Search PubMed⌕ Search

Biomedical subjects

M Kasuga

Publications and source records attributed to M Kasuga.

At least 217 records · Page 12Linked to original sources
PubMed1995

A dominant-negative mutant of mSOS1 inhibits insulin-induced Ras activation and reveals Ras-dependent and -independent insulin signaling pathways.

The role of the Grb2-SOS complex in insulin signal transduction was investigated with a deletion mutant of mSOS1 that lacks the guanine nucleotide exchange domain of the wild-type protein. Cells over-expressing either wild-type (CHO-IR/SOS cells) or mutant (CHO-IR/delta SOS cells) mSOS1 were established by transfection of Chinese hamster ovary cells that express human insulin receptors (CHO-IR cells) with the appropriate expression plasmid. The mutant mSOS1 protein did not contain the guanine nucleotide exchange activity in vitro and associated with Grb2 both in vivo and in vitro. In both CHO-IR and CHO-IR/SOS cells, insulin rapidly stimulated the formation of GTP-bound Ras and the phosphorylation of mitogen-activated protein (MAP) kinase; both these effects of insulin were markedly inhibited in CHO-IR/delta SOS cells. Insulin-induced glycogen synthase and 70-kDa S6 kinase activities were not affected by expression of either wild-type or mutant mSOS1. These results show that the mutant mSOS1 acts in a dominant-negative manner and suggest that the Grb2-SOS complex mediates, at least in part, insulin-induced activation of Ras in intact cells. The data also indicate that Ras activation is not required for insulin-induced stimulation of glycogen synthase and 70-kDa S6 kinase.

Animals↗
PubMed1995

Roles of 1-phosphatidylinositol 3-kinase and ras in regulating translocation of GLUT4 in transfected rat adipose cells.

Insulin stimulates glucose transport in insulin target tissues by recruiting glucose transporters (primarily GLUT4) from an intracellular compartment to the cell surface. Previous studies have demonstrated that insulin receptor tyrosine kinase activity and subsequent phosphorylation of insulin receptor substrate 1 (IRS-1) contribute to mediating the effect of insulin on glucose transport. We have now investigated the roles of 1-phosphatidylinositol 3-kinase (PI 3-kinase) and ras, two signaling proteins located downstream from tyrosine phosphorylation. Rat adipose cells were cotransfected with expression vectors that allowed transient expression of epitope-tagged GLUT4 and the other genes of interest. Overexpression of a mutant p85 regulatory subunit of PI 3-kinase lacking the ability to bind and activate the p110 catalytic subunit exerted a dominant negative effect to inhibit insulin-stimulated translocation of epitope-tagged GLUT4 to the cell surface. In addition, treatment of control cells with wortmannin (an inhibitor of PI 3-kinase) abolished the ability of insulin to recruit epitope-tagged GLUT4 to the cell surface. Thus, our data suggest that PI 3-kinase plays an essential role in insulin-stimulated GLUT4 recruitment in insulin target tissues. In contrast, over-expression of a constitutively active mutant of ras (L61-ras) resulted in high levels of cell surface GLUT4 in the absence of insulin that were comparable to levels seen in control cells treated with a maximally stimulating dose of insulin. However, wortmannin treatment of cells overexpressing L61-ras resulted in only a small decrease in the amount of cell surface GLUT4 compared with that of the same cells in the absence of wortmannin. Therefore, while activated ras is sufficient to recruit GLUT4 to the cell surface, it does so by a different mechanism that is probably not involved in the mechanism by which insulin stimulates GLUT4 translocation in physiological target tissues.

Adipocytes↗
PubMed1995

Quantitation of hepatitis C viral RNA in liver and serum samples using competitive polymerase chain reaction.

AIMS: To investigate whether the amount of hepatitis C viral RNA (HCV-RNA) in liver and serum can predict the effectiveness of interferon treatment in patients with chronic hepatitis C. METHODS: The amount of HCV-RNA in frozen liver tissues and sera of 22 patients with chronic hepatitis C was determined before and after interferon alfa treatment by the competitive reverse transcriptional polymerase chain reaction method. RESULTS: Patients with small amounts of HCV-RNA in serum before treatment showed a significantly more effective response to interferon treatment. After treatment, HCV-RNA disappeared from both the liver and serum of all patients who sustained complete response (n = 11); in contrast, HCV-RNA decreased but persisted in the liver of all those who relapsed after cessation of treatment (n = 11). CONCLUSIONS: The elimination of hepatitis C virus from the liver as well as from the serum seems to be essential for sustained remission. The quantitation of HCV-RNA in liver biopsy specimens obtained after treatment would be a highly accurate predictor of whether relapse is likely to occur.

Base Sequence↗
PubMed1995

Amino acid polymorphisms of the insulin receptor substrate-1 in Japanese noninsulin-dependent diabetes mellitus.

By using polymerase chain reaction-restriction length polymorphism and polymerase chain reaction-single-strand conformation polymorphism analysis, we screened 283 Japanese subjects [226 noninsulin-dependent diabetes mellitus (NIDDM), 12 impaired glucose tolerance, and 45 normal controls] for 2 amino acid polymorphisms, Ala513Pro and Gly972Arg, of the insulin receptor substrate-1. Only 8 NIDDM, 1 impaired glucose tolerance, and 1 normal subject were identified to be heterozygous for the Gly972Arg mutation, whereas no subject had an Ala513Pro polymorphism. The frequency of Gly972Arg was lower than recently reported in Danish and Finnish populations and was in good agreement with that previously reported in another Japanese cohort. Analysis of 1 pedigree of 1 NIDDM patient with a Gly972Arg showed no co-segregation between this polymorphism and the onset of NIDDM. Our results suggest that the Gly972Arg polymorphism does not play an important role in the pathogenesis of NIDDM in Japanese patients.

Adolescent↗
PubMed1995

Hypertrophic cardiomyopathy in patients with diabetes mellitus associated with mitochondrial tRNA(Leu)(UUR) gene mutation.

Left ventricular function and morphology were assessed using M-mode echocardiography in 3 patients with diabetes mellitus associated with mitochondrial tRNA(Leu)(UUR) gene mutation, who were free of clinical, electrocardiographic, or thallium scan evidence of ischemic heart disease. Echocardiograms revealed hypertrophic cardiomyopathy in all 3 patients. Hypertrophy of the interventricular septum was mild in Cases 1 and 3 (12 and 13 mm, respectively) and severe in Case 2 (22 mm) (normal 7-10 mm). When they had neither signs nor symptoms suggestive of congestive heart failure, percentage fractional shortening (%FS), an index of wall motion of the left ventricle (normal > 28%), was normal in Cases 2 and 3 (28 and 32%, respectively) whereas it was slightly decreased in Case 1 (22%). In Case 1 with mild hypertrophy, the development of congestive heart failure was associated with a marked decrease in %FS to 13%; this patient responded well to diuretics and captopril and %FS rose to 22%. However, a mild decrease in %FS to 21% caused congestive heart failure in Case 2 with severe hypertrophy. His response to treatment was marginal. The present study indicates that mitochondrial DNA analysis should be done in patients with diabetic cardiomyopathy, and that sequential echocardiography is invaluable for the detection of hypertrophic cardiomyopathy and the management of subsequent myocardial dysfunction in patients with mitochondrial diabetes mellitus and cardiomyopathy.

Adult↗
PubMed1994

Grb2/Ash binds directly to tyrosines 1068 and 1086 and indirectly to tyrosine 1148 of activated human epidermal growth factor receptors in intact cells.

The activation of receptor tyrosine kinases generates tyrosine-phosphorylated recognition motifs for the binding of signaling proteins containing Src homology 2 domains. We determined the binding sites of Grb2/Ash, an Src homology 2 domain-containing adaptor protein, within epidermal growth factor (EGF) receptors, using Chinese hamster ovary cells overexpressing human EGF receptor mutants in which one of the autophosphorylation sites was retained. In intact cells, the amount of Grb2/Ash coimmunoprecipitated with mutant receptors retaining tyrosines 992, 1068, 1086, 1148, or 1173 was approximately 10, 85, 55, 50, or 20% of wild-type levels, respectively. The association of Grb2/Ash with in vitro autophosphorylated EGF receptor mutants was detectable in those retaining either tyrosines 1068 or 1086 but not in other mutants including those retaining tyrosine 1148. In peptide inhibition assay, phosphorylated peptides representing tyrosines 1068 and 1086 inhibited the binding of Grb2/Ash to in vitro autophosphorylated wild-type EGF receptors, whereas the other peptides representing tyrosines 992, 1148, and 1173 failed to inhibit the binding. Given that tyrosine 1148 of the activated EGF receptor is a major binding site of Shc (Okabayashi, Y., Kido, Y., Okutani, T., Sugimoto, Y., Sakaguchi, K., and Kasuga, M. (1994) J. Biol. Chem. 269, 18674-18678), these results indicate that tyrosines 1068 and 1086 of activated human EGF receptors are direct high affinity binding sites of Grb2/Ash and that tyrosine 1148 is an indirect binding site through Shc in intact cells.

Adaptor Proteins, Signal Transducing↗
PubMed1994

Src kinase tyrosine phosphorylates PTP1C, a protein tyrosine phosphatase containing Src homology-2 domains that down-regulates cell proliferation.

PTP1C is a non-transmembrane-type protein-tyrosine phosphatase and contains two Src homology-2 (SH2) domains. PTP1C was tyrosine-phosphorylated in SR-3Y1, a v-Src-transformed rat fibroblast cell line. Tyrosine phosphorylation of PTP1C was more prominent when PTP1C was overexpressed in SR-3Y1 cells. PTP1C lacking SH2 domains was also tyrosine-phosphorylated in SR-3Y1 cells, indicating that SH2 domains of PTP1C are not required for tyrosine phosphorylation of PTP1C by v-Src kinase. V-Src kinase catalyzed the phosphorylation of PTP1C in a cell-free system. The growth rate of SR-3Y1 was reduced by the expression of PTP1C in the low-serum medium. Furthermore, overexpression of PTP1C suppressed the anchorage-independent colony formation of SR-3Y1 cells. These results suggest that PTP1C is a direct target for v-Src kinase and may down-regulate the proliferation of cells.

Animals↗
PubMed1994

Expression of insulin receptor substrate-1 in hepatocytes: an investigation using monoclonal antibodies.

To investigate the expression and subcellular distribution of insulin receptor substrate-1 in hepatocytes, which are major targets of insulin along with muscle and adipose tissue, we obtained monoclonal antibodies by immunizing mice with a fusion protein consisting of the C-terminal portion of the human insulin receptor substrate-1 and glutathione-S-transferase. Two of the monoclonal antibodies (designated as 7B3 and 6G5) were found to be useful for immunohistochemical studies. Using 6G5 we demonstrate a high level of expression of insulin receptor substrate-1 in liver cirrhosis hepatocytes and variable expression in hepatocellular carcinoma cells. These results suggest that insulin receptor substrate-1 may play a role in liver regeneration during cirrhosis and that an insulin signaling cascade may be involved in hepatocarcinogenesis.

Animals↗
PubMed1994

Increase in 3-deoxyglucosone levels in diabetic rat plasma. Specific in vivo determination of intermediate in advanced Maillard reaction.

A specific assay of 3-deoxyglucosone (3-DG) was developed in our laboratory to help elucidate the relationship between advanced Maillard reaction and diabetic complications. 3-DG is known as a highly reactive intermediate of the reaction in vitro and a precursor of advanced glycosylation end products such as pyrraline and pentosidine, which have been previously detected in vivo. 3-DG was converted to a stable compound, 2-(2,3,4-trihydroxybutyl)-benzo[g]quinoxaline, by reacting with 2,3-diaminonaphthalene. Since the derivative had a characteristic UV spectrum, it was determined at 268 nm by high performance liquid chromatography. This method was sensitive enough to detect 10 ng/ml (61.7 nM) of 3-DG in vitro. A slight modification to this method allowed in vivo detection of small amounts of 3-DG. Plasma free 3-DG levels were significantly higher in streptozotocin-induced diabetic rats compared with controls (918 +/- 134 nM versus 379 +/- 69 nM, p < 0.001) and were suppressed with the administration of aminoguanidine, an inhibitor of Maillard reaction. Plasma pyrraline levels in diabetic rats also increased in parallel with elevated 3-DG levels but were only marginally suppressed by administration of aminoguanidine. Our results indicate that 3-DG is present in vivo under normal conditions and that its level increases in diabetic subjects. Determination of 3-DG represents a good tool to predict development and progression of diabetic complications and to assess the efficiency of inhibitors to Maillard reaction.

2-Naphthylamine↗
PubMed1994

Tyrosines 1148 and 1173 of activated human epidermal growth factor receptors are binding sites of Shc in intact cells.

Autophosphorylation of receptor tyrosine kinases provides binding sites for signaling proteins containing Src homology 2 (SH2) domains. We determined the binding sites of Shc, SH2-containing adaptor protein, within epidermal growth factor (EGF) receptors, using Chinese hamster ovary cells overexpressing EGF receptor mutants in which autophosphorylation sites, either alone or in combination, were replaced by phenylalanine. Binding of Shc to EGF receptor mutants lacking single tyrosine residues at 1148 or 1173 decreased by approximately 60 or approximately 15%, respectively, whereas other single point mutants bound the wild-type level of Shc. Binding of Shc markedly decreased in mutants lacking both tyrosine residues at 1148 and 1173. In peptide inhibition assay, phosphorylated nonameric peptide representing tyrosine 1148, DNPDpYQQDF, but not pentameric peptide, pYQQDF, inhibited the binding of glutathione S-transferase-Shc SH2 domain fusion protein to in vitro autophosphorylated EGF receptors, suggesting that N-terminal sequences adjacent to phosphotyrosine are necessary for the association of Shc. Based on results of peptide inhibition assays in which phosphorylated peptides representing tyrosines 992, 1148, and 1173 inhibited Shc binding to the receptor, we constructed another EGF receptor mutant in which one of these tyrosine residues was retained. The amount of Shc bound to mutant receptors retaining tyrosines 1148, 1173, or 992 was approximately 80, approximately 40, or approximately 10% of wild-type level, respectively. These results indicate that tyrosine 1148 of activated human EGF receptors is a major binding site of Shc and tyrosine 1173 is a secondary binding site in intact cells.

Amino Acid Sequence↗
PubMed1994

Inhibition of the translocation of GLUT1 and GLUT4 in 3T3-L1 cells by the phosphatidylinositol 3-kinase inhibitor, wortmannin.

Wortmannin is a potent and reversible inhibitor of insulin-stimulated PtdIns 3-kinase activity in 3T3-L1 cells (IC50 = 2.6 +/- 0.8 nM). Wortmannin inhibits the PtdIns 3-kinase activity which is precipitated with antibodies against insulin receptor substrate 1 and against the alpha-p85 subunit of PtdIns 3-kinase. These observations suggest that wortmannin inhibits at the p110 catalytic subunit of PtdIns 3-kinase. Insulin stimulation of glucose transport in permeabilized 3T3-L1 cells is also inhibited by wortmannin (IC50 = 6.4 +/- 1.4 nM). Wortmannin did not inhibit basal glucose transport activity. The close similarity of the IC50 values for wortmannin inhibition of insulin-stimulated PtdIns 3-kinase and glucose transport activities suggests that the PtdIns 3-kinase is a key intermediate in insulin signalling of glucose-transport stimulation. The wortmannin inhibitory effect on transport is associated with a reduction in the cell-surface, but not the total cellular, levels of both GLUT1 and GLUT4 glucose transporter isoforms that are accessible to the cell-impermeant photolabel, ATB-BMPA. These photolabelling results suggest that the glucose transporter translocation process is dependent upon PtdIns 3-kinase activity. The stimulatory effect of guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) on glucose transport activity in permeabilized cells is only partially blocked by concentrations of wortmannin that completely inhibit the stimulatory effect of insulin. The residual stimulatory effect of GTP gamma S that occurs in the presence of wortmannin suggests that at least part of the GTP gamma S effect is mediated at a signalling site that is downstream of the site at which wortmannin inhibits the insulin stimulation of PtdIns 3-kinase and glucose transport activities.

3T3 Cells↗
PubMed1994

Activation of phosphoinositide 3-kinase is required for PDGF-stimulated membrane ruffling.

BACKGROUND: There is substantial evidence that phosphoinositide 3-kinase (PI 3-kinase) is a critical component of signalling pathways used by the cell-surface receptors for a variety of mammalian growth factors and other hormones. The physiological product of this enzyme is a highly polar membrane lipid called phosphatidylinositol (3,4,5)-trisphosphate This lipid has been postulated to act as a second-messenger in cells but its putative targets are still unknown. RESULTS: A particular rearrangement of actin filaments, which results in membrane ruffling, is elicited by the activation of PDGF beta-receptors expressed in cultured porcine aortic endothelial cells. We have found that this consequence of PDGF beta-receptor activation is inhibited by three independent manipulations of PI 3-kinase activity: firstly, by the deletion of tyrosine residues in the PDGF beta-receptor to which PI 3-kinase binds; secondly, by the overexpression of a mutant 85 kD PI 3-kinase regulatory subunit to which the catalytic kinase subunit cannot bind; and thirdly, by the addition of the fungal metabolite wortmannin, which is a potent inhibitor of the catalytic activity of PI 3-kinase. CONCLUSIONS: These results argue strongly that phosphatidylinositol (3,4,5)-trisphosphate synthesis is required for growth-factor-stimulated membrane ruffling in porcine aortic endothelial cells, and suggest that synthesis of this lipid may be part of a signalling pathway leading to direct or indirect activation of the small GTP-binding protein Rac.

Androstadienes↗
PubMed1994

Insulin stimulates the phosphorylation of Tyr538 and the catalytic activity of PTP1C, a protein tyrosine phosphatase with Src homology-2 domains.

PTP1C is a non-transmembrane protein-tyrosine phosphatase and contains two Src homology-2 (SH2) domains. Insulin stimulated the tyrosine phosphorylation of PTP1C in human 1M-9 lymphoblast cells, in rat H35 hepatoma cells and in Chinese hamster ovary cells over-expressing both insulin receptors and PTP1C. Insulin also stimulated the tyrosine phosphorylation of a mutant PTP1C lacking SH2 domains in Chinese hamster ovary cells, suggesting that the SH2 domains are not required for insulin-stimulated tyrosine phosphorylation of PTP1C. The insulin receptor tyrosine kinase catalyzed the tyrosine phosphorylation of PTP1C in a cell-free system. Peptide mapping of phosphorylated PTP1C showed that Tyr538 in the C-terminal region was phosphorylated in response to insulin. The tyrosine phosphorylation of PTP1C by the insulin receptor kinase increased phosphatase activity. Furthermore, PTP1C was shown to bind to autophosphorylated insulin receptors through its C-terminal region, but PTP1C did not bind to unphosphorylated receptors. These results suggest that PTP1C is a target protein for the insulin receptor tyrosine kinase and that the C-terminal region of PTP1C may function both in the regulation of phosphatase activity and in the association of PTP1C with autophosphorylated insulin receptors.

Amino Acid Sequence↗
PubMed1994

Substitution of glutamine for arginine 1131. A newly identified mutation in the catalytic loop of the tyrosine kinase domain of the human insulin receptor.

We studied a patient with severe insulin resistance and a remarkable decrease in the in vivo autophosphorylation of the insulin receptor. Using a polymerase chain reaction-single strand conformation polymorphism method and direct sequencing, we identified a heterozygous mutation substituting Gln for Arg1131 in the putative "catalytic loop" of the tyrosine kinase domain of the insulin receptor gene. The Gln1131 mutant receptor was expressed by transfection in Chinese hamster ovary cells and compared with cells expressing the wild-type insulin receptor. Both mutant and wild-type receptors were expressed on the cell surface and displayed similar insulin-binding affinity. The Gln1131 mutation impaired the activity of the receptor tyrosine kinase and inhibited the ability of insulin to phosphorylate the endogenous substrate insulin receptor substrate-I. In addition, the Gln1131 mutant receptor exhibited diminished tyrosine-phosphorylated phosphatidylinositol 3-kinase and myelin basic protein kinase activities compared with the wild-type cells. It also demonstrated a defective mediation of the insulin signal stimulating 2-deoxy-D-glucose transport and thymidine incorporation, resistance to endocytosis, and insulin-induced down-regulation. Unlike a previously described mutation in the putative catalytic loop of the receptor that substituted Glu for Ala1135, the Gln1131 mutation retained proteolytic cleavage of the proreceptor into separate subunits. Our results demonstrate that a naturally occurring mutation (R1131Q) in the putative catalytic loop of the insulin receptor results in severe impairment of the tyrosine kinase function in our patient. In addition, our results indicate that Arg1131 is important for receptor-mediated insulin action in vivo and suggest that the amino acids constituting the catalytic loop of protein kinases may possess different modes in order to retain kinase function.

Amino Acid Sequence↗