Amino Acid-Induced mTORC1 Signaling in the Maintenance of Skeletal Muscle Protein Synthesis
Restricted (Penn State Only)
- Author:
- Kincheloe, Gregory
- Graduate Program:
- Anatomy
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- June 07, 2023
- Committee Members:
- Patricia Mclaughlin, Program Head/Chair
Scot Kimball, Chair & Dissertation Advisor
Patricia Mclaughlin, Outside Unit Member
Charles Lang, Major Field Member
Christopher Yengo, Major Field Member
Michael Dennis, Outside Field Member - Keywords:
- mTORC1
Muscle Atrophy
Protein Balance
Muscle Metabolism
4E-BP1/2
Rag GTPases - Abstract:
- Disuse-induced skeletal muscle atrophy is a serious comorbidity often seen in patients who must undergo periods of bedrest, limb immobilization, or limb suspension. Because the extent of muscle wasting is often correlated with worsened health outcomes, skeletal muscle metabolism in response to immobilization is an important issue to address. Heavily implicated in disuse-induced muscle atrophy is the master regulator for mRNA translation, Mechanistic Target of Rapamycin in Complex 1 (mTORC1), which is activated in healthy conditions by nutrients such as amino acids and hormones such as insulin and insulin-like growth factor 1. However, upon limb immobilization, mTORC1 in muscle exhibits blunted nutrient and hormone-induced activation, in a state that is commonly termed “anabolic resistance.” During anabolic resistance, pathways both upstream and downstream of mTORC1 are negatively affected, resulting in diminished rates of protein synthesis and a resultant imbalance that produces a net increase in muscle protein degradation and muscle atrophy. Three of the most well-known downstream targets of mTORC1, the 70kDa Ribosomal Protein S6 Kinase (p70S6K1) and Eukaryotic Initiation Factor (eIF) 4E Binding Proteins 1 and 2 (4E-BP1 and 4E-BP2, respectively) work to regulate translation in separate ways. Upon mTORC1-mediated phosphorylation, p70S6K1 proceeds to phosphorylate and activate additional proteins that assist in translation, while mTORC1-phosphorylated 4E-BP1/2 cease their sequestration of the important and limiting mRNA 5’ cap-binding protein eIF4E, allowing it to be utilized for translation initiation. To identify future therapeutic targets for treatment of disuse-induced muscle atrophy, 4E-BP1/2 double-knockout (DKO) mouse hindlimb muscles were investigated after three days of unilateral hindlimb immobilization with the overarching hypothesis that protein synthesis rates would be increased and as a result skeletal muscle atrophy would be decreased. Though these mice exhibited higher basal rates of protein synthesis in the muscles of their immobilized hindlimbs, they also showed equal rates of muscle atrophy as the control mice. In addition, in contrast to wild type (WT) mice, oral leucine administration did not increase the rate of muscle protein synthesis in DKO mice. A possible explanation for these findings is the notable downregulation of the protein eIF4E that manifests in DKO mice, a critical component in the initiation of translation, which may create a “ceiling” on rates of protein synthesis that depend on the diminished availability of the protein. To investigate differences in newly synthesized proteins in DKO and WT mice after oral leucine (a potent mTORC1 stimulator) administration, the methionine analog, azidohomoalanine (AHA), was injected into mice and proteins containing AHA were subsequently isolated for further proteomic analysis. This analysis of the effects of immobilization on newly synthesized proteins in DKO muscle showed diminished protein expression of key regulators of glycolysis as well. A separate study investigating proteins directly upstream of mTORC1 focused on the four Ras-Related GTP-binding proteins (Rag GTPases) which function to activate mTORC1 in a nutrient sensing pathway activated by amino acids. These Rag GTPases form a heterodimer containing one small Rag GTPase (RagA or RagB) and one large Rag GTPase (RagC or RagD) that function to localize mTORC1 to the lysosomal membrane for subsequent activation by GTP-bound Rat Homolog Enriched in Brain (Rheb-GTP), which is downstream of a separate signaling pathway. Until recently, these pairs of proteins were thought to be functionally redundant to each other until recent studies showed that different combinations of the Rag isoforms differentially affect mTORC1 activation and selection of downstream target substrates. After examination of both the mRNA and protein expression of the Rag GTPases in skeletal muscle, heart, liver, kidney, and brain, we found large discrepancies between mRNA expression and protein expression in each of the tissues. Notably however, skeletal muscle expressed predominantly RagA over RagB protein, while other tissues expressed primarily more RagB. In addition, all five of the studied tissue types expressed primarily RagD compared to RagC protein, which was also contrary to what was observed for the mRNAs. Interestingly, further investigation into the Rag GTPase expression in L6 myoblasts and differentiated L6 myotubes showed a transition to further predominance of RagB expression over RagA after differentiation, contrasting with what was observed in skeletal muscle in vivo. Differentiated L6 myotubes only slightly increased protein expression of RagD over RagC, which was consistent with the mRNA findings. The results illustrated in this thesis serve to paint a wider picture of the metabolic mechanisms at work in immobilized skeletal muscle, as the eIF4E downregulation in response to 4E-BP1/2 deletion greatly affects the potential for protein synthesis and may serve as a therapeutic target for future studies in ameliorating skeletal muscle atrophy. In addition, the findings of the tissue-specific expressions of the Rag GTPases may serve as a steppingstone for future studies that may be able to selectively promote activation or expression of one Rag isoform over the other in a specific tissue type. With certain specific mutations in Rag GTPases and their regulators playing roles in disease states such as cardiomyopathies, epilepsies, and cancer, further exploration of the Rag isoforms may prove useful in the development of future therapies.