AMPK: Methods and Protocols

Description

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 γ2 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. Front Matter ....Pages i-xvii Production and Crystallization of Full-Length Human AMP-Activated Protein Kinase (α1β1γ1) (Julia A. Hubbard, Bing Xiao, Jon R. Wilson)....Pages 1-14 Visualizing AMPK Drug Binding Sites Through Crystallization of Full-Length Phosphorylated α2β1γ1 Heterotrimer (Christopher G. Langendorf, Jonathan S. Oakhill, Bruce E. Kemp)....Pages 15-27 Biophysical Interactions of Direct AMPK Activators (Ravi G. Kurumbail, Graham M. West, Venkatasubramanian Dharmarajan, Kris A. Borzilleri, Jane M. Withka, Jessica Ward et al.)....Pages 29-55 Biochemical Measurement of Glycogen: Method to Investigate the AMPK-Glycogen Relationship (Elite Possik, Arnim Pause)....Pages 57-67 Cell-Free Assays to Measure Effects of Regulatory Ligands on AMPK (Fiona A. Fyffe, Simon A. Hawley, Alexander Gray, D. Grahame Hardie)....Pages 69-86 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, Jesse I. Mobbs)....Pages 87-98 Bioinformatics Approach to Identify Novel AMPK Targets (Brendan Gongol, Traci Marin, David A. Johnson, John Y.-J. Shyy)....Pages 99-109 Studying AMPK in an Evolutionary Context (Arpit Jain, Valentin Roustan, Wolfram Weckwerth, Ingo Ebersberger)....Pages 111-142 AMPK Protein Interaction Analyses by Yeast Two-Hybrid (Pascual Sanz, Rosa Viana, Maria Adelaida Garcia-Gimeno)....Pages 143-157 Transient Expression of AMPK Heterotrimer Complexes in Mammalian Cells (Jonathan S. Oakhill, John W. Scott, Toby A. Dite)....Pages 159-169 Knockdown of Human AMPK Using the CRISPR/Cas9 Genome-Editing System (Adrien Grenier, Pierre Sujobert, Séverine Olivier, Hélène Guermouche, Johanna Mondésir, Olivier Kosmider et al.)....Pages 171-194 Compound C/Dorsomorphin: Its Use and Misuse as an AMPK Inhibitor (Biplab Dasgupta, William Seibel)....Pages 195-202 Identifying the Heterotrimeric Complex Stoichiometry of AMPK in Skeletal Muscle by Immunoprecipitation (Jesper B. Birk, Jørgen F. P. Wojtaszewski)....Pages 203-213 Kinase Activity Determination of Specific AMPK Complexes/Heterotrimers in the Skeletal Muscle (Jesper B. Birk, Jørgen F. P. Wojtaszewski)....Pages 215-228 Determination of Adenine Nucleotide Concentrations in Cells and Tissues by High-Performance Liquid Chromatography (Noemí García-Tardón, Bruno Guigas)....Pages 229-237 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, D. Grahame Hardie)....Pages 239-253 Cellular Application of Genetically Encoded Sensors and Impeders of AMPK (Takafumi Miyamoto, Elmer Rho, Allen Kim, Takanari Inoue)....Pages 255-272 Assessing Mitochondrial Bioenergetics by Respirometry in Cells or Isolated Organelles (Guillaume Vial, Bruno Guigas)....Pages 273-287 Study of AMPK-Regulated Metabolic Fluxes in Neurons Using the Seahorse XFe Analyzer (Claudia Marinangeli, Jérome Kluza, Philippe Marchetti, Luc Buée, Valérie Vingtdeux)....Pages 289-305 Investigating the Role of AMPK in Inflammation (Sarah J. Mancini, Ian P. Salt)....Pages 307-319 Studying the Role of AMPK in Cardiac Hypertrophy and Protein Synthesis (Florence Mailleux, Christophe Beauloye, Jean-Luc Balligand, Sandrine Horman, Luc Bertrand)....Pages 321-342 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, Miranda Nabben)....Pages 343-361 Measurement of AMPK-Induced Inhibition of Lipid Synthesis Flux in Cultured Cells (Marc Foretz, Benoit Viollet)....Pages 363-371 Studying the Role of AMPK in Autophagy (Sarah Krieg, Bernhard Lüscher, Jörg Vervoorts, Marc Dohmen)....Pages 373-391 Determining AMPK Activation via the Lysosomal v-ATPase-Ragulator-AXIN/LKB1 Axis (Chen-Song Zhang, Mengqi Li, Yue Zong, Sheng-Cai Lin)....Pages 393-411 Manipulation and Measurement of AMPK Activity in Pancreatic Islets (Aida Martinez-Sanchez, Marie-Sophie Nguyen-Tu, Isabelle Leclerc, Guy A. Rutter)....Pages 413-431 Analyzing AMPK Function in the Hypothalamus (Patricia Seoane-Collazo, Miguel López)....Pages 433-448 Using Ex Vivo Kidney Slices to Study AMPK Effects on Kidney Proteins (Renee Rao, Kazuhiro Omi, Roshan Rajani, Hui Li, Nuria M. Pastor-Soler)....Pages 449-464 A Flow Cytometry-Based Protocol to Measure Lymphocyte Viability Upon Metabolic Stress (Sébastien Denanglaire, Tiphène Pirnay, Oberdan Leo, Fabienne Andris)....Pages 465-476 Methods to Evaluate AMPK Regulation of Macrophage Cholesterol Homeostasis (Nicholas D. LeBlond, Morgan D. Fullerton)....Pages 477-493 Modulation of Vascular Function by AMPK: Assessment of NO Bioavailability and Surrogates of Oxidative Stress (Swenja Kröller-Schön, Andreas Daiber, Eberhard Schulz)....Pages 495-506 Measurement of Reactive Oxygen Species (ROS) and Mitochondrial ROS in AMPK Knockout Mice Blood Vessels (Qilong Wang, Ming-Hui Zou)....Pages 507-517 Studying the Role of AMPK in Angiogenesis (Katrin Spengler, Silke Große, Nderim Kryeziu, Anne Knierim, Regine Heller)....Pages 519-537 Analysis of Muscle Stem Cell Fate Through Modulation of AMPK Activity (Marine Theret, Linda Gsaier, Sabrina Ben Larbi, Michèle Weiss-Gayet, Rémi Mounier)....Pages 539-549 Evaluating the Role of Host AMPK in Leishmania Burden (Diana Moreira, Jérôme Estaquier, Anabela Cordeiro-da-Silva, Ricardo Silvestre)....Pages 551-563 Analysis of Transgenerational Phenotypes Following Acute Starvation in AMPK-Deficient C. elegans (Emilie Demoinet, Richard Roy)....Pages 565-579 Human γ2-AMPK Mutations (Arash Yavari, Dhruv Sarma, Eduardo B. Sternick)....Pages 581-619 Back Matter ....Pages 621-626