Effect of lipid peroxidation on the insulin receptor in rat adipocytes

Lipid peroxidation is studied for its effect on insulin receptors in isolated rat adipocytes. The results suggest that addition of two peroxidants (3 mM cumene hydroperoxide and 0.2 mM Fe2+) leads to malondialdehyde accumulation and binding inhibition through insulin receptors quantity and affinity decrease.     

Effect of acute zinc deficiency on insulin receptor binding in rat adipocytes

Considerable data have been reported on the relationship between insulin resistance and zinc deficiency. In this study, insulin receptor binding was measured in isolated rat adipocytes. Two assays were carried out at 37°C (binding and internalization) and 16°C (binding) using¹²⁵I insulin 0.05–20 nM. A decreased insulin receptor binding was observed in zinc-deficient rat adipocytes, but we could not make any distinction between the specific zinc depletion effects and the effects of the caloric restriction induced by zinc deficiency.     

Recycling of the glucose transporter, the insulin receptor, and insulin in rat adipocytes. Effect of acidtropic agents

The notion of an insulin-dependent translocation of the glucose transporter in rat adipocytes was confirmed by immunoblotting and reconstitution of glucose transport activity of subcellular fractions. Quantitatively, however, significantly different results were obtained with these two techniques; when compared with reconstitution, immunoblotting detected translocation of a larger amount of the transporter from a low density microsome fraction to a plasma membrane fraction. The acidtropic agents chloroquine and dibucaine, which have been reported to inhibit the recycling of various receptors, were utilized to study the detailed translocation mechanism of the glucose transporter and the insulin receptor. These acidtropic agents caused accumulation of 125I-insulin in a subcellular fraction probably corresponding to lysosomes. They did not, however, significantly affect either the insulin-induced activation of glucose transport or the recycling of the transporter and the insulin receptor as detected by immunoblotting. About 50% of radioactivity released from adipocytes which were allowed to internalize insulin was due to intact insulin, and chloroquine did not change the release rate of intact insulin, raising the possibility of receptor-mediated exocytosis of insulin. The release of degraded insulin decreased with chloroquine treatment. The results are consistent with the idea that these acidtropic agents mainly act to inhibit degradation of insulin in lysosomes, and their effect on the recycling of the glucose transporter and the insulin receptor is minimal, indicating that the recycling of these membrane proteins proceeds irrespective of organelle acidification. Electron micrographs showed vesicles underneath the plasma membranes, with sizes similar to those of the low density microsome fraction where the internalized glucose transporter and the insulin receptor were located.     

The role of insulin receptor autophosphorylation in signal transduction.

We have examined the role of autophosphorylation in insulin signal transmission by oligonucleotide directed mutagenesis of seven potential tyrosine autophosphorylation sites in the human insulin receptor. Chinese hamster ovary cells transfected with these receptors were analyzed for insulin stimulated 2-deoxyglucose uptake, thymidine incorporation, endogenous substrate phosphorylation, and in vitro kinase activity. We found that phosphorylation on tyrosine residues 953, 1316, and 1322 were not necessary for receptor-mediated signal transduction. Mutation of tyrosine 960 reduced but did not abolish the signaling capabilities of the receptor. Finally, the simultaneous mutation of tyrosine residues 1146, 1150, and 1151 (the numbering system is that of Ullrich et al. (Ullrich, A., Bell, J. R., Chen, E. Y., Herrera, R., Petruzzelli, L. M., Dull, T. J., Gray, A., Coussens, L., Liao, Y. C., Tsubokawa, M., Mason, A., Seeburg, P.H., Grunfeld, C., Rosen, O. M., and Ramachandran, J. (1985) Nature 313, 756-761) resulted in a biologically inactive receptor, suggesting that the insulin receptor can be inactivated by removal of key autophosphorylation sites.     

Autoantibodies to the insulin receptor activate glycogen synthase in rat adipocytes

Summary Autoantibodies to the insulin receptor mimic the effects of insulin on glycogen synthase and phosphorylase. The interaction of antibodies with adipocyte cell surface insulin receptors seems sufficient to promote stable changes in the activities of these intracellular enzymes, suggesting that internalization or processing of insulin is not important in the generation of these biological responses.     

Genistein differentially inhibits postreceptor effects of insulin in rat adipocytes without inhibiting the insulin receptor kinase.

Genistein, an isoflavone putative tyrosine kinase inhibitor, was used to investigate the coupling of insulin receptor tyrosine kinase activation to four metabolic effects of insulin in the isolated rat adipocyte. Genistein inhibited insulin-stimulated glucose oxidation in a concentration-dependent manner with an ID50 of 25 micrograms/ml and complete inhibition at 100 micrograms/ml. Genistein also prevented insulin's (10(-9) M) inhibition of isoproterenol-stimulated lipolysis with an ID50 of 15 micrograms/ml and a complete effect at 50 micrograms/ml. The effect of genistein (25 micrograms/ml) was not reversed by supraphysiological (10(-7) M) insulin levels. In contrast, genistein up to 100 micrograms/ml had no effect on insulin's (10(-9) M) stimulation of either pyruvate dehydrogenase or glycogen synthase activity. We determined whether genistein influenced insulin receptor beta-subunit autophosphorylation or tyrosine kinase substrate phosphorylation either in vivo or in vitro by anti-phosphotyrosine immunoblotting. Genistein at 100 micrograms/ml did not inhibit insulin's (10(-7) M) stimulation of insulin receptor tyrosine autophosphorylation or tyrosine phosphorylation of the cellular substrates pp185 and pp60. Also, genistein did not prevent insulin-stimulated autophosphorylation of partially purified human insulin receptors from NIH 3T3/HIR 3.5 cells or the phosphorylation of histones by the activated receptor tyrosine kinase. In control experiments using either NIH 3T3 fibroblasts or partially purified membranes from these cells, genistein did inhibit platelet-derived growth factor's stimulation of its receptor autophosphorylation. These findings indicate the following: (a) Genistein can inhibit certain responses to insulin without blocking insulin's stimulation of its receptor tyrosine autophosphorylation or of the receptor kinase substrate tyrosine phosphorylation. (b) In adipocytes genistein must block the stimulation of glucose oxidation and the antilipolytic effects of insulin at site(s) downstream from the insulin receptor tyrosine kinase. (c) The inhibitory effects of genistein on hormonal signal transduction cannot necessarily be attributed to inhibition of tyrosine kinase activity, unless specifically demonstrated.     

Reversible reduction of insulin receptor affinity by ATP depletion in rat adipocytes.

The effects of the metabolic inhibitor NaN3 on insulin receptors in isolated rat fat-cells were investigated. The agent reduced insulin binding in parallel to a decrease of the ATP content of cells. Both effects were observed in the same concentration range of NaN3, and were fully reversible. According to the binding curves the affinity rather than the number of receptors was reduced. Kinetic experiments revealed an increased dissociation rate of the insulin-receptor complex. The effects outlasted cell disruption, since the receptor affinity was still lowered in plasma membranes obtained from NaN3-treated cells. Thus an inhibition of insulin internalization could not account for the observed effects. It is suggested that the observed ATP-dependence of insulin receptor affinity reflects a reversible structural alteration of the receptor, or of some closely related membrane protein.     

Insulin receptor kinase activity in rat adipocytes is decreased during aging

The tyrosine kinase activity of the insulin receptor derived from rat adipocyte plasma membranes was examined during aging. In the absence of insulin, autophosphorylation and histone H2B phosphorylation activities, measured with equal numbers of insulin receptors, were comparable among 3- and 24-month-old rats. In contrast, insulin-stimulated kinase activity was significantly reduced in the old animals. We have also found that the insulin dependent phosphorylation of a putative endogenous substrate of 60 kDa was drastically reduced in old animals. These results suggest that the decrease in kinase activity in old rats could be related with the insulin resistance of aging.     

Cascade of autophosphorylation in the beta-subunit of the insulin receptor

Insulin stimulated autophosphorylation of the beta-subunit of the insulin receptor purified from Fao hepatoma cells or purified from Chinese hamster ovary (CHO/HIRC) or Swiss 3T3 (3T3/HIRC) cells transfected with the wild-type human insulin receptor cDNA. Autophosphorylation of the purified receptor occurred in at least two regions of the beta-subunit: the regulatory region containing Tyr-1146, Tyr-1150, and Tyr-1151, and the C-terminus containing Tyr-1316 and Tyr-1322. In the presence of antiphosphotyrosine antibody (alpha-PY), autophosphorylation of the purified receptor was inhibited nearly 80% during insulin stimulation. Tryptic peptide mapping showed that alpha-PY inhibited autophosphorylation of both tyrosyl residues in the C-terminus and one tyrosyl residue in the regulatory region, either Tyr-1150 or Tyr-1151. Thus, a bis-phosphorylated form of the regulatory region accumulated in the presence of alpha-PY, which contained Tyr(P)-1146 and either Tyr(P)-1150 or 1151. In intact Fao, CHO/HIRC, and 3T3/HIRC cells, insulin stimulated tyrosyl phosphorylation of the beta-subunit of the insulin receptor. Tryptic peptide mapping indicated that the regulatory region of the beta-subunit was mainly (greater than 80%) bis-phosphorylated; however, all three tyrosyl residues of the regulatory region were phosphorylated in about 20% of the receptors. As the phosphotransferase was activated by tris-phosphorylation but not bis-phosphorylation of the regulatory region of the beta-subunit (White et al.: Journal of Biological Chemistry 263:2969-2980, 1988), the extent of autophosphorylation in the regulatory region may play an important regulatory role during signal transmission in the intact cell.     

A mutation in the extracellular domain of the insulin receptor impairs the ability of insulin to stimulate receptor autophosphorylation.

Mutations of the insulin receptor gene have been shown to cause insulin-resistant diabetes in patients with genetic forms of insulin resistance. We have previously reported that a mutation substituting valine for Phe382 in the alpha-subunit of the insulin receptor is associated with impaired transport of the mutant receptor to the plasma membrane (Accili, D., Frapier, C., Mosthaf, L., McKeon, C., Elbein, S. C., Permutt, M. A., Ramos, E., Lander, E. S., Ullrich, A., and Taylor, S. I. (1989) EMBO J. 8, 2509-2517). In this study, we demonstrate that the Val382 mutation impairs the ability of insulin to activate receptor autophosphorylation. Furthermore, the Val382 receptor has reduced activity to phosphorylate other peptide substrates in the presence of insulin. Nevertheless, when the Val382 mutant and wild-type receptors are mixed together, the wild-type human insulin receptor is able to phosphorylate the Val382 mutant receptor, thereby activating the tyrosine kinase activity of the mutant receptor. Thus, the conformational change caused by the Val382 mutation compromises the ability of the receptor to transmit a signal across the plasma membrane. Furthermore, our observations suggest that receptor phosphorylation by an intermolecular mechanism (i.e. transphosphorylation) may play a role in mediating the action of insulin upon the target cell.     

Cytoskeletal components enhance the autophosphorylation of retinal insulin receptor

Insulin receptor (IR) signaling provides a trophic signal for transformed retinal neurons in culture, and we recently reported that deletion of IR from rod photoreceptors resulted in stress-induced photoreceptor degeneration. Retinal insulin receptor has a high basal level autophosphorylation compared to liver and the reasons for higher autophosphorylation are not known. In the current study we report a novel finding that cytoplasmic actin associates with and activates the retinal IR in vivo. Similar to insulin, actin also induced autophosphorylation at tyrosines 1158, 1162 and 1163 in the catalytic loop of IR. Our studies also suggest that globular actin activates the retinal IR more effectively than does filamentous actin. Retinal IR kinase activity has been shown to decrease in hyperglycemia and we found a decreased binding of actin to the IR under hyperglycemia. This is the first study which demonstrates that cytoplasmic actin regulates autophosphorylation of the retinal IR.     

Separate intracellular pathways for insulin receptor recycling and insulin degradation in isolated rat adipocytes

To gain insight into the sequence of events that follow endocytotic uptake of insulin receptor complexes, we have examined the interrelationship between the degradative pathway of the insulin ligand and the recycling pathway of the insulin receptor. Tris(hydroxymethyl)aminomethane and other nonamphoteric amines were found to selectively impair insulin receptor recycling while leaving the insulin-degradative pathway intact. In contrast, low concentrations of the lysosomotropic agent chloroquine markedly inhibited intracellular insulin degradation but had little or no affect on the recycling of internalized receptors. Thus, we conclude: (1) that insulin dissociates from its receptor after endocytotic uptake and both receptor and ligand follow a separate intracellular pathway; and (2) that receptor recycling and insulin degradation can be selectively inhibited by Tris and chloroquine, respectively, highlighting the potential usefulness of these agents as intracellular probes in the study of receptor-ligand metabolism.     

Effect of lactation on insulin sensitivity of glucose metabolism in rat adipocytes

During lactation glucose metabolism in paraovarian adipocytes is characterized by a 40 and 80% decrease of glucose incorporation into CO2 and fatty acids in the presence of insulin. In contrast with the stimulation by insulin of glucose incorporation into lactate, glycerol remains unchanged. As a result, insulin sensitivity of total glucose metabolism (oxidation and lipid synthesis) is not altered in adipocytes from lactating rats.     

Quercetin selectively inhibits insulin receptor function in vitro and the bioresponses of insulin and insulinomimetic agents in rat adipocytes.

We report here that quercetin, a naturally occurring bioflavonoid, is an effective blocker of insulin receptor tyrosine kinase-catalyzed phosphorylation of exogenous substrate. The ID50 was estimated to be 2 +/- 0.2 microM in cell-free experiments, using a partially purified insulin receptor and a random copolymer of glutamic acid and tyrosine as a substrate. Insulin-stimulated autophosphorylation of the receptor itself was not blocked by quercetin (up to 500 microM). In intact rat adipocytes, quercetin inhibited insulin-stimulating effects on glucose transport, oxidation, and its incorporation into lipids. Inhibition of lipogenesis (50%) occurred at 47 +/- 4 microM, whereas full inhibition was evident at 110 +/- 10 microM quercetin. In contrast, the effect of insulin in inhibiting lipolysis remained unaltered in quercetin-treated adipocytes. The inhibitor was devoid of general adverse cell affects. Basal activities and the ability of lipolytic agents to stimulate lipolysis were not affected. Inhibition by quercetin enabled us to evaluate which insulinomimetic agents are dependent on tyrosine phosphorylation of endogenous substrates for stimulating glucose metabolism. Quercetin blocked lipogenesis mediated by insulin, wheat germ agglutinin, and concanavalin A. The lipogenic effect of Zn2+ and Mn2+ was partially blocked, whereas that of vanadate was not affected at all.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pertussis toxin inhibits autophosphorylation and activation of the insulin receptor kinase.

Pertussis toxin is an ADP-ribosyltransferase which alters the function of some of the GTP-binding proteins and inhibits some actions of insulin. In vivo, pertussis toxin (2 micrograms/ml/2h) inhibited insulin-stimulated tyrosyl autophosphorylation of the insulin receptor by 50% in FaO cells, and nearly completely inhibited phosphorylation of the cellular insulin receptor substrate pp185. Similarly, insulin-stimulated autophosphorylation and kinase activity of the insulin receptor purified on wheat germ agglutinin-agarose from pertussis toxin-treated FaO cells was diminished 50%; however, treatment of cells with the catalytically inactive B-oligomer of the toxin had no effect on receptor tyrosine kinase activity in vitro. Pertussis toxin did not alter insulin binding or the cellular levels of ATP, cAMP, and cGMP. Furthermore, immunoprecipitation of the insulin receptor from intact cells with anti-insulin receptor antibodies showed that pertussis toxin did not increase the phosphorylation of serine or threonine residues in the insulin receptor. These results suggest that pertussis toxin can modulate signal transduction of insulin at the level of the insulin receptor kinase.     

Effect of insulin on glucagon binding to isolated rat epididymal adipocytes

Effect of insulin on glucagon binding to rat epididymal adipocytes was studied in vitro. [125I]iodoglucagon binding to isolated adipocytes was increased by preincubation of the cells with insulin. Maximal increase was observed with 7 X 10(-10) M insulin. In Scatchard analysis, [125I]iodoglucagon competition data generated one binding site with a single affinity for glucagon binding in the cells pretreated with buffer alone. Pretreatment of the cells with insulin increased the affinity without changes in the number of binding sites. [125I]iodoglucagon binding to isolated adipocytes was not affected by pretreatment of the cells with luteinizing hormone, follicle-stimulating hormone, growth hormone, or with prolactin. These results suggest that insulin stimulates glucagon binding to adipocytes.     

Effects of fluorescein isothiocyanate on insulin actions in rat adipocytes.

The effects of fluorescein isothiocyanate II (FITC) on the actions of insulin in rat adipocytes were studied. When adipocytes were incubated with FITC at pH 7.4 (2 mM agent, 8 min), the cells were completely deprived of their specific insulin-binding activity and rendered unresponsive to the hormone. The effect of FITC on the insulin-binding activity was milder at pH 9.0, and cAMP phosphodiesterase in cells exposed to FITC at pH 9.0 was maximally stimulated if the insulin concentration was increased to 100 nM. Under identical conditions, however, glucose transport activity was rendered not only less sensitive but also less responsive to the hormone. When FITC was added to cells after insulin at pH 9.0, the glucose transport activity that had been stimulated by the hormone was considerably reduced. This reduction was largely, but not entirely, prevented if the cells were deprived of ATP, suggesting that FITC (a) elicited the ATP-dependent reversal of the hormonal effect and, simultaneously, (b) mildly inhibited the transport activity per se. Western blot assay of GLUT-4 (a major isoform of glucose transporter in adipocytes) indicated that FITC (a) partially blocked insulin-dependent translocation of GLUT-4 from the intracellular site to the plasma membrane while it (b) induced a mild "insulin-like" effect. It is concluded that FITC at pH 9.0 (a) renders both glucose transport and phosphodiesterase activities less insulin sensitive presumably by modifying the cellular hormone receptor and (b) makes glucose transport activity less responsive to insulin presumably by (i) blocking hormone-dependent translocation of glucose transporter and (ii) mildly inhibiting intrinsic glucose transport activity.     

Autophosphorylation of the insulin receptor in rat adipocytes is modulated by thyroid hormone status

The effect of thyroid status on the in vitro autophosphorylation of the insulin receptors was studied in triton-solubilized adipocyte plasma membranes obtained from normal and thyroidectomized rats. Thyroidectomy results in an increase (two to three times) of the in vitro insulin-dependent phosphorylation of the insulin beta-subunit receptor. Phosphorylation occurred on tyrosine residues. In vivo injection of triiodothyronine to thyroidectomized rats restored plasma membranes autophosphorylation of the beta-subunit to the values obtained for control euthyroid rats. This effect was independent of the number and affinity of the insulin receptors, which were not modified regardless of thyroid status.     

A thiol-sensitive degradative process of liver uncouples autophosphorylation of the insulin receptor from insulin binding.

Insulin receptors derived from highly purified rat liver plasma membranes and Golgi membranes showed differences in insulin-mediated receptor autophosphorylation, even though their insulin-binding characteristics were similar. This difference was related to the generation of a Mr-84,000 fragment of the Mr-90,000 beta subunit of the plasma-membrane receptor, a fragment that was not present in the receptor from Golgi membranes, in the absence of a change in the insulin-binding alpha subunit. When autophosphorylation activity was based on insulin binding, the activity of the plasma-membrane-derived insulin receptor was decreased to 25-30% that of the Golgi-derived receptor. Endoglycosidase F digestion produced changes in the Mr values for both species, but they were not converted into a single subunit, thereby suggesting differences in the protein component of the two subunits. Although the proteinase inhibitors phenylmethanesulphonyl fluoride, ovomucoid and aprotinin failed to block the formation of the Mr-84,000 fragment, the presence of iodoacetamide or EDTA during liver homogenization markedly inhibited fragment generation and allowed the plasma-membrane insulin receptor to retain an autophosphorylation activity comparable with that present in insulin receptors from Golgi membranes. Thus a thiol-sensitive, cation-dependent, degrading activity has been identified that can uncouple the insulin-binding activity of the plasma-membrane insulin receptor from its tyrosine kinase activity.