AMPK

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.

Includes cutting-edge methods and protocols Provides step-by-step detail essential for reproducible results Contains key notes and implementation advice from the experts

Inhalt
Production and Crystallization of Full-Length Human AMP-Activated Protein Kinase (a1b1y1).- Visualizing AMPK Drug Binding Sites through Crystallization of Full Length Phosphorylated a2b1y1 Heterotrimer.- Biophysical Interactions of Direct AMPK Activators.- Biochemical Measurement of Glycogen: Method to Investigate the AMPK-Glycogen Relationship.- Cell-Free Assays to Measure Effects of Regulatory Ligands on AMPK.- Applications of NMR and ITC for the Study of the Kinetics of Carbohydrate Binding by AMPK -Subunit Carbohydrate Binding Modules.- Bioinformatics Approach to Identify Novel AMPK Targets.- Studying AMPK in an Evolutionary Context.- AMPK Protein Interaction Analyses by Yeast Two-Hybrid.- Transient Expression of AMPK Heterotrimer Complexes in Mammalian Cells.- Knockdown of Human AMPK using the CRISPR-Cas9 Genome-Editing System.- Compound C/Dorsomorphin Its Use and Misuse as an AMPK Inhibitor.- Identifying the Heterotrimeric Complex Stoichiometry of AMPK in Skeletal Muscleby Immunoprecipitation.- Kinase Activity Determination of Specific AMPK Complexes/Heterotrimers in Skeletal Muscle.- Determination of Adenine Nucleotide Concentrations in Cells and Tissues by High Performance Liquid Chromatography.- Intact Cell Assays to Monitor AMPK and Determine the Contribution of the AMP-Binding or ADaM Sites to Activation.- Cellular Application of Genetically-Encoded Sensors and Impeders of AMPK.- Assessing Mitochondrial Bioenergetics by Respirometry in Cells or Isolated Organelles.- Study of AMPK Regulated Metabolic Fluxes in Neurons using the Seahorse XFe Analyzer.- Investigating the Role of AMPK in Inflammation.- Studying the Role of AMPK in Cardiac Hypertrophy and Protein Synthesis.- Assessment of AMPK-Stimulated Cellular Long-Chain Fatty Acid and Glucose Uptake.- Measurement of AMPK-Induced Inhibition of Lipid Synthesis Flux in Cultured Cells.- Studying the Role of AMPK in Autophagy.- Determining AMPK Activation via the Lysosomal v-ATPase-Ragulator-AXIN/LKB1Axis.- Manipulation and Measurement of AMPK Activity in Pancreatic Islets.- Analyzing AMPK Function in the Hypothalamus.- Using Ex Vivo Kidney Slices to Study AMPK Effects on Kidney Proteins.- A Flow Cytometry-Based Protocol to Measure Lymphocyte Viability upon Metabolic Stress.- Methods to Evaluate AMPK Regulation of Macrophage Cholesterol Homeostasis.- Modulation of Vascular Function by AMPK: Assessment of NO Bioavailability and Surrogates of Oxidative Stress.- Measurement of Reactive Oxygen Species (ROS) and Mitochondrial ROS in AMPK Knockout Mice Blood Vessels.- Studying the Role of AMPK in Angiogenesis.- Analysis of Muscle Stem Cell Fate through Modulation of AMPK Activity.- Evaluating the Role of Host AMPK in Leishmania Burden.- Analysis of Transgenerational Phenotypes Following Acute Atarvation in AMPK-Deficient C. elegans.- Human 2 AMPK Mutations.