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• W2080981566 abstract "AMP-activated protein kinase (AMPK) is activated in adipocytes during exercise and other states in which lipolysis is stimulated. However, the mechanism(s) responsible for this effect and its physiological relevance are unclear. To examine these questions, 3T3-L1 adipocytes were treated with cAMP-inducing agents (isoproterenol, forskolin, and isobutylmethylxanthine), which stimulate lipolysis and activate AMPK. When lipolysis was partially inhibited with the general lipase inhibitor orlistat, AMPK activation by these agents was also partially reduced, but the increases in cAMP levels and cAMP-dependent protein kinase (PKA) activity were unaffected. Likewise, small hairpin RNA-mediated silencing of adipose tissue triglyceride lipase inhibited both forskolin-stimulated lipolysis and AMPK activation but not that of PKA. Forskolin treatment increased the AMP:ATP ratio, and this too was reduced by orlistat. When acyl-CoA synthetase, which catalyzes the conversion of fatty acids to fatty acyl-CoA, was inhibited with triacsin C, the increases in both AMPK activity and AMP:ATP ratio were blunted. Isoproterenol-stimulated lipolysis was accompanied by an increase in oxidative stress, an effect that was quintupled in cells incubated with the AMPK inhibitor compound C. The isoproterenol-induced increase in the AMP:ATP ratio was also much greater in these cells. In conclusion, the results indicate that activation of AMPK in adipocytes by cAMP-inducing agents is a consequence of lipolysis and not of PKA activation. They suggest that AMPK activation in this setting is caused by an increase in the AMP:ATP ratio that appears to be due, at least in part, to the acylation of fatty acids. Finally, this AMPK activation appears to restrain the energy depletion and oxidative stress caused by lipolysis. AMP-activated protein kinase (AMPK) is activated in adipocytes during exercise and other states in which lipolysis is stimulated. However, the mechanism(s) responsible for this effect and its physiological relevance are unclear. To examine these questions, 3T3-L1 adipocytes were treated with cAMP-inducing agents (isoproterenol, forskolin, and isobutylmethylxanthine), which stimulate lipolysis and activate AMPK. When lipolysis was partially inhibited with the general lipase inhibitor orlistat, AMPK activation by these agents was also partially reduced, but the increases in cAMP levels and cAMP-dependent protein kinase (PKA) activity were unaffected. Likewise, small hairpin RNA-mediated silencing of adipose tissue triglyceride lipase inhibited both forskolin-stimulated lipolysis and AMPK activation but not that of PKA. Forskolin treatment increased the AMP:ATP ratio, and this too was reduced by orlistat. When acyl-CoA synthetase, which catalyzes the conversion of fatty acids to fatty acyl-CoA, was inhibited with triacsin C, the increases in both AMPK activity and AMP:ATP ratio were blunted. Isoproterenol-stimulated lipolysis was accompanied by an increase in oxidative stress, an effect that was quintupled in cells incubated with the AMPK inhibitor compound C. The isoproterenol-induced increase in the AMP:ATP ratio was also much greater in these cells. In conclusion, the results indicate that activation of AMPK in adipocytes by cAMP-inducing agents is a consequence of lipolysis and not of PKA activation. They suggest that AMPK activation in this setting is caused by an increase in the AMP:ATP ratio that appears to be due, at least in part, to the acylation of fatty acids. Finally, this AMPK activation appears to restrain the energy depletion and oxidative stress caused by lipolysis. AMP-activated protein kinase (AMPK) 2Theabbreviationsusedare:AMPK,AMP-activatedproteinkinase; PKA, cAMP-dependent protein kinase; shRNA, small hairpin RNA; DMEM, Dulbecco's modified Eagle's medium; IBMX, isobutylmethylxanthine; BSA, bovine serum albumin; FFA, free fatty acid; GFP, green fluorescent protein; MEF, mouse embryonic fibroblast; WT, wild type; ACS, acyl-CoA synthetase; DCF, 5-(6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate; ROS, reactive oxygen species; CREB, cAMP-response element-binding protein; P-CREB, phospho-CREB; CC, compound C; ATGL, adipose-tissue triglyceride lipase; Peri A, perilipin A; ACC, acetyl-CoA carboxylase. 2Theabbreviationsusedare:AMPK,AMP-activatedproteinkinase; PKA, cAMP-dependent protein kinase; shRNA, small hairpin RNA; DMEM, Dulbecco's modified Eagle's medium; IBMX, isobutylmethylxanthine; BSA, bovine serum albumin; FFA, free fatty acid; GFP, green fluorescent protein; MEF, mouse embryonic fibroblast; WT, wild type; ACS, acyl-CoA synthetase; DCF, 5-(6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate; ROS, reactive oxygen species; CREB, cAMP-response element-binding protein; P-CREB, phospho-CREB; CC, compound C; ATGL, adipose-tissue triglyceride lipase; Peri A, perilipin A; ACC, acetyl-CoA carboxylase. is a sensor of cellular energy state that responds to metabolic stresses and other regulatory signals. The mechanism of activation of AMPK is a complex phenomenon and is still not fully understood, although it is recognized that it involves phosphorylation of the critical Thr-172 residue of its α catalytic subunit by upstream kinases such as LKB1, Ca2+-calmodulin-dependent protein kinase kinase, and possibly Tak1, a member of the mitogen-activated protein kinase kinase kinase family (1Hawley S.A. Boudeau J. Reid J.L. Mustard K.J. Udd L. Makela T.P. Alessi D.R. Hardie D.G. J. Biol. 2003; 2: 28Crossref PubMed Google Scholar, 2Woods A. Johnstone S.R. Dickerson K. Leiper F.C. Fryer L.G. Neumann D. Schlattner U. Wallimann T. Carlson M. Carling D. Curr. Biol. 2003; 13: 2004-2008Abstract Full Text Full Text PDF PubMed Scopus (1332) Google Scholar, 3Woods A. Dickerson K. Heath R. Hong S.P. Momcilovic M. Johnstone S.R. Carlson M. Carling D. Cell Metab. 2005; 2: 21-33Abstract Full Text Full Text PDF PubMed Scopus (1066) Google Scholar, 4Hawley S.A. Pan D.A. Mustard K.J. Ross L. Bain J. Edelman A.M. Frenguelli B.G. Hardie D.G. Cell Metab. 2005; 2: 9-19Abstract Full Text Full Text PDF PubMed Scopus (1272) Google Scholar, 5Momcilovic M. Hong S.-P. Carlson M. J. Biol. Chem. 2006; 281: 25336-25343Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). AMPK is also regulated by AMP allosterically and the current view is that this both increases AMPK activity directly and makes it a poorer substrate for phosphatases (6Hardie D.G. Annu. Rev. Pharmacol. Toxicol. 2007; 47: 185-210Crossref PubMed Scopus (355) Google Scholar). The role and regulation of AMPK in muscle, liver, and various cultured cells have been extensively studied. It is now well established that energy depletion because of starvation, hypoxia, and exercise increases the intracellular AMP:ATP ratio and secondarily AMPK activity (7Hardie D.G. Hawley S.A. Scott J.W. J. Physiol. (Lond.). 2006; 574: 7-15Crossref Scopus (649) Google Scholar). Upon its activation, a major role of AMPK is to replete cellular energy stores by stimulating processes that generate ATP, such as fatty acid oxidation, and inhibiting ATP-consuming pathways (e.g. lipogenesis, triglyceride synthesis, and gluconeogenesis) that are not acutely needed for cell survival (7Hardie D.G. Hawley S.A. Scott J.W. J. Physiol. (Lond.). 2006; 574: 7-15Crossref Scopus (649) Google Scholar). In addition, changes in the levels of various hormones and fuels have been demonstrated to activate or inhibit AMPK suggesting that its regulation and physiological relevance are more complex than initially appreciated. In keeping with this notion, AMPK has also been shown to protect cells by reducing or even preventing a variety of stress responses, including the generation of free radicals and inflammation (8Cacicedo J.M. Gauthier M.-S. Ruderman N.B. Ido Y. Diabetes. 2007; 56 (Abstr. 2039): A515Google Scholar, 9Cacicedo J.M. Yagihashi N. Keaney Jr., J.F. Ruderman N.B. Ido Y. Biochem. Biophys. Res. Commun. 2004; 324: 1204-1209Crossref PubMed Scopus (209) Google Scholar, 10Lee W.J. Lee I.K. Kim H.S. Kim Y.M. Koh E.H. Won J.C. Han S.M. Kim M.-S. Jo I. Oh G.T. Park I.-S. Youn J.H. Park S.-W. Lee K.-U. Park J.-Y. Arterioscler. Thromb. Vasc. Biol. 2005; 25: 2488-2494Crossref PubMed Scopus (117) Google Scholar, 11Pilon G. Dallaire P. Marette A. J. Biol. Chem. 2004; 279: 20767-20774Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 12Alba G. El Bekay R. Alvarez-Maqueda M. Chacon P. Vega A. Monteseirin J. Santa Maria C. Pintado E. Bedoya F.J. Bartrons R. Sobrino F. FEBS Lett. 2004; 573: 219-225Crossref PubMed Scopus (78) Google Scholar). Adipose tissue is a key player in whole-body energy regulation. One of its major roles is to provide free fatty acids (FFA) as a fuel for other tissues in times of need. In these circumstances, catecholamines, and possibly other hormones and neurotransmitters, rapidly activate β-adrenergic receptors and the cAMP-dependent protein kinase (PKA) axis. This results in the stimulation of lipolysis by a complex regulatory mechanism that appears to involve hormone-sensitive lipase, adipose-tissue triglyceride lipase (ATGL), and perilipin, a lipid droplet-associated phosphoprotein that has proven to be essential for β-adrenergic stimulation of lipolysis (13Miyoshi H. Perfield II, J.W. Souza S.C. Shen W.-J. Zhang H.-H. Stancheva Z.S. Kraemer F.B. Obin M.S. Greenberg A.S. J. Biol. Chem. 2007; 282: 996-1002Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 14Miyoshi H. Souza S.C. Zhang H.-H. Strissel K.J. Christoffolete M.A. Kovsan J. Rudich A. Kraemer F.B. Bianco A.C. Obin M.S. Greenberg A.S. J. Biol. Chem. 2006; 281: 15837-15844Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 15Souza S.C. Muliro K.V. Liscum L. Lien P. Yamamoto M.T. Schaffer J.E. Dallal G.E. Wang X. Kraemer F.B. Obin M. Greenberg A.S. J. Biol. Chem. 2002; 277: 8267-8272Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 16Souza S.C. de Vargas L.M. Yamamoto M.T. Lien P. Franciosa M.D. Moss L.G. Greenberg A.S. J. Biol. Chem. 1998; 273: 24665-24669Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 17Greenberg A. Egan J. Wek S. Garty N. Blanchette-Mackie E. Londos C. J. Biol. Chem. 1991; 266: 11341-11346Abstract Full Text PDF PubMed Google Scholar). In recent years, ATGL has been identified as the predominant lipase responsible for the initial step in hydrolyzing triglycerides, at least in rodents (18Zimmermann R. Strauss J.G. Haemmerle G. Schoiswohl G. Birner-Gruenberger R. Riederer M. Lass A. Neuberger G. Eisenhaber F. Hermetter A. Zechner R. Science. 2004; 306: 1383-1386Crossref PubMed Scopus (1504) Google Scholar, 19Zechner R. Strauss J.G. Haemmerle G. Lass A. Zimmermann R. Curr. Opin. Lipidol. 2005; 16: 333-340Crossref PubMed Scopus (222) Google Scholar, 20Haemmerle G. Lass A. Zimmermann R. Gorkiewicz G. Meyer C. Rozman J. Heldmaier G. Maier R. Theussl C. Eder S. Kratky D. Wagner E.F. Klingenspor M. Hoefler G. Zechner R. Science. 2006; 312: 734-737Crossref PubMed Scopus (1011) Google Scholar). Despite its prominent role in energy metabolism, few investigations have addressed the regulation of AMPK in adipose tissue. Previous studies conducted in our laboratory first showed that in rodents exercise increases AMPK activity in intra-abdominal adipose tissue (21Park H. Kaushik V.K. Constant S. Prentki M. Przybytkowski E. Ruderman N.B. Saha A.K. J. Biol. Chem. 2002; 277: 32571-32577Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, 22Kelly M. Keller C. Avilucea P.R. Keller P. Luo Z. Xiang X. Giralt M. Hidalgo J. Saha A.K. Pedersen B.K. Ruderman N.B. Biochem. Biophys. Res. Commun. 2004; 320: 449-454Crossref PubMed Scopus (225) Google Scholar). Acute exercise is known to increase the release of catecholamines, which participate in the lipolytic response of adipose tissue to exercise. In keeping with this notion, β-adrenergic agonists and agents that increase cAMP have been found to stimulate both lipolysis and AMPK activation in cultured adipocytes (23Moule S.K. Denton R.M. FEBS Lett. 1998; 439: 287-290Crossref PubMed Scopus (103) Google Scholar, 24Yin W. Mu J. Birnbaum M.J. J. Biol. Chem. 2003; 278: 43074-43080Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar), and to cause a decrease in their cellular energy state in vitro (25Angel A. Desai K.S. Halperin M.L. J. Lipid Res. 1971; 12: 203-213Abstract Full Text PDF PubMed Google Scholar, 26Angel A. Desai K. Halperin M.L. Metabolism. 1971; 20: 87-99Abstract Full Text PDF PubMed Scopus (26) Google Scholar, 27Vallano M.L. Lee M.Y. Sonenberg M. Am. J. Physiol. 1983; 245: E266-E272PubMed Google Scholar, 28Kather H. J. Clin. Investig. 1990; 85: 106-114Crossref PubMed Scopus (19) Google Scholar) and in rats in vivo (29Koh H.-J. Hirshman M.F. He H. Li Y. Manabe Y. Balschi J.A. Goodyear L.J. Biochem. J. 2007; 403: 473-481Crossref PubMed Scopus (98) Google Scholar). Despite this, the mechanism(s) by which these agents activate AMPK, and whether it is dependent on their ability to stimulate lipolysis is unknown, as is the physiological significance of AMPK activation in this setting. To address these questions, we first examined activation of AMPK by agents that increase cAMP in cultured adipocytes in situations in which their ability to stimulate lipolysis was inhibited downstream of PKA. We then investigated the consequences of inhibiting AMPK activation on reactive oxygen species (ROS) generation and energy depletion in these cells. Culture of 3T3-L1 Adipocytes—3T3-L1 preadipocytes were purchased from American Type Culture Collection (Manassas, VA). 3T3-L1 preadipocytes placed in 12-well plates were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% bovine calf serum and differentiated to adipocytes using standard protocols (30Kohanski R. Frost S. Lane M. J. Biol. Chem. 1986; 261: 12272-12281Abstract Full Text PDF PubMed Google Scholar). 3T3-L1 attained a fully differentiated state on day 10 after the initiation of the differentiation protocol, and experiments were done on days 12–16. DMEM, bovine calf serum, and fetal bovine serum were purchased from Invitrogen. Generation and Differentiation of Stable Lines of Peri-/- Mouse Embryonic Fibroblast Adipocytes—Stable lines of MEFs were generated from embryos of perilipin null mice (Peri-/-) as described previously (13Miyoshi H. Perfield II, J.W. Souza S.C. Shen W.-J. Zhang H.-H. Stancheva Z.S. Kraemer F.B. Obin M.S. Greenberg A.S. J. Biol. Chem. 2007; 282: 996-1002Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 14Miyoshi H. Souza S.C. Zhang H.-H. Strissel K.J. Christoffolete M.A. Kovsan J. Rudich A. Kraemer F.B. Bianco A.C. Obin M.S. Greenberg A.S. J. Biol. Chem. 2006; 281: 15837-15844Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 31Rosen E.D. Hsu C.-H. Wang X. Sakai S. Freeman M.W. Gonzalez F.J. Spiegelman B.M. Genes Dev. 2002; 16: 22-26Crossref PubMed Scopus (1091) Google Scholar). MEF adipocytes were generated by retroviral expression of peroxisome proliferator-activated receptor-γ (31Rosen E.D. Hsu C.-H. Wang X. Sakai S. Freeman M.W. Gonzalez F.J. Spiegelman B.M. Genes Dev. 2002; 16: 22-26Crossref PubMed Scopus (1091) Google Scholar) followed by selection, expansion, and differentiation using a standard differentiation medium (13Miyoshi H. Perfield II, J.W. Souza S.C. Shen W.-J. Zhang H.-H. Stancheva Z.S. Kraemer F.B. Obin M.S. Greenberg A.S. J. Biol. Chem. 2007; 282: 996-1002Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 14Miyoshi H. Souza S.C. Zhang H.-H. Strissel K.J. Christoffolete M.A. Kovsan J. Rudich A. Kraemer F.B. Bianco A.C. Obin M.S. Greenberg A.S. J. Biol. Chem. 2006; 281: 15837-15844Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). MEFs attained a differentiated adipocyte phenotype within 7 days of culturing in differentiation medium (13Miyoshi H. Perfield II, J.W. Souza S.C. Shen W.-J. Zhang H.-H. Stancheva Z.S. Kraemer F.B. Obin M.S. Greenberg A.S. J. Biol. Chem. 2007; 282: 996-1002Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 14Miyoshi H. Souza S.C. Zhang H.-H. Strissel K.J. Christoffolete M.A. Kovsan J. Rudich A. Kraemer F.B. Bianco A.C. Obin M.S. Greenberg A.S. J. Biol. Chem. 2006; 281: 15837-15844Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). Adenoviral Expression of GFP and Peri A Constructs in Peri-/-MEF Adipocytes—Adenoviruses expressing Aequoria victoria green fluorescent protein (Ad-GFP) or wild-type perilipin A (Ad-WT Peri A) were generated and verified as described previously (13Miyoshi H. Perfield II, J.W. Souza S.C. Shen W.-J. Zhang H.-H. Stancheva Z.S. Kraemer F.B. Obin M.S. Greenberg A.S. J. Biol. Chem. 2007; 282: 996-1002Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 14Miyoshi H. Souza S.C. Zhang H.-H. Strissel K.J. Christoffolete M.A. Kovsan J. Rudich A. Kraemer F.B. Bianco A.C. Obin M.S. Greenberg A.S. J. Biol. Chem. 2006; 281: 15837-15844Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 32Zhang H.H. Souza S.C. Muliro K.V. Kraemer F.B. Obin M.S. Greenberg A.S. J. Biol. Chem. 2003; 278: 51535-51542Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Recombinant adenovirus was transduced into Peri-/- MEFs with Lipofectamine Plus™ (Invitrogen) on day 3 after induction of differentiation. The amount of each adenovirus used was selected to ensure comparable levels of expression of the different Peri A constructs, which was confirmed by Western blots and densitometry (13Miyoshi H. Perfield II, J.W. Souza S.C. Shen W.-J. Zhang H.-H. Stancheva Z.S. Kraemer F.B. Obin M.S. Greenberg A.S. J. Biol. Chem. 2007; 282: 996-1002Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 14Miyoshi H. Souza S.C. Zhang H.-H. Strissel K.J. Christoffolete M.A. Kovsan J. Rudich A. Kraemer F.B. Bianco A.C. Obin M.S. Greenberg A.S. J. Biol. Chem. 2006; 281: 15837-15844Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 32Zhang H.H. Souza S.C. Muliro K.V. Kraemer F.B. Obin M.S. Greenberg A.S. J. Biol. Chem. 2003; 278: 51535-51542Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). GFP was transduced in Peri-/- MEFs as a negative control. Recombinant Adenoviruses Expressing Small Hairpin RNA (shRNA) Directed Against Murine ATGL—ATGL shRNA design was based on GenBank™ accession number NM025802 (sequence GGAGAGAACGTCATCATAT) as described (13Miyoshi H. Perfield II, J.W. Souza S.C. Shen W.-J. Zhang H.-H. Stancheva Z.S. Kraemer F.B. Obin M.S. Greenberg A.S. J. Biol. Chem. 2007; 282: 996-1002Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). A “scrambled” version of these shRNA (CGCGCTTTGTAGGATTCA) was generated as a control for nonspecific effects of shRNA. All shRNAs were cloned into the pQuiet vector to generate recombinant adenoviruses. Recombinant adenovirus expressing shRNAs was transduced into MEFs cells with Lipofectamine Plus™ on day 2 after induction of differentiation. Lipolysis Assays—Lipolysis was stimulated by addition of the β-adrenergic agonist isoproterenol (10 μm), the adenylate cyclase activator forskolin (20 μm), or the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) (0.5 mm) into serum-free culture medium containing 0.5% fatty acid-free bovine serum albumin (Celliance, Toronto, Canada) in the presence or absence of the general lipase inhibitor orlistat (100 μm), the ACS inhibitor triacsin C (6 μm), or the AMPK inhibitor compound C (50 μm). Lipolysis was assessed from the release of glycerol and FFA in the culture medium as described previously (14Miyoshi H. Souza S.C. Zhang H.-H. Strissel K.J. Christoffolete M.A. Kovsan J. Rudich A. Kraemer F.B. Bianco A.C. Obin M.S. Greenberg A.S. J. Biol. Chem. 2006; 281: 15837-15844Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar) using free glycerol reagent (Sigma) and nonesterified fatty acids measurement kit (Waco Diagnostics, Richmond, VA). Triacsin C was purchased from Biomol (Plymouth Meeting, PA), compound C from Calbiochem, and other compounds from Sigma. Immunoblot Analysis—Cultured cells were scraped on ice in cell lysis buffer (plus 1 mm phenylmethylsulfonyl fluoride), sonicated, and centrifuged (14,000 × g for 15 min at 4 °C). Protein concentrations of cell supernatants were determined using the bicinchoninic acid (BCA) reagents (Pierce) using bovine serum albumin as the standard. Proteins (20 μg) were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Amersham Biosciences). Membranes were blocked in Tris-buffered saline (pH 7.5) containing 0.05% Tween 20 (TBST) and 5% milk for 1 h at room temperature and then probed with antibodies specific to β-actin, perilipin, or ATGL for 1 h at room temperature, or specific to P-AMPK Thr-172, pan-αAMPK, P-ACC Ser-79, P-LKB1 Ser-431, P-CREB Ser-133, phospho-(Ser/Thr)-PKA substrate overnight at 4 °C. Bound antibodies were detected with the appropriate horseradish peroxidase-linked whole secondary antibodies. Protein immunoblots were visualized by enhanced chemiluminescence, and bands were quantified with scanning densitometry. Antibodies for pan-α-AMPK, P-AMPK Thr-172, and P-CREB Ser-133, phospho-(Ser/Thr)-PKA substrate, and cell lysis buffer were purchased from Cell Signaling Technology (Beverly, MA); P-ACC Ser-79 was from Upstate Biotechnology, Inc. (Lake Placid, NY); β-actin was from Sigma; P-LKB1 Ser-431 and mouse and rabbit antibodies conjugated to horseradish peroxidase were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Specific polyclonal ant-rabbit ATGL and perilipin antibody were generated and affinity-purified as described (13Miyoshi H. Perfield II, J.W. Souza S.C. Shen W.-J. Zhang H.-H. Stancheva Z.S. Kraemer F.B. Obin M.S. Greenberg A.S. J. Biol. Chem. 2007; 282: 996-1002Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 14Miyoshi H. Souza S.C. Zhang H.-H. Strissel K.J. Christoffolete M.A. Kovsan J. Rudich A. Kraemer F.B. Bianco A.C. Obin M.S. Greenberg A.S. J. Biol. Chem. 2006; 281: 15837-15844Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 15Souza S.C. Muliro K.V. Liscum L. Lien P. Yamamoto M.T. Schaffer J.E. Dallal G.E. Wang X. Kraemer F.B. Obin M. Greenberg A.S. J. Biol. Chem. 2002; 277: 8267-8272Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Determination of AMPK Activity—3T3-L1 express mainly the α1 isoform of AMPK (data not shown and see Ref. 33Daval M. Diot-Dupuy F. Bazin R. Hainault I. Viollet B. Vaulont S. Hajduch E. Ferre P. Foufelle F. J. Biol. Chem. 2005; 280: 25250-25257Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). AMPK α1 activity was measured after 1 h of treatment with or without isoproterenol (10 μm) in 3T3-L1 adipocytes. In addition, a possible nonspecific direct effect of orlistat or triacsin C on AMPK activity was ruled out by measuring AMPK activity in a cell-free system with/without the presence of orlistat (100 μm) or triacsin C (6 μm). To do so, untreated 3T3-L1 lysates containing 100 μg of protein were immunoprecipitated with AMPKα1-specific polyclonal antibody (Bethyl Laboratories Inc., Montgomery, TX) and protein A/G-agarose beads. Beads were washed, and the immobilized enzyme was assayed based on the phosphorylation of SAMS peptide (HMRSAMSGLHLVKRR) (0.2 mmol/liter) by 0.2 mmol/liter ATP (containing 2 μCi of [γ-32P]ATP) in the presence and absence of 0.2 mmol/liter AMP and the presence or absence of orlistat (100 μm) or triacsin C (6 μm). Label incorporation into the SAMS peptide was measured using a scintillation counter. Nucleotide Studies in 3T3-L1 Adipocytes—Cellular levels of ATP, ADP, and AMP were measured spectrophotometrically as described previously (34Maizels E.Z. Ruderman N.B. Goodman M.N. Lau D. Biochem. J. 1977; 162: 557-568Crossref PubMed Scopus (108) Google Scholar, 35Lowry O.H. Passoneau J.V. A Flexible System of Enzymatic Analysis. Academic Press, New York1972: 147-156Google Scholar). In brief, 3T3-L1 adipocytes were treated as described under “Lipolysis Assays.” After the incubation period, the media were kept for glycerol measurements, and the cells were washed once with cold phosphate-buffered saline, and proteins were precipitated with 1% trichloroacetic acid. The acid was then removed from the aqueous phase of the trichloroacetic acid extracts with repeated washes with ether. The samples were then dried to powder and kept at -80 °C until the assay. Determination of Intracellular cAMP Content in 3T3-L1 Adipocytes—3T3-L1 adipocytes were treated as described under “Lipolysis Assays.” After the incubation period, cAMP was extracted and measured using a commercially available cAMP EIA kit according to the instructions provided by the manufacturer (Biomedical Technologies Inc., Stoughton, MA). Detection of Reactive Oxygen Species—3T3-L1 adipocytes were incubated overnight with compound C or vehicle (DMSO) and thereafter treated for 1 h with isoproterenol and loaded with the redox-sensitive dye 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (DCF, Invitrogen) during the last 30 min of this incubation period. After the loading period, the media were kept for glycerol assay, and cells were washed with and incubated in pre-warmed Hanks' buffered saline solution at 37 °C. Reactive oxygen species (H2O2 and OH) formation was measured for 1 h in a multiwell fluorescence plate reader (Fluoroskan Ascent, Labsystems) set at 37 °C using excitation and emission filters of 485 and 538 nm, respectively. At the end of the DCF experiment, tert-butyl hydroperoxide (1 mm) (Sigma) was added to the incubation media to verify that the measurement maximal capacity of the system had not been reached during the experiment. Statistical Analysis—Results were analyzed using Statview version 5.0.1 and are presented as means ± S.E. of at least three independent experiments. Statistical significance was determined by a one-way or two-way analysis of variance for nonrepeated or repeated measures, as appropriate. Fisher's protected least significant difference post hoc test was used in the event of a significant (p < 0.05) ratio. Agents That Increase cAMP Stimulate Both Lipolysis and AMPK Activation in Adipocytes—Incubation of 3T3-L1 adipocytes with a β-adrenergic agonist (isoproterenol, 10 μm) and an adenylyl cyclase activator (forskolin, 20 μm) caused a 20-fold increase in lipolysis (Fig. 1B). In confirmation of previous reports (23Moule S.K. Denton R.M. FEBS Lett. 1998; 439: 287-290Crossref PubMed Scopus (103) Google Scholar, 24Yin W. Mu J. Birnbaum M.J. J. Biol. Chem. 2003; 278: 43074-43080Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar), these agents also activated AMPK as reflected by increases in the phosphorylation of its α-subunit at Thr-172 and of its downstream target ACC at Ser-79 (Fig. 1A). That isoproterenol activates AMPK was confirmed by measurements of the activity of AMPK α1, the main isoform expressed in 3T3-L1 adipocytes (data not shown). Incubation of the cells with the phosphodiesterase inhibitor IBMX (0.5 mm) also stimulated lipolysis and increased AMPK and ACC phosphorylation (Fig. 1, A and B). Because the phosphodiesterase catalyzes the conversion of cAMP to AMP, this essentially rules out this source of AMP as a major cause of AMPK activation during lipolysis. Iinhibition of Lipolysis by Orlistat Reduces AMPK Activation by Agents That Increase cAMP—We next sought to determine whether AMPK activation by isoproterenol, forskolin, and IBMX is related to their ability to stimulate lipolysis. To test this possibility, we incubated adipocytes with orlistat, a general lipase inhibitor (36Nolan C.J. Leahy J.L. Delghingaro-Augusto V. Moibi J. Soni K. Peyot M.L. Fortier M. Guay C. Lamontagne J. Barbeau A. Przybytkowski E. Joly E. Masiello P. Wang S. Mitchell G.A. Prentki M. Diabetologia. 2006; 49: 2120-2130Crossref PubMed Scopus (98) Google Scholar). As shown in Fig. 2, orlistat inhibited the ability of all three lipolytic agents to stimulate lipolysis by ∼50% (Fig. 2, A and B), and it had a similar inhibitory effect on AMPK activation (Fig. 2, C–E). The possibility that orlistat could have a direct inhibitory action on AMPK was ruled out by its lack of effect on AMPK activity in a cell-free system (data not shown, see under “Experimental Procedures”). Regression analyses revealed a strong positive correlation between both P-AMPK Thr-172 and lipolysis (r2 = 0.44; p < 0.0001) and between P-ACC Ser-79 and lipolysis (r2 = 0.64; p < 0.0001) (Fig. 3, A and B).FIGURE 1Agonists that increase cAMP stimulate both lipolysis and AMPK activation. L1 adipocytes were treated with the phosphodiesterase inhibitor IBMX (0.5 mm), the β-agonist isoproterenol (Iso, 10 μm), or the adenylyl cyclase activator forskolin (FKN, 20 μm) for 1 h, following a 4-h preincubation period in serum-free DMEM containing 0.5% fatty acid-free BSA. Ctrl, control. A, activation of AMPK as assessed from immunoblots of phospho-AMPK Thr-172 and P-ACC Ser-79. IB, immunoblot. B, lipolysis was quantified on the basis of glycerol release into the incubation media. Results for glycerol release are means ± S.E. (n = 9) and were obtained in three independent experiments. Immunoblots shown are representative of those obtained in a total of nine lysates. Significantly different from control group: ***, p < 0.001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3Correlation of measures of AMPK activation and lipolysis in adipocytes stimulated by agents that increase cAMP. Lipolysis was assessed on the basis of glycerol release and was correlated with the abundance of P-AMPK Thr" @default.
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• W2080981566 date "2008-06-01" @default.
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• W2080981566 title "AMP-activated Protein Kinase Is Activated as a Consequence of Lipolysis in the Adipocyte" @default.
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• W2080981566 doi "https://doi.org/10.1074/jbc.m708177200" @default.
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