Analysis of a genetic region affecting mouse body weight.

Genetic factors affect an individual's risk of developing obesity, but in most cases each genetic variant has a small effect. Discovery of genes that regulate obesity may provide clues about its underlying biological processes and point to new ways the disease can be treated. Preclinical animal models facilitate genetic discovery in obesity because environmental factors can be better controlled compared with the human population. We studied inbred mouse strains to identify novel genes affecting obesity and glucose metabolism. BTBR T+ Itpr3tf/J (BTBR) mice are fatter and more glucose intolerant than C57BL/6J (B6) mice. Prior genetic studies of these strains identified an obesity locus on chromosome 2. Using congenic mice, we found that obesity was affected by a ∼316 kb region, with only two known genes, pyruvate dehydrogenase kinase 1 (Pdk1) and integrin α 6 (Itga6). Both genes had mutations affecting their amino acid sequence and reducing mRNA levels. Both genes have known functions that could modulate obesity, lipid metabolism, insulin secretion, and/or glucose homeostasis. We hypothesized that genetic variation in or near Pdk1 or Itga6 causing reduced Pdk1 and Itga6 expression would promote obesity and impaired glucose tolerance. We used knockout mice lacking Pdk1 or Itga6 fed an obesigenic diet to test this hypothesis. Under the conditions we studied, we were unable to detect an individual contribution of either Pdk1 or Itga6 to body weight. During our studies, with conditions outside our control, we were unable to reproduce some of our previous body weight data. However, we identified a previously unknown role for Pdk1 in cardiac cholesterol metabolism providing the basis for future investigations. The studies described in this paper highlight the importance and the challenge using physiological outcomes to study obesity genes in mice.

Sex differences in the HPA axis.

The hypothalamic-pituitary-adrenal (HPA) axis is a major component of the systems that respond to stress, by coordinating the neuroendocrine and autonomic responses. Tightly controlled regulation of HPA responses is critical for maintaining mental and physical health, as hyper- and hypo-activity have been linked to disease states. A long history of research has revealed sex differences in numerous components of the HPA stress system and its responses, which may partially form the basis for sex disparities in disease development. Despite this, many studies use male subjects exclusively, while fewer reports involve females or provide direct sex comparisons. The purpose of this article is to present sex comparisons in the functional and molecular aspects of the HPA axis, through various phases of activity, including basal, acute stress, and chronic stress conditions. The HPA axis in females initiates more rapidly and produces a greater output of stress hormones. This review focuses on the interactions between the gonadal hormone system and the HPA axis as the key mediators of these sex differences, whereby androgens increase and estrogens decrease HPA activity in adulthood. In addition to the effects of gonadal hormones on the adult response, morphological impacts of hormone exposure during development are also involved in mediating sex differences. Additional systems impinging on the HPA axis that contribute to sex differences include the monoamine neurotransmitters norepinephrine and serotonin. Diverse signals originating from the brain and periphery are integrated to determine the level of HPA axis activity, and these signals are, in many cases, sex-specific.

Central vasopressin V1A receptor blockade alters patterns of cellular activation and prevents glucocorticoid habituation to repeated restraint stress exposure.

Our previous experiments implicated a role for the arginine vasopressin (AVP) V1A receptor subtype in mediating the normal decline (habituation) of hypothalamic-pituitary-adrenal (HPA) axis responses to repeated restraint exposure. To explore pathways mediating the endogenous effects of central AVP on stress HPA axis habituation, here we compared cellular (Fos) and hormone responses in male rats receiving chronic icv infusion of vehicle or a V1A receptor antagonist that began 7 d before stress testing, continued through the duration of acute and repeat restraint exposure. As a group, rats with V1A antagonism displayed a modest reduction in ACTH habituation, whereas the decline in corticosterone was completely prevented. V1A antagonized rats also showed reduced evidence of habituated Fos responses in the paraventricular nucleus of the hypothalamus, medial amygdala, and within the anterior division of the bed nucleus of the stria terminalis. Based on these cellular and neuroendocrine responses, we then examined whether repeated restraint is reflected by changes in V1A receptor binding. Relative to stress naïve animals, repeatedly exposed rats showed lower levels of V1A binding in the dentate gyrus of the hippocampus, thalamus and central amygdala, but higher levels in the septum and anterior BST. Taken together, these findings suggest that AVP may act within multiple targets to regulate the normal decline in stress-induced drive to the HPA axis, and that this may involve the net of V1A receptor stimulatory and inhibitory influences on neuroendocrine habituation.

Sex differences in serotonin (5-HT) 1A receptor regulation of HPA axis and dorsal raphe responses to acute restraint.

The serotonin (5-HT) 1A receptor subtype has been implicated as an important mediator for the stimulatory influence of serotonin on stress hypothalamic-pituitary-adrenal (HPA) activity, at least in males. Females show greater HPA axis responses to stress compared to males. To determine the nature by which the 5-HT 1A receptor contributes to the sex difference in stress, we examined neuroendocrine and cellular (Fos) responses in male and female rats receiving systemic injections of the 5-HT 1A receptor antagonist, WAY 100635, prior to acute restraint exposure. WAY decreased the corticosterone response in males, but not in females. In the paraventricular nucleus of the hypothalamus (PVH), WAY produced similar decrements in the restraint-induced activation (Fos) of neuroendocrine neurons in males and females. In contrast to the PVH, WAY administration increased total Fos activation in the dorsal raphe nucleus, but only in males. WAY also provoked higher Fos responses within subsets of dorsal raphe cells identified as serotonergic (tryptophan hydroxylase-, TPH-ir) in both males and females. These data provide evidence to suggest a differential influence of presynaptic 5-HT 1A receptors to regulate the stress-induced recruitment of non-serotonergic dorsal raphe neurons in males and females. At present, we cannot rule out a possible role for estrogen in females to alter 5-HT outflow to the HPA axis. There was a negative correlation between estrogen and Fos responses within TPH-positive cells in the dorsal raphe of WAY-administered females, whereas a positive correlation was found between estrogen and 5-HT 1A mRNA expression localized to the region of the zona incerta in close proximity to the PVH. As the raphe complex and 5-HT system impinge on several central autonomic, behavioral and neuroendocrine control systems, the current findings provide an important framework for future studies directed at sex differences in adaptive homeostatic responses.

Central vasopressin V1A receptor blockade impedes hypothalamic-pituitary-adrenal habituation to repeated restraint stress exposure in adult male rats.

Previous studies suggest that central arginine vasopressin (AVP) signaling can inhibit the hypothalamic-pituitary-adrenal (HPA) axis. To test a role for the AVP V1A receptor in stress HPA axis habituation, adult male rats were exposed to 5 consecutive days of 3 h restraint with or without continuous intracerebroventricular infusion of the V1A receptor antagonist d(CH2)5Tyr(Me)AVP (10 µg/day). Assessment of neuropeptide expression and HPA output under basal conditions revealed no effects of V1A receptor antagonism in stress naive animals. Between the first and last day of restraint exposure, controls showed marked declines in ACTH and corticosterone responses, and maintained plasma concentrations of testosterone. In contrast, V1A receptor antagonized animals displayed significantly smaller declines in ACTH and corticosterone responses, and a decrease in plasma testosterone. Despite their reduced expression of HPA axis habituation, antagonized animals continued to show stress-induced increases in AVP mRNA in the hypothalamic paraventricular nucleus and bed nucleus of the stria terminalis, and even higher levels of AVP expression in the medial amygdala relative to controls. The data leave open the nature and extent to which these and other AVP-containing pathways are recruited during repeated restraint, but nevertheless reveal a critical role for central V1A receptors in stress adaptation. As the effects of V1A receptor antagonism were restricted to the repeated restraint condition, we conclude that normal adaptation to stress involves a shift toward enhanced AVP utilization and/or V1A receptor signaling.

Postnatal aromatase blockade increases c-fos mRNA responses to acute restraint stress in adult male rats.

Recent evidence suggests that the aromatization of testosterone to estrogen is important for the organizing effects of neonatal testosterone on neuroendocrine responses to acute challenges. However, the extent to which neonatal inhibition of aromatase alters the stress-induced activation of neural pathways has not been examined. Here we assessed central patterns of c-fos mRNA induced by 30 min of restraint in 65-d-old adult male rats that were implanted with sc capsules of the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD), introduced within 12 h of birth and removed on d 21 of weaning. Neonatal ATD decreased the expression of arginine vasopressin within extrahypothalamic regions in adults, confirming reduced estrogen exposure during development. As adults, ATD-treated animals showed higher corticosterone responses at 30 min of restraint exposure compared with control animals as well as higher c-fos expression levels in the paraventricular nucleus of the hypothalamus. ATD treatment also increased stress-induced c-fos within several limbic regions of the forebrain, in addition to areas involved in somatosensory processing. Based on these results, we propose that the conversion of testosterone to estrogen during the neonatal period exerts marked, system-wide effects to organize adult neuroendocrine responses to homeostatic threat.

Androgen receptors in the posterior bed nucleus of the stria terminalis increase neuropeptide expression and the stress-induced activation of the paraventricular nucleus of the hypothalamus.

The posterior bed nuclei of the stria terminalis (BST) are important neural substrate for relaying limbic influences to the paraventricular nucleus (PVN) of the hypothalamus to inhibit hypothalamic-pituitary-adrenal (HPA) axis responses to emotional stress. Androgen receptor-expressing cells within the posterior BST have been identified as projecting to the PVN region. To test a role for androgen receptors in the posterior BST to inhibit PVN motor neurons, we compared the effects of the non-aromatizable androgen dihydrotestosterone (DHT), the androgen receptor antagonist hydroxyflutamide (HF), or a combination of both drugs implanted unilaterally within the posterior BST. Rats bearing unilateral implants were analyzed for PVN Fos induction in response to acute-restraint stress and relative levels of corticotrophin-releasing hormone and arginine vasopressin (AVP) mRNA. Glutamic acid decarboxylase (GAD) 65 and GAD 67 mRNA were analyzed in the posterior BST to test a local involvement of GABA. There were no changes in GAD expression to support a GABA-related mechanism in the BST. For PVN neuropeptide expression and Fos responses, basic effects were lateralized to the sides of the PVN ipsilateral to the implants. However, opposite to our expectations of an inhibitory influence of androgen receptors in the posterior BST, PVN AVP mRNA and stress-induced Fos were augmented in response to DHT and attenuated in response to HF. These results suggest that a subset of androgen receptor-expressing cells within the posterior BST region may be responsible for increasing the biosynthetic capacity and stress-induced drive of PVN motor neurons.

The medial preoptic nucleus integrates the central influences of testosterone on the paraventricular nucleus of the hypothalamus and its extended circuitries.

Testosterone contributes to sex differences in hypothalamic-pituitary-adrenal (HPA) function in humans and rodents, but the central organization of this regulation remains unclear. The medial preoptic nucleus (MPN) stands out as an important candidate in this regard because it contains androgen receptors and projects to forebrain nuclei integrating cognitive-affective information and regulating HPA responses to homeostatic threat. These include the HPA effector neurons of the paraventricular nucleus (PVN) of the hypothalamus, medial amygdala, and lateral septum. To test the extent to which androgen receptors in the MPN engage these cell groups, we compared in adult male rats the effects of unilateral microimplants of testosterone and the androgen receptor antagonist hydroxyflutamide into the MPN on acute restraint induced activation and/or neuropeptide expression levels. The basic effects of these implants were lateralized to the sides of the nuclei ipsilateral to the implants. Testosterone, but not hydroxyflutamide implants, decreased stress-induced Fos and arginine vasopressin (AVP) heteronuclear RNA expression in the PVN, as well as Fos expression in the lateral septum. In unstressed animals, AVP mRNA expression in the PVN decreased and increased in response to testosterone and hydroxflutamide MPN implants, respectively. The differential influences of these implants on AVP mRNA expression were opposite in the medial amygdala. These results confirm a role for androgen receptors in the MPN to concurrently modulate neuropeptide expression and activational responses in the PVN and its extended circuitries. This suggests that the MPN is capable of bridging converging limbic influences to the HPA axis with changes in gonadal status.