Skeletal muscle conveys several of the health-promoting effects of exercise; yet the underlying mechanisms are not fully elucidated. Studying skeletal muscle is challenging due to its different fiber types and the presence of non-muscle cells. This can be circumvented by isolation of single muscle fibers. Here, we develop a workflow enabling proteomics analysis of pools of isolated muscle fibers from freeze-dried human muscle biopsies. We identify more than 4000 proteins in slow- and fast-twitch muscle fibers. Exercise training alters expression of 237 and 172 proteins in slow- and fast-twitch muscle fibers, respectively. Interestingly, expression levels of secreted proteins and proteins involved in transcription, mitochondrial metabolism, Ca2+ signaling, and fat and glucose metabolism adapts to training in a fiber type-specific manner. Our data provide a resource to elucidate molecular mechanisms underlying muscle function and health, and our workflow allows fiber type-specific proteomic analyses of snap-frozen non-embedded human muscle biopsies.

Key points A single bout of exercise is capable of increasing insulin sensitivity in human skeletal muscle. Whether this ability is affected by training status is not clear. Studies in mice suggest that the AMPK‐TBC1D4 signalling axis is important for the increased insulin‐stimulated glucose uptake after a single bout of exercise. The present study is the first longitudinal intervention study to show that, although exercise training increases insulin‐stimulated glucose uptake in skeletal muscle at rest, it diminishes the ability of a single bout of exercise to enhance muscle insulin‐stimulated glucose uptake. The present study provides novel data indicating that AMPK in human skeletal muscle is important for the insulin‐sensitizing effect of a single bout of exercise. Abstract Not only chronic exercise training, but also a single bout of exercise, increases insulin‐stimulated glucose uptake in skeletal muscle. However, it is not well described how adaptations to exercise training affect the ability of a single bout of exercise to increase insulin sensitivity. Rodent studies suggest that the insulin‐sensitizing effect of a single bout of exercise is AMPK‐dependent (presumably via the α2β2γ3 AMPK complex). Whether this is also the case in humans is unknown. Previous studies have shown that exercise training decreases the expression of the α2β2γ3 AMPK complex and diminishes the activation of this complex during exercise. Thus, we hypothesized that exercise training diminishes the ability of a single bout of exercise to enhance muscle insulin sensitivity. We investigated nine healthy male subjects who performed one‐legged knee‐extensor exercise at the same relative intensity before and after 12 weeks of exercise training. Training increased V̇O2 peak and expression of mitochondrial proteins in muscle, whereas the expression of AMPKγ3 was decreased. Training also increased whole body and muscle insulin sensitivity. Interestingly, insulin‐stimulated glucose uptake in the acutely exercised leg was not enhanced further by training. Thus, the increase in insulin‐stimulated glucose uptake following a single bout of one‐legged exercise was lower in the trained vs. untrained state. This was associated with reduced signalling via confirmed α2β2γ3 AMPK downstream targets (ACC and TBC1D4). These results suggest that the insulin‐sensitizing effect of a single bout of exercise is also AMPK‐dependent in human skeletal muscle.

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A single bout of exercise enhances insulin action in the exercised muscle. However, not all human studies find that this translates into increased whole-body insulin action, suggesting that insulin action in rested muscle or other organs may be decreased by exercise. To investigate this, eight healthy men underwent a euglycemic-hyperinsulinemic clamp on 2 separate days: one day with prior one-legged knee-extensor exercise to local exhaustion (∼2.5 h) and another day without exercise. Whole-body glucose disposal was ∼18% lower on the exercise day as compared with the resting day due to decreased (∼37%) insulin-stimulated glucose uptake in the nonexercised muscle. Insulin signaling at the level of Akt2 was impaired in the nonexercised muscle on the exercise day, suggesting that decreased insulin action in nonexercised muscle may reduce GLUT4 translocation in response to insulin. Thus, the effect of a single bout of exercise on whole-body insulin action depends on the balance between local effects increasing and systemic effects decreasing insulin action. Physiologically, this mechanism may serve to direct glucose into the muscles in need of glycogen replenishment. For insulin-treated patients, this complex relationship may explain the difficulties in predicting the adequate insulin dose for maintaining glucose homeostasis following physical activity.

Insulin sensitivity was reduced in early postmenopausal women. However, postmenopausal women increased peripheral insulin sensitivity, skeletal muscle insulin-stimulated glucose uptake, and skeletal muscle mass to the same extent as premenopausal women after 3 months of high-intensity exercise training.