Agonism and Antagonism at the Insulin Receptor

Insulin can trigger metabolic as well as mitogenic effects, the latter being pharmaceutically undesirable. An understanding of the structure/function relationships between insulin receptor (IR) binding and mitogenic/metabolic signalling would greatly facilitate the preclinical development of new insulin analogues. The occurrence of ligand agonism and antagonism is well described for G protein-coupled receptors (GPCRs) and other receptors but in general, with the exception of antibodies, not for receptor tyrosine kinases (RTKs). In the case of the IR, no natural ligand or insulin analogue has been shown to exhibit antagonistic properties, with the exception of a crosslinked insulin dimer (B29-B’29). However, synthetic monomeric or dimeric peptides targeting sites 1 or 2 of the IR were shown to be either agonists or antagonists. We found here that the S961 peptide, previously described to be an IR antagonist, exhibited partial agonistic effects in the 1–10 nM range, showing altogether a bell-shaped dose-response curve. Intriguingly, the agonistic effects of S961 were seen only on mitogenic endpoints (³H-thymidine incorporation), and not on metabolic endpoints (¹⁴C-glucose incorporation in adipocytes and muscle cells). The agonistic effects of S961 were observed in 3 independent cell lines, with complete concordance between mitogenicity (³H-thymidine incorporation) and phosphorylation of the IR and Akt. Together with the B29-B’29 crosslinked dimer, S961 is a rare example of a mixed agonist/antagonist for the human IR. A plausible mechanistic explanation based on the bivalent crosslinking model of IR activation is proposed.     

Hyaluronan substratum holds mesenchymal stem cells in slow-cycling mode by prolonging G<Subscript>1</Subscript> phase

We examined, in vitro, whether hyaluronan induces slow cycling in placenta-derived mesenchymal stem cells (PDMSCs) by comparing cell growth on a hyaluronan-coated surface with cell growth on a tissue-culture polystyrene surface. The hyaluronan-coated surface significantly downregulated the proliferation of PDMSCs, more of which were maintained in the G₀/G₁ phases than were cells on the tissue-culture polystyrene surface. Both PKH-26 labeling and BrdU incorporation assays showed that most PDMSCs grown on a hyaluronan-coated surface duplicated during cultivation indicating that the hyaluronan-coated surface did not inhibit PDMSCs from entering the cell cycle. Mitotic synchronization showed that the G₁-phase transit was prolonged in PDMSCs growing on a hyaluronan-coated surface. Increases in p27^Kip1 and p130 were the crucial factors that allowed hyaluronan to lengthen the G₁ phase. Thus, hyaluronan might be a promising candidate for maintaining stem cells in slow-cycling mode by prolonging their G₁-phase transit. This work was supported by research grant NSC95-2745-B-006-003-MY2 from the National Science Council, Taiwan, and by Landmark Project Grant A25, funded by the Taiwan Ministry of Education, from National Cheng Kung University.     

Insulin increases H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced pancreatic beta cell death

Insulin resistance results, in part, from impaired insulin signaling in insulin target tissues. Consequently, increased levels of insulin are necessary to control plasma glucose levels. The effects of elevated insulin levels on pancreatic beta (β) cell function, however, are unclear. In this study, we investigated the possibility that insulin may influence survival of pancreatic β cells. Studies were conducted on RINm, RINm5F and Min-6 pancreatic β-cells. Cell death was induced by treatment with H₂O₂, and was estimated by measurements of LDH levels, viability assay (Cell-Titer Blue), propidium iodide staining and FACS analysis, and mitochondrial membrane potential (JC-1). In addition, levels of cleaved caspase-3 and caspase activity were determined. Treatment with H₂O₂ increased cell death; this effect was increased by simultaneous treatment of cells with insulin. Insulin treatment alone caused a slight increase in cell death. Inhibition of caspase-3 reduced the effect of insulin to increase H₂O₂-induced cell death. Insulin increased ROS production by pancreatic β cells and increased the effect of H₂O₂. These effects were increased by inhibition of IR signaling, indicative of an effect independent of the IR cascade. We conclude that elevated levels of insulin may act to exacerbate cell death induced by H₂O₂ and, perhaps, other inducers of apoptosis.     

An improved method for the cell cycle synchronization of <Emphasis Type="Italic">Vicia faba</Emphasis> root meristem cells

Plant root meristem cells divide asynchronously which makes biochemical analysis of cell cycle regulation particularly difficult. In the present article a high level of cell cycle synchronization in Vicia faba root meristems was obtained by using a rich medium (HNS), special culture conditions and a double-block method with replication inhibitor—hydroxyurea (HU). Two HU concentrations were tested and different periods of the first and the second synchronization, and of cycle recommencement between the first and the second blockage. The level of synchronization was estimated on the basis of ³H-thymidine labeling indices, mitotic, and phase indices and indices determining the percentage of G1 and G2 cells, which were identified by cytophotometric measurements of DNA content in individual nuclei. The highest level of cell cycle synchronization was obtained after double treatment of meristems with 1.25 mM HU (18 and 12 h) separated by 6-h incubation in HNS without HU. During the second postincubation in HNS in subsequent hours: 4, 7, 10, 11, over 90% of cells in the S phase, nearly 70% in G2 phase, 86% in mitosis, and nearly 70% in G1 phase were received, respectively. The use of 2.5 mM HU in a similar experimental procedure caused disturbed divisions.     

Aluminum ions stimulate DNA synthesis in quiescent cultures of Swiss 3T3 and 3T6 cells

Micromolar concentrations of AI3+ are shown to be strongly mitogenic for quiescent cultures of Swiss 3T3 and 3T6 cells. AI3+ caused a striking shift in the dose-response curve for the effect of fetal bovine serum on 3H-thymidine incorporation. In the absence of serum the mitogenic effect of aluminum was greatly potentiated by insulin or cholera toxin, but not epidermal growth factor or 12-0-tetradecanoyl-phorbol-13-acetate. The stimulation of DNA synthesis was maximal by 15-20 microM AI3+ X AI3+ at 100 microM had no inhibitory effect on DNA synthesis. AI3+ had no significant effect on cellular cyclic adenosine monophosphate in the presence or absence of insulin or an inhibitor of cyclic nucleotide phosphodiesterases.     

Effect of insulin on sulfated proteoglycan synthesis in cultured smooth muscle cells from pig aorta

The effect of insulin upon proteoglycan synthesis was studied in cultured smooth muscle cells from pig aorta blocked in the G0 phase by serum deprivation. Insulin enhanced [35S]sulfate incorporation into cell layer and medium-secreted proteoglycans. The increase in incorporation of the precursor was not due to a mitogenic response by smooth muscle cells to the hormone and the specific radioactivity of proteoglycans showed that the stimulation reflected a real increase in sulfated proteoglycan synthesis. Maximal stimulation was observed, for the cell layer as well as for the medium, 40 h after the addition of 1.7 x 10(-7) M insulin and reached respectively 65 and 53%. This stimulation was about 80 and 60% of the level achieved with 10% fetal calf serum for cell layer and medium-secreted proteoglycans, respectively. The half-maximal effect was attained, for both the cell layer and the medium, in the presence of 2.1 x 10(-9) M insulin. Proteoglycans secreted into the medium, in the presence of 1.7 x 10(-8) M insulin for 40 h, showed a higher proportion of complexes (24%) than those synthesized in control medium (11%) and at least 95% of the monomers from culture treated with insulin were characterized by a smaller hydrodynamic size than those synthesized by cells maintained in control medium. This decrease in the size of proteoglycans was partly due to a decrease in the size of their glycanic chains.     

Regulation of the Akt/Glycogen synthase kinase-3 axis by insulin-like growth factor-II via activation of the human insulin receptor isoform-A

Insulin-like growth factor II (IGF-II) plays a key role in mitogenesis during development and tumorigenesis and is believed to exert its mitogenic functions mainly through the IGF-I receptor. Recently, we identified the insulin receptor isoform A (IR(A)) as an additional high affinity receptor for IGF-II in both fetal and cancer cells. Here we investigated the mitogenic signaling of IGF-II via the Akt/Glycogen synthase kinase 3 (Gsk3) axis employing R-IR(A) cells that are IGF-I receptor null mouse embryonic fibroblasts expressing the human IR(A). IGF-II induced activation of the proto-oncogenic serine kinase Akt, reaching maximal at 5-10 min. IGF-II also caused the rapid and sustained deactivation of glycogen synthase kinase 3-beta (Gsk3beta), reaching maximal at 1-3 min, shortly preceding, therefore, maximal activation of Akt. Under our conditions, IGF-II and insulin induced 70-80% inhibition of Gsk3betaactivity. In these cells IGF-II also deactivated Gsk3alpha although less effectively than Gsk3beta. In parallel experiments, we found that IGF-II induced transient activation of extracellular-signal-regulated kinases (Erk) reaching maximal at 5-10 min and decreasing thereafter. Time courses and potencies of regulation of both mitogenic pathways (Akt/Gsk3beta and Erk) by IGF-II via IR(A) were similar to those of insulin. Furthermore, IGF-II like insulin effectively stimulated cell cycle progression from the G0/G1 to the S and G2/M phases. Interestingly, AP-1-mediated gene expression, that was reported to be negatively regulated by Gsk3beta was only weakly increased after IGF-II stimulation. Our present data suggest that the coordinated activation or deactivation of Akt, Gsk3beta, and Erk may account for IGF-II mitogenic effects and support an active role for IR(A) in IGF-II action.     

Mitochondrial H<Subscript>2</Subscript>O<Subscript>2</Subscript> as an enable signal for triggering autophosphorylation of insulin receptor in neurons

Background

Insulin receptors are widely distributed in the brain, where they play roles in synaptic function, memory formation, and neuroprotection. Autophosphorylation of the receptor in response to insulin stimulation is a critical step in receptor activation. In neurons, insulin stimulation leads to a rise in mitochondrial H₂O₂ production, which plays a role in receptor autophosphorylation. However, the kinetic characteristics of the H₂O₂ signal and its functional relationships with the insulin receptor during the autophosphorylation process in neurons remain unexplored to date.

Results

Experiments were carried out in culture of rat cerebellar granule neurons. Kinetic study showed that the insulin-induced H₂O₂ signal precedes receptor autophosphorylation and represents a single spike with a peak at 5–10 s and duration of less than 30 s. Mitochondrial complexes II and, to a lesser extent, I are involved in generation of the H₂O₂ signal. The mechanism by which insulin triggers the H₂O₂ signal involves modulation of succinate dehydrogenase activity. Insulin dose–response for receptor autophosphorylation is well described by hyperbolic function (Hill coefficient, n_H, of 1.1±0.1; R²=0.99). N-acetylcysteine (NAC), a scavenger of H₂O₂, dose-dependently inhibited receptor autophosphorylation. The observed dose response is highly sigmoidal (Hill coefficient, n_H, of 8.0±2.3; R²=0.97), signifying that insulin receptor autophosphorylation is highly ultrasensitive to the H₂O₂ signal. These results suggest that autophosphorylation occurred as a gradual response to increasing insulin concentrations, only if the H₂O₂ signal exceeded a certain threshold. Both insulin-stimulated receptor autophosphorylation and H₂O₂ generation were inhibited by pertussis toxin, suggesting that a pertussis toxin-sensitive G protein may link the insulin receptor to the H₂O₂-generating system in neurons during the autophosphorylation process.

Conclusions

In this study, we demonstrated for the first time that the receptor autophosphorylation occurs only if mitochondrial H₂O₂ signal exceeds a certain threshold. This finding provides novel insights into the mechanisms underlying neuronal response to insulin. The neuronal insulin receptor is activated if two conditions are met: 1) insulin binds to the receptor, and 2) the H₂O₂ signal surpasses a certain threshold, thus, enabling receptor autophosphorylation in all-or-nothing manner. Although the physiological rationale for this control remains to be determined, we propose that malfunction of mitochondrial H₂O₂ signaling may lead to the development of cerebral insulin resistance.

     

Evidence for the possible involvement of the P2Y<Subscript>6</Subscript> receptor in Ca<Superscript>2+</Superscript> mobilization and insulin secretion in mouse pancreatic islets

Subtypes of purinergic receptors involved in modulation of cytoplasmic calcium ion concentration ([Ca²⁺]_i) and insulin release in mouse pancreatic β-cells were examined in two systems, pancreatic islets in primary culture and beta-TC6 insulinoma cells. Both systems exhibited some physiological responses such as acetylcholine-stimulated [Ca²⁺]_i rise via cytoplasmic Ca²⁺ mobilization. Addition of ATP, ADP, and 2-MeSADP (each 100 μM) transiently increased [Ca²⁺]_i in single islets cultured in the presence of 5.5 mM (normal) glucose. The potent P2Y₁ receptor agonist 2-MeSADP reduced insulin secretion significantly in islets cultured in the presence of high glucose (16.7 mM), whereas a slight stimulation occurred at 5.5 mM glucose. The selective P2Y₆ receptor agonist UDP (200 μM) transiently increased [Ca²⁺]_i and reduced insulin secretion at high glucose, whereas the P2Y_2/4 receptor agonist UTP and adenosine receptor agonist NECA were inactive. [Ca²⁺]_i transients induced by 2-MeSADP and UDP were antagonized by suramin (100 μM), U73122 (2 μM, PLC inhibitor), and 2-APB (10 or 30 μM, IP₃ receptor antagonist), but neither by staurosporine (1 μM, PKC inhibitor) nor depletion of extracellular Ca²⁺. The effect of 2-MeSADP on [Ca²⁺]_i was also significantly inhibited by MRS2500, a P2Y₁ receptor antagonist. These results suggested that P2Y₁ and P2Y₆ receptor subtypes are involved in Ca²⁺ mobilization from intracellular stores and insulin release in mouse islets. In beta-TC6 cells, ATP, ADP, 2-MeSADP, and UDP transiently elevated [Ca²⁺]_i and slightly decreased insulin secretion at normal glucose, while UTP and NECA were inactive. RT-PCR analysis detected mRNAs of P2Y₁ and P2Y₆, but not P2Y₂ and P2Y₄ receptors.     

Different ligand affinities of the two human insulin receptor splice variants are reflected in parallel changes in sensitivity for insulin action

The human insulin receptor (hIR) is expressed in two variant forms that are generated by tissue-specific alternative splicing of the 11th exon of the IR gene. This leads to receptors that differ in their affinities for insulin based on the absence (hIR-A) or presence (hIR-B) of a 12-amino acid insert near the C-terminus of the alpha-subunit. To explore further the functional significance of the difference in these receptor subtypes, the properties of hIR-A(exon 11-) and hIR-B(exon 11+) receptors have been compared in parallel. Despite their different affinities for insulin, the receptor variants retain equivalent acid sensitivity for insulin binding and bind proinsulin with the same relative affinity. Both hIR-A and hIR-B are able to signal a variety of insulin's actions, but the insulin dose-response curves for receptor autophosphorylation and for mitogenesis and glycogen synthase stimulation in cells are shifted to the right for hIR-B receptors compared to hIR-A receptors. The magnitude of these rightward shifts, 1.5- to 3-fold in the assays listed above, are similar to and presumably accounted for by the 2-fold difference in insulin affinity exhibited by the receptor variants. Occupied hIR-A and hIR-B receptors undergo indistinguishable endocytotic itineraries after insulin binding. Both lead to insulin degradation that is quantitatively and kinetically similar, and both down-regulate when exposed to saturating insulin for 24 h. Thus, the functional consequences of the alternative splicing of IRs are limited to those related to the variants' differing affinities for insulin.     

Cellular Interactions and the Secondary Response <Emphasis Type="Italic">in vitro</Emphasis>

ALTHOUGH the role of cellular cooperation in the induction of the immune response has become firmly established only recently¹, morphological evidence suggesting that such cooperation takes place is quite old. Reports² of the aggregation of lymphoid cells around macrophages³ have been confirmed: “islets”, “rosettes” or “clusters” in cultures of cells (derived from humans⁴, guinea-pigs⁵, rabbits⁶ or mice⁷) stimulated with antigen or PHA⁸ were reported. We have investigated cluster formation to ascertain its relationship, if any, to the antigen-induced stimulation of sensitized cells⁹. We used peripheral blood leucocytes from rabbits immunized to bovine serum albumin (BSA) or to human red blood cells (HRBC). The BSA was given in complete Freund's adjuvant (three intramuscular injections of 7.5 mg BSA each, into the hind legs at weekly intervals). HRBC (1 ml.) was given once into the ear vein, as a 20% suspension in saline. Cell cultures and ³H-thymidine incorporation were measured as before¹⁰. To prepare cell smears, cells were washed three times and suspended in one drop of normal rabbit serum and 1 µl. of the suspension was spread on a microscope slide. This ensured a reasonably constant number of cells per slide and made possible comparisons between different experiments. Smears were fixed with methanol and stained with Giemsa.     

The effect of phorbol esters on the proliferation of C3H-10T1/2 mouse fibroblasts: Consideration of both stimulatory and inhibitory effects

TPA stimulates cell cycle activation in both serum-deprived and density-inhibited cultures. The cells reestablish cycle arrest after no more than one generation, and addition of fresh drug produces no further response. However, cells freshly trypsinized can respond with a series of repetitive generations resulting in 3.5–4.0 population doublings over 72 hrs. In kinetic pulse experiments TPA enhanced ³H-thymidine incorporation in densityinhibited cells stimulated by fresh serum but only after markedly suppressing incorporation 8–13 hrs after serum stimulation. When cells arrested by serum deprivation were pretreated with TPA, fresh serum stimulation led to initiation of ³H-TdR incorporation 5 hrs earlier than untreated controls. However, TPA addition at the time of serum stimulation did not lead to a suppression at 8–13 hrs, whereas enhancement was observed during peak incorporation times regardless of whether the cells were pretreated with TPA during serum deprivation. The results support the concept that there can exist within G₁ multiple states of responsiveness to phorbol esters. These pharmacologically induced states may be correlated with corresponding physiological states of the G₁ phase of cell cycle.     

Intracellular signaling by a mutant human insulin receptor lacking the carboxyl-terminal tyrosine autophosphorylation sites.

We have recently characterized a mutant insulin receptor (Y/F2) in which the two tyrosines in the carboxyl terminus (Tyr1316, Tyr1322) were mutated to phenylalanine. Compared with wild type receptors, the Y/F2 receptor exhibited markedly enhanced sensitivity to insulin-stimulated DNA synthesis with normal insulin-stimulated glucose uptake (Takata, Y., Webster, N. J. G., and Olefsky, J. M. (1991) J. Biol. Chem. 266, 9135-9139). In this paper, we present further evidence for the divergence of the metabolic and mitogenic signaling pathways utilized by the insulin receptor. The mutant receptor showed normal sensitivity and responsiveness for insulin-stimulated glucose incorporation into glycogen. The insulin sensitivity for phosphorylation of two substrates (pp180 and pp220) was the same in both Y/F2 cells and HIRc cells. Phosphotyrosine content, however, was greater in Y/F2 cells than in HIRc cells, especially in the basal state. Insulin stimulated S6 kinase activity 2-6-fold, with an ED50 of -10 nM in Rat 1 cells and 0.5 nM in HIRc cells. The sensitivity to insulin was enhanced in Y/F2 cells with an ED50 of 0.1 nM. These effects were insulin-specific, since insulin-like growth factor (IGF)-I-stimulated mitogenesis was normal. In summary: 1) Y/F2 receptors exhibit normal metabolic and enhanced mitogenic signaling; 2) the enhanced mitogenic signaling is specific for the insulin receptor in the Y/F2 cells, since IGF-I-stimulated mitogenesis is normal; 3) Y/F2 cells display increased endogenous substrate phosphorylation and augmented insulin-stimulated S6 kinase activity placing these responses among insulin's mitogenic effects; and 4) these results are consistent with the concept that the COOH-terminal tyrosine residues of the insulin receptor are normally inhibitory to mitogenic signaling.     

Glucocorticoids enhance the mitogenic action of insulin in serum-free cultures of chick embryo cells.

Addition of insulin to nonproliferating serum-free cultures of secondary chicken embryo (CE) cells caused a 30% to 50% increase in cell number. Addition of any one of several glucocorticoids (dexamethasone, cortisol, or corticosterone) to the cultures two days before insulin addition increased the mitogenic effect of insulin by about twofold at each insulin concentration tested. This glucocorticoid stimulation of cell proliferation was “permissive” because in the absence of insulin glucocorticoids caused little increase in cell number (usually less than 15%). Glucocorticoids were maximally active at low concentrations (e.g., 10^?10 M dexamethasone). Steroids without glucocorticoid activity were inactive over a wide range of concentrations. Glucocorticoids increased the mitogenic response to insulin largely by increasing the percentage of cells that insulin stimulated to synthesize DNA. The maximum mitogenic effect of insulin upon CE cells rapidly decreased after the cells were serially subcultured. After only nine population doublings (4 passages) in culture, the response to insulin was diminished by about 70%. The mitogenic effect of insulin plus dexamethasone declined similarly during serial subculture, and was always about twofold greater than the effect of insulin alone. The cells maintained their mitogenic responsiveness to serum as these responses decreased. In contrast to the growth promoting influence of glucocorticoids in the presence of insulin, glucocorticoids inhibited the mitogenic response of CE cells to serum. This result may resolve our above findings with reports that glucocorticoids inhibit the proliferation of CE cells.     

Dietary Protein-induced Changes of the Erythropoietin Level in Rat Serum

Erythropoietin, a glycoprotein, is the primary regulator of erythropoiesis. The most convenient and sensitive assay for active erythropoietin is to measure its stimulatory effect on in vitro ³H-thymidine incorporation into DNA of erythropoietin-responsive cells. An attempt with this method to estimate the erythropoietin level in rat serum, however, was unsuccesful because of the presence of inhibitory substance(s) and non-erythropoietic factor(s) stimulating ³H-thymidine incorporation. Pretreatment of the serum by heating, extraction of erythropoietin from denatured-protein aggregates, and subsequent concentration of erythropoietin in the extract with alcohol precipitation made it possible to measure the serum erythropoietin levels. Rabbit anti-erythropoietin antibody was used for a quantitative estimation of erythropoietin in the concentrated extracts. Erythropoietin levels in sera of rats fed on varied amounts of casein for 7 days were measured with these procedures to find if the impairment of erythropoiesis upon protein deprivation was due to changes in the erythropoietin level. We found that the level in protein-deprived rats was less than 1/8 that of 20% casein-fed rats, a level undetectable by the present assay, and that the serum erythropoietin increased as the protein content in the diet was increased up to 20%, then leveled off. The erythropoietin in serum decreased rapidly after protein deprivation; the level at 12hr after deprivation began was about 1/5 that in 20% casein-fed rats. Thus, the depression of erythropoiesis upon protein deprivation is primarily caused by the lowered level of erythropoietin.     

Tyrosine phosphorylation of phosphoinositide-dependent kinase 1 by the insulin receptor is necessary for insulin metabolic signaling

In L6 myoblasts, insulin receptors with deletion of the C-terminal 43 amino acids (IR(Delta43)) exhibited normal autophosphorylation and IRS-1/2 tyrosine phosphorylation. The L6 cells expressing IR(Delta43) (L6(IRDelta43)) also showed no insulin effect on glucose uptake and glycogen synthase, accompanied by a >80% decrease in insulin induction of 3-phosphoinositide-dependent protein kinase 1 (PDK-1) activity and tyrosine phosphorylation and of protein kinase B (PKB) phosphorylation at Thr(308). Insulin induced the phosphatidylinositol 3 kinase-dependent coprecipitation of PDK-1 with wild-type IR (IR(WT)), but not IR(Delta43). Based on overlay blotting, PDK-1 directly bound IR(WT), but not IR(Delta43). Insulin-activated IR(WT), and not IR(Delta43), phosphorylated PDK-1 at tyrosines 9, 373, and 376. The IR C-terminal 43-amino-acid peptide (C-terminal peptide) inhibited in vitro PDK-1 tyrosine phosphorylation by the IR. Tyr-->Phe substitution prevented this inhibitory action. In the L6(hIR) cells, the C-terminal peptide coprecipitated with PDK-1 in an insulin-stimulated fashion. This peptide simultaneously impaired the insulin effect on PDK-1 coprecipitation with IR(WT), on PDK-1 tyrosine phosphorylation, on PKB phosphorylation at Thr(308), and on glucose uptake. Upon insulin exposure, PDK-1 membrane persistence was significantly reduced in L6(IRDelta43) compared to control cells. In L6 cells expressing IR(WT), the C-terminal peptide also impaired insulin-dependent PDK-1 membrane persistence. Thus, PDK-1 directly binds to the insulin receptor, followed by PDK-1 activation and insulin metabolic effects.     

Glucose regulates insulin mitogenic effect by modulating SHP-2 activation and localization in JAr cells

The glucose effect on cell growth has been investigated in the JAr human choriocarcinoma cells. When JAr cells were cultured in the presence of 6 mm glucose (LG), proliferation and thymidine incorporation were induced by serum, epidermal growth factor, and insulin-like growth factor 1 but not by insulin. In contrast, at 25 mm glucose (HG), proliferation and thymidine incorporation were stimulated by insulin, serum, epidermal growth factor, and insulin-like growth factor 1 to a comparable extent, whereas basal levels were 25% lower than those in LG. HG culturing also enhanced insulin-stimulated insulin receptor and insulin receptor substrate 1 (IRS1) tyrosine phosphorylations while decreasing basal phosphorylations. These actions of glucose were accompanied by an increase in cellular tyrosine phosphatase activity. The activity of SHP-2 in HG-treated JAr cells was 400% of that measured in LG-treated cells. SHP-2 co-precipitation with IRS1 was also increased in HG-treated cells. SHP-2 was mainly cytosolic in LG-treated cells. However, HG culturing largely redistributed SHP-2 to the internal membrane compartment, where tyrosine-phosphorylated IRS1 predominantly localizes. Further exposure to insulin rescued SHP-2 cytosolic localization, thereby preventing its interaction with IRS1. Antisense inhibition of SHP-2 reverted the effect of HG on basal and insulin-stimulated insulin receptor and IRS1 phosphorylation as well as that on thymidine incorporation. Thus, in JAr cells, glucose modulates insulin mitogenic action by modulating SHP-2 activity and intracellular localization.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C,and <Superscript>15</Superscript>N backbone chemical shift assignments of 4E-BP1<Subscript>44–87</Subscript> and 4E-BP1<Subscript>44–87</Subscript> bound to eIF4E

The eukaryotic translational initiation factor 4G (eIF4G) interacts with the cap-binding protein eIF4E through a consensus binding motif, Y(X)₄LΦ (where X is any amino acid and Φ is a hydrophobic residue). 4E binding proteins (4E-BPs), which also contain a Y(X)₄LΦ motif, regulate the eIF4E/eIF4G interaction. The non- or minimally-phosphorylated form of 4E-BP1 binds eIF4E, preventing eIF4E from interacting with eIF4G, thus inhibiting translation initiation. 4EGI-1, a small molecule inhibitor of the eIF4E/eIF4G interaction that is under investigation as a novel anti-cancer drug, has a dual activity; it disrupts the eIF4E/eIF4G interaction and stabilizes the binding of 4E-BP1 to eIF4E. Here, we report the complete backbone NMR resonance assignment of an unliganded 4E-BP1 fragment (4E-BP1_44–87). We also report the near complete backbone assignment of the same fragment in complex to eIF4E/m⁷GTP (excluding the assignment of the last C-terminus residue, D87). The chemical shift data constitute a prerequisite to understanding the mechanism of action of translation initiation inhibitors, including 4EGI-1, that modulate the eIF4E/4E-BP1 interaction.