AMPK alpha1 activation is required for stimulation of glucose uptake by twitch contraction, but not by H2O2, in mouse skeletal muscle

Abstract

Background: AMPK is a promising pharmacological target in relation to metabolic disorders partly due to its non-insulin dependent glucose uptake promoting role in skeletal muscle. Of the 2 catalytic alpha-AMPK isoforms, alpha(2) AMPK is clearly required for stimulation of glucose transport into muscle by certain stimuli. In contrast, no clear function has yet been determined for alpha(1) AMPK in skeletal muscle, possibly due to alpha-AMPK isoform signaling redundancy. By applying low-intensity twitch-contraction and H(2)O(2) stimulation to activate alpha(1) AMPK, but not alpha(2) AMPK, in wildtype and alpha-AMPK transgenic mouse muscles, this study aimed to define conditions where alpha(1) AMPK is required to increase muscle glucose uptake.

Methodology/principal findings: Following stimulation with H(2)O(2) (3 mM, 20 min) or twitch-contraction (0.1 ms pulse, 2 Hz, 2 min), signaling and 2-deoxyglucose uptake were measured in incubated soleus muscles from wildtype and muscle-specific kinase-dead AMPK (KD), alpha(1) AMPK knockout or alpha(2) AMPK knockout mice. H(2)O(2) increased the activity of both alpha(1) and alpha(2) AMPK in addition to Akt phosphorylation, and H(2)O(2)-stimulated glucose uptake was not reduced in any of the AMPK transgenic mouse models compared with wild type. In contrast, twitch-contraction increased the activity of alpha(1) AMPK, but not alpha(2) AMPK activity nor Akt or AS160 phosphorylation. Glucose uptake was markedly lower in alpha(1) AMPK knockout and KD AMPK muscles, but not in alpha(2) AMPK knockout muscles, following twitch stimulation.

Conclusions/significance: These results provide strong genetic evidence that alpha(1) AMPK, but not alpha(2) AMPK, Akt or AS160, is necessary for regulation of twitch-contraction stimulated glucose uptake. To our knowledge, this is the first report to show a major and essential role of alpha(1) AMPK in regulating a physiological endpoint in skeletal muscle. In contrast, AMPK is not essential for H(2)O(2)-stimulated muscle glucose uptake, as proposed by recent studies.

Figure 1. Tension-measurements.

Tension-development during A) H2O2 stimulation (3 mM, 20 min) and B) twitch (white bars: 0.1 ms, 2 Hz, 2 min) and tetanic (black bars: 0.2 ms, 100 Hz, 1s/15s, 2 min) stimulation in mouse soleus muscles (n =  5–8). *** p<0.001 vs. pre.

Figure 2. H₂O₂ and twitch-contraction stimulated 2-deoxyglucose (2DG) uptake and signaling in mouse soleus muscle.

A) 2DG uptake in basal vs. H₂O₂ (3 mM, 20 min), twitch contraction (0.1 ms, 2 Hz, 2 min) or tetanic contraction (0.2 ms, 100 Hz, 1s/15s, 10 min)-stimulated muscles (n = 5–12). B) α₁ AMPK and C) α₂ AMPK activities in basal vs. H₂O₂ with same conditions as in panel A (n = 6–16). D) Akt Ser473 phosphorylation in basal, twitch-contracted and H₂O₂-stimulated soleus and EDL muscles (n = 6). E) H₂O₂-stimulated 2DG uptake in wildtype vs. kinase-dead (KD) AMPK muscles (n =  5–6) and F) wildtype vs. α₁ AMPK KO muscles (n = 8). **/*** p<0.01/0.001 vs. basal.

Figure 3. Twitch-contraction (0.1 ms, 2 Hz, 2 min) signaling in AMPK transgenic soleus muscles.

A) α₁ AMPK and B) α₂ AMPK activities in wildtype vs. kinase-dead (KD) AMPK muscles (n = 7–8), C ) α₁ AMPK and D) α₂ AMPK activities in wildtype vs. α₁ AMPK KO muscles (n = 7–10), E ) α₁ AMPK and F) α₂ AMPK activities E) and F) wildtype vs. α₂ AMPK (n = 10). G) Basal and twitch-stimulated AS160 phosphorylation in wildtype vs. AMPK transgenic muscles (n = 6–9). As a positive control, insulin-stimulated (2000 µU/ml, 15 min) soleus was included (n = 1). * p<0.05 vs. basal. ††/††† p<0.01/0.001 genotype main effect.

Figure 4. Twitch-stimulated (0.1 ms, 2 Hz, 2 min) AMPK and ACCβ phosphorylation in mouse soleus muscles.

AMPK Thr172 phosphorylation in. A) wildtype vs. kinase-dead (KD) AMPK muscles (n = 7–8), C) wildtype vs. α₁ AMPK KO muscles (n = 9–10) E) wildtype vs. α₂ AMPK KO muscles (n = 10). ACCβ Ser221 phosphorylation in B) wildtype vs. kinase-dead (KD) AMPK muscles (n = 7–8), D) wildtype vs. α₁ AMPK KO muscles (n = 9–10) F) wildtype vs. α₂ AMPK KO muscles (n = 10). */** p<0.05/0.01 vs. basal. ††/††† p<0.01/0.001 genotype-effect. (†) indicates borderline significant genotype-effect, p = 0.06. G) Representative blots

Figure 5. Twitch-contraction requires α₁ AMPK to stimulate glucose uptake.

Twitch contraction (0.1 ms, 2 Hz, 2 min) stimulated 2-deoxyglucose in mouse soleus muscles from either A) wildtype vs. kinase-dead AMPK muscles (n = 10–13) B) wildtype vs. α₁ AMPK KO muscles (n = 16–17) C) wildtype vs. α₂ AMPK KO muscles (n = 11–12). Absolute values are shown on the left and the corresponding percentage increase above basal for paired muscles is shown on the right. */** p<0.05/0.01 vs. wildtype or basal, † genotype x contraction p<0.05.