Genes Associated with Amino Acid Sensing in Human Skeletal Muscle are Altered by Amino Acid Availability and Acute Lysosomal Perturbation
Authors: Graber Ted G , Borack Michael S , Reidy Paul T , Volpi Elena , Rasmussen Blake B 
Published: November 2023

Amino acid availability stimulates protein synthesis via the mTORC1 signaling pathway. Recent evidence supports the critical role of the lysosome in the activation of mTORC1. In response to an increase in cellular amino acid availability, translocation of cytosolic mTORC1 to the lysosomal surface (where it interacts with v‐ATPase, the Ragulator complex (p14, p18, MP1, HBXIP, Corf59; aka: LAMTOR1‐5), the RAG heterodimers (RAG A/B; RAG C/D), and RHEB) is required to activate mTOR kinase. Chloroquine (CQ) is a lysosomotropic agent known to acutely disrupt lysosomal function. Therefore, we hypothesized that an acute disruption in lysosome function via administration of chloroquine would interfere with amino acid sensing in human skeletal muscle. The specific aims of this study were to determine: 1) whether an increase in amino acid availability altered mRNA expression of genes associated with cellular sensing of amino acids; 2) whether an acute perturbation in lysosomal function would disrupt muscle amino acid sensing. We recruited 13 young adults and randomized them to one of two groups: 10 g ingestion of essential amino acids (EAA) or 10 g ingestion of EAA in the presence of chloroquine (EAA+CQ). The study was approved by our local IRB and all subjects gave written, informed consent. The subjects from each group had biopsies of the vastus lateralis taken before and after amino acid ingestion, with samples quickly flash frozen in liquid nitrogen. After isolating RNA and producing cDNA, we determined the relative mRNA expression of ~50 genes related to amino acid sensing and mTORC1 signaling using q‐RT‐PCR (BioRad CFX) on custom‐designed BioRad PrimePCR plates to detect relative fold changes using the Livak method (housekeeping genes were B2M and GAPDH). We discovered differential mRNA expression in 15 genes, with 12 mRNAs changing statistically after amino acid ingestion in EAA, versus 5 in EAA+CQ. In addition, 7 out of the 15 genes had statistically significant differences in the percent change in response to the chloroquine intervention. Highlights of the mRNA changes included: the SLC7A8 amino acid transporter (LAT2) increased 28±15% in EAA, but decreased 13±6% in the EAA+CQ (41% difference, p=0.012); DDIT4L (REDD2), an mTOR inhibitor, had a −39% difference in expression in the EAA group (p=0.043); AKT1S1 (PRAS40) had a 28±6% increase in EAA (p=0.007) but no change in EAA+CQ (p=0.565); and components of Ragulator also changed (LAMTOR1 and 2 increased with EAA, p=0.038 and 0.016, but not in EAA+CQ, p=0.651 and 0.830, and LAMTOR 3 decreased 14±5% in EAA+CQ, p=0.031, but with no change in EAA, p=0.545). We conclude that mRNA expression of specific genes associated with amino acid sensing in human skeletal muscle are responsive to an increase in amino acid availability. Furthermore, an acute disruption in lysosomal function interferes with amino acid sensing gene expression highlighting the importance of the lysosome in amino acid sensing within human skeletal muscle cells.Support or Funding InformationGrant Support: NIH R01 AR049877; R56 AG051267