1st Edition - September 25, 2012

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  • Editors: Masayasu Kojima, Kenji Kangawa
  • eBook ISBN: 9780123812735
  • Hardcover ISBN: 9780123812728

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This new volume of Methods in Enzymology continues the legacy of this premier serial by containing quality chapters authored by leaders in the field. The volume covers ghrelin, and has chapters on such topics as orphan gpcrs and methods for identifying their ligands, ghrelin o-acyltransferase assays and inhibition, and thermogenic characterization of ghrelin receptor null mice.

Key Features

  • Contains quality chapters authored by leaders in the field
  • Has chapters on such topics as orphan gpcrs and methods for identifying their ligands, ghrelin o-acyltransferase assays and inhibition, and thermogenic characterization of ghrelin receptor null mice


Biochemists, biophysicists, molecular biologists, analytical chemists, and physiologists

Table of Contents

  • Series Page



    Methods in Enzymology

    Chapter One History to the Discovery of Ghrelin

    1 Introduction/Pre-GHRP Studies

    2 GHRP Beginning

    3 GHRP-6 Active In Vitro and In Vivo (1982)

    4 GHRH Isolation and Identification (1982)

    5 GHRP Versus GHRH (1981–1984)

    6 GHRP Reflects Activity of a New Hormone and New Receptor—Hypothalamic–Pituitary Models (1984–1991)

    7 GHRP/GHS Receptor Assays (1989–2000)

    8 GHS/GHRP Receptor (1996–1998)

    9 Clinical Results in Humans

    10 Summary/Conclusion

    Chapter Two Orphan GPCRs and Methods for Identifying Their Ligands

    1 Introduction

    2 Strategies for Identifying Ligands of Orphan GPCRs

    3 Selection of Target Orphan GPCRs

    4 Recombinant Expression Systems for Orphan GPCRs

    5 Sources of Potential Ligands of Orphan GPCRs

    6 Functional Assays for Screening Candidate Ligands of Orphan GPCRs

    7 Summary

    Chapter Three Purification of Rat and Human Ghrelins

    1 Introduction

    2 GHS and Its Receptor

    3 Purification of Rat Ghrelin

    4 Purification of Human Ghrelin

    5 Purification of Rat des-Gln14-ghrelin

    6 Obestatin, an Anorexigenic Hormone or a Digested Peptide Fragment from a Ghrelin Precursor?

    7 Conclusion

    Chapter Four Variety of Acyl Modifications in Mammalian Ghrelins

    1 Introduction

    2 Purification of Mammalian Ghrelin

    3 GH-Releasing Activity of Ghrelin in Mammals

    4 Summary

    Chapter Five Determination of Nonmammalian Ghrelin

    1 Introduction

    2 Purification of Ghrelin

    3 Measurement of Ghrelin Activity

    4 CDNA Cloning and Determination of Amino Acid Sequence of Prepro-Ghrelin

    5 Determination of Species of Fatty Acid Modification by Mass Spectrometric Analysis

    6 Summary

    Chapter Six Morphological Analysis of Ghrelin Neurons in the Hypothalamus

    1 Introduction

    2 Observation of Ghrelin-Containing Cells at the Light Microscope Level

    3 Observation of Ghrelin-Containing Cells at Electron Microscopic Level

    4 Summary

    Chapter Seven High-Performance Liquid Chromatography Analysis of Hypothalamic Ghrelin

    1 Introduction

    2 Identification of Hypothalamic Ghrelin

    3 Quantification of Immunoreactive Ghrelin in Rats

    4 Quantification of Rat Ghrelin mRNA in Hypothalamic Ghrelin in Rats

    5 Identification of Ghrelin-Producing Neurons in Rats

    6 Summary

    Chapter Eight Standard Sample Collections for Blood Ghrelin Measurements

    1 Introduction

    2 Materials and Methods

    3 Study Protocols

    4 Results

    5 Discussion

    Chapter Nine From Ghrelin to Ghrelin’s O-Acyl Transferase

    1 Introduction

    2 Quantitative MALDI-ToF MS Methodology for Ghrelin in Biological Matrices

    3 Stabilizing Ghrelin in Biological Matrices

    4 TT Cell Culture System for Ghrelin Production

    5 Functional Screening for Ghrelin’s O-Acyl Transferase

    6 GOAT is Ghrelin’s Acyl Transferase

    7 Summary

    Chapter Ten Enzymatic Characterization of GOAT, ghrelin O-acyltransferase

    1 Introduction

    2 GOAT Enzymatic Assay

    3 Detecting the Molecular Forms of Ghrelin Generated by In Vitro GOAT Enzymatic Assays

    4 Enzymatic Characterization of GOAT

    5 Alterations in GOAT mRNA Expression in the Stomach Under Fasting Conditions

    6 Conclusion

    Chapter Eleven The Study of Ghrelin Deacylation Enzymes

    1 Introduction

    2 Processing of Ghrelin

    3 Determination of Ghrelin Deacylation Activity

    4 Future Directions

    Chapter Twelve Synthesis of Ghrelin

    1 Introduction

    2 Protocols of Chemical Synthesis of Human Ghrelin

    3 Protocols of Semisynthesis of Human Ghrelin

    4 Conclusion

    Chapter Thirteen Ghrelin O-Acyltransferase Assays and Inhibition

    1 Introduction

    2 Analysis of Acyl Ghrelin Levels and GOAT Activity

    3 GOAT Inhibitor Discovery

    4 Challenges and Future Directions

    Chapter Fourteen Model-Based Evaluation of Growth Hormone Secretion

    1 Introduction

    2 Ensemble GH-Regulation MODEL

    3 Technical Assessment of Multipathway GH Regulation

    4 Specific Methodological Strategies

    5 Summary

    Chapter Fifteen Ghrelin in the Control of Energy, Lipid, and Glucose Metabolism

    1 Introduction

    2 Central Administration of Ghrelin Increases Acute Food Intake in Rats

    3 Chronic Central Infusion of Ghrelin Generates a Positive Energy Balance in Rodents

    4 Ghrelin Regulates Glucose Homeostasis

    5 Summary

    Chapter Sixteen Ghrelin and the Vagus Nerve

    1 Introduction

    2 Anatomy of the Vagus Nerve

    3 GHS-R in the Vagal Afferent Neurons

    4 Action of Ghrelin via the Vagus Nerve

    5 Summary

    Chapter Seventeen Measurement of AMP-Activated Protein Kinase Activity and Expression in Response to Ghrelin

    1 Introduction

    2 The AMPK Assay Methodology

    3 Immunoblotting pAMPK

    4 AMPK Gene Expression

    5 Summary

    Chapter Eighteen Ghrelin and Gastrointestinal Movement

    1 Introduction

    2 The Role of Ghrelin in Gastroduodenal Motility

    3 The GI Motor Effect of Ghrelin Mediated by the Gut–Brain Axis

    4 Summary

    Chapter Nineteen Ghrelin Acylation by Ingestion of Medium-Chain Fatty Acids

    1 Introduction

    2 Preparing Food and Water Containing MCFAs or MCTs

    3 Samples for Ghrelin Measurement

    4 Measurement of Ghrelins Modified with or without the n-Octanoyl Group

    5 Purification and Characterization of Acyl Ghrelins from Stomachs

    6 Summary

    Chapter Twenty Islet β-Cell Ghrelin Signaling for Inhibition of Insulin Secretion

    1 Introduction

    2 Ghrelin Is Released in the Pancreatic Islets

    3 Insulinostatic Function of Islet-Derived Ghrelin

    4 Ghrelin Signaling in Islet β-Cells

    5 Conclusion

    Chapter Twenty-One Rikkunshito as a Ghrelin Enhancer

    1 Introduction

    2 Cisplatin-Induced Anorexia

    3 Anorexia of Aging

    4 Stress

    5 Ghrelin-Degrading Enzyme

    Chapter Twenty-Two Thermogenic Characterization of Ghrelin Receptor Null Mice

    1 Introduction

    2 In Vivo Metabolic and Thermogenic Characterizations

    3 Hormonal Characterization: Thyroid Hormones and Catecholamines

    4 Gene/Protein Expression Profiles and Ex Vivo Lipolysis of BAT

    5 Isolation and Characterization of Brown Adipocytes

    6 Summary

    Chapter Twenty-Three Transgenic Mice Overexpressing Ghrelin or Ghrelin Analog

    1 Introduction

    2 Des-Acyl Ghrelin Tg Mice

    3 Tg Mice Overexpressing a Ghrelin Analog

    4 Tg Mice Overexpressing Both Human GOAT and Ghrelin

    5 Summary

    Chapter Twenty-Four Therapeutic Potential of Ghrelin in Restricting-Type Anorexia Nervosa

    1 Introduction

    2 Pathophysiology of AN

    3 Plasma Ghrelin in AN

    4 Clinical Application of Ghrelin in Patients with AN

    5 Conclusions

    Chapter Twenty-Five Clinical Application of Ghrelin for Chronic Respiratory Diseases

    1 Introduction

    2 Clinical Application of Ghrelin for Chronic Respiratory Disease

    3 Summary

    Chapter Twenty-Six Clinical Trial of Ghrelin Synthesis Administration for Upper GI Surgery

    1 Introduction

    2 Ghrelin Replacement Therapy After TG and Esophagectomy

    3 Anti-inflammatory Role of Ghrelin After Esophageal Surgery

    4 Ghrelin Replacement Therapy During Chemotherapy in Patients with Esophageal Cancer

    Author Index

    Subject Index

Product details

  • No. of pages: 512
  • Language: English
  • Copyright: © Academic Press 2012
  • Published: September 25, 2012
  • Imprint: Academic Press
  • eBook ISBN: 9780123812735
  • Hardcover ISBN: 9780123812728

About the Serial Volume Editors

Masayasu Kojima

Affiliations and Expertise

California Institute of Technology, Division of Biology, Pasadena, U.S.A.

Kenji Kangawa

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