Publications by authors named "Claire Chaussade"

22 Publications

  • Page 1 of 1

Vps34 PI 3-kinase inactivation enhances insulin sensitivity through reprogramming of mitochondrial metabolism.

Nat Commun 2017 11 27;8(1):1804. Epub 2017 Nov 27.

UCL Cancer Institute, University College London, 72 Huntley Street, London, WC1E 6DD, UK.

Vps34 PI3K is thought to be the main producer of phosphatidylinositol-3-monophosphate, a lipid that controls intracellular vesicular trafficking. The organismal impact of systemic inhibition of Vps34 kinase activity is not completely understood. Here we show that heterozygous Vps34 kinase-dead mice are healthy and display a robustly enhanced insulin sensitivity and glucose tolerance, phenotypes mimicked by a selective Vps34 inhibitor in wild-type mice. The underlying mechanism of insulin sensitization is multifactorial and not through the canonical insulin/Akt pathway. Vps34 inhibition alters cellular energy metabolism, activating the AMPK pathway in liver and muscle. In liver, Vps34 inactivation mildly dampens autophagy, limiting substrate availability for mitochondrial respiration and reducing gluconeogenesis. In muscle, Vps34 inactivation triggers a metabolic switch from oxidative phosphorylation towards glycolysis and enhanced glucose uptake. Our study identifies Vps34 as a new drug target for insulin resistance in Type-2 diabetes, in which the unmet therapeutic need remains substantial.
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http://dx.doi.org/10.1038/s41467-017-01969-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5703854PMC
November 2017

Novel pyrazolo[1,5-a]pyridines with improved aqueous solubility as p110α-selective PI3 kinase inhibitors.

Bioorg Med Chem Lett 2017 01 25;27(2):187-190. Epub 2016 Nov 25.

Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand; Department of Molecular Medicine and Pathology, School of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand.

As part of our investigation into pyrazolo[1,5-a]pyridines as novel p110α selective PI3 kinase inhibitors, we report a range of analogues with improved aqueous solubility by the addition of a basic amine. The compounds demonstrated comparable p110α potency and selectivity to earlier compounds but with up to 1000× greater aqueous solubility, as the hydrochloride salts. The compounds also displayed good activity in a cellular assay of PI3 kinase activity.
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http://dx.doi.org/10.1016/j.bmcl.2016.11.078DOI Listing
January 2017

Inactivation of class II PI3K-C2α induces leptin resistance, age-dependent insulin resistance and obesity in male mice.

Diabetologia 2016 07 30;59(7):1503-1512. Epub 2016 Apr 30.

UCL Cancer Institute, University College London, 72 Huntley Street, London, WC1E 6DD, UK.

Aims/hypothesis: While the class I phosphoinositide 3-kinases (PI3Ks) are well-documented positive regulators of metabolism, the involvement of class II PI3K isoforms (PI3K-C2α, -C2β and -C2γ) in metabolic regulation is just emerging. Organismal inactivation of PI3K-C2β increases insulin signalling and sensitivity, whereas PI3K-C2γ inactivation has a negative metabolic impact. In contrast, the role of PI3K-C2α in organismal metabolism remains unexplored. In this study, we investigated whether kinase inactivation of PI3K-C2α affects glucose metabolism in mice.

Methods: We have generated and characterised a mouse line with a constitutive inactivating knock-in (KI) mutation in the kinase domain of the gene encoding PI3K-C2α (Pik3c2a).

Results: While homozygosity for kinase-dead PI3K-C2α was embryonic lethal, heterozygous PI3K-C2α KI mice were viable and fertile, with no significant histopathological findings. However, male heterozygous mice showed early onset leptin resistance, with a defect in leptin signalling in the hypothalamus, correlating with a mild, age-dependent obesity, insulin resistance and glucose intolerance. Insulin signalling was unaffected in insulin target tissues of PI3K-C2α KI mice, in contrast to previous reports in which downregulation of PI3K-C2α in cell lines was shown to dampen insulin signalling. Interestingly, no metabolic phenotypes were detected in female PI3K-C2α KI mice at any age.

Conclusions/interpretation: Our data uncover a sex-dependent role for PI3K-C2α in the modulation of hypothalamic leptin action and systemic glucose homeostasis.

Access To Research Materials: All reagents are available upon request.
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http://dx.doi.org/10.1007/s00125-016-3963-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4901096PMC
July 2016

Inactivation of the Class II PI3K-C2β Potentiates Insulin Signaling and Sensitivity.

Cell Rep 2015 Dec 19;13(9):1881-94. Epub 2015 Nov 19.

UCL Cancer Institute, University College London, 72 Huntley Street, London WC1E 6DD, UK. Electronic address:

In contrast to the class I phosphoinositide 3-kinases (PI3Ks), the organismal roles of the kinase activity of the class II PI3Ks are less clear. Here, we report that class II PI3K-C2β kinase-dead mice are viable and healthy but display an unanticipated enhanced insulin sensitivity and glucose tolerance, as well as protection against high-fat-diet-induced liver steatosis. Despite having a broad tissue distribution, systemic PI3K-C2β inhibition selectively enhances insulin signaling only in metabolic tissues. In a primary hepatocyte model, basal PI3P lipid levels are reduced by 60% upon PI3K-C2β inhibition. This results in an expansion of the very early APPL1-positive endosomal compartment and altered insulin receptor trafficking, correlating with an amplification of insulin-induced, class I PI3K-dependent Akt signaling, without impacting MAPK activity. These data reveal PI3K-C2β as a critical regulator of endosomal trafficking, specifically in insulin signaling, and identify PI3K-C2β as a potential drug target for insulin sensitization.
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http://dx.doi.org/10.1016/j.celrep.2015.10.052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4675724PMC
December 2015

Essential role of class II PI3K-C2α in platelet membrane morphology.

Blood 2015 Aug 24;126(9):1128-37. Epub 2015 Jun 24.

Inserm, U1048-Université Toulouse III, Institut des Maladies Métaboliques et Cardiovasculaires, Toulouse, France;

The physiologic roles of the class II phosphoinositide 3-kinases (PI3Ks) and their contributions to phosphatidylinositol 3-monophosphate (PI3P) and PI(3,4)P2 production remain elusive. Here we report that mice heterozygous for a constitutively kinase-dead PI3K-C2α display aberrant platelet morphology with an elevated number of barbell-shaped proplatelets, a recently discovered intermediate stage in the final process of platelet production. Platelets with heterozygous PI3K-C2α inactivation have critical defects in α-granules and membrane structure that are associated with modifications in megakaryocytes. These platelets are more rigid and unable to form filopodia after stimulation. Heterozygous PI3K-C2α inactivation in platelets led to a significant reduction in the basal pool of PI3P and a mislocalization of several membrane skeleton proteins known to control the interactions between the plasma membrane and cytoskeleton. These alterations had repercussions on the performance of platelet responses with delay in the time of arterial occlusion in an in vivo model of thrombosis and defect in thrombus formation in an ex vivo blood flow system. These data uncover a key role for PI3K-C2α activity in the generation of a basal housekeeping PI3P pool and in the control of membrane remodeling, critical for megakaryocytopoiesis and normal platelet production and function.
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http://dx.doi.org/10.1182/blood-2015-03-636670DOI Listing
August 2015

Patent foramen ovale and stroke in intermediate-risk pulmonary embolism.

Chest 2014 Oct;146(4):967-973

Department of Cardiology, Pasteur University Hospital, Nice, France.

Background: Patent foramen ovale (PFO) in pulmonary embolism (PE) is associated with an increased risk of complications. However, little is known about PFO and ischemic stroke prevalence, particularly in acute intermediate-risk PE. In addition, in this context, the so-called "gold standard" method of PFO diagnosis remains unknown. We aimed to evaluate PFO and ischemic stroke prevalence and determine which of transesophageal echocardiography (TEE) or transthoracic echocardiography (TTE) is the best PFO diagnostic method in this context.

Methods: We conducted a prospective monocentric study of consecutive patients with intermediate-risk PE in whom a TEE and TTE with contrast were performed. Brain MRI was used to confirm clinically obvious strokes or to diagnose subclinical ones.

Results: Forty-one patients with intermediate-risk PE were identified over a 9-month period. Contrast TEE revealed PFO in 56.1%, whereas contrast TTE showed PFO in only 19.5% (P < .001). Of note, all PFOs observed with TTE were also diagnosed by TEE. Ischemic stroke occurred in 17.1% and was always associated with PFO and large shunt.

Conclusions: PFO and related ischemic strokes are frequent in intermediate-risk PE. TEE is much more efficient than TTE for PFO diagnosis. Considering the high risk of intracranial bleeding with thrombolysis in PE, which may be partly due to hemorrhagic transformation of subclinical strokes, screening PFO with TEE should be considered in intermediate-risk PE when thrombolytic treatment is discussed.
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http://dx.doi.org/10.1378/chest.14-0100DOI Listing
October 2014

Cardiac biomarkers in Takotsubo cardiomyopathy.

Int J Cardiol 2014 Jul 18;174(3):798-801. Epub 2014 Apr 18.

Cardiology Department, Pasteur University Hospital, Nice, France.

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http://dx.doi.org/10.1016/j.ijcard.2014.04.120DOI Listing
July 2014

Imaging in pulmonary hypertension: Focus on the role of echocardiography.

Arch Cardiovasc Dis 2014 Apr 18;107(4):261-71. Epub 2014 Apr 18.

Service de cardiologie, hôpital Pasteur, CHU de Nice, 30, avenue de la Voie-Romaine, BP69, CS 51069, 06001 Nice cedex 1, France.

Patients with pulmonary hypertension must be evaluated using a multimodality approach to ensure a correct diagnosis and basal evaluation as well as a prognostic assessment. Beyond the assessment of pulmonary pressures, the echocardiographical examination allows the evaluation of right ventricular adaptation to elevated afterload. Numbers of variables are commonly used in the assessment of the pulmonary hypertension patient in order to detect changes in right heart geometry, right-to-left interaction and right ventricular dysfunction. Whereas an isolated change in one echocardiographical variable is not meaningful, multiple echocardiographical variable modifications together provide accurate information. In this review, we will link pulmonary hypertension pathophysiological changes with echocardiographical indices and describe the clinical implications of echocardiographical findings.
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http://dx.doi.org/10.1016/j.acvd.2014.02.005DOI Listing
April 2014

Novel pyrazolo[1,5-a]pyridines as p110α-selective PI3 kinase inhibitors: Exploring the benzenesulfonohydrazide SAR.

Bioorg Med Chem 2012 Jan 25;20(1):58-68. Epub 2011 Nov 25.

Auckland Cancer Society Research Centre, School of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand.

Structure-activity relationship studies of the pyrazolo[1,5-a]pyridine class of PI3 kinase inhibitors show that substitution off the hydrazone nitrogen and replacement of the sulfonyl both gave a loss of p110α selectivity, with the exception of an N-hydroxyethyl analogue. Limited substitutions were tolerated around the phenyl ring; in particular the 2,5-substitution pattern was important for PI3 kinase activity. The N-hydroxyethyl compound also showed good inhibition of cell proliferation and inhibition of phosphorylation of Akt/PKB, a downstream marker of PI3 kinase activity. It had suitable pharmacokinetics for evaluation in vivo, and showed tumour growth inhibition in two human tumour cell lines in xenograft studies. This work has provided suggestions for the design of more soluble analogues.
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http://dx.doi.org/10.1016/j.bmc.2011.11.031DOI Listing
January 2012

Discovery of pyrazolo[1,5-a]pyridines as p110α-selective PI3 kinase inhibitors.

Bioorg Med Chem 2012 Jan 25;20(1):69-85. Epub 2011 Nov 25.

Auckland Cancer Society Research Centre, School of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand.

We have made a novel series of pyrazolo[1,5-a]pyridines as PI3 kinase inhibitors, and demonstrated their selectivity for the p110α isoform over the other Class Ia PI3 kinases. We investigated the SAR around the pyrazolo[1,5-a]pyridine ring system, and found compound 5x to be a particularly potent example (p110α IC(50) 0.9nM). This compound inhibits cell proliferation and phosphorylation of Akt/PKB, a downstream marker of PI3 kinase activity, and showed in vivo activity in an HCT-116 human xenograft model.
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http://dx.doi.org/10.1016/j.bmc.2011.11.029DOI Listing
January 2012

Calpain interacts with class IA phosphoinositide 3-kinases regulating their stability and signaling activity.

Proc Natl Acad Sci U S A 2011 Sep 19;108(39):16217-22. Epub 2011 Sep 19.

Analytical Signaling Group, Centre for Cell Signaling, Barts Cancer Institute, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, United Kingdom.

Class IA phosphoinositide 3-kinases (PI3Ks) are signaling enzymes with key roles in the regulation of essential cellular functions and disease, including cancer. Accordingly, their activity is tightly controlled in cells to maintain homeostasis. The formation of multiprotein complexes is a ubiquitous mechanism to regulate enzyme activity but the contribution of protein-protein interactions to the regulation of PI3K signaling is not fully understood. We designed an affinity purification quantitative mass spectrometry strategy to identify proteins interacting dynamically with PI3K in response to pathway activation, with the view that such binding partners may have a functional role in pathway regulation. Our study reveals that calpain small subunit 1 interacts with PI3K and that the association between these proteins is lower in cells stimulated with serum compared to starved cells. Calpain and PI3K activity assays confirmed these results, thus demonstrating that active calpain heterodimers associate dynamically with PI3K. In addition, calpains were found to cleave PI3K proteins in vitro (resulting in a reduction of PI3K lipid kinase activity) and to regulate endogenous PI3K protein levels in vivo. Further investigations revealed that calpains have a role in the negative regulation of PI3K/Akt pathway activity (as measured by Akt and ribosomal S6 phosphorylation) and that their inhibition promotes cell survival during serum starvation. These results indicate that the interaction between calpain and PI3K is a novel mechanism for the regulation of class IA PI3K stability and activity.
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http://dx.doi.org/10.1073/pnas.1107692108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3182684PMC
September 2011

Syndecan-2 is a novel ligand for the protein tyrosine phosphatase receptor CD148.

Mol Biol Cell 2011 Oct 3;22(19):3609-24. Epub 2011 Aug 3.

Centre for Microvascular Research, William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London EC1M 6BQ, United Kingdom.

Syndecan-2 is a heparan sulfate proteoglycan that has a cell adhesion regulatory domain contained within its extracellular core protein. Cell adhesion to the syndecan-2 extracellular domain (S2ED) is β1 integrin dependent; however, syndecan-2 is not an integrin ligand. Here the protein tyrosine phosphatase receptor CD148 is shown to be a key intermediary in cell adhesion to S2ED, with downstream β1 integrin-mediated adhesion and cytoskeletal organization. We show that S2ED is a novel ligand for CD148 and identify the region proximal to the transmembrane domain of syndecan-2 as the site of interaction with CD148. A mechanism for the transduction of the signal from CD148 to β1 integrins is elucidated requiring Src kinase and potential implication of the C2β isoform of phosphatidylinositol 3 kinase. Our data uncover a novel pathway for β1 integrin-mediated adhesion of importance in cellular processes such as angiogenesis and inflammation.
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http://dx.doi.org/10.1091/mbc.E11-02-0099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3183016PMC
October 2011

Evidence for a role for the p110-alpha isoform of PI3K in skeletal function.

Biochem Biophys Res Commun 2010 Jan 24;391(1):564-9. Epub 2009 Nov 24.

Department of Medicine, University of Auckland, Auckland, New Zealand.

Signaling through phosphatidylinositol-3 kinases (PI3K) regulates fundamental cellular processes such as survival and growth, and these lipid kinases are currently being investigated as therapeutic targets in several contexts. In skeletal tissue, experiments using pan-specific PI3K inhibitors have suggested that PI3K signaling influences both osteoclast and osteoblast function, but the contributions of specific PI3K isoforms to these effects have not been examined. In the current work, we assessed the effects of pharmacological inhibitors of the class Ia PI3Ks, alpha, beta, and delta, on bone cell growth, differentiation and function in vitro. Each of the class Ia PI3K isoforms is expressed and functionally active in bone cells. No consistent effects of inhibitors of p110-beta or p110-delta on bone cells were observed. Inhibitors of p110-alpha decreased osteoclastogenesis by 60-80% (p<0.001 vs control) by direct actions on osteoclast precursors, and decreased the resorptive activity of mature osteoclasts by 60% (p<0.01 vs control). The p110-alpha inhibitors also decreased the growth of osteoblastic and stromal cells (p<0.001 vs control), and decreased differentiated osteoblast function by 30% (p<0.05 vs control). These data suggest that signaling through the p110-alpha isoform of class Ia PI3Ks positively regulates the development and function of both osteoblasts and osteoclasts. Therapeutic agents that target this enzyme have the potential to significantly affect bone homeostasis, and evaluation of skeletal endpoints in clinical trials of such agents is warranted.
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http://dx.doi.org/10.1016/j.bbrc.2009.11.099DOI Listing
January 2010

Phosphoinositide-3-kinase (PI3K) inhibitors: identification of new scaffolds using virtual screening.

Bioorg Med Chem Lett 2009 Oct 29;19(20):5842-7. Epub 2009 Aug 29.

Auckland Cancer Society Research Centre (ACSRC), School of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland 1020, New Zealand.

In the present work, we used virtual screening (VS) of the ZINC database of 2.5 million compounds to seek new PI3K inhibitory scaffolds. The VS flowchart implemented various filters, including a 3D-database screen, and extensive docking studies, to derive 89 derivatives that were experimentally assayed against the four PI3K isoforms. Seven compounds showed inhibitory activities between 1 and 100 microM, with four being sufficiently potent to constitute potential new scaffolds. The binding conformations of these four were analyzed to provide a rationalization of their activity profile.
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http://dx.doi.org/10.1016/j.bmcl.2009.08.087DOI Listing
October 2009

Functional differences between two classes of oncogenic mutation in the PIK3CA gene.

Biochem Biophys Res Commun 2009 Apr 20;381(4):577-81. Epub 2009 Feb 20.

Department of Molecular Medicine, University of Auckland, New Zealand.

PIK3CA codes for the p110alpha isoform of class-IA PI 3-kinase and oncogenic mutations in the helical domain and kinase domain are common in several cancers. We studied the biochemical properties of a common helical domain mutant (E545K) and a common kinase domain mutant (H1047R). Both retain the ability to autophosphorylate Ser608 of p85alpha and are also inhibited by a range of PI 3-kinase inhibitors (Wortmannin, LY294002, PI-103 and PIK-75) to a similar extent as WT p110alpha. Both mutants display an increased V(max) but while a PDGF derived diphosphotyrosylpeptide caused an increase in V(max) for WT p85alpha/p110alpha it did not for the E545K variant and actually decreased V(max) for the H1047R variant. Further, the E545K mutant was activated by H-Ras whereas the H1047R mutant was not. Together these results suggest helical domain mutants are in a state mimicking activation by growth factors whereas kinase domain mutants mimic the state activated by H-Ras.
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http://dx.doi.org/10.1016/j.bbrc.2009.02.081DOI Listing
April 2009

Investigating the role of class-IA PI 3-kinase isoforms in adipocyte differentiation.

Biochem Biophys Res Commun 2009 Feb 27;379(4):830-4. Epub 2008 Dec 27.

Dept. of Molecular Medicine and Pathology and Maurice Wilkins Center for Molecular Biodiscovery, University of Auckland, Private Bag 92019, Auckland, New Zealand.

PI 3-kinases, in particular class-IA, are key signalling molecules controlling many cellular processes including growth, proliferation, migration and differentiation. In this study, we have used a collection of isoform selective PI 3-kinase inhibitors to determine whether attenuation of signalling through class-IA PI 3-kinase isoforms will impact adipocyte differentiation. First, we analysed the expression profiles and found that fibroblastic pre-adipocytes express detectable levels of p110alpha and p110delta and that after differentiation, p110delta levels fall while p110alpha levels rise, together with C/EBPalpha and PPARgamma. When using specific inhibitors during the differentiation process, we observed that neither p110beta nor p110delta inhibition, had any significant effect. In contrast PIK-75, a selective p110alpha inhibitor completely abolished adipocyte differentiation as assessed by morphology, transcript and protein levels of adipocyte markers. These results indicate that long term treatment with p110alpha inhibitors could potentially have a severe impact on fat cell numbers in vivo.
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http://dx.doi.org/10.1016/j.bbrc.2008.12.089DOI Listing
February 2009

Synthesis, biological evaluation and molecular modelling of sulfonohydrazides as selective PI3K p110alpha inhibitors.

Bioorg Med Chem 2007 Dec 4;15(24):7677-87. Epub 2007 Sep 4.

Auckland Cancer Society Research Centre, School of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland 1020, New Zealand.

A series of 2-methyl-5-nitrobenzenesulfonohydrazides were prepared and evaluated as inhibitors of PI3K. An isoquinoline derivative shows good selectivity for the p110alpha isoform over p110beta and p110delta, and also demonstrates good in vitro activity in a cell proliferation assay. Molecular modelling provides a rationalisation for the observed SAR.
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http://dx.doi.org/10.1016/j.bmc.2007.08.062DOI Listing
December 2007

Evidence for functional redundancy of class IA PI3K isoforms in insulin signalling.

Biochem J 2007 Jun;404(3):449-58

Maurice Wilkins Centre for Molecular Biodiscovery, Department of Molecular Medicine and Pathology, University of Auckland, Private Bag 92019, Auckland, New Zealand.

Recent genetic knock-in and pharmacological approaches have suggested that, of class IA PI3Ks (phosphatidylinositol 3-kinases), it is the p110alpha isoform (PIK3CA) that plays the predominant role in insulin signalling. We have used isoform-selective inhibitors of class IA PI3K to dissect further the roles of individual p110 isoforms in insulin signalling. These include a p110alpha-specific inhibitor (PIK-75), a p110alpha-selective inhibitor (PI-103), a p110beta-specific inhibitor (TGX-221) and a p110delta-specific inhibitor (IC87114). Although we find that p110alpha is necessary for insulin-stimulated phosphorylation of PKB (protein kinase B) in several cell lines, we find that this is not the case in HepG2 hepatoma cells. Inhibition of p110beta or p110delta alone was also not sufficient to block insulin signalling to PKB in these cells, but, when added in combination with p110alpha inhibitors, they are able to significantly attenuate insulin signalling. Surprisingly, in J774.2 macrophage cells, insulin signalling to PKB was inhibited to a similar extent by inhibitors of p110alpha, p110beta or p110delta. These results provide evidence that p110beta and p110delta can play a role in insulin signalling and also provide the first evidence that there can be functional redundancy between p110 isoforms. Further, our results indicate that the degree of functional redundancy is linked to the relative levels of expression of each isoform in the target cells.
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http://dx.doi.org/10.1042/BJ20070003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1896275PMC
June 2007

Nuclear forkhead box O1 controls and integrates key signaling pathways in hepatocytes.

Endocrinology 2007 May 15;148(5):2424-34. Epub 2007 Feb 15.

Institut National de la Santé et de la Recherche Médicale U145, Institut Federatif de Recherche 50, Faculté de Médecine, Avenue de Valombrose, F-06107 Nice, France.

Insulin inhibits forkhead O class (FoxO) transcription factors, which down-regulate the expression of genes involved in metabolism, cell cycle arrest, and apoptosis. After being phosphorylated by protein kinase B (PKB) on S253 in its DNA-binding domain, Foxo1 is phosphorylated on T24 and additional sites, which overall triggers its nuclear exclusion. During this process, Foxo1 is thought to retain some transcriptional activity and signaling potential. To evaluate this Foxo1 action, we used a Foxo1-ADA mutant that is constitutively nuclear due to mutation of T24 and S316 to A and harbors a mutation of S253 to D. Adenoviral-mediated expression of Foxo1-ADA in hepatocytes activates PKB and MAPK pathways more than expression of wild-type or of a transactivation domain-deleted mutant (Delta256). PKB activation cannot be accounted for by a Foxo1-mediated increase in upstream signaling components such as insulin receptor substrate 1 or 2 or by Foxo1-mediated down-regulation of Tribbles homolog 3. In contrast, Foxo1-ADA increases p38 activity, and p38 is required for effects of Foxo1 on PKB, at least in part. We propose that Foxo1 turns on a feed-forward loop, relayed by p38 and acting to amplify both PKB activation and Foxo1 inhibition. To conclude, key signaling pathways are activated in hepatocytes through nuclear Foxo1.
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http://dx.doi.org/10.1210/en.2006-1411DOI Listing
May 2007

Production of phosphatidylinositol 5-phosphate by the phosphoinositide 3-phosphatase myotubularin in mammalian cells.

J Biol Chem 2004 Feb 1;279(8):7304-12. Epub 2003 Dec 1.

INSERM U563, Département d'Oncogenèse et Signalisation dans les Cellules Hématopoïétiques, CPTP, IFR 30, Hôpital Purpan, 31059 Toulouse Cedex, France.

MTM1, the gene encoding myotubularin (MTM1), is mutated in the X-linked myotubular myopathy (XLMTM), a severe genetic muscular disorder. MTM1 is a phosphoinositide phosphatase hydrolyzing phosphatidylinositol 3-phosphate (PtdIns(3)P) in yeast and in vitro. Because this lipid is implicated in the regulation of vesicular trafficking, we used established cell lines from XLMTM patients to evaluate whether the lack of endogenous MTM1 expression could affect PtdIns(3)P labeling patterns. Our results showed that the vesicular trafficking related to early endosomes was not significantly affected in the XLMTM cell lines compared with control cells. However, in addition to PtdIns(3)P, we found that MTM1 can hydrolyze phosphatidylinositol 3,5-bisphosphate both in vitro and in mammalian cells. Using a mass assay, we demonstrated that the product generated is phosphatidylinositol 5-phosphate (PtdIns(5)P), a recently discovered phosphoinositide, the function of which is still unknown. In L6 myotubes overexpressing MTM1, hyperosmotic shock induced an increase in the mass level of PtdIns(5)P that was reduced by 50% upon overexpression of the MTM1 inactive mutant D278A. These data demonstrate for the first time a role for MTM1 in the production of PtdIns(5)P in mammalian cells, suggesting that the lack of transformation of phosphatidylinositol 3,5-bisphosphate into PtdIns(5)P might be an important component in the etiology of myotubular myopathy.
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http://dx.doi.org/10.1074/jbc.M311071200DOI Listing
February 2004

Expression of myotubularin by an adenoviral vector demonstrates its function as a phosphatidylinositol 3-phosphate [PtdIns(3)P] phosphatase in muscle cell lines: involvement of PtdIns(3)P in insulin-stimulated glucose transport.

Mol Endocrinol 2003 Dec 18;17(12):2448-60. Epub 2003 Sep 18.

Institut National de la Santé et de la Recherche Medicale, Unité 145, 28, avenue de Valombrose, 06 107 Nice Cedex 2, France.

X-linked myotubular myopathy is a muscle disorder caused by mutations on the myotubular myopathy-1 (MTM-1) gene, coding for myotubularin a 65-kDa polypeptide similar to protein phosphatases. Biochemical and in vivo studies define myotubularin as a phosphatidylinositol 3-phosphate [PtdIns(3)P] phosphatase. To efficiently express myotubularin in muscle cell lines and adipocytes, we used an adenoviral genome recombinogenic to pcDNA3, and to other widely used expression vectors, to produce adenoviruses expressing wild-type (wt), catalytically inactive C375S, and substrate trap D278A myotubularin.[32P]Orthophosphate labeling followed by phosphoinositide analysis of differentiated L6 and C2C12 cells expressing myotubularin demonstrated increased PtdIns(3)P levels upon expression of the C375S and D278A mutants. In keeping with its biochemical function, overexpression of wt myotubularin as an enhanced green fluorescent protein fusion disrupted the endosomal punctuated staining of the FYVE (Fab1p/YOTB Vac1p/EEA1)-domain-containing PtdIns(3)P binding protein early endosomal antigen 1 as well as of a gluathione-S-transferase-FYVE probe directed to PtdIns(3)P. Expression of wt myotubularin, although not affecting activation of proximal insulin signal transduction targets such as protein kinase B and MAPK, induced a decrease in insulin-induced glucose uptake, whereas basal glucose uptake was augmented by expression of D278A (DA) and C375S (CS) mutants. Moreover, overexpression of myotubularin in 3T3-L1 adipocytes impaired insulin-induced translocation at the plasma membrane of green fluorescent protein-tagged glucose transporter 4. These data indicate that PtdIns(3)P is required to direct glucose transporter 4 to insulin-responsive compartments and/or to allow the translocation of the latter at the plasma membrane. We conclude that myotubularin, by modulating the intracellular levels of PtdIns(3)P, plays a role in the control of vesicular traffic related to glucose transport, by counteracting the activities of the PtdIns(3)P-producing phosphatidylinositol 3-kinases.
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http://dx.doi.org/10.1210/me.2003-0261DOI Listing
December 2003

Phosphoinositide 3-kinase-mediated reduction of insulin receptor substrate-1/2 protein expression via different mechanisms contributes to the insulin-induced desensitization of its signaling pathways in L6 muscle cells.

J Biol Chem 2003 May 18;278(18):15641-51. Epub 2003 Feb 18.

INSERM U145, IFR50, Faculté de Médecine, 06107 Nice Cedex 2, France.

Impaired glucose tolerance precedes type 2 diabetes and is characterized by hyperinsulinemia, which develops to balance peripheral insulin resistance. To gain insight into the deleterious effects of hyperinsulinemia on skeletal muscle, we studied the consequences of prolonged insulin treatment of L6 myoblasts on insulin-dependent signaling pathways. A 24-h long insulin treatment desensitized the phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB) and p42/p44 MAPK pathways toward a second stimulation with insulin or insulin-like growth factor-1 and led to decreased insulin-induced glucose uptake. Desensitization was correlated to a reduction in insulin receptor substrate (IRS)-1 and IRS-2 protein levels, which was reversed by the PI3K inhibitor LY294002. Co-treatment of cells with insulin and LY294002, while reducing total IRS-1 phosphorylation, increased its phosphotyrosine content, enhancing IRS-1/PI3K association. PDK1, mTOR, and MAPK inhibitors did not block insulin-induced reduction of IRS-1, suggesting that the PI3K serine-kinase activity causes IRS-1 serine phosphorylation and its commitment to proteasomal degradation. Contrarily, insulin-induced IRS-2 down-regulation occurred via a PI3K/mTOR pathway. Suppression of IRS-1/2 down-regulation by LY294002 rescued the responsiveness of PKB and MAPK toward acute insulin stimulation. Conversely, adenoviral-driven expression of constitutively active PI3K induced an insulin-independent reduction in IRS-1/2 protein levels. IRS-2 appears to be the chief molecule responsible for MAPK and PKB activation by insulin, as knockdown of IRS-2 (but not IRS-1) by RNA interference severely impaired activation of both kinases. In summary, (i) PI3K mediates insulin-induced reduction of IRS-1 by phosphorylating it while a PI3K/mTOR pathway controls insulin-induced reduction of IRS-2, (ii) in L6 cells, IRS-2 is the major adapter molecule linking the insulin receptor to activation of PKB and MAPK, (iii) the mechanism of IRS-1/2 down-regulation is different in L6 cells compared with 3T3-L1 adipocytes. In conclusion, the reduction in IRS proteins via different PI3K-mediated mechanisms contributes to the development of an insulin-resistant state in L6 myoblasts.
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http://dx.doi.org/10.1074/jbc.M208984200DOI Listing
May 2003
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