Role of 5α-reductase type 1 in modifying anxiety, appetite and the HPA axis

Abstract

Glucocorticoid excess is associated with adverse effects on a number of physiological parameters, leading to obesity, dysfunction of the hypothalamic-pituitary- adrenal (HPA) axis and behavioural changes such as anxiety and impaired learning and memory. Circulating and local tissue glucocorticoid levels are tightly controlled by the HPA axis but an additional level of control exists in tissues such as brain, liver and adipose tissue. In these structures, enzymes including 5α-reductase 1 (5αR1), catalyse the conversion of corticosterone to A-ring reduced metabolites, which have a different spectrum of activities. This thesis investigates the role of 5αR1 in regulating central glucocorticoid actions which control HPA axis function and behaviour in a mouse model with genetic disruption of 5αR1 (5αR1-KO). Preliminary data showed 5αR1-KO mice were susceptible to developing insulin resistance and obesity and had reduced HPA axis responses to acute stress. Additionally, male 5αR1-KO mice were more prone to obesity than wild-type (WT) when fed a high-fat diet whilst female 5αR1-KO mice gained more weight than WT even on a normal chow diet. Intriguingly, female 5αR1-KO mice subjected to social isolation stress lost this extra weight and became comparable to WT controls. This study tested the hypothesis that 5αR1-KO mice are less able to inactivate glucocorticoids in the periphery and within tissues, resulting in a predisposition to metabolic disturbances and behavioural alterations. These were hypothesised to include hyperphagia, weight gain, impaired stress responses, anxiety (exacerbated by environmental stress) and cognitive deficits. It was also thought that many of these features would be more pronounced in female vs. male mice. The main aims of this study were to determine if 5αR1-KO induced weight gain and if this was correlated to altered gene expression of key hypothalamic neuropeptides which regulate appetite, to determine the central mechanisms which underpin attenuated HPA axis responses to acute stress and to determine whether behaviours such as anxiety and learning and memory ability are affected by global 5αR1 loss. It was hypothesised that female 5αR1-KO mice have increased appetite and reduced locomotor activity compared with WT and male 5αR1-KOs. However, male 5αR1- KO mice (on a mixed genetic background, C57Bl/6j/SvEv/129) were hyperphagic on a normal chow diet but did not gain extra weight, while female 5αR1-KO mice gained more weight vs. WT despite hypophagia. Free ambulatory activity was unaffected by genotype in either sex. Male 5αR1-KO mice appeared less anxious but responses of female 5αR1-KO mice in tests of anxiety did not differ from WT controls. Mice lacking 5αR1 generally had a poorer metabolic profile with impaired glucose tolerance and hyperinsulinaemia; with hepatic steatosis evident in female mice. There was evidence of compensatory changes in hypothalamic orexigenic and anorexigenic peptides. Phenotypes were sexually dimorphic such that male mice had a poorer metabolic profile vs. females, which was particularly marked in male 5αR1- KO animals. 5αR1-KO mice were previously shown to have attenuated HPA axis responses to acute stress and it was hypothesised that disruption of 5αR1 would result in altered expression of genes related HPA axis regulation with a view to increased negative feedback. Here, male and female 5αR1-KO mice demonstrated altered corticosteroid receptor expression within the hippocampus and the pituitary, two key structures in the HPA cascade. In situ hybridisation showed reduced mRNA for MR in the hippocampus and for Crh in the hypothalamus of 5αR1-KO mice. These modifications along with decreased Crhr-1 mRNA (CRH‘s main receptor) may be due to a lack of corticosterone metabolism within the brain resulting in enhanced negative feedback and reduced HPA axial drive. In order to study behaviour in detail and also to test whether potential central glucocorticoid excess may predispose to cognitive decline with ageing, a separate cohort of female 5αR1-KO backcrossed onto a uniform C57Bl/6j background was studied both when young (6 months) and when aged (14-15 months). Additionally, mice were housed in either groups or singly (social isolation) to investigate the potentially additive effects of environmental stress. It was hypothesised that local glucocorticoid increases in the brains of 5αR1-KO mice would be associated with anxiety and cognitive deficiencies and that these phenotypes would be exaggerated by the stress of social isolation as well as ageing. Behavioural differences were not observed at 6 months of age. However aged, 5αR1-KO mice housed singly showed increased anxiety and had higher plasma corticosterone levels than group-housed mice. Moreover, aged mice lacking 5αR1 performed less well than WT in tests of memory and had a marginally greater cognitive decline when learning ability at 14- 15 months old was compared to that of the same animals tested at 6 months old. Overall, mice with global 5αR1 loss appeared susceptible to anxiety as well as some degree of age-associated cognitive impairment, but only when subjected to social isolation stress which is a known chronic stressor. The final set of experiments aimed to determine the effect of mouse strain on 5αR1- KO phenotypes. It was hypothesised that glucocorticoid clearance would be attenuated to a lesser degree in 5αR1-KO mice bred onto a congenic C57Bl/6j strain compared to those of the mixed strain and that this would manifest as less disruption of metabolism and less suppression of HPA axis stress responses. Although social isolation again induced weight-loss in female mice and more so in 5αR1-KO animals, mice on the C57Bl/6j background strain did not show dampened HPA axis responses to acute stress as seen previously. It was subsequently shown in adrenalectomised mice that animals bred on the C57Bl/6j strain cleared active corticosterone from plasma and liver faster than mixed strain mice. This may have rendered mixed strain 5αR1-KO mice more susceptible to excessive corticosterone levels producing a more exaggerated phenotype in this group. In conclusion, these data suggest a role for the enzyme 5αR1 in modifying glucocorticoid concentrations in the brain and liver, influencing not only metabolic and peripheral effects such as weight gain and insulin resistance, but also in modifying cognition, appetite stimulation and affective behaviours. It has been highlighted that outside factors such as housing and age can modify these phenotypes and are important considerations for future studies. This study has also highlighted the importance of choosing an appropriate genetic background for genetically modified animals since phenotypes can be enhanced or attenuated depending on strain. Finally, 5αR inhibitors are used to treat disorders such as benign prostatic hyperplasia in men, and it is important to consider that these drugs may have a wide array of associated side effects both systemically and in the central nervous system.