Central glucocorticoid administration promotes weight gain and increased 11β-hydroxysteroid dehydrogenase type 1 expression in white adipose tissue

Glucocorticoids (GCs) are involved in multiple metabolic processes, including the regulation of insulin sensitivity and adipogenesis. Their action partly depends on their intracellular activation by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). We previously demonstrated that central GC administration promotes hyperphagia, body weight gain, hyperinsulinemia and marked insulin resistance at the level of skeletal muscles. Similar dysfunctions have been reported to occur upon specific overexpression of 11β-HSD1 in adipose tissue. The aim of the present study was therefore to determine whether the effects of central GC infusion may enhance local GC activation in white adipose tissue. Male Wistar and Sprague Dawley (SD) rats were intracerebroventricularly infused with GCs for 2 to 3 days. Body weight, food intake and metabolic parameters were measured, and expression of enzymes regulating 11β-HSD1, as well as that of genes regulated by GCs, were quantified. Central GC administration induced a significant increase in body weight gain and in 11β-HSD1 and resistin expression in adipose tissue. A decrease 11β-HSD1 expression was noticed in the liver of SD rats, as a partial compensatory mechanism. Such effects of GCs are centrally elicited. This model of icv dexamethasone infusion thus appears to be a valuable acute model, that helps delineating the initial metabolic defects occurring in obesity. An impaired downregulation of intracellular GC activation in adipose tissue may be important for the development of insulin resistance.     

Overexpression of CD39 in Mouse Airways Promotes Bacteria-Induced Inflammation

In airways, the ecto-nucleoside triphosphate diphosphohydrolase CD39 plays a central role in the regulation of physiological mucosal nucleotide concentrations and likely contributes to the control of inflammation because accelerated ATP metabolism occurs in chronic inflammatory lung diseases. We sought to determine whether constant elevated CD39 activity in lung epithelia is sufficient to cause inflammation and whether this affects the response to acute LPS or Pseudomonas aeruginosa exposure. We generated transgenic mice overexpressing human CD39 under the control of the airway-specific Clara cell 10-kDa protein gene promoter. Transgenic mice did not develop any spontaneous lung inflammation. However, intratracheal instillation of LPS resulted in accelerated recruitment of neutrophils to the airways of transgenic mice. Macrophage clearance was delayed, and the amounts of CD8(+) T and B cells were augmented. Increased levels of keratinocyte chemoattractant, IL-6, and RANTES were produced in transgenic lungs. Similarly, higher numbers of neutrophils and macrophages were found in the lungs of transgenic mice infected with P. aeruginosa, which correlated with improved bacteria clearance. The transgenic phenotype was partially and differentially restored by coinstillation of P2X(1) or P2X(7) receptor antagonists or of caffeine with LPS. Thus, a chronic increase of epithelial CD39 expression and activity promotes airway inflammation in response to bacterial challenge by enhancing P1 and P2 receptor activation.     

Probing the Acceptor Active Site Organization of the Human Recombinant β1,4-Galactosyltransferase 7 and Design of Xyloside-based Inhibitors

Among glycosaminoglycan (GAG) biosynthetic enzymes, the human β1,4-galactosyltransferase 7 (hβ4GalT7) is characterized by its unique capacity to take over xyloside derivatives linked to a hydrophobic aglycone as substrates and/or inhibitors. This glycosyltransferase is thus a prime target for the development of regulators of GAG synthesis in therapeutics. Here, we report the structure-guided design of hβ4GalT7 inhibitors. By combining molecular modeling, in vitro mutagenesis, and kinetic measurements, and in cellulo analysis of GAG anabolism and decorin glycosylation, we mapped the organization of the acceptor binding pocket, in complex with 4-methylumbelliferone-xylopyranoside as prototype substrate. We show that its organization is governed, on one side, by three tyrosine residues, Tyr¹⁹⁴, Tyr¹⁹⁶, and Tyr¹⁹⁹, which create a hydrophobic environment and provide stacking interactions with both xylopyranoside and aglycone rings. On the opposite side, a hydrogen-bond network is established between the charged amino acids Asp²²⁸, Asp²²⁹, and Arg²²⁶, and the hydroxyl groups of xylose. We identified two key structural features, i.e. the strategic position of Tyr¹⁹⁴ forming stacking interactions with the aglycone, and the hydrogen bond between the His¹⁹⁵ nitrogen backbone and the carbonyl group of the coumarinyl molecule to develop a tight binder of hβ4GalT7. This led to the synthesis of 4-deoxy-4-fluoroxylose linked to 4-methylumbelliferone that inhibited hβ4GalT7 activity in vitro with a K_i 10 times lower than the K_m value and efficiently impaired GAG synthesis in a cell assay. This study provides a valuable probe for the investigation of GAG biology and opens avenues toward the development of bioactive compounds to correct GAG synthesis disorders implicated in different types of malignancies.     

Sphingosine 1-phosphate, present in serum-derived lipoproteins, activates matriptase.

We describe here a novel biological function of sphingosine 1-phosphate (S1P): the activation of a serine protease, matriptase. Matriptase is a type II integral membrane serine protease, expressed on the surface of a variety of epithelial cells; it may play an important role in tissue remodeling. We have previously reported that the activation of matriptase is regulated by serum. We have now identified the bioactive component from serum. First, the activity was observed to co-purify with lipoproteins by conventional liquid chromatography and immunoaffinity chromatography. The ability of lipoproteins to induce the activation of matriptase was further confirmed with commercial preparations of low density lipoprotein (LDL) and very low density lipoprotein (VLDL). Next, we observed that the bioactive component of LDL is associated with the phospholipid components of LDL. Fractionation of lipid components of LDL by thin layer chromatography (TLC) revealed that the bioactive component of LDL comigrates with S1P. Nanomolar concentrations of commercially obtained S1P were then observed to induce the rapid activation of matriptase on the surfaces of nontransformed human mammary epithelial cells. Other structurally related sphingolipids, including dihydro-S1P, ceramide 1-phosphates, and sphingosine phosphocholine as well as lysophosphatidic acid, can also induce the activation of matriptase, but at significantly higher concentrations than S1P. Furthermore, S1P-dependent matriptase activation is dependent on Ca(2+) but not via G(i) protein-coupled receptors. Our results demonstrate that bioactive phospholipids can function as nonprotein activators of a cell surface protease, suggesting a possible mechanistic link between S1P and normal and possibly pathologic tissue remodeling.     

Did the Quaternary climatic fluctuations really influence the tempo and mode of diversification in European rodents?

The objective of this study was to establish whether the Quaternary climatic fluctuations influenced the tempo and mode of diversification in European rodents. Our case study is the subgenus Microtus (Terricola) distributed from western Europe to the Caucasus. Mitochondrial cytochrome b gene sequences from several representatives of all the species were used to generate maximum‐likelihood and Bayesian phylogenetic trees, to estimate divergence times, to identify biogeographic ancestral areas and to study the rate of diversification. Results showed that phylogenetic tree topologies were similar to previous published studies but with a better resolution at some nodes. The origin of Microtus (Terricola) is dated back to approximately 4.05 Myr in the Early Pliocene, and molecular dating for most Terricola species corresponds to several glacial periods of the Pleistocene. Results of the biogeographic ancestral area reconstruction suggest that Microtus (Terricola) diversified from the Caucasus/Turkey/Iran area through western Europe. Several periods of diversity variation were highlighted as follows: two period of diversity increase, between 3 and 2 Myr, and after 1 Myr; two periods of diversity decrease, before 3 Myr, and between 2 and 1 Myr. The diversification rate of Microtus (Terricola) was 0.353 ± 0.004 event/Myr, a rate similar to that of the Muridae family. To conclude, although the Pleistocene glacial conditions had an impact on the speciation events, the Quaternary does not appear however as a period with an exceptional rate of diversification for European rodents.     

Ac His1 [D-Phe2, K15, R16, L27] VIP (3-7)/GRF (8-27)--a VPAC1 receptor antagonist--is an inverse agonist on two constitutively active truncated VPAC1 receptors

C-terminally truncated human VPAC(1) receptors were constructed and stably transfected in Chinese hamster ovary (CHO) cells. Selected clones expressing comparable receptor densities were studied for ligand's binding properties, basal and stimulated adenylate cyclase activity. The wild-type (1-457) receptor served as reference. The binding properties of all the constructions were preserved. As judged by the intrinsic activity of the partial agonist Q(3)-VIP, the shortest receptors have a moderate impairment of the coupling efficacy to G(alpha s) protein. Cells expressing the VPAC(1) (1-436) and (1-441) truncated receptors had a two- to three-fold higher basal adenylate cyclase activity than those expressing the wild-type or the VPAC(1) (1-444), (1-433), (1-429), (1-421) and (1-398) receptor. The stimulatory effect of VIP and other agonist was preserved. This suggested that VPAC(1) (1-436) and (1-441) receptors had a constitutive activity. The selective VPAC(1) receptor antagonist Ac His(1) [D-Phe(2), K(15), R(16), L(27)] VIP (3-7)/GRF (8-27) reduced by 60% the basal activity with an EC(50) value of 3 nM comparable to its IC(50) value for binding. This agonist behaved thus like an inverse agonist on the constitutively active VPAC(1) receptors generated by C-terminal truncation and expressed in CHO cells.     

Regulatory role of arginine 204 in the catalytic activity of rat alloantigens ART2a and ART2b

ART2a (RT6.1) and ART2b (RT6.2) are NAD glycohydrolases (NADases) that are linked to T lymphocytes by glycosylphosphatidylinositol anchors. Although both mature proteins possess three conserved regions (I, II, III) that form the NAD-binding site and differ by only ten amino acids, only ART2b is auto-ADP-ribosylated and only ART2a is glycosylated. To investigate the structural basis for these differences, wild-type and mutant ART2a and ART2b were expressed in rat mammary adenocarcinoma (NMU) cells and released with phosphatidylinositol-specific phospholipase C. All mutants were immunoreactive NADases. Arginine 204 (Arg204), NH2-terminal to essential glutamate 209 in Region III, is found in ART2b, but not ART2a. Replacement of Arg204 in ART2b with lysine, tyrosine, or glutamate abolished auto-ADP-ribosylation. Unlike wild-type ART2a, ART2a(Y204R) was auto-ADP-ribosylated. The tryptophan mutant ART2b(R204W) was auto-ADP-ribosylated and exhibited enhanced NADase activity. Incubation with NAD and auto-ADP-ribosylation decreased the NADase activities of wild-type ART2b and ART2b (R204W), whereas activity of ART2b(R204K), which is not auto-modified, was unchanged by NAD. Facilitation of auto-ADP-ribosylation by tryptophan 204 suggests that the hydrophobic amino acid mimics an ADP-ribosylated arginine. Thus, Arg204 in ART2b serves as a regulatory switch whose presence is required for additional auto-ADP-ribosylation and regulation of catalytic activity.