Muscle Disuse Atrophy Clinical Trial
Official title:
Can Supplemental Leucine Offset Disuse-induced Muscle Atrophy?
Ageing is associated with a gradual decline in muscle mass that is detrimental to both
physical function and metabolic health, increasing the risk of morbidity and mortality. The
loss of protein muscle mass with ageing is poorly understood, but it may partly relate to
inactivity/disuse (i.e. during injury or hospitalization). Periods of inactivity/disuse blunt
the ability of muscle to grow (termed anabolic blunting), leading to a loss of muscle mass
and strength. An accumulation of these periods over a lifetime promotes the devastating loss
of muscle protein mass and strength seen with ageing.
Disuse-induced muscle loss is underpinned by a blunted muscle anabolic response to protein
nutrition. Supplementing the diet with the amino acid leucine may offer a potential solution
to alleviate muscle mass and strength loss during disuse. In fact, leucine is suggested to
promote muscle protein growth and reduce muscle protein loss during disuse in rats, but this
is yet to be shown in humans. Accordingly, the proposed study will investigate whether
leucine supplementation can offset muscle and strength loss during short-term disuse.
Twenty-four healthy (non-obese, non-diabetic, non-smokers) men aged 18-35 years will
initially complete a lower-limb strength assessment and undergo a body composition scan three
days later. The following morning, participants will be randomly assigned to ingest either 5g
of leucine (n=12) or a caloric-matched placebo (n=12) with each meal over a 7 d period of a
single-leg immobilisation. Immediately following immobilisation participants will undergo
another body composition scan. Additionally, a stable isotope infusion will be combined with
serial muscle biopsies from the thigh of each leg to determine the measure rates of muscle
protein synthesis in the fasted state and in the 'early' and 'late' phase of feeding. A day
later, the assessment of muscle strength will be repeated.
It is well established that skeletal muscle mass progressively declines with healthy ageing,
a phenomenon commonly termed sarcopenia. Overtime, this leads to a loss in functional
independence and provides an increased risk of developing a co-morbidity. Recent estimates
suggest that 30% of those aged 75-84 years suffer from sarcopenia. As the 85-and-over
population are rapidly expanding, sarcopenia places a considerable socio-economic burden to
healthcare services. From a physiological perspective, the maintenance of muscle mass is
dependent on a fine balance between muscle protein synthesis (MPS) and breakdown (MPB) rates.
In old age, the MPS response to nutrition is impaired (termed 'anabolic resistance'), leaving
muscle in a chronic catabolic state and ultimately underpinning the progression of
sarcopenia.
Age-associated muscle anabolic resistance may stem from an accumulation of periods of
inactivity/disuse, which are strongly associated with a decline in muscle mass (atrophy) in
both the young and old. In fact, the associated loss of muscle mass is the most rapid at the
onset of muscle disuse, with significant declines in muscle mass occurring after just 5 days
of disuse. This is of particular importance since the average length of hospitalization in
older individuals in 5-6 days. Disuse-induced muscle deconditioning is attributed to declines
in MPS in response to nutrition, and potentially an elevated MPB. Therefore, an accumulation
of these periods of disuse over a lifetime may lead to an acceleration in the trajectory of
sarcopenia that is commonly seen with ageing. Consequently, nutritional strategies to
maximize healthy musculoskeletal ageing should focus on alleviating muscle metabolic
dysregulation during short-term disuse.
One approach to counteract muscle deterioration during short-term disuse is to increase
dietary protein consumption. In fact, adequate protein intake is a necessity for muscle to
remain in a positive net protein balance, with the importance of this nutritional
intervention increasing during periods of disuse. However, this approach is not always
feasible as activity levels are more limited during periods of disuse and individuals are
thus less likely to consume adequate nutritional intake. A more feasible approach may be to
enhance the anabolic potency of sub-optimal protein doses through supplementation with the
amino acid, leucine. Leucine is unique in its ability to increase the protein balance more so
than any other amino acid through stimulation of MPS and suppression of MPB. Leucine provides
a strong anti-catabolic effect during hind limb immobilization in rats, which has yet to be
examined in humans. Therefore, it is clear that leucine represents a viable strategy to
offset the dysregulation of MPS and MPB and preserve musculoskeletal health during short-term
disuse in both young and old individuals and thus warrants further investigation.
Participants:
Twenty-four healthy (non-obese, non-diabetic, non-smokers) males aged 18-35 years will be
recruited to participate in the study. The current study will be a placebo controlled
double-blind study with two intervention groups, in which, participants will be randomly
assigned to receive supplemental leucine (LEU; n=12) or placebo (PLA; n=12) during 7 days of
unilateral leg immobilization. Participants will be active, but athletically untrained (i.e.
exercise ≤3x/week). All study procedures will be clearly explained and participants will
provide written informed consent prior to obtaining baseline measures.
Enrolment:
Participants will be invited to the University of Birmingham's School of Sport, Exercise and
Rehabilitation Sciences. Upon arrival, a researcher involved in the trial will explain the
study design and interventions to the participant. Participants will be given the opportunity
to ask any questions related to the study and will be given time to decide whether or not to
take part. If the participant is happy to take part the participant will be asked to sign a
consent form and fill in a health questionnaire. When the participant has left, the
participant will be categorized at random to the leucine or placebo group. Participants will
be asked to fill in a three-day diet diary to assess habitual dietary intake.
Preliminary assessments:
Following explanation of the study and the acquisition of informed consent, participants will
report to the laboratory at 0800-0900h having fasted for 10 hours and refrained from vigorous
exercise and alcohol for 24 hours previously. Participants baseline muscle function will be
acquired through measures of maximal isometric and isokinetic strength of the knee extensor
and flexor muscles using an isokinetic dynamometer. Following strength testing the
investigators will provide individuals with a three-day diet diary and activity monitor.
Three days following the baseline strength measures, participants will once again report to
the laboratory at 0800-0900h following a 10h overnight fast. Participants will be weighed on
a digital scale to the nearest 0.1kg in light clothing. Dual x-ray absorptiometry (DXA) and
ultrasound scans will be conducted to determine the composition of the thigh (fat and fat
free mass).
Immobilization:
At 0800-0900h on the morning following preliminary assessments, participants will undergo 7
days of unilateral leg immobilization. A single leg will be randomly selected
(counterbalanced left/right) and placed in a full-leg knee brace. Participants will ambulate
using crutches and perform daily ankle exercises to minimize the risk of deep vein
thrombosis, as indicated by a qualified physiotherapist. Participants will also be permitted
to remove the knee brace during overnight sleep. A trained physiotherapist will instruct the
participant on the safe use of crutches (i.e climbing stairs). During each main meal in the
immobilization period, participants will consume 5 g of a powdered LEU or PLA supplement with
250-300 ml of water, which is within the safe limits. Dietary intake will be controlled
throughout the immobilization period according to the individuals total calorie intake
(derived from standardized equations), with a macro-nutrient composition of 55% carbohydrate,
30% fat and 15% protein. The total quantity of protein ingestion will equate to 1.0g/kg/day.
Participant's activity levels will be monitored throughout the 7 day period through a
wrist-worn activity monitor.
Experimental trials:
On the morning marking the end of the 7 d immobilization period participants will report to
the laboratory at 0600-0700h following an overnight fast. Ultrasound and DXA assessments will
be repeated, immediately after which a catheter will be inserted into a forearm vein of both
arms for i) frequent blood sampling at -150, -90, 0, 20, 40, 60, 80, 120, 180, 240-minutes of
the experimental trial (∼100mL in total) and ii) a continuous infusion of a stable isotope
amino acid tracer (L- [ring] 13C6 phenylalanine). Participants will remain in a supine
position throughout the trial. After 150 min of infusion, a muscle biopsy will be obtained
from the quadriceps muscle of immobilized and non-immobilized legs under local anaesthetic.
Participants will then consume a 20g milk protein beverage. Further biopsies will be obtained
from both legs after 270-min and 390-mins of infusion. Thus, a total of 6 invasive muscle
biopsies will be obtained during the trial (3 from each leg), with each biopsy obtained from
a separate incision spaced ∼3cm apart. This two- stage stable-isotope infusion design allows
efficient assessment of MPS in the transition from post-absorptive (0-120 min) to
postprandial conditions (120-240 min). Leg strength will be reassessed a day following the
stable isotope infusion at 0800-0900h following a 10 hr fast to avoid any interference effect
of prior contraction on muscle protein turnover.
Data Analysis:
To calculate myofibrillar and mitochondrial protein synthesis, the investigators will adopt
typical sophisticated mass spectrometry techniques to determine isotopic tracer enrichment in
biopsy-isolated muscle proteins and plasma. Intramuscular "anabolic signals" (in the mTORC1
pathway) and "catabolic" signals will be determined via western blot and q-rtPCR analysis.
High resolution respirometry will also be used to assess the mitochondrial function of
biopsy-isolated skeletal muscle tissue in both the control and immobilized limbs.
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