Activation of Wnt/β-Catenin Signaling Increases Insulin Sensitivity through a Reciprocal Regulation of Wnt10b and SREBP-1c in Skeletal Muscle Cells

Background

Intramyocellular lipid accumulation is strongly related to insulin resistance in humans, and we have shown that high glucose concentration induced de novo lipogenesis and insulin resistance in murin muscle cells. Alterations in Wnt signaling impact the balance between myogenic and adipogenic programs in myoblasts, partly due to the decrease of Wnt10b protein. As recent studies point towards a role for Wnt signaling in the pathogenesis of type 2 diabetes, we hypothesized that activation of Wnt signaling could play a crucial role in muscle insulin sensitivity.

Methodology/Principal Findings

Here we demonstrate that SREBP-1c and Wnt10b display inverse expression patterns during muscle ontogenesis and regeneration, as well as during satellite cells differentiation. The Wnt/β-catenin pathway was reactivated in contracting myotubes using siRNA mediated SREBP-1 knockdown, Wnt10b over-expression or inhibition of GSK-3β, whereas Wnt signaling was inhibited in myoblasts through silencing of Wnt10b. SREBP-1 knockdown was sufficient to induce Wnt10b protein expression in contracting myotubes and to activate the Wnt/β-catenin pathway. Conversely, silencing Wnt10b in myoblasts induced SREBP-1c protein expression, suggesting a reciprocal regulation. Stimulation of the Wnt/β-catenin pathway i) drastically decreased SREBP-1c protein and intramyocellular lipid deposition in myotubes; ii) increased basal glucose transport in both insulin-sensitive and insulin-resistant myotubes through a differential activation of Akt and AMPK pathways; iii) restored insulin sensitivity in insulin-resistant myotubes.

Conclusions/Significance

We conclude that activation of Wnt/β-catenin signaling in skeletal muscle cells improved insulin sensitivity by i) decreasing intramyocellular lipid deposition through downregulation of SREBP-1c; ii) increasing insulin effects through a differential activation of the Akt/PKB and AMPK pathways; iii) inhibiting the MAPK pathway. A crosstalk between these pathways and Wnt/β-catenin signaling in skeletal muscle opens the exciting possibility that organ-selective modulation of Wnt signaling might become an attractive therapeutic target in regenerative medicine and to treat obese and diabetic populations.     

Structural and Functional Investigations of Matrilin-1 A-domains Reveal Insights into Their Role in Cartilage ECM Assembly

Matrilin-1 is expressed predominantly in cartilage and co-localizes with matrilin-3 with which it can form hetero-oligomers. We recently described novel structural and functional features of the matrilin-3 A-domain (M3A) and demonstrated that it bound with high affinity to type II and IX collagens. Interactions preferentially occurred in the presence of Zn²⁺ suggesting that matrilin-3 has acquired a requirement for specific metal ions for activation and/or molecular associations. To understand the interdependence of matrilin-1/-3 hetero-oligomers in extracellular matrix (ECM) interactions, we have extended these studies to include the two matrilin-1 A-domains (i.e. M1A1 and M1A2 respectively). In this study we have identified new characteristics of the matrilin-1 A-domains by describing their glycosylation state and the effect of N-glycan chains on their structure, thermal stability, and protein-protein interactions. Initial characterization revealed that N-glycosylation did not affect secretion of these two proteins, nor did it alter their folding characteristics. However, removal of the glycosylation decreased their thermal stability. We then compared the effect of different cations on binding between both M1A domains and type II and IX collagens and showed that Zn²⁺ also supports their interactions. Finally, we have demonstrated that both M1A1 domains and biglycan are essential for the association of the type II·VI collagen complex. We predict that a potential role of the matrilin-1/-3 hetero-oligomer might be to increase multivalency, and therefore the ability to connect various ECM components. Differing affinities could act to regulate the integrated network, thus coordinating the organization of the macromolecular structures in the cartilage ECM.     

ICEEc2, a New Integrative and Conjugative Element Belonging to the pKLC102/PAGI-2 Family,Identified in Escherichia coli Strain BEN374

The diversity of the Escherichia coli species is in part due to the large number of mobile genetic elements that are exchanged between strains. We report here the identification of a new integrative and conjugative element (ICE) of the pKLC102/PAGI-2 family located downstream of the tRNA gene pheU in the E. coli strain BEN374. Indeed, this new region, which we called ICEEc2, can be transferred by conjugation from strain BEN374 to the E. coli strain C600. We were also able to transfer this region into a Salmonella enterica serovar Typhimurium strain and into a Yersinia pseudotuberculosis strain. This transfer was then followed by the integration of ICEEc2 into the host chromosome downstream of a phe tRNA gene. Our data indicated that this transfer involved a set of three genes encoding DNA mobility enzymes and a type IV pilus encoded by genes present on ICEEc2. Given the wide distribution of members of this family, these mobile genetic elements are likely to play an important role in the diversification of bacteria.The fantastic diversity of the Escherichia coli species has been known for a long time. With modern sequencing strategies, the molecular bases of this diversity are now being unraveled (49). Analyzing the genome of 20 E. coli strains, Touchon et al. recently showed that only a minority of genes, approximately 1,900 genes, were shared by all E. coli strains and constituted the core genome of the E. coli species (50). Additionally, the total number of genes found in all E. coli strains, the pan-genome, is an order of magnitude larger than this core genome (50). The non-core genome of a strain, also called flexible gene pool, is therefore made of a wide diversity of genes. This genetic diversity of the E. coli species translates into a diversity of phenotypic properties. While most E. coli strains are commensal of the gastrointestinal tract of humans and warm-blooded animals, a significant number are responsible for different diseases in humans and animals (22), including extraintestinal infections in chickens; strains isolated from such cases are designated by the term APEC for avian pathogenic E. coli (10).This diversity arises from frequent horizontal gene transfers of mobile genetic elements such as transposons, plasmids, phages, genomic islands, or integrative and conjugative elements (ICEs) (11, 21, 34). Among these mobile genetic elements, ICEs have a particular place as they share properties with both plasmids, genomic islands, and transposons; they can be defined as elements that encode all the necessary machineries that allow their excision from the chromosome, their transfer to a recipient strain, and their integration into the recipient strain''s genome (5, 6, 46, 54). Well-known representatives of this class of genetic elements include Tn916 discovered in Enterococcus faecalis, the conjugative transposon CTnDOT in Bacteroides thetaiotaomicron, ICEKp1 in Klebsiella pneumoniae, SXT/R391-related elements, PFGI-1 in Pseudomonas fluorescens, and the clc element in Pseudomonas sp. strain B13 as well as ICEBs1 in Bacillus subtilis and ICEEc1 in the E. coli strain ECOR31 (1, 39, 44, 46, 54). Typically, ICEs contain at least three modules that are required for key steps in the ICE''s life cycle: an excision/integration module, a transfer module, and a regulation module (54). Besides these, ICEs often contain cargo regions that confer on their host a diverse array of properties, such as virulence properties (ICEEc1), antibiotic resistance (SXT), or degradation of chemical compounds (clc). Because of their self-transfer abilities and their diverse accessory gene repertoires, ICEs are very likely to play a major role in bacteria evolution (46).A new family of ICEs has recently gained interest and was named the pKLC102/PAGI-2 family. The first element of this family, the clc element, was discovered in Pseudomonas sp. strain B13 and confers on the bacteria the possibility to degrade aromatic compounds (42). The transfer of this element was discovered long before its complete sequence was characterized (16). Other members of this family include several elements present in Pseudomonas strains such as PAGI-1 and PAGI-2 as well as the pKLC102 element first considered to be a plasmid but later on shown to be an ICE because of its ability to integrate into the chromosome of its host (23, 52). pKLC102/PAGI-2 elements share a set of core genes (33) and, like most ICEs and genomic islands, are all integrated downstream of tRNA genes (26, 52). The transfer between strains has been demonstrated, albeit with different frequencies, for only a few members, such as the clc element, Pseudomonas aeruginosa pathogenicity island 1 (PAPI-1), and ICEHin1056 from Haemophilus influenzae (20, 37, 41); this transfer involves the type IV pilus (20), the integrase (40), and in some cases the formation of a circular intermediate of the excised ICE (24).In order to identify new accessory genes of APEC strains, we previously described tRNA loci in the E. coli genome that could represent potential insertion sites for new genomic islands (18). We had already used this strategy to characterize the AGI-3 region that is involved in the virulence of an avian pathogenic E. coli strain and that confers the ability to grow on fructooligosaccharides (7, 43). During this tRNA screening, we showed that genomic islands might potentially be present downstream of the tRNA genes argW, leuX, pheU, pheV, selC, serU, and thrW in several APEC strains.In this report, we describe the identification of a new genomic island located downstream of pheU in the APEC strain BEN374. This region, which we named ICEEc2, was fully sequenced, and its properties were analyzed in detail; ICEEc2 is a new ICE found in E. coli and belongs to the pKLC102/PAGI-2 family described above.     

Changes to the Natural Killer Cell Repertoire after Therapeutic Hepatitis B DNA Vaccination

Background

Improvements to the outcome of adaptive immune responses could be achieved by inducing specific natural killer (NK) cell subsets which can cooperate with dendritic cells to select efficient T cell responses. We previously reported the induction or reactivation of T cell responses in chronic hepatitis B patients vaccinated with a DNA encoding hepatitis B envelope proteins during a phase I clinical trial.

Methodology/Principal Findings

In this study, we examined changes in the peripheral NK cell populations occurring during this vaccine trial using flow cytometry analysis. Despite a constant number of NK cells in the periphery, a significant increase in the CD56^bright population was observed after each vaccination and during the follow up. Among the 13 different NK cell markers studied by flow cytometry analysis, the expression of CD244 and NKG2D increased significantly in the CD56^bright NK population. The ex vivo CD107a expression by CD56^bright NK cells progressively increased in the vaccinated patients to reach levels that were significantly higher compared to chronically HBV-infected controls. Furthermore, modifications to the percentage of the CD56^bright NK cell population were correlated with HBV-specific T cell responses detected by the ELISPOT assay.

Conclusions/Significance

These changes in the CD56^bright population may suggest a NK helper effect on T cell adaptive responses. Activation of the innate and adaptive arms of the immune system by DNA immunization may be of particular importance to the efficacy of therapeutic interventions in a context of chronic infections.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00988767","term_id":"NCT00988767"}}NCT00988767     

The Lectin ArtinM Induces Recruitment of Rat Mast Cells from the Bone Marrow to the Peritoneal Cavity

Background

The D-mannose binding lectin ArtinM is known to recruit neutrophils, to degranulate mast cells and may have potential therapeutic applications. However, the effect of ArtinM on mast cell recruitment has not been investigated.

Methodology

Male Wistar rats were injected i.p. with ArtinM or ConA (control). The ability of the lectin to degranulate peritoneal and mesenteric mast cells was examined. Recruitment of mast cells to the peritoneal cavity and mesentery after ArtinM injection was examined with or without depletion of peritoneal mast cells by distilled water.

Results

ArtinM degranulated both peritoneal and mesentery mast cells in vitro. Three days after i.p. injection of the lectin there were reduced numbers of mast cells in the peritoneal lavage, while at 7 days post injection of ArtinM, the number of peritoneal mast cells was close to control values. Since immature mast cells are recruited from the bone marrow, the effect of the lectin on bone marrow mast cells was examined. Injection of ArtinM resulted in an increased number of mast cells in the bone marrow. To determine if degranulation of mast cells in the peritoneal cavity was required for the increase in bone marrow mast cells, the peritoneal cavity was depleted of mast cells with ultrapure water. Exposure to ArtinM increased the number of mast cells in the bone marrow of rats depleted of peritoneal mast cells.

Conclusions

The ArtinM induced recruitment of mast cells from the bone marrow to the peritoneal cavity may partially explain the therapeutic actions of ArtinM.     

Adaptive properties of living beings: proposal for a generic mechanism. (Self-programming machines III)

Living systems are capable to have appropriate responses to unpredictable environment. This kind of self-organization seems to operate as a self-programming machine, i.e. an organization able to modify itself. Until now the models of self-organization of living beings proposed are functions solutions of differential systems or transition functions of automata. These functions are fixed and these models are therefore unable to modify their organization. On the other hand, computer science propose a lot of models having the properties of adaptive systems of living beings, but all these models depend on the comparison between a goal and the results and ingenious choices of parameters by programmers, whereas there are no programmer's intention nor choice in the living systems. From two best known examples of adaptive systems of living beings, nervous system and immune system that have in common that the external signals modify the rewriting of their organization and therefore work as self-organizing machines, we devised machines with a finite set of inputs, based upon a recurrence, are able to rewrite their organization (Self-programming machines or m(sp)) whenever external conditions vary and have striking properties of adaptation. M(sp) have similar properties whatever the operation defining the recurrence maybe. These results bring us to make the following statement: adaptive properties of living systems can be explained by their ability to rewrite their organization whenever external conditions vary under the only assumption that the rewriting mechanism be a deterministic constant recurrence in a finite state set.     

Overexpression and characterization of two human salivary proline rich proteins

Proline rich proteins (PRP) are among major human saliva constituents and are known to interact with wine tannins that are involved in astringency. To characterize these interactions, a human salivary proline rich pro-protein, PRB4S, was overexpressed in Pichia pastoris. Six recombinant proteins resulting from maturation in bioreactor were detected by SDS-PAGE analysis between 15 and 45 kDa (apparent molecular weight). Two of them, the 45 and the 15 kDa ones, were isolated from culture supernatant by adsorption and permeation chromatography. They were characterized by N-terminal sequencing and MALDI-TOF analysis after trypsic digestion. The 45 kDa protein is glycosylated while the 15 kDa one was obtained after a furin-like proteolysis. Both of them are similar to human whole saliva PRP resulting from proteolysis of PRB4S pro-protein in Golgi network and known as II-1 and IB-5. Because of their sensitivity to proteolysis or their unusual mobility on SDS-PAGE gel, these recombinant proteins seem to be intrinsically unstructured proteins.     

Synthesis of peptide aldehydes.

The functionalization of peptides and proteins by aldehyde groups has become the subject of intensive research since the discovery of the inhibition properties of peptide aldehydes towards various enzymes. Furthermore, peptide aldehydes are of great interest for peptide backbone modification or ligation reactions. This review focuses upon their synthesis, which has been developed following two main strategies. The first strategy consists of prior synthesis of the peptide, followed by the introduction of the aldehyde function. The second possible strategy uses alpha-amino aldehydes as starting materials. After protection of the aldehyde, peptide elongation occurs. At the end of the synthesis, the aldehyde function can be unmasked.     

Trans-inactivation of receptor tyrosine kinases by novel angiotensin II AT2 receptor-interacting protein, ATIP

Negative regulation of mitogenic pathways is a fundamental process that remains poorly characterized. The angiotensin II AT2 receptor is a rare example of a 7-transmembrane domain receptor that negatively cross-talks with receptor tyrosine kinases to inhibit cell growth. In the present study, we report the molecular cloning of a novel protein, ATIP1 (AT2-interacting protein), which interacts with the C-terminal tail of the AT2 receptor, but not with those of other receptors such as angiotensin AT1, bradykinin BK2, and adrenergic beta(2) receptor. ATIP1 defines a family of at least four members that possess the same domain of interaction with the AT2 receptor, contain a large coiled-coil region, and are able to dimerize. Ectopic expression of ATIP1 in eukaryotic cells leads to inhibition of insulin, basic fibroblast growth factor, and epidermal growth factor-induced ERK2 activation and DNA synthesis, and attenuates insulin receptor autophosphorylation, in the same way as the AT2 receptor. The inhibitory effect of ATIP1 requires expression, but not ligand activation, of the AT2 receptor and is further increased in the presence of Ang II, indicating that ATIP1 cooperates with AT2 to transinactivate receptor tyrosine kinases. Our findings therefore identify ATIP1 as a novel early component of growth inhibitory signaling cascade.