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Y Ebina

Publications and source records attributed to Y Ebina.

At least 55 records · Page 3Linked to original sources
PubMed1998

A method for motion compensation of a moving nematode Caenorhabditis elegans and its application to frequency analysis of pharyngeal pulsation.

A new sequential image processing method for motion compensation of a moving object with stringy shape has been developed for estimating the pharyngeal pulsation of the nematode Caenorhabditis elegans under several environmental conditions. The method is based on the pixel data transfer on a new image frame while changing the boundary shape and the position but preserving the conformation of the inner structure of an object. All digitized image frames of C. elegans were first converted to motion-compensated images to arrange the pulsation site in the same region of the every transformed frame. The pulsation site was then automatically detected by determining the pixels where the temporal brightness variation was much larger than that of the other pixels. Finally, the pulsation frequency was determined by the Fourier analysis. The validity of our method has been confirmed by analyzing various test data, and the method has been applied for detecting the pharyngeal pulsation frequencies of C. elegans on some environmental conditions, i.e. feed bacteria-free/rich, doping of nerve inactivating ethyl-alcohol and nerve stimulant neurochemical substance of serotonin. The motion compensation method automatically provided reasonable pulsation frequencies which were found to be comparable to those obtained by manual counting. Thus the method is useful for systematic investigations on the variation of pharyngeal pulsation associated with the activity change of the nervous system in environments.

Algorithms↗
PubMed1998

GLUT4 translocation by insulin in intact muscle cells: detection by a fast and quantitative assay.

We report a rapid and sensitive colorimetric approach to quantitate the amount of glucose transporters exposed at the surface of intact cells, using L6 muscle cells expressing GLUT4 containing an exofacial myc epitope. Unstimulated cells exposed to the surface 5 fmol GLUT4myc per mg protein. This value increased to 10 fmol/mg protein in response to insulin as 2-deoxyglucose (10 microM) uptake doubled. The results are substantiated by immunofluorescent detection of GLUT4myc in unpermeabilized cells and by subcellular fractionation. We further show that wortmannin and the cytoskeleton disruptors cytochalasin D and latrunculin B completely blocked these insulin effects. The rapid quantitative assay described here could be of high value to study insulin signals and to screen for potential anti-diabetic drugs.

Androstadienes↗
PubMed1998

Insulin enhances macrophage scavenger receptor-mediated endocytic uptake of advanced glycation end products.

Hyperglycemia accelerates the formation and accumulation of advanced glycation end products (AGE) in plasma and tissue, which may cause diabetic vascular complications. We recently reported that scavenger receptors expressed by liver endothelial cells (LECs) dominantly mediate the endocytic uptake of AGE proteins from plasma, suggesting its potential role as an eliminating system for AGE proteins in vivo (Smedsrod, B., Melkko, J., Araki, N., Sano, H., and Horiuchi, S. (1997) Biochem. J. 322, 567-573). In the present study we examined the effects of insulin on macrophage scavenger receptor (MSR)-mediated endocytic uptake of AGE proteins. LECs expressing MSR showed an insulin-sensitive increase of endocytic uptake of AGE-bovine serum albumin (AGE-BSA). Next, RAW 264.7 cells expressing a high amount of MSR were overexpressed with human insulin receptor (HIR). Insulin caused a 3.7-fold increase in endocytic uptake of 125I-AGE-BSA by these cells. The effect of insulin was inhibited by wortmannin, a phosphatidylinositol-3-OH kinase (PI3 kinase) inhibitor. To examine at a molecular level the relationship between insulin signal and MSR function, Chinese hamster ovary (CHO) cells expressing a negligible level of MSR were cotransfected with both MSR and HIR. Insulin caused a 1.7-fold increase in the endocytic degradation of 125I-AGE-BSA by these cells, the effect of which was also inhibited by wortmannin and LY294002, another PI3 kinase inhibitor. Transfection of CHO cells overexpressing MSR with two HIR mutants, a kinase-deficient mutant, and another lacking the binding site for insulin receptor substrates (IRS) resulted in disappearance of the stimulatory effect of insulin on endocytic uptake of AGE proteins. The present results indicate that insulin may accelerate MSR-mediated endocytic uptake of AGE proteins through an IRS/PI3 kinase pathway.

Androstadienes↗
PubMed1998

Impaired glucose tolerance in mice with a targeted impairment of insulin action in muscle and adipose tissue.

Type 2 diabetes is a complex metabolic disorder characterized by peripheral insulin resistance and impaired beta cell function. Insulin resistance is inherited as a non-mendelian trait. In genetically predisposed individuals, resistance of skeletal muscle and adipose tissue to insulin action precedes the onset of clinical diabetes, and is thought to contribute to hyperglycaemia by leading to impaired beta cell function and increased hepatic glucose production. It is not clear whether beta cell and liver defects are also genetically determined. To test the hypothesis that insulin resistance in muscle and fat is sufficient to cause type 2 diabetes in the absence of intrinsic beta cell and liver abnormality, we generated transgenic mice that were insulin-resistant in skeletal muscle and adipose tissue. These mice developed all the prodromal features of type 2 diabetes but, despite the compounded effect of peripheral insulin resistance and a mild impairment of beta cell function, failed to become diabetic. These findings indicate the need for a critical re-examination of the primary site(s) of insulin resistance in diabetes.

Adipose Tissue↗
PubMed1998

Rapid stimulation of glucose transport by mitochondrial uncoupling depends in part on cytosolic Ca2+ and cPKC.

2,4-Dinitrophenol (DNP) uncouples the mitochondrial oxidative chain from ATP production, preventing oxidative metabolism. The consequent increase in energy demand is, however, contested by cells increasing glucose uptake to produce ATP via glycolysis. In L6 skeletal muscle cells, DNP rapidly doubles glucose transport, reminiscent of the effect of insulin. However, glucose transport stimulation by DNP does not require insulin receptor substrate-1 phosphorylation and is wortmannin insensitive. We report here that, unlike insulin, DNP does not activate phosphatidylinositol 3-kinase, protein kinase B/Akt, or p70 S6 kinase. However, chelation of intra- and extracellular Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid-AM in conjunction with EGTA inhibited DNP-stimulated glucose uptake by 78.9 +/- 3.5%. Because Ca2+-sensitive, conventional protein kinase C (cPKC) can activate glucose transport in L6 muscle cells, we examined whether cPKC may be translocated and activated in response to DNP in L6 myotubes. Acute DNP treatment led to translocation of cPKCs to plasma membrane. cPKC immunoprecipitated from plasma membranes exhibited a twofold increase in kinase activity in response to DNP. Overnight treatment with 4-phorbol 12-myristate 13-acetate downregulated cPKC isoforms alpha, beta, and gamma and partially inhibited (45.0 +/- 3.6%) DNP- but not insulin-stimulated glucose uptake. Consistent with this, the PKC inhibitor bisindolylmaleimide I blocked PKC enzyme activity at the plasma membrane (100%) and inhibited DNP-stimulated 2-[3H]deoxyglucose uptake (61.2 +/- 2.4%) with no effect on the stimulation of glucose transport by insulin. Finally, the selective PKC-beta inhibitor LY-379196 partially inhibited DNP effects on glucose uptake (66.7 +/- 1.6%). The results suggest interfering with mitochondrial ATP production acts on a signal transduction pathway independent from that of insulin and partly mediated by Ca2+ and cPKCs, of which PKC-beta likely plays a significant role.

2,4-Dinitrophenol↗
PubMed1998

Bradykinin directly triggers GLUT4 translocation via an insulin-independent pathway.

Physical exercise induces translocation of GLUT4 from an intracellular pool to the cell surface in skeletal muscles and increases glucose uptake via an insulin-independent pathway. However, the molecular mechanism remains to be identified. Some studies have suggested that bradykinin is locally released from contracting muscles and may be responsible for GLUT4 translocation and the increase of glucose transport in skeletal muscles. To determine whether bradykinin directly triggers GLUT4 translocation, we established L6 myotubes, 3T3-L1 adipocytes, and Chinese hamster ovary cells stably expressing c-myc epitope-tagged GLUT4 (GLUT4myc) and bradykinin B2 receptors. We found that bradykinin directly triggered GLUT4myc translocation and increased the rate of glucose uptake in a dose-dependent manner in these cells. The translocation with bradykinin occurred even after pretreatment with an islet-activating protein, wortmannin, and phorbol 12,13-dibutyrate. The signaling pathway does not seem to be mediated by Gi, phosphatidylinositol 3-kinase, or protein kinase C. It is insulin-independent and via trimeric G-protein Gq. Bradykinin is probably one of the factors responsible for exercise-stimulated glucose uptake in skeletal muscles.

3T3 Cells↗
PubMed1997

Transcriptional activation of the Glut1 gene in response to oxidative stress in L6 myotubes.

Exposure of L6 myotubes to prolonged low grade oxidative stress results in increased Glut1 expression at both the protein and mRNA levels, leading to elevated glucose transport activity. To further understand the cellular mechanisms responsible for this adaptive response, the Glut1 transcription rate and mRNA stability were assessed. Nuclear run-on assays revealed 2.0- and 2.4-fold increases in Glut1 transcription rates in glucose oxidase- and xanthine/xanthine oxidase-pretreated cells, respectively. Glut1 mRNA stability was increased with both treatments compared with the control (t1/2 = 7.8 +/- 1.3, 6.0 +/- 2.0, and 2.4 +/- 0.5 h, respectively). The serum-responsive element and AP-1 (but not the cAMP-responsive element) showed increased binding capacity following oxidative stress. Both activation of AP-1 binding and elevation of Glut1 mRNA were prevented by cycloheximide. The involvement of enhancer 1 of the Glut1 gene was demonstrated using transfected 293 cells. Induction of Glut1 mRNA in response to oxidative stress differed from its activation by chronic insulin exposure as demonstrated by the ability of rapamycin to inhibit the latter without an effect on the former. In conclusion, oxidative stress increases the Glut1 transcription rate by mechanisms that may involve activation of AP-1 binding to enhancer 1 of the Glut1 gene.

Animals↗
PubMed1997

Hyperinsulinemia but no diabetes in transgenic mice homozygously expressing the tyrosine kinase-deficient human insulin receptor.

We generated transgenic mice homozygous for the tyrosine kinase-deficient human insulin receptor (hIRK1030M(+/+)) under control of the insulin receptor promoter. Similar growth patterns and results of glucose tolerance tests were observed among normal, heterozygous, and homozygous mice. Insulin tolerance test indicated no significant difference in the hypoglycemic response to insulin among the three genotypes. However, the serum insulin levels of the homozygous mice before and after glucose loading (201.42 +/- 58.15 pg/ml to 578.57 +/- 49.03 pg/ml) were significantly higher than in the control mice (100.92 +/- 19.55 pg/ml to 356.36 +/- 55.08 pg/ml; p < 0.01 and p < 0.01, respectively) and heterozygous mice (74.46 +/- 18.55 pg/ml to 352.33 +/- 52.43 pg/ml; p < 0.005 and p < 0.01, respectively). Immunohistological evidence of pancreatic islets showed no significant difference among the three genotypes. Taken together, these results suggest that the tyrosine kinase-deficient insulin receptor causes hyperinsulinemia but not diabetes in these homozygous transgenic mice.

Animals↗
PubMed1997

Lectin affinity electrophoresis in a yolk sac tumour in the vagina with yolk sac tumour-type glycoform of alpha fetoprotein.

AIMS: To investigate a potential diagnostic use of alpha fetoprotein (alpha FP) isoform analysis by lectin affinity electrophoresis to distinguish between endodermal sinus tumours arising in the vagina in infants from those at other sites. METHODS: alpha FP in the serum of a patient with a vaginal endodermal sinus tumour was analysed for its isoforms by lectin affinity electrophoresis. The isoforms were compared with that of cord serum, sera of hepatoid adenocarcinoma of the uterus, and endodermal sinus tumour of the ovary. RESULTS: The isoforms of alpha FP obtained by lectin affinity electrophoresis in the serum of the patient with vaginal endodermal sinus tumour differed from the isoforms of alpha FP in the cord serum of normal neonates, and sera of patients with hepatoid adenocarcinoma of the uterus or endodermal sinus tumour of the ovary. CONCLUSIONS: Endodermal sinus tumour arising in the vagina could be distinguished from that in the ovary by the lectin affinity electrophoresis, and a potential diagnostic use of alpha FP isoform analysis by the lectin affinity electrophoresis for the detection of the endodermal sinus tumour in infants was demonstrated.

Diagnosis, Differential↗
PubMed1996

Gq-coupled receptors transmit the signal for GLUT4 translocation via an insulin-independent pathway.

Guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) induces the translocation of glucose transporter type 4 (GLUT4) from an intracellular pool to the cell surface and increases glucose uptake in adipocytes. The GTP-binding protein(s) responsible for the translocation has remained to be identified. Using a sensitive and quantitative method to assess the translocation of c-MYC epitope-tagged GLUT4, we obtained evidence that the activation of receptor-coupled Gq (neither Gi nor Gs) triggered GLUT4 translocation in cells, independently of insulin signaling pathway(s). Platelet-activating factor (PAF) induced GLUT4 translocation in the cells expressing the Gi- and Gq-coupled PAF receptor, but the translocation was induced even after pretreatment with wortmannin, an islet-activating protein and phorbol 12, 13-dibutyrate. Norepinephrine triggered GLUT4 translocation in cells expressing the Gq-coupled alpha1-adrenergic receptor, but prostaglandin E2 did not cause GLUT4 translocation in cells expressing the Gs-coupled EP4 receptor or the Gi-coupled EP3alpha receptor. The norepinephrine-stimulated GLUT4 translocation and glucose uptake via Gq may possibly contribute to the fuel supply required for thermogenesis in brown adipocytes and for the enhanced contractility in cardiomyocytes, both of which have an abundant endogenous GLUT4.

3T3 Cells↗
PubMed1996

Roles of insulin, guanosine 5'-[gamma-thio]triphosphate and phorbol 12-myristate 13-acetate in signalling pathways of GLUT4 translocation.

Insulin, guanosine 5'-[gamma-thio]triphosphate (GTP[S] and phorbol 12-myristate 13-acetate (PMA) trigger the translocation of Gl UT4 (type 4 glucose transporter; insulin-sensitive glucose transporter) from an intracellular pool to the cell surface. We have developed a highly sensitive and quantitative method to detect GLUT4 immunologically on the surface of intact 3T3-L1 adipocytes and Chinese hamster ovary (CHO) cells, using c-myc epitope-tagged GLUT4 (GLUT4myc). We examined the roles of insulin, GTP[S] and PMA in the signalling pathways of GLUT4 translocation in the CHO cell system. Among small molecular GTP-binding proteins, ras, rab3D, rad and rho seem to be candidates as signal transmitters of insulin-stimulated GLUT4 translocation. Overexpression of wild-type H-ras and the dominant negative mutant H-rass17N in our cell system respectively enhanced and blocked insulin-stimulated activation of mitogen-activated protein kinase, but did not affect insulin-stimulated GLUT4 translocation. Overexpression of rab3D or rad in the cells did not affect GLUT4 translocation triggered by insulin, GTP[S] or PMA. Treatment with Botulinum C3 exoenzyme, a specific inhibitor of rho, had no effect on GLUT4 translocation induced by insulin, GTP[S] or PMA. Therefore these small molecular GTP-binding proteins are not likely to be involved in GLUT4 translocation. In addition, insulin, GTP[S] and PMA apparently stimulate GLUT4 translocation through independent pathways.

3T3 Cells↗
PubMed1996

A phenomenological model describing pharyngeal pulsing in the nematode Caenorhabditis elegans anesthetized by alcohol.

The recovery process of the suppressed pharyngeal pulsation in the nematode has been investigated for several concentrations of a homologous primary alcohol series (CnH2n-1OH, n = 1,2,3). A mathematical model describing the time course of the recovery process is phenomenologically constructed by using two time constants of delay time tD and recovery time tau. The values of tD and tau are obtained by fitting the equation to experimental data. The obtained values increase with increasing alcohol concentration. To observe the characteristics of tD and tau against the alcohol of order n, the inverse of these time constants are computed at 25 v/v% concentration and plotted on a semi-logarithmic scale. The plot curves decrease non-linearly and are dissimilar to the well-known curves illustrating the importance of lipid solubility in the cell membrane in anesthetic phenomena.

Alcohols↗
PubMed1995

Cloning of a cDNA encoding a 190-kDa insulin receptor substrate-1-like protein of simian COS cells.

Major insulin signals such as stimulation of glucose uptake and DNA synthesis and modification of hexose metabolism are mediated by the tyrosine-phosphorylated insulin receptor substrate-1 (IRS-1; pp180) in many species of cells. We cloned cDNA encoding a 190-kDa IRS-1-like protein (pp190) in simian COS cells and which is slightly larger than IRS-1 (pp180) of human, rat, and mouse cells. The deduced amino acid sequence of COS pp190 consisted of 1251 amino acids and was 96.4%, 87.9% and 88.7% identical to human, mouse and rat IRS-1. The COS pp190 bound to SH2 (src-homology 2) domains of p85, Grb2/Ash, and SH-PTP2, as did IRS-1. In IRS-1-knockout mice, insulin signals are thought to be mediated by IRS-2 (pp190), which is an alternative signaling molecule and is slightly larger than IRS-1. However, the COS pp190 may be a simian homologue of IRS-1, but not of IRS-2. The results of Southern blotting suggested the possibility that Chinese hamster ovary (CHO) cells have not only the IRS-1 gene but also a gene related to the COS pp190.

Amino Acid Sequence↗
PubMed1995

Possible domains responsible for intracellular targeting and insulin-dependent translocation of glucose transporter type 4.

Translocation of the type 4 glucose transporter (GLUT4) to the cell surface from an intracellular pool is the major mechanism of insulin-stimulated glucose uptake in insulin-target cells. We developed a highly sensitive and quantitative method to detect GLUT4 immunologically on the surface of intact cells, using c-myc epitope-tagged GLUT4 (GLUT4myc). We constructed c-myc epitope-tagged glucose transporter type 1 (GLUT1myc) and found that the GLUT1myc was also translocated to the cell surface of Chinese hamster ovary cells, 3T3-L1 fibroblasts and NIH 3T3 cells, in response to insulin, but the degree of translocation was less than that of GLUT4myc. Since GLUT1 and GLUT4 have different intracellular distributions and different degrees of insulin-stimulated translocation, we examined the domains of GLUT4, using c-myc epitope-tagged chimeric glucose transporters between these two isoforms. The results indicated that, (1) all the cytoplasmic N-terminal region, middle intracellular loop and cytoplasmic C-terminal region of GLUT4 have independent intracellular targeting signals, (2) these sequences for intracellular targeting of GLUT4 were not sufficient to determine GLUT4 translocation in response to insulin, and (3) the N-terminal half of GLUT4 devoid both of cytoplasmic N-terminus and of middle intracellular loop seems to be necessary for insulin-stimulated GLUT4 translocation.

3T3 Cells↗
PubMed1995

Platelet-derived growth factor triggers translocation of the insulin-regulatable glucose transporter (type 4) predominantly through phosphatidylinositol 3-kinase binding sites on the receptor.

Insulin is the only known hormone which rapidly stimulates glucose uptake in target tissues, mainly by translocation to the cell surface of the intracellular insulin-regulatable glucose transporter (glucose transporter type 4, GLUT4). We have developed a cell line for direct, sensitive detection of GLUT4 on the cell surface. We have suggested that insulin-activated phosphatidylinositol (PI) 3-kinase may be involved in the signaling pathway of insulin-stimulated GLUT4 translocation. We report that platelet-derived growth factor (PDGF), which stimulates PI 3-kinase activity, triggers GLUT4 translocation in Chinese hamster ovary (CHO) cells stably overexpressing the PDGF receptor and in 3T3-L1 mouse adipocytes. Using mutant PDGF receptors that cannot bind to Ras-GTPase-activating protein, phospholipase C-gamma, and PI 3-kinase, respectively, we obtained evidence that PI 3-kinase binding sites play a key role in the signaling pathway of PDGF-stimulated GLUT4 translocation in the CHO cell system.

3T3 Cells↗
PubMed1995

Developmental change in fast Na channel properties in embryonic chick ventricular heart cells.

To assess development changes in kinetic properties of the cardiac sodium current, whole-cell voltage-clamp experiments were conducted using 3-, 10-, and 17-day-old embryonic chick ventricular heart cells. Experimental data were quantified according to the Hodgkin-Huxley model. While the Na current density, as examined by the maximal conductance, drastically increased (six- to seven-fold) with development, other current - voltage parameters remained unchanged. Whereas the activation time constant and the steady-state activation characteristics were comparable among the three age groups, the voltage dependence of the inactivation time constant and the steady-state inactivation underwent a shift in the voltage dependence toward negative potentials during embryonic development. Consequently, the steady-state (window current) conductance, which was sufficient to induce automatic activity in the young embryos, was progressively reduced with age.

Animals↗