Effect of mTORC Agonism via MHY1485 with and without Rapamycin on C2C12 Myotube Metabolism

Abstract

The mechanistic target of rapamycin complex (mTORC) regulates protein synthesis and can be activated by branched-chain amino acids (BCAAs). mTORC has also been implicated in the regulation of mitochondrial metabolism and BCAA catabolism. Some speculate that mTORC overactivation by BCAAs may contribute to insulin resistance. The present experiments assessed the effect of mTORC activation on myotube metabolism and insulin sensitivity using the mTORC agonist MHY1485, which does not share structural similarities with BCAAs.

Methods: C2C12 myotubes were treated with MHY1485 or DMSO control both with and without rapamycin. Gene expression was assessed using qRT-PCR and insulin sensitivity and protein expression by western blot. Glycolytic and mitochondrial metabolism were measured by extracellular acidification rate and oxygen consumption. Mitochondrial and lipid content were analyzed by fluorescent staining. Liquid chromatography-mass spectrometry was used to assess extracellular BCAAs.

Results: Rapamycin reduced p-mTORC expression, mitochondrial content, and mitochondrial function. Surprisingly, MHY1485 did not alter p-mTORC expression or cell metabolism. Neither treatment altered indicators of BCAA metabolism or extracellular BCAA content.

Conclusion: Collectively, inhibition of mTORC via rapamycin reduces myotube metabolism and mitochondrial content but not BCAA metabolism. The lack of p-mTORC activation by MHY1485 is a limitation of these experiments and warrants additional investigation.

Keywords: branched-chain amino acids; isoleucine; leucine; mitochondrial function; skeletal muscle; valine.

Figure 1

Effect of MHY1485 on myotube viability and differentiation. (a) Effect of MHY1485 (MHY) at 10 μM both with and without rapamycin (RAPA) at 100 nM (final concentration of DMSO at 0.2% for all samples) for 24 h on cell viability presented as time trial (left) or end point (right). (b) Effect of MHY with or without RAPA for 24 h on p-MTORC expression. (c) Effect of MHY with or without RAPA for 24 h on myosin heavy chain 7 (Myh7) mRNA expression. (d) Effect of MHY with or without RAPA for 24 h on MYH3 protein expression. (e) Effect of MHY with or without RAPA for 24 h on myotube fusion index. NOTES: Data were analyzed using two-way ANOVA followed by one-way ANOVA with Bonferroni’s correction for multiple comparisons to assess differences in each outcome. * Indicates a significant difference between groups upon pair-wise comparisons. Data were generated from three replicates per group across two independent experiments with n = 6 for the final analysis. Target gene expression was normalized to TATA-binding protein (Tbp), which did not differ between groups (Figure S1). Protein expression was normalized to β-Actin, which did not differ between groups (Figure S2). Myotube fusion was quantified by two blinded members of the research team. No differences were observed between any groups. Images in (e) of representative individual myotubes were taken using the 20× objective.

Figure 2

Effect of MHY1485 on mitochondrial content and function. (a) Time course of the effect of MHY1485 (MHY) at 10 μM both with and without rapamycin (RAPA) at 100 nM (final concentration of DMSO at 0.2% for all samples) for 24 h on mitochondrial function. (b,c) Effect of treatment as described in (a) on basal (b) and peak (c) mitochondrial metabolism following normalization to cell nuclei content (presented in Figure S3). (d) Mitochondrial content of cells described in (a) indicated by NAO staining following normalization to cell nuclei content (presented in Figure S3). NOTES: Data were analyzed using two-way ANOVA followed by one-way ANOVA with Bonferroni’s correction for multiple comparisons to assess differences in each outcome. * Indicates a significant difference between groups upon pair-wise comparisons. States of mitochondrial metabolism were calculated by subtracting non-mitochondrial respiration from basal or FCCP-induced peak oxygen consumption. Metabolic measurements were performed using n = 23 individual replicates per treatment condition and were repeated across two independent experiments with n = 46 per group in the final analyses. No wells responded with negative raw values. Mitochondrial staining was performed using n = 23 individual replicates per treatment condition and were repeated across two independent experiments with n = 46 per group in the final analyses using the average of three measurements per experiment less background. Images in (d) of representative individual myotubes were taken using the 20× objective.

Figure 3

Effect of MHY1485 on mitochondrial biogenic signaling. (a) Effect of MHY1485 (MHY) at 10 μM both with and without rapamycin (RAPA) at 100 nM (final concentration of DMSO at 0.2% for all samples) for 24 h on mRNA expression of mitochondrial biogenesis including peroxisome proliferator-activated receptor-gamma coactivator-1alpha (Ppargc1a), nuclear respiratory factor 1 (Nrf1), mitochondrial transcription factor A (Tfam), and citrate synthase (Cs). (b) Effect of MHY with or without RAPA for 24 h on protein expression of peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1α), nuclear respiratory factor 1 (NRF1), mitochondrial transcription factor A (TFAM), and citrate synthase (CS). NOTES: Data were analyzed using two-way ANOVA followed by one-way ANOVA with Bonferroni’s correction for multiple comparisons to assess differences in each outcome. Data were generated from three replicates per group across two independent experiments with n = 6 for the final analysis. Target gene expression was normalized to TATA-binding protein (Tbp), which did not differ between groups (Figure S1). Protein expression was normalized to β-Actin, which did not differ between groups (Figure S2).

Figure 4

Effect of MHY1485 on glycolytic metabolism. (a) Time course of the effect of MHY1485 (MHY) at 10 μM both with and without rapamycin (RAPA) at 100 nM (final concentration of DMSO at 0.2% for all samples) for 24 h on glycolytic metabolism. (b,c) Effect of treatment as described in (a) on basal (b) and peak (c) glycolytic metabolism following normalization to cell nuclei content (presented in Figure S3). (d) Effect of treatment as described in (a) on mRNA expression of glycolytic metabolism including lactate dehydrogenase a (Ldha), lactate dehydrogenase b (Ldhb), pyruvate dehydrogenase (Pdh), and glucose transporter 4 (Slc2a4/Glut4). NOTES: Data were analyzed using two-way ANOVA followed by one-way ANOVA with Bonferroni’s correction for multiple comparisons to assess differences in each outcome. * Indicates a significant difference between groups upon pair-wise comparisons. Metabolic measurements were performed using n = 23 individual replicates per treatment condition and were repeated across two independent experiments with n = 46 per group in the final analyses. No wells responded with negative raw values. Gene expression data were generated from three replicates per group across two independent experiments with n = 6 for the final analysis. Target gene expression was normalized to TATA-binding protein (Tbp), which did not differ between groups (Figure S1).

Figure 5

Effect of MHY1485 on insulin sensitivity. The effect of MHY1485 (MHY) at 10 μM both with and without rapamycin (RAPA) at 100 nM (final concentration of DMSO at 0.2% for all samples) for 24 h followed by insulin stimulation at 100 nM for 30 min on pIRS1 (Tyr612) expression and pAkt (Ser473) expression. NOTES: Data were analyzed using two-way ANOVA followed by one-way ANOVA with Bonferroni’s correction for multiple comparisons to assess differences in each outcome. * Indicates a significant difference between groups upon pair-wise comparisons. Data were generated from three replicates per group across two independent experiments with n = 6 for the final analysis. Protein expression of phosphorylated protein targets was normalized to either total IRS1 or total Akt, and total IRS1 and total Akt expression were normalized to β-Actin. Non-stimulated (NS) negative control is also displayed on the far-right lane of each blot.

Figure 6

Effect of MHY1485 on BCAA catabolism. (a) Effect of MHY1485 (MHY) at 10 μM both with and without rapamycin (RAPA) at 100 nM (final concentration of DMSO at 0.2% for all samples) for 24 h on mRNA expression of branched-chain alpha-keto acid dehydrogenase (Bckdha) and branched-chain amino transaminase 2 (Bcat2). (b) Effect of MHY with or without RAPA for 24 h on protein expression of phospho-branched-chain alpha-keto acid dehydrogenase (pBCKDHa) and branched-chain amino transaminase 2 (BCAT2). (c) Effect of MHY with and without RAPA as described in (a) on extracellular BCAA content following normalization to relative nuclei content (Figure S4). NOTES: Data were analyzed using two-way ANOVA followed by one-way ANOVA with Bonferroni’s correction for multiple comparisons to assess differences in each outcome. Data were generated from three replicates per group across two independent experiments with n = 6 for the final analysis. Target gene expression was normalized to TATA-binding protein (Tbp), which did not differ between groups (Figure S1). Protein expression was normalized to β-Actin, which did not differ between groups (Figure S2). Extracellular BCAAs were normalized to nuclei content (Figure S4).