Long-Acting Glucagon-Like Peptide-1 Receptor Agonists Suppress Voluntary Alcohol Intake in Male Wistar Rats

Alcohol use disorder (AUD) is a chronic relapsing condition characterized by compulsive alcohol-seeking, loss of control in limiting alcohol intake, and the emergence of a negative emotional state that leads to dependence on alcohol and serious detrimental health consequences (Rehm et al., 2009; Koob, 2015). The Food and Drug Administration (FDA) approved medications to treat AUD have shown efficacy, but their effect sizes are sub-optimal. Therefore, there is a critical need for investigating new pharmacotherapies targeting alternative pathways involved in the neurobiology of AUD (Klein, 2016; Farokhnia et al., 2019a; Witkiewitz et al., 2019).

Glucagon-like peptide-1 (GLP-1) is a gut neuropeptide hormone involved in the regulation of food intake and glucose homeostasis via GLP-1 receptors (GLP-1Rs). GLP-1 is produced both in the periphery, mainly by intestinal L-cells (Novak et al., 1987), and in several brain regions including the nucleus tractus solitarius (NTS), mainly by preproglucagon (PPG) neurons (Larsen et al., 1997; Merchenthaler et al., 1999). In vivo, the half-life of GLP-1 is very short (1.5 min following intravenous dosing and 1.5 h following subcutaneous dosing in humans), mainly due to degradation by the dipeptidyl peptidase-4 (DPP-4) enzyme (Deacon et al., 1995a, b). GLP-1R is a member of the secretin-like class B family of G-protein coupled receptors (GPCRs) expressed in various peripheral tissues, including vagal afferents, hepatic portal system and pancreatic β-cells, where they regulate food intake and glycemia by delaying gastric emptying, decreasing glucagon release and glucose-dependent stimulation of insulin secretion (Orskov et al., 1988; Flint et al., 2001; Kanoski et al., 2011). The critical role of GLP-1 in glucose homeostasis, food intake, and energy metabolism has led to the development of multiple FDA-approved GLP-1 analogs, such as exenatide, liraglutide, and semaglutide, for type 2 diabetes treatment (Agersø et al., 2002; Estall and Drucker, 2006; Eng et al., 2014; Lau et al., 2015). Liraglutide is also approved for the treatment of obesity. To overcome the rapid enzymatic degradation of GLP-1 and to prolong GLP-1R activation, liraglutide and semaglutide were designed by adding fatty-acid chains to the GLP-1 peptide, enhancing their binding affinity to albumin and protecting them against DPP-4 enzymatic degradation and renal filtration, while preserving their GLP-1R potency (Knudsen and Lau, 2019). Due to their prolonged half-lives, liraglutide and semaglutide are referred to as long-acting GLP-1 analogs, and are prescribed once daily and once weekly, respectively (Knudsen and Lau, 2019).

In the central nervous system (CNS), GLP-1Rs are expressed in several brain regions involved in regulating metabolism and energy balance, with preferential localization in inhibitory GABAergic neurons over excitatory glutamatergic neurons (Hayes et al., 2010; Cork et al., 2015; Fortin et al., 2020; Graham et al., 2020). In the hypothalamus, activation of GLP-1Rs has been shown to regulate food intake, glycemia, and stress responses (Kinzig et al., 2003; Sandoval et al., 2008; Hayes et al., 2010; Ghosal et al., 2017). GLP-1Rs are also found in reward-related brain regions that regulate appetitive and consummatory behaviors, such as the nucleus accumbens (NAc), ventral tegmental area (VTA), and amygdala (Göke et al., 1995; Alvarez et al., 1996; Merchenthaler et al., 1999; Cork et al., 2015; Graham et al., 2020). Recently, rodent studies have shown that GLP-1 analogs reduce the rewarding effects of several drugs of abuse, including alcohol, primarily via activation of central GLP-1Rs (Egecioglu et al., 2013a, b; Shirazi et al., 2013; Suchankova et al., 2015; Sirohi et al., 2016; Vallöf et al., 2016, 2019b). In humans, genetic variation in GLP-1R was found to be associated with increased risk of AUD, increased alcohol self-administration in a laboratory setting, and brain activity when being notified about the receipt of a monetary reward (Suchankova et al., 2015). Hence, GLP-1 analogs, already deemed to have a favorable safety profile in humans, are increasingly gaining attention as a novel therapeutic approach to treat AUD.

Recent studies have also renewed interest in the G protein-coupled receptor 119 (GPR119), a member of the class A of rhodopsin-like GPCRs, for its role in glucose homeostasis (Fredriksson et al., 2003; Soga et al., 2005). GPR119 is expressed in limited tissues, including the pancreatic islets and gastrointestinal tract. Activation of GPR119 via both direct actions on pancreatic β-cells and indirect actions on intestinal L-cells results in increased glucose-dependent insulin release and increased gene expression of proglucagon, a biosynthetic precursor of GLP-1, with subsequent release of endogenous GLP-1, glucose-dependent insulinotropic peptide, neurotensin, and peptide YY, leading to improvement of glucose tolerance in rodents and humans (Overton et al., 2006; Chu et al., 2007, 2008; Hansen et al., 2011; Katz et al., 2011; Lan et al., 2012; Chepurny et al., 2013; Mandøe et al., 2015; Hassing et al., 2016; Han et al., 2018; Matsumoto et al., 2018). Its restricted tissue distribution, relative selectivity in enhancing GLP-1 release, and tolerability of its agonists have made GPR119 an attractive therapeutic target for developing orally bioavailable agonists for type 2 diabetes treatment (Terauchi et al., 2018; Yang et al., 2018), and potentially for other related disorders.

In the present study we investigated the effects of different pharmacological manipulations of the GLP-1 system on escalated voluntary ethanol intake and preference, water intake, and body weight in male Wistar rats, in order to determine the potential utility of these compounds in the treatment of AUD. Specifically, we tested two GPR119 agonists, AR231453↗ and APD668, two long-acting GLP-1 analogs, liraglutide and semaglutide, a blocker of DPP-4, sitagliptin, and another peptide putatively working as a GLP-1R antagonist, exendin 9-39 (Ex9-39).

Increasing evidence points to an important role of gut-brain peptides, including GLP-1, in modulating the biobehavioral correlates of alcohol use (Thorsell and Mathé, 2017; Jerlhag, 2018; Farokhnia et al., 2019b; von Holstein-Rathlou and Gillum, 2019; Hopf, 2020). Here, we found that GLP-1 analogs, liraglutide and semaglutide, potently, albeit transiently, decreased voluntary EtOH intake. Notably, semaglutide also reduced alcohol preference. Administration of Ex9-39, an inhibitor of GLP-1R, failed to prevent liraglutide- and semaglutide-induced decreases in EtOH intake. Sitagliptin, a blocker of DPP-4, the enzyme responsible for GLP-1 degradation, did not affect EtOH intake or preference. Similarly, activation of GPR119, which has been shown to promote endogenous GLP-1 release, had no effect on EtOH intake. Altogether, our findings support the hypothesis that direct pharmacological agonism of the GLP-1 receptor may represent an effective approach for AUD treatment.

Several recent studies in rodents have highlighted the role of the GLP-1 system in regulating addictive behaviors related to different drugs of abuse, including nicotine, cocaine, opioids, and alcohol, suggesting a new and promising pharmacotherapeutic target for drug addiction (Egecioglu et al., 2013a, b; Graham et al., 2013; Shirazi et al., 2013; Harasta et al., 2015; Suchankova et al., 2015; Sørensen et al., 2015; Sirohi et al., 2016; Vallöf et al., 2016, 2019b, 2020; Fortin and Roitman, 2017; Tuesta et al., 2017; Bornebusch et al., 2019; Zhang et al., 2020). Here, we described, for the first time, that liraglutide and semaglutide had similar transient inhibitory effects on EtOH intake, but different effects on EtOH preference. In humans, liraglutide and semaglutide have an estimated half-life of 13 and 165 h, respectively (Agersø et al., 2002; Kapitza et al., 2015). Because of its longer half-life and long-acting properties, semaglutide is injected once weekly, as opposed to liraglutide which is injected once daily (Hall et al., 2018). In animals, liraglutide and semaglutide have an estimated half-life of 23 and 64 h, respectively, after subcutaneous administration (Lau et al., 2015). Although previous studies have investigated acute administration of liraglutide, they only reported its effects over a 24-h period, leaving room for speculation about possible time course and persistence of its effect on EtOH intake (Shirazi et al., 2013; Sirohi et al., 2016; Vallöf et al., 2016). The lack of long-lasting effects of GLP-1 analogs on EtOH intake after acute injection could be due to a faster metabolism of GLP-1 analogs in EtOH drinking animals and/or the need for repeated injections. In support of this hypothesis, long-lasting effects of liraglutide and dulaglutide on EtOH intake have been demonstrated after their daily administration for several consecutive weeks (Vallöf et al., 2016, 2020). However, relatively long-lasting effects of GLP-1 analogs on body weight were observed in the present study. Altogether, these findings suggest that the suppressing effects of GLP-1 analogs on EtOH intake and body weight may be mediated through different mechanisms – a question beyond the scope of the present study.

Malaise and nausea are common side effects of GLP-1 analogs (Buse et al., 2009; Hall et al., 2018), and could be, at least partially, responsible for their effects on EtOH intake, food intake, and body weight. Indeed, concentration higher than 50 μg/kg (i.p.) of liraglutide induces conditioned taste aversion (CTA) (Kanoski et al., 2012). However, lower dose of liraglutide (10 μg/kg, i.p.) has been found to reduce chow consumption without inducing CTA, suggesting that nausea does not solely account for the effects of higher concentration of liraglutide on food intake (Kanoski et al., 2012). In this study, we found that liraglutide significantly decreased EtOH intake without affecting EtOH preference on the day of injection. However, liraglutide significantly decreased EtOH preference at 2-day post-injection, while concomitantly increasing water intake at this time-point. These data tentatively suggest that liraglutide’s effects may not be selective for EtOH. Indeed, liraglutide has been shown to affect water intake after peritoneal injection (McKay et al., 2011). In contrast to liraglutide, semaglutide significantly decreased EtOH preference and did not affect water intake, suggesting that its effects are more selective for EtOH. Consistent with our findings, previous studies suggest that the effects of GLP-1 analogs in reducing EtOH intake are due to reduced EtOH rewarding properties rather than nausea, which is a common side effect of GLP-1 analogs (Buse et al., 2009; Egecioglu et al., 2013b; Shirazi et al., 2013; Sirohi et al., 2016; Vallöf et al., 2016, 2019a, 2020; Hall et al., 2018). Altogether, our results suggest that semaglutide is more specific for alcohol than liraglutide, and given its more favorable prescription dosing (i.e., once a week), may be better suited for use in patients with AUD. However, clinical trials will be needed to test and compare the safety, efficacy, and patients’ compliance to semaglutide and liraglutide as AUD treatment options.

Previous findings that the inhibitory effect of exendin-4 on EtOH intake was blunted in mice lacking GLP-1Rs in the CNS suggest that central GLP-1Rs play a key role in mediating the effects of GLP-1 analogs on EtOH intake (Sirohi et al., 2016). A recent study demonstrated that microinjection of Ex9-39 into the NTS was able to prevent the suppressing effect of exendin-4 on EtOH-induced increase in locomotor activity (Vallöf et al., 2019a). Here, we showed that co-administration of Ex9-39 with liraglutide or semaglutide did not prevent the reduction in EtOH intake and weight. The lack of inhibitory effects of Ex9-39 on GLP-1 analog actions could be due to differences in the half-lives of the drugs. While the exact metabolism profile of Ex9-39 is unclear, it is expected that its half-life would be similar to that of its parent peptide, exendin-4 (Göke et al., 1993), which is 33 min after intravenous and 2.5 h after subcutaneous administration in humans (Edwards et al., 1999; Kolterman et al., 2005). The putatively shorter half-life of Ex9-39, compared to both liraglutide and semaglutide (see above), could provide an explanation for the lack of effectiveness of Ex9-39 in blocking the effects of GLP-1 analogs in this study. The lack of inhibitory effect of Ex9-39 could also be due to insufficient blockade of central GLP-1Rs, which are critical in mediating reduced EtOH intake (Sirohi et al., 2016). Because Ex9-39 was administered intraperitoneally, it is possible that Ex9-39 had limited access to the brain leading to insufficient inhibition of central GLP-1Rs. A previous study showed that Ex9-39 administration increased EtOH intake (Shirazi et al., 2013). Here, we found that Ex9-39, administered alone, did not affect EtOH intake, which may also suggest a lack of relative effectiveness of the drug, at least in the context of our experiments. The discrepancy in the effects of Ex9-39 in Shirazi et al. (2013) and our study could also be due to differences in experimental designs. In our study, each rat was its own control (within-subject design); therefore, the EtOH/water intake over the 24-h period after injection was directly compared to each subject’s basal intake. In contrast, the between-subject comparisons performed by Shirazi et al. (2013) compared EtOH/water intake from two different groups of animals. The within-subject design of this study allowed us to control the potential confounding factors and account for individual differences in baseline EtOH consumption. Additional studies are needed to further investigate the effects of systemic GLP-1R antagonism and its effectiveness in blocking central GLP-1R-mediated effects on alcohol consummatory behaviors.

Several studies have started to unravel the putative neuronal networks and cellular pathways underlying the beneficial effects of GLP-1 analogs in reducing alcohol intake. Initially, GLP-1 analogs were thought to cross the blood brain barrier (BBB), but increasing evidence suggest that this may not be the case, and instead, they may reach the CNS via the circumventricular organs (CVOs), characterized by loose and permeable BBB (Kastin and Akerstrom, 2003; Hunter and Hölscher, 2012; Secher et al., 2014; Ast et al., 2020; Gabery et al., 2020). The effects of GLP-1 analogs on alcohol-related behaviors have been attributed to the activation of central, not peripheral, GLP-1Rs (Sirohi et al., 2016). GLP-1Rs are expressed in brain areas involved in the development and maintenance of AUD, such as NAc, VTA, amygdala, and paraventricular nucleus of the hypothalamus (PVN) (Cork et al., 2015; Graham et al., 2020). Activation of GLP-1Rs has been shown to modulate the increased activity of the mesolimbic dopamine system induced by alcohol (Egecioglu et al., 2013b; Vallöf et al., 2016, 2019a). Peripheral administration of liraglutide prevents alcohol-induced increase in dopamine release in the NAc in rats (Vallöf et al., 2016), while microinjection of exendin-4 into the NAc shell was shown to reduce EtOH intake and block EtOH-induced increase in locomotor activity (Vallöf et al., 2019b). These findings suggest that the effects of GLP-1 analogs on alcohol consumption may result from decreased alcohol-induced dopamine release in the NAc, which in turn, attenuates the rewarding properties of alcohol.

Recent studies have highlighted the importance of NTS GLP-1Rs in mediating the effects of GLP-1 analogs. Selective knockdown of NTS GLP-1Rs attenuates liraglutide-induced decreases in food intake and body weight (Fortin et al., 2020). Moreover, local activation of NTS GLP-1Rs decreases EtOH intake and prevents EtOH-induced accumbal dopamine release (Vallöf et al., 2019a). Located in the brainstem, the NTS is a key regulator of the gut-brain axis, the immune system, and the integration of autonomic functions (Andresen and Kunze, 1994; Marty et al., 2008; Zoccal et al., 2014). The NTS is the primary source of GLP-1-producing neurons, also known as preproglucagon (PPG) neurons, in the CNS, which are believed to play a critical role in addictive behaviors, as they project to and receive inputs from several regions involved in reward processing and consummatory behaviors (Larsen et al., 1997; Merchenthaler et al., 1999; Llewellyn-Smith et al., 2011; Holt et al., 2019). Selective activation of NTS PPG neurons, using chemogenetic techniques, has been shown to mimic the suppressing effects of GLP-1 on food consumption (Gaykema et al., 2017; Holt et al., 2019). Additional optogenetic studies found that the NTS-to-PVN GLP-1 neural pathway is critical for anorectic properties of PPG neurons (Liu et al., 2017). Altogether, these findings suggest that the effects of GLP-1 analogs on alcohol intake may be mediated by NTS GLP-1R-expressing neurons, while their effects on food intake may be mediated by NTS PPG neurons, which could also explain the transient vs. long-lasting effects of liraglutide and semaglutide on EtOH intake vs. body weight, respectively.

Due to the difficulties in developing orally bioavailable non-peptide agonists at GLP-1Rs, and class B GPCRs of the secretin family in general, numerous studies have focused their interest on identifying novel class A rhodopsin-like GPCRs with similar pancreatic β-cells expression and G-protein coupling to GLP-1R. From this effort, GPR119 emerged as a promising therapeutic target for the development of orally active, non-peptide, small-molecule agonists to treat type 2 diabetes and obesity (Koole et al., 2010; Yoshida et al., 2010; Semple et al., 2011, 2012; Melancon et al., 2012; Hothersall et al., 2015; Spasov et al., 2017; Fang et al., 2020a). Administration of GPR119 agonists has been shown to increase plasma GLP-1 and insulin, resulting in improved glucose homeostasis in rodents and humans (Chu et al., 2008; Hansen et al., 2011; Katz et al., 2011; Mandøe et al., 2015; Han et al., 2018; Matsumoto et al., 2018). Here, we tested two different synthetic GPR119 agonists. In contrast to GLP-1 analogs, GPR119 agonists failed to reduce EtOH intake and body weight. Several explanations could account for the lack of effectiveness of GPR199 agonists in the present study. Since we did not measure blood GLP-1 levels, it is possible that the injections of GPR119 agonists did not lead to physiologically relevant increases in blood GLP-1. Because of the relatively short half-lives of AR231453↗ and APD668, estimated at around 1 h after oral and intravenous administration, respectively, it is also possible that GPR119 agonists may not be able to produce sustained increases in endogenous GLP-1 levels necessary to mimic the effects of GLP-1 analogs (Chu et al., 2008; Semple et al., 2008, 2011). In support of this hypothesis, studies have demonstrated that co-administration of GPR119 agonists with a DPP-4 inhibitor, to prevent rapid degradation of GLP-1, allows for a greater and sustained increase in blood GLP-1 and enhanced effects on glucose homeostasis (Chu et al., 2008; Bahirat et al., 2017; Park et al., 2017). Although we showed that inhibition of DPP-4 with sitagliptin did not affect EtOH intake, co-injection of DPP-4 with GPR119 agonists could be more effective in increasing endogenous GLP-1 levels (Chu et al., 2008) and potentially influencing EtOH drinking. Future studies will be needed to investigate the effects of newly developed compounds with both potent DPP-4 inhibition and GPR119 agonistic activity (Fang et al., 2020b; Li et al., 2020). It is also possible that exposure to alcohol via the IA2BC paradigm may damage GLP-1 producing intestinal L-cells. Alcohol administration was shown to induce intestinal hyperpermeability and endotoxemia (Keshavarzian et al., 2009), which could result in deficient release of endogenous GLP-1 and subsequently blunt the potential effects of endogenous GLP-1 stimulators on alcohol-related outcomes.

In summary, findings of the present study suggest that, among different classes of drugs with modulatory effects on the GLP-1 system, GLP-1 analogs hold the strongest promise to be effective in reducing alcohol consumption, and semaglutide may overall be a better drug candidate than liraglutide in this regard. Preclinical studies are still needed to further characterize the cellular mechanisms and neuronal circuitry which mediate the suppressant effects of GLP-1 analogs on alcohol intake. It is also important to note that chronic exposure to alcohol and alcohol dependence lead to dysregulation of several neural circuits, such as the mesolimbic dopamine and the corticotropin releasing factor systems (Diana et al., 1993; Weiss et al., 1996; Volkow et al., 2007; Roberto et al., 2010; Marty and Spigelman, 2012; Liang et al., 2014a, b; Herman et al., 2016; Marty et al., 2020), which could alter the beneficial effects of GLP-1 analogs observed in non-dependent subjects – an important question that should be explored in future studies. Overall, the present study adds to the growing literature suggesting that the GLP-1 receptor could be a novel and promising pharmacotherapeutic target for AUD. If the results are confirmed in proof-of-concept human studies, repurposing of the readily available FDA-approved medications acting on the GLP-1 system would allow for a quick and efficient medication development process to add new options to the armamentarium of pharmacotherapies for AUD.

The original contributions presented in the study are included in the article/, further inquiries can be directed to the corresponding author/s. Supplementary Material

The animal study was reviewed and approved by the University of California, Los Angeles, Animal Research Committee.

VM, MF, LL, and IS designed the experiments. VM, JM, and YM conducted the experiments and analyzed the data. VM prepared and wrote the manuscript. MF, LL, and IS prepared and edited the manuscript. LL and IS supervised the project. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.