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. 2007 Jul 17;104(29):12017-22.
doi: 10.1073/pnas.0705070104. Epub 2007 Jul 3.

AMP-activated protein kinase (AMPK) action in skeletal muscle via direct phosphorylation of PGC-1alpha

Sibylle Jäger  1 , Christoph Handschin, Julie St-Pierre, Bruce M Spiegelman
Affiliations

AMP-activated protein kinase (AMPK) action in skeletal muscle via direct phosphorylation of PGC-1alpha

Sibylle Jäger et al. Proc Natl Acad Sci U S A. .

Abstract

Activation of AMP-activated kinase (AMPK) in skeletal muscle increases glucose uptake, fatty acid oxidation, and mitochondrial biogenesis by increasing gene expression in these pathways. However, the transcriptional components that are directly targeted by AMPK are still elusive. The peroxisome-proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha) has emerged as a master regulator of mitochondrial biogenesis; furthermore, it has been shown that PGC-1alpha gene expression is induced by exercise and by chemical activation of AMPK in skeletal muscle. Using primary muscle cells and mice deficient in PGC-1alpha, we found that the effects of AMPK on gene expression of glucose transporter 4, mitochondrial genes, and PGC-1alpha itself are almost entirely dependent on the function of PGC-1alpha protein. Furthermore, AMPK phosphorylates PGC-1alpha directly both in vitro and in cells. These direct phosphorylations of the PGC-1alpha protein at threonine-177 and serine-538 are required for the PGC-1alpha-dependent induction of the PGC-1alpha promoter. These data indicate that AMPK phosphorylation of PGC-1alpha initiates many of the important gene regulatory functions of AMPK in skeletal muscle.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
AMPK-driven increase in PGC-1α and GLUT4 gene expression requires PGC-1α protein. (A) AMPK is activated in AICAR and metformin-treated primary myotubes. Comparison of AMPKα protein phosphorylated at threonine-172 (pT172) with total levels of AMPKα protein in WT and PGC-1α knockout (KO) primary myotubes treated with vehicle, AICAR, or metformin for 1 h is shown. A also shows the levels of ACC protein phosphorylated at serine-79: loading control (γ-tubulin). (B) AICAR and metformin treatments elevate the expression of PGC-1α in WT but not in PGC-1α −/− cells. The inhibitor of AMPK, 8-BrAMP, blocks this increase. (C) mRNA levels of GLUT4. (D) The relative gene expression of PGC-1β does not change under the same conditions. Primary myotubes were treated with vehicle, 500 μM AICAR, 1 mM metformin, 1 mM 8-BrAMP, 8-BrAMP/AICAR, and 8-BrAMP/metformin in DMEM/0.5% BSA for 16 h. The relative PGC-1α mRNA levels were determined with primers in exon2, which is present in WT and PGC-1α −/− cells, by using semiquantitative PCR.
Fig. 2.
Fig. 2.
AMPK-driven increase in expression of mitochondrial genes and in respiration requires PGC-1α. Primary myotubes were treated as in Fig. 1, and mRNA levels of cytochrome c (A), UCP-3 (B), and UCP-2 (C) were determined by using semiquantitative PCR. KO, knockout. (D) Oxygen consumption was measured in WT and PGC-1α −/− primary myotubes treated with AICAR in DMEM/5% HS for 2 days. At day 3, cells were shifted to DMEM/0.5% BSA and treated for an additional 16 h before oxygen consumption was measured as described in Materials and Methods.
Fig. 3.
Fig. 3.
AMPK-driven increase in PGC-1α, GLUT4, and cytochrome c gene expression requires PGC-1α protein in vivo. (A) AMPK is activated in skeletal muscle of AICAR-injected mice. Levels of ACC protein phosphorylated at serine-79 in gastrocnemeus muscle from WT and PGC-1α muscle-specific knockout (KO) mice, injected with saline or AICAR are shown. (B) Injection of AICAR induces the mRNA expression of PGC-1α and cytochrome c but not PGC-1β in the skeletal muscle of WT mice (∗, P < 0.01); this induction does not occur in the skeletal muscle of the muscle-specific PGC-1α −/− mice. UCP-3 gene expression is also increased in the muscle-specific PGC-1α −/− mice (∗∗, P < 0.05). (C) Injection of AICAR induces the mRNA expression of GLUT4 (∗, P < 0.01) and PDK4 (∗∗, P < 0.1) but not of hexokinase in the skeletal muscle of WT mice. PDK4 gene expression increases also in the muscle-specific PGC-1α −/− mice. Female mice were injected with either saline or 250 mg/kg AICAR. Skeletal muscle was harvested after 6 h, and gene expression was measured by using semiquantitative PCR (n = 5–7).
Fig. 4.
Fig. 4.
AMPK phosphorylates PGC-1α at threonine-177 and serine-538 in vitro and in cells. (A) PGC-1α interacts with AMPKα2 in cells. Expression vectors for FLAG-PGC-1α and Myc-AMPKα2 were transfected into BOSC cells, as indicated. Coimmunoprecipitation was performed as described in Materials and Methods. (B) Primary PGC-1α −/− myotubes stably expressing PGC-1α and PGC-1α T177A/S538A, respectively, were treated with vehicle or AICAR for 1 h in the presence of 32P. (C) Purified recombinant GST-PGC-1α fragments (full-length 1–797, 1–190, 200–400, 395–565, 551–797) were incubated with purified AMPK, and phosphorylation was determined by incorporation of [γ-32P]ATP. (D) Mass spectrometry identified threonine-177 and serine-538 as phosphorylated residues. The GST-PGC-1α fragment containing amino acid 1–190 T177A and the GST-PGC-1α fragment containing amino acids 395–565 S538A are not phosphorylated by AMPK. (E) The phosphorylation of PGC-1α protein by AMPK is required for elevated PGC-1α-dependent activity of the PGC-1α promoter. C2C12 muscle cells were transfected with a 2-kb PGC-1α promoter construct and expression plasmids for PGC-1α or PGC-1α T177A/S538A, respectively. After transfection, cells were differentiated for 1 day and treated with AICAR for 7.5 h before reporter gene levels were determined (∗, P < 0.01).
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