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. 2006 Dec 15;577(Pt 3):1021-32.
doi: 10.1113/jphysiol.2006.120972. Epub 2006 Oct 12.

Predominant alpha2/beta2/gamma3 AMPK activation during exercise in human skeletal muscle

J B Birk  1 , J F P Wojtaszewski
Affiliations

Predominant alpha2/beta2/gamma3 AMPK activation during exercise in human skeletal muscle

J B Birk et al. J Physiol. .

Abstract

5'AMP-activated protein kinase (AMPK) is a key regulator of cellular metabolism and is regulated in muscle during exercise. We have previously established that only three of 12 possible AMPK alpha/beta/gamma-heterotrimers are present in human skeletal muscle. Previous studies describe discrepancies between total AMPK activity and regulation of its target acetyl-CoA-carboxylase (ACC)beta. Also, exercise training decreases expression of the regulatory gamma3 AMPK subunit and attenuates alpha2 AMPK activity during exercise. We hypothesize that these observations reflect a differential regulation of the AMPK heterotrimers. We provide evidence here that only the alpha2/beta2/gamma3 subunit is phosphorylated and activated during high-intensity exercise in vivo. The activity associated with the remaining two AMPK heterotrimers, alpha1/beta2/gamma1 and alpha2/beta2/gamma1, is either unchanged (20 min, 80% maximal oxygen uptake ) or decreased (30 or 120 s sprint-exercise). The differential activity of the heterotrimers leads to a total alpha-AMPK activity, that is decreased (30 s trial), unchanged (120 s trial) and increased (20 min trial). AMPK activity associated with the alpha2/beta2/gamma3 heterotrimer was strongly correlated to gamma3-associated alpha-Thr-172 AMPK phosphorylation (r(2) = 0.84, P < 0.001) and to ACCbeta Ser-221 phosphorylation (r(2) = 0.65, P < 0.001). These data single out the alpha2/beta2/gamma3 heterotrimer as an important actor in exercise-regulated AMPK signalling in human skeletal muscle, probably mediating phosphorylation of ACCbeta.

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Figures

Figure 1
Figure 1. AMPK heterotrimer composition and phosphorylation in human skeletal muscle
Lysates were prepared from biopsies taken in the vastus lateralis muscle before and after 20 min of exercise at 80%V˙O2,peak (n = 11). From 400 μg of lysate AMPK α1 (A), α2 (B), phospho α-Thr-172 (C) or γ3 (D) was immunoprecipitated (ip). The figure shows representative blots of the IP, post-IP and pre-IP (lysate) in the rested and exercised state. One-eighth of the IP corresponding to 50 μg was loaded together with 20 μg of the post- and pre-IPs. The blotted membranes were analysed with anti-α1, -α2, -phospho α-Thr-172, -β2, -γ1 and -γ3 as indicated to the far right. The small arrow indicates IgG light and heavy chains on the blots.
Figure 2
Figure 2. Thr-172 α-AMPK phosphorylation
Lysates are from the vastus lateralis muscle before and after exercise for 30 s (n = 10), 120 s (n = 9) and 20 min (n = 11), respectively; 25 μg of lysate were run on SDS-PAGE and blotted for phosphorylation of Thr-172 α-AMPK. Results are means ±s.e.m., ***P < 0.001, difference between rest and exercise.
Figure 3
Figure 3. AMPK subunit-associated activity in response to exercise
Lysates are from the vastus lateralis muscle before and after exercise for A, 20 min (n = 11); B, 120 s (n = 9) and C, 30 s (n = 10). AMPK α1 (α1/β2/γ1), α2 (α2/β2/γ1 + α2/β2/γ3) or β2 α1/β2/γ1 + α2/β2/γ1 + α2/β2/γ3) were immunoprecipitated from 200 μg of lysate, and activity against the AMARA peptide was measured in the presence of 200 μm AMP. Results are means ±s.e.m., *P < 0.05, **P < 0.01 and ***P < 0.001, significant differences between rest and exercise.
Figure 4
Figure 4. AMPK subunit-associated activity in response to exercise
Lysates are from the vastus lateralis muscle before and after exercise for A, 20 min (n = 11); B, 120 s (n = 9) and C, 30 s (n = 10). AMPK γ3 (α2/β2/γ3) was immunoprecipitated from 200 μg of lysate, and activity against the AMARA peptide was measured in the presence of 200 μm AMP. The α2/β2/γ1-associated activity was determined by IP of α2 after immunodepleting the lysate for γ3. Results are means ±s.e.m., *P < 0.05, **P < 0.01 and ***P < 0.001, significant differences between rest and exercise.
Figure 5
Figure 5. AMPK phosphorylation in relation to AMPK γ3-associated activity
Lysates are from the vastus lateralis muscle before and after exercise for 30 s (n = 10), 120 s (n = 9) and 20 min (n = 11), respectively. A, 25 μg of lysate were run on SDS-PAGE and blotted for Thr-172 phosphorylation of α-AMPK. These signal intensities were multiplied with the amount of Thr-172 phosphorylated α-AMPK co-immunoprecipitated with γ3 to get the level of phosphorylation associated with the γ3 trimers. B, relationship between the γ3-associated level of Thr-172 α-AMPK phosphorylation and the AMPK activity of α2/β2/γ3. •= 30 s trial (n = 10); ▴= 120 s trial (n = 9) and ▪= 20 min trial (n = 11). A significant coefficient was found with linear regression analysis (r2 = 0.84, P < 0.001). Results are means ±s.e.m., ***P < 0.001, difference between rest and exercise, †††P < 0.001, significantly different from the 20 min trial.
Figure 6
Figure 6. ACCβ phosphorylation in relation to AMPK γ3-associated activity
Lysates are from the vastus lateralis muscle before and after exercise for 30 s (n = 10), 120 s (n = 9) and 20 min (n = 11), respectively. A, 25 μg of lysate were run on SDS-PAGE and blotted for phospho Ser-221 ACCβ. B, relationship between the level of ACCβ phosphorylation and the AMPK activity of α2/β2/γ3. •= 30 s (n = 10), ▴= 120 s (n = 9) and ▪= 20 min (n = 11). A significant coefficient was found with linear regression analysis (r2 = 0.65, P < 0.001). Results are means ±s.e.m., ***P < 0.001, difference between rest and exercise, †P < 0.05 and †††P < 0.001, significantly different from the 20 min trial.
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