Cycle training increased GLUT4 and activation of mammalian target of rapamycin in fast twitch muscle fibers

Abstract

Purpose: To determine whether cycle training of sedentary subjects would increase the expression of the principle muscle glucose transporters, six volunteers completed 6 wk of progressively increasing intensity stationary cycle cycling.

Methods: In vastus lateralis muscle biopsies, changes in expression of GLUT1, GLUT4, GLUT5, and GLUT12 were compared using quantitative immunoblots with specific protein standards. Regulatory pathway components were evaluated by immunoblots of muscle homogenates and immunohistochemistry of microscopic sections.

Results: GLUT1 was unchanged, GLUT4 increased 66%, GLUT12 increased 104%, and GLUT5 decreased 72%. A mitochondrial marker (cytochrome c) and regulators of mitochondrial biogenesis (peroxisome proliferator-activated receptor gamma coactivator 1 alpha and phospho-5'-adenosine monophosphate-activated protein kinase) were unchanged, but the muscle hypertrophy pathway component, phospho-mammalian target of rapamycin (mTOR), increased 83% after the exercise program. In baseline biopsies, GLUT4 by immunohistochemical techniques was 37% greater in Type I (slow twitch, red) muscle fibers, but the exercise training increased GLUT4 expression in Type II (fast twitch, white) fibers by 50%, achieving parity with the Type I fibers. Baseline phospho-mTOR expression was 50% higher in Type II fibers and increased more in Type II fibers (62%) with training but also increased in Type I fibers (34%).

Conclusion: Progressive intensity stationary cycle training of previously sedentary subjects increased muscle insulin-responsive glucose transporters (GLUT4 and GLUT12) and decreased the fructose transporter (GLUT5). The increase in GLUT4 occurred primarily in Type II muscle fibers, and this coincided with activation of the mTOR muscle hypertrophy pathway. There was little impact on Type I fiber GLUT4 expression and no evidence of change in mitochondrial biogenesis.

Figure 1. Serial dilutions of ova-GLUT1 and ova-GLUT4 on immunoblots

Shown here are serial dilution immunoblots of ova-GLUT1 and ova-GLUT4. The first lane of each blot contains 10 μL of the cell free translation protein production as described in Methods, diluted 1:1,000. The fmoles/lane as indicated was calculated based on the protein content of the specific band determined by the Agilent Bioanalyzer 2100e and a deduced molecular weight of 44,244.

Figure 2. Change in muscle Glut4 expression by stationary cycle training

Six subjects underwent muscle biopsy before and after six weeks of supervised, increasing intensity training on a stationary bike. Panel A. RNA was isolated and mRNA was quantified using real-time quantitative PCR as described. One sample was degraded and not usable. The data shown here are means of three separate assays for each sample. The 22% increase was significant at p=0.05 by paired t-test. Panel B. Immunoblots of muscle homogenates and GLUT4 standards were quantified by image analysis in four separate experiments. The amount of muscle homogenate applied to each lane for GLUT4 measurements was 10 μg membrane protein. Each subject is indicated by his/her subject code (EX01 through EX06). The baseline sample is indicated by “A” and the post-training sample by “B” for each subject. The blot shown is typical and the graph shows the means of four studies for each sample. The 66% increase was significant at the p<0.01 level with paired t-test. Panel C. Panel C displays images from one subject. The top two images are from the pre-training biopsy and the bottom two are from the post-training biopsy. The red image is tagged using anti-hGLUT4 as the primary antibody and the blue image from the same section was probed with the anti-human fast myosin monoclonal antibody. The most intense staining in the top GLUT4 image is in the Type I fibers (unstained in the blue image) and the most intense GLUT4 signal in the post-training image is in the Type II fibers (blue positive fibers). The images for each subject were from two corresponding sections prepared and incubated simultaneously on the same slide. The confocal microscope settings were identical for all twelve images shown here. Panel D. This panel shows the results of image analysis assessment of the intensity of the fluorescent signal in Type I and Type II fibers. Ten fibers of each type in each image were assessed for average intensity using the Quantity One software and the average intensity from those ten fibers was plotted for each data point shown in Panel D. Paired t-tests gave p < 0.01 for both Type I and Type II changes.

Figure 3. Changes in GLUT5 and GLUT12 induced by six weeks of stationary cycle training

Similar to the studies of GLUT4 expression, GLUT1, GLUT5, and GLUT12 were quantified on immunoblots with specific protein standards. The blots shown are typical of several studies. Each subject is indicated by his/her subject code (EX01 through EX06). These blots included 20 μg membrane protein in each lane. The graphs (Panels B, C, and D) represent means of four separate analyses for GLUT5 and GLUT12, and three for GLUT1. The 104% increase in GLUT12 expression and 72% decrease in GLUT5 were each significant at p<0.01 by paired t-test.

Figure 4. Comparison of changes in muscle glucose transporter expression

Shown here are the quantification of the glucose transporter protein as fmoles per 20 μg membrane protein applied to the polyacrylamide gel electrophoresis system for immunoblotting. Since each lane for GLUT4 measurement as shown in Figure 3 contained 10 μg, the GLUT4 data in this figure are adjusted to allow direct comparison. Each of these three hexose transporters were significantly different in expression after the training protocol at p<0.01 as indicated by the asterisk. The data presented are compiled from at least three separate determinations for each glucose transporter.

Figure 5. Changes in cytochrome c and PGC-1α by an exercise training program

Immunoblots of cytochrome c and PGC-1α are shown along with a blot showing actin expression as a housekeeper protein. Each subject is indicated by his/her subject code (EX01 through EX06). The baseline sample is indicated by “A” and the post-training sample by “B” for each subject. Cytochrome c expression was not altered by the stationary cycle training program, nor was the mitochondrial biogenesis coactivator, PGC-1α, suggesting that there was little or no aerobic training-related muscle adaptation.

Figure 6. Changes in phospho-AMPK and phospho-mTOR induced by six weeks of cycle training of sedentary volunteers

Immunoblots of muscle homogenate from vastus lateralis biopsies were probed with antibodies against phospho-AMPK and phospho-mTOR to determine which of these two protein kinase systems were activated by six weeks of progressive training on stationary cycles. Each subject is indicated by his/her subject code (EX01 through EX06). The baseline sample is indicated by “A” and the post-training sample by “B” for each subject. Image analysis showed a non-significant 12% drop in AMPK phosphorylation (Panel B), but phospho-mTOR increased by 83% (Panel C). The data shown in Panel B are means from three separate experiments and those in Panel C represent the mean of two separate studies for each data point. The increase in phospho-mTOR was significant at p<0.01 by paired t-test.

Figure 7. Muscle fiber-specific changes in phospho-mTOR from cycle training

Panel A is similar to Panel C of Figure 2 above, except that the primary antibody in the red image was anti-phospho-mTOR. Panel B shows the results of quantitative image analysis assessment of the intensity of the fluorescent signal in Type I and Type II fibers. The image analysis revealed increased signal in both fiber types, but the increase was greater in the Type II fibers (+62%) than in the Type I fibers (+34%). Paired t-tests gave p < 0.01 for both Type I and Type II changes.