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Biomedical subjects

S I Taylor

Publications and source records attributed to S I Taylor.

At least 19 recordsLinked to original sources
PubMed1997

Development of a novel polygenic model of NIDDM in mice heterozygous for IR and IRS-1 null alleles.

NIDDM is a polygenic disease characterized by insulin resistance in muscle, fat, and liver, followed by a failure of pancreatic beta cells to adequately compensate for this resistance despite increased insulin secretion. Mice double heterozygous for null alleles in the insulin receptor and insulin receptor substrate-1 genes exhibit the expected approximately 50% reduction in expression of these two proteins, but a synergism at a level of insulin resistance with 5- to 50-fold elevated plasma insulin levels and comparable levels of beta cell hyperplasia. At 4-6 months of age, 40% of these double heterozygotes become overtly diabetic. This NIDDM mouse model in which diabetes arises in an age-dependent manner from the interaction between two genetically determined, subclinical defects in the insulin signaling cascade demonstrates the role of epistatic interactions in the pathogenesis of common diseases with non-Mendelian genetics.

Alleles↗
PubMed1996

Characterization of a hybrid receptor formed by dimerization of the insulin receptor-related receptor (IRR) with the insulin receptor (IR): coexpression of cDNAs encoding human IRR and human IR in NIH-3T3 cells.

In many tissues, the insulin receptor-related receptor (IRR) is colocalized with the homologous receptors for insulin and insulin-like growth factor-I (IGF-I). Since a ligand for the IRR has not yet been identified, it has been proposed previously that IRR may be activated and transduce its signal via formation of hybrids with the insulin and IGF-I receptors. To test this hypothesis, we have coexpressed the human IRR and the human insulin receptor (IR) in NIH-3T3 cells. Although IRR/IR hybrid receptors were detected in these cells by using immunoprecipitation techniques, only a small proportion of each receptor was assembled into hybrids. While insulin was capable of stimulating insulin receptors autophosphorylation in these cells, there was no detectable increase in the total phosphotyrosine content of IRR. We conclude that the IRR/IR hybrid receptor does not play a major role in IRR signal transduction in response to insulin in NIH-3T3-hIRR/hIR cells.

3T3 Cells↗
PubMed1996

Cloning of the chicken insulin receptor substrate 1 gene.

The action of insulin, IGF-1, and IGF-2 is mediated via two receptor tyrosine kinases, the insulin and IGF-1 receptors. Upon ligand binding, these receptors become active kinases, undergoing autophosphorylation and phosphorylating cellular substrates, including insulin receptor substrate-1 (IRS-1). IRS-1 acts as a docking protein and mediates multiple interactions among other proteins, resulting in transduction of the metabolic and mitogenic signals. The IRS-1 gene has been cloned from four species (human, rat, mouse, and frog). In the present study, the chicken IRS-1 gene was cloned. Chicken, as is true of birds in general, have a higher fasting and fed blood glucose than do mammals. Chicken IRS-1 DNA sequence encodes a 1240 amino acid protein. The most conserved regions were the IRS homology-2 (IH-2), the pleckstrin homology, and the shc and IRS-1 NPXY-binding (SAIN) domains. Twelve of the cIRS-1 tyrosine residues are in sequence motifs that, when phosphorylated, could interact with proteins containing SH2 domains. All twelve of these motifs were conserved. IRS-1 mRNA is expressed during embryogenesis in chicken and persists after hatching. In LMH cells, derived from a chicken hepatoma, two bands were tyrosine phosphorylated in an insulin-dependent manner: IRS-1 (approximately 180 kDa) and the insulin receptor beta subunit (approximately 95 kDa). Chicken IRS-1 is structurally and functionally similar to its human homolog, despite the difference in blood glucose levels and the evolutionary distance between birds and mammals.

Amino Acid Sequence↗
PubMed1996

Effects of overexpressing wild-type and mutant PDGF receptors on translocation of GLUT4 in transfected rat adipose cells.

Activation of phosphatidylinositol 3-kinase (PI3K) by insulin is necessary for the effect of insulin to recruit GLUT4 to the cell surface in insulin target cells. In adipose cells, stimulation of endogenous PDGF receptors (PDGF-R) results in increased PI3K activity without causing recruitment of GLUT4. We overexpressed wild-type or mutant forms of the PDGF-R in rat adipose cells and examined their effects on PDGF- and insulin-stimulated recruitment of co-transfected epitope-tagged GLUT4. Control cells expressing only tagged GLUT4 had a 3-fold increase in cell surface GLUT4 upon insulin stimulation but no response to PDGF. Cells overexpressing wild-type PDGF-R maintained insulin responsiveness and, in addition, acquired the ability to recruit GLUT4 in response to PDGF. Surprisingly, overexpression of F740/ F751 (mutant PDGF-R unable to directly activate PI3K) led to similar results. Nevertheless, wortmannin (an inhibitor of PI3K) blocked effects of both PDGF and insulin to recruit GLUT4. Our data suggest that overexpression of PDGF-R mediates positive effects on GLUT4 translocation by a wortmannin sensitive pathway not dependent on direct interaction of the PDGF-R with PI3K.

Adipose Tissue↗
PubMed1995

Insulin-induced activation of phosphatidylinositol (PI) 3-kinase. Insulin-induced phosphorylation of insulin receptors and insulin receptor substrate-1 displaces phosphorylated platelet-derived growth factor receptors from binding sites on PI 3-kinase.

Phosphatidylinositol (PI) 3-kinase is an enzyme that functions in the signaling pathways downstream from multiple cell surface receptors. The p85 regulatory subunit of PI 3-kinase binds to phosphotyrosine residues of various phosphoproteins including the platelet-derived growth factor (PDGF) receptor, the insulin receptor, and insulin receptor substrate-1 (IRS-1). Using NIH-3T3 cells overexpressing the human insulin receptor, we demonstrate that the p85 regulatory subunit of PI 3-kinase binds to phosphorylated PDGF receptor in cells incubated in the absence of insulin. When insulin is added, p85 is released from phosphorylated PDGF receptors and binds to phosphorylated insulin receptors and insulin receptor substrate-1. Moreover, insulin-induced dissociation of PDGF receptors from binding sites on PI 3-kinase requires a functional insulin receptor and is not prevented by vanadate treatment. In contrast, insulin activation does not displace PDGF receptors from binding sites on Ras GTPase-activating protein. This competition for binding to PI 3-kinase provides a mechanism for cross-talk among signaling pathways initiated by distinct peptide hormones and growth factors such as insulin and PDGF.

3T3 Cells↗
PubMed1995

Tyrosine phosphorylation of insulin receptor substrate-1 in vivo depends upon the presence of its pleckstrin homology region.

To characterize the structural basis for the interactions between the insulin receptor (IR) and its major substrate, insulin receptor substrate-1 (IRS-1), a segment of the NH2-terminal region of IRS-1 (Pro5-Pro65) was deleted. This region contains the first four conserved boxes of a pleckstrin homology (PH) domain, located at the NH2-terminal part of IRS-1. COS-7 cells were then cotransfected with the genes coding for IR and a wild-type (WT) or a mutated form of IRS-1. IRS-1 delta PH underwent significantly reduced insulin-dependent tyrosine phosphorylation compared with WT IRS-1. The reduced in vivo tyrosine phosphorylation of IRS-1 delta PH was accompanied by reduced association between IRS-1 delta PH and its downstream effector p85 regulatory subunit of phosphatidylinositol-3 kinase. In contrast, both WT IRS-1 and IRS-1 delta PH underwent comparable insulin-dependent tyrosine phosphorylation in vitro when incubated with partially purified insulin receptor kinase. These findings suggest that the overall structure of IRS-1 is not altered by deletion of its PH domain and that the PH domain is not the main site for protein-protein interactions between the insulin receptor and IRS-1, at least in vitro. In conclusion, the PH region might facilitate in vivo binding of IRS-1 to membrane phospholipids or other cellular constituents in close proximity to the IR, whereas the actual interactions with the IR are presumably mediated through other domains of the IRS-1 molecule. This could account for the fact that partial deletion of the PH domain selectively impairs the in vivo interactions between the insulin receptor and IRS-1, whereas their in vitro interactions remain unaffected.

Amino Acid Sequence↗
PubMed1995

Insulin-stimulated phosphorylation of recombinant pp120/HA4, an endogenous substrate of the insulin receptor tyrosine kinase.

Insulin binding to the alpha-subunit of its receptor stimulates the receptor tyrosine kinase to phosphorylate the beta-subunit and several endogenous protein substrates, including pp120/HA4, a liver-specific plasma membrane glycoprotein of M(r) 20,000. Analysis of the deduced amino acid sequence of rat liver pp120/HA4 revealed two potential sites for tyrosine phosphorylation in the cytoplasmic domain (Tyr488 and Tyr513), as well as a potential cAMP-dependent protein kinase phosphorylation site (Ser503). To determine which of these sites is phosphorylated in response to insulin, each of these amino acid residues was altered by site-directed mutagenesis. Mutant cDNAs were then expressed by stable transfection in NIH 3T3 cells. Two mutations (Phe488 and Ala503) impaired insulin-induced phosphorylation of pp120/HA4, suggesting that pp120/HA4 undergoes multisite phosphorylation. It seems likely that Tyr488 is phosphorylated by the insulin receptor kinase, and phosphorylation of Ser513 may contribute to the regulation of tyrosine phosphorylation. Since pp120/HA4 is believed to be associated with a Ca2+/Mg(2+)-dependent ecto-ATPase activity, we determined the effects of insulin-induced phosphorylation on this enzymatic activity. In NIH 3T3 cells co-expressing the insulin receptor and pp120/HA4, insulin caused a 2-fold increase in ecto-ATPase activity. Moreover, elimination of the phosphorylation sites of pp120/HA4 impaired the ability of insulin to stimulate the ecto-ATPase activity. These data suggest that tyrosine phosphorylation of pp120/HA4 may regulate Ca2+/Mg(2+)-dependent ecto-ATPase activity.

3T3 Cells↗
PubMed1995

Homozygosity for a null allele of the insulin receptor gene in a patient with leprechaunism.

Mutations in the insulin receptor gene can cause genetic syndromes associated with extreme insulin resistance. We have investigated a patient with leprechaunism (leprechaun/Qatar-1) born of a consanguineous marriage. Postnatally, the proband had episodes of severe hypoglycemia and hyperinsulinernia, with blood glucose levels ranging from 0.9 to 9.9 mmol/L. The C peptide concentration with 1880 nmol/L, and the total insulin concentration was 1409 mU/L. The patient died outside the hospital at the age of four months. All 22 exons of the patient's insulin receptor gene were screened for mutations using denaturing gradient gel electrophoresis. Thereafter, the nucleotide sequences of selected exons were determined directly. The patient was homozygous for a mutation in exon 13; thirteen base pairs were deleted and replaced by a 5 b.p. sequence. This mutation shifts the reading frame and introduces a premature chain termination codon downstream in exon 13. Thus, the mutant allele is predicted to be a null allele that encodes a truncated receptor lacking both transmembrane and tyrosine kinase domains.

Alleles↗
PubMed1995

Two mutant alleles of the insulin receptor gene in a family with a genetic form of insulin resistance: a 10 base pair deletion in exon 1 and a mutation substituting serine for asparagine-462.

Mutations in the insulin receptor gene cause several genetic syndromes associated with extreme insulin resistance. We have studied three insulin resistant siblings with acanthosis nigricans, dental abnormalities, and acral hypertrophy. The female patient also had primary amenorrhea due to hyperandrogenism. All three patients were compound heterozygotes with two mutant alleles of the insulin receptor gene. One allele had a 10-bp deletion in the region of exon 1 encoding the hydrophobic signal peptide; this leads to a frameshift and premature chain termination at codon 61. The deletion occurs at the site of a direct repeat of a hexanucleotide sequence interrupted by a tetranucleotide sequence; the deletion may have resulted from recombination between the upstream and downstream hexanucleotide repeats. In the other mutant allele, there is a missense mutation substituting serine for Asn462-a mutation identified previously in one allele of the insulin receptor gene in a patient with type-A insulin resistance. The Ser462 mutation impaired the ability of acidic pH to dissociate insulin from the receptor. Thus, Thus, like the previously described Glu460 mutation, the Ser462 mutation may retard dissociation of insulin from the receptor in the acidic compartment of the endosome and may, as a result, accelerate the rate of receptor degradation.

Alleles↗
PubMed1995

Roles of 1-phosphatidylinositol 3-kinase and ras in regulating translocation of GLUT4 in transfected rat adipose cells.

Insulin stimulates glucose transport in insulin target tissues by recruiting glucose transporters (primarily GLUT4) from an intracellular compartment to the cell surface. Previous studies have demonstrated that insulin receptor tyrosine kinase activity and subsequent phosphorylation of insulin receptor substrate 1 (IRS-1) contribute to mediating the effect of insulin on glucose transport. We have now investigated the roles of 1-phosphatidylinositol 3-kinase (PI 3-kinase) and ras, two signaling proteins located downstream from tyrosine phosphorylation. Rat adipose cells were cotransfected with expression vectors that allowed transient expression of epitope-tagged GLUT4 and the other genes of interest. Overexpression of a mutant p85 regulatory subunit of PI 3-kinase lacking the ability to bind and activate the p110 catalytic subunit exerted a dominant negative effect to inhibit insulin-stimulated translocation of epitope-tagged GLUT4 to the cell surface. In addition, treatment of control cells with wortmannin (an inhibitor of PI 3-kinase) abolished the ability of insulin to recruit epitope-tagged GLUT4 to the cell surface. Thus, our data suggest that PI 3-kinase plays an essential role in insulin-stimulated GLUT4 recruitment in insulin target tissues. In contrast, over-expression of a constitutively active mutant of ras (L61-ras) resulted in high levels of cell surface GLUT4 in the absence of insulin that were comparable to levels seen in control cells treated with a maximally stimulating dose of insulin. However, wortmannin treatment of cells overexpressing L61-ras resulted in only a small decrease in the amount of cell surface GLUT4 compared with that of the same cells in the absence of wortmannin. Therefore, while activated ras is sufficient to recruit GLUT4 to the cell surface, it does so by a different mechanism that is probably not involved in the mechanism by which insulin stimulates GLUT4 translocation in physiological target tissues.

Adipocytes↗
PubMed1995

Insulin resistance associated with decreased levels of insulin-receptor messenger ribonucleic acid: evidence of a de novo mutation in the maternal allele.

Mutations in the insulin receptor gene may lead to insulin resistance and diabetes mellitus in some patients. We have studied an insulin-resistant patient with leprechaunism. Insulin binding to the patient's fibroblasts was markedly decreased. Determination of the nucleotide sequence of the patient's insulin receptor gene revealed heterozygosity for a 2-basepair deletion in exon 15. If the premessenger ribonucleic acid (pre-mRNA) is spliced normally, it causes a replacement of codon 970 in the beta-subunit with a premature chain termination codon, thereby deleting most of the intracellular domain of the receptor. The mRNA transcribed from the allele with a 2-base-pair deletion is likely to be unstable because mRNA transcripts from this allele could not be detected by complementary DNA sequencing. Northern blot analysis showed that the patient's insulin receptor mRNA was decreased by 90% compared with that of a control subject, thus suggesting that the patient is a compound heterozygote for two mutations that decrease levels of insulin receptor mRNA. This deletion mutation in exon 15 seems to be a de novo mutation, because it was not detected in either parent. Investigation of the inheritance of a silent sequence polymorphism in exon 17 provided that the deletion occurred in the maternal allele. Furthermore, linkage analysis suggests that the second mutation is derived from the patient's father, although we could not directly identify it by sequencing the coding region of the insulin receptor gene. Therefore, it is possible that this mutation is present in a regulatory domain of the insulin receptor gene, acting in cis-dominant fashion to reduce the levels of insulin receptor mRNA. Analyses of the hypervariable region in the myoglobin and pMCT118 loci were consistent with the assumption that the father and mother studied here are indeed the biological parents of the diseased patient. We hereby conclude that the patient is a compound heterozygote for two mutant alleles, both of which are responsible for the reduced levels of insulin receptor mRNA and insulin binding.

Adult↗
PubMed1994

Insulin-induced activation of phosphatidyl inositol 3-kinase. Demonstration that the p85 subunit binds directly to the COOH terminus of the insulin receptor in intact cells.

Insulin activates the insulin receptor tyrosine kinase to phosphorylate signaling molecules such as insulin receptor substrate-1 (IRS-1). Phosphorylated IRS-1 binds to SH2 domains in the p85 regulatory subunit of phosphatidyl inositol (PI) 3-kinase, thereby stimulating the catalytic activity of PI 3-kinase. For most growth factor receptor tyrosine kinases (including receptors for epidermal growth factor and platelet-derived growth factor), the p85 regulatory subunit of PI 3-kinase binds directly to phosphorylated YXXM motifs contained in the cytoplasmic domain of the receptor itself. Previous studies in cell-free systems have shown that the phosphorylated YHTM sequence (amino acid residues 1322-1325) in the COOH terminus of the insulin receptor has the ability to bind to the p85 subunit of PI 3-kinase, thereby activating the enzyme. In this investigation, we demonstrate the occurrence of the same direct binding interaction in intact cells. Subsequent to insulin-stimulated phosphorylation of the insulin receptor, a complex is formed that contains the insulin receptor and PI 3-kinase. This complex can be immunoprecipitated by antibodies directed against either the insulin receptor or the p85 subunit of PI 3-kinase. The delta 43 mutant insulin receptor that lacks 43 amino acids at the COOH terminus does not bind p85. In addition, the delta 43 truncation impairs the ability of the receptor to mediate the activation of PI 3-kinase. Thus, by binding directly to p85, the phosphorylated YHTM motif in the COOH terminus of the insulin receptor contributes partially to mediating the effect of insulin to activate PI 3-kinase.

3T3 Cells↗
PubMed1994

Insulin receptor substrate 1 mediates the stimulatory effect of insulin on GLUT4 translocation in transfected rat adipose cells.

Insulin signaling is initiated at least in part by activation of the insulin receptor tyrosine kinase and subsequent phosphorylation of cellular substrates such as insulin receptor substrate 1 (IRS-1). Previous studies have focused on the role of IRS-1 in the mitogenic actions of insulin. We have now investigated the possible role of IRS-1 in mediating the effect of insulin to stimulate glucose transport in a physiologically relevant insulin target tissue. In this study, we transfected rat adipose cells in primary culture with an antisense ribozyme directed against rat IRS-1. Expression of the ribozyme in these cells caused a 4.4-fold increase in the concentration of insulin required to achieve half-maximal stimulation of the translocation of cotransfected epitope-tagged GLUT4 without changing the maximal insulin response. Overexpression of human IRS-1 increased the basal cell surface GLUT4 to nearly the maximal level in the absence of insulin. When the ribozyme (specific to rat IRS-1) was cotransfected along with human IRS-1, the insulin dose-response curve was shifted to the left when compared with cells transfected with the ribozyme alone. These data provide strong support for the hypothesis that IRS-1 plays a role in insulin-stimulated glucose transport in insulin-responsive cells.

Adipose Tissue↗
PubMed1994

Rescue and activation of a binding-deficient insulin receptor. Evidence for intermolecular transphosphorylation.

Binding of insulin to the alpha subunit of the insulin receptor (IR) leads to autophosphorylation of the beta subunit. The reaction proceeds as intramolecular transphosphorylation between alpha beta half-receptors of the heterotetrameric receptor dimer (alpha 2 beta 2). Since IRs are mobile in the plane of the plasma membrane, it is also possible that transphosphorylation may occur between adjacent holoreceptors (alpha 2 beta 2) by an intermolecular reaction. To address this question, we cotransfected NIH-3T3 cells with two IR cDNA constructs: a truncated but functionally normal IR lacking the C-terminal 43 amino acids (delta 43) and a full-length Leu323 mutant receptor that is expressed on the cell surface but that does not bind insulin. A clonal cell line was selected from cells cotransfected with a 1/5 ratio of delta 43 cDNA/Leu323 cDNA. The two homodimers (Leu323 and delta 43) were expressed without detectable formation of hybrid receptors. By using specific antibodies, we demonstrate that in cells coexpressing both homodimers, the Leu323 mutant receptor was phosphorylated in vivo by the delta 43 IR in an insulin-dependent manner. However, when the Leu323 mutant receptor was expressed alone, no phosphorylation was detected. In addition, we demonstrate the association of the phosphorylated Leu323 mutant receptor with insulin receptor substrate-1 and with phosphatidylinositol 3-kinase. These findings indicate that insulin binding is not required for phosphorylation of the Leu323 mutant receptor, that the phosphorylation of the Leu323 mutant receptor occurs by an intermolecular transphosphorylation mechanism, and, finally, that the Leu323 mutant receptor, once phosphorylated, can associate with insulin receptor substrate-1 and phosphatidylinositol 3-kinase.

3T3 Cells↗