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. 2007 Jun;27(12):4317-27.
doi: 10.1128/MCB.02222-06. Epub 2007 Apr 9.

Leptin stimulates fatty acid oxidation and peroxisome proliferator-activated receptor alpha gene expression in mouse C2C12 myoblasts by changing the subcellular localization of the alpha2 form of AMP-activated protein kinase

Atsushi Suzuki  1 , Shiki Okamoto, Suni Lee, Kumiko Saito, Tetsuya Shiuchi, Yasuhiko Minokoshi
Affiliations

Leptin stimulates fatty acid oxidation and peroxisome proliferator-activated receptor alpha gene expression in mouse C2C12 myoblasts by changing the subcellular localization of the alpha2 form of AMP-activated protein kinase

Atsushi Suzuki et al. Mol Cell Biol. 2007 Jun.

Abstract

Leptin stimulates fatty acid oxidation in skeletal muscle through the activation of AMP-activated protein kinase (AMPK) and the induction of gene expression, such as that for peroxisome proliferator-activated receptor alpha (PPARalpha). We now show that leptin stimulates fatty acid oxidation and PPARalpha gene expression in the C2C12 muscle cell line through the activation of AMPK containing the alpha2 subunit (alpha2AMPK) and through changes in the subcellular localization of this enzyme. Activated alpha2AMPK containing the beta1 subunit was shown to be retained in the cytoplasm, where it phosphorylated acetyl coenzyme A carboxylase and thereby stimulated fatty acid oxidation. In contrast, alpha2AMPK containing the beta2 subunit transiently increased fatty acid oxidation but underwent rapid translocation to the nucleus, where it induced PPARalpha gene transcription. A nuclear localization signal and Thr(172) phosphorylation of alpha2 were found to be essential for nuclear translocation of alpha2AMPK, whereas the myristoylation of beta1 anchors alpha2AMPK in the cytoplasm. The prevention of alpha2AMPK activation and the change in its subcellular localization inhibited the metabolic effects of leptin. Our data thus suggest that the activation of and changes in the subcellular localization of alpha2AMPK are required for leptin-induced stimulation of fatty acid oxidation and PPARalpha gene expression in muscle cells.

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Figures

FIG. 1.
FIG. 1.
Leptin stimulates fatty acid oxidation through specific activation of α2AMPK in C2C12 cells. (A) Total RNA extracted from C2C12 cells or mouse skeletal muscle (soleus or white gastrocnemius) was subjected to RT-PCR analysis of Ob-Ra, Ob-Rb, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, internal control) mRNAs. The soleus and white gastrocnemius were used as exemplars of red and white types of skeletal muscle, respectively. (B) Cells were exposed to leptin (10 ng/ml) for the indicated times, after which cell extracts were subjected to immunoblot analysis with antibodies to Thr172-phosphorylated (pThr172) AMPK or total α subunits of AMPK or to Ser79-phosphorylated (pACC) or total ACC (tACC). (C) Cells stably expressing Flag-α1 or Flag-α2 were stimulated with leptin for the indicated times, after which cell extracts were subjected to immunoprecipitation with antibodies to Flag. The resulting precipitates were subjected to immunoblot analysis with antibodies to Thr172-phosphorylated AMPK or total α subunits of AMPK. (D) Parental cells and cells stably expressing Flag-α1 or -α2 were treated with leptin for the indicated times, after which AMPK activity in extracts of the parental cells and in immunoprecipitates (IP) prepared with anti-Flag from the transfected cells was measured. Data are means ± SEM (error bars) from three independent experiments. *, P was <0.05 versus the corresponding value for time zero. (E) Cells were transfected with siRNAs specific for α1 or α2 subunits of AMPK (+) or with a control siRNA (−). After 48 h, total RNA was isolated from the cells and subjected to RT-PCR analysis of α1, α2, and GAPDH mRNAs (upper panels) and cell extracts were subjected to immunoblot analysis (IB) of α1, α2, and β-actin (internal control). (F) Cells transfected with control, α1, or α2 siRNAs were stimulated with leptin for the indicated times, and the rate of fatty acid (FA) oxidation was measured. Data are means ± SEM (error bars) from three independent experiments. *, P was <0.05 versus the corresponding value for time zero.
FIG. 2.
FIG. 2.
Leptin induces subcellular redistribution of α2AMPK. (A and B) C2C12 cells stably expressing Flag-α1 or -α2 were treated with leptin for 0 (control), 1, or 6 h, fixed, immunostained with antibodies to Flag, and examined with a fluorescence microscope at a magnification of ×400 (A). Immunostained cells expressing Flag-α2 were also examined at a magnification of ×200 (B). (C) C2C12 cells stably expressing Flag-α1 or -α2 were exposed to leptin for the indicated times, fixed, immunostained with anti-Flag, and examined by fluorescence microscopy. The numbers of cells in which Flag-α2 was detected in the cytoplasm, the nucleus, or both the cytoplasm and the nucleus were counted (total of 500 cells in each well). Data are means ± SEM (error bars) from three independent experiments. *, P was <0.05 versus the corresponding value for time zero. (D) C2C12 cells stably expressing Flag-α1 or -α2 were stimulated with leptin for the indicated times, after which cell lysates were separated into cytoplasmic and nuclear fractions. The cytoplasmic and nuclear fractions as well as anti-Flag immunoprecipitates (IP) of cell extracts were subjected to immunoblot analysis with antibodies to Thr172-phosphorylated AMPK or total α subunits of AMPK, to β-actin (cytoplasmic marker), or to lamin B (nuclear marker), as indicated. (E) C2C12 cells were treated with leptin for the indicated times, after which cell lysates were separated into cytoplasmic and nuclear fractions. Each fraction was subjected to immunoblot analysis with antibodies to α1, to α2, to β-actin, or to lamin B. (F) C2C12 cells stably expressing Flag-α1 or -α2 were exposed to adiponectin (100 ng/ml) or to glucose-free medium for the indicated times, after which cell lysates were separated into cytoplasmic and nuclear fractions. The cytoplasmic and nuclear fractions were subjected to immunoblot analysis with antibodies to Flag, to β-actin, or to lamin B, as indicated. Cell extracts were also subjected to immunoprecipitation with anti-Flag, and the resulting precipitates were subjected to immunoblot analysis with antibodies to Thr172-phosphorylated AMPK or total α subunits of AMPK. (G and H) Mouse fibroblast 3T3-L1 (G) or rat myoblastoma L6 (H) cells transiently expressing Flag-α1 or -α2 were incubated in the absence (−) or presence (+) of 0.5 mM AICAR for 1 h. Cell lysates were then separated into cytoplasmic and nuclear fractions, and these fractions as well as anti-Flag immunoprecipitates of cell extracts were subjected to immunoblot analysis (left panels) as described for panel F. Cells treated with AICAR were also fixed and immunostained with anti-Flag (right panels).
FIG. 3.
FIG. 3.
Nuclear translocation of α2AMPK is dependent on an NLS in the α2 subunit. (A) Amino acid sequences of putative NLSs in human α2. The filled and striped boxes in the schematic depiction of the α subunit represent the catalytic and regulatory domains, respectively. *1 and *2, amino acid sequences of putative NLSs in human α2. (B) C2C12 cells transiently expressing Flag-α2 (WT, K224A, or K399A) were treated (+) or not treated (−) with leptin for 1 h, after which cell extracts were subjected to immunoprecipitation with anti-Flag and the resulting precipitates were subjected to immunoblot analysis with antibodies to Thr172-phosphorylated or total α subunits. (C) C2C12 cells transiently expressing Flag-α2 (WT, K224A, or K399A) were exposed (+) or not exposed (−) to leptin for 1 h, after which cell lysates were separated into cytoplasmic and nuclear fractions. Each fraction was then subjected to immunoblot analysis with antibodies to Flag, to β-actin, or to lamin B, as indicated. (D) C2C12 cells transiently expressing Flag-α2 (WT, K224A, or K399A) were incubated in the absence (−) or presence (+) of leptin for 1 h and then examined by immunofluorescence analysis with anti-Flag. (E) The numbers of cells in which Flag-α2 was detected in the cytoplasm or nucleus after treatment and analysis, as described for panel D, were counted (total of 500 cells per well). Data are means ± SEM (error bars) from three independent experiments. *, P was <0.05 versus the corresponding value for cells not exposed to leptin.
FIG. 4.
FIG. 4.
Phosphorylation of α2 on Thr172 and the presence of β and γ regulatory subunits are necessary for the leptin-induced changes in the subcellular localization of α2AMPK in C2C12 cells. (A) Cells transiently expressing Flag-α2 (WT, T172D, or T172A) were treated with leptin for the indicated times, after which cell lysates were separated into cytoplasmic and nuclear fractions. Each fraction was then subjected to immunoblot analysis with antibodies to Flag, to β-actin, or to lamin B, as indicated. (B) Cells transiently expressing Flag-α2 (WT, T172A, or T172D) were treated with leptin for the indicated times, fixed, and immunostained with anti-Flag. The numbers of cells in which Flag-α2 was detected in the cytoplasm or in the nucleus were counted (total of 500 cells per well). Data are means ± SEM (error bars) from three independent experiments. *, P was <0.05 versus the corresponding value for time zero. (C) Cells were treated with leptin for the indicated times, after which total RNA was isolated and subjected to RT-PCR analysis of AMPK-subunit and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs (left panel); total RNA from mouse skeletal muscle (mixture of soleus and red and white types of gastrocnemius) was used as a positive control (PC). Alternatively, extracts of the leptin-treated cells were subjected to immunoblot (IB) analysis with antibodies to β1, β2, or γ1 subunits of AMPK and to β-actin. (D) Cells transfected with control, β1, β2, or γ1 siRNAs or parental cells were treated with leptin for the indicated times, after which total RNA was isolated and subjected to RT-PCR analysis of β1, β2, γ1, or GAPDH mRNAs. (E) Cells stably expressing Flag-α2 were transfected with control, β1, or β2 siRNAs and then treated with leptin for the indicated times, after which cell lysates were separated into cytoplasmic and nuclear fractions. These fractions were subjected to immunoblot analysis with antibodies to β-actin or to lamin B, respectively. The fractions were also subjected to immunoprecipitation (IP) with anti-Flag, and the resulting precipitates were subjected to immunoblot analysis with antibodies to Thr172-phosphorylated α subunits of AMPK and to α2. (F) Cells stably expressing Flag-α2 were transfected with control or γ1 siRNAs and treated with leptin for the indicated times, after which cell extracts were subjected to immunoprecipitation with anti-Flag and the resulting precipitates were subjected to immunoblot analysis with antibodies to Thr172-phosphorylated α subunits of AMPK and to α2. (G) Cells stably expressing Flag-α2 were transfected with control or γ1 siRNAs and treated with leptin for the indicated times, after which cell lysates were separated into cytoplasmic and nuclear fractions. Each fraction was subjected to immunoblot analysis with anti-Flag.
FIG. 5.
FIG. 5.
Role of the β1 subunit in cytoplasmic anchoring of α2AMPK and persistent ACC phosphorylation in C2C12 cells. (A) Cells stably expressing Flag-α1 or Flag-α2 were treated with leptin for the indicated times, after which cell lysates were separated into cytoplasmic and nuclear fractions. Each fraction was subjected to immunoprecipitation (IP) with anti-Flag, and the resulting precipitates were subjected to immunoblot analysis with antibodies to Thr172-phosphorylated α subunits of AMPK and to α2. The cytoplasmic and nuclear fractions were also subjected to immunoblot analysis with antibodies to β-actin and to lamin B, respectively. In addition, cell extracts were subjected to immunoblot analysis with antibodies to phosphorylated or total ACC as well as with those to Thr172-phosphorylated α subunits of AMPK and to α2. (B) Cells were treated with leptin for 0, 1, or 6 h, fixed, and immunostained with antibodies to β1 or to β2. (C) Cells transiently expressing HA-tagged β1(WT) or β1(G2A) were treated with leptin for 0, 1, or 6 h, fixed, and immunostained with anti-HA. (D) Cells stably expressing Flag-α2 were transfected with vectors for HA-tagged β1(WT) or β1(G2A), stimulated with leptin, fixed, and immunostained with anti-Flag. (E) Cells stably expressing Flag-α2 were transfected with vectors for β1(WT) or β1(G2A) or with the corresponding empty vector. They were then treated with leptin, fixed, and immunostained with anti-Flag, and the cells in which Flag-α2 was detected in the cytoplasm or in the nucleus were counted (total of 500 cells per well). Data are means ± SEM (error bars) from three independent experiments. *, P was <0.05 versus the corresponding value for time zero. (F) Cells stably expressing Flag-α2 were transfected with vectors for β1(WT) or β1(G2A) or with the corresponding empty vector. They were then treated with leptin, after which cell lysates were separated into cytoplasmic and nuclear fractions. Cell extracts were subjected to immunoblot analysis with antibodies to phosphorylated or total ACC, whereas the cytoplasmic and nuclear fractions were subjected to immunoblot analysis with antibodies to Flag, to β-actin, or to lamin B. (G) Cells stably expressing Flag-α2 were incubated in the absence (control) or presence of leptin for 6 h, after which cell lysates were separated into nuclear and cytoplasmic fractions and the cytoplasmic fraction was further separated into soluble and insoluble portions. Cell extracts and subcellular fractions were subjected to immunoblot analysis with antibodies to Flag and to phosphorylated or total ACC. (H) Cells stably expressing Flag-α2 were transfected with vectors for β1(WT) or β1(G2A) and then treated with leptin for 1 h. Cell lysates were separated into nuclear and cytoplasmic fractions, and the cytoplasmic fraction was further separated into soluble and insoluble portions. Cell extracts and subcellular fractions were subjected to immunoblot analysis with antibodies to Flag and to phosphorylated or total ACC. The blots were also probed with antibodies to cytochrome c, to β-actin, to lamin B, and to Bcl-2 as markers for mitochondria, the cytoplasm, the nucleus, and both the nucleus and mitochondria, respectively.
FIG. 6.
FIG. 6.
Nuclear α2AMPK induces PPARα gene expression in C2C12 cells. (A) Cells were exposed to leptin for the indicated times, after which total RNA was isolated and subjected to RT-PCR analysis of PPARα and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs. (B) Cells were transiently transfected with vectors for Flag-α2(T172D) or Flag-α2(T172A) or with the corresponding empty vector and were then treated with leptin for the indicated times. Total RNA was isolated and subjected to RT-PCR analysis of PPARα and GAPDH mRNAs. (C) Cells were transiently transfected with vectors for β1(WT) or β1(G2A) or with the corresponding empty vector and were then treated with leptin for the indicated times, after which total RNA was subjected to RT-PCR analysis of PPARα and GAPDH mRNAs.
FIG. 7.
FIG. 7.
Proposed model for the signaling pathway by which leptin stimulates fatty acid oxidation and PPARα gene expression in C2C12 cells. Long-term stimulation with leptin induces a biphasic activation of α2AMPK. Leptin first activates α2AMPK containing β2 and γ1 subunits, resulting in the stimulation of fatty acid (FA) oxidation through the phosphorylation of ACC and depletion of malonyl-CoA. However, this promotion of fatty acid oxidation is transient because α2AMPK containing β2 and γ1 rapidly undergoes translocation to the nucleus (within 1 h) as a result of an NLS in the α2 subunit. The active α2AMPK in the nucleus then induces the transcription of the PPARα gene. Stimulation with leptin for at least 6 h triggers a second wave of α2AMPK activation, in part attributable to the induction of transcription of the gene for the β1 subunit. Active α2AMPK containing β1 and γ1 is retained in the cytoplasm as a result of myristoylation of β1; it phosphorylates ACC, thereby stimulating fatty acid oxidation in mitochondria. Active α2AMPK containing β2 and γ1 again translocates to the nucleus and increases PPARα gene expression.
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