Laura A. Dollar

Insulin-induced Loss of Insulin-like Growth Factor-I Receptors on IM-9 Lymphocytes

Preexposure of IM-9 lymphocytes to the somatomedin peptide insulin-like growth factor-I (IGF-I) results in a time- and concentration-dependent reduction in specific receptors for IGF-I. Since insulin and proinsulin are structurally homologous to IGF-I, we investigated the ability of insulin analogues to compete for occupancy and to directly modulate IGF-I receptor concentrations. IGF-I binds rapidly and reversibly to IM-9 cells at 15°C, with half-maximal displacement of 125I-I-IGF-I at IGF-I concentrations of 3.6 × 10−9 M and insulin concentrations of 5 × 10−7 M. Preexposure of cells at 37°C to either IGF-I or insulin produced a concentration-dependent reduction in binding of 125I-IGF-I. A 50% decrease in binding was observed following preincubation of cells with IGF-I at 2.5 × 10−9 M and insulin at 2 × 10−7 M. At higher insulin concentrations (10−6–10−5 M), up to 70% reduction in 125I-IGF-I binding occurred. Bovine proinsulin and guinea pig insulin competed less potently than porcine insulin for the IGF-I receptor, and produced receptor loss in proportion to their ability to occupy the IGF-I receptor. Scatchard analysis indicated that at all insulin concentrations, the decrease in binding was secondary to loss of available IGF-I receptors, with no change in affinity. Receptor loss was evident following 1–2 h preexposure to insulin, with a t½ of 4 h and maximal receptor loss within 10 h. Similarly, IGF-I and IGF-II competed for occupancy of the IM-9 insulin receptor, with 50% displacement of 125I-insulin occurring at peptide concentrations of 3.5 × 10−9 M (insulin), 3.5 × 10−8 M (IGF-II), and 3 × 10−7 M (IGF-I). Preexposure of cells to these peptides at 37°C for 20 h resulted in a concentration-dependent reduction in binding of 125I-insulin, with the order of analogue effectiveness being insulin > IGF-II > IGF-I. These data emphasize the structural and functional homology of insulin and the somatomedin peptides, IGF-I and II, as well as their respective receptors. Additionally, the data support the conclusion that the insulin and somatomedin peptides not only bind to both receptors, but downregulate each receptor in proportion to their ability to occupy that receptor.

Recombinant DNA-derived methionyl human growth hormone is similar in membrane binding properties to human pituitary growth hormone

Abstract N-terminal methionyl human growth hormone (met-hGH), expressed in Escherechia coli by recombinant DNA techniques, was compared to native human pituitary growth hormone in receptor-binding activity. Met-hGH was indistinguishable from pituitary hGH in binding to specific hGH receptors on cultured IM-9 lymphocytes and to “lactogenic” receptors on pregnant rat liver membranes. Additionally, met-hGH and pituitary hGH were equivalent in ability to induce hGH receptor loss in IM-9 cells, with an 80% decrease in membrane binding sites following 20 hour preincubation with 10 ng/ml of either met-hGH or pituitary hGH. We conclude that the receptor-binding activity of bacterially synthesized met-hGH and pituitary hGH are identical, suggesting a common tertiary structure.

Simultaneous Inhibition of Insulin and Somatomedin-C Binding to Cultured IM-9 Lymphocytes by Naturally Occurring Antireceptor Antibodies

A 53-yr-old male patient with insulin-resistant diabetes was found to have circulating inhibitors of both insulin and somatomedin-C binding. Serum obtained from the patient at the time of initial presentation inhibited 50% of both 125I-insulin and 125I-SM-C binding to IM-9 lymphocytes at dilutions of 1:150. Spontaneous improvement in the patients diabetic state was associated with a simultaneous and equal decrease in the serum inhibitory titers for both radioligands. Scatchard analysis indicated that the observed serum-induced decrease in both insulin and SM-C binding was due to decreased receptor affinity, with no alteration in receptor number. The serum inhibitors of both insulin and SM-C binding were precipitated equally by Staph-A and also by 40% ammonium sulfate, suggesting they were immunoglobulins. The observation of naturally occurring autoantibodies against both the insulin and SM-C receptors suggests a structural homology between the two receptors.