Inhibition of phosphatidylinositol 3-kinase activity by adenovirus-mediated gene transfer and its effect on insulin action

Phosphatidylinositol 3-kinase (PI 3-K) is implicated in cellular events including glucose transport, glycogen synthesis, and protein synthesis. It is activated in insulin-stimulated cells by binding of the Src homology 2 (SH2) domains in its 85-kDa regulatory subunit to insulin receptor substrate-1 (IRS-1), and, others. We have previously shown that IRS-1-associated PI 3-kinase activity is not essential for insulin-stimulated glucose transport in 3T3-L1 adipocytes, and that alternate pathways exist in these cells. We now show that adenovirus-mediated overexpression of the p85N-SH2 domain in these cells behaves in a dominant-negative manner, interfering with complex formation between endogenous PI 3-K and its SH2 binding targets. This not only inhibited insulin-stimulated IRS-1-associated PI 3-kinase activity, but also completely blocked anti-phosphotyrosine-associated PI 3-kinase activity, which would include the non-IRS-1-associated activity. This resulted in inhibition of insulin-stimulated glucose transport, glycogen synthase activity and DNA synthesis. Further, Ser/Thr phosphorylation of downstream molecules Akt and p70 S6 kinase was inhibited. However, co-expression of a membrane-targeted p110(C) with the p85N-SH2 protein rescued glucose transport, supporting our argument that the p85N-SH2 protein specifically blocks insulin-mediated PI 3-kinase activity, and, that the signaling pathways downstream of PI 3-kinase are intact. Unexpectedly, GTP-bound Ras was elevated in the basal state. Since p85 is known to interact with GTPase-activating protein in 3T3-L1 adipocytes, the overexpressed p85N-SH2 peptide could titrate out cellular GTPase-activating protein by direct association, such that it is unavailable to hydrolyze GTP-bound Ras. However, insulin-induced mitogen-activated protein kinase phosphorylation was inhibited. Thus, PI 3-kinase may be required for this action at a step independent of and downstream of Ras. We conclude that, in 3T3-L1 adipocytes, non-IRS-1-associated PI 3-kinase activity is crucial for insulin's metabolic signaling, and that overexpressed p85N-SH2 protein inhibits a variety of insulin's ultimate biological effects.     

Insulin stimulates sequestration of beta-adrenergic receptors and enhanced association of beta-adrenergic receptors with Grb2 via tyrosine 350

G-protein-linked receptors, such as the beta2-adrenergic receptor, are substrates for growth factor receptors with intrinsic tyrosine kinase activity (Karoor, V., Baltensperger, K., Paul, H., Czech, M. P., and Malbon C. C. (1995) J. Biol. Chem. 270, 25305-25308). In the present work, the counter-regulatory action of insulin on catecholamine action is shown to stimulate enhanced sequestration of beta2-adrenergic receptors in either DDT1MF-2 smooth muscle cells or Chinese hamster ovary cells stably expressing beta2-adrenergic receptors. Both insulin and insulin-like growth factor-1 stimulate internalization of beta-adrenergic receptors, contributing to the counter-regulatory effects of these growth factors on catecholamine action. In combination with beta-adrenergic agonists, insulin stimulates internalization of 50-60% of the complement of beta-adrenergic receptors. Insulin administration in vitro and in vivo stimulates phosphorylation of Tyr-350 of the beta-adrenergic receptor, creating an Src homology 2 domain available for binding of the adaptor molecule Grb2. The association of Grb2 with beta-adrenergic receptors was established using antibodies to Grb2 as well as a Grb2-glutathione S-transferase fusion protein. Insulin treatment of cells provokes binding of Grb2 to beta2-adrenergic receptors. Insulin also stimulates association of phosphatidylinositol 3-kinase and dynamin, via the Src homology 3 domain of Grb2. Both these interactions as well as internalization of the beta-adrenergic receptor are shown to be enhanced by insulin, beta-agonist, or both. The Tyr-350 --> Phe mutant form of the beta2-adrenergic receptor, lacking the site for tyrosine phosphorylation, fails to bind Grb2 in response to insulin, fails to display internalization of beta2-adrenergic receptor in response to insulin, and is no longer subject to the counter-regulatory effects of insulin on cyclic AMP accumulation. These data are the first to demonstrate the ability of a growth factor insulin to counter-regulate G-protein-linked receptor, the beta-adrenergic receptor, via a new mechanism, i.e. internalization.     

Regulation of the major detoxication functions by phenobarbital and 3-methylcholanthrene in co-cultures of rat hepatocytes and liver epithelial cells

We have examined the rotational diffusion of the luteinizing hormone (LH) receptors binding human chorionic gonadotropin (hCG) or ovine luteinizing hormone (oLH) in MA-10 Leydig tumor cells using time-resolved phosphorescence anisotropy techniques. LH receptors binding erythrosin isothiocyanate (ErITC)-derivatized oLH were rotationally mobile with rotational correlation times of 62 micros, 48 micros, 38 micros, and 29 micros at 4 degrees C, 15 degrees C, 25 degrees C, and 37 degrees C, respectively. ErITC-hCG bound to the LH receptor was rotationally immobile, showing no anisotropy decay at 4 degrees C, 15 degrees C, 25 degrees C, and 37 degrees C. To determine whether cytoskeletal components influenced the rotational diffusion of LH receptors, we measured rotational diffusion of LH receptors on MA-10 cells treated with 20 microg/ml cytochalasin D and on plasma membrane preparations. Following 1 h exposure to cytochalasin D, the rotational correlation times for hCG-occupied LH receptors were typically 11 micros at 37 degrees C compared to > 1000 micros on untreated cells. Treatment of MA-10 cells with cytochalasin B or colchicine had no affect on LH receptor rotational diffusion. Rotational correlation times for LH-occupied receptors decreased from 29 micros to 12 micros at 37 degrees C following cytochalasin D treatment. The rotational diffusion of LH receptors on plasma membrane preparations was similar to that observed for LH- and hCG-occupied receptors on intact cells treated with cytochalasin D. These various results indicate that there are differential effects of LH and hCG binding on the interactions of LH receptors with plasma membrane proteins and that microfilaments anchor the hCG- and LH-occupied receptors.     

Insulin signal transduction by a mutant human insulin receptor lacking the NPEY sequence. Evidence for an alternate mitogenic signaling pathway that is independent of Shc phosphorylation

The cytoplasmic juxtamembrane domain of the human insulin receptor (hIR) contains a single copy of the tetrameric amino acid sequence Asn-Pro-Glu-Tyr (NPEY) (residues 969-972 in the exon 11-containing B-isoform), which exists in the context of NPXY. In this study, we examined the role of NPEY972 in mediating insulin signal transduction and cellular biological effects. Transfected Chinese hamster ovary cell lines expressing either the wild-type hIR-B isoform (hIR.WT) or a mutant receptor lacking the NPEY972 sequence (hIRDeltaNPEY) and control Chinese hamster ovary.Neo cells were used to comparatively analyze the following insulin effects: in vivo receptor tyrosine autophosphorylation and kinase activity, signal transduction to downstream signaling molecules, and stimulation of glycogen and DNA synthesis. The results showed that in comparison to hIR.WT, the hIRDeltaNPEY mutant demonstrated the following: (a) normal insulin-mediated receptor tyrosine phosphorylation, but approximately 50% reduction in phosphorylation of p185-(insulin receptor substrate-1) and binding of the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase), (b) an enhanced stimulation of PI 3-kinase enzymatic activity, (c) a complete inability to phosphorylate Shc, (d) minimal impairment of insulin sensitivity for glycogen synthesis, and (e) an augmented response to insulin-stimulated DNA synthesis via a high capacity, low sensitivity pathway. These results demonstrate the following: 1) the NPEY972 sequence is important but not absolutely essential for coupling of hIR kinase to insulin receptor substrate-1 and p85 or for mediating insulin's metabolic and mitogenic effects, 2) the NPEY972 sequence is necessary for Shc phosphorylation, and 3) the absence of Shc phosphorylation releases the constraints on maximal insulin-stimulated mitogenic response, thus indicating that alternate signaling pathway(s) exist for this insulin action. This alternate pathway appears to be associated with enhanced activation of PI 3-kinase and is of high capacity and low sensitivity.     

IFN-gamma treatment increases insulin binding and MHC class I expression in erythroleukemia cells

We have investigated if interferon-gamma (IFN-gamma) treatment of human K562 tumor cells, which upregulates the expression of MHC class I antigens (MHC-I), simultaneously would influence insulin binding. Treatment of K562 cells with recombinant human IFN-gamma for 48 h caused a significant increase of insulin binding at 37 degrees C. Recombinant human tumor necrosis factor-alpha (TNF-alpha) alone had no effect but acted synergistically with IFN-gamma, leading to a two-fold increase of insulin binding. No change in affinity, number of binding sites or cell surface expression of insulin receptors (IR) after IFN-gamma treatment could be detected. The increased insulin binding observed at 37 degrees C was not seen at 4 degrees C, suggesting alteration of insulin internalization. The dose-response curve, as well as the time curve, for the increase in insulin binding after IFN-gamma treatment correlated with enhanced cell surface expression of MHC-I antigens. However, the correlation was not absolute. Our results show that IFN-gamma treatment alone or together with TNF-alpha, can alter the insulin binding to K562 cells without changing the expression or affinity of the IR. This correlates with the effect of IFN-gamma on MHC-I expression. These results support the findings that MHC-I molecules associate and interact with the IR at the cell surface.     

Post-translational glycosylation-induced activation of aglycoinsulin receptor accumulated during tunicamycin treatment

Tunicamycin, which inhibits N-linked oligosaccharide chain addition to nascent polypeptides, interrupts glycosylation of the insulin receptor in 3T3-L1 adipocytes giving rise to inactive receptors. Chronic exposure of cells to low levels (100 ng/ml) of high performance liquid chromatography-purified tunicamycin causes a greater than or equal to 90% depletion of insulin binding to cell surface and Triton X-100-extractable receptors and a 93% inhibition of [3H]glucosamine incorporation into protein in alkali-stable form. Under identical conditions, protein synthesis was inhibited less than 10%. Recovery of insulin binding activity after the removal of tunicamycin achieves 70-80% of control activity within 36 h. Concomitant with the withdrawal of tunicamycin, cells were shifted to medium containing heavy (greater than 95% 15N, 13C, 2H) amino acids after which Triton X-100-solubilized "light" and "heavy" insulin receptors were separated isopycnically on CsCl density gradients. A kinetic analysis of the recovery of functional receptors revealed that the initial appearance of previously synthesized light receptor was followed, after a short lag, by newly synthesized heavy receptor. Similar levels of light receptor, but no new heavy receptor, accrue in the presence of cycloheximide. This strongly suggests that inactive aglycoinsulin receptor accumulated post-translationally during chronic treatment with tunicamycin and then re-entered the glycosylation pathway when the inhibitor was removed giving rise to a functional insulin receptor.     

Detection of naturally expressed receptors for gastrin-releasing peptide and tachykinins using cyanine 3-labelled neuropeptides

Peptides labelled with the fluorophore cyanine 3 were used to study naturally expressed neuropeptide receptors by confocal microscopy in continuous cell lines, primary cultures, and unfixed tissue. Swiss 3T3 fibroblasts bound cyanine 3-gastrin-releasing peptide at 4 degrees C, and internalized the peptide after 10 min at 37 degrees C. Internalization was specific, since it was blocked by incubation with unlabelled peptide. Primary cultures of myenteric neurons of the guinea pig incubated with cyanine 3-substance P at 4 degrees C had specific surface labelling. After 30 s at 37 degrees C, the peptide was internalized into vesicles in both the soma and neurites. Direct observation of live neurons showed movement of fluorescent vesicles to a perinuclear region after 30 min. Endocytosis was associated with a loss of surface binding sites. Unfixed whole mounts of guinea pig and rat ileum were incubated with cyanine 3-neurokinin A at 4 degrees C. After 5 min at 37 degrees C, Cy3-neurokinin A was specifically internalized in neurons and smooth muscle cells. After 30 min, a perinuclear labelling occurred in some cells. Labelling in rat neurons was diminished by the NK3-R antagonist SR142801. Thus, cyanine 3-neuropeptides are valuable tools to study expression and endocytosis of naturally expressed receptors.     

Insulin and insulin-like growth factor-I signal transduction requires p21ras

We have investigated the role of cellular p21ras protein in insulin and insulin-like growth factor-I (IGF-I) signaling pathways. Insulin stimulation increased Ras-GTP formation in Rat-1 fibroblasts overexpressing normal human insulin receptors (HIRc-B), far greater than in parental Rat-1 fibroblasts, indicating that competent insulin receptors mediate this response. Cellular microinjection of a dominant-negative mutant p21ras protein (N17 ras) or anti-p21ras monoclonal antibody (Y13-259) into HIRc-B cells reduced insulin- and IGF-I-stimulated DNA synthesis by 75-90%. Insulin-induced c-fos protein expression was also inhibited by 74%. Microinjection of oncogenic p21ras (T-24 ras) into HIRc-B cells activated the mitogenic pathway, and coinjection of N17 ras and T-24 ras showed that oncogenic p21ras rescued the cells from the N17 ras blockade. This later finding indicates that T-24 ras acts downstream of N17 ras. In conclusion, 1) microinjection of a dominant interferring ras mutant into quiescent cells abrogated subsequent insulin and IGF-I mitogenic signaling; 2) oncogenic ras protein rescued cells from the N17 ras blockade, indicating that T24 ras action is downstream of the site of N17 inhibition; and 3) p21ras is an intermediate signaling molecule in the insulin/IGF-I signal transduction pathway and is required for gene expression and DNA synthesis.     

Adrenomedullin stimulates DNA synthesis and cell proliferation via elevation of cAMP in Swiss 3T3 cells

Our results demonstrate that the novel vasoactive regulatory peptide adrenomedullin is a potent mitogen for Swiss 3T3 cells. Acting via a specific adrenomedullin receptor, it stimulates a dose-dependent increase in DNA synthesis in synergy with insulin. Additionally, adrenomedullin stimulates further progression through the cell cycle resulting in cell proliferation, an effect that was further enhanced by the presence of insulin. Adrenomedullin rapidly induces accumulation of intracellular cAMP but does not stimulate an increase in intracellular Ca2+, activation of protein kinase C, or tyrosine phosphorylation of intracellular substrates. Adrenomedullin-stimulated mitogenesis is markedly enhanced in Swiss 3T3 cells stably transfected with a constitutively activated Gs alpha, which are highly sensitive to agents that elevate cAMP, and is inhibited by the PKA inhibitor H-89. Adrenomedullin is, thus, identified as a novel mitogenic regulatory peptide acting via cAMP.     

Insulin stimulates testosterone biosynthesis by human thecal cells from women with polycystic ovary syndrome by activating its own receptor and using inositolglycan mediators as the signal transduction system

To determine whether insulin stimulates human ovarian testosterone production in the polycystic ovary syndrome by activating its own receptor and using inositolglycan mediators as the signal transduction system, thecal cells from polycystic ovary syndrome women were isolated and cultured. Insulin and insulin-like growth factor I stimulated thecal testosterone biosynthesis. Antibody blockade of the insulin receptor abolished insulin's stimulatory action, whereas effective antibody blockade of the insulin-like growth factor I receptor did not alter insulin's stimulation of thecal testosterone biosynthesis. A chiro-inositol containing glycan (INS-2) increased thecal testosterone biosynthesis. Preincubation of cells with an antiinositolglycan antibody (A23939 or alpha IGP) abolished insulin's stimulatory effect, but not that of hCG. These findings suggest that inositolglycans serve as the signal transduction system for insulin's stimulation of human thecal testosterone biosynthesis.     

Indirect effect of insulin to suppress endogenous glucose production is dominant, even with hyperglucagonemia

Suppression of endogenous glucose production (EGP) is one of insulin's primary metabolic effects and failure of this action is a major contributor to fasting hyperglycemia of type 2 diabetes mellitus. Classically, insulin was thought to suppress the liver directly, via hyperinsulinemia in the portal vein. Recently, however, we and others have demonstrated that at least part, and possibly most of insulin's action to suppress EGP is normally mediated via an extrahepatic (i.e., indirect) mechanism. We have suggested that this mechanism involves insulin suppression of adipocyte lipolysis, leading to lowered FFA and reduced EGP ("Single Gateway Hypothesis"). Previous studies of the indirect insulin effect from this laboratory were done under conditions of lowered portal glucagon. Because of the possibility that the direct (i.e., portal) effect of insulin may have been underestimated with hypoglucagonemia, these studies examined the relative importance of portal insulin, versus peripheral insulin (administered at one-half the dose to equalize peripheral insulin levels) at four rates of portal glucagon infusion: 0, 0.65 (under-), 1.5 (basal-), and 3.0 ng/kg per min (over-replacement). Portal versus peripheral insulin suppressed steady-state EGP to the same extent (52%), confirming that the primary effect of insulin to suppress EGP is via the peripheral mechanism. This conclusion was maintained regardless of portal glucagonemia, although there was some evidence for an increase in the direct insulin effect at hyperglucagonemia. The indirect effect of insulin is the primary mechanism of steady-state EGP suppression under normal conditions. The direct effect increases with hyperglucagonemia; however, the indirect effect remains predominant even under those conditions.     

Heparinization on pericardial substitutes can reduce adhesion and epicardial inflammation in the dog

Some patients with severe insulin resistance develop pathological tissue growth reminiscent of acromegaly. Previous studies of such patients have suggested the presence of a selective postreceptor defect of insulin signaling, resulting in the impairment of metabolic but preservation of mitogenic signaling. As the activation of phosphoinositide 3-kinase (PI 3-kinase) is considered essential for insulin's metabolic signaling, we have examined insulin-stimulated PI 3-kinase activity in anti-insulin receptor substrate (IRS)-1 immunoprecipitates from cultured dermal fibroblasts obtained from pseudoacromegalic (PA) patients and controls. At a concentration of insulin (1 nM) similar to that seen in vivo in PA patients, the activation of IRS-1-associated PI 3-kinase was reduced markedly in fibroblasts from the PA patients (32+/-7% of the activity of normal controls, P < 0.01). Genetic and biochemical studies indicated that this impairment was not secondary to a defect in the structure, expression, or activation of the insulin receptor, IRS-1, or p85alpha. Insulin stimulation of mitogenesis in PA fibroblasts, as determined by thymidine incorporation, was indistinguishable from controls, as was mitogen-activated protein kinase phosphorylation, confirming the integrity of insulin's mitogenic signaling pathways in this condition. These findings support the existence of an intrinsic defect of postreceptor insulin signaling in the PA subtype of insulin resistance, which involves impairment of the activation of PI 3-kinase. The PA tissue growth seen in such patients is likely to result from severe in vivo hyperinsulinemia activating intact mitogenic signaling pathways emanating from the insulin receptor.     

Do adipocytes contain high affinity sulfonylurea receptors

Sulfonylureas interact with specific, high affinity receptors on the pancreatic beta-cell to close ATP-sensitive K+ channels, depolarize the cell, activate Ca2+ influx through voltage-dependent Ca2+ channels, and trigger insulin secretion. We tested the hypothesis that sulfonylureas promote glucose uptake into 3T3-L1 cells or isolated rat adipocytes by similar mechanisms. Using 125I-labeled 5-iodo-2-hydroxyglyburide and either equilibrium binding or photoaffinity labeling, a high affinity sulfonylurea receptor was not found on plasma membranes of either the 3T3-L1 cells or rat adipocytes. Furthermore, glyburide did not inhibit 86Rb+ efflux (a marker for ATP-sensitive K+ channel conductance), increase free cytosolic calcium in adipocytes or 3T3-L1 cells, or increase basal or insulin-stimulated glucose uptake into 3T3-L1 cells or rat adipocytes. Parallel studies using a hamster insulin-secreting tumor cell line (HIT cells) easily demonstrated both the receptor and biological effects of glyburide on free cytosolic calcium and insulin secretion. Thus, rat adipocytes and 3T3-L1 cells do not possess the high affinity sulfonylurea receptor or respond to glyburide alone. We conclude that the antidiabetogenic effects of sulfonylureas are not mediated by a direct action of sulfonylureas to increase glucose uptake into adipose tissue and suggest that the major locus for sulfonylurea action is the beta-cell.     

Endogenous serotonin-2A and -2C receptors in Balb/c-3T3 cells revealed in serotonin-free medium: desensitization and down-regulation by serotonin

We studied the endogenous expression of the serotonin-2A (5-hydroxytryptamine2A, 5-HT2A) 5-HT2C, and a splice-variant of the 5-HT2C receptor in murine Balb/c-3T3 fibroblast cells that is revealed when these cells are maintained in medium containing 5-HT-free serum. RNA editing of the 5-HT2C receptor was exclusively at a single brain-specific site. Addition of 5-HT (EC50 = 23 +/- 2.9 nM) induced an immediate release of calcium from an ionomycin-sensitive intracellular store by coupling to a pertussis toxin-insensitive pathway. The 5-HT-induced calcium mobilization displayed a 5-HT-2-like pharmacology, and ligand binding analyses indicated the presence of specific binding sites (27.5 +/- 2 fmol/mg protein) with a 5-HT2A-like pharmacology. Although the 5-HT2A receptor site was predominant, the smaller component of 5-HT2C receptors alone was sufficient to mediate a maximal calcium response. The 5-HT-induced increase in [Ca2+]i was reversibly inhibited by >75% following a 12-hr pretreatment (T1/2 = 2 hr) with 5-HT (EC50 = 400 nM). Extended treatment (24-96 hr) with 5-HT induced a complete functional desensitization that was associated with a partial (60%) reduction in 5-HT2 receptor number, implicating both receptor down-regulation and post-receptor mechanisms in 5-HT-induced desensitization. Long-term (hours to days) treatment with 5-HT did not modulate DNA synthesis, cell proliferation, or transformation in Balb/c-3T3 cells. These results demonstrate that Balb/c-3T3 cells express endogenous 5-HT2 receptors that are desensitized by the 5-HT present in normal serum, illustrating the importance of growth conditions in the identification of receptor responsiveness. The lack of proliferative response to 5-HT in Balb/c-3T3 suggests a putative role of desensitization as a "safety valve" to prevent abnormal cell growth during sustained 5-HT2 receptor activation.     

Cations affect [3H]mazindol and [3H]WIN 35,428 binding to the human dopamine transporter in a similar fashion

The present study addresses the possibility that there are different cocaine-related and mazindol-related binding domains on the dopamine transporter (DAT) that show differential sensitivity to cations. The effects of Zn2+, Mg2+, Hg2+, Li+, K+, and Na+ were assessed on the binding of [3H]mazindol and [3H]WIN 35,428 to the human (h) DAT expressed in C6 glioma cells under identical conditions for intact cell and membrane assays. The latter were performed at both 0 and 21 degrees C. Zn2+ (30-100 microM) stimulated binding of both radioligands to membranes, with a relatively smaller effect for [3H]mazindol; Mg2+ (0.1-100 microM) had no effect; Hg2+ at approximately 3 microM stimulated binding to membranes, with a relatively smaller effect for [3H]mazindol than [3H]WIN 35,428 at 0 degrees C, and at 30-100 microM inhibited both intact cell and membrane binding; Li+ and K+ substitution (30-100 mM) inhibited binding to membranes more severely than to intact cells; and Na+ substitution was strongly stimulatory. With only a few exceptions, the patterns of ion effects were remarkably similar for both radioligands at both 0 and 21 degrees C, suggesting the involvement of common binding domains on the hDAT impacted similarly by cations. Therefore, if there are different binding domains for WIN 35,428 and mazindol, these are not affected differentially by the cations studied in the present experiments, except for the stimulatory effect of Zn2+ at 0 and 21 degrees C and Hg2+ at 0 degrees C.     

Mechanism of impaired metabolic signaling by a truncated human insulin receptor. Decreased activation of protein phosphatase 1 by insulin

Previous studies have shown that a human insulin receptor lacking the COOH-terminal 43-amino acid domain (HIR delta CT) displays a compromised ability to stimulate glucose transport and glycogen synthase, whereas mitogenic signaling and stimulation of the insulin receptor tyrosine kinase activity remain intact (Maegawa, H., McClain, D. A., Freidenberg, G., Olefsky, J. M., Napier, M., Lipari, T., Dull, T. J., Lee, J., and Ullrich, A. (1988) J. Biol. Chem. 263, 8912-8917). In this study, we examined the effect of insulin on protein phosphatase 1 (PP-1) activity and phosphorylation in cells expressing wild-type human insulin receptor (HIRc) and HIR delta CT cells using phosphorylase alpha as substrate in the presence of 3 nM okadaic acid. Basal PP-1 activity was significantly lower in HIR delta CT than in HIRc cells (p < 0.05). Insulin stimulated PP-1 activity in HIRc cells (25-30% increase over basal activity) in a time- and dose-dependent manner. Insulin failed to stimulate PP-1 activity in HIR delta CT cells. Western blotting with the catalytic subunit antibody and the regulatory subunit antibody revealed similar amounts of the 37-kDa band (catalytic subunit) and the 160-kDa band (presumed regulatory subunit) in HIRc and HIR delta CT cells. We conclude that the COOH-terminal domain of the insulin receptor is an important element in mediating the effect of insulin on PP-1 and suggest that activation of PP-1 may be linked to signaling insulin's metabolic actions.     

Insulin-induced activation of NADPH-dependent H2O2 generation in human adipocyte plasma membranes is mediated by Galphai2

Human fat cells possess a multireceptor-linked H2O2-generating system that is activated by insulin. Previous studies revealed that manganese was the sole cofactor required for a hormonal regulation of NADPH-dependent H2O2 generation in vitro. In this report it is shown that the synergistic activation of NADPH-dependent H2O2 generation by Mn2+ and insulin was blocked by GDPbetaS (guanosine 5'-O-(2-thiodiphosphate)), pertussis toxin and COOH-terminal anti-Galphai1-2 or the corresponding peptide. Consistently, manganese could be replaced by micromolar concentrations of GTPgammaS (guanosine 5'-O-(3-thiotriphosphate)), which increased NADPH-dependent H2O2 generation by 20-40%. Insulin shifted the dose response curve for GTPgammaS to the left (>10-fold) and increased the maximal response. In the presence of 10 microM GTPgammaS, the hormone was active at picomolar concentrations, indicating that insulin acted via its cognate receptor. The insulin receptor and Gi were co-adsorbed on anti-Galphai and anti-insulin receptor beta-subunit (anti-IRbeta) affinity columns. Partially purified insulin receptor preparations contained Galphas, Galphai2, and Gbetagamma (but no Galphai1 or Galphai3). The functional nature of the insulin receptor-Gi2 complex was made evident by insulin's ability to modulate labeling of Gi by bacterial toxins. Insulin action was mimicked by activated Galphai, but not by Galphao or Gbetagamma, indicating that insulin's signal was transduced via Galphai2. Thus, NADPH oxidase is the first example of an effector system that is coupled to the insulin receptor via a heterotrimeric G protein.     

Desensitization of hepatoma cells to insulin action. Evidence for a post-receptor mechanism

We have previously reported that incubation of rat hepatoma cells with insulin causes a complete and reversible loss of responsiveness to insulin. In order to determine the role of the insulin receptor in desensitization, we have examined the effect of insulin on insulin binding. Exposure of rat hepatoma cells to insulin causes a time-dependent decrease in insulin binding capacity which is detectable at 30 min and maximal at 4-6 h, after which time insulin binding remains 40-50% that of untreated cells. Scatchard analysis indicates that insulin causes a decrease in the number of receptors with little change in the binding affinity. Insulin-induced down regulation of receptors, observable at insulin concentrations as low as 3 ng/ml, is half-maximal at 10-20 ng/ml and is maximal at 100 ng of insulin/ml. When insulin is removed from the culture medium, the cells slowly recover insulin binding capacity; recovery is minimal at 2-4 h but nearly complete after 24 h. Recovery of insulin responsiveness, in contrast, is complete as early as 2 h after insulin is removed. The extent of down regulation of receptors (50-60%) is not sufficient to account for the complete insulin desensitization. In addition, recovery of maximal responsiveness to insulin occurs long before recovery of insulin binding. Therefore, insulin-induced desensitization to insulin is not caused by down regulation of receptors but must involve a post-receptor mechanism.