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• W2106761656 abstract "Fas (CD95) is a member of the tumor necrosis factor (TNF) receptor superfamily and plays a crucial role in the induction of apoptosis. However, like TNF, Fas can induce nonapoptotic signaling pathways. We previously demonstrated that mice lacking Fas specifically in adipocytes are partly protected from diet-induced insulin resistance, potentially via decreased delivery of FAs to the liver, as manifested by lower total liver ceramide content. In the present study, we aimed to delineate the signaling pathway involved in Fas-mediated adipocyte lipid mobilization. Treatment of differentiated 3T3-L1 adipocytes with membrane-bound Fas ligand (FasL) significantly increased lipolysis after 12 h without inducing apoptosis. In parallel, Fas activation increased phosphorylation of ERK1/2, and FasL-induced lipolysis was blunted in the presence of the ERK-inhibitor U0126 or in ERK1/2-depleted adipocytes. Furthermore, Fas activation increased phosphorylation of the Ca2+/calmodulin-dependent protein kinases II (CaMKII), and blocking of the CaMKII-pathway (either by the Ca2+ chelator BAPTA or by the CaMKII inhibitor KN62) blunted FasL-induced ERK1/2 phosphorylation and glycerol release. In conclusion, we propose a novel role for CaMKII in promoting lipolysis in adipocytes. Fas (CD95) is a member of the tumor necrosis factor (TNF) receptor superfamily and plays a crucial role in the induction of apoptosis. However, like TNF, Fas can induce nonapoptotic signaling pathways. We previously demonstrated that mice lacking Fas specifically in adipocytes are partly protected from diet-induced insulin resistance, potentially via decreased delivery of FAs to the liver, as manifested by lower total liver ceramide content. In the present study, we aimed to delineate the signaling pathway involved in Fas-mediated adipocyte lipid mobilization. Treatment of differentiated 3T3-L1 adipocytes with membrane-bound Fas ligand (FasL) significantly increased lipolysis after 12 h without inducing apoptosis. In parallel, Fas activation increased phosphorylation of ERK1/2, and FasL-induced lipolysis was blunted in the presence of the ERK-inhibitor U0126 or in ERK1/2-depleted adipocytes. Furthermore, Fas activation increased phosphorylation of the Ca2+/calmodulin-dependent protein kinases II (CaMKII), and blocking of the CaMKII-pathway (either by the Ca2+ chelator BAPTA or by the CaMKII inhibitor KN62) blunted FasL-induced ERK1/2 phosphorylation and glycerol release. In conclusion, we propose a novel role for CaMKII in promoting lipolysis in adipocytes. adipose triglyceride lipase calmodulin calmodulin-dependent protein kinases II endoplasmic reticulum fetal calf serum Fas ligand hormone-sensitive lipase nerve growth factor protein kinase A tumor necrosis factor thiazolidinedione white adipose tissue White adipose tissue (WAT) has major metabolic and endocrine functions mediated by secretion of different adipokines and fat-derived metabolites such as NEFAs. These molecules regulate food intake, energy expenditure, and glucose homeostasis (1Ahima R.S. Flier J.S. Adipose tissue as an endocrine organ.Trends Endocrinol. Metab. 2000; 11: 327-332Abstract Full Text Full Text PDF PubMed Scopus (1195) Google Scholar, 2Frühbeck G. Gomez-Ambrosi J. Muruzabal F.J. Burrell M.A. The adipocyte: a model for integration of endocrine and metabolic signaling in energy metabolism regulation.Am. J. Physiol. Endocrinol. Metab. 2001; 280: E827-E847Crossref PubMed Google Scholar). In obesity, excess WAT accumulation is accompanied by local infiltration of macrophages and other inflammatory cells secreting different cytokines such as IL-1α, IL-1β, IL-6, IL-8 (KC), and MCP-1, which in turn alter the expression and secretion pattern of adipokines and cytokines and stimulate the release of NEFAs by elevating basal lipolysis. All of these changes contribute to the development of detrimental complications of obesity such as insulin resistance and diabetes mellitus (2Frühbeck G. Gomez-Ambrosi J. Muruzabal F.J. Burrell M.A. The adipocyte: a model for integration of endocrine and metabolic signaling in energy metabolism regulation.Am. J. Physiol. Endocrinol. Metab. 2001; 280: E827-E847Crossref PubMed Google Scholar, 3Lazar M.A. How obesity causes diabetes: not a tall tale.Science. 2005; 307: 373-375Crossref PubMed Scopus (446) Google Scholar). Lipolysis is physiologically stimulated by the catecholamine hormones noradrenaline and adrenaline through activation of protein kinase A (PKA). Further downstream, PKA activates hormone-sensitive lipase (HSL), perilipin 1, and adipose triglyceride lipase (ATGL), resulting in triglyceride breakdown (4Girousse A. Langin D. Adipocyte lipases and lipid droplet-associated proteins: insight from transgenic mouse models.Int. J. Obes. (Lond.). 2012; 36: 581-594Crossref PubMed Scopus (69) Google Scholar). Besides this well-established regulation of lipolysis by catecholamines, tumor necrosis factor α (TNFα) is another potent inducer of lipolysis in adipocytes (5Souza S.C. Palmer H.J. Kang Y.H. Yamamoto M.T. Muliro K.V. Paulson K.E. Greenberg A.S. TNF-alpha induction of lipolysis is mediated through activation of the extracellular signal related kinase pathway in 3T3-L1 adipocytes.J. Cell. Biochem. 2003; 89: 1077-1086Crossref PubMed Scopus (152) Google Scholar). TNFα-mediated lipolysis involves activation of the p44/42 MAP kinases (ERK1/2) leading to downregulation of the lipid droplet coating protein perilipin (6Carmen G.Y. Victor S.M. Signalling mechanisms regulating lipolysis.Cell. Signal. 2006; 18: 401-408Crossref PubMed Scopus (347) Google Scholar). Fas (FasR, CD95, apo-1) is a type I transmembrane protein that belongs to the tumor necrosis factor (TNF)/nerve growth factor (NGF) receptor superfamily (7Itoh N. Yonehara S. Ishii A. Yonehara M. Mizushima S. Sameshima M. Hase A. Seto Y. Nagata S. The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis.Cell. 1991; 66: 233-243Abstract Full Text PDF PubMed Scopus (2674) Google Scholar) and is activated by Fas ligand (FasL, CD95L), a type II membrane protein (8Suda T. Takahashi T. Golstein P. Nagata S. Molecular cloning and expression of the Fas ligand, a novel member of the tumor necrosis factor family.Cell. 1993; 75: 1169-1178Abstract Full Text PDF PubMed Scopus (2444) Google Scholar). Fas was first described in 1989 as a surface molecule on lymphocytes that can trigger cell death (9Lambert C. Landau A.M. Desbarats J. Fas-beyond death: a regenerative role for Fas in the nervous system.Apoptosis. 2003; 8: 551-562Crossref PubMed Scopus (74) Google Scholar). In the adult mouse, Fas and FasL are expressed in several tissues including WAT (10French L.E. Hahne M. Viard I. Radlgruber G. Zanone R. Becker K. Muller C. Tschopp J. Fas and Fas ligand in embryos and adult mice: ligand expression in several immune-privileged tissues and coexpression in adult tissues characterized by apoptotic cell turnover.J. Cell Biol. 1996; 133: 335-343Crossref PubMed Scopus (443) Google Scholar, 11Wueest S. Rapold R.A. Schumann D.M. Rytka J.M. Schildknecht A. Nov O. Chervonsky A.V. Rudich A. Schoenle E.J. Donath M.Y. et al.Deletion of Fas in adipocytes relieves adipose tissue inflammation and hepatic manifestations of obesity in mice.J. Clin. Invest. 2010; 120: 191-202Crossref PubMed Scopus (118) Google Scholar). Upon binding of FasL, preformed trimeric Fas complexes undergo a conformational change that results in the formation of a death-inducing signaling complex and activation of downstream pathways leading to apoptosis (9Lambert C. Landau A.M. Desbarats J. Fas-beyond death: a regenerative role for Fas in the nervous system.Apoptosis. 2003; 8: 551-562Crossref PubMed Scopus (74) Google Scholar, 12Peter M.E. Budd R.C. Desbarats J. Hedrick S.M. Hueber A.O. Newell M.K. Owen L.B. Pope R.M. Tschopp J. Wajant H. et al.The CD95 receptor: apoptosis revisited.Cell. 2007; 129: 447-450Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). However, in addition to this well-established role of Fas in apoptosis, Fas activation contributes to nonapoptotic signaling pathways, including cell proliferation (9Lambert C. Landau A.M. Desbarats J. Fas-beyond death: a regenerative role for Fas in the nervous system.Apoptosis. 2003; 8: 551-562Crossref PubMed Scopus (74) Google Scholar, 13Wajant H. Pfizenmaier K. Scheurich P. Non-apoptotic Fas signaling.Cytokine Growth Factor Rev. 2003; 14: 53-66Crossref PubMed Scopus (171) Google Scholar) and the induction of inflammatory responses in different cell types (14Abreu-Martin M.T. Vidrich A. Lynch D.H. Targan S.R. Divergent induction of apoptosis and IL-8 secretion in HT-29 cells in response to TNF-alpha and ligation of Fas antigen.J. Immunol. 1995; 155: 4147-4154PubMed Google Scholar–18Sekine C. Yagita H. Kobata T. Hasunuma T. Nishioka K. Okumura K. Fas-mediated stimulation induces IL-8 secretion by rheumatoid arthritis synoviocytes independently of CPP32-mediated apoptosis.Biochem. Biophys. Res. Commun. 1996; 228: 14-20Crossref PubMed Scopus (62) Google Scholar). Moreover, we have recently reported that Fas is increasingly expressed in WAT of obese subjects and that Fas-deficient and adipocyte-specific Fas knockout mice are partly protected from high-fat diet-induced insulin resistance (11Wueest S. Rapold R.A. Schumann D.M. Rytka J.M. Schildknecht A. Nov O. Chervonsky A.V. Rudich A. Schoenle E.J. Donath M.Y. et al.Deletion of Fas in adipocytes relieves adipose tissue inflammation and hepatic manifestations of obesity in mice.J. Clin. Invest. 2010; 120: 191-202Crossref PubMed Scopus (118) Google Scholar, 19Wueest S. Rapold R.A. Schoenle E.J. Konrad D. Fas activation in adipocytes impairs insulin-stimulated glucose uptake by reducing Akt.FEBS Lett. 2010; 584: 4187-4192Crossref PubMed Scopus (12) Google Scholar), implicating a role for Fas in the pathogenesis of obesity-associated insulin resistance. Although the underlying mechanism remains incompletely understood, livers of adipocyte-specific Fas knockout mice had lower levels of total ceramides, which are potentially metabolized from NEFA delivered to the liver from adipose tissue. Thus, in the present study, we hypothesized that in adipocytes, Fas activation can, independently of its pro-apoptotic effects, directly induce the hydrolysis of triglycerides (i.e., increase basal lipolysis), and aimed to investigate the intracellular signaling pathway(s) involved. 3T3-L1 adipocytes were cultured in DMEM (Invitrogen; Basel, Switzerland) containing 25 mM glucose (high glucose), supplemented with 10% fetal calf serum (FCS; Socochim SA, Lausanne) and antibiotics (Invitrogen). Forty-eight hours after reaching confluence (day 0, D0), cells were treated with a mixture of 500 μM methylisobutylxanthine, 1 μM dexamethasone, 1.7 μM insulin (all from Sigma; Buchs, Switzerland), and 1 μM rosiglitazone (Alexis Biochemicals) to induce differentiation. Two days later (D2) the medium was changed to high-glucose culture medium containing insulin (0.5 μM). Another 2 days later (D4), the medium was replaced by culture medium without insulin. The culture medium was replaced every other day and changed to culture medium containing 5.5 mM glucose (low glucose) after 4 days (D8). Cells were kept at least 2 days on low glucose before experiments were performed. Membrane-bound Fas ligand (Upstate; Lake Placid, NY) was added to low-glucose serum-free medium as indicated. For pretreatment experiments with rosiglitazone (Enzo Life Science; Lausen, Switzerland), mature adipocytes were incubated for 48 h with 5 μM of the compound in low-glucose medium. Thereafter, FasL, together with rosiglitazone, was applied for another 6 h or 12 h. For siRNA-mediated ERK1/2 knock down, mature 3T3-L1 adipocyte cells were treated with siRNA (target sequences for ERK1/2: 5′ ACAAGCGCATCACAGTAGA 3′, 5′ GAACCCTAAGAGAGATAAA 3′ scrambled sequences for ERK1/2: 5′ GCAACCGAACGGAAACATT 3′, 5′ GAACAAAGTAACGGTAACA 3′) in a transfection mixture containing Lipofectamin 2000 (Invitrogen) in culture medium (low-glucose, without FCS, without penicillin/streptomycin) according to the manufacturer's instructions. Adipocytes were lysed in ice-cold lysis buffer containing 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 1 mM EGTA, 1% NP-40, 0.25% sodium deoxycholate, 1 mM sodium vanadate, 1 mM NaF, 10 mM sodium β-glycerophosphate, 100 nM okadaic acid, 0.2 mM PMSF, and a 1:1,000 dilution of protease inhibitor cocktail (Sigma). Protein concentration was determined using a bicinchoninic acid assay (Pierce; Rockford, IL). Equal amounts of protein were resolved by lithium dodecyl sulfate PAGE (4–12% gel; NuPAGE, Invitrogen) and electro-transferred onto nitrocellulose membranes (0.2 μm; BioRad, Reinach, Switzerland). Protein content on membranes was checked by Ponceau S staining. Blots were blocked in tris-buffered saline containing 0.1% Tween (TBS-T) supplemented with 5% nonfat dry milk. Primary antibody was applied in the same buffer in a dilution of 1:1,000, secondary antibody in a dilution of 1:5,000. Primary antibodies were purchased either from Cell Signaling Technology [phospho-p44/42 and total MAPKs, phospho-(Ser/Thr) PKA substrate, phospho-Ser 563 and total HSL, ATGL], MBL International (perilipin A), Millipore [Actin, Fas (CD95)], Vala Sciences (phospho-Perilipin Ser 522) or Santa Cruz Biotechnology [PPARg2, C/EBPa, pCaMKIIα (Thr 286)]. Signal was generated based on chemiluminescence and detected with the Fuji LAS-3000 image reader. Cells were incubated in DMEM without FCS and treated with 2 ng/ml FasL or 1 µM isoproterenol in the presence or absence of 50 μM of the mitogen-activated protein kinase kinase inhibitor U0126 (Sigma), of 50 μM of the intracellular calcium chelator BAPTA/AM (Calbiochem), or of 50 μM of CaMKII inhibitor KN62 (Sigma), as indicated. Inhibitors or BAPTA/AM were added to the cells at the same time as FasL. Thereafter, cells were washed in PBS, and NEFA and glycerol were collected in Krebs-Ringer-HEPES buffer supplemented with 0.1% FA-free BSA for 1 h. NEFAs in the supernatant were measured with the NEFA kit from Wako (Neuss, Germany), and glycerol with the free glycerol reagent from Sigma. Fractional re-esterification was calculated as previously described (20Rosenstock M. Greenberg A.S. Rudich A. Distinct long-term regulation of glycerol and non-esterified fatty acid release by insulin and TNF-alpha in 3T3-L1 adipocytes.Diabetologia. 2001; 44: 55-62Crossref PubMed Scopus (35) Google Scholar). Data are presented as means ± SEM and were analyzed by a one-sample t-test or ANOVA with a Newman-Keuls multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001. The Fas receptor (CD95) is expressed in 3T3-L1 preadipocytes and decreases during adipocyte differentiation, but is still clearly expressed in mature adipocytes (Fig. 1). Expression levels of the adipocyte-specific proteins PPARγ2, C/EBPα, and perilipin reflect the respective stages of differentiation. Treatment of differentiated 3T3-L1 adipocytes with 2 ng/ml FasL for 12 h significantly increased lipolysis (Fig. 2), consistent with our earlier observation (11Wueest S. Rapold R.A. Schumann D.M. Rytka J.M. Schildknecht A. Nov O. Chervonsky A.V. Rudich A. Schoenle E.J. Donath M.Y. et al.Deletion of Fas in adipocytes relieves adipose tissue inflammation and hepatic manifestations of obesity in mice.J. Clin. Invest. 2010; 120: 191-202Crossref PubMed Scopus (118) Google Scholar), and without affecting their viability as assessed by Terminal deoxynucleotidyltransferase dUTP nick end labeling assay (see supplementary Fig. I) and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide determination (19Wueest S. Rapold R.A. Schoenle E.J. Konrad D. Fas activation in adipocytes impairs insulin-stimulated glucose uptake by reducing Akt.FEBS Lett. 2010; 584: 4187-4192Crossref PubMed Scopus (12) Google Scholar). Fractional reesterification did not decrease upon FasL incubation (basal: 68.0 ± 2.6%; 6 h FasL: 64.0 ± 6.0%; 12 h FasL: 63.6 ± 5.5%; P = 0.8). Such data suggest that Fas activation increases lipolysis by increased triglyceride hydrolysis rather than by decreased reesterification. Shorter incubation periods with FasL (≤6 h) and lower concentrations of FasL did not increase lipolysis to a significant degree (see supplementary Fig. II). Moreover, blunted Fas-stimulated lipolysis in Fas-depleted 3T3-L1 adipocytes suggests that membrane-bound FasL signals via the Fas receptor (see supplementary Fig. III).Fig. 2Fas stimulation induces lipolysis in 3T3-L1 adipocytes. Fully differentiated 3T3-L1 adipocytes were treated with FasL (2 ng/ml) for the indicated time periods. NEFA (A) and glycerol (B) concentrations were determined in the supernatant collected for 1 h as described in Materials and Methods. Results represent the means ± SEM of six independent experiments. *P < 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Beta adrenergic receptor agonists such as catecholamines stimulate lipolysis via adenylate cyclase-dependent activation of PKA and consecutive activation of HSL, perilipin 1, and ATGL. Inhibiting such effect, insulin activates phosphodiesterase 3, which converts cAMP to 5′-AMP, thereby diminishing cAMP-mediated PKA activity, which results in inhibition of lipolysis. To examine whether Fas-mediated lipolysis comprises activation of PKA, phosphorylation of PKA substrates was determined in 3T3-L1 adipocytes. As expected, the β1,2 receptor agonist isoproterenol increased phosphorylation of PKA substrates significantly. In contrast, treatment with FasL had no effect on the abundance of phosphorylated PKA substrates (Fig. 3A). However, even though not detected by the PKA substrate antibody, incubation of 3T3-L1 adipocytes with FasL for 6 h and 12 h significantly increased phosphorylation of HSL at Ser563 (Fig. 3B), whereas it had no effect on total HSL protein levels (see supplementary Fig. IV) and phosphorylation of perilipin (Fig. 3B). In addition, ATGL protein levels were slightly but not significantly upregulated upon Fas incubation (Fig. 3C). Thus, Fas-mediated lipolysis may depend on activation of HSL and/or ATGL. An alternative signaling pathway to activate lipolysis in adipocytes involves the p44/42 MAP kinases (ERK1/2), as was shown for TNFα (5Souza S.C. Palmer H.J. Kang Y.H. Yamamoto M.T. Muliro K.V. Paulson K.E. Greenberg A.S. TNF-alpha induction of lipolysis is mediated through activation of the extracellular signal related kinase pathway in 3T3-L1 adipocytes.J. Cell. Biochem. 2003; 89: 1077-1086Crossref PubMed Scopus (152) Google Scholar). Because Fas belongs to the TNF receptor superfamily, we postulated that FasL-induced lipolysis is mediated via ERK1/2 activation. Incubation of mature 3T3-L1 adipocytes with 2 ng/ml FasL increased phosphorylation of ERK1/2 significantly after 6 h and 12 h, whereas total protein concentration of ERK1/2 was not affected (Fig. 4A). To exclude the possibility that the effects of FasL treatment were due to a FasL-mediated increase in TNFα secretion and, thus, to a paracrine regulatory loop mediated by this cytokine, TNFα concentration was determined in the supernatant. As depicted (see supplementary Fig. V), incubation with 2 ng/ml FasL for 12 h did not lead to increased TNFα secretion to the medium. We therefore concluded that Fas-mediated ERK1/2 activation was independent of TNFα. To assess whether Fas-induced lipolysis is dependent on ERK1/2 activation, we incubated 3T3-L1 cells in the presence or absence of the MEK1/2 inhibitor U0126. Inhibition of the MAP kinase pathway with U0126 (50 μM) completely blocked Fas-induced ERK1/2 phosphorylation and lipolysis (Fig. 4B). To further strengthen a role of the p44/42 MAP kinase pathway in Fas-mediated lipolysis, 3T3-L1 adipocytes were treated with targeted or scrambled siRNA against ERK1/2, respectively. Compared with scrambled siRNA control, siRNA targeted toward ERK1/2 decreased ERK1/2 protein by about 50%, an effect that was associated with a significant blunting of FasL-stimulated lipolysis (Fig. 4C). Thus, Fas-stimulated lipolysis is dependent on ERK1/2 activation. To further corroborate a Fas-ERK1/2-lipolysis pathway in adipocytes, we tested whether PPARγ agonists such as thiazolidinediones (TZDs), which were demonstrated to inhibit whole-body lipolysis in patients with type 2 diabetes (21Gastaldelli A. Casolaro A. Ciociaro D. Frascerra S. Nannipieri M. Buzzigoli E. Ferrannini E. Decreased whole body lipolysis as a mechanism of the lipid-lowering effect of pioglitazone in type 2 diabetic patients.Am. J. Physiol. Endocrinol. Metab. 2009; 297: E225-E230Crossref PubMed Scopus (26) Google Scholar), could inhibit Fas-induced lipolysis and if so, whether activation of ERK1/2 was also diminished. As shown in Fig. 4D, FasL-stimulated ERK1/2 phosphorylation was reduced in the presence of rosiglitazone without affecting total ERK1/2 protein content. Correspondingly, rosiglitazone also reduced FasL-mediated lipolysis (Fig. 4D). CaMKII was previously shown to mediate magnolol-triggered lipolysis in sterol ester-loaded 3T3-L1 preadipocytes in an ERK1/2-dependent fashion (22Huang S.H. Shen W.J. Yeo H.L. Wang S.M. Signaling pathway of magnolol-stimulated lipolysis in sterol ester-loaded 3T3-L1 preadipocyes.J. Cell. Biochem. 2004; 91: 1021-1029Crossref PubMed Scopus (21) Google Scholar). We therefore postulated that FasL-induced lipolysis may be dependent on intracellular changes in calcium levels, because it is well accepted that Fas activation can increase intracellular calcium levels (23Vacher P. Khadra N. Vacher A.M. Charles E. Bresson-Bepoldin L. Legembre P. Does calcium contribute to the CD95 signaling pathway?.Anticancer Drugs. 2011; 22: 481-487Crossref PubMed Scopus (8) Google Scholar). Indeed, preincubation of 3T3-L1 adipocytes with the intracellular calcium chelator BAPTA/AM prevented both FasL-induced ERK activation and lipolysis (Fig. 5A), suggesting that both effects of Fas activation are dependent on an intracellular calcium rise. In response to the latter, the developing calcium/calmodulin complex may activate CaMKII, leading to intramolecular autophosphorylation at several sites, including Thr286, Thr305, and Thr306 (24Wayman G.A. Tokumitsu H. Davare M.A. Soderling T.R. Analysis of CaM-kinase signaling in cells.Cell Calcium. 2011; 50: 1-8Crossref PubMed Scopus (94) Google Scholar). As depicted in Fig. 5B, incubation of mature 3T3-L1 adipocytes with 2 ng/ml FasL increased phosphorylation of CaMKII at Thr286 significantly after 6 h and 12 h. Moreover, pretreatment with the CaMKII inhibitor KN62 reduced FasL-mediated ERK1/2 activation as well as lipolysis (Fig. 5C). These data strongly suggest that Ca2+-mediated activation of CaMKII is a proximal signaling response to Fas activation, which is propagated further downstream via ERK1/2 to induce basal lipolysis. In obesity, higher basal lipolysis rate resulting in increased release of NEFAs into the circulation contributes to the development of hepatic and total body insulin resistance (25Delarue J. Magnan C. Free fatty acids and insulin resistance.Curr. Opin. Clin. Nutr. Metab. Care. 2007; 10: 142-148Crossref PubMed Scopus (341) Google Scholar). Several factors contribute to such increase in lipolysis. First, obesity is associated with a persistent low-grade inflammation of adipose tissue, as manifested by an increased production and secretion of proinflammatory cytokines such as TNFα and IL-6 (26Fantuzzi G. Adipose tissue, adipokines, and inflammation.J. Allergy Clin. Immunol. 2005; 115: 911-919Abstract Full Text Full Text PDF PubMed Scopus (1946) Google Scholar). The latter in turn can directly stimulate adipocyte lipolysis, even as isolated factors (5Souza S.C. Palmer H.J. Kang Y.H. Yamamoto M.T. Muliro K.V. Paulson K.E. Greenberg A.S. TNF-alpha induction of lipolysis is mediated through activation of the extracellular signal related kinase pathway in 3T3-L1 adipocytes.J. Cell. Biochem. 2003; 89: 1077-1086Crossref PubMed Scopus (152) Google Scholar, 27Petersen E.W. Carey A.L. Sacchetti M. Steinberg G.R. Macaulay S.L. Febbraio M.A. Pedersen B.K. Acute IL-6 treatment increases fatty acid turnover in elderly humans in vivo and in tissue culture in vitro.Am. J. Physiol. Endocrinol. Metab. 2005; 288: E155-E162Crossref PubMed Scopus (243) Google Scholar, 28Zhang H.H. Halbleib M. Ahmad F. Manganiello V.C. Greenberg A.S. Tumor necrosis factor-alpha stimulates lipolysis in differentiated human adipocytes through activation of extracellular signal-related kinase and elevation of intracellular cAMP.Diabetes. 2002; 51: 2929-2935Crossref PubMed Scopus (327) Google Scholar). Second, hypertrophic adipocytes are characterized by an elevated rate of basal lipolysis (29Wueest S. Rapold R.A. Rytka J.M. Schoenle E.J. Konrad D. Basal lipolysis, not the degree of insulin resistance, differentiates large from small isolated adipocytes in high-fat fed mice.Diabetologia. 2009; 52: 541-546Crossref PubMed Scopus (62) Google Scholar), which might be at least partly mediated by self-production of inflammatory cytokines acting in an autocrine manner, possibly as a self-protective cellular mechanism against excessive cellular over-growth. Third, because insulin is the major anti-lipolytic hormone, insulin resistance at the adipocyte level results in increased lipolysis, creating a vicious cycle between hypertrophy, inflammation, lipolysis, and insulin resistance. Fas may be a key component of such dysregulation: Fas is activated by FasL, which can be produced by inflammatory cells infiltrating adipose tissue in obesity. Moreover, expression of Fas is increased in adipose tissue of obese humans and in isolated adipocytes of obese and diabetic mice, and intriguingly, its protein expression correlates with adipocyte size and is therefore increased in hypertrophic adipocytes (11Wueest S. Rapold R.A. Schumann D.M. Rytka J.M. Schildknecht A. Nov O. Chervonsky A.V. Rudich A. Schoenle E.J. Donath M.Y. et al.Deletion of Fas in adipocytes relieves adipose tissue inflammation and hepatic manifestations of obesity in mice.J. Clin. Invest. 2010; 120: 191-202Crossref PubMed Scopus (118) Google Scholar). In agreement with such notion, we show herein that chronic stimulation of the death receptor Fas induced lipolysis in 3T3-L1 adipocytes. Importantly, this effect occurred under conditions that did not induce apoptosis (11Wueest S. Rapold R.A. Schumann D.M. Rytka J.M. Schildknecht A. Nov O. Chervonsky A.V. Rudich A. Schoenle E.J. Donath M.Y. et al.Deletion of Fas in adipocytes relieves adipose tissue inflammation and hepatic manifestations of obesity in mice.J. Clin. Invest. 2010; 120: 191-202Crossref PubMed Scopus (118) Google Scholar). Like TNFα, FasL stimulates lipolysis through activation of the ERK1/2 MAP kinases, because Fas activation led to increased phosphorylation of ERK1/2 and pretreatment with the MEK1/2 inhibitor U0126 or siRNA-mediated downregulation of ERK1/2 blocked Fas-induced lipolysis. In the case of TNFα, it was proposed that ERK1/2-dependent downregulation of the lipid droplet coating protein perilipin is responsible for the increase in lipolysis (5Souza S.C. Palmer H.J. Kang Y.H. Yamamoto M.T. Muliro K.V. Paulson K.E. Greenberg A.S. TNF-alpha induction of lipolysis is mediated through activation of the extracellular signal related kinase pathway in 3T3-L1 adipocytes.J. Cell. Biochem. 2003; 89: 1077-1086Crossref PubMed Scopus (152) Google Scholar). We also observed a downregulation of perilipin in cells treated with FasL for 12 h. However, in contrast to TNFα, such effect was not mediated by ERK1/2, because FasL-induced decrease in perilipin expression was not prevented by ERK inhibition (see supplementary Fig. VI). Moreover, 6 h of FasL incubation did not decrease perilipin protein content (data not shown) but increased FFA and glycerol release. All these results suggest that Fas-mediated lipolysis is independent of a decrease in perilipin expression. Herein, we present evidence for calcium-triggered activation of ERK in Fas activation-mediated lipolysis. Fas activation in cells was previously shown to raise intracellular free Ca2+ levels (23Vacher P. Khadra N. Vacher A.M. Charles E. Bresson-Bepoldin L. Legembre P. Does calcium contribute to the CD95 signaling pathway?.Anticancer Drugs. 2011; 22: 481-487Crossref PubMed Scopus (8) Google Scholar). Similarly, increased intracellular Ca2+ levels induced by endoplasmic reticulum (ER) stress were shown to induce lipolysis in adipocytes ERK dependently (30Deng J. Liu S. Zou L. Xu C. Geng B. Xu G. Lipolysis response to endoplasmic reticulum stress in adipose cells.J. Biol. Chem. 2012; 287: 6240-6249Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Hence, Fas-induced ERK activation may be mediated by ER stress-triggered Ca2+ release. However, as shown in supplementary Fig. VII, FasL stimulation of 3T3-L1 adipocytes did not provoke ER stress. Moreover, the extracellular Ca2+-chelator EDTA blunted Fas-induced ERK activation and lipolysis similar to the intracellular chelator BAPTA (see supplementary Fig. VIII). Such data suggest that extracellular Ca2+ influx rather than Ca2+ release from ER is involved in Fas-induced lipolysis. Increased intracellular Ca2+ is bound by the calcium-binding protein calmodulin (CaM) forming a complex. The latter then binds to and thereby activates CaMKII. Activation of CaMKII by the Ca2+/CaM complex allows intramolecular autophosphorylation at several sites, including Thr286. This generates calcium-independent activity that persists after dissociation of calcium/CaM, allowing transient calcium elevation to promote prolonged kinase activation (24Wayman G.A. Tokumitsu H. Davare M.A. Soderling T.R. Analysis of CaM-kinase signaling in cells.Cell Calcium. 2011; 50: 1-8Crossref PubMed Scopus (94) Google Scholar). We found that FasL treatment increased phosphorylation at Thr286 of CaMKII. Moreover, Fas activation-induced lipolysis was prevented in the presence of the CaMKII inhibitor as well as of the Ca2+ chelator, BAPTA. Thus, we postulate that Fas activation in adipocytes increases intracellular Ca2+ levels, leading to activation of CaMKII, which, in turn, activates ERK1/2 and, thus, lipolysis. Accordingly, it was previously reported that trans-10, cis-12-conjugated linoleic acid-induced ERK1/2 activation in adipocytes is dependent on a rise in intracellular free Ca2+ levels and consecutive activation of CaMKII (31Kennedy A. Martinez K. Chung S. LaPoint K. Hopkins R. Schmidt S.F. Andersen K. Mandrup S. McIntosh M. Inflammation and insulin resistance induced by trans-10, cis-12 conjugated linoleic acid depend on intracellular calcium levels in primary cultures of human adipocytes.J. Lipid Res. 2010; 51: 1906-1917Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Unfortunately, lipolysis was not addressed in this study. The data presented herein may suggest a novel pathway for lipolysis in adipocytes via CaMKII-dependent activation of ERK. Interestingly, such a pathway may be very ancient and evolutionarily preserved, because CaMKII-mediated release of NEFAs was recently demonstrated to play a role in pheromone biosynthesis in insects (32Ohnishi A. Hull J.J. Kaji M. Hashimoto K. Lee J.M. Tsuneizumi K. Suzuki T. Dohmae N. Matsumoto S. Hormone signaling linked to silkmoth sex pheromone biosynthesis involves Ca2+/calmodulin-dependent protein kinase II-mediated phosphorylation of the insect PAT family protein Bombyx mori lipid storage droplet protein-1 (BmLsd1).J. Biol. Chem. 2011; 286: 24101-24112Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Interestingly, the TZD rosiglitazone reduced both Fas-mediated ERK1/2 phosphorylation and lipolysis. PPARγ agonists have profound effects on adipocyte metabolism and were shown to improve insulin sensitivity in patients with type 2 diabetes. Moreover, in a recent paper, treatment with another TZD, pioglitazone, was able to reduce whole-body lipolysis in type 2 diabetic patients (21Gastaldelli A. Casolaro A. Ciociaro D. Frascerra S. Nannipieri M. Buzzigoli E. Ferrannini E. Decreased whole body lipolysis as a mechanism of the lipid-lowering effect of pioglitazone in type 2 diabetic patients.Am. J. Physiol. Endocrinol. Metab. 2009; 297: E225-E230Crossref PubMed Scopus (26) Google Scholar). We previously described a potential role for Fas activation in obesity-associated insulin resistance (11Wueest S. Rapold R.A. Schumann D.M. Rytka J.M. Schildknecht A. Nov O. Chervonsky A.V. Rudich A. Schoenle E.J. Donath M.Y. et al.Deletion of Fas in adipocytes relieves adipose tissue inflammation and hepatic manifestations of obesity in mice.J. Clin. Invest. 2010; 120: 191-202Crossref PubMed Scopus (118) Google Scholar) and show herein that Fas activation leads to increased lipolysis. Thus, our experiments may point to a role of TZDs in counteracting Fas-induced metabolic changes in adipocytes and further underscore the importance of ERK-activation for FasL-induced lipolysis. Similarly, TZDs were previously found to antagonize the effects of TNFα on adipocytes (33Souza S.C. Yamamoto M.T. Franciosa M.D. Lien P. Greenberg A.S. BRL 49653 blocks the lipolytic actions of tumor necrosis factor-alpha: a potential new insulin-sensitizing mechanism for thiazolidinediones.Diabetes. 1998; 47: 691-695Crossref PubMed Scopus (153) Google Scholar). We recently showed that treatment of 3T3-L1 adipocytes with 2 ng/ml FasL for 12 h reduced protein levels of Akt/PKB (protein kinase B) (19Wueest S. Rapold R.A. Schoenle E.J. Konrad D. Fas activation in adipocytes impairs insulin-stimulated glucose uptake by reducing Akt.FEBS Lett. 2010; 584: 4187-4192Crossref PubMed Scopus (12) Google Scholar). Moreover, a recent publication reported that deletion of rictor, an essential component of the Akt kinase mammalian target of rapamycin complex 2 (mTORC2), increases basal lipolysis as well as phosphorylation of HSL at Ser563 in adipocytes (34Kumar A. Lawrence Jr, J.C. Jung D.Y. Ko H.J. Keller S.R. Kim J.K. Magnuson M.A. Harris T.E. Fat cell-specific ablation of rictor in mice impairs insulin-regulated fat cell and whole-body glucose and lipid metabolism.Diabetes. 2010; 59: 1397-1406Crossref PubMed Scopus (202) Google Scholar). Hence, it is conceivable that Fas-mediated reduction in Akt protein levels contributed to FasL-induced lipolysis, potentially via increased phosphorylation of HSL at serine residue 563. Moreover, ERK1/2-mediated phosphorylation of HSL at Ser600 (35Greenberg A.S. Shen W.J. Muliro K. Patel S. Souza S.C. Roth R.A. Kraemer F.B. Stimulation of lipolysis and hormone-sensitive lipase via the extracellular signal-regulated kinase pathway.J. Biol. Chem. 2001; 276: 45456-45461Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar) might contribute to Fas-induced lipolysis. However, such a corresponding anti-phospho-HSLSer600 antibody is not commercially available and, thus, not available to us for evaluating a potential role of HSLSer600 phosphorylation in this study. Besides activation of HSL, we cannot exclude the involvement of ATGL to Fas activation-induced lipolysis, because ATGL protein levels were slight, albeit not significantly increased, after FasL treatment. In summary, activation of the Fas receptor alters the metabolism of adipocytes and leads to ERK1/2-mediated lipolysis, which may be triggered by a Fas-induced increase in intracellular calcium levels and hence, autophosphorylation of the Ca2+/calmodulin-dependent protein kinases II. Thus, our findings suggest an important role of the Fas receptor in the development of adipose tissue dysfunction in the context of obesity. The authors thank Dr. Assaf Rudich from the Department of Clinical Biochemistry and the S. Daniel Centre for Health and Nutrition, Ben-Gurion University, Beer-Sheva, Israel, for helpful discussion." @default.
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