Insulin Receptor Occupancy Research Articles

Systematic Evaluation of the Metabolic to Mitogenic Potency Ratio for B10-Substituted Insulin Analogues

BackgroundInsulin analogues comprising acidic amino acid substitutions at position B10 have previously been shown to display increased mitogenic potencies compared to human insulin and the underlying molecular mechanisms have been subject to much scrutiny and debate. However, B10 is still an attractive position for amino acid substitutions given its important role in hexamer formation. The aim of this study was to investigate the relationships between the receptor binding properties as well as the metabolic and mitogenic potencies of a series of insulin analogues with different amino acid substitutions at position B10 and to identify a B10-substituted insulin analogue without an increased mitogenic to metabolic potency ratio.Methodology/Principal FindingsA panel of ten singly-substituted B10 insulin analogues with different amino acid side chain characteristics were prepared and insulin receptor (both isoforms) and IGF-I receptor binding affinities using purified receptors, insulin receptor dissociation rates using BHK cells over-expressing the human insulin receptor, metabolic potencies by lipogenesis in isolated rat adipocytes, and mitogenic potencies using two different cell types predominantly expressing either the insulin or the IGF-I receptor were systematically investigated. Only analogues B10D and B10E with significantly increased insulin and IGF-I receptor affinities as well as decreased insulin receptor dissociation rates displayed enhanced mitogenic potencies in both cell types employed. For the remaining analogues with less pronounced changes in receptor affinities and insulin receptor dissociation rates, no apparent correlation between insulin receptor occupancy time and mitogenicity was observed.Conclusions/SignificanceSeveral B10-substituted insulin analogues devoid of disproportionate increases in mitogenic compared to metabolic potencies were identified. In the present study, receptor binding affinity rather than insulin receptor off-rate appears to be the major determinant of both metabolic and mitogenic potency. Our results also suggest that the increased mitogenic potency is attributable to both insulin and IGF-I receptor activation.

Insulin Does Not Induce the Hydrolysis of a Glycosyl Phosphatidylinositol in Rat Fetal Hepatocytes

An inositol phosphoglycan that is the polar head group of a glycosyl phosphatidylinositol has been considered as a putative mediator of insulin action. To gain insight into the functions of this hormone during development, the relationships between insulin, insulin receptors, glycosyl phosphatidylinositol, and inositol phosphoglycan were studied. Glycosyl phosphatidylinositol was isolated and characterized in fetal liver as early as day 15 of intrauterine life. In isolated hepatocytes from fetal and adult rats labeled with [3H]glucosamine, [3H]galactose, or [3H]myo-inositol, these molecules were incorporated into glycosyl phosphatidylinositol. In hepatocytes labeled with [3H]glucosamine and then allowed to react with [1-14C]IAI, the [3H]glycosyl phosphatidylinositol was purified as the 14C-labeled amidinated lipid. Glycosyl phosphatidylinositol molecules from fetal and adult cells were sensitive to hydrolysis by a phosphatidylinositol-specific phospholipase C from B. cereus. The product of this hydrolysis inhibits the activity of a cAMP-dependent protein kinase, whereas this effect was abolished by nitrous acid deamination. In isolated hepatocytes from adult animals, an inverse correlation between extracellular insulin and the number of insulin receptors and the cellular content of glycosyl phosphatidylinositol was observed. However, in fetal hepatocytes insulin failed to reduce the glycosyl-phosphatidylinositol content when labeled either with [1-14C]isethionyl acetimidate or [3H]glucosamine, whereas insulin-like growth factor I produced a significant hydrolysis of glycosyl phosphatidylinositol. Fetal and adult hepatocytes were incubated with insulin or inositol phosphoglycan after which glycogen phosphorylase activities were determined. Inositol phosphoglycan mimicked the action of insulin on both forms of the enzyme from adult hepatocytes, whereas in fetal cells insulin did not change, and purified inositol phosphoglycan reduced the activities of glycogen phosphorylase. These findings suggest a dissociation between insulin receptor occupancy and the expected hormonal effects in fetal hepatocytes. This could be related to alterations at a postreceptor level.

Quantitative dissociation of glucose transport stimulation and insulin receptor tyrosine kinase activation in isolated adipocytes with a covalent insulin dimer (B29,B29′-sunberoyl-insulin)

The covalent insulin dimer B29,B29′-suberoyl-insulin was investigated for its effects on insulin receptor binding, insulin receptor tyrosine kinase activity and glucose transport in isolated adipose cells. The dimer stimulated glucose transport (initial 3- O-methylglucose uptake rate) to the same extent as insulin did (basal rate, 35 ± 3 pmol/sec/ μl lipid; insulin, 380 ± 27; B29,B29′-suberoyl-insulin, 369 ± 24, means ± S.E.), although at higher concentrations ( ec 50 1.94 ± 0.64nM versus 0.1 ± 0.02 with insulin). In contrast, the dimer only partially (23%) mimicked insulin's effect on phosphate incorporation into insulin receptors immunoprecipitated after equilibration of cells with [ 32P]phosphate. Similarly, insulin receptor tyrosine kinase as assessed by receptor autophosphorylation and phosphorylation of the substrate poly-(Glu/Tyr) was not fully activated by treatment of cells with the insulin dimer (31 and 42% of the effect of insulin, respectively) in concentrations which maximally activate glucose transport and give rise to full insulin receptor occupancy (5 × 10 −7M). Further, the dimer activated the receptor tyrosine kinase in solubilized purified insulin receptor preparations from adipose cells to only 25% of the effect of insulin ( ec 50) 32.0 ± 16 versus 1.9 ± 1.0 nM with insulin) in spite of full receptor occupancy. Binding of the dimer to insulin receptors followed single site binding kinetics, indicating that the derivative is unable to induce negative cooperativity of the insulin receptor. It is concluded that a partial phosphorylation of insulin receptors and a submaximal tyrosine kinase activation are sufficient for full stimulation of glucose transport in the adipocyte. Further, it is suggested that negative cooperativity of the insulin receptor and activation of its tyrosine kinase require a similar conformational change of the receptor protein.

Characterization of insulin binding in the UMR-106 rat osteoblastic osteosarcoma cell.

The correlation of insulin receptor occupancy with classic insulin effects, such as stimulation of glucose uptake, have not been examined in osteoblastlike cells. Accordingly, we characterized insulin binding and examined its relationship to stimulation of glucose analog transport in the UMR-106 rat osteoblastic osteosarcoma cell line. Insulin binding in UMR-106 cells was found to be pH sensitive, temperature dependent, saturable, and specific. Proinsulin was 100-fold less effective than insulin in displacing specific [125I]insulin binding in these cells, whereas IGF-I at concentrations between 0.1 and 10 nM produced no displacement of [125I]insulin but did produce significant displacement of insulin binding at 100 and 1000 nM. Insulin receptor downregulation was observed after exposure to 100 nM insulin for 6 h at 37 degrees C and was temperature dependent. Insulin binding was reversible after 24 h at 4 degrees C. Insulin binding correlated directly with stimulation of 2-deoxyglucose uptake at insulin concentrations between 0.1 and 100 nM, with a half-maximal concentration (ED50) of 0.9 nM for both [125I]insulin binding displacement and stimulation of 2-deoxyglucose uptake. Hence, there was no evidence for spare insulin receptors with regard to stimulation of glucose analog transport. Scatchard analysis of insulin binding kinetics yielded a curvilinear plot, suggesting negative cooperativity. Analysis of insulin binding kinetics using a two-site model yielded a KD of 0.9 nM for the apparent high-affinity binding site and an estimated 80,000 high-affinity binding sites per cell. These findings demonstrate that osteoblastlike cells exhibit a relationship between insulin binding and glucose transport stimulation that is similar to that in liver cells and other insulin-sensitive tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin receptor kinase following internalization in isolated rat adipocytes.

We have studied how insulin-mediated internalization of insulin receptors and insulin activation of the insulin receptor kinase might be inter-related. Isolated rat adipocytes were exposed to 0, 6, or 500 ng/ml insulin for 40 min at 37 degrees C. Subsequently, plasma membrane, low-density microsomal membrane and high-density microsomal membrane subcellular fractions were prepared. Measurement of insulin binding to insulin receptors isolated from the membrane fractions revealed that exposure of cells to insulin resulted in a loss of binding activity (13% at 6 ng/ml, 27% at 500 ng/ml insulin) from the plasma membranes which was completely accounted for by the appearance of receptors in the low-density and high-density microsomal membrane fractions, indicating that insulin had induced translocation of insulin receptors from the surface to the cell interior. Measurement of kinase activity of the isolated receptors revealed that exposure of intact cells to 500 ng/ml insulin resulted in as much as a 35-fold increase in the intrinsic kinase activity of receptors from subcellular fractions. The kinase activity per receptor was equal in all fractions at 3-4 min but by 20 min the activity of the internalized receptors fell approximately 40% to a steady state; plasma membrane receptors, on the other hand, remained fully active over time. This indicates that newly internalized receptors retain their kinase activity but undergo subsequent deactivation. Following exposure of cells to 6 ng/ml insulin, the degree of activation of the insulin receptor kinase was lower in the plasma membrane fraction (24% of the insulin effect at 500 ng/ml) than in the low-density and high-density microsomal membrane fractions (54 and 77%, respectively, of the insulin effect at 500 ng/ml). These results suggest that receptors with an activated kinase are preferentially internalized. We conclude that exposure of adipocytes to insulin causes endocytosis of insulin receptors and activation of insulin receptor kinase, newly internalized receptors are fully active tyrosine kinases but are deactivated as they traverse the intracellular organelles represented by low-density and high-density microsomal membranes, and insulin receptor occupancy, possibly by stimulating phosphorylation and activating the insulin receptor kinase, is important for targeting insulin receptors for internalization.

Effect of insulin receptor autophosphorylation on insulin receptor binding

Insulin receptor beta-subunit autophosphorylation, the first event occurring after insulin binding, plays a crucial role in modulation of receptor-associated kinase activity towards exogenous substrates and possibly in the transmission of biological signals of insulin. Receptor autophosphorylation strongly depends on insulin receptor occupancy. Till now the effects of receptor phosphorylation on insulin binding itself have not been clarified. In the present report we demonstrate the absence of any feedback mechanism by which insulin receptor activation by phosphorylation affects binding affinity of insulin receptor itself.

Influence of changes in insulin receptor binding during insulin infusions on the shape of the insulin dose-response curve for glucose disposal in man.

To determine the influence of insulin infusions used in dose-response studies on monocyte insulin binding, monocyte insulin binding and glucose disposal were measured in six normal subjects before and at the end of each of four sequential 2-h insulin infusions (0.4, 1.0, 2.0, and 10 mU kg-1 min-1). Monocyte insulin binding was unaltered at the end of the first three infusions (plasma insulin, 31 +/- 2 (SEM), 77 +/- 3, and 184 +/- 10 microU/ml) but was decreased after the last infusion (plasma insulin, 1730 +/- 125 microU/ml) at 0.2 through 10.2 ng/ml insulin concentrations in the binding assay (P less than 0.01). Using a one-site model, this could be ascribed to a decrease in insulin receptor affinity (1.54 +/- 0.26 vs. 2.27 +/- 0.48 X 10(8) M-1, P less than 0.05), whereas in a two-site model this appeared to be due to a decrease in high affinity binding sites (1,868 +/- 228 vs. 2,387 +/- 207, P less than 0.02). Nevertheless, insulin receptor occupancies estimated to occur during the insulin infusions were virtually identical whether preinsulin infusion binding data (745 +/- 72, 1,383 +/- 117, 2,572 +/- 302, and 10,092 +/- 1,708) or binding data at the end of each infusion (702 +/- 56, 1,367 +/- 150, 2,383 +/- 318, and 9,158 +/- 2,023) were used to calculate occupancy. These results indicate that although monocyte insulin binding decreased during dose-response experiments using sequential infusions of insulin, due to the concentrations of insulin at which this occurs this decrease did not alter the shape of the dose-response curve relating glucose disposal to monocyte insulin receptor occupancy.

Effects of growth hormone on insulin action in man. Mechanisms of insulin resistance, impaired suppression of glucose production, and impaired stimulation of glucose utilization.

The present studies were undertaken to assess the mechanisms responsible for growth hormone-induced insulin resistance in man. The insulin dose-response characteristics for suppression of glucose production and stimulation of glucose utilization and their relationship to monocyte insulin binding were determined in six normal volunteers after 12-h infusion of growth hormone and 12-h infusion of saline. The infusion of growth hormone (2 micrograms . kg-1 . h-1) increased plasma growth hormone nearly threefold (to congruent to 9 ng/ml) within the range observed during sleep and exercise. This increased plasma insulin (14 +/- 1 versus 8 +/- 1 microunits/ml, P less than 0.005) concentrations without significantly altering plasma glucose concentrations or basal rates of glucose production and utilization. Insulin dose-response curves for both suppression of glucose production (half-maximal response at 37 +/- 3 versus 20 +/- 3 microunits/ml, P less than 0.01) and stimulation of glucose utilization (half-maximal response at 98 +/- 8 versus 52 +/- 8 microunits/ml, P less than 0.01) were shifted to the right with preservation of normal maximal responses to insulin. Monocyte insulin binding was unaffected. Thus, except at near maximal insulin receptor occupancy, the action of insulin on glucose production and utilization per number of monocyte insulin receptors occupied was decreased. These results indicate that increases in plasma growth hormone within the physiologic range can cause insulin resistance in man, which is due to decreases in both hepatic and extrahepatic effects of insulin. Assuming that insulin binding to monocytes reflects insulin binding in insulin sensitive tissues, this decrease in insulin action can be explained on the basis of a postreceptor defect.

Kinetic relationships between insulin receptor binding and effects on glucose transport in isolated rat adipocytes.

The role of insulin receptor occupancy in the stimulation of glucose transport has been studied in isolated rat adipocytes. At 37 degrees C, under steady-state conditions, the time needed to fill the fraction of receptors (less than 1%) required for an initial measurable effect varied with insulin concentration from less than 10 s at 100 ng/mL to 90 s at 0.5 ng/mL. However, at all insulin concentrations there was an initial lag period before any activation was seen. The length of the initial lag was inversely related to the insulin concentration, lasting 2 min at 0.5 ng/mL and only 30-40 s at 5-500 ng/mL (maximal levels). A similar discrepancy was noted between dissociation of prebound insulin and the loss of insulin's effects on transport. At an insulin concentration of 0.3 ng/mL, half of the insulin effect was lost within 12 min; the t1/2 of dissociation was 8 min. When the insulin concentration was increased to 10 ng/mL, the t1/2 of dissociation increased only to 10 min while the t1/2 of deactivation was now 60 min. In conclusion, (1) kinetic studies reveal a time-requiring step between insulin binding and early effects on glucose transport, (2) a low level of insulin binding (less than 1% occupancy) is all that is necessary to initiate the insulin stimulus-response sequence, and (3) the rate of deactivation is closely related to the steady-state level of insulin binding, and with increasing insulin concentrations this rate slows and diverges from the rate of dissociation of insulin from receptors.

Relation of insulin receptor occupancy and deactivation of glucose transport.

Kinetics of association and dissociation of 125I-insulin and of activation and deactivation of 3-O-methylglucose transport were determined in isolated rat fat cells. Equilibrium bound insulin (7.5, 25, 100 microunits/ml) dissociated with a t1/2 of 2 min (100 microunits/ml), 4 min (25 microunits/ml), and 16 min (7.5 microunits/ml). Consecutive deactivation of transport is observed only in the presence of glucose (1 mM); the t1/2 of deactivation is approximately 60 min (100 and 25 microunits/ml) and 20 min (7.5 microunits/ml). At 15 degrees C, the t1/2 of dissociation (7.5 microunits/ml) is 25 min, and deactivation is not observed. Addition of dithioerythritol (5 mM) during the association of insulin decreased the binding rapidly; however, a reduced insulin effect was only seen if the binding decreased during the early activation phase of transport. In conclusion, the maintenance of the insulin effect on transport does not require persistent receptor occupancy; dissociation and deactivation are, with respect to kinetics, temperature dependency and requirement of glucose, independent processes. Receptor occupancy probably only controls the activation of transport; deactivation seems to be controlled by postreceptor processes.

Cortisol-induced insulin resistance in man: impaired suppression of glucose production and stimulation of glucose utilization due to a postreceptor detect of insulin action.

The present studies were undertaken to assess the mechanisms responsible for cortisol-induced insulin resistance in man. The insulin dose-response characteristics for suppression of glucose production and stimulation of glucose utilization and their relationship to monocyte and erythrocyte insulin receptor binding were determined in six normal volunteers after 24-h infusion of cortisol and 24-h infusion of saline. The infusion of cortisol (2 microgram kg-1 min-1) increased the plasma cortisol concentration approximately 4-fold (37 +/- 3 vs. 14 +/- 1 microgram/dl; P less than 0.01) to values observed during moderately severe stress in man. This hypercortisolemia increased postabsorptive plasma glucose (126 +/- 2 vs. 97 +/- 2 mg/dl; P less than 0.01) and plasma insulin (16 +/- 2 vs. 10 +/- 2 microU/ml; P less than 0.01) concentrations and rates of glucose production (2.4 +/- 0.1 vs. 2.1 +/- -0.1 mg kg-1 min-1; P less than 0.01) and utilization (2.5 +/- 0.1 vs. 2.1 +/- 0.1 mg kg-1 min -1; P less than 0.01). Insulin dose-response curves for both suppression of glucose production (half-maximal response at 81 +/- 19 vs. 31 +/ 5 microU/ml; P less than 0.05) and stimulation of glucose utilization (half-maximal response at 104 +/- 9 vs. 64 +/- 7 microU/ml; P less than 0.01) were shifted to the right, with preservation of normal maximal responses to insulin. Neither monocyte nor erythrocyte insulin binding was decreased. However, except at near-maximal insulin receptor occupancy, the action of insulin on glucose production and utilization per number of monocyte and erythrocyte insulin receptors occupied was decreased. These results indicate that the cortisol-induced insulin resistance in man is due to the decrease in both hepatic and extrahepatic sensitivity to insulin. Assuming that insulin binding to monocytes and erythrocytes reflects insulin binding in insulin-sensitive tissues, this decrease in insulin action can be explained on the basis of a postreceptor defect.

Insulin receptors convert to a higher affinity state subsequent to hormone binding. A two-state model for the insulin receptor.

Kinetic experiments were performed to determine the effects of insulin receptor occupancy on insulin binding. The following results were obtained: (a) the rate constant (k1) for uptake of 125I-insulin by liver plasma membranes was 2 X 10(6) M-1 s-1 and invariant at applied hormone concentrations of 7.5 to 100 X 10(-11) M. 125I-Insulin dissociated from membranes in a biphasic manner with rapid (k-1 = 2-4 X 10(-3) s-1) and slow (k-1 = 2-3 X 10(-4) s-1) components of release when dissociation was initiated by dilution into excess medium. Under all dissociation conditions employed, 125I-insulin was the radioactive species bound to and released from membranes. (b) Native insulin (100 nM) or 131I-insulin (5 nM) in the dissociation medium enhanced the dilution-induced dissociation of bound 125I-insulin. In the latter experiment, total receptor occupancy (bound 125I-insulin and 131I-insulin) decreased during dissociation. The enhanced dissociation effect was therefore not necessarily due to increased site occupancy. (c) As association time prior to dissociation was increased, the dissociability of bound 125I-insulin diminished. Decreased dissociability resulted from an increase in the slow component of hormone release at the expense of the rapid component. 131I-insulin was bound to membranes to which 125I-insulin had been prebound. The dissociation of 131I-insulin had been prebound. The dissociation of 131I-insulin was unaffected by the presence of 125I-insulin dissociating at either rapid or slow rates. The data suggest that there are no cooperative interactions between binding sites and that the hormone-receptor complex converts to a higher affinity state subsequent to occupancy since KD = k-1/k1 and K-1 decreased. A two-state model for the hepatic insulin receptor is proposed.

Dose-response characteristics for effects of insulin on production and utilization of glucose in man.

To determine the dose-response characteristics for the effects of insulin on glucose production, glucose utilization, and overall glucose metabolism in normal man, 15 healthy subjects were infused with insulin for 8 h at sequential rates ranging from 0.2 to 5.0 mU.kg-1.min-1; each rate was used for 2 h. Glucose production and utilization were measured isotopically ([3-3H]glucose). Tissue insulin receptor occupancy was estimated from erythrocyte insulin binding. Glucose production was completely suppressed at plasma insulin concentrations of approximately 60 microunits/ml. Maximal glucose utilization (10-11 mg.kg-1.min-1) occurred at insulin concentrations of 200-700 microunits/ml. The concentration of insulin causing half-maximal glucose utilization (55 + 7 microunits/ml) was significantly greater than that required for half-maximal suppression of glucose production (29 +/- 2 microunits/ml, P less than 0.01). Maximal effects of insulin on glucose production and utilization occurred at plasma insulin concentrations causing 11 and 49% insulin receptor occupancy, respectively. The above dose-response relationships indicate that in man 1) glucose production is more sensitive to changes in plasma insulin concentration than is glucose utilization; 2) both hepatic and peripheral tissues may contain "spare" insulin receptors; and 3) relatively minor changes in plasma insulin concentration or insulin receptor function can cause appreciable alterations in glucose metabolism.