The insulin receptor of the turkey erythrocyte. Characterization of the membrane-bound receptor.

The insulin receptor of the turkey erythrocyte has previously been shown to be very similar to that of the mammalian insulin receptors. As a first step in the isolation of this receptor a highly purified plasma membrane fraction has been prepared. The binding characteristics of the purified membrane-bound receptor were identical to those found with intact erythrocytes, but the membrane preparation had very little insulin-degrading activity. Isolation of the membrane by the methods described gave a 100-fold purification of the insulin receptor with 67% yield.

Modification of membrane physical properties, biological response and insulin binding in Friend cells by low serum concentration.

The effect of low serum concentration on plasma membrane fluidity and lipid composition, differentiation and insulin binding was investigated in three Friend erythroleukemia clones. Both FLC (clones No. 745) and F(+) (Ostertag F4N) Friend erythroleukemia cells can be induced to differentiate and to produce hemoglobin when exposed to DMSO. Clone R(3) (Ostertag F4-D5-1) is a DMSO-resistant clone when grown under normal conditions (15% serum) but could undergo differentiation with accumulation of protoporphyrin IX upon induction with DMSO when grown in low serum concentration (2.5% serum). Electron spin resonance measurements of the order parameters (S) and S(T parallel) demonstrate that R(3) has a more fluid plasma membrane than the FLC and F(+). The order parameters of the outer hyperfine splittings S(T parallel) at 37 degrees C are 0.60 +/- 0.009, 0.62 +/- 0.008 and 0.64 +/- 0.009 for R(3), F(+) and FLC, respectively. We have used the insulin receptors as a model for a polypeptide hormone receptor associated with the plasma membrane of the Friend clones. Insulin binding studies demonstrated that the receptor of R(3) had a decreased affinity for insulin manifest as a 10-fold increase in the amount of insulin required to compete for half of the tracer binding (18 nM for R(3) vs. 2 nM for FLC and F(+)). Computer-fit Scatchard plot analysis by the negative cooperativity model reveal that R(3) possessed a similar number of sites/cell (about 70,000) as the FLC or F(+) cells, with similar high and low affinities. Growing the DMSO-resistant clone R(3) in low serum concentration caused a decrease in receptor number by 35%, and an increase in receptor affinity to that seen with the differentiable clones. Thus, the abnormal properties of the plasma membrane and insulin receptor of the DMSO-resistant clone in our earlier report (Simon et al. (1984) Biochim. Biophys. Acta 803, 39-47) were partially reversed by growing the cells in a low serum concentration, restoring the cellular response to the differentiation agent.

Abnormal insulin binding and membrane physical properties of a Friend erythroleukemia clone resistant to dimethylsulfoxide-induced differentiation.

We have compared insulin binding, plasma membrane fluidity, and phospholipid composition of three different Friend erythroleukemia clones, a wild type (FLC) a mutant (R3) and the revertant to wild type F+. The R3 clone is a non-differentiating DMSO-resistant clone (R3) and has altered membrane fluidity and dramatically altered insulin-binding properties. The receptor of R3 bound insulin as if it possessed a single class of low affinity receptors that lacks the property of negative cooperativity. The Scatchard plot is linear and there is no ligand-induced acceleration of dissociation. The Hill coefficient for R3 is 1, implying 'no cooperativity', whereas the Hill coefficient for the two DMSO-inducible clones, (FLC and F+) is 0.3, implying 'negative cooperativity'. In addition, the insulin receptor of R3 has a decreased affinity for insulin, manifested as a 40-fold increase in the amount of insulin required to compete for half of the tracer binding (41 nM for R3 vs. 1 nM for FLC and F+). Computer-fitted Scatchard plots analyzed by the negative cooperativity model reveal that R3 has 95 000 receptor sites/cell, with a high affinity constant Ke of 0.016 nM-1, and a low affinity constant, Kf of 0.012 nM-1. Both DMSO-inducible clones have about 40 000 receptor sites/cell with Ke of 0.11 nM-1 and Kf of 0.02 nM-1. Electron spin resonance measurements with the 5-nitroxy stearate spin probe demonstrate that R3 had a more fluid plasma membrane than the FLC and F+ clones. The lipid composition of R3 is different from that of the DMSO-inducible clones. The weight ratio for unsaturated fatty acids to saturated fatty acids for R3 is 2.5, and the FLC clone has a lower ratio of 1.9. These results are consistent with our earlier findings in FLC that very high membrane fluidity is associated with alterations in the binding properties of the insulin receptor.

Effect of alterations in membrane lipid unsaturation on the properties of the insulin receptor of Ehrlich ascites cells.

We have altered the phospholipid composition of the plasma membranes of Ehrlich ascites cells, grown in mice and studied the effects on the properties of the insulin receptor of this cell. The insulin receptor of the Ehrlich cell demonstrated all of the binding characteristics of mammalian insulin receptors: specificity for insulin and insulin analogs, saturability, inverse relationship of steady-state binding levels to temperature, and negative cooperativity. Cellular phospholipids enriched in monounsaturated fatty acyl groups were produced by growth in animals that were maintained on a diet rich in coconut oil; cellular phospholipids enriched in polyunsaturated fatty acyl groups were produced in animals fed sunflower oil. Insulin receptors were present in the normal cells at 180,000 sites/cell but this fell to 125000 (P less than 0.001) in cells enriched in monounsaturated fatty acids and rose to 386,000 (P less than 0.001) in cells enriched in polyunsaturated fatty acids. The normal cells had affinity constants (Ke and Kf) of 0.03 and 0.01 nM-1. The cells enriched in monounsaturated fatty acids had an increase in these affinity constants to 0.06 and 0.03 nM-1 whereas values of 0.01 and 0.005 mM-1 were obtained in the cells enriched in polyunsaturated fatty acids (all comparison P less than 0.001). Thus, increased unsaturation of plasma membrane phospholipids, produced by dietary manipulations, was associated with an increase in insulin receptor number but a decrease in binding affinity. In contrast, increased saturation of the phospholipids of the plasma membrane was associated with a decrease in receptor number and an increase in affinity. The results can be explained by a model in which the insulin receptor is assumed to be multimeric.

Properties and partial purification of the detergent-solubilized insulin receptor: a demonstration of negative cooperativity in micellar solution.

Turkey erythrocytes possess insulin receptors with binding properties very similar to those of mammalian insulin receptors. In the present study, the insulin receptor of the avian erythrocyte has been solubilized in Triton X-100, extensively characterized and partially purified, and its properties compared to those of the membrane-bound receptor. The solubilized insulin receptor has a Stokes radius of 70 A and an apparent molecular weight of 300 000 in 0.05% Triton. The binding of insulin to the soluble receptor was very similar to the binding observed with the membrane-bound receptor. Thus, binding was markedly temperature dependent for both the soluble and membrane-bound forms, although the kinetics of binding were slower with the soluble receptor. Both forms of the receptor also showed a sharp pH optimum; however, solubilization produced a shift from maximal binding at pH 7.8 to pH 7.3. The soluble receptor also retained insulin analog specificity, ion sensitivity and negative cooperativity. The soluble receptor did not appear to degrade either bound or free insulin. On DEAE-cellulose chromatography the receptor eluted as a single peak. The specific activity of this partially purified preparation was 25--30 pmol/mg protein (about 500-fold enrichment over crude extract and 5-fold over highly purified membranes). Extensive attempts to purify further the receptor by gel filtration, carboxymethyl-cellulose chromatography and affinity chromatography resulted in either a very low yield or only modest enrichment. Purification was also complicated because the receptor was easily denatured; about 40% of the activity was lost after a 90-min exposure to 3 M urea or pH 4.5. These data suggest that the insulin receptor retains its properties in the absence of the lipid bilayer of the membrane. Complete purification will be difficult due to a lack of stability under a number of conditions.

Decrease in insulin receptors during Friend erythroleukemia cell differentiation.

The Friend erythroleukemia cell has an insulin receptor with all the properties of mammalian insulin receptors: rapid, reversible, and saturable binding of insulin; specific for insulin and insulin analogs; inversely proportional to temperatures; sharply pH dependent (optimum = 8.0); and demonstrated ligand-induced accelerated dissociation consistent with negative cooperativity. There were 17,200 sites per cell. After induction by dimethylsulfoxide, 80% of the cells became benzidine positive (i.e., contained hemoglobin). The receptor concentration dropped to 4300 sites per cell, while the remaining receptors retained all the initial binding characteristics. This loss of receptors could not be attributed directly to either dimethylsulfoxide or changes in cell size. Thus, during the process of differentiation, the concentration of insulin receptors in the Friend erythroleukemia cell decreases.

Insulin-induced desensitization at the receptor and postreceptor level in mitogen-activated human T-lymphocytes.

Human T-lymphocytes activated by phytohemagglutin acquire insulin receptors in culture. Saturation analysis of insulin-binding activity in the presence of competing ligand revealed curvilinear Scatchard plots. Insulin receptors were not regulated by insulin before mitogen activation and culture of T-lymphocytes. However, insulin-induced downregulation of insulin receptors was: (1) demonstrable in receptor-positive cells, (2) dependent on insulin concentration, (3) temporally unrelated to insulin internalization, and (4) prevented by culture at 4 degrees C but not by cycloheximide at 37 degrees C. Recovery of insulin receptors required further culture of cells in media depleted of insulin for 24 h. Scatchard analysis revealed loss of receptor number without changes in receptor affinity. Insulin-induced increases in glucose transport and oxidation were demonstrable in receptor-positive cells but not in receptor-negative cells. However, these effects were extremely time-dependent. After a 2-h exposure of cells to 10(-8) M insulin, increases in glucose transport were no longer demonstrable. Elution of bound insulin from these cells followed by re-exposure to insulin depressed glucose transport in them. Recovery from this hyporesponsive, desensitized state required a 6-h culture in insulin-depleted media. Glucose oxidation of desensitized cells could be stimulated by spermine but not by insulin. These studies demonstrate the activated human T-lymphocyte is an insulin-sensitive tissue that is capable of limiting its physiologic response to insulin by receptor- and postreceptor-mediated mechanisms.

Anomalous glucose and insulin responses in patients with insulinoma. Caveats for diagnosis.

Two patients with insulinomas had unusual glucose and insulin-secretory dynamics in response to prolonged fasting. In patient 1, low insulin values persisted throughout three separate supervised fasts without a steady rise in the insulin-glucose ratio. In patient 2, a rising insulin-glucose ratio during a fast returned to normal after a documented catecholamine surge following a transient hypoglycemic episode. While patient 1 had clearly elevated proinsulin values of 52% to 57%, patient 2 had a near-normal value of 23%. The diagnosis of an insulinoma can usually be made by obtaining simultaneous glucose and insulin values during a prolonged supervised fast. Rarely, however, anomalous results may be obtained during supervised fasts of patients with insulinoma, and a broader range of diagnostic tests will be required to establish the correct diagnosis.

The role of technology in diabetes therapy.

This decade will bring major changes to the therapy of diabetes. New drugs are likely to include monomeric insulins, fatty-acid-oxidation inhibitors, insulin-secretion inducers, and nutrition modifiers. Likely new devices include improved insulin pens, less invasive methods of insulin administration, and noninvasive blood glucose monitoring. The use of computers will integrate this care, and artificial intelligence will provide new approaches to all of health care. An integrated system for using these new technologies, such as staged diabetes management, will ensure an orderly, cost-effective transition in therapy by the entire health-care community.

A new consensus error grid to evaluate the clinical significance of inaccuracies in the measurement of blood glucose.

OBJECTIVE: The objectives of this study were 1) to construct new error grids (EGs) for blood glucose (BG) self-monitoring by using the expertise of a large panel of clinicians and 2) to use the new EGs to evaluate the accuracy of BG measurements made by patients. RESEARCH DESIGN AND METHODS: To construct new EGs for type 1 and type 2 diabetic patients, a total of 100 experts of diabetes were asked to assign any error in BG measurement to 1 of 5 risk categories. We used these EGs to evaluate the accuracy of self-monitoring of blood glucose (SMBG) levels in 152 diabetic patients. The SMBG data were used to compare the new type 1 diabetes EG with a traditional EG. RESULTS: Both the type 1 and type 2 diabetes EGs divide the risk plane into 8 concentric zones with no discontinuities. The new EGs are similar to each other, but they differ from the traditional EG in several significant ways. When used to evaluate a data set of measurements made by a sample of patients experienced in SMBG, the new type 1 diabetes EG rated 98.6% of their measurements as clinically acceptable, compared with 95% for the traditional EG. CONCLUSIONS: The consensus EGs furnish a new tool for evaluating errors in the measurement of BG for patients with type 1 and type 2 diabetes.

The insulin receptor of the turkey erythrocyte: similarity to mammalian insulin receptors.

Avian erythrocytes possess insulin receptors which have binding properties that are virtually identical to those of the well studied mammalian insulin receptors. The affinity for porcine insulin was identical for the turkey and mammalian receptors over the entire range of insulin concentrations, as was the affinity of each of four insulin analogues which differed 300-fold in biological potency. Insulin induced acceleration of dissociation (i.e., the negatively cooperativite site-site interaction) was indistinguishable over a 10(6) range of insulin concentrations. Sharp pH dependence of binding was identical for turkey and mammalian receptors. The effects of temperature on association, dissociation and steady state binding were also identical. Thus, although birds and mammals have evolved separately for 300 million years there has been little change in the properties of the insulin receptor over this time period.

Rapid transport of biologically intact insulin through cultured endothelial cells.

Mono A14-[125I]-iodoinsulin was incubated with cultured endothelial cells derived from bovine pulmonary arteries at physiologic conditions. The processing of the cell-bound A14-[125I]-iodoinsulin was evaluated by trichloroacetic acid precipitation, gel filtration and high performance liquid chromatography. In contrast to insulin processing in many other cell types, approximately 95% of cell bound insulin was dissociated from the cells in less than 15 minutes, and biologically intact insulin rapidly passed through the endothelial cells. The unique location of endothelial cells coupled with the ability of rapid transport of intact insulin are consistent with an endothelial role for either the transport of insulin out of the bloodstream or as an extra-pancreatic storage area for insulin.

Comparison of insulin binding to cells of fed and fasted obese patients: results in erythrocytes and monocytes.

The erythrocyte (RBC) has received recent interest as a cell model to examine insulin receptor status in humans. In the present study we have compared the insulin receptors on mature RBCs and monocytes of four hyperinsulinemic obese patients in the fed state and after a 14-day fast (less than 50 cal/day). Insulin binding in the basal (fed) state was described in RBC and monocytes due predominantly to a decrease in the receptor concentration in both cell types. After a 14-day fast, insulin binding to both RBCs and monocytes increased significantly in each patient. Maximal binding of [125I]iodoinsulin to RBCs increased by 29% (range, 20-46%), and binding to monocytes increased by 116% (range, 46-321%). In response to the fast, the concentration of insulin needed to inhibit binding by 50% decreased from 5 to 2 ng/ml in RBC and from 3 to 1 ng/ml in monocytes. Conventional and computer-fitted Scatchard analyses demonstrated no change in the receptor concentration of RBCs of any patient, whereas the receptor concentration of monocytes increased by more than 50% in two of the four patients and by 40% for the group. Thus, in response to the fast, the direction of the change in insulin binding was similar in the RBCs and monocytes, whereas the magnitude and, in certain patients, the mechanism of the binding increase differed.

Protamine-induced fatal anaphylaxis. Prevalence of antiprotamine immunoglobulin E antibody.

Protamine is used widely to reverse the anticoagulant effects of heparin and to delay the absorption of insulin. Although adverse reactions to protamine are reported infrequently and are usually mild, we recently observed the first fatal case of type I anaphylaxis resulting from protamine. This patient had previously been sensitized to protamine during cardiac catheterization and had high levels of protamine-specific immunoglobulin E in the serum. In a prospective study, we found that 10 of 19 diabetic patients (53%) who had received insulin containing insulin also had high levels of antiprotamine immunoglobulin E. In contrast, none of 27 nondiabetic healthy normal controls or 10 diabetics who had never received protamine or protamine-containing insulin had levels of antiprotamine immunoglobulin E over background. This study underscores the risks of routinely administering protamine to susceptible individuals and the need for alternative therapies.

Regulation of insulin receptors in erythroid cells.

Previous studies using inhibitors have suggested that protein synthesis is necessary for "down-regulation" of insulin receptors. We have tested this hypothesis without the use of inhibitors by studying the ability of cells of the erythroid series to down-regulate their insulin receptors in vitro. The cells tested include mature erythrocytes and reticulocytes from rabbits and Friend erythroleukemia cells (a model for the basophilic erythroblast, a primitive nucleated erythrocyte). All cells were maintained at 37 degrees C for 18 hr +/- insulin (10(-8)M). Cultures were then incubated with phosphate buffered salines (pH 7.0) at 30 degrees C for 40 min to remove bound insulin. Receptors were quantitated by computerized analysis of Scatchard plots of subsequent insulin binding studies. Cells fully capable of both mRNA synthesis and protein synthesis, such as the undifferentiated and differentiated Friend erythroleukemia cell, had reduction of insulin receptors at 60% and 43%, respectively. Reticulocytes, which were capable of protein synthesis but not mRNA synthesis, had decreases of 25%-30% in 8 separate experiments. Mature erythrocytes, capable of neither RNA nor protein synthesis had no significant changes in receptor concentrations. Since mature erythrocytes do not "down-regulate" their insulin receptor concentration, studies of these receptors in erythrocytes of patients should be interpreted with caution.

Tunicamycin blocks the emergence and maintenance of insulin receptors on mitogen-activated human T lymphocytes.

Treatment of phytohemagglutinin (PHA) activated human T lymphocytes with tunicamycin, an antibiotic that specifically inhibits asparagine-linked N-glycosylation of proteins, totally blocked the normal emergence of insulin receptors on these lymphocytes and their cellular proliferation during culture in a dose-dependent manner. Carbohydrate incorporation into protein was inhibited 82% by 0.5 microgram/mL while leucine incorporation was unaffected. Tunicamycin exposure of activated T lymphocytes, which had acquired insulin receptors during culture, reduced cellular insulin binding by 35% to 84% and reduced PHA binding to 40% of control levels within 24 hours. Scatchard analysis revealed decreases in insulin binding capacity but not affinity. Similar treatment with cycloheximide only decreased insulin binding by 12%. These findings suggest N-glycosylation of proteins is a necessary biochemical event (1) for the emergence and maintenance of insulin receptors on mitogen activated T lymphocytes, and (2) for mitogen activated T lymphocytes to undergo cell division.

Role of injection technique in use of insulin pens: prospective evaluation of a 31-gauge, 8-mm insulin pen needle.

OBJECTIVE: To evaluate the effectiveness, comfort, and ease of use of insulin pen injections with a 31-gauge, 8-mm needle. METHODS: In 50 study subjects (24 patients with type 1 insulin-dependent diabetes and 26 insulin-using patients with type 2 diabetes), we assessed the delivery of insulin, residual insulin leakage, glycemic control, plunger depression pressure, and perceived pain associated with the B-D 31-gauge, 8-mm pen needles in comparison with the B-D conventional 30-gauge, 8-mm pen needles, while the patient used their own insulin pens (Novo or B-D). The study subjects injected their usual dose of regular and NPH insulin using the 30-gauge, 8-mm needle during the first 3 weeks of the study. This period was followed by two 3-week crossover segments of the study with either needle assigned in random sequence. RESULTS: No statistically significant differences were noted in glycemic control or perceived pain of injection between the two needles. The interaction between the two needles and the two insulin pen brands on glycemic control was not statistically significant. Plunger depression pressure increased with the increase in the gauge of the needle and with increases in size of dose of injected insulin (P<0.01). B-D pen users reported lower plunger pressure ratings in comparison with Novo pen users (P<0.01), regardless of the needle type and dose range. Both the insulin pen type and the needle type individually had statistically significant (P<0.01) effects on the residual insulin leakage from the needle tip after injection; however, their interaction was not statistically significant. Insulin doses greater than 30 units were associated with increased leakage (P<0.01). As needle retention time decreased, residual insulin leakage from the needle tip after injection increased (P<0.01), regardless of the needle used. CONCLUSION: The 31-gauge insulin pen needles are safe and effective for the delivery of insulin. With both 30-gauge and 31-gauge needles, attention to injection technique is essential to ensure complete delivery of insulin, particularly with administration of large doses.