Nocturnal protein supply during sleep as a dietary strategy to improve muscle mass in elderly

Muscle loss, which begins after the age of 55 years, is one of the most
important factors of disability in elderly people. This age-related loss of
muscle mass and function, or sarcopenia, has substantial health consequences.
The decline in lean body mass is accompanied by reduced physical performance,
the loss of functional capacity and an increased risk of developing chronic
metabolic diseases, like obesity and type 2 diabetes. Food intake and in
particular the ingestion of protein or amino acids has been shown to be a
powerful stimulus to promote net muscle protein anabolism by providing ample
amino acids as precursors for protein assembly. However, dietary protein/amino
acids should not be regarded as merely the building blocks of body tissue
proteins. A growing body of evidence indicates that essential amino acids
(EAA), and leucine in particular, function as potent nutritional signalling
molecules with an active regulatory role in muscle protein metabolism. It is
believed that EAA are likely to be responsible for the amino acid induced
stimulation of muscle protein synthesis.

So far, only few studies have focused on overnight muscle protein synthesis.
Stimulating muscle protein synthesis during sleep, might augment muscle
hypertrophy, increase mitochondrial mass, and/or improve muscle tissue repair.

Overall, the goal of this proposal will be to provide further insight into the
responsiveness of the muscle protein synthetic machinery to food intake at
night in relation to age.

The aim of this proposal is to investigate the nocturnal muscle protein
synthesis rates in elderly men in response to a meal-like protein bolus at
night.

To test the hypothesis that ingesting a nocturnal bolus of intact casein
stimulates muscle protein synthesis in elderly.

At 18.00 h subjects will report to the laboratory, where a standardized meal
will be provided. Before 19.00 h, a Teflon catheter will be inserted into an
antecubital vein for starting a primed, continuous infusion of stable isotope
labelled phenylalanine and tyrosine. A second Teflon catheter will be inserted
in the contra lateral hand vein for arterialised blood sampling. Blood will be
arterialised by placing the hand in a hot-box and a resting/background blood
sample will be drawn. At 19.00 h, tracer infusion will be started. At 21.00 a
commercially available nasogastric stomach feeding catheter (Bengmark, Flocare,
Zoetermeer, the Netherlands) will be inserted by a physician. The catheter will
be placed with the tube tip located in the stomach, according to the
manufacturers instructions (see attachment and MEC NL24018.068.08). This
catheter will be used to administer protein beverages during sleep. At 23.00 h
the first muscle biopsy will be taken. Subsequently, subjects prepare to sleep
at 0:00 h. This will be followed by a sleeping period of 7 h. The second muscle
biopsy will be obtained at 7.00 h. Blood samples (8ml) will subsequently be
taken from the arterialised hand vein at t = -420, -360, -300, -240, -180,
-120, -60, 0, 30, 60, 90, 120, 180, 240 and 300 minutes. Muscle biopsies will
be taken at t = -3 h, and 5 h after protein ingestion by nasogastric tube.

At 02.00 h, half of the subjects will receive a bolus of protein beverage by
their nasogastric tube. The same protein as has been used previously [MEC
07-3-086], will be used in this study. Subjects receive a beverage volume of
500 ml water with the addition of 40 g intact casein protein (CAS).
L-[1-13C]phenylalanine labelled milk proteins are produced in collaboration
with Dr Boirie (Clermont-Ferrand, France) using registered cattle for milk
production applying quality process #0000640 in accordance with standard NF V
01-005. Milk proteins will be processed to obtain purified CAS by DSM Food
Specialties (Delft, The Netherlands). During the processing of the milk, total
plate count and several tests for micro-organisms will be performed (B. cereus,
St. aureus, coliforms, moulds yeast, salmonellae and lysteria) to assure that
no contamination occurs during processing. The intact casein protein will be
tested according to DSM*s manufacturing specifications conforms to the
performance typical for this grade and product description, before clearance
will be given for use in human subjects. Beverages will be uniformly flavoured
by adding 0.2 g sodium-saccharinate solution (25% w/w), 1.8 g citric acid
solution (50% w/w) and 5 g of cream vanilla flavour for each litre of beverage.
Drinks will be prepared according to Standard Operating Procedure #RM001,
routinely applied in the kitchen of the departments of Human Biology and
Movement Sciences the evening before each test. After preparation, drinks will
be stored at 4 ºC until utilization the next morning.

The risks involved in participating in this experiment are minimal. Insertion
of the catheters in a vein is comparable to a normal blood drawn and the only
risk is of a small local haematoma. This is the same for the muscle biopsy. The
incision made for obtaining the muscle biopsy (performed by an experienced
physician) will heal completely. The labelled amino acid tracers applied in
this experiment are not radioactive and are completely safe. The test beverage
contains intrinsically labelled dietary protein, which is safe for human
consumption (see attachment) and has been used in previous studies [MEC
07-3-086]. The drinks are made from normal nutritional ingredients and do not
impose any health risks.