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. Author manuscript; available in PMC: 2020 Nov 1.
Published in final edited form as: Neuropharmacology. 2019 Jul 13;158:107711. doi: 10.1016/j.neuropharm.2019.107711

Intravenous administration of ghrelin increases serum cortisol and aldosterone concentrations in heavy-drinking alcohol-dependent individuals: results from a double-blind, placebo-controlled human laboratory study

Carolina L Haass-Koffler 1,2,3,*, Victoria M Long 2, Mehdi Farokhnia 3, Molly Magill 2, George A Kenna 1, Robert M Swift 1,4, Lorenzo Leggio 2,3,5,*
PMCID: PMC6745267  NIHMSID: NIHMS1535113  PMID: 31310775

Abstract

Increasing evidence supports the role of appetite-regulating hormones, including ghrelin, in alcohol use disorder (AUD). Effects of ghrelin administration on cortisol and aldosterone, two hormones known to influence the development and maintenance of AUD, have been observed in ghrelin-exposed tissues or cells, as well as rodents and healthy volunteers, however whether these effects replicate in individuals with AUD is unknown. Here, we tested the hypothesis that intravenous administration of ghrelin leads to increase in endogenous serum cortisol and aldosterone concentrations in alcohol-dependent, heavy drinking individuals, and that these changes may predict ghrelin-induced alcohol craving. This was a double-blind, placebo-controlled human laboratory study in non-treatment-seeking, heavy-drinking, alcohol-dependent individuals randomized to receive either placebo, 1 mcg/kg or 3 mcg/kg of intravenous ghrelin. Then, participants underwent a cue-reactivity procedure in a bar-like setting, which included exposure to both neutral (juice) and alcohol cues. Repeated blood samples were collected and used to measure endogenous cortisol and aldosterone serum concentrations, in response to exogenous ghrelin administration. Furthermore, cortisol and aldosterone serum concentrations were used to develop a model to predict the effect of exogenous ghrelin administration on alcohol craving. Intravenous ghrelin administration increased endogenous cortisol and aldosterone serum concentrations. While the effects on cortisol were greater than those on aldosterone, only the ghrelin-induced changes in aldosterone serum concentrations predicted craving. These findings provide initial evidence of ghrelin effects on glucocorticoids and mineralocorticoids in individuals with AUD, thereby providing additional information on the potential mechanisms by which the ghrelin system may play a role in alcohol craving and seeking in AUD.

Keywords: Alcohol Use Disorder, aldosterone, cortisol, ghrelin, craving

Graphical Abstract

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INTRODUCTION

Ghrelin is a 28-amino acid residue peptide originally discovered for its ability to facilitate growth hormone release via activation of its receptor, the growth hormone (GH) secretagogue receptor (GHS-R1a) (Kojima et al. 1999). Ghrelin is produced by endocrine cells located primarily in the stomach. In order to activate the GHS-R1a receptor, ghrelin needs to be acylated, a process that is facilitated by the ghrelin-O-acyltransferase (GOAT) enzyme (Gutierrez et al. 2008).

Acyl-ghrelin (hereby referred to as ghrelin) plays well-known roles in increasing appetite, meal initiation and food intake (Skibicka and Dickson 2013) by stimulating orexigenic and inhibiting anorexigenic pathways (Tschop et al. 2000). Ghrelin also increases food reward and motivation for food by modulating activity in the midbrain dopaminergic neurons (Abizaid et al. 2006).

Growing preclinical and human work supports a role of the ghrelin system in addictive behaviors, for reviews see: (Farokhnia et al. 2019; Morris et al. 2018; Zallar et al. 2017). In different rodent models, ghrelin increases, while ghrelin receptor antagonism decreases, accumbal dopamine release, condition place preference for alcohol and alcohol drinking, alcohol preference and operant self-administration (for reviews, see: (Engel and Jerlhag 2014; Leggio 2010; Zallar et al. 2017)). Several, but not all, human studies with alcohol-dependent or moderate-drinking individuals have shown a positive relationship between blood ghrelin levels and alcohol craving, as well as risk of relapse and subjective effects of acute alcohol administration (for review see (Zallar et al. 2017)). Furthermore, two double-blind, placebo-controlled human laboratory studies with intravenous (IV) ghrelin administration have been conducted in alcohol-dependent, heavy drinkers. These studies indicated that IV administration of exogenous ghrelin acutely increases cue-induced alcohol craving (Leggio et al. 2014), modulates brain functional activity in response to alcohol reward anticipation and increases alcohol self-administration (Farokhnia et al. 2018).

Several pathways involved in the regulation of stress and emotionality are also involved in the mechanisms related to uncontrolled drinking and vulnerability to relapse during abstinence (Koob and Le Moal 1997). These systems include both the hypothalamic–pituitary–adrenal (HPA) axis, as well as extra-hypothalamic brain pathways. The activity of the HPA axis is primarily regulated by the glucocorticoid and mineralocorticoid pathways (Harris et al. 2013).

Within the HPA axis, cortisol, the main glucocorticoid in humans, produced in the zona fasciculata of the adrenal cortex within the adrenal glands (Tsigos et al. 2000), has been extensively studied in AUD (Stephens and Wand 2012). Dysregulation of the HPA axis may contribute to initial vulnerability to AUD, even before alcohol exposure (Wand et al. 2001; Wand and Dobs 1991). Furthermore, acute alcohol consumption, similarly to stress exposure, activates the HPA axis by resulting in elevated concentrations of cortisol and other glucocorticoids (Richardson et al. 2008). Repeated exposure to high concentrations of alcohol (e.g., binge-drinking episodes and intoxication), followed by withdrawal, also elevates blood cortisol concentrations (Adinoff et al. 1998). The transition to AUD, however, is accompanied by low cortisol responsivity, due to an allostatic shift in HPA axis functioning (Koob and Le Moal 1997).

Outside the HPA axis, glucocorticoids have positive reinforcement properties, themselves, and seem to play a critical role in the dopamine reward circuitry (Fahlke et al. 1996; Pruessner et al. 2004). Notably, pharmacological blockade of the glucocorticoid receptor via mifepristone, or other more selective blockers, reduces stress-induced alcohol-seeking and other alcohol-related behaviors, both in animal models of, and individuals with, AUD (Simms et al. 2012; Vendruscolo et al. 2015).

Aldosterone, a steroid hormone of the mineralocorticoid family, is produced by the zona glomerulosa of the adrenal cortex in the adrenal gland. The major function of peripheral aldosterone is to regulate electrolytes and fluid homeostasis. In the central nervous system (CNS), mineralocorticoid receptors are expressed in key regions involved in the development and maintenance of excessive alcohol consumption (Edwards and Koob 2010; Reul and de Kloet 1985). In alcohol-dependent patients, plasma aldosterone concentrations increase during early alcohol withdrawal and normalize during recovery (Kovacs 2000). Furthermore, plasma aldosterone concentrations have been positively correlated with craving and anxiety in abstinent, alcohol-dependent patients (Leggio et al. 2008). These latter results were recently replicated in an independent cohort of alcohol-dependent patients, and further corroborated by rat and monkey data indicating a relationship between peripheral aldosterone concentrations, expression of the mineralocorticoid receptor in the amygdala and alcohol drinking (Aoun et al. 2018).

A relationship between ghrelin and both cortisol and aldosterone has been previously described. In particular, the effects of ghrelin on cortisol concentration has been observed in ghrelin-exposed tissues or cells (Kageyama et al. 2011; Nemoto et al. 2011; Rucinski et al. 2009), rodents (Asakawa et al. 2001; Mozid et al. 2003; Ruter et al. 2003) and healthy volunteers (Giordano et al. 2006; Giordano et al. 2004). Ghrelin acts at the hypothalamic paraventricular nucleus (PVN), where it stimulates the release of neuropeptide Y, which in turn inhibits gamma-aminobutyric acid (GABA) and corticotropin releasing factor (CRF) release (Morris et al. 2018). In parallel, increased peripheral blood cortisol increases peripheral acyl-ghrelin (Azzam et al. 2017). This effect suggests a link between stress, ghrelin and cortisol (for review, see (Morris et al. 2018)). Also, pharmacological manipulation using administration of adrenocorticotropic hormone (ACTH), cortisone or the cortisol synthesis inhibitor metyrapone further support the relationship between ghrelin and cortisol (Azzam et al. 2017). Both rat and human studies show that increase of ACTH and cortisol leads to an increase in circulating ghrelin, indicating that HPA-axis activation leads to an increase in ghrelin concentrations (Azzam et al. 2017). Preliminary studies have shown that ghrelin may also contribute to increasing aldosterone concentration. In fact, ghrelin in its acylated form (but not des-acyl-ghrelin) increases blood aldosterone concentrations both in rats (Milosevic et al. 2010) and healthy individuals (Zhang et al. 2017). Furthermore, in rats, aldosterone (as well as corticosterone) concentrations are reduced by the inhibition of the GOAT enzyme (Rucinski et al. 2012), which is highly expressed in zona glomerulosa of the adrenal glands (Rucinski et al. 2009).

In summary, preclinical and initial human studies indicate that manipulations of the ghrelin system may lead to changes in cortisol and aldosterone. However, there is no information on the effects of ghrelin manipulations on both cortisol and aldosterone concentrations in individuals with AUD. This question is relevant from both mechanistic and clinical standpoints, given that the ghrelin system is involved in alcohol seeking and consummatory behaviors and, on the other hand, the HPA axis is altered in AUD patients (Stephens and Wand 2012). Therefore, in the present study, we conducted a secondary analysis of a previously published human laboratory study (Leggio et al. 2014), in order to investigate the potential effects of ghrelin administration on peripheral cortisol and aldosterone concentrations in AUD individuals. This double-blind, placebo-controlled human laboratory study was conducted in heavy-drinking, alcohol-dependent individuals who received either placebo, IV ghrelin 1 mcg/kg or IV ghrelin 3 mcg/kg (Leggio et al. 2014). Consistent with the previous literature summarized above, we hypothesized that IV ghrelin administration would increase cortisol and aldosterone serum concentrations, and that these increases in cortisol and aldosterone would moderate ghrelin’s effect on alcohol craving during the human laboratory experiment.

MATERIALS AND METHODS

Setting

This study was conducted at the Brown University Center for Alcohol and Addiction Studies, Providence, RI. The study was approved by the Brown University Institutional Review Board and registered at the clinicaltrials.gov (). The administration of synthetic human acyl-ghrelin was conducted under an approved Food and Drug Administration Investigational New Drug (IND#109,242).

Parent Study

The parent study (Leggio et al. 2014) was a between-subject, double-blind, placebo-controlled, randomized human laboratory study with forty-five non-treatment-seeking, heavy-drinking, alcohol-dependent individuals. All participants signed an informed consent prior to participation. In brief, eligible participants had a Diagnostic and Statistical Manual of Mental Disorders-IV (DSM-IV) diagnosis of current alcohol dependence and heavy drinking levels, the latter defined as consuming on average ≥ 4 standard drinks/day for women or ≥ 5 standard drinks/day for men, during the 90-day period before screening, as assessed by the Timeline Followback (TLFB). For additional details on the inclusion and exclusion criteria, please also see the description of the parent study in (Leggio et al. 2014). A breath alcohol concentration of 0.00 and fasting overnight were required on the day of the human laboratory experiment. An IV cannula was placed, and a fixed light breakfast of ~700 kJ (approximately 62%, carbohydrate, 13% protein and 25% fat) was served. The experiment started at 12:00, participants then received a ~10-min administration of IV ghrelin at a dose of either 3 mcg/kg, 1 mcg/kg or 0 mcg/kg (placebo). Following the IV infusion, participants were exposed to a neutral (juice) cues trial and an alcohol cues trial in a bar-like lab setting. At the end of each trial, a visual analogue scale (VAS), rated on an 11-point anchored Likert-type scale, was administered to assess the participant’s urge to drink juice (J-VAS) or alcohol (A-VAS) (Rohsenow et al. 2000). Then, another juice and alcohol cues trial was conducted. Blood samples for hormones were collected at six time points during the experiment, i.e., at baseline (before the infusion), immediately following each of the four trials and finally prior to discharge.

The main finding of the parent study was a significant effect of ghrelin 3 mcg/kg versus placebo in increasing alcohol craving, but not juice craving. For additional details on the parent study, see: (Leggio et al. 2014).

Description of the sample

Out of the 45 participants in the parent study, blood samples for 37 subjects were available for this analysis. There were no significant differences in the demographics and baseline characteristics, including basal ghrelin concentrations, between groups in the main sample of the parent study and the sample of this analysis (p’s>0.05).

Blood Samples Analysis

The IV administration of either ghrelin or placebo took place from −10 min to 0 min. Blood sample time points were at baseline (−10 min), after the first juice trial (+6 min), after the first alcohol trial (+17 min), after the second juice trial (+23 min), after the second alcohol trial (+29 min) and post-experiment (+48 min).

Blood samples were centrifuged, serum was extracted and serum aliquots were stored at −80 °C until analysis. Cortisol and aldosterone serum c oncentrations were measured at a Clinical Laboratory Improvement Amendments (CLIA) certified laboratory (East Side Clinical Laboratory, Providence, RI). The cortisol serum samples were processed by Roche Cobas Electrochemiluminescence. The aldosterone serum samples were processed by Liaison XL Chemiluminescence. Levels of both hormones were expressed as mcg/dL.

Data Analytic Strategy

Distributional characteristics of outcome measures were examined to evaluate similarity to the normal distribution. While aldosterone concentrations had a slightly larger kurtosis (George and Mallery 2003), there was not difference in the baseline serum levels between groups. Outliers were taken into account as we did in other secondary analyses (Haass-Koffler et al. 2015; Haass-Koffler et al. 2016), and as previously suggested (Tabachnick and Fidell 2001). For the serum level, values below the lower limit of the quantitation were set to ½ of the detected lower limit of the assay quantitation level (Beal 2001).

Generalized Estimating Equation (GEE) models (Liang and Zeger 1986) were used to determine the effects of IV ghrelin administration (0 mcg/kg, 1 mcg/kg and 3 mcg/kg) on hormone (cortisol and aldosterone) serum concentrations and on alcohol craving. The experimental procedures (baseline and cue-reactivity trials) were set as a categorical variable with the intervals that corresponded to the time for blood collection: IV ghrelin pre-infusion (baseline, BL) and IV ghrelin post-infusion (T1-T5: +6 min; +17 min; +23 min; +29 min and +48 min), where T1 and T3 correspond to the juice trials, T2 and T4 correspond to the alcohol trials and T5 the end of the experiment. To evaluate the effects of ghrelin administration on endogenous cortisol and aldosterone serum concentrations, the GEE model examined the main effect of each of the two doses of ghrelin compared to placebo. Next, the GEE analysis included the ghrelin x trial interaction, which indicated if there was a difference in ghrelin effect on cortisol and aldosterone serum concentrations at each post-infusion alcohol trial (Singer et al. 2003).

For the alcohol and juice craving analysis, the main effect of ghrelin dose on post-infusion craving are reported in the parent paper (Leggio et al. 2014). Here, the GEE model examined the ghrelin x hormone (cortisol and aldosterone) interaction, which indicated whether the main effect of ghrelin on alcohol and juice craving outcomes differed by cortisol or aldosterone serum concentration. The final GEE analysis examined whether the effect of ghrelin on alcohol craving differed by trial during the experimental procedures: where BL is the baseline (pre-infusion), T1 and T3 correspond to the juice trials, T2 and T4 correspond to the alcohol trials and T5 the end of the experiment. All GEE models were run with an unstructured correlation structure. The variables were run in linear GEE models. Pictorial results were expressed as Means (M) and Standard Errors of the mean (SEMs). All statistical tests were two-sided, and statistical significance was accepted if a p-value of < 0.05 was obtained. Statistical Package for the Social Sciences (SPSSv.24) (Armonk, NY, U.S.) was used to conduct the analysis and GraphPad Prism (v.7) was used to generate the figures (La Jolla, CA, U.S.).

RESULTS

Effects of exogenous ghrelin administration on endogenous cortisol and aldosterone serum concentrations

The change in endogenous cortisol and aldosterone serum concentrations after exogenous ghrelin administration, as compared to placebo, are depicted in Figure 1.

Figure 1. Endogenous cortisol and aldosterone concentrations after IV ghrelin administration.

Figure 1.

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(A) Cortisol response. There was a significant and positive effect for ghrelin vs placebo for both the 1 mcg/kg (B1=3.236, p=0.008) and 3 mcg/kg (B1=3.631, p=0.002) dose across the post-infusion trials. The ghrelin by trial interaction model showed the magnitude of these effects varied by trial. There was a significant increase post-infusion for the 1 mcg/kg (B1=4.253, p<0.001) and 3 mcg/kg (B1=7.092, p<0.001) dose. There was also a significant change in intercept post-infusion, compared to placebo, for each trial during the experimental procedures that concluded at +48 min after the IV ghrelin infusion, which corresponds to the end of the experiment. (B) Aldosterone response. There was no significant slope for ghrelin vs placebo for either the 1 mcg/kg or the 3 mcg/kg (p’s>0.05) doses. The ghrelin × trial interaction showed a significant change in the intercept post-infusion for the 1 mcg/kg (B1=2.378, p=0.001), but not for the 3 mcg/kg (p>0.05) dose. There was an increment of significant change in intercept post-infusion, compared to placebo, only for the T2. Results are represented by the M±SEM, **(p<0.01), ***(p<0.001), not significant (p>0.05).

The main effect GEE model for cortisol serum concentrations showed a significant and positive effect for ghrelin vs placebo for both the 1 mcg/kg (B1=3.236, p=0.008) and 3 mcg/kg (B1=3.631, p=0.002) doses across the post-infusion, cue-reactivity trials. The ghrelin by trial interaction model showed the magnitude of these effects varied by experimental procedures. Specifically, Figure 1A depicts a significant increase post-infusion for the 1 mcg/kg (B1=4.253, p<0.001) and 3 mcg/kg (B1=7.092, p<0.001) doses, suggesting that there was a difference in cortisol serum concentration pre- vs post-infusion at the ghrelin doses tested here. There was also a significant change in intercept post-infusion, compared to placebo, for each trial during the experimental procedures that concluded at +48 min after the IV ghrelin infusion, which corresponds to the end of the experiment (Table 1).

Table 1 –

Cortisol and aldosterone serum concentrations: ghrelin by trial interaction at each trial of the cue-reactivity procedures.

Experimental points Procedures Dose (mcg/kg) Parameter p
Cortisol
BL Pre-ghrelin/placebo infusion 1 and 3 - -
1 Post-Juice Trial 1 1
3		 B10=4.253
B10=7.092		 <0.001
<0.001
2 Post-Alcohol Trial 1 1
3		 B10=5.747
B19=8.732		 <0.001
<0.001
3 Post-Juice Trial 2 1
3		 B10=6.487
B10=10.107		 <0.001
<0.001
4 Post-Alcohol Trial 2 1
3		 B10=7.615
B10=10.05		 <0.001
<0.001
5 Post-experiment 1
3		 B10=3.622
B10=8.517		 =0.026
<0.001
Aldosterone
BL Pre-ghrelin/placebo infusion 1 and 3 - -
1 Post-Juice Trial 1 1
3		 B10=2.378
B10=−1.177		 >0.05
<0.001
2 Post-Alcohol Trial 1 1
3		 B10=2.059
B10=−1.194		 >0.05
=0.015
3 Post-Juice Trial 2 1
3		 B10=1.570
B10=−1.181		 >0.05
>0.05
4 Post-Alcohol Trial 2 1
3		 B10=−0.074
B10=−1.116		 >0.05
>0.05
5 Post-experiment 1
3		 B10=1.233
B10=0.987		 >0.05
>0.05
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Note: in bold are statistically significant results, trend reported numerically, not significant as p > 0.05. BL: baseline

The main effect GEE model for the aldosterone serum concentrations did not show a significant effect for ghrelin vs placebo, for either the 1 mcg/kg or the 3 mcg/kg (p’s>0.05) doses, across the post-infusion, cue-reactivity trials. However, the ghrelin × trial interaction model sho wed a significant change in the intercept post-infusion for the 1 mcg/kg (B1=2.378, p=0.001), but not for the 3 mcg/kg (p>0.05) dose (Figure 1B), suggesting that there was a difference in aldosterone serum concentration pre- vs post-infusion only for the 1 mcg/kg ghrelin dose. As depicted in Figure 1B, there was an increment of significant change in intercept post-infusion, compared to placebo, only up to T2 at dose 1 mcg/kg (B1=2.059, p=0.015), suggesting that there was a difference in aldosterone serum concentration up to 17 min post-ghrelin infusion (Table 1).

Cortisol and aldosterone serum concentrations as moderators of alcohol craving

Next, we tested if cortisol or aldosterone serum concentration were moderators of ghrelin-induced, alcohol craving evaluated in the parent paper (Leggio et al. 2014). The GEE models included cortisol and aldosterone as moderators of ghrelin dose effect (ghrelin x hormone) on alcohol and juice craving outcomes. Then, we included the experimental procedures (ghrelin x moderator x trial) to test whether the moderated effect varied with the cue-reactivity.

There was no significant moderating effect for ghrelin vs placebo, at either the 1 mcg/kg or the 3 mcg/kg (p’s>0.05) doses, by cortisol concentration on alcohol craving outcomes over time. Also there was no significant change in intercept post-infusion, compared to placebo, for either the 1 mcg/kg or the 3 mcg/kg (p’s>0.05) doses at any trial during the cue reactivity of both alcohol trials, indicating that cortisol concentrations showed no additional influence on ghrelin’s effects in alcohol craving. In contrast, there was a significant moderating effect for ghrelin vs placebo, at the 3 mcg/kg (B2=0.649, p<0.001), but not at the 1 mcg/kg dose (p>0.05), by cortisol concentration on juice craving outcomes. However, there was no significant change in intercept post-infusion, compared to placebo, for the 3 mcg/kg (p’s>0.05) doses at any trial during the cue reactivity of both juice trials, indicating that cortisol concentrations showed no additional influence on ghrelin’s effects on juice craving.

There was no significant moderated effect for ghrelin vs placebo, at either the 1 mcg/kg or the 3 mcg/kg (p’s>0.05) doses, by aldosterone concentration on alcohol craving outcomes. The ghrelin by aldosterone × trial interaction, however, showed a significant change in the intercept post-infusion for the 1 mcg/kg dose (B1=1.397, p=0.004), with significant effect both at the first (B1=0.922, p=0.002) and second alcohol trial (B1=0.754, p=0.015). There was no significant change in the intercept post-infusion for the 3 mcg/kg dose, with no effect at any trial during the cue reactivity. As depicted in Figure 2A, the three-way interaction (ghrelin x aldosterone x trial) indicates that the aldosterone influenced ghrelin’s effects in alcohol craving (in both trials) only at the 1 mcg/kg dose. There was, however, a significant moderating effect for ghrelin vs placebo, at the 1 mcg/kg (B2=0.525, p=0.015), but not at the 3 mcg/kg dose, by aldosterone concentration on juice craving outcomes. The ghrelin x aldosterone × trial interaction, showed a significa nt change in the intercept post-infusion for the 1 mcg/kg dose (B1=1.540, p=0.008), with significant effect both at the first (B1=1.296, p=0.004) and second juice trials (B1=1.422, p=0.003) (Figure 2B). There was no significant change in the intercept post-infusion for the 3 mcg/kg dose, with no effect at any trial during the juice cue reactivity.

Figure 2. Three-way interaction (ghrelin x aldosterone x trial) after IV ghrelin administration and during the alcohol and juice cue reactivity procedure.

Figure 2.

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(A) The ghrelin by aldosterone × trial interaction showed a signifi cant change in the intercept post-infusion for the 1 mcg/kg dose (B1=1.397, p=0.004), with significant effect both at the first (B1=0.922, p=0.002) and second alcohol trial (B1=0.754, p=0.015). There was not increment of significant change in intercept post-infusion, compared to placebo, for the 3 mcg/kg dose. (B) The ghrelin by aldosterone × trial interaction sh owed a significant change in the intercept post-infusion for the 1 mcg/kg dose (B1=1.540, p=0.008), with significant effect both at the first (B1=1.296, p=0.004) and second juice trial (B1=1.422, p=0.003). There was not increment of significant change in intercept post-infusion, compared to placebo, for the 3 mcg/kg dose. Results are represented by the M±SEM, **(p<0.01), ***(p<0.001), not significant (p>0.05). BL; baseline (pre-infusion), IV ghrelin post-infusion (T1-T5: +6 min; +17 min; +23 min; +29 min and +48 min), where T1 and T3 correspond to the juice trials, T2 and T4 correspond to the alcohol trials and T5 the end of the experiment.

DISCUSSION

Consistent with our hypothesis, exogenous IV ghrelin administration increased endogenous cortisol and aldosterone serum concentrations. From a statistical standpoint, the effects on cortisol were greater than those on aldosterone, but on the other hand, only the aldosterone serum concentration was a moderator of ghrelin-induced craving.

The possibility of a cross-talk between the ghrelin system and cortisol and aldosterone pathways, with possible effects in modulating craving response, hold important clinical value because craving may be associated with relapse. Indeed, craving is a component of the DSM-5 diagnostic criteria for AUD and has been proposed as a clinically relevant endophenotype able to predict alcohol-related outcomes (Haass-Koffler and Kenna 2013; Haass-Koffler et al. 2014). Notably, cue-reactivity has demonstrated utility in eliciting urge to drink in alcohol-dependent individuals, and several medications that reduce alcohol craving in cue-reactivity human laboratory studies also reduce alcohol consumption in the real world (Ray et al. 2010).

We previously demonstrated in the parent study that administration of IV ghrelin resulted in an acute increase in cue-induced urge to drink alcohol, with a significant positive correlation between the post-infusion serum ghrelin concentrations and the increase in urge to drink alcohol (Leggio et al. 2014). The findings of the present analysis add potentially important clinical information. Here we report that aldosterone increased after IV ghrelin infusion and its increase significantly predicted the urge to drink. It is intriguing to speculate that ghrelin’s effects on craving might be mediated, at least in part, via its effects in increasing aldosterone concentrations, which in turn may affect drinking behaviors (Aoun et al. 2018). However, it is important to point out that while only the highest dose of ghrelin increased alcohol craving (Leggio et al. 2014), here the effect of IV ghrelin on aldosterone levels was limited to the lower dose of ghrelin. Indeed, the fact that we did not detect an interaction between the higher dose of ghrelin, aldosterone and alcohol craving needs to be taken cautiously. While there was not a statistical difference at the baseline aldosterone serum levels between groups, the aldosterone serum level of the 3 mcg/kg group was higher than the other two groups. The higher level of aldosterone could be attributed to other alcohol-related factors that were not accounted for in this laboratory study and the change from baseline was not detectable in the limited sample size of this experiment. As such, while these data suggest a potential ghrelin-aldosterone cross-talk, it remains to be determined whether this interaction plays any role in alcohol-seeking behaviors, and translational studies are needed to confirm such a link to fully address the potential mechanisms. Regarding the juice craving response detected at the 1 mcg/kg ghrelin dose, but not at the 3 mcg/kg ghrelin dose, it is possible that the time between trials (10 min) was not sufficient to detect the change of aldosterone between the experimental procedures (alcohol vs juice). Also, juice may not be an entirely neutral stimulus. The study was designed to measure alcohol craving; juice is liquid and has caloric content, so its related cues could have effects on the hormones that are not dissociable from alcohol craving.

We also found that IV ghrelin infusion increased blood cortisol concentrations, which did not moderate the effects of ghrelin on the increased craving for alcohol or juice. Previous work shows that dexamethasone increases ghrelin mRNA levels in hypothalamic cells, and the glucocorticoid receptor antagonist mifepristone blocks dexamethasone-induced increases in ghrelin mRNA levels (Kageyama et al. 2012). Dexamethasone also stimulates ghrelin receptor mRNA and protein concentrations. The role of ghrelin within the HPA-axis is supported by the fact that ghrelin increases CRF mRNA levels, and that co-application of both dexamethasone and ghrelin produces an additive effect on CRF and ghrelin mRNA levels (Kageyama et al. 2012). The hypothesis of a glucocorticoid-mediated hypothalamic control of ghrelin signaling still needs to be fully evaluated. From the parent study (Leggio et al. 2014), the cue reactivity was sufficient to elicit alcohol craving in alcohol dependent individuals. However, we believe that because the ghrelin-induced increase of cortisol levels did not exceed physiological conditions (normal range of cortisol is between 5–20 mcg/dL), the cortisol levels were not sufficient to elicit alcohol-related behaviors as tested in two-way (ghrelin x cortisol) and three-way (ghrelin x cortisol x trial) interactions. We speculate that either the cue reactivity or the short infusion of ghrelin were not sufficient to activate cortisol centrally via a negative feedback loop, or it is possible that, as cortisol acts centrally via a negative feedback loop regulating stress activation, ghrelin does not interact meaningfully with this neuroendocrinological pathway in absence of stress stimuli.

It is also important to point out that, while we are measuring peripheral cortisol concentrations as a surrogate of HPA-axis activity, and we are studying the HPA axis as a proxy of stress activity, it is plausible that extra-hypothalamic pathways are also involved. For example, a role of the amygdala in the complex interactions between ghrelin, alcohol and GABA signaling was shown (Cruz et al. 2013). Furthermore, we have recently shown that IV ghrelin administration, as compared to placebo, leads to increased amygdala activation in presence of alcohol cues and administration in alcohol-dependent, heavy drinkers during a neuroimaging experiment (Farokhnia et al. 2018).

This study has several strengths: (1) it was the first human research to evaluate glucocorticoid and mineralocorticoid response to an IV ghrelin infusion, compared with placebo, specifically in a sample of heavy-drinking, alcohol-dependent individuals; (2) the study was conducted in a well-controlled environment, i.e., real time, cue-elicited alcohol craving. The latter is particularly critical given that cortisol concentrations follow the circadian cycle and some individuals with primary aldosteronism have a circadian rhythm of aldosterone mediated by changes in ACTH (Kem et al. 1973); and (3) the IV ghrelin dose was calculated based on weight. Limitations of this study include: (1) the small sample size; (2) the short period of ghrelin infusion; (3) the limited time (10 min) between the alcohol and juice sessions; and (4) the difference of baseline aldosterone serum level (albeit not statistically significant) of the ghrelin 3 mcg/kg group, which might be responsible, at least in part, for the counterintuitive findings that 1 mcg/kg ghrelin, but not the higher dose, exhibited an effect on aldosterone levels. However, ghrelin remains in the system for almost one hour, and similar time was used for in vitro work demonstrating the increased concentration of both cortisol and aldosterone from cultured rat adrenocortical cells after ghrelin exposure (Rucinski et al. 2009). Future work could take into account pharmacological manipulations at the glucocorticoid and mineralocorticoid receptor level to further understand the complex relationship between the ghrelin system and both cortisol and aldosterone pathways.

In conclusion, the present study provides preliminary evidence of a cross-talk between ghrelin and both cortisol and aldosterone in AUD individuals. The present results also suggest some differences in the ghrelin/cortisol interaction versus the ghrelin/aldosterone interaction, and how they may interplay in predicting alcohol craving. While intriguing, this hypothesis is merely speculative and future translational research is needed to shed light on the potential role of the interaction of these two pathways in alcohol craving and drinking, and on the underlying mechanisms.

HIGHLIGHTS.

  • Evidence supports the role of appetite-regulating hormones in alcohol use disorder (AUD).

  • Preclinical and human studies support a role of the ghrelin system in addictive behaviors.

  • Endogenous cortisol and aldosterone serum concentrations increased after exogenous ghrelin administration, as compared to placebo.

  • Only the ghrelin-induced changes in aldosterone serum concentrations predicted craving.

ACKNOWLEDGMENT

The images of the stomach and kidney in the graphical abstract were obtained from ClipArtMag.com (free clip art).

FUNDING

Dr. Haass-Koffler is supported by the National Institute on Alcohol Abuse and Alcoholism (K01AA023867; PI: Haass-Koffler). Drs. Farokhnia and Leggio are supported by the National Institute on Alcohol Abuse and Alcoholism Division of Intramural Clinical and Biological Research and the National Institute on Drug Abuse Intramural Research Program (ZIA-AA000218, Section on Clinical Psychoneuroendocrinology and Neuropsychopharmacology; PI: Leggio). The parent study was funded by the National Institute on Alcohol Abuse and Alcoholism (R21AA019709; PIs: Leggio and Kenna). The content of this article is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or the Department of Veterans Affairs.

Footnotes

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CONFLICTS OF INTEREST

The authors report no biomedical financial interests or potential conflicts of interest.

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