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Clustering of cell-surface insulin receptors has led to the speculation that intermolecular phosphorylation of unoccupied receptors catalyzed by ligand-occupied receptors within the cluster could be a mechanism by which the insulin-binding signal is amplified. We examined whether insulin receptors can be phosphorylated by an intermolecular mechanism. In this study, we used highly purified insulin receptors isolated from rat liver plasma membranes and human placental membranes. Rat liver insulin receptors were "activated" by incubation with 10 nM insulin in the presence of ATP. Subsequent to removal of insulin by immunodepletion, these receptors were used as an enzyme source to study phosphorylation of unphosphorylated "substrate" human receptors. Initially, we found no evidence that the addition of activated rat receptors increased phosphorylation of human receptors, when assessed by immunoprecipitation with a human-specific monoclonal antibody. To examine the possibility that these negative results were due to insufficient receptor concentration, activated human receptors were mixed with unphosphorylated substrate receptors at concentrations up to 60 micrograms/ml. In this study, we found that addition of activated receptors resulted in increased phosphorylation of the substrate receptors at the highest concentrations employed. These are the first data indicating that insulin receptors per se are capable of intermolecular phosphorylation. In vivo, this could be the initial step in amplifying the insulin-binding signal.

Prior to investigating the role of individual glycosylation sites in insulin receptor function, we are mapping the sites of glycosylation in the receptor. We report here a generally applicable methodology for the isolation and identification of glycosylation sites in cell surface glycoproteins. Human insulin receptors were labeled with [3H]-sugars using a CHO cell line transfected with the human receptor cDNA. Labelled receptors were mixed with receptors purified from human placental membranes and tryptic peptides prepared. Peptides were fractionated by gel filtration chromatography to limit the number of non-glycopeptides present. Peptides were then separated by reverse phase HPLC and glycopeptides identified by scintillation counting. Using this technique we have shown the insulin receptor to be glycosylated at Asn 397 and Asn 881. This increase the known number of occupied glycosylation sites to five.

In an attempt to elucidate the cellular mechanism(s) by which sulfonylureas exert their extrapancreatic hypoglycemic effects, various parameters of insulin action were examined in vitro, using rat adipocytes maintained in a biochemically defined medium. Cells were maintained for 20 hours in the absence or presence of 175 micrograms/mL chlorpropamide and insulin binding, hexose transport, glucose metabolism, and insulin receptor tyrosine kinase activity were compared. Chlorpropamide treatment had no effect on insulin binding, altering neither receptor number nor affinity. However, the sulfonylurea did enhance 2-deoxyglucose transport in both the absence (17%, P less than .01) and presence (20%, P less than .01) of insulin. Furthermore, glucose metabolism as measured by the conversion of glucose (0.2 mmol/L) to CO2 and total lipids was also significantly increased by chlorpropamide treatment in both the absence (30%, P less than .01) and presence (31%, P less than .05) of insulin. Potentiation of insulin-stimulated transport or metabolism was not explained by an increase in the basal state alone because the incremental responses to 40 ng/mL insulin were potentiated by 19% (P less than .01) and 25% (P less than .05), respectively. Activity of the insulin receptor kinase was unchanged as evaluated by autophosphorylation of partially purified receptors, phosphorylation of an artificial substrate and by phosphorylation of the receptor in situ. These studies demonstrate that the sulfonylurea, chlorpropamide, stimulates glucose transport and potentiates insulin's effect on this process by acting at a site(s) beyond insulin receptor binding and phosphorylation.

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.

Removal of cell surface sialic acid from adipocytes with neuraminidase inhibits insulin action. Here, we have examined the effects of mild neuraminidase treatment (5 milliunits/ml, 12 degrees C, 15 min) on insulin receptor structure and function. Neuraminidase treatment sufficient to cause greater than 90% loss of insulin stimulatable lipogenesis had no effect on 125I-insulin binding, tyrosine kinase activity of partially purified insulin receptors, insulin receptor phosphorylation in intact cells, or insulin-induced receptor internalization. However, recycling of the insulin receptor to the plasma membrane was inhibited by 50%. Recycled receptors in neuraminidase-treated cells were unable to mediate insulin action in contrast to recycled receptors from non-neuraminidase-treated cells. Furthermore, when insulin receptors were protected from exposure to neuraminidase, by inducing receptor internalization prior to neuraminidase treatment, the cells were still unable to respond to insulin. Analysis of the alpha and beta subunits of the receptor from neuraminidase-treated cells, affinity-labeled with 125I-insulin, or labeled by autophosphorylation, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis failed to indicate any changes in the holoreceptor or the individual subunits. This suggests there was no detectable release of sialic acid from the receptor. From this data we conclude that loss of sialic acid from nonreceptor glycoconjugates leads to loss of insulin action and inhibition of receptor recycling. The insulin receptor does not appear to be involved in this inhibitory effect. These findings suggest that an uncharacterized plasma membrane glycoprotein is essential in transmitting the "signal" of insulin binding to the cellular effector system.

The cellular mechanism(s) by which the biguanide, metformin, exerts its antihyperglycaemic effect was investigated. Rat adipocytes were either treated acutely (2 h) or maintained in a biochemically defined medium (20 h) in the presence or absence of metformin (1 X 10(-4) mol/l). Exposure to the drug resulted in a significant enhancement (p less than 0.01) of hexose transport in both the absence (basal) and presence of insulin. Stimulation of transport was not explained by the increase in the basal state alone, since the incremental response to maximally effective concentrations of insulin was significantly enhanced p less than 0.025. Insulin-receptor tyrosine kinase activity was examined under the same experimental conditions. Activity of the kinase was unaltered as evaluated by phosphorylation of an artificial substrate and by phosphorylation of the receptor in situ. Furthermore, in this investigation neither insulin receptor number nor affinity was changed in adipose tissue treated with metformin. These studies indicate that metformin potentiates the effect of insulin on glucose transport at a site(s) beyond insulin receptor binding and phosphorylation.

We evaluated the possibility that impaired insulin-receptor kinase activity contributes to insulin resistance by examining in vitro receptor tyrosine kinase activity and in situ receptor phosphorylation in four models of insulin resistance. Adipocytes from streptozocin-induced nonketotic diabetic (STZ-D), glucocorticoid-treated, fasted, and chronically uremic rats showed reduced basal and maximally insulin-stimulated 2-deoxy-D-glucose transport compared with matched controls. Adipocytes from these models were also resistant to stimulation of hexose transport by hydrogen peroxide, a postbinding insulin mimicker. Changes in the number of insulin receptors per cell could not account for these alterations in transport. Cell surface 125I-labeled insulin binding was 142% of control in STZ-D and 129% with fasting and unchanged in glucocorticoid excess and chronic uremia. Insulin-stimulated tyrosine kinase was measured by means of a synthetic substrate, Glu80Tyr20. Partially purified receptors from these resistant models had unaltered kinase activity when normalized to soluble 125I-insulin binding. In situ stimulation of receptor phosphorylation by 7 and 100 nM insulin was determined after equilibration of adipocytes with 32PO4. Compared with matched controls, these intact cells, from all four resistant models, had insulin-stimulated receptor phosphorylation that was unchanged per unit of cell surface binding. Similar to results with insulin, hydrogen peroxide stimulation of in situ receptor phosphorylation was unchanged in each model. Thus, both in vitro and in situ measures of receptor phosphorylation suggest that the cellular alterations leading to insulin resistance in these adipocytes resides beyond phosphorylation of the insulin receptor.

The oxidant H2O2 has many insulin-like effects in rat adipocytes. To determine whether these effects could be mediated by the tyrosine kinase activity of the insulin receptor, the ability of H2O2 to stimulate receptor phosphorylation in intact adipocytes and partially purified insulin receptors has been examined. Phosphorylation of the beta subunit of the insulin receptor was increased approximately 2-fold by treatment of intact cells with 3 mM H2O2, a concentration that maximally stimulates 2-deoxyglucose uptake. Stimulation of receptor phosphorylation was rapid, reaching maximal levels within 5 min, and preceded activation of glucose transport. Phosphoamino acid analysis of insulin receptors from H2O2-treated adipocytes showed that 32P incorporation into phosphotyrosine and phosphoserine residues of the beta subunit was enhanced. Furthermore, partially purified receptors from H2O2-treated cells exhibit increased tyrosine kinase activity, as measured by phosphorylation of the peptide Glu80Tyr20. In contrast, the direct addition of H2O2 to partially purified insulin receptors did not stimulate tyrosine kinase activity or insulin receptor autophosphorylation. This was not due to breakdown of H2O2 or oxidation of ATP or the required divalent cations. To define the factors involved in H2O2's effect, we have examined receptor phosphorylation in fat cell homogenates and purified plasma membranes. Although insulin stimulated receptor phosphorylation in both of these systems, H2O2 was only effective in the cell homogenates. These data demonstrate that, under certain conditions, H2O2 stimulates insulin receptor phosphorylation and tyrosine kinase activity, suggesting that the insulin-like effects of H2O2 may be mediated by stimulation of insulin receptor phosphorylation. This does not appear to be a direct effect of H2O2 on the insulin receptor and requires nonplasma membrane cellular constituents.