Metabolic changes contribute to maladaptive right ventricular hypertrophy in pulmonary hypertension beyond pressure overload: an integrative imaging and omics investigation.

Right ventricular (RV) failure remains the strongest determinant of survival in pulmonary hypertension (PH). We aimed to identify relevant mechanisms, beyond pressure overload, associated with maladaptive RV hypertrophy in PH. To separate the effect of pressure overload from other potential mechanisms, we developed in pigs two experimental models of PH (M1, by pulmonary vein banding and M2, by aorto-pulmonary shunting) and compared them with a model of pure pressure overload (M3, pulmonary artery banding) and a sham-operated group. Animals were assessed at 1 and 8 months by right heart catheterization, cardiac magnetic resonance and blood sampling, and myocardial tissue was analyzed. Plasma unbiased proteomic and metabolomic data were compared among groups and integrated by an interaction network analysis. A total of 33 pigs completed follow-up (M1, n = 8; M2, n = 6; M3, n = 10; and M0, n = 9). M1 and M2 animals developed PH and reduced RV systolic function, whereas animals in M3 showed increased RV systolic pressure but maintained normal function. Significant plasma arginine and histidine deficiency and complement system activation were observed in both PH models (M1&M2), with additional alterations to taurine and purine pathways in M2. Changes in lipid metabolism were very remarkable, particularly the elevation of free fatty acids in M2. In the integrative analysis, arginine-histidine-purines deficiency, complement activation, and fatty acid accumulation were significantly associated with maladaptive RV hypertrophy. Our study integrating imaging and omics in large-animal experimental models demonstrates that, beyond pressure overload, metabolic alterations play a relevant role in RV dysfunction in PH.

Neurons regulate the esterification of bioactive lipid mediators in the brain of acid sphingomyelinase deficient mice.

Acid sphingomyelinase deficiency is a neurodegenerative lysosomal storage disorder caused by mutations in the sphingomyelin-degrading enzyme acid sphingomyelinase (ASM) gene. Upregulated neuroinflammation has been well-characterized in an ASM knockout mouse model of acid sphingomyelinase deficiency disease, but lipid mediator pathways involved in 'mediating' inflammation and inflammation-resolution have yet to be characterized. In this study, we 1) measured free (bioactive) and esterified (inactive) lipid mediators involved in inflammation and inflammation resolution in cerebellum and neuronal cultures of ASM knockout (ASMko) mice and wildtype (WT) controls, and 2) quantified the esterification of labeled pro-resolving free d11-14(15)-epoxyeicosatrienoic acid in cultured neurons from ASMko and WT mice. We found elevated concentrations of esterified pro-resolving lipid mediators and hydroxyeicosatrienoic acids typically destined for pro-resolving lipid mediator synthesis (e.g. lipoxins) in the cerebellum and neurons of ASMko mice compared to controls. Free d11-14(15)-epoxyeicosatrienoic acid esterification within neurons of ASMko mice was significantly elevated compared to WT. Our findings show evidence of increased inactivation of free pro-resolving lipid mediators through esterification in ASMko mice, suggesting impaired resolution as a new pathway underlying ASM deficiency pathogenesis.

IF1 ablation prevents ATP synthase oligomerization, enhances mitochondrial ATP turnover and promotes an adenosine-mediated pro-inflammatory phenotype.

ATPase Inhibitory Factor 1 (IF1) regulates the activity of mitochondrial ATP synthase. The expression of IF1 in differentiated human and mouse cells is highly variable. In intestinal cells, the overexpression of IF1 protects against colon inflammation. Herein, we have developed a conditional IF1-knockout mouse model in intestinal epithelium to investigate the role of IF1 in mitochondrial function and tissue homeostasis. The results show that IF1-ablated mice have increased ATP synthase/hydrolase activities, leading to profound mitochondrial dysfunction and a pro-inflammatory phenotype that impairs the permeability of the intestinal barrier compromising mouse survival upon inflammation. Deletion of IF1 prevents the formation of oligomeric assemblies of ATP synthase and alters cristae structure and the electron transport chain. Moreover, lack of IF1 promotes an intramitochondrial Ca2+ overload in vivo, minimizing the threshold to Ca2+-induced permeability transition (mPT). Removal of IF1 in cell lines also prevents the formation of oligomeric assemblies of ATP synthase, minimizing the threshold to Ca2+-induced mPT. Metabolomic analyses of mice serum and colon tissue highlight that IF1 ablation promotes the activation of de novo purine and salvage pathways. Mechanistically, lack of IF1 in cell lines increases ATP synthase/hydrolase activities and installs futile ATP hydrolysis in mitochondria, resulting in the activation of purine metabolism and in the accumulation of adenosine, both in culture medium and in mice serum. Adenosine, through ADORA2B receptors, promotes an autoimmune phenotype in mice, stressing the role of the IF1/ATP synthase axis in tissue immune responses. Overall, the results highlight that IF1 is required for ATP synthase oligomerization and that it acts as a brake to prevent ATP hydrolysis under in vivo phosphorylating conditions in intestinal cells.

Integrating the potential of ion mobility spectrometry-mass spectrometry in the separation and structural characterisation of lipid isomers.

It is increasingly evident that a more detailed molecular structure analysis of isomeric lipids is critical to better understand their roles in biological processes. The occurrence of isomeric interference complicates conventional tandem mass spectrometry (MS/MS)-based determination, necessitating the development of more specialised methodologies to separate lipid isomers. The present review examines and discusses recent lipidomic studies based on ion mobility spectrometry combined with mass spectrometry (IMS-MS). Selected examples of the separation and elucidation of structural and stereoisomers of lipids are described based on their ion mobility behaviour. These include fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, and sterol lipids. Recent approaches for specific applications to improve isomeric lipid structural information using direct infusion, coupling imaging, or liquid chromatographic separation workflows prior to IMS-MS are also discussed, including: 1) strategies to improve ion mobility shifts; 2) advanced tandem MS methods based on activation of lipid ions with electrons or photons, or gas-phase ion-molecule reactions; and 3) the use of chemical derivatisation techniques for lipid characterisation.

Glutamine Metabolism Supports the Functional Activity of Immune Cells against Aspergillus fumigatus.

The reprogramming of cellular metabolism of immune cells is an essential process in the regulation of antifungal immune responses. In particular, glucose metabolism has been shown to be required for protective immunity against infection with Aspergillus fumigatus. However, given the intricate cross talk between multiple metabolic networks and signals, it is likely that cellular metabolic pathways other than glycolysis are also relevant during fungal infection. In this study, we demonstrate that glutamine metabolism is required for the activation of macrophage effector functions against A. fumigatus. Glutamine metabolism was found to be upregulated early after fungal infection and glutamine depletion or the pharmacological inhibition of enzymes involved in its metabolism impaired phagocytosis and the production of both proinflammatory and T-cell-derived cytokines. In an in vivo model, inhibition of glutaminase increased susceptibility to experimental aspergillosis, as revealed by the increased fungal burden and inflammatory pathology, and the defective cytokine production in the lungs. Moreover, genetic variants in glutamine metabolism genes were found to regulate cytokine production in response to A. fumigatus stimulation. Taken together, our results demonstrate that glutamine metabolism represents an important component of the immunometabolic response of macrophages against A. fumigatus both in vitro and in vivo. IMPORTANCE The fungal pathogen Aspergillus fumigatus can cause severe and life-threatening forms of infection in immunocompromised patients. The reprogramming of cellular metabolism is essential for innate immune cells to mount effective antifungal responses. In this study, we report the pivotal contribution of glutaminolysis to the host defense against A. fumigatus. Glutamine metabolism was essential both in vitro as well as in in vivo models of infection, and genetic variants in human glutamine metabolism genes regulated cytokine production in response to fungal stimulation. This work highlights the relevance of glutaminolysis to the pathogenesis of aspergillosis and supports a role for interindividual genetic variation influencing glutamine metabolism in susceptibility to infection.

Optimization of sample extraction and injection-related parameters in HILIC performance for polar metabolite analysis. Application to the study of a model of pulmonary hypertension.

HILIC is a separation technique increasingly used for the study of polar metabolites. However, HILIC suffers of a drawback related to the solvents used for these analyses: acetonitrile as sample solvent leads to the best chromatographic peaks, but it is not capable to extract/dissolve the most polar compounds. In this work we evaluated the use of several strategies for the extraction of polar compounds from plasma samples and, although methanol was the ideal solvent for analyte extraction, distorted peaks were obtained in the chromatography. Different strategies were tested included changing the solvent after extraction or modifying the injection volume and type. Finally, the best solution was using the lowest injection volume possible and to employ a simple sandwich injection including acetonitrile during the injection of the sample. This remarkably improves the peak shape when methanol is used in the sample. We evaluated and applied two HILIC-MS methods, in positive and negative ionization modes, on plasma samples from pigs with pulmonary hypertension produced by aorto-pulmonary shunting to identify metabolic signatures underlying damage to the right heart. Our analyses revealed altered relevant metabolic pathways, suggestive of oxidative stress and reduced energy demands.

Identification of a broad lipid repertoire associated to the endothelial cell protein C receptor (EPCR).

Evidence is mounting that the nature of the lipid bound to the endothelial cell protein C receptor (EPCR) has an impact on its biological roles, as observed in anticoagulation and more recently, in autoimmune disease. Phosphatidylethanolamine and phosphatidylcholine species dominate the EPCR lipid cargo, yet, the extent of diversity in the EPCR-associated lipid repertoire is still unknown and remains to be uncovered. We undertook mass spectrometry analyses to decipher the EPCR lipidome, and identified species not yet described as EPCR ligands, such as phosphatidylinositols and phosphatidylserines. Remarkably, we found further, more structurally divergent lipids classes, represented by ceramides and sphingomyelins, both in less abundant quantities. In support of our mass spectrometry results and previous studies, high-resolution crystal structures of EPCR in three different space groups point to a prevalent diacyl phospholipid moiety in EPCR's pocket but a mobile and ambiguous lipid polar head group. In sum, these studies indicate that EPCR can associate with varied lipid classes, which might impact its properties in anticoagulation and the onset of autoimmune disease.

Plasma lipidomics in subjects with combat posttraumatic stress disorder.

Posttraumatic stress disorder (PTSD) is complex neuropsychiatric disorder triggered by a traumatic event and characterized by the symptoms that represent large burden to patients, as well as to society. Lipidomic approach can be applied as a useful tool for discovery of novel diagnostic, prognostic and therapeutic lipid biomarkers of various disorders, whose etiology is complex and still unknown, including PTSD. Since changes in the levels of lipid metabolites might indicate impairments in various metabolic pathways and cellular processes, the aim of this lipidomic study was to determine altered levels of lipid compounds in PTSD. The study enrolled 235 male patients with combat PTSD and 241 healthy male control subjects. Targeted lipidomic analysis of plasma samples was conducted using reverse-phase liquid chromatography coupled with mass spectrometry. Lipids that have been analyzed belong to the group of ceramides, cholesterol esters, diacylglycerols, lysophosphatidylcholines, lysophosphatidylethanolamines, phosphatidylcholines, phosphatidylethanolamines, sphingomyelins and triglycerides. The levels of fifteen lipid compounds were found to be significantly different between PTSD patients and healthy control subjects, including four phosphatidylcholines, two phosphatidylethanolamines, five sphingomyelins, two cholesterol esters and two ceramides. The lipid metabolites whose levels significantly differed between patients with PTSD and control subjects are associated with various biological processes, including impairments of membrane integrity and function, mitochondrial dysfunction, inflammation and oxidative stress. As these processes might be associated with development and progression of PTSD, altered lipid compounds represent potential biomarkers that could facilitate the diagnosis of PTSD, prediction of the disease, as well as identification of novel treatment approaches in PTSD.

Recent developments in data acquisition, treatment and analysis with ion mobility-mass spectrometry for lipidomics.

Lipids are involved in many biological processes and their study is constantly increasing. To identify a lipid among thousand requires of reliable methods and techniques. Ion Mobility (IM) can be coupled with Mass Spectrometry (MS) to increase analytical selectivity in lipid analysis of lipids. IM-MS has experienced an enormous development in several aspects, including instrumentation, sensitivity, amount of information collected and lipid identification capabilities. This review summarizes the latest developments in IM-MS analyses for lipidomics and focuses on the current acquisition modes in IM-MS, the approaches for the pre-treatment of the acquired data and the subsequent data analysis. Methods and tools for the calculation of Collision Cross Section (CCS) values of analytes are also reviewed. CCS values are commonly studied to support the identification of lipids, providing a quasi-orthogonal property that increases the confidence level in the annotation of compounds and can be matched in CCS databases. The information contained in this review might be of help to new users of IM-MS to decide the adequate instrumentation and software to perform IM-MS experiments for lipid analyses, but also for other experienced researchers that can reconsider their routines and protocols.

A Set of Reliable Samples for the Study of Biomarkers for the Early Diagnosis of Parkinson's Disease.

Background: Parkinson's disease (PD) is a progressive neurodegenerative disorder, diagnosed according to the clinical criteria that occur in already advanced stages of PD. The definition of biomarkers for the early diagnosis of PD represents a challenge that might improve treatment and avoid complications in this disease. Therefore, we propose a set of reliable samples for the identification of altered metabolites to find potential prognostic biomarkers for early PD. Methods: This case-control study included plasma samples of 12 patients with PD and 21 control subjects, from the Spanish European Prospective Investigation into Cancer and Nutrition (EPIC)-Navarra cohort, part of the EPIC-Spain study. All the case samples were provided by healthy volunteers who were followed-up for 15.9 (±4.1) years and developed PD disease later on, after the sample collection. Liquid chromatography coupled to tandem mass spectrometry was used for the analysis of samples. Results: Out of 40 that were selected and studied due to their involvement in established cases of PD, seven significantly different metabolites between PD cases and healthy control subjects were obtained in this study (benzoic acid, palmitic acid, oleic acid, stearic acid, myo-inositol, sorbitol, and quinolinic acid). These metabolites are related to mitochondrial dysfunction, the oxidative stress, and the mechanisms of energy production. Conclusion: We propose the samples from the EPIC study as reliable and invaluable samples for the search of early biomarkers of PD. Likewise, this study might also be a starting point in the establishment of a well-founded panel of metabolites that can be used for the early detection of this disease.

Metabolomics and biochemical alterations caused by pleiotrophin in the 6-hydroxydopamine mouse model of Parkinson's disease.

Pleiotrophin (PTN) is a cytokine involved in nerve tissue repair processes, neuroinflammation and neuronal survival. PTN expression levels are upregulated in the nigrostriatal pathway of Parkinson's Disease (PD) patients. We aimed to characterize the dopaminergic injury and glial responses in the nigrostriatal pathway of mice with transgenic Ptn overexpression in the brain (Ptn-Tg) after intrastriatal injection of the catecholaminergic toxic 6-hydroxydopamine (6-OHDA) at a low dose (5 µg). Ten days after surgery, the injection of 6-OHDA induced a significant decrease of the number of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra and of the striatal TH contents in Wild type (Wt) mice. In contrast, these effects of 6-OHDA were absent in Ptn-Tg mice. When the striatal Iba1 and GFAP immunoreactivity was studied, no statistical differences were found between vehicle-injected Wt and Ptn-Tg mice. Furthermore, 6-OHDA did not cause robust glial responses neither on Wt or Ptn-Tg mice 10 days after injections. In metabolomics studies, we detected interesting metabolites that significantly discriminate the more injured 6-OHDA-injected Wt striatum and the more protected 6-OHDA-injected Ptn-Tg striatum. Particularly, we detected groups of metabolites, mostly corresponding to phospholipids, whose trends were opposite in both groups. In summary, the data confirm lower 6-OHDA-induced decreases of TH contents in the nigrostriatal pathway of Ptn-Tg mice, suggesting a neuroprotective effect of brain PTN overexpression in this mouse model of PD. New lipid-related PD drug candidates emerge from this study and the data presented here support the increasingly recognized "lipid cascade" in PD.

Metformin Treatment or PRODH/POX-Knock out Similarly Induces Apoptosis by Reprograming of Amino Acid Metabolism, TCA, Urea Cycle and Pentose Phosphate Pathway in MCF-7 Breast Cancer Cells.

It has been considered that proline dehydrogenase/proline oxidase (PRODH/POX) is involved in antineoplastic activity of metformin (MET). The aim of this study is identification of key metabolites of glycolysis, pentose phosphate pathway (PPP), tricarboxylic acids (TCA), urea cycles (UC) and some amino acids in MET-treated MCF-7 cells and PRODH/POX-knocked out MCF-7 (MCF-7crPOX) cells. MCF-7crPOX cells were generated by using CRISPR-Cas9. Targeted metabolomics was performed by LC-MS/MS/QqQ. Expression of pro-apoptotic proteins was evaluated by Western blot. In the absence of glutamine, MET treatment or PRODH/POX-knock out of MCF-7 cells contributed to similar inhibition of glycolysis (drastic increase in intracellular glucose and pyruvate) and increase in the utilization of phospho-enol-pyruvic acid, glucose-6-phosphate and some metabolites of TCA and UC, contributing to apoptosis. However, in the presence of glutamine, MET treatment or PRODH/POX-knock out of MCF-7 cells contributed to utilization of some studied metabolites (except glucose), facilitating pro-survival phenotype of MCF-7 cells in these conditions. It suggests that MET treatment or PRODH/POX-knock out induce similar metabolic effects (glucose starvation) and glycolysis is tightly linked to glutamine metabolism in MCF-7 breast cancer cells. The data provide insight into mechanism of anticancer activity of MET as an approach to further studies on experimental breast cancer therapy.

Prognostic biomarkers of Parkinson's disease in the Spanish EPIC cohort: a multiplatform metabolomics approach.

The lack of knowledge about the onset and progression of Parkinson's disease (PD) hampers its early diagnosis and treatment. Metabolomics might shed light on the PD imprint seeking a broader view of the biochemical remodeling induced by this disease in an early and pre-symptomatic stage and unveiling potential biomarkers. To achieve this goal, we took advantage of the great potential of the European Prospective Study on Nutrition and Cancer (EPIC) cohort to apply metabolomics searching for early diagnostic PD markers. This cohort consisted of healthy volunteers that were followed for around 15 years until June 2011 to ascertain incident PD. For this untargeted metabolomics-based study, baseline preclinical plasma samples of 39 randomly selected individuals that developed PD (Pre-PD group) and the corresponding control group were analyzed using a multiplatform approach. Data were statistically analyzed and exposed alterations in 33 metabolites levels, including significantly lower levels of free fatty acids (FFAs) in the preclinical samples from PD subjects. These results were then validated by adopting a targeted HPLC-QqQ-MS approach. After integrating all the metabolites affected, our finding revealed alterations in FFAs metabolism, mitochondrial dysfunction, oxidative stress, and gut-brain axis dysregulation long before the development of PD hallmarks. Although the biological purpose of these events is still unknown, the remodeled metabolic pathways highlighted in this work might be considered worthy prognostic biomarkers of early prodromal PD. The findings revealed by this work are of inestimable value since this is the first study conducted with samples collected many years before the disease development.

Combined Genome, Transcriptome and Metabolome Analysis in the Diagnosis of Childhood Cerebellar Ataxia.

Ataxia in children is a common clinical sign of numerous neurological disorders consisting of impaired coordination of voluntary muscle movement. Its most common form, cerebellar ataxia, describes a heterogeneous array of neurologic conditions with uncountable causes broadly divided as acquired or genetic. Numerous genetic disorders are associated with chronic progressive ataxia, which complicates clinical management, particularly on the diagnostic stage. Advances in omics technologies enable improvements in clinical practice and research, so we proposed a multi-omics approach to aid in the genetic diagnosis and molecular elucidation of an undiagnosed infantile condition of chronic progressive cerebellar ataxia. Using whole-exome sequencing, RNA-seq, and untargeted metabolomics, we identified three clinically relevant mutations (rs141471029, rs191582628 and rs398124292) and an altered metabolic profile in our patient. Two POLR1C diagnostic variants already classified as pathogenic were found, and a diagnosis of hypomyelinating leukodystrophy was achieved. A mutation on the MMACHC gene, known to be associated with methylmalonic aciduria and homocystinuria cblC type, was also found. Additionally, preliminary metabolome analysis revealed alterations in our patient's amino acid, fatty acid and carbohydrate metabolism. Our findings provided a definitive genetic diagnosis reinforcing the association between POLR1C mutations and hypomyelinating leukodystrophy and highlighted the relevance of multi-omics approaches to the disease.

Metabolomics analysis of microbiota-gut-brain axis in neurodegenerative and psychiatric diseases.

Gut microbiota represents a complex physiological ecosystem that influences the host health. Alterations in the microbiome metabolism affect the body homeostasis and they have been associated with the development of different human neurodegenerative and neuropsychiatric disorders, such as Alzheimer's disease, autism spectrum disorder, bipolar disorder, depression, Huntington's disease, Parkinson's disease, posttraumatic stress disorder and schizophrenia. The development of these complex diseases is influenced by various factors, including genetic predisposition and environmental triggers. Gut microbiota has recently emerged as an important actor in their physiopathology that has been shown to play a role in inflammation, oxidative stress, and gut permeability. Therefore, targeting the metabolites that are produced by or associated with the gut microbiota may help us understand how imbalance in the gut-brain axis affects human health. This review offers a comprehensive overview of the literature on this matter, offering the readers an insight in the state-of-art metabolic measurements of the gut-brain axis in various brain-related diseases.

Metabolomics in posttraumatic stress disorder: Untargeted metabolomic analysis of plasma samples from Croatian war veterans.

Posttraumatic stress disorder (PTSD) is a severe, multifactorial and debilitating neuropsychiatric disorder, which can develop in a subset of individuals as a result of the exposure to severe stress or trauma. Such traumatic experiences have a major impact on molecular, biochemical and cellular systems, causing psychological and somatic alterations that affect the whole organism. Although the etiology of PTSD is still unclear, it seems to involve complex interaction between various biological genetic and environmental factors. Metabolomics, as one of the rapidly developing "omics" techniques, might be a useful tool for determining altered metabolic pathways and stress-related metabolites as new potential biomarkers of PTSD. The aim of our study was to identify metabolites whose altered levels allow us to differentiate between patients with PTSD and healthy control individuals. The study included two cohorts. The first, exploratory, group included 50 Croatian veterans with PTSD and 50 healthy control subjects, whereas a validation group consisted of 52 veterans with PTSD and 52 control subjects. The metabolomic analysis of plasma samples was conducted using liquid chromatography coupled with mass spectrometry (LC-MS), as well as gas chromatography coupled with mass spectrometry (GC-MS). The LC-MS analysis determined significantly different levels of two glycerophospholipids, PE(18:1/0:0) and PC(18:1/0:0), between control subjects and PTSD patients in both cohorts. The altered metabolites might play a role in multiple cellular processes, including inflammation, mitochondrial dysfunction, membrane breakdown, oxidative stress and neurotoxicity, which could be associated with PTSD pathogenesis.

Impact of global PTP1B deficiency on the gut barrier permeability during NASH in mice.

OBJECTIVE: Non-alcoholic steatohepatitis (NASH) is characterized by a robust pro-inflammatory component at both hepatic and systemic levels together with a disease-specific gut microbiome signature. Protein tyrosine phosphatase 1 B (PTP1B) plays distinct roles in non-immune and immune cells, in the latter inhibiting pro-inflammatory signaling cascades. In this study, we have explored the role of PTP1B in the composition of gut microbiota and gut barrier dynamics in methionine and choline-deficient (MCD) diet-induced NASH in mice. METHODS: Gut features and barrier permeability were characterized in wild-type (PTP1B WT) and PTP1B-deficient knockout (PTP1B KO) mice fed a chow or methionine/choline-deficient (MCD) diet for 4 weeks. The impact of inflammation was studied in intestinal epithelial and enteroendocrine cells. The secretion of GLP-1 was evaluated in primary colonic cultures and plasma of mice. RESULTS: We found that a shift in the gut microbiota shape, disruption of gut barrier function, higher levels of serum bile acids, and decreased circulating glucagon-like peptide (GLP)-1 are features during NASH. Surprisingly, despite the pro-inflammatory phenotype of global PTP1B-deficient mice, they were partly protected against the alterations in gut microbiota composition during NASH and presented better gut barrier integrity and less permeability under this pathological condition. These effects concurred with higher colonic mucosal inflammation, decreased serum bile acids, and protection against the decrease in circulating GLP-1 levels during NASH compared with their WT counterparts together with increased expression of GLP-2-sensitive genes in the gut. At the molecular level, stimulation of enteroendocrine STC-1 cells with a pro-inflammatory conditioned medium (CM) from lipopolysaccharide (LPS)-stimulated macrophages triggered pro-inflammatory signaling cascades that were further exacerbated by a PTP1B inhibitor. Likewise, the pro-inflammatory CM induced GLP-1 secretion in primary colonic cultures, an effect augmented by PTP1B inhibition. CONCLUSION: Altogether our results have unraveled a potential role of PTP1B in the gut-liver axis during NASH, likely mediated by increased sensitivity to GLPs, with potential therapeutic value.

Insulin receptor substrate 2 (IRS2) deficiency delays liver fibrosis associated with cholestatic injury.

Insulin receptor substrate 2 (IRS2) is a key downstream mediator of insulin and insulin-like growth factor 1 (IGF1) signalling pathways and plays a major role in liver metabolism. The aim of this study was to investigate whether IRS2 had an impact on the hepatic fibrotic process associated with cholestatic injury. Bile duct ligation (BDL) was performed in wild-type (WT) and Irs2-deficient (IRS2KO) female mice. Histological and biochemical analyses, together with fibrogenic and inflammatory responses were evaluated in livers from mice at 3, 7 and 28 days following BDL. We also explored whether activation of human hepatic stellate cells (HSCs) induced by IGF1 was modulated by IRS2. IRS2KO mice displayed reduced disruption of liver histology, such hepatocyte damage and excess deposition of extracellular matrix components, compared with WT mice at 3 and 7 days post-BDL. However, no histological differences between genotypes were found at 28 days post-BDL. The less pro-inflammatory profile of bile acids accumulated in the gallbladder of IRS2KO mice after BDL corresponded with the reduced expression of pro-inflammatory markers in these mice. Stable silencing of IRS2 or inhibition of ERK1/2 reduced the activation of human LX2 cells and also reduced induction of MMP9 upon IGF1 stimulation. Furthermore, hepatic MMP9 expression was strongly induced after BDL in WT mice, but only a slight increase was found in mice lacking IRS2. Our results have unravelled the signalling pathway mediated by IGF1R-IRS2-ERK1/2-MMP9 as a key axis in regulating HSC activation, which might be therapeutically relevant for targeting liver fibrosis.

How to tackle chemical communication? Relative proportions versus semiquantitative determination of compounds in lizard chemical secretions.

Knowledge about chemical communication in some vertebrates is still relatively limited. Squamates are a glaring example of this, even when recent evidences indicate that scents are involved in social and sexual interactions. In lizards, where our understanding of chemical communication has considerably progressed in the last few years, many questions about chemical interactions remain unanswered. A potential reason for this is the inherent complexity and technical limitations that some methodologies embody when analyzing the compounds used to convey information. We provide here a straightforward procedure to analyze lizard chemical secretions based on gas chromatography coupled to mass spectrometry that uses an internal standard for the semiquantification of compounds. We compare the results of this method with those obtained by the traditional procedure of calculating relative proportions of compounds. For such purpose, we designed two experiments to investigate if these procedures allowed revealing changes in chemical secretions 1) when lizards received previously a vitamin dietary supplementation or 2) when the chemical secretions were exposed to high temperatures. Our results show that the procedure based on relative proportions is useful to describe the overall chemical profile, or changes in it, at population or species levels. On the other hand, the use of the procedure based on semiquantitative determination can be applied when the target of study is the variation in one or more particular compounds of the sample, as it has proved more accurate detecting quantitative variations in the secretions. This method would reveal new aspects produced by, for example, the effects of different physiological and climatic factors that the traditional method does not show.