Papers by Hiroshi Nagasaki
Frontiers in Endocrinology
Human stem cell-derived organoid culture enables the in vitro analysis of the cellular function i... more Human stem cell-derived organoid culture enables the in vitro analysis of the cellular function in three-dimensional aggregates mimicking native organs, and also provides a valuable source of specific cell types in the human body. We previously established organoid models of the hypothalamic-pituitary (HP) complex using human pluripotent stem cells. Although the models are suitable for investigating developmental and functional HP interactions, we consider that isolated pituitary cells are also useful for basic and translational research on the pituitary gland, such as stem cell biology and regenerative medicine. To develop a method for the purification of pituitary cells in HP organoids, we performed surface marker profiling of organoid cells derived from human induced pluripotent stem cells (iPSCs). Screening of 332 human cell surface markers and a subsequent immunohistochemical analysis identified epithelial cell adhesion molecule (EpCAM) as a surface marker of anterior pituitary...
Experimental and Clinical Endocrinology & Diabetes, 2011
Angiopoietin-like protein 4 (Angptl4) is thought to cause an increase in serum triglyceride level... more Angiopoietin-like protein 4 (Angptl4) is thought to cause an increase in serum triglyceride levels. In the present study, we elucidated Angptl4 expression in the mouse models of type 1 and type 2 diabetes mellitus, and investigated the possible mechanisms involved.Type 1 diabetes was induced in C57BL/6 J mice by treating them with streptozotocin (STZ). Type 2 diabetes was induced by feeding the mice a high-fat diet (HFD) for 18 weeks.The levels of Angptl4 mRNA expression in liver, white adipose tissue (WAT), and brown adipose tissue (BAT) were found to increase in the STZ diabetic mice relative to control mice. This effect was attenuated by insulin administration. In the HFD diabetic mice, the Angptl4 mRNA expression levels were increased in liver, WAT, and BAT. Treatment with metformin for 4 weeks attenuated the increased levels of Angptl4 mRNA. Fatty acids (FAs) such as palmitate and linoleate induced Angptl4 mRNA expression in H4IIE hepatoma cells and 3T3-L1 adipocytes. Treatment...
PLOS ONE
The hypothalamus is comprised of heterogenous cell populations and includes highly complex neural... more The hypothalamus is comprised of heterogenous cell populations and includes highly complex neural circuits that regulate the autonomic nerve system. Its dysfunction therefore results in severe endocrine disorders. Although recent experiments have been conducted for in vitro organogenesis of hypothalamic neurons from embryonic stem (ES) or induced pluripotent stem (iPS) cells, whether these stem cell-derived hypothalamic neurons can be useful for regenerative medicine remains unclear. We therefore performed orthotopic transplantation of mouse ES cell (mESC)-derived hypothalamic neurons into adult mouse brains. We generated electrophysiologically functional hypothalamic neurons from mESCs and transplanted them into the supraoptic nucleus of mice. Grafts extended their axons along hypothalamic nerve bundles in host brain, and some of them even projected into the posterior pituitary (PPit), which consists of distal axons of the magnocellular neurons located in hypothalamic supraoptic an...
Nihon Naika Gakkai Zasshi, 1994
The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 21, 2005
Neuropeptide Y (NPY) in the arcuate nucleus is an orexigenic hormone of which levels are regulate... more Neuropeptide Y (NPY) in the arcuate nucleus is an orexigenic hormone of which levels are regulated by humoral as well as neural signals. In this study, we examined the regulation of NPY gene expression in the arcuate nucleus in hypothalamic organotypic cultures. Dexamethasone (DEX) (10(-9) to 10(-7) M) significantly increased NPY mRNA expression, and the effects were not influenced by coincubation with the sodium channel blocker tetrodotoxin (TTX), indicating that the action of DEX is independent of action potentials. Conversely, insulin (10(-11) to 10(-9) M) significantly inhibited NPY expression stimulated by DEX, and the inhibitory action of insulin was abolished in the presence of TTX. Because GABA and its receptors are expressed in the arcuate nucleus in vivo, we examined whether GABAergic systems were involved in the insulin action. The GABAB agonist baclofen significantly inhibited NPY expression stimulated by DEX, and the inhibitory action of insulin was completely abolished...
Journal of Neuroendocrinology, 2001
The plasma concentration of arginine vasopressin (AVP) is closely regulated by plasma osmolality.... more The plasma concentration of arginine vasopressin (AVP) is closely regulated by plasma osmolality. In this study, we used intronic in-situ hybridization to investigate the transcriptional activity of the AVP gene in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) after intraperitoneal (i.p.) injection of hypertonic saline inducing small changes in plasma osmolality in rats. After ip injection of 900 mOsm/kg saline (2% BW), plasma [Na] reached the highest level at 10 min (900 mOsm/kg, 146±1 mEq/l; 290 mOsm/kg, 139±1 mEq/l, P<0.01) and maintained that level until 30 min. The expression of AVP heteronuclear (hn) RNA in the SON and PVN increased significantly as early as 10 min and peaked 30 min after ip injection of the hypertonic saline. AVP hnRNA expression showed a significant increase even after a 2 mEq/L elevation in plasma [Na] concentration, and increased dose-dependently in response to the concentration of saline injection (290-900 mOsm/kg). These results demonstrate that, similar to AVP secretion, AVP gene transcriptional activity in the SON and PVN is closely regulated by plasma osmolality.
Journal of Endocrinology, 2002
Arginine vasopressin (AVP) is a major antidiuretic hormone, the overproduction of which causes di... more Arginine vasopressin (AVP) is a major antidiuretic hormone, the overproduction of which causes diluting hyponatremia in humans and is called the syndrome of inappropriate antidiuresis (SIAD). To study physiological changes resulting from AVP overproduction and to develop an animal model of hyponatremia, the human AVP gene was expressed under the control of the metallothionein promoter in transgenic (Tg) rats. Analyses of AVP immunoreactivity (irAVP) in the tissues revealed that the transgene is expressed mainly in the central nervous system. Gel filtration showed that irAVP in the brain and plasma was properly processed AVP. AVP purified from the brains of both Tg and control rats also exerted equal bioactivity to generate cAMP in LLC-PK1 cells. The founder rats did not show any physical or anatomical abnormalities. Under basal conditions, Tg rats had high plasma AVP levels (Tg 13.8 +/- 2.5 pg/ml; control 2.7 +/- 1.2 pg/ml; n=6 in both groups; means +/- S.E.M.), decreased urine volu...
Experimental and Clinical Endocrinology &amp; Diabetes, 2010
Hedgehog (Hh) signaling plays an important role in pancreas development. However, its role in the... more Hedgehog (Hh) signaling plays an important role in pancreas development. However, its role in the developed endocrine pancreas remains to be elucidated. To clarify whether Hh signaling participates in beta-cell survival, we investigated the role of Hh signaling in cytokine-induced apoptosis in pancreatic beta-cells. Insulin-producing INS-1E cells were transfected with Sonic Hh (Shh) expression vector or siRNA against Indian Hh (siIhh). The Hh signal inhibitor cyclopamine were pretreated in INS-1E cells and rat islets. The cells were exposed to 200 U/ml IL-1β and 200 U/ml IFN-γ for 48 h. Apoptosis was estimated by flow cytometory and immunofluorescence staining for cleaved caspase-3. Nitric oxide generation was measured by Griess reaction. We found that exposure to proinflammatory cytokines increased Ihh expression in rat islets and INS-1E cells. Overexpression of Shh reduced cytokine-induced apoptosis. By contrast, treatment with cyclopamine increased cytokine-induced apoptosis in INS-1E cells and rat islets. Treatment with the siIhh showed same results in INS-1E cells. Forced expression of Shh suppressed cytokine-induced nuclear factor-κB promoter activity, leading to attenuation of nitric oxide synthase 2 expression and nitric oxide production, while Ihh knockdown enhanced this pathway in INS-1E cells. Our findings suggest that Hh signaling is implicated in protecting beta-cells from cytokine-induced cytotoxicity.
Cell Reports, 2020
Highlights d Hypothalamic-pituitary functional units can be generated from 3D-cultured human iPS ... more Highlights d Hypothalamic-pituitary functional units can be generated from 3D-cultured human iPS cells d Maturation of induced pituitary through simultaneous culture with hypothalamic neurons d ACTH secretion ability of induced pituitary is comparable with adult mice d Induced pituitary cells respond to low-glucose stimulation through the CRH-ACTH pathway
Journal of Neural Transmission
Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis, and its stab... more Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis, and its stability is a fundamental factor to maintain the level of the catecholamines in cells. However, the intracellular stability of TH determined by the degradation remains unknown; although the TH molecule phosphorylated at its Ser19 was observed in the nucleus, and the phosphorylation suspected to trigger its proteasome-mediated degradation. Computer-assisted analysis using the cNLS Mapper program predicted that two sequences of nuclear localization signals (NLS) exist in the N-terminus of TH molecule containing the phosphorylation sites Ser19, Ser31, and Ser40 (Pro-Arg and Lys-Ile): the NLS scores indicated that TH could become localized in both nucleus and cytoplasm. Moreover, inhibition of the importin α/β-mediated nuclear import pathway increased the level of TH phosphorylated at its Ser19 in PC12D cells. The results suggest that TH might be imported to nucleus from cytoplasm to be degraded. Recent studies revealed that proteasomes predominantly exist in the nucleus rather than in the cytoplasm to degrade the nuclear proteins related to cell-cycle progression, gene expression, DNA damage, and DNA repair. Therefore, these studies suggest that the relationship between the phosphorylation and the nuclear localization of the TH molecule should be a matter of focus to understand the mechanism of proteasome-mediated degradation of the enzyme as a first priority.
Journal of intensive care, 2016
The aim of this study was to investigate the effects of recombinant human-soluble thrombomodulin ... more The aim of this study was to investigate the effects of recombinant human-soluble thrombomodulin (rTM) on lipopolysaccharide (LPS)-induced, platelet-dependent neutrophil extracellular trap (NET) formation (NETosis). Human peripheral blood neutrophils and platelets were co-incubated with or without LPS (0.2 μg/ml) in the presence and absence of rTM (2 μg/ml). NETosis was confirmed by immunostaining and confocal microscopy. In the absence of platelets, LPS did not induce NETosis in the neutrophils. NETosis, however, was induced by LPS when neutrophils were co-cultured with platelets (64 % of neutrophils). Notably, rTM was able to fully inhibit NETosis in neutrophils cultured with platelets and in the presence of LPS. rTM did not induce NETosis in this co-culture system (p < 0.01 versus LPS in the absence of rTM). These results show that rTM can suppress LPS-induced platelet-dependent NETosis in vitro.
Nippon Rinsho Japanese Journal of Clinical Medicine, Sep 1, 2005
Interdisciplinary Information Sciences, 2015
Recently, various hypothalamic neurons have been successfully engineered from pluripotent stem ce... more Recently, various hypothalamic neurons have been successfully engineered from pluripotent stem cells, including mouse and human embryonic stem cells. Because pluripotent cells need to undergo stepwise changes during organogenesis, developmental analyses on the hypothalamus have been inevitable for numerous transcription factors that determine specification, survival, and migration during the formation of specific neurons. Hypothalamic progenitor cells arise from the retina and anterior neural fold homeobox (Rax) þ ventral part of the ventricular zone at embryonic day 10.5 (E10.5), and orthopedia (Otp) and steroidgenic factor-1 (SF-1) respectively appear in the dorsal and ventral regions at E13.5, which subsequently produce specific transcription factors required for the final maturation of hypothalamic neurons. In the pluripotent stem cells, rostrodorsal hypothalamus-like progenitors expressing retina and anterior neural fold homeobox are generated from floating aggregates in serum-free conditions with minimized exogenous patterning signaling. A certain population of the Rax þ progenitors generate Otp þ neuronal precursors, which subsequently develop into various dorsal and lateral hypothalamic neurons, including arginine vasopressin (AVP) and oxytocin neurons. Alternatively, early exposure to sonic hedgehog (Shh) induces differentiation markers including SF-1, specific for rostral-ventral hypothalamiclike precursors that eventually produce neuropeptide Y (NPY) and pro-opio-melanocortin (POMC). In conclusion, it is now possible to induce most types of hypothalamic neurons from pluripotent stem cells. Application of these cells would have advantages for studies on specification, migration, drug development, and regenerative medicine.
Journal of neural transmission (Vienna, Austria : 1996), 2015
We previously reported that an optimal dose of lipopolysaccharide (LPS) markedly extends the life... more We previously reported that an optimal dose of lipopolysaccharide (LPS) markedly extends the lifespan of murine primary-cultured microglia by suppressing cell death pathways. In this study, we investigated the effects of LPS pretreatment on UV light-induced apoptosis of cells from the microglial cell line BV-2. More than half of BV-2 cells were apoptotic, and procaspase-3 was cleaved into its active form at 3 h of UV irradiation. In contrast, in BV-2 cells treated with LPS for 24 h, UV irradiation caused neither apoptosis nor procaspase-3 cleavage. LPS treatment arrested the cell cycle in G1 phase and upregulated cyclin-dependent kinase inhibitor p21(Waf1/Cip1) and growth arrest and DNA damage-inducible (GADD) 45α in BV-2 cells. When p21(Waf1/Cip1) and GADD45α were knocked down by small interfering RNA, procaspase-3 was cleaved into its active form to induce apoptosis. Our findings suggest that LPS inhibits UV-induced apoptosis in BV-2 cells through arrest of the cell cycle in G1 ph...
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Papers by Hiroshi Nagasaki