Plasma pregnenolone levels in cynomolgus monkeys following pharmacological challenges of the hypothalamic–pituitary–adrenal axis

Abstract

Pregnenolone (PREG) is an endogenous neuroactive steroid that is increased in rodent brain and plasma after hypothalamic–pituitary–adrenal (HPA) activation by acute stress or ethanol administration. Plasma levels of PREG metabolites are altered by pharmacological challenges of the HPA axis, however little is known about HPA regulation of PREG levels in monkeys. PREG concentrations were determined by radioimmunoassay in plasma samples from cynomolgus monkeys, following challenge with naloxone (125 and 375 μg/kg), corticotropin-releasing factor (CRF; 1 μg/kg), dexamethasone (130 μg/kg), adrenocorticotropic hormone (ACTH; 10 ng/kg; 4–6 h after 0.5 mg/kg dexamethasone) and ethanol (1.0 and 1.5 g/kg). Naloxone increased PREG levels, while CRF appeared to increase metabolism of PREG to deoxycorticosterone (DOC). ACTH, administered after dexamethasone, reduced PREG levels, despite an increase in plasma cortisol. Ethanol did not alter PREG levels. Changes in PREG levels were correlated with changes in DOC levels after naloxone 125 μg/kg, CRF, ethanol 1.5 g/kg, and dexamethasone challenges. Furthermore, dexamethasone-induced changes in PREG levels were correlated with subsequent alcohol intake. These data suggest that PREG responses to dexamethasone challenge may represent a trait marker of alcohol drinking. The lack of effect of ethanol on PREG levels suggests differential regulation in non-human primates vs. rodents.

Introduction

Pregnenolone (PREG) is a steroid hormone synthesized from cholesterol in a reaction catalyzed by the mitochondrial enzyme CYP11A (cholesterol side-chain cleavage or P450scc). The conversion of cholesterol into PREG is the first and rate limiting step of steroidogenesis, hence PREG is the precursor of numerous different steroid hormones, including glucocorticoids (cortisol in humans and non-human primates and corticosterone in rodents) and mineralocorticoids (aldosterone) at the level of the adrenal gland, or the 5α/5β-reduced neuroactive steroids 3α-hydroxy-5α-pregnan-20-one (3α,5α-THP or allopregnanolone), 3α-hydroxy-5β-pregnan-20-one (3α,5β-THP or pregnanolone), 3α,21-dihydroxy-5α-pregnan-20-one (3α,5α-THDOC or allotetrahydrodeoxycorticosterone) and 3α,5β-THDOC.

PREG has been suggested to have effects on both cognition and mood. PREG and its sulfated derivative enhance learning and memory in rodent models and pregnenolone sulfate administration to aged rats reverses age-related cognitive decline (Flood et al., 1992, Flood et al., 1995, Vallée et al., 1997, Vallée et al., 2001). PREG induces anxiogenic effects in mice (Melchior and Ritzman, 1994), and it has recently been shown to attenuate diazepam-induced sedation (Meieran et al., 2004). PREG levels are altered in patients with anxiety-depressive disorder (George et al., 1994) and PMS (Wang et al., 1996), and they are elevated in postmortem brain of subjects with schizophrenia and bipolar disorder (Marx et al., 2006).

The brain and plasma concentrations of PREG and its neuroactive metabolites are increased by acute stress in rodents (Barbaccia et al., 1996, Purdy et al., 1991). Similarly, stress elevates levels of 3α,5α-THP in human subjects (Droogleever Fortuyn et al., 2004, Girdler et al., 2001). The increase in neuroactive steroid levels elicited by stress appears to be mediated by activation of the hypothalamus–pituitary–adrenal (HPA) axis, since it is no longer apparent in adrenalectomized animals (Purdy et al., 1991).

Activation of the HPA axis under acute stress triggers the release of corticotropin releasing factor (CRF) from the hypothalamus. CRF induces the synthesis of adrenocorticotropic hormone (ACTH) from the pituitary, which stimulates the adrenal cortex to release glucocorticoids, their precursors progesterone and deoxycorticosterone (DOC) and also neuroactive steroids. Glucocorticoids, as well as 3α,5α-THP and 3α,5α-THDOC, have a negative feedback upon the hypothalamus and pituitary, thus inhibiting CRF production, release, ACTH release and subsequent corticosterone levels in rodents (Owens et al., 1992, Patchev et al., 1994, Patchev et al., 1996).

We have recently demonstrated that DOC levels in plasma from cynomolgus monkeys are regulated by naloxone, CRF and dexamethasone challenges to the HPA axis. However, circulating DOC levels did not change in response to acute challenges of 1.0 g/kg or 1.5 g/kg ethanol (i.g.), even though average blood ethanol levels were as high as 147 mg/dl (Porcu et al., 2006). In contrast, DOC levels are markedly increased in rat brain and plasma following acute ethanol administration (Khisti et al., 2005), suggesting that higher doses of alcohol are necessary to activate the HPA axis in monkeys, or perhaps that DOC synthesis is differentially regulated in non-human primates compared to rodents (Porcu et al., 2006).

It is well known that systemic administration of ethanol increases both plasma and brain levels of PREG and its neuroactive metabolites 3α,5α-THP and 3α,5α-THDOC in rodents (Barbaccia et al., 1999, Korneyev et al., 1993, Morrow et al., 1999, O'Dell et al., 2004, VanDoren et al., 2000). Laboratory administration of low doses of ethanol has recently been reported to increase PREG levels in healthy human subjects (Pierucci-Lagha et al., 2006), while 3α,5α-THP levels are decreased in the same subjects. In contrast, other studies have reported increased plasma 3α,5α-THP levels in human adolescent emergency room patients with signs of ethanol intoxication (Torres and Ortega, 2003, Torres and Ortega, 2004). The increase in PREG and its neuroactive metabolites, induced by acute ethanol in rats, is prevented by adrenalectomy/orchiectomy, thus, suggesting that ethanol activates the HPA axis to modulate neuroactive steroids synthesis (Korneyev et al., 1993, O'Dell et al., 2004).

Chronic ethanol consumption results in adaptation of the HPA axis causing decreased levels of corticosterone (Spencer and McEwen, 1990) and blunted elevation of cerebral cortical 3α,5α-THP (Morrow et al., 2001) and plasma and brain DOC levels (Khisti et al., 2005) in rats. Similar alterations in the HPA axis are observed in actively drinking or alcohol dependent human subjects. These patients show attenuated responsiveness of the HPA axis following stimulation with naloxone, CRF, exogenous ACTH administered after dexamethasone, and they exhibit a greater suppression of cortisol and ACTH levels following dexamethasone suppression (Adinoff et al., 2005a, Adinoff et al., 2005b, Inder et al., 1995, Wand and Dobs, 1991).

The aim of this study was to characterize plasma PREG levels following pharmacological challenges of the HPA axis, since PREG has effects on cognition and mood and is the precursor of other neuroactive steroids and hormones that are regulated by HPA axis modulation. Furthermore, PREG and its metabolites can penetrate the blood brain barrier and thus influence the overall concentration of brain neuroactive steroids; therefore changes in peripheral PREG levels might reflect changes in its brain content. Given that both stress and ethanol modulate neuroactive steroids and given the role played by the HPA axis in the etiology of alcoholism, the regulation of plasma PREG levels by the HPA axis was studied in eleven male cynomolgus monkeys before they were enrolled in a protocol of ethanol self-administration (Vivian et al., 2001). We hypothesized that PREG levels in monkey plasma would be regulated by pharmacological challenge of the HPA axis.