Thermogenic Fat: Methods and Protocols

Description

This volume presents a broad collection of state-of-the-art methods to study the biology of thermogenic fat using in vitro cell culture and animal models.  Chapters guide the readers through protocols on differentiation of human pluripotent stem cells and murine adipocyte precursors; methods for measuring mitochondrial respiration, heat generation, brown fat activation, and effects on energy metabolism in mice; and techniques for AAV-mediated gene delivery, transplantation of adipose tissue, isolation of adipose tissue immune cells and extracellular vesicles, and mass spectrometry-based profiling of brown fat lipids.  Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials and methods, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols.   Authoritative and cutting-edge, Thermogenic Fat: Methods and Protocols aims to be comprehensive guide for researchers in the field. Preface Contents Contributors Chapter 1: Differentiation of Human Pluripotent Stem Cells (hPSCs) into Brown-Like Adipocytes 1 Introduction 2 Materials 2.1 Cell Culture and Differentiation Reagents 2.2 Functional Assessment 2.3 Software 3 Methods 3.1 Matrigel Coating of 12 Well Plates 3.2 Seeding (see Note 2) 3.3 Differentiation (see Note 2) 3.4 Functional Assessment of Lipid Accumulation 4 Notes References Chapter 2: Culture and Differentiation of Primary Preadipocytes from Mouse Subcutaneous White Adipose Tissue 1 Introduction 2 Materials 2.1 Stromal Vascular Cell Isolation 2.2 Plating and Proliferation 2.3 Adipogenic Differentiation 2.4 Equipment 3 Methods 3.1 SVF Isolation 3.1.1 Preparation for SVF Isolation 3.1.2 Mouse Sacrifice 3.1.3 Tissue Digest 3.1.4 Washing of SVF Cells 3.2 Plating and Proliferation 3.3 Differentiation (see Note 21) 4 Notes References Chapter 3: Isolation and Differentiation of Adipocyte Precursors Derived from Neonatal Murine Brown Adipose Tissue 1 Introduction 2 Materials 2.1 Collagen Coating of Plates 2.2 Adipocyte Precursor Isolation 2.3 Differentiation 3 Methods 3.1 Collagen Coating of Plates 3.2 Adipocyte Precursor Isolation 3.3 Differentiation 4 Notes References Chapter 4: Determination of Adipocyte Size and Number 1 Introduction 2 Materials 2.1 Osmium-Tetroxide Fixation for Adipocyte Size Determination 2.1.1 Solutions 2.1.2 Equipment 2.2 Adipocyte Size Determination Using Collagenase Digestion 2.2.1 Solutions 2.2.2 Equipment 2.3 Adipocyte Size Determination Using Histological Sections 3 Methods 3.1 Osmium-Tetroxide Fixation and Coulter Counter Analysis for Adipocyte Size Determination 3.1.1 Adipocyte Fixation with Osmium-Tetroxide 3.1.2 Analysis of Osmium-Labeled Adipocyte by Beckman Coulter Multisizer 3.2 Adipocyte Isolation by Collagenase Digestion and Size Determination Using ImageJ 3.2.1 Adipocyte Isolation by Collagenase Digestion 3.2.2 Analysis of Isolated Adipocyte Size with ImageJ 3.2.3 Determination of Adipocyte Size Using Histological Sections and ImageJ 4 Notes References Chapter 5: Measuring Mitochondrial Uncoupling in Mouse Primary Brown Adipocytes Differentiated Ex Vivo 1 Introduction 2 Materials 2.1 Preadipocyte Isolation 2.1.1 Isolation from BAT of Neonates 2.1.2 Isolation from BAT of Three- to Four-Week-Old Mice 2.2 Differentiation Protocol 2.2.1 Differentiation from Neonatal Preadipocytes 2.2.2 Differentiation Protocol from Three- to Four-Week-Old Mice Preadipocytes 2.3 Respirometry 3 Methods 3.1 Preadipocyte Isolation 3.1.1 Isolation from Neonatal BAT (see Note 7) 3.1.2 Isolation from BAT of Three- to Four-Week-Old Mice (see Note 6) 3.2 Differentiation Protocol (see Note 8) 3.2.1 Neonatal Preadipocytes 3.2.2 Differentiation Protocol from Three- to Four-Week-Old Mice Preadipocytes 3.3 Respirometry Using XF24 Agilent Seahorse Extracellular Analyzer 3.4 Normalization of Respirometry per Cell Count and Data Analysis 4 Notes References Chapter 6: Assessment of Bioenergetics in BAT Mitochondria 1 Introduction 2 Materials 2.1 Equipment 2.2 Buffers (see Note 1) 2.3 Reagents for Respirometry and Fluorometry 3 Methods 3.1 BAT Mitochondrial Isolation 3.2 UCP1-Dependent Respiration (Pyruvate) 3.3 UCP1-Dependent Respiration (Palmitate) 3.4 ADP-Dependent Respiration 3.5 ATP Production (High-Resolution Fluorometry with Fluoromax-4) 3.6 Determining ATP/O Ratio 4 Notes References Chapter 7: Use of Isothermal Microcalorimetry to Measure Cellular Heat Production in Thermogenic Adipocytes 1 Introduction 2 Materials 2.1 Isolation of Adipocytes 2.2 Isolation of Mature Brown Adipocytes 2.3 Isolation of Mature White/Brite Adipocytes 2.4 Culture of hMADS Cells 2.5 Isothermal Microcalorimeter Assay 2.6 Data Analysis 3 Methods 3.1 Isolation Mature Brown Adipocytes 3.2 Isolation Mature White/Brite Adipocytes 3.3 Culture of hMAD Cells 3.4 Isothermal Microcalorimetry 3.5 Data Analysis 4 Notes References Chapter 8: Measurement of Intracellular Temperature in Brown Adipocytes Using a Cationic Fluorescent Polymeric Thermometer 1 Introduction 2 Materials 2.1 Biological and Other Materials 2.2 Instruments and Supplies 2.3 Cell Culture and Primary Cell Preparation Reagents 2.4 Cell Culture Media 2.5 Primary Brown Adipocyte Preparation and Calibration Curve 2.6 Stimulation of Brown Adipocytes for Intracellular Heat Generation 2.7 Software 3 Methods 3.1 Culture of Primary Brown Adipocyte 3.2 Optimization of Incorporation of Ratiometric FPT into Live Cultured BACs 3.3 Obtaining a Calibration Curve Under Fluorescence Microscopy 3.3.1 Preparation of BAC Extract 3.3.2 Obtaining a Calibration Curve 3.4 Temperature Imaging in Ratiometric FPT-Incorporated BACs 3.5 Analysis of the Fluorescence Ratio of Ratiometric FPT in BACs 4 Notes References Chapter 9: Indirect Calorimetry to Assess Energy Balance in Mice: Measurement and Data Analysis 1 Materials 1.1 Example: Differences in Energy Balance Prompted by Cold Versus Thermoneutral Housing Temperatures 2 Importing Data into CalR 3 Data Inspection Using Time Plots 4 Food Intake 5 Energy Expenditure 6 Energy Balance 7 Change in Mass and Quality Control 8 Excluding Suspect Mice and Rerunning Analysis 9 Details of Calculating Energy Balance 10 Statistically Analyzing Energy Balance References Chapter 10: Thermogenic Phenotyping in Mice 1 Introduction 2 Materials 2.1 Measurement of Body Temperature in Mice 2.2 Measurement of β3-Adrenoceptor Agonist-Induced Oxygen Consumption 3 Methods 3.1 Measurement of Body Temperature in Mice Using Implantable Transponders 3.1.1 Animal Anesthesia 3.1.2 Transponder Implantation 3.1.3 Temperature Measurement 3.2 Use of Indirect Calorimetry to Measure β3-Adrenoceptor Agonist-Induced Oxygen Consumption 3.2.1 Metabolic Cage Setup 3.2.2 Starting the PhenoMaster Indirect Calorimetry System 3.2.3 Measurement of Oxygen Consumption After Administration of CL-316,243 4 Notes References Chapter 11: Calculating Diet-Induced Thermogenesis in Mice 1 Introduction 2 Materials 2.1 Animals 2.2 Diet 2.3 Calorimeter for Mice 3 Methods 3.1 Measurement of Activity and EE of Fasted Mice 3.2 Measurement of DIT 4 Notes References Chapter 12: Assessment of Brown and Beige Adipose Tissue Activation in Mice Using PET/CT Imaging 1 Introduction 2 Materials 2.1 Cold Acclimation in Mice 2.2 PET/CT 2.3 H&E Staining 3 Methods 3.1 Cold Acclimation in Mice 3.2 PET/CT 3.3 H&E Staining 4 Notes References Chapter 13: Imaging Brown Adipose Tissue with TSPO PET Tracers in Preclinical Animal Studies 1 Introduction 2 Materials 2.1 Instruments and Software 2.2 Reagents 3 Methods 3.1 Preparation of Precursor for 18F-Labeling (Fig. 1) 3.2 18F-Labeling (Fig. 2) 3.3 In Vivo PET Imaging with [18F]-DPA (Fig. 3) 3.4 Biodistribution Studies 4 Notes References Chapter 14: Use of CIDEA Reporter Mouse Model for Screening Thermogenic Fat-Activating Drugs 1 Introduction 2 Materials 2.1 Animals 2.2 Genotyping 2.3 Dissociation of Adipose Tissue and Stromal Vascular Cell Isolation 2.4 Fluorescence Microscopy 2.5 Bioluminescence Imaging 2.6 Luciferase Assay 3 Methods 3.1 Genotyping 3.2 Establishment of In Vitro CIDEA Reporter Preadipocytes 3.3 In Vitro Drug Screening 3.3.1 In Vitro Fluorescence Imaging 3.3.2 In Vitro Luminescence Imaging 3.3.3 In Vitro Luciferase Assay 3.4 In Vivo Drug Screening 3.4.1 In Vivo Bioluminescence Imaging 3.4.2 Ex Vivo Bioluminescence Imaging 3.4.3 Tissue Lysate Luciferase Assay 4 Notes References Chapter 15: AAV-Mediated Gene Delivery to Mouse Brown Adipose Tissue 1 Introduction 2 Materials 2.1 rAAV Production 2.2 Interscapular Brown Adipose Tissue Injection 2.3 Oral Gavage 3 Methods 3.1 rAAV Production 3.1.1 Transfection and Cell Lysis 3.1.2 Iodixanol Purification and Concentration 3.1.3 AAV Titration 3.2 Mouse Interscapular BAT Injection 3.3 Oral Gavage 4 Notes References Chapter 16: Subcutaneous Transplantation of White Adipose Tissue 1 Introduction 2 Materials 2.1 Mouse 2.2 Chemicals 2.3 Tools and Equipment 3 Methods 3.1 Preparation of the Donor Fat Grafts 3.2 Fat Graft Transplantation 3.3 Postsurgical Care 3.4 Confirmation of Fat Graft Success 4 Notes References Chapter 17: Brown Adipose Tissue Transplantation 1 Introduction 2 Materials 2.1 Equipment and Tools 2.2 Drugs 2.3 Supplies 3 Methods 3.1 Maintain Aseptic Conditions 3.2 Anesthesia (See Note 1) 3.3 Isolation of Donor Tissue (See Note 3) 3.3.1 Adult BAT (Fig. 1) 3.3.2 Embryonic BAT 3.4 Transplantation (Fig. 2) 3.4.1 Prep (See Notes 2, 5, and 6) 3.4.2 Surgery 3.4.3 Postoperative Care 4 Notes References Chapter 18: Isolation and Characterization of Brown Adipose Tissue T Cells 1 Introduction 2 Materials 2.1 Animals 2.2 Tools 2.3 Buffer and Culture Medium 2.4 Antibodies 3 Methods 3.1 Isolation of Stromal Vascular Fractions (SVFs) from BAT 3.2 Characterization of Brown Adipose Tissue T Cells 4 Notes References Chapter 19: Isolation of Adipose Tissue Extracellular Vesicles 1 Introduction 2 Materials 2.1 Buffers 2.2 Size Exclusion Column and EV Isolation 2.3 Cardiac Perfusion and Tissue Harvest 2.4 Adipose Tissue Dissociation and Sample Clarification 2.5 Antibodies 3 Methods 3.1 Preparation of the Size Exclusion Columns (see Note 2) 3.2 Setup for Tissue Harvest 3.3 Cardiac Perfusion 3.4 Adipose Tissue Harvest and Dissociation 3.5 EV Isolation and Characterization 4 Notes References Chapter 20: Isolation and Mass Spectrometry-Based Profiling of Major Lipids in Brown Adipose Tissue 1 Introduction 2 Materials 2.1 Sample Preparation 2.2 Mass Spectrometric Analysis 3 Methods 3.1 Preparation of Fatty Acid Internal Standard Solutions (see Note 1) 3.1.1 Fatty Acid Internal Standard Solution 3.1.2 Phospholipid Internal Standards 3.2 Isolation and Separation of Lipid Species from BAT 3.2.1 Lipid Extraction by Folch Method with Modification (see Note 2) 3.2.2 Fractionation of Total Lipid Extract Using SPE Column (see Note 8) 3.2.3 Hydrolysis of Esterified Fatty Acids to Nonesterified Fatty Acids (see Note 12) 3.3 Mass Spectrometric Analysis 3.3.1 Mass spectrometric Analysis Using Linked Scannings on a TSQ Instrument (see Note 14) 3.3.2 High-Resolution (HR) Mass Spectrometric Analysis 4 Notes References Chapter 21: Methods for Single Cell Transcriptomic Analysis of Adipose Tissue 1 Introduction 2 Materials 2.1 Isolation of the Stromal Vascular Fraction from Mouse BAT or WAT for Single Cell Transcriptomics 2.2 Isolation of Cell Type-/Lineage-Specific Nuclei Using NuTRAP Strain 3 Method 3.1 Isolation of the Stromal Vascular Fraction from Mouse BAT or WAT for Single Cell Transcriptomics 3.2 Isolation of Cell Type-/Lineage-Specific Nuclei Using NuTRAP Strain 3.2.1 Tissue Homogenization and Nuclei Isolation 3.2.2 Labeling and Purification of Biotinylated Nuclei 3.2.3 Magnetic Separation with MS Columns 4 Notes References Index