Characterization of phthalate presence in cattle environment and its effects on epigenetic markers expression and steroidogenesis in bovine granulosa cells

Farm animals are now more likely to be exposed to environmental pollutants like phthalates as result of intensifying livestock production systems. Phthalate exposure may cause impairments in human and animal health and fertility. However, research into the occurrence of phthalates in cow operations is scarce. Also, studies on the impact of Di-isobutyl phthalate (DIBP) on female fertility are limited in phthalate research. The first project aimed to determine the effects of DIBP in bovine granulosa cells (GC) proliferation, steroidogenesis, and epigenetic and inflammation pathways. Using in vitro cell culture of bovine GC, DIBP treatment at levels detected in human blood (0 ng/mL, 1 ng/mL, 10 ng/mL, 100 ng/mL) combined with hormones that regulate ovarian folliculogenesis: Follicle-Stimulating Hormone (FSH) or FSH + Insulin Growth Factor 1 (IGF1) were tested. Estradiol production was assessed by ELISA, cell proliferation was determined by cell counting, and relative mRNA abundance was evaluated by RT-qPCR. Cell proliferation and estradiol production were not affected by DIBP treatments in combination with FSH or FSH+IGF1. DIBP treatment in combination with FSH+IGF1 decreased mRNA levels of METTL14 (P<0.01), an RNA N6 methyladenosine (m6A) methylation methyltransferase, TRDMT1 (P<0.05), an RNA 5-methylcytosine (m5C) methylation methyltransferase, and ASC (P<0.05), an NLRP3 inflammasome player. These results indicate that DIBP at environmentally relevant doses regulates gene expression of epigenetic and inflammatory pathways but does not alter GC proliferation or steroidogenesis. The second project aimed to characterize the phthalate concentrations on a livestock farm. Phthalate concentrations were measured in alfalfa hay, corn silage, and pasture. Lactating multiparous Angus cows were fed with alfalfa hay or corn silage for 49 hours. Cows were maintained on pasture prior to exposure to other types of feed. Urine and blood samples were collected before and during feedstuff exposure to examine phthalate metabolite levels. Silage presented higher concentrations of Di-ethyl phthalate (DEP), Benzylbutyl phthalate (BBP), and Di-isononyl phthalate (DINP) than pasture and hay (P<0.05), and hay presented higher concentrations of Di-n-octyl phthalate (DOP) and DINP than pasture (P<0.05). During the trial, hay-fed group had higher urine concentrations of Mono-isobutyl phthalate (MIBP), Mono-n-butyl phthalate (MBP), Mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP), Mono-2-ethylhexyl terephthalate (MEHtP) compared with silage-fed group (P<0.05), and silage fed group had higher urine concentrations of Mono-n-pentyl phthalate (MPP), Mono-cyclohexyl phthalate (MCHP), Mono-2-ethylhexyl phthalate (MEHP), Mono-isononyl phthalate (MINP) (P<0.05). Urine concentrations of MCHP and Mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) were higher in silage-fed group at the end of the trial compared with prior to the start of the trial (P<0.05). Urine concentrations of MIBP and MEOHP were higher in hay fed group at the end of the trial compared with prior to the start of the trial (P<0.05). No differences in plasma phthalate metabolite concentrations were found between hay-fed group and silage-fed group after 49 hours of feeding. However, because Mono-benzyl phthalate (MBzP) had the highest plasma concentrations, it was selected to determine its effects on in vitro cultured bovine GC function. Bovine GC were treated with MBzP at concentrations found in the plasma in the animal trial combined with FSH+IGF1. Bovine GC steroidogenesis or proliferation were not affected by MBzP treatments. The findings demonstrated that silage and, to a lesser extent, hay are possible sources of phthalate exposure for cattle and that feeding them silage or hay resulted in greater amounts of several phthalates. Taken together, these studies reinforce the importance of monitoring environmental contaminants in livestock production and assessing the effects of phthalates at environmentally relevant doses on human and animal reproduction

Implicaciones de la hormona de crecimiento, hormonas gastrointestinales, irisina y "microARNs" en la obesidad

[Resumo]A obesidade é unha das principais preocupacións relacionadas coa saúde pública. Na obesidade prodúcense diferentes alteracións hormonais, como o aumento da leptina e a dlminución da hormona de crecemento (GH) e a ghrelina. Entre os estímulos secretorios de GH que se conserva na obesidad e é destacable o que se presenta de forma tardía tras a sobrecarga oral de glucosa (SOG). A ghrelina ten unha potente acción como factor oresixénico e poderla ser un sinal fundamental relacionando a inxestión co desenvolvemento da obesidade. Existen outras hormonas importantes na regulación da inxestión, entre elas o péptido Y (PYV). Ademais o descubrimento recente dunha nova hormona segregada polo músculo esquelético (irisina) abre o abano no estudo das alteracions hormonais na obesidade. Por outra parte, os microARNs (mIARNs) postuláronse como posibles reguladores dunha serie de procesos biolóxicos, incluida a adipoxénese. Neste contexto, o obxectivo global desta tese é avanzar na caracterización do papel da GH, PYV, irisina e ghrelina en pacientes normais, obesos e hipopituitarios e por outra parte estudar o papel dos miARNs hipotalámicos nun modelo animal na regulaci6n de diversos problemas metabóicos asociados coa obesidade como a resistencia á insulina e á leptina. Podemos concluir que tras a SOG os niveis de GH e ghrelina total están diminuidos nos pacientes obesos con respecto aos controis, ademais en mulleres encontramos que a ghrellna circulante é un importante regulador da secreción da GH, que os valores de irisina son superiores en obesos e que existen varias familias de miARNs relacionados coas rutas de sinalizaci6n da leptina e a insulina que se atopan alterados a nivel hipotalámico en ratas obesas, anoréxicas e/ou tratadas con leptina.[Resumen]La obesidad es una de las principales preocupaciones relacionadas con la salud pública. En la obesidad se producen diferentes alteraciones hormonales, como el aumento de la leptina y la disminución de la hormona de crecimiento (GH) y la ghrellna. Entre los estlmulos secretorios de GH que se conservan en la obesidad es destacable el que se presenta de forma tardla tras la sobrecarga oral de glucosa (SOG). La ghrelina tiene una potente acción como factor orexigénico y podrfa ser una seílal fundamental relacionando la ingesta con el desarrollo de la obesidad. Existen otras hormonas importantes en la regulación de la ingesta, entre ellas el péptido Y (PVY). Además el descubrimiento reciente de una nueva hormona secretada por el músculo esquelético (irisina) abre el abanico en el estudio de las alteraciones hormonales en la obesidad. Por otra parte, los microARNs (miARNs) se postularon como posibles reguladores de una serie de procesos biológicos, incluida la adipogénesis. En este contexto, el objetivo global de esta tesis es avanzar en la caracterización del papel de la GH, PVY, irisina y ghrelina en pacientes normales, obesos e hipopituitarios y por otra parte estudiar el papel de los miARNs hlpotalámlcos en un modelo animal en la regulación de diversos problemas metabólicos asociados con la obesidad como la resistencia a la insulina ya la leptina. Podemos concluir que tras la SOG los niveles de GH V ghrelina total están disminuidos en los pacientes obesos con respecto a los controles, además en mujeres encontramos que la ghrelina circulante es un importante regulador de la secreción de la GH, que los valores de irisina son superiores en obesos V que existen varias familias de miARNs relacionados con las rutas de seílalización de la leptina V la insulina que se encuentran alterados a nivel hipotalámico en ratas obesas, anoréxicas V/o tratadas con leptina[Abstract]Obesity is one of the main concerns related to the public health. In the obesity different hormonal alteratlons, as the increase of leptin and the decrease of growth hormone (GH) and ghrelin, take place. Among the GH secretory stimuli that are preserved in the obesity is noteworthy which occurs at a late stage after the oral glucose overload (OGO). Ghrelin has a powerful action as orexlgenic factor and could be a basic signal relating the food intake to the development of the obesity. There are other important hormones in the regulation of the ingestion, among them the peptide Y (PYY). Besides the recent discovery of a new hormone secreted by the skeletal muscle (irlsin) opens the range in the study of the hormonal alterations in the obesity. On the other hand, the microRNAs (miRNAs) were postulated as possible regulators of a series of biological processes, included adipogenesis. In thls context, the global goal of thls thesis is to advance in the characterization of role the GH, PYY, irisin and ghrelin in normal, obese and hypopituitary patients and on the other hand, to study the role of hypothalamic miRNAs in an animal model in the regulation of different metabolic problems related with the obesity like insulin and leptin resistance. We can conclude that after the OGO GH and total ghrelin levels are decreased in the obese patients with regard to the controls, besides in women we find that circulating ghrelin levels are an important regulator of GH secretion, that blood irisin levels are superior In obese and moreover several famlies of miRNAs related with leptin and insulin signaling pathways show hypothalamic expression levels altered in obese, anorexic and/or with leptin treatment

Perturbation of hypothalamic MicroRNA expression patterns in male rats after metabolic distress: impact of obesity and conditions of negative energy balance

[Abstract] The hypothalamus plays a crucial role in body weight homeostasis through an intricate network of neuronal circuits that are under the precise regulation of peripheral hormones and central transmitters. Although deregulated function of such circuits might be a major contributing factor in obesity, the molecular mechanisms responsible for the hypothalamic control of energy balance remain partially unknown. MicroRNAs (miRNAs) have been recognized as key regulators of different biological processes, including insulin sensitivity and glucose metabolism. However, the roles of miRNA pathways in the control of metabolism have been mostly addressed in peripheral tissues, whereas the potential deregulation of miRNA expression in the hypothalamus in conditions of metabolic distress remains as yet unexplored. In this work, we used high-throughput screening to define to what extent the hypothalamic profiles of miRNA expression are perturbed in two extreme conditions of nutritional stress in male rats, namely chronic caloric restriction and high-fat diet–induced obesity. Our analyses allowed the identification of sets of miRNAs, including let-7a, mir-9*, mir-30e, mir-132, mir-145, mir-200a, and mir-218, whose expression patterns in the hypothalamus were jointly altered by caloric restriction and/or a high-fat diet. The predicted targets of these miRNAs include several elements of key inflammatory and metabolic pathways, including insulin and leptin. Our study is the first to disclose the impact of nutritional challenges on the hypothalamic miRNA expression profiles. These data will help to characterize the molecular miRNA signature of the hypothalamus in extreme metabolic conditions and pave the way for targeted mechanistic analyses of the involvement of deregulated central miRNAs pathways in the pathogenesis of obesity and related disorders.Instituto de Salud Carlos III; PI10/00088Ministerio de Economia y Competitividad; IN845B-2010/187Instituto de Salud Carlos III; PI13/00322FISXunta de Galicia; 10CSA916014PRXunta de Galicia; EM2013/011Ministerio de Ciencia e Innovación; BFU 2011-2502

Place of Preoperative Treatment of Acromegaly with Somatostatin Analog on Surgical Outcome: A Systematic Review and Meta-Analysis

Context: Transsphenoidal neurosurgery is the accepted first-line treatment of acromegaly in the majority of patients. Previous studies addressing preoperative somatostatin analog (SSA) treatment and subsequent surgical cure rates are conflicting, reporting either benefits or no significant differences. Objective: The aim of this study, based on a meta-analysis of all published reports, was to investigate whether treatment with SSA before surgery improves the surgical outcome of acromegaly. Data sources: All studies of preoperative treatment of acromegaly with SSA were systematically reviewed up to December 2011. We searched the Medline, Embase, Cochrane and Google Scholar electronic databases. Study selection: The primary endpoint was the biochemical postoperative cure rate. We identified 286 studies, out of which 10 studies (3.49%) fulfilling the eligibility criteria were selected for analysis; five retrospective studies with a control group, two prospective non-randomized trials, and three prospective controlled trials. The meta-analysis was conducted using the random-effects model. Data extraction: Data were extracted from published reports by two independent observers. Data synthesis: A borderline effect was detected in the analysis of all of the trials with control groups, with a pooled odds ratio (OR) for biochemical cure with SSA treatment of 1.62 (95% CI, 0.93-2.82). In the analysis of the three prospective controlled trials, a statistically significant effect was identified OR: 3.62 (95% CI, 1.88-6.96). Conclusions: Preoperative treatment with SSA og GH-secreting pituitary adenomas shows a significant improvement on surgical results. This meta-analysis suggests that in centers without optimal results all patients with a GH-secreting pituitary macroadenoma should be treated with a long-acting SSA prior to surgical treatment

La expresión génica de FNDC5 en distintos tejidos, y los niveles circulantes de irisina, están modificados por la dieta y las condiciones hormonales

ResumenXunta de Galicia; EM2013/011Instituto de Salud Carlos III; PI13/0032

Alteración de la expresión génica de Sor1 y RB1 en distintos modelos de estrés metabólico

AbstractXunta de Galicia; EM2013/011Instituto de Salud Carlos III; PI13/0032

FNDC5 expression and circulating irisin levels are modified by diet and hormonal conditions in hypothalamus, adipose tissue and muscle

[Abstract] Irisin is processed from fibronectin type III domain-containing protein 5 (FNDC5). However, a controversy exists concerning irisin origin, regulation and function. To elucidate the relationship between serum irisin and FNDC5 mRNA expression levels, we evaluated plasma irisin levels and FNDC5 gene expression in the hypothalamus, gastrocnemius muscle and different depots of adipose tissue in models of altered metabolism. In normal rats, blood irisin levels diminished after 48-h fast and with leptin, insulin and alloxan treatments, and serum irisin concentrations increased in diabetic rats after insulin treatment and acute treatments of irisin increased blood insulin levels. No changes were observed during long-term experiments with different diets. We suggested that levels of circulating irisin are the result of the sum of the irisin produced by different depots of adipose tissue and skeletal muscle. This study shows for the first time that there are differences in FNDC5 expression depending on white adipose tissue depots. Moreover, a considerable decrease in visceral and epididymal adipose tissue depots correlated with increased FNDC5 mRNA expression levels, probably in an attempt to compensate the decrease that occurs in their mass. Hypothalamic FNDC5 expression did not change for any of the tested diets but increased with leptin, insulin and metformin treatments suggesting that the regulation of central and peripheral FNDC5/irisin expression and functions are different.Galicia. Consellería de Cultura, Educación e Ordenación Universitaria; EM2013/011Instituto de Salud Carlos III; PI13/0032

Niveles séricos de la adipomioquina irisina en pacientes con enfermedad renal crónica

[Abstract] Background. Irisin is an adipomyokine with claimed anti-obesity and anti-diabetic effects. This hormone has been insufficiently studied in patients with advanced chronic kidney disease (CKD). Objective. To perform an exploratory analysis of serum irisin levels in patients undergoing different CKD treatments. Method. Following a cross-sectional design, we estimated serum levels of irisin in 95 patients with CKD managed conservatively (advanced CKD), with peritoneal dialysis (PD) or with haemodialysis, and compared our findings with a control group of 40 healthy individuals. We investigated the correlations between serum irisin and demographic, clinical, body composition and metabolic variables. Results. Irisin levels were lower in all the CKD groups than in the control group. The univariate analysis revealed limited correlations between irisin, on the one hand, and fat (but not lean) mass, glomerular filtration rate (GFR) and plasma albumin and bicarbonate, on the other. The multivariate analysis confirmed that advanced CKD patients managed conservatively (difference 111.1 ng/mL), with PD (25.9 ng/mL) or haemodialysis (61.4 ng/mL) (all p < .0005) presented lower irisin levels than the control group. Furthermore, PD patients presented higher serum levels of irisin than those on haemodialysis (difference 39.4 ng/mL, p = .002) or those managed conservatively (24.4 ng/mL, p = .036). The multivariate analysis also identified plasma bicarbonate (B = 3.90 per mM/l, p = .001) and GFR (B = 1.89 per mL/min, p = .003) as independent predictors of irisin levels. Conversely, no adjusted correlation between irisin and body composition markers was found. Conclusions. Serum irisin levels are low in patients with CKD and show a consistent correlation with GFR and plasma bicarbonate levels. PD patients present higher levels of irisin than those managed conservatively or with haemodialysis. Our study confirms a general inconsistency of the association between serum irisin levels, on the one hand, and body composition and metabolic markers, on the other.[Resumen] Antecedentes. La irisina es una adipomioquina con posibles efectos antiobesidad y antidiabéticos. Esta hormona ha sido insuficientemente estudiada en pacientes con enfermedad renal crónica (ERC) avanzada. Objetivo. Realizar un análisis exploratorio de los niveles séricos de irisina en pacientes con diferentes modalidades de tratamiento de la ERC. Método. Según diseño transversal, estimamos niveles de irisina en 95 pacientes con ERC manejados conservadoramente (ERCA), con diálisis peritoneal (DP) o con hemodiálisis, comparándolos con un grupo control de 40 individuos sanos. También investigamos las correlaciones entre irisina sérica y variables demográficas, clínicas, metabólicas y de composición corporal. Resultados. Los niveles de irisina fueron más bajos en cualquier grupo de pacientes que en los controles. El análisis univariante desveló correlaciones moderadas entre irisina, por un lado, y masa grasa (pero no magra), filtrado glomerular (GFR) y albúmina y bicarbonato plasmático, por otro. El análisis multivariante confirmó que los pacientes con ERCA (diferencia 111,1 ng/mL), en DP (25,9 ng/mL) o hemodiálisis (61,4 ng/mL) (todos p < 0,0005) presentaban niveles ajustados más bajos de irisina que los controles. Asimismo, los pacientes en DP presentaban niveles más altos de la hormona que los de hemodiálisis (diferencia 39,4 ng/mL; p = 0,002) o ERCA (24,4 ng/mL; p = 0,036). El análisis multivariante también identificó bicarbonato plasmático (B = 3,90 por mM/L; p = 0,001) y GFR (B = 1,89 por mL/min; p = 0,003) como predictores independientes de los niveles de irisina. Por el contrario, no observamos correlación ajustada entre irisina y marcadores de composición corporal. Conclusiones. Los niveles de irisina son bajos en pacientes con ERC, y muestran correlación consistente con GFR y bicarbonato plasmático. Los pacientes en DP presentan niveles más altos de irisina que los manejados conservadoramente o con hemodiálisis. Nuestro estudio confirma una inconsistencia general en los análisis de asociación entre irisina sérica, por un lado, y marcadores metabólicos y de composición corporal, por otro.Instituto de Salud Carlos III; PI10/00088Instituto de Salud Carlos III; PI13/00322Xunta de Galicia; IN845B-2010/187Xunta de Galicia; 10CSA916014PRXunta de Galciia; CN2012/31

Sexual dimorphism on growth hormone secretion after oral glucose administration

[Abstract] Sexual dimorphism of GH secretion is unclear in humans. There is evidence that oral glucose (OG) administration initially decreases and subsequently stimulates GH secretion. Our aim was to study fasting GH concentrations and their response to OG administration in obese and healthy women and men, in order to elucidate the mechanism of sexual dimorphism of GH secretion and the possible contribution of ghrelin. We selected 33 women and 11 men as obese and healthy subjects. After an overnight fast, 75 g of oral glucose were administered; glucose, insulin, ghrelin, and PYY1-36 were obtained at baseline and during 300 min. Fasting GH (μg/l) was higher in women than men; 1.3 ± 0.3 vs. 0.2 ± 0.1, p=0.009, for women and men, respectively. The area under the curve between 0 and 150 min (AUC) of GH (μg/l · min) was higher in women than men; 98.2 ± 25.9 vs. 41.5 ± 28.6, p=0.002, for women and men, respectively. The AUC of total ghrelin (pg/ml · min, mean ± SEM) between 0 and 150 min was borderline and significantly higher in women than men; 128 562.3 ± 8 335.9 vs. 98 839.1 ± 7 668.6, p=0.069, for women and men, respectively. Several initial time points were higher in women than men. Glucose, insulin, and PYY1-36 were similar in women and men after OG. There were significant correlations between indices of post-oral glucose GH and ghrelin secretion. Fasting and initial GH secretion is higher in women than men, in contrast to peak and late GH secretion, which is similar in both cases. Sexual dimorphism in the regulation of GH secretion probably involves ghrelin.Instituto de Salud Carlos III; PI070413Instituto de Salud Carlos III; PI10/00088Xunta de Galicia; PS07/12Xunta de Galicia; INCITE08ENA916110ESXunta de Galicia; INCITE09E1R91634ESXunta de Galicia; IN845B-2010/187Xunta de Galicia; 10CSA916014P