Short Term Soft Pellet Diet Changes Intestinal Characteristics in MiceKaren Miyakawa; Jin Huang; Toru Tanaka; Ichiro Sakata
Journal of Animal Physiology and Animal Nutrition, 2025年03月,
［査読有り］,
［責任著者］ABSTRACT
Diet alters the function and composition of small intestinal epithelial cells, making the relationship between diet and the intestine a focus of much research. This study aimed to clarify the effects of a soft diet on the small intestine. We fed mice a soft pellet diet (SP) and a control hard pellet diet (CD) for 14 days and examined changes in the epithelial cells of the small intestine. We found that the body weights of SP‐fed mice were lower than those of CD‐fed mice. SP did not alter the length of the small intestine, crypt to villus, or the number of Paneth and Goblet cells, but decreased the expression of small intestinal epithelial cell markers. We also found that SP did not change the copy number of mitochondrial DNA, but decreased the mRNA expression of mitochondrial metabolism genes in SP‐fed mice. In addition, we found that E‐cadherin, a cell adhesion factor, was decreased in SP‐fed mice and that the composition of their microbiota was different from that of CD mice. Our study suggests that SP may impair the homeostasis of small intestine epithelial cells, reinforcing the need for further research on how food texture affects intestinal health.
Wiley, 研究論文（学術雑誌）
DOI：https://doi.org/10.1111/jpn.14117DOI ID：10.1111/jpn.14117,
ISSN：0931-2439,
eISSN：1439-0396 Action of cocaine- and amphetamine-regulated transcript (CART) peptide to attenuate cisplatin-induced emesis in Suncus murinus (house musk shrew) Zengbing Lu; Sze Wa Chan; Bin Jiang; Dexuan Cui; Ichiro Sakata; Takafumi Sakai; Xiaofei Huang; Julia Yuen Hang Liu; Tak Wah Dominic Chan; John A. Rudd
European Journal of Pharmacology,
巻：984,
開始ページ：177072,
終了ページ：177072, 2024年12月,
［査読有り］Elsevier BV, 研究論文（学術雑誌）
DOI：https://doi.org/10.1016/j.ejphar.2024.177072DOI ID：10.1016/j.ejphar.2024.177072,
ISSN：0014-2999 Exploring the role of ghrelin and des-acyl ghrelin in chemotherapy-induced nausea and vomiting Lingqing Yang; Charmaine J.S. Kung; Zengbing Lu; Julia Y.H. Liu; Man Piu Ngan; Takafumi Sakai; Ichiro Sakata; Sze Wa Chan; Longlong Tu; John A. Rudd
Neuropharmacology,
巻：251,
開始ページ：109919,
終了ページ：109919, 2024年06月,
［査読有り］Elsevier BV, 研究論文（学術雑誌）
DOI：https://doi.org/10.1016/j.neuropharm.2024.109919DOI ID：10.1016/j.neuropharm.2024.109919,
ISSN：0028-3908 Identification of glucagon like peptide-1 (GLP-1) in mice stomach Manami Otsuka; Jin Huang; Toru Tanaka; Ichiro Sakata
Biochemical and Biophysical Research Communications,
巻：704,
開始ページ：149708,
終了ページ：149708, 2024年04月,
［査読有り］,
［責任著者］Elsevier BV, 研究論文（学術雑誌）
DOI：https://doi.org/10.1016/j.bbrc.2024.149708DOI ID：10.1016/j.bbrc.2024.149708,
ISSN：0006-291X The role of free fatty acid receptor-1 in gastric contractions in Suncus murinusJin Huang; Miu Suzuki; Ami Endo; Ayumi Watanabe; Ichiro Sakata
Food & Function,
巻：15,
号：4,
開始ページ：2221,
終了ページ：2233, 2024年02月,
［査読有り］,
［責任著者］Free fatty acid receptor-1 is involved in the regulation of gastric motility during the interdigestive and postprandial state in Suncus murinus.
Royal Society of Chemistry (RSC), 研究論文（学術雑誌）
DOI：https://doi.org/10.1039/d3fo03565dDOI ID：10.1039/d3fo03565d,
ISSN：2042-6496,
eISSN：2042-650X Involvement of the autonomic nervous system in colonic contractions in conscious Suncus murinusMiu Suzuki; Ayumi Watanabe; Jin Huang; Yuki Kobayashi; Ichiro Sakata
Neurogastroenterology & Motility,
巻：36,
号：2, 2023年11月,
［査読有り］,
［責任著者］Abstract
Background
Colonic motility is regulated by various factors along the gut‐brain axis; however, detailed mechanisms are unknown. This study aimed to examine the involvement of the autonomic nervous system in colonic motility. Suncus murinus (suncus) is a small laboratory mammal suitable for gastrointestinal motility studies.
Methods
Colonic motility and concomitant feeding and defecation behaviors in vagotomized and reserpine‐administered suncus were recorded simultaneously for 24 h. Furthermore, we performed immunohistochemistry on tyrosine hydroxylase (TH) and in situ hybridization on corticotropin‐releasing hormone (CRH) in suncus brain. Additionally, we examined c‐Fos expression in the brain using immunohistochemistry in conscious suncus with colorectal distension.
Key Results
In vagotomized suncus, clustered giant migrating contractions (GMCs), consisting of strong contractions occurring in a short time, were observed, and the percentage of GMCs without defecation increased. The frequency of GMCs in the reserpine‐administered suncus increased during a light period (ZT0‐4, 4–8) and decreased during a dark period (ZT16‐20, 20–24) compared to a vehicle group. Additionally, the percentage of GMCs without defecation in the reserpine‐administered suncus increased. Suncus TH‐immunopositive neurons were found in the locus coeruleus (LC), as shown in rodents. In contrast, CRH mRNA‐expressing cells were not observed in a region assumed to be the Barrington's nucleus (Bar). Furthermore, colorectal distension in conscious suncus induced c‐Fos expression in LC TH neurons.
Conclusions & Inferences
Our results suggest that the vagus and sympathetic nerves are not required for induction of GMCs in vivo. However, they are likely to exert a modulatory role in control of GMC frequency in Suncus murinus.
Wiley, 研究論文（学術雑誌）
DOI：https://doi.org/10.1111/nmo.14716DOI ID：10.1111/nmo.14716,
ISSN：1350-1925,
eISSN：1365-2982 Effect of cholecystokinin on small intestinal motility in suncus murinus Naho Yokota; Shota Takemi; Ichiro Sakata
General and Comparative Endocrinology,
巻：342,
開始ページ：114352,
終了ページ：114352, 2023年10月,
［査読有り］,
［責任著者］Elsevier BV, 研究論文（学術雑誌）
DOI：https://doi.org/10.1016/j.ygcen.2023.114352DOI ID：10.1016/j.ygcen.2023.114352,
ISSN：0016-6480 Molecular cloning and analysis of the ghrelin/GHSR system in Xenopus tropicalis. Reiko Wada; Shota Takemi; Mio Matsumoto; Mio Iijima; Takafumi Sakai; Ichiro Sakata
General and comparative endocrinology,
巻：331,
開始ページ：114167,
終了ページ：114167, 2023年01月,
［査読有り］,
［責任著者］,
［国際誌］Ghrelin is a gut-derived peptide with several physiological functions, including feeding, gastrointestinal motility, and hormonal secretion. Recently, a host defense peptide, liver-expressed antimicrobial peptide-2 (LEAP2), was reported as an endogenous antagonist of growth hormone secretagogue receptor (GHS-R). The physiological relevance of the molecular LEAP2-GHS-R interaction in mammals has been explored; however, studies on non-mammals are limited. Here, we report the identification and functional characterization of ghrelin and its related molecules in Western clawed frog (Xenopus tropicalis), a known model organism. We first identified cDNA encoding X. tropicalis ghrelin and GHS-R. RT-qPCR revealed that ghrelin mRNA expression was most abundant in the stomach. GHS-R mRNA was widely distributed in the brain and peripheral tissues, and a relatively strong signal was observed in the stomach and intestine. In addition, LEAP2 was mainly expressed in intestinal tissues at higher levels than in the liver. In functional analysis, X. tropicalis ghrelin and human ghrelin induced intracellular Ca2+ mobilization with EC50 values in the low nanomolar range in CHO-K1 cells expressing X. tropicalis GHS-R. Furthermore, ghrelin-induced GHS-R activation was antagonized with IC50 values in the nanomolar range by heterologous human LEAP2. We also validated the expression of ghrelin and feeding-related factors under fasting conditions. After 2 days of fasting, no changes in ghrelin mRNA levels were observed in the stomach, but GHS-R mRNA levels were significantly increased, associated with significant downregulation of nucb2. In addition, LEAP2 upregulation was observed in the duodenum. These results provide the first evidence that LEAP2 functions as an antagonist of GHS-R in the anuran amphibian X. tropicalis. It has also been suggested that the ghrelin/GHS-R/LEAP2 system may be involved in energy homeostasis in X. tropicalis.
英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1016/j.ygcen.2022.114167DOI ID：10.1016/j.ygcen.2022.114167,
PubMed ID：36402245 Identification of motilin in Japanese fire bellied newt. Mio Matsumoto; Shota Takemi; Takafumi Sakai; Ichiro Sakata
General and comparative endocrinology,
巻：323-324,
開始ページ：114031,
終了ページ：114031, 2022年07月,
［査読有り］,
［責任著者］,
［国際誌］Motilin, a peptide hormone consisting of 22 amino acid residues, was identified in the duodenum of pigs in the 1970s. It is known to induce gastrointestinal contractions during the interdigestive state in mammals. Although the motilin gene has been identified in various animal species, it has not been studied in amphibians. Here, we identified the motilin gene in the Japanese fire bellied newt (Cynops pyrrhogaster), and conducted an analysis of tissue distribution, morphological observations, and physiological experiments. The deduced mature newt motilin comprises 22 amino acid residues, like in mammals and birds. The C-terminus of the newt motilin showed high homology with motilin from other species compared to the N-terminus region, which is considered the bioactive site. Motilin mRNA expression in newts was abundant in the upper small intestine, with notably high motilin mRNA expression found in the pancreas. Motilin-producing cells were found in the mucosal layer of the upper small intestine and existed as two cell types: open-and closed-type cells. Motilin-producing cells in the pancreas were also found to produce insulin but not glucagon. Newt motilin stimulated gastric contractions but not in other parts of the intestines in vitro, and motilin-induced gastric contraction was significantly inhibited by treatment with atropine, a muscarinic acetylcholine receptor antagonist. These results indicate that motilin is also present in amphibians, and that its gastrointestinal contractile effects are conserved in mammals, birds, and amphibians. Additionally, we demonstrated for the first time the existence of pancreatic motilin, suggesting that newt motilin has an additional unknown physiological role.
英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1016/j.ygcen.2022.114031DOI ID：10.1016/j.ygcen.2022.114031,
PubMed ID：35331740 Molecular cloning of cholecystokinin (CCK) and CCK-A receptor and mechanism of CCK-induced gastrointestinal motility in Suncus murinus. Shota Takemi; Wataru Honda; Naho Yokota; Haruka Sekiya; Takashi Miura; Reiko Wada; Takafumi Sakai; Ichiro Sakata
General and comparative endocrinology,
巻：327,
開始ページ：114074,
終了ページ：114074, 2022年06月,
［査読有り］,
［責任著者］,
［国際誌］Cholecystokinin (CCK) is a peptide hormone mainly secreted by small intestinal endocrine I-cells and functions as a regulator of gallbladder contraction, gastric emptying, gastrointestinal (GI) motility, and satiety. The cellular effects of CCK in these peripheral tissues are predominantly mediated via CCK-A receptors which are found in smooth muscles, enteric neurons, and vagal afferent neurons in humans and animal models. Although various functions of CCK have been reported to be neurally mediated, it can also stimulate contraction via the CCK receptor on the smooth muscle. However, the entire underlying neural and cellular mechanisms involved in CCK-induced GI contractions are not clearly understood. Here, we first determined the cDNA and amino acid sequences of CCK and CCK-A receptor along with the distributions of cck mRNA and CCK-producing cells in house musk shrew (Suncus murinus, the laboratory strain named as suncus) and examined the mechanism of CCK-induced contraction in the GI tract. Mature suncus CCK-8 was identical to other mammalian species tested here, and suncus CCK-A receptor presented high nucleotide and amino acid homology with that of human, dog, mouse, and rat, respectively. Suncus CCK mRNA and CCK-producing cells were found mainly in small intestine and colon. In the organ bath study, CCK-8 induced dose-dependent contractions in the suncus stomach, duodenum, and jejunum, and these contractions were inhibited by atropine and CCK-A receptor antagonist. These results suggest that CCK-8-induced contraction is mediated in the myenteric cholinergic neural network and that CCK-A receptor is partly responsible for CCK-8-induced contractions. This study indicates that suncus is a useful animal model to study the functions of CCK involved in GI motility.
英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1016/j.ygcen.2022.114074DOI ID：10.1016/j.ygcen.2022.114074,
PubMed ID：35700795 The Actions of Centrally Administered Nesfatin-1 on Emesis, Feeding, and Locomotor Activity in Suncus murinus (House Musk Shrew). Zengbing Lu; Dexuan Cui; Julia Yuen Hang Liu; Bin Jiang; Man Piu Ngan; Ichiro Sakata; Shota Takemi; Takafumi Sakai; Ge Lin; Sze Wa Chan; John A Rudd
Frontiers in pharmacology,
巻：13,
開始ページ：858522,
終了ページ：858522, 2022年,
［査読有り］,
［国際誌］Nesfatin-1 is an anorectic peptide expressed in both peripheral tissues and brain areas involved in the regulation of feeding, emotion and emesis. The aim of the present study is to characterize the distribution of NUCB2/nesfatin-1 in Suncus murinus and to investigate the actions of nesfatin-1 to affect gastrointestinal contractility, emesis, food and water intake, and locomotor activity. The deduced amino acid sequence of S. murinus nesfatin-1 using in silico cloning showed high homology with humans and rodents. NUCB2 mRNA was detected throughout the entire brain and in the gastrointestinal tract, including the stomach and gut. Western blot analysis and immunohistochemistry confirmed the expression of nesfatin-1 protein in these regions. The NUCB2 mRNA levels in the hypothalamus, hippocampus and brainstem were significantly decreased, whereas that in the striatum were increased after 24 h starvation compared to ad libitum-fed animals (p < 0.05). In in vitro studies, nesfatin-1 (0.3-1,000 pM) failed to contract or relax the isolated gastric antrum and intestinal segments. In conscious, freely moving animals, intracerebroventricular administration of nesfatin-1 (1-50 pmol) induced emesis (p < 0.05) and suppressed 6-h cumulative food intake (p < 0.05), without affecting the latency to feeding. Nesfatin-1 (25 pmol, i.c.v.) decreased 24-h cumulative food and water intake by 28.3 and 35.4%, respectively (p < 0.01). No significant differences in locomotor activity were observed. In conclusion, NUCB2/nesfatin-1 might be a potent regulator of feeding and emesis in S. murinus. Further studies are required to elucidate the mechanism of actions of this peptide as a mediator linking the brainstem NUCB2/nesfatin-1 to forebrain system.
英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.3389/fphar.2022.858522DOI ID：10.3389/fphar.2022.858522,
PubMed ID：35462894,
PubMed Central ID：PMC9019301 Molecular characterization and expression analysis of the regenerating islet-derived protein 3 alpha from Suncus murinus Shota Takemi; Takashi Miura; Toru Tanaka; Ichiro Sakata
GENE REPORTS,
巻：25, 2021年12月,
［査読有り］The regenerating islet-derived protein (Reg) 3 family of C-type lectins is primarily produced in the intestinal epithelial cells and exert several different physiological functions including as antimicrobial agents. The Reg3 family includes Reg3 alpha, Reg3 beta, Reg3 gamma, and Reg3 delta in mice, while only Reg3 alpha and Reg3 gamma are found in humans. Accumulating evidence suggests that Reg3 proteins play several critical roles in maintaining host-bacterial homeostasis in the mammalian intestine, but little information is available on the mechanism regulating their expression in other organisms. In an effort to gain some insight into the evolutionary features of Reg3 proteins in mammals, we cloned Reg3 alpha from suncus (Suncus murinus), which belongs to the Insectivora, and examined the tissue specific gene expression of Reg3 alpha. Although suncus Reg3 alpha lacks the bacterial binding/killing motifs, the deduced peptide of Reg3 alpha do exhibit the other characteristic features of Reg3 family members, including several disulfide bonds and trypsin-dependent proteolytic processing, suggesting their functionality. Reg3 alpha mRNA was found to be most abundant in the pancreas and highly expressed in the intestine. Reg3 alpha function is not limited to its bactericidal effect; it is also known to attenuate intestinal inflammation. Given that the expression of Reg3a alpha mRNA in DSS-treated suncus was significantly greater than that in the control, we hypothesized that it plays a protective role during inflammation. This study provides a basis for further investigation of the expression and physiological role of Reg proteins in other mammals and broadens our understanding of the divergence and evolutionary conservation of Reg proteins in various mammalian species.
ELSEVIER, 英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1016/j.genrep.2021.101400DOI ID：10.1016/j.genrep.2021.101400,
eISSN：2452-0144,
Web of Science ID：WOS:000710737500005 The suppressive effect of REVERBs on ghrelin and GOAT transcription in gastric ghrelin-producing cells Mio Iijima; Shota Takemi; Sayaka Aizawa; Takafumi Sakai; Ichiro Sakata
Neuropeptides,
巻：90,
開始ページ：102187,
終了ページ：102187, 2021年12月,
［査読有り］,
［責任著者］Elsevier BV, 研究論文（学術雑誌）
DOI：https://doi.org/10.1016/j.npep.2021.102187DOI ID：10.1016/j.npep.2021.102187,
ISSN：0143-4179 Diurnal changes of colonic motility and regulatory factors for colonic motility in Suncus murinus Yuki Kobayashi; Shota Takemi; Takafumi Sakai; Chikashi Shibata; Ichiro Sakata
NEUROGASTROENTEROLOGY AND MOTILITY, 2021年11月,
［査読有り］,
［責任著者］Background The aim of this study was to investigate the fundamental mechanisms of colonic motility in the house musk suncus (Suncus murinus) as an established animal model of gut motility. Methods To measure gut motility in free-moving conscious suncus, strain gauge force transducers were implanted on the serosa of the colon and gastric body. Key Results We recorded diurnal changes in colonic motility and observed the relationship between feeding and colonic motility. Giant migrating contractions (GMCs) of the colon were invariably detected during defecation and tended to increase during the dark period, thereby indicating that colonic motility has a circadian rhythm. Given that GMCs in the suncus were observed immediately after feeding during the dark period, we assume the occurrence of a gastrocolic reflex in suncus, similar to that observed in humans and dogs. We also examined the factors that regulate suncus GMCs. Intravenous administration of 5-HT (100 mu g/kg), substance P (10 and 100 mu g/kg), calcitonin gene-related peptide (10 mu g/kg), and alpha 2 adrenergic receptor antagonist yohimbine (0.5, 1, and 3 mg/kg) induced GMC-like contractions, as did intragastric and intracolonic administration of the transient receptor potential vanilloid 1 agonist, capsaicin (1 mg/kg). Conclusions & Inferences These results indicate that the fundamental mechanisms of colonic motility in suncus are similar to those in humans and dogs, and we thus propose that suncus could serve as a novel small animal model for studying colonic motility.
WILEY, 英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1111/nmo.14302DOI ID：10.1111/nmo.14302,
ISSN：1350-1925,
eISSN：1365-2982,
Web of Science ID：WOS:000723663300001 Pyridoxine stimulates filaggrin production in human epidermal keratinocytes. Miyuki Fujishiro; Shoichi Yahagi; Shota Takemi; Mio Nakahara; Takafumi Sakai; Ichiro Sakata
Molecular biology reports,
巻：48,
号：7,
開始ページ：5513,
終了ページ：5518, 2021年07月,
［査読有り］,
［責任著者］,
［国際誌］Pyridoxine (PN), one of the vitamers of vitamin B6, plays an important role in the maintenance of epidermal function and is used to treat acne and rough skin. Clinical studies have revealed that PN deficiency causes skin problems such as seborrheic dermatitis and stomatitis. However, the detailed effects of PN and its mechanism of action in epidermal function are poorly understood. In this study, we examined the effects of PN on epidermal function in normal human epidermal keratinocytes and found that PN specifically causes an increase in the expression of profilaggrin mRNA, among marker genes of terminal epidermal differentiation. In addition, PN treatment caused an increase in the production of filaggrin protein in a concentration-dependent manner. Treatment with P2x purinoceptor antagonists, namely, pyridoxal phosphate-6-azo (benzene-2,4-disulfonic acid) tetrasodium salt hydrate and TNP-ATP hydrate, induced an increase in the filaggrin protein levels. Moreover, we showed that elevated filaggrin production induced upon PN treatment was suppressed by ATP (known as P2x purinoceptor agonist). This study is the first to report that PN causes an increase in filaggrin transcription and production, and these results suggest that PN-induced filaggrin production may be a useful target as a daily care component in atopic dermatitis, wherein filaggrin levels are specifically reduced.
英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1007/s11033-021-06563-yDOI ID：10.1007/s11033-021-06563-y,
PubMed ID：34302584 Ghrelin-cell physiology and role in the gastrointestinal tract. Ichiro Sakata; Shota Takemi
Current opinion in endocrinology, diabetes, and obesity,
巻：28,
号：2,
開始ページ：238,
終了ページ：242, 2021年04月,
［査読有り］,
［筆頭著者］,
［国際誌］PURPOSE OF REVIEW: Ghrelin was discovered in 1999; extensive research and clinical studies on ghrelin have been published in the last 20 years. Physiological research on ghrelin ranges from its appetite-stimulating effects to its association with energy homeostasis. The physiological effects of ghrelin in the gastrointestinal tract and its relevance in the pathological conditions of the gastrointestinal tract have gradually become clearer. The purpose of the review is to provide current information on ghrelin cell biology and physiology, particularly in the gastrointestinal tract. RECENT FINDINGS: Ghrelin-producing cells in the stomach are characterized as X/A-like cells, but immunohistochemical analyses have revealed co-expression of several secreted proteins and hormones in ghrelin-producing cells such as nesfatin-1, somatostatin, and pancreastatin. Furthermore, the local physiological roles and/or mechanisms of ghrelin in gastrointestinal functions such as gastric motility and inflammation are discussed. SUMMARY: Ghrelin is a brain-gut hormone with a wide range of physiological actions; hence, it is important to understand its effects on the physiological functions of the gastrointestinal tract to elucidate the biological significance of ghrelin.
英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1097/MED.0000000000000610DOI ID：10.1097/MED.0000000000000610,
PubMed ID：33394720 The role of central corticotrophin-releasing factor receptor signalling in plasma glucose maintenance through ghrelin secretion in calorie-restricted mice. Risa Kimura; Daisuke Kondo; Shota Takemi; Miyuki Fujishiro; Shinji Tsukahara; Takafumi Sakai; Ichiro Sakata
Journal of neuroendocrinology,
巻：33,
号：3,
開始ページ：e12961, 2021年03月,
［査読有り］,
［責任著者］,
［国際誌］Under severe calorie restriction (CR), the ghrelin-growth hormone axis in mice is involved in the maintenance of plasma glucose levels. Ghrelin, a stomach-derived acylated peptide, is up-regulated by the sympathetic nerve in the negative energy status. Central corticotrophin-releasing factor receptor (CRF-R) signalling stimulates the sympathetic tone. The present study aimed to examine the effect of central CRF-R signalling on the maintenance of plasma glucose concentrations in severe calorie-restricted mice with the involvement of ghrelin. Intracerebroventricular injections of urocorin-1 and urocorin-2, which are natural ligands for CRF-R1 and CRF-R2, elevated plasma ghrelin concentrations and ghrelin elevation with an i.c.v. injection of urocorin-1 was cancelled by atenolol (β1 adrenergic receptor antagonist) administration. We then established a mice model of 60% CR and found that the administration of [d-Lys3]-GHRP-6 (a ghrelin receptor antagonist) in mice under 60% CR reduced the plasma glucose concentration more compared to the vehicle mice. Similarly, the atenolol injection in mice under 60% CR significantly reduced the plasma glucose concentration, which was rescued by the co-administration of ghrelin. An i.c.v. injection of the alpha helical CRH, a non-selective corticotrophin-releasing factor receptor antagonist, in mice under 60% CR significantly reduced the plasma glucose concentration, although the co-administration of α-helical CRH with ghrelin maintained plasma glucose levels. These results suggest that central CRF-R signalling is involved in the maintenance of plasma glucose levels in mice under severe CR via the sympathetic-ghrelin pathway.
英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1111/jne.12961DOI ID：10.1111/jne.12961,
PubMed ID：33675127 [The basics of the study of gastrointestinal motility]. Shota Takemi; Ichiro Sakata; Takafumi Sakai
Nihon Shokakibyo Gakkai zasshi = The Japanese journal of gastro-enterology,
巻：118,
号：2,
開始ページ：107,
終了ページ：113, 2021年,
［国内誌］日本語, 研究論文（学術雑誌）
DOI：https://doi.org/10.11405/nisshoshi.118.107DOI ID：10.11405/nisshoshi.118.107,
PubMed ID：33563849 The inhibitory effect of somatostatin on gastric motility in Suncus murinus. Haruka Sekiya; Naho Yokota; Shota Takemi; Keiji Nakayama; Hiroki Okada; Takafumi Sakai; Ichiro Sakata
Journal of smooth muscle research = Nihon Heikatsukin Gakkai kikanshi,
巻：56,
号：0,
開始ページ：69,
終了ページ：81, 2020年,
［査読有り］,
［責任著者］,
［国内誌］Gastric contractions show two specific patterns in many species, migrating motor contractions (MMC) and postprandial contractions (PPCs), that occur in the fasted and fed states, respectively. In this study, we examined the role of somatostatin (SST) in gastric motility both in vivo and in vitro using the Asian house shrew (Suncus murinus). We performed in vivo recordings of gastric motility and in vitro organ bath experiments using S. murinus, which was recently established as a small laboratory animal for use in tests of gastrointestinal motility. SST (1.65 µg kg-1 min-1) was intravenously administered during phase II of MMC and PPCs. Next, the effect of SST on motilin-induced gastric contractions at phase I of MMC was measured. Cyclosomatostatin (CSST), an SST receptor antagonist, was administered at the peak of phase III of MMC. In addition, the effect of SST (10-11-10-9 M) on motilin-induced gastric contractions was evaluated using an organ bath experiment in vitro. In conscious, free-moving S. murinus, the administration of SST decreased the occurrence of the spontaneous phase II of MMC and PPCs. Pretreatment with SST and octreotide suppressed the induction of motilin-induced gastric contractions both in vivo and in vitro. Administration of CSST before the peak of spontaneous phase III contractions had no effect on gastric contractions. Endogenous SST is not involved in the regulation of gastric MMC and PPCs, but exogenous SST suppresses spontaneous gastric contractions. Thus, SST would be good for treating abnormal gastrointestinal motility disorders.
英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1540/jsmr.56.69DOI ID：10.1540/jsmr.56.69,
PubMed ID：33473062,
PubMed Central ID：PMC7817339 Identification of pheasant ghrelin and motilin and their actions on contractility of the isolated gastrointestinal tract Shuangyi Zhang; Yuji Okuhara; Mio Iijima; Shota Takemi; Ichiro Sakata; Hiroyuki Kaiya; Hiroki Teraoka; Takio Kitazawa
General and Comparative Endocrinology,
巻：285,
開始ページ：113294,
終了ページ：113294, 2020年01月,
［査読有り］Elsevier BV, 研究論文（学術雑誌）
DOI：https://doi.org/10.1016/j.ygcen.2019.113294DOI ID：10.1016/j.ygcen.2019.113294,
ISSN：0016-6480 Molecular cloning and analysis of Suncus murinus group IIA secretary phospholipase A2 expression. Shota Takemi; Ryo Nishio; Hayato Taguchi; Shiomi Ojima; Mio Matsumoto; Takafumi Sakai; Ichiro Sakata
Developmental and comparative immunology,
巻：100,
開始ページ：103427,
終了ページ：103427, 2019年11月,
［国際誌］The intestinal epithelial monolayer forms a mucosal barrier between the gut microbes and the host tissue. The mucosal barrier is composed of mucins and antimicrobial peptides and proteins (AMPs). Several animal studies have reported that Paneth cells, which occupy the base of intestinal crypts, play an important role in the intestinal innate immunity by producing AMPs, such as lysozyme, Reg3 lectins, α-defensins, and group IIA secretory phospholipase A2 (GIIA sPLA2). The house musk shrew (Suncus murinus) has only a few intestinal commensal bacteria and is reported to lack Paneth cells in the intestine. Although the expression of lysozyme was reported in the suncus intestine, the expression of other AMPs has not yet been reported. Therefore, the current study was focused on GIIA sPLA2 expression in Suncus murinus. GIIA sPLA2 mRNA was found to be most abundant in the spleen and also highly expressed in the intestine. Cells expressing GIIA sPLA2 mRNA were distributed not only in the crypt, but also in the villi. In addition, intragastric injection of lipopolysaccharide increased GIIA sPLA2 expression in the small intestine of suncus. These results suggest that suncus may host unique AMP-secreting cells in the intestine.
英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1016/j.dci.2019.103427DOI ID：10.1016/j.dci.2019.103427,
PubMed ID：31278953 Adenosine stimulates neuromedin U mRNA expression in the rat pars tuberalis. Sayaka Aizawa; Tingting Gu; Arisa Kaminoda; Ryuya Fujioka; Fumiya Ojima; Ichiro Sakata; Takafumi Sakai; Maho Ogoshi; Sumio Takahashi; Sakae Takeuchi
Molecular and cellular endocrinology,
巻：496,
号：496,
開始ページ：110518,
終了ページ：110518, 2019年10月,
［査読有り］,
［国際誌］Neuromedin U (NMU) shows circadian expression in the rat pars tuberalis (PT), and is known to be suppressed by melatonin. Here we examined the involvement of adenosine in the regulation of Nmu expression. We found that the rat PT expressed adenosine receptor A2b and that an adenosine receptor agonist, NECA, stimulated Nmu expression in brain slice cultures. In vitro promoter assays revealed that NECA stimulated Nmu promoter activity via a cAMP response element (CRE) in the presence of adenosine receptor A2b. NECA also increased the levels of phosphorylated CRE-binding protein. These findings suggest that adenosine stimulates Nmu expression by activating the cAMP signaling pathway through adenosine receptor A2b in the rat PT. This is the first report to demonstrate that Nmu expression in the PT is regulated by adenosine, which acts as an intravital central metabolic signal, in addition to melatonin, which acts as an external photoperiodic environmental signal.
英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1016/j.mce.2019.110518DOI ID：10.1016/j.mce.2019.110518,
ISSN：0303-7207,
PubMed ID：31344393 Circulating messenger for neuroprotection induced by molecular hydrogen. Mami Noda; Yuya Uemura; Yusuke Yoshii; Taichi Horita; Shota Takemi; Ichiro Sakata; Takafumi Sakai
Canadian journal of physiology and pharmacology,
巻：97,
号：10,
開始ページ：909,
終了ページ：915, 2019年10月,
［国際誌］Molecular hydrogen (H2) showed protection against various kinds of oxidative-stress-related diseases. First, it was reported that the mechanism of therapeutic effects of H2 was antioxidative effect due to inhibition of the most cytotoxic reactive oxygen species, hydroxy radical (•OH). However, after chronic administration of H2 in drinking water, oxidative-stress-induced nerve injury is significantly attenuated even in the absence of H2. It suggests indirect signaling of H2 and gastrointestinal tract is involved. Indirect effects of H2 could be tested by giving H2 water only before nerve injury, as preconditioning. For example, preconditioning of H2 for certain a period (∼7 days) in Parkinson's disease model mice shows significant neuroprotection. As the mechanism of indirect effect, H2 in drinking water induces ghrelin production and release from the stomach via β1-adrenergic receptor stimulation. Released ghrelin circulates in the body, being transported across the blood-brain barrier, activates its receptor, growth-hormone secretagogue receptor. H2-induced upregulation of ghrelin mRNA is also shown in ghrelin-producing cell line, SG-1. These observations help with understanding the chronic effects of H2 and raise intriguing preventive and therapeutic options using H2.
英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1139/cjpp-2019-0098DOI ID：10.1139/cjpp-2019-0098,
PubMed ID：31100203 Generation and characterization of Suncus murinus intestinal organoid: a useful tool for studying motilin secretion. Natsumi Takakura; Shota Takemi; Shunsuke Kumaki; Mio Matsumoto; Takafumi Sakai; Ken Iwatsuki; Ichiro Sakata
Cell biology international, 2019年07月,
［査読有り］,
［責任著者］,
［国際誌］Motilin, a 22-amino-acid peptide produced in the upper small intestine, induces strong gastric contraction in fasted state. In many rodents, motilin and its cognate receptors exist as pseudogenes, which has delayed motilin research in the past decades. Recently, the house musk shrew (Suncus murinus) was developed as a useful model for studying motilin and gastrointestinal motility. However, due to a lack of motilin-producing cell lines and difficulties in culturing small intestinal cells, the regulatory mechanisms of motilin secretion and its messenger RNA (mRNA) transcription have remained largely unclear. In this study, we generated small intestinal organoids from S. murinus for the first time. Using methods similar to mouse organoid generation, we found crypt-like budding structures 3 days after isolating intestinal tissues. The organoids grew gradually with time. In addition, the generated organoids were able to be passaged and maintained for 6 months or longer. Motilin messenger RNA (mRNA) and immunopositive cells were observed in both S. murinus intestinal organoids and primary tissues. This is the first report of intestinal organoids in S. murinus, and our results suggest that S. murinus intestinal organoids could be useful for analyzing motilin secretion and transcription.
英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1002/cbin.11201DOI ID：10.1002/cbin.11201,
PubMed ID：31293061 Identification and characterization of an antimicrobial peptide, lysozyme, from Suncus murinus. Shota Takemi; Shiomi Ojima; Toru Tanaka; Takafumi Sakai; Ichiro Sakata
Cell and tissue research,
巻：376,
号：3,
開始ページ：401,
終了ページ：412, 2019年06月,
［国際誌］Lysozyme is one of the most prominent antimicrobial peptides and has been identified from many mammalian species. However, this enzyme has not been studied in the order Insectivora, which includes the most primitive placental mammals. Here, we done the lysozyme cDNA from Suncus murinus (referred to as suncus, its laboratory name) and compare the predicted amino acid sequence to those from other mammalian species. Quantitative PCR analysis revealed a relatively higher expression of this gene in the spleen and gastrointestinal tract of suncus. The lysozyme-immunopositive (ip) cells were found mainly in the red pulp of the spleen and in the mucosa of the whole small intestine, including the follicle-associated epithelium and subepithelial dome of Peyer's patches. The lysozyme-ip cells in the small intestine were mostly distributed in the intestinal crypt, although lysozyme-expressing cells were found not only in the crypt but also in the villi. On the other hand, only a few lysozyme-ip cells were found in the villi and some granules showing intense fluorescence were located toward the lumen. As reported for other mammals, Ki67-ip cells were localized in the crypt and did not co-localize with the lysozyme-ip cells. Moreover, fasting induced a decrease in the mRNA levels of lysozyme in the intestine of suncus. In conclusion, we firstly identified the lysozyme mRNA sequence, clarified expression profile of lysozyme transcripts in suncus and found a unique distribution of lysozyme-producing cells in the suncus intestine.
英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1007/s00441-019-02991-2DOI ID：10.1007/s00441-019-02991-2,
PubMed ID：30680460 A verification study of gastrointestinal motility-stimulating action of guinea-pig motilin using isolated gastrointestinal strips from rabbits and guinea-pigs Takio Kitazawa; Rio Harada; Ichiro Sakata; Takafumi Sakai; Hiroyuki Kaiya
General and Comparative Endocrinology,
巻：274,
開始ページ：106,
終了ページ：112, 2019年04月
© 2019 Elsevier Inc. Motilin (MLN), a 22-amino-acid peptide hormone, is generally present in the mucosa of the upper gastrointestinal (GI) tract, mainly the duodenum of mammals, and it regulates GI motility, especially that related to interdigestive migrating contraction. However, MLN and its receptor are absent in mice and rats, and MLN does not cause any mechanical responses in the rat and mouse GI tracts. The guinea-pig is also a rodent, but expression of the MLN gene in the guinea-pig has been reported. In the present study, two guinea-pig MLNs, FIPIFTYSELRRTQEREQNKGL found in the Ensemble Genome Database (gpMLN-1) and FVPIFTYSELRRTQEREQNKRL reported by Xu et al. (2001) (gpMLN-2), were synthesized, and their biological activities were evaluated in the rabbit duodenum and guinea-pig GI tract in vitro. Both gpMLNs showed contractile activity in longitudinal muscle strips of the rabbit duodenum. The EC 50 values of gpMLN-1 and gpMLN-2 were slightly higher than that of human MLN (hMLN), but the maximum contractions were as same as that of hMLN. Treatment with GM109 and hMLN-induced receptor desensitization decreased the contractile activity of both gpMLNs, indicating that the two gpMLN candidates are able to activate the MLN receptor (MLN-R) of the rabbit duodenum. In guinea-pig GI preparations, hMLN and gpMLNs did not show any mechanical responses in circular muscle strips from the gastric antrum or in longitudinal strips of the duodenum, ileum and colon although acetylcholine and 1,1-dimethyl-4-phenylpiperazinium (DMPP) caused definite mechanical responses. The DMPP-induced neural responses in the gastric circular muscle and ileal longitudinal muscles were not modified by gpMLN-1. Even in the gastric and ileal strips with intact mucosa, no mechanical responses were seen with either of the gpMLNs. Furthermore, RT-PCR using various primer sets failed to amplify the gpMLN-2 mRNA. In conclusion, gpMLNs including one that was already reported and the other that was newly found in a database were effective to the rabbit MLN-R, whereas they did not cause any contractions or modification of neural responses in the guinea-pig GI tract, indicating that the MLN system is vestigial and not functional in regulation of GI motility in the guinea-pig as well as in other rodents such as rats and mice.
研究論文（学術雑誌）
DOI：https://doi.org/10.1016/j.ygcen.2019.01.010Scopus：https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85060522122&origin=inwardScopus Citedby：https://www.scopus.com/inward/citedby.uri?partnerID=HzOxMe3b&scp=85060522122&origin=inwardDOI ID：10.1016/j.ygcen.2019.01.010,
ISSN：0016-6480,
eISSN：1095-6840,
PubMed ID：30677392,
SCOPUS ID：85060522122 β-Oxidation in ghrelin-producing cells is important for ghrelin acyl-modification. Chika Ikenoya; Shota Takemi; Arisa Kaminoda; Sayaka Aizawa; Shiomi Ojima; Zhi Gong; Rakhi Chacrabati; Daisuke Kondo; Reiko Wada; Toru Tanaka; Sachiko Tsuda; Takafumi Sakai; Ichiro Sakata
Scientific reports,
巻：8,
号：1,
開始ページ：9176,
終了ページ：9176, 2018年06月,
［国際誌］Ghrelin is a unique fatty acid-modified peptide hormone produced in the stomach and has important roles in energy homeostasis and gastrointestinal motility. However, the medium-chain fatty acid source for ghrelin acyl-modification is not known. We found that a fat-free diet and the removal of intestinal microbiota did not decrease acyl-ghrelin production in the stomach or plasma acyl-ghrelin levels in mice. RT-PCR analysis showed that genes involving fatty acid synthesis, metabolism, and transport were expressed in pancreas-derived ghrelinoma (PG-1) cells. Treatment with an irreversible inhibitor of carnitine palmitoyltransferase-1 (CPT-1) strongly decreased acylated ghrelin levels but did not affect ghrelin or ghrelin o-acyl transferase (GOAT) mRNA levels in PG-1 cells. Our results suggest that the medium-chain fatty acid used for the acyl-modification of ghrelin is produced in ghrelin-producing cells themselves by β-oxidation of long-chain fatty acids provided from the circulation.
英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1038/s41598-018-27458-2DOI ID：10.1038/s41598-018-27458-2,
PubMed ID：29907775,
PubMed Central ID：PMC6003948 Study of termination of postprandial gastric contractions in humans, dogs and Suncus murinus: role of motilin- and ghrelin-induced strong contraction T. Mikami; K. Ito; H. O. Diaz-Tartera; P. M. Hellström; E. Mochiki; S. Takemi; T. Tanaka; S. Tsuda; T. Jogahara; I. Sakata; T. Sakai
Acta Physiologica,
巻：222,
号：2, 2018年02月,
［査読有り］Aim: Stomach contractions show two types of specific patterns in many species, that is migrating motor contraction (MMC) and postprandial contractions (PPCs), in the fasting and fed states respectively. We found gastric PPCs terminated with migrating strong contractions in humans, dogs and suncus. In this study, we reveal the detailed characteristics and physiological implications of these strong contractions of PPC. Methods: Human, suncus and canine gastric contractions were recorded with a motility-monitoring ingestible capsule and a strain-gauge force transducer. The response of motilin and ghrelin and its receptor antagonist on the contractions were studied by using free-moving suncus. Results: Strong gastric contractions were observed at the end of a PPC in human, dog and suncus models, and we tentatively designated this contraction to be a postprandial giant contraction (PPGC). In the suncus, the PPGC showed the same property as those of a phase III contraction of MMC (PIII-MMC) in the duration, motility index and response to motilin or ghrelin antagonist administration. Ghrelin antagonist administration in the latter half of the PPC (LH-PPC) attenuated gastric contraction prolonged the duration of occurrence of PPGC, as found in PII-MMC. Conclusion: It is thought that the first half of the PPC changed to PII-MMC and then terminated with PIII-MMC, suggesting that PPC consists of a digestive phase (the first half of the PPC) and a discharge phase (LH-PPC) and that LH-PPC is coincident with MMC. In this study, we propose a new approach for the understanding of postprandial contractions.
Blackwell Publishing Ltd, 英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1111/apha.12933DOI ID：10.1111/apha.12933,
ISSN：1748-1716,
PubMed ID：28786555,
SCOPUS ID：85029411372 GABAergic and glutamatergic neurons in the brain regulate phase II of migrating motor contractions in the Suncus murinus. Taichi Horita; Kouhei Koyama; Shota Takemi; Toru Tanaka; Takafumi Sakai; Ichiro Sakata
Journal of smooth muscle research = Nihon Heikatsukin Gakkai kikanshi,
巻：54,
号：0,
開始ページ：91,
終了ページ：99, 2018年,
［査読有り］,
［責任著者］,
［国内誌］Gastric contractions exhibit characteristic motor patterns in the fasted state, known as migrating motor contractions (MMC). MMC consist of three periodically repeated phases (phase I, II and III) and are known to be regulated by hormones and the autonomic and enteric nervous systems. However, the central regulation of gastric contractions in the fasted state is not completely understood. Here, we have examined the central effects of motilin, ghrelin, γ-aminobutyric acid (GABA) and L-glutamate signaling on gastric MMC by using suncus (Suncus murinus) as an animal model, because of their similar gastric motor patterns to those observed in humans and dogs. Intracerebroventricular (i.c.v.) administration of motilin and ghrelin had no effect on phase I and II contractions, respectively. Conversely, i.c.v. administration of GABAA receptor antagonist, during phase I of the MMC, evoked phase II-like contractions and significantly increased the motility index (MI). This was compared with the i.c.v. administration of GABA which inhibited spontaneous phase II contractions with a significantly decreased MI. In addition, i.c.v. administration of L-glutamate during phase I also induced phase II-like irregular contractions with a significant increase in the MI. Taken together with previous findings, these results suggest that central GABAergic and glutamatergic signaling, with the coordination of both peripheral motilin and ghrelin, regulate phase II contractions of MMC in the fasted state.
英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1540/jsmr.54.91DOI ID：10.1540/jsmr.54.91,
PubMed ID：30787212,
PubMed Central ID：PMC6380905 Milk basic protein increases ghrelin secretion and bone mineral density in rodents Yuko Ishida; Rakhi Chacrabati; Aiko Ono-Ohmachi; Zhi Gong; Chika Ikenoya; Sayaka Aizawa; Takayuki Y. Nara; Yoshikazu Morita; Ken Kato; Takafumi Sakai; Ichiro Sakata
Nutrition,
巻：39-40,
開始ページ：15,
終了ページ：19, 2017年07月
Elsevier BV, 研究論文（学術雑誌）
DOI：https://doi.org/10.1016/j.nut.2017.02.003DOI ID：10.1016/j.nut.2017.02.003,
ISSN：0899-9007 Underlying mechanism of the cyclic migrating motor complex in Suncus murinus: a change in gastrointestinal pH is the key regulator. Anupom Mondal; Kouhei Koyama; Takashi Mikami; Taichi Horita; Shota Takemi; Sachiko Tsuda; Ichiro Sakata; Takafumi Sakai
Physiological reports,
巻：5,
号：1, 2017年01月,
［国際誌］In the fasted gastrointestinal (GI) tract, a characteristic cyclical rhythmic migrating motor complex (MMC) occurs in an ultradian rhythm, at 90-120 min time intervals, in many species. However, the underlying mechanism directing this ultradian rhythmic MMC pattern is yet to be completely elucidated. Therefore, this study aimed to identify the possible causes or factors that involve in the occurrence of the fasting gastric contractions by using Suncus murinus a small model animal featuring almost the same rhythmic MMC as that found in humans and dogs. We observed that either intraduodenal infusion of saline at pH 8 evoked the strong gastric contraction or continuously lowering duodenal pH to 3-evoked gastric phase II-like and phase III-like contractions, and both strong contractions were essentially abolished by the intravenous administration of MA 2029 (motilin receptor antagonist) and D-Lys3-GHRP6 (ghrelin receptor antagonist) in a vagus-independent manner. Moreover, we observed that the prostaglandin E2-alpha (PGE2-α) and serotonin type 4 (5HT4) receptors play important roles as intermediate molecules in changes in GI pH and motilin release. These results suggest a clear insight mechanism that change in the duodenal pH to alkaline condition is an essential factor for stimulating the endogenous release of motilin and governs the fasting MMC in a vagus-independent manner. Finally, we believe that the changes in duodenal pH triggered by flowing gastric acid and the release of duodenal bicarbonate through the involvement of PGE2-α and 5HT4 receptor are the key events in the occurrence of the MMC.
英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.14814/phy2.13105DOI ID：10.14814/phy2.13105,
PubMed ID：28082431,
PubMed Central ID：PMC5256163 The study of ghrelin secretion and acyl-modification using mice and ghrelinoma cell lines
Ichiro Sakata; Zhi Gong; Chika Ikenoya; Shota Takemi; Takafumi Sakai
ENDOCRINE JOURNAL, 巻：64, 開始ページ：S27, 終了ページ：S29, 2017年, ［査読有り］
Ghrelin is a peptide hormone with a unique structure comprising a medium chain fatty acid modification. Ghrelin cells are known to be abundantly localized in the gastric mucosa and are released into the blood stream to exert their multifunctional physiological effects. To elucidate the regulatory mechanisms of ghrelin secretion and acyl-modification, we developed novel ghrelin-producing cell lines. Using ghrelinoma cell lines, we focused on the mechanisms of ghrelin secretion and found that several GPCRs were highly expressed in ghrelin cells. Then, we showed that noradrenaline treatment stimulated ghrelin secretion via beta 1-adrenergic receptor, and fasting-induced ghrelin elevation was completely inhibited by the beta 1-adrenergic receptor antagonist in mice. In addition, we demonstrated that long chain fatty acids, glucose, and L-glutamate significantly inhibited ghrelin secretion. Furthermore, we recently revealed that the genes involved in fatty acid synthesis and long chain fatty acid metabolism were expressed in ghrelin cells, and that CPT-1 inhibitor treatment dramatically decreased the levels of acyl-modified ghrelin. Here, we introduce the current knowledge of the mechanisms involving ghrelin secretion and its acyl-modification.
JAPAN ENDOCRINE SOC, 英語, 研究論文（学術雑誌）
ISSN：0918-8959, eISSN：1348-4540, Web of Science ID：WOS:000414045000007
The important role of ghrelin on gastric contraction in Suncus murinus
Shota Takemi; Ichiro Sakata; Kayuri Kuroda; Yuki Miyano; Anupon Mondal; Takafumi Sakai
ENDOCRINE JOURNAL, 巻：64, 開始ページ：S11, 終了ページ：S14, 2017年, ［査読有り］
Ghrelin, a peptide hormone produced in the stomach, has been known to be involved in the regulation of gastric contraction in humans and rodents. To elucidate the detailed mechanisms of ghrelin on gastric contractions, we used Suncus murinus, a recently established small animal model for gastrointestinal motility. S. murinus produces motilin, a family peptide of ghrelin, and its stomach anatomy and physiological patterns of gastric contractions, in fed and fasted states, are closely similar to humans. Ghrelin administration in phase II, and latter half of phase I, of the migrating motor contractions (MMC) enhanced gastric motility in S. murinus. In addition, we showed that ghrelin and motilin coordinately stimulated strong gastric contractions in vitro and in vivo. We also demonstrated that a pretreatment with a ghrelin antagonist, D-Lys3-GHRP6, inhibited the effects of motilin-induced gastric contractions, and a gamma-aminobutyric acid (GABA) antagonist reversed this inhibition. Our results suggest that ghrelin is essential for motilin-induced gastric contractions and that ghrelin-mediated GABAergic neurons are involved in this neural pathway.
JAPAN ENDOCRINE SOC, 英語, 研究論文（学術雑誌）
ISSN：0918-8959, eISSN：1348-4540, Web of Science ID：WOS:000414045000003
Identification of marker genes for pars tuberalis morphogenesis in chick embryo: expression of Cytokine-like 1 and Gap junction protein alpha 5 in pars tuberalis.
Sayaka Aizawa; Yuriko Higaki; Amrita Dudaui; Mai Nagasaka; Sumio Takahashi; Ichiro Sakata; Takafumi Sakai
Cell and tissue research, 巻：366, 号：3, 開始ページ：721, 終了ページ：731, 2016年12月, ［国際誌］
The adenohypophysis is formed from the oral ectoderm and consists of the pars distalis (PD), pars intermedia, and pars tuberalis (PT). The mechanisms of PD development have been extensively studied, and the cellular differentiation of the PD is well understood. However, the morphogenesis and differentiation of the PT are still unclear, and the genes expressed during PT development remain largely unknown. We have explored genes specifically expressed in the PT during embryonic development and analyzed their spatiotemporal expression patterns. Microarray analysis of laser-captured PT and PD tissues obtained from chick embryos on embryonic day 10 (E10.0) has shown high expression of Cytokine-like 1 (CYTL1) and Gap junction protein alpha 5 (GJA5) genes in the PT. Detailed analysis of these spatiotemporal expression patterns during chick embryo development by in situ hybridization has revealed that CYTL1 mRNA first appears in the lateral head ectoderm and ventral head ectoderm at E1.5. The expression of CYTL1 moves into Rathke's pouch at E2.5 and is then localized in the PT primordium where it is continuously expressed until E12.0. GJA5 mRNA is transiently detected in the PT primordium from E6.0 to E12.0, whereas its expression is not detected in the PD during development. Thus, these genes might be involved in the regulation mechanisms of PT development and could be useful markers for PT development.
英語, 研究論文（学術雑誌）
eISSN：1432-0878, PubMed ID：27590887
A Sexually Dimorphic Area of the Dorsal Hypothalamus in Mice and Common Marmosets Yadanar Moe; Chaw Kyi-Tha-Thu; Tomoko Tanaka; Hiroto Ito; Satowa Yahashi; Ken-Ichi Matsuda; Mitsuhiro Kawata; Goro Katsuura; Fumihiro Iwashige; Ichiro Sakata; Atsushi Akune; Akio Inui; Takafumi Sakai; Sonoko Ogawa; Shinji Tsukahara
ENDOCRINOLOGY,
巻：157,
号：12,
開始ページ：4817,
終了ページ：4828, 2016年12月,
［査読有り］We found a novel sexually dimorphic area (SDA) in the dorsal hypothalamus (DH) of mice. The SDA-DH was sandwiched between 2 known male-biased sexually dimorphic nuclei, the principal nucleus of the bed nucleus of the stria terminalis and the calbindin-sexually dimorphic nucleus, and exhibited a female-biased sex difference in neuronal cell density. The density of neurons in the SDA-DH was increased in male mice by orchidectomy on the day of birth and decreased in female mice by treatment with testosterone, dihydrotestosterone, or estradiol within 5 days after birth. These findings indicate that the SDA-DH is defeminized under the influence of testicular testosterone, which acts via both directly by binding to the androgen receptor, and indirectly by binding to the estrogen receptor after aromatization. We measured the activity of SDA-DH neurons with c-Fos, a neuronal activity marker, in female mice during maternal and sexual behaviors. The number of c-Fos-expressing neurons in the SDA-DH of female mice was negatively correlated with maternal behavior performance. However, the number of c-Fos-expressing neurons did not change during female sexual behavior. These findings suggest that the SDA-DH contains a neuronal cell population, the activity of which decreases in females exhibiting higher performance of maternal behavior, but it may contribute less to female sexual behavior. Additionally, we examined the brain of common marmosets and found an area that appears to be homologous with the mouse SDA-DH. The sexually dimorphic structure identified in this study is not specific to mice and may be found in other species.
ENDOCRINE SOC, 英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1210/en.2016-1428DOI ID：10.1210/en.2016-1428,
ISSN：0013-7227,
eISSN：1945-7170,
Web of Science ID：WOS:000392840400032 Molecular Cloning of Ghrelin and Characteristics of Ghrelin-Producing Cells in the Gastrointestinal Tract of the Common Marmoset (Callithrix jacchus) Shota Takemi; Ichiro Sakata; Auvijit Saha Apu; Shinji Tsukahara; Satowa Yahashi; Goro Katsuura; Fumihiro Iwashige; Atsushi Akune; Akio Inui; Takafumi Sakai
ZOOLOGICAL SCIENCE,
巻：33,
号：5,
開始ページ：497,
終了ページ：504, 2016年10月,
［査読有り］Ghrelin was first isolated from human and rat as an endogenous ligand for the growth hormone secretagogue receptor (GHS-R). In the present study, we determined the ghrelin cDNA sequence of the common marmoset (Callithrix jacchus), a small-bodied New World monkey, and investigated the distribution of ghrelin-producing cells in the gastrointestinal tract and localization profiles with somatostatin-producing cells. The marmoset ghrelin cDNA coding region was 354 base pairs, and showed high homology to that in human, rhesus monkey, and mouse. Marmoset ghrelin consists of 28 amino acids, and the N-terminal region is highly conserved as found in other mammalian species. Marmoset preproghrelin and mature ghrelin have 86.3% and 92.9% homology, respectively, to their human counterparts. Quantitative RT-PCR analysis showed that marmoset ghrelin mRNA is highly expressed in the stomach, but it is not detected in other tissues of the gastrointestinal tract. In addition, a large number of ghrelin mRNA-expressing cells and ghrelin-immunopositive cells were detected in the mucosal layer of the stomach, but not in the myenteric plexus. Moreover, all the ghrelin cells examined in the stomach were observed to be closed-type. Double staining showed that somatostatin-immunopositive cells were not co-localized with ghrelin-producing cells; however, a subset of somatostatin-immunopositive cells is directly adjacent to ghrelin-immunopositive cells. These findings suggest that the distribution of ghrelin cells in marmoset differs from that in rodents, and thus the marmoset may be a more useful model for the translational study of ghrelin in primates. In conclusion, we have clarified the expression and cell distribution of ghrelin in marmoset, which may represent a useful model in translational study.
ZOOLOGICAL SOC JAPAN, 英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.2108/zs160020DOI ID：10.2108/zs160020,
ISSN：0289-0003,
Web of Science ID：WOS:000385345800007 A comparative study of sex difference in calbindin neurons among mice, musk shrews, and Japanese quails Yadanar Moe; Tomoko Tanaka; Masahiro Morishita; Ryoko Ohata; Chihiro Nakahara; Takaharu Kawashima; Fumihiko Maekawa; Ichiro Sakata; Takafumi Sakai; Shinji Tsukahara
NEUROSCIENCE LETTERS,
巻：631,
開始ページ：63,
終了ページ：69, 2016年09月,
［査読有り］The medial preoptic nucleus (MPN) and the bed nucleus of the stria terminalis (BNST) of mice contain sexually dimorphic nuclei (SDNs) that are larger and have more neurons expressing calbindin D-28K (CB), a calcium-binding protein, in males than females. However, it is largely unknown whether such SDNs exist in species other than rodents. In this study, we performed an immunohistochemical study of CB in the MPN and BNST of musk shrews and Japanese quails to examine the existence of homologs of SDNs in mice. Like mice, musk shrews had a SDN exhibiting male-biased sex differences in volume and CB-immunoreactive (ir) cell number in the MPN. The BNST of musk shrews also contained a male-biased SDN, but consisted of non-CB neurons. The paratenial thalamic nucleus of musk shrews, but not mice, had more CB-ir cells in males than females. In Japanese quails of both sexes, CB-ir cells in the MPN and BNST were extremely small in number and did not cluster. These results suggest that the distribution of CB neurons differs among these species. Musk shrews may have a homolog of the SDN composed of CB neurons in the MPN of mice. (C) 2016 Elsevier Ireland Ltd. All rights reserved.
ELSEVIER IRELAND LTD, 英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1016/j.neulet.2016.08.018DOI ID：10.1016/j.neulet.2016.08.018,
ISSN：0304-3940,
eISSN：1872-7972,
Web of Science ID：WOS:000384786600011 Molecular cloning of motilin and mechanism of motilin-induced gastrointestinal motility in Japanese quail. Auvijit Saha Apu; Anupom Mondal; Takio Kitazawa; Shota Takemi; Takafumi Sakai; Ichiro Sakata
General and comparative endocrinology,
巻：233,
開始ページ：53,
終了ページ：62, 2016年07月,
［国際誌］Motilin, a peptide hormone produced in the upper intestinal mucosa, plays an important role in the regulation of gastrointestinal (GI) motility. In the present study, we first determined the cDNA and amino acid sequences of motilin in the Japanese quail and studied the distribution of motilin-producing cells in the gastrointestinal tract. We also examined the motilin-induced contractile properties of quail GI tracts using an in vitro organ bath, and then elucidated the mechanisms of motilin-induced contraction in the proventriculus and duodenum of the quail. Mature quail motilin was composed of 22 amino acid residues, which showed high homology with chicken (95.4%), human (72.7%), and dog (72.7%) motilin. Immunohistochemical analysis showed that motilin-immunopositive cells were present in the mucosal layer of the duodenum (23.4±4.6cells/mm(2)), jejunum (15.2±0.8cells/mm(2)), and ileum (2.5±0.7cells/mm(2)), but were not observed in the crop, proventriculus, and colon. In the organ bath study, chicken motilin induced dose-dependent contraction in the proventriculus and small intestine. On the other hand, chicken ghrelin had no effect on contraction in the GI tract. Motilin-induced contraction in the duodenum was not inhibited by atropine, hexamethonium, ritanserin, ondansetron, or tetrodotoxin. However, motilin-induced contractions in the proventriculus were significantly inhibited by atropine and tetrodotoxin. These results suggest that motilin is the major stimulant of GI contraction in quail, as it is in mammals and the site of action of motilin is different between small intestine and proventriculus.
英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1016/j.ygcen.2016.05.017DOI ID：10.1016/j.ygcen.2016.05.017,
PubMed ID：27179882 The proximal gastric corpus is the most responsive site of motilin-induced contractions in the stomach of the Asian house shrew. Amrita Dudani; Sayaka Aizawa; Gong Zhi; Toru Tanaka; Takamichi Jogahara; Ichiro Sakata; Takafumi Sakai
Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology,
巻：186,
号：5,
開始ページ：665,
終了ページ：75, 2016年07月,
［国際誌］The migrating motor complex (MMC) is responsible for emptying the stomach during the interdigestive period, in preparation for the next meal. It is known that gastric phase III of MMC starts from the proximal stomach and propagates the contraction downwards. We hypothesized that a certain region of the stomach must be more responsive to motilin than others, and that motilin-induced strong gastric contractions propagate from that site. Stomachs of the Suncus or Asian house shrew, a small insectivorous mammal, were dissected and the fundus, proximal corpus, distal corpus, and antrum were examined to study the effect of motilin using an organ bath experiment. Motilin-induced contractions differed in different parts of the stomach. Only the proximal corpus induced gastric contraction even at motilin 10(-10) M, and strong contraction was induced by motilin 10(-9) M in all parts of the stomach. The GPR38 mRNA expression was also higher in the proximal corpus than in the other sections, and the lowest expression was observed in the antrum. GPR38 mRNA expression varied with low expression in the mucosal layer and high expression in the muscle layer. Additionally, motilin-induced contractions in each dissected part of the stomach were inhibited by tetrodotoxin and atropine pretreatment. These results suggest that motilin reactivity is not consistent throughout the stomach, and an area of the proximal corpus including the cardia is the most sensitive to motilin.
英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1007/s00360-016-0985-1DOI ID：10.1007/s00360-016-0985-1,
PubMed ID：27062028 Involvement of Transient Receptor Potential Vanilloid Receptor 1, (TRPV1)-Expressing Vagal Nerve in the Inhibitory Effect of Gastric Acidification on Exogenous Motilin-Induced Gastric Phase III Contractions in Suncus murinus Makoto Yoshimura; Takashi Mikami; Kayuri Kuroda; Maki Nishida; Kazuma Ito; Anupom Mondal; Kouhei Koyama; Takamichi Jogahara; Ichiro Sakata; Takafumi Sakai
DIGESTIVE DISEASES AND SCIENCES,
巻：61,
号：6,
開始ページ：1501,
終了ページ：1511, 2016年06月,
［査読有り］Gastric acidification inhibits motilin-induced gastric phase III contractions. However, the underlying mechanism has not been thoroughly investigated. Here, we studied the inhibitory mechanism by gastric acidification on motilin-induced contraction in Suncus murinus (S. murinus).
We measured interdigestive gastric phase III contractions in conscious, freely moving S. murinus, and examined the inhibitory effect of gastric acidification on motilin action and the involvement of the vagus nerve and transient receptor potential vanilloid receptor 1 (TRPV1) in the inhibitory mechanism.
A bolus injection of motilin evoked phase III-like contractions during intravenous infusion of saline. Intragastric acidification (pH 1.5-2.5) inhibited motilin-induced phase III contractions in a pH-dependent manner and significantly decreased the motility index at a pH below 2.0. In contrast, intraduodenal acidification (pH 2.0) failed to inhibit motilin-induced contractions. Vagotomy significantly alleviated the suppression of motilin-induced gastric contractions under acidic conditions (pH 2.0), suggesting vagus nerve involvement. Moreover, intragastric acidification (pH 2.0) significantly increased the number of c-Fos-positive cells in the nucleus tractus solitarii. In vagotomized S. murinus, the number of c-Fos-positive cells did not change, even under gastric acidification conditions. TRPV1 mRNA was highly expressed in the muscle and mucosal regions of the antrum and the nodose ganglion, whereas was not detected in the upper small intestine. Capsazepin, a TRPV1 antagonist, completely rescued the inhibitory effect of gastric acidification.
Gastric acidification in S. murinus inhibits motilin-induced contractions, a finding similar to results observed in humans, while TRPV1-expressing vagus nerves play a role in the inhibitory mechanism.
SPRINGER, 英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1007/s10620-015-4023-zDOI ID：10.1007/s10620-015-4023-z,
ISSN：0163-2116,
eISSN：1573-2568,
PubMed ID：26860510,
Web of Science ID：WOS:000376587600016 Ghrelin Is an Essential Factor for Motilin-Induced Gastric Contraction in Suncus murinus. Kayuri Kuroda; Huang Hequing; Anupom Mondal; Makoto Yoshimura; Kazuma Ito; Takashi Mikami; Shota Takemi; Takamichi Jogahara; Ichiro Sakata; Takafumi Sakai
Endocrinology,
巻：156,
号：12,
開始ページ：4437,
終了ページ：47, 2015年12月,
［国際誌］Motilin was discovered in the 1970s as the most important hormone for stimulating strong gastric contractions; however, the mechanisms by which motilin causes gastric contraction are not clearly understood. Here, we determined the coordinated action of motilin and ghrelin on gastric motility during fasted and postprandial contractions by using house musk shrew (Suncus murinus; order: Insectivora, suncus named as the laboratory strain). Motilin-induced gastric contractions at phases I and II of the migrating motor complex were inhibited by pretreatment with (D-Lys(3))-GHRP-6 (6 mg/kg/h), a ghrelin receptor antagonist. Administration of the motilin receptor antagonist MA-2029 (0.1 mg/kg) and/or (D-Lys(3))-GHRP-6 (0.6 mg/kg) at the peak of phase III abolished the spontaneous gastric phase III contractions in vivo. Motilin did not stimulate gastric contractions in the postprandial state. However, in the presence of a low dose of ghrelin, motilin evoked phase III-like gastric contractions even in the postprandial state, and postprandial gastric emptying was accelerated. In addition, pretreatment with (D-Lys(3))-GHRP-6 blocked the motilin-induced gastric contraction in vitro and in vivo, and a γ-aminobutyric acid (GABA) antagonist reversed this block in gastric contraction. These results indicate that blockade of the GABAergic pathway by ghrelin is essential for motilin-induced gastric contraction.
英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1210/en.2015-1561DOI ID：10.1210/en.2015-1561,
PubMed ID：26441238 Motilin stimulates pepsinogen secretion in Suncus murinus Chayon Goswami; Toru Tanaka; Takamichi Jogahara; Takafumi Sakai; Ichiro Sakata
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS,
巻：462,
号：3,
開始ページ：263,
終了ページ：268, 2015年07月,
［査読有り］Motilin and ghrelin are gastrointestinal hormones that stimulate the migrating motor complex (MMC) of gastrointestinal motility during the fasting state. In this study, we examined the effect of motilin and ghrelin on pepsinogen secretion in anesthetized suncus (house musk shrew, Suncus murinus), a ghrelin-and motilin-producing mammal. By using a gastric lumen-perfusion system, we found that the intravenous administration of carbachol and motilin stimulated pepsinogen secretion, the latter in a dose-dependent manner, whereas ghrelin had no effect. We then investigated the pathways of motilin-induced pepsinogen secretion using acetylcholine receptor antagonists. Treatment with atropine, a muscarinic acetylcholine receptor antagonist, completely inhibited both carbachol and motilin-induced pepsinogen secretion. Motilin-induced pepsinogen secretion was observed in the vagotomized suncus. This is the first report demonstrating that motilin stimulates pepsinogen secretion, and suggest that this effect occurs through a cholinergic pathway in suncus. (C) 2015 Elsevier Inc. All rights reserved.
ACADEMIC PRESS INC ELSEVIER SCIENCE, 英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1016/j.bbrc.2015.04.129DOI ID：10.1016/j.bbrc.2015.04.129,
ISSN：0006-291X,
eISSN：1090-2104,
PubMed ID：25957475,
Web of Science ID：WOS:000356319900016 Rikkunshito induces gastric relaxation via the β-adrenergic pathway in Suncus murinus A. Mondal; A. Takehara; S. Aizawa; T. Tanaka; N. Fujitsuka; T. Hattori; Takafumi Sakai; Ichiro Sakata
Neurogastroenterology and Motility,
巻：27,
号：6,
開始ページ：875,
終了ページ：884, 2015年06月
Background: Rikkunshito (RKT) is a gastroprotective herbal medicine. In this study, we investigated the role of RKT in the relaxation of the gastric body (fundus and corpus) and antrum. Methods: We used Suncus murinus, a unique small model animal with similar gastrointestinal motility to humans and dogs. RKT was added at 0.1, 1.0, and 5.0 mg/mL to induce relaxation in vitro
the outcome measure was the intensity of relaxation. The number of spontaneous antral contractions in the absence or the presence of RKT was also counted. Key Results: Rikkunshito induced the relaxation of the gastric body and antrum and decreased the number of spontaneous antral contractions in a dose-dependent manner. The responses to RKT (1.0 mg/mL) were not affected by pretreatment with atropine, N-nitro-l-arginine methyl ester, ritanserin, or ondansetron. On the other hand, timolol almost completely reversed the relaxation induced by RKT (1.0 mg/mL) on the gastric body and antrum and the occurrence of the spontaneous antral contractions. Both butoxamine, a β2-adrenoreceptor antagonist, and L 748337, a β3-adrenoreceptor antagonist, but not CGP 20712, a β1-adrenoreceptor antagonist, significantly reversed the RKT-induced (1.0 mg/mL) gastric relaxation. Conclusions &
Inferences: These results indicate that RKT stimulates and modulates gastric relaxation through β2- and β3-adrenergic, but not β1-adrenergic, pathways in S. murinus.
英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1111/nmo.12564DOI ID：10.1111/nmo.12564,
ISSN：1365-2982,
PubMed ID：25846270,
SCOPUS ID：84929656075 Regulation of LH/FSH expression by secretoglobin 3A2 in the mouse pituitary gland Yuki Miyano; Shigeyuki Tahara; Ichiro Sakata; Takafumi Sakai; Hiroyuki Abe; Shioko Kimura; Reiko Kurotani
CELL AND TISSUE RESEARCH,
巻：356,
号：1,
開始ページ：253,
終了ページ：260, 2014年04月,
［査読有り］Secretoglobin (SCGB) 3A2 was originally identified as a downstream target for the homeodomain transcription factor NKX2-1 in the lung. NKX2-1 plays a role in the genesis and expression of genes in the thyroid, lung and ventral forebrain; Nkx2-1-null mice have no thyroid and pituitary and severely hypoplastic lungs and hypothalamus. To demonstrate whether SCGB3A2 plays any role in pituitary hormone production, NKX2-1 and SCGB3A2 expression in the mouse pituitary gland was examined by immunohistochemical analysis and RT-PCR. NKX2-1 was localized in the posterior pituitary lobe, whereas SCGB3A2 was observed in both anterior and posterior lobes as shown by immunohistochemistry and RT-PCR. Expression of CCAAT-enhancer binding proteins (C/EBPs), which regulate mouse Scgb3a2 transcription, was also examined by RT-PCR. C/EBP beta, gamma, delta and zeta were expressed in the adult mouse pituitary gland. SCGB3A2 was expressed in the anterior and posterior lobes from postnatal days 1 and 5, respectively and the areas where SCGB3A2 expression was found coincided with the area where FSH-secreting cells were found. Double-staining for SCGB3A2 and pituitary hormones revealed that SCGB3A2 was mainly localized in gonadotrophs in 49 % of FSH-secreting cells and 47 % of LH-secreting cells. In addition, SCGB3A2 dramatically inhibited LH and FSH mRNA expression in rat pituitary primary cell cultures. These results suggest that SCGB3A2 regulates FSH/LH production in the anterior pituitary lobe and that transcription factors other than NKX2-1 may regulate SCGB3A2 expression.
SPRINGER, 英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1007/s00441-014-1794-zDOI ID：10.1007/s00441-014-1794-z,
ISSN：0302-766X,
eISSN：1432-0878,
PubMed ID：24514953,
Web of Science ID：WOS:000334175100023 G protein-coupled receptor 120 signaling regulates ghrelin secretion in vivo and in vitro. Zhi Gong; Makoto Yoshimura; Sayaka Aizawa; Reiko Kurotani; Jeffrey M Zigman; Takafumi Sakai; Ichiro Sakata
American journal of physiology. Endocrinology and metabolism,
巻：306,
号：1,
開始ページ：E28-35, 2014年01月,
［国際誌］Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor, is produced predominantly in the stomach. It has been reported that endogenous ghrelin levels are increased by fasting and decreased immediately after feeding and that fasting-induced ghrelin release is controlled by the sympathetic nervous system. However, the mechanisms of plasma ghrelin decrement after feeding are poorly understood. Here, we studied the control of ghrelin secretion using ghrelin-producing cell lines and found that these cells express high levels of mRNA encoding G-protein coupled receptor 120 (GPR120). Addition of GW-9508 (a GPR120 chemical agonist) and α-linolenic acid (a natural ligand for GPR120) inhibited the secretion of ghrelin by ∼50 and 70%, respectively. However, the expression levels of preproghrelin and ghrelin O-acyltransferase (GOAT) mRNAs were not influenced by GW-9508. In contrast, the expression levels of prohormone convertase 1 were decreased significantly by GW-9508 incubation. Moreover, we observed that the inhibitory effect of GW-9508 on ghrelin secretion was blocked by a small interfering RNA (siRNA) targeting the sequence of GPR120. Furthermore, pretreatment with GW-9508 blocked the effect of the norepinephrine (NE)-induced ghrelin elevation in ghrelin cell lines. In addition, we showed that GW-9508 inhibited ghrelin secretion via extracellular signal-regulated kinase activity in ghrelin cell lines. Finally, we found that GW-9508 decreased plasma ghrelin levels in mice. These results suggest that the decrease of ghrelin secretion after feeding is induced partially by long-chain fatty acids that act directly on gastric GPR120-expressing ghrelin cells.
英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1152/ajpendo.00306.2013DOI ID：10.1152/ajpendo.00306.2013,
PubMed ID：24222669 Detailed morphogenetic analysis of the embryonic chicken pars tuberalis as glycoprotein alpha subunit positive region. Makiko Inoue; Sayaka Aizawa; Yuriko Higaki; Akira Kawashima; Kanako Koike; Hiroyasu Takagi; Takafumi Sakai; Ichiro Sakata
Journal of molecular histology,
巻：44,
号：4,
開始ページ：401,
終了ページ：9, 2013年08月,
［国際誌］The pars tuberalis (PT) is a part of the anterior pituitary gland that is located as a thin cell layer surrounding the median eminence. The characteristics of PT, including cell shape and cell composition, differ from those of the pars distalis (PD), suggesting that PT has unique physiological functions and different morphogenesis compared to PD. In this study, we used chicken embryos and showed for the first time that most hormone-producing cells in PT at embryonic day (E) 20.0 were only glycoprotein α subunit (αGSU)-positive staining cells. Then, using serial frontal and sagittal sections, we examined the detailed distribution of the αGSU mRNA-expressing region, as a marker of PT in the chicken embryonic pituitary gland during the E3.0-20.0 period. This three-dimensional expression pattern analysis clarified that αGSU mRNA expression initially appeared only in the bilateral regions of the Rathke's recess (RR) at E3.5, and this region expanded and showed a ring-like structure on RR. Subsequently, this αGSU mRNA-expressing region gradually expanded upward and reached the diencephalon at E8.0. This region became thinner as it surrounded the base of the diencephalon from E12.0 to E20.0. In this study, we demonstrated the detailed morphological changes of the chicken PT primordium by detecting αGSU mRNA, and we also showed that PT is a unique region in the early developmental stage.
英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1007/s10735-012-9479-yDOI ID：10.1007/s10735-012-9479-y,
PubMed ID：23269505 Mechanism of ghrelin-induced gastric contractions in Suncus murinus (house musk shrew): involvement of intrinsic primary afferent neurons. Anupom Mondal; Sayaka Aizawa; Ichiro Sakata; Chayon Goswami; Sen-ichi Oda; Takafumi Sakai
PloS one,
巻：8,
号：4,
開始ページ：e60365, 2013年,
［国際誌］Here, we have reported that motilin can induce contractions in a dose-dependent manner in isolated Suncus murinus (house musk shrew) stomach. We have also shown that after pretreatment with a low dose of motilin (10(-10) M), ghrelin also induces gastric contractions at levels of 10(-10) M to 10(-7) M. However, the neural mechanism of ghrelin action in the stomach has not been fully revealed. In the present study, we studied the mechanism of ghrelin-induced contraction in vitro using a pharmacological method. The responses to ghrelin in the stomach were almost completely abolished by hexamethonium and were significantly suppressed by the administration of phentolamine, prazosin, ondansetron, and naloxone. Additionally, N-nitro-l-arginine methylester significantly potentiated the contractions. Importantly, the mucosa is essential for ghrelin-induced, but not motilin-induced, gastric contractions. To evaluate the involvement of intrinsic primary afferent neurons (IPANs), which are multiaxonal neurons that pass signals from the mucosa to the myenteric plexus, we examined the effect of the IPAN-related pathway on ghrelin-induced contractions and found that pretreatment with adenosine and tachykinergic receptor 3 antagonists (SR142801) significantly eliminated the contractions and GR113808 (5-hydroxytryptamine receptor 4 antagonist) almost completely eliminated it. The results indicate that ghrelin stimulates and modulates suncus gastric contractions through cholinergic, adrenergic, serotonergic, opioidergic neurons and nitric oxide synthases in the myenteric plexus. The mucosa is also important for ghrelin-induced gastric contractions, and IPANs may be the important interneurons that pass the signal from the mucosa to the myenteric plexus.
英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1371/journal.pone.0060365DOI ID：10.1371/journal.pone.0060365,
PubMed ID：23565235,
PubMed Central ID：PMC3614873 The role of the vagus nerve in the migrating motor complex and ghrelin- and motilin-induced gastric contraction in suncus. Yuki Miyano; Ichiro Sakata; Kayuri Kuroda; Sayaka Aizawa; Toru Tanaka; Takamichi Jogahara; Reiko Kurotani; Takafumi Sakai
PloS one,
巻：8,
号：5,
開始ページ：e64777, 2013年,
［国際誌］The upper gastrointestinal (GI) tract undergoes a temporally coordinated cyclic motor pattern known as the migrating motor complex (MMC) in both dogs and humans during the fasted state. Feeding results in replacement of the MMC by a pattern of noncyclic, intermittent contractile activity termed as postprandial contractions. Although the MMC is known to be stimulated by motilin, recent studies have shown that ghrelin, which is from the same peptide family as motilin, is also involved in the regulation of the MMC. In the present study, we investigated the role of the vagus nerve on gastric motility using conscious suncus-a motilin- and ghrelin-producing small animal. During the fasted state, cyclic MMC comprising phases I, II, and III was observed in both sham-operated and vagotomized suncus; however, the duration and motility index (MI) of phase II was significantly decreased in vagotomized animals. Motilin infusion (50 ng·kg(-1)·min(-1) for 10 min) during phase I had induced phase III-like contractions in both sham-operated and vagotomized animals. Ghrelin infusion (0.1, 0.3, 1, 3, or 10 µg·kg(-1)·min(-1) for 10 min) enhanced the amplitude of phase II MMC in sham-operated animals, but not in vagotomized animals. After feeding, phase I was replaced by postprandial contractions, and motilin infusion (50 ng·kg(-1)·min(-1) for 10 min) did not induce phase III-like contractions in sham-operated suncus. However, in vagotomized suncus, feeding did not evoke postprandial contractions, but exogenous motilin injection strongly induced phase III-like contractions, as noted during the phase I period. Thus, the results indicate that ghrelin stimulates phase II of the MMC via the vagus nerve in suncus. Furthermore, the vagus nerve is essential for initiating postprandial contractions, and inhibition of the phase III-like contractions induced by motilin is highly dependent on the vagus nerve.
英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1371/journal.pone.0064777DOI ID：10.1371/journal.pone.0064777,
PubMed ID：23724093,
PubMed Central ID：PMC3665597 Negative regulation of neuromedin U mRNA expression in the rat pars tuberalis by melatonin. Sayaka Aizawa; Ichiro Sakata; Mai Nagasaka; Yuriko Higaki; Takafumi Sakai
PloS one,
巻：8,
号：7,
開始ページ：e67118, 2013年,
［国際誌］The pars tuberalis (PT) is part of the anterior pituitary gland surrounding the median eminence as a thin cell layer. The characteristics of PT differ from those of the pars distalis (PD), such as cell composition and gene expression, suggesting that the PT has a unique physiological function compared to the PD. Because the PT highly expresses melatonin receptor type 1, it is considered a mediator of seasonal and/or circadian signals of melatonin. Expression of neuromedin U (NMU) that is known to regulate energy balance has been previously reported in the rat PT; however, the regulatory mechanism of NMU mRNA expression and secretion in the PT are still obscure. In this study, we examined both the diurnal change of NMU mRNA expression in the rat PT and the effects of melatonin on NMU in vivo. In situ hybridization and quantitative PCR analysis of laser microdissected PT samples revealed that NMU mRNA expression in the PT has diurnal variation that is high during the light phase and low during the dark phase. Furthermore, melatonin administration significantly suppressed NMU mRNA expression in the PT in vivo. On the other hand, 48 h fasting did not have an effect on PT-NMU mRNA expression, and the diurnal change of NMU mRNA expression was maintained. We also found the highest expression of neuromedin U receptor type 2 (NMUR2) mRNA in the third ventricle ependymal cell layer, followed by the arcuate nucleus and the spinal cord. These results suggest that NMU mRNA expression in the PT is downregulated by melatonin during the dark phase and shows diurnal change. Considering that NMU mRNA in the PT showed the highest expression level in the brain, PT-NMU may act on NMUR2 in the brain, especially in the third ventricle ependymal cell layer, with a circadian rhythm.
英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1371/journal.pone.0067118DOI ID：10.1371/journal.pone.0067118,
PubMed ID：23843987,
PubMed Central ID：PMC3699551 Ghrelin increases intracellular Ca²⁺ concentration in the various hormone-producing cell types of the rat pituitary gland. Mami Yamazaki; Sayaka Aizawa; Toru Tanaka; Takafumi Sakai; Ichiro Sakata
Neuroscience letters,
巻：526,
号：1,
開始ページ：29,
終了ページ：32, 2012年09月,
［国際誌］Ghrelin, isolated from the stomach as an endogenous ligand for the growth hormone secretagogue receptor (GHS-R), has potent growth hormone release ability in vivo and in vitro. Although GHS-R is abundantly expressed in the pituitary gland, there is no direct evidence of a relationship between hormone-producing cells and functional GHS-R in the pituitary gland. The aim of this study was to determine which anterior pituitary cells respond to ghrelin stimulation in male rats. We performed Fura-2 Ca(2+) imaging analysis using isolated pituitary cells, and performed immunocytochemistry to identify the type of pituitary hormone-producing cells. In Fura-2 Ca(2+) imaging analysis, ghrelin administration increased the intracellular Ca(2+) concentration in approximately 50% of total isolated anterior pituitary cells, and 20% of these cells strongly responded to ghrelin. Immunocytochemical analysis revealed that 82.9 ± 1.3% of cells that responded to ghrelin stimulation were GH-immunopositive. On the other hand, PRL-, LH-, and ACTH-immunopositive cells constituted 2.0 ± 0.3%, 12.6 ± 0.3%, and 2.5 ± 0.8% of ghrelin-responding pituitary cells, respectively. TSH-immunopositive cells did not respond to ghrelin treatment. These results suggest that ghrelin directly acts not only on somatotrophs, but also on mammotrophs, gonadotrophs, and corticotrophs in the rat pituitary gland.
英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1016/j.neulet.2012.07.063DOI ID：10.1016/j.neulet.2012.07.063,
PubMed ID：22897875 In vitro selection of a peptide antagonist of growth hormone secretagogue receptor using cDNA display Shingo Ueno; Sayaka Yoshida; Anupom Mondal; Kazuya Nishina; Makoto Koyama; Ichiro Sakata; Kenju Miura; Yujiro Hayashi; Naoto Nemoto; Koichi Nishigaki; Takafumi Sakai
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA,
巻：109,
号：28,
開始ページ：11121,
終了ページ：11126, 2012年07月,
［査読有り］G protein-coupled receptors (GPCRs) are major drug targets, and their ligands are currently being explored and developed by many pharmaceutical companies and independent researchers. Class A (rhodopsin-like) GPCRs compose a predominant GPCR family; therefore, class A GPCR ligands are in demand. Growth hormone secretagogue receptor (GHS-R) is a class A GPCR that stimulates food intake by binding to its peptide ligand, ghrelin. Therefore, antagonists of GHS-R are expected to exert antiobesity function. In this article, we describe the use of cDNA display to screen for successfully and identify an antagonistic peptide of GHS-R. The antagonistic peptide inhibited the ghrelin-induced increase in intracellular Ca2+ in vitro (IC50 = approximately 10 mu M) and repressed the contraction of isolated animal stomach in response to ghrelin. Furthermore, peripheral administration of the peptide inhibited the food intake of mice. This work provides new insight into the development of antiobesity drugs and describes a method for the discovery of unique peptide ligands for class A GPCRs.
NATL ACAD SCIENCES, 英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1073/pnas.1203561109DOI ID：10.1073/pnas.1203561109,
ISSN：0027-8424,
PubMed ID：22723348,
Web of Science ID：WOS:000306642100026 Molecular identification of GHS-R and GPR38 in Suncus murinus. Airi Suzuki; Yuko Ishida; Sayaka Aizawa; Ichiro Sakata; Chihiro Tsutsui; Anupom Mondal; Koike Kanako; Takafumi Sakai
Peptides,
巻：36,
号：1,
開始ページ：29,
終了ページ：38, 2012年07月,
［国際誌］We previously identified ghrelin and motilin genes in Suncus murinus (suncus), and also revealed that motilin induces phase III-like strong contractions in the suncus stomach in vivo, as observed in humans and dogs. Moreover, repeated migrating motor complexes were found in the gastrointestinal tract of suncus at regular 120-min intervals. We therefore proposed suncus as a small laboratory animal model for the study of gastrointestinal motility. In the present study, we identified growth hormone secretagogue receptor (GHS-R) and motilin receptor (GPR38) genes in the suncus. We also examined their tissue distribution throughout the body. The amino acids of suncus GHS-R and GPR38 showed high homology with those of other mammals and shared 42% amino acid identity. RT-PCR showed that both the receptors were expressed in the hypothalamus, medulla oblongata, pituitary gland and the nodose ganglion in the central nervous system. In addition, GHS-R mRNA expressions were detected throughout the stomach and intestine, whereas GPR38 was expressed in the gastric muscle layer, lower intestine, lungs, heart, and pituitary gland. These results suggest that ghrelin and motilin affect gut motility and energy metabolism via specific receptors expressed in the gastrointestinal tract and/or in the central nervous system of suncus.
英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1016/j.peptides.2012.04.019DOI ID：10.1016/j.peptides.2012.04.019,
PubMed ID：22579813 Detailed analysis of the δ-crystallin mRNA-expressing region in early development of the chick pituitary gland. Makiko Inoue; Tomoya Shiina; Sayaka Aizawa; Ichiro Sakata; Hiroyasu Takagi; Takafumi Sakai
Journal of molecular histology,
巻：43,
号：3,
開始ページ：273,
終了ページ：80, 2012年06月,
［国際誌］Although δ-crystallin (δ-crys), also known as lens protein, is transiently expressed in Rathke's pouch (RP) of the chick embryo, detailed temporal and spatial expression patterns have been obscure. In this study, to understand the relationship between the δ-crys mRNA-expressing region and RP formation, we examined the embryonic expression pattern of δ-crys mRNA in the primordium of the adenohypophysis. δ-crys mRNA expression was initially found at stage 15 anterior to the foregut and posterior to the invaginated oral ectoderm. After RP formation, the δ-crys mRNA was expressed in the post-ventral region of RP and the anterior region of RP. δ-crys mRNA expression was then restricted to the cephalic lobe of the pituitary gland. From stage 20, the δ-crys and alpha-glycoprotein subunit (αGSU) mRNA-expressing regions were almost completely overlapping. The αGSU mRNA-expressing region is thought to be the primordium of the pars tuberalis, and these regions were overlapped with the Lhx3 mRNA-expressing region. The intensity of δ-crys mRNA expression gradually decreased with development and completely disappeared by stage 34. These results suggest that the embryonic chick pituitary gland consists of two different regions labeled with δ-crys and Lhx3.
英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1007/s10735-012-9407-1DOI ID：10.1007/s10735-012-9407-1,
PubMed ID：22461196 Coordination of motilin and ghrelin regulates the migrating motor complex of gastrointestinal motility in Suncus murinus. Anupom Mondal; Zuoyun Xie; Yuki Miyano; Chihiro Tsutsui; Ichiro Sakata; Yoichi Kawamoto; Sayaka Aizawa; Toru Tanaka; Sen-ichi Oda; Takafumi Sakai
American journal of physiology. Gastrointestinal and liver physiology,
巻：302,
号：10,
開始ページ：G1207-15, 2012年05月,
［国際誌］Motilin and ghrelin are the gastrointestinal (GI) hormones released in a fasting state to stimulate the GI motility of the migrating motor complex (MMC). We focused on coordination of the ghrelin/motilin family in gastric contraction in vivo and in vitro using the house musk shrew (Suncus murinus), a ghrelin- and motilin-producing mammal. To measure the contractile activity of the stomach in vivo, we recorded GI contractions either in the free-moving conscious or anesthetized S. murinus and examined the effects of administration of motilin and/or ghrelin on spontaneous MMC in the fasting state. In the in vitro study, we also studied the coordinative effect of these hormones on the isolated stomach using an organ bath. In the fasting state, phase I, II, and III contractions were clearly recorded in the gastric body (as observed in humans and dogs). Intravenous infusion of ghrelin stimulated gastric contraction in the latter half of phase I and in the phase II in a dose-dependent manner. Continuous intravenous infusion of ghrelin antagonist (d-Lys3-GHRP6) significantly suppressed spontaneous phase II contractions and prolonged the time of occurrence of the peak of phase III contractions. However, intravenous infusion of motilin antagonist (MA-2029) did not inhibit phase II contractions but delayed the occurrence of phase III contractions of the MMC. In the in vitro study, even though a high dose of ghrelin did not stimulate contraction of stomach preparations, ghrelin administration (10(-10)-10(-7) M) with pretreatment of a low dose of motilin (10(-10) M) induced gastric contraction in a dose-dependent manner. Pretreatment with 10(-8) M ghrelin enhanced motilin-stimulated gastric contractions by 10 times. The interrelation of these peptides was also demonstrated in the anesthetized S. murinus. The results suggest that ghrelin is important for the phase II contraction and that coordination of motilin and ghrelin are necessary to initiate phase III contraction of the MMC.
英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1152/ajpgi.00379.2011DOI ID：10.1152/ajpgi.00379.2011,
PubMed ID：22383491 Glutamine and glutamic acid enhance thyroid-stimulating hormone beta subunit mRNA expression in the rat pars tuberalis Sayaka Aizawa; Takafumi Sakai; Ichiro Sakata
JOURNAL OF ENDOCRINOLOGY,
巻：212,
号：3,
開始ページ：383,
終了ページ：394, 2012年03月,
［査読有り］Thyroid-stimulating hormone (TSH)-producing cells of the pars tuberalis (PT) display distinct characteristics that differ from those of the pars distalis (PD). The mRNA expression of TSH beta and alpha GSU in PT has a circadian rhythm and is inhibited by melatonin via melatonin receptor type 1; however, the detailed regulatory mechanism for TSH beta expression in the PT remains unclear. To identify the factors that affect PT, a microarray analysis was performed on laser-captured PT tissue to screen for genes coding for receptors that are abundantly expressed in the PT. In the PT, we found high expression of the KA2, which is an ionotropic glutamic acid receptor (iGluR). In addition, the amino acid transporter A2 (ATA2), also known as the glutamine transporter, and glutaminase (GLS), as well as GLS2, were highly expressed in the PT compared to the PD. We examined the effects of glutamine and glutamic acid on TSH beta expression and alpha GSU expression in PT slice cultures. L-Glutamine and L-glutamic acid significantly stimulated TSH beta expression in PT slices after 2- and 4-h treatments, and the effect of L-glutamic acid was stronger than that of L-glutamine. In contrast, treatment with glutamine and glutamic acid did not affect alpha GSU expression in the PT or the expression of TSHb or alpha GSU in the PD. These results strongly suggest that glutamine is taken up by PT cells through ATA2 and that glutamic acid locally converted from glutamine by Gls induces TSH beta expression via the KA2 in an autocrine and/or paracrine manner in the PT. Journal of Endocrinology (2012) 212, 383-394
BIOSCIENTIFICA LTD, 英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1530/JOE-11-0388DOI ID：10.1530/JOE-11-0388,
ISSN：0022-0795,
Web of Science ID：WOS:000302116400013 Physiological characteristics of gastric contractions and circadian gastric motility in the free-moving conscious house musk shrew (Suncus murinus) Satoshi Sakahara; Zuoyun Xie; Kanako Koike; Satoya Hoshino; Ichiro Sakata; Sen-Ichi Oda; Toku Takahashi; Takafumi Sakai
American Journal of Physiology - Regulatory Integrative and Comparative Physiology,
巻：299,
号：4,
開始ページ：R1106,
終了ページ：R1113, 2010年10月
Although many studies have demonstrated the physiological action of motilin on the migrating motor complex, the precise mechanisms remain obscure. To obtain new insights into the mechanisms, we focused on the house musk shrew (Suncus murinus, suncus used as a laboratory name) as a small model animal for in vivo motilin study, and we studied the physiological characteristics of suncus gastrointestinal motility. Strain gauge transducers were implanted on the serosa of the gastric body and duodenum, and we recorded gastrointestinal contractions in the free-moving conscious suncus and also examined the effects of intravenous infusion of various agents on gastrointestinal motility. During the fasted state, the suncus stomach and duodenum showed clear migrating phase III contractions (intervals of 80-150 min) as found in humans and dogs. Motilin (bolus injection, 100-300 ng/kg
continuous infusion, 10-100 ng·kg -1·min-1) and erythromycin (80 μg·kg -1·min-1) induced gastric phase III contractions, and motilin injection also increased the gastric motility index in a dose-dependent manner (P <
0.05, vs. saline). Pretreatment with atropine completely abolished the motilin-induced gastric phase III contractions. On the other hand, in the free-feeding condition, the suncus showed a relatively long fasting period in the light phase followed by spontaneous gastric phase III contractions. The results suggest that the suncus has almost the same gastrointestinal motility and motilin response as those found in humans and dogs, and we propose the suncus as a new small model animal for studying gastrointestinal motility and motilin in vivo.
英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1152/ajpregu.00278.2010DOI ID：10.1152/ajpregu.00278.2010,
ISSN：0363-6119,
PubMed ID：20686171,
SCOPUS ID：77957604330 Identification of ghrelin in the house musk shrew (Suncus murinus): cDNA cloning, peptide purification and tissue distribution Yuko Ishida; Satoshi Sakahara; Chihiro Tsutsui; Hiroyuki Kaiya; Ichiro Sakata; Sen ichi Oda; Takafumi Sakai
Peptides,
巻：30,
号：5,
開始ページ：982,
終了ページ：990, 2009年05月
Ghrelin is the endogenous ligand for the growth hormone (GH) secretagogue receptor, and the sequence of ghrelin has been determined in many species from fish to mammals. In the present study, to reveal the production of ghrelin in the house musk shrew (Suncus murinus, order: Insectivora, suncus is used as a laboratory name), we determined the cDNA sequence and structure of suncus ghrelin and also demonstrated the ghrelin-producing cells in the gastrointestinal tract. Results of cDNA cloning and mass spectrometry analysis revealed that suncus ghrelin is composed of 18 or 26 amino acid residues and that the 3rd Ser was acylated mainly by n-octanoic acid. The 10 amino acids of the N-terminal region of suncus mature ghrelin were consistent with those of other mammals. Quantitative RT-PCR revealed that suncus ghrelin mRNA is highly expressed in the gastric corpus and pyloric antrum, and low expression levels were found in various tissues, including the intestinal tract. Ghrelin cells were found only in the corpus and antrum by immunohistochemistry and in situ hybridization, and most of the ghrelin cells were closed-type cells with relatively rich cytoplasm and scattered in the glandular body and base of the gastric mucosa. The density of ghrelin cells in the corpus was significantly greater than that in the antrum. The results of this study together with our recent results regarding motilin production in the suncus indicate that the suncus will be a useful model animal for study of physiological function of the motilin/ghrelin family. © 2009 Elsevier Inc. All rights reserved.
研究論文（学術雑誌）
DOI：https://doi.org/10.1016/j.peptides.2009.01.006Scopus：https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=67349120118&origin=inwardScopus Citedby：https://www.scopus.com/inward/citedby.uri?partnerID=HzOxMe3b&scp=67349120118&origin=inwardDOI ID：10.1016/j.peptides.2009.01.006,
ISSN：0196-9781,
PubMed ID：19428777,
SCOPUS ID：67349120118 Detailed analysis of formation of chicken pituitary primordium in early embryonic development Hiroyasu Takagi; Keiko Nagashima; Makiko Inoue; Ichiro Sakata; Takafumi Sakai
CELL AND TISSUE RESEARCH,
巻：333,
号：3,
開始ページ：417,
終了ページ：426, 2008年09月,
［査読有り］The primordium of the mammalian adenohypophysis derived from Rathke's pouch (RP) is known to be formed by oral ectoderm invagination. However, in the early phase of pituitary development, the detailed process by which the oral ectoderm develops into the adenohypophysis remains largely unknown. Using high-resolution non-radiolabeled in situ hybridization and the BrdU and TUNEL methods, we have examined the detailed expression pattern of factors involved in the formation of RP of chicken and the changes in the mitotic and apoptotic cell regions in RP. In the chicken embryo, Sonic hedgehog (Shh) mRNA was initially expressed in the stomodeal plate but not in the oral ectoderm. After prospective diencephalon had detached from the oral ectoderm, another Shh-expressing region appeared in the most rostral part of the recess. LIM homeobox gene 3 (Lhx3) mRNA first appeared in the anterior area of Rathke's recess, and expression then spread to the caudal region. alpha GSU mRNA-expressing cells were observed at both ends of the Lhx3-expressing region, and thereafter the expression area moved to the posterior region. Furthermore, a close overlap was found between the proliferating region and Lhx3 mRNA-expressing area, and TUNEL-positive cells appeared in Seessel's pouch derived from the foregut. Thus, the primordium of the pituitary gland corresponding to the Lhx3-expressing region is surrounded by the Shh-expressing region, which appears in two steps, and the mass growth and invagination of RP of chicken result from the coordination of the dorsal extension of the anterior region and the ventral extension of the posterior region of RP.
SPRINGER, 英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1007/s00441-008-0647-zDOI ID：10.1007/s00441-008-0647-z,
ISSN：0302-766X,
Web of Science ID：WOS:000258528200006 Gastric leptin, but not estrogen and somatostatin, contributes to the elevation of ghrelin mRNA expression level in fasted rats Zheng Zhao; Ichiro Sakata; Yusuke Okubo; Kanako Koike; Kenji Kangawa; Takafumi Sakai
JOURNAL OF ENDOCRINOLOGY,
巻：196,
号：3,
開始ページ：529,
終了ページ：538, 2008年03月,
［査読有り］Ghrelin, an endogenous ligand for the GH secretagog receptor, is predominantly produced in the stomach. It has been reported that endogenous ghrelin levels are increased by fasting and decreased after refeeding. It has also been reported that estrogen upregulates ghrelin expression and production and that somatostatin inhibits ghrelin secretion, whereas leptin has a paradoxical effect. Recently, several studies have shown that estrogen, somatostatin, and leptin are produced in the stomach, but the direct effects of these gastric hormones on ghrelin expression in a fasting state remain obscure. In this study, we examined the mRNA expression levels of gastric ghrelin, aromatase (estrogen synthetase), leptin and somatostatin, and concentrations of stomach leptin and portal vein 17 beta-estradiol in fasted male rats. After 48 h of fasting, although gastric ghrelin mRNA level was significantly increased, both gastric leptin mRNA level and leptin content were decreased. Further, refeeding of fasted rats resulted in a decrease in ghrelin expression level and an increase in leptin expression level. On the other hand, gastric estrogen and somatostatin levels did not change after fasting. In vitro studies revealed that leptin dose-dependently inhibited ghrelin expression and also inhibited estrogen-stimulated ghrelin expression. Moreover, ghrelin cells were found to be tightly surrounded by leptin cells. RT-PCR analysis clearly showed that long and short forms of the leptin receptor are expressed in the rat stomach. These results strongly suggest that an elevated gastric ghrelin expression level in a fasting state is regulated by attenuated restraint from decreased gastric leptin level.
SOC ENDOCRINOLOGY, 英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1677/JOE-07-0300DOI ID：10.1677/JOE-07-0300,
ISSN：0022-0795,
Web of Science ID：WOS:000254297800009 DNA introduction into living cells by water droplet impact with an electrospray process Yusuke Okubo; Kazuto Ikemoto; Kanako Koike; Chihiro Tsutsui; Ichiro Sakata; Osamu Takei; Akihito Adachi; Takafumi Sakai
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION,
巻：47,
号：8,
開始ページ：1429,
終了ページ：1431, 2008年,
［査読有り］WILEY-V C H VERLAG GMBH, 英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1002/anie.200704429DOI ID：10.1002/anie.200704429,
ISSN：1433-7851,
PubMed ID：18203224,
Web of Science ID：WOS:000253345700013 Identification of immunoreactive plasma and stomach ghrelin, and expression of stomach ghrelin mRNA in the bullfrog, Rana catesbeiana Hiroyuki Kaiya; Ichiro Sakata; Kazutoshi Yamamoto; Aya Koda; Takafumi Sakai; Kenji Kangawa; Sakae Kikuyama
General and Comparative Endocrinology,
巻：148,
号：2,
開始ページ：236,
終了ページ：244, 2006年09月
In this study, we established a radioimmunoassay (RIA) specific for ghrelin from the bullfrog Rana catesbeiana using a novel antibody raised against the C-terminal amino acid sequence of bullfrog ghrelin [13-28]. We also examined the distribution of ghrelin-producing cells in the stomachs of bullfrogs using this antibody and a cRNA probe specific for the bullfrog ghrelin gene. Ghrelin levels in plasma and stomach extracts were approximately 150 fmol/ml and 83-135 fmol/mg wet tissue, respectively. Reverse-phase high performance liquid chromatographic analysis, combined with bullfrog ghrelin RIA, revealed that ghrelin immunoreactivity in the stomach was composed of non-acylated ghrelin (des-acyl ghrelin) and several acylated forms of ghrelin bearing different fatty acid modifications, which could induce increases in intracellular Ca2+ in cells expressing the rat GH secretagogue receptor. In the stomach, the major storage form was acylated ghrelin. In bullfrog plasma, however, the majority of ghrelin immunoreactivity was des-acyl ghrelin and C-terminal fragments of frog ghrelin. Acylated ghrelin forms comprised only minor peaks. Ghrelin-immunopositive and ghrelin mRNA-expressing cells were observed within the mucosal layer of the stomach. Following starvation, significant increases in plasma ghrelin levels and stomach ghrelin mRNA levels were observed as early as 10 days after starvation. These results indicate that ghrelin is present in the stomach and plasma of the bullfrog, which can be detected with our novel antibody. Interestingly, the primary storage form of ghrelin in the stomach differed from the circulating form dominating in the plasma. Furthermore, increases in ghrelin levels in plasma and mRNA levels in the stomach after starvation suggest the possible involvement of ghrelin in energy homeostasis in the bullfrog. © 2006 Elsevier Inc. All rights reserved.
研究論文（学術雑誌）
DOI：https://doi.org/10.1016/j.ygcen.2006.03.008Scopus：https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=33746564831&origin=inwardScopus Citedby：https://www.scopus.com/inward/citedby.uri?partnerID=HzOxMe3b&scp=33746564831&origin=inwardDOI ID：10.1016/j.ygcen.2006.03.008,
ISSN：0016-6480,
eISSN：1095-6840,
PubMed ID：16630619,
SCOPUS ID：33746564831 Exogenous administration of octanoic acid accelerates octanoylated ghrelin production in the proventriculus of neonatal chicks Maya Yamato; Ichiro Sakata; Reiko Wada; Hiroyuki Kaiya; Takafumi Sakai
Biochemical and Biophysical Research Communications,
巻：333,
号：2,
開始ページ：583,
終了ページ：589, 2005年07月
Ghrelin is modified by fatty acid at the third serine residue. In this study, derivation of fatty acid for acylation of ghrelin was investigated using a hatchling chicken model. We first studied ghrelin gene expression and production in the neonatal chick proventriculus and then investigated the effect of exogenous octanoic acid (OA) administration on acylated ghrelin production. In a free-feeding condition on day 2.5 after hatching, the density of ghrelin mRNA-expressing (ghrelin-ex) cells was greater than that of ghrelin- immunopositive (ghrelin-ip) cells, but no difference was found between those densities in adult chickens. Intraperitoneal or oral administration of OA for a few days significantly increased the density of ghrelin-ip cells without any changes in ghrelin-ex cells and elevated only octanoylated ghrelin levels in the proventriculus. The results indicate that fatty acid absorbed from food is directly utilized in acylated ghrelin production in the chicken. © 2005 Elsevier Inc. All rights reserved.
研究論文（学術雑誌）
DOI：https://doi.org/10.1016/j.bbrc.2005.05.107Scopus：https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=20544444427&origin=inwardScopus Citedby：https://www.scopus.com/inward/citedby.uri?partnerID=HzOxMe3b&scp=20544444427&origin=inwardDOI ID：10.1016/j.bbrc.2005.05.107,
ISSN：0006-291X,
PubMed ID：15953586,
SCOPUS ID：20544444427 Structural determination and histochemical localization of ghrelin in the red-eared slider turtle, Trachemys scripta elegans Hiroyuki Kaiya; Ichiro Sakata; Masayasu Kojima; Hiroshi Hosoda; Takafumi Sakai; Kenji Kangawa
General and Comparative Endocrinology,
巻：138,
号：1,
開始ページ：50,
終了ページ：57, 2004年08月
We purified ghrelin peptide and determined the cDNA sequence encoding the precursor protein from the stomach of the red-eared slider turtle, Trachemys scripta elegans. The Trachemys ghrelin is comprised of 25-amino acids and has the sequence GSSFLSPEYQNTQQRKDPKKHTKLN. The third serine residue was modified by n-octanoic (C8:0), decanoic (C10:0) or unsaturated decanoic acid (C10:1). The carboxyl-terminal end of the peptide was not amidated, as seen in the ghrelins of other land vertebrates. Quantitative real-time PCR analysis revealed high levels of gene expression in the stomach and moderate levels in the large intestine and pancreas. Histochemical studies of turtle stomach revealed that ghrelin-immunopositive (ghrelin-ip) cells, which were small and round, were observed in the mucosal layer of the stomach but not in the myenteric plexus, and ghrelin-mRNA-expressing (ghrelin-ex) cells detected by in situ hybridization were scattered in a similar distribution as ghrelin-ip cells. These results indicate that ghrelin is present in reptiles. © 2004 Elsevier Inc. All rights reserved.
研究論文（学術雑誌）
DOI：https://doi.org/10.1016/j.ygcen.2004.05.005Scopus：https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=3042746882&origin=inwardScopus Citedby：https://www.scopus.com/inward/citedby.uri?partnerID=HzOxMe3b&scp=3042746882&origin=inwardDOI ID：10.1016/j.ygcen.2004.05.005,
ISSN：0016-6480,
eISSN：1095-6840,
PubMed ID：15242751,
SCOPUS ID：3042746882 Localization of ghrelin-producing cells in the stomach of the rainbow trout (Oncorhynchus mykiss) Ichiro Sakata; Tsukasa Mori; Hiroyuki Kaiya; Mami Yamazaki; Kenji Kangawa; Kinji Inoue; Takafumi Sakai
Zoological Science,
巻：21,
号：7,
開始ページ：757,
終了ページ：762, 2004年07月
Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor (GHS-R), was isolated from the rat stomach and determined to be n-octanoylated 28-amino-acid peptide. In this study, we studied the distribution of ghrelin-producing cells (ghrelin cells) in the gastrointestinal tract of male and female rainbow trout (Oncorhynchus mykiss) by immunohistochemistry using N-terminal region-recognizing antibody and also by in situ hybridization using a trout ghrelin-specific cRNA probe. Ghrelin cells were found in the mucosal layer of the stomach but not in the myenteric plexus, and no ghrelin cells were observed in other regions of the gastrointestinal tract. Ghrelin cells could be classified into two types: closed- and opened-type cells. The density of ghrelin cells increased gradually in the direction from the cardiac to pyloric portions of the stomach in both sexes. The number of ghrelin cells per unit area seemed to be higher in females than in males. In conclusion, trout ghrelin cells exist in the stomach and are classified into two types of cells, closed- and opened-type cells.
研究論文（学術雑誌）
DOI：https://doi.org/10.2108/zsj.21.757Scopus：https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=4344635899&origin=inwardScopus Citedby：https://www.scopus.com/inward/citedby.uri?partnerID=HzOxMe3b&scp=4344635899&origin=inwardDOI ID：10.2108/zsj.21.757,
ISSN：0289-0003,
PubMed ID：15277719,
SCOPUS ID：4344635899 Existence of ghrelin-immunopositive and -expressing cells in the proventriculus of the hatching and adult chicken Reiko Wada; Ichiro Sakata; Hiroyuki Kaiya; Kazuaki Nakamura; Yujiro Hayashi; Kenji Kangawa; Takafumi Sakai
Regulatory Peptides,
巻：111,
号：1-3,
開始ページ：123,
終了ページ：128, 2003年03月
Ghrelin was isolated from the rat stomach as an endogenous ligand for the growth hormone secretagogue receptor (GHS-R) and has been found in the gastrointestinal tract of many vertebrates. Although the sequence and structure of chicken ghrelin has recently been determined, morphological characteristics of ghrelin cells in the chicken gastrointestinal tract are still obscure. In this study, we investigated ghrelin expression and distribution of ghrelin-producing cells in the hatching and adult chicken gastrointestinal tract by RT-PCR, immunohistochemistry and in situ hybridization. Ghrelin mRNA expression was observed mainly in the proventriculus in the hatching chicken and in the proventriculus, pylorus and duodenum of the adult chicken by RT-PCR. Ghrelin-immunopositive (ghrelin-ip) cells in the proventriculus were located at the mucosal layer but not in the myenteric plexus or smooth muscle layer. The number of ghrelin-ip cells in the adult chicken was greater than that in the hatching chicken. Interestingly, in the adult chicken, the number of ghrelin-ip cells were almost the same as that of ghrelin mRNA-expressing (ghrelin-ex) cells; however, in the hatching chicken, the number of ghrelin-ex cells was greater than that of ghrelin-ip cells. These results clearly demonstrate that ghrelin-producing cells exist in the chicken gastrointestinal tract, especially in the proventriculus, from hatching to adult stages of development, as well as in mammals. © 2002 Elsevier Science B.V. All rights reserved.
研究論文（学術雑誌）
DOI：https://doi.org/10.1016/S0167-0115(02)00265-3Scopus：https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=0037471360&origin=inwardScopus Citedby：https://www.scopus.com/inward/citedby.uri?partnerID=HzOxMe3b&scp=0037471360&origin=inwardDOI ID：10.1016/S0167-0115(02)00265-3,
ISSN：0167-0115,
PubMed ID：12609759,
SCOPUS ID：0037471360 Ghrelin-producing cells exist as two types of cells, closed- and opened-type cells, in the rat gastrointestinal tract.
Ichiro Sakata; Kazuaki Nakamura; Mami Yamazaki; Maki Matsubara; Yuijiro Hayashi; Kenji Kangawa; Takafumi Sakai
Peptides, 巻：23, 号：3, 開始ページ：531, 終了ページ：6, 2002年03月, ［国際誌］
Ghrelin was recently isolated from the rat stomach as an endogenous ligand for the growth-hormone secretagogue receptor (GHS-R) and is known to exist in the gastrointestinal tract and hypothalamus. In this study, we investigated in detail the distribution and morphologic characteristics of ghrelin-containing cells (ghrelin cells) in the gastrointestinal tract by immunohistochemistry and in situ hybridization. Ghrelin cells were found to be localized in the mucous membrane of the stomach, duodenum, ileum, cecum and colon but not in myenteric plexus, and they can be classified into open- and closed-type cells. The greatest number of ghrelin cells was found in the stomach, and it was found that the number of the opened-type cells gradually increased in the direction from stomach to the lower gastrointestinal tract. These results suggest that the two types of ghrelin cells may be distinctly regulated and play different physiological roles in various regions of the gastrointestinal tract.
英語, 研究論文（学術雑誌）
ISSN：0196-9781, PubMed ID：11836003
Utility of animal gastrointestinal motility and transit models in functional gastrointestinal disorders Ahmad Al-Saffar; Shota Takemi; Hiwa K. Saaed; Ichiro Sakata; Takafumi Sakai
巻：40-41, 2019年06月
Alteration in the gastrointestinal (GI) motility and transit comprises an important component of the functional gastrointestinal disorders (FGID). Available animal GI motility and transit models are to study symptoms (delayed gastric emptying, constipation, diarrhea) rather than biological markers to develop an effective treatment that targets the underlying mechanism of altered GI motility in patients. Animal data generated from commonly used methods in human like scintigraphy, breath test and wireless motility capsule may directly translate to the clinic. However, species differences in the control mechanism or pharmacological responses of GI motility may compromise the predictive and translational value of the preclinical data to human. In this review we aim to provide a summary on animal models used to mimic GI motility alteration in FGID, and the impact of the species differences in the physiological and pharmacological responses on the translation of animal GI motility and transit data to human. (C) 2019 Elsevier Ltd. All rights reserved.
英語, 書評論文,書評,文献紹介等
DOI：https://doi.org/10.1016/j.bpg.2019.101633DOI ID：10.1016/j.bpg.2019.101633,
ISSN：1521-6918,
eISSN：1532-1916,
Web of Science ID：WOS:000517974000011 Mechanism of Rikkunshito-Induced Gastric Relaxation in Suncus Murinus Through the beta-Adrenergic Pathway
Anupom Mondal; Amane Takehara; Sayaka Aizawa; Toru Tanaka; Naoki Fujitsuka; Tomohisa Hattori; Takafumi Sakai; Ichiro Sakata
巻：148, 号：4, 開始ページ：S893, 終了ページ：S893, 2015年04月
英語, 研究発表ペーパー・要旨（国際会議）
ISSN：0016-5085, eISSN：1528-0012, Web of Science ID：WOS:000360120300167
Motilin and Ghrelin Additively Stimulate Gastric Acid Secretion in Suncus Murinus
Ichiro Sakata; Yoshiaki Shimada; Toru Tanaka; Kanako Koike; Sayaka Aizawa; Takafumi Sakai
巻：144, 号：5, 開始ページ：S711, 終了ページ：S711, 2013年05月
英語, 研究発表ペーパー・要旨（国際会議）
ISSN：0016-5085, eISSN：1528-0012, Web of Science ID：WOS:000322997204148
Ghrelin cells in the gastrointestinal tract Ichiro Sakata; Takafumi Sakai
巻：2010, 2010年
Ghrelin is 28-amino-acid peptide that was discovered from the rat and human stomach in 1999. Since the discovery of ghrelin, various functions of ghrelin, including growth hormone release, feeding behavior, glucose metabolism, memory, and also antidepressant effects, have been studied. It has also been reported that ghrelin in the gastrointestinal tract has an important physiological effect on gastric acid secretion and gastrointestinal motility. Ghrelin has a unique structure that is modified by O -acylation with n-octanoic acid at third serine residues, and this modification enzyme has recently been identified and named ghrelin O -acyl transferase (GOAT). Ghrelin is considered to be a gut-brain peptide and is abundantly produced from endocrine cells in the gastrointestinal mucosa. In the gastrointestinal tract, ghrelin cells are most abundant in the stomach and are localized in gastric mucosal layers. Ghrelin cells are also widely distributed throughout the gastrointestinal tract. In addition, abundance of ghrelin cells in the gastric mucosa is evolutionally conserved from mammals to lower vertebrates, indicating that gastric ghrelin plays important roles for fundamental physiological functions. Ghrelin cells in the gastrointestinal tract are a major source of circulating plasma ghrelin, and thus understanding the physiology of these cells would reveal the biological significance of ghrelin. Copyright © 2010 I. Sakata and T. Sakai.
英語, 書評論文,書評,文献紹介等
DOI：https://doi.org/10.1155/2010/945056DOI ID：10.1155/2010/945056,
ISSN：1687-9767,
SCOPUS ID：79952041715 House musk shrew (Suncus murinus, order: Insectivora) as a new model animal for motilin study Chihiro Tsutsui; Kie Kajihara; Takatsugu Yanaka; Ichiro Sakata; Zen Itoh; Sen-ichi Oda; Takafumi Sakai
巻：30,
号：2,
開始ページ：318,
終了ページ：329, 2009年02月
Although many studies have demonstrated the action of motilin on migrating motor complex by using human subjects and relatively large animals, the precise physiological mechanisms of motilin remain obscure. One reason for the lack of progress in this research field is that large animals are generally not suitable for molecular-level study. To overcome this problem, in this study, we focused on the house musk shrew (Suncus murinus, order: insectivora, suncus named as laboratory strain) as a small model animal, and we present here the results of motilin gene cloning and its availability for motilin study. The motilin gene has a high homology sequence with that of other mammals, including humans. Suncus motilin is predicted to exist as a 117-residue prepropeptide that undergoes proteolytic cleavage to form a 22-amino-acid mature peptide. The results of RT-PCR showed that motilin mRNA is highly expressed in the upper small intestine, and low levels of expression were found in many tissues. Morphological analysis revealed that suncus motilin-producing cells were present in the upper small intestinal mucosal layer but not in the myenteric plexus. Administration of suncus motilin to prepared muscle strips of rabbit duodenum showed almost the same contractile effect as that of human motilin. Moreover, suncus stomach preparations clearly responded to suncus or human motilin stimulation. To our knowledge, this is the first report that physiological active motilin was determined in small laboratory animals, and the results of this study suggest that suncus is a suitable model animal for studying the motilin-ghrelin family. (C) 2008 Elsevier Inc. All rights reserved.
英語
DOI：https://doi.org/10.1016/j.peptides.2008.10.006DOI ID：10.1016/j.peptides.2008.10.006,
ISSN：0196-9781,
Web of Science ID：WOS:000263447700016 Gastric estrogen directly induces ghrelin expression and production in the rat stomach Ichiro Sakata; Toru Tanaka; Mami Yamazaki; Takashi Tanizaki; Zhao Zheng; Takafumi Sakai
巻：190,
号：3,
開始ページ：749,
終了ページ：757, 2006年09月
Ghrelin, an endogenous ligand for the GH secretagogue receptor, is predominantly produced in the stomach. Little is known about the regulation mechanism of gastric ghrelin. Here, we report that estrogen synthesized in the stomach induces rat gastric ghrelin gene expression and production. We established a gastric ghrelin cell enrichment method using Percoll centrifugation and then studied the effect of estrogen and/or its antagonist on ghrelin expression and production. Treatment with estrogen for 8 h significantly increased the level of ghrelin expression, and ICI-182 780, an estrogen receptor (ER) antagonist, completely reversed this effect. Reverse transcriptase-PCR analysis clearly showed that ER alpha and aromatase are expressed in the female rat stomach. Moreover, treatment with an aromatase inhibitor, 4-hydroxyandrostenedione (formestane), significantly decreased the level of ghrelin mRNA expression in minced stomach tissue. In vivo studies revealed that the ghrelin mRNA expression and production did not change in gonadectomized rat 3 weeks after surgery. These results strongly suggest that estrogen produced in the stomach directly induces ghrelin expression and production in both female and male rat stomachs.
英語
DOI：https://doi.org/10.1677/joe.1.06808DOI ID：10.1677/joe.1.06808,
ISSN：0022-0795,
Web of Science ID：WOS:000241324000020 Caspase-3 sensitive signaling in vivo in apoptotic HeLa cells by chemically engineered intramolecular fluorescence resonance energy transfer mutants of green fluorescent protein M Suzuki; Y Ito; Sakata, I; T Sakai; Y Husimi; KT Douglas
巻：330,
号：2,
開始ページ：454,
終了ページ：460, 2005年05月
Green fluorescent protein (UV5) was re-engineered to remove native cysteine residues, and a new cysteine was introduced near the C-terminus, similar to 20 angstrom from the native fluorophore, for site-specific attachment of chemical fluorophores. The resultant efficient intramolecular FRET quenched GFP emission and gave a new emission band from the conjugated fluorophore. Caspase-3 cleavage of constructs with a caspase-3 sequence near the C-terminus in the sequence between the native fluorophore and the new cysteine, located C-terminal to the caspase site, destroyed the FRET, the emitted color reverting to that of unmodified GFP. This process was demonstrated in vitro with caspase-3 and lysates from cells undergoing apoptosis. Real-time emission changes for the Alexa Fluor 532 conjugate of this GFP, studied quantitatively in vivo for single HeLa cells using the ratios of fluorescence at the red and green maxima by confocal microscopy, showed that caspase-3 action in the cytosol preceded that in the nucleus. (c) 2005 Elsevier Inc. All rights reserved.
英語
DOI：https://doi.org/10.1016/j.bbrc.2005.02.178DOI ID：10.1016/j.bbrc.2005.02.178,
ISSN：0006-291X,
CiNii Articles ID：80017288069,
PubMed ID：15796904,
Web of Science ID：WOS:000228315800015 Influence of sex steroids on ghrelin-induced growth hornione secretion in male rats
Takashi Tanizaki; Mami Yamazaki; Ichiro Sakata; Hisae Kobayashi; Takafumi Sakai
巻：21, 号：12, 開始ページ：1337, 終了ページ：1337, 2004年12月
英語, 研究発表ペーパー・要旨（国際会議）
ISSN：0289-0003, Web of Science ID：WOS:000237651300643
Octanoic acid administration increases the production of acylated ghrelin in the hatched chicken proventriculus
Maya Yamato; Ichiro Sakata; Reiko Wada; Hiroyuki Kaiya; Takafumi Sakai
巻：21, 号：12, 開始ページ：1337, 終了ページ：1337, 2004年12月
英語, 研究発表ペーパー・要旨（国際会議）
ISSN：0289-0003, Web of Science ID：WOS:000237651300642
Estrogen modulates ghrelin expression in the female rat stomach M Matsubara; Sakata, I; R Wada; M Yamazaki; K Inoue; T Sakai
巻：25,
号：2,
開始ページ：289,
終了ページ：297, 2004年02月
Ghrelin was recently identified as an endogenous ligand for GH secretagogue receptor. In this study, we investigated the effects of ovariectomy on the numbers of ghrelin-immunopositive and -expressing cells, ghrelin mRNA levels, and plasma ghrelin concentrations in 4- and 9-week-old female rats. Three days after ovariectomy, the number of ghrelin cells and plasma ghrelin level significantly increased in both 4- and 9-week-old rats and the ghrelin mRNA level also increased in 4-week-old rats. These responses were reversed by 17beta-estradiol replacement. We also found that ghrelin-immunopositive cells express estrogen receptor alpha. These results suggested that estrogen is involved in the regulation of ghrelin expression. (C) 2004 Elsevier Inc. All rights reserved.
英語
DOI：https://doi.org/10.1016/j.peptides.2003.12.020DOI ID：10.1016/j.peptides.2003.12.020,
ISSN：0196-9781,
Web of Science ID：WOS:000220943100018 Growth hormone secretagogue receptor expression in the cells of the stomach-projected afferent nerve in the rat nodose ganglion Sakata, I; M Yamazaki; K Inoue; Y Hayashi; K Kangawa; T Sakai
巻：342,
号：3,
開始ページ：183,
終了ページ：186, 2003年05月
Growth hormone secretagogue receptor (GHS-R) is widely expressed in various regions of the body, such as the brain, pituitary gland, heart and gastrointestinal tract. Recently, ghrelin, an endogenous ligand for GHS-R, was found in the rat stomach, and several studies have suggested that ghrelin acts via the vagal afferent nerve. In this study, we studied the expression of GHS-R mRNA in the rat nodose ganglion by reverse transcriptase-polymerase chain reaction and in situ hybridization, the results of which clearly demonstrated the presence of GHSR mRNA and GHS-R producing cells in the rat nodose ganglion. We also studied the retrograde tracing of nodose ganglion cells to the stomach and found that some GHS-R mRNA-expressing cells contain the retrograde rebelling. Our results provide direct morphological evidence that GHS-R is produced in afferent neurons of the nodose ganglion and suggest that ghrelin signals from the stomach are transmitted to the brain via vagal afferent nerves. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved.
英語
DOI：https://doi.org/10.1016/S0304-3940(03)00294-5DOI ID：10.1016/S0304-3940(03)00294-5,
ISSN：0304-3940,
Web of Science ID：WOS:000183093200012
大腸強収縮運動及び排便を制御する脳神経核の同定と神経回路の解析
日本学術振興会, 科学研究費助成事業, 基盤研究(C), 2023年04月 - 2026年03月
坂田 一郎, 埼玉大学
配分額（総額）：4680000, 配分額（直接経費）：3600000, 配分額（間接経費）：1080000
課題番号：23K07348
胃腸管収縮ホルモンとして知られるモチリンの新規生理作用の解明
日本学術振興会, 科学研究費助成事業, 基盤研究(B), 2022年04月 - 2025年03月
海谷 啓之; 今野 紀文; 東 森生; 坂田 一郎; 竹見 祥大, 富山大学
配分額（総額）：16770000, 配分額（直接経費）：12900000, 配分額（間接経費）：3870000
課題番号：22H02659
サーカディアンリズムによる消化管運動調節機構の解明
日本学術振興会, 科学研究費助成事業, 基盤研究(C), 2020年04月 - 2023年03月
坂井 貴文; 坂田 一郎; 竹見 祥大, 埼玉大学
配分額（総額）：4290000, 配分額（直接経費）：3300000, 配分額（間接経費）：990000
本研究は、食虫目スンクスを用いて生物リズムと脳腸相関軸の基幹をなすホルモンと自律神経の関連性、すなわち「ウルトラ―サーカディアン」軸の機能連関による消化管運動調節機構を明らかにすることを目的としている。昨年度の検討により、スンクスでは排便時には必ず大腸で強収縮（GMC）が見られることが明らかとなったので、本年度は、スンクス排便と摂食との関連を検討した。自由摂食下で排便は明期よりも活動期である暗期で多く観察され、特に摂食頻度が増加するZT 20-24で高頻度に排便していた。明暗周期は変更せずに給餌時間をZT 1-10に限定する（時限給餌）と、翌日から排便が暗期では減少、明期では増加した。特に、明期後半（ZT 13-18）で排便が多く観察された。さらに時限給餌を4日間継続した後、自由給餌に戻すと、排便はその翌日から暗期で増加し、明期で減少した。また、時限給餌によって排便時間が暗期から明期にシフトした要因を検討するために、大腸での時計遺伝子発現（bmal1, per1, cry1）を定量PCR法で検討した。その結果、自由給餌群でbmal1、per1、cry1はそれぞれZT 24,ZT 12、ZT 18でピークとなる発現リズムを示したが、時限給餌群ではそのリズムが消失した。以上の結果から、スンクスの排便行動は摂食による支配が大きいこと、及び体内時計が関与していることが示唆された。これらの結果は、時差ボケやナイトシフトワークによる大腸運動機能不全の理解に繋がると考えられる。
課題番号：20K06714
スンクスを用いた摂食調節ー消化管運動機能軸の比較内分泌学的解析
日本学術振興会, 科学研究費助成事業, 基盤研究(C), 2018年04月 - 2021年03月
坂田 一郎; 坂井 貴文, 埼玉大学
配分額（総額）：4550000, 配分額（直接経費）：3500000, 配分額（間接経費）：1050000
小型哺乳動物であるスンクスを用いて、消化管ホルモンによる消化管運動と摂食行動への影響とその作用経路を明らかにすることを目的に研究を行った。胃運動を刺激するモチリンアゴニストの慢性投与は摂食量と体重を増加させなかったが、グレリンの慢性投与は明期での摂食量をわずかに増加させた。一方、グレリンアンタゴニストの長期投与はスンクスの体重に影響を与えなかった。行動解析の結果、オープンフィールドテストではグレリン投与によって探索行動が増加したが、高架式十字迷路による抗不安作用が見られず、グレリンの行動に及ぼす影響はスンクスとげっ歯類では異なることが示された。
課題番号：18K06309
脳腸相関制御による大腸運動の解明ー新規大腸運動機能改善剤の開発に向けてー
日本学術振興会, 科学研究費助成事業, 基盤研究(C), 2017年04月 - 2020年03月
柴田 近; 小川 仁; 坂井 貴文; 坂田 一郎, 東北医科薬科大学
配分額（総額）：4680000, 配分額（直接経費）：3600000, 配分額（間接経費）：1080000
大腸運動異常は、QOLを低下させる疾患を引き起こすことから、大腸運動制御機構の解明が求められている。本研究は、食虫目スンクスを用いて、小型の埋め込み型strain gauge force transducerで胃及び大腸収縮運動と行動を同時に24時間連続観察できる実験系を確立した。その結果、排便の際には必ず巨大伝搬性収縮波が観察され、排便の前後に高確率で摂食や飲水が見られることを明らかにした。また、消化管ホルモンのモチリンは大腸運動を刺激しなかったが、セロトニンやノルアドレナリン受容体阻害剤であるヨヒンビンは巨大伝搬性収縮波及び排便を引き起こすことを明らかにした。
課題番号：17K10648
空腹期消化管運動に見られるウルトラディアンリズム機構の解明
日本学術振興会, 科学研究費助成事業, 基盤研究(B), 2016年04月 - 2020年03月
坂井 貴文; 坂田 一郎; MONDAL ANUPOM, 埼玉大学
配分額（総額）：14300000, 配分額（直接経費）：11000000, 配分額（間接経費）：3300000
本研究は、スンクスを用いて、空腹時に90分から120分間隔のウルトラディアンリズムで胃から小腸へと伝播する伝播性空腹期収縮（MMC）の駆動メカニズムを明らかにすることを目的とした。グレリンやモチリンに加えて、コレシストキニンは胃収縮運動を刺激することを明らかにした。一方、ソマトスタチンは胃収縮運動を抑制した。また、交感神経は空腹期胃強収縮を抑制し、モチリンの胃収縮刺激作用はGABA作動性神経を介して抑制されることを示した。さらに、小腸内のpHによって強収縮が制御されることを明らかした。本研究はウルトラディアンリズムを示すMMCの制御機構に新たな知見を与えた。
課題番号：16H04811
グレリンの脂肪酸修飾機構の研究-中鎖脂肪酸起源の同定-
日本学術振興会, 科学研究費助成事業, 挑戦的萌芽研究, 2015年04月 - 2017年03月
坂田 一郎; 坂井 貴文, 埼玉大学
配分額（総額）：3250000, 配分額（直接経費）：2500000, 配分額（間接経費）：750000
グレリンは28アミノ酸残基からなるペプチドホルモンである。3番目のセリン残基には中鎖脂肪酸が結合しているが、中鎖脂肪酸の由来については不明な点が多い。普通食と無脂肪食を与えたマウスの血漿アシルグレリン濃度と胃のアシルグレリン細胞数を比較した結果、普通食群と無脂肪食群の間に差は認められなかった。腸内細菌除去したマウスでは血中アシルグレリン量と胃のアシルグレリン細胞数は減少しなかった。グレリン産生細胞株（PG-1）にβ酸化阻害剤であるエトモキシルを処理すると、アシルグレリン産生が抑制された。以上の結果は、グレリン細胞によって長鎖脂肪酸を分解することで産生される中鎖脂肪酸を利用することが示された。
課題番号：15K14557
下垂体隆起部分泌因子の脳内への作用経路の解明
日本学術振興会, 科学研究費助成事業, 挑戦的萌芽研究, 2014年04月 - 2016年03月
坂井 貴文; 坂田 一郎, 埼玉大学
配分額（総額）：3120000, 配分額（直接経費）：2400000, 配分額（間接経費）：720000
大槽内へのhorseradish peroxidase (HRP)投与により、HRPの脳実質内への浸透度が、時間経過に従い有意に上昇した。HRPシグナルは隆起部周辺においてLobule 構造を取り囲むように見られ、第三脳室上衣細胞層及び松果体周辺でも認められた。一方、頸静脈内へのHRP投与では脳実質内でHRPのシグナルが検出されなかった。タンパク質質量分析では、3 kDa~16 kDaの範囲において、隆起部ペプチド抽出液で観測された因子が脳脊髄液にも存在することを確認した。
課題番号：26650109
中枢及び末梢制御によるグレリン分泌調節の解明
日本学術振興会, 科学研究費助成事業, 若手研究(B), 2012年04月 - 2015年03月
坂田 一郎, 埼玉大学
配分額（総額）：4290000, 配分額（直接経費）：3300000, 配分額（間接経費）：990000
本研究では、グレリンの分泌制御機構について検討を行った。グレリン産生細胞に長鎖脂肪酸の受容体であるGPR120が発現していることを見出した。グレリン産生細胞株及びマウス胃粘膜初代培養細胞を用いてGPR120のアゴニスト処理によってグレリン分泌が抑制されることを明らかにした。また、GPR120 アゴニストは extracellular signal-regulated kinase (ERK) の リン酸化を介してグレリン分泌を抑制することを示した。さらに、グレリン分泌を制御する脳内神経核の同定を目的に、交感神経起始核の電気破壊実験を行い、視床下部傍室核がグレリン分泌を制御する可能性を示唆した。
課題番号：24790941
グレリン分泌を制御する神経ネットワークの解明
日本学術振興会, 科学研究費助成事業, 研究活動スタート支援, 2011年08月 - 2013年03月
坂田 一郎, 埼玉大学
配分額（総額）：3250000, 配分額（直接経費）：2500000, 配分額（間接経費）：750000
グレリンは胃で産生されるホルモンで、成長ホルモン分泌刺激、摂食亢進、血糖値調節そして消化管運動刺激に関与するペプチドホルモンである。血中グレリン濃度は絶食やストレスなどで増加することが知られているが、その制御機構は不明である。申請者は、グレリン放出がノルアドレナリンの刺激により調節されていることをこれまでに明らかにしている。そこで、本研究はグレリン分泌に関与する神経ネットワーク機構を明らかにすることを目的としている。本年度はまず、交感神経系の関与を検証するために、視床下部神経核の破壊実験系の確立を行った。現在までに、視床下部神経核(背内側核、外側野など)を電気刺激で破壊する系を確立している。現在、これらの個体を用いて絶食誘導性グレリン分泌を検討中である。
さらに、絶食時におけるグレリン分泌作用機序を明らかにするために、マウス胃粘膜細胞培養系を用いてグレリン分泌における培地中のグルコース濃度の影響について検討を行った。胃粘膜単離細胞を1mM、5mMもしくは10mMのグルコースを含むDMEM培地で培養したところ、グルコース濃度の増加に伴いグレリン分泌が有意に抑制された。さらに、2DGを培養液中に添加するとグルコース誘導性グレリン分泌の抑制がブロックされた。また、定量的PCR法を用いた解析により、グレリン細胞にはグルコーストランスポーター(GLUT4及びGLUT5)、Glucokinase、hexokinases、Kir6.2そしてSURIが高発現していることを明らかにした。
課題番号：23890028
モチリンによる空腹期収縮機構の解明-新たなモデル動物を用いたアプローチ-
日本学術振興会, 科学研究費助成事業, 基盤研究(C), 2009年 - 2011年
坂井 貴文; 足立 明人; 坂田 一郎, 埼玉大学
配分額（総額）：4680000, 配分額（直接経費）：3600000, 配分額（間接経費）：1080000
スンクスの空腹期消化管運動はヒトやイヌと同様に80-150分周期で強収縮が起こり、また、外因性モチリンの投与はphaseIII様収縮を刺激した。また、モチリンアンタゴニストの投与は、phaseIII収縮の出現を阻害した。迷走神経切除はMMCの出現に影響を与えなかったが、phaseIIの長さと運動性は有意に減少させた。迷走神経切除群では摂食後に食後期収縮が起こらず、モチリン投与によって自発性phaseIIIと同様の強収縮が惹起された。(214字)
課題番号：21590785
性ホルモンによる胃グレリン発現動態の検討
日本学術振興会, 科学研究費助成事業, 特別研究員奨励費, 2005年 - 2006年
坂田 一郎, 埼玉大学
配分額（総額）：1900000, 配分額（直接経費）：1900000
17年度に、胃で産生されたエストロゲンが直接胃グレリン発現に関与し、その主要な起源は血液中に存在するエストロゲンではなく、胃で産生されたエストロゲンが直接胃グレリン発現に関係していることを明らかにした。この研究成果は、Journal of Endocrinologyに発表しな。18年度は、さらに胃グレリン発現調節機構を解明するために引き続き研究を行った。グレリン発現、分泌はレプチン、ソマトスタチンそしてインスリンなどにより抑制され、絶食により胃グレリンmRNA発現が増加することが知られている。また、エストロゲン、レプチン、ソマトスタチンは、胃で産生されることが報告されている。そこで、まず絶食時における胃のグレリン発現調節機構のメカニズムを明らかにするために、絶食ラット胃におけるエストロゲン合成酵素、レプチンそしてソマトスタチン発現を定量PCR法を用いて検討した。その結果、エストロゲン合成酵素及びソマトスタチンmRNAレベルは絶食において変化しなかったが、レプチンmRNAレベルは絶食により有意に減少した。また、胃におけるレプチン産生細胞とグレリン産生細胞の局在を2重免疫組織化学により検討したところ、レプチン産生細胞とグレリン産生細胞は近接して存在していた。以上の結果は、絶食時に胃で産生されたレプチンの減少が胃グレリン発現を増加させることを強く示唆するものである。本研究で得られた胃で産生されたレプチンがグレリン発現に関与するという結果は、現在外国雑誌に投稿準備中である。
課題番号：05J03100
