Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2021 Jun 1.
Published in final edited form as: Alcohol. 2020 Jan 7;85:111–118. doi: 10.1016/j.alcohol.2019.12.006

Effects of Ethanol on Plasma Ghrelin levels in the Rat During Early and Late Adolescence

Kati L Healey 1, Justine D Landin 2, Kira Dubester 1, Sandra Kibble 1, Kristin Marquardt 2, Julianna N Brutman 3, Jon F Davis 3, H Scott Swartzwelder 1, L Judson Chandler 2
PMCID: PMC7238721  NIHMSID: NIHMS1582555  PMID: 31923560

Abstract

Ghrelin is an appetite-regulating peptide that is primarily secreted by endocrine cells in the stomach and is implicated in regulation of alcohol consumption and alcohol-reinforced behaviors. In the present study, adolescent Sprague-Dawley rats received intermittent ethanol (AIE) exposure by intragastric intubation (5 g/kg) or vapor inhalation, manipulations conducted between postnatal days (PD) 28–43. On the first and last day of AIE exposure, the level of intoxication was examined 1 hr after ethanol gavage or upon removal from the vapor chamber. This was immediately followed by a blood draw for determination of the blood ethanol concentration (BEC) and plasma levels of acylated ghrelin (acyl-ghrelin; active). On PD 29, plasma levels of acyl-ghrelin were significantly elevated in male (but not female) rats in response to acute ethanol exposure by both gastric gavage and vapor inhalation. Importantly, assessment of plasma acyl-ghrelin in response to repeated ethanol exposure revealed a complex interaction of both sex and method of AIE exposure. On PD 43, vapor inhalation increased plasma acyl-ghrelin in both males and females compared to their air control counterparts, whereas there was no change in plasma levels of acyl-ghrelin in either male or female rats in response to exposure by intragastric gavage. Assessment of plasma acyl-ghrelin following a 30-day ethanol-free period revealed AIE exposure did not produce a change in basal levels. In addition, an acute ethanol challenge in adult rats of 5g/kg via gastric gavage had no effect on plasma ghrelin levels when assessed one hr after initiation of exposure. Collectively, these observations suggest that acyl-ghrelin, a primary gut-brain signaling hormone, is elevated by ethanol during early adolescence independent of administration route, and in gender-dependent fashion.

Keywords: Alcohol, Adolescence, Ghrelin, Intragastric Ethanol, Ethanol Vapor, Intermittent Ethanol

Introduction

Alcohol is the most widely abused drug during adolescence and is typically consumed in binge-like patterns that are associated with high levels of intoxication (Witt 2010). In addition, the age of initiation of alcohol use is one of the strongest predictors of whether an individual will develop an alcohol use disorder (AUD) at some point in their life (Grant and Dawson 1997). Adolescence is a critical period for brain development and maturation, and accumulating evidence from both human studies and animal models indicates that the abuse of alcohol during this period is associated with changes in adult brain structure and function. For example, preclinical studies have shown that binge-like adolescent intermittent ethanol (AIE) exposure results in enduring changes in alcohol’s interoceptive effects as well as on the structure and function of multiple brain regions, including the hippocampus, prefrontal cortex and amygdala (Spear 2018).

It is well established that the intake of food and addictive drugs are controlled by a shared set of neuronal circuits (Volkow, Wang et al. 2013). In this context, feeding peptides are stimulated not only by energy balance, but also cognitive-emotional processes, and they target the brain to regulate monoamine release, appetitive, and consummatory behaviors (Barson, Morganstern et al. 2012). Ghrelin is an orexigenic peptide secreted by endocrine cells localized primarily in the stomach. Once released, the ghrelin peptide is acylated by ghrelin-O-acyltransferase to yield acyl-ghrelin, which is the active form of ghrelin that acts upon growth-hormone secretagogue receptors [(GHS-R1A; (Kojima, Hosoda et al. 1999, Zigman, Nakano et al. 2005)] to stimulate biological actions. Interestingly, a number of recent studies have suggested that ghrelin may play an important role in addiction and have implicated the ghrelin system as a potential pharmacological target to reduce alcohol drinking. Preclinical studies indicate that genetic reduction of GHSR-1a abolishes ethanol intake in mice (Jerlhag, Egecioglu et al. 2009) while the administration of ghrelin directly into mesolimbic brain regions augments ethanol intake (Jerlhag, Egecioglu et al. 2009). Supporting these observations in animal models, clinical studies have shown that circulating levels of ghrelin are increased in non-treatment seeking individuals with an AUD (Hillemacher, Kraus et al. 2007, Koopmann, von der Goltz et al. 2012). A positive association between ghrelin and alcohol craving has also been reported clinically, implicating a potential role of ghrelin signaling in alcohol withdrawal. In alcohol dependent male and females greater circulating ghrelin levels have been linked to increased alcohol craving (Addolorato, Capristo et al. 2006, Wurst, Graf et al. 2007, Leggio, Ferrulli et al. 2011, Koopmann, von der Goltz et al. 2012). Thus, ghrelin signaling may regulate multiple phases of alcohol addiction.

While the above studies suggest that the ghrelin system may be an important regulator of alcohol reward, little is known regarding the potential impact of alcohol use during adolescence on ghrelin signaling. As an initial step toward gaining a greater understanding of the impact of adolescent alcohol abuse on the ghrelin system, the goal of the present study was to determine the effects of AIE in rats on circulating acyl-ghrelin levels during alcohol exposure and following protracted abstinence in adulthood. The experimental approach involved assessment of changes in the plasma levels of active ghrelin when alcohol was administered intermittently during adolescence by either intragastric intubation or inhalation of ethanol vapor, two widely employed exposure models that involve distinctly different routes of alcohol absorption.

Materials and Methods

Adolescent rats were subjected to AIE exposure by either intragastric intubation or by vapor inhalation. The two procedures were conducted at separate institutions with the studies involving intragastric intubation conducted at Duke University (Duke; Durham, NC) and the studies involving vapor inhalation conducted at the Medical University of South Carolina (MUSC; Charleston, SC). The studies were run in parallel at the two institutions and attempts were made to standardize the experimental design and variables between the two institutions as much as possible. All of the procedures used were conducted in accordance with the guidelines of the American Association for the Accreditation of Laboratory Animal Care and the National Research Council’s Guide for Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committees of both Duke and MUSC.

Animals

Sprague-Dawley dams with pups were obtained by each institution from Envigo (Indianapolis, IN). Dams were shipped with 8 (Duke University) or 10 (MUSC) pups (male and female) that were postnatal day (PD) 15 upon arrival. After acclimation to the vivarium, pups were weaned on PD21 and pair-housed with same sex littermates. Rats were maintained on a 12 hr/12 hr reverse light/dark cycle (lights on at 1900 hr Duke and 2100 hr MUSC) with ad libitum access to food (MUSC: Teklab Global 18% Protein Rodent Diet 2918, Envigo, Madison, WI; Duke: Laboratory Rodent Chow 5001, Purina, St Hubert, Quebec) and water and remained pair-housed for the duration of the study. At the time of weaning, rats were randomly assigned to either an experimental or control group, and male/female and control/experimental groups were counterbalanced and run in tandem as separate cohorts of animals.

AIE Exposure

As depicted in the schematic in Figure 1, adolescent rats underwent intermittent binge-like exposure to ethanol by either intragastric gavage or vapor inhalation. Both adolescent exposure paradigms involved 10 episodes of ethanol exposure with the first exposure on PD28 and the last on PD43, corresponding with early and late adolescence, respectively (Spear 2000). The exposures were separated into 5 cycles, with each cycle consisting of 2 consecutive days of ethanol exposure followed by non-exposure day(s). The adult (PD 71–72) ethanol challenge experiment consisted of one exposure to intragastric gavage.

Figure 1. Experimental design and time-line.

Figure 1.

Open in a new tab

A) Schematic depiction of the time-line for adolescent ethanol exposure and obtaining blood for measurement of ethanol and/or acyl-ghrelin (active ghrelin) levels. B) Schematic depiction of the daily sequence of adolescent intermittent ethanol exposure with the first exposure occurring on PD28 and the last on PD43. During the adolescent period, rats were exposed to 5 intermittent ethanol exposure cycles with each cycle consisting of 2 consecutive days of exposure by either vapor inhalation or intragastric gavage (5 g/kg) followed by ethanol free day(s). Each day of vapor exposure consisted of 14 hrs of ethanol vapor exposure. Each episode of gastric ethanol administration is indicated by the vertical arrows. Alcohol exposure by intragastric gavage (n=8–10) (C) or vapor inhalation (n=9–10) (D) did not alter the developmental time-course of body weight gain in either male or female rats.

Intragastric AIE exposure or adult acute exposure involved a well-characterized procedure in which rats were administered a dose of 5 g/kg ethanol (35% v/v in water at 18.12 ml/kg) or isovolumetric water (control) at 1000 hours on each exposure day (Risher, Fleming et al. 2015). Ethanol exposure by vapor inhalation also involved a well-characterized procedure in which rats in standard polycarbonate housing cages (with bedding, food and water) were placed into clear acrylic chambers containing vaporized ethanol (Gass, Glen et al. 2014). Each vapor exposure period consisted of 14 hours in the ethanol chambers followed by 10 hours out of the chambers. Rats were placed into the chambers with same-sex littermates at 1800 hours and removed at 0800 hours.

Intoxication scores were taken either 1 hour after each dose of intragastric ethanol or immediately upon removal from the vapor chambers. Intoxication rating involved a subjective 5-point scale (Nixon and Crews 2002, Monti, Miranda et al. 2005, Gass, Glen et al. 2014) where 1 = no signs of intoxication; 2 = slight motor impairment; 3 = obvious motor impairment but able to walk; 4 = dragging abdomen, loss of righting reflex; 5 = loss of righting and eyeblink reflexes. Tail blood (40–50 μLs) was collected immediately after intoxication scoring for measurement of blood ethanol concentrations (BECs) using an Analox alcohol analyzer (Analox Instruments, Atlanta, GA). As the results from a pilot study conducted in a separate group of animals, as well as previously published work (Risher, Fleming et al. 2015), indicated that the BEC 1 hr after an intragastric ethanol dose of 5g/kg is in the 150–200 mg/dl range, the ethanol flow into the vapor chambers was adjusted daily in order to target a similar level of alcohol exposure.

Measurement of Acyl-Ghrelin (Active)

For analysis of circulating levels of acyl-ghrelin, blood samples were collected after the first exposure to ethanol (PD28) and the last exposure (PD43). Given that vapor exposure involved a 14-hr exposure period, blood samples were taken on PD29 and PD44. However, the PD43 time-point when exposure was initiated for both procedures will be used for data presentation purposes. To assess enduring effects of AIE on the basal levels of circulating ghrelin, blood was also drawn in adulthood at PD73. An additional adult cohort was acutely exposed to ethanol or water via intragastric gavage on PD71–72 and blood samples were drawn 1 hour following gavage. For sampling at PD28, blood (200–400 μL) was obtained from the tail vein, whereas for PD43 and PD71–73 blood draws it was obtained from the saphenous vein. Blood samples were collected in tubes containing EDTA (1.86 mg/mL) and Pefabloc (1.0 mg/mL), and then centrifuged at 10,000 RPM for 15 minutes at 4° C. Plasma was transferred into a separate tube on ice containing 10 μL 1N HCl (10.0% total volume). Samples were subsequently shipped overnight on dry ice to Washington State University for measurement of acylated ghrelin levels using commercially available ELISA assay plates for Rat/Mouse Ghrelin (EZRGRA-90K, Millipore Sigma, Darmstadt, Germany).

Data Analysis

A three-way ANOVA was conducted to determine the impact of AIE on body weight across sex and postnatal day (PD). A two-way ANOVA indicated there was no main effect of sex on AIE-induced blood ethanol content (BEC) or intoxication scores. As a result, data were collapsed across sex, and a two-tailed student’s t-test was used to assess BEC and intoxication scores at different stages of ethanol exposure. The effect of AIE exposure on circulating acyl-ghrelin levels at PD28 was analyzed using a two-tailed student’s t-test. The effect of AIE exposure on circulating acyl-ghrelin levels at PD43 and 73 was analyzed using a one-tailed student’s t-test. The effect of acute ethanol exposure on circulating acyl-ghrelin levels in adults was analyzed using a two-tailed t-test.

Results

The present study investigated the impact of AIE exposure on the levels of plasma acyl-ghrelin using two established rodent models of alcohol exposure. Levels of ethanol and acyl-ghrelin were measured after the first (PD28) and last (PD43) day of AIE exposure. Immediately prior to each blood draw, behavioral intoxication was assessed using a subjective 5-point rating scale. As shown in Figure 1C & 1D, neither method of AIE exposure altered the developmental progression of body weight gain when compared to the sex and aged-matched non-ethanol exposure control group.

As there were no significant differences in the BEC between males and females (BEC main effect of sex: Duke F(1,25)= 0.13, p = 0.72, MUSC F(1,35)= 1.13, p = 0.30; Intoxication main effect of sex: Duke F(1,18)= 0.50, p = 0.46, MUSC F(1,35)= 1.00, p = 0.33). Therefore, Figure 2 represents data collapsed across sex. One hour after intragastric administration of 5 g/kg ethanol, the BECs were similar on both PD28 (219.1 ± 11.9 mg/dl) and PD43 (238.8 ± 17.8 mg/dl) (Figure 2A). While the BEC levels following vapor exposure were slightly lower compared to the levels associated with the intragastric 5 g/kg dose, they were similar across both exposure days (PD28 = 175.7 ± 14.3 mg/dl; PD43 = 143.4 ± 18.3 mg/dl) (Figure 2A). Consistent with previous studies (Nixon and Crews 2002, Gass, Glen et al. 2014) assessment of the level of intoxication using the 5-point rating scale revealed that at PD28, rats exhibited either low levels of intoxication following intragastric exposure (1.421 ± 0.116) or no signs of intoxication following vapor exposure (1.000 ± 0.000) (Figure 2B). Interestingly, when assessed at PD43, the rats exposed to ethanol by intragastric gavage were significantly more sensitive to the intoxicating effect of ethanol (3.421 ± 0.221) (t = 8.29, p<0.01) while the rats subjected to vapor inhalation continued to exhibit few signs of intoxication (1.050 ± 0.050). Although these studies were not designed to make a direct comparison between intragastric gavage and vapor inhalation and therefore were treated statistically as separate and independent studies, these observations may indicate that over the time-course of AIE exposure, Sprague Dawley rats develop differential sensitivity to the intoxicating effects of ethanol as a function of the method of AIE exposure.

Figure 2. BEC and intoxication scores for the first and last day of AIE.

Figure 2.

Open in a new tab

A) Blood ethanol concentration (BEC) during the first (PD28) and last (PD43) day of ethanol exposure. BEC levels were obtained 1 hr after intragastric gavage (n = 12–17/group) of 5 g/kg or upon removal of rats from the vapor (n = 19–20/group) exposure chambers. B) Immediately prior to the blood draw for measurement of BEC, the level of intoxication was assessed using a 5-point intoxication rating scale (* p < 0.01, intragastric gavage n = 19/group, vapor n = 20/group).

Plasma levels of acyl-ghrelin were assessed 1 hr after the first intragastric dose of ethanol or immediately upon removal of rats from ethanol vapor chambers following the first 14-hr period of exposure. As shown in Figures 3A & 3C, plasma levels of acyl-ghrelin in male rats were significantly elevated in both AIE exposure paradigms compared to their non-exposed litter mate controls (intragastric: t=2.34, p<0.05; vapor: t=2.70, p<0.02). In contrast, there were no changes in acyl-ghrelin levels in female rats following either intragastric (t=0.49, p=0.63) or vapor (t=0.20, p=0.84) exposure (Figures 3B & 3D). Therefore, early adolescent Sprague-Dawley male, but not female, rats exhibited an increase in plasma acyl-ghrelin in response to acute ethanol that was not dependent upon the method of ethanol administration.

Figure 3. Sex-specific increase in plasma ghrelin levels during the first episode of ethanol exposure.

Figure 3.

Open in a new tab

Plasma levels of acyl-ghrelin were significantly increased in male rats during the initial episode of ethanol exposure at PD28 following either gastric gavage (5 g/kg) (A) or vapor inhalation (C) compared to their respective ethanol-naive controls (intragastric gavage n = 7–9/group, vapor n = 9/group). In contrast, the levels of acyl-ghrelin were not altered in female rats following either gastric gavage (B) or vapor inhalation (D). (* p < 0.05)

To determine the effect of repeated cycles of ethanol exposure on acyl-ghrelin levels during the early to late period of adolescence, we next obtained blood following the last ethanol exposure period on PD43. There was no change in plasma levels of acyl-ghrelin in either male (t=0.74, p=0.47; Figure 4A) or female (t= 1.40, p=0.19; Figure 4B) rats compared to controls during the final episode of ethanol exposure by intragastric gavage. In contrast, following the last episode of AIE exposure by vapor inhalation, both male (t=1.79, p<0.05, Figure 4C) and female (t=2.54, p<0.05, Figure 4D) rats exhibited significant increases in acyl-ghrelin compared to their air control counterparts.

Figure 4. Ethanol-induced increases in plasma ghrelin following AIE is dependent upon sex and method of alcohol exposure.

Figure 4.

Open in a new tab

Plasma ghrelin levels were not significantly altered in either male (A) or female (B) rats during the last episode of AIE exposure by intragastric administration of ethanol (5g/kg) on PD43 (male n = 6–9/group, female n = 5–8/group) In contrast, plasma levels of acyl-ghrelin were significantly increased in both male (n = 8–9/group) (C) and female (n = 9–10/group) (D) rats following the last episode of ethanol exposure by vapor inhalation. (* p < 0.05).

To determine whether AIE exposure resulted in alterations in the baseline levels of plasma acyl-ghrelin after an extended period of ethanol absence, blood samples were obtained from these same rats at PD73. As shown in Figure 5, there were no enduring effects of AIE exposure by either gavage or vapor on plasma acyl-ghrelin levels in adult rats (PD73) compared to age-matched controls (p>0.05).

Figure 5. AIE had no effect on basal levels of plasma ghrelin in adulthood.

Figure 5.

Open in a new tab

Thirty days after the last episode of AIE exposure by gastric gavage, basal levels of plasma acyl-ghrelin were not significantly different in either male (n = 8–9/group) (A) or female (n = 7–9/group) (B) adult (PD73) rats. Similarly, basal levels of plasma acyl-ghrelin were also not significantly different in either male (n = 9–10/group) (C) or female (n = 8–9/group) (D) adult rats that had been subjected to AIE exposure by vapor inhalation (n = 7–10/group).

To determine whether adult rats exhibit a similar sex-dependent increase in plasma levels of acyl-ghrelin following an acute ethanol challenge, as was observed in adolescent rats, we next exposed an ethanol naive group of adult (PD70) male (n =6–7) and female (n=5–8) Sprague-Dawley rats to an acute intragastric gavage of either water or ethanol (5g/kg) and obtained blood 1 hr later for analysis. As shown in Figure 6, the plasma levels of acyl-ghrelin levels were not altered during this time frame in either adult male (1.098, p=0.30) of female (t=0.12, p=0.91) rats. Therefore, the sex-dependent increase in plasma acyl-ghrelin that was observed 1 hour after an acute intragastric challenge of ethanol was not observed in adult rats.

Figure 6. Acute ethanol challenge does not alter plasma ghrelin levels in adulthood.

Figure 6.

Open in a new tab

Adult (PD70) male and female rats were subjected gavage of either water or ethanol (5g/kg). Measurement of plasma acyl-ghrelin from blood obtained one hour later revealed there was no change in levels in either male (n = 6–7/group) or female (n = 5–8/group) rats.

Discussion

The majority of studies that have investigated the effects of ethanol on gut-brain signaling have focused on adult ethanol exposure, and therefore very little is known about alterations of this process during adolescent alcohol exposure. In the present study, we observed that acute ethanol exposure of young adolescent (PD28) male but not female rats increased the plasma levels of the active form of ghrelin. This sex-specific increase was observed when the blood was obtained either 1 hr after intragastric administration of ethanol (5 g/kg) or immediately following a 14-hr period of exposure by ethanol vapor inhalation. However, when male and female rats were subjected to 10 episodes of intermittent ethanol, with the first exposure occurring on PD28 and the last on PD43, ethanol-induced alterations in plasma acyl-ghrelin depended upon the method of AIE exposure. With vapor inhalation, ghrelin levels were significantly increased during the last episode of ethanol exposure in both male and female rats. In contrast, no changes were observed during the last episode of ethanol exposure by intragastric gavage. Taken together, these results reveal that the effect of ethanol on the levels of plasma ghrelin during adolescence is complex and dependent on sex and route/duration of alcohol exposure.

The present findings of increased plasma acyl-ghrelin following acute AIE in early adolescence infers that gastric ghrelin secretion is enhanced by acute ethanol exposure. This observation is inconsistent with a recent study conducted in both 1 month old and adult mice, which observed reduced levels of plasma ghrelin 0.5–4 hrs after an acute intraperitoneal (i.p.) injection of ethanol (2 g/kg). However, following 20 daily episodes of ethanol exposure by vapor inhalation (8 hrs/day), an acute ethanol challenge (i.p.) led to increased plasma ghrelin (Yoshimoto, Nagao et al. 2017). It should be considered that in addition to species differences, other experimental variables such as the duration of ethanol exposure and inclusion of ethanol challenge [e.g., intraperitoneal injection of ethanol (2 g/kg)] likely also contributed to the discrepant observations between the two studies.

It is important to note that the aforementioned studies and the current study relied upon involuntary methods of ethanol administration. Previous studies have linked increased ghrelin signaling with increased motivation to consume alcohol in both humans (Koopmann, von der Goltz et al. 2012, Leggio, Ferrulli et al. 2012, Leggio, Zywiak et al. 2014) and animals (Jerlhag, Egecioglu et al. 2009, Gomez, Cunningham et al. 2015). However, the majority of these studies have been conducted in subjects that have a history of alcohol exposure, and it is unclear whether ghrelin levels or alcohol craving would similarly be impacted in alcohol-naïve animals or non-drinking individuals. While the present study was not designed to test this hypothesis, it is important to determine whether initial voluntary consumption of alcohol during adolescence increases circulating ghrelin levels in a similar manner to intragastric gavage or vapor exposure. In addition, future studies are needed to clarify whether increased plasma ghrelin levels following AIE provoke anticipatory seeking behaviors for alcohol and/or food. In this regard, previous studies indicate that antagonism of ghrelin receptor (GHSR-1a) in alcohol-dependent rodents reduced the amount of alcohol voluntarily consumed without affecting feeding behavior (Gomez, Cunningham et al. 2015). Therefore, separate from the ghrelin ligand, targeted antagonism of GHSR-1a may have the potential to influence behavioral mechanisms that support escalation of drinking post AIE.

As stated above, few studies have investigated the relationship between adolescent ethanol exposure and ghrelin in animal models. Although the relationship between ethanol and ghrelin has been studied in both healthy adults and adults with alcohol use disorders (AUD), to our knowledge there have been no studies investigating ethanol consumption and ghrelin in adolescent humans. In adults, it has been repeatedly reported that total and acyl-ghrelin are reduced following acute alcohol exposure in healthy adults (Calissendorff, Danielsson et al. 2005, Calissendorff, Danielsson et al. 2006, Zimmermann, Buchmann et al. 2007, Leggio, Schwandt et al. 2013, Ralevski, Horvath et al. 2017). To our knowledge, there are no studies in adolescent rodents that have examined the effect of an acute ethanol challenge on plasma levels of ghrelin, and the few adult rodent studies have conflicting findings (Szulc, Mikolajczak et al. 2013, Yoshimoto, Nagao et al. 2017). The current study suggests that there are no differences in adult rats following acute ethanol or water challenge on plasma levels of ghrelin. This may indicate that the increases in plasma ghrelin levels following adolescent ethanol exposure are specific to this developmental period. While the observation in human adults would appear inconsistent with our current finding that adolescent rats showed increased ghrelin following initial exposure and after repeated ethanol exposure, these discrepancies could be a result of differences in ghrelin signaling in adolescence, compared to adulthood. Future studies will be needed to address this possibility.

In contrast to healthy adults, those with AUD exhibit increased circulating ghrelin during acute alcohol intoxication (Kraus, Schanze et al. 2005). This indicates divergent processes for alcohol-ghrelin interactions in individuals with AUD compared to their healthy counterparts and suggest that similar divergent processes may underlie the present findings in adolescent rats. Additionally, early abstaining humans (abstinent for 72 hours) also display elevated levels of circulating ghrelin compared to controls and acutely intoxicated patients with AUDs (Kraus, Schanze et al. 2005). Further, among individuals with AUD, ghrelin levels increase for up to 14 days after the initiation of abstinence (Koopmann, von der Goltz et al. 2012). However, this relationship is complex, as some studies have reported decreased fasting levels of plasma ghrelin in AUD early abstainers compared to healthy controls (Addolorato, Capristo et al. 2006, Badaoui, De Saeger et al. 2008). Notably, infusion of exogenous ghrelin in non-treatment seeking alcohol-dependent individuals potentiates alcohol cravings (Leggio, Zywiak et al. 2014), increases intravenous self-administration of alcohol (Farokhnia, Grodin et al. 2018), reduces latency to first alcohol drink (Farokhnia, Grodin et al. 2018), and attenuates hangover symptoms (Farokhnia, Lee et al. 2018).

The high comorbidity of AUD and eating disorders suggests a link between alcohol use and food intake regulation (Goldbloom, Naranjo et al. 1992, Dansky, Brewerton et al. 2000, Bulik, Klump et al. 2004). Following the ingestion of food, healthy adults exhibit reduced plasma ghrelin (Tschop, Wawarta et al. 2001), and it seems reasonable to suggest that ethanol’s caloric value could contribute to the decrease in total ghrelin levels observed in healthy individuals. Although speculative, it is possible that ghrelin activation in individuals with an AUD may respond more strongly to the reinforcing properties of ethanol than to its caloric value. If so, that could explain why individuals with AUD show an increase in plasma ghrelin following alcohol. Consistent with this hypothesis, social drinkers with elevated levels of plasma ghrelin after fasting experience more intense and longer subjective effects of alcohol (Ralevski, Horvath et al. 2017) and consume more alcohol than those with lower ghrelin levels (Ralevski, Horvath et al. 2017). Further, social drinkers with elevated plasma ghrelin are more sensitive to reward and more impulsive due to reduced self-control (Ralevski, Shanabrough et al. 2018). Since adolescent animals are more sensitive than adults to the reinforcing properties of various stimuli, including alcohol, it is possible that the reinforcing properties of alcohol are a more salient driver of its effects on ghrelin than its caloric value.

The results of the present study also indicate that baseline levels of plasma acyl-ghrelin were not altered in adult rats 30 days after the last episode of either intragastric or vapor ethanol exposure during adolescence. This is in contrast to a previous report in human alcohol dependent individuals where ghrelin levels continued to rise between abstinence days 1 and 14 (Koopmann, von der Goltz et al. 2012). However, the extant literature is inconsistent with respect to whether baseline levels of plasma ghrelin are altered in abstinent, alcohol-dependent individuals compared to controls (Kim, Yoon et al. 2005, Kraus, Schanze et al. 2005, Addolorato, Capristo et al. 2006, Koopmann, von der Goltz et al. 2012). The apparently conflicting observations could be due to the length of abstinence (currently drinking, withdrawal or abstinence) or the type of ghrelin measured (total versus acyl-ghrelin) or the age of the subjects during ethanol exposure (adolescence versus adulthood). Notably, many of these studies, clinical and preclinical, investigated total ghrelin, which includes activated and inactivated forms of ghrelin, whereas in our study we measured activated acyl-ghrelin only. While some studies report that changes in total and acyl-ghrelin follow a similar pattern of change (Ralevski, Shanabrough et al. 2018), this similarity is not always observed [for review see: (Jerlhag 2018, Koopmann, Bach et al. 2018)], which underscores the importance of measuring acyl-ghrelin instead of total ghrelin.

In summary, the present study demonstrates that the acute exposure of early adolescent rats to ethanol by either intragastric gavage or vapor inhalation leads to an increase in the levels of plasma acyl-ghrelin in male but not female rats. However, assessment of plasma acyl-ghrelin in response to repeated ethanol exposure revealed a complex interaction of both sex and route of ethanol exposure. While additional studies are clearly needed to more fully define the interaction of age, sex and ethanol exposure methodology, these studies provide additional support for the accumulating clinical data suggesting that the ghrelin system could represent a novel therapeutic target for the treatment and prevention of AUD and may be an influencing factor in adolescent alcohol use.

Highlights.

  • Intermittent ethanol exposure by both intragastric gavage and vapor inhalation increased plasma levels of acyl-ghrelin (activated) in male, but not female, early adolescent rats.

  • Only intermittent ethanol exposure through vapor inhalation led to a sex-independent increase in acyl-ghrelin during late adolescence.

  • Adolescent intermittent ethanol exposure did not alter basal levels of plasma acyl-ghrelin following an extended period of alcohol absence.

Acknowledgements

The authors thank Sierra Hodges, Amanda Nilsen, and Aly Selchick, and Kristin Marquardt for technical assistance. This work was supported by NIH grants NIAAA NADIA AA010983 (LJC), AA019925 (HSS).

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  1. Addolorato G, Capristo E, Leggio L, Ferrulli A, Abenavoli L, Malandrino N, Farnetti S, Domenicali M, D’Angelo C, Vonghia L, Mirijello A, Cardone S and Gasbarrini G (2006). “Relationship Between Ghrelin Levels, Alcohol Craving, and Nutritional Status in Current Alcoholic Patients.” Alcoholism: Clinical and Experimental Research 30(11): 1933–1937. [DOI] [PubMed] [Google Scholar]
  2. Badaoui A, De Saeger C, Duchemin J, Gihousse D, De Timary P and Stärkel P (2008). “Alcohol dependence is associated with reduced plasma and fundic ghrelin levels.” European Journal of Clinical Investigation 38(6): 397–403. [DOI] [PubMed] [Google Scholar]
  3. Barson JR, Morganstern I and Leibowitz SF (2012). “Neurobiology of consummatory behavior: mechanisms underlying overeating and drug use.” ILAR J 53(1): 35–58. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bulik CM, Klump KL, Thornton L, Kaplan AS, Devlin B, Fichter MM, Halmi KA, Strober M, Woodside DB, Crow S, Mitchell JE, Rotondo A, Mauri M, Cassano GB, Keel PK, Berrettini WH and Kaye WH (2004). “Alcohol use disorder comorbidity in eating disorders: a multicenter study.” J Clin Psychiatry 65(7): 1000–1006. [DOI] [PubMed] [Google Scholar]
  5. Calissendorff J, Danielsson O, Brismar K and Röjdmark S (2006). “Alcohol ingestion does not affect serum levels of peptide YY but decreases both total and octanoylated ghrelin levels in healthy subjects.” Metabolism - Clinical and Experimental 55(12): 1625–1629. [DOI] [PubMed] [Google Scholar]
  6. Calissendorff J, Danielsson O, Brismar K and Röjdmark S (2005). “Inhibitory effect of alcohol on ghrelin secretion in normal man.” European Journal of Endocrinology eur j endocrinol 152(5): 743–747. [DOI] [PubMed] [Google Scholar]
  7. Dansky BS, Brewerton TD and Kilpatrick DG (2000). “Comorbidity of bulimia nervosa and alcohol use disorders: Results from the National Women’s Study.” 27(2): 180–190. [DOI] [PubMed] [Google Scholar]
  8. Farokhnia M, Grodin EN, Lee MR, Oot EN, Blackburn AN, Stangl BL, Schwandt ML, Farinelli LA, Momenan R, Ramchandani VA and Leggio L (2018). “Exogenous ghrelin administration increases alcohol self-administration and modulates brain functional activity in heavy-drinking alcohol-dependent individuals.” Mol Psychiatry 23(10): 2029–2038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Farokhnia M, Lee MR, Farinelli LA, Ramchandani VA, Akhlaghi F and Leggio L (2018). “Pharmacological manipulation of the ghrelin system and alcohol hangover symptoms in heavy drinking individuals: Is there a link?” Pharmacology Biochemistry and Behavior 172: 39–49. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gass JT, Glen WB Jr., McGonigal JT, Trantham-Davidson H, Lopez MF, Randall PK, Yaxley R, Floresco SB and Chandler LJ (2014). “Adolescent alcohol exposure reduces behavioral flexibility, promotes disinhibition, and increases resistance to extinction of ethanol self-administration in adulthood.” Neuropsychopharmacology 39(11): 2570–2583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Goldbloom DS, Naranjo CA, Bremner KE and Hicks LK (1992). “Eating disorders and alcohol abuse in women.” 87(6): 913–920. [DOI] [PubMed] [Google Scholar]
  12. Gomez JL, Cunningham CL, Finn DA, Young EA, Helpenstell LK, Schuette LM, Fidler TL, Kosten TA and Ryabinin AE (2015). “Differential effects of ghrelin antagonists on alcohol drinking and reinforcement in mouse and rat models of alcohol dependence.” Neuropharmacology 97: 182–193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Grant BF and Dawson DA (1997). “Age at onset of alcohol use and its association with DSM-IV alcohol abuse and dependence: results from the National Longitudinal Alcohol Epidemiologic Survey.” J Subst Abuse 9: 103–110. [DOI] [PubMed] [Google Scholar]
  14. Hillemacher T, Kraus T, Rauh J, Weiß J, Schanze A, Frieling H, Wilhelm J, Heberlein A, Gröschl M, Sperling W, Kornhuber J and Bleich S (2007). “Role of Appetite-Regulating Peptides in Alcohol Craving: An Analysis in Respect to Subtypes and Different Consumption Patterns in Alcoholism.” Alcoholism: Clinical and Experimental Research 31(6): 950–954. [DOI] [PubMed] [Google Scholar]
  15. Holst B and Schwartz TW (2004). “Constitutive ghrelin receptor activity as a signaling set-point in appetite regulation.” Trends Pharmacol Sci 25(3): 113–117. [DOI] [PubMed] [Google Scholar]
  16. Jerlhag E (2018). “Gut-brain axis and addictive disorders: A review with focus on alcohol and drugs of abuse.” Pharmacology & Therapeutics. [DOI] [PubMed] [Google Scholar]
  17. Jerlhag E, Egecioglu E, Landgren S, Salome N, Heilig M, Moechars D, Datta R, Perrissoud D, Dickson SL and Engel JA (2009). “Requirement of central ghrelin signaling for alcohol reward.” Proc Natl Acad Sci U S A 106(27): 11318–11323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jerlhag E, Ivanoff L, Vater A and Engel JA (2014). “Peripherally circulating ghrelin does not mediate alcohol-induced reward and alcohol intake in rodents.” Alcohol Clin Exp Res 38(4): 959–968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kim DJ, Yoon SJ, Choi B, Kim TS, Woo YS, Kim W, Myrick H, Peterson BS, Choi YB, Kim YK and Jeong J (2005). “Increased fasting plasma ghrelin levels during alcohol abstinence.” Alcohol Alcohol 40(1): 76–79. [DOI] [PubMed] [Google Scholar]
  20. Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H and Kangawa K (1999). “Ghrelin is a growth-hormone-releasing acylated peptide from stomach.” Nature 402: 656. [DOI] [PubMed] [Google Scholar]
  21. Koopmann A, Bach P, Schuster R, Bumb JM, Vollstadt-Klein S, Reinhard I, Rietschel M, Witt SH, Wiedemann K and Kiefer F (2018). “Ghrelin modulates mesolimbic reactivity to alcohol cues in alcohol-addicted subjects: a functional imaging study.” Addiction Biology 0(0). [DOI] [PubMed] [Google Scholar]
  22. Koopmann A, von der Goltz C, Grosshans M, Dinter C, Vitale M, Wiedemann K and Kiefer F (2012). “The association of the appetitive peptide acetylated ghrelin with alcohol craving in early abstinent alcohol dependent individuals.” Psychoneuroendocrinology 37(7): 980–986. [DOI] [PubMed] [Google Scholar]
  23. Kraus T, Schanze A, Groschl M, Bayerlein K, Hillemacher T, Reulbach U, Kornhuber J and Bleich S (2005). “Ghrelin levels are increased in alcoholism.” Alcohol Clin Exp Res 29(12): 2154–2157. [DOI] [PubMed] [Google Scholar]
  24. Leggio L, Ferrulli A, Cardone S, Nesci A, Miceli A, Malandrino N, Capristo E, Canestrelli B, Monteleone P, Kenna GA, Swift RM and Addolorato G (2011). “Ghrelin system in alcohol-dependent subjects: role of plasma ghrelin levels in alcohol drinking and craving.” Addiction Biology 17(2): 452–464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Leggio L, Ferrulli A, Cardone S, Nesci A, Miceli A, Malandrino N, Capristo E, Canestrelli B, Monteleone P, Kenna GA, Swift RM and Addolorato G (2012). “Ghrelin system in alcohol-dependent subjects: role of plasma ghrelin levels in alcohol drinking and craving.” Addict Biol 17(2): 452–464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Leggio L, Schwandt ML, Oot EN, Dias AA and Ramchandani VA (2013). “Fasting-induced increase in plasma ghrelin is blunted by intravenous alcohol administration: a within-subject placebo-controlled study.” Psychoneuroendocrinology 38(12): 3085–3091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Leggio L, Zywiak WH, Fricchione SR, Edwards SM, de la Monte SM, Swift RM and Kenna GA (2014). “Intravenous Ghrelin Administration Increases Alcohol Craving in Alcohol-Dependent Heavy Drinkers: A Preliminary Investigation.” Biological Psychiatry 76(9): 734–741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Leggio L, Zywiak WH, Fricchione SR, Edwards SM, de la Monte SM, Swift RM and Kenna GA (2014). “Intravenous ghrelin administration increases alcohol craving in alcohol-dependent heavy drinkers: a preliminary investigation.” Biol Psychiatry 76(9): 734–741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Monti PM, Miranda R Jr., Nixon K, Sher KJ, Swartzwelder HS, Tapert SF, White A and Crews FT (2005). “Adolescence: booze, brains, and behavior.” Alcohol Clin Exp Res 29(2): 207–220. [DOI] [PubMed] [Google Scholar]
  30. Nixon K and Crews FT (2002). “Binge ethanol exposure decreases neurogenesis in adult rat hippocampus.” 83(5): 1087–1093. [DOI] [PubMed] [Google Scholar]
  31. Ralevski E, Horvath TL, Shanabrough M, Hayden R, Newcomb J and Petrakis I (2017). “Ghrelin is Supressed by Intravenous Alcohol and is Related to Stimulant and Sedative Effects of Alcohol.” Alcohol and Alcoholism 52(4): 431–438. [DOI] [PubMed] [Google Scholar]
  32. Ralevski E, Shanabrough M, Newcomb J, Gandelman E, Hayden R, Horvath TL and Petrakis I (2018). “Ghrelin is Related to Personality Differences in Reward Sensitivity and Impulsivity.” Alcohol and Alcoholism 53(1): 52–56. [DOI] [PubMed] [Google Scholar]
  33. Risher ML, Fleming RL, Risher WC, Miller KM, Klein RC, Wills T, Acheson SK, Moore SD, Wilson WA, Eroglu C and Swartzwelder HS (2015). “Adolescent intermittent alcohol exposure: persistence of structural and functional hippocampal abnormalities into adulthood.” Alcohol Clin Exp Res 39(6): 989–997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sirohi S, Richardson BD, Lugo JM, Rossi DJ and Davis JF (2017). “Impact of Roux-en-Y gastric bypass surgery on appetite, alcohol intake behaviors, and midbrain ghrelin signaling in the rat.” Obesity (Silver Spring) 25(7): 1228–1236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sirohi S, Van Cleef A and Davis JF (2017). “Intermittent access to a nutritionally complete high-fat diet attenuates alcohol drinking in rats.” Pharmacol Biochem Behav 153: 105–115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Spear LP (2000). “The adolescent brain and age-related behavioral manifestations.” Neurosci Biobehav Rev 24(4): 417–463. [DOI] [PubMed] [Google Scholar]
  37. Spear LP (2018). “Effects of adolescent alcohol consumption on the brain and behaviour.” Nature Reviews Neuroscience 19: 197. [DOI] [PubMed] [Google Scholar]
  38. Szulc M, Mikolajczak PL, Geppert B, Wachowiak R, Dyr W and Bobkiewicz-Kozlowska T (2013). “Ethanol affects acylated and total ghrelin levels in peripheral blood of alcohol-dependent rats.” Addict Biol 18(4): 689–701. [DOI] [PubMed] [Google Scholar]
  39. Tschop M, Wawarta R, Riepl RL, Friedrich S, Bidlingmaier M, Landgraf R and Folwaczny C (2001). “Post-prandial decrease of circulating human ghrelin levels.” J Endocrinol Invest 24(6): RC19–21. [DOI] [PubMed] [Google Scholar]
  40. Volkow ND, Wang GJ, Tomasi D and Baler RD (2013). “Obesity and addiction: neurobiological overlaps.” Obes Rev 14(1): 2–18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Witt ED (2010). “Research on alcohol and adolescent brain development: opportunities and future directions.” Alcohol 44(1): 119–124. [DOI] [PubMed] [Google Scholar]
  42. Wurst FM, Graf I, Ehrenthal HD, Klein S, Backhaus J, Blank S, Graf M, Pridzun L, Wiesbeck GA and Junghanns K (2007). “Gender Differences for Ghrelin Levels in Alcohol-Dependent Patients and Differences Between Alcoholics and Healthy Controls.” Alcoholism: Clinical and Experimental Research 31(12): 2006–2011. [DOI] [PubMed] [Google Scholar]
  43. Yoshimoto K, Nagao M, Watanabe Y, Yamaguchi T, Ueda S, Kitamura Y, Nishimura K, Inden M, Marunaka Y, Hattori H, Murakami K, Tokaji M and Ochi K (2017). “Enhanced alcohol-drinking behavior associated with active ghrelinergic and serotoninergic neurons in the lateral hypothalamus and amygdala.” Pharmacology Biochemistry and Behavior 153: 1–11. [DOI] [PubMed] [Google Scholar]
  44. Zigman JM, Nakano Y, Coppari R, Balthasar N, Marcus JN, Lee CE, Jones JE, Deysher AE, Waxman AR, White RD, Williams TD, Lachey JL, Seeley RJ, Lowell BB and Elmquist JK (2005). “Mice lacking ghrelin receptors resist the development of diet-induced obesity.” J Clin Invest 115(12): 3564–3572. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Zimmermann US, Buchmann A, Steffin B, Dieterle C and Uhr M (2007). “CLINICAL STUDY: Alcohol administration acutely inhibits ghrelin secretion in an experiment involving psychosocial stress.” Addiction Biology 12(1): 17–21. [DOI] [PubMed] [Google Scholar]

RESOURCES