Involvement of 4E-BP1 in the protection induced by HDLs on pancreatic beta cells.

High-density lipoproteins (HDLs) protect pancreatic beta cells against apoptosis. This property might relate to the increased risk to develop diabetes in patients with low HDL blood levels. The mechanisms by which HDLs protect beta cells are poorly characterized however. Here we used a transcriptomic approach to identify genes differentially modulated by HDLs in beta cells subjected to apoptotic stimuli. The transcript encoding 4E-BP1 was up-regulated by serum starvation and HDLs blocked this increase. 4E-BP1 inhibits cap-dependent translation in its non- or hypo-phosphorylated state but it looses this ability when hyper-phosphorylated. At the protein level, 4E-BP1 was also up-regulated in response to starvation and IL1beta and this was blunted by HDLs. While an ectopic increase of 4E-BP1 expression induced beta cell death, silencing 4E-BP1 increase with shRNAs inhibited the apoptotic-inducing capacities of starvation. HDLs can therefore protect beta cells by blocking 4E-BP1 protein expression but this is not the sole protective mechanism activated by HDLs. Indeed, HDLs blocked apoptosis induced by ER stress with no associated decrease in total 4E-BP1 induction. Although, HDLs favored the phosphorylation, and hence the inactivation of 4E-BP1 in these conditions, this appeared not to be required for HDL protection. Our results indicate that HDLs can protect beta cells through modulation of 4E-BP1 depending on the type of stress stimuli.

The nuclear hormone receptor peroxisome proliferator-activated receptor beta/delta potentiates cell chemotactism, polarization, and migration.

After an injury, keratinocytes acquire the plasticity necessary for the reepithelialization of the wound. Here, we identify a novel pathway by which a nuclear hormone receptor, until now better known for its metabolic functions, potentiates cell migration. We show that peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) enhances two phosphatidylinositol 3-kinase-dependent pathways, namely, the Akt and the Rho-GTPase pathways. This PPARbeta/delta activity amplifies the response of keratinocytes to a chemotactic signal, promotes integrin recycling and remodeling of the actin cytoskeleton, and thereby favors cell migration. Using three-dimensional wound reconstructions, we demonstrate that these defects have a strong impact on in vivo skin healing, since PPARbeta/delta-/- mice show an unexpected and rare epithelialization phenotype. Our findings demonstrate that nuclear hormone receptors not only regulate intercellular communication at the organism level but also participate in cell responses to a chemotactic signal. The implications of our findings may be far-reaching, considering that the mechanisms described here are important in many physiological and pathological situations.

Genetic removal of tri-unsaturated fatty acids suppresses developmental and molecular phenotypes of an Arabidopsis tocopherol-deficient mutant. Whole-body mapping of malondialdehyde pools in a complex eukaryote.

Malondialdehyde (MDA) is a small, ubiquitous, and potentially toxic aldehyde that is produced in vivo by lipid oxidation and that is able to affect gene expression. Tocopherol deficiency in the vitamin E2 mutant vte2-1 of Arabidopsis thaliana leads to massive lipid oxidation and MDA accumulation shortly after germination. MDA accumulation correlates with a strong visual phenotype (growth reduction, cotyledon bleaching) and aberrant GST1 (glutathione S-transferase 1) expression. We suppressed MDA accumulation in the vte2-1 background by genetically removing tri-unsaturated fatty acids. The resulting quadruple mutant, fad3-2 fad7-2 fad8 vte2-1, did not display the visual phenotype or the aberrant GST1 expression observed in vte2-1. Moreover, cotyledon bleaching in vte2-1 was chemically phenocopied by treatment of wild-type plants with MDA. These data suggest that products of tri-unsaturated fatty acid oxidation underlie the vte2-1 seedling phenotype, including cellular toxicity and gene regulation properties. Generation of the quadruple mutant facilitated the development of an in situ fluorescence assay based on the formation of adducts of MDA with 2-thiobarbituric acid at 37 degrees C. Specificity was verified by measuring pentafluorophenylhydrazine derivatives of MDA and by liquid chromatography analysis of MDA-2-thiobarbituric acid adducts. Potentially applicable to other organisms, this method allowed the localization of MDA pools throughout the body of Arabidopsis and revealed an undiscovered pool of the compound unlikely to be derived from trienoic fatty acids in the vicinity of the root tip quiescent center.

Generation of a tightly regulated all-cis beta cell-specific tetracycline-inducible vector.

Ability to induce protein expression at will in a cell is a powerful strategy used by scientists to better understand the function of a protein of interest. Various inducible systems have been designed in eukaryotic cells to achieve this goal. Most of them rely on two distinct vectors, one encoding a protein that can regulate transcription by binding a compound X, and one hosting the cDNA encoding the protein of interest placed downstream of promoter sequences that can bind the protein regulated by compound X (e.g., tetracycline, ecdysone). The commercially available systems are not designed to allow cell- or tissue-specific regulated expression. Additionally, although these systems can be used to generate stable clones that can be induced to express a given protein, extensive screening is often required to eliminate the clones that display poor induction or high basal levels. In the present report, we aimed to design a pancreatic beta cell-specific tetracycline-inducible system. Since the classical two-vector based tetracycline-inducible system proved to be unsatisfactory in our hands, a single vector was eventually designed that allowed tight beta cell-specific tetracycline induction in unselected cell populations.

Cardiovascular response to beta-adrenergic blockade or activation in 23 inbred mouse strains.

We report the characterisation of 27 cardiovascular-related traits in 23 inbred mouse strains. Mice were phenotyped either in response to chronic administration of a single dose of the beta-adrenergic receptor blocker atenolol or under a low and a high dose of the beta-agonist isoproterenol and compared to baseline condition. The robustness of our data is supported by high trait heritabilities (typically H(2)>0.7) and significant correlations of trait values measured in baseline condition with independent multistrain datasets of the Mouse Phenome Database. We then focused on the drug-, dose-, and strain-specific responses to beta-stimulation and beta-blockade of a selection of traits including heart rate, systolic blood pressure, cardiac weight indices, ECG parameters and body weight. Because of the wealth of data accumulated, we applied integrative analyses such as comprehensive bi-clustering to investigate the structure of the response across the different phenotypes, strains and experimental conditions. Information extracted from these analyses is discussed in terms of novelty and biological implications. For example, we observe that traits related to ventricular weight in most strains respond only to the high dose of isoproterenol, while heart rate and atrial weight are already affected by the low dose. Finally, we observe little concordance between strain similarity based on the phenotypes and genotypic relatedness computed from genomic SNP profiles. This indicates that cardiovascular phenotypes are unlikely to segregate according to global phylogeny, but rather be governed by smaller, local differences in the genetic architecture of the various strains.

Constitutively active mutants of the beta1-adrenergic receptor.

We provide the first evidence that point mutations can constitutively activate the beta(1)-adrenergic receptor (AR). Leucine 322 of the beta(1)-AR in the C-terminal portion of its third intracellular loop was replaced with seven amino acids (I, T, E, F, C, A and K) differing in their physico-chemical properties. The beta(1)-AR mutants expressed in HEK-293 cells displayed various levels of constitutive activity which could be partially inhibited by some beta-blockers. The results of this study might have interesting implications for future studies aiming at elucidating the activation process of the beta(1)-AR as well as the mechanism of action of beta-blockers.

Effects of the PPAR-beta agonist GW501516 in an in vitro model of brain inflammation and antibody-induced demyelination.

BACKGROUND: Brain inflammation plays a central role in numerous brain pathologies, including multiple sclerosis (MS). Microglial cells and astrocytes are the effector cells of neuroinflammation. They can be activated also by agents such as interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS). Peroxisome proliferator-associated receptor (PPAR) pathways are involved in the control of the inflammatory processes, and PPAR-beta seems to play an important role in the regulation of central inflammation. In addition, PPAR-beta agonists were shown to have trophic effects on oligodendrocytes in vitro, and to confer partial protection in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. In the present work, a three-dimensional brain cell culture system was used as in vitro model to study antibody-induced demyelination and inflammatory responses. GW 501516, a specific PPAR-beta agonist, was examined for its capacity to protect from antibody-mediated demyelination and to prevent inflammatory responses induced by IFN-gamma and LPS. METHODS: Aggregating brain cells cultures were prepared from embryonal rat brain, and used to study the inflammatory responses triggered by IFN-gamma and LPS and by antibody-mediated demyelination induced by antibodies directed against myelin-oligodendrocyte glycoprotein (MOG). The effects of GW 501516 on cellular responses were characterized by the quantification of the mRNA expression of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), inducible NO synthase (i-NOS), PPAR-beta, PPAR-gamma, glial fibrillary acidic protein (GFAP), myelin basic protein (MBP), and high molecular weight neurofilament protein (NF-H). GFAP expression was also examined by immunocytochemistry, and microglial cells were visualized by isolectin B4 (IB4) and ED1 labeling. RESULTS: GW 501516 decreased the IFN-gamma-induced up-regulation of TNF-alpha and iNOS in accord with the proposed anti-inflammatory effects of this PPAR-beta agonist. However, it increased IL-6 m-RNA expression. In demyelinating cultures, reactivity of both microglial cells and astrocytes was observed, while the expression of the inflammatory cytokines and iNOS remained unaffected. Furthermore, GW 501516 did not protect against the demyelination-induced changes in gene expression. CONCLUSION: Although GW 501516 showed anti-inflammatory activity, it did not protect against antibody-mediated demyelination. This suggests that the protective effects of PPAR-beta agonists observed in vivo can be attributed to their anti-inflammatory properties rather than to a direct protective or trophic effect on oligodendrocytes.

Identification of muscle-specific calpain and beta-sarcoglycan genes in progressive autosomal recessive muscular dystrophies.

The autosomal recessive forms of limb-girdle muscular dystrophies are encoded by at least five distinct genes. The work performed towards the identification of two of these is summarized in this report. This success illustrates the growing importance of genetics in modern nosology.

Loss of glucose-induced insulin secretion and GLUT2 expression in transplanted beta-cells.

Either 200 or 400 syngeneic islets were transplanted under the kidney capsule of normal or streptozocin-induced diabetic B6/AF1 mice. The diabetic mice with 400 islets became normoglycemic, but those with 200 islets, an insufficient number, were still diabetic after the transplantation (Tx). Two weeks after Tx, GLUT2 expression in the islet grafts was evaluated by immunofluorescence and Western blots, and graft function was examined by perfusion of the graft-bearing kidney. Immunofluorescence for GLUT2 was dramatically reduced in the beta-cells of grafts with 200 islets exposed to hyperglycemia. However, it was plentiful in grafts with 400 islets in a normoglycemic environment. Densitometric analysis of Western blots on graft homogenates demonstrated that GLUT2 protein levels in the islets, when exposed to chronic hyperglycemia for 2 weeks, were decreased to 16% of those of normal recipients. Moreover, these grafts had defective glucose-induced insulin secretion, while the effects of arginine were preserved. We conclude that GLUT2 expression in normal beta-cells is promptly down-regulated during exposure to hyperglycemia and may contribute to the loss of glucose-induced secretion of diabetes.

Thermodynamic Characterization of Humic Acid-surfactant Interaction: New Insights into the Characteristics and Structure of Humic Acids

ABSTRACT Humic acids (HA) are a component of humic substances (HS), which are found in nearly all soils, sediments, and waters. They play a key role in many, if not most, chemical and physical properties in their environment. Despite the importance of HA, their high complexity makes them a poorly understood system. Therefore, understanding the physicochemical properties and interactions of HA is crucial for determining their fundamental role and obtaining structural details. Cationic surfactants are known to interact electrostatically and hydrophobically with HA. Because they are a very well-known and characterized system, they offer a good choice as molecular probes for studying HA. The objective of this study was to evaluate the interaction between cationic surfactants and HA through isothermal titration calorimetry in a thermodynamic manner, aiming to obtain information about the basic structure of HA, the nature of this interaction, and if HA from different origins show different basic structures. Contrary to what the supramolecular model asserts, HA structure is not loosely held, though it may separate depending on the conditions the HA are subjected to in their milieu. It did not show any division or conformational change when interacting with surfactants. The basic structure of the HA remains virtually the same regardless of the different sources and compositions of these HA.

Muscle protein waste in tumor-bearing rats is effectively antagonized by a beta 2-adrenergic agonist (clenbuterol). Role of the ATP-ubiquitin-dependent proteolytic pathway.

Tissue protein hypercatabolism (TPH) is a most important feature in cancer cachexia, particularly with regard to the skeletal muscle. The rat ascites hepatoma Yoshida AH-130 is a very suitable model system for studying the mechanisms involved in the processes that lead to tissue depletion, since it induces in the host a rapid and progressive muscle waste mainly due to TPH (Tessitore, L., G. Bonelli, and F. M. Baccino. 1987. Biochem. J. 241:153-159). Detectable plasma levels of tumor necrosis factor-alpha associated with marked perturbations in the hormonal homeostasis have been shown to concur in forcing metabolism into a catabolic setting (Tessitore, L., P. Costelli, and F. M. Baccino. 1993. Br. J. Cancer. 67:15-23). The present study was directed to investigate if beta 2-adrenergic agonists, which are known to favor skeletal muscle hypertrophy, could effectively antagonize the enhanced muscle protein breakdown in this cancer cachexia model. One such agent, i.e., clenbuterol, indeed largely prevented skeletal muscle waste in AH-130-bearing rats by restoring protein degradative rates close to control values. This normalization of protein breakdown rates was achieved through a decrease of the hyperactivation of the ATP-ubiquitin-dependent proteolytic pathway, as previously demonstrated in our laboratory (Llovera, M., C. García-Martínez, N. Agell, M. Marzábal, F. J. López-Soriano, and J. M. Argilés. 1994. FEBS (Fed. Eur. Biochem. Soc.) Lett. 338:311-318). By contrast, the drug did not exert any measurable effect on various parenchymal organs, nor did it modify the plasma level of corticosterone and insulin, which were increased and decreased, respectively, in the tumor hosts. The present data give new insights into the mechanisms by which clenbuterol exerts its preventive effect on muscle protein waste and seem to warrant the implementation of experimental protocols involving the use of clenbuterol or alike drugs in the treatment of pathological states involving TPH, particularly in skeletal muscle and heart, such as in the present model of cancer cachexia.

Effects of dexamethasone on the metabolic responses to mental stress in humans.

The haemodynamic effects of the sympathetic nervous system (SNS) activations elicited by hypoglycaemia, acute alcohol administration, or insulin can be prevented by a pretreatment with dexamethasone in humans. This suggests a possible role of central corticotropin releasing hormone (GRIT) release. Mental stress activates the SNS, and decreases systemic vascular resistances though a beta-adrenergic-mediated vasodilation thought to involve vascular nitric oxide release. It also increases insulin-mediated glucose disposal, an effect presumably related to vasodilation. In order to evaluate whether activation of SNS by mental stress is glucocorticoid-sensitive, we monitored the haemodynamic and metabolic effects of mental stress during hyperinsulinaemia in healthy humans with and without a 2-day treatment with 8 mg day(-1) dexamethasone. Mental stress decreased systemic vascular resistances by 21.9% and increased insulin-mediated glucose disposal by 2 8.4% without dexamethasone pretreatment. After 2 days of dexamethasone treatment, whole body insulin-mediated glucose disposal was decreased by 40.8%. The haemodynainic effects of mental stress were however, not affected. Mental stress acutely increased insulin-mediated glucose disposal by 28.0%. This indicates that mental stress elicits a stimulation of SNS through dexamethasone-insensitive pathway, distinct of those activated by insulin, alcohol, or hyperglycaemia.

A V beta 4-specific superantigen encoded by a new exogenous mouse mammary tumor virus.

The superantigen (SAg) expressed by mouse mammary tumor virus (MMTV) has been shown to play an essential role in the course of the viral life cycle. In the present study, we describe a V beta 4-specific SAg encoded by a new exogenous MMTV carried by the SIM mouse strain. This is the first report of a viral or bacterial SAg reacting with mouse V beta 4+ T cells. Injection of MMTV(SIM) into adult BALB/c mice leads to a rapid and strong stimulation of V beta 4+ CD4+ T cells, followed by a slow deletion of these cells. Neonatal exposure to the virus also leads to a progressive deletion of V beta 4+ T cells. In contrast to other strong MMTV SAg, this new SAg requires the presence of major histocompatibility complex class II I-E molecules to be presented efficiently to T cells. Sequence analysis revealed a new predicted amino acid sequence in the C-terminal polymorphic region of this SAg. Furthermore, sequence comparisons to the most closely related SAg with different V beta specificities hint at the specific residues involved in the interaction with the T cell receptor.

Induction of the acyl-coenzyme A synthetase gene by fibrates and fatty acids is mediated by a peroxisome proliferator response element in the C promoter.

The long-chain acyl-coenzyme A synthetase (ACS) gene gives rise to three transcripts containing different first exons preceded by specific regulatory regions A, B, and C. Exon-specific oligonucleotide hybridization indicated that only A-ACS mRNA is expressed in rat liver. Fibrate administration induced liver C-ACS strongly and A-ACS mRNA to a lesser extent. B-ACS mRNA remained undetectable. In primary rat hepatocytes and Fa-32 hepatoma cells C-ACS mRNA increased after treatment with fenofibric acid, alpha-bromopalmitate, tetradecylthioacetic acid, or alpha-linolenic acid. Nuclear run-on experiments indicated that fenofibric acid and alpha-bromopalmitate act at the transcriptional level. Transient transfections showed a 3.4-, 2.3-, and 2.2-fold induction of C-ACS promoter activity after fenofibric acid, alpha-bromopalmitate, and tetradecylthioacetic acid, respectively. Unilateral deletion and site-directed mutagenesis identified a peroxisome proliferator activator receptor (PPAR)-responsive element (PPRE) mediating the responsiveness to fibrates and fatty acids. This ACS PPRE contains three imperfect half sites spaced by 1 and 3 oligonucleotides and binds PPAR.retinoid X receptor heterodimers in gel retardation assays. In conclusion, the regulation of C-ACS mRNA expression by fibrates and fatty acids is mediated by PPAR.retinoid X receptor heterodimers interacting through a PPRE in the C-ACS promoters. PPAR therefore occupies a key position in the transcriptional control of a pivotal enzyme controlling the channeling of fatty acids into various metabolic pathways.

Interleukins (IL)-1 and IL-2 control IL-2 receptor alpha and beta expression in immature thymocytes.

Functional high-affinity interleukin-2 receptors (IL-2R) contain three transmembrane proteins, IL-2R alpha, beta and gamma. We have investigated the expression of IL-2R alpha and beta genes in immature mouse thymocytes. Previous work has shown that during differentiation these cells transiently express IL-2R alpha on their surface. Stimulation of IL-2R alpha+ and IL-2R alpha- immature thymocytes with phorbol 12-myristate 13-acetate and calcium ionophore induces synthesis of IL-2R alpha and IL-2R beta mRNA. Most of this response depends on autocrine stimulation by IL-2. IL-1 synergizes with IL-2 to induce a 120-fold increase in IL-2R alpha mRNA and a 14-fold increase in IL-2R beta mRNA levels. A large proportion of the stimulated cells contains both transcripts. These interleukins do not induce any differentiation to more mature phenotypes. Collectively, these results show that IL-2 plays a major role in the regulation of IL-2R expression in normal immature thymocyte. We suggest that this response to interleukins may be part of a homeostatic mechanism to increase the production of immature thymocytes during stress.