Evidence that extrapancreatic GLUT2-dependent glucose sensors control glucagon secretion.

GLUT2-/- mice reexpressing GLUT1 or GLUT2 in their beta-cells (RIPGLUT1 x GLUT2-/- or RIPGLUT2 x GLUT2-/- mice) have nearly normal glucose-stimulated insulin secretion but show high glucagonemia in the fed state. Because this suggested impaired control of glucagon secretion, we set out to directly evaluate the control of glucagonemia by variations in blood glucose concentrations. Using fasted RIPGLUT1 x GLUT2-/- mice, we showed that glucagonemia was no longer increased by hypoglycemic (2.5 mmol/l glucose) clamps or suppressed by hyperglycemic (10 and 20 mmol/l glucose) clamps. However, an increase in plasma glucagon levels was detected when glycemia was decreased to < or =1 mmol/l, indicating preserved glucagon secretory ability, but of reduced sensitivity to glucopenia. To evaluate whether the high-fed glucagonemia could be due to an abnormally increased tone of the autonomic nervous system, fed mutant mice were injected with the ganglionic blockers hexamethonium and chlorisondamine. Both drugs lead to a rapid return of glucagonemia to the levels found in control fed mice. We conclude that 1) in the absence of GLUT2, there is an impaired control of glucagon secretion by low or high glucose; 2) this impaired glucagon secretory activity cannot be due to absence of GLUT2 from alpha-cells because these cells do not normally express this transporter; 3) this dysregulation may be due to inactivation of GLUT2-dependent glucose sensors located outside the endocrine pancreas and controlling glucagon secretion; and 4) because fed hyperglucagonemia is rapidly reversed by ganglionic blockers, this suggests that in the absence of GLUT2, there is an increased activity of the autonomic nervous system stimulating glucagon secretion during the fed state.

Discordant increases in CD4+ T cells in human immunodeficiency virus-infected patients experiencing virologic treatment failure: role of changes in thymic output and T cell death.

Some patients infected with human immunodeficiency virus (HIV) who are experiencing antiretroviral treatment failure have persistent improvement in CD4+ T cell counts despite high plasma viremia. To explore the mechanisms responsible for this phenomenon, 2 parameters influencing the dynamics of CD4+ T cells were evaluated: death of mature CD4+ T cells and replenishment of the CD4+ T cell pool by the thymus. The improvement in CD4+ T cells observed in patients with treatment failure was not correlated with spontaneous, Fas ligand-induced, or activation-induced T cell death. In contrast, a significant correlation between the improvement in CD4+ T cell counts and thymic output, as assessed by measurement of T cell receptor excision circles, was observed. These observations suggest that increased thymic output contributes to the dissociation between CD4+ T cell counts and viremia in patients failing antiretroviral therapy and support a model in which drug-resistant HIV strains may have reduced replication rates and pathogenicity in the thymus.

Use of the FACTS solvation model for protein-ligand docking calculations. Application to EADock.

Protein-ligand docking has made important progress during the last decade and has become a powerful tool for drug development, opening the way to virtual high throughput screening and in silico structure-based ligand design. Despite the flattering picture that has been drawn, recent publications have shown that the docking problem is far from being solved, and that more developments are still needed to achieve high successful prediction rates and accuracy. Introducing an accurate description of the solvation effect upon binding is thought to be essential to achieve this goal. In particular, EADock uses the Generalized Born Molecular Volume 2 (GBMV2) solvent model, which has been shown to reproduce accurately the desolvation energies calculated by solving the Poisson equation. Here, the implementation of the Fast Analytical Continuum Treatment of Solvation (FACTS) as an implicit solvation model in small molecules docking calculations has been assessed using the EADock docking program. Our results strongly support the use of FACTS for docking. The success rates of EADock/FACTS and EADock/GBMV2 are similar, i.e. around 75% for local docking and 65% for blind docking. However, these results come at a much lower computational cost: FACTS is 10 times faster than GBMV2 in calculating the total electrostatic energy, and allows a speed up of EADock by a factor of 4. This study also supports the EADock development strategy relying on the CHARMM package for energy calculations, which enables straightforward implementation and testing of the latest developments in the field of Molecular Modeling.

Induction of the alternative NF-κB pathway by lymphotoxin αβ (LTαβ) relies on internalization of LTβ receptor.

Several tumor necrosis factor receptor (TNFR) family members activate both the classical and the alternative NF-κB pathways. However, how a single receptor engages these two distinct pathways is still poorly understood. Using lymphotoxin β receptor (LTβR) as a prototype, we showed that activation of the alternative, but not the classical, NF-κB pathway relied on internalization of the receptor. Further molecular analyses revealed a specific cytosolic region of LTβR essential for its internalization, TRAF3 recruitment, and p100 processing. Interestingly, we found that dynamin-dependent, but clathrin-independent, internalization of LTβR appeared to be required for the activation of the alternative, but not the classical, NF-κB pathway. In vivo, ligand-induced internalization of LTβR in mesenteric lymph node stromal cells correlated with induction of alternative NF-κB target genes. Thus, our data shed light on LTβR cellular trafficking as a process required for specific biological functions of NF-κB.

Constitutively active mutants of the alpha 2-adrenergic receptor.

We have mutated a single residue, Thr373 [corrected], in the C-terminal portion of the third intracellular loop of the alpha 2C10-adrenergic receptor into five different amino acids. In analogy with the effect of similar mutations in the alpha 1B- and beta 2-adrenergic receptors, these substitutions resulted in two major biochemical modifications: 1) increased constitutive activity of the alpha 2-adrenergic receptor leading to agonist-independent inhibition of adenylyl cyclase and 2) increased affinity of the receptor for binding agonist but not antagonists. The increased constitutive activity of the mutated alpha 2-adrenergic receptors could be inhibited by pertussis toxin, clearly indicating that it results from spontaneous ligand-independent receptor coupling to Gi. In contrast, the increased affinity of the mutant receptors for binding agonists was unaffected by pertussis toxin treatment, indicating that this is an inherent property of the receptors not dependent on interaction with Gi. Coexpression of the receptor mutants with the receptor-specific kinase, beta ARK1, indicated that the constitutively active alpha 2-adrenergic receptors are substrates for beta-adrenergic receptor kinase (beta ARK)-mediated phosphorylation even in the absence of agonist. These findings strengthen the idea that constitutively active adrenergic receptors mimic the "active" state of a G protein-coupled receptor adopting conformations similar to those induced by agonist when it binds to wild type receptors. In addition, these results extend the notion that in the adrenergic receptor family the C-terminal portion of the third intracellular loop plays a general role in the processes involved in receptor activation.

The chemokines CCL19 and CCL21 and their role in T and dendritic cell biology

Summary : The chemokines CCL19 and CCL21 and their common receptor CCR7 attract antigenpresenting dendritic cells (DCs) and naive T cells into the T zone of secondary lymphoid organs (SLO) and are therefore critically involved in homeostatic T cell recirculation and the initiation of adaptive immune responses. In addition. CCR7 ligands were proposed to mediate T cell exit from neonatal thymus, allowing colonization of T zones in SLOB. The relative contribution of CCL19 and CCL21 to these processes has remained unclear, as they were studied in mouse models lacking either CCR7 or both ligands. The aim of my thesis was to characterize Cc119-' mice and thereby investigate the relative roles of the two CCR7 ligands in development, homeostasis and immune response. The first study addressed the role of CCR7 ligands in DC biology. We found that CCL19 is dispensable for DC migration to lymph nodes and their localization to T zones. Furthermore, a CCL19-deficient environment did not lead to a defect in DC maturation or T cell priming. Therefore, CCL21 is sufficient to mediate CCR7-dependent processes during the initiation of adaptive immune responses. In the second study we investigated how the two CCR7 ligands affect CCR7 expression and function on naive T cells. We found that in SLOB CCR7 is constantly occupied with CCL19 and CCL21, eventually leading to its internalization. The reduced level of free CCR7 on these cells led to diminished ligand sensitivity and consequently impaired chemotactic responses. This effect was reversible by passage through aCCR7 ligand-free environment like the blood circulation. We propose that the different states of ligand sensitivity in SLOB and blood are important to allow for proper T cell recirculation. In the third study the role of CCL19 in neonatal thymus and spleen was analyzed. While neonatal Cc119-!- mice had no defect in thymic egress, we observed reduced T cell accumulation in the spleen but not lymph nodes. We identified reticular stromal cells in the developing white pulp (WP) as the major CCL 19 source. The development of these WP stromal cells as well as their CCL19 expression were dependent on LTalß2+ B cells. In conclusion, we have found that CCL21 can mostly compensate for lack of CCL19 in homeostasis and immunity. In contrast, during development. CCL19 has anon-redundant function for T cell colonization of the spleen.

Macrophage migration inhibitory factor deficiency leads to age-dependent impairment of glucose homeostasis in mice.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine produced by many cells and tissues including pancreatic beta-cells, liver, skeletal muscle, and adipocytes. This study investigates the potential role of MIF in carbohydrate homeostasis in a physiological setting outside of severe inflammation, utilizing Mif knockout (MIF-/-) mice. Compared with wild-type (WT) mice, MIF-/- mice had a lower body weight, from birth until 4 months of age, but subsequently gained weight faster, resulting in a higher body weight at 12 months of age. The lower weight in young mice was related to a higher energy expenditure, and the higher weight in older mice was related to an increased food intake and a higher fat mass. Fasting blood insulin level was higher in MIF-/- mice compared with WT mice at any age. After i.p. glucose injection, the elevation of blood insulin level was higher in MIF-/- mice compared with WT mice, at 2 months of age, but was lower in 12-month-old MIF-/- mice. As a result, the glucose clearance during intraperitoneal glucose tolerance tests was higher in MIF-/- mice compared with WT mice until 4 months of age, and was lower in 12-month-old MIF-/- mice. Insulin resistance was estimated (euglycemic-hyperinsulinemic clamp tests), and the phosphorylation activity of AKT was similar in MIF-/- mice and WT mice. In conclusion, this mouse model provides evidence for the role of MIF in the control of glucose homeostasis.

PDMP blocks brefeldin a-induced retrograde membrane transport from Golgi to ER: evidence for involvement of calcium homeostasis and dissociation from sphingolipid metabolism

In this study, we show that an inhibitor of sphingolipid biosynthesis, d,l-threo-1-phenyl-2- decanoylamino-3-morpholino-1-propanol (PDMP), inhibits brefeldin A (BFA)-induced retrograde membrane transport from Golgi to endoplasmic reticulum (ER). If BFA treatment was combined with or preceded by PDMP administration to cells, disappearance of discrete Golgi structures did not occur. However, when BFA was allowed to exert its effect before PDMP addition, PDMP could not ¿rescue¿ the Golgi compartment. Evidence is presented showing that this action of PDMP is indirect, which means that the direct target is not sphingolipid metabolism at the Golgi apparatus. A fluorescent analogue of PDMP, 6-(N-[7-nitro-2,1,3-benzoxadiazol-4-yl]amino)hexanoyl-PDMP (C6-NBD-PDMP), did not localize in the Golgi apparatus. Moreover, the effect of PDMP on membrane flow did not correlate with impaired C6-NBD-sphingomyelin biosynthesis and was not mimicked by exogenous C6-ceramide addition or counteracted by exogenous C6-glucosylceramide addition. On the other hand, the PDMP effect was mimicked by the multidrug resistance protein inhibitor MK571. The effect of PDMP on membrane transport correlated with modulation of calcium homeostasis, which occurred in a similar concentration range. PDMP released calcium from at least two independent calcium stores and blocked calcium influx induced by either extracellular ATP or thapsigargin. Thus, the biological effects of PDMP revealed a relation between three important physiological processes of multidrug resistance, calcium homeostasis, and membrane flow in the ER/ Golgi system.

Activation of Srk1 by the Mitogen-activated Protein Kinase Sty1/Spc1 Precedes Its Dissociation from the Kinase and Signals Its Degradation

Control of cell cycle progression by stress-activated protein kinases (SAPKs) is essential for cell adaptation to extracellular stimuli. The Schizosaccharomyces pombe SAPK Sty1/Spc1 orchestrates general changes in gene expression in response to diverse forms of cytotoxic stress. Here we show that Sty1/Spc1 is bound to its target, the Srk1 kinase, when the signaling pathway is inactive. In response to stress, Sty1/Spc1 phosphorylates Srk1 at threonine 463 of the regulatory domain, inducing both activation of Srk1 kinase, which negatively regulates cell cycle progression by inhibiting Cdc25, and dissociation of Srk1 from the SAPK, which leads to Srk1 degradation by the proteasome.

A clathrin-dependent pathway leads to KRas signaling on late endosomes en route to lysosomes

Ras proteins are small guanosine triphosphatases involved in the regulation of important cellular functions such as proliferation, differentiation, and apoptosis. Understanding the intracellular trafficking of Ras proteins is crucial to identify novel Ras signaling platforms. In this study, we report that epidermal growth factor triggers Kirsten Ras (KRas) translocation onto endosomal membranes (independently of calmodulin and protein kinase C phosphorylation) through a clathrin-dependent pathway. From early endosomes, KRas but not Harvey Ras or neuroblastoma Ras is sorted and transported to late endosomes (LEs) and lysosomes. Using yellow fluorescent protein¿Raf1 and the Raichu-KRas probe, we identified for the first time in vivo¿active KRas on Rab7 LEs, eliciting a signal output through Raf1. On these LEs, we also identified the p14¿MP1 scaffolding complex and activated extracellular signal-regulated kinase 1/2. Abrogation of lysosomal function leads to a sustained late endosomal mitogen-activated protein kinase signal output. Altogether, this study reveals novel aspects about KRas intracellular trafficking and signaling, shedding new light on the mechanisms controlling Ras regulation in the cell.

PCAF regulates the stability of the transcriptional regulator and cyclin-dependent kinase inhibitor p27Kip1

P27(Kip1) (p27) is a member of the Cip/Kip family of cyclin-dependent kinase inhibitors. Recently, a new function of p27 as transcriptional regulator has been reported. It has been shown that p27 regulates the expression of target genes mostly involved in splicing, cell cycle, respiration and translation. We report here that p27 directly binds to the transcriptional coactivator PCAF by a region including amino acids 91-120. PCAF associates with p27 through its catalytic domain and acetylates p27 at lysine 100. Our data showed that overexpression of PCAF induces the degradation of p27 whereas in contrast, the knockdown of PCAF stabilizes the protein. A p27 mutant in which K100 was substituted by arginine (p27-K100R) cannot be acetylated by PCAF and has a half-life much higher than that of p27WT. Moreover, p27-K100R remains stable along cell-cycle progression. Ubiquitylation assays and the use of proteasome inhibitors indicate that PCAF induces p27 degradation via proteasome. We also observed that knockdown of skp2 did not affect the PCAF induced degradation of p27. In conclusion, our data suggest that the p27 acetylation by PCAF regulates its stability.

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.

Inhibition of estrogen-responsive gene activation by the retinoid X receptor beta: evidence for multiple inhibitory pathways.

The retinoid X receptor beta (RXR beta; H-2RIIBP) forms heterodimers with various nuclear hormone receptors and binds multiple hormone response elements, including the estrogen response element (ERE). In this report, we show that endogenous RXR beta contributes to ERE binding activity in nuclear extracts of the human breast cancer cell line MCF-7. To define a possible regulatory role of RXR beta regarding estrogen-responsive transcription in breast cancer cells, RXR beta and a reporter gene driven by the vitellogenin A2 ERE were transfected into estrogen-treated MCF-7 cells. RXR beta inhibited ERE-driven reporter activity in a dose-dependent and element-specific fashion. This inhibition occurred in the absence of the RXR ligand 9-cis retinoic acid. The RXR beta-induced inhibition was specific for estrogen receptor (ER)-mediated ERE activation because inhibition was observed in ER-negative MDA-MB-231 cells only following transfection of the estrogen-activated ER. No inhibition of the basal reporter activity was observed. The inhibition was not caused by simple competition of RXR beta with the ER for ERE binding, since deletion mutants retaining DNA binding activity but lacking the N-terminal or C-terminal domain failed to inhibit reporter activity. In addition, cross-linking studies indicated the presence of an auxiliary nuclear factor present in MCF-7 cells that contributed to RXR beta binding of the ERE. Studies using known heterodimerization partners of RXR beta confirmed that RXR beta/triiodothyronine receptor alpha heterodimers avidly bind the ERE but revealed the existence of another triiodothyronine-independent pathway of ERE inhibition. These results indicate that estrogen-responsive genes may be negatively regulated by RXR beta through two distinct pathways.