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AMPK pp 551-563 | Cite as

Evaluating the Role of Host AMPK in Leishmania Burden

  • Diana Moreira
  • Jérôme Estaquier
  • Anabela Cordeiro-da-Silva
  • Ricardo Silvestre
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1732)

Abstract

The study of host AMP-activated protein kinase (AMPK) activation during Leishmania infection imposes distinct types of techniques to measure protein expression and activation, as well as to quantify, at transcription and translational levels, its downstream targets. The investigation of host AMPK protein modulation during Leishmania infection should primarily be assessed during in vitro infections using as a host murine bone marrow-derived macrophages (BMMos). The infection outcome is assessed measuring the percentage of infected cells in the context of BMMos. To evaluate AMPK activity during infection, the expression of AMPK phosphorylated at Thr172 as well as the transcription and translational levels of its downstream targets are evaluated by quantitative PCR and immunoblotting. The modulation of AMPK activity in vivo is determined specifically in sorted splenic macrophages harboring Leishmania parasites recovered from infected mice using fluorescent-labeled parasites in the infectious inocolum. The modulation of AMPK activity was assessed by AMPK activators and inhibitors and also using AMPK, SIRT1, or LKB1 KO mice models. The infection outcome in BMMos and in vivo was further determined using these two different approaches. To finally understand the metabolic impact of AMPK during infection, in vitro metabolic assays in infected BMMos were measured in the bioenergetic profile using an extracellular flux analyzer.

Key words

Leishmania AMPK Bioenergetic profile Extracellular flux analyzer AMPK activators and inhibitors SIRT1 Mitochondria Cell metabolism Macrophages 

Notes

Acknowledgments

This work was supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) (NORTE-01-0145-FEDER-000013) and the Fundação para a Ciência e Tecnologia (FCT) (contract IF/00021/2014 to R.S.), by FEDER funds through the Operational Competitiveness Programme (COMPETE), and by national funds through FCT (Fundação para a Ciência e a Tecnologia) under the project FCOMP-01-0124-FEDER-011054 (PTDC/SAU-FCF/100749/2008) and PTDC/BIA-MIC/118644/2010. The research leading to these results has also received funding from the European community’s Seventh Framework Programme under grant agreement No.602773 to JE and ACS (Project KINDRED). DM was supported by SFRH/BD/91543/2012. JE thanks the Canada Research Chair program for their supports.

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Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Diana Moreira
    • 1
    • 4
    • 5
  • Jérôme Estaquier
    • 2
    • 3
  • Anabela Cordeiro-da-Silva
    • 1
  • Ricardo Silvestre
    • 4
    • 5
  1. 1.Parasite Disease Group, IBMC-Instituto de Biologia Molecular e Celular; Instituto de Investigação e Inovação em Saúde, and Departamento de Ciências Biológicas, Faculdade de FarmáciaUniversidade do PortoPortoPortugal
  2. 2.CNRS FR 3636Université Paris DescartesParisFrance
  3. 3.Centre de Recherche du CHU de QuébecUniversité LavalLavalCanada
  4. 4.Life and Health Sciences Research Institute (ICVS), School of MedicineUniversity of MinhoBragaPortugal
  5. 5.ICVS/3Bs-PT Government Associate LaboratoryGuimarãesPortugal

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