Abstracts of 6th Symposium on Biomedical Research

e00001
Novel molecular pathways for the central control of puberty: Basic aspects and translational implications.

Manuel Tena-Sempere1*.

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00001

Copyright: © 2019 Manuel Tena-Sempere. This is an open-access article distributed under the terms of the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and
source are credited.

Cite as: Tena-Sempere, M. Novel molecular pathways for the central control of puberty: Basic aspects and translational implications.
IBJ Plus 2019 S(3):e0001 doi: 10.24217/2531-0151.19v1s3.00001.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Puberty is crucial developmental event, driven by a large set of sophisticated regulatory networks, which are capable to
integrate internal and peripheral signals to precisely and timely activate the brain centers governing the reproductive
axis. In humans and other mammalian species, the timing of puberty is genetically determined. Yet, puberty is also
highly sensitive to numerous internal and external cues, among which metabolic and nutritional signals are especially
prominent. Compelling epidemiological evidence suggests that alterations of the age of puberty are becoming more
frequent in humans, via as yet unknown mechanisms; yet, the escalating prevalence of obesity and other metabolic/
feeding disorders has been pointed out as major contributing factor. Notably, this phenomenon is likely to have
translational implications, since alterations in the timing of puberty have been associated to adverse health outcomes
later in life, including higher risk of earlier all-cause mortality. This urges for a better understanding of the neurohormonal
basis of normal puberty and the mechanisms of its deviations.

In recently years, compelling evidence has documented the master role of hypothalamic neurons producing kisspeptins,
which are encoded by the Kiss1 gene and operate via de G-protein coupled receptor, Gpr54, in the neuroendocrine
pathways controlling puberty, with a particularly prominent function of Kiss1 neurons located in the arcuate nucleus
(ARC). Other transmitters, such as tachykinins (e.g., Neurokinin B), opioids (e.g., dynorphin) and melanocortins,
cooperate with kisspeptins in the precise regulation of puberty. In addition, Kiss1 neurons seemingly participate in
transmitting at least part of the regulatory actions of the signals responsible for the metabolic control of puberty. In
this context, recent work from our group has unveiled the important role of key cellular metabolic sensors, such as
AMP-activated protein kinase (AMPK), the master cellular sensor activated in conditions of energy insufficiency, and the
fuel-sensing deacetylase, Sirt1, as major components for the metabolic modulation of female puberty onset. Our data
indicate that AMPK and Sirt1, acting in ARC Kiss1 neurons, are major molecular effectors for the metabolic control of
Kiss1 and, thereby, puberty onset, whose alteration may contribute to the perturbations of pubertal timing frequently
linked to conditions of metabolic stress, such as sub-nutrition and obesity.

e00002
To sleep or not to sleep: how the brain decides to wake us up.

Luis de Lecea1*

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00002

Copyright: © 2019 Luis de Lecea. This is an open-access article distributed under the terms of the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are
credited.

Cite as: de Lecea, L. To sleep or not to sleep: how the brain decides to wake us up. IBJ Plus 2019 S(3):e0002 doi: 10.24217/2531-
0151.19v1s3.00002.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

The arousal construct underlies a spectrum of behaviors that include sleep, exploration, feeding, sexual activity and
adaptive stress. Pathological arousal conditions include stress, anxiety disorders, and addiction. In the past few years we
have used optogenetics to identify and interrogate neuronal circuits underlying transitions between arousal states. In
the first presentation, I will talk about how the hypocretin system coordinates and makes the decisions about when to
mark the transition between sleep and wakefulness. The dynamics between arousal state transitions are also modulated
by norepinephrine neurons in the locus coeruleus, histaminergic neurons in the hypothalamus, dopaminergic neurons
in the mesencephalon and cholinergic neurons in the basal forebrain. I will introduce our own model of sleep/wake
dynamics using probabilistic estimates of neurotransmitter function based on optogenetic stimulations. I will also
discuss how these models can help characterize dissociated arousal states and develop treatments for neuropsychiatric
disorders.

e00003
Emerging genes and pathways in thyroid differentiation and
tumorigenesis.

Pilar Santisteban1*

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00003

Copyright: © 2019 Pilar Santisteban. This is an open-access article distributed under the terms of the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are
credited.

Cite as: Santisteban, P. Emerging genes and pathways in thyroid differentiation and tumorigenesis. IBJ Plus 2019 S(3):e0003 doi:
10.24217/2531-0151.19v1s3.00003.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Thyroid cancer remains the most common endocrine malignancy worldwide and its incidence and mortality has increased steadily over the last four decades. In general, it has a good outcome; however, some patients develop aggressive forms of thyroid cancer that are untreatable and the molecular bases are poorly understood. These aggressive forms have lost NIS (Na/I Symporter) function, one of the most important hallmarks during thyroid cancer progression, as it leads to radioiodine-resistant metastatic disease. Our work has contributed to understand the mechanisms involved in iodide uptake repression and tumor progression. We have found that BRAF activation decreases NIS expression and impairs NIS trafficking to the membrane of follicular thyroid cells, and accordingly causes (RAI)-refractory metastatic disease in patients with papillary thyroid cancer. We have demonstrated that the mechanism by which BRAF impairs NIS function is mediated by a TGFβ autocrine loop. Furthermore, by next-generation sequencing and gene expression analysis we have identified a master miRNA (miR) regulatory network involved in essential biological process such as thyroid differentiation. Among those miRNAs, the most abundantly expressed in thyroid tumors is the miR-146b and we found that it binds to the 3’-UTR region of thyroid differentiation genes such PAX8 and NIS, leading to impaired protein translation and subsequently a reduction of iodide uptake. Besides, we show that miR-146b and PAX8 regulate each other sharing common target genes, thus highlighting a novel regulatory circuit that govern differentiated phenotype in thyroid tumors. Furthermore, we have shown that the overexpression of miR-146b induces an hyperactivation of the PI3K/AKT pathway, via PTEN suppression, leading to a more aggressive tumoral behavior. In summary, our work described molecular determinants that may be exploited therapeutically to modulate thyroid cell differentiation and iodide uptake for improved treatment of advanced thyroid cancer.

e00004
Adipose tissue expandability, lipotoxicity and the metabolic syndrome.

Antonio Vidal-Puig1*

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00004

Copyright: © 2019 Antonio Vidal-Puig. This is an open-access article distributed under the terms of the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are
credited.

Cite as: Vidal-Puig, A. Adipose tissue expandability, lipotoxicity and the metabolic syndrome. IBJ Plus 2019 S(3):e0004 doi:
10.24217/2531-0151.19v1s3.00004.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

The link between obesity and type 2 diabetes is clear on an epidemiological level, however the mechanism linking
these two common disorders is not well defined. One hypothesis linking obesity to type 2 diabetes is the adipose tissue
expandability hypothesis. The adipose tissue expandability hypothesis states that a failure in the capacity for adipose
tissue expansion, rather than obesity per se is the key factor linking positive energy balance and type 2 diabetes. All
individuals possess a maximum capacity for adipose expansion which is determined by both genetic and environmental
factors. Once the adipose tissue expansion limit is reached, adipose tissue ceases to store energy efficiently and lipids
begin to accumulate in other tissues. Ectopic lipid accumulation in non-adipocyte cells causes lipotoxic insults including
insulin resistance, apoptosis and inflammation. This article discusses the links between adipokines, inflammation,
adipose tissue expandability and lipotoxicity. Finally, we will discuss how considering the concept of allostasis may
enable a better understanding of how diabetes develops and allow the rational design of new anti diabetic treatments.

e00005
Regulation of tissue macrophage functions by LXR nuclear receptor
dependent transcriptional pathways.

Antonio Castrillo1*

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00005

Copyright: © 2019 Antonio Castrillo. This is an open-access article distributed under the terms of the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are
credited.

Cite as: Castrillo, A. Regulation of tissue macrophage functions by LXR nuclear receptor dependent transcriptional pathways. IBJ Plus
2019 S(3):e0005 doi: 10.24217/2531-0151.19v1s3.00005..

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Macrophages are professional phagocytic cells that play crucial roles in immune processes, but they also perform other important functions in the regulation of metabolism and maintenance of tissue homeostasis. A simple definition of the term “macrophage” is currently a challenge due to the continuous advances in the field, with the classification of their multiple origins, the study of their reprogramming capacities during homeostasis and disease. Indeed, exciting research findings have emerged during the last several years, in which macrophage heterogeneity is now believed to be determined by a combination of signals governed by the cellular origin and others coming from the environment. We have studied the transcriptional regulatory circuits that control macrophage behaviour in response to different physiological or pathological situations. A fraction of the transcriptional control of macrophage functions is achieved by the Liver X receptors, LXRα and LXRβ, which are transcription factors that belong to the nuclear hormone receptor superfamily. LXRs are involved in the regulation of cholesterol, fatty acid and phospholipid metabolism. In addition to their role in sterol metabolism, LXRs are important for the immune response against microbial pathogens. LXRα and LXRβ are highly similar in sequence and most of their reported functions are substantially overlapping. In this symposium, we will discuss our recent advances in LXR biology. With a combination of functional assays, expression profiling and ChIPsequencing data, we will shed light on our understanding of LXR´s cell-specific targets and the specific actions of LXRα and LXRβ in macrophage immune responses. Collectively we will discuss the current view of LXR transcription factors in immune processes and their impact in macrophage biological processes.

e00006
Genetic determinants of thyroid function: novel insights and new approaches.

Marco Medici1*

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00006

Copyright: © 2019 Marco Medici. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Medici, M. Genetic determinants of thyroid function: novel insights and new approaches. IBJ Plus 2019 S(3):e0006 doi: 10.24217/2531-0151.19v1s3.00006.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

In the last few years studies have shown that subtle variations in thyroid function, including subclinical thyroid dysfunction, and even variation in thyroid function within the normal range, are associated with morbidity and mortality. It is estimated that 40-65% of the inter-individual variation in serum TSH and FT4 levels is determined by genetic factors. To identify these factors, various linkage and candidate gene studies have been performed in the past, which have identified only a few genes. In the last decade, genome-wide association studies identified >100 genetic variants, while whole exome and whole genome sequencing studies are expected to further clarify the genetic basis of thyroid function in the near future. The identification of these genes has paved the way for various lines of research. Examples of followup in vitro characterization studies of these genes include the identification of SLC17A4 as a novel thyroid hormone transporter and AADAT as a novel thyroid hormone metabolizing enzyme. Furthermore, mendelian randomization studies are expected to clarify whether the observed associations between minor variations in thyroid function and clinical endpoints are causal or not, which is key when considering treatment for these mild variations in thyroid function. Finally, these genetic markers have been associated with normal range thyroid function as well as thyroid dysfunction, thereby possibly serving as predictive markers for the individual thyroid setpoint and for thyroid disease.

e00007
The role of endocrine disruptors in the etiology of diabetes mellitus.

Marco Medici1*

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00007

Copyright: © 2019 Ángel Nadal. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Nadal, A. The role of endocrine disruptors in the etiology of diabetes mellitus. IBJ Plus 2019 S(3):e0007 doi: 10.24217/25310151.19v1s3.00007.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

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e00008
Pathogenesis of autoimmune thyroid diseases: role of cellular immunity.

Mónica Marazuela1*

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00008

Copyright:© 2019 Mónica Marazuela. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Marazuela, M. Pathogenesis of autoimmune thyroid diseases: role of cellular immunity. IBJ Plus 2019 S(3):e0008 doi: 10.24217/2531-0151.19v1s3.00008.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Autoimmune thyroid disease (AITD), which includes Hashimoto’s thyroiditis (HT) and Graves’ disease (GD) are one of the most prevalent organ-specific autoimmune diseases which are characterized by circulating antibodies and lymphocyte infiltration of the thyroid gland. The etiology of AITD is multifactorial due to a complex interplay of specific susceptibility genes and environmental exposures and is characterized by reactivity to self-thyroid antigens due to autoreactive lymphocytes escaping tolerance. Different adhesion molecules, cytokines and chemokines help lymphocyte migration to the thyroid in AITD. Although humoral and cellular mechanisms have been classically considered separately in AITD, recent research suggests a close reciprocal relationship between these two immune pathways. The simple dichotomy Th1/Th2 has been outweighed by the recently described T cells subtypes Treg and Th17 have an essential role in the pathogenesis of AITD. Although several types of CD4 Treg cells (Foxp3, CD69, Tr1), which are able to prevent the appearance of autoimmune diseases, are found in peripheral blood and thyroid tissue from patients with AITD, these cells are apparently unable to put down the autoimmune process. In addition, many reports indicate the involvement of different Th17 cells in AITD. Recently, specific miRNA have been involved in the disregulation of immune mechanisms in AITD probably through differential Treg and Th17 differentiation. Nowadays, both Treg and Th17 cells must be considered as key elements in the pathogenesis of AITD as well as plausible potential targets for the next generation of therapeutic options of this condition.

e00009
Urinary concentration of Iodotyrosines correlates with the severity of iodine deficiency in Dehal1 knockout mice.

Mónica Marazuela1*

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00009

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: González-Guerrero C., Borso M., García-Giménez J., Salas-Lucia F., Alikhani P., Saba A., Zucchi R., Moreno JC.. Urinary concentration of Iodotyrosines correlates with the severity of iodine deficiency in Dehal1 knockout mice. IBJ Plus 2019 S(3):e0009 doi: 10.24217/2531-0151.19v1s3.00009.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Thyroid hormone (TH) synthesis requires iodine, a scarce element whose recycling is mediated by DEHAL1 through deiodination of iodotyrosines MIT and DIT. In humans, DEHAL1 defects lead to severe congenital hypothyroidism (CH) non-detected by neonatal screening programs, which involves the risk of mental retardation in infants. The timing for establishment of this hypothyroidism remains unknown, but environmental iodine deficiency may represent a triggering factor. While urinary loss of iodotyrosines could represent early biomarkers for the disorder, its determination in fluids remains a technical challenge. To measure urinary concentrations of iodotyrosines and test their correlation with urinary iodide in Dehal1 knockout mice under experimentally controlled iodine deficiency.

Dehal (-/-) and wild type (wt) mice were subjected to normal, low, and very low iodine diets (NID, LID, VLID) containing 5.6, 1 and 0.25µgI/day, for 28 days. At d0, d15 and d28, urinary iodine concentration (UIC) was determined by Kolthoff method and MIT, DIT determined in urine using a novel LC/MS-MS protocol.

At baseline and NID, MIT and DIT urinary levels were statistically higher in Dehal1(-/-) (5 and 8ng/ml) than in wt (1 and 0.5ng/ml), respectively (p<0.02). Accordingly, UIC was increased in Dehal1 (-/-) (30mg/dl) compared to wt (15mg/dl) (p<0.05), suggesting that iodide content of iodotyrosines is freed during Kolthoff procedure and adds to the general iodide pool in the urine. This situation persists in time till d28. Under LID, MIT and DIT concentrations remain elevated in Dehal1(-/-) (5.5 and 8ng/ml) compared to wt (0.5 and 0.5ng/ml) (p<0.05) at d15, but start to decrease in both genotypes at d28. UIC was still higher in Dehal1(-/-) (4 mg/dl) versus wt (1mg/dl) (p<0.05) at d15 and d28, but decreased with respect to NID. Under VLID, MIT and DIT levels were still remained significantly higher in Dehal1 (-/-) (4 and 3ng/ ml) compared to wt (0.5 and 0.25ng/ml (p<0.05) at d15. At d28, iodotyrosines lowered to barely detectable levels in wt mice.

Finally, UIC levels in Dehal1(-/-) were intensely decreased (0.5 mg/dl) at d15 and d28, while wt mice showed undetectable levels, reflecting stringent iodine restriction. Our data strongly suggest that the accurate measurement of increased urinary loss of MIT and DIT correlates with UIC increment in Dehal1 deficient mice, which is triggered by iodine deficient intake. Therefore, urinary iodotyrosines may represent pre-clinical biomarkers for early detection and treatment of DEHAL1 deficiency and prevention of mental retardation risks related to late diagnosis of CH.

e00010
3D mapping and in silico predictions of the DEHAL1 enzyme as a tool to discriminate pathogenic mutations from non-functional variants in hypothyroidism.

Jorge García-Giménez1, Ángel González Wong2, Cristian González-Guerrero1*, Ainhoa Iglesias1, Emily Styers1, José Cocho3, Leonardo Pardo2, José C. Moreno1.

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00010

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: García-Giménez J., González Wong A., González-Guerrero C., Iglesias A., Styers E., Cocho J., Pardo L., Moreno JC. 3D mapping and in silico predictions of the DEHAL1 enzyme as a tool to discriminate pathogenic mutations from non-functional variants in hypothyroidism. IBJ Plus 2019 S(3):e0010 doi: 10.24217/2531-0151.19v1s3.00010.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Next Generation Sequencing (NGS) is becoming widely used for genetic diagnosis. While its capacity for detection of human genetic variations (GV) is outstanding, drawbacks is the identification of numerous GV of which functional significance cannot be predicted in silico by computer programs (variants of uncertain significance, VUS). Currently, only sensitive (but also expensive and time-demanding) in vitro cell assays, can trustfully ascertain pathogenicity of VUSs, generally of selected proteins o signalling pathways, seriously hampering efficiency of genetic diagnostic for the clinic.

To apply 3D mapping and in silico predictions as tools to inform pathogenicity of GVs identified in hypothyroid patients with alleged iodotyrosine deiodinase deficiency, as compared with in vitro dehalogenation assays. Nine missense DEHAL1 variants identified in goitrous hypothyroid patients were subjected to both in vitro functional testing and in silico 3D modelling and docking with substrates (MIT, DIT) and cofactor (FMN), using available X-Ray crystallographic information of dimeric DEHAL1. Three (K258N, V265M and R279S) were identified in patients with undisputed diagnosis of iodotyrosine deiodinase deficiency through in vivo 123I-MITdeiodination test. The rest (N108S, A202T, R246Q, L260P and E271K) are harboured by patients with clinical suspicion of DEHAL1 defect. The functional assay involved mutagenesis of DEHAL1 variants in expression vectors, transfection in HEK293 cells, addition of MIT, FMN and NADPH to culture medium and determination of % MIT decrease by LC/MS-MS. The functional assay showed significant decrease of deiodination for K258N (60%) and R279S (58%) versus WT. However, V265M did not, suggesting limitations of our assay to detect less severe mutations. The rest of variants showed normal deiodination with the exception of L260P, which behaved as deleterious (10%).Interestingly, in silico studies on K258N, R279S, V265M and L260P revealed that the amino acids changes had damaging effects on the structural stability, electrostatic properties or interaction with cofactor and substrates (FMN and MIT) on the DEHAL1, and provide a structural explanation for the iodotyrosine deiodinase deficiency (Figure 1). Besides, the peripheral location of N108S, A202T, R246Q, and E271K variants in the model correlates with the benign nature of such changes in the assay (Figure 1). Structural modelling and molecular dynamics is a valuable tool to discriminate pathogenic versus VUS changes in human DEHAL1, showing consistency with the in vitro assay and superior sensitivity to detect some pathogenic changes. An additional advantage of this filtering approach is the prediction of the intrinsic molecular mechanism driving the functional damage of mutations.

e00011
Mice deficient in Mct8 and Dio2 as a new tool to go in depth in the neuropathology of human MCT8 deficiency and to explore new therapeutic strategies.

Soledad Bárez-López1,2, Carmen Grijota-Martínez1,2, Eva Ausó1,3, Mario Fernández-de Frutos1,4, Ana Montero-Pedrazuela1 and Ana Guadaño-Ferraz1,2*

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00011

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Bárez-López S., Grijota-Martínez C., Ausó E., Fernández-de Frutos M., Montero-Pedrazuela A., Guadaño-Ferraz A., Mice deficient in Mct8 and Dio2 as a new tool to go in depth in the neuropathology of human MCT8 deficiency and to explore new therapeutic strategies. IBJ Plus 2019 S(3):e0011 doi: 10.24217/2531-0151.19v1s3.00011.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Allan-Herndon-Dudley syndrome is a rare disease caused by mutations in the specific thyroid hormone transporter monocarboxylate transporter 8 (MCT8). Defects in this transporter cause peripheral hyperthyroidism and profound psychomotor alterations in humans. Mice lacking Mct8 were the first murine model used for the study of this disease; they presented peripheral hyperthyroidism but unfortunately no gross neurological abnormalities. This seems to be due to a compensatory mechanism in brain involving the enzyme deiodinase type 2 (Dio2) which converts T4 into T3. Therefore, a new animal model has been proposed: mice lacking both Mct8 transporter and Dio2 enzyme. Here we have analysed the endocrine and neurologic phenotype of these mice at 3 and 6 months of age. Our results show that these animals present peripheral hyperthyroidism and brain hypothyroidism that seems to be permanent and to vary across regions, being the striatum the most sensitive area. Immunohistochemical studies in brain show alterations compatible with thyroid hormone deficiency. We have also found alterations in motor skills evaluations. All these data support the potential of Mct8/Dio2-deficient mice as a new tool to understand the mechanism underlying the pathophysiology of human Mct8 deficiency and to explore new therapeutic strategies.

e00012
Interplay between TGFβ and the Hippo mediator TAZ leads to Sodium-iodide symporter inhibition.

Celia Fernández-Méndez1, Pilar Santisteban1, 2*

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00012

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Fernández-Méndez C., Santisteban P., Interplay between TGFβ and the Hippo mediator TAZ leads to Sodium-iodide symporter inhibition. IBJ Plus 2019 S(3):e0012 doi: 10.24217/2531-0151.19v1s3.00012. 

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

TAZ is a well-known coactivator downstream of the Hippo signaling pathway. Together with its paralogous YAP and the transcription factors of the TEAD family, TAZ plays a key role in the regulation of differentiation, among other cellular processes. Little is known about the involvement of this signaling pathway in the function of the thyroid gland, although TAZ has been described as a coactivator of Pax8 on the thyroglobulin promoter. Pax8 has a predominant role in thyroid differentiation by regulating the transcription of many crucial genes, such as sodium-iodide symporter (NIS). The aim of this work was to study the role of the Hippo pathway, and particularly its mediator TAZ, in NIS expression, and hence in thyroid differentiation.

By western blot and immunofluorescence, we show TAZ nuclear expression is promoted by TGFβ signalling through its downstream mediator p38. We demonstrated that nuclear TAZ exerts a marked role as NIS repressor, decreasing its expression levels, affecting its presence in the celular membrane and thus impairing iodide uptake. By chromatin immunoprecipitation (ChIP) and luciferase reporter assays, we demonstrated that TAZ strongly suppresses Pax8 activation of the NIS promoter by decreasing its binding to the NIS Upstream Enhancer (NUE). In fact, the absence of TAZ directly increases Pax8 nuclear levels. RNAi assays of other Hippo effectors were performed in order to check their participation in this process. Although YAP and TEAD1 could have additive effects, TAZ seems to be the main requirement for NIS downregulation. TGFβ role in thyroid differentiation is well establish, however here we describe a novel crosstalk with the Hippo Pathway and its mediator TAZ, remarkably involved in the control of the sodium iodide symporter expression.

e00013
Molecular Basis of Deafness Caused by Insulin-like Growth Factor Type 1 Deficiency.

Lourdes Rodríguez-de la Rosa*1,2,3, Jose M. Bermúdez-Muñoz1,2, Marina López1, Almudena Sanz1, Melanie Mertens1, Adelaida M. Celaya1, Jose M. Morales3,4, Miryam Calvino3,4, Luis Lassaletta1,2,3,4, Rafael Cediel1,2,5, Julio Contreras1,2,5, Isabel Varela-Nieto1,2,3 and Silvia Murillo-Cuesta1, 2, 3.

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00013

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Rodríguez-de la Rosa L., Bermúdez-Muñoz JM., López M., Sanz A., Mertens M., Celaya AM., Morales JM., Calvino M., Lassaletta L., Cediel R., Contreras J., Isabel Varela-Nieto I., Murillo-Cuesta S., Molecular Basis of Deafness Caused by Insulin-like Growth Factor Type 1 Deficiency. IBJ Plus 2019 S(3):e0013 doi: 10.24217/2531-0151.19v1s3.00013.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

World Health Organization estimates that around 466 million people worldwide have disabling hearing loss (HL), and 34 million of these are children (1). The most common is sensorineural hearing loss (SNHL), a heterogeneous disorder which is produced mainly by the irreversible loss of sensory cells or neurons in the cochlea. Insulin like growth factor type 1 (IGF-1) is a neurotrophic factor key for the regulation of postnatal cochlear growth and differentiation. Human IGF-1 deficiency is a rare disease (ORPHA73272) associated with growth retardation, microcephaly and SNHL. The mouse model lacking the Igf1 gene reproduces the syndrome and presents dwarfism and SNHL (2). IGF-1 deficiency causes important cellular alterations in the mouse cochlea, such as the early apoptosis of auditory neurons and the deficit in myelination (3). Analysis of downstream signalling in the Igf1-/- cochlea has shown the activation of p38 MAPK pathway, involved in response to stress, whereas ERK1/2 and AKT pathways, which regulate proliferation and survival, are impaired (4).

A transcriptomic study carried out in the Igf1-/- showed the altered expression of the cell cycle modulator Foxm1 and of the myocyte enhancer factor-2 (Mef2), a key factor for cellular differentiation, during inner ear development and early postnatal ages (4). IGF-1 haploinsufficiency has been also associated with growth retardation and HL in human genetic disorders such as Laron syndrome (5). In contrast, the Igf1+/- mouse does not show congenital HL, but adult mice hearing thresholds progressively increase with ageing and mice show increased susceptibility to noise insult (6). Adult Igf1+/- cochleae show unbalanced redox and inflammation biomarkers, as well as altered IGF-1 downstream signalling, which have been proposed as molecular mechanism underlying susceptibility (7).

Mouse models of IGF-1 deficiency constitute a valuable tool to study the molecular bases of deafness and to identify potential targets to develop new HL therapies.

(1) World Health Organization. https://www.who.int/pbd/deafness/estimates/en/. Published 2018.

(2) Cediel et al., Eur J Neurosci. 2006 Jan;23(2):587-90.

(3) Camarero et al., J Neurosci. 2001 Oct 1;21(19):7630-41.

(4) Sanchez-Calderon et al., PLoS One. 2010 Jan 25;5(1):e8699.

(5) Varela-Nieto et al., Ped Endocrinol Rev. 2013; 10(4):460-72.

(6) Rodríguez-de la Rosa et al., Neurobiol Dis. 2012 May;46(2):476-85.

(7) Celaya et al., 2019. Submitted.

e00014
Insulin-like growth factor-1 regulates survival and differentiation in otic cells.

Sara Pulido1,2*, Ángela García-Mato1,2, Blanca Cervantes1,2, Marina López1, Yolanda León1,3, Isabel Varela-Nieto1,2,4, Marta Magariños1,2,3, Lourdes Rodríguez de la Rosa1,2,4

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00014

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Pulido S., García-Mato A., Cervantes B., López M., León Y., Varela-Nieto I., Magariños M., Rodríguez de la Rosa L., Insulin-like growth factor-1 regulates survival and differentiation in otic cells. IBJ Plus 2019 S(3):e0014 doi: 10.24217/2531-0151.19v1s3.00014.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Insulin-like growth factor-1 (IGF-1) is a key element in chicken otic development, and its deficit causes syndromic deafness in mice and men (1). Early steps of inner ear development involve the formation of an autonomous embryonic structure, the otic vesicle or otocyst. Otic vesicle development requires the coordinated actions of apoptosis, proliferation, senescence, cell differentiation and autophagy (2). Autophagy is a catabolic process essential for vertebrate development and homeostasis. We have reported that autophagy is required to facilitate the clearance of apoptotic cells and neuronal differentiation during early otic development (3). In other cellular context, IGF-1 has been shown to regulate autophagy (4). To better understand the molecular actions of IGF-1 and its potential modulation of autophagy in otic cells, we have used the cell line HEI-OC1 (derived from the auditory organ of the transgenic mouse Immortomouse™) and ex vivo cultures of chicken otocysts.

IGF-1 signaling pathway was studied by Western blotting of main IGF-1 targets. Proliferation was measured by incorporation of EdU. Apoptosis was assessed by TUNEL labelling and flow cytometry. Autophagy flux was analyzed by measuring LC3 and p62 relative protein levels. Our results show that IGF-1 promotes cell survival and cell proliferation while reduces cell death in both experimental models. IGF-1 is also playing a role in neuronal differentiation in the otic vesicle, maintaining otic neuroblasts in an undifferentiated and proliferative state. Furthermore IGF-1 actions include the downregulation of autophagy, which is induced in response to starvation and is essential for development and differentiation of otic cells.

(1) Rodríguez-de la Rosa, L., Lassaletta, L., Calvino, M., Murillo-Cuesta, S., & Varela-Nieto, I. (2017). The Role of Insulin-Like Growth Factor 1 in the Progression of Age-Related Hearing Loss. Frontiers in aging neuroscience, 9, 411. doi:10.3389/ fnagi.2017.00411

(2) Varela-Nieto, I., Palmero, I., Magariños, M. (2019). Complementary and distinct roles of autophagy, apoptosis and senescence during early inner ear development. Hearing Research, 376, 86-96. doi: 0.1016/j.heares.2019.01.014

(3) Aburto, M. R., Sánchez-Calderón, H., Hurlé, J. M., Varela-Nieto, I., & Magariños, M. (2012). Early otic development depends on autophagy for apoptotic cell clearance and neural differentiation. Cell death & disease, 3(10), e394. doi:10.1038/cddis.2012.132

(4) Renna, M., Bento, C. F., Fleming, A., Menzies, F. M., Siddiqi, F. H., Ravikumar, B., … Rubinsztein, D. C. (2013). IGF-1 receptor antagonism inhibits autophagy. Human molecular genetics, 22(22), 4528–4544. doi:10.1093/hmg/ddt300

e00015
Ghrelin modulates hippocampal plasticity changing density and morphology of dendritic spines.

Perea Vega ML1, Sánchez M.2, De Barioglio S. R.1

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00015

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Perea Vega ML., Sánchez M., De Barioglio S. R., Ghrelin modulates hippocampal plasticity changing density and morphology of dendritic spines. IBJ Plus 2019 S(3):e0015 doi: 10.24217/2531-0151.19v1s3.00015.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Ghrelin (Gr) is a peptide involved in the modulation of various biological processes. In previous works we have demonstrated that intracerebroventricular or hippocampal (Hp) Gr administration improves memory retention in a dose-dependent manner in rats and mice using different behavioral paradigms that evaluate memory. In addition, we also showed that Gr decreases the threshold for inducing long-term potentiation (LTP, a process underlying memory formation), increases glutamate release and the expression of the NR2B subunit of NMDA receptor.

It is well known that neuronal plasticity correlated with changes on the level of the dendritic spines (DS). It has been demonstrated a correlation between the structure and number of DS and mechanisms related to learning and memory. The density of DS is related to the amount of connectivity between neurons and indicates the numbers of synaptic excitatory inputs onto a particular neuron. DS are largely heterogeneous and can be classified into three classes according the morphology: stubby, thin, and mushroom spines. DS are highly dynamic in the mature nervous system and could be modulated by inputs from the environment in the form of synaptic activity which is central to memory formation. Investigating the molecular mechanisms that underlie structural plasticity of synapses will therefore be crucial not only in understanding the brain functions but should also provide important insights on identifying therapeutic targets for various neurological disorders.

Taking into account the functional effects of Gr previously described, the purpose of this study was to investigate morphological and quantitative changes in DS after Gr administration (2 different doses) in primary hippocampal cultures from rats.

The analysis of the DS showed that Gr increases the total density of DS (+150%) in relation to non-treated cells (control). In relation to the morphology the mushroom type was the most increased type. These results indicate that Gr promotes the formation of new spines as well as the enlargement and stabilization of these spines.

e00016
Identification of primary cilia targeting sequences in HTR6 and SSTR3.

Pablo Barbeito1,3, Paula Moreno1,3, Raquel Martin-Morales1,3, Maria Belen Sierra-Rodero1,3 and Francesc R. Garcia-Gonzalo1-3,*.

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00016

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Barbeito P., Moreno P., Martin-Morales R., Sierra-Rodero MB. and Garcia-Gonzalo FR., Identification of primary cilia targeting sequences in HTR6 and SSTR3. IBJ Plus 2019 S(3):e0016 doi: 10.24217/2531-0151.19v1s3.00016.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Serotonin receptor 6 (Htr6) is a neuronal G protein-coupled receptor (GPCR) involved in multiple aspects of brain pathophysiology. Htr6 localizes to primary cilia, microtubule-based plasma membrane protrusions that act as signaling platforms. Htr6 contains a ciliary targeting sequence (CTS) in its third intracellular loop. This CTS (CTS1) is sufficient to confer cilia localization to non-ciliary proteins such as Htr7, a non-ciliary GPCR, or CD8, a single pass transmembrane protein. Surprisingly, however, CTS1 is not required for Htr6 itself to localize to cilia, suggesting that Htr6 contains another CTS. Here, we have identified a second CTS (CTS2) in the C-terminal tail of Htr6. Like CTS1, the novel CTS2 is sufficient but not necessary for cilia localization. When both CTS1 and CTS2 are mutated, Htr6 no longer accumulates in cilia. Thus, CTS1 and CTS2 act redundantly and are, in combination, both necessary and sufficient for Htr6 ciliary targeting. Furthermore, we have found that the same pattern applies to another ciliary GPCR, namely, Somatostatin receptor 3 (Sstr3). We are now elucidating the mechanisms of action of these novel CTSs.

e00017
Oxidative stress, altered dopamine neurotransmission and increased susceptibility to nigrostriatal neurodegeneration in diabetic mice.

Pérez-Taboada, I1, Moratalla, R2 and Vallejo, M.1, *.

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00017

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Pérez-Taboada I., Moratalla R., Vallejo M., Oxidative stress, altered dopamine neurotransmission and increased susceptibility to nigrostriatal neurodegeneration in diabetic mice. IBJ Plus 2019 S(3):e0017 doi: 10.24217/2531-0151.19v1s3.00017.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Diabetes has been associated with increased risk of neurodegenerative diseases in a number of epidemiological studies, but the underlying mechanisms are unknown. In the case of Parkinson’s disease, this association has been confirmed in an increasing number of reports, although other fail to find a link. Here, using a mouse model of diabetes, we describe oxidative stress and neurotransmission alterations in the nigrostriatal system, and investigate whether diabetes increases the vulnerability of substantia nigra neurons to neurodegeneration. We used C57BL/6 mice rendered diabetic by streptozocin injections. Dopaminergic neuron and oxidative stress markers were determined by RT-qPCR or western blot. Striatal neurotransmitter and related metabolite levels were monitored by HPLC. Oxidative stress in the striatum was determined using commercial tests. [3H]-Dopamine uptake was assessed using striatal synaptosomes. After unilateral striatal injections of a sub-threshold dose of 6OHDA, motor performance was assessed using standard tests and neurodegenerative damage was histologically studied. The expression of genes involved in the regulation of the defense response against oxidative stress, including Nrf2, Keap1, Foxo1, catalase and SOD2 was altered in the substantia nigra of diabetic mice. SOD2 protein levels were unaffected, but catalase levels and activity were reduced. Oxidative stress was confirmed by elevated 4-HNE in the striatum. Diabetes resulted in decreased levels of dopamine and their metabolites in the striatum, without affecting those of noradrenaline or serotonine. Functional changes in neurotransmission were documented by reduced levels of the dopamine transporter DAT, the potassium channel Girk2, and the presynaptic vesicle markers VMAT2 and Syb2. However, dopamine reuptake was not significantly affected in diabetic animals. Finally, histological and motor performance tests revealed increased damage and motor impairment after 6OHDA injections in diabetic mice. Our data indicate that diabetic mice develop striatal oxidative stress and present altered striatal dopaminergic neurotransmission as well as increased vulnerability of nigrostriatal neurons to neurodegenerative damage, providing new insights into the pathophysiology of a possible relationship between diabetes and Parkinson’s disease.

e00018
Effect of melatonin administration on the 24-hour variations of prolactin secretion in bilaterally gangliectomized adult male rats.

Diego Fajardo-Puig1, Pilar Fernández-Mateos2,3, Vanesa Jiménez-Ortega1,3, Pilar Cano-Barquilla1,3, Juliana Pérez-Miguelsanz3,4 and Esquifino AI1,3*.

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00018

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Fajardo-Puig D., Fernández-Mateos P., Jiménez-Ortega V., Cano-Barquilla P., Pérez-Miguelsanz J., Esquifino AI., Effect of melatonin administration on the 24-hour variations of prolactin secretion in bilaterally gangliectomized adult male rats. IBJ Plus 2019 S(3):e0018 doi: 10.24217/2531-0151.19v1s3.00018.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: The superior cervical ganglion (SCG) is responsible of innervating intracranial which includes the hypothalamus or pineal gland. Its surgical removal leaves these areas without sympathetic innervation. Unilateral denervation does not generate important effects on noradrenergic function because the non-operated side can partially compensate its loss. However, if the extirpation is bilateral (SCGx), it does generate alterations in pituitary and pineal hormone secre-tion. The bilateral SCGx generates a decrease in the production of melatonin by the pineal gland due to the decreased noradrenaline arrival to the gland, which is the key role for its synthesis. This lack of melatonin leads to changes in the daily patterns of secretion of hormones like prolactin, ACTH or GH. Also neurotransmitters involved in pituitary hormones such as serotonin, GABA or dopamine may be changed as well. With all data mentioned above, this work was designed to analyse whether the administration of melatonin in vivo is able to reverse the alterations observed in the hypothalamic-pituitary axis in SCGx male rats in autumn.

Material and methods: Eight 60 days old male rats of the Wistar strain, SCGx, per group were used at 09:00, 13:00, 17:00, 21:00, 01:00 and 05:00. 30μg of melatonin were administered in 200μL of vehicle one hour before lights switch off for 11 days with the subsequent slaughter of the animals by decapitation without previous anaesthesia, to avoid stress conditions that may interfere with the prolactin secretory mechanism, at the different times studied throughout the day. Control and SCGx rats were administered either with the vehicle or melatonin. Plasma prolactin levels were measured by radioimmunoassay and dopamine was by HPLC, both techniques in routine in our laboratory.

Results and conclusions: Bilateral gangliectomy effectively reduced both noradrenalin arrival to the pineal gland together with a marked decrease in melatonin secretion (see fig.). Besides, it did not modify the 24-h pattern of prolactin. However, when melatonin is exogenous replaced, a phase advance in the secretory rhythm of prolactin occurs, showing a plat-eau between 5:00 and 9:00 p.m., that resembles the circadian rhythm of summer for this hormone. Melatonin induced an increase in dopamine content in median eminence (p<0.05) with respect to control SCGx rats, with a plateau between 01:00 and 05:00 similar to prolactin. In addition, there is an interaction between the factors time, surgery and administra-tion of melatonin for both dopamine (p <0.0001) and prolactin (p <0.0115). It may be concluded that the presence of the autonomic innervation is a key factor for melatonin effect at the hypothalamus pituitary axis.

e00019
Role of the Prolactin Releasing Peptide (PrRP) in the neuroendocrine regulation of thermogenesis.

Diego Fajardo Puig1*, David Bolado López de Andújar2*, Lorena Caballero Sánchez3, Francisco Javier Martínez Picabea4 .

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00019

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Fajardo Puig D., López de Andújar DB., Caballero Sánchez L., Martínez Picabea FJ., Role of the Prolactin Releasing Peptide
(PrRP) in the neuroendocrine regulation of thermogenesis. IBJ Plus 2019 S(3):e0019 doi: 10.24217/2531-0151.19v1s3.00019.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

The prolactin-releasing peptide (PrRP) is involved in the regulation of appetite and regulates prolactin secretion. This
molecule was discovered as a ligand of the orphan GRP10 / hGR3 receptors in man and UHR1 in the rat, in the year 1998
and it was very important to identify new functions of prolactin.

This molecule is synthesized in the ventromedial, ventrolateral hypothalamus and in the nucleus of the solitary tract
and is involved in the regulation of energy expenditure. PrPR interacts with the hypothalamic-hypophysis-adrenal
axis exerting a similar effect on energy metabolism, producing an anorectic effect. Likewise, PrRP also interacts with
melanocortin alfa and beta (MSH), reducing the intake. The PrRP is directly related to an increase in energy expenditure,
through an increase in oxygen consumption, producing a decrease in weight or preventing its increase. The increase in
energy expenditure would be related to the increase in the degree of expression of UCP1 in brown adipose tissue (BAT).
In fact, as already known, the intra-cerebro-ventricular administration (ICV) of PrRP at a concentration of 4 nmolar
generated a hyperthermia that correlates with the increased expression of the UCP1 gene. Likewise, the dietary intake
of rats treated with PrRP is significantly reduced, which would confirm its anorexigenic effect, its maximum effects being
in the first 24 hours after its administration, producing a gradual refractoriness to the administration of this peptide. This
desensitization seems to be mediated by the neuroendocrine changes produced by PrRP on cholecystokinin and leptin.
It is also possible that, due to continuous injections to administer PrPR, the mouse raised its stress level noticeably and
any minimal modification generated by the hormone could have been masked. In addition, in situations of stress, the
decrease in secretion of PrRP from the nucleus of the solitary tract attenuates ACTH / corticosterone, prolactin and
oxytocin responses due to a lack of interaction with the amygdala and, therefore, its anorexic effect. This effect could be
potentiated because its stimulating effect on the hypothalamic-pituitary-adrenal axis is attenuated, since, as previously
mentioned, ACTH produces an anorexigenic effect. It should be noted that, in addition to the aforementioned hormones,
the existence of mechanisms of interaction between leptin and PrRP has been demonstrated. This is due to the fact that
both leptin and PrRP produce an anorectic effect and are involved in the regulation of responses to cold and heat. The
blocking of the PrRP gene annuls the thermoregulatory effect of leptin, so its presence is essential for leptin to exert its
thermoregulatory role. In these effects, the interactions between the mechanisms of secretion of cholecystokinin, leptin
and PrRP for the control of intake and thermoregulation must be taken into account.

e00020
Investigating the role of molting hormones in adult stages.

Gil-Marti B1, Barredo CG.2, Rodríguez-Navas C.3, Martín F.A. 4*.

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00020

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Gil-Marti B., Barredo CG., Rodríguez-Navas C., Martín FA., Investigating the role of molting hormones in adult stages. IBJ Plus
2019 S(3):e0020 doi: 10.24217/2531-0151.19v1s3.00020.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Juvenile forms of insects express the non-steroid Prothoracicotropic Hormone (PTTH) (1). As documented in Drosophila,
the established function takes place during metamorphosis triggering the molt throughout larval and pupal stages (2).
However, the expression of both ptth and its receptor torso in the adult brain suggests additional functions beyond
the larval stage. Indeed, the observation that PTTH immune-signal is detected in the antennal lobe indicates that this
hormone could play a role in olfaction. Thus, our biological question in this project is: does Drosophila need PTTH to
smell?
Using individual Y-maze and collective T-maze assays, we analyze the response of control and ptth mutant flies to
different odors. Current results indicate that adult PTTH modulates olfaction. In particular, it modulates OR (typical from
insects) and IR (present in all protostomes) olfactory subsystems (3).
Further odorants and ages are now under analysis. We hypothesize that this study will provide a novel insight to
neurohormone mediated olfaction, and its evolution given the functional homology between fly PTTH and vertebrate
Gonadotropin Release Hormone (GnRH).

e00021
Uncovering sexual dimorphism in type 2 diabetes by redox proteomics.

María Gómez-Serrano1,2, Emilio Camafeita1,2, Jesús Vázquez1,2, Belén Peral3*.

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00021

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Gómez-Serrano M., Camafeita E., Vázquez J., Belén Peral B., Uncovering sexual dimorphism in type 2 diabetes by redox
proteomics. IBJ Plus 2019 S(3):e0021 doi: 10.24217/2531-0151.19v1s3.00021.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

The prevalence of type 2 diabetes (T2DM) is rising globally due to, among other factors, obesity, physical inactivity, and
aging superimposed on a genetic predisposition. Despite that the NIH recently highlighted the importance of sexual
dimorphism and dictated inclusion of both sexes in clinical trials and basic research, little is known about the role of
gender in adipose tissue biology.

We have recently validated that antioxidant response is one of the gender-specific hallmarks of sexual dimorphism in
adipose tissue. Since mitochondria is recognized as the main source of oxidative stress, in this work we aimed to better
understand the impact of gender on adipocyte mitochondria by quantitating not only protein abundance changes, but
also redox alterations undergone by Cys protein residues.

Mitochondrial samples were obtained from adipocytes previously isolated from visceral adipose tissue biopsies
collected from morbidly obese patients (BMI > 35 kg/m2) suffering T2DM who were submitted for bariatric surgery.
Protein abundance and redox differences between women and men suffering T2DM were assessed following a highthroughput
approach encompassing differential alkylation of reduced and reversibly oxidized Cys (FASILOX), isobaric
labelling (iTRAQ) and LC-MS/MS.
LC-MS/MS analysis allowed the identification of 15,763 peptides at 1% FDR, corresponding to 2,895 proteins in the
mitochondrial extracts. Of note, 754 proteins were annotated in the current version of Human MitoCarta v.2 (68%
coverage). FASILOX technology allowed the identification of 277 oxidized and 1990 reduced Cys-containing peptides.

Cys oxidation was globally increased in diabetic men compared to women. Enrichment analyses showed that oxidation
was focus on the oxidative phosphorylation (OXPHOS) system, especially in Complex I (p <0.001, FDR 1%) which showed
diminished abundance in men as compared to women.
In conclusion, male patients displayed higher oxidation than females in adipocyte mitochondria under T2DM, most likely
revealing a substantial oxidative stress on OXPHOS machinery.

These results provide the first evidence for a decreased
efficiency in the antioxidant response of diabetic men as compared to women. The identification of gender-matched
oxidation targets in T2DM may help to improve the therapeutic approaches currently available for these patients.

e00022
The new role of RXR in podocytes: implications in renal disease
associated to obesity and Type 2 diabetes.

Almudena García-Carrasco1, Borja Lanzón-García1, Adriana Izquierdo-Lahuerta1, Daniel Horrillo-Novero1, Mercedes Ricote2 , Manuel Ros-Pérez1
Gema Medina-Gómez1*.

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00022

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: García-Carrasco A., Lanzón-García B., Izquierdo-Lahuerta A., Horrillo-Novero D., Ricote M., Manuel Ros-Pérez M., Gema
Medina-Gómez G. The new role of RXR in podocytes: implications in renal disease associated to obesity and Type 2 diabetes. IBJ Plus
2019 S(3):e0022 doi: 10.24217/2531-0151.19v1s3.00022.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Background: Obese patients show other comorbidities such as hypertension, heart disease, dyslipidemia, type 2 diabetes
and renal disease. Recently, evidences suggest that renal lipid accumulation leads to glomerular damage and more
specifically, whether this accumulation produces podocyte dysfunction. Nuclear receptors (NRs) are ligand-dependent
transcription factors, which can activate or suppress the transcription of target genes in lipid metabolism and inflammation.
We have shown that the mice with specific deletion of isoform gamma 2 of Peroxisome proliferator-activated
receptor (PPARƴ2KO) in an obese background, exhibits IR, an alteration of lipid metabolism and glomeruli damage associated
with significantly increased inflammation. RXRs are ligand-dependent NRs that play an important role as a heterodimeric
partner for other NRs, including PPARs. The aim of this study was to analyze the role of RXR isoforms in the
integrity of the podocyte and the involvement in the maintenance of Glomerular Filtration Barrier.

Methods: Mice with specific deletion of RXRα and β in podocyte (podRXRKO) were generated. At 3 months of age, serum
and urine were collected; kidneys were removed for histological and molecular studies.

Results: Lack of RXR induced a reduction of the glomerulus size associated to the presence of fibrosis. No changes in
blood pressure were shown. Podocyte effacement was observed in 3-month-old males. Moreover, females also showed
a decrease in the urine volume.

Conclusion: This study suggests a crucial role of RXR in the integrity of the podocyte and its possible implication as a
target in renal disease.

e00023
Bioavailability of the multitasking hormone melatonin from
lentil sprouts and its role on plasmatic antioxidant status in rats..

Miguel Rebollo-Hernanz1,2, Yolanda Aguilera1,2, Teresa Herrera2, L. Tábata Cayuelas2, Pilar Rodríguez-Rodríguez3, David Ramiro3, Ángel L. López de
Pablo3, Silvia M. Arribas3, María A. Martín-Cabrejas*1,2.

Miguel Rebollo-Hernanz1,2, Yolanda Aguilera1,2, Teresa Herrera2, L. Tábata Cayuelas2, Pilar Rodríguez-Rodríguez3, David Ramiro3, Ángel L. López de
Pablo3, Silvia M. Arribas3, María A. Martín-Cabrejas*1,2.

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00023

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Rebollo-Hernanz M., Aguilera Y., Herrera T., Tábata Cayuelas L., Rodríguez-Rodríguez P., Ramiro D., López de Pablo A.L., Arribas
S.M., Martín-Cabrejas M.A., Bioavailability of the multitasking hormone melatonin from lentil sprouts and its role on plasmatic
antioxidant status in rats. IBJ Plus 2019 S(3):e0023 doi: 10.24217/2531-0151.19v1s3.00023.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Melatonin is an endogenous antioxidant neurohormone present in the organism in decreasing concentrations with
aging. These low plasmatic concentrations are associated with diverse chronic diseases, including hypertension, type 2
diabetes, and obesity. Plant foods could serve as sources of this indolamine, being legumes a sustainable and healthy
option for this purpose. Germination of legumes enhances melatonin concentration, which renders sprouts as a suitable
food source of this hormone.

Since little information on melatonin bioavailability from food sources has been generated to date, we aimed to evaluate
the effect lentil sprouts intake on the plasmatic levels of melatonin and metabolically related compounds (plasmatic
serotonin and urinary 6-sulfatoxymelatonin), total phenolic compounds, and plasmatic antioxidant status and compare
it with synthetic melatonin bioavailability.

Male young Sprague Dawley rats (n = 10 per group) were used. Pharmacokinetic profile of melatonin was investigated
after oral administration of lentil sprouts at a dose 50 μg of melatonin/kg of body weight. Blood samples were drawn
before and sequentially within the 4 h period after ingestion of the extract. Likewise, blood and urine samples were
obtained from rats before four experimental conditions (Control: ad libitum feeding; Fasting: 12h fasting; Lentil Sprouts:
12h fasting + gavage administration of lentil sprouts extract; and MEL: 12h fasting + gavage administration of melatonin
solution). Melatonin and its related biomarkers were determined by ELISA; antioxidant capacity of the plasma was
measured using colorimetric/fluorimetric techniques (FRAP and ORAC).

Melatonin showed maximum concentration 1.5 h (Tmax) after lentil sprouts administration (Cmax = 45.4 pg/mL);
mean residence time was 3.2 h. Moreover, variations of the plasmatic antioxidant capacity measure by FRAP followed
a similar behavior. The plasmatic melatonin concentration increased after lentil sprouts intake (70%, p < 0.05) respect
to the control, 1.2-fold more than after synthetic melatonin ingestion. These increments correlated with the urinary
6-sulfatoxymelatonin content (p < 0.05), a key biomarker of plasmatic melatonin, as well as with plasmatic serotonin
concentration (p < 0.05). Nonetheless, the phenolic compounds content did not exhibit any significant variation.
Plasmatic antioxidant status evidenced increases in the antioxidant capacity upon both lentil sprouts and synthetic
melatonin administration.

For the first time, we investigated the bioavailability of melatonin from lentil sprouts and its role plasmatic antioxidant
status. From results, we conclude that lentils sprouts are a good source of bioavailable dietary melatonin which intake
could attenuate plasmatic oxidative stress and protect the organism against aging among other potential health benefits.

e00024
Phenolic compounds from cocoa shell prevent inflammation,
mitochondrial dysfunction, and insulin resistance via activation
of insulin/PI3K/AKT signaling pathways in 3T3-L1 adipocytes.

Miguel Rebollo-Hernanz1,2,3, Qiaozhi Zhang3,4, Yolanda Aguilera1,2, Elvira González de Mejía3, María A. Martín-Cabrejas*1,2.

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00024

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Rebollo-Hernanz M., Zhang Q., Aguilera Y., González de Mejía E., Martín-Cabrejas M.A., Phenolic compounds from cocoa shell
prevent inflammation, mitochondrial dysfunction, and insulin resistance via activation of insulin/PI3K/AKT signaling pathways in 3T3-
L1 adipocytes. IBJ Plus 2019 S(3):e0024 doi: 10.24217/2531-0151.19v1s3.00024.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

The incidence of these metabolic disorders is increasing worldwide primarily due to poor lifestyle habits. Obesity is caused
by the imbalance between energy intake and expenditure and entails the storage of excess amounts of triglycerides,
associated with the production of adipokines involved in inflammation. Likewise, the accumulation of macrophages in
the adipose tissue results in the secretion of a variety of pro-inflammatory cytokines and chemokines. This inflammation
triggers mitochondrial dysfunction and insulin resistance.
We aimed to investigate the effect of an aqueous extract from cocoa shell (CAE) and its main phenolic compounds
(protocatechuic acid, procyanidin B2, and epicatechin) on the prevention of obesity-induced mitochondrial dysfunction
and insulin resistance, targeting the inflammatory crosstalk between macrophages and adipocytes in vitro.

CAE and its main phenolic compounds were tested in cell culture of RAW264.7 macrophages and 3T3-L1 mature
adipocytes. Macrophages were stimulated with lipopolysaccharide (LPS, 1 μg/mL) and the media was recovered as
macrophages-conditioned media (CM). Adipocytes were treated with CM in the presence/absence of CAE and pure
phenolics. Biomarkers for inflammation, mitochondrial dysfunction, and insulin resistance were determined after 24 h
co-treatment with CAE or pure phenolics and CM using chemical and immunochemical techniques.
The inflammatory crosstalk created by stimulating adipocytes with CM was arrested by the treatment with CAE and
pure phenolics. CAE reduced tumor necrosis factor-α (TNF-α, 67%, p < 0.05) and highly stimulate adiponectin secretion
(12.3-fold, p < 0.05). Mitochondrial dysfunction, measured through reactive oxygen species production, mitochondrial
content, and activity, was attenuated in CM-treated adipocytes via the up-regulation of peroxisome proliferator-activated
receptor gamma coactivator 1-α expression (PGC1-α, 46%, p < 0.05). Significant increases in insulin receptor (INSR, 9-fold,
p < 0.05), phosphoinositide 3-kinase (PI3K, 3-fold, p < 0.05), protein kinase B (AKT, 4-fold, p < 0.05) phosphorylation and
a diminished insulin receptor substrate 1 serine phosphorylation promoted glucose uptake (IRS-1, 34%, p < 0.05) and
glucose transporter 4 translocation (GLUT4, 14-fold, p < 0.05) in 3T3-L1 adipocytes under CM-triggered inflammatory
conditions.

Cocoa shell aqueous extract and its main phenolic compounds blocked macrophages-adipocytes inflammatory interplay
preventing mitochondrial dysfunction and insulin resistance via activation of insulin/PI3K/AKT signaling pathways in 3T3-
L1 adipocytes. For the first time, CAE phenolics exhibited a positive effect on inflammatory obesity-related disorders.

e00025
PTP1B deficiency protects mice against metabolic
dysfunction in glucose homeostasis upon Olanzapine
intraperitoneal treatment.

Vítor Ferreira1,2*, Patrícia Rada1,2, Diana Grajales1,2, Irma García-Martinez1,2, Ángela M. Valverde1,2.

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00025

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Ferreira V., Rada P., Grajales D., García-Martinez I., Valverde A.M., PTP1B deficiency protects mice against metabolic dysfunction
in glucose homeostasis upon Olanzapine intraperitoneal treatment. IBJ Plus 2019 S(3):e0025 doi: 10.24217/2531-0151.19v1s3.00025.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

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.

e00026
TGFβ3 as a novel target in obesity-associated kidney failure.

Elia Escasany1, Adriana Izquiero-Lahuerta1, Patricia Corrales1, Almudena García-Carrasco1, Lucía Torres1, Yamila López1,2, Paul Petrus3, Estela
Maldonado2, Concha Martínez Álvarez2, Manuel Ros1, Peter Arner3, Miakel Ryden3, Gema Medina-Gómez1.

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00026

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Escasany E., Izquiero-Lahuerta A., Corrales P., García-Carrasco A., Torres L., López Y., Petrus P., Maldonado E., Martínez Álvarez
C., Ros M., Arner P., Ryden M., Medina-Gómez G., TGFβ3 as a novel target in obesity-associated kidney failure. IBJ Plus 2019 S(3):e0026
doi: 10.24217/2531-0151.19v1s3.00026

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Obesity is associated with development of lipotoxicity in metabolically important organs such as the kidney leading to
apoptosis and dedifferentiation, which contributes to fibrosis and chronic kidney diseases progression. Glomerulosclerosis
and tubulointerstitial fibrosis are the reversible common final pathological features of chronic kidney diseases (CKD),
regarding the aetiology making it an appealing target of study for both finding new biomarkers and developing
therapeutic strategies for CKD. Transforming growth factor β (TGFβ) is involved in differentiation, apoptosis and fibrosis.
TGFβ1 has long been associated with renal fibrosis; however, the role for TGFβ3 is not so clear.
Our aim was to unravel the role of TGFβ3 in the pathophysiology of the kidney. We have used in vivo and in vitro lipotoxic
models. Regarding the in vitro model, we have used heterozygous (HZ) TGFβ3 mice, due to lethality of the homozygous
TGFβ3 mutant mice. These mice were subjected to either a control (CD) or a high fat diet (HFD) and sacrificed at 16
weeks of age.
In HZ mice, lower levels of TGFβ3 did not affect body weight; however, under HFD, HZ mice showed glucose intolerance
but no insulin resistance, with higher basal levels of glucose and insulin. Despite similar renal hypertrophy index,
glomerular area or blood pressure, HZ mice under CD already showed decreased 24h urine volume and a tendency
towards higher albumin/creatinine ratio under both diets, which was aggravated under HFD.
Histological analysis (PAS and α-SMA IHC) showed renal fibrosis in HZ mice under both diets confirmed by increased
fibronectin, osteopontin and N-cadherin and decreased E-cadherin and β-catenin mRNA levels. Partial lack of TGFβ3 lead
to higher oxidative stress (ROS), thickening of the glomerular basement membrane (GBM), foot process effacement and
loss of podocytes, as observed by electronic microscopy and flow cytometry.
Kidneys of HZ mice showed altered expression of lipid metabolism genes, with an increase in lipid synthesis genes (ACC
and SREBP1) and a decrease in β-oxidation genes (PPARα, CPT1, PGC1α and PGC1β). This translated into an accumulation
of lipids inside the kidney as observed histologically (Oil Red) and by flow cytometry experiments (Bodipy staining).
Altogether, these data suggest a tight link between TGFβ3 and lipid metabolism at the renal levels as well as a possible
important role of TGFβ3 as anti-fibrotic in the development of renal fibrosis under lipotoxic conditions.

e00027
Initial age-associated metabolic alterations in white and brown fat depots: effect of long-term caloric restriction.

Patricia Corrales1, Yurena Vivas1, Adriana Izquierdo-Lahuerta1, Daniel Horrillo1, Patricia Seoane-Collazo2, Ismael Velasco1, Lucia Torres1, Yamila
Lopez1, Carmen Martínez1, Miguel López2, Manuel Ros1, Maria Jesús Obregón3, Gema Medina-Gómez1.

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00027

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Corrales P., Vivas Y., Izquierdo-Lahuerta A., Horrillo D., Seoane-Collazo P., Velasco I., Torres L., Lopez Y., Martínez C., López M.,
Ros M., Obregón MJ., Medina-Gómez G., Initial age-associated metabolic alterations in white and brown fat depots: effect of longterm
caloric restriction. IBJ Plus 2019 S(3):e0027 doi: 10.24217/2531-0151.19v1s3.00027.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Changes in the body composition such as high adiposity and sarcopeny occur during aging. These changes are usually
associated to metabolic alterations like insulin resistance, type 2 diabetes and metabolic syndrome. Adipose tissue is not
homogeneous and dysfunction of white adipose tissue (WAT) and brown adipose tissue (BAT) can develop differently
during aging. Our aim is to study different WAT and BAT depots at middle-age to detect the initial signals of alteration.
Moreover, we have studied the effect of the long-term caloric restriction (CR) and its benefits.

Sv129 mice 3 and 12-month-old mice fed ad libitum and 12-month-old mice fed with a 20% CR (3m, 12m and 12mCR,
respectively) have been used. RT-PCR has been employed to study lipid metabolism gene expression in eWAT, scWAT and
BAT. Moreover, circulating serum and BAT thyroid hormones levels were measured by radioimmunoassay (RIA).

12m animals showed insulin resistance and lower adiponectin levels in plasma compared with 3m. Expression of insulin
pathway (Glut4, Irs1), lipogenesis (Fas, Scd1) and lipolysis (Hsl, Atgl, Pparα) genes was decreased in scWAT of 12m
animals, but not in eWAT. Furthermore, UCP-1 expression was significantly lower in scWAT of aged mice compared
with younger mice, suggesting a decrease in beige adipose tissue associated to aging. On the other hand, we found
decreased expression of some BAT-selective genes12m animals such as Ucp1, βAr. CR recovered the expression levels to
those of 3m mice. Thyroxine (T4) in plasma did not reach significance among middle-aged and young animals, however
triiodothyronine (T3) levels were lower in 12m than 3m and 12mCR mice. Dio2 expression, T4 and T3 concentrations in
BAT followed a similar pattern, a decrease in 12m mice and an increase in CR mice.

Although eWAT has been described as the most dangerous tissue from the metabolic point of view, our data could
suggest an earlier metabolic alteration in scWAT than in eWAT. Our findings prompt that CR could revert the initial agerelated
alterations in scWAT. In agreement, we point to alteration of thyroid axis status with age is an important factor
contributing to BAT dysfunction at middle-age, which can be ameliorated by CR.

e00028
Microsomal Prostaglandin E Synthase-1 (mPGES-1) mediates the development of metabolic and cardiovascular alterations associated to obesity.

Ballesteros-Martinez C1, Rodrigues-Diez R1,2,3, Gonzalez-Amor M1, Salaices M1,2,3, Briones AM1,2,3.

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00028

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Ballesteros-Martinez C., Rodrigues-Diez R., Gonzalez-Amor M., Salaices M., Briones AM., Microsomal Prostaglandin E
Synthase-1 (mPGES-1) mediates the development of metabolic and cardiovascular alterations associated to obesity. IBJ Plus 2019
S(3):e0028 doi: 10.24217/2531-0151.19v1s3.00028.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: Obesity is linked to chronic low-grade inflammation. Adipose tissue responds to metabolic signals, being
also metabolically active secreting peptides, hormones and adipokines that, under obesity situation, participate in the
development of cardiovascular and metabolic alterations such as endothelial dysfunction, arterial stiffness and insulin
resistance. Microsomal prostaglandin E Synthase-1 (mPGES-1) is an inducible isomerase responsible for the production
of prostaglandin E2 (PGE2) under inflammatory conditions. PGE2 is a key lipid mediator implicated in the regulation of
adipose tissue homeostasis. It is also involved in vascular remodeling in different inflammatory pathologies.

Objective: To evaluate the role of mPGES-1 in the development of obesity and in the metabolic and cardiovascular
alterations associated.

Methods: We have developed a model of high fat diet (HFD, 60% fat)-induced obesity in mPGES-1-/- and their control
littermates mPGES-1+/+ mice. Body weight was measured twice a week and organs and adipose tissue weights were
measured at the end of the experiment. The glycemic profile was studied by the glucose tolerance test (GTT) and insulin
tolerance test (ITT). Arterial pressure was measured by tail-cuff plethysmography. Aortic vascular function and structural
and mechanical properties of mesenteric resistance arteries where evaluated by isometric and perfusion myographs
respectively. Histological analysis was also performed. Changes in mRNA expression were studied by q-RT- PCR.

Results: Our results show that mPGES-1-/- mice fed with a HFD are protected against body weight gain and present
reduced adiposity with less grade of macrophage infiltration. In addition, mPGES-1 deficient mice have better glycemic
profile compared to wild type mice. At cardiovascular level, mPGES-1+/+ but not mPGES-1-/- mice on HFD, develop an
increase in the size of cardiomyocytes, which results in cardiac hypertrophy, show aortic endothelial dysfunction and
vascular insulin resistance, and show structural alterations in resistance arteries.

Conclusion: Our data suggest that the lack of mPGES-1 protects against some of the cardiovascular and metabolic
alterations associated to obesity.

e00029
Lack of improvement in sperm quality and changes in reproductive hormones in obese mice and patients males after obesity surgery and diet.

Casado ME1,2, Calderón B2,4, Huerta L1, Botella-Carretero JI2,3 and Martín-Hidalgo A1,2.

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00029

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Casado ME., Calderón B., Huerta L., Botella-Carretero JI. and Martín-Hidalgo A., Lack of improvement in sperm quality and
changes in reproductive hormones in obese mice and patients males after obesity surgery and diet. IBJ Plus 2019 S(3):e0029 doi:
10.24217/2531-0151.19v1s3.00029.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Obesity in men is associated with a decrease in sperm concentration, motility, alterations in sperm morphology, chromatin
integrity and hormonal profile that leads to infertility. Even though obesity surgery normalizes circulating testosterone
concentrations in males with obesity-associated secondary hypogonadism, its impact on spermatogenesis remains
controversial. We aimed to evaluate sperm characteristics and changes in reproductive hormones in obese men after
bariatric surgery and obese mice after low fat diet.

Methods: Twenty severely obese men (body mass index (BMI) ≥ 35 kg/m2) were evaluated before and 2 years after bariatric
surgery. We assayed insulin, leptin, kisspeptin, and inhibin B in the serum of the patients. The obese mice model
included the following experimental groups: 1) LFD 10 weeks; 2) HFD 10 weeks; 3) LFD 18 weeks; 4) HFD 18 weeks; 5)
HFD 10 weeks and LFD 8 weeks. The seminal fluid was get from epididymis for spermatozoa counts and motility. We
studied the mice at 10 and 18 weeks after the different diets. We assayed glucose, insulin and testosterone in plasma.
Homeostasis model assessment of insulin resistance (HOMA-IR) was estimated. We used World Health Organization
reference values for sperm analysis.

Results: Before surgery, serum total testosterone, calculated free testosterone, leptin, glucose and fasting insulin, HOMA-
IR, increased in obese patients. After surgery, serum total testosterone, calculated free testosterone, inhibin B, and
kisspeptin increased, whereas fasting insulin, HOMA-IR, and leptin concentrations decreased in the patients. Despite
these improvements, sperm volume showed a small decrease after surgery, while the rest of sperm characteristics
remained mostly unchanged. Abnormal sperm concentration persisted in 60% of the obese patients. In mice, plasma
glucose and insulin increased in HFD compared with LFD at 10 and 18 weeks. These values decreased in the group of
HFD+LFD at 18 weeks. The HFD mice have insulin resistance compared with LFD mice. Plasma testosterone decreased in
HFD compared to LFD at 10 and 18 weeks; and remained low in HFD+LFD. There was not reversion of plasma testosterone
levels with the change of the diet. Spermatozoa concentration decreased in HFD compared to LFD mice, and there
was not reversion of sperm concentration after the change of HFD to LFD at 18 weeks.

Conclusions: Sperm characteristics may not improve after bariatric surgery despite the beneficial changes of reproductive
hormones in obese patients. There is not improvement in sperm quality and testosterone in obese mice after diet.

e00030
The short-term dietary impact on the central and circulating IGF systems in rats.

Santiago Guerra-Cantera1,2,3*, Francisca Díaz2,3, Purificación Ros1,4, Alejandra Freire-Regatillo1,2,3, Laura M. Frago1,2,3, Vicente Barrios2,3, Jesús
Argente1,2,3,5, Julie Chowen2,3,5.

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00030

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Guerra-Cantera S., Díaz F., Ros P., Freire-Regatillo A., Frago L.M., Barrios V., Argente J., Chowen J., The short-term dietary
impact on the central and circulating IGF systems in rats. IBJ Plus 2019 S(3):e0030 doi: 10.24217/2531-0151.19v1s3.00030.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

The insulin-like growth factor system (IGF) exerts a wide range of functions in the organism, participating in linear bone
growth, metabolism and neuroprotection. Poor nutrition and obesity can alter this system; however, little is known
regarding the effects of short-term dietary changes on the IGF axis. Our aim was to determine if a 1-week high-fat diet
(HFD) or a low-fat diet (LFD) modifies the central and peripheral IGF systems.

Adult male and female Wistar rats were given a HFD (60% fat, 8.9% sucrose, 5.1 kcal/g), LFD (10% fat, 33.1% sucrose, 3.76
kcal/g) or standard rodent chow (3.1% fat, 0.9% sucrose, 3.41 kcal/g) for 1 week. Serum hormone levels were measured
by ELISA and tissue gene expression by RT-qPCR. Hepatic glucose-6-phosphate dehydrogenase (G6PDH) activity was
evaluated (Sigma-Aldrich) and malic enzyme activity was determined as described by Geer et al (1).
HFD increased body weight (p<0.05) exclusively in males. In serum, males had higher levels of free and total IGF1,
IGF-binding protein (IGFBP) 3, IGFBP5, insulin, leptin and triglycerides compared to females (p<0.001) and pregnancyassociated
plasma protein-A2 (PAPP-A2) was higher in females (p<0.05), with no dietary effect on these parameters.

In females, serum IGF2 levels increased on the LFD (p<0.01). Hepatic IGF1 and IGFBP3 mRNA levels were affected by
both sex and diet (p<0.01) and IGF2 mRNA levels were increased in response to HFD, reaching significance in females
(p<0.01). Hepatic IGFBP2 mRNA levels were increased in response to HFD in both sexes (p<0.001) with sex differences,
as levels were higher in females than males (p<0.001). Liver G6PDH activity was higher on the LFD compared to chow in
both sexes (p<0.001), whereas in females HFD also increased G6PDH activity. Liver malic enzyme activity was increased
in response to LFD compared to chow in both sexes (p<0.001).
Hypothalamic mRNA levels of IGF2 (p<0.01) and IGFBP2 (p<0.001) increased after LFD intake, but only in males. No
effects were found on the remaining members of the IGF system. In males, LFD also increased the mRNA levels of
neuropeptide-Y (p<0.01) and Agouti-related peptide (p<0.05), with no changes in proopiomelanocrotin or cocaine- and
amphetamine-regulated transcript mRNA levels.
In conclusion, a short-term dietary change can modify both the systemic and central IGF systems, with these modifications
differing between males and females. Thus, this system could be involved in the sex difference in weight gain in response
to poor dietary habits. Further investigation is warranted to determine the role of IGF2 and IGFBP2 in metabolism.
(1) Geer et al., Comp. Biochem. Physiol., 1980. Vol. 65B, pp. 25-34.

e00031
Myeloid SAPKs in the control of obesity and BAT thermogenesis.

Maria Crespo1, Ivana Nikolic1, Aránzazu Pintor1, Alfonso Mora1, Elena Rodríguez1, Luis Leiva-Vega1, Magdalena Leiva1*, Guadalupe Sabio1*..

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00031

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Crespo M., Nikolic I., Pintor A., Mora A., Rodríguez E., Leiva-Vega L., Leiva M., Sabio G., Myeloid SAPKs in the control of obesity
and BAT thermogenesis. IBJ Plus 2019 S(3):e0031 doi: 10.24217/2531-0151.19v1s3.00031.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Chronic inflammation of white adipose tissue during obesity is a main driver of insulin resistance and type 2 diabetes
(T2D). Among this inflammatory scenario, macrophages play important roles. Stress activated kinases (SAPKs) are
key members of the signaling pathways implicated in inflammatory response, function and cytokine production in
macrophages. Brown adipose tissue (BAT) is a potential therapeutic target for obesity because of its capacity to burn
fat during thermogenesis, thus increasing energy expenditure. M1 (pro-inflammatory or classically activated) and M2
(anti-inflammatory or alternatively activated) macrophages are important for the control of BAT thermogenic activity,
by blocking or promoting it, respectively. Using a mice model lacking the main upstream activators of some SAPKs in the
myeloid lineage we found that this pathway is crucial for M2, but not M1 macrophage polarization. Moreover, this mice
model is more sensitive to high-fat diet (HFD)-induced obesity as they have more body weight and liver steatosis, they
are more insulin resistant. This correlates with the less energy expenditure, BAT thermogenic activity and subcutaneous
browning found in this mice model. Accordingly with this, we found there is a significant depletion of M2 macrophage
population in BAT from these mice. All this data suggests the crucial role of myeloid SAPKs in the protection against
obesity and in the control of BAT thermogenesis during HFD.

e00032
Intermittent Hypoxia a powerful stimulus of the endocrine system:
Anabolic/Catabolic Hormone response in Athletes.

Fernández-Lázaro Diego1, Fernández-Lázaro César Ignacio1,2, Mielgo-Ayuso Juan3, Caballero García Alberto4, Pascual Fernández Jorge5, Córdova
Martínez Alfredo3.

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00032

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Fernández-Lázaro D., Fernández-Lázaro César I., Mielgo-Ayuso J., Caballero García A., Pascual Fernández J., Córdova Martínez
A., Intermittent Hypoxia a powerful stimulus of the endocrine system: Anabolic/Catabolic Hormone response in Athletes. IBJ Plus
2019 S(3):e0032 doi: 10.24217/2531-0151.19v1s3.00032.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: Exposure to intermittent hypoxia (IH), that is used as a complement to training to increase sports performance,
affects the function on the hypothamulus-pitutary-adrenal axis, increases the corticotroph number and the levels of plasma
ACTH and stimulates the expression of the steroidogenic acute regulatory protein and enhances the secretion of glucocorticoids
as cortisol (C) (1). Also, the extreme physical effort induces a powerful stimulus of the endocrine system, that generates a
hormonal response (2,3). Therefore, we investigated the effects of acute short duration IH along a macrocycle of training, over
variations and behavior of anabolic/catabolic hormones. In this context we wont to evaluate also the influence of IH on specific
test of performance. Material and Methods: We investigated the effects of IH along 4 weeks training on endocrine response by
anabolic/catabolic hormones, Testosterone (T) and C. We studied professional male athletes exposed (PA) (n=12) or not (n=12)
to IH, at three different moments: baseline (M1) (i.e., under resting conditions immediately before start the study); after two
weeks of IH (M2); and at the end of the 4-week IH exposure period (M3). Physical performance was assessed by individual test:
aerobic power, lactic power and speed were performed in the athletics track, over distances of 1000 meters (m), 400m and
60m respectively. For the strength test, force of the quadriceps with a dynamometer was measured. The tests were performed
on M1 and M3. Results: Significant differences were observed throughout the study (M1 to M3) in T and T/C between groups.
In PA group T increase significantly (P<0.001), however the tendency of C was to increase but no significantly. With respect T/C
ratio increase (p<0.01) along of study (M1 to M3). In CG both, T and T/C were showed a significant decrease along the study
(p<0.05), and T/C ratio decrease significantly in M2 and M3 with respect baseline (M1). The stepwise discriminant function
analysis resulted indicated athletes who use hypoxia (centroid = 1,793) had higher Δ T values, whereas the athletes who use
normoxia (centroid = -1,569) had lower Δ T values. The results, of physical parameters, were better in PA group that in CG,
being significant the differences in anaerobic (P=0.003) and aerobic (P>0.001) power between both groups. Conclusion: We
have observed that IH might potentially stimulate performance through an anabolic effect. The testosterone is a determinant
informative to evaluate the answer to exercise in PA. For all this we recommended performing the resistance training under the
intermittent hypoxia to induce anabolic hormone responses and after improve the physical performances.
References:
1.Kujala UM, Alen M, Huhtaniemi IT. Gonadotrophin-releasing hormone and human chorionic gonadotrophin tests reveal that
both hypothalamic and testicular endocrine functions are suppressed during acute prolonged physical exercise. Clin Endocrinol
(Oxf). 1990; 33: 219-225.
2.Cordova A, Fernandez-Lazaro D, Mielgo J, Seco J, Caballero A. Effect of magnesium supplementation on muscular damage
markers in basketball players during a full season. Magnes Res. 2017; 1; 30: 61-70.
3.Navarrete-Opazo A, Mitchell GS. Therapeutic potential of intermittent hypoxia: A matter of dose. Am J Physiol Regul Integr
Comp Physiol. 2014; 307: 1181-1197.

e00033
Influence of malnutrition during pregnancy on maternal and fetus
plasmatic corticosterone concentrations.

Andrea Gila-Diaz1* , Cynthia Guadalupe Reyes-Hernández1, David Ramiro-Cortijo1, Pilar Rodríguez-Rodríguez1, Angel Luis López de Pablo1, Mª del
Rosario López-Giménez2, Mª del Carmen González1, Silvia M. Arribas1.

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00033

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Gila-Diaz A., Reyes-Hernández C.G., Ramiro-Cortijo D., Rodríguez-Rodríguez P., López de Pablo A.L., López-Giménez M.R.,
González M.C., Arribas S.M., Influence of malnutrition during pregnancy on maternal and fetus plasmatic corticosterone concentrations.
IBJ Plus 2019 S(3):e0033 doi: 10.24217/2531-0151.19v1s3.00033.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: Exposure to stress factors during gestation can negatively influence fetal development leading to low birth weight (LBW), which has been associated with later development of cardiometabolic diseases (fetal programming). Malnutrition and exposure to excess glucocorticoids during gestation are some of the stress factors that negatively influence fetal development. In situations of maternal undernutrition, the stress induced by reduced nutrient availability may elevate maternal cortisol levels. Under normal conditions, cortisol concentrations in the fetus are low due to the placental enzymatic barrier (11-β-HSD2) that converts cortisol to inactive cortisone. However, this barrier may be inhibited in malnutrition situations.

Hypothesis: Maternal undernutrition during gestation may induce a poor fetal development, in part due to increased access of cortisol to the fetus.

Objective: To analyze if undernutrition during gestation in the rat increases fetal corticosterone levels and to assess the relationship with fetal growth. Material and methods: Dams were fed ad libitum (Control) or with 50% of the control diet (Maternal undernutrition, MUN) during the second half of gestation. At the end of gestation (gestational day 20) a blood sample was obtained from the dam and the fetuses were extracted. The fetuses were weighted, and blood was collected after decapitation. Plasma corticosterone levels were assessed by ELISA; maternal protein levels by Bradford and glucose levels with a glucometer. Mann-Whitney U and Student’s t test were used for statistical analysis, as appropriate.

Results: In MUN dams, plasma protein levels were significantly lower, glycaemia was not statistically different and corticosterone levels were significantly higher compared to control dams. MUN fetuses exhibited significantly lower weight and length compared to controls, while plasma corticosterone was significantly higher.

Conclusions: Nutritional restriction during gestation in the rat produces a situation of stress that: 1) increases maternal plasma corticosterone, which can contribute to low protein availability through its catabolic effects; 2) increases passage of corticosterone to the fetus, which may contribute to the deficient fetal growth. Evaluation of 11-β-HSD2 enzymatic activity or protein expression in the placenta is needed to elucidate if these alterations are related to an alteration of this enzymatic barrier in situations of malnutrition.

e00034
Maternal cortisol in early pregnancy as potential biomarker of fetal complications.

David Ramiro-Cortijo1, Andrea Gila-Diaz1, Pilar Rodríguez-Rodríguez1, Ángel L. López de Pablo1, Mª Carmen González1, María de la Calle2, Rubén Gómez-Rioja3, Silvia M. Arribas1..

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00034

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Ramiro-Cortijo D., Gila-Diaz A., Rodríguez-Rodríguez P., López de Pablo A.L., González M.C., De la Calle M., Gómez-Rioja R., Arribas S.M., Maternal cortisol in early pregnancy as potential biomarker of fetal complications. IBJ Plus 2019 S(3):e0034 doi: 10.24217/2531-0151.19v1s3.00034.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: Maternity pattern has changed in Spain, leading to an increase in the age of first pregnancy and the used of assisted reproduction techniques (ART). A relevant consequence is the increase in fetal complications, such as small for gestational age (SGA) and preterm birth, both of which lead to low birth weight (LBW). LBW has a negative impact for both neonatal and long term health, being a risk factor for cardiometabolic disease. Delayed maternity and ART are associated with maternal psychological stress. We have evidenced that stress during pregnancy is associated with LBW. A possible mechanism could be an excess in the level of cortisol access to the fetus that may be deleterious for fetal growth. We hypothesized that maternal plasma cortisol at the beginning of pregnancy, associated with fetal complications and LBW and, therefore, it can be used as early diagnostic tool.

Material and Methods: The study included 200 healthy pregnant women, being attended at the Obstetrics and Gynecology Service from La Paz University Hospital (Madrid, Spain). Women were appointed for blood sample extraction at 10th week of gestation and followed-up until delivery, recording: 1) development of fetal complications including SGA (growth <p3th or <p10th with hemodynamic alterations by Echo-Doppler) and preterm birth (<37gestational age) and 2) offspring birth weight. Blood samples were centrifuged to obtain plasma and cortisol was assessed by competitive immunoassay using a direct chemiluminescent assay. Data were expressed as mean +- standard error of mean or relative frequencies (%). Statistical differences were assessed by Student´s t test with Confidence Interval at 95%. Pearson test (r) was used for correlation analysis, stablishing significance differences at p-value<0.05.

Results: Age of the pregnant women was 34.4+-0.33 years old. The prevalence of fetal complications was 26.5% and LBW was 28.8%. Both were significantly higher in older women (fetal complications=95% CI [-3.25; -0.18]; LBW=95% CI [-3.03; -0.11]). Maternal cortisol, in the first trimester of pregnancy, exhibited a positive correlation with maternal age (r=0.225; p=0.008) and a negative correlation with birth weight (r=-0.326; p=0.006). Plasma cortisol was significantly higher in women who developed fetal complications (95% CI [-5.24; -0.16]) and also in women with premature delivery (95% CI [-6.61; -1.44]).

Conclusions: Maternal plasma cortisol at the beginning of pregnancy is related to the age of maternity and fetal complications leading to LBW. Plasma cortisol could be used as potential biomarker to predict fetal complications particularly in delayed maternity context.

e00035
Metabolic response to diets enriched with sunflower seed oil or palm seed oil.

María Valencia-Avezuela1, Victor Naranjo2, Ana Conreras2, Santiago Guerra-Cantera1,3, Sandra Canelles1,3, Jesús Argente1,3,4, Nuria Del Olmo2, Laura M. Frago1,3, Mariano Ruiz-Gayo2, Julie Chowen1,3,4 .

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00035

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Valencia-Avezuela M., Naranjo V., Conreras A., Guerra-Cantera S., Canelles S., Argente J., Del Olmo N., Frago L.M., Ruiz-
Gayo M., Chowen J. Metabolic response to diets enriched with sunflower seed oil or palm seed oil. IBJ Plus 2019 S(3):e0035 doi:
10.24217/2531-0151.19v1s3.00035.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

The hypothalamus is the main integrating center for metabolic control, regulating the response to nutrients and other
metabolic signals to maintain energy homeostasis. This response includes not only neuronal circuits that control appetite,
but also glial cells that are involved in nutrient processing and inflammatory responses to high fat intake. However, it is
now clear that the metabolic response is different depending on the type of fatty acid consumed. Our aim was to analyze
the hypothalamic response to the consumption of a diet enriched with sunflower seed oil or palm seed oil.
Adult male C57/BL6J mice were given ad libitum access to a normal rodent chow diet enriched with sunflower seed oil
with high oleic content (SO) or with palm seed oil (PO), or to a standard rodent chow (SD) for 8 weeks. Energy intake and
body weight were monitored weekly. Relative protein expression was analyzed by Western blotting and relative gene
expression by RT-PCR.

Both the SO and PO diets increased body weight and energy intake compared to SD (p<0.01). In the hypothalamus
neuropeptide Y (NPY) mRNA levels were lower in mice consuming the SO diet compared to SD (p<0.05). No dietary
effects were found on the neuropeptides Agouti-related peptide (AgRP) or pro-opiomelanocortin (POMC). Expression of
the leptin receptor did not change. There was no change in the relative protein levels of ionized calcium-binding adaptor
molecule 1 (Iba 1), a marker of microglia, or of glial fibrillary acidic protein (GFAP), a marker of astrocytes. We found no
significant effect on hypothalamic mRNA levels of the cytokine interleukin-6 (IL-6), although there was a clear tendency
to increase on the PO diet. There was also no change in the cytokine TNFα or the cell stress marker DNA damageinducible
transcript 3 (CHOP). No activation of c-Jun N-terminal kinases (JNK) was found. To explore hypothalamic fatty
acid metabolism, we analyzed the relative expression of fatty acid synthase (FASn) and carnitine palmitoyltransferase
(Cpt1a). PA decreased Cpt1a mRNA expression compared to SD (p<0.01), with no change in FASn.

In conclusion, although both diets enriched in fat induce weight gain, the central response to these diets differs. The
mRNA levels of the orexigenic neuropeptide NPY were lower on the SO diet, which may suggest that this diet could
be more effective in reducing food intake, at least at this time-point. In contrast, the PO diet decreased Cpt1a mRNA
expression, indicating a possible reduction in hypothalamic fatty acid oxidation that could lead to stress of the system.
Further studies are necessary to determine the secondary complications as a result of these specific high fat diets.

e00036
Skeletal muscle kinases in the regulation of energy balance.

Leticia Herrera-Melle1, Alfonso Mora1, Juan Antonio López2, Sara Cogliati3, María Elena Rodríguez1, Luis Leiva-Vega1, Daniel Beiroa4, Cintia Folgueira4,
José Antonio Enríquez3, Jesús Vázquez2, Rubén Nogueiras4, Guadalupe Sabio1*.

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00036

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Herrera-Melle L., Mora A., López J.A., Cogliati S., Rodríguez M.E., Leiva-Vega L., Beiroa D., Folgueira C., Enríquez J.A., Vázquez
J., Nogueiras R., Sabio G., Skeletal muscle kinases in the regulation of energy balance. IBJ Plus 2019 S(3):e0036 doi: 10.24217/2531-
0151.19v1s3.00036.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Obesity is a well-known risk factor for several chronic diseases. Physical inactivity and changes in dietary habits have
increased the incidence and prevalence of obesity in current societies, which has become a major health problem.
Although several pharmacological treatments to lose weight through a reduction in food intake have been proposed,
understanding the molecular mechanisms that lead to increased energy expenditure may help to find new therapies. In
fact, exercise has the capacity to improve the metabolic status in obesity and type 2 diabetes, being skeletal muscle an
important regulator of glucose homeostasis and an essential contributor to exercise-induced changes in metabolism.

In this work, using a conditional mouse model lacking a kinase in striated muscle, we have attempted to increase
knowledge about the role of this protein both at the local and the systemic level. Our results show that the deficiency
of this kinase in striated muscle decreases body weight and protects mice against high-fat diet (HFD)-induced obesity
by increasing energy expenditure. The molecular alterations caused by this deficiency lead to skeletal muscle metabolic
remodeling, increasing mitochondrial oxidative metabolism. Altogether, these molecular and metabolic changes in
skeletal muscle are physiologically manifested as enhanced locomotor activity as well as improved glucose homeostasis,
decreasing the risk of developing diabetes and liver steatosis, therefore linking local and systemic manifestations of the
deficiency of this protein.

e00037
Role of pleiotrophin in hepatic metabolism: characterization in
an aging model.

Begoña Zapatería1, María Gracia Sánchez1, Julio Sevillano1, Gonzalo Herradón2, María del Pilar Ramos1.

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00037

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Zapatería B., Sánchez M.G., Sevillano J., Herradón G., Ramos M.P., Role of pleiotrophin in hepatic metabolism: characterization
in an aging model. IBJ Plus 2019 S(3):e0037 doi: 10.24217/2531-0151.19v1s3.00037.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Pleiotrophin (PTN) belongs to a family of heparin-binding growth factors involved in regulation of cell growth and
angiogenesis. Recently we have shown that PTN also modulates thermogenesis and adipose tissue plasticity. However,
the function of this cytokine in hepatic metabolism is completely unknown.

Methods: To determine if pleiotrophin plays a role in the metabolism of the liver, we used a mouse model in which
pleiotrophin has been deleted (Ptn-/-) and its corresponding controls (Ptn+/+). Glucose and lipid metabolism were studied
including glucose intolerance, liver weight and lipid content, as well as gene expression of membrane transporters and
key enzymes of glucose and lipid metabolic pathways in those mice at 3, 6, 12 and 15 months of age.

Results and discussion: Ptn-/- mice were smaller and present lower circulating triglycerides (TG). Both liver weight and
total lipid content were significantly lower than in their corresponding controls. On the other hand, in Ptn+/+ mice,
aging was accompanied by an increase in hepatic lipogenesis, while deletion of Ptn produces a marked reduction in the
expression of all enzymes involved in fatty acid synthesis and esterification, which could be related with the reduced TG
content in the liver of these animals. In addition, the expression of AQP 9 was clearly diminished in Ptn-/-, a fact that
could suggest a lower uptake of glycerol in these animals than in the control mice.

Although, no significant changes were found in mRNA levels of most of the enzymes of glycolysis and gluconeogenesis,
their enzymatic activity indicates that gluconeogenesis is significantly enhanced in Ptn-/- mice with aging, which
correlates with the glucose intolerance of these animals. Finally, in the cycle of tricarboxylic acids, the activity of the
enzymes involved is higher in Ptn-/- mice and in the oldest ones.
These results are the first ones showing the involvement of pleiotrophin in the control of hepatic homeostasis, which
postulates pleiotrophin as a potential therapeutic target for the treatment of several metabolic disorders such as
dyslipidaemia or NAFLD.

e00038
A possible role of Gonadotropin Releasing Hormone functional
homolog, prothoracicotropic hormone (ptth), in metabolism.

Celia G. Barredo1, Beatriz Gil-Marti2, Carmen Rodriguez-Navas3 and Francisco A. Martin4.

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00038

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Barredo, C.G. Gil-Marti B., Rodriguez-Navas C. and Martin F.A., A possible role of Gonadotropin Releasing Hormone functional
homolog, prothoracicotropic hormone (ptth), in metabolism. IBJ Plus 2019 S(3):e0038 doi: 10.24217/2531-0151.19v1s3.00038.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Besides its classical role on the control of adult transition, Gonadotropin Releasing Hormone (GnRH) influences
sleep, memory and energetic metabolism during adulthood. However, mechanisms are not really known. We are
interested in uncovering how GnRH regulates these behaviors. Functional GnRH homolog in Drosophila melanogaster
is the prothoracicotropic hormone (ptth). Ptth might be involved in regulating the metabolic state, as suggested by its
expression in two brain structures such as the Ellipsoid Body and the Mushroom body. Interestingly, torso (ptth receptor)
is expressed in the pars intercerebralis and fat cells. Animals mutant for ptth are bigger in size. To assess ptth implications
in starvation resistance, we used Drosophila Activity Monitors (DAMs-Trikinetics). DAMs quantify the movement of flies,
so it can be used to determine when the animal stops moving for a long period of time, meaning that it is dead. We have
also used the CAFE assay (CApillary FEeder assay) to quantify the feeding amount. Our results show that ptth mutant flies
survive longer than controls under starvation. However, the way ptth mutant animals feed and move is indistinguishable
from control flies. In conclusion, loss of ptth prolongs the survival of starving animals despite their normal activity and
food ingestion, suggesting a role for ptth in regulating metabolism per se.

e00039
Alx3 deficiency in the hypothalamic arcuate nucleus alters adipose
tissue distribution, feeding and body mass composition.

M. Mirasierra, A. Fernández-Pérez and M. Vallejo*.

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00039

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Mirasierra M., Fernández-Pérez A. and Vallejo M., Alx3 deficiency in the hypothalamic arcuate nucleus alters adipose tissue
distribution, feeding and body mass composition. IBJ Plus 2019 S(3):e0039 doi: 10.24217/2531-0151.19v1s3.00039.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Alx3, an aristaless-type homeodomain transcription factor expressed in pancreatic islets, regulates glucose homeostasis
by stimulating the expression of the insulin gene in beta cells, or by coordinately inhibiting that of the glucagon gene in
alfa cells in a glucose-dependent manner. Our previous studies demonstrated that Alx3 deficiency leads to enhanced
islet cell apoptosis and mild hyperglycemia without reaching levels characteristic of overt diabetes. Independently of
its pancreatic functions, we have previously reported using data from positron emission tomography and diffusionweighted
magnetic resonance imaging that Alx3-deficient mice show signs of hypothalamic dysfunction. Indeed, the
presence of Alx3 in neurons of the arcuate nucleus was demonstrated by immunofluorescence studies. In addition,
Alx3-deficient mice exhibit decreased oxygen consumption, energy expenditure and respiratory exchange ratio. These
studies strongly suggest that Alx3 may contribute to the systemic regulation of metabolic homeostasis by acting at the
hypothalamic level independently of its functions in the pancreas. In the present study, we investigated whether the
expression of genes regulating food intake in the arcuate nucleus of Alx3-deficient mice is altered, leading to changes in
feeding and accumulation of fat.

Alx3-knockout and control mice of similar age and body weight were used. Gene expression was determined by RT-qPCR
in freshly microdissected blocks of hypothalamic tissue containing the arcuate nucleus. Body mass composition was
analyzed by magnetic resonance imaging. Muscle function was assessed by treadmill, rotarod and strength tests.
We found that Alx3-deficient mice exhibit reduced food intake but similar body weight relative to wild type animals.
Expression of Alx3 in the arcuate nucleus was detected by immunofluorescence and confirmed by RT-qPCR. The
expression of the orexigenic genes Npy and Agrp in response to fasting was similar in wild type and Alx3-deficient mice,
but the expression of the anorexigenic genes Pomc and Cart was altered. Fasting and refeeding experiments showed
that, after an overnight fasting, Alx3-deficient mice stopped eating shortly after the onset of the refeeding period.
This coincided with elevated expression of Cart in the arcuate nucleus. Afterwards, Alx3-decicient animals proceeded
to eat at a lower rate than controls. We found no differences in the hypothalamic expression of leptin, ghrelin and
erythropoietin receptors. The expression of the alfaMSH receptor MC4R was similar in Alx3-mutant and in control mice,
but the expression of MC3R was increased in Alx3-deficient animals. Relative to body weight, Alx3-deficient animals
showed increased proportion of fat mass and reduced proportion of lean mass, as assessed by magnetic resonance
imaging. In consonance with these results, muscle function (motor coordination and strength) was reduced in mutant
mice relative to controls. Our results indicate that Alx3 in the arcuate nucleus of the hypothalamus regulates food intake
and body mass composition.

e00040
p38γ and p38δ control postnatal heart metabolism through
glycogen synthase.

Bárbara González-Terán1, Valle Montalvo-Romeral1, Cristina Casanueva Benítez1, Ayelén M. Santamans1, Alfonso Mora1, Elena Rodríguez1, Rebeca
Acín-Pérez1, Luis Leiva-Vega1, Jesús Jiménez-Borreguero1, José Antonio Enríquez1,2 and Guadalupe Sabio1*.

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00040

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: González-Terán B., Montalvo-Romeral V., Casanueva Benítez C., Santamans A.M., Mora A., Rodríguez E., Acín-Pérez R., Leiva-
Vega L., Jiménez-Borreguero J., Enríquez J.A. and Sabio G., p38γ and p38δ control postnatal heart metabolism through glycogen
synthase. IBJ Plus 2019 S(3):e0040 doi: 10.24217/2531-0151.19v1s3.00040

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Rationale– After birth, high-energy demand and increasing levels of circulating free fatty acids
trigger a profound shift in cardiomyocyte metabolism to a predominant reliance on oxidative fatty
acid metabolism, which consumes more oxygen but yields more ATP per molecule. This critical
metabolic switch from glycolytic to oxidative metabolism is a key
transition in cardiomyocyte maturation during heart development. Stress-activated protein kinases
(SAPK) p38g and p38d expression and activation increase after birth promoting cardiomyocyte
hypertrophic growth and might be involved in these metabolic changes.

Objective– Understand whether p38g and p38d are implicated in the critical maturational changes
that occur in cardiac energy metabolism during heart development.

Methods and Results– Using adeno-associated viral vectors, we have overexpressed in cardiomyocytes
at postnatal day 1 (P1) the active forms of p38g and p38d. Cardiac function and whole body
metabolism were evaluated. We found that postnatal p38γ and p38δ expression in the heart produces a
reduction in glycogen content through the
phosphorylation and subsequent inhibition of the muscle-specific glycogen synthase 1 (GYS1), the
main enzyme involved in cardiac glycogen production. Mice with reduced cardiac glycogen levels
during the first two weeks after birth, showed increased lipid storage in the heart and developed
dilated cardiomyopathy. Moreover, these changes in
cardiac energy metabolism resulted in whole body metabolism dysregulations. Interestingly, although
the systemic metabolism is normalized after cardiac metabolic shift into fatty acid oxidation, the
heart physiology alterations persisted in adult mice.

Conclusions– p38g and p38d function in a cooperative manner to inhibit GYS1 activity through
phosphorylation, blocking glycogen production. Both kinases are involved in the metabolic switch of
cardiomyocytes after birth. Our results point out these defects in cardiac fuel during the early
postnatal development might cause cardiac dysfunction and deleterious
metabolic consequences in whole body metabolism.

e00041
MKK6: a novel player in cardiac hypertrophy and sudden cardiac
death.

Bárbara González-Terán1#, Rafael Romero-Becerra1#, Daniela Ponce-Balbuena2, Roberto Ramos Mondragon2, Eric Jimenez Vazquez2, Elisa Manieri1,
Laura Sanz1, Ivana Nikolic1, María del Valle Montalvo1, Ayelen Santamans1, Maria Elena Rodriguez1, Luis Leiva-Vega1, Víctor Buendía1, Guadalupe
Guerrero Serna2, David Filgueiras1,3, Luis Jesús Jiménez Borreguero1, José Jalife1,2,3* & Guadalupe Sabio1*.

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00041

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: González-Terán B., Romero-Becerra R., Ponce-Balbuena D., Ramos Mondragon R., Jimenez Vazquez E., Manieri E., Sanz
L., Nikolic I., Montalvo MV., Santamans A., Rodriguez M.E., Leiva-Vega L., Buendía V., Guerrero Serna G., Filgueiras D., Jiménez
Borreguero L.J., Jalife J. & Sabio G., MKK6: a novel player in cardiac hypertrophy and sudden cardiac death. IBJ Plus 2019 S(3):e0041
doi: 10.24217/2531-0151.19v1s3.00041.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Knowledge about signaling pathways associated with hypertrophic cardiomyopathy and the risk of sudden cardiac death
remains incomplete. Mitogen activated protein kinases kinases MKK6 and MKK3 have been suggested to be involved in
cardiac growth. However, it is unclear how these kinases control the activation of the different p38 mitogen-activated
protein kinase (MAPK) isoforms in cardiomyocytes and whether they are implicated in pathological cardiac hypertrophy
and sudden cardiac death. Our results show that MKK3 is the main activator of p38γ and p38δ in the heart, whereas
MKK6 activates cardiac p38α. Mice lacking MKK3 show a reduction in postnatal cardiac growth due to reduced p38γ
and p38δ activation, while lack of MKK6-p38α activation leads to MKK3-p38γ/δ hyperactivation and increased mTOR
signaling, resulting in physiological cardiac hypertrophy. Cardiac hypertrophy in Mkk6-/- mice can be reverted by knocking
out MKK3, and by inhibiting mTOR signaling. Interestingly, when MKK6-deficient mice were challenged with chronic
endurance training, the QT interval was prolonged and mice died prematurely during or just after exercise. In addition,
Mkk6-/- mice showed cardiac ion channel dysfunction, prolonged action potential duration and increased arrhythmia
susceptibility on programmed electrical stimulation, particularly in the presence of β-adrenergic stimulation. MKK6
deletion and p38γ activation also promotes the hyperphosphorylation of hDlg/SAP97, a scaffolding protein involved in
the assembly and functionality of cardiac ion channels in the T tubule. SAP97 hyperphosphorylation results in altered
levels and function of cardiac ion channels, likely because and increased phosphorylation of these channels by PKA and
CAMKII, two other proteins that also interact with SAP97. These results indicate that the MKK3/6-p38γ/δ pathway is
implicated in cardiac hypertrophy and cardiac ion channel dysfunction, which promotes QT prolongation and sudden
cardiac death.

e00042
Nuclear receptors are required for transcriptional control of
hematopoietic stem cells and myeloid differentiation.

Jesús Porcuna 1*, María Piedad Menéndez-Gutiérrez 1* and Mercedes Ricote1.*.

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00042

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Porcuna J., Menéndez-Gutiérrez MP. and Ricote M., Nuclear receptors are required for transcriptional control of hematopoietic
stem cells and myeloid differentiation. IBJ Plus 2019 S(3):e0042 doi: 10.24217/2531-0151.19v1s3.00042.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Hematopoietic stem cells (HSCs) maintain the hematopoietic system balance during the life of an organism. HSC fate and differentiation
are based on gene expression, which is the result of an orchestrated transcriptional regulation. However, the transcription factors that
govern HSC homeostasis remain poorly studied. Retinoid X Receptors (RXRs) are ligand-dependent transcription factors that respond to
vitamin A derivatives. The Vitamin A signaling pathway activation has been demonstrated to regulate HSC differentiation and stem cell
plasticity.
In this project, we aim to uncover the role of RXRs in the differentiation and identity of HSCs. We have generated conditional HSC
RXRα and RXRβ knock out mice (KO) to study the RXRα and RXRβ specific contribution to HSCs. Our findings show that RXRs control
myeloid differentiation and HSC survival. Flow cytometry analysis of bone marrow and spleen demonstrated an increase of myeloid
progenitor cells LKs (Linage-/c-kit+/sca1-) and LSKs (Linage-/c-kit+/sca1+) in KO compared to WT mice. Interestingly, the myeloid-biased
LSK subpopulation was largely increased indicating first, a tendency to myeloid differentiation and, second, a hematopoietic aged
phenotype in these mice. Transplantation experiments suggested that KO-associated hematopoietic defects are serial and long-term
transplantable, and showed that the effect of RXR in myelopoiesis is bone marrow niche-dependent. To further study the RXRs role in
myeloid differentiation, we have developed conditional myeloid RXRα and RXRβ knock out mice. Flow cytometry data have proven that
the lack of RXRs in myeloid cells led to the dysregulation of specific tissue resident macrophage populations.
In summary, we have proved that RXRα and RXRβ are novel and key regulators of HSC homeostasis, identity and differentiation, that the
hematopoietic absence of RXRs leads to a myeloid-biased hematopoietic phenotype, and that RXRs are key controllers of specific tissue
resident macrophages.

e00043
Quantitative analysis of cardiac receptors of the renin angiotensin
system (RAS) in a fetal programming hypertension rat model.

Rodríguez-Rodríguez P1, Vieira-Rocha MS2, Quintana-Villamandos B3, Monedero-Cobeta I1, González MC1, López de Pablo AL1, Morato M2, Diniz C2,
Arribas SM1.

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00043

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Rodríguez-Rodríguez P., Vieira-Rocha MS., Quintana-Villamandos B., Monedero-Cobeta I., González MC., López de Pablo
AL., Morato M., Diniz C., Arribas SM., Quantitative analysis of cardiac receptors of the renin angiotensin system (RAS) in a fetal
programming hypertension rat model. IBJ Plus 2019 S(3):e0043 doi: 10.24217/2531-0151.19v1s3.00043.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Purpose. Maternal undernutrition during pregnancy is associated with low birth weight of the offspring, which in adult
life develop hypertension and heart disease (fetal programming of cardiovascular diseases). We have evidence that male
offspring from rats exposed to undernutrition during gestation (MUN) exhibits enlarged heart at weaning and cardiac
dysfunction in ageing (Rodríguez-Rodriguez P. 2017). One of the potential mechanisms implicated in fetal programming
is an alteration in renin angiotensin system (RAS).

Hypothesis. We hypothesized that perinatal alterations in RAS components are implicated in early cardiac hypertrophy
and fibrosis, setting the basis for later occurrence of heart diseases.

Methods. Sprague Dawley rats were used. Control (C) dams were fed ad libitum during pregnancy and lactation. MUN
dams were fed 50% of the calculated ad libitum daily intake during the second half of gestation and returned to ad
libitum diet during lactation. All experimental procedures were performed at weaning (21 day-old) in male offspring
from C and MUN groups. We analyzed : 1) plasma Ang II concentrations (by solid phase extraction and ELISA kit), 2)
expression of AT1, AT2, Mas and MrgD receptors in myocardium and intramyocardial artery (immunohistochemistry), 3)
intramyocardial artery (IMA) structure (histology), 4) Cardiac structure and function (echocardiography).

Results. Plasma concentration of Ang II was markedly higher in MUN rats compared to controls (Control=0.87±0.38 pg/
ml; MUN=59.42±17.70 pg/ml, n=5; p<0.05).
Immunoreactivity against the AT1, AT2, Mas and MrgD receptors was observed in the myocardium from MUN and C rats.
No statistical differences between C and MUN rats were detected in AT1, AT2, Mas receptors, while there was a lower
expression of MrgD receptors in MUN rats. IMA from MUN rats exhibited a significantly higher wall/lumen ratio, collagen
content and expression of AT1 and Mas receptors than controls. By contrast, AT2 and MrgD receptor expression was
lower in MUN when compared to C rats.
Compared to C offspring, MUN rats exhibited an increased Left ventricular mass and reduced E/A ratio (parameter of
diastolic function) but normal ejection fraction (EF, which evaluates systolic function. No statistical differences were
detected in LVEF.

Conclusions. Fetal undernutrition induces perinatal alterations in RAS, which are implicated in heart hypertrophic
responses and early signs of diastolic dysfunction. These findings indicate that fetal programming of cardiac dysfunction
starts in perinatal life, suggesting that lactation period is an opportunity window for prevention, being the RAS a potential
target.

e00044
Modulation of mitochondrial activity in heart failure.

Ayelén M. Santamans1*, Aránzazu Pintor Chocano1, Alfonso Mora2, Elena Rodríguez1, Ainoa Caballero, María Villalba-Orero, Pablo Hernansanz-
Agustín, José Antonio Enriquez1, Mercedes Rincón2, Guadalupe Sabio1.

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00044

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: M. Santamans A., Pintor Chocano A., Mora A., Rodríguez E., Caballero A., Villalba-Orero, M. Hernansanz-Agustín P., Enriquez
JA., Rincón M., Guadalupe Sabio G., Modulation of mitochondrial activity in heart failure. IBJ Plus 2019 S(3):e0044 doi: 10.24217/2531-
0151.19v1s3.00044.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

During cardiac hypertrophy it is vital for the heart to adapt to an increasing energy demand by improving mitochondrial
activity and proliferation. The defective capacity of mitochondria to respond to these demands could result in heart
failure. We found that modulation of mitochondria complex I activity during heart injury could be beneficial. Our
results suggest that ATP availability decreases during heart injury. Genetic modification of the mice to increase this
ATP availability resulted in protection against hypertrophy and left ventricle dilatation. Genetic modified animals are
protected against diastolic dysfunction and showed less inflammation markers. In addition, the deposit of extracellular
matrix is differential as there is an increase in the perivascular area in WT mice after cardiac injury, not observed in the
genetically modified animals.

Altogether, these results suggest that mitochondria activity modulation could be a possible mechanism for the treatment
or prevention of heart failure by improving cardiological parameters and leading to a differential cardiac remodeling by
altering the mitochondrial metabolism.

e00045
DICER1 downregulation and impaired miRNA processing
in thyroid tumorigenesis.

Julia Ramírez-Moya1,2, León Wert-Lamas1,3, Garcilaso Riesco-Eizaguirre1,2,3, Pilar Santisteban1,2..

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00045

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Ramírez-Moya J., Wert-Lamas L., Riesco-Eizaguirre G., Santisteban P., DICER1 downregulation and impaired miRNA processing
in thyroid tumorigenesis. IBJ Plus 2019 S(3):e0045 doi: 10.24217/2531-0151.19v1s3.00045.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Thyroid cancer is a common endocrine malignancy that has rapidly increased its global incidence in recent decades.
Currently the important role of microRNAs (miRNAs) in cancer has been described, including thyroid neoplasms. It is
known that miRNAs are important modulators of cancer progression and are globally downregulated in several human
tumor types, through mechanisms that remain largely undefined. Functional maturation of most miRNAs requires
processing of the primary transcript by DICER1, an RNAse III-type enzyme essential for both mammalian development
and cell differentiation. Here we identified DICER1 and miRNA downregulation as sentinel events in thyroid cancer,
providing a clear cut evidence of the molecular mechanisms underlying this effect. We demonstrated that this key miRNA-
processing enzyme is a target of the most upregulated miRNAs in thyroid cancer. Specifically, miR-146b reduced
DICER1 expression and DICER1 overexpression inhibited all the miR-146b-induced aggressive phenotypes, both in cells
and in tumor models. Our analysis of The Cancer Genome Atlas revealed a general decrease in DICER1 expression in
thyroid cancer and suggested a clinical association between DICER1 and patient prognosis. Notably, DICER1 downregulation
promoted proliferation, migration, invasion and epithelial-mesenchymal transition in thyroid cancer cell lines,
whereas it suppressed the expression of pro-differentiation transcription factors. Interestingly, these factors transcriptionally
upregulated DICER1 expression, supporting the existence of a positive feedback loop. Finally, administration of
the small molecule enoxacin to promote DICER1-complex activity reduced cell aggressiveness in vitro and tumor growth
in vivo. Overall, our data establish DICER1 as a new tumor suppressor in thyroid cancer, and highlight a potential
therapeutic approach of RNA-based therapies including antagomiRs and restoration of the biogenesis machinery may
provide treatments for thyroid cancer and other cancers.

e00046
Antitumor activity of the ERK inhibitor DEL22379 against
BRAF-like cells in thyroid cancer.

Adrián Acuña-Ruiz1,2, Miguel Angel Zaballos1,2, Garcilaso Riesco-Eizaguirre2,3, Piero Crespo 2,4, Pilar Santisteban1,2,*.

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00046

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Acuña-Ruiz A., Zaballos MA., Riesco-Eizaguirre G., Crespo P., Santisteban P., Antitumor activity of the ERK inhibitor DEL22379
against BRAF-like cells in thyroid cancer. IBJ Plus 2019 S(3):e0046 doi: 10.24217/2531-0151.19v1s3.00046.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Thyroid cancer is the most prevalent human carcinoma of endocrine origin and its incidence is continuously increasing.
Around 80% of all the somatic mutations are accumulated in only five driver genes (BRAF, H/N/K-RAS and RET) which
converge in the hyperactivation of the MAPK signalling pathway. Given the central role of this pathway in thyroid tumorigenesis,
thyroid cancer research is focused on the identification of new drugs that inhibit the different kinases of the pathway.
DEL22379 is an inhibitor of ERK dimerization previously characterized in melanoma and colorectal cancer-derived cell lines as
a new approach to partially inhibit ERK activation. It has been reported that ERK dimerization contributes to the activation of
ERK cytoplasmic effectors, such as RSK, and enhances malignant cell features.
Our objective was to analyse the antitumor effect of the inhibitor of ERK dimerization DEL22379 as a possible therapeutic
compound in thyroid cancer.
In vitro assays were made with different thyroid cancer cell lines with mutations in BRAF, H/N/K-RAS or RET. To evaluate cell
proliferation, migration and invasion; we have used cell cycle, wound healing and matrigel invasion assays, respectively. We
also have developed an orthotopic mouse model of anaplastic thyroid cancer by the generation of the 8505c-luc cell line. In
vivo tumour progression was monitored by measuring luciferase activity.
Treatment with DEL22379 impairs ERK dimers formation in BRAF-like thyroid cells whereas Ras-like cells were unaffected.
Unlike melanoma, phosphorylated ERK levels were substantially reduced after treatment with the inhibitor. Inhibition of
ERK dimerization and phosphorylation correlated with diminished RSK phosphorylation. We also observed a decrease in cell
proliferation, migration and invasion in the anaplastic thyroid cell lines 8505c and OCUT2. DEL22379 treatment significantly
reduced tumour growth and formation of lung metastases in an orthotopic mouse model.
We propose DEL22379 as a new effective molecule for the inhibition of MAPK signalling and the reduction of cell malignant
behaviour, tumour growth and metastasis in vivo, specifically in BRAF-like thyroid tumours. It promises to be another step
forward in the achievement to cure thyroid cancer.

e00047
Role of the IQGAP1 scaffold protein in thyroid cancer.

Carlos Carrasco-López1, 2, Miguel Angel Zaballos1, 2, Pilar Santisteban1, 2, *.

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00047

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Carrasco-López C., Zaballos MA., Santisteban P., Role of the IQGAP1 scaffold protein in thyroid cancer. IBJ Plus 2019 S(3):e0047
doi: 10.24217/2531-0151.19v1s3.00047.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

The IQGAP family of scaffold proteins comprises three members which have been described to interact with numerous
proteins in several biological processes and participate in the signaling cascade of multiple pathways. Among these
proteins IQGAP1 has been the most deeply studied, having characterized its function as a scaffold protein in the MAPK
pathway and its interaction with actin leading to regulation of the cytoskeleton. Because of these functions it has been
studied in multiple malignancies, where it has been assigned a tumorigenic effect.
We have studied IQGAP1 expression using different stablished cell lines of human thyroid carcinomas. Our results
indicate that these carcinoma cell lines have variable levels of IQGAP1 but in general these levels are higher when
compared to the normal thyroid cell line control. The results obtained are in agreement with the TCGA and Oncomine
databases. Using IQGAP1 silenced cell lines we tried to elucidate how this protein was affecting tumor hallmarks such
as proliferation, invasion, cell migration and epithelial mesenchymal transition. The effects of silencing IQGAP1 were
different depending on the cell line and the oncogenic signature. While in some of them silencing produced an increase
in the migration rate of the cells, others had the opposite behavior. We also found these differences when studying
EMT markers such as E-cadherin and N-cadherin. Furthermore, cell viability was unaffected by IQGAP1 silencing and
consequently the size of the primary tumor was not altered when cells were xenografted in a chicken embryo model.
Importantly IQGAP1 silencing greatly decreased the ability of the tumor cells to intravasate and metastasize to distant
organs such as lungs and brain.
Further studies will focus on trying to discover the underlying mechanisms responsible for the differences observed
between cell lines. Our results indicate a role forIQGAP1 in thyroid tumorigenesis and underscore the importance
of defining the oncogenic signature and particularities of a given tumor type in order to design effective therapeutic
strategies.

e00048
Proteomic analysis of fine-needle aspirates (FNA) for the molecular
characterization of non-diagnostic follicular neoplasia.

Vanessa Arosa1, Mikel Azkargorta2, Leire Isasa1, Carmen Fernández1, Leire Pérez1, María Angeles Antón1, Gonzalo Maldonado1.

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00048

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Arosa V., Azkargorta M., Isasa L., Fernández C., Pérez L., Antón MA., Maldonado G., Santisteban P., Proteomic analysis of
fine-needle aspirates (FNA) for the molecular characterization of non-diagnostic follicular neoplasia. IBJ Plus 2019 S(3):e0048 doi:
10.24217/2531-0151.19v1s3.00048.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Thyroid cancer is the most prevalent malignant endocrine pathology. Differentiated thyroid cancer represents more than
90% of these cases, and 12% of these are follicular carcinomas. Currently, the diagnosis of thyroid nodules is based on the
cytologic analysis of fine-needle aspirates (FNA) aided by echography. Around 10% of the cases provided are categorized
as IV in the Bethesda system. Since malignancy cannot be ruled out, these cases are subjected to local surgery. The
post-operatory analysis of these samples reveals that in 70-85% of them this intervention was not necessary. Given this,
additional tools for sample categorization would be welcome.
Mass spectrometry-based proteomics provides a number of tools for the large-scale analysis of proteins in biological
samples. Current technology allows the analysis of the presence and/or relative abundance of hundreds/thousands of
proteins in a given sample; thus quantitative proteomics allows the comparison of protein levels between conditions
(i.e.: healthy vs. disease). Further bioinformatics analyses help deepen into the molecular functions and pathways
these proteins are involved in, providing additional information for the understanding of the disease. Thus, proteomics
provides a very valuable tool for the molecular characterization of a disease, as well as for biomarker discovery.
In this work we present preliminary results on the analysis of FNA samples by proteomics. The aspirates were centrifuged,
and both the resulting supernatant and the pellet containing aspirated cells and debris were processed and loaded into
an LTQ Orbitrap XL-ETD mass spectrometer for protein identification. Identified proteins were further characterized
using bioinformatic tools. The methods and results presented in this poster lay foundation for coming analyses in the
context of the molecular characterization of these poorly defined cases, being our main goal the search for FNA markers
that help distinguish follicular adenomas from carcinomas and thus avoid surgery.

e00049
Radioiodide therapy in ovarian cancer.

Mielu Lidia Mirela1,2, Fajardo-Delgado Dánae2, Hortigüela Rafael2, Diego-Hernández Cristina3, García-Jiménez Custodia4, Martín-Duque Pilar5, and
De la Vieja Antonio1,2.

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00049

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Mielu Lidia M., Fajardo-Delgado D., Hortigüela R., Diego-Hernández C., García-Jiménez C., Martín-Duque P., and De la Vieja A.,
Radioiodide therapy in ovarian cancer. IBJ Plus 2019 S(3):e0049 doi: 10.24217/2531-0151.19v1s3.00049.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: Ovarian cancer is the most lethal gynecological malignancy. Early diagnosis has a survival rate of 90%.
Unfortunately, more than 70% of cases are diagnosed when the cancer has already metastasized, and survival rates do
not exceed 30% in these cases. The sodium iodide symporter (NIS) mediates active transport of iodide into the thyroid
as a fist steps in thyroid hormone biosynthesis, but also in others tissues like salivary gland, lactating mammary gland
and stomach. Our group has demonstrated that NIS is expressed in ovarian surface epithelium and is overexpressed
in human epithelial ovarian cancer, establishing NIS as a tumor marker. The aim of this study is to determine whether
overexpression of NIS in ovarian cancer can be use as therapeutic tool using radioiodide therapy (RAI) in ovarian tumors.

Materials and Methods: serous ovarian cancer cell line (SKOV3) was transfected permanently with exogenous NIS (SKOV3-
hNIS) and in vitro characterized by different techniques (western-blot, PCR, flow cytometry, immunofluorescence, iodide
uptake and proliferation, migration, invasion, and adhesion assays). In vivo, NIS transfected cells and no transfected was
injected into the flanks of nude and NSGs mice. Tumor growth was monitored by measurements with caliber and IVIS.
The expression of NIS in tumors was analyzed by different molecular biology techniques and NIS functionality in animal
models was measured by SPECT-CT.

Results: PCR and western-blot show NIS expression in both in vitro cancer cells and in vivo with xenotransplanted cells in
animal models. Immunofluorescence and immunohistochemistry show that NIS expression occurs in plasma membrane,
and iodide uptake assays show that the expression of NIS in plasma membrane is functional in vitro and in SPECT-CT
assay in vivo. Additionally, NIS presence alters in vitro and in vivo proliferation, migration, invasion and adhesion of
tumor cell properties.

Conclusion: NIS expression in human ovarian cancer cell lines is functional in vitro and in vivo, targeted to the plasma
membrane and able to accumulate iodide. The high expression levels are correlated with worse prognosis pointing NIS
as a future therapeutic approach in treatment of ovarian cancer.

e00050
Metabolic adaptations in spontaneously immortalized PGC-1α
knock-out mouse embryonic fibroblasts increase their oncogenic
potential.

Ignacio Prieto1, Carmen Rubio-Alarcón1, Raquel García-Gómez1, Rebeca Berdún2, Manuel Portero2, Reinald Pamplona2, Antonio Martínez-Ruiz3,4,
José Ignacio Ruiz-Sanz5, Mª Begoña Ruiz-Larrea5, Mariona Jove2, Sebastian Cerdán1, María Monsalve1*..

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00050

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Prieto I., Rubio-Alarcón C., García-Gómez R., Berdún R., Portero M., Pamplona R., Antonio Martínez-Ruiz A., Ruiz-Sanz J.I., Ruiz-
Larrea MB., Jove M., Cerdán S., Monsalve M., Santisteban P., Metabolic adaptations in spontaneously immortalized PGC-1α knock-out
mouse embryonic fibroblasts increase their oncogenic potential. IBJ Plus 2019 S(3):e0050 doi: 10.24217/2531-0151.19v1s3.00050.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

PGC-1α 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-1α in cancer. To investigate how loss of PGC-1α 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-1α knockout (KO) mice
and analyzed their oncogenic potential in vivo and in vitro. We found that PGC-1α 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 reduced growth factor
dependence. The mechanistic basis of this phenotype is likely associated with the observed higher levels of nuclear
β-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 aminoacid 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-1α, restraining metabolic adaptations in cancer.

e00051
Gene regulatory and phenotypic effects of calcitriol and canonical
WNT in human colon fibroblasts.

Gemma Ferrer-Mayorga1,2,3, Núria Niell1,4,^, Ramón Cantero2,5, José Manuel González-Sancho1,3,4, Luis del Peso1,2,4,6, Alberto Muñoz1,2,3,* &
María Jesús Larriba1,2,3,*.

Published May, 2019.

DOI: 10.24217/2531-0151.19v1s3.00051

Copyright: © 2019 Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Ferrer-Mayorga G., Niell N., Cantero R., González-Sancho J.M., Del Peso L., Muñoz A. & Larriba MJ., Gene regulatory and
phenotypic effects of calcitriol and canonical WNT in human colon fibroblasts. IBJ Plus 2019 S(3):e0051 doi: 10.24217/2531-
0151.19v1s3.00051.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Vitamin D3 (cholecalciferol) is synthesized in the skin by action of solar UV radiation or
incorporated via diet, and is the inactive precursor of 1α,25-dihydroxyvitamin D3 (calcitriol), a
major pleiotropic hormone in the human organism. By binding and activation of vitamin D receptor
(VDR), a member of the superfamily of nuclear receptors, calcitriol regulates the expression of
hundreds of genes in a tissue- and cell-type specific manner by transcriptional and
posttranscriptional mechanisms.

Human WNT factors are a family of 19 members of secreted proteins that regulate crucial processes
in many tissues and organs during development and adult life. Some (canonical) WNTs act by
modulating the transcriptional activity of β-catenin whereas other (non-canonical) WNTs affect
different signal transducers (Ca2+, Rho, JNK…) independently of β-catenin.

Colorectal cancer (CRC) is one of the most important neoplasias worldwide in terms of incidence and
mortality. The key role of the constitutive activation of the WNT/β-catenin signaling pathway
promoting CRC and the protective effect of VDR agonists, in part by antagonizing the WNT/β-catenin
pathway, have been widely described. However, the effects of calcitriol and canonical WNTs on
stromal fibroblasts, which are important players in CRC with protumorigenic action, remain mostly
unknown.

We have studied the effect of calcitriol and WNT3A on the human CCD-18Co colonic fibroblast cell
line and on primary colon fibroblasts isolated from CRC patients. Data from global transcriptomic
analyses by RNA-sequencing and also functional assays indicate that calcitriol and WNT3A exert a
wide regulatory action on the gene expression and phenotype of colonic fibroblasts. Remarkably, a
complex interplay exists between the two agents: while calcitriol and WNT3A act cooperatively in
some effects, antagonistic actions are found in others.

Our results show that calcitriol and WNT3A are important modulators of human colon fibroblasts,
with potential
implications in colon homeostasis and neoplastic transformation.