Acute exercise and GLUT4 expression in human skeletal muscle: influence of exercise intensity

To examine the influence of exercise intensity on the increases in vastus lateralis GLUT4 mRNA and protein after exercise, six untrained men exercised for 60 min at 39 ± 3% peak oxygen consumption (VO2 peak) (Lo) or 27 ± 2 min at 83 ± 2% VO2 peak (Hi) in counterbalanced order. Preexercise muscle glycogen levels were not different between trials (Lo: 408 ± 35 mmol/kg dry mass; Hi: 420 ± 43 mmol/kg dry mass); however, postexercise levels were lower (P < 0.05) in Hi (169 ± 18 mmol/kg dry mass) compared with Lo (262 ± 35 mmol/kg dry mass). Thus calculated muscle glycogen utilization was greater (P < 0.05) in Hi (251 ± 24 mmol/kg) than in Lo (146 ± 34). Exercise resulted in similar increases in GLUT4 gene expression in both trials. GLUT4 mRNA was increased immediately at the end of exercise (~2-fold; P < 0.05) and remained elevated after 3 h of postexercise recovery. When measured 3 h after exercise, total crude membrane GLUT4 protein levels were 106% higher in Lo (3.3 ± 0.7 vs. 1.6 ± 0.3 arbitrary units) and 61% higher in Hi (2.9 ± 0.5 vs. 1.8 ± 0.5 arbitrary units) relative to preexercise levels. A main effect for exercise was observed, with no significant differences between trials. In conclusion, exercise at ~40 and ~80% VO2 peak, with total work equal, increased GLUT4 mRNA and GLUT4 protein in human skeletal muscle to a similar extent, despite differences in exercise intensity and duration.

Regulation of metabolic transcriptional co-activators and transcription factors with acute exercise

Endurance exercise improves insulin sensitivity and increases fat oxidation, which are partly facilitated by the induction of metabolic transcription factors. Next to exercise, increased levels of FFA's also increase the gene expression of transcription factors, hence making it difficult to discern the effects from contractile signals produced during exercise, from those produced by increased circulatory FFA's. We aimed to investigate, in human skeletal muscle, whether acute exercise affects gene expression of metabolic transcriptional co-activators and transcription factors, including PGC-1α, PRC, PPARα, β/δ, and γ and RXR, SREBP-1c and FKHR, and to discern the effect of exercise per se from those of elevated levels of FFA. Two hours of endurance exercise was performed either in the fasted state, or following carbohydrate ingestion prior to and during exercise, thereby blunting the fasting-induced increase in FA availability and oxidation. Of the genes measured, PGC-1α and PRC mRNA increased immediately after, while PPARβ/δ and FKHR mRNA increased 1–4 h after exercise, irrespective of the increases in FFA's. Our results suggest that the induction in vivo of metabolic transcription factors implicated in mitochondrial biogenesis are under the control of inherent signals, (PGC-1α, PRC), while those implicated in substrate selection are under the control of associated signals (PPARβ/δ, FKHR) stimulated from the contracting skeletal muscle that are independent of circulating FFA levels.

Increased insulin-stimulated Akt pSer473 and cytosolic SHP2 protein abundance in human skeletal muscle following acute exercise and short-term training.

The purpose of the present study was to determine in human skeletal muscle whether a single exercise bout and 7 days of consecutive endurance (cycling) training 1) increased insulin-stimulated Akt pSer473and 2) altered the abundance of the protein tyrosine phosphatases (PTPases), PTP1B and SHP2. In healthy, untrained men (n = 8; 24 ± 1 yr), glucose infusion rate during a hyperinsulinemic euglycemic clamp, when compared with untrained values, was not improved 24 h following a single 60-min bout of endurance cycling but was significantly increased (~30%; P < 0.05) 24 h following completion of 7 days of exercise training. Insulin-stimulated Akt pSer473was ~50% higher (P < 0.05) 24 h following the acute bout of exercise, with this effect remaining after 7 days of training (P < 0.05). Insulin-stimulated insulin receptor and insulin receptor substrate-1 tyrosine phosphorylation were not altered 24 h after acute exercise and short-term training. Insulin did not acutely regulate the localization of the PTPases, PTP1B or SHP2, although cytosolic protein abundance of SHP2 was increased (P < 0.05; main effect) 24 h following acute exercise and short-term training. In conclusion, insulin-sensitive Akt pSer473and cytosolic SHP2 protein abundance are higher after acute exercise and short-term training, and this effect appears largely due to the residual effects of the last bout of prior exercise. The significance of exercise-induced alterations in cytosolic SHP2 and insulin-stimulated Akt pSer473on the improvement in insulin sensitivity requires further elucidation.

Real time RT-PCR analysis of housekeeping genes in human skeletal muscle following acute exercise

Studies examining gene expression with RT-PCR typically normalize their mRNA data to a constitutively expressed housekeeping gene. The validity of a particular housekeeping gene must be determined for each experimental intervention. We examined the expression of various housekeeping genes following an acute bout of endurance (END) or resistance (RES) exercise. Twenty-four healthy subjects performed either a interval-type cycle ergometry workout to exhaustion (~75 min; END) or 300 single-leg eccentric contractions (RES). Muscle biopsies were taken before exercise and 3 h and 48 h following exercise. Real-time RT-PCR was performed on ß-actin, cyclophilin (CYC), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and ß2-microglobulin (ß2M). In a second study, 10 healthy subjects performed 90 min of cycle ergometry at ~65% of O2 max, and we examined a fifth housekeeping gene, 28S rRNA, and reexamined ß2M, from muscle biopsy samples taken immediately postexercise. We showed that CYC increased 48 h following both END and RES exercise (3- and 5-fold, respectively; P < 0.01), and 28S rRNA increased immediately following END exercise (2-fold; P = 0.02). ß-Actin trended toward an increase following END exercise (1.85-fold collapsed across time; P = 0.13), and GAPDH trended toward a small yet robust increase at 3 h following RES exercise (1.4-fold; P = 0.067). In contrast, ß2M was not altered at any time point postexercise. We conclude that ß2M and ß-actin are the most stably expressed housekeeping genes in skeletal muscle following RES exercise, whereas ß2M and GAPDH are the most stably expressed following END exercise.

The effect of acute exercise on undercarboxylated osteocalcin in obese men

Summary : The purpose of this study was to examine if the reduction in glucose post-exercise is mediated by undercarboxylated osteocalcin (unOC). Obese men were randomly assigned to do aerobic or power exercises. The change in unOC levels was correlated with the change in glucose levels post-exercise. The reduction in glucose post-acute exercise may be partly related to increased unOC.

Introduction : Osteocalcin (OC) in its undercarboxylated (unOC) form may contribute to the regulation of glucose homeostasis. As exercise reduces serum glucose and improves insulin sensitivity in obese individuals and individuals with type 2 diabetes (T2DM), we hypothesised that this benefit was partly mediated by unOC.

Methods : Twenty-eight middle-aged (52.4 ± 1.2 years, mean ± SEM), obese (BMI = 32.1 ± 0.9 kg m⁻²) men were randomly assigned to do either 45 min of aerobic (cycling at 75% of VO2peak) or power (leg press at 75% of one repetition maximum plus jumping sequence) exercises. Blood samples were taken at baseline and up to 2 h post-exercise.

Results : At baseline, unOC was negatively correlated with glucose levels (r = −0.53, p = 0.003) and glycosylated haemoglobin (HbA1c) (r = −0.37, p = 0.035). Both aerobic and power exercises reduced serum glucose (from 7.4 ± 1.2 to 5.1 ± 0.5 mmol L⁻¹, p = 0.01 and 8.5 ± 1.2 to 6.0 ± 0.6 mmol L⁻¹, p = 0.01, respectively). Aerobic exercise significantly increased OC, unOC and high-molecular-weight adiponectin, while power exercise had a limited effect on OC and unOC. Overall, those with higher baseline glucose and HbA1c had greater reductions in glucose levels after exercise (r = −0.46, p = 0.013 and r = −0.43, p = 0.019, respectively). In a sub-group of obese people with T2DM, the percentage change in unOC levels was correlated with the percentage change in glucose levels post-exercise (r = −0.51, p = 0.038).

Conclusions : This study reports that the reduction in serum glucose post-acute exercise (especially aerobic exercise) may be partly related to increased unOC.r exercises. The change in unOC levels was correlated with the change in glucose levels post-exercise. The reduction in glucose post-acute exercise may be partly related to increased unOC.

Xanthine oxidase inhibition attenuates skeletal muscle signaling following acute exercise but does not impair mitochondrial adaptations to endurance training.

The aim of this research was to examine the impact of the xanthine oxidase (XO) inhibitor allopurinol on the skeletal muscle activation of cell signaling kinases' and adaptations to mitochondrial proteins and antioxidant enzymes following acute endurance exercise and endurance training. Male Sprague-Dawley rats performed either acute exercise (60 min of treadmill running, 27 m/min, 5% incline) or 6 wk of endurance training (5 days/wk) while receiving allopurinol or vehicle. Allopurinol treatment reduced XO activity to 5% of the basal levels (P < 0.05), with skeletal muscle uric acid levels being almost undetectable. Following acute exercise, skeletal muscle oxidized glutathione (GSSG) significantly increased in allopurinol- and vehicle-treated groups despite XO activity and uric acid levels being unaltered by acute exercise (P < 0.05). This suggests that the source of ROS was not from XO. Surprisingly, muscle GSSG levels were significantly increased following allopurinol treatment. Following acute exercise, allopurinol treatment prevented the increase in p38 MAPK and ERK phosphorylation and attenuated the increase in mitochondrial transcription factor A (mtTFA) mRNA (P < 0.05) but had no effect on the increase in peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), nuclear respiratory factor-2, GLUT4, or superoxide dismutase mRNA. Allopurinol also had no impact on the endurance training-induced increases in PGC-1α, mtTFA, and mitochondrial proteins including cytochrome c, citrate synthase, and β-hydroxyacyl-CoA dehydrogenase. In conclusion, although allopurinol inhibits cell signaling pathways in response to acute exercise, the inhibitory effects of allopurinol appear unrelated to exercise-induced ROS production by XO. Allopurinol also has little effect on increases in mitochondrial proteins following endurance training.

Regular exercise participation mediates the affective response to acute bouts of vigorous exercise

Physical inactivity is a leading factor associated with cardiovascular disease and a major contributor to the global burden of disease in developed countries. Subjective mood states associated with acute exercise are likely to influence future exercise adherence and warrant further investigation. The present study examined the effects of a single bout of vigorous exercise on mood and anxiety between individuals with substantially different exercise participation histories. Mood and anxiety were assessed one day before an exercise test (baseline), 5 minutes before (pre-test) and again 10 and 25 minutes post-exercise. Participants were 31 university students (16 males, 15 females; Age M = 20), with 16 participants reporting a history of regular exercise with the remaining 15 reporting to not exercise regularly. Each participant completed an incremental exercise test on a Monark cycle ergometer to volitional exhaustion. Regular exercisers reported significant post-exercise improvements in mood and reductions in state anxiety. By contrast, non-regular exercisers reported an initial decline in post-exercise mood and increased anxiety, followed by an improvement in mood and reduction in anxiety back to pre-exercise levels. Our findings suggest that previous exercise participation mediates affective responses to acute bouts of vigorous exercise. We suggest that to maximise positive mood changes following exercise, practitioners should carefully consider the individual's exercise participation history before prescribing new regimes.

Regulation of miRNAs in human skeletal muscle following acute endurance exercise and short-term endurance training

The identification of microRNAs (miRNAs) has established new mechanisms that control skeletal muscle adaptation to exercise. The present study investigated the mRNA regulation of components of the miRNA biogenesis pathway (Drosha, Dicer and Exportin-5), muscle enriched miRNAs, (miR-1, -133a, -133b and -206), and several miRNAs dysregulated in muscle myopathies (miR-9, -23, -29, -31 and -181). Measurements were made in muscle biopsies from nine healthy untrained males at rest, 3 h following an acute bout of moderate-intensity endurance cycling and following 10 days of endurance training. Bioinformatics analysis was used to predict potential miRNA targets. In the 3 h period following the acute exercise bout, Drosha, Dicer and Exportin-5, as well as miR-1, -133a, -133-b and -181a were all increased. In contrast miR-9, -23a, -23b and -31 were decreased. Short-term training increased miR-1 and -29b, while miR-31 remained decreased. Negative correlations were observed between miR-9 and HDAC4 protein (r=-0.71; P= 0.04), miR-31 and HDAC4 protein (r =-0.87; P= 0.026) and miR-31 and NRF1 protein (r =-0.77; P= 0.01) 3 h following exercise. miR-31 binding to the HDAC4 and NRF1 3′ untranslated region (UTR) reduced luciferase reporter activity. Exercise rapidly and transiently regulates several miRNA species in muscle. Several of these miRNAs may be involved in the regulation of skeletal muscle regeneration, gene transcription and mitochondrial biogenesis. Identifying endurance exercise-mediated stress signals regulating skeletal muscle miRNAs, as well as validating their targets and regulatory pathways post exercise, will advance our understanding of their potential role/s in human health

Short-term intensified cycle training alters acute and chronic responses of PGC1α and cytochrome C oxidase IV to exercise in human skeletal muscle

Reduced activation of exercise responsive signalling pathways have been reported in response to acute exercise after training; however little is known about the adaptive responses of the mitochondria. Accordingly, we investigated changes in mitochondrial gene expression and protein abundance in response to the same acute exercise before and after 10-d of intensive cycle training.

Nine untrained, healthy participants (mean±SD; VO2peak 44.1±17.6 ml/kg/min) performed a 60 min bout of cycling exercise at 164±18 W (72% of pre-training VO2peak). Muscle biopsies were obtained from the vastus lateralis muscle at rest, immediately and 3 h after exercise. The participants then underwent 10-d of cycle training which included four high-intensity interval training sessions (6×5 min; 90–100% VO2peak) and six prolonged moderate-intensity sessions (45–90 min; 75% VO2peak). Participants repeated the pre-training exercise trial at the same absolute work load (64% of pre-training VO2peak). Muscle PGC1-α mRNA expression was attenuated as it increased by 11- and 4- fold (P<0.001) after exercise pre- and post-training, respectively. PGC1-α protein expression increased 1.5 fold (P<0.05) in response to exercise pre-training with no further increases after the post-training exercise bout. RIP140 protein abundance was responsive to acute exercise only (P<0.01). COXIV mRNA (1.6 fold; P<0.01) and COXIV protein expression (1.5 fold; P<0.05) were increased by training but COXIV protein expression was decreased (20%; P<0.01) by acute exercise pre- and post-training.

These findings demonstrate that short-term intensified training promotes increased mitochondrial gene expression and protein abundance. Furthermore, acute indicators of exercise-induced mitochondrial adaptation appear to be blunted in response to exercise at the same absolute intensity following short-term training.

Differential effects of exercise on insulin-signaling gene expression in human skeletal muscle.

Skeletal muscle insulin sensitivity is enhanced after acute exercise and short-term endurance training. We investigated the impact of exercise on the gene expression of key insulin-signaling proteins in humans. Seven untrained subjects (4 women and 3 men) completed 9 days of cycling at 63 ± 2% of peak O₂ uptake for 60 min/day. Muscle biopsies were taken before, immediately after, and 3 h after the exercise bouts (on days 1 and 9). The gene expression of insulin receptor substrate-2 and the p85α subunit of phosphatidylinositol 3-kinase was significantly higher 3 h after a single exercise bout, although short-term training ameliorated this effect. Gene expression of insulin receptor and insulin receptor substrate-1 was not significantly altered at any time point. These results suggest that exercise may have a transitory impact on the expression of insulin receptor substrate-2 and phosphatidylinositol 3-kinase; however, the predominant actions of exercise on insulin sensitivity appear not to reside in the transcriptional activation of the genes encoding major insulin-signaling proteins.

The effect of insulin and exercise on c-Cbl protein abundance and phosphorylation in insulin-resistant skeletal muscle in lean and obese Zucker rats.

Aims/hypothesis: Recruitment of the protein c-Cbl to the insulin receptor (IR) and its tyrosine phosphorylation via a pathway that is independent from phosphatidylinositol 3prime-kinase is necessary for insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. The activation of this pathway by insulin or exercise has yet to be reported in skeletal muscle. Methods: Lean and obese Zucker rats were randomly assigned to one of three treatment groups: (i) control, (ii) insulin-stimulated or (iii) acute, exhaustive exercise. Hind limb skeletal muscle was removed and the phosphorylation state of IR, Akt and c-Cbl measured.  Results:   Insulin receptor phosphorylation was increased 12-fold after insulin stimulation (p<0.0001) in lean rats and threefold in obese rats. Acute exercise had no effect on IR tyrosine phosphorylation. Similar results were found for serine phosphorylation of Akt. Exercise did not alter c-Cbl tyrosine phosphorylation in skeletal muscle of lean or obese rats. However, in contrast to previous studies in adipocytes, c-Cbl tyrosine phosphorylation was reduced after insulin treatment (p<0.001). Conclusions/interpretation: We also found that c-Cbl associating protein expression is relatively low in skeletal muscle of Zucker rats compared to 3T3-L1 adipocytes and this could account for the reduced c-Cbl tyrosine phosphorylation after insulin treatment. Interestingly, basal levels of c-Cbl tyrosine phosphorylation were higher in skeletal muscle from insulin-resistant Zucker rats (p<0.05), but the physiological relevance is not clear. We conclude that the regulation of c-Cbl phosphorylation in skeletal muscle differs from that previously reported in adipocytes.

Muscle Na+ -K+ -ATPase activity and isoform adaptions to intense interval exercise and training in well-trained athletes

The Na⁺-K⁺-ATPase enzyme is vital in skeletal muscle function. We investigated the effects of acute high-intensity interval exercise, before and following high-intensity training (HIT), on muscle Na⁺-K⁺-ATPase maximal activity, content, and isoform mRNA expression and protein abundance. Twelve endurance-trained athletes were tested at baseline, pretrain, and after 3 wk of HIT (posttrain), which comprised seven sessions of 8 x 5-min interval cycling at 80% peak power output. Vastus lateralis muscle was biopsied at rest (baseline) and both at rest and immediately postexercise during the first (pretrain) and seventh (posttrain) training sessions. Muscle was analyzed for Na⁺-K⁺-ATPase maximal activity (3-O-MFPase), content ([³H]ouabain binding), isoform mRNA expression (RT-PCR), and protein abundance (Western blotting). All baseline-to-pretrain measures were stable. Pretrain, acute exercise decreased 3-O-MFPase activity [12.7% (SD 5.1), P < 0.05], increased α₁, α₂, and α₃ mRNA expression (1.4-, 2.8-, and 3.4-fold, respectively, P < 0.05) with unchanged ß-isoform mRNA or protein abundance of any isoform. In resting muscle, HIT increased (P < 0.05) 3-O-MFPase activity by 5.5% (SD 2.9), and α₃ and ß₃ mRNA expression by 3.0- and 0.5-fold, respectively, with unchanged Na+-K+-ATPase content or isoform protein abundance. Posttrain, the acute exercise induced decline in 3-O-MFPase activity and increase in α₁ and α₃ mRNA each persisted (P < 0.05); the postexercise 3-O-MFPase activity was also higher after HIT (P < 0.05). Thus HIT augmented Na⁺-K⁺-ATPase maximal activity despite unchanged total content and isoform protein abundance. Elevated Na⁺-K⁺-ATPase activity postexercise may contribute to reduced fatigue after training. The Na⁺-K⁺-ATPase mRNA response to interval exercise of increased α - but not ß-mRNA was largely preserved posttrain, suggesting a functional role of α mRNA upregulation.