We have proposed to test if a 20g protein dose of cheddar cheese would acutely increase the blood branch-chained amino acids (particularly leucine) and muscle mTORC1 signaling compared to baseline. Furthermore, we have proposed to compare the blood branch-chained amino acid and muscle mTORC1 response to a comparable protein dose in the form of milk.
General objective: To determine the blood amino acid and muscle cellular signaling response to cheese and milk ingestion.
Blood samples will be assessed for plasma essential and non-essential amino acids before and after nutrient ingestion using EZ:faast amino acid analysis kit (Phenomenex). Plasma insulin and free fatty acids will be determined by ELISA according to manufacturer procedures. Muscle biopsy samples will be assessed for mTORC1 signaling protein expression at specific phosphorylation sites (mTOR, P70S6K1, and RPS6) using standard western blotting procedures. mTORC1 co-localization to the lysosome will be determined in histological specimens using a mTOR-LAMP2 IHC co-stain.