Little is known about HPA axis regulation of neuroactive steroid synthesis in humans. In this study, we show that PREG-S levels are regulated by adrenal activation of the HPA axis and are sensitive to negative feedback mechanisms; however, they do not exhibit diurnal variation or significantly change after naloxone and oCRH infusions. In contrast, deoxycorticosterone levels exhibit diurnal variations and are regulated by the HPA axis at all levels examined. Abstinent alcohol-dependent patients, primarily nicotine dependent, have blunted PREG-S response to adrenal stimulation with cosyntropin, butno other alterations in PREG-S and deoxycorticosterone responses to HPA axis challenges were found (see for recapitulative results). To the best of our knowledge, this is the first comprehensive study of the selective modulation of the HPA axis with naloxone, oCRH, cosyntropin and dexamethasone on PREG-S and deoxycorticosterone levels in men.
Recapitulative table of results.
The finding that naloxone or oCRH challenges did not alter PREG-S levels could indicate that PREG-S levels are independent of hypothalamic or pituitary activation of the HPA axis. This would explain the lack of diurnal changes in circulating PREG-S levels that we and others (Laatikainen and Vihko, 1968
) have observed in humans. The lack of change in PREG-S levels following naloxone or oCRH stimulation in humans could be due to the preferential metabolism of pregnenolone to progesterone (ratherthan PREG-S) mediated by an effect of naloxone or oCRH on the steroidogenic enzymes sulfotransferase and sulfatase that interconvert pregnenolone and PREG-S. Indeed, naloxone, but not CRH, increases plasma pregnenolone levels in cynomolgus monkeys (Porcu et al., 2006c
). However, CRH does increase progesterone and its neuroactive metabolite 3α,5α-THP in humans (Genazzani et al., 1998
), suggesting that CRH may promote pregnenolone metabolism to progesterone rather than PREG-S. Alternatively, it is possible that activation of the HPA axis via hypothalamic or pituitary stimulation induces the release of other hormones, in addition to ACTH, that could interfere with PREG-S biosynthesis. For instance, it is known that ACTH derives from a large precursor molecule, pro-opiomelanocortin, that gives rise to numerous hormones, including opioid peptides and melanocyte stimulating hormone (Raffin-Sanson et al., 2003
In contrast to PREG-S, deoxycorticosterone had a circadian variation (Tan and Mulrow, 1975
) which is similar to that observed for ACTH and cortisol in the same subjects (Adinoff et al., 2005a
). Furthermore, plasma deoxycorticosterone and cortisol appear to be similarly regulated by challenges to the HPA axis, as shown by the correlations in deoxycorticosterone and cortisol responses following naloxone, oCRH, and cosyntropin challenges. In addition, naloxone, oCRH, cosyntropin and cosyntropin following dexamethasone all significantly increase deoxycorticosterone levels and dexamethasone decreases deoxycorticosterone levels. These findings are consistent with other studies assessing deoxycorticosterone response to pharmacological challenges (Brown and Strott, 1971
; Tan and Mulrow, 1975
; Kater et al., 1990
The second aim of this study was to explore putative differences in basal and stimulated PREG-S and deoxycorticosterone levels between control subjects and abstinent alcohol-dependent patients. The finding that basal PREG-S did not differ between healthy controls and abstinent alcohol-dependent patients contrasts with a previous study showing higher plasma PREG-S levels in premenopausal women undergoing alcohol detoxification treatment compared to controls (Hill et al., 2005
). Either sex differences or length of abstinence could be responsible for this discrepancy. The blunted PREG-S response in alcohol-dependent patients following cosyntropin is similar to the blunted cortisol response observed in the same subjects by Adinoff et al. (2005a)
(following dexamethasone pretreatment) and Wand and Dobs (1991)
(without dexamethasone pretreatment). Other experimental evidence suggests that suppressed PREG-S levels might be relevant in alcoholism. Administration of PREG-S to rodents improves cognition (Vallee et al., 2001
) and cognitive impairment has been observed inalcoholics (Bates et al., 2002
). In agreement, PREG-S prevents the memory-impairing effects of ethanol in mice (Melchior and Ritzmann, 1996
). Therefore, diminished PREG-S responses to adrenal activation may contribute to cognitive impairment in alcoholism.
Overall, we did not observe significant differences in basal or stimulated deoxycorticosterone levels between controls and alcohol-dependent subjects. Basal deoxycorticosterone levels have never previously been measured in alcohol-dependent subjects. In cynomolgus monkeys basal deoxycorticosterone levels were dramatically increased after long-term alcohol exposure (Porcu et al., 2006b
). Furthermore, cynomolgus monkeys show a blunted deoxycorticosterone response to CRH and dexamethasone challenges (Porcu et al., 2006b
). In addition, rats after chronic ethanol exposure had blunted deoxycorticosterone responses to an acute ethanol challenge (Khisti et al., 2005
). These discrepancies might be the result of species differences or they might be due to different experimental conditions: assessment immediately following chronic alcohol exposure in rats and cynomolgus monkeys versus one-month abstinence in humans. In addition, more subtle findings of deoxycorticosterone alterations in the one-month abstinent alcohol-dependent patients, including a delayed response to oCRH and a non-statistically significant (p=0.055) suppressed response to cosyntropin, may yield significant differences in future studies with a more thorough time course and increased group size.
A methodological strength of this study is the population. Patients and controls were similar in age and race. Although there was a statistically significant difference in age between the two groups, the HPA axis remains functioning stable over this age range. Neither group had significant medical disorders nor was taking any psychotropic drugs or other medications. Period of abstinence in alcohol-dependent patients was controlled such that the study occurred within four-six weeks after the last drink. However, alcohol-dependent patients were almost all nicotine-dependent while controls were not. Although a previous study reported that cigarette smoking in abstinent alcohol-dependent subjects did not alter the pituitary-adrenal response to a pharmacological challenge (Anthenelli et al., 2001
), a more recent study found that 3α,5α-THP levels may be up-regulated in smokers and dehydroepiandrosterone sulfate levels may predict nicotine dependence (Marx et al., 2006
). Indeed, acute administration of nicotine alters pregnenolone, progesterone, 3α,5α-THP and 3α,5α-THDOC levels in rat brain and plasma (Porcu et al., 2003
The major limitation of the study is that we had only few time points available; thus, our basal measures preceded the challenge infusions by 20 to 45 min and time points after the infusion were analyzed based on plasma availability. Therefore, it is possible that we may have missed the actual peak level of PREG-S and deoxycorticosterone. Other limitations include the high prevalence of psychologicalstress (divorced, unemployed, homeless) in the alcohol-dependent group, although these subjects did not meet criterion for depression or anxiety disorders. Stress is known to interfere with HPA axis functioning and we cannot rule out the possibility that the alterations in PREG-S and deoxycorticosterone responses to HPA axis challenge seen in these patients are strictly related to the alcohol-dependence. Also, recent findings using highly sensitive mass spectrometry techniques suggest that PREG-S is not present in rat brain and plasma (Liu et al., 2003
; Liere et al., 2004
). However, these authors were able to measure PREG-S levels in human plasma and values are comparable to the ones we obtained using a radioimmunoassay with a commercially available antibody.
In conclusion, we have shown that HPA axis modulation by pharmacological challenges differentially regulates the neuroactive steroids PREG-S and deoxycorticosterone in humans. The small number of subjects is a limitation that precludes definitive conclusions and argues for further studies in alcohol-dependent subjects, examining other stages of alcohol dependence (we only looked at one-month abstinent alcohol-dependent patients) and women. Moreover, since deoxycorticosterone is the precursor of other GABAergic neuroactive steroids, it is possible that its metabolites, or other pregnenolone metabolites, may be altered in alcohol dependence. Nonetheless, these results add new information on neuroactive steroids in alcohol-dependent subjects; since neuroactive steroids influence ethanol sensitivity (Morrow et al., 2006
), they might contribute to the underlying pathology.