Insulin and insulin-like growth factor-1 binding specificity is determined by distinct regions of their cognate receptors.

Chimeric insulin/insulin-like growth factor-1 receptors and insulin receptor alpha-subunit point mutants were characterized with respect to their binding properties for insulin and insulin-like growth factor-1 (IGF-1) and their ability to translate ligand interaction into tyrosine kinase activation in intact cells. We found that replacement of the amino-terminal 137 amino acids of the insulin receptor (IR) with the corresponding 131 amino acids of the IGF-1 receptor (IGF-1R) resulted in loss of affinity for both ligands. Further replacement of the adjacent cysteine region with IGF-1R sequences fully reconstituted affinity for IGF-1, but only marginally for insulin. Unexpectedly, replacement of the IR cysteine-rich domain alone by IGF-1R sequences created a high affinity receptor for both insulin and IGF-1. The binding characteristics of all receptor chimeras reflected the potential of both ligands to regulate the receptor tyrosine kinase activity in intact cells. Our chimeric receptor data, in conjunction with IR amino-terminal domain point mutants, strongly suggest major contributions of structural determinants in both amino- and carboxyl-terminal IR alpha-subunit regions for the formation of the insulin-binding pocket, whereas, surprisingly, the residues defining IGF-1 binding are present predominantly in the cysteine-rich domain of the IGF-1R.     

Regulation of vascular endothelial growth factor-dependent retinal neovascularization by insulin-like growth factor-1 receptor

Although insulin-like growth factor 1 (IGF-1) has been associated with retinopathy, proof of a direct relationship has been lacking. Here we show that an IGF-1 receptor antagonist suppresses retinal neovascularization in vivo, and infer that interactions between IGF-1 and the IGF-1 receptor are necessary for induction of maximal neovascularization by vascular endothelial growth factor (VEGF). IGF-1 receptor regulation of VEGF action is mediated at least in part through control of VEGF activation of p44/42 mitogen-activated protein kinase, establishing a hierarchical relationship between IGF-1 and VEGF receptors. These findings establish an essential role for IGF-1 in angiogenesis and demonstrate a new target for control of retinopathy. They also explain why diabetic retinopathy initially increases with the onset of insulin treatment. IGF-1 levels, low in untreated diabetes, rise with insulin therapy, permitting VEGF-induced retinopathy.     

Mediation of the actions of insulin and insulin-like growth factor-1 on preimplantation mouse embryos in vitro.

Previous studies showed that both insulin and insulin-like growth factor-1 (IGF-1) stimulate metabolism and growth of preimplantation embryos. Because the effects of insulin occur with very low doses, it was suggested that its effects were mediated by its own receptors. However, the effects of IGF-1 occurred at higher doses, suggestive of cross reaction with the insulin receptor but still in the range for mediation via its own receptor. The aim of this study was to investigate the mediation of the metabolic and growth effects of insulin and IGF-1 using a specific insulin receptor antagonist. The antagonistic B-10 Fab fragment (B-10f) completely blocked stimulation of protein synthesis by both insulin and IGF-1, indicating that the insulin receptor mediates this action of both hormones. Alternately, only insulin's stimulation of inner cell mass mitogenesis and morphological development was inhibited by the B-10 Fab fragment. This showed that growth stimulation by insulin and IGF-1 was mediated via different receptors, insulin through its own receptor and IGF-1 through some other receptor. However, mediation via the IGF-2 receptor is not excluded since IGF-1 stimulates compaction when there is evidence for only the presence of the IGF-2 receptor. In summary, insulin or IGF-1 at physiological concentrations stimulates preimplantation mouse embryos, suggesting an important role for both these growth factors in early development.     

Modelling of the disulphide-swapped isomer of human insulin-like growth factor-1: implications for receptor binding.

Insulin-like growth factor-1 (IGF-1) is a serum protein which unexpectedly folds to yield two stable tertiary structures with different disulphide connectivities; native IGF-1 [18-61,6-48,47-52] and IGF-1 swap [18-61,6-47, 48-52]. Here we demonstrate in detail the biological properties of recombinant human native IGF-1 and IGF-1 swap secreted from Saccharomyces cerevisiae. IGF-1 swap had a approximately 30 fold loss in affinity for the IGF-1 receptor overexpressed on BHK cells compared with native IGF-1.The parallel increase in dose required to induce negative cooperativity together with the parallel loss in mitogenicity in NIH 3T3 cells implies that disruption of the IGF-1 receptor binding interaction rather than restriction of a post-binding conformational change is responsible for the reduction in biological activity of IGF-1 swap. Interestingly, the affinity of IGF-1 swap for the insulin receptor was approximately 200 fold lower than that of native IGF-1 indicating that the binding surface complementary to the insulin receptor (or the ability to attain it) is disturbed to a greater extent than that to the IGF-1 receptor. A 1.0 ns high-temperature molecular dynamics study of the local energy landscape of IGF-1 swap resulted in uncoiling of the first A-region alpha-helix and a rearrangement in the relative orientation of the A- and B-regions. The model of IGF-1 swap is structurally homologous to the NMR structure of insulin swap and CD spectra consistent with the model are presented. However, in the model of IGF-1 swap the C-region has filled the space where the first A-region alpha-helix has uncoiled and this may be hindering interaction of Val44 with the second insulin receptor binding pocket.     

Specific binding of insulin-like growth factors 1 and 2 to the type 1 and type 2 receptors respectively.

1. Competitive binding and receptor cross-linking experiments have been used to examine the receptor-ligand interactions between three bovine insulin-like growth factors (IGF) and monolayer cultures of myoblasts and fibroblasts. 2. Labelled IGF-2 bound predominantly to the type 2 receptor with negligible label cross-linked to the type 1 receptor, notwithstanding the ability of IGF-2 to compete effectively for the binding of IGF-1 to the type 1 receptor. Approx. 100-fold higher concentrations of IGF-1 or the N-terminal truncated (des-Gly-Pro-Glu) IGF-1 (-3N:IGF-1) were required to produce competition equivalent to IGF-2. 3. All IGF peptides, but especially IGF-1, enhanced the binding of labelled IGF-2 to the type 2 receptor of lung fibroblasts. This unusual effect was probably a consequence of the displacement of labelled IGF-2 otherwise bound to a medium protein, a conclusion supported by the demonstration of a 38 kDa membrane protein cross-linked to labelled IGF-2. 4. Both IGF-1 and -3N:IGF-1 bound only to the type 1 IGF receptor in L6 myoblasts, rat vascular smooth-muscle cells and human lung fibroblasts. The peptides competed for labelled IGF-1 binding with potencies in the order -3N:IGF-1 greater than IGF-1 greater than IGF-2 much greater than insulin. Since the IGF peptides were equipotent in skin fibroblasts, it was proposed that the apparently higher affinity of -3N:IGF-1 for receptors in the other cell types was instead a consequence of a low affinity of this peptide for the competing 38 kDa binding protein.     

The insulin-like growth factor type 1 and insulin-like growth factor type 2/mannose-6-phosphate receptors independently regulate ERK1/2 activity in HEK293 cells

Insulin-like growth factor types 1 and 2 (IGF-1; IGF-2) and insulin-like peptides are all members of the insulin superfamily of peptide hormones but bind to several distinct classes of membrane receptor. Like the insulin receptor, the IGF-1 receptor is a heterotetrameric receptor tyrosine kinase, whereas the IGF-2/ mannose 6-phosphate receptor is a single transmembrane domain protein that is thought to function primarily as clearance receptors. We recently reported that IGF-1 and IGF-2 stimulate the ERK1/2 cascade by triggering sphingosine kinase-dependent "transactivation" of G protein-coupled sphingosine-1-phosphate receptors. To determine which IGF receptors mediate this effect, we tested seven insulin family peptides, IGF-1, IGF-2, insulin, and insulin-like peptides 3, 4, 6, and 7, for the ability to activate ERK1/2 in HEK293 cells. Only IGF-1 and IGF-2 potently activated ERK1/2. Although IGF-2 was predictably less potent than IGF-1 in activating the IGF-1 receptor, they were equipotent stimulators of ERK1/2. Knockdown of IGF-1 receptor expression by RNA interference reduced the IGF-1 response to a greater extent than the IGF-2 response, suggesting that IGF-2 did not signal exclusively via the IGF-1 receptor. In contrast, IGF-2 receptor knockdown markedly reduced IGF-2-stimulated ERK1/2 phosphorylation, with no effect on the IGF-1 response. As observed previously, both the IGF-1 and the IGF-2 responses were sensitive to pertussis toxin and the sphingosine kinase inhibitor, dimethylsphingosine. These data indicate that endogenous IGF-1 and IGF-2 receptors can independently initiate ERK1/2 signaling and point to a potential physiologic role for IGF-2 receptors in the cellular response to IGF-2.     

Activation of the insulin receptor (IR) by insulin and a synthetic peptide has different effects on gene expression in IR-transfected L6 myoblasts

Single-chain peptides have been recently produced that display either mimetic or antagonistic properties against the insulin and IGF-1 (insulin-like growth factor 1) receptors. We have shown previously that the insulin mimetic peptide S597 leads to significant differences in receptor activation and initiation of downstream signalling cascades despite similar binding affinity and in vivo hypoglycaemic potency. It is still unclear how two ligands can initiate different signalling responses through the IR (insulin receptor). To investigate further how the activation of the IR by insulin and S597 differentially activates post-receptor signalling, we studied the gene expression profile in response to IR activation by either insulin or S597 using microarray technology. We found striking differences between the patterns induced by these two ligands. Most remarkable was that almost half of the genes differentially regulated by insulin and S597 were involved in cell proliferation and growth. Insulin either selectively regulated the expression of these genes or was a more potent regulator. Furthermore, we found that half of the differentially regulated genes interact with the genes involved with the MAPK (mitogen-activated protein kinase) pathway. These findings support our signalling results obtained previously and confirm that the main difference between S597 and insulin stimulation resides in the activation of the MAPK pathway. In conclusion, we show that insulin and S597 acting via the same receptor differentially affect gene expression in cells, resulting in a different mitogenicity of the two ligands, a finding which has critical therapeutic implications.     

The insulin-like growth factor 1 (IGF-1) receptor is responsible for mediating the effects of insulin, IGF-1, and IGF-2 in Xenopus laevis oocytes.

Competitive hormone binding studies with membrane and partially purified receptors from Xenopus laevis oocytes revealed that the oocyte possesses high affinity (KD = 1-3 nM) binding sites for both insulin growth factors 1 and 2 (IGF-1 and IGF-2), but not for insulin. Consistent with these findings, IGF-1 activates hexose uptake by Xenopus oocytes with a KA (3 nM) identical with its KD, while IGF-2 and insulin activate hexose uptake with KA values of 50 nM and 200-250 nM, respectively, suggesting activation mediated through an IGF-1 receptor. Both IGF-1 and insulin activate receptor beta-subunit autophosphorylation and, thereby, protein substrate (reduced and carboxyamidomethylated lysozyme, i.e. RCAM-lysozyme) phosphorylation with KA values comparable to their respective KD values for ligand binding and KA values for activation of hexose uptake. The autophosphorylated beta-subunit(s) of the receptor were resolved into two discrete components, beta 1 and beta 2 (108 kDa and 94 kDa, respectively), which were phosphorylated exclusively on tyrosine and which exhibited similar extents of IGF-1-activated autophosphorylation. When added prior to autophosphorylation, RCAM-lysozyme blocks IGF-1-activated autophosphorylation and, thereby, IGF-1-activated protein substrate (RCAM-lysozyme) phosphorylation. Based on these findings, we conclude that IGF-1-stimulated autophosphorylation of its receptor is a prerequisite for catalysis of protein substrate phosphorylation by the receptor's tyrosine-specific protein kinase. The IGF-1 receptor kinase is implicated in signal transmission from the receptor, since anti-tyrosine kinase domain antibody blocks IGF-1-stimulated kinase activity in vitro and, when microinjected into intact oocytes, prevents IGF-1-stimulated hexose uptake.     

Binding and action of insulin-like growth factors and insulin in bovine luteal tissue during the oestrous cycle.

The effect of insulin-like growth factors (IGFs) and insulin on the release of progesterone and oxytocin from bovine corpus luteum was investigated at early (days 5-7), mid- (days 8-12) and late (days 15-18) luteal phases of the oestrous cycle in an in vitro microdialysis system. The expression of specific receptors was evaluated in bovine corpora lutea of the respective luteal stages. A 30 min infusion of IGF-1, IGF-2 (1.3, 13 and 130 nmol l-1) or insulin (13, 130 and 1300 nmol l-1) caused a stimulation of the release of progesterone (P < 0.05). IGF-1 was most effective in releasing progesterone. Oxytocin release from corpora lutea was stimulated by insulin at all doses tested (13-1300 nmol l-1), whereas the IGFs were only effective at the highest dose (130 nmol l-1) applied. The high doses of IGFs (130 nmol l-1) and insulin (1300 nmol l-1) stimulated the release of progesterone and oxytocin throughout the luteal phase (P < 0.05). For all three peptides, greatest stimulation was seen during the late luteal phase (days 15-18 of the oestrous cycle) with the peak of progesterone release directly related to peptide infusion (P < 0.05). In addition, IGF-1 stimulated total release of progesterone (units in 4 h) after the beginning of the stimulation during this phase (P < 0.05). IGF-1 caused a gradual increase of progesterone even beyond the time of peptide perfusion, whereas IGF-2 and insulin stimulated progesterone release only during the peptide perfusion. Distinct receptors for IGF-1 and IGF-2 were present in corpora lutea membrane preparations at all stages investigated. Specific binding for insulin was also seen in all stages of the cycle without any cycle-dependent changes in the amount of binding. The displacement of labelled insulin by unlabelled IGF-1 and IGF-2 did not show the rank of order that has been described as typical for insulin receptors (i.e. insulin > IGF-1 > IGF-2), but comparable binding affinities were observed for the three unlabelled ligands. Specific binding of IGF-2 was markedly higher than that of IGF-1 or insulin throughout the cycle (1.9- and 4.9-fold higher compared with IGF-1 and insulin, respectively). Receptor specificity did not change during luteal development. Binding affinity and capacity of IGF-1 receptor was constant throughout the oestrous cycle. Specific IGF-2 binding increased and showed a positive co-operativity towards the end of the cycle. Specific binding of insulin was not significantly different in the three luteal stages examined.     

Structural determinants for high-affinity binding of insulin-like growth factor II to insulin receptor (IR)-A, the exon 11 minus isoform of the IR

The insulin receptor (IR) lacking the alternatively spliced exon 11 (IR-A) is preferentially expressed in fetal and cancer cells. The IR-A has been identified as a high-affinity receptor for insulin and IGF-II but not IGF-I, which it binds with substantially lower affinity. Several cancer cell types that express the IR-A also overexpress IGF-II, suggesting a possible autocrine proliferative loop. To determine the regions of IGF-I and IGF-II responsible for this differential affinity, chimeras were made where the C and D domains were exchanged between IGF-I and IGF-II either singly or together. The abilities of these chimeras to bind to, and activate, the IR-A were investigated. We also investigated the ability of these chimeras to bind and activate the IR exon 11+ isoform (IR-B) and as a positive control, the IGF-I receptor (IGF-1R). We show that the C domain and, to a lesser extent, the D domains represent the principal determinants of the binding differences between IGF-I and IGF-II to IR-A. The C and D domains of IGF-II promote higher affinity binding to the IR-A than the equivalent domains of IGF-I, resulting in an affinity close to that of insulin for the IR-A. The C and D domains also regulate the IR-B binding specificity of the IGFs in a similar manner, although the level of binding for all IGF ligands to IR-B is lower than to IR-A. In contrast, the C and D domains of IGF-I allow higher affinity binding to the IGF-1R than the analogous domains of IGF-II. Activation of IGF-1R by the chimeras reflected their binding affinities whereas the phosphorylation of the two IR isoforms was more complex.     

胰岛素受体家族的结构与功能研究

胰岛素（insulin）与胰岛素样生长因子-1（IGF-1）分别是由胰岛β细胞和肝细胞分泌的 多肽类激素.它们通过结合并激活位于细胞膜上的受体酪氨酸激酶(RTKs),发挥重要的生理作用. 作为起始信号传导的第一步,胰岛素与IGF-1是如何与各自受体的膜外区域（ectodomain） 结合并进一步激活受体的细胞膜内酪氨酸激酶活性一直属于科学研究的关键基础问题.本文 概述了胰岛素受体家族（IR和IGF-1R）及其配体的结构与功能的特点和关系,并重点介绍 了近年来国内外在胰岛素受体家族复合体结构和功能上的研究手段和取得的突破性进展.     

Coupling of insulin-responsive glucose transport to receptors for insulin-like growth factor 1 in primary human fibroblasts

We have recently described an insulin-resistant patient with leprechaunism (leprechaun G.) having a homozygous leucine----proline mutation at amino acid position 233 in the alpha-chain of the insulin receptor. The mutation results in a loss of insulin binding to cultured fibroblasts. Fibroblasts from the patient and control individuals were used to quantify the stimulation of 2-deoxyglucose uptake by insulin and insulin-like growth factor 1 (IGF-1). Insulin hardly stimulates basal 2-deoxyglucose uptake in the patient's fibroblasts whereas in control fibroblasts the uptake of 2-deoxyglucose is stimulated by insulin approximately 1.7 times. In contrast, IGF-1 stimulates hexose uptake in the patient's fibroblasts 1.8 times, a similar value to that obtained by stimulation of control fibroblasts with insulin or IGF-1. With both types of fibroblasts, maximal IGF-1 response is reached at about 10 nM IGF-1, the ED50 being approximately 4 nM. The results indicate that the insulin responsive glucose transport in primary fibroblasts is functionally linked to the receptor for IGF-1. Insulin binds with an approximately 200-fold lower affinity to IGF-1 receptors, compared to homologous IGF-1 binding. As an insulin concentration of 10 microM is unable to give maximal stimulation of glucose uptake in the patient's fibroblasts, which is already seen with 10 nM IGF-1, it seems that occupation of IGF-1 receptors by insulin on the patient's cells is less efficient at stimulating hexose uptake compared to homologous activation.     

Insulin receptor structure and its implications for the IGF-1 receptor

The insulin receptor (isoforms IR-A and IR-B) and the type-I insulin-like growth factor receptor (IGF-1R) are homologous, multi-domain tyrosine kinases that bind insulin and IGF-1 with differing specificity. IR is involved in metabolic regulation and IGF-1R in normal growth and development. IR-A also binds IGF-2 with an affinity comparable to IGF-1R and, like the latter, is implicated in a range of cancers. The recent structure of the IR ectodomain dimer explains many features of ligand-receptor binding and provides insight into the structure of the intact ligand-binding site in both receptors. The structures of the L1-CR-L2 fragments of IR and IGF-1R reveal major differences in the regions that govern ligand specificity. The IR ectodomain X-ray structure raises doubts about that obtained by STEM reconstruction.     

Insulin-like growth factor II (IGF-II). Its role among regulatory peptides of the insulin superfamily

The review presents data on the insulin-like growth factor-II (IGF-II), a regulatory peptide included in the insulin superfamily, as its structure and function are the closest to those of insulin and IGF-I. The last decade investigations revealed the biological properties of IGF-II which distinguish it from related peptides. The primary sequence of the IGF-II structure has peculiar differences from those of insulin but insignificant ones from IGF-I. The tertiary structure of IGF-II is similar to that of the related peptide molecules, but a peculiar receptor-binding domain in the IGF-II molecule provides for its unique capability of interacting with receptors. IGF-II interacts with three types of receptors: receptors of IGF-I, IGF-2, and insulin. IGF-II has the highest affinity to IGF-2 receptors but its mitogenic effects are mediated by IGF-I receptors (i.e., the phenomenon of divergence of binding and biological activities). The arguments obtainedin vitro andin vivo are presented, which confirm propagation of mitogenic effects by IGF-I receptors but deny participation of IGF-2 receptors. The structural and functional bivalency of the M6P/IGF-2 receptor (a peculiar form of the M6P receptor in mammals) is considered in detail. The results of interactions of IGF-II and the M6P/IGF-2 receptors are not yet known. The primary function of the M6P/IGF-2 receptor (sorting and transport of the lysosomal enzymes) is likely to be due to the peptides inactivation and does not imply its participation in the IGF-II signaling. However, several data do not permit ruling out participation of the IGF-2 receptor in the IGF-II effects different from mitogenic ones. The organization of related peptide gene in the lancelet allows us to suggest the appearance of the IGF-II gene at the initial steps of the vertebrate evolution and to trace all stages of formation of two separate IGF genes up to the mammalian IGF-II and IGF-I genes with different structural organizations. The IGF-II expression by embryonic tissues is revealed earlier than that of other related peptides and reaches the highest level at the embryonal period. The general regularities of the IGF-II regulatory activity in embryogenesis and of the growth hormone effect on the IGF-II expression in embryonal tissues are considered.     

An investigation of the ligand binding properties and negative cooperativity of soluble insulin-like growth factor receptors

To investigate the interaction of the insulin-like growth factor (IGF) ligands with the insulin-like growth factor type 1 receptor (IGF-1R), we have generated two soluble variants of the IGF-1R. We have recombinantly expressed the ectodomain of IGF-1R or fused this domain to the constant domain from the Fc fragment of mouse immunoglobulin. The ligand binding properties of these soluble IGF-1Rs for IGF-I and IGF-II were investigated using conventional ligand competition assays and BIAcore biosensor technology. In ligand competition assays, the soluble IGF-1Rs both bound IGF-I with similar affinities and a 5-fold lower affinity than that seen for the wild type receptor. In addition, both soluble receptors bound IGF-II with similar affinities to the wild type receptor. BIAcore analyses showed that both soluble IGF-1Rs exhibited similar ligand-specific association and dissociation rates for IGF-I and for IGF-II. The soluble IGF-1R proteins both exhibited negative cooperativity for IGF-I, IGF-II, and the 24-60 antibody, which binds to the IGF-1R cysteine-rich domain. We conclude that the addition of the self-associating Fc domain to the IGF-1R ectodomain does not affect ligand binding affinity, which is in contrast to the soluble ectodomain of the IR. This study highlights some significant differences in ligand binding modes between the IGF-1R and the insulin receptor, which may ultimately contribute to the different biological activities conferred by the two receptors.     

pI-shifted insulin analogs with extended in vivo time action and favorable receptor selectivity

A long-acting (basal) insulin capable of delivering flat, sustained, reproducible glycemic control with once daily administration represents an improvement in the treatment paradigm for both type 1 and type 2 diabetes. Optimization of insulin pharmacodynamics is achievable through structural modification, but often at the expense of alterations in receptor affinity and selectivity. A series of isoelectric point (pI)-shifted insulin analogs based on the human insulin sequence or the GlyA21 acid stable variant were prepared by semi-synthetic methods. The pI shift was achieved through systematic addition of one or more arginine (Arg) or lysine (Lys) residues at the N terminus of the A chain, the N terminus of the B chain, the C terminus of the B chain, or through a combination of additions at two of the three sites. The analogs were evaluated for their affinity for the insulin and IGF-1 receptors, and aqueous solubility under physiological pH conditions. Notably, the presence of positively charged amino acid residues at the N terminus of the A chain was consistently associated with an enhanced insulin to IGF-1 receptor selectivity profile. Increased IGF-1 receptor affinity that results from Arg addition to the C terminus of the B chain was attenuated by cationic extension at the N terminus of the A chain. Analogs 10, 17, and 18 displayed in vitro receptor selectivity similar to that of native insulin and solubility at physiological pH that suggested the potential for extended time action. Accordingly, the in vivo pharmacokinetic and pharmacodynamic profiles of these analogs were established in a somatostatin-induced diabetic dog model. Analog 18 (A0:Arg, A21:Gly, B31:Arg, B32:Arg human insulin) exhibited a pharmacological profile comparable to that of analog 15 (insulin glargine) but with a 4.5-fold more favorable insulin:IGF-1 receptor selectivity. These results demonstrate that the selective combination of positive charge to the N terminus of the A chain and the C terminus of the B chain generates an insulin with sustained pharmacology and a near-native receptor selectivity profile.     

A panel of monoclonal antibodies for the type I insulin-like growth factor receptor. Epitope mapping, effects on ligand binding, and biological activity.

We obtained 20 mouse monoclonal antibodies specific for human type I insulin-like growth factor (IGF) receptors, using transfected cells expressing high levels of receptors (IGF-1R/3T3 cells) as immunogen. The antibodies immunoprecipitated receptor.125I-IGF-I complexes and biosynthetically labeled receptors from IGF-1R/3T3 cells but did not react with human insulin receptors or rat type I IGF receptors. Several antibodies stimulated DNA synthesis in IGF-1R/3T3 cells, but the maximum stimulation was only 25% of that produced by IGF-I. The antibodies fell into seven groups recognizing distinct epitopes and with different effects on receptor function. All the antibodies reacted with the extracellular portion of the receptor, and epitopes were localized to specific domains by investigating their reaction with a series of chimeric IGF/insulin receptor constructs. Binding of IGF-I was inhibited up to 90% by antibody 24-60 reacting in the region 184-283, and by antibody 24-57 reacting in the region 440-586. IGF-I binding was stimulated up to 2.5-fold by antibodies 4-52 and 16-13 reacting in the region 62-184, and by antibody 26-3 reacting downstream of 283. The latter two groups of antibodies also dramatically stimulated insulin binding to intact IGF-1R/3T3 cells (by up to 50-fold), and potentiated insulin stimulation of DNA synthesis. Scatchard analysis indicated that in the presence of these antibodies, the affinity of the type I IGF receptor for insulin was comparable with that of the insulin receptor. These data indicate that regions both within and outside the cysteine-rich domain of the receptor alpha-subunit are important in determining the affinity and specificity of ligand binding. These antibodies promise to be valuable tools in resolving issues of IGF-I receptor heterogeneity and in studying the structure and function of classical type I receptors and insulin/IGF receptor hybrids.     

Seguridad de los análogos de insulina: qué evaluar,cómo hacerlo y cómo interpretar los resultados

The widespread use of insulin analogues is based not only on the pharmacokinetics of these preparations, which is much closer to the physiology of insulin secretion under normal conditions, but also on their safety and effectiveness. The publication of a possible association between the use of a long-acting insulin analogue (glargine) and breast cancer has caused uneasiness among the medical community regarding the safety of these analogues.The mechanism of increased tumor activity of insulin analogues is explained by the fact that they act through insulin receptors (IR) and insulin-like growth factor-1 (IGF-1R), stimulating cell growth and inhibiting apoptosis. There are two major mechanisms: an increase in the binding time of insulin to IR and increased activation of IGF-1R. Therefore, to evaluate the safety of an analogue, the slower dissociation rate from its insulin receptor must be excluded, as well as the increased affinity for the IGF-1 receptor. This is equivalent to an index of mitogenic/metabolic activity of less than 1. These aspects can only be evaluated through study of cell lines and animal testing, which are reductionist models that cannot always be extrapolated to humans. To date, there are no data to question the safety of insulin analogues in general. However, the results of observational studies and some in vitro studies, suggesting a potential risk of mitogenicity with the administration of glargine, have caused some alarm among the medical community. Until now, there are no data to refute or confirm this risk and, therefore, evaluation of the existing data is crucial to obtain objective information.     

Disruption of the insulin-like growth factor type 1 receptor in osteoblasts enhances insulin signaling and action

Defective bone formation is common in patients with diabetes, suggesting that insulin normally exerts anabolic actions in bone. However, because insulin can cross-activate the insulin-like growth factor type 1 receptor (IGF-1R), which also functions in bone, it has been difficult to establish the direct (IGF-1-independent) actions of insulin in osteoblasts. To overcome this problem, we examined insulin signaling and action in primary osteoblasts engineered for conditional disruption of the IGF-1 receptor (DeltaIGF-1R). Calvarial osteoblasts from mice carrying floxed IGF-1R alleles were infected with adenoviral vectors expressing the Cre recombinase (Ad-Cre) or green fluorescent protein (Ad-GFP) as control. Disruption of IGF-1R mRNA (>90%) eliminated IGF-1R without affecting insulin receptor (IR) mRNA and protein expression and eliminated IGF-1R/IR hybrids. In DeltaIGF-1R osteoblasts, insulin signaling was markedly increased as evidenced by increased phosphorylation of insulin receptor substrate 1/2 and enhanced ERK/Akt activation. Microarray analysis of RNA samples from insulin-treated, DeltaIGF-1R osteoblasts revealed striking changes in several genes known to be downstream of ERK including Glut-1 and c-fos. Treatment of osteoblasts with insulin induced Glut-1 mRNA, increased 2-[1,2-(3)H]-deoxy-d-glucose uptake, and enhanced proliferation. Moreover, insulin treatment rescued the defective differentiation and mineralization of DeltaIGF-1R osteoblasts, suggesting that IR signaling can compensate, at least in part, for loss of IGF-1R signaling. We conclude that insulin exerts direct anabolic actions in osteoblasts by activation of its cognate receptor and that the strength of insulin-generated signals is tempered through interactions with the IGF-1R.     

Insulin-like growth factors mediate heterotrimeric G protein-dependent ERK1/2 activation by transactivating sphingosine 1-phosphate receptors

Although several studies have shown that a subset of insulin-like growth factor (IGF) signals require the activation of heterotrimeric G proteins, the molecular mechanisms underlying IGF-stimulated G protein signaling remain poorly understood. Here, we have studied the mechanism by which endogenous IGF receptors activate the ERK1/2 mitogen-activated protein kinase cascade in HEK293 cells. In these cells, treatment with pertussis toxin and expression of a Galpha(q/11)-(305-359) peptide that inhibits G(q/11) signaling additively inhibited IGF-stimulated ERK1/2 activation, indicating that the signal was almost completely G protein-dependent. Treatment with IGF-1 or IGF-2 promoted translocation of green fluorescent protein (GFP)-tagged sphingosine kinase (SK) 1 from the cytosol to the plasma membrane, increased endogenous SK activity within 30 s of stimulation, and caused a statistically significant increase in intracellular and extracellular sphingosine 1-phosphate (S1P) concentration. Using a GFP-tagged S1P1 receptor as a biological sensor for the generation of physiologically relevant S1P levels, we found that IGF-1 and IGF-2 induced GFP-S1P receptor internalization and that the effect was blocked by pretreatment with the SK inhibitor, dimethylsphingosine. Treating cells with dimethylsphingosine, silencing SK1 expression by RNA interference, and blocking endogenous S1P receptors with the competitive antagonist VPC23019 all significantly inhibited IGF-stimulated ERK1/2 activation, suggesting that IGFs elicit G protein-dependent ERK1/2 activation by stimulating SK1-dependent transactivation of S1P receptors. Given the ubiquity of SK and S1P receptor expression, S1P receptor transactivation may represent a general mechanism for G protein-dependent signaling by non-G protein-coupled receptors.