Regulation of biological functions by an insulin receptor monoclonal antibody in insulin receptor beta-subunit mutants.

We investigated the effects of MA-5, a human-specific monoclonal antibody to the insulin receptor alpha-subunit, on transmembrane signaling in cell lines transfected with and expressing both normal human insulin receptors and receptors mutated in their beta-subunit tyrosine kinase domains. In cell lines expressing normal human insulin receptors, MA-5 stimulated three biological functions: aminoisobutyric acid (AIB) uptake, thymidine incorporation, and S6 kinase activation. Under conditions where these biological functions were stimulated, there was no detectable stimulation of receptor tyrosine kinase. We then combined the use of this monoclonal antibody with cells expressing insulin receptors with mutations in the beta-subunit tyrosine kinase domain; two of ATP binding site mutants V1008 (Gly----Val) and M1030 (Lys----Met) and one triple-tyrosine autophosphorylation site mutant F3 (Tyr----Phe at 1158, 1162, and 1163). In cells expressing V1008 receptors, none of the three biological functions of insulin was stimulated. In cells expressing M1030 receptors, AIB uptake was stimulated to a small, but significant, extent whereas the other two functions were not. In cells expressing F3 receptors, AIB uptake and S6 kinase activation, but not thymidine incorporation, were fully stimulated. The data suggest, therefore, that (1) activation of insulin receptor tyrosine kinase may not be a prerequisite for signaling of all the actions of insulin and (2) there may be multiple signal transduction pathways to account for the biological actions of insulin.     

Evidence that insulin plus ATP may induce a conformational change in the beta subunit of the insulin receptor without inducing receptor autophosphorylation.

The effect of insulin and ATP on insulin receptor beta subunit conformation was studied in vitro with radioiodinated monoclonal antibodies directed at several regions of the receptor beta subunit. Insulin plus ATP inhibited their binding to the receptor. The greatest inhibitory effect of insulin and ATP was seen with antibody 17A3 which recognizes a domain of the beta subunit that is near the major tyrosine autophosphorylation sites at residues 1158, 1162, and 1163. ATP alone inhibited 17A3 binding with a one-half maximal ATP inhibitory concentration of 186 +/- 7 microM. Insulin at concentrations as low as 100 pM potentiated the effect of ATP; at 100 nM where insulin had its maximal effect, insulin lowered the one-half maximal inhibitory concentration of ATP to 16 +/- 6 microM. At 1 mM CTP, GTP, ITP, TTP, and AMP were without effect in either the presence or absence of insulin; in contrast, ADP was inhibitory in the presence of insulin. Of major interest was adenyl-5'-yl imidodiphosphate (AMP-PNP). This nonhydrolyzable analog of ATP inhibited 17A3 binding, and the effect of AMP-PNP (like ATP) was potentiated by insulin. Two insulin receptor beta subunit mutants then were studied. Mutant receptor F3, where the major tyrosine autophosphorylation sites at residues 1158, 1162, and 1163 were changed to phenylalanines, bound to 17A3; antibody binding was inhibited by insulin and ATP in a manner similar to normal receptors. In contrast, mutant receptor M1030, where the lysine in the ATP binding site at residue 1030 was changed to methionine, bound 17A3, but unlike either normal receptors or F3 receptors, the binding of 17A3 was not inhibited by insulin and ATP. Therefore, these studies raise the possibility that, in vivo, ATP binding in the presence of insulin may induce a conformational change in the insulin receptor beta subunit which in turn signals some of the biological effects of insulin.     

Alterations in the tyrosine kinase activity of the insulin receptor produced by in vitro hyperinsulinemia

The tyrosine kinase activity of the insulin receptor was examined in fat cells made insulin resistant by hyperinsulinemia. Adipocytes previously treated for 16 h in vitro with 0.1 microM insulin were markedly insensitive to insulin as shown by the requirement of 2.3-fold higher concentration of insulin to stimulate half-maximal activation of glucose metabolism (glucose oxidation and glucose conversion to lipids). A similar rightward shift in the insulin dose-response curve was also evident for an insulin action not dependent on glucose metabolism, i.e. the inhibition of hormone-stimulated lipolysis. The almost 60% loss of insulin sensitivity occurred in conjunction with a 37% loss in insulin-binding activity, an alteration caused by a reduction in the number of insulin receptors. Studies of tyrosine kinase activity demonstrated a preferential alteration in this insulin receptor property, in addition to the receptor loss. The insulin-stimulated Vmax for the phosphorylation of the artificial substrate, Glu80-Tyr20, was significantly reduced by almost 40%, when the activity was expressed per insulin binding and measured in a receptor preparation purified by wheat germ affinity chromatography and immunoprecipitation. An elevation in basal tyrosine kinase activity was also present, which correlated with a lower apparent Km for ATP (0.025 mM) in comparison to the Km of 0.070 mM for the receptors from control cells. These findings indicate the presence of two types of alterations that involve the insulin receptor and hyperinsulinemia: one constituted by a modest reduction in receptor number and a second by modifications in the tyrosine kinase activity of the remaining receptors. Both alterations are required to explain the magnitude of the insulin resistance that develops after 16 h of insulin treatment.     

Regulation of the insulin receptor kinase by hyperinsulinism

A murine fibroblast cell line transfected with human insulin receptor cDNA, NIH 3T3 HIR3.5, was observed to display insulin-induced down-regulation of insulin-binding activity in a time- and concentration-dependent manner. Maximal inhibition of insulin-binding activity (54%) occurred within 16 h of exposure to 100 nM insulin in vivo, where in vivo refers to intact cells in tissue culture. The decrease in cellular insulin-binding activity was the consequence of a decrease in the number of cell-associated insulin receptors as determined by Scatchard analysis of insulin binding, 125I-insulin affinity cross-linking, and Western blotting of the insulin receptor beta subunit. Acute insulin treatment in vivo (1-60 min) resulted in the activation of the insulin receptor protein tyrosine kinase as determined by in vitro phosphorylation of glutamic acid:tyrosine (4:1), where in vitro refers to broken cell preparations. This acute in vivo insulin activation of the insulin receptor tyrosine kinase resulted in a greater stimulation (1.4-1.9-fold) of tyrosine kinase activity in the glutamic acid:tyrosine (4:1) assay than the maximal stimulation produced by insulin treatment in vitro. In contrast, long term (24 h) insulin treatment in vivo resulted in a 50-70% decrease in intrinsic protein tyrosine kinase activity of the insulin receptors compared with that of acutely activated (1 min) insulin receptors. Under these conditions, the insulin receptor protein kinase activity remained insulin independent in the in vitro substrate kinase assay. Surprisingly, the insulin-independent activated (1 min in vivo insulin-treated) and uncoupled (24 h in vivo insulin-treated) insulin receptors displayed similar stoichiometries of 32P incorporation into the beta subunit by in vitro autophosphorylation when compared with the control insulin receptors, ranging from 1.5 to 1.8 mol of phosphate incorporated/mol of insulin receptor. Phosphoamino acid analysis demonstrated that the phosphoserine/phosphothreonine content of in vivo 32P-labeled insulin receptors increased markedly within a 1-h exposure to insulin in vivo, whereas insulin-induced receptor desensitization was not apparent until 10-24 h after exposure to insulin. These data suggest that insulin treatment in vivo results initially in the activation of the insulin receptor kinase followed by a subsequent uncoupling of protein kinase activity. This insulin-induced desensitization of the insulin receptor kinase does not correlate with the extent of beta subunit serine/threonine phosphorylation.     

Vanadate down-regulates cell surface insulin and growth hormone receptors and inhibits insulin receptor degradation in cultured human lymphocytes

Insulin is able to down-regulate its specific cell surface receptor in cultured human lymphocytes. The effect of vanadate, a known insulinomimetic agent, was examined to determine whether it could mimic insulin to down-regulate the insulin receptor. Exposure of cultured human lymphocytes (IM-9) to vanadate (0-200 microM) resulted in a time- and dose-dependent decrease in cell surface insulin receptors to 60% of control, while insulin (100 nM) down-regulated to 40%. The vanadate effect, in contrast to the rapid effect of insulin, was slow to develop (4-6 h). Surface receptor recovery after 18 h exposure was rapid after vanadate removal (20 min), but it required hours after insulin suggesting the presence of an intracellular (cryptic) pool of receptors after vanadate treatment. Insulin binding to Triton X-100-solubilized whole cells after 18 h treatment revealed that total cell receptors had decreased to 50% of control after insulin but increased to 120 and 189% of control after 100 and 200 microM vanadate, respectively. Furthermore, vanadate inhibited the insulin-mediated loss of total cell receptors from 50 to 28%. Removal of cell surface receptors by trypsin before cell solubilization revealed that 100 microM vanadate increased insulin binding to 321% of control indicating an accumulation of intracellular receptors. Labeling of cell surface proteins with Na125I and lactoperoxidase followed by immunoprecipitation of solubilized receptors with anti-receptor antibody after incubation for various times up to 20 h and quantitation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that, while insulin shortened t1/2 from 7.3 to 5.3 h, vanadate prolonged receptor t1/2 to 14 h. No effect of vanadate was detected on insulin receptor tyrosine kinase activity with up to 4 h incubation at the vanadate concentrations used in this study. Furthermore, human growth hormone surface receptors were similarly down-regulated by vanadate. We conclude that 1) vanadate has an apparent insulin-like effect to down-regulate cell surface insulin receptors in cultured human lymphocytes; 2) in contrast to insulin-induced down-regulation which is associated with receptor degradation vanadate causes an accumulation of intracellular (cryptic) receptors and inhibits insulin receptor degradation; and 3) these effects of vanadate may be exerted on other cell surface receptors.     

Mutation of the high cysteine region of the human insulin receptor alpha-subunit increases insulin receptor binding affinity and transmembrane signaling

Our previous studies indicated that amino acid residues 240-250 in the cysteine-rich region of the human insulin receptor alpha-subunit constitute a site in which insulin binds (Yip, C. C., Hsu, H., Patel, R. G., Hawley, D. M., Maddux, B. A., and Goldfine, I. D. (1988) Biochem. Biophys. Res. Commun. 157, 321-329). We have now constructed a human insulin receptor mutant in which 3 residues in this sequence were altered (Thr-Cys-Pro-Pro-Pro-Tyr-Tyr-His-Phe-Gln-Asp to Thr-Cys-Pro-Arg-Arg-Tyr-Tyr-Asp-Phe-Gln-Asp) and have expressed this mutant in rat hepatoma (HTC) cells. When compared with cells transfected with normal insulin receptors, cells transfected with mutant receptors had an increase in insulin-binding affinity and a decrease in the dissociation of bound 125I-insulin. Studies using solubilized receptors also demonstrated that mutant receptors had a higher binding affinity than normal receptors. In contrast, cells transfected with either mutant or normal receptors bound monoclonal antibodies against the receptor alpha-subunit with equal affinity. When receptor tyrosine kinase activity and alpha-aminoisobutyric acid uptake were measured, cells transfected with mutant insulin receptors were more sensitive to insulin than cells transfected with normal receptors. These findings lend further support therefore to the hypothesis that amino acid sequence 240-250 of the human insulin receptor alpha-subunit constitutes one site that interacts with insulin, and they indicate that mutations in this site can influence insulin receptor binding and transmembrane signaling.     

Insulin binding on cultured chick muscle cells: decrease in binding associated with cell fusion

Binding of 125I-bovine and chicken insulin to cultured embryonic chick skeletal muscle cells was studied. Bovine and chicken insulin bound cultured cells with high affinities of 2.4 X 10(9)M-1 and 4.8 X 10(9)M-1 and low affinities of 2.4 X 10(7)M-1 and 3.7 X 10(7)M-1, respectively. Maximum insulin binding was achieved after 90 min of incubation at 20 degrees C and the maximum value was maintained for an additional 3 hr. Insulin binding increased in a linear manner with increasing nuclei number over a 5-fold range. Maximum insulin binding per nuclei decreased as cell fusion increased between 24 and 72 hr in culture, primarily due to a decrease in the number of low affinity insulin receptors.     

Phosphorylation of insulin-like growth factor I receptor by insulin receptor tyrosine kinase in intact cultured skeletal muscle cells

The interaction between insulin and insulin-like growth factor I (IGF I) receptors was examined by determining the ability of each receptor type to phosphorylate tyrosine residues on the other receptor in intact L6 skeletal muscle cells. This was made possible through a sequential immunoprecipitation method with two different antibodies that effectively separated the phosphorylated insulin and IGF I receptors. After incubation of intact L6 cells with various concentrations of insulin or IGF I in the presence of [32P]orthophosphate, insulin receptors were precipitated with one of two human polyclonal anti-insulin receptor antibodies (B2 or B9). Phosphorylated IGF I receptors remained in solution and were subsequently precipitated by anti-phosphotyrosine antibodies. The identities of the insulin and IGF I receptor beta-subunits in the two immunoprecipitates were confirmed by binding affinity, by phosphopeptide mapping after trypsin digestion, and by the distinct patterns of expression of the two receptors during differentiation. Stimulated phosphorylation of the beta-subunit of the insulin receptor correlated with occupancy of the beta-subunit of the insulin receptor by either insulin or IGF I as determined by affinity cross-linking. Similarly, stimulation of phosphorylation of the beta-subunit of the IGF I receptor by IGF I correlated with IGF I receptor occupancy. In contrast, insulin stimulated phosphorylation of the beta-subunit of the IGF I receptor at hormone concentrations that were associated with significant occupancy of the insulin receptor but negligible IGF I receptor occupancy. These findings indicate that the IGF I receptor can be a substrate for the hormone-activated insulin receptor tyrosine kinase activity in intact L6 skeletal muscle cells.     

Acute effects of insulin on surface insulin receptors in isolated hepatocytes. Evidence for a recycling pathway

Isolated rat hepatocytes were incubated for 1 h at 37 degrees C with 10 nM insulin. Following washout of insulin, cells were incubated with [125I] monoiodoinsulin at 15 degrees C to assess surface insulin binding. Preincubation with 10 nM insulin did not cause a decrease in insulin binding. Scatchard analysis confirmed that insulin receptor number remained constant. In the presence of 200 microM chloroquine or 25 microM monensin, surface insulin binding after preincubation with 10 nM insulin fell to 81.1 +/- 1.2% or 39.0 +/- 2.7% of control, respectively. It is suggested that the maintenance of insulin receptor number following acute insulin treatment in vitro is due to an insulin receptor recycling pathway, possibly involving lysosomes and/or the Golgi apparatus.     

Glucose regulation of insulin receptor affinity in primary cultured adipocytes

Treatment of primary cultured adipocytes with 20 mM glucose resulted in a progressive increase in specific 125I-insulin binding that began almost immediately (no lag period) and culminated in a 60% increase by 24 h. This effect was dose-dependent (glucose ED50 of 4.6 mM) and mediated by an increase in insulin receptor affinity. Moreover, it appears that glucose modulates insulin receptor affinity through de novo protein synthesis rather than through covalent modification of receptors, since cycloheximide selectively inhibited the glucose-induced increase in insulin binding capacity (ED50 of 360 ng/ml) and restored receptor affinity to control values. Importantly, insulin sensitivity of the glucose transport system was increased by glucose treatment (63%) to an extent comparable with the enhancement in receptor affinity, thus indicating a functional coupling between insulin binding and insulin action. When the long term effects of insulin were assessed (24 h), we found that insulin treatment reduced 125I-insulin binding by greater than 60% by down-regulating the number of cell surface receptors in a dose-dependent manner (insulin ED50 of 7.4 ng/ml). On the basis of these studies, we conclude that 1) insulin binding is subject to dual regulation (glucose controls insulin action by enhancing receptor affinity, whereas insulin controls the number of cell surface receptors); and 2) glucose appears to modulate insulin receptor affinity through the rapid biosynthesis of an affinity regulatory protein.     

Insulin receptor cycling and insulin action in the rat adipocyte

The possibility that the insulin receptor of adipocytes undergoes cycling was examined by a method involving pronase digestion at 12 degrees C, followed by insulin binding studies to determine receptor location and quantity. In the absence of insulin treatment, the amount of internal receptors (i.e. protected from pronase) was 10% of total receptor content. Following a 30-min insulin treatment (0.1 microM) at 37 degrees C, the internal receptor content increased 2-fold (206 +/- 12% of control, 100%). This effect was rapid, and maximum internalization was approached by 5 min of insulin treatment. Warming pronase-digested cells to 37 degrees C allowed the internal receptors to move to the cell surface. This movement was rapid also, and expansion of the internal pool by insulin pretreatment provided a 2.4-fold increase in the reinsertion of cell-surface receptors (238 +/- 28% of nontreated cells, 100%). Insulin-pretreated and nontreated cells had approximately 13 and 6%, respectively, of their original cell-surface receptor content, i.e. their content before pronase digestion. These receptors appeared intact after the cycling process, as judged by affinity labeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the receptor and its binding subunit. The ability of the recycled receptor to respond to insulin was examined by studies of glucose incorporation into lipids and the inhibition of isoproterenol-stimulated lipolysis. Cells pretreated with insulin and allowed to recycle (e.g. 13% of normal receptor content) were 2-3-fold more responsive and 7-fold more sensitive to subsequent insulin stimulation than nontreated cells (e.g. 6% of normal receptor content), indicating that the recycled receptors are biologically active and coupled to cellular effector systems.     

Effect of internalization and degradation of insulin on rat adipocyte insulin receptor binding kinetics

We have assessed the influence of nondisplaceable (internalized) insulin and insulin degradation during binding reactions at 37 degrees C on the numbers and affinities of insulin binding sites on isolated rat adipocytes. Corrections for nondisplaceable insulin caused a 33% reduction in the number of the high affinity sites (p less than 0.01) and a 24% reduction (p less than 0.01) in the number of the low affinity sites which was associated with a 20% increase (p less than 0.01) in affinity when a two-site model was applied. With a one-site model, the number of insulin receptors decreased by approximately 33% (p less than 0.01), but the affinity did not change. These results indicate that the internalization and degradation of insulin that occurs during the binding reaction can significantly affect the estimation of insulin binding kinetics. Potential variations in internalization and degradation of insulin by cells obtained under various physiological or pathologic conditions should, therefore, be taken into consideration in the interpretation of insulin binding data.     

Regulation of insulin receptor function by a small molecule insulin receptor activator

In type 2 diabetes mellitus, impaired insulin signaling leads to hyperglycemia and other metabolic abnormalities. TLK19780, a non-peptide small molecule, is a new member of a novel class of anti-diabetic agents that function as activators of the insulin receptor (IR) beta-subunit tyrosine kinase. In HTC-IR cells, 20 microm TLK19780 enhanced maximal insulin-stimulated IR autophosphorylation 2-fold and increased insulin sensitivity 2-3-fold. In contrast, TLK19780 did not potentiate the action of insulin-like growth factor-1, indicating the selectivity of TLK19780 toward the IR. The predominant effect of TLK19780 was to increase the number of IR that underwent autophosphorylation. Kinetic studies indicated that TLK19780 acted very rapidly, with a maximal effect observed 2 min after addition to insulin-stimulated cells. In 3T3-L1 adipocytes, 5 microm TLK19780 enhanced insulin-stimulated glucose transport, increasing both the sensitivity and maximal responsiveness to insulin. These studies indicate that at low micromolar levels small IR activator molecules can enhance insulin action in various cultured cells and suggest that this effect is mediated by increasing the number of IR that are tyrosine-phosphorylated in response to insulin. These studies suggest that these types of molecules could be developed to treat type 2 diabetes and other clinical conditions associated with insulin resistance.     

Insulin receptor substrates 1 and 2 but not Shc can activate the insulin receptor independent of insulin and induce proliferation in CHO-IR cells

Ligand-activated insulin receptor (IR) attracts and phosphorylates various substrates such as insulin receptor substrates 1-4 (IRS) and Shc. To investigate how binding affinity for substrate affects signalling we generated chimeric receptors with the beta-chain of the insulin receptor containing NPXY motives with different affinities for receptor substrates. We found that the extent of receptor tyrosine phosphorylation positively correlates with binding affinity towards IRS1/2 but not towards Shc. Moreover, overexpression of IRS1 or IRS2 but not of Shc increased IR tyrosine phosphorylation in a dose-dependent manner, also independent of insulin. Molecular truncations of IRS1 revealed that neither the isolated PH and PTB domains nor the C-terminus with the tyrosine phosphorylation sites alone are sufficient for substrate-dependent receptor activation. Overexpression of IRS1 and IRS2 impaired insulin-induced internalization of the IR in a dose-dependent manner suggesting that IRS proteins prevent endosome-associated receptor dephosphorylation/inactivation. IRS1 and IRS2 could therefore target the activated IR to different cellular compartments. Overexpression of IRS1 and IRS2 inhibited insulin-stimulated activation of the MAP kinases Erk1/2 while it increased/induced activation of Akt/PKB. Finally, overexpression of IRS1 and IRS2 but not of Shc induced DNA synthesis in starved CHO-IR cells independent of exogenous growth factors. Our results demonstrate that variations in cellular IRS1 and IRS2 concentration affect insulin signalling both upstream and downstream and that IRS proteins could play instructive rather than just permissive roles in signal transmission.     

Two subpopulations of alpha 1-adrenergic receptors with high and low affinity for agonists: short-term exposure to agonists reduced the high-affinity sites

Short-term receptor regulation by agonists is a well-known phenomenon for a number of receptors, including beta-adrenergic receptors, and has been associated with receptor changes revealed by radioligand binding. In the present study, we investigated the rapid changes in alpha 1-adrenergic receptors induced by agonists. alpha 1-receptors were studied on DDT1 MF-2 smooth muscle cells (DDT1-MF-2 cells) by specific [3H]prazosin binding. In competition binding on membranes and on intact cells at 4 degrees C or at 37 degrees C in 1-min assays, agonists competed for a single class of sites with relatively high affinity. By contrast, in equilibrium binding at 37 degrees C on intact cells agonists competed with two receptor forms (high- and low-affinity). We quantified the receptors in the high-affinity form by measuring the [3H]prazosin binding inhibited by 20 microM norepinephrine (this concentration selectively saturated the high-affinity sites). The low-affinity sites were measured by subtracting the binding of [3H]prazosin to the high-affinity sites from the total specific binding. High-affinity receptors were 85% of the total sites in binding experiments at 4 degrees C, but only 30% at 37 degrees C. On DDT1-MF-2 cells preequilibrated with [3H]prazosin at 4 degrees C, and then shifted to 37 degrees C for a few minutes, norepinephrine selectively reduced the high-affinity sites by 30%. We suggest that at 4 degrees C it is the native form of alpha 1-receptors that is measured, with most of the sites in the high-affinity form, while during incubation at 37 degrees C the norepinephrine present in the binding assay converts most of the receptors to an apparent low-affinity form, so that they are no longer recognized by 20 microM norepinephrine. The nature of this low-affinity form was further investigated. On DDT1-MF-2 cells preincubated with the agonist and then extensively washed at 4 degrees C (to maintain the receptor changes induced by the agonist) the number of receptors recognized by [3H]prazosin at 4 degrees C was reduced by 38%. After fragmentation of the cells, the number of receptors measured at 4 degrees C was the same in control and norepinephrine-treated cells, suggesting that the disruption of cellular integrity might expose the receptors which are probably sequestered after agonist treatment. In conclusion, the appearance of the low affinity for agonists at 37 degrees C may be due to the agonist-induced sequestration of alpha 1-adrenergic receptors, resulting in a limited accessibility to hydrophilic ligands.     

Insulin Binding and Effects on Pyrimidine Nucleoside Uptake and Incorporation in Cultured Mouse Astrocytes

Binding of 125I-insulin to primary cultures of differentiated mouse astrocytes was time-dependent, reaching equilibrium after 2 h at 22 degrees C, with equilibrium binding corresponding to 20.79 fmol/mg of protein, representing approximately 5,000 occupied binding sites/cell. The half-life of 125I-insulin dissociation at 22 degrees C was 2 min, with an initial dissociation rate constant of 4.12 X 10(-2) s-1. Dissociation of bound 125I-insulin was not accelerated significantly in the presence of unlabeled insulin (16.7 microM). Porcine and desoctapeptide insulins competed for specific 125I-insulin binding in a dose-dependent manner, whereas growth hormone, glucagon, and somatostatin did not. For porcine insulin, Scatchard analysis suggested multiple-affinity binding sites (high-affinity Ka = 4.92 X 10(8) M-1; low-affinity Ka = 0.95 X 10(7) M-1). After incubation with insulin (0.5 microM) for 2 h at 37 degrees C, increases above basal values of 254 +/- 23 and 189 +/- 34% for [3H]uridine uptake and incorporation, respectively, were observed. After incubation with insulin (0.5 microM) for 24 h at 37 degrees C, there were increases of 145 +/- 6% for [3H]thymidine uptake and 166 +/- 11% for thymidine incorporation. Basal and stimulated uridine and thymidine uptake and incorporation were inhibited by 50 microM dipyridamole. These studies confirm that mouse astrocytes in vitro possess specific insulin receptors and demonstrate an effect of insulin on pyrimidine nucleoside uptake and incorporation.     

Modulation of the insulin growth factor II/mannose 6-phosphate receptor in microvascular endothelial cells by phorbol ester via protein kinase C

Phosphorylation of hormone receptors by protein kinase C (PKC) may be involved in the regulation of receptor recycling. We have studied the recycling and the phosphorylation state of the insulin growth factor (IGF) II/mannose 6-phosphate (Man-6-P) receptor in microvascular endothelial cells from rat adipose tissue. Scatchard analysis showed these cells have over 2 x 10(6) receptors/cell with an affinity constant of 1 x 10(9) M-1. In the presence of phorbol myristate acetate (PMA), an activator of PKC and analog of diacylglycerol, IGF-II receptor number increased in the plasma membrane by 60% without changes in the binding affinity. This increase in cell surface receptor number was confirmed by affinity cross-linking and 125I-surface labeling studies, occurred with a half-time of 20 min, and was reversible upon withdrawal of PMA. The redistribution of IGF-II/Man-6-P receptors was not due to an inhibition of internalization which was in fact stimulated by PMA. The effect of PMA on IGF-II receptor recycling correlated with its stimulation of PKC activity. Furthermore, after down-regulation of cellular PKC levels by preincubation with PMA, PMA was unable to activate residual PKC activity in the membranous pool or increase IGF-II receptor number at the cell surface. The phosphorylation state of the IGF-II/Man-6-P receptor was determined by 32P labeling of intact cells and immunoprecipitation with anti-receptor antibodies. In the basal state, the receptor was phosphorylated only on serine residues which was increased by 75% after treatment with PMA. In contrast, IGF-II decreased receptor phosphorylation and plasma membrane binding in a parallel and dose-dependent manner. Thus, PKC-stimulated serine phosphorylation of IGF-II/Man-6-P receptor may promote the translocation of the receptor to the cell surface, whereas IGF-II-stimulated dephosphorylation of the receptor may lead to a decrease in the number of cell surface receptors. These data suggest a role for PKC-mediated serine phosphorylation in the regulation of intracellular trafficking of receptors in endothelial cells.     

Ontogenesis of the insulin receptor in the rabbit brain

We delineated the ontogeny of the brain insulin binding, insulin receptor number and affinity using plasma membranes isolated from the rabbit. Specific 125I-insulin binding and receptor number expressed per milligram of protein increased from the 20 day gestation fetus to the 1-day-old newborn, declining thereafter to attain adult values by day 6 of postnatal life. Specific 125I-insulin binding and the receptor number in the adult brain was less than the fetal and neonatal (1 day) brain receptors. Although a similar trend was observed specifically during fetal development, the changes in receptor number expressed per microgram DNA were not significant in the neonatal period. The adult brain insulin receptor number was higher than the 20- to 27-day fetus and similar to that of the 30-day fetus and the 1- to 5-day newborns. The total receptor number correlated linearly with the brain plasma membrane protein increment velocity. The affinity of the receptors increased during early fetal development (20-27 days) and remained constant thereafter in the postnatal period. We conclude that the ontogenic changes of the brain insulin receptors are similar to the ontogenic changes of brain plasma membrane protein. The developmental changes are more pronounced when the receptor number is expressed per milligram protein versus microgram DNA.     

Insulin Binding to Human Astrocytoma Cells and Its Effect on Uridine Incorporation into Nucleic Acid

Binding of [125I]monoiodoinsulin to human astrocytoma cells (U-373 MG) was time dependent, reaching equilibrium after 1 h at 22 degrees C with equilibrium binding corresponding to 2.2 fmol/mg protein: this represents approximately 2,000 occupied binding sites per cell. The t1/2 of 125I-insulin dissociation at 22 degrees C was 10 min; the dissociation rate constant of 1.1 X 10(-2) s-1 was unaffected by a high concentration of unlabeled insulin (16.7 microM). Porcine insulin competed for specific 125I-insulin binding in a dose-dependent manner and Scatchard analysis suggested multiple affinity binding sites (higher affinity Ka = 4.4 X 10(8) M-1 and lower affinity Ka = 7.4 X 10(6) M-1). Glucagon and somatostatin did not compete for specific insulin binding. Incubation of cells with insulin (0.5 microM) for 2 h at 37 degrees C increased [2-14C]uridine incorporation into nucleic acid by 62 +/- 2% (n = 3) above basal. Cyclic AMP, in the absence of insulin, also stimulated nucleoside incorporation into nucleic acid [65 +/- 1% (n = 3)] above basal. Preincubation with cyclic AMP followed by insulin had an additive effect on nucleoside incorporation [160 +/- 4% (n = 3) above basal]. Dipyridamole (50 microM), a nucleoside transport inhibitor, blocked both basal and stimulated uridine incorporation. These studies confirm that human astrocytoma cells possess specific insulin receptors with a demonstrable effect of ligand binding on uridine incorporation into nucleic acid.     

The effect of high-glucose condition on insulin binding to IM-9 lymphocytes

The long-term effect of high-glucose condition on insulin binding to IM-9 cells was studied by incubating the cells in RPMI-1640 medium containing 450 mg/dl of glucose. Insulin binding began to decrease after incubation for 6 days in high-glucose-treated cells, and significantly decreased after 14 days due to the reduction of receptor affinity. The number of binding sites for insulin did not change by the treatment. Thus, high-glucose condition per se does not appear to induce the decrease of the number of insulin receptors on target cells, as observed in diabetic patients.