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C R Kahn

Publications and source records attributed to C R Kahn.

At least 307 records · Page 17Linked to original sources
PubMed1983

Biosynthesis and glycosylation of the insulin receptor. Evidence for a single polypeptide precursor of the two major subunits.

The biosynthesis and carbohydrate processing of the insulin receptor were studied in cultured human lymphocytes by means of metabolic and cell surface labeling, immunoprecipitation with anti-receptor autoantibodies, and analysis on sodium dodecyl sulfate-polyacrylamide gels under reducing conditions. In addition to the two major subunits of Mr = 135,000 and Mr = 95,000, two higher molecular weight bands were detected of Mr = 210,000 and Mr = 190,000. The Mr = 210,000 band and the two major subunits were labeled by [3H]mannose, [3H]glucosamine, [3H]galactose, and [3H]fucose, and were bound by immobilized lentil, wheat germ, and ricin I lectins. On the other hand, the Mr = 190,000 band was labeled only by [3H]mannose and [3H]glucosamine and was bound only by lentil lectin. All four components could be labeled with [35S] methionine; however, in contrast with the other three polypeptides, the Mr = 190,000 band was not labeled by cell surface iodination with lactoperoxidase, suggesting that it is not exposed at the outer surface of the plasma membrane. Pulse-chase studies with [3H]mannose showed that the Mr = 190,000 was the earliest labeled component of the receptor; radioactivity in this band reached a maximum 1 h after the pulse, clearly preceded the appearance of the other components, and had a very brief half-life (t1/2 = 2.5 h). The Mr = 210,000, Mr = 135,000, and Mr = 95,000 bands were next in appearance and reached a maximum 6 h in the chase period. Monensin, an ionophore which interferes with maturation of some proteins, blocked both the disappearance of the Mr = 190,000 protein and the appearance of the Mr = 135,000 and Mr = 95,000 subunits. The mannose incorporated in the Mr = 190,000 component was fully sensitive to treatment with endoglycosidase H while that in the Mr = 210,000 band and the two major subunits was only partially sensitive. Tryptic fingerprints of the 125I-labeled Mr = 210,000 band suggested that this component contains peptides of both the Mr = 135,000 and Mr = 95,000 subunits. In conclusion, the Mr = 190,000 component appears to represent the high mannose precursor form of the insulin receptor that undergoes carbohydrate processing and proteolytic cleavage to generate the two major subunits. In addition, the Mr = 210,000 band is probably the fully glycosylated form of the precursor that escapes cleavage and is expressed in the plasma membrane.

Cell Line↗
PubMed1983

Expression of insulin receptors and insulin action in cell hybrids and cybrids.

The expression of insulin receptors and insulin action was studied in cell hybrids and cybrids produced by fusion of the BWIJ mouse hepatoma cell line with nucleated and enucleated mouse L-cells (LEA-2A) respectively. The BWIJ parent and the cybrids expressed high numbers of insulin receptors, whereas the hybrids resembled the L-cell parent with low numbers of receptors. Likewise, the hybrids resembled the LEA-2A cells with high levels of glycogen synthase, whereas the BWIJ cells and cybrids had much lower levels. Both parents, the cybrids, and the hybrids, expressed insulin stimulation of alpha-aminoisobutyric acid influx, but the dose-response curves indicated an increased insulin sensitivity in the cells with the higher receptor concentration. Insulin also stimulated 86Rb+ uptake in the hepatoma parent, hybrids and cybrids, but not in the L-cell parent. These data suggest that insulin receptors, like other hepatoma-specific properties, behave as a 'luxury function' of the hepatoma cell line and are extinguished when the hepatoma cell is fused with a less differentiated cell type. The biological activities associated with insulin action, on the other hand, are much more complex in their expression and probably the result of the interaction of multiple factors that vary in their expression in cell hybrids and cybrids.

Amino Acids↗
PubMed1983

Characterization of a membrane regulator of insulin receptor affinity.

Using the technique of radiation inactivation we have previously shown that the insulin receptor behaves as if it is composed of at least two functional components: a binding component (Mr approximately equal to 100,000) and an affinity regulatory component (Mr approximately equal to 300,000). The interaction between the affinity regulator and binding component results in a decrease in the affinity of the receptor for insulin. To examine in more detail the interaction between this "affinity regulator" and the binding component we have studied the insulin receptor by radiation inactivation under conditions which alter receptor concentration or receptor affinity. Liver membranes of ob/ob mice exhibit a decrease in insulin binding when compared to their lean litter mates which is due to a decrease in receptor concentration. When studied by radiation inactivation, however, there was no detectable change in the interaction or size of the two receptor components. By contrast, under circumstances in which the affinity of the receptor was increased (treatment with high salt, high pH, 1 mM dithiothreitol, 1-5 micrograms/ml of trypsin), the interaction between the regulatory and binding components was either decreased or absent, i.e. there was no increase in binding with irradiation. Conversely, conditions which produce a decrease in receptor affinity resulted in an increase in the interaction between the regulatory and binding components. The changes in receptor affinity and interactions of the two components produced by either high salt or pH were reversible. Partial purification of the solubilized receptor on lectin affinity columns resulted in the apparent removal of the affinity regulator, i.e. receptor affinity was increased. In this state, radiation inactivation studies revealed a monoexponential decay indicating no interaction between binding and regulatory components. Taken together, these results suggest that the affinity regulator is a membrane protein which is both trypsin-sensitive and has disulfide bond(s) essential for its function. The interaction between the affinity regulator and binding component is not via a covalent bond and the two components appear to be separated by lectin chromatography. The interaction between these components appears to be altered in most states associated with altered receptor affinity.

Animals↗
PubMed1983

Characterization of insulin-mediated phosphorylation of the insulin receptor in a cell-free system.

Insulin stimulates phosphorylation of both alpha- and beta- subunits of its own receptor in a cell-free system. A solubilized lectin-purified preparation of insulin receptors from rat liver membranes was preincubated with or without insulin at 4 degrees C and labeled for 10 min with Mn[gamma- 32P]ATP; the receptor subunits were isolated by specific immunoprecipitation with anti-receptor antibodies, followed by gel electrophoresis in sodium dodecyl sulfate. In gels run under reduced conditions, two bands (Mr = 135,000 and 95,000) were selectively labeled. These correspond exactly to the position of the alpha- and beta-subunits of the insulin receptor. Labeling of the Mr = 95,000 band was approximately 5-fold that of the Mr = 135,000 band. No labeled bands were detected when identical samples were immunoprecipitated in control serum. Phosphorylation of the receptor subunits required the presence of the divalent cation Mn2+ or Co2+; other cations such as Mg2+, Cr3+, Ca2+, and Zn2+ were ineffective. [gamma- 32P]ATP served as the 32P donor, whereas [gamma- 32P]GTP was ineffective. Phosphorylation of both subunits was stimulated 4-6-fold after a 60-min exposure to 10(-7) M pork insulin. Insulin-stimulated phosphorylation was half-maximal after 5 min of incubation with 10(-7) M insulin or after 18 h with 3 X 10(-10) M hormone. The enhanced phosphorylation was specific for insulin and its analogs; guinea pig insulin was about 2% as potent as pork insulin, whereas epidermal growth factor, adrenocorticotropic hormone, and glucagon, as well as cAMP, were ineffective. The rapidity and specificity of this reaction, as well as the presence of all necessary components in the plasma membrane, suggest that insulin-mediated receptor phosphorylation is one of the earliest biochemical steps following insulin binding.

Adenosine Triphosphate↗
PubMed1983

Glucagon regulation of amino acid transport in hepatocytes: effect of cell enucleation.

Glucagon and cAMP analogs stimulate amino acid transport in freshly isolated hepatocytes by inducing the synthesis of new transport proteins. The role of the cell nucleus in the glucagon regulation of amino acid transport has been studied in rat hepatocytes enucleated by centrifugation through a discontinuous Ficoll gradient in the presence of cytochalasin B. Enucleated hepatocytes take up alpha-aminoisobutyric acid (AIB) through a Na+-dependent transport component with kinetic properties similar to those found in intact hepatocytes. Cytoplasts prepared from glucagon-stimulated cells retain the increase AIB transport induced by the hormone in the intact cells. The direct addition of glucagon to cytoplasts has no effect on AIB transport, in spite of the fact that the cytoplasts exhibit a higher capacity to bind glucagon than their nucleated counterparts. These data indicate that the nucleus is required for the glucagon stimulation of amino acid transport in isolated hepatocytes.

Amino Acids↗
PubMed1983

The binding of insulin to mouse leucocytes during viral infections.

The effect of viral infections on insulin binding in vivo was evaluated by measuring the binding of 125I-insulin to several different tissues. We found that splenic leucocytes from mice infected with either the diabetogenic (D) or non-diabetogenic (B) variants of encephalomyocarditis virus, herpes simplex virus, or lactic dehydrogenase virus showed up to a 130% increase in insulin binding. As much as a 300% increase in the binding of 125I-insulin to splenic leucocytes was observed in mice given bacterial lipopolysaccharide. In neither virus-infected nor lipopolysaccharide-treated mice was there any substantial change in insulin receptors on thymocytes, liver membranes, or peripheral erythrocytes. Thus, the increased binding of insulin appears to be limited to leucocytes and does not appear to represent a generalized metabolic alteration. These experiments suggest that during infection, the binding of insulin to leucocytes, which is widely used to measure insulin receptors, may not always accurately reflect the insulin receptor status of other tissues.

Animals↗
PubMed1983

Cultured quail neural crest cells attain competence for terminal differentiation into melanocytes before competence to terminal differentiation into adrenergic neurons.

At the onset of migration the quail neural crest contains pluripotent progenitor cells that give rise to both melanocytes and adrenergic neurons as well as progenitor cells that are already committed to the melanogenic or the neuronal pathway. In this paper we show that melanogenic progenitors attain the competence for terminal differentiation prior to adrenergic progenitors. The adrenergic phenotype was only expressed when the crest cells were allowed to proliferate in vitro for at least 3 days. Differentiation into melanocytes, however, occurred even when proliferation was blocked with cytosine arabinoside immediately after explanation of the neural tube.

Animals↗
PubMed1983

Sharing of biological effect and receptors between guinea pig insulin and platelet-derived growth factor.

Insulins from the hystricomorphs (guinea pig, porcupine, coypu, and casiragua) at high concentration stimulate DNA synthesis in human fibroblasts to a greater level than other mammalian insulins or insulin-like growth factors (IGFs). 125I-Labeled guinea pig insulin binds to a specific receptor and this binding is competed for by hystricomorph insulins but not by porcine insulin or IGFs. Fetal bovine serum also inhibits the binding of 125I-labeled guinea pig insulin and is more potent than fetal bovine plasma, in concordance with their relative potencies for growth stimulation in human fibroblasts. Of several other known growth factors tested, only platelet-derived growth factor (PDGF) inhibits binding of 125I-labeled guinea pig insulin. Four preparations of PDGF that vary in purity and potency for the stimulation of DNA synthesis in human fibroblasts over a 1,000-fold range compete with binding of 125I-labeled guinea pig insulin in proportion to their biological potencies. The purest preparation of PDGF is able to inhibit binding of 125I-labeled guinea pig insulin by 50% at 15 ng/ml (0.25 nM). Biologically, guinea pig insulin, like PDGF, exhibits a synergistic effect with plasma in initiating DNA synthesis in human fibroblasts; this effect is not observed with other mammalian insulins or IGFs. Thus, hystricomorph insulins appear to be mediating their growth-promoting effect through a different receptor and mechanism than other mammalian insulins or IGFs. Further, hystricomorph insulins may be sharing the mechanism of action for their growth effects with PDGF, perhaps suggesting some relationship between these peptides from very different sources.

Animals↗
PubMed1983

Tyrosine-specific protein kinase activity is associated with the purified insulin receptor.

Highly purified human placental insulin receptors were obtained by sequential affinity chromatography on wheat germ agglutinin and insulin-agarose. The preparation had an insulin binding capacity of 4,700 pmol/mg of protein approaching theoretical purity. The purified receptor revealed three major bands of Mr 135,000, 95,000, and 52,000 in NaDodSO4/polyacrylamide gel electrophoresis after reduction by dithiothreitol. All three bands were immunoprecipitated by anti-insulin-receptor antibodies. When this preparation was incubated with [gamma-32P]ATP in the presence of MnCl2 (2 mM) and analyzed in NaDodSO4/acrylamide gel electrophoresis, only the Mr 95,000 band was labeled. Preincubation with several concentrations of insulin increased the 32P incorporation into this peptide in dose-dependent fashion, whereas insulin-like growth factors were approximately equal to 2% as potent and epidermal growth factor had little or no effect, consistent with their known affinities for the insulin receptor. Insulin stimulation of phosphorylation of the Mr 95,000 subunit of the receptor was observed also in immunoprecipitates of this highly purified insulin receptor by anti-insulin-receptor antibodies. Phosphoamino acid determination revealed only phosphotyrosine in both the basal and insulin-stimulated states. These data suggest that a tyrosine-specific protein kinase activity is closely associated with insulin receptor, and this may be important in the signal transmission required for insulin action.

Female↗
PubMed1983

Differential responsiveness to insulin of endothelial and support cells from micro- and macrovessels.

The pathologies of diabetic micro- and macroangiopathy are different, suggesting that diabetes affects these two types of vascular tissue in a dissimilar manner. We have compared insulin receptors and the effects of insulin on cultured endothelium from calf retinal capillaries and aorta, and the vascular supporting cells, retinal pericytes, and aortic smooth muscle cells. 125I-insulin binds to high affinity insulin receptors on all four cell types. Receptor concentrations were similar except for aortic smooth muscle cells, which have 10-fold fewer receptors than the other cell types. Insulin at a concentration of 10 ng/ml stimulated [14C]glucose incorporation into glycogen in retinal endothelial cells and pericytes and aortic smooth muscle cells, but had no effect on aortic endothelium. Insulin over a concentration range of 10 ng/ml-10 microgram/ml, stimulated [3H]thymidine incorporation into the DNA of retinal pericytes, and endothelial cells and aortic smooth muscle cells but had no effect on aortic endothelial cells. These data suggested that a differential response to insulin may exist between endothelium of micro- and macrovasculature, and suggest that retinal capillary endothelium and retinal pericytes are both very insulin-sensitive tissues.

Animals↗
PubMed1983

Specific plasma membrane receptors for reovirus on rat pituitary cells in culture.

Specific cellular and host tropism is a characteristic property of many viruses mediated by the interaction of viral attachment proteins with components of the plasma membrane of the cell. We have studied the binding of virus to cells quantitatively by using type 3 reovirus labeled with 125I and GH4C1 pituitary cells in culture. Binding was rapid at both 4 degrees and 15 degrees C and was stable over a 9-h period. Unlabeled virus inhibited binding of the labeled virus in a dose-dependent manner. Scatchard analysis revealed 4,200 viral binding sites/cell with an apparent affinity of 1.2 X 10(-11) M. Also, binding of type 3 reovirus was inhibited by antibodies directed against the viral hemagglutinin and partially inhibited by type 2 reovirus, but was unaffected by type 1 reovirus or a variety of other ligands that bind to receptors on GH4C1 cells. These data indicate that reovirus binds to a high affinity, specific receptor on target cells, which may control its tropism and ultimate disease expression.

Animals↗
PubMed1983

In vitro lymphocyte proliferation response to therapeutic insulin components. Evidence for genetic control by the human major histocompatibility complex.

Genes in the major histocompatibility complex of mice and guinea pigs control immunologic responsiveness to insulins from other animal species. In order to determine if similar genetic control exists in man, we have examined lymphocyte proliferation responses to components of therapeutic insulins by employing lymphocytes from diabetic patients that receive insulin. Distinct groups of individuals demonstrated positive lymphocyte proliferative responses to beef insulin, beef and pork insulin, beef proinsulin, pork proinsulin, and protamine. Lymphocytes from the patient population were typed for the HLA-A, B, C, and DR antigens. An increased frequency of certain HLA antigens was found in those individuals that responded to the following therapeutic insulin components: beef, HLA-DR4; beef and pork, HLA-DR3; beef proinsulin, HLA-BW4, CW2, CW5, DR2, and DR5; protamine, HLA-CW3, CW5, and DR7. The results demonstrate that the human immune system recognized the structural differences between human and beef and/or pork insulin. These differences are two amino acids in the A chain, alpha loop, of beef insulin and the single terminal amino acid, alanine, which is common to pork and beef insulins. Positive responses to both beef proinsulin and pork proinsulin demonstrated the capability of restricted recognition of more complex proteins represented by the C-peptide in these insulin preparations. Lymphocyte proliferative responses to protamine were also restricted, which suggests a genetic control to this antigen. The association of these responses with HLA alloantigens strongly suggests that genes within the human major histocompatibility complex control recognition and lymphocyte response to therapeutic insulin components.

Adult↗
PubMed1983

Antireceptor antibodies as probes of insulinlike growth factor receptor structure.

Insulin receptors and Type I insulinlike growth factor (IGF) receptors have a similar structure with a major binding subunit of Mr approximately 130,000 linked by disulfide bonds to other membrane proteins to form a Mr greater than 300,000 complex. Both insulin and Type I IGF receptors also interact with both insulin and IGF, although with different binding affinities. We used a panel of human and rabbit sera containing antibodies to insulin receptors to determine whether these sera also interact with Type I IGF receptors. Immunoglobulins from five of five human sera inhibited binding of 125I-insulin and 125I-IGF-I to insulin receptors and Type I IGF receptors in human placenta and human lymphocytes. The rank order of reactivity with both receptors was the same; two sera, however, appeared to be selectively less reactive with the Type I IGF receptor, especially in placenta. Sera from five of seven patients and from a rabbit immunized with purified insulin receptor effectively immunoprecipitated both placental insulin receptors and Type I IGF receptors. Of the remaining sera, one had only a low titer against the insulin receptor and did not immunoprecipitate the IGF receptor, whereas the second serum effectively immunoprecipitated cross-linked and surface-iodinated insulin receptors, but had negligible reactivity against the Type I IGF receptor. These results suggest that most antisera to the insulin receptor also contain antibodies to Type I IGF receptors. Whether both specificities are inherent in the same or different antibody molecules remains to be determined. These data support the hypothesis that the insulin and IGF-I receptors are separate but related molecules, although there remains a small possibility that both receptors are domains on the same protein.

Animals↗
PubMed1983

Analysis of insulin action using differentiated and dedifferentiated hepatoma cells.

Hepatoma cells in culture exhibit a range of differentiated functions. Well differentiated hepatoma cells retain most of the functions of the adult liver, whereas dedifferentiated cells have lost most of them. In the present study, insulin binding and insulin effects were studied in four differentiated and two dedifferentiated cell lines derived from the Reuber hepatoma and in a partially differentiated cell line, HTC, derived from the Morris 7288C hepatoma. Specific insulin binding was lower in dedifferentiated cells than in partially differentiated and differentiated ones. In all cell lines, analysis of insulin binding yielded linear Scatchard plots. Although some variations in affinity of insulin for its receptor were observed, most of the differences in binding were accounted for by differences in insulin receptor number. In differentiated hepatoma cells, tyrosine aminotransferase (TAT)-specific activity was easily detectable, and insulin produced a 2- to 3-fold increase in enzyme activity within 4-6 h. By contrast, TAT activity in the dedifferentiated cells was low and did not respond to insulin. In the partially differentiated hepatoma HTC, insulin stimulated TAT only after basal TAT activity was induced by glucocorticoid treatment. Glycogen synthase (I and D forms) activities were detectable in all cell lines. In both differentiated and dedifferentiated Reuber hepatoma cells, insulin increased the I-form of glycogen synthase within 15 min. This effect of insulin was observed at lower insulin concentrations than stimulation of TAT activity. By contrast, insulin at any concentration was totally ineffective in stimulating glycogen synthase in glucocorticoid-treated and untreated HTC cells. These results indicate that in hepatoma cells, 1) insulin receptor number and insulin effect on TAT are modulated by the degree of cell differentiation; 2) the pathways of insulin action on TAT and glycogen synthase diverge in some postreceptor step(s) which is under independent control in differentiation; and 3) receptor and postreceptor defects exist in hepatoma cell lines which may be useful in dissecting the pathways of insulin action.

Animals↗
PubMed1983

[B17-D-leucine]insulin and [B17-norleucine]insulin: synthesis and biological properties.

The chemical synthesis of two porcine insulin analogues is described. Leucine in position B17 of the native molecule was substituted by its D-enantiomer and by L-norleucine, respectively. Both B-chain derivatives were synthesized by fragment condensation and purified as di-S-sulphonates by gel filtration followed by ion exchange chromatography on SP-Sephadex at pH3. Combination with native sulphhydryl A-chain yielded [DLeuB17]insulin and [NleB17]insulin. Both insulin analogues were isolated by gel filtration followed by ion exchange chromatography on CM-cellulose at pH 4.0. Biological activities of the analogues were determined relative to native pork insulin: 1) glucose oxidation in rat epididymal adipocytes was 6% for [DLeuB17]insulin and 16% for [NleB17]insulin, 2) receptor-binding affinity tested with cultured human fibroblasts and with rat adipocytes was 3% for [DLeuB17]insulin and 26% for [NleB17]insulin, and 3) thymidine incorporation into DNA of human fibroblasts was 35% for [DLeuB17]insulin and 100% for [NleB17]insulin.

Adipose Tissue↗
PubMed1983

Reactive hypoglycemia and insulin autoantibodies in drug-induced lupus erythematosus.

An 82-year-old woman developed symptomatic reactive hypoglycemia in the same year she developed a lupus-like syndrome, probably secondary to the administration of procainamide or hydralazine. Reactive hypoglycemia was confirmed by an oral glucose tolerance test, in which plasma glucose decreased from a fasting level of 87 mg/dL to 32 mg/dL at 3 hours and 23 mg/dL at 4 hours, the last value being associated with loss of consciousness. The patient awoke after the intravenous administration of dextrose. Sensitivity to exogenous insulin was normal or increased. Attempts to measure plasma insulin levels led to the finding of anti-insulin antibodies in the patient's serum; these antibodies were of relatively low titer, were IgG, and not associated with antibodies to the insulin receptor. The patient had no history of exogenous insulin use. Her reactive hypoglycemia appeared due to the autoimmune insulin syndrome, which developed in association with drug-induced lupus erythematosus.

Aged↗