Henriette Pilegaard

Exercise induces transient transcriptional activation of the PGC‐1α gene in human skeletal muscle

Endurance exercise training induces mitochondrial biogenesis in skeletal muscle. The peroxisome proliferator activated receptor co‐activator 1α (PGC‐1α) has recently been identified as a nuclear factor critical for coordinating the activation of genes required for mitochondrial biogenesis in cell culture and rodent skeletal muscle. To determine whether PGC‐1α transcription is regulated by acute exercise and exercise training in human skeletal muscle, seven male subjects performed 4 weeks of one‐legged knee extensor exercise training. At the end of training, subjects completed 3 h of two‐legged knee extensor exercise. Biopsies were obtained from the vastus lateralis muscle of both the untrained and trained legs before exercise and after 0, 2, 6 and 24 h of recovery. Time to exhaustion (2 min maximum resistance), as well as hexokinase II (HKII), citrate synthase and 3‐hydroxyacyl‐CoA dehydrogenase mRNA, were higher in the trained than the untrained leg prior to exercise. Exercise induced a marked transient increase (P < 0.05) in PGC‐1α transcription (10‐ to > 40‐fold) and mRNA content (7‐ to 10‐fold), peaking within 2 h after exercise. Activation of PGC‐1α was greater in the trained leg despite the lower relative workload. Interestingly, exercise did not affect nuclear respiratory factor 1 (NRF‐1) mRNA, a gene induced by PGC‐1α in cell culture. HKII, mitochondrial transcription factor A, peroxisome proliferator activated receptor α, and calcineurin Aα and Aβ mRNA were elevated (≈2‐ to 6‐fold; P < 0.05) at 6 h of recovery in the untrained leg but did not change in the trained leg. The present data demonstrate that exercise induces a dramatic transient increase in PGC‐1α transcription and mRNA content in human skeletal muscle. Consistent with its role as a transcriptional coactivator, these findings suggest that PGC‐1α may coordinate the activation of metabolic genes in human muscle in response to exercise.

Evidence for a release of brain‐derived neurotrophic factor from the brain during exercise

Brain‐derived neurotrophic factor (BDNF) has an important role in regulating maintenance, growth and survival of neurons. However, the main source of circulating BDNF in response to exercise is unknown. To identify whether the brain is a source of BDNF during exercise, eight volunteers rowed for 4 h while simultaneous blood samples were obtained from the radial artery and the internal jugular vein. To further identify putative cerebral region(s) responsible for BDNF release, mouse brains were dissected and analysed for BDNF mRNA expression following treadmill exercise. In humans, a BDNF release from the brain was observed at rest (P < 0.05), and increased two‐ to threefold during exercise (P < 0.05). Both at rest and during exercise, the brain contributed 70–80% of circulating BDNF, while that contribution decreased following 1 h of recovery. In mice, exercise induced a three‐ to fivefold increase in BDNF mRNA expression in the hippocampus and cortex, peaking 2 h after the termination of exercise. These results suggest that the brain is a major but not the sole contributor to circulating BDNF. Moreover, the importance of the cortex and hippocampus as a source for plasma BDNF becomes even more prominent in response to exercise.

Transcriptional activation of the IL-6 gene in human contracting skeletal muscle: influence of muscle glycogen content

In humans, the plasma interleukin 6 (IL‐6) concentration increases dramatically during low‐intensity exercise. Measurements across the working limb indicate that skeletal muscle is the source of IL‐6 production. To determine whether energy availability influences the regulation of IL‐6 expression during prolonged exercise, six male subjects completed two trials consisting of 180 min of two‐legged dynamic knee extensor with either normal or low (~60% of control) pre‐exercise muscle glycogen levels. Increases in plasma IL‐6 during exercise were significantly higher (P < 0.05) in the low‐glycogen (16‐fold) trial verses the control (10‐fold) trial. Transcriptional activation of the IL‐6 gene in skeletal muscle was also higher in the low‐glycogen trial; it increased by about 40‐fold after 90 min of exercise and about 60‐fold after 180 min of exercise. Muscle IL‐6 mRNA followed a similar but delayed pattern, increasing by more than 100‐fold in the low‐glycogen trial and by about 30‐fold in the control trial. These data demonstrate that exercise activates transcription of the IL‐6 gene in working skeletal muscle, a response that is dramatically enhanced when glycogen levels are low. These findings also support the hypothesis that IL‐6 may be produced by contracting myofibers when glycogen levels become critically low as a means of signaling the liver to increase glucose production.

Brain-derived neurotrophic factor (BDNF) and type 2 diabetes

Aims/hypothesisDecreased levels of brain-derived neurotrophic factor (BDNF) have been implicated in the pathogenesis of Alzheimer’s disease and depression. These disorders are associated with type 2 diabetes, and animal models suggest that BDNF plays a role in insulin resistance. We therefore explored whether BDNF plays a role in human glucose metabolism.Subjects and methodsWe included (Study 1) 233 humans divided into four groups depending on presence or absence of type 2 diabetes and presence or absence of obesity; and (Study 2) seven healthy volunteers who underwent both a hyperglycaemic and a hyperinsulinaemic–euglycaemic clamp.ResultsPlasma levels of BDNF in Study 1 were decreased in humans with type 2 diabetes independently of obesity. Plasma BDNF was inversely associated with fasting plasma glucose, but not with insulin. No association was found between the BDNF G196A (Val66Met) polymorphism and diabetes or obesity. In Study 2 an output of BDNF from the human brain was detected at basal conditions. This output was inhibited when blood glucose levels were elevated. In contrast, when plasma insulin was increased while maintaining normal blood glucose, the cerebral output of BDNF was not inhibited, indicating that high levels of glucose, but not insulin, inhibit the output of BDNF from the human brain.Conclusions/interpretationLow levels of BDNF accompany impaired glucose metabolism. Decreased BDNF may be a pathogenetic factor involved not only in dementia and depression, but also in type 2 diabetes, potentially explaining the clustering of these conditions in epidemiological studies.

Lactic Acid Efflux from White Skeletal Muscle Is Catalyzed by the Monocarboxylate Transporter Isoform MCT3

The newly cloned proton-linked monocarboxylate transporter MCT3 was shown by Western blotting and immunofluorescence confocal microscopy to be expressed in all muscle fibers. In contrast, MCT1 is expressed most abundantly in oxidative fibers but is almost totally absent in fast-twitch glycolytic fibers. Thus MCT3 appears to be the major MCT isoform responsible for efflux of glycolytically derived lactic acid from white skeletal muscle. MCT3 is also expressed in several other tissues requiring rapid lactic acid efflux. The expression of both MCT3 and MCT1 was decreased by 40–60% 3 weeks after denervation of rat hind limb muscles, whereas chronic stimulation of the muscles for 7 days increased expression of MCT1 2–3-fold but had no effect on MCT3 expression. The kinetics and substrate and inhibitor specificities of monocarboxylate transport into cell lines expressing only MCT3 or MCT1 have been determined. Differences in the properties of MCT1 and MCT3 are relatively modest, suggesting that the significance of the two isoforms may be related to their regulation rather than their intrinsic properties.

Endurance training enhances BDNF release from the human brain

The circulating level of brain-derived neurotrophic factor (BDNF) is reduced in patients with major depression and type-2 diabetes. Because acute exercise increases BDNF production in the hippocampus and cerebral cortex, we hypothesized that endurance training would enhance the release of BDNF from the human brain as detected from arterial and internal jugular venous blood samples. In a randomized controlled study, 12 healthy sedentary males carried out 3 mo of endurance training (n = 7) or served as controls (n = 5). Before and after the intervention, blood samples were obtained at rest and during exercise. At baseline, the training group (58 + or - 106 ng x 100 g(-1) x min(-1), means + or - SD) and the control group (12 + or - 17 ng x 100 g(-1) x min(-1)) had a similar release of BDNF from the brain at rest. Three months of endurance training enhanced the resting release of BDNF to 206 + or - 108 ng x 100 g(-1) x min(-1) (P < 0.05), with no significant change in the control subjects, but there was no training-induced increase in the release of BDNF during exercise. Additionally, eight mice completed a 5-wk treadmill running training protocol that increased the BDNF mRNA expression in the hippocampus (4.5 + or - 1.6 vs. 1.4 + or - 1.1 mRNA/ssDNA; P < 0.05), but not in the cerebral cortex (4.0 + or - 1.4 vs. 4.6 + or - 1.4 mRNA/ssDNA) compared with untrained mice. The increased BDNF expression in the hippocampus and the enhanced release of BDNF from the human brain following training suggest that endurance training promotes brain health.

Effects of α-AMPK knockout on exercise-induced gene activation in mouse skeletal muscle

We tested the hypothesis that 5′AMP‐activated protein kinase (AMPK) plays an important role in regulating the acute, exercise‐induced activation of metabolic genes in skeletal muscle, which were dissected from whole‐body α2‐ and α1‐AMPK knockout (KO) and wild‐type (WT) mice at rest, after treadmill running (90 min), and in recovery. Running increased α1‐AMPK kinase activity, phosphorylation (P) of AMPK, and acetyl‐CoA carboxylase (ACC)β in α2‐WT and α2‐KO muscles and increased α2‐AMPK kinase activity in α2‐WT. In α2‐KO muscles, AMPK‐P and ACCβ‐P were markedly lower compared with α2‐WT. However, in α1‐WT and α1‐KO muscles, AMPK‐P and ACCβ‐P levels were identical at rest and increased similarly during exercise in the two genotypes. The α2‐KO decreased peroxisome‐proliferator‐activated receptor γ coactivator (PGC)‐1α, uncoupling protein‐3 (UCP3), and hexokinase II (HKII) transcription at rest but did not affect exercise‐induced transcription. Exercise increased the mRNA content of PGC‐1α, Forkhead box class O (FOXO)1, HKII, and pyruvate dehydrogenase kinase 4 (PDK4) similarly in α2‐WT and α2‐KO mice, whereas glucose transporter GLUT 4, carnitine palmitoyltransferase 1 (CPTI), lipoprotein lipase, and UCP3 mRNA were unchanged by exercise in both genotypes. CPTI mRNA was lower in α2‐KO muscles than in α2‐WT muscles at all time‐points. In α1‐WT and α1‐KO muscles, running increased the mRNA content of PGC‐1α and FOXOl similarly. The α2‐KO was associated with lower muscle adenosine 5′‐triphosphate content, and the inosine monophosphate content increased substantially at the end of exercise only in α2‐KO muscles. In addition, subcutaneous injection of 5‐aminoimidazole‐4‐carboxamide‐1‐β‐4‐ribofuranoside (AICAR) increased the mRNA content of PGC‐1α, HKII, FOXO1, PDK4, and UCP3, and α2‐KO abolished the AICAR‐induced increases in PGC‐1α and HKII mRNA. In conclusion, KO of the α2‐ but not the α1‐AMPK isoform markedly diminished AMPK activation during running. Nevertheless, exercise‐induced activation of the investigated genes in mouse skeletal muscle was not impaired in α1‐ or α2‐AMPK KO muscles. Although it cannot be ruled out that activation of the remaining α‐isoform is sufficient to increase gene activation during exercise, the present data do not support an essential role of AMPK in regulating exercise‐induced gene activation in skeletal muscle.

Effect of high-intensity exercise training on lactate/H+ transport capacity in human skeletal muscle.

The present study examined the effect of high-intensity exercise training on muscle sarcolemmal lactate/H+ transport and the monocarboxylate transporters (MCT1 and MCT4) as well as lactate and H+ release during intense exercise in humans. One-legged knee-extensor exercise training was performed for 8 wk, and biopsies were obtained from untrained and trained vastus lateralis muscle. The rate of lactate/H+ transport determined in sarcolemmal giant vesicles was 12% higher ( P < 0.05) in the trained than in untrained muscle ( n = 7). The content of MCT1 and MCT4 protein was also higher (76 and 32%, respectively; n = 4) in trained muscle. Release of lactate and H+ from the quadriceps muscle at the end of intense exhaustive knee-extensor exercise was similar in the trained and untrained leg, although the estimated muscle intracellular-to-interstitial gradients of lactate and H+ were lower ( P < 0.05) in the trained than in the untrained muscle. The present data show that intense exercise training can increase lactate/H+transport capacity in human skeletal muscle as well as improve the ability of the muscle to release lactate and H+ during contractions.

Influence of pre-exercise muscle glycogen content on exercise-induced transcriptional regulation of metabolic genes.

Transcription of metabolic genes is transiently induced during recovery from exercise in skeletal muscle of humans. To determine whether pre‐exercise muscle glycogen content influences the magnitude and/or duration of this adaptive response, six male subjects performed one‐legged cycling exercise to lower muscle glycogen content in one leg and then, the following day, completed 2.5 h low intensity two‐legged cycling exercise. Nuclei and mRNA were isolated from biopsies obtained from the vastus lateralis muscle of the control and reduced glycogen (pre‐exercise glycogen = 609 ± 47 and 337 ± 33 mmol kg−1 dry weight, respectively) legs before and after 0, 2 and 5 h of recovery. Exercise induced a significant (P < 0.05) increase (2‐ to 3‐fold) in transcription of the pyruvate dehydrogenase kinase 4 (PDK4) and uncoupling protein 3 (UCP3) genes in the reduced glycogen leg only. Although PDK4, lipoprotein lipase (LPL) and hexokinase II (HKII) mRNA were elevated in the reduced glycogen leg before exercise, no consistent difference was found between the two legs in response to exercise. In a second study, six subjects completed two trials (separated by 2 weeks) consisting of 3 h of two‐legged knee extensor exercise with either control (398 ± 52 mmol kg−1 dry weight) or low (240 ± 38 mmol kg−1 dry weight) pre‐exercise muscle glycogen. Exercise induced a significantly greater increase in PDK4 transcription in the low glycogen (> 6‐fold) than in the control (< 3‐fold) trial. Induction of PDK4 and UCP3 mRNA in response to exercise was also signficantly higher in the low glycogen (11.4‐ and 3.5‐fold, respectively) than in the control (5.0‐ and 1.7‐fold, respectively) trial. These data indicate that low muscle glycogen content enhances the transcriptional activation of some metabolic genes in response to exercise, raising the possibility that signalling mechanisms sensitive to glycogen content and/or FFA availability may be linked to the transcriptional control of exercise‐responsive genes.

Expression of interleukin-15 in human skeletal muscle – effect of exercise and muscle fibre type composition

The cytokine interleukin‐15 (IL‐15) has been demonstrated to have anabolic effects in cell culture systems. We tested the hypothesis that IL‐15 is predominantly expressed by type 2 skeletal muscle fibres, and that resistance exercise regulates IL‐15 expression in muscle. Triceps brachii, vastus lateralis quadriceps and soleus muscle biopsies were obtained from normally physically active, healthy, young male volunteers (n= 14), because these muscles are characterized by having different fibre‐type compositions. In addition, healthy, normally physically active male subjects (n= 8) not involved in any kind of resistance exercise underwent a heavy resistance exercise protocol that stimulated the vastus lateralis muscle and biopsies were obtained from this muscle pre‐exercise as well as 6, 24 and 48 h post‐exercise. IL‐15 mRNA levels were twofold higher in the triceps (type 2 fibre dominance) compared with the soleus muscle (type 1 fibre dominance), but Western blotting and immunohistochemistry revealed that muscle IL‐15 protein content did not differ between triceps brachii, quadriceps and soleus muscles. Following resistance exercise, IL‐15 mRNA levels were up‐regulated twofold at 24 h of recovery without any changes in muscle IL‐15 protein content or plasma IL‐15 at any of the investigated time points. In conclusion, IL‐15 mRNA level is enhanced in skeletal muscles dominated by type 2 fibres and resistance exercise induces increased muscular IL‐15 mRNA levels. IL‐15 mRNA levels in skeletal muscle were not paralleled by similar changes in muscular IL‐15 protein expression suggesting that muscle IL‐15 may exist in a translationally inactive pool.