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F Grigorescu

Publications and source records attributed to F Grigorescu.

At least 19 recordsLinked to original sources
PubMed2000

Studies of the variability of the genes encoding the insulin-like growth factor I receptor and its ligand in relation to type 2 diabetes mellitus.

Insulin-like growth factor I (IGF-I) is an important regulator of many aspects of growth, differentiation, and development, and as low birth weight has been associated with impaired glucose tolerance and overt type 2 diabetes in adult life, we considered the genes encoding the IGF-I and the IGF-I receptor (IGF-IR) as candidates for low birth weight, insulin resistance, and type 2 diabetes. Here we report the mutational analysis of the coding regions of the IGF-I and IGF-IR performed on genomic DNA from probands of 82 Danish type 2 diabetic families. No mutations predicting changes in the amino acid sequences of the IGF-I or IGF-IR genes were detected, but several silent and intronic polymorphisms were found. The impact of the most prevalent polymorphism, GAG1013GAA of the IGF-IR, was evaluated in a population-based sample of 349 young healthy subjects, where the variant had an allele frequency of 0.44 (95% confidence interval, 0.40-0.48). No significant relationships between this variant and birth weight, birth length, or insulin sensitivity index were detected. In addition, we did not observe any significant differences in allelic frequencies of the codon 1013 variant between 395 type 2 diabetic patients (allele frequency, 0.52; 95% confidence interval, 0.49-0.55) and 238 matched glucose-tolerant control subjects (allelic frequency, 0.47; 95% confidence interval, 0.43-0.50). In conclusion, variability in the coding regions of IGF-I and the IGF-IR does not associate with reduced birth weight, insulin sensitivity index, or type 2 diabetes in the Danish population.

Adult↗
PubMed1999

Congenital insulin resistance associated with a conformational alteration in a conserved beta-sheet in the insulin receptor L1 domain.

The hormone binding site of members of the insulin receptor family is contained within a highly conserved extracellular region of the receptor. Recent crystallization of the N-terminal region of the binding site revealed two large domains (L1, L2), each organized as a single-stranded right-handed beta-helix, connected by a rod-shaped cysteine-rich domain. Here, we analyze two new naturally occurring mutations in a single beta-sheet within L1, D59G and L62P, that we previously identified in a young woman with classic congenital insulin resistance (type A). Substitution of D59G, a beta-sheet connecting loop residue, caused decreased hormone binding but did not disrupt overall folding, assembly, or movement to the cell surface. In contrast, replacement of the adjacent residue L62P, which is located within the beta-sheet, and positioned in a hormone binding surface, completely disrupted intracellular folding, oligomerization, and trafficking and resulted in aberrant proteolytic degradation. Immunohistochemistry in combination with biosynthetic studies showed that misfolded receptors were retained in an incorrect cellular location and that they colocalized with the resident endoplasmic reticulum chaperone calnexin. This study, together with other mutagenesis data, shows that formation of beta-sheet elements within the L1 beta-helix are critical for the folding of the entire extracellular domain of the receptor and that the hormone contact site is composed in part by residues in this domain.

Biotinylation↗
PubMed1999

Identification by RT-PCR and immunolocalization of arginine vasopressin in rat pancreas.

Arginine vasopressin (AVP), a hormone of the hypothalamic pituitary axis, has been described in several peripheral tissues, including pancreas. To demonstrate the ectopic synthesis of AVP at the pancreatic level, we explored the expression of the AVP-neurophysin-II (AVP-NP-II) precursor gene by reverse-transcriptase polymerase chain reaction (RT-PCR) and sequencing and attempted to localise the peptide by immunocytochemistry in normal rat pancreas. Primers designed at the 3' and 5' ends of the AVP-NP-II gene, RT-PCR, and automatic sequencing of PCR products from rat pancreas revealed transcripts of the predicted size with an identical sequence to those from the hypothalamus. In addition, AVP antiserum revealed immunoreactive material of perivascular localisation. These data provide the first direct evidence for the presence of AVP transcripts in rat pancreatic tissue, whereas concurrent immunodetection of this hormone offers further support for the potential role of ectopic AVP in local regulation of the secretory activity of the pancreas.

Animals↗
PubMed1999

[Insulin resistance: from clinical diagnosis to molecular genetics. Implications in diabetes mellitus].

Insulin resistance is observed in several diseases such as non insulin dependent diabetes mellitus (NIDDM) or polycystic ovarian syndrome (PCOS). To understand genetic determinism of this abnormality we have developed a multidisciplinary approach including selection of phenotypes with insulin resistance confirmed in vivo by minimal model of Bergman and characterization of cellular defects in insulin action on circulating erythrocytes and monocytes. Exploration of variability in candidate genes by direct sequencing in some genetic syndromes of severe insulin resistance and acanthosis nigricans (mainly the Type A syndrome) revealed mutations of the insulin receptor gene associated with major defects in insulin binding or kinase activity. In other rare genetic syndromes or patients affected by NIDDM or PCOS defects appear to be located at post-receptor level, where IRS (insulin receptor substrate) genes are the most attractive candidates. Prevalence of some allelic variants suggested a potential role of IRS genes in insulin resistance, although their involvement in the pathogenesis of NIDDM remains controversial. Genotype-phenotype correlations in first degree relatives of an index case caring the Type A syndrome, suggested that association of allelic variants of IRS-1 and IRS-2 with insulin receptor mutations contribute, by synergistic effects, to phenotypic expression of defects in signal transduction. These mechanisms through genetic epistasis, involving several genes in insulin action, fit better with the polygenic nature of current forms of NIDDM and represent a good model in the study of pathogenesis of insulin resistance.

Acanthosis Nigricans↗
PubMed1998

Refinement of genetic localization of the Alström syndrome on chromosome 2p12-13 by linkage analysis in a North African family.

Alström syndrome is a rare autosomal recessive disorder characterized by retinal pigment degeneration, neurogenic deafness, infantile obesity, hyperlipidemia, and non-insulin-dependent diabetes mellitus. While the disease-related gene remains unknown, studies of the genetic isolate of French Acadians provisionally locate the Alström syndrome on chromosome 2p12-13 within a 14.9-cM interval. To confirm this finding in another ethnic population and refine the candidate region we investigated by linkage analysis a consanguineous family of North African origin, in which three of seven siblings displayed all major neurological and metabolic features of Alström syndrome. Genotyping was performed on an ABI377 DNA automatic sequencer and LOD scores were obtained with the Fastlink program. Five markers previously investigated in French Acadians confirmed the involvement of the candidate region, although pairwise LOD scores were of poor significance (Zmax = 2.9). To further confirm homogeneity and refine the candidate region, 20 additional markers were investigated. Haplotype analysis and allele segregation revealed that affected children shared a single haplotype and were homozygous for the eight most centromeric markers (D2S291-D2S2114), over a 6.1-cM interval. Significative multipoint LOD scores (Zmax = 3.96) were obtained between markers D2S2110/145 and D2S286. Two clusters of known genes are present in this refined region of chromosome 2p, the most attractive candidate being the hexokinase II gene. However, except for several known polymorphisms, no mutations were detected in the coding region of this gene. In conclusion, the location of Alström syndrome on chromosome 2p12-13 is confirmed, reducing the genetic interval to 6.1 cM.

Abnormalities, Multiple↗
PubMed1998

Identification and sequence analysis of arginine vasopressin mRNA in normal and Brattleboro rat aortic tissue.

Arginine vasopressin (AVP), a hormone of the hypothalamic-pituitary axis, was also localized in peripheral tissues. To explore AVP precursor gene expression at the vascular level, we have investigated gene transcripts by reverse transcription-polymerase chain reaction (RT-PCR) and sequencing in aortic tissue of normal rat and in the particular genetic condition of the homozygous (di/di) Brattleboro rat strain suffering from diabetes insipidus. In these rats, a gene deletion induces an unprocessed AVP precursor in the hypothalamus with undetectable immunoreactive AVP, in contrast to the detection of immunoreactive material at the vascular level. In normal rats, using primers complementary to exon 1 and 3 of the AVP neurophysin precursor gene, RT-PCR and sequencing revealed transcripts of the expected size from aorta, mesenteric artery and hypothalamus with normal, authentic sequences. Removal of aortic endothelium severely reduced the amounts of transcripts, suggesting their main endothelial origin. In Brattleboro rats, transcripts of similar size were obtained from aorta and hypothalamus and sequencing revealed the homozygous deletion (deltaG316) in both tissues, identical to that found in genomic DNA (deltaG1864). While sequence data from normal rats provide the first direct evidence for the presence of AVP precursor transcripts in rat aortic tissue, identification of the deleted sequence of transcripts in Brattleboro rat aorta suggests that tissue-specific mechanisms are operating for the expression of vasopressin neurophysin precursor in peripheral vascular tissue compared with the hypothalamus.

Animals↗
PubMed1997

Identification of two novel insulin receptor mutations, Asp59Gly and Leu62Pro, in type A syndrome of extreme insulin resistance.

To elucidate genetic determinants of insulin resistance, we investigated insulin receptor (IR) and insulin receptor substrate-1 (IRS-1) genes, in vitro IR function and in vivo insulin sensitivity in a family with Type A syndrome. Two missense IR mutations (Asp59Gly and Leu62Pro) found in the proband, resulted in reduction by 90% of insulin binding to erythrocytes, decreased receptor autophosphorylation and a dramatic reduction of insulin sensitivity. The proband and mother were heterozygote for Gly972Arg IRS-1 variant. Asp59Gly mutation, also carried by proband's brother with no consequence on insulin sensitivity, was inherited from the mother who is diabetic and insulin resistant and Leu62Pro was from the father. We conclude that severity of insulin resistance in the proband may be explained by the genetic condition of compound heterozygote for IR mutations while severe insulin resistance in the mother raises the possibility that other genetic factors, like IRS-1 polymorphisms, may contribute to the phenotypic expression of IR mutations.

Amino Acid Sequence↗
PubMed1997

Characterization of vanadyl sulfate effect on vascular contraction: roles of calcium and tyrosine phosphorylation.

In order to explore the mechanism of action of vanadyl sulfate (VOSO4), previously described as an antidiabetic and antihypertensive agent, we have investigated the role of calcium and tyrosine phosphorylation in the contractile responses of rat aorta or skinned rabbit mesenteric artery rings. VOSO4 induced a concentration-dependent contraction of aorta (pD2 = 3.2), which was potentiated by endothelium removal (pD2 = 4.2). After a first exposure to VOSO4, no change in responsiveness was observed even though high vanadium concentrations had accumulated in the aortic tissue (approximately 4 x 10(-3) M). VOSO4 induced, in calcium-free medium, a significant response that, relative to contractions measured in Krebs-Henseleit buffer, was higher (36%) than norepinephrine (16%)-, arginine-vasopressin (8%)- or KCI (5%)-induced responses. 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8), an intracellular calcium release inhibitor, did not modify VOSO4-induced response either in the presence or in the absence of ambient calcium. On skinned preparations, VOSO4 antagonized Ca++-induced contraction. The tyrosine kinase inhibitors tyrphostin 23 (T23) and tyrphostin 47 (T47) potentiated by 4- and 14-fold, respectively, the activity of VOSO4, in contrast to the lack of effect of T47 on pervanadate-induced contraction. When phosphotyrosine content was revealed by Western blotting, VOSO4 had no effect alone, but in the presence of T47, it dramatically increased the phosphotyrosine content. This result contrasts again with PV-induced tyrosine phosphorylation, which was blocked by T47. These data suggest that the signaling events involved in vascular effects of VOSO4, although they depend little on calcium mobilization, are related to tyrosine phosphorylation, likewise through a pathway different from that of pervanadate.

Animals↗
PubMed1997

Decrease in insulin and insulin-like growth factor I (IGF-I) binding to erythrocytes from patients with cystic fibrosis.

Cystic fibrosis, an autosomal recessive disease affecting exocrine glands, is associated in many cases with a severe undernutritional state, growth retardation and glucose intolerance. To obtain a better definition of the possible defects of insulin and insulin-like growth factor I (IGF-I) receptors, we investigated 125I-insulin and 125I-IGF-I binding to erythrocytes from patients with cystic fibrosis (n = 23) and controls (n = 13). Erythrocytes were isolated by Ficoll-Hypaque gradient centrifugation, and hormone binding was performed in cell suspensions of 3 x 10(9) cells/ml. Cystic fibrosis patients displayed a statistically significant 33% and 40% (p < 0.05) decrease of insulin and IGF-I binding, respectively, compared to controls. These alterations were due to an almost 50% reduction in the binding capacity of the high-affinity receptor compartment. Affinity constants were modified to a lesser extent, except for a two-fold decrease in K1 of the high-affinity compartment of insulin receptors. Interestingly, the decrease in insulin binding was proportional to the degree of growth failure. The statistical significance of hormone binding alterations was assessed in terms of the graphic distribution of individual affinity constants and binding capacity values. Although variable, 50 to 60% of cystic fibrosis patients displayed alterations in stoichiometric binding parameters located outside the area described by the 95% tolerance interval of controls. A major reduction in insulin and IGF-I binding in conditions of low and normal insulin and IGF-I plasma levels, respectively, as well as the correlation with the degree of growth failure in patients with cystic fibrosis, may contribute to an understanding of the pathogenesis of insulin resistance and glucose abnormalities in undernutritional states.

Adolescent↗
PubMed1995

Insulin-dependent phosphatidylinositol 3'-kinase activity co-precipitates with insulin receptor in human circulating mononuclear cells.

In order to establish a working cellular model for the study of post-receptor signaling events, insulin-dependent phosphatidylinositol 3' (PtdIns 3')-kinase activity was investigated in circulating mononuclear cells from normal subjects. The p85 alpha regulatory subunit of PtdIns 3'-kinase co-precipitated with activated insulin receptor as revealed by immunoblotting. Whereas insulin receptor substrate-1 was weakly detected, insulin increased 5.5 +/- 1.5-fold (mean +/- SD) PtdIns 3'-kinase activity in receptor immunoprecipitates. We conclude that insulin-stimulated PtdIns 3'-kinase activity is measurable in circulating mononuclear cells which may constitute an easily available cellular model for the detection of post-receptor defects in insulin-resistant states.

Adult↗
PubMed1994

Involvement of phosphoinositide 3-kinase in insulin- or IGF-1-induced membrane ruffling.

Insulin, IGF-1 or EGF induce membrane ruffling through their respective tyrosine kinase receptors. To elucidate the molecular link between receptor activation and membrane ruffling, we microinjected phosphorylated peptides containing YMXM motifs or a mutant 85 kDa subunit of phosphoinositide (PI) 3-kinase (delta p85) which lacks a binding site for the catalytic 110 kDa subunit of PI 3-kinase into the cytoplasm of human epidermoid carcinoma KB cells. Both inhibited the association of insulin receptor substrate-1 (IRS-1) with PI 3-kinase in a cell-free system and also inhibited insulin- or IGF-1-induced, but not EGF-induced, membrane ruffling in KB cells. Microinjection of nonphosphorylated analogues, phosphorylated peptides containing the EYYE motif or wild-type 85 kDa subunit (Wp85), all of which did not inhibit the association of IRS-1 with PI 3-kinase in a cell-free system, did not inhibit membrane ruffling in KB cells. In addition, wortmannin, an inhibitor of PI 3-kinase activity, inhibited insulin- or IGF-1-induced membrane ruffling. These results suggest that the association of IRS-1 with PI 3-kinase followed by the activation of PI 3-kinase are required for insulin- or IGF-1-induced, but not for EGF-induced, membrane ruffling.

Amino Acid Sequence↗
PubMed1994

Insulin and IGF-1 signaling in oocyte maturation.

Xenopus laevis oocytes possess insulin and/or insulin-like growth factor-1 (IGF-1) receptors and respond to respective hormones by increasing glucose transport and progressing from the G2 to M phase of the cell cycle (maturation). While molecular transduction mechanisms involving mitogen-activating kinases and cyclin-dependent kinases begin to be elucidated, missing links remain between the initial receptor tyrosine phosphorylation events and downstream signaling. The discovery that phosphotyrosines produced by receptor autophosphorylation or during substrate phosphorylation serve as an anchor for src homology 2 domains of several signaling proteins had a major impact on understanding how cytoplasmic enzymes are recruited at the level of the plasma membrane for subsequent activation.

Animals↗
PubMed1994

Prevalence of mutations in the insulin receptor gene in subjects with features of the type A syndrome of insulin resistance.

Mutations of the insulin receptor gene are a cause of the type A syndrome of extreme insulin resistance. This study assessed the prevalence of such mutations in women with clinical features of the type A syndrome including ovarian hyperandrogenism, moderate-to-severe degrees of insulin resistance, and acanthosis nigricans. We studied 22 unrelated women with insulin resistance (fasting insulin > 300 pM [50 microU/ml] and/or peak during an oral glucose tolerance test (OGTT) > 1,800 pM [300 microU/ml]), acanthosis nigricans, and the polycystic ovary syndrome (hyperandrogenemia, oligoamenorrhea, and hirsutism). Two insulin-resistant probands with congenital generalized lipodystrophy and one male proband with severe insulin resistance also were included in the study. Southern blotting experiments were performed to exclude gross gene deletions, insertions, or rearrangements. Exons 2-22 of the insulin receptor gene were polymerase chain reaction (PCR) amplified from genomic DNA and screened for nucleotide variation using single-strand conformation polymorphism (SSCP). No nucleotide variation between study subjects was detected in exons 4-6, 10-12, 15, 16, 18, 19, or 21. Sequencing of amplified DNA revealed that SSCP variants in exons 2, 3, 8, 9, and 17 corresponded to known silent polymorphisms within the coding region. Variants in exons 2, 9, 13, and 14 were caused by novel silent polymorphisms; variants in exons 7 and 22 were caused by nucleotide substitutions in flanking introns. One proband was found to have a heterozygous point mutation in exon 20 (CGG-->CAG, Arg1174-->Gln) that involves the intracellular receptor beta-subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Acanthosis Nigricans↗
PubMed1993

Resin immobilized synthetic peptides used to characterize phosphorylation and antigenic properties of insulin receptor autophosphorylation domains.

To develop a common strategy in peptide design for kinase assay, antibody production and affinity purification, we investigated phosphorylation and antigenic properties of peptides immobilized on an aminated polyacrylic resin (Expansin) corresponding to autophosphorylation domains of the insulin receptor tyrosine kinase. Immobilized peptides (1143-1155) and peptide (1314-1330), designated p1151 and p1322, respectively, were good substrates for the insulin receptor with Km of 0.74 and 0.78 mM. By contrast, peptide (952-963), designated p960, was poorly phosphorylated. p1151 showed distinctive behaviour as a substrate, displaying a higher basal phosphorylation, a leftward shift of the insulin dose-response curve (ED50 = 0.7 ng mL-1 insulin compared to 20 ng mL-1 for other substrates) and an inhibition by 90% of receptor autophosphorylation (ID50 = 0.5 mM). Similar substrate behaviour was observed with another tyrosine kinase, the pp60c-src. Antibodies against P1151 and p1322 have comparable reactivity in ELISA, but the antibody against p960 was poor. While purified immunoglobulins (IgG) against both p1151 and p1322 were inhibitors of receptor autophosphorylation and kinase, in immunoprecipitation the IgG against p1151 mainly interacted with the phosphorylated receptor and that against p1322 with non-phosphorylated forms. Functional mapping of the receptor with oligoclonal 1322-antibody revealed inhibition of phosphate transfer to exogenous substrate poly(Glu,Tyr) (4:1) but not towards immobilized p1151. These data provide further support for the distinctive features of endogenous phosphorylation domain 1151. We conclude that immobilized peptides on polyacrylic resin offer a major new potential for use in kinase assays, immunization, immunoabsorbent techniques and purification of well defined oligoclonal antibodies.

Amino Acid Sequence↗
PubMed1993

Insulin-mediated pseudoacromegaly: clinical and biochemical characterization of a syndrome of selective insulin resistance.

We have performed clinical, physiological, in vitro biochemical and genetic studies of a patient with severe insulin resistance associated with the phenotype of "pseudoacromegaly," defined as the presence of acromegaloid features in the absence of elevated levels of GH or insulin-like growth factor-I (IGF-I). Despite marked hyperinsulinemia, insulin and IGF-I binding to circulating blood cells and cultured skin fibroblasts was normal. Insulin and IGF-I-stimulated autophosphorylation of their respective receptors in cultured skin fibroblasts was also normal. However, neither insulin nor IGF-I were able to stimulate 2-deoxy D-glucose uptake by cultured skin fibroblasts. In contrast, the ability of insulin and IGF-I (or IGF-II) to stimulate amino acid uptake and thymidine incorporation into DNA was not impaired. This unique discordant signaling defect through both insulin and IGF-I receptors appeared not to be the consequence of altered expression or primary structure of the insulin receptor or the GLUT-4 glucose transporter, as assessed by several genetic and biochemical techniques. GLUT-4 expression in muscle was normal on Western blots, and SSCP screening of all 11 exons of the gene for nucleotide variation revealed no variations from normal. DNA sequencing and SSCP screening of exons 2-22 of the insulin receptor gene revealed only one variation predicted to alter the amino acid sequence (Val985-->Met). No functional differences between Met985 and wild-type human insulin receptors were evident in studies performed with Chinese hamster ovary cell transfectants that overexpress either receptor. This data combined with our previously published epidemiological data concerning the frequency of the Met985 allele, indicate that this variant insulin receptor is not responsible for the insulin resistant glucose uptake or the clinical syndrome of pseudoacromegaly. We conclude that: 1) The molecular lesion responsible for the selective biochemical defect in this individual appears to involve a signaling intermediate required for insulin and IGF-I regulation of glucose transport, and/or an effector mechanism operative in this process. 2) Cells derived from this patient may be a valuable tool in the search for such molecular mechanisms. 3) The Met985 allele is a relatively common variant which has no demonstrable adverse consequences for insulin receptor function. 4) Pseudoacromegaly can be viewed as the expected result of hyperinsulinemia driving the unopposed mitogenic and anabolic actions of insulin.

Acromegaly↗
PubMed1992

Kinase inhibition by a phosphorylated peptide corresponding to the major insulin receptor autophosphorylation domain.

We studied the inhibitory effect of non-phosphorylated and triphosphorylated synthetic peptides, corresponding to amino acids 1143-1155 of the insulin proreceptor (domain 1151) on autophosphorylation and kinase of the insulin receptor. Tyrosine-phosphorylated peptides were synthesized using the N-(9-fluorenylmethoxycarbonyl)-O-dibenzylphosphono-L- tyrosine. The triphosphorylated peptide (1151-P3) and the non-phosphorylated peptide (1151-NP), respectively, inhibited insulin receptor autophosphorylation by 65% and 70%, in a dose-dependent and additive manner. When the receptor was pre-phosphorylated for 1 min with [gamma-32P]ATP, 1151-P3 decreased autophosphorylation to 60% of maximum, whereas 1151-NP had no further effect. In both non-activated and preactivated receptors, 1151-P3 inhibition of receptor autophosphorylation was prevented by adding 2 mM vanadate. Kinase activity towards exogenous substrate poly(Glu4, Tyr) was dose-dependently inhibited by both analogues. This effect was independent of the state of receptor phosphorylation or the addition of vanadate. Since 1151-P3 inhibited the exogenous kinase without altering receptor endogenous autophosphorylation after the addition of vanadate, we investigated 1151-NP and 1151-P3 competition for the phosphorylation of a resin-immobilized 1151 peptide. While 1151-NP (at 2 mM) was highly competitive, inhibiting phosphate incorporation by 70%, 1151-P3 caused a four-fold increase in the phosphorylation of 1151-NP--resin. The receptor underwent conformational changes during autophosphorylation and an antibody directed against a peptide corresponding to amino acids 1314-1330 of the proreceptor (1322Ab) was previously shown to immunoprecipitate specifically the non-phosphorylated receptor forms. Nevertheless, the 1322Ab immunoprecipitated a fully autophosphorylated receptor in the presence of 1151-NP, but not of 1151-P3, thus suggesting a conformational change induced by the non-phosphorylated peptide. In conclusion, kinase inhibition was still observed after the addition of phosphate groups to three 1151-peptide tyrosines, but the peptide effect on receptor autophosphorylation, phosphorylation of homologous 1151-NP--resin and conformational changes induced in the receptor was altered dramatically. These data may provide a basis for further understanding the role of tyrosine phosphorylation in insulin receptor kinase activation or regulation.

Amino Acid Sequence↗