@phdthesis{oai:sucra.repo.nii.ac.jp:00017917,
 author = {RAKHI, CHACRABATI},
 month = {},
 note = {xi, 76 ｐ., 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　I
ABSTRACT　II
TABLE OF CONTENTS　IV
LIST OF FIGURES　　VIII
Chapter 1　1-17
1.1. Introduction　1
1.1.1. Ghrelin　1
1.1.2. Regulatory mechanisms of ghrelin secretion　2
1.1.3. Stomach ghrelinoma cells (SG-1) and pancreatic ghrelinoma cells (PG-1)　4
1.1.4. G protein-coupled receptor, family C, group 5, member B (GPRC5B)　4
1.2. Materials and Methods　6
1.2.1. Cell lines　6
1.2.2. Chemicals　6
1.2.3. RT-PCR　6
1.2.4. Primary culture of gastric mucosal cells　7
1.2.5. Ghrelin secretion experiment in SG-1 cells and PG-1 cells　8
1.2.6. Quantitative RT-PCR　9
1.2.7. Measurement of ghrelin concentrations　10
1.2.8. Statistical analysis　10
1.3. Results　11
1.3.1. GPRC5B mRNA expression in SG-1 cells, PG-1 cells, and stomach tissue.　11
1.3.2. Effect of glutamate on ghrelin secretion in SG1 cells, PG-1 cells, and primary gastric mucosal cells　11
1.3.3. Role of glutamate on preproghrelin, GOAT, and PC1 mRNA expression in SG-1 cells　12
1.3.4. Effect of glutamate on preproghrelin, GOAT, and PC1 mRNA expression in PG-1 cells　12
1.3.5. Effect of D-glutamate on mRNA expression of preproghrelin and acyl-ghrelin secretion in PG-1 cells　12
1.4. Discussion　14
1.4.1. GPRC5B regulates ghrelin secretion in gastric ghrelin cells　14
1.5. Summary　17
Chapter 2　18-28
2.1. Introduction　18
2.2. Materials and methods　20
2.2.1. Cell lines　20
2.2.2. Chemicals　20
2.2.3. small-interfering RNA (siRNA) knockdown of GPRC5B　20
2.2.4. In vivo experiments　21
2.2.5. Measurement of ghrelin concentrations　21
2.2.6. Statistical analysis　22
2.3.Results　23
2.3.1. GPRC5B siRNA knockdown of ghrelin secretion in PG-1 cells　23
2.3.2. Effect of glutamate on food intake and plasma acyl-ghrelin levels in mice　23
2.3.3. Dose-dependent effect of glutamate on food intake and plasma acyl ghrelin levels in mice　23
2.4. Discussion　25
2.5. Summary　28
Chapter 3　29-40
3.1. Introduction　29
3.2. Materials and methods　31
3.2.1. Cell lines　31
3.2.2. Chemicals　31
3.2.3. Ghrelin secretion experiment　31
3.2.4. Western Blotting　32
3.2.5. Quantitative RT-PCR　33
3.2.6. Measurement of ghrelin concentrations　34
3.2.7. Statistical analysis　34
3.3. Results　35
3.3.1. Intracellular signaling in ghrelin secretion　35
3.3.2. Role of glutamate in regulating phosphorylated ERK levels in PG-1 cells and SG-1 cells　35
3.3.3. Effect of glutamate on mRNA expression of CREB1 and CREB2 in PG-1 cells and SG-1 cells　36
3.3.4. Effect of GPRC5B signaling on NE-induced ghrelin elevation　36
3.4. Discussion　37
3.4.1. GPRC5B signaling mechanisms involved in ghrelin secretion　37
3.4.2. Crosstalk between GPRC5B and adrenergic receptor signaling pathways in regulating ghrelin secretion　37
3.4.3. The role of CREB2 in GPRC5B-mediated inhibition of ghrelin secretion　38
3.5. Summary　40
4.0. CONCLUSION　41
ABBREVIATIONS　42
ACKNOWLEDGEMENTS　46
REFERENCES　47
FIGURES, 主指導教員 : 坂田一郎, text, application/pdf},
 school = {埼玉大学},
 title = {A novel role of G protein coupled receptor, family C, group 5, member B (GPRC5B) in the regulation of ghrelin expression and secretion},
 year = {2017},
 yomi = {ラキ, チャックラーバティ}
}