Generic placeholder image

Endocrine, Metabolic & Immune Disorders - Drug Targets

Author(s): Yao Wang, Hui Lian, Xitong Wang, Tianyu Zheng, Xiaoxiao Yu, Ruzhang Chen, Zhiyong Huang, Yinxiang Lv, Ai Zhao* and Jimin Gao*

DOI: 10.2174/1871530320666200121100449

DownloadDownload PDF Flyer Cite As
Characterization of the Active Components of the Multimerized sTNFRII-Adiponectin Fusion Protein Showing Both TNFα-Antagonizing and Glucose Uptake-Promoting Activities

Page: [1081 - 1089] Pages: 9

  • * (Excluding Mailing and Handling)

Abstract

Background: The sTNFRII-adiponectin fusion protein previously showed strong TNFα antagonistic activity. However, the fusion protein exists as mixture of different multimers. The aim of the present study was to characterize its active components.

Methods: In this study, the fusion protein was isolated and purified by Ni-NTA affinity and gel exclusion chromatography, and further identified by Coomassie staining and western blotting. The TNFα antagonistic and glucose uptake-promoting activities were determined in vitro. The glucose detection kit and 2- NBDG (2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose) were used to measure their effects on glucose metabolism (including glucose consumption and glucose uptake in HepG2 and H9C2 cells). The effect of the fusion protein on glucose uptake was also examined in free fatty acid (FFA)- induced insulin resistance cell model.

Results: The sTNFRII-adiponectin fusion protein was found to exist in three forms: 250 kDa (hexamer), 130 kDa (trimer), and 60 kDa (monomer), with the final purity of 90.2%, 60.1%, and 81.6%, respectively. The fusion protein could effectively antagonize the killing effect of TNFα in L929 cells, and the multimer was found to be superior to the monomer. In addition, the fusion protein could increase glucose consumption without impacting the number of cells (HepG2, H9C2 cells) in a dosedependent manner. Mechanistically, glucose uptake was found to be enhanced by the translocation of GLUT4. However, it could not improve glucose uptake in the cell model of insulin resistance.

Conclusion: In summary, the active components of the fusion protein are hexamers and trimers. The hexamer and trimer of sTNFRII-adiponectin fusion protein had both TNFα-antagonizing and glucose uptake-promoting activities, although neither of them could improve glucose uptake in the cell model of insulin resistance.

Keywords: Fusion protein, soluble TNFRII, adiponectin, TNFα antagonist, multimer, insulin resistance.

Graphical Abstract

[1]
Achari, A.E.; Jain, S.K. Adiponectin, a Therapeutic Target for Obesity, Diabetes, and Endothelial Dysfunction. Int. J. Mol. Sci., 2017, 18(6)E1321
[http://dx.doi.org/10.3390/ijms18061321] [PMID: 28635626]
[2]
Odegaard, J.I.; Chawla, A. Pleiotropic actions of insulin resistance and inflammation in metabolic homeostasis. Science, 2013, 339(6116), 172-177.
[http://dx.doi.org/10.1126/science.1230721] [PMID: 23307735]
[3]
Esser, N.; Legrand-Poels, S.; Piette, J.; Scheen, A.J.; Paquot, N. Inflammation as a link between obesity, metabolic syndrome and type 2 diabetes. Diabetes Res. Clin. Pract., 2014, 105(2), 141-150.
[http://dx.doi.org/10.1016/j.diabres.2014.04.006] [PMID: 24798950]
[4]
Borst, S.E. The role of TNF-alpha in insulin resistance. Endocrine, 2004, 23(2-3), 177-182.
[http://dx.doi.org/10.1385/ENDO:23:2-3:177] [PMID: 15146098]
[5]
Hotamisligil, G.S. Inflammation and metabolic disorders. Nature, 2006, 444(7121), 860-867.
[http://dx.doi.org/10.1038/nature05485] [PMID: 17167474]
[6]
Afshin, A.; Forouzanfar, M.H.; Reitsma, M.B.; Sur, P.; Estep, K.; Lee, A.; Marczak, L.; Mokdad, A.H.; Moradi-Lakeh, M.; Naghavi, M.; Salama, J.S.; Vos, T.; Abate, K.H.; Abbafati, C.; Ahmed, M.B.; Al-Aly, Z.; Alkerwi, A.; Al-Raddadi, R.; Amare, A.T.; Amberbir, A.; Amegah, A.K.; Amini, E.; Amrock, S.M.; Anjana, R.M.; Ärnlöv, J.; Asayesh, H.; Banerjee, A.; Barac, A.; Baye, E.; Bennett, D.A.; Beyene, A.S.; Biadgilign, S.; Biryukov, S.; Bjertness, E.; Boneya, D.J.; Campos-Nonato, I.; Carrero, J.J.; Cecilio, P.; Cercy, K.; Ciobanu, L.G.; Cornaby, L.; Damtew, S.A.; Dandona, L.; Dandona, R.; Dharmaratne, S.D.; Duncan, B.B.; Eshrati, B.; Esteghamati, A.; Feigin, V.L.; Fernandes, J.C.; Fürst, T.; Gebrehiwot, T.T.; Gold, A.; Gona, P.N.; Goto, A.; Habtewold, T.D.; Hadush, K.T.; Hafezi-Nejad, N.; Hay, S.I.; Horino, M.; Islami, F.; Kamal, R.; Kasaeian, A.; Katikireddi, S.V.; Kengne, A.P.; Kesavachandran, C.N.; Khader, Y.S.; Khang, Y.H.; Khubchandani, J.; Kim, D.; Kim, Y.J.; Kinfu, Y.; Kosen, S.; Ku, T.; Defo, B.K.; Kumar, G.A.; Larson, H.J.; Leinsalu, M.; Liang, X.; Lim, S.S.; Liu, P.; Lopez, A.D.; Lozano, R.; Majeed, A.; Malekzadeh, R.; Malta, D.C.; Mazidi, M.; McAlinden, C.; McGarvey, S.T.; Mengistu, D.T.; Mensah, G.A.; Mensink, G.B.M.; Mezgebe, H.B.; Mirrakhimov, E.M.; Mueller, U.O.; Noubiap, J.J.; Obermeyer, C.M.; Ogbo, F.A.; Owolabi, M.O.; Patton, G.C.; Pourmalek, F.; Qorbani, M.; Rafay, A.; Rai, R.K.; Ranabhat, C.L.; Reinig, N.; Safiri, S.; Salomon, J.A.; Sanabria, J.R.; Santos, I.S.; Sartorius, B.; Sawhney, M.; Schmidhuber, J.; Schutte, A.E.; Schmidt, M.I.; Sepanlou, S.G.; Shamsizadeh, M.; Sheikhbahaei, S.; Shin, M.J.; Shiri, R.; Shiue, I.; Roba, H.S.; Silva, D.A.S.; Silverberg, J.I.; Singh, J.A.; Stranges, S.; Swaminathan, S.; Tabarés-Seisdedos, R.; Tadese, F.; Tedla, B.A.; Tegegne, B.S.; Terkawi, A.S.; Thakur, J.S.; Tonelli, M.; Topor-Madry, R.; Tyrovolas, S.; Ukwaja, K.N.; Uthman, O.A.; Vaezghasemi, M.; Vasankari, T.; Vlassov, V.V.; Vollset, S.E.; Weiderpass, E.; Werdecker, A.; Wesana, J.; Westerman, R.; Yano, Y.; Yonemoto, N.; Yonga, G.; Zaidi, Z.; Zenebe, Z.M.; Zipkin, B.; Murray, C.J.L. GBD 2015 Obesity Collaborators. Health Effects of Overweight and Obesity in 195 Countries over 25 Years. N. Engl. J. Med., 2017, 377(1), 13-27.
[http://dx.doi.org/10.1056/NEJMoa1614362] [PMID: 28604169]
[7]
Tzanavari, T.; Giannogonas, P.; Karalis, K.P. TNF-alpha and obesity. Curr. Dir. Autoimmun., 2010, 11, 145-156.
[http://dx.doi.org/10.1159/000289203] [PMID: 20173393]
[8]
Akash, M.S.H.; Rehman, K.; Liaqat, A. Tumor Necrosis Factor-Alpha: Role in Development of Insulin Resistance and Pathogenesis of Type 2 Diabetes Mellitus. J. Cell. Biochem., 2018, 119(1), 105-110.
[http://dx.doi.org/10.1002/jcb.26174] [PMID: 28569437]
[9]
Stanley, T.L.; Zanni, M.V.; Johnsen, S.; Rasheed, S.; Makimura, H.; Lee, H.; Khor, V.K.; Ahima, R.S.; Grinspoon, S.K. TNF-alpha antagonism with etanercept decreases glucose and increases the proportion of high molecular weight adiponectin in obese subjects with features of the metabolic syndrome. J. Clin. Endocrinol. Metab., 2011, 96(1), E146-E150.
[http://dx.doi.org/10.1210/jc.2010-1170] [PMID: 21047923]
[10]
Esser, N.; Paquot, N.; Scheen, A.J. Anti-inflammatory agents to treat or prevent type 2 diabetes, metabolic syndrome and cardiovascular disease. Expert Opin. Investig. Drugs, 2015, 24(3), 283-307.
[http://dx.doi.org/10.1517/13543784.2015.974804] [PMID: 25345753]
[11]
Liu, C.; Feng, X.; Li, Q.; Wang, Y.; Li, Q.; Hua, M. Adiponectin, TNF-α and inflammatory cytokines and risk of type 2 diabetes: A systematic review and meta-analysis. Cytokine, 2016, 86, 100-109.
[http://dx.doi.org/10.1016/j.cyto.2016.06.028] [PMID: 27498215]
[12]
Monaco, C.; Nanchahal, J.; Taylor, P.; Feldmann, M. Anti-TNF therapy: past, present and future. Int. Immunol., 2015, 27(1), 55-62.
[http://dx.doi.org/10.1093/intimm/dxu102] [PMID: 25411043]
[13]
Serio, I.; Tovoli, F. Rheumatoid arthritis: new monoclonal antibodies Drugs of today (Barcelona, Spain : 1998) 2018, 54(3), 219-230.
[http://dx.doi.org/10.1358/dot.2018.54.3.2788019]
[14]
Combs, T.P.; Marliss, E.B. Adiponectin signaling in the liver. Rev. Endocr. Metab. Disord., 2014, 15(2), 137-147.
[http://dx.doi.org/10.1007/s11154-013-9280-6] [PMID: 24297186]
[15]
Adolph, T.E.; Grander, C.; Grabherr, F.; Tilg, H. Adipokines and Non-Alcoholic Fatty Liver Disease: Multiple Interactions. Int. J. Mol. Sci., 2017, 18(8)E1649
[http://dx.doi.org/10.3390/ijms18081649] [PMID: 28758929]
[16]
Ghadge, A.A.; Khaire, A.A.; Kuvalekar, A.A. Adiponectin: A potential therapeutic target for metabolic syndrome. Cytokine Growth Factor Rev., 2018, 39, 151-158.
[http://dx.doi.org/10.1016/j.cytogfr.2018.01.004] [PMID: 29395659]
[17]
Goto, M.; Goto, A.; Morita, A.; Deura, K.; Sasaki, S.; Aiba, N.; Shimbo, T.; Terauchi, Y.; Miyachi, M.; Noda, M.; Watanabe, S. Saku Cohort Study Group. Low-molecular-weight adiponectin and high-molecular-weight adiponectin levels in relation to diabetes. Obesity (Silver Spring), 2014, 22(2), 401-407.
[http://dx.doi.org/10.1002/oby.20553] [PMID: 23818415]
[18]
Croft, M.; Siegel, R.M.; Beyond, T.N.F. Beyond TNF: TNF superfamily cytokines as targets for the treatment of rheumatic diseases. Nat. Rev. Rheumatol., 2017, 13(4), 217-233.
[http://dx.doi.org/10.1038/nrrheum.2017.22] [PMID: 28275260]
[19]
Yanai, H.; Yoshida, H. Beneficial Effects of Adiponectin on Glucose and Lipid Metabolism and Atherosclerotic Progression: Mechanisms and Perspectives. Int. J. Mol. Sci., 2019, 20(5)E1190
[http://dx.doi.org/10.3390/ijms20051190] [PMID: 30857216]
[20]
Lihn, A.S.; Pedersen, S.B.; Richelsen, B. Adiponectin: action, regulation and association to insulin sensitivity. Obes. Rev., 2005, 6(1), 13-21.
[http://dx.doi.org/10.1111/j.1467-789X.2005.00159.x] [PMID: 15655035]
[21]
Wang, Z.V.; Scherer, P.E. Adiponectin, the past two decades. J. Mol. Cell Biol., 2016, 8(2), 93-100.
[http://dx.doi.org/10.1093/jmcb/mjw011] [PMID: 26993047]
[22]
Liu, M.; Liu, F. Regulation of adiponectin multimerization, signaling and function. Best Pract. Res. Clin. Endocrinol. Metab., 2014, 28(1), 25-31.
[http://dx.doi.org/10.1016/j.beem.2013.06.003] [PMID: 24417943]
[23]
Liu, M.; Liu, F. Transcriptional and post-translational regulation of adiponectin. Biochem. J., 2009, 425(1), 41-52.
[http://dx.doi.org/10.1042/BJ20091045] [PMID: 20001961]
[24]
Qiang, L.; Wang, H.; Farmer, S.R. Adiponectin secretion is regulated by SIRT1 and the endoplasmic reticulum oxidoreductase Ero1-L alpha. Mol. Cell. Biol., 2007, 27(13), 4698-4707.
[http://dx.doi.org/10.1128/MCB.02279-06] [PMID: 17452443]
[25]
Wang, Z.V.; Scherer, P.E. DsbA-L is a versatile player in adiponectin secretion. Proc. Natl. Acad. Sci. USA, 2008, 105(47), 18077-18078.
[http://dx.doi.org/10.1073/pnas.0810027105] [PMID: 19020096]
[26]
Mai, S.; Walker, G.E.; Brunani, A.; Guzzaloni, G.; Grossi, G.; Oldani, A.; Aimaretti, G.; Scacchi, M.; Marzullo, P. Inherent insulin sensitivity is a major determinant of multimeric adiponectin responsiveness to short-term weight loss in extreme obesity. Sci. Rep., 2014, 4, 5803.
[http://dx.doi.org/10.1038/srep05803] [PMID: 25056918]
[27]
Sowers, J.R. Endocrine functions of adipose tissue: focus on adiponectin. Clin. Cornerstone, 2008, 9(1), 32-38.
[http://dx.doi.org/10.1016/S1098-3597(08)60026-5] [PMID: 19046738]
[28]
Semaan, D.G.; Igoli, J.O.; Young, L.; Gray, A.I.; Rowan, E.G.; Marrero, E. In vitro anti-diabetic effect of flavonoids and pheophytins from Allophylus cominia Sw. on the glucose uptake assays by HepG2, L6, 3T3-L1 and fat accumulation in 3T3-L1 adipocytes. J. Ethnopharmacol., 2018, 216, 8-17.
[http://dx.doi.org/10.1016/j.jep.2018.01.014] [PMID: 29339110]
[29]
Zhang, X.; Wang, S.; Li, Y.; Zhao, D.; An, N.; Wu, J.; Zhang, T.; Wu, C.; Li, Y. Tadehaginoside modulates lipogenesis and glucose consumption in HepG2 cells. Nat. Prod. Res., 2015, 29(24), 2287-2290.
[http://dx.doi.org/10.1080/14786419.2014.1001387] [PMID: 25589148]
[30]
Cammisotto, P.G.; Bendayan, M. Adiponectin stimulates phosphorylation of AMP-activated protein kinase alpha in renal glomeruli. J. Mol. Histol., 2008, 39(6), 579-584.
[http://dx.doi.org/10.1007/s10735-008-9198-6] [PMID: 18941912]
[31]
Iwabu, M.; Yamauchi, T.; Okada-Iwabu, M.; Sato, K.; Nakagawa, T.; Funata, M.; Yamaguchi, M.; Namiki, S.; Nakayama, R.; Tabata, M.; Ogata, H.; Kubota, N.; Takamoto, I.; Hayashi, Y.K.; Yamauchi, N.; Waki, H.; Fukayama, M.; Nishino, I.; Tokuyama, K.; Ueki, K.; Oike, Y.; Ishii, S.; Hirose, K.; Shimizu, T.; Touhara, K.; Kadowaki, T. Adiponectin and AdipoR1 regulate PGC-1alpha and mitochondria by Ca(2+) and AMPK/SIRT1. Nature, 2010, 464(7293), 1313-1319.
[http://dx.doi.org/10.1038/nature08991] [PMID: 20357764]
[32]
Han, P.; Zhang, Y.Y.; Lu, Y.; He, B.; Zhang, W.; Xia, F. Effects of different free fatty acids on insulin resistance in rats. HBPD INT, 2008, 7(1), 91-96.
[PMID: 18234646]
[33]
Luo, Y.; Rana, P.; Will, Y. Palmitate increases the susceptibility of cells to drug-induced toxicity: an in vitro method to identify drugs with potential contraindications in patients with metabolic disease Toxico-logical sciences An official journal of the Society of Toxicology, 2012, 192(2), 346-62.
[34]
Legrand-Poels, S.; Esser, N.; L’homme, L.; Scheen, A.; Paquot, N.; Piette, J. Free fatty acids as modulators of the NLRP3 inflammasome in obesity/type 2 diabetes. Biochem. Pharmacol., 2014, 92(1), 131-141.
[http://dx.doi.org/10.1016/j.bcp.2014.08.013] [PMID: 25175736]
[35]
Delarue, J.; Magnan, C. Free fatty acids and insulin resistance. Curr. Opin. Clin. Nutr. Metab. Care, 2007, 10(2), 142-148.
[http://dx.doi.org/10.1097/MCO.0b013e328042ba90] [PMID: 17285001]
[36]
Gutiérrez-Rodelo, C.; Roura-Guiberna, A.; Olivares-Reyes, J.A. Molecular Mechanisms of Insulin Resistance: An Update Gac. Med. Mex., 2017, 153(2), 214-228.
[PMID: 28474708]
[37]
Jaiswal, N.; Gunaganti, N.; Maurya, C.K.; Narender, T.; Tamrakar, A.K. Free fatty acid induced impairment of insulin signaling is prevented by the diastereomeric mixture of calophyllic acid and isocalophyllic acid in skeletal muscle cells. Eur. J. Pharmacol., 2015, 746, 70-77.
[http://dx.doi.org/10.1016/j.ejphar.2014.10.049] [PMID: 25445050]
[38]
Armoni, M.; Harel, C.; Bar-Yoseph, F.; Milo, S.; Karnieli, E. Free fatty acids repress the GLUT4 gene expression in cardiac muscle via novel response elements. J. Biol. Chem., 2005, 280(41), 34786-34795.
[http://dx.doi.org/10.1074/jbc.M502740200] [PMID: 16096283]
[39]
Alam, F.; Islam, M.A.; Khalil, M.I.; Gan, S.H. Metabolic Control of Type 2 Diabetes by Targeting the GLUT4 Glucose Transporter: Intervention Approaches. Curr. Pharm. Des., 2016, 22(20), 3034-3049.
[http://dx.doi.org/10.2174/1381612822666160307145801] [PMID: 26951104]