Brown Adipose Tissue: Methods and Protocols

Description

This detailed volume explores techniques for researching brown adipose tissue (BAT) and the fascinating biology and therapeutic potential of thermogenic adipocytes. The content reflects the advancing technologies in genetics, imaging, and 'omics strategies that are allowing researchers to probe BAT biology at unprecedented depths and detail, yet it also presents classic physiology principles, which remain the core tenets of BAT biology. Written for the highly successful Methods in Molecular Biology series, 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.  Authoritative and practical, Brown Adipose Tissue: Methods and Protocols provides perspectives and detailed protocols for the benefit of both new BAT researchers looking for guidance as well as seasoned researchers who would like to expand their toolkits.  Chapter 12 is available open access under a Creative Commons Attribution 4.0 International License via link.springer.com. Foreword Preface Contents Contributors Chapter 1: Brown Adipose Tissue: A Short Historical Perspective 1 Introduction 2 Early History 2.1 Brown Fat as the Major Thermogenic Organ 2.2 How Heat Is Generated in Brown Adipose Tissue 2.3 UCP1 in the Identification of BAT 3 BAT in Nutritional Energetics and Obesity 4 Brown Fat in Humans 5 Renaissance: Brown Fat in Humans 6 Cellular Heterogeneity and Metabolic Roles 6.1 Cells 6.2 Metabolic Roles 7 Concluding Comments References Chapter 2: Brown Fat Anatomy in Humans and Rodents 1 Introduction 2 Materials 2.1 BAT Sampling and Tissue Preparation 2.1.1 Animal Perfusion and Tissue Preparation 2.1.2 Human Tissue Preparation 2.2 BAT Immunoperoxidase Staining and Morphometry 2.2.1 Light Microscopy on Paraffin-Embedded Samples and Cryosections 2.3 BAT Immunofluorescence 2.3.1 Confocal Microscopy on Cryosections 2.3.2 Confocal Microscopy on Paraffin-Embedded Samples 2.4 Ultrastructural Analyses 2.4.1 Transmission Electron Microscopy on Epoxy-Resin Embedded Samples 2.4.2 Pre-embedding Immunoperoxidase Reactions for TEM Analyses 2.4.3 Scanning Electron Microscopy 2.4.4 High-Resolution Scanning Electron Microscopy 3 Methods 3.1 BAT Sampling and Tissue Preparation 3.1.1 Rodents BAT Sampling 3.1.2 Humans Tissue Preparation 3.2 BAT Immunoperoxidase Staining and Morphometry 3.2.1 Sample Preparation: Dehydration, Embedding and Sectioning 3.2.2 Routine Staining 3.2.3 Immunoperoxidase Staining 3.2.4 Morphometric Analysis 3.2.5 Immunoperoxidase Staining on Cryosections 3.3 BAT Immunofluorescence 3.3.1 Sample Preparation: Dehydration, Embedding, and Sectioning 3.3.2 Immunofluorescence Reactions on Cryosections and Paraffin-Embedded Samples 3.4 Ultrastructural Analyses 3.4.1 Transmission Electron microscopy on Epoxy-Resin Embedded Samples 3.4.2 Morphometric Study of Mitochondrial Features 3.4.3 Pre-embedding Immunoperoxidase Reactions for TEM Analyses 3.4.4 Scanning Electron Microscopy 3.4.5 High-Resolution Scanning Electron Microscopy 4 Notes References Chapter 3: Analysis of Thermogenesis Experiments with CalR 1 Introduction 2 Thermogenesis Experimental Examples Presented in this Chapter 3 Materials 4 Methods 4.1 Preparing Data for CalR 4.1.1 Promethion 4.1.2 CLAMS 4.1.3 PhenoMaster 4.2 Example 1 4.2.1 Thermogenesis in the Hyperthyroid State 4.2.2 Graphing CalR Data 4.2.3 Data Interpretation and Quality Control 4.2.4 View and Download Group Averages 4.2.5 Weight Plot 4.2.6 Regression Plots 4.2.7 Analysis/Interpretation 4.3 Example 2 4.3.1 Beta-Adrenergic Agonism 4.3.2 Loading and Visualizing Experimental Data 4.3.3 Experimental Analysis/Interpretation 4.4 Example 3 4.4.1 Thermoneutrality (30 C), Mild Cold Exposure (23 C) and Cold Challenge (4 C) References Chapter 4: A Clearing Method for Three-Dimensional Imaging of Adipose Tissue 1 Introduction 2 Materials 3 Methods 3.1 Tissue Preparation 3.2 Delipidation and Permeabilization 3.3 Whole Mount Immunostaining 3.4 Optional: Agarose Embedding (see Note 20) 3.5 Tissue Clearing 3.6 Microscopy 4 Notes References Chapter 5: Activating Human Adipose Tissue with the β3-Adrenergic Agonist Mirabegron 1 Introduction 2 Materials 2.1 Pharmacology 2.2 Subjects 2.3 Metabolic Measures 2.4 Imaging 3 Methods 3.1 Screening and Preparation of Subjects 3.2 Acute Activation with Mirabegron (Fig. 1) 3.3 Chronic Activation with Mirabegron 4 Notes References Chapter 6: Fluorescent and Luminescent Methods to Detect Lipolysis 1 Introduction 2 Materials 2.1 Fluorescent Fatty Acid and Colorimetric Glycerol Detection 2.1.1 Cell Culture-Brown Adipocytes 2.1.2 Fluorescent Fatty Acid Detection 2.1.3 Glycerol Colorimetric Detection 2.2 Luminescent Fatty Acid Detection 2.2.1 Transient Transfection 2.2.2 Stable Cell Line Generation 2.2.3 Luminescent Detection 3 Methods 3.1 Fluorescent Fatty Acid Detection 3.1.1 Growth, Differentiation and Assay of Brown Adipocytes 3.1.2 Fluorescent Free Fatty Acid Detection 3.1.3 Glycerol Colorimetric Detection 3.2 Luminescent Fatty Acid Detection 3.2.1 Transient Transfection 3.2.2 Stable Cell Line Generation 3.2.3 Detection for Luminescent Fatty Acid Sensor 4 Notes 4.1 Fluorescent Fatty Acid Detection 4.2 Luminescent Fatty Acid Detection References Chapter 7: Metabolic Turnover Studies to Quantify Energy Uptake by Thermogenic Adipose Tissues of Mice 1 Introduction 2 Materials 2.1 Radioactive Tracer Solutions 2.2 Solutions 2.3 Instruments 3 Methods 3.1 Production of Radiolabeled Triglyceride-Rich Lipoproteins (TRL) 3.1.1 Isolation of Triglyceride-Rich Lipoproteins from Hyperlipidemic Plasma 3.1.2 Lipid Extraction of TRL According to the Method of Folch 3.1.3 Preparation of Radioactive Labeled Recombinant TRL 3.2 Metabolic Clearance Studies of Radiolabeled Recombinant Triglyceride-Rich Lipoproteins 3.3 Oral Fat Tolerance Test (OFTT) or Glucose and Fat Tolerance Test (OGFT) 4 Notes References Chapter 8: Stable Isotope Tracing and Metabolomics to Study In Vivo Brown Adipose Tissue Metabolic Fluxes 1 Introduction 2 Materials 2.1 Mice 2.2 Isotope Tracer 2.3 Tissue Harvesting/Processing 2.4 Extraction and LC-MS 3 Methods 3.1 Temperature Challenge 3.2 Tracer Delivery 3.3 Tissue Harvest 3.4 Tissue/Serum Processing and LC-MS 3.5 LC-MS Data Analysis 4 Notes References Chapter 9: Activation of UCP1-Independent Ca2+ Cycling Thermogenesis by Wireless Optogenetics 1 Introduction 2 Materials 2.1 Animal 2.2 AAV Delivery 2.3 Wireless Optogenetics Device 2.4 Recording of Tissue Temperature 2.5 Calcium Imaging 3 Methods 3.1 Implantation of Wireless Optogenetics Device 3.2 Recording of Tissue Temperature 3.3 Administration of AAV Vectors to the Subcutaneous Adipose Tissue 3.4 Intracellular Ca2+ Imaging 4 Notes References Chapter 10: Measurement of Futile Creatine Cycling Using Respirometry 1 Introduction 1.1 Health Impact of the obesity Epidemic 1.2 Control of Obesity and Metabolic Health by Brown Adipose Tissue 1.3 Principles of Non-shivering Thermogenesis 1.4 Futile Creatine Cycle 2 Materials 2.1 Brown Adipocyte Differentiation 2.2 Mitochondrial Isolation 2.3 Oxygen Electrode 3 Methods 3.1 Brown Adipocyte Differentiation 3.2 Mitochondrial Isolation 3.3 Oxygen Electrode Setup (Fig. 1) 3.4 Respiration Measurements 3.5 Data Analysis 3.5.1 Calculation of Stoichiometry of Creatine-Dependent ADP Liberation (Futile Creatine Cycling) 4 Notes References Chapter 11: Analysis of Enhancers and Transcriptional Networks in Thermogenic Adipocytes 1 Introduction 1.1 Genome-Wide Profiling of the Cellular Transcriptome and Regulatory Genomic Regions 1.1.1 Transcriptome Analysis 1.1.2 Profiling of Location and/or Activity of Regulatory Genomic Elements 1.2 Prediction of Transcription Factors Regulating Gene Expression and Cellular Function 1.2.1 IMAGE Predicts Causal TFs Regulating Enhancer Activity and Gene Expression 2 Materials 2.1 Software 2.2 Input Files 3 Methods: Example Application 3.1 Generation of Transcriptome and Enhancer Data in White and Brown Adipocytes 3.2 Preparing RNA and DNase-Seq Libraries from White and Brown Adipocytes 3.3 Preprocessing and QC of Sequencing Files 3.4 Generating Count Matrices of Gene Expression and Enhancer Data 3.5 Using IMAGE to Predict TFs Controlling Gene Expression in White and Brown Adipocytes 3.5.1 Determining Differential Motif Activities Between Conditions 3.5.2 Prediction of Target Sites and Target Genes in White and Brown Adipocytes 3.5.3 Limitations of IMAGE Analyses 4 Notes References Chapter 12: Diet-Induced Thermogenesis: Principles and Pitfalls 1 Introduction 2 Principles 2.1 Obligatory Diet-Induced Thermogenesis 2.2 Facultative Adaptive Diet-Induced Thermogenesis 2.3 Where Is Diet-Induced Thermogenesis Located? 2.4 Why Would Diet-Induced Thermogenesis Exist? 2.5 The Experimental Models 3 Possible Pitfalls 3.1 Metabolic Characterization 3.1.1 Perform Experiments at Thermoneutrality 3.1.2 Food Intake Measurements Are Essential but Difficult to Perform 3.1.3 Indirect Calorimetry Chambers Are Stressful 3.1.4 Do Not Divide by Body Weight 3.1.5 In an Equation, the Two Sides Must Be Expressed Equally 3.1.6 Determination of Metabolic Capacity for Diet-Induced Thermogenesis 3.1.7 Determination of Physiologically Relevant Metabolic Capacity 3.2 Tissue Characterization 3.2.1 Do Not Express Results per Square Centimeter 3.2.2 Do Not Express Results (Only) per Cubic Centimeter 3.2.3 Do Not Express Enzymatic Results per g Tissue Weight 3.2.4 Do Not Express Apparent thermogenesis per g Tissue Weight 3.2.5 Do Not Express Results (Only) per mg Protein 3.2.6 To Express UCP1 Levels Only per mg Tissue Protein Can Be Misleading 3.2.7 UCP1 Protein Levels Should Be Expressed per Total Adipose Tissue Depot 3.2.8 Use the Same (Arbitrary) Units to Express UCP1 Protein Amounts in Different Tissues 3.2.9 In Brown Adipose Tissue, it May Be Difficult to Identify a ``Normalizing´´ Protein 3.2.10 To Express UCP1 mRNA as a Proxy for UCP1 Protein (Thermogenic Capacity) Can Be Misleading 3.2.11 Compare to the Same Standard Sample for mRNA Levels when Analyzing Different Tissues 3.2.12 Beware of the ``Divisor´´ Normalization 3.2.13 Total UCP1 mRNA Levels per Tissue Depot Are Physiologically Meaningful 3.2.14 To Determine UCP1 Recruitment by Immunohistochemistry Is Difficult 3.3 Blood Glucose Estimation 3.3.1 For Glucose Tolerance Tests, Do Not Inject Glucose in Proportion to Body Weight 3.3.2 For Euglycemic, Hyperinsulinemic Clamps, Do Not Express the Results per g Body Weight 4 Final Remarks References Chapter 13: Fluorescent Genetic Tools for Studying Brown Fat Development and Function in Mice 1 Introduction 2 Materials 2.1 Brown Adipocyte Precursors Flow Analysis 2.2 Whole Mount Confocal Microscopy 2.3 Adipose Tissue Clearing 3 Methods 3.1 Adipocyte Precursors Flow Analysis of R26R-mTmG Mice 3.1.1 Fat Pad Dissection and Digestion into Single Cells 3.1.2 Stromal Vascular Fraction Isolation 3.1.3 Adipocyte Precursor Labeling 3.1.4 FACS Data Analysis 3.2 Whole Mount Confocal Microscopy of R26R-mTmG Mice 3.2.1 Fat Pad Dissection and Mounting 3.2.2 Fat Pad Confocal Imaging 3.2.3 Imaging Analysis 3.3 Adipose Tissue Clearing for Immunostaining 3.3.1 Tissue Preparation 3.3.2 Dehydration and Delipidation 3.3.3 Rehydration and Permeabilization 3.3.4 Immunostaining 3.3.5 Dehydration and Clearing 3.3.6 Mounting 3.3.7 Confocal Imaging and Processing 3.4 Adipose Tissue Clearing for Endogenous Fluorescence 3.4.1 Tissue Preparation 3.4.2 Dehydration and Delipidation 3.4.3 Dehydration and Clearing 3.4.4 Mounting, Confocal Imaging, and Processing 4 Notes References Chapter 14: Isolation and Characterization of Human Brown Adipocytes 1 Introduction 1.1 BAT in Human Metabolism 1.2 Thermogenic Plasticity of BAT 1.3 Heterogeneity of BAT 1.4 Human BAT In Vitro Models 2 Materials 2.1 Isolating Progenitors from Adipose Tissue Biopsies 2.2 Differentiation of Progenitors into Lipid Droplet Containing Cells 2.3 Characterization of Brown Adipocyte Phenotype Using qPCR 2.4 Measure Oxygen Consumption Rate Using the Seahorse XFe96 Analyzer 2.5 UCP1 Induction Using FISH/RNAscope 3 Methods 3.1 Isolating Progenitors from Adipose Tissue Biopsies 3.2 Differentiation of Progenitors into Lipid Droplet Containing Cells 3.3 Characterization of Brown Adipocyte Phenotype Using qPCR 3.4 Characterization of Brown Adipocyte Phenotype Using the XFe96 Seahorse Bioscience Analyzer 3.5 Characterization of UCP1 Induction Using FISH 4 Notes References Chapter 15: Setting Ambient Temperature Conditions to Optimize Translation of Molecular Work from the Mouse to Human: The ``Go... 1 Introduction 2 Effects of Ambient Temperature on Metabolic Rates of Endotherms 3 To Translate Work to Humans, Mice Should Not Be Kept at 20 C 4 Should Mice Be Kept at 30 C 4.1 Humans Live at Their Thermoneutral Temperature. 4.2 Mouse Thermoneutral is 30 C 4.3 Mice Themselves Choose 30 C or Above If Given the Choice 4.4 Measures of Physiological Parameters Differ Between 30 and 20-21 C. 5 An Ambient Temperature of 25-26 C Is Probably Optimal 6 What Humans Are We Trying to Mimic? 7 Group Housing 8 Reproductive Performance 9 Conclusions References Chapter 16: Signaling Lipidomic Analysis of Thermogenic Adipocytes 1 Introduction 2 Materials 2.1 Human Preadipocyte Differentiation (see Notes 1 and 2) 2.2 Bicinchoninic Acid (BCA) Assay 2.3 Signaling Lipid Extraction 2.4 Equipment 2.5 Software 3 Methods 3.1 Culture of Human Brown Preadipocytes and Differentiation into Mature Adipocytes (see Notes 4-6) 3.2 Treatment and Sample Collection for Analysis 3.3 Protein Quantification for Cell Lysate Normalization 3.4 Sample Preparation for LC-MS/MS Analysis 3.5 12,13-diHOME and 12-HEPE Standard Curve Preparation for LC-MS/MS Analysis 3.6 LC-MS/MS Analysis 3.7 Integrating Peak Areas from LC-MS/MS Analysis 3.8 Processing and Analysis of Data Using MetaboDiff (see Note 17) 4 Notes References Chapter 17: Blood Sampling for Arteriovenous Difference Measurements Across Interscapular Brown Adipose Tissue in Rat 1 Introduction 2 Materials 2.1 Animals 2.2 Anesthesia 2.3 Surgery and Blood Collection 3 Methods 3.1 Preparation 3.2 Anesthesia Administration 3.3 Sulzer´s Vein Blood Collection 3.4 Arterial Blood Collection 3.5 Plasma Isolation 4 Notes References Chapter 18: In Vivo Imaging of Brown Adipose Tissue in Humans: FDG-PET/CT and Beyond 1 Introduction 2 Methods 2.1 Preparations of the Study Subject for the Scan 2.2 Scanning of BAT (and Other Tissues) 2.3 Analysis of the Image Data References Chapter 19: Analysis of Single-Cell/Nucleus Transcriptome Data in Adipose Tissue 1 Introduction 2 Materials 3 Methods 3.1 Get FASTQ Files 3.2 Align Reads to Genome 3.3 Eliminate Technical Noise 3.4 Filtering Low Quality Cells 3.5 Remove Doublets 3.6 Normalize Raw Reads 3.7 Identify Highly Variable Genes for Clustering 3.8 Scale Data 3.9 Dimension Reduction 3.10 Cluster Cells 3.11 Visualize Data in UMAP 3.12 Find Cluster-Specific Markers 3.13 Visualize Gene Expression 3.14 Annotate Cell Type to Each Cluster 3.15 Subcluster a Cell Type 4 Notes References Index