All the Sarcoplasm Is a Stage; and All the Anabolic and Catabolic Complexes Merely Players; They Have Their Exits and Entrances An Analysis of How Proteolytic Pathways Impact Protein Synthesis Capacity in C2C12 Myotubes

Abstract

The control of anabolism and catabolism in muscle, termed protein turnover, is a heavily studied research area. It is known that steps in anabolism involve the mechanistic Target of Rapamycin (mTOR) and can have a direct impact on catabolic pathways. While we acknowledge that catabolism within the cell provides an intracellular source of amino acids (AA) to support anabolism, lesser known is whether catabolic pathways within skeletal muscle have a direct impact on anabolic capacity. To better understand how catabolic pathways impact anabolism, these studies used key anabolic and/or catabolic inhibitors to determine their role on 24-h fractional protein synthesis rates (FSR) and anabolic signaling in cultured C2C12 myotubes. The underlying hypothesis for the present study was catabolic processes directly affect the anabolic capacity by affecting anabolic signal transduction. To complete these studies, specialized inhibitors designed to block the autophagic (NSC 185058) and/or proteasomal (MG-262) catabolic pathways and/or anabolic signaling through mTOR were utilized to delineate contributions of the respective catabolic pathways on mTOR activity and FSR. Application of MG-262 had no statistical impact on FSR. However, incubation of cells with NSC 185058 yielded blunted FSR (p=0.0146), similar to that of complete mTOR inhibition using Torin 1 (p=0.0012). When Torin 1 and NSC 185058 were combined, FSR was lowered beyond single inhibitor application levels (vs Control; p<0.0001). Western blot analyses revealed that NSC 185058 had a differential effect on the activation of two mTOR targets, showing a decrease in activation of p70S6K (involved with ribosomal biogenesis) with no change in eIF4EBP (involved with CAP-dependent mRNA translation). This effect persisted when NSC 185058 was combined with either Rapamycin, MG-262, or Torin 1. Both p70S6K and eIF4EBP1 responded as expected to incubation with Torin 1, regardless of combination with either catabolic inhibitor. These results collectively suggest that there is a direct contribution of autophagic processes on normal anabolism in the cell affecting FSR, which could be mediated through disrupted mTOR signal transduction through a yet to be defined mechanism affecting ribosomal biogenesis. Additionally, the data indicate that autophagic contribution to cellular protein turnover outweighs the proteasomal contribution to intracellular AA utilized in anabolism.