Adaptations of Amino Acid Transporters and Sestrins to Exercise, Nutrition and Ageing in Human Skeletal Muscle

Abstract

Human skeletal muscle plays a fundamental role in mobility, metabolism and disease prevention. Protein metabolism is critical for the regulation of skeletal muscle mass. The mammalian target of rapamycin complex 1 (mTORC1) is proposed to be essential in the regulation of both protein synthesis and degradation, but the mechanistic details of the upstream events that are responsible for amino acid (AA)-dependent mTORC1 activation remains elusive in vivo. Many pathways have been proposed via in vitro models and it appears that AA transporters (AAT), sensors and mediators are the key players. Limited studies have attempted to translate and validate the knowledge derived from in vitro models to human skeletal muscle. Therefore, this thesis explored the effects of ageing, nutrition and resistance exercise (RE) on AAT, sensors and mediators in an attempt to provide a better understanding of how AA stimulate mTORC1 activation in human skeletal muscle. Ageing is associated with a gradual and involuntary loss of skeletal muscle mass and function, a condition known as sarcopenia. Habitual dietary protein intake is essential for the maintenance of muscle mass. However, providing a complete diet containing either 0.8 g protein ∙ kg-1 ∙ d-1 or 1.6 g protein ∙ kg-1 ∙ d-1 to older men (70‒81 y) for ten weeks did not alter the expression of AAT, sensors, mediators and mTORC1 anabolic signalling. Contrarily, the combination of a single bout of RE and dietary protein supplementation in middle-aged men (40‒55 y) robustly induced the transcriptional response of AAT. However, a direct translation of some in vitro pathways was not achieved in human skeletal muscle, thereby highlighting the complexity of the mechanisms regulating AAdependent mTORC1 activation. While validating the relevance of putative AA sensors in human skeletal muscle, unexpected findings from Sestrin2 were generated. Being a purported leucine sensor, Sestrin2 has been proposed to regulate mTORC1, AMP-dependent protein kinase (AMPK), autophagy, oxidative stress, metabolism and muscle development. By treating samples with lambda phosphatase, evidence was generated to show that Sestrin2 is a phosphoprotein in human skeletal muscle. Phosphorylation is one of the most important protein modifications in signal transduction pathways. Hence the focus of this thesis has been shifted to gain a better understanding of this multifunctional protein. Included in the complex functionality of Sestrin2, mammals express three paralogs, Sestrin1/2/3. Whether they share redundant functions in human skeletal muscle is unknown. By comparing the effects of RE, nutrition and ageing on the Sestrin family, it was concluded that despite the strong sequence homology, they were differentially regulated. Sestrin2 was transiently hyperphosphorylated after RE but not after dietary protein supplementation and there was no evidence of either Sestrin1 and 3 being phosphoproteins. RE did not affect the expression of Sestrin3, but it was upregulated after carbohydrate supplementation. Compared to Sestrin2 and 3, Sestrin1 mRNA was abundantly expressed in human skeletal muscle. Further, Sestrin1 protein was upregulated after 12 weeks of resistance training in young men but downregulated with increasing age. The complete sequence of studies in this thesis highlighted the convoluted pathways involved in AA-dependent mTORC1 activation in human skeletal muscle. While many mechanistic pathways have been proposed in vitro, controversies between theories still exist. Hence making it difficult to interpret the results generated from human studies. Similarly, there is an ongoing debate on the ability of Sestrin2 to sense leucine. Its relevance as a leucine sensor in skeletal muscle is unresolved. However, new data were generated indicating that Sestrin2 is a phosphoprotein regulated by RE and could potentially mediate an adaptive mechanism to regulate redox homeostasis. These findings offer an avenue for future research on the roles of Sestrins in muscle physiology.