Effect of A-769662, a direct AMPK activator, on Tlr-4 expression and activity in mice heart tissue

Document Type: Original Article

Authors

1 Department of Pharmacology and Toxicology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran

2 Student Research Committee, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran

3 Department of Pharmacology, Faculty of Pharmacy, Urmia University of Medical Sciences, Urmia, Iran

4 Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran

Abstract

Objective(s): TLR-4 activates a number of inflammatory signaling pathways. Also, AMPK could be involved in anti-inflammatory signaling. The aim of this study was to identify whether stimulation of AMPK could inhibit LPS-induced Tlr-4 gene expression in mice hearts.
Materials and methods: Heart AMPK activity and/or Tlr-4 expression was stimulated in different mice groups, using respectively IP injection of A-769662 (10 mg/kg) and LPS (2 mg/kg) or a combination of both agents. Moreover, compound-C (20 mg/kg), as an AMPK antagonist, was intraperitoneally co-administrated with both A-769662 and LPS in another group to investigate the role of AMPK activity on Tlr-4 regulation. After 8 hr, in addition to peripheral neutrophil cell count, myocardial p-AMPK, p-ACC as well as MyD88 protein contents and Tlr-4 expression was assessed by Western blotting and real-time qRT-PCR, respectively. TNF-α and IL-6 expression levels were also determined by ELISA.
Results: LPS induced heart Tlr-4 expression (P<0.001) associating with an increase in the myocardial MyD88 protein content (P<0.001), elevation of heart TNF-α (P<0.01) and IL-6 (P<0.05) concentrations, and rise in the peripheral neutrophil cell count (P<0.001). Administration of A-769662 decreased LPS-induced Tlr-4 expression (P<0.01) and alleviated peripheral neutrophil cell count (P<0.01). The inhibitory effect of A-769662 on LPS-induced Tlr-4 expression was reversed by antagonizing AMPK with compound-C (P<0.001) which reduced p-AMPK (P<0.05) and p-ACC (P<0.01) myocardial protein contents in the LPS+A-769662 group.
Conclusion: This study demonstrated that activation of AMPK, by A-769662 agent, could inhibit Tlr-4 expression and activity, suggesting a link between AMPK and Tlr-4 in heart tissue.

Keywords


1.   Fang Y, Hu J. Toll-like receptor and its roles in myocardial ischemic/reperfusion injury. Medical science monitor : international medical journal of experimental and clinical research 2011;17:RA100-109.

2.   Timmers L, Pasterkamp G, de Hoog VC, Arslan F, Appelman Y, de Kleijn DP. The innate immune response in reperfused myocardium. Cardiovasc Res 2012; 94:276-283.

3. Chao W. Toll-like receptor signaling: a critical modulator of cell survival and ischemic injury in the heart. Am J Physiol-Heart C 2009;296:H1-H12.

4. Jiang D, Liang J, Fan J, Yu S, Chen S, Luo Y, et al. Regulation of lung injury and repair by Toll-like receptors and hyaluronan. Nat Med 2005;11:1173-1179.

5. Prince JM, Levy RM, Yang R, Mollen KP, Fink MP, Vodovotz Y, et al. Toll-like receptor-4 signaling mediates hepatic injury and systemic inflammation in hemorrhagic shock. J Am Coll Surg 2006;202:407-417.

6. Tang SC, Arumugam TV, Xu X, Cheng A, Mughal MR, Jo DG, et al. Pivotal role for neuronal Toll-like receptors in ischemic brain injury and functional deficits. Proc Natl Acad Sci U S A 2007;104:13798-13803.

7. Arumugam TV, Okun E, Tang S-C, Thundyil J, Taylor SM, Woodruff TM. Toll-like receptors in ischemia-reperfusion injury. Shock 2009;32:4-16.

8. Edfeldt K, Swedenborg J, Hansson GK, Yan ZQ. Expression of toll-like receptors in human atherosclerotic lesions: a possible pathway for plaque activation. Circulation 2002;105:1158-1161.

9. Bagheri B, Sohrabi B, Movassaghpur A, Mashayekhi S, Garjani A, Shokri M, et al. Association of monoctye expression of Toll-like receptor 4 and its related cytokines with coronary luminal stenosis. Adv Biosci Biotechnol 2013;04:19-25.

10. Bagheri B, Sohrabi B, Movassaghpur A, Mashayekhi S, Garjani A, Shokri M, et al. Monocyte expression of Toll-like receptor-4 in patients with stable angina undergoing percutanoeus coronary intervention. Iran J Immunol 2012;9:149-158.

11. Rameshrad M, Maleki-Dizaji N, Vaez H, Soraya H, Nakhlband A, Garjani A. Lipopolysaccharide induced activation of toll like receptor 4 in isolated rat heart suggests a local immune response in myocardium. Iran J Immunol 2015;12:104-116.

12. Soraya H, Farajnia S, Khani S, Rameshrad M, Khorrami A, Banani A, et al. Short-term treatment with metformin suppresses toll like receptors (TLRs) activity in isoproterenol-induced myocardial infarction in rat: are AMPK and TLRs connected? Int Immunopharmacol 2012; 14:785-791.

13. Soraya H, Clanachan AS, Rameshrad M, Maleki-Dizaji N, Ghazi-Khansari M, Garjani A. Chronic treatment with metformin suppresses toll-like receptor 4 signaling and attenuates left ventricular dysfunction following myocardial infarction. Eur J Pharmacol 2014; 737:77-84.

14. Rameshrad M, Soraya H, Maleki-Dizaji N, Vaez H, Garjani A. A769662, a direct AMPK activator, attenuates lipopoly-saccharideinduced acute heart and lung inflammation in rats. Mol Med Rep 2016; 13:2843-2849.

15. Shirwany NA, Zou MH. AMPK in cardiovascular health and disease. Acta pharmacologica Sinica 2010;31:1075-1084.

16. Young LH, Li J, Baron SJ, Russell RR. AMP-activated protein kinase: A key stress signaling pathway in the heart. Trends Cardiovas Med 2005;15:110-118.

17. Santos GA, Moura RF, Vitorino DC, Roman EA, Torsoni AS, Velloso LA, et al. Hypothalamic AMPK activation blocks lipopolysaccharide inhibition of glucose production in mice liver. Mol Cell Endocrinol 2013;381:88-96.

18. Park DW, Jiang S, Tadie JM, Stigler WS, Gao Y, Abraham E, et al. Activation of AMPK enhances neutrophil chemotaxis and bacterial killing. Mol Med 2013;1:00065.

19. Zhao X, Zmijewski JW, Lorne E, Liu G, Park YJ, Tsuruta Y, et al. Activation of AMPK attenuates neutrophil proinflammatory activity and decreases the severity of acute lung injury. Am J Physiol Lung Cell Mol Physiol 2008;295:27.

20. Tadie JM, Bae HB, Deshane JS, Bell CP, Lazarowski ER, Chaplin DD, et al. Toll-like receptor 4 engagement inhibits adenosine 5'-monophosphate-activated protein kinase activation through a high mobility group box 1 protein-dependent mechanism. Mol Med 2012;18:659-668.

21. Abraham E, Carmody A, Shenkar R, Arcaroli J. Neutrophils as early immunologic effectors in hemorrhage- or endotoxemia-induced acute lung injury. Am J Physiol Lung Cell Mol Physiol 2000;279:L1137-1145.

22. Ao L, Song Y, Fullerton DA, Dinarello CA, Meng X. The interaction between myocardial depressant factors in endotoxemic cardiac dysfunction: role of TNF-alpha in TLR4-mediated ICAM-1 expression. Cytokine 2007;38:124-129.

23. Abdulrahman RM, Boon MR, Sips HC, Guigas B, Rensen PC, Smit JW, et al. Impact of Metformin and compound-C on NIS expression and iodine uptake in vitro and in vivo: a role for CRE in AMPK modulation of thyroid function. Thyroid 2014;24:78-87.

24. Cool B, Zinker B, Chiou W, Kifle L, Cao N, Perham M, et al. Identification and characterization of a small molecule AMPK activator that treats key components of type 2 diabetes and the metabolic syndrome. Cell metabolism 2006;3:403-416.

25. Huang X, Wullschleger S, Shpiro N, McGuire VA, Sakamoto K, Woods YL, et al. Important role of the LKB1-AMPK pathway in suppressing tumorigenesis in PTEN-deficient mice. Biochem J 2008; 412:211-221.

26. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001;25:402-408.

27. Takeda K. Evolution and integration of innate immune recognition systems: the Toll-like receptors. J Endotoxin Res 2005; 11:51-55.

28. Kim AS, Miller EJ, Wright TM, Li J, Qi D, Atsina K, et al. A small molecule AMPK activator protects the heart against ischemia-reperfusion injury. J Mol Cell Cardiol 2011;51:24-32.

29. Hardie DG, Carling D, Gamblin SJ. AMP-activated protein kinase: also regulated by ADP? Trends Biochem Sci 2011;36:470-477.

30.Goransson O, McBride A, Hawley SA, Ross FA, Shpiro N, Foretz M, et al. Mechanism of action of A-769662, a valuable tool for activation of AMP-activated protein kinase. J Biol Chem 2007;282:32549-32560.

31. Bai A, Ma AG, Yong M, Weiss CR, Ma Y, Guan Q, et al. AMPK agonist downregulates innate and adaptive immune responses in TNBS-induced murine acute and relapsing colitis. Biochem Pharmacol 2010;80:1708-1717.

32. Nath N, Giri S, Prasad R, Salem ML, Singh AK, Singh I. 5-aminoimidazole-4-carboxamide ribonucleoside: a novel immunomodulator with therapeutic efficacy in experimental autoimmune encephalomyelitis. J Immunol 2005;175:566-574.

33. Arslan F, de Kleijn DP, Pasterkamp G. Innate immune signaling in cardiac ischemia. Nat Rev Cardiol 2011;8:292-300.

34. Zhou G, Myers R, Li Y, Chen Y, Shen X, Fenyk-Melody J, et al. Role of AMP-activated protein kinase in mechanism of metformin action. J Clin Invest 2001; 108:1167-1174.

35.Fryer LG, Parbu-Patel A, Carling D. Protein kinase inhibitors block the stimulation of the AMP-activated protein kinase by 5-amino-4-imidazolecarboxamide riboside. FEBS letters 2002;531:189-192.

36.Labuzek K, Liber S, Gabryel B, Buldak L, Okopien B. Ambivalent effects of compound-C (dorsomorphin) on inflammatory response in LPS-stimulated rat primary microglial cultures. Naunyn Schmiedebergs Arch Pharmacol 2010; 381:41-57.

37.Kim D, Kang D, Martin EA, Kim I, Carroll JL. Effects of modulators of AMP-activated protein kinase on TASK-1/3 and intracellular Ca(2+) concentration in rat carotid body glomus cells. Respir Physiol Neurobiol 2014; 195:19-26.

38.Wu W, Tang S, Shi J, Yin W, Cao S, Bu R, et al. Metformin attenuates palmitic acid-induced insulin resistance in L6 cells through the AMP-activated protein kinase/sterol regulatory element-binding protein-1c pathway. Int J Mol Med 2015; 35:1734-1740.

39.Nguyen TM, Seigneurin F, Froment P, Combarnous Y, Blesbois E. The 5'-AMP-activated protein kinase (AMPK) is involved in the augmentation of antioxidant defenses in cryopreserved chicken sperm. PloS one 2015;10:e0134420.

40.Akira S, Takeda K. Toll-like receptor signalling. Nat Rev Immunol 2004; 4:499-511.

41.Bosisio D, Polentarutti N, Sironi M, Bernasconi S, Miyake K, Webb GR, et al. Stimulation of toll-like receptor 4 expression in human mononuclear phagocytes by interferon-γ: a molecular basis for priming and synergism with bacterial lipopolysaccharide. Blood 2002;99:3427-3431.

42. Zhao P, Wang J, He L, Ma H, Zhang X, Zhu X, et al. Deficiency in TLR4 signal transduction ameliorates cardiac injury and cardiomyocyte contractile dysfunction during ischemia. J Cell Mol Med 2009;13:1513-1525.

43. Dong B, Qi D, Yang L, Huang Y, Xiao X, Tai N, et al. TLR4 regulates cardiac lipid accumulation and diabetic heart disease in the nonobese diabetic mouse model of type 1 diabetes. Am J Physiol Heart Circ Physiol 2012;303:H732-742.

44. Vaez H, Rameshrad M, Najafi M, Barar J, Barzegari A, Garjani A. Cardioprotective effect of metformin in lipopolysaccharide-induced sepsis via suppression of toll-like receptor 4 (TLR4) in heart. Eur J Pharmacol 2015; 772:115-123.

45. Soraya H, Rameshrad M, Mokarizadeh A, Garjani A. Metformin attenuates myocardial remodeling and neutrophil recruitment after myocardial infarction in rat. BioImpacts : BI 2015;5:3-8.

46. Guma M, Wang Y, Viollet B, Liu-Bryan R. AMPK activation by A-769662 controls IL-6 expression in inflammatory arthritis. PloS one 2015;10:e0140452.