【博士論文】学術データベース

博士論文 / A novel role of G protein coupled receptor, family C, group 5, member B (GPRC5B) in the regulation of ghrelin expression and secretion グレリン発現と分泌におけるGPRC5Bの役割に関する研究

著者

書誌事項

タイトル

A novel role of G protein coupled receptor, family C, group 5, member B (GPRC5B) in the regulation of ghrelin expression and secretion

タイトル別名

グレリン発現と分泌におけるGPRC5Bの役割に関する研究

著者名

RAKHI CHACRABATI

学位授与大学

埼玉大学 (大学ID:0019) (CAT機関ID:KI00020X)

取得学位

博士(学術)

学位授与番号

甲第1051号

学位授与年月日

2017-03-22

注記・抄録

Ghrelin is a 28-amino acid peptide that is mainly produced in the stomach and is purified as an endogenous ligand of the growth hormone secretagogue receptor (GHSR). Ghrelin plays many important roles in the regulation of feeding behavior and energy homeostasis. Ghrelin plasma levels change depending on the nutritional and metabolic status of the body; ghrelin levels increase dramatically before each meal and decline rapidly after feeding. However, ghrelin secretion mechanisms are not fully understood. To determine the novel factors that are involved in the regulation of ghrelin secretion, we focused on the G protein-coupled receptor, family C, group 5, member B (GPRC5B). We studied the role of GPRC5B on ghrelin expression and secretion in stomach ghrelinoma (SG-1), pancreatic ghrelinoma (PG-1), and primary gastric mucosal cells. Results of quantitative RT-PCR analysis revealed that SG-1, PG-1, and stomach tissue exhibit transcriptional expression of GPRC5B strongly. Glutamate, a putative GPRC5B agonist candidate, inhibited ghrelin secretion in SG-1, PG-1, and primary cells in a dose-dependent manner. However, glutamate treatment significantly inhibited mRNA expression of ghrelin, but not ghrelin O-acyltransferase (GOAT) and prohormone convertase 1 (PC1). L-glutamate treatment significantly decreased ghrelin expression, but D-glutamate did not affect ghrelin expression and secretion. Small interfering RNA (siRNA) targeting GPRC5B blocked the inhibitory effect of glutamate on ghrelin secretion. Furthermore, pretreatment with glutamate blocked the effect of the norepinephrine (NE)-induced ghrelin upregulation in primary cultures of gastric mucosal cells. Additionally, the results showed that glutamate treatment significantly increased the levels of phosphorylated extracellular signal-regulated kinase (ERK) compared to the control group. Co-administration of glutamate with an ERK pathway inhibitor reversed the inhibitory effect of glutamate on acyl-ghrelin secretion in PG-1 and SG-1 cells. Glutamate treatment increased mRNA expression levels of cAMP response element-binding protein2 (CREB2) in both SG-1 and PG-1 cell lines. Furthermore, glutamate decreased both food intake and plasma acyl-ghrelin concentrations in mice. These results suggest that glutamate is an important regulator of ghrelin signaling and that GPRC5B signaling is involved in the inhibition of ghrelin expression and secretion in gastric ghrelin cells.

DECLARATION IABSTRACT IITABLE OF CONTENTS IVLIST OF FIGURES VIIIChapter 1 1-171.1. Introduction 11.1.1. Ghrelin 11.1.2. Regulatory mechanisms of ghrelin secretion 21.1.3. Stomach ghrelinoma cells (SG-1) and pancreatic ghrelinoma cells (PG-1) 41.1.4. G protein-coupled receptor, family C, group 5, member B (GPRC5B) 41.2. Materials and Methods 61.2.1. Cell lines 61.2.2. Chemicals 61.2.3. RT-PCR 61.2.4. Primary culture of gastric mucosal cells 71.2.5. Ghrelin secretion experiment in SG-1 cells and PG-1 cells 81.2.6. Quantitative RT-PCR 91.2.7. Measurement of ghrelin concentrations 101.2.8. Statistical analysis 101.3. Results 111.3.1. GPRC5B mRNA expression in SG-1 cells, PG-1 cells, and stomach tissue. 111.3.2. Effect of glutamate on ghrelin secretion in SG1 cells, PG-1 cells, and primary gastric mucosal cells 111.3.3. Role of glutamate on preproghrelin, GOAT, and PC1 mRNA expression in SG-1 cells 121.3.4. Effect of glutamate on preproghrelin, GOAT, and PC1 mRNA expression in PG-1 cells 121.3.5. Effect of D-glutamate on mRNA expression of preproghrelin and acyl-ghrelin secretion in PG-1 cells 121.4. Discussion 141.4.1. GPRC5B regulates ghrelin secretion in gastric ghrelin cells 141.5. Summary 17Chapter 2 18-282.1. Introduction 182.2. Materials and methods 202.2.1. Cell lines 202.2.2. Chemicals 202.2.3. small-interfering RNA (siRNA) knockdown of GPRC5B 202.2.4. In vivo experiments 212.2.5. Measurement of ghrelin concentrations 212.2.6. Statistical analysis 222.3.Results 232.3.1. GPRC5B siRNA knockdown of ghrelin secretion in PG-1 cells 232.3.2. Effect of glutamate on food intake and plasma acyl-ghrelin levels in mice 232.3.3. Dose-dependent effect of glutamate on food intake and plasma acyl ghrelin levels in mice 232.4. Discussion 252.5. Summary 28Chapter 3 29-403.1. Introduction 293.2. Materials and methods 313.2.1. Cell lines 313.2.2. Chemicals 313.2.3. Ghrelin secretion experiment 313.2.4. Western Blotting 323.2.5. Quantitative RT-PCR 333.2.6. Measurement of ghrelin concentrations 343.2.7. Statistical analysis 343.3. Results 353.3.1. Intracellular signaling in ghrelin secretion 353.3.2. Role of glutamate in regulating phosphorylated ERK levels in PG-1 cells and SG-1 cells 353.3.3. Effect of glutamate on mRNA expression of CREB1 and CREB2 in PG-1 cells and SG-1 cells 363.3.4. Effect of GPRC5B signaling on NE-induced ghrelin elevation 363.4. Discussion 373.4.1. GPRC5B signaling mechanisms involved in ghrelin secretion 373.4.2. Crosstalk between GPRC5B and adrenergic receptor signaling pathways in regulating ghrelin secretion 373.4.3. The role of CREB2 in GPRC5B-mediated inhibition of ghrelin secretion 383.5. Summary 404.0. CONCLUSION 41ABBREVIATIONS 42ACKNOWLEDGEMENTS 46REFERENCES 47FIGURES

主指導教員 : 坂田一郎

博士の専攻分野の名称 : 博士(学術)学位授与年月日 : 平成29年3月22日

各種コード

NII論文ID(NAID)

500001055008

NII著者ID(NRID)
  • 8000001169329
本文言語コード

eng

データ提供元

機関リポジトリ / NDLデジタルコレクション

博士論文 / 埼玉大学 / 学術

博士論文 / 埼玉大学

博士論文 / 学術

関連著者

博士論文 / 大学

博士論文 / 学位