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M Kasuga

Publications and source records attributed to M Kasuga.

At least 379 records · Page 21Linked to original sources
PubMed1982

The structure of insulin receptor and its subunits. Evidence for multiple nonreduced forms and a 210,000 possible proreceptor.

We have identified the subunits of the insulin receptor using immunoprecipitation by antibodies to the insulin receptor after either biosynthetic or surface labeling of cultured human lymphocytes (IM-9). With this approach, we have found there are two major, Mr = 135,000 (alpha), Mr = 95,000 (beta) and one minor, Mr = 210,000 (gamma) subunit. Peptide mapping clearly demonstrates that the major peptides of the alpha and beta subunits are different, whereas similarities exist in the peptide fragments of the gamma subunit and the alpha and beta subunits after limited proteolysis. The gamma subunit, however, is not simply a disulfide heterodimer of alpha and beta subunits, since this subunit was not reduced by 100 mM dithiothreitol plus 5% 2-mercaptoethanol, or even under more potent denaturing conditions, such as 8 M guanidine-HCL and mercaptoethanol at pH 10.5. In nonreduced gels, free insulin receptor subunits are observed, as well as two higher molecular weight bands of Mr = 520,000 and 350,000. On reduction, the 520,000 band was composed primarily of Mr = 210,000 and 95,000 subunits, whereas the 350,000 band was composed primarily of Mr = 135,000 and 95,000 subunits. These data suggest that the two major subunits of the insulin receptor (alpha and beta) are distinct. In addition, there is a third component of the receptor identifiable of 210,000 which may be a proreceptor or some closely associated effector protein. Furthermore, it appears that in the native state several kinds of disulfide oligomers of these subunits exist. These findings suggest a complex model for insulin receptor synthesis and insertion into the membrane.

Cell Line↗
PubMed1982

Insulin stimulates the phosphorylation of the 95,000-dalton subunit of its own receptor.

Cultured human lymphocytes and rat hepatoma cells were labeled with [32P]orthophosphate and the insulin receptor subunits identified by immunoprecipitation and sodium dodecyl sulfate-gel electrophoreses. In both cell types the 95,000-dalton (beta) subunit of the insulin receptor was selectively phosphorylated. Phosphorylation was specifically stimulated by insulin in a dose-dependent fashion after 1 and 15 minutes of hormone treatment, whereas human growth hormone was without effect. This phosphorylation may be a very early event in insulin action.

Animals↗
PubMed1982

Autoantibodies to insulin receptors in man: immunological determinants and mechanism of action.

The insulin receptor is a membrane glycoprotein of high Mr which binds insulin with high affinity and specificity and transmits some intracellular signal(s) that initiate(s) insulin action. Antibodies to the receptor have been identified in patients with a syndrome characterized by severe resistance to endogenous and exogenous insulin, varying degrees of glucose intolerance, and the skin lesion acanthosis nigricans. The syndrome is most common in non-Caucasian, middle-aged women, but occurs in patients of all races, both sexes, and spanning the ages of 12-62. Most patients have evidence of other autoimmune disease with increased erythrocyte sedimentation rate and gamma globulins, anti-DNA and anti-nuclear antibodies, leucopenia, and other signs and symptoms of autoimmune disease. Antibodies to the insulin receptor are detected by their ability to inhibit 125I-insulin binding or to immunoprecipitate solubilized insulin receptors. In vitro these antibodies acutely mimic most of insulin's metabolic effects. This insulin-like activity depends on antibody bivalence; monovalent Fab fragments block insulin binding and action but lack intrinsic activity. With prolonged exposure of cells to anti-receptor antibody the insulin-like effect is lost and a state of insulin resistance ensues. This is due to both a blockage of insulin binding and a form of post-receptor desensitization. The possible causation of anti-receptor antibodies in this condition is discussed.

Antibody Specificity↗
PubMed1982

The insulin receptor on the human lymphocyte: insulin-induced down-regulation of 126,000 and 90,000 glycosylated subunits.

Cultured human lymphoblastoid B lymphocytes were surface-labelled with iodine125 and solubilized in 1% Triton X-100 in the presence of protease inhibitors. After purification of labelled glycoproteins by elution from immobilized wheat germ lectin with 0.3 mol/l N-acetyl-D-glucosamine, insulin receptors were quantitatively immunoprecipitated using IgG receptor auto-antibodies. The overall recovery of labelled glycoprotein was 0.02-0.04%; analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography under reducing conditions revealed two major bands with molecular weights of 126,000 and 90,000, and a minor band of 67,000 daltons. The mobilities of both major receptor subunits were increased after treatment with neuraminidase. When lymphocyte receptor binding was 'down-regulated' before surface labelling, there was a concomitant decrease in the recovery of both the 126,000 and 90,000 subunits. These data indicate that 'down-regulation' of binding probably involves degradation of the receptor molecule.

Autoantibodies↗
PubMed1982

Structure of the insulin-like growth factor receptor in chicken embryo fibroblasts.

The insulin-like growth factors (IGFs) and insulin stimulate DNA synthesis and cell multiplication in chicken embryo fibroblasts in culture. This response appears to be mediated by interaction with a single type of IGF receptor. The present study examines the subunit structure of this receptor by covalently crosslinking two 125I-labeled IGFs, IGF-I and multiplication-stimulating activity (MSA), to chicken embryo fibroblasts by using disuccinimidyl suberate. After solubilization, NaDodSO4/polyacrylamide gel electrophoresis, and autoradiography, IGF receptor complexes of appropriate specificity were identified; they had Mr approximately 130,000 (major band) and approximately 260,000 (minor band) under reducing conditions and Mr greater than 300,000 without disulfide reduction. The proportion of the Mr 260,000 component increased with increasing concentration of crosslinking agent, suggesting that it was formed from smaller proteins during the crosslinking procedure. The IGF receptor in chicken embryo fibroblasts resembles the insulin receptor in size and structure but can be distinguished by a higher affinity for IGF-I and MSA than for insulin. Although IGF receptors with different structure and specificity have been recognized in other tissues, the function of these binding sites is unknown. The present study demonstrates that the IGF receptor of chicken embryo fibroblasts that appears to mediate the growth-promoting effects of the IGFs contains a Mr approximately 130,000 binding subunit and exists as a native receptor complex of Mr greater than 300,000.

Animals↗
PubMed1982

Decreased insulin binding in cultured lymphocytes from two patients with extreme insulin resistance.

[125I]Insulin binding has been studied in two patients with extreme insulin resistance using cultured B-lymphocytes transformed with Epstein-Barr virus. A cell line from a female infant with leprechaunism had insulin binding which was decreased 90% below the lower limit of normal. Lymphocytes from a young woman with type A extreme insulin resistance (associated with acanthosis nigricans and virilization) had insulin binding which was 80% depressed. In both cases, the defect in binding resulted from a decrease in the number of receptors per cell. The remaining receptors had normal properties, including a normal affinity for insulin and a normal specificity for insulin analogs. Insulin binding in cultured lymphocytes from these two insulin-resistant patients was also inhibited normally by antibodies to the insulin receptor. Immunological assays using anti-receptor antibodies confirmed the conclusion that the number of receptors was decreased. Affinity labeling of the leprechaun insulin receptor with [125I]insulin demonstrated the existence of an alpha-subunit with apparently normal molecular weight (130,000 daltons). However, the number of receptor molecules per cell appeared reduced.

Adolescent↗
PubMed1982

Intratumor immunochemotherapy with 5-fluorouracil emulsion and BCG in C3H/HE mice transplanted with MH134.

Responses to intratumor immunochemotherapy by intralesional injections of BCG and 5-fluorouracil (5-FU) emulsion, prior to resection of a transplanted primary tumor, were investigated using a host-tumor system consisting of C3H/He mice and MH134. A significant prolongation of survival and suppression of lymph node metastasis were attained by the combined use of BCG and 5-FU emulsion, compared to treatment with either BCG or 5-FU emulsion alone. However, this did not alter the survival rate of the combined regimen group as compared with those of the latter treatment groups. When rechallenged with the same tumor cells after resection of the primary lesion, mice treated with BCG alone exhibited a marked tumor growth suppression while the control and BCG + 5-FU emulsion group showed a less effective suppression. No such effects were observed in the group given 5-FU emulsion alone. Assessment by the splenic plaque-forming cell assay with sheep erythrocytes revealed a marked inhibition in the development of humoral immunity in the animals treated with 5-FU emulsion alone. Concomitant administration of BCG was effective to some extent in preventing the depression of these immune functions. The data obtained indicate that the clinical response to immunochemotherapy is determined by a balance between the antitumor effect of the antitumor agent administered and its influence on the host's immune functions.

Animals↗
PubMed1982

[Experimental study on local immunochemotherapy].

The purposes of this work were twofold: firstly to determine whether intratumor chemoimmunotherapy was more effective than either treatment alone or systemic therapy and; secondly to study how the intratumor therapy affected on the development of the tumor-specific immunity. Inbred male C3H/He mice and mouse ascited hepatoma 134 (MH 134) of C3H origin were used as host-tumor system. Mitomycin C was used as the chemotherapeutic agent and BCG as the immunopotentiating agent. Intratumor treatment of MMC + BCG led to complete cure in 85 percent of the mice. The lymph node metastases were markedly inhibited in the group treated with MMC + BCG compared to the groups treated with MMC alone or BCG alone. The growth of rechallenged tumor was investigated; 79% of mice treated with MMC + BCG were immune to rechallenge, whereas 57% of mice treated with BCG alone. The number of PFC and DTH against SRBC of the mice treated with MMC intraperitoneally significantly decreased compared to that treated with MMC intratumorally.

Animals↗
PubMed1982

[Clinical evaluation of the serum level of immunosuppressive acidic protein (IAP) and immunosuppressive substance (IS) in gastric cancer patients].

Recently, immunosuppressive acidic protein (IAP) and immunosuppressive substance (IS) were reported by two institutions, and they have been currently under extensive clinical investigations. As these two acidic proteins seemed to be very similar glycoproteins, we tried to elucidate clinically the dissimilarities between these two substances. The mean value of IAP of 106 healthy donors was 378 +/- 100 micrograms/ml, and as 95% of them was under 550 micrograms/ml, therefore, the upper limit of normal value of IAP was determined to be 550 micrograms/ml. On the other hand, the mean value of IS was 553 +/- 125 micrograms/ml and the upper limit of normal value was 750 micrograms/ml. In 110 gastric cancer patients, the incidence of the abnormal value of IS (43%) was higher than that of IAP (33%), and abnormal values were observed at earlier stage in IS than that observed in IAP. The reaction of skin tests was more highly correlated with the value of IS than the value of IAP. From these facts, it was suggested that the value of IS was more predictable than that of IAP as the parameter of the immunosuppression of the gastric cancer patients.

Adolescent↗
PubMed1982

[Effect of PSK on DMH (dimethyl hydrazine) induction of cancer].

The effect of PSK on the induction of intestinal tumors by DMH in Wistar rats was examined. One hundred and fifty-one rats were divided into two groups. Seventy-two rats were treated with DMH alone and 79 rats were treated with DMH and PSK. All animals were subjected to a sequential autopsy and all lesions within the GI tract were examined macroscopically and histologically. Tumor incidence in the DMH and PSK treated group was significantly lower than that in the DMH treated alone, and peritoneal disseminations and distant metastases were also significantly decreased in the PSK treated rats. The number of circulating lymphocytes dropped in the 25th and 35th week in the group treated with DMH alone, but the drop in the PSK treated group was not remarkable. DTH reaction to PPD and antibody forming capacity to SRBC were well maintained in the PSK treated rats than in those treated with DMH alone. The most interesting findings in these experiments were the differences concerning the serum blocking activities and serum immunosuppressive factor in these two groups; these were markedly reduced in PSK-treated rats. From these experiments, the mechanisms may be explained by the competitive action of PSK against the immunosuppressive factor produced by a tumor-bearing host.

Adjuvants, Immunologic↗
PubMed1981

Demonstration of two subtypes of insulin-like growth factor receptors by affinity cross-linking.

The structure of receptors for insulin-like growth factors in rat liver plasma membranes and the BRL 3A2 rat liver cell line has been examined by chemical cross-linking with disuccinimidyl suberate and sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. Two receptor subtypes have been identified: (i) 125I multiplication-stimulating activity cross-linked to liver membranes or intact cells appeared in a complex of Mr = 260,000 (reduced) and 220,000 (nonreduced) and (ii) 125I-insulin-like growth factor I cross-linked to BRL 3A2 cells appeared predominantly in two bands of Mr greater than 300,000 without disulfide reduction and in a Mr = 130,000 complex following reduction. The two subtypes of insulin-like growth factor receptors identified by structural analysis correspond to previously observed differences in their specificity for insulin and insulin-like growth factors.

Animals↗
PubMed1981

Monocyte count, monocyte chemotaxis and chemotactic factor inactivator in gastric cancer patients.

The monocyte count in the peripheral blood, chemotactic responsiveness (MCR) and chemotactic factor inactivator (CFI) were measured in 66 patients with gastric cancer. Monocyte counts in advanced gastric cancer patients were significantly increased. MCR was significantly depressed in gastric cancer patients in the advanced stage of the disease, as compared with findings in the control groups. Sixty-seven percent of the patients with stage IV carcinoma had abnormally depressed responses. The CFIs were significantly higher in gastric cancer patients than in the control groups, and there was a positive correlation between the degree of inhibition and extent of cancer progress. These data support the hypothesis that qualitative and quantitative abnormalities of circulating monocytes, as macrophage precursors, may hinder immunological host defense mechanisms.

Adult↗
PubMed1981

Insulin-induced receptor loss in cultured human lymphocytes is due to accelerated receptor degradation.

We have measured the turnover rate of the polypeptide subunits of the insulin receptor in cultured human lymphocytes (IM-9 line) and have investigated the mechanism of insulin-induced receptor loss. To estimate the rate of receptor degradation, lymphocytes were either pulse-labeled with [35S]methionine or surface labeled with Na125I and lactoperoxidase. The insulin receptor was isolated by immunoprecipitation with anti-receptor antibody, and the rate of loss of radioactivity from each receptor subunit was determined after sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Two major (Mr 135,000 and 95,000) and one minor (Mr 210,000) subunits were found. By both labeling methods, the half-lives of the major insulin receptor subunits were 9--12 hr in normal media. When the cells were cultured in media containing 1 microM insulin the turnover was accelerated 2.5- to 3.5-fold (half-life approximately 3 hr). The increase in degradation rate was dependent on the insulin concentration and correlated well with the ability to "down-regulate" the receptor. Guinea pig insulin was about 2% as active as porcine insulin in accelerating degradation, and human growth hormone was without effect. The acceleration of receptor degradation induced by insulin was partially blocked by 100 microM cycloheximide. The rate of biosynthesis of the insulin receptor did not appear to be altered in the presence of 1 microM insulin after correction for the change in degradation rate. In conclusion, these data demonstrate that insulin-induced receptor loss in cultured lymphocytes is due to accelerated receptor degradation.

Animals↗
PubMed1981

Direct demonstration of glycosylation of insulin receptor subunits by biosynthetic and external labeling: evidence for heterogeneity.

Insulin receptors of human lymphocytes (IM-9 line) were biosynthetically labeled with [3H]glucosamine, [3H]galactose, [3H]fucose, or [3H]mannose. After solubilization in Triton X-100, cell extracts were immunoprecipitated with serum from a patient containing autoantibodies to the insulin receptor. Na-DodSO4/polyacrylamide gel electrophoresis of the immunoprecipitates under reducing conditions showed the presence of major labeled subunits of apparent Mr 134,000 and 98,000 and a minor component of Mr 206,000. The ratio of activity in the 134,000 versus 98,000 Mr bands varied from 2:1 for mannose to 1.2:1 for galactose. In addition, the receptor subunits could be demonstrated when the cell surface of intact lymphocytes was labeled with NaB3H4 by using either the galactose oxidase (acts on nonreducing terminal galactose and N-acetylgalactosamine) technique or the periodate (oxidizes sialic acid) technique. With the periodate treatment, NaB3H4 labeled preferentially the Mr 98,000 band. With the galactose oxidase procedure, on the other hand, NaB3H4 labeled only the Mr 134,000 band; prior treatment with neuraminidase increased the labeling of this band and also revealed the Mr 98,000 subunit. These data demonstrate that the major subunits of the insulin receptor are complex glycoproteins that have differences in the nonreducing ends of the carbohydrate chains. In the Mr 134,000 subunit, there appear to be more exposed galactosyl or N-acetylgalactosaminyl (or both) residues, whereas the Mr 98,000 subunit appears to have a higher degree of sialylation. These labeling techniques provide new tools to examine the role of the carbohydrate moiety in insulin receptor function and turnover.

Cells, Cultured↗
PubMed1981

Autoantibodies against the insulin receptor recognize the insulin binding subunits of an oligomeric receptor.

125I-insulin was specifically cross-linked to membranes of human cultured lymphocytes (IM-9 line) using disuccinimidyl suberate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and autoradiography of this preparation revealed a major 125I-labeled band with an apparent mol wt of 130,000 and minor bands of about 300,000 and 95,000. Labeling of these bands was inhibited by incubation of membranes with either unlabeled insulin or autoantibodies to the insulin receptor. The bands were also observed after the 125I-insulin cross-linked preparation was solubilized and immunoprecipitated with a panel of autoantibodies to the insulin receptor. However, immunoprecipitation of the 125I-insulin-receptor complex was inhibited by preincubation with excess unlabeled insulin. Finally, 125I-Fab fragments of mol wt 50,000 prepared from anti-receptor antibodies and cross-linked to membranes were resolved into a major complex of mol wt 180,000 and a minor band of 125,000. Neither band was observed when 125I-Fab fragments were cross-linked to membranes in the presence of an excess of unlabeled insulin. These findings indicate that autoantibodies to the insulin receptor are directed against the insulin binding subunits of an oligomeric receptor.

Autoantibodies↗