Yin-Yang Regulation of Adiponectin Signaling by APPL Isoforms in Muscle Cells
2009; Elsevier BV; Volume: 284; Issue: 46 Linguagem: Inglês
10.1074/jbc.m109.010355
ISSN1083-351X
AutoresChanghua Wang, Xiaoban Xin, RuiHua Xiang, Fresnida J. Ramos, Meilian Liu, Hak Joo Lee, Hongzhi Chen, Xuming Mao, Chintan K. Kikani, Feng Liu, Lily Dong,
Tópico(s)Retinoids in leukemia and cellular processes
ResumoAPPL1 is a newly identified adiponectin receptor-binding protein that positively mediates adiponectin signaling in cells. Here we report that APPL2, an isoform of APPL1 that forms a dimer with APPL1, can interacts with both AdipoR1 and AdipoR2 and acts as a negative regulator of adiponectin signaling in muscle cells. Overexpression of APPL2 inhibits the interaction between APPL1 and AdipoR1, leading to down-regulation of adiponectin signaling in C2C12 myotubes. In contrast, suppressing APPL2 expression by RNAi significantly enhances adiponectin-stimulated glucose uptake and fatty acid oxidation. In addition to targeting directly to and competing with APPL1 in binding with the adiponectin receptors, APPL2 also suppresses adiponectin and insulin signaling by sequestrating APPL1 from these two pathways. In addition to adiponectin, metformin also induces APPL1-APPL2 dissociation. Taken together, our results reveal that APPL isoforms function as an integrated Yin-Yang regulator of adiponectin signaling and mediate the cross-talk between adiponectin and insulin signaling pathways in muscle cells. APPL1 is a newly identified adiponectin receptor-binding protein that positively mediates adiponectin signaling in cells. Here we report that APPL2, an isoform of APPL1 that forms a dimer with APPL1, can interacts with both AdipoR1 and AdipoR2 and acts as a negative regulator of adiponectin signaling in muscle cells. Overexpression of APPL2 inhibits the interaction between APPL1 and AdipoR1, leading to down-regulation of adiponectin signaling in C2C12 myotubes. In contrast, suppressing APPL2 expression by RNAi significantly enhances adiponectin-stimulated glucose uptake and fatty acid oxidation. In addition to targeting directly to and competing with APPL1 in binding with the adiponectin receptors, APPL2 also suppresses adiponectin and insulin signaling by sequestrating APPL1 from these two pathways. In addition to adiponectin, metformin also induces APPL1-APPL2 dissociation. Taken together, our results reveal that APPL isoforms function as an integrated Yin-Yang regulator of adiponectin signaling and mediate the cross-talk between adiponectin and insulin signaling pathways in muscle cells. IntroductionAdiponectin, an adipocyte-secreted hormone that regulates energy homeostasis and insulin sensitivity, has been shown to be a promising therapeutic drug target for the treatment of type 2 diabetes (1.Kadowaki T. Yamauchi T. Kubota N. Hara K. Ueki K. Tobe K. J. Clin. Invest. 2006; 116: 1784-1792Crossref PubMed Scopus (2204) Google Scholar, 2.Mao X. Hong J.Y. Dong L.Q. Mini. Rev. Med. Chem. 2006; 6: 1331-1440Crossref PubMed Scopus (26) Google Scholar, 3.Kim J.Y. van de Wall E. Laplante M. Azzara A. Trujillo M.E. Hofmann S.M. Schraw T. Durand J.L. Li H. Li G. Jelicks L.A. Mehler M.F. Hui D.Y. Deshaies Y. Shulman G.I. Schwartz G.J. Scherer P.E. J. Clin. Invest. 2007; 117: 2621-2637Crossref PubMed Scopus (990) Google Scholar). Adiponectin binds to its membrane receptors (AdipoR1 and AdipoR2) 3The abbreviations used are: AdipoRadiponectin receptorAMPKAMP-activated protein kinaseAPPLadaptor protein containing PH domain, PTB domain, and leucine zipper motifIRS-1insulin receptor substrate-1MAPKmitogen-activated protein kinaseGSTglutathione S-transferaseMOImultiplicity of infectionDAPI4′,6-diamidino-2-phenylindoleHAhemagglutininPIphosphatidylinositol. and regulates lipid and glucose metabolism by activating downstream signaling molecules, such as AMP-activated protein kinase (AMPK), p38 MAP kinase (MAPK), and PPARα, in the muscle and liver (1.Kadowaki T. Yamauchi T. Kubota N. Hara K. Ueki K. Tobe K. J. Clin. Invest. 2006; 116: 1784-1792Crossref PubMed Scopus (2204) Google Scholar, 4.Yamauchi T. Kamon J. Ito Y. Tsuchida A. Yokomizo T. Kita S. Sugiyama T. Miyagishi M. Hara K. Tsunoda M. Murakami K. Ohteki T. Uchida S. Takekawa S. Waki H. Tsuno N.H. Shibata Y. Terauchi Y. Froguel P. Tobe K. Koyasu S. Taira K. Kitamura T. Shimizu T. Nagai R. Kadowaki T. Nature. 2003; 423: 762-769Crossref PubMed Scopus (2614) Google Scholar). Activation of AMPK by adiponectin reduces S6 kinase-mediated IRS-1 serine phosphorylation and increases IRS-1 tyrosine phosphorylation thus sensitizes insulin signaling in C2C12 myotubes (5.Wang C. Mao X. Wang L. Liu M. Wetzel M.D. Guan K.L. Dong L.Q. Liu F. J. Biol. Chem. 2007; 282: 7991-7996Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar), suggesting a direct cross-talk between the adiponectin and insulin signaling pathways.We have recently identified APPL1 (adaptor protein-containing PH domain, PTB domain, and leucine zipper motif) as a signaling protein immediately downstream of adiponectin receptors and positively mediates adiponectin signaling in muscle cells (6.Mao X. Kikani C.K. Riojas R.A. Langlais P. Wang L. Ramos F.J. Fang Q. Christ-Roberts C.Y. Hong J.Y. Kim R.Y. Liu F. Dong L.Q. Nat. Cell Biol. 2006; 8: 516-523Crossref PubMed Scopus (535) Google Scholar). This adaptor protein was previously shown to interact with the catalytic subunit of PI 3-kinase (p110) and Akt, which are two key kinases in the PI 3-kinase pathway downstream of the insulin receptor (7.Mitsuuchi Y. Johnson S.W. Sonoda G. Tanno S. Golemis E.A. Testa J.R. Oncogene. 1999; 18: 4891-4898Crossref PubMed Scopus (172) Google Scholar). The interaction between APPL1 and Akt is required for insulin-stimulated GLUT4 translocation (8.Saito T. Jones C.C. Huang S. Czech M.P. Pilch P.F. J. Biol. Chem. 2007; 282: 32280-32287Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar) and for controlling Akt substrate selectivity (9.Schenck A. Goto-Silva L. Collinet C. Rhinn M. Giner A. Habermann B. Brand M. Zerial M. Cell. 2008; 133: 486-497Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). It has been shown that APPL1-potentiated Akt activity to suppress androgen receptor transactivation in prostate cancer cells (10.Yang L. Lin H.K. Altuwaijri S. Xie S. Wang L. Chang C. J. Biol. Chem. 2003; 278: 16820-16827Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). APPL1 has also been suggested to function as an adaptor protein in regulating follicle-stimulated hormone (FSH)-mediated PI 3-kinase/Akt signaling pathway (11.Nechamen C.A. Thomas R.M. Dias J.A. Mol. Cell. Endocrinol. 2007; 260–262: 93-99Crossref PubMed Scopus (77) Google Scholar, 12.Nechamen C.A. Thomas R.M. Cohen B.D. Acevedo G. Poulikakos P.I. Testa J.R. Dias J.A. Biol. Reprod. 2004; 71: 629-636Crossref PubMed Scopus (92) Google Scholar). Our results showed that APPL1 binds directly to the intracellular part of the adiponectin receptors and positively mediates adiponectin signaling to the AMPK and p38 MAPK pathways, leading to increased glucose uptake and fatty acid oxidation in muscle cells (6.Mao X. Kikani C.K. Riojas R.A. Langlais P. Wang L. Ramos F.J. Fang Q. Christ-Roberts C.Y. Hong J.Y. Kim R.Y. Liu F. Dong L.Q. Nat. Cell Biol. 2006; 8: 516-523Crossref PubMed Scopus (535) Google Scholar). In addition, we found that APPL1 plays a critical role in regulating the cross-talk between adiponectin and insulin signaling pathways (6.Mao X. Kikani C.K. Riojas R.A. Langlais P. Wang L. Ramos F.J. Fang Q. Christ-Roberts C.Y. Hong J.Y. Kim R.Y. Liu F. Dong L.Q. Nat. Cell Biol. 2006; 8: 516-523Crossref PubMed Scopus (535) Google Scholar). APPL1 has since been found to mediate adiponectin signaling in other types of cells such as endothelial cells to regulate nitric oxide production and endothelium-dependent vasodilation (13.Cheng K.K. Lam K.S. Wang Y. Huang Y. Carling D. Wu D. Wong C. Xu A. Diabetes. 2007; 56: 1387-1394Crossref PubMed Scopus (273) Google Scholar) and to protect from IL-18-mediated cell death (14.Chandrasekar B. Boylston W.H. Venkatachalam K. Webster N.J. Prabhu S.D. Valente A.J. J. Biol. Chem. 2008; 283: 24889-24898Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). A recent study also suggested that APPL1 has a potentiating effect on insulin-stimulated suppression of hepatic glucose production in mice (15.Cheng K.K. Iglesia M.A. Lam K.S. Wang Y. Sweeney G. Zhu W. Vanhoutte P.M. Kraegen E.W. Xu A. Cell Metab. 2009; 9: 417-427Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). These evidence suggest that APPL1 is an essential mediator in both adiponectin and insulin signaling, which are the two major pathways regulating energy homeostasis.APPL2 is an isoform of APPL1, and these two proteins display 54% identity in protein sequences (6.Mao X. Kikani C.K. Riojas R.A. Langlais P. Wang L. Ramos F.J. Fang Q. Christ-Roberts C.Y. Hong J.Y. Kim R.Y. Liu F. Dong L.Q. Nat. Cell Biol. 2006; 8: 516-523Crossref PubMed Scopus (535) Google Scholar, 16.Miaczynska M. Christoforidis S. Giner A. Shevchenko A. Uttenweiler-Joseph S. Habermann B. Wilm M. Parton R.G. Zerial M. Cell. 2004; 116: 445-456Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar). Similar to APPL1, APPL2 contains an N-terminal BAR domain, a central PH domain, and a C-terminal PTB domain. It has been shown that APPL2 is essential for cell proliferation and embryonic development (9.Schenck A. Goto-Silva L. Collinet C. Rhinn M. Giner A. Habermann B. Brand M. Zerial M. Cell. 2008; 133: 486-497Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 16.Miaczynska M. Christoforidis S. Giner A. Shevchenko A. Uttenweiler-Joseph S. Habermann B. Wilm M. Parton R.G. Zerial M. Cell. 2004; 116: 445-456Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar). Recent studies indicated that APPL2 regulates FSH signaling by forming a dimer with APPL1 via the BAR domains of the isoforms, leading to the formation of a complex with the FSH receptor, APPL1 and Akt2. However, unlike APPL1, APPL2 does not directly interact with Akt2 (11.Nechamen C.A. Thomas R.M. Dias J.A. Mol. Cell. Endocrinol. 2007; 260–262: 93-99Crossref PubMed Scopus (77) Google Scholar, 17.Chial H.J. Wu R. Ustach C.V. McPhail L.C. Mobley W.C. Chen Y.Q. Traffic. 2008; 9: 215-229Crossref PubMed Scopus (44) Google Scholar). The role of APPL2 in adiponectin and insulin signaling remains largely unknown.In the present study, we show that APPL2 functions as a negative regulator in adiponectin signaling. APPL2 competes with APPL1 in the binding to AdipoR1 and blocks adiponectin signaling in muscle cells. In addition, APPL2 also sequesters APPL1 from adiponectin signal pathway by forming a heterodimer with APPL1. Adiponectin as well as metformin induces a dissociation of this complex. These results provide a mechanism for negative regulation of adiponectin signaling and adiponectin-regulated glucose and lipid metabolism in muscle cells. Furthermore, APPL2 blocks APPL1-mediated insulin-sensitizing effect of adiponectin in muscle cells by heterodimerizing with APPL1 to inhibit insulin-stimulated Akt activation. Our data indicate that APPL isoforms function as an integrated Yin-Yang regulator in adiponectin signaling and reveals a novel molecular mechanism for adiponectin and insulin resistance.DISCUSSIONWe have recently identified APPL1 as a critical signaling molecule that positively mediates adiponectin signaling and action (6.Mao X. Kikani C.K. Riojas R.A. Langlais P. Wang L. Ramos F.J. Fang Q. Christ-Roberts C.Y. Hong J.Y. Kim R.Y. Liu F. Dong L.Q. Nat. Cell Biol. 2006; 8: 516-523Crossref PubMed Scopus (535) Google Scholar). However, the role of APPL2, a protein that shares a 54% identity in amino acid sequence with APPL1 (6.Mao X. Kikani C.K. Riojas R.A. Langlais P. Wang L. Ramos F.J. Fang Q. Christ-Roberts C.Y. Hong J.Y. Kim R.Y. Liu F. Dong L.Q. Nat. Cell Biol. 2006; 8: 516-523Crossref PubMed Scopus (535) Google Scholar, 16.Miaczynska M. Christoforidis S. Giner A. Shevchenko A. Uttenweiler-Joseph S. Habermann B. Wilm M. Parton R.G. Zerial M. Cell. 2004; 116: 445-456Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar), was unclear. It is interesting to notice that APPL1 but not APPL2 interacts directly with Akt (11.Nechamen C.A. Thomas R.M. Dias J.A. Mol. Cell. Endocrinol. 2007; 260–262: 93-99Crossref PubMed Scopus (77) Google Scholar). Thus, the two APPL isoforms may play distinct roles in adiponectin downstream signaling and action.In the present study, we show that APPL2 negatively regulates adiponectin signaling in muscle cells (Fig. 7A). Under the basal condition, APPL2 interacts with AdipoR1 via its BAR domain (Fig. 1). The binding of APPL2 to AdipoR1 hinders the accessibility of APPL1 to AdipoR1 (Fig. 4, C and D) and thus inhibits the phosphorylation and activation of AMPK and p38 MAPK (Fig. 2). Adiponectin treatment stimulates APPL2 dissociating from AdipoR1 (Fig. 1C and supplemental Fig. S2), hence facilitates the recruitment of APPL1 to AdipoR1 (Fig. 4C, E, and F and supplemental Fig. S1), which is a critical step for adiponectin signaling transduction (6.Mao X. Kikani C.K. Riojas R.A. Langlais P. Wang L. Ramos F.J. Fang Q. Christ-Roberts C.Y. Hong J.Y. Kim R.Y. Liu F. Dong L.Q. Nat. Cell Biol. 2006; 8: 516-523Crossref PubMed Scopus (535) Google Scholar).In addition to interacting with AdipoR1, APPL2 also undergoes heterodimerization with APPL1. The interaction between APPL2 and APPL1 could negatively regulate adiponectin signaling by sequestering APPL1 from the adiponectin receptor (Fig. 7A). Treating cells with adiponectin or metformin promoted the dissociation between APPL1 and APPL2 (Fig. 4, A and B), suggesting that the heterodimer could be a central node in the regulation of adiponectin signaling. Taken together with the findings that APPL1 and APPL2 migrated to distinct cellular compartments under the adiponectin/metformin-stimulatory conditions (Fig. 3), it is conceivable that a dynamic balance between association of APPL2 with APPL1 and with other molecules in the adiponectin signaling pathways could be a key mechanism regulating adiponectin signaling in cells. Interestingly, both APPL2 and AdipoR1 were simultaneously co-immunoprecipitated with APPL1 at endogenous level (supplemental Fig. S1), suggesting that these three molecules could form a complex in cells. Further studies will be needed to elucidate these possibilities.In addition to negatively regulating adiponectin signaling, APPL2 also plays an inhibitory role in mediating the insulin sensitizing effect of adiponectin in muscle cells (Fig. 7B). Overexpression of APPL2 blocks the synergistic effect of adiponectin on insulin-stimulated Akt activation and the effects of insulin itself on this kinase (Fig. 6A). Suppression of APPL2 expression levels, on the other hand, greatly enhanced both the sensitizer effect of adiponectin on insulin signaling and the direct insulin-simulated activation of Akt in C2C12 myotubes (Fig. 6B). However, unlike APPL1, APPL2 does not interact with Akt (11.Nechamen C.A. Thomas R.M. Dias J.A. Mol. Cell. Endocrinol. 2007; 260–262: 93-99Crossref PubMed Scopus (77) Google Scholar) or PI 3-kinase (data not shown), suggesting that APPL2 may negatively regulate insulin signaling through an indirect mechanism, probably via dimerization with APPL1. It is interesting to notice that adiponectin treatment inhibits APPL1/APPL2 dimerization (Fig. 4A) and promotes APPL1 plasma membrane translocation (Fig. 3, A and C), suggesting that the adiponectin-induced dissociation of APPL1-APPL2 heterodimer may facilitate APPL1 translocation to the plasma membrane where APPL1 interacts with PI 3-kinase and Akt and promotes insulin signaling (Fig. 7B). In addition to a direct interaction with signaling molecules in the insulin signaling pathway, membrane association of APPL1 may also sensitize insulin signaling by promoting adiponectin-stimulated AMPK signaling pathway, which has been shown to suppress S6-kinase-mediated IRS-1 serine phosphorylation, leading to increased IRS-1 tyrosine phosphorylation and Akt phosphorylation in response to insulin stimulation (5.Wang C. Mao X. Wang L. Liu M. Wetzel M.D. Guan K.L. Dong L.Q. Liu F. J. Biol. Chem. 2007; 282: 7991-7996Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar).Adiponectin resistance has been shown to contribute significantly to insulin resistance and its associated metabolic diseases (1.Kadowaki T. Yamauchi T. Kubota N. Hara K. Ueki K. Tobe K. J. Clin. Invest. 2006; 116: 1784-1792Crossref PubMed Scopus (2204) Google Scholar, 19.Mullen K.L. Pritchard J. Ritchie I. Snook L.A. Chabowski A. Bonen A. Wright D. Dyck D.J. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2009; 296: R243-R251Crossref PubMed Scopus (105) Google Scholar, 20.Lin H.V. Kim J.Y. Pocai A. Rossetti L. Shapiro L. Scherer P.E. Accili D. Diabetes. 2007; 56: 1969-1976Crossref PubMed Scopus (76) Google Scholar). Identification of APPL isoforms as a Yin-Yang regulator of adiponectin signaling and the adiponectin-insulin cross-talk in muscle cells provides insight not only to the molecular mechanism by which adiponectin signaling is regulated in cells but also on the identification of a potential therapeutic drug target for the treatment of insulin resistance and related disorders. IntroductionAdiponectin, an adipocyte-secreted hormone that regulates energy homeostasis and insulin sensitivity, has been shown to be a promising therapeutic drug target for the treatment of type 2 diabetes (1.Kadowaki T. Yamauchi T. Kubota N. Hara K. Ueki K. Tobe K. J. Clin. Invest. 2006; 116: 1784-1792Crossref PubMed Scopus (2204) Google Scholar, 2.Mao X. Hong J.Y. Dong L.Q. Mini. Rev. Med. Chem. 2006; 6: 1331-1440Crossref PubMed Scopus (26) Google Scholar, 3.Kim J.Y. van de Wall E. Laplante M. Azzara A. Trujillo M.E. Hofmann S.M. Schraw T. Durand J.L. Li H. Li G. Jelicks L.A. Mehler M.F. Hui D.Y. Deshaies Y. Shulman G.I. Schwartz G.J. Scherer P.E. J. Clin. Invest. 2007; 117: 2621-2637Crossref PubMed Scopus (990) Google Scholar). Adiponectin binds to its membrane receptors (AdipoR1 and AdipoR2) 3The abbreviations used are: AdipoRadiponectin receptorAMPKAMP-activated protein kinaseAPPLadaptor protein containing PH domain, PTB domain, and leucine zipper motifIRS-1insulin receptor substrate-1MAPKmitogen-activated protein kinaseGSTglutathione S-transferaseMOImultiplicity of infectionDAPI4′,6-diamidino-2-phenylindoleHAhemagglutininPIphosphatidylinositol. and regulates lipid and glucose metabolism by activating downstream signaling molecules, such as AMP-activated protein kinase (AMPK), p38 MAP kinase (MAPK), and PPARα, in the muscle and liver (1.Kadowaki T. Yamauchi T. Kubota N. Hara K. Ueki K. Tobe K. J. Clin. Invest. 2006; 116: 1784-1792Crossref PubMed Scopus (2204) Google Scholar, 4.Yamauchi T. Kamon J. Ito Y. Tsuchida A. Yokomizo T. Kita S. Sugiyama T. Miyagishi M. Hara K. Tsunoda M. Murakami K. Ohteki T. Uchida S. Takekawa S. Waki H. Tsuno N.H. Shibata Y. Terauchi Y. Froguel P. Tobe K. Koyasu S. Taira K. Kitamura T. Shimizu T. Nagai R. Kadowaki T. Nature. 2003; 423: 762-769Crossref PubMed Scopus (2614) Google Scholar). Activation of AMPK by adiponectin reduces S6 kinase-mediated IRS-1 serine phosphorylation and increases IRS-1 tyrosine phosphorylation thus sensitizes insulin signaling in C2C12 myotubes (5.Wang C. Mao X. Wang L. Liu M. Wetzel M.D. Guan K.L. Dong L.Q. Liu F. J. Biol. Chem. 2007; 282: 7991-7996Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar), suggesting a direct cross-talk between the adiponectin and insulin signaling pathways.We have recently identified APPL1 (adaptor protein-containing PH domain, PTB domain, and leucine zipper motif) as a signaling protein immediately downstream of adiponectin receptors and positively mediates adiponectin signaling in muscle cells (6.Mao X. Kikani C.K. Riojas R.A. Langlais P. Wang L. Ramos F.J. Fang Q. Christ-Roberts C.Y. Hong J.Y. Kim R.Y. Liu F. Dong L.Q. Nat. Cell Biol. 2006; 8: 516-523Crossref PubMed Scopus (535) Google Scholar). This adaptor protein was previously shown to interact with the catalytic subunit of PI 3-kinase (p110) and Akt, which are two key kinases in the PI 3-kinase pathway downstream of the insulin receptor (7.Mitsuuchi Y. Johnson S.W. Sonoda G. Tanno S. Golemis E.A. Testa J.R. Oncogene. 1999; 18: 4891-4898Crossref PubMed Scopus (172) Google Scholar). The interaction between APPL1 and Akt is required for insulin-stimulated GLUT4 translocation (8.Saito T. Jones C.C. Huang S. Czech M.P. Pilch P.F. J. Biol. Chem. 2007; 282: 32280-32287Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar) and for controlling Akt substrate selectivity (9.Schenck A. Goto-Silva L. Collinet C. Rhinn M. Giner A. Habermann B. Brand M. Zerial M. Cell. 2008; 133: 486-497Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). It has been shown that APPL1-potentiated Akt activity to suppress androgen receptor transactivation in prostate cancer cells (10.Yang L. Lin H.K. Altuwaijri S. Xie S. Wang L. Chang C. J. Biol. Chem. 2003; 278: 16820-16827Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). APPL1 has also been suggested to function as an adaptor protein in regulating follicle-stimulated hormone (FSH)-mediated PI 3-kinase/Akt signaling pathway (11.Nechamen C.A. Thomas R.M. Dias J.A. Mol. Cell. Endocrinol. 2007; 260–262: 93-99Crossref PubMed Scopus (77) Google Scholar, 12.Nechamen C.A. Thomas R.M. Cohen B.D. Acevedo G. Poulikakos P.I. Testa J.R. Dias J.A. Biol. Reprod. 2004; 71: 629-636Crossref PubMed Scopus (92) Google Scholar). Our results showed that APPL1 binds directly to the intracellular part of the adiponectin receptors and positively mediates adiponectin signaling to the AMPK and p38 MAPK pathways, leading to increased glucose uptake and fatty acid oxidation in muscle cells (6.Mao X. Kikani C.K. Riojas R.A. Langlais P. Wang L. Ramos F.J. Fang Q. Christ-Roberts C.Y. Hong J.Y. Kim R.Y. Liu F. Dong L.Q. Nat. Cell Biol. 2006; 8: 516-523Crossref PubMed Scopus (535) Google Scholar). In addition, we found that APPL1 plays a critical role in regulating the cross-talk between adiponectin and insulin signaling pathways (6.Mao X. Kikani C.K. Riojas R.A. Langlais P. Wang L. Ramos F.J. Fang Q. Christ-Roberts C.Y. Hong J.Y. Kim R.Y. Liu F. Dong L.Q. Nat. Cell Biol. 2006; 8: 516-523Crossref PubMed Scopus (535) Google Scholar). APPL1 has since been found to mediate adiponectin signaling in other types of cells such as endothelial cells to regulate nitric oxide production and endothelium-dependent vasodilation (13.Cheng K.K. Lam K.S. Wang Y. Huang Y. Carling D. Wu D. Wong C. Xu A. Diabetes. 2007; 56: 1387-1394Crossref PubMed Scopus (273) Google Scholar) and to protect from IL-18-mediated cell death (14.Chandrasekar B. Boylston W.H. Venkatachalam K. Webster N.J. Prabhu S.D. Valente A.J. J. Biol. Chem. 2008; 283: 24889-24898Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). A recent study also suggested that APPL1 has a potentiating effect on insulin-stimulated suppression of hepatic glucose production in mice (15.Cheng K.K. Iglesia M.A. Lam K.S. Wang Y. Sweeney G. Zhu W. Vanhoutte P.M. Kraegen E.W. Xu A. Cell Metab. 2009; 9: 417-427Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). These evidence suggest that APPL1 is an essential mediator in both adiponectin and insulin signaling, which are the two major pathways regulating energy homeostasis.APPL2 is an isoform of APPL1, and these two proteins display 54% identity in protein sequences (6.Mao X. Kikani C.K. Riojas R.A. Langlais P. Wang L. Ramos F.J. Fang Q. Christ-Roberts C.Y. Hong J.Y. Kim R.Y. Liu F. Dong L.Q. Nat. Cell Biol. 2006; 8: 516-523Crossref PubMed Scopus (535) Google Scholar, 16.Miaczynska M. Christoforidis S. Giner A. Shevchenko A. Uttenweiler-Joseph S. Habermann B. Wilm M. Parton R.G. Zerial M. Cell. 2004; 116: 445-456Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar). Similar to APPL1, APPL2 contains an N-terminal BAR domain, a central PH domain, and a C-terminal PTB domain. It has been shown that APPL2 is essential for cell proliferation and embryonic development (9.Schenck A. Goto-Silva L. Collinet C. Rhinn M. Giner A. Habermann B. Brand M. Zerial M. Cell. 2008; 133: 486-497Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 16.Miaczynska M. Christoforidis S. Giner A. Shevchenko A. Uttenweiler-Joseph S. Habermann B. Wilm M. Parton R.G. Zerial M. Cell. 2004; 116: 445-456Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar). Recent studies indicated that APPL2 regulates FSH signaling by forming a dimer with APPL1 via the BAR domains of the isoforms, leading to the formation of a complex with the FSH receptor, APPL1 and Akt2. However, unlike APPL1, APPL2 does not directly interact with Akt2 (11.Nechamen C.A. Thomas R.M. Dias J.A. Mol. Cell. Endocrinol. 2007; 260–262: 93-99Crossref PubMed Scopus (77) Google Scholar, 17.Chial H.J. Wu R. Ustach C.V. McPhail L.C. Mobley W.C. Chen Y.Q. Traffic. 2008; 9: 215-229Crossref PubMed Scopus (44) Google Scholar). The role of APPL2 in adiponectin and insulin signaling remains largely unknown.In the present study, we show that APPL2 functions as a negative regulator in adiponectin signaling. APPL2 competes with APPL1 in the binding to AdipoR1 and blocks adiponectin signaling in muscle cells. In addition, APPL2 also sequesters APPL1 from adiponectin signal pathway by forming a heterodimer with APPL1. Adiponectin as well as metformin induces a dissociation of this complex. These results provide a mechanism for negative regulation of adiponectin signaling and adiponectin-regulated glucose and lipid metabolism in muscle cells. Furthermore, APPL2 blocks APPL1-mediated insulin-sensitizing effect of adiponectin in muscle cells by heterodimerizing with APPL1 to inhibit insulin-stimulated Akt activation. Our data indicate that APPL isoforms function as an integrated Yin-Yang regulator in adiponectin signaling and reveals a novel molecular mechanism for adiponectin and insulin resistance.
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