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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online May 5, 2005 as doi:10.1096/fj.04-3144fje. |
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-AMPK knockout on exercise-induced gene activation in mouse skeletal muscle
* Copenhagen Muscle Research Centre, Department of Human Physiology, Institute of Exercise and Sport Sciences, University of Copenhagen, Copenhagen, Denmark;
Institut Cochin, INSERM, CNRS, Rene Descartes University, Department of Genetic, Development and Molecular Pathology, Paris, France;
Copenhagen Muscle Research Centre, Department for Molecular Muscle Biology, Rigshospitalet, Copenhagen, Denmark;
The John B. Pierce Laboratory, Department of Cellular and Molecular Physiology, Yale University, New Haven, Connecticut, USA; and
|| Copenhagen Muscle Research Centre, University of Copenhagen, Copenhagen, Denmark
1 Correspondence: Copenhagen Muscle Research Centre, Dept. of Human Physiology, Inst. of Exercise and Sport Sciences, 13-Universitetsparken. University of Copenhagen, Copenhagen 2100, Denmark. E-mail: sbjorgensen{at}ifi.ku.dk
SPECIFIC AIMS
The aim of the study was to investigate whether the exercise-induced activation of genes encoding metabolic proteins was reduced when the principal catalytic isoform (
2) of AMPK in muscle was knocked out. Selected data were also collected in mice in which the 1-AMPK subunit was knocked out. Because evidence indicates that AMPK may be important in regulating the energy balance in exercising muscle, adenine nucleotide levels were measured. Mice were exercised 1.5 h by treadmill running and hindlimb muscles were removed from whole-body 1 or 2-AMPK knockout (KO) or wild-type (WT) mice either at rest or 0, 1, or 3 h after running. In an additional experiment, WT and 2-AMPK KO mice were injected with saline or AICAR and muscles were removed 6.5 h after injection to investigate the role of 2-AMPK in gene activation in resting muscles. AMPK kinase activity was measured in vitro and by phosphorylation of -AMPK thr172 and ACCß ser227. Gene transcription was measured by a RT-PCR based nuclear run-on technique and mRNA content by RT-PCR using real time PCR. Adenine nucleotides were measured by HPLC in extracts from freeze dried muscles.
PRINCIPAL FINDINGS
1. AMPK activity was markedly reduced both at rest and during exercise in 2-AMPK knockout muscles
Judged from -AMPK phosphorylation, exercise increased (P<0.05, main effect) AMPK activity in both 2-WT (240%) and 2-KO (510%) quadriceps muscles, confirming that AMPK was activated during treadmill running. When the 2-isoform was lacking, AMPK phosphorylation was reduced on average by 69% (P<0.05, main effect) at rest, during running, and in recovery but still displayed the same relative response to exercise. Phosphorylation of the AMPK substrate ACCß showed essentially the same pattern (P<0.05, main effect). This was seen although the remaining 1-protein content increased (67%, P<0.05). Despite the increased 1-AMPK protein expression, the 1-AMPK activity was increased (P<0.05, main effect) to the same extent in 2-WT (226%) and 2-KO (256%) muscles in response to running. The 2-AMPK activity in 2-WT muscles increased (180%, P<0.05) in response to running. In 1-AMPK KO muscles, the increase in AMPK-P and ACCß-P during running was similar to the increase in WT muscles. The markedly decreased phosphorylation of AMPK and ACCß in 2-AMPK KO muscles shows that the 2-AMPK isoform is the main donor of AMPK activity in exercising mouse skeletal muscle in vivo.
2. Gene transcription was reduced in 2-AMPK knockout muscles
Exercise increased (P<0.05, main effect) HKII gene transcription similarly in 2-WT (119%) and in 2-KO (204%) muscles, and PGC-1 transcription tended (P=0.08) to increase in 2-WT (203%) and 2-KO (512%) muscles 1 h after treadmill running with no difference between the genotypes (Fig. 1
). The 2-knockout was associated with a reduction (P<0.05, main effects) in transcriptional activity of PGC-1 (66%) and UCP3 (58%), whereas HKII transcription tended (44%, P=0.08) to be decreased (Fig. 1)
. This novel finding indicates that AMPK plays a role in regulating basal transcription of genes encoding metabolic enzymes in skeletal muscles.
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3. The 2-AMPK knockout did not reduce the exercise-induced increases in mRNA but abolished AICAR-induced increases in PGC-1 and HKI mRNA in muscle
Treadmill running increased (P<0.05, main effects) the mRNA content of PGC-1 (
300–500%), FOXO1 (50-200%), HKII (40-70%), and PDK4 (80-140%) after 1 h and 3 h of recovery, and FOXO1 mRNA was also increased during exercise. These exercise-induced mRNA responses were, however, similar in both 2-WT and 2-KO muscles (Fig. 2
), indicating that 2-AMPK is not essential for the exercise-induced increase in mRNA of the investigated metabolic genes. Exercise-induced increases of mRNA coding for the four proteins mentioned above were also unaffected by knockout of the 1-AMPK subunit. The CPT1 mRNA content was reduced (26%, P<0.05, main effect) in 2-KO muscle at all time points compared with 2-WT muscles, indicating that AMPK is involved in regulating the basal expression of this mitochondrial protein. When AICAR was injected subcutaneously increases (P<0.05) in mRNA content of PGC-1 (164%) and HKII (539%) occurred in 2-WT muscles compared with saline controls but the AICAR response was abolished in 2-KO muscles. In response to AICAR, FOXO1 (90%-100%), PDK4 (300%-500%), and UCP3 (50%-100%) mRNA content were all increased (P<0.05, main effects) in both 2-WT and 2-KO muscles compared with the saline group. This indicates that the 2-isoform is indispensable for AICAR-induced increases in PGC-1 and HKII mRNA but not for FOXO1, PDK4 and UCP3 mRNA.
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4. The 2-knockout was associated with a decreased ATP content and increased IMP content during exercise
Muscle content of ATP, ADP, AMP, and inosine monophosphate (IMP) was measured to evaluate the metabolic stress induced by treadmill running and the impact of the 2-knockout on nucleotide balance. A notable finding was that the exercise bout induced a transient reduction (22%, P<0.05) in ATP content only in 2-KO muscles while the ATP content in 2-WT muscles was unaffected. Another observation was that the lack of the 2-isoform was associated with a lower (12%, P<0.05) ATP content in both nonexercising and recovering muscles. The ATP reduction in KO muscles during exercise (P<0.05) was paralleled with a dramatic increase (P<0.05) of the AMP deamination product IMP. IMP was raised by 28.1-fold above the detection limit (0.2 µmol g-1 d.w.) during exercise only in the 2-KO muscles, while no IMP was detected in 2-WT muscles at any time.
CONCLUSIONS AND SIGNIFICANCE
The observations that AMPK is activated in skeletal muscle during exercise and that pharmacological activation of AMPK is associated with increases in mRNA content of exercise-responsive enzymes suggest that AMPK is a likely candidate involved in transmitting an exercise signal to the nucleus of the muscle fiber. The genes investigated in the present study have all previously been demonstrated to be activated in response to exercise in human and/or rodent skeletal muscle. In the present study knockout of the 1- or 2-AMPK isoform had no effect on the exercise-induced increases in transcription or mRNA content of PGC-1, HKII, PDK4, and FOXO1 observed in WT mice, providing evidence that neither 1- nor 2-AMPK activation is essential for the acute responses of these genes to exercise. In resting muscle, previous studies have shown that chronic activation of AMPK with AICAR or ß-GPA activates several genes (GLUT4, UCP3, PGC-1, and HKII). In the present study we show that the 2-isoform is solely responsible for activation of the PGC-1 and HKII gene in response to AICAR. While these findings collectively illustrate that stimulating AMPK activity in resting muscle can activate genes, our data also demonstrate that neither 1- nor 2-AMPK is mandatory for exercise-induced activation of several metabolic genes. The reason for the different results in resting and exercising muscles may be explained by additional signaling induced by exercise (e.g., a calcium- or MAPK-dependent signaling) (Fig. 3
).
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The 2-knockout was associated with a reduced transcription of PGC-1, HKII, and UCP3 both at rest and after exercise. This is a novel finding and may, in contrast to previous beliefs, indicate that AMPK is more important in resting than exercising muscles in mediating gene activation. The finding that the 2-knockout decreased gene transcription was not followed by a decreased mRNA content suggests that AMPK also influences mRNA stability, maybe via its interactions with the mRNA stabilizing protein HuR.
Several studies suggest that AMPK is of potential importance in regulating muscle metabolism during and after exercise. In the present study, the 2-KO muscles had a lower resting ATP content which was even further reduced during running while 2-WT ATP content was maintained at resting levels. The observed reduction of ATP content in 2-KO muscles during running indicates that 2-KO muscles had a reduced capacity to rephosphorylate ATP during exercise. The finding of a substantial increase in IMP content during exercise only in 2-KO muscle supports the notion that 2-KO muscles were more metabolically stressed than 2-WT muscles during exercise.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3144fje; doi: 10.1096/fj.04-3144fje
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