will participate in 1 experimental test day lasting ~8.5 h. During
the test day, subjects will perform a single bout of concurrent exercise (both
endurance and resistance) and will ingest their randomly assigned nutrient
treatment beverage. The use of [13C6] phenylalanine and (3,5-D2)-tyrosine will
allow us to assess mixed, myofibrillar, and mitochondrial protein synthesis
rates (FSR) during the post-exercise recovery period in an in vivo human
setting.
Protocol
An outline of the study protocol is shown in Figure 1. At 8.00 am, following an
overnight fast, subjects will arrive at the laboratory by car or public
transportation. Subjects will rest in a supine position and a Teflon catheter
will be inserted into an antecubital vein for intravenous stable isotope
infusion. A second Teflon catheter will be inserted in a heated dorsal hand
vein of the contralateral arm and placed in a hot-box (60°C) for arterialized
blood sampling. Following basal blood collection (8 mL; t=-150 min), the plasma
phenylalanine and tyrosine pool will be primed with a single intravenous dose
of tracer and a continuous tracer infusion will commence. Arterialized blood
samples (8 mL) will then be drawn at t= -90, -30, and 0. Starting at -45,
subjects will perform a bout of resistance exercise consisting of two different
leg exercises (seated knee extension, supine leg press) on a guided-motion
exercise machine for 4 sets at a load they can lift for 10 - 12 repetitions.
Subjects will be allowed to rest 2 minutes in between each exercise set and the
load will be adjusted to maintain the desired 10-12 repetitions. Immediately
following the resistance exercise, subjects will perform a bout of
endurance-type exercise on a cycle ergometer at ~60% of maximum wattage for 30
minutes. Immediately after the exercise bout subjects will return to the
resting supine position and arterialized blood sample will be drawn.
Afterwards, a muscle biopsy will be collected (t=0 min). Subjects will then
receive their respective nutritional treatment (t= 0). Arterialized blood
samples (8 ml) will be collected at t= 15, 30, 60, 90 and 120 min during the
postprandial (fed) period. At 120 min in the postprandial period, the second
muscle biopsy will be taken from the same leg as the last biopsy but from a
different incision. Subsequently, arterialized blood samples (8 ml) will be
collected at t=150, 180, 240, 300, and 360 min. Finally, at 360 min a third
muscle biopsy will be taken. In total, three muscle biopsies will be taken
through three separate incisions during the trial. The muscle biopsies
(immediately, 2 h, and 6 h after exercise) will allow us to measure the
temporal response of muscle protein synthesis between the different consumed
protein sources after exercise. It is generally assumed that *peak* stimulation
of muscle protein synthesis rates is more meaningful in predicting phenotypic
outcomes (muscle hypertrophy). However, peak muscle protein synthesis rates may
appear at different time points depending on the protein source consumed.
Obtaining a muscle biopsy at 2 h post-exercise will allow us to determine peak
muscle protein synthesis rates between the different consumed protein sources.
However, resistance exercise-induced muscle protein synthesis rates can extend
beyond this 2 h time point and thus obtaining an third muscle biopsy at 6 h
will also allow us to obtain physiological relevant information with regards to
whether there are differences between protein sources in the duration of the
anabolic response to combined resistance and endurance exercise.

Randomized, double-blind, parallel group design in which subjects are randomly
assigned to one of the 6 different nutrition treatment groups: carbohydrate
(placebo), whey protein, casein protein, milk protein, soy protein, soy protein
+ additional free leucine.

DUAL-ENERGY X-RAY ABSORPTIOMETRY (DXA) SCAN
A dual-energy x-ray absorptiometry (DXA) scan will be used to asses the amount
of muscle in your whole body. This is a painless non-invasive procedure that
uses a small amount of radiation to assess how much fat, bone, and lean tissue
you have in your entire body. This procedure will take place at the McMaster
University Medical Centre and you will be scanned once before starting the
study.

Potential Risks. The DXA scan involves exposure to radiation. The radiation
dose is ~0.001 microSieverts, which is about the amount of radiation an average
person receives every 24 hours from natural radiation in our environment (i.e.,
from the sun, television and computer screens etc).

MUSCLE BIOPSY SAMPLING
This procedure involves the removal of a small piece of muscle tissue using a
sterile hollow needle. A trained investigator will clean an area over your
quadriceps muscle (vastus lateralis) with antiseptic solution and then inject a
small amount of local anaesthetic ("freezing") into and under your skin. The
investigator will then make a small incision (~4-5 mm) in your skin in order to
create an opening through which to put the biopsy needle into your thigh
muscle. There will be a small amount of bleeding from the incision, but this
will be minimal. The investigator will then quickly cut off a very small piece
of muscle (~150 mg; about the size of half of a pea or an eraser on the end of
a pencil) and remove the needle from your leg. During the time that the sample
is being taken (~5 sec), you may feel the sensation of deep pressure in your
thigh and on some occasions this is moderately painful. However, the discomfort
very quickly passes and you are quite capable of performing exercise and daily
activities. In some cases, you may experience dizziness, cold sweat, increased
heart rate or difficulty in breathing as a reaction to the needle prick when
anaesthetizing your thigh or the insertion of the biopsy needle, similar to the
reactions that you might have when giving blood. However, these sensations will
subside fairly quickly with rest and elevation of your feet. If you do feel
these sensations or in any way faint at all then please let the investigators
know as soon as possible.

Following the biopsies, the incisions will be closed with a steri-strip, and
wrapped with a tensor bandage. You should refrain from excessive muscle use for
the remainder of the day. Once the anaesthetic wears off, your leg may feel
tight and often there is the sensation of a deep bruise or "Charlie Horse".
Analgesics (pain killers) such as Tylenol or Ibuprofen (Motrin) are acceptable
if you experience significant pain associated with the biopsy. It is also
beneficial to periodically apply an ice pack to the biopsy site during 24-48
hours after the biopsy, as this will help to reduce any swelling and any
residual soreness. The following day your leg may feel uncomfortable when going
down stairs. The tightness in the muscle usually disappears within 2 days and
subjects routinely begin exercising at normal capacity within a day. In order
to allow the incisions to heal properly and minimize any risk of infection, you
should avoid prolonged submersion in water for 2-3 days. Daily showers are
acceptable so long as you pat the area dry following the shower, but baths,
swimming, saunas, or any water immersion should be avoided for at least 5 days
following the biopsy procedure.
Potential Risks. The biopsy technique is routinely used in physiological
research, and complications are rare provided that proper precautions are
taken. However, there is a risk of internal bleeding at the site of the biopsy,
which can result in bruising and temporary discoloration of the skin. On
occasion a small lump may form under the site of the incision, but this
normally disappears within 2-3 weeks. As with any incision there is also a risk
of infection, however this risk is virtually eliminated through proper
cleansing of the area and daily changing of wound coverings. If the incision
does not heal within a few days or you are in any way concerned about
inflammation or infection (usually this means that the incision site is red,
hot, swollen and/or itchy), please remove the stitch, clean the cut and contact
us immediately. In very rare occasions, there can be damage to a superficial
sensory nerve, which will result in temporary numbness in the area. There is
also an extremely remote chance that you will be allergic to the local
anaesthetic (lidocaine); the real incidence of lidocaine allergy is unknown.

In past experience with healthy young subjects, approximately 1 in 2,200 have
experienced a local skin infection; 1 in 500 have experienced a small lump at
the site of the biopsy (in all cases this disappeared within ~2-3 weeks using
local massage); 1 in 1,500 have experienced a temporary loss of sensation in
the skin at the site of incision (an area of numbness about the size of a
quarter which lasted up to 3-4 months), and 1 in 30 have experienced bruising
around the site of incision which lasted for ~4-5 days. To the best of our
knowledge, no adverse reactions have been reported by older subjects who have
undergone the muscle biopsy procedure. While there is also a theoretical risk
of damage to a small motor nerve branch (that is used to allow your muscle to
move) of the medial vastus lateralis, this has never been seen in over 9,500
biopsies performed by the investigators at McMaster University in Canada
(Suction-modified Bergström muscle biopsy technique: experience with 13,500
procedures. Tarnopolsky MA, Pearce E, Smith K, Lach B. Muscle Nerve. 2011
May;43(5):717-25). Hence, the risk of damaging a small motor nerve branch is
remote.

FOREARM VENOUS CATHETERIZATION & STABLE ISOTOPE-LABELLED AMINO ACID INFUSION
A study investigator familiar with this procedure will first place a needle
into veins in both of your arms (blood vessels that takes blood from your arms
to your heart) to take a sample of your blood. The needles will be placed in
what are called your antecubital veins. In this experiment, the total amount of
blood taken from the antecubital veins will be 112 ml, which is ~1/5 of a cup
of blood. You will then receive, through a small catheter placed in your arm,
an infusion (slow measured amount) of an amino acid (a small component of
protein) solution. The amino acid will be dissolved in saline (a salt solution
similar to your blood). The amino acid will be labelled with a stable isotope
of carbon, hydrogen, or nitrogen. An isotope is slightly heavier form of these
elements. Since the isotope is stable (i.e., non-radioactive) it poses no
health risk to you due to radioactive exposure. In addition, a certain fraction
of all of the carbon, hydrogen, and nitrogen within your body is already in the
same form as that of the stable isotope. Hence, the infusion of the stable
isotope-labelled amino acid will simply result in a slight increase in the
amount of stable isotope within your body; we refer to this as "enriching" the
amount of stable isotope within your body. This enrichment will not remain
high, however, and will be back to pre-infusion levels within a few days. All
of the infused solutions are prepared under sterile conditions and are filtered
through a very selective filter prior to entering your body. All solutions that
enter your body do not contain, except for the amino acid, anything that will
affect your health.

Potential Risks. The insertion of catheters for blood sampling is a common
medical practice and involves few risks if proper precautions are taken. The
catheters are inserted under completely sterile conditions; however, there is a
theoretical risk of infection. There is also a chance of internal bleeding if
adequate pressure is not maintained upon removal of the catheter. This may
cause some minor discomfort and could result in bruising/skin discoloration,
which could last for up to a few weeks. In very rare occasions, trauma to the
vessel wall could result in the formation of a small blood clot, which could
travel through the bloodstream and become lodged in a smaller vessel. However,
we have never experienced such a complication after several thousand catheter
placements. Despite all precautions, there is a theoretical risk (less than 1
in 1,000,000) that you could have a rapid drop in blood pressure due to some
small bacterial contamination of the infusion solution (infusate). This has
never occurred in our experience.

BENEFITS:
There are no direct benefits to the subjects.