PubMed Health⌕ Search

Biomedical subjects

J M Tavaré

Publications and source records attributed to J M Tavaré.

At least 19 recordsLinked to original sources
PubMed2006

Regulation of small GTP-binding proteins by insulin.

Several members of the extensive family of small GTP-binding proteins are regulated by insulin, and have been implicated in insulin action on glucose uptake. These proteins are themselves negatively regulated by a series of specific GAPs (GTPase-activating proteins). Interestingly, there is increasing evidence to suggest that PKB (protein kinase B)-dependent phosphorylation of some GAPs may relieve this negative regulation and so lead to the activation of the target small GTP-binding protein. We review recent evidence that this may be the case, and place specific emphasis on the role of these pathways in insulin-stimulated glucose uptake.

Animals↗
PubMed2005

Role of protein kinase B in insulin-regulated glucose uptake.

The activation of protein kinase B (or Akt) plays a central role in the stimulation of glucose uptake by insulin. Currently, however, numerous questions remain unanswered regarding the role of this kinase in bringing about this effect. For example, we do not know precisely where in the GLUT4 trafficking pathway this kinase acts. Nor do we know which protein substrates are responsible for mediating the effects of protein kinase B, although two recently identified proteins (AS160 and PIKfyve) may play a role. This paper addresses these important questions by reviewing recent progress in the field.

Animals↗
PubMed2002

Molecular mechanisms of insulin-stimulated glucose uptake in adipocytes.

The stimulation of muscle and adipose tissue glucose metabolism, which is ultimately responsible for bringing about post-absorptive blood glucose clearance, is the primary clinically relevant action of insulin. Insulin acts on many steps of glucose metabolism, but one of the most important effects is its ability to increase the rate of cellular glucose transport. This results from the translocation of the insulin-responsive transporter isoform, GLUT4, from intra-cellular vesicular storage sites to the plasma membrane. In adipocytes, a substantial amount of cellular GLUT4 is located in a specific highly insulin-responsive storage pool, termed GLUT4 Storage Vesicles (GSVs). GLUT4 can also translocate to the plasma membrane from the recycling endosomal pool which also additionally contains the GLUT1 isoform of glucose transporter and the transferrin receptor. In this article we review the molecular mechanism by which insulin stimulates GLUT4 translocation in adipose cells, including the nature of the signaling pathways involved and the role of the cytoskeleton.

Adipocytes↗
PubMed2001

Lighting up insulin action.

Understanding the mechanism of insulin action remains one of the most important challenges in modern medical biology. Recent advances in cell imaging techniques, increased processing power of computers and the internet, and the introduction of novel fluorescent reagents such as green fluorescent proteins (GFPs) have revolutionized our ability to scrutinize insulin action by time-lapse microscopy at the single-cell level. This article outlines some of the advances made in the authors' laboratory, with particular reference to imaging the movements of the insulin-sensitive glucose transporter, GLUT4, and the generation of phosphoinositide lipids.

Animals↗
PubMed2001

Using green fluorescent protein to study intracellular signalling.

Subcellular compartmentalisation of signalling molecules is an important phenomenon not only in defining how a signalling pathway is activated but also in influencing the desired physiological output of that pathway (e.g. cell growth or differentiation, regulation of metabolism, cytoskeletal changes etc.). Biochemical analyses of protein and lipid compartmentalisation by, for example, subcellular fractionation presents many technical difficulties. However, this aspect of cell signalling research has seen a major revolution thanks to the cloning and availability of a variety of mutant green fluorescent protein derivatives with distinct molecular properties. Mutants with increased brightness, altered excitation and emission maxima, altered stability and differential sensitivity to pH, are now in widespread use for following the trafficking and function of proteins in living cells and for monitoring the intracellular environment. In this review we focus on some of the recent developments in the use of green fluorescent proteins for studying intracellular signalling pathways often with special reference to the actions of insulin. We also discuss the future utility of these proteins to analyse protein--protein interactions in signalling pathways using fluorescence resonance energy transfer.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2000

Role for the microtubule cytoskeleton in GLUT4 vesicle trafficking and in the regulation of insulin-stimulated glucose uptake.

Insulin stimulates glucose uptake into adipocytes by promoting the translocation of the glucose transporter isoform 4 (GLUT4) from intracellular vesicles to the plasma membrane. In 3T3-L1 adipocytes GLUT4 resides both in an endosomal pool, together with transferrin receptors, and in a unique pool termed 'GLUT4 storage vesicles' (GSVs), which excludes endosomal proteins. The trafficking of GLUT4 vesicles was studied in living 3T3-L1 adipocytes by time-lapse confocal microscopy of GLUT4 tagged with green fluorescent protein. GLUT4 vesicles exhibited two types of motion: rapid vibrations around a point and short (generally less than 10 microm) linear movements. The linear movements were completely blocked by incubation of the cells in the presence of microtubule-depolymerizing agents. This suggests that a subpopulation of GLUT4 vesicles can exhibit motor-driven movements along microtubules. Upon further examination, microtubule depolymerization inhibited insulin-stimulated glucose uptake and GLUT4 translocation to the plasma membrane by approx. 40%, but had no effect on insulin-induced translocation of the transferrin receptor to the plasma membrane from endosomes. We propose that an intact microtubule cytoskeleton may be required for optimal trafficking of GLUT4 present in the GSV pool, but not that resident in the endosomal pool.

3T3 Cells↗
PubMed2000

Rapid caspase-3 activation during apoptosis revealed using fluorescence-resonance energy transfer.

Caspase-3 is a crucial component of the apoptotic machinery in many cell types. Here, we report the timescale of caspase-3 activation in single living cells undergoing apoptosis. This was achieved by measuring the extent of fluorescence resonance energy transfer within a recombinant substrate containing cyan fluorescent protein (CFP) linked by a short peptide possessing the caspase-3 cleavage sequence, DEVD, to yellow fluorescent protein (YFP; i.e. CFP-DEVD-YFP). We demonstrate that, once initiated, the activation of caspase-3 is a very rapid process, taking 5 min or less to reach completion. Furthermore, this process occurs almost simultaneously with a depolarization of the mitochondrial membrane potential. These events occur just prior to the characteristic morphological changes associated with apoptosis. Our results clearly demonstrate that, once initiated, the commitment of cells to apoptosis is a remarkably rapid event when visualized at the single cell level.

Animals↗
PubMed1999

Confocal imaging of the subcellular distribution of phosphatidylinositol 3,4,5-trisphosphate in insulin- and PDGF-stimulated 3T3-L1 adipocytes.

The activation of phosphatidylinositol 3-kinase (PI 3-kinase) and production of PtdIns(3,4,5)P(3) is crucial in the actions of numerous extracellular stimuli, including insulin-stimulated glucose uptake. Platelet-derived growth factor (PDGF) also stimulates PI 3-kinase, but only weakly promotes glucose uptake when compared with insulin. Insulin and PDGF have thus been proposed to have differential effects on the subcellular targeting of PI 3-kinase. However, owing to a lack of suitable methodologies, the subcellular localization of the PtdIns(3,4,5)P(3) generated has not been examined. The pleckstrin-homology (PH) domains of the nucleotide exchange factors, ADP-ribosylation factor nucleotide-binding-site opener (ARNO) and general receptor for 3-phosphoinositides (GRP1), which have a high affinity and specificity for PtdIns(3,4,5)P(3), were fused to green fluorescent protein and used to examine the subcellular localization of PtdIns(3,4,5)P(3) generation in living 3T3-L1 adipocytes. PtdIns(3,4,5)P(3) was produced almost exclusively in the plasma membrane in response to both agonists, although the response to insulin was greater in magnitude and occurred in considerably more cells. The results suggest that the greater ability of insulin to stimulate glucose uptake may be the result of its ability to generate significantly more plasma-membrane PtdIns(3, 4,5)P(3) than PDGF. ARNO and GRP1 are nucleotide exchange factors for the small GTP-binding protein ADP-ribosylation factor 6 (ARF6). The inability of a constitutively active GTPase-deficient mutant of ARF6 (ARF6-Q67L; Gln(67)-->Leu) to cause glucose transporter GLUT4 translocation suggests that activation of this pathway is not sufficient to cause GLUT4 translocation.

3T3 Cells↗
PubMed1999

Trafficking of Glut4-green fluorescent protein chimaeras in 3T3-L1 adipocytes suggests distinct internalization mechanisms regulating cell surface glut4 levels.

Insulin stimulates glucose transport in adipose and muscle tissue by stimulating the movement ('translocation') of an intracellular pool of glucose transporters (the Glut4 isoform) to the plasma membrane. We have engineered a series of chimaeras between Glut4 and green fluorescent protein (GFP) from Aequoria victoria and expressed these proteins in 3T3-L1 adipocytes by microinjection of plasmid cDNA. In the absence of insulin, GFP-Glut4 is localized intracellularly within a perinuclear compartment and multiple intracellular punctate structures. In response to insulin, chimaeric GFP-Glut4 species exhibit a profound redistribution to the cell surface with kinetics comparable with the endogenous protein. The intracellular localization of GFP-Glut4 overlaps partially with compartments labelled with Texas Red transferrin, but is largely distinct from intracellular structures identified using Lysotracker-Red(R). K(+)-depletion resulted in the accumulation of GFP-Glut4 at the cell surface, but to an lesser extent than that observed in response to insulin. In contrast with native Glut4, removal of the insulin stimulus or treatment of insulin-stimulated cells with phosphatidylinositol 3'-kinase inhibitors did not result in re-internalization of the chimaeric GFP-Glut4 from the plasma membrane, suggesting that the recycling properties of this species differ from the native Glut4 molecule. We suggest that the recycling pathway utilized by GFP-Glut4 in the absence of insulin is distinct from that used to internalize GFP-Glut4 from the plasma membrane after withdrawal of the insulin stimulus, which may reflect distinct pathways for internalization of endogenous Glut4 in the presence or absence of insulin.

3T3 Cells↗
PubMed1999

The protein kinase C inhibitors bisindolylmaleimide I (GF 109203x) and IX (Ro 31-8220) are potent inhibitors of glycogen synthase kinase-3 activity.

Here we report that the widely used protein kinase C inhibitors, bisindolylmaleimide I and IX, are potent inhibitors of glycogen synthase kinase-3 (GSK-3). Bisindolylmaleimide I and IX inhibited GSK-3 in vitro, when assayed either in cell lysates (IC(50) 360 nM and 6.8 nM, respectively) or in GSK-3beta immunoprecipitates (IC(50) 170 nM and 2.8 nM, respectively) derived from rat epididymal adipocytes. Pretreatment of adipocytes with bisindolylmaleimide I (5 microM) and IX (2 microM) reduced GSK-3 activity in total cell lysates, to 25.1+/-4.3% and 12.9+/-3.0% of control, respectively. By contrast, bisindolylmaleimide V (5 microM), which lacks the functional groups present on bisindolylmaleimide I and IX, had little apparent effect. We propose that bisindolylmaleimide I and IX can directly inhibit GSK-3, and that this may explain some of the previously reported insulin-like effects on glycogen synthase activity.

Adipocytes↗
PubMed1999

Protein kinase B stimulates the translocation of GLUT4 but not GLUT1 or transferrin receptors in 3T3-L1 adipocytes by a pathway involving SNAP-23, synaptobrevin-2, and/or cellubrevin.

An interaction of SNAP-23 and syntaxin 4 on the plasma membrane with vesicle-associated synaptobrevin-2 and/or cellubrevin, known as SNAP (soluble N-ethyl-maleimide-sensitive factor attachment protein) receptors or SNAREs, has been proposed to provide the targeting and/or fusion apparatus for insulin-stimulated translocation of the GLUT4 isoform of glucose transporter to the plasma membrane. By microinjecting 3T3-L1 adipocytes with the Clostridium botulinum toxin B or E, which proteolyzed synaptobrevin-2/cellubrevin and SNAP-23, respectively, we investigated the role of these SNAREs in GLUT4, GLUT1, and transferrin receptor trafficking. As expected, insulin stimulated the translocation of GLUT4, GLUT1, and transferrin receptors to the plasma membrane. By contrast, a constitutively active protein kinase B (PKB-DD) only stimulated a translocation of GLUT4 and not GLUT1 or the transferrin receptor. The GLUT4 response to PKB-DD was abolished by toxins B or E, whereas the insulin-evoked translocation of GLUT4 was inhibited by approximately 65%. These toxins had no significant effect on insulin-stimulated transferrin receptor appearance at the cell surface. Thus, insulin appears to induce GLUT4 translocation via two distinct routes, only one of which involves SNAP-23 and synaptobrevin-2/cellubrevin, and can be mobilized by PKB-DD. The PKB-, SNAP-23-, and synaptobrevin-2/cellubrevin-independent GLUT4 translocation pathway may involve movement through recycling endosomes, together with GLUT1 and transferrin receptors.

3T3 Cells↗
PubMed1999

Identification of centaurin-alpha1 as a potential in vivo phosphatidylinositol 3,4,5-trisphosphate-binding protein that is functionally homologous to the yeast ADP-ribosylation factor (ARF) GTPase-activating protein, Gcs1.

Centaurin-alpha is a 46 kDa in vitro binding protein for the lipid second messenger PtdIns(3,4,5)P3. In this report we have addressed whether centaurin-alpha1, a human homologue of centaurin-alpha, binds PtdIns(3,4,5)P3 in vivo and furthermore, identified a potential physiological function for centaurin-alpha1. Using confocal microscopy of live PC12 cells, transiently transfected with a chimera of green fluorescent protein (GFP) fused to the N-terminus of centaurin-alpha1 (GFP-centaurin-alpha1), we demonstrated the rapid plasma membrane recruitment of cytosolic GFP-centaurin-alpha1 following stimulation with either nerve growth factor or epidermal growth factor. This recruitment was dependent on the centaurin-alpha1 pleckstrin homology domains and was blocked by the PtdIns(4,5)P2 3-kinase (PI 3-kinase) inhibitors wortmannin (100 nM) and LY294002 (50 microM), and also by co-expression with a dominant negative p85. Functionally, we demonstrated that centaurin-alpha1 could complement a yeast strain deficient in the ADP-ribosylation factor (ARF) GTPase-activating protein Gcs1; a complementation that was blocked by mutagenesis of conserved cysteine residues within the ARF GTPase-activating protein analogous domain of centaurin-alpha1. Taken together, our data demonstrated that centaurin-alpha1 could potentially function as an ARF GTPase-activating protein that, on agonist stimulation, was recruited to the plasma membrane possibly through an ability to interact with PtdIns(3,4,5)P3.

ADP-Ribosylation Factors↗
PubMed1999

Sustained phosphorylation and activation of protein kinase B correlates with brain-derived neurotrophic factor and insulin stimulated survival of cerebellar granule cells.

Brain-derived neurotrophic factor (BDNF) and insulin promote the survival of 6-7 day old post-natal rat cerebellar granule cells. Previous studies using the PI3 kinase inhibitor, wortmannin and the over-expression of protein kinase B (PKB) have indicated that both PI3 kinase and PKB activation are central for insulin-stimulated survival of these neurones. Here we report that BDNF, insulin and epidermal growth factor (EGF) all cause the phosphorylation and stimulation of endogenous PKB activity, though with differing profiles. The addition of BDNF, or insulin resulted in a rapid and sustained phosphorylation and stimulation of PKB activity, whilst EGF stimulation, which does not promote survival, caused a more transient phosphorylation and stimulation of PKB activity. We also investigated the involvement of the PKB substrate, glycogen synthase kinase 3 (GSK 3). All three growth factors caused the inactivation of GSK-3beta, suggesting that the inactivation of GSK-3beta does not correlate with survival.

Animals↗
PubMed1999

EGF-and NGF-stimulated translocation of cytohesin-1 to the plasma membrane of PC12 cells requires PI 3-kinase activation and a functional cytohesin-1 PH domain.

ADP-ribosylation factors (ARFs) are small GTP-binding proteins that function as regulators of eukaryotic vesicle trafficking. Cytohesin-1 is a member of a family of ARF guanine nucleotide-exchange factors that contain a C-terminal pleckstrin homology (PH) domain which has been proposed to bind the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3). Here we demonstrate that in vitro, recombinant cytohesin-1 binds, via its PH domain, the inositol head group of PIP3, inositol 1,3,4, 5-tetrakisphosphate (IP4), with an affinity greater than 200-fold higher than the inositol head group of either phosphatidylinositol 4, 5-bisphosphate or phosphatidylinositol 3,4-bisphosphate. Moreover, addition of glycerol or diacetylglycerol to the 1-phosphate of IP4 does not alter the ability to interact with cytohesin-1, data which is entirely consistent with cytohesin-1 functioning as a putative PIP3 receptor. To address whether cytohesin-1 binds PIP3 in vivo, we have expressed a chimera of green fluorescent protein (GFP) fused to the N terminus of cytohesin-1 in PC12 cells. Using laser scanning confocal microscopy we demonstrate that either EGF- or NGF-stimulation of transiently transfected PC12 cells results in a rapid translocation of GFP-cytohesin-1 from the cytosol to the plasma membrane. This translocation is dependent on the cytohesin-1 PH domain and occurs with a time course that parallels the rate of plasma membrane PIP3 production. Furthermore, the translocation requires the ability of either agonist to activate PI 3-kinase, since it is inhibited by wortmannin (100 nM), LY294002 (50 microM) and by coexpression with a dominant negative p85. This data therefore suggests that in vivo cytohesin-1 can interact with PIP3 via its PH domain.

Animals↗
PubMed1998

Insulin-stimulated expression of c-fos, fra1 and c-jun accompanies the activation of the activator protein-1 (AP-1) transcriptional complex.

The activator protein-1 (AP-1) transcriptional complex is made up of members of the Fos (c-Fos, FosB, Fra1, Fra2) and Jun (c-Jun, JunB, JunD) families and is stimulated by insulin in several cell types. The mechanism by which insulin activates this complex is not well understood but it is dependent on the activation of the Erk1 and Erk2 isoforms of mitogen-activated protein kinases. In the current study we show that the AP-1 complex isolated from insulin-stimulated cells contained c-Fos, Fra1, c-Jun and JunB. The activation of the AP-1 complex by insulin was accompanied by (i) a transient increase in c-fos expression, and the transactivation of the ternary complex factors Elk1 and Sap1a, in an Erk1/Erk2-dependent fashion; (ii) a substantial increase in the expression of Fra1 protein and mRNA, which was preceded by a transient decrease in its electrophoretic mobility upon SDS/PAGE, indicative of phosphorylation; and (iii) a sustained increase in c-jun expression without increasing c-Jun phosphorylation on serines 63 and 73 or activation of the stress-activated kinase JNK/SAPK. In conclusion, insulin appears to stimulate the activity of the AP-1 complex primarily through a change in the abundance of the components of this complex, although there may be an additional role for Fra1 phosphorylation.

Animals↗
PubMed1998

Nerve growth factor- and epidermal growth factor-stimulated translocation of the ADP-ribosylation factor-exchange factor GRP1 to the plasma membrane of PC12 cells requires activation of phosphatidylinositol 3-kinase and the GRP1 pleckstrin homology domain.

ADP-ribosylation factors (ARFs) are small GTP-binding proteins that are regulators of vesicle trafficking in eukaryotic cells. GRP1 is a member of a family of ARF guanine-nucleotide-exchange factors that binds in vitro the lipid second messenger phosphatidylinositol 3,4, 5-trisphosphate [PtdIns(3,4,5)P3]. In order to study the effects of PtdIns(3,4,5)P3 on the function of GRP1, we have cloned the human homologue of GRP1, encoding for a protein which is 98.8% identical to mouse brain GRP1. Human GRP1 binds, via its pleckstrin homology (PH) domain, the inositol head group of PtdIns(3,4,5)P3, inositol 1, 3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4], with high affinity (Kd 32. 2+/-5.2 nM) and inositol phosphate specificity [Kd values for Ins(1, 3,4,5,6)P5, InsP6, Ins(1,3,4)P3 and Ins(1,4,5)P3: 283+/-32, >10000, >10000 and >10000 nM, respectively). Furthermore, GRP1 can accommodate addition of glycerol or diacetylglycerol to the 1-phosphate of Ins(1,3,4,5)P4, data that are consistent with its proposed role as a putative PtdIns(3,4,5)P3 receptor. To address whether GRP1 binds PtdIns(3,4,5)P3 in vivo, we have expressed a chimaera of green fluorescent protein (GFP) fused to the N-terminus of GRP1 in PC12 cells and, using confocal microscopy, examined its resultant localization in live cells. Stimulation with either nerve growth factor or epidermal growth factor (both at 100 ng/ml) results in a rapid, PH-domain dependent, translocation of GFP-GRP1 from the cytosol to the plasma membrane, which occurs with a time course that parallels the production of PtdIns(3,4,5)P3. This translocation is dependent on the activation of phosphatidylinositol 3-kinase, since it is inhibited by wortmannin (100 nM), LY294002 (50 microM) and by the co-expression with dominant negative p85. Taken together these data strongly suggest that GRP1 interacts in vivo with plasma membrane-located PtdIns(3,4,5)P3 and hence constitutes a true PtdIns(3,4,5)P3 receptor.

ADP-Ribosylation Factors↗