Publications by authors named "Kanako Iwasaki"

24 Publications

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Sensitive and selective methanol biosensor using two-enzyme cascade reaction and fluorometry for non-invasive assessment of intestinal bacteria activity.

Biosens Bioelectron 2021 Jun 4;181:113136. Epub 2021 Mar 4.

Department of Biomedical Devices and Instrumentation, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo, 101-0062, Japan; Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan. Electronic address:

For understanding the status of intestinal flora non-invasively, methanol (MeOH) has been attracting the attention. In this study, we have developed and compared two different liquid-phase methanol biosensors. One, referred to as the AOD electrosensor, utilized alcohol oxidase (AOD) and an oxygen electrode. It electrochemically measured the decrease in oxygen through AOD-catalyzed oxidation of MeOH. The other, referred to as the AOD-FALDH fluorosensor, exploited a cascade reaction of AOD and formaldehyde dehydrogenase (FALDH) in conjunction with a fiber-optic sensor. It measured increase in the fluorescence from reduced form of β-nicotinamide adenine dinucleotide (NADH) that was a final product of the two-enzyme cascade reaction. Due to the cascade reaction, the AOD-FALDH fluorosensor showed 3 times better sensitivity along with 335 times wider dynamic range (494 nM-100 mM) than those of the AOD electrosensor (1.5-300 μM). The selectivity to MeOH was also improved by the cascade reaction with AOD-FALDH as no sensor output was observed from other aliphatic alcohols than MeOH in contrast to the AOD electrosensor. Although the use of FALDH resulted in the increase in the sensor output from aldehydes, such as acetaldehyde and formaldehyde, considering their concentrations in body fluids, the influence on the sensor output is limited. These results indicate that incorporating the cascade reaction into fluorometry enables enhanced biosensing of MeOH that will be useful for assessment of intestinal flora with little burden.
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http://dx.doi.org/10.1016/j.bios.2021.113136DOI Listing
June 2021

Induction of Mutant Allele in Neurons in Late Infancy Increases Sleep Need.

J Neurosci 2021 Mar 8;41(12):2733-2746. Epub 2021 Feb 8.

International Institute for Integrative Sleep Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan

Sleep is regulated in a homeostatic manner. Sleep deprivation increases sleep need, which is compensated mainly by increased EEG δ power during non-rapid eye movement sleep (NREMS) and, to a lesser extent, by increased sleep amount. Although genetic factors determine the constitutive level of sleep need and sleep amount in mice and humans, the molecular entity behind sleep need remains unknown. Recently, we found that a gain-of-function () mutation in the () gene, which produces the mutant SIK3(SLP) protein, leads to an increase in NREMS EEG δ power and sleep amount. Since mice express SIK3(SLP) in various types of cells in the brain as well as multiple peripheral tissues from the embryonic stage, the cell type and developmental stage responsible for the sleep phenotype in mice remain to be elucidated. Here, we generated two mouse lines, and mice, which enable inducible Cre-mediated, conditional expression of SIK3(SLP) in neurons on tamoxifen administration. Administration of tamoxifen to mice during late infancy resulted in higher recombination efficiency than administration during adolescence. SIK3(SLP) expression after late infancy increased NREMS and NREMS δ power in male mice. The expression of SIK3(SLP) after adolescence led to a higher NREMS δ power without a significant change in NREMS amounts. Thus, neuron-specific expression of SIK3(SLP) after late infancy is sufficient to increase sleep. The propensity to accumulate sleep need during wakefulness and to dissipate it during sleep underlies the homeostatic regulation of sleep. However, little is known about the developmental stage and cell types involved in determining the homeostatic regulation of sleep. Here, we show that allele induction in mature neurons in late infancy is sufficient to increase non-rapid eye movement sleep amount and non-rapid eye movement sleep δ power. SIK3 signaling in neurons constitutes an intracellular mechanism to increase sleep.
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http://dx.doi.org/10.1523/JNEUROSCI.1004-20.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8018738PMC
March 2021

Carbonic anhydrase 8 (CAR8) negatively regulates GLP-1 secretion from enteroendocrine cells in response to long-chain fatty acids.

Am J Physiol Gastrointest Liver Physiol 2021 04 3;320(4):G617-G626. Epub 2021 Feb 3.

Department of Diabetes, Endocrinology, and Nutrition, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

Glucagon-like peptide-1 (GLP-1) is an incretin secreted from enteroendocrine preproglucagon (PPG)-expressing cells (traditionally known as L cells) in response to luminal nutrients that potentiates insulin secretion. Augmentation of endogenous GLP-1 secretion might well represent a novel therapeutic target for diabetes treatment in addition to the incretin-associated drugs currently in use. In this study, we found that PPG cells substantially express carbonic anhydrase 8 (CAR8), which has been reported to inhibit inositol 1,4,5-trisphosphate (IP3) binding to the IP3 receptor and subsequent Ca efflux from the endoplasmic reticulum in neuronal cells. In vitro experiments using STC-1 cells demonstrated that knockdown increases long-chain fatty acid (LCFA)-stimulated GLP-1 secretion. This effect was reduced in the presence of phospholipase C (PLC) inhibitor; in addition, knockdown increased the intracellular Ca elevation caused by α-linolenic acid, indicating that CAR8 exerts its effect on GLP-1 secretion via the PLC/IP3/Ca pathway. null mutant mice showed significant increase in GLP-1 response to oral corn oil administration compared with that in wild-type littermates, with no significant change in intestinal GLP-1 content. These results demonstrate that CAR8 negatively regulates GLP-1 secretion from PPG cells in response to LCFAs, suggesting the possibility of augmentation of postprandial GLP-1 secretion by CAR8 inhibition. This study focused on the physiological significance of carbonic anhydrase 8 (CAR8) in GLP-1 secretion from enteroendocrine preproglucagon (PPG)-expressing cells. We found an inhibitory role of CAR8 in LCFA-induced GLP-1 secretion in vitro and in vivo, suggesting a novel therapeutic approach to diabetes and obesity through augmentation of postprandial GLP-1 secretion by CAR8 inhibition.
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http://dx.doi.org/10.1152/ajpgi.00312.2020DOI Listing
April 2021

Generation of a p16 Reporter Mouse and Its Use to Characterize and Target p16 Cells In Vivo.

Cell Metab 2020 11 18;32(5):814-828.e6. Epub 2020 Sep 18.

Division of Molecular Regulation of Inflammatory and Immune Diseases, Research Institute of Biomedical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-0022, Japan.

Cell senescence plays a key role in age-associated organ dysfunction, but the in vivo pathogenesis is largely unclear. Here, we generated a p16-Cre-tdTomato mouse model to analyze the in vivo characteristics of p16 cells at a single-cell level. We found tdTomato-positive p16 cells detectable in all organs, which were enriched with age. We also found that these cells failed to proliferate and had half-lives ranging from 2.6 to 4.2 months, depending on the tissue examined. Single-cell transcriptomics in the liver and kidneys revealed that p16 cells were present in various cell types, though most dominant in hepatic endothelium and in renal proximal and distal tubule epithelia, and that these cells exhibited heterogeneous senescence-associated phenotypes. Further, elimination of p16 cells ameliorated nonalcoholic steatohepatitis-related hepatic lipidosis and immune cell infiltration. Our new mouse model and single-cell analysis provide a powerful resource to enable the discovery of previously unidentified senescence functions in vivo.
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http://dx.doi.org/10.1016/j.cmet.2020.09.006DOI Listing
November 2020

-Mediated Gene Deletion and Loss of Heterozygosity in a Patient with Multiple Endocrine Neoplasia Type 1.

J Endocr Soc 2020 Aug 9;4(8):bvaa051. Epub 2020 May 9.

Department of Diabetes and Endocrinology, Tazuke Kofukai Medical Research Institute, Kitano Hospital, Osaka, Japan.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder caused by mutations of the tumor suppressor gene . Most of the germline gene mutations have been small mutations, and the whole gene deletion is rarely observed. In the present study, we revealed retrotransposon-mediated germline deletion of the whole gene and somatic copy-neutral loss of heterozygosity (LOH) in a patient with MEN1. The patient is a 39-year-old woman who was referred to our department for the management of prolactinoma. She was also diagnosed with primary hyperparathyroidism and suspected of MEN1. Although nucleotide sequencing did not detect any gene mutations, multiplex ligation-dependent probe amplification (MLPA) revealed a large germline deletion of the gene. Subsequent quantitative polymerase chain reaction (qPCR)-based copy number mapping showed a monoallelic loss of approximately 18.5-kilobase region containing the whole gene. Intriguingly, the 2 breakpoints were flanked by repetitive elements, suggesting the contribution of /-mediated rearrangements (AAMR) to the whole gene deletion. Furthermore, copy number mapping using MLPA and qPCR in combination with single nucleotide polymorphism analysis revealed copy-neutral LOH as a somatic event for parathyroid tumorigenesis. In conclusion, copy number mapping revealed a novel combination of /-mediated germline deletion of the gene and somatic copy-neutral LOH as a cytogenetic basis for the MEN1 pathogenesis. Moreover, subsequent in silico analysis highlighted the possible predisposition of the gene to retrotransposon-mediated genomic deletion.
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http://dx.doi.org/10.1210/jendso/bvaa051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7371388PMC
August 2020

Vasodilator-stimulated phosphoprotein (VASP) is recruited into dendritic spines via G-actin-dependent mechanism and contributes to spine enlargement and stabilization.

Eur J Neurosci 2020 02 18;51(3):806-821. Epub 2019 Dec 18.

Department of Cellular Neurobiology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

Actin organization and dynamics are modulated by diverse actin regulators during dendritic spine development. To understand the molecular network that regulates actin organization and spine morphology, it is important to investigate dynamic redistribution of actin regulators during spine development. One of the actin regulators, vasodilator-stimulated phosphoprotein (VASP), has multiple functions in actin regulation and is known to regulate spine morphology. However, dynamics and temporal regulation of VASP during spine development have not been clarified. In this study, we performed time-lapse imaging of mouse hippocampal dissociated neurons to analyse the change in localization of VASP during spine development. We found that accumulation of VASP within spines precedes the start of persistent F-actin increase, which are temporally coupled with spine enlargement. Using domain deletion or mutation constructs of VASP, we revealed that the interaction with G-actin is important for the preceding accumulation of VASP. Furthermore, we showed that accumulation of VASP contributes to actin enrichment within spines and stabilization of spine morphology by dominant negative experiments. These data suggest that G-actin-dependent VASP recruitment has dual functions in spine development, enlargement and stabilization, through the interaction with actin and other cytoskeletal regulators.
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http://dx.doi.org/10.1111/ejn.14634DOI Listing
February 2020

Facilitating screening of Klinefelter syndrome among patients with diabetes.

J Diabetes Investig 2020 Mar 7;11(2):506-507. Epub 2019 Aug 7.

Center for Diabetes and Endocrinology, Tazuke Kofukai Medical Research Institute Kitano Hospital, Osaka, Japan.

Klinefelter syndrome (KS) is frequently complicated by diabetes. However, it is severely underdiagnosed due to a lack of reliable screening methods. We diagnosed two patients with KS at the Center for Diabetes and Endocrinology, Tazuke Kofukai Medical Research Institute Kitano Hospital, Osaka, Japan. By comparing the patients with 39 non-KS patients with diabetes, we propose a screening tool for KS in patients with diabetes.
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http://dx.doi.org/10.1111/jdi.13113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7078131PMC
March 2020

Free fatty acid receptors, G protein-coupled receptor 120 and G protein-coupled receptor 40, are essential for oil-induced gastric inhibitory polypeptide secretion.

J Diabetes Investig 2019 Nov 7;10(6):1430-1437. Epub 2019 May 7.

Department of Diabetes, Endocrinology and Nutrition, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

Aims/introduction: Incretin hormone glucose-dependent insulinotropic polypeptide/gastric inhibitory polypeptide (GIP) plays a key role in high-fat diet-induced obesity and insulin resistance. GIP is strongly secreted from enteroendocrine K cells by oil ingestion. G protein-coupled receptor (GPR)120 and GPR40 are two major receptors for long chain fatty acids, and are expressed in enteroendocrine K cells. In the present study, we investigated the effect of the two receptors on oil-induced GIP secretion using GPR120- and GPR40-double knockout (DKO) mice.

Materials And Methods: Global knockout mice of GPR120 and GPR40 were crossbred to generate DKO mice. Oral glucose tolerance test and oral corn oil tolerance test were carried out. For analysis of the number of K cells and gene expression in K cells, DKO mice were crossbred with GIP-green fluorescent protein knock-in mice in which visualization and isolation of K cells can be achieved.

Results: Double knockout mice showed normal glucose-induced GIP secretion, but no GIP secretion by oil. We then investigated the number of K cells and gene characteristics in K cells isolated from GIP-green fluorescent protein knock-in mice. Deficiency of both receptors did not affect the number of K cells in the small intestine or expression of GIP messenger ribonucleic acid in K cells. Furthermore, there was no significant difference in the expression of the genes associated with lipid absorption or GIP secretion in K cells between wild-type and DKO mice.

Conclusions: Oil-induced GIP secretion is triggered by the two major fatty acid receptors, GPR120 and GPR40, without changing K-cell number or K-cell characteristics.
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http://dx.doi.org/10.1111/jdi.13059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6825923PMC
November 2019

Medium-chain triglyceride diet stimulates less GIP secretion and suppresses body weight and fat mass gain compared with long-chain triglyceride diet.

Am J Physiol Endocrinol Metab 2019 07 16;317(1):E53-E64. Epub 2019 Apr 16.

Department of Diabetes, Endocrinology, and Nutrition, Graduate School of Medicine, Kyoto University , Kyoto , Japan.

Gastric inhibitory polypeptide (GIP) is an incretin secreted from enteroendocrine K cells and potentiates insulin secretion from pancreatic β-cells. GIP also enhances long-chain triglyceride (LCT) diet-induced obesity and insulin resistance. Long-term intake of medium-chain triglyceride (MCT) diet is known to induce less body weight and fat mass gain than that of LCT diet. However, the effect of MCT diet feeding on GIP secretion and the effect of GIP on body weight and fat mass under MCT diet-feeding condition are unknown. In this study, we evaluated the effect of single MCT oil administration on GIP secretion and compared the effect of long-term MCT and LCT diet on body weight and fat mass gain in wild-type (WT) and GIP-knockout (GIP KO) mice. Single administration of LCT oil induced GIP secretion but that of MCT oil did not in WT mice. Long-term intake of LCT diet induced GIP hypersecretion and significant body weight and fat mass gain compared with that of control fat (CF) diet in WT mice. In contrast, MCT diet did not induce GIP hypersecretion, and MCT diet-fed mice showed smaller increase in body weight and fat mass gain compared with CF diet-fed mice. In GIP KO mice, body weight and fat mass were markedly attenuated in LCT diet-fed mice but not in MCT diet-fed mice. Our results suggest that long-term intake of MCT diet stimulates less GIP secretion and suppresses body weight and fat mass gain compared with that of LCT diet.
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http://dx.doi.org/10.1152/ajpendo.00200.2018DOI Listing
July 2019

"Switched" metabolic acidosis in mitochondrial diabetes mellitus.

J Diabetes Investig 2019 Jul 18;10(4):1116-1117. Epub 2019 Jan 18.

Center for Diabetes and Endocrinology, The Tazuke Kofukai Medical Research Institute Kitano Hospital, Osaka, Japan.

A patient with mitochondrial diabetes mellitus developed diabetic ketoacidosis. During insulin treatment, although diabetic ketoacidosis improved, lactic acidosis unexpectedly worsened. This clinical course, named "switched metabolic acidosis," could reflect the unique pathophysiology of the mitochondrial disorder.
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http://dx.doi.org/10.1111/jdi.12992DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6626961PMC
July 2019

Ablation of Central Serotonergic Neurons Decreased REM Sleep and Attenuated Arousal Response.

Front Neurosci 2018 7;12:535. Epub 2018 Aug 7.

International Institute for Integrative Sleep Medicine (WPI-IIIS), University of Tsukuba, Tsukuba, Japan.

Sleep/wake behavior is regulated by distinct groups of neurons, such as dopaminergic, noradrenergic, and orexinergic neurons. Although monoaminergic neurons are usually considered to be wake-promoting, the role of serotonergic neurons in sleep/wake behavior remains inconclusive because of the effect of serotonin (5-HT)-deficiency on brain development and the compensation for inborn 5-HT deficiency by other sleep/wake-regulating neurons. Here, we performed selective ablation of central 5-HT neurons in the newly developed mouse line that was crossed with mice to examine the role of 5-HT neurons in the sleep/wake behavior of adult mice. Intracerebroventricular administration of diphtheria toxin completely ablated tdTomato-positive cells in mice. Electroencephalogram/electromyogram-based sleep/wake analysis demonstrated that central 5-HT neuron ablation in adult mice decreased the time spent in rapid eye movement (REM) sleep, which was associated with fewer transitions from non-REM (NREM) sleep to REM sleep than in control mice. Central 5-HT neuron-ablated mice showed attenuated wake response to a novel environment and increased theta power during wakefulness compared to control mice. The current findings indicated that adult 5-HT neurons work to support wakefulness and regulate REM sleep time through a biased transition from NREM sleep to REM sleep.
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http://dx.doi.org/10.3389/fnins.2018.00535DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6090062PMC
August 2018

Transcriptional factor Pdx1 is involved in age-related GIP hypersecretion in mice.

Am J Physiol Gastrointest Liver Physiol 2018 08 3;315(2):G272-G282. Epub 2018 May 3.

Department of Diabetes, Endocrinology and Nutrition, Graduate School of Medicine, Kyoto University , Kyoto , Japan.

Fat accumulation with aging is a serious problem; glucose-dependent insulinotropic polypeptide/gastric inhibitory polypeptide (GIP) is an incretin that plays an important role in fat accumulation. GIP receptor knockout mice show reduced fat mass and improved insulin sensitivity associated with aging. Therefore, GIP is involved in fat accumulation and insulin resistance with aging. However, age-related changes of GIP secretion remain unclear. The present study aimed to elucidate age-related changes of GIP secretion and enteroendocrine K cells using GIP reporter [GIP-green fluorescent protein (GFP) knock-in heterozygous (GIP)] mice. Aged 1-yr-old GIP mice exhibited a phenotype of fat accumulation, insulin resistance, and GIP hypersecretion compared with young (3-4 mo old) GIP mice. In aged mice, K-cell number in the small intestine and the mRNA expression levels of GIP and transcriptional factor pancreatic and duodenal homeobox-1 (Pdx1) in K cells were increased. K-cell number, GIP mRNA expression and content in small intestine, and GIP secretion were decreased after posteriori suppression of Pdx1 using intestine-specific gene transfer. Thus, Pdx1 positively regulates GIP mRNA and K-cell number in small intestine. Increased Pdx1 expression might be involved in GIP hypersecretion with aging. NEW & NOTEWORTHY Age-related changes of glucose-dependent insulinotropic polypeptide/gastric inhibitory polypeptide (GIP) secretion and K cells were investigated. We found that K-cell number and GIP and pancreatic and duodenal homeobox-1 (Pdx1) expression in K cells were increased in aged mice, which showed greater GIP secretion compared with young mice. In addition, we have succeeded in posteriori suppression of Pdx1 in small intestine using the method of intestine-specific gene transfer, and showed that K-cell number, GIP expression, and GIP secretion were decreased in the Pdx1-knockdown intestine.
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http://dx.doi.org/10.1152/ajpgi.00054.2018DOI Listing
August 2018

Sleep/Wake Behaviors in Mice During Pregnancy and Pregnancy-Associated Hypertensive Mice.

Sleep 2018 03;41(3)

International Institute for Integrative Sleep Medicine (WPI-IIIS), University of Tsukuba, Tsukuba, Ibaraki, Japan.

Study Objectives: In humans and other mammals, sleep is altered during pregnancy. However, no studies have been conducted on sleep/wakefulness during pregnancy in mice. In this study, we examined sleep/wakefulness in female C57BL/6 mice during pregnancy. We also examined sleep/wake behaviors in an animal model of preeclampsia, pregnancy-associated hypertensive (PAH) mice, in which increased angiotensin causes hypertension.

Methods: Sleep/wake behaviors of female C57BL/6 and PAH mice were examined based on electroencephalogram (EEG) or electromyogram recordings before, during, and after pregnancy. To examine whether high blood pressure disrupts the integrity of the blood-brain barrier in PAH mice, Evans blue dye was injected intravenously. Angiotensin II receptor blocker (olmesartan)-administered PAH mice and female Tsukuba hypertensive mice were also examined.

Results: C57BL/6 mice showed a decreased total wake time and increased nonrapid eye movement (NREM) sleep time during late pregnancy. Rapid eye movement (REM) sleep time did not change during the course of pregnancy. PAH mice exhibited a general slowing of EEG during late pregnancy and subsequently returned to apparently normal sleep/wakefulness after delivery. All PAH mice exhibited multiple focal leakages of Evans blue dye in the brain. Spike-and-wave discharges were observed in 50% of PAH mice. Olmesartan-administered PAH mice did not show general slowing of EEG. Tsukuba hypertensive mice showed a normal time spent in wakefulness and NREM sleep and a decreased total REM sleep time.

Conclusions: This study showed pregnant-stage-specific changes in sleep/wakefulness in C57BL/6 mice. Furthermore, PAH mice may be useful as an animal model for eclampsia.
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http://dx.doi.org/10.1093/sleep/zsx209DOI Listing
March 2018

Diverse metabolic effects of O-GlcNAcylation in the pancreas but limited effects in insulin-sensitive organs in mice.

Diabetologia 2017 09 22;60(9):1761-1769. Epub 2017 Jun 22.

Department of Medicine, Shiga University of Medical Science, Tsukinowa-cho, Seta, Otsu, Shiga, 520-2192, Japan.

Aims/hypothesis: O-GlcNAcylation is characterised by the addition of N-acetylglucosamine to various proteins by O-GlcNAc transferase (OGT) and serves in sensing intracellular nutrients by modulating various cellular processes. Although it has been speculated that O-GlcNAcylation is associated with glucose metabolism, its exact role in whole body glucose metabolism has not been fully elucidated. Here, we investigated whether loss of O-GlcNAcylation globally and in specific organs affected glucose metabolism in mammals under physiological conditions.

Methods: Tamoxifen-inducible global Ogt-knockout (Ogt-KO) mice were generated by crossbreeding Ogt-flox mice with R26-Cre-ER mice. Liver, skeletal muscle, adipose tissue and pancreatic beta cell-specific Ogt-KO mice were generated by crossbreeding Ogt-flox mice with Alb-Cre, Mlc1f-Cre, Adipoq-Cre and Pdx1 -CreER™ mice, respectively. Glucose metabolism was evaluated by i.p. glucose and insulin tolerance tests.

Results: Tamoxifen-inducible global Ogt-KO mice exhibited a lethal phenotype from 4 weeks post injection, suggesting that O-GlcNAcylation is essential for survival in adult mice. Tissue-specific Ogt deletion from insulin-sensitive organs, including liver, skeletal muscle and adipose tissue, had little impact on glucose metabolism under physiological conditions. However, pancreatic beta cell-specific Ogt-KO mice displayed transient hypoglycaemia (Ogt-flox 5.46 ± 0.41 vs Ogt-βKO 3.88 ± 0.26 mmol/l) associated with about twofold higher insulin secretion and accelerated adiposity, followed by subsequent hyperglycaemia (Ogt-flox 6.34 ± 0.32 vs Ogt-βKO 26.4 ± 2.37 mmol/l) with insulin depletion accompanied by beta cell apoptosis.

Conclusions/interpretation: These findings suggest that O-GlcNAcylation has little effect on glucose metabolism in insulin-sensitive tissues but plays a crucial role in pancreatic beta cell function and survival under physiological conditions. Our results provide novel insight into O-GlcNAc biology and physiology in glucose metabolism.
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http://dx.doi.org/10.1007/s00125-017-4327-yDOI Listing
September 2017

Distribution and hormonal characterization of primary murine L cells throughout the gastrointestinal tract.

J Diabetes Investig 2018 Jan 29;9(1):25-32. Epub 2017 May 29.

Department of Diabetes, Endocrinology and Nutrition, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

Aims/introduction: Glucagon-like peptide-1 (GLP-1) secreted from enteroendocrine L cells is an incretin that potentiates insulin secretion and is already applied in therapies for type 2 diabetes. However, detailed examination of L cells throughout the gastrointestinal tract remains unclear, because of difficulties in purifying scattered L cells from other cells. In the present study, we identified characteristics of L cells of the upper small intestine (UI), the lower small intestine (LI) and the colon using glucagon-green fluorescent protein-expressing mice that express GFP driven by the proglucagon promoter.

Materials And Methods: The localization and density of primary L cells were evaluated by anti-green fluorescent protein antibody reactivity. GLP-1 content, messenger ribonucleic acid (mRNA) expression levels and secretion in purified L cells were measured.

Results: The number of L cells significantly increased toward the colon. In contrast, the GLP-1 content and secretion from L cells were higher in the UI than in the LI and colon. L cells from the UI and LI expressed notably high mRNA levels of the transcription factor, islet 1. The mRNA expression levels of peptide YY in L cells were higher in the LI than in the UI and colon. The mRNA expression levels of gastric inhibitory polypeptide in L cells from the UI were significantly higher compared with those from the LI and colon.

Conclusions: L cells show different numbers and characteristics throughout the gut, and they express different mRNA levels of transcription factors and gastrointestinal hormones. These results contribute to the therapeutic application of promoting GLP-1 release from L cells for the treatment of type 2 diabetes.
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http://dx.doi.org/10.1111/jdi.12681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5754545PMC
January 2018

Long-Chain Free Fatty Acid Receptor GPR120 Mediates Oil-Induced GIP Secretion Through CCK in Male Mice.

Endocrinology 2017 05;158(5):1172-1180

Department of Diabetes, Endocrinology and Nutrition, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.

Free fatty acid receptors GPR120 and GPR40 are involved in the secretion of gut hormones. GPR120 and GPR40 are expressed in enteroendocrine K cells, and their activation induces the secretion of the incretin glucose-dependent insulinotropic polypeptide (GIP). However, the role of these receptors in fat-induced GIP secretion in vivo and the associated mechanisms are unclear. In this study, we investigated corn oil-induced GIP secretion in GPR120-knockout (GPR120-/-) and GPR40-knockout (GPR40-/-) mice. Oil-induced GIP secretion was reduced by 50% and 80% in GPR120-/- and GPR40-/- mice, respectively, compared with wild-type mice. This was not associated with a significant difference in K-cell number or GIP content in K cells, nor messenger RNA levels of the lipid receptor GPR119, nor bile acid receptors TGR5 and farnesoid X receptor. GPR120-/- and GPR40-/- mice also exhibited substantially decreased levels of cholecystokinin (CCK), a hormone from I cells that promotes bile and pancreatic lipase secretion, and this decrease was associated with impaired gallbladder contraction. Notably, treatment with a CCK analog resulted in recovery of oil-induced GIP secretion in GPR120-/- mice but not in GPR40-/- mice. These results indicate that corn oil-induced GIP secretion from K cells involves both GPR120 and GPR40 signaling pathways, and GPR120-induced GIP secretion is indirectly mediated by CCK.
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http://dx.doi.org/10.1210/en.2017-00090DOI Listing
May 2017

Inhibition of Gastric Inhibitory Polypeptide Receptor Signaling in Adipose Tissue Reduces Insulin Resistance and Hepatic Steatosis in High-Fat Diet-Fed Mice.

Diabetes 2017 04 17;66(4):868-879. Epub 2017 Jan 17.

Department of Diabetes, Endocrinology and Nutrition, Graduate School of Medicine, Kyoto University, Kyoto, Japan

Gastric inhibitory polypeptide receptor (GIPR) directly induces energy accumulation in adipose tissue in vitro. However, the importance of the direct effect of GIPR signaling on adipose tissue in vivo remains unclear. In the current study, we generated adipose tissue-specific GIPR knockout (GIPR) mice and investigated the direct actions of GIP in adipose tissue. Under high-fat diet (HFD)-fed conditions, GIPR mice had significantly lower body weight and lean body mass compared with those in floxed GIPR (GIPR) mice, although the fat volume was not significantly different between the two groups. Interestingly, insulin resistance, liver weight, and hepatic steatosis were reduced in HFD-fed GIPR mice. Plasma levels of interleukin-6 (IL-6), a proinflammatory cytokine that induces insulin resistance, were reduced in HFD-fed GIPR mice compared with those in HFD-fed GIPR mice. Suppressor of cytokine signaling 3 (SOCS3) signaling is located downstream of the IL-6 receptor and is associated with insulin resistance and hepatic steatosis. Expression levels of SOCS3 mRNA were significantly lower in adipose and liver tissues of HFD-fed GIPR mice compared with those of HFD-fed GIPR mice. Thus, GIPR signaling in adipose tissue plays a critical role in HFD-induced insulin resistance and hepatic steatosis in vivo, which may involve IL-6 signaling.
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http://dx.doi.org/10.2337/db16-0758DOI Listing
April 2017

Nardilysin Is Required for Maintaining Pancreatic β-Cell Function.

Diabetes 2016 10 6;65(10):3015-27. Epub 2016 Jul 6.

Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto, Japan

Type 2 diabetes (T2D) is associated with pancreatic β-cell dysfunction, manifested by reduced glucose-stimulated insulin secretion (GSIS). Several transcription factors enriched in β-cells, such as MafA, control β-cell function by organizing genes involved in GSIS. Here we demonstrate that nardilysin (N-arginine dibasic convertase; Nrd1 and NRDc) critically regulates β-cell function through MafA. Nrd1(-/-) mice showed glucose intolerance and severely decreased GSIS. Islets isolated from Nrd1(-/-) mice exhibited reduced insulin content and impaired GSIS in vitro. Moreover, β-cell-specific NRDc-deficient (Nrd1(delβ)) mice showed a diabetic phenotype with markedly reduced GSIS. MafA was specifically downregulated in islets from Nrd1(delβ) mice, whereas overexpression of NRDc upregulated MafA and insulin expression in INS832/13 cells. Chromatin immunoprecipitation assay revealed that NRDc is associated with Islet-1 in the enhancer region of MafA, where NRDc controls the recruitment of Islet-1 and MafA transcription. Our findings demonstrate that NRDc controls β-cell function via regulation of the Islet-1-MafA pathway.
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http://dx.doi.org/10.2337/db16-0178DOI Listing
October 2016

Enteral supplementation with glutamine, fiber, and oligosaccharide modulates incretin and glucagon-like peptide-2 secretion.

J Diabetes Investig 2015 May 23;6(3):302-8. Epub 2014 Oct 23.

Department of Diabetes, Endocrinology and Nutrition, Graduate School of Medicine, Kyoto University Kyoto, Japan.

Aims/introduction: A dietary supplementation product enriched with glutamine, dietary fiber and oligosaccharide (GFO) is widely applied for enteral nutrition support in Japan. The aim of the present study was to evaluate the effects of GFO ingestion on secretion of incretins, gastric inhibitory polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), and glucagon-like peptide-2 (GLP-2).

Materials And Methods: We carried out a cross-over study involving 20 healthy Japanese volunteers. The participants received GFO or 17 g of glucose, the equivalent carbohydrate in GFO as the control. Plasma glucose, serum insulin, and plasma total GIP, total GLP-1 and total GLP-2 levels during GFO or glucose loading were determined.

Results: GFO loading produced significantly higher plasma GLP-1 levels at 30 min and 60 min, area under the curve-GLP-1 value, and area under the curve-GLP-2 value after administration compared with those by glucose loading. In contrast, plasma GIP levels at both 30 and 60 min, and area under the curve-GIP value after glucose loading were significantly higher than those after GFO loading.

Conclusions: These results show that GFO ingestion stimulates GLP-1 and GLP-2 secretion, and reduces GIP secretion compared with glucose ingestion. Therefore, GFO could have an intestinotrophic effect as well as an ameliorating effect on metabolic disorders through modification of release of gut hormones.
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http://dx.doi.org/10.1111/jdi.12289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4420562PMC
May 2015

Fatty acid-binding protein 5 regulates diet-induced obesity via GIP secretion from enteroendocrine K cells in response to fat ingestion.

Am J Physiol Endocrinol Metab 2015 Apr 27;308(7):E583-91. Epub 2015 Jan 27.

Department of Diabetes, Endocrinology and Nutrition, Graduate School of Medicine, Kyoto University, Kyoto, Japan;

Gastric inhibitory polypeptide (GIP) is an incretin released from enteroendocrine K cells in response to nutrient intake, especially fat. GIP is one of the contributing factors inducing fat accumulation that results in obesity. A recent study shows that fatty acid-binding protein 5 (FABP5) is expressed in murine K cells and is involved in fat-induced GIP secretion. We investigated the mechanism of fat-induced GIP secretion and the impact of FABP5-related GIP response on diet-induced obesity (DIO). Single oral administration of glucose and fat resulted in a 40% reduction of GIP response to fat but not to glucose in whole body FABP5-knockout (FABP5(-/-)) mice, with no change in K cell count or GIP content in K cells. In an ex vivo experiment using isolated upper small intestine, oleic acid induced only a slight increase in GIP release, which was markedly enhanced by coadministration of bile and oleic acid together with attenuated GIP response in the FABP5(-/-) sample. FABP5(-/-) mice exhibited a 24% reduction in body weight gain and body fat mass under a high-fat diet compared with wild-type (FABP5(+/+)) mice; the difference was not observed between GIP-GFP homozygous knock-in (GIP(gfp/gfp))-FABP5(+/+) mice and GIP(gfp/gfp)-FABP5(-/-) mice, in which GIP is genetically deleted. These results demonstrate that bile efficiently amplifies fat-induced GIP secretion and that FABP5 contributes to the development of DIO in a GIP-dependent manner.
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http://dx.doi.org/10.1152/ajpendo.00543.2014DOI Listing
April 2015

Free fatty acid receptor GPR120 is highly expressed in enteroendocrine K cells of the upper small intestine and has a critical role in GIP secretion after fat ingestion.

Endocrinology 2015 Mar 23;156(3):837-46. Epub 2014 Dec 23.

Department of Diabetes, Endocrinology and Nutrition (K.Iw., N.H., K.Sa., S.Y., K.Su., A.Ha., D.N., K.Sh., E.J., T.Har., N.I.), Graduate School of Medicine, and Department of Genomic Drug Discovery Science (K.Ii., A.Hi.), Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8507, Japan; and Faculty of Pharmaceutical Sciences (T.Has., Y.A.), Tokushima Bunri University, Tokushima 770-8514, Japan.

Gastric inhibitory polypeptide (GIP) is an incretin secreted from enteroendocrine K cells in response to meal ingestion. Recently free fatty acid receptor G protein-coupled receptor (GPR) 120 was identified as a lipid sensor involved in glucagon-like peptide-1 secretion. However, Gpr 120 gene expression and its role in K cells remain unclear, partly due to difficulties in separation of K cells from other intestinal epithelial cells. In this study, we purified K cells using GIP-green fluorescent protein (GFP) knock-in mice, in which K cells can be visualized by GFP fluorescence. GFP-positive cells (K cells) were observed in the small intestine but not in the stomach and colon. K cell number and GIP content in K cells were significantly higher in the upper small intestine than those in the lower small intestine. We also examined the expression levels of several free fatty acid receptors in K cells. Among free fatty acid receptors, GPR120 was highly expressed in the K cells of the upper small intestine compared with the lower small intestine. To clarify the role of GPR120 on K cells in vivo, we used GPR120-deficient mice (GPR120(-/-)). GPR120(-/-) exhibited significantly lower GIP secretion (75% reduction, P < .01) after lard oil ingestion compared with that in wild-type mice. Consistently, pharmacological inhibition of GPR120 with grifolic acid methyl ether in wild-type mice significantly attenuated lard oil-induced GIP secretion. In conclusion, GPR120 is expressed abundantly in K cells of the upper small intestine and plays a critical role in lipid-induced GIP secretion.
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http://dx.doi.org/10.1210/en.2014-1653DOI Listing
March 2015

Chronic reduction of GIP secretion alleviates obesity and insulin resistance under high-fat diet conditions.

Diabetes 2014 Jul 28;63(7):2332-43. Epub 2014 Feb 28.

Department of Diabetes, Endocrinology and Nutrition, Graduate School of Medicine, Kyoto University, Kyoto, Japan

Gastric inhibitory polypeptide (GIP) exhibits potent insulinotropic effects on β-cells and anabolic effects on bone formation and fat accumulation. We explored the impact of reduced GIP levels in vivo on glucose homeostasis, bone formation, and fat accumulation in a novel GIP-GFP knock-in (KI) mouse. We generated GIP-GFP KI mice with a truncated prepro-GIP gene. The phenotype was assessed in heterozygous and homozygous states in mice on a control fat diet and a high-fat diet (HFD) in vivo and in vitro. Heterozygous GIP-GFP KI mice (GIP-reduced mice [GIP(gfp/+)]) exhibited reduced GIP secretion; in the homozygous state (GIP-lacking mice [GIP(gfp/gfp)]), GIP secretion was undetectable. When fed standard chow, GIP(gfp/+) and GIP(gfp/gfp) mice showed mild glucose intolerance with decreased insulin levels; bone volume was decreased in GIP(gfp/gfp) mice and preserved in GIP(gfp/+) mice. Under an HFD, glucose levels during an oral glucose tolerance test were similar in wild-type, GIP(gfp/+), and GIP(gfp/gfp) mice, while insulin secretion remained lower. GIP(gfp/+) and GIP(gfp/gfp) mice showed reduced obesity and reduced insulin resistance, accompanied by higher fat oxidation and energy expenditure. GIP-reduced mice demonstrate that partial reduction of GIP does not extensively alter glucose tolerance, but it alleviates obesity and lessens the degree of insulin resistance under HFD conditions, suggesting a potential therapeutic value.
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http://dx.doi.org/10.2337/db13-1563DOI Listing
July 2014

Vital capacity induction with 8% sevoflurane and N2o causes cerebral hyperemia.

J Anesth 2003 ;17(1):3-7

Department of Anesthesia, Kanto Rosai Hospital, 2035 Kizukisumiyoshi-cho, Nakahara-ku, Kawasaki 211-8501, Japan.

Purpose: Little is known about the influence of high-dose sevoflurane on cerebral volume. We evaluated induction time and cerebral blood volume with 8% sevoflurane using the "vital capacity induction" technique.

Methods: Thirty-four patients were randomly allocated into three groups. Group P received 2.0 mg x kg(-1) of propofol i.v. and inhalation of 67% N2o/O2, whereas group S5 and group S8 received inhalation of primed 5% and 8% sevoflurane in 67% N2O/O2, respectively. Induction time was measured as the time from the start of inhalation, or from the end of injection, until loss of eyelash reflex. Near-infrared spectroscopy and bispectral index (BIS) were monitored continuously until 3 min after tracheal intubation.

Results: Induction time was less in group S8 (17.3 +/- 6.4s, mean +/- SD) than in groups P (25.7 +/- 8.2s) and S5 (33.0 +/- 16.8s). There was a significant increase in cerebral blood volume after intubation in group S8, as suggested by higher cerebral oxyhemoglobin and total hemoglobin levels. There were no differences in BIS scores among the groups during the study period.

Conclusion: Vital capacity inhalation of 8% sevoflurane produces a faster loss of eyelash reflex than does 5% sevoflurane or propofol, but increases cerebral blood volume.
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http://dx.doi.org/10.1007/s005400300001DOI Listing
December 2003