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Defining the role of lysosomal trafficking in age-associated anabolic resistance in human skeletal muscle

Periodic Reporting for period 1 - LYSOTRACK (Defining the role of lysosomal trafficking in age-associated anabolic resistance in human skeletal muscle)

Reporting period: 2016-09-01 to 2018-08-31

Ageing is associated with a progressive loss of skeletal muscle mass and strength, which ultimately results in a condition termed as sarcopenia. While variable between individuals, typically, muscle mass remains relatively stable until the age of ~50 years, after which it deteriorates at a rate of approximately 0.5-1% per year. However, associated losses in muscle strength occur more rapidly which results in older muscles becoming disproportionately weak. Sarcopenia occurs independently of health status and leads to increased frailty, loss of mobility, an increased risk of falls and fractures, a diminished quality of life, and in some cases, premature mortality.

Maintenance or growth of muscle mass is mainly driven by increased muscle protein synthesis (i.e. the generation of new muscle protein) in response to exercise and consumption of high-quality proteins rich in essential amino acids. However, several studies have shown that elderly individuals have a blunted protein synthesis response following amino acid administration. Similarly, the elderly also elicit a dampened protein synthesis response following resistance exercise, compared to the young. This inability of the elderly to properly respond to growth-promoting (i.e. anabolic) stimuli has been termed anabolic resistance and plays a significant role in the development of sarcopenia. However, while age-associated anabolic resistance is a well-established physiological phenomenon, the precise mechanisms underpinning anabolic resistance are unknown.

At the molecular level, skeletal muscle protein synthesis is primarily regulated by a protein complex called mTOR Complex 1 (mTORC1). The precise mechanisms by which anabolic stimuli activate mTORC1 in humans are not known, however recent studies in cells have identified the lysosome as a being vital for the activation of mTORC1 and thus, protein synthesis. The lysosome is the organelle within cells responsible for breaking down proteins and recycling the amino acids (i.e. the building blocks of proteins).

Lysosomes have the ability to move from one part of the cell to another, and based on recent evidence, this lysosomal movement (i.e. lysosomal trafficking) in itself, seems to be vital for the activation of protein synthesis. Thus, age-associated impairments in the movement of lysosomes within aged muscle cells, may represent a possible explanation for the development of anabolic resistance in elderly individuals.

Therefore the primary aim of this project was to examine the involvement of lysosomal movement in the regulation of protein synthesis in human skeletal muscle in the context of age-associated anabolic resistance.
To investigate the role of lysosomal movement in age-associated anabolic resistance, a pre-clinical study was undertaken in which 10 young (mean age: 22±1 years) and 10 elderly (mean age: 70±1 years) healthy, normal weight and non-smoking male participants took part. Each participant visited the laboratory on three different occasions to undergo measurements of body weight, body height, body composition and leg strength, as well as to perform resistance exercise. During a fourth and final visit, each participant underwent an experimental trial which included infusion of a stable isotopically labelled amino acid (13C6-phenylalanine) for the measurement of skeletal muscle protein synthesis, one-legged resistance exercise, ingestion of an amino acid drink as well as blood and muscle tissue sampling. All in all, 80 laboratory visits were conducted during the pre-clinical study.
During each experimental trial and for each participant, 18 blood samples and 7 muscle tissue samples were collected totalling 360 blood samples and 140 muscle tissue samples for the whole study. Six out of seven muscle biopsies were collected for measuring baseline levels of muscle protein synthesis and protein synthesis levels in the rested and exercised leg following amino acid ingestion. This experimental design allowed the examination of muscle protein synthesis in 1) the rested and fasted state; 2) in the fed state (i.e. in the rested leg following amino acid ingestion); and 3) in the fed and exercised state (i.e. in the exercised leg following amino acid ingestion).
The remaining muscle tissue sample (i.e. one muscle sample out of seven) from each participant was used for the isolation and expansion of primary myoblasts. Myoblasts are precursor (immature) muscle cells that can be found dormant within muscle tissue. Once they have been isolated, they can be grown in cell culture dishes and stimulated so that they develop into myotubes (a precursor state of the muscle fiber which makes up skeletal muscle tissue). This experimental model is used to mimic skeletal muscle tissue outside of the human body and may be used for experiments that for ethical and safety reasons cannot be performed in human participants (for instance turning genes and proteins on or off).
To examine the role of lysosomal movement in human skeletal muscle, two innovative methodological approaches have been employed which go beyond the current state of the art; 1) utilizing immunohistofluorescence techniques to examine the localization, interactions and movements of lysosomes and mTORC1 components in young and elderly human skeletal tissue; and 2) isolating primary myoblasts from young and elderly skeletal muscle to determine if anabolic resistance is retained in elderly muscle cells compared to young, and if so, if the anabolic resistance can be overcome by targeting various genes and proteins involved in lysosomal movement.
According to the World Health Organization, the world’s population of people aged 60 or older currently number 900 million and those aged 80 or older number 125 million. The number of individuals aged 60 years and over is expected to more than triple by the year of 2050, and for individuals over 80, almost quadruple. It has been estimated that ~30% of those aged 75-84 suffer from sarcopenia. As such, the project has the potential for a large societal impact in that it can provide novel insights into the age-associated dysregulation of protein synthesis and therefore stimulate the development of life quality-enhancing treatments for very large number of individuals. It may also open up possibilities to develop treatments beneficial for additional groups such as patients with type 2 diabetes, obesity and cancer, all of which are diseases that include components of anabolic resistance.
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