Relationships of dehydroepiandrosterone sulfate in the elderly with functional, psychological, and mental status, and short-term mortality: A French community-based study

In human beings of both sexes, dehydroepiandrosterone sulfate (DHEAS) circulating in blood is mostly an adrenally secreted steroid whose serum concentration (in the micromolar range and 30–50% higher in men than in women) decreases with age, toward ≈20–10% of its value in young adults during the 8th and 9th decades. The mechanism of action of DHEA and DHEAS is poorly known and may include partial transformation into sex steroids, increase of bioavailable insulin-like growth factor I, and effects on neurotransmitter receptors. Whether there is a cause-to-effect relationship between the decreasing levels of DHEAS with age and physiological and pathological manifestations of aging is still undecided, but this is of obvious theoretical and practical interest in view of the easy restoration by DHEA administration. Here we report on 622 subjects over 65 years of age, studied for the 4 years since DHEAS baseline values had been obtained, in the frame of the PAQUID program, analyzing the functional, psychological, and mental status of a community-based population in the south-west of France. We confirm the continuing decrease of DHEAS serum concentration with age, more in men than in women, even if men retain higher levels. Significantly lower values of baseline DHEAS were recorded in women in cases of functional limitation (Instrumental Activities of Daily Living), confinement, dyspnea, depressive symptomatology, poor subjective perception of health and life satisfaction, and usage of various medications. In men, there was a trend for the same correlations, even though not statistically significant in most categories. No differences in DHEAS levels were found in cases of incident dementia in the following 4 years. In men (but not in women), lower DHEAS was significantly associated with increased short-term mortality at 2 and 4 years after baseline measurement. These results, statistically established by taking into account corrections for age, sex, and health indicators, suggest the need for further careful trials of the administration of replacement doses of DHEA in aging humans. Indeed, the first noted results of such “treatment” are consistent with correlations observed here between functional and psychological status and endogenous steroid serum concentrations.

Cyp7b, a novel brain cytochrome P450, catalyzes the synthesis of neurosteroids 7α-hydroxy dehydroepiandrosterone and 7α-hydroxy pregnenolone

Steroids produced locally in brain (neurosteroids), including dehydroepiandrosterone (DHEA), influence cognition and behavior. We previously described a novel cytochrome P450, Cyp7b, strongly expressed in rat and mouse brain, particularly in hippocampus. Cyp7b is most similar to steroidogenic P450s and potentially could play a role in neurosteroid metabolism. To examine the catalytic activity of the enzyme mouse Cyp7b cDNA was introduced into a vaccinia virus vector. Extracts from cells infected with the recombinant showed NADPH-dependent conversion of DHEA (Km, 13.6 μM) and pregnenolone (Km, 4.0 μM) to slower migrating forms on thin layer chromatography. The expressed enzyme was less active against 25-hydroxycholesterol, 17β-estradiol and 5α-androstane-3β,17β-diol, with low to undetectable activity against progesterone, corticosterone, and testosterone. On gas chromatography and mass spectrometry of the Cyp7b metabolite of DHEA the retention time and fragmentation patterns were identical to those obtained with authentic 7α-hydroxy DHEA. The reaction product also comigrated on thin layer chromatography with 7α-hydroxy DHEA but not with 7β-hydroxy DHEA; when [7α-3H]pregnenolone was incubated with Cyp7b extracts the extent of release of radioactivity into the medium suggested that hydroxylation was preferentially at the 7α position. Brain extracts also efficiently liberated tritium from [7α-3H]pregnenolone and converted DHEA to a product with a chromatographic mobility indistinguishable from 7α-hydroxy DHEA. We conclude that Cyp7b is a 7α-hydroxylase participating in the synthesis, in brain, of neurosteroids 7α-hydroxy DHEA, and 7α-hydroxy pregnenolone.

Hyposecretion of the adrenal androgen dehydroepiandrosterone sulfate and its relation to clinical variables in inflammatory arthritis

Hypothalamic–pituitary–adrenal underactivity has been reported in rheumatoid arthritis (RA). This phenomenon has implications with regard to the pathogenesis and treatment of the disease. The present study was designed to evaluate the secretion of the adrenal androgen dehydroepiandrosterone sulfate (DHEAS) and its relation to clinical variables in RA, spondyloarthropathy (Spa), and undifferentiated inflammatory arthritis (UIA). Eighty-seven patients (38 with RA, 29 with Spa, and 20 with UIA) were studied, of whom 54 were women. Only 12 patients (14%) had taken glucocorticoids previously. Age-matched, healthy women (134) and men (149) served as controls. Fasting blood samples were taken for determination of the erythrocyte sedimentation rate (ESR), serum DHEAS and insulin, and plasma glucose. Insulin resistance was estimated by the homeostasis-model assessment (HOMAIR). DHEAS concentrations were significantly decreased in both women and men with inflammatory arthritis (IA) (P < 0.001). In 24 patients (28%), DHEAS levels were below the lower extreme ranges found for controls. Multiple intergroup comparisons revealed similarly decreased concentrations in each disease subset in both women and men. After the ESR, previous glucocorticoid usage, current treatment with nonsteroidal anti-inflammatory drugs, duration of disease and HOMAIR were controlled for, the differences in DHEAS levels between patients and controls were markedly attenuated in women (P = 0.050) and were no longer present in men (P = 0.133). We concluded that low DHEAS concentrations are commonly encountered in IA and, in women, this may not be fully explainable by disease-related parameters. The role of hypoadrenalism in the pathophysiology of IA deserves further elucidation. DHEA replacement may be indicated in many patients with IA, even in those not taking glucocorticoids.

Ergosteroids: induction of thermogenic enzymes in liver of rats treated with steroids derived from dehydroepiandrosterone.

Dehydroepiandrosterone (DHEA), an intermediate in the biosynthesis of testosterone and estrogens, exerts several physiological effects not involving the sex hormones. When fed to rats it induces the thermogenic enzymes mitochondrial sn-glycerol-3-phosphate dehydrogenase and cytosolic malic enzyme in their livers. Animals and humans, and their excised tissues, are known to hydroxylate DHEA at several positions and to interconvert 7 alpha-hydroxy-DHEA, 7 beta-hydroxy-DHEA, 7-oxo-DHEA, and the corresponding derivatives of androst-5-enediol. We report here that these 7-oxygenated derivatives are active inducers of these thermogenic enzymes in rats and that the 7-oxo derivatives are more active than the parent steroids. We postulate that the 7 alpha-hydroxy and 7-oxo derivatives are on a metabolic pathway from DHEA to more active steroid hormones. These 7-oxo steroids have potential as therapeutic agents because of their increased activity and because they are not convertible to either testosterone or estrogens.

Neurosteroids, via sigma receptors, modulate the [3H]norepinephrine release evoked by N-methyl-D-aspartate in the rat hippocampus.

N-Methyl-D-aspartate (NMDA, 200 microM) evokes the release of [3H]norepinephrine ([3H]NE) from preloaded hippocampal slices. This effect is potentiated by dehydroepiandrosterone sulfate (DHEA S), whereas it is inhibited by pregnenolone sulfate (PREG S) and the high-affinity sigma inverse agonist 1,3-di(2-tolyl)guanidine, at concentrations of > or = 100 nM. Neither 3 alpha-hydroxy-5 alpha-pregnan-20-one nor its sulfate ester modified NMDA-evoked [3H]NE overflow. The sigma antagonists haloperidol and 1-[2-(3,4-dichlorophenyl)-ethyl]-4-methylpiperazine, although inactive by themselves, completely prevented the effects of DHEA S, PREG S, and 1,3-di(2-tolyl)guanidine on NMDA-evoked [3H]NE release. Progesterone (100 nM) mimicked the antagonistic effect of haloperidol and 1-[2-(3,4-dichlorophenyl)ethyl]-4-methyl-piperazine. These results indicate that the tested steroid sulfate esters differentially affected the NMDA response in vitro and suggest that DHEA S acts as a sigma agonist, that PREG S acts as a sigma inverse agonist, and that progesterone may act as a sigma antagonist. Pertussis toxin, which inactivates the Gi/o types of guanine nucleotide-binding protein (Gi/o protein) function, suppresses both effects of DHEA S and PREG S. Since sigma 1 but not sigma 2 receptors are coupled to Gi/o proteins, the present results suggest that DHEA S and PREG S control the NMDA response via sigma 1 receptors.

Effects of follicular versus luteal phase-based strength training in young women

Hormonal variations during the menstrual cycle (MC) may influence trainability of strength. We investigated the effects of a follicular phase-based strength training (FT) on muscle strength, muscle volume and microscopic parameters, comparing it to a luteal phase-based training (LT). Eumenorrheic women without oral contraception (OC) (N = 20, age: 25.9 ± 4.5 yr, height: 164.2 ± 5.5 cm, weight: 60.6 ± 7.8 kg) completed strength training on a leg press for three MC, and 9 of them participated in muscle biopsies. One leg had eight training sessions in the follicular phases (FP) and only two sessions in the luteal phases (LP) for follicular phase-based training (FT), while the other leg had eight training sessions in LP and only two sessions in FP for luteal phase-based training (LT). Estradiol (E2), progesterone (P4), total testosterone (T), free testosterone (free T) and DHEA-s were analysed once during FP (around day 11) and once during LP (around day 25). Maximum isometric force (Fmax), muscle diameter (Mdm), muscle fibre composition (No), fibre diameter (Fdm) and cell nuclei-to-fibre ratio (N/F) were analysed before and after the training intervention. T and free T were higher in FP compared to LP prior to the training intervention (P < 0.05). The increase in Fmax after FT was higher compared to LT (P <0.05). FT also showed a higher increase in Mdm than LT (P < 0.05). Moreover, we found significant increases in Fdm of fibre type ΙΙ and in N/F only after FT; however, there was no significant difference from LT. With regard to change in fibre composition, no differences were observed between FT and LT. FT showed a higher gain in muscle strength and muscle diameter than LT. As a result, we recommend that eumenorrheic females without OC should base the periodization of their strength training on their individual MC.

Terapêutica da osteopénia e osteoporose em mulheres com anorexia nervosa

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Oxidation of protein in human low-density lipoprotein exposed to peroxyl radicals facilitates uptake by monocytes; protection by antioxidants in vitro

Generation of neoepitopes on apolipoprotein B within oxidised low-density lipoprotein (LDL) is important in the unregulated uptake of LDL by monocytic scavenger receptors (CD36, SR-AI, LOX-1). Freshly isolated LDL was oxidised by peroxyl radicals generated from the thermal decomposition of an aqueous azo-compound. We describe that formation of carbonyl groups on the protein component is early as protein oxidation was seen after 90min. This is associated with an increased propensity for LDL uptake by U937 monocytes. Three classes of antioxidants (quercetin, dehydroepiandrosterone (DHEA) and ascorbic acid) have been examined for their capacity to inhibit AAPH-induced protein oxidation, (protein carbonyls, Δ electrophoretic mobility and LDL uptake by U937 monocytes). CD36 expression was assessed by flow cytometry and was seen to be unaltered by oxidised LDL uptake. All three classes were effective antioxidants, quercetin (P<0.01), ascorbic acid (P<0.01), DHEA (P<0.05). As LDL protein is the control point for LDL metabolism, the degree of oxidation and protection by antioxidants is likely to be of great importance for (patho)-physiological uptake of LDL by monocytes. © 2003 Elsevier B.V. All rights reserved.

Dehdyroepiandrosterone sulfate directly activates protein kinase C-β to increase human neutrophil superoxide generation

Dehydroepiandrosterone sulfate (DHEAS) is the most abundant steroid in the human circulation and is secreted by the adrenals in an age-dependent fashion, with maximum levels during the third decade and very low levels in old age. DHEAS is considered an inactive metabolite, whereas cleavage of the sulfate group generates dehydroepiandrosterone (DHEA), a crucial sex steroid precursor. However, here we show that DHEAS, but not DHEA, increases superoxide generation in primed human neutrophils in a dose-dependent fashion, thereby impacting on a key bactericidal mechanism. This effect was not prevented by coincubation with androgen and estrogen receptor antagonists but was reversed by the protein kinase C inhibitor Bisindolylmaleimide 1. Moreover, we found that neutrophils are unique among leukocytes in expressing an organic anion-transporting polypeptide D, able to mediate active DHEAS influx transport whereas they did not express steroid sulfatase that activates DHEAS to DHEA. A specific receptor for DHEAS has not yet been identified, but we show that DHEAS directly activated recombinant protein kinase C-ß (PKC-ß) in a cell-free assay. Enhanced PKC-ß activation by DHEAS resulted in increased phosphorylation of p47phox, a crucial component of the active reduced nicotinamide adenine dinucleotide phosphate complex responsible for neutrophil superoxide generation. Our results demonstrate that PKC-ß acts as an intracellular receptor for DHEAS in human neutrophils, a signaling mechanism entirely distinct from the role of DHEA as sex steroid precursor and with important implications for immunesenescence, which includes reduced neutrophil superoxide generation in response to pathogens.

Salmeterol enhances the cardiac response to gene therapy in Pompe disease.

Enzyme replacement therapy (ERT) with recombinant human (rh) acid α-glucosidase (GAA) has prolonged the survival of patients. However, the paucity of cation-independent mannose-6-phosphate receptor (CI-MPR) in skeletal muscle, where it is needed to take up rhGAA, correlated with a poor response to ERT by muscle in Pompe disease. Clenbuterol, a selective β2 receptor agonist, enhanced the CI-MPR expression in striated muscle through Igf-1 mediated muscle hypertrophy, which correlated with increased CI-MPR (also the Igf-2 receptor) expression. In this study we have evaluated 4 new drugs in GAA knockout (KO) mice in combination with an adeno-associated virus (AAV) vector encoding human GAA, 3 alternative β2 agonists and dehydroepiandrosterone (DHEA). Mice were injected with AAV2/9-CBhGAA (1E+11 vector particles) at a dose that was not effective at clearing glycogen storage from the heart. Heart GAA activity was significantly increased by either salmeterol (p<0.01) or DHEA (p<0.05), in comparison with untreated mice. Furthermore, glycogen content was reduced in the heart by treatment with DHEA (p<0.001), salmeterol (p<0.05), formoterol (p<0.01), or clenbuterol (p<0.01) in combination with the AAV vector, in comparison with untreated GAA-KO mice. Wirehang testing revealed that salmeterol and the AAV vector significantly increased performance, in comparison with the AAV vector alone (p<0.001). Similarly, salmeterol with the vector increased performance significantly more than any of the other drugs. The most effective individual drugs had no significant effect in absence of vector, in comparison with untreated mice. Thus, salmeterol should be further developed as adjunctive therapy in combination with either ERT or gene therapy for Pompe disease.

Impacts moléculaires d’un excès d’acides gras sur l’androgenèse des cellules surrénaliennes

Le syndrome des ovaires polykystiques (SOPK) touche entre 5 à 10 % des femmes en âge de procréer et est associé à de nombreuses complications. Ce désordre endocrinien est caractérisé par des niveaux circulants élevés d’androgènes, dont la production est principalement modulée par la P450c17 et son cofacteur, soit la P450oxydoréductase (POR). Plusieurs études démontrent que l’hyperandrogénie présente chez les femmes SOPK pourrait être causée par la formation de phénomènes toxiques survenant à la suite de l’exposition des tissus non adipeux à un excès d’acides gras non estérifiés (AGNE), appelé lipotoxicité. Ainsi, l’objectif de cette étude est de déterminer les mécanismes cellulaires sous-jacents à l’hyperandrogénie induite par la surexposition des cellules productrices d’androgènes aux AGNE. Pour y arriver, les cellules surrénaliennes bovines (CSB) ont été exposées en présence de forskoline (Fsk; 1X/2 jours; 10 µM; activateur des adénylates cyclases) et d’oléate (acide gras monoinsaturé, 2X/jour; 200 µM) durant 48 heures. Par la suite, le milieu de culture a été prélevé afin de doser le DHEA (principal androgène surrénalien) par ELISA. De plus, les protéines ont été récoltées afin de déterminer l’expression protéique de la P450c17 et de POR par Western blot. Finalement, pour déterminer les activités 17αhydroxylase et 17,20-lyase de la P450c17, la concentration de plusieurs stéroïdes a été déterminé par LC-MS/MS et le ratio produit/substrat a été effectué. Les résultats sont présentés en moyenne ± SEM. Ainsi, sous stimulation à la Fsk, la présence de 200 µM d’oléate (vs absence d’oléate) augmente la production de DHEA de 114% par les CSB (n=17; 214 ± 20% vs 100 ± 0%; p<0,0001). De plus, l’ajout d’oléate n’affecte pas l’expression de la P450c17 (n=8; 98 ± 6% vs 100 ± 0%; p=0,74) et de POR (n=7; 119 ± 13% vs 100 ± 0%; p= 0,22). Finalement, la présence d’oléate augmente l’activité 17αhydroxylase de la P450c17 de 124% (n=7; 224 ± 19% vs 100 ± 0%; p= 0,02) et tends à augmenter l’activité 17,20-lyase de la P450c17 de 81% (n=7; 181 ± 28% vs 100 ± 0%; p= 0,08). Ainsi, l’augmentation de la production des androgènes induite par les AGNE pourrait être due principalement à leurs effets sur l’augmentation des deux activités enzymatiques de la P450c17, sans effet significatifs sur l’expression de la P450c17 ni de son cofacteur POR. Les mécanismes sous-jacents à l’augmentation des activités de la P450c17 demeurent à élucider.

Role of 17β-Hydroxysteroid Dehydrogenase Type 5 in Breast Cancer Studied by Intracrinology

Dans cette thèse, je présente une étude (1) du rôle de la 17β-HSD5 dans la modulation des taux d’hormones et dans la prolifération, et l’impact de l’expression de la 17β-HSD5 sur d’autres protéines de BC cellules; (2) une étude comparative sur trois enzymes (17β-HSD1, 17β-HSD7 et 3α-HSD3) avec la provision de DHEA et ses substrats directes soit l’E1 ou la DHT. Les principaux résultats obtenus dans cette étude sont les suivants: (1) en utilisant l’ARN d’interférence de la 17β-HSD5, des immunodosages enzymatiques et des tests de prolifération de cellules démontrent que l’expression de la 17β-HSD5 est positivement corrélée à un niveau de T et de DHT dans les BCC, mais négativement corrélée pour l’E2 et la prolifération des cellules de BC (2) les analyses quantitatives de PCR en temps réel et de Western blot ont démontré que l’inhibition de l’expression de la 17β-HSD5 régule à la hausse l’expression de l’aromatase dans les cellules MCF-7. (3) L’analyse d’ELISA de la prostaglandine E2 a vérifié que l’expression accrue de l’aromatase a été modulée par des niveaux élevés de PGE2 après l’inactivation de l’expression du gène de la 17β-HSD5. (4) Le test de cicatrisation a montré que l’inactivation de l’expression du gène de la 17β-HSD5 favorise l’augmentation de la migration cellulaire. (5) L’expression du gène 17β-HSD5 dans des échantillons cliniques, à partir de l’analyse de base de données ONCOMINE, a montré que sa plus faible expression a été corrélée avec le statut de l’HER-2 et de la métastase de la tumeur. (6) Les données protéomiques révèlent également que des protéines impliquées dans les voies métaboliques sont fortement exprimées dans les cellules MCF-7 après l’inactivation de l’expression du gène de la 17β-HSD5. (7) L’étude n’a démontré aucune différence dans la fonction biologique de la 17β-HSD1 et de la 17β-HSD7 lorsqu’elles sont cultivées avec différentes stéroïdes: tel que les niveaux de stéroides, la prolifération cellulaire et les protéines régulées. (8) Toutefois, la supplémentation du milieu de culture se révèle avoir un impact marqué sur l’étude de la 3α-HSD3. (9). Nous avons proposé que l’utilisation de la DHEA comme source d’hormone puisse entraîner une meilleure imitation des conditions physiologiques post-ménopausales en culture cellulaire selon l’intracrinologie.

Multidisciplinary approach to horse welfare in different management systems

From its domestication until nowadays, the horse has assumed multiple roles in human society. Over time, this condition and the lack of specific regulation have led to the development of different kinds of management systems for this species. This Ph.D. research project aims to investigate horses' welfare in different management practices and housing systems, considering a multidisciplinary approach, taking into account biological function, naturalness, and affective dimension. The results are presented in five articles that evidence risk factors that can mine horse welfare, and examine tools and parameters that can be employed for its assessment. Our research shows the importance of considering the evolutionary history and the species-specific and behavioural needs of horses in their management and housing. Sociality, free movement, diet composition and foraging routine, and the workload that these animals undergo are important factors that should be taken into account. Furthermore, this research has evidenced the importance of employing different parameters (e.g., behaviour, endocrinological parameters, and immune activity) in welfare assessment and proposes the use of horsehair DHEA (dehydroepiandrosterone) as a possible useful additional non-invasive measure for the investigation of long-term stress conditions. Finally, our results underline the importance of considering the affective dimension in welfare research. Recently, Judgement Bias Tests (JBT), which are based on the influence of affective states on the decision-making process, have been widely employed in animal welfare research. However, our studies show that the use of spatial JBT in horses can have some limitations. Still today several management systems do not fulfill species-specific needs of horses, thus the implementation of specific regulations could ameliorate horse welfare. A multidisciplinary approach to welfare assessment is fundamental, but it should be always remembered the individual and its own characteristics, which can influence not only physiological, immunological, and behavioural responses but also emotional and cognitive dimensions.

Ações preventivas em saúde do idoso: prevenção primária

Aborda as ações preventivas primárias em saúde do idoso, com enfoque na Imunização: Influenza ou gripe Myxovirus influenza, vacina anti-pneumocócia, vacina contra tétano e difteria; Quimioproteção: vitamina A e carotenoides, vitamina E, vitamina C e vitaminas do complexo B, vitamina B12, vitamina D, cálcio, selênio, ácidos graxos essenciais - Ômega3; e Hormônios: GH (hormônio de crescimento), dehidroepiandrosterona (DHEA) e sulfato-DHEA (DHEAS), testosterona e estrogênios. Curso de Especialização em Saúde da Pessoa Idosa (unidade 03, do módulo 03).