67 results on '"Toshiyuki Obata"'
Search Results
2. IgM-mediated Warm Autoimmune Hemolytic Anemia: An Autopsy Report
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Keiko Takashiro, Toshiyuki Obata, Takahiko Ito, Noriko Takahara, Kouhei Yoshimura, Natsuka Tojo, Kazuyo Yamamoto, Shigeo Hara, Kouhei Takesue, and Naoko Yoshimatsu
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Male ,Hemolytic anemia ,Case Report ,Autopsy ,030204 cardiovascular system & hematology ,03 medical and health sciences ,Fatal Outcome ,0302 clinical medicine ,Internal Medicine ,medicine ,Humans ,Lupus Erythematosus, Systemic ,autoimmune hemolytic anemia ,warm antibody ,Aged ,business.industry ,General Medicine ,medicine.disease ,Antibodies, Anti-Idiotypic ,Complement system ,Impaired consciousness ,Agglutination (biology) ,Immunoglobulin M ,Immunology ,Autopsy report ,030211 gastroenterology & hepatology ,Rituximab ,Anemia, Hemolytic, Autoimmune ,Autoimmune hemolytic anemia ,business ,medicine.drug - Abstract
A 79-year-old man with Sjögren's syndrome and systemic lupus erythematosus developed acute impaired consciousness and hemolytic anemia. The patient's red blood cells agglutinated spontaneously at 25-37°C. The treatment of red blood cells with 2-mercaptoethanol resulted in the loss of spontaneous agglutination. A diagnosis of IgM-mediated warm autoimmune hemolytic anemia was made. The patient received steroid pulse and plasma exchange therapies. Rituximab was also administered. However, the patient died from multiple organ failure at six days from the symptom onset. The clinical progress of the patient and autopsy findings suggested that complement activation might have been associated with the pathology.
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- 2019
3. A Case of Type B Insulin-resistance Syndrome Ameliorated with Immune-suppression Therapies
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Naohi Isse, Toshiyuki Obata, Kohei Takesue, Yuuki Tachibana, Noriko Takahara, Naoko Yoshimatsu, and Takahiko Ito
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business.industry ,General Medicine ,030204 cardiovascular system & hematology ,medicine.disease ,03 medical and health sciences ,Remission induction ,0302 clinical medicine ,Insulin resistance ,Immune system ,Immunology ,Medicine ,030212 general & internal medicine ,business ,Receptor ,Insulin metabolism - Published
- 2016
4. The Influence of Statin Treatment on the Effect of Sitagliptin in Obese Patients with Diabetes
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Toru Shinmen, Hisae Nakata, Masayo Shioe, Keisuke Yonezawa, Nobuyuki Muroi, Toshiyuki Obata, Naotake Takase, Yuka Onoue, Ikuko Miki, Shunichi Gouda, and Noriko Takahara
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medicine.medical_specialty ,business.industry ,Internal medicine ,Sitagliptin ,Diabetes mellitus ,medicine ,Statin treatment ,medicine.disease ,business ,medicine.drug - Published
- 2015
5. [Case Report; A case of type B insulin-resistance syndrome ameliorated with immune-suppression therapies]
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Kohei, Takesue, Toshiyuki, Obata, Naohi, Isse, Takahiko, Ito, Naoko, Yoshimatsu, Yuuki, Tachibana, and Noriko, Takahara
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Blood Glucose ,Drug Combinations ,Remission Induction ,Humans ,Female ,Insulin Resistance ,Middle Aged ,Immunosuppressive Agents ,Receptor, Insulin - Published
- 2016
6. Relation of the Expression of Transcriptional Factor TFAP2B to That of Adipokines in Subcutaneous and Omental Adipose Tissues
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Toshiyuki Obata, Yoshihiko Nishio, Shiro Maeda, Kazuhiro Ikeda, Motoyuki Kondo, Masaaki Kobayashi, Hiroshi Maegawa, Takeshi Yoshizaki, Hiroshi Yamamoto, Satoshi Ugi, Hiroyuki Naitoh, Toru Tani, Atsunori Kashiwagi, Katsutaro Morino, Shuichi Tsukada, and Takashi Uzu
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Leptin ,Male ,medicine.medical_specialty ,Endocrinology, Diabetes and Metabolism ,Abdominal Fat ,Subcutaneous Fat ,Medicine (miscellaneous) ,Adipose tissue ,Adipokine ,Body Mass Index ,Endocrinology ,In vivo ,Internal medicine ,Blood plasma ,medicine ,Humans ,RNA, Messenger ,Interleukin 6 ,Aged ,Aged, 80 and over ,Metabolic Syndrome ,Messenger RNA ,Nutrition and Dietetics ,biology ,Adiponectin ,Interleukin-6 ,Middle Aged ,Diabetes Mellitus, Type 2 ,Transcription Factor AP-2 ,biology.protein ,Female ,Omentum - Abstract
To determine the potential role of the transcriptional factor-activating enhancer-binding protein-2beta (TFAP2B) in the regulation of expression of adipokines, adiponectin, leptin, and interleukin-6 (IL-6) in vivo, we quantified the mRNA expression levels of these adipokines and TFAP2B in visceral (omental) and abdominal subcutaneous adipose tissues of 66 individuals with variable degree of adiposity and studied their correlations with BMI and their plasma concentrations. We found that BMI correlated negatively with plasma adiponectin levels and positively with those of leptin. Adiponection mRNA expression in subcutaneous fat correlated negatively with BMI, whereas leptin mRNA levels in the omentum correlated with plasma leptin levels and BMI. In contrast, IL-6 mRNA levels in subcutaneous and omental fat did not correlate with BMI. IL-6 mRNA levels in the omental fat correlated with plasma IL-6 levels. Whereas TFAP2B mRNA expression did not correlate with BMI, it correlated negatively with adiponectin expression in the subcutaneous adipose tissue. Furthermore, TFAP2B mRNA expression correlated negatively with leptin and positively with IL-6 expression in both subcutaneous and omental adipose tissues. These relationships are consistent with our in vitro observations and indicate that TFAP2B seems to regulate the expression of various adipokines in vivo.
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- 2010
7. Transcription factor AP-2β: A negative regulator of IRS-1 gene expression
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Eiichi Araki, Motoyuki Kondo, Toshiyuki Obata, Xiangning Meng, Atsunori Kashiwagi, Katsutaro Morino, Satoshi Ugi, Shiro Maeda, Yoshihiko Nishio, Hiroshi Maegawa, Tomoya Fuke, and Takeshi Yoshizaki
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Biophysics ,Electrophoretic Mobility Shift Assay ,Mice, Inbred Strains ,Response Elements ,Biochemistry ,Mice ,3T3-L1 Cells ,Gene expression ,Transcriptional regulation ,Animals ,Electrophoretic mobility shift assay ,RNA, Messenger ,Binding site ,Promoter Regions, Genetic ,Molecular Biology ,Transcription factor ,Regulation of gene expression ,biology ,Promoter ,Cell Biology ,Molecular biology ,Insulin receptor ,Gene Expression Regulation ,Transcription Factor AP-2 ,Gene Knockdown Techniques ,Mutation ,Insulin Receptor Substrate Proteins ,biology.protein ,Nucleic Acid Conformation ,Insulin Resistance - Abstract
Down-regulation of insulin receptor substrate-1 (IRS-1) expression could modify the ability of IRS-1 to fulfill its functions. It has been proposed that the phosphorylation of IRS-1 on serine residues could promote its degradation. However, few studies have investigated the transcriptional regulation of IRS-1 in the pathogenesis of insulin resistance. Genotyping for genome-wide single nucleotide polymorphisms revealed that the transcription factor activating enhancer-binding protein-2beta (AP-2beta) is a novel candidate gene for conferring susceptibility to obesity and type 2 diabetes. AP-2beta is expressed in adipose tissue and its expression is increased during the maturation of adipocytes. Overexpression of AP-2beta leads to adipocyte hypertrophy, directly inhibits adiponectin expression, and enhanced the expression of inflammatory adipokines such as IL-6 and MCP-1. In this study, we found that overexpression of AP-2beta in 3T3-L1 adipocytes impaired the promoter activity of IRS-1, and subsequently decreased mRNA and protein expression. Electrophoretic mobility shift assays showed that AP-2beta bound specifically to the IRS-1 promoter region. Furthermore, site-directed mutagenesis of the AP-2 binding site located at -362 to -351, relative to the transcription start site, markedly decreased AP-2-induced suppression of IRS-1 promoter activity, whereas other putative AP-2 binding sites did not. Our results clearly showed that AP-2beta directly decreased IRS-1 expression by binding to its promoter. Based on these findings, we speculate that the AP-2beta transcriptional factor is a unique regulator of IRS-1 and a candidate gene for insulin resistance.
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- 2010
8. Transcription factor AP-2β inhibits expression and secretion of leptin, an insulin-sensitizing hormone, in 3T3-L1 adipocytes
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Yoshihiko Nishio, Toshiyuki Obata, Atsunori Kashiwagi, Takeshi Yoshizaki, Katsutaro Morino, Hiroshi Maegawa, Motoyuki Kondo, Tomoya Fuke, Satoshi Ugi, and Shiro Maeda
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Leptin ,medicine.medical_specialty ,Endocrinology, Diabetes and Metabolism ,Medicine (miscellaneous) ,Adipokine ,Adipose tissue ,Mice ,chemistry.chemical_compound ,3T3-L1 Cells ,Internal medicine ,Adipocyte ,Adipocytes ,medicine ,Animals ,Humans ,RNA, Messenger ,Promoter Regions, Genetic ,Transcription factor ,Pancreatic hormone ,Nutrition and Dietetics ,Leptin receptor ,digestive, oral, and skin physiology ,Biological Transport ,Promoter ,Endocrinology ,Adipose Tissue ,Gene Expression Regulation ,Transcription Factor AP-2 ,chemistry ,Mutagenesis, Site-Directed ,Insulin Resistance ,hormones, hormone substitutes, and hormone antagonists - Abstract
We have previously reported an association between the activator protein-2beta (AP-2beta) transcription factor gene and type 2 diabetes. This gene is preferentially expressed in adipose tissue, and subjects with a disease-susceptible allele of AP-2beta showed stronger AP-2beta expression in adipose tissue than those without the susceptible allele. Furthermore, overexpression of AP-2beta led to lipid accumulation and induced insulin resistance in 3T3-L1 adipocytes.We found that overexpression of AP-2beta in 3T3-L1 adipocytes decreased the promoter activity of leptin, and subsequently decreased both messenger RNA (mRNA) and protein expression and secretion. Furthermore, knockdown of endogenous AP-2beta by RNA-interference increased mRNA and protein expression of leptin. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed specific binding of AP-2beta to leptin promoter regions in vitro and in vivo. In addition, site-directed mutagenesis of the AP-2-binding site located between position +34 and +42 relative to the transcription start site abolished the inhibitory effect of AP-2beta. Our results clearly showed that AP-2beta directly inhibited insulin-sensitizing hormone leptin expression by binding to its promoter.AP-2beta modulated the expression of leptin through direct interaction with its promoter region.
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- 2010
9. A novel ultra-sensitive enzyme immunoassay for soluble human insulin receptor ectodomain and its measurement in urine from healthy subjects and patients with diabetes mellitus
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Asako Umehara, Hiroshi Shiota, Seiichi Hashida, Toshiyuki Obata, Yousuke Ebina, and Mamiko Nishioka
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Adult ,Blood Glucose ,Leptin ,Male ,medicine.medical_specialty ,Diabetes risk ,Adolescent ,medicine.medical_treatment ,Urinary system ,Clinical Biochemistry ,Urine ,Sensitivity and Specificity ,Immunoenzyme Techniques ,Young Adult ,chemistry.chemical_compound ,Antigens, CD ,Internal medicine ,Diabetes mellitus ,parasitic diseases ,Diabetes Mellitus ,medicine ,Humans ,Insulin ,Resistin ,Obesity ,Creatinine ,Binding Sites ,biology ,medicine.diagnostic_test ,business.industry ,Reproducibility of Results ,General Medicine ,Glucose Tolerance Test ,Middle Aged ,medicine.disease ,Receptor, Insulin ,Circadian Rhythm ,Insulin receptor ,Endocrinology ,chemistry ,Immunoassay ,Calibration ,biology.protein ,Female ,business - Abstract
Objective For the early identification of patients at risk of developing diabetes mellitus, and to prevent the onset of diabetes by performing dietary counseling and exercise guidance, we have developed an ultra-sensitive immune complex transfer enzyme immunoassay (ICT-EIA) to measure soluble human insulin receptor ectodomain (sIRα) in urine which is collected non-invasively. Design and methods We developed ICT-EIA for sIRα and measured urinary sIRα from 106 healthy volunteers, 35 obese volunteers and 42 patients with diabetes. Results The detection limit of ICT-EIA (0.04 pg/mL), using a urine sample of as little as 100 μL, was a few hundred-fold higher than that of conventional ELISA. Using ICT-EIA, the urinary sIRα level in patients with diabetes (9.7 ± 20.1 pg/mg creatinine) was significantly higher than those in healthy volunteers (1.4 ± 0.9; P Conclusion ICT-EIA for sIRα may be useful as a good marker for evaluating diabetes risk.
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- 2009
10. SAFB1, an RBMX-binding protein, is a newly identified regulator of hepatic SREBP-1c gene
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Hiroshi Maegawa, Satoshi Ugi, Chikako Ikeuchi, Yoshihiko Nishio, Osamu Sekine, Tadashi Takemoto, Atsunori Kashiwagi, Katsutaro Morino, Hiroshi Kimura, Yasushi Omura, Yasuhiro Maeno, and Toshiyuki Obata
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Male ,Chromatin Immunoprecipitation ,Immunoprecipitation ,Biology ,Biochemistry ,Heterogeneous-Nuclear Ribonucleoproteins ,Mice ,RNA interference ,Cell Line, Tumor ,Two-Hybrid System Techniques ,Gene expression ,polycyclic compounds ,Transcriptional regulation ,Animals ,Promoter Regions, Genetic ,Molecular Biology ,Gene ,Regulation of gene expression ,Binding protein ,RNA-Binding Proteins ,food and beverages ,General Medicine ,Molecular biology ,Rats ,DNA-Binding Proteins ,lipids (amino acids, peptides, and proteins) ,Sterol Regulatory Element Binding Protein 1 ,Chromatin immunoprecipitation ,Protein Binding - Abstract
Sterol regulatory element-binding protein (SREBP)-1c plays a crucial role in the regulation of lipogenic enzymes in the liver. We previously reported that an X-chromosome-linked RNA binding motif (RBMX) regulates the promoter activity of Srebp-1c. However, still unknown was how it regulates the gene expression. To elucidate this mechanism, we screened the cDNA library from mouse liver by yeast two-hybrid assay using RBMX as bait and identified scaffold attachment factor B1 (SAFB1). Immunoprecipitation assay demonstrated binding of SAFB1 to RBMX. Chromatin immunoprecipitation assay showed binding of both SAFB1 and RBMX to the upstream region of Srebp-1c gene. RNA interference of Safb1 reduced the basal and RBMX-induced Srebp-1c promoter activities, resulting in reduced Srebp-1c gene expression. The effect of SAFB1 overexpression on Srebp-1c promoter was found only in the presence of RBMX. These results indicate a major role for SAFB1 in the activation of Srebp-1c through its interaction with RBMX.
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- 2009
11. The Rab GTPase-Activating Protein AS160 as a Common Regulator of Insulin- and G.ALPHA.q-Mediated Intracellular GLUT4 Vesicle Distribution
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Toshiyuki Obata, Tomoyuki Yuasa, Yousuke Ebina, Hiroyuki Sano, Keiji Uchiyama, Kazushiro Kishi, Kiyoshi Teshigawara, Yoshinori Tanaka, Toshio Hosaka, Yuko Ogura, and Masafumi Kimura
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endocrine system diseases ,Endocrinology, Diabetes and Metabolism ,Chromosomal translocation ,CHO Cells ,AMP-Activated Protein Kinases ,Mice ,Cricetulus ,Endocrinology ,3T3-L1 Cells ,Cricetinae ,Animals ,Humans ,Insulin ,Protein kinase A ,Protein kinase B ,Glucose Transporter Type 4 ,biology ,GTPase-Activating Proteins ,Glucose transporter ,nutritional and metabolic diseases ,musculoskeletal system ,Rats ,Cell biology ,biology.protein ,GTP-Binding Protein alpha Subunits, Gq-G11 ,Phosphorylation ,Rab ,hormones, hormone substitutes, and hormone antagonists ,GLUT4 ,Intracellular - Abstract
Akt substrate of 160kDa (AS160) is a Rab GTPase activating protein (GAP) and was recently identified as a component of the insulin signaling pathway of glucose transporter type 4 (GLUT4) translocation. We and others, previously reported that the activation of Galphaq protein-coupled receptors (GalphaqPCRs) also stimulated GLUT4 translocation and glucose uptake in several cell lines. Here, we report that the activation of GalphaqPCRs also promoted phosphorylation of AS160 by the 5'-AMP activated protein kinase (AMPK). The suppression of AS160 phosphorylation by the siRNA mediated AMPKalpha1 subunit knockdown promoted GLUT4 vesicle retention in intracellular compartments. This suppression did not affect the ratio of non-induced cell surface GLUT4 to Galphaq-induced it. Rat 3Y1 cells lacking AS160 did not show insulin-induced GLUT4 translocation. The cells stably expressing GLUT4 revealed GLUT4 vesicles that were mainly localized in the perinuclear region and less frequently on the cell surface. After expression of exogenous AS160, GLUT4 on the cell surface decreased and GLUT4 vesicles were redistributed throughout the cytoplasm. Although PMA-induced or sodium fluoride-induced GLUT4 translocation was significantly increased in these cells, insulin did not affect GLUT4 translocation. These results suggest that AS160 is a common regulator of insulin- and GalphaqPCR activation-mediated GLUT4 distribution in the cells.
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- 2009
12. Transcription Factor Activating Protein-2β: A Positive Regulator of Monocyte Chemoattractant Protein-1 Gene Expression
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Yukie Nakai, Shiro Maeda, Yoshihiko Nishio, Satoshi Ugi, Motoyuki Kondo, Toshiyuki Obata, Hiroshi Maegawa, Atsunori Kashiwagi, Katsutaro Morino, and Kazuhiro Ikeda
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Chromatin Immunoprecipitation ,Blotting, Western ,Genetic Vectors ,Adipose tissue ,Electrophoretic Mobility Shift Assay ,Enzyme-Linked Immunosorbent Assay ,Response Elements ,Adenoviridae ,Mice ,Endocrinology ,3T3-L1 Cells ,Gene expression ,Animals ,Humans ,RNA, Small Interfering ,Promoter Regions, Genetic ,Transcription factor ,Chemokine CCL2 ,ATF3 ,Gene knockdown ,biology ,Reverse Transcriptase Polymerase Chain Reaction ,DNA ,TCF4 ,Molecular biology ,Activating transcription factor 2 ,Gene Expression Regulation ,Transcription Factor AP-2 ,TAF2 ,biology.protein ,Plasmids ,Protein Binding - Abstract
We previously reported an association between the activating protein (AP)-2beta transcription factor gene and type 2 diabetes. This gene is preferentially expressed in adipose tissue, and subjects with a disease-susceptible allele of AP-2beta showed stronger AP-2beta expression in adipose tissue than those without the susceptible allele. Furthermore, overexpression of AP-2beta leads to lipid accumulation by enhancing glucose transport and inducing insulin resistance in 3T3-L1 adipocytes. In this study, we found that overexpression of AP-2beta in 3T3-L1 adipocytes accelerated the promoter activity of monocyte chemoattractant protein-1 (MCP-1) and subsequently increased both mRNA and protein expression and protein secretion. Furthermore, knockdown of endogenous AP-2beta by RNA interference reduced the mRNA and the protein expression of MCP-1. EMSAs and chromatin immunoprecipitation assays revealed specific binding of AP-2beta to MCP-1 promoter regions, in vitro and in vivo. Additionally, site-directed mutagenesis of the AP-2 binding site located at -137 to -129 relative to the transcription start site markedly diminished MCP-1 promoter activity, whereas other putative AP-2 binding sites did not. Our results clearly show that AP-2beta directly enhanced MCP-1 secretion by binding to its promoter. Thus, we propose that AP-2beta positively regulates MCP-1 expression; subsequently contributes to the infiltration of macrophages to adipose tissue; and leads to insulin resistance, type 2 diabetes, and cardiovascular diseases.
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- 2008
13. Regulation of the PI3K-Akt Network: Current Status and a Promise for the Treatment of Human Diseases
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Masayuki Noguchi, Toshiyuki Obata, and Futoshi Suizu
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Endocrinology ,Chemistry ,Pharmacology (medical) ,Current (fluid) ,Neuroscience ,Protein kinase B ,PI3K/AKT/mTOR pathway - Published
- 2008
14. Investigation of defects in GaN with varying Mg doping concentrations
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Yoshinobu Aoyagi, Naoki Matsumura, Hideki Hirayama, Toshiyuki Obata, Koji Ishibashi, and Kiyoshi Ogiwara
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Condensed Matter::Materials Science ,chemistry ,Impurity ,Doping ,Analytical chemistry ,chemistry.chemical_element ,Chemical vapor deposition ,Conductivity ,Condensed Matter Physics ,Spectroscopy ,Rutherford backscattering spectrometry ,Oxygen ,Nitrogen - Abstract
We report on the electrical and lattice properties of heavily Mg-doped GaN in which the conductivity is inverted to n-type, as grown by low-pressure (LP) metalorganic chemical vapour deposition. We suggested that n-type inverted conductivity is caused by self-compensation effects induced by nitrogen vacancies, and not by unintentionally-doped impurities such as oxygen or carbon by performing Hall-effect and secondary-ion-mass spectroscopy (SIMS) measurements. We directly observed the configurations of N and Ga atoms in Mg-doped GaN by using the Rutherford backscattering spectrometry (RBS)-channeling method. We found that Mg-doping causes the degradation of both the Ga and N lattice properties. We observed for the first time that the density or the lattice relaxation amplitude of N atoms was larger than that of Ga atoms. (© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)
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- 2006
15. Intra-islet insulin suppresses glucagon release via GABA-GABAA receptor system
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Yousuke Ebina, Shiying Liu, Qinghua Wang, Toshiyuki Obata, Matthias Braun, Shaoping Deng, Anna Wendt, Mohan Kumar, William Ju, Yi Zhang, Michael B. Wheeler, Nina Zhang, and Elaine Xu
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Male ,endocrine system ,medicine.medical_specialty ,Physiology ,medicine.medical_treatment ,Guinea Pigs ,HUMDISEASE ,030209 endocrinology & metabolism ,Biology ,Glucagon ,Models, Biological ,Alpha cell ,Rats, Sprague-Dawley ,03 medical and health sciences ,Islets of Langerhans ,0302 clinical medicine ,Insulin resistance ,Internal medicine ,medicine ,Animals ,Humans ,Insulin ,GABA-A Receptor Antagonists ,Protein kinase B ,Molecular Biology ,Glucagon-like peptide 1 receptor ,030304 developmental biology ,0303 health sciences ,Glucagon secretion ,Membrane hyperpolarization ,Cell Biology ,medicine.disease ,Receptors, GABA-A ,Rats ,Endocrinology ,SIGNALING ,Glucagon-Secreting Cells ,CELLBIO ,Female ,Insulin Resistance ,hormones, hormone substitutes, and hormone antagonists - Abstract
Excessive secretion of glucagon is a major contributor to the development of diabetic hyperglycemia. Secretion of glucagon is regulated by various nutrients, with glucose being a primary determinant of the rate of alpha cell glucagon secretion. The intra-islet action of insulin is essential to exert the effect of glucose on the alpha cells since, in the absence of insulin, glucose is not able to suppress glucagon release in vivo. However, the precise mechanism by which insulin suppresses glucagon secretion from alpha cells is unknown. In this study, we show that insulin induces activation of GABAA receptors in the alpha cells by receptor translocation via an Akt kinase-dependent pathway. This leads to membrane hyperpolarization in the alpha cells and, ultimately, suppression of glucagon secretion. We propose that defects in this pathway(s) contribute to diabetic hyperglycemia.
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- 2006
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16. Inhibition of Akt Kinase Activity by a Peptide Spanning the βA Strand of the Proto-oncogene TCL1
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Futoshi Okada, Masayuki Noguchi, Makoto Hiromura, Toshiyuki Obata, Daniel Auguin, Takeshi Shibata, and Christian Roumestand
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Models, Molecular ,Magnetic Resonance Spectroscopy ,Time Factors ,Protein Conformation ,AKT1 ,Apoptosis ,Phosphatidylinositols ,Proto-Oncogene Mas ,Biochemistry ,Mice ,Neoplasms ,Enzyme Inhibitors ,Phosphorylation ,Glutathione Transferase ,Blood Proteins ,Mitochondria ,Cell biology ,Pleckstrin homology domain ,Protein Transport ,Protein Binding ,Molecular Sequence Data ,Antineoplastic Agents ,Protein Serine-Threonine Kinases ,Biology ,Binding, Competitive ,Permeability ,Cell Line ,Cell Line, Tumor ,Proto-Oncogene Proteins ,Animals ,Humans ,Immunoprecipitation ,Amino Acid Sequence ,Kinase activity ,Molecular Biology ,Protein kinase B ,PI3K/AKT/mTOR pathway ,Cell Proliferation ,Dose-Response Relationship, Drug ,Sequence Homology, Amino Acid ,Cell growth ,Akt/PKB signaling pathway ,Cell Membrane ,Cell Biology ,Lipid Metabolism ,Phosphoproteins ,Protein Structure, Tertiary ,Mice, Inbred C57BL ,Kinetics ,Microscopy, Fluorescence ,Peptides ,Proto-Oncogene Proteins c-akt ,Neoplasm Transplantation - Abstract
Akt plays a central role in the regulation of cellular anti-apoptosis underlying various human neoplastic diseases. We have demonstrated previously that TCL1 (a proto-oncogene underlying human T cell prolymphocytic leukemia) interacts with Akt and functions as an Akt kinase co-activator. With the aim to develop an Akt kinase inhibitor, we hypothesized that a peptide, which spans the Akt-binding site, binds to Akt and modulates Akt kinase activity and its downstream biological responses. Indeed, we demonstrated that a peptide, named "Akt-in" (Akt inhibitor, NH(2)-AVTDHPDRLWAWEKF-COOH, encompassing the betaA strand of human TCL1), interacted with Akt and specifically inhibited its kinase activity. Nuclear magnetic resonance studies suggested that interaction of Akt-in with the pleckstrin homology domain (PH) of Akt caused conformational changes on the variable loop 1 of Akt, the locus mediating phosphoinositide binding. Consistently, interaction of Akt-in with the Akt PH domain prevented phosphoinositide binding and hence inhibited membrane translocation and activation of Akt. Moreover, Akt-in inhibited not only cellular proliferation and anti-apoptosis in vitro but also in vivo tumor growth without any adverse effect. The roles of Akt, which possesses a PH domain, in intracellular signaling were well established. Hence, Akt inhibitors create an attractive target for anticancer therapy. However, no effective inhibitors specific for Akt have been developed. Akt-in, which inhibits association of phosphatidylinositol with Akt, is the first molecule to demonstrate specific Akt kinase inhibition potency. This observation will facilitate the design of specific inhibitors for Akt, a core intracellular survival factor underlying various human neoplastic diseases.
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- 2004
17. Protein Phosphatase 2A Negatively Regulates Insulin's Metabolic Signaling Pathway by Inhibiting Akt (Protein Kinase B) Activity in 3T3-L1 Adipocytes
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Yousuke Ebina, Toshiyuki Obata, Jerrold M. Olefsky, Kun Shi, Takeshi Yoshizaki, Takeshi Imamura, Satoshi Ugi, Katsuya Egawa, Atsunori Kashiwagi, and Hiroshi Maegawa
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Protein Serine-Threonine Kinases ,environment and public health ,3-Phosphoinositide-Dependent Protein Kinases ,Glycogen Synthase Kinase 3 ,Phosphatidylinositol 3-Kinases ,GSK-3 ,Proto-Oncogene Proteins ,Adipocytes ,Phosphoprotein Phosphatases ,Humans ,Insulin ,Protein Phosphatase 2 ,Phosphorylation ,Antigens, Viral, Tumor ,Cell Growth and Development ,Molecular Biology ,Protein kinase B ,Protein kinase C ,PI3K/AKT/mTOR pathway ,Glycogen Synthase Kinase 3 beta ,biology ,Akt/PKB signaling pathway ,Cell Biology ,Protein phosphatase 2 ,Phosphoproteins ,Molecular biology ,Insulin Receptor Substrate Proteins ,biology.protein ,Mitogen-Activated Protein Kinases ,Proto-Oncogene Proteins c-akt ,GLUT4 ,Signal Transduction - Abstract
Protein phosphatase 2A (PP2A) is a multimeric serine/threonine phosphatase which has multiple functions, including inhibition of the mitogen-activated protein (MAP) kinase pathway. Simian virus 40 small t antigen specifically inhibits PP2A function by binding to the PP2A regulatory subunit, interfering with the ability of PP2A to associate with its cellular substrates. We have reported that the expression of small t antigen inhibits PP2A association with Shc, leading to augmentation of insulin and epidermal growth factor-induced Shc phosphorylation with enhanced activation of the Ras/MAP kinase pathway. However, the potential involvement of PP2A in insulin's metabolic signaling pathway is presently unknown. To assess this, we overexpressed small t antigen in 3T3-L1 adipocytes by adenovirus-mediated gene transfer and found that the phosphorylation of Akt and its downstream target, glycogen synthase kinase 3beta, were enhanced both in the absence and in the presence of insulin. Furthermore, protein kinase C lambda (PKC lambda) activity was also augmented in small-t-antigen-expressing 3T3-L1 adipocytes. Consistent with this result, both basal and insulin-stimulated glucose uptake were enhanced in these cells. In support of this result, when inhibitory anti-PP2A antibody was microinjected into 3T3-L1 adipocytes, we found a twofold increase in GLUT4 translocation in the absence of insulin. The small-t-antigen-induced increase in Akt and PKC lambda activities was not inhibited by wortmannin, while the ability of small t antigen to enhance glucose transport was inhibited by dominant negative Akt (DN-Akt) expression and Akt small interfering RNA (siRNA) but not by DN-PKC lambda expression or PKC lambda siRNA. We conclude that PP2A is a negative regulator of insulin's metabolic signaling pathway by promoting dephosphorylation and inactivation of Akt and PKC lambda and that most of the effects of PP2A to inhibit glucose transport are mediated through Akt.
- Published
- 2004
18. Growth and annealing conditions of high Al-content p-type AlGaN for deep-UV LEDs
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Koji Ishibashi, Toshiyuki Obata, Yoshinobu Aoyagi, and Hideki Hirayama
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Materials science ,Annealing (metallurgy) ,business.industry ,Doping ,Chemical vapor deposition ,Condensed Matter Physics ,Laser ,Electronic, Optical and Magnetic Materials ,law.invention ,Hall effect ,law ,Optoelectronics ,Metalorganic vapour phase epitaxy ,business ,Diode ,Light-emitting diode - Abstract
We report on growth and annealing conditions for high Al-content p-type AlGaN that is required to achieve deep-ultraviolet (UV) light-emitting diodes (LEDs) or laser diodes (LDs). Mg-doped AlGaN epilayers with the Al-contents between 0 and 40% were grown by low-pressure (LP) metalorganic chemical vapor deposition (MOCVD). We found that the suitable annealing temperature for p-Al0.22Ga0.78N is around 900 °C, which is approximately 50 °C higher than that for p-GaN. We also demonstrated that the use of a lower V/III ratio and a higher growth temperature are preferable for the growth of high Al-content p-AlGaN, in comparison with the case of p-GaN. By controlling the growth and annealing conditions, we have achieved hole concentrations as high as 2.0 × 1018 cm–3 for Mg-doped AlGaN with an Al-content of 32% as determined by Hall effect measurement. (© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)
- Published
- 2004
19. The phosphatidylinositol-3 kinase pathway regulates bladder cancer cell invasion
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X. Wu, R. W. De Vere White, Colleen A Sweeney, R.A. Highshaw, Toshiyuki Obata, and Q. Khan
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Pathology ,medicine.medical_specialty ,Morpholines ,Urology ,Phosphatidylinositol 3-Kinases ,Cell Line, Tumor ,medicine ,Humans ,PTEN ,Neoplasm Invasiveness ,Enzyme Inhibitors ,Protein kinase B ,PI3K/AKT/mTOR pathway ,Urinary bladder ,Bladder cancer ,biology ,Akt/PKB signaling pathway ,business.industry ,Tumor Suppressor Proteins ,PTEN Phosphohydrolase ,Cancer ,medicine.disease ,Phosphoric Monoester Hydrolases ,medicine.anatomical_structure ,Urinary Bladder Neoplasms ,Chromones ,Cancer cell ,biology.protein ,Cancer research ,business - Abstract
Three of the studies described in this section relate to bladder cancer. The first of these concerns the PI-3 kinase pathway, which has been a topic of interest in cancer in general. The authors from Sacramento suggest that it may regulate cancer cell invasion, and hope that this may lead to translational therapeutic uses. Another study describes the pharmacological characteristics of Ro115-1240, which is a selective alpha1A/1L adrenoceptor partial agonist, a compound which may have a future in treating stress urinary incontinence. OBJECTIVES To investigate the role of the phosphatidylinositol (PI)-3 kinase pathway in the invasion of bladder cancer cell lines, and to assess the activation of this pathway in primary human bladder tumours. MATERIALS AND METHODS Human bladder cancer cells were treated with pathway specific inhibitors or were transfected with PI-3 kinase pathway components. The invasion of cultured bladder cancer cells was analysed by an invasion assay. Bladder cancer cells lines and primary human bladder tumours were analysed for pathway activation by western blotting. RESULTS A specific inhibitor of PI-3 kinase enzyme activity, Ly294002, potently suppressed the invasive properties of three highly invasive bladder tumour cell lines. Restoration of the PTEN gene to invasive UM-UC-3 bladder tumour cells or expression of a dominant-negative version of the PI-3 kinase target, Akt, also potently inhibited invasion, indicating a central role for the PI-3 kinase/Akt pathway in this process. In addition, 55% of primary tumours from patients with bladder cancer had markedly high levels of phosphorylated Akt. CONCLUSION Pharmacological or biochemical inhibition of the PI-3 kinase pathway drastically reduced the invasive capacity of bladder cancer cell lines; over half of primary human bladder tumours had high Akt phosphorylation, suggesting that the aberrant activation of this pathway may contribute to the invasion of a significant subset of bladder cancers.
- Published
- 2004
20. ZFH4 protein is expressed in many neurons of developing rat brain
- Author
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Masahiko Kanamori, Kiyoshi Takagawa, Makoto Kawaguchi, Masakiyo Sasahara, Toshiyuki Obata, Yoko Ishii, Shigeharu Nogami, Hemragul Sabit, Tomoatsu Kimura, Nobuo Sakata, and Takeshi Oya
- Subjects
Cellular differentiation ,Blotting, Western ,Mesenchymal cell differentiation ,Nerve Tissue Proteins ,Biology ,medicine ,Animals ,Tissue Distribution ,Northern blot ,Rats, Wistar ,Homeodomain Proteins ,Regulation of gene expression ,Zinc finger ,General Neuroscience ,Neurogenesis ,Brain ,Gene Expression Regulation, Developmental ,Cell Differentiation ,Zinc Fingers ,Blotting, Northern ,Immunohistochemistry ,Molecular biology ,Rats ,medicine.anatomical_structure ,Cerebral cortex ,Subcommissural organ ,Transcription Factors - Abstract
The zinc finger-homeodomain (ZFH) transcription factors contain a zinc finger motif and a homeodomain that might regulate neural and mesenchymal cell differentiation. We have cloned the ZFH4 gene that encodes a protein with structures closely related to ATBF1. In order to study the expression pattern of ZFH4 in the developing rat brain, we raised an antibody against a glutathione-S-transferase (GST) fusion protein of ZFH4. Western blotting with this antibody identified a gene product of 390 kDa in the normal rat brain. Levels of the protein were high in the brainstem at embryonic and neonatal periods and in the midbrain and diencephalon in neonatal rat brain. In addition, the corresponding mRNA of 12.5 kb was detected by Northern blotting. An immunolocalization study showed that postmitotic neurons in the brainstem were the major site of ZFH4 expression, and the levels of expression varied depending on age and anatomical sites. Expression was transient and weak in precursor cells at early neurogenesis. Although ZFH4 levels decreased after birth, ZFH4 continued to be expressed in the mature neurons including DOPA decarboxylase-positive neurons. High levels of expression were also detected in non-neuronal cells of the subcommissural organ, but the expression was almost undetectable throughout precursor cells to mature neurons in the cerebral cortex and hippocampus. The spatial and temporal expression patterns closely resembled those of ATBF1, and we detected neurons that expressed ZFH4, ATBF1, or both. We postulate that ZFH4 participates in the regulation of neural cell maturation or of region-specific differentiation of the brain.
- Published
- 2004
21. Use of RNA Interference-mediated Gene Silencing and Adenoviral Overexpression to Elucidate the Roles of AKT/Protein Kinase B Isoforms in Insulin Actions
- Author
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Yukiko Gotoh, Kazuhiro Kishi, Lewis C. Cantley, Norihisa Masuyama, Hiroshi Shiota, Yousuke Ebina, Rie Matsushima, Takashi Katome, and Toshiyuki Obata
- Subjects
Time Factors ,Monosaccharide Transport Proteins ,Immunoblotting ,Molecular Sequence Data ,Muscle Proteins ,AKT1 ,AKT2 ,CHO Cells ,Deoxyglucose ,Protein Serine-Threonine Kinases ,Biochemistry ,AKT3 ,Adenoviridae ,Mice ,Cricetinae ,Proto-Oncogene Proteins ,Animals ,Insulin ,Protein Isoforms ,Glucose homeostasis ,Gene Silencing ,Luciferases ,Glycogen synthase ,Molecular Biology ,Protein kinase B ,PI3K/AKT/mTOR pathway ,Gene Library ,Glucose Transporter Type 4 ,Base Sequence ,Dose-Response Relationship, Drug ,biology ,Brain ,3T3 Cells ,Cell Biology ,Precipitin Tests ,Molecular biology ,Rats ,Protein Transport ,Insulin receptor ,Genetic Techniques ,COS Cells ,biology.protein ,RNA Interference ,Proto-Oncogene Proteins c-akt ,Glycogen ,Plasmids - Abstract
Insulin plays a central role in the regulation of glucose homeostasis in part by stimulating glucose uptake and glycogen synthesis. The serine/threonine protein kinase Akt has been proposed to mediate insulin signaling in several processes. However, it is unclear whether Akt is involved in insulin-stimulated glucose uptake and which isoforms of Akt are responsible for each insulin action. We confirmed that expression of a constitutively active Akt, using an adenoviral expression vector, promoted translocation of glucose transporter 4 (GLUT4) to plasma membrane, 2-deoxyglucose (2-DG) uptake, and glycogen synthesis in both Chinese hamster ovary cells and 3T3-L1 adipocytes. Inhibition of Akt either by adenoviral expression of a dominant negative Akt or by the introduction of synthetic 21-mer short interference RNA against Akt markedly reduced insulin-stimulated GLUT4 translocation, 2-DG uptake, and glycogen synthesis. Experiments with isoform-specific short interference RNA revealed that Akt2, and Akt1 to a lesser extent, has an essential role in insulin-stimulated GLUT4 translocation and 2-DG uptake in both cell lines, whereas Akt1 and Akt2 contribute equally to insulin-stimulated glycogen synthesis. These data suggest a prerequisite role of Akt in insulin-stimulated glucose uptake and distinct functions among Akt isoforms.
- Published
- 2003
22. Akt Enhances Mdm2-mediated Ubiquitination and Degradation of p53
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Toshiyuki Obata, Yuko Isazawa, Keiji Tanaka, Toshiaki Suzuki, Yoko Ogawara, Shohei Kishishita, Yukiko Gotoh, and Norihisa Masuyama
- Subjects
Time Factors ,Cell Survival ,Morpholines ,Blotting, Western ,Retroviridae Proteins, Oncogenic ,Apoptosis ,Protein Serine-Threonine Kinases ,Biochemistry ,Phosphatidylinositol 3-Kinases ,chemistry.chemical_compound ,Ubiquitin ,Proto-Oncogene Proteins ,Serine ,Tumor Cells, Cultured ,Humans ,LY294002 ,RNA, Messenger ,Phosphatidylinositol ,Enzyme Inhibitors ,Phosphorylation ,Molecular Biology ,Protein kinase B ,PI3K/AKT/mTOR pathway ,Dose-Response Relationship, Drug ,biology ,Reverse Transcriptase Polymerase Chain Reaction ,Chemistry ,Nuclear Proteins ,Proto-Oncogene Proteins c-mdm2 ,Cell Biology ,Precipitin Tests ,Recombinant Proteins ,Ubiquitin ligase ,Cell biology ,Oncogene Protein v-akt ,enzymes and coenzymes (carbohydrates) ,Microscopy, Fluorescence ,Chromones ,biology.protein ,Cancer research ,Mdm2 ,Tumor Suppressor Protein p53 ,Proto-Oncogene Proteins c-akt ,Plasmids ,Protein Binding ,Subcellular Fractions - Abstract
p53 plays a key role in DNA damage-induced apoptosis. Recent studies have reported that the phosphatidylinositol 3-OH-kinase-Akt pathway inhibits p53-mediated transcription and apoptosis, although the underlying mechanisms have yet to be determined. Mdm2, a ubiquitin ligase for p53, plays a central role in regulation of the stability of p53 and serves as a good substrate for Akt. In this study, we find that expression of Akt reduces the protein levels of p53, at least in part by enhancing the degradation of p53. Both Akt expression and serum treatment induced phosphorylation of Mdm2 at Ser186. Akt-mediated phosphorylation of Mdm2 at Ser186 had little effect on the subcellular localization of Mdm2. However, both Akt expression and serum treatment increased Mdm2 ubiquitination of p53. The serum-induced increase in p53 ubiquitination was blocked by LY294002, a phosphatidylinositol 3-OH-kinase inhibitor. Moreover, when Ser186 was replaced by Ala, Mdm2 became resistant to Akt enhancement of p53 ubiquitination and degradation. Collectively, these results suggest that Akt enhances the ubiquitination-promoting function of Mdm2 by phosphorylation of Ser186, which results in reduction of p53 protein. This study may shed light on the mechanisms by which Akt promotes survival, proliferation, and tumorigenesis.
- Published
- 2002
23. Electrical determination of current injection and internal quantum efficiencies in AlGaN-based deep-ultraviolet light-emitting diodes
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Toru Kinoshita, Naoki Tamari, Guo-Dong Hao, Toshiyuki Obata, and Shin-ichiro Inoue
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010302 applied physics ,Materials science ,business.industry ,02 engineering and technology ,Chemical vapor deposition ,Rate equation ,Electroluminescence ,021001 nanoscience & nanotechnology ,01 natural sciences ,Atomic and Molecular Physics, and Optics ,law.invention ,Optics ,law ,0103 physical sciences ,Optoelectronics ,Quantum efficiency ,Current (fluid) ,0210 nano-technology ,business ,Current density ,Light-emitting diode ,Diode - Abstract
We propose a method to determine the current injection efficiency (CIE) and internal quantum efficiency (IQE) of light-emitting diodes (LEDs) during current injection. The method is based on fourth-order polynomial fitting of a modified rate equation to electroluminescence data. Our method can extract the CIE at low injection current densities, unlike conventional methods that generally assume the CIE to be unity. We apply the method to AlGaN-based deep-ultraviolet LEDs. Results show that the CIE was only approximately 51% at low injection current densities and was almost independent of injection current density up to 100 A/cm2. The peak IQE was 77%.
- Published
- 2017
24. A motif-based profile scanning approach for genome-wide prediction of signaling pathways
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Lewis C. Cantley, Jack Lai, Toshiyuki Obata, Michael B. Yaffe, German Leparc, and Stefano Volinia
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Threonine ,Cell signaling ,Databases, Factual ,Amino Acid Motifs ,Molecular Sequence Data ,Biomedical Engineering ,Bioengineering ,Genomics ,Computational biology ,Biology ,Applied Microbiology and Biotechnology ,Genome ,Mice ,Serine ,Animals ,Humans ,Amino Acid Sequence ,Peptide library ,Genetics ,Internet ,Rats ,Proteome ,Tyrosine ,Molecular Medicine ,Phosphorylation ,Cattle ,Signal transduction ,Sequence motif ,Algorithms ,Software ,Signal Transduction ,Biotechnology - Abstract
The rapid increase in genomic information requires new techniques to infer protein function and predict protein-protein interactions. Bioinformatics identifies modular signaling domains within protein sequences with a high degree of accuracy. In contrast, little success has been achieved in predicting short linear sequence motifs within proteins targeted by these domains to form complex signaling networks. Here we describe a peptide library-based searching algorithm, accessible over the World Wide Web, that identifies sequence motifs likely to bind to specific protein domains such as 14-3-3, SH2, and SH3 domains, or likely to be phosphorylated by specific protein kinases such as Src and AKT. Predictions from database searches for proteins containing motifs matching two different domains in a common signaling pathway provides a much higher success rate. This technology facilitates prediction of cell signaling networks within proteomes, and could aid in the identification of drug targets for the treatment of human diseases.
- Published
- 2001
25. Peptide and Protein Library Screening Defines Optimal Substrate Motifs for AKT/PKB
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Lewis C. Cantley, German Leparc, Atsunori Kashiwagi, Toshiyuki Obata, Elizabeth T. Piro, Hiroshi Maegawa, Michael B. Yaffe, and Ryuichi Kikkawa
- Subjects
Models, Molecular ,Tyrosine 3-Monooxygenase ,Proto-Oncogene Proteins c-akt ,Amino Acid Motifs ,Molecular Sequence Data ,AKT1 ,Protein Serine-Threonine Kinases ,Biology ,Proto-Oncogene Mas ,Biochemistry ,Substrate Specificity ,Mice ,Peptide Library ,Proto-Oncogene Proteins ,Consensus Sequence ,Animals ,Humans ,Amino Acid Sequence ,Amino Acids ,Cloning, Molecular ,Phosphorylation ,Phosphoamino Acids ,Protein kinase A ,Peptide library ,Molecular Biology ,Protein kinase B ,Gene Library ,Binding Sites ,Akt/PKB signaling pathway ,Kinase ,Cyclin-dependent kinase 2 ,Cell Biology ,Recombinant Proteins ,Protein Structure, Tertiary ,Kinetics ,14-3-3 Proteins ,biology.protein ,Peptides ,HeLa Cells - Abstract
AKT was originally identified as a proto-oncogene with a pleckstrin homology and Ser/Thr protein kinase domains. Recent studies revealed that AKT regulates a variety of cellular functions including cell survival, cell growth, cell differentiation, cell cycle progression, transcription, translation, and cellular metabolism. To clarify the substrate specificity of AKT, we have used an oriented peptide library approach to determine optimal amino acids at positions N-terminal and C-terminal to the site of phosphorylation. The predicted optimal peptide substrate (Arg-Lys-Arg-Xaa-Arg-Thr-Tyr-Ser*-Phe-Gly where Ser* is the phosphorylation site) has similarities to but is distinct from optimal substrates that we previously defined for related basophilic protein kinases such as protein kinase A, Ser/Arg-rich kinases, and protein kinase C family members. The positions most important for high V(max)/K(m) ratio were Arg-3>Arg-5>Arg-7. The substrate specificity of AKT was further investigated by screening a lambdaGEX phage HeLa cell cDNA expression library. All of the substrates identified by this procedure contained Arg-Xaa-Arg-Xaa-Xaa-(Ser/Thr) motifs and were in close agreement with the motif identified by peptide library screening. The results of this study should help in prediction of likely AKT substrates from primary sequences.
- Published
- 2000
26. Expression of a Dominant Negative SHP-2 in Transgenic Mice Induces Insulin Resistance
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Takahiko Sakamoto, Ryuichi Kikkawa, Masakazu Haneda, Yoshihiko Nishio, Hiroshi Maegawa, Toshiyuki Obata, Atsunori Kashiwagi, Katsutaro Morino, Masaaki Hasegawa, Katsuya Egawa, Satoshi Ugi, Satoshi Sugai, Hideto Kojima, Hitoshi Yasuda, and Toshiki Fujita
- Subjects
Male ,medicine.medical_specialty ,medicine.medical_treatment ,Glucose uptake ,Mice, Transgenic ,Protein Tyrosine Phosphatase, Non-Receptor Type 11 ,Carbohydrate metabolism ,Biochemistry ,Mice ,chemistry.chemical_compound ,Insulin resistance ,Internal medicine ,medicine ,Animals ,Insulin ,Muscle, Skeletal ,Glycogen synthase ,Molecular Biology ,Genes, Dominant ,biology ,Protein Tyrosine Phosphatase, Non-Receptor Type 6 ,Intracellular Signaling Peptides and Proteins ,Tyrosine phosphorylation ,Cell Biology ,medicine.disease ,IRS1 ,Enzyme Activation ,Insulin receptor ,Glucose ,Glycogen Synthase ,Endocrinology ,chemistry ,biology.protein ,Insulin Resistance ,Mitogen-Activated Protein Kinases ,Protein Tyrosine Phosphatases ,Signal Transduction - Abstract
To elucidate the roles of SHP-2, we generated transgenic (Tg) mice expressing a dominant negative mutant lacking protein tyrosine phosphatase domain (DeltaPTP). On examining two lines of Tg mice identified by Southern blot, the transgene product was expressed in skeletal muscle, liver, and adipose tissues, and insulin-induced association of insulin receptor substrate 1 with endogenous SHP-2 was inhibited, confirming that DeltaPTP has a dominant negative property. The intraperitoneal glucose loading test demonstrated an increase in blood glucose levels in Tg mice. Plasma insulin levels in Tg mice after 4 h fasting were 3 times greater with comparable blood glucose levels. To estimate insulin sensitivity by a constant glucose, insulin, and somatostatin infusion, steady state blood glucose levels were higher, suggesting the presence of insulin resistance. Furthermore, we observed the impairment of insulin-stimulated glucose uptake in muscle and adipocytes in the presence of physiological concentrations of insulin. Moreover, tyrosine phosphorylation of insulin receptor substrate-1 and stimulation of phosphatidylinositol 3-kinase and Akt kinase activities by insulin were attenuated in muscle and liver. These results indicate that the inhibition of endogenous SHP-2 function by the overexpression of a dominant negative mutant may lead to impaired insulin sensitivity of glucose metabolism, and thus SHP-2 may function to modulate insulin signaling in target tissues.
- Published
- 1999
27. High Glucose-Induced Abnormal Epidermal Growth Factor Signaling
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Ryuichi Kikkawa, Atsunori Kashiwagi, Hiroshi Maegawa, Tahir S Pillay, and Toshiyuki Obata
- Subjects
medicine.medical_specialty ,medicine.drug_class ,medicine.medical_treatment ,Phosphatase ,Protein tyrosine phosphatase ,Biology ,Biochemistry ,chemistry.chemical_compound ,Cytosol ,Epidermal growth factor ,Internal medicine ,medicine ,Animals ,Humans ,Hypoglycemic Agents ,RNA, Messenger ,Phosphatidylinositol ,Phosphorylation ,Thiazolidinedione ,Molecular Biology ,Cells, Cultured ,Epidermal Growth Factor ,Pioglitazone ,Insulin ,Cell Membrane ,General Medicine ,Fibroblasts ,Receptor, Insulin ,Rats ,Isoenzymes ,Thiazoles ,Insulin receptor ,Glucose ,Endocrinology ,chemistry ,Calcium-Calmodulin-Dependent Protein Kinases ,biology.protein ,Thiazolidinediones ,Protein Tyrosine Phosphatases ,Signal transduction ,hormones, hormone substitutes, and hormone antagonists ,Signal Transduction - Abstract
We have reported that high glucose conditions (27 mM for 4 days) induces activation of protein tyrosine phosphatases (PTPases) which are associated with impaired insulin signaling in Rat 1 fibroblasts expressing human insulin receptors [Maegawa, H. et al. (1995) J. Biol. Chem. 270, 7724-7730]. In this study, we found increased mRNA-levels of a non-receptor type PTPase, protein tyrosine phosphatase 1B (PTP1B), and receptor type PTPases, leukocyte common antigen-related phosphatase (LAR), and LAR-related phosphatase (LRP), under high glucose conditions. In accordance with these results, LAR content was significantly increased, whereas LRP content was not increased. Cytosolic PTP1B content was increased, but membrane-associated PTP1B content showed no detectable change. Pioglitazone, a thiazolidinedione, normalized increased cytosolic PTPase activity through reduction of cytosolic PTP1B content, but it had no effect on mRNA levels of these PTPases. Under the high glucose condition, we also found that epidermal growth factor (EGF)-stimulated signaling, including tyrosine-phosphorylation of EGF receptor and phosphatidylinositol 3'-kinase activities, was attenuated. Nevertheless, pioglitazone failed to restore the attenuated EGF-signaling. These results indicate that the high glucose conditions cause dysfunction of EGF receptor. However, the increased cytosolic PTP1B content is not involved in the abnormal regulation of EGF-signaling, in contrast to insulin-signaling.
- Published
- 1998
28. PO187 RAPID GLUCOSE LOWERING EFFECT OF SITAGLIPTIN IN POORLY CONTROLLED TYPE 2 DIABETIC PATIENTS (SUNSHINE STUDY)
- Author
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Yoshihiko Nishio, Toshiyuki Obata, Satoshi Ugi, M. Nishimura, Hiroshi Maegawa, Takaaki Nakamura, A. Kishi, Atsunori Kashiwagi, Katsutaro Morino, S. Yamada, Yasuo Kida, Yasushi Omura, M. Yamanaka, and Katsuya Egawa
- Subjects
Glucose lowering ,medicine.medical_specialty ,business.industry ,Endocrinology, Diabetes and Metabolism ,General Medicine ,medicine.disease ,Endocrinology ,Sitagliptin ,Internal medicine ,Diabetes mellitus ,Internal Medicine ,medicine ,business ,medicine.drug - Published
- 2014
29. Pyruvate improves deleterious effects of high glucose on activation of pentose phosphate pathway and glutathione redox cycle in endothelial cells
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Toshiyuki Obata, Noriko Takahara, Atsunori Kashiwagi, Hideki Hidaka, Ryuichi Kikkawa, Natsuki Harada, Takayuki Asahina, Motoyoshi Ikebuchi, Yasushi Tanaka, Hideki Taki, Yoshihiko Nishio, and Yukikazu Saeki
- Subjects
medicine.medical_specialty ,Umbilical Veins ,Endocrinology, Diabetes and Metabolism ,Pentose phosphate pathway ,Biology ,medicine.disease_cause ,Umbilical vein ,Pentose Phosphate Pathway ,chemistry.chemical_compound ,Adenosine Triphosphate ,Diabetes mellitus ,Internal medicine ,Pyruvic Acid ,medicine ,Fructosediphosphates ,Internal Medicine ,Humans ,Lactic Acid ,Cells, Cultured ,Glutathione ,Hydrogen Peroxide ,medicine.disease ,NAD ,In vitro ,Endothelial stem cell ,Endocrinology ,Glucose ,L-Glucose ,chemistry ,Endothelium, Vascular ,Oxidation-Reduction ,Oxidative stress ,NADP - Abstract
In our previous study (Diabetes 44:520–526, 1995), endothelial cells cultured in high glucose condition showed impairment of an oxidant-induced activation of the pentose phosphate pathway (PPP) and a reduced supply of NADPH to the glutathione redox cycle. To gain insight into the mechanisms of this impairment, the protective effect of pyruvate was studied in human umbilical vein endothelial cells cultured in either 5.5 mmol/l glucose (normal glucose [NG] condition) or 33 mmol/l glucose (high glucose [HG] condition). Through pretreatment of cells with 0.2 mmol/l pyruvate for 5–7 days in the HG condition, glucose oxidation through the PPP and total cellular NADPH content in the presence of 0.2 mmol/l H2O2 were increased by 54 (P < 0.05) and 34%, respectively, and glutathione-dependent degradation of H2O2 in HG cells was enhanced by 41% (P < 0.01), when compared with those cells to which pyruvate was not added. The addition of pyruvate significantly reduced the fructose 1,6-bisphosphate (FDP) content and free cytoplasmic NADH/NAD ratio, estimated by increased pyruvate/lactate ratio in NG and HG cells exposed to H2O2. Furthermore, the addition of pyruvate also showed a 46% reduction (P < 0.01) of endothelial cell damage induced by H2O2 in HG cells. These results indicate that abnormalities in PPP activation and glutathione redox cycle activity induced by H2O2 in HG cells are compensated, and that the accentuated reductive stress is improved by an addition of pyruvate. These pyruvate effects are associated with protection against an oxidant-induced endothelial cell injury in the high glucose condition.
- Published
- 1997
30. SHPTP2 Serves Adapter Protein Linking between Janus Kinase 2 and Insulin Receptor Substrates
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Toshiki Fujita, Masaaki Hasegawa, Hiroshi Maegawa, Yoshihiko Nishio, Atsunori Kashiwagi, Ryuichi Kikkawa, Hideto Kojima, Toshiyuki Obata, Satoshi Ugi, and Hideki Hidaka
- Subjects
Insulin Receptor Substrate Proteins ,Biophysics ,Protein Tyrosine Phosphatase, Non-Receptor Type 11 ,CHO Cells ,Biochemistry ,Cell Line ,Cricetinae ,Proto-Oncogene Proteins ,hemic and lymphatic diseases ,Insulin receptor substrate ,Animals ,Insulin ,Phosphorylation ,Molecular Biology ,Protein kinase B ,Janus kinase 2 ,biology ,Chemistry ,Protein Tyrosine Phosphatase, Non-Receptor Type 6 ,GRB10 ,Intracellular Signaling Peptides and Proteins ,food and beverages ,Janus Kinase 1 ,Cell Biology ,Janus Kinase 2 ,Protein-Tyrosine Kinases ,Phosphoproteins ,Receptor, Insulin ,IRS2 ,Rats ,Insulin receptor ,biology.protein ,Protein Tyrosine Phosphatases ,Signal Transduction ,Janus Kinase Family - Abstract
To investigate the role of Janus kinase family (JAK1 and JAK2) in insulin signaling, we assessed their insulin-induced associations with other molecules in the cells overexpressing insulin receptors (HIRc and CHO-IR). After insulin stimulation, pp185 proteins (insulin receptor substrate, IRS) were co-immunoprecipitated with both kinases by alpha JAK1 and alpha JAK2 antibodies. However, JAK2 constitutively associated with pp95 protein (IR beta). Moreover, JAK2 also constitutively bound to a protein tyrosine phosphatase containing Src 2 regions (SHPTP2), but JAK1 did not. In HIRc cells expressing PTPase-negative mutant SHPTP2, no association of JAK2 with either pp185 or pp95 was detected. Thus, SHPTP2 might serve as an adapter protein linking between JAK2 and IRS. These results suggest that JAK1 and JAK2 behave differently and they may constitute a new regulatory component in insulin signaling.
- Published
- 1996
31. Postprandial activation of protein kinase C� regulates the expression of adipocytokines via the transcription factor AP-2β
- Author
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Atsunori Kashiwagi, Takeshi Yoshizaki, Katsutaro Morino, Hiroshi Maegawa, Satoshi Ugi, Toshiyuki Obata, Motoyuki Kondo, Shiro Maeda, Tomoya Fuke, and Yoshihiko Nishio
- Subjects
Male ,Transcriptional Activation ,medicine.medical_specialty ,Adipokine ,Biology ,Mice ,Insulin resistance ,Adipokines ,3T3-L1 Cells ,Internal medicine ,Adipocytes ,Genetics ,medicine ,Animals ,E2F1 ,Protein kinase A ,Transcription factor ,Chemokine CCL2 ,Protein Kinase C ,Protein kinase C ,Adiponectin ,Interleukin-6 ,General Medicine ,Postprandial Period ,medicine.disease ,Mice, Inbred C57BL ,Endocrinology ,Postprandial ,Transcription Factor AP-2 ,Cancer research - Abstract
Abnormal secretion of adipocytokines promotes atherosclerosis, diabetes and insulin resistance, and is mainly induced by adipocyte hypertrophy. Recently, the circulating adipocytokine concentrations were reported to change in the postprandial period, as the levels of TNFα, IL-6 IL-8 and MCP-1 increased after a meal, whereas that of adiponectin decreased. These data suggest that prandial modulation of cytokines may be involved in the pathogenesis of atherosclerosis in type 2 diabetes. However, the regulatory mechanism of such change is still unclear. In the present study, we identified this mechanism with a special focus on the functions of protein kinase C (PKC) and of the transcription factor AP-2β, both of which are associated with the pathophysiology of adipocytokine regulation. PKCµ was highly phosphorylated in the re-feeding condition compared to the fasting condition in mouse adipose tissue, while other PKC isoforms remained unchanged. Furthermore, overexpression of PKCµ in 3T3-L1 adipocytes, but not other PKC isoforms, positively regulated the mRNA expression and promoter activity of MCP-1 and IL-6, and negatively regulated those of adiponectin. AP-2β had similar effects on the expression and promoter activity of these adipocytokines. Interestingly, overexpression of PKCµ enhanced the stimulatory and inhibitory effects of AP-2β on the expression of these adipocytokines. Finally, PKCµ could not activate a mutant MCP-1 promoter lacking the AP-2β binding domain. Our results suggest that postprandial activation of PKCµ plays a role in disordered postprandial adipocytokine expression through AP-2β.
- Published
- 2011
32. Association between serum soluble TNFα receptors and renal dysfunction in type 2 diabetic patients without proteinuria
- Author
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Hiromichi Kawai, Satoshi Ugi, Atsunori Kashiwagi, Shin-ichi Araki, Katsutaro Morino, Takeshi Yoshizaki, Daisuke Koya, Takashi Uzu, Yoshihiko Nishio, Hiroshi Maegawa, Toshiyuki Obata, Itsuko Miyazawa, Masakazu Haneda, and Aya Kadota
- Subjects
Male ,medicine.medical_specialty ,Endocrinology, Diabetes and Metabolism ,Renal function ,Enzyme-Linked Immunosorbent Assay ,Gastroenterology ,Endocrinology ,Sex Factors ,Diabetes mellitus ,Internal medicine ,Internal Medicine ,medicine ,Humans ,Receptors, Tumor Necrosis Factor, Type II ,Receptor ,Tumor necrosis factor α ,Aged ,Proteinuria ,business.industry ,Tumor Necrosis Factor-alpha ,General Medicine ,Middle Aged ,medicine.disease ,Diabetes Mellitus, Type 2 ,Receptors, Tumor Necrosis Factor, Type I ,Tumor necrosis factor alpha ,Female ,medicine.symptom ,business ,Glomerular Filtration Rate - Abstract
Aim The aim of our study was to investigate whether serum levels of soluble tumor necrosis factor α receptor (sTNFR) 1 and 2 are markers for renal dysfunction in type 2 diabetic patients without overt proteinuria. Methods Japanese type 2 diabetic patients without overt proteinuria (n = 168) enrolled in the prospective observational follow-up study in 2001 were retrospectively analyzed. At baseline, the serum levels of sTNFR1 and sTNFR2 were measured by sandwich ELISA. The associations between these markers and change in estimated glomerular filtration rate (eGFR) after 5 years were evaluated. Results The levels of sTNFR1 and sTNFR2 closely correlated. At baseline, sTNFR1 and sTNFR2 associated inversely with eGFR. After 5 years, patients with high level of both sTNFR1 and sTNFR2 showed a greater decline in eGFR (−13.8 ± 15.5% versus −8.5 ± 11.8%, P = 0.027) and a 4-fold higher risk for a GFR decline of ≥25% than those with high level of only one receptor or low level of both receptors. These associations were enhanced in diabetic women. Conclusions The higher levels of sTNFR1 and sTNFR2 were associated with a greater decline in eGFR in type 2 diabetic patients without proteinuria, especially in diabetic women.
- Published
- 2010
33. Hypoxia prevents seizures and neuronal damages of the hippocampus induced by kainic acid in rats
- Author
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Toshiyuki Obata, Shigeru Amano, Morimi Shimada, Nishizawa Kashiro, and Fumitada Hazama
- Subjects
Male ,medicine.medical_specialty ,Kainic acid ,Blood Pressure ,Endogeny ,Hippocampus ,chemistry.chemical_compound ,Seizures ,Neuronal damage ,Internal medicine ,medicine ,Animals ,Hypoxia ,Molecular Biology ,Neurons ,Kainic Acid ,Behavior, Animal ,General Neuroscience ,Rats, Inbred Strains ,Carbon Dioxide ,Hydrogen-Ion Concentration ,Hypoxia (medical) ,Adenosine ,Rats ,Oxygen ,Blood pressure ,Endocrinology ,nervous system ,chemistry ,Moderate hypoxia ,Neurology (clinical) ,medicine.symptom ,Neuroscience ,Developmental Biology ,medicine.drug - Abstract
The effects of the hypoxia on the epileptic seizures and neuronal damages induced by kainic acid were studied in rats using hypoic chamber equipment. Rats treated with kainic acid and placed in atmospheric pressure showed typical limbic seizures and regressive neuronal changes in CA3 and CA4 of the hippocampus, while those kept in a hypoxic chamber with 8.5% O 2 and 91.5% N 2 showed moderate hypoxia and a lsight decline of mean arterial blood pressure. In these hypoxic rats, seizures were completely prevented and there was remarkably less regressive neuronal injury of the hippocampus. Thus hypoxia has a rather ameliorative effect of the occurrence od seizures and excitotoxic neuronal injuries induced by kainic acid. The contribution of oxygen radicals and endogenous adenosine to preventing excitotoxic neuronal damages by kainic acid was discussed.
- Published
- 1990
34. Deficiency of Cbl-b gene enhances infiltration and activation of macrophages in adipose tissue and causes peripheral insulin resistance in mice
- Author
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Takeshi Nikawa, Shin'ichi Takeda, Yutaka Nakaya, Nagakatsu Harada, Katsuya Hirasaka, Hua Gu, Toshio Hosaka, Shohei Kohno, Jumpei Goto, Kazumi Ishidoh, Toshiyuki Obata, Kyoichi Kishi, Kazuaki Mawatari, Yousuke Ebina, and Harumi Furochi
- Subjects
Blood Glucose ,medicine.medical_specialty ,Endocrinology, Diabetes and Metabolism ,medicine.medical_treatment ,Glucose uptake ,Adipose tissue ,White adipose tissue ,Biology ,environment and public health ,Mice ,Insulin resistance ,hemic and lymphatic diseases ,Internal medicine ,Glucose Intolerance ,Internal Medicine ,medicine ,Adipocytes ,Animals ,Homeostasis ,Insulin ,Proto-Oncogene Proteins c-cbl ,Crosses, Genetic ,Adaptor Proteins, Signal Transducing ,Mice, Knockout ,fungi ,Biological Transport ,3T3 Cells ,Glucose Tolerance Test ,Macrophage Activation ,medicine.disease ,Flow Cytometry ,Coculture Techniques ,IRS1 ,Mice, Inbred C57BL ,enzymes and coenzymes (carbohydrates) ,Endocrinology ,Glucose ,Adipose Tissue ,Tumor necrosis factor alpha ,Insulin Resistance ,Energy Metabolism ,hormones, hormone substitutes, and hormone antagonists - Abstract
OBJECTIVE—c-Cbl plays an important role in whole-body fuel homeostasis by regulating insulin action. In the present study, we examined the role of Cbl-b, another member of the Cbl family, in insulin action. RESEARCH DESIGN AND METHODS—C57BL/6 (Cbl-b+/+) or Cbl-b-deficient (Cbl-b−/−) mice were subjected to insulin and glucose tolerance tests and a hyperinsulinemic-euglycemic clamp test. Infiltration of macrophages into white adipose tissue (WAT) was assessed by immunohistochemistry and flow cytometry. We examined macrophage activation using co-cultures of 3T3-L1 adipocytes and peritoneal macrophages. RESULTS—Elderly Cbl-b−/− mice developed glucose intolerance and peripheral insulin resistance; serum insulin concentrations after a glucose challenge were always higher in elderly Cbl-b−/− mice than age-matched Cbl-b+/+ mice. Deficiency of the Cbl-b gene significantly decreased the uptake of 2-deoxyglucose into WAT and glucose infusion rate, whereas fatty liver was apparent in elderly Cbl-b−/− mice. Cbl-b deficiency was associated with infiltration of macrophages into the WAT and expression of cytokines, such as tumor necrosis factor-α, interleukin-6, and monocyte chemoattractant protein (MCP)-1. Co-culture of Cbl-b−/− macrophages with 3T3-L1 adipocytes induced leptin expression and dephosphorylation of insulin receptor substrate 1, leading to impaired glucose uptake in adipocytes. Furthermore, Vav1, a key factor in macrophage activation, was highly phosphorylated in peritoneal Cbl-b−/− macrophages compared with Cbl-b+/+ macrophages. Treatment with a neutralizing anti–MCP-1 antibody improved peripheral insulin resistance and macrophage infiltration into WAT in elderly Cbl-b−/− mice. CONCLUSIONS—Cbl-b is a negative regulator of macrophage infiltration and activation, and macrophage activation by Cbl-b deficiency contributes to the peripheral insulin resistance and glucose intolerance via cytokines secreted from macrophages.
- Published
- 2007
35. Soluble insulin receptor ectodomain is elevated in the plasma of patients with diabetes
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Eiji Okamoto, Yousuke Ebina, Yoshiko Kanezaki, Kiyoshi Teshigawara, Ichiro Yokota, Toshiyuki Obata, Kazuhiro Kishi, Atsushi Hattori, Toshio Matsumoto, Fumiko Hirota, Tomoyuki Yuasa, Mitsuru Matsumoto, Atsunori Kashiwagi, Kazuhiko Masuda, Kazuhiro Yokoyama, Seiichi Hashida, Yoshinori Tanaka, and Hiroshi Maegawa
- Subjects
medicine.medical_specialty ,Chemistry ,Endocrinology, Diabetes and Metabolism ,Enzyme-Linked Immunosorbent Assay ,Mice, Transgenic ,medicine.disease ,Receptor, Insulin ,Streptozocin ,Diabetes Mellitus, Experimental ,Mice ,Endocrinology ,Diabetes Mellitus, Type 1 ,Ectodomain ,Diabetes Mellitus, Type 2 ,Solubility ,Diabetes mellitus ,Internal medicine ,Soluble insulin ,Internal Medicine ,medicine ,Diabetes Mellitus ,Animals ,Humans ,Receptor - Abstract
OBJECTIVE—Insulin binds to the α-subunit of the insulin receptor (IRα) and subsequently exerts its effects in the cells. The soluble ectodomains of several receptors have been found to circulate in the plasma. Therefore, we hypothesized that soluble human insulin receptor (hIR) ectodomain (α-subunit and a part of β-subunit) may exist in the plasma of diabetic patients. RESEARCH DESIGN AND METHODS—We identified soluble hIR ectodomain in human plasma by a two-step purification followed by immunoblotting and gel-filtration chromatography. Furthermore, we established an hIRα-specific enzyme-linked immunosorbent assay and measured the plasma IRα levels in patients with diabetes. We also investigated this phenomenon in streptozotocin-induced diabetic hIR transgenic mice. RESULTS—Soluble hIRα, but not intact hIRβ or whole hIR, exists in human plasma. The plasma IRα levels were significantly higher in type 1 (2.00 ± 0.60 ng/ml; n = 53) and type 2 (2.26 ± 0.80; n = 473) diabetic patients than in control subjects (1.59 ± 0.40 ng/ml; n = 123 (P < 0.001 vs. control). Plasma IRα level was positively correlated with blood glucose level, and 10–20% of the insulin in plasma bound to hIRα. In the in vivo experiments using diabetic hIR transgenic mice, hyperglycemia was confirmed to increase the plasma hIRα level and the half-life estimated to be ∼6 h. CONCLUSIONS—We propose that the increased soluble IR ectodomain level appears to be a more rapid glycemic marker than A1C or glycoalbumin.
- Published
- 2007
36. AKT1 overexpression in endothelial cells leads to the development of cutaneous vascular malformations in vivo
- Author
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Jacqueline Banyard, Randy Watnick, Allie Sohn, David N. Brindley, Toshiyuki Obata, Elizabeth R. McLaughlin, Lewis C. Cantley, Jack L. Arbiser, Betsy N. Perry, and Cynthia Cohen
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Male ,Pathology ,medicine.medical_specialty ,AKT1 ,Mice, Nude ,Dermatology ,Vascular anomaly ,Cell Line ,Mice ,medicine ,Animals ,Protein kinase B ,PI3K/AKT/mTOR pathway ,Skin ,business.industry ,Vascular malformation ,Endothelial Cells ,General Medicine ,medicine.disease ,Endothelial stem cell ,Vascular endothelial growth factor B ,medicine.anatomical_structure ,Blood Vessels ,Endothelium, Vascular ,business ,Proto-Oncogene Proteins c-akt ,Blood vessel - Abstract
Background Vascular malformations are clinical disorders in which endothelial cells fail to remodel and/or undergo programmed cell death, leading to abnormal persistence of blood vessels. The abnormal persistence of vessels makes therapy difficult because these lesions are resistant to interventions that are effective against hemangiomas. Akt1 is a serine-threonine protein kinase, which is a key mediator of resistance to programmed cell death. Our objective was to determine whether sustained activation of Akt1 could lead to vascular malformation in mice. Observations We examined the effect of constitutive activation of Akt1 in murine endothelial cells (MS1 cells). Overexpression of active AKT1 in MS1 cells led to the development of vascular malformations, characterized by wide endothelial lumens and minimal investment of smooth muscle surrounding the vessels. The histologic features of these vascular malformations is distinct from ras -transformed MS1 cells (angiosarcoma) and suggest that differing signal abnormalities give rise to human vascular malformations vs malignant vascular tumors. Conclusions Inhibition of Akt signaling may be useful in the treatment of vascular malformations. Examination of problematic hemangiomas and vascular malformations for the presence of activated Akt or downstream targets of Akt, such as mammalian target of rapamycin (mTOR), may predict response to treatment with Akt inhibitors or rapamycin. This study provides a potential rationale for the systemic and topical use of these inhibitors for vascular malformations and hemangiomas.
- Published
- 2007
37. Erratum. Increased Insulin Sensitivity and Hypoinsulinemia in APS Knockout Mice. Diabetes 2003;52:2657—2665
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Kazuhiro Kishi, Kiyoshi Takatsu, Sanae Hayashi, Mitsuru Matsumoto, Asako Minami, Fumiko Kajiura, Masanori Iseki, Mizuki Yamada, Yousuke Ebina, Yoshimi Bando, Keisuke Izumi, Yutaka Nakaya, Makoto Ogura, Noboru Furukawa, Miao Wang, Shonna Moodie, Satoshi Takaki, Yukari Takeshita, and Toshiyuki Obata
- Subjects
medicine.medical_specialty ,Endocrinology ,business.industry ,Endocrinology, Diabetes and Metabolism ,Diabetes mellitus ,Internal medicine ,Knockout mouse ,Internal Medicine ,Medicine ,Insulin sensitivity ,business ,medicine.disease ,Hypoinsulinemia - Published
- 2015
38. Fabrication of vertical Schottky barrier diodes on n-type freestanding AlN substrates grown by hydride vapor phase epitaxy
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Toshiyuki Obata, Rie Togashi, Raoul Schlesser, Toru Kinoshita, Yoshinao Kumagai, Shinya Takashima, Zlatko Sitar, Akinori Koukitu, Toru Nagashima, Reo Yamamoto, and Ramόn Collazo
- Subjects
Fabrication ,Materials science ,business.industry ,Hydride ,Schottky barrier ,General Engineering ,General Physics and Astronomy ,Schottky diode ,Substrate (electronics) ,Epitaxy ,Optoelectronics ,Breakdown voltage ,business ,Diode - Abstract
Thick Si-doped AlN layers were homoepitaxially grown by hydride vapor phase epitaxy on AlN(0001) seed substrates. Following the removal of the seed substrate, an n-type AlN substrate with a carrier concentration of 2.4 × 1014 cm−3 was obtained. Vertical Schottky barrier diodes were fabricated by depositing Ni/Au Schottky contacts on the N-polar surface of the substrate. High rectification with a turn-on voltage of approximately 2.2 V was observed. The ideality factor of the diode at room temperature was estimated to be ~8. The reverse breakdown voltage, defined as the leakage current level of 10−3 A/cm2, ranged from 550 to 770 V.
- Published
- 2015
39. Light extraction enhancement of 265 nm deep-ultraviolet light-emitting diodes with over 90 mW output power via an AlN hybrid nanostructure
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Hiroyuki Yanagi, Shin-ichiro Inoue, Toru Kinoshita, Toshiyuki Obata, and Tamari Naoki
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Nanostructure ,Materials science ,Physics and Astronomy (miscellaneous) ,business.industry ,Wide-bandgap semiconductor ,Nanophotonics ,Nitride ,law.invention ,Wavelength ,law ,Optoelectronics ,business ,Photonic crystal ,Light-emitting diode ,Diode - Abstract
Deep-ultraviolet (DUV) aluminum gallium nitride-based light-emitting diodes (LEDs) on transparent aluminum nitride (AlN) substrates with high light extraction efficiency and high power are proposed and demonstrated. The AlN bottom side surface configuration, which is composed of a hybrid structure of photonic crystals and subwavelength nanostructures, has been designed using finite-difference time-domain calculations to enhance light extraction. We have experimentally demonstrated an output power improvement of up to 196% as a result of the use of the embedded high-light-extraction hybrid nanophotonic structure. The DUV-LEDs produced have demonstrated output power as high as 90 mW in DC operation at a peak emission wavelength of 265 nm.
- Published
- 2015
40. Functional analysis of PIK3CA gene mutations in human colorectal cancer
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Masayuki Matsumura, Keisuke Tateishi, Amarsanaa Jazag, Toshiyuki Obata, Tsuneo Ikenoue, Yohko Hikiba, Jun Imamura, Bayasi Guleng, Masao Omata, Takao Kawabe, Yoshinari Asaoka, Miki Ohta, Fumihiko Kanai, and Yasuo Tanaka
- Subjects
Cancer Research ,Class I Phosphatidylinositol 3-Kinases ,Protein Conformation ,Mutant ,Lipid kinase activity ,Cell Growth Processes ,P110α ,Biology ,Protein Serine-Threonine Kinases ,medicine.disease_cause ,Mice ,Phosphatidylinositol 3-Kinases ,Proto-Oncogene Proteins ,medicine ,Biomarkers, Tumor ,Cell Adhesion ,Animals ,Humans ,Kinase activity ,Protein kinase B ,PI3K/AKT/mTOR pathway ,Genetics ,Kinase ,Ribosomal Protein S6 Kinases, 70-kDa ,Molecular biology ,Protein Structure, Tertiary ,Enzyme Activation ,Oncology ,NIH 3T3 Cells ,Carcinogenesis ,Colorectal Neoplasms ,Proto-Oncogene Proteins c-akt - Abstract
Mutations in the PIK3CA gene, which encodes the p110α catalytic subunit of phosphatidylinositol 3-kinase (PI3K), have been reported in human cancers, including colorectal cancer. Most of the mutations cluster at hotspots within the helical and kinase domains. Whereas H1047R, one of the hotspot mutants, is reported to have elevated lipid kinase activity, the functional consequences of other mutations have not been examined. In this study, we examined the effects of colon cancer–associated PIK3CA mutations on the lipid kinase activity in vitro, activation of the downstream targets Akt and p70S6K in vivo and NIH 3T3-transforming ability. Of eight mutations examined, all showed increased lipid kinase activity compared with wild-type p110α. All the mutants strongly activated Akt and p70S6K compared with wild-type p110α as determined by immunoblotting using phospho-specific antibodies. These mutants also induced morphologic changes, loss of contact inhibition, and anchorage-independent growth of NIH 3T3 cells. The hotspot mutations examined in this study, E542K, E545K, and H1047R, all had high enzymatic and transforming activities. These results show that almost all the colon cancer–associated PIK3CA mutations are functionally active so that they are likely to be involved in carcinogenesis.
- Published
- 2005
41. ERK1/2 activation by angiotensin II inhibits insulin-induced glucose uptake in vascular smooth muscle cells
- Author
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Keisuke Ishizawa, Yousuke Ebina, Yoshiko Fujita, Shuhei Tomita, Masanori Yoshizumi, Yuki Izawa, Koichiro Tsuchiya, Yasuhisa Kanematsu, Toshiaki Tamaki, Toshiyuki Obata, and Nermin Ali
- Subjects
Male ,medicine.medical_specialty ,Cytoplasm ,Vascular smooth muscle ,Monosaccharide Transport Proteins ,Glucose uptake ,Myocytes, Smooth Muscle ,Muscle Proteins ,Biology ,Protein Serine-Threonine Kinases ,Rats, Sprague-Dawley ,Insulin resistance ,Internal medicine ,Proto-Oncogene Proteins ,medicine ,Serine ,Animals ,Insulin ,Enzyme Inhibitors ,Phosphorylation ,Protein kinase B ,Mitogen-Activated Protein Kinase 1 ,Glucose Transporter Type 4 ,Mitogen-Activated Protein Kinase 3 ,Angiotensin II ,Cell Membrane ,JNK Mitogen-Activated Protein Kinases ,Cell Biology ,medicine.disease ,Phosphoproteins ,Rats ,Enzyme Activation ,Insulin receptor ,Protein Transport ,Endocrinology ,Glucose ,Mitogen-activated protein kinase ,Hypertension ,biology.protein ,Insulin Receptor Substrate Proteins ,Blood Vessels ,Tyrosine ,Insulin Resistance ,Proto-Oncogene Proteins c-akt ,hormones, hormone substitutes, and hormone antagonists - Abstract
Clinical evidence suggests a relationship between hypertension and insulin resistance, and cross-talk between angiotensin II (Ang II) and insulin signaling pathways may take place. We now report the effect of Ang II on insulin-induced glucose uptake and its intracellular mechanisms in vascular smooth muscle cells (VSMC). We examined the translocation of glucose transporter-4 (GLUT-4) and glucose uptake in rat aortic smooth muscle cells (RASMC). Mitogen-activated protein (MAP) kinases and Akt activities, and phosphorylation of insulin receptor substrate-1 (IRS-1) at the serine and tyrosine residues were measured by immunoprecipitation and immunoblotting. As a result, Ang II inhibited insulin-induced GLUT-4 translocation from cytoplasm to the plasma membrane in RASMC. Ang II induced extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK) activation and IRS-1 phosphorylation at Ser307 and Ser616. Ang II-induced Ser307 and Ser616 phophorylation of IRS-1 was inhibited by a MEK inhibitor, PD98059, and a JNK inhibitor, SP600125. Ang II inhibition of insulin-stimulated IRS-1 tyrosyl phophorylation and Akt activation were reversed by PD98059 but not by SP600125. Ang II inhibited insulin-induced glucose uptake, which was also reversed by PD98059 but not by SP600125. It is shown that Ang II-induced ERK1/2 activation inhibits insulin-dependent glucose uptake through serine phophorylation of IRS-1 in RASMC.
- Published
- 2005
42. Platelet-derived growth factor stimulates glucose transport in skeletal muscles of transgenic mice specifically expressing platelet-derived growth factor receptor in the muscle, but it does not affect blood glucose levels
- Author
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Rei Kakuhata, Tomoyuki Yuasa, Mitsuru Matsumoto, Kazuhiro Kishi, Keisuke Izumi, Yousuke Ebina, Yasuo Shinohara, Fumiko Kajiura, Yoshimi Bando, and Toshiyuki Obata
- Subjects
Blood Glucose ,medicine.medical_specialty ,Platelet-derived growth factor ,Endocrinology, Diabetes and Metabolism ,Glucose uptake ,medicine.medical_treatment ,Mice, Transgenic ,Receptor, Platelet-Derived Growth Factor beta ,chemistry.chemical_compound ,Mice ,Epidermal growth factor ,Internal medicine ,Internal Medicine ,medicine ,Animals ,Insulin ,Muscle, Skeletal ,Protein kinase B ,Platelet-Derived Growth Factor ,biology ,Glucose transporter ,Skeletal muscle ,Biological Transport ,Heart ,Recombinant Proteins ,medicine.anatomical_structure ,Endocrinology ,Glucose ,chemistry ,biology.protein ,GLUT4 ,Signal Transduction - Abstract
Insulin stimulates the disposal of blood glucose into skeletal muscle and adipose tissues by the translocation of GLUT4 from intracellular pools to the plasma membrane, and consequently the concentration of blood glucose levels decreases rapidly in vivo. Phosphatidylinositol (PI) 3-kinase and Akt play a pivotal role in the stimulation of glucose transport by insulin, but detailed mechanisms are unknown. We and others reported that not only insulin but also platelet-derived growth factor (PDGF) and epidermal growth factor facilitate glucose uptake through GLUT4 translocation by activation of PI 3-kinase and Akt in cultured cells. However, opposite results were also reported. We generated transgenic mice that specifically express the PDGF receptor in skeletal muscle. In these mice, PDGF stimulated glucose transport into skeletal muscle in vitro and in vivo. Thus, PDGF apparently shares with insulin some of the signaling molecules needed for the stimulation of glucose transport. The degree of glucose uptake in vivo reached ∼60% of that by insulin injection in skeletal muscle, but blood glucose levels were not decreased by PDGF in these mice. Therefore, PDGF-induced disposal of blood glucose into skeletal muscle is insufficient for rapid decrease of blood glucose levels.
- Published
- 2004
43. K(ATP) channel knockout mice crossbred with transgenic mice expressing a dominant-negative form of human insulin receptor have glucose intolerance but not diabetes
- Author
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Kazuhiro Kishi, Toshio Matsumoto, Yousuke Ebina, Mitsuru Matsumoto, Yoshiko Kanezaki, Yoshimi Bando, Toshiyuki Obata, Asako Minami, Tasuku Mitani, Keisuke Izumi, Yutaka Nakaya, Hisanori Uehara, Rie Matsushima, Tomoyuki Yuasa, and Yukari Takeshita
- Subjects
Genetically modified mouse ,Blood Glucose ,Leptin ,Male ,endocrine system ,medicine.medical_specialty ,Genotype ,Endocrinology, Diabetes and Metabolism ,Mice, Transgenic ,Protein Serine-Threonine Kinases ,Impaired glucose tolerance ,Mice ,Endocrinology ,Insulin resistance ,Internal medicine ,Diabetes mellitus ,Proto-Oncogene Proteins ,Glucose Intolerance ,Insulin Secretion ,medicine ,Animals ,Humans ,Insulin ,Potassium Channels, Inwardly Rectifying ,Genes, Dominant ,Epididymis ,Mice, Knockout ,Glucose tolerance test ,medicine.diagnostic_test ,Chemistry ,Type 2 Diabetes Mellitus ,Fasting ,Organ Size ,Glucose clamp technique ,Glucose Tolerance Test ,medicine.disease ,Postprandial Period ,Receptor, Insulin ,Enzyme Activation ,Glucose ,Adipose Tissue ,Liver ,Knockout mouse ,cardiovascular system ,Glucose Clamp Technique ,Proto-Oncogene Proteins c-akt - Abstract
Impaired insulin secretion and insulin resistance are thought to be two major causes of type 2 diabetes mellitus. There are two kinds of diabetic model mice: one is a K(ATP) channel knockout (Kir6.2KO) mouse which is defective in glucose-induced insulin secretion, and the other is a transgenic mouse expressing the tyrosine kinase-deficient (dominant-negative form of) human insulin receptor (hIR(KM)TG), and which has insulin resistance in muscle and fat. However, all of these mice have no evidence of overt diabetes. To determine if the double mutant Kir6.2KO/hIR(KM)TG mice would have diabetes, we generated mutant mice by crossbreeding, which would show both impaired glucose-induced insulin secretion and insulin resistance in muscle and fat. We report here that: 1) blood glucose levels of randomly fed and 6 h fasted double mutant (Kir6.2KO/hIR(KM)TG) mice were comparable with those of wild type mice; 2) in intraperitoneal glucose tolerance test (ipGTT), Kir6.2KO/hIR(KM)TG mice had an impaired glucose tolerance; and 3) during ipGTT, insulin secretion was not induced in either Kir6.2KO/hIR(KM)TG or Kir6.2KO mice, while the hIR(KM)TG mice showed a more prolonged insulin secretion than did wild type mice; 4) hyperinsulinemic euglycemic clamp test revealed that Kir6.2KO, Kir6.2KO/hIR(KM)TG and hIR(KM)TG mice, showed decreased whole-body glucose disposal compared with wild type mice; 5) Kir6.2KO, but not Kir6.2KO/hIR(KM)TG mice had some obesity and hyperleptinemia compared with wild type mice. Thus, the defects in glucose-induced insulin secretion (Kir6.2KO) and an insulin resistance in muscle and fat (hIR(KM)TG) were not sufficient to lead to overt diabetes.
- Published
- 2004
44. Increased insulin sensitivity and hypoinsulinemia in APS knockout mice
- Author
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Fumiko Kajiura, Mizuki Yamada, Miao Wang, Yousuke Ebina, Yukari Takeshita, Asako Minami, Kazuhiro Kishi, Makoto Ogura, Mitsuru Matsumoto, Keisuke Izumi, Noboru Furukawa, Yutaka Nakaya, Masanori Iseki, Kiyoshi Takatsu, Sanae Hayashi, Yoshimi Bando, Satoshi Takaki, Toshiyuki Obata, and Shonna Moodie
- Subjects
Blood Glucose ,Leptin ,medicine.medical_specialty ,Endocrinology, Diabetes and Metabolism ,medicine.medical_treatment ,Glucose uptake ,Mice ,Internal medicine ,Hyperinsulinism ,Internal Medicine ,medicine ,Adipocytes ,Animals ,Insulin ,Protein kinase B ,Triglycerides ,Adaptor Proteins, Signal Transducing ,Mice, Knockout ,Glucose tolerance test ,biology ,Adiponectin ,medicine.diagnostic_test ,Body Weight ,Glucose transporter ,Proteins ,3T3 Cells ,Glucagon ,Receptor, Insulin ,Insulin receptor ,Adaptor Proteins, Vesicular Transport ,Endocrinology ,Glucose ,biology.protein ,Glucose Clamp Technique ,Intercellular Signaling Peptides and Proteins ,Energy Intake ,GLUT4 - Abstract
A tyrosine kinase adaptor protein containing pleckstrin homology and SH2 domains (APS) is rapidly and strongly tyrosine phosphorylated by insulin receptor kinase upon insulin stimulation. The function of APS in insulin signaling has heretofore remained unknown. APS -deficient ( APS −/− ) mice were used to investigate its function in vivo. The blood glucose-lowering effect of insulin, as assessed by the intraperitoneal insulin tolerance test, was increased in APS −/− mice. Plasma insulin levels during fasting and in the intraperitoneal glucose tolerance test were lower in APS −/− mice. APS −/− mice showed an increase in the whole-body glucose infusion rate as assessed by the hyperinsulinemic-euglycemic clamp test. These findings indicated that APS −/− mice exhibited increased sensitivity to insulin. However, overexpression of wild-type or dominant-negative APS in 3T3L1 adipocytes did not affect insulin receptor numbers, phosphorylations of insulin receptor, insulin receptor substrate-1, or Akt and mitogen-activated protein kinase. The glucose uptake and GLUT4 translocation were not affected by insulin stimulation in these cells. Nevertheless, the insulin-stimulated glucose transport in isolated adipocytes of APS −/− mice was increased over that of APS +/+ mice. APS −/− mice also showed increased serum levels of leptin and adiponectin, which might explain the increased insulin sensitivity of adipocytes.
- Published
- 2003
45. Injection of the insulin receptor alpha subunit increases blood glucose levels in mice
- Author
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Toshio Matsumoto, Rie Matsushima, Yoshiko Kanezaki, Yutaka Nakaya, Toshiyuki Obata, and Yousuke Ebina
- Subjects
Blood Glucose ,medicine.medical_specialty ,Protein Folding ,Glycosylation ,medicine.medical_treatment ,Recombinant Fusion Proteins ,Biophysics ,CHO Cells ,Biology ,Biochemistry ,Mice ,Internal medicine ,Insulin receptor substrate ,Cricetinae ,medicine ,Animals ,Humans ,Insulin ,Phosphorylation ,Receptor ,Molecular Biology ,Glucose tolerance test ,Expression vector ,medicine.diagnostic_test ,Chinese hamster ovary cell ,Cell Biology ,Glucose Tolerance Test ,Receptor, Insulin ,Insulin receptor ,Protein Subunits ,Endocrinology ,Cell culture ,biology.protein ,Injections, Intraperitoneal - Abstract
Using the expression vector of the truncated human insulin receptor (hIR), we have constructed a stable Chinese hamster ovary (CHO) cell line which secretes the His-tagged alpha subunit (insulin-binding domain) of hIR into medium. To examine characteristics of the His-tagged hIRalpha, we purified the protein secreted from the CHO cells. The His-tagged hIRalpha was glycosylated and processed a dimer. The molecule bound insulin with an affinity similar to that of the intact hIR. The His-tagged full length of hIR was autophosphorylated by insulin stimulation in CHO cells. Injection of the purified His-tagged hIRalpha into veins of mice increased in the concentration of blood glucose within 30 min. The intraperitoneal glucose tolerance test (ipGTT) done after injection of the purified His-tagged hIRalpha showed evidence of a marked hyperglycemia. These findings provide direct evidence that the presence of hIRalpha in the blood stream inhibits insulin actions by binding with plasma insulin.
- Published
- 2003
46. Membrane localization of 3-phosphoinositide-dependent protein kinase-1 stimulates activities of Akt and atypical protein kinase C but does not stimulate glucose transport and glycogen synthesis in 3T3-L1 adipocytes
- Author
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Takaaki Nakamura, Atsunori Kashiwagi, Takeshi Yoshizaki, Katsutaro Morino, Hiroshi Maegawa, Katsuya Egawa, Eiji Suzuki, Toshiyuki Obata, Kun Shi, Shinya Shimizu, and Yoshihiko Nishio
- Subjects
Male ,Monosaccharide Transport Proteins ,Recombinant Fusion Proteins ,Molecular Sequence Data ,Muscle Proteins ,Biology ,Protein Serine-Threonine Kinases ,Biochemistry ,3-Phosphoinositide-Dependent Protein Kinases ,Rats, Sprague-Dawley ,chemistry.chemical_compound ,Mice ,Phosphatidylinositol Phosphates ,Proto-Oncogene Proteins ,Adipocytes ,Animals ,Humans ,Insulin ,Phosphorylation ,Protein kinase A ,Glycogen synthase ,Molecular Biology ,Protein kinase B ,Protein kinase C ,Protein Kinase C ,Glucose Transporter Type 4 ,Akt/PKB signaling pathway ,Brain ,Tyrosine phosphorylation ,Biological Transport ,Cell Biology ,3T3 Cells ,Cell biology ,Rats ,Isoenzymes ,Insulin receptor ,Glucose ,chemistry ,biology.protein ,Hepatocytes ,Mitogen-Activated Protein Kinases ,Proto-Oncogene Proteins c-akt ,Glycogen ,Signal Transduction - Abstract
It is reported that 3-phosphoinositide-dependent protein kinase-1 (PDK-1) is activated in a phosphatidylinositol 3,4,5-trisphosphate-dependent manner and phosphorylates Akt, p70S6 kinase, and atypical protein kinase C (PKC), but its function on insulin signaling is still unclear. We cloned a full-length pdk-1 cDNA from a human brain cDNA library, and the adenovirus to overexpress wild type PDK-1 (PDK-1WT) or membrane-targeted PDK-1 (PDK-1CAAX) was constructed. Overexpressed PDK-1WT existed mainly at cytosol, and PDK-1CAAX was located at the plasma membrane. In 3T3-L1 adipocytes, insulin induced mobility shift of PDK-1 protein, but overexpressed PDK-1WT and CAAX were shifted at the basal state. Insulin stimulated tyrosine phosphorylation of PDK-1WT, but PDK-1CAAX was already tyrosine-phosphorylated at the basal state. Overexpression of PDK-1WT led to a full activation of PKC zeta/lambda without insulin stimulation but showed only the minimum effects to stimulate phosphorylation of Akt and GSK-3. In contrast, the overexpression of PDK-1CAAX caused phosphorylation of Akt and GSK-3 more strongly without insulin stimulation. However, PDK-1CAAX did not affect 2-deoxyglucose uptake and inhibited glycogen synthesis, surprisingly. Finally, PDK-1CAAX expression inhibited insulin-induced ERK1/2 phosphorylation in a dose-dependent manner. Taken together, the translocation of PDK-1 from cytosol to the plasma membrane is critical for Akt and GSK-3 activation. On the other hand, only atypical PKC and Akt activation was insufficient for stimulation of glucose transport, and constitutive activation of Akt-GSK-3 pathway may inhibit glycogen synthesis and MAPK cascade in 3T3-L1 adipocytes.
- Published
- 2002
47. Akt/protein kinase B promotes organ growth in transgenic mice
- Author
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Julie R. McMullen, Thomas F. Franke, Pamela S. Douglas, Tetsuo Shioi, Peter M. Kang, Toshiyuki Obata, Lewis C. Cantley, and Seigo Izumo
- Subjects
Genetically modified mouse ,Heart growth ,Cardiomegaly ,Mice, Transgenic ,Growth ,Protein Serine-Threonine Kinases ,Ventricular Function, Left ,Mice ,Phosphatidylinositol 3-Kinases ,Mutant protein ,Proto-Oncogene Proteins ,medicine ,Animals ,Molecular Biology ,Protein kinase B ,Cell Growth and Development ,PI3K/AKT/mTOR pathway ,Sirolimus ,biology ,Kinase ,Myocardium ,Heart ,Cell Biology ,Organ Size ,Molecular biology ,Cell biology ,Insulin receptor ,Mutation ,biology.protein ,Proto-Oncogene Proteins c-akt ,medicine.drug - Abstract
One of the least-understood areas in biology is the determination of the size of animals and their organs. In Drosophila, components of the insulin receptor phosphoinositide 3-kinase (PI3K) pathway determine body, organ, and cell size. Several biochemical studies have suggested that Akt/protein kinase B is one of the important downstream targets of PI3K. To examine the role of Akt in the regulation of organ size in mammals, we have generated and characterized transgenic mice expressing constitutively active Akt (caAkt) or kinase-deficient Akt (kdAkt) specifically in the heart. The heart weight of caAkt transgenic mice was increased 2.0-fold compared with that of nontransgenic mice. The increase in heart size was associated with a comparable increase in myocyte cell size in caAkt mice. The kdAkt mutant protein attenuated the constitutively active PI3K-induced overgrowth of the heart, and the caAkt mutant protein circumvented cardiac growth retardation induced by a kinase-deficient PI3K mutant protein. Rapamycin attenuated caAkt-induced overgrowth of the heart, suggesting that the mammalian target of rapamycin (mTOR) or effectors of mTOR mediated caAkt-induced heart growth. In conclusion, Akt is sufficient to induce a marked increase in heart size and is likely to be one of the effectors of the PI3K pathway in mediating heart growth.
- Published
- 2002
48. Differential regulation of Akt kinase isoforms by the members of the TCL1 oncogene family
- Author
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Gerald Künstle, Masayuki Noguchi, Jarmo Laine, and Toshiyuki Obata
- Subjects
Recombinant Fusion Proteins ,T-Lymphocytes ,Restriction Mapping ,Mitogen-activated protein kinase kinase ,Protein Serine-Threonine Kinases ,Transfection ,Biochemistry ,Polymerase Chain Reaction ,Proto-Oncogene Mas ,Gene Expression Regulation, Enzymologic ,MAP2K7 ,Cell Line ,Proto-Oncogene Proteins ,Proto-Oncogenes ,Animals ,Humans ,ASK1 ,Kinase activity ,Cloning, Molecular ,Phosphorylation ,Molecular Biology ,Protein kinase B ,DNA Primers ,Glutathione Transferase ,Mammals ,MAP kinase kinase kinase ,biology ,Cyclin-dependent kinase 4 ,Cell Biology ,Protein-Tyrosine Kinases ,Molecular biology ,Kinetics ,embryonic structures ,biology.protein ,Cyclin-dependent kinase 9 ,Proto-Oncogene Proteins c-akt ,Protein Binding - Abstract
The members of the TCL1 proto-oncogene family (TCL1, MTCP1, and TCL1b) bind to Akt1, increasing its phosphorylation status and kinase activity. This is thought to be secondary to the formation of TCL1-Akt oligomers within which Akt is preferentially phosphorylated. Here we show that, in contrast to Akt1 and Akt2, which bind to all members of the TCL1 family, Akt3 specifically interacts with TCL1 but not with MTCP1 or TCL1b. This association is functional, as the presence of TCL1 but not MTCP1 or TCL1b increased Akt3 kinase activity in in vitro kinase assays. Functional specificity is determined by the Akt pleckstrin homology domain as chimeric Akt1, where Akt1 PH domain was replaced by that of Akt3 was no longer able to interact with MTCP1 or TCL1b and its kinase activity was solely enhanced by TCL1. Moreover, we show that, in TCL1-overexpressing SUPT-11 T-cell leukemia and P3HR-1 Burkitt's lymphoma cell lines, TCL1 interacts with endogenous Akt1, Akt2, and Akt3. TCL1 enhanced hetero-oligomerization of Akt1 with Akt3 and as a consequence facilitated transphosphorylation of Akt molecules, which may contribute to Akt activation and TCL1-induced leukemogenesis in vivo.
- Published
- 2001
49. The protooncogene TCL1 is an Akt kinase coactivator
- Author
-
Gerald Künstle, Jarmo Laine, Masayuki Noguchi, Ma Sha, and Toshiyuki Obata
- Subjects
X Chromosome ,T-Lymphocytes ,AKT1 ,Biology ,Protein Serine-Threonine Kinases ,Spodoptera ,Lymphocyte Activation ,Transfection ,Translocation, Genetic ,Cell Line ,Membrane Potentials ,src Homology Domains ,Substrate-level phosphorylation ,Glycogen Synthase Kinase 3 ,In vivo ,Proto-Oncogene Proteins ,Coactivator ,Proto-Oncogenes ,Tumor Cells, Cultured ,Animals ,Humans ,Cloning, Molecular ,Molecular Biology ,Protein kinase B ,PI3K/AKT/mTOR pathway ,Chromosomes, Human, Pair 14 ,Binding Sites ,Cell Death ,Cell growth ,Cell Biology ,Intracellular Membranes ,Recombinant Proteins ,Cell biology ,Mitochondria ,Pleckstrin homology domain ,Calcium-Calmodulin-Dependent Protein Kinases ,Cancer research ,bcl-Associated Death Protein ,Carrier Proteins ,Proto-Oncogene Proteins c-akt ,Cell Division - Abstract
Human T cell prolymphocytic leukemia can result from chromosomal translocations involving 14q32.1 or Xq28 regions. The regions encode a family of protooncogenes (TCL1, MTCP1, and TCL1b) of unknown function. In yeast two-hybrid screening, we found that TCL1 interacts with Akt. All TCL1 isoforms bind to the Akt pleckstrin homology domain. Both in vitro and in vivo TCL1 increases Akt kinase activity and as a consequence enhances substrate phosphorylation. In vivo, TCL1 stabilizes the mitochondrial transmembrane potential and enhances cell proliferation and survival. In vivo, TCL1 forms trimers, which associate with Akt. TCL1 facilitates the oligomerization and activation of Akt. Our data show that TCL1 is a novel Akt kinase coactivator, which promotes Akt-induced cell survival and proliferation.
- Published
- 2000
50. MAP kinase pathways activated by stress: the p38 MAPK pathway
- Author
-
Glenn E. Brown, Michael B. Yaffe, and Toshiyuki Obata
- Subjects
biology ,MAP kinase kinase kinase ,Transcription, Genetic ,business.industry ,MAP Kinase Signaling System ,Critical Illness ,Cyclin-dependent kinase 2 ,Apoptosis ,Mitogen-activated protein kinase kinase ,Critical Care and Intensive Care Medicine ,Protein kinase R ,p38 Mitogen-Activated Protein Kinases ,MAP2K7 ,Cell biology ,Stress, Physiological ,Immunology ,biology.protein ,Medicine ,Humans ,Cyclin-dependent kinase 9 ,ASK1 ,c-Raf ,Enzyme Inhibitors ,Mitogen-Activated Protein Kinases ,business - Abstract
A stress-activated serine/threonine protein kinase, p38 mitogen-activated protein kinase (p38 MAPK), belongs to the MAP kinase superfamily. Diverse extracellular stimuli, including ultraviolet light, irradiation, heat shock, high osmotic stress, proinflammatory cytokines and certain mitogens, trigger a stress-regulated protein kinase cascade culminating in activation of p38 MAPK through phosphorylation on a TGY motif within the kinase activation loop. p38 MAPK appears to play a major role in apoptosis, cytokine production, transcriptional regulation, and cytoskeletal reorganization, and has been causally implicated in sepsis, ischemic heart disease, arthritis, human immunodeficiency virus infection, and Alzheimer's disease. The availability of specific inhibitors helps to clarify the role that p38 MAPK plays in these processes, and may ultimately offer therapeutic benefit for certain critically ill patients.
- Published
- 2000
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