Ghrelin

Series Page

Contributors

Preface

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