Leptin Stimulates Fatty Acid Oxidation and Peroxisome Proliferator-Activated Receptor {alpha} Gene Expression in Mouse C2C12 Myoblasts by Changing the Subcellular Localization of the {alpha}2 Form of AMP-Activated Protein Kinase

Leptin stimulates fatty acid oxidation in skeletal muscle throughthe activation of AMP-activated protein kinase (AMPK) and theinduction of gene expression, such as that for peroxisome proliferator-activatedreceptor ${alpha}$ (PPAR ${alpha}$ ). We now show that leptin stimulates fatty acidoxidation and PPAR ${alpha}$ gene expression in the C2C12 muscle cellline through the activation of AMPK containing the ${alpha}$ 2 subunit( ${alpha}$ 2AMPK) and through changes in the subcellular localizationof this enzyme. Activated ${alpha}$ 2AMPK containing the ß1subunit was shown to be retained in the cytoplasm, where itphosphorylated acetyl coenzyme A carboxylase and thereby stimulatedfatty acid oxidation. In contrast, ${alpha}$ 2AMPK containing the ß2subunit transiently increased fatty acid oxidation but underwentrapid translocation to the nucleus, where it induced PPAR ${alpha}$ genetranscription. A nuclear localization signal and Thr¹⁷² phosphorylationof ${alpha}$ 2 were found to be essential for nuclear translocation of ${alpha}$ 2AMPK, whereas the myristoylation of ß1 anchors ${alpha}$ 2AMPKin the cytoplasm. The prevention of ${alpha}$ 2AMPK activation and thechange in its subcellular localization inhibited the metaboliceffects of leptin. Our data thus suggest that the activationof and changes in the subcellular localization of ${alpha}$ 2AMPK arerequired for leptin-induced stimulation of fatty acid oxidationand PPAR ${alpha}$ gene expression in muscle cells.

INTRODUCTION

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INTRODUCTION

AMP-activated protein kinase (AMPK) is a serine/threonine kinasethat is thought to function in mammalian cells to yeast as acellular "fuel gauge," signaling when energy stores are fullor depleted (9, 16). The activation of AMPK results in the phosphorylationof several target molecules and consequent stimulation of fattyacid oxidation, glucose transport in muscle, and cardiac glycolysisas well as the inhibition of anabolic processes and ion channelactivities (9, 16). AMPK is a heterotrimer consisting of a catalytic ${alpha}$ subunit ( ${alpha}$ 1 or ${alpha}$ 2) and regulatory ß (ß1 orß2) and ${gamma}$ ( ${gamma}$ 1, ${gamma}$ 2, or ${gamma}$ 3) subunits (9, 16). Phosphorylationof the ${alpha}$ subunit (on Thr¹⁷²) by the upstream kinase AMPKK orallosteric modulation by AMP binding is essential for the activationof AMPK (8, 38). Four kinases, LKB1 (1), Ca²⁺/calmodulin-dependentprotein kinase kinase (10, 13, 39), ATM (32), and TAK1 (24),have been proposed to function as AMPKKs.

The activity of AMPK is regulated by hormones (21, 22, 41) andgrowth factors (32, 33) as well as by changes in cellular energystatus. Leptin is a hormone produced by adipocytes that regulatesfood intake and fuel metabolism (3). Leptin exerts metaboliceffects in peripheral tissues both directly and through thecentral nervous system (22, 25). We have previously shown thatleptin stimulates fatty acid oxidation in skeletal muscle byactivating the ${alpha}$ 2 subunit-containing AMPK ( ${alpha}$ 2AMPK) both directlyat the muscle level and indirectly through the hypothalamusand the sympathetic nervous system (22). Activated ${alpha}$ 2AMPK phosphorylatesacetyl coenzyme A (CoA) carboxylase (ACC), resulting in theinhibition of its activity and reduced formation of malonyl-CoA.The latter effect in turn results in the activation of carnitinepalmitoyltransferase 1, a step required for the stimulationof fatty acid oxidation in mitochondria (27). In addition, leptininduces the expression of genes whose products contribute tofatty acid oxidation, at least in part in a manner dependenton the transactivation activity of peroxisome proliferator-activatedreceptor ${alpha}$ (PPAR ${alpha}$ ) (18, 36). The activation of AMPK increasesPPAR ${alpha}$ gene expression in muscle cells (19). However, the molecularmechanisms by which leptin activates fatty acid oxidation through ${alpha}$ 2AMPK and induces PPAR ${alpha}$ gene expression have remained unknown.

We have now examined how leptin increases fatty acid oxidationand PPAR ${alpha}$ gene expression via AMPK activation in the mouse musclecell line C2C12. We found that leptin achieves these effectsthrough the activation of ${alpha}$ 2AMPK, but not through that of ${alpha}$ 1AMPK.Activated AMPK consisting of ${alpha}$ 2, ß1, and ${gamma}$ 1 subunitswas shown to phosphorylate ACC, thereby stimulating fatty acidoxidation, whereas activated AMPK consisting of ${alpha}$ 2, ß2,and ${gamma}$ 1 subunits translocates to the nucleus and induces PPAR ${alpha}$ gene expression. These results indicate that leptin stimulatesfatty acid oxidation and gene expression in muscle cells byactivating ${alpha}$ 2AMPK and changing its subcellular localization,with the regulatory ß subunits of AMPK playing animportant role in this process.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Cell lines, culture, and transfection. The mouse myoblastoma C2C12 cell line as well as 3T3-L1 andL6 cells were obtained from the American Type Culture Collection.Cells were cultured in six-well plates and maintained in Dulbecco'smodified Eagle's medium (DMEM) containing glucose at 4.5 mg/ml(Sigma) and were supplemented with 10% heat-inactivated fetalbovine serum (Invitrogen) and nonessential amino acids (Sigma).Transient transfection (5 µg of DNA per well) of C2C12cells was performed with the TransFast transfection reagent(Promega) in serum-free DMEM for 4 h; cells were subjected toexperiments after 48 h. The transfection efficiency was assessedwith a vector encoding green fluorescent protein and was foundto be >90%. To generate C2C12 cells that stably express Flagepitope-tagged ${alpha}$ 1 or ${alpha}$ 2 subunits of human AMPK, we seeded cellsat a density of 2.5 x 10⁵ per well and subjected them to transfectionas described above; after 48 h, the cells were harvested byexposure to trypsin, seeded in 100-cm² culture dishes, and culturedin the presence of G418 (1 mg/ml). G418-resistant cells expressingFlag- ${alpha}$ 1 or Flag- ${alpha}$ 2 were maintained in the G418-containing medium.For glucose deprivation, C2C12 cells were incubated for 1 or6 h in glucose-free DMEM (Invitrogen) supplemented with 10%dialyzed fetal bovine serum and nonessential amino acids. Cellswere stimulated with leptin, adiponectin, or 5-amino-4-imidazolecarboxamideribose (AICAR) at a concentration of 10 ng/ml, 100 ng/ml, or500 µM, respectively.

Antibodies, recombinant proteins, and plasmids. Antibodies to the ${alpha}$ subunits of AMPK (total ${alpha}$ or Thr¹⁷² phosphorylated),to ß-actin, or to cytochrome c were obtained fromCell Signaling Technology; those to ACC (total ACC or Ser⁷⁹phosphorylated) were from Upstate Biotechnology; those to Flagor to the hemagglutinin epitope (HA) were from Sigma; thoseto the ß1 or ß2 subunits of AMPK or to laminB were from Santa Cruz Biotechnology; and those to Bcl-2 werefrom Transduction Laboratories. We also prepared affinity-purifiedantibodies that recognize either ${alpha}$ 1 or ${alpha}$ 2 subunits of AMPK byinjecting rabbits with subunit-specific peptides (H₂N-Cys-TSPPDSFLDDHHLTR-COOHfor ${alpha}$ 1; H₂N-Cys-MDDSAMHIPPGLKPH-COOH for ${alpha}$ 2) (40). Human recombinantleptin and human recombinant adiponectin were obtained fromPeproTech and Alexis, respectively. AICAR was obtained fromSigma. Expression vectors [pcDNA3.1(+)] containing cDNAs forFlag-tagged (NH₂-terminal) human ${alpha}$ 1 or ${alpha}$ 2 subunits of AMPK andfor the nontagged mouse ß1 subunit were kindly providedby H. Esumi (National Cancer Center, Kashiwa, Japan) and T.Ogura (Hokuriku University, Japan). The pCR2.1-Topo TA cloningvector and pLITMUS-28i vector were obtained from Invitrogenand New England Biolabs, respectively.

Immunofluorescence staining. Cells were fixed with 4% paraformaldehyde for 20 min at roomtemperature. Nonspecific sites were blocked with fetal bovineserum in phosphate-buffered saline (PBS), and the cells werethen incubated for 16 h at 4°C with rabbit polyclonal antibodiesto Flag, to HA, or to ß1 or ß2 subunitsof AMPK, washed with PBS, and incubated for 1 h at 37°Cwith fluorescein isothiocyanate-conjugated goat antibodies torabbit immunoglobulin G (Santa-Cruz). The cells were then washedtwice with PBS and observed with a fluorescence microscope (IX70and DP Controller; Olympus).

RNA extraction and RT-PCR. Total RNA was extracted from cells with Isogen (Wako), and portionsof the RNA (100 ng) were subjected to reverse transcription(RT) with avian myeloblastosis virus reverse transcriptase andan oligo(dT)₁₆ primer (Takara Biochemicals). The resulting first-strandcDNA was subjected to PCR with an LA PCR kit, version 2.1 (Takara),and gene-specific primers (see Table S1 in the supplementalmaterial).

Preparation of siRNAs. Small interfering RNAs (siRNAs) specific for each subunit ofAMPK were prepared with a HiScribe RNA interference (RNAi) transcriptionkit (New England Biolabs). PCR was performed with first-strandDNA from C2C12 cells as a template and specific primer pairs(see Table S1 in the supplemental material), and the PCR productswere subcloned into the pCR2.1-Topo vector. After sequencing,the vectors were digested with BamHI and XhoI, and the insertfragments were ligated into the pLITMUS-28i vector for RNAi.Double-stranded RNA was prepared from the resulting vectors(or from the empty vector as a control) by in vitro transcriptionwith T7 RNA polymerase and was digested with RNase III (NewEngland Biolabs) before application to cells. The efficacy ofRNAi was assessed on the basis of the depletion of the targetmRNA and protein as monitored by RT-PCR and immunoblot analyses.

Mutagenesis. Mutagenesis was performed by PCR. PCR products were obtainedwith a reverse mutagenesis primer (see Table S2 in the supplementalmaterial) and a T7 promoter primer, with the corresponding pcDNA3.1(+)vector as a template, as well as with a forward mutagenesisprimer (see Table S2 in the supplemental material) and a reverseprimer specific for the polyadenylation signal of bovine growthhormone cDNA, again with the corresponding pcDNA3.1(+) vectoras a template. Both PCR products were purified and then mixed,annealed, and amplified by PCR with the T7 promoter and bovinegrowth hormone reverse primers. The resulting PCR product waspurified, digested with restriction enzymes, and ligated intopcDNA3.1(+).

Subcellular fractionation. Cells were suspended in a solution containing 2 mM EDTA and10 mM Tris-HCl (pH 7.5) and incubated on ice for 10 min, afterwhich an equal volume of a solution containing 0.5 M sucrose,0.1 M KCl, 10 mM MgCl₂, 2 mM CaCl₂, 2 mM EDTA, and 10 mM Tris-HCl(pH 7.5) was added. The cell lysate was centrifuged at 2,700rpm (700 x g) for 10 min at 4°C, and the resulting pelletand supernatant were collected as the nuclear and cytoplasmicfractions, respectively. The cytoplasmic fraction was then furthercentrifuged at 15,000 rpm (20,000 x g) for 3 h at 4°C, andthe resulting supernatant and pellet were saved as the solubleand insoluble fractions, respectively.

Immunoprecipitation. Cell lysates prepared by incubation of cells for 30 min at 4°Cwith 0.1% NP-40 in PBS were centrifuged at 15,000 x g for 15min at 4°C, and the resulting supernatants (1 mg of protein)were subjected to immunoprecipitation with specific antibodiesand protein G-Sepharose (Amersham). The beads were separatedby centrifugation, washed six times with 0.1% NP-40 in PBS,and subjected to immunoblot analysis or an assay of kinase activity.

Immunoblot analysis. Cells were lysed as for immunoprecipitation with the exceptionthat the NP-40 concentration was 1%, and the supernatants weresubjected to immunoblot analysis with specific primary antibodies.Immune complexes were detected with horseradish peroxidase-conjugatedsecondary antibodies (Santa Cruz) and ECL reagents (Amersham).

Assay of AMPK activity. The AMPK activities of cell extracts and immunoprecipitateswere measured with the SAMS peptide and [ ${gamma}$ -³²P]ATP as describedpreviously (17), with a small modification. A recombinant proteincomprising maltose binding protein (MBP) fused with three copiesof the SAMS peptide (MBP-SAMS) was used as the substrate. Afterincubation with [ ${gamma}$ -³²P]ATP, the fusion protein was purified withamylose resin (Amersham) and the associated radioactivity wasmeasured with a scintillation counter (Beckman Coulter). Weconfirmed that the efficiency of phosphorylation of the MBP-SAMSprotein by AMPK was similar to that of phosphorylation of theSAMS peptide fused to glutathione S-transferase (17).

Measurement of fatty acid oxidation. Cells were exposed to [¹⁴C]palmitic acid (American RadiolabeledChemicals, Inc.) for 30 min after incubation with or withoutleptin. The culture supernatant was then transferred to a 50-mltube (Falcon) and mixed with a 1/10 volume of 1 M HCl. The ¹⁴CO₂produced during incubation of the mixture for 50 min at 30°Cwas trapped with a paper filter soaked with NaOH. The paperfilter was then transferred to 1 ml of H₂O and agitated for5 min, after which the radioactivity in the H₂O was measuredwith a scintillation counter.

Statistics. All experiments were repeated at least three times, and eachexperiment was performed with duplicate or triplicate samples.Data are presented as means ± standard errors of themeans (SEM). Statistical analysis was performed by analysisof variance, followed by Dunnett's posthoc test. Data (suchas cell number) expressed as a percentage were analyzed afterthe arcsine transformation (30). A P value of <0.05 was consideredstatistically significant.

RESULTS

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RESULTS

Leptin stimulates fatty acid oxidation by activating ${alpha}$ 2AMPK. We previously showed that leptin activates ${alpha}$ 2AMPK and therebystimulates fatty acid oxidation in red muscle types, includingthe soleus (22). Similar to the soleus muscle, the mouse myoblastomaC2C12 cell line was found to express the leptin receptor Ob-Rb(Fig. 1A). Leptin (10 ng/ml) induced the phosphorylation ofthe catalytic ${alpha}$ subunit of AMPK on Thr¹⁷² in C2C12 cells; thiseffect was biphasic, with the early phase peaking at 30 minto 1 h and the second phase manifesting between 6 and 24 h (Fig.1B). The phosphorylation of ACC (on Ser⁷⁹) also increased ina biphasic manner in parallel with that of the ${alpha}$ subunit of AMPK(Fig. 1B). A Flag-tagged version of the human ${alpha}$ 2 subunit of AMPK(Flag- ${alpha}$ 2), but not Flag- ${alpha}$ 1, was also phosphorylated on Thr¹⁷²(Fig. 1C) and activated (Fig. 1D) in C2C12 cells at both 1 and6 h after leptin stimulation. To examine the relative rolesof ${alpha}$ 2 and ${alpha}$ 1 in leptin-induced fatty acid oxidation in C2C12 cells,we depleted cells of each subunit individually by RNAi (Fig.1E). The siRNA specific for ${alpha}$ 2, but not that for ${alpha}$ 1, abolishedleptin-induced fatty acid oxidation in C2C12 cells (Fig. 1F).These data thus indicated that leptin stimulates fatty acidoxidation in C2C12 cells directly by activating ${alpha}$ 2AMPK. Theyfurther showed that long-term stimulation with leptin activates ${alpha}$ 2AMPK in a biphasic manner.

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FIG. 1. Leptin stimulates fatty acid oxidation through specific activation of ${alpha}$ 2AMPK 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 ${alpha}$ subunits of AMPK or to Ser⁷⁹-phosphorylated (pACC) or total ACC (tACC). (C) Cells stably expressing Flag- ${alpha}$ 1 or Flag- ${alpha}$ 2 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 ${alpha}$ subunits of AMPK. (D) Parental cells and cells stably expressing Flag- ${alpha}$ 1 or - ${alpha}$ 2 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 ${alpha}$ 1 or ${alpha}$ 2 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 ${alpha}$ 1, ${alpha}$ 2, and GAPDH mRNAs (upper panels) and cell extracts were subjected to immunoblot analysis (IB) of ${alpha}$ 1, ${alpha}$ 2, and ß-actin (internal control). (F) Cells transfected with control, ${alpha}$ 1, or ${alpha}$ 2 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.

${alpha}$ 2AMPK translocates to the nucleus concomitant with its activation. In a variety of cell types, including cell lines (29) and cellsof the central nervous system (6, 34), immunofluorescence studieshave revealed that ${alpha}$ 2 localizes to the nucleus or cytoplasm (orboth), whereas ${alpha}$ 1 is restricted to the cytoplasm. With immunofluorescenceanalysis, we found that both Flag- ${alpha}$ 2 and - ${alpha}$ 1 were present in thecytoplasm of C2C12 cells under basal conditions (Fig. 2A and B).In cells exposed to leptin, however, Flag- ${alpha}$ 2 was detected inthe nucleus at 1 h, had returned to the cytoplasm by 3 h, andwas present in both the cytoplasm and the nucleus at 6 h afterstimulation (Fig. 2A and B). Flag- ${alpha}$ 1, which was not phosphorylatedon Thr¹⁷² in response to leptin stimulation (Fig. 1C), remainedin the cytoplasm in leptin-treated cells (Fig. 2A). To quantifythe changes in the subcellular localization of Flag- ${alpha}$ 2, we countedthe number of cells in which Flag- ${alpha}$ 2 was detected in the cytoplasm,nucleus, or both compartments (Fig. 2C). The translocation ofFlag- ${alpha}$ 2 to the nucleus at 1 h and its return to the cytoplasmby 3 h after leptin stimulation were apparent in >90% ofcells. At 6 or 12 h after stimulation, Flag- ${alpha}$ 2 was detected inthe nucleus of $~$ 40% of cells and in both the cytoplasm and thenucleus of $~$ 25 to 30% of cells. Similarly, immunoblot analysisrevealed that Flag- ${alpha}$ 2, but not Flag- ${alpha}$ 1, translocated to the nucleusin a biphasic manner in parallel with Thr¹⁷² phosphorylationof the ${alpha}$ subunit (Fig. 2D). Furthermore, endogenous ${alpha}$ 2, but not ${alpha}$ 1, had translocated to the nucleus at 1 h and appeared in boththe cytoplasm and nucleus by 6 h after leptin stimulation (Fig.2E). These observations thus showed that leptin changes thesubcellular localization of ${alpha}$ 2 concomitant with its inductionof Thr¹⁷² phosphorylation of ${alpha}$ 2 and its activation of ${alpha}$ 2AMPK.

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FIG. 2. Leptin induces subcellular redistribution of ${alpha}$ 2AMPK. (A and B) C2C12 cells stably expressing Flag- ${alpha}$ 1 or - ${alpha}$ 2 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 x400 (A). Immunostained cells expressing Flag- ${alpha}$ 2 were also examined at a magnification of x200 (B). (C) C2C12 cells stably expressing Flag- ${alpha}$ 1 or - ${alpha}$ 2 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- ${alpha}$ 2 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- ${alpha}$ 1 or - ${alpha}$ 2 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 ${alpha}$ subunits of AMPK, to ß-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 ${alpha}$ 1, to ${alpha}$ 2, to ß-actin, or to lamin B. (F) C2C12 cells stably expressing Flag- ${alpha}$ 1 or - ${alpha}$ 2 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 ß-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 ${alpha}$ subunits of AMPK. (G and H) Mouse fibroblast 3T3-L1 (G) or rat myoblastoma L6 (H) cells transiently expressing Flag- ${alpha}$ 1 or - ${alpha}$ 2 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).

To examine whether the induction of the subcellular redistributionof ${alpha}$ 2 is specific to leptin, we examined the effects of adiponectinand glucose deprivation on the subcellular localization of Flag- ${alpha}$ 1and - ${alpha}$ 2 in C2C12 cells (Fig. 2F). Adiponectin-induced Thr¹⁷²phosphorylation of Flag- ${alpha}$ 2, but not that of Flag- ${alpha}$ 1, was apparentat 1 and 6 h after stimulation, whereas glucose deprivationinduced the phosphorylation of Flag- ${alpha}$ 1 but not that of Flag- ${alpha}$ 2.Similar to the effect of leptin, adiponectin induced translocationof Flag- ${alpha}$ 2, but not that of Flag- ${alpha}$ 1, to the nucleus in cells stimulatedfor 1 h and Flag- ${alpha}$ 2 was localized to both the nucleus and cytoplasmat 6 h after stimulation (Fig. 2F). In contrast, glucose deprivationdid not affect the subcellular localization of either Flag- ${alpha}$ 1or - ${alpha}$ 2. AICAR, an activator of ${alpha}$ 1AMPK and ${alpha}$ 2AMPK (8), also inducedthe translocation of Flag- ${alpha}$ 2, but not that of Flag- ${alpha}$ 1, to thenucleus of 3T3-L1 (Fig. 2G), L6 (Fig. 2H), and C2C12 (data notshown) cells stimulated for 1 h. It also induced Thr¹⁷² phosphorylationof both Flag- ${alpha}$ 1 and - ${alpha}$ 2. These results thus showed that ${alpha}$ 2 translocatesto the nucleus in a variety of cell types in parallel with itsphosphorylation on Thr¹⁷², whereas ${alpha}$ 1 does not translocate tothe nucleus even when activated.

Nuclear translocation of ${alpha}$ 2AMPK is dependent on a nuclear localization signal (NLS) in the ${alpha}$ 2 subunit. To explore the mechanism by which ${alpha}$ 2 is preferentially translocatedto the nucleus, we searched for an NLS in ${alpha}$ 1 and ${alpha}$ 2. The minimumrequirement for an NLS is Lys-(Lys/Arg)-X-(Lys/Arg) (12), andtwo amino acid sequences in human ${alpha}$ 2, but none in ${alpha}$ 1, met thiscriterion. One of these two sequences is present in the catalyticdomain (Lys²²³ to Arg²²⁶) of ${alpha}$ 2, and the other is in the regulatorydomain (Lys³⁹⁸ to Lys⁴⁰¹) of ${alpha}$ 2 (Fig. 3A). These sequences arealso conserved in mouse and rat ${alpha}$ 2 (data not shown). We constructedNLS mutants (K224A and K399A) of human ${alpha}$ 2 to examine the roleof these sequences in the leptin-induced nuclear translocationof ${alpha}$ 2AMPK. Leptin induced Thr¹⁷² phosphorylation of both mutantproteins to a level similar to that observed with the wild-typeprotein [ ${alpha}$ 2(WT)] (Fig. 3B). However, whereas leptin stimulationfor 1 h induced the nuclear translocation of Flag- ${alpha}$ 2(K399A),it did not trigger that of Flag- ${alpha}$ 2(K224A) (Fig. 3C to E). Theseresults thus indicated that the NLS at Lys²²³ to Arg²²⁶ of ${alpha}$ 2is essential for nuclear translocation in response to leptin.

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FIG. 3. Nuclear translocation of ${alpha}$ 2AMPK is dependent on an NLS in the ${alpha}$ 2 subunit. (A) Amino acid sequences of putative NLSs in human ${alpha}$ 2. The filled and striped boxes in the schematic depiction of the ${alpha}$ subunit represent the catalytic and regulatory domains, respectively. *1 and *2, amino acid sequences of putative NLSs in human ${alpha}$ 2. (B) C2C12 cells transiently expressing Flag- ${alpha}$ 2 (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 ${alpha}$ subunits. (C) C2C12 cells transiently expressing Flag- ${alpha}$ 2 (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 ß-actin, or to lamin B, as indicated. (D) C2C12 cells transiently expressing Flag- ${alpha}$ 2 (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- ${alpha}$ 2 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.

Phosphorylation of ${alpha}$ 2 on Thr¹⁷² and the presence of ß and ${gamma}$ subunits are essential for nuclear translocation of ${alpha}$ 2AMPK. We next examined whether Thr¹⁷² phosphorylation is requiredfor the nuclear translocation of ${alpha}$ 2 in response to leptin stimulation.We constructed two Thr¹⁷² mutants of human ${alpha}$ 2, ${alpha}$ 2(T172D) and ${alpha}$ 2(T172A),which mimic the phosphorylated and nonphosphorylated forms ofthe protein, respectively. In cells stimulated with leptin,Flag- ${alpha}$ 2(WT) had translocated to the nucleus at 1 h and was apparentin both the cytoplasm and nucleus at 6 h, whereas Flag- ${alpha}$ 2(T172A)did not translocate to the nucleus in response to leptin (Fig.4A and B). In contrast, Flag- ${alpha}$ 2(T172D), which was present inthe cytoplasm before leptin stimulation, had translocated tothe nucleus at 1 h but had not relocated to the cytoplasm at6 h after exposure of cells to leptin. These results thus indicatedthat Thr¹⁷² phosphorylation is essential for the nuclear translocationof ${alpha}$ 2 in response to leptin and that the subsequent dephosphorylationof this residue is necessary for the relocation of ${alpha}$ 2 to thecytoplasm apparent at 6 h. Furthermore, the cytoplasmic localizationof Flag- ${alpha}$ 2(T172D) apparent before leptin stimulation indicatedthat some other factor in addition to Thr¹⁷² phosphorylationis required for leptin-induced nuclear translocation of ${alpha}$ 2.

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FIG. 4. Phosphorylation of ${alpha}$ 2 on Thr¹⁷² and the presence of ß and ${gamma}$ regulatory subunits are necessary for the leptin-induced changes in the subcellular localization of ${alpha}$ 2AMPK in C2C12 cells. (A) Cells transiently expressing Flag- ${alpha}$ 2 (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 ß-actin, or to lamin B, as indicated. (B) Cells transiently expressing Flag- ${alpha}$ 2 (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- ${alpha}$ 2 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 ß1, ß2, or ${gamma}$ 1 subunits of AMPK and to ß-actin. (D) Cells transfected with control, ß1, ß2, or ${gamma}$ 1 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 ß1, ß2, ${gamma}$ 1, or GAPDH mRNAs. (E) Cells stably expressing Flag- ${alpha}$ 2 were transfected with control, ß1, or ß2 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 ß-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 ${alpha}$ subunits of AMPK and to ${alpha}$ 2. (F) Cells stably expressing Flag- ${alpha}$ 2 were transfected with control or ${gamma}$ 1 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 ${alpha}$ subunits of AMPK and to ${alpha}$ 2. (G) Cells stably expressing Flag- ${alpha}$ 2 were transfected with control or ${gamma}$ 1 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.

We next examined the potential role of regulatory subunits ofAMPK in the changes in the subcellular localization of ${alpha}$ 2AMPKin response to leptin. C2C12 cells express endogenous ${alpha}$ 1, ${alpha}$ 2,ß2, and ${gamma}$ 1 subunits of AMPK before and after leptinstimulation (Fig. 4C). In addition, leptin induced ß1expression at the mRNA and protein levels after stimulationfor 6 h. We constructed siRNAs for ß1, ß2,and ${gamma}$ 1 subunits and showed that these siRNAs effectively depletedC2C12 cells of the corresponding mRNAs (Fig. 4D). The siRNAfor ß1 inhibited the relocalization of ${alpha}$ 2 to the cytoplasmnormally apparent at 6 h after leptin stimulation, whereas thatfor ß2 inhibited the nuclear translocation of ${alpha}$ 2 at1 or 6 h (Fig. 4E). The siRNA for ß2 also abolishedthe leptin-induced phosphorylation of ${alpha}$ 2 on Thr¹⁷² at 1 h, atwhich time it was the only ß subunit expressed inthese cells. These results indicated that ß1 mediatesthe cytoplasmic relocalization of ${alpha}$ 2 apparent at 6 h after leptinstimulation, whereas ß2 contributes to the nucleartranslocation of ${alpha}$ 2. In addition, the siRNA for ${gamma}$ 1, which is theonly ${gamma}$ subunit expressed in C2C12 cells, abolished both Thr¹⁷²phosphorylation (Fig. 4F) and nuclear translocation (Fig. 4G)of ${alpha}$ 2 in response to leptin. Both Thr¹⁷² phosphorylation andß ${gamma}$ regulatory subunits are thus essential for the leptin-inducednuclear translocation of ${alpha}$ 2. Furthermore, ß1 and ß2appear to determine the subcellular localization of ${alpha}$ 2AMPK inleptin-treated cells.

The NH₂-terminal myristoylation site of the ß1 subunit is necessary for cytoplasmic anchoring of ${alpha}$ 2AMPK and persistent ACC phosphorylation. To explore the role of the ß subunits of AMPK in theregulation of fatty acid oxidation, we examined the relationshipbetween ACC phosphorylation (on Ser⁷⁹) and the subcellular localizationof the ${alpha}$ 2, ß1, and ß2 subunits of AMPK inleptin-treated cells. In parallel with the phosphorylation ofcytoplasmic ${alpha}$ 2 on Thr¹⁷², the level of ACC phosphorylation wasincreased from 6 to 24 h after leptin stimulation (Fig. 5A).Endogenous ß1 was present in the cytoplasm at 6 hafter leptin stimulation, whereas ß2 had translocatedto the nucleus at both 1 and 6 h (Fig. 5B). These results suggestedthat ${alpha}$ 2AMPK containing ß1 mediates ACC phosphorylationin the cytoplasm during the late phase of ${alpha}$ 2AMPK activation inresponse to leptin.

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FIG. 5. Role of the ß1 subunit in cytoplasmic anchoring of ${alpha}$ 2AMPK and persistent ACC phosphorylation in C2C12 cells. (A) Cells stably expressing Flag- ${alpha}$ 1 or Flag- ${alpha}$ 2 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 ${alpha}$ subunits of AMPK and to ${alpha}$ 2. The cytoplasmic and nuclear fractions were also subjected to immunoblot analysis with antibodies to ß-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 ${alpha}$ subunits of AMPK and to ${alpha}$ 2. (B) Cells were treated with leptin for 0, 1, or 6 h, fixed, and immunostained with antibodies to ß1 or to ß2. (C) Cells transiently expressing HA-tagged ß1(WT) or ß1(G2A) were treated with leptin for 0, 1, or 6 h, fixed, and immunostained with anti-HA. (D) Cells stably expressing Flag- ${alpha}$ 2 were transfected with vectors for HA-tagged ß1(WT) or ß1(G2A), stimulated with leptin, fixed, and immunostained with anti-Flag. (E) Cells stably expressing Flag- ${alpha}$ 2 were transfected with vectors for ß1(WT) or ß1(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- ${alpha}$ 2 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- ${alpha}$ 2 were transfected with vectors for ß1(WT) or ß1(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 ß-actin, or to lamin B. (G) Cells stably expressing Flag- ${alpha}$ 2 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- ${alpha}$ 2 were transfected with vectors for ß1(WT) or ß1(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 ß-actin, to lamin B, and to Bcl-2 as markers for mitochondria, the cytoplasm, the nucleus, and both the nucleus and mitochondria, respectively.

The ß1 subunit of AMPK contains an NH₂-terminal consensussequence for myristoylation, with Gly at position 2 and Serat position 6 (residues 1 to 6: MGNTSS) (23, 37). To examinethe role of this myristoylation site of ß1 in thecytoplasmic localization of ${alpha}$ 2AMPK, we constructed wild-typeand G2A mutant versions of mouse ß1 with an HA tagat the COOH terminus. ß1(WT) was detected in the cytoplasmconstitutively before and after leptin stimulation, whereasß1(G2A) had translocated from the cytoplasm to thenucleus at 1 and 6 h after leptin stimulation (Fig. 5C). Furthermore,the expression of ß1(WT) inhibited the nuclear translocationof ${alpha}$ 2 at 1 and 6 h, whereas that of ß1(G2A) promotedthis process (Fig. 5D to F). The expression of ß1(WT)also increased ACC phosphorylation at both 1 and 6 h after leptinstimulation, whereas the expression of ß1(G2A) inhibitedACC phosphorylation (Fig. 5F). Whereas ${alpha}$ 2AMPK (Flag- ${alpha}$ 2) was presentin the soluble portion of the cytoplasmic fraction before leptinstimulation, it was detected in the insoluble portion of thisfraction 6 h after leptin stimulation (Fig. 5G). Consistentwith this change in the subcellular localization of ${alpha}$ 2, phosphorylatedACC was detected in the insoluble portion of the cytoplasmicfraction, not in the soluble portion, at this time. Furthermore,the expression of ß1(WT) resulted in the localizationof ${alpha}$ 2 in the insoluble portion of the cytoplasmic fraction 1h after leptin stimulation, in contrast with the nuclear localizationof ${alpha}$ 2 apparent at this time in cells expressing ß1(G2A)(Fig. 5H). The amount of phosphorylated ACC was also increasedin the insoluble portion of the cytoplasmic fraction at thistime in cells expressing ß1(WT) but not in those expressingß1(G2A). The NH₂-terminal myristoylation site of ß1thus appears to be necessary both for anchoring ${alpha}$ 2AMPK to organellesin the cytoplasm such as mitochondria and for persistent ACCphosphorylation during the late phase of ${alpha}$ 2AMPK activation inresponse to leptin.

Nuclear ${alpha}$ 2AMPK induces PPAR ${alpha}$ gene expression. Leptin and AMPK stimulate the expression of genes whose productscontribute to the regulation of fatty acid oxidation (18, 20,42). Among these genes, we found that leptin increased the amountof PPAR ${alpha}$ mRNA in C2C12 cells in a biphasic manner (Fig. 6A).The expression of Flag- ${alpha}$ 2(T172D) enhanced this effect of leptin,whereas the expression of Flag- ${alpha}$ 2(T172A) blocked it (Fig. 6B).Furthermore, the expression of ß1(WT) suppressed theup-regulation of PPAR ${alpha}$ mRNA by leptin, whereas the expressionof ß1(G2A) promoted it (Fig. 6C). The induction ofPPAR ${alpha}$ gene expression thus correlated well with the nuclear localizationof ${alpha}$ 2AMPK in leptin-stimulated cells.

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FIG. 6. Nuclear ${alpha}$ 2AMPK induces PPAR ${alpha}$ 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 PPAR ${alpha}$ and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs. (B) Cells were transiently transfected with vectors for Flag- ${alpha}$ 2(T172D) or Flag- ${alpha}$ 2(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 PPAR ${alpha}$ and GAPDH mRNAs. (C) Cells were transiently transfected with vectors for ß1(WT) or ß1(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 PPAR ${alpha}$ and GAPDH mRNAs.

DISCUSSION

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DISCUSSION

We have shown that changes in the subcellular localization of ${alpha}$ 2AMPK mediated by the ${alpha}$ 2 and ß1 and ß2 regulatorysubunits play an important role in the metabolic effects ofleptin in C2C12 cells. Previous studies demonstrated that bothleptin and adiponectin activate only ${alpha}$ 2AMPK, whereas glucosedeprivation activates only ${alpha}$ 1AMPK and AICAR activates both forms(2, 21, 22, 41). Furthermore, we have now shown that leptin-inducedactivation of ${alpha}$ 2AMPK occurs in a biphasic manner in C2C12 cells.At early time points after leptin stimulation, the ${alpha}$ 2 subunitof AMPK bound to the ß2 subunit translocates to thenucleus in a manner dependent on an NLS that is present in ${alpha}$ 2but not in ${alpha}$ 1; the ${alpha}$ 2ß2 form of AMPK in the nucleusthen rapidly induces transcription of the PPAR ${alpha}$ gene. The exposureof the cells to leptin for 6 h triggered transcription of thegene for the ß1 subunit of AMPK, and the encoded protein,together with ${alpha}$ 2, became localized to an insoluble fraction ofthe cytoplasm as a result of a myristoylation sequence at itsNH₂ terminus. The ${alpha}$ 2ß1 form of AMPK then phosphorylatedACC and stimulated fatty acid oxidation. Our data thus indicatethat ${alpha}$ 2ß1 and ${alpha}$ 2ß2 forms of AMPK play distinctroles in mediating the metabolic actions of leptin. The mechanismby which leptin activates the expression of the ß1-subunitgene remains unclear. However, the exposure of C2C12 cells toadiponectin or AICAR also induced ß1 gene expression(data not shown), suggesting that ${alpha}$ 2AMPK may contribute to thiseffect. The anchoring of some ${alpha}$ 2ß1 molecules to theouter mitochondrial membrane through the myristoylation of ß1might be expected to result in highly efficient depletion ofmalonyl-CoA, the activation of carnitine palmitoyltransferase1, and the stimulation of fatty acid oxidation in mitochondria.Our proposed model for the mechanism by which ${alpha}$ 2AMPK mediatesthe stimulatory effect of leptin on fatty acid oxidation issummarized in Fig. 7.

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FIG. 7. Proposed model for the signaling pathway by which leptin stimulates fatty acid oxidation and PPAR ${alpha}$ gene expression in C2C12 cells. Long-term stimulation with leptin induces a biphasic activation of ${alpha}$ 2AMPK. Leptin first activates ${alpha}$ 2AMPK containing ß2 and ${gamma}$ 1 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 ${alpha}$ 2AMPK containing ß2 and ${gamma}$ 1 rapidly undergoes translocation to the nucleus (within 1 h) as a result of an NLS in the ${alpha}$ 2 subunit. The active ${alpha}$ 2AMPK in the nucleus then induces the transcription of the PPAR ${alpha}$ gene. Stimulation with leptin for at least 6 h triggers a second wave of ${alpha}$ 2AMPK activation, in part attributable to the induction of transcription of the gene for the ß1 subunit. Active ${alpha}$ 2AMPK containing ß1 and ${gamma}$ 1 is retained in the cytoplasm as a result of myristoylation of ß1; it phosphorylates ACC, thereby stimulating fatty acid oxidation in mitochondria. Active ${alpha}$ 2AMPK containing ß2 and ${gamma}$ 1 again translocates to the nucleus and increases PPAR ${alpha}$ gene expression.

We previously showed that leptin activates ${alpha}$ 2AMPK in the soleusmuscle in a biphasic manner in vivo (22). We injected leptinin a bolus through an intravenous catheter, which resulted ina transient increase in the concentration of leptin in plasma(22). This transient increase in plasma leptin level resultedin an early transient activation of ${alpha}$ 2AMPK in the soleus by adirect action of leptin, with the second phase of activationbeing mediated through the hypothalamus and the sympatheticnervous system. Our present data suggest that long-term exposureto leptin is necessary for a biphasic activation of ${alpha}$ 2AMPK mediatedby a direct action of the hormone. Indeed, short-term treatment(1 h) with leptin did not induce a second phase of ${alpha}$ 2AMPK activationin C2C12 cells, but it did activate ß1 gene expressionat 6 h (data not shown).

Changes in the subcellular localization of ${alpha}$ 2AMPK in responseto specific stimuli appear to be conserved from yeast to mammals.The Snf1 kinase complex is an AMPK homolog in yeast that isrequired for the cellular response to glucose limitation (4,9). The Snf1 kinase complex is also activated by upstream kinases(11) and comprises a catalytic ${alpha}$ subunit (Snf1), one of threerelated ß subunits (Gal83, Sip1, or Sip2) (14), anda ${gamma}$ subunit (Snf4) (15). The three ß subunits showdistinct patterns of localization to the nucleus, vacuole, andcytoplasm, respectively. Gal83 is required for the nuclear localizationof Snf1 in a glucose-regulated manner (35). Consistent withour present findings and previous observations with the Snf1kinase complex, a recent study showed that the activation of ${alpha}$ 2AMPK is accompanied by its translocation to the nucleus inskeletal muscle cells in vivo (31). Our data also show thatboth AICAR and adiponectin induce the nuclear translocationof ${alpha}$ 2AMPK. Changes in the subcellular localization of ${alpha}$ 2AMPK thatare dependent on the ß subunits of this enzyme thusappear to explain, at least in part, the multifunctional effectsof AMPK on metabolism. The expression of the ß1 andß2 subunits and the myristoylation of ß1vary among different types of skeletal muscle and other tissues(5, 6, 23, 37). The activation of AMPK may elicit distinct metaboliceffects in tissues and cells depending on the expression ofthe different ß- and ${alpha}$ -subunit isoforms.

Our present study also revealed that ${alpha}$ 2AMPK that has translocatedto the nucleus induces transcription of the PPAR ${alpha}$ gene. It ispossible that this effect of nuclear ${alpha}$ 2AMPK is mediated by theSp1-CRSP complex. The 5' flanking region of the PPAR ${alpha}$ gene (includingthat in mouse) contains several putative Sp1 binding sites (7).The CRSP complex (also known as CRSP130, SUR2, MED23, and DRIP130)directs transcriptional initiation by the RNA polymerase IIapparatus and is required for efficient transcriptional activationby Sp1 (26, 28). Our preliminary data show that siRNAs for Sp1or CRSP3, both of which are expressed in C2C12 cells, suppressedleptin-induced expression of the PPAR ${alpha}$ gene in these cells andthat ${alpha}$ 2AMPK phosphorylates CRSP3 in vitro (unpublished data).The role of the Sp1-CRSP complex in the regulation of PPAR ${alpha}$ geneexpression by ${alpha}$ 2AMPK thus warrants further investigation.

In conclusion, our data indicate that the metabolic effectsof leptin are controlled by changes in the subcellular localizationof ${alpha}$ 2AMPK. As in yeast, the subcellular localization of ${alpha}$ 2AMPKin mammalian cells appears to be determined by the regulatoryß subunits. Our results provide new insight into themolecular mechanism by which energy metabolism is regulatedby AMPK in response to leptin and other specific stimuli.

ACKNOWLEDGMENTS

This work was supported by a Grant-in-Aid for Scientific Research(B) (16390062) (to Y.M.), a Grant-in-Aid for Exploratory Research(17659069) (to Y.M.), a Grant-in-Aid for Young Scientists (A)(18689023) (to A.S.), and a Grant-in-Aid for Young Scientists(B) (18790629) (to S.O.) from the Ministry of Education, Culture,Sports, Science, and Technology of Japan; a research award fromDiko Foundation (to Y.M.); the Smoking Research Foundation (toY.M.); and the Japan Foundation for Applied Enzymology (to Y.M.).

We thank H. Esumi and T. Ogura for expression plasmids, K. Kameda(Ehime University, Japan) for a constitutively active form ofthe ${alpha}$ 1 subunit of AMPK used as a positive control for the AMPKactivity assay, and the Center for Analytical Instruments atthe National Institute for Basic Biology (Okazaki) for DNA sequencing.

FOOTNOTES

* Corresponding author. Mailing address: Division of Endocrinology and Metabolism, Department of Developmental Physiology, National Institute for Physiological Sciences, 38 Nishigonaka, Myodaiji, Okazaki, Aichi 444-8585, Japan. Phone: 81-564-55-7745. Fax: 81-564-55-7741. E-mail: minokosh{at}nips.ac.jp

Published ahead of print on 9 April 2007.

Supplemental material for this article may be found at http://mcb.asm.org/.

REFERENCES

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