[Development of insulin resistance in muscle and adipose tissue during obesity in the mouse. Role of phosphatidylinositol 3-kinase].
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Adipose Tissue↗
Biomedical subjects
N Gautier
Publications and source records attributed to N Gautier.
Structures of the glycopeptidolipid antigens of Mycobacterium abscessus and Mycobacterium chelonae and possible chemical basis of the serological cross-reactions in the Mycobacterium fortuitum complex.
Mycobacterium abscessus and Mycobacterium chelonae, two members of the Mycobacterium fortuitum complex, contain five major glycolipids. A combination of NMR spectroscopy, fast atom bombardment mass spectrometry and chemical degradation was used to elucidate their structures. All the compounds belong to the family of glycopeptidolipids. A 6-deoxy-alpha-L-talosyl unit, which may bear one or two acetyl groups, invariably occupies the site of glycosylation on the threonine residue in the various compounds. A 3,4-di-O-methyl- or 2,3,4-tri-O-methyl-alpha-L-rhamnosyl unit modifies the alaninol end of the diglycosylated molecules. Both species also contain a multiglycosylated compound consisting of alpha-L-rhamnosyl-(1-->2)-3,4-di-O-methyl-alpha-L-rhamnosyl linked to alaninol, which belongs to the class of new variants of glycopeptidolipids recently described. Using an ELISA, the latter glycolipid as well as the diglycosylated ones (not previously reported to be antigenic), were shown to react with the serum raised against the whole lipid antigens of M. chelonae. A comparative serologic study of the native and chemically modified glycopeptidolipid antigens allowed the identification of their epitope as the 3,4-di-O-methyl-alpha-L-rhamnosyl residue. Similar experiments conducted on the glycopeptidolipids isolated from the serologically cross-reacting species M. peregrinum led to the conclusion that the epitope identified in M. chelonae and M. abscessus was involved in the cross-reactions and demonstrated the existence of a second haptenic moiety in the glycolipids of M. peregrinum, the 3-O-methyl-alpha-L-rhamnosyl unit. In addition to this latter non-shared epitope, the recently described sulfated glycopeptidolipid antigen of M. peregrinum did not react with the M. chelonae serum, thus further explaining the difference in the seroreactivity within the complex.
Amino Acid Sequence↗
Differential effects of okadaic acid on insulin-stimulated glucose and amino acid uptake and phosphatidylinositol 3-kinase activity.
The effect of okadaic acid, a serine/threonine phosphatase inhibitor, was analyzed in two insulin-responsive systems, the isolated mouse soleus muscle and 3T3-L1 adipocytes. While okadaic acid alone was a potent stimulator of glucose transport in both systems, it prevented transport stimulation by insulin. To gain insight into this inhibitory action, the activation of phosphatidylinositol 3-kinase (PI3-kinase), one of the earliest postreceptor steps identified so far, was studied. In 3T3-L1 adipocytes and muscle, insulin increased PI3-kinase activity in immunoprecipitates obtained with antibodies to phosphotyrosine. Okadaic acid alone had no effect but strongly inhibited this hormonal action. Okadaic acid treatment did not interfere with insulin-induced receptor autophosphorylation or with its tyrosine kinase activity toward artificial substrates. In contrast, in the presence of the phosphatase inhibitor, we did not observe tyrosine phosphorylation of the insulin receptor cellular substrate p185 (IRS-1) or immunoprecipitation of PI3-kinase by antibodies to phosphotyrosine. These results suggest that okadaic acid interferes with insulin's stimulation of glucose transport by inhibiting IRS-1 phosphorylation and its association with PI3-kinase and/or other signaling molecules. However, okadaic acid did not block the insulin stimulation of aminoisobutyric acid uptake in muscle. This would indicate that IRS-1 phosphorylation and PI3-kinase activation are not required for all the effects of insulin and that the serine/threonine phosphorylation events implicated in the translocation of glucose transporters are not controlling amino acid transport in muscle.
3T3 Cells↗
Antibodies to the extracellular receptor domain restore the hormone-insensitive kinase and conformation of the mutant insulin receptor valine 382.
A mutation substituting a valine for phenylalanine at residue 382 in the insulin receptor alpha-subunit has been found in two sisters with a genetic form of extreme insulin resistance. This receptor mutation impairs the ability of the hormone to activate autophosphorylation of solubilized receptors and phosphorylation of substrates (Accili, D., Mosthaf, L., Ullrich, A., and Taylor, S. I. (1991) J. Biol. Chem. 266, 434-439). We have previously demonstrated that in native receptors insulin induces a conformational change in the receptor beta-subunit, which is thought to be necessary for receptor activation (Baron, V., Gautier, N., Komoriya, A., Hainaut, P., Scimeca, J. C., Mervic, M., Lavielle, S., Dolais-Kitabgi, J., and Van Obberghen, E. (1990) Biochemistry 29, 4634-4641). Hence, it was thought that a defect in this conformational change might explain the functional defect of the mutant receptor. This appears to be the case, as we demonstrate here that the mutant receptor is locked in its inactive configuration. However, we found two monoclonal antibodies, directed to the extracellular domain, which are capable of restoring the mutant receptor kinase activity. The activation of the mutant receptor was accompanied by restoration of conformational changes in the beta-subunit C terminus. From these data, we draw the two following conclusions. (i) A causal link exists between receptor kinase activation and the occurrence of conformational changes. (ii) Ligands other than insulin, such as antibodies, which perturb the extracellular domain, can function as alternative ways to restore the mutant receptor kinase.
3T3 Cells↗
Expression of the glucose transporter GLUT4 in the muscular dystrophic mdx mouse.
Glucose transporter protein levels have been investigated in mdx and control (C57Bl/10) mice. Crude membrane fractions (microsomes plus plasma membranes) were prepared from skeletal muscle, heart, diaphragm and brain of 5-6-week-old and 6-7-month-old control and mdx mice. Using Western blot analysis with C-terminal-specific anti-peptide antibodies, we investigated the glucose transporters GLUT4 in the different muscle tissues and GLUT1 in brain. In skeletal tissue from the hindlegs, GLUT4 was increased by approximately 55% in mdx mice compared with control mice at both ages studied. In the diaphragm, the amount of GLUT4 protein was unchanged in young mdx mice, and was decreased by 37.4 +/- 4.7% in older mice compared with age-matched control mice. No difference was observed between mdx and control mice in the amounts of GLUT4 and GLUT1 in heart and brain preparations respectively. To determine whether the change in GLUT4 protein observed in the diaphragm and skeletal muscle of mdx mice was regulated through changes at the level of glucose transporter mRNA, Northern blot analyses were performed. In skeletal muscle, GLUT4 mRNA level per tissue was not different between the two groups of mice at both ages studied. In contrast, the decrease in the amount of GLUT4 protein observed in the diaphragm of 6-7-month-old mdx mice was accompanied by a decrease in the GLUT4 mRNA level. In conclusion, the levels of GLUT4 protein were modified in muscle tissues from mdx compared with control mice, and these modifications were different depending on the muscle involved and the age of the mice. An increase in the amount of GLUT4 protein in the skeletal muscle of mdx mice was not due to changes at the mRNA level. The diaphragms of 6-7-month-old mdx mice exhibited decreases in GLUT4 protein and mRNA levels that were not detected in young animals (5-6 weeks old).
Animals↗
Defect in skeletal muscle phosphatidylinositol-3-kinase in obese insulin-resistant mice.
Activation of phosphatidylinositol-3-kinase (PI3K) is one of the earliest postreceptor events in the insulin signaling pathway. Incubation of soleus muscles from lean mice with 50 nM insulin caused a 3-10-fold increase in antiphosphotyrosine-immunoprecipitable PI3K (antiPTyr-PI3K) activity within 2 min in muscle homogenates as well as both the cytosolic and membrane fractions. Insulin did not affect total PI3K activity. Both the antiPTyr-PI3K stimulation and activation of insulin receptor tyrosine kinase were dependent on hormone concentration. In muscles from obese, insulin-resistant mice, there was a 40-60% decrease in antiPTyr-PI3K activity after 2 min of insulin that was present equally in the cytosolic and membrane fractions. A significant reduction in insulin sensitivity was also observed. The defect appears to result from alterations in both insulin receptor and postreceptor signaling. Starvation of obese mice for 48 h, which is known to reverse insulin resistance, normalized the insulin response of both PI3K and the receptor tyrosine kinase. The results demonstrate that: (a) antiPTyr-PI3K activity is responsive to insulin in mouse skeletal muscle, (b) both the insulin responsiveness and sensitivity of this activity are blunted in insulin-resistant muscles from obese mice, (c) these alterations result from a combination of insulin receptor and postreceptor defects, and (d) starvation restores normal insulin responses.
Animals↗
The insulin receptor activation process involves localized conformational changes.
The molecular process by which insulin binding to the receptor alpha-subunit induces activation of the receptor beta-subunit with ensuing substrate phosphorylation remains unclear. In this study, we aimed at approaching this molecular mechanism of signal transduction and at delineating the cytoplasmic domains implied in this process. To do this, we used antipeptide antibodies to the following sequences of the receptor beta-subunit: (i) positions 962-972 in the juxtamembrane domain, (ii) positions 1247-1261 at the end of the kinase domain, and (iii) positions 1294-1317 and (iv) positions 1309-1326, both in the receptor C terminus. We have previously shown that insulin binding to its receptor induces a conformational change in the beta-subunit C terminus. Here, we demonstrate that receptor autophosphorylation induces an additional conformational change. This process appears to be distinct from the one produced by ligand binding and can be detected in at least three different beta-subunit regions: the juxtamembrane domain, the kinase domain, and the C terminus. Hence, the cytoplasmic part of the receptor beta-subunit appears to undergo an extended conformational change upon autophosphorylation. By contrast, the insulin-induced change does not affect the juxtamembrane domain 962-972 nor the kinase domain 1247-1261 and may be limited to the receptor C terminus. Further, we show that the hormone-dependent conformational change is maintained in a kinase-deficient receptor due to a mutation at lysine 1018. Therefore, during receptor activation, the ligand-induced change could precede ATP binding and receptor autophosphorylation. We propose that insulin binding leads to a transient receptor form that may allow ATP binding and, subsequently, autophosphorylation. The second conformational change could unmask substrate-binding sites and stabilize the receptor in an active conformation.
Animals↗
Structure of mycoside F, a family of trehalose-containing glycolipids of Mycobacterium fortuitum.
Nuclear magnetic resonance spectroscopy, fast-atom bombardment mass spectrometry, gas chromatography-mass spectrometry, as well as chemical degradations were used to elucidate the structure of the major glycolipids of Mycobacterium fortuitum. Three main glycoconjugates were detected and their structures established as 2,3-diacyl, 2,3,4- and 2,3,6-triacyl trehalose. The characteristic infrared spectrum which led to their original designation as mycoside F, a family of glycolipids limited in distribution to M. fortuitum, was due to the nature of the fatty acyl substiuents identified primarily as 2-methyl-octadecen-2-oyl. The antigenic glycolipids typified the biovar. fortuitum, thus allowing its easy recognition from the C-mycoside glycopeptidolipid-containing biovar. peregrinum.
Fatty Acids↗
Antipeptide antibody to the insulin-like growth factor-I receptor sequence 1232-1246 inhibits the receptor kinase activity.
To approach the question of why insulin-like growth factor-I (IGF-I) and insulin have different physiological actions, we developed antibodies directed against cytoplasmic regions of the IGF-I receptor exhibiting a low degree of homology with the corresponding sequences of the insulin receptor. We found that an antipeptide antibody directed against the beta-subunit carboxyl-terminal sequence (1232-1246) of the IGF-I receptor significantly reduced the in vitro receptor autophosphorylation. The ability of the synthetic peptide corresponding to the IGF-I receptor sequence 1232-1246 to abolish this inhibitory effect reflects the specific nature of the antibody interaction with the targeted domain in the receptor. Antipeptide antibody to IGF-I receptor sequence 1232-1246 also decreased receptor phosphorylation activity toward the exogenous substrate poly(Glu/Tyr). The reduction in poly(Glu/Tyr) phosphorylation was seen even when the antibody was incubated with a receptor previously activated and phosphorylated. Therefore, the inhibitory action on substrate phosphorylation is likely to be unrelated to the antibody reduction of receptor autophosphorylation but rather results from a global decrease in receptor enzymatic activity. The effect of the antipeptide antibody on receptor tyrosine kinase cannot be accounted for by a lowering of the receptor Km for ATP or of its affinity for the substrate poly(Glu/Tyr). Moreover, the interaction of the antibody with the receptor had no repercussion on the ligand binding site as shown by the unaltered IGF-I binding. Taken together our data suggest that the beta-subunit carboxyl-terminal domain of the IGF-I receptor plays a key role in regulating its kinase activity and that the particular sequence recognized by our antipeptide antibody could be involved in negative regulation of receptor functioning.
3T3 Cells↗
Potential involvement of the carboxy-terminus of the Glut 1 transporter in glucose transport.
The role of the carboxy-terminal domain of the Glut 1 glucose transporter was investigated using an antipeptide antibody to the C-terminal part of the molecule. The study was performed in fibroblasts transfected with the cDNA coding for the human insulin receptor. These cells acutely respond to insulin for glucose transport. Using antipeptide antibodies to Glut 1 and Glut 4, we first established that these cells expressed only Glut 1. Then, to define the role of the C-terminal part of Glut 1 in glucose transport, the antibodies were loaded into the cells by electroporation. When anti-Glut 1 immunoglobulins were introduced into the cells, a 60% increase in basal deoxyglucose and 3-O-methylglucose transport was observed compared to that in cells electroporated with nonimmune immunoglobulins. The stimulatory action of the antipeptide was not due to an increase in the total amount of transporters. It was found only at low glucose concentrations, suggesting that the affinity of the transporter, rather than its maximal capacity, was changed. Finally, the effect of antibody was additive to that of insulin. The interaction between the anti-Glut 1 antibody and the carboxy-tail of the transporter seems to lead to an increase in the intrinsic activity of the transporter, suggesting that this part of the molecule could be implicated in the regulation of glucose uptake.
3-O-Methylglucose↗
Activation and regulation of the insulin receptor kinase.
For the insulin receptor and the EGF receptor it is believed that ligand occupancy results in interactions within the heterotetrameric alpha 2 beta 2 insulin receptor or between monomeric EGF receptors. These interactions then activate the intracellular receptor tyrosine kinase which induces receptor autophosphorylation and phosphorylation of cellular substrates. In the present study we have approached the nature of this receptor activation and autophosphorylation. We have investigated whether these phenomena occur via an intra--or an intermolecular process. To this end the following receptor model system consisting of two receptors was co-expressed in NIH 3T3 cells: a kinase inactive human insulin receptor (HIR K1018A) and a chimeric (EIR) receptor corresponding to the extracellular and transmembrane domains of the human EGF receptor and the cytosolic domain of the human insulin receptor beta subunit. Using this system we found that stimulation of the cells with EGF induced tyrosine autophosphorylation of the EGF-insulin receptor chimera (150 kd) and tyrosine phosphorylation of the beta-subunit of the kinase-deficient insulin receptor (95 kd). The phosphopeptides of the autophosphorylated cytoplasmic domain of the EGF-insulin receptor chimera were comparable to those of the transphosphorylated beta subunit of the kinase-deficient insulin receptor and the wild type human insulin receptor. When immunoaffinity purified EGF-insulin receptor hybrids and kinase-deficient insulin receptors were used in a cell lysate phosphorylation assay, it was found that addition of EGF produced [32P]-labeling of both receptor species. In conclusion, we have shown that tyrosine transphosphorylation can occur between homologous receptor domains. This transphosphorylation and transactivation could be a possible mechanism for signal amplification.2+ domain could influence interactions between the receptor and cellular structures and, as such, play a key role in signal transduction.
Animals↗
The carboxyl-terminal domain of the insulin receptor: its potential role in growth-promoting effects.
The role of the insulin receptor carboxyl-terminal domain in regulation of insulin signal transduction was studied with antipeptide antibodies against the sequence 1321-1338, which contains two autophosphorylation sites, tyrosine 1328 and tyrosine 1334. The antibodies were introduced by electroporation in murine fibroblasts transfected with an expression plasmid encoding the human insulin receptor. We found that introduction of these antipeptides into cells stimulated cellular proliferation, compared to cells loaded with nonimmune Ig. In contrast, neither glucose transport nor amino acid transport was stimulated by the antibodies. Despite its stimulatory effect on cell growth, the injected antipeptide did not enhance phosphorylation of ribosomal protein S6. In vitro, anti-C1 antipeptide stimulated insulin receptor autophosphorylation but did not increase receptor-mediated phosphorylation of the copolymer (glutamate/tyrosine, 4/1), while histone phosphorylation was increased. We interpret our results to mean that perturbation of the receptor C-terminus could lead to phosphorylation of selected substrates, which may be involved in cell growth regulation. Taken together, our data suggest that (i) insulin receptor mediated stimulation of cell growth and stimulation of ribosomal protein S6 phosphorylation result from divergent signaling pathways and (ii) the insulin receptor C-terminal domain exerts an inhibition on the growth signal mediated by the receptor. This inhibition appears to be released upon insulin binding to receptor or by interaction of the antipeptide with the receptor.
Amino Acids↗
Effects of insulin, insulin-like growth factor-I (IGF-I) and progesterone on glucose and amino acid uptake in Xenopus laevis oocytes.
In Xenopus oocytes, insulin or IGF-I activated glucose transport and maturation, but not amino acid transport, as measured by the uptake of alanine. Glucose transporter identical or closely related to the mammalian erythroid/brain transporter (Glut-1/EGT) were found in oocyte membranes. The EC50 for stimulation of glucose uptake and of maturation were similar (1-5 x 10(-8) M for insulin and 2-8 x 10(-10) M for IGF-I), confirming that these effects were mediated through IGF-I receptors. Other agents, such as phorbol 12-myristate 13-acetate (TPA) (0.5 microM) and vanadate (2 mM) evoked only part of the insulin effect on glucose uptake (50% and 65%, respectively), without being additive to insulin. In contrast, progesterone at 1 microM, a potent inducer of maturation, inhibited at least partially the insulin-induced glucose uptake. Uptake of alanine and glucose was decreased after prolonged incubations (3-6 h) with agents that trigger maturation, and was dramatically reduced in oocytes that have undergone maturation (unfertilized eggs). Maturation is thus accompanied by a reduction in glucose and amino acid transports. These result further document the validity of Xenopus oocytes as a model to study insulin and IGF-I signalling.
Alanine↗
Insulin binding to its receptor induces a conformational change in the receptor C-terminus.
Antibodies against peptides corresponding to sequences in the C-terminus of the insulin receptor beta-subunit were used to approach the putative role of this receptor domain in signal generation. Two sequences were chosen and correspond to peptide C1, comprising amino acids 1309-1326, and peptide C2, comprising amino acids 1294-1317. The two antibodies produced distinct immunoprecipitation patterns as a function of the insulin receptor form and recognized changes in the insulin receptor molecule induced by ligand binding and autophosphorylation. Both antipeptides, anti-C1 and anti-C2, showed an important decrease in their recognition capacity for the receptor occupied by insulin when compared to the empty receptor. Further, anti-C1 had a lower affinity for the phosphorylated receptor compared to the unphosphorylated receptor and failed to recognize a fraction of the phosphoreceptor population. In contrast, anti-C2 had similar affinities for the phosphorylated and unphosphorylated receptors but was unable to interact with part of the unphosphorylated receptors. Finally, using immunoblotting of the receptor to analyze the denatured molecules, we showed that the phosphorylation-induced changes detected by anti-C1 are retained, suggesting that they are likely not of a conformational nature. In contrast, the insulin-induced changes in the receptor molecule disappear with receptor denaturation which points to their reversible nature. We conclude from these data that (i) antipeptides against the receptor C-terminal sequence are able to distinguish between phosphorylated and unphosphorylated receptor forms and (ii) binding of insulin to its receptor leads to a reversible, phosphorylation-independent, and possibly conformational change at the level of the receptor C-terminal domain.
Animals↗
Object-centered representation for species systematics and identification in living systems in nature.
An artificial intelligence application to identification in biological taxonomy is presented. An object-centered representation is described using SHIRKA, a knowledge-based system developed in France. The capacities of this system, relating to a classification algorithm, are described. An application to the identification of the trees and lianas of the evergreen forests of the Western Ghats (India) is presented to illustrate the problems encountered in the development of the knowledge base. A model of a systematist's reasoning, using evolutionary principles, is the basis of this work. Finally the efficiency of the system is discussed.
Artificial Intelligence↗
Antibodies to insulin receptor tyrosine kinase stimulate its activity towards exogenous substrates without inducing receptor autophosphorylation.
Anti-peptide antibodies directed against a highly-conserved sequence of the insulin receptor tyrosine kinase domain have been used to study the relationship between this specific region and kinase activation. Antibodies have been prepared by the injection into a rabbit of a synthetic peptide (P2) corresponding to residues 1110-1125 of the proreceptor. The peptide exhibits 88-95% sequence similarity with the corresponding sequence in the v-ros protein and in receptors for epidermal growth factor and for insulin-like growth factor 1. Two antibodies with different specificities could be separated from total antiserum obtained after immunization with P2. One antibody [anti-(P-Tyr)] cross-reacted with phosphotyrosine and immunoprecipitated solely autophosphorylated receptors. This antibody was shown to increase or decrease the receptor tyrosine kinase activity depending on its concentration. In all circumstances receptor autophosphorylation and substrate phosphorylation were modulated in a parallel fashion. The second antibody (anti-P2) failed to immunoprecipitate the insulin receptor, but was found to interact with both the peptide and the receptor by e.l.i.s.a. assay. Using a tyrosine co-polymer we found that anti-P2 activated the insulin receptor kinase leading to substrate phosphorylation at a level similar to that observed with insulin. This effect was additive to the hormonal effect. In contrast, receptor autophosphorylation was not modified by the anti-peptide. The differential effect of this anti-peptide further supports the idea that receptor autophosphorylation and kinase activity towards exogenous substrates might be independently regulated. Finally, our data suggest that conformational changes in the receptor tyrosine kinase domain may be sufficient for activation of its enzymic activity.
Animals↗
Use of an anti-insulin receptor antibody to discriminate between metabolic and mitogenic effects of insulin: correlation with receptor autophosphorylation.
In a previous report we described the properties of a rabbit anti-insulin receptor antibody (RAIR-IgG) and its effects on the autophosphorylation and kinase activity of human insulin receptors. The present study was carried out on the hepatoma cell line Fao. We tested the mimetic effects of RAIR-IgG on different biological parameters known to be stimulated by insulin, receptor autophosphorylation and kinase activity. RAIR-IgG stimulated the metabolic effects (glucose and amino acid transport) but, unlike insulin, was unable to promote cell proliferation. These data clearly demonstrated the existence of two distinctly controlled pathways in the mediation of the hormonal response. When we investigated the effects of this antibody at the molecular level we found that in a cell-free system RAIR-IgG weakly stimulated receptor autophosphorylation on non-regulatory sites and failed to stimulate tyrosine kinase activity toward exogenous substrates. Accordingly, RAIR-IgG did not stimulate receptor autophosphorylation in 32P-labelled intact cells. Interestingly, under similar conditions RAIR-IgG elicited ribosomal S6 protein phosphorylation, as did insulin. The possibility that RAIR-IgG activated a cryptic tyrosine kinase activity is discussed.
Amino Acid Sequence↗
Insulin and rabbit anti-insulin receptor antibodies stimulate additively the intrinsic receptor kinase activity.
This paper describes the properties of rabbit polyclonal antibodies directed against purified human insulin receptor which strongly stimulate the intrinsic tyrosine kinase activity. The stimulatory effect of the antibodies on the kinase activity was obtained on the insulin receptor autophosphorylation as well as on the kinase activity towards a synthetic substrate. This stimulation is additive to that induced by insulin. Moreover, rabbit antibodies do not impair insulin binding. These data strongly suggest that antibodies and insulin act through separate pathways. This conclusion is reinforced by the differences observed on the phosphopeptide maps of the receptor's beta subunit whose phosphorylation was performed either in the presence of insulin or rabbit antibodies. Interestingly, these polyclonal antibodies can also induce an activation of the receptor autophosphorylation by interacting only with extracellular determinants. The anti-insulin receptor antibodies mimic insulin in their stimulatory effect on amino acid (AIB) uptake, but they have a different effect to that found on the kinase activity; the simultaneous addition of the antiserum and insulin failed to stimulate this amino acid transport over the level induced by a saturating concentration of hormone.
Antibodies↗