Impact of Roux en Y Gastric Bypass Surgery on Appetite, Alcohol Intake Behaviors, and Midbrain Ghrelin Signaling in the Rat

Abstract

Objective

Roux en Y Gastric Bypass surgery (RYGB) reduces appetite and stimulates new onset alcohol misuse, however the genesis of these behavioral changes is unclear. We hypothesized that new onset alcohol intake is a behavioral adaptation that occurs secondary to reduced appetite and correlates with altered central ghrelin signaling.

Methods

We evaluated hedonic high fat diet (HFD) intake prior to assessing alcohol intake behaviors in RYGB and control rats. We also measured circulating ghrelin, and ghrelin receptor (GHSR) regulation of neuronal firing in ventral tegmental area (VTA) dopamine (DA) neurons.

Results

RYGB rats displayed reduced HFD intake relative to controls. Sham and RYGB rats consumed more alcohol and preferred lower concentrations of alcohol whereas only RYGB rats escalated alcohol intake during acute withdrawal. Remarkably, GHSR activity, independent of peripheral ghrelin release, set the tonic firing tone of VTA DA neurons, a response selectively diminished in RYGB rats.

Conclusions

This study indicates that gut manipulations lead to increased alcohol intake whereas RYGB promotes behaviors that may maintain alcohol misuse. Reductions in hedonic feeding and diminished GHSR control of VTA firing further distinguish gut manipulation from complete bypass and present a potential mechanism linking reduced appetite with alcohol misuse after RYGB surgery.

Introduction

Roux en Y Gastric Bypass (RYGB) surgery [1] is a reliable therapy that produces sustained body weight loss and remission of associated metabolic complications. However, the positive benefits of the RYGB procedure are not risk free. For example, new onset alcohol misuse and suicide are potential outcomes of RYGB [2–9]. Thus, understanding how physiological and psychological mechanisms adapt to RYGB surgery and how these changes affect long-term behavioral outcomes is essential for successful recovery in bariatric patients.

A key feature of the RYGB procedure is reduced appetite [10], and this change in feeding behavior is hypothesized to stimulate new onset alcohol intake once the reinforcing properties of palatable food have been diminished [11]. Presently, the mechanisms responsible for new onset of alcohol misuse, its relation to feeding behavior are unknown.

Prior work has established that the gastrointestinal (GI) feeding peptide ghrelin and activity of its receptor (GHSR) in the ventral tegmental area (VTA) stimulates palatable food and alcohol intake [12,13]. In addition, a collection of recent preclinical data suggest that central GHSR activity, in the absence of GI ghrelin secretion, can regulate both food and alcohol intake [14,15]. These observations suggest that altered ghrelin and/or GHSR activity within the VTA may be potential mechanisms explaining changes in appetite and alcohol intake after RYGB.

We hypothesized that modification of appetite is a prerequisite for new onset alcohol intake after RYGB, and that alterations in VTA ghrelin signaling correlate with surgical-induced drinking. To address this contention, we utilized a multidisciplinary approach to evaluate hedonic feeding behavior, alcohol intake behaviors, and VTA GHSR activity in a rodent model of RYGB.

Materials & Methods

Animals

Male Long-Evans rats (Harlan, IN) housed in an environmentally controlled vivarium on a reverse light cycle (lights off at 7 a.m.) were used as experimental subjects. Food and water were available ad libitum, except when indicated. All work adhered to Institutional Animal Care and Use Committee guidelines at Washington State University. All rats were maintained on a high fat diet (HFD; Research Diets, New Brunswick, NJ, 4.41 kcal/g, 1.71 kcal/g from fat) for a period of 2 months, prior to any surgical manipulations. Subsequently, rats received RYGB (n=9), sham (n=9) or no surgery (n=8) and were maintained on chow diet (Teklad, 3.41 kcal/g, 0.51 kcal/g from fat) throughout the remainder of the study.

Surgery

All rats were fasted 24hr before surgery as described previously [5]. For RYGB a gastric pouch (~ 20 % of total stomach volume) was created by two straight triple-staple lines between lesser and greater curvature using a stapler (Ethicon, Ithaca, New York). A jejunal transection was created ~40 cm from the ileocecal value followed by end-to-side gastrojejunostomy, and an end-to-side jejuno-jejunostomy 25 cm from the ileocecal valve, creating a 15-cm long Roux limb, a 25-cm long common limb, and a roughly 40-cm long biliopancreatic limb. For sham surgery, the small intestines were removed from the body cavity and an end-to-end jejunostomy was created. All rats were allowed to recover until body weight was stable. Food intake was recorded daily.

Hedonic Feeding

Rats were food deprived for 21hrs and the next day, pre-weighed chow hoppers were placed in each cage and subsequently reweighed each hour for 2hrs. Following the second hour of chow access, a separate set of food hoppers containing HFD was introduced. HFD access after re-feeding on chow constitutes the hedonic portion of this test.

Alcohol Intake

Rats were provided with unsweetened alcohol solutions (5, 8, or 10% v/v) on alternating days in counterbalanced fashion using a two-bottle choice paradigm. Alcohol bottles were introduced at the onset of their subjective dark cycle and 24hr alcohol and water intake was recorded. Bottles were weighed, gently placed in the cages and re-weighed manually following each session to evaluate alcohol intake (g/kg).

Measurement of Plasma Ghrelin

All rats were fasted overnight and blood samples were collected before and in 15min increments following 5 ml of 20 % ethanol gavage. Blood samples were collected in tubes containing EDTA and Pefabloc at concentration 1.86 mg/ml and 1.0 mg/ml of blood, respectively, on ice. All samples were centrifuged at 21000g for 20 min and plasma was transferred into a separate tube on ice and immediately treated with 1 N HCL. Plasma acyl-ghrelin levels were detected using a MILLIPLEX MAP Rat Metabolic Hormone Magnetic Bead Panel -Metabolism Multiplex Assay kit (RMHMAG-84K, EMD Millipore Corporation).

Acute Withdrawal Measures

All rats were given a 30 min alcohol (8% v/v) intake session to establish an acute baseline intake. Next, all rats were exposed to control (# F1259SP, Shake and Pour, BioServ) or alcohol liquid diet (# F1258SP -Rodent Liquid Diet, Lieber-DeCarli’82, 36% kcal from ethanol, Shake and Pour, BioServ) [16] in their home cage, which was the sole source of calories for next 7 days. Alcohol liquid diet was gradually introduced by altering the concentration of alcohol (0, 2.2, 4.3 and 6.4%). Tail blood samples were collected at various time-points during liquid diet presentation and analyzed using an Analox microstat GL5 (Analox Instruments Ltd., Lunenberg, MA). Following 7 days of alcohol liquid diet access, alcohol intake (8% v/v, over 30 min) was recorded 5hrs after removing alcohol liquid diet.

Body Composition Analysis

Body composition analysis (total, lean and fat body mass) was measured using a Bruker Minispec LF110 NMR Body Composition Rat Analyzer (Bruker Biospin, Rheinstetten, Germany).

Ex Vivo Electrophysiology

VTA brain slice preparation

Following completion of the alcohol withdrawal experiment brain slices containing the ventral tegmental area (VTA) were collected for whole-cell recording experiments (see supplemental methods for VTA sectioning).

Whole-cell recoding and data analysis

Individual brain slices containing the VTA were transferred to a custom recording chamber, and continuously perfused at a rate of ~3–4ml/min with ACSF at 32–35°C. Putative VTA dopaminergic neurons were identified by their characteristic large soma size and irregular soma shape. Whole-cell recordings of these cells were made with borosilicate glass pipettes (3–6 MΩ) filled with an internal solution containing (in mM): 130 K-gluconate, 5 KCl, 2 MgCl₂, 10 HEPES, 0.1 EGTA, 2 Mg-ATP, 0.3 Na-GTP and 30 μM Alexa488 or Alexa568 with pH adjusted to 7.2–7.3 and an osmolarity of 290–300 mOsm. Recordings were digitized at 20kHz, and cells were rejected if access resistance was > 30MΩ. The recorded membrane potential was corrected off-line for a calculated 14.9 mV liquid junction potential. All recordings were performed in current-clamp mode and each cell was subject to an initial current injection protocol with 20 pA steps (1 sec duration) starting with hyperpolarizing pulses sufficient to bring the membrane potential to at least −85 mV Figure 5A & C. Putative dopaminergic neurons were further identified by the Ih-mediated “sag” in the membrane potential when hyperpolarized by a somatic current injection Figure 5A, and an action potential half-width of greater than 0.75 msec Figure 5B. Following the step protocol, no further current was injected for the remainder of the experiment and only the endogenous resting potential/spontaneous firing activity of each neuron was monitored. Beginning at least 90 sec after the last current injection, the endogenous firing frequency of each neuron was evaluated over a 3–10 min period. To determine the contribution of constitutive ghrelin receptor activity for action potential firing of VTA dopaminergic neurons, the action potential firing rate was assessed over a 120–240 sec period immediately before, during and after application of the ghrelin receptor antagonist, [D-Lys3]-GHRP-6 (25μM). [D-Lys3]-GHRP-6 was only applied once to a given brain slice. [D-Lys3]-GHRP-6 (Tocris Bioscience) was dissolved directly into ACSF and was always used within 30hrs following reconstitution.

Figure 5. Circulating Ghrelin Levels Inversely Correlate with RYGB-Induced Alcohol Intake.

A) A univariate ANOVA revealed that fasting plasma acyl-ghrelin levels were significantly (F_{2.0, 12.0 =} 4.06, p= 0.04) different between groups. Post hoc analysis further identified that RYGB rats had significantly (p=0.02) lower fasting acyl-ghrelin compared to sham and PF rats. We further analyzed plasma acyl-ghrelin levels following ethanol gavage and compared these to baseline fasting levels using mixed-model ANOVA. A main effect of time (F_{3.0, 36.0 =} 5.19, p= 0.004) and significant (F_{2.0, 12.0 =} 10.17, p= 0.003) between group differences were identified. Post-hoc analysis further revealed that fasting acyl-ghrelin levels were significantly lower in the RYGB rats relative to the sham (p=0.001) and PF (p=0.007) controls. **p=0.003, main effect of exposure. B) When all measured time points were collapsed, a univariate ANOVA further confirmed a significant between group effect (F_{2.0, 12.0 =} 10.228, p= 0.003). **p=0.007 and κκ p=0.001 compared to PF and sham, indicating that acyl-ghrelin was significantly reduced in RYGB rats compared to sham and PF controls.

Statistical Analysis

Body weight, food intake, alcohol intake, blood alcohol data, and plasma ghrelin were analyzed by a mixed-model two-way ANOVA, with post-hoc tests to compare within group effects. The within-subject variable was time intervals (time or conditions of measurements) and the between-groups variable was surgical procedure (Pair-fed, sham or RYGB surgery). Body composition data was analyzed by univariate ANOVA. Electrophysiological data were analyzed using Clampfit software and statistical significance was determined using a one-way ANOVA or one-tailed paired t-test. All statistical comparisons were conducted at 0.05 α level.

Results

RYGB Surgery Attenuates Hedonic Feeding

RYGB led to decreased body weight Figure 1A and body fat relative to control rats Figure 1A (inset). The intakes of RYGB rats during the 45-day recovery period were used to pair-fed (PF) un-operated rats, which resulted in a 75gm reduction of body weight for the PF group. Differences in body weight across groups persisted across time and study manipulations Figure 1B. Daily chow intake was not different between groups after recovery from surgery. Fig 2A–B. In addition, fasting-induced chow intake was similar across all groups Fig 2C. However, re-feeding from HFD after consumption of the chow pre-load was significantly reduced in RYGB relative to controls Fig 2D.

Figure 1. RYGB Surgery Promotes Sustained Decreases in Body Weight & Body Fat.

A) RYGB led to a significant decrease in body weight (F_{2.0, 17.0} = 5.0, p= 0.02) relative to sham controls. Sham rats lost an average of 10 gm (~ 0.9%) of their initial body weight, whereas RYGB rats lost an average of 91.0 gm (~16%) of their initial body weight in the first five weeks of recovery. Rats in the un-operated pair-fed (PF) group lost an average of 75 gm (~12%) of their initial body weight under an identical time timeframe through nutrient restriction and regained this body weight by the time behavioral testing began. A mixed-model ANOVA identified a main effect of time (F_{3.7, 62.84} = 57.05, p= 0.000), significant time and treatment interaction (F_7.4,62.84 = 12.09, p= 0.000) and a significant between group differences (F_{2.0, 17.0} = 5.0, p= 0.02) for body weight among groups. *p<0.05 main effect of exposure. Post-hoc analysis further confirmed that A-Inset) A significant (F_{2.0, 12.0} = 8.68, p= 0.005) reduction in the body fat was also observed in RYGB rats compared to the sham (p=0.005) and PF (p=0.003) rats. **p<0.01 and κκp<0.01 compared to PF and sham, respectively. B) Significant (p<0.000) body weight differences existed between sham and RYGB groups at each manipulation interval after surgery, whereas no differences existed between sham and PF rats. ***p=0.000 main effect of exposure. In addition, a significant body weight loss was observed within each group following exposure to liquid diet to induce alcohol dependence (p=0.0001), however between group differences in body weight were maintained.

Figure 2. RYGB Surgery Selectively Attenuates Hedonic Intake of Palatable Food.

No differences in A) daily or B) cumulative chow intake were evident across experimental groups. C) When exposed to chow following an overnight deprivation, a mixed model ANOVA confirmed a significant effect of time (F_{2.0, 38.0 =} 127.40, p= 0.000), but no interaction or between groups differences were evident indicating that all groups consumed equivalent amounts of chow during the re-feeding period. ***p<0.001 main effect of time. D) In contrast, when HFD was presented during 3^rd hr, after re-feeding had ceased, RYGB rats displayed a significant reduction in HFD intake (F_{2.0, 19.0 =} 5.21, p= 0.016) compared to PF and sham controls as identified by a univariate ANOVA. *p<0.05 main effect of exposure.

Gut Manipulation Induces Alcohol Consumption and Increases Alcohol Preference

Clinical observations of increased alcohol intake have been documented as early as 1-year post surgery, [4] and consistently at 2 years post-surgery [2,3]. In an effort to model this time course in a preclinical rodent model, rats were offered alcohol 5 months after surgery, a time that approximates clinical observations of increased alcohol intake after RYGB.

Results indicated that RYGB and sham operated rats consumed more alcohol at each concentration tested relative to PF control rats Fig 3AB. Importantly, the ratio of alcohol intake to water was significantly increased after RYGB and sham surgery relative to PF controls Fig 3C–D. Water intake was equivalent across groups when alcohol was not present Fig 3H. However, when both were presented, RYGB and sham rats selectively decreased water intake and increased intake of alcohol Fig 3EF. When intakes across concentrations were collapsed, both sham and RYGB rats displayed increased mean alcohol intakes and increased alcohol preference compared to PF rats Fig 3B–D.

Figure 3. RYGB Surgery and GI manipulation Increase Alcohol Intake & Alcohol Preference.

A–B) Data from alcohol (5–10%) drinking sessions were analyzed by a mixed model ANOVA which identified a main effect of session (F_{2.0, 34.0 =} 9.13, p= 0.001) and a significant between group (F_{2.0, 17.0 =} 5.16, p= 0.018) difference in alcohol preference. Post-hoc analysis further confirmed that alcohol intake was significantly (p=0.008) elevated in RYGB rats compared to PF controls, although sham and RYGB intakes did not differ. *p<0.05 main effect of exposure. C and D) Similar results were obtained when alcohol/water ratio data were analyzed. A mixed model ANOVA further revealed a main effect of session (F_{2.0, 34.0 =} 30.11, p= 0.000) and a significant between group (F_{2.0, 17.0 =} 17.15, p= 0.000) differences in alcohol preference. Post-hoc analysis further identified that the alcohol/water intake ratio was significantly (p=0.000) elevated in RYGB rats compared to shams (8% only, p<0.005) and PF controls (p<0.000). ***p<0.001 main effect of exposure. E–F) Water intake was significantly decreased in RYGB rats relative to sham (p<0.05) and PF controls (p<0.000). ***p<0.001 main effect of exposure. G) whereas no differences were observed in the total fluid intake (water + alcohol) during alcohol drinking sessions. H) No between group differences existed in the water intake on non-alcohol drinking days.

RYGB Surgery Enhances Alcohol Intake During Acute Withdrawal

To determine the long-term behavioral adaptations that maintain elevated levels of alcohol intake, we measured escalation of alcohol intake following acute withdrawal from alcohol liquid diet. When an alcohol liquid diet served as the only source of calories, equivalent volumes of alcohol intake were observed across treatment groups Figure 4A. Maintenance on this diet produced significant elevations in blood ethanol concentrations in all groups Figure 4B. Notably, exposure to alcohol liquid diet resulted in significant body weight loss in all groups (as illustrated in Figure 1B). Following removal from this diet, BEC levels were statistically reduced in all groups and under these conditions, only RYGB rats escalated alcohol intake Figure 4C.

Figure 4. RYGB Surgery Augments Alcohol Intake During Acute Withdrawal.

A) To induce alcohol dependence, rats were maintained on alcohol liquid diet for 7 days. On an average all rats (PF = 131.5 ml/kg ± 3.2; Sham = 137.1 ml/kg ± 8.7; RYGB = 134.8 ml/kg ± 24.0) consumed equivalent amounts of alcohol liquid diet and no statistical significant differences existed between groups in terms of amount of liquid diet consumption or the amount of alcohol (g/kg) ingested. B) To validate the liquid diet to increase blood ethanol concentration (BEC), we collected and analyzed blood samples from each group at various time points during alcohol liquid diet exposure. A mixed-model ANOVA revealed a main effect of time (F_{2.0, 26.0 =} 13.703, p= 0.000), a significant time*exposure interaction (F_{4.0, 26.0 =} 3.719, p= 0.016). However, no between group differences existed. ***p=0.000 main effect of time. C) A mixed model ANOVA revealed a main effect of time (F_{1.0, 13.0 =} 6.231, p= 0.03). A repeated measure ANOVA further revealed that alcohol intake in RYGB rats was (F_{2.0, 8.0 =} 11.81, p= 0.0041) increased during acute withdrawal from liquid diet, whereas no significant differences were observed in PF or sham rats under identical conditions, when compared to the baseline intakes. **p=0.0041 compared to respective baseline intakes. Note: baseline 30min alcohol intakes did not differ between groups.

RYGB-Induced Alcohol Intake Occurs in the Presence of Reduced Ghrelin

We measured acyl ghrelin levels under two conditions: 1) following an overnight fast, a condition that elevates acyl-ghrelin [17] and 2) after exposure to alcohol, a condition that produces decreases in circulating acyl-ghrelin levels [18]. Acyl-ghrelin levels in RYGB rats were significantly reduced relative to sham and PF control rats after fasting Figure 5A–B. Acute alcohol exposure suppressed circulating ghrelin levels in all groups, an affect that was more pronounced in RYGB rats Figure 5A. When all measurements were collapsed across time, acyl-ghrelin levels were significantly reduced in RYGB rats relative to sham and PF controls Figure 5B.

RYGB Surgery Alters GHSR Activity in VTA DA Neurons

To determine the relationship between RYGB and VTA GHSR activity, we evaluated spontaneous activity of putative VTA dopaminergic (DA) neurons Figure 6A, B and changes in this activity following application of the GHSR antagonist D-Lys-3 GHRP-6 (25μM) or ghrelin (20nM). Evoked VTA DA neuron firing was similar across all groups Figure 6C and spontaneous firing rates were decreased in PF and RYGB rats relative to sham controls, but this difference was not statistically reliable Figure 6D. Surprisingly, application of D-Lys-3 GHRP-6 (25μM) significantly reduced VTA DA neuronal firing in sham and PF rats Figure 6E, F, suggesting that GHSR activity drives tonic VTA DA neuronal firing. Remarkably, the ability of GHSR antagonism to decrease VTA DA firing was absent in RYGB rats Figure 6E, F. To determine if RYGB rats were responsive to circulating ghrelin, we bath applied ghrelin (20nM) following washout of the antagonist. Indeed, ghrelin was still capable of enhancing VTA DA firing rates in all cells tested (n=3 cells) by at least 50% (data not shown).

Figure 6. RYGB Surgery Alters GHSR Activity in Mesolimbic Dopamine Neurons.

(A & B) Representative current-clamp trace showing response to hyperpolarizing and depolarizing current injections (±100 pA) in a VTA dopamine neuron. Note I_h-like ‘sag’ in the membrane potential when hyperpolarized (A) and long duration (>750 msec) action potentials (B; horizontal line: half-width = 1.15 msec) used as identifying hallmarks to identify VTA dopamine (DA) neurons. Action potential half-width ranged from 0.80–1.90 msec (mean: 1.09 ± 0.05 msec; n = 24 cells). (C) Plot of action potential number elicited in response to increasing levels of current injection for VTA DA neurons from PF (black closed circles), Sham (open circles), and RYGB (triangles) rats. (D) Plot of mean spontaneous action potential firing rate for VTA DA neurons from PF (black), Sham (striped), and RYGB (gray) rats (H_{2 =} 2.795, p= 0.247). (E) Representative current-clamp traces showing action potential firing in VTA neurons at baseline (left), after application of the ghrelin receptor antagonist ([D-Lys3]-GHRP-6 (25μM), center), and after wash (right), in PF (top), Sham (middle), and RYGB (bottom) rats. (F) Plot of percent reduction of action potential firing rate induced by [D-Lys3]-GHRP-6 (25μM) in VTA DA neurons indicates that ghrelin receptor (GHSR) blockade significantly reduced DA firing in PF (black; t (2) = 3.027, p = 0.047) and Sham (striped; t (4) = 3.043, p = 0.019) rats, but not in RYGB (gray; t (3) = 0.614, p = 0.292) rats. *p<0.05 using paired t-test.

Discussion

The goal of this manuscript was to determine if changes in hedonic appetite precede problematic alcohol intake induced by RYGB and if this behavioral adaptation correlated with altered VTA GHSR activity. In this regard, several notable findings emerged. First, RYGB rats display attenuated hedonic intake of palatable food despite having normal homeostatic re-feeding responses. In addition, RYGB and sham operated rats both consumed more alcohol, and displayed increased preference for alcohol relative to un-operated controls, suggesting that manipulations of the small intestine also provoke increased alcohol intake. Importantly, RYGB rats consumed more alcohol during acute withdrawal relative to sham or PF controls, highlighting long-term consequences of alcohol intake after bypass surgery in particular. Lastly, RYGB rats displayed reduced but not increased GHSR activity in midbrain DA neurons, an effect that correlated with decreased circulating levels of plasma ghrelin. Collectively these results indicate that gut manipulation can induce alcohol intake, a process distinguishable from alcohol-dependent behaviors, hedonic food intake, circulating ghrelin, and VTA GHSR activity.

Increasing evidence suggests that palatable food and alcohol intake are controlled by a shared set of neuronal circuitry that is responsive to GI peptides [19]. However, the relationships between RYGB-induced changes in hedonic intake of palatable food, alcohol use, and their control by GI peptides have yet to be explored in a cohesive manner. Here, we provide insight into these phenomena by demonstrating that reduction of hedonic-based feeding couples with elevated alcohol intake in RYGB rats. Specifically, reduction of palatable food intake following consumption of a caloric preload was significantly reduced in RYGB rats, suggesting that hedonic-based feeding is attenuated by this surgical procedure. However, future studies are required to determine if this phenomena persists after repeated bouts of alcohol ingestion.

It is notable that sham operated rats displayed increased alcohol intake, and increased alcohol preference relative to un-operated control rats. We previously observed increased consumption of a 10% alcohol solution relative to un-operated control rats when alcohol was administered intermittently over a 2-week period [5]. In the current manuscript, rats received multiple concentrations of alcohol in counterbalanced fashion. Under these conditions, both RYGB and sham rats displayed increased alcohol intake and preference for alcohol, as evidenced by selective reduction of water intake in the presence of alcohol. Similar studies from independent laboratories have utilized sham surgery that does not entail anastomoses of the small intestine [6] or only compared RYGB-induced alcohol intake with sham rats only [7,8]. Thus, to the best of our knowledge, the current data represent the first comparison of small intestine anastomoses with RYGB and un-operated rats. Prolonged access to HFD reduces reward-related behaviors such as amphetamine conditioned place preference, and operant responding for sucrose [20]. In the current manuscript this dietary constraint was removed which may have further revealed increased alcohol intake in the sham condition. Importantly, previous studies indicate that mechanical stimulation of the gut in rats increases nutrient transit through the small bowel and facilitates gastrointestinal (GI) hormone release [21]. Thus, our manipulations that included complete removal of the small intestine from the body cavity, cutting and suturing the jejunum, may have altered GI function or produced secondary adaptive responses that promote excess alcohol intake. This raises critical questions that will require future experimental validation. When viewed collectively, this observation indicates that complete bypass and GI manipulation without bypass can lead to increased alcohol intake in rats that are non-preferring at baseline.

When considering the long-terms consequence of alcohol use after RYGB or GI manipulation, we detected significant body weight loss in all rats following 7-day exposure to a liquid diet that significantly elevated blood alcohol concentrations. Metabolic complications and body weight loss are common symptoms in alcohol-dependent patients [22] and out data indicate that these changes are unaltered by surgical manipulation. Importantly, reductions in BEC secondary to liquid diet removal led to an escalation of alcohol intake in RYGB rats, an effect that was not present in sham or PF rats. These data indicate that sustained elevation of BEC in the RYGB condition can promote behaviors common to alcohol dependence. Moreover, this finding distinguishes the impact of continued alcohol intake following GI manipulation from complete bypass. The implication being that RYGB surgery has the potential to maintain alcohol intake through promoting excess intake during acute withdrawal from alcohol.

Prior clinical studies report reduced circulating levels of acyl-ghrelin after RYGB [23] while others report no change [24] or even increases [25]. These discrepant findings may be due to variations in surgical procedure or time of sampling. More recent preclinical studies indicate that the GI cells that produce ghrelin undergo extensive adaptations after RYGB favoring increased ghrelin release 30 days after surgery [25]. This finding is in agreement with previous preclinical work from our group indicates that acyl-ghrelin levels were significantly elevated 30 days after RYGB surgery [5]. However, acyl-ghrelin levels were reduced 110 days after surgery in the same cohort of rats, a finding in agreement with previous rodent studies at this time point [26]. In our previous study, we detected increased alcohol intake 90 days after RYGB, a time that correlated with reduced acyl-ghrelin. However, that particular cohort of rats was not behaviorally characterized for alcohol intake. Here, we extend these findings by demonstrating that RYGB-induced alcohol intake occurs in a context where the physiological response of acyl-ghrelin after fasting is reduced. In addition, our data indicate that alcohol exposure depressed ghrelin levels to a greater degree in RYGB rats relative to sham and PF controls, suggesting that continued alcohol consumption may further decrease circulating ghrelin levels in RYGB patients. Although the GI ghrelin system is incapable of responding to a robust physiological challenge in place to stimulate homeostatic feeding, our data indicate that RYGB rats are capable of displaying rebound hyperphagic feeding after deprivation. These findings become relevant when considering the regulation of GHSR by circulating ghrelin. For example, fasting-induced increases in ghrelin are associated with increased central expression of GHSR [27]. Thus, post-surgical fluctuations (acute increases followed by sharp decreases) in circulating ghrelin may lead to altered central expression and/or activity of GHSR.

In addition to the physiological relationship of ghrelin and GHSR signaling, prior studies have now highlighted the ability of GHSR to act independent of circulating ghrelin to control both food and alcohol intake [14,15]. These seminal findings suggest that altered central GHSR activity alone may control changes in food and alcohol intake after RYGB. Regarding the neural substrate where GHSR activity may be most relevant, VTA DA neurons have received much attention for their participation in control of food and alcohol intake. Previous work has established that VTA DA neurons are susceptible to cellular adaptations that occur secondary to intake of palatable food [28] or following chronic alcohol intake [29]. Importantly, pharmacological activation of GHSR in VTA DA neurons stimulates neuronal firing, promotes food-seeking behavior, and controls alcohol intake [30, 12, 13]. In the current study, we provide novel evidence that pharmacological blockade of GHSR diminishes tonic neuronal firing in VTA DA neurons. In the VTA, tonic firing represents baseline activity of dopaminergic neurons and this process sets the basal tone of DA in the striatum [31]. DA release in the striatum plays a pivotal role in regulating palatable food and alcohol intake [31, 32], and pathologies associated with each process. Thus, control of tonic DA firing by VTA GHSR activity, irrespective of circulating ghrelin levels, may offer new insights regarding psychopathologies associated with feeding and alcohol misuse behavior. It is remarkable that the ability of GHSR antagonism to reduce VTA dopaminergic firing was absent following RYGB that displayed reduced hedonic feeding. It has been proposed that conditions of hypodopamine promote behaviors that elicit mesolimbic DA release in effort to experience pleasure [11], raising the possibility that new onset alcohol misuse after RYGB could derive from reduced GHSR control of VTA DA neuronal firing. This concept is supported by the observation that alcohol stimulates VTA DA neuronal firing [33], a process that occurs in isolation from neuroendocrine, neuropeptide, or neurotransmitter signaling mechanisms and is dependent on activation of voltage gated ion channels [34]. In addition, rodents readily self-administer alcohol directly into the VTA [35]. Thus, alcohol misuse after RYGB could be a behavioral adaptation that occurs to stimulate VTA DA firing, and restore brain DA levels secondary to dampened GHSR activity. However, this conjecture requires experimental validation.

Conclusions

In summary, our data suggest that reduction of hedonic feeding precedes increased alcohol intake after RYGB, leading to facilitated alcohol intake and dependence. This is distinguishable from increased alcohol intake observed after GI manipulations. Reduction in GHSR control of VTA DA neuronal firing, a process that occurs in the context of low circulating ghrelin, provides a novel future mechanism for RYGB-induced changes in food and alcohol intake. Overall, our collection of findings provides new insights that underscore how adaptations in the gut-brain signaling axis influence neurobiology of addictive behavior.

• RYGB increases alcohol intake in patients and rodents.

• This phenomenon occurs in patients and rodents that are non-preferring at baseline.

• RYGB-induced alcohol intake is associated with changes in the mRNA expression of central orexigenic neuropeptides.

• This study indicates the RYGB-induced alcohol intake occurs in rats that display decreased hedonic feeding, indicating that reduction in appetite may contribute to new onset alcohol intake.

• Moreover, the current data indicate that RYGB rats facilitate to alcohol dependence more rapidly, highlighting behavioral adaptations that maintain elevated alcohol intake in patients.

• Finally, our data indicate that RYGB reduces midbrain ghrelin signaling, an event that could explain reduced appetite and increased alcohol intake after RYGB.