Joji Hari

The receptor for insulin-like growth factor II mediates an insulin-like response.

Insulin‐like growth factor II (IGF‐II) shares sequence homology and predicted three‐dimensional structure with insulin and IGF‐I. IGF‐II can bind, therefore, to a limited extent with the receptors for these two other hormones, as well as to a distinct receptor for IGF‐II. Previous studies have been unable to attribute a particular response of IGF‐II through its own receptor. In the present studies, the IGF‐II receptor is shown to mediate the stimulation of glycogen synthesis in human hepatoma cells since: (i) IGF‐II is found to be capable of stimulating a response at concentrations in which it would primarily interact with its own receptor; (ii) the response to IGF‐II was not blocked by monoclonal antibodies which inhibit the responses of cells through the insulin and IGF‐I receptors; and (iii) polyclonal antibodies to the IGF‐II receptor were found to mimic the ability of IGF‐II to stimulate glycogen synthesis. These results indicate that the IGF‐II receptor mediates a particular biological response–stimulation of glycogen synthesis in hepatoma cells. Furthermore, a monovalent Fab fragment of the polyclonal antibody to the IGF‐II receptor was also shown to stimulate glycogen synthesis in these cells. These data indicate that clustering of the IGF‐II receptor is not required to stimulate a biological response.

Prevention of Cyclophosphamide-Induced and Spontaneous Diabetes in NOD/Shi/Kbe Mice by Anti-MHC Class I Kd Monoclonal Antibody

The immune mechanisms directly responsible for β-cell destruction in insulin-dependent diabetes are undefined. We studied the role of MHC class I–restricted T lymphocytes in the development of diabetes in cyclophosphamide (CY)-treated male and untreated female NOD mice (H-2Kd,Db). After administration of CY to 10-wk-old male NOD/Shi/Kbe mice, 37 of 64 (58%) phosphate-buffered saline–injected control mice and 13 of 22 (59%) anti-Kb and 12 of 27 (44%) anti-Db monoclonal antibody (MoAb)-injected mice became diabetic by 14 wk of age, whereas only 3 of 38 (8%) anti-Kd and 2 of 13 (15%) anti-Lyt-2 MoAb-injected mice did. In untreated female NOD/Shi/Kbe mice, 30 of 46 (65%) mice developed spontaneous diabetes by 30 wk of age, whereas none of 9 anti-Kd MoAb-injected mice became diabetic. Immunohistochemical studies showed that islet-infiltrating cells in CY-treated control mice were composed mainly of both L3T4+ and Lyt-2+ T lymphocytes, whereas many L3T4+ and very few Lyt-2+ lymphocytes infiltrated within the islets in anti-Kd MoAb-injected mice. Administration of anti-Lyt-2 MoAb induced the absence of Lyt-2+ T lymphocytes in the islet and spleen. However, anti-Kd MoAb did not change the number of spleen cells or the T-lymphocyte subset and response to concanavalin A. These results suggest that MHC class I Kd-restricted Lyt-2+ T lymphocytes play an important role as direct effector cells in destruction of β-cells in NOD/Shi/Kbe mice.

Production of monoclonal antibodies to islet cell surface antigens using hybridization of spleen lymphocytes from non-obese diabetic mice.

SummaryNon-obese diabetic mice display a syndrome with dramatic clinical and pathological features similar to those of Type 1 (insulin-dependent) diabetes in man. Circulating autoantibodies to the surface of islet cells were demonstrated in some of these mice by a protein A radioligand assay. To produce monoclonal antibodies to islet cell surface antigens, therefore, we took the spleens of non-obese diabetic mice, transferred the spleen cells into non-immunized recipient mice, which were made immunologically incompetent by a large dose of X-irradiation, and then fused their lymphocytes with FO mouse myeloma cells. After screening the resultant hybrids, one stable hybridoma (3A4) that produced a monoclonal antibody (IgG1) specifically bound to the surface of islet cells was obtained. The purified monoclonal antibody was bound to the surface of transplantable Syrian golden hamster insulinoma cells sevenfold more than control antibody. Adsorption of the antibody on mouse spleen lymphocytes or thymocytes resulted in only a slight decrease in 125I-protein A binding to insulinoma cells. This antibody also reacted with the surface of mouse and rat islet cells, but not with that of rat spleen cells or hepatocytes. A spectrophotometric assay for peroxidase activity demonstrated that six times more peroxidase bound to insulinoma cells incubated with the antibody than to cells treated with control antibody. Furthermore, this antibody could be visually detected in the immunoenzymatic labelling of the surface of insulinoma cells. In summary, we have developed a novel method of producing monoclonal antibodies to the surface of islet cells for probing into the pathogenesis of Type 1 diabetes.

Insulin-degrading enzyme is capable of degrading receptor-bound insulin

In the investigation of the intracellular sites of insulin degradation, it might be important whether receptor-bound insulin could be a substrate for insulin-degrading enzyme (IDE). Insulin receptor and IDE were purified from rat liver using a wheat germ agglutinin column and monoclonal anti-IDE antibody affinity column, respectively. [125I]insulin-receptor complex was incubated with various amounts of IDE at 0 degree C in the presence of disuccinimidyl suberate and analyzed by reduced 7.5% SDS-PAGE and autoradiography. With increasing amounts of IDE, the radioactivity of 135 kd band (insulin receptor alpha-subunit) decreased, whereas that of 110 kd band (IDE) appeared then gradually increased, suggesting that IDE could bind to receptor-bound insulin. During incubation of insulin-receptor complex with IDE at 37 degrees C, about half of the [125I]insulin was dissociated from the complex. However, the time course of [125I]insulin degradation in this incubation was essentially identical to that of free [125I]insulin degradation. Cross-linked, non-dissociable receptor-bound [125I]insulin was also degraded by IDE. Rebinding studies to IM-9 cells showed that the receptor binding activity of dissociated [125I]insulin from insulin-receptor complex incubated with IDE was significantly (p less than 0.001) decreased as compared with that without the enzyme. These results, therefore, show that IDE could recognize and degrade receptor-bound insulin, and suggest that IDE may be involved in insulin metabolism during receptor-mediated endocytosis through the degradation of receptor-bound insulin in early neutral vesicles before their internal pH is acidified.

Anti-interleukin 2 receptor antibody attenuates low-dose streptozotocin-induced diabetes in mice

SummaryRecent evidence indicates that activated T cells and macrophages play an important role in the induction of insulitis and diabetes in certain strains of mice treated with multiple subdiabetogenic doses of streptozotocin. In the present study, we treated C57BL/6J mice with five daily doses of 40 mg/ml streptozotocin and examined the prophylactic effect of an anti-interleukin 2 receptor monoclonal antibody (PC61). In mice treated with streptozotocin, interleukin 2 receptor-positive mononuclear cells were shown to infiltrate into the islets and soluble interleukin 2 receptors in the sera were significantly increased compared with control mice. The administration of PC61 to the mice attenuated the insulitis, and diminished interleukin 2 receptor-positive cells from islets and soluble interleukin 2 receptors in the sera. Moreover, the administration of PC61 significantly reduced the development of hyperglycaemia shown in these mice (12.8±1.1 mmol/l vs 18.5±0.7 mmol/l, p<0.005). As judged by flow cytometric analysis, this antibody did not cause any changes in either spleen cell counts or T cell subsets. Interleukin 2 receptors were expressed on a minor population of spleen cells regardless of treatment with PC61 (STZ + normal rat IgG: 2.1±0.3%, STZ + PC61: 2.4±0.3%). Even after stimulation of spleen cells with concanavalin A or alloantigen, interleukin 2 receptor expression was not significantly different between the two groups. Our studies suggest that interleukin 2 receptor-positive activated T cells or macrophages are important in the development of multi-low-dose streptozotocin diabetes and that an anti-interleukin 2 receptor antibody can attenuate this process.

Possible role of cell surface insulin degrading enzyme in cultured human lymphocytes

SummaryThe kinetic changes of insulin receptors and cell surface insulin degrading enzyme were examined in Bri-7 cultured human lymphocytes after preincubation with or without insulin. The concentration of cell surface insulin degrading enzyme was determined by immunoenzymatic labeling method using a polyclonal antiserum to insulin degrading enzyme. In Bri-7 cells preincubated with 10−10 to 10−5mol/l insulin for 18h, the surface insulin receptors and insulin degrading enzyme decreased progressively as a function of the concentration of insulin in the preincubation medium. The surface insulin receptors and insulin degrading enzyme of cells preincubated with 10−6mol/l insulin were decreased to 25 and 35% of the control respectively. In Bri-7 cells preincubated with 10−6 mol/l insulin for 30 min to 18 h, the loss of surface insulin degrading enzyme was slightly slower than that of the receptors; however, the curves were essentially parallel to each other. Thus, the treatment of Bri-7 cells with insulin caused down-regulation of insulin receptors in a dose- and time-dependent manner. Cell surface insulin degrading enzyme also decreased simultaneously. A combination of several insulin degradation assays (trichloroacetic acid precipitation, gel filtration and receptor rebinding) demonstrated that cell surface bound insulin remained intact, and that the degradation in Bri-7 cells seemed to be a limiting proteolysis of insulin. Furthermore, by the receptor rebinding method insulin degrading activity in cells after preincubation with 10−6 mol/l insulin (19.6±4.6%) was decreased, although not significantly, as compared with cells after preincubation without insulin (24.6±4.8%). These results suggest a possible hypothesis that cell surface insulin degrading enzyme may be internalized with the insulin-receptor complex, and that it may degrade insulin during the intracellular process.

Internalization and degradation of insulin by a human insulin receptor-v-ros hybrid in Chinese hamster ovary cells

Chinese hamster ovary cell lines expressing either the wild-type human insulin receptor or a hybrid molecule in which the tyrosine kinase domain of the insulin receptor is replaced with that of the oncogene, v-ros were examined for their ability to internalize and degrade insulin. Cells expressing the hybrid receptor were found to internalize and degrade insulin at approximately half the rate of cells expressing the native insulin receptor. Moreover, insulin was incapable of inducing the internalization of the cell-surface hybrid molecule. In contrast, the constitutive rate of receptor internalization was found to be the same for the hybrid and wild-type receptors. These results obtained were similar to those with cells expressing either wild-type or mutant receptors lacking kinase activity. In conclusion, the substitution of the specificity of tyrosine kinase of the insulin receptor with that of the v-ros oncogene product results in defective internalization and degradation of insulin, and loss of ligand-induced receptor internalization.

Regulation of biological functions by insulin and insulin-like growth factor I via their own distinct receptors

Insulin and insulin-like growth factor I (IGF-I) are related hormones that have a high degree (50%) of amino acid sequence homology and a similar spectrum of biological responses [l-3]. They are considered to be vertebrate hormones, although the basic structure of these molecules is present even in hormones isolated from invertebrates [4]. In man, both of these polypeptide hormones have been well characterized. However, their relative potencies differ. The physiological role of insulin is primarily to regulate short-term metabolic effects, such as glucose uptake into fat and muscle cells [5,6]. IGF-I, on the other hand. has a more potent long-term response. the growth of the organism [7,8]. Both polypeptide hormones bind with high affinity to their own distinct cell surface receptors, but each can also bind to the heterologous receptor with substantially reduced binding affinity [9]. As do insulin and IGF-I, their receptors for these hor-

Effect of antiserum to monoclonal anti-islet cell surface antibody on pancreatic insulitis in non-obese diabetic mice

Immunotherapeutic intervention has been studied in non-obese diabetic (NOD) mice. Twenty-five male NOD mice aged 6 weeks were treated with anti-mouse T lymphocyte serum (ATS), N-(2-carboxyphenyl)-4-chloroanthranilic acid disodium salt (CCA); a non-specific immunostimulant which seems to potentiate the suppressor T cell activity, and antiserum raised against previously reported monoclonal antibody 3A4 to the surface of islet cells. Pancreatic islets from NOD mice sacrificed at 12 weeks of age were scored morphologically for the severity and the frequency of insulitis. Both the severity of insulitis in each islet and the frequency of insulitis-positive islets in each pancreas were reduced in the groups treated with ATS (group B), antiserum to 3A4 (group D) and a combination of antiserum plus CCA (group E) in comparison with other groups (control: group A and CCA: group C). At 8 weeks of age, the binding capacities of sera to insulinoma cells measured by protein A radioligand assay were significantly decreased in the groups treated with antiserum to 3A4 (groups D and E) as compared with those in the other groups. These results suggest that both ATS and antiserum to 3A4 prevent the occurrence and the progress of insulitis in NOD mice, but the immunosuppressive mechanisms differ from each other; therefore, combination therapy with these suppressants may be more effective in the prevention of Type 1 diabetes mellitus.