Satoshi Okano

Publications (7)15.11 Total impact

• Article: Non-obese early onset diabetes mellitus in mutant cryptochrome1 transgenic mice.

  Satoshi Okano, Kiyoshi Hayasaka, Masahiko Igarashi, Harue Iwai, Yoshiyuki Togashi, Osamu Nakajima
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  ABSTRACT: An earlier report described that transgenic mice ubiquitously expressing cryptochrome1 (CRY1) with a mutation in cystein414 (CRY1-AP Tg mice) display diabetes mellitus in addition to anomalous circadian behaviours. This study examined characteristic aspects of symptoms to clarify the diabetes type and pathogenesis. The body weights and blood glucose levels of CRY1-AP Tg mice were measured for 7weeks starting at 3weeks after birth. Glucose tolerance test for the mice of various ages and insulin tolerance test at 6weeks of age were conducted. Immunohistochemical analysis of islets was carried out for the mice of 19 and 40weeks of age. Basal and glucose-stimulated serum insulin levels of mice at 27weeks were also measured. Three-week-old CRY1-AP Tg mice, which showed mild retardation in growth, already displayed glucose intolerance. Hyperglycaemia progressed with age, without accompanying insulin resistance. Insulin-stained areas in islets in CRY1-AP Tg mice were smaller than that in wild-type controls. Both basal and glucose-stimulated secretion of insulin decreased in CRY1-AP Tg mice. The symptoms of diabetes in CRY1-AP Tg mice turned out to be similar to those of maturity onset diabetes of the young (MODY) in humans in terms of early onset, non-obesity and primary dysfunction of beta cells. The CRY1-AP Tg mice might serve as an animal model of early onset insulin-secretory defect of diabetes.
  European Journal of Clinical Investigation 11/2010; 40(11):1011-7. · 3.02 Impact Factor
• Article: Indispensable function for embryogenesis, expression and regulation of the nonspecific form of the 5‐aminolevulinate synthase gene in mouse

  Satoshi Okano, Lingyun Zhou, Toshimasa Kusaka, Kazuhide Shibata, Kazuhiro Shimizu, Xu Gao, Yuko Kikuchi, Yoshiyuki Togashi, Tomonori Hosoya, Satoru Takahashi, Osamu Nakajima, Masayuki Yamamoto
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  ABSTRACT: The first step of heme biosynthesis in animals is catalyzed by 5-aminolevulinate synthase (ALAS), which controls heme supply in various tissues. To clarify the roles that the nonspecific isoform of ALAS (ALAS-N) plays in vivo, we prepared a green fluorescent protein (GFP) knock-in mouse line in which the Alas1 gene (encoding ALAS-N) is replaced with a gfp gene. We found that mice bearing a homozygous knock-in allele (Alas1GFP/GFP) were lethal by embryonic day 8.5, demonstrating that ALAS-N is essential for early embryogenesis. Fluorescence microscopic and flow cytometric analyses of heterozygous mouse (Alas1+/GFP) tissues showed that the Alas1 expression level differs substantially in tissues; Alas1 is highly expressed in testis Leydig cells, exocrine glands (including submandibular and parotid glands), endocrine glands (such as adrenal and thyroid glands) and hematopoietic lineage cells (including neutrophils and eosinophils). Quantitative analyses of GFP mRNA and ALAS-N mRNA in various tissues of Alas1+/GFP mice suggested that the destabilization of ALAS-N mRNA was not uniform in the various tissues. These results thus lay bare that elaborate control of the endogenous heme supply operates in various mouse tissues through regulation of the ALAS-N expression level and that this control is essential for heme homeostasis in animals.
  Genes to Cells 12/2009; 15(1):77 - 89. · 2.68 Impact Factor
• Article: Unusual circadian locomotor activity and pathophysiology in mutant CRY1 transgenic mice.

  Satoshi Okano, Makoto Akashi, Kiyoshi Hayasaka, Osamu Nakajima
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  ABSTRACT: In the widely accepted molecular model underlying mammalian circadian rhythm, cryptochrome proteins (CRYs) play indispensable roles as inhibitive components of the CLOCK-BMAL1-mediated transcriptional-translational negative feedback loop. In order to clarify yet uncovered aspects of mammalian CRYs in vivo, we generated transgenic (Tg) mice ubiquitously overexpressing CRY1 as well as CRY1 having a mutation in the dipeptide motif of cysteine and proline that is conserved beyond evolutional divergence among animal CRYs: cysteine414 of the motif was replaced with alanine (CRY1-AP). The mice overexpressing CRY1 (CRY1 Tg) exhibited robust circadian rhythms of locomotor activity. In sharp contrast, the mice overexpressing CRY1-AP (CRY1-AP Tg) displayed a unique circadian phenotype. Their locomotor free-running periods were very long (around 28h) with rhythm splitting: the bout of activity of CRY1-AP Tg mice was split into two equal components in constant darkness. Moreover, CRY1-AP Tg mice displayed abnormal entrainment behavior: their bout of activity shifted immediately in response to a shift of the light-dark cycles. In addition, we found that CRY1-AP Tg mice showed symptoms characteristic of diabetes mellitus. The results indicate that the motif of CRY1 is crucial to the mammalian clock system and physiology.
  Neuroscience Letters 02/2009; 451(3):246-51. · 2.11 Impact Factor
• Article: In vivo oxygen imaging using green fluorescent protein.

  Eiji Takahashi, Tomohiro Takano, Yasutomo Nomura, Satoshi Okano, Osamu Nakajima, Michihiko Sato
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  ABSTRACT: In vivo oxygen measurement is the key to understanding how biological systems dynamically adapt to reductions in oxygen supply. High spatial resolution oxygen imaging is of particular importance because recent studies address the significance of within-tissue and within-cell heterogeneities in oxygen concentration in health and disease. Here, we report a new technique for in vivo molecular imaging of oxygen in organs using green fluorescent protein (GFP). GFP-expressing COS-7 cells were briefly photoactivated with a strong blue light while lowering the oxygen concentration from 10% to <0.001%. Red fluorescence (excitation 520-550 nm, emission >580 nm) appeared after photoactivation at <2% oxygen (the red shift of GFP fluorescence). The red shift disappeared after reoxygenation of the cell, indicating that the red shift is stable as long as the cell is hypoxic. The red shift of GFP fluorescence was also demonstrated in single cardiomyocytes isolated from the GFP knock-in mouse (green mouse) heart. Then, we tried in vivo molecular imaging of hypoxia in organs. The red shift could be imaged in the ischemic liver and kidney in the green mouse using macroscopic optics provided that oxygen diffusion from the atmospheric air was prevented. In crystalloid-perfused beating heart isolated from the green mouse, significant spatial heterogeneities in the red shift were demonstrated in the epicardium distal to the coronary artery ligation. We conclude that the present technique using GFP as an oxygen indicator may allow in vivo molecular imaging of oxygen in organs.
  AJP Cell Physiology 10/2006; 291(4):C781-7. · 3.54 Impact Factor
• Article: Transgenic rescue of erythroid 5-aminolevulinate synthase-deficient mice results in the formation of ring sideroblasts and siderocytes.

  Osamu Nakajima, Satoshi Okano, Hiroko Harada, Toshimasa Kusaka, Xu Gao, Tomonori Hosoya, Norio Suzuki, Satoru Takahashi, Masayuki Yamamoto
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  ABSTRACT: Molecular defects in erythroid 5-aminolevulinate synthase (ALAS-E), the first enzyme in the heme biosynthetic pathway, cause X-linked sideroblastic anemia (XLSA). However, ring sideroblasts, the hallmark of XLSA, were not found in ALAS-E-deficient mouse embryos, indicating that simple ALAS-E-deficiency is not sufficient for ring sideroblast formation. To investigate the developmental stage-specific pathogenesis caused by heme-depletion, we attempted a complementation rescue of ALAS-E-deficiency. We exploited transgenic mouse lines expressing human ALAS-E at approximately half that of wild-type levels. In these hypomorphic embryos, most of the primitive erythroid cells were transformed into ring sideroblasts. The majority of the circulating definitive erythroid cells became siderocytes, enucleated erythrocytes containing iron deposits, and definitive ring sideroblasts were also observed. These iron-overloaded cells suffered from an alpha/beta globin chain imbalance. Despite the iron overload, transferrin receptors were highly expressed in the erythroid cells, suggesting they contribute to the formation of ring sideroblasts and siderocytes. These results indicate that a partially depleted heme supply provokes ring sideroblast formation. The experimental generation of ring sideroblasts in animals would contribute to our understanding of the iron metabolism and its disorder in erythroid cells.
  Genes to Cells 07/2006; 11(6):685-700. · 2.68 Impact Factor
• Chapter: Genetic Oxygen Sensor

  Eiji Takahashi, Tomohiro Takano, Aya Numata, Natsuho Hayashi, Satoshi Okano, Osamu Nakajima, Yasutomo Nomura, Michihiko Sato
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  ABSTRACT: We report in this article a new method for in vivo oxygen measurement using green fluorescence protein (GFP). COS7 cells were transiently transfected with an expression vector, pCMX-GFP, using a polyethylenimine reagent and cultured for 48 hrs. After exposure of the cell to anoxic gas (O2<.001%), a 1 min illumination of the cell to strong 470–490 nm light evoked a significant red fluorescence (excitation 520–550 nm, emission >580 nm) that had been negligible before the photoactivation. This red shift of (green) GFP fluorescence was never observed in normoxia. We then examined the validity of this method in transgenic mice in which GFP is stably expressed (green mice). All the ventricular myocytes isolated from the green mice showed significant green fluorescence, although the intensity was ∼1/200 of the transiently GFP-expressing COS7 cells. The photoactivation in anoxia increased the red fluorescence in these cells, but the magnitude was much smaller than expected. In summary, GFP can be used as an in situ probe for hypoxia. In GFP-expressing transgenic animals, in vivo imaging of anoxic loci with a submicron spatial resolution may be possible.
  06/2006: pages 39-44;
• Article: Genetic oxygen sensor: GFP as an indicator of intracellular oxygenation.

  Eiji Takahashi, Tomohiro Takano, Aya Numata, Natsuho Hayashi, Satoshi Okano, Osamu Nakajima, Yasutomo Nomura, Michihiko Sato
  [show abstract] [hide abstract]
  ABSTRACT: We report in this article a new method for in vivo oxygen measurement using green fluorescence protein (GFP). COS7 cells were transiently transfected with an expression vector, pCMX-GFP, using a polyethylenimine reagent and cultured for 48 hrs. After exposure of the cell to anoxic gas (O2 < .001%), a 1 min illumination of the cell to strong 470-490 nm light evoked a significant red fluorescence (excitation 520-550 nm, emission > 580 nm) that had been negligible before the photoactivation. This red shift of (green) GFP fluorescence was never observed in normoxia. We then examined the validity of this method in transgenic mice in which GFP is stably expressed (green mice). All the ventricular myocytes isolated from the green mice showed significant green fluorescence, although the intensity was approximately 1/200 of the transiently GFP-expressing COS7 cells. The photoactivation in anoxia increased the red fluorescence in these cells, but the magnitude was much smaller than expected. In summary, GFP can be used as an in situ probe for hypoxia. In GFP-expressing transgenic animals, in vivo imaging of anoxic loci with a submicron spatial resolution may be possible.
  Advances in experimental medicine and biology 01/2005; 566:39-44. · 1.09 Impact Factor