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C R Kahn

Publications and source records attributed to C R Kahn.

At least 325 records · Page 18Linked to original sources
PubMed1982

Insulin stimulation of phosphorylation of the beta subunit of the insulin receptor. Formation of both phosphoserine and phosphotyrosine.

Rat hepatoma cells were labeled with [32P]orthophosphate and the insulin receptor subunits were identified by immunoprecipitation and sodium dodecyl sulfate-acrylamide gel electrophoresis. In the basal state, only the Mr = 95,000 (beta) subunit of the insulin receptor was phosphorylated. The covalent labeling with 32P of this subunit was stimulated about 3-fold by insulin (10(-6) M). This stimulation was due to an increase in the content of phosphoserine, the appearance of phosphotyrosine, and a possible increase in phosphothreonine as well. These results suggest phosphorylation of the insulin receptor at multiple sites is an early event in insulin action.

Amino Acids↗
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

Subpopulations of antibodies directed against evolutionarily conserved regions of the insulin molecule in insulin-treated patients.

In the present study, we attempted to define possible subpopulations of antibodies which theoretically could be directed against evolutionarily conserved regions of the insulin molecule in sera from insulin-treated diabetic patients using a variety of labelled and unlabelled insulins which differ widely in structure but are very similar in functional properties. Ten high titre human insulin antisera from patients treated with mixed beef-pork insulin were examined. All sera were found to bind 125I-pork insulin better than labelled chicken insulin which bound better than labelled fish insulin. Detailed studies were conducted with four of the antisera using the pork and fish tracers. With two of the antisera, a subpopulation of antibody could be detected with 125I-fish insulin which had similar affinity for both fish and pork insulin, but reacted much less well with guinea pig insulin and the desoctapeptide derivative of porcine insulin. Based on the known properties of these four insulins, the data provide suggestive evidence consistent with the hypothesis that there are subpopulations of antibodies recognizing regions on the insulin molecule that are well conserved, possibly the region involved in the formation of insulin dimers or receptor binding.

Animals↗
PubMed1982

Insulin binding and activation of glycogen synthase in fibroblasts from Type 1 (insulin-dependent) diabetic patients.

125I-Insulin binding and insulin stimulation of glycogen synthase were examined in fibroblasts cultured from nine Type 1 (insulin-dependent) diabetic patients with age of onset of less than 42 years. In all cases specific insulin binding was qualitatively and quantitatively normal. Total 125I-insulin binding was elevated in cells from three patients with early onset diabetes (two with onset before age 1 year) due to an increase in 'non-specific' binding. When the ability of insulin to stimulate the conversion of the glucose-6-phosphate dependent to the glucose-6-phosphate independent form of glycogen synthase was measured, all cell lines responded, albeit to differing degrees. In general, the response of cells from diabetic donors was more variable than that of control fibroblasts. A slightly lower level of cellular glycogen was evident in the cells of the diabetic patients, and this was mirrored in slightly higher levels of the independent form of the enzyme. The average maximal level of the independent form of the enzyme also was higher in the diabetic patients' cells. Fibroblasts from one of the patients with very early onset diabetes had glycogen synthase levels that were markedly lower than in any other cell line examined. In summary, fibroblasts cultured from Type 1 diabetic patients do not show major defects in either insulin binding or action. A suggestion of subtle differences in the cells from the diabetic patients, particularly those with very early onset, is evident, however. Whether these are secondary to some primary genetic defect or represent some selection during culture remains to be determined.

Cells, Cultured↗
PubMed1982

Effect of cross-linking agents on insulin associated responses in adipocytes.

The effect of 10 bifunctional cross-linking agents and four monofunctional analogues was studied on isolated adipocytes. [125I]Insulin binding and degradation, basal and insulin-stimulated glucose oxidation, and 3-O-methyl glucose uptake were measured. Two cross-linkers, which possess succinimide ester residues (disuccinimidyl suberate and dithiobis(succinimidyl propionate)) and react selectively with amino groups, appeared to react relatively specifically with the insulin receptor. Both produced a slight stimulation of basal glucose transport and metabolism, a marked inhibition of insulin-stimulated glucose transport and metabolism, and a marked decrease in insulin binding. Pretreatment of cells with unlabelled insulin partially blocked the effect of disuccinimidyl suberate, and as has been previously shown, disuccinimidyl suberate cross-linked insulin to its receptor. A monofunctional analogue of these compounds was 100-fold less active in altering cellular metabolic activity. Bisimidates, such as dimethyl suberimidate, dimethyl adipimidate, and dimethyl dithiobispropionimidate, also react with free amino groups but are more hydrophilic. These agents produced similar effects on glucose oxidation as the succinimide esters, but had little or no effect on insulin binding. The effects of these agents are not blocked by insulin and they do not cross-link insulin to its receptor. Mixed bifunctional reagents containing either a succinimide ester or an imidate and a group which reacts with thiols produced effects similar to the cross-linkers containing two succinimide groups or bisimidates, respectively. The bifunctional arylating agents difluorodinitrobenzene and bis(fluoronitrophenyl)sulfone produce marked effects on insulin binding and glucose oxidation at micromolar concentrations, but the monofunctional analogue fluorodinitrobenzene is almost equally active suggesting that with these compounds chemical modifications and not cross-linking was important. With neither the mixed bifunctional reagents, nor the arylating agents, did insulin pretreatment alter the effect of cross-linker and none of these agents cross-linked [125I]insulin to its receptor. These data suggest that the insulin receptor possesses a free amino group in a hydrophobic environment in its active site. A reactive amino group in a hydrophilic environment as well as other reactive groups are also present in some component of the insulin receptor-effector complex. Chemical modification or cross-linking of these functional groups results in an inhibition or mimicking of insulin action. Further study will be required to identify the exact locus of these sites.

Adipose Tissue↗
PubMed1982

Autoantibodies to the insulin receptor and insulin-resistant diabetes.

Autoantibodies to the insulin receptor are a rare cause of insulin-resistant diabetes, but when they occur they produce a profound clinical syndrome. These antibodies block insulin binding, immunoprecipitate solubilized insulin receptors, and their acute effect is to mimic the biological effects of insulin. However, prolonged exposure of cells to these antibodies produces a state of insulin resistance. Since the antigen to which the antibody is directed is relatively well-characterized, many of the observations in this syndrome can serve as a model for elucidating molecular mechanisms in other diseases with antibodies against membrane components. The autoantibodies to the insulin receptor have also provided valuable probes in the study of insulin receptor structure and insulin action.

Adipose Tissue↗
PubMed1982

Insulin radioreceptor assay on murine splenic leukocytes and peripheral erythrocytes.

Insulin radioreceptor assays were developed using splenic leukocytes and peripheral erythrocytes from individual mice. Splenic leukocytes were prepared using an NH4Cl buffer which did not alter insulin binding, but gave much higher yields than density gradient methods. Mouse erythrocytes were isolated from heparinized blood by three passages over a Boyum gradient, and a similar buffer was used to separate cells from free [125I]iodoinsulin at the end of the binding incubation. Insulin binding to both splenic leukocytes and peripheral erythrocytes had typical pH, temperature, and time dependencies, and increased linearly with an increased number of cells. Optimal conditions for the splenic leukocytes (6 X 10(7)/ml) consisted of incubation with [125I]iodoinsulin at 15 C for 2 h in Hepes buffer, pH 8.0. In cells from 20 individual mice, the specific [125I]iodoinsulin binding was 2.6 +/- 0.1% (SEM), and nonspecific binding was 0.3 +/- 0.04% (10.6% of total binding). Erythrocytes (2.8 X 10(9)/ml) were incubated with [125I]iodoinsulin at 15 C for 2 h in Hepes buffer, pH 8.2. In cells from 25 individual mice, the specific [125I]iodoinsulin binding was 4.5 +/- 0.2%, and nonspecific binding was 0.7 +/- 0.03% (13.6% of total binding). In both splenic leukocytes and peripheral erythrocytes, analysis of equilibrium binding data produced curvilinear Scatchard plots with approximately 3500 binding sites/leukocyte and 20 binding sites/erythrocyte. These data demonstrate that adequate numbers of splenic leukocytes and peripheral erythrocytes can be obtained from individual mice to study insulin binding in a precise and reproducible manner. This should facilitate direct comparisons of these cells with classical target cells in a variety of mouse model diseases.

Ammonium Chloride↗
PubMed1982

[B22-D-arginine]insulin: synthesis and biological properties.

An analogue of porcine insulin which differs from the native molecule in that the amino-acid residue B22-L-arginine is replaced by its D-enantiomer has been synthesized. The [D ArgB22]B-chain was synthesized by the segment condensation method and purified as the di-S-sulfonate by ion exchange chromatoggraphy on SP-Sephadex at pH 3.5. Combination with native porcine sulfhydryl A-chain gave [DArgB22]insulin which was purified by ion exchange chromatography on SP-Sephadex at pH 4.5 with a linear NaCl gradient. The biological activity of this analogue as measured by glucose oxidation in rat epididymal adipocytes was 2%. Thymidine incorporation into DNA of human fibroblast was 16%. The immunoreactivity using antipork insulin antibody in a double antibody immunoassay was 4%. The receptor-binding affinity as measured by radioreceptor assays was 2% with cultured human fibroblasts and 1% with rat adipocytes. These results suggest that the L-configuration at B22-arginine is essential for retaining the biological, immunological and receptor-binding properties of the hormone.

Adipose Tissue↗