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Catecholamine-stimulated lipolysis occurs by activating adenylate cyclase and raising cAMP levels, thereby increasing protein kinase A (PKA) activity. This results in phosphorylation and modulated activity of several key lipolytic proteins. Adipose triglyceride lipase (ATGL) is the primary lipase for the initial step in triacylglycerol hydrolysis, and ATGL activity is increased during stimulated lipolysis. Here, we demonstrate that murine ATGL is phosphorylated by PKA at several serine residues in vitro and identify Ser⁴⁰⁶ as a functionally important site. ATGL null adipocytes expressing ATGL S406A (nonphosphorylatable) had reduced stimulated lipolysis. Studies in mice demonstrated increased ATGL Ser⁴⁰⁶ phosphorylation during fasting and moderate intensity exercise, conditions associated with elevated lipolytic rates. ATGL Ser⁴⁰⁴ (corresponding to murine Ser⁴⁰⁶) phosphorylation was increased by β-adrenergic stimulation but not 5′AMP-activated protein kinase activation in human subcutaneous adipose tissue explants, which correlated with lipolysis rates. Our studies suggest that β-adrenergic activation can result in PKA-mediated phosphorylation of ATGL Ser⁴⁰⁶, to moderately increase ATGL-mediated lipolysis.

Free fatty acids stored in adipocyte triacylglycerol (TG) are the largest energy depot in mammals and provide an important energy substrate for many cell types, particularly during times of physiological stress, such as fasting, cold exposure, and exercise (1). The release of free fatty acids is dependent on the lipolysis of stored TG, which is tightly controlled by neural regulation and several hormones to meet energy demands. The catecholamines are the most important lipolytic stimuli and mediate their effects by activating adenylate cyclase and raising cAMP levels, thereby causing activation of protein kinase A (PKA).

PKA phosphorylation of several proteins is critical for efficient lipolysis. Hormone-sensitive lipase (HSL) is a key enzyme controlling lipolysis, and PKA phosphorylates HSL at residues Ser⁵⁶³, Ser⁶⁵⁹, and Ser⁶⁶⁰, resulting in modest increases in HSL activity (2). PKA phosphorylation also promotes the translocation of HSL from the cytosol to the lipid droplet containing its lipid substrates (3, 4). Perilipin 1 resides at the surface of lipid droplets in adipocytes and is a major facilitator of stimulated TG lipolysis (5, 6). Perilipin 1 contains six consensus sites for serine phosphorylation by PKA, although phosphorylation at all sites has not been confirmed. It is evident that phosphorylation in at least three of these sites is required to facilitate HSL access to lipid substrates (7) and that phosphorylation of Ser⁴⁹² is required for dispersion of large lipid droplets into minidroplets (8) and maximal lipolysis (9).

Until recently, HSL was presumed to be the rate-limiting enzyme for TG lipolysis. However, the identification of adipose triglyceride lipase (ATGL) (10–12) has altered the understanding of TG metabolism. ATGL shares several common motifs with other known TG lipases, including a GXSXG motif with N-terminal active sites, Ser⁴⁷ and Asp¹⁶⁶, which are both critical for TG hydrolysis (13), an α/β hydrolase fold and an N-terminal “patatin” homology domain, which is common to plant and mammalian proteins with acyl-hydrolase activity toward phospholipid, monoglyceride, and DG substrates (12, 14). ATGL exerts a key role in lipid droplet degradation in adipocytes and nonadipocyte cells (15–18), and ATGL null mice are obese due to defective lipolysis (19). ATGL exhibits high substrate specificity for TG (10, 12), and full activation requires interaction with the protein comparative gene identification (CGI)-58 (20). Very recent studies also suggest that perilipin 1 binds CGI-58 under nonstimulated conditions and that phosphorylation of perilipin 1 on Ser⁴⁹² or Ser⁵¹⁷ releases CGI-58 from perilipin 1, allowing CGI-58 to directly interact with ATGL at the lipid droplet (21, 22).

The role of phosphorylation in ATGL is poorly described. ATGL gene silencing or ablation reduced basal and PKA-induced lipolysis (23, 24), whereas ATGL^−/− mice are unable to increase lipolysis during physiological conditions characterized by marked β-adrenergic stimulation, such as fasting and exercise (19, 25). Hence, ATGL is essential for both basal and β-adrenergic-stimulated lipolysis. ATGL is phosphorylated by unknown endogenous kinases in HepG2 cells (12), and human phosphoprotein proteomic mass spectrometry analysis detected phosphorylation at Ser⁴⁰⁴ and Ser⁴²⁸ (corresponding to murine Ser⁴⁰⁶ and Ser⁴³⁰) (26). Phosphorylation of ATGL by AMP-activated protein kinase (AMPK) in Caenorhabditis elegans (27) and cyclin-dependent kinase 1 in yeast reduces lipolysis (28), whereas AMPK induces the opposite effect in murine ATGL, that is, phosphorylation of ATGL Ser⁴⁰⁶ increases lipolysis (29). Thus, ATGL activity is subject to phospho-regulation.

Given the prominent role of ATGL in adipose tissue lipolysis and of PKA in β-adrenergic-stimulated lipolysis, the aim of this study was to examine the regulation of ATGL by PKA-mediated phosphorylation. We report here the identification of a regulatory PKA phosphorylation site in ATGL that stimulates adipocyte lipolysis in vitro and is modified under physiological settings in vivo.

Materials and Methods

Expression and purification of murine ATGL

Total RNA was isolated from mouse adipose tissue using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturers protocol. RNA (3 μg) was reverse transcribed using the Thermoscript RT-PCR system (Invitrogen) and oligo dT primers. ATGL cDNA was PCR amplified using Amplitaq Gold DNA polymerase (Applied Biosystems, Foster City, CA) with the following primers: mouse ATGL forward, 5′-GGTACCGTTCCCGAGGGAGACCAAGTGGA-3′ and mouse ATGL reverse, 5′-CCTCGAGCGCAAGGCGGGAGGCCAGGT-3′.

The PCR products were gel purified, subjected to restriction digestion (KpnI/XhoI), subcloned into the multiple cloning site of the pcDNA/HisMax plasmid vector (Invitrogen), and sequence verified.

Sf21 cell culture, expression, and protein purification

The PCR product was subcloned into the pFASTBAC-FLAG-Tev vector (BamHI/SalI). Sf21 insect cells were grown in Sf-900 II medium at 27 C, 130 rpm. Recombinant FLAG-ATGL baculovirus was plaque purified and amplified to high titer. For expression, cells were infected using an MOI of 2 and harvested at 20% cell death, at 48–72 h after infection. Expressed protein was extracted from 480 ml of cell pellet on FLAG antibody resin. Cells were harvested by centrifugation at 1500 × g for 15 min and the pellets sonicated in 40 ml of 50 mm HEPES buffer (pH 7.2), 150 mm NaCl, 1% Triton X-100, 1 mm EGTA, 10% glycerol, 2 mm phenylmethylsulfonylfluoride, 10 μg/ml leupeptin, 1 μg/ml pepstatin, and 0.01% sodium azide, 3 × 30 sec at setting 3 with a Branson probe sonifier. The cell lysate was cleared by centrifugation at 20,000 × g for 20 min and incubated with 2 ml of FLAG antibody resin 3.1 mg/ml overnight at 4 C. FLAG-ATGL was eluted with 0.25 mg/ml FLAG peptide (DYKDDDDK) in Tris-buffered saline 10% glycerol and 0.01% Triton X-100. The preparation was concentrated by ultrafiltration at 4 C on an Amicon 10 Ultra to 2 mg/ml.

Phosphorylation of ATGL

Recombinant murine ATGL (45 μm) was phosphorylated by cAMP-dependent protein kinase (PKA), catalytic subunit (P6000S, 250 U/reaction; Sigma, St. Louis, MO), or purified liver AMPK in kinase assay buffer [50 mm HEPES (pH 7.5), 10 mm MgCl₂, 5% glycerol, 1 mm dithiothreitol, and 0.05% Triton X-100] containing 250 μm [γ-³²P]ATP (5000 cpm/pmol) for 2 h at 30 C. The protein mixture was separated by SDS-PAGE, and the ³²P-labeled ATGL was detected by phosphorimaging.

Phosphopeptide extraction and purification of tryptic digests

Tryptic peptides of ATGL gel bands were prepared as described previously (30) and extracted with consecutive washes in 2% trifluoroacetic acid (TFA), 0.1% TFA in 30% acetonitrile, then 0.1% TFA in 60% acetonitrile in a tube floated on a sonicating water bath. The digest was almost dried in a centrifugal freeze drier. The peptides were desalted by C18-ZipTip chromatography (Millipore, Bedford, MA) into 10 μl of 80% acetonitrile and 0.1% TFA before phosphate release sequencing or MALDI-TOF mass spectrometry.

Peptide sequencing and phosphate release

Peptides were sequenced with a Hewlett Packard G1000A protein sequencer (Hewlett Packard, Palo Alto, CA) using Routine 3.5 Edman degradation chemistry as recommended by the manufacturer. Phosphopeptides were covalently linked to Sequelon AAfilters (PerSeptive Biosystems, Framingham, MA), and phosphate was extracted at each cycle with 3 × 0.5-ml volumes of 90% methanol, 0.015% phosphoric acid as the solvent in the Routine 3.1 PVDF method. Extracted cycles were diverted via valve number RV6 (line 61), collected directly into fraction collector tubes, and counted on a liquid scintillation counter.

Mass spectrometry analysis of phosphopeptides

Proteins were extracted from sodium dodecyl sulfate gel slices after digestion with trypsin (Promega, Madison, WI). Phosphopeptides were purified on Phostrap beads (PerkinElmer, Waltham, MA). The purified phosphopeptides were dried under vacuum and resuspended in 20 μl of 2% formic acid and analyzed by LC-MS. Reversed phase chromatography was performed on a 20-cm × 75-μm column packed with Pepmap300 C18 (LC-Packings, Sunnyvale, CA) at a flow rate of 500 nl/min on a Dionex Ultimate 3000 nano-LC (Dionex, Rockford, IL). Samples were ionized with a microionspray source (Applied Biosystems) and mass spectrometry performed on a QSTAR Pulsar I (Applied Biosystems). Data were acquired using a data dependent acquisition program and peptide spectra interpreted by MASCOT provided by the Australian Proteomic Computing Facility. All spectra identified by MASCOT were manually validated.

Cell culture

Cos-1 were purchased from American Type Culture Collection (Manassas, VA) and grown in DMEM, 10% fetal bovine serum, and 0.5% penicillin-streptomycin. Cells were transfected with vectors containing wild-type ATGL and mutant inserts using Lipofectamine 2000 plus reagent. Experiments were performed after 24 h. For lipolysis experiments, cells were pulsed with 1 mm [1-¹⁴C]oleate (1 μCi/ml) for 4 h, washed, and incubated in the presence of Triacsin C, an inhibitor of acyl coenzyme A (CoA) synthetases that prevents reesterification of fatty acids. The rate of ¹⁴C-oleate appearance in the culture media was determined after 8 h (LS6500 Scintillantion Counter; Beckman Coulter, Inc., Brea, CA).

Retroviral constructs

Murine ATGL underwent Site-Directed Mutagenesis using pfx polymerase (Invitrogen) to generate S406A (forward-ACGTGCCCAGGCTCTGCCCTCTGTG, reverse-CACAGAGGGCAGAGCCTGGGCACGT). The wild-type and mutant murine ATGL were excised using KpnI and ApaI and ligated into pEGFP C1 vector (CLONTECH, Mountain View, CA). The green fluorescent protein (GFP)-ATGL inserts were excised with AgeI and SmaI, treated with Klenow (NEB) and ligated into SnaB1 (NEB)-digested pBABE-puro vector (Cell Biolabs, Inc., San Diego, CA). The constructs were sequence verified using BigDye terminator mix (Applied Biosystems).

Isolation of murine embryonic fibroblasts (MEF) from wild-type and ATGL^−/− mice

MEF (E14) were derived from ATGL^−/− and wild-type mice by digesting with 0.02% collagenase (Sigma) for 1 h at 37 C. The cells were washed and plated overnight. Cells were used up to passage 3 for experiments.

Transfection of retroviral plasmids and infection of MEF

Retroviral vectors containing wild-type ATGL and mutant inserts were transfected using Lipofectamine 2000 plus reagent (Invitrogen) into Platinum E cells (Cell Biolabs, Inc.) as previously described (31). The viral supernatant was collected after 48 h and passed through a 0.45-μm filter. MEF were infected with viral supernatant containing 3.3 mg/ml Polybrene (Sigma) for 5 h at 37 C. The viral supernatant was removed, and the infected MEF were selected using 2.5 μg/ml puromycin (Invitrogen) as determined by a puromycin kill curve. Noninfected cells died within 2 d and the infected cells proliferated.

Differentiation of MEF into adipocytes

Infected MEF at 2 × 10⁵ to 2.5 × 10⁵ cells per well of a six-well dish were grown to confluence in MEF medium [high glucose DMEM (Invitrogen), 10% fetal bovine serum (Invitrogen), 0.5% penicillin-streptomycin (Invitrogen), and 200 μm ascorbic acid (Sigma)]. To initiate differentiation, cells were cultured in MEF medium containing 2 μm insulin (Sigma) and 1 nm T₃ (Sigma-Aldrich, Castle Hill, New South Wales, Australia) for 3 d, then incubated for 3 d in the additional presence of 250 nm dexamethasone (Sigma), 0.5 mm isobutylmethylxanthine (Sigma), and 125 nm indomethacine (Wako Pure Chemical Industries Ltd., Osaka, Japan). Cells were then returned to MEF medium alone and incubated for a further 4 d before performing experiments.

Animal studies

All experimental procedures were approved by the Monash University School of Biomedical Science Animal Ethics Committee and were in accordance with the National Health and Medical Research Council of Australia's guidelines on animal experimentation. Animals received humane care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals. Male C57BL/6 mice aged 8 wk were purchased from Monash Animal Services. All mice were bred and housed under controlled temperature (22 C) and lighting (12-h light, 12-h dark cycle) and had free access to water and standard rodent chow (specialty feeds irradiated rat and mouse pellets; 14.3 MJ/kg digestible energy). Mice were subjected to one of several perturbations: ad libitum feeding, fasting from 1600–0800 h, or exercise for 30 min at a speed of 16 m/min at a 5% grade. Mice were killed by cervical dislocation and the epididymal fat pad removed and rapidly frozen in liquid nitrogen for later analysis. In some studies, epididymal adipose tissue explants (∼40 mg) were incubated in Krebs buffer containing 2% BSA and 5 mm glucose for 1 h without or with 20 μm H-89 (EnoGene Biotech, New York, NY), a cell permeable PKA inhibitor. Tissues were then treated without or with 20 μm forskolin for 20 min (signaling experiments) or 1 h (lipolysis determination by glycerol release). ATGL^−/− mice were obtained from R. Zechner (University of Graz, Graz, Austria).

Human experiments

The Monash University Human Research Ethics Committee approved the human experiments and informed consent was obtained from patients before surgery. Abdominal subcutaneous adipose tissue was obtained from anesthetized subjects undergoing bariatric surgery. The adipose tissue was transferred to oxygenated Krebs buffer containing 2% BSA and 5 mm glucose, cut into approximately 30-mg pieces, incubated for 20 min (immunoblot analysis) or 2 h (lipolysis measure) in the presence of saline, isoproterenol, or AICAR at the indicated concentrations, and then snap frozen in liquid nitrogen.

Immunoblot analysis

Tissues or cells were homogenized in ice cold RIPA lysis buffer containing protease inhibitor cocktail (Roche, Mannheim, Germany), phosphostop (Roche), and 1 mm dithiothreitol, centrifuged, and the supernatant fraction was resolved by SDS-PAGE and immunoblotted. A rabbit polyclonal antibody raised against the phosphopeptide based on the amino acid sequence of murine ATGL (400–414) pS406 LRRAQpSLPSVPLSC was purified as described previously (32). This site is conserved in humans at Ser⁴⁰⁴. Primary antibodies for anti-ATGL, anti-pAMPK Thr¹⁷², anti-AMPK, antiacetyl CoA carboxylase (ACC), and anti-pACCβ Ser²²¹ were purchased from Cell Signaling (Danvers, MA), anti-α-actin and antiperilipin A/B from Sigma-Aldrich, and anti-HSL from Abcam (Cambridge, UK). The anti-HSL Ser⁵⁵⁴ and CGI-58 antibody were produced in-house as described (33).

Lipolysis

Glycerol (free glycerol reagent; Sigma-Aldrich, St. Louis, MO) and free fatty acids (Wako Chemicals; Wako, Richmond, VA) released from 3T3-L1 or human adipose tissue explants into the media was measured using commercial assays.

Immunofluorescence microscopy

MEF were grown and differentiated on 0.17-mm glass coverslips. Cells were fixed with 4% paraformaldehyde for 30 min at room temperature. The lipid droplets were stained with HCS Lipidtox deep red (Invitrogen), the nucleus was stained with Hoescht 33342 (no. H21492; Invitrogen), and the ATGL-GFP with anti-GFP antibody (no. 2956; Cell Signaling) followed by antirabbit Alexa Fluor 488 (no. A11008; Invitrogen). Coverslips were mounted on glass slides and imaged with the Leica SP5 5 Channel microscope (Leica, Heerbrugg, Switzerland).

Triglyceride hydrolase and AMPK activity assays

TG hydrolase activity was assessed in cell/tissue homogenates against a [9,10-³H]triolein tracer as described previously (19). AMPK activity was assessed by radiometric method as described (34).

Statistics

All data are presented as mean ± sem. Statistical analysis was performed using either an unpaired Student's t test or one-way or two-way ANOVA. Bonferroni post hoc analysis was performed where appropriate. Statistical significance was set a priori at P < 0.05.

Results

Identification of PKA phosphorylation sites in ATGL

A phosphoprotein proteomic mass spectrometry screen of lipid droplet-associated proteins previously detected phosphorylation at Ser⁴⁰⁴ and Ser⁴²⁸ in human ATGL (corresponding to murine Ser⁴⁰⁶ and Ser⁴³⁰) (26). In silico analysis of murine ATGL predicted Ser⁴⁰⁶ to be a putative PKA site (also PKC and AMPK), with several other potential PKA sites based on the consensus sequence R(R/K)SS, including Ser⁴³⁰ (www.cbs.dtu.dk/services/NetPhosK). To identify PKA phosphorylation sites in ATGL, purified recombinant murine ATGL was incubated in the presence of PKA and [γ-³²P]ATP. Incubation of recombinant murine ATGL with PKA in vitro increased phosphorylation above no kinase alone (Fig. 1A). Phosphoamino analysis of the ATGL tryptic digest revealed that the phosphopeptide/s were serine (data not shown). Edman degradation showed that the radioactivity was released at cycles 3–5 (Fig. 1B), and in silico enzymatic digestion of the recombinant ATGL protein sequence predicted that the phosphorylation sites were Ser⁴⁰⁶ (cycle 3), Ser¹¹, Ser¹¹⁷, Ser⁴³⁰, Ser⁴⁶⁸ (all cycle 4), Ser²¹¹, and Ser³⁷⁴ (cycle 5). Mass spectrometry analysis of PKA phosphorylated murine recombinant ATGL identified Ser³⁷⁴, Ser³⁹⁶, Ser⁴⁰⁶, Ser⁴³⁰, and Ser⁴⁶⁸ or Thr³⁷⁶ (three independent experiments). The phosphopeptides are shown in Table 1 and Fig. 1C. Ser³⁷⁴, Ser⁴⁰⁶, and Ser⁴³⁰ are conserved in humans (Fig. 1D). We examined the role of Ser³⁹⁶ and Ser⁴⁰⁶ based on the higher abundance of phosphopeptides detected by mass spectrometry (Supplemental Fig. 1, published on The Endocrine Society's Journals Online web site at http://endo.endojournals.org) and the suggested functional role of Ser⁴⁰⁶ (29). ATGL Ser³⁷⁴ was not examined due to very low coverage in the mass spectra.

Identification of PKA phosphorylation sites in ATGL. A, Recombinant murine ATGL was phosphorylated by cAMP-dependent protein kinase (PKA) catalytic subunit in kinase assay buffer containing [γ-³²P]ATP. The protein mixture was separated by SDS-PAGE (upper band) and the ³²P-labeled ATGL was detected by phosphorimaging (lower band). B, Tryptic peptides of phosphorylated ATGL were subjected to Edman degradation. Phosphate release showed that the radioactivity was contained in fractions 3–5 with the potential phosphorylation sites being Ser⁴⁰⁶ (cycle 3), Ser¹¹, Ser¹¹⁷, Ser⁴³⁰, Ser⁴⁶⁸ (all cycle 4), Ser²¹¹, and Ser³⁷⁴ (cycle 5). C, Domain organization and phosphorylation sites in murine ATGL. D, Sequence comparison between murine and human ATGL. Letters in bold-type indicate the identified PKA phosphorylation sites.

Identification of PKA phosphorylation sites in ATGL. A, Recombinant murine ATGL was phosphorylated by cAMP-dependent protein kinase (PKA) catalytic subunit in kinase assay buffer containing [γ-³²P]ATP. The protein mixture was separated by SDS-PAGE (upper band) and the ³²P-labeled ATGL was detected by phosphorimaging (lower band). B, Tryptic peptides of phosphorylated ATGL were subjected to Edman degradation. Phosphate release showed that the radioactivity was contained in fractions 3–5 with the potential phosphorylation sites being Ser⁴⁰⁶ (cycle 3), Ser¹¹, Ser¹¹⁷, Ser⁴³⁰, Ser⁴⁶⁸ (all cycle 4), Ser²¹¹, and Ser³⁷⁴ (cycle 5). C, Domain organization and phosphorylation sites in murine ATGL. D, Sequence comparison between murine and human ATGL. Letters in bold-type indicate the identified PKA phosphorylation sites.

Identification of PKA phosphorylation sites in recombinant murine ATGL by mass spectrometry

Peptide(s) identified and phosphorylation site

QTG374SICQYLVMR

KLGDHLP396SRLSEQVELR (both PKA and AMPK)

AQ406SLPSVPLSCATYSEALPNWVR (both)

NNL430SLGDALAK

MRAPA468SPTAADPATPQDPPGLPPC

Peptide(s) identified and phosphorylation site

QTG374SICQYLVMR

KLGDHLP396SRLSEQVELR (both PKA and AMPK)

AQ406SLPSVPLSCATYSEALPNWVR (both)

NNL430SLGDALAK

MRAPA468SPTAADPATPQDPPGLPPC

The peptides were identified at least twice in independent experiments.

Identification of PKA phosphorylation sites in recombinant murine ATGL by mass spectrometry

Peptide(s) identified and phosphorylation site

QTG374SICQYLVMR

KLGDHLP396SRLSEQVELR (both PKA and AMPK)

AQ406SLPSVPLSCATYSEALPNWVR (both)

NNL430SLGDALAK

MRAPA468SPTAADPATPQDPPGLPPC

Peptide(s) identified and phosphorylation site

QTG374SICQYLVMR

KLGDHLP396SRLSEQVELR (both PKA and AMPK)

AQ406SLPSVPLSCATYSEALPNWVR (both)

NNL430SLGDALAK

MRAPA468SPTAADPATPQDPPGLPPC

The peptides were identified at least twice in independent experiments.

Analysis of ATGL phosphorylation mutants S396A and S406A in COS1 cells

Site-Directed Mutagenesis to convert the individual serine residues into alanine was used to test the functional and regulatory roles of the identified PKA phosphorylation sites. The mutated GFP-tagged ATGL proteins were expressed in COS1 cells (which contain almost undetectable levels of ATGL), and expression was confirmed by Western blotting (Fig. 2A). We combined extracts from cells transfected with wild-type or mutant ATGL and CGI-58 and performed TG hydrolase assays. All data in COS1 cells were normalized to total ATGL content determined by Western blot analysis and densitometry, because total ATGL protein influences lipolysis (23). The combination of wild-type ATGL and CGI-58 extracts increased the TG hydrolase activity 4-fold above ATGL alone (data not shown). TG hydrolase activity was reduced by 23 and 31% in ATGL Ser³⁹⁶ and Ser⁴⁰⁶ mutants compared with wild-type ATGL (Fig. 2B). To determine whether the reduction in ATGL TG hydrolase activity reduces lipolysis, COS1 cells were transfected with wild-type or mutant ATGL and CGI-58 cDNA. Twenty-four hours after transfection, cells were pulsed with ¹⁴C-oleate for 4 h, washed, and incubated in the presence of Triacsin C, an inhibitor of acyl CoA synthetases that prevents reesterification of fatty acids. The rate of ¹⁴C-oleate appearance in the culture media was reduced in cells transfected with ATGL Ser⁴⁰⁶ mutants (Fig. 2C). Thus, a mutation in the PKA phosphorylation site ATGL Ser⁴⁰⁶ reduces both ATGL TG hydrolase activity determined in vitro and lipolysis determined in vivo. The mismatch between TG hydrolase activity and lipolysis in the ATGL S396A cells suggests that factors other than TG hydrolase activity are required to regulate lipolytic activity in vivo. This might include translocation of ATGL to the substrate (which is not a limitation of in vitro TG hydrolase assays) and protein-protein interactions. Although these experiments in COS1 cells provide important proof-of-concept information, the absence of the essential lipolytic protein, perilipin 1, in nonadipocyte cells (Fig. 2D) confounds the interpretation for the regulation of adipocyte lipolysis (9).

Analysis of ATGL phosphorylation mutants S396A and S406A in COS1 cells. A, Representative immunoblot of ATGL expression in COS1 cells transfected with various ATGL constructs. B, TG hydrolase activity was assessed in cell lysates of CGI-58 combined with either ATGL, s396A, or S406A using a triolein substrate. *, P < 0.05 vs. ATGL. Measurements were from three independent plates from two independent experiments (n = 6 per condition). ND, Not determined because ATGL content was too low for assessment by immunoblot. C, COS1 cells were transfected with wild-type or mutant ATGL and CGI-58 cDNA. Twenty-four hours after transfection, cells were pulsed with ¹⁴C-oleate for 4 h, washed, and incubated in the presence of Triacsin C. ¹⁴C-oleate appearance was measured in the culture media. *, P < 0.05 vs. ATGL. Measurements are representative of two independent experiments (n = 6 per condition). TG hydrolase activity and lipolysis were normalized to total ATGL content within each condition. D, Immunoblot showing the absence of perilipin 1 in COS1 cells.

Analysis of ATGL phosphorylation mutants S396A and S406A in COS1 cells. A, Representative immunoblot of ATGL expression in COS1 cells transfected with various ATGL constructs. B, TG hydrolase activity was assessed in cell lysates of CGI-58 combined with either ATGL, s396A, or S406A using a triolein substrate. *, P < 0.05 vs. ATGL. Measurements were from three independent plates from two independent experiments (n = 6 per condition). ND, Not determined because ATGL content was too low for assessment by immunoblot. C, COS1 cells were transfected with wild-type or mutant ATGL and CGI-58 cDNA. Twenty-four hours after transfection, cells were pulsed with ¹⁴C-oleate for 4 h, washed, and incubated in the presence of Triacsin C. ¹⁴C-oleate appearance was measured in the culture media. *, P < 0.05 vs. ATGL. Measurements are representative of two independent experiments (n = 6 per condition). TG hydrolase activity and lipolysis were normalized to total ATGL content within each condition. D, Immunoblot showing the absence of perilipin 1 in COS1 cells.

Functional characterization of ATGL phosphorylation mutant S406A in differentiated MEF from ATGL null mice

Wild-type and S406A forms of ATGL were introduced into MEF obtained from ATGL null mice by retroviral transduction. GFP was introduced into MEF from wild-type mice as a control. ATGL wild-type and ATGL S406A proteins were expressed at similar ratios in ATGL null MEF (Fig. 3, A and B), and the key lipolytic proteins perilipin 1, CGI-58, and HSL were similarly expressed in differentiated MEF (Fig. 3A). Retroviral infection did not restrain adipogenesis during differentiation as indicated by normal lipid droplet number and gross morphology (Fig. 3C). Adipocytes were treated without or with forskolin, an adenylyl cyclase stimulator that activates the cAMP-PKA pathway, and the culture medium was assessed for released glycerol as a marker of complete lipolysis. Figure 3D shows that basal and stimulated lipolysis are reduced in ATGL null vs. wild-type MEF and that forced expression of ATGL increases lipolysis in ATGL null MEF. ATGL S406A did not affect basal lipolysis but decreased forskolin-induced lipolysis by 21% compared with ATGL (Fig. 3D). Lipolysis in ATGL null MEF with ATGL reintroduction was lower than wild-type MEF due to reduced total ATGL expression (Fig. 3B). Fatty acids are sequentially hydrolyzed from TG by the lipases ATGL, HSL, and MAGL, and FFA release from adipocytes is thereby thought to more accurately reflect ATGL action. Figure 3D shows that fatty acid release in response to cAMP-PKA stimulation is reduced by 20% in ATGL S406A adipocytes. The majority of ATGL is localized to the cytoplasm in adipocytes with small amounts at the lipid droplet surface under nonstimulated conditions (11, 12, 21). Some ATGL translocates to the surface of small lipid droplets containing perilipin 1 upon β-adrenergic signaling (12, 21, 23), which is consistent with a role in stimulated lipolysis. We analyzed ATGL localization by confocal microscopy and report no changes in cellular localization with ATGL S406A (Fig. 3C). In addition, TG hydrolase activity of cell lysates was not altered in ATGL S406A MEF adipocytes (data not shown). Collectively, these data demonstrate that phosphorylation of ATGL Ser⁴⁰⁶ reduces cAMP-PKA-stimulated lipolysis in adipocytes by undefined mechanisms.

Functional characterization of the ATGL phosphorylation mutant S406A in MEF adipocytes. MEF obtained from wild-type mice were infected with GFP cDNA and MEF obtained from ATGL null mice were infected with GFP, GFP-ATGL, or GFP-ATGL S406A. MEF were differentiated into adipocytes for 7–10 d before experiments. A, Protein expression of lipolytic proteins. Note for ATGL, endogenous ATGL at 54 kDa and GFP-ATGL at 82 kDa. B, Quantification of ATGL (n = 6–12 per group from two independent experiments). C, Cellular localization of ATGL phosphorylation mutants. MEF adipocytes expressing GFP, GFP-ATGL, or GFP-ATGL S406A were fixed, probed for GFP (GFP, green), Lipidtox deep red (lipid droplet, red), and 4′,6-diamidino-2-phenylindole (DAPI) (nuclear stain, blue) and visualized by confocal microscopy. D, Lipolysis was assessed by glycerol (left) and free fatty acid (right) release into the media. Open bars, Spontaneous lipolysis; closed bars, stimulated lipolysis. *, P < 0.05 vs. ATGL, n = 6 from two independent experiments.

Functional characterization of the ATGL phosphorylation mutant S406A in MEF adipocytes. MEF obtained from wild-type mice were infected with GFP cDNA and MEF obtained from ATGL null mice were infected with GFP, GFP-ATGL, or GFP-ATGL S406A. MEF were differentiated into adipocytes for 7–10 d before experiments. A, Protein expression of lipolytic proteins. Note for ATGL, endogenous ATGL at 54 kDa and GFP-ATGL at 82 kDa. B, Quantification of ATGL (n = 6–12 per group from two independent experiments). C, Cellular localization of ATGL phosphorylation mutants. MEF adipocytes expressing GFP, GFP-ATGL, or GFP-ATGL S406A were fixed, probed for GFP (GFP, green), Lipidtox deep red (lipid droplet, red), and 4′,6-diamidino-2-phenylindole (DAPI) (nuclear stain, blue) and visualized by confocal microscopy. D, Lipolysis was assessed by glycerol (left) and free fatty acid (right) release into the media. Open bars, Spontaneous lipolysis; closed bars, stimulated lipolysis. *, P < 0.05 vs. ATGL, n = 6 from two independent experiments.

ATGL Ser⁴⁰⁶ phosphorylation is increased during fasting and exercise in mice

We next evaluated the physiological relevance of the ATGL Ser⁴⁰⁶ site in vivo. The polyclonal antibody was first validated using cells expressing ATGL or alanine mutations at specific serine residues (Fig. 4, A and B). No signal was detected in the GFP control or ATGL S406A-expressing lines, demonstrating specificity of the antibody. Furthermore, no immunoreactive band was detected in adipose tissue lysates from ATGL^−/− mice (Fig. 4C). To test whether PKA phosphorylates ATGL in the physiological condition of stimulated lipolysis, we knocked down PKA signaling using the pharmacological inhibitor H-89. Both basal and stimulated adipose tissue lipolysis were reduced in adipose explants (Fig. 4D), and this coincided with a complete attenuation of ATGL Ser⁴⁰⁶ phosphorylation upon PKA stimulation (Fig. 4E). To test the in vivo relevance of the ATGL Ser⁴⁰⁶ site, mice were subjected to fasting or treadmill running, physiological situations that are characterized by increased lipolysis. ATGL Ser⁴⁰⁶ phosphorylation was low in ad libitum-fed mice and increased with prolonged fasting (Fig. 4F) and during moderate intensity exercise (Fig. 4G). AMPK activity (P = 0.31) (Fig. 4H) and AMPK Thr¹⁷² phosphorylation were not different in fasted vs. fed mice (Fig. 4H), and exercise did not affect AMPK Thr¹⁷² or ACCβ Ser²²¹ phosphorylation (Fig. 4H, quantification not shown).

ATGL Ser⁴⁰⁶ phosphorylation is increased during fasting and exercise in mice. A, Immunoblot of COS1 cell lysates transfected with ATGL mutants using a polyclonal antibody against ATGL pSer⁴⁰⁶. Note the absence of an immunoreactive band in the S406A cell lysates. B, 3T3-L1 adipocytes were stably infected with GFP-ATGL phosphorylation mutants S396A, S406A, and S430A. The ATGL pSer⁴⁰⁶ polyclonal antibody did not detect an immunoreactive band in the S406A cell lysates. C, No evidence of an immunoreactive band in adipose tissue lysates prepared from ATGL^−/− mice with either the ATGL total or pSer⁴⁰⁶ antibodies. Note, images from separate immunoblots. D, Adipose tissue explants were incubated without (open bars) or with (closed bars) the PKA inhibitor H-89 before stimulation with 20 μm forskolin. Lipolysis was assessed as glycerol release into the culture medium (n = 4 per condition). E, ATGL Ser⁴⁰⁶ phosphorylation in H-89-treated adipose explants (n = 4 per condition). Right, Representative immunoblot. F, Epididymal white adipose tissue was excised from mice fed ad libitum or after an overnight fast. G, Epididymal white adipose tissue was excised from mice fed ad libitum (rest) or after treadmill running (run, 30 min at 16 m/min; 5% grade). Immunoblot using antibody against ATGL Ser⁴⁰⁶ and ATGL. Below, Quantification of blots. Top, Representative immunoblots. *, P < 0.05, n = 8–13 per group. H, AMPK activity in adipose tissue was not affected by fasting or exercise (n = 5 per group). Below, Immunoblot showing AMPK Thr¹⁷² and ACCβ Ser²²¹ phosphorylation.

ATGL Ser⁴⁰⁶ phosphorylation is increased during fasting and exercise in mice. A, Immunoblot of COS1 cell lysates transfected with ATGL mutants using a polyclonal antibody against ATGL pSer⁴⁰⁶. Note the absence of an immunoreactive band in the S406A cell lysates. B, 3T3-L1 adipocytes were stably infected with GFP-ATGL phosphorylation mutants S396A, S406A, and S430A. The ATGL pSer⁴⁰⁶ polyclonal antibody did not detect an immunoreactive band in the S406A cell lysates. C, No evidence of an immunoreactive band in adipose tissue lysates prepared from ATGL^−/− mice with either the ATGL total or pSer⁴⁰⁶ antibodies. Note, images from separate immunoblots. D, Adipose tissue explants were incubated without (open bars) or with (closed bars) the PKA inhibitor H-89 before stimulation with 20 μm forskolin. Lipolysis was assessed as glycerol release into the culture medium (n = 4 per condition). E, ATGL Ser⁴⁰⁶ phosphorylation in H-89-treated adipose explants (n = 4 per condition). Right, Representative immunoblot. F, Epididymal white adipose tissue was excised from mice fed ad libitum or after an overnight fast. G, Epididymal white adipose tissue was excised from mice fed ad libitum (rest) or after treadmill running (run, 30 min at 16 m/min; 5% grade). Immunoblot using antibody against ATGL Ser⁴⁰⁶ and ATGL. Below, Quantification of blots. Top, Representative immunoblots. *, P < 0.05, n = 8–13 per group. H, AMPK activity in adipose tissue was not affected by fasting or exercise (n = 5 per group). Below, Immunoblot showing AMPK Thr¹⁷² and ACCβ Ser²²¹ phosphorylation.

ATGL Ser⁴⁰⁴ phosphorylation is increased by β-adrenergic regulation, but not AMPK, in human adipose tissue

Browser based games like diablo. Subcutaneous adipose tissue was obtained from obese subjects undergoing bariatric surgery. Subject characteristics are listed in Table 2. Adipocyte lipolysis was assessed ex vivo and was increased with β-adrenergic stimulation as expected (Fig. 5A). ATGL Ser⁴⁰⁴ (corresponding to Ser⁴⁰⁶ in mice) phosphorylation was dose dependently increased by isoproterenol (Fig. 5B) and correlated with adipocyte lipolysis (r² = 0.62, P = 0.004). These data show that activation of cAMP-PKA signaling concomitantly increases lipolysis and ATGL Ser⁴⁰⁴ phosphorylation in human adipose tissue.

Baseline clinical characteristics of subjects

50 ± 4

Sex (m/f)	2/5
Mass (kg)	114 ± 5
Body mass index (kg/m2)	42.7 ± 2.0
Blood glucose (mmol/liter)	5.1 ± 0.2
HOMA-IR	3.3 ± 0.6
HbA1c (%)	5.6 ± 0.1
Total cholesterol (mmol/liter)	5.6 ± 0.6
HDL cholesterol (mmol/liter)	1.3 ± 0.1
LDL cholesterol (mmol/liter)	3.7 ± 0.5
Plasma triglycerides (mmol/liter)	1.4 ± 0.3

50 ± 4

Sex (m/f)	2/5
Mass (kg)	114 ± 5
Body mass index (kg/m2)	42.7 ± 2.0
Blood glucose (mmol/liter)	5.1 ± 0.2
HOMA-IR	3.3 ± 0.6
HbA1c (%)	5.6 ± 0.1
Total cholesterol (mmol/liter)	5.6 ± 0.6
HDL cholesterol (mmol/liter)	1.3 ± 0.1
LDL cholesterol (mmol/liter)	3.7 ± 0.5
Plasma triglycerides (mmol/liter)	1.4 ± 0.3

HDL, High density lipoprotein; HOMA-IR, homeostasis model assessment of insulin resistance; LDL, low density lipoprotein.

Baseline clinical characteristics of subjects

50 ± 4

Sex (m/f)	2/5
Mass (kg)	114 ± 5
Body mass index (kg/m2)	42.7 ± 2.0
Blood glucose (mmol/liter)	5.1 ± 0.2
HOMA-IR	3.3 ± 0.6
HbA1c (%)	5.6 ± 0.1
Total cholesterol (mmol/liter)	5.6 ± 0.6
HDL cholesterol (mmol/liter)	1.3 ± 0.1
LDL cholesterol (mmol/liter)	3.7 ± 0.5
Plasma triglycerides (mmol/liter)	1.4 ± 0.3

50 ± 4

Sex (m/f)	2/5
Mass (kg)	114 ± 5
Body mass index (kg/m2)	42.7 ± 2.0
Blood glucose (mmol/liter)	5.1 ± 0.2
HOMA-IR	3.3 ± 0.6
HbA1c (%)	5.6 ± 0.1
Total cholesterol (mmol/liter)	5.6 ± 0.6
HDL cholesterol (mmol/liter)	1.3 ± 0.1
LDL cholesterol (mmol/liter)	3.7 ± 0.5
Plasma triglycerides (mmol/liter)	1.4 ± 0.3

HDL, High density lipoprotein; HOMA-IR, homeostasis model assessment of insulin resistance; LDL, low density lipoprotein.

ATGL Ser⁴⁰⁴ phosphorylation is increased by β-adrenergic regulation in human adipose tissue. Human sc adipose tissue was incubated in oxygenated Krebs buffer for 4 h with no additions [vehicle (Veh)], 0.1 or 1 μm isoproterenol, or 2 mm AICAR. A, Lipolysis was assessed as glycerol release into the culture medium. Quantification of ATGL Ser⁴⁰⁴ phosphorylation (B), AMPK Thr¹⁷² phosphorylation (C), and HSL Ser⁵⁶⁵ phosphorylation (D) after 20 min of incubation under the above conditions. *, P < 0.05, n = 7 per group. Representative immunoblots shown above columns.

ATGL Ser⁴⁰⁴ phosphorylation is increased by β-adrenergic regulation in human adipose tissue. Human sc adipose tissue was incubated in oxygenated Krebs buffer for 4 h with no additions [vehicle (Veh)], 0.1 or 1 μm isoproterenol, or 2 mm AICAR. A, Lipolysis was assessed as glycerol release into the culture medium. Quantification of ATGL Ser⁴⁰⁴ phosphorylation (B), AMPK Thr¹⁷² phosphorylation (C), and HSL Ser⁵⁶⁵ phosphorylation (D) after 20 min of incubation under the above conditions. *, P < 0.05, n = 7 per group. Representative immunoblots shown above columns.

The amino acid sequence surrounding Ser⁴⁰⁴ is a consensus site for AMPK and a recent study in mice suggests that ATGL can be phosphorylated in Ser⁴⁰⁶ by AMPK (29). In vitro kinase assays demonstrated phosphorylation of murine ATGL by AMPK (Supplemental Fig. 2A). Phosphate release showed that the radioactivity was released at cycles 3 and 4, corresponding to Ser⁴⁰⁶ (cycle 3), Ser¹¹, Ser¹¹⁷, Ser⁴³⁰, and Ser⁴⁶⁸ (cycle 4) (Supplemental Fig. 2B). Of these sites, only Ser⁴⁰⁶ was confirmed by mass spectrometry. The detected phosphopeptides were AQ⁴⁰⁶SLPSVPL ⁴¹³SCATYSEALPNWVR and ITVSPFSGESDICPQD¹⁹⁸SSTNIHELR. Although AMPK phosphorylated recombinant ATGL in vitro, ATGL Ser⁴⁰⁴ phosphorylation was not increased with pharmacological AMPK activation (Fig. 5C) in human adipose tissue (Fig. 5B). Notably, phosphorylation of the documented AMPK site in HSL Ser⁵⁵⁴ (corresponding to murine HSL Ser⁵⁶⁵) was increased with AMPK activation (Fig. 5D).

Discussion

The mobilization of free fatty acids via adipocyte TG lipolysis is a key event in maintaining energy homeostasis. During periods of fasting or increased energy demand, adipocyte lipolysis is increased to release fatty acids into the circulation to provide fuel for other tissues. Lipolysis is stimulated by the actions of the catecholamines that act by increasing cAMP and eventually PKA, and PKA phosphorylation of several lipolytic proteins is a hallmark of β-adrenergic-stimulated lipolysis (35). ATGL is the predominant TG lipase in adipocytes and is essential for basal and stimulated lipolysis (12, 16, 36). These observations are highlighted by loss-of-function experiments showing reduced adipose tissue lipolysis and tissue TG accumulation in all species examined to date (15, 17, 19, 37, 38). The factors regulating ATGL action remain uncertain, particularly in the setting of β-adrenergic regulation. In this study, we identified ATGL Ser⁴⁰⁶ as a PKA target in murine and ATGL Ser⁴⁰⁴ in human adipocytes that plays a role in ATGL-mediated lipolysis.

Ongoing studies are unraveling ATGL regulation during basal (spontaneous) and PKA stimulated conditions. Under basal conditions, ATGL is dispersed throughout the cytosol, and some localize to the lipid droplets, with evidence that these droplets contain perilipin 1 (12, 13, 21, 39). The C-terminal region of ATGL is required for localization of ATGL to the lipid droplet (39, 40). PKA stimulation induces some translocation of ATGL to smaller lipid droplets (21, 23, 41) and increases the association of ATGL with its coactivator CGI-58, thereby increasing TG lipase activity (20). It appears that PKA-induced phosphorylation of perilipin 1 causes the release of CGI-58 from perilipin 1, and phosphorylation of Ser⁵¹⁷ of perilipin 1 is implicated in this process (9). Finally, binding of the protein G_o/G₁ switch gene 2 to the catalytic patatin-like domain of ATGL inhibits its activity, although this regulation is not PKA dependent (41). Thus, ATGL is known to be regulated by a complex interaction between translocation and protein-protein interactions, which may be dependent upon PKA-mediated phosphorylation of the protein targets.

Previous studies have reported phosphorylation in ATGL. Zimmermann et al. (12) first reported phosphorylation of ATGL under basal conditions in cellular extracts of HepG2 cells overexpressing ATGL, whereas Ser⁴⁰⁴ and Ser⁴²⁸ (murine Ser⁴⁰⁶ and Ser⁴³⁰) of ATGL were later identified in a mass spectrometry screen of phosphorylated lipid droplet-associated proteins (26). Neither study reported the upstream kinase/s. Although unequivocal evidence points to a role for ATGL in stimulated lipolysis, it was previously reported that ATGL was not a direct PKA target (12). This was a curious observation given the presence of several putative PKA sites contained within ATGL and the critical role of PKA phosphorylation in regulating other key lipolytic proteins (e.g. HSL and perilipin 1). Our studies using both protein chemistry and mass spectrometry approaches show that recombinant murine ATGL can be phosphorylated by PKA at multiple sites in vitro (Ser³⁷⁴), Ser³⁹⁶, Ser⁴⁰⁶, and Ser⁴³⁰, and several factors indicate that ATGL Ser⁴⁰⁶ regulates lipolysis in vivo. Alanine mutation of ATGL Ser⁴⁰⁶ reduced TG hydrolase activity and basal lipolysis in COS1 cells, suggesting that some phosphorylation of ATGL Ser⁴⁰⁶ is required to maintain basal lipolysis in nonadipocytes. This finding is in partial agreement with previous work demonstrating decreased TG hydrolase activity in human embryonic kidney cells with Ser⁴⁰⁶ alanine mutation (29), but contrast, previous work by the same group showing that phosphorylation of ATGL at Ser⁴⁰⁶ is not essential for activity or localization in the 293FT human embryonic kidney cell line (13). Others have suggested that phosphorylation of perilipin 1 at Ser⁵¹⁷ is required for ATGL activation (9), although our own studies showing effects of ATGL Ser⁴⁰⁶ phosphorylation in COS1 cells argue against a requisite role of perilipin 1. It is likely that ATGL, or other lipolytic proteins, can interact with other members of the perilipin family to influence ATGL action. Indeed, CGI-58 interacts with perilipin 5 (also known as OXPAT, LSDP5) to modulate ATGL activity in nonadipocytes (42), whereas perilipin 2 (ADRP) overexpression reduces ATGL binding to lipid droplets (43).

Studies in adipocytes revealed a slightly different regulation. Basal lipolysis was unchanged, whereas stimulated lipolysis was reduced by 20% when the ATGL Ser⁴⁰⁶ site was rendered inactive. Although phosphorylation of ATGL Ser⁴⁰⁶ is important for adipocyte lipolysis, we were unable to determine the mechanism/s mediating this effect. Phosphorylation did not influence cellular localization or reduce TG hydrolase activity (assessed ex vivo). The absence of a putative effect on TG hydrolase activity in MEF adipocytes was surprising given that TG hydrolase activity was reduced in COS cells expressing ATGL S406A. This difference may be explained by the use of MEF adipocyte lysates that contain HSL (and other lipases) that contribute to TG hydrolase activity and may mask a subtle effect of ATGL S406 on total activity. In contrast, COS cells have extremely low HSL expression, which obviates this issue. Schweiger et al. (39) showed that the C-terminal region of ATGL suppressed enzyme activity and proposed that conformational changes in the C-terminal region is required for full enzyme activation and association with CGI-58. The involvement of ATGL Ser⁴⁰⁶ in this putative regulation warrants further examination.

We also examined the physiological significance of phosphorylation by PKA in vivo. ATGL Ser⁴⁰⁶ phosphorylation was low in tissues obtained from resting, ad libitum-fed mice and was increased by moderate intensity exercise and prolonged fasting, two stresses associated with β-adrenergic activation and increased lipolysis. ATGL Ser⁴⁰⁴ phosphorylation was also increased in human adipose tissue in response to β-adrenergic stimulation and correlated tightly with lipolysis rates.

AMPK is a critical energy sensor that regulates several proteins involved in substrate metabolism. The role of AMPK in lipolysis regulation is controversial. Some evidence demonstrates that AMPK partially inhibits β-adrenoreceptor-stimulated lipolysis via phosphorylation of HSL Ser⁵⁶⁵ (33, 44–47), which is thought to prevent phosphorylation of the active Ser⁵⁶³, Ser⁶⁵⁹, and Ser⁶⁶⁰ sites and inhibit translocation of HSL to the lipid droplet. Conversely, other studies have shown that activation of β-adrenergic signaling stimulates AMPK phosphorylation and activity (48, 49) and that AMPK actually enhances lipolysis (29, 49, 50). We identified ATGL as a substrate for AMPK in vitro (Supplemental Fig. 2A), and AMPK-mediated phosphorylation of ATGL (Ser³⁰³) reduces lipolysis in C. elegans during dauer (fasting) (27). Although this critical Ser³⁰³ is not conserved in mammalian ATGL, we predicted that AMPK would negatively regulate ATGL action. While this manuscript was in preparation, Ahmadian et al. (29) reported phosphorylation of ATGL Ser⁴⁰⁶ by AMPK, which stimulated lipolysis. We show that although both PKA and AMPK can phosphorylate ATGL Ser⁴⁰⁶in vitro, only PKA appears to phosphorylate ATGL Ser⁴⁰⁶in vivo. In support of this observation, pharmacological inhibition of PKA prevented the β-adrenergic-stimulated increase in ATGL Ser⁴⁰⁶. Next, AMPK activity was not enhanced in murine adipose tissue during fasting (Fig. 4), yet ATGL Ser⁴⁰⁶ phosphorylation was increased. Also, AMPK activation does not increase lipolysis in HSL-deficient mice that are heavily reliant upon ATGL for lipolysis (Supplemental Fig. 3B) or in human adipose tissue with pharmacological HSL inhibition (S3C). Thus, our results demonstrate that AMPK phosphorylates ATGL Ser⁴⁰⁶in vitro but indicate that AMPK is not an important mediator of ATGL-mediated lipolysis in intact cells or during physiological stress in vivo. Future studies examining lipolysis in mice with adipocyte AMPK deletion will definitively address this issue.

In conclusion, this study is the first direct demonstration of phosphorylation-dependent regulation of ATGL in mammalian adipose tissue by PKA. β-Adrenergic activation results in PKA-mediated phosphorylation of ATGL Ser⁴⁰⁶, which moderately increases ATGL-mediated lipolysis. This observation provides new insights into the control of adipocyte lipolysis, an evolutionarily conserved process essential for survival.

Acknowledgments

We thank the technical support of J. Boon, L. Castelli, Z. P. Chen, C. Garcia-Rudaz, A. Hoy, F. Katsis, F. Ke, B. Kemp, and R. Steele; R. Zechner for the ATGL^−/− and HSL−/− mice; and C. Fledelius for the NN076-0079 compound.

This work was supported by the National Health and Medical Research Council (NHMRC) of Australia (M.J.W.), the Australian Research Council (M.J.W.), a Monash fellowship (M.J.W.), and a Monash University Faculty of Medicine Strategic grants scheme (M.J.W. and P.E.B.). T.T. and M.J.W. are supported by research fellowships from the NHMRC.

Disclosure Summary: The authors have nothing to disclose.

Abbreviations

• Acetyl CoA carboxylase

• AMP-activated protein kinase

• adipose triglyceride lipase

• comparative gene identification

• coenzyme A

• green fluorescent protein

• hormone-sensitive lipase

• murine embryonic fibroblast

• protein kinase A

• trifluoroacetic acid

• triacylglycerol.

References

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Supplementary data