Ting in a significant main effect of training (p,0.05; With an altered PKM2 expression were established using RNA interference (PKM Figure 1C). Maximal activity of bHAD tended to be higher post-training (p = 0.07) in both the LO (Pretest: 2.361.5 mmol/min/g, Post-test: 2.761.9 mmol/min/g) and HI (Pre-test: 2.760.7 mmol/min/g, Post-test: 3.160.4 mmol/min/ g) groups (Figure 1C). No group by time interaction effects were observed for any marker of skeletal muscle oxidative capacity.Western Blot Analysis30?0 mg of frozen muscle tissue was homogenized in prechilled lysis buffer supplemented with Halt Protease Inhibitor Cocktail (100X, Thermo Fisher Scientific, Rockford, IL). Protein concentrations were determined by protein assay (Pierce, Rockford, IL) and equal amounts of total protein were loaded and separated by SDS-PAGE using an 8.0 (PGC-1a, AMPKa), 10.0 (COX I, COX IV), or 12.0 (SIRT1) polyacrylamide gel before subsequent transfer to a polyvinylidene difluoride membrane. Commercially available antibodies were used for the detection of PGC-1a (Calbiochem, San Diego, CA), AMPKa, GAPDH (Millipore, Temecula, CA), COX I, COX IV (Cell Signalling, Danvers, MA), and SIRT1 (Abcam, Cambridge, MA).Interval Training in Overweight/Obese MenFigure 2. Effects of HI and LO on PGC-1a, AMPK, and SIRT1 protein content. Changes in the protein content of PGC-1a, AMPK and SIRT1 (A). Representative western blots, including loading controls, are also shown (B). * Significant (p,0.05) effect of training. { Significant (p,0.05) interaction. doi:10.1371/journal.pone.0068091.g002 Figure 1. Effect of HI and LO on markers of skeletal muscle oxidative capacity. Changes in protein content of COX I and COX IV (A) and the maximal enzyme activities of citrate synthase (CS) and bhydroxyacyl-CoA dehydrogenase (bHAD) (C). Representative western blots, including loading controls, are also shown (B). { Significant (p,0.05) effect of training. ` Non-significant (p = 0.07) effect of training. doi:10.1371/journal.pone.0068091.gVO2peak and Submaximal Exercise PerformanceOne participant from the LO group was unable to complete VO2peak testing due to intolerability of the apparatus. A significant main effect of training (p,0.001) and a significant group by time interaction effect (p,0.05) were observed for VO2peak (Table 1; Figure 3A). Further, only 5 participants in the LO group demonstrated an elevated VO2peak following training compared to all 9 participants in the HI group (Figure 3C). A significant main effect of training (p,0.05) was also observed for peak power and peak HR during the ramp protocol (Table 1). A main effect of training (p,0.001) was demonstrated for the time to complete 500 kcal test (Table 1; Figure 3B). The group by time interaction effect did not reach statistical significance (p = 0.07), but indicates a trend towards differing adaptations between groups similar to that observed for VO2peak. Significant (p,0.05) effects of training and a group by time interaction effect were observed for peak O2 pulse (Table 1; Figure 4).Regulators of Mitochondrial Title Loaded From File BiogenesisThere was a main effect of training for PGC-1a whole muscle protein content (LO, Pre-test: 160.06 AU, Post-test: 1.2460.17 AU; HI, Pre-test: 160.08 AU, Post-test: 1.2260.09 AU; p,0.05; Figure 2A). A significant effect of training (p,0.05) was also observed for AMPK (LO, Pre-test: 160.05 AU, Post-test: 0.9460.03 AU; HI, Pre-test: 160.06 AU, Post-test: 0.8860.03 AU) and SIRT1 (LO, Pre-test: 160.09 AU, Post-test: 1.1060.07 AU; HI, Pre-test: 160.06 AU, Post-test: 1.4360.15 AU) pr.Ting in a significant main effect of training (p,0.05; Figure 1C). Maximal activity of bHAD tended to be higher post-training (p = 0.07) in both the LO (Pretest: 2.361.5 mmol/min/g, Post-test: 2.761.9 mmol/min/g) and HI (Pre-test: 2.760.7 mmol/min/g, Post-test: 3.160.4 mmol/min/ g) groups (Figure 1C). No group by time interaction effects were observed for any marker of skeletal muscle oxidative capacity.Western Blot Analysis30?0 mg of frozen muscle tissue was homogenized in prechilled lysis buffer supplemented with Halt Protease Inhibitor Cocktail (100X, Thermo Fisher Scientific, Rockford, IL). Protein concentrations were determined by protein assay (Pierce, Rockford, IL) and equal amounts of total protein were loaded and separated by SDS-PAGE using an 8.0 (PGC-1a, AMPKa), 10.0 (COX I, COX IV), or 12.0 (SIRT1) polyacrylamide gel before subsequent transfer to a polyvinylidene difluoride membrane. Commercially available antibodies were used for the detection of PGC-1a (Calbiochem, San Diego, CA), AMPKa, GAPDH (Millipore, Temecula, CA), COX I, COX IV (Cell Signalling, Danvers, MA), and SIRT1 (Abcam, Cambridge, MA).Interval Training in Overweight/Obese MenFigure 2. Effects of HI and LO on PGC-1a, AMPK, and SIRT1 protein content. Changes in the protein content of PGC-1a, AMPK and SIRT1 (A). Representative western blots, including loading controls, are also shown (B). * Significant (p,0.05) effect of training. { Significant (p,0.05) interaction. doi:10.1371/journal.pone.0068091.g002 Figure 1. Effect of HI and LO on markers of skeletal muscle oxidative capacity. Changes in protein content of COX I and COX IV (A) and the maximal enzyme activities of citrate synthase (CS) and bhydroxyacyl-CoA dehydrogenase (bHAD) (C). Representative western blots, including loading controls, are also shown (B). { Significant (p,0.05) effect of training. ` Non-significant (p = 0.07) effect of training. doi:10.1371/journal.pone.0068091.gVO2peak and Submaximal Exercise PerformanceOne participant from the LO group was unable to complete VO2peak testing due to intolerability of the apparatus. A significant main effect of training (p,0.001) and a significant group by time interaction effect (p,0.05) were observed for VO2peak (Table 1; Figure 3A). Further, only 5 participants in the LO group demonstrated an elevated VO2peak following training compared to all 9 participants in the HI group (Figure 3C). A significant main effect of training (p,0.05) was also observed for peak power and peak HR during the ramp protocol (Table 1). A main effect of training (p,0.001) was demonstrated for the time to complete 500 kcal test (Table 1; Figure 3B). The group by time interaction effect did not reach statistical significance (p = 0.07), but indicates a trend towards differing adaptations between groups similar to that observed for VO2peak. Significant (p,0.05) effects of training and a group by time interaction effect were observed for peak O2 pulse (Table 1; Figure 4).Regulators of Mitochondrial BiogenesisThere was a main effect of training for PGC-1a whole muscle protein content (LO, Pre-test: 160.06 AU, Post-test: 1.2460.17 AU; HI, Pre-test: 160.08 AU, Post-test: 1.2260.09 AU; p,0.05; Figure 2A). A significant effect of training (p,0.05) was also observed for AMPK (LO, Pre-test: 160.05 AU, Post-test: 0.9460.03 AU; HI, Pre-test: 160.06 AU, Post-test: 0.8860.03 AU) and SIRT1 (LO, Pre-test: 160.09 AU, Post-test: 1.1060.07 AU; HI, Pre-test: 160.06 AU, Post-test: 1.4360.15 AU) pr.
