AMPK, 1st ed. 2018 Methods and Protocols Methods in Molecular Biology Series, Vol. 1732

This volume explores the latest methods used to study AMPK by computational, biochemical, biophysical, cellular, and ex vivo and in vivo approaches. The chapters in this book cover specific topics, such as methods to measure change in cellular energy metabolism and analyze metabolic pathways regulated by AMPK; bioinformatics tools to identify AMPK targets; knockdown of AMPK by CRISPR-Cas9; production and crystallization of full-length human AMP-activated protein kinase; cell-free assays to measure the effects of regulatory ligands on AMPK; use of sensors of AMPK activity; AMPK protein interaction by yeast two-hybrid; the role of AMPK in inflammation and autophagy; analyzing the AMPK function in C. elegans and mammals (with special focus on skeletal muscle, blood vessels, kidneys, pancreatic islets and hypothalamus); and human ?2 AMPK mutations. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Cutting edge and thorough, AMPK: Methods and Protocols is a valuable resource for graduate students, postdoctoral researchers, and established investigators who are interested in the richly diverse AMPK field.

Preface…

Table of Contents…

Contributing Authors…

1. Production and Crystallization of Full-Length Human AMP-Activated Protein Kinase (a1b1y1)

Julia A. Hubbard, Bing Xiao, and Jon R. Wilson

2. Visualizing AMPK Drug Binding Sites through Crystallization of Full Length Phosphorylated a2b1y1 Heterotrimer

Christopher G. Langendorf, Jonathan S. Oakhill, and Bruce E. Kemp

3. Biophysical Interactions of Direct AMPK Activators

Ravi G. Kurumbail, Graham M. West, Venkatasubramanian Dharmarajan, Kris A. Borzilleri, Jane M. Withka, Jessica Ward, Allan R. Reyes, Francis Rajamohan, Patrick R. Griffin, and Matthew F. Calabrese

4. Biochemical Measurement of Glycogen: Method to Investigate the AMPK-Glycogen Relationship

Elite Possik and Arnim Pause

5. Cell-Free Assays to Measure Effects of Regulatory Ligands on AMPK

Fiona A. Fyffe, Simon A. Hawley, Alexander Gray, and D. Grahame Hardie

6. Applications of NMR and ITC for the Study of the Kinetics of Carbohydrate Binding by AMPK β-Subunit Carbohydrate Binding Modules

Paul R. Gooley, Ann Koay, and Jesse I. Mobbs

7. Bioinformatics Approach to Identify Novel AMPK Targets

Brendan Gongol, Traci Marin, David A. Johnson, and John Y-J. Shyy

8. Studying AMPK in an Evolutionary Context

Arpit Jain, Valentin Roustan, Wolfram Weckwerth, and Ingo Ebersberger

9. AMPK Protein Interaction Analyses by Yeast Two-Hybrid

Pascual Sanz, Rosa Viana, and Maria Adelaida Garcia-Gimeno

10. Transient Expression of AMPK Heterotrimer Complexes in Mammalian Cells

Jonathan S. Oakhill, John W. Scott, and Toby A. Dite

11. Knockdown of Human AMPK using the CRISPR-Cas9 Genome-Editing System

Adrien Grenier, Pierre Sujibert, Séverine Olivier, Hélène Guermouche, Johanna Mondésir, Olivier Kosmider, Benoit Viollet, and Jérôme Tamburini

12. Compound C/Dorsomorphin – Its Use and Misuse as an AMPK Inhibitor

Biplab Dasgupta and William Seibel

13. Identifying the Heterotrimeric Complex Stoichiometry of AMPK in Skeletal Muscle by Immunoprecipitation

Jesper B. Birk and Jørgen F. P. Wojtaszewski

14. Kinase Activity Determination of Specific AMPK Complexes/Heterotrimers in Skeletal Muscle

Jesper B. Birk and Jørgen F. P. Wojtaszewski

15. Determination of Adenine Nucleotide Concentrations in Cells and Tissues by High Performance Liquid Chromatography

Noemí García-Tardón and Bruno Guigas

16. Intact Cell Assays to Monitor AMPK and Determine the Contribution of the AMP-Binding or ADaM Sites to Activation

Simon A. Hawley, Fiona A. Fyffe, Fiona M. Russell, Graeme J. Gowans, and D. Grahame Hardie

17. Cellular Application of Genetically-Encoded Sensors and Impeders of AMPK

Takafumi Miyamoto, Elmer Rho, Allen Kim, and Takanari Inoue

18. Assessing Mitochondrial Bioenergetics by Respirometry in Cells or Isolated Organelles

Guillaume Vial and Bruno Guigas

19. Study of AMPK Regulated Metabolic Fluxes in Neurons using the Seahorse XFe Analyzer

Claudia Marinangeli, Jérome Kluza, Philippe Marchetti, Luc Buée, and Valérie Vingtdeux

20. Investigating the Role of AMPK in Inflammation

Sarah J. Mancini and Ian P. Salt

Florence Mailleux, Christophe Beauloye, Jean-Luc Balligand, Sandrine Horman, and Luc Bertrand

22. Assessment of AMPK-Stimulated Cellular Long-Chain Fatty Acid and Glucose Uptake

Joost J.F.P. Luiken, Dietbert Neumann, Jan F.C. Glatz, Will A. Coumans, Dipanjan Chanda, and Miranda Nabben

Marc Foretz and Benoit Viollet

24. Studying the Role of AMPK in Autophagy

Sarah Krieg, Bernhard Lüscher, Jörg Vervoorts, and Marc Dohmen

25. Determining AMPK Activation via the Lysosomal v-ATPase-Ragulator-AXIN/LKB1 Axis

Chen-Song Zhang, Mengqi Li, Yue Zong, and Sheng-Cai Lin

26. Manipulation and Measurement of AMPK Activity in Pancreatic Islets

Aida Martinez-Sanchez, Marie-Sophie Nguyen-Tu, Isabelle Leclerc, and Guy A. Rutter

27. Analyzing AMPK Function in the Hypothalamus

Patricia Seoane-Collazo and Miguel López

28. Using Ex Vivo Kidney Slices to Study AMPK Effects on Kidney Proteins

Renee Rao, Kazuhiro Omi, Roshan Rajani, Hui Li, and Nuria M. Pastor-Soler

29. A Flow Cytometry-Based Protocol to Measure Lymphocyte Viability upon Metabolic Stress

Sébastien Denanglaire, Tiphène Pirnay, Oberdan Leo, and Fabienne Andris

30. Methods to Evaluate AMPK Regulation of Macrophage Cholesterol Homeostasis

Nicholas D. LeBlond and Morgan D. Fullerton

31. Modulation of Vascular Function by AMPK: Assessment of NO Bioavailability and Surrogates of Oxidative Stress

Swenja Kröller-Schön, Andreas Daiber, and Eberhard Schulz

32. Measurement of Reactive Oxygen Species (ROS) and Mitochondrial ROS in AMPK Knockout Mice Blood Vessels

Qilong Wang and Ming-Hui Zou

33. Studying the Role of AMPK in Angiogenesis

Katrin Spengler, Silke Lindenmüller, Nderim Kryeziu, Anne Knierim, and Regine Heller

34. Analysis of Muscle Stem Cell Fate through Modulation of AMPK Activity

Marine Theret, Linda Gsaier, Sabrina Ben Larbi, Michèle Weiss-Gayet, and Rémi Mounier

35. Evaluating the Role of Host AMPK in Leishmania Burden

Diana Moreira, Jérôme Estaquier, Anabela Cordeiro-da-Silva, and Ricardo Silvestre

36. Analysis of Transgenerational Phenotypes Following Acute Atarvation in AMPK-Deficient C. elegans

Emilie Demoinet and Richard Roy

37. Human γ2 AMPK Mutations

Arash Yavari, Dhruv Sarma, and Eduardo B. Sternick 

Includes cutting-edge methods and protocols

Provides step-by-step detail essential for reproducible results

Contains key notes and implementation advice from the experts