PPAR beta a player in astrocyte metabolism and morphology

AbstractPPARP is a nuclear receptor responding in vivo to several free fatty acids, and implicated in cell metabolism, differentiation and survival. PPARp is ubiquitously expressed but shows high expression in the developing and adult brain. PPARp is expressed in different cell types such as neurons and astrocytes, where it might play a role in metabolism. To study this nuclear receptor the laboratory engineered a PPARP -/- mouse model. The aim of my PhD was to dissect the role of PPARP in astrocytes.Experiments in primary culture revealed that cortical astrocytes from PPARP -/- mouse have an impaired energetic metabolism. Unstimulated PPARP -/- astrocytes exhibit a 30% diminution in glucose uptake, correlating to a 30% decrease in lactate release and intracellular glucose. After acute stimulation by D- aspartate mimicking glutamate exposure, both WT and -/- astrocytes up-regulate their metabolism to respond to the increasing energy needed (ATP) for glutamate uptake. According to the Astrocyte Neuron Lactate Shuttle Hypothesis (ANLSH), the ratio between glucose uptake/ lactate release is 1. However, stimulated PPARp -/- astrocytes display a higher increase in lactate release than glucose uptake which remains lower than in WT. The extra glucose equivalents could come from the degradation of intra cellular glycogen stores, which indeed decrease in PPARP -/- cells upon stimulation. Lower glucose metabolism correlates with a decreased acute glutamate uptake in PPARP -/- astrocytes. Reciprocally, we also observed an increase of glutamate uptake and ATP production after treatment of WT astrocytes with a PPARp agonist. Glutamate transporter protein expression is not affected. However, their trafficking and localization might be altered as PPARp -/- astrocytes have higher cholesterol levels, which may also affect proper transporter structure in the membrane.Metabolism, transporter localization and cholesterol levels are respectively linked to cell mobility, cell cytoskeleton and cellular membrane composition. All three functions are important in astrocytes to in vivo acquire star shaped morphology, in a process known as stellation. PPARP -/- astrocytes showed an impaired acquired stellation in presence of neurons or chemical stimuli, as well as more actin stress fibers and cell adhesion structures. While non stellation of astrocytes is mainly an in vitro phenomenon, it reveals PPARp -/- primary astrocytes inability to respond to different exterior stimuli. These morphological phenotypes correlate with a slower migration in cell culture wound healing assays.This thesis work demonstrates that PPARp is implicated in cortical astrocyte glucose metabolism. PPARp absence leads to an unusual intracellular glycogen use. Added to the effect on acute glutamate uptake and astrocyte migration, PPARp could be an interesting target for neuroprotection therapies.RésuméPPARP est un récepteur nucléaire qui a pour ligands naturels certains acides gras libres. Il est impliqué dans le métabolisme, la différentiation et la survie des cellules. PPARP est ubiquitaire, et a une expression élevée dans le cerveau en développement ainsi qu'adulte. PPARp est exprimé dans différents types cellulaires tels que les neurones et les astrocytes, où il régule potentiellement leurs métabolismes. Pour étudier ce récepteur nucléaire, le laboratoire a créé un modèle de souris PPARp -/-. L'objectif de ma thèse est de comprendre le rôle de PPARp dans les astrocytes.Les expériences montrent un défaut du métabolisme énergétique dans les astrocytes corticaux primaires tirés de souris PPARp -/-. Sans stimulation, l'entrée du glucose dans les astrocytes PPARP -/- est diminuée de 30% ce qui correspond à une diminution de 30% du relargage du lactate. Après stimulation par du D-Aspartate qui mime une exposition au glutamate, les astrocytes WT et -/- augmentent leur métabolisme en réponse à la demande accrue en énergie (ATP) due à l'entrée du glutamate. D'après l'Astrocyte Neuron Lactate Shuttle Hypothesis (ANLSH), le ratio entre le glucose entrant et le lactate sortant est de 1. Cependant le relargage du lactate dans les astrocytes PPARP-/- est plus élevé que l'entrée du glucose. L'apport supplémentaire de glucose transformé en lactate pourrait provenir de la dégradation des stocks de glycogène intracellulaire, qui sont partiellement diminués après stimulation dans les cellules PPARP -/-. Un métabolisme plus faible du glucose corrèle avec une réduction de l'import du glutamate dans les astrocytes PPARp -/-. Réciproquement, nous observons une augmentation de l'import du glutamate et de la production d'ATP après traitement avec l'agoniste pour PPARp. Bien que l'expression des transporteurs de glutamate ne soit pas affectée, nous ne pouvons pas exclure que leur localisation et leur structure soient altérées du fait du niveau élevé de cholestérol dans les astrocytes PPARp -/-.Le métabolisme, la localisation des transporteurs et le niveau de cholestérol sont tous liés au cytosquelette, à la mobilité, et à la composition des membranes cellulaires. Toutes ces fonctions sont importantes pour les astrocytes pour acquérir leur morphologie in vivo. Les astrocytes PPARP -/- présentent un défaut de stellation, aussi bien en présence de neurones que de stimuli chimiques, ainsi qu'un plus grand nombre de fibres de stress (actine) et de structures d'adhésion cellulaire. Bien que les astrocytes non stellaires soient principalement observés in vitro, le défaut de stellation des astrocytes primaires PPARp -/- indique une incapacité à répondre aux différents stimuli extérieurs. Ces phénotypes morphologiques corrèlent avec une migration plus lente en cas de lésion de la culture.Ce travail de thèse a permis de démontrer l'implication de PPARP dans le métabolisme du glucose des astrocytes corticaux. L'absence de ce récepteur nucléaire amène à l'utilisation du glucose intracellulaire, auquel s'ajoutent les effets sur l'import du glutamate et la migration des astrocytes. PPARp aurait des effets neuroprotecteurs, et de ce fait pourrait être utilisé à des fins thérapeutiques.

Role of prostacyclin versus peroxisome proliferator-activated receptor beta receptors in prostacyclin sensing by lung fibroblasts.

Prostacyclin and its mimetics are used therapeutically for the treatment of pulmonary hypertension. These drugs act via cell surface prostacyclin receptors (IP receptors); however, some of them can also activate the nuclear receptor peroxisome proliferator-activated receptor beta (PPARbeta). We examined the possibility that PPARbeta is a therapeutic target for the treatment of pulmonary hypertension. Using the newly approved (for pulmonary hypertension) prostacyclin mimetic treprostinil sodium, reporter gene assays for PPARbeta activation and measurement of lung fibroblast proliferation were analyzed. Treprostinil sodium was found to activate PPARbeta in reporter gene assays and to inhibit proliferation of human lung fibroblasts at concentrations consistent with an effect on PPARs but not on IP receptors. The effects of treprostinil sodium on human lung cell proliferation are mimicked by those of the highly selective PPARbeta ligand GW0742. There are no receptor antagonists for PPARbeta or for IP receptors, but by using lung fibroblasts cultured from mice lacking PPARbeta (PPARbeta-/-) or IP (IP-/-), we demonstrate that the antiproliferative effects of treprostinil sodium are mediated by PPARbeta and not IP in lung fibroblasts. These observations suggest that some of the local, longer-term benefits of treprostinil sodium on reducing the remodeling associated with pulmonary hypertension may be mediated by PPARbeta. This study is the first to identify PPARbeta as a potential therapeutic target for the treatment of pulmonary hypertension, which is important because orally active PPARbeta ligands have been developed for the treatment of dyslipidemia.

IL-4 rapidly produced by V beta 4 V alpha 8 CD4+ T cells instructs Th2 development and susceptibility to Leishmania major in BALB/c mice.

BALB/c mice develop aberrant T helper 2 (Th2) responses and suffer progressive disease after infection with Leishmania major. These outcomes depend on the production of interleukin-4 (IL-4) early after infection. Here we demonstrate that the burst of IL-4 mRNA, peaking in draining lymph nodes of BALB/c mice 16 hr after infection, occurs within CD4+ T cells that express V beta 4 V alpha 8 T cell receptors. In contrast to control and V beta 6-deficient BALB/c mice, V beta 4-deficient BALB/c mice were resistant to infection, demonstrating the role of these cells in Th2 development. The early IL-4 response was absent in these mice, and T helper 1 responses occurred following infection. Recombinant LACK antigen from L. major induced comparable IL-4 production in V beta 4 V alpha 8 CD4+ cells. Thus, the IL-4 required for Th2 development and susceptibility to L. major is produced by a restricted population of V beta 4 V alpha 8 CD4+ T cells after cognate interaction with a single antigen from this complex organism.

Peroxisome proliferator-activated receptor-beta as a target for wound healing drugs.

Healing of cutaneous wounds, which is crucial for survival after an injury, proceeds via a well-tuned pattern of events including inflammation, re-epithelialisation, and matrix and tissue remodelling. These events are regulated spatio-temporally by a variety of growth factors and cytokines. The inflammation that immediately follows injury increases the expression of peroxisome proliferator-activated receptor (PPAR)-beta in the wound edge keratinocytes and triggers the production of endogenous PPARbeta ligands that activate the newly produced receptor. This elevated PPARbeta activity results in increased resistance of the keratinocytes to the apoptotic signals released during wounding, allowing faster re-epithelialisation. The authors speculate that, in parallel, ligand activation of PPARbeta in infiltrated macrophages attenuates the inflammatory response, which also promotes repair. Thus, current understanding of the roles of PPARbeta in different cell types implicated in tissue repair has revealed an intriguing intercellular cross-talk that coordinates, spatially and temporally, inflammation, keratinocyte survival, proliferation and migration, which are all essential for efficient wound repair. These novel insights into the orchestrating roles of PPARbeta during wound healing may be helpful in the development of drugs for acute and chronic wound disorders.

GLUT2 surface expression and intracellular transport via the constitutive pathway in pancreatic beta cells and insulinoma: evidence for a block in trans-Golgi network exit by brefeldin A.

The biosynthesis, intracellular transport, and surface expression of the beta cell glucose transporter GLUT2 was investigated in isolated islets and insulinoma cells. Using a trypsin sensitivity assay to measure cell surface expression, we determined that: (a) greater than 95% of GLUT2 was expressed on the plasma membrane; (b) GLUT2 did not recycle in intracellular vesicles; and (c) after trypsin treatment, reexpression of the intact transporter occurred with a t1/2 of approximately 7 h. Kinetics of intracellular transport of GLUT2 was investigated in pulse-labeling experiments combined with glycosidase treatment and the trypsin sensitivity assay. We determined that transport from the endoplasmic reticulum to the trans-Golgi network (TGN) occurred with a t1/2 of 15 min and that transport from the TGN to the plasma membrane required a similar half-time. When added at the start of a pulse-labeling experiment, brefeldin A prevented exit of GLUT2 from the endoplasmic reticulum. When the transporter was first accumulated in the TGN during a 15-min period of chase, but not following a low temperature (22 degrees C) incubation, addition of brefeldin A (BFA) prevented subsequent surface expression of the transporter. This indicated that brefeldin A prevented GLUT2 exit from the TGN by acting at a site proximal to the 22 degrees C block. Together, these data demonstrate that GLUT2 surface expression in beta cells is via the constitutive pathway, that transport can be blocked by BFA at two distinct steps and that once on the surface, GLUT2 does not recycle in intracellular vesicles.

Increased α2- and β1-adrenoceptor densities in postmortem brain of subjects with depression: differential effect of antidepressant treatment.

BACKGROUND: Brain α2- and β-adrenoceptor alterations have been suggested in suicide and major depressive disorder. METHODS: The densities of α2-, β1- and β2-adrenoceptors in postmortem prefrontal cortex of 26 subjects with depression were compared with those of age-, gender- and postmortem delay-matched controls. The effect of antidepressant treatment on α2- and β-adrenoceptor densities was also evaluated. α2- and β-adrenoceptor densities were measured by saturation experiments with respective radioligands [(3)H]UK14304 and [(3)H]CGP12177. β1- and β2-adrenoceptor subtype densities were dissected by means of β1-adrenoceptor selective antagonist CGP20712A. RESULTS: Both, α2- and β1-adrenoceptors densities were higher in antidepressant-free depressed subjects (n=14) than those in matched controls (Δ~24%, p=0.013 and Δ~20%, p=0.044, respectively). In antidepressant-treated subjects (n=12), α2-adrenoceptor density remained increased over that in controls (Δ~20%), suggesting a resistance of α2-adrenoceptors to the down-regulatory effect of antidepressants. By contrast, β1-adrenoceptor density in antidepressant-treated depressed subjects was not different from controls, suggesting a possible down-regulation by antidepressants. The down-regulation of β1-adrenoceptor density in antidepressant-treated depressed subjects differs from the unaltered β1-adrenoceptor density observed in citalopram-treated rats and in a group of non-depressed subjects also treated with antidepressants (n=6). β2-adrenoceptor density was not altered in depressed subjects independently of treatment. LIMITATIONS: Antidepressant-treated subjects had been treated with a heterogeneous variety of antidepressant drugs. The results should be understood in the context of suicide victims with depression. CONCLUSIONS: These results show the up-regulation of brain α2- and β1-adrenoceptors in depression and suggest that the regulation induced by chronic antidepressant treatment would be altered in these subjects.

Allergie aux beta-lactamines [Allergy to beta-lactam antibiotics].

Beta-lactam antibiotics allergies are common. Up to 10% of the population describe a former allergy to penicillins. However only 10 to 15% of these individuals are actually allergic. In most cases, beta-lactam antibiotics will be avoided and replaced by other antibiotics such as quinolones. This fear of a serious allergic reaction has an economic impact and may lead to the emergence of antibiotic resistance. A thorough allergic work-up can accurately determine true allergic patients. Most of the patients with a proven allergy will be able to tolerate other antibiotics belonging to the beta-lactam family. This article focuses on the management of beta-lactam allergic patients.

Incretin receptor null mice reveal key role of GLP-1 but not GIP in pancreatic beta cell adaptation to pregnancy.

Islet adaptations to pregnancy were explored in C57BL6/J mice lacking functional receptors for glucagon-like peptide 1 (GLP-1) and gastric inhibitory polypeptide (GIP). Pregnant wild type mice and GIPRKO mice exhibited marked increases in islet and beta cell area, numbers of medium/large sized islets, with positive effects on Ki67/Tunel ratio favouring beta cell growth and enhanced pancreatic insulin content. Alpha cell area and glucagon content were unchanged but prohormone convertases PC2 and PC1/3 together with significant amounts of GLP-1 and GIP were detected in alpha cells. Knockout of GLP-1R abolished these islet adaptations and paradoxically decreased pancreatic insulin, GLP-1 and GIP. This was associated with abolition of normal pregnancy-induced increases in plasma GIP, L-cell numbers, and intestinal GIP and GLP-1 stores. These data indicate that GLP-1 but not GIP is a key mediator of beta cell mass expansion and related adaptations in pregnancy, triggered in part by generation of intra-islet GLP-1.

Phylogenetic alpha and beta diversities of butterfly communities correlate with climate in the western Swiss Alps

The observation of non-random phylogenetic distribution of traits in communities provides evidence for niche-based community assembly. Environment may influence the phylogenetic structure of communities because traits determining how species respond to prevailing conditions can be phylogenetically conserved. In this study, we investigate the variation of butterfly species richness and of phylogenetic - and -diversities along temperature and plant species richness gradients. Our study indicates that butterfly richness is independently positively correlated to temperature and plant species richness in the study area. However, the variation of phylogenetic - and -diversities is only correlated to temperature. The significant phylogenetic clustering at high elevation suggests that cold temperature filters butterfly lineages, leading to communities mostly composed of closely related species adapted to those climatic conditions. These results suggest that in colder and more severe conditions at high elevations deterministic processes and not purely stochastic events drive the assemblage of butterfly communities.

Altered expression of beta-galactosidase-1-like protein 3 (Glb1l3) in the retinal pigment epithelium (RPE)-specific 65-kDa protein knockout mouse model of Leber's congenital amaurosis

Purpose: In this study, we investigated the expression of the gene encoding beta-galactosidase (Glb)-1-like protein 3 (Glb1l3), a member of the glycosyl hydrolase 35 family, during retinal degeneration in the retinal pigment epithelium (RPE)-specific 65-kDa protein knockout (Rpe65(-/-)) mouse model of Leber congenital amaurosis (LCA). Additionally, we assessed the expression of the other members of this protein family, including beta-galactosidase-1 (Glb1), beta-galactosidase-1-like (Glb1l), and beta-galactosidase-1-like protein 2 (Glb1l2).Methods: The structural features of Glb1l3 were assessed using bioinformatic tools. mRNA expression of Glb-related genes was investigated by oligonucleotide microarray, real-time PCR, and reverse transcription (RT) -PCR. The localized expression of Glb1l3 was assessed by combined in situ hybridization and immunohistochemistry.Results: Glb1l3 was the only Glb-related member strongly downregulated in Rpe65(-/-) retinas before the onset and during progression of the disease. Glb1l3 mRNA was only expressed in the retinal layers and the RPE/choroid. The other Glb-related genes were ubiquitously expressed in different ocular tissues, including the cornea and lens. In the healthy retina, expression of Glb1l3 was strongly induced during postnatal retinal development; age-related increased expression persisted during adulthood and aging.Conclusions: These data highlight early-onset downregulation of Glb1l3 in Rpe65-related disease. They further indicate that impaired expression of Glb1l3 is mostly due to the absence of the chromophore 11-cis retinal, suggesting that Rpe65 deficiency may have many metabolic consequences in the underlying neuroretina.

GLP-1 protects beta-cells against apoptosis by enhancing the activity of an IGF-2/IGF-1 receptor autocrine loop

GLP-1 protects β-cells against apoptosis by still incompletely understood mechanisms. In a recent study, we searched for novel anti-apoptotic pathways by performing comparative transcriptomic analysis of islets from Gipr-/-;Glp-1r-/- mice, which show increased susceptibility to cytokine-induced apoptosis. We observed a strong reduction in IGF-1R expression in the knockout islets suggesting a link between the gluco-incretin and IGF-1R signaling pathways. Using MIN6 and primary islet cells, we demonstrated that GLP-1 strongly stimulates IGF-1R expression and that activation of the IGF-1R/Akt signaling pathway required active secretion of IGF-2 by the β-cells. We showed that inactivation of the IGF-1 receptor gene in β-cells or preventing its up-regulation by GLP-1, as well as suppressing IGF-2 expression or action, blocked the protective effect of GLP-1 against cytokine-induced apoptosis. Thus, an IGF-2/IGF-1 receptor autocrine loop operates in β-cells and GLP-1 increases its activity by enhancing IGF-1R expression and by stimulating IGF-2 secretion. This mechanism is required for GLP-1 to protect β-cells against apoptosis.

Exendin-(9-39) is an inverse agonist of the murine glucagon-like peptide-1 receptor: implications for basal intracellular cyclic adenosine 3',5'-monophosphate levels and beta-cell glucose competence.

The effect of exendin-(9-39), a described antagonist of the glucagon-like peptide-1 (GLP-1) receptor, was evaluated on the formation of cAMP- and glucose-stimulated insulin secretion (GSIS) by the conditionally immortalized murine betaTC-Tet cells. These cells have a basal intracellular cAMP level that can be increased by GLP-1 with an EC50 of approximately 1 nM and can be decreased dose dependently by exendin-(9-39). This latter effect was receptor dependent, as a beta-cell line not expressing the GLP-1 receptor was not affected by exendin-(9-39). It was also not due to the endogenous production of GLP-1, because this effect was observed in the absence of detectable preproglucagon messenger RNA levels and radioimmunoassayable GLP-1. Importantly, GSIS was shown to be sensitive to this basal level of cAMP, as perifusion of betaTC-Tet cells in the presence of exendin-(9-39) strongly reduced insulin secretion. This reduction of GSIS, however, was observed only with growth-arrested, not proliferating, betaTC-Tet cells; it was also seen with nontransformed mouse beta-cells perifused in similar conditions. These data therefore demonstrated that 1) exendin-(9-39) is an inverse agonist of the murine GLP-1 receptor; 2) the decreased basal cAMP levels induced by this peptide inhibit the secretory response of betaTC-Tet cells and mouse pancreatic islets to glucose; 3) as this effect was observed only with growth-arrested cells, this indicates that the mechanism by which cAMP leads to potentiation of insulin secretion is different in proliferating and growth-arrested cells; and 4) the presence of the GLP-1 receptor, even in the absence of bound peptide, is important for maintaining elevated intracellular cAMP levels and, therefore, the glucose competence of the beta-cells.

The scaffold protein IB1/JIP-1 is a critical mediator of cytokine-induced apoptosis in pancreatic beta cells.

In insulin-secreting cells, cytokines activate the c-Jun N-terminal kinase (JNK), which contributes to a cell signaling towards apoptosis. The JNK activation requires the presence of the murine scaffold protein JNK-interacting protein 1 (JIP-1) or human Islet-brain 1(IB1), which organizes MLK3, MKK7 and JNK for proper signaling specificity. Here, we used adenovirus-mediated gene transfer to modulate IB1/JIP-1 cellular content in order to investigate the contribution of IB1/JIP-1 to beta-cell survival. Exposure of the insulin-producing cell line INS-1 or isolated rat pancreatic islets to cytokines (interferon-gamma, tumor necrosis factor-alpha and interleukin-1beta) induced a marked reduction of IB1/JIP-1 content and a concomitant increase in JNK activity and apoptosis rate. This JNK-induced pro-apoptotic program was prevented in INS-1 cells by overproducing IB1/JIP-1 and this effect was associated with inhibition of caspase-3 cleavage. Conversely, reducing IB1/JIP-1 content in INS-1 cells and isolated pancreatic islets induced a robust increase in basal and cytokine-stimulated apoptosis. In heterozygous mice carrying a selective disruption of the IB1/JIP-1 gene, the reduction in IB1/JIP-1 content in happloinsufficient isolated pancreatic islets was associated with an increased JNK activity and basal apoptosis. These data demonstrate that modulation of the IB1-JIP-1 content in beta cells is a crucial regulator of JNK signaling pathway and of cytokine-induced apoptosis.