Pyroglutamated Apelin-13 Inhibits Lipopolysaccharide-Induced Production of Pro-Inflammatory Cytokines in Murine Macrophage J774.1 Cells


Apelin, recently identified as an endogenous ligand of the orphan G protein-coupled receptor APJ, has multiple pathophysiological properties. In the present study, we investigated whether pyroglutamated apelin-13 ([Pyr1]-apelin-13), the most highly active isoform among the mature apelin peptide family, modulates the effect of bacterial lipopolysaccharide (LPS) on cytokine induction in a murine macrophage-like cell line, J774.1 cells. J774.1 cells expressed the APJ protein in a stationary state, and the expression of APJ was not affected by LPS stimulation. No significant effect of [Pyr1]-apelin-13 treatment alone was observed on the proliferation or cytokine production of J774.1 cells in the stationary state. However, prior to LPS stimulation, pretreatment with [Pyr1]apelin-13 for 16 h significantly diminished mRNA expression and protein secretion of inflammatory cytokine interleukin-6, which was confirmed by RT-PCR and ELISA, respectively. Western blot analysis revealed that the phosphorylation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase, but not extracellular signal-regulated kinase, which was induced by LPS, significantly decreased in [Pyr1]-apelin-13-pretreated J774.1 cells compared with untreated cells. These observations suggest that [Pyr1]-apelin-13 functions as a negative regulator of LPS-mediated pro-inflammatory responses in macrophages.

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Obara, S. , Akifusa, S. , Ariyoshi, W. , Okinaga, T. , Usui, M. , Nakashima, K. and Nishihara, T. (2014) Pyroglutamated Apelin-13 Inhibits Lipopolysaccharide-Induced Production of Pro-Inflammatory Cytokines in Murine Macrophage J774.1 Cells. Modern Research in Inflammation, 3, 59-66. doi: 10.4236/mri.2014.32007.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Pitkin, S.L., Maguire, J.J., Bonner, T.I. and Davenport, A.P. (2010) International Union of Basic and Clinical Pharmacology. LXXIV. Apelin Receptor Nomenclature, Distribution, Pharmacology, and Function. Pharmacological Review, 62, 331-342.
[2] O’Carroll, A.M., Lolait, S.J., Harris, L.E. and Pope, G.R. (2013) The Apelin Receptor APJ: Journey from an Orphan to a Multifaceted Regulator of Homeostasis. Journal of Endocrinology, 219, R13-R35.
[3] Kalin, R.E., Kretz, M.P., Meyer, A.M., Kispert, A., Heppner, F.L. and Brandli, A.W. (2007) Paracrine and Autocrine Mechanisms of Apelin Signaling Govern Embryonic and Tumor Angiogenesis. Developmental Biology, 305, 599-614.
[4] Carpene, C., Dray, C., Attane, C., Valet, P., Portillo, M.P., Churruca, I., Milagro, F.I. and Castan-Laurell, I. (2007) Expanding Role for the Apelin/APJ System in Physiopathology. Journal of Physiology and Biochemistry, 63, 359-373.
[5] Medhurst, A.D., Jennings, C.A., Robbins, M.J., Davis, R.P., Ellis, C., Winborn, K.Y., Lawrie, K.W., Hervieu, G., Riley, G., Bolaky, J.E., Herrity, N.C., Murdock, P. and Darker, J.G. (2003) Pharmacological and Immunohistochemical Characterization of the APJ Receptor and Its Endogenous Ligand Apelin. Journal of Neurochemistry, 84, 1162-1172.
[6] O’Carroll, A.M., Selby, T.L., Palkovits, M. and Lolait, S.J. (2000) Distribution of mRNA Encoding B78/apj, the Rat Homologue of the Human APJ Receptor, and Its Endogenous Ligand Apelin in Brain and Peripheral Tissues. Biochimica et Biophysica Acta, 1492, 72-80.
[7] Falcao-Pires, I., Ladeiras-Lopes, R. and Leite-Moreira, A.F. (2010) The Apelinergic System: A Promising Therapeutic Target. Expert Opinion on Therapeutic Targets, 14, 633-645.
[8] Boucher, J., Masri, B., Daviaud, D., Gesta, S., Guigne, C., Mazzucotelli, A., Castan-Laurell, I., Tack, I., Knibiehler, B., Carpene, C., Audigier, Y., Saulnier-Blache, J.S. and Valet, P. (2005) Apelin, a Newly Identified Adipokine Up-Regulated by Insulin and Obesity. Endocrinology, 146, 1764-1771.
[9] Ishida, J., Hashimoto, T., Hashimoto, Y., Nishiwaki, S., Iguchi, T., Harada, S., Sugaya, T., Matsuzaki, H., Yamamoto, R., Shiota, N., Okunishi, H., Kihara, M., Umemura, S., Sugiyama, F., Yagami, K., Kasuya, Y., Mochizuki, N. and Fukamizu, A. (2004) Regulatory Roles for APJ, a Seven-Transmembrane Receptor Related to Angiotensin-Type 1 Receptor in Blood Pressure in Vivo. The Journal of Biological Chemistry, 279, 26274-26279.
[10] Salcedo, A., Garijo, J., Monge, L., Fernandez, N., Luis Garcia-Villalon, A., Sanchez Turrion, V., Cuervas-Mons, V. and Dieguez, G. (2007) Apelin Effects in Human Splanchnic Arteries. Role of Nitric Oxide and Prostanoids. Regulatory Peptides, 144, 50-55.
[11] Sorli, S.C., van den Berghe, L., Masri, B., Knibiehler, B. and Audigier, Y. (2006) Therapeutic Potential of Interfering with Apelin Signalling. Drug Discovery Today, 11, 1100-1106.
[12] Zhu, S., Sun, F., Li, W., Cao, Y., Wang, C., Wang, Y., Liang, D., Zhang, R., Zhang, S., Wang, H. and Cao, F. (2011) Apelin Stimulates Glucose Uptake through the PI3K/Akt Pathway and Improves Insulin Resistance in 3T3-L1 Adipocytes. Molecular and Cellular Biochemistry, 353, 305-313.
[13] Castan-Laurell, I., Vitkova, M., Daviaud, D., Dray, C., Kovacikova, M., Kovacova, Z., Hejnova, J., Stich, V. and Valet, P. (2008) Effect of Hypocaloric Diet-Induced Weight Loss in Obese Women on Plasma Apelin and Adipose Tissue Expression of Apelin and APJ. European Journal of Endocrinology, 158, 905-910.
[14] Li, L., Yang, G., Li, Q., Tang, Y., Yang, M., Yang, H. and Li, K. (2006) Changes and Relations of Circulating Visfatin, Apelin, and Resistin Levels in Normal, Impaired Glucose Tolerance, and Type 2 Diabetic Subjects. Experimental and Clinical Endocrinology & Diabetes, 114, 544-548.
[15] Habata, Y., Fujii, R., Hosoya, M., Fukusumi, S., Kawamata, Y., Hinuma, S., Kitada, C., Nishizawa, N., Murosaki, S., Kurokawa, T., Onda, H., Tatemoto, K. and Fujino, M. (1999) Apelin, the Natural Ligand of the Orphan Receptor APJ, Is Abundantly Secreted in the Colostrum. Biochimica et Biophysica Acta, 1452, 25-35.
[16] Horiuchi, Y., Fujii, T., Kamimura, Y. and Kawashima, K. (2003) The Endogenous, Immunologically Active Peptide Apelin Inhibits Lymphocytic Cholinergic Activity during Immunological Responses. Journal of Neuroimmunology, 144, 46-52.
[17] Choe, H., Farzan, M., Konkel, M., Martin, K., Sun, Y., Marcon, L., Cayabyab, M., Berman, M., Dorf, M.E., Gerard, N., Gerard, C. and Sodroski, J. (1998) The Orphan Seven-Transmembrane Receptor Apj Supports the Entry of Primary T-Cell-Line-Tropic and Dualtropic Human Immunodeficiency Virus Type 1. Journal of Virology, 72, 6113-6118.
[18] Leeper, N.J., Tedesco, M.M., Kojima, Y., Schultz, G.M., Kundu, R.K., Ashley, E.A., Tsao, P.S., Dalman, R.L. and Quertermous, T. (2009) Apelin Prevents Aortic Aneurysm Formation by Inhibiting Macrophage Inflammation. American Journal of Physiology-Heart and Circulatory Physiology, 296, H1329-H1335.
[19] Lu, H., Gauvreau, D., Tom, F.Q., Lapointe, M., Luo, X.P. and Cianflone, K. (2013) Inflammatory Markers and Adipokines Alter Adipocyte-Derived ASP Production through Direct and Indirect Immune Interaction. Experimental and Clinical Endocrinology & Diabetes, 121, 194-200.
[20] Liu, X.Y., Lu, Q., Ouyang, X.P., Tang, S.L., Zhao, G.J., Lv, Y.C., He, P.P., Kuang, H.J., Tang, Y.Y., Fu, Y., Zhang, D.W. and Tang, C.K. (2013) Apelin-13 Increases Expression of ATP-Binding Cassette Transporter A1 via Activating Protein Kinase C Alpha Signaling in THP-1 Macrophage-Derived Foam Cells. Atherosclerosis, 226, 398-407.
[21] Lago, F., Dieguez, C., Gomez-Reino, J. and Gualillo, O. (2007) Adipokines as Emerging Mediators of Immune Response and Inflammation. Nature Clinical Practice Rheumatology, 3, 716-724.
[22] Falcao-Pires, I., Castro-Chaves, P., Miranda-Silva, D., Lourenco, A.P. and Leite-Moreira, A.F. (2012) Physiological, Pathological and Potential Therapeutic Roles of Adipokines. Drug Discovery Today, 17, 880-889.
[23] Chi, H., Barry, S.P., Roth, R.J., Wu, J.J., Jones, E.A., Bennett, A.M. and Flavell, R.A. (2006) Dynamic Regulation of Pro- and Anti-Inflammatory Cytokines by MAPK Phosphatase 1 (MKP-1) in Innate Immune Responses. Proceedings of the National Academy of Sciences of the United States of America, 103, 2274-2279.
[24] Chan, E.D. and Riches, D.W. (2001) IFN-Gamma + LPS Induction of iNOS Is Modulated by ERK, JNK/SAPK, and p38(mapk) in a Mouse Macrophage Cell Line. American Journal of Physiology. Cell Physiology, 280, C441-C450.
[25] Folco, E.J., Rocha, V.Z., Lopez-Ilasaca, M. and Libby, P. (2009) Adiponectin Inhibits Pro-Inflammatory Signaling in Human Macrophages Independent of Interleukin-10. Journal of Biological Chemistry, 284, 25569-25575.
[26] Kamio, N., Akifusa, S., Yamaguchi, N., Nonaka, K. and Yamashita, Y. (2009) Anti-Inflammatory Activity of a Globular Adiponectin Function on RAW 264 Cells Stimulated by Lipopolysaccharide from Aggregatibacter actinomycetemcomitans. FEMS Immunology & Medical Microbiology, 56, 241-247.
[27] Park, P.H., Huang, H., McMullen, M.R., Mandal, P., Sun, L. and Nagy, L.E. (2008) Suppression of LipopolysaccharideStimulated Tumor Necrosis Factor-Alpha Production by Adiponectin Is Mediated by Transcriptional and Post-Transcriptional Mechanisms. Journal of Biological Chemistry, 283, 26850-26858.
[28] Mandal, P., Park, P.H., McMullen, M.R., Pratt, B.T. and Nagy, L.E. (2010) The Anti-Inflammatory Effects of Adiponectin Are Mediated via a Heme Oxygenase-1-Dependent Pathway in Rat Kupffer Cells. Hepatology, 51, 1420-1429.
[29] Mandal, P., Pratt, B.T., Barnes, M., McMullen, M.R. and Nagy, L.E. (2011) Molecular Mechanism for AdiponectinDependent M2 Macrophage Polarization: Link between the Metabolic and Innate Immune Activity of Full-Length Adiponectin. Journal of Biological Chemistry, 286, 13460-13469.

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