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Biomedical subjects

M Kasuga

Publications and source records attributed to M Kasuga.

At least 235 records · Page 13Linked to original sources
PubMed1994

Characterization of a 60-kilodalton substrate of the insulin receptor kinase.

A 60-kDa tyrosine-phosphorylated protein has been observed after insulin treatment of cells in immunoprecipitations of the GTPase-activating protein of Ras (called GAP) as well as the phosphatidylinositol 3-kinase. In the present studies, these two 60-kDa proteins have been shown to differ by limited proteolytic digestions as well as by immunoprecipitation with a monoclonal antibody. This monoclonal antibody was also utilized to show that the 60-kDa GAP-associated protein was rapidly phosphorylated in intact cells after insulin stimulation and to associate with GAP only after insulin treatment of the cells. In addition, the 60-kDa protein was found to be phosphorylated in vitro by the insulin receptor. Finally, the 60-kDa protein immunoprecipitated by this antibody was found not to react with a polyclonal antibody directed against a 62-kDa tyrosine-phosphorylated GAP-associated protein previously observed in src-transformed cells. These studies indicate that insulin stimulates the tyrosine phosphorylation of at least two distinct 60-kDa proteins, one that becomes associated with GAP and appears to be a direct substrate of the insulin receptor kinase and another that associates with the phosphatidylinositol 3-kinase.

Animals↗
PubMed1994

Substitution of tyrosine 293 of GLUT1 locks the transporter into an outward facing conformation.

Tyrosines 292 and 293 in the mammalian glucose transporter GLUT1 have been substituted by either isoleucine or phenylalanine. Chinese hamster ovary clones that were transfected with Tyr-292-->Ile, Tyr-292-->Phe, Tyr-293-->Ile, and Tyr-293-->Phe constructs of GLUT1 were shown, by Western blotting and cell surface carbohydrate labeling, to have expression levels that were comparable with the wild type. The Vmax for 2-deoxy-D-glucose transport was markedly reduced only as a result of the Tyr-293-->Ile mutation. The ability of the Tyr-293-->Ile mutated GLUT1 to bind the exofacial ligand 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos- 4-yloxy)-2- propylamine (ATB-BMPA) and the endofacial ligand cytochalasin B were assessed by photolabeling procedures. The ability to bind the bis-mannose compound was unimpaired, whereas the ability to bind cytochalasin B was totally abolished, and the level of labeling was lower than in the nontransfected clone. Affinities of the wild-type and Tyr-293-->Ile GLUT1 for D-glucose, the exofacial ligands (ATB-BMPA and 4,6-O-ethylidene-D-glucose), and the endofacial ligand (cytochalasin B) were assessed by the ability of these agents to displace the radioactive ATB-BMPA photolabel. These data indicated that the Tyr-293-->Ile substitution produced no change in the affinity for D-glucose, a relatively small enhancement in the affinity for exofacial ligands, but a large approximately 300-fold reduction in affinity for cytochalasin B, suggesting that the mutated GLUT1 is locked in an outward facing conformation. The observation that the Tyr-293-->Ile mutant transporter can bind nontransported C4 and C6 substituted hexose analogues but cannot catalyze transport is interpreted as indicating that Tyr-293 is involved in closing the exofacial site around C4 and C6 of D-glucose in the transport catalysis process.

Affinity Labels↗
PubMed1994

PI 3-kinase: structural and functional analysis of intersubunit interactions.

Phosphatidylinositol (PI) 3-kinase has an 85 kDa subunit (p85 alpha) which mediates its association with activated protein tyrosine kinase receptors through SH2 domains, and an 110 kDa subunit (p110) which has intrinsic catalytic activity. Here p85 alpha and a related protein p85 beta are shown to form stable complexes with recombinant p110 in vivo and in vitro. Using a panel of glutathione S-transferase (GST) fusion proteins of the inter-SH2 region of p85, 104 amino acids were found to bind directly the p110 protein, while deletion mutants within this region further defined the binding site to a sequence of 35 amino acids. Transient expression of the mutant p85 alpha protein in mouse L cells showed it was unable to bind PI 3-kinase activity in vivo. Mapping of the complementary site of interaction on the p110 protein defined 88 amino acids in the N-terminal region of p110 which mediate the binding of this subunit to either the p85 alpha or the p85 beta proteins. The inter-SH2 region of p85 is predicted to be an independently folded module of a coiled-coil of two long anti-parallel alpha-helices. The predicted structure of p85 suggests a basis for the intersubunit interaction and the relevance of this interaction with respect to the regulation of the PI 3-kinase complex is discussed.

Amino Acid Sequence↗
PubMed1994

Substitution at Pro385 of GLUT1 perturbs the glucose transport function by reducing conformational flexibility.

The mammalian glucose transporter, GLUT1, is capable of alternating between two conformations which expose either an outward- or inward-facing ligand binding site. The possibility that these conformational changes are related to the presence of prolines and glycines in transmembrane region 10 was investigated by site-directed mutagenesis. Chinese hamster ovary clones which were transfected with Pro385-->Ile and Pro385-->glycine mutations of GLUT1 were shown, by Western blotting and cell surface carbohydrate labelling, to have expression levels which were comparable with the wild type. The transport activity was markedly reduced as a result of the Pro385-->isoleucine but not in the Pro385-->glycine mutation. The loss of transport activity in the Pro385-->isoleucine clone was associated with loss of labeling by the exofacial photoaffinity ligand, 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannos-4 -yloxy)-2- propylamine (ATB-BMPA), but there was no loss in labeling by the inside site-directed ligand cytochalasin B. These results suggest that the transporter cannot adopt the outward-directed conformation in the Pro385-->isoleucine clone. By contrast, the glycine substitution for proline at this position resulted in a retention of the ligand binding properties at both inside and outside sites. We suggest a putative mode of operation of the transporter which involves conformational flexibility about the prolines in transmembrane segment 10 such that helices 11 and 12 can alternately either pack against the outside (ATB-BMPA binding) site in helices 7, 8, and 9 or against the inner (cytochalasin B binding) site at the base of transmembrane segment 10.

Amino Acid Sequence↗
PubMed1994

A convulsant, 3-mercaptopropionic acid, decreases the level of GABA in pancreatic islets of rat as well as that of brain.

To investigate the properties of the gamma-aminobutyric acid (GABA) synthesizing enzyme, glutamate decarboxylase (GAD), in the brain and the pancreatic islets of the rat, GABA concentration in the brain and the pancreatic islets was measured after administration of 3-mercaptopropionic acid (3-MP) at 25 mg/kg intraperitoneally. Sixty minutes after the administration of 3-MP, GABA concentration in the hypothalamus, the superior colliculus and the hippocampus of the brain decreased by 20-30% and in the pancreatic islets by 35%. The activities of GAD in the pancreatic islets and brain can be modified by a convulsant, in this case 3-MP. These results suggest the properties of GAD may be similar in the pancreatic islets and brain.

3-Mercaptopropionic Acid↗
PubMed1994

Insulin receptor disorders in Japan.

The clinical features of insulin receptor disorders found in Japan are summarized. About 20 cases of primary receptor mutations (type A syndrome of insulin resistance, leprechaunism and Rabson-Mendenhall syndrome) and 16 cases of autoantibodies against insulin receptor (type B syndrome of insulin resistance) are described in Japan. There was a trial of IGF-1 for some of the patients, revealing its usefulness.

Acanthosis Nigricans↗
PubMed1994

Negative and positive effects of intracerebroventricular scopolamine on memory in mice undergoing passive avoidance and escape tests.

The effects of intracerebroventricular administration of scopolamine on memory and learning in the conscious, freely moving mouse were evaluated using step-down passive avoidance and water maze tests. A new technique was used that allows convenient injection into the cerebral ventricles without disturbing the animal's behavior. No significant changes in locomotor activity were observed after low doses of scopolamine (0.1 and 1.0 microgram). However, 10 micrograms produced an increase in locomotor activity, while 100 micrograms caused an initial decrease followed by an increase in activity. In the passive avoidance test, scopolamine significantly impaired memory acquisition at doses higher than 1.0 microgram, consolidation at a dose of 100 micrograms, and retrieval at doses of 10 and 100 micrograms. In contrast, a dose of 0.1 microgram significantly improved consolidation and retrieval. In the water maze with a bridge, scopolamine either impaired memory acquisition, consolidation, and retrieval, or had no significant effect in the dose range tested. These results suggest that there are differences in the process of memory formation in the passive avoidance and escape tests.

Animals↗
PubMed1994

Potentiating effect of insulin on exocrine secretory function in isolated rat pancreatic acini.

BACKGROUND/AIMS: Insulin is shown to exert various regulatory effects on the exocrine pancreatic function. We investigated the direct effect of insulin on exocrine pancreatic secretion. METHODS: The effects of insulin on amylase release, 125I-secretin binding and Na(+)- and K(+)-activated adenosine triphosphate phosphohydrolase (Na+,K(+)-ATPase) activity were measured using the isolated rat pancreatic acini. RESULTS: Insulin potentiated the amylase release elicited by secretin plus cholecystokinin (CCK), but not by either secretin or CCK alone. The potentiating effect of insulin was dependent on the concentration and preincubation time. Insulin had no effect on 125I-secretin binding. Ouabain, a specific Na+,K(+)-ATPase inhibitor, caused a concentration-dependent inhibition of the potentiated secretion by insulin without affecting the secretory response to secretin plus CCK. In membranes prepared from acini treated with insulin, Na+,K(+)-ATPase activity was significantly increased. Similar results were obtained when acini were treated with insulin in combination with secretin plus CCK. CONCLUSIONS: Insulin exerts a direct effect on pancreatic acinar cells and potentiates exocrine secretion elicited by secretin in combination with CCK, in part, by increasing Na+,K(+)-ATPase activity.

Amylases↗
PubMed1994

Role of SH-PTP2, a protein-tyrosine phosphatase with Src homology 2 domains, in insulin-stimulated Ras activation.

SH-PTP2 is a nontransmembrane human protein-tyrosine phosphatase that contains two Src homology 2 (SH2) domains and binds to insulin receptor substrate 1 (IRS-1) via these domains in response to insulin. The expression of a catalytically inactive mutant of SH-PTP2 (containing the mutation Cys-459-->Ser) in Chinese hamster ovary cells that overexpress human insulin receptors (CHO-IR cells) markedly attenuated insulin-stimulated Ras activation. Expression of mutant SH-PTP2 also inhibited MAP kinase activation in response to insulin but not in response to 12-O-tetradecanoyl phorbol-13-acetate. In contrast, the insulin-induced association of phosphoinositide 3-kinase activity with IRS-1 was not affected by the expression of inactive SH-PTP2. Furthermore, the expression of mutant SH-PTP2 had no effect on the binding of Grb2 to IRS-1, on the tyrosine phosphorylation of Shc, or on the formation of the complex between Shc and Grb2 in response to insulin. However, the amount of SH-PTP2 bound to IRS-1 in insulin-treated CHO-IR cells expressing mutant SH-PTP2 was greater than that observed in CHO-IR cells overexpressing wild-type SH-PTP2. Recombinant SH-PTP2 specifically dephosphorylated a synthetic phosphopeptide corresponding to the sequence surrounding Tyr-1172 of IRS-1, a putative binding site for SH-PTP2. Additionally, phenylarsine oxide, an inhibitor of protein-tyrosine phosphatases, inactivated SH-PTP2 in vitro and increased the insulin-induced association of SH-PTP2 with IRS-1. These results suggest that SH-PTP2 may regulate an upstream element necessary for Ras activation in response to insulin and that this upstream element may be required for the Grb2- or Shc-dependent pathway. Furthermore, these results are consistent with the notion that SH-PTP2 may bind to IRS-1 through its SH2 domains in response to insulin and dephosphorylate the phosphotyrosine residue to which it binds, thereby regulating its association with IRS-1.

Adaptor Proteins, Signal Transducing↗
PubMed1994

A 3-basepair in-frame deletion (delta Leu999) in exon 17 of the insulin receptor gene in a family with insulin resistance.

We studied a woman with acanthosis nigricans and insulin resistance. The patient's Epstein-Barr virus-transformed lymphocytes revealed slightly decreased insulin binding and markedly decreased insulin-stimulated autophosphorylation of the insulin receptor. The nucleotide sequence analysis of the patient's genomic DNA revealed a 3-basepair in-frame deletion in one allele, resulting in the loss of leucine at position 999 of the insulin receptor (delta Leu999). The messenger ribonucleic acid transcripts from the mutant allele in the patient's lymphocytes were not decreased. Insulin-stimulated autophosphorylation of the insulin receptor from cells expressing delta Leu999 mutant insulin receptor complementary DNA was markedly decreased. The proband, her mother, elder brother, and younger brother, who were heterozygous for this mutation, showed moderate or marked hyperinsulinemia during oral glucose tolerance tests. Although fasting glucose levels were normal and fasting insulin values were preserved in all subjects with the mutation for the 8-yr period of observation, a tendency of progressive increase in postload glucose levels was observed. These results suggest that the delta Leu999 mutation, which reduces tyrosine kinase activity, was responsible for insulin resistance and contributed to postload hyperglycemia.

Acanthosis Nigricans↗
PubMed1994

Severe insulin-resistant diabetes mellitus associated with hypereosinophilic syndrome.

We describe a 52-year-old male manifesting severe insulin resistance associated with hypereosinophilic syndrome (HES). Diabetes mellitus was initially well-controlled by an oral hypoglycemic agent, and thereafter by human insulin. Due to the progression of hypereosinophilia, hepatosplenomegaly and peripheral lymphoadenopathy, severe insulin resistance associated with diabetic ketoacidosis occurred repeatedly, despite intravenous administration of over 1,000 U per day of human insulin. A high plasma insulin-binding capacity as determined by Scatchard analysis was consistent with insulin antibody-mediated resistance. The diagnosis of HES was made due to the persistent elevation of eosinophil count and associated liver and cardiac damage. Glucocorticoid therapy successfully achieved both reducing clinical symptoms and improving glycemic control.

Diabetes Complications↗
PubMed1994

Japanese family with a deficiency of lecithin:cholesterol acyltransferase (LCAT).

We present findings in the ninth known Japanese family with lecithin:cholesterol acyltransferase (LCAT) deficiency. A 54-year-old man (proband) and his 58-year-old brother presented with corneal opacity. Both subjects showed a marked decrease in serum high density lipoprotein (HDL)-cholesterol and in the cholesteryl ester ratio. Although apo A-I and A-II were low, apo E tended to be high. Serum LCAT activity and mass were not detectable. Urinary examination showed microhematuria or proteinuria. Renal function was normal and no anemia was demonstrated, but blood smears showed poikilocytosis with target cells. The serum LCAT activity of the proband's three sons, obligate heterozygotes of LCAT deficiency, was about one-half the normal level, and HDL-cholesterol and apo A-I levels were low normal.

Adult↗
PubMed1994

EGF stimulates both cyclooxygenase activity and cell proliferation of cultured guinea pig gastric mucous cells.

Epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and insulin all dose-dependently stimulated [3H]-thymidine incorporation into guinea pig gastric mucous cells cultured in vitro. On the other hand, other growth factors, e.g., platelet-derived growth factor (PDGF) and gastrointestinal hormones, such as gastrin, had no effect on DNA synthesis in these cells. Exposure of the cells to EGF at concentrations which stimulated DNA synthesis caused increases in both prostaglandin (PG) E2 release and cyclooxygenase (COX) enzyme protein synthesis, as evaluated by Western blot analysis. These results suggest that such increases in DNA synthesis and PGE2 release may be involved, at least in part, in the mechanism of EGF-induced local regulation of gastric mucosal integrity.

Animals↗
PubMed1994

[Hereditary spherocytosis associated with non-Hodgkin's lymphoma in the spleen].

A 60-year-old female was admitted complaining of anemia. We diagnosed her hereditary spherocytosis (HS) from spherocytosis and family history and found a tumor in her enlarged spleen. Splenectomy was performed and swollen paraaortic lymph nodes were found at laparotomy. The tumor in the spleen was diagnosed as Non-Hodgkin's lymphoma (follicular mixed type). After CHOP therapy she entered complete remission. Though the relationship between HS and malignant lymphoma was not clear, splenomegaly due to hemolysis inducing chronic stimulation might have resulted in malignant lymphoma.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1994

[The structure function relationship in the human insulin receptor].

The human insulin receptor gene is composed of 22 exons and spans in excess of 130 kb, on chromosome 19. The basic structure of the insulin receptor is a disulfide-linked tetramer, composed of the alpha subunit (135 kDa), which is extracellular and provides the binding site for insulin, and the beta subunit (95 kDa), contains the transmembrane domain, tyrosine kinase domain and C-terminal domain. Insulin binding to the alpha subunit causes the activation of the receptor tyrosine kinase activity that plays a critical role in mediating insulin signal transduction. Site-directed mutagenesis or the gene analysis of the patients with insulin resistant diabetes mellitus has revealed the structure and functional relationship of the insulin receptor to some extent but further investigations required.

Amino Acid Sequence↗
PubMed1994

Effects of pancreatic polypeptide family peptides on feeding and learning behavior in mice.

We studied the effects of intra-third cerebroventricular administration of neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP) on the locomotor activity and the feeding and learning behavior of mice. NPY (0.3-10 micrograms), PYY (0.1-10 micrograms) and PP (3.0-10 micrograms) produced significant increases in locomotor activity. A significant decrease was then observed 15 min after administration of 10 micrograms of PYY. NPY, PYY and PP significantly increased food intake at 20 min and this effect continued for 2 to 4 hr at the high doses. The feeding response to PP family peptides were quite similar to that in locomotor activity with respect to dose-response, time course and peptide specificity. Learning behaviors were evaluated at three different stages of memory processing, acquisition, consolidation and retrieval, in a battery of step-down type passive avoidance tests. NPY and PYY had no effect on acquisition, but significantly improved consolidation at a dose of 0.03 and 0.3 microgram, respectively. NPY also improved retrieval at a dose of 0.03 microgram. The ranking order of potency in stimulating feeding and locomotor activity was PYY > NPY > PP, and in improving memory consolidation NPY > PYY >> PP. These observations suggest that NPY and PYY influence different neural substrates in the brain involved in feeding and learning.

Animals↗
PubMed1993

[Mechanism of insulin action--signal transductions after binding to insulin receptor].

Insulin binds to the alpha subunit of the insulin receptor which activates the tyrosine kinase in the beta subunit and tyrosine-phosphorylates the insulin receptor substrates-1 (IRS-1). Insulin promotes the formation of a complex between tyrosine-phosphorylated IRS-1 and several proteins including phosphoinositide(PI) 3-kinase, a heterodimer consisting of regulatory 85-kDa (p85) and catalytic 110-kDa (p110) subunits, GRB2 and Syp via the Src homology region 2 (SH2) domains. Recently, it was suggested that GRB2-Sos complex binding to IRS-1 was linked to Ras activation and that PI 3-kinase binding to IRS-1 was linked to activation of glucose transport. Since the mechanism of insulin-stimulated glucose uptake is mainly due to translocation of glucose transporters from an intracellular vesicle pool to the plasma membrane, PI 3-kinase activity may be involved in vesicle transport in mammalian cells.

Adaptor Proteins, Signal Transducing↗