研究者情報

マスター

アカウント（マスター）

• 氏名

  竹見　祥大(タケミ　シヨウタ), タケミ　シヨウタ

所属（マスター）

• 先端生命科学研究院　生命機能科学研究部門　発生工学分野

所属（マスター）

• 先端生命科学研究院　生命機能科学研究部門　発生工学分野

researchmap

プロフィール情報

所属

• 北海道大学, 大学院先端生命科学研究院, 特任助教

学位

• 理学博士（2019年03月 埼玉大学）

プロフィール情報

• 姓

  竹見, タケミ

• 名

  祥大, ショウタ

• ID各種

  202001011803505061

所属

• 北海道大学, 大学院先端生命科学研究院, 特任助教

業績リスト

研究キーワード

• 小腸Paneth細胞、抗菌ペプチド、粘膜免疫   

研究分野

• ライフサイエンス / 生理学

経歴

• 2023年07月 - 現在 北海道大学 大学院先端生命科学研究院 特任助教
• 2020年01月 - 2023年06月 埼玉大学 大学院理工学研究科 助教
• 2019年05月 - 2019年12月 University of Texas Southwestern Medical Center Hypothalamic Research Center Postdoctoral researcher
• 2019年04月 - 2019年05月 独立行政法人日本学術振興会 特別研究員PD
• 2018年04月 - 2019年03月 独立行政法人日本学術振興会 特別研究員DC2

受賞

• 2019年08月 日本平滑筋学会 学術交流特別賞
  

論文

• Effect of cholecystokinin on small intestinal motility in suncus murinus

  Naho Yokota, Shota Takemi, Ichiro Sakata

  General and Comparative Endocrinology 342 114352 - 114352 2023年10月
• 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 331 114167 - 114167 2023年01月15日 

  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.
• Low Surface Potential with Glycoconjugates Determines Insect Cell Adhesion at Room Temperature.

  Takahisa Matsuzaki, Daigo Terutsuki, Shoma Sato, Kohei Ikarashi, Kohei Sato, Hidefumi Mitsuno, Ryu Okumura, Yudai Yoshimura, Shigeyoshi Usami, Yusuke Mori, Mai Fujii, Shota Takemi, Seiichiro Nakabayashi, Hiroshi Y Yoshikawa, Ryohei Kanzaki

  The journal of physical chemistry letters 13 40 9494 - 9500 2022年10月13日 

  Cell-coupled field-effect transistor (FET) biosensors have attracted considerable attention because of their high sensitivity to biomolecules. The use of insect cells (Sf21) as a core sensor element is advantageous due to their stable adhesion to sensors at room temperature. Although visualization of the insect cell-substrate interface leads to logical amplification of signals, the spatiotemporal processes at the interfaces have not yet been elucidated. We quantitatively monitored the adhesion dynamics of Sf21 using interference reflection microscopy (IRM). Specific adhesion signatures with ring-like patches along the cellular periphery were detected. A combination of zeta potential measurements and lectin staining identified specific glycoconjugates with low electrostatic potentials. The ring-like structures were disrupted after cholesterol depletion, suggesting a raft domain along the cell periphery. Our results indicate dynamic and asymmetric cell adhesion is due to low electrostatic repulsion with fluidic sugar rafts. We envision the logical design of cell-sensor interfaces with an electrical model that accounts for actual adhesion interfaces.
• Identification of motilin in Japanese fire bellied newt.

  Mio Matsumoto, Shota Takemi, Takafumi Sakai, Ichiro Sakata

  General and comparative endocrinology 323-324 114031 - 114031 2022年07月01日 

  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.
• 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 327 114074 - 114074 2022年06月11日 

  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.
• 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 13 858522 - 858522 2022年 

  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.
• 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 90 102187 - 102187 2021年12月 

  Ghrelin is a multifunctional gut peptide with a unique structure, which is modified by a medium chain fatty acid at the third serine by ghrelin O-acyl transferase (GOAT). It is well known that the major source of plasma ghrelin is the stomach, but the transcriptional regulation of gastric ghrelin and GOAT is incompletely understood. Here, we studied the involvement of the nuclear receptors REV-ERBα and REV-ERBβ on ghrelin and GOAT gene expression in vivo and in vitro. Reverse-transcriptase polymerase chain reaction analysis showed that REV-ERBα and REV-ERBβ mRNAs were expressed in the stomach and a stomach-derived ghrelin cell line (SG-1 cells). In vivo experiments with mice revealed the circadian rhythm of ghrelin, GOAT, and REV-ERBs. The peak expression of ghrelin and GOAT mRNAs occurred at Zeitgeber time (ZT) 4, whereas that of REV-ERBα and REV-ERBβ was observed at ZT8 and ZT12, respectively. Treatment of SG-1 cells with SR9009, a REV-ERB agonist, led to a significant reduction in ghrelin and GOAT mRNA levels. Overexpression of REV-ERBα and REV-ERBβ decreased ghrelin and GOAT mRNA levels in SG-1 cells. In contrast, small-interfering RNA (siRNA)-mediated double-knockdown of REV-ERBα and REV-ERBβ in SG-1 cells led to the upregulation in the expression of ghrelin and GOAT mRNAs. These results suggest that REV-ERBs suppress ghrelin and GOAT mRNA expression.
• 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 25 2021年12月 

  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.
• 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 

  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.
• LEAP2 deletion in mice enhances ghrelin's actions as an orexigen and growth hormone secretagogue

  Kripa Shankar, Nathan P. Metzger, Omprakash Singh, Bharath K. Mani, Sherri Osborne-Lawrence, Salil Varshney, Deepali Gupta, Sean B. Ogden, Shota Takemi, Corine P. Richard, Karabi Nandy, Chen Liu, Jeffrey M. Zigman

  MOLECULAR METABOLISM 53 2021年11月 

  Objective: The hormone liver-expressed antimicrobial peptide-2 (LEAP2) is a recently identified antagonist and an inverse agonist of the growth hormone secretagogue receptor (GHSR). GHSR's other well-known endogenous ligand, acyl-ghrelin, increases food intake, body weight, and GH secretion and is lowered in obesity but elevated upon fasting. In contrast, LEAP2 reduces acyl-ghrelin-induced food intake and GH secretion and is found elevated in obesity but lowered upon fasting. Thus, the plasma LEAP2/acyl-ghrelin molar ratio could be a key determinant modulating GHSR signaling in response to changes in body mass and feeding status. In particular, LEAP2 may serve to dampen acyl-ghrelin action in the setting of obesity, which is associated with ghrelin resistance. Here, we sought to determine the metabolic effects of genetic LEAP2 deletion. Methods: We generated the first known LEAP2-KO mouse line. Food intake, GH secretion, and cellular activation (c-fos induction) in different brain regions following s.c. acyl-ghrelin administration in LEAP2-KO mice and wild-type littermates were determined. LEAP2-KO mice and wild-type littermates were submitted to a battery of tests (such as measurements of body weight, food intake, and body composition; indirect calorimetry, determination of locomotor activity, and meal patterning while housed in metabolic cages) over the course of 16 weeks of high-fat diet and/or standard chow feeding. Fat accumulation was assessed in hematoxylin & eosin-stained and oil red O-stained liver sections from these mice. Results: LEAP2-KO mice were more sensitive to s.c. ghrelin. In particular, acyl-ghrelin acutely stimulated food intake at a dose of 0.5 mg/kg BW in standard chow-fed LEAP2-KO mice while a 2x higher dose was required by wild-type littermates. Also, acyl-ghrelin stimulated food intake at a dose of 1 mg/kg BW in high-fat diet-fed LEAP2-KO mice while not even a 10x higher dose was effective in wild-type littermates. Acyl-ghrelin induced a 90.9% higher plasma GH level and 77.2-119.7% higher numbers of c-fos-immunoreactive cells in the arcuate nucleus and olfactory bulb, respectively, in LEAP2-KO mice than in wild-type littermates. LEAP2 deletion raised body weight (by 15.0%), food intake (by 18.4%), lean mass (by 6.1%), hepatic fat (by 42.1%), and body length (by 1.7%) in females on long-term high-fat diet as compared to wild-type littermates. After only 4 weeks on the high-fat diet, female LEAP2-KO mice exhibited lower O2 consumption (by 13%), heat production (by 9.5%), and locomotor activity (by 49%) than by wild-type littermates during the first part of the dark period. These genotype-dependent differences were not observed in high-fat diet-exposed males or female and male mice exposed for long term to standard chow diet. Conclusions: LEAP2 deletion sensitizes lean and obese mice to the acute effects of administered acyl-ghrelin on food intake and GH secretion. LEAP2 deletion increases body weight in females chronically fed a high-fat diet as a result of lowered energy expenditure, reduced locomotor activity, and increased food intake. Furthermore, in female mice, LEAP2 deletion increases body length and exaggerates the hepatic fat accumulation normally associated with chronic high-fat diet feeding. (c) 2021 The Author(s). Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
• Ghrelin cell-expressed insulin receptors mediate meal- and obesity-induced declines in plasma ghrelin

  Kripa Shankar, Shota Takemi, Deepali Gupta, Salil Varshney, Bharath K. Mani, Sherri Osborne-Lawrence, Nathan P. Metzger, Corine P. Richard, Eric D. Berglund, Jeffrey M. Zigman

  JCI INSIGHT 6 18 2021年09月 

  Mechanisms underlying postprandial and obesity-associated plasma ghrelin reductions are incompletely understood. Here, using ghrelin cell-selective insulin receptor-KO (GhIRKO) mice, we tested the impact of insulin, acting via ghrelin cell-expressed insulin receptors (IRs), to suppress ghrelin secretion. Insulin reduced ghrelin secretion from cultured gastric mucosal cells of control mice but not from those of GhIRKO mice. Acute insulin challenge and insulin infusion during both hyperinsulinemic-hypoglycemic clamps and hyperinsulinemic-euglycemic clamps lowered plasma ghrelin in control mice but not GhIRKO mice. Thus, ghrelin cell-expressed IRs are required for insulin-mediated reductions in plasma ghrelin. Furthermore, interventions that naturally raise insulin (glucose gavage, refeeding following fasting, and chronic high-fat diet) also lowered plasma ghrelin only in control mice - not GhIRKO mice. Thus, meal-and obesity-associated increases in insulin, acting via ghrelin cell-expressed IRs, represent a major, direct negative modulator of ghrelin secretion in vivo, as opposed to ingested or metabolized macronutrients. Refed GhIRKO mice exhibited reduced plasma insulin, highlighting ghrelin's actions to inhibit insulin release via a feedback loop. Moreover, GhIRKO mice required reduced glucose infusion rates during hyperinsulinemic-hypoglycemic clamps, suggesting that suppressed ghrelin release resulting from direct insulin action on ghrelin cells usually limits ghrelin's full potential to protect against insulin induced hypoglycemia.
• Pyridoxine stimulates filaggrin production in human epidermal keratinocytes.

  Miyuki Fujishiro, Shoichi Yahagi, Shota Takemi, Mio Nakahara, Takafumi Sakai, Ichiro Sakata

  Molecular biology reports 48 7 5513 - 5518 2021年07月 

  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.
• Ghrelin-cell physiology and role in the gastrointestinal tract.

  Ichiro Sakata, Shota Takemi

  Current opinion in endocrinology, diabetes, and obesity 28 2 238 - 242 2021年04月01日 

  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.
• 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 33 3 e12961  2021年03月 

  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.
• [The basics of the study of gastrointestinal motility].

  Shota Takemi, Ichiro Sakata, Takafumi Sakai

  Nihon Shokakibyo Gakkai zasshi = The Japanese journal of gastro-enterology 118 2 107 - 113 2021年
• 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 285 113294 - 113294 2020年01月01日 

  Motilin and ghrelin were identified in the pheasant by molecular cloning, and the actions of both peptides on the contractility of gastrointestinal (GI) strips were examined in vitro. Molecular cloning indicated that the deduced amino acid sequences of the pheasant motilin and ghrelin were a 22-amino acid peptide, FVPFFTQSDIQKMQEKERIKGQ, and a 26-amino acid peptide, GSSFLSPAYKNIQQQKDTRKPTGRLH, respectively. In in vitro studies using pheasant GI strips, chicken motilin caused contraction of the proventriculus and small intestine, whereas the crop and colon were insensitive. Human motilin, but not erythromycin, caused contraction of small intestine. Chicken motilin-induced contractions in the proventriculus and ileum were not inhibited by a mammalian motilin receptor antagonist, GM109. Neither atropine (a cholinergic receptor antagonist) nor tetrodotoxin (a neuron blocker) inhibited the responses of chicken motilin in the ileum but both drugs decreased the responses to motilin in the proventriculus, suggesting that the contractile mechanisms of motilin in the proventriculus was neurogenic, different from that of the small intestine (myogenic). On the other hand, chicken and quail ghrelin did not cause contraction in any regions of pheasant GI tract. Since interaction of ghrelin and motilin has been reported in the house musk shrew, interaction of two peptides was examined. The chicken motilin-induced contractions were not modified by ghrelin, and ghrelin also did not cause any contraction under the presence of motilin, suggesting the absence of interaction in both peptides. In conclusion, both the motilin system and ghrelin system are present in the pheasant. Regulation of GI motility by motilin might be common in avian species. However, absence of ghrelin actions in any GI regions suggests the avian species-related difference in regulation of GI contractility by ghrelin.
• 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 56 0 69 - 81 2020年 

  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.
• 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 100 103427 - 103427 2019年11月 

  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.
• 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 97 10 909 - 915 2019年10月 

  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.
• 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10日 

  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.
• Utility of animal gastrointestinal motility and transit models in functional gastrointestinal disorders

  Ahmad Al-Saffar, Shota Takemi, Hiwa K. Saaed, Ichiro Sakata, Takafumi Sakai

  BEST PRACTICE & RESEARCH CLINICAL GASTROENTEROLOGY 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.
• 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 376 3 401 - 412 2019年06月 

  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.
• 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 8 1 9176 - 9176 2018年06月15日 

  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.
• 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 (Oxford, England) 222 2 2018年02月 

  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.
• 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 54 0 91 - 99 2018年 

  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.
• 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 5 1 2017年01月 

  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.
• 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 64 Suppl. S11-S14  2017年 

  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 γ-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.
• 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 64 Suppl. S27-S29  2017年 

  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 β1-adrenergic receptor, and fasting-induced ghrelin elevation was completely inhibited by the β1-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.
• 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 33 5 497 - 504 2016年10月 

  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.
• 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 233 53 - 62 2016年07月01日 

  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.
• 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 156 12 4437 - 47 2015年12月 

  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.

書籍等出版物

• 動物/疾患モデルの作製技術・病態解析・評価手法

  技術情報協会 
  技術情報協会 2017年08月 (ISBN: 9784861046650) 428p

講演・口頭発表等

• Study of gastrointestinal motility using Suncus murinus  [招待講演]

  Takemi S, Sakata I

  International Workshop on Emergence of Life-Nano-Bio Science 2021年03月 シンポジウム・ワークショップパネル（指名）
• 食虫目スンクス（Suncus murinus）を用いた消化管運動研究の展開  [招待講演]

  竹見祥大, 坂田一郎, 坂井貴文

  第61回日本平滑筋学会総会 2019年08月 口頭発表（招待・特別）

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• 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)

  研究期間 : 2020年04月 -2023年03月 

  代表者 : 坂井 貴文, 坂田 一郎, 竹見 祥大

   

  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。
• インスリンによる抗菌ペプチド産生・分泌を介した新たな腸内細菌制御機構の解明

  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

  研究期間 : 2020年09月 -2022年03月 

  代表者 : 竹見 祥大

   

  本研究は、小腸の陰窩に位置するPaneth細胞が分泌する抗菌ペプチドおよびタンパク質の新たな産生・分泌制御機構を探索することを目的とした。これまでの研究代表者の実験から、インスリンがそれに関与するとの仮説をたて研究を行った。その結果、マウスへのインスリンの投与によって、Lysozyme（Paneth細胞から分泌される抗菌タンパク質）の染色性が減少した。加えて、マウス小腸から作製したオルガノイドへインスリンを処理したところ、Paneth細胞からの小胞の分泌が見られた。
• 若齢期の栄養失調が及ぼす腸内免疫への影響の検討

  三島海運記念財団：学術研究奨励金

  研究期間 : 2020年06月 -2021年06月
• スンクス空腹期モチリン分泌機構の解明‐約１００分周期はどのように制御されるのか‐

  日本学術振興会：科学研究費助成事業 特別研究員奨励費

  研究期間 : 2018年04月 -2020年03月 

  代表者 : 竹見 祥大

   

  本研究は、空腹時に胃から下部小腸へと約100分周期で伝播する伝播性空腹期収縮（Migrating Motor Contraction; MMC）がどのように制御されるか明らかにすることを目的としており、そのために平成31年度は昨年度に引き続き、MMCの強収縮を引き起こすモチリンの転写・分泌制御機構についてスンクス小腸オルガノイドを用いて検討した。これまで、モチリンはげっ歯類では偽遺伝子化していることから研究が進んでおらず、本研究ではモチリンを産生する小型実験動物であるスンクスを用いている。これまで、スンクスで初めて小腸からオルガノイドを作製することに成功し、また安定して長期間培養することにも成功した。 今年度は、モチリン産生株化細胞をスンクス小腸オルガノイドから作出するために、IRES-T antigen遺伝子をモチリン遺伝子下流にノックインすることを目的とした。IRES-T antigenを含むベクターコンストラクトを作製し、Cas9、guideRNAとともにスンクス小腸オルガノイドへトランスフェクションした。トランスフェクションしたベクター由来の蛍光タンパク質は確認できているが、未だモチリン遺伝子へのノックインは成功していない。 それに加えて、蛍光レポーターであるtdTomato遺伝子をモチリン遺伝子下流にノックインするため、tdTomato遺伝子及びネオマイシン耐性遺伝子を含むベクターを作製し、guideRNA、Cas9とともにスンクス小腸オルガノイドへトランスフェクションすることも試みた。ベクターの作製は終了したが、トランスフェクションは成功していない。
• 重篤な低血糖下でのグレリンによる血糖調節機構の 解明

  上原記念生命科学財団：海外留学助成金ポストドクトラルフェローシップ

  研究期間 : 2019年05月 -2019年12月
• 小型実験動物ｽﾝｸｽを用いた精細胞を介した新たな遺伝子改変個体の作製

  公益財団法人日本科学協会：笹川科学研究助成

  研究期間 : 2017年04月 -2018年03月