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P De Meyts

Publications and source records attributed to P De Meyts.

At least 19 recordsLinked to original sources
PubMed2002

An arginine to cysteine(252) mutation in insulin receptors from a patient with severe insulin resistance inhibits receptor internalisation but preserves signalling events.

AIMS/HYPOTHESIS: We examined the properties of a mutant insulin receptor (IR) with an Arg(252) to Cys (IR(R252C)) substitution in the alpha-subunit originally identified in a patient with extreme insulin resistance and acanthosis nigricans. METHODS: We studied IR cell biology and signalling pathways in Chinese Hamster Ovary cells overexpressing this IR(R252C). RESULTS: Our investigation showed an impairment in insulin binding to IR(R252C) related mostly to a reduced affinity of the receptor for insulin and to a reduced rate of IR(R252C) maturation; an inhibition of IR(R252C)-mediated endocytosis resulting in a decreased insulin degradation and insulin-induced receptor down-regulation; a maintenance of IR(R252C) on microvilli even in the presence of insulin; a similar autophosphorylation of mutant IR(R252C) followed by IRS 1/IRS 2 phosphorylation, p85 association with IRS 1 and IRS 2 and Akt phosphorylation similar to those observed in cells expressing wild type IR (IRwt); and finally, a reduced insulin-induced Shc phosphorylation accompanied by decreased ERK1/2 phosphorylation and activity and of thymidine incorporation into DNA in cells expressing IR(R252C) as compared to cells expressing IRwt. CONCLUSION/INTERPRETATION: These observations suggest that: parameters other than tyrosine kinase activation participate in or control the first steps of IR internalisation or both; IR-mediated IRS 1/2 phosphorylation can be achieved from the cell surface and microvilli in particular; Shc phosphorylation and its subsequent signalling pathway might require IR internalisation; defective IR endocytosis correlates with an enhancement of some biological responses to insulin and attenuation of others.

Acanthosis Nigricans↗
PubMed2002

Growth failure in a child showing characteristics of Seckel syndrome: possible effects of IGF-I and endogenous IGFBP-3.

Seckel syndrome is an autosomal-recessive disorder with a frequency of less than 1/10 000 births in which there are multiple malformations including severe short stature. We report on a patient with Seckel syndrome with a current body height of -7.5 SDS. Laboratory investigations at the age of 19 months revealed high levels of IGF-I, IGF-II and IGFBP-3. These data suggested the existence of IGF-I resistance possibly caused by impairment of the IGF-I receptor (IGF-IR) or altered IGFBPs. The purpose of this investigation was to examine whether the growth retardation in a Seckel syndrome patient is related to an alteration in the IGF system. Analysis of IGF-IR mRNA of patient's and control fibroblasts by solution hybridization/RNase protection assay did not show differences of IGF-IR transcript expression or size. Affinity crosslinking studies using [125I]-IGF-I showed normal-sized IGF-IR-ligand complexes. Mutation analysis of the complete coding regions of the IGF-I and IGF-IR genes showed no evidence of genetic alterations. Ligand blot analysis of IGFBPs secreted by the patient's fibroblasts showed stronger signals than control cells. Quantitative measurement of IGFBP-3 in cell-conditioned media was performed by radioimmunoassay (RIA) and revealed a sixfold increase when compared to control fibroblasts. We conclude that in this patient with Seckel syndrome and severe growth impairment IGF-I resistance is possibly related to altered production of IGFBP-3.

Abnormalities, Multiple↗
PubMed2001

Structure-function studies of an IGF-I analogue that can be chemically cleaved to a two-chain mini-IGF-I.

The structure and biological activities of two disulphide isomers of a C-region deletion mutant of insulin-like growth factor-I (IGF-I) which has an Asn--Gly link engineered at the junction of the A- and B-regions were studied before and after chemical cleavage. Circular dichroism (CD) spectra and binding affinity to IGF binding protein 3 (IGFBP3) indicated that the treatment with hydroxylamine did not disrupt the overall tertiary fold of the hormones. Cleavage restored some binding affinity for the IGF-I receptor in both isomers and weakly restored the ability to stimulate incorporation of tritiated thymidine into DNA in NIH 3T3 fibroblasts transfected with the human IGF-I receptor. Cleavage also restored metabolic capacity, as measured by the ability of the isomers to promote lipogenesis in isolated rat adipocytes through the insulin receptor. These results are consistent with the theory that binding of IGF-I to the IGF-I receptor requires a conformational change similar to that involved in insulin binding the insulin receptor. The weak affinity for the IGF-I receptor after cleavage is consistent with the belief that residues in the C-region interact with the IGF-I receptor. This structural difference between insulin and IGF-I gives each a higher binding affinity for its own receptor.

3T3 Cells↗
PubMed2000

Timing-dependent modulation of insulin mitogenic versus metabolic signalling.

This chapter will not deal sensu stricto with the mechanisms and biological significance of pulsatile hormone secretion, the general theme of this book. Rather, we will attempt to demonstrate that timing events at the receiving end of the hormonal signal, i.e. the kinetics and duration of receptor activation in target cells and subsequent downstream signalling, can play an equally important role as that of the timing aspects of secretion, in determining the qualitative and quantitative aspects of hormonal responses. We will focus on the mechanisms that determine signalling specificity by the receptor tyrosine kinases, especially the insulin receptor and the type I insulin-like growth factor receptors (IGF-I receptor). We will be succinct and refer the reader to our recent reviews and publications on this topic and references therein.

Animals↗
PubMed1999

Timing-dependence of insulin-receptor mitogenic versus metabolic signalling: a plausible model based on coincidence of hormone and effector binding.

Mitogenic signalling through the insulin receptor is enhanced compared with metabolic signalling for insulin analogues having slower dissociation kinetics than insulin itself. A plausible explanation in molecular terms of this timing-dependent specificity is lacking. We show here that if signalling is transmitted through a single effector, binding coincidentally with hormone to the insulin receptor and whose association and dissociation kinetics are slow relative to the hormone dissociation rate, the resulting biological effect is predicted to be dependent on hormone-binding kinetics. However, known primary effector molecules associating with the insulin receptor bind and interact rapidly with the receptor, contrary to the assumptions of the single-effector model. A model with two effectors which must bind coincidentally with hormone for signalling to occur also gives the required dependence of signalling on hormone-binding kinetics, provided that at least one of the effectors has slow binding kinetics relative to hormone binding. In this case, the other effector can have rapid kinetics, which is consistent with the properties of the major known substrates of the insulin receptor, such as the insulin receptor substrate (IRS) molecules.

Binding Sites↗
PubMed1999

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.

3T3 Cells↗
PubMed1999

Alternative splicing of exon 17 and a missense mutation in exon 20 of the insulin receptor gene in two brothers with a novel syndrome of insulin resistance (congenital fiber-type disproportion myopathy).

The insulin receptor (IR) in two brothers with a rare syndrome of congenital muscle fiber type disproportion myopathy (CFTDM) associated with diabetes and severe insulin resistance was studied. By direct sequencing of Epstein-Barr virus-transformed lymphocytes both patients were found to be compound heterozygotes for mutations in the IR gene. The maternal allele was alternatively spliced in exon 17 due to a point mutation in the -1 donor splice site of the exon. The abnormal skipping of exon 17 shifts the amino acid reading frame and leads to a truncated IR, missing the entire tyrosine kinase domain. In the correct spliced variant, the point mutation is silent and results in a normally translated IR. The paternal allele carries a missense mutation in the tyrosine kinase domain. All three cDNA variants were present in the lymphocytes of the patients. Purified IR from 293 cells overexpressing either of the two mutated receptors lacked basal or stimulated IR beta-subunit autophosphorylation. A third brother who inherited both normal alleles has an normal muscle phenotype and insulin sensitivity, suggesting a direct linkage of these IR mutations with the CFTDM phenotype.

Adolescent↗
PubMed1998

Genetic engineering in mice: impact on insulin signalling and action.

The expression of a number of genes encoding key players in insulin signalling and action, including insulin, insulin receptor (IR), downstream signalling molecules such as insulin receptor substrate-1 (IRS-1) and IRS-2, glucose transporters (GLUT4, GLUT2) and important metabolic enzymes such as glucokinase, has now been altered in transgenic or knockout mice. Such mice presented with phenotypes ranging from mild defects, revealing complementarity between key molecules or pathways, to severe diabetes with ketoacidosis and early postnatal death. Insulin action could also be improved by overproduction of proteins acting at regulatory steps. The development of diabetes by combining mutations, which alone do not lead to major metabolic alterations, validated the 'diabetogenes' concept of non-insulin-dependent diabetes mellitus. Genes encoding insulin-like growth factors (IGF-I and IGF-II) and their type I receptor (IGF-IR) have also been disrupted. It appears that although IR and IGF-IR are both capable of metabolic and mitogenic signalling, they are not fully redundant. However, IR could replace IGF-IR if efficiently activated by IGF-II. Studies with cell lines lacking IR or IGF-IR lend support to such conclusions. Concerning the issues of specificity and redundancy, studies with cell lines derived from IRS-1-deficient mice showed that IRS-1 and IRS-2 are also not completely interchangeable.

Animals↗
PubMed1998

Insulin and insulin-like growth factor-I receptor mediated differentiation of 3T3-F442A cells into adipocytes: effect of PI 3-kinase inhibition.

The ability of insulin and insulin-like growth factors (IGF-I and IGF-II) to induce differentiation of 3T3-F442A cells into adipocytes was examined at various hormone concentrations. Both insulin and the IGFs promoted differentiation at concentrations compatible with binding to their cognate receptors, suggesting that both insulin and IGF-I receptors are capable of promoting this differentiation. Adipocyte conversion of 3T3-F442A cells was completely blocked in the presence of LY294002, a specific inhibitor of PI 3-kinase, indicating that PI 3-kinase activity plays a crucial role in the initial signalling events that trigger this differentiation process.

3T3 Cells↗
PubMed1998

Insulin receptor-deficient cells as a new tool for dissecting complex interplay in insulin and insulin-like growth factors.

Cell systems derived from knockout mice for the insulin receptor (IR) or the IGF-1 receptor (IGF-1R) represent unique tools for dissecting complex interplay in the actions of insulin and insulin-like growth factors through their cognate versus non-cognate receptor. In this study, we used a fibroblast cell line derived from IR-deficient mice to investigate metabolic and mitogenic effects of IGF-1 and insulin. IGF-1 was able to stimulate glucose uptake, glucose incorporation into glycogen and thymidine incorporation in such cells. Phosphatidylinositol 3-kinase and mitogen-activated protein kinase, two enzymes of major metabolic-mitogenic signaling pathways, were activated upon stimulating these cells with IGF-1. All these effects were also achieved when IR-deficient cells were stimulated with insulin. Thus, IGF-1R can represent an alternative receptor through which insulin might exert some of its effects.

Animals↗
PubMed1998

Inhibition by insulin of glucocorticoid-induced gene transcription: involvement of the ligand-binding domain of the glucocorticoid receptor and independence from the phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways.

Insulin can inhibit the stimulatory effect of glucocorticoid hormones on the transcription of genes coding for enzymes involved in glucose metabolism. We reported earlier that insulin inhibits the glucocorticoid-stimulated transcription of the gene coding for liver 6-phosphofructo-2-kinase (PFK-2). To elucidate the mechanism of these hormonal effects, we have studied the regulatory regions of the PFK-2 gene in transfection experiments. We found that both glucocorticoids and insulin act via the glucocorticoid response unit (GRU) located in the first intron. Footprinting experiments showed that the GRU binds not only the glucocorticoid receptor (GR), but also ubiquitous [nuclear factor I (NF-I)] and liver-enriched [hepatocyte nuclear factor (HNF)-3, HNF-6, CAAT/enhancer binding protein (C/EBP)] transcription factors. Site-directed mutational analysis of the GRU revealed that these factors modulate glucocorticoid action but that none of them seems to be individually involved in the inhibitory effect of insulin. We did not find an insulin response element in the GRU, but we showed that insulin targets the GR. Insulin-induced inhibition of the glucocorticoid stimulation required the ligand-binding domain of the GR. Finally, the insulin-signaling cascade involved was independent of the phosphatidylinositol-3-kinase and mitogen-activated protein kinase pathways. Together, these results suggest that insulin acts on the PFK-2 gene via another pathway and targets either the GR in its ligand-binding domain or a cofactor interacting with this domain.

Animals↗
PubMed1997

Logical analysis of timing-dependent receptor signalling specificity: application to the insulin receptor metabolic and mitogenic signalling pathways.

We present a method for logical analysis of signal-transduction networks, focusing on metabolic and mitogenic signalling by the insulin receptor, with specific emphasis on dependence of the signalling properties on the timing of binding events. We discuss a basic model which demonstrates this dependence (hormone binding leads to activation of the receptor which can lead to a commitment to mitogenic signalling), and show how residence time of the hormone on the receptor can determine the specificity of signalling between the alternative metabolic or mitogenic pathways. The method gives conditions for the selection of specific branches in the signalling pathway expressed in terms of inequalities among the characteristic activation or deactivation times of components of that pathway. In this way, the conditions for mitogenic signalling can be given in terms of a required range of values of the hormone residence time on the receptor, which is directly related to the kinetic dissociation rate.

Animals↗
PubMed1997

Mitogenic properties of insulin and insulin analogues mediated by the insulin receptor.

Insulin has traditionally been considered as a hormone essential for metabolic regulation, while the insulin-like growth factors (IGF-I and IGF-II) are postulated to be more specifically involved in growth regulation. The conventional wisdom is that they share each other's effects only at high concentrations, due to their weak affinity for the heterologous receptor. We discuss here the evidence that in the proper cellular context, insulin can be mitogenic at physiologic concentrations through its own receptor. We studied the insulin and IGF-I binding characteristics of a new model suitable for analysing insulin receptor mediated mitogenesis; that is, a T-cell lymphoma line that depends on insulin for growth, but is unresponsive to IGFs. The cells showed no specific binding of 125I-IGF-I and furthermore, no IGF-I receptor mRNA was detected by RNAse protection assay in the LB cells, in contrast with mouse brain and thymus. The cells bound at saturation about 3000 insulin molecules to receptors that had normal characteristics in terms of affinity, kinetics, pH dependence and negative co-operativity. A series of insulin analogues competed for 125I-insulin binding with relative potencies comparable to those observed in other insulin target cells. The full sequence of the insulin receptor cDNA was determined and found to be identical to the published sequence of the murine insulin receptor cDNA. The LB cell line is therefore an ideal model with which to investigate insulin mitogenic signalling without interference from the IGF-I receptor. Using this model, we have started approaching the molecular basis of insulin-induced mitogenesis, in particular the role of signalling kinetics in choosing between mitogenic and metabolic pathways.

Animals↗
PubMed1996

Biosensor measurement of the binding of insulin-like growth factors I and II and their analogues to the insulin-like growth factor-binding protein-3.

Most insulin-like growth factor (IGF) molecules in the circulation are found in a 150-kDa complex containing IGF-binding protein-3 (IGFBP-3) and an acid-labile subunit, which does not itself bind IGF. Affinities (Kd values) between 0.03 and 0.5 nM have been reported for IGF-I/IGFBP-3 binding, but no kinetic data are available. In this study we measured the high affinity binding of unlabeled IGFs and IGF analogues to recombinant unglycosylated IGFBP-3, using a BIAcoretrade mark instrument (Pharmacia Biosensor AB). IGF-I binding showed fast association and slow non-first-order dissociation kinetics, and an equilibrium Kd of 0.23 nM. IGF-II had similar kinetics with slightly higher affinity. Analogues with mutations in the first 3 amino acids of the B-region (des(1-3) IGF-I and long IGF-I) showed 25 and 50 times lower affinity than IGF-I. Replacement of residues 28-37 by Gly-Gly-Gly-Gly or deletion of residues 29-41 in the C-region had little effect on the kinetic parameters, contrasting with the markedly impaired binding of these analogues to the IGF-I receptor. Swapping of the disulfide bridges in IGF-I and the C-region mutants decreased the affinity dramatically for IGFBP-3, primarily by decreasing the association rate. Insulin had approximately 1000 times lower affinity than IGF-I.

Biosensing Techniques↗
PubMed1996

Biological effects of human growth hormone in rat adipocyte precursor cells and newly differentiated adipocytes in primary culture.

The effects of human growth hormone (hGH) on proliferation and differentiation of primary adipocyte precursor cells isolated from rat epididymal fat pads were studied under serum-free culture conditions. hGH markedly reduced the formation of new fat cells and the expression of glycerophosphate dehydrogenase activity, a marker enzyme of adipose differentiation, in a dose-dependent manner. To find an explanation for this inhibitory effect, we investigated the action of GH on (1) cell proliferation and on (2) lipid accumulation, the latter in the absence and presence of corticosterone. In undifferentiated cells, 5 nmol/L hGH increased both cell number and [3H]-thymidine incorporation (1.3- and 2.6-fold over basal, respectively). This effect was mediated by insulin-like growth factor-I (IGF-I), since hGH stimulated IGF-I production in undifferentiated cells by 12-fold and addition of an anti-IGF-I monoclonal antibody (IGF-I MAb) abolished the mitogenic effect of hGH but did not prevent hGH-induced suppression of adipose differentiation. In developing fat cells, hGH significantly reduced cellular 2-deoxyglucose uptake and glucose incorporation into lipids. In addition, hGH exhibited a lipolytic action in the presence of insulin and triiodothyronine. These effects were not prevented by IGF-I MAb. Specific binding of [125I]-hGH to precursor cells increased significantly during adipose conversion. In differentiated cells Scatchard analysis yielded linear plots with an apparent Kd of 0.16 nmol/L and 8,400 sites per cell. Taken together, these data show that hGH reduces adipose conversion in primary cultures of rat adipocyte precursor cells while promoting cell proliferation through an increase in IGF-I production.

Adipocytes↗
PubMed1996

Engineering the C-region of human insulin-like growth factor-1: implications for receptor binding.

Recombinant wild-type human IGF-1 and a C-region mutant in which residues 28-37 have been replaced by a 4-glycine bridge (4-Gly IGF-1) were secreted and purified from yeast. An IGF-1 analogue in which residues 29-41 of the C-region have been deleted (mini IGF-1) was created by site-directed mutagenesis and also expressed. All three proteins adopted the insulin-fold as determined by circular dichroism. The significantly raised expression levels of mini IGF-1 allowed the recording of two-dimensional NMR spectra. The affinity of 4-Gly IGF-1 for the IGF-1 receptor was approximately 100-fold lower than that of wild-type IGF-1 and the affinity for the insulin receptor was approximately 10-fold lower. Mini IGF-1 showed no affinity for either receptor. Not only does the C-region of IGF-1 contribute directly to the free energy of binding to the IGF-1 receptor, but also the absence of flexibility in this region eliminates binding altogether. As postulated for the binding of insulin to its own receptor, it is proposed that binding of IGF-1 to the IGF-1 receptor also involves a conformational change in which the C-terminal B-region residues detach from the body of the molecule to expose the underlying A-region residues.

Amino Acid Sequence↗
PubMed1996

Mitogenic and antiadipogenic properties of human growth hormone in differentiating human adipocyte precursor cells in primary culture.

Children with GH deficiency have enlarged fat cells but a reduced number of fat cells compared with healthy children. After treatment with human GH (hGH) both fat cell volume and number are shifted toward normal. To clarify the role of hGH in fat cell formation in human adipose tissue, we investigated the effect of hGH on the proliferation and the differentiation of cultured human adipocyte precursor cells obtained from five children and 10 adults. In a chemically defined serum-free medium treatment of adipocyte precursor cells with hGH led to an increase in IGF-I production and a stimulation of cell proliferation, which could be blocked by a MAb raised against human IGF-I. hGH dose-dependently reduced the number of differentiating cells and suppressed the expression of glycerol-3-phosphate dehydrogenase (GPDH), a marker of adipose differentiation. No significant differences in the hGH effects on proliferation and differentiation capacities were seen between cultures obtained from children and adults. In newly differentiated adipocytes, hGH inhibited glucose uptake and lipogenesis, and stimulated lipolysis. Scatchard analysis of hGH competition experiments using 125I-labeled hGH yielded a linear plot with an apparent Kd of 1.08 nM and an estimated number of 7000 hGH receptors per cell. These data suggest that hGH is able to enlarge the human adipocyte precursor pool via induction of IGF-I synthesis but exhibits a direct antiadipogenic activity. hGH is also able to reduce fat cell volume by reducing lipogenesis and increasing lipolysis.

Adipocytes↗