The integrin beta1 modulator Tirofiban prevents adipogenesis and obesity by the overexpression of integrin-linked kinase: a pre-clinical approach in vitro and in vivo.

BACKGROUND: Obesity is caused by the enlargement of the white adipose tissue (WAT) depots, characterized by the hypertrophic enlargement of malfunctioning adipocytes within WAT which increases the storage of triglycerides (TG) in the lipid droplets (LD). Adipogenesis pathways as well as the expression and activity of some extracellular matrix receptors integrins are upregulated. Integrinß1 (INTB1) is the main isoform involved in WAT remodeling during obesity and insulin resistance-related diseases. We recently described Integrin Linked Kinase (ILK), a scaffold protein recruited by INTB1, as an important mediator of WAT remodeling and insulin resistance. As the few approved drugs to fight obesity have brought long-term cardiovascular side effects and given that the consideration of INTB1 and/or ILK modulation as anti-obesogenic strategies remains unexplored, we aimed to evaluate the anti-obesogenic capacity of the clinically approved anticoagulant Tirofiban (TF), stated in preclinical studies as a cardiovascular protector. METHODS: Fully differentiated adipocytes originating from C3H10T1/2 were exposed to TF and were co-treated with specific INTB1 blockers or with siRNA-based knockdown ILK expression. Lipid-specific dyes were used to determine the TG content in LD. The genetic expression pattern of ILK, pro-inflammatory cytokines (MCP1, IL6), adipogenesis (PPARγ, Leptin), thermogenesis (UCP1), proliferation (PCNA), lipid metabolism (FASN, HSL, ATGL), and metabolite transporters (FABP4, FAT, AQP7) were detected using quantitative PCR. Cytoskeletal actin polymerization was detected by confocal microscopy. Immunoblotting was performed to detect INTB1 phosphorylation at Thr788/9 and ILK activity as phosphorylation levels of protein kinase B (AKT) in Ser473 and glycogen synthase kinase 3ß (GSK3ß) at Ser9. TF was intraperitoneally administered once per day to wildtype and ILK knockdown mice (cKDILK) challenged with a high-fat diet (HFD) or control diet (STD) for 2 weeks. Body and WAT weight gains were compared. The expression of ILK and other markers was determined in the visceral epididymal (epi) and inguinal subcutaneous (sc) WAT. RESULTS: TF reduced TG content and the expression of adipogenesis markers and transporters in adipocytes, while UCP-1 expression was increased and the expression of lipases, cytokines or PCNA was not affected. Mechanistically, TF rapidly increased and faded the intracellular phosphorylation of INTB1 but not AKT or GSK3ß. F-actin levels were rapidly decreased, and INTB1 blockade avoided the TF effect. After 24 h, ILK expression and phosphorylation rates of AKT and GSK3ß were upregulated, while ILK silencing increased TG content. INTB1 blockade and ILK silencing avoided TF effects on the TG content and the transcriptional expression of PPARγ and UCP1. In HFD-challenged mice, the systemic administration of TF for several days reduced the weight gain on WAT depots. TF reduced adipogenesis and pro-inflammatory biomarkers and increased lipolysis markers HSL and FAT in epiWAT from HFD, while increased UCP1 in scWAT. In both WATs, TF upregulated ILK expression and activity, while no changes were observed in other tissues. In HFD-fed cKDILK, the blunted ILK in epiWAT worsened weight gain and avoided the anti-obesogenic effect of in vivo TF administration. CONCLUSIONS: ILK downregulation in WAT can be considered a biomarker of obesity establishment. Via an INTB1-ILK axis, TF restores malfunctioning hypertrophied WAT by changing the expression of adipocyte-related genes, increasing ILK expression and activity, and reducing TG storage. TF prevents obesity, a property to be added to its anticoagulant and cardiovascular protective advantages.

A rural worker infected with a bovine-prevalent genotype of Campylobacter fetus subsp. fetus supports zoonotic transmission and inconsistency of MLST and whole-genome typing.

Whole-genome characterisation in clinical microbiology enables to detect trends in infection dynamics and disease transmission. Here, we report a case of bacteraemia due to Campylobacter fetus subsp. fetus in a rural worker under cancer treatment that was diagnosed with cellulitis; the patient was treated with antibiotics and recovered. The routine typing methods were not able to identify the microorganism causing the infection, so it was further analysed by molecular methods and whole-genome sequencing. The multi-locus sequence typing (MLST) revealed the presence of the bovine-associated ST-4 genotype. Whole-genome comparisons with other C. fetus strains revealed an inconsistent phylogenetic position based on the core genome, discordant with previous ST-4 strains. To the best of our knowledge, this is the first C. fetus subsp. fetus carrying the ST-4 isolated from humans and represents a probable case of zoonotic transmission from cattle.

Integrin-linked kinase mediates the hydrogen peroxide-dependent transforming growth factor-ß1 up-regulation.

Transforming growth factor type-ß1 (TGF-ß1) has been recognized as a central mediator in many pathological events related to extracellular matrix (ECM) proteins accumulation, where their locally increased expression has been implicated in the fibrosis process of numerous organs, including glomerular fibrosis in the kidney. We and others have reported the TGF-ß1 synthesis regulation by reactive oxygen species (ROS), and moreover we also described the implication of integrin-linked kinase (ILK) in the AP-1-dependent TGF-ß1 up-regulation. Thus, we propose here that hydrogen peroxide (H2O2)-dependent TGF-ß1 regulation may be mediated by ILK activation. First we confirmed the increase in TGF-ß1 expression in human mesangial cells (HMC) after treatment with H2O2 or with an alternative H2O2-generating system such as the glucose-oxidase enzyme (GOX). By using immunoblotting, immunofluorescence, and ELISA techniques, we demonstrate that extracellular H2O2 up-regulates TGF-ß1 transcription, as well as increases TGF-ß1 promoter activity. Furthermore, catalase-decreased intracellular H2O2 abolished TGF-ß1 up-regulation. The use of pharmacological inhibitors as well as knockdown of ILK with small interfering RNA (siRNA) demonstrated the implication of a PI3K/ILK/AKT/ERK MAPK signaling pathway axis in the H2O2-induced TGF-ß1 overexpression. Finally, we explored the physiological relevance of these findings by treating HMC with angiotensin II, a known stimuli of H2O2 synthesis. Our results confirm the relevance of previous findings after a more physiological stimulus. In summary, our results provide evidence that ILK activity changes may act as a mechanism in response to different stimuli such as H2O2 in the induced TGF-ß1 up-regulation in pathological or even physiological conditions.

Cytogenetics and genome evolution in the subfamily Triatominae (Hemiptera, Reduviidae).

The subfamily Triatominae (Hemiptera, Reduviidae), vectors of Chagas disease, includes over 140 species. Karyotypic information is currently available for 80 of these species. This paper summarizes the chromosomal variability of the subfamily and how it may reveal aspects of genome evolution in this group. The Triatominae present a highly conserved chromosome number. All species, except 3, present 20 autosomes. The differences in chromosome number are mainly caused by variation in the number of sex chromosomes, due to the existence of 3 sex systems in males (XY, X(1)X(2)Y and X(1)X(2)X(3)Y). However, inter- and intraspecific differences in the position, quantity and meiotic behavior of constitutive heterochromatin, in the total genome size, and in the location of ribosomal 45S rRNA clusters, have revealed considerable cytogenetic variability within the subfamily. This cytogenetic diversity offers the opportunity to perform cytotaxonomic and phylogenetic studies, as well as structural, evolutionary, and functional analyses of the genome. The imminent availability of the complete genome of Rhodnius prolixus also opens new perspectives for understanding the evolution and genome expression of triatomines. The application of fluorescence in situ hybridization for the mapping of genes and sequences, as well as comparative analyses of genome homology by comparative genomic hybridization will be useful tools for understanding the genomic changes in relation to evolutionary processes such as speciation and adaptation to different environments.

Systematics of Mepraia (Hemiptera-Reduviidae): cytogenetic and molecular variation.

The haematophagous insects of the subfamily Triatominae (Hemiptera-Reduviidae) have great epidemiological importance as vectors of Trypanosoma cruzi, the causative agent of Chagas disease. Mepraia was originally described as a monotypic genus comprised of Mepraia spinolai, distributed along coastal areas of northern Chile (from Region I to the Metropolitan Region). Recently, some M. spinolai populations have been ranked as a new species named Mepraia gajardoi. Several populations along the distribution range of the genus were sampled, and genetic differentiation was studied based upon the analysis of three molecular markers: cytogenetics (karyotype and chromosome behaviour during meiosis using the C-banding technique), mitochondrial DNA (a cytochrome oxidase I gene fragment), and nuclear ribosomal DNA (intergenic region including the two internal transcribed spacers ITS-1 and ITS-2 and the 5.8S rRNA gene). The data here presented indicate that populations within the Mepraia genus (excluding Region II specimens) can be divided into two separate lineages. One lineage is comprised of specimens from the northernmost Region I and represents M. gajardoi. The other includes samples from the southern III, IV and the Metropolitan Regions, and represents M. spinolai. Region II individuals deserve particular attention as their relationship to the two identified lineages is not clear-cut. While they appear to belong to M. spinolai based on cytogenetics and rDNA markers, COI results indicate a closer relationship to M. gajardoi. This disagreement can be due to mitochondrial DNA introgression or the retention of ancestral polymorphisms.

Hydrogen peroxide down-regulates inositol 1,4,5-trisphosphate receptor content through proteasome activation.

Hydrogen peroxide (H(2)O(2)) is implicated in the regulation of signaling pathways leading to changes in vascular smooth muscle function. Contractile effects produced by H(2)O(2) are due to the phosphorylation of myosin light chain kinase triggered by increases in intracellular calcium (Ca(2+)) from intracellular stores or influx of extracellular Ca(2+). One mechanism for mobilizing such stores involves the phosphoinositide pathway. Inositol 1,4,5-trisphosphate (IP(3)) mobilizes intracellular Ca(2+) by binding to a family of receptors (IP(3)Rs) on the endoplasmic-sarcoplasmic reticulum that act as ligand-gated Ca(2+) channels. IP(3)Rs can be rapidly ubiquitinated and degraded by the proteasome, causing a decrease in cellular IP(3)R content. In this study we show that IP(3)R(1) and IP(3)R(3) are down-regulated when vascular smooth muscle cells (VSMC) are stimulated by H(2)O(2), through an increase in proteasome activity. Moreover, we demonstrate that the decrease in IP(3)R by H(2)O(2) is accompanied by a reduction in calcium efflux induced by IP(3) in VSMC. Also, we observed that angiotensin II (ANGII) induces a decrease in IP(3)R by activation of NADPH oxidase and that preincubation with H(2)O(2) decreases ANGII-mediated calcium efflux and planar cell surface area in VSMC. The decreased IP(3) receptor content observed in cells was also found in aortic rings, which exhibited a decreased ANGII-dependent contraction after treatment with H(2)O(2). Altogether, these results suggest that H(2)O(2) mediates IP(3)R down-regulation via proteasome activity.