Exercise-induced alterations in diurnal rates of muscle protein synthesis in young men

From p10 of C1 protocol document:

Skeletal muscle plays an important role as a metabolic organ and accounts for
30-45% of whole body protein metabolism. In addition, skeletal muscle plays a
central role in the regulation of whole body glucose regulation. Aging and
disease, such as cancer or COPD, are associated with significant losses of
muscle mass. Such losses of muscle mass is associated with poor physical
performance and increases the likelihood of developing type II diabetes type,
obesity and osteoporosis. Nutritional and/or physical activity interventions
can influence skeletal muscle metabolism to achieve a more anabolic state,
generally allowing for an increase or maintenance of muscle over a prolonged
duration. Within skeletal muscle tissue, such an anabolic state is achieved
when the on-going process of muscle protein synthesis occurs at a faster rate
than the process of muscle protein breakdown. Thus, a key analytical target of
research into the regulation of skeletal muscle mass is skeletal muscle protein
synthesis, which is most credibly obtained by the use of stable isotope
infusion techniques. Currently, the use of short-term (<9 hr) stable
isotopically labelled amino acid tracer infusions are utilized to provide a
mechanistic assessment of the shifts in rates of muscle protein synthesis that
accompany certain phenotypic states (young vs. aged vs. disease) or various
nutritional and physical activity interventions. However, the assessment of
muscle protein synthesis using amino acid tracer infusions becomes much less
reliable after a period longer than approximately 12 hrs and requires a high
level of standardization in that test subjects must stay within the laboratory
setting for the duration of the research protocol. While amino acid tracers can
reliably assess acute interactions (e.g., single meal-based), such limitations
in the amino acid tracer infusion method prevent scientific investigation into
the impact of nutritional and physical activity interventions in the context
real-life conditions carried out over an extended period of time (days-weeks).

From p12 of C1 protocol document:

To assess how diurnal rates of muscle protein synthesis are altered during a
short-term (3-day) implementation of resistance exercise training when compared
to normal daily activity.

From p12-14 of C1 protocol document:

3.1 Screening

When volunteers respond to the advertisement, we will 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 screening at 08:30. 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 will
assess body composition by performing a dual-energy X-ray absorptiometry (DEXA)
scan and measure body height and body weight. 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.

Following the DEXA scan and anthropometric measurements, subjects will be
familiarized and tested for strength on the exercise machines. Subjects will
then be instructed on proper single legged weight-lifting technique on each
exercise machine (leg-press and leg-extension) and complete a standardized
testing protocol to determine a measurement of maximal strength (1RM) for each
leg 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. Following the determination of
1RM, subjects will be scheduled for their experimental testing days (dosing
day, muscle biopsies, exercise sessions) before leaving for home.

3.2 Experimental trial

The experimental protocol consists of a 7-day testing period (Figure 1).
Subjects will begin on day 1 by tracking normal dietary intake and daily
activity using diary entries and will also being tracking steps taken with an
*Actical* accelerometer which will be worn on the their wrist. On day 2,
subjects will initiate the heavy water ingestion protocol, which will last for
the remaining 6 days of the experiment. A detailed methodological description
of the heavy water approach to measuring muscle protein synthesis is written in
section 65.3.4. Subjects will report to the University at 0800 on the first day
of the heavy water dosing protocol for the purpose of gathering baseline
samples and supervising of the initiation of the heavy water (deuterium oxide)
dosing protocol. This *dosing day* consists of the ingestion of 50ml of 70 APE
enriched heavy water 8 times during waking hours, at: 0830, 0930, 1100, 1230,
1400, 1530, 1700, 1830 hr. Saliva and blood samples will be taken 4 times
during the dosing day (0800 * basal, 1100, 1400, 1530) to track the rise in
body water and plasma enrichment. After ingesting the 6th of 8 doses of heavy
water, at approximately 1530 h, subjects will be free to go home where they
will ingest the final 2 heavy water doses of the dosing day. For the remainder
of the experiment, subjects will ingest one 50ml dose of 70 APE enriched heavy
water each day upon waking to maintain 1-2 APE enrichment in body water (see
section 5.3.4). On day 4 of the experiment, subjects will report back to the
University at 1100 to have a muscle biopsy taken from each leg, to have plasma
and saliva samples taken and to complete the first unilateral resistance
exercise session, beginning at 1200. Subjects will report to the University at
1200 on the following 2 days to have saliva and blood sampled and to complete
the same unilateral exercise protocol. Subjects will complete, in total, 3
consecutive sessions of unilateral resistance exercise. Subjects will be
provided with a drink containing 30g whey protein dissolved in 350ml water
after each exercise training session and can go home afterwards. On the last
experimental day (day 7), subjects will ingest their final dose of heavy water
before coming into the University at 1100 to have their final saliva and blood
samples collected as well as their final two biopsies. The experiment is
finalized once the samples have been collected.

From p17 of C1 protocol document:

To maximize muscle protein accretion, participants will perform a 3 day
unilateral lower body resistance-type exercise training protocol under personal
supervision. The training consists of a 5 min warm-up on a cycle ergometer and
4 sets on the leg-press and leg-extension machines (Technogym, Rotterdam, the
Netherlands). The workload is set at 80% of the subjects* one-repetition
maximum (8*10 repetitions) to stimulate muscle hypertrophy. Resting periods of
2 min are allowed between sets and 2 min between exercises.

The burden and risks associated with participation are minimal. Saliva sampling
(9 samples) is risk-free. Blood sampling is minimal (9 x 10mL total) and will
be conducted using catheter placement during the dosing day (4 blood samples)
and with the single venipuncture method on each of the following 5 days (1
sample per day). Muscle biopsies will be taken under local anesthesia by an
experienced physician, but may cause some minor discomfort up to 24 h after
completion. The discomfort is comparable to muscle soreness or the pain one has
after bumping into a table. Participants will come to the University for seven
visits: 1 screening visit (1.5 hours), 1 dosing day (day 2 - ~9 hours) and 5
times (1 hour each) for tissue sampling (blood, saliva, muscle) and/or the
exercise sessions. During the screening visit, we will perform a DEXA scan and
determine maximal strength (1RM) on each exercise machine. Furthermore, we will
ask the participants to fill out a medical questionnaire. For the duration of
the study protocol, subjects will ingest doses of 50ml of 70% deuterium oxide
(2H2O) to enrich the body water pool to approximately 1 APE (atom percent
excess, similar to percent). Deuterium oxide dosing to achieve a body water
enrichment of 1 APE is completely safe as it is far below the threshold for
biological toxicity in humans (approximately 20 APE) and will be returned to
baseline enrichments within 30 days.
Deuterium oxide intake in small amounts increases the presence of deuterium on
H2O molecules in the body water pool of the test subject. The deuterium is
available for transfer from the D2O molecule for incorporation into any
endogenously synthesized substrate utilizing H2O as reagent in the biochemical
reaction. For the aims of this study, the endogenously synthesized amino acid
alanine is of primary analytical interest. Once a deuterium atom has been
incorporated into newly formed alanine, the amount of alanine incorporated into
proteins can be accurately measured. There are no adverse health effects
associated with deuterium oxide dosing within the ranges of the present study.
However, a small fraction of the studies using heavy water in humans have
suggested that short-term vertigo (similar to dizziness) may occur in some
subjects during the initial phases of heavy water dosing if the doses are taken
too rapidly. The study in which the current dosing protocol was based off of
reported that only one subject experienced any form of dizziness, which
subsided within 30 minutes. We will be monitoring subjects during the initial
dosing phase in the current study in case any such dizziness occurs and do not
expect any complications.

Subjects will be required to fill in activity and dietary records on each day
of the protocol and one day prior (7 days total). Furthermore, subjects will
wear accelerometer watches for the purpose of quantitatively tracking physical
activity levels throughout the day. There is no direct benefit for the
participants except for their contribution to the scientific knowledge of
exercise and nutritional strategies to improve muscle mass, which will be
obtained from this study and used in the future.