Effects of a selective long‐acting amylin receptor agonist on alcohol consumption, food intake and body weight in male and female rats

Alcohol use disorder is a complex neuropsychiatric disorder affecting both males and females worldwide; however, the efficacy of current pharmacotherapies varies. Recent advances show that gut‐brain peptides, like amylin, regulate alcohol behavioural responses by acting on brain areas involved in alcohol reward processes. Thus, the activation of amylin receptors (AMYRs) by salmon calcitonin (sCT) decreases alcohol behaviours in male rodents. Given that sCT also activates the sole calcitonin receptor (CTR), studies of more selective AMYR agonists in both male and female rodents are needed to explore amylinergic modulation of alcohol behaviours. Therefore, we investigated the effects of repeated administration of a selective long‐acting AMYR agonist, NNC0174‐1213 (AM1213), on alcohol, water and food intake, as well as body weight in male and female rats chronically exposed to alcohol. We confirm our previous studies with sCT in male rats, as repeated AM1213 administration for 2 weeks initially decreased alcohol intake in both male and female rats. However, this reduction ceases in both sexes on later sessions, accompanied by an increase in males. AM1213 reduced food intake and body weight in both male and female rats, with sustained body weight loss in males after discontinuation of the treatment. Moreover, AM1213 administration for 3 or 7 days, differentially altered dopamine, serotonin and their metabolites in the reward‐related areas in males and females, providing tentative, but different, downstream mechanism through which selective activation of AMYR may alter alcohol intake. Our data provide clarified insight into the importance of AMYRs for alcohol intake regulation in both sexes.


| INTRODUCTION
Although alcohol abuse can ultimately lead to the development of alcohol use disorder (AUD), a great burden both for the ailing and the society, 1 current pharmacotherapies for the treatment of this disorder vary in efficiency between patients. 2 In attempts to identify new neurochemical alcohol targets that could serve as potential treatments, studies have evaluated the neurochemical mechanisms through which alcohol activates brain areas involved in reward. 3 These mechanisms include activation of interconnected brain areas like the laterodorsal tegmental area (LDTg), the ventral tegmental area (VTA) [4][5][6] and the nucleus accumbens (NAc). 4,7,8 Despite the complexity of these mechanisms, recent advances have pinpointed gut-brain peptides as modulators of alcohol-mediated behaviours and the development of AUD. 9 The gut-brain peptide amylin decreases food intake 10 and acts as a satiation signal via central amylin receptors (AMYRs) in male rats. 11,12 The amylinergic pathway is involved not only in food regulation but also in the expression of alcohol-related behaviours (for review 9 ). Accordingly, differential expression of the components of the AMYR1, that is, of the calcitonin receptor (CTR) and the receptor activity-modifying protein (RAMP) 1, has been shown in the NAc of male rats consuming high amounts of alcohol compared to low alcohol-consuming rats. 13 Furthermore, salmon calcitonin (sCT), an AMYR agonist, inhibits alcohol induced reward-related behaviours in male mice. 14 Likewise, sCT decreases alcohol intake, prevents relapse drinking and reduces operant self-administration of alcohol in male rats chronically exposed to alcohol. 13 In addition to AMYRs, 15 sCT also activates the (CTR) 16 and has a relatively short half-life, 17 increasing the need of testing a more selective and long-acting AMYR agonist in the investigation of amylinergic regulation of alcohol-mediated behaviours. Furthermore, the recent studies showing that sCT attenuates alcohol-mediated behaviours in rodents have been solely conducted on male animals, despite AUD's prevalence for both males and females. 18,19 Hence, we investigated the effect of repeated administration of the long-acting, and selective over the calcitonin receptor (CTR), AMYR agonist NNC0174-1213 (AM1213), 20 at a dose without behavioural effects per se, on alcohol, water, food intake and body weight in both male and female rats exposed to the intermittent alcohol access paradigm. Additionally, we performed ex vivo biochemical experiments investigating monoamines and their metabolites in the LDTg, VTA and NAc following short-and long-term administration of AM1213. Although preliminary, these data could identify possible neurochemical mechanisms involved in the ability of AM1213 to regulate alcohol intake in male and female rats.

| Animals
Male and female RccHan Wistar rats (Envigo, Horst, The Netherlands) weighing 180 g at the day of arrival were used for the intermittent alcohol access and ex vivo biochemistry experiments. In the intermittent alcohol access experiment, the individually housed (high Macrolon III cages) rats were maintained on a 12-h light/dark cycle (a reversed cycle with the lights off at 9 AM was applied only for the intermittent alcohol access experiment) in rooms with 20 o C and 50% humidity with ad libitum food access. For the ex vivo biochemical experiments, the same rat strain was used, and the animals were maintained under the same conditions as described above.
Individually housed male Sprague Dawley rats (Charles River Laboratories, Calco, Italy) were used for the Irwin test (Supporting Information).
The female rats used in the study were intact and not controlled for estrous cycle as recent literature suggests that the female estrous cycle does not have any effect in voluntary alcohol intake paradigms similar to the one used in our studies. 21 Moreover, in the context of more complex behavioural tasks, like operant sucrose selfadministration, the female estrous cycle does not appear to influence data variability. 22 Taken together, the observed response differences in female rats in this study are likely not attributed to hormonal differences.

| Drugs
The long-acting recombinant amylin analogue, AM1213, 20 was used in the present studies. The compound was diluted in vehicle solution (sodium acetate, glycerol and sterile water solution; pH 4.00 ± 0.05).
It was administered subcutaneously (SC) at the dose of 0.3 mg/kg 120 min prior to dark cycle onset. Alcohol (95%, Solveco, Stockholm, Sweden) was diluted in tap water to 20% v/v for oral consumption. As shown by the in vitro binding affinity and functional properties of AM1213 on rat AMYR and CTR, AM1213 has higher selectivity for the AMYR over the CTR (Supporting Information).

| Irwin test
The Irwin tests evaluate the main central and peripheral nervous system functions 23  2.4.1 | Ten-week baseline alcohol drinking After 1 week of habituation to the animal facilities, the rats were given free access to one bottle of 20% alcohol and one bottle of water during three 24-h sessions per week (Mondays, Wednesdays and Fridays). The rats had unlimited access to two bottles of water on the non-alcohol days (Tuesday, Thursday and weekends). All bottles were weighed daily, 24 h after their presentation to the cages. The rats' body weight was registered once weekly, prior to bottle presentation.
In these rats, a stable alcohol intake was established and was maintained for a 10-week period prior to the initiation of the treatments.

| Two-week AM1213 intervention
After 10 weeks of alcohol intake, the males and females were assigned to treatment groups in a balanced design. Rats were injected with AM1213 or vehicle solution (sodium acetate, glycerol and sterile water solution; pH 4.00 ± 0.05, SC) Monday through Saturday. Measurements of bottles, food intake and body weight were done daily. On alcohol days (Monday, Wednesday and Friday), values for the time points of 1 and 4 h were additionally registered.
Preference for alcohol over water (the ratio of alcohol to total fluid intake), water, total fluid and food intake was calculated for all time points.
To evaluate the effects of AM1213 on the calories obtained from alcohol and food, we calculated the caloric content of alcohol (1.8 kcal/g) and food (3.2 kcal/g) for all the measured time points of 1, 4 and 24 h. Moreover, in order to explore the degree of responsiveness to AM1213 between males and females in regards to alcohol intake, food intake and body weight, we analysed the area under the curve (AUC). The AUC was calculated as the 24-h AM1213 values of each measured parameter subtracted from the vehicle values. For the alcohol intake AUC analysis, we compared the AUC between sexes for sessions 1-3 and for 4-6 separately, because the initial decrease in alcohol intake (sessions 1-3) was followed by return to the baseline (sessions 4-6).

| Two-week washout period
Following the injection period, the rats were not given any drugs for two more weeks and all 24-h values were registered as described above.

| Ex vivo biochemical analysis of monoamines and their metabolites
For this experiment, separate groups of alcohol-naïve male and female rats were used, and the aim was to preliminarily identify the possible role of monoamines modulating the behavioural outcome of AM1213.
Based on the outcome of the intermittent access experiment, two separate setups were included in the ex vivo biochemical test. The In addition, in order to explore possible basal differences between sexes, we analysed the levels of monoamines and their metabolites in the aforementioned brain regions, in the vehicle treated male versus female rats of the short-term administration experiment.

| Tissue isolation
The brains were isolated, rapidly transferred into plastic tubes and were snap frozen in dry ice (stored in −80 C). The frozen brains were placed in a cold rat brain matrix (Zivic instruments, Pittsburgh, PA, USA) and coronally sectioned in 1-mm slices rostral and caudal to the fusion of the optic nerves with the optic chiasm according to the brain atlas. 24 The desired section was placed under a stereoscope on a very cold glass plate (mix of dry ice and regular ice) to avoid tissue degradation. The LDTg, VTA and NAc were subsequently isolated from both sides, using a tissue biopsy punch.

| Statistical analysis
The baseline alcohol intake values between treatment groups and per sex were calculated with an unpaired t test. The data from the 2-week treatment period in the intermittent alcohol access paradigm were assessed with two-way repeated measures ANOVA, followed by a Bonferroni's test for multiple comparisons for the effect of treatment and measured time points (1, 4 and 24 h). While using similar statistics for the washout period data, only the 24-h values were analysed. The levels of monoamines and their metabolites were evaluated by an unpaired, two-tailed test, for comparisons between vehicle and drug conditions per monoamine, brain region and sex. The AUC was calculated as the 24-h AM1213 values subtracted from the vehicle values for each measured parameter. The alcohol intake, food intake and body weight AUC data were further analysed using a t test for differences between sexes. A probability value of P < 0.05 was considered as statistically significant. Data are represented as mean ± SEM.

| AM1213 decreases alcohol consumption, food intake and body weight in male rats
The average daily alcohol intake values during the 10 weeks prior to vehicle or treatment administration did not differ between rats later assigned to AM1213 (4.1 ± 1.3 g/kg; N = 10) and vehicle (4.2 ± 1.3 g/kg; N = 10) groups (P = 0.9752).

| Two-week AM1213 intervention
The overall statistical analysis of all the measured parameters in all time points in male rats is shown in Table 1.
A significant interaction effect was noted for alcohol intake in all measured time points (1, 4 and 24 h). Post hoc analysis revealed that AM1213 (0.3 mg/kg, SC), increased alcohol intake at alcohol session 4 (P = 0.0064) for the 1-h time point ( Figure 1A).
For the 4-h time point, AM1213 decreased alcohol intake at alcohol session 1 (P = 0.0400; Figure 1B). AM1213 treatment decreased alcohol intake at alcohol session 1 (P = 0.0009) and increased the intake at alcohol session 4 (P = 0.0328) for the 24-h time point ( Figure 1C).
The alcohol preference scores were not altered by treatment at the 1-h ( Figure 1D) or 4-h ( Figure 1E) time points. Albeit, a significant interaction effect at the 24-h ( Figure 1F) time point, post hoc analysis did not reveal differences between specific alcohol sessions.
Even though a significant treatment effect at the 1-hour ( Figure 1G) time point, where the water intake for the vehicle is higher than in AM1213 treated rats, post hoc analysis did not reveal any differences at any specific alcohol session. There were no differences in water intake at the 4-h ( Figure 1H) time point. There was a significant interaction effect at the 24-h ( Figure 1I) time point, where the water intake values were higher in the vehicle compared to AM1213 treated group of rats; post hoc analysis showed no differences at any specific alcohol session.
There were no differences in total fluid intake at the time point of 1 h ( Figure 1J), but a decrease was noted on the 4-h total fluid intake at alcohol sessions 1 (P = 0.0235) and 2 (P = 0.0032), after AM1213 treatment ( Figure 1K). A significant interaction effect on 24-h values of total fluid intake was evident; however, post hoc analysis did not reveal any differences at any specific alcohol session ( Figure 1L).

| Two-week washout period
The overall statistical analysis of all the measured parameters in all time points in male rats is shown in Table 2.
As shown in Figure S2A-E, during washout, previous AM1213 treatment had no effect on alcohol intake and alcohol preference.
Albeit a significant interaction effect on water intake, post hoc analysis revealed no significant differences at any specific alcohol session.
There were no differences between the previous treatment groups on total fluid intake or food intake after treatment discontinuation. However, AM1213 retained an inhibitory effect on the rats' body weight at alcohol sessions 1-4 (P = 0.0038, P = 0.0094, P = 0.0218, P = 0.0357 respectively; Figure S2F).

| AM1213 repeated administration decreases alcohol consumption, food intake and body weight in female rats
The average daily alcohol intake values during the 10 weeks prior to vehicle or treatment administration did not differ between rats later assigned to AM1213 (4.6 ± 1.5 g/kg; N = 10) and vehicle

| Two-week AM1213 intervention
The overall statistical analysis of all the measured parameters in all time points in female rats is shown in Table 3.
Although an overall interaction effect on alcohol caloric intake in all measured time pints, AM1213 did not affect alcohol caloric intake at the 1-h time point (Figure S3A), as shown by post hoc analysis.

| Two-week washout period
The overall statistical analysis of all the measured parameters in all time points in female rats is shown in Table 4.
Following treatment discontinuation, there was an interaction effect on alcohol preference, water intake and total fluid intake in the measured 24-h time point. As shown in Figures S4A-D and 4F, after post hoc analysis, AM1213 had no effect on any measured parameter at any specific alcohol session. However, food intake was affected by treatment and time × treatment interaction and that was accompanied by significant increase of food intake in the AM1213 group, at alcohol sessions 4 (P = 0.0007) and 5 (P = 0.0167) as shown in Figure S4E.

| Analysis of the degree of responsiveness to AM1213 between male and female rats
The overall statistical analysis is shown in Table 5.
A separate analysis of the AUC revealed no significant differences between alcohol intake AUC for sessions 1-3 between males and female rats, similarly to the alcohol intake AUC comparison for sessions 4-6. However, there was significant difference between food intake AUC in male and female rats, similarly to the body weight AUC.

| Effects of short-term administration of AM1213 on monoamine levels in male rats
There was no effect of 3-day AM1213 administration on the levels of monoamines in the area of the LDTg (vehicle: N = 8, AM1213: N = 7; Figure 3A). In the VTA (vehicle: N = 6, AM1213: N = 5; Figure 3B

| Effects of long-term administration of AM1213 on monoamine levels in male rats
There was no effect of 7-day AM1213 administration on the levels of

| Effects of short-term administration of AM1213 on monoamine levels in female rats
In the LDTg of female rats (vehicle: N = 8, AM1213: N = 7; Figure 4A Table 7. Figure 4A demonstrates the data obtained from the LDTg, Figure 4B the data from the VTA and Figure 4C the data from the NAc.

| Effects of long-term administration of AM1213 on monoamine levels in female rats
In the LDTg ( Figure S4A) of female rats (vehicle: N = 8, AM1213: N = 6), long-term treatment with AM1213 revealed no significant differences in the levels of monoamines. Analysis of the VTA ( Figure S4B) data of monoamine levels showed no differences after long-term treatment with AM1213 in any monoamine measured (N = 8 per treatment group). AM1213 significantly increased the levels  Table 7. NAc between male and female rats are shown in Table 8.

| DISCUSSION
Previous data have showed that sCT, an AMYR agonist with affinity for the CTR, reduces alcohol-mediated behaviours in male rodents. 13,14 To expand these data, we herein further identify the importance of amylin signaling for alcohol and food intake as well as body weight modulation in both sexes, by using a long-acting selective AMYR analogue. We also indicate a tentative mechanism of action implicated in the outcomes on alcohol intake, by determining the effects of short-and long-term administration of the same compound on ex vivo monoamine levels and their metabolites in reward-related areas in both male and female rats.
AM1213 decreased alcohol intake during the first two alcohol sessions in males and females. Statistical analysis of the AM1213 AUC for alcohol intake revealed a similar response to AM1213 in both sexes. Overall, these data clarify the importance of selective AMYR activation in alcohol intake reduction, which is independent of sex.
Previous studies show that both sCT and calcitonin administration initially reduce alcohol intake. 13,14,25 Given that these compounds also activate the CTR alone, distinguishing the exact role of AMYRs or CTRs in alcohol intake regulation is challenging. Therefore, future studies including knockout models of the AMYR components (i.e., CTR and RAMPs) will further elucidate the role of the CTR, RAMPs and AMYRs in alcohol-related behaviours.
In male rats, the initial decrease in alcohol intake following to reduce alcohol intake. It should however be noted that intrahypothalamic amylin, at least in pharmacologically suprathreshold high doses, changes the levels of serotonin in the brain 29 and that CTR mRNA is colocalised with the serotonin transporter mRNA in the mouse brain. 30 Nevertheless, functional studies indicating that amylin-mediated effects involve the serotonergic system are limited and more studies are needed in order to clarify the role of serotonin in those behaviours.
After the initial decrease, alcohol consumption returned to vehicle baseline on the second alcohol session in male and the third session in female rats. These data are in accordance with our previous studies in male rats, showing that repeated sCT administration decreased alcohol intake during the initial two alcohol sessions, but showed a tolerance effect on the third alcohol drinking session. 13 Interestingly, a similar alcohol drinking pattern is also observed following repeated calcitonin administration in male rats 25 and by glucagon-like peptide-1, another gut-brain peptide. 31,32 During the later alcohol drinking sessions, a visual but not statistically significant biphasic effect on alcohol intake is observed, as we show a discrepancy in response to AM1213 between male and female rats. Interestingly, AM1213 increased alcohol intake on session four in male rats and this consumption visually appears to be elevated on sessions five and six. On the contrary, alcohol intake was at baseline level during sessions 4-6 in female rats. The context of the noted differential effect on alcohol intake between male and female rats remains unknown.
The visually observed difference in the drinking pattern between sexes could possibly be explained by our ex vivo biochemical data. In male rats, which displayed a later increase in alcohol intake, we found an opposite effect on the monoamine levels in the VTA after longcompared to short-term treatment. In fact, in the same area, the dopamine and serotonin turnover are increased, accompanied by reduced serotonin levels. In females, after long-term AM1213 treatment, fewer changes in monoamine levels and their metabolites were noted, which is in line with the observed alcohol-drinking pattern. It should also be noted that after discontinuation of treatment there were no differences in alcohol intake in male or female rats previously treated with AM1213 compared to vehicle.
AM1213 decreased the 24-h water intake, accompanied by reduced total fluid intake in both male and female rats. This is in contrast with previous data showing that single and repeated sCT administration increases water intake in male rats 13 and can possibly be explained by the potentially different mechanisms of action between AM1213 and sCT, as the latter has been attributed diuretic properties 33 that might lead to compensatory increased T A B L E 6 Effects of short-and long-term AM1213 versus vehicle treatment on monoamines and their metabolites in brain areas in male rats  water intake. After discontinuation of treatment, water intake remained lower in females but was slightly elevated in male rats, indicating differences in response between sexes after AM1213 treatment termination.
We further found that repeated AM1213 reduced food intake in both sexes, showing a tolerance pattern in later sessions. AM1213 robustly reduced the initial food intake in both male and female rat.
Notably, food intake returned to baseline on the last session in males, whereas this return to baseline was observed on the fourth session in females. Interestingly, the food intake AUC analysis revealed a higher reduction in males than females. The tolerance pattern following the initial food intake reduction is in accordance with our previous data showing that repeated treatment with sCT reduced food intake during the three alcohol session days, 13 and with other studies showing robust food intake drop after acute and chronic activation of AMYRs in rats. 34,35 Similarly to our results, long-term amylin administration reduces food intake in female rats, most profoundly during the first week of treatment, which corresponds to three alcohol sessions in our study, followed by a return to the baseline at later sessions. 36 This tolerant pattern is evident in other studies, where repeated sCT infusions for 7 weeks did not sustain food intake reduction for more than 5 days in male diet-induced obese rats. 37 Interestingly, following discontinuation of treatment, AM1213 treated females showed increase in food intake on discontinuation sessions 4 and 5, not accompanied by body weight differences.
We here found a robust body weight reduction during the entire treatment period in both sexes. Given the tolerance effect on food intake by AM1213, the prolonged body weight reduction in males plausibly reflects long-term metabolic effects on adiposity, rather than food intake changes. Previous studies in obese male rats showed that daily infusions of amylin and leptin are needed in order to sustain body weight loss. 38 Interestingly, in our studies, AM1213 alone sustained body weight loss in male rats even after its discontinuation, demonstrating a robust long-term effect. Supportively, repeated administration of sCT or amylin reduces body weight in male rats 11,13,39 and amylin causes a sustained decrease in body weight in high-fat fed female rats. 36 The body weight decrease was more profound in males as the analysis of the AUC revealed. Moreover, the body weight reduction was sustained during the washout period in male rats, although this is not observed in females. The protracted effects noted in males suggest that AM1213 acts differently on male and female rats. The observed sex differences could also potentially be attributed to the diverse molecular background between sexes, as female rats have higher mRNA levels of endogenous amylin in the T A B L E 7 Results of short-and long-term AM1213 versus vehicle treatment in monoamines and their metabolites in brain areas in female rats

T A B L E 8
Results of basal monoamine levels and their metabolites in brain areas in males versus female rats Note: Significance level of P < 0.05 after unpaired t test for comparison of monoamines (after vehicle treatment) in brain areas in males versus female rats; N/A, not available comparison. The values emphasised in Bold are values were P < 0.05 (statistically significant effect).
brain than males. 40 This could imply physiologically higher levels of endogenous amylin, which can tentatively influence AMYR activity and thus, in turn, influence the AMYR agonizing effect of AM1213 in females in particular. Another possibility could be the observed difference in basal monoamine levels, as we found that males have altered monoaminergic signalling in the LDTg, VTA as well as NAc compared to females. Nevertheless, given the preliminary nature of the study in regards to differential sex responses to AM1213, further behavioural and molecular experiments are warranted in order to further clarify the mechanisms underlying those sex differences.
The present study shows that AM1213, a selective AMYR agonist, initially reduced alcohol and food intake in male and female rats. The initial reduction in food and alcohol intake is in line with our previous data showing amylinergic regulation of alcohol-mediated behaviours. 13,14 AM1213 decreased body weight in both sexes and this effect is observed even after discontinuation of treatment in male rats. Notably, our present results that AM1213 differentially modulates alcohol, food intake as well as body weight, implies the involvement of distinctive amylinergic modulation of these behaviours, which are different between sexes. Overall, our results expand the significance of AMYRs in the regulation of alcohol intake and pinpoint their importance in alcohol intake reduction in both sexes.