The DAergic system in the brain consists of four different pathways, the mesolimbic pathway, the nigrostriatal pathway, the mesocortical pathway and the tuberoinfundibular pathway. In the context of AN, the motivational pathway (mesocorticolimbic; combination of mesolimbic and mesocortical pathway) is most relevant, due to its regulatory effect on motivation or “wanting” of food (37, 38). Additionally, the nigrostriatal pathway must be involved too, as food seeking behavior is not only the motivation to obtain food, but also the locomotive behavior to obtain it. Furthermore, DAergic signaling, as ghrelin signaling, generally has modulatory effects on anxiety and gut motility (39–41). The DAergic neuronal circuits are complex systems which show a high interconnective capacity (30, 42). For these neuronal circuits DA is the main neurotransmitter which can activate different signal pathways depending on the activated neuronal sub-populations or the receptor type DA binds to.

DA binds to and acts via two different receptor classes, the D1 receptor class (D1R and D5R) and the D2 receptor class (D2R, D3R and D4R). Both receptor classes are G protein-coupled receptors (GPCRs) but they activate opposite intracellular signaling pathways. D1R is Gαs-coupled, and its activation will lead to adenylate cyclase activation which results in the accumulation of 3′,5′-cyclic adenosine monophosphate (cAMP) and activation of the neuron it is expressed on. D2R is Gαi/o coupled, which leads to a decrease in cAMP and inhibition of the neuron (43). Both D1R and D2R can recruit β-Arestin’s (43). D2R is expressed in multiple brain areas and can be expressed both pre- and post-synaptic. The presynaptic expression of D2R induces a negative feedback loop and its postsynaptic expression inhibits neurotransmitter release from both DAergic and non-DAergic neurons (44). D1R is only expressed post-synaptic. D1R and D2R are differently expressed throughout brain regions and neurons but can be co-expressed. In the VTA, both D2R and D1R are expressed. However, D1R is most highly expressed in the dorsal and ventral striatum. D2R is expressed on presynaptic neurons in the VTA and on postsynaptic neurons, e.g. on a subset of medium spiny neurons in the NAc (part of the ventral striatum), which are targeted by DAergic projections from the VTA (44).

Individuals affected by AN display a behavior that appears to be closely related to dysregulation of the mesolimbic system and motivational control. AN patients generally show a high rate of self-control and are less sensitive to rewards and more sensitive to punishment (9, 45). However, other studies have found increased brain activity of the DA-mediated reward system in AN patients compared to healthy controls when exposing the subjects to situations that require reward-related learning (46). A recent study has observed that the altered reward response is inversely associated with the body mass index in eating disorder patients, including AN patients. Further, they report changed signaling of reward and food intake brain circuits in AN patients compared to healthy controls (47). Overall, indications of altered DA signaling in AN patients are given. Nevertheless, it is difficult to determine whether the DA signaling is generally increased or decreased. For instance, in the previously described study with DA deficient mice, the mice showed both hypoactivity and hypophagic behavior before dying when three weeks old due to lack of food seeking behavior (18). These findings are both parallel and antiparallel to typical AN behavior as the patients show lack of food seeking behavior, however AN is associated with increased activity levels (6, 35, 36). A recent fMRI study in women demonstrated that the VTA activity, measured with the blood flow to the VTA, in AN patients is reduced compared to bulimic patients when exposed to food odors, which may indicate the potential dysregulation of VTA activity in AN patients (9). Additionally, a retrograde viral injected, chemogenetic induction the Gαq activity in projections from the VTA to NAc led to increased food intake and food anticipatory behavior in ABA rats. This neuron activation was mainly due to activated DAergic neurons (11). These results imply that a decreased activation of the DAergic projections from VTA to the NAc, which are known to be activated by ghrelin in the presence of food, may be part of the AN etiology and an activation/modulation of these projections may reduce AN development (28). However, it was observed that a knockout of the DA transporter, leading to an increase in extracellular DA, led to a stronger progression of AN-associated phenotypes in female ABA mice. This indicates that an increase in DA release might be relevant for the initial development of AN, which suggests a dual role of DA in the etiology of AN (48). Due to the promising link between AN and the DAergic system, various studies have looked into the potential of antipsychotic drugs (D2R receptor antagonists). However, no overall positive effect was observed in a recent meta-analysis (3).

Ghrelin is most prominently studied in regard to its growth hormone-releasing, food intake- and energy balance-regulating functions. However, ghrelin is a hormone with various physiological and behavioral modulating effects. Most interestingly, some of the physiological functions of ghrelin are highly connected to characteristic AN symptoms. Ghrelin is known to increase food intake by modulating the homeostatic and hedonic systems and it is also known to increase gut motility. Furthermore, ghrelin is involved in stress and reward-oriented behaviors and regulates anxiety and locomotion (54, 55). Since ghrelin specifically increases the pleasure and motivation related to food intake, which is relevant for AN patients, it is possible that an agonist of the GhrR could be a potential pharmacological target. Studies examining the effect of GhrR targeting in cancer induced cachexia have shown promising results regarding increasing body weight (BW) and muscle strength (56, 57). However, clinical trials with AN patients have demonstrated that the hunger-inducing effect of the full GhrR agonists in this sensitive population of patients may not be tolerated unless it is accompanied by other beneficial effects, such as alleviation of anxiety and gastro-intestinal dysfunction (34). Accordingly, more research must be done to find out how to target the GhrR pathways in order to enhance gut motility, lower anxiety and keep locomotion at least neutral in AN patients while still enhancing food intake in a healthy way that is tolerable to the patients. However, many of the studies examining the exact physiological and behavioral functions of ghrelin still show inconsistent results.

The GhrR is expressed in different tissues both peripherally, e.g. in the pituitary gland or pancreatic islets and centrally, most importantly for AN in brain regions related to food intake and anxiety behavior, such as the hypothalamus, the VTA and the amygdala (50, 58–60). The GhrR is a member of the Class A Rhodopsin-like GPCRs and the signaling of the GhrR is very complex (61, 62). The first discovered and best studied pathway is the increased Ca²⁺ mobilization after activation of the GhrR when inducing growth hormone release in the pituitary gland (63). This effect was traced back to the activation of the Gαq/11-coupled pathway, which initiates the activation of phospholipase C, which will then activate the inositol 1,4,5-trisphosphate and diacylglycerol pathways, which both ultimately result in increased Ca²⁺ mobilization, which increases neurotransmitter release in neurons (64, 65). This discovery was followed by further observation demonstrating that GhrR signaling pathways additionally include Gαi/o, Gα12/13 and the recruitment of β-arrestin ( Figure 2 ), where Gαi/o-coupling may initiate neuronal inhibition. Signaling of Gα12/13 is often considered as one category even though they have different signaling properties (66, 67). It has been shown that Gα12/13 activity is often involved in cytoskeletal rearrangement (68). β-arrestin recruitment is part of a negative feedback mechanism and is not mediated via the G proteins associated with the receptor, however previous G protein coupling may be required (69). β-arrestins are also known to activate the mitogen-activated protein kinase pathway, which will influence gene expression (66, 67). The GhrR full agonist ghrelin can activate all these pathways, but it is unknown whether ghrelin activates all pathways to a similar degree in different cell types and tissues (64, 70, 71). Furthermore, the GhrR has a very high and physiologically relevant constitutive activity of approximately 50% of its maximum capacity and it is able to heterodimerize with other GPCRs such as both DA receptor types (70, 72–74). This is particularly interesting as the high constitutive GhrR activity and the heterodimerization potential may be crucial for GhrR signaling in the VTA, as it is still controversial whether peripheral ghrelin can reach the central nervous system (54).

Even though the complexity of GhrR signaling is well established, still very little is known about physiologically relevant actions and pathways of the GhrR in the DAergic system, even though many studies have shown the importance of the GhrR in reward-related behavior [e.g. (75–77)]. Furthermore, only very few studies have attempted to translate the different pathways into physiological functions. This is an obstacle when it comes to designing GhrR targeting drugs as a high pleiotropic signaling could mediate a higher risk of developing adverse effects. Understanding the exact physiological effects of signaling via the different GhrR pathways would allow the design of biased agonists or antagonists with fewer adverse effects, e.g. in the treatment of AN. It could further open the possibility to target selectively the primary symptoms of AN, such as increase of motivation to eat and food intake in addition to the secondary symptoms, such as impaired gut motility, anxiety and locomotion ( Figure 2 ). Being able to target those secondary symptoms is particularly valuable as clinical trials with AN patients have demonstrated that the patients’ mental state can be negatively affected or sensitive to the increase in appetite and food intake induced by GhrR ligands. Having the increase in food intake accompanied by the attenuation of symptoms that affect the subjective well-being of the patients, such as anxiety and impaired gut motility could lead to a higher compliance to the drug within the AN patient population (34).

As pointed out before, a stable energy balance is important for every living organism and long-term imbalance will lead to changes and impairments of the organism (15). Prolonged fasting induces alterations in the endocrine system, as the body tries to regulate the extreme situation and an increase in systemic ghrelin levels is one of those (78, 79). A systematic meta-analysis showed that all subtypes of AN lead to an increase in ghrelin levels in the acute/fasting state. After receiving treatment, all AN subtypes had decreased ghrelin levels compared to before the treatment, although still increased compared to healthy controls (80). These findings make ghrelin a possible biomarker for AN. Whether the increase in circulating ghrelin is a cause or result of AN is still not understood.

A recent study showed that in female AN patients the decrease in ghrelin levels 30 min after eating breakfast showed a similar pattern of food-induced decrease to the healthy controls. However, the general ghrelin levels were still higher than in healthy controls. Furthermore, a larger percentage decrease in circulating ghrelin after eating (T30-T0) was associated with a larger decrease in the wanting/motivation to eat food (measured with Visual Analog Scales). This effect was only observed in AN patients and not in the healthy controls. Thus, it seems that AN patients are more sensitive to the decrease in ghrelin levels after eating compared to healthy controls (81). Studies investigating AN commonly use healthy controls as their control group, which hinders examining if alterations are AN-specific or due to acute fasting. A recent study investigated the ghrelin levels in young females including AN patients, healthy controls and non-anorexic individuals with food avoidant behavior. This latter group of individuals had the same body mass index as the AN patients, however without the anxiety of weight gain or preoccupation with body shape AN patients have. AN patients demonstrated increased ghrelin levels compared to both control groups. This hints towards a disorder-specific alteration that may be due to differences in the respective motivational drive (82).

That the effects of GhrR signaling show sex-dependent differences has been observed over the last years (83). However, inconsistencies can be found for the food increasing effect of ghrelin. A study observed that female mice were more sensitive to stress-induced restricted food intake and peripheral ghrelin-administration effects including increased food intake, the latter was reduced when the mice were exposed to stress (84). In contrast, another study observed that with ghrelin administered into the hypothalamus, there was no sex-dependent difference in the effect of ghrelin on food intake (85). It was discussed that the sex-dependent differences of ghrelin-effects may be specific for peripherally administered ghrelin and not relevant for centrally administered ghrelin (83). Examining sex-differences of AN in mouse models has been proven to be difficult. With rodent ABA model studies both males and females have been shown to be more prone to develop the AN-like phenotype and no sex-dependent increased vulnerability to the AN-like phenotype was observed in previous studies (86–88). These inconsistencies increase the difficulty to investigate the pathology of sex-differences in AN, which are however an important part of AN, as females are more prone to develop AN and are generally used more for AN studies (2, 89). An important first step would be to include both/all sexes in any study regarding AN, as more data is needed, particularly in regard to animal models.

Particularly interesting is the current discussion of ghrelin as a survival hormone that is responsible for maintaining body weight (BW) in metabolically and psychologically challenging situations. This is considered in the larger context that includes not only food intake regulation, but also glucose homeostasis or effects of chronic stress (90, 91). Studies have shown that under severe caloric restriction a knockout of the GhrR or GOAT leads to an impaired blood glucose maintenance, a stronger decrease in BW and a reduced survival chance (49, 53, 92, 93). These results indicate the importance of the GhrR in blood glucose and BW control under extreme circumstances. Therefore, it has been suggested that the increase in ghrelin levels in AN patients is a survival response to reduce detrimental effects, e.g. BW loss and anxiety (94). This hypothesis is supported by the fact that functional AgRP neurons, which are activated by ghrelin, are important for energy mobilization and survival during the high activity and low energy states responsible for phenotype in the ABA model (95).

Ghrelin is the only known peptide hormone from the gut that has an orexigenic effect (54). The most extensively studied orexigenic ghrelin activity is via the homeostatic system. Ghrelin signaling can reach the hypothalamus both via vagal innervation and via the circulation. It is well established that endocrine ghrelin binds to GhrRs on NPY/AgRP neurons in the ARC, activates them and induces their orexigenic effects, which can lead to increased food intake and adiposity (97–99).

Regarding transgenic ghrelin- or GhrR-deficiency models, no big effect on food intake or BW can usually be observed under basal conditions. However, when the model is metabolically challenged, the importance of the ghrelin pathway can often be observed. For instance, it has been shown that transgenic mice with knockout of the GhrR have a decreased calorie consumption when on a HFD, compared to wild type mice (100). As most studies were performed with male mice, sex-dependent differences of food intake in GhrR knockout mice are only recently explored. It was demonstrated that in males the reduction of mean meal number due to a GhrR knockout was significant while in females no significant mean meal number reduction was observed. However, other food-related parameters showed no genotype x sex interaction effect (101). In one study, a mouse model with a selective knockout of the Gαq/11 subunits in AgRP neurons was generated to gain an insight on the relevance of this specific ghrelin-activated pathway in the context of appetite regulation. The study showed that under basal conditions no difference in food intake could be seen in the knockout mice compared to wild type mice. However, with intracerebroventricular (ICV) administration of ghrelin both knockout and wild type mice showed a significant increase in food intake 60min after administration. Nevertheless, ghrelin-induced food intake of the knockout mice was significantly reduced compared to the food intake in the wild type mice. This indicates the importance of the Gαq/11 pathway in ARC AgRP neurons in ghrelin-induced food intake ( Figure 3 ). The importance of the Gαq/11 pathway was further supported with the biased GhrR agonists Abb13d and YIL781. The inverse agonist for Gαq/11 signaling, Abb13d led to a decrease in food intake during dark phase. The partial agonist YIL781 led to no difference during dark phase, but to an increase in food intake during light phase. This demonstrates that even a partial Gαq/11 activation can induce an increase in food intake, at least during light phase when endogenous ghrelin is low, whereas an inhibition of the Gαq/11 pathway in AgRP neurons will result in reduced food intake (62).

Excessive locomotion in the form of overexercising is a pivotal part of AN, particularly in the restricted type (35). Ghrelin has been shown to be important in the anticipatory increase in locomotion for scheduled meals in rodents. The anticipatory locomotion is positively correlated with ghrelin plasma levels in ABA rats. The same study looked at the differences in anticipatory behavior of ABA rats after GhrR antagonist treatments or in GhrR knockouts. Both the antagonists and knockout of the GhrR led to a decrease in anticipatory activity (102). Contradictory, ghrelin knockout mice fed with a high-fat diet (HFD) had an increase in energy expenditure and locomotor activity compared to wild type mice, which indicates a different effect of ghrelin and GhrR signaling depending on the metabolic state (103). Investigating how GhrR signaling is involved in increased locomotion, particularly in the VTA, may contribute to understanding the etiology of AN. Furthermore, when designing a potential AN drug targeting the GhrR a further increase of locomotion and therefore energy expenditure in the patients could counteract the therapy.

That ghrelin plays a role in anxiety regulation has been shown in several studies, however with contradictory results; both anxiolytic and anxiogenic properties have been attributed to GhrR signaling (104–106). All these studies have been performed under slightly different conditions, which could imply that differently modulated GhrR pathways were initiated. Results from a study provided insight on the importance of GhrR expression sites in anxiety modulation. The study demonstrated a general anxiolytic effect of high peripheral ghrelin doses. Additionally, the authors found that an overexpression of the GhrR led to a weak or strong anxiolytic effect, depending on the expression level in the lateral nuclei of the amygdala, which could indicate that GhrR effects on anxiety depend on the nuclei in which it is expressed (58). However, no study has connected the anxiety-modulating effects to a specific GhrR pathway. Examining which of the GhrR signaling pathways are involved in the anxiety-modulating effects may help to understand the contradictory results so far. The possibility of translating a GhrR pathway to the potential anxiolytic effects of ghrelin may allow a reduction of anxiety in AN patients with a GhrR targeting drug. Furthermore, it could help to investigate if GhrR:DA receptor heterodimerization occurs in the amygdala and which DA receptor types and pathways are involved.

AN patients suffer from an impairment in gut motility, which is reported as one of the main factors reducing the quality of life (107, 108). GhrR signaling in the gastrointestinal tract leads to increased gastric emptying/gut motility (109). In a GhrR biased signaling translation study, two GhrR agonists were used with different properties; Abb13d is an inverse agonist for Gαq/11 and YIL781 is a partial agonist for the same pathway. Both agonists do not or barely activate other GhrR pathways, including β-arrestin recruitment. Both biased agonists led to a decrease in gastric emptying compared to vehicle administration, which indicates that the Gαq/11 pathway is not responsible for inducing gastric emptying/gut motility via the GhrR (62) ( Figure 3 ). It was observed that administration of YIL781 also interferes with defecation induction of intrathecal administered DA. This study further demonstrated a GhrR and D2R co-expression in autonomic neurons. As it is generally assumed that ghrelin is not reaching the central nervous system, these results suggest that a GhrR and D2R signaling interaction, potentially heterodimerization, is necessary for DA-induced defecation (110).