BUSCADOR RECOLECTA

Objectives: Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in Western countries. Protein tyrosine phosphatase 1B (PTP1B), a negative modulator of insulin and cytokine signaling, is a therapeutic target for type 2 diabetes and obesity. We investigated the impact of PTP1B deficiency during NAFLD, particularly in non-alcoholic steatohepatitis (NASH).
Methods: NASH features were evaluated in livers from wild-type (PTP1BWT) and PTP1B-deficient (PTP1BKO) mice fed methionine/cholinedeficient diet (MCD) for 8 weeks. A recovery model was established by replacing MCD to chow diet (CHD) for 2e7 days. Non-parenchymal liver cells (NPCs) were analyzed by flow cytometry. Oval cells markers were measured in human and mouse livers with NASH, and in oval cells from PTP1BWT and PTP1BKO mice.
Results: PTP1BWT mice fed MCD for 8 weeks exhibited NASH, NPCs infiltration, and elevated Fgf21, Il6 and Il1b mRNAs. These parameters
decreased after switching to CHD. PTP1B deficiency accelerated MCD-induced NASH. Conversely, after switching to CHD, PTP1BKO mice rapidly
reverted NASH compared to PTP1BWT mice in parallel to the normalization of serum triglycerides (TG) levels. Among NPCs, a drop in cytotoxic
natural killer T (NKT) subpopulation was detected in PTP1BKO livers during recovery, and in these conditions M2 macrophage markers were upregulated. Oval cells markers (EpCAM and cytokeratin 19) significantly increased during NASH only in PTP1B-deficient livers. HGF-mediated
signaling and proliferative capacity were enhanced in PTP1BKO oval cells. In NASH patients, oval cells markers were also elevated.
Conclusions: PTP1B elicits a dual role in NASH progression and reversion. Additionally, our results support a new role for PTP1B in oval cell
proliferation during NAFLD.

PGC-1 alpha controls, to a large extent, the capacity of cells to respond to changing nutritional requirements and energetic demands. The key role of metabolic reprogramming in tumor development has highlighted the potential role of PGC-1 alpha in cancer. To investigate how loss of PGC-1 alpha activity in primary cells impacts the oncogenic characteristics of spontaneously immortalized cells, and the mechanisms involved, we used the classic 3T3 protocol to generate spontaneously immortalized mouse embryonic fibroblasts (iMEFs) from wild-type (WT) and PGC-1 alpha knockout (KO) mice and analyzed their oncogenic potential in vivo and in vitro. We found that PGC-1 alpha KO iMEFs formed larger and more proliferative primary tumors than WT counterparts, and fostered the formation of lung metastasis by B16 melanoma cells. These characteristics were associated with the reduced capacity of KO iMEFs to respond to cell contact inhibition, in addition to an increased ability to form colonies in soft agar, an enhanced migratory capacity, and a reduced growth factor dependence. The mechanistic basis of this phenotype is likely associated with the observed higher levels of nuclear beta-catenin and c-myc in KO iMEFs. Evaluation of the metabolic adaptations of the immortalized cell lines identified a decrease in oxidative metabolism and an increase in glycolytic flux in KO iMEFs, which were also more dependent on glutamine for their survival. Furthermore, glucose oxidation and tricarboxylic acid cycle forward flux were reduced in KO iMEF, resulting in the induction of compensatory anaplerotic pathways. Indeed, analysis of amino acid and lipid patterns supported the efficient use of tricarboxylic acid cycle intermediates to synthesize lipids and proteins to support elevated cell growth rates. All these characteristics have been observed in aggressive tumors and support a tumor suppressor role for PGC-1 alpha, restraining metabolic adaptations in cancer., This work was funded by grants from the Spanish "Ministerio de Ciencia, Innovacion y Universidades" (MICINN) and ERDF/FEDER funds, SAF2012-37693, SAF2015-63904-R, SAF2015-71521-REDC, RTI2018-093864-B-I00 to M.M., SAF2017-83043-R and B2017/BMD-3724 to S.C., PI15/00107 to A.M.R, the University of the Basque Country UPV/EHU grant GIU16/62) to J.l.R.S. and M.B.R.L., and the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement 721236-TREATMENT to M.M.

Schizophrenia is a neuropsychiatric disorder that chronically affects 21 million people worldwide. Secondgeneration
antipsychotics (SGAs) are the cornerstone in the management of schizophrenia. However,
despite their efficacy in counteracting both positive and negative symptomatology of schizophrenia, recent clinical
observations have described an increase in the prevalence of metabolic disturbances in patients treated with
SGAs, including abnormal weight gain, hyperglycemia and dyslipidemia. While the molecular mechanisms responsible
for these side-effects remain poorly understood, increasing evidence points to a link between SGAs and
adipose tissue depots of white, brown and beige adipocytes. In this review, we survey the present knowledge in
this area, with a particular focus on the molecular aspects of adipocyte biology including differentiation, lipid metabolism, thermogenic function and the browning/beiging process, This work was funded by H2020 Marie Skłodowska-Curie
ActionsITN-TREATMENT (Grant Agreement 721236, European
Commission). We also acknowledge grants RTI2018-094052-B-100
(MICINN/FEDER, Spain), S2017/BMD-3684 MOIR2-CM (Comunidad
de Madrid, Spain) and CIBERdem (ISCIII, Spain).

Aims: Pupillography is a noninvasive and cost-effective method to determine autonomic nerve activity. Genetic variants in cytochrome P450 (CYP), dopamine receptor (DRD2, DRD3), serotonin receptor (HTR2A, HTR2C) and ATP-binding cassette subfamily B (ABCB1) genes, among others, were previously associated with the pharmacokinetics and pharmacodynamics of antipsychotic drugs. Our aim was to evaluate the effects of aripiprazole and olanzapine on pupillary light reflex related to pharmacogenetics. Methods: Twenty-four healthy volunteers receiving 5 oral doses of 10 mg aripiprazole and 5 mg olanzapine tablets were genotyped for 46 polymorphisms by quantitative polymerase chain reaction. Pupil examination was performed by automated pupillometry. Aripiprazole, dehydro-aripiprazole and olanzapine plasma concentrations were measured by high-performance liquid chromatography–tandem mass spectrometry. Results: Aripiprazole affected pupil contraction: it caused dilatation after the administration of the first dose, then caused constriction after each dosing. It induced changes in all pupillometric parameters (P '.05). Olanzapine only altered minimum pupil size (P =.046). Polymorphisms in CYP3A, HTR2A, UGT1A1, DRD2 and ABCB1 affected pupil size, the time of onset of constriction, pupil recovery and constriction velocity. Aripiprazole, dehydro-aripiprazole and olanzapine pharmacokinetics were significantly affected by polymorphisms in CYP2D6, CYP3A, CYP1A2, ABCB1 and UGT1A1 genes. Conclusions: In conclusion, aripiprazole and its main metabolite, dehydro-aripiprazole altered pupil contraction, but olanzapine did not have such an effect. Many polymorphisms may influence pupillometric parameters and several polymorphisms had an effect on aripiprazole, dehydro-aripiprazole and olanzapine pharmacokinetics. Pupillography could be a useful tool for the determination of autonomic nerve activity during antipsychotic treatment., Consejería de Educación, Juventud y Deporte, Comunidad de Madrid, Grant/Award Number: PEJD-2017-PRE/BMD-4164; H2020 Marie Skłodowska-Curie Actions, Grant/Award Number: 721236

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., This work was funded by grants SAF2015-65267-R (MINECO/FEDER,
UE), RTI2018-094052-B-100 (MCI/AEI/FEDER, UE), S2010/BMD-2423,
S2017/BMD-3684 (Comunidad de Madrid, Spain), CIBERdem, CIBERhed
and PI 16/02096 (ISCIII, Spain). We also acknowledge grants H2020
Marie Sklodowska-Curie ITN-TREATMENT (Grant Agreement 721236,
European Commission), Spanish Ministry of Education-MECD (FPU13/05802 grant to C.R) and Fundación Ramón Areces (to A.M.V.). CBMSO is
recipient of institutional aid fromFundación Ramón Areces. VBL,FMGand
FR received funding from a Wellcome Trust joint investigator award
(106262/Z/14/Z and 106263/Z/14/Z) and a joint MRC programme within
the Metabolic Diseases Unit (MRC_MC_UU_12012/3), United Kingdom.
VBL received a project support grant from the Society for Endocrinology,
United Kingdom. The MRL Histology and Biochemistry Assay Lab Core
facilities received funding from the MRC Metabolic Diseases Unit
[MRC_MC_UU_12012/5] and the Imaging Core through a Wellcome
Trust Strategic Award [100574/Z/12/Z], United Kingdom

Mitophagy is a selective autophagic process, essential for cellular homeostasis, that eliminates dysfunctional mitochondria. Activated by inner membrane depolarization, it plays an important role during development and is fundamental in highly differentiated post‐mitotic cells that are highly dependent on aerobic metabolism, such as neurons, muscle cells, and hepatocytes. Both defective and excessive mitophagy have been proposed to contribute to age‐related neurodegener-ative diseases, such as Parkinson’s and Alzheimer’s diseases, metabolic diseases, vascular complications of diabetes, myocardial injury, muscle dystrophy, and liver disease, among others. Pharmacological or dietary interventions that restore mitophagy homeostasis and facilitate the elimination of irreversibly damaged mitochondria, thus, could serve as potential therapies in several chronic diseases. However, despite extraordinary advances in this field, mainly derived from in vitro and preclinical animal models, human applications based on the regulation of mitochondrial quality in patients have not yet been approved. In this review, we summarize the key selective mitochondrial autophagy pathways and their role in prevalent chronic human diseases and highlight the potential use of specific interventions., This research was funded by the Spanish “Ministerio de Ciencia, Innovación y Universidades”
(MICIU) and ERDF/FEDER funds, grant number RTI2018-093864-B-I00, and the European
Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement 721236-TREATMENT to M.M.

Background and Objectives
Aripiprazole is an atypical antipsychotic drug that is metabolized by cytochrome P450 (CYP) 2D6 and CYP3A4, which mainly form its active metabolite dehydro-aripiprazole. Because of the genetic polymorphism of CYP2D6, plasma concentrations are highly variable between different phenotypes. In this study, phenotype-related physiologically based pharmacokinetic models were developed and evaluated to suggest phenotype-guided dose adjustments.

Methods
Physiologically based pharmacokinetic models for single dose (oral and orodispersible formulation), multiple dose, and steady-state condition were built using trial data from genotyped healthy volunteers. Based on evaluated models, dose adjustments were simulated to compensate for genetically caused differences.

Results
Physiologically based pharmacokinetic models were able to accurately predict the pharmacokinetics of aripiprazole and dehydro-aripiprazole according to CYP2D6 phenotypes, illustrated by a minimal bias and a good precision. For single-dose administration, 92.5% (oral formulation) and 79.3% (orodispersible formulation) of the plasma concentrations of aripiprazole were within the 1.25-fold error range. In addition, physiologically based pharmacokinetic steady-state simulations demonstrate that the daily dose for poor metabolizer should be adjusted, resulting in a maximum recommended dose of 10 mg, but no adjustment is necessary for intermediate and ultra-rapid metabolizers.

Conclusions
In clinical practice, CYP2D6 genotyping in combination with therapeutic drug monitoring should be considered to personalize aripiprazole dosing, especially in CYP2D6 poor metabolizers, to ensure therapy effectiveness and safety, Open Access funding enabled and organized by Projekt DEAL. Dora Koller and the multiple-dose phase I trial (EUDRA-CT: 2018-000744-26) were co-financed by the H2020 Marie Sklodowska-Curie Innovative Training Network 721236 grant

The mealworm (Tenebrio molitor Linnaeus 1758) is gaining importance as one of the most popular edible insects. Studies focusing on its bioactivities are increasing, although alternative forms of consumption other than the whole insect or flour, such as bioactive non-protein extracts, remain underexplored. Furthermore, the incidence of metabolic syndrome-related pathologies keeps increasing, hence the importance of seeking novel natural sources for reducing the impact of certain risk factors. The aim was to study the potential of a non-protein mealworm extract on metabolic syndrome-related pathologies, obtained with ethanol:water (1:1, v/v) by ultrasound-assisted extraction. We characterized the extract by gas-chromatography mass-spectrometry and assessed its hypolipidemic potential, its ability to scavenger free radicals, to attenuate the inflammatory response in microglial cells, to affect mitochondrial respiration and to enhance insulin sensitivity in mouse hepatocytes. The extract contained fatty acids, monoglycerides, amino acids, certain acids and sugars. The mealworm extract caused a 30% pancreatic lipase inhibition, 80% DPPH· scavenging activity and 55.9% reduction in the bioaccessibility of cholesterol (p = 0.009). The extract was effective in decreasing iNOS levels, increasing basal, maximal and ATP coupled respiration as well as enhancing insulin-mediated AKT phosphorylation at low insulin concentrations (p < 0.05). The potential of a non-protein bioactive mealworm extract against metabolic syndrome-related pathologies is shown, although further studies are needed to elucidate the mechanisms and relationship with compounds

Aims/hypothesis: Second-generation antipsychotic (SGA) drugs have been associated with the development of type 2 diabetes and the metabolic syndrome in patients with schizophrenia. In this study, we aimed to investigate the effects of two different SGA drugs, olanzapine and aripiprazole, on metabolic state and islet function and plasticity. Methods: We analysed the functional adaptation of beta cells in 12-week-old B6;129 female mice fed an olanzapine- or aripiprazole-supplemented diet (5.5–6.0 mg kg−1 day−1) for 6 months. Glucose and insulin tolerance tests, in vivo glucose-stimulated insulin secretion and indirect calorimetry were performed at the end of the study. The effects of SGAs on beta cell plasticity and islet serotonin levels were assessed by transcriptomic analysis and immunofluorescence. Insulin secretion was assessed by static incubations and Ca2+ fluxes by imaging techniques. Results: Treatment of female mice with olanzapine or aripiprazole for 6 months induced weight gain (p<0.01 and p<0.05, respectively), glucose intolerance (p<0.01) and impaired insulin secretion (p<0.05) vs mice fed a control chow diet. Aripiprazole, but not olanzapine, induced serotonin production in beta cells vs controls, likely by increasing tryptophan hydroxylase 1 (TPH1) expression, and inhibited Ca2+ flux. Of note, aripiprazole increased beta cell size (p<0.05) and mass (p<0.01) vs mice fed a control chow diet, along with activation of mechanistic target of rapamycin complex 1 (mTORC1)/S6 signalling, without preventing beta cell dysfunction. Conclusions/interpretation: Both SGAs induced weight gain and beta cell dysfunction, leading to glucose intolerance; however, aripiprazole had a more potent effect in terms of metabolic alterations, which was likely a result of its ability to modulate the serotonergic system. The deleterious metabolic effects of SGAs on islet function should be considered while treating patients as these drugs may increase the risk for development of the metabolic syndrome and diabetes, Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This work was funded by H2020
Marie Sklodowska-Curie ITN-TREATMENT (Grant Agreement 721236, European Commission). We also acknowledge grants
RTI2018-094052-B-100/ AEI/10.13039/501100011033 (Ministerio de Ciencia e Innovación y Fondo Europeo de Desarrollo Regional [FEDER]) and S2017/BMD-3684 (Comunidad de Madrid, Spain), and grants from Fundación Ramón Areces (Spain) and CIBERDEM (ISCIII, Spain)

Second-generation antipsychotics (SGAs) are a mainstay therapy for schizophrenia. SGA-treated patients present higher risk for weight gain, dyslipidemia and hyperglycemia. Herein, we evaluated the effects of olanzapine (OLA), widely prescribed SGA, in mice focusing on changes in body weight and energy balance. We further explored OLA effects in protein tyrosine phosphatase-1B deficient (PTP1B-KO) mice, a preclinical model of leptin hypersensitivity protected against obesity. Wild-type (WT) and PTP1B-KO mice were fed an OLA-supplemented diet (5 mg/kg/day, 7 months) or treated with OLA via intraperitoneal (i.p.) injection or by oral gavage (10 mg/kg/day, 8 weeks). Readouts of the crosstalk between hypothalamus and brown or subcutaneous white adipose tissue (BAT and iWAT, respectively) were assessed. The effects of intrahypothalamic administration of OLA with adenoviruses expressing constitutive active AMPKα1 in mice were also analyzed. Both WT and PTP1B-KO mice receiving OLA-supplemented diet presented hyperphagia, but weight gain was enhanced only in WT mice. Unexpectedly, all mice receiving OLA via i.p. lost weight without changes in food intake, but with increased energy expenditure (EE). In these mice, reduced hypothalamic AMPK phosphorylation concurred with elevations in UCP-1 and temperature in BAT. These effects were also found by intrahypothalamic OLA injection and were abolished by constitutive activation of AMPK in the hypothalamus. Additionally, OLA i.p. treatment was associated with enhanced Tyrosine Hydroxylase (TH)-positive innervation and less sympathetic neuron-associated macrophages in iWAT. Both central and i.p. OLA injections increased UCP-1 and TH in iWAT, an effect also prevented by hypothalamic AMPK activation. By contrast, in mice fed an OLA-supplemented diet, BAT thermogenesis was only enhanced in those lacking PTP1B. Our results shed light for the first time that a threshold of OLA levels reaching the hypothalamus is required to activate the hypothalamus BAT/iWAT axis and, therefore, avoid weight gain. Our results have unraveled an unexpected metabolic rewiring controlled by hypothalamic AMPK that avoids weight gain in male mice treated i.p. with OLA by activating BAT thermogenesis and iWAT browning and a potential benefit of PTP1B inhibition against OLA-induced weight gain upon oral treatment, This work was funded by grants PID-2021-122766OB-100 (to AMV) and PID2019-104399RB-I00 (to GS) funded by Ministerio de Ciencia e Innovacion/Agencia Estatal de Investigacion /10.13039/5011000110 33 and “ERDF A way of making Europe” by the European Union. We also acknowledge grants H2020 Marie Sklodowska-Curie ITNTREATMENT (Grant Agreement 721236, European Commission), S2017/BMD-3684 (Comunidad de Madrid, Spain), Fundacion ´ Ramon ´ Areces (Spain) and CIBERdem (ISCIII, Spain) to AMV. JWE was funded by the Swedish Diabetes Foundation and the Novo Nordisk Foundation (NNF20OC0063864). VF was a recipient of a contract from ITNTREATMENT and is currently a PhD fellow from the Portuguese Foundation for Science and Technology (FCT, Portugal)/ERDF (2020.08388.BD). CF was awarded with Sara Borrell contract (CD19/00078, ISCIII,Spain)

Presentation at the Kick-off meeting of H2020-funded Metabolic Dysfunctions associated with Pharmacological Treatment of Schizophrenia project (Treatment) held in Madrid at the CSIC Institute de Investigaciones Biomédicas Alberto Sols on June 15-16, 2017., No

The liver is a main target tissue of the biguanide metformin which activates AMP-activated protein kinase (AMPK). We previously reported that administration of metformin glycinate showed a greater decrease of glycated hemoglobin A1c than a placebo in patients with type 2 diabetes mellitus (T2DM). In this study, we compared the effects of metformin hydrochloride, the oral antidiabetic drug of first choice, with those of metformin glycinate in hepatocytes from non-diabetic and diabetic mice and humans. Both formulations were equally potent regard to the reduction of basal and glucagon-induced glucose production and mRNA levels of gluconeogenic enzymes (Pck1 and G6pc) in hepatocytes from C57/Bl6 mice and humans. On the contrary, phosphorylation of AMPK and its substrate acetyl CoA carboxylase (ACC) was faster in hepatocytes treated with metformin glycinate. Likewise, we found stronger reduction in hepatocytes from obese/diabetic db/db mice of glucagon-induced glucose output and more sustained AMPK phosphorylation after treatment with metformin glycinate. Importantly, insulin sensitization regarding phosphorylation of AKT (Ser473) was enhanced in hepatocytes from db/db mice or humans pretreated with metformin glycinate. In conclusion, our data indicate that metformin glycinate may be an alternative therapy against insulin resistance during obesity and/or T2DM., Financial support was received from Laboratorios Silanes S.A. de C.V. (Project reference 20159770) as a research agreement with the Instituto de Investigaciones Biomedicas Alberto Sols (CSIC). This work was also funded by grants SAF2015-65267-R (MINECO/FEDER), S2017/BMD-3684 MOIR2-CM (Comunidad de Madrid, Spain), CIBERdem (ISCIII, Spain), and H2020 Marie Sklodowska-Curie ITN-TREATMENT (Grant Agreement number 721236) (European Commission) to A.M.V. and IJCI-2014-19381 (MINECO/FEDER) to P.R and A.M.V., Peer reviewed

Neuroinflammation is an early event during diabetic retinopathy (DR) that impacts the dynamics of microglia polarization. Gliosis is a hallmark of DR and we have reported the beneficial effects of 1R-DSO-ONJ, a member of the sp2-iminosugar glycolipid (sp2-IGL) family, in targeting microglia and reducing gliosis in diabetic db/db mice. Herein, we analyzed the effect of DSO2-ONJ, another family compound incorporating a sulfone group that better mimics the phosphate group of phosphatidylinositol ether lipid analogues (PIAs), in Bv.2 microglial cells treated with bacterial lipopolysaccaride (LPS) and in retinal explants from db/db mice. In addition to decreasing iNOS and inflammasome activation, the anti-inflammatory effect of DSO2-ONJ was mediated by direct p38α MAPK activation. Computational docking experiments demonstrated that DSO2-ONJ binds to p38α MAPK at the same site where PIAs and the alkyl phospholipid perifosine activators do, suggesting similar mechanism of action. Moreover, treatment of microglial cells with DSO2-ONJ increased both heme-oxygenase (HO)-1 and Il10 expression regardless the presence of LPS. In retinal explants from db/db mice, DSO2-ONJ also induced HO-1 and reduced gliosis. Since IL-10-mediated induction of HO-1 expression is mediated by p38α MAPK activation, our results suggest that this molecular mechanism is involved in the anti-inflammatory effects of DSO2-ONJ in microglia., This work was supported by grants from the Spanish Ministry of Economy and Competitiveness: SAF2015-65267-R (MINECO/FEDER), SAF2016-76083-R (MINECO-FEDER), CTQ2015-64425-C2-1-R (MINECO-FEDER) and CTQ2014-53144-P and grants from the Spanish ISCIII (CIBERdem) and INFLAMES (ISCIII PIE14/00045, co-funded by ERDF, “Investing in your future”). We also acknowledge the European Union project H2020-MSCA-ITN TREATMENT Grant Agreement number: 721236. L.M. thanks the Universitat Autònoma de Barcelona-Banco Santander Program for financial support., Peer reviewed

ARS Open., [Aims]: Sirtuin 1 (SIRT1) is a key player in liver physiology and a therapeutic target against hepatic inflammation. We evaluated the role of SIRT1 in the proinflammatory context and oxidative stress during acetaminophen (APAP)-mediated hepatotoxicity., [Results]: SIRT1 protein levels decreased in human and mouse livers following APAP overdose. SIRT1-Tg mice maintained higher levels of SIRT1 on APAP injection than wild-type mice and were protected against hepatotoxicity by modulation of antioxidant systems and restrained inflammatory responses, with decreased oxidative stress, proinflammatory cytokine messenger RNA levels, nuclear factor kappa B (NFκB) signaling, and cell death. Mouse hepatocytes stimulated with conditioned medium of APAP-treated macrophages (APAP-CM) showed decreased SIRT1 levels; an effect mimicked by interleukin (IL)1β, an activator of NFκB. This negative modulation was abolished by neutralizing IL1β in APAP-CM or silencing p65-NFκB in hepatocytes. APAP-CM of macrophages from SIRT1-Tg mice failed to downregulate SIRT1 protein levels in hepatocytes. In vivo administration of the NFκB inhibitor BAY 11-7082 preserved SIRT1 levels and protected from APAP-mediated hepatotoxicity., [Inovation]: Our work evidenced the unique role of SIRT1 in APAP hepatoprotection by targeting oxidative stress and inflammation., [Conclusion]: SIRT1 protein levels are downregulated by IL1β/NFκB signaling in APAP hepatotoxicity, resulting in inflammation and oxidative stress. Thus, maintenance of SIRT1 during APAP overdose by inhibiting NFκB might be clinically relevant., [Rebound Track]: This work was rejected during standard peer review and rescued by Rebound Peer Review (Antioxid Redox Signal 16:293-296, 2012) with the following serving as open reviewers: Rafael de Cabo, Joaquim Ros, Kalervo Hiltunen, and Neil Kaplowitz. Antioxid. Redox Signal. 28, 1187-1208., This work was funded by SAF2015-65267-R (MINECO/FEDER), CIBERdem (ISCIII, Spain), and INFLAMES (ISCIII PIE14/00045, cofunded by ERDF, ‘‘Investing in your
future’’) to A.M.V.; IJCI-2014-19381 (MINECO/FEDER) to P.R. and A.M.V.; SAF2014-52009-R (MINECO/FEDER) to S.A.; SAF2015-63904-R (MINECO/FEDER) to M.M.; CP14/00181 (ISCIII/FEDER) to A.G.-R.; and PI13/00021 (ISCIII/FEDER) to J.M. CIBERehd (ISCIII) to A.G.-R. and J.M.; Grant 37/371 from Al Jouf University to M.A.M. We also acknowledge H2020 Marie Sklodowska-Curie ITNTREATMENT
(Grant Agreement No. 721236) (European Commission)., Peer Reviewed

Fentanyl is an agonist of the μ‐opioid receptor commonly used in the treatment of moderate‐severe pain. In order to study whether pharmacogenetics explains some of the variability in the response to fentanyl, several genes related to fentanyl receptors, transporters and metabolic enzymes have been analysed. Thirty‐five healthy volunteers (19 men and 16 women) receiving a single 300 μg oral dose of fentanyl were genotyped for 9 polymorphisms in cytochrome P450 (CYP) enzymes (CYP3A4 and CYP3A5), ATP‐binding cassette subfamily B member 1 (ABCB1), opioid receptor mu 1 (OPRM1), catechol‐O‐methyltransferase (COMT) and adrenoceptor beta 2 (ADRB2) by real‐time PCR. Fentanyl concentrations were measured by ultra‐performance liquid chromatography combined with tandem mass spectrometry (UPLC‐MS/MS). Fentanyl pharmacokinetics is affected by sex. Carriers of the CYP3A4*22 allele, which is known to reduce the mRNA expression, showed higher area under the concentration‐time curve (AUC) and lower clearance (Cl) values. Although this finding might be of importance, its validity needs to be confirmed in other similar settings. Furthermore, carriers of the ABCB1 C1236T T/T genotype presented a lower AUC and higher Cl, as well as lower half‐life (T1/2). As volunteers were blocked with naltrexone, the effect of fentanyl on pharmacodynamics might be biased; however, we could observe that fentanyl had a hypotensive effect. Moreover, ADRB2 C523A A allele carriers showed a tendency towards reducing systolic blood pressure. Likewise, OPRM1 and COMT minor allele variants were risk factors for the development of somnolence. CYP3A5*3, ABCB1 C3435T and ABCB1 G2677T/A were not associated with fentanyl's pharmacokinetics, pharmacodynamics and safety profile., F. Abad‐Santos and D. Ochoa have been consultants or investigators in clinical trials sponsored by the following pharmaceutical companies: Abbott, Alter, Chemo, Cinfa, FAES, Farmalíder, Ferrer, GlaxoSmithKline, Galenicum, Gilead, Janssen‐Cilag, Kern, Normon, Novartis, Servier, Silverpharma, Teva and Zambon. D. Koller is co‐financed by the H2020 Marie Sklodowska‐Curie Innovative Training Network 721236 grant. P. Zubiaur is co‐financed by Consejería de Educación, Juventud y Deporte from Comunidad de Madrid and Fondo Social Europeo., Peer reviewed

A simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for simultaneous quantification of aripiprazole and its active metabolite, dehydro-aripiprazole, in human plasma. Stable isotopically labeled aripiprazole, aripiprazole-D8, has been used as the internal standard (IS) for both analytes. Only 200 μl of human plasma was needed for analyte extraction, using effective phospholipids-eliminating three-step microelution-solid-phase extraction (SPE, Oasis PRiME HLB 96-well μElution Plate). An ACE C18-PFP column was applied for chromatographic separation at 25 °C, protected by a 0.2-μm on-line filter. A combination of ammonium formate (5 mM)-acetonitrile (pH 4.0; 65:35, v/v) was used as mobile phase and the chromatogram was run under gradient conditions at a flow rate of 0.6 ml/min. Run time lasted 5 min, followed by a re-equilibration time of 3 min, to give a total run time of 8 min. Five μl of the sample was injected into the chromatographic system. Aripiprazole, dehydro-aripiprazole and IS were detected using the mode multiple reaction monitoring in the positive ionization mode. The method was linear in the concentration range of 0.18–110 ng/ml and 0.35–100 ng/ml for aripiprazole and dehydro-aripiprazole, respectively. Our method has been validated according to the recommendations of regulatory agencies through tests of precision, accuracy, recovery, matrix effect, stability, sensitivity, selectivity and carry-over. Our microelution-SPE method removes more than 99% of main plasma phospholipids compared to protein precipitation and was successfully applied to several bioequivalence studies., The authors wish to thank Instituto-Fundación Teófilo Hernando for its continued financial support. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 721236. This work was also supported by FPU12/02220 from MECD to AW., Peer reviewed

This observational retrospective study assessed the antiplatelet response and clinical events after clopidogrel treatment in patients who underwent percutaneous neurointervention, related to CYP2C19 metabolizer status (normal (NM), intermediate/poor (IM‐PM), and ultrarapid (UM); inferred from *2, *3, and *17 allele determination). From 123 patients, IM‐PM had a higher aggregation value (201.1 vs. 137.6 NM, 149.4 UM, P < 0.05) and lower response rate (37.5% vs. 69.8% NM, 61.1% UM), along with higher treatment change rate (25% vs. 5.7% NM, 10.5% UM). The highest ischemic events incidence occurred in NM (11.3% vs. 6.3% IM, 10.5% UM) and hemorrhagic events in UM (13.2% vs. 0% IM and 3.8% NM). No differences were found regarding ischemic event onset time, while hemorrhagic event frequency in UM was higher with shorter onset time (P = 0.047). CYP2C19 no‐function and increased function alleles defined the clopidogrel response. UM patients had increased bleeding risk. Therapeutic recommendations should include dose reduction or treatment change in UM., M. Saiz-Rodriguez is co-financed by Consejería de Educación, Juventud y Deporte from Comunidad de Madrid and Fondo Social Europeo. D. Koller is co-financed by the H2020 Marie Sklodowska-Curie Innovative Training Network 721236 grant., Peer reviewed

Taurine (TAU) is a sulfur-containing beta amino acid that is not involved in protein composition and anabolism, conditionally essential in mammals provided through diet. Growing evidence supports a protective role of TAU supply in osmoregulation, calcium flux, and reduction of inflammation and oxidant damage in renal diseases like diabetes. Endoplasmic reticulum (ER) stress, due to abnormal proteostasis, is a contributor to nephrotic syndrome and related renal damage. Here, we investigated the effect of dietary TAU (1.5% in drinking water for 15 days) in an established rat model that mimics human minimal change nephrosis, consisting of a single puromycin aminonucleoside (PAN) injection (intraperitoneally 15 mg/100 g body weight), with sacrifice after eight days. TAU limited proteinuria and podocytes foot processes effacement, and balanced slit diaphragm nephrin and glomerular claudin 1 expressions. In cortical proximal tubules, TAU improved lysosomal density, ER perimeter, restored proper ER-mitochondria tethering and mitochondrial cristae, and decreased inflammation. Remarkably, TAU downregulated glomerular ER stress markers (GRP78, GRP94), pro-apoptotic C/EBP homologous protein, activated caspase 3, tubular caspase1, and mitochondrial chaperone GRP75, but maintained anti-apoptotic HSP25. In conclusion, TAU, by targeting upstream ER stress separate from mitochondria dysfunctions at crucial renal sites, might be a promising dietary supplement in the treatment of the drug-resistant nephrotic syndrome., This study was supported by New Pet Food Italia S.r.l. (Italy) donation, ex 60% grants from the University of Brescia (Italy), FFARB 2017, SAF2015-63904-R grant from the Spanish Ministerio de Economía Industria y Competitividad (MINEICO), FEDER funds and MSCA-ITN-2016-721236 from the European Commission H2020-MSCA-ITN program., Peer Reviewed

Protein tyrosine phosphatase 1B (PTP1B) is widely expressed in mammalian tissues, in particular in immune cells, and plays a pleiotropic role in dephosphorylating many substrates. Moreover, PTP1B expression is enhanced in response to pro-inflammatory stimuli and to different cell stressors. Taking advantage of the use of mice deficient in PTP1B we have investigated the effect of γ-radiation in these animals and found enhanced lethality and decreased respiratory exchange ratio vs. the corresponding wild type animals. Using bone-marrow derived macrophages and mouse embryonic fibroblasts (MEFs) from wild-type and PTP1B-deficient mice, we observed a differential response to various cell stressors. PTP1B-deficient macrophages exhibited an enhanced response to γ-radiation, UV-light, LPS and S-nitroso-glutathione. Macrophages exposed to γ-radiation show DNA damage and fragmentation, increased ROS production, a lack in GSH elevation and enhanced acidic β-galactosidase activity. Interestingly, these differences were not observed in MEFs. Differential gene expression analysis of WT and KO macrophages revealed that the main pathways affected after irradiation were an up-regulation of protein secretion, TGF-β signaling and angiogenesis among other, and downregulation of Myc targets and Hedgehog signaling. These results demonstrate a key role for PTP1B in the protection against the cytotoxicity of irradiation in intact animal and in macrophages, which might be therapeutically relevant., This work was supported by grants SAF2017-82436R, SAF2016-75004R and SAF2015-65267 from MINIECO, S2017/BMD-3686 and BMD3684 from Comunidad de Madrid, Fundación Ramón Areces (2016/CIVP18A3864) and Cibercv, Ciberdem and Ciberehd (funded by the Instituto de Salud Carlos III and by Fondos FEDER). We also acknowledge H2020 Marie Sklodowska-Curie ITN-TREATMENT (Grant Agreement number 721236) (European Commission). P.R. was recipient a Juan de la Cierva postdoctoral contract from MINIECO (IJCI-2014-19381)., Peer Reviewed

Retinal degenerative diseases are a group of heterogeneous diseases that include age-related macular degeneration (AMD), retinitis pigmentosa (RP), and diabetic retinopathy (DR). The progressive degeneration of the retinal neurons results in a severe deterioration of the visual function. Neuroinflammation is an early hallmark of many neurodegenerative disorders of the retina including AMD, RP and DR. Microglial cells, key components of the retinal immune defense system, are activated in retinal degenerative diseases. In the microglia the interplay between the proinflammatory/classically activated or antiinflammatory/alternatively activated phenotypes is a complex dynamic process that occurs during the course of disease due to the different environmental signals related to pathophysiological conditions. In this regard, an adequate transition from the proinflammatory to the anti-inflammatory response is necessary to counteract retinal neurodegeneration and its subsequent damage that leads to the loss of visual function. Insulin like-growth factor-1 (IGF-1) has been considered as a pleiotropic factor in the retina under health or disease conditions and several effects of IGF-1 in retinal immune modulation have been described. In this review, we provide recent insights of inflammation as a common feature of retinal diseases (AMD, RP and RD) highlighting the role of microglia, exosomes and IGF-1 in this process., The work of AIA and ÁMV has been funded by grants from European Union (project H2020-MSCA-ITN TREATMENT Grant Agreement number: 721236 and project EUROCONDOR FP7 Grant Agreement number 278040), grant from the Spanish Ministry of Economy and Competitiveness: SAF2015-65267-R (MINECO/FEDER) and
grants from the Spanish ISCIII (CIBERdem) and INFLAMES (ISCIII PIE14/00045, co-funded by ERDF, Investing in your future)., Peer Reviewed

[Background]: Pupillometry is used for the detection of autonomic dysfunction related to numerous diseases and drug administration. Genetic variants in cytochrome P450 (CYP2D6, CYP3A4), dopamine receptor (DRD2, DRD3), serotonin receptor (HTR2A, HTR2C) and ATP-binding cassette subfamily B (ABCB1) genes were previously associated with aripiprazole response., [Aims]: Our aim was to evaluate if aripiprazole affects pupil contraction and its relationship with pharmacokinetics and pharmacogenetics., [Methods]: Thirty-two healthy volunteers receiving a 10 mg single oral dose of aripiprazole were genotyped for 15 polymorphisms in ABCB1, CYP2D6, DRD2, DRD3, HTR2A and HTR2C genes by reverse transcription polymerase chain reaction. Aripiprazole and dehydro-aripiprazole plasma concentrations were measured by high-performance liquid chromatography tandem mass spectrometry. Pupil examination was performed by automated pupillometry., [Results]: Aripiprazole caused pupil constriction and reached the peak value at Cmax. HTR2A rs6313 T allele carriers and HTR2C rs3813929 C/T subjects showed higher maximum constriction velocity and maximum pupil diameter. Besides, Gly/Gly homozygotes for DRD3 rs6280 showed significantly lower maximum constriction velocity values. A/G heterozygotes for DRD2 rs6277 showed higher total time taken by the pupil to recover 75% of the initial resting size values. CYP2D6 intermediate metabolisers showed higher area under the curve, Cmax and T1/2 than extensive metabolisers. ABCB1 G2677T/A A/A homozygotes had greater T1/2 in comparison with C/C homozygotes. ABCB1 C3435T T allele carriers and C1236T C/T subjects showed greater area under the curve than C/C homozygotes., [Conclusions]: Aripiprazole affects pupil contraction, which could be a secondary effect through dopamine and serotonin receptors. Pupillometry could be a useful tool to assess autonomic nervous system activity during antipsychotic treatment., D. Koller is co-financed by the H2020 Marie Sklodowska-Curie Innovative Training Network 721236 grant. M. Saiz-Rodríguez and P. Zubiaur are co-financed by Consejería de Educación, Juventud y Deporte from Comunidad de Madrid and Fondo Social Europeo., Peer reviewed

Resumen del trabajo presentado al 5th Symposium on Biomedical Research: "Advances and Perspectives In Pharmacology, Drug Toxicity and Pharmacogenetics", celebrado en Madrid del 15 al 16 de marzo de 2018., [Introduction]: Second-generation Antipsychotics (SGAs) have become the treatment of choice over the typical antipsychotics as they provide excellent efficacy and fewer extrapyramidal symptoms. However, the compliance of the patients to SGAs is negatively affected by their ability to induce or aggravate metabolic syndrome, namely, weight gain, insulin resistance, and Type 2 Diabetes. The exact underlying mechanism of metabolic effects of SGAs is not fully elucidated and it is assumed to
be at least partially due to their effect on central nervous system. However, whether SGAs have a direct effect on insulin action in the tissues is still to be elucidated. The effect of SGAs on body metabolism varies and we have chosen two drugs, Olanzapine and Aripiprazole, which are associated with high and low risk of metabolic side-effects, respectively. Our research is focused on studying the effect of both SGAs on insulin resistance in human adipose tissue. Aside from lipid storage function, adipose tissue has been recognised, as an endocrine organ, producing hormones, such as adiponectin and leptin, indispensable for energy homeostasis. The set of experiments performed as a part of this study includes measuring the effect of Olanzapine and Aripiprazole on the lipolysis in human isolated adipocytes., [Methods]: Biopsies of subcutaneous adipose tissue (SAT) were collected from 6 patients (3 men, 3 women; age: 20-76 years; BMI: 20.9-34.5 kg/m2). Subjects were free of antidepressants or antipsychotics treatment. At the moment, only the effect of Olanzapine has been tested and measured, the experiments with Aripiprazole are in progress. A 6% adipocyte suspension was incubated with olanzapine (0.004, 0.04, 0.1, 0.2, 2 and 20 μM) or aripiprazole (0.02, 0.2, 0.5, 1, 10 and 100 μM). This was followed by 10 minutes incubation with 4 concentrations of insulin (0.1 μU; 1.0 μU; 10 μU; 100
μU) and then incubated with 0.5 μM ß-adrenergic receptor agonist, Isopretenerol, for 1h 50 min. ß-adrenergic stimulation activates hormone-sensitive lipase (HSL) enzyme via cAMP-dependent pathway. HSL, in turn, hydrolyses tritriacylglycerol (TAG), diacylglycerol (DAG) or monoacylglycerol (MAG) molecules producing free fatty acids and glycerol. The supernatant was then collected and used for glycerol measurement., [Results]: Short incubations of adipocytes with therapeutic concentrations of Olanzapine show no effect in lipolysis. The highest concentration of the drug hints at a reduced rate of lipolysis in adipocytes by more than 50% for each insulin concentration (p<0.0001) and in control conditions (p<0.01)., [Conclusions]: Therefore, it seems that short-term incubation of adipocytes with 20 μM Olanzapine reduces the rate of lipolysis, while the therapeutic concentrations do not seem to alter lipolysis in adipocytes., This work is being supported by Marie Skłodowska Curie Actions (H2020-MSCA-ITN-2016)., Peer reviewed

Resumen del trabajo presentado al 5th Symposium on Biomedical Research: "Advances and Perspectives In Pharmacology, Drug Toxicity and Pharmacogenetics", celebrado en Madrid del 15 al 16 de marzo de 2018., [Introduction]: Second-generation Antipsychotics (SGAs) are preferable pharmacological treatment for patients with Schizophrenia, mainly due to their efficacy and reduced risk of extrapyramidal effects when compared with first generation antipsychotics. However, there are metabolic side effects associated with the administration of SGAs, namely weight gain, dyslipidaemia and impaired glucose metabolism. Literature reports that even in the absence of antipsychotic treatment patients with Schizophrenia have a propensity to develop metabolic changes that can lead to cardiovascular diseases, insulin resistance, obesity and Type 2 Diabetes. Olanzapine and aripiprazole belong to SGAs and have been reported as drugs with the highest and the lowest risk of inducing metabolic changes, respectively. However, the pharmacological mechanisms underlying their metabolic side effects remain unclear and they will be the main focus of our study. Adipose tissue is not only specialized in storing lipids but it is also an endocrine organ that produces and secretes numerous
biological active compounds that regulate metabolic homeostasis. Our aim is to evaluate the effect of olanzapine and aripiprazole in the glucose uptake on human isolated adipocytes., [Methods]: Biopsies of subcutaneous adipose tissue were collected from 16 healthy volunteers (4 men, 12 women; age: 20-76 years; BMI: 20.9-34.5 kg/m2). Subjects taking antidepressants or antipsychotics were not included. The effect of short-term incubation (30 min) with different concentrations of olanzapine (0.004, 0.04, 0.1, 0.2, 2 and 20 μM) or aripiprazole (0.02, 0.2, 0.5, 1, 10 and 100 μM) on basal and insulin-stimulated (25 and 1000 μU/ml) D-[U-14C]-glucose uptake of isolated adipocytes
was measured and compared with control., [Results]: Short incubation of adipocytes with olanzapine or aripiprazole showed no effect on basal or insulin-stimulated glucose uptake, with the exception of supra-physiological concentrations of aripiprazole (10 and 100 μM) where we see a systematic decrease of basal and insulin-stimulated glucose uptake; 10 μM by ͌20-25% (p<0.05) and 100 μM by ͌60-70% (p<0.01)., [Conclusions]: Short-term treatment of isolated adipocytes with therapeutic doses of olanzapine or aripiprazole did not affect glucose uptake, suggesting no acute alteration in insulin activity. These data suggest that the plasma glucose increase seen in patients taking Olanzapine cannot be justified by acute alteration in insulin-signalling in adipocytes. Incubation with aripiprazole at 10 and 100 μM decreased the adipocyte glucose uptake but this might be justified by cell death, which will be explored in future experiments., This work is being supported by Marie Skłodowska Curie Actions (H2020-MSCA-ITN-2016)., Peer reviewed

Resumen del trabajo presentado al 5th Symposium on Biomedical Research: "Advances and Perspectives In Pharmacology, Drug Toxicity and Pharmacogenetics", celebrado en Madrid del 15 al 16 de marzo de 2018., [Introduction]: The liver is the main organ in charge of drug catabolism and also the major site susceptible to drug injury. Sirtuin 1 (SIRT1), a NAD+-dependent histone deacetylase, is a key player in liver physiology and a therapeutic target against hepatic inflammation. In this study, we evaluated the role of SIRT1 in the pro-inflammatory context and oxidative stress during acetaminophen (APAP)-mediated hepatotoxicity., [Material and Methods]: SIRT1 expression was analyzed in APAP-induced liver failure in humans and mice. Hepatotoxicity was assessed in wild-type and transgenic mice overexpressing SIRT1 (SIRT1 Tg) poisoned with APAP (300 mg/kg). Raw 264.7 and peritoneal macrophages were treated with APAP and conditioned medium (CM) was added to mouse hepatocytes. siRNA was used to reduce inflammatory mediators in hepatocytes., [Results]: SIRT1 protein levels decreased in human and mouse livers following APAP overdose. SIRT1-Tg mice maintained higher levels of SIRT1 upon APAP injection than wild-type mice and were protected against hepatotoxicity by modulation of antioxidant systems and restrained inflammatory responses, with decreased oxidative stress, pro-inflammatory cytokine mRNA levels, nuclear factor kappa B (NFκB) signaling, and cell death. Mouse hepatocytes stimulated with conditioned medium of APAP-treated macrophages (APAP-CM) showed decreased SIRT1 levels; an effect mimicked by interleukin 1β (IL1β), an activator of NFκB. This negative modulation was abolished by neutralizing IL1β in APAP-CM or silencing p65-NFκB in hepatocytes. APAP-CM of macrophages from SIRT1-Tg mice failed to downregulate SIRT1 protein levels in hepatocytes. In vivo administration of the NFκB inhibitor BAY 11-7082 preserved SIRT1 levels and protected from APAP-mediated hepatotoxicity., [Conclusion]: SIRT1 protein levels are downregulated by IL1β/NFκB signaling in APAP hepatotoxicity, resulting in inflammation and oxidative stress. Thus, maintenance of SIRT1 during APAP overdose by inhibiting NFκB might be clinically relevant. Our work evidenced the unique role of SIRT1 in APAP hepatoprotection by targeting oxidative stress and inflammation., This work was funded by SAF2015-65267-R (MINECO/FEDER), CIBERdem (ISCIII, Spain), INFLAMES (ISCIII PIE14/00045, co-funded by ERDF, “Investing in your future”) to A.M.V; IJCI-2014-19381 (MINECO/FEDER) to P.R and A.M.V; SAF2014-52009-R (MINECO/FEDER) to S.A; SAF2015-63904-R (MINECO/FEDER) to M.M.; CP14/00181 (ISCIII/FEDER) to A. G-R; PI13/00021 (ISCIII/FEDER) to J.M. CIBERehd (ISCIII, Spain) to A. G-R and J.M; Grant 37/371 from Al Jouf University (to M.A.M). We also acknowledge H2020 Marie Sklodowska-Curie ITN-TREATMENT (Grant Agreement number 721236) (European Commission)., Peer reviewed

Resumen del trabajo presentado al 5th Symposium on Biomedical Research: "Advances and Perspectives In Pharmacology, Drug Toxicity and Pharmacogenetics", celebrado en Madrid del 15 al 16 de marzo de 2018., [Introduction]: Schizophrenia (SCZ) is a chronic brain disorder with symptoms of hallucinations, delusions, disturbed behavior and emotional and cognitive impairment. These symptoms are managed by administration of antipsychotic drugs (APDs), either as a single APD or a combination of different APDs depending on the individual. It is always recommended to take a maintenance dose of APD even after the reduction of SCZ symptoms which makes it a lifelong treatment. APD treatment is currently known to be the only effective treatment against SCZ. However, they are observed to trigger metabolic
dysfunctions such as insulin resistance, obesity, coronary heart diseases and dyslipedemia in SCZ patients at the later stage during APD treatment. The main aim of the project is to identify a biomarker signature that could be detected in blood to diagnose the metabolic dysfunction due to APD administration at an early stage. The talk will provide an insight into omic approaches for biomarker discovery and a worked example of these approaches using relevant existing data sets from public
repositories., [Materials and Methods]: The public repository Array Express (AE) (https://www.ebi.ac.uk/arrayexpress/) was used to shortlist the datasets from transcriptomic studies of primary human hepatocytes (PHH) that has been treated with any antipsychotic drug. R studio was used to process the data and shortlist genes that had a minimum fold change of 2. Bioinfominer online tool (https://bioinfominer.com/) was used for Gene ontology enrichment analysis., [Results and conclusion]: We found a microarray dataset of PHH that has been treated with Chlorpromazine (APD) from AE (AE id: E-MTAB-1747). The deregulated gene list from PHH that were treated with CPZ was significantly grouped under inflammatory and immune response from the gene ontology term enrichment analysis. Since the gene products of the inflammatory/immune response are mostly secreted, they are likely to be detected in blood which makes them potential biomarker candidates. However, these candidates were identified from PHH isolated from a single donor, so more work would be required to confirm them as valid biomarkers. Despite that caveat, the gene list can be considered as a useful reference list for future experiments., H2020-MSCA-ETN-2016-72123., Peer reviewed

Resumen del trabajo presentado al 5th Symposium on Biomedical Research: "Advances and Perspectives In Pharmacology, Drug Toxicity and Pharmacogenetics", celebrado en Madrid del 15 al 16 de marzo de 2018., [Introduction]: The P-glycoprotein (P-gp) is an efflux pump widely expressed in the organism that exports xenobiotic compounds out of the tissue where it is expressed. It plays a central role in the Blood-Brain Barrier permeability, being responsible of Central Nervous System side-effects or ineffectiveness of many drugs. Several single-nucleotide polymorphisms (SNPs) in ABCB1 (the gene
encoding for P-gp) have been identified. The most relevant ones, C3435T, C2677T/A and C1236T have been associated with variable pharmacokinetic parameters in healthy volunteers that received single oral doses of antidepressants and antipsychotics. There is no consensus regarding the in vivo effect they have. Here, we compile relevant information in order to simplify the understanding of
materials, methods and cell lines classically used to assess polymorphic P-gp activity in in vitro cell culture models., [Methods]: A comprehensive research of the studies performed in this regard has been accomplished. More than 389 articles have been reviewed, corresponding to the topics “ABCB1 polymorphisms”, “assessment of P-gp function” and “P-gp expression in cell cultures” published in PubMed Search Engine., [Results]: Twenty-four articles have been summarised and classified. Site-directed mutagenesis has been acknowledged as a convenient approach to obtain cell lines expressing mutant P-gp. Ten different techniques have been identified as key in the assessment of P-gp function: Transfection; Western Blot; Flow Cytometry; Transcriptional Analysis; TEER measurements; Calcein-AM, Rhodamine-123 or Radioactivity based accumulation and transport assays; Transwell® inserts; MTT viability assays., [Conclusion]: Site-directed mutagenesis performed in plasmids that contain wild-type ABCB1, followed by transfection of the plasmid into cells (HeLa, Caco-2 cells) may lead to cell lines expressing P-gp with the SNPs of interest (C3435T, C2677T/A and C1236T). Assessment of P-gp function may be accomplished by Calcein-AM or Rhodamine-123 accumulation assays. The actual effect of these SNPs in P-gp on antidepressants or antipsychotics efflux through membranes could be assessed by Transwell® insert transport assays., P. Zubiaur is co-financed by Consejería de Educación, Juventud y Deporte from Comunidad de Madrid and Fondo Social Europeo. D. Koller is co-financed by the H2020 Marie Sklodowska-Curie Innovative Training Network 721236 grant., Peer reviewed

Obesity is associated with local and systemic complications in acute pancreatitis. PPARγ coactivator 1α (PGC-1α) is a transcriptional coactivator and master regulator of mitochondrial biogenesis that exhibits dysregulation in obese subjects. Our aims were: (1) to study PGC-1α levels in pancreas from lean or obese rats and mice with acute pancreatitis; and (2) to determine the role of PGC-1α in the inflammatory response during acute pancreatitis elucidating the signaling pathways regulated by PGC-1α. Lean and obese Zucker rats and lean and obese C57BL6 mice were used first; subsequently, wild-type and PGC-1α knockout (KO) mice with cerulein-induced pancreatitis were used to assess the inflammatory response and expression of target genes. Ppargc1a mRNA and protein levels were markedly downregulated in pancreas of obese rats and mice versus lean animals. PGC-1α protein levels increased in pancreas of lean mice with acute pancreatitis, but not in obese mice with pancreatitis. Interleukin-6 (Il6) mRNA levels were dramatically upregulated in pancreas of PGC-1α KO mice after cerulein-induced pancreatitis in comparison with wild-type mice with pancreatitis. Edema and the inflammatory infiltrate were more intense in pancreas from PGC-1α KO mice than in wild-type mice. The lack of PGC-1α markedly enhanced nuclear translocation of phospho-p65 and recruitment of p65 to Il6 promoter. PGC-1α bound phospho-p65 in pancreas during pancreatitis in wild-type mice. Glutathione depletion in cerulein-induced pancreatitis was more severe in KO mice than in wild-type mice. PGC-1α KO mice with pancreatitis, but not wild-type mice, exhibited increased myeloperoxidase activity in the lungs, together with alveolar wall thickening and collapse, which were abrogated by blockade of the IL-6 receptor glycoprotein 130 with LMT-28. In conclusion, obese rodents exhibit PGC-1α deficiency in the pancreas. PGC-1α acts as selective repressor of nuclear factor-κB (NF-κB) towards IL-6 in pancreas. PGC-1α deficiency markedly enhanced NF-κB-mediated upregulation of Il6 in pancreas in pancreatitis, leading to a severe inflammatory response., This work was supported by grants SAF2009‐09500 and SAF2015–71208‐R with FEDER funds from the Spanish Ministry of Economy and Competitiveness to JS and by grants SAF2015‐63904‐R with FEDER funds from the Spanish Ministry of Economy and Competitiveness and EC MSCA‐ITN‐2016‐721236 to MM. IF was recipient of a fellowship from ‘Programa de Pós‐Doutorado no Exterior (PDE)’ that belongs to the ‘Conselho Nacional de Desenvolvimento Científico e Tecnológico’ (CNPq)., Peer Reviewed

Capítulo 6, Parte I: Biochemical and Molecular Methods., All four FOXO isoforms have been shown to respond to changes in the cellular redox status of the cell, and regulate the expression of target genes that in turn can modulate the cellular oxidative status. However, the mechanisms involved are still controversial. It is clear though that redox regulation of FOXO factors occurs at different levels. The proteins themselves are redox-sensitive and their capacity to bind their target sites seems to be at least partially dependent on their oxidative status. Importantly, several of the cofactors that are known to regulate FOXO transcriptional activity are also sensitive to changes in the cellular redox status, in particular the deacetylase SirT1 is activated in response to reduced levels of reducing equivalents (increased NAD+/NADH+ ratio) and the coactivator PGC-1α is induced in response to increased cellular oxidative stress. Furthermore, nuclear localization of FOXO factors is also regulated by proteins that, like AKT, are themselves regulated directly or indirectly by the cellular levels of reactive oxygen and nitrogen species. In this technical review, we aim to update the current status of our knowledge of how to handle redox-regulated FOXO factor research in order to better understand FOXO biology., This work was supported by grants from the Spanish “Ministerio de Economía Industria y Competitividad” (MINEICO) and FEDER funds [Grant numbers SAF2015-63904-R, SAF2015-71521-REDC] and from the EU H2020 framework programm Grant MSCA-ITN-2016-721236., Peer reviewed

[Objectives]: Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in Western countries. Protein tyrosine phosphatase 1B (PTP1B), a negative modulator of insulin and cytokine signaling, is a therapeutic target for type 2 diabetes and obesity. We investigated the impact of PTP1B deficiency during NAFLD, particularly in non-alcoholic steatohepatitis (NASH). [Methods]: NASH features were evaluated in livers from wild-type (PTP1BWT) and PTP1B-deficient (PTP1BKO) mice fed methionine/choline-deficient diet (MCD) for 8 weeks. A recovery model was established by replacing MCD to chow diet (CHD) for 2–7 days. Non-parenchymal liver cells (NPCs) were analyzed by flow cytometry. Oval cells markers were measured in human and mouse livers with NASH, and in oval cells from PTP1BWT and PTP1BKO mice. [Results]: PTP1BWT mice fed MCD for 8 weeks exhibited NASH, NPCs infiltration, and elevated Fgf21, Il6 and Il1b mRNAs. These parameters decreased after switching to CHD. PTP1B deficiency accelerated MCD-induced NASH. Conversely, after switching to CHD, PTP1BKO mice rapidly reverted NASH compared to PTP1BWT mice in parallel to the normalization of serum triglycerides (TG) levels. Among NPCs, a drop in cytotoxic natural killer T (NKT) subpopulation was detected in PTP1BKO livers during recovery, and in these conditions M2 macrophage markers were up-regulated. Oval cells markers (EpCAM and cytokeratin 19) significantly increased during NASH only in PTP1B-deficient livers. HGF-mediated signaling and proliferative capacity were enhanced in PTP1BKO oval cells. In NASH patients, oval cells markers were also elevated. [Conclusions]: PTP1B elicits a dual role in NASH progression and reversion. Additionally, our results support a new role for PTP1B in oval cell proliferation during NAFLD., This work was funded by SAF2015-65267-R (MINECO/FEDER), CIBERdem (ISCIII, Spain), INFLAMES (ISCIII PIE14/00045, co-funded by ERDF, “Investing in your future”) to A.M.V; IJCI-2014-19381 (MINECO/FEDER) to P.R and A.M.V; CP14/00181 and PI16/00823 (ISCIII/FEDER) and CIBERehd (ISCIII, Spain) to A.G-R; BFU2015-65937-R (MINECO/FEDER), 67-2015 (Department of Health, Navarra Government) and CIBERobn (ISCIII, Spain) to M.J.M; SAF2015-69145-R (MINECO/FEDER) to A.S. A.A. was recipient of a Marie Curie ESR contract from IT-LIVER (Marie Curie Action of the FP7-2012, grant agreement 316549). We also acknowledge the European Union, project H2020-MSCA-ITN TREATMENT Grant Agreement number: 721236., Peer Reviewed

A simple and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated in human plasma for the simultaneous determination of aripiprazole (ARI) and its metabolite dehydro-aripiprazole (DARI); olanzapine (OLA), risperidone (RIS), paliperidone (PAL), quetiapine (QUE), clozapine (CLO) and caffeine (CAF). CAF is included to the method because it can have an influence on drug metabolism due to competitive inhibition. The above mentioned compounds and their isotope-labeled internal standards were extracted from 200 µL human plasma samples by both, effective phospholipids-eliminating three-step microelution-solid-phase extraction (µ-SPE) and protein precipitation (PPT) for comparison. A combination of formic acid (0.2%)-acetonitrile (pH 3.0; 65:35, v/v) was used as mobile phase and the chromatogram was run under gradient conditions at a flow rate of 0.6 mL/min. Run time lasted 6 min, followed by a re-equilibration time of 3 min. All analytes were monitored by mass spectrometric detection operating in multiple reaction monitoring mode and the method was validated covering the corresponding therapeutic ranges: 0.18–120 ng/mL for ARI, 0.25–80 ng/mL for DARI, 1.00–100 ng/mL for OLA, 0.70–60 ng/mL for RIS, 0.20–30 ng/mL for PAL, 0.50–160 ng/mL for QUE, 0.50–1000 ng/mL for CLO, and finally 1200–3700 ng/mL for CAF. The method was validated based on the recommendations of regulatory agencies through tests of precision, accuracy, extraction recovery, identity confirmation, trueness, matrix effect, process efficiency, stability, selectivity, linearity and carry-over effect fulfilling the guideline requirements. Our µ-SPE method results in the elimination of more than 99% of early eluting and more than 92% of late-eluting phospholipids compared to PPT. Additionally, the method was successfully applied for quantifying ARI and OLA plasma concentrations from healthy volunteers., This work and D. Koller were supported by the H2020 Marie Skłodowska-Curie Actions Innovative Training Network 721236 grant. P. Zubiaur is co-financed by “Consejería de Educación, Juventud y Deporte” from “Comunidad de Madrid” and “Fondo Social Europeo”., Peer reviewed

[Purpose]: Clopidogrel is a thienopyridine prodrug that inhibits platelet aggregation. It is prescribed to prevent atherothrombotic and thromboembolic events in patients receiving a stent implant in carotid, vertebral, or cranial arteries. The influence of cytochrome P-450 (CYP) 2C19 on the response to clopidogrel has been widely studied; however, the effect of other genes involved in clopidogrel absorption and metabolism has not been established in this cohort of patients., [Methods]: This observational retrospective study assessed the antiplatelet response and the prevalence of hemorrhagic or ischemic events after percutaneous neurointervention in clopidogrel-treated patients, related to 35 polymorphisms in the genes encoding the clopidogrel-metabolizing enzymes (CYP2C19, CYP1A2, CYP2B6, CYP2C9, CYP2C9, CYP3A4, CYP3A5, carboxylesterase-1 [CES1], and paraoxonase-1 [PON1]), P-glycoprotein transporter (ABCB1), and platelet receptor P2Y12. Polymorphisms were analyzed by quantitative real-time polymerase chain reaction and matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry. Antiplatelet response was documented with the VerifyNow system (Accriva, San Diego, California)., [Findings]: We confirmed that CYP2C19 is the most important enzyme involved in clopidogrel response. The carriage of the CYP2C19*2 allele was strongly associated with hyporesponse to clopidogrel, while the CYP2C19*17 allele was a protective factor for the development of ischemic events (odds ratio = 0.149; P = 0.002) but a risk factor for bleeding (odds ratio = 3.60; P = 0.038). Patients carrying ABCB1 mutated alleles showed lower aggregation values, suggesting that clopidogrel absorption is influenced by P-glycoprotein. In fact, the percentage of responders was significantly higher in the group carrying the mutated haplotype compared to the wild type (80.8% vs 43.3%; P = 0.009). Patients with the CES1 G143E C/T genotype showed a considerably lower, aggregation value versus wild-type patients, although the difference was not significant likely due to the small sample size (59.0 [21.2] vs 165.2 [86.0] PRU; P = 0.084), which suggests an increased active metabolite formation. No relationship was found between polymorphisms in other CYP genes, PON1, or P2RY12 and response to clopidogrel in patients subjected to neurointervention procedures., [Implications]: Therapeutic guidelines recommend that CYP2C19 intermediate and poor metabolizers with acute coronary syndromes undergoing percutaneous coronary intervention receive an alternative antiplatelet therapy; however, genotype-guided therapy is not a standard recommendation for neurovascular conditions. This is the first study to carry out a joint analysis of CYP2C19 and other genes involved in clopidogrel treatment in patients receiving percutaneous neurointervention. Our findings support routine genotyping in clopidogrel-treated patients. Moreover, we encourage considering an alternative antiplatelet therapy in CYP2C19 intermediate, poor and ultrarapid metabolizers. Additionally, ABCB1 polymorphisms could be considered for a better pharmacogenetic approach., D. Koller has received research funding from H2020 Marie Sklodowska-Curie Innovative Training Network grant 721236. P. Zubiaur has received research funding from the Department of Education, Community Youth and Sports of Madrid and the European Social Fund. The MassArray genotyping service was carried out at CEGEN-PRB3-ISCIII, which is supported by PE I+D+i 2013–2016 grant PT17/0019, funded by Instituto de Salud Carlos III (ISCIII) and European Regional Development Fund (ERDF)., Peer reviewed

[Background]: Agomelatine is an agonist of the melatoninergic receptors used for the treatment of depression. Our aim was to evaluate the effect of genetic polymorphisms in metabolising enzymes and the P-glycoprotein transporter on agomelatine pharmacokinetics and pharmacodynamics., [Methods]: Twenty-eight healthy volunteers receiving a single 25 mg oral dose of agomelatine, were genotyped for nine polymorphisms in cytochrome P450 enzymes (CYP1A2, CYP2C9 and CYP2C19) and adenosine triphosphate-binding cassette subfamily B member 1 (ABCB1), by real-time polymerase chain reaction. Agomelatine concentrations were measured by high performance liquid chromatography coupled to a tandem mass spectrometry detector., [Results]: We calculated a CYP1A2 activity score that was directly correlated with agomelatine pharmacokinetics. Individuals with a decreased enzyme activity (*1C carriers) had a lower clearance and accumulated higher concentrations of agomelatine. In contrast, individuals with a high CYP1A2 inducibility (*1F or *1B carriers) showed an extensive clearance and lower agomelatine concentrations. The apparently marked differences between races were due to the different CYP1A2 genotype distribution. CYP2C9 intermediate/poor metabolisers showed a higher area under the concentration-time curve and maximum concentration. ABCB1 G2677T/A polymorphism affected the time to reach maximum concentration, as subjects carrying A/A+A/T genotypes showed higher values. No association was found for CYP2C19 phenotype. Agomelatine did not produce any change in blood pressure, heart rate or QT interval., [Conclusions]: CYP1A2 polymorphisms affect agomelatine pharmacokinetics. CYP1A2 phenotype inferred from the genotyping of CYP1A2*1C, *1F and *1B alleles might be a potential predictor of agomelatine exposure. ABCB1 G2677T/A could affect agomelatine absorption, as subjects with A/A+A/T genotypes had lower agomelatine concentration and they take more time to reach the maximum concentration., D. Koller is co-financed by the H2020 Marie Sklodowska-Curie Innovative Training Network 721236 grant. P. Zubiaur is co-financed by Consejería de Educación, Juventud y Deporte from Comunidad de Madrid and Fondo Social Europeo, Peer reviewed

This article belongs to the Special Issue Plant Antioxidant for Application in Food and Nutraceutical Industries., N-acetylcysteine (NAC), a plant antioxidant naturally found in onion, is a precursor to glutathione. It has been used as a drug since the 1960s and is listed on the World Health Organization (WHO) Model List of Essential Medicines as an antidote in poisonings. There are numerous other uses or proposed uses in medicine that are still in preclinical and clinical investigations. NAC is also used in food supplements and cosmetics. Despite its abundant use, there are projections that the NAC global market will grow in the next five years; therefore, the purpose of this work is to provide a balanced view of further uses of NAC as a dietary supplement. Although NAC is considered a safe substance, the results among clinical trials are sometimes controversial or incomplete, like for many other antioxidants. More clinical trials are underway that will improve our understanding of NAC applicability., B.P. and I.M. are partially supported by Slovenian Research Agency (research core funding No. P3-0388 and P3-0019, respectively). B.K. is supported by the H2020-MSCA-ITN:721236 TREATMENT project and T.P.M. by the Slovenian Research Agency Early Stage Researcher Scheme., Peer reviewed

Inflammation is typically associated with the development of fibrosis, cirrhosis and hepatocellular carcinoma. The key role of protein tyrosine phosphatase 1B (PTP1B) in inflammatory responses has focused this study in understanding its implication in liver fibrosis. Here we show that hepatic PTP1B mRNA expression increased after bile duct ligation (BDL), while BDL-induced liver fibrosis was markedly reduced in mice lacking Ptpn1 (PTP1B−/−) as assessed by decreased collagen deposition and α-smooth muscle actin (α-SMA) expression. PTP1B−/− mice also showed a significant increase in mRNA levels of key markers of monocytes recruitment (Cd68, Adgre1 and Ccl2) compared to their wild-type (PTP1B+/+) littermates at early stages of injury after BDL. Interestingly, the lack of PTP1B strongly increased the NADPH oxidase (NOX) subunits Nox1/Nox4 ratio and downregulated Cybb expression after BDL, revealing a pro-survival pattern of NADPH oxidase induction in response to liver injury. Chimeric mice generated by transplantation of PTP1B−/− bone marrow (BM) into irradiated PTP1B+/+ mice revealed similar hepatic expression profile of NOX subunits than PTP1B−/− mice while these animals did not show differences in infiltration of myeloid cells at 7 days post-BDL, suggesting that PTP1B deletion in other liver cells is necessary for boosting the early inflammatory response to the BDL. PTP1B−/− BM transplantation into PTP1B+/+ mice also led to a blockade of TGF-β and α-SMA induction after BDL. In vitro experiments demonstrated that deficiency of PTP1B in hepatocytes protects against bile acid-induced apoptosis and abrogates hepatic stellate cells (HSC) activation, an effect ameliorated by NOX1 inhibition. In conclusion, our results have revealed that the lack of PTP1B switches NOX expression pattern in response to liver injury after BDL and reduces HSC activation and liver fibrosis., This work was funded by grants SAF2015-65267-R and RTI2018-094052-B-100 (MICINN/FEDER, Spain), S2017/BMD-3684 MOIR2-CM (Comunidad de Madrid, Spain) and CIBERdem (ISCIII, Spain) to A.M.V; IJCI-2014-19381 (MINECO/FEDER, Spain) to P.R and A.M.V. We also acknowledge H2020 Marie Sklodowska-Curie ITN-TREATMENT (Grant Agreement 721236, European Commission)., Peer reviewed

Non-alcoholic fatty liver disease (NAFLD) is associated with post-operative liver failure (PLF) and impaired liver regeneration. We investigated the effects of a glucagon-like peptide-1 (GLP-1) receptor agonist on NAFLD, PLF and liver regeneration in mice fed chow diet or methionine/choline-deficient diet (MCD) or high fat diet (HFD). Fc-GLP-1 decreased transaminases, reduced intrahepatic triglycerides (TG) and improved MCD-induced liver dysfuction. Macrophage/Kupffer cell-related markers were also reduced although Fc-GLP-1 increased expression of genes related to natural killer (NK), cytotoxic T lymphocytes and hepatic stellate cell (HSC) activation. After partial hepatectomy (PH), survival rates increased in mice receiving Fc-GLP-1 on chow or MCD diet. However, the benefit of Fc-GLP-1 on NASH-like features was attenuated 2 weeks post-PH and liver mass restoration was not improved. At this time-period, markers of NK cells and cytotoxic T lymphocytes were further elevated in Fc-GLP-1 treated mice. Increased HSC related gene expression in livers was observed together with decreased retinyl ester content and increased retinal and retinoic acid, reflecting HSC activation. Similar effects were found in mice fed HFD receiving Fc-GLP-1. Our results shed light on the differential effects of a long-acting GLP-1R agonist in improving NAFLD and PLF, but not enhancing liver regeneration in mice., This work was supported by grants SAF2015-65267-R (MINECO, Spain), S2017/BMD-3684 MOIR2-CM (Comunidad de Madrid, Spain), CIBERdem (ISCIII, Spain), INFLAMES (ISCIII PIE14/00045, co-funded by ERDF, “Investing in your future”), European Foundation for the Study of Diabetes (EFSD) and H2020 Marie Sklodowska-Curie ITN-TREATMENT (Grant Agreement Number 721236) (European Commission) to A.M.V. Financial support was also received from MedImmune (MA-416191) as a research agreement with the Instituto de Investigaciones Biomedicas Alberto Sols., Peer reviewed

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., This work was supported by the Instituto de Salud Carlos III (ISCIII/FEDER) (PI17/00535 and CIBEREHD) to C.G.-M.; Ministerio de Ciencia e Innovación (MICINN/FEDER) (RTI2018-094052-B-100), Comunidad de Madrid (S2017/BMD-3684 MOIR2-CM), Instituto de Salud Carlos III (ISCIII/FEDER) (CIBERDEM) and H2020 Marie Sklodowska-Curie ITN-TREATMENT (Grant agreement number 721236) to A.M.V.; Instituto de Salud Carlos III (ISCIII/FEDER) (CP14/00181 and PI16/00823) and Beca Eduardo Gallego 2016 (Fundación Francisco Cobos) to A.G.-R., Peer reviewed

[Aims]: Oxidative stress is known to induce early replicative senescence. Senescence has been proposed to work as a barrier to immortalization and tumor development. Here, we aimed to evaluate the impact of the loss of peroxisome proliferator activated receptor γ co-activator 1α (PGC-1α), a master regulator of oxidative metabolism and mitochondrial reactive oxygen species (ROS) generation, on replicative senescence and immortalization in mouse embryonic fibroblasts (MEFs)., [Results]: We found that primary MEFs lacking PGC-1α showed higher levels of ROS than wild-type MEFs at all cell passages tested. The elevated production of ROS was associated with higher levels of oxidative DNA damage and the increased formation of DNA double-strand breaks. Evaluation of the induction of DNA repair systems in response to γ-radiation indicated that the loss of PGC-1α also resulted in a small but significant reduction in their activity. DNA damage induced the early activation of senescence markers, including an increase in the number of β-galactosidase-positive cells, the induction of p53 phosphorylation, and the increase in p16 and p19 protein. These changes were, however, not sufficient to reduce proliferation rates of PGC-1α-deficient MEFs at any cell passage tested. Moreover, PGC-1α-deficient cells escaped replicative senescence., [Innovation & conclusion]: PGC-1α plays an important role in the control of cellular senescence and immortalization., This work was supported by grants from the Spanish Ministerio de Economía Industria y Competitividad (MINEICO) and FEDER funds [Grant numbers SAF2012-37693, SAF2015-63904-R, SAF2015-71521-REDC, to M.M., BFU-2014-53610-P to A.A.], from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Action grant agreement 721236-TREATMENT to M.M. and MPY-1038/14 and MPY-1146/16 from ISCIII to A.Z., Peer reviewed

This article belongs to the Special Issue Cellular Stress Response in Health and Disease., The interplay of mitochondria with the endoplasmic reticulum and their connections, called mitochondria-ER contacts (MERCs) or mitochondria-associated ER membranes (MAMs), are crucial hubs in cellular stress. These sites are essential for the passage of calcium ions, reactive oxygen species delivery, the sorting of lipids in whole-body metabolism. In this perspective article, we focus on microscopic evidences of the pivotal role of MERCs/MAMs and their changes in metabolic diseases, like obesity, diabetes, and neurodegeneration., This research was funded by FFARB 2017 of A.S., ex 60% grants of University of Brescia (Italy) of A.S. and R.R. and Spanish “Ministerio de Economía Industria y Competitividad” (MINEICO) that includes FEDER funds (SAF2015-63904-R) and from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie agreement 721236-TREATMENT of M.M., Peer reviewed

PGC‐1α (peroxisome proliferator‐activated receptor gamma coactivator‐1α, PPARGC1A) regulates the expression of genes involved in energy homeostasis and mitochondrial biogenesis. Here we identify inactivation of the transcriptional regulator PGC‐1α as a landmark for experimental nephrotoxic acute kidney injury (AKI) and describe the in vivo consequences of PGC‐1α deficiency over inflammation and cell death in kidney injury. Kidney transcriptomic analyses of WT mice with folic acid‐induced AKI revealed 1398 up‐ and 1627 downregulated genes. Upstream transcriptional regulator analyses pointed to PGC‐1α as the transcription factor potentially driving the observed expression changes with the highest reduction in activity. Reduced PGC‐1α expression was shared by human kidney injury. Ppargc1a−/− mice had spontaneous subclinical kidney injury characterized by tubulointerstitial inflammation and increased Ngal expression. Upon AKI, Ppargc1a−/− mice had lower survival and more severe loss of renal function, tubular injury, and reduction in expression of mitochondrial PGC‐1α‐dependent genes in the kidney, and an earlier decrease in mitochondrial mass than WT mice. Additionally, surviving Ppargc1a−/− mice showed higher rates of tubular cell death, compensatory proliferation, expression of proinflammatory cytokines, NF‐κB activation, and interstitial inflammatory cell infiltration. Specifically, Ppargc1a−/− mice displayed increased M1 and decreased M2 responses and expression of the anti‐inflammatory cytokine IL‐10. In cultured renal tubular cells, PGC‐1α targeting promoted spontaneous cell death and proinflammatory responses. In conclusion, PGC‐1α inactivation is a key driver of the gene expression response in nephrotoxic AKI and PGC‐1α deficiency promotes a spontaneous inflammatory kidney response that is magnified during AKI., Sandra Zazo and Federico Rojo of the IIS‐FJD Biobank (B.0000647). FIS/Fondos FEDER (PI15/00298, CP14/00133, PI16/02057, PI16/01900, ISCIII‐RETIC REDinREN RD016/0009), Sociedad Española de Nefrología, FRIAT, Comunidad de Madrid en Biomedicina B2017/BMD‐3686 CIFRA2‐CM. Grants from the Spanish ‘Ministerio de Economía Industria y Competitividad’ (MINEICO) and FEDER funds (Grant numbers SAF2015‐63904‐R, SAF2015‐71521‐REDC), and from the EC H2020 framework program Grant MSCA‐ITN‐2016‐721236 to MM. Salary support: ISCIII Miguel Servet and to ABS and MDS‐N. ISCIII Sara Borrell to JM‐MM, and Fundación Conchita Rabago to DMS. Consejería de Educación, Juventud y Deporte (Comunidad de Madrid/FSE) to MF‐B., Peer reviewed

Ever since Claude Bernards discovery in the mid 19th-century that a lesion in the floor of the third ventricle in dogs led to altered systemic glucose levels, a role of the CNS in whole-body glucose regulation has been acknowledged. However, this finding was later overshadowed by the isolation of pancreatic hormones in the 20th century. Since then, the understanding of glucose homeostasis and pathology has primarily evolved around peripheral mechanism. Due to scientific advances over these last few decades, however, increasing attention has been given to the possibility of the brain as a key player in glucose regulation and the pathogenesis of metabolic disorders such as type 2 diabetes. Studies of animals have enabled detailed neuroanatomical mapping of CNS structures involved in glucose regulation and key neuronal circuits and intracellular pathways have been identified. Furthermore, the development of neuroimaging techniques has provided methods to measure changes of activity in specific CNS regions upon diverse metabolic challenges in humans. In this narrative review, we discuss the available evidence on the topic. We conclude that there is much evidence in favor of active CNS involvement in glucose homeostasis but the relative importance of central vs. peripheral mechanisms remains to be elucidated. An increased understanding of this field may lead to new CNS-focusing pharmacologic strategies in the treatment of type 2 diabetes., This review was funded by grants from the Swedish Diabetes Foundation, Ernfors foundation, ALF (Swedish Government Research Fund), and the European Union’s Horizon 2020 Research and Innovation Programme under the Marie Skłodowska-Curie grant agreement 721236-TREATMENT., Peer reviewed

Resumen del póster presentado al 6th Symposium on Biomedical Research: Advances and Perspectives in Molecular Endocrinology "In Homage to Gabriella Morreale", celebrado en el Instituto de Investigaciones Biomédicas Alberto Sols (IIBM-CSIC) el 31 de mayo de 2019., Schizophrenia is a chronic mental disorder that affects approximately 50 million people worldwide. Also, it is associated with psychotic experiences that not allow patients to have a normal life. Fortunately, treatments used can suppress the symptomatology and lead to a productive life and integration in the society. According to the current guidelines, the first line in schizophrenia’s therapy are the second generation antipsychotic drugs (SGA). Besides being D2 receptors antagonists/partial agonists, the SGAs have the ability to interact also with serotonergic, histaminergic and other receptors; minimizing
the symptomatology and not resulting in neurological side effects (contrarily to first generation antipsychotic drugs). Nevertheless, recent clinical observations show a variety of metabolic dysfunctions in patients under SGAs treatment, such as abnormal gain weight, hyperglycemia and dyslipidemia. However, the molecular mechanisms behind these alterations are still very poorly understood. Having this in consideration, the objective of this work was to study the effect of Olanzapine administration in wild-type (WT) and PTP1B-deficient (KO) mice on glucose homeostasis and the mechanisms involved. Of interest, PTP1B is the main tyrosine phosphatase of the insulin receptor, and PTP1B KO mice are protected against insulin resistance and development of type 2 diabetes mellitus. After administration of 10 mg/kg/day of Olanzapine for 8 weeks to WT and PTP1B-KO mice, the glucose homeostasis tests suggest that Olanzapine induced insulin resistance only in WT mice. Also, primary hepatocytes from WT mice treated with Olanzapine showed lower glucose uptake than those from non-treated WT mice. In contrast, Olanzapine treatment of PTP1B KO mice significantly increased hepatocyte glucose uptake, suggesting that Olanzapine-induced insulin resistance is at least in part dependent on PTP1B. These results support the relevance of PTP1B in
Olanzapine-induced insulin resistance in hepatocytes., European Union's EU Framework Programme for Research and Innovation Horizon 2020. ITN-TREATMENT under Grant Agreement No GA 721236., Peer reviewed

Melatonin, an indole produced by pineal and extrapineal tissues, but also taken with a vegetarian diet, has strong anti-oxidant, anti-inflammatory and anti-obesogenic potentials. Non-alcoholic fatty liver disease (NAFLD) is the hepatic side of the metabolic syndrome. NAFLD is a still reversible phase but may evolve into steatohepatitis (NASH), cirrhosis and carcinoma. Currently, an effective therapy for blocking NAFLD staging is lacking. Silent information regulator 1 (SIRT1), a NAD+ dependent histone deacetylase, modulates the energetic metabolism in the liver. Micro-RNA-34a-5p, a direct inhibitor of SIRT1, is an emerging indicator of NAFLD grading. Thus, here we analyzed the effects of oral melatonin against NAFLD and underlying molecular mechanisms, focusing on steatosis, ER stress, mitochondrial shape and autophagy. Male C57BL/6J (WT) and SIRT1 heterozygous (HET) mice were placed either on a high-fat diet (58.4% energy from lard) (HFD) or on a standard maintenance diet (8.4% energy from lipids) for 16 weeks, drinking melatonin (10 mg/kg) or not. Indirect calorimetry, glucose tolerance, steatosis, inflammation, ER stress, mitochondrial changes, autophagy and microRNA-34a-5p expression were estimated. Melatonin improved hepatic metabolism and steatosis, influenced ER stress and mitochondrial shape, and promoted autophagy in WT HFD mice. Conversely, melatonin was ineffective in HET HFD mice, maintaining NASH changes. Indeed, autophagy was inconsistent in HET HFD or starved mice, as indicated by LC3II/LC3I ratio, p62/SQSTM1 and autophagosomes estimation. The beneficial role of melatonin in dietary induced NAFLD/NASH in mice was related to reduced expression of microRNA-34a-5p and sterol regulatory element-binding protein (SREBP1) but only in the presence of full SIRT1 availability., This research was funded by FFABR 2017, 60% from grants of University of Brescia (Italy) and grants by the Spanish “Ministerio de Economía Industria y Competitividad” (MINEICO) including FEDER funds (SAF2015-63904-R) and the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie agreement 721236-TREATMENT to M.M., Peer reviewed

Las alteraciones identificables tanto en estructura como en marcadores moleculares mediante análisis histológico de muestras de tejido tiroideo son esenciales tanto para su clasificación como para la estadificación del riesgo., Este trabajo ha sido realizado con fondos aportados por proyectos de investigación del Ministerio de Economía Industria y Competitividad (MINEICO) y fondos FEDER [proyectos con referencias
SAF2012-37693, SAF2015-63904-R, SAF2015-71521-REDC] así como por el proyecto europeo 721236-TREATMENT del programa marco Horizon 2020., Peer reviewed

[Objective] Metabolic syndrome is prevalent in up to 50% of schizophrenia patients, which reduces their quality of life and their compliance with the treatment. It is unclear whether metabolic adverse effects of these agents are due to their direct effect on insulin-sensitive tissues or are secondary to increased adiposity. The study aimed to investigate the direct effects of the second-generation antipsychotics olanzapine and aripiprazole on human subcutaneous adipose tissue and isolated adipocyte metabolism., [Methods] Abdominal subcutaneous adipose tissue needle biopsies were taken from 72 healthy subjects (49 F/23 M; age: 19–78 yr; BMI: 20.0–35.6 kg/m2). Isolated adipocytes or adipose tissue were respectively pre-incubated short- (30 min) and long-term (24 h, 72 h) with or without olanzapine (0.004 μM – 20 μM) and aripiprazole (0.002 μM – 100 μM). Pre-incubated adipose tissue was then snap-frozen for mRNA expression analysis of adipokines genes and genes involved in inflammation, adipogenesis, and mitochondrial function. Isolated adipocytes were used to measure basal and insulin-stimulated glucose uptake and lipolysis., [Results] Acute treatment with a therapeutic concentration of olanzapine decreases basal lipolysis in isolated adipocytes; this effect was not observed after long-term incubation with the drug. Supra-therapeutic concentration of aripiprazole reduced basal and insulin-stimulated glucose uptake after short- and long-term pre-incubation. Both drugs at supra-therapeutic concentrations downregulated the expression of the pro-inflammatory cytokines IL6 and IL1B genes after 72 h incubation. Similarly, supra-therapeutic concentrations of both drugs and therapeutic concentration of olanzapine, reduced the expression of PPARGC1A, PDK4, and CPT1B genes involved in the regulation of mitochondrial functions. Neither of the antipsychotics affected the expression of the main adipokines LEP and ADIPOQ, genes involved in the regulation of lipid metabolism, LPL and FASN, nor the master adipogenesis regulator, PPARG., [Conclusion] Therapheutic concentrations of olanzapine and aripiprazole have a moderate direct effect on adipocyte lipid and glucose metabolism, respectively. At supra-therapeutic concentrations, both of the antipsychotics seem to act as anti-inflammatory agents and mildly suppressed genes involved in the regulation of mitochondrial functions, which could potentially contribute to metabolic adverse effects. Alternatively, second-generation antipsychotics could induce metabolic side effects via acting on other insulin-sensitive tissues and central nervous system., This work was supported by research grants from the European Commision via the Marie Sklodowska Curie Innovative Training Network TREATMENT (H2020-MSCA-ITN-721236), the Uppsala University Hospital ALF grants, Svenska Sällskapet för Medicinsk Forskning (Swedish Society for Medical Research), the Diabetes Foundation (Swedish Diabetes Association); the Ernfors Foundation; and the Excellence of Diabetes Research in Sweden (EXODIAB)., Peer reviewed

[Introduction] Metabolic syndrome and associated diseases are a global health problem. Detection of early metabolic modifications that may lead to metabolic syndrome would enable timely introduction of preventive lifestyle modifications., [Material and methods] In total 103 young, healthy adults were assessed for indicators of metabolic alterations. Anthropometric, lifestyle, genetic and biochemical parameters were assessed. Individuals who fulfilled at least one criterion for diagnosis of metabolic syndrome were assigned to the group with the higher metabolic syndrome burden (B-MeS)., [Results] The 34 young healthy individuals who were assigned to the B-MeS group had lower fat-free mass, higher body mass index, waist-to-hip ratio, fat mass, and blood pressure, more visceral fat, they were less physically active, had higher C-reactive protein values and higher catalase activity. Their phenotype was more similar to that of patients diagnosed with metabolic syndrome than the rest of the population., [Conclusions] Simple anthropometric measurements, lifestyle assessment and basic biochemical measurements can be used to identify young healthy individuals with increased risk for metabolic syndrome. These assessments can be performed at periodic check-ups of the healthy population so that timely diagnosis of B-MeS can be made. As lifestyle factors have a big influence on development or improvement of the MeS, the timely diagnosis for B-MeS would enable an early opportunity for intervention for lifestyle modification in the still healthy population, saving costs and reducing disability adjusted life years., The authors acknowledge the financial support from the Slovenian Research Agency (research core funding No. P1-0170 and P3-0019). This study was also partially supported by the H2020-MSCA-ITN:721236 TREATMENT project., Peer reviewed

This article belongs to the Special Issue Oxidative stress and Applied Biology., High levels of reactive oxygen species (ROS) can lead to impairment of cell structure, biomolecules' loss of function and cell death and are associated with liver diseases. Cells that survive increased ROS often undergo malignant transformation. Many cancer cells tolerate high levels of ROS. Here we report a transiently increased production of H2O2 and concomitant upregulation of antioxidative enzymes triggered by hepatocyte isolation; the H2O2 levels revert in about two days in culture. Three-day survival rate of the isolated cells in the presence of 2.5-fold increase of H2O2 is almost 80%. Apoptosis activation through the mitochondrial pathway is meanwhile reduced by inhibition of caspase-9 triggering. This reduction depends on the amount of H2O2 production, as decreased production of H2O2 in the presence of an antioxidant results in increased apoptosis triggering. These stress adaptations do not influence urea production, which is unchanged throughout the normal and stress adapted phases. We conclude that hepatocytes' stress adaptation is mediated by increased ROS production. In this case, high ROS improve cell survival., This research was funded by the Slovenian Research Agency (research core funding No. P3-0019).
This work was also partially supported by the H2020-MSCA-ITN:721236 TREATMENT project., Peer reviewed

Schizophrenia is a neuropsychiatric disorder that chronically affects 21 million people worldwide. Second-generation antipsychotics (SGAs) are the cornerstone in the management of schizophrenia. However, despite their efficacy in counteracting both positive and negative symptomatology of schizophrenia, recent clinical observations have described an increase in the prevalence of metabolic disturbances in patients treated with SGAs, including abnormal weight gain, hyperglycemia and dyslipidemia. While the molecular mechanisms responsible for these side-effects remain poorly understood, increasing evidence points to a link between SGAs and adipose tissue depots of white, brown and beige adipocytes. In this review, we survey the present knowledge in this area, with a particular focus on the molecular aspects of adipocyte biology including differentiation, lipid metabolism, thermogenic function and the browning/beiging process., This work was funded by H2020 Marie Skłodowska-Curie ActionsITN-TREATMENT (Grant Agreement 721236, European Commission). We also acknowledge grants RTI2018-094052-B-100 (MICINN/FEDER, Spain), S2017/BMD-3684 MOIR2-CM (Comunidad de Madrid, Spain) and CIBERdem (ISCIII, Spain)., Peer reviewed

Schizophrenia is a mental disease that results in decreased life expectancy and wellbeing, by promoting obesity and sedentary lifestyles. Schizophrenia is treated by antipsychotic drugs. While the second generation of antipsychotics (SGA), Olanzapine and Aripiprazole are more effective in treating schizophrenia, they display a higher risk of metabolic side effects, mostly by development of diabetes and insulin resistance, weight gain as well as dyslipidemia. Endoplasmic reticulum (ER) stress is induced when ER homeostasis of lipid biosynthesis and protein folding is impaired. This leads to the activation of the unfolded protein response (UPR), a signaling cascade that aims to restore ER homeostasis or initiate cell death. Chronic conditions of ER stress in the liver are associated with diabetes and perturbed lipid metabolism. These metabolic dysfunctions resemble the pharmacological side effects of SGAs. We, therefore, investigated whether SGAs promote the UPR in human and mouse hepatocytes. We observed full-fledged activation of ER stress by Aripiprazole, not by Olanzapine. This occurred at low micromolar concentrations and to variable intensities in different cell types, such as hepatocellular carcinoma, melanoma and glioblastoma. Mechanistically, Aripiprazole caused depletion of ER calcium, leading to activation of IRE1 and PERK, two major transducers of the UPR. Cells underwent apoptosis upon Aripiprazole treatment, which coincided with UPR induction, and this effect was reduced by adding glutathione without affecting UPR itself. Deletion of IRE1 from HepG2 cells protected cells from Aripiprazole toxicity. Our study reveals for the first time a cytotoxic effect of Aripiprazole that involves the induction of ER stress., FF, PD and VF are Marie Curie fellows [Treatment H2020-MSCA-ITN721236]. AMV, PR and IGM were supported by grants [RTI2018-094052-B-100] (MCIU/AEI/FEDER, UE)) and [S2017/BMD-3684] MOIR2-CM (Comunidad de Madrid, Spain) and CIBERdem (ISCIII, Spain)., Peer reviewed

[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., This work was funded by grants SAF2015-65267-R (MINECO/FEDER, UE), RTI2018-094052-B-100 (MCI/AEI/FEDER, UE), S2010/BMD-2423, S2017/BMD-3684 (Comunidad de Madrid, Spain), CIBERdem, CIBERhed and PI 16/02096 (ISCIII, Spain). We also acknowledge grants H2020 Marie Sklodowska-Curie ITN-TREATMENT (Grant Agreement 721236, European Commission), Spanish Ministry of Education-MECD (FPU13/05802 grant to C.R) and Fundación Ramón Areces (to A.M.V.). CBMSO is recipient of institutional aid from Fundación Ramón Areces. VBL, FMG and FR received funding from a Wellcome Trust joint investigator award (106262/Z/14/Z and 106263/Z/14/Z) and a joint MRC programme within the Metabolic Diseases Unit (MRC_MC_UU_12012/3), United Kingdom. VBL received a project support grant from the Society for Endocrinology, United Kingdom. The MRL Histology and Biochemistry Assay Lab Core facilities received funding from the MRC Metabolic Diseases Unit [MRC_MC_UU_12012/5] and the Imaging Core through a Wellcome Trust Strategic Award [100574/Z/12/Z], United Kingdom., Peer reviewed

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease around the world estimated to affect up to one-third of the adult population and is expected to continue rising in the coming years. Nonalcoholic fatty liver disease is considered as the hepatic manifestation of the metabolic syndrome because it is strongly associated with obesity, insulin resistance, type 2 diabetes mellitus, and cardiovascular complications. Despite its high prevalence, factors leading to NAFLD progression from simple steatosis to nonalcoholic steatohepatitis, cirrhosis, and, ultimately hepatocellular carcinoma remain poorly understood. To date, no treatment has proven efficacy, and also no reliable method is currently available for diagnosis or staging of NAFLD beyond the highly invasive liver biopsy. Recently, extracellular vesicles (EVs) have emerged as potential candidate biomarkers for the diagnosis of NAFLD. Extracellular vesicles are circulating, cell-derived vesicles containing proteins and nucleic acids, among other components, that interact with and trigger a plethora of responses in neighbor or distant target cells. Several mechanisms implicated in NAFLD progression, such as inflammation, fibrosis, and angiogenesis, all related to metabolic syndrome–associated lipotoxicity, trigger EV production and release by liver cells. As hepatocytes represent ~80% of the liver volume, in this review we will focus on hepatocyte-derived EVs as drivers of the interactome between different liver cell types in NAFLD pathogenesis, as well as in their role as noninvasive biomarkers for NAFLD diagnosis and progression. Based on that, we will highlight the research that is currently available on EVs in this topic, the current limitations, and future directions for implementation in a clinical setting as biomarkers or targets of liver disease., We acknowledge grant RTI2018-094052-B-100 (MCIU/AEI/FEDER, UE), Fundación Ramón Areces (Spain), S2017/BMD-3684 (Comunidad de Madrid, Spain), and H2020 Marie Sklodowska-Curie ITN-TREATMENT (Grant Agreement 721236, European Commission)., Peer reviewed