Steroid 21-hydroxylase expression in cultured rat astrocytes

Over the past decade or so it has become widely recognised that the brain is a significant steroidogenic organ. Many publications have highlighted the ability of the brain to synthesise and interconvert a large number of steroid products including cholesterol, progesterone and testosterone. In this study, in vitro experiments were performed to determine if 21-hydroxylation of steroids is undertaken by rat brain astrocytes in culture. This is a common reaction that occurs in the adrenal gland and other organs in mammals, catalysing the conversion of pregn-4-ene-3,20-dione (progesterone) to 21-hydroxypregn-4-ene-3,20-dione (deoxycorticosterone).

Previous reports have indicated that 21-hydroxylation occurs within the rat brain, however, the precise identity of the cells expressing 21-hydroxylase has not yet been determined. Several metabolites, such as 5α-pregnan-3α-ol-20-one (tetrahydroprogesterone) and 3α,21-dihydroxy-5-pregnan-20-one (tetrahydrodeoxycorticosterone) were of particular interest because of their modulatory role in neuronal function, such as their agonist activity at γ-aminobutyric acid (GABA_A) receptors.

Evidence was obtained for the expression of peripheral 21-hydroxylase enzyme (P450c21) in cultured rat brain astrocytes by a combination of mass spectroscopy and molecular biology techniques. This is a significant finding as expression of 21-hydroxylase within astrocytes may be indicative of a wider role for these cells in modulating neuronal behaviour.

1-14C-Linoleic Acid distrubution in various tissue lipids of guinea pigs following an oral dose

A recent study on the metabolism of 1-¹⁴C-α-linolenic acid in the guinea pig revealed that the fur had the highest specific activity of all tissues examined, 48 h after dosing. The present study investigated the pattern of tissue lipid labeling following an oral dose of 1-¹⁴C-linoleic acid after the animals had been dosed for the same time as above. Guinea pigs were fed one of two diets with a constant linoleic acid content (18% total fatty acids) and a different content of α-linolenic acid (0.3 or 17.3%) from weaning for 3 wk and 1-¹⁴C-linoleic acid was given orally to each animal for 48 h prior to sacrifice. The most highly labeled tissues (dpm/mg of linoleic acid) were liver, followed by brain, lung and spleen, heart, kidney and adrenal and intestines, in both diet groups. The liver had almost a three-fold higher specific activity than skin and fur which was more extensively labeled than the adipose and carcass. Approximately two-thirds of the label in skin plus fur was found in the fur which, because of a low lipid mass, would indicate that the fur was highly labeled. All tissues derived from animals on the diet with the low α-linolenic acid level were significantly more labeled than the tissues from the animals on the high α-linolenic acid diet, by a factor of 1.5 to 3. The phospholipid fraction was the most highly labeled fraction in the liver, free fatty acids were the most labeled fraction in skin & fur, while triacyglycerols were the most labeled in the carcass and adipose tissue. In these tissues, more than 90% of the radioactivity was found in fatty acids with 2-double bonds in the tissue lipids. These data indicate that the majority of label found in guinea pig tissues 48 h after dosing was still associated with a fatty acid fraction with 2-double bonds, which suggests there was little metabolism of linoleic acid to more highly unsaturated fatty acids in this time frame. In this study, the labeling of guinea pig tissues with linoleic acid, 48 h after dosing, was quite different from the labeling with α-linolenic acid reported previously. The retention of the administered radioactivity from ¹⁴C-linoleic acid in the whole body lipids was 1.6 times higher in the group fed the low α-linolenic acid diet (diet contained a total of 1.8 g PUFA/100 g diet)compared with the group fed the high α-linolenic acid diet (diet contained 3.6 g PUFA/100 g diet). The lack of retention of ¹⁴C-labeled lipids in the whole body would be consistent with an increased rate of β-oxidation of the labeled fatty acid on the diet rich in PUFA, a result supported by other studies using direct measurement of labeled carbon dioxide.

Dietary electrolytes are related to mood

Dietary therapies are routinely recommended to reduce disease risk; however, there is concern they may adversely affect mood. We compared the effect on mood of a low-sodium, high-potassium diet (LNAHK) and a high-calcium diet (HC) with a moderate-sodium, high-potassium, high-calcium Dietary Approaches to Stop Hypertension (DASH)-type diet (OD). We also assessed the relationship between dietary electrolytes and cortisol, a stress hormone and marker of hypothalamic–pituitary–adrenal (HPA) axis activity. In a crossover design, subjects were randomized to two diets for 4 weeks, the OD and either LNAHK or HC, each preceded by a 2-week control diet (CD). Dietary compliance was assessed by 24 h urine collections. Mood was measured weekly by the Profile of Mood States (POMS). Saliva samples were collected to measure cortisol. The change in mood between the preceding CD and the test diet (LNAHK or HC) was compared with the change between the CD and OD. Of the thirty-eight women and fifty-six men (mean age 56·3 (sem 9·8) years) that completed the OD, forty-three completed the LNAHK and forty-eight the HC. There was a greater improvement in depression, tension, vigour and the POMS global score for the LNAHK diet compared to OD (P < 0·05). Higher cortisol levels were weakly associated with greater vigour, lower fatigue, and higher levels of urinary potassium and magnesium (r 0·1–0·2, P < 0·05 for all). In conclusion, a LNAHK diet appeared to have a positive effect on overall mood.

Mate choice in zebra finches: does corticosterone play a role?

The importance of stress as a factor in influencing life history strategies has received considerable attention in recent years, because it appears to have a substantial impact on an individual's behaviour and physiology. Birds respond to environmental and social stressors by the production of corticosterone, a glucocorticoid hormone released by the adrenal gland. In this experiment, we tested whether female zebra finches preferred males selected to produce low or high peak levels of circulating plasma corticosterone. Plasma corticosterone and testosterone levels of the males were recorded, as were morphometric measurements and perch activity. Spectrophotometric measurements were also taken from several putatively sexually selected regions of the males. The females preferred the males from the low corticosterone lines to the high corticosterone males. In addition to, and consistent with this effect, females preferred males with the lowest corticosterone titres. Male activity, testosterone level, body size and mass had no effect on female preference. Leg and beak brightness were important, however, as were the brightness and chromaticity of the male cheek patch. These results are discussed in relation to contemporary hypotheses in sexual selection, particularly in the context of stress-mediated signalling.

Stress, cortisol and reproduction in female pigs

Two key hypotheses emerge in the literature regarding the impact of stress on reproduction in females of any species. First, prolonged stress impairs reproduction in females. Secondly, acute stress impairs reproduction, if it occurs at a critical time during the precisely timed series of endocrine events that induce oestrus and ovulation. We reviewed studies conducted in female pigs to find support or opposition for these hypotheses in female pigs. We also considered the role of cortisol. We found confirmation that prolonged stress or the prolonged elevation of cortisol can impair reproductive processes in female pigs, but also found that there appear to be some female pigs in which reproduction is resistant to such treatments. Reproduction in female pigs appears to be resistant to acute or repeated acute stress or elevation of cortisol, even if these occur during the series of precisely timed endocrine events that induce oestrus and ovulation. Thus, we propose modified versions of the above hypotheses that are specific to female pigs. Furthermore, while cortisol may mediate the effects of prolonged stress on reproduction in female pigs, there is evidence that, in female pigs, ACTH may require the presence of the adrenal glands to impair reproduction rather than having direct effects.

A sex difference in the cortisol response to tail docking and ACTH develops between 1 and 8 weeks of age in lambs

It is important to understand factors that may influence responses to stress, as these factors may also influence vulnerability to pathologies that can develop when stress responses are excessive or prolonged. It is clear that, in adults, the sex of an individual can influence the cortisol response to stress in a stressor specific manner. Nevertheless, the stage of development at which these sex differences emerge is unknown. We tested the hypothesis that there are sex differences in the cortisol response to tail docking and ACTH in lambs of 1 and 8 weeks of age. We also established cortisol responses in males when tail docking was imposed alone and in combination with castration at these ages. In experiment 1, 1 and 8 week old male and female lambs were subjected to sham handling, tail docking or, in males, a combination of tail docking and castration. In experiment 2, we administered ACTH (1.0 IU/kg) to male and female lambs at 1 and 8 weeks of age. There were significant cortisol responses to all treatments at both ages. Sex differences in the cortisol responses to tail docking and ACTH developed between 1 and 8 weeks of age, with females having greater responses than males. The data suggest that the mechanism for the sex difference in response to tail docking may involve the adrenal glands. At both ages, in males, the cortisol response to the combined treatment of tail docking and castration was significantly greater than that for tail docking alone.

Projections to the preoptic area from the paraventricular nucleus, acuate nucleus and the bed nucleus of the stria terminalis are unlikely to be involved in stress-induced suppression of GnRH secretion in sheep

Stress compromises reproductive function and the major physiological system activated during stress is the hypothalamo-pituitary-adrenal axis. Corticotrophin-releasing hormone and arginine vasopressin (AVP), which are produced in neurones of the paraventricular nucleus (PVN), drive the hypothalamo-pituitary-adrenal axis and are also implicated in the suppression of the reproductive axis. We used retrograde tracing and Fos labelling to map the projections from the PVN to the preoptic area (POA) where most gonadotrophin releasing hormone (GnRH) neurones are found. Fluorogold (FG) injections were made into the POA of gonadectomised male and female sheep (n = 5/sex), the animals were stressed and the brains recovered for histochemistry. All animals responded to stress with an increase in the number of Fos-labelled nuclei in the PVN. Few retrogradely labelled cells of the PVN were activated by stress. Dual labelling showed that very few FG-labelled cells also stained for corticotrophin-releasing hormone, none for AVP or enkephalin. Dual labelling for FG and Fos in the bed nucleus of the stria terminalis (BNST) and the arcuate nucleus showed that no FG-labelled cells in the BNST and only few in the ARC were activated by stress. No sex differences were observed in the activation of FG-labelled cells in any of the nuclei examined. We conclude that, although cells of the PVN, BNST and/or arcuate nucleus may affect reproduction via the GnRH cells of the POA, this is unlikely to involve direct input to the POA. If cells of these regions are involved in GnRH suppression during stress, this may occur via interneuronal pathways.

Co-localization and distribution of corticotrophin-releasing hormone, arginine vasopressin and enkephalin in the paraventricular nucleus of sheep : a sex comparison

The paraventricular nucleus (PVN) is integral to regulation of the hypothalamo-pituitary-adrenal (HPA) axis and contains cells producing corticotrophin-releasing hormone (CRH), arginine vasopressin (AVP) and enkephalin. We used immunohistochemistry to map these peptides and to resolve the extent of co-localization within PVN cells in intact and gonadectomized male and female sheep. Immunoreactive (ir) CRH, AVP and enkephalin cells were mapped in two rams and two ewes at 180 μm intervals throughout the rostro-caudal extent of the PVN. Similar distributions of AVP-ir cells occurred in both sexes whereas CRH-ir and enkephalin-ir cells extended more rostrally in rams. In groups (n=4) of intact and gonadectomized sheep of both sexes, co-localization and distribution of neuropeptides was influenced by sex and gonadectomy. Males had more AVP and CRH cells than females. Intact animals had more AVP cells than gonadectomized animals. There were no differences between groups in the number or percentage of cells that stained for both CRH and AVP or in the number of cells that stained for both CRH and enkephalin. Differences were observed in the percentage of enkephalin cells that contained CRH with males having a greater percentage of co-localized cells than did females. Differences were also observed in the number and percentage of cells that stained for both enkephalin and AVP; the number of cells that stained for both neuropeptides was greater in males than in females and greater in intact animals than in gonadectomized animals. Differences were observed in the percentage of AVP cells that contained enkephalin, and in the percentage of enkephalin cells that contained AVP with males having a greater percentage of co-localized cells than did females. We conclude that sex and gonadal status affect peptide distribution in the PVN of the sheep which may provide an anatomical basis for sex differences in HPA axis

Seasonal differences in the effect of isolation and restraint stress on the luteinizing hormone response to gonadotropin-releasing hormone in hypothalamopituitary disconnected, gonadectomized rams and ewes

Stress responses are thought to act within the hypothalamopituitary unit to impair the reproductive system, and the sites of action may differ between sexes. The effect of isolation and restraint stress on pituitary responsiveness to GnRH in sheep was investigated, with emphasis on possible sex differences. Experiments were conducted during the breeding season and the nonbreeding season. In both experiments, 125 ng of GnRH was injected i.v. every 2 h into hypothalamopituitary disconnected, gonadectomized rams and ewes on 3 experimental days, with each day divided into two periods. During the second period on Day 2, isolation and restraint stress was imposed for 5.5 h. Plasma concentrations of LH and cortisol were measured in samples of blood collected from the jugular vein. In the second experiment (nonbreeding season), plasma concentrations of epinephrine, norepinephrine, 3,4-dihydroxyphenylalanine, and 3,4-dihydroxyphenylglycol were also measured. In both experiments, there was no effect of isolation and restraint stress on plasma concentrations of cortisol in either sex. During the breeding season, there was no effect of isolation and restraint stress on plasma concentrations of LH in either sex. During the nonbreeding season, the amplitude of the first LH pulse after the commencement of stress was significantly reduced (P < 0.05) in rams and ewes. In the second experiment, during stress there was a significant increase (P < 0.05) in plasma concentrations of epinephrine in rams and ewes and significantly higher (P < 0.05) basal concentrations of norepinephrine in ewes than in rams. These results suggest that in sheep stress reduces responsiveness of the pituitary gland to exogenous GnRH during the nonbreeding season but not during the breeding season, possibly because of mediators of the stress response other than those of the hypothalamus-pituitary-adrenal gland axis.

Susceptibility of reproduction in female pigs to impairment by stress or elevation of cortisol

It is generally agreed that stress can impair reproduction. Furthermore, it is often thought that cortisol, which is secreted during stress as a result of activation of the hypothalamo-pituitary adrenal axis, is associated with this stress-induced impairment of reproduction. It has been hypothesized that reproduction in females is particularly susceptible to disruption by acute stress during the series of endocrine events that induce estrus and ovulation. Nevertheless, we found no support for this conjecture when we subjected female pigs to repeated acute stress or repeated acute elevation of cortisol during the period leading up to estrus and ovulation. Conversely, studies have demonstrated that prolonged stress and sustained elevation of cortisol can disrupt reproductive processes in female pigs. Nevertheless, in each study that demonstrated this effect, there were some animals subjected to the prolonged stressor or the sustained elevation of cortisol in which the reproductive parameters that were measured were not affected by the treatment. We propose that reproduction in female pigs is resistant to the effects of acute or repeated acute stress or acute or repeated acute elevation of cortisol even if these occur during the series of endocrine events that induce estrus and ovulation. Furthermore, while reproductive processes in some individuals are compromised, reproduction in a proportion of female pigs appears to be resistant to the effects of prolonged stress or sustained elevation of cortisol.

Androgen metabolism in the Australian lizard Tiliqua rugosa

Nonmammalian vertebrates possess some unusual features in their hormonal systems/ when compared to mammals. As a consequence, they can make an important contribution in investigations concerning the fundamental mechanisms operating in endocrinology. Such studies concerning androgens include inter alia their effects on developmental aspects in the brain of birds and related singing behaviour; the role of neural enzymes in reproductive processes in fish; and the relation between androgens and the stages of spermatogenesis in amphibia, The present thesis examines the biochemistry of androgens in the Australian lizard Tiliqua rugosa. The major compounds studied were testosterone and epitestosterone, which are known to be present in high concentrations in the plasma of the male animal. Previous investigations are expanded, particularly in the areas of steroid identification and testicular biosynthesis. In addition, preliminary studies on the metabolism in the brain (and other tissues) and plasma protein binding are reported. The presence of epitestosterone as a major free androgen in the plasma of the male lizard was confirmed. Other steroids were found in the sulphate fraction. Testosterone sulphate was the most rigorously identified compound, while some evidence was also found for the presence of conjugated 5-androstene-3β,17-diols, etiocholanolone and dehydroepiandrosterone (DHA). Epitestosterone does not appear to be extensively conjugated in this animal. Steroids were not found to be conjugated as glucuronides. The identification studies employed a novel method of electrochemical detection of steroids. This technique was investigated and extended in the current thesis. Biosynthetic studies were carried out on androgen interconversions in the testis, in vitro. The major enzyme activities detected were 17α-arid 17β-oxidoreductases (17α-OR and l7β-OR) and 3β-hydroxysteroid dehydrogenase (3β-HSD)/isonerase. No evidence was found for the presence of a steroid-17-epimerase that would directly interconvert testosterone and epitestosterone. The 17-oxidoreductases were found to be dependent on the cofactor NBDFH. Testosterone appears to be formed mainly via the 4-ene pathway, whereas epitestosterone is formed from both the 4- and 5-ene routes. The compound 5-androstene-3β, 17α-diol was found to be an intermediate in the synthesis of epitestosterone from DHA. Temperature was found to significantly affect 17α-OR activity (maximum at 32°C). In contrast,17β-OR activity was independent of this factor in the testis. Androgen metabolism in the testis was found to be regulated by cofactors, temperature and season. The major enzyme activities found in the male brain were 17α- and 17β-OR. 3βHSD/isomerase was not found; however a low activity of 5α-reductase was identified. Aromatase activity was not positively identified, but preliminary results suggest that it may be present at low levels. The 17-oxidoreductases were widespread throughout the brain. The 17α-OR was significantly lower in the forebrain than other brain sections. The 170-OR activity did not vary significantly throughout the organ, although there was a trend for its activity to be higher in the midbrain region (containing the hypothalamus in these sections). The concentration of endogenous steroids in brain tissue was estimated by radioimmunoassay. Epitestosterone was found throughout the organ structure, whereas testosterone was found mainly in the midbrain (containing hypothalamic regions in these sections). Correlations between enzyme activities and steroid concentrations in brain regions suggested that the main function of 17α-OR is to produce epitestosterone, whereas the 17β-OR may catalyse a more reversible reaction in vivo. Temperature was found to significantly affect both 17α- and 17β-OR activities in the brain. In contrast to the testis, the maximum activity of the brain enzymes occurred at 37°C. The level of 17α-OR activity in the male lizard (100 nmol/g tissue/h) is the highest reported for this enzyme in vertebrates. Both activities were found to be quantitatively similar in the whole brain homogenates of male and female animals, and did not vary seasonally when examined in the male. The 17-oxidoreductases were also found in most other tissues in T.rugosa, including epididymis, adrenal, kidney and liver (but not blood). This suggests that the high activities of both 17α-OR and 17β-OR are dominant features of the steroid system in this animal. The formation of 11-oxygenated compounds was found in the adrenal, in addition to the formation of polar metabolites in the kidney and liver (possibly polyhydroxylated and conjugated steroids). A preliminary investigation into the plasma binding of androgens was carried out. The insults suggest that there are several binding sites for testosterone; one with high affinity and low capacity; the other with low affinity and high capacity. Binding experiments were carried out at 32°C. At this temperature, specific binding was greater than at 25 or 37°C. From the results of competition studies it was suggested that epitestosterone (with a K(i)= 3 X 10 (-6)M for testosterone binding) regulates the binding of testosterone (K(i)=10(-7)M) and hence the concentrations of the latter steroid as a free compound in plasma. In general, the study has shown that the biochemistry of androgens in the reptile T.rugosa is largely similar to that found in other vertebrates. The major difference is a greatly increased activity of 17α-OR, which causes a higher concentration of 17α-compounds to be present in the tissues of this lizard. The physiological roles for epitestosterone are not yet clear. However it appears from this study that this steroid regulates testosterone concentrations in several tissues by either steroidogenic or binding mechanisms. Several major influences on this regulation include temperature, availability of cofactors and seasonal effects.

Does stress induce salt intake?

Psychological stress is a common feature of modern day societies, and contributes to the global burden of disease. It was proposed by Henry over 20 years ago that the salt intake of a society reflects the level of stress, and that stress, through its effect on increasing salt intake, is an important factor in the development of hypertension. This review evaluates the evidence from animal and human studies to determine if stress does induce a salt appetite and increase salt consumption in human subjects. Findings from animal studies suggest that stress may drive salt intake, with evidence for a potential mechanism via the sympatho-adrenal medullary system and/or the hypothalamo–pituitary–adrenal axis. In contrast, in the few laboratory studies conducted in human subjects, none has found that acute stress affects salt intake. However, one study demonstrated that life stress (chronic stress) was associated with increased consumption of snack foods, which included, but not specifically, highly salty snacks. Studies investigating the influence of chronic stress on eating behaviours are required, including consumption of salty foods. From the available evidence, we can conclude that in free-living, Na-replete individuals, consuming Na in excess of physiological requirements, stress is unlikely to be a major contributor to salt intake.

Depression and pain : an overview

Background: Depression and pain are both burdensome ailments that affect a major proportion of the population. It is evident that depression and pain frequently coexist, with treatment and outcome implications.

Objective: To review the literature on the nature, prevalence and co-morbidity of depression and pain, the biological and psychological mechanisms involved and treatment options, thus presenting a broad overview of the current information available.

Methods: Relevant sources were identified from PubMed and Medline databases using a combination of keywords including depression, pain, prevalence, co-morbidity, biological and psychological mechanisms, serotonin (5-HT), norepinephrine (NE), hypothalamic-pituitary-adrenal (HPA) axis, amygdala, functional magnetic resonance imaging (fMRI), antidepressant and psychological therapy.

Results: It is evident from the research that depression and pain are common co-morbidities. Pain as a physical symptom of depression affects approximately 65% of patients, leading to less favourable outcomes and greater health care utilization. Moreover, depression is a common feature in chronic pain patients and can affect pain threshold and tolerance. Evidence from biological and psychological studies has revealed mechanisms that link chronic pain to depression. Several classes of anti-depressants and psychological interventions have been used successfully in the treatment of somatic symptoms of depression and for a variety of pain syndromes.

Conclusions: Pain and depression are linked by overlapping phenomenology, neurobiology and therapy. They are mutually interacting, and the interaction has significant treatment and outcome implications.

Reducing psychological distress and obesity in Australian farmers by promoting physical activity

Background: Studies have confirmed that the rate of mental illness is no higher in rural Australians than that of urban Australians. However, the rate of poor mental health outcomes, and in particular suicide, is significantly raised in rural populations. This is thought to be due to lack of early diagnosis, health service access, the distance-decay effect, poor physical health determinants and access to firearms. Research conducted by the National Centre for Farmer Health between 2004 and 2009 reveals that there is a correlation between obesity and psychological distress among the farming community where suicide rates are recognised as high. Chronic stress overstimulates the regulation of the hypothalamic-pituitary-adrenal (HPA) axis that is associated with abdominal obesity. Increasing physical activity may block negative thoughts, increase social contact, positively influence brain chemistry and improve both physical and mental health. This paper describes the design of the Farming Fit study that aims to identify the effect of physical activity on psychological distress, obesity and health behaviours such as diet patterns and smoking in farm men and women.
Methods/Design: For this quasi-experimental (convenience sample) control-intervention study, overweight (Body Mass Index ≥25 kg/m2) farm men and women will be recruited from Sustainable Farm Families™ (SFF) programs held across Victoria, Australia. Baseline demographic data, health data, depression anxiety stress scale (DASS) scores, dietary information, physical activity data, anthropometric data, blood pressure and biochemical analysis of plasma and salivary cortisol levels will be collected. The intervention group will receive an exercise program and regular phone coaching in order to increase their physical activity. Analysis will evaluate the impact of the intervention by longitudinal data (baseline and post intervention) comparison of intervention and control groups.
Discussion: This study is designed to examine the effect of physical activity on psychological health and other comorbidities such as obesity, impaired glucose tolerance, hypertension and dyslipidaemia within a high-risk cohort. The outcomes of this research will be relevant to further research and service delivery programs, in particular those tailored to rural communities.